Biomarkers in Kidney Disease PDF

Biomarkers in Disease:
Methods, Discoveries and Applications

Series Editor: Victor R. Preedy
Vinood B. Patel
Victor R. Preedy Editors
Biomarkers in
Kidney Disease
Biomarkers in Disease: Methods, Discoveries
and Applications
Series Editor
Victor R. Preedy
Department of Nutrition and Dietetics
Division of Diabetes and Nutritional Sciences
Faculty of Life Sciences and Medicine
King’s College London
London, UK
In the past decade there has been a sea change in the way disease is diagnosed and
investigated due to the advent of high throughput technologies, such as microarrays,
lab on a chip, proteomics, genomics, lipomics, metabolomics, etc. These advances
have enabled the discovery of new and novel markers of disease relating to autoim-
mune disorders, cancers, endocrine diseases, genetic disorders, sensory damage,
intestinal diseases, etc. In many instances these developments have gone hand in
hand with the discovery of biomarkers elucidated via traditional or conventional
methods, such as histopathology or clinical biochemistry. Together with
microprocessor-based data analysis, advanced statistics and bioinformatics these
markers have been used to identify individuals with active disease or pathology as
well as those who are refractory or have distinguishing pathologies. Unfortunately
techniques and methods have not been readily transferable to other disease states and
sometimes diagnosis still relies on single analytes rather than a cohort of markers.
Furthermore, the discovery of many new markers have not been put into clinical
practice, partly because of their cost and partly because some scientists are unaware
of their existence or the evidence is still at the preclinical stage. In some cases the
work needs further scientific scrutiny. There is thus a demand for a comprehensive
and focused evidenced-based text and scientific literature that addresses these issues.
Hence the formulation of Biomarkers in Disease: Methods, Discoveries and Appli-
cations. The series covers a wide number of areas including for example, nutrition,
cancer, endocrinology, cardiology, addictions, immunology, birth defects, genetics
and so on. The chapters are written by national or international experts and
specialists.
Series Titles
1. General Methods in Biomarker Research and Their Applications

2. Biomarkers in Cancer
3. Biomarkers in Cardiovascular Disease
4. Biomarkers in Kidney Disease
5. Biomarkers in Bone Disease
6. Biomarkers in Liver Disease
More information about this series at http://www.springer.com/series/13842

Vinood B. Patel • Victor R. Preedy
Editors
Biomarkers in Kidney
Disease
With 208 Figures and 142 Tables

Editors
Vinood B. Patel Victor R. Preedy
Department of Biomedical Sciences Department of Nutrition and Dietetics
Faculty of Science and Technology Division of Diabetes and Nutritional
University of Westminster Sciences
London, UK Faculty of Life Sciences and Medicine
King’s College London
London, UK
ISBN 978-94-007-7698-2 ISBN 978-94-007-7699-9 (eBook)

ISBN 978-94-007-7700-2 (print and electronic bundle)
DOI 10.1007/978-94-007-7699-9
Library of Congress Control Number: 2016939598
# Springer Science+Business Media Dordrecht 2016

This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the
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The publisher, the authors and the editors are safe to assume that the advice and information in this book
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editors give a warranty, express or implied, with respect to the material contained herein or for any errors
or omissions that may have been made.
Printed on acid-free paper
This Springer imprint is published by Springer Nature

The registered company is Springer Science+Business Media B.V. Dordrecht
Volume Preface
In the present volume, Biomarkers in Kidney Disease, we have sections on
• General Aspects
• Circulating and Body Fluid Biomarkers
• Specific Diseases and Conditions
• Molecular, Cellular, and Histological Variables
• Functional and Structural Variables
• Resources
While the Editors recognize the difficulties in assigning particular chapters

to particular sections, the book has enormously wide coverage and includes the
following areas, analytes, and conditions: osmolal gap, metabolic acidosis,
metabolomics, hypoxia, micro-RNAs, creatinine, spot urine markers, neutrophil
gelatinase-associated lipocalin (NGAL), chemokines, angiotensinogen, flow
cytometry, leucocytes, lymphocytes, exosomes, N-acetyl-beta-D-glucosaminidase
(NAG), endothelin, methylated arginines, albuminuria, cystatin C,
homocysteinemia, fetal beta2-microglobulin, proteinuric biomarkers, apelin,
copeptin, BLyS and APRIL cytokines, glutathione transferase, growth arrest-specific
protein 6 (Gas6), urokinase receptor, urea nitrogen, allograft damage index (CADI),
antibody arrays, malondialdehyde, matrix metalloproteinase-2 (MMP-2), plasmino-
gen activator inhibitor-1 (PAI-1), fibrosis, kidney biopsies, next generation sequenc-
ing (NGS), cell-cycle arrest biomarkers, integrin-linked kinase (ILK), molecular
biomarkers, M-type phospholipase A2 receptor, ultrasound elastography, aortic
pulse wave velocity, renal arterial resistance index, pulmonary pressure, glomerular
filtration rates, and erythrocyte width. A wide spectrum of acute and chronic
conditions are described including, nephritis, neoplastic disease, transplantation,
allograft damage, cystic fibrosis, diabetes, IgA nephropathy, focal segmental
glomerulosclerosis, renal microthrombosis, and dialysis.
There are also many other analytes and conditions described within this volume.
Finally, the last chapter is devoted to locating resource material for biomarker
discovery and applications.
v
vi Volume Preface
The chapters are written by national or international experts and specialist. This
book is specifically designed for clinical biochemists, nephrologists, specialists
working within the field of kidney disease and treatments, health scientists, epide-
miologists, and doctors and nurses, from students to practioners at the higher level.
It is also designed to be suitable for lecturers and teachers in health care and libraries
as a reference guide.
April 2015 Victor R. Preedy

London Vinood B. Patel
Series Preface
In the past decade, there has been a sea change in the way disease is diagnosed and
investigated due to the advent of high-throughput technologies and advances in
chemistry and physics, leading to the development of microarrays, lab-on-a-chip,
proteomics, genomics, lipomics, metabolomics, etc. These advances have enabled
the discovery of new and novel markers of disease relating to autoimmune disorders,
cancers, endocrine diseases, genetic disorders, sensory damage, intestinal diseases,
and many other conditions too numerous to list here. In many instances, these
developments have gone hand in hand with the discovery of biomarkers elucidated
via traditional or conventional methods, such as histopathology, immunoassays, or
clinical biochemistry. Together with microprocessor-based data analysis, advanced
statistics, and bioinformatics these markers have been used to identify individuals
with active disease as well as those who are refractory or have distinguishing
pathologies.
Unfortunately, techniques and methods have not been readily transferable to other
disease states, and sometimes diagnosis still relies on a single analyte rather than a
cohort of markers. Furthermore, the discovery of many new markers has not been
put into clinical practice partly because of their cost and partly because some
scientists are unaware of their existence or the evidence is still at the preclinical
stage. There is thus a demand for a comprehensive and focused evidenced-based text
and scientific literature that addresses these issues. Hence the book series Bio-
markers in Disease: Methods, Discoveries and Applications. It imparts holistic
information on the scientific basis of health and biomarkers and covers the latest
knowledge, trends, and treatments. It links conventional approaches with new
platforms. The ability to transcend the intellectual divide is aided by the fact that
each chapter has:
• Key Facts (areas of focus explained for the lay person)

• Definitions of Words and Terms
• Potential Applications to Prognosis, Other Diseases, or Conditions
• Summary Points
vii
viii Series Preface
The material in Potential Applications to Prognosis, Other Diseases, or Con-

ditions pertains to speculative or proposed areas of research, cross-transference to
other diseases or stages of the disease, translational issues, and other areas of wide
applicability.
The Series is expected to prove useful for clinicians, scientists, epidemiologists,
doctors, and nurses, and also academicians and students at an advanced level.
April 2015 Victor R. Preedy

London
Contents
Volume 1
Part I General Aspects .................................... 1
1 Biomarkers in Kidney Transplantation ..................... 3

Mohsen Nafar and Shiva Samavat
2 Diagnostic Biomarkers of Acute Kidney Injury in Newborns . . . . 27
Athanasios Chalkias and Nicoletta Iacovidou
3 Osmolal Gap as a Biomarker in Kidney Injury: Focusing on the
Differential Diagnosis of Metabolic Acidosis . . . . . . . . . . . . . . . . . 41
Jeonghwan Lee, Nam Ju Heo, and Jin Suk Han
4 Peritoneal Effluent Biomarker Discovery in Peritoneal Dialysis:
The Omics Era . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Deirisa Lopes Barreto and Dirk G. Struijk
5 Metabolomics and Stages of Chronic Kidney Disease .......... 69
Toshihiro Kobayashi
6 Hypoxia as a Biomarker of Kidney Disease . . . . . . . . . . . . . . . . . . 83
Roger G. Evans, Julian A. Smith, Bruce S. Gardiner,
David W. Smith, Amanda G. Thrift, Clive N. May,
Yugeesh R. Lankadeeva, and Andrew D. Cochrane
7 MicroRNAs in Kidney Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
Grazia Serino, Fabio Sallustio, and Francesco Paolo Schena
8 Serum Creatinine Trajectories in Kidney Disease . . . . . . . . . . . . . 139
Macaulay Onuigbo, Nneoma Agbasi, Ogonna Oguejiofor,
Emmanuel Okocha, Chinawaeze Aneke, and Charles Odenigbo
9 Random Spot Urine Markers for Kidney and Their
Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171
Maria Guedes-Marques, Carlos Botelho, Pedro Maia,
Teresa Mendes, and Armando Carreira
ix
x Contents
10 Overview of Neutrophil Gelatinase-Associated Lipocalin (NGAL)

as a Biomarker in Nephrology . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Valeria Cernaro, Davide Bolignano, Antoine Buemi,
Antonio Lacquaniti, Domenico Santoro, and Michele Buemi
11 Chemokines as Potential Markers in Pediatric Renal
Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229
Ana Cristina Simões e Silva, André Barreto Pereira, Mauro
Martins Teixeira, and Antônio Lúcio Teixeira
12 Kidney and Neoplastic Disease: Overview with a Particular
Interest to Interpretation of Cancer Biomarkers . . . . . . . . . . . . . . 249
Giuseppe Coppolino, Mariadelina Simeoni, Laura Rivoli,
Chiara Summaria, and Davide Bolignano
Part II Circulating and Body Fluid Biomarkers . . . . . . . . . . . . . . . . 269
13 Creatinine Assays in Early Infancy: How to Aim for

a Moving Target . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 271
Karel Allegaert
14 Urinary Angiotensinogen as a Biomarker for Renal Disease . . . . . 301
Zeynep Kendi Celebi, Siyar Erdogmus, and Sule Sengul
15 Flow Cytometry of Urinary Leukocytes and Lymphocytes as
a Biomarker of Renal Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
Philipp Enghard, Birgit Rudolph, Jan Klocke, and
Gabriela Riemekasten
16 Proteome of Human Urinary Exosomes in Diabetic
Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
Gloria Alvarez-Llamas and Irene Zubiri
17 Renal Biomarkers N-Acetyl-Beta-D-Glucosaminidase (NAG),
Endothelin, and Their Application . . . . . . . . . . . . . . . . . . . . . . . . . 369
Serap Çuhadar and Tuna Semerci
18 Methylated Arginines as Biomarkers in Renal Disease . . . . . . . . . 397
Arduino A. Mangoni, Angelo Zinellu, Salvatore Sotgia,
Andrew Rowland, and Ciriaco Carru
19 Albuminuria as a Biomarker of the Renal Disease . . . . . . . . . . . . . 427
Visnja Lezaic
20 Serum Cystatin C as a Biomarker . . . . . . . . . . . . . . . . . . . . . . . . . 445
Serap Çuhadar
21 Homocysteinemia as a Biomarker in Kidney Disease . . . . . . . . . . . 463
Velibor Čabarkapa and Mirjana Đerić
Contents xi
22 Fetal Beta2-Microglobulin as a Biomarker of Kidney Disease . . . . 491

Gilles Grangé, Marie Clémence Leguy, Vassilis Tsatsaris, and
Jean Guibourdenche
23 Proteinuric Biomarkers in Chronic Kidney Disease . . . . . . . . . . . . 515
Claudio Bazzi and Omran Bakoush
24 Apelin and Copeptin as Biomarkers of Kidney Disease . . . . . . . . . 535
Antonio Lacquaniti, Valeria Chirico, Valeria Cernaro,
Rosaria Lupica, Antonio David, and Michele Buemi
25 BLyS and APRIL Cytokines as Biomarkers of Kidney
Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557
26 Erythrocyte Glutathione Transferase as a Biomarker in Kidney
Health and Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577
Alessio Bocedi, Annalisa Noce, Raffaele Fabrini, Nicola Di Daniele,
Francesco Galli, and Giorgio Ricci
27 Plasma Growth Arrest-Specific Protein 6 (Gas6) as a Biomarker
of Renal Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 599
Aybala Erek Toprak
28 Soluble Urokinase Receptor as a Biomarker in Kidney
Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 623
Takehiko Wada
29 Salivary Urea Nitrogen as a Biomarker for Renal Dysfunction . . . 647
Viviane Calice-Silva, Jochen G. Raimann, Wen Wu,
Roberto Pecoits-Filho, Peter Kotanko, and Nathan Levin
Volume 2
Part III Specific Diseases and Conditions .................... 667
30 Chronic Allograft Damage Index (CADI) as a Biomarker in

Kidney Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 669
Ilkka Helanter€a, Fernanda Ortiz, and Petri Koskinen
31 Biomarkers for Kidney Injury in Cystic Fibrosis . . . . . . . . . . . . . . 689
Kevin J. Downes and Stuart L. Goldstein
32 Biomarkers in IgA Nephropathy .......................... 719
33 MicroRNAs as Biomarkers of Diabetic Nephropathy .......... 749
Aaron D. McClelland and Phillip Kantharidis
34 Biomarkers in Focal Segmental Glomerulosclerosis . . . . . . . . . . . . 779
Mohsen Nafar and Shiva Kalantari
xii Contents
35 Biomarkers of Renal Microthrombosis in Lupus Nephritis . . . . . . 811

María Galindo-Izquierdo, Elena Gonzalo-Gil, Oscar Toldos, and
José Luis Pablos-Álvarez
36 Planar Antibody Arrays for Biomarkers in Nephritis . . . . . . . . . . 831

Christer Wingren
37 Malondialdehyde as a Biomarker in Kidney Transplantation . . . . 849

Isabel Fonseca
38 Utility of Neutrophil Gelatinase-Associated Lipocalin in

Kidney Transplantation: Detailed Review . . . . . . . . . . . . . . . . . . . 875
Juan C. Ramirez-Sandoval, William Herrington, and
Luis E. Morales-Buenrostro
39 miR-210 as a Biomarker in Renal Carcinoma . . . . . . . . . . . . . . . . 895

Hideto Iwamoto, Mitsuhiko Osaki, Masashi Honda,
Takehiro Sejima, Atsushi Takenaka, and Futoshi Okada
40 Matrix Metalloproteinase-2 (MMP-2) and Plasminogen Activator

Inhibitor-1 (PAI-1) in Peritoneal Dialysis: Biological Implications
and Clinical Utility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 911
Deirisa Lopes Barreto and Raymond T. Krediet
Part IV Molecular, Cellular, and Histological Variables . . . . . . . . . 931
41 Assessing Fibrosis in Kidney Biopsies . . . . . . . . . . . . . . . . . . . . . . . 933

Behtash Ghazi Nezami and Alton B. Farris
42 Next-Generation Sequencing (NGS) in Biomarker Discovery

and Applications in Nephrology . . . . . . . . . . . . . . . . . . . . . . . . . . . 955
Imari Mimura and Masaomi Nangaku
43 Cell Cycle Arrest Biomarkers in Kidney Disease . . . . . . . . . . . . . . 977

Kianoush Kashani, Erin N. Frazee, and John A. Kellum
44 Integrin-Linked Kinase (ILK) Expression as a Biomarker in

Cancer of the Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 991
Miriam de Fatima Brasil Engelman and
Gustavo Gonçalves Engelman
45 Molecular Biomarkers and Treatments for Renal

Cell Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1015
46 M-Type Phospholipase A2 Receptor as a Biomarker in

Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1033
Elion Hoxha and Rolf A.K. Stahl
Contents xiii
Part V Functional and Structural Variables .................. 1049
47 Ultrasound Elastography in Kidney Disease . . . . . . . . . . . . . . . . . 1051

Fuat Ozkan, Cemil Goya, Sema Yildiz, Mahmut Duymus,
Mehmet Sait Menzilcioglu, Serhat Avcu, and Mehmet Fatih Inci
48 Aortic Pulse Wave Velocity as a Biomarker in Chronic Dialysis
Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1077
Petar Avramovski and Aleksandar Sikole
49 Renal Arterial Resistance Index . . . . . . . . . . . . . . . . . . . . . . . . . . . 1101
Massimo Iacoviello, Valeria Antoncecchi, Marta Leone, and
Marco Matteo Ciccone
50 Pulmonary Pressure as a Novel Prognostic Biomarker in Renal
Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1121
Davide Bolignano, Francesco Mattace-Raso, Eric J. Sijbrands,
Anna Pisano, and Giuseppe Coppolino
51 Estimation of Glomerular Filtration Rate . . . . . . . . . . . . . . . . . . . 1143
Antonín Jabor, Janka Franeková, and Lenka Hošková
52 Red Blood Cell Distribution Width: Useful Predictor for
Treatment Response in Primary Glomerular Diseases . . . . . . . . . . 1175
Kenan Turgutalp, Simge Bardak, Serap Demir, and
Ahmet Kıykım
Part VI Resources ....................................... 1193
53 Recommended Resources on Biomarkers in Kidney Disease . . . . . 1195

Rajkumar Rajendram, Vinood B. Patel, and Victor R. Preedy
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1205
About the Editors
Vinood B. Patel
Reader in Clinical Biochemistry
Course Leader for MSc Clinical Biochemistry
Department of Biomedical Science, Faculty of Science
and Technology
University of Westminster
London, UK
Dr. Vinood B. Patel B.Sc., Ph.D., FRSC is currently a

Reader in Clinical Biochemistry at the University of Westminster and honorary
fellow at King’s College London. He presently directs studies on metabolic path-
ways involved in liver disease, particularly related to mitochondrial energy regula-
tion and cell death. Research is being undertaken to study the role of nutrients,
antioxidants, phytochemicals, iron, alcohol, and fatty acids in the pathophysiology
of liver disease. Other areas of interest are identifying new biomarkers that can be
used for diagnosis and prognosis of liver disease, understanding mitochondrial
oxidative stress in Alzheimer’s disease, and gastrointestinal dysfunction in autism.
Dr. Patel graduated from the University of Portsmouth with a degree in Pharmacol-
ogy and completed his Ph.D. in Protein Metabolism from King’s College London in
1997. His postdoctoral work was carried out at Wake Forest University Baptist
Medical School studying structural-functional alterations to mitochondrial ribo-
somes, where he developed novel techniques to characterize their biophysical
properties. Dr. Patel is a nationally and internationally recognized liver researcher
and was involved in several NIH-funded biomedical grants related to alcoholic liver
disease. Dr. Patel has edited biomedical books in the area of nutrition and health
prevention, autism, and biomarkers and has published over 150 articles, and in 2014
he was elected as a Fellow to The Royal Society of Chemistry.
Victor R. Preedy B.Sc., Ph.D., D.Sc., FRSB, FRSH, FRIPHH, FRSPH,

FRCPath, FRSC is a senior member of King’s College London (Professor of
Nutritional Biochemistry) and King’s College Hospital (Professor of Clinical Bio-
chemistry; Honorary). He is attached to both the Diabetes and Nutritional Sciences
Division and the Department of Nutrition and Dietetics. He is also founding and
xv
xvi About the Editors
current Director of the Genomics Centre and a member of the School of Medicine.
Professor Preedy graduated in 1974 with an Honours Degree in Biology and
Physiology with Pharmacology. He gained his University of London Ph.D. in
1981. In 1992, he received his Membership of the Royal College of Pathologists,
and in 1993 he gained his second doctoral degree, for his contribution to the science
of protein metabolism in health and disease. Professor Preedy was elected as a
Fellow of the Institute of Biology (Society of Biology) in 1995 and to the Royal
College of Pathologists in 2000. He was then elected as a Fellow to the Royal
Society for the Promotion of Health (2004) and The Royal Institute of Public Health
and Hygiene (2004). In 2009, Professor Preedy became a Fellow of the Royal
Society for Public Health, and in 2012 a Fellow of the Royal Society of Chemistry.
In 2015, the Society of Biology received its Royal Charter, so Professor Preedy
became an FRSB. In his career, Professor Preedy worked at the National Heart
Hospital (part of Imperial College London) and the MRC Centre at Northwick Park
Hospital. He has collaborated with research groups in Finland, Japan, Australia,
USA, and Germany. He is a leading expert on biomedical sciences and has a long-
standing interest in analytical methods and biomarkers, especially their applications
to the study of health and disease. He has lectured nationally and internationally. To
his credit, Professor Preedy has published over 500 articles, which includes peer-
reviewed manuscripts based on original research, reviews, abstracts, and numerous
books and volumes.
Editorial Advisors
Caroline J. Hollins Martin School of Nursing, Midwifery and Social Care Edin-
burgh Napier University (Sighthill Campus), Midlothian, UK
Ross J. Hunter The Barts Heart Centre, St Bartholomew’s Hospital, London, UK
Colin R. Martin Faculty of Society and Health, Buckinghamshire New University,
Uxbridge, Middlesex, UK
Rajkumar Rajendram Division of Diabetes and Nutritional Sciences, Faculty of
Life Sciences and Medicine, King’s College London, London, UK
Department of Anaesthesia and Intensive Care, Stoke Mandeville Hospital,
Aylesbury, UK
xvii
Contributors
Nneoma Agbasi North East London NHS Foundation Trust, Ilford, Essex, UK
Karel Allegaert Department of Development and Regeneration, KU Leuven,
Leuven, Belgium
Neonatal Intensive Care Unit, University Hospitals Leuven, Leuven, Belgium
Intensive Care and Surgery, Erasmus MC, Sophia Children’s Hospital, Rotterdam,
The Netherlands
Gloria Alvarez-Llamas Department of Immunology, IIS-Fundación Jiménez Díaz,
UAM, Madrid, Spain
Chinawaeze Aneke Department of Medicine, Nnamdi Azikiwe Teaching Hospital,
Awka, Nigeria
Valeria Antoncecchi Cardiology Unit, School of Cardiology, Department of
Emergency and Organ Transplantation, University of Bari, Bari, Italy
Serhat Avcu Department of Radiology, Faculty of Medicine, Gazi University,
Ankara, Turkey
Yingyos Avihingsanon Division of Nephrology, Department of Medicine, Faculty
of Medicine, Chulalongkorn University and King Chulalongkorn Memorial
Hospital, Bangkok, Thailand
Center of Excellence in Immunology and Immune-Mediated Diseases, Faculty of
Medicine, Chulalongkorn University, Bangkok, Thailand
Petar Avramovski Department of Internal Medicine, Clinical Hospital “D-r Trifun
Panovski”, Bitola, Macedonia
Omran Bakoush Department of Nephrology, Lund University, Lund, Sweden
Department of Internal Medicine, UAE University, Al Ain, United Arab Emirates
Simge Bardak Division of Nephrology, Department of Internal Medicine, School
of Medicine, Mersin University, Mersin, Turkey
Claudio Bazzi D’Amico Foundation for Renal Disease Research, Milan, Italy
xix
xx Contributors
Alessio Bocedi Department of Chemical Sciences and Technologies, University of

Rome “Tor Vergata”, Rome, Italy
Davide Bolignano CNR – Institute of Clinical Physiology, Reggio Calabria, Italy
Carlos Botelho Nephrology Department, Centro Hospitalar e Universitário de
Coimbra – Hospital dos Covões, Coimbra, Portugal
Antoine Buemi Department of Clinical and Experimental Medicine, University of
Messina, Messina, Italy
Michele Buemi Department of Clinical and Experimental Medicine, University
Hospital “G. Martino”, Messina, Italy
Viviane Calice-Silva School of Medicine, Pontifícia Universidade Católica do
Paraná, Curitiba, PR, Brazil
Armando Carreira Nephrology Department, Centro Hospitalar e Universitário de
Ciriaco Carru Department of Biomedical Sciences, University of Sassari, Sassari,
Italy
Velibor Čabarkapa Department of Pathophysiology, Medical Faculty Novi Sad,
University of Novi Sad, Novi Sad, Serbia
Zeynep Kendi Celebi Nephrology Department, Ankara University School of
Medicine, Ibni Sina Hospital, Ankara, Turkey
Valeria Cernaro Department of Clinical and Experimental Medicine, University
Athanasios Chalkias University of Athens, Medical School, MSc “Cardiopulmo-
nary Resuscitation”, Piraeus, Greece
Hellenic Society of Cardiopulmonary Resuscitation, Athens, Greece
Juan Chipollini Department of Urology, University of Miami Miller School of
Medicine, Miami, FL, USA
Valeria Chirico Department of Pediatrics, University Hospital “G. Martino”,
Messina, Italy
Marco Matteo Ciccone Cardiology Unit, School of Cardiology, Department of
Emergency and Organ Transplantation, University of Bari, Bari, Italy
Andrew D. Cochrane Department of Surgery, Monash University, Melbourne,
VIC, Australia
Giuseppe Coppolino Nephrology and Dialysis Unit, University Hospital of
Catanzaro, Catanzaro, Italy
Serap Çuhadar Department of Medical Biochemistry, Ataturk Training and
Research Hospital, Izmir, Turkey
Contributors xxi
Nicola Di Daniele Department of Systems Medicine, Nephrology and Hyperten-

sion Unit, University of Rome “Tor Vergata”, Rome, Italy
Antonio David Department of Neuroscience and Anesthesiology, University
Serap Demir Division of Nephrology, Department of Internal Medicine, School of
Medicine, Mersin University, Mersin, Turkey
Mirjana Đerić Department of Pathophysiology, Medical Faculty Novi Sad,
University of Novi Sad, Novi Sad, Serbia
Kevin J. Downes Department of Pediatrics, Perelman School of Medicine, The
University of Pennsylvania, Philadelphia, PA, USA
Division of Infectious Diseases, The Children’s Hospital of Philadelphia,
Philadelphia, PA, USA
Mahmut Duymus Department of Radiology, Faculty of Medicine, Gazi Univer-
sity, Ankara, Turkey
Miriam de Fatima Brasil Engelman Department of Pathology, Faculdade de
Ciências da Saúde Dr. José Antônio Garcia Coutinho, Universidade do Vale do
Sapucaí (UNIVÁS), Pouso Alegre, Minas Gerais, Brazil
Gustavo Gonçalves Engelman Faculdade de Medicina Universidade José do
Rosário Vellano (UNIFENAS), Alfenas, Minas Gerais, Brazil
Philipp Enghard Klinik mit Schwerpunkt Nephrologie und internistische
Intensivmedizin, Charité Berlin, Berlin, Germany
Klinik mit Schwerpunkt Rheumatologie und klinische Immunologie, Charité Berlin,
Berlin, Germany
Siyar Erdogmus Nephrology Department, Ankara University School of Medicine,
Ibni Sina Hospital, Ankara, Turkey
Roger G. Evans Department of Physiology, Monash University, Melbourne, VIC,
Australia
Raffaele Fabrini Department of Chemical Sciences and Technologies, University
of Rome “Tor Vergata”, Rome, Italy
Alton B. Farris Department of Pathology and Laboratory Medicine, Emory
University Hospital, Atlanta, GA, USA
Isabel Fonseca Department of Nephrology and Kidney Transplantation, Centro
Hospitalar do Porto, Hospital de Santo António, Porto, Portugal
Janka Franeková Department of Laboratory Methods, Institute for Clinical and
Experimental Medicine, CZ, Prague, Czech Republic
Erin N. Frazee Hospital Pharmacy Services, Mayo Clinic, Rochester, MN, USA
xxii Contributors
María Galindo-Izquierdo Rheumatology Department, Hospital Universitario

12 de Octubre, Madrid, Spain
Francesco Galli Department of Pharmaceutical Sciences, University of Perugia,
Perugia, Italy
Bruce S. Gardiner School of Engineering and Information Technology, Murdoch
University, Perth, WA, Australia
Stuart L. Goldstein Division of Nephrology and Hypertension, Center for Acute
Care Nephrology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH,
USA
Elena Gonzalo-Gil Rheumatology Department, Hospital Universitario 12 de
Octubre, Madrid, Spain
Cemil Goya Department of Radiology, Faculty of Medicine, Dicle University,
Diyarbakir, Turkey
Gilles Grangé CHU Cochin, AP-HP, Maternité Port Royal, Paris, France
Maria Guedes-Marques Nephrology Department, Centro Hospitalar e
Universitário de Coimbra – Hospital dos Covões, Coimbra, Portugal
Jean Guibourdenche CHU Cochin, AP-HP, Biologie hormonale, INSERM
U1139, Physiologie, Faculté de Pharmacie, Université Paris Descartes, Paris, France
Jin Suk Han Department of Internal Medicine, Seoul National University, College
of Medicine, Seoul, Republic of Korea
Ilkka Helanter€ a Transplantation and Liver Surgery, Helsinki University Hospital,
Helsinki, Finland
Martin J. P. Hennig Department of Urology, University of L€ubeck, L€ubeck,
Germany
Nam Ju Heo Department of Internal Medicine, Healthcare System Gangnam
Center, Seoul National University Hospital, Seoul, Republic of Korea
William Herrington Oxford Kidney Unit, Oxford University Hospitals NHS
Trust, Churchill Hospital, Oxford, UK
Masashi Honda Division of Urology, Faculty of Medicine, Tottori University,
Yonago, Tottori, Japan
Lenka Hošková Cardiology Clinic, Institute for Clinical and Experimental Medi-
cine, CZ, Prague, Czech Republic
Elion Hoxha University Medical Center Hamburg-Eppendorf, Hamburg, Germany
Nicoletta Iacovidou Hellenic Society of Cardiopulmonary Resuscitation, Athens,
Greece
Contributors xxiii
University of Athens, Medical School, Aretaieio Hospital, Department of Neonatol-

ogy, Athens, Greece
Massimo Iacoviello Cardiology Unit, Cardiothoracic Department, University

Hospital Policlinico Consorziale of Bari, Bari, Italy
Mehmet Fatih Inci Department of Radiology, Izmir Katip Celebi University,
School of Medicine, Izmir, Turkey
Hideto Iwamoto Division of Urology, Faculty of Medicine, Tottori University,

Antonín Jabor Department of Laboratory Methods, Institute for Clinical and

Experimental Medicine, CZ, Prague, Czech Republic
Shiva Kalantari Chronic Kidney Disease Research Center (CKDRC), Shahid
Beheshti University of Medical Sciences, Tehran, Iran
Department of Basic Science, Faculty of Paramedical Sciences, Shahid Beheshti
University of Medical Sciences, Tehran, Iran
Phillip Kantharidis JDRF Danielle Alberti Memorial Centre for Diabetes Com-
plications, Diabetes Domain, Baker IDI Heart and Diabetes Institute, Melbourne,
VIC, Australia
Kianoush Kashani Division of Nephrology and Hypertension, Department of
Medicine, Mayo Clinic, Rochester, MN, USA
Division of Pulmonary and Critical Care Medicine, Department of Medicine, Mayo
Clinic, Rochester, MN, USA
John A. Kellum The Center for Critical Care Nephrology, CRISMA, Department
of Critical Care Medicine, University of Pittsburgh School of Medicine, Pittsburgh,
PA, USA
Ahmet Kıykım Division of Nephrology, Department of Internal Medicine, School
of Medicine, Mersin University, Mersin, Turkey
Jan Klocke Klinik mit Schwerpunkt Rheumatologie und klinische Immunologie,

Charité Berlin, Berlin, Germany
Toshihiro Kobayashi College of Nutrition, Koshien University, Takarazuka,
Hyogo, Japan
Petri Koskinen Department of Medicine, Division of Nephrology, Helsinki Uni-
versity Hospital, Helsinki, Finland
Peter Kotanko Renal Research Institute, New York, NY, USA

Raymond T. Krediet Division of Nephrology, Academic Medical Center –
University of Amsterdam, Amsterdam, The Netherlands
xxiv Contributors
Antonio Lacquaniti Department of Clinical and Experimental Medicine,

University Hospital “G. Martino”, Messina, Italy
Yugeesh R. Lankadeeva Department of Physiology, Monash University,
Melbourne, VIC, Australia
Florey Institute of Neuroscience and Mental Health, University of Melbourne,
Parkville, VIC, Australia
Jeonghwan Lee Department of Internal Medicine, Hallym University Hangang
Sacred Heart Hospital, Seoul, Republic of Korea
Asada Leelahavanichakul Division of Immunology, Department of Microbiology,
Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
Center of Excellence in Immunology and Immune-Mediated Diseases, Faculty of
Medicine, Chulalongkorn University, Bangkok, Thailand
Marie Clémence Leguy CHU Cochin, AP-HP, Biologie hormonale, Paris, France
Marta Leone Cardiology Unit, School of Cardiology, Department of Emergency
and Organ Transplantation, University of Bari, Bari, Italy
Nathan Levin Renal Research Institute, New York, NY, USA
Visnja Lezaic Faculty of medicine, University of Belgrade, Belgrade, Serbia
Vinata B. Lokeshwar Department of Biochemistry and Molecular Biology,
Medical College of Georgia, Georgia Regents University, Augusta, GA, USA
Deirisa Lopes Barreto Division of Nephrology, Academic Medical Center,
University of Amsterdam, Amsterdam, The Netherlands
Rosaria Lupica Department of Clinical and Experimental Medicine, University
Pedro Maia Nephrology Department, Centro Hospitalar e Universitário de
Arduino A. Mangoni Department of Clinical Pharmacology, School of Medicine,
Flinders University, Adelaide, Australia
Francesco Mattace-Raso Department of Internal Medicine, Erasmus University
Medical Center, Rotterdam, The Netherlands
Clive N. May Florey Institute of Neuroscience and Mental Health, University of
Melbourne, Parkville, VIC, Australia
Aaron D. McClelland JDRF Danielle Alberti Memorial Centre for Diabetes Com-
plications, Diabetes Domain, Baker IDI Heart and Diabetes Institute, Melbourne,
VIC, Australia
Teresa Mendes Nephrology Department, Centro Hospitalar e Universitário de
Contributors xxv
Mehmet Sait Menzilcioglu Department of Radiology, Faculty of Medicine, Gazi

University, Ankara, Turkey
Imari Mimura Division of Nephrology and Endocrinology, The University of
Tokyo, Tokyo, Japan
Luis E. Morales-Buenrostro Department of Nephrology and Mineral Metabolism,
National Institute of Medical Sciences and Nutrition Salvador Zubirán, D.F., Mexico
Mohsen Nafar Department of Nephrology, Labbafinejad Medical Center, Shahid
Chronic Kidney Disease Research Center (CKDRC), Shahid Beheshti University of
Medical Sciences, Tehran, Iran
Masaomi Nangaku Division of Nephrology and Endocrinology, The University of
Tokyo, Tokyo, Japan
Behtash Ghazi Nezami Department of Digestive Diseases, Department of Medi-
cine, Emory University School of Medicine, Atlanta, GA, USA
Department of Pathology and Laboratory Medicine, Emory University Hospital,
Atlanta, GA, USA
Annalisa Noce Department of Systems Medicine, Nephrology and Hypertension
Unit, University of Rome “Tor Vergata”, Rome, Italy
Charles Odenigbo Department of Medicine, Nnamdi Azikiwe Teaching Hospital,
Awka, Nigeria
Ogonna Oguejiofor Department of Medicine, Nnamdi Azikiwe Teaching Hospi-
tal, Awka, Nigeria
Futoshi Okada Division of Pathological Biochemistry, Faculty of Medicine,
Tottori University, Yonago, Tottori, Japan
Emmanuel Okocha Department of Medicine, Nnamdi Azikiwe Teaching Hospital,
Awka, Nigeria
Macaulay Onuigbo Department of Medicine, College of Medicine, Mayo Clinic,
Rochester, MN, USA
Department of Nephrology, Mayo Clinic Health System, Eau Claire, WI, USA
Fernanda Ortiz Department of Medicine, Division of Nephrology, Helsinki
University Hospital, Helsinki, Finland
Mitsuhiko Osaki Division of Pathological Biochemistry, Faculty of Medicine,
Tottori University, Yonago, Tottori, Japan
Fuat Ozkan Department of Radiology, Faculty of Medicine, Gazi University,
Ankara, Turkey
xxvi Contributors
José Luis Pablos-Álvarez Rheumatology Department, Hospital Universitario

12 de Octubre, Madrid, Spain
Vinood B. Patel Department of Biomedical Sciences, Faculty of Science and
Technology, University of Westminster, London, UK
Roberto Pecoits-Filho School of Medicine, Pontifícia Universidade Católica do
Paraná, Curitiba, PR, Brazil
André Barreto Pereira Department of Molecular Medicine, Faculty of Medicine,
Interdisciplinary Laboratory of Medical Investigation, Federal University of Minas
Gerais, Belo Horizonte, MG, Brazil
Anna Pisano CNR – Institute of Clinical Physiology, Reggio Calabria, Italy
Wannarat Pongpirul Division of Nephrology, Department of Medicine, Faculty of
Medicine, Chulalongkorn University and King Chulalongkorn Memorial Hospital,
Bangkok, Thailand
Department of Medicine, Bamrasnaradura Infectious Diseases Institute, Ministry of
Public Health, Nonthaburi, Thailand
Victor R. Preedy Department of Nutrition and Dietetics, Division of Diabetes
and Nutritional Sciences, Faculty of Life Sciences and Medicine, King’s College
London, London, UK
Jochen G. Raimann Renal Research Institute, New York, NY, USA
Rajkumar Rajendram Division of Diabetes and Nutritional Sciences, Faculty of
Department of Anaesthesia and Intensive Care, Stoke Mandeville Hospital,
Aylesbury, UK
Juan C. Ramirez-Sandoval Department of Nephrology and Mineral Metabolism,
National Institute of Medical Sciences and Nutrition Salvador Zubirán, D.F., Mexico
Giorgio Ricci Department of Chemical Sciences and Technologies, University of
Rome “Tor Vergata”, Rome, Italy
Gabriela Riemekasten Rheumatologie, Universit€at L€ubeck, L€ubeck, Germany
Laura Rivoli Nephrology and Dialysis Unit, University Hospital of Catanzaro,
Catanzaro, Italy
Andrew Rowland Department of Clinical Pharmacology, School of Medicine,
Flinders University, Adelaide, Australia
Birgit Rudolph Institut f€ur Pathologie, Charité Berlin, Berlin, Germany
Fabio Sallustio Department of Emergency and Organ Transplantation, University
of Bari, Bari, Italy
Contributors xxvii
Shiva Samavat Department of Nephrology, Labbafinejad Medical Center, Shahid

Domenico Santoro Department of Clinical and Experimental Medicine, University
of Messina, Messina, Italy
Francesco Paolo Schena Department of Emergency and Organ Transplantation,
University of Bari, Bari, Italy
C.A.R.S.O. Consortium, University of Bari, Bari, Italy
Takehiro Sejima Division of Urology, Faculty of Medicine, Tottori University,
Tuna Semerci Department of Medical Biochemistry, Giresun University, Giresun,
Turkey
Sule Sengul Nephrology Department, Ankara University School of Medicine, Ibni
Sina Hospital, Ankara, Turkey
Grazia Serino Laboratory of Experimental Immunopathology, IRCCS “de Bellis”,
Castellana Grotte, BA, Italy
Eric J. Sijbrands Department of Internal Medicine, Erasmus University Medical
Center, Rotterdam, The Netherlands
Aleksandar Sikole Medical Faculty “Ss. Cyril and Methodius University” Skopje,
University Clinic of Nephrology, Skopje, Republic of Macedonia
Mariadelina Simeoni Nephrology and Dialysis Unit, University Hospital of
Catanzaro, Catanzaro, Italy
Ana Cristina Simões e Silva Unit of Pediatric Nephrology, Department of Pediat-
rics, Faculty of Medicine, Interdisciplinary Laboratory of Medical Investigation,
Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
Julian A. Smith Department of Surgery, Monash University, Melbourne, VIC,
Australia
David W. Smith School of Computer Science and Software Engineering, The
University of Western Australia, Perth, WA, Australia
Salvatore Sotgia Department of Biomedical Sciences, University of Sassari, Sas-
sari, Italy
Rolf A.K. Stahl University Medical Center Hamburg-Eppendorf, Hamburg,
Germany
Dirk G. Struijk Division of Nephrology, Academic Medical Center, University of
Amsterdam, Amsterdam, The Netherlands
xxviii Contributors
Chiara Summaria Nephrology and Dialysis Unit, University Hospital of Catan-

zaro, Catanzaro, Italy
Atsushi Takenaka Division of Urology, Faculty of Medicine, Tottori University,
Mauro Martins Teixeira Department of Biochemistry and Immunology, Institute
of Biological Sciences, Laboratory of Immunopharmacology, Federal University of
Minas Gerais, Belo Horizonte, MG, Brazil
Antônio Lúcio Teixeira Department of Medicine, Faculty of Medicine, Interdisci-
plinary Laboratory of Medical Investigation, Federal University of Minas Gerais,
Belo Horizonte, MG, Brazil
Amanda G. Thrift Department of Medicine, Monash University (Monash Medical
Centre), Melbourne, VIC, Australia
Oscar Toldos Pathology Department, Hospital Universitario 12 de Octubre,
Madrid, Spain
Aybala Erek Toprak Göztepe Training and Research Hospital, Medical Biochem-
istry Lab, Istanbul Medeniyet University School of Medicine, Kadıkoy/Istanbul,
Turkey
Natavudh Townamchai Division of Nephrology, Department of Medicine, Fac-
ulty of Medicine, Chulalongkorn University and King Chulalongkorn Memorial
Hospital, Bangkok, Thailand
Vassilis Tsatsaris CHU Cochin, AP-HP, Maternité Port Royal, Paris, France
Kenan Turgutalp Division of Nephrology, Department of Internal Medicine,
School of Medicine, Mersin University, Mersin, Turkey
Takehiko Wada Division of Nephrology, Endocrinology and Metabolism, Tokai
University School of Medicine, Isehara, Japan
Christer Wingren Department of Immunotechnology and CREATE Health, Lund
University, Lund, Sweden
Wen Wu Integrated Biomedical Technology, Elkhart, IN, USA
Sema Yildiz Department of Radiology, Faculty of Medicine, Gazi University,
Ankara, Turkey
Angelo Zinellu Department of Biomedical Sciences, University of Sassari, Sassari,
Italy
Irene Zubiri Queen Mary University of London, London, UK
Part I
General Aspects
Biomarkers in Kidney Transplantation
1
Contents
Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Key Facts of Operational Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Key Facts of Costimulatory Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
Biomarkers of Allograft Rejection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Biomarkers of Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Gene Expression Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
Potential Applications to Prognosis, Other Diseases, or Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
Abstract
Kidney transplantation is the optimal renal replacement therapy. The progressions
in immunosuppressive drugs improved the short-term survival, but 10-year graft
survival is about 50 %, only. Acute or chronic rejection, drug nephrotoxicity, and
transplant glomerulopathy all have adverse impacts on graft survival. Most of
these events are the result of over- or under-immunosuppression.
On the other hand, tolerance as a state of no immunosuppression in the
presence of functioning graft is an ultimate goal of transplantation.
M. Nafar • S. Samavat (*)

Department of nephrology, Labbafinejad medical center, Shahid Beheshti University of
e-mail: nafar@sbmu.ac.ir; m.nafar.md@gmail.com; shsamavat@gmail.com;
sh_samavat@yahoo.com
# Springer Science+Business Media Dordrecht 2016 3

V.B. Patel, V.R. Preedy (eds.), Biomarkers in Kidney Disease, Biomarkers in Disease:
Methods, Discoveries and Applications, DOI 10.1007/978-94-007-7699-9_29
4 M. Nafar and S. Samavat
In order to individualize treatments and recognize the optimal level of immu-

nosuppression, noninvasive methods for diagnosis of acute rejection and toler-
ance have been developed, and biomarkers in the shade of technological advances
would help physician in this way. Peripheral blood cell, plasma, and urine are
readily accessible and perfect specimens for identification of biomarkers. This
review is focused on recently developed biomarkers in acute rejection and
tolerance as the two most important processes in decision-making about immu-
nosuppressive therapy. The clinical utilities and limitations of these markers are
discussed in details.
Keywords
Kidney transplantation • Acute rejection • Tolerance • Biomarker • Genomics •
Proteomics • miRNA
Abbreviations
AR Acute rejection
ATI Acute tubular injury
ATN Acute tubular necrosis
AUC Area under the curve
BPAR Biopsy-proven acute rejection
CAD Chronic allograft dysfunction
CAMR Chronic antibody-mediated rejection
CE-MS Capillary electrophoresis mass spectrometry
CMV Cytomegalovirus
COT Clinical operational tolerance
Cr Creatinine
CXCL-10 C-X-C motif chemokine 10
DGF Delayed graft function
eGFR Estimated glomerular filtration rate
ELISA Enzyme-linked immunosorbent assay
Foxp3 Forkhead/winged helix transcription factor
IF/TA Interstitial fibrosis/tubular atrophy
IRI Ischemia-reperfusion injury
IS Immunosuppression
LC-MS Liquid chromatography-mass spectrometry
LC-MS/MS Liquid chromatography-tandem mass spectrometry
MMP-8 Matrix metalloproteinase-8
NPV Negative predictive value
PBMC Peripheral blood mononuclear cell
PCR Polymerase chain reaction
PPV Positive predictive value
qPCR Quantitative polymerase chain reaction
RT-qPCR Real-time quantitative polymerase chain reaction
SELDI-TOF-MS Surface-enhanced laser desorption/ionization time-of-flight
mass spectrometry
1 Biomarkers in Kidney Transplantation 5
TCMR T-cell-mediated rejection

TG Transplant glomerulopathy
TOL Tolerance
Treg Regulatory T-cells
UMOD Uromodulin
UTI Urinary tract infection
VEGF Vascular endothelial growth factor
Key Facts
Key Facts of Operational Tolerance
• Operational tolerance is a state of stable graft function despite cessation of

immunosuppressive drug for more than a year without evidences of chronic
rejection.
• Most cases were reported in liver transplantation.
• The majority of cases in renal transplantation are due to noncompliance or
intentional withdrawal due to lymphoproliferative disorders.
• Lack of donor-specific antibodies and donors of young age are related to opera-
tional tolerance.
Key Facts of Costimulatory Signal
• T-cell activation requires two signals.

• Signal 1 is an antigen-specific pathway that involves T-cell receptor and major
histocompatibility complex.
• Signal 2 is the result of other T-cell surface receptors and their ligands on antigen-
presenting cell.
• Cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) and CD28 and their
ligands B7-1 and B7-2 are the major receptors involved.
• CTLA-4 binding to B7-1 and B7-2 is an inhibitory signal and leads to anergy.
• Abatacept and belatacept are CTLA4Igs that block costimulatory signal.
• CTLA4Ig is a competitive inhibitor of CD28 binding.
• Targeting receptors and/or ligands in costimulatory pathway is a way to increase
graft survival.
Definitions
ELISA The enzyme-linked immunosorbent assay is based on antigen and antibody

interaction and enzyme-induced color changes in substrate. Antigens are attached
into wells in a plate. Then an antibody that can bind to the antigen and is linked to an
enzyme is added. The next step is the addition of substrate. The reaction causes color
change in the substrate, and the intensity of the color signal is indicative of the
amount of antigen present.
Genomics Genomics is a combination of genome detection methods (polymerase

chain reaction) and bioinformatics to detect the whole genome in a cell and to
identify the function and pathways that are involved.
Microarray Microarray is one of the tools in genomics, which is consisted of a

glass slide with DNA molecules attached to it in specific spots. It detects gene
expression, and the data is processed and normalized and the results are expressed in
a gene expression matrix. The information from microarray studies is presented
either in absolute measures or expression ratio.
MicroRNAs MicroRNAs (miRNAs) are 21–23-nucleotide noncoding RNAs that

regulate posttranscriptional gene expression by binding to target mRNA leading to
either the degradation of mRNA or inhibition of their transcription.
Proteomics Proteomics is the analysis of the whole protein content of a biofluid.

The changes in the proteomes are caused by changes in synthesis or modifications
during the course of biologic or pathologic processes. These modifications can be
used as specific markers of the process.
SELDI-TOF technique One of the proteomic techniques for profiling the prote-
ome of different types of samples using mass spectrometer. This technique does not
need sample preparation procedure and may serve as a diagnostic tool. Low resolu-
tion and lack of reproducibility are some of the limitations of this technique.
Introduction
Kidney transplantation is the most physiologic renal replacement therapy. Despite

significant improvement in 1-year graft survival, long-term graft survival improve-
ment was minimally increased (Hariharan et al. 2000).
During the early phase (first 2 weeks, mostly) of kidney transplantation, factors
affecting the outcome are those related to the status of the donated kidney, ischemia-
reperfusion injury, acute tubular necrosis (ATN), and the resulting delayed graft function
(DGF). Acute rejections whether antibody-mediated or cell-mediated ones are other
determinants of graft survival especially during the first posttransplant year.
During recent years, advances in immunosuppressive protocols lead to better
short-term graft survival. On the contrary, the incidence of highly sensitized recip-
ients, extended criteria donors, and marginal kidney quality are rising, and therefore
detecting patients at higher risk of acute rejection and prompt intervention is critical
to save the organ.
The early-phase insults might occur subclinically and consequently cause chronic
allograft rejection, transplant glomerulopathy, and end in chronic allograft loss.
Detecting rejection based on currently available techniques (increased serum

creatinine or allograft biopsy) is either inaccurate, late, or invasive.
There is an urgent need for markers of graft status from early to late phase of
transplantation to ensure timely diagnosis of events before irreversible histologic
damage occurred.
On the other hand, overzealous immunosuppression causes infection and malignan-
cies in the long term. It would be wise to adjust immunosuppressive regimes according
to the immunologic risk of each individual patient (Lodhi and Meier-Kriesche 2011).
In order to define a biomarker or a panel of biomarkers for a specific process,
apart from accuracy, precision, and validity, one must describe the clinical utility of
the marker, such as when to evaluate and the frequency of assessments. Additionally,
these biomarkers must be clinically available and cost effective.
Biofluids such as blood and urine are readily available and relatively noninvasive
samples with the ability of repeated sampling and follow-up monitoring.
Finding and proving the clinical use of biomarkers of ATN, DGF, acute rejection,
transplant glomerulopathy (TG), chronic allograft dysfunction (CAD), and tolerance
would help to prolong allograft survival. In the following sections, biomarkers of
acute rejection and allograft tolerance will be discussed as a guide for immunosup-
pression therapy.
Biomarkers of Allograft Rejection
Diagnosis of acute rejection is currently based on histologic assessment of allograft

sample, which is invasive and has a minor risk of bleeding complications. Additionally,
current markers such as serum creatinine cannot detect subclinical rejections (Rush
et al. 1994). To improve clinical outcome, there is a need to find markers that predict
events before histopathologic and mostly irreversible evidences of rejection become
evident and have the ability to differentiate rejection from other causes of allograft
inflammation and dysfunction such as pyelonephritis, viral infection, and ATN.
Differentially expressed proteins in blood or urine sample of transplant patients
might help to have early diagnosis, predict outcome, and response to therapy in a
noninvasive way.
Urine Biomarkers
Urine is an easily accessible biofluid, which allows repeated sampling and reflects
intrarenal processes.
Perforin, Granzyme B, and Fas-L mRNA

The major players in cell-mediated rejection are cytotoxic T-cells. CD8+ T-cells are
first cells that appear at the scene of rejection. Activated cytotoxic T-cells release
granzyme B and perforin. Perforin allows granzyme B to enter the target cells and
lead to cell death via mitochondrial apoptotic pathways. Additionally, a small
portion of endothelial cell death is mediated by Fas-ligand (Fas-L) pathway (Choy
2010). Apart from CD8+ T-cells, CD30+ T-cells have been proven to be involved in
alloimmunity, and CD30 acts as a costimulatory molecule (S€usal et al. 2011). Thus,
urinary cytotoxic markers might be helpful in diagnosis of acute rejection.
Urinary concentration of perforin and granzyme B mRNA was elevated in
24 patients with biopsy-proven acute rejection (BPAR) compared with 22 patients
with other diagnoses (chronic allograft nephropathy, toxic tubulopathy, ATN, and
nonspecific findings). The ROC curve for perforin mRNA at the cutoff of 0.9 fg per
microgram of total RNA showed 83 % sensitivity and specificity for diagnosis of
acute rejection. At the cutoff point of 0.4 fg per microgram of total RNA for
granzyme B mRNA, granzyme B had 79 % sensitivity and 77 % specificity in
identifying acute rejection (Li et al. 2001). These data demonstrate diagnostic value
of cytotoxic markers; however, the question is whether they could distinguish acute
rejection from other etiologies of inflammation. In a study, urinary mRNA levels of
perforin, granzyme B, and Fas-L were followed longitudinally in 37 cadaveric
transplant patients by the means of real-time PCR assay. Urine samples were
collected during the episodes of BPAR, cytomegalovirus (CMV) infection and
disease, urinary tract infection (UTI), DGF, and CAD. Perforin, granzyme B, and
Fas-L mRNA levels were significantly higher in BPAR than controls with stable
graft function. Interestingly, the urinary levels of markers were not significantly
different among patients with BPAR, UTI, CMV infection or disease, and DGF
(Yannaraki et al. 2006). Therefore these markers are not specific for acute rejection
and are evidences of graft inflammation.
Granzyme A mRNA
Granzyme A along with granzyme B is the most abundant cytolytic molecules of the
effector T-cells. It also triggers inflammation by induction of cytokines. Its role as a
biomarker of subclinical and clinical T-cell-mediated rejection (TCMR) has been
evaluated in a study on 60 patients in six different groups, including those with stable
graft function, CMV infection, calcineurin inhibitor toxicity, subclinical rejection
(SCR), TCMR-I (with prominent tubulitis), and TCMR-II (with moderate or severe
intimal arteritis and tubulitis). High urinary granzyme A mRNA was able to differ-
entiate patients with SCR and TCMR-I from those stable graft function and
calcineurin inhibitor toxicity. However, this marker was also elevated in patients
with CMV infection; thus, confronting an increased urinary granzyme A, one must
rule out the presence of CMV infection by CMV-PCR (van Ham et al. 2010).
It seems that granzyme A could be a useful marker in diagnosis of subclinical
rejection after exclusion of CMV infection and gives the clinician enough time to
promptly treat the patients before occurrence of irreversible damage.
Foxp3 mRNA
Regulatory T-cells are known since 1975 and have regulatory role in immune
response and are involved in tolerance. In the biopsy samples of acute rejection,
increased infiltration of Tregs along with effector T-cells has been shown.
The immunoregulatory role of Tregs was proven in acute rejections as they con-
trolled further damage. Forkhead/winged helix transcription factor (Foxp3) is
expressed by Tregs and could be used as a marker of their presence and activity
(Brown and Wong 2008).
Urinary expressions of Foxp3 mRNA along with CD3E, perforin, and CD25 were
significantly higher in patients with biopsy-proven acute rejection compared with
those with chronic allograft nephropathy and stable graft function. Foxp3 mRNA
level was inversely correlated with severity of acute rejection. Interestingly, there
was no correlation among other markers (perforin, CD3E, and CD25) and serum
creatinine in patients with acute rejection. Urinary Foxp3 mRNA was predictive of
acute rejection episode reversibility, and at the cutoff of 3.46, it had a sensitivity of
90 % and specificity of 73 % in prediction of reversal of graft function. Furthermore,
the combination of serum creatinine and the Foxp3 mRNA level was more accurate
in predicting the reversal of acute rejection with 96 % specificity. The results indicate
that the higher the Foxp3 mRNA level, the greater the chance of reversal of acute
rejection. These are all in line with damage controlling role of Tregs (Muthukumar
et al. 2005).
Thus, increased urinary Foxp3 mRNA is useful in diagnosis as well as predicting
the outcome of acute rejection.
Cytokine/Chemokine mRNA
Cytokines and chemokines (chemotactic cytokines) play a major role in the inflam-
matory cascade. Each cytokine represents activation of a specific pathway.
C-X-C motif chemokine 10 (CXCL-10) also known as interferon gamma-induced
protein 10 (IP-10) is secreted by monocytes, endothelial cells, and renal tubular and
mesangial cells in response to interferon-γ (IFNγ). CXCL-10 by binding to its
receptor CXCR-3 on activated T-cells and natural killer cells leads to leukocyte
recruitment during acute rejection (Ho et al. 2011).
Data suggested that urinary CXCL-10 elevation preceded serum creatinine rise.
Urine CXCL-10 can be used as a marker of inflammation and can distinguish
tubulitis (histologic characteristic of cellular rejection) from fibrosis. In a study of
91 patients with a wide range of histologic findings from normal to various degrees
of tubulitis (borderline, subclinical, and clinical tubulitis) and those with interstitial
fibrosis and tubular atrophy (IF/TA), urine CXCL-10-to-creatinine (CXCL-10/Cr)
ratio at the cutoff of 2.87 ng/mmol had 81.8 % sensitivity and 86.4 % specificity in
differentiating normal histology from subclinical and clinical tubulitis. At the lower
cutoff of 1.97 ng CXCL-10/mmol Cr, the sensitivity and specificity for diagnosis of
normal histology versus borderline or subclinical tubulitis were 73.3 % and 72.7 %,
respectively (Ho et al. 2011).
Along with CXCL-10, the other CXCR-3 ligand, CXCL-9, was shown to be
correlated with subclinical rejection. At the cutoff of 7.5 ng/mmol Cr, CXCL-9 had
86 % sensitivity and 64 % specificity in diagnosis of subclinical tubulitis from
normal histology or borderline tubulitis. Urinary CXCL-10 and CXCL-9 were not
elevated in those with IF/TA as a sole histologic finding (Schaub et al. 2009).
The advantage of these chemokines is earlier appearance in urine than CXCR-3,
perforin, and granzyme B and therefore timely recognition of subclinical tubulitis.
These chemokines have same accuracy in pediatric as well as adult transplant

patients (Jackson et al. 2011).
Unlike granzyme B and perforin, urine CXCL-10 level is not increased in other
inflammatory processes such as UTI and CMV infection (Ho et al. 2011). Tubuloin-
terstitial inflammation by BK virus and ischemia-reperfusion injury (IRI) might
increase urinary levels of CXCL-9 and CXCL-10. Therefore, it is necessary to
exclude BK virus infection by plasma PCR. The effects of IRI would not last
more than 2 months, and thereafter urine chemokines could be reliable markers of
tubulitis due to rejection (Schaub et al. 2009). The influence of UTI on urine
chemokines is controversial; thus, to be on the safe side, it is better to rule out UTI
by negative urine cultures.
A group evaluated the clinical utility of CXCL-9 in risk stratification, prediction
of acute rejection in patients with acute graft dysfunction, and prediction of late graft
loss. In the setting of acute graft dysfunction, urinary levels of CXCL-9 mRNA had a
negative predictive value (NPV) of more than 92 % in putting acute rejection aside.
As its positive predictive value (PPV) was about 61–67 %, this biomarker could not
be used instead of the gold standard tissue biopsy, but the high NPV might help to
avoid the unnecessary invasive kidney biopsy. The NPV was independent of recip-
ient age, HLA mismatch, and de novo donor-specific antibodies. The elevated urine
CXCL-9 mRNA level preceded the serum creatinine increment by almost 30 days,
and thus it could be used as a predictor of intragraft inflammation days before the
clinically evident increase in serum creatinine and as a guide for prompt treatment.
Additionally, high urine CXCL-9 mRNA level at 6 months posttransplantation could
predict >30 % decrement in estimated glomerular filtration rate (eGFR) at 24 months
posttransplantation. In this study, urinary level of CXCL-9 was higher in patients
with acute rejection than in those with BK virus infection (Hricik et al. 2013).
Briefly, the urinary mRNA of CXCL-9 is a promising marker to rule out acute
rejection and graft inflammation based on its high NPV. As measurement of CXCL-9
protein by ELISA is easier and more reliable in clinical settings, according to the
current data, its use to exclude acute rejection is suggested.
OX40/OX40-L mRNA
During T-cell activation along with T-cell receptor (TCR) and major histocompati-
bility complex (MHC) interaction on antigen-presenting cells (APC), there are
second regulatory signals consisted of costimulatory and co-inhibitory pathways
(Fig. 1). The major molecular players of these pathways are from either the immu-
noglobulin superfamily (CD28, CTLA-4, CD80 and CD86, PD-1, and PD-L) or the
TNF family (CD40, CD40L, OX40, and OX40-L) (Ford et al. 2014).
OX40 interaction with its ligand causes memory T-cell generation and cytokine
production and results in Th2 response and leads to acute rejection. On the contrary,
PD-1 and PD-L ligation acts as an inhibitory signaling pathway on T-cells. In a
study, the urinary mRNA expression of costimulatory pathway members was com-
pared between patient with stable graft function and those with biopsy-proven acute
rejection. The group reported significantly increased levels of OX40, OX40-L, and
PD-1 mRNA in urinary cells of patients with acute rejection. PD-1L levels were not
T-Cell APC
Negative
PD-1 PD-L
CTLA-4 CD80/86
TCR MHC
CD28 CD80/86
Positive
OX40 OX40-L
Fig. 1 The costimulatory pathway. Costimulatory signaling results from interaction of ligands on
antigen-presenting cells (APCs) and the related protein on T-cells. Signals with positive effect lead
to T-cell proliferation and cytokine production, and signals with negative effects cause anergy and
apoptosis. CTLA-4 cytotoxic T-lymphocyte-associated protein 4, MHC major histocompatibility
complex, PD-1 programmed cell death protein 1, PD-L programmed cell death protein 1 ligand,
TCR T-cell receptor
different between the two groups. OX40 mRNA level alone at a cutoff of 5.98 had a
sensitivity of 81 % and specificity of 88 % in diagnosis of acute rejection. When
combined with urinary levels of mRNA for OX40-L, PD-1, and Foxp3, the sensi-
tivity and specificity would rise to 95 % and 92 %, respectively. Also the higher
OX40-L mRNA level (cutoff value of 3.79) predicted the higher probability of
reversal of acute rejection (sensitivity of 69 % and specificity of 100 %) (Afaneh
et al. 2010).
Thus, OX40 and its ligand might be used as diagnostic and also predictive
biomarker of acute rejection.
mRNA Signature
In a recent study, investigators introduced a urinary mRNA profile instead of a single
mRNA in approach to kidney transplant patient with acute graft dysfunction by the
means of RT-qPCR. They suggested an mRNA signature with the ability to differ-
entiate acute rejection (AR) from acute tubular injury (ATI).
Combination of urinary values of CD3E, CD105, TLR4, CD14, complement
factor B, and vimentin mRNAs formed a diagnostic signature that differentiated AR
from ATI. Data suggested that using this signature decreases the unnecessary
allograft biopsies. Among patients with AR, a five-mRNA diagnostic model was
developed that differentiated acute cellular rejection (ACR) from antibody-mediated
rejection (AMR). This model was consisted of CD3E, CD105, CD14, CD46, and
18S rRNA with the area under the curve of 0.81 (95 % confidence interval,
0.68–0.93). Decision curve analysis to assess the clinical benefit was performed in
this study (Matignon et al. 2014).
Briefly, using the signature model of mRNAs helps decreasing the number of
biopsies in patients with acute graft dysfunction.
Urine miRNAs
MicroRNAs (miRNAs) are 21–23-nucleotide noncoding RNAs that regulate post-
transcriptional gene expression by binding to target mRNA leading to either the
degradation of mRNA or inhibition of their transcription. They play a role in almost
every cellular pathway, and each cell type has its own miRNA pattern. The miRNA
profile is representative of the ongoing biologic process and could be evaluated in
different biofluids such as urine, blood, and other body fluids. Despite its high cost,
RT-qPCR has the ability of detecting a wide range of miRNA when compared with
microarray (Mas et al. 2013).
Lorenzen et al. were the first group evaluating the diagnostic role of urine miRNA
in acute rejection. Using RT-qPCR, urine samples of 62 patients with biopsy-proven
acute rejection were compared with those of patients with stable graft function. The
initial data found 21 differentially expressed miRNAs among patients and controls.
Among these miRNAs, miR-210 and miR-10b were downregulated, and miR-10a
was upregulated in patients with acute rejection compared to the controls with stable
graft function. Lower levels of miR-201 were correlated with faster eGFR decline
and more severe rejection. Successful reversal of acute rejection normalized the
miR-210 and miR-10b levels. The variations in urine levels of miR-210 were
independent of the presence of leukocyturia and UTI and age (Lorenzen
et al. 2011). If further validation studies confirm these findings, miR-210 could
serve as a noninvasive biomarker in diagnosis of acute rejection. However, based on
the results from samples collected before evolution of rejection, miR-210 could not
predict the impending episodes of acute rejection.
Urine Proteomics
In search for biomarkers, urine proteome profile comes to the center of attention. It is
the indicator of local processes in kidney and systemic events that might change
urine proteins. In order to characterize urine proteome profile in acute rejection,
several studies have been performed (Table 1). Some are discussed in more details.
In a study on 73 patients with graft dysfunction who underwent indication biopsy,
by the means of surface-enhanced laser desorption/ionization time-of-flight mass
spectrometry (SELDI-TOF-MS), two differentially expressed peptides were identi-
fied. In patients with acute rejection compared with other causes of graft dysfunction,
urinary expression of human β-defensin-1 (HBD-1) was reduced, and urinary
expression of α-1-antichymotrypsin (ACT) was elevated. Both of these markers
are part of inflammatory and immune responses. When used in combination, the
elevated ACT and decreased HBD-1 levels, the sensitivity and specificity for
diagnosis of acute rejection would be 85.7 % and 80.2 %, respectively (O’Riordan
et al. 2007).
Table 1 Selected urine biomarker for acute allograft rejection

Detection
Biomarker method Cutoff AUC Sensitivity (%) Specificity (%) Reference
sHLA-DR ELISA 15 U/mL 0.88 80 98 Ting
et al. (2010)
sUPAR ELISA NA NA NA NA Roelofs
et al. (2003)
VEGF ELISA 3.64 pg/μmol Cr 0.871 85.1 78.4 Peng
et al. (2008)
MASP2 LC-MS/MS NA NA NA NA Loftheim
et al. (2012)
CD103 RT-qPCR 8.16 copies/μg Cr 0.73 59 75 Ding
mRNA et al. (2003)
TIM-3 RT-qPCR 1.2a 0.96 84 96 Manfro
mRNA et al. (2008)
ChrY dPCR 3 copies of 0.80 81 75 Sigdel
dd-cfDNAb ChrY/K μg Cr et al. (2013)
AUC area under curve, CE-MS capillary electrophoresis mass spectrometry, ChrY dd-cfDNA chromosome
Y donor-derived cell-free DNA, Cr creatinine, dPCR digital polymerase chain reaction, MASP2 isoform
2 of mannan-binding lectin serine protease 2, MMP-8 matrix metalloproteinase-8, NA not available,
RT-qPCR real-time quantitative polymerase chain reaction, sUPAR soluble urokinase-type plasminogen
activator receptor, TIM-3 T-cell immunoglobulin mucin domain 3, VEGF vascular endothelial growth
factor
a
By the relative quantification method 2ΔΔCT
b
It is a sensitive marker for diagnosis of acute allograft injury, but it is not that specific to distinguish acute
rejection from BK virus nephropathy
Metzger et al. conducted a multicenter study on 103 transplant patients to identify

biomarkers of acute subclinical and clinical rejection and the role of confounding
conditions such CMV infection, BK virus infection, and UTI. Capillary electropho-
resis mass spectrometry (CE-MS) analyses were used to evaluate urine peptide
pattern. Not a single peptide was able to discriminate rejection from other clinical
conditions with an acceptable specificity, but a panel of 14 differentially expressed
peptides was extracted with the area under the curve (AUC) of 0.89. In order to
further validate the panel, the group used it in a validation set and reached an AUC of
0.91 and 93 % sensitivity and 78 % specificity. The presence of UTI and CMV
infection did not cause any misclassification. Most of the peptides in this panel were
collagen α-1 fragments, which could be an indicator of extracellular matrix degra-
dation and matrix metalloproteinase-8 (MMP-8) activity (Metzger et al. 2011).
Sigdel et al. conducted a shotgun proteomic study with capillary LC-MS/MS on
92 urine samples of patients, including those with biopsy-proven acute rejection,
stable graft function, nephrotic syndrome, and healthy controls. The advantage of
this study is that they further validated the identified markers by ELISA in an
independent set of samples, which is more cost effective, and affordable assay for
clinical use. Most of the discriminating proteins in the acute rejection group were
MHC antigens, complement pathway proteins, and extracellular matrix proteins.
Applying ELISA, they reported significantly decreased uromodulin (UMOD) (AUC
= 84.6 %) and CD44 (AUC = 97.3 %) in those with acute rejection with a
Table 2 Urine biomarker panels in diagnosis of acute allograft rejection

Biomarker panel Detection method Reference
ANXA11 ("), integrin α3 ("), Antibody microarrays and reverse Srivastava
integrin β3 ("), TNF-α (") capture protein microarray et al. (2011)
IP-10 ("), MIG ("), I-TAC (") Luminex assays Huang et al. (2014)
UMOD (#), SERPINF1 ("), LC-MS/MS Sigdel et al. (2010)
CD44 (")
COL1A2, COL3A1, UMOD, LC-MS and multiple reaction Ling et al. (2010)
MMP-7, SERPING1, TIMP1a monitoring (MRM)
HLA-DRB1 ("), fibrinogen beta iTRAQ Sigdel et al. (2014)
("), fibrinogen gamma (")
ID-3796 peptide and 13 collagen α CE-MS Metzger
(I, III) fragments et al. (2011)
CLCA1 ("), PROS1 ("), and 2D-LC-MS/MS Sigdel
KIAA0753 (")b et al. (2014b)
ANXA11 annexin A 11, COL1A collagen type 1 α, CLCA1 calcium-activated chloride channel
regulator-1, IP-10 IFN-induced protein 10, I-TAC IFN-induced T-cell chemoattractant, MIG
monokine induced by IFNγ, MMP-7 matrix metalloproteinase-7, PROS1 vitamin K-dependent
protein S, SERPINF1 pigment epithelium-derived factor (PEDF), SERPING1 serpin peptidase
inhibitor, TIMP1 tissue inhibitor of metalloproteinase 1, TNF-α tumor necrosis factor-α, UMOD
uromodulin
a
Gene expression
b
Exosomal proteins
correlation coefficient of 0.99 and 0.84, respectively, and significantly elevated

pigment epithelium-derived factor (PEDF, SERPINF1) levels (AUC = 93.2 %)
with a correlation coefficient of 0.78. Thus, this pattern of peptides could verify
acute rejection in transplant patients with high sensitivity and specificity independent
of age, proteinuria, and immunosuppression protocol (Sigdel et al. 2010) (Table 2).
As there are concerns about the confounding factors such as the amount of
proteins in urine (the effect of highly abundant proteins on identification of proteins
with lower abundance) and BK virus nephropathy (a pathologically challenging
diagnosis), the group conducted a study based on urine peptidomic analysis by
LC-MS and multiple reaction monitoring (MRM) on 70 urine samples from 50 trans-
plant patients. Peptidomic analysis provides information about disease-related mod-
ification on proteins (proteolytic and antiproteolytic activities). The abundance of
UMOD and collagen peptides (COL1A2 and COL3A1) in urine was lower in
patients with acute rejection. Evaluating the transcriptome in kidney tissue of these
patients demonstrated higher gene expression for matrix metalloproteinase-7
(MMP-7), tissue inhibitor of metalloproteinase 1 (TIMP1), and the serpin peptidase
inhibitor (SERPING1) in patients with acute rejection. The abovementioned changes
were independent of the presence of BK nephropathy. Apart from being a specific
biomarker profile, this panel sheds light on the underlying mechanism of injury
during acute rejection and subsequent chronic graft fibrosis: the collagen cascade
(Ling et al. 2010).
Recently, the isobaric tags for relative and absolute quantitation (iTRAQ) prote-
omic technique was used to identify biomarkers of acute rejection. The proteins then
were validated by ELISA. Of a total of 389 measured proteins, nine were highly
specific for acute rejection. These were identified as: HLA class II protein
HLA-DRB1, keratin-14 (KRT14), histone H4 (HIST1H4B), fibrinogen gamma
(FGG), actin-beta (ACTB), fibrinogen beta (FGB), fibrinogen alpha (FGA),
keratin-7 (KRT7), and dipeptidyl-peptidase-4 (DPP4). These markers could differ-
entiate acute rejection from chronic allograft injury and BK virus nephropathy.
Further validation, by ELISA in independent samples, showed increased urinary
levels of HLA-DRB1, fibrinogen beta, and fibrinogen gamma (Sigdel et al. 2014a).
Overall, urine peptidomics and proteomics are raising horizon in the land of
biomarker studies. The identified profile needs to be validated by a less time and
cost-consuming technique such as ELISA for routine clinical utility.
Blood Biomarkers
Evaluating blood biomarkers is also a minimally invasive way to diagnose acute
rejection. However, the diagnostic profile might be confounded by systemic milieu,
and its sensitivity and specificity might decline. Numerous markers were introduced
by different studies using various techniques, but clinical validation is needed before
routine application (Table 3).
Table 3 Selected serum biomarker for acute allograft rejection

Biomarker Method Sample Reference
Granzyme B, RT-PCR PBL Vasconcellos et al. (1998)
perforin, Fas-L
Foxp3 RT-PCR PBL Aquino-Dias et al. (2008)
IFNγ – producing ELISPOT Pretransplant PBML Nickel et al. (2004)
memory T-cell
Nitric oxide Serum Bellos et al. (2011) and
Masin-Spasovska
et al. (2013)
PECAM1 ELISA Serum Chen et al. (2010)
HLA class I (ABC) Flow Peripheral blood CD3 Tian et al. (2009)
cytometry +/CD8+ T
lymphocytes
Titin, kininogen-1, iTRAQ Plasma Freue et al. (2010)
and LPS-BP
IL-1R antagonist, Luminex™ Serum Xu et al. (2013)
IL-20, and sCD40 bead array
ligand analysis
ELISA enzyme-linked immunosorbent assay, ELISPOT enzyme-linked immunosorbent spot, IL-1R
interleukin-1 receptor, iTRAQ isobaric tagging for relative and absolute protein quantification, LPS-
BP lipopolysaccharide-binding protein, PBL peripheral blood leukocytes, PBML peripheral blood
mononuclear cells, PECAM1 platelet endothelial cell adhesion molecule 1
CD30
CD30 as a marker of Th2-type immune response has been shown to be associated
with allograft outcome (Pelzl et al. 2002). Soluble CD30 (sCD30) as a potential
marker of an alloimmunity reaction was evaluated in 203 living kidney transplant
patients before, on the fifth day posttransplantation, and at the time of acute increase
in serum creatinine with ELISA kit. sCD30 levels among patients with BPAR were
compared with those of patients with stable graft function and non-rejection cause of
acute allograft dysfunction (including CMV infection, ATN, and calcineurin inhib-
itor toxicity). sCD30 level on the fifth day posttransplantation with the cutoff value
of 41 U/ml predicted the occurrence of acute rejection in the first 6 months with a
sensitivity and specificity of 70 % and 71.7 %, respectively. It could not predict the
2-year graft survival. Pretransplant sCD30 level could not predict acute rejection,
and there was a significant elevation in sCD30 level during the episodes of BPAR.
Thus, sCD30 level after transplantation and its changes could be used as a predictor
of acute rejection (Nafar et al. 2009). In a multicenter study on 2,322 transplant
patients, investigators demonstrated an association between day 30 posttransplant
CD30 level and 3-year graft survival. CD30 levels 40 U/ml on day 30 were
associated with high anti-HLA antibody activity and could be considered as a marker
of alloimmunity (S€usal et al. 2011). Same results were obtained in an earlier study, of
course with smaller sample size but longer follow-up of 5 years posttransplantation
(Delgado et al. 2009). Thus, posttransplant CD30 level might be utilized as a marker
of increased alloimmunity and if proved by clinical trials might be used as a guide to
immunosuppressive dose adjustment.
Genomics
In order to enhance the sensitivity and specificity of peripheral blood diagnostic
tests, transcriptional profile (genomics) was utilized by the means of microarray
studies. Gene expression in peripheral blood samples was extensively evaluated in
association with acute rejection. Since 1998 that Vasconcellos et al. described the
correlation of cytotoxic lymphocyte gene expression (perforin, granzyme B, and
Fas-ligand) and acute rejection (Vasconcellos et al. 1998), there are a wide range of
studies evaluating gene expression of various effector molecules in diagnosis and
prediction of rejection.
T-cell immunoglobulin mucin domain 3 (TIM-3) is a membrane glycoprotein
expressed on Th1 cells, cytotoxic T-cells, natural killer cells, and Th17. It has a
known role in inducing tolerance. TIM-3 binding to its ligand, galectin-9, results in
reduction of cytotoxicity of CD8+ T-cells. TIM-3 mRNA level is proposed as a
biomarker of effector T-cell activation and was evaluated in 24 patients with
acute rejection, 20 patients with ATN, and 18 patients with stable graft function
by the means of RT-PCR. Peripheral blood cell TIM-3 mRNA was significantly
higher among patients with acute rejection, and this increased level was not
due to decreased GFR. At the threshold of 1.58, TIM-3 mRNA had 100 %
sensitivity and 87.5 % specificity in discriminating acute rejection from ATN. The
TIM-3 mRNA level did not differentiate refractory from responsive acute rejection
(sensitivity of 66.7 % and specificity of 57.1 %). Despite encouraging results, a lack
of biopsy-proven acute rejection in all the cases and exclusion of infective causes of
impaired renal function (CMV infection, UTI) brings up the need for further
validation of the marker (Luo et al. 2011).
In order to bring biomarkers from bench to beside and assessing their clinical
utilities and their limitations, recently the gene expression profiles of patients were
studied.
In a large cohort, 367 blood samples from pediatric transplant patients, including
115 patients with biopsy-proven acute rejection, 180 cases with stable graft function,
and 72 cases with other causes of graft dysfunction (chronic allograft injury, viral or
bacterial infection, calcineurin inhibitor toxicity, and borderline acute rejection),
microarray analysis and subsequent quantitative PCR led to the discovery of a
five-gene panel. This gene panel consisted of DUSP1, MAPK9, NKTR, PBEF1,
and PSEN1. The gene profile is representative of immunologic activity and injury:
leukocyte recruitment; B-cell, T-cell, and monocyte activation; oxidative stress;
apoptosis; IL-2 pathway activation; increased adhesion; and vascular smooth muscle
cell injury. Except MAPK9 and NKTR, which were under-expressed, the remaining
three genes were overexpressed in patients with acute rejection. The data was further
validated in an independent cohort.
The five-gene model can discriminate acute rejection from those with stable graft
function with a sensitivity of 91 % and specificity of 94 % and a NPV of 97 % (AUC
0.955). It also has the ability to separate acute rejection from other causes of graft
dysfunction with 91 % and 90 % sensitivity and specificity, respectively. None of the
confounding factors affected the results, and the high NPV in the setting of graft
dysfunction might decrease the unnecessary biopsies. The downside of the five-gene
profile is its inability in detecting borderline rejection and distinguishing humoral
from cellular rejection (Li et al. 2012). Further validation for clinical utility in adult
recipients is required.
To validate the five-gene panel (DUSP1, MAPK9, NKTR, PBEF1, and PSEN1)
in Korean patients, Lee et al. conducted a study on 143 recipients. Patients with acute
cellular rejection had significantly lower levels of MAPK9 and higher PSEN1 than
controls. However, patients with acute antibody mediated had the similar profile
with controls and those with other graft injuries (BK nephropathy, calcineurin
inhibitor toxicity, glomerulonephritis, and ATN). Conversely, PSEN1 level was
lower and MAPK9 level was higher in patients with other graft injuries. The
two-gene set alone had 73.33 % sensitivity and 75 % specificity (AUC, 0.841) in
discriminating acute cellular rejection from other causes of graft injury. However, the
five-gene set in combination with clinical variables had 90 % sensitivity and
specificity (AUC, 0.964) and PPV of 93.1 and NPV of 85.1. Therefore, this five-
gene panel is a promising tool for diagnosis of acute cellular rejection from other
causes of graft dysfunction (Lee et al. 2014).
Recently, Roedder et al. studied blood gene expression on 558 blood samples of
436 transplant patients both pediatric and adults in a multicenter study. Using real-
time quantitative PCR (RT-qPCR), patients with acute rejection were compared
with patients with other causes of graft dysfunction (chronic allograft injury, chronic
calcineurin inhibitor toxicity, BK virus infection, and acute tubular nephritis).
They utilized the previously reported ten-gene panel (DUSP1, CFLAR, ITGAX,
NAMPT, MAPK9, RNF130, IFNGR1, PSEN1, RYBP, and NKTR) (Li et al. 2012)
and added seven genes (SLC25A37, CEACAM4, RARA, RXRA, EPOR, GZMK,
RHEB). This 17-gene panel showed a significantly higher sensitivity (82.98 %) and
specificity (90.63 %), with an AUC of 0.94 (95 % CI 0.91–0.98, p < 0.001).
The 17-gene panel identified as the Kidney Solid Organ Response Test (kSORT)
was validated in a 124 sample independent cohort, and a further cross-validation was
performed on 100 samples. In the validation group, the mean predicted probability of
acute rejection was significantly different between the two groups as reported in the
training set. The kSORT is a sensitive and specific noninvasive test to detect acute
rejection whether cellular or antibody mediated. Its high specificity and NPV
(91.58 %) make it a valuable marker with a utility as a negative predictor of
rejection. As most of the genes in the panel are related to monocyte activation, and
monocyte activation is evident in both cellular- and antibody-mediated rejection, one
of the limitations of kSORT is its inability to differentiate between these two types of
rejection. In order to be used as a predictor of acute rejection, the group designed a
longitudinal multicenter study and evaluated 191 blood samples before, at the time,
and after acute rejection in an independent cohort. kSORT could predict clinical
acute rejection in more than 60 % of samples up to 3 months before the clinical or
histological event. After further validations, this panel might replace the invasive
protocol biopsy in prediction of subclinical rejection. The group also created a risk
score for acute rejection called kSAS (kSORT analysis suite). kSAS algorithm is able
to categorize patients according to the risk of acute rejection: high risk for AR (risk
score 9), low risk for AR (risk score 9), and indeterminate (risk score <9 and
> 9) (Roedder et al. 2015).
It seems that after further validation in clinical trials, kSORT could be used as a
diagnostic and predictive marker of acute rejection.
miRNAs
Like urine samples and tissue biopsies, peripheral blood samples could be assessed
for the presence of miRNAs with the ability to diagnose acute rejection.
miRNAs were evaluated in 32 renal transplant patients including 11 patients with
biopsy-proven acute rejection. Both intragraft and peripheral blood mononuclear
cells (PBMCs) were evaluated for miRNA expression. miR-142-5p, miR-155, and
miR-223 were overexpressed both in biopsy samples and in the peripheral blood
(Angelicheau et al. 2009). The study showed correlation between tissue and serum
markers, which could be the base for further investigations.
In a study on 12 transplant patients, eight of which had an episode biopsy-proven
acute rejection, expression of miRNAs was analyzed in serum by qPCR. miR-223
and miR-10a were significantly reduced among patients with acute rejection.
Although the results are encouraging, they must be interpreted keeping in mind
the small number of cases (Betts et al. 2014). On the contrary, Lui et al. in their
report on 12 transplant patients with acute rejection (in a cohort of 33 patients)
demonstrated elevated levels of miR-223 in PBMCs at the time of rejection
with a sensitivity of 92 % and specificity of 90 % in diagnosis of acute rejection
(Scian et al. 2013). Small number of cases and different study design may explain
the discrepancies.
In a cohort of 112 transplant patients and 11 healthy controls, the miRNA profile
of patients with chronic antibody-mediated rejection (CAMR) differed from that of
acute rejection. Increased expression of miR-142-5p in PBMCs has been reported in
CAMR. It was also reported to be able to discriminate CAMR from those with stable
renal function (AUC, 0.74) (Danger et al. 2013).
As mentioned above, most of the miRNA studies are on urine samples, and the
recent data opens new fields in biomarker studies in PBMCs or blood samples.
Overall, biomarker identification is a science in evolution, and there is a long way
ahead in order to introduce a biomarker or a panel of biomarkers with accurate
clinical utility to substitute the invasive gold standard “allograft biopsy.”
Biomarkers of Tolerance
Allograft transplantation is the treatment of choice in patients with end-stage renal

disease. The downside of transplantation is the long-term need for immunosuppres-
sion with infections, malignancies, and nephrotoxicity of drugs as the main side
effects. Tolerance gives the opportunity to cease the immunosuppression or to
minimize it. Attempts to induce tolerance were not a great success. In order to
identify patients who are candidates for immunosuppression minimization or with-
drawal, biomarkers of tolerance have been evaluated among patients with “clinical
operational tolerance (COT).” COT is a state of tolerating the allograft in the absence
of immunosuppressive drugs without pathologic evidences of rejection for at least
1 year. About 100 patients with kidney transplantation have been reported to be at
the state of COT, mostly due to noncompliance or lymphoproliferative disorders
(Orlando et al. 2010). The clinicians need an assay to guide them in safe reduction in
immunosuppression in selected patients without increasing the risk of acute rejec-
tion; thus, most of the studies conducted on patients with COT. To evaluate bio-
markers, a sample size of at least 200 is needed, and in order to compensate the lack
of adequate sample size, studies were conducted on training set, validation set, and
cross-validation sets. Data on urine biomarkers are rare. Most promising data come
from gene expression studies in peripheral blood, although flow cytometry and
ELISA methods also have been used (Gökmen and Hernandez-Fuentes 2013)
(Table 4).
Gene Expression Studies
Gene expression microarray assays using RT-qPCR are valuable tools for biomarker
discovery and extracting functional and biological role of the marker by the means of
bioinformatics.
One of the earliest studies on biomarkers of tolerance was conducted by Brouard
et al. They performed a microarray study on a group of 17 COT patients (5 in
Table 4 Biomarkers of tolerance

Biomarker set Detection method Reference
IGKV4-1, IGLL1, IGKV1D-13a Multiplex real- Newell
time PCR et al. (2010)
CD79B, TCL1A, HS3ST1, SH2D1B, MS4A1, TLR5, Microarray, Sagoo
FCRL1, PNOC, SLC8A1, FCRL2a RT-PCR et al. (2010)
Foxp3, CCL20, TLE4, CDH2, PARVG, SPON1, RAB30, Microarray, Brouard
BTLA, SMILE, SOX3, CHEK1, HBB, DEPDC1, CDC2a RT-qPCR et al. (2007)
KLF6, BNC2, CYP1B1a Microarray, Roedder
qPCR et al. (2015)
miR-142-3p Microarray Danger
et al. (2012)
Urine CD20 RT-qPCR Newell
et al. (2010)
qPCR quantitative polymerase chain reaction, RT-qPCR real-time quantitative polymerase chain
reaction
a
Gene set as a biomarker
training group and 12 in test group) and compared the results with healthy controls
and those with various graft statuses (chronic rejection, stable graft function on
immunosuppressive therapy, and those on steroid monotherapy). A set of 49 genes
was identified as the footprint of tolerance. Among these genes, 33 distinguished
tolerance from chronic rejection with 86 % sensitivity and 99 % specificity. The
identified genes were involved in costimulatory signaling and memory T-cell
response. They also suggested a role for transforming growth factor-β pathways. If
validated in larger cohorts, this panel could be used as a guide for immunosuppres-
sion reduction (Brouard et al. 2007).
In a cohort of 25 COT patients (off immunosuppressive drug for at least a year,
20 due to noncompliance), 33 patients with stable graft function, and 42 healthy
controls, a microarray study conducted on whole-blood total RNA. A set of five
genes were differentially expressed between COT and stable patients – TUBB2A,
TCL1A, BRDG1, HTPAP, and PPPAPDC1B – all of which were involved in B-cell
activation. The COT group had higher expression of CD20 transcript in urine
sediment compared to those on immunosuppressive drugs. After performing mul-
tiplex RT-PCR, a 3-gene set found to predict tolerance – IGKV4-1, IGLL1, and
IGKV1D-13 – with PPV of 83 % and NPV of 84 %. Whole-blood flow cytometry
confirmed a significantly higher number of total B-cells, naïve B-cells, and tran-
sitional B-cells (CD19+CD38+CD24+IgD+) in COT patients than in those with
stable graft function on drugs. Among the flow cytometry results, transitional
B-cell had the highest predictive value for COT (85 % and 96 % PPV and NPV,
respectively). These results pointed out the important role of B-cell in tolerance
and introduced the 3-gene set as a predictive marker of tolerance (Newell
et al. 2010).
Following this study, Sagoo et al. studied a cohort of 71 kidney transplant patients –
11 patients with COT, 11 patients on low-dose prednisolone only, 40 patients on full
immunosuppression, and 9 patients with pathologic evidence of chronic rejection.
Interestingly, the COT group had the highest degree of HLA mismatch but
undetectable donor-specific anti-HLA antibodies. Microarray, RT-qPCR, and flow
cytometry techniques were applied on peripheral blood monocyte cells (PBMCs).
Recipients with COT (like healthy controls) had the highest B-cell-to-T-cell ratio,
which was the result of an elevated number of B-cells rather than reduction of T-cell
population. COT patients had decreased proportion of memory B-cells and activated
T-cells and increased proportion of transitional B-cells as previously reported by
Newell et al. Additionally, tolerant patients had a high ratio of Foxp3/α-1,2-
mannosidase in peripheral blood. The microarray data and RT-qPCR resulted in a
10-gene set with diagnostic capability (See Table 4). The set could discriminate COT
from non-tolerant transplant patients with 80.6 % sensitivity, 89 % specificity, and
93 % NPV (Sagoo et al. 2010).
Overall, these studies pointed out the significance of B-cell and natural killer cell
(NK cell) expansion in tolerant patients. The B-cell signature of tolerance has been
developed in two independent cohorts (increased number of naïve and transitional
B-cells), and after further validation in larger cohorts, this could be used to choose
patients for immunosuppression minimization or cessation. In a prospective obser-
vational study, Viklicky et al. tried to validate the abovementioned gene set as a
guide in immunosuppression minimization. They compared operational tolerance-
associated transcripts (MS4A1, CD79B, TCL1A, TMEM176B, Foxp3, TOAG-1,
MAN1A1 (α-1,2-mannosidase), and TLR5) in patients with and without acute
rejection in the first day posttransplantation. The expressions of MS4A1 (CD20),
CD79B, TCL1A, and TOAG-1 as markers of naïve and immature B-cells were
significantly higher in patients without acute rejection, as well as the
Foxp3/α-1,2-mannosidase ratio. The expression of TLR5 was not different between
the examined groups, and TMEM176B expression was higher in rejection group
(Viklicky et al. 2013).
It seems that these seven genes have the capacity to be used as criteria to select
patients who are still on immunosuppressive regimes for drug minimization or
withdrawal.
In the most recent study, with the aim of providing a highly cross-validated COT
gene signature in blood samples and estimating the frequency of the gene signature
in patients on immunosuppressive drug, 571 peripheral blood samples were assessed
by microarray, qPCR, and flow cytometric analysis (cross-platform) in a four-stage
study design. The smallest gene set with the best performance in detection of COT
was a three-gene set, KLF6, BNC2, and CYP1B1, with 84.6 % and 90.2 % sensi-
tivity and specificity, respectively. Besides the strong B-cell signature in tolerance,
the flow cytometric analysis results demonstrated decreased total number of T-cells
and CD4/CD3+ T-cells in COT patients. On the other hand, monocytes and
dendritic cells were significantly increased in COT patients.
The study cross-validated the previous findings and introduced a highly validated
assay to recognize patients with tolerance, which are still on immunosuppressive
drugs and are targets of immunosuppression reduction. The frequency of predicted
accommodation was 7.3 % by this assay (Roedder et al. 2015).
Briefly, all the abovementioned genomic studies and the cross-platform bio-
markers of tolerance (gene expression, flow cytometry, anti-donor immune response,
and anti-donor antibodies) are the first few steps in the long way of establishing
personalized transplantation medicine.
Potential Applications to Prognosis, Other Diseases,

or Conditions
Two major treats in the allograft patients are rejection and infection. In order to
counteract these treats, balanced immunosuppression is needed. Determining the
immunologic risk of every patient, and adjusting the immunosuppressive regime
according to it, is the optimal way to face this issue.
In the light of novel biomarkers, physicians will be able to estimate the immu-
nologic risk in the pretransplant period and prescribe initial immunosuppression in a
personalized fashion rather than in a protocol-wise manner. Biomarkers of acute
rejection after transplantation can be utilized to identify subclinical rejections and
provide timely intervention before clinical and yet irreversible histological changes
occur. The absence of these biomarkers helps in choosing appropriate patients for
immunosuppressive withdrawal in the presence of the tolerance molecular signature.
Both the tolerance signatures and the rejection predictors are beneficial during the
follow-up of patients with minimization or cessation of immunosuppressive agents.
Figure 2 is a schematic plan of what would be the treatment approach if we have
validated and easily performed biomarkers in future.
Summary Points
• This chapter is focused on novel biomarkers in diagnosis of acute rejection and

tolerance among kidney transplant patients.
• Subclinical acute rejection could only be diagnosed by protocol biopsy, which is
an invasive procedure.
• Biomarkers with the ability to diagnose subclinical rejection and guide therapy
might improve long-term graft survival.
• High urinary granzyme A mRNA is able to differentiate patients with subclinical
rejection and mild T-cell-mediated rejection from those with stable graft function.
But CMV infection must be ruled out.
• Urine mRNA of CXCL-9 with its high negative predictive value is a useful
biomarker in excluding acute rejection as the cause of impaired graft function.
• Urine mRNA of Foxp3 and OX40 predict occurrence of rejection.
Pre-transplantation
Evaluation of Biomarkers
of Immunologic Status &
Risk Stratification
Transplantation
Initiate IS
According
to Risk
Low Risk for AR & High Risk for AR &

Serial Evaluation of markers of AR
Positive for TOL Negative for TOL
& TOL in Stable Graft Function
Signature Signature
Withdraw IS Continue IS
Positive
Serial Evaluation of markers of Marker of AR Allograft Biopsy
AR & TOL Signature or Loss of TOL & Resume IS
Signature
Negative
Marker of AR
& Positive for
TOL Signature
Fig. 2 The proposed clinical utility for biomarkers in individualization of immunosuppressive

therapy. With the goal for individualization of IS therapy, biomarkers could be used in pretransplant
period to identify high immunologic risk patients in need of strong IS regimes and low-risk patients
who might benefit from withdrawal of IS. During the posttransplantation period, biomarkers of
acute rejection help recognizing the subclinical rejections. If the patient has the TOL signature and
devoid markers of rejection, he/she would be considered for IS withdrawal. After withdrawal serial
assessment of biomarkers would be mandatory for timely diagnosis of loss of tolerance and
predicting rejection. AR acute rejection, IS immunosuppression, TOL tolerance
• Urine proteomic and genomic (mRNA and miRNA) studies are in the path of
evolution and soon be used clinically in predicting and detecting acute rejection.
• Posttransplant serum level of CD30 might be utilized as a marker of increased
alloimmunity and a guide to immunosuppressive dose adjustment.
• The 17-gene panel identified as the Kidney Solid Organ Response Test (kSORT)
is the best genomic marker of acute rejection till now.
• Increased expression of miR-142-5p in peripheral blood cells is diagnostic for
chronic antibody rejection.
• Clinical operational tolerance is a state of tolerating the allograft in the absence of
immunosuppressive drugs without pathologic evidences of rejection for at least
1 year.
• The tolerance signature was introduced based on genomic studies.

• Different cross-platform studies identified gene sets for diagnosis of tolerance.
• The identified gene sets pointed out the role of naïve and transitional B-cells and
natural killer cells in maintenance of tolerance against allograft.
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Diagnostic Biomarkers of Acute Kidney
Injury in Newborns 2
Athanasios Chalkias and Nicoletta Iacovidou
Contents
Key Facts of Diagnostic Biomarkers of Acute Kidney Injury in Newborns . . . . . . . . . . . . . . . . . . . . 28
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
Biomarkers of Acute Kidney Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
Cystatin C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
Neutrophil Gelatinase-Associated Lipocalin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Interleukin-18 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Kidney Injury Molecule-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Fibroblast Growth Factor-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Urinary Epidermal Growth Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Urine Metabolomic Profiling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Potential Applications to Prognosis, Other Diseases, or Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
A. Chalkias (*)
University of Athens, Medical School, MSc “Cardiopulmonary Resuscitation”, Piraeus, Greece
e-mail: thanoschalkias@yahoo.gr
N. Iacovidou
University of Athens, Medical School, Aretaieio Hospital, Department of Neonatology, Athens,
Greece
e-mail: niciac58@gmail.com

28 A. Chalkias and N. Iacovidou
Abstract
Acute kidney injury refers to the rapid loss of renal function. In newborns,
although the precise incidence of acute kidney injury is unknown, research has
shown that 8–24 % of all critically ill newborns in neonatal intensive care units
may develop the condition. Although traditional markers of acute kidney injury
lack sensitivity and specificity for early diagnosis in the neonatal period, several
novel serum and urinary biomarkers are under intense scrutiny for their role as
noninvasive indicators of early acute kidney injury. The most promising bio-
markers are cystatin C, neutrophil gelatinase-associated lipocalin, interleukin-18,
and kidney injury molecule-1.
Keywords
Acute kidney injury • Newborn • Neonatal period • Biomarkers • Diagnosis
Abbreviations
AKI Acute kidney injury
CysC Cystatin C
EGF Epidermal growth factor
FGF-2 Fibroblast growth factor-2
GFR Glomerular filtration rate
IL-18 Interleukin-18
KIM-1 Kidney injury molecule-1
NGAL Neutrophil gelatinase-associated lipocalin
NICUs Neonatal intensive care units
sCysC Serum CysC
sNGAL Serum neutrophil gelatinase-associated lipocalin
uCysC Urinary CysC
uIL-18 Urinary interleukin-18
uKIM-1 Urine kidney injury molecule-1
uNGAL Urinary neutrophil gelatinase-associated lipocalin
Key Facts of Diagnostic Biomarkers of Acute Kidney Injury

in Newborns
• A major concern for neonatologists is the disturbance of renal function during the
neonatal period, i.e., acute kidney injury.
• The diagnosis of acute kidney injury is not easy and is based mainly on urine
production and various serum markers.
• The traditional markers are not so specific for early diagnosis of neonatal acute
kidney injury.
• Research has shown that some novel biomarkers may be much better than the
traditional.
2 Diagnostic Biomarkers of Acute Kidney Injury in Newborns 29
• The novel biomarkers are cystatin C, neutrophil gelatinase-associated lipocalin,

interleukin-18, and kidney injury molecule-1.
• Despite the encouraging data, further research is necessary in order to find the
most reliable biomarker for timely detection of the condition.
Definitions
Apoptosis The process of programmed cell death.
Metabolomics The scientific study of chemical processes involving metabolites.
Necrosis The premature death of cells and living tissue.
Proteomics The large-scale study of proteins, particularly their structures and

functions.
Proximal tubules The proximal tubule is the portion of the duct system of the
nephron of the kidney which leads from Bowman’s capsule to the loop of Henle.
Reperfusion injury The tissue damage caused when blood supply returns to the
tissue after a period of ischemia or lack of oxygen.
Very low-birth-weight infants Infants who are born weighing less than 1,000 g.
Introduction
Acute kidney injury (AKI) is defined by an acute and reversible increase in serum
creatinine levels associated or not with a reduction in urine output (Singbartl
et al. 2012). It is an important cause of morbidity and mortality in newborns and is
directly associated with poor outcomes (Singbartl and Kellum 2012; Siew and Deger
2012; Akcan-Arikan et al. 2007; Zappitelli et al. 2008). The overall incidence of AKI
in critically ill newborns in NICUs is 8–24 %. In neonates with perinatal asphyxia, the
incidence of AKI may increase to 56.3 % (Durkan and Alexander 2011). Other
conditions that may lead to AKI in the perinatal period include prematurity, congenital
diseases, sepsis, or administration of nephrotoxic agents (Andreoli 2004).
The newborn’s kidneys are more susceptible to injury due to increased vulnerability
to hypoperfusion, as well as due to low glomerular filtration rate, high renal vascular
resistance, high plasma renin activity, decreased intercortical perfusion, and decreased
reabsorption of sodium in the proximal tubules (Liborio et al. 2014). The prognosis of
AKI in newborns depends on its etiology and on gestational age; however, 25–50 % of
neonates with AKI die, and long-term problems may appear in the survivors (Andreoli
2004; Askenazi et al. 2009a; Agras et al. 2004; Abitbol et al. 2003).
Table 1 Classification and staging systems for acute kidney injury

Urine output
System Serum creatinine criteria criteria
AKIN stage
1 Serum creatinine increase 26.5 μmol/l (0.3 mg/dl) or increase to <0.5 ml/kg/h
1.5–2.0-fold from baseline for 6 h
2 Serum creatinine increase >2.0–3.0-fold from baseline <0.5 ml/kg/h
for 12 h
3 Serum creatinine increase >3.0-fold from baseline or serum Anuria for 12 h
creatinine 354 μmol/l (4.0 mg/dl) with an acute increase of at
least 44 μmol/l (0.5 mg/dl) or need for renal replacement therapy
RIFLE class
Risk Serum creatinine increase to 1.5-fold or glomerular filtration rate <0.5 ml/kg/h
decrease >25 % from baseline for 6 h
Injury Serum creatinine increase to 2.0-fold or glomerular filtration rate <0.5 ml/kg/h
decrease >50 % from baseline for 12 h
Failure Serum creatinine increase to 3.0-fold or glomerular filtration rate Anuria for 12 h
decrease >75 % from baseline or serum creatinine 354 μmol/l
(4 mg/dl) with an acute increase of at least 44 μmol/l (0.5 mg/dl)
AKIN AKI Network; RIFLE Risk, Injury Failure, Loss, End-stage renal disease
On the other hand, diagnosis of neonatal AKI is highly challenging and is based
on urine output, serum creatinine, and glomerular filtration rate (GFR) values
(Table 1). Unfortunately, the diagnosis may be delayed due to abnormal or immature
tubular function especially in premature neonates and the limitations in the use of
serum creatinine as diagnostic marker; serum creatinine is not increased until 50 %
of kidney function has been lost, while its levels are affected by renal and nonrenal
factors (Argyri et al. 2013; Askenazi et al. 2009a; Bariciak et al. 2011; Druker and
Guignard 2002).
Although traditional markers of AKI lack sensitivity and specificity for early
diagnosis in the neonatal period, several novel serum and urinary biomarkers are
under intense scrutiny for their role as noninvasive indicators of early AKI (Parikh
and Devarajan 2008). These novel biomarkers will be presented in the present
chapter, and their role in early diagnosis and prognosis of AKI in newborns will
be discussed.
Biomarkers of Acute Kidney Injury
Several biomarkers have been so far associated with AKI in newborns (Alge
et al. 2013). The most promising are cystatin C (CysC), neutrophil gelatinase-
associated lipocalin (NGAL), interleukin-18 (IL-18), and fibroblast growth
factor-2 (FGF-2) in serum and CysC, NGAL, kidney injury molecule-1
(KIM-1), IL-18, and urinary epidermal growth factor (EGF) in urine (Table 2).
Table 2 Biomarkers associated with acute kidney injury in newborns

Serum Urine
Cystatin C Cystatin C
Neutrophil gelatinase-associated lipocalin Neutrophil gelatinase-associated lipocalin
Interleukin-18 Kidney injury molecule-1
Fibroblast growth factor-2 Interleukin-18
Urinary epidermal growth factor
Cystatin C
Cystatin C (CysC) is a low-molecular-weight (13 kDa) cysteine proteinase inhibitor

expressed in all nucleated cells. It is produced at a constant rate and cleared exclusively
by the kidney. Cystatin C does not cross the placenta and, thus, reflects the renal
function of the neonates in early postnatal life regardless of body composition and size
(Dinarello et al. 1998; Sharma et al. 2009). Due to its small size, CysC is normally
filtered by the glomerulus and is completely reabsorbed and catabolized by the
proximal tubule without significant secretion by the renal tubules (Westhuyzen
2006). Serum CysC (sCysC) levels increase in term newborns and decrease over the
first 5 days of life, remaining stable up to day 28 of life (Plebani et al. 1997; Cataldi
et al. 1999; Novo et al. 2011). Due to this, sCysC has been used as a biomarker of renal
tolerance of ibuprofen treatment in very low-birth-weight infants (Gokmen et al. 2011).
Serum CysC was measured in 62 preterm neonates with respiratory distress syndrome
and 34 control neonates and was identified as an earlier marker of AKI before serum
creatinine increase (Elmas et al. 2013). Moreover, sCysC has been used as a predictive
biomarker of AKI in preterm very low-birth-weight infants who received indomethacin
for duct closure followed by furosemide or placebo administration (Lee et al. 2010). In
this study, infants of the furosemide group were more likely to develop AKI and had
higher levels of serum creatinine and CysC than the placebo group.
Regarding its role as a biomarker in perinatal asphyxia, not only sCysC but also
urinary CysC (uCysC) predicted AKI in neonates with a 5-min Apgar score <7
(Askenazi et al. 2012). Contrarily, sCysC was not a useful marker of renal function
in neonates with sepsis (Maruniak-Chudek et al. 2012). Newborns with septic shock
had lower levels of sCysC than those with sepsis. The uCysC was also predictive of
AKI, in non-septic critically ill newborns (Li et al. 2012). Although other studies have
evaluated CysC in older children, it is difficult to extend their results to the neonatal
population, considering that normal values of uCysC decrease with tubular maturation.
Evidence has shown so far that sCysC is not more sensitive than serum creatinine
as a marker of GFR in newborns (Treiber et al. 2006). Both sCysC and creatinine
levels at birth and 3 days after birth have been reported to be independent of sex,
gestational age, birth weight, bilirubin levels, and hydration state (Treiber
et al. 2006; Armangil et al. 2008). In addition, sCysC was correlated with the degree
of congenital renal abnormalities; a significant increase of sCysC was observed in
neonates with bilateral congenital kidney anomalies in contrast to those with unilat-
eral kidney abnormalities or without kidney malformation (Parvex et al. 2012).
Neutrophil Gelatinase-Associated Lipocalin
Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa protein of the

lipocalin superfamily (Bolignano et al. 2008). NGAL is expressed in renal tubules
after an ischemic or toxic insult and following transplantation (Mishra et al. 2003,
2004, 2006). Immediately after the onset of AKI, NGAL mRNA is rapidly
upregulated in the loop of Henle and proximal tubules, causing an increase in the
synthesis and excretion of NGAL into the urine by the proximal tubule (Gupta
et al. 2005; Alge et al. 2013). However, NGAL is released in plasma by other injured
tissues, and NGAL-based diagnosis of AKI may be challenging.
The possible use of NGAL in toxic AKI was studied both in animals and humans.
In a rodent study, the authors administered an intraperitoneal dose of cisplatin
(20 mg/kg) and reported that tubule cell necrosis and apoptosis, as well as expression
of NGAL in renal tissue, occurred within 3 h, while NGAL was detected in urine in a
dose- and duration-dependent manner (Mishra et al. 2003). In a human study,
Wasilewska et al. reported that both serum and urinary NGAL levels increased
during the course of cyclosporine treatment (Wasilewska et al. 2010). In this study,
however, no firm conclusion could be drawn on urinary NGAL’s (uNGAL) possible
role as a predictor of cyclosporine nephropathy due to the small sample size. Despite
this finding, NGAL has been recently associated with inflammation; expression of
NGAL in trophoblast tissues of term placenta increased after intra-amniotic infection
in vivo and after exposure to mediators of inflammation (Tadesse et al. 2011).
Urinary NGAL was also increased in septic very low-birth-weight infants and
predicted the renal function of approximately the first month of life in this age
group (Parravicini et al. 2010; La Manna et al. 2011).
Of note, uNGAL has been characterized as the most promising AKI biomarker
(Liborio et al. 2014). Urinary NGAL is the most strikingly upregulated gene and
overexpressed protein in the kidney after ischemia and depends on birth weight and
gestational age (Askenazi et al. 2009a; Mishra et al. 2004). In neonates, a 2-h serum
NGAL (sNGAL) threshold of 100 ng/mL and a 2-h uNGAL of 185 ng/mL were the
best cutoff values with improved sensitivity and specificity for early diagnosis of
AKI compared with creatinine, whereas in children this threshold was set at 50 ng/
Ml (Argyri et al. 2013). Nevertheless, a single measurement of sNGAL in neonates
may be not sufficient for the prediction of AKI (Koch et al. 2011); the combination
of NGAL with other biomarkers may be of value in the early diagnosis of AKI
(Sarafidis et al. 2012). Askenazi et al. (2011) evaluated NGAL, IL-18, KIM-1, and
CysC and reported that they may predict AKI and mortality in very low-birth-weight
infants.
Interleukin-18
Interleukin-18 is a pro-inflammatory cytokine released by macrophages. It stimu-

lates the synthesis of other inflammatory molecules and enhances the maturation of
T and natural killer cells, as well as the infiltration of neutrophils and macrophage
accumulation (Dinarello et al. 1998). Interleukin-18 is cleaved by caspase-1 and is
released in the proximal tubule after AKI (Melnikov et al. 2002); this biochemical
behavior is responsible for its deleterious effects on renal function.
In an experimental model of ischemia-reperfusion injury, IL-18-deficient mice
were protected from acute renal failure compared with wild-type controls (Melnikov
et al. 2001). In the same study, the IL-18-deficient mice had attenuated production of
pro-inflammatory molecules within the kidney at 24 h. Similar results were observed
in neonatal septic mice, in which neutralization of IL-18 significantly increased
mortality, while administration or recombinant IL-18 improved survival (Cusumano
et al. 2004).
In humans, serum IL-18 was significantly increased in infected newborns com-
pared with noninfected neonates, although IL-18 did not predict early onset neonatal
sepsis (Bender et al. 2008). In this study, however, very few infants had septic shock,
which might be the reason for the difference between the results. Li et al. enrolled
62 non-septic critically ill neonates of which they collected urine every 48–72 h
during the first 10 days of life (Li et al. 2012). They reported that both urinary IL-18
(uIL-18) and uCysC were independently associated with AKI. Interestingly, uCysC
levels may decrease with increasing renal maturity, but this may not occur with
uIL-18 levels. Interleukin-18 has been reported to have a potential advantage of not
changing its normal value with increasing renal maturity but can be influenced by
sepsis, reducing its ability to detect AKI (Liborio et al. 2014).
Kidney Injury Molecule-1
KIM-1 is a type 1 transmembrane glycoprotein that is not detected in urine under

normal conditions but is markedly increased after kidney ischemic or toxic injury,
characterized by epithelial cell differentiation (Argyri et al. 2013). It has significant
immunomodulatory properties as it confers the ability on epithelial cells to phago-
cytose dead cells and transforms them into phagocytes (Bonventre 2009). In kid-
neys, KIM-1 is shed from proximal tubule cells and secreted in urine.
In animals exposed to various nephrotoxic agents, as well as to gentamicin,
S-(1,1,2,2-tetrafluoroethyl)-l-cysteine, and folic acid, urine KIM-1 (uKIM-1) has
been reported to be a more sensitive and specific biomarker of AKI than creatinine.
In rats, it was reported to detect renal injury following ischemia-reperfusion
(Vaidya et al. 2006). In humans, uNGAL predicted AKI and uKIM-1 correlated
with mortality in very low-birth-weight infants, independent of gestational age and
birth weight (Askenazi et al. 2011). In addition, newborns that developed AKI had
higher levels of uKIM-1 and uNGAL than those without AKI, but this difference was
not statistically significant (Askenazi et al. 2012). Moreover, the predictive value of
uKIM-1 in premature neonates showed that the biomarker predicted aminoglycoside-
induced nephrotoxicity (Argyri et al. 2013), while in contrast, a reduction in serum
creatinine was also observed during treatment (McWilliam et al. 2011).
Fibroblast Growth Factor-2
Various studies have demonstrated that fibroblast growth factor-2 (FGF-2) may be a
good urinary candidate biomarker for children with AKI secondary to renal endo-
thelial injury (Ray et al. 1999, 2002, 2006). Recently, Hoffman et al. evaluated
urinary FGF-2 as a marker of AKI in neonates and reported that the combination of
NGAL and FGF-2 improved the specificity to identify newborns at risk of develop-
ing AKI. However, FGF-2 did not differentiate those with AKI among those at risk
(Hoffman et al. 2013). Although the role of FGF-2 as a marker of AKI has not been
fully established so far, it has a promising role as a late biomarker for detecting the
recovery of renal function.
Urinary Epidermal Growth Factor
Urinary epidermal growth factor (EGF) may be a reliable biomarker to follow the
outcome of infants with AKI (Askenazi et al. 2012; Soler-Garcia et al. 2009; Tsau
et al. 1996; Chen and Liu 1997), while the urinary levels of EGF appear to be a
predictor of renal function recovery in adults with AKI (Kwon et al. 2010). Consid-
ering that the urinary excretion of EGF is more dependent on its renal pool
(Watanabe et al. 1989; Evans et al. 1986; Di Paolo et al. 1997), it may be a more
specific biomarker to identify ongoing renal injury in critically ill neonates than
serum creatinine.
Urine Metabolomic Profiling
In an experimental study on newborn rats, nephrotoxicity induced by gentamicin

was associated with different urinary patterns of metabolites (Fanos et al. 2014). In
total, glucose, galactose, N-acetylglucosamine, myoinositol, butanoic acid,
pseudouridine, and 3-hydroxybutyrate were significantly increased following
administration of gentamicin.
Atzori et al. investigated the possibility of kidney injuries being associated with a
well-defined metabolic pattern using 1H nuclear magnetic resonance spectroscopy
technology (Atzori et al. 2011). They analyzed the urine metabolic profile of
21 children affected by nephropathies and compared it with that of 19 healthy
controls. They reported that renal and urinary tract malformations were related to a
specific metabolic profile belonging to the nuclear magnetic resonance spectroscopy
regions [3.5–3.9], [4.1–4.4], and [8.2–8.6], suggesting the localization of the damage
(cortexes 1, 2, and 3, medullary). In particular, multivariate analysis of urine spectral
data revealed a clustered distribution with a significant separation of the term infant
samples and preterm infant samples. This study is of major importance because data
analysis enabled the identification of the main discriminating low-molecular-weight
metabolites, suggesting that amino acid biosynthesis and metabolism are the key
metabolic mechanisms underlying fetal and perinatal maturation processes. In
conclusion, 1H-NMR metabolomic analysis of urine appears to be able to assess the

metabolic status of preterm and term infants at birth. Nevertheless, the findings of
Atzori et al. (2011) should be interpreted with caution because their results may be
biased by variables such as maternal pathological conditions, mode of delivery,
perinatal and neonatal disorders, as well as by iatrogenic factors. In either case,
further large-scale prospective clinical metabolomic studies are needed in order to
confirm the aforementioned findings. Particularly, research should focus on the
investigation of selected groups of neonates affected by specific disorders or under
particular conditions (Atzori et al. 2011).
Discussion
Until now, neither serum creatinine nor urine output meets the characteristics of the
ideal biomarker for early diagnosis of AKI (Askenazi et al. 2009b; Bariciak
et al. 2011; Drukker and Guignard 2002; Murray et al. 2002). The biomarkers
discussed can be measured in serum and in urine. Each one of these biomarkers
has some advantages.
On the other hand, sCysC does not reflect maternal values in contrast to serum
creatinine (Plebani et al. 1997; Cataldi et al. 1999), but its role in estimating GFR in
newborns still remains controversial (Treiber et al. 2006). NGAL seems to be the
most promising biomarker of AKI in newborns. It increases earlier in ischemic AKI
than other emerging markers (Krawczeski et al. 2011). Nevertheless, both serum and
urinary NGAL are increased at 2 h after cardiopulmonary bypass in neonates, while
sCysC, uKIM-1, and uIL-18 are markedly increased at 12–24, 6–12, and 4–6 h,
respectively, in children undergoing the procedure (Krawczeski et al. 2010; Han
et al. 2008; Parikh et al. 2006). In addition, both uNGAL and uKIM-1 seem to be
promising biomarkers of toxic AKI (Mishra et al. 2004; Wasilewska et al. 2010;
Vaidya et al. 2006; McWilliam et al. 2011; Zhou et al. 2008), while uKIM-1 and
sIL-18 have been positively correlated with adverse clinical outcome (Argyri
et al. 2013).
Despite the increase in evidence, further research and extensive comparison of
each new marker with both traditional and other novel markers of AKI are necessary
before any of these biomarkers can be used in everyday clinical practice for
monitoring AKI. In addition, the range of baseline values of the aforementioned
biomarkers has to be established. Given the fact that AKI is associated with high
mortality, especially in very low-birth-weight infants (Koralkar et al. 2011), it is
unquestionably important to establish biomarkers for early diagnosis. As none of the
above biomarkers have all the characteristics required for a substance to be charac-
terized as an ideal biomarker of AKI, their combination might be a better diagnostic
and prognostic tool of the condition than each one separately (Argyri et al. 2013).
A combination of these biomarkers could make a “biomarker panel” for the early
diagnosis and severity stratification of AKI in newborns with high sensitivity,
specificity, and predictive value, especially when combined with the metabolomic
profile. Recently, serum and urine samples have been analyzed by the “omics”
techniques. Not only proteomics but also metabolomics seems to be a promising tool
in identifying diagnostic biomarkers, which in turn assist timely intervention
(Lindon et al. 2004). However, although kidney disease is an appropriate field for
mass spectrometry and 1H nuclear magnetic resonance spectroscopy studies, the
pathophysiology of kidney injury is still unclear (Pan and Raftery 2007; Weiss and
Kim 2012).
Conclusions
Several biomarkers have been proposed as predictors of neonatal AKI. However,

further prospective studies in large populations of neonates have to be conducted
before any of these biomarkers are used in everyday practice. Comparison of each
new marker with both traditional and other novel markers of AKI is necessary to find
the most reliable biomarker for timely detection of the condition. A combination of
the described biomarkers with metabolomics could make a “biomarker-metabolomic
panel” for the early diagnosis and severity stratification of AKI in newborns with
high sensitivity, specificity, and predictive value.
Potential Applications to Prognosis, Other Diseases, or

Conditions
Critically ill newborns with acute kidney injury may develop hypertension, partic-
ularly those requiring intensive care. Hypertension increases the risk for early onset
cardiovascular disease and requires careful diagnostic evaluation and prompt treat-
ment. Moreover, acute kidney injury may evolve to multiple organ failure and death.
Although in newborns, renal dysfunction did not seem to have a critical impact on
mortality until now, in a recent study, the authors showed that severity of renal failure
was strongly associated with mortality in infants who were treated with extracorpo-
real membrane oxygenation (Zwiers et al. 2013). This finding suggests that there
should be a cutoff value at which the severity of acute kidney injury directly impacts
mortality.
In general, acute kidney injury is an independent risk factor for poor outcomes in
critically ill neonates, and prognosis is variable. Infants with prerenal acute kidney
injury who receive prompt treatment for renal hypoperfusion have an excellent
prognosis, while those with postrenal acute kidney injury related to congenital
urinary tract obstruction have a variable outcome. Also, infants with intrinsic acute
kidney injury have significant risks of morbidity and mortality.
Despite advantages in renal medicine, renal pathophysiology of critically ill
newborn patients makes it difficult to interpret urine output and serum creatinine
levels to diagnose acute kidney injury. Considering that renal dysfunction may be
reversible if early diagnosed, improving our diagnostic methods with better acute
kidney injury biomarkers will allow us to intervene much earlier in the disease
course.
Summary Points
• Eight to twenty-four percent of all critically ill newborns in neonatal intensive

care units may develop acute kidney injury.
• Diagnosis of neonatal acute kidney injury is highly challenging and is based on
urine output, serum creatinine, and glomerular filtration rate values.
• Traditional markers of acute kidney injury lack sensitivity and specificity for early
diagnosis in the neonatal period.
• Several novel serum and urinary biomarkers are under intense scrutiny for their
role as noninvasive indicators of early acute kidney injury.
• The most promising biomarkers are cystatin C, neutrophil gelatinase-associated
lipocalin, interleukin-18, and kidney injury molecule-1.
• A combination of the described biomarkers with metabolomics could make a
“biomarker-metabolomic panel” for the early diagnosis and severity stratification
of acute kidney injury in newborns with high sensitivity, specificity, and predic-
tive value.
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Osmolal Gap as a Biomarker in Kidney
Injury: Focusing on the Differential 3
Diagnosis of Metabolic Acidosis
Jeonghwan Lee, Nam Ju Heo, and Jin Suk Han
Contents
Key Facts of the Osmolal Gap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Estimation of the Osmolal Gap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Measured Osmolality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Calculation of Osmolality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Serum or Urine Osmolal Gap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Clinical Utility of the Serum and Urine Osmolal Gap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Serum Osmolal Gap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Urine Osmolal Gap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Limitations to the Clinical Utility of the Osmolal Gap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Various Causes of an Osmolal Gap . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Limitations of Methods of Osmometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
J. Lee
Department of Internal Medicine, Hallym University Hangang Sacred Heart Hospital, Seoul,
Republic of Korea
e-mail: jeonghwan@hallym.or.kr; woogaelee@gmail.com
N.J. Heo
Department of Internal Medicine, Healthcare System Gangnam Center, Seoul National University
Hospital, Seoul, Republic of Korea
e-mail: njheo@snuh.org
J.S. Han (*)
Department of Internal Medicine, Seoul National University, College of Medicine, Seoul, Republic
of Korea
e-mail: jshan@snu.ac.kr

42 J. Lee et al.
Abstract
Although numerous osmoles contribute to the measured value of osmolality in
serum and urine, most clinical laboratories cannot measure these individually.
The most part of osmolality of serum or urine is determined by five or six
major effective osmoles. Formulae for calculating serum and urine osmolality
derive from these osmoles. The difference between the measured and calcu-
lated osmolality in serum and urine is defined as the osmolal gap.
The serum osmolal gap is useful in the differential diagnosis of high anion gap
metabolic acidosis and can be used as a screening test for intoxication with active
osmoles, including ethanol or other toxic alcohols. In addition, serum osmolal
gap can be increased in ketoacidosis, lactic acidosis, and advanced chronic kidney
disease. The urine osmolal gap is correlated with ammonium (NH4+) excretion
and is useful in the differential diagnosis of normal anion gap metabolic acidosis.
A decreased urine osmolal gap can be observed in patients with distal renal
tubular acidosis, early chronic renal failure, and aldosterone deficiency or
resistance.
Keywords
Serum osmolal gap • Urine osmolal gap • Metabolic acidosis
Abbreviations
Cl Chloride
H+ Acid
HCO3 Bicarbonate
K+ Potassium
Na+ Sodium
NH4+ Ammonium
Key Facts of the Osmolal Gap
• Although there are numerous osmoles that contribute to the actual measured value
of osmolality in serum and urine, these cannot be measured individually in
standard clinical laboratories.
• Since the osmolality of serum or urine is largely determined by five or six major
effective osmoles, formulae for the calculation of serum and urine osmolality
derive from these osmoles.
• The osmolal gap refers to the difference between the measured and calculated
osmolality of serum or urine.
• The most common cause of an increased serum osmolal gap is the addition of
osmotically active substances, including ethanol, toxic alcohols such as methanol,
glycols, or drug metabolites.
3 Osmolal Gap as a Biomarker in Kidney Injury: Focusing on the Differential. . . 43
• A decreased urine osmolal gap is associated with conditions including distal renal
tubular acidosis, chronic renal failure, aldosterone deficiency, and aldosterone
resistance; in these conditions, urine ammonium excretion or production is low
due to defective renal acidification.
Definitions
Osmolarity The numbers of osmoles of solute per liter of solution (mmol or

mOsmol/L).
Osmolality Osmoles of solute per kg of solvent, expressed as mmol or mOsmol/

kgH2O.
Measured osmolality Osmolality which can be measured directly by osmometry.
Calculated osmolality Osmolality which can be estimated by using formulae

measuring the concentrations of the major effective solutes.
The osmolal gap Difference between the measured and calculated osmolality.
Metabolic acidosis Conditions with the gain of acid or loss of alkali due to the
metabolic causes.
Introduction
The concentration of the osmotic particles (osmoles) of a solute is expressed as either

osmolarity or osmolality. Osmolarity refers to the numbers of osmoles of solute per
liter of solution (mmol or mOsmol/L), while osmolality refers to osmoles of solute
per kg of solvent, expressed as mmol or mOsmol/kgH2O. Most available osmom-
eters measure the osmolality of a solution.
In serum or urine, there are numerous osmoles, which cannot be measured indi-
vidually in standard clinical laboratories. In serum and urine, only five or six major
effective (osmotically active) osmoles determine the osmolality, and some of these are
routinely measured in the clinical laboratory. Once the concentration of the measurable
principal osmoles is established, it is possible to approximately calculate the osmolal-
ity of serum or urine. The difference between the measured and calculated osmolality
is defined as an osmolal gap. In serum, this refers to the concentration of trivial solutes,
while in urine it comprises immeasurable major osmoles, namely, ammonium (NH4+).
An increase in the serum osmolal gap is associated with conditions in which
abnormal solutes are either produced endogenously or gained exogenously. Heavy
alcohol ingestion or toxic alcohol intoxication is one of the common causes of an
increased serum osmolal gap.
44 J. Lee et al.
A decreased urine osmolal gap is associated with conditions in which

urinary ammonium excretion or production is low, due to defective renal
acidification that occurred in distal renal tubular acidosis or chronic kidney disease.
Definition
Serum and urine osmolality can be measured by osmometry (the “measured osmo-
lality”) or estimated by using formulae measuring the concentrations of the major
effective solutes of serum or urine (the “calculated osmolality”). The osmolal gap is
the difference between the measured and calculated osmolality (Koga et al. 2004).
Estimation of the Osmolal Gap
Measured Osmolality
The osmolality of serum or urine is measured by an osmometer. The most commonly

used osmometer is freezing point osmometry, which is useful for the majority of
samples, which have an osmolality of approximately 500 mOsmol (or mmol)/
kgH2O. Vapor pressure measurement is suitable for samples with osmolalities in the
range of 100–3000 mOsmol (or mmol)/kgH2O and not affected by sample viscosity
or the presence of suspended particles (Walker et al. 1986; Sweeney et al. 1993.
Calculation of Osmolality
Calculated Serum Osmolality

The serum osmolality is estimated by the concentrations of the solutes in the serum.
In normal subjects, total serum solute concentration is determined by the five major
effective osmoles: sodium (Na+), chloride (Cl), bicarbonate (HCO3), glucose, and
urea. Since sodium ions are counterbalanced by Cl and HCO3, only Na+, glucose,
and urea need to be measured to calculate the serum osmolality (Rasouli and
Kalantari 2005; Gennari 1984).
A variety of formulae have been proposed for use in the prediction of the serum
osmolality. Most studies, however, have concluded that the calculated serum osmo-
lality can be best estimated from the following formulae (Worthley et al. 1987;
Purssel et al. 2001; Bhagat et al. 1984):
(a) Calculated serum osmolality (mOsmol/kgH2O) = 2 [Na+] + [glucose]/18 +

[blood urea nitrogen]/2.8
Note: in standard clinical laboratory, measured [Na+] and [K+] are in mmol/L,
while [glucose] and [urea] are in mg/dL. The divisors 18 and 2.8 convert units of
mg/dL into mOsmol/kg or mmol/L.
(b) Calculated serum osmolality (mOsmol/kgH2O) = 2 [Na] + [glucose] +
[blood urea nitrogen] (in mmol/L)
Calculated Urine Osmolality

The calculation of the urine osmolality is possible by the concentrations of the
solutes in the urine. In normal subjects, total urine solute concentration is determined
by six major osmoles: Na+, K+, Cl, glucose, urea, and ammonium (NH4+).
Under normal physiologic conditions, there is a large amount of K+, NH4+, and
undetectable HCO3. Nearly all of the filtered HCO3 is reabsorbed in the proximal
tubules, and 1 mmol/kg of acid (H+) per day should be excreted from the intercalated
cells of the collecting ducts. The acid excreted is combined with luminal NH3 and
Cl (Figs. 1, 2). Since at least 1 mmol/kg/day of NH4Cl is excreted in the urine,
NH4+ becomes the major cation in urine. Given the abundance of K+ in food, a
mechanism is required to maintain serum concentration; the kidney is genetically
adapted to excrete the excess K+ into the urine. Together Na+, NH4+, and K+ which
are counterbalanced by Cl are the major cations in urine. Since NH4+ cannot be
measured easily in most standard clinical laboratory, only Na+, K+, glucose, and urea
are measured to calculate urine osmolality.
The calculated urine osmolality can be estimated by the following formulae:
(a) Calculated urine osmolality (mOsmol/kgH2O) = 2 {[Na+] + [K+]} +

[glucose]/18 + [urea]/2.8
Note: in standard clinical laboratory, measured [Na+] and [K+] are in mmol/L,
while [glucose] and [urea] are in mg/dL. The divisors 18 and 2.8 convert units of
mg/dL into mOsmol/kg or mmol/L.
Fig. 1 Acidification in proximal tubule. Acid (H+) is excreted into the lumen of the proximal
tubule by Na+-H+ exchanger (NHE) or H+-ATPase. In lumen, H+ is combined to HCO3 forming
dissolved CO2 in water by carbonic anhydrase (CA). Dissolved CO2 is reabsorbed by aquaporin
(AQP) 1, water channel. In the proximal cell, it is cleaved to acid and HCO3 by CA. Along with
Na+, nearly all of HCO3 is reabsorbed to blood by Na+-bicarbonate cotransporter (NBC)
46 J. Lee et al.
Lumen 3Na+ Blood

PT
HPO4 2- Na+ ATP
NHE 2K+
H+ HCO3 -
H2CO3 NBC
CA Na+
NaH2PO4 CO2 + H2O
(Titratable acid)
CD
NH3 H+ ATP H+ HCO3-

H2CO3 AE1
CA Cl-
NH4Cl H2O + CO2 Aldosterone
(Ammonium)
Fig. 2 Urine acidification by acid excretion and equimolar alkali (HCO3) reclamation in the
kidney. The remnant H+ exceeding the limit that can be combined to HCO3 in the lumen of the
proximal tubule should be combined to phosphate buffer forming titratable acidity. In the proximal
tubule, nearly all of phosphate is reabsorbed by Na+-phosphate transporter (NPT). In the collecting
ducts, daily load of H+ should be excreted by either H+-ATPase or H+-K+-ATPase under the control
of aldosterone. In the lumen, the excreted acid is combined to NH3 and Cl forming NH4Cl. Urine
NH4+ reflects on the amount of acid excretion. In each process, H+ excretion is accompanied by an
equimolar reabsorption of HCO3 by NBC and AE (anion exchanger)
(b) Calculated urine osmolality (mOsmol/kgH2O) = 2 {[Na+] + [K+]} +

[glucose] + [urea] (in mmol/L)
Serum or Urine Osmolal Gap
The serum osmolal gap refers to the concentration of trivial solutes in serum. In
clinical practice, the normal serum osmolal gap is below 10–15 mOsmol/kgH2O.
The urine osmolal gap refers to the concentration of immeasurable major
osmoles, namely, NH4+, present in the urine (Fig. 3). In clinical practice, the normal
urine osmolal gap is above 80–100 mOsmol/kgH2O (Halperin et al. 1988).
Clinical Utility of the Serum and Urine Osmolal Gap
Serum Osmolal Gap
The mechanism of an increased serum osmolal gap is either the increase in measured
osmolality or a decrease in calculated osmolality. An increase in unmeasured solutes
in the serum results in an increase of the serum osmolality and an osmolal gap. In the
case of pseudohyponatremia accompanying hyperlipidemia or hyperproteinemia,
Measured Osm = 2{[Na+]+[ K+ ]+[ NH4+]}+[Urea]+[Glucose]+ immeasurable Osm

- Calculated Osm = 2{[Na+]+[ K+]} +[Urea]+[ Glucose]
Osmolal gap = 2 [NH4+] + immeasurable Osm
Na+
K+ d Cl -
NH4+
C+ A-
Fig. 3 Compositions of urine osmoles and urine osmolal gap (C+ immeasurable cations, A
immeasurable anions). In normal subjects, total urine solute concentration is determined by six
major osmoles: Na+, K+, Cl, glucose, urea, and NH4+. The urine osmolal gap refers to the
concentration of NH4+ present in the urine
Table 1 Serum osmolal gap. In normal subjects, total serum osmolality is determined by five
major effective osmoles: sodium (Na+), chloride (Cl), bicarbonate (HCO3), glucose, and urea.
Since sodium ions are counterbalanced by Cl and HCO3, only Na+, glucose, and urea need to be
measured to calculate the serum osmolality. The osmolal gap is the difference between the measured
and calculated osmolality (Osm osmolality)
Measured Osm ¼ 2 ½Naþ þ Glucose
18 þ 2:8 þ immeasurable Osm
BUN
Calculated Osm ¼ 2 ½Naþ þ Glucose

18 þ 2:8
BUN
Osmolal gap = Immeasurable Osm (<10–15 mOsmol/kgH2O)

*Osm osmolality
the calculated serum osmolality decreases, while the serum osmolal gap increases
(Tables 1 and 2).
Estimation of the serum osmolal gap is useful in the differential diagnosis of high
anion gap metabolic acidosis (Kraut and Xing 2011). The serum osmolal gap can be
used as a rapid screening test for the detection of active osmoles, including ethanol or
other toxic alcohols, such as methanol, ethylene glycol, diethylene glycol, propylene
glycol, and isopropanol. Ethanol ingestion is one of the most common causes of an
elevated serum osmolal gap (Shull and Rapoport 2010), and results in ketoacidosis
with a high serum anion gap (Almaghamsi and Yeung 1997). The osmolality of
ethanol can be calculated by the following formulae: (1) osmolal gap = ethanol
48 J. Lee et al.
Table 2 Conditions with an increased serum osmolal gap. Increased osmolal gap is observed in
several clinical conditions including intoxication of alcohol, toxin, or drugs
Alcohol
Ethanol
Toxic alcohols: methanol, (di)ethylene glycol, propylene glycol, isopropanol
Lactic acidosis, diabetic ketoacidosis
Hyperlipidemia, hyperproteinemia
Kidney disease (renal failure): acute, chronic
Drugs or metabolites: mannitol, radiocontrast dye, salicylate, acetaminophen
SIADH, diabetes insipidus
Trauma, crush injury
Circulatory collapse: severe heart failure, sepsis
Table 3 Causes of metabolic acidosis. The serum osmolal gap is high in patients with acidosis due
to exogenous solutes gain. A low urine osmolal gap reflects on a low urinary excretion of
ammonium (NH4+), in the patients with acidosis due to defective acid excretion of the kidney.
This kind of acidosis can be easily detected by low urine osmolal gap (*GI gastrointestinal)
I. Acid gain (production)
1. Endogenous
Lactic acidosis, diabetic ketoacidosis, renal failure
2. Exogenous: high serum osmolal gap
Methanol, ethanol, ethylene glycol, paraldehyde
II. Alkali (HCO3-) loss
1. Direct loss
1. GI* loss: diarrhea, pancreatic/biliary drainage, short bowel, ileostomy
2. Renal loss: proximal renal tubular acidosis
2. Indirect loss: low urine osmolal gap
Defective HCO3 reclamation due to defective H+ excretion
Distal renal tubular acidosis, hypoaldosteronism, Gordon syndrome, mild chronic renal
failure
*GI gastrointestinal
(mg/dL)/3.7–0.35 or (2) 1.25 ethanol (mmol/L) – 0.35 (Purssell et al. 2001). A

high anion gap metabolic acidosis with a high osmolal gap may also be caused by the
ingestion of toxic alcohol/glycols, such as methanol and ethylene glycols (Kraut
et al. 2013). A serum osmolal gap >10 mOsmol/kgH2O can be used as a reliable
screening test for methanol or ethylene glycol ingestion (Lynd et al. 2008). Other
alcohol derivatives, such as diethylene glycol and propylene glycol, can cause a high
anion gap metabolic acidosis with a high serum osmolal gap. In contrast, isopropyl
alcohol (isopropanol or rubbing alcohol), which is metabolized to acetone, results in
a high osmolal gap without metabolic acidosis (Slaughter et al. 2014; Table 3).
In ketoacidosis, lactic acidosis, and advanced chronic kidney disease (renal
failure), the serum osmolal gap may be slightly increased; a typical osmolal gap in
100 800
600
UOG (mosmol/kgH2O)
50
UAG (mmol/L)
400
0
200
-50 0
Fig. 4 Urine anion gap and osmolal gap in chronic renal failure and distal renal tubular
acidosis patients and in acid-loaded normal controls. Patients with chronic renal failure (solid
circle) and distal renal tubular acidosis (triangle) had defects in acid excretion in the collecting
ducts. The urine osmolal gaps were very low in those patients due to decreased NH4+ excretion in
urine compared to the acid-loaded normal controls (open circle) (Kim et al. 1996)
these conditions is in the region of 10 mOsmol/kgH2O, and it is rarely >20 mOsmol/

kgH2O (Dursun et al. 2007).
Estimation of the serum osmolal gap can serve a variety of purposes. It is a useful
indicator for the initiation and termination of dialysis in methanol intoxication
(Hunderi et al. 2006). The increased serum osmolal gap is predictive of contrast-
induced acute kidney injury (Kim et al. 2012; Ford et al. 2013). An increased
osmolal gap (>10 mOsmol/kgH2O) has been reported in patients with a wide variety
of conditions including acute renal failure (associated with sepsis, abdominal sur-
gery, or acute pancreatitis), abdominal sepsis with liver damage, severe heart failure,
syndrome of inappropriate antidiuretic hormone (SIADH), acquired diabetes
insipidus, trauma or crushing injuries, lactic acidosis, and ingestion of drugs includ-
ing salicylate, acetaminophen, and lorazepam (Tomey 1997; Shull 1978).
Urine Osmolal Gap
In clinical practice, the urine osmolal gap is useful in the differential diagnosis of
normal anion gap metabolic acidosis.
Normal individuals excrete over 75 mmol/L (150 mOsmol/kgH2O) of urine
NH4+ per day, and this increases further in metabolic acidosis. The reference value of
the urine osmolal gap for the individual with metabolic acidosis whose acid excre-
tion of the kidney is normal is up to 150–200 mOsmol/kgH2O (Kim et al. 1996;
Halperin et al. 2004); (Fig. 4). A urine osmolal gap below normal limits indicates
that urinary NH4+ excretion is low. A decreased urinary NH4+ excretion may result
50 J. Lee et al.
from defective renal acid excretion which will be observed in patients with distal
renal tubular acidosis, early chronic renal failure, and resistance to or deficiency of
aldosterone. Impaired urine acidification results in a decrease in urinary NH4+ and
net acid excretion (NAE). Distal renal tubular acidosis, early chronic renal failure, or
defective response to aldosterone with impaired urinary acid excretion is associated
with a high urine anion gap and a low urine osmolal gap.
In patients with a high anion gap metabolic acidosis, the urine osmolal gap would
be high due to urinary loss of organic acid anions (i.e., ketones in diabetic
ketoacidosis).
Limitations to the Clinical Utility of the Osmolal Gap
Various Causes of an Osmolal Gap
An elevated serum osmolal gap can occur in a variety of clinical settings, other than
metabolic acidosis. Causes of an osmolal gap include sick cell syndrome in patients
with multiorgan failure (Guglielminotti et al. 2002), absorption of glycine, and
intravenous infusion of hypertonic mannitol (Dorman et al. 1990). After ingestion
or intoxication, toxic alcohol/glycols are metabolized from uncharged active
osmoles to charged molecules such as formate or glycolate; the latter molecules
have no effect on osmolality or the osmolal gap. Therefore, the diagnostic usefulness
of the serum osmolal gap is limited in the acute phase of intoxication with ethanol or
toxic alcohols.
Limitations of Methods of Osmometry
Accurate measurements of osmolality using osmometry require that the solutions

have specific characteristics. Freezing point osmometry cannot be performed on
samples containing particles, which can act as crystallization nuclei, or on samples
with viscosities that are different to water. Vapor pressure osmometry is less useful in
certain circumstances, particularly for samples containing volatile solutes (Walker
et al. 1986; Sweeney et al. 1993).
Summary Points
• This chapter focuses on the clinical usefulness of the serum or urine osmolal gap
in the differential diagnosis of metabolic acidosis.
• Metabolic acidosis is the process of acid (H+) gain or alkali (HCO3) loss.
• In metabolic acidosis, the associated anions are increased in the blood due to
acid gain.
• Acids are converted from endogenous solutes or exogenously inflowing solutes.
• Acids and associated anions converted from endogenous solutes or exogenously
inflowing solutes act as osmoles.
• In the case of metabolic acidosis, there is an additional increase in osmoles in the

serum due to ingestion of toxins or drugs (exogenous solutes).
• Decreased renal excretion of acid results in a low urinary excretion of ammonium
NH4+ and defective urinary acidification: distal renal tubular acidosis, early
chronic renal failure, and resistance to or deficiency of aldosterone.
• Since the majority of the urine osmolal gap consists of NH4+, a low urine osmolal
gap may be useful in screening for defective urinary acidification.
References
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Kim GH, Han JS, Kim YS, et al. Evaluation of urine acidification by urine anion gap and urine
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Peritoneal Effluent Biomarker Discovery
in Peritoneal Dialysis: The Omics Era 4
Deirisa Lopes Barreto and Dirk G. Struijk
Contents
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Methodological Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Analytical Validity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Study Designs and Population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Omics Data Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Current Developments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Proteomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
Genomics and Metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
Potential Applications to Prognosis, Other Diseases, or Conditions . . . . . . . . . . . . . . . . . . . . . . . . 64
Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Abstract
One of the main renal replacement treatment modalities for patients with
end-stage renal diseases is peritoneal dialysis (PD). In PD therapy, the peritoneum
is used as an intracorporeal dialysis system. The monitoring of intraperitoneal
events is hampered by the absence of serial peritoneal biopsies. However, the
acquisition of peritoneal effluent is simple and usually occurs after a predefined
dwell or if possible after a standardized peritoneal function test. This peritoneal
effluent is composed of several proteins and metabolites, which modifies accord-
ingly due to intraperitoneal events. To date, peritoneal effluent biomarker discov-
ery is evolving with a holistic perspective. The rise of applying suffix -omics
technologies within PD therapy introduced a more exploratory approach for the
D. Lopes Barreto (*) • D.G. Struijk

Division of Nephrology, Academic Medical Center, University of Amsterdam, Amsterdam,
The Netherlands
e-mail: D.LopesBarreto@amc.uva.nl; D.G.Struijk@amc.uva.nl

54 D. Lopes Barreto and D.G. Struijk
identification of candidate effluent biomarkers. The application of genomics,

metabolomics, and proteomics with the peritoneal effluent as biospecimen is
however still in its infancy.
The emerging field of omics techniques as tools for peritoneal effluent
biomarker discovery is presented in this chapter. The high sensitivity of omics
technologies requires stringent conditions, and therefore methodological pre-
cautions must be undertaken on laboratory technical level, appropriate selection
of study design and population, as well as data analysis. For this reason,
methodological considerations for conducting omics-based PD research and
the current developments with regard to the usage of these disciplines are
addressed. Lastly, a summary is given on the available literature concerning
the usage of omics techniques with the peritoneal effluent as a liquid biopsy
within PD therapy.
Keywords
Biomarker • Discovery • Effluent biomarker • Genomics • Metabolomics •
Peritoneal dialysate • Peritoneal dialysis • Peritoneal effluent • Proteomics
Abbreviations
2D-DIGE Two-dimensional difference gel electrophoresis
Biobank Biological bank
Biomarker Biological marker
CAPD Continuous ambulatory peritoneal dialysis
CKD Chronic kidney disease
CRP C-reactive protein
CV Coefficient of variation
DN Diabetic nephropathy
DNA Deoxyribonucleic acid
ELISA Enzyme-linked immuno assay
EPS Encapsulating peritoneal sclerosis
GN Glomerulonephritis
GWAS Genome-wide association studies
Ig Immunoglobulin
IL-6 Interleukin-6
MS Mass spectrometry
NECOSAD Netherlands Cooperative Study on the Adequacy of Dialysis
NMR Nuclear magnetic resonance
NRI Net reclassification index
PD Peritoneal dialysis
RNA Ribonucleic acid
ROC curve Receiver operating characteristic curve
SNPs Single nucleotide polymorphisms
SOP Standard operating procedures
4 Peritoneal Effluent Biomarker Discovery in Peritoneal Dialysis: The Omics Era 55
Definitions
Biological bank (biobank) An archive containing human biospecimens that may

include blood, effluent, serum, or tissue samples. The storage of these samples occurs
preferably under standardized conditions within a single or multicenter study cohort.
Encapsulating peritoneal sclerosis (EPS) EPS is the most devastating complica-

tion of PD therapy that occurs in 3–7 % of PD patients. EPS is characterized by a
dense cocoon of fibrous tissue that covers the abdomen. High mortality rates and
severe morbidity are present for patients diagnosed with EPS.
Free water transport (FWT) Aquaporin-1 mediated transport of water, without

dissolved solutes and electrolytes. FWT is one of the peritoneal transport parameters
that become impaired in long-term PD patients.
Genomics Laboratory strategy that investigates genes and their functions. Utilized
methods comprise DNA sequencing and fingerprinting. Genomics is furthermore
subdivided into various disciplines such as epigenomics and metagenomics and
functional genomics and structural genomics.
Metabolomics Laboratory method for the identification and quantification of

metabolite levels. Chromatography is one of the primary steps used as a separation
technique. Thereafter, the metabolites may be detected by means of mass spectrom-
etry (MS) or nuclear magnetic resonance (NMR) spectroscopy.
Peritoneal dialysis (PD) therapy One of the renal replacement therapies for
end-stage renal disease patients for over almost five decades. The proportion of
PD patients in the worldwide dialysis population covers 11 %, and the 5-year
survival has increased to 41 %. In many countries, PD therapy is presented as
primary dialysis modality choice, as there is a survival benefit over hemodialysis
in the first 3 years.
Peritoneal function test A standardized peritoneal test to assess the function status
of the peritoneal membrane. Additionally, the tests provide insight into the magni-
tude of small-solute removal, fluid transport, and ultrafiltration capacity. Several
forms of peritoneal function tests are available such as a standardized peritoneal
permeability analysis or (modified) peritoneal equilibration test. However, all of
them are characterized by a predefined dwell time with or without intermediate
sampling of the peritoneal effluent.
Peritoneal membrane The peritoneal membrane is described to be a semiperme-

able membrane that consists of three main layers. Firstly, the mesothelial cell layer is
encountered, followed by the interstitium in which the peritoneal capillaries are
imbedded. Long-term and continuous exposure of PD solutions to this membrane
may lead to functional and morphological alterations.
Proteomics Laboratory discipline for analyzing structure, conformation, and bio-

logical function of proteins. In brief, proteomic analyses encompass separation of the
proteins by gel electrophoresis methods, followed by MS-based analyses.
Systems biology A holistic point of view for gaining insight into biological mech-
anisms within cells, organisms, or species. Systems biology has been applied from
the early 1900. Genomics, metabolomics, and proteomics are disciplines within
systems biology that seek to qualify and quantify the genome, metabolome, or
proteome, respectively.
Introduction
The field of omics is expanding at a rapid pace as novel instruments for biological
marker (biomarker) discovery and unraveling pathophysiological mechanisms.
Within peritoneal dialysis (PD) therapy, the application of high-throughput technol-
ogies is still in its infancy. Especially, the peritoneal effluent as biospecimen, which
contains a variety of proteins either due to transperitoneal transport or local produc-
tion, has not yet been explored extensively (Table 1). Numerous biomarker consor-
tia, working groups, and scientists contributed to the diversity of existing biomarker
definitions with different classifications and applications (Atkinson et al. 2001;
Table 1 Key facts of peritoneal dialysis and peritoneal effluent biomarkers

In PD therapy, the peritoneum serves as a biological semipermeable dialysis membrane. Through
this membrane, toxic waste products and excess fluid are removed from the circulation into the
peritoneal cavity. During the day, 3–5 daily exchanges of PD solutions take place. These
exchanges vary in dose and duration of the dwell. After a predefined dwell time, the infused
dialysis solution is drained. This drained fluid is called the peritoneal effluent or peritoneal
dialysate, which contains several substances
Proteomic profiling and characterization of the peritoneal effluent indicated that the substances
represent merely extracellular proteins. In addition, the circulation is found to be responsible for
the presence of a majority of peritoneal effluent constituents. Substances or proteins in the
peritoneal effluent can only be eligible as effluent biomarker within PD if they are locally
produced within the peritoneal cavity
Unfortunately, no direct visualization of the peritoneal membrane is possible without invasive
procedures. Furthermore, computed tomography scans are unable to detect anatomical
modifications timely. Peritoneal effluent biomarkers are considered as noninvasive instruments for
screening or diagnostic purposes. They would allow uncomplicated monitoring of the integrity of
the peritoneal membrane. For this reason, the peritoneal effluent is regarded as the most clinically
relevant specimen within PD therapy. However, the implementation of the more established
peritoneal effluent biomarkers in routine PD patient care is fairly small
The discovery of peritoneal effluent biomarkers was previously based on pathophysiological
knowledge and hypothesis driven. The first omics-conducted research within PD therapy
originates from 2007. Effluent biomarker discovery through omics technologies is aimed at
providing proxies for specific anatomical alterations. This holistic approach is expected to deliver
insight into the sequence of intraperitoneal events. Nevertheless, current omics studies within PD
require further calibration and standardization
PD peritoneal dialysis
Peritoneal membrane
modifications
Functional Morphological
Increase vascular Loss ultrafiltration Loss free Peritoneal

Interstitium Mesothelium
surface area capacity water transport capillaries
Mesothelial cell
Peritoneal transport parameters Neoangiogenesis Fibrosis
loss
Peritoneal effluent biomarkers
Fig. 1 Peritoneal membrane modifications. The peritoneal membrane consists of three main layers:
mesothelium, interstitium, and peritoneal capillaries. In all of these layers, morphological modifi-
cations are observed for which effluent biomarkers could be utilized for diagnostic or prognostic
purposes. However, not all of these layers are considered as prominent barriers to the peritoneal
membrane transport. The increase in perfused peritoneal capillaries leads to a rapid dissipation of
glucose and consequently may evolve in a decreased ultrafiltration capacity. In a progressive stage,
the fluid transport through the water channels, e.g., aquaporins, may become impaired
Hulka et al. 1990; Perera and Weinstein 2000; Colburn 2000; Jain 2010). Overall,
the essence of molecular biomarkers is to provide noninvasive, cost-effective tools
for screening or diagnostic purposes and disease prognosis and surveillance. Addi-
tionally, molecular biomarkers may also be intended to assess therapeutic respon-
siveness or offer novel intervention strategies (Atkinson et al. 2001).
The main purpose of PD therapy as renal replacement treatment modality is to
remove toxins and excess fluid from the body. For this intention, a permanent
catheter is inserted into the peritoneal cavity through which dialysis solutions can
be instilled. The removal of these toxic waist products and excess fluid, from the
circulation into the peritoneal cavity, occurs mainly by means of diffusion through
the peritoneal membrane. Therefore, the efficacy of PD therapy is highly dependent
on the biological condition of the peritoneal membrane. The continuous exposure of
dialysis solutions to the peritoneal membrane causes however several functional and
morphological modifications (Fig. 1). The most encountered functional changes are
the rapid dissipation of the osmotic gradient and a decrease in ultrafiltration capacity.
Furthermore, some of the long-term PD patients also present with an impairment of
free water transport. Especially in patients who develop EPS, free water transport
appears to be the only peritoneal transport parameter, which can distinguish patients
with this severe complication from patient with a long PD therapy duration (Lopes
Barreto et al. 2014). The functionality of the peritoneal membrane can be monitored
incessantly by peritoneal permeability tests (Coester et al. 2009), whereas the
progression of morphological modifications remains unrevealed because no serial
peritoneal biopsies can be performed. More importantly, the functionality of the
peritoneal membrane is not inherent to the observed morphologic modifications. In
Table 2 Prerequisites for a peritoneal effluent biomarker

1. Detectable in peritoneal effluent by means of omics technologies or other protein detection
methods
2. Computational assessment of local release or production within the peritoneal cavity
3. Involvement in pathological pathway of the peritoneal membrane
4. Good measures of diagnostic accuracy for PD-related outcomes (e.g., high sensitivity/
specificity)
this perspective, more emphasis is placed on potential markers that are present in the
peritoneal effluent and mirror the integrity of peritoneal tissues. Thus, promising
peritoneal effluent substances have to meet several prerequisites before the acknowl-
edgment of a peritoneal effluent biomarker is given to them (Table 2). Prior to the
rise of suffix -omics technologies, effluent biomarker discovery in PD was merely
based on hypothesis-driven research. Even though this is still valid within these
translational scientific disciplines, at present the discovery of effluent biomarkers is
evolving with a more global and exploratory perspective. The application of high-
throughput laboratory techniques offers a great opportunity to gain insight in the
peritoneal alterations that occur over time due to PD treatment and identify clinically
relevant effluent substances that may serve as biomarkers. The vast majority of high-
dimensional methodologies applied to the peritoneal effluent consist of genomics,
metabolomics, and proteomics. Previous reviews have acknowledged the potential
use of proteomics for effluent biomarker discovery and elucidation of pathophysio-
logical processes of the peritoneal membrane (Brewis and Topley 2010;
Thongboonkerd 2010). The present chapter highlights the evolving landscape of
omics, in which the human peritoneal effluent is regarded as central specimen.
Methodological considerations in effluent biomarker discovery are provided, and
an overview is given to illustrate the application and progression of omics-conducted
research within PD therapy.
Methodological Considerations
As a consequence of the large amount of data that is acquired by suffix -omics analyses,
several methodological precautions have to be taken. These preventative measures
include the optimization of analytical validity, well-designed study and selection of
study population, as well as proper data analysis. This section describes these afore-
mentioned considerations and provides examples in PD-conducted omics studies.
Analytical Validity
In biomarker discovery studies, three main phases are encountered on a laboratory

technical level that might alter research findings and thwart the interpretation of the
acquired data: pre-analytical, analytical, and post-analytical.
The pre-analytical phase evolves sample handling and has a direct influence on the
accuracy and reproducibility. This is especially of importance when investigating the
proteome, metabolites, or candidate effluent biomarkers, which are influenced by
structural peritoneal membrane modifications over time. One of the bias-introducing
factors could be the effect of storage on the assembled effluent biospecimens. DNA
extraction and genotyping from frozen peritoneal effluent samples up to 7 years at
20 C indicated no influence of storage duration (Gillerot et al. 2004). However, it is
not known whether long-term storage of the effluent at lower temperatures is superior
to temperatures of at least 20 C. Moreover, the stability of metabolites and proteins
present in peritoneal effluent and the effect of repeated freeze-thaw cycles have not
been investigated yet. To preserve the quality of the effluent and reduce the amount of
variability due to incorrect or unbalanced sample handling, standard operating pro-
cedures (SOPs) are needed. Included elements in a SOP for local or multicenter
biorepositories should at least cover the amount and volume of aliquots, storage
conditions, e.g., minimal temperature of 20 C, mechanical freezer, or directly frozen
by liquid nitrogen, and the necessity to document the number of freeze-thaw cycles.
From the omics studies within the discipline of PD, only some of the articles provided
detailed information on sample collection and archiving comprising momentum of
effluent withdrawal, storage temperature, and time frame of sample processing. For
comparability and assessment of study quality, reporting this information is essential.
Intra-analytical inaccuracies may contribute to random or systematic errors.
These errors occur during assaying of effluent constituents, which could be
influenced by sampling handling of the laboratory personnel, measurement appara-
tus, or reagents. By performing the experiments in dupli- or triplicate, the degree of
precision can be assessed. Also, the range of standard reference curves should be
wisely chosen in order to determine the appropriate detection limit of an assay.
Typically acceptable coefficients of variation (CV) lie beneath 20 % for which one
should strive for CVs of 5 %. Furthermore, in the validation phase of biomarker
discovery by means of enzyme-linked immunosorbent assays (ELISAs) or Western
blots, one could reduce intra- and inter-variability by even distribution of the study
groups within and throughout batches. Preferably, the laboratory technician should be
blinded for the outcome of interest as well. Recently, a methodological article proposed
an optimal high-resolution effluent protein separation technique by two-dimensional
gel electrophoresis (2D-DIGE) intended for proteomic analysis (Zhang et al. 2013).
The authors investigated five precipitation methods followed by constraining abundant
proteins of the peritoneal effluent. Such attempts are in favor of reducing sample
preparation heterogeneity and enhance critical appraisal of the used high-throughput
methodologies across studies analyzing the peritoneal effluent.
Incorrect post-analytical inferences may lead to differential misclassification bias.
For example, if threshold values of the peritoneal effluent are inappropriately
determined, the contrast between groups could be augmented or diminished. As a
consequence, the results could indicate nonexisting differences between the group
with the outcome of interest and those without. Eventually, this nonrandom mea-
surement error may contribute to biased estimates of association.
Study Designs and Population
The majority of the omics studies with the peritoneal effluent follow a cross-
sectional design. Generally, in these studies, the profiles of PD patients with the
characteristic or clinical endpoint of interest are compared to PD patients with a
stable condition. Omics studies have to be well designed, especially with regard to
the study subjects where one should strive for a homogeneous population, as the
peritoneal metabolome and proteome of PD patients are likely to be susceptive to
posttranslational modifications due to patient-related and external factors. Therefore,
the selection of patients as well as a priori specification of a validation subset is of
similar or even of greater importance when compared to other epidemiologic studies.
Unfortunately, often clinical and demographic data of the study population or
independent validation sample is lacking in omics-conducted PD research. The
sparse number of cohort and nested case-control studies is presumably the result
of impracticable specimen collection or cost related. This is however a great loss, as
the presence of a local biological bank with repetitive effluent specimens would
enable trend analyses. In addition, the sample size is relatively small in the majority
of the omics studies with PD therapy. Lastly, it is questionable whether the follow-up
duration in a number of studies is sufficient to measure difference in peritoneal
membrane alterations. Especially, since the factual peritoneal membrane dysfunction
is usually observed after a therapy duration of at least 2 years.
Another important factor of the peritoneal effluent is its origin. As the peritoneal
effluent can be obtained right after a regular PD exchange or after a standardized
peritoneal function test, it follows from this that the biological variability increases
concordantly. Therefore, the withdrawal of effluent samples in studies should be
harmonized within or between centers. Regrettably, not all omics studies within PD
report whether the peritoneal effluent is derived from a regular dwell of after a
standardized peritoneal function test.
Omics Data Analysis
An effluent biomarker can be an outcome to monitor the progression of PD therapy as

well as a prognostic factor of various PD-related complications. When a biomarker is
intended as a diagnostic or prognostic instrument, receiver operating characteristic
(ROC) curve and C-statistics are used in order to evaluate the discriminative power.
Additionally, optimal threshold values can be estimated based on the sensitivity and
specificity of a biomarker. However, these measures are highly dependent on the base
study population, and misclassification may arise due to patient demographics, labo-
ratory measurement errors, and the degree of the exposure to dialysis solutions. Hence,
one has to be cautious with the definition and selection of a correct clinical endpoint in
biomarker research. Nevertheless for that reason, the net reclassification index (NRI)
was introduced suggesting a method to gain prognostic accuracy of a biomarker
(Wilson et al. 2008). The interpretation of omics-derived findings usually requires the
use bioinformatics or the application of molecular epidemiology. In general, the sample
Fig. 2 Proportion of omics

studies with the human
peritoneal effluent as
biospecimen. The number of
studies applying omics
technique with the human
peritoneal effluent as central
biospecimen is still limited.
Proteomics attributed to 81 %
of all studies followed by
genomics (13 %) and
metabolomics (6 %)
sizes of the current omics studies in PD are small. However, omics studies generate
enormous amount of data that are subjective to false-positive or false-negative results
when not handled as appropriate. Therefore, correction for multiple comparisons or
adjustments with regard to p-value thresholds should be applied.
Current Developments
Proteomics is the main applied high-throughput technology followed by genomics

and metabolomics for peritoneal effluent biomarker discovery (Fig. 2). These fields
have the potential to elucidate underlying molecular mechanisms that are involved in
the pathophysiology of the peritoneum. Moreover, they can empirically provide
novel diagnostic and therapeutic biomarkers based on genome, metabolite, or
protein profiles of PD patients. Nevertheless, the number of studies is still modest;
a summary is given on the recent developments and main findings.
Proteomics
In nephrology, proteomics is merely applied on plasma, serum and urinary samples,

or renal tissue. The number of proteomics studies with the peritoneal effluent as
sample type is rising, and investigations have been executed from various perspec-
tives other than the identification of novel biomarkers for PD (Fig. 3). The main
obstacle for the discovery of effluent biomarkers includes the dynamic range in
proteomic analyses.
Uremia in itself is suggested to alter the functional and structural organization of the
peritoneal membrane. Therefore, the effect of a uremic environment was investigated
Fig. 3 Areas of interest 8% Areas of interest proteomic

within proteomics studies. studies:
The proof-of-principle studies 15%
Uraemic effect
applying proteomic analyses
have been executed with 23%
interests in the effect of Glucose effect
uremia (8 %) and glucose
(15 %), peritoneal transport Peritoneal transport status
status (23 %), and
characterization and profiling 54% Characterization peritoneal
of the human peritoneal
effluent
effluent (54 %)
by proteomic analysis in chronic kidney disease patients (stage five) versus patients
with normal renal function (Wang et al. 2012). A number of protein alterations were
found including elevated levels of vascular endothelial growth-A (VEGF-A).
The majority of proteomics research is aimed at the characterization and profiling
of the peritoneal effluent in order to identify potential biomarkers. This discovery-
based approach has been adapted in the peritoneal effluent of incident and prevalent
adult PD patients (Wu et al. 2013; Cuccurullo et al. 2011). The study by Wu
et al. included a number of ten incident PD patients for whom three patients were
used for validation. The peritoneal effluent was assembled at start of PD therapy and
once again after 1 year. Validation by Western blots showed elevated levels of
immunoglobulin (Ig) μ chain, fibrinogen γ chain, and C-reactive protein (CRP) at
baseline and Ig δ, α-1 antitrypsin, histidine-rich glycoprotein, apolipoprotein A1,
and serum amyloid P-component after 1 year of PD therapy duration. The authors
speculated that elevation in the protein levels that were measured after 1 year could
indicate markers for early peritoneal membrane injury. The second study consisted of
15 prevalent PD patients with varying PD therapy duration ranging from 1 to 84 months.
A subgroup was additionally defined to study the effect of glucose in PD solutions.
Profiling and characterization of the peritoneal effluent has also been investigated in
nine pediatric PD patients (Raaijmakers et al. 2008). A number of 88-shared proteins
were identified. All of the abovementioned studies indicated that the effluent of PD
patients is merely from systematic origin and reflects extracellular proteins. Charac-
terization of the human effluent has furthermore been performed with regard to
diabetic PD patients (Yang et al. 2013; Wang et al. 2010) and before and after a
peritonitis episode (Lin et al. 2008; Tyan et al. 2013). Validation by Western blots
was performed within the same study populations. The latter study identified up to
41 proteins with shared alterations in haptoglobin expression and revealed in an area
under the receiver operating characteristic curve of 0.92.
Higher levels of glucose and osmolarity in PD solutions are known to induce a
greater removal of excess fluid and toxic waste products. However, continuous
exposure of these PD solutions high in glucose leads to damage to the peritoneal
membrane. In this respect, proteomic analyses have been performed as well. An
increased appearance of advanced glycosylation end products was shown when

patients were infused with higher percentages of glucose-based dialysis solutions
(Pešić et al. 2011). Moreover, a number of nonredundant proteins including
cystatin C, collagen, fibronectin, matrix metalloproteinase-2, plasminogen, and
vitronectin were identified (Cuccurullo et al. 2011; Pešić et al. 2011). Additionally,
an under-expression was found for α-1 antitrypsin, apolipoprotein A-IV, fibrinogen
β-chain, and transthyretin in patients treated with the highest glucose concentration
(4.25 %) of PD solutions when compared to those treated with 1.5 % or 2.5 %
glucose-containing PD solutions (Cuccurullo et al. 2011).
Due to the large interindividual variation in peritoneal transport status at initiation
of PD therapy, comparative analyses of the peritoneal effluent in PD patients with
different peritoneal transport characteristics have been studied as well. In these
studies, the authors found increased protein losses for PD patients with a fast
transport status as compared to patients characterized by slow peritoneal transport
rates (Wen et al. 2013; Sritippayawan et al. 2007). External validation by ELISA
confirmed elevated levels of complement 4A and IgG in the fast transporters
(Sritippayawan et al. 2007). Overall, the heterogeneity in study populations and
practical laboratory techniques contributes to the variety of identified proteins and
complexes inferences throughout studies.
Genomics and Metabolomics
Genomic biomarkers have not yet been identified, but intriguing single nucleotide
polymorphisms (SNPs) have been found in the C/C genotype on the interleukin-6
(IL-6)-174G/C loci (Verduijn et al. 2012). The base population for this study
originated from a Dutch multicenter cohort, also known as the Netherlands Coop-
erative Study on the Adequacy of Dialysis, archiving peritoneal effluent and serum
of incident dialysis patients. Additionally, two external cohorts were used for
independent data replication. A significant increased risk for mortality was associ-
ated with this IL-6 gene variant in adult patients who survived PD treatment over a
period of at least 2 years. Albeit external cohorts were defined to authenticate these
findings, further validation is necessary. Polymorphisms in the promoter region of
VEGF have also been associated with an increased risk for mortality (Szeto
et al. 2004). This was found in a prospective cohort study in 135 continuous
ambulatory PD (CAPD) patients who had a follow-up duration of 1 year. The
effluent was obtained within 2 months after start of PD and after 12 months.
Furthermore, effluent levels of VEGF measured by ELISA showed a tendency
toward lower levels in patients with the CC genotype when compared to those
with an AA/AC genotype. In contrast, messenger RNA expression was significantly
lower in PD patients with the CC genotype.
The GLOBAL Fluid Study group is a prospective longitudinal worldwide
biobank within PD that serially collects peritoneal effluent alongside serum samples.
The effluent samples within the cohort are all obtained at the end of a 4-h peritoneal
equilibration test in incident PD patients and repeated thereafter at predefined

intervals. This group recently published a study in which an attempt was made to
identify metabolic profiles specific for PD patients who developed EPS (Dunn
et al. 2012). As no previous study investigated metabolites within the peritoneal
effluent, this study secondly aimed to provide optimal strategies for analyzing the
metabolome alongside the identification of differences in effluent composition. The
authors found that the peritoneal effluent consists grossly of low molecular weight
metabolites. Moreover, prior to the diagnosis of EPS, modifications in several amino
and short-chain fatty acids and its derivates were present. The abovementioned
studies demonstrate the capability and importance of longitudinal (multicenter)
study cohorts containing effluent specimens.
Potential Applications to Prognosis, Other Diseases, or Conditions
Within the discipline of nephrology, suffix -omics technologies are widely

applied. The earliest studies originate from 1997 with an increasing tendency in
the number of published articles ever since. Genome-wide association studies
(GWASs) have contributed greatly to the understanding of chronic kidney dis-
eases (CKDs) as well as the main kidney failure diseases: renovascular disease,
diabetic nephropathy (DN), and glomerulonephritis (GN) (Atzler et al. 2014;
Kottgen 2010). The reviews on the recent developments of genomic and
metabolomic analyses uncovered that over 98 % of the estimated heritability
within nephrological diseases remains to be revealed. From this, it can be con-
cluded that a majority of the pathophysiological mechanisms of kidney diseases
and function still warrants elucidation. Proteomics have been applied widely as
well with similar intentions such as biomarker discovery, to reveal causal path-
ways of kidney diseases and to indicate potential therapeutic targets (Bonomini
et al. 2012). Proteomic studies within nephrology have focused on urinary bio-
markers for DN, IgA nephropathy, lupus nephritis, and rejection of renal trans-
plants (Papale et al. 2010; Rocchetti et al. 2008; Zhang et al. 2008; Metzger
et al. 2011). However, harmonization of the analytical procedures is warranted.
Another commonly mentioned issue encompasses the study designs, which are
merely represented by transversal research rather than longitudinal cohorts inves-
tigating the course of events. Moreover, the sample sizes of some studies are
relatively small. These tendencies are in line with the current advances observed
in omics-conducted PD research.
The utilized biospecimens within nephrology range from blood and serum sam-
ples to urine and renal tissue. Within nephrology, these various specimens have been
investigated extensively. However, the clinical application of potential biomarkers
still fails to appear despite their promising results. One of the aspects that also should
not be overseen is the collaboration between the clinicians, epidemiologists, (labo-
ratory) scientists, and healthcare funding organizations to enable a more rapid
integration and implementation of biomarkers.
- Coefficient of variation
Computational based (inter & intra)
on:
molecular weight or - Influence of freeze-thaw Multi- or single center
free diffusion coefficient cycles and temperature cohort studies
Omics technologies
Assessment of Time course assessment &

Detection in Evaluation biological
local peritoneal associations with peritoneal
peritoneal effluent variability & stability
production transport parameters
Biomarker identification:
- Discriminative capacity
-Association with PD-related outcomes
Independent Establishment
validation threshold values
- External study population - Effluent biomarker for

screening, diagnosis or
prognosis
- Experimental models
(in vitro / in vivo)
- Histological
Fig. 4 Suggested flowchart for peritoneal effluent biomarker discovery. A flowchart is suggested
for effluent biomarker discovery that are detected by means of omics technologies. Some of the
suggested phases are based on the requirements of a peritoneal effluent biomarker. These comprise
the detection of the substance in peritoneal effluent, which should be locally produced within the
peritoneal cavity. Furthermore, the candidate peritoneal effluent marker is involved in the pathology
of peritoneal membrane and related to peritoneal dialysis-related clinical outcomes
Future Directions
As blood, serum, and urine samples are not representative for intraperitoneal events,
the central emphasis remains to be on the peritoneal effluent. PD treatment induces a
complex and multifactorial pathogenesis of the peritoneal membrane. The deficiency
of this non-defined common pathway contributes to the difficulties in effluent
biomarker discovery. These challenges can possibly be overcome with the unbiased
field of omics technologies, where associations between clinical and expression data
from confirmatory investigations may lead to the perception of underlying biological
processes preceding peritoneal injury and novel biomarker identification. A
suggested flowchart for effluent biomarkers discovery is depicted in Fig. 4 in
which their prerequisites are integrated. To our knowledge, unfortunately no perito-
neal tissue of patients treated with PD has been investigated by means of omics
technologies. The presence of serial peritoneal tissue alongside peritoneal effluents
of PD patients would be of great additive value and a prerequisite for omics-
conducted PD investigation. Nevertheless, it is doubtful that an individual peritoneal
effluent biomarker possesses the ability to mirror or predict all of these processes.
More likely is that a synergism of effluent biomarkers will eventually be identified in
order to predict clinically relevant PD outcomes. Thus, large multicenter biobanks
with preferably longitudinal data would contribute significantly to the discovery of
novel effluent biomarkers and their validation. The number of omics-based research
in PD is still limited, and the early phase of high-throughput technologies warrants
standardization and calibration. Essential in the conductance of omics studies is
systematic collection and storage of peritoneal effluent. To date, the absence of
longitudinal effluent biobanks including peritoneal tissue and a small sample size
has prevented the analysis of modifications in proteomic profiles over time. How-
ever, longitudinal multicenter studies such as the GLOBAL Fluid Study or within
center biobanks will hopefully bridge this gap. Furthermore, collaborations are
necessary to facilitate independent replication and validation of candidate effluent
biomarkers.
In summary, peritoneal effluent biomarker discovery is moving toward system
biology where a holistic approach may eventually lead to personalized-guided
medicine within PD. Nevertheless, the challenge remains, and the actual application
and implementation of omics-discovered effluent biomarkers is a process that may
encompass decades.
Summary Points
• This chapter focuses on the emerging field of omics technologies within perito-
neal dialysis (PD) therapy for the discovery of peritoneal effluent biomarkers.
• The peritoneal effluent can be regarded as a noninvasive liquid biopsy within PD
therapy that is easily acquired after a (standardized) predefined PD exchange.
• The peritoneal effluent contains clinically relevant proteins and substances such
as lymphocytes, macrophages, and a variety of proteins that mirror intraperitoneal
events.
• The peritoneal effluent is susceptible to posttranslational modifications.
• Methodological precautions on a laboratory level as well as computational
techniques are essential for proper assessment and interpretation of omics-
derived data.
• The number of studies that apply high-throughput laboratory techniques with the
peritoneal effluent as biospecimen within PD therapy is still limited.
• Peritoneal effluent biomarker discovery is moving toward systems biology.
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Metabolomics and Stages of Chronic
Kidney Disease 5
Toshihiro Kobayashi
Contents
Key Facts About Metabolomics and Stages of Chronic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . 70
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Why Use Metabolomics to Diagnose CKD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
Using Mass Spectrometry for Metabolite Exploration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
Nontarget Analysis and Target Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Nontarget Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
Target Analysis (Multi-target Analysis) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
Future of Metabolomics in the Diagnosis of Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
Potential Applications to Prognosis and Other Diseases and Conditions . . . . . . . . . . . . . . . . . . . . . . . 80
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Abstract
The kidneys are critical for the secretion of cytokines and hormones, the excretion
of waste metabolites, and the homeostasis of electrolytes. Chronic kidney disease
(CKD) is a major epidemiologic problem and a risk factor for cardiovascular
events and cerebrovascular accidents. At present, renal function is generally
evaluated by measuring estimated glomerular filtration rate (eGFR). However,
this method has low sensitivity during the early stages of CKD. A new biomarker
that can detect CKD during its early stages is eagerly awaited: mass spectrometry
(MS), an effective technology for the discovery of biomarkers due to its high
sensitivity to detect many compounds, seems to fit these conditions.
Metabolomics using mass spectrometry is a powerful strategy for profiling
metabolites and can be used to effectively explore unknown compounds that
T. Kobayashi (*)
College of Nutrition, Koshien University, Takarazuka, Hyogo, Japan
e-mail: kobayapi1973@yahoo.co.jp; t-kobayashi@koshien.ac.jp

70 T. Kobayashi
change in abundance with respect to disease condition. Recently, many

researchers have endeavored to apply metabolomics techniques to diagnose
various diseases, including CKD. Some metabolites that can serve as biomarkers
for CKD severity have been discovered, thanks to their efforts.
This chapter reviews metabolomics techniques and their potential to be
applied to CKD diagnosis.
Keywords
Chronic kidney disease • Metabolomics • Biomarker • Multivariate statistics •
Mass spectrometry
Abbreviations
CE-MS Capillary electrophoresis-mass spectrometry
CysC Cystatin C
DMSO Dimethyl sulfoxide
ESRD End-stage renal disease
FT-ICR-MS Fourier transform-ion cyclotron resonance-mass spectrometry
GC-MS Gas chromatography-mass spectrometry
HMDB Human metabolome database
KEGG Kyoto encyclopedia of genes and genomes
LC-MS Liquid chromatography-mass spectrometry
MS/MS Tandem mass spectrometry
PLS Partial least squares
SFC-MS Supercritical fluid chromatography-mass spectrometry
TOC Total organic carbon
TOF-MS Time-of-flight-mass spectrometry
Key Facts About Metabolomics and Stages of Chronic Kidney

Disease
• Chronic kidney disease (CKD) is a concept that was advocated for the first time in
the United States in 2002.
• A lifestyle-related disease, CKD has attracted concern and has entered the
medical consciousness worldwide.
• Left untreated, CKD aggravates irreversibly and finally degenerates into
end-stage renal disease (ESRD), requiring either dialysis therapy or renal
transplantation.
• Dialysis therapy is effective in removing uremic toxins from the blood but has no
effect on anemia or electrolyte disturbance, resulting in impaired quality of life.
• ESRD also is a risk factor of cardiovascular disease.
• Thus, early detection and treatment of CKD is not only an important issue for the
health of individuals but also a wider problem in the medical economy.
5 Metabolomics and Stages of Chronic Kidney Disease 71
Definitions
Chronic kidney disease (CKD) and glomerular filtration rate (GFR) CKD is
defined by the following two criteria. (1) Kidney damage confirmed by urine tests,
imaging tests, and blood tests. Especially, albuminuria is a typical symptom.
(2) Reduced glomerular filtration rate (GFR) less than 60 mL/min/1.73 m2 over
3 months.
CKD is classified into six stages according to GFR (see table below) (Levey
et al. 2011). Stage 1 and Stage 2 do not equate to CKD if kidney damage is not
comorbid. Stage 5 is equivalent to end-stage renal disease.
GFR (mL/min/1.73 m2) CKD stage

90> 1
60–89 2
45–59 3a
30–44 3b
15–29 4
15< 5
Estimated glomerular filtration rate (eGFR) Since direct measurements of GFR

are invasive and time-consuming, GFR is typically estimated using blood creatinine,
age, and gender. There are various formulas for calculating eGFR: the Cockcroft–
Gault equation, the Modification of Diet in Renal Disease (MDRD) Study equation,
the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation, the
Japanese Society of Nephrology Chronic Kidney Disease Initiative (JSN-CKDI)
equation, and so on. Because blood creatinine is proportional to the muscle mass, the
formula used differs depending on the patient’s race and gender.
Introduction
Mass spectrometry (MS), which is capable of quantitative and qualitative analyses

with high sensitivity, is a useful technique for determining the presence of various
trace metabolites in body fluids like blood or urine. Taking advantage of MS’s utility,
many researchers have attempted to perform definitive diagnosis or early detection
of diseases using it, including metabolic abnormalities, cancers in various organs,
and chronic kidney disease (CKD). In fact, mass spectrometry is already used to find
metabolic abnormalities in neonatal mass screening programs.
Because healthy kidneys have a large reserve capacity, the early stages of CKD
are difficult to identify by subjective symptoms. CKD is frequently detected by
symptoms of significantly reduced glomerular filtration rate (GFR) or increased
albumin concentration in urine (Levey et al. 2011).
Though glomerular filtration rate can be measured rigorously in terms of inulin
clearance according to international criteria, the method is very intricate and difficult
72 T. Kobayashi
to routinely use clinically. Thus, creatinine clearance and estimated GFR (eGFR) are
generally used as surrogate markers of inulin clearance.
However, creatinine is known to be produced by the degradation of the muscle
and secreted from the proximal renal tubule. Therefore, in aged people, low levels of
blood creatinine may instead be a result of low muscle mass, which creates the
problem of consequent overestimation of renal function. To avoid this confusion,
cystatin C (CysC) has been frequently used as an alternative clinical index for
creatinine in recent years.
CysC is not affected by muscle mass or dietary protein but is affected by
hyperthyroidism, obesity, and steroid therapy (Inker et al. 2012). Thus, CysC
alone is not a sufficient index for GFR, and further studies are needed to determine
whether to include this measurement in standard criteria (Peralta et al. 2011b).
Given this background, some researchers are exploring biomarkers that reflect
renal function loss with the use of mass spectrometry and have proposed some
candidate markers (Niwa 1997, 2009; Toyohara et al. 2010; Kikuchi et al. 2010; Sato
et al. 2011; Kobayashi et al. 2014a).
This chapter outlines search methods for biomarkers that are concerned with renal
function loss using LC-MS and evaluation methods for CKD severity using these
biomarkers.
Why Use Metabolomics to Diagnose CKD
CKD is definitively diagnosed by the following symptoms: abnormally low glomer-

ular filtration rate, existence of albuminuria, and apparent nephropathy as deter-
mined by imaging or pathological examination. These are not preventive indices of
CKD, but definitive criteria.
Representative evaluative indices for GFR are serum creatinine, CysC concen-
trations, and inulin clearance (Peralta et al. 2011a, b; Horio et al. 2013). However,
serum creatinine and CysC have a so-called blind area in that their concentrations are
not increased immediately in the early stages of CKD, which causes difficulty in
detecting CKD in the early stages using creatinine and CysC as indices of CKD
(Herget-Rosenthal et al. 2007; Tangri et al. 2011). While inulin clearance is the gold
standard method for determining GFR (Levey et al. 2005), the procedure for
measuring it is invasive and cumbersome, and so it is not suitable for screening use.
Therefore, from the viewpoint of early discovery and prevention of CKD, it is
necessary to employ indicators that more sensitively reflect decreased renal function
than these CKD diagnostic criteria commonly adopted today. Because healthy
kidneys have a large reserve capacity, even if the concentration of metabolites
in vivo fluctuates due to the pathology of CKD, it is reasonable to believe that the
degree of fluctuation is negligible in its initial stages. Therefore, the use of MS has
been promoted in order to detect very slight changes in the concentrations of
compounds in samples.
The relatively new field of metabolomics has been developed in recent years,
stimulated by the spread of mass spectrometry (Kell 2004). At first, it was proposed
as a means to comprehensively analyze the various metabolic products present in the

living body. Many attempts to assess renal function using metabolomics techniques
have been made to date (Niwa 1997, 2009; Toyohara et al. 2010; Kikuchi et al. 2010;
Sato et al. 2011; Kobayashi et al. 2014a, b).
Using Mass Spectrometry for Metabolite Exploration
Since LC-MS has high sensitivity, the results of metabolomics analysis might be
affected by even slight changes in conditions. Thus, conditions surrounding the
sample vial, sample handling, and analytic method need to be kept constant as much
as possible. These measurement methods, including sample collection and
pretreatment, must be examined beforehand in house with respect to procedures
detailed in previous studies and should not be changed during a series of
experiments.
Pollution of the LC-MS probe and mass detector during repeated analysis,
including by biological samples and contaminants, causes poor peak detection and
high background noise. In addition, column deterioration and slight compositional
changes or flow rate fluctuation of the solvent affect peak retention times. In order to
obtain reliable results, because contaminants in water are a major cause of noise
peaks, the total organic carbon (TOC) in a solvent used for LC-MS should be lower
than 1 ppb if using an in-house ultrapure water generator. In addition, although it is a
very basic precaution, moving phase solvents should be obtained from reliable
reagent suppliers, and once reagent bottles are opened, it is better to consume
them as soon as possible.
Moreover, in the case of performing a quantitative analysis, it is necessary to
analyze specimens with respect to reference standards every time and determine the
concentration of each compound of interest based on its calibration curve.
LC-MS can analyze a wide variety of metabolites, including refractory or ther-
molabile substances, if they are soluble in the solvent. This is an advantage in
LC-MS, but not in GC-MS. An additional concern is that even when using a
LC-MS device at the same settings as in a previous study, the retention time of
metabolites is often not consistent, for various reasons, e.g., differences in the
plumbing head of the devices, in the state of the separation column, and in the purity
of solvent. Therefore, even if a given peak has the same mass unit and the same
retention time as a known compound as indicated in a previous study or databases,
these compounds cannot be said to be identical purely on that basis. It is necessary to
confirm the spectrum of the reference standards with the same device used for that
analysis and to acquire spectral data in house.
GC-MS is characterized by a high compatibility between devices compared with
LC-MS or CE-MS. Since commercial and open-access databases of GC-MS libraries
(i.e., compound databases) are well developed, it is advantageous to identify metab-
olites by qualitative analyses. However, in profiling metabolites exhaustively using
body fluid samples such as blood or urine, a derivatization step is necessary to
vaporize metabolites, which creates difficulties because of differences in volatility of
74 T. Kobayashi
the constituent compounds. Because the derivatization efficiency of all compounds

is not constant, quantitative discussions should be careful when interpreting results.
More recently, newer analyses using CE-MS and SFC-MS have become possible
with advances in analytic devices; some researchers have reported studies that
employ these cutting-edge systems (Toyohara et al. 2010; Taguchi et al. 2014).
CE-MS shows promise in effectively analyzing ionic metabolites that are hardly
retained on the reverse-phase columns widely used in LC-MS, whereas using it to
analyze lipophilic and neutral metabolites is difficult in principle. On the other hand,
SFC-MS is feasible for and capable of analyzing refractory and heat-labile com-
pounds that GC-MS can hardly detect and also boasts higher throughput than
LC-MS. These new devices will be powerful tools when widely used in the future.
However, even among the analytical methods described above, it is impossible to
find all metabolites comprehensively in a sample using a single analytical instru-
ment. It is thus necessary to select the device according to the purpose of a study or to
use multiple devices. Prerequisites for metabolite identification using MS have been
discussed in some review papers (Zhao 2013; Sumner et al. 2007; Yoshida
et al. 2012).
In general, target compounds explored by LC-MS-based metabolomics are low-
molecular-weight compounds of less than 1000 Da. Typical target biological sam-
ples – serum, plasma, and urine – require pretreatment steps to remove inhibitory
components such as proteins and minerals.
Preprocessing methods are mainly divided into methods to denature proteins
using solvents (such as methanol, chloroform, and acetonitrile) and methods to
separate the sample physicochemically by solid-phase extraction. Neither
trifluoroacetic acid nor DMSO is unequivocally superior for use as a solvent in
LC-MS pretreatment, because the former inhibits the ionization process and the
latter causes background noise peaks. Additionally, ultrafiltration is also available,
but it seems to not be used widely owing to its high cost compared with solvent
extraction methods.
Solvent extraction methods are widely used because of their low cost and
simplicity, but they still have disadvantages: centrifugation and collection of super-
natant are necessary, and working time increases in proportion to the number of
samples. In addition, the pretreatment procedure for biological fluids such as plasma
needs a large amount of organic solvent, and thereby the extracted volume will be
increased above the starting volume, diluting the concentration of target metabolites.
Solid phase extraction methods are profitable because they can analyze a large
number of samples and are able to concentrate the target metabolites by controlling
elution volume (Mallet et al. 2004); however, they demand higher costs than solvent
extraction methods do and suffer from the possibilities that some kinds of metabo-
lites can be absorbed or, conversely, hardly retained by the column and that condi-
tions in the housing unit of the column can affect following analyses.
In other words, each kind of method has advantages and disadvantages. In
addition, for both methods, the recovery efficiency changes depending on the solvent
and column selected. The important thing is that users must refer to a pretreatment
method used by a previous study and examine its applicability in advance by using
their own MS. Sharp signal peaks, large signal/noise ratio (e.g., S/N >10), and low
background peaks are good criteria for this prior examination.
Metabolites known to reflect disease severity by alterations in their blood levels,
such as indoxyl sulfate in the case of CKD (Barreto et al. 2009; Kikuchi et al. 2010),
can be used as a positive control when analyzed quantitatively. Thus, when consid-
ering the pretreatment method, one good strategy would be to carry out the proce-
dure with the addition of such an authentic sample and to select an extraction method
in which the recovery rate is close to 100 %.
After determining the pretreatment method, it is necessary to confirm the dynamic
range of the device using an authentic sample of the compounds targeted for
detection before performing quantitative analysis. Usually, it is necessary to select
two or more kinds of targeted compounds. Even for metabolites whose blood or
urine levels change according to CKD severity, their concentration is not in all cases
regulated directly by CKD. For example, kynurenine is known as a metabolite
whose concentration in the blood increases with CKD exacerbation (Saito
et al. 2000; Kobayashi et al. 2014b), but its increase is also known to reflect staging
of colon cancer (Nishiumi et al. 2012). In addition, plasma kynurenine has been
reported to be elevated in major depression patients (Dahl et al. 2015). Thus,
estimating CKD stages using only one metabolite is prone to low reliability. Since
LC-MS is able to detect multiple compounds at the same time in a single injection,
the time cost is almost the same for one target metabolite as for two or more. In fact,
in many previous studies, four or more metabolites with different masses and
retention times were selected (Toyohara et al. 2010; Sato et al. 2011; Kobayashi
et al. 2014b).
Because a biological sample such as the plasma, serum, and urine contains
various compounds, some sorts of contaminants will remain even after pretreatment.
These contaminants sometimes inhibit ionization of target metabolites (the so-called
matrix effect). Because each metabolite has a different ionization efficiency, internal
standard samples of targeted metabolites are needed when doing quantitative anal-
ysis. Stable isotope-labeled compounds have equal ionization efficiency to their
non-labeled equivalents, making them the most reasonable internal standards for
quantitative analysis. They can be employed effectively by adding a known concen-
tration of labeled compounds to samples prior to pretreatment procedures. It is
preferable to analyze corresponding labeled compounds for every targeted metabo-
lite; however, when obtaining some compounds is difficult, the labeled compound
should be prepared at as many concentrations as possible, with the remainder
quantified using a calibration curve method for the non-labeled standard while
paying attention to quantitative accuracy.
For this calibration curve method, first, the compounds are dissolved in solvent
and mixed in one vial. Then, sequential dilutions are prepared from this vial. In
doing so, researchers should be careful to reconcile the solvent component of the
diluent with that of the pretreated specimens. When analyzing the dilution series,
obtained peaks should be manually checked for good separation, sharp shape, and
minimal tailing. Next, the peak area is plotted for the prepared diluents at measured
concentrations, followed by construction of a calibration curve. The range of the
76 T. Kobayashi
calibration curve should be linear; the range over which the linearity of the calibra-
tion curve is preserved indicates the concentrations at which metabolites can be
analyzed quantitatively.
Nontarget Analysis and Target Analysis
Biomarker analysis using MS is useful for disease exploration and medical

checkups. To conduct it, two ways are possible: one is revealing previously
unknown metabolites that can serve as disease markers (nontarget analysis), and
the other is quantitative analysis of predefined metabolites and estimating disease
severity based on them (target analysis or multi-target analysis).
When using nontarget analysis methods, unknown metabolites are detected in the
biological samples in addition to well-known metabolites. Accurate mass measure-
ments are necessary when detecting unreported, new metabolites and many kinds of
known metabolites alike: thus, high-resolution time of flight-MS (TOF-MS) or
fourier transform-ion cyclotron resonance-MS (FT-ICR-MS) is essential for nontar-
get analysis.
Since the main purpose of nontarget analysis is the screening and detection of
unknown metabolites, the most important factors are mass accuracy and spectral
resolution. Therefore, the concentration data of metabolites are typically shown in
terms of relative quantitation. On the other hand, when using the multi-target
analysis approach, the target compounds are limited to several metabolites. Thus,
multi-target analysis is usually performed using quantitative analysis with a stable
isotope-labeled compound or authentic sample as the internal standard.
Nontarget Analysis
Since a meaningful spectrum must be selected for nontarget analysis – that is, a
spectrum in which background and noise peaks are excluded and large quantities of
mass spectrum data are accurately profiled – a software that can help extract significant
peaks and construct matrix data is necessary. Unlike GC-MS, a universal mass spectral
library is insufficient for LC-MS, because it often causes undetectable peaks to appear
when the matrix data were derived from values obtained by LC-MS/MS analysis. In
order to solve this problem, many mass spectra databases have been established,
including MassBank, HMDB, KNApSAcK, and METLIN, which are able to predict
(or identify) constituent metabolites based on LC-MS/MS spectra. An outline of the
nontarget analysis procedure is shown in Fig. 1. In the metabolite identification step,
the following requirements must be met: the sample and reference standards must be
analyzed under the same conditions, and the retention time and mass spectrum used
for each analyte must be ensured to be identical (Sumner et al. 2007).
To date, many studies have reported alterations in the concentrations of various
blood metabolites, along with a decline in renal function, in outpatients with CKD
(Niwa 2011; Toyohara et al. 2010), hemodialysis patients (Niwa 2011; Saito
Fig. 1 Outline of nontarget analysis: Searching for early detection markers of CKD. 1 LC-MS/MS
analysis of plasma samples, peak selection, and matrix generation (by software to analyze device
data, additional purchase may be required). 2 Multivariate analysis (by commercial software) and
selection of candidate peaks for markers (part of the red circle). 3 Evaluation of chronological
change of the candidate peaks. 4 Identification of metabolite(s) using analyzed data: mass number,
retention time, and MS/MS spectrum
et al. 2000; Sato et al. 2011), rats with adenine-induced CKD (Zhao et al. 2012;
Kobayashi et al. 2014a), and partially nephrectomized rats (Saito et al. 2000;
Kikuchi et al. 2010), suggesting that the concentrations of several of these metab-
olites can be used to construct a quantitative index of renal function. Metabolomics
approaches are most suitable for targeting low-molecular-weight compounds
(approx. <1000 Da) whose molecular structures and characters are the same
between animal species. This is one advantage of exploring metabolites versus
genes or proteins. Thus, common MS programs can be used even when animal
species are different. However, the human metabolic system is nevertheless different
from those of other experimental animals. As a simple example, hyperuricemia,
78 T. Kobayashi
which is speculated to be related with CKD, shows the following features. Since
Homo sapiens and some other primates do not have uricase (Yeldandi et al. 1991),
the end product of adenosine and guanosine metabolic systems is a urate. Low urate
excretion from the kidney or excess intake of purine bases from diets causes
hyperuricemia in Homo sapiens and some other primates. In contrast, some other
mammals, typified by rodents, have active urate oxidase that metabolizes uric acid to
allantoin (Ackroyd 1914). Thus, these animals are able to excrete excess uric acid,
and blood uric acid levels are not easily affected by experimental CKD induction.
This observation shows the importance of understanding metabolic pathways when
applying the results of animal experiments to human samples. In this respect, it is
best to use open databases such as the KEGG pathway website where many useful
pathway maps for Homo sapiens and other animal species are published.
In order to explore disease biomarkers by the use of nontarget analysis, namely,
through metabolomics, understanding of metabolic systems in vivo and knowledge
of mass spectrometry and bioinformatics are also necessary. In addition, the mass
spectrometer employed should have high enough resolving power to measure mass
accurately. Thus, it might be difficult for novices to conduct metabolomics studies.
In part, however, how to perform data mining and extract useful peaks from the sea
of spectral data obtained by MS to identify new metabolites and how to predict the
onset of a disease by observing the layout of mass spectra are processes that depend
on the ideas of individual researchers. The field of metabolomics is awaiting
breakthroughs from new researchers; this field could take a quantum leap forward
by their contributions.
Target Analysis (Multi-target Analysis)
In target analysis, metabolites to be monitored are selected prior to the analyses. If

the target metabolite has been previously identified, its peak can be detected during
the analysis with mass spectrometer based on its retention time and mass number.
The peaks, which were considered meaningful for the study, can be selected as an
examination target, even if their corresponding compound is unidentified at first.
However, for practical purposes, determining which peaks is derived from what kind
of metabolite is an essential objective.
Uremic toxins, which are metabolites that are excreted under normal circum-
stances, accumulate in vivo because of renal function loss and have been inferred to
harm many tissues (Vanholder et al. 2003; Richard and Shaul 2012). Therefore,
uremic toxins of low molecular weight can be adopted as candidate biomarkers that
reflect renal function loss in metabolomics studies. However, the extent by which a
uremic toxin increases in the blood according to CKD stage differs among toxins.
Candidate biomarkers should exhibit more remarkably altered blood levels in the
early stages of CKD than those of existing biomarkers (i.e., creatinine and CysC) and
have high sensitivity and reproducibility. Otherwise, they have limited utility
(although knowledge about such compounds is important). This is because the
existing biochemical indices such as creatinine and CysC have already spread
widely, and biomarkers that do not fulfill the conditions above cannot compete with
them to be employed as standards in analysis techniques using the MS because of
issues of measurement cost, convenience, and specificity.
In addition, metabolites that exhibit decreased blood levels according to the
extent of renal function loss (Toyohara et al. 2010) could be used as CKD bio-
markers in the same way as uremic toxins.
Though several specified metabolites are quantified in a target analysis as men-
tioned above, in most cases, the concentrations of a plurality of metabolites that can
reflect the severity of CKD do not change according to the same ratio. Thus, in many
cases, a quantified concentration of given metabolites is not informative or useful for
predicting severity of CKD directly. By using multivariate analyses, a speculative
regression equation can be constructed based on the prospective contribution ratios
of the metabolites identified in a multi-target analysis. In this process, logistic
regression or partial least squares (PLS) regression are often used, and the derived
equation should correlate with CKD stage as tightly as possible (Kobayashi
et al. 2014b). Next, unused sample sets should be used as the validation set to
validate the accuracy of the derived equation. The validation set should be input into
the equation, and the CKD stage estimated. By this process, whether the equation is
able to estimate CKD stage can be validated before it is used.
Future of Metabolomics in the Diagnosis of Disease
There are several reports on new biomarkers for estimating CKD severity that have
been found through metabolomics studies (Zhao 2013; Rhee et al. 2013; Shah
et al. 2013; Duranton et al. 2014; Kobayashi et al. 2014b). The predictive formula
and combination of metabolites used differ variously between each report. In other
words, these are method development studies, and their methods have not yet been
validated sufficiently for use in clinical settings. Progress in metabolomics tech-
niques and parameters would enable the establishment of reliable biomarkers to
predict CKD stage, as well as the discovery of CKD in earlier stages.
This chapter introduced metabolomics in the context of evaluating CKD stage.
There are other studies that have established diagnostic methods for other diseases
using metabolomics as well (Nishiumi et al. 2010; Soga et al. 2011; Ikeda et al. 2012;
Nishiumi et al. 2012; Maekawa et al. 2013; Yokokura et al. 2014). Since diagnostic
strategies for various diseases using metabolomics are currently in development,
metabolomics techniques are not fully refined. Recently, new researchers who start
metabolomics studies as well as research reports have been increasing every year. In
addition, the performance of mass spectrometry, which plays an important role in
metabolite identification, is improving day by day. Accordingly, metabolomics is a
field that is expected to be developed more and more into the future. Through its
development, after biomarkers are established by which practitioners are able to
detect many diseases in their early stages, including CKD, collecting one drop of
blood and analyzing it using mass spectrometry would enable the diagnosis of many
critical illness all at once.
80 T. Kobayashi
Potential Applications to Prognosis and Other Diseases

and Conditions
Though this chapter described applications of metabolomics to CKD diagnosis,

diseases that can potentially be targeted are not limited to CKD. Metabolomics is
used to study various other diseases as well, including gastrointestinal cancer (Ikeda
et al. 2012), colorectal cancer (Nishiumi et al. 2012), pancreatic cancer (Nishiumi
et al. 2010), inflammation (Yokokura et al. 2014), dilated cardiomyopathy
(Maekawa et al. 2013), and different forms of liver disease (Soga et al. 2011).
Summary Points
• CKD is the irreversible loss of kidney function, often progressing to ESRD and
resulting in impaired quality of life.
• Although estimated GFR, a commonly used marker for CKD is convenient and
useful for the initiation of dialysis, the indicator cannot detect CKD in its early
stages because of its low sensitivity.
• Metabolites indicative of CKD have been identified using metabolomics.
• CKD stages can be estimated by metabolite concentrations after carrying out
statistical processing.
• Metabolomics shows promise as a novel method for identifying patients with
early stage CKD.
• In addition, metabolomics can be applied not only to CKD but to various other
diseases.
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Hypoxia as a Biomarker of Kidney Disease
6
Roger G. Evans, Julian A. Smith, Bruce S. Gardiner, David W. Smith,
Amanda G. Thrift, Clive N. May, Yugeesh R. Lankadeeva,
and Andrew D. Cochrane
Contents
Key Facts of Kidney Oxygenation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
Chronic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
R.G. Evans (*)

Department of Physiology, Monash University, Melbourne, VIC, Australia
e-mail: roger.evans@monash.edu
J.A. Smith • A.D. Cochrane
Department of Surgery, Monash University, Melbourne, VIC, Australia
e-mail: julian.smith@monash.edu; andrew.cochrane@monashhealth.org
B.S. Gardiner (*)
School of Engineering and Information Technology, Murdoch University, Perth, WA, Australia
e-mail: B.Gardiner@murdoch.edu.au
D.W. Smith
School of Computer Science and Software Engineering, The University of Western Australia, Perth,
WA, Australia
e-mail: david.smith@uwa.edu.au
A.G. Thrift
Department of Medicine, Monash University (Monash Medical Centre), Melbourne, VIC, Australia
e-mail: amanda.thrift@monash.edu
C.N. May
Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC,
Australia
e-mail: clive.may@florey.edu.au
Y.R. Lankadeeva
Department of Physiology, Monash University, Melbourne, VIC, Australia
Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville, VIC,
Australia
e-mail: yugeesh.lankadeva@florey.edu.au

84 R.G. Evans et al.
The Role of Hypoxia in the Pathogenesis of Chronic Kidney Disease . . . . . . . . . . . . . . . . . . . . 89

The Clinical Problem: Early Diagnosis and Instigation of Therapy . . . . . . . . . . . . . . . . . . . . . . . . 89
Assessment of Kidney Oxygenation by BOLD MRI in Chronic Kidney Disease . . . . . . . . . 89
Acute Kidney Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
The Role of Hypoxia in the Pathogenesis of Acute Kidney Injury . . . . . . . . . . . . . . . . . . . . . . . . 91
The Clinical Problem: Prevention of Acute Kidney Injury in the Hospital Setting . . . . . . . . 92
Urinary PO2 as a Diagnostic Biomarker in Acute Kidney Injury . . . . . . . . . . . . . . . . . . . . . . . . . . 95
Potential Applications to Prognosis, Other Disease and Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . 100
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Abstract
All established (e.g., serum creatinine, albuminuria) and emerging (e.g., neutro-
phil gelatinase-associated lipocalin, cystatin C) biomarkers of kidney disease
suffer from the disadvantage that they are markers of damage to the kidney or
loss of renal function. Tissue hypoxia is believed to be an initiating factor, in both
chronic kidney disease (CKD) and acute kidney injury (AKI), so may provide a
physiological biomarker for early diagnosis of both conditions. Currently blood
oxygen dependent magnetic resonance imaging (BOLD MRI) appears to have little
diagnostic value in human CKD. On the other hand, the measurement of urinary
oxygen tension (PO2) has potential as a biomarker of risk of AKI in a hospital
setting because: (i) Hypoxia in the renal medulla plays a central role in AKI of
multiple causes; (ii) The vasa recta are closely associated with collecting ducts in
the medulla so that pelvic urinary PO2 would be expected to equilibrate with
medullary tissue PO2; (iii) The PO2 of urine in both the renal pelvis and the bladder
varies in response to stimuli that would be expected to alter medullary tissue PO2;
and (iv) New fibre-optic methods make it feasible to measure bladder urine PO2 in
patients with a bladder catheter. But translation of this approach to hospital practice
requires: (i) A quantitative understanding of the impact of oxygen transport across
the epithelium of the ureter and bladder on urinary PO2 measured from the bladder,
(ii) confirmation that changes in urinary PO2 parallel those in medullary PO2 in
physiology and pathology, and (iii) Studies of the prognostic utility of urinary PO2
in hospital settings associated with risk of AKI, such as in patients undergoing
cardiac surgery with cardiopulmonary bypass, those at risk of sepsis, and those
undergoing imaging procedures requiring administration of radiocontrast agents.
Keywords
Acute kidney injury • BOLD MRI • Cardiopulmonary bypass • Chronic kidney
disease • Intensive care • Sepsis • Urinary oxygen tension
Abbreviations
BOLD Blood oxygen dependent
CPB Cardiopulmonary bypass
6 Hypoxia as a Biomarker of Kidney Disease 85

MRI Magnetic resonance imaging
RBF Renal blood flow
Key Facts of Kidney Oxygenation
• Although renal blood flow represents a large proportion (25 %) of cardiac

output, the kidneys are susceptible to hypoxia.
• Oxygenation of kidney tissue is determined by the balance between oxygen
delivery and oxygen consumption.
• Oxygen is delivered to the kidney through renal blood flow. Blood flow to the
renal medulla is in part regulated independently of blood flow to the bulk of the
renal cortex. Blood flow per unit tissue volume is much less in the renal medulla
than in the renal cortex.
• Oxygen delivery to kidney tissue is limited by diffusive oxygen shunting in the
renal cortex and medulla. In the medulla, oxygen delivery to the thick ascending
limbs of the loop of Henle is also limited by their anatomical position, at the
periphery of the vascular bundles
• Approximately 80 % of renal oxygen consumption is used to power tubular
reabsorption of sodium. The proximal tubules and thick ascending limbs of the
loop of Henle are the major sites of renal sodium reabsorption.
Definitions
Acute kidney injury (AKI) Some loss of function of the kidney, usually assessed
initially as a reduction in glomerular filtration rate. It can be relatively mild, in which case
complete recovery can occur in as little as a few days, or be so severe that renal
replacement therapy (dialysis or a kidney transplant) is required (end-stage renal disease).
Cardiopulmonary bypass (CPB) To perform open heart surgery, in most cases the
heart must be stopped and the patient’s circulation supported by a ‘bypass circula-
tion’ incorporating a pump to do the job of the heart and an oxygenator to do the job
of the lungs.
Chronic kidney disease (CKD) A condition where the function of the kidney
gradually declines over many years. In some cases, this leads to end stage renal
disease. Chronic kidney disease is usually first diagnosed when glomerular filtration
rate is found to be less than the normal range.
Clearance This is a technique used for measurement of certain kidney functions. It

relies on the ability to measure the concentrations of a marker in the plasma and urine
and measurement of the amount of urine produced over a given time period.
Cortex The outer part of an organ (e.g., kidney cortex).
End-stage renal disease A condition in which the kidneys are no longer able to
function sufficiently to maintain the patients overall health. In such cases renal replace-
ment therapy is required, in the form of dialysis therapy or kidney transplantation.
Fibrosis Many disease conditions, including chronic kidney disease, are character-
ized by the build-up of connective tissue fibres. In the kidney, fibrosis reduces the
ability of oxygen to get to cells.
Fluorescence lifetime oximetry A technique for measuring the partial pressure of

oxygen which relies on the ability of oxygen to quench the fluorescence emitted by
certain molecules.
Glomerular filtration rate The amount of plasma filtered by the kidney each
minute.
Haemoglobin The oxygen carrying molecules that are packed inside red blood
cells. When haemoglobin is not carrying oxygen it is called deoxyhaemoglobin.
Hypoxia Low levels of oxygen.
Ischaemia Inadequate blood supply to an organ or part of the body.
Magnetic resonance imaging (MRI) A medical imaging technique that can pro-
duce three dimensional images of anatomy. It can also be used to map functional
indices such as the concentration of deoxyhaemoglobin (blood oxygen level depen-
dent – BOLD MRI).
Medulla The inner part of an organ (e.g., kidney medulla).
PO2 The partial pressure of oxygen. This is how we measure the concentration of
dissolved oxygen in biological fluids and tissue, in the units of millimetres of
mercury (mmHg).
Radiocontrast An agent which is administered to patients undergoing some x-ray

procedures to assist visualisation of body structures.
Renal replacement therapy Patients who suffer from end-stage renal disease
require renal replacement therapy, either in the form of renal dialysis therapy or a
kidney transplant.
Risk factor A risk factor is anything that is found to be associated with the
development of disease. Some of these are non-modifiable (e.g., age, gender)
while others are potentially modifiable (e.g., smoking, alcohol use etc.).
Sepsis An inflammatory condition that results from severe bacterial infection.
Ureter The tube in which urine travels from the kidney to the urinary bladder.
Vasa recta A series of straight blood vessels in the kidney medulla.
Introduction
Kidney disease is broadly classified into two major types; acute kidney injury (AKI)
and chronic kidney disease (CKD). CKD is characterized by a gradual decline in
glomerular filtration rate (GFR) over many years. AKI, on the other hand, is
characterized by a rapid decline in GFR over a 24–48 h period. These two forms
of kidney disease share some similarities. Both are traditionally diagnosed by
measurement of serum concentrations of creatinine, or by the renal clearance of
creatinine, to give estimates of GFR. As we will outline in detail herein, renal
hypoxia is also a common feature of both AKI and CKD (Mimura and Nangaku
2010; Heyman et al 2012; Evans et al. 2013). However, the challenges for early
diagnosis and accurate prognosis for these two conditions contrast strikingly.
Early diagnosis of CKD may provide an opportunity for early intervention with
therapies to slow disease progression. However, this is made more difficult by the
fact that measurable deficits in GFR occur well into disease progression (Fig. 1a).
This is because, as nephrons are lost during the disease process, filtration in the
remaining nephrons increases (Fong et al. 2014). Thus, as CKD progresses GFR can
remain stable because reserve filtration capacity is consumed as single nephron GFR
increases. It is only when these compensatory processes are exhausted that clinically
significant deficits in GFR can be detected. Thus, early diagnosis of CKD requires
minimally invasive methods to assess renal function at the single nephron level,
and/or methods to detect the upstream events that damage nephrons. In this context,
it is worth considering the evidence (vide infra) that renal tissue hypoxia precedes
and drives progression of CKD. Thus, minimally invasive methods for assessing
kidney oxygenation may provide a means for early diagnosis of CKD.
In AKI, the challenge is to reach a diagnosis before the onset of irreversible damage
(Fig. 1b). The ideal would be to identify patients who are at risk of developing AKI and
intervene to manage their kidney health before injury occurs. The traditional biomarker
serum creatinine, which is used to estimate GFR, can show that a patient has developed
AKI, but not that they are currently at risk of developing AKI. Other plasma and
urinary biomarkers, including neutrophil gelatinase-associated lipocalin (NGAL) and
cystatin C, have shown promise in the early detection of AKI (Haase et al. 2010), but
they are markers of renal damage, so diagnosis still lags hours behind the initiating
insult. As with CKD, assessment of kidney oxygenation may provide a method for
early diagnosis of AKI. Importantly, in the hospital setting, including during surgery, a
measure of kidney oxygenation could provide an approach to identify patients at risk of
developing AKI, so that steps can be taken to reduce that risk.
a Glomerular injury
CKD Hypoxia
Renal Dysfunction
100
Glomerular Loss of Loss of
Filtration Nephrons Function
Rate
(% baseline)
0
Early Progression of Disease End Stage
b
Initiating Factors
AKI
Hypoxia
Renal Dysfunction
Glomerular
Filtration 100
Rate At Risk
(% baseline)
Repair
Functional Extension
Maint-
enance
Initiation
0
Early Time after Insult Late
Fig. 1 Proposed timelines in the initiation and progression of chronic kidney disease (CKD) and
acute kidney injury (AKI). (a) According to the chronic hypoxia hypothesis, proposed initially by
Fine and colleagues (1998) and Fine and Norman (2008), in CKD primary glomerular injury leads
to insufficiency of post glomerular capillary blood flow, and thus tubulointersitial hypoxia.
Tubulointerstitial hypoxia in turn drives fibrosis, capillary rarefaction and tubular atrophy, and
thus progressive glomerular injury. However, because surviving nephrons hyperfilter, a deficit in
glomerular filtration is not observed until the disease process is well advanced. (b) Initiating factors
such as cardiopulmonary bypass, sepsis or nephrotoxins lead to renal medullary ischaemia and/or
hypoxia, which in turn may contribute to loss of renal functional capacity. This concept is related to
the established clinical phases of AKI (Adapted from Okusa et al. (2013)). In both CKD and AKI it
has been proposed that hypoxia occurs before, and contributes to, loss of renal function. Thus,
hypoxia may be a useful biomarker for kidney disease
This chapter presents the argument that assessment of kidney oxygenation, and
thus detection of renal hypoxia, may provide a means for early diagnosis of AKI and
CKD. Early diagnosis would allow more reliable prognosis and improved manage-
ment of renal health. The optimal diagnostic approach is likely to differ between
CKD and AKI. For CKD, blood oxygen dependent (BOLD) magnetic resonance
imaging (MRI) has been proposed as a useful tool for early diagnosis. This approach
is non-invasive so can be applied periodically in out-patients. Unfortunately, despite
promising observations in animal models of CKD, available evidence indicates that
this approach has little clinical utility. For AKI, a method for continuous assessment
of kidney oxygenation, in the operating theatre or bedside, is required for rapid
identification of patients at risk of developing AKI. Continuous measurement of
urinary PO2 may provide this information. But the reader must first be convinced
that renal hypoxia is a common feature of both CKD and AKI, and that it precedes,
and perhaps even drives, the disease process.
Chronic Kidney Disease
The Role of Hypoxia in the Pathogenesis of Chronic Kidney Disease
Renal tissue hypoxia has been observed in all experimental forms of CKD examined to
date, including those initiated by renovascular disease (Evans and O’Connor 2014),
diabetic nephropathy (Hansell et al. 2013), five sixths nephrectomy (the remnant kidney
model) (Manotham et al. 2004), polycystic kidney disease (Ow et al. 2014), and CKD
after long-term recovery from ischaemia-reperfusion injury (Basile et al. 2003).
Fine and colleagues first developed the chronic hypoxia hypothesis regarding the
initiation and progression of CKD more than 15 years ago (Fine et al. 1998; Fine and
Norman 2008). In brief, they propose that primary injury to glomeruli results in
increased metabolic work in remaining nephrons to reabsorb the filtered load of
sodium. The resultant hypoxia initiates inflammatory and fibrotic pathways, which in
turn drive rarefaction of the microcirculation, so exacerbating hypoxia (Fig. 1a).
Evidence in support of this hypothesis includes the observation of tubulointerstitial
hypoxia preceding evidence of nephropathy in the remnant kidney model (Manotham
et al. 2004) and in diabetes (dos Santos et al. 2007; Rosenberger et al. 2008). Further-
more, CKD can be induced by treatments that promote renal hypoxia, even in the
absence of confounding effects such as oxidative stress (Friederich-Persson et al. 2013).
The Clinical Problem: Early Diagnosis and Instigation of Therapy
CKD cannot yet be cured. Current therapies are palliative, so aimed at slowing the
progression of disease rather than reversing it (Lambers Heerspink and de Zeeuw
2013). The major flaw of the current diagnostic approach is that deficits in GFR are
only observed once disease processes are well advanced (Fig. 1a). Diagnostic methods
capable of detecting the disease process in its early stages would represent a significant
advance, as this would support early intervention to prevent progression to end-stage
renal disease. Could diagnosis of kidney hypoxia provide such an advance?
Assessment of Kidney Oxygenation by BOLD MRI in Chronic Kidney

Disease
There has recently been an explosion of interest in the use of BOLD MRI for
assessment of kidney oxygenation in patients with CKD. BOLD MRI provides a
measure of deoxygenated haemoglobin, so is an indirect measure of tissue oxygen-

ation (Evans et al. 2008b). Nevertheless, changes in the BOLD signal have been
shown to follow changes in more direct measures of renal tissue oxygenation in
response to multiple physiological manoeuvres in multiple species of healthy ani-
mals (Pedersen et al. 2005; Pohlmann et al. 2013; Zhang et al. 2014). Furthermore,
renal hypoxia has been detected using BOLD MRI in the early stages of diabetic
nephropathy (dos Santos et al. 2007; Prasad et al. 2010) and in severe renovascular
disease (Gloviczki et al. 2011) in humans. Yet two relatively recent and large clinical
studies failed to detect differences in the BOLD signal, in either the cortex or
medulla, between patients with CKD and controls at rest (Michaely et al. 2012;
Pruijm et al. 2014). In both studies, which included a total of 400 (Michaely
et al. 2012) and 195 (Pruijm et al. 2014) patients respectively, no relationship
could be detected between BOLD signals and GFR estimated from creatinine
clearance or serum creatinine concentration at rest (Michaely et al. 2012). Inoue
and colleagues were also unable to demonstrate a relationship between the BOLD
signal and GFR in 43 patients with diabetic nephropathy, although they could detect
a relationship in 76 subjects with CKD chiefly due to glomerulonephritis or hyper-
tensive nephrosclerosis (Inoue et al. 2011). Furthermore, in a relatively small study,
hypoxia could not be detected using BOLD MRI in patients with moderate renal
artery stenosis (Gloviczki et al. 2010).
How can the disparity in the experimental and clinical findings described above
be explained? One possibility is that established CKD in humans, unlike in exper-
imental animals, is not associated with renal tissue hypoxia. This possibility cannot
be completely discounted, since reductions in GFR would be expected to lead to
reduced renal oxygen consumption, as the major source of oxygen demand in the
kidney is for generation of ATP for tubular Na/K-ATPase activity (Evans
et al. 2014a). For example, reduced renal oxygen demand likely explains why the
BOLD MRI signal reflects increased renal oxygenation in chronic allograft rejection
(Djamali et al. 2007). Part of the explanation might also lie with the heterogeneous
nature of CKD, and the way in which renal oxygen supply and demand change over
the course of development of the various forms of CKD (Neugarten 2012). However,
it seems likely that much of the problem lies with the indirect nature of the BOLD
signal in relation to tissue oxygenation. The BOLD signal is heavily weighted
towards venous blood, because it chiefly provides a measure of deoxyhaemoglobin
concentration. Furthermore, renal tissue oxygenation does not necessarily reflect
renal blood oxygenation, in part because of the phenomenon of arterial-to-venous
oxygen shunting (O’Connor et al. 2006a; Evans et al. 2008a). Indeed, renal venous
PO2 and renal tissue PO2 can change independently in the kidney, providing direct
evidence that changes in the BOLD MRI signal do not necessarily reflect changes in
tissue oxygenation (O’Connor et al. 2006a; Evans et al. 2008a). Regardless, avail-
able evidence indicates that measurement of baseline BOLD signals provide little
diagnostic or prognostic information in patients with CKD.
A more promising approach may be to use BOLD MRI in conjunction with a
physiological challenge to kidney function. Recently it was demonstrated, in a
cohort of 195 subjects, that the response of the medullary BOLD signal to furose-
mide is markedly blunted in CKD (Pruijm et al. 2014). Furosemide inhibits sodium
reabsorption, and thus oxygen consumption, in the loop of Henle. Thus, in normal
subjects, renal oxygenation (as assessed by BOLD MRI and more direct methods)
increases markedly after furosemide administration. The fact that this response is
blunted in patients with CKD provides a diagnostic opportunity. The critical
unresolved question is whether this change in the response to furosemide occurs
before, or only after CKD can be diagnosed by conventional methods. This question
could only be answered through prospective studies.
Acute Kidney Injury
The Role of Hypoxia in the Pathogenesis of Acute Kidney Injury
The kidneys receive a quarter of the cardiac output, so are very highly perfused in
relation to their demand for oxygen (Evans et al. 2008a). It is rather paradoxical,
then, that they are susceptible to hypoxic damage in both CKD (Fine and Norman
2008) and AKI (Heyman et al. 2012). A number of factors conspire to render the
medulla particularly susceptible to hypoxia. Firstly, blood flow per unit volume of
tissue is considerably less in the medulla than the cortex (Evans et al. 2013).
Secondly, oxygen delivery to medullary tissue is limited by counter-current shunting
of oxygen, between arteries and veins in the cortex (Evans et al. 2013) and between
descending and ascending vasa recta in the medulla (Zhang and Edwards 2002).
Thirdly, in the outer medulla the thick ascending limbs of Henle’s loop, which
require oxygen to reabsorb sodium, are located at the periphery of the vascular
bundles (Chen et al. 2009a, b). This topographic arrangement limits oxygen delivery
from vasa recta to the thick ascending limbs (Chen et al. 2009a, b). These factors
likely explain why the outer medullary thick ascending limbs are often found to be
damaged in human AKI (Heyman et al. 2010).
Tissue hypoxia has been proposed to be a common pathway in the pathogenesis
and progression of AKI (Aksu et al. 2011; Heyman et al. 2012). It is consistently
observed in AKI of multiple aetiologies (Heyman et al. 2012; Evans et al. 2013).
Furthermore, hypoxia can initiate signalling pathways that drive renal tissue dys-
function and damage (Heyman et al. 2012; Evans et al. 2013). These pathways
include those driven by transforming growth factor β and Smads (inflammation and
fibrosis), depletion of cellular ATP (apoptosis and necrosis) and oxidative stress.
Tissue hypoxia is then further exacerbated as a consequence of initiation of these
pathways. Hypoxia inducible factors can provide some protection against mild
and/or brief hypoxia, but not when hypoxia is profound and/or prolonged (Heyman
et al. 2012). Hypoxia in the renal medulla may initiate a vicious cycle of damage and
dysfunction, which drives further hypoxia. Early disruption of these processes could
be beneficial by preventing renal medullary hypoxia. In order to prevent medullary
hypoxia, a means to evaluate it is required.
The Clinical Problem: Prevention of Acute Kidney Injury

in the Hospital Setting
Approximately 5 % of hospitalized patients develop AKI, so it is a major burden on

health systems (Mehta and Chertow 2003). AKI is chiefly an iatrogenic illness. It is
often associated with situations in which the patient and/or their kidneys are exposed
to hypoxia (Evans et al. 2013).
Cardiac Surgery
AKI is a prevalent complication of major surgery. After cardiac surgery, up to one
third of patients experience some degree of AKI (Karkouti et al. 2009). Even mild
AKI following surgery performed on cardiopulmonary bypass (CPB) is
prognostically important, being associated with more than a fourfold increase in
the risk of in-hospital death as well as extended hospitalisation (Karkouti
et al. 2009). Mortality rate exceeds 60 % when AKI is so severe that renal replace-
ment therapy is required (in 1–2 % of patients after CPB) (Lenihan et al. 2013). The
scale of the problem is receiving growing recognition, with the most recent evidence
showing that the problem is becoming worse rather than better (Lenihan et al. 2013).
There are currently no effective pharmacological approaches for preventing AKI
after CPB (Patel et al. 2011b). Thus, the best approach to prevention is avoidance of
known modifiable risk factors in at-risk patients. Consequently, AKI risk scoring
models have been developed (Huen and Parikh 2012). These tools are used in practice
to help identify at-risk patients, but they only have limited predictive efficacy (Huen
and Parikh 2012). They also provide little guidance for the medical team to know
what actions are needed before, during and after surgery to ameliorate the risk of AKI.
Clinical findings indicate that prevention of kidney ischaemia and hypoxia during
CPB should reduce the risk of AKI. Fundamental to this argument is the observation
that most potentially modifiable risk factors for AKI after CPB are associated with
reduced renal oxygen delivery (Karkouti et al. 2009). For example, a haematocrit of
less than 21 % and whole body oxygen delivery less than 262 ml/min/m2 are risk
factors for AKI following cardiac surgery (Rosner et al. 2008). This clinical finding
accords with the prediction that pre-operative anaemia and intraoperative
haemodilution reduce blood oxygen carrying capacity and so reduce renal oxygen
delivery (Evans et al. 2013). Chronic obstructive pulmonary disease causes systemic
hypoxaemia (and thus reduced renal oxygen delivery) and is also a risk factor for
AKI in patients undergoing cardiac surgery (Rosner et al. 2008). During CPB, the
autoregulatory capacity of the renal circulation is blunted, so that renal blood flow
(RBF) and thus renal oxygen delivery is limited by the level of pump flow, and thus
the level of arterial pressure (Andersson et al. 1994). Indeed, computational model-
ling indicates that the combined effects of haemodilution and non-pulsatile blood
flow during CPB render the kidney susceptible to hypoxia if arterial pressure falls
below 50 mmHg (Sgouralis et al. 2014), a relatively common occurrence in clinical
practice (Rosner et al. 2008). It is also salient to consider that patients with heart
disease will often have marked activation of the renin-angiotensin and sympathetic
nervous systems, which will further promote renal vasoconstriction and thus reduced
renal oxygen delivery (Evans et al. 2013). Furthermore, anaesthesia itself tends to
reduce RBF, and thus renal oxygen delivery (Ullman et al. 2001).
It is not currently feasible to directly assess kidney oxygenation in patients on
CPB, but this has been achieved in experimental animals. In pigs, medullary and
urinary hypoxia (PO2 1–5 mmHg for both) were observed during CPB (Stafford-
Smith and Grocott 2005) and profound medullary hypoxia was observed 24 h after
CPB (Patel et al. 2011a, c). Treatments that ameliorated medullary hypoxia amelio-
rated AKI (Patel et al. 2011a, c). Medullary hypoxia was also observed in rats during
CPB (Darby et al. 2013). Critically, as discussed in detail later in this chapter, data
from humans show that low intra-operative urinary PO2 and a poor recovery of
urinary PO2 after CPB is associated with increased post-operative serum creatinine
and thus reduced GFR (Kainuma et al. 1996).
A unique aspect of CPB as a cause of AKI is that the surgical team, including
perfusionists and anaesthetists, have the means to modify systemic and renal
haemodynamic function when the patient is at risk of developing AKI. The funda-
mental problem is that different tissues have different perfusion needs. Consequently
a single group of macro-cardiovascular parameters may not provide suitable rates of
perfusion for all tissues. The optimal choice of these macro-cardiovascular param-
eters is not clear because, unfortunately, surgical teams currently have little tissue
specific information upon which to base these important decisions.
Sepsis
Ten to 50 % of patients with sepsis develop AKI. Indeed, sepsis is responsible for
approximately 50 % of cases of AKI in patients who are critically ill (Zarjou and
Agarwal 2011). Furthermore, outcomes are often worse for patients with septic AKI
than for those with non-septic AKI (Parmar et al. 2009).
It is difficult to predict which patients with sepsis will go on to develop AKI.
Furthermore, despite recent advances in urinary and plasma biomarkers, once
patients have developed sepsis the only established read-outs clinicians have to
determine the impact of the therapies they institute on renal function are systemic
haemodynamics, creatinine clearance and urine flow (Zarjou and Agarwal 2011).
The ‘real time’ evaluation of interventions to raise arterial pressure such as volume
resuscitation and vasoconstrictor therapy (Mori et al. 2010), is thus mainly limited to
assessment of their effects on systemic haemodynamics and blood chemistry, rather
than organ-specific effects on perfusion and oxygenation. Thus, septic AKI is
another example in which a continuous measure of kidney health should allow
considerable improvement in the management of patients at risk of, or who have
already developed, AKI. Could renal hypoxia be a useful biomarker in this respect?
Renal hypoxia has been observed in most, although not all, models of sepsis in
experimental animals (Evans et al. 2013). The potential mechanisms that drive renal
hypoxia in sepsis have recently been reviewed in detail (Aksu et al. 2011). In brief,
evidence from rodent models indicates that the process is initiated by hypoperfusion,
hypoxia, and/or inflammatory activation. The delicate balance between the bioavail-
ability of oxygen, nitric oxide and oxygen radical species is then disrupted, resulting
in reduced oxygen delivery to tissue and inappropriately high renal oxygen con-
sumption. However, there is currently considerable controversy as to the relative
merits of the various animal models of sepsis. Most models in rodents are associated
with renal ischaemia, so the presence of renal hypoxia comes as little surprise. It has
been argued that sepsis in humans is often associated with a hyperdynamic circula-
tion, so that renal blood flow is maintained or even increased from baseline
(Langenberg et al. 2005). Recently, medullary hypoxia and ischaemia was observed
in an ovine model of septic AKI in which total renal blood flow is actually increased
during sepsis (Calzavacca et al. 2015). These observations provide further support
for the notion that medullary hypoxia contributes to the maintenance and progres-
sion of septic AKI. Thus, a real time measure of renal medullary oxygenation would
potentially provide a method for early diagnosis and better management of septic
AKI, with potential for limiting the progression of septic AKI.
Radiocontrast Agents and Other Nephrotoxins

Radio-contrast administration is the third most common cause of hospital acquired
AKI (11 % of cases) (McCullough 2008). It adds considerably to both in-hospital
costs and the expenses patients face after they are discharged (McCullough 2008).
Perhaps most concerning is the fact that radio-contrast induced AKI is associated
with a four to sixfold increase in the risk of death during and after hospitalization
(McCullough 2008). AKI can also arise in (and outside) a hospital setting due to the
nephrotoxicity of a range of drugs (Perazella 2009). The countercurrent exchange
mechanisms in the renal medulla may concentrate contrast agents and other
nephrotoxins, and thus exacerbate the risk of AKI.
Radiocontrast-induced AKI probably progresses via multiple pathways. Never-
theless, one of these pathways is likely to be medullary hypoxia (Heyman
et al. 2008). Medullary hypoxia has been observed after radiocontrast administration
in anaesthetized rats (Heyman et al. 1991; Liss et al. 1998; Prasad et al. 2001) and
humans (Hofmann et al. 2006). Hypoxia is driven by a range of factors, including
activation of vasoconstrictor factors, the haemodynamic consequences of the vis-
cosity of contrast agents, and increased medullary tissue oxygen consumption due to
the combined effects of increased solute delivery to the distal nephron segments and
oxidative stress (Heyman et al. 2008; Evans et al. 2013).
Standard practice is to volume load patients thought to be at risk of AKI, with
isotonic saline, prior to radiocontrast administration (McCullough 2008). This is the
only prophylactic treatment that has been conclusively shown to be effective.
As is the situation with CPB and sepsis, a major challenge for management of
renal health in patients receiving radiocontrast agents or other potential
nephrotoxins, is the development of methods for real-time assessment of kidney
health. In the following section, the argument is developed that continuous mea-
surement of urinary PO2, to monitor changes in medullary oxygenation, would
provide the critical information needed to manage patients at risk of developing of
AKI in a hospital setting.
Urinary PO2 as a Diagnostic Biomarker in Acute Kidney Injury
The evidence that (bladder) urinary PO2 can provide prognostic information regard-
ing kidney health in a hospital setting has recently been reviewed in detail (Evans
et al. 2014b). The argument relies on five lines of evidence: that (i) the anatomy of
the renal medulla promotes diffusion of oxygen between the collecting ducts and the
vasa recta, so that (ii) oxygen tension in urine in the renal pelvis is in equilibrium
with that in medullary tissue, (iii) that oxygen diffusion across the epithelium of the
ureter and bladder only partially confounds the relationship between medullary PO2
and the PO2 of urine in the bladder, (iv) that bladder urine PO2 provides
prognostically useful information, and (v) that new fibre optic methods make
continuous measurement of urinary PO2 in patients with a bladder catheter relatively
straight forward and risk free. Each of these lines of argument is described in turn.
(i) Renal medullary anatomy promotes diffusion of oxygen between the

collecting ducts and the vasa recta: Detailed anatomical information regarding
the arrangement of various vascular and tubular elements within the rat renal
medulla indicates that, in the inner medulla, four ascending vasa recta usually
abut each collecting duct (Pannabecker and Dantzler 2006). This intimate
association likely promotes oxygen diffusion between collecting ducts and
ascending vasa recta, making it likely that urinary oxygen content equilibrates
with inner medullary interstitial oxygen, as shown in three human subjects
(Leonhardt et al. 1965) (Fig. 2).
(ii) Oxygen in the renal pelvis is in equilibrium with that in medullary tissue: In
the In the 1950s it was shown in anaesthetized dogs that the PO2 of pelvic urine
is rather low; certainly much lower than that of renal venous blood (Reeves
et al. 1957; Rennie et al. 1958). Later studies in anaesthetized dogs demon-
strated that experimental manoeuvres aimed at altering oxygen delivery to the
medulla, or medullary oxygen consumption, could alter the PO2 of pelvic urine
(Aukland and Krog 1961; Aukland 1962; Washington and Holland 1966). In
humans, it was shown that diuresis could increase pelvic urinary PO2, while
infusion of hypertonic saline reduced it. Furthermore, pelvic urinary PO2 fell in
response to vasopressin administration (Leonhardt and Landes 1965), a treat-
ment associated with reduced medullary perfusion and oxygenation (O’Connor
et al. 2006b). The response of human pelvic urinary PO2 to inspiration of 100 %
oxygen was also studied (Leonhardt and Landes 1965). This response was
found to be altered in a number of disease states, including chronic pyelone-
phritis, hydronephrosis, essential hypertension, arteriolar nephrosclerosis and
renal artery stenosis. The authors argued that breathing 100 % oxygen would
increase medullary oxygen delivery but not oxygen consumption. Thus, the
degree to which urinary PO2 increases upon breathing 100 % oxygen, and the
rate at which it increases, may provide an indirect measure of medullary
perfusion.
d
Urinary Oxygen Tension (mmHg)
100
80
60
40
20
0
0 20 40 60 80 100
a b Mean Medullary Oxygen Tension (mmHg)
Fig. 2 The relationship between oxygen tension (PO2) in the renal medulla and in pelvic urine.
Panels (a–c), originally reproduced from Pannabecker and Dantzler (2006) (with permission from
the American Physiological Society), show the close spatial relationships between ascending vasa
recta) and the collecting ducts. (a, b) Three dimensional reconstructions of the inner medulla of the
rat. Scale bars = 500 μm. In (a), descending vasa recta are shown in green, descending thin limbs of
the loop of Henle are shown in red or grey, and collecting ducts are shown in blue. In (b), collecting
ducts are shown in blue, ascending vasa recta are shown in red and ascending thin limbs are shown
in green. (c) Shows a transverse section near the base of the inner medulla. Ascending vasa recta,
shown in red, are seen to be positioned around collecting ducts which are shown in blue.
Descending vasa recta are shown in green. Scale bar = 30 μm. Panel (d) was redrawn from
Leonhardt et al. (1965). It shows nine paired measurements of urinary PO2 and mean medullary
PO2 made in a total of three human subjects. The dashed line is the line of identity. The entire figure
is reproduced with permission from the American Physiological Society (Evans et al. 2014b)
a b
Urinary Oxygen Tension (mmHg)
50 100
Urinary Oxygen Tension in the

40
Bladder (mmHg)
30
50
20
10
0 0
0 5 10 15 20 25 30 0 50 100
Distance from the Renal Pelvis (cm) Urinary Oxygen Tension in the Ureter (mmHg)
Fig. 3 Changes in urinary oxygen tension along the upper urinary tract. (a) Mean (n = 5) oxygen
tension of ureteral urine at various distances from the renal pelvis in human subjects (Data redrawn
from Leonhardt and Landes (1963)). (b) Scattergram with line of best fit for measures of
oxygen tension in the ureter and bladder in two anaesthetized dogs in which renal blood flow was
altered by infusion of dobutamine and propranolol (Figure redrawn from Kitashiro et al. (1995))
Taken collectively, the studies described in the paragraph above provide indi-
rect evidence that the PO2 of pelvic urine provides a useful index of medullary
oxygenation. Nevertheless, there is limited direct evidence to support
this proposition. The only published observations are data from three patients
(Leonhardt et al. 1965), in which there was excellent agreement between
pelvic urinary PO2 and medullary tissue PO2 (Fig. 2). Thus, there remains a
need for studies in experimental animals to better define the relationship
between medullary tissue PO2 and pelvic urinary PO2 in the healthy state and
in disease.
(iii) Oxygen tension of urine in the bladder: There are a number of factors that might
confound measurement of urinary PO2 in the bladder. These include the presence
of reducing agents such as ascorbic acid, thiols and polyphenols that can lead to
oxygen consumption (Rennie et al. 1958). Diffusion of oxygen between the urine
and the epithelium of the ureter and bladder might also be a confounding factor
(Rennie et al. 1958). However, experiments in both experimental animals and
humans have provided evidence and optimism that these confounding effects do
not invalidate the use of bladder urine PO2 as a measure of medullary tissue PO2.
For example, the PO2 of urine in the bladder of anaesthetised dogs was consis-
tently less (12
3 mmHg) than the PO2 of urine in the ureter (Kitashiro
et al. 1995). Nevertheless, when these animals were subjected to treatments
that altered cardiac output, the PO2 of urine in the bladder was found to correlate
closely with the PO2 of urine in the ureter (Fig. 3). Similarly, the PO2 of urine was
found to fall in humans, from 48 mmHg at the level of the renal pelvis to
33 mmHg in the bladder (Leonhardt and Landes 1963; Fig. 3). Nevertheless, in
the same individuals the PO2 of voided urine consistently increased in response
to hydration and fell in response to dehydration (Leonhardt and Landes 1963).
Fig. 4 Oxygen tension in the 140

urine of two patients a
undergoing cardiac surgery 120
and cardiopulmonary bypass.
In (a), note the fall in urinary 80
oxygen tension after bypass
commenced and the gradual
Bladder Urine PO2 (mmHg)

increase when the patient was 40
weaned from bypass. In (b),
note the fall in urinary PO2 0
after weaning from bypass
(Redrawn from Kainuma
Cardiopulmonary 30 min
et al. (1996)) Calibrate bypass
140
b
120
80
40
Cardiopulmonary 30 min
Calibrate bypass
The PO2 of urine from the bladder of patients with circulatory shock was also
found to be low (10–15 mmHg), but could be increased by fluid loading. Thus,
although oxygen transport between urine and the epithelium of the ureter and
bladder alters urinary PO2, changes in the PO2 of bladder urine do appear to
reflect those in pelvic urine (Fig. 3).
(iv) Prognostic value of urinary oxygen tension: In patients, bladder urine PO2 has
been shown to fall during surgical procedures associated with renal dysfunction
(Koivusalo et al. 1998), but not during surgical procedures associated with well-
maintained renal function (Laisalmi et al. 2001). It has also been shown to
increase in response to blood transfusion in anaemic patients (Valente
et al. 2008), and when tubular oxygen consumption is reduced by diuretic
therapy (Morelli et al. 2003). In some patients undergoing cardiac surgery
requiring CPB, urinary PO2 progressively decreased after the start of CPB and
then partially recovered during weaning from CPB (Kainuma et al. 1996; Fig. 4).
In others, it was found to be well maintained during CPB but to then fall after the
patient was weaned from CPB. Crucially, post-operative peak serum creatinine
was higher in patients whose bladder urine PO2 decreased following CPB
(Fig. 5). Importantly, post-operative recovery of urinary PO2 predicted post-
operative renal function (Fig. 5) better than pre-operative serum creatinine,
Bladder Urine PO2 (mmHg)

Post-operative Change in
50
-50
0 2 4 6
Post-Operative Peak Serum Creatinine (mg/dl)
Fig. 5 Scatterplot and line of best fit for the relationship between the peak serum creatinine
concentration versus the change in PO2 of bladder urine in the post-operative period after cardio-
pulmonary bypass (Redrawn from Kainuma et al. (1996))
blood urea-nitrogen, duration of CPB, cardiac index or urine flow after CPB. In
another study, a fall in the PO2 of bladder urine was also observed during CPB
(Farahani et al. 2010). Thus, there is considerable evidence that bladder urine
PO2 can provide prognostically useful information in the setting of surgery. We
are not aware of any measurements of urinary PO2 in patients with septic AKI or
AKI induced by radiocontrast agents or other nephrotoxins.
(v) New methods for measurement of urinary PO2: Most previous clinical and
experimental studies in which urinary PO2 has been measured have employed
either fragile polarographic electrodes (Aukland and Krog 1961; Leonhardt and
Landes 1965; Washington and Holland 1966; Kainuma et al. 1990; Kitashiro
et al. 1995; Morelli et al. 2003) or collection of urine for measurement of PO2
using a standard blood gas analyser (Valente et al. 2008). MRI could be used to
measure urinary PO2 in a non-invasive manner (Wang et al. 2008), but is not
feasible for continuous monitoring of patients.
Fluorescence lifetime oximetry provides an opportunity for continuous measure-
ment of urinary PO2 via a fibre optic probe. The probe can be inserted into a
bladder catheter and advanced to its tip, so as to be in direct contact with bladder
urine. These optical fibres can be very thin, so risk of obstruction of the bladder
catheter is minimal (Fig. 6). Other advantages of this approach are that the fibre
optic probes are sturdy; they come pre-calibrated by the manufacturer, and can be
operated with complete electrical isolation from the patient. They have been
extensively validated (Evans et al. 2008b) and applied to in vivo measurement of
the PO2 in a range of tissues including the kidney (Evans et al. 2011) and in body
fluids (e.g., oviductal fluid (Rafferty et al. 2013) and blood (Abdelkader
et al. 2014)). However, they are currently not approved for human use.
Fig. 6 A fibre optic probe for fluorescence lifetime oximetry. (a) Coiled probe with inset (b)
showing the tip of the probe. (c) Close up of the probe tip. This particular probe (NX-LAS-1/O/E) is
commercially available (Oxford Optronix Milton Park, United Kingdom; http://www.oxford-
optronix.com). Note that these probes are not approved for human use (Images were provided
courtesy of Oxford Optronix and are reproduced with their permission)
Potential Applications to Prognosis, Other Disease

and Conditions
The potential approaches to the use of hypoxia as a biomarker for kidney disease will
depend on the specific situation. CKD develops slowly, so a method that can be
applied to outpatients and provides relatively good reproducibility within subjects
would be ideal. BOLD MRI has these characteristics, but has proved so far to be
rather disappointing as a marker of CKD in human subjects. As things currently
stand, the balance of evidence does not support the use of BOLD MRI as a
diagnostic and prognostic tool in CKD. It is also impractical for continuous moni-
toring of patients in a hospital setting.
AKI often occurs in a hospital setting. Because of its rapid onset, and because
bladder catheterization is standard practice in many of the clinical situations in which
patients are at risk of AKI, continuous measurement of urinary PO2 by fluorescence
lifetime oximetry has the potential to provide real time information regarding the risk
of AKI. Continuous measurement of urinary PO2 might be particularly useful in
patients undergoing cardiac surgery requiring CPB and those in the intensive care
unit. This information could inform clinical decisions, particularly those regarding
renal or haemodynamic support. They could also be incorporated into a computa-
tional model of the kidney during surgery (Sgouralis et al. 2014). Such an approach
would allow real-time feedback-driven control of circulatory function to optimise
management of renal health. However, at least three gaps exist in the evidential basis
for such an approach. Firstly, understanding of the roles of tissue hypoxia in the
initiation and progression of AKI remains rudimentary (Evans et al. 2013). Sec-
ondly, there is limited information regarding the relationship between the PO2 of
urine in the bladder and renal medullary PO2 (Evans et al. 2014b). Thirdly, although
available information regarding the prognostic value of urinary PO2 are promising,
they are limited to a single study of 98 patients undergoing cardiac surgery while on
CPB (Kainuma et al. 1996).
Summary Points
• Current methods for diagnosis of chronic kidney disease and acute kidney injury
detect disease only once it is well progressed.
• There is evidence that renal hypoxia, a hallmark of both chronic kidney disease
and acute kidney injury, both initiates and drives progression of renal disease.
• Blood oxygen level dependent magnetic resonance imaging provides a measure of the
deoxyhaemoglobin content of renal tissue, so an indirect measure of renal hypoxia.
• In recent clinical studies, the blood oxygen level dependent magnetic resonance
imaging signal has been found to correlate poorly with the degree of renal
impairment in chronic kidney disease, so is unlikely to be useful for early
diagnosis of chronic kidney disease.
• There is strong evidence that oxygen in urine in the renal pelvis is in equilibrium
with oxygen in the renal medulla.
• Despite oxygen exchange between urine and the epithelium of the ureter and
bladder, changes in the oxygen tension of urine in the bladder appear to reflect
changes in the oxygen tension of pelvic urine, and thus medullary tissue.
• In patients undergoing cardiac surgery requiring cardiopulmonary bypass, post-
operative recovery of urinary oxygen tension predicted post-operative renal
function better than pre-operative serum creatinine or blood urea-nitrogen, dura-
tion of cardiopulmonary bypass, or cardiac index and urine flow after cardiopul-
monary bypass.
• Fibre-optic probes using fluorescence lifetime oximetry provide a safe and
technically simple method for continuous measurement of the oxygen tension
of bladder urine in patients at risk of acute kidney injury in a hospital setting.
• Continuous measurement of urinary oxygen tension may aid in management of
patients at risk of developing acute kidney injury, particularly in surgical and
intensive care settings.
Acknowledgments The authors’ work is supported by grants from the National Health and
Medical Research Council of Australia (606601, 1024575, 1042600, 1050672) and the
Australian Research Council (DP140103045).
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MicroRNAs in Kidney Diseases
7
Grazia Serino, Fabio Sallustio, and Francesco Paolo Schena
Contents
Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Key Facts of Renal Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Key Facts of PKD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Key Facts of Primary and Secondary Glomerulonephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
Key Facts of Kidney Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Key Facts of miRNA Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
Introduction: Biogenesis of miRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
New Strategies for Detection of Renal miRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114
MiRNAs in Renal Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Blood Flow and Oxygen Supply . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
Glomerular Filtration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Tubular Reabsorption and Excretion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Interstitial Osmolarity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
MiRNAs in Polycystic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
MiRNAs in Primary and Secondary Glomerulonephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 123
MiRNAs in Kidney Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
G. Serino
Laboratory of Experimental Immunopathology, IRCCS “de Bellis”, Castellana Grotte, BA, Italy
e-mail: grazia.serino@uniba.it
F. Sallustio
Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy
e-mail: fabio.sallustio@uniba.it; fabsal74@gmail.com
F.P. Schena (*)
Department of Emergency and Organ Transplantation, University of Bari, Bari, Italy
C.A.R.S.O. Consortium, University of Bari, Bari, Italy
e-mail: paolo.schena@uniba.it

108 G. Serino et al.
Potential Applications to Prognosis and Other Diseases or Conditions . . . . . . . . . . . . . . . . . . . . . . . 130

MiRNAs as Diagnostic Tools in Blood and Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
New Approaches for miRNA Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131
Challenges and Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
Abstract
MicroRNAs (miRNAs) play an important role in physiological and pathological
condition in human organs including kidneys. Their dysregulation on one hand
can induce the onset of a specific disease and on the other hand may represent
potential biomarkers for the diagnosis and therapy. In this chapter, the miRNA
functions and modulations are described in several kidney diseases such as
polycystic kidney disease, primary and secondary glomerulonephritides and
renal transplantation. After the overview on the approach to identify and study
miRNAs in nephrology, the miRNA meaning in renal physiology is illustrated.
Also, the attractive perspectives of the use of miRNAs as diagnostic tools or for
the specific treatment of kidney disease are taken into consideration.
Keywords
MicroRNA • Kidney physiology • Polycystic kidney disease • Glomerulonephri-
tis • Kidney transplantation
Abbreviations
AGO Argonaute
ANA Autoantibodies to nuclear antigen
ASO Antisense oligonucleotide
Bicc1 Bicaudal C. homolog 1
CAMR Chronic antibody mediated rejection
Cdc25A Cell division cycle 25A
cDNA Complementary DNA
DGCR8 RNA-binding protein DiGeorge syndrome critical region gene 8
EMT Epithelial-to-mesenchymal transition
FPC Fibrocystin
GTP Guanosine triphosphate
HBEGF Heparin binding epidermal growth factor
HCV Hepatitis C virus
HIVAN HIV associated nephropathy
IF Interstitial fibrosis
IFN Interferon
IRAK1 IL-1 receptor associated kinase1
IRI Ischemia-reperfusion injury
7 MicroRNAs in Kidney Diseases 109
LN Lupus nephritis
miRNA MicroRNA
MRE MiRNA recognition elements
OREB Osmotic response element binding protein
PAZ Piwi-Argonaute-Zwille
PBMCs Peripheral blood mononuclear cells
PC Polycystin
PKD Polycystic kidney disease
pri-miRNA Primary miRNA transcript
RISC RNA-induced silencing complex
shRNA Small hairpin RNA
SLE Systemic lupus erythematosus
TA Tubular atrophy
TAL Thick ascending limb
TGF Transforming growth factor
TNF Tumor necrosis factor
TRAF6 Tumor necrosis factor receptor-associated factor 6
TRBP Transactivator RNA binding protein
UTR Untranslated region
VLP Virus like particles
WMLK With-no-lysine kinase
Key Facts
Key Facts of Renal Physiology
• The juxtaglomerular apparatus, located in the wall of the afferent arterioles of

glomeruli, regulates the blood flow at renal level and controls the blood pressure
by the release of renin.
• Acute kidney injury is the consequence of reduced or fallen arterial blood
pressure caused by hemorrhagic shock or of toxic effect of agents or drugs.
• Chronic renal failure is the consequence of chronic kidney diseases characterized
by persistent hypoxic injury that causes renal fibrosis.
• Ischemia/reperfusion injury is a process occurring in kidney transplantation.
The hypoxic injury followed by fluid reperfusion leads to the activation of innate
and adaptive immune responses and to the delivery of cytokines and growth
factors.
• Glomerular filtration is the process of blood ultrafiltration at the level of glomer-
ular membrane that is formed by three layers: (i) endothelial cells; (ii) capillary
basement membrane; (iii) visceral epithelial cells (podocytes). Filtration is deter-
mined by the molecular size and charge of proteins.
• The entire tubular part of nephron provides the salt and water reabsorption and
electrolyte excretion by regulating the electrolyte homeostasis.
• The medullary part of the kidney modulates the osmoregulation response and
water handling under physiological and pathological conditions.
Key Facts of PKD
• The autosomal dominant polycystic kidney disease (ADPKD) is characterized by

cysts in kidneys and sometimes in liver and/or other organs (pancreas, arachnoid
membrane). This common genetic disorder clinically manifests in adulthood,
often leading to end-stage kidney disease with renal replacement therapy (dialysis
or kidney transplantation).
• The autosomal recessive polycystic kidney disease (ARPKD) is a rare cystic
disease that affects the renal tubules and the biliary tract. This rare genetic
disorder typically begins in utero and it is associated with congenital hepatic
fibrosis.
• The mechanosensory organelle localized in the apical surface of the cilia in
tubular cells has the important role in regulating the fluid flow and in signaling
the intracellular calcium.
• In the tubular cells of PKD there is the dysregulation of some intracellular
pathways like MAPK, ERK and AKT/mTOR leading to abnormality in cell
proliferation and cyst expansion.
Key Facts of Primary and Secondary Glomerulonephritis
• IgA Nephropathy (IgAN) is characterized by mesangial proliferative lesions

associated with diffuse deposition of IgA1-IgG immune complexes. The clinical
symptoms are recurrent episodes of gross hematuria in concomitance of upper
respiratory tract infections or permanent benign microscopic hematuria.
• Minimal change disease (MCD) is a glomerular lesion characterized by fusion of
epithelial cell foot processes that is responsible for an abnormal filtration of
albumin and transferrin through the filtration barrier. It is the principal cause of
nephrotic syndrome in children younger than 10 years. May be corticosteroid
responsive, non-responsive or dependent.
• Focal Segmental Glomerulosclerosis (FSGS) is characterized by a glomerulo-
sclerotic lesion that involves a minority of glomeruli located in deep section
of renal cortex and a portion of the glomerular tuft. For this reason the renal lesion
is defined focal and segmental. It is more frequent in adults than in children.
• Membranous glomerulonephritis (MGN) is an immune complex disease in which
deposits of immunoglobulins and complement are located in the subepithelial
surface of the glomerular capillary wall. The increasing thickness of the glomer-
ular basement membrane causes proteinuria with development of nephrotic
syndrome. It is more frequent in adults than in children.
• C3-glomerulopathy is a glomerular disease characterized by deposits of C3 alone

associated with mesangial and endocapillary proliferation and crescents. The
clinical manifestations are proteinuria and microscopic hematuria, but in two
thirds of patients is present nephrotic syndrome.
• Crescentic glomerulonephritis (CGN) is characterized by abnormal formation of
crescents (more than 50 %) that may occur in different forms of glomerulone-
phritis. The crescentic lesion is formed by epithelial proliferation of the Bowman
capsule delimiting the glomerular tuft.
• Lupus nephritis (LN) is the kidney involvement of systemic lupus erythematosus
caused by glomerular deposition of circulating immune complexes. The poly-
morphism of renal lesions and the different stages of the disease have been
included in the international classification of LN for the reading of the renal
biopsy. This approach is necessary for diagnosis, prognosis and therapy.
• HIV-associated nephropathy is a collapsing glomerulopathy characterized by
involvement of glomeruli (proliferation and dysregulation of podocytes, and
glomerular collapse), tubules (microcystic changes) and interstitium (chronic
inflammation and fibrosis). Nephrotic syndrome and failure of the renal function
are the clinical manifestations.
Key Facts of Kidney Transplantation
• Ischemia-reperfusion injury (IRI) is a damage in the tissue due to the blood supply
return to the tissue after a period of ischemia or lack of oxygen.
• Delayed graft function is a form of acute renal failure resulting in low output of
urine after transplantation.
• Interstitial fibrosis and tubular atrophy (IF/TA) is a common histological abnor-
mality of kidney transplants in which an expanded interstitial space replaces
normal cortical structures.
• Chronic antibody mediated rejection (CAMR) is an important cause of allograft
dysfunction and graft loss.
Key Facts of miRNA Therapy
• The antimiR oligonucleotides are chemically modified molecules in which sugar

modifications are included for increasing the duplex melting temperature and the
nuclear resistance.
• The locked nucleic acid is a bicyclic RNA analogue in which the introduced 20 -O,
40 -C methylene locks the ribose.
• Morpholino oligomer is a six-numbered morpholine ring that replaces the sugar in
the oligo nucleotide thus increasing the binding affinity with the cognate miRNA.
• MiRNA mimic is composed of an antisense (guide) that is identical to the cognate
miRNA, whereas the sense strand (passenger) is modified and linked to a
macromolecule to enhance the uptake.
Definitions
Antisense oligonucleotide inhibitors Antisense oligonucleotide inhibitors are

unmodified or chemically modified single-stranded molecules that specifically
bind mature miRNAs and inhibit their function.
Argonaute Argonaute is a family of proteins involved in RNA silencing process.

They are components of RNA-induced silencing complex (RISC) in which have the
role to cleave the target mRNA strand complementary to the bound miRNA.
Dicer Dicer is a large protein that contains several domains: ATPase/RNA helicase,
a DUF283 (Domain of unknown function) domain, a PAZ (Piwi, Argonaut and
Zwille) domain, two catalytic RNase III domains (RIIIa and RIIIb), and a C-terminal
double-stranded RNA-binding domain (dsRBD) is an enzyme with endonuclease
activity that cleaves the precursor-miRNA in double strand miRNA.
Locked nucleic acid (LNA) Locked nucleic acid (LNA) is an oligonucleotide that
contains one or more RNA modified nucleotides in which the ribose moiety is
modified with an extra methylene bridge that connects the 20 oxygen and 40 carbon.
This type of oligonucleotide offers an increased affinity for its complementary
strand.
miRNAsponges miRNA sponges are transcripts with repeated miRNA antisense

sequences that contain complementary binding sites to a miRNA of interest. As with
most miRNA target genes, sponge’s binding sites are specific to the miRNA seed
region, which allows them to block a whole family of related miRNAs.
Ran-GTP Ran (RAs-related Nuclear) protein is a small nuclear protein that is

required for the translocation of RNA and/or proteins through the nuclear mem-
brane. Ran binds either GTP and GDP, in particular nuclear Ran is mainly in the GTP
form and cytoplasmic Ran is in the GDP-bound form.
Transfection Transfection is a process that permits the introduction of nucleic acids

into cells.
Introduction: Biogenesis of miRNAs
MicroRNAs (miRNAs) are a class of small non-coding single strand RNAs (19–25
nucleotides) that regulate gene expression at the post-transcriptional level by
targeting messenger RNAs for translation repression or, less frequently, degradation
(Ambros 2004). MiRNAs represent the 1–5 % of all genes and are present in the
genome as independent genes or in intron regions of coding genes. The high
sequence conservation of many miRNAs among distantly organisms suggests strong
evolutionary pressure and participation in essential processes.
miRNA genes
RNA polimerase II
Drosha-DGCR8
5’ Cap (A)n
Pri-miRNAs Pre-miRNA
Nucleus
Cytoplasm
Exportin-5
DICER-TRBP
Pre-miRNA
miRNA duplex
RISC Asymmetric
unwinding
mature miRNA
Perfect complementarity Partial complementarity
mRNA degradation Translational repression
Fig. 1 Biogenesis of miRNAs
The biogenesis of miRNAs starts from the transcription of miRNA genes in the
nucleus (Fig. 1). MiRNA genes are transcribed by RNA polymerase II in primary
miRNA transcript (pri-miRNA); some miRNAs are transcribed by RNA polymerase
III in repetitive regions of genome. Pri-miRNAs contain a stem loop structure that
encodes the functional miRNA sequences in the stem. This stem loop structure is
precisely cut by the nuclear RNAse III type enzyme called Drosha, as part of a
complex called Microprocessor that also contains the RNA-binding protein
DiGeorge syndrome critical region gene 8 (DGCR8). DGCR8 recognizes and
interacts with the hairpin structure in the pri-miRNAs and recruits Drosha. These
pri-miRNAs are processed in fragments of RNA composed of 70 nucleotides,
called precursor miRNAs or pre-miRNAs. The pre-miRNAs are transported from the
nucleus to the cytoplasm by Exportin-5, a nuclear export factor that specifically

binds to the pre-miRNA in a Ran-GTP dependent manner. Within the cytoplasm, a
second cleavage event occurs via the RNAse III enzyme Dicer, which forms the
mature double stranded miRNA composed of 22-nucleotides. Like Drosha, Dicer
is associated with dsRNA-binding domain containing a protein partner known as
Loquacious/TRBP (transactivator RNA binding protein). In addition to its involve-
ment in mature miRNA generation, the Dicer-containing protein complex regulates
also the assembly of mature miRNA into the effector complex called RNA-induced
silencing complex (RISC)-like ribonucleoprotein particle (miRNP).
After strand separation of the duplexes, the mature single-stranded miRNA (guide
strand) is incorporated into RISC, whereas the other strand is often degraded. The
main constituents of the RISC are members of the Argonaute (AGO) family, which in
humans include eight members, of which four are ubiquitously expressed and are
associated with siRNA and miRNA, and four seem restricted to expression in the germ
line, whose function is poorly understood. Argonaute is a basic protein consisting of a
Piwi-Argonaute-Zwille (PAZ) domain that mediates nucleic acid binding, a middle
domain that seems to be critical association between the RNA and AGO, and a PIWI
domain that contains endonuclease activity. This complex inhibits mRNA translation
or reduces mRNA stability following imperfect binding of the guide strand to miRNA
recognition elements (MRE) within the 30 -untraslated region (UTR) of target genes.
MiRNAs bind their target RNAs in the 30 UTR through RNA-RNA base pairing that
involves not only the Watson-Crick pairs but also the G:U pair. The miRNA-binding
sequence in the target is mediated by the “seed” region localized at residues 2–8 at the
50 -end, although it also appears to be influenced by additional factors such as the
presence and cooperation between multiple MREs, the spacing between MREs, the
proximity to the stop codon, position within the 30 UTR, AU composition, and target
mRNA secondary structure. Since the specificity between miRNA and mRNA is
mainly determined by Watson-Crick base pairing, each miRNA can potentially regu-
lates the translation of a large number of different mRNAs, and each mRNA can
possess multiple binding sites for a single or for many different miRNAs.
New Strategies for Detection of Renal miRNA
The approach to identify and study miRNAs in kidney physiology and renal diseases
is summarized in Fig. 2. The experimental procedure can be divided into three steps:
(i) detection of miRNAs, (ii) individuation of miRNA target genes and (iii) identi-
fication of cellular processes affected by specific miRNAs and functional studies.
These experimental methods have been extensively used to identify miRNA regu-
lation in renal and other pathophysiological conditions.
The first step of miRNA research is their detection. Since the expression of
miRNA genes does not correspond to the expression level of mature miRNAs,
only the quantification of the mature miRNA level allows to determine whether a
miRNA is present and/or is regulated in a cell or a tissue. After the first miRNA
discovery, the quantification of miRNA sequences has been challenged; nowadays,
Fig. 2 Flow-chart that describes the steps to study miRNA in renal physiology and kidney
diseases. The experimental procedure can be divided into three sections: detection of miRNAs,
individuation of miRNA target genes and identification of cellular processes affected by specific
miRNAs and functional studies
advanced technologies facilitate their detection. The expression profiles of different

miRNAs in parallel can be measured by microarray analysis or deep sequencing,
whereas quantitative real-time RT-PCR and Northern blotting can be used to deter-
mine the level of individual miRNAs.
Microarray technology is a high-throughput method that allows the parallel screening
of thousands of miRNAs in one sample. Although miRNA microarray offers a good tool
to examine the miRNA that are expressed or deregulated in a cell or tissue of interest,
these data should be considered as a guide and should be validated by other methods.
In addition to using microarray analysis, in the last years, next generation
sequencing platforms allow the sequencing of small RNA molecules, including
miRNAs. Deep sequencing utilizes massively parallel sequencing, generating mil-
lions of small RNA sequence reads from a given sample. The sensitivity of deep
sequencing offers several advantages over microarray technology. First, unlike
microarray, sequencing is not limited to detecting miRNAs that correspond to
existing sequences, but this allows the discovery of novel miRNAs and could reveal
variations in the sequence. Second, sequencing has very low background noise and
cross hybridization; however, since deep-sequencing experiments produce a lot of
sequence data, many bioinformatic skills are necessary for their analysis.
To date the common method to detect the level of specific miRNAs is real-time
RT-PCR that has high sensitivity, specificity and it is cheap. There are two different
approaches for this reaction; the first one comprises double-stranded DNA interca-
lating molecules, such as SYBR Green I and EvaGreen; the second one includes
fluorophore-labeled oligonucleotides. A miRNA real-time reaction starts with RNA
that is reverse transcribed into cDNA. The short length of the mature miRNA, the
lack of a common sequence like a poly(A) tail, and the fact that the mature miRNA
sequence is also present in the pri- and pre-miRNA transcript mean that the reaction
is different from the classical reverse transcription. Currently, two methods are used
for the reverse transcription: miRNA-specific and universal reverse transcription. In
the first method, individual miRNAs in tissue or in cells are reverse transcribed
specifically by using stem-loop-specific reverse transcription primers. Stem-loop
primers are designed to have a short single-stranded region that is complementary to
the known sequence on the 30 end of the miRNA, a double-stranded part (the stem),
and the loop that contains the universal primer-binding sequence. The resulting cDNA
is then used as a template for the real-time RT-PCR with a miRNA-specific primer and
a second universal primer. The second approach initially adds a poli-A tail to all
miRNAs and then reverse transcribes all miRNAs by means of a universal primer.
A primer consisting of an oligo(dT) sequence with a universal primer-binding
sequence is then used to amplify the specific miRNA in the real-time RT-PCR.
More recently, real time PCR has been adapted to quantify also precursors and
primary transcripts. Moreover, miRNA real-time PCR array has been developed for
the screening of a small number of miRNAs especially in samples from body fluids
(blood and urine). The miRNA profiles depict the possibility to discover disease
biomarkers that can easily measured in samples collected in a non-invasively manner
and can promote the monitoring of the disease outcome.
An approach to visualize specific mature miRNAs and pre-miRNAs is the Northern
blotting technique. It involves the use of electrophoresis to separate RNA samples by
size and detection with a hybridization probe complementary to miRNA sequence.
The profile of miRNAs varies greatly between tissues (organ-specific), cells (cell-
specific) and during the phase of activity in a cell (phase-specific). The average half-
life of miRNA is approximately 5 days.
MiRNAs have been also studied in renal biopsy (frozen or formalin-fixed paraffin-
embedded renal tissue) through combined approach of “in situ” hybridization and
immunohistochemistry. Mostly biopsy samples exist in formalin-fixed paraffin-embed-
ded archives and the possibility to use this enormous resource could permit to organize
mechanistic studies in humans. Another interesting approach is the study of miRNA
expression in isolated glomeruli or tubules obtained by laser-captured kidney tissue.
After the individuation of a list of miRNAs that differentiate two conditions
(e.g. normal and disease), the most important step is to predict and then to confirm
the miRNA target genes (Fig. 2). First, several bioinformatic programs or databases,
as miRBase (http://microrna.sanger.ac.uk), TargetScan (http://www.targetscan.org),
PicTar (http://pictar.org), and miRWalk (http://www.ma.uni-heidelberg.de) are avail-
able to predict the potential target genes for each miRNA. Since each miRNA can
regulate the translation of numerous mRNAs, and each mRNA possess multiple
binding sites for a single or for many different miRNAs, these algorithms generate
numerous putative target genes (Brodersen and Voinnet 2009). Second, after finding
potential targets for miRNAs, it is essential to validate biologically the relationship
between a miRNA and a mRNA. To deepen the study of the pathophysiological role of
an identified miRNAs, it is possible to up-regulate specific miRNA levels in cells or
knockdown specific miRNA activity (Fig. 2). There are two main approaches to study
experimentally the regulation of a specific gene by miRNA: the use of a miRNA mimic
or a miRNA inhibitor. MiRNA mimics are small, chemically modified double-stranded
RNAs that mimic mature endogenous miRNAs after transfection into cells. Instead,
miRNA inhibitors are antisense oligonucleotides, chemically synthesized, which spe-
cifically inhibit endogenous miRNA function after transfection into cells. After trans-
fection, the miRNA target gene must be measured at mRNA and protein levels.
Once the target protein has been biologically validated, it is important to identify
its functional role in the pathophysiological conditions in order to provide a com-
prehensive view of the pathogenesis and leads to the development of novel thera-
peutic approaches.
MiRNAs in Renal Physiology
The expression profile of miRNAs is different in organs. Kidney compared to other

organs has high expression of miR-192, miR-194, miR-204, miR-215 and miR-216
(Sun et al. 2004); however other miRNAs expressed in other organs are present at
lower level. Tian et al. (2008) demonstrated a different distribution of miRNAs in
renal cortex and medulla of Sprague Dawley rats; these miRNAs have different
target genes and explain the different function in the sections of kidney.
In this section the different expression of miRNAs that contribute to the normal
renal physiology like blood flow, glomerular filtration, tubular reabsorption and
excretion, and interstitial osmolarity are described.
Blood Flow and Oxygen Supply
Blood pressure is modulated at renal level by the juxtaglomerular cell apparatus that
produces renin. Sequeira-Lopez et al. (2010) studied the participation of miRNAs in
hypertensive kidney disease by inducing the ablation of the Dicer enzyme in the
juxtaglomerular cells (Fig. 3). They obtained a reduced number of juxtaglomerular
cells with low expression of renin genes (Ren1 and Ren2), decreased plasma renin
and low blood pressure in the knockout Dicer mouse that developed nephrovascular
abnormalities and corticomedullary fibrosis.
Kidney hypoxia is characterized by a reduction of oxygen in site. It occurs either
in the acute process of ischemia (duration: seconds/minutes) leading to the acute
kidney injury or in the chronic process (duration: hours/days) leading to chronic
renal failure and end-stage kidney disease. Many investigators have demonstrated
that intracellular signaling pathways involved in hypoxia are modulated by miRNAs
miR-210----|EphrinA3
Blood flow
Dicer Deletion
In JXT cells
⊥
Ren1;Ren2
Collecting
duct
Cortex
Medulla
Interstitial osmolarity
Hypoxia
↑miR-34a HIF-1
miR-156
miR-16 miR-200b OREB
miR-20a,b miR-717 OREB
miR-499 a/b PAI-1 miR-8 family Nherf1
↓miR-21 miR-192 WNK1
miR-127 miR-802 caveolin-1
miR-210 VEGF miR-194 intersectin-1
Tubular Reabsorption
+ +
↑miR-192 Na /K Atp 1b1
↑miR-9, miR-374 Claudin 14
Fig. 3 Participation of miRNAs in modulating the kidney functions
(Fig. 3). Hypoxia causes renal damage because the activation of HIF-1 signal in
epithelial tubular cells under low oxygen induces downregulation of miR-34a
expression and activation of Notch signaling pathway leading to epithelial-
mesenchymal transition process and renal fibrosis (Du et al. 2012).
The participation of miRNAs in hypoxic injury followed by subsequent reperfu-
sion has been studied in kidneys of C57BL/6 mice by Godwin et al. (2010). They
found a miRNA pattern characterized by 9 miRNAs differently expressed. Among
these, miR-21, abnormally expressed in mice and in tubular epitelial cells, was
defined an important player in protecting cells from death.
A protective effect of miRNAs in the ischemia/reperfusion injury has been
described by some investigators. Aguado-Fraile et al. (2013) demonstrated that
miR-127 is a regulator of the proximal tubular cell response to the injury (prevention
of the focal adhesion complex disassembly and tight junction disruption). Recently,
Bijkerk et al (2014) showed the protective role of miR-216 in mice that, before the
ischemia/reperfusion injury, received bone marrow transplantation containing hema-
topoietic cells with overexpression of this miRNA. MiR-216 promoted progenitor
cell mobilization, vascular integrity and recovery of the renal damage after the
ischemia/reperfusion injury.
Another miRNA, modulated by hypoxia, is miR-210 that is involved in the
process of renal angiogenesis during the acute ischemia/reperfusion injury (Liu
et al. 2012). This miRNA mediates the activation of VEGF signaling pathway.
Finally, Wei et al. (2010) showed that Dicer deletion of proximal tubular cells
protected against renal ischemia-reperfusion injury. They used a proximal tubule
specific Dicer KO mice model that was resistant to hypoxia showing better renal
function, reduced tissue damage, lower apoptosis of tubular cells and higher survival
rate. During the 12–48 h of reperfusion there was a change of miR-132, miR-362,
miR-379, miR-668 and miR-687.
Glomerular Filtration
MiRNAs modulate the development and function of podocytes and consequently the
glomerular filtration. The ablation of Dicer enzyme in podocytes of mice caused
significant proteinuria by 2 weeks after birth and death within 4 weeks (Harvey
et al. 2008; Ho et al. 2008; Shi et al. 2008).
Tubular Reabsorption and Excretion
Salt and fluid reabsorption at the thick ascending limb (TAL) level of Henle loop are
modulated by miR-192 that targets the Na+/k+-ATPase beta1 subunit gene (Atp1b1)
through the 50 UTR (Fig. 3). High salt intake increases the expression of this miRNA thus
suppressing the Atp1b1 gene function and promoting diuresis (Mladinov et al. 2013).
Claudin-14 is localized in the TAL and it is important for the Ca2+ reabsorption
in the kidney. MiR-9 and miR-374 recognize the 30 UTR of claudin-14 mRNA. High
intake of Ca2+ downregulates the expression levels of these two miRNAs in TAL
cells that in turns induce an increase of claudin 14 expression. Claudin 14, included
in the complex claudin 16–11 proteins, inhibits the reabsorption of urinary Ca2+ in
the TAL of nephron (Gong et al. 2012). Recently, the same group demonstrated that
treatment with histone deacetylase inhibitors stimulated miR-9 and miR-374, that
downregulated the renal Claudin 14 mRNA. These findings suggest a novel
approach for treating hypercalciuria (Gong et al. 2014).
Interstitial Osmolarity
Osmotic response element binding protein (OREB) is a transcription factor that

regulates the cellular osmoregulation in kidneys. Huang et al. (2011) demonstrated
that in mice miR-200b and miR-717 modulated the common gene target OREB
(Fig. 3). In fact, the depletion of these miRNAs by knocking-down Dicer increased
significantly OREB causing more renal tonicity, whereas their overexpression
decreased the hypertonicity.
The osmolarity has also been studied in zebrafish embryos. Flynt et al. (2009)
showed that the miR-8 family regulated the osmoregulation because these miRNAs
are expressed in ionocytes where they modulate the expression of Nherf1, a regulator
of apical trafficking of transmembrane ion transporters. Deletion of the miR-8 family
caused an inability of response to the osmotic stress and blocked the properties of the
transmembrane glycoproteins at the apical surface of ionocytes.
The distal nephron, formed by the distal convoluted tubule, the connecting tubule
and the collecting duct, plays an important role in modulating the interstitial
osmolarity. In this area two miRNAs participate to the regulation of sodium and
potassium ion transport. MiR-192 modulates the target gene serine-threonine kinase
WNK1 that is also regulated by aldosterone, thus it contributes to the sodium
reabsorption (Elvira-Matelot et al. 2010). MiR-802 modulates the caveolin-1 gene
expression that regulates the renal outer medullary channel located in the collecting
duct and involves the urinary potassium secretion (Lin et al. 2011). Therefore, high
intake of potassium rich diet induces upregulation of miR-802 which decreases its
gene target (caveolin-1) leading more excretion of potassium ions. Recently, Lin
et al. (2014) discovered another miRNA, miR-194, that regulates the renal outer
medullary potassium channel activity by targeting the intersectin-1 and modulating
the With-No-Lysine Kinase (WMLK)-induced endocytosis at cellular level.
MiRNAs in Polycystic Kidney Disease
Polycystic kidney disease (PKD) is a congenital disorder characterized by the

presence of numerous cysts arising from the tubules in the renal parenchyma.
PKD may be transmitted as autosomal dominant (ADPKD) or autosomal recessive
(ARPKD) disease caused by mutations of three genes like PKD1 (located on
chromosome 16p13.3), PKD2 (located on chromosome 4q21) and PKHD1 (located
on chromosome 6) that encode the corresponding proteins, polycystin-1 (PC-1),
polycystin-2 (PC-2) and fibrocystin (FPC), respectively. These proteins are present
in the primary cilium that is a sensory organelle localized in the apical surface of the
tubular cells (Fig. 4). The abnormal function of these proteins induces abnormal cell
division and proliferation leading to the formation of cysts in kidneys and liver
(ARPKD). Polycystins and other proteins constitute a complex mechanoreceptor
that senses fluid flow in lumen of tubules, triggering Ca2+ influx through the TRFP2
channel, thus affecting the fluid secretion. The development of renal cysts is the
result of a process based on two hits. The first one is the inheritance of the germline
mutation; the second one is the somatic mutation that inactivates PKD1 or PKD2
allele gene in tubular cells thus reducing the expression levels of polycystin. This
hypothesis is supported by studies carried out in genetically engineered mouse
models with targeted disruption of PKD1 and PKD2 genes. However, the inactiva-
tion of PKD genes in tubular epithelial cells is not sufficient to initiate the cyst
formation, therefore an additional factor may be required (third hit) and it may be the
renal injury or miRNAs described in this section. In conclusion, several pathways
including cell polarity, fluid secretion, cell proliferation and apoptosis participate in
the cyst formation.
Pandey et al. (2008) used a microarray-based approach to study the mRNA and
miRNA expression patterns in PKD of Hannover rat (Han: Sprague Dawley cy/+
rat) that develops by 8 weeks an ADPKD characterized by cyst formation and
progressive renal failure. The combinatorial approach of mRNA and miRNA
study demonstrated 935 dysregulated genes, 29 downregulated miRNAs and the
upregulated miR-21 (Fig. 4). Several dysregulated genes were targets of some
miRNAs like miR-21, miR-31, miR-128, miR-147 and miR-217. Later, Pandey
≠miR-17-92 cluster
PKD1 ≠miR-21
ØmiR-200 (a,b,c)
PKD2 ≠miR-17
ØmiR-15a
PKHD1
≠miR-365-1
TUBULAR CELL
PKD Tubular cyst
Fig. 4 Modulation of PKD genes by miRNAs in polycystic kidney disease (PKD)
et al. (2011) examined the global gene-expression profiling of renal cyst formation
and growth in embryonic kidneys of PKD-null type mice at days 14.5 and 17.5.
They, using computational analyses, predicted miRNAs that targeted the differently
expressed mRNAs, confirmed by quantitative real-time PCR analysis. At days 14.5
miR-204 and miR-488 were downregulated whereas miR-10a, miR-30a-5p,
miR-126-5p, miR-182, miR-200a and miR-429 were significantly upregulated. At
days 17.5 miR-10a, miR-126-5p and miR-425 were significantly downregulated
while miR-96, miR-182, miR-30a-5p were upregulated. These findings indicate that
several miRNAs are involved in the development of cysts. Since each miRNA
targets more genes and each gene is targeted by more than one miRNA the inves-
tigators found a cascade of dysregulated pathways (MAPK, JAK-STAT, Ca2+
signaling and WnT) leading to renal failure in PKD null type mice.
Dweep et al. (2013) studied the regulatory role of miRNAs in another model of
PKD/Mhn (cy/+) rat used for understanding the biological processes in cyst forma-
tion in ADPKD. The profile of abnormal expression of mRNA and miRNA revealed
3,333 deregulated genes and 8 upregulated miRNAs (miR-214, miR-34a, miR-199a-
5p, miR-146b, miR-503, miR-31, miR-132, and miR-21). These miRNA regulated
23 gene pathways that participate in the cyst formation and expansion.
Patel et al. (2012) generated a transgenic mouse with the ablation of the Dicer
enzyme in miRNA for studying the renal tubular maturation. This model had the
targeted deletion of the enzyme only in the section of nephros generating renal
tubules and collecting ducts. The Dicer mutant mice developed cysts at tubular level
and the microarray analysis of the kidneys showed a downregulation of miR-200
family (miR-200a, miR-200b and miR-200c) and an upregulation of PKD1 gene
(Fig. 4). These findings were confirmed by in vitro study on cultured renal epithelial
cells in which the inhibition of miR-200 dysrupted tubulogenesis and upregulated

PKD1. In conclusion, PKD1 gene is a target of miR-200 in the process of renal
tubule maturation and dowregulation of this miRNA causes cyst initiation and
formation. Recently, same group of investigators (Patel et al. 2013) demonstrated
the upregulation of the miR-17-92 cluster (miR-17, miR-18 and miR-20a) in a
mouse model of PKD; its inactivation reduced the kidney cyst growth, improved
renal function and prolonged survival (Fig. 4). This miRNA cluster promotes cell
proliferation through the post-transcriptional repression of PKD1 and PKD2 genes
and the hepatocyte nuclear factor-1β.
Two independent groups of investigators (Sun et al. 2010; Tran et al. 2010) have
demonstrated the translational repression of PKD2 gene by miR-17 (Fig. 4); the
ectopic expression of this miRNA can promote the proliferation of HEK cells by
targeting PKD2. After bioinformatics analysis miR-17 was found to be a regulator of
PKD2 gene expression. Sun et al. (2010), used stable cell lines with overexpression
of miR-17 for demonstrating that this miRNA modulates PKD2 gene in 30 UTR thus
repressing the expression of mRNA and promoting cell proliferation. Therefore,
miR-17 may be considered an important player in the mechanism of cystogenesis
and at the same time a therapeutic target for the growth of cyst and progression of
renal damage in ADPKD. The gene Bicaudal C. homolog 1 (Bicc 1), located on
chromosome 10 may have some mutations in mouse that can be responsible for renal
cyst formation; Tran et al. (2010) demonstrated that Bicc1, a key regulator of
embryonic development, acts as a post-transcriptional regulator upstream of
PKD2, thus regulating the stability of PKD2 mRNA, and its translation efficiency.
In addition, Bicc1 antagonizes the repressive activity of the miR-17 on the 30 UTR of
PKD2 mRNA. Therefore, mouse with Bicc1 mutants develop cysts in organs like
kidneys, liver and pancreas.
Lee et al. (2008), using microarray on RNA isolated from cholangiocyte cell
lines of PKD rats (a model of ARPDK), demonstrated a decreased expression of
miR-15a associated with upregulation of its target, the cell-cycle regulator cell
division cycle 25A (Cdc25A). The depression of this miRNA in normal rat caused
accelerated cell proliferation and hepatic cystic formation while overexpression of
miR-15a in PKD-CCL cells decreased Cdc25A, inhibited cell proliferation and
reduced cyst growth (Fig. 4). These results indicate that the dysregulation of
miR-15c/Cdc25A complex may be an important player in the hepatic cyst growth
and proliferation. Recently, Duan et al. (2012) identified by bioinformatics analysis
the miR-365-1 as a regulator of the 30 UTR of PKHD1 gene of which some
mutations contribute to the development of ARPKD (Fig. 4). The expression of
PKHD1, post transcriptionally regulated by miR-365-1, was validated in cell lines;
miR-365-1 modulated PKHD1 gene suppressing cell-cell adhesion through
E-cadherin. This implies that dysregulation of this miRNA may be relevant for
the ARPKD onset.
In conclusion, data reported in the literature support the hypothesis that abnormal
expression of some miRNAs may alter the function of PKD genes thus contributing
to the cyst development and growth in kidneys and/or liver.
IgAN MCD FSGS MGN

Blood ↑let-7b GALNT2 Kidney ↓miR-30a annexin V Blood ↑miR-15; miR-152
↑miR-148b C1GALT1 ↑miR-193 WT1 ↓miR-82; miR-84
Kidney ↑miR-21 fibrinogen Urine ↑miR-200c ↓miR-89; miR-98
↓miR-223 ICAM1 ↑miR-30a-5p
↓miR-148b TPM1 miR-196b
↓miR-29c COL2A1 miR-490
C3-GN CGN
Kidney miR-1207-5p HBEGF miR-155 Th17
LN HIVAN
Blood miR-371-5p; miR-423-5p; miR-1224-3p Kidney ↓miR-200b,c TGFβ
⊥ ↓miR-33
IFN pathway
↑miR-21; miR-148a; miR-126
⊥
DNA methylation
miR-146 IRAK1, TRAF6
↓miR-125a
T reg
↑miR-155
↑miR-21 PDCD4
Profiling: miR-142-3p; miR-637
Kidney miR-516-5p; miR-637
↓miR-638 ↑miR-146a; miR-198
Urine miR-146a; miR-155
miR-150
Fig. 5 MiRNAs involved in the pathogenesis of primary and secondary glomerulonephritides.

HIVAN histology picture has been kindly given by A. Fogo, Dept of Pathology, Vanderbilt
University
MiRNAs in Primary and Secondary Glomerulonephritis
This section describes the role of miRNAs involved in different forms of glomeru-
lonephritis (Fig. 5).
IgA Nephropathy (IgAN) is the most common primary glomerulonephritis in
the world. It is characterized by deposition of immune complexes (IgA1-anti IgA1)
or polymeric IgA1 in the mesangial area of glomeruli. IgA1 in its deglycosylated
form is the first hit of this disease because it is recognized like a non-self antigen by
the immune system that produces IgG or IgA antibodies against the deglycosylated
IgA1. Serino et al. (2012, 2015) demonstrated that two miRNAs modulate the
process of IgA1 glycosylation, let7 and miR-148b, that have as gene targets the
enzyme N-acetylgalactosamyltransferase 2 (GALNT2) and core 1, β1,
3-galactosyltransferase1 (C1GALT1), respectively (Fig. 5). The overexpression of
these miRNAs reduced the activity of the two enzymes which participate in the
process of IgA1 glycosylation.
Bao et al. (2014a, b) demonstrated that polymeric IgA obtained from serum of
IgAN patients stimulated in vitro human mesangial cells to produce abnormal
amount of TGFβ and TNFα that upregulated miR-21 that was found at glomerular
and tubular level in kidney biopsies of IgAN patients. In addition, a downregulation
of miR-223 in glomerular endothelial cells was responsible of high expression of
ICAM-1 and monocyte-endothelial adhesion that participate in the progression of
renal damage. The downregulation of other miRNAs, like miR-148b and miR-29c
caused the high expression of some molecule targets (TPM1, COL2A1), that
participate in production of collagen and sequential glomerular sclerosis and inter-
stitial fibrosis.
Minimal change disease (MCD) and focal segmental glomerulosclerosis
(FSGS) occur more frequently in children and boys. This disease is characterized
by an extensive flattening of podocyte foot processes in the first phase (MCD), then
glomerular sclerosis, tubular atrophy and interstitial fibrosis appear in FSGS.
A downregulation of the miR-30 family has been found in microdissected glomeruli
of renal biopsies from patients with FSGS; this alteration explains the abnormal
function and structure of podocytes (Wu et al. 2014; Fig. 5). An important role of
miR-193a has been shown in the destabilization of podocyte foot processes
(Gebeshuber et al. 2013). The upregulation of this miRNA suppressed the transcrip-
tion factor WT1 that participates in the maturation process of podocytes, thus
causing downregulation of podocalyxin, nephrin and podocin with collapse of the
podocyte structure.
After the first report of Cai et al. (2013), who described high serum levels of
miR-192 and miR-205 in patients with FSGS, a recent retrospective study of Zhang
et al. (2014) has shown high values of miR-125b, miR-186 and miR-193a-3p in the
plasma of patients with disease in active phase. MiR-125b and miR-186 declined
markedly in patients who were responsive to corticosteroid therapy and miR-186
correlated with the degree of proteinuria.
The evaluation of miRNAs in the urine of patients with FSGS has been done by
Wang et al. (2013) who found increased expression of miR-200c in the urinary cells
but only a few cases were analyzed and data were not conclusive. Recently, Zhang
et al. (2014) identified a panel of four urinary miRNAs (miR-155, miR-196a,
miR-30a-5p and miR-490) that were significantly higher in patients with active
FSGS than patients in remission. In addition, three combined miRNAs (miR-30a-
5p, miR-196a and miR-490) formed a signature with a discriminating AUC of 95 %.
This pattern was validated in a prospective study that indicated this signature as
biomarker to discriminate patients with FSGS in active phase from those in remis-
sion. Finally, urinary miR-30a-5p levels significantly reduced after corticosteroid
therapy thus predicting the positive response to treatment.
Membranous glomerulonephrits (MGN) is characterized by a thickening of the

capillary wall in the glomeruli after deposition of immune complexes. Recently,
Chen et al. (2014) studied the miRNA profiling in peripheral blood mononuclear
cells (PBMCs) of 30 patients with MGN. They found a list of 40 miRNAs with the
highest fold change in expression of which 20 downregulated and 20 upregulated.
Furthermore, they reported four novel miRNAs downregulated (miR-82, miR-84,
miR-89 and miR-98) and two novel miRNAs upregulated (miR-15 and miR-152)
(Fig. 5). The authors concluded that these miRNAs for their higher difference in
expression may be involved in the pathogenesis of the disease.
SNPs can occur in the sequential phases of miRNA biogenesis (pri-miRNA,
pre-miRNA and mature miRNA) causing the deregulation of the correlated gene
targets and development of a disease. This situation has been observed in
C3-glomerulopathy, that is characterized by a common exon 2–3 heterozygous
duplication of CFHR5 gene. Papagregoriou et al. (2012) evidenced the important
role of miR-1207-5p that regulates the heparin binding epidermal growth factor
(HBEGF), expressed in the podocytes, as gene target (Fig. 5). This regulation
was abolished by the presence of the C 1936 T SNP in the miR-1207-5p. This
variant was evaluated in a cohort of 78 patients with biopsy-proven C3
glomerulopathy and it indicated the progression to chronic renal failure in the
long-term follow-up.
Autoimmunity is an interesting field that involves many investigators because
many renal diseases have an autoimmune origin. Recently, Krebs et al. (2013) have
studied the role of miR-155 in a mouse model of crescentic glomerulonephritis
(CGN) induced by sheep globulins. They demonstrated that this miRNA drives renal
injury through the recruitment of nephritogenetic Th17 cells (Fig. 5). The potential
role of miR-155 was confirmed in nephritis in induced miR/ mice in which the
systemic and renal nephritogenic Th17 immune response was markedly decreased
and mice developed a less severe nephritis. These results suggest that miR-155 may
be considered a potential therapeutic target in autoimmune nephritis; in fact, pre-
ventive treatment of wild-type mice with a miR-155 antagonist decreased the
recruitment of Th17 at renal level and reduced the renal damage. Finally, the
investigators studied the expression of miR-155 in renal biopsies of patients with
ANCA-associated glomerulonephritis and found high expression of this miRNA that
correlated with severity of renal damage.
Lupus nephritis (LN) is the manifestation of renal damage occurring in systemic
lupus erythematosus (SLE) that is an autoimmune disorder characterized by the loss
of immune tolerance to nuclear self antigens with consequent formation of circulat-
ing immune complexes that deposit in kidneys. Innate and adaptive immunity are
involved in this disease and miRNAs participate as potential modulators of this
immune response.
First, Dai et al. (2007) described the miRNA pattern in PBMCs of patients with
SLE and identified 16 miRNAs of which 9 upregulated and 7 downregulated. Later,
Te et al. (2010) described the miRNA pattern of LN (miR-371-5p, miR-423-5p and
miR-1224-3p). Bioinformatic analysis demonstrated that the IFN signaling pathway
was modulated by these miRNAs.
MiRNA participate in DNA hypomethylation of CD4+ T cells in this disease; this

phenomenon is responsible for T cell autoreactivity. Three upregulated miRNAs
(miR-21, miR-148a and miR-126) inhibited the DNA methylation process in CD4+
T cells of SLE patients (Pan et al. 2010; Zhao et al. 2011). It has been demonstrated
that a genetic variant (rs57095329) in the promoter region of miR-146 gene is
responsible for a reduced expression of this miRNA that targets some genes of the
innate immunity like IL-1 receptor associated kinase1 (IRAK1) and tumor necrosis
factor receptor-associated factor 6 (TRAF6) that are two signal transducers in the
NFkB pathway.
Other dysregulated miRNAs contribute to the development of SLE, like a
downregulation of miR-125a (Zhao et al. 2010) and an overexpression of miR-21
(Stagakis et al. 2011) and miR-155 (Divekar et al. 2011). They modulate the activity
of T cells, mainly T reg.
The presence of miRNAs in serum or plasma of patients with SLE has also been
studied by two groups of Chinese investigators. The Szeto’s group (Wang
et al. 2010b, c) proposed the downregulated miR-200 family, miR-205, miR-192,
miR-146a and miR-155 as biomarkers of disease activity. In addition, an improve-
ment of miR-146a was observed after administration of calcitriol that may be
considered an immune modulator because the low expression of miR-146a promoted
the type 1 IFN pathway activation.
The Dai’s group (Wang et al. 2012) identified the circulating miR-126 specifically
higher in SLE patients and other 3 miRNAs (miR-125a-3p, miR-155 and miR-146a)
downregulated. They suggested this miRNA signature as a potential biomarker for
monitoring SLE. Since bioinformatic analysis identified a number of significant
pathways, they considered these miRNAs an additional step for studying the path-
ogenetic mechanisms of the disease (Sui et al. 2014).
Recently, Carlsen et al. (2013) reported a miRNA signature (miR-142-3p,
miR-106a, miR-17 and miR-20a) as a classifier for analyzing the SLE risk. Interest-
ingly, these results were validated in two independent cohorts of SLE patients from
Denmark and Sweeden, respectively. The target genes of these miRNAs are involved
in the inflammatory pathogenesis of the disease.
Reports of scientists on the miRNA expression in kidney biopsies of SLE patients
are very few (Fig. 5). The transcriptomic analysis of Dai et al. (2009; Sui et al. 2014)
indicated two validated miRNAs (miR-516-5p and miR-637) whereas Lu
et al. (2012) found a downregulated expression of miR-638 at glomerular level
and an upregulation of miR-146a and miR-198 in the glomerular and tubuloin-
terstitial compartments. Fibronectin and CXCR3 were found as the target genes of
these miRNAs.
Szeto’s group (Wang et al. 2010b) reported additional data of miRNAs in urine of
patients with SLE. This team of investigators indicated two urinary miRNAs
(miR-146a and miR-155) as expression of the disease because they are regulators
of the immune system. Finally, Zhou et al. (2013) indicated the upregulated miR-150
expression at renal level as a potential biomarker for evaluating the renal outcome in
SLE patients.
The role of miRNAs has been studied in the pathogenesis of HIV associated
nephropathy (HIVAN), a collapsing focal segmental glomerulosclerosis with
microcystic dilatation of tubules, by Cheng et al. (2013a) in the HIV-1 transgenic
mouse. Thirteen miRNAs belonging to 11 miRNA families were found
downregulated in kidney sections. They were validated in vitro in HIV-1 transduced
human podocytes and a notable downregulation of miR-200b and c and miR-33
expression was found in podocytes. The authors concluded that these miRNAs
contribute to the development of the renal damage because miR-200 could inhibit
TGFβ- induced EMT at tubular level. Later, the same investigators (Cheng
et al. 2013b) evaluated the effect of rapamycin administration on miRNA expression
pattern in HIVAN mice. They observed an attenuation of renal lesions because
rapamycin reversed the expression of downregulated miRNAs, mainly those of
miR-200 family. In conclusion, this agent attenuates the renal cell EMT through
the modulation of miR-200 expression and these results could be taken into consid-
eration for improving the outcome of HIVAN.
MiRNAs in Kidney Transplantation
MiRNAs can participate to several physiopathological mechanisms that characterize

the kidney transplantation: the ischemia-reperfusion injury (IRI), the acute rejection
and the chronic allograft dysfunction (Table 1).
The IRI is one of the main causes of acute kidney injury (AKI) in kidney
transplantation. It is due to a decreased blood supply followed by a
re-establishment of the normal bloodstream that led to hypoxia, vascular dysfunction
and immune response activation. IRI have a major role in the development of
delayed graft function (DGF) following kidney transplantation (Jang et al. 2009;
Eltzschig and Eckle 2011).
One of the miRNAs more involved in the IRI is miR-21. It has been found
upregulated in murine kidney injury model, performed by unilateral ureteral obstruc-
tion, at 3 and 7 days after IRI. The ureteral obstruction caused a renal fibrosis
whereas the in vivo inhibition of miR-21 led to a decrease of expression of
TGF-β, α-SMA, PAI-1, col1A1, col1A2 and fibronectin (Godwin et al. 2010; Sha-
piro et al. 2011; Zarjou et al. 2011). Also the macrophage infiltration was reduced
and further miRNAs were found modulated in this process: miR-142–3p,
miR-142–5p, miR-214, miR-223, miR-101a, miR-193 and miR-218 (Shapiro
et al. 2011). Most of these miRNAs did not regulate processes involving lympho-
cytes since they are regulated also in immunodeficient mice (Godwin et al. 2010). In
addition to these miRNAs, also miR-155 and miR-18a have been found upregulated
in rat kidneys following IRI-induced tubular injury, even if in blood and urine they
resulted at the same time downregulated (Saikumar et al. 2012), suggesting a specific
pattern of expression, depending on the type of cells.
To understand how the miRNAs influence the injured human kidney, a prospec-
tive cohort study investigated zero-hour and protocol allograft biopsies from
Table 1 miRNAs involved in kidney transplantation

Pathology Modulated miRNA Involved pathway Expressed in
Ischemia- miR-142–3p, miR-142–5p, Modulation of TGF-β, Renal tissue/
reperfusion miR-214, miR-223, miR-101a, α-SMA, PAI-1, cells
injury miR-193, miR-218 miR-155 and collagens and
miR-18a fibronectin
Acute miR-10a, miR-10b and miR-210, / Urinary cell
rejection miR-142–5p, miR-155, pellets;
miR-223, miR-10b, miR-30a-3p PBMCs; renal
and let-7C, miR-10a, miR-10b tissue/cells
and miR-210
Chronic miR-204, miR-21, miR-142-5p, Smad /TGFB signalling Renal tissue;
allograft miR-142-3p, miR-506 miR-30b PBMCs
dysfunction and miR-30c
with IF/TA
Allograft miR-125b, miR-203miR-142- Regulation of Renal tissue
chronic 3p, miR-204, miR-211 inflammation and
dysfunction fibrosis development
CAMR with miR-142-5p Immune-regulation Renal tissue;
IF/TA PBMCs
AKI miR-182-5p and miR-21-3p Homeostasis of cells of Renal tissue
the immune system;
apoptosis and
proliferation
166 patients. Eight cases with AKI and ten matched allografts without pathology,
used as control group, were followed-up within the first 12 days after engraftment. In
these samples miRNA and mRNA profiles were analyzed and, following the base-
line adjustment for zero-hour biopsy expression levels, a specific molecular AKI
signature of 20 mRNAs and 2 miRNAs (miR-182-5p and miR-21-3p) were identi-
fied. These miRNAs could describe the evolution of events during acute injury. The
miR-182-5p seems be the main controller of the kidney tissue injury; it can be
activated by IL-2 and STAT5 and it inhibits FOXO1 expression that regulates
homeostasis of cells of the immune system such as T-cells, B-cells and neutrophils.
MiR-182-5p could regulate also molecular processes related to the AKI, as apoptosis
and proliferation (Wilflingseder et al. 2013, 2014).
The screening of miRNAs in acute rejection biopsies revealed three most impor-
tant miRNAs that were overexpressed (miR-142-5p, miR-155 and miR-223) and
three miRNAs downregulated (miR-10b, miR-30a-3p and let-7c). The three
miRNAs upregulated in the renal tissue were also highly expressed in PBMCs and
the stimulation with the mitogen phytohaemagglutinin led to increased levels of
miR-155 and lower levels of miR-223 and let-7c. Moreover, intragraft levels of
miR-142-5p or miR-155 can accurately predict the acute rejection (100 % sensitivity
and 95 % specificity) (Anglicheau et al. 2009). Therefore, these miRNAs could be
potentially considered as non-invasive diagnostic biomarkers of acute rejection.
In patients with acute rejection, the levels of miR-10a, miR-10b and miR-210
have been found modulated in urinary cell pellet. Specifically, miR-10a was
upregulated, whereas miR-10b and miR-210 were downregulated (Lorenzen

et al. 2011). However, the data on miR-210 are discordant considering another
study on the expression of miRNAs isolated from plasma of patients with AKI, in
which it was found upregulated (Lorenzen et al. 2011). This inconsistency can be
ascribed to the different type of samples or conditions.
A study of the miRNA signature in chronic allograft dysfunction has been
performed comparing patients with interstitial fibrosis (IF) and tubular atrophy
(TA) with patients with normal allografts and correlating miRNA profiles derived
from allograft biopsies with IF/TA with urinary profiles. Three miRNAs, miR-142-
3p, miR-204 and miR-21, were found differentially expressed in tissue and urine and
were confirmed in an independent set of samples (Scian et al. 2011). Another study
has been carried out on eight human kidney allograft biopsies, four IF/TA and four
normal biopsies, and confirmed on further ten IF/TA and eight normal samples.
Ben-Dov et al. (2012) found miR-21, miR-142-5p, miR-142-3p and miR-506
upregulated and miR-30b and miR-30c downregulated, compared with normal
kidneys.
Even if the studies on miRNAs expression in the renal tissue can be useful to
understand the process correlated to the rejection or fibrosis subsequent the trans-
plantation, they assume a greater clinical significance when are performed on body
fluids as plasma or urine because the investigated miRNAs could be used as
non-invasive biomarkers.
Several studies have investigated this issue. Twenty-two miRNAs have been
found able to discriminate between renal allograft recipients with chronic dysfunc-
tion and well-functioning controls. This study was performed on urinary cell pellets
on a total of 191 samples. The identified miRNAs are involved in the regulation of
inflammation and fibrosis development pathways and the panel of urine miRNAs, if
further validated, could be useful in allograft for monitoring graft function and for
the prediction of progression to chronic allograft dysfunction (Maluf et al. 2014).
Interestingly, the miR-142-5p, previously described as upregulated in allograft
biopsies with IF/TA (Ben-Dov et al. 2012) has been found overexpressed also in a
further study both in PBMCs and in renal tissue of patients with chronic antibody
mediated rejection (CAMR) as well as in a rodent model of CAMR. The miR-142-5p
was not modulated by immunosuppressive therapy, suggesting that its expression
was not induced by treatment. Moreover, when PBMCs of those patients were
activated with phytohemagglutinin A, the miR-142-5p expression decreased and
was not increased in the blood of patients with acute rejection. This miRNA may be
considered an excellent CAMR biomarker because it has been validated in PBMCs
of an independent cohort of patients discriminating those with CAMR (AUC = 0.74;
p = 0.0056) (Danger et al. 2013). Moreover, this miRNA modulates the expression
of genes belonging to the category of immune-regulation and could be used as a
marker of tolerance in B-cells of operationally tolerant patients (Danger et al. 2012).
Taking into account all these studies, we can conclude that the analysis of the
expression of certain miRNAs is a promising methodology to impact clinical
decisions. The miRNAs, in addition to provide further discernment into specific
pathophysiological mechanisms, are good candidate molecular biomarkers since
they are stable overtime and can be detected in body fluids and in sample sources
with degraded RNA.
Potential Applications to Prognosis and Other Diseases or

Conditions
Since some diseases are characterized by an abnormal miRNAs expression, the

identification of miRNA signature for specific disease will be helpful in early and
differential diagnosis. There are some studies that provide evidence on the potential
use of circulating miRNAs in different body fluids as biomarkers for disease state
and progression. The application of miRNAs in diagnosis and prognosis of disease
are described in detail below in paragraph “MiRNAs as diagnostic tools in blood and
urine.”
Moreover, miRNAs have some properties that make them useful as targets for
therapeutic applications. First, miRNA signature differentiates various disease;
this allow the identification of specific miRNAs that could be manipulated to
control gene regulation. Second, miRNAs, as small entities, are able to be
in vivo easily delivered. Finally, miRNAs have multiple gene targets, some of
that work simultaneously to control a common pathway or biological process.
However, this could also be a disadvantage due the “off-target” side effects. The
application of miRNAs in therapy and the description of different approaches
used are well explained below in paragraph “New approaches for miRNA
therapy.”
MiRNAs as Diagnostic Tools in Blood and Urine
In the last years, a particular attention was given to the identification of novel and
reliable biomarkers for renal diseases. Recent studies, mainly in the cancer field,
have provided evidence on the potential use of miRNAs as new diagnostic tool. As
described above, miRNA studies in renal diseases have demonstrated that miRNAs
have both an important role in the pathogenesis and in the diagnosis of many renal
diseases.
The first evidence regarding the extraction and determination of cell-free miRNA
content in body fluids was shown by Chen et al. (2008). Cell-free miRNAs in body
fluids are stable under not easy conditions including boiling, low/high pH, extended
storage, multiple freeze-thaw cycles. Moreover, they are resistant to endogenous
RNase for their small size and perhaps for packaging inside lipid or lipoprotein
complexes such as microvesicles/microparticles or exosomes (Mitchell et al. 2008).
It has been also hypothesized that circulating miRNAs have a role in cell-to-cell
communication; in fact, they could transport information from a donor to a recipient
cell. Instead, in urine, miRNAs may be filtered and excreted by, or directly from, the
kidney and/or urinary tract.
All these characteristics and the non-invasive nature make blood- and urine-
circulating miRNAs as ideal and potential biomarkers to detect or monitor various
human diseases.
Since the levels of circulating miRNAs are very low, the real-time RT-PCR is well
adapted for the analysis of circulating miRNA profiles for its sensitivity. However,
one of the major challenge in the analysis of circulating miRNAs is the suitable
method of normalization. To date, an established housekeeping gene to normalize
the expression of circulating miRNAs is lacking. Consequently, researchers have
proposed other methods of normalization. Some researchers have added spiked-in
control miRNAs during the RNA purification process in order to resolve differences
in recovery during the purification procedure and amplification efficiency.
Synthetic C. elegans miRNAs are usually used as spiked-in control miRNAs.
Other groups have used different miRNAs as normalizing controls such as miR-16
or miR-17. However, one needs to be mindful that miRNAs used as normalizers are
highly and equally expressed in the samples analyzed and they are stable in different
disease conditions. In fact, levels of miR-16 have been shown to be increased in
critical limb ischemia patients (Spinetti et al. 2013), miR-17 is reduced in patients
with systemic lupus erythematosus (Carlsen et al. 2013).
In conclusion, although circulating miRNAs potentially could be used as disease
biomarker, for their introduction in clinical practice, a method that replaces the time-
consuming RNA isolation, reverse transcription and quantitative PCR analysis is
needed. Moreover, currently studies published on circulating miRNAs in kidney
diseases have enrolled a low number of patients and the use of miRNAs as prog-
nostic biomarkers is still limited. Future studies involving larger cohorts of patients
and different ethnic groups will be useful.
New Approaches for miRNA Therapy
Therapeutic Approaches
MiRNA dysregulation occurs in many kidney diseases. The main therapeutic
approach is to normalize miRNA expression values in blood, kidney or urine.
There are two ways for modulating the dysregulated miRNAs: (i) inhibition of the
upregulated miRNAs; (ii) restoration of the activity in downregulated miRNAs
(Table 2).
(i) miRNA antagonists

First, the inhibition of miRNA activity can be obtained using small interfering
RNA (siRNA) against the sequential components of miRNA biogenesis that are
the two enzymes, Drosha and Dicer/DGCR8, and the RISC-miRNA complex.
The ablations of Dicer, Drosha or RISC in animal models have generated kidney
malformations or genetic diseases.
Second, the inhibition of upregulated miRNA activity can be obtained by
several methods like chemically modified antisense oligonucleotide inhibitors
(antagomirs or anti-miRs) or introducing a tandem miRNA-binding site repeats
Table 2 Strategies to MiRNA antagonists

modulate miRNAs in the
Small interfering RNAs (siRNAs)
clinic
Antagomirs/anti-miRs (ASO): 20 -O-methyl RNA (20 -O-Me)
20 -O-methoxyethyl (20 -MOE)
20 -fluoromodification
Sponge/decoy miRNAs locked-nucleic acid
Phosphorotionate linkage
MiRNA erasers
MiRNA target occupiers
MiRNA mimics
MiRNA sharing
Small hairpin RNAs (ShRNAs)
Scaffold miRNAs
Artificial miRNAs
(Sponge or decoy miRNAs). The antisense oligonucleotide (ASO) has a com-

plementary sequence to the mature miRNA thus it binds and sequesters the
cognate miRNA; this approach has been used in cell cultures for repressing the
mRNA gene target. The antagomirs may have different chemical modifications
such as 20 -O-methylRNA, 20 -fluomodification, LNA (locked-nucleic acid) mod-
ifications, phosphorotionate linkage. The LNA modifications provide long-term
and efficient suppression of miRNAs. MiRNA sponges consist of multiple
tandem binding sites into the 30 UTR of cognate endogenous miRNAs. Other
approaches are the miRNA erasers and the miRNA target occupiers.
(ii) miRNA mimics
The restoring of downregulated miRNAs can be obtained introducing miRNA
mimics that share the structure of miRNA or employing small hairpin RNA
(shRNA) or using miRNA scaffolds and artificial miRNAs.
MiRNAs as Potential Therapeutic Strategies

(i) Animal models
The ablation of Dicer or Drosha or the introduction of siRNA against these two
enzymes can cause death of animals or kidney malformations. This means that
miRNAs play an important role in embryo development and organ formation.
Details of these experimental studies can be found in the section “miRNAs and
kidney development.”
Transgenic mice with ablation of a miRNA gene or introduction of
overexpressed miRNA gene have been realized for studying the biological
properties of a cognate miRNA.
Manipulation of the downregulated miR-146a has been done in lupus-prone
BXSB mice by Pan et al. (2012) administering virus like particles (VLP) containing
miR146a. The 20-week-old BXSB mice showed an increase of the cognate miRNA
expression in PBMCs, kidney, spleen and lung associated with reduction of
anti-dsDNA antibodies and ANA. Furthermore, the investigators observed reduced

expression of inflammatory cytokine SLE-related (IFNα, IL-1β, IL-6). These
results suggest a therapeutic approach for mice predisposed to develop SLE.
Since miR-192 is overexpressed in streptozotocin (STZ)-induced diabetic
mice (type 1 Diabetic Nephropathy) and in diabetic db/db mice (type 2 DN),
Putta et al. (2012) evaluated the efficacy of the anti-miR-192 in C57BL/6
diabetic mice. They observed a decreased expression of extracellular matrix
associated with reduced expression of profibrotic genes (TGFβ, CTGF, fibro-
nectin, COL 1α2 and COL 4α1). Furthermore, they found reduced albuminuria
in diabetic mice.
Qin et al. (2011) administered a double-stranded miRNA mimics like
miR-29b in a rat model of obstructive nephropaty to block renal fibrosis. The
ultrasound-mediated gene delivery of miR-29b blocked the progression of renal
fibrosis by inhibiting the Smad 3/TGF-β pathway.
(ii) Human diseases
The use of miRNA blockers or enhancers in humans with non-renal diseases is
entering in a crucial phase of development and application. An extensive review
on this topic has recently been published by Van Rooij E and Kauppinen S
(2014). We have focused in this section only the recent progresses on the
therapeutic application of Miravisen in patients with hepatitis C virus (HCV)
infection because these individuals could potentially develop secondary
cryoglobulinemic glomerulonephritis. This locked nucleic acid-modified DNA
phosphorothioate antisense oligonucleotide sequesters the oligomeric miR-122-
HCV complex that protects the HCV genome from nucleolytic degradation.
Two drug companies, Santaris Pharma and Regulus Therapeutics, are cur-
rently moving to phase III studies with Miravisen in patients with HCV infection
after the recent published data of Janssen et al. (2013) obtained from the first
phase II study. Thirty-six patients received Miravisen subcutaneously at doses
of 3, 5 or 7 mg/kg or placebo for a total of 5 weeks. Treatment with this antimiR-
122 produced a dose-dependent and long-lasting anti-viral activity with reduc-
tion of HCV RNA levels. Remission was obtained in a large number of treated
patients with only rare mild side-effects. The therapeutic results are promising
but remain to be validated in large cohorts of patients.
Challenges and Future Perspectives
Despite the research on miRNAs in renal pathophysiology is very interesting, it is

highly challenging. Firstly, it is not completely understood the mechanism of regu-
lation of miRNA biogenesis. In fact a lot of miRNAs are located within the introns of
genes, but their expression regularly doesn’t correlate with that of host gene demon-
strating additional regulatory mechanisms after the miRNA transcription. For many
miRNAs several gene targets are unknown. Bioinformatic analyses have predicted
many thousands of target genes for each miRNA but only a small number of these has
been validated biologically. MiRNAs have a tissue and cell-specific expression,
but kidneys are constituted by different types of cells which could be diversely affected
in various renal diseases. So, to validate experimentally the role of a miRNA, it is
necessary to identify the cell types in which a miRNA is expressed in a particular
pathologic condition. As described in detail above, the main system to study experi-
mentally the link between a miRNA and a target gene is the transfection of miRNA
mimics and inhibitors. Sometimes cell lines are difficult to transfect and the introduced
oligonucleotides may have non-specific effect when used in the in vivo models.
Although several challenges exist in miRNA research, progress in technological
advances will help to overcome these difficulties and permit us to understand the
effect of miRNAs on pathogenesis of renal diseases. In the future, animal models
that enhance or silence a particular miRNA could provide the best model to study the
function of miRNAs. Moreover, the detection of circulating miRNAs could help the
clinicians in the diagnosis, prognosis and therapeutic response of kidney diseases.
Finally, the most attractive perspective is the use of miRNAs for the specific
treatment of kidney diseases even if the development of safe and reliable organ
and cell-specific delivery systems, the limitation of toxicity derived from off-target
effects and from the activation of immune response are needed.
Summary Points
• This chapter focuses on the role of microRNAs (miRNAs) in kidneys.

• MiRNAs are small RNA molecules that can regulate gene expression at post-
transcriptional level.
• MiRNAs play an important role in essential biological processes of renal
physiology.
• MiRNA dysregulations are involved in the pathogenesis of several kidney dis-
eases such as polycystic kidney disease, primary and secondary glomerulone-
phritides and renal transplantation.
• Thus, they represent an important tool for the diagnosis of renal diseases and
could be used as new targets for the treatment of kidney diseases.
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Serum Creatinine Trajectories in Kidney
Disease 8
Macaulay Onuigbo, Nneoma Agbasi, Ogonna Oguejiofor,
Emmanuel Okocha, Chinawaeze Aneke, and Charles Odenigbo
Contents
Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 140
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
Creatinine: Metabolism, Chemical Structure, and Excretion in the Urine . . . . . . . . . . . . . . . . . 142
Serum Creatinine Trajectories in Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Serum Creatinine Trajectories in Acute Kidney Injury (AKI): The Rainbow
Spectrum of Renal Outcomes Following AKI in CKD Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Serum Creatinine Trajectories in Adult Polycystic Kidney Disease (ADPKD):
An Nnamdi Azikiwe University Teaching Hospital, Nnewi, Nigeria Renal Clinic
Experience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Serum Creatinine Trajectories in Chronic Kidney Disease: The NKF KDOQI
CKD Staging Paradigm Revisited: CKD Prediction Is an Inexact Science: The Novel
Concept of CKD “Progressors” and CKD “Nonprogessors” . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
Two Divergent Patterns of CKD to ESRD Progression: The “Classic” Pattern and the
Pattern of the Syndrome of Rapid-Onset End-Stage Renal Disease (SORO-ESRD) . . . . . . 153
Late-Onset End-Stage Renal Failure from Angiotensin Blockade (LORFFAB) . . . . . . . . . . . 155
Serum Creatinine Trajectories in a Case of “Quadruple Whammy” . . . . . . . . . . . . . . . . . . . . . . . 157
Perioperative AKI and Concurrent Angiotensin Inhibition Alone: A Case
Presentation from Mayo Clinic Health System, Eau Claire, Wisconsin, USA . . . . . . . . . . . . . 158
M. Onuigbo (*)
Department of Medicine, College of Medicine, Mayo Clinic, Rochester, MN, USA
Department of Nephrology, Mayo Clinic Health System, Eau Claire, WI, USA
e-mail: onuigbo.macaulay@mayo.edu; monuigbo27@hotmail.com
N. Agbasi
North East London NHS Foundation Trust, Ilford, Essex, UK
e-mail: nnoms@aol.com
O. Oguejiofor • E. Okocha • C. Aneke • C. Odenigbo
Department of Medicine, Nnamdi Azikiwe Teaching Hospital, Awka, Nigeria
e-mail: cogobrus@yahoo.com; onyichideokocha@yahoo.com; anekejc@gmail.com;
codenigbo@hotmail.com

140 M. Onuigbo et al.
Serum Creatinine Trajectories in HIV-Associated Nephropathy: An Nnamdi

Azikiwe University Teaching Hospital, Nnewi, Nigeria, Renal Clinic Experience . . . . . . . . 160
Serum Creatinine Trajectories in Sickle Cell Disease Nephropathy: An Nnamdi Azikiwe
University Teaching Hospital, Nnewi, Nigeria, Experience . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
Conclusions: A Need for More Preventative Renal Medicine: Renoprevention Revisited.
The Introduction of the New Innovative CKD Express # IT Software . . . . . . . . . . . . . . . . . . . . . . . 163
Potential Applications of Serum Creatinine Trajectories in Kidney Disease Prognostication
and Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166
Abstract
Creatinine is the end product of the metabolism of creatine phosphate, a by-product
of skeletal muscle metabolism. It is excreted mainly via the kidneys, primarily by
glomerular filtration. It is therefore the most widely used blood assay to measure the
presence and progression of chronic kidney disease. The real-time monitoring of
serum creatinine translations at the individual patent level, the so-called serum
creatinine trajectories, offers a fascinating methodology of the study of kidney
function and disease. In this review, we have examined and analyzed the serum
creatinine trajectories in kidney disease, ranging from acute kidney injury (AKI) with
its multifarious rainbow spectrum of renal outcomes in AKI, through the titillating
vicissitudes of the different patterns of CKD to ESRD progression including a
description of the syndrome of rapid-onset end-stage renal disease (SORO-ESRD)
and the syndrome of late-onset renal failure from angiotensin blockade (LORFFAB)
to the serum creatinine trajectories of some specific renal syndromes including adult
polycystic kidney disease, HIV nephropathy, and sickle cell disease. These patients
represent representative cases of the named renal states as managed at the Renal Unit
of the Mayo Clinic Health System, Eau Claire, Northwestern Wisconsin, USA, and
the Renal Clinic of Nnamdi Azikiwe University Teaching Hospital, Nnewi, Nigeria.
We surmise that the study of individual patient-level serum creatinine trajectories, an
evolving area of current nephrology practice, can indeed provide additional diag-
nostic and prognostic insights in the management of the nephrology patient.
Keywords
Acute kidney injury (AKI) • Chronic kidney disease (CKD) • End-stage renal
disease (ESRD) • Late-onset renal failure from angiotensin blockade
(LORFFAB) • Renal replacement therapy (RRT) • Renoprevention • Serum
creatinine • Serum creatinine trajectories • Syndrome of rapid-onset end-stage
renal disease (SORO-ESRD)
Key Facts
• Serum creatinine is a measure of kidney function in patients with kidney disease.

• With kidney disease and the resulting failing kidney function, the serum creati-
nine concentration rises.
8 Serum Creatinine Trajectories in Kidney Disease 141
• The estimated glomerular filtration rate (eGFR) derived from serum creatinine
using prespecified formulas gives GFR values equivalent to specific serum
creatinine concentrations for a particular patient at any point in time and is
expressed in ml/min/1.73 m2 BSA.
• The graphical representation and analysis of the real-time translations in serum creat-
inine at the individual patient level constitute the science of serum creatinine trajectories.
• Serum creatinine trajectories can provide exciting and scintillating new insights
into renal disease diagnosis and prognostication.
• Angiotensin-converting enzyme inhibitors and angiotensin receptor blockers are
the most studied pharmaceutical agents which by blocking the renin angiotensin
aldosterone system are able to slow down the progression of kidney disease
especially in proteinuric CKD patients, diabetic and nondiabetic.
• There are recent reports of a potential nephrotoxic effect of angiotensin-converting
enzyme inhibitors or angiotensin receptor blockers especially at higher doses in
older (>65-year-old) later (>CKD III) patients, as typified in the newly described
syndrome of late-onset renal failure from angiotensin blockade (LORFFAB).
Definitions
Acute kidney injury (AKI) This is any disease state or condition that results in an
acute new-onset injury to the kidneys, usually leading to an increase in measured
serum creatinine concentration.
Chronic kidney disease (CKD) This describes a chronic and sometimes often
stable state of chronic disease affectation of the kidneys by medical conditions
such as hypertension and diabetes mellitus and is characteristically categorized
into five stages, CKD I, II, III, IV, and V, according to prespecified eGFR cutoff
ranges with CKD V being the worst group with eGFR of <15 ml/min/1.73 m2 BSA.
Creatinine This is the end product of skeletal muscle creatine phosphate metabo-
lism that enters the blood stream and is excreted from the body via the kidneys in the
urine; hence the serum concentration of creatinine is a measure of kidney function in
health and in disease.
End-stage renal disease (ESRD) This is the terminal state of usually irreversible
kidney disease resulting from disease conditions such as hypertension and diabetes
mellitus that require renal replacement therapy.
Renal replacement therapy (RRT) This refers to alternative lifesaving interven-

tions used to prolong life in patients with advanced kidney failure and include
hemodialysis, peritoneal dialysis, and kidney transplantation.
Renoprevention This is the practice of calculated and preemptive avoidance or

minimization of nephrotoxic exposure in CKD patients undergoing major surgery,
prior to iodinated contrast administration and during critical illness, and the agents to
avoid include nephrotoxic antimicrobials such as aminoglycosides, NSAIDs and cox
II inhibitors, diuretics, and angiotensin-converting enzyme inhibitors or angiotensin
receptor blockers (“triple whammy” medications).
Quadruple whammy This is the name of a newly described syndrome of postop-

erative AKI occurring in CKD patients on concurrent “triple whammy” medications.
Syndrome of rapid-onset end-stage renal disease (SORO-ESRD) This is a

newly described syndrome of rapid unanticipated acute-onset yet irreversible kidney
failure requiring renal replacement therapy indefinitely, following episodes of acute
kidney injury from medical and surgical factors.
Introduction
Creatinine: Metabolism, Chemical Structure, and Excretion

in the Urine
Creatinine is the end product of the breakdown of creatine phosphate in muscle

metabolism. It is usually produced at a fairly constant rate by the body, depending
on the individual’s muscle mass. It is produced via a biological system involving
creatine, phosphocreatine (also known as creatine phosphate), and adenosine triphos-
phate (ATP, the body’s immediate energy supply). Creatine is synthesized primarily in
the liver from the methylation of glycocyamine (guanidinoacetate, synthesized in the
kidney from the amino acids, arginine, and glycine) by S-adenosylmethionine. It is
then transported through the blood to the other organs, muscle, and the brain, where,
through phosphorylation, it becomes the high-energy compound phosphocreatine
(Taylor 1989). During the reaction, creatine and phosphocreatine are catalyzed by
creatine kinase, and a spontaneous nonenzymatic conversion process to creatinine may
occur (Allen 2012). Degradation consists of cyclization of creatine to form creatinine
(Wyss and Kaddurah-Daouk 2000). The degradation process is dependent on both pH
and temperature. If creatine is in the presence of basic solutions, it will remain as
creatine, if the temperature remains low (Wyss and Kaddurah-Daouk 2000). Con-
versely, if creatine is in the presence of an acidic solution with high temperatures, it
will be converted to creatinine. Creatinine is eventually excreted from the body
through the urine. It is removed from the blood chiefly by the kidneys, primarily by
glomerular filtration, but also by proximal tubular secretion. Little or no tubular
reabsorption of creatinine occurs. Thus, the level of the serum creatinine in a subject
is a general reflection of the level of kidney function. With kidney disease and loss of
nephrons, the level of serum creatinine would therefore show an upward trend.
Conversely, with improving kidney function, say following acute kidney injury
(AKI), the level of serum creatinine will then trend downwards. Accordingly, serum
creatinine is the most widely used assay to measure the presence and progression of
chronic kidney disease (Levey 1990).
Serum Creatinine Trajectories in Kidney Disease
Serum Creatinine Trajectories in Acute Kidney Injury (AKI): The

Rainbow Spectrum of Renal Outcomes Following AKI in CKD Patients
Whereas nephrologists and physicians are generally conversant with the common
diagnosis of acute kidney injury (AKI) in patients with chronic kidney disease
(CKD), the so-called phenomenon of acute-on-chronic renal disease (AKI-on-
CKD), nevertheless, the common consensus is that the impact of AKI on renal
function is usually short-lived and fleeting, with typical expected recovery of renal
function in most instances (Ponte et al. 2008; Wald et al. 2009; Ishani et al. 2009;
Onuigbo and Achebe 2013). Nonetheless, mutually anecdotal as well as objective
evidence in the nephrology literature support a contrarian notion that quite often,
much less renal recovery follows these AKI on CKD events (Onuigbo and Achebe
2013). Indeed, there is new and cumulative evidence in the AKI literature demon-
strating that AKI not only leads to and propagates CKD but that AKI could also
directly lead to irreversible ESRD and the need for permanent renal replacement
therapy, the so-called newly described syndrome of rapid-onset end-stage renal
disease (Onuigbo 2010).
The following are four case reports with representative graphs of serum creatinine
trajectories of patients seen and managed at the Renal Unit of the Mayo Clinic
Health System in Northwestern Wisconsin, USA, who typify the varying spectrum
of renal outcomes following AKI in patients with CKD.
Rapid and Full Recovery of Renal Function Following AKI on CKD

A 48-year-old obese hypertensive Caucasian male patient, with previously stable
stage II CKD, with a baseline serum creatinine of 1.2 mg/dL and estimated GFR
(eGFR) of >60 ml/min/1.73 m2 BSA in January 2013, developed AKI on CKD
following fever of unknown origin (FUO) complicating methicillin-resistant Staph-
ylococcus aureus (MRSA) bacteremia (Onuigbo and Achebe 2013). ACE inhibi-
tion with benazepril was discontinued due to AKI presentation, whereas amlodipine
was continued for hypertension control. He had iodinated contrast administration as
part of work-up of the bacteremia. He received multiple courses of different
parenteral antibiotics including nafcillin, vancomycin, and Cubicin, as prescribed
by Infectious Disease Consultation. A kidney biopsy revealed acute interstitial
nephritis, without evidence for contrast-induced nephropathy, nor glomerulone-
phritis. Serum creatinine peaked at 3.3 mg/dL (eGFR of 20 ml/min/1.73 m2 BSA)
(Fig. 1). He was treated conservatively and did not need renal replacement therapy
(RRT). He rapidly improved and exhibited full recovery of his renal function within
a month (Fig. 1).
Partial Recovery of Renal Function Following AKI on CKD

An 83-year-old white woman with hypertension and otherwise stable CKD III
underwent a right hemicolectomy procedure together with the resection of about
4 Creatinine
3
mg/dL
Generalized normal hight
1
Generalized normal low
0
13
13
13
13
13
13
13
13
13
13
20
20
20
20
20
20
20
20
20
20
6/
3/
0/
7/
3/
0/
7/
4/
3/
0/
/0
/1
/2
/2
/0
/1
/1
/2
/0
/1
01
01
01
01
02
02
02
02
03
03
Fig. 1 Serum creatinine trajectory showing rapid full recovery of renal function following AKI
on CKD
150 cm of her small intestine with end-to-end ileocolic anastomosis for colon cancer
in October 2011. This was complicated by diarrhea and dehydration. Furthermore,
there was the need for a second laparoscopic procedure in November 2011.
She experienced AKI in October 2011 following the initial surgical intervention.
She experienced yet a second AKI episode the following month in November 2011
from hypovolemic dehydration with peak creatinine values as shown in Fig. 2. She
partially recovered kidney function and since December 2011 has maintained a
higher new baseline serum creatinine. She otherwise remains asymptomatic and
continues to feel great at her current age of 85 years. Her previous baseline serum
creatinine was 1.2 mg/dL in January 2006, 1.3–1.4 mg/dL in 2010–2011 (CKD stage
III, eGFR approximately 36–38 mL/min per 1.73 m2 BSA). Between December
2011 and March 2014, the timeline for this review, she has maintained a new higher
but stable baseline serum creatinine of approximately 2.0–2.2 mg/dl (eGFR
20–25 mL/min per 1.73 m2 BSA), CKD stage IV (Fig. 2).
Rapid-Onset Yet Irreversible ESRD or the Syndrome of Rapid-Onset

ESRD (SORO-ESRD) Following AKI on CKD in a Patient with Native
Kidneys
An 81-year-old type 2 diabetic hypertensive Caucasian female patient on losartan
50 mg daily for hypertension control was evaluated for acute worsening dyspnea in
December 2004 (Onuigbo and Achebe 2013). This was associated with a large right-
sided pleural effusion. Critical Care Pulmonary Medicine consultation ordered a
contrast-enhanced chest CT examination to rule out pulmonary embolism. She did
not have pulmonary embolism. An echocardiogram showed stable left ventricular
ejection fraction of 45–50 %, unchanged from a previous examination completed
Fig. 2 Serum creatinine trajectory showing partial recovery of renal function following postop-
erative AKI on CKD in an 83-year-old Caucasian woman
earlier in April 2003. However, the patient very rapidly, within 24 h, following
iodinated contrast exposure, developed oliguric AKI on CKD. Serum creatinine had
more than doubled, from a baseline of 1.6 mg/dL, quickly up to 3.7 mg/dL, in
association with acutely worsening new-onset dipstick proteinuria. Serum creatinine
continued to rise with falling urine output mandating the initiation of RRT (Onuigbo
and Achebe 2013). She was started on hemodialysis, just 3 days following iodinated
contrast exposure. She remained oligoanuric and was on maintenance hemodialysis
up until October 2012, nearly 8 years later. She died in October 2012, at the age of
89 years, serum creatinine about 4 mg/dL, from hypotensive shock following
urosepsis, overall cachexia, and failure to thrive. This picture of precipitate acute
unanticipated but yet irreversible AKI resulting in ESRD needing permanent RRT
was termed the syndrome of rapid-onset ESRD (SORO-ESRD), a newly described
syndrome that we first reported in 2010 (Onuigbo 2010; Onuigbo et al. 2013d,
2014).
Rapid-Onset Yet Irreversible ESRD or the Syndrome of Rapid-Onset

ESRD (SORO-ESRD) Following AKI on CKD in a Renal Transplant
Recipient
A 53-year-old Caucasian female patient, with type 1 diabetes mellitus, hyperten-
sion, and diabetic gastroparesis, had received a simultaneous pancreas-kidney
transplantation (SPK) in 2000 for ESRD. She was on maintenance of immunosup-
pression with tacrolimus, mycophenolate mofetil, and prednisone. Through 2010,
she had maintained a baseline serum creatinine of 1.6–1.8 mg/dL, eGFR 34 ml/
min/1.73 m2 BSA, consistent with stable renal allograft stage III CKD (Onuigbo
EGFR
Transplant Pyelonephritis
40
Dehydration
Gastroenteritis
January 3, 2011
30
mL/min per 1.73 m2
SORO-ESRD
20
10
0
10
10
10
10
10
10
11
11
20
20
20
20
20
20
20
20
1/
1/
1/
9/
9/
8/
7/
0/
/0
/0
/3
/2
/2
/2
/2
/3
02
10
10
11
12
01
02
03
Fig. 3 eGFR trajectory in a renal transplant recipient demonstrating rapid-onset yet irreversible
ESRD following AKI on CKD requiring permanent RRT following transplant pyelonephritis and
concomitant dehydration
and Achebe 2013). In January 2011, she presented with symptomatic acute trans-
plant pyelonephritis from Escherichia coli, further complicated by dehydration
following 1 week of nausea, vomiting, and diarrhea (Onuigbo 2013d; 2014). She
quickly developed worsening AKI on CKD with worsening oliguria. Serum creat-
inine quickly increased within days to 5.16 mg/dL (Fig. 3). She soon needed the
initiation of RRT for progressive oliguric AKI with anorexia and volume overload.
Emergent RRT was started as hemodialysis via a tunneled central dialysis catheter
on January 8, 2011. She was then referred to Mayo Clinic, Rochester, for continued
care. Renal allograft biopsy, carried out the following week at Mayo Clinic,
Rochester, revealed acute tubular necrosis and chronic transplant glomerulopathy,
but without rejection (Onuigbo 2013; Onuigbo et al. 2014). She remained on
maintenance outpatient in-center, three times weekly, hemodialysis, for oliguric
irreversible ESRD, for 1 year. In January 2012, exactly 1 year since the AKI on
CKD event which led to the rapid-onset ESRD or SORO-ESRD, she received a
second living-related renal allograft from her then 32-year-old son, again at Mayo
Clinic, Rochester.
Remarkably, throughout all of these events, her pancreas allograft, part of the
SPK from 2000, has nevertheless remained perfectly functional. She has continued
to maintain excellent renal allograft function, with current baseline serum creatinine
in May 2014 of 0.88 mg/dL, eGFR >60 ml/min/1.73 m2 BSA and a current A1c of
4.8 %. Again, this picture of precipitate acute unanticipated but yet irreversible AKI
resulting in ESRD needing permanent RRT was termed the syndrome of rapid-onset
ESRD (SORO-ESRD), a newly described syndrome that we first reported in 2010
(Onuigbo 2010; Onuigbo et al. 2013, 2014).
Serum Creatinine Trajectories in Adult Polycystic Kidney Disease

(ADPKD): An Nnamdi Azikiwe University Teaching Hospital, Nnewi,
Nigeria Renal Clinic Experience
Case I
A 50-year-old obese Nigerian female patient, with a 4-year history of uncontrolled
hypertension, on four different antihypertensive drugs, was evaluated in the Renal
Clinic of Nnamdi Azikiwe University Teaching Hospital, Nnewi, Nigeria, in July
2008, following the development of early morning facial puffiness. Her initial serum
creatinine was 1.6 mg/dL, eGFR of 46 ml/min/1.73 m2 BSA, CKD stage III. There
was a strong family history of hypertension and an older brother had died of a
hemorrhagic stroke. She had hepatomegaly of 8 cm. Both kidneys were ballottable.
Chest radiograph showed features of hypertensive heart disease, EKG revealed left
ventricular hypertrophy, and abdominal ultrasound demonstrated enlarged kidneys
with multiple cysts as well as hepatic cysts. A brain CT scan was normal. There was 2+
proteinuria. A diagnosis of autosomal dominant polycystic kidney disease (ADPKD)
was made. Her antihypertensive drugs were adjusted to include lisinopril 20 mg daily.
Serum creatinine remained stable. After 13 months follow-up, in August 2009, she
was admitted with complicated acute pyelonephritis. Urine culture showed coliform
organisms, sensitive to ciprofloxacin. She experienced AKI with serum creatinine
peaking at 5.2 mg/dL (Fig. 4). She improved and was discharged after 6 days. Kidney
function improved and serum creatinine decreased to 3.0 mg/dL a month later (Fig. 4).
Serum creatinine trajectory in a 50-yo female with ADPKD over 54 months

9
7
Serum creatinine (mg/dL)
0
0 200 400 600 800 1000 1200 1400
Time (Days)
Fig. 4 Serum creatinine trajectory in a 50-year-old female with ADPKD over 54 months
Afterward, for almost a year, serum creatinine remained stable at 3.0–3.2 mg/dL,
equivalent to eGFR of 21 ml/min/1.73 m2 BSA, consistent with otherwise stable CKD
IV (Fig. 4). Nevertheless, for unknown reasons, in late 2010, she developed progres-
sively worsening azotemia and anemia, without clinical evidence of fluid overload.
Serum creatinine had risen to 8.2 mg/dL by January 2012 (Fig. 4). She started renal
replacement therapy in January 2012, but died after just 6 months on hemodialysis.
Case II
A 62-year-old grandmother presented to the Renal Clinic, Nnamdi Azikiwe Univer-
sity Teaching Hospital, Nnewi, Nigeria, in February 2014, with a history of loin pain
and gross hematuria following a fall while on a visit to her daughter’s house. She had
been hypertensive for the preceding 6 years and had been compliant with her
antihypertensive medications. In previous months, prior to her presentation, her
blood pressure had been poorly controlled despite continuing her usual antihyper-
tensive agents. Family history of hypertension was present in both parents. She had
tender ballotable kidneys but no other palpable abdominal organs. Chest radiograph
was normal, but EKG showed changes of the left ventricular hypertrophy. Serum
creatinine was normal, at 0.8 mg/dL, eGFR of 92 ml/min/1.73 m2 BSA, consistent
with CKD stage I. Renal ultrasound showed bilateral multiple cysts in both kidneys.
The liver, pancreas, and other abdominal organs were otherwise normal. Again, a
diagnosis of ADPKD was made. She was admitted for bed rest, analgesics, and
intravenous fluids. She made an uneventful recovery and has since continued her
outpatient visits and her serum creatinine has remained otherwise stable (Fig. 5).
Autosomal dominant polycystic kidney disease is the most common inherited renal
cystic disease. It occurs worldwide, in all races, but appears less common in black
individuals (Yersin et al. 1997). Although, it has been suggested that ADPKD is rare in
Africans, the reality is that the paucity of reports from Africa is most likely attributable
to a low index of suspicion and inadequate diagnosis (Fary Ka et al. 2010). The first
sonographic family study of ADPKD in a Nigerian family was described in 1991,
following the introduction of the first functional ultrasound unit in Southeastern
Nigeria in Enugu (Onuigbo et al. 1991). This was the first Nigerian publication on a
family study of ADPKD that emphasized the need and foundational relevance of
ultrasonographic family screening to increase the premorbid diagnosis of ADPKD
among Nigerians (Onuigbo et al. 1991). With the increasing availability of ultrasound
and other imaging methods, more cases are clearly now being recognized in Africa. A
recent report from Ilorin in Northern Nigeria showed that 8 % of renal cases visiting
the renal clinics over a 10-year period have ADPKD (Chijioke et al. 2010).
Hypertension occurs in about 50 % of young adults with ADPKD and normal
renal function. Its frequency increases as renal function deteriorates and almost
approaches 100 % of patients at ESRD (Kelleher et al. 2004). The development of
renal failure is highly variable. In most patients renal function is maintained within
normal range, despite relentless cyst growth until the fourth to sixth decade of life.
By the time decline in renal function begins, cyst growth is extensive with little
recognizable renal parenchyma. When it starts, the rate of decline of renal function is
about 4.4–5.9 ml/min/year (Klahr et al. 1995). Risk factors for decline in renal
function include PKD1 gene, male sex, black race, first episode of hematuria before
30 years, onset of hypertension before 35 years, hyperlipidemia, low HDL concen-
tration, and sickle cell trait (Johnson and Gabow 1997).
The first case of ADPKD presented in this review of serum creatinine trajectories
in kidney disease clearly demonstrated the various vicissitudes of serum creatinine
translations in CKD patients (Fig. 4) (Onuigbo and Agbasi 2014). Earlier on, the
patient was able to maintain a stable CKD stage III status, with serum creatinine of
about 1.6 mg/dL, eGFR of 46 ml/min/1.73 m2 BSA. However, following compli-
cated acute coliform pyelonephritis, she experienced AKI on CKD, serum creatinine
peaked at 5.2 mg/dL. After antibiotic therapy, she had experienced partial recovery
from the AKI episode, with a new albeit higher baseline serum creatinine of 3.0 mg/
dL (Onuigbo and Agbasi 2014) (Fig. 4). Nevertheless, for almost a year following
this, she maintained stable serum creatinine of 3.0–3.2 mg/dL, eGFR 21 ml/min/
1.73 m2 BSA, consistent with otherwise stable CKD IV (Onuigbo and Agbasi 2014).
However, after mid-2010, for unclear reasons, she now developed slowly but
relentlessly progressive kidney failure, inexorably reaching ESRD and the need for
RRT in January 2012, with serum creatinine reaching 8.2 mg/dL (Fig. 4).
The second case of ADPKD, on the other hand, despite several years of hyper-
tension, on antihypertensive agents, often uncontrolled, and despite an episode of
gross hematuria following a fall in February 2014, had continued to maintain stable
excellent renal function, in November 2014, with a serum creatinine of 0.86 mg/dL,
eGFR of 92 ml/min/1.73 m2 BSA, CKD stage I (Fig. 5).
Serum creatinine trajectory in a 63-yo grandmother

with ADPKD over 9 months
1
0.9
0.8
0.7
0.6
0.5
0 50 100 150 200 250 300
Time (Days)
Fig. 5 Serum creatinine trajectory in a 63-year-old female with ADPKD over 9 months
Serum Creatinine Trajectories in Chronic Kidney Disease: The NKF

KDOQI CKD Staging Paradigm Revisited: CKD Prediction Is an Inexact
Science: The Novel Concept of CKD “Progressors” and CKD
“Nonprogessors”
The National Kidney Foundation Kidney Disease Outcomes Quality Initiative (NKF
KDOQI) expert committee in 2002 instituted and established new guidelines that
established a novel chronic kidney disease (CKD) staging paradigm (NKF K/DOQI
2002; Levey et al. 2003). In this CKD prototype model, using ranges of prespecified
estimated glomerular filtration rates (eGFR), CKD was characterized into five stages: I,
II, III, IV, and V (NKF K/DOQI 2002; Levey et al. 2003). Clearly, the principle behind
this NKF K/DOQI CKD staging archetype solely rests on the absolute assumption that
serum creatinine and eGFR trajectories in CKD patients generally follow a linear,
predictable, smoothly progressive, and time-dependent curve to advance through the
increasing CKD stages I through V before inexorably reaching ESRD and the need for
renal replacement therapy (RRT) (NKF K/DOQI 2002; Levey et al. 2003; Chiu
et al. 2008; Onuigbo and Agbasi 2014). Nonetheless, it must be recognized that such
proposition of predictable, linear, time-dependent progressive step-wise decline in
kidney function, with mathematically linear falling eGFR over time, and with eGFR
methodically marching through these incremental projected CKD stages I through V,
and finally inexorably ending in symptomatic ESRD and the need for RRT, is
unproven, untested, and potentially flawed (Ballardie et al. 1983; Walser et al. 1989;
Shah and Levey 1992; Onuigbo 2009a, 2013; Onuigbo et al. 2013; Onuigbo and
Agbasi 2014, 32). Moreover, a 2011 Canadian retrospective analysis, which analyzed
the longitudinal changes during a 1.1-year observation period of eGFR and CKD
stages, demonstrated CKD stage variability (defined by changes in CKD stages)
among 1262 patients, mean age 71.25 years, drawn from two large Canadian renal
clinics (Sikaneta et al. 2012). This study reported that CKD stage changed in 40 % of
the cohort (including 7.4 % in whom CKD stage improved), whereas CKD stage
remained static in 762 (60.4 %) patients, the majority of this CKD cohort (Sikaneta
et al. 2012). Another earlier Canadian study examined 4231 CKD IV patients charac-
terized by an index eGFR of <30 mL/min per 1.73 m2 BSA, with at least three
subsequent eGFR values available for analysis, and no <4 months of follow-up
between January 2000 and January 2004 (Levin et al. 2008). The conclusion was that
the clinical course of patients with CKD stage 4 was unpredictably variable (Levin
et al. 2008). Furthermore, a 2012 retrospective report from South Korea examined
347 CKD III patients, enrolled between January 1997 and December 1999, who were
followed up through June 2010, a period of 10 years (Baek et al. 2012). One hundred
and sixty-seven patients (48.1 %) did not progress, 60 (17.3 %) progressed to stage
4, and 120 (34.6 %) progressed to stage 5, with 91 (26.2 %) starting dialysis (Baek
et al. 2012). Besides, recently, French investigators examined 406 patients in the
NephroTest cohort with measured glomerular filtration rates (mGFRs) measured by
51Cr-EDTA clearance at least three times during at least 2 years of follow-up (Weis
et al. 2013). The individual examination of mGFR trajectories by four independent
nephrologists classified patients as “improvers,” defined as those showing a sustained
mGFR increase, or “nonimprovers” (Weis et al. 2013). Measured GFR improved over
time in 62 patients (15.3 %). Their median mGFR slope was + 1.88 (IQR, 1.38, 3.55)
mL/min per year; it was 22.23 (23.9, 20.91) for the 332 “nonimprovers.” The conclu-
sion from this French study was that GFR improvement is possible in CKD patients at
any CKD stage through stages 4–5 (Weis et al. 2013). In a previous 2013 review and in
a more recent wide-ranging review article published in 2014, we have exhaustively
reexamined these phenomena regarding CKD behavior and again proposed the nomen-
clature of CKD “progressors” and CKD “nonprogressors” (Onuigbo et al. 2013;
Onuigbo and Agbasi 2014).
We shall now describe some selected CKD patients seen and managed at the
Mayo Clinic Health System Renal Unit in Northwestern Wisconsin who showed
stable and unchanged kidney functional states at CKD stages III, IV, and V, respec-
tively, over several years with no perceptible changes in eGFR, despite the advanced
ages of the majority of these patients, mostly >75 years of age, the so-called CKD
nonprogressors (Onuigbo et al. 2013; Onuigbo and Agbasi 2014).
Stable CKD V Over 7 Years in a Now 78-Year-Old Caucasian Hypertensive

Diabetic Male
As at February 2014, a now 78 year-old obese hypertensive type II diabetic white
male has over the last 7 years, between 2006 and 2013, despite a serum creatinine of
4.5–5.5 mg/dL, eGFR 8–11 mL/min per 1.73 m2 BSA, stage V CKD, remained
otherwise asymptomatic (Fig. 6) (Onuigbo and Agbasi 2014). He has continued for
the past 4 years on alternate monthly courses of prophylactic short-duration oral
Creatinine
6
4
mg/dL
2
Generalized normal high
1
Generalized normal low
0
01/01/2009 01/25/2010 02/18/2011 03/14/2012 04/07/2013 05/01/2014
Fig. 6 Serum creatinine trajectory in a now 78-year-old Caucasian hypertensive diabetic male with
stable CKD V between 2006 and 2013, serum creatinine of 4.5–5.5 mg/dL, eGFR 8–11 mL/min per
1.73 m2 BSA
levofloxacin for recurrent UTI prophylaxis. He has not progressed any further in the
last 7 years and has not needed renal replacement therapy.
Stable CKD IV Over 8 Years in a Now 78-Year-Old Caucasian

Hypertensive Diabetic Male
A then 69-year-old Caucasian male was diagnosed with Wegener’s
granulomatosis complicated by AKI on CKD in 2005 (Fig. 7). Serum creatinine
then had increased from 2.0 mg/dL to 3.5–4.0 mg/dL (Onuigbo and Agbasi
2014). The Wegener’s granulomatosis was treated with standard immunosup-
pressive therapy using prednisone. He had remained in remission on low-dose
prednisone since 2006, albeit with a new higher baseline serum creatinine of
3.5–4.0 mg/dL, eGFR 16–22 mL/min per 1.73 m2 BSA, stage IV CKD. He, now
aged 78 years in 2014, has remained otherwise an asymptomatic CKD IV patient
for 8 years (Fig. 7).
Stable CKD III Over Nearly 10 Years in a Now 87-Year-Old Caucasian

Hypertensive Male
An 87-year-old obese Caucasian hypertensive male patient, with a history of a
cerebrovascular accident following right carotid surgery in 2001, has generally
maintained a baseline serum creatinine of 1.6–2.0 mg/dL, eGFR 31–40 ml/min/
1.73 m2 BSA, CKD stage III, between October 2005 and September 2014, the only
period that we have available serum creatinine values.
Creatinine
5
4
mg/dL
1
05
06
07
08
09
11
12
3
01
01
20
20
20
20
20
20
0
/2
/2
/2
1/
5/
8/
4/
7/
6/
1
2
/0
/2
/1
/1
/0
/0
/2
/1
/1
01
01
02
03
04
05
05
06
07
Fig. 7 Serum creatinine trajectory in a now 78-year-old Caucasian male with Wegener’s
granulomatosis in remission, otherwise stable CKD IV between 2006 and 2014, serum creatinine
of 3.5–4.0 mg/dL, eGFR 16–22 mL/min per 1.73 m2 BSA
Two Divergent Patterns of CKD to ESRD Progression: The “Classic”

Pattern and the Pattern of the Syndrome of Rapid-Onset End-Stage
Renal Disease (SORO-ESRD)
Generally, it is a commonly held consensus opinion among practicing nephrologists,

in particular, and physicians, in general, that the propagation of CKD to ESRD is that
of a predictable, linear, progressive, relentless, time-dependent, and knowable
decline in renal function, with predictably increasing serum creatinine or falling
eGFR, leading inexorably to ESRD and the need for RRT (Rutherford 1977; NKF
K/DOQI 2002; Levey et al. 2003; Chiu et al. 2008). This universally accepted
paradigm of CKD-ESRD progression will be referred to in this review as the
“classic” pattern of CKD to ESRD progression (Onuigbo and Agbasi 2014). Nev-
ertheless, various observations and reports in the nephrology literature, to the
contrary, have demonstrated that quite often, the path from a priori stable CKD to
irreversible ESRD can be precipitate, acute, and unpredictable (Merino et al. 1975;
Bonomini et al. 1984; Bhandari and Turney 1996; Firth 1996; Onuigbo 2009, 2010,
2013; Onuigbo and Onuigbo 2011, 2012; Onuigbo et al. 2013, 2014). In 2010, we
first described the previously unrecognized syndrome of rapid-onset end-stage renal
disease or SORO-ESRD in the journal, Renal Failure (Onuigbo 2010). SORO-
ESRD, the syndrome of rapid-onset ESRD, is the unpredictable, unanticipated,
and accelerated progression from a priori stable CKD to irreversible ESRD, requir-
ing permanent RRT, following a new episode of AKI precipitated by antecedent new
medical/surgical events, with the interval between AKI and the need for RRT
represented by a period of often <2 weeks, usually measured only in days following
surgically induced AKI (Onuigbo 2010, 2013; Onuigbo et al. 2014; Onuigbo and
Agbasi 2014).
In the following section, we would present selected case reports demonstrating
the features of “classic” CKD-ESRD progression pattern and the CKD-ESRD
progression pattern of the syndrome of rapid-onset ESRD.
“Classic” Pattern of CKD-ESRD Progression in a Hypertensive

Caucasian Male
A 52-year-old Caucasian hypertensive male with low ejection fraction ischemic
cardiomyopathy, and hypothyroidism, had developed progressively worsening
renal failure with predictable linear increases in serum creatinine after November
2007 when serum creatinine was 1.9 mg/dL through to December 2010 when serum
creatinine exceeded 7.0 mg/dL, and he developed features of uremia and started
renal replacement therapy in the form of in-center outpatient hemodialysis (Onuigbo
and Agbasi 2014) (Fig. 8). He has continued on maintenance hemodialysis through
March 2014, when this review was completed.
“Classic” Pattern of CKD-ESRD Progression in a Caucasian Hypertensive

Diabetic Male
An 82-year-old Caucasian man with past medical history for hypertension, low
ejection fraction ischemic cardiomyopathy, type II diabetes mellitus, coronary
Fig. 8 Serum creatinine trajectory in a 52-year-old Caucasian hypertensive male who developed
predictable linear and progressive time-dependent CKD to ESRD, 2007–2010, and has remained on
maintenance hemodialysis 2010–2014
artery disease, obesity, and hypothyroidism developed progressively worsening

renal failure with a time-dependent predictable linear increase in serum creatinine
from May 2006. Serum creatinine was 2.6 mg/dL in May 2006 and progressively
increased through to January 2010 when serum creatinine exceeded 6.0 mg/dL and
he had then developed features of uremia. He therefore initiated renal replacement
therapy in the form of in-center outpatient hemodialysis in January 2010. He
however died in early 2014, while still on maintenance hemodialysis, from failure
to thrive. Regrettably, there were no laboratory data for serum creatinine available
for this patient before May 2006.
Pattern of Syndrome of Rapid-Onset ESRD in a Caucasian Hypertensive

Diabetic Male
A 73-year-old obese hypertensive type II diabetic male patient with a stable
baseline serum creatinine of approximately 1.7 mg/dL, eGFR 43 ml/min/1.73 m2
BSA, stable CKD stage III, between 2010 and 2012, on concurrent ACE inhibition
with lisinopril 40 mg daily, was admitted to the coronary care unit in February
2012 with acute decompensated heart failure (Onuigbo and Agbasi 2014). Acute
coronary syndrome was ruled out by investigation, and the patient subsequently
underwent minimally invasive aortic valve replacement for symptomatic aortic
stenosis. A 25 mm St. Jude Epic Stented Tissue Valve was deployed on March
2, 2012, by his cardiothoracic surgeon. He rapidly developed postoperative AKI on
CKD and required hemodialysis on the first postoperative day with worsening
oliguria and associated severe volume overload (Fig. 9). He never recovered any
kidney function, and he has since then remained on outpatient in-center mainte-
nance, three times weekly, hemodialysis for ESRD, over two and half years later in
November 2014. His current serum creatinine in November 2014 is 9.88 mg/dL
(Fig. 9).
Late-Onset End-Stage Renal Failure from Angiotensin Blockade

(LORFFAB)
We described, for the first time in 2005, the syndrome of late-onset renal failure from
angiotensin blockade (LORFFAB) (Onuigbo and Onuigbo 2005, 2008). This is
defined as the accelerated but potentially reversible iatrogenic renal failure from
concurrent angiotensin blockade, which occurs in usually older CKD patients,
despite normal renal arteries, absent traditionally acknowledged precipitating risk
factors, and while remaining on the same dose of angiotensin blockade during the
preceding 3 months or greater (Onuigbo and Onuigbo 2005, 2008; Onuigbo and
Achebe 2013).
Fig. 9 Serum creatinine trajectory in a 75-year-old Caucasian obese hypertensive diabetic male
who developed acute unpredictable yet irreversible AKI needing RRT consistent with the syndrome
of rapid-onset ESRD (SORO-ESRD), following minimally invasive aortic valve replacement in
March 2012; he has remained on maintenance hemodialysis through November 2014
The following case presentation is an illustration of the impact of LORFFAB on

serum creatinine trajectories in CKD patients.
In April 2004, a 77-year-old diabetic hypertensive Caucasian male patient
was admitted to our Renal Unit in Northwestern Wisconsin with symptomatic
oliguric renal failure, serum creatinine of 4.6 mg/dL, eGFR of 13 ml/min/
1.73 m2 BSA, associated with dyspnea and volume overload (Onuigbo and
Onuigbo 2008). He needed the initiation of emergent hemodialysis with ultra-
filtration via a dialysis catheter. Lisinopril, 20 mg daily, which he had received
for 21 months prior to presentation, was promptly discontinued. Amlodipine,
5 mg BID, was substituted for antihypertensive therapy. He continued on
maintenance hemodialysis for 10.5 months and his serum creatinine improved
slowly with increasing urine output (Fig. 10). By February 2005, his serum
creatinine had stabilized at about 3 mg/dL and hemodialysis was discontinued
(Fig. 10). He remained hemodialysis independent for almost 2 years. Unfortu-
nately, in January 2007, at the age of 79 years, still off hemodialysis, with
otherwise stable CKD IV, he suffered a heart attack and underwent a cardiac
catheterization, followed by a four-vessel coronary artery bypass graft proce-
dure. As a result of worsening postoperative AKI, he again needed hemodialysis
postoperatively (Fig. 10). He subsequently was transferred out to Minneapolis,
MN, for cardiac rehabilitation and was lost to our follow-up (Onuigbo and
Achebe 2013).
LVEF 55%-60%
70 LVEF 50%
LVEF 55%
HD started April 2004 LVEF 50%
60
ACEI Stopped
eGFR (ml/min/1.73 sq m BSA)
Stopped HD
50
Palmoeary edema Feb 2005
Volume overload
January 2007
Aoocexia
40 Acute MI
Cardiac cath
4-vessel CABG
30
20
10 Lisinopril 20 mg QD
Captopril 50 mg TID
HD started again
May 1999 Jan 2007
0
0 20 40 60 80 100 120
eGFR in patient with progressive renal failure while on ACE inhibitors (LORFFAB) despite normal EF and normal MRA
Months
Fig. 10 Serum creatinine trajectory in the then 77-year-old diabetic hypertensive Caucasian male
patient with features of LORFFAB who in 2004 required temporary hemodialysis for 11 months,
was hemodialysis independent 2005–2007 following discontinuation of lisinopril, but developed
postoperative AKI again requiring RRT in January 2007 following cardiac catheterization and four-
vessel coronary artery bypass graft procedure
Serum Creatinine Trajectories in a Case of “Quadruple Whammy”
We recently described a new syndrome of accelerated postoperative AKI on CKD

occurring in previously stable CKD patients who were concurrently at the time of the
surgery on triple whammy medications of angiotensin blockade, a diuretic and an
NSAID or a COX-2 inhibitor (Onuigbo and Onuigbo 2013; Onuigbo et al. 2013c;
Onuigbo and Agbasi 2014). We present in the following section a representative case
report.
In the Spring of 2013, our nephrology service was consulted on a 46-year-old
morbidly obese (170 Kg) hypertensive Caucasian male patient, current smoker, with
previously otherwise stable stage III CKD, serum creatinine of 1.21 mg/dL, eGFR of
70 ml/min/1.73 m2 BSA. He had developed accelerated AKI on CKD following an
elective right hip arthroplasty for symptomatic degenerative joint disease (Onuigbo
and Onuigbo 2013; Onuigbo and Agbasi 2014) (Fig. 11). He was on lisinopril 40 mg
daily and hydrochlorothiazide 25 mg daily. Incidentally, the patient also received a
preoperative dose of celecoxib (Celebrex ®) per orthopedic unit “operative analgesia
protocol,” a COX-2 inhibitor, thus completing the “triple whammy circle” (Onuigbo
and Onuigbo 2013). Within 36 h, postoperatively, he quickly developed oliguric
AKI on CKD, complicated by metabolic acidosis. Serum creatinine had more than
doubled to 2.58 mg/dL, eGFR of 28 ml/min/1.73 m2 BSA (Fig. 11). Notably, there
was evidence of significant intraoperative as well as postoperative hypotension,
despite several liters of infused normal saline (Fig. 11). Lisinopril and
Creatinine
3.0
2.5
2.0
mg/dL
1.5
Generalized Normal
High
1.0
Generalized Normal
Low
0.5
0.0
11/01/2012 11/30/2012 12/30/2012 01/29/2013 02/28/2013 03/31/2013
Fig. 11 Serum creatinine trajectory showing rapidly rising serum creatinine within 36 h in CKD III
patient on “triple whammy” medications following elective right hip arthroplasty procedure
consistent with the “quadruple whammy” syndrome
hydrochlorothiazide were promptly discontinued. He also developed postoperative

anemia (hemoglobin down to 10.4 g/L from 15.8 g/L). He received more intravenous
normal saline for hypotension and was started on intravenous furosemide due to
persistent oliguria. Subsequently, urine output improved and serum creatinine started
to fall (Fig. 11). He improved and was soon discharged from the hospital, on
postoperative day 3 on amlodipine 10 mg daily and furosemide 40 mg daily for
hypertension. Serum creatinine, from March 25, 2013, approximately 1 month
following the hip arthroplasty, was 0.99 mg/dL, lower than his premorbid levels,
with an improved eGFR of 85 ml/min/1.73 m2 BSA (Fig. 11).
Recently, in a 2014 publication in the journal, Nigerian Journal of Clinical
Practice, we elaborated in great detail the typical presentations of patients who
experience this newly described syndrome of “quadruple whammy” (Onuigbo and
Agbasi 2014). We indeed further hypothesized on a plausible role of concomitant
obesity in the pathogenesis of this syndrome (Onuigbo and Agbasi 2014). For
purposes of limiting AKI in CKD patients, more so in the perioperative period, we
once again call on physicians, in general, and nephrologists, in particular, to pre-
emptively discontinue “triple whammy” medications before any major surgical
interventions (Onuigbo 2009, 2013; Onuigbo and Agbasi 2014). This is one of the
cardinal principles of our “Renoprevention” mantra (Onuigbo 2009, 2013; Onuigbo
and Agbasi 2014).
Perioperative AKI and Concurrent Angiotensin Inhibition

Alone: A Case Presentation from Mayo Clinic Health System,
Eau Claire, Wisconsin, USA
The role of concurrent angiotensin inhibition alone, without the involvement of

the combination “triple whammy” medications in exacerbating postoperative
AKI, continues to generate significant controversy in the nephrology literature
(Onuigbo 2011, 2013, 2014; Nielson et al. 2014). However, it would appear that on
the balance, accruing evidence favors the notion that more so with prevalent
intraoperative hypotension, that concurrent angiotensin inhibition results in higher
rates of postoperative AKI when compared to situations with its preemptive
withdrawal preoperatively (Onuigbo 2011, 2013, 2014; Nielson et al. 2014). The
later approach of a preoperative preemptive temporary withdrawal of angiotensin
inhibition especially in the older (>65-year-old and later stage CKD) patients is
indeed one of the cornerstones of our newly introduced concept of
“Renoprevention” (Onuigbo 2009, 2013; Onuigbo and Agbasi 2014). We now
report here a case of postoperative AKI apparently exacerbated by concurrent
angiotensin inhibition.
A 57-year-old obese hypertensive type II diabetic Caucasian male patient
underwent an elective pulmonary vein isolation and ablation procedure for symp-
tomatic atrial fibrillation in early July 2014. Concurrent outpatient oral medica-
tions included chlorthalidone, metformin 1 g BID, and lisinopril 40 mg daily.
Postoperative AKI triggered a nephrology consultation. Baseline serum creatinine
was 1.0 mg/dL, GFR 81 ml/min/1.73 m2 BSA, CKD stage II. On postoperative day
1, serum creatinine had quickly increased to 1.96 mg/dL (Fig. 12a). At the time of
the nephrology consultation the following day, on postoperative day 1, he was
normotensive, and all available medical floor blood pressure recordings before and
after the procedure were noted to be normal. We had been informed that the
procedure in the operating room “went well without complications.” However,
our urgent and meticulous review and analysis of the operating room intraoperative
anesthesia records revealed significant hypotension during the over 4-h surgical
procedure (Fig. 12b). He was nonoliguric and otherwise asymptomatic except for
mild lightheadedness. The patient was therefore managed conservatively.
Lisinopril and metformin were promptly discontinued. Kidney function subse-
quently quickly improved. A few days later, with improved and stable kidney
function, he was placed back on the lisinopril, at discharge. His subsequent serum
creatinine in late July 2014 was 0.91 mg/dL, eGFR of 91 mL/min/1.73 m2 BSA
(Fig. 12a).
a SERUM CREATININE
2.5
1.5
mg/dL
0.5
0
0 50 100 150 200 250
Days
b Systolic BP following induction of anesthesia
200
Blood pressure (mm Hg)
150
100
50
0
0 50 100 150 200
Minutes
Fig. 12 (a) Serum creatinine trajectory 2 weeks following elective ablation procedure for symp-
tomatic atrial fibrillation with complete recovery from AKI. (b) Intraoperative systolic blood
pressure changes following induction of anesthesia during elective ablation procedure for symp-
tomatic atrial fibrillation
Serum Creatinine Trajectories in HIV-Associated Nephropathy: An

Nnamdi Azikiwe University Teaching Hospital, Nnewi, Nigeria, Renal
Clinic Experience
HIV-infected individuals present with a variety of renal syndromes manifesting as

acute renal failure or chronic renal failure (Weiner et al. 2003). HIV-associated
nephropathy (HIVAN) is diagnosed to be present when a seropositive person pre-
sents with persistent proteinuria, progressively worsening azotemia with preserva-
tion of normal kidney size (or even enlarged kidneys), and renal histological findings
of focal segmental glomerulosclerosis on renal biopsy. These subjects usually have
relatively normal blood pressure and often exhibit rapid progression to renal failure
and end-stage renal disease.
Among the various glomerulopathies documented in HIV or AIDS subjects, the
commonest is the classic HIV-associated nephropathy with focal glomerulosclerosis
(Klotman 1999). This occurs in 3–10 % of HIV-infected persons with high preva-
lence reported in black males with low socioeconomic status (Winston et al. 1998). It
is also the third leading cause of ESRD among African Americans aged 20–64 years
(United States Renal Data System USRDS, 1992–1999).
In Nigeria, the prevalence of HIV has risen exponentially from <1 % to 5.8 %,
values from different communities and different states ranging from 2 % to 15 %
(National AIDS/HIV/STD Control Program, Technical Reports 1997–2003), with
HIVAN contributing significantly to the overall burden and magnitude of CKD and
ESRD in Nigeria. Studies from Nigeria have reported high prevalence rates of renal
function impairment of 53.3 % in South-South Nigeria (Okafor et al. 2011) and
52.0 % in North-Central Nigeria (Agaba et al. 2003). Another study from 2008
demonstrated the occurrence of elevated serum creatinine in a high proportion of
HIV-infected Nigerian patients (Emem et al. 2008). The presentation of two HIV
AIDS patients managed for HIVAN at Nnamdi Azikiwe University Teaching Hos-
pital, Nnewi, Nigeria, is presented below to document the patterns of serum creat-
inine trajectories of such subjects in Nnewi, in Southeastern Nigeria.
Case I
A 25-year-old male Nigerian trader resident in Aba, Abia State, Southeastern
Nigeria, presented to the Medical Outpatient Department (MOPD) of Nnamdi
Azikiwe Teaching Hospital, Nnewi, Nigeria, in September 2013 with 2-month
history of shortness of breath, intermittent leg swelling associated with facial
puffiness, and diarrhea. He was recently diagnosed with HIV disease as the
index illness and had been commenced on highly active antiretroviral therapy
(HAART) (Abacavir, Lamivudine and Efavirenz) for about 2 weeks prior to
presentation, on account of very low CD4 count of five cells/mm3. He was
chronically ill-looking and pale. Pulse was 100/min and blood pressure was
140/80 mmHg. He was dehydrated and was managed as a case of HIVAN with
gastroenteritis. Pertinent test results included elevated serum creatinine, 5.9 mg/
dL, metabolic acidosis with bicarbonate level of 10 mmol/L, normokalemia at
3.8 mEq/L, and albumin 26 g/dL. He demonstrated 2+ proteinuria and severe
a 7
Serum creatinine trajectory in HIVAN with AKI
6
5
0
0 2 4 6 8 10 12 14
Admission Days
b Serum creatinine trajectory in CKD from HIVAN

10
9
8
7
6
5
4
3
2
1
0
0 200 400 600 800 1000
Time (Days)
Fig. 13 (a) Serum creatinine trajectory in a 25-year-old Nigerian male patient who developed AKI
with HIVAN and died 16 days into hospital admission. (b) Serum creatinine in HIV-associated CKD
on conservative management with HAART
anemia, with a hematocrit of 18 %. HIV serology was positive, but serology tests
for hepatitis B and C were nonreactive. He was transfused with five units of blood
but developed seizures 13 days into his admission. He died on the 16th day. Kidney
function tests had however started to improve prior to the patient’s death on
conservative management (Fig. 13a).
Case II
A 26-year-old Nigerian female student from Anambra State, Southeastern Nigeria,
presented to the MOPD of Nnamdi Azikiwe Teaching Hospital, Nnewi, Nigeria, in
September 2010 with 6 weeks’ history of generalized body swelling and fatigue of
4 weeks’ duration, with associated decrease in urine output and increased frothi-
ness of urine. She also had dyspnea, orthopnea, and paroxysmal nocturnal dys-
pnea. She had been diagnosed with HIV disease 4 years previously and was
commenced on HAART (zidovudine, lamivudine, and nevirapine) shortly after
the diagnosis. She is not diabetic nor hypertensive. Examination showed
respiratory distress, pallor, and bilateral pitting edema. Pulse was 84/min and blood
pressure was 110/60 mmHg. There was a pansystolic murmur loudest at the apex,
fine bibasal crepitations, and a tender hepatomegaly, measured 4 cm below the
right costal margin, together with ascites. Serum creatinine was 8.7 mg/dL, potas-
sium 3.9 mEq/L, bicarbonate 14 mEq/L, and hematocrit of only 18 %. She was
managed conservatively for chronic kidney disease secondary to HIVAN and
anemic heart failure. She responded to conservative management at the MOPD
after initial discharge. Her serum creatinine trajectory between August 2010 and
February 2014 is shown above (Fig. 13b). She has not yet required renal replace-
ment therapy.
The first patient with HIVAN developed symptomatic AKI while on HAART and
died 13 days into the admission despite improving renal function (Fig. 13a). The
second patient has features of CKD V secondary to HIVAN and on HAART
(Fig. 13b). Despite the elevated serum creatinine values, she has continued to remain
otherwise asymptomatic on conservative medical management without requiring
renal replacement therapy (Onuigbo 2013; Onuigbo and Agbasi 2014).
Serum Creatinine Trajectories in Sickle Cell Disease Nephropathy: An

Nnamdi Azikiwe University Teaching Hospital, Nnewi, Nigeria,
Experience
A 26-year-old Nigerian male patient with sickle cell anemia (HbSS) was well
until 24 months before presentation to Nnamdi Azikiwe University Teaching
Hospital, Nnewi, Nigeria, in February 2013, when he woke up with
lightheadedness and dyspnea. Prior to this, he had experienced frequent epi-
sodes of bone pain crises for which he was in and out of hospital admissions
and needed regular use of opiates and other analgesics for pain control. He had
earlier been placed on hydroxyurea, over a 3-month period for management of
recurrent bone pain crises. Further evaluation revealed severe hypertension for
which he was placed on nifedipine. Laboratory work-up revealed severe anemia,
proteinuria, and azotemia, serum creatinine 4.9 mg/dL, eGFR 13 ml/min/1.73 m2
BSA, consistent with CKD V. A diagnosis of sickle cell nephropathy was conse-
quently made and he was then referred to the renal clinic. Following conservative
management including transfusion of blood for symptomatic anemia and improved
hypertension control, his CKD status improved, without a need for renal replace-
ment therapy. The trajectory of his serum creatinine over a period of 21 months is
shown in Fig. 14 above.
Sickle cell nephropathy encompasses the spectrum of morphologic, laboratory,
and clinical changes due to kidney pathology associated with sickle cell disease
(SCD). It specifically includes papillary necrosis, hyposthenuria, impaired renal
acidification, proteinuria, hematuria, supranormal proximal tubular function, and
renal failure (Stuart and Nagel 2004). It is a major risk factor for early mortality in
SCD (Platt et al. 1994), hence its importance in patient evaluation and management.
The magnitude of sickle cell nephropathy in Nigeria had been emphasized in
Fig. 14 Serum creatinine trajectory in a 26-year-old Nigerian male with sickle cell nephropathy
and CKD IV–V, over 21 months, 2013–2014
previous reports where up to 47 %, 69.4 %, and 37.2 % of patients with SCD had
chronic kidney disease (CKD), respectively (Aneke et al. 2014; Bolarinwa
et al. 2012; Arogundade et al. 2011). Similarly, the median age of onset of significant
renal impairment in SCD was 23.1 years, while the median age at the time of death
was 27 years (Powars et al. 1991).
Serum creatinine measurement and urinalysis for proteinuria are important
laboratory work-up for patients with sickle cell nephropathy; indeed they are
frontline investigations in our facility for patients with this condition. Our index
patient presented with biochemical evidence of nephropathy (proteinuria and
deranged serum creatinine) in addition to severe anemia. Here again, a
patient with SCD and sickle cell nephropathy has exhibited often unpredictable
CKD stage changes during his conservative management (Onuigbo and Agbasi
2014).
Conclusions: A Need for More Preventative Renal Medicine:

Renoprevention Revisited. The Introduction of the New
Innovative CKD Express # IT Software
In 2002, the National Kidney Foundation established a novel chronic kidney disease
(CKD) staging paradigm (NKF K/DOQI 2002; Levey et al. 2003). In 2012, the
authoritative United States Preventive Task Force questioned the validity of asymp-
tomatic CKD screening (Moyer 2012). The American Society of Nephrology and the
American College of Physicians have opposite recommendations regarding this
controversy (ASN 2013; ACP 2013). Moreover, CKD prediction and prognostica-
tion is clearly an inexact science (Onuigbo and Agbasi 2014, 32). CKD care must
therefore be individualized. We recently developed a new IT software, the CKD
Express #, currently in US Patent Application (Onuigbo 2012; Onuigbo et al. 2013;
Onuigbo and Agbasi 2014). The incorporation of this yet to be patented IT software
into existing electronic medical record (EMR) systems would go a long way in
bridging this yawning gap in our knowledge and understanding of CKD initiation,
propagation, and progression.
Preventative medicine is a neglected art in modern-day practice of medicine.
Prevention is always better than cure. According to Yach and Calitz, the greatest
increase in healthcare spending between 2000 and 2011 was attributable to drugs,
medical devices, and hospital care, with the cost of treating noncommunicable
diseases (NCDs) estimated to exceed 80 % of annual healthcare expenditure,
whereas 3 % was spent on public health and disease prevention programs. The
National Institutes of Health estimates that 20 % of its $30 billion annual budget is
allocated to prevention; however, <10 % is spent on human behavioral interventions
that target the major modifiable risk factors. More investment in prevention science
could lead to greater health gains at lower cost (Yach and Calitz 2014). We support
such reengineering of nephrology practice whereby a lot more emphasis would now
be placed on preventative nephrology (Onuigbo 2009, 2013; Onuigbo and
Agbasi 2014).
The role of perioperative, and more so intraoperative hypotension, in the patho-
genesis of postoperative AKI has been variously described. Despite the intensive use
of vasopressors including multiple infusions of intravenous phenylephrine during
the ablation procedure, our patient clearly experienced significant intraoperative
hypotension. Without accessing intraoperative anesthesia records to demonstrate
significant hypotension, the cause of the AKI would have remained speculative.
Furthermore, the fact that the patient had remained on concurrent angiotensin
inhibition up until the first postoperative day could possibly have contributed to
the severity of postoperative AKI in our patient (Onuigbo 2011, 2014 (2); Nielson
et al. 2014). Nielson et al. (2014) in a study of 1,154 surgical patients demonstrated
that surgical candidates who receive preoperative angiotensin blockade have an
associated increased risk of post-induction hypotension and postoperative AKI
resulting in a greater hospital length of stay. From a perspective of
“Renoprevention,” we again posit here that such patients should have the angioten-
sin blockade preemptively withheld 3–5 days prior to the surgery and angiotensin
blockade can be restarted postoperatively if kidney function remained stable
(Onuigbo 2009, 2011, 2013, 2014 (2); Onuigbo and Agbasi 2014). Furthermore,
more efforts must be applied in the operating room to maintain more normal blood
pressures during surgical procedures. This way, we would prevent more postopera-
tive AKI events, reduce hospital length of stay, and help save scarce US healthcare
dollars (Onuigbo 2013).
Finally, we must note here that two recent large multicenter randomized clinical
trials failed to demonstrate any therapeutic benefits of perioperative use of vasodi-
lators, aspirin, clonidine, or fenoldopam to mitigate against noncardiac postoperative
AKI (Bove et al. 2014; Garg et al. 2014; Winkelmayer and Finkel 2014). We
strongly advocate and submit that our suggested approach, heretofore, to reduce
intraoperative hypotension while at the same time limiting exposure to potential
nephrotoxics including angiotensin inhibition, would be far cheaper and more
effective in limiting postoperative AKI in our CKD patients than any esoteric
pharmaceutical intervention (Jo et al. 2014; Onuigbo 2009, 2011, 2013, 2014;
Nielson et al. 2014; Onuigbo and Agbasi 2014).
Potential Applications of Serum Creatinine Trajectories in Kidney

Disease Prognostication and Management
In this review of serum creatinine trajectories in kidney disease, we have examined

and reported on the multifarious behavior of serum creatinine in acute kidney injury,
in CKD to ESRD progression, in otherwise nonprogressive CKD, in late-onset renal
failure from angiotensin blockade (LORFFAB), in other specifically named renal
syndromes, and in postoperative acute kidney injury. Clearly, there is a broad
spectrum of renal outcomes following acute kidney injury, and indeed CKD in all
its stages can be “progressors” vs. “nonprogressors” or “nonimprovers”
vs. “improvers” (Onuigbo and Agbasi 2014).
From this analysis of the graphical representation and analysis of real-time
translations in serum creatinine trajectories at the individual patient level, the
science of serum creatinine trajectories, we submit that the science of serum
creatinine trajectories is a neglected paradigm of modern nephrology practice
(Onuigbo and Agbasi 2014). The study of individual patient-level serum creatinine
trajectories, whether real-time, concurrent, or in retrospective analysis, can indeed
provide exciting and sometimes scintillating new insights in our understanding of
kidney disease diagnosis and prognostication (Onuigbo and Agbasi 2014). We, at
the Renal Unit of the Mayo Clinic Health System, in Northwestern Wisconsin,
USA, have extensively and consistently enabled the utility of this science of
individual patient-level serum creatinine trajectories in the definition of two
newly described renal syndromes, the syndrome of late-onset renal failure from
angiotensin blockade (LORFFAB) in 2005 and the syndrome of rapid-onset
end-stage renal disease (SORO-ESRD) in 2010 (Onuigbo and Onuigbo 2005;
Onuigbo 2010).
Summary Points
• The analysis of serum creatinine trajectories at the individual patient level pro-
vides additional diagnostic and prognostic insights in patient care.
• We have described the different varied patterns of renal outcomes and the
resulting serum creatinine trajectories in acute kidney injury (AKI).
• We also established that CKD staging and prognostication are an inexact science
and that the commonly applied 2002 NKF K/DOQI CKD staging paradigms must
be seen as what they are – clinical guidelines, only.
• We further described the serum creatinine trajectory patterns in CKD to ESRD
progression including the syndrome of rapid-onset end-stage renal disease
(SORO-ESRD).
• Additionally, we demonstrated the patterns of serum creatinine trajectories in

patients with some specific renal syndromes including autosomal dominant
polycystic kidney disease (ADPKD).
• We recommend an increasing utilization of this often-neglected area of nephrol-
ogy practice, especially for individualized CKD care, including CKD prognosti-
cation at the individual patient level.
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Random Spot Urine Markers for Kidney
and Their Applications 9
Maria Guedes-Marques, Carlos Botelho, Pedro Maia,
Teresa Mendes, and Armando Carreira
Contents
Key Facts of Urinalysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
The Specimen for Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176
Physical Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Color . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Odor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Turbidity or Clarity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 177
Relative Density . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
Chemical Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179
Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 180
pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181
Bilirubin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Urobilinogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182
Ketones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Nitrites and Leukocyte Esterase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184
Albuminuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186
Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Uric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
Electrolytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192
Sodium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Potassium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Phosphate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Others . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193
Microscopic Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194
Erythrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
M. Guedes-Marques (*) • C. Botelho • P. Maia • T. Mendes • A. Carreira

Nephrology Department, Centro Hospitalar e Universitário de Coimbra – Hospital dos Covões,
Coimbra, Portugal
e-mail: mariaguedesmarques@gmail.com; mpekenita@hotmail.com; medmigui@hotmail.com;
pmai@sapo.pt; mariateresa.cpfm@gmail.com; armando.carreira@sapo.pt

172 M. Guedes-Marques et al.
Leukocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 195
Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Casts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Crystals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196
Organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Potential Applications to Prognosis, Other Diseases, or Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . 199
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200
Abstract
Over the few last decades, several additional urinary biomarkers have been
correlated to other organ or system abnormalities other than kidney disease. In
this setting, abnormal findings on a routine urinalysis, often in an otherwise
asymptomatic patient, may be the first evidence of underlying kidney disease
and even other diseases or conditions like a higher cardiovascular risk set.
A 24-h urine evaluation is still considered the gold standard method for the
quantification of important urinary biomarkers like proteinuria. Nevertheless, its
collection is laborious and entails significant errors that could compromise the
accuracy of this method. Therefore, random spot urine assessment was developed
to estimate quantitative measurements of 24-h collections. Many trials have been
conducted to determine which formulae (correction for other parameters) and
methods (including voiding of the day or technical procedures) are better to
minimize sources of false results and enhance correlation with the gold standard.
Currently, random spot urine examination is already mentioned in some interna-
tional guidelines as an alternative analysis to diagnose and monitor several
diseases. It allows the identification of multiple markers, which can be organized
into three groups, according to their laboratory method assessment: physical,
chemical, and microscopic characters. However, evidence regarding its role and
power is still not unanimous, at least in some diseases, and future trials to prove
how best to apply it are needed.
This review outlines random spot urine biomarkers for the kidney and their
applications in clinical practice.
Keywords
Urinary biomarker • Kidney disease • Cardiovascular disease • Random spot
urine • Urinalysis • Proteinuria • Hematuria • Sediment
Abbreviations
ATN Acute tubular necrosis
Ca Calcium
CuSO4 Copper sulfate
9 Random Spot Urine Markers for Kidney and Their Applications 173
DBDH Diisopropylbenzene dihydroperoxide

DIDNTB bis(30 ,300 -Diiodo-40 ,400 -dihydroxy-50 ,500 -dinitrophenyl)-3,4,5,6-
tetrabromo-sulfonephthalein
eAER Estimated albumin excretion rate
FCU Fractional renal clearance of urate
INTERSALT International study of electrolyte excretion and blood pressure
IRMA-2 Irbesartan in Patients with Type 2 Diabetes and Microalbuminuria
K Potassium
KDIGO Kidney Disease: Improving Global Outcomes
LE Leukocyte esterase
LN Lupus nephritis
MESNA Mercaptoethane sulfonate sodium
Mg Magnesium
Na Sodium
P/C Protein/creatinine ratio
Ph Phosphate
PREVEND Prevention of Renal and Vascular End-Stage Disease
RBCs Red blood cells
SG Specific gravity
SGLT2 Sodium/glucose cotransporter 2
SLC5A2 Solute carrier family 5
SNP Single-nucleotide polymorphisms
SSA Sulfosalicylic acid
STENO-2 Effect of a Multifactorial Intervention on Mortality in Type
2 Diabetes
TMB 3,30 ,5,50 -Tetramethylbenzidine
UACR Urinary albumin/protein ratio
UTI Urinary tract infection
WBCs White blood cells
Zn Zinc
Key Facts of Urinalysis
• Among other characteristics, an ideal biomarker should be easily accessible and

noninvasive, which could be achieved with urinalysis.
• Although the 24-h urine evaluation is the gold standard for most quantitative
parameters, the random spot urinalysis may give a result that correlates with this
technique over a wide range of measurements.
• Random spot sample repeated measurements can be easily obtained to ascertain
disease activity and response to treatment. However, the results can be seriously
affected by several variables that depend on the patient and the technique itself, so
these must be regularly confirmed.
• Several formulae and methods have successfully been developed to improve
acuity of measurements in a random spot urine sample.
• Currently, guidelines already recommend protein or albumin/creatinine ratio in a
random spot evaluation as a first option to monitor diseases like chronic kidney
disease and lupus nephritis.
• Despite these recommendations, evidence on reliability is not unanimous in all
diseases (e.g., lupus nephritis), so it should be applied carefully, especially if
therapeutic changes are about to be made.
Definitions
Biomarker The Oxford dictionary describes a biomarker as “a measurable sub-

stance in an organism whose presence is indicative of some phenomenon such as
disease, infection, or environmental exposure.”
Chronic kidney disease Kidney damage or an estimated glomerular filtration rate

below 60 mL/min/1.73 m2 persisting for 3 months or more, irrespective of the cause.
Diabetes Group of metabolic diseases characterized by hyperglycemia resulting

from defects in insulin secretion, insulin action, or both. Chronic hyperglycemia is
associated with long-term damage, dysfunction, and failure of various organs,
especially the eyes, kidneys, nerves, heart, and blood vessels.
Dipstick A thin, plastic stick with strips of chemicals on it, which is placed in urine
to detect abnormalities. The chemical strips change color if certain substances are
present or if their levels are above normal.
Glomerulonephritis Disease that primarily affects the glomerulus. It has clinical

presentations that vary from the asymptomatic individual who is found to have
hypertension, edema, hematuria, or proteinuria at a routine medical checkup to a
patient who has fulminant disease, with acute kidney injury, possibly associated with
life-threatening extrarenal disease.
Lupus nephritis Immune complex glomerulonephritis that is a common and seri-

ous feature of systemic lupus erythematosus.
Mercaptoethane sulfonate sodium (MESNA) A sulfhydryl compound that is

used to reduce the incidence of hemorrhagic cystitis associated with certain chemo-
therapeutic agents, like cyclophosphamide.
Random spot urine Occasional, one-off, urine collection.
Systemic lupus erythematosus Multisystem, autoimmune disease, whose diagno-

sis demands a combination of clinical and laboratory criteria defined by the Amer-
ican College of Rheumatology.
Sodium/glucose cotransporter 2 (SLGT2) Protein encoded by the SLC5A2 gene

and member of the sodium glucose cotransporter family involved in glucose
reabsorption in the kidney.
Solute carrier family 5 (SLC5A2) Gene that encodes the SGLT2 protein.
Urocrit Similarly to the hematocrit word, it has been used to define the propor-
tion of the urine that is composed of red blood cells. It is measured by centrifu-
gation technique and is used when erythrocyturia is too high to count by
microscopy.
Introduction
Urinalysis has historically been defined as a test to evaluate kidney and urinary tract
disease. However, during the last few decades, several additional urinary biomarkers
have been correlated to other organ or system abnormalities other than kidney disease.
It is well known that an ideal biomarker should be easily accessible and nonin-
vasive, among other characteristics. This profile perfectly matches a random spot
urine marker because patients with nephropathy who often progress to end-stage
renal disease (ESRD) and depend on hemodialysis treatment will require a vascular
access (ideally, arteriovenous fistula or graft) whose correct function depends on
good vessel conditions, for which venous punctures to collect blood samples are
harmful and should be avoided. In this setting, abnormal findings on a urinalysis may
be the first evidence of underlying kidney disease and may be a suitable test for
monitoring several conditions, thereby minimizing the number of blood tests.
Twenty-four-hour urine samples used to be the only reliable samples for most of
the quantitative analysis. Nevertheless, collecting them is laborious and unfeasible
for many population groups, like young working adults with hard schedules and
multiple workplaces, elderly people with memory problems and urinary inconti-
nence, as well as children. In these cases, random spot urine assessment has been
successfully used. Although this method may be limited in some quantitative
parameters and more vulnerable to factors related to the patient’s lifestyle, some
correcting formulae have been developed to achieve quantitative results that are
highly correlated with timed specimens. This finding supports some current guide-
lines that already recommend random spot urinalysis instead of the 24-h specimen
for screening, diagnosis, and monitoring of several diseases like diabetes or systemic
lupus erythematosus (SLE) (Engelgau et al. 2000; Hahn et al. 2012).
Random spot urine markers can be organized into three groups, according to their
laboratory method assessment: physical, chemical, and microscopic markers. Phys-
ical markers include color, odor, clarity, and specific gravity (SG). Chemical exam-
ination includes the identification of protein, blood, glucose, pH, bilirubin,
urobilinogen, ketones, nitrites, and leukocyte esterase (LE). Finally, microscopic
evaluation entails the detection of crystals, cells, casts, and organisms. Chemical
results are the most often widely used, probably because of their (semi)quantitative
presentation and possible correlation with quantitative timed collections, but phys-
ical and microscopic ones may play a key role in many diagnoses and monitoring.
This review outlines random spot urine biomarkers for the kidney and their
applications in both research and clinical practice.
The Specimen for Analysis
Urine collection requires standard handling guidelines to minimize unwanted

sources of variability that could compromise the acuity of test results. Good practice
prevents potential sources of error; thus, patient preparation and clear information is
essential for a correct collection (Strasinger and Di Lorenzo 2014). Both the suit-
ability of a specimen and the rejection criteria must be determined. Following
collection, specimens should be delivered and tested within 2 h because changes
in urine composition start immediately after the voiding (Strasinger and Di Lorenzo
2014). Bacteria may proliferate and alter pH, casts may dissolve, and crystals may be
lost. Refrigeration may cause precipitation of orange red crystals of uric acid, which
can be redissolved by rewarming the urine (Strasinger and Di Lorenzo 2014).
Additionally, urine analysis can be seriously affected by other variables that depend
on the patient, the techniques, and the medical team.
Although this type of specimen is the easiest and most convenient for the patient
because it can be collected at any time, some basic recommendations should be
followed. Vigorous physical exercise should be avoided for at least 24 h before the
collection to minimize exercise-induced proteinuria, hematuria, or cylindruria (John-
son et al. 2014). Women should avoid collection during menstruation because of
blood contamination. A midstream clean-catch specimen provides a suitable spec-
imen for routine urinalysis and bacterial culture because it is less contaminated by
epithelial cells and bacteria and, therefore, is more representative of the actual urine
(Strasinger and Di Lorenzo 2014).
Regarding collection schedule, the first morning void is the most ideal to prevent
false-negative pregnancy tests and to evaluate orthostatic proteinuria, because it is a
more concentrated specimen, thereby assuring the detection of lower levels of
substances otherwise missed in a more diluted specimen (Strasinger and Di Lorenzo
2014). On the other hand, lysis of cells and casts may occur in the bladder overnight,
which may lead to false-negative results. In these cases, a second morning urine is
more appropriate.
Finally, before reviewing each biomarker analysis and applications, it should be

remembered that urine normally contains 95 % water and 5 % solutes. As stated above,
considerable variations in these solute concentrations can occur in healthy people
owing to the influence of normal biological factors, but they could also be markers of
pathogenic processes or pharmacological responses to a therapeutic intervention.
Physical Markers
All routine urinalysis should begin with a physical examination of the sample which
includes description of color, odor, clarity, and SG.
Color
Normal urine ranges from pale to dark yellow, which is mainly due to the urochrome
pigment, a product of endogenous metabolism, but also to other chemical concen-
trations, and pH. Changes in color can be caused by physical activity, food, drugs, or
pathological conditions. The most frequent pathological conditions that can cause
color changes of the urine are gross hematuria, hemoglobinuria, or myoglobinuria
(pink, red, brown, or black); bilirubinuria (dark yellow to brown); and urinary
infections (dark yellow, white, green, or even purple, according to germs). Less
frequent causes include uric acid crystalluria (pink) and porphyrinuria and
alkaptonuria (red, turning black on standing). The main drugs responsible for
abnormal urine color are rifampin, phenazopyridine, and phenindione (yellow
orange); desferrioxamine (pinkish); phenytoin (red); chloroquine and nitrofurantoin
(brown); amitriptyline, triamterene, propofol, and blue dyes of enteral feeds (green);
methylene blue (blue); and metronidazole, methyldopa, phenol derivatives, argyrol,
and imipenem–cilastatin (darkening on standing). Some examples among food
include beetroot (red), senna and rhubarb (yellow to brown or red), and carotene
(brown) (Strasinger and Di Lorenzo 2014; Johnson et al. 2014).
Odor
A change in urine odor may be caused by the ingestion of some food, urinary tract
infection (UTI), maple syrup urine disease, phenylketonuria, isovaleric academia,
and hypermethioninemia.
Turbidity or Clarity
The transparency or turbidity of a urine specimen is determined by visually exam-

ining the mixed specimen in a clear container while holding it in front of a light
source. Freshly voided normal urine is usually clear, particularly if it is a midstream
clean-catch specimen.
Urine turbidity can have multiple causes. The most frequent non-pathological
causes include squamous epithelial cells, mucous, amorphous phosphates, carbon-
ates, urates, semen, fecal contamination, radiographical contrast media, talcum
powder, and vaginal creams. On the other hand, the most common pathological
causes are red blood cells (RBCs), white blood cells (WBCs), bacteria,
non-squamous epithelial cells, yeast, abnormal crystals, lymph fluid, and lipids.
Clear urine is not always normal; thus, chemical analysis will increase the acuity of
physical markers in detecting certain abnormalities (Strasinger and Di Lorenzo 2014).
Relative Density
The evaluation of urine concentration is made by measuring the SG, which correlates
with urine osmolality, rising by approximately 0.001 for every 35–40 mosmol/kg
increase in osmolality. Thus, a urine osmolality of 280 mosmol/kg (isosmotic to
plasma) is usually associated with a urine SG of 1.008 or 1.009 (Wald 2014). It refers
to the weight of a urine volume compared with the weight of the same volume of
distilled water and depends on the mass and number of the dissolved particles. It can
be evaluated by dipstick, which provides a rapid semiquantitative result, as well as
by other urinary markers on a series of test pads embedded on a reagent strip (Wald
2014). This method can be influenced by urine pH and non-ionized molecules:
underestimation occurs with pH above 6.5, whereas overestimation is found with
urine protein concentration above 7.0 g/L (Assadi and Fornell 1986). Additionally,
non-ionized molecules, such as glucose and urea, are not detected by the dipstick, so
this method may not strictly correlate with the results obtained by refractometry and
osmolality (Siegrist et al. 1993). Refractometry measures all solutes, rather than just
ionic substances, and is therefore more accurate than dipstick. Temperature-
compensated equipment eliminates the influence of temperature, and therefore it is
recommended for use in everyday practice (Strasinger and Di Lorenzo 2014).
Nevertheless, this method can be affected by protein, glucose, mannitol, dextrans,
diuretics, radiographical contrast media, and some antibiotics (Siegrist et al. 1993).
Osmolality is measured by an osmometer and depends only on the number of
particles present; it is not influenced by urine temperature or molecule size.
Regarding clinical application, SG gives important insight into the patient’s
hydration status and the concentrating ability of the kidneys (Simerville
et al. 2005). It can be used to crudely estimate how the concentration of other
urine constituents may reflect total excretion of those constituents (Jung 1991)
because SG correlates inversely with a 24-h urine volume (McCormack
et al. 1991). An important additional function of this parameter is to determine
whether specimen concentration is adequate to ensure the accuracy of chemical tests
because values that are extremely outside the normal range may induce false results.
Most random specimens fall between 1.015 and 1.030. A SG of 1.000 to 1.003 is
consistent with marked urinary dilution, as observed in patients with diabetes
insipidus or water intoxication. Specific gravity of 1.010 is often called isosthenuric
urine because it is similar to plasma, so it is often observed in conditions in which
urinary concentration is impaired, such as acute tubular necrosis (ATN) and chronic
kidney disease (CKD). Specific gravity above 1.040 almost always indicates the
presence of some extrinsic osmotic agent, such as radiocontrast. Indeed, self-
monitoring this parameter may be useful for stone-forming patients, who benefit
from maintaining dilute urine (Siegrist et al. 1993).
In conclusion, dipstick is the least accurate method, so it should be replaced by
refractometry if a precise measurement is needed or error interference factors are
suspected. Osmolality is more reliable than both methods, and most clinical deci-
sions should be based on this determination when evaluating pathological urine.
Chemical Markers
Chemical urinary markers are probably most useful in daily practice, because their
value in the diagnosis, monitoring, and prognosis of glomerular diseases has long
been proven. This subgroup also contains multiple methods, discussed below, to
measure each type of marker.
Reagent strips currently provide a simple, affordable, and rapid evaluation. As
written above, they consist of absorbent pads impregnated with chemicals and
attached to a plastic strip. A color-producing chemical reaction takes place when
the absorbent pad comes in contact with urine (Fig. 1). The timing for reactions to
take place varies between tests and manufacturers and ranges from an immediate
reaction for pH to 120 s for LE. The results are interpreted by comparing the
color within a chart supplied by the manufacturer. A semiquantitative value of
trace, 1+, 2+, 3+, or 4+, can be reported, and a corresponding estimation of the
milligrams per deciliter is available for appropriate testing areas (Strasinger and Di
Lorenzo 2014). Improved specificity and sensitivity of the most recent dipsticks, as
well as the use of automated strip readers, have reduced the need for routine use of
confirmatory tests of this method.
Fig. 1 Dipstick image. The first strip (A) shows a regular pad before being dipped in urine. The
second strip (B) has been dipped in a random spot urine sample of a patient with glomerulonephritis,
detecting trace hematuria (a), 3+ proteinuria (b), and 2+ leukocyturia (c)
Blood
Blood may be present in the urine, either in the form of intact RBCs (hematuria) or as
the product of RBC destruction, hemoglobin (hemoglobinuria). Therefore, chemical
tests for hemoglobin provide the most accurate means of determining the presence of
blood.
Hematuria can be measured quantitatively by any of the following: indirect
examination by dipstick, determination of RBC number through chamber count,
and direct examination of urinary sediment.
The dipstick method is based on the pseudoperoxidase activity of the heme,
which catalyzes the reaction of peroxide and a chromogen to form a colored product
(green to blue). Red blood cells, hemoglobin, and myoglobin will catalyze this
reaction, so a positive test result may indicate hematuria, hemoglobinuria, or
myoglobinuria, respectively (Brenner et al. 2011). This latest condition may be
suspected when a red-brown urine is seen. Dipsticks can detect concentrations as
low as five RBCs per microliter. The terms trace, small, moderate, and large or trace,
1+, 2+, and 3+ are used for reporting (Strasinger and Di Lorenzo 2014). A positive
test with green spots mostly results from intact erythrocytes. On the other hand, a
homogeneous, diffuse green pattern can result from three main situations: marked
hematuria with a high number of erythrocytes that cover the whole pad surface; lysis
of erythrocytes favored by delayed examination, alkaline urine pH, or low SG, as
well as hemoglobinuria secondary to intravascular hemolysis. False-negative results
are mainly caused by ascorbic acid (reducing agent) (Rauta et al. 2002) and high
SG. On the other hand, false-positive results can occur due to myoglobinuria and to a
high concentration of bacteria with pseudoperoxidase activity (Enterobacteriaceae,
Staphylococci, Streptococci) (Lam 1995). Given the limited specificity of the dip-
stick method (65–99 % for 2–5 RBCs per high-power microscopic field), an initial
positive result should be confirmed by microscopic evaluation (Sutton 1990).
The chamber count method measures the number of RBCs per milliliter and has
been suggested to have greater precision and sensitivity than the sediment count
(Grossfeld et al. 2001). Although microscopic examination is only a semiquantita-
tive method of determining the degree of hematuria, it is easier to perform, less time-
consuming, and more cost-effective than the chamber count. Furthermore, it allows
different elements to be distinguished by their appearance, enabling the source of the
hematuria and its main etiologies to be pointed out (Johnson et al. 2014). However,
both chamber and sediment counts have been shown to correlate with acceptable
sensitivity (Grossfeld et al. 2001).
Regarding clinical significance, hematuria is most closely related to disorders of
renal or genitourinary origin that can be caused by glomerular, non-glomerular
(tubulointerstitial (Ballarin et al. 2011), toxic chemicals, anticoagulant-related
nephropathy (Brodsky et al. 2011), renovascular or metabolic abnormalities),
and urological (tumors, especially bladder cancer (Muto et al. 2014), trauma,
pyelonephritis, or renal calculi) diseases. Glomerular hematuria is typically asso-
ciated with significant proteinuria (Simerville et al. 2005). However, 20 % of
patients with biopsy-proven glomerulonephritis (GN) present hematuria alone
(Fassett et al. 1982), among which IgA nephropathy is the most common
(Simerville et al. 2005). Hematuria of non-pathological significance is observed
following strenuous exercise and during menstruation. In both cases, results of
repeated urinalysis after 48–72 h should be negative. On the other hand, hemo-
globinuria may result from RBC lysis after urination or intravascular hemolysis
(hemolytic anemia, transfusion reactions, severe burns, spider bites and infec-
tions). Distinctively, myoglobinuria due to rhabdomyolysis can happen in condi-
tions like strenuous exercise, trauma, prolonged coma, convulsions, muscle-
wasting diseases, alcoholism, as well as from statin medication and several kinds
of drug abuse.
Besides the multiple conditions stated above, where hematuria has an “injury
marker” value, recent information has presented its prognostic value in diseases like
IgA nephropathy (Moreno et al. 2012) and acute kidney injury (AKI) (Gutierrez
et al. 2007). An association with long-term incidence of ESRD has also been
established (“risk factor” value) (Vivante et al. 2011). Despite this evidence, there are
pitfalls in quantifying hematuria, rendering it more difficult to objectively assess the
impact of therapy and to predict the outcome. Firstly, this is because it is not routinely
quantified as accurately as albuminuria and proteinuria are (Moreno et al. 2012), and
secondly, the assessment of glomerular hematuria may be interfered with by the
presence of the non-glomerular one. Because of the absence of hard data, at the present
time, it is difficult to make recommendations on the specific target values of hematuria
that should influence treatment decision making.
pH
Urine pH reflects the presence of hydrogen ions, but this does not necessarily reflect
the overall acid load in urine because most of the acid is excreted as ammonia
(Johnson et al. 2014) and a very small amount as weak organic acids. The normal
range for urine pH is 4.5–7.8.
As above, it is usually measured with a reagent test strip. Most commonly, methyl
red and bromthymol blue double indicators are used to give a broad range of colors
at different pH values (Brenner et al. 2011), covering a range from 5.0 to 9.0. A more
precise method, pH meter, measures the concentration of hydrogen ions, whose
positive charge creates an electrical potential detected by an electrode.
The importance of urinary pH is primarily as an aid in the differential diagnosis of
acid–base disorders. Moreover, this marker can be used to monitor conditions that
require urine to be maintained at a specific pH, to prevent drug nephrotoxicity, like
that due to precipitation of methotrexate in the renal tubules, or to inhibit renal
calculi formation (Sand and Jacobsen 1981). Regarding the second disorder, an
alkaline pH favors the crystallization of calcium- and phosphate-containing stones,
whereas an acidic pH promotes uric acid or cystine stones (Wagner and Mohebbi
2010). For that reason, pH control is essential to assess treatment response. Urine pH
measurement is also mandatory if an accurate crystal microscopic evaluation is
necessary.
However, pH by itself provides little useful diagnostic information, and it must be

considered in conjunction with other information about the patient because many of
the deviations can be explained by non-pathological conditions. A low pH is
observed in metabolic acidosis, one of the main causes of which is high-protein
meals (Brenner et al. 2011), and hypovolemia, in which aldosterone stimulation
increases hydrogen secretion in the distal tubules. Indeed, low urine pH may help
distinguish prerenal AKI from ATN, which is typically associated with higher
values. Other conditions with alkaline pH are infection by urease-positive organism
(Proteus), renal tubular acidosis, vomiting, and gastric suction. Non-pathological
causes include vegetarian diet (minimal nitrogen and acid generation), diuretic, and
alkali therapy.
Bilirubin
Bilirubin is a highly pigmented yellow compound that results from hemoglobin

degradation. Its appearance in urine may provide an early indication of liver disease
due to obstructive disease or hepatocellular injury, but not hemolysis, because only
conjugated bilirubin is water soluble and renal excreted.
Although reagent test strips are very sensitive to bilirubin, detecting as little as
0.05 mg/dL, this method is not very sensitive for detecting hepatic abnormalities. In
addition, measurement errors often occur, firstly, due to false-positive results because
of urine contamination with stool and other pigments and, secondly, due to false
negatives because of prolonged sample storage and exposure to light, promoting
bilirubin photo-oxidation to biliverdin (Young 1990). Consequently, since the intro-
duction of serum tests of liver enzyme function, this measurement has lost its clinical
application.
Urobilinogen
Urobilinogen appears in urine because as it circulates in the blood back to the liver, it
passes through the kidney and is filtered by the glomerulus. A small amount of
urobilinogen (<1 mg/dL) is normally found in urine.
This strip test reaction sensitivity increases with temperature (ideally room temper-
ature). False-positive reactions may occur due to porphobilinogen, p-aminosalicylic
acid, sulfonamides, methyldopa, procaine, and chlorpromazine compounds, among
others. False-negative results occur most frequently when specimens are improperly
preserved (photo-oxidation of urobilin) or formalin is used as a preservative.
Although this parameter also has low clinical application, it can be slightly
increased in chronic constipation, and main pathological causes are liver disease
and hemolytic disorders (Strasinger and Di Lorenzo 2014). Some authors
(Gorchynski et al. 2009) tried to prove its utility for adult blunt abdominal trauma
patients, but they found it was a poor predictor for intra-abdominal injury, thus not
clinically useful.
Ketones
Ketones represent intermediate products of fat metabolism, namely, acetone (2 %),

acetoacetic acid (20 %), and β-hydroxybutyrate (78 %). Normally, measurable
amounts of ketones do not appear in urine, because all the metabolized fat is
completely broken down into carbon dioxide and water. However, when the use of
carbohydrate is compromised, body fat stores are metabolized to supply energy,
increasing ketones in the blood, which may lead to dehydration, electrolyte imbal-
ance, acidosis, and coma.
With the exception of β-hydroxybutyrate, they can be detected in urine through
the dipstick nitroprusside reaction (Jacobs et al. 1990), where acetoacetic acid reacts
with sodium nitroprusside to produce a purple color. Results are reported qualita-
tively as negative, trace, small (1+), moderate (2+), or large (3+), or semiquantita-
tively as negative, trace (5 mg/dL), small (15 mg/dL), moderate (40 mg/dL), or large
(80–160 mg/dL) (Strasinger and Di Lorenzo 2014).
Positive reactions can occur in prolonged fasting or starvation, strenuous exercise,
malabsorption, vomiting, and alcoholic or diabetic ketoacidosis. Published data
showed a good correlation between urinary and capillary blood ketones for lower
values of glycemia, but not for higher. As a result, this marker can be used to exclude
ketosis, but not to confirm ketoacidosis (Taboulet et al. 2007). Therefore, it is valuable
in the management and monitoring of type 1 diabetes mellitus because ketonuria is an
early indicator of insufficient insulin, indicating the need to upregulate dosage, as well
as other concurrent events. False-positive reactions can be due to late readings after
long sample standing and the presence of mercaptoethane sulfonate sodium (MESNA),
captopril (sulfhydryl groups), levodopa, ascorbic acid, or phenazopyridine, among
others. False-negative tests can happen if specimens are not preserved properly,
allowing the metabolization and volatilization of ketone compounds.
Nitrites and Leukocyte Esterase
Nitrites result from the conversion of nitrates in the presence of bacteriuria. This
marker is highly specific for bacteriuria, but several uropathogens other than
Enterobacteriaceae do not reduce nitrate to nitrite (Patel et al. 2005); thus, a negative
test does not necessarily mean that the urine is free of bacteria.
Nitrite is detected by the Griess reaction, in which nitrite at an acidic pH reacts
with an aromatic amine to form a diazonium compound that then reacts with
tetrahydrobenzoquinolin compounds to produce a pink color (positive). Different
shades of pink may be produced, but they do not reflect the degree of bacteriuria.
Optimal results for a urinary nitrite test are obtained by analyzing an early morning
specimen that has been incubating in the bladder for 4 h or more. A clean midstream
specimen is important to reduce bacterial contamination.
Apart from the first situation mentioned above, false-negative results may also
occur when urine has been in the bladder for a short period, dietary nitrite is absent,
nitrate reduction has gone beyond the nitrite stage to form nitrogen (Garingalo-
Molina 2000), and bacterial metabolism is inhibited by the presence of antibiotics or

large amounts of ascorbic acid (Strasinger and Di Lorenzo 2014). On the other hand,
false-positive tests can result from improperly preserved specimens and highly
pigmented urine (Strasinger and Di Lorenzo 2014).
Although a semiquantitative culture of a urine specimen is the only method that
can provide detailed documentation of a bacterial UTI, it is costly and takes at least
24 h. Thus, a screening test could be carried out using dipstick, where nitrites and LE
accuracy have been correlated to culture (Lohr 1991).
The LE test detects esterase, an enzyme released by both intact and lysed WBCs
(except lymphocytes), which catalyze the hydrolysis of an acid ester to produce an
aromatic purple azo dye. Because it also detects lysed cells, it could be more accurate
than microscopic evaluation, but should be quantified using this second method. It
should be performed on a fresh specimen and reactions are reported as trace, small,
moderate, and large or trace, 1+, 2+, and 3+ (1). In addition to UTI screening, it also
detects infections caused by Trichomonas, Chlamydia (Rahman et al. 2014), and
inflammation of renal tissues (like interstitial nephritis) that produce leukocyturia
without bacteriuria.
False-positive reactions can be due to the presence of strong oxidizing agents,
formalin in the container, highly pigmented urine, and nitrofurantoin. False-negative
results may occur with high SG urine, antibiotics like gentamicin, cephalexin,
cephalothin, and tetracycline, as well as high concentrations of protein (>500 mg/
dL), glucose (>3 g/dL), oxalic, and ascorbic acid (Strasinger and Di Lorenzo 2014).
Although several studies have confirmed the low sensitivity and specificity of the
dipstick test, these two positive reactions suggestive of UTI can be used to determine
empirical treatment, as well as in follow-up (Demilie et al. 2014), because their
combination improves each other’s performance (Semeniuk and Church 1999). In
contrast, a negative reaction should be an indication for culture if clinical features are
present. Even though they are not intended to replace urine culture, they can be cost-
effective in reducing the necessity to perform these cultures (Wise et al. 1984).
Currently, they are often ordered to screen high-risk patients who are frequently
asymptomatic, like pregnant women, children, the elderly, and patients with recur-
rent UTI, CKD, and diabetes.
Glucose
Glycosuria is the most frequently performed chemical analysis on urine.

One of the oldest methods available is based on copper reduction, where glucose
reduces copper sulfate to cuprous oxide in the presence of alkali and heat (Benedict
reaction). Color ranges from a negative blue through a positive green to orange/red
and should be compared with the manufacturer’s chart to estimate quantitative
measurement of glucose. Its sensitivity is reduced to a minimum of 200 mg/dL,
but at high glucose levels, a “pass through” phenomenon may occur and a return to a
negative color can happen without reporting the previous temporary positive result
(Strasinger and Di Lorenzo 2014). In contrast to the causes of false-negative results,
this test is not specific for glucose, so other reducing substances, such as sugars
including galactose, lactose, fructose, maltose, and pentose, ascorbic acid, certain
drug metabolites, containers oxidizing detergents, and antibiotics, like cephalospo-
rins, may induce false-positive reactions (Brigden et al. 1992). Although it has a low
sensitivity and specificity to detect glycosuria, this method detects urine galactose;
thus, it can be used to screen newborns for “error of metabolism,” in which a lack of
the enzyme galactose-1-phosphate uridyl transferase prevents breakdown of
ingested galactose (Strasinger and Di Lorenzo 2014).
A more recent method is performed through a dipstick impregnated with a
mixture of components, including glucose oxidase, which promotes a double
sequential enzyme reaction, since glucose triggers the production of gluconic acid
and peroxide, which in turn catalyzes the oxidation of a chromogen to form a colored
compound that will vary according to the chromogen used by each manufacturer.
Under ideal conditions, the intensity of the color should be directly proportional to
glucose concentration, thereby allowing a quantitative estimation (Gray and Millar
1953). Here, interference by other reducing agents does not occur thanks to the
additional chemicals in the strip. Results are reported in terms of negative, trace, 1+,
2+, 3+, and 4+, which correspond to quantitative measurements ranging from
100 mg/dL to 2 g/dL, or 0.1 % to 2 %, provided by the manufacturers (Strasinger
and Di Lorenzo 2014). Although its sensitivity decreases with high SG and low
temperature, the greatest source of false-negative results is the long-standing sam-
ples subjecting glucose to bacterial degradation. High ketones also may interfere
with the results but only in the presence of low glycosuria levels, which rarely
happens.
Glycosuria clinical application occurs when kidney proximal tubules are unable
to reabsorb all the filtered glucose, despite normal plasma glucose levels, or urinary
leak occurs due to hyperglycemia above 180 mg/dL (10 mmol/L), when tubular
reabsorption of glucose has reached its threshold. In the first setting, glycosuria can
occur in an isolated setting in ESRD and cystinosis, but usually occurs with Fanconi
syndrome (phosphaturia, uricosuria, and aminoaciduria). Possible causes are multi-
ple myeloma, heavy metal exposure, and treatment with certain medications includ-
ing tenofovir, lamivudine, cisplatin, valproic acid, and aminoglycosides (Haque
et al. 2012). In rarer situations, it may also be an isolated defect associated with
genetic mutations, as in solute carrier family 5 (SLC5A2) gene encoding sodium/
glucose cotransporter 2 (SGLT2), which affects renal glucose transport (Magen
et al. 2005). Hyperglycemic scenarios are mostly caused by diabetes, but a minority
can be due to pancreatitis, acromegaly, Cushing syndrome, hyperthyroidism, pheo-
chromocytoma, and thyrotoxicosis. Nevertheless, healthy people can have glycos-
uria caused by a temporary lowering of the renal threshold during pregnancy and
after a high glucose content meal. Therefore, collection conditions should be con-
trolled (previous 12-h fasting recommended) and results interpreted according to
settings. The use of currently available reagent strip methods for both blood and
urine glucose testing has greatly increased early diagnosis of diabetes, allowed
patients to monitor themselves at home, and improved outcomes. For purposes of
diabetes monitoring, specimens are usually tested 2 h after meals. Additionally, a
first morning specimen does not always represent a fasting specimen because
glucose from an evening meal may remain in the bladder overnight, so patients
should be advised to empty the bladder and collect the second specimen. Overall,
fasting glycosuria testing has a specificity of 98 % and a sensitivity of 17 % (Singer
et al. 1989) as a screening test for diabetes.
Albuminuria
Albuminuria has been the hallmark used to develop clinical practice and research
in the area of diabetic nephropathy, CKD, and cardiovascular disease, among
others.
The normal rate of albumin excretion is less than 30 mg/day (20 mcg/min), about
4–7 mg/day (3–5 mcg/min) in healthy young adults, and increases with age and with
body weight. Microalbuminuria is currently defined as a persistent albumin excretion of
between 30 and 300 mg/day (20–200 mcg/min) or a urinary albumin/creatinine ratio
(UACR) of 2.5–35 mg/mmol in male subjects and 3.5–35 mg/mmol in female subjects
(KDIGO 2013; Stevens and Levin 2013). Albumin excretion above 300 mg/day
(200 mcg/min) is considered macroalbuminuria. Previous reviews and clinical practice
guidelines have called for the abandonment of the term “microalbuminuria”
(Ruggenenti and Remuzzi 2006). The Kidney Disease: Improving Global Outcomes
(KDIGO) 2012 clinical practice guideline for the evaluation and management of CKD
discourages the use of the term when classifying patients to a CKD stage according to
their level of albuminuria. The suggested approach is to slot albuminuria into three
categories: A1, normal to mildly increased (instead of normoalbuminuria); A2, mod-
erately increased (instead of microalbuminuria); and A3, severely increased (instead of
macroalbuminuria or proteinuria) (KDIGO 2013).
Transient elevations in the excretion of albumin can be seen in fever, infection,
exercise, heart failure, nonspecific joint inflammation, poor glycemic control (hemo-
globin A1c >8 %), obesity, and hyperlipidemia (low-density lipoprotein cholesterol
>120 mg/dL) (Wald 2014). Posture, smoking, and diet may also influence albumin
excretion rate (MacIsaac et al. 2014). However, persistent microalbuminuria has not
only been consistently associated with diabetic nephropathy, but also with hyperten-
sion (>160/100 mmHg), coronary heart disease, and overall cardiovascular disease,
in both diabetic and nondiabetic patients (Newman et al. 2005). As illustrated in the
Prevention of Renal and Vascular End-Stage Disease (PREVEND) study
(Abdelmalek et al. 2014), it also enhances the predictive value of ST and T wave
changes for cardiovascular disease. Concerning kidney disease, it is predictive of
developing clinical proteinuria, faster decline in glomerular filtration rate (GFR), and
higher risk of ESRD (Newman et al. 2005). Microalbuminuria has generally been
accepted as the first injury marker of diabetic nephropathy. One other study showed
that cardiovascular risk for patients with type 2 diabetes starts to increase with urinary
albumin excretion levels above 1 mcg/min, even before the upper limit of
normoalbuminuria is reached (Ruggenenti et al. 2012). In addition to its “injury
marker” value, it may also be useful in monitoring treatment. Additionally to this
evidence, both Effect of a Multifactorial Intervention on Mortality in Type 2 Diabetes

(STENO-2) and Irbesartan in Patients with Type 2 Diabetes and Microalbuminuria
(IRMA-2 trials) trials concluded that early intervention with anti-albuminuric drugs
and monitoring is cost-effective, leading to amelioration of GFR loss, decrease in
ESRD incidence, and prolongation of life. A similar association between those drug
effects and other well-accepted surrogate cardiovascular end points, such as blood
pressure and cholesterol, has been reported (Roscioni et al. 2014). However, it should
be noticed that there is no test capable of distinguishing between microalbuminuria
linked to cardiovascular disease and that linked to renal disease (MacIsaac et al. 2014).
Regarding its measurement, although a 24-h urine collection is the gold standard
for the detection of microalbuminuria (Bennett et al. 1995), it has been suggested
that screening can be more simply achieved through a random spot specimen.
Albumin dipsticks are based on binding antibody to the strip to form a conjugated
enzyme, which reacts with the substrate to produce a color range according to pH
changes. Colors range from pale green to aqua blue; intensity is proportional to the
concentration of albumin itself and is graded on a scale from 0 to 4+ (Lamb et al. 2009),
which can be correlated to a quantitative scale supplied by the manufacturers. Although
urine concentration may strongly influence this marker result, limited data suggest that
correcting this value to urine SG improves ability to identify abnormal results
(Constantiner et al. 2005). Other false positives may occur after the use of iodinated
radiocontrast agents (Morcos et al. 1992), with a highly alkaline urine (pH >8)
(Simerville et al. 2005), with gross hematuria or a urocrit >1 % (Tapp and Copley
1988), and when specific antiseptics (chlorhexidine, benzalkonium) are used before
collection (Magen et al. 2005). Abnormally high values should always be confirmed by
repeated measurements (KDIGO 2013), and if a true positive is confirmed, accurate
quantification by another method is needed. On the other hand, other factors unrelated
to low SG may be associated with false-negative rates, as some albumin components
are not immunologically reactive with the strip component (Busby and Bakris 2004).
Overall, albumin dipstick test is a very specific but not sensitive method, because
many patients with microalbuminuria may be missed unless their urine is highly
concentrated. Still, more reliable strips have been designed and been successful in
detecting moderately increased albuminuria with a sensitivity and specificity of
80–97 % and 33–80 %, respectively (Comper and Osicka 2005).
In addition to this method, the high-performance liquid chromatography tech-
nique is able to assess all intact urinary albumin, even non-immunoreactive albumin,
thereby increasing sensitivity and specificity (Busby and Bakris 2004) and proving
itself useful for earlier detection. Other types of reagent strip provide simultaneous
measurement of albumin (or protein) and creatinine, thereby allowing the compar-
ison between their excretion and producing a semiquantitative UACR, which corre-
sponds to a more accurate estimation of the 24-h excretion.
Conventional reagent pads detect a minimum of 30 mg/dL and may include other
proteins besides albumin. Strips using dye bis(30 ,300 -diiodo-40 ,400 -dihydroxy-
50 ,500 -dinitrophenyl)-3,4,5,6-tetrabromo-sulfonephthalein (DIDNTB) can measure
albumin as low as 8 mg/dL, without the inclusion of other proteins, increasing
overall sensitivity and specificity. Reaction interference by highly alkaline urine
can be also controlled with bis-(heptapropylene glycol) carbonate reagent

(Strasinger and Di Lorenzo 2014).
As stated before, limitations regarding the 24-h collection adequacy are not negligi-
ble, so this vital step can be checked by quantifying the 24-h urine creatinine and
comparing this value to the expected one. As a general rule, in adults under the age of
50, daily creatinine excretion should be 20–25 mg/kg (177–221 micromol/kg) of lean
body weight in men and 15–20 mg/kg (133–177 micromol/kg) of lean body weight in
women, figures which progressively decline to 50 % in their 90s. Due to these limitations
and the need for laborious control, some alternatives have been proposed. Most com-
monly, random spot UACR is used to estimate a 24-h proteinuria and to follow the
effects of treatment in patients with cardiovascular and albuminuric kidney diseases.
Reagent strips containing copper sulfate (CuSO4), 3,30 ,5,50 -tetramethylbenzidine
(TMB), and diisopropylbenzene dihydroperoxide (DBDH) allow for the detection of
creatinine based on the pseudoperoxidase activity of copper–creatinine complexes,
producing a color change from orange through green blue. Quantitative results may be
estimated according to charts as 10, 50, 100, 200, or 300 mg/dL and 0.9, 4.4, 8.8, 17.7,
or 26.5 mmol/L. Falsely elevated results can be caused by bloody or colored urine and
cimetidine. The absence of creatinine in readings is considered abnormal, as it is
usually found in concentrations of 10–300 mg/dL (Strasinger and Di Lorenzo 2014).
According to several studies, UACR measured in a morning spot urine correlates
well with the timed excretion rate (Ginsberg et al. 1983; Heerspink et al. 2010). One
trial reported that a random UACR above 30 mg/g had a sensitivity of 100 % for the
detection of moderately increased albuminuria (Nathan et al. 1987). Usually, albu-
min concentration is measured in mg/dL and is divided by the creatinine concentra-
tion also in mg/dL, yielding a dimensionless number that estimates the 24-h albumin
excretion in grams per day (Shidham and Hebert 2006). If SI units (mg/mmol) are
used, the value is divided by 8.8.
The quantification of UACR is mandatory in patients with AKI and CKD and all
patients with a previously elevated semiquantitative result. On the one hand, it is
imperative to exclude a false positive; on the other hand, the quantification of
excretion is essential in establishing the diagnosis, prognosis, and monitoring
response to treatment.
The optimal time to measure the UACR remains uncertain (Wald 2014). In an
initial study, the best correlation with a 24-h collection occurred with first morning
and before bedtime samples (Ginsberg et al. 1983). A larger study also found the best
correlation for the first morning void, although the difference regarding other timings
was not significant (Witte et al. 2009). There is some evidence that random untimed
samples may be more prone to error because they generally show higher intra- and
interindividual coefficients of variation than those found for the first or second
morning samples (Witte et al. 2009; Naresh et al. 2012). In this setting, one report
(Saydah et al. 2013) showed that random urine samples appear to overestimate the
prevalence of albuminuria compared to first morning collection. Additionally, cre-
atinine excretion variance may also influence both random spot and 24-h sample
results. According to the rates reported before, the average 24-h urine creatinine
excretion is approximately 1000 mg/day (11.4 mmol/day) per 1.73 m2, so this ratio
accuracy is diminished if creatinine excretion is either markedly higher or lower than

the average population. The groups more prone to error are individuals with large
muscle mass, male gender, black/hispanic race, and younger in age, who may exhibit
a much higher creatinine excretion rate than 1000 mg/day, and spot UACR will
underestimate a 24-h albuminuria, while among patients who have small muscle
mass like cachectic, older, and females, creatinine excretion may be much lower, and
24-h results overestimated. Given these UACR limitations, several investigators
have developed an “estimated albumin excretion rate” (eAER) to more accurately
predict the 24-h albumin excretion (Abdelmalek et al. 2014; Fotheringham
et al. 2014). The eAER can be calculated by multiplying the spot UACR by the
expected 24-h creatinine generation. This method may be particularly important in
the groups mentioned above.
In conclusion, KDIGO Guidelines recommend screening for moderately
increased albuminuria among diabetic patients for early detection of nephropathy
and nondiabetic patients at increased risk for CKD or cardiovascular disease, such as
those with hypertension and metabolic syndrome (KDIGO 2013).
Protein
Proteinuria is the hallmark of renal disease because it often implies an increase in

glomerular permeability, which allows the filtration of normally non-filtered macro-
molecules. According to the previous evidence described for albuminuria, protein-
uria of increasing severity is associated with a faster rate of decline in GFR,
regardless of its baseline (Turin et al. 2013), is an independent risk predictor for
ESRD (Iseki 2013), and is associated with the risk of myocardial infarction and
mortality (Hemmelgarn et al. 2010).
Proteinuria is defined as urinary protein excretion of more than 150 mg/day
(10–20 mg/dL). Normal urinary proteins include albumin, serum globulins, and
Tamm–Horsfall protein (uromodulin) secreted by the renal distal tubular epithelial
cells. Proteinuria can be organized into different categories according to the source
and pathogenesis of the defect, as well as to its clinical significance. It may be divided
into glomerular, tubular, overflow, and post-renal proteinuria. Patients may have more
than one type of proteinuria. Glomerular proteinuria is due to increased filtration through
the glomerular capillary wall. It is a sensitive marker of glomerular disease and hyper-
tension, where increased pressure from the blood entering the glomerulus overrides the
selective filtration of the glomerulus, as well as all types of GN where different kinds of
membrane lesions occur. More benign causes, such as orthostatic, high fever, or
exercise-induced proteinuria, usually cause an isolated, transient, and asymptomatic
proteinuria, less than 1–2 g/day (Wald 2014). Differential diagnosis of orthostatic
proteinuria is carried out when a patient is requested to empty the bladder before
going to bed, to collect a specimen immediately upon rising in the morning (negative),
and to collect a second one after standing for several hours (positive) (Strasinger and Di
Lorenzo 2014). Tubular proteinuria includes low-molecular-weight proteins, such as
beta2-microglobulin, immunoglobulin light chains, retinol-binding protein, and
polypeptides derived from the breakdown of albumin, which have molecular weights
under 25,000 Daltons in comparison to the 69,000 Daltons of albumin. These smaller
proteins can be filtered through the glomerulus and almost completely reabsorbed in the
proximal tubule. Interference with proximal tubular reabsorption due to tubulointerstitial
diseases, or even some primary glomerular syndromes, can lead to increased excretion of
these smaller proteins (Carter et al 2006). Tubular proteinuria often fails to be diagnosed
since dipstick is not sensitive for non-albumin proteins, and they are usually found in low
concentrations. Mild increased excretion of immunoglobulin light chains (Bence Jones
proteins) is polyclonal (Kappa and Lambda), and not injurious to the kidney.
In contrast, the monoclonal nature of the light chains in the overflow proteinuria
seen in multiple myeloma is highly nephrotoxic (Wald 2014). This type of proteinuria
results from conditions affecting the plasma prior to reaching the kidney and is
therefore not indicative of actual renal disease, so it may also be classified as prerenal
proteinuria. The most common conditions that lead to overflow proteinuria are fre-
quently transient. It is caused by increased levels of low-molecular-weight plasma
proteins that exceed tubular reabsorptive capacity, like acute phase reactants in infec-
tion and inflammation. Common examples, other than myeloma kidney, are due to
lysozyme in acute myelomonocytic leukemia, myoglobin in rhabdomyolysis, and free
hemoglobin in intravascular hemolysis (Barratt and Topham 2007). Patients with
myeloma kidney may also develop a component of tubular proteinuria since the
excreted light chains may be toxic to the tubules, leading to diminished reabsorption
(Wald 2014). Based on false-negative dipstick results, suspected cases of multiple
myeloma must be diagnosed by performing serum and urinary immunoelectrophoresis.
Post-renal proteinuria may be caused by inflammation in the urinary tract (often
in UTI, but also in nephrolithiasis or tumors), although the mechanism is unclear.
The excreted proteins are often non-albumin (often IgA or IgG), in small amounts,
and usually accompanied by leukocyturia (Wald 2014).
Regarding measurement techniques, there are two semiquantitative methods to
screen patients for proteinuria: dipstick (similar to albuminuria) and sulfosalicylic
acid (SSA) precipitation.
Protein dipstick principles and main interference conditions are similar to those
described above in the section on “Albuminuria.” Although very specific, the dipstick
test is not sensitive to low levels of proteinuria (<10–20 mg/dL) or low concentra-
tions of γ-globulins and Bence Jones proteins. A result of 1+ corresponds to approx-
imately 30 mg/dL, 2+ to 100 mg/dL, 3+ to 300 mg/dL, and 4+ to 1,000 mg/dL (House
and Cattran 2002). Dipstick urinalysis can reliably predict proteinuria with sensitiv-
ities and specificities of greater than 99 % (Woolhandler et al. 1989).
In contrast to the dipstick test, which primarily detects albumin, SSA is a cold
precipitation test that detects all proteins in urine at a sensitivity of 5–10 mg/dL (Rose
1987). A significantly positive SSA test in conjunction with a negative dipstick often
indicates immunoglobulin light chain excretion caused by dysproteinemias. Various
concentrations and amounts of SSA can be used to precipitate protein, and methods
vary greatly among laboratories. It is mostly performed by mixing one part of urine
supernatant (2.5 mL) with three parts of 3 % SSA and grading the resultant turbidity
according to one scheme given by the manufacturer: 0 or no turbidity corresponds to
0 mg/dL; trace or slight turbidity to 1–10 mg/dL; 1+ or mild turbidity to 15–30 mg/
dL; 2+ or white cloud without precipitate to 40–100 mg/dL; 3+ or white cloud with
fine precipitate to 150–350 mg/dL; and 4+ or flocculent precipitate to >500 mg/dL
(Strasinger and Di Lorenzo 2014). The SSA test will be overestimated by as much as
1.5–2 g/L in the presence of iodinated radiocontrast agents (Morcos et al. 1992),
penicillins, sulfisoxazole, and with gross hematuria (Simerville et al. 2005).
Both the SSA and dipstick test can detect urinary lysozyme. Total lysozyme excretion
is usually below 1 g/day but can exceed 4.5 g/day in some patients with acute monocytic
or myelocytic leukemia (Mok et al. 1994). Thus, lysozyme excretion should be mea-
sured in patients who have a persistently positive urine dipstick for proteinuria in the
absence of albuminuria, particularly if other signs of the nephrotic syndrome are absent.
Once again, patients with persistent proteinuria should undergo a quantitative
measurement. As for albuminuria, the 24-h sample is the gold standard method.
Nevertheless, based on the assumption that the potential error in determining pro-
teins in a spot urine sample does not exceed the 24-h sample error (Morales
et al. 2004), the protein/creatinine ratio (P/C) in spot urine was developed as an
alternative (Methven et al. 2010). The same sample can even be used for microscopic
investigation. Nevertheless, although the correlation between P/C ratio and 24-h
proteinuria has been established, authors are not unanimous (Birmingham
et al. 2007), because the results may be influenced by factors like a creatinine
excretion deviation from the average. Other authors suggest that this correlation
varies in accordance with different levels of proteinuria (Methven et al. 2011), with
the majority finding acceptable agreement in the range 0.5–2 g/day (Leung
et al. 2007), but not for nephrotic range (Antunes et al. 2008). Moreover, some
data also revealed that this correlation is not reliable for some diseases. One example
is lupus nephritis (LN), where the accurate evaluation of proteinuria is critical to
clinical management, because it is currently the most important available biomarker
of disease activity and renal prognosis. Furthermore, proteinuria is often a primary
end point in clinical trials of new therapies and therefore must be measured with
precision. Based on the possible lack of correlation in this disease, short-interval
timed urine collections have been studied as a surrogate for 24-h collections to
increase patient compliance and improve accuracy of the results. One study found
that a 12-h overnight collection provides a more accurate result than shorter ones
(Fine et al. 2009). Although a random spot P/C measurement may not be reliable to
make decisions for patients with LN, it may be useful as a screening and monitoring
test justified by its advantages concerning facility, reliability, accuracy, and diagnos-
tic speed (Guedes-Marques et al. 2013). It could be used as the preferential marker in
subgroups of subjects who find it more difficult to properly collect 24-h urine, such
as children, elderly people, patients with intellectual disabilities, incompatible pro-
fessional activities, or lack of adherence. Another factor to be considered is the
timing of the sample, which is influenced by the daily circadian fluctuation of both
protein and creatinine excretion. According to some data, the best estimation is
probably obtained with morning samples, but not the first void (Saydah et al. 2013).
The technical and methodological details about spot P/C ratio are described in the
section on “Albuminuria.”
Proteinuria selectivity can be assessed in nephrotic patients through the ratio of

IgG clearance (160,000 D) to transferrin clearance (88,000 D). Although infre-
quently used, highly selective proteinuria (ratio <0.1) in nephrotic children suggests
the diagnosis of minimal change disease and predicts corticosteroid responsiveness
(Johnson et al. 2014).
In conclusion, some authors consider that a normal spot P/C ratio is sufficient to
rule out pathological proteinuria, but that an elevated value should be confirmed and
quantified with a 24-h collection (Price et al. 2005). Other investigators have
reported a poor correlation at high levels of proteinuria, as well as in some diseases
like LN, where this ratio is useful to monitor so a 24-h ratio is recommended before
treatment decisions are made.
Uric Acid
Hyperuricemia and gout correlate with risk factors for cardiovascular disease. They
are caused by an overproduction and/or inefficient renal clearance of urate.
The fractional renal clearance of urate (FCU, renal clearance of urate/renal
clearance of creatinine) expresses urate clearance as a fraction of creatinine clear-
ance. It provides information about the efficiency of the renal tubular mechanisms of
urate clearance by correcting for the effect of the glomerular filtration rate
(Kannangara et al. 2012). This spot-FCU has been demonstrated to be a convenient,
valid, and reliable indicator of the efficiency of the kidney in removing urate from
the system. Spot-FCU has been used in studies investigating molecular mechanisms
of kidney clearing urate from the blood and so identifying people at increased risk
for cardiovascular disease.
Furthermore, urinary uric acid assessment is necessary for the early diagnosis and
adequate treatment of urolithiasis. A recent paper (Sáez-Torres et al. 2014) proposed
the analysis of late-afternoon spot urine collection as an appropriate sample to
evaluate patient-specific urinary risk factors.
Uric acid excretion is more favorable in alkaline urine. Based on this evidence,
dietary intervention in metabolic syndrome, focused on hyperuricemia, recommends
taking alkaline-rich fruit and vegetables which could be monitored through urinary
pH and uric acid excretion in a random spot sample.
Electrolytes
Urinary excretion of electrolytes can be very useful for diagnosing and monitoring
several diseases, as it mainly distinguishes between systemic and kidney-limited
disorders. The first condition is caused by abnormal levels of serum electrolytes, to
which the kidney tries to adapt its function to maintain homeostasis. The second
condition is found with renal tubular disorders with abnormal electrolyte excretion.
Once more, the electrolyte/creatinine ratio in a random spot urine was found to be
an alternative to 24-h measurements. Furthermore, as described for uric acid, a
random spot urine sample may also be used to measure the fractional renal clearance
of an electrolyte, which expresses its excretion corrected for creatinine clearance.
The main practical disadvantage of this method is that a blood sample is required,
which is invasive and inconvenient to the patient.
Sodium
In steady-state conditions, the kidneys handle most of the sodium (Na) consumed in a
day, and the majority (up to 95 %) is excreted in urine within 24 h. A systematic review
of studies comparing 24-h and spot urine collections for estimating population salt
intake (Ji et al. 2012) found that most available studies are heterogeneous and there is
no uniform pool of data to assess the suitability of alternative random spot samples for
measuring 24-h Na excretion (salt intake estimation). However, most studies found
acceptable reproducibility. In this setting, most authors believe that using random spot
urine samples requires a greater number of collections, but it would still be more
convenient and feasible for monitoring. So, regarding programs of population salt
reduction, this method is less desirable to provide an initial absolute measure of salt
intake, but it may be useful in following repeated assessments.
According to one study (Mann and Gerber 2010), late-afternoon or early-evening
samples are more reliable in predicting 24-h Na excretion and may be a cost-effective
alternative in salt intake assessment in clinical practice and epidemiological studies.
Potassium
A study with the INTERSALT trial population (Tanaka et al. 2002) demonstrated that
the ratio of Na or potassium (K) to creatinine concentration in a random spot specimen is
directly proportional to the 24-h ratio. Furthermore, a Na:K ratio may be more reliable in
estimating increased risk in cardiovascular disorders such as arterial hypertension.
Phosphate
Calcium (Ca)/creatinine and phosphate (Ph)/creatinine ratios in random spot urine

specimens were also found to be a reliable method for estimating daily urinary Ca and
Ph excretion (Gokçe et al. 1991) useful in evaluating mineral bone disease in CKD,
diagnosis and monitoring of urolithiasis and in the differential diagnosis of hereditary
diseases of renal tubular transport (Bartter, Gitelman and Fancony syndrom).
Others
One study (Ilich et al. 2009) compared the concentrations of essential elements
magnesium (Mg) and zinc (Zn) in 24-h and spot urine samples and found that
although spot urine sampling might not replace 24-h measurements in all cases, it
could be a reliable alternative in those evaluated. Urinay chloride measured in a spot
sample is quite useful to evaluate volume and acid-base status and establish the
differential diagnosis between extra-renal losses of Na (very low chloride like 1
mmol/L) and renal tubular disorders (> 20 mmol/L).
A significant number of other substances may be detected from random spot
urinalysis. This is useful for detecting drug consumption or toxicity (alcohol,
benzodiazepines, others), fibroblast growth factor 23 (FGF23), and a phosphaturic
hormone to assess bone mineral disease in CKD, among many other approaches less
widely used and explored.
Microscopic Markers
Microscopic evaluation is an essential part of spot urine analysis because it provides

confirmation of urine dipstick findings and also allows the identification of formed
elements that are not evaluated through other methods. Their quantification is mostly
semiquantitative, and there is a general interobserver variability, which fails to make
them ideal markers. As an example, one study shows only fair to moderate agree-
ment among nephrologists in identifying important structures (Wald et al. 2009).
Nevertheless, their presence in urine can be evaluated through a random spot sample
and is often crucial to point toward a specific diagnosis.
Regarding specimen handling, it should be examined while fresh or adequately
preserved, because elements like RBCs or WBCs and hyaline casts disintegrate
rapidly, particularly in dilute alkaline urine. Refrigeration may cause precipitation of
amorphous urates, phosphates, and other non-pathological crystals that can obscure
other elements in the urine sediment.
The second morning voiding is the most appropriate to obtain because it avoids
the lysis of particles that can occur in the bladder overnight. The midstream clean-
catch specimen minimizes external contamination and bacterial growth without
pathological significance. As for other techniques, diluted specimens may cause
false-negative readings.
Concerning sample preparation, after centrifugation, the supernatant is removed.
After this, the sediment is resuspended, then transferred to the slide, and prepared
using a coverslip. Phase contrast microscopy is recommended because it improves
the identification of almost all particles, whereas polarized light is mandatory for the
correct identification of some lipids and crystals (Fogazzi 2010). For correct exam-
ination, both pH and SG of the sample should be known, because both alkaline pH
(7.0) and low SG (especially <1.010) favor the lysis of cells, which causes
discrepancies between dipstick and microscopic examination. The various elements
observed are quantified as number per microscopic field, and if counting chambers
are used, the elements are quantified as number per milliliter (very precise, but rarely
used in everyday practice) (Strasinger and Di Lorenzo 2014).
The main elements formed are cells (erythrocytes, leukocytes, and epithelial),
lipids, casts and crystals.
Fig. 2 Microscopic
observation of dysmorphic
erythrocytes. Microscopic
observation (400) of
dysmorphic erythrocytes in a
random spot urine sample of a
patient with
glomerulonephritis (Kindly
provided by Drª. Fernanda
Carvalho from Curry Cabral
Hospital)
Erythrocytes
The assessment of these elements may play a key role in the isolated hematuria
diagnostic approach. Although there is no agreement on hematuria definition criteria,
it is commonly defined as the presence of two or more RBCs per high-powered field
(Cohen and Brown 2003). Additionally, microscopic evaluation may distinguish two
main types of erythrocytes: isomorphic, with regular shapes and contours, derived
from the urinary excretory system, and dysmorphic, with irregular shapes and
contours, which have a glomerular origin (Fig. 2). Thus, according to their relative
proportion, hematuria may be defined as non-glomerular or glomerular, respectively.
However, this proportion threshold is not unanimous between authors, either.
As was already described above (see chemical parameters – blood, above),
among the most common pathological causes of microscopic hematuria are kidney
stones, malignancy, and glomerular disease.
Leukocytes
More than simply identifying leukocytes by chemical analysis, it is valuable to

distinguish the different types that exist because they may point to specific diseases.
Neutrophils usually mean bacterial UTI, but they may also result from urine contam-
ination caused by genital secretions, especially in young women. Nevertheless, they
can also be found in interstitial nephritis and proliferative GN. In both conditions they
are lower in number, and in GN they are mostly accompanied by dysmorphic
erythrocytes and proteinuria. Eosinophils can be detected by applying Wright’s or
Hansel’s stain to the urine sediment (Nolan et al. 1986) and are currently seen as a
nonspecific finding because they may be present in various types of GN, prostatitis,
chronic pyelonephritis, urinary schistosomiasis, and cholesterol embolism. Lympho-
cytes may indicate acute cellular rejection in renal allograft recipients. Macrophages
may be engorged with lipid droplets, appearing as “oval fat bodies,” usually seen in
nephrotic syndrome, but may also appear in other settings like Fabry disease.
Cells
Different kinds of cells (renal tubular epithelial, transitional epithelial, and squa-
mous) can be found in normal or pathological conditions according to their
proportion and presentation. Epithelial cells may appear in the urine after being
shed from anywhere within the genitourinary tract and are mostly common
in ATN.
Lipids
Lipids are found in urine as drops of different sizes that can be isolated or in clusters,
as oval fat bodies (Hotta et al. 2000). All these particles mainly contain cholesterol
esters and free cholesterol, showing a Maltese cross appearance with symmetric
arms, under polarized light. These lipids are typical of glomerular diseases associ-
ated with nephrotic range proteinuria and Fabry disease.
Casts
Casts are cylindrical structures formed in the lumen of distal renal tubules and
collecting ducts (Fig. 3). Their matrix is made of Tamm–Horsfall glycoprotein
(uromodulin), and they have different appearances, each of which has different
features and a specific clinical significance summarized in Table 1.
Crystals
Correct identification of urine crystals requires a wide knowledge of crystal morphol-

ogy and appearance under polarizing light. Whether crystals form in the urine depends
upon a variety of factors, including relative concentration of constituent molecules,
Fig. 3 Microscopic
observation of a large
granulous cast. Microscopic
observation (400) of a large
granulous cast in a random
spot urine sample of a patient
with chronic kidney disease
(Kindly provided by Drª.
Fernanda Carvalho from
Curry Cabral Hospital)
Table 1 Individual features and clinical significance of urinary casts

Cast Appearance Associated conditions
Hyaline Uniform pale pink or purple High concentrated
urine (normal
conditions)
Diuretic therapy
Nonspecific
Hyaline–granular Fine purple granules in pale pink or purple matrix Normal
Granular Dark purple granules in dark purple matrix ATN
Waxy Homogeneous pale pink or faint yellow (darker Nephrotic syndrome
than hyaline) with sharp indentations and darker CKD
edges Nonspecific (acute
and chronic
conditions)
Erythrocyte Pink to orange red Glomerulonephritis
(glomerular
hematuria)
Hemoglobin Uniform orange red Intravascular
hemolysis
Leukocyte Mostly granular aspect Interstitial nephritis
Pyelonephritis
Glomerulonephritis
Epithelial Cellular bodies in a hyaline matrix ATN
Acute interstitial
nephritis
Glomerulonephritis
Broad Larger casts CKD
Myoglobin Dark pigments Rhabdomyolysis
Bilirubin Greenish yellow Jaundice (liver
disease)
Microorganisms Pure or mixed Bacterial or fungal
urinary tract infection
ATN acute tubular necrosis, CKD chronic kidney disease
urine pH, and the presence of crystallization inhibitors. Examination of urine for
crystals is informative in the assessment of patients with stone disease, some rare
inherited metabolic disorders (like cystinuria, oxalosis, phosphoribosyltransferase
deficiency), and suspected drug nephrotoxicity (Fogazzi 2010). The main individual
features and clinical significance of crystals are summarized in Table 2.
Organisms
Bacteria are often seen in urine, but their clinical significance is generally guided by
patient symptoms and other urinary features like the presence of neutrophils (Fig. 4).
Fungi are also frequent (Fig. 4). Examiners should be aware that specimen handling is
vital to this examination because a sample that is not fresh can be contaminated.
198
Table 2 Individual features and clinical significance of urinary crystals

Urine
Crystal Composition Appearance conditions Associated disorders
Uric acid Amorphous urates Rhomboids and barrels (polychromatic pH Transient supersaturation of urine (no significance)
polarize) 5.0–5.8 Mild dehydration
(acid) Urate nephropathy/tumor lysis syndrome (AKI)
Calcium oxalate Bihydrated Bipyramidal (no polarize) pH Transient supersaturation of urine (no significance)
Monohydrated Ovoid or biconcave (polarize) 5.4–6.7 Mild dehydration
(acid) Calcium lithiasis
Ethylene glycol intoxication (AKI)
Brushite (calcium Amorphous phosphates Pleomorphic: prism, stars, needles pH 7 Transient supersaturation of urine (no significance)
phosphate) (polarize), granular plates (no polarize)) (alkaline) Mild dehydration
Struvite Magnesium ammonium phosphate Coffin lids (polarize) pH 7 Urinary tract infection
(triple phosphate) and calcium carbonate apatite (alkaline) (Ureaplasma urealyticum and Corynebacterium
urealyticum)
Cholesterol Cholesterol and other lipids Thin, transparent plates
Cystine Cystine Hexagonal irregular plates Acid pH Cystinuria
2,8- 2,8-Dihydroxyadenine Spherical, brownish, central umbilicus, Homozygotic deficiency of adenine
Dihydroxyadenine birefringent cross-like appearance phosphoribosyltransferase
(polarize)
Tyrosine Tyrosine Acute liver disease
Tyrosinemia
Leucine Leucine Acute liver disease
Drugs Drug Unusual morphology Drug overdose, dehydration, or hypoalbuminemia
Calcium oxalate (naftidrofuryl (sulfadiazine, amoxicillin, ciprofloxacin, acyclovir,
oxalate, orlistat, and vitamin C) indinavir, pyridoxylate, naftidrofuryl oxalate,
primidone, felbamate, orlistat, intravenous vitamin C)
AKI acute kidney injury
M. Guedes-Marques et al.
Fig. 4 Microscopic
observation of fungus,
bacteria, and leukocytes.
Microscopic observation
(400) of fungus, bacteria,
and leukocytes in a random
spot urine of a patient with
sepsis and acute kidney injury
(Kindly provided by Drª.
Fernanda Carvalho from
Curry Cabral Hospital)
Conclusions
It is unusual to only focus on a single risk marker for the development and progression
of a disease. In this setting, random spot urine analysis may provide the identification
of several markers at just one moment, without an invasive procedure. There is a need
to develop broader models of progressive kidney diseases and the relationship they
have with the cardiovascular background, which include novel pathways and risk
markers apart from those related to the traditional proteinuric pathway. In addition,
more trials are needed to find out which sample is most appropriate for analysis and if
this alternative method is reliable in all types of diseases.
Finally, many other biomarkers, like genetic fragments and cytokines,
among others, can already be measured in a random spot urine sample, but their
comparison with a 24-h sample measurement and many of their applications is still
not reported.

Conditions
Random spot urine examination is already much more than a complementary exam
limited to the nephrology field. In this setting, currently identified urinary markers
have been found to be associated to other diseases, other than kidney abnormalities.
Moreover, new biomarkers are being identified to help in the early detection of
cancer, tuberculosis, HIV, malaria, and potentially many other diseases.
In the future, random spot urinary genetic markers, as well as others, may predict
chronic or acute events, even before traditional laboratorial and clinical signs
become positive. Adding to this “risk” and “diagnostic” value, future biomarkers
may contribute to novel disease pathogenesis models, which could be crucial in
developing new therapeutic targets and activity markers.
Maybe one day, one simple occasional urine sample could be used to screen for
diseases like oncological, autoimmune, metabolic, or other diseases, immediately
after birth. Additionally, other markers could equally be associated to future specific
therapy responses, helping to decide the best treatment for each patient according to
their individual profile.
Summary Points
• This chapter focuses on the analysis of random spot urine, which can be easily
obtained at any time of the day with only one urination. It allows several,
otherwise asymptomatic, markers to be identified and avoids 24-h collection-
related errors.
• The collection and handling of the urine sample is crucial to avoid errors and both
the suitability and the rejection criteria must be determined.
• Random spot urine markers can be organized into three groups, according to their
laboratory method assessment: physical, chemical, and microscopic markers.
• Physical markers include color, odor, clarity, and specific gravity.
• Chemical examination includes the identification of protein, blood, glucose, pH,
bilirubin, urobilinogen, ketones, nitrites, and leukocyte esterase. They are prob-
ably the most useful in daily practice, because their value in the diagnosis,
monitoring, and prognosis of glomerular diseases has long been proven.
• Microscopic evaluation entails the detection of crystals, cells, casts, and
organisms.
• Chemical results are the most often widely used, because of their (semi)quanti-
tative presentation and possible correlation with quantitative timed collections,
but physical and microscopic ones may play a key role in many differential
diagnoses and monitoring.
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Overview of Neutrophil Gelatinase-
Associated Lipocalin (NGAL) as a Biomarker 10
in Nephrology
Valeria Cernaro, Davide Bolignano, Antoine Buemi,

Antonio Lacquaniti, Domenico Santoro, and Michele Buemi
Contents
Key Facts of Creatinine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 206
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208
NGAL and Acute Kidney Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 210
NGAL and Autosomal-Dominant Polycystic Kidney Disease (ADPKD) . . . . . . . . . . . . . . . . . . . . . 213
NGAL and Chronic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
NGAL in Kidney Transplant Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
Abstract
Neutrophil gelatinase-associated lipocalin (NGAL) is a small 25-kD peptide belong-
ing to the lipocalin superfamily. It mainly binds to siderophores, iron-carrying
molecules which are essential to bacterial and eukaryotic cells to meet their needs
of this mineral and then ensure survival. Indeed, NGAL primarily acts as an innate
nonspecific antibacterial factor, since it prevents bacteria to internalize iron thus
inhibiting their growth. However, serum NGAL levels rise not only in the course
of infective processes but also in other pathological conditions, as the result of an
V. Cernaro • A. Lacquaniti • M. Buemi (*)

Department of Clinical and Experimental Medicine, University Hospital “G. Martino”, Messina, Italy
e-mail: valecern82@virgilio.it; ant.lacq@gmail.com; alacquaniti@unime.it; buemim@unime.it
D. Bolignano
CNR – Institute of Clinical Physiology, Reggio Calabria, Italy
e-mail: davide.bolignano@gmail.com
A. Buemi • D. Santoro
Department of Clinical and Experimental Medicine, University of Messina, Messina, Italy
e-mail: antoinebuemi@hotmail.it; santisi@hotmail.com

206 V. Cernaro et al.
increased production and release from several tissues after an injury. Numerous
experimental and clinical data support a role for NGAL as a biomarker in nephrology.
It has been demonstrated that NGAL predicts the onset of acute kidney injury (AKI),
closely correlates with the extent of renal impairment in patients with chronic kidney
disease (CKD) irrespective of the underlying renal disease; it is higher in kidney
transplant patients who will develop a delayed graft function (DGF) compared to
those who will not experience this complication. Furthermore, NGAL predicts CKD
progression and may represent a precocious marker of therapeutic response in
different clinical situations. A number of studies also established the involvement
of this molecule in carcinogenesis and progression of several human tumors, in the
regulation of erythropoiesis and in the pathophysiology of cardiovascular diseases.
The aim of the present review has been therefore to summarize all the most recent
findings concerning the potential use of NGAL as a diagnostic and prognostic marker
as well as a marker of therapeutic response in patients with renal diseases.
Keywords
Acute kidney injury • Biomarker • Chronic kidney disease • Neutrophil
gelatinase-associated lipocalin • Renal transplantation
Abbreviations
ADPKD Autosomal-dominant polycystic kidney disease
CIN Contrast-induced nephropathy
CPB Cardiopulmonary bypass
CT Computed tomography
FABP Fatty acid-binding protein
FeNGAL Fractional excretion of NGAL
NAG N-acetyl-β-D-glucosaminidase
PCI Percutaneous coronary intervention
shRNA Short hairpin RNA
sNGAL Serum NGAL
uNGAL Urinary NGAL
Key Facts of Creatinine
• Creatinine is an endogenous substance produced by the muscle metabolism of

creatine that, phosphorylated by creatine kinase to creatine phosphate, acts as a
reserve of energy for muscle contraction.
10 Overview of Neutrophil Gelatinase-Associated Lipocalin (NGAL) as a. . . 207
• Creatinine production is poorly correlated to diet and physical exercise, whereas it

depends on the development of the muscular masses.
• In adults, serum creatinine values range between 0.8 and 1.3 mg/dl in men and
between 0.6 and 1.1 mg/dl in women.
• The glomerular function is evaluated primarily by the determination of creatinine
clearance.
• The diagnosis of acute kidney injury (AKI) is mainly based on increased serum
creatinine, which is not an ideal indicator of renal impairment. Serum creatinine is
produced endogenously, excreted by the kidney, easily measured in body fluids,
and inexpensive. However, its levels vary slowly and are influenced by several
factors including sex, age, nutritional status, muscle mass, blood volume, and
some drugs. Besides, during AKI, the tubular secretion of creatinine is increased;
in this condition, it cannot therefore be regarded as a reliable marker of the actual
glomerular filtration rate.
• Serum creatinine proves not to be an ideal marker even in the course of chronic
kidney disease: indeed, it is kept within the normal range until the glomerular
filtration rate is not reduced up to about 50 ml/min. Therefore, its serum concen-
tration may remain unchanged as long as a substantial percentage of renal
function does not become compromised.
• Since creatinine is not an ideal marker, researchers are trying to identify new
markers of renal function, which have the characteristic to change early on and to
predict the evolution of renal disease with sufficient accuracy. Among them, one
of the most promising is neutrophil gelatinase-associated lipocalin (NGAL).
Definitions
Acute kidney injury (AKI) Sudden decline, from 1 to 7 days, of renal function
with retention of nitrogen products (urea, creatinine), metabolic acidosis,
hyperkalemia, salt and water retention, decreased urine flow, and anuria.
Autosomal-dominant polycystic kidney disease (ADPKD) The most common

hereditary renal disease; it is characterized by the progressive cystic dilatation of
renal tubules with volume increase of the kidneys and gradual progression to
end-stage renal disease.
Biomarker Molecular substance or characteristic that can be used as an indicator in

a living organism. Its presence, absence, or modification, detectable and quantifiable
using appropriate tests, are the expression of a normal or pathological biological
process or the response to a certain pharmacological treatment.
Cold ischemia time Time elapsed between the start of perfusion with cold solutions
in the explantation operating room and the extraction of the organ from the ice in
which it is preserved (or from other refrigerated systems of preservation) at the
moment of transplantation.
Contrast-induced nephropathy (CIN) Absolute (0.5 mg/dl) or relative increase

(25 %) in serum creatinine level at 48–72 h after exposure to a contrast agent
compared to serum creatinine at baseline.
Delayed graft function Dialysis requirement during the first week after kidney
transplantation.
Siderophores Iron-carrying molecules which are essential to bacterial and eukary-

otic cells to meet their needs of this mineral and then ensure survival.
Introduction
NGAL (neutrophil gelatinase-associated lipocalin), also referred to as lipocalin-2,

siderocalin, uterocalin, and 24p3, is a small 25-kD peptide belonging to the lipocalin
superfamily. The latter includes molecules with a three-dimensional structure char-
acterized by an eight-stranded antiparallel b-barrel surrounding a central pocket;
through this central calyx, they interact with different kinds of low-molecular-weight
ligands (Flower 1996; Kjeldsen et al. 2000; Bolignano et al. 2008a).
NGAL mainly binds to siderophores (Goetz et al. 2000), small iron-carrying
molecules which are essential to bacterial and eukaryotic cells to meet their needs of
this mineral and then ensure survival.
NGAL name originates from the fact that this protein was firstly discovered in
activated neutrophils where it is covalently bound to the enzyme gelatinase present
in the cytoplasmic azurophilic granules, and serum NGAL mainly derives from these
cells under normal conditions. It is also produced by other elements of the immune
system, such as macrophages, in response to the addition of lipopolysaccharide and
other bacterial products. Indeed, NGAL primarily acts as an innate nonspecific
antibacterial factor, since it prevents bacteria to internalize iron thus inhibiting
their growth (Meheus et al. 1993; Malyszko et al. 2010; Helanova et al. 2014).
The interesting observation that viral infections were able to stimulate NGAL
production also in cells not belonging to the immune system, as demonstrated in
in vitro models of murine kidney cells infected with strains of SV-40 virus (Hraba-
Renevey et al. 1989), has for the first time raised the hypothesis that this protein
could be much more than just a factor involved in nonspecific mechanisms of
cellular immunity. This theory was further supported by the demonstration that
NGAL was able to interact with many ligands other than siderophores such as
hepatocyte growth factor, gelatinase-B, different kinases, and proteins of intra- and
extracellular matrix (Kjeldsen et al. 1993; Yan et al. 2001). Moreover, later studies
revealed that the promoter region of the NGAL gene is subject to transcriptional
regulation by various factors including, for example, NF-kB (Cowland and
Borregaard 1997; Cowland et al. 2003), a key element involved in tissue prolifera-
tion, in tumor growth, in the inflammatory acute phase mechanisms, and, most
importantly, in the cellular response to and defense against various types of acute
and chronic injuries such as ischemia and oxidative stress.
Fig. 1 Molecular mechanisms of action of NGAL at the cellular level. NGAL can be internalized
into the cell either alone (Apo-NGAL) or as a complex with the iron-binding siderophores (Holo-
NGAL) (Bolignano and Buemi 2009; with permission from Publisher Nuova Editoriale Bios)
NGAL cellular activities are closely influenced by the interaction with specific
membrane receptors present on almost all human cells; among them are 24p3R,
which belongs to the family of brain-type organic cation transporters, and the multi-
scavenger complex of megalin that is mainly found on the brush border of renal
tubular cells (Devireddy et al. 2005; Hvidberg et al. 2005).
Following the interaction with these receptors, NGAL can be internalized into the
cell either alone (Apo-NGAL) or as a complex with the iron-binding siderophores
(Holo-NGAL). The different configuration with which NGAL enters the cell is crucial
because this may lead to diametrically opposite effects. Holo-NGAL is captured
within endosomal vesicles and transported into the cytoplasm where it can release
its iron-siderophore complexes, so enhancing specific iron-mediated intracellular
activation pathways such as cell growth or mitigation of oxidative stress. The protein
core can at this stage be degraded or expelled outside the cell as Apo-NGAL.
Conversely, Apo-NGAL, once internalized, is able to attract the intracellular iron
and release it outside the cell with resulting depletion of iron reserves; this condition
can sometimes stimulate gene pathways producing exactly opposite effects compared
to the previous ones including the induction of apoptosis and subsequent programmed
cell death (Devireddy et al. 2005; Schmidt-Ott et al. 2007; Fig. 1).
Most likely, this dual cellular action ascribed to NGAL is at the base of the numerous
inconsistencies reported by various studies with the consequent impossibility, some-
times, to assign a univocal role to this protein in the pathophysiology of some diseases.
According to what just said above, serum NGAL levels raise not only in the
course of infective processes but also in other pathological conditions, as the result
of an increased production and release from several tissues after an injury. For
instance, NGAL is significantly high during inflammatory diseases involving intes-
tinal epithelium and endothelium (Playford et al. 2006), the skin, and distal and
proximal airways (Cowland et al. 2003); it is also hyper-expressed in atherosclerotic
plaques and infarcted myocardium (Hemdahl et al. 2006).
A special attention should be paid to the numerous experimental and clinical data
supporting a role for NGAL as a biomarker in nephrology. Indeed, several studies
have been performed to evaluate the usefulness of NGAL in the clinical settings of
acute kidney injury (AKI), chronic kidney disease (CKD), autosomal-dominant
polycystic kidney disease (ADPKD), and renal transplantation.
The aim of the present review has been therefore to summarize all the most recent
findings concerning the potential use of NGAL as a diagnostic and prognostic
marker as well as a marker of therapeutic response in patients with renal diseases.
NGAL and Acute Kidney Injury
In current clinical practice, AKI is typically diagnosed by measuring the concentra-

tions of serum creatinine. This parameter, however, is not very reliable in detecting
acute changes in renal function for several reasons: it can significantly vary with age,
sex, muscle mass, and hydration status and can be distorted by particular drug
treatments, and especially it may remain unchanged as long as a substantial percent-
age of renal function does not become compromised. In addition, it is always
necessary to attain a steady state in glomerular filtration before the levels of
creatinine rise, and this, in most cases, may take several days.
Unfortunately, also other parameters such as the amount of diuresis, the urinary
levels of certain high-molecular-weight proteins (e.g., beta-2-microglobulin, fatty
acid-binding protein [FABP], etc.), and the calculation of the excreted sodium
fraction or that of the cylinders in the sediment are not very sensitive or specific
for the early detection of AKI; hence, the need of finding new biomarkers able to
overcome such limitations. To date, the ideal biomarker of AKI still does not exist. It
should first be precocious, that is, detectable long before the positivization of the
most common parameters of renal impairment (urine output, creatinine, etc.), sensi-
tive, able of minimizing the possibility of false negatives, as specific as possible for
AKI, easily available and analyzable, and of course inexpensive.
The surprising predictive properties of NGAL for the onset of AKI have been for
the first time revealed by Mishra et al. (2005) in a famous study published in Lancet.
The authors analyzed NGAL serum (sNGAL) and urinary (uNGAL) levels in a
cohort of 71 children undergoing cardiopulmonary bypass (CPB) surgery, a proce-
dure known to be burdened with high risk of postoperative AKI. The 20 subjects
who developed this complication showed a significant increase of NGAL already 2 h

after the end of the surgical intervention (whereas creatinine increased only after
1–3 days), unlike the others whose NGAL levels remained almost unchanged
throughout the entire length of hospital stay. Multivariate analyses confirmed the
validity of NGAL assay in serum and (especially) in the urine as an independent
predictor of AKI, and the use of ROC curves showed an excellent diagnostic power
of uNGAL considering a cutoff of 50 ng/ml (specificity 98 %, sensitivity 100 %).
Two subsequent studies, extended to more numerous cohorts of pediatric patients
subjected to the same type of surgery (CPB), have clearly validated these prelimi-
nary results. In the first case, Dent et al. (2007) confirmed the predictive power of
sNGAL through its measurement in 120 subjects, of which 45 (37 %) developed
AKI. In these patients, an increase of sNGAL levels up to three times was observed
already 2 h after surgery; in multivariate analysis, sNGAL value measured after 2 h
also turned out to be the most significant independent predictor of AKI (beta =
0.004, p <0.0001), furthermore showing a great diagnostic power with an AUC of
0.96, a sensitivity of 0.84, and a specificity of 0.94 for a cutoff of 150 ng/ml. Similar
results were reported by Bennett et al. (2008), this time taking into consideration
uNGAL measurement in 196 children undergoing CPB. In those who developed
AKI (51 % of patients), uNGAL levels increased by approximately 15 times 4 h after
surgery and even up to 25 times after 6 h, while creatinine began to increase only in
the second day. The ROC analysis showed the excellent diagnostic power of uNGAL
at 2 h in predicting the future onset of AKI (cutoff 100 ng/ml, AUC 0.95, sensitivity
82 %, specificity 90 %). This parameter also appeared to be directly and indepen-
dently correlated with the severity and duration of AKI, length of hospitalization,
need for replacement therapy, and even mortality.
Later studies have attempted to extend these findings also to populations of adult
patients; in an interesting work, Haase-Fielitz et al. (2009a) have indeed confirmed
that NGAL plasma levels are independent predictors of AKI development
(established as a 50 % increase in serum creatinine), as well as of a composite
endpoint represented by the need of dialysis treatment and the inhospital mortality, in
patients undergoing major heart surgery. The predictive ability of NGAL also
increased with the severity of AKI and was directly related to the stage of renal
function impairment according to the RIFLE or AKIN criteria, the two main scales
of AKI severity used by the intensive care nephrologists (Haase-Fielitz et al. 2009b).
Wagener et al. (2008) have instead analyzed uNGAL levels in a much larger
cohort of patients (n. 426) undergoing major heart surgery. These values increased
immediately after the procedure, remained significantly elevated up to 24 h after the
operation, and were also related to the duration of the intervention and that of aortic
clamping. uNGAL showed however a poor diagnostic power at each measurement
time (3, 18, and 24 h) in predicting the future onset of AKI (AUC from 0.573 to
0.584), defined by an increase in serum creatinine >50 % or >0.3 mg/dl within 48 h.
On the contrary, the diagnostic performance of uNGAL significantly improved if the
measurement of this parameter was supplemented by that of other similar bio-
markers, such as kidney injury molecule-1 (KIM-1) and N-acetyl-β-D-
glucosaminidase (NAG) (AUC 0.780) (Han et al. 2009).
The interesting NGAL predictive abilities toward the onset of AKI have recently
led to the launch of specific automated “point-of-care” systems able to provide a
rapid quantification of NGAL in biological samples obtained from patients (for the
most critical) at high risk of developing AKI. Similarly to what already happens
long since for troponin (as a marker of myocardial infarction) and, more recently, for
BNP (whose levels seem able to discriminate a cardiogenic dyspnea from those of
other causes), the purpose of these tools would be to provide the intensivist taking
charge of a patient at risk of AKI with a single datum in real time that indicates the
presence or absence of such a complication in an immediate way. The fundamental
premise for this is of course to have two important validated parameters: the best
timing of measurement and the exact threshold value which discriminates the
impending AKI from its sure absence. If for the first parameter “pathophysiological”
prerequisites exist that address to NGAL measurement from 2 to 6 h after the alleged
traumatic event for the kidney (there is a strong clinical and experimental agreement
on the peak of release by the tubular cells), probably it has been instead tried to
establish the elusive discriminating threshold value with too much “haste.” In the
study of Haase-Fielitz et al. (2009a), for instance, a value of 150 ng/ml (sNGAL) is
proposed as a cutoff capable of distinguishing, through a single measurement, the
imminent onset of AKI in the patient just arrived in the intensive care unit. This would
allow the “preventive” implementation of therapeutic supports (vasotonic drugs,
volume expansion, or even dialysis) able to avoid the complication in question.
Since the critical patient often presents different conditions or comorbidities (infec-
tions, malignancies, anemia, chronic inflammations, atherosclerosis, diabetes, vascular
disease, chronic kidney diseases, or recent treatment with steroids) that are causes per
se of increased NGAL levels well beyond the recently proposed threshold values, the
idea to report the presence of looming AKI to a single measurement (e.g., <150 ng/ml
= no AKI, >150 ng/ml = ongoing AKI) has been recently criticized, as potentially
affected by too many conditions (Bolignano et al. 2009a). Conversely, the evaluation of
a “delta” value (i.e., the analysis of the pre- and postoperative change in NGAL levels)
might be most useful and reliable: the presence of a documentable sharp increase of
NGAL values would in fact reflect more realistically an underway kidney suffering.
Anyway, the predictive abilities of NGAL toward the onset of AKI have recently
been extended also to other types of risk patients with similar interesting results.
Makris et al. (2009) analyzed uNGAL levels in 31 patients with multiple trauma,
respectively, at the admission to the intensive care unit, 24 and 48 h after. uNGAL had
a great diagnostic power (AUC 0.977) in predicting the future onset of AKI defined
according to the RIFLE criteria, with an optimal cutoff of 25 ng/ml (sensitivity 0.91,
specificity 0.95). In agreement with the previously mentioned concerns, the authors
excluded from the study patients with preexisting heart or chronic kidney disease.
Contrast-induced nephropathy (CIN) is another important cause of AKI. The
identification of predisposing conditions such as the use of iodinated hyperosmolar
contrast media, the presence of diabetes or preexisting renal impairment,
hypovolemia, hematological disorders, and concomitant nephrotoxic drugs intake
proved not to be sufficient alone to drastically reduce the incidence of this compli-
cation. Moreover, to date there is not even a consensus on possible preventive and/or
precocious therapeutic strategies (volume expansion, intravenous, and/or oral bicar-

bonates, N-acetylcysteine, etc.) to be put in place to counteract the evolution of this
threat, with the result that the risk of CIN still remains, especially in the renal patient,
one of the most serious contraindications to the execution of important diagnostic-
therapeutic procedures such as coronary arteriography and computed tomography
(CT).
It has been shown how, in a cohort of mostly diabetic patients undergoing
percutaneous coronary intervention (PCI), sNGAL and uNGAL levels increased,
respectively, from 2 to 4 h and from 4 to 12 h after the end of this procedure
(Bachorzewska-Gajewska et al. 2006). Despite not considering the relationship
between NGAL values and outcome (CIN or non-CIN), nevertheless this study
has the merit to have demonstrated for the first time that the contrastographic
procedure induces an important alteration in NGAL levels, suggesting that this
reflects an early renal suffering still not detectable by the increase in serum
creatinine.
Similar observations were afterward reported by another work that has taken in
consideration not only the urinary values of NGAL but also those of IL-18, another
potential biomarker of AKI (Ling et al. 2008). Twenty-four hours after the execution
of a coronary angiography, the levels of both biomarkers were significantly
increased from baseline in subjects who would have later developed CIN and then
AKI. NGAL also showed a diagnostic ability toward the outcome significantly better
than that of serum creatinine, and IL-18 resulted even independently associated with
late cardiovascular events in the course of a follow-up of 17 months. In another
interesting work, Hirsch et al. (2007) enrolled 91 pediatric patients with major heart
malformations requiring elective cardiac catheterization and angiography with
administration of iodinated contrast medium (ioversol). In the 11 subjects (12 %)
who developed CIN, defined as an increase >50 % in serum creatinine from
baseline, sNGAL and uNGAL levels increased significantly already 2 h after the
end of the procedure, whereas the increase of creatinine appeared only after 24 h.
The values of uNGAL and sNGAL at 2 h revealed to be independent predictors of
CIN with an optimal cutoff of 100 ng/ml and an AUC of 0.92 (sensitivity 73 %,
specificity 100 %) and 0.91 (sensitivity 73 %, specificity 100 %), respectively.
More recently, it has been demonstrated that NGAL revealed CIN within 8 h after
iodinated contrast material injection and that therapy with N-acetylcysteine, sodium
bicarbonate, or physiologic saline immediately before and after administration of the
contrast agent did not influence NGAL values (Lacquaniti et al. 2013).
NGAL and Autosomal-Dominant Polycystic Kidney Disease

(ADPKD)
The autosomal-dominant polycystic kidney disease (ADPKD) is a congenital disor-

der characterized by an alteration of the renal parenchyma which is progressively
replaced by multiple bilateral cysts of tubular origin, with resulting disruption of the
normal corticomedullary architecture (Santoro et al. 2015). Despite the genetic bases
of this condition are well known (mutation of the genes PKD1, PKD2, PKD3 with
formation of abnormal polycystins), it is still unclear why some patients present a
greater tendency to develop complications (arterial hypertension, polyglobulia, etc.)
and a more rapid progression toward terminal uremia, whereas others, the cystic
development being equal, maintain a perfectly preserved renal function over time.
Patients with ADPKD showed noticeably increased NGAL values compared to
healthy subjects (Bolignano et al. 2007). These levels were also closely related to the
amount of residual renal function, showing a direct correlation with the values of
creatinine and an inverse relationship with those of estimated GFR (sNGAL/GFR,
R = 0.81, p = 0.006; sNGAL/creatinine, R = 0.90, p = 0.007; uNGAL/GFR,
R = 0.49, p = 0.05; uNGAL/creatinine, R = 0.84, p = 0.001). Unexpectedly, if
patients were categorized into two groups based on ultrasound criteria of phenotypic
expression of the disease (number of cysts < or >10; renal length < or >16 cm),
regardless of residual renal function subjects with larger kidneys and more signifi-
cant cystic development showed NGAL levels statistically higher than the others,
thus suggesting a possible involvement of NGAL in the pathophysiology of the
adaptations to polycystic disease. It must be remembered that in physiological
conditions, NGAL is an important differentiating and structuring factor for the
renal tubular epithelium, since the suppression of its gene expression in the embry-
onic organ leads to the formation of cystic-looking poorly organized structures
instead of mature tubules (Gwira et al. 2005).
Starting from this assumption, Wei F et al. (2008) provided, through an experi-
mental model, interesting pathophysiological explanations to the clinical observa-
tions on patients with ADPKD; the addition of recombinant NGAL in cultures of
PKD1/ polycystic kidney cells proved in fact to be able to significantly reduce
both the area and the volume of the cystic development, whereas, on the contrary, the
silencing of NGAL gene expression through the use of specific short hairpin RNAs
(shRNAs) markedly increased the absolute number of these cells and their prolifer-
ative index. All this would suggest that the significant increase in the biological
levels of NGAL found in patients with ADPKD could represent the exasperation of a
compensatory mechanism aimed at restricting cystic growth, assuming a possible,
faraway therapeutic use of this protein in the context of a disease which to date
knows only symptomatic treatments.
NGAL and Chronic Kidney Disease
The potential involvement of NGAL in the pathophysiology of chronic kidney

diseases does not appear to be limited to ADPKD. In particular, the close parallelism
between NGAL levels and the extent of renal impairment, observed for the first time
in patients with polycystic kidney disease (Bolignano et al. 2007), is evident in an
unequivocal manner in any patient with CKD, irrespective of the underlying renal
disease. More specifically, serum and urine levels of NGAL were evaluated in
69 patients with varying degrees of CKD secondary to different etiologies such as
glomerulonephritis, nephroangiosclerosis, and obstructive nephropathy (Bolignano
a 1400 b 1000 *
1200
p<0.005 * p<0.004
750
(ng/g creatinine)
1000
urinary NGAL
serum NGAl
(ng/mL)
800
500
600
400
250
200
0 0
Controls CKD Controls CKD
c R=−0.739 ; p<0.0001 d R=−0.771 ; p<0.0001

80
90
80 70
GFR Cockcroft/Gault
GFR Cockcroft/Gault
70 60
60
(mL/min)
50
(mL/min)
50 40
40
30
30
20
20
10 10
0 0
1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 1.75 2.00 2.25 2.50 2.75 3.00 3.25
Log sNGAL (ng/mL) Log uNGAL (ng/g creatinine )
Fig. 2 Serum and urine levels of NGAL in patients with varying degrees of chronic kidney disease
(CKD) secondary to different etiologies. Comparison of serum (a) and urinary (b) NGAL levels
between controls and patients with CKD. Statistical correlations between glomerular filtration rate
(GFR) and log-transformed sNGAL (c) and between GFR and log-transformed uNGAL (d) in CKD
patients (Bolignano and Buemi 2009; with permission from Publisher Nuova Editoriale Bios)
et al. 2008b). These levels and the fractional excretion of NGAL (FeNGAL),
calculated according to the formula uNGAL/sNGAL x serum creatinine/urine cre-
atinine 100, were significantly increased compared with control subjects. Univar-
iate correlations were also described between GFR values (Cockcroft-Gault formula/
MDRD formula) and, respectively, those of sNGAL (R = 0.739, p <0.0001/
R = 0.732, p <0.0001), uNGAL (R = 0.771, p <0.0001/R = 0.769,
p <0.0001), and FeNGAL (R = 0.452, p <0.001/R = 0.450, p <0.001) as well
as between the values of creatinine and those of sNGAL (R = 0.445, p <0.001) and
uNGAL (R = 0.399, p <0.001). In addition, after multivariate adjustment, the
correlations between GFR values and those of sNGAL (= 0.645, p <0.01) and
uNGAL (= 0.688, p <0.005) remained significant, thus confirming to be inde-
pendent (Fig. 2). In two other studies, the close relations between NGAL and degree
of renal impairment were confirmed also in cohorts of patients much more homo-
geneous for etiology of CKD, e.g., membranous glomerulonephritis (Bolignano
et al. 2008c) or diabetic nephropathy (Bolignano et al. 2009b): this further reinforces
the idea that the relationship between NGAL and residual renal function is anything
but random, representing on the contrary a constant that accompanies the progres-
sion of chronic renal failure. Considering the close correlations between NGAL and
creatinine as well as those even more significant (and inverse) between NGAL and
GFR, one might first assume that the increased levels of this protein are simply due
Fig. 3 Forest fire theory. According to this theory, the increased NGAL values during CKD, which
is comparable to a forest fire, are the result of an active and sustained production by residual tubular
cells still viable but “inflamed” by the surrounding renal disease, where instead the reduction in
GFR and the consequent increase in serum creatinine are only the passive consequence of the
functional loss of nephron units (and then tubules) already “burned” by the forest fire (Bolignano
and Buemi 2009; with permission from Publisher Nuova Editoriale Bios)
to the reduction in the ability of renal clearance, thus representing nothing more than
a mere surrogate of the function of this organ similarly to what happens for
creatinine.
Mori and Nakao (2007) have instead provided an interesting alternative expla-
nation on the meaning of the correlations between NGAL biological levels and
renal function indices, developing the so-called theory of the burning forest
(Forest Fire Theory; Fig. 3). According to this theory, the increase in NGAL
values during CKD, comparable to a forest fire, is the result of an active and
sustained production by residual tubular cells still viable but “inflamed” by the
surrounding renal disease, where instead the reduction in GFR and the consequent
increase in serum creatinine are only the passive consequence of the functional
loss of nephron units (and then tubules) already “burned” by the forest fire. From
this point of view, NGAL production by tubular cells (with a defensive meaning
very similar to that described in conditions of AKI) would then represent a kind of
“real-time” indicator of how much damage and active suffering are present within
chronic renal impairment. Although it cannot ruled out that other sources (acti-
vated neutrophils, systemic inflammation, atherosclerosis, etc.) can contribute to
the increase in NGAL levels in the course of chronic kidney diseases, there is
further evidence supporting the central role of the renal tubular cells in the active
production and disposal of blood and urinary NGAL. In a cohort of patients
affected by childhood-onset systemic lupus erythematosus with renal involve-
ment, Brunner et al. (2006) observed close correlations between NGAL levels and
severity of the activity and chronicity scores of the disease evaluated on samples
of renal biopsies. These correlations proved to be also much stronger than those
seen with any other renal and extrarenal index of disease severity. Ding
et al. (2007) have instead evaluated NGAL tissue expression in a cohort of
patients suffering from IgA nephropathy with varying degrees of biopsy severity
(Lee classification), observing a very strong histological positivity, localized
predominantly at the tubular level, in patients with high-grade kidney injury
(Lee grade III). The amount of such tissue expression also strongly correlated
with uNGAL levels, confirming the idea of an active production by suffering
tubular cells.
As previously discussed (Bolignano et al. 2008b), CKD patients have, as well as
an increase of sNGAL and uNGAL absolute values, also an elevation of FeNGAL
compared to healthy subjects. Normally, the calculation of the fractional excretion
is applied to sodium balance (FeNa) in subjects showing an acute diuresis contrac-
tion, in order to discriminate the renal or prerenal origin of the functional block of
this organ. More in detail, a FeNa <1 % suggests a conserved ability of the kidney
to reabsorb sodium aimed at preserving the hemodynamic stability threatened by
various causes (cardiogenic or circulatory shock, hypovolemia, etc.): this orients
toward the presence of a prerenal oliguria. Conversely, a high FeNa (>1 %) gives
evidence of renal parenchymal damage with consequent active loss of this electro-
lyte through the tubular structures altered by different potential causes (ischemia,
drugs, infections, etc.): the so-called renal or organic oliguria. By extending this
formula to NGAL balance, it has been noted that not only patients with CKD
present a FeNGAL significantly increased compared to healthy controls, but also
this invariably results well above the value of 1 %, once again supporting the idea
that the damaged kidney actively supports the increased biological levels of this
protein.
As a further confirmation, even patients with diabetic nephropathy have increased
values of FeNGAL compared to controls, but only those with severe renal functional
impairment (macroalbuminuria and reduced GFR) show a FeNGAL >1 %
(Bolignano et al. 2009b).
However, the relationships between NGAL and diabetic nephropathy go far
beyond this observation, and this study designed specifically on patients with this
condition has clearly demonstrated as the diagnostic potentialities (and with them,
the pathophysiological significance) of NGAL in the field of chronic kidney diseases
can take a particular interest precisely in those subjects who, while not having yet
developed an overt CKD (then with GFR >90 ml/min/1.73 m2, stage I according to
the recent NKF-KDOQI guidelines), show however early signs of renal involvement
(e.g. microalbuminuria or microproteinuria). In this regard, it was observed that in
patients with type 2 diabetes mellitus treated with insulin, categorized according to
the extent of albumin urinary excretion in normoalbuminuric (albuminuria/
creatinuria ratio <30), microalbuminuric (ratio >30 but <300), and with overt
diabetic nephropathy (ratio >300 and reduced GFR), NGAL levels increase in
parallel to the extent of the glomerular damage resulting highest in subjects with
reduced GFR and macroalbuminuria (Fig. 4). Very interestingly, patients with
normoalbuminuria, so devoid of any clinical/laboratory sign of renal involvement
Fig. 4 In patients with type 2 diabetes mellitus, NGAL levels increase in parallel to the extent of
the glomerular damage resulting highest in subjects with reduced GFR and macroalbuminuria. (a)
sNGAL levels in healthy controls and diabetic patients with normoalbuminuria (NA),
microalbuminuria (MA), and overt diabetic nephropathy (DN). #, p <0.01 versus controls; *,
p <0.001 versus controls; **, p <0.01 versus NA; ***, p <0.01 versus MA. (b) uNGAL values
in control subjects and diabetic patients with NA, MA, and DN. #, p <0.01 versus controls; §,
p <0.05 versus NA; *, p <0.001 versus controls; **, p <0.01 versus NA; ***, p <0.05 versus MA
(Bolignano and Buemi 2009; with permission from Publisher Nuova Editoriale Bios)
from diabetes, had sNGAL and uNGAL levels already significantly increased
compared to controls. NGAL also showed an excellent diagnostic ability in detecting
the presence of diabetes among all subjects with normal albumin urinary excretion
(controls + normoalbuminuric patients) with an AUC of 0.969 and a cutoff >88 ng/ml
for sNGAL and an AUC of 0.910 and a cutoff >22 ng/ml for uNGAL. These
unexpected and surprising findings indicate that NGAL may represent a biomarker of
kidney (tubular) damage from diabetes even more precocious than microalbuminuria
(glomerular), supporting those recent theories that sustain the existence of a phase of
renal tubule suffering that temporally precedes the appearance of the best known
glomerular changes responsible for microalbuminuria and proteinuria (Thomas
et al. 2005).
Anyway, it is clear that even in the absence of a GFR reduction, and then of an
overt renal failure, significant alterations of NGAL biological levels can already be
identified, although it is not yet possible to ascribe a precise pathophysiological

significance to this phenomenon.
It is no coincidence that the most recent NKF-KDOQI US guidelines have revised
the classification of CKD severity by including already in stage I those subjects with
still preserved GFR (>90 ml/min) but with persistence of clinical/laboratory signs of
CKD such as hematuria and especially persistent proteinuria.
Furthermore, it is known that the latter condition, regardless of the underlying
disease (e.g., diabetes or primary and secondary glomerulonephritis, etc.), represents
not only an expression of glomerular damage but also a cause per se of progression
of nephropathy toward the establishment of an evident organ failure. The persistence
of blood proteins in the tubular lumen (especially if proteinuria is in the nephrotic
range, >3.5 g/24 h) is in fact harmful to the epithelial cells through activation of the
complement cascade and subsequent immune-mediated damage. This leads to the
appearance of tubular atrophy and interstitial fibrosis, irreversible lesions that mark
the onset of a functional deficit of the nephron units and, with it, of the reduction of
renal clearance ability (Abbate et al. 2006; Morita et al. 2000).
Patients with macroproteinuria due to membranous glomerulonephritis have high
urinary NGAL excretion compared to healthy subjects even in the presence of
normal GFR (Bolignano et al. 2008d), and, when the daily proteinuria becomes
particularly severe (e.g., in the nephrotic range), uNGAL levels may increase over
500 times the norm, becoming also closely related to the values of proteinuria itself
(Bolignano et al. 2008c, e).
The reasons of such an important increase, despite the absence of overt renal
failure, are not as yet known; however, considering the renal turnover of NGAL in
normal and pathological conditions, several hypotheses can be postulated (Fig. 5).
In the healthy adult kidney, NGAL is freely filtered through the glomerulus and
physiologically almost completely reabsorbed in the proximal tubular tract
(Helanova et al. 2014). This reabsorption is mediated by the coupling with the
membrane transporter cubilin-megalin, particularly expressed on the surface of the
cellular brush border. Afterward, NGAL is incorporated by endocytosis and
removed from the tubular lumen, so that only small amounts (approximately 5 ng/
ml) of this protein are found in the urine of healthy subjects (Cowland et al. 2003).
An initial hypothesis that would justify the high NGAL levels in the urine of
proteinuric patients may be represented by an increased loss of circulating NGAL
through the damaged glomerulus, as it happens with other plasma proteins; this
concept would be partially supported by the observed correlations between NGAL
levels and amount (g/24 h) of daily proteinuria as well as between sNGAL and
uNGAL values (Bolignano et al. 2008c, d, e, 2009b).
Although plausible, this model does not exclude, however, that at least in part the
same tubular cells may just as significantly contribute to the massive elevation of
uNGAL: from this point of view, at least two mechanisms could be conceivable.
Firstly, the aforementioned cubilin-megalin carrier, responsible for NGAL resorp-
tion, acts through a mechanism of nonspecific protein endocytosis that recognizes
several other serum ligands such as albumin, beta2-microglobulin, and serum
immunoglobulins. In confirmation of this, murine models of knockout mice for
Fig. 5 Possible explanations for the increased uNGAL levels in patients with proteinuria. (a)
Physiologically, sNGAL is filtered through the glomerulus and almost completely reabsorbed by the
proximal tubule through the cubilin-megalin carrier. (b) In proteinuric diseases, an increased
amount of NGAL could be lost through the damaged glomerulus similarly to what happens for
other circulating proteins. Besides, the megalin carrier is rapidly saturated by the considerable
protein tubular overload, further decreasing the ability to reabsorb NGAL. (c) Megalin function
would be further compromised by the injured tubular brush border due to the complement cascade
activation caused by persistent proteinuria. (d) Damaged tubular epithelial cells themselves may
actively produce and release NGAL as a defense mechanism against oxidative stress and comple-
ment activation (Bolignano and Buemi 2009; with permission from Publisher Nuova Editoriale
Bios)
megalin soon develop a condition of severe nonselective proteinuria, even in the

absence of documentable glomerular histological lesions (Leheste et al. 1999).
Under conditions of sustained proteinuria (e.g., nephrotic syndrome), this
nonspecific carrier soon becomes saturated because of the massive tubular protein
overload, causing further loss of plasma proteins that in part contributes to determine
the extent of final proteinuria (Christensen and Gburek 2004). It is also to be
considered that the main site of the damage induced by proteinuria through comple-
ment activation is precisely the brush border of the tubular cells, where the most of
the cubilin-megalin complexes are located: this condition would further contribute to
compromise NGAL endocytosis by its carrier. Ultimately, it cannot be excluded,
however, that the same tubular cells, subjected to stress from the insult caused by the
activity of complement factors, actively produce and release high amounts of NGAL
with a defensive significance similar to what observed in experimental models of
acute kidney damage. In accordance with this, previous studies have shown that the
tubular epithelium responds to a sustained load of plasma proteins through the

release of multiple “stress proteins” including KIM-1, whose urinary levels accord-
ingly rise in a dramatic way (van Timmeren et al. 2006).
In conclusion, waiting for specific studies doing more light on the topic, it is not
to exclude that, in the end, both the passive loss and the active tubular production
contribute in net measure to the increase of NGAL levels observed in the urine of
proteinuric patients.
NGAL in Kidney Transplant Patients
Another interesting field of application of NGAL seems to be represented by the

measurement of its biological levels in patients just subjected to kidney transplan-
tation. Even in these patients, the risk of AKI is particularly fearful, not so much as
the result of an acute rejection (which is now well prevented by the massive
immunosuppressive therapy) as for a possible late organ functional recovery that
leads to the need of a temporary hemodialysis support (the so-called delayed graft
function, DGF); moreover, this condition is much more frequent if the transplanted
kidney is from a cadaver than from a living donor, especially in relation to the
occurrence of a more or less prolonged cold ischemia.
Patients waiting for a kidney, and then subjected to chronic hemodialysis treat-
ment, have NGAL levels significantly increased compared to healthy controls
(Bolignano et al. 2009c, d, 2010a; Kusaka et al. 2008). It has been shown that
since the first day after transplantation, NGAL values tend to decrease progressively
and in a dramatic way, preceding the recovery of normal diuresis and lowering of
serum creatinine: this, however, does not happen in those subjects who will subse-
quently develop a DGF with the need to receive renal replacement therapy (Kusaka
et al. 2008; Lebkowska et al. 2009).
In biopsy samples from just implanted organs, the levels of NGAL tissue expres-
sion are also significantly higher in cadaver than in living donor kidneys, correlating
in the former with the time of cold ischemia, the postoperative creatinine peak
(which occurs with a latency of several days), and the need for hemodialysis due
to the appearance of a DGF (Mishra et al. 2006).
In another interesting work, Parikh et al. (2006) assessed urine NGAL and IL-18
levels in 53 kidney transplant patients immediately after surgery. In the ten patients
who developed a DGF over the 2–4 following days, the basal urinary values of both
biomarkers were significantly higher than those observed in patients with normal
postoperative course, and both NGAL and IL-18 showed a considerable diagnostic
power (AUC 0.9) in predicting the future occurrence of DGF. Nevertheless, in
another study, uNGAL levels did not prove to be equally able to distinguish the
histological presence of a subclinical tubular injury from a stable transplant with
normal tubular histology (Schaub et al. 2007): this would slow, at least for the
moment and waiting for more specific studies on the topic, the possibility of
extending the measurement of NGAL in the diagnosis of chronic rejection with
the same success.
Furthermore, in a very recent study (Buemi et al. 2014), plasma and urine NGAL
levels were assessed in a cohort of kidney donors before organ explantation and in
recipients before transplantation and then 6, 24, and 48 h after the surgical interven-
tion, in order to evaluate the ability of this biomarker to predict DGF occurrence and
posttransplant function restoration. The authors observed that recipient but not donor
plasma NGAL values seem to predict DGF incidence and renal function recovery,
however, too long for an interval to be able to compete with the markers of kidney
function at present employed in clinical practice.

Conditions
The potential use of NGAL as a biomarker in clinical practice appears not to be

limited to the early diagnosis of kidney diseases (Mishra et al. 2005; Bolignano
et al. 2009b; Lacquaniti et al. 2012).
This protein has been also demonstrated to predict CKD progression (Bolignano
et al. 2009e; Lin et al. 2015), in such a way becoming a reliable prognostic factor
which might help nephrologists in the risk stratification and in the implementation of
appropriate preventive therapeutic strategies with the ultimate purpose of improving
the management of nephropathic patients.
Moreover, NGAL may represent a precocious marker of therapeutic response
(Cernaro et al. 2011), as observed in patients suffering from severe proteinuria
secondary to idiopathic membranous nephropathy and treated with a single high-
dose bolus of intravenous immunoglobulin (Bolignano et al. 2008d) and in patients
affected by Crohn’s disease and receiving infliximab (Bolignano et al. 2010b). In
both cases, a marked reduction was found in NGAL levels after the administration of
the respective drugs. This suggests the possible application of NGAL measurement
in monitoring the effectiveness of a particular treatment and predicting different
clinical outcomes in the course of renal as well as extrarenal diseases characterized
by an increase in the serum and urinary levels of this protein (Cernaro et al. 2011).
Indeed, recent studies propose for NGAL a role of “biomarker beyond the confines
of nephrology” (Bolignano et al. 2010c). This molecule is involved, for instance, in
carcinogenesis and progression of several human tumors (Bolignano et al. 2010d;
Barresi et al. 2010; Lippi et al. 2014), in the regulation of red blood cell growth by
inhibiting the maturation and differentiation of bone marrow erythroid precursors
(Bolignano et al. 2010e), and in the pathophysiology of cardiovascular diseases
(atherosclerosis, acute myocardial infarction, heart failure) (Bolignano et al. 2010c).
Although the oncological and cardiovascular settings constitute two promising
horizons of application, to date, nephrology represents the field with the most
imminent opportunities of NGAL transposition in daily clinical practice. In particular,
the existence of a close involvement of this protein in the maturation of the embryonic
kidney, as well as in defense mechanisms against acute and chronic stress in the adult
kidney, has led to the real possibility in the next future of using measurement of
NGAL biological levels for previously unimaginable purposes: evaluation of AKI
risk in critically ill patients with the chance of carrying out early therapeutic inter-
ventions that radically alter the incidence of adverse outcomes, stratification of the
risk of progression to end-stage renal disease (ESRD) in CKD patients, monitoring of
renal and systemic response to different therapeutic regimens, evaluation of iron
balance, and dialysis adequacy in uremic patients on hemodialysis.
As previously described, in patients with renal disease, uNGAL levels have
increasingly gained importance in addition to serum values. This has raised the
problem of establishing the origin, renal or systemic, of NGAL dosed in the urine.
The issue can be solved by assessing FeNGAL, similarly to what is done for sodium
in order to determine the prerenal or organic etiology of AKI.
Moreover, a distinction have been made among the monomeric form of NGAL
secreted by injured renal tubular epithelial cells, the homodimeric form released by
activated neutrophils, and the heterodimeric form that is specific for tubular cells but
seems to be produced in very low amounts even during AKI (Cai et al. 2010).
The currently available commercial assays and point-of-care devices probably
measure a mixture of different forms of NGAL, having a limited ability to discrim-
inate among them. This compromises NGAL specificity for AKI, especially in
patients with systemic inflammation and several comorbidities. Such considerations
have recently led some authors to express concerns about the diagnostic and clinical
value of this biomarker (Mårtensson and Bellomo 2014).
In conclusion, several studies have shown the excellent ability of urinary and
serum NGAL levels to predict AKI onset or CKD progression in selected
populations of patients (Ronco et al. 2014). Nevertheless, the extension of these
experimental results to clinical practice requires assays able to specifically measure
the different forms of NGAL as well as larger trials which better define the normal
range of NGAL values and the correct interpretation of their alterations, in order to
confirm NGAL as the troponin of the kidney (Devarajan 2010) according to the
hypothesis that has been postulated from the beginning.
Summary Points
• Neutrophil gelatinase-associated lipocalin (NGAL) is a small 25-kD peptide

belonging to the lipocalin superfamily.
• Numerous experimental and clinical data support a role for NGAL as a biomarker
in nephrology.
• NGAL predicts the onset of acute kidney injury (AKI) better than serum creat-
inine and urine output.
• Patients with autosomal-dominant polycystic kidney disease (ADPKD) show
markedly increased NGAL values compared to healthy subjects.
• In chronic kidney disease (CKD) patients, NGAL closely correlates with the
extent of renal impairment irrespective of the underlying kidney disease and
predicts CKD progression.
• Since the first day after transplantation, NGAL values tend to decrease progres-
sively, preceding the recovery of normal urine output and lowering of serum
creatinine; this does not happen in those patients who will subsequently develop a
delayed graft function (DGF) with the need to receive renal replacement therapy.
• NGAL may represent a precocious marker of therapeutic response in different
clinical situations.
• NGAL exists in three forms: the monomeric form secreted by injured renal
tubular epithelial cells, the homodimeric form released by activated neutrophils,
and the heterodimeric form that is specific for tubular cells but seems to be
produced in very low amounts even during AKI.
• The currently available commercial assays and point-of-care systems probably
measure a mixture of different forms of NGAL.
• The extension of the experimental results to clinical practice requires assays able
to specifically measure the different forms of NGAL as well as larger trials which
better define the normal range of NGAL values and the correct interpretation of
their alterations.
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Chemokines as Potential Markers
in Pediatric Renal Diseases 11
Ana Cristina Simões e Silva, André Barreto Pereira,
Mauro Martins Teixeira, and Antônio Lúcio Teixeira
Contents
Key Facts of Chemokines in Pediatric Renal Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 231
Chemokines: General Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232
Chemokines in Renal Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233
Chemokines in Glomerular Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238
Chemokines in Congenital Uropathies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 239
Ureteropelvic Junction Obstruction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240
Vesicoureteral Reflux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
Potential Applications to Prognosis, Other Diseases or Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 244
A.C. Simões e Silva (*)

Unit of Pediatric Nephrology, Department of Pediatrics, Faculty of Medicine, Interdisciplinary
Laboratory of Medical Investigation, Federal University of Minas Gerais, Belo Horizonte, MG,
Brazil
e-mail: acssilva@hotmail.com; ana@medicina.ufmg.br
A.B. Pereira
Department of Molecular Medicine, Faculty of Medicine, Interdisciplinary Laboratory of Medical
Investigation, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
e-mail: andrebarper@yahoo.com.br
M.M. Teixeira
Department of Biochemistry and Immunology, Institute of Biological Sciences, Laboratory of
Immunopharmacology, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
e-mail: mmtex.ufmg@gmail.com; mmtex@icb.ufmg.br
A.L. Teixeira
Department of Medicine, Faculty of Medicine, Interdisciplinary Laboratory of Medical
Investigation, Federal University of Minas Gerais, Belo Horizonte, MG, Brazil
e-mail: altexr@gmail.com; altexr@medicina.ufmg.br

230 A.C. Simões e Silva et al.
Abstract
Recently, biomarkers have become a focus of clinical research as potentially useful
diagnostic and prognostic tools in many diseases. Among several putative bio-
markers, chemokines emerge as promising molecules since they play relevant roles
in the pathophysiology of chronic kidney disease. In pediatric patients, glomerular
diseases and congenital anomalies of the kidney and urinary tract are the
commonest cause of chronic kidney disease. The evaluation of these inflammatory
mediators might help the management of diverse renal diseases in children and the
detection of patients at high risk to develop chronic kidney disease. The aim of this
chapter is to revise general aspects of chemokines and the potential link between
chemokines and the most common causes of pediatric chronic kidney disease. The
literature revision showed that the chemokines more commonly associated with
pediatric renal diseases were CCL2/MCP-1 and CXCL8/IL-8. In glomerular dis-
eases, high urinary levels of CCL2/MCP-1 have been associated to FSGS, lupus
nephritis and IgA nephropathy. On the other hand, urinary levels of CXCL8/IL-
8 positively correlated with proteinuria in pediatric patients with primary nephrotic
syndrome, suggesting a role in glomerular permeability changes. With reference to
congenital anomalies of the kidney and urinary tract, the chemokine CCL2/MCP-1
has been associated with urinary tract obstruction, whereas high urinary levels of
CXCL8/IL-8 were found in patients with vesicoureteral reflux and correlated with
renal scarring and renal function deterioration.
Keywords
Chemokines • Biomarker • Glomerular diseases • Congenital anomalies of the
kidney and urinary tract • Chronic kidney disease
Abbreviations
CAKUT Congenital anomalies of the kidney and urinary tract
CCL1/TCA-3 T cell activation-3
CCL11/eotaxin Eosinophil chemotactic protein
CCL2/MCP-1 Monocyte chemotactic protein-1
CCL3/MIP-1a Macrophage inflammatory protein-1 alpha
CCL3/MIP-1α Macrophage inflammatory protein 1 alfa
CCL4/MIP-1b Macrophage inflammatory protein-1 beta
CCL5/RANTES Regulated on activation, normal T Expressed and Secreted
CX3CL1/fractalkine Chemokine (C-X3-C motif) ligand 1
CXCL10/IP-10 ɣ-interferon-inducible protein
CXCL13/BLC Serum B lymphocyte chemoattractant
CXCL2/MIP-2 Macrophage inflammatory protein-2
CXCL8/IL8 Interleukin 8
FSGS Focal segmental glomerulosclerosis
INS Idiopathic nephrotic syndrome
11 Chemokines as Potential Markers in Pediatric Renal Diseases 231
MCNS Minimal change nephrotic syndrome

NF-κB Nuclear factor-kappa B
PBMC Peripheral blood mononuclear cell
UPJO Ureteropelvic junction obstruction
VUR Vesicoureteral reflux
Key Facts of Chemokines in Pediatric Renal Disease
• Urinary levels of CXCL8/IL-8 positively correlated with 24-h proteinuria in

pediatric patients with idiopathic nephrotic syndrome.
• Urinary levels of CXCL8/IL-8 seem to be a marker of disease activity in pediatric
patients with lupus nephritis.
• Urinary levels of CCL2/MCP-1 were increased in pediatric patients with focal
segmental glomerulosclerosis and correlated with plasma cholesterol and
triglycerides.
• Urinary levels of CCL2/MCP-1 from voided urine before and after surgery and
from the affected pelvis of pediatric patients with ureteropelvic junction obstruc-
tion were higher than non-surgically managed cases and healthy controls.
• Urinary measurement of CXCL8/IL-8 seems to be useful for the diagnostic of
vesicoureteral reflux at a cutt-off concentration of 5 pg/μmol with sensitivity of
88 % and specificity of 69 %.
• Urinary levels of CXCL8/IL-8 negatively correlated with glomerular filtration
rate in pediatric patients with chronic kidney disease due to congenital anomalies
of the kidney and urinary tract.
Definitions
Chemokine A large family of low molecular-weight cytokines whose main action

is the recruitment of leukocyte subsets under homeostatic and pathological
conditions.
Chronic kidney disease Progressive deterioration of renal function with glomeru-

lar filtration reduced for more than 3 months.
Congenital anomalies of the kidney and urinary tract This term comprises a
wide spectrum of malformations that occur at the level of the kidney, collecting
system, bladder, or urethra.
Focal segmental glomerulosclerosis This is a possible histopathology profile

observed in idiopathic nephrotic syndrome, which is characterized by partial fibrosis
of some of the glomerulus in renal biopsy.
Idiopathic nephrotic syndrome Primary glomerular disease characterized by pro-

teinuria, edema, low plasma albumin levels and dyslipidemia.
IgA nephropathy Glomerular disease diagnosed by the predominance of Immu-

noglobulin A deposits in the glomerular mesangium.
Minimal change nephrotic syndrome This is the nephrotic syndrome due to an

abnormal fusion of pedicles from podocytes.
Systemic lupus erythematosus This is a multisystemic autoimmune disease affect-

ing predominantly women and two thirds of cases occur in the first two decades of life.
Ureteropelvic junction obstruction This is the most common cause of severe

hydronephrosis in children due to a narrowing and/or obstruction of variable degrees
at the ureteropelvic junction.
Vesicoureteral reflux This is a congenital anomaly of the urinary tract due to an

anomalous implantation of the ureters into the bladder wall, leading to the return of
urine to renal pelvis.
Chemokines: General Concepts
Chemokines constitute a large family of low molecular-weight cytokines whose main

action is the recruitment of leukocyte subsets under homeostatic and pathological
conditions – the word “chemokine” is the contraction of the terms “chemotactic” and
“cytokine” (Charo and Ransohoff 2006). More specifically, leukocyte arrest during the
rolling phase of its recruitment cascade is rapidly triggered by chemokines. After
binding to specific seven transmembrane-domain G-protein-coupled receptors,
chemokines regulate integrin-mediated adhesion among other effects (Ley et al.
2007). The expression of variable concentrations of different chemokines contributes
to the diversity of leukocyte subsets present in inflamed tissues.
To date, approximately 50 chemokines and 20 receptors have been described in
humans (Charo and Ransohoff 2006; Ransohoff 2009; Sereger and Alpers 2003).
Chemokines are divided in four families based on differences in structure and
function. The largest family comprises CC chemokines, so named because the first
two cysteine residues are adjacent to each other, which are primarily involved in the
attraction of mononuclear cells to sites of chronic inflammation. CXC family, in
which the first two cysteine residues are separated by a single aminoacid, consists of
two subfamilies based on the presence of a characteristic glutamic acid-leucine-
arginine (ELR) motif near the N terminal of the molecule. ELR (+) CXC
chemokines, of which CXCL8/IL-8 (IUPHAR nomenclature/original name) is the
prototype molecule, attract polymorphonuclear leukocytes to sites of acute inflam-
mation. Conversely ELR (-) CXC chemokines, like CXCL9/MIG, CXCL10/IP-10
and CXCL11/I-TAC, are IFN-γ inducible chemokines, being involved in the
recruitment of Th1 lymphocytes among other cell types. The remaining chemokines
families comprise CX3C (with three amino acids separating the first two cysteine
residues) with a single member CX3CL1/fractalkine, and XC (with a single cysteine
residue) with XCL1/lymphotactin and XCL2 (Charo and Ransohoff 2006;
Ransohoff 2009; Sereger and Alpers 2003).
In the context of leukocyte trafficking, chemokines can be functionally grouped as
‘homeostatic’, i.e., chemokines constitutively expressed in organs, like CXCL12/SDF-
1, and ‘inflammatory’, i.e., chemokines induced on inflamed sites, which are bound by
cognate receptors on infiltrating leukocytes (Charo and Ransohoff 2006; Ransohoff
2009). Although certain chemokines may be stored in granules of cells, such as platelets
and mast cells, most chemokine expression is newly generated and released on-demand
at inflammatory sites (Sereger and Alpers 2003. Indeed chemokines have been impli-
cated in several diseases in which inflammatory mechanisms are involved, including
renal diseases. Table 1 shows the subtypes of chemokines reported in humans.
With respect to renal diseases, there is much evidence that leukocyte infiltration is
mediated by inflammatory chemokines released by various cell types (Sereger and
Alpers 2003; Sereger et al. 2000). Infiltrating leukocytes produce chemokines that
may amplify inflammatory responses in the kidney. Tubular epithelial cells can release
inflammatory chemokines as CCL5/RANTES (Regulated on activation, normal T
Expressed and Secreted), CCL2/MCP-1 (Monocyte chemotactic protein-1), CCL3/
MIP-1α (Macrophage inflammatory protein 1 alfa), CX3CL1/fractalkine and CXCL8/
IL8 (Interleukin 8) (Sereger et al. 2000). Tubular epithelial cells are also targets for
chemokines since these cells respond to CCL2/MCP1 stimulation by releasing
interleukin-6 and intracellular adhesion molecule-1 (Viedt et al. 2002). Messenger
RNA of chemokines receptors can also be detected in podocytes and glomeruli
(Sereger and Alpers 2003).
There are several techniques to measure chemokine – protein or mRNA – in tissues
and body fluids. For example, chemokines could be directly measured in renal tissue by
immunohistochemical or immunofluorescent techniques or by evaluating their levels in
supernatants of homogenized tissues (by ELISA). In patients with renal diseases, the
direct exam of tissue samples would be ideal since it may evaluate the affected organ.
However, kidney biopsy is an aggressive procedure and could be harmful. On the other
hand, ELISA or flow cytometry based techniques are less invasive and more useful for
clinical purposes by measuring the levels of chemokines in urine or blood samples
(Souto et al. 2008; Arraya et al. 2009; Vasconcelos et al. 2011; Pereira et al. 2012;
Santos Jr et al. 2012; Vianna et al. 2013). Alternatively, chemokine mRNA can be
measured by polymerase chain reaction or microarray in tissues or leukocytes of
patients (Arraya et al. 2009; Sellares et al. 2013; Halloran et al. 2013).
Chemokines in Renal Diseases
Several studies have shown the involvement of chemokines in the inflammatory

process responsible for the progression of chronic kidney disease (CKD) and renal
transplant rejection (for review, see refs. Vianna et al. 2011; Pereira et al. 2009). It
Table 1 Subtypes of chemokines reported in humans (IUPHAR nomenclature/original name)

Chemokine Main
subtype Biochemical structure Function molecules
CC First two cysteine residues Recruitment of mononuclear CCL2/
chemokines are adjacent to each other cells to sites of chronic MCP-1
inflammation CCL3/
MIP-1α
CCL5/
RANTES
CXC First two cysteine residues Recruitment of CXCL8/IL-8
chemokines – are separated by a single polymorphonuclear
ELR (+) aminoacid with a glutamic leukocytes to sites of acute
acid-leucine-arginine (ELR) inflammation
motif near the N terminal of
the molecule
CXC First two cysteine residues IFN-γ inducible chemokines CXCL9/MIG
chemokines – are separated by a single responsible for the CXCL10/IP-
ELR (-) aminoacid without ELR recruitment of Th1 10
motif lymphocytes CXCL11/I-
TAC
CX3C First two cysteine residues Chemokines expressed on CX3CL1/
chemokines are separated by three activated endothelial cells fractalkine
aminoacids responsible for leucocyte
adhesion and migration
XC With a single cysteine Recruitment of certain XCL1/
chemokines residue subsets of T-cells and natural lymphotactin-
killer cells α
XCL2/
lymphotactin-
β
CCL2/MCP-1 Monocyte chemotactic protein-1, CCL3/MIP-1α Macrophage inflammatory protein
1 alfa, CCL5/RANTES Regulated on activation, normal T Expressed and Secreted, CXCL8/IL-8
interleukin-8, CXCL9/MIG Monokine induced by gamma interferon, CXCL10/IP-10 Interferon
gamma-induced protein 10, CXCL11/I-TAC interferon-inducible T-cell alpha chemoattractant
was also suggested that the chemokines might act as biomarkers of renal disease
progression (Vianna et al. 2013) and as predictors of graft function in renal trans-
plantation (Pereira et al. 2012).
Many chemokines were measured in patients with nephrotic syndrome or in
animal models of the disease (for review, see refs. Pereira et al. 2014, 2015). Some
of them were correlated with proteinuria and suggested as candidates for promoting
glomerular permeability and, as consequence, proteinuria (for review, see ref.
Moreno et al. 2014). In animal models of nephrotic syndrome, increased renal
concentrations of the chemokines CCL5/RANTES, CCL11/eotaxin, CCL1/TCA-3
(T cell activation-3), CCL2/MCP-1, CCL3/MIP-1a (macrophage inflammatory
protein-1 alpha) and CCL4/MIP-1b (macrophage inflammatory protein-1 beta)
have been reported (Vielhauer et al. 2004). Blockade of the CCR1 chemokine
receptor reduced the infiltration of macrophages, lymphocytes and fibroblasts in
renal tissue (Vielhauer et al. 2004). Wang and co-workers (1997) showed that high
concentrations of albumin stimulated proximal tubular cells in culture increase the
production and secretion of CCL2/MCP-1. Rats immunized with CCL2/MCP-1 and
CCL5/RANTES DNA before the induction of nephrotic syndrome by doxorubicin
presented low chemotaxis of monocytes/macrophages and improvement in biochem-
ical changes and renal histopathology (Wu et al. 2005). In addition, the measurement
of urinary, plasma and renal tissue levels of chemokines has been used to diagnosis
and monitor various renal diseases (Souto et al. 2008; Arraya et al. 2009; Vasconcelos
et al. 2011; Pereira et al. 2012; Santos Jr et al. 2012; Vianna et al. 2013). We reported
below the chemokines most frequently associated to renal diseases.
CXCL8, also known IL-8, is an inflammatory chemokine that activates CXCR1 and
CXCR2, and promotes neutrophil infiltration and activation in proteinuria-associated
renal diseases (Souto et al. 2008; Moreno et al. 2014; Tang et al. 2003; Yokoyama
et al. 1998). CXCL8/IL-8 expression was increased in tubules of patients with heavy
proteinuria and non-proliferative glomerulopathy (Souto et al. 2008; Tang et al. 2003)
and IgA nephropathy (Yokoyama et al. 1998). Several studies have also demonstrated
high concentrations of IL-8/CXCL-8 in serum (Garin et al. 1994; Kanai et al. 2009;
Garin et al. 1998) and urine (Souto et al. 2008) of patients with primary nephrotic
syndrome, as well as increased levels of mRNA for CXCL-8/IL-8 in peripheral blood
mononuclear cell (PBMC) culture of these patients (Garin et al. 1998). The CXCL-8/
IL-8 present in PBMC culture supernatant from patients with nephrotic syndrome alters
the sulfated component metabolism at the glomerular basement membrane in rats
(Garin et al. 1994, 1998). In vitro studies in tubular epithelial cells have shown that
albumin increases CXCL8/IL-8 through the activation of the nuclear factor-kappa B
(NF-κB) transcription factor. CXCL8/IL-8 urine levels have been used as a marker of
renal disease progression (Tang et al. 2003; Yokoyama et al. 1998). Increased urinary
levels of CXCL8/IL-8 were observed in idiopathic nephrotic syndrome during relapses
(Souto et al. 2008), IgA nephropathy (Yokoyama et al. 1998), lupus nephritis and
membranoproliferative glomerulonephritis (Wada et al. 1994). In lupus nephritis,
urinary levels of CXCL8/IL-8 were used as a marker of disease activity (Rovin
et al. 2005). Urinary CXCL8/IL-8 levels were higher in patients with glomerular
leukocyte infiltration than in those without (Rovin et al. 2005). Moreover, patients in
remission showed lower CXCL8/IL-8 levels than patients with idiopathic nephrotic
syndrome in relapse, and levels correlated with proteinuria values (Souto et al. 2008).
CXCL8/IL-8 is also responsible for neutrophil infiltration into the urinary tract
with an important role in acute pyelonephritis (Sheu et al. 2006; Artifoni et al. 2007).
In this regard, gene polymorphisms of this chemokine seem to increase the suscep-
tibility for acute pyelonephritis (Sheu et al. 2006). For instance, the presence of the
IL-8-251A allele in the genotype of children with urinary tract infection without
vesicoureteral reflux has increased the risk of pyelonephritis (Artifoni et al. 2007).
Table 2 summarizes the studies about CXCL-8/Il-8 in renal diseases.
Chemokine C–C motif ligand 2 (CCL2), also known as monocyte
chemoattractant protein 1 (MCP-1), is one of the most widely studied chemokines.
CCL2/MCP-1 is involved in the recruitment of monocytes and lymphocytes to sites
of inflammation through the activation of several receptors, mainly CCR2
Table 2 Studies reporting potential role for Interleukin-8 (CXCL8/IL-8) in renal diseases
Author Year Main findings
Garin et al. 1994 High levels of mRNA for CXCL-8/IL-8 in PBMC culture of patients with
nephrotic syndrome
Garin et al. 1998 CXCL-8/IL-8 present in the PBMC culture supernatant from patients
with nephrotic syndrome alters the sulfated component metabolism at the
glomerular basement membrane in rats
Yokoyama 1998 Urinary levels of chemokines, including CXCL8/IL-8, reflect distinct
et al. disease activities and phases of human IgA nephropathy
Tang et al. 2003 Albumin increases CXCL8/IL-8 through the activation of the NF-κB
transcription factor in proximal tubular cells in vivo and in vitro
Rovin et al. 2005 Urine chemokine measurements have a potential role as biomarkers of
lupus nephritis activity
Sheu et al. 2006 Gene polymorphisms of CXCL8/IL-8 seem to increase the susceptibility
for acute pyelonephritis
Artifoni 2007 Presence of the IL-8-251A allele in the genotype of children with urinary
et al. tract infection without vesicoureteral reflux increases the risk of
pyelonephritis
Souto et al. 2008 Urinary levels of CXCL8/IL-8 are positively correlated with 24-h
proteinuria in pediatric patients with primary nephrotic syndrome
Kanai et al. 2009 Urinary levels of CXCL8/IL-8 are associated with idiopathic steroid
sensitive nephrotic syndrome
Vianna 2013 Urinary levels of CXCL8/IL-8 inversely correlate with GFR in pediatric
et al. CKD patients due to CAKUT
PBMC peripheral blood mononuclear cell, NF-κB nuclear factor-kappaB, GFR glomerular filtration
rate, CKD chronic kidney disease, CAKUT congenital anomalies of the kidney and urinary tract
(Viedt et al. 2002; Pereira et al. 2012; Vianna et al. 2013; Yadav et al. 2010; Kim and
Tam 2011; Marks et al. 2010; Wada et al. 2000; Bobkova et al. 2006; Eddy and
Warren 1996; Stangou et al. 2009; Giunti et al. 2006; Kolattukudy and Niu 2012;
Dubinski et al. 2008; Ho et al. 2013). Many studies have associated MCP-1/CCL2 to
glomerulopathies (Viedt et al. 2002; Vianna et al. 2013; Marks et al. 2010; Wada
et al. 2000; Bobkova et al. 2006; Eddy and Warren 1996; Stangou et al. 2009) and to
renal transplantation (Pereira et al. 2012; Dubinski et al. 2008; Ho et al. 2013). Wada
and co-workers found significantly elevated urinary levels of this chemokine in
adults with diabetic nephropathy, whereas serum levels remained similar to those
of healthy volunteers (Wada et al. 2000). Patients with active proteinuric forms of
chronic glomerulonephritis have higher urine excretion of CCL2/MCP-1 than
healthy controls (Bobkova et al. 2006). In pediatric lupus nephritis, it was recently
shown that increased urinary, but not plasma, CCL2/MCP-1 levels correlated with
disease activity (Marks et al. 2010). Taken together, these studies indicate a potential
role for CCL2/MCP-1 in glomerular inflammation. Concerning renal transplanta-
tion, urinary levels of CCL2/MCP-1 were significantly higher in patients with acute
rejection and a significant reduction of this chemokine was found in patients who
responded to anti-rejection treatment (Dubinski et al. 2008). In addition, increased
urinary levels CCL2/MCP-1 at 6 months after renal transplantation might predict
Table 3 Studies reporting potential role for monocyte chemoattractant protein 1 (CCL2/MCP-1) in
renal diseases
Eddy et al. 1996 MCP-1 gene and protein expression are increased in the kidneys of rats
with aminonucleoside nephrosis
Wada et al. 2000 Adults with diabetic nephropathy have elevated urinary levels of CCL2/
MCP-1
Viedt et al. 2002 CCL2/MCP-1 induces inflammatory activation of human tubular
epithelial cells
Bobkova 2006 Patients with active proteinuric forms of chronic glomerulonephritis have
et al. higher urine excretion of CCL2/MCP-1 than healthy controls
Giunt et al. 2006 The MCP-1/CCR2 system has direct proinflammatory effects in human
mesangial cells
Dubinski 2008 Urinary levels of CCL2/MCP-1 are higher in patients with acute rejection
et al. and the reduction of this chemokine occurs in response to anti-rejection
treatment
Stangou 2009 Up-regulation of urinary CCL2/MCP-1 predicts outcome in IgA
et al. nephropathy
Marks 2010 Increased urinary levels of CCL2/MCP-1 correlate with lupus nephritis
et al. activity in pediatric patients
Pereira 2012 Urinary levels of CCL2/MCP-1 significantly reduce from 30 to 300 days
et al. after renal transplantation in live donors subgroup
Ho et al. 2013 Increased urinary CCL2:Cr ratio at 6 months is associated with late renal
allograft loss
Vianna 2013 Urinary CCL2/MCP-1 levels are increased in FSGS patients and
et al. positively correlated with plasma cholesterol
CCR2 receptor for the monocyte chemoattractant protein 1, Cr creatinin, FSGS focal segmental
glomerulosclerosis
renal allograft loss (Ho et al. 2013). Urinary MCP-1/CCL2 measurements may be an
early marker of therapy responsiveness in patients with acute rejection. Table 3
displays the main findings of the studies of CCL2/MCP-1.
Chemokine C–C motif ligand 5 (CCL5), also known as RANTES, and its specific
receptors CCR1, CCR3 and CCR5 have also been associated with renal diseases
(Krensky and Ahn 2007). CCL5/RANTES is a chemokine produced by human T
lymphocytes at a ‘late’ stage (3–5 days) after activation through their T-cell recep-
tors. It is broadly chemoattractive for T lymphocytes, monocytes, natural killer cells,
basophils and eosinophils, and can also activate immune cells. This chemokine is
involved in AIDS, cancer, atherosclerosis, asthma, organ transplantation, and auto-
immune diseases such as arthritis, diabetes and glomerulonephritis (Krensky and
Ahn 2007). Albumin and other filtered urinary proteins, such as IgA and IgG, can
induce up-regulation of CCL5/RANTES. As compared with CCL2/MCP-1, CCL5/
RANTES induction is delayed, but remains increased for days. CCL5/RANTES was
expressed by tubular epithelial cells during proteinuria, and its expression was
associated with interstitial CCR5-positive mononuclear cells (predominantly
CD3-positive T lymphocytes) and fibrosis in patients with acute interstitial nephritis
(Sereger et al. 1999). CCL5/RANTES levels are found increased in kidneys of

autoimmune nephritis that is characterized by proteinuria and monocyte recruitment
(Xie et al. 2011).
Chemokines in Glomerular Diseases
In the last 5 years, several studies measuring chemokines in different forms of

glomerular diseases such as IgA nephropathy, lupus nephritis and idiopathic
nephrotic syndrome (INS) including minimal change lesion (MCNS) and focal
segmental glomerulosclerosis (FSGS) have been performed.
IgA nephropathy is diagnosed by the predominance of IgA deposits in the
glomerular mesangium and is present in around 13.8 % of renal biopsies of children,
being the second more frequent renal disease, and the mesangioproliferative glo-
merulonephritis is the most common histological presentation (Bazina et al. 2007).
Chemokines have been evaluated in IgA patients and some of them seemed to
predict the outcome of the disease (Stangou et al. 2013).
Systemic lupus erythematosus (SLE) is a multisystemic autoimmune disease
affecting predominantly women and two thirds of cases occur in the first two decades
of life. Lupus nephritis is a very common condition in 50–67 % of children with SLE
(Mina and Brunner 2010). A wide range of chemokines (CXCL10/IP-10, CCL3/MIP-
1α, CCL5/RANTES, CXCL8/IL-8, CCL2/MCP-1 and CCL4/MIP-1β) was tested for
correlation with lupus activity as assessed by the Systemic Lupus Erythematosus
Disease Activity Index (SLEDAI-1) (Barbado et al. 2012). The chemokine CCL2/
MCP-1 was the best biomarker of SLE activity (Barbado et al. 2012). Regarding
lupus nephritis, the serum B lymphocyte chemoattractant (CXCL13/BLC) was
increased in 31 adult patients in comparison with 60 SLE patients without renal
involvement and might be a surrogate marker as well (Schiffer et al. 2009).
MCNS is the most common cause of nephrotic syndrome in children. This disease
reflects a disorder of T lymphocytes and some patients can progress to FSGS (Cho
et al. 2007). Our research group measured plasma and urinary chemokines in 32 chil-
dren with INS divided according to steroid responsiveness and 12 healthy controls
(Souto et al. 2008). We found increased levels of urinary CXCL8/IL-8 in relapsed
steroid resistant children when compared to steroid sensitive patients in remission,
with a positive correlation with urinary protein levels (Souto et al. 2008). These
findings suggest that the renal release of the chemokine CXCL8/IL-8 might be
associated with changes in glomerular permeability (Souto et al. 2008). More recently,
by studying a group of pediatric patients at stages 2–4 of CKD, we detected higher
levels of urinary CCL2/MCP-1 in patients with FSGS than in cases of uropathies at the
same stage of CKD (Vianna et al. 2013). In addition, urinary levels of CCL2/MCP-1
positively correlated with serum total cholesterol and triglycerides concentrations
(Vianna et al. 2013). This study supports the concept that differences in chemokine
profile may be related to CKD etiology and disease-associated changes.
Figure 1 shows inflammatory mechanisms that linked chemokines and the emer-
gence of CKD in glomerular diseases.
Immune stimulus
Normal glomerulus
Release of cytokines
and chemokines
Iniltration of leukocytes
Mesangial proliferation
Inlamed glomerulus
Injury of podocytes
Altered glomerular permeability
Proteinuria
Deposition of collagen
Fibrotic glomerulus
Fig. 1 Inflammatory mechanisms related to the emergence of chronic kidney disease in

glomerulopathies
Chemokines in Congenital Uropathies
Congenital anomalies of the kidney and urinary tract (CAKUT) comprise a spectrum
of malformations that occur at the level of the kidney (e.g., hypoplasia and dyspla-
sia), collecting system (e.g., idiopathic hydronephrosis, ureteropelvic junction
obstruction, and megaureter), bladder (e.g., ureterocele and vesicoureteral reflux),
or urethra (e.g., posterior urethral valves) (Carr and Kim 2010). A variety of
intrarenal factors lead to progressive interstitial and renal parenchyma fibrosis in

patients with CAKUT, including growth factors, cytokines, chemokines and adhe-
sion molecules (Klahr and Morrisey 2002). Altered renal expression of these factors
modulates cell death by apoptosis and/or phenotypic transition of glomerular,
tubular, and vascular cells. Mediators of cellular injury include hypoxia, ischemia,
and reactive oxygen species, while fibroblasts undergo myofibroblast transformation
with increased deposition of extracellular matrix. On the other hand, a number of
endogenous antifibrotic counter-regulatory molecules has been identified, opening
the possibility of enhancing the kidney’s own defenses against progressive fibrosis
(Klahr and Morrisey 2002; Chevalier et al. 2010).
In this regard, chemokines like CCL2/MCP-1, CCL5/RANTES, macrophage
inflammatory protein-2 (CXCL2/MIP-2) and ɣ-interferon-inducible protein
(CXCL10/IP-10) have been evaluated in experimental hydronephrosis (Stephan
et al. 2002; Vielhauer et al. 2001; Crisman et al. 2001; Madsen 2013). Stephan
and co-workers produced partial or complete ureteral obstruction in 28-day-old
Wistar rats (Stephan et al. 2002). CCL2/MCP-1 mRNA expression was moderately
increased in partial ureteral obstruction, whereas kidneys without significant damage
did not show any up-regulation of this chemokine. The study qualified CCL2/MCP-
1 mRNA expression as a prognostic marker of partial ureteral obstruction (Stephan
et al. 2002). In addition, Vielhauer and co-workers found an increased expression of
the CC chemokines, CCL2/MCP-1 and CCL5/RANTES, at sites of progressive
tubulointerstitial damage in murine obstructive nephropathy model (Vielhauer
et al. 2001). It was also observed an interstitial infiltration of macrophages and T
lymphocytes, which differentially expressed CCR2 receptors. These data suggest
that CCR2- and CCR5-positive monocytes and CCR5-positive lymphocytes are
attracted by locally released CCL2/MCP-1 and CCL5/RANTES, resulting in chronic
interstitial inflammation (Vielhauer et al. 2001). Crisman and co-workers detected
the expression of CCL2/MCP-1, CCL5/RANTES and CXCL10/IP-10 at 1 day of
unilateral ureteral obstruction in mice (Crisman et al. 2001). At 7 days, CCL5/
RANTES became the most abundant chemokine in the obstructed kidney and the
cortical tubular cells significantly contributed to this elevation (Crisman et al. 2001).
The study of chemokines in hydronephrosis might provide new insights for treat-
ment or novel ways to blunt renal damage in obstructive nephropathy (Madsen
2013). Figure 2 proposes a link between chemokine release and CKD in patients
with congenital uropathies.
Very few data about the role of chemokines in CAKUT have been provided by
clinical studies and the majority of them evaluated ureteropelvic junction obstruction
(UPJO) and vesicoureteral reflux (VUR).
Ureteropelvic Junction Obstruction
UPJO is the most common cause of severe hydronephrosis in children (Quirino

et al. 2012). Postnatal differentiation between obstructive and non-obstructive
hydronephrosis is quite difficult (Dias et al. 2013). Several studies have been made
Congenital urinary tract obstruction
Ischemia Cell stretching Release of cytokines Release of Reactive Hypoxia

and chemokines Oxigen Species
Renal tubule cell injury
RANTE MCP-1 Angiotensin II TGF- b1
Macrophage Release of ciytokines Stimulation of Fibroblasts

accumulation And chemokines Collagen deposition
Chronic interstitial inlammation
Apoptosis and Fibrosis
Fig. 2 Schematic view of chemokine and fibrogenic factors release at renal tissue in congenital
uropathies
in patients with UPJO in order to find out noninvasive biomarkers to allow the
diagnosis and treatment of these patients (Decramer et al. 2007; Lee 2009). Specif-
ically for chemokines, the most promising results were obtained with CCL2/MCP-1
(Grandaliano et al. 2000; Bartoli et al. 2011; Madsen et al. 2013).
Healthy children presented high expression of epidermal growth factor (EGF)
mRNA in renal tissue, whereas CCL2/MCP-1 mRNA was normally undetectable. In
UPJO patients, CCL2/MCP-1 gene expression was strikingly increased at the
tubulointerstitial level, while the EGF gene expression was markedly reduced.
Moreover, the interstitial mononuclear cell infiltrate in UPJO patients correlated
with the degree of tubulointerstitial damage (Grandaliano et al. 2000). Accordingly,
urinary concentrations of EGF were reduced in UPJO patients, whereas the CCL2/
MCP-1 levels were increased (Grandaliano et al. 2000; Madsen et al. 2013). After
Table 4 Studies reporting potential role for monocyte chemoattractant protein 1 (CCL2/MCP-1) in
ureteropelvic junction obstruction (UPJO)
Grandaliano 2000 Urinary levels of CCL2/MCP-1 are increased in UPJO patients before
et al. surgery and the levels reduce after the surgical procedure
Bartoli et al. 2011 Urinary levels of CCL2/MCP-1 increase in patients with UPJO
compared with healthy controls and significantly reduce after surgical
treatment
Madsen et al. 2012 Urinary levels of CCL2/MCP-1 are increased in preoperative samples
collected in UPJO patients before surgical procedure in comparison to
urine from healthy children
Taranta- 2012 Urinary levels of CCL2/MCP-1 from voided urine before and after
Janusz et al. surgery and from the affected pelvis are higher than non-surgically
managed cases of UPJO and control group
surgical correction of UPJO, there was a significant reduction in urinary levels of

CCL2/MCP-1 accompanied by a marked increase in EGF concentration. Therefore,
these two biomarkers could be useful for the follow-up of obstructed patients
(Grandaliano et al. 2000). In a prospective study, Madsen and co-workers reported
that urinary concentrations of EGF and CCL2/MCP-1 were significantly increased in
preoperative samples collected in UPJO patients before surgical procedure in com-
parison to urine from healthy children (Madsen et al. 2013). At the same study, the
concentrations of CCL2/MCP-1, CCL3/MIP-1α, CXCL10/IP-10 and CCL5/
RANTES were increased in the urine from the obstructed kidney compared to the
urine from the contralateral non-obstructed kidney (Madsen et al. 2013). These urine
samples were collected during surgical procedure. One year after surgery, the
concentrations of these chemokines decreased to levels comparable to healthy
controls (Madsen et al. 2013).
Taranta-Janusz and co-workers compared obstructed prenatal hydronephrosis
cases (who underwent surgery) with non-surgically managed cases and healthy
subjects (control group) (Taranta-Janusz et al. 2012). Urinary levels of CCL2/
MCP-1 from voided urine before and after surgery and from the affected pelvis
were significantly higher than non-surgically managed cases and control group
(Taranta-Janusz et al. 2012). The authors also studied the level of CCL5/RANTES
in urine samples, which were significantly higher in urine samples from affected
pelvis collected during surgery than in voided urine before pyeloplasty (Taranta-
Janusz et al. 2012). Three months after surgery, urinary levels of these biomarkers
did not return to control values (Taranta-Janusz et al. 2012). Table 4 shows clinical
studies of chemokines in UPJO.
Vesicoureteral Reflux
VUR is a congenital anomaly that increases the risk of repeated pyelonephritis and,
consequently, can result in renal scarring, renin-mediated hypertension, and, in some
Table 5 Studies reporting potential role for Interleukin-8 (CXCL8/IL-8) in vesicoureteral reflux
(VUR)
Haraoka 1996 Urinary levels of CXCL8/IL-8 are higher in children with renal scarring
et al. than without and in patients with VUR than without
Galanakis 2006 Urinary measurement of CXCL8/IL-8 is used for the diagnostic of VUR
et al. at a cutt-off concentration of 5 pg/μmol with sensitivity of 88 % and
specificity of 69 %
Merrikhi 2012 Urinary levels of CXCL8/IL-8 are higher in children with VUR than
et al. without and this chemokine could be a marker of renal scarring due to
VUR
Vianna 2013 Urinary levels of CXCL8/IL-8 negatively correlate with GFR in CAKUT
et al. patients, suggesting that this chemokine might be associated to renal
scarring
GFR glomerular filtration rate, CAKUT congenital anomalies of the kidney and urinary tract
cases, renal insufficiency (Silva et al. 2006, 2009; Simões e Silva et al. 2007). There
is persistent inflammatory reaction in VUR despite the occurrence or not of urinary
tract infection (UTI). The elevated urinary level of CXCL8/IL-8 in children with
reflux and without UTI might contribute to reflux nephropathy (Haraoka et al. 1996;
Galanakis et al. 2006; Merrikhi et al. 2012). Haraoka and co-workers found a
significant difference between urinary levels of CXCL8/IL-8 in children with and
without renal scarring and in patients with and without VUR (Haraoka et al. 1996).
Merrikhi and co-workers also showed significantly higher levels of CXCL8/IL-8 in
patients with VUR than in those without VUR (Merrikhi et al. 2012). This finding
suggests that urinary CXCL8/IL-8 measurements could be useful to detect VUR
patients with more pronounced renal damage and who need strict follow-up
(Merrikhi et al. 2012). Galanakis and co-workers proposed the use of CXCL8/IL-
8 as a biomarker for the diagnostic of VUR (Galanakis et al. 2006). A cutt-off
concentration of 5 pg/μmol has a sensitivity of 88 % and a specificity of 69 %
(Galanakis et al. 2006). Our research group has recently reported a correlation
between high urinary levels of CXCL8/IL-8 and reduced glomerular filtration rate
in CAKUT patients, suggesting that this chemokine might be associated to renal
scarring and CKD (Vianna et al. 2013). Table 5 shows clinical studies of chemokines
in VUR.
Potential Applications to Prognosis, Other Diseases or Conditions
Clinical and experimental evidence leaves no doubt about the role of inflammation in
renal diseases. Understanding the effects of chemokines on the onset and progres-
sion of renal injury is of great interest as new diagnostic and prognostic markers, and
maybe as alternative therapeutic targets.
CCL2/MCP-1 and CXCL8/IL-8 are the chemokines more commonly associated
with pediatric renal diseases. In glomerular diseases, high urinary levels of CCL2/
MCP-1 have been associated to FSGS, lupus nephritis and IgA nephropathy. There
is also evidence of a potential link between monocyte recruitment and dyslipidemia
in pediatric patients with CKD due to FSGS. On the other hand, urinary levels of
CXCL8/IL-8 positively correlated with proteinuria in pediatric patients with primary
nephrotic syndrome, suggesting a role in glomerular permeability changes. With
reference to CAKUT, the chemokine CCL2/MCP-1 has been associated with urinary
tract obstruction in patients with UPJO, whereas high urinary levels of CXCL8/IL-8
were found in patients with VUR and correlated with renal scarring and renal
function deterioration.
What is more, new roles are emerging for chemokines outside the renal disease
field: for example, in Parkinson disease, high plasma levels of the chemokine
CXCL10/IP-10 seem to be associated with worse performance on cognitive tests
(Rocha et al. 2014) and, in bipolar disorder, plasma levels of CXCL8/IL-8 was
decreased, whereas CCL11, CCL24 and CXCL10/IP-10 concentrations were
increased in comparison to healthy controls (Barbosa et al. 2013).
Finally, despite great advances in our knowledge on the pathways linking
chemokines to renal diseases, much still remains to be elucidated.
Summary Points
• Chemokines are responsible for recruitment of leukocytes.

• There are several techniques to measure chemokines.
• Chemokines seem to have a role in the pathogenesis of renal diseases.
• CCL2/MCP-1 and CXCL8/IL-8 are the chemokines more commonly associated
with pediatric renal diseases.
• High urinary levels of CCL2/MCP-1 is found in glomerular diseases.
• CXCL8/IL-8 seems to have a role in glomerular permeability changes and in
renal scarring.
Acknowledgments This study was partially supported by CNPq (Conselho Nacional de

Desenvolvimento Científico e Tecnológico, Brazil) and FAPEMIG (Fundação de Amparo à
Pesquisa do Estado de Minas Gerais, Brazil) by the Grant INCT-MM (Instituto Nacional de Ciência
e Tecnologia – Medicina Molecular: FAPEMIG: CBB-APQ-00075-09/CNPq 573646/2008-2).
Dr. AC Simões e Silva, Dr AL Teixeira and Dr MM Teixeira received a research grant from CNPq
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Kidney and Neoplastic Disease: Overview
with a Particular Interest to Interpretation 12
of Cancer Biomarkers
Giuseppe Coppolino, Mariadelina Simeoni, Laura Rivoli,

Chiara Summaria, and Davide Bolignano
Contents
Key Facts of Renal Disease Subsequent to Neoplasia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
Key Facts of Neoplasia Risk in Renal Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Key Facts of Neoplasia Risk in Renal Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
Renal Disease Associated with Malignancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252
Malignancy Associated with Renal Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 254
Breast Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255
Colorectal Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 256
Women-Specific Tumoral Diseases: Cervical Cancer and Ovary Cancer . . . . . . . . . . . . . . . . . . . . . . 256
Lung Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Liver Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
Prostate Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 258
Tumors of the Urinary Tract: Kidney, Ureters, and Bladder . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
Markers of Cancer and Renal Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 259
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 262
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 264
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265
G. Coppolino (*)
Nephrology and Dialysis Unit, University Hospital of Catanzaro, Catanzaro, Italy
e-mail: gcoppolino@hotmail.it
M. Simeoni • L. Rivoli • C. Summaria
e-mail: adelina_simeoni@yahoo.it; rivoli.laura@gmail.com; chiara.summaria@hotmail.com
D. Bolignano
e-mail: davide.bolignano@gmail.com

250 G. Coppolino et al.
Abstract
Tumor markers represent useful tools in diagnosis and clinical management of
patients with cancer, because they are easy to use, minimally invasive, and easily
measured either by blood or urine. Unfortunately, such an ideal marker, as yet,
does not exist. Different pathological states may increase the level of a tumor
marker in the absence of any neoplasia, or alternatively during these conditions,
not every subject with cancer has abnormally high levels of the tumor marker
usually associated with that neoplasia. We aimed at reviewing study literature
examining the association between tumor markers and different renal impairment
conditions. Each tumor marker was found to be differently influenced by these
criteria; additionally, we revealed in many cases a lacking of available
published data.
Keywords
Tumor markers • Chronic renal failure • Renal impairment • Hemodialysis •
Transplant
Abbreviations
ADPKD Autosomal dominant polycystic kidney disease
AFP Alpha-fetoprotein
ARF Acute renal failure
Beta-hCG Beta-human chorionic gonadotropin
CAPD Continuous ambulatory peritoneal dialysis
CEA Carcinoembryonic antigen
CRF Chronic renal failure
NSAIDs Nonsteroidal anti-inflammatory drugs
NSE Neuron-specific enolase
PSA Prostate-specific antigen
PTH Parathyroid hormone
Key Facts of Renal Disease Subsequent to Neoplasia
• Renal impairment of different origin and severity occurs in patients affected by a

neoplasia.
• It could be caused by a prerenal volume depletion, for example, due to the
vomiting and diarrhea associated with chemotherapy.
• It may be originated by a postrenal compression to excretory system but also by
multiple intrarenal causes of kidney function impairment, including glomerular,
tubulointerstitial, and vascular diseases.
12 Kidney and Neoplastic Disease: Overview with a Particular Interest to. . . 251
Key Facts of Neoplasia Risk in Renal Patients
• Patients in different stages of chronic kidney disease, but in particular in renal

failure requiring substitutive therapy with hemodialysis or peritoneal dialysis,
have an increased risk.
• Probably as a result of the accumulation of uremic toxins, patients in chronic renal
failure are in immunosuppressed state and more prone to translate into an
increased incidence of cancer.
• In renal transplant patients, the risk is furtherly increased by immunosuppression
therapy.
Key Facts of Neoplasia Risk in Renal Patients
• Elevated levels of several tumor markers can be frequently detected in patients

with impaired kidney function because their renal elimination is retarded.
• In other cases, neoplasia is really present, given the higher risk of developing
malignancies in these patients.
• During renal replacement therapy by hemodialysis or peritoneal dialysis,
tumor markers could result even lower because of removal by the dialysis procedure.
• Consequently, while cancer screening and surveillance are important in this
population, the possibility of false-positive results notably reduces the diagnostic
value of those markers that are mainly eliminated by renal excretion.
Definitions
Chronic kidney disease (CKD) A pathological condition characterized by a

reduced renal function with consequent alterations in fluid regulation, blood pressure
control, waste product elimination, and metabolic alterations. CKD may progress,
more or less rapidly, to end stages.
End-stage renal disease (ESRD) The final stage of CKD, also known as terminal
uremia. Residual renal function is not anymore sufficient to control the body
homeostasis so that patients need to start in due course chronic hemodialysis
treatment or be transplanted.
Hemodialysis (HD) Chronic or acute therapy for replacing renal function. ESKD
patients usually undergo chronic hemodialysis thrice a week. Each HD session lasts
3.5–4 h. During the HD session, fluid and electrolyte excess and waste products are
removed from the blood circulation.
Peritoneal dialysis (PD) Alternative technique to hemodialysis consisting in using

the patient’s own peritoneal membrane as an exchange surface for balancing fluid,
electrolyte, and waste homeostasis. The peritoneal cavity is accessed via a
permanent catheter placed in the patient’s abdomen that is linked to an external

machine. Exchanges are induced and regulated according to diffusion and convec-
tion principles by fluid bags with established content.
Introduction
Cancer biomarkers are useful tools for cancer diagnosis and clinical management of
neoplastic patients. Additionally, their determination involves mini-invasivity for the
patient and simple lab procedures. Although reference ranges have been developed
for the correct interpretation of cancer biomarkers, kidney function often represent a
confounder, due to several different aspects influencing renal load and clearance in
the course of neoplasia. In fact, renal involvement is a frequent event associated with
cancer. By contrast, a preexisting renal impairment represents an independent-risk
factor for cancer development. Finally, in the presence of all cause of renal function
decline, cancer markers raise without a clinical meaning (Coppolino et al. 2014). In
the following paragraphs discussed in detail are all these issues, with particular
attention to the interpretation of cancer markers as diagnostic and prognostic lab
parameters influenced by the renal function.
Renal Disease Associated with Malignancies
The occurrence of renal failure is an additional morbidity and mortality risk factor in
the course of neoplasia (Lameire et al. 2005). Renal involvement occurs as conse-
quence of the neoplasia or due to the nephrotoxicity of antitumoral drugs (Table 1).
Acute renal failure (ARF) is the most frequent renal complication induced by chemo-
therapeutic treatment occurring in 12–49 % of terminal cancer patients. Although a
preexisting renal impairment might influence the outcome, it is estimated that 9–32 %
of ARF cancer patients need hemodialysis showing a high mortality rate (72–85 %)
(Darmon et al. 2006). Chronic renal failure (CRF) accompanied with clinical appear-
ance of nephrotic syndrome or isolated proteinuria or tubulopathy is another frequent
renal complication associated with cancer and its treatment (Humphreys et al. 2005).
More often than in noncancer-associated CRF, electrolyte disorders occur. Hypercal-
cemia is mainly due to local osteolysis but can also depend on the ectopic production
of PTH-like peptides and/or calcitriol (Stewart 2005). Sodium homeostasis is often
altered for several reasons such as paraneoplastic antidiuretic hormone inappropriate
secretion, gastrointestinal losses, and/or diabetes insipidus. Kalemia and magnesemia
abnormalities might occur due to associated ARF or to electrolyte loss in the renal
tubule or in the gastrointestinal tract. A milder presentation of renal involvement is
common in oncohematologic diseases, showing trend to reversibility after treatment
suspension and/or disease remission (Manning et al. 1996).
The tumor lysis syndrome is another remarkable cause of renal impairment. The
syndrome is determined by metabolic abnormalities such as hyperuricemia,
Table 1 Causes of renal failure in cancer patients

Prerenal
Extracellular fluid depletion (vomiting, diarrhea, and hyperkalemia)
Hepatorenal syndrome (VOD and hepatic resection)
Drugs (calcineurin inhibitors and NSAIDs)
Renal
Glomerular membranous nephropathy amyloidosis (MM)
Pamidronate-associated glomerulopathy (incidence unknown) LCDD
Acute tubulointerstitial necrosis (toxic/ischemic)
Lymphomatous renal infiltration
LCDD
Drug (cisplatin and ifosfamide) endovenous contrast media
Cast nephropathy (MM)
Vascular thrombotic thrombocytopenic purpura/hemolytic uremic syndrome
Tumor infiltration (renal cell carcinoma with renal vein thrombosis)
Postrenal
Intratubular obstruction uric acid nephropathy Methotrexate
Cast nephropathy (MM) extrarenal obstruction
Ureteral diseases (primary diseases and retroperitoneal lymphadenopathies)
Retroperitoneal fibrosis
LCDD light-chain deposition disease, MM multiple myeloma
hyperkalemia, hypocalcemia, hyperphosphatemia, and renal failure related to rapid

tumoral cell lysis due to apoptosis or chemotherapy.
With regard to pathophysiological mechanisms determining renal involvement in
the course of neoplastic disease, it is practical and useful to look for obstructive,
parenchymal, or prerenal primum movens for a correct diagnosis and treatment
modulation. In more than half cases, vomiting or/and diarrhea related to antineoplastic
treatment, especially if not balanced by appropriate liquid intake due to ureteral
obstruction or to renal hypoperfusion, induce renal damage. The evidence of acute
postrenal failure due to bilateral obstruction in patients not diagnosed for cancer and in
the absence of lithiasis has to be highly suspected for a neoplastic obstructive cause
(Rosad et al. 2014). The obstruction can affect any urinary segment, even in the
absence of hydronephrosis, and can depend on extrinsic mechanical obstructive causes
(retroperitoneal lymphadenopathy, metastases of urogynecological cancers) (Wong
et al. 2007) or on ab intrinseco blockage to urinary flow. As for intrinsic obstruction,
regardless of the cause (tubular deposit of urate or calcium phosphate crystals in high-
turnover cancers; direct crystallization of methotrexate), the general indication is to
maintain an adequate urine output, evaluating the administration of urate-lowering
therapy and urinary alkalizing agents, aimed at implementing renal damage prophy-
laxis (Kjellstrand et al. 1974; Thomas and Chisholm 1973; Buemi et al. 2009).
Renal parenchymal damage occurs with a variable frequency (6–60 %) in cancer
patients. Microangiopathy, disseminated intravascular coagulation, tumoral infiltra-
tion of the renal tissue, and tumor lysis are typical causes of parenchymal kidney
impairment strictly dependent on tumoral disease. Also, severe forms of acute leuke-
mia or lymphoma induce parenchymal impairment when able to infiltrate both kidneys
causing a nephromegaly without hydronephrosis, renal function loss, and/or urinary
abnormalities (Simsek et al. 2003). Solid tumors infrequently infiltrate or metastasize
the kidney. However, primary lung, breast, and stomach tumors are described for
metastasizing the kidney (Wagle et al. 1975). Another interesting modality for renal
damage classification in neoplastic patients is based on determining the nephron portion
primarily affected. Glomerular paraneoplastic syndromes are generally due to the
intraglomerular deposition of amyloid or neoplastic antigens. In most cases, a second-
ary membranous glomerulonephritis can be detected, representing 9 % of all biopsies
diagnostic for glomerulonephritis. Therefore, the finding of a membranous glomerulo-
nephritis could be a “spy” for occult malignancy in adults and should be followed by
neoplastic screening, especially looking for lung and gastrointestinal cancer (Birkeland
and Storm 2003; Burstein et al. 1993). Beside cancer-related parenchymal causes, the
chemotherapeutic agents’ nephrotoxicity is a major factor for renal involvement in the
course of cancer. In fact, antitumoral drugs, such as bisphosphonates and mitomycin C,
can induce a specific glomerular damage. Pamidronate at high-dose infusion commonly
induces collapsing focal and segmental glomerulosclerosis (Perazella and Markowitz
2008), while treatment of solid tumors with mitomycin C significantly correlates with
thrombotic microangiopathy occurrence (Antman et al. 1979). Tubulointerstitial
nephropathy secondary to cancer is a common example of tubular involvement mainly
depending on cisplatin and ifosfamide effects on proximal tubule and being potentially
age dependent (Kintzel 2001). A well-established correlation between neoplasia and
tubulointerstitial damage is also described for multiple myeloma with remarkable
prevalence (20 %). Moreover, renal involvement in the course of multiple myeloma
has to be considered as a marker of disease severity and predictor of mortality (Blade
et al. 1998). Although multiple myeloma represents 1 % of all-type cancers, end-stage
renal disease (ESRD) secondary to multiple myeloma has accounted for 58 % of all
cases of cancer-related kidney damage between 1997 and 2001. The renal deposition of
monoclonal light chains in the course of multiple myeloma induces three different
mechanisms of renal damage, depending on amyloidosis, light-chain deposition dis-
ease, or cast nephropathy (2003; Tang et al. 1989).
Malignancy Associated with Renal Disease
Development of cardiovascular and infectious complications is the main contributor

for high mortality in patients with renal failure. However, the occurrence of cancer
remains a major concerning comorbidity factor increasing mortality rate in both
ARF and CRF. Several data suggest a role for CRF as an independent-risk factor for
cancer development, with high incidence rates (Vajdic et al. 2006; Wong et al. 2009).
According to the Michigan Kidney Registry collected between 1973 and 1984,
prostate cancer, renal cell carcinoma, and cervical carcinoma are the most common
tumoral diseases occurring in CRF patients (Arican et al. 1999). Even neoplasms
arising in other organs such as the liver, thyroid, and tongue resulted more frequently
in CRF patients than in the general population. Among oncohematologic diseases,

multiple myeloma and non-Hodgkin lymphoma are the most commonly associated
with CRF, especially in patients with glomerulonephritis. Bladder transitional cancer
has also being found associated with nonsteroidal anti-inflammatory drug (NSAID)
nephropathy (Maisonneuve et al. 1999). Not surprisingly, a different geographical
distribution has been found for kidney and urinary tract tumors. Kidney cancers are
more common in Europe, Australia, and New Zealand, while urinary tract cancers
show prevalence peaks in Taiwan and in the Balkan nephropathy-endemic area,
mostly affecting females and localizing in the upper urinary tract (Wang et al. 2014).
ESRD patients undergoing renal replacement treatment are considered at high-risk
population for developing cancer with an overall standardized cancer incidence of
1:18. Moreover, cancer incidence in ESRD patients appears age correlated showing
prevalence three times higher in over 65-year-old patients compared to younger
patients. Dialysis age is an additional risk factor for cancer-related mortality with
highest incidence of neoplasm occurrence after 3 years of dialytic treatment (Collins
et al. 2003). A specific tumoral disease associated with kidney transplant is Kaposi
sarcoma. Kidney transplant together with glomerulonephritis treated with immuno-
suppressants is a condition at higher risk than renal insufficiency alone for cancer
development. Latrogenic influence by immunosuppressant agents in fact adds the
impairment of DNA repair mechanisms and alterations of immunosurveillance pro-
cesses, to the accumulation of carcinogenic agents and the reduced antioxidant
response due to the renal impairment (Kooman et al. 2014; Heidland et al. 2000;
Wang et al. 2014). Another renal disease showing an increased risk for neoplastic
transformation is the autosomal dominant polycystic kidney disease (ADPKD) due to
the tendence to malignant evolution of renal cysts. An additional factor predisposing to
cancer in ADPKD is represented by long-term abuse of NSAIDs as painkillers, which
contributes to metaplasia occurrence in the bladder (Stewart et al. 2003).
Breast Cancer
Breast cancer is the most common cancer in the world, although females are
prevalently affected. In 2008, more than one million of cases of breast cancer were
diagnosed, with a higher incidence in industrialized areas (North America, Australia,
New Zealand, Northwest Europe, South Asia, and sub-Saharan Africa), confirming
the influence of environmental risk factors in cancer pathogenesis (Parkin
et al. 2005; Kajbaf et al. 2002). The prevalence of breast cancer in women with
CRF is comparable to the general population. However, life expectancy is reduced
due to the comorbidity influence on death rate. It remains valid the recommendation
for annual mammography screening after menopause with anticipation in women
over 40 years old on hemodialysis treatment and waiting for kidney transplantation
(Holley and Von Roenn 2010). However, mammography is more difficult to interpret
in these patients because of the interference of vascular calcifications (Castellanos
et al. 2006; Siegel et al. 2013). Breast cancer biomarkers, CA 15-3, CA 27.29, and
CEA, cannot be used for diagnostic purpose in renal patients due to the tendence to
accumulation. However, they could find space as recurrence markers in the follow-up.
Other tissutal markers, particularly estrogen and progesterone receptors, were identified
as prognostic indices for predicting tumor aggressiveness and response to treatment.
Colorectal Cancer
Colorectal cancer is the second tumor most diagnosed in women and the third in
men. However, men are more affected than women in absolute. In recent years, a
trend to reduction of prevalence and incidence has been registered, probably due to
screening programs and improved therapy efficacy. However, in 2008 over one
million of incident cases and over 600,000 deaths for colorectal cancer were reported
with variable geographical distribution (Jemal et al. 2011, 2013). According to
general data, colorectal cancer has a higher incidence in kidney transplant recipients,
overlapping that of 60-year-old subjects that are reported to have the highest risk for
colorectal cancer. Hence, the current recommendation is for starting antineoplastic
screening in all 40-year-old kidney transplant recipients or, regardless of anagraphic
age, at 5-year after transplant (Park et al. 2010). Patients on chronic hemodialysis
have also a greater risk for developing colorectal cancer. However, reliability of fecal
occult blood for determining eventual indication for colonoscopy is limited in this
population due to the high frequency of gastritis and gastrointestinal telangiectasias
(Ajam et al. 1990). On the other hand, not even cancer markers are reliable tools for
diagnosing colorectal cancer in both general population and patients with renal
failure. CEA and CA 19-9, in fact, result altered in the advanced tumoral disease,
limiting their usefulness to the follow-up. Moreover, CA 19-9 specificity is low,
being shared as a marker also by pancreatic cancer.
Women-Specific Tumoral Diseases: Cervical Cancer and Ovary

Cancer
The uterine cervical cancer is the third most common cancer in sexually active
women, because it is closely related to HPV infection. Being considered a sexually
transmitted disease, about 85 % of new cases occur in developing countries. The
squamous cell variant has a higher prevalence compared to adenocarcinoma. In the
United States, 12,360 new cases were reported in 2014, accounting for an estimation
of 4,020 cancer-related deaths, the equivalent of about 1.5 % of cancer deaths in
women (Siegel et al. 2014; Jemal et al. 2011). Screening for uterine cervical cancer is
entrusted to cervical cytology and to the HPV DNA individuation. Controversial is
the utility of the HPV vaccination aimed at protecting women from the high-risk
viral genotypes responsible for 70 % of uterine cervical cancer (Ault 2006). In CKD
patients and in hemodialysis patients waiting for a renal transplant, HPV vaccination
has not yet a strong indication. However, in over 21-year-old renal transplant
candidates, a strong recommendation applies to annual Pap test and HPV DNA
assay (Kajbaf et al. 2002). The second most common gynecological neoplasia is
ovarian cancer, although it belongs to the group of rare cancers. In industrialized

countries, it is estimated that ovarian cancer incidence is equivalent to 9,4 new cases
per 100,000, and mortality rate reaches 5.1 related deaths per 100,000. Furthermore,
it is consistent that the risk for ovarian cancer development increases with age. The
epithelial variant of ovarian cancer is the most common accounting for 95 % of
cases. The most specific marker for epithelial ovarian cancer is CA 125, although CA
72-4, CEA, and LASA-P are other less-specific but useful markers for such variant.
The germ cell variant of ovarian cancer is more sporadic and correlates with the
increase of human chorionic gonadotropin (hCG) and AFP. However, regardless of
the variant, the diagnosis of ovarian cancer is often tardive due to its radiological
escape, and not so rarely, the diagnosis is based on the exploratory surgery (Siegel
et al. 2013). However, the use of the high-specific markers hCG and AFP appears
useful not only in the follow-up but also in the diagnosis of the germ cell variant,
while CA 125 is validated only for the follow-up of the epithelial variant of ovarian
cancer.
Lung Cancer
The lung cancer is the leading cause of death for cancer in men and the second in
women with a variable incidence not only by gender but also with respect to the
geographical area and the smoking cigarette habit (Parsons et al. 2010). It is
remarkable that most data on lung cancer come from industrialized countries,
where industrial fumes and environmental pollution could play a major role. A
previous renal impairment does not appear as a significant risk factor for lung cancer,
while renal complications due to chemotherapy are very common. Apart from
carcinoembryonic antigen (CEA) in ongoing non-small cell lung cancer and
neuron-specific enolase (NSE) in small-cell lung cancer in patients with intact
renal function, lab markers show low specificity and sensibility in early detecting
lung cancer. On the contrary, tomography screening is recognized to be effective and
reliable and, thus, is recommended in all cigarette smokers with more than 30 years
of exposure (Boiselle 2013).
Liver Cancer
Liver cancer is another leading form of neoplastic disease with different incidence
within men and women. Liver cancer is fifth neoplastic disease diagnosed in men
and the second leading cause of death for cancer. Instead, in women, it represents the
seventh carcinoma for prevalence and the sixth causing death for cancer. The
estimated incidence in the United States in 2010 was six cases per 100,000 in adults
and 0.05 cases per 100,000 in children (El-Serag and Kanwal 2014). Because of his
aggressiveness, the incidence of liver cancer virtually coincides with the associated
mortality rate. Chronic HBV or HCV infection is the major causal determinant of
hepatocellular carcinoma (Perz et al. 2006; Allan et al. 2014). The cornerstone of
liver cancer screening and follow-up, especially in patients with cirrhosis and/or
hepatitis, is alpha-fetoprotein (AFP). However, the routinary use of AFP as screen-
ing has been found more useful in Asia, where there is a greater spread of liver
cancer.
Prostate Cancer
Prostate cancer is the most common neoplasm in men and in patients with CKD (Port
et al. 1989). Overall, prostate cancer is the most diagnosed within visceral cancers
with a report in the United States of 200,000 new cases and 30,000 related deaths in
2014. On this basis, all American men show an estimated risk for developing
prostate cancer of 16 % and a risk for prostate cancer-related mortality of 2.9 %
(Siegel et al. 2014). Consequently, prostate cancer is the leading cause of cancer-
related death in men after nonmelanoma skin cancer and lung cancer. Furthermore,
epidemiological data tend to underestimate the magnitude of the problem. In fact,
slow-progressor patients can remain clinically silent, and prostate cancer is often
found incidentally at autopsy (Dorr et al. 1993). The 5-year prognosis for prostate
cancer is strongly influenced by the timing of the diagnosis, being very favorable in
the case of local extension of the cancer and extremely poor for metastatic tumors. In
the past, the obsolete prostatic acid phosphatase was the only available prostate
cancer marker but showed very low sensitivity. Since 1990, prostate-specific antigen
(PSA) has been validated as the cornerstone marker in the diagnosis and follow-up of
prostate cancer. In 1992, in fact the ability of PSA in early detecting the tumor
resulted in a peak of prostate cancer diagnosis mainly at a localized stage. However,
PSA in rare variants of prostate cancer (e.g., small-cell neuroendocrine tumor) is not
affected and could represent a confounder delaying the diagnosis. Instead, the most
reliable cancer marker for these atypical prostate tumors is chromogranin A. These
are tumors responsive to hormone therapy, which profit instead of chemotherapy.
Their diagnosis may be delayed only by the observation of the PSA or, on the
contrary, confirmed by an alteration in the levels of chromogranin A. PSA appears
also as a very useful tool in the assessment of the response to treatment or relapse. An
increase of PSA after prostatectomy is a sign of relapse of the disease. A different
behavior is observed after radiotherapy. PSA values, in fact, undergo a drastic
reduction after treatment with a gradual return to normal levels within a few years,
and the recovery is identified only with a further PSA increase exceeding the
standard range. Unfortunately, PSA levels are influenced by the renal function;
therefore, its use as routinary screening is controversial in CKD patients. A cost-
benefit balance should be assessed between the trend to overdiagnosis leading to
improper prostate biopsies and the recognized advantage deriving from an early
detection of prostate cancer (Smith et al. 2001; Khairullah et al. 2004). However, the
utility of PSA as routinary screening is more consolidated in kidney transplant and in
young patients undergoing dialysis, although in these patients cannot be considered
as a marker of tumor aggressiveness and extension (Joseph et al. 2010).
Tumors of the Urinary Tract: Kidney, Ureters, and Bladder
Renal cell carcinoma affects preferentially male subjects in old age and has a high
incidence in the Czech Republic and North America (Siegel et al. 2014). The kidney
cancer tends to develop from a renal cyst especially in the context of a hereditary
renal cystic disease with a risk about 30 times higher than the general population. In
contrast, patients with acquired renal cysts seem to have a risk of neoplastic
transformation approximately comparable to the general population (6 %) (Brennan
et al. 1991; Truong et al. 1995). Also, in patients with a cystic hereditary disease, the
tumor assumes clinical characteristics that differ from sporadic renal tumors, having
a multicentric and bilateral localization multicentric with a sarcomatoid aspect
(Keith et al. 1994). Upper urinary tract cancer and CKD appear bidirectionally
related probably due to the action of some oncogenic nephrotoxins, such as
aristolochic acids and analgesics. These cancerogenic agents not only increase the
risk for cancer development but also are responsible for chronic interstitial nephritis,
accounting for renal failure occurrence in 80 % of cases with progression to ESRD in
10 % of patients. Hematuria is the main clinical symptom of urinary tract cancer;
however, its onset in patients on hemodialysis cannot be considered as highly
significant due to additional causes of hematuria such as intradialytic heparinization.
Furthermore, urinary cytology and cystoscopy, especially in anuric patients, show
low sensitivity and require the integration with imaging for the diagnosis definition.
In specific geographical areas in Asia (China and Taiwan), the prevalence of CKD in
patients with upper urinary tract carcinoma exceeds 55 %, and a strong correlation
has been reported with all age, aristolochic acid nephropathy and previous nephrour-
eterectomy. Moreover, Hung et al. demonstrated a linear correlation between the
prevalence of cancer and CKD severity, being prevalence increased up to 55 % and
71 % in CKD stages 4 and 5, respectively. Such correlation was found stronger in
female patients being on chronic hemodialysis treatment or undergone renal trans-
plantation (Wang et al. 2014). Bladder cancer is the fifth most common of cancer in
the United States, and 13,000 related deaths are reported annually. Standard detec-
tion methods for bladder cancer diagnosis include urine cytology and cystoscopy.
Only an early detection of bladder cancer leads to a favorable prognosis, while
cytology and cystoscopy are generally performed at advanced stages of the disease.
Specific urinary cancer markers such as bladder tumor antigen (BTA) and nuclear
matrix protein-22 (NMP22) increase the accuracy of detection of bladder cancer
when coupled with cystoscopy. In addition, BTA and NMP22, used alone, are useful
for addressing instrumental control timing during the follow-up (Grossman
et al. 2005).
Markers of Cancer and Renal Function
Among different hormones, metabolites, immunoglobulins, and antigens recognized

as markers of neoplasia, the majority consists of relatively high molecular weight
glycoproteins (3,400–5,000 kD) undergoing renal and/or liver metabolism. Their use
CEA Lung
CYFRA 21-1 CA 15-3
NSE
Breast
CA 27.29
CEA
Liver
AFP Colorectal CEA
CA 19-9 CA 125
Bladder Bladder CA 72-4
tumor
antigen (BTA)
Cervical & Overy CEA
Prostate LASA-P
tPSA
hCG
fPSA
AFP
Cromogranin A
Fig. 1 Representation of main tumor markers on body
in screening for cancer the general population is progressively decreasing due to a

poor sensitivity and specificity (Fig. 1). Furthermore, if a renal impairment is present
at cancer biomarker determination, their predictive validity and specificity are even
more limited due to kinetics alterations (Tzitzikos et al. 2010). Therefore, CRF
should be considered as a nonneoplastic disease responsible for altering tumor
marker blood concentration. Although the span of error in cancer biomarker deter-
mination according to various stages of renal failure has not been quantified, it is
notorious that the specificity of some markers and renal failure stage are inversely
correlated (Xiaofang et al. 2007). Consequently, cancer biomarker use could result in
misleading the diagnostic process or the follow-up if a renal impairment coexists. As
consequence, tumor markers should be classified as stable or unstable with respect to
renal function. However, many unstable tumor markers tend to restabilize with
dialysis, and a relative reduction of their concentration compared to pre-dialysis
values should not be interpreted as an improvement of the neoplastic disease (Arik
et al. 1996). Among the different variables influencing cancer, biomarker dosing
should be considered the pro-inflammatory state associated with CRF, which could
influence many biomarker levels. Furthermore, other than CFR, renal diseases can
alter the interpretation of tumor markers: proteinuria affects metabolism and excre-
tion of those protein structured; nephrolithiasis can trigger the production of certain
Table 2 Chemical structure and biological function of main tumor markers

Tumor marker Chemical structure Biological function
CEA Glycoprotein Membrane carcinoembryonic antigen
AFP Glycoprotein Alpha-fetoprotein
PSA Protease Prostate-specific antigen
CA125 Glycoprotein Carbohydratic antigen (mucins)
CA19.9 Glycoprotein Carbohydratic antigen (mucins)
CA15.3 Glycoprotein Carbohydratic antigen (mucins)
CYFRA Cytokeratin Tissue polypeptide antigen
β- hCG Glycoprotein Hormonal function
ones already produced in small concentrations from normal tissues. Moreover, an

analytical variability intrinsic to the dialysis process should be considered when
determining cancer biomarkers. In fact, certain markers tend to accumulate or
rapidly decrease based on several treatment parameters such as length, type of
dialysis, and hemodialysis membrane characteristics (Lye et al. 1994). Cancer bio-
markers have different molecular weights, and this represents an additional reason
for the variable behavior of certain biomarkers in hemodialysis patients. In deep,
cancer markers having a small molecular weight (<5–50 kD) easily undergo dialysis
clearance, while those with a higher molecular weight are only partially removed by
dialysis processes (Xiaofang et al. 2007; Table 2). The variability further increases
with interindividual, demographic characteristics and comorbidity of the population
undergoing treatment (Soletormos et al. 1993). It is particularly useful to evaluate for
each patient on dialysis the different comorbidities able to cause an increased tumor
marker concentration: inflammatory states of bowel or interesting coelomic epithe-
lium tissues (pleura, pericardium, and peritoneum) and liver disease secondary to
viral hepatitis B and C (Arican et al. 1999). This may result in false positive and false
negative, with a significant reduction of the specificity of each marker. A remarkable
example is CEA, a 180-kDa glycoprotein produced exclusively during fetal devel-
opment, used as a marker of relapse in several malignancies. Because of its molec-
ular weight, CEA is not removed by any dialysis modality (Arican et al. 1999). On
the contrary, despite its hepatic metabolism, CEA level is affected by renal clearance
(Lye et al. 1994). Moreover, smoking habit, chronic obstructive pulmonary disease,
and chronic inflammatory bowel disease tend to increase CEA levels with high
possibility of false-positive results. Biomarkers with independent metabolism are
also available. AFP, a 65-kDa-oncofetal protein produced by the fetal liver, yolk sac,
and adult liver, is considered as a “historical” marker, which is altered in 80 % of
hepatomas and in 60 % of germ cell tumors. AFP is used for the management of
hepatocarcinoma, liver metastasis, or non-seminoma testicular cancer. Nonetheless,
as expressed by multiple tumors, it shows reduced specificity and can rise even in the
course of ovarian cancer. A further example of stable cancer biomarker is PSA, a
serine protease regulated by androgens and having a molecular weight of 33-kD.
Differentiated cells of ductal and alveolar prostatic epithelium provide to PSA
physiological secretion. However, PSA level increases at prostate cancer develop-
ment resulting the main lab parameter useful for screening, diagnosis, risk
stratification, and follow-up of one of the most incident tumors, even in the dialysis
population (Lindblom and Liljegren 2000). Unfortunately, several confounding
variables can influence PSA, and recent prostatitis, digital prostatic examination,
transrectal ultrasound, and colonoscopy should be excluded before PSA dosing for
preserving specificity and lowering risk of false-positive results. A recent optimization
in PSA lab determination has been introduced. Circulating PSA exists as free or
complexed antigen, and both forms are detectable separately with specific laboratory
techniques. This advancement allows to distinguish increases in PSA due to benign
causes from prostate cancer. In the absence of the malignancy, an increase in total PSA
due to diagnostic procedures or benign prostatic hypertrophy can be individualized in
the isolated increase of free PSA (fPSA) levels (Robitaille et al. 2006). Thereby, current
indication is for referring to fPSA in general population screening. However, in patients
with reduced renal clearance, even fPSA has low specificity. Regardless of race and
age, in fact, fPSA tends to accumulate with glomerular filtration rate (GFR) reduction.
Similarly, in hemodialysis, patient fPSA accuracy is not optimal due to its low
molecular weight and high permeability to dialysis filtration membranes (Joseph
et al. 2010). A different class of cancer markers is that of carbohydratic antigens CA
19-9, CA 15.3, and CA 125, for which renal function influence on specificity is not
clearly determined. CA19-9 belongs to Lewis blood type carbohydratic antigens and
has low specificity (70 %) and high sensitivity (90 %) for malignancies of the
gastrointestinal tract and pancreas. CA 15.3 is mainly useful in the surveillance of
metastatic breast cancer. However, CA 15.3 specificity is affected by the coexistence of
HCV infection and pregnancy, although with less interference from renal function (Han
et al. 2012). CA 125 is a 90-kD membrane protein ubiquitary expressed in the eye,
respiratory tract, and female reproductive epithelium. CA 125 is widely validated for
ovarian cancer screening showing high sensitivity but limited specificity. CA 125 levels
are in fact influenced by alterations in serous membranes, a condition frequently
associated to both chronic hemodialysis and CRF. Although increased, CA 125 remains
more stable during conservative phase of CRF and in hemodialysis, while in peritoneal
dialysis, it has an oscillating trend. Finally, after renal transplantation, CA125 levels
tend to normalization. This behavior, indeed, suggests a strict participation to CA125
metabolism and/or clearance. Moreover, CA 125 is gender specific and is higher in
male CRF patients on conservative treatment (Sevinc et al. 2000). As for peritoneal
dialysis, it is the particular case where a chronic stimulation on peritoneum induces a
specific CA 125 production even in the absence of ovarian cancer. However, the CA
125 level increase stops when peritoneal sclerosis occurs in response to peritoneal
dialysis age progression, and an underlying malignancy could be masked (Tables 3 and
4, and Fig. 2).
Conclusions
Among the wide availability of tumor markers, only a few are clinically relevant due
to several reasons. Firstly, most cancer biomarkers are considered tumor associated
rather than tumor specific. Their diagnostic power is often garbled, and further
Table 3 Summary of main variations of tumor markers levels in CKD, dialysis, and kidney
transplantation (Coppolino et al. 2014)
Kidney
CKD Hemodialysis Peritoneal dialysis transplantation
Alpha- = = = =
fetoprotein
(AFP)
Beta-2- " " " "
microglobulin
(B2M)
Beta-HCG " " – –
CA 15-3 and CA " "* ; = * – =
27.29
CA 125 = = " In case of peritonitis or PD =
catheter placement
CA 19-9 =*; "* – – –
Total tPSA = # = –
Free fPSA " " – –
Chromogranin A " " – "
Legend: = unvaried with respect to patients with normal renal function; " increased;# decreased;
– no sufficient data; * see text
Table 4 Hemodialysis removal of main tumor markers

Tumor marker Molecular weight Hemodialysis removal (cutoff: <5–50 kDa)
CEA 180 kDa Not removed
AFP 65 kDa Not removed
PSA 33 kDa Removed
CA 125 90 kDa Not removed
CA 19.9 360 kDa Not removed
CA 15.3 300–400 kDa Not removed
CYFRA 40 kDa Removed
β- hCG 40 kDa (19 kDa for b subunit) Removed
investigations are usually needed to confirm or countermand the diagnosis. Simi-

larly, cancer biomarkers show a limited ability in monitoring the effectiveness of
antineoplastic therapies or malignancy recurrence. Another important issue involves
the appropriateness of antineoplastic screening in CRF. To correctly approach the
problem, a distinction between CRF stages is mandatory. In young CFR patients on
conservative treatment having a positive family history or clinical suspicion for
malignancy, screening for cancer finds its rationale as in the general population.
Conversely, patients on chronic dialysis should not be routinary screened for malig-
nancy, unless they are a candidate for kidney transplantation. It is well established
that patients undergoing chronic dialysis have a shortened life expectancy for
different reasons, and the advantage in reducing neoplastic related mortality is not
Fig. 2 Method of assay for tumor markers. LIA luminescence immunoenzymometric assay, ELISA
enzyme-linked immunosorbent assay, RIA radioimmunoassay, NM microarray nanotechnology
relevant (0.2 %) compared to the costs (Kajbaf et al. 2002). Opposite is the case of
dialysis patients on the waiting list for kidney transplant, in which, regardless of
renal function, it is essential to discover any cancer or precancerous lesion before
transplant, as the antirejection therapy may be precipitant. However, in all patients
eligible for cancer biomarker screening, the following step consists in the correct
interpretation of lab results in consideration of the residual renal function. Accord-
ingly, the most reliable cancer markers are AFP, hCG, and PSA to a certain extent. In
fact, compared to CEA and carbohydrates antigens, AFP, hCG, and PSA are not
virtually affected by coexisting renal failure or liver infectious diseases or serous
membrane diseases. An ideal cancer biomarker should be independent of renal
function, stable, sensitive, and specific for achieving the goal of the reliability in
the early diagnosis, staging, and monitoring of the neoplastic disease. So far, none of
the available cancer biomarkers simultaneously has all these requirements. Hope-
fully, most advanced research in the future will allow the early detection of cancer
and the management of antineoplastic therapy with a simple blood or urine test.
Summary Points
• There is a correlation between renal damage, in its various forms, and neoplastic
disease.
• Chronic renal failure predisposes to a higher incidence of cancer than the general
population.
• The tumor and/or its treatment can cause kidney damage, both acute and chronic.
• Tumor markers are affected in great part by renal metabolism and, therefore, by
alterations of renal function whether preexisting or arisen ex novo.
• AFP is the only reliable tumor marker in case of renal damage.
• The antineoplastic screening in patients with impaired renal function should be
individualized to expect life and concomitant risk factors for the development of
neoplasia and then interpreted in a critical manner in relation to the behavior of
each marker.
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Part II
Circulating and Body Fluid Biomarkers
Creatinine Assays in Early Infancy: How
to Aim for a Moving Target 13
Karel Allegaert
Contents
Key Facts of Creatinine Assays and Their Clinical Relevance in Early Infancy . . . . . . . . . . . . . . 273
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
Neonatal Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
Bio-Analysis-Related Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 275
Development of Glomerular Filtration Rate in Early Infancy
and Its Covariates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
Urinary Creatinine Measurements in Early Infancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 277
The Impact of the Introduction of IDMS-Traceable Assays on Creatinine
Reference Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Between-Assay Differences of IDMS-Traceable Creatinine Assays in Early Infancy . . . . . 286
Cystatin C to Assess Glomerular Filtration Rate in Early Infancy . . . . . . . . . . . . . . . . . . . . . . . . . 289
From Biochemical Quantification to Clinical Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 295
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 298
Abstract
Glomerular filtration rate (GFR) in neonates is very low and can only be
maintained due to a delicate balance between both vasodilatory effects at the
afferent and vasoconstrictor effects at the efferent glomerular arteriole. Despite
this low clearance capacity, interindividual variability is already extensive and
K. Allegaert (*)
Department of Development and Regeneration, KU Leuven, Leuven, Belgium
Neonatal Intensive Care Unit, University Hospitals Leuven, Leuven, Belgium
Intensive Care and Surgery, Erasmus MC, Sophia Children’s Hospital, Rotterdam, The Netherlands
e-mail: karel.allegaert@uzleuven.be; allegaertkarel@hotmail.com

272 K. Allegaert
can be predicted by covariates (gestational age, birth weight, postnatal age, drugs,
growth restriction, or peripartal asphyxia).
We still commonly used creatinine as a proxy for renal clearance capacity.
However, before creatinine values can be used to estimate renal elimination
capacity, there are some issues that need to be considered related to physiol-
ogy and methodology. Creatinine at birth does not yet reflect neonatal but
maternal creatinine clearance, and because of passive tubular back leak
instead of active secretion, creatinine clearance does not yet fully reflect
GFR. Trends will be described. Moreover, absolute creatinine values also
depend on the technique. The move toward harmonization through isotope
dilution mass spectrometry (IDMS) traceability has helped but has not
completely solved this problem. In line with recent observations in adults,
more research is needed to document the potential add on the benefit of
advanced biomarkers (e.g., cystatin C). In the meanwhile, IDMS-traceable
creatinine observations, compared to age-dependent, assay-specific reference
values, should be used to support clinical decisions.
Keywords
Assay • Clearance • Creatinine • Enzymatic • Glomerular filtration rate • Infant •
Isotope dilution mass spectrometry (IDMS) • Jaffe • Maturational changes •
Newborn • Preterm • Term
Abbreviations
95 % CI 95 % confidence intervals
CALIPER Canadian laboratory initiative on pediatric reference intervals
cGFR Calculated glomerular filtration rate
CLcrea Creatinine clearance
CLSI Clinical and laboratory standards institute
51
Cr-EDTA Chrome ethylene diamine tetra acetic acid
CysC Cystatin C
ELBW Extreme low birth weight infants (i.e., <1,000 g at birth)
ELISA Enzyme linked immunosorbent assay
IDMS Isotope dilution mass spectrometry
IgG Immunoglobulin G
kDa Kilodalton
NKDEP National Kidney Disease Education Program
PENIA Particle-enhanced nephelometric immunoassay
PETIA Particle-enhanced turbidimetric immunoassay
pRIFLE Pediatric risk, injury, failure, loss, end-stage (renal risk score)
13 Creatinine Assays in Early Infancy: How to Aim for a Moving Target 273
Scr Serum creatinine

SD Standard deviation
99m
Tc-DTPA Technecium diethylene triamine penta acetic acid
Key Facts of Creatinine Assays and Their Clinical Relevance

in Early Infancy
• The assessment of renal function is of clinical relevance. This is because it guides

short-term clinical decisions (e.g., pharmacotherapy) and determines long-term
outcome (e.g., cardiovascular or renal failure) and prognosis. In clinical practice,
this is commonly performed by a single-point serum creatinine (Scr) measure-
ment. These values are subsequent conversion to estimated or calculated glomer-
ular filtration rate (eGFR and cGFR). Obviously, this needs an accurate Scr
measurement to maintain the variability due to estimations more limited.
• Historically, serum creatinine is commonly quantified by Jaffe creatinine assays.
It is a colorimetric technique method to determine creatinine, based on a propor-
tional color change following a chemical reaction between picric acid and
creatinine. However, it is well known that these techniques suffer from interfer-
ences with endogenous or exogenous compounds present in the serum. Among
those interfering substances, bilirubin is the most prominent compound, but also
proteins and drugs can affect the color change. More recently, efforts have been
made to reduce these interferences and to standardize through isotope dilution
mass spectroscopy (IDMS) traceability.
• Enzymatic methods have been developed more recently to quantify creatinine.
Although theoretically more specific, these enzymatic methods can also display
interferences. Compared to Jaffe, these reactions suffer less from associated
bilirubin. Enzymatic methods use creatininase and creatinine hydrolase to convert
creatinine to creatine, with subsequent additional reactions and a color conver-
sion. Similar to Jaffe reactions, efforts have been made to reduce these interfer-
ences and to standardize through isotope dilution mass spectroscopy (IDMS)
traceability.
• The creatinine clearance rate is the blood volume that is completely cleared of
creatinine per unit time (/min or/h) and is commonly used to estimate GFR. In
adults, reference values are 90–120 ml/min. These maturational trends also in part
depend on the denominator (e.g., weight, body surface area) applied. In early
infancy, creatinine clearance is mainly driven by weight at birth (maturation until
birth) and by postnatal covariates (postnatal age, perinatal asphyxia, congenital
renal malformations, nephrotoxic drugs). In the term neonate, it is 20–45 ml/min/
1.73 m2, with a subsequent fast increase to reach (when/m2) adult reference
values in the second part of the first year of life. In preterm newborns, GFR is
much lower at birth (7.9 ml/kg/1.73 m2), with a similar subsequent fast increase
throughout the first weeks and months of life.
274 K. Allegaert
• In essence, the between- and within-patient variability of creatinine clearance in

early infancy displays at least a fivefold variability. This variability is of utmost
importance since this will guide clinical practices (e.g., fluid therapy, pharmaco-
therapy). Moreover, impaired renal function in early infancy has also been linked
to long-term morbidity characteristics (e.g., persistent renal failure, cognitive
outcome, retinopathy of prematurity).
Definitions
Neonatal Terminology
Newborn Any child in the first 28 days of postnatal life. This time interval is commonly
further subdivided in early neonatal (<day 8) and late neonatal life (day 8–28).
Infant Any child beyond the first 28 days of postnatal life but not yet 365 days old
(<1 year).
Preterm Newborn born too soon; normal pregnancy takes 40 weeks of gestational age
until delivery. Preterm delivery is any delivery before 37 weeks of gestational age.
ELBW Within the preterm neonates, there is a further subdivision, either based on
gestational age or on weight. Extreme low birth weight infants have a birth weight
below 1,000 g at delivery.
Bio-Analysis-Related Terminology
Creatinine Creatinine is a degradation product from creatine and is produced at a

fairly constant rate, reflecting muscle mass. It is commonly measured as a reflection
of renal function.
Creatinine assays The Jaffe quantification is a colorimetric reaction method using

alkaline picrate. Jaffe assays suffer from interference by endogenous (e.g., pseudo-
creatinines, hemoglobin F, bilirubin) and exogenous (e.g., cephalosporins). More recently,
enzymatic methods were introduced. These assays are less prone to such interference-
related errors and thus seem more suitable. Nevertheless, enzymatic assays can also be
affected by interferences. It is generally accepted that uncompensated Jaffe overestimates
Scr and fixed corrections (e.g., 0.2 or 0.3 mg/dl), or adaptations in the analytical procedure
(e.g., rate blanking) have been suggested to adapt Jaffe assays observations.
Matrix effect This term refers to the change or the measurement bias of a given
quantification method caused by (differences) in the non-analyte matrix. This may be
of specific relevance in human biology since the matrix in neonates (e.g., blood)
commonly differs (e.g., albumin, bilirubin, hemoglobin F) from other populations.

These differences may affect measurement results and/or accuracy.
Clearance Clearance is defined as the volume of fluid that – for a given time
interval – is completely cleared of a specific compound. Creatinine clearance hereby
reflects the renal elimination capacity or the glomerular filtration rate.
IDMS traceability Isotope dilution mass spectrometry traceability. This has been
introduced to adapt for the differences between different creatinine assays and serves
as a golden standard to all currently marketed creatinine assays. This became even
more important, since estimated glomerular filtration (eGFR) values are extrapolated
from single-serum creatinine (Scr) measurements.
Introduction
Renal function covers glomerular filtration rate (GFR), renal tubular transport activity
(excretion, absorption), and diuresis, but serum creatinine (Scr) is a commonly measured
and readily accessible biomarker to estimate renal (dys)function. To a certain extent, this
reflects the general condition of an individual patient and supports the clinician in the
decision-making process to adapt drug dosing, to formulate a prognosis, and to individ-
ualize clinical follow-up and monitoring (Levey 2014). This is also true in early infancy
since renal dysfunction (reduced clearance, raised creatinine) is associated with higher
mortality and morbidity (e.g., retinopathy of prematurity, neurodevelopmental impair-
ment) and is crucial to tailor pharmacotherapy or fluid exposure to the individual
newborn (Guignard and Gouyon 2008). Furthermore, an association of extreme preterm
birth and renal dysfunction in later life has been reported. More adequate detection of
renal dysfunction or impaired clearance in early infancy may hereby serve as an indicator
for focused renal follow-up in specific patients (Walker 2011).
Besides clearance assessment based on exogenous compounds like inulin, iohexol,
99m
Tc-DTPA (diethylene triamine penta acetic acid), or 51Cr-EDTA (ethylene diamine
tetra acetic acid) with consecutive, timed sampling, creatinine clearance or estimated
creatinine clearance can be used to quantify GFR. Creatinine clearance (CLcrea) can be
calculated based on serum and urine creatinine measurements [CLcrea = Creaur (mg/dl)
x urine volume (ml/min)/Scr], preferably based on 24 h urine collections (Levey 2014;
Guignard and Gouyon 2008). Because of the maturational GFR changes in early life,
population-specific reference values are needed. Reported reference values on creat-
inine clearance in neonates, developmental aspects of urine creatinine values, and
related to this, the impact on assays will be covered in this chapter (2.).
Indirect calculations based on single Scr values are also commonly used and are
much more convenient. Estimated (eGFR) or calculated GFR (cGFR) is commonly
used to screen renal function or to monitor high-risk patients (e.g., hypertension,
diabetes mellitus) consecutively. The National Kidney Foundation even strongly
recommends automatic calculation every time a serum creatinine measurement has
been performed and has provided kidney damage stages (stage 1–5), reflecting
276 K. Allegaert
Table 1 Creatinine clearance (CLcrea) from single-serum creatinine (Scr) values can be performed,
based on Schwartz estimates. Besides isotope dilution mass spectrometry (IDMS) traceability, these
Schwartz estimates further consider maturational aspects (k factor)
Original Schwartz estimate: serum creatinine (Scr) is measured based on a non-IDMS-traceable
assay, most likely Jaffe colorimetric technique
CLcrea ¼ ½k height=Scr
k , age-related factor 0.33, in low birth weight infant, <1 year
0.45, in term infant, <1 year
0.55, child or adolescent girl
0.70, adolescent boy
Revised Schwartz estimate: serum creatinine (Scr) is measured based on an IDMS-traceable assay,
most likely enzymatic technique
CLcrea ¼ ½0:413 height=Scr
normal or near-normal eGFR (stage 1, >90 ml/min; stage 2, 60–89, both with
protein and albumin in urine, cells, or casts) or decreased eGFR (stage
3, 30–59 ml/min; stage 4, 15–29 ml/min; stage 5, <15 ml/min) (US Department
of Health and Human Services 2012). Obviously, once again, population-specific
reference values are needed since these adult values do not yet apply to early infancy.
A major hurdle to implement the eGFR calculation concept based on single-
serum creatinine measurement is the obvious need for standardization and accuracy
to reduce inter-assay and intra-assay variation. This is because the assays (Jaffe,
enzymatic) for creatinine quantification are commonly biased by various interfering
substances, both endogenous (bilirubin, hemoglobin F) and exogenous (cephalospo-
rins, dopamine). This standardization has been driven by the creatinine standardization
program as created by the National Kidney Disease Education Program (NKDEP)
laboratory working group (US Department of Health and Human Services 2012).
Improvement has been made based on calibration traceability, standard reference
materials, and isotope dilution mass spectrometry (IDMS) reference measurement
for both Jaffe and enzymatic assays (Levey 2014). Once calibrated, creatinine
clearance (CLcrea) from single-serum creatinine (Scr) values can be performed,
based on Schwartz estimates (Delanaye and Ebert 2012). These Schwartz estimates
further consider maturational issues, as summarized in Table 1.
Similar to the reference values or intervals in other populations, this standardization
process also altered the reference values in young infants and resulted in the need to
adapt, e.g., creatinine observations driven dosing regimens and to generate assay-
specific reference values (Ceriotti et al. 2008; Ceriotti 2012). The impact of covariates
(gestational age, postnatal age, weight) on serum creatinine reference values for both
an uncompensated Jaffe and an IDMS-traceable enzymatic assay is discussed (3.).
Because of the specific characteristics of the neonatal serum matrix, it was
hypothesized that – even after IDMS standardization – the remaining Scr between-
assay differences (IDMS Jaffe versus IDMS enzymatic assay) is affected by
population-specific aspects (e.g., bilirubin, albumin, protein) (4.).
Finally, it has been suggested that eGFR values based on cystatin C (CysC)
strengthens the association between this marker and the risk of death or end-stage
renal disease across diverse adult populations (Shlipak et al. 2013). The available
data on CysC in neonates were therefore discussed in the final part of this chapter
(5.). Before we discuss these topics, we will first explore the development of
glomerular filtration rate in early infancy, with emphasis on renal maturation (phys-
iology) and covariates that affect this maturational pattern (pathophysiology) (1.).
Development of Glomerular Filtration Rate in Early Infancy

and Its Covariates
Glomerular filtration rate depends on the net ultrafiltration pressure across the glomerulus.
The kidney of a term neonate already possesses a full set of nephrons, approximately
850,000–1,200,000 per kidney. Despite this, the GFR in neonates is still very low (2–4 ml/
min or 20 ml/min/1.73 m2) and can only be maintained due to a delicate balance between
vasodilatory effects at the afferent and vasoconstrictor effects at the efferent glomerular
arteriole (Guignard and Gouyon 2008). These forces recruit maximal transmembrane
filtration pressure in a setting of overall low mean arterial blood pressure (30–40 mmHg)
and high intrarenal vascular resistance. The postnatal changes are mainly due to changes in
renal and intrarenal blood perfusion. The main drivers of renal function at birth besides age
are the impressive renal and extra-renal hemodynamic changes and include increased renal
blood flow, alterations of intrarenal blood flow, increased blood pressure, and raised
cardiac output. This delicate balance however results in relative limited capacity to adapt
to endogenous (e.g., peripartal asphyxia, hypotension, sepsis) or exogenous (e.g., neph-
rotoxic drugs like aminoglycosides or nonsteroidal anti-inflammatory drugs) stressors in
early life (Guignard and Gouyon 2008; Smits et al. 2012).
To further put this into some perspective, the GFR is 20–45 ml/min/1.73 m2 in the
term neonate, with a subsequent progressive increase of 5–10 ml/min/1.73 m2/week.
Similar, in the preterm neonate, median GFR reference values in infants aged
27–31 weeks gestation ranged from 7.9 to 30.3 ml/min/1.73 m2 on day
7, 10.7–33.1 ml/min/1.73 m2 on day 14, 12.5–34.9 ml/min/1.73 m2 on day
21, and 15.5–37.9 ml/min/1.73 m2 on day 28 (fivefold increase) (Guignard and
Gouyon 2008). When corrected for body surface area, GFR values reach adult values
in the second half of infancy (8 months) (Rhodin et al. 2009). In contrast, when
expressed in a weight-corrected approach (/kg), GFR relative to age is severalfold
higher in toddlers. Similarly, renal tubular functions (secretion, absorption) also
display maturation, but with a somewhat later onset and rate, to reach adult capacity
at the end of the first year of life (Guignard and Gouyon 2008; Smits et al. 2012).
Urinary Creatinine Measurements in Early Infancy
Similar to serum, creatinine can also be quantified by Jaffe or enzymatic assays in

urine. Urinary creatinine is hereby needed to calculate creatinine clearance as a
reflection of GFR or as denominator to quantify urinary excretion (Shlipak
et al. 2013; Junge et al. 2004).
278 K. Allegaert
In clinical practice, creatinine clearance (CLcrea) is commonly used as an indicator of

the glomerular filtration rate. In adults, CLcrea somewhat overestimates GFR because of
the additional active renal tubular secretion of creatinine. In preterm and term neonates,
active renal tubular secretion still displays ontogeny, i.e., age-related activity. In
addition and due to less effective tight junctions at the level of the apical renal tubular
membranes, there is passive back leak of filtered creatinine across the immature leaky
tubules. The need for a timed, complete urine collection, e.g., for 12 or 24 h, is another
important practical hurdle and limits its use mainly to clinical research (Guignard and
Gouyon 2008). Using this approach and based on a standardized Jaffe method, a
CLcrea-based GFR nomogram for preterm neonates (27–31 weeks) in the first month
of life has recently been reported (Vieux et al. 2010). These data and other CLcrea-based
GFR estimates (all expressed by ml/min/1.73 m2) in different cohorts as reported in the
literature are provided in Table 2 (Vieux et al. 2010; Gordjani et al. 1988; Sonntag
et al. 1996; Bueva and Guignard 2004; Gallini et al. 2000). In essence, these pooled
cohort data nicely illustrate that GFR estimates depend on both gestational age and birth
weight (reflecting renal function at birth), as well as postnatal age. Such reference
values in healthy neonates can subsequently be used to quantify the impact of diseases
(congenital renal malformation, peripartal asphyxia) or treatment modalities (e.g.,
indomethacin, ibuprofen) (Rhodin et al. 2009; Smits et al. 2012).
Urinary creatinine is also commonly used as denominator to quantify the urinary
excretion or loss of electrolytes, amino acids, or proteins (Allegaert et al. 2014b). An
alternative denominator is urine-specific gravity, i.e., the ratio of urine density
compared to water density (1.0), with a reference range in adults between 1.005
and 1.030. However, urine density will also be affected by exogenous (e.g., drugs)
and endogenous (e.g., glucosuria) compounds not routinely retrieved in urine (Jung
1991). In neonates, the urine-specific gravity reference range is 1.002–1.004 and
1.002–1.006 in infants. Beyond infancy, reference values (1.001–1.030) are similar
to adult values (Guignard and Gouyon 2008). These maturational changes largely
reflect the progressive increase in concentration capacity throughout infancy. The
same pattern, with increasing concentration capacity and increasing renal elimina-
tion capacity throughout infancy, can be observed in reference urine creatinine
values, as reflected in Table 3. This table summarizes observations on urine creat-
inine concentrations as reported in cohort of neonates, children, or adults (Sonntag
et al. 1996; Srivastava et al. 2008; Van Lente and Suit 1989; Apple et al. 1986;
Allegaert et al. 2014b). This maturational trend, with much lower creatinine (5- to
20-fold) values in neonatal urine, may also be of relevance when we consider the
creatinine quantification assays (Tables 2 and 3).
Indeed – irrespective of its application (CLcrea estimates or denominator) –
adequate standardization of creatinine quantification is of utmost importance for
accurate and interchangeable test results and reference values. This is also true for
the urine analysis. Historically, creatinine assays showed great variation depending
on, e.g., reaction mechanism (Jaffe or enzymatic) or manufacturer, but also differ-
ences in the concentration range need to be considered (Table 2) (van Lente en Suit
1989; Apple et al. 1986). However, IDMS calibration has been more extensively
verified for serum than urine matrix. Moreover, standardization itself does not
eliminate reaction mechanism-related differences. Jaffe methods interfere with
Table 2 Glomerular filtration rate reference values, based on creatinine clearance estimates as
reported in different neonatal cohorts
Creatinine clearance
Reference Assay Clinical characteristics estimates
Vieux et al. (2010) Jaffe 146/275 healthy neonates,
27–31 weeks
Day 7 19.95 ml/min/
(SD 9.30) 1.73 m2
Day 14 22.11 ml/min/
(SD 14.90) 1.73 m2
(SD 10.88) 1.73 m2
(SD 12.66) 1.73 m2
Gordjani Jaffe 12 neonates, 28–32 weeks at
et al. (1988) birth
Day 4–5 10.7 (range ml/min/
9.40–15.3) 1.73 m2
Day 8–10 (7/12) 14.2 (range ml/min/
12.1–28.7) 1.73 m2
13 neonates, 33–37 weeks at
birth
Day 4–5 20.6 (range ml/min/
14.4–31.6) 1.73 m2
Day 8–10 (4/13) 33.3 (range ml/min/
23.1–43.4) 1.73 m2
Sonntag Jaffe 34 neonates, 26–34 weeks at
et al. (1996) birth
First week 12.5 (range ml/min/
7–22) 1.73 m2
Second week 15.7 (range ml/min/
10–28) 1.73 m2
3–4 week 19.7 (range ml/min/
11–34) 1.73 m2
Bueva and Jaffe 66 healthy neonates, Trends in median estimates
Guignard (2004) 29–40 weeks at birth
Term cases Day 2–9–15 29 to 46 to 68 ml/min/
1.73 m2
2,000–2,500 g Day 2–9–15 18 to 32 to 38 ml/min/
1.73 m2
1,500–2,000 g Day 2–9–15 11 to 23 to 32 ml/min/
1.73 m2
1,000–1,500 g Day 2–9–15 8 to 17 to 19 ml/min/
1.73 m2
Gallini et al. (2000) Jaffe 83 preterm neonates Trends in median estimates
<27 weeks Day 6 to 11 to 12 to ml/min/
3–10–17–31 21 1.73 m2
27–28 weeks Day 8 to 15 to 20 to ml/min/
3–10–17–31 24 1.73 m2
29–30 weeks Day 12 to 22 to ml/min/
3–10–17–31 24 to 29 1.73 m2
31–32 weeks Day 13 to 23 to ml/min/
3–10–17–31 26 to 32 1.73 m2
280 K. Allegaert
exogenous (e.g., cephalosporins) or endogenous (e.g., pseudo-creatinines, bilirubin,

glucose) substances. Enzymatic methods are less prone but also suffer from inter-
ferences (e.g., dopamine, bilirubin). Consequently, maturational changes (e.g., bil-
irubin production, creatinine concentrations) as well as treatment modalities may
have a population-specific impact (Guignard and Gouyon 2008; Ceriotti 2012;
Allegaert et al. 2014b). Given the method and assay-specific interferences, urinary
creatinine may still vary in a method and assay-specific way with between-assay
differences related to the composition of neonatal urine and despite the use of IDMS-
traceable assays. Taking these abovementioned remarks into account, the inter-
changeability of two IDMS-traceable assays (Jaffe and enzymatic) was verified in
84 urine samples, collected in 23 neonates (Allegaert et al. 2014b).
Samples were analyzed using two clinical IDMS-traceable assays, commercial
available analytic methods [Roche Diagnostics Jaffe (urine application without
compensation factor and a sample dilution factor of 1:25) and enzymatic method
(dilution factor of 1:50)], and were measured on a Cobas c702 module. The total
imprecision (expressed as CV) of the Jaffe assay determined according to Clinical and
Laboratory Standards Institute (CLSI) guidelines was 2.2 % at 61.5 mg/dl and 2.2 %
at 138.9 mg/dl with a measuring range from 4.2 to 622 mg/dl. For the enzymatic
assay, the total imprecision was 1.4 % at 64.8 mg/dl and 1.2 % at 145.8 mg/dl with a
measuring range from 1.1 to 610 mg/dl. Internal quality control was performed with
commercial control material (Bio-Rad unassayed) using simplified Westgard Rules
for statistical process control and biological critical acceptance limits; external quality
control was based on the Bio-Rad Unity urine chemistry report. The median and
range in neonates for both techniques are reported in Table 3. Paired measurement of
urinary creatinine in neonates based on Jaffe and enzymatic IDMS-traceable assays
Table 3 Assay specific urine creatinine concentrations as reported in neonates, children, or adults.
Data reported by median and range or mean and standard deviation
Reference Clinical characteristics Assay Observations
Allegaert 23 neonates, 24–41 weeks Jaffe, 9.25 (3.7–42.2) mg/dl
et al. (2014a, b) gestational age modified
Postnatal age 1–26 days, Enzymatic 9.15 (3.8–42.9) mg/dl
paired analysis
Sonntag 34 preterm neonates, Jaffe, 9.7 (3.5–17.7) mg/dl
et al. (1996) <1,500 g modified
First 9 weeks postnatal life
Srivastava 18 children, 40 urine Jaffe, 69.3 (SD 66.2) mg/dl
et al. (2008) samples modified
Enzymatic 75.7 (SD 69.7) mg/dl
Van Lente 100 adults, 15–71 years Jaffe 269.8 (SD 199.8) mg/dl
et al. (1989) Including cases with renal Enzymatic 231 (SD 178.7) mg/dl
impairment
Apple 17 healthy adult volunteers Jaffe 166 (SD 65) mg/dl
et al. (1986) Enzymatic 138 (SD 49) mg/dl
15
10 +1.96 SD
9,6
crea Jaffe - crea enzym, %
Mean
0
0,4
-5
-1.96 SD
-10 -8,9
-15
0 10 20 30 40 50
crea Jaffe (mg/dL)
Fig. 1 Bland-Altman plot, illustrating a mean difference of 0.4 % (equal to 0.2 mg/dl) following
paired measurement of urinary creatinine in neonates based on Jaffe and enzymatic IDMS-traceable
assays
resulted in a limited mean difference of 0.2 mg/dl, and the regression line (y = 0.28 +
0.95, 95 % CI slope 0.93–0.98) suggests that the enzymatic assay is proportionately
about 5 % higher compared to Jaffe. Figure 1 is a Bland-Altman plot, illustrating a
mean difference of 0.4 % (absolute mean difference = 0.2 mg/dl).
Although the overall difference between both assays is very limited, there seems
to be a urine creatinine concentration-dependent impact on the extent and the
direction of the difference between both assays. At low concentration (<10 mg/dl),
the Jaffe assay is higher compared to the enzymatic assay (up to 15 %), while the
reverse is observed at urine creatinine concentrations above 25 mg/dl (5–10 %)
(Allegaert et al. 2014b). Taking some limitations (e.g., limited number of cases with
hyperbilirubin >16 mg/dl, only two specific assays) of this study into account,
differences between IDMS-traceable Jaffe and enzymatic assays were limited, even
in a heterogeneous group of neonates with still an assay-specific impact of bilirubin.
The Impact of the Introduction of IDMS-Traceable Assays

on Creatinine Reference Values
It is generally accepted that uncompensated Jaffe overestimates Scr and fixed

corrections (e.g., 0.2 or 0.3 mg/dl) or adaptations in the analytical procedure (e.g.,
282 K. Allegaert
Fig. 2 Postnatal trends of serum creatinine (Scr) in 151 extreme low birth weight neonates were
combined with data earlier reported by Bueva and Guignard (2004)
rate blanking) have been suggested to adapt or convert Jaffe assay observations
(Kaiser et al. 2014; US Department of Health and Human Services 2012; Myers
et al. 2006). Because of the specific aspects of neonatal serum matrix (bilirubin,
protein, albumin), such a conversion factor should not be taken for granted to apply
earlier reported creatinine reference intervals or threshold to new assays. Such
reference values are further complicated by the extensive inter- and intra-patient
variability in early infancy (Ceriotti 2012). There is an initial increase in the first days
of life, most pronounced in the most immature neonates, with a subsequent progres-
sive decrease throughout infancy, most blunted in the most immature neonates, as
illustrated in Fig. 2 (Bueva and Guignard 2004; George et al. 2011).
This extensive variability very likely also explains the absence of a universally
adopted definition of acute kidney injury (AKI) in infants. Different threshold Scr
values (1.13–2 mg/dl) are suggested, while the modified pediatric RIFLE (pRIFLE,
pediatric risk, injury, failure, loss, end-stage) recommends diagnostic criteria based
on the Scr increase (stage 1, + 0.3 mg/dl or 150–200 % increase from through Scr
value; stage 2, increase of 200–300 % from through Scr value; stage 3, increase Scr
>300 %, or 2.5 mg/dl or dialysis) and the urine output (Akcan-Arikan et al. 2007). In
essence, it remains difficult to disentangle physiology from pathophysiology. Nei-
ther an absolute value nor a proportional increase is unrelated to the maturational
changes, and, e.g., stage 1 is almost “common” in extreme low birth weight infants
(George et al. 2011; Allegaert et al. 2012). It seems much more reasonable to use
age-dependent assay-specific reference intervals or centiles as threshold values.
To illustrate the impact of the introduction of an IDMS-traceable enzymatic assay

compared to an uncompensated Jaffe assay on reference ranges, postnatal trends in
serum creatinine in two cohorts of neonates before and after switch in assay
(uncompensated Jaffe to IDMS-traceable enzymatic assay) were collected, with
emphasis on extreme low birth weight (ELBW, i.e., <1,000 g) neonates in the first
6 weeks of postnatal life (Allegaert et al. 2012). Based on 1 883 (uncompensated
Jaffe assay) and 1 295 (IDMS traceable enzymatic assay) Scr observations in 151 and
116 ELBW infants, respectively, reference values for postnatal serum creatinine
values in ELBW infants during the first 6 weeks of postnatal life determined by
either Jaffe or enzymatic Scr quantification were generated.
For both datasets, gestational age, postnatal age, ibuprofen co-administration, and
other indicators of disease severity (e.g., the need for respiratory support, low Apgar
score) were covariates of Scr observations. To illustrate the dynamics and the impact
of both postnatal age and disease characteristics, median creatinine values on day
1, 3, 14, and 28 for either the Jaffe (not IDMS traceable) or the IDMS-traceable
enzymatic assay in ELBW infants either or not exposure to ibuprofen (Fig. 3) are
1.2
jaffe + ibuprofen
1.1 jaffe - ibuprofen
enzymatic + ibuprofen
enzymatic - ibuprofen
1
0.9
Serum creatinine (mg/dl)
0.8
0.7
0.6
0.5
0.4
0.3
day day day day day
Fig. 3 Median creatinine values on day 1, 3, 14, and 28 for either the uncompensated Jaffe or
isotope dilution mass spectrometry (IDMS)-traceable enzymatic assay in extreme low birth weight
(ELBW, i.e., <1,000 g at birth) infants either or not exposure to ibuprofen
284 K. Allegaert
provided. In addition and based on the differences in reference values in the two
consecutive cohorts, we confirm that conversion based on a single fixed value is not
appropriate in early infancy. This is because such a fixed value cannot account for the
variability in neonatal serum composition (e.g., albumin, IgG, bilirubin). Conse-
quently, enzymatic quantification is the preferred method for use in early life.
Unfortunately, these enzymatic reaction methods are more expensive.
To further assess the impact of the use of an IDMS-traceable enzymatic assay
versus an uncompensated Jaffe quantification method, a retrospective study on Scr
values obtained by the Jaffe method in 1,140 neonates admitted between 2001 and
2006 was analyzed and compared to values obtained by using the enzymatic method
in 1,023 neonates admitted between 2007 and 2011 in the same unit. The pooled
results on postnatal trends for both cohorts (<1 kg, 1–2 kg, 2–3 kg and >3 kg) in
whom either the Jaffe or enzymatic assay is provided in Table 4 (median values and
difference between both values) and Fig. 4 (Allegaert et al. 2011). In essence, Jaffe is
always higher compared to enzymatic techniques, but the differences in median
values vary between both techniques (0.1–0.26 mg/dl).
Age- and weight-based reference values and the development of age-corrected
centiles or Z scores similar to body weight or length obviously cannot be generated
based on single-unit observations and re-illustrates the challenge to establish pedi-
atric reference values for laboratory values for early infancy. Initiatives like the
CALIPER (Canadian Laboratory Initiative on Pediatric Reference Intervals) provide
online creatinine data for both Jaffe and enzymatic assays but limit this to “neonates”
in the first 14 days of postnatal life (Colantonio et al. 2012). To illustrate at least the
potential impact of age-corrected centiles, we refer to recently reported examples on
(i) the use of Scr centile values in early infancy as a predictor for neonates with
posterior urethral valves (Lemmens et al. 2014) and on (ii) the use of Scr centile
values to further explore the variability in amikacin clearance in preterm neonates
(Smits et al. 2013).
Most authors agree that the “nadir” Scr (i.e., the lowest value, threshold 1 mg/dl)
in infancy accurately predicts long-term renal prognosis in neonates with posterior
urethral valves (Pohl et al. 2012). However, the determination of the nadir Scr
requires meticulous follow-up, and it is not yet available in early infancy. Using
population-specific Scr centiles in neonates with posterior urethral valves, peak Scr
but also Scr values between day 9 and day 42 above the 75th centile already
predicted unfavorable renal outcome (GFR <60 ml/min/1.73 m2) at 2 years of
life (Lemmens et al. 2014). Similarly, birth weight and postnatal age but not
creatinine were significant covariates of amikacin clearance in neonates, despite
the fact that there is claimed similarity between amikacin clearance and GFR
(Smits et al. 2012). However, estimation of GFR in individual cases based on
weight- and postnatal age-dependent threshold (<P25th, 25–75th or >75th centile)
creatinine values for extreme low birth weight (ELBW <1,000 g) infants can be
used as additional biomarker of amikacin clearance instead of an absolute value
once birth weight and postnatal age have been considered (Smits et al. 2013). As
illustrated in Fig. 5, an age-corrected creatinine value below the 25th centile
(reflecting “improved” renal function) was associated with significantly higher
13
Table 4 Postnatal trends (median, mg/dl) in serum creatinine values for consecutive birth weight categories. Serum creatinine (Scr) was quantified by either
enzymatic analysis or Jaffe. The differences in median values are between 0.1 and 0.245 mg/dl
<1 kg D1 D2 D3 D4 D5 D6 D7 D8 D14 D21 D28 D42
Enzyme 0,58 0,85 0,91 0,88 0,825 0,82 0,75 0,73 0,695 0,58 0,465 0,42
Jaffe 0,75 1 1,09 1,09 0,98 0,93 0,88 0,84 0,765 0,68 0,61 0,54
Difference 0,17 0,15 0,18 0,21 0,155 0,11 0,13 0,11 0,07 0,1 0,145 0,12
1–2 kg
Enzyme 0,61 0,82 0,77 0,68 0,63 0,64 0,59 0,585 0,59 0,51 0,43 0,375
Jaffe 0,78 0,97 0,95 0,88 0,85 0,81 0,77 0,775 0,72 0,63 0,58 0,52
Difference 0,17 0,15 0,18 0,2 0,22 0,17 0,18 0,19 0,13 0,12 0,15 0,145
2–3 kg
Enzyme 0,64 0,79 0,68 0,56 0,49 0,46 0,48 0,45 0,47 0,37 0,34 0,29
Jaffe 0,8 0,95 0,88 0,78 0,74 0,705 0,65 0,66 0,62 0,6 0,535 0,46
Difference 0,16 0,16 0,2 0,22 0,25 0,245 0,17 0,21 0,15 0,23 0,195 0,17
>3 kg
Enzyme 0,65 0,71 0,63 0,5 0,45 0,41 0,4 0,4 0,375 0,35 0,3 0,27
Creatinine Assays in Early Infancy: How to Aim for a Moving Target
Jaffe 0,88 0,935 0,81 0,71 0,66 0,67 0,65 0,63 0,59 0,55 0,505 0,51
Difference 0,23 0,225 0,18 0,21 0,21 0,26 0,25 0,23 0,215 0,2 0,205 0,24
285
286 K. Allegaert
Fig. 4 Postnatal trends for both cohorts (<1 kg, 1–2 kg, 2–3 kg and >3 kg) in whom either the
uncompensated Jaffe or the isotope dilution mass spectrometry (IDMS)-traceable enzymatic assay is
provided. In essence, Jaffe is always higher compared to enzymatic techniques, but the differences
in median values differ between both techniques
amikacin clearance. This remained an independent covariate of amikacin clear-

ance, also when birth weight and postnatal age were considered. Both observations
at least suggest that further exploration of such a “threshold” or centile approach is
warranted, but we do need a “neonatal” CALIPER approach to further develop and
validate neonatal reference values in both preterm and term neonates (Ceriotti
2012; Colantonio et al. 2012).
Between-Assay Differences of IDMS-Traceable Creatinine Assays

in Early Infancy
Initially, Scr reference values were based on uncompensated Jaffe quantification, a

colorimetric reaction method using alkaline picrate. Jaffe assays are known to suffer
from interference by multiple endogenous (e.g., hemoglobin F, IgG, bilirubin) and
exogenous (e.g., cephalosporins, dopamine) substances, of which some are com-
monly found in the serum of neonates (Guignard and Gouyon 2008; Junge
et al. 2004). Driven by the need to improve estimated GFR performance, Jaffe and
enzymatic quantification methods were calibrated to IDMS (US Department of
2,5
+ +
+
2,0
amikacin clearance (ml/kg/min)
1,5
+
1,0
0,5
0,0
1 2 3
creatinine [1=<25th centile, 2 = 25-75th centiel, 3 = >75th centile]
Fig. 5 In a dataset on individual amikacin clearance calculations in 175 preterm neonates, serum
creatinine (Scr) was a significant covariate of clearance, when creatinine age-specific reference
values (<25th centile, 25–75th centile, or >75th centile) were used. Such reference values are
displayed in Table 4 and Fig. 4
Health and Human Services 2012; Myers et al. 2006). To explore if IDMS-based Scr
standardization program affects the remaining Scr between-assay differences with a
population-specific impact, a study compared Scr values measured by Jaffe and
enzymatic methods in paired neonatal serum samples and linked this with clinical
characteristics (Allegaert et al. 2014a). Scr was measured in blood samples collected
from 129 neonates [gestational age range 24–41 weeks, weight range 0.62–4.55 kg,
albumin range 21–67 g/l, and total bilirubin range 0.2–16.1 mg/dl]. The median Jaffe
Scr was 0.50 (range 0.17–1.16) mg/dl, and median enzymatic Scr was 0.48 (range
0.06–1.11) mg/dl, resulting in a mean difference in Scr of 0.013 (range 0.2 to 0.11)
mg/dl (equal to 3.9 %). Bland-Altman analysis hereby illustrates that there is no Scr
concentration-related impact on this difference (Fig. 6). Bilirubin (total, direct) and
albumin (Fig. 7) had a significant correlation with the Scr difference. Using multiple
regression, only total bilirubin remained a significant covariate (r2 = 0.24).
In essence, this study observed a statistical significant but clinical likely irrelevant
mean difference of 0.013 mg/dl between IDMS-traceable Jaffe and enzymatic
assays. The extent of this difference was in part explained by bilirubin. There are
different reports that document that both Jaffe and enzymatic assays are affected by
bilirubin, although interference of enzymatic methods has been minimized (Myers
288 K. Allegaert
40
+1.96 SD
20
21,0
(enzymatic - jaffe) / enzymatic (%)
0 Mean
– 3,9
–20
– 1.96 SD
– 28,8
–40
–60
–80
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
enzymatic (mg/dL)
Fig. 6 Bland-Altman plot of paired serum samples from 129 (pre)term neonates. There is a mean
difference in Scr of 3,9 %, equal to 0.013 (range 0.2 to 0.11) mg/dl
70
60
albumin (g/L)
50
40
30
20
–0.1 0.0 0.1 0.2
difference (Jaffe - enzymatic) (mg/dL)
Fig. 7 The absolute mean difference in Scr measurement in 129 paired serum samples in (pre)term
neonates was 0.013 (range 0.2 to 0.11) mg/dl. A significant effect of albumin on the difference
between both assays was documented ( p = 0.0016)
et al. 2006). With increasing bilirubin (up to 16.1 mg/dl), Jaffe results are relatively
(up to 0.1 mg/dl) lower compared to enzymatic measurements, most likely explained
by overcorrection of the Jaffe assay. Bilirubin-related interference is caused by an
oxidation of bilirubin to biliverdin in the alkaline Jaffe reaction medium. The Roche
Jaffe assay uses rate blanking to compensate for this interference. Prior to adding
picric acid (and starting the reaction), alkaline is added to the sample and the
bilirubin oxidation rate is measured and later on subtracted from the measured
reaction rate of creatinine with alkaline picric acid. As the oxidation of bilirubin in
alkaline is curvilinear, overcorrection can occur with increasing bilirubin concentra-
tion (Apple et al. 1986; Junge et al. 2004). In clinical laboratory practice, Jaffe
creatinine results from samples with high bilirubin concentrations (>10 mg/dl)
should either not be reported or at least a warning should be added. In line with
the earlier reports in patients with hypoalbuminemia, we confirm an albumin
concentration-related effect on the difference between both assays, although the
overall strength and trend of the impact is even more limited when compared to
bilirubin. In conclusion and taking the limitations of this single study (only two
specific assays, range of bilirubin not above 16 mg/dl, limited number of samples)
into account, it seems that the differences between IDMS-traceable Jaffe and enzy-
matic assays are limited, even for a heterogeneous group of neonates. Only an assay-
specific impact of bilirubin could be demonstrated (Allegaert et al. 2014a).
Cystatin C to Assess Glomerular Filtration Rate in Early Infancy
As discussed earlier in this chapter, there are limitations on the use of creatinine as a
robust and sensitive marker for alterations in glomerular filtration rate (GFR) (Levey
et al. 2014; Delanaye and Ebert 2012; Shlipak et al. 2013). Moreover, some of the
creatinine-related limitations (e.g., passive tubular back leak, bilirubin,
hemoglobin F, lower creatinine concentration range in urine or serum) are more
relevant in perinatal life, even after IDMS traceability for the different assays has
been introduced (Guignard and Gouyon 2008; Vieux et al. 2010; Allegaert
et al. 2012). Low molecular weight proteins (e.g., cystatin C, beta-trace protein,
beta-2 microglobulin, alpha-1 microglobulin) have been suggested as valid alterna-
tives and biomarkers (Levey et al. 2014; Zwiers et al. 2014; Zaffanello et al. 2009). It
is claimed that cystatin C (CysC) is not affected by body muscle mass, age, gender,
inflammation, or nutritional conditions (Shlipak et al. 2013).
CysC is a low molecular mass (13 kDa) basic protein and belongs to the cystatin
superfamily of reversible inhibitors of cysteine proteases. It is a proteinase inhibitor
involved in normal intracellular protein turnover. Consequently, CysC is produced at
a constant rate by any nucleated cell and is eliminated exclusively through glomer-
ular filtration. After ultrafiltration through the basal membrane of the glomerulus,
CysC is completely metabolized in the proximal renal tubular cell following
megalin-mediated endocytosis at the apical renal tubular cell brush border (Levey
et al. 2014; Delanaye and Mariat 2013). Consequently, serum CysC will reflect the
glomerular filtration rate, while urinary CysC’s presence is a reflection of renal
tubular dysfunction (Levey et al. 2014; Delanaye and Mariat 2013).
290 K. Allegaert
There are at present at least three different registered methods for serum analysis.
Besides particle-enhanced nephelometric immunoassays (PENIAs), the use of
particle-enhanced turbidimetric immunoassays (PETIAs) and ELISA methods has
been reported (Hossain et al. 2009). For both PENIA and PETIA, specific GFR
estimators have been developed (Delanaye and Mariat 2013; Levey et al. 2014; Li
et al. 2010). Unfortunately and similar to creatinine assays, the diagnostic perfor-
mance at present is still suboptimal because of higher imprecision, between-assay
differences or over-recovery or between laboratory differences. This resulted in the
suggestion of Li et al. to apply assay-specific cystatin C-based GFR equations or
estimators, until an international calibration for CysC – similar to the IDMS
approach for creatinine – has been developed (Li et al. 2010).
Assay-specific reference values for healthy term (Abitbol et al. 2014; Lee
et al. 2013; Finney et al. 2000; Harmoinen et al. 2000; Bahar et al. 2003; Treiber
et al. 2006; Bariciak et al. 2011; Dorum et al. 2012; Cataldi et al. 1999; Randers
et al. 1998), healthy preterm (Abitbol et al. 2014; Lee et al., 2013; Finney et al. 2000;
Harmoinen et al. 2000; Armangil et al. 2008; Bariciak et al. 2011; Dorum et al. 2012;
Demirel et al. 2013), and neonates with specific perinatal disease characteristics are
summarized in Tables 5, 6, and 7, respectively. We hereby also added information on
the assay used, since we are unaware of any between-assay assessment in neonatal
samples. In essence (i) CysC concentrations are higher at birth, with a subsequent
decrease in both term and preterm neonates; (ii) CysC concentrations are higher in
preterm neonates when compared to term neonates, with the highest values in the
most immature preterms; (iii) disease characteristics like respiratory distress (Elmas
et al. 2013), bilateral kidney abnormalities (Parvex et al. 2012), peripartal asphyxia
(Treiber et al. 2014), aminoglycoside exposure (Abitbol et al. 2014), renal dysfunc-
tion (Elmas et al. 2013; Montini et al. 2001), or sepsis (Maruniak-Chudek
et al. 2012). Surprisingly, the CysC concentrations were lower in the septic shock
newborns; (iv) the assay seems also to be of relevance in neonatal samples. The
mean/median values for CysC measured on day 1 in term neonates are 1.64, 1.70, or
2.11 for PETIA measurements and 1.6, 1.97, 1.84, and 1.21 mg/l for PENIA
quantifications. Furthermore, Fig. 8 shows all mean/median values reported in
Tables 5 and 6, strongly suggesting an assay-mediated difference in mean/median
values in neonatal serum samples, similar to creatinine.
Besides these analytical issues, we also have to take perinatal renal physiology into
account before we consider the introduction of CysC to estimate perinatal GFR. In
contrast to creatinine and because of its size, CysC does not cross the placental barrier.
Consequently, there is no correlation between maternal and umbilical cord blood CysC
observations (Cataldi et al. 1999; Kuppens et al. 2012). Different authors described a
progressive physiological decrease in CysC values throughout infancy, with subse-
quent stabilization to age-independent reference values after the first year of life
(0.57–1.12 mg/l) (Randers et al. 1999). This means that – similar to creatinine –
age-specific reference values in early life are needed before we can consider to integrate
this biomarker into neonatal practice as a sensitive and specific marker to discriminate
between normal physiology and renal dysfunction (Ceriotti 2012; Colantonio
et al. 2012). Finally – following ultrafiltration – CysC undergoes megalin-mediated
Table 5 Plasma cystatin C (CysC) values in healthy term neonates

Reference Assay Clinical characteristics CysC values
Abitbol PENIA n = 48, term, 39 (SD 1) weeks 1.33 mg/l
et al. (2014) Second part of the first week of life (SD 0.18)
Lee et al. (2013) PETIA n = 119 cases
Day 0–3 1.64 mg/l
(SD 0.32)
Day 4–6 1.42 mg/l
(SD 0.26)
Day 7–10 1.51 mg/l
(SD 0.23)
Day 11–15 1.55 mg/l
(SD 0.29)
Day 16–21 1.55 mg/l
(SD 0.34)
Day 22–30 1.56 mg/l
(SD 0.39)
Finney PENIA n = 50 cases, 0–3 months 1.37 mg/l
et al. (2000) (0.81–2.32)
Harmoinen PETIA n = 50, term cases 1.70 mg/l
et al. (2000) (SD 0.26)
n = 65, >8 days–1 year 0.75–1.87 mg/l
Bahar et al. (2003) PENIA n = 112, umbilical blood at birth 1.36 mg/l
(SD 0.35)
n = 98, venous blood, day 3 1.35 mg/l
(SD 0.33)
Treiber PENIA n = 75, umbilical blood at birth 1.97 mg/l
et al. (2006) (34–41 weeks) (SD 0.60)
n = 75, venous blood, day 1.93 mg/l
3 (34–41 weeks) (SD 0.33)
Bariciak PENIA 36 weeks, n = 24, postnatal day 1 1.84 mg/l
et al. (2011) (1.32–2.63)
36 weeks, n = 21, postnatal day 3 1.58 mg/l
(1.16–1.95)
Dorum PENIA At delivery, term cases (n = 33) 1.21 mg/l
et al. (2012) (SD 0.31)
Cataldi PETIA 88 term cases, at delivery 2.11 mg/l
et al. (1999) (1.17–3.06)
Term cases, day 3 1.75 mg/l
(0.75–2.7)
Term cases, day 5 1.63 mg/l
(0.66–2.15)
Randers PENIA 12 term cases, <1 month 1.63 mg/l
et al. (1998) (SD 0.26)
PENIA particle-enhanced nephelometric immunoassay, PETIA particle-enhanced turbidimetric
immunoassay, SD standard deviation
292 K. Allegaert
Table 6 Plasma cystatin C (CysC) values in healthy preterm neonates

Abitbol PENIA n = 60, preterm, 34 (SD 3) 1.42 mg/l
et al. (2014) weeks (SD 0.21)
Second part of the first week of
life
Lee et al. (2013 PETIA n = 72 cases, 33–36 weeks
gestational age
Day 0–3 1.67 mg/l
(SD 0.25)
Day 4–6 1.68 mg/l
(SD 0.27)
Day 7–10 1.69 mg/l
(SD 0.32)
Day 11–15 1.72 mg/l
(SD 0.24)
Day 16–21 1.81 mg/l
(SD 0.22)
Day 22–30 1.64 mg/l
(SD 0.23)
n = 40 cases, 29–32 weeks
gestational age
Day 0–3 1.56 mg/l
(SD 0.28)
Day 4–6 1.53 mg/l
(SD 0.21)
Day 7–10 1.75 mg/l
(SD 0.29)
Day 11–15 1.87 mg/l
(SD 0.31)
Day 16–21 1.68 mg/l
(SD 0.31)
Day 22–30 1.84 mg/l
(SD 0.27)
n = 40 cases, 28 weeks
gestational age
Day 0–3 1.60 mg/l
(SD 0.21)
Day 4–6 1.55 mg/l
(SD 0.28)
Day 7–10 1.73 mg/l
(SD 0.41)
Day 11–15 1.87 mg/l
(SD 0.26)
Day 16–21 1.80 mg/l
(SD 0.28)
Day 22–30 2.02 mg/l
(SD 0.42)
(continued)
Table 6 (continued)
Finney In-house n = 14, 29–36 weeks, day 1 1.65 mg/l
et al. (2000) immunoassay (0.62–4.42)
n = 16, 24–28 weeks, day 1 1.48 mg/l
(0.65–3.37)
Harmoinen PETIA n = 58, preterm cases 1.88 mg/l
et al. (2000) (SD 0.36)
Armangil PENIA n = 108, (32.5, SD 2.6 weeks), 1.80 mg/l
et al. (2008) day 1 (SD 0.3)
n = 108, day 3 1.65 mg/l
(SD 0.3)
Bariciak PENIA 32–36 weeks, n = 29, postnatal 1.89 mg/l
et al. (2011) day 1 (0.58–2.93)
32–36 weeks, n = 37, postnatal 1.64 mg/l
day 3 (1.17–2.19)
day 1 (1.05–2.41)
day 3 (1.07–2.17)
day 1 (1.17–2.24)
day 3 (1.14–2.08)
Dorum PENIA At delivery, 33–36 weeks, n = 1.22 mg/l
et al. (2012) 30 (SD 0.30)
At delivery, 28–32 weeks, n = 1.41 mg/l
25 (SD 0.27)
Demirel PETIA 30–32 weeks, day 1 1.79 mg/l
et al. (2013) (0.68–2.31)
30–32 weeks, day 3 1.61 mg/l
(0.92–2.21)
28–30 weeks, day 1 1.80 mg/l
(0.65–2.48)
28–30 weeks, day 3 1.70 mg/l
(0.56–2.31)
26–28 weeks, day 1 1.80 mg/l
(1.51–3.19)
26–28 weeks, day 3 1.61 mg/l
(1.10–3.41)
24–26 weeks, day 1 1.80 mg/l
(0.80–2.20)
24–26 weeks, day 3 1.52 mg/l
(0.54–2.0)
PENIA particle-enhanced nephelometric immunoassay, PETIA particle-enhanced turbidimetric
immunoassay, SD standard deviation
294 K. Allegaert
Table 7 Plasma cystatin C (CysC) values in neonates with specific perinatal disease characteristics
Abitbol et al. (2014) PENIA Preterm, 34 (SD 3) weeks 1.35 (SD 0.19)–1.47 mg/l
Second part of first week of life (SD 0.21)
26/60 exposed to gentamicin
(CysC lower)
Treiber et al. (2014) PENIA 50 term cases perinatal
asphyxia/controls
At birth (umbilical cord blood) 2.12 (SD 0.53)–1.39 mg/l
(SD 0.19)
On day 3 of life (venous blood) 1.56 (SD 0.32)–1.34 mg/l
(SD 0.21)
28–34 weeks, birth weight
910–2,250 g
Montini et al. (2001) PETIA 20 preterms, postnatal 4–7 days 1.88 (1.2–2.3) mg/l
Treiber et al. (2006) PENIA n = 75, umbilical blood at birth 1.97 (SD 0.60) mg/l
(34–41 weeks)
Raised CysC with pH <7.2 at
birth
Correlation (r = 0.28) with
hemoglobin
Parvex et al. (2012) PENIA 100 controls (term), all 2.02 (1.54–2.64) mg/l
umbilical cord blood
13 congenital renal anomaly 2.52 (1.80–3.50) mg/l
cases, bilateral (+24.5 %)
Elmas et al. (2013) PENIA 34 cases without respiratory 1.30 (SD 0.2) mg/l
distress, day 3
28 cases with respiratory
distress, day 3
No acute kidney injury (22/28) 1.14 (SD 0.1) mg/l
Acute kidney injury (6/28) 1.49 (SD 0.09) mg/l
34 cases without respiratory 1.29 (0.68–1.67) mg/l
distress, day 30
28 cases with respiratory
distress, day 30
No acute kidney injury (22/28) 1.40 (1.01–1.89) mg/l
Acute kidney injury (6/28) 1.51 (1.16–1.70) mg/l
Marunika-Chudek ELISA 32 neonates, 34–40 weeks,
et al. (2012) 0 and 48 h
Sepsis 1.47 (1.01–1.9)–1.43 mg/l
(1.05–1.81)
Severe sepsis 1.5 (1.12–1.87)–1.31 mg/l
(1.05–1.58)
Septic shock 1.23 (0.92–1.54)–1.21 mg/l
(0.95–1.47)
PENIA particle-enhanced nephelometric immunoassay, PETIA particle-enhanced turbidimetric immuno-
assay, SD standard deviation
Fig. 8 Median cystatin C values as reported in different cohort of healthy (pre)term neonates.
When displayed in an assay-specific way, this strongly suggests a between-assay difference, similar
to what has been observed in adults. Median and range and clinical characteristics for these cohorts
are provided in Tables 5 and 6 (PENIA particle-enhanced nephelometric immunoassay, PETIA
particle-enhanced turbidimetric immunoassay)
endocytosis and metabolism in the proximal renal tubular cell, but little is known on the
ontogeny of the megalin receptor and the maturation of CysC metabolism.
In conclusion and based on the currently available information, it seems that the
range (four- to fivefold) in serum CysC observations in early infancy is extensive and
only in part explained by renal (patho)physiology. This likely relates at least in part
to the ontogeny of both CysC synthesis and metabolism, with an additional between-
assay variability. This can be further improved by the use of assay-specific reference
values, adapted to the clinical characteristics (e.g., weight, gestational age, postnatal
age) and compared to inulin clearance or similar as golden standard for GFR
estimation. Until such information becomes available and taking also the differences
in analytical costs into account, the use of serum creatinine still remains a somewhat
crude but useful biomarker of GFR (dys)function in perinatal life.
From Biochemical Quantification to Clinical Application
Neonatal care critically depends on the availability of reference intervals for any
specific laboratory test or biomarker to support clinical decision-making, to tailor
296 K. Allegaert
therapy to the individual needs, or to support prognosis (Ceriotti 2012; Colantonio

et al. 2012). However, children are not small adults and neither are newborns small
children. Since maturational physiological changes are most prominent in early
infancy, variability is the key feature in this population: developmental physiology
drives developmental laboratory medicine. This is also reflected by the extensive
inter- and intra-individual variability in Scr and eGFR documented in early infancy
and described in this chapter. This variability is in part related to physiological
changes (e.g., birth weight, gestational age, postnatal age) as well as changes related
to pathophysiology (e.g., peripartal asphyxia, co-medication).
The basic problem is to read and recognize the signal in this noise. Consequently,
clinicians are still struggling with the translations of the (patho)physiology to the
clinical setting, as, e.g., reflected in the inadequate definitions of AKI or pRIFLE
(Akcan-Arikan 2007). Another interesting and emerging pattern that is not yet well
explored during critical illness in adults and not yet even considered in neonates is
“hyperfiltration”: a disease state where renal clearance capacity is actually higher
than anticipated. We commonly search for decreased renal clearance, while, e.g.,
ineffective pharmacotherapy may also relate to increased clearance (Grootaert
2012). Strategies to further improve the current setting either relate to novelties or
to improve the use of the already available tools.
The search for novel biomarkers of renal “(dys)function” (e.g., cystatin C, beta-
trace protein, beta-2 microglobulin, alpha-1 microglobulin) is valuable but will need
focused studies in specific cohorts of (pre)term neonates once the within-assay issues
have been considered (Levey et al. 2014; Zwiers et al. 2014; Zaffanello et al. 2009).
This has been illustrated in this chapter based on the reported observations on CysC
(Tables 5, 6, and 7) in neonates. Besides the search for new biomarkers, we have the
strong opinion that the efforts made to standardize creatinine assays toward IDMS
hold a unique opportunity to develop reference values and centiles for creatinine in
(pre)term neonates. An effort similar to the CALIPER initiative but focused to early
infancy is needed (Ceriotti 2012; Colantonio et al. 2012). At least, we have provided
preliminary evidence that such an approach may be beneficial to improve prognosis
(urethral valves and outcome) (Lemmens et al. 2014) or to tailor pharmacotherapy
(Scr centiles and amikacin clearance) (Smits et al. 2013). Further exploration of such
a “threshold” or centile approach is warranted, but we do need a “neonatal”
CALIPER approach to develop and validate neonatal reference values in both
preterm and term neonates.

Conditions
Maturational physiological changes are most prominent in early infancy. Conse-

quently, variability is the key feature in early infancy. This is not limited to creatinine
but is also reflected in age-specific reference intervals for other commonly measured
biomarkers (e.g., glycemia, aminotransferases, alkaline phosphatases, hormones).
Neonatal care critically depends on the availability of reference intervals for any
specific laboratory test or biomarker to support clinical decision-making, to tailor
therapy to the individual needs, or to support prognosis. This is also true but not
limited to creatinine measurements. A recent example is the paper on plasma
aminotransferase concentrations in preterm infants (Victor et al. 2011). Extreme
preterm neonates had higher plasma aminotransferase levels compared to near-term
cases, again reflecting either maturational issues or disease-related differences.
For those interested in bio-analysis, we would like to make the point that this
chapter also re-illustrates the relevance of the matrix in which the measurement is
performed. Please take into account that the matrix matters: assays may be affected
by specific composition of the matrix (serum, urine) in neonates. This is not limited
to bilirubin but may also be related to differences in albumin (e.g., binding capacity),
immunoglobulins, or free fatty acids.
The impact of matrix-related measurement is not limited to early infancy. We
use the example of bilirubin to illustrate this. Although raised bilirubin
(unconjugated) is indeed common in early infancy, it is not limited to newborns,
since raised bilirubin (conjugated) is a well-known biomarker of cholestasis and
liver disease, as recently reported in jaundiced adults (Kaiser 2014). In these
patients, bilirubin-related interferences may also affect Scr measurements. This
may be of clinical relevance for both issues related to diagnosis (e.g., hepato-renal
syndrome) and treatment modalities (e.g., Scr is a biomarker to decide on liver
transplantation) (Kaiser 2014).
Summary Points
• This chapter focuses on specific aspects of creatinine assays in early infancy. This
includes both analytical aspects (between-assay differences) and clinical issues
(extensive variability).
• The concept of isotope dilution mass spectrometry (IDMS) traceability improved
the measurement but affected the reference values for serum and urine creatinine
in early infancy. Moreover, the difference between different assays (Jaffe versus
enzymatic “conversion”) in neonatal matrix is not a single absolute value.
• Reference values for creatinine urine measurement are much lower in neonatal
urine compared to children and adults, but the difference between IDMS-
traceable enzymatic and Jaffe assays is very limited and not clinically significant.
• After the introduction of IDMS traceability of serum creatinine (Scr) measure-
ment, there is still a minor difference between IDMS-traceable enzymatic and
Jaffe assays, in part explained by bilirubin.
• Cystatin C also displays assay-related differences, and the number of reference
values in neonates is still very limited.
• Despite the limitations, serum creatinine (Scr) therefore still remains the most com-
monly used biomarker for glomerular filtration rate (GFR) assessment in neonates.
298 K. Allegaert
• To improve the clinical application following IDMS traceability, a concerted

effort is urgently needed to develop and validate neonatal reference values in
both preterm and term neonates.
Acknowledgment Karel Allegaert is supported by the Fund for Scientific Research, Flanders
(Fundamental Clinical Investigatorship 1800214N), and by an IWT-SBO project (130033).
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Urinary Angiotensinogen as a Biomarker
for Renal Disease 14
Zeynep Kendi Celebi, Siyar Erdogmus, and Sule Sengul
Contents
Key Facts of Urinary Angiotensinogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303
Intrarenal RAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 304
Does uAGT Reflect Intrarenal RAS? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 305
Animal Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 306
Human Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 311
The Importance of uAGT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
Abstract
Chronic kidney disease (CKD) is an emergent worldwide epidemic, and hyper-
tension is a major cause of this condition. The renin-angiotensin system (RAS) is
likely the most important factor in the development and maintenance of
hypertension.
Recently, two distinct RASs were identified: first, the systemic or circulatory
RAS and, second, the local or tissue-specific RAS. The local RAS is found in
many tissues such as those of the heart, brain, kidney, and adrenal glands. The
most important tissue containing the local RAS is that of the kidney because these
organs contain all of the RAS components necessary to produce angiotensin II
Z.K. Celebi (*) • S. Erdogmus • S. Sengul

Nephrology Department, Ankara University School of Medicine, Ibni Sina Hospital, Ankara,
Turkey
e-mail: zeynepkendi@yahoo.com; zeynepkendi@gmail.com; si.yar21@hotmail.com;
sengul@medicine.ankara.edu.tr

302 Z.K. Celebi et al.
(Ang II), a molecule that has many direct and indirect effects on blood pressure
and water homeostasis.
Animal studies have guided the definition of the RAS components in the
kidney and have helped us to understand their mechanisms of action.
Angiotensinogen (AGT), renin, and angiotensin-converting enzyme (ACE) are
abundant in the tubular system. Most studies have shown that AGT is produced in
the proximal tubule and secreted to the tubular lumen. AGT cleavage with renin
results in the generation of angiotensin I (Ang I). Ang I is then transformed into
Ang II with ACE, the latter of which is the active metabolite of the RAS that
contributes to sodium homeostasis and the blood pressure response.
In humans, clinical studies have shown that urinary AGT (uAGT) is correlated
with both systolic and diastolic blood pressure (SBP and DBP, respectively) as well
as urinary protein, in patients with CKD and in patients with hypertension. uAGT is
also a risk factor for the progression of CKD. Recently, uAGT was studied in
patients with glomerulonephritis, amyloidosis, and autosomal-dominant polycystic
kidney disease, as well as in renal transplant recipients. Limited information exists
with regard to the use of uAGT for acute kidney injury (AKI).
The current chapter describes uAGT in detail. Growing evidence shows that
uAGT reflects the intrarenal RAS. Some authors have also reported that uAGT is
a marker of glomerular injury. Nevertheless, additional studies are needed to
recognize uAGT as a biomarker.
Keywords
Angiotensin II • Angiotensinogen • Blood pressure • Chronic kidney disease •
Renin-angiotensin system • Urinary angiotensinogen • Urinary protein
Abbreviations
ACE Angiotensin-converting enzyme
ACEi Angiotensin-converting enzyme inhibitor
ADPKD Autosomal-dominant polycystic kidney disease
AGT Angiotensinogen
AGT mRNA Angiotensinogen messenger ribonucleic acid
Ang I Angiotensin I
Ang II Angiotensin II
ARB Angiotensin II type 1 receptor blocker
AT1R Angiotensin II type 1 receptor
BMI Body mass index
DBP Diastolic blood pressure
IgA Immunoglobulin A
MGA Minimal glomerular abnormality
PRA Plasma renin activity
RAS Renin-angiotensin system
14 Urinary Angiotensinogen as a Biomarker for Renal Disease 303
SBP Systolic blood pressure

uAGT Urinary angiotensinogen
uAGT/uCre Urinary angiotensinogen/urinary creatinine ratio
uAlb/uCre Urinary albumin/urinary creatinine ratio
uPro/uCre Urinary protein/urinary creatinine ratio
Key Facts of Urinary Angiotensinogen
• AGT is a 60-kD glycoprotein of the alpha-2 globulin fraction of plasma proteins,

which is produced mainly by the liver.
• uAGT was first described in 1972, and after the definition of local RAS, uAGT
was used as a measurable parameter of intrarenal RAS activity.
• uAGT can be measured by commercially available ELISA kits.
• uAGT excretion is correlated with blood pressure levels and proteinuria in renal
diseases (e.g., CKD, diabetic nephropathy, chronic glomerulonephritis).
• In the future, uAGT may be used as a prognostic biomarker in patients with renal
diseases.
Definitions
LMB2 LMB2 (anti-Tac (Fv)-PE38) is an immunotoxin with specific binding to

human CD25 and induces progressive nonselective proteinuria, ascites, and edema
in NEP25 mice.
Megalin Megalin, a member of the low-density lipoprotein receptor gene family, is

a multiligand receptor expressed in the apical membrane of proximal-tubule epithe-
lial cells that plays a central role in the endocytic process.
RAS The renin-angiotensin system is the main pathway that regulates blood pres-
sure and fluid and sodium homeostasis by producing Ang II from AGT.
Streptozocin Streptozocin is an antibiotic with toxic effects that induce type

1 diabetes in animals.
Introduction
The renin-angiotensin system (RAS) plays a critical role in blood pressure control,
fluid and electrolyte homeostasis, and progression of renal disease (Navar
et al. 1999). Knowledge of the RAS began with a study in 1898, which describes
that the infusion of renal extracts obtained from the renal vein of a rabbit increased
the arterial pressure of nephrectomized rabbits. These authors named this pressor
compound “renin” (Tigerstedt and Bergman 1898). Subsequent experiments showed
that the blood that came from the renal veins of dogs after acute ischemia had a
strong pressor effect and showed different characteristics from renin; this substance
was called “hypertensin” (Braun-Menéndez et al. 1939). During the same years,
another study group discovered a crystalline pressor substance, which was cleaved
by renin, and they named this substance “angiotonin” (Page and Helmer 1940).
Later, investigators decided that unique nomenclature was necessary for additional
studies, and angiotensinogen (AGT) was defined as the only substrate of renin (ISH
1978). Favaro et al. first described the excretion of AGT in 1972 among patients with
glomerulonephritis (1972); however, the source of urinary AGT (uAGT) and its
pathophysiological role remained unknown.
Over the last few decades, research has shown that different tissues have elements
of the RAS, and the kidney is the only organ to have all of them, primarily in the
tubular system. Although the liver produces circulating angiotensinogen, studies
have indicated that the kidneys produce it as well.
Recently, the focus of interest on the RAS has shifted toward the role that the local
RAS plays in specific tissues. Considerable attention has been paid to the signifi-
cance of the tissue RAS in the brain, heart, adrenal glands, vasculature, and kidney.
Intrarenal RAS
Studies have shown that a major source of intrarenal Ang II is AGT, which is locally
produced by the proximal tubular cells. Animal studies have found AGT mRNA and
protein in proximal-tubule segments (Darby et al. 1994; Kamiyama et al. 2012a).
The AGT produced in proximal-tubule cells and its formed metabolites are likely
secreted directly into the tubular lumen (Rohrwasser et al. 1999). Renin is secreted
from the juxtaglomerular apparatus, and it might be the source of both circulating
and intrarenal levels of AGT. However, Prieto-Carrasquero MC et al. (2004) also
showed that Ang-II-infused rats demonstrated suppressed renin immunostaining in
the juxtaglomerular apparatus cells and increased immunostaining in the distal
tubules. The renal interstitium and vascular structure also contains renin and serves
as a pathway in the generation of Ang I. ACE is abundant throughout the nephron,
except for the late distal nephron segments. Furthermore, ACE is important in
transforming Ang I into Ang II. A schematic illustration of intrarenal RAS is
shown in Fig. 1. Locally formed Ang II has many direct and indirect effects in the
kidney (Kobori et al. 2007a).
Ang II has direct constrictor effects on the glomerular arterioles and mesangial
cells, and it indirectly enhances the tubuloglomerular feedback response (Seeliger
et al. 2009). Blockage of the RAS attenuates the tubuloglomerular feedback
response and increases the distal nephron perfusion rate. Ang II increases sodium
and bicarbonate reabsorption at proximal and distal tubules (Hashimoto et al. 2005;
Dibona and Sawin 2004). Ang II also stimulates the zona glomerulosa and indirectly
increases distal tubule sodium reabsorption through aldosterone.
Fig. 1 Local production of

RAS components in kidney.
The angiotensinogen
produced in proximal-tubule
cell is secreted to the tubular
lumen and is cleaved by renin,
which is secreted from
juxtaglomerular apparatus.
The angiotensin I formed from
the angiotensinogen is then
converted to angiotensin II
through the angiotensin-
converting enzyme, which is
abundant in the tubular
system. The final product,
angiotensin II, has many
direct and indirect effects in
the kidney and blood pressure
control (RAS renin-
angiotensin system, ACE
angiotensin-converting
enzyme)
Does uAGT Reflect Intrarenal RAS?
Multiphoton imaging of glomerular permeability has shown that under physiological

and pathological conditions, systemic AGT does not cross through the glomerular
filtration barrier. After glomerular damage, however, the uAGT increased, and the
majority of this increase was endogenous, not the labeled AGT. These results
suggest that tubules can produce AGT, and uAGT is a marker of intrarenal RAS
(Nakano et al. 2012). In contrast to this study, Pohl M. et al. (2010) showed that
signals of labeled AGT and AGT content in rat proximal convoluted tubule consist of
the endocytic uptake from the glomerular filtrate, and this uptake is mediated
through the megalin-a scavenger receptor. Mosaic megalin-deficient mice showed
subapical endosomal or lysosomal accumulation of AGT in megalin-positive cells;
however, megalin-deficient cells were AGT negative. These mice had similar plasma
AGT levels to megalin-positive mice. Megalin-deficient mice revealed substantial
uAGT excretion, but uAGT was undetectable in megalin-positive mice. AGT mRNA
was detected in the proximal straight tubules, but the proximal convoluted tubules
expressed weaker AGT mRNA signals. These studies supported that both early
proximal convoluted tubule compartments expressed low levels of AGT mRNA,
and intensive endocytosis of filtrated systemic AGT demonstrates that excreted
uAGT is derived from the proximal straight tubules or more distal components of
the nephron containing local RAS components.
Epidemiologic studies have shown that increased dietary salt intake is associated
with increased prevalence and progression of hypertension. Salt sensitivity is vari-
able among adults; however, patients with essential hypertension are more likely to
have salt sensitivity. Salt restriction decreases plasma AGT, stimulates PRA, and
increases plasma Ang I and Ang II (Ingert et al. 2000). A study regarding the effects
of a low sodium diet on the intratubular RAS demonstrated that despite an increase
in PRA, plasma Ang II, and kidney Ang II levels, uAGT and urinary Ang II
excretion did not increase. Therefore, the physiologic stimulation of the RAS does
not explain the increasing levels of uAGT. Rather, increased uAGT might merely
reflect pathological stimulations of the intrarenal RAS (Shao et al. 2013).
Animal Studies
Animal studies have provided specific models to test the effects of many RAS
components. These models include transgenic mice and enable the examination of
the specific effect of a protein such as AGT as well as the observation of hypertensive
or diabetic animals and animals with glomerulonephritis. In addition, sexual differ-
ences can be described using appropriate models. These models even allow for the
description of treatment mechanisms and drug effects.
Single transgenic mice that expressed human AGT only in the kidney (group A),
double transgenic mice expressing systemic human renin and human AGT in the
kidney (group D) and wild-type mice (group W) all received an infusion of human
AGT. Renin has a high specificity for species, and exogenously administered human
AGT cannot be cleaved by mouse renin. This transgenic mouse model enabled us to
test the intrarenal mouse AGT expression induced by the selective intrarenal
overproduction of Ang II. Exogenous human AGT mRNA and protein was
expressed in the kidneys of groups A and D. Mice liver did not express human
AGT mRNA or protein in any group. Plasma Ang II levels were similar across the
groups, but renal Ang II levels were higher in group D. Groups A and D had
detectable human uAGT, but group W had no detectable levels of human AGT in
the urine. Group D showed increased endogenous mouse AGT mRNA and protein.
Group D showed kidney damage parameters with an increase in interstitial collagen,
interstitial macrophage, and monocyte infiltration and afferent arteriolar hypertro-
phy. These findings suggest that increased selective intrarenal Ang II generation
produces endogenous AGT mRNA, increases intrarenal AGT, and contributes to
progressive hypertension with renal damage (Kobori et al. 2007b).
Chronic Ang II infusion enables the creation of a useful animal model of Ang-II-
dependent hypertension. Chronic Ang II infusion with a high salt diet increases
blood pressure and augments renal AGT mRNA and protein, plasma AGT, and liver-
AGT mRNA and protein, despite suppressing plasma renin activity (Kobori
et al. 2001). Ang-II-infused mice on a high salt diet showed an increase in kidney
Ang II content, and this increase was correlated with uAGT; however, no correlation
was found between plasma Ang II and uAGT (Kobori et al. 2002). Chronic Ang II
infusion creates time- and dose-dependent increases in uAGT and protein. Although
higher urinary protein excretion rates were observed in the volume-dependent

hypertension group (high salt plus deoxycorticosterone acetate [DOCA] salt
group), an increase in uAGT was not observed in this group. In addition, Ang-II-
infused mice and sham-operated mice received human AGT, but a urinary analysis
did not show any human AGT; moreover, these mice did not show alterations in
blood pressure. These data indicate that glomerular permeability prevents AGT from
crossing the plasma to the tubules (Kobori et al. 2003a).
Ang-II-infused rats also showed increased urinary renin and Ang II levels.
Suppressed juxtaglomerular apparatus and low levels of plasma renin activity
(PRA) make filtration of the circulating renin unlikely, and urinary renin likely
reflects the secreted renin from the collecting ducts and provides a pathway for
augmented Ang II levels in the tubules. Therefore, renin might play a part in the
maintenance of hypertension. Despite the increased PRA, the urinary renin level
decreased after candesartan treatment, further supporting the tubular secretion of
renin hypothesis (Liu et al. 2011).
Dahl salt-sensitive rats are genetically predisposed to hypertension, and salt
loading causes the development of hypertension. A strong correlation between
uAGT/renal AGT content and systolic blood pressure (SBP) was observed among
Dahl salt-sensitive rats on a high salt diet. These rats have low PRA levels; thus, their
systemic RAS is suppressed. Nevertheless, their intrarenal RAS seemed to be
activated because of increased renal Ang II and renal AGT contents. In addition,
these rats did not show Ang II type 1 receptor (AT1R) suppression, and the increase
in kidney-AGT content caused inappropriate augmentation in the intrarenal RAS
activity and contributed to the development and maintenance of hypertension
(Kobori et al. 2003b).
Mouse AGT cDNA knock-in mice exhibiting an overexpression of AGT in the
proximal tubules showed higher uAGT and urinary Ang II excretion rates and higher
daytime and nighttime SBP with a higher salt diet than controls. On a normal salt
diet, plasma renin content and PRA were similar between groups; with a high salt
intake, however, the plasma renin content was lower in the targeted animals than
controls. PRA was undetectable in both groups. This study revealed that proximal-
tubule-derived AGT causes hypertension, and this effect is independent of the
systemic RAS because low levels of PRA suggest that the systemic RAS is
suppressed (Ying et al. 2012).
Spontaneously hypertensive rats on a high salt diet developed increased plasma
Ang II, uAGT, and urinary protein excretion as well as glomerular and tubular
damage. AT1R blockage with losartan did not reduce blood pressure but did reduce
plasma Ang II in rats with normal salt diets. However, reductions in blood pressure
and plasma Ang II were not observed in rats with high salt diets treated with losartan.
Glomerular and tubulointerstitial changes decreased with AT1R blockage. Renal
AGT mRNA expression was similar between groups. Despite low levels of renal
AGT mRNA, the increased uAGT excretion can be explained due to the leakage
from the damaged glomerulus or the increased intratubular formation of AGT.
Losartan, independent of lowered blood pressure, decreased the structural and
functional effects of Ang II. High blood pressure among rats with a high salt diet
might be the result of volume overload due to their diet. Furthermore, renal injury
likely develops through AT1R activation (Susic et al. 2011). High salt intake also
increased oxidative stress in Ang-II-infused rats, and these effects were reversed
with AT1R blocker (ARB) treatment (Lara et al. 2012). These studies support the
theory that a high salt intake exacerbates the renal damage in Ang-II-dependent
hypertension and ARB treatment ameliorates the renal damage and its associated
parameters.
In malignant hypertension, the kidneys exhibit myointimal proliferation and
fibrinoid necrosis. Increased hydrostatic pressure enhances Ang II production, and
this increase results in endothelial and epithelial cell apoptosis and proliferation
(Efrati et al. 2007). CYP1A1-REN2 transgenic rats with malignant hypertension
showed increased proteinuria and uAGT excretion, and these effects were amelio-
rated after candesartan treatment (Milani et al. 2010). uAGT might reflect increased
intraluminal AGT, which contributes to intrarenal Ang II augmentation. Intrarenal
Ang II also plays an important role in malignant hypertension.
Both systemic and intrarenal RAS have effects on the regulation of blood
pressure. Ramkumar N et al. (2013) examined the effects of hepatic overexpression,
kidney overexpression, or both with regard to AGT. Proximal-tubule-overexpressing
mice were labeled “group K.” liver-overexpressing mice were labeled “group L.”
both liver- and proximal-tubule-overexpressing mice were labeled “group KL,” and
the control group was labeled “group W.” The mean arterial pressure was
KL>K>L>W on a normal sodium diet. High sodium intake increased the mean
arterial pressure in group L mice. Plasma AGT levels were increased in
L>KL>K>W mice with normal or high sodium intake. Plasma renin concentrations
were similar in the groups on a normal sodium diet. A high sodium diet decreased
plasma renin concentrations similarly in all groups. The uAGT associated with a
normal sodium intake was higher in groups KL and K and similar in groups L and
W. uAGT increased with a high sodium intake and was higher in KL>K>L>W. The
results demonstrated that uAGT levels were correlated with hypertension. Liver-
AGT overexpression only caused hypertension in the presence of a high sodium
intake, and proximal-tubule AGT overexpression caused hypertension, regardless of
liver-AGT overexpression.
To summarize the intrarenal RAS studies regarding hypertension, Ang-II-depen-
dent hypertension might be the result of increased intrarenal RAS activity, and Ang
II likely plays a major role in the maintenance of hypertension and the progression of
renal damage in its volume-dependent forms.
Although the specific pathological feature of diabetic nephropathy is nodular
glomerulosclerosis, most biopsy specimens show excessive mesangial matrix depo-
sitions. Primary cultures of rat mesangial cells with high glucose concentrations
showed increased intracellular renin activity as well as ACE mRNA and AGT
mRNA expression, which resulted in the augmentation of Ang II. Increased Ang
II might cause cell proliferation and mesangial matrix deposition with intracrine
actions (Vidotti et al. 2004).
Intrarenal RAS activity plays an important role in the pathogenesis and progres-
sion of diabetic nephropathy, and urinary albumin excretion is a prognostic factor for
renal function impairment. Streptozocin-induced type 1 diabetic rats without com-

plications were followed up after the onset of diabetes; some rats received insulin
treatment. An increase in uAGT developed before an increase in urinary albumin.
Insulin treatment ameliorated urinary albumin and uAGT excretion, and AGT
mRNA expression was weaker in the treated group (Kamiyama et al. 2012b). Future
comprehensive studies may use uAGT as an early biomarker of intrarenal RAS
activation in patients with diabetes to help anticipate the development of diabetic
nephropathy.
It has been shown that patients with glomerular diseases treated with
Ang-converting enzyme inhibitor (ACEi) or ARB have better renal outcomes; this
finding might be related to increased intrarenal RAS activity. Rats with anti-
thymocyte serum nephritis developed proteinuria, and their renal histology showed
mesangial proliferation. Renal AGT, Ang II expression, and uAGT excretion
increased. uAGT excretion was positively correlated with glomerular damage,
proteinuria, and renal AGT content. After treatment with olmesartan, uAGT, renal
AGT, and Ang II expression were attenuated; however, plasma Ang II and PRA
increased. These results suggest that the intrarenal RAS is regulated independent of
the systemic RAS in glomerular diseases (Ohashi et al. 2008).
Another glomerulonephritis model of rats showed increased PRA, plasma Ang II,
renal cortical, and medullar Ang II levels; proteinuria, microalbuminuria, and uAGT
excretion; and renal AGT mRNA expression. These effects and the pathologic
features of glomerulonephritis (defined as mesangial matrix expansion), except for
the renal cortex Ang II content, diminished with aliskiren treatment (Miyata
et al. 2014).
Renal Ang II generation in the liver and kidney or dual AGT of knockout mice
was examined in the context of glomerular diseases featuring podocyte injury; the
results were compared with those of control mice. LMB2-injected mice developed
massive proteinuria. After podocyte injury, kidney-AGT knockout mice showed
similar increases of renal AGT and Ang II content as well as uAGT excretion rates
as the control group. Liver-AGT knockout mice showed decreased renal AGT and
Ang II content and decreased uAGT excretion rates similar to the dual-AGT
knockout mice. Liver-AGT and dual-AGT knockout mice showed more severe
tubulointerstitial damage than the kidney-AGT knockout and control mice. This
animal model suggests that impaired glomerular permeability allows liver-derived
systemic AGT to pass through the glomerular filtration barrier. Moreover, uAGT
primarily consists of liver-derived AGT (Matsusaka et al. 2014).
Premenopausal women have lower blood pressure and a reduced incidence of
cardiovascular disease than age-matched men. Similar sex differences have been
observed across species and in multiple animal models of hypertension. Female sex
hormones, especially estrogen, modulate the renin-angiotensin-aldosterone system.
Dahl salt-sensitive rats on a high salt diet exhibited sex-dependent changes in renal
AGT mRNA and protein, with higher levels found in male rats. After castration,
blood pressure increases, and urinary protein and albumin excretion, glomerular
sclerosis, AGT mRNA, and protein levels are attenuated in male rats. Testosterone
replacement after castration showed elevated blood pressure and increased renal
damage with a rise in renal AGT mRNA and protein (Yanes et al. 2009). The effects
of estrogen depletion and dietary salt intake were studied with regard to AGT
expression in the mRen (2) Lewis strain. The results showed that male rats on a
normal salt diet exhibited a correlation between uAGT and proteinuria; however,
those on a high salt diet did not show relationships among uAGT, SBP, and renal
AGT expression. Female rats on a high salt diet showed the highest SBP, following
ovariectomized rats and rats on a normal salt diet. Plasma AGT was not influenced
by ovariectomy or salt; however, uAGT increased 180-fold among the high salt-fed
female rats and 16-fold among ovariectomized rats. These rates were higher than
those in the male rats on a high salt diet. Ovariectomy did not change protein
excretion in female rats but did increase uAGT excretion. Although a high salt diet
resulted in increased uAGT and protein excretion in both female and male rats, the
expected increase in renal AGT content and AGT gene expression was not observed.
These results suggest that uAGT increases because of the increase in systemic RAS
activity. Male rats’ higher levels of uAGT and protein excretion might reflect
additional renal injury response to hypertension (Cohen et al. 2010). Another
study regarding the sexual dimorphism of the intrarenal RAS showed that both
salt-induced and Ang-II-dependent hypertension result in higher uAGT, protein
excretion, and blood pressure responses among male rats; furthermore, they exhibit
significantly more AGT gene expression in their kidneys. These results suggest that
males have higher intrarenal RAS activity and are more likely to have kidney
damage with hypertension (Rands et al. 2012). The increased RAS activity among
males might be because of the androgen-dependent dimorphic expression of AGT
mRNA in the kidney (Wang et al. 1994).
Most studies have shown that uAGT reflects intrarenal RAS activity and is
generated locally in the proximal tubules; however, two studies have shown that
uAGT originates from the liver (Matsusaka et al. 2012, 2014). To determine the
source of uAGT, liver-AGT knockout mice, kidney-AGT knockout mice, and
dual-AGT knockout mice were compared with the control group. Liver and
dual-AGT knockout mice exhibited similar SBPs, which was lower than those of
the kidney knockout mice and control group. The former mice groups also showed
increased urine volumes, higher PRA, and renal renin activity than kidney knock-
out mice and the control group. Liver-AGT mRNA and protein, plasma AGT,
kidney-AGT protein, and Ang II content was lower; however, kidney-AGT mRNA
was higher in the liver and dual-AGT knockout mice compared with kidney-AGT
knockout and control mice. Histological examinations of the mice kidneys
revealed medial hyperplasia, juxtaglomerular cell hypertrophy, and mesangial
matrix expansion in the liver knockout mice as well as hypoplastic papillae,
tubular dilatation, and interstitial fibrosis in the dual-AGT knockout mice.
Kidney-AGT knockout mice showed normal renal morphologies. These results
suggest that liver-derived AGT is the major source of uAGT (Matsusaka
et al. 2012). Furthermore, the systemic RAS is likely important in maintaining
normal blood pressure response and water balance. In the absence of the intrarenal
RAS activity observed in the kidney-AGT knockout mice, systemic RAS activity
might not lead to severe renal damage.
Rats showed an increase in uAGT, SBP, renal cortex AGT, and renal Ang II
content after Ang II infusion. Treatment with olmesartan resulted in an increase of
plasma Ang II; however, renal Ang II, uAGT, and urinary protein excretion was
attenuated. Intrarenal AGT increases were associated with AT1R activation in Ang-
II-dependent hypertension, and AT1R blockage decreased uAGT, urinary protein,
and SBP (Kobori et al. 2004). When lisinopril (ACEi) was added to the treatment of
Ang-II-infused rats, unlike AT1R blockage, plasma and intrarenal Ang II levels also
decreased; however, this study did not explore the effects on uAGT (Gonzalez-
Villalobos et al. 2009). Low-dose Ang-II-infused mice exhibited similar results with
a high dose Ang II infusion, leading to remarkably increased intrarenal AGT
expression, except for a slower rise in the blood pressure response. Treatment with
olmesartan diminished both intrarenal AGT expression and blood pressure levels
(Gonzalez-Villalobos et al. 2008). In the kidneys and adrenal glands, the intracellular
uptake of Ang II appears to be mediated by AT1R. When transgenic rats exhibiting a
lack of AT1R were compared with wild-type rats, 80 % of the intracellular uptake of
labeled Ang II occurred through AT1R activation. The remaining 20 % of the uptake
was not mediated by the receptor (Li and Zhuo 2008). All of these findings suggest
that treatment with an ACEi or AT1R blocker not only ameliorates hypertension but
also improves the renal histology and decreases uAGT and urinary protein excretion;
these effects were not associated with blood pressure levels.
Human Studies
Twenty years after the study by Favaro et al. (1972), in the 1990s, many studies
made important advances in the understanding of the pathophysiology and compli-
cations of hypertension. The chief portion of this development was with regard to the
RAS. After defining the components of the RAS, their relationships and functions
were examined using animal models. The results of these studies were hopeful;
thereafter, many clinical studies in humans were designed to study the RAS and its
components. The relationship between the intrarenal RAS and uAGT in animal
models was well defined. Human genetic studies have revealed a link between the
AGT gene and hypertension (Inoue et al. 1997). In addition, various clinical studies
have shown that increased uAGT levels indicate the intrarenal RAS status in
different populations such as patients with hypertension, type 1 and type 2 diabetes
mellitus, chronic glomerulonephritis, immunoglobulin A (IgA) nephropathy,
chronic kidney disease (CKD), renal amyloidosis or autosomal-dominant polycystic
kidney disease (ADPKD), as well as renal transplant recipients. In 2007, Katsurada
and colleagues developed a novel sandwich ELISA for human AGT. The methods
and uAGT results of the discussed studies are given in Tables 1, 2, and 3.
In various studies, the relationship between uAGT and hypertension has been
discussed. Kobori et al. (2009) investigated the uAGT levels in 70 patients with
hypertension and 36 normotensive controls. Elevated uAGT levels were reported in
patients with hypertension compared with controls. In addition, these authors dem-
onstrated that uAGT was significantly and positively correlated with SBP, diastolic
Table 1 Human AGT results in urine

Study Population Method uAGT Comment
Katsurada Healthy ELISA 7.1–35 ng/ml Human AGT ELISA might be a
et al. (2007) volunteers useful disease marker
Mills Patients ELISA CKD: uAGT might identify patients with
et al. (2012) with CKD 45.4 μg/24 h CKD
Controls:
7.4 μg/24 h
AGT angiotensinogen, CKD chronic kidney disease, uAGT urinary angiotensinogen
Table 2 Studies using log uAGT/uCre and their results

Study Population Method Log uAGT/uCre Comment
Kobori Patients with ELISA CKD: 1.8801
0.0885 uAGT might be a
et al. (2008) CKD Controls: biomarker of CKD
0.9417
0.1048 severity, and the
ELISA method is
valid for measuring
uAGT
Xu Patients with ELISA CKD: 2.02
0.55 ng/mg uAGT reflects
et al. (2015) CKD Control 1.77
0.40 ng/mg intrarenal Ang II
activity in patients
with CKD
Kutlugun Amyloidosis ELISA Amyloidosis: uAGT is correlated
et al. (2012a) patients 1.88
0.92 μg/g with proteinuria in
Controls: 1.25
0.70 μg/g renal amyloidosis
AA
AGT angiotensinogen, CKD chronic kidney disease, uAGT urinary angiotensinogen
blood pressure (DBP), the urinary albumin/creatinine ratio (uAlb/uCre), and the
urinary protein/creatinine ratio (uPro/uCre). uAGT was not related to race, age,
gender, height, body weight, body mass index (BMI), serum sodium levels, serum
potassium levels, serum creatinine levels, the urinary sodium/creatinine ratio, the
urinary potassium/creatinine ratio, the fractional excretion of sodium, plasma AGT
levels, or the estimated glomerular filtration rate (eGFR). They also observed that
uAGT levels were significantly higher among patients with hypertension without
RAS blockage compared with normotensive participants. Importantly, patients
treated with RAS blockers demonstrated a prominent attenuation of this augmenta-
tion. These data suggest that the efficacy of the RAS blockage seeking to reduce
intrarenal RAS activity can be assessed by measuring uAGT excretion.
Another study reported that uAGT levels were higher in 55 patients with con-
firmed primary hypertension than a reference group consisting of 33 participants
with white-coat hypertension. This study also reported that the increased excretion of
uAGT was correlated with hyperuricemia in adolescents with primary hypertension
(Kuroczycka-Saniutycz et al. 2013).
Table 3 uAGT/uCre results in human studies

Study Population Method uAGT/uCre Comment
Kobori Patients with ELISA Hypertension with RAS Treatment with RAS
et al. (2009) hypertension blocker: blockers suppresses
25.00
4.96 μg/g uAGT
Normotensive controls:
13.70
2.33 μg/g
Zou Patients with ELISA 0.72 μg/g versus uAGT is positively
et al. (2012) hypertension >0.72 μg/g correlated with clinic
and ambulatory
blood pressure
Kuroczycka- Adolescents with ELISA Hypertension: uAGT is correlated
Saniutycz hypertension 0–2.28 ng/mg Cre with hyperuricemia
et al. (2013) Controls: 0–0.44 ng/mg Cre
Saito Patients with type ELISA Type 1 diabetes: uAGT might be an
et al. (2009) 1 diabetes 12.1
3.2 μg/g early marker of
Controls: 4.2
0.7 μg/g diabetic nephropathy
and the initial sign of
proteinuria or
microalbuminuria
Terami Patients with type ELISA Normoalbuminuria: uAGT is a marker of
et al. (2013) 2 diabetes at 6.8
11.6 μg/g Cre tubular injuries in the
various stages of Microalbuminuria: early stages of
nephropathy 8.5
9.9 μg/g Cre diabetic nephropathy
Macroalbuminuria:
73.3
95.2 μg/g Cre
Sawaguchi Patients with type ELISA Patients with albuminuria: Higher levels of
et al. (2012) 2 diabetes mellitus 62.0 μg/g Cre uAGT among
(interquartile range: patients with type
25.4–146.5) 2 diabetic and
Patients with albuminuria is a
normoalbuminuria: strong risk factor for
17.5 μg/g Cre worsening renal and
(interquartile range cardiovascular
11.4–28.2) complications
Soltysiak Normoalbuminuric ELISA Children with type uAGT might reflect
et al. (2014) children with 1 diabetes: 0.00 and early renal
diabetes 1.76 ng/mg involvement and
Controls: 0.00 and precede the onset of
0.00 ng/mg microalbuminuria
and hypertension
Urushihara Patients with ELISA Chronic GN: Treatment with RAS
et al. (2010) chronic GN 19.79
3.70 μg/g blockers suppresses
Chronic GN+ARB: uAGT
10.58
1.23 μg/g
Controls: 6.22
0.98 μg/g
(continued)
Table 3 (continued)
Study Population Method uAGT/uCre Comment
Nishiyama Patients with IgA ELISA IgA nephropathy: uAGT is powerful
et al. (2011) nephropathy 39
31 μg/g marker of intrarenal
Healthy controls: RAS status and
10
4 μg/g associated renal
derangement
Kim Patients with IgA ELISA IgA nephropathy: uAGT increased and
et al. (2011) nephropathy 104.96
23.23 ng/mg Cre was positively
Healthy controls: correlated with uPro/
6.71
1.13 ng/mg Cre uCre; patients with
uAGT levels
>100 ng/mg Cre
might have poor
renal function
Yamamoto Patients with CKD RIA <3 nmol Ang I Eq/g Cre uAGT >3 nmol
et al. (2007) versus Ang I Eq/g Cre is a
>3 nmol Ang I Eq/g Cre predictive marker for
the deterioration of
renal function
Mills Patients with CKD ELISA CKD: 26.3 μg/g uAGT might identify
et al. (2012) Controls: 4.4 μg/g patients with CKD
Kocyigit Patients with ELISA Patients with ADPKD and uAGT is a potential
et al. (2013) ADPKD with or hypertension: biomarker of
without 23.7
8.4 μg/g intrarenal RAS
hypertension Patients with ADPKD status among
without hypertension: patients with
16.6
5.2 μg/g ADPKD and
Healthy controls: hypertension
6.9
3.3 μg/g
Kurultak Patients with ELISA Patients with ADPKD: uAGT might be a
et al. (2014) ADPKD 19.35
6.06 μg/g useful marker for
Healthy controls: risk classification
11.65
1.66 μg/g among patients with
ADPKD
Kutlugun Renal transplant ELISA Patients with uAGT can help to
et al. patients with/ hypertension: evaluate intrarenal
(2012b) without 8.98
6.89 μg/g RAS activation in
hypertension Patients with renal transplant
normotension: patients
5.48
3.33 μg/g
Erdogmus Renal transplant ELISA Renal transplant patients: uAGT is associated
et al. (2013) patients 70.6
11.3 μg/g with increased
Healthy controls: proteinuria among
9.3
2.35 μg/g patients without
chronic allograft
injury
ADPKD autosomal-dominant polycystic kidney disease, CKD chronic kidney disease, GN glomer-
ulonephritis, uAGT urinary angiotensinogen, RAS renin-angiotensin system
In the Bogalusa Heart Study, Kobori and colleagues (2010) evaluated the rela-
tionship between uAGT and traditional cardiovascular disease risk factors among
asymptomatic young adults. They demonstrated that uAGT levels did not differ with
regard to race or sex but were significantly correlated with SBP, DBP, uAlb/uCre,
and uPro/uCre. Moreover, high correlations were found among men, especially
those of African descent.
Jun Zou et al. investigated the association between uAGT excretion and ambu-
latory blood pressure levels (2012). They found that uAGT/uCre was significantly
and positively associated with clinical and ambulatory blood pressure levels. uAGT
excretion was high with a greater urinary sodium excretion as well as associated with
clinical and ambulatory blood pressure measurements.
Identifying the sensitive biomarkers that can predict microalbuminuria or diabetic
nephropathy during, an earlier stage of diabetes might provide meaningful informa-
tion concerning early pathophysiology and an earlier clinical approach to the
diagnosis and treatment of diabetic nephropathy. In this regard, a study (performed
with youths with type 1 diabetes and control participants) found important results to
support the hypothesis that enhanced uAGT levels are not a consequence of pro-
teinuria. Specifically, both groups showed similar uAlb/uCre and uPro/uCre values,
and these parameters were in the normal range; however, the former was signifi-
cantly higher among patients with type 1 diabetes than controls. Importantly, AGT
was not increased in their plasma. These data indicate that uAGT can be used as an
early biomarker of diabetic nephropathy during the premicroalbuminuric phase
among patients with type 1 diabetes (Saito et al. 2009). In another study conducted
with children with type 1 diabetes, researchers evaluated the associations among
blood pressure, uAGT, and renal sodium excretion. They showed that uAGT levels
were significantly higher among patients than controls. uAGT levels were positively
correlated with 24-h ambulatory SBP, DBP, and mean arterial pressure. The signif-
icant increase in uAGT was observed even in patients with prehypertension. Inter-
estingly, significant correlations were not found between uAGT and uAlb/uCre or
between uAGT and eGFR. Furthermore, uAGT, uAlb/uCre, and eGFR were nega-
tively correlated with renal sodium excretion (Soltysiak et al. 2014). Given these
results, the authors suggested that elevated levels of uAGT reflect early renal
involvement and should be considered a new marker of hypertension in
normoalbuminuric children with type 1 diabetes.
A study of Japanese patients with type 2 diabetes (at various stages of nephrop-
athy) demonstrated that uAGT levels were positively correlated with uAlb/uCre and
urinary alpha-1-microglobulin as well as negatively correlated with eGFR. Never-
theless, a significant and high correlation between uAGT levels and alpha-1-
microglobulin was demonstrated during the normoalbuminuric stage. In addition,
uAlb/uCre was higher among patients receiving an RAS inhibitor; furthermore,
urinary alpha-1-microglobulin and AGT did not denote a significant increase in
this group (Terami et al. 2013). This clinical study found that uAGT is a marker for
tubular injuries of early-stage diabetic nephropathy in patients with type 2 diabetes.
Sawaguchi and colleagues (2012) showed that baseline uAGT levels were pos-
itively correlated with uAlb/uCre and urinary beta-2-microglobulin in patients with
type 2 diabetes with normo- and microalbuminuria. However, plasma AGT levels
were not correlated with these renal factors or uAGT levels. Moreover, uAGT was
negatively correlated with the annual decrease of eGFR, and patients with high
levels of uAGT and albuminuria demonstrated a progressive decline in eGFR and a
higher incidence of renal and cardiovascular composite endpoints. These data
suggest that a high level of uAGT in patients with type 2 diabetic and albuminuria
is a risk factor for renal and cardiovascular complications. More recently, AGT
mRNA and protein were found to be significantly higher in patients with diabetes
than control participants. Moreover, the AGT mRNA levels in tubules were nega-
tively correlated with eGFR in patients with diabetes. Kidney biopsy specimens of
patients with diabetes also showed increased expression of 4-hydroxy-2-nonenal and
heme oxygenase-1 than a control group (Kamiyama et al. 2013). These data suggest
that intrarenal RAS activation and oxidative stress play important roles in the
development of diabetic nephropathy.
Heuvel et al. (2011) studied 101 diabetic and nondiabetic patients with or
without hypertension. These authors observed that RAS blockage using either
ACEi or ARB increases plasma renin concentration and decreases urinary renin.
The decrease in urinary renin levels after RAS blockage, which occurred indepen-
dent of the plasma renin levels, reflects the activated renal RAS in patients with
diabetes and the success of the RAS blockage in the kidney. Therefore, urinary
renin more closely reflects renal RAS activity than uAGT. The same investigators
designed a new study to confirm their findings; they measured uAGT and plasma
AGT levels as well as renin and albumin in 22 patients with type 2 diabetes and
hypertension accompanied by albuminuria over a 2-month treatment period com-
pared with a placebo or RAS blocker group. They found that the urine/plasma renin
ratio, not only urinary renin, reflects the renal efficacy of the RAS blockage
(Persson et al. 2013). This study elucidated the origin of uAGT and renin; specif-
ically, uAGT might be a marker of filtration barrier damage rather than intrarenal
RAS activity.
CKD is a major public health problem worldwide. The investigators designed
many studies to investigate the role of uAGT levels on the progression of CKD.
Yamamoto et al. (2007) found that uAGT levels were higher in patients with low
eGFR. Elevated urinary protein and type IV collagen excretion were also correlated
with uAGT levels. These authors also reported that uAGT levels were positively
correlated with renal Ang II and type I collagen immunostaining intensities. More-
over, treatment with losartan reduced uAGT, plasma AGT, urinary protein, and tip
IV collagen and SBP despite concomitant increases in plasma renin and Ang II. A
higher level of uAGT (>3.0 nmol Ang I Eq/g Cre) was a risk factor for progressive
renal dysfunction in patients with CKD.
In 2008, Kobori and colleagues reported that uAGT levels were significantly
higher in 80 patients with CKD than in seven healthy volunteers. They also reported
that uAGT levels were significantly and positively correlated with uAlb/uCre and
uPro/uCre. Furthermore, they found an inverse association between uAGT and
eGFR. In contrast, the uAGT levels in patients with minimal change disease were
similar to those in control participants, although patients with minimal change
disease had severe proteinuria. Furthermore, the uAGT levels were not correlated
with gender, age, BMI, SBP, DBP, PRA, or plasma AGT levels.
In another study, uAGT levels were positively correlated with intrarenal
immunostaining intensities of AGT, Ang II, and Ang II type 1 receptors in Chinese
patients with CKD. Furthermore, a multiple regression analysis indicated that high
urinary protein, urinary Ang II, and urinary collagen IV excretion were significantly
correlated with high uAGT (Xu et al. 2015). Collectively, these findings indicate the
potential of uAGT as a marker of intrarenal Ang II activity in patients with CKD
(intrarenal Ang II activity increases in parallel with the severity of fibrotic renal
damage among these patients). These data also suggest that the levels of uAGT
should help to identify patients with CKD who are at increased risk for progressive
renal failure.
Recently, Nakano et al. (2012) showed that the vast majority of uAGT originates
from the tubules rather than via glomerular filtration using multiphoton fluorescence
microscopic imaging of the glomerular permeability of AGT. These findings
strongly support the hypothesis that the increase in uAGT reflects the de novo
production of AGT in the kidney and marks the activation of the intrarenal RAS.
Mills et al. (2012) found that uAGT levels were positively correlated with
albuminuria but negatively correlated with eGFR among 201 patients with CKD.
They also reported that uAGT levels were significantly higher in patients with CKD
than in controls. These associations were independent of established risk factors for
CKD and the use of RAS inhibitors. These findings indicate that an association
exists between uAGT excretion and CKD that is independent of albuminuria, a
sensitive marker for kidney structure damage, and urinary protein leakage. There-
fore, uAGT excretion might provide additional information for risk classification and
prediction among patients with CKD.
In a recent clinical study, Nishijima and colleagues (2012) examined the preser-
vation conditions of the measurements of uAGT concentrations and the ultradian
rhythm of uAGT excretion in healthy individuals. They found that the preservation
conditions did not change the uAGT concentration measurements. In addition,
uAGT excretion in healthy volunteers did not show an ultradian change during the
daytime. In a later study, Nishijima et al. (2014) investigated the relationship
between circadian rhythms and plasma AGT and uAGT in healthy volunteers and
patients with CKD. They did not find evidence that AGT has a circadian rhythm
under any condition.
Chronic glomerulonephritis is one of the common causes of end-stage renal
disease. In 2010, Urushihara and colleagues performed a study to evaluate the
uAGT levels in chronic glomerulonephritis patients (26 with IgA nephropathy,
24 with purpura nephritis, 8 with lupus nephritis, 7 with focal segmental glomerulo-
sclerosis, and 5 with non-IgA mesangial proliferative glomerulonephritis). uAGT
levels were significantly increased among patients with chronic glomerulonephritis
without RAS blockage, compared with control participants. Importantly, patients
with glomerulonephritis treated with RAS blockers showed a prominent suppression
of uAGT. Moreover, the uAGT levels were positively correlated with uAlb/uCre and
uPro/uCre.
Nishiyama et al. (2011) reported that uAGT levels were correlated with aug-
mented intrarenal AGT gene expression and Ang II levels in normotensive patients
with moderately proteinuric IgA nephropathy. They also reported that uAGT levels
did not differ between healthy volunteers and patients with minimal glomerular
abnormality (MGA). However, uAGT levels, renal tissue AGT expression, and Ang
II immunoreactivity were significantly higher among patients with IgA nephropathy
than those with MGA. Furthermore, treatment with an Ang II receptor blocker
reduced uAGT levels, renal tissue AGT gene expression, and Ang II immunoreac-
tivity in patients with IgA nephropathy.
In another study, Kim and colleagues (2011) sought to determine the role of
uAGT as a predictive marker in patients with IgA nephropathy. They found that
uAGT levels were significantly higher among patients with IgA nephropathy and
non-IgA nephropathy than healthy participants. Using a univariate regression anal-
ysis, they found that uPro/uCre, serum creatinine, SBP, and DBP were positively
correlated with uAGT. They did not find any correlations among uAGT, PRA, and
aldosterone levels. In addition, a multivariate regression analysis revealed that
uAGT was positively related to uPro/uCre. Few studies have addressed the cut-off
value of uAGT in patients with CKD. The current authors divided patients into two
groups based on uAGT levels (100 ng/ml Cr). Patients with uAGT levels >100 ng/
ml Cr had higher SBP, baseline uPro/uCre, and serum creatinine levels than those
who had lower uAGT levels. These results suggest that uAGT levels can assess the
local activity of the RAS in the kidney and identify patients at risk for renal disease
progression.
Secondary amyloidosis is a rare form of CKD in many countries; however,
patients, especially those living in Mediterranean regions, are at increased risk for
developing amyloidosis because of familial Mediterranean fever. Kutlugun et al. from
Turkey reported that logarithmic transformation of the uAGT/uCre levels was sig-
nificantly higher in 32 patients with renal AA amyloidosis than in 16 healthy controls
(2012a). One of the most important findings in that study was the significant and
positive correlation between uAGT and daily protein excretion among the patients
with renal AA amyloidosis. However, log uAGT/UCre levels were not significantly
correlated with patient age, gender, serum creatinine, eGFR, BMI, SBP, or DBP. They
did not observe a significant difference in log uAGT/UCre levels between patients
with and without hypertension. These findings suggest that uAGT is not a nonspecific
result of proteinuria. The activated intrarenal RAS might play a role in the pathogen-
esis and development of proteinuria in patients with renal AA amyloidosis.
ADPKD is a multisystem disorder characterized by bilateral, multiple cysts in the
kidneys and other organs such as the liver, pancreas, and arachnoid membranes. It is
an inherited disease, and patients usually develop CKD during follow-up. Kurultak
et al. (2014) demonstrated that uAGT/uCre levels were positively correlated with
uPro/uCre and uAlb/uCre in patients with normotensive autosomal-dominant
polycystic kidney disease (ADPKD). They also found that the other parameters such
as patient age, gender, serum creatinine, sodium, potassium, uric acid, eGFR, BMI,
SBP, and DBP were not correlated with uAGT/uCre levels. In a similar study,
Kocyigit et al. (2013) reported that uAGT/uCre levels were significantly higher
among 43 patients with hypertensive ADPKD compared with 41 normotensive
ADPKD patients and 40 normotensive healthy controls. In addition, uAGT/uCre
levels were correlated with the uPro/uCre levels and 24-h DBP. They also observed
that the uAGT/uCre and uPro/uCre levels were significantly higher in patients with
hypertensive ADPKD without RAS blockage. Moreover, patients with hypertension
and RAS blockage did not have this augmentation. In light of these findings, uAGT
levels might indicate the risk classification of ADPKD, particularly for patients in the
early stage of disease and during the early onset of hypertension.
In kidney transplantation, the activation of the intrarenal RAS might play a role in
the development and progression of chronic allograft injury. Erdogmus et al. (2013)
reported that uAGT levels were significantly higher in 70 renal transplant patients
compared with 21 healthy volunteers. We also reported a significant positive corre-
lation between uAGT excretion and proteinuria and a negative correlation between
uAGT excretion and eGFR in renal transplant patients without overt chronic allo-
graft injury. Moreover, uAGT levels were not associated with other patient param-
eters including age, sex, height, BMI, serum sodium, potassium, chloride, uric acid,
creatinine or urinary sodium, potassium, or blood pressure levels in renal transplants
patients. In addition, we did not observe any effect of RAS inhibitors or other
antihypertensive drugs on uAGT excretion in this relatively small group of renal
transplant recipients. These results suggest that uAGT excretion, which denotes the
activation of the intrarenal RAS, plays a role in the development of chronic allograft
injury among renal transplant recipients. Likewise, Naganuma and colleagues
(2014) demonstrated that uAGT levels were higher among renal transplant recipients
than renal transplant donors. They also reported that uAGT levels were significantly
and positively correlated with uAlb/uCre but negatively correlated with eGFR.
uAGT levels were higher in renal transplant patients with hypertension than those
who were normotensive. uAGT levels were not significantly correlated with patient
age, gender, serum creatinine, eGFR, BMI, SBP, DBP, dialysis modality, duration of
dialysis before transplantation, or time since transplantation. These authors also
showed that uAGT levels were correlated with urinary protein excretion in renal
transplant recipients with hypertension; however, this correlation was not found
among those who were normotensive (Kutlugun et al. 2012a).
There is limited data with respect to employing uAGT in AKI as a predictive
biomarker. Alge et al. (2013a) demonstrated that uAGT could be used as a prognos-
tic biomarker of acute kidney injury (AKI) in patients who developed this condition
after cardiac surgery. In another study, these authors reported that uAGT could be
used as a prognostic AKI biomarker in intensive care units. Increased uAGT
was associated with adverse events in AKI patients in the intensive care unit
(Alge et al. 2013b).
The Importance of uAGT
A biomarker is a characteristic that is objectively measured and evaluated to indicate

normal biological processes, pathogenic processes, or pharmacologic responses to
an intervention (National Research Council 2010). Urinary Ang II is unstable and
therefore cannot be used as a reliable marker of intrarenal RAS activity in clinical
studies. uAGT might reflect intrarenal Ang II levels, and it is the only known
noninvasively measured marker for assessing the activity of intrarenal RAS (Kobori
et al. 2002, 2003b).
The development of an ELISA kit for uAGT measurement would enable sensitive
and specific quantifications as well as the evaluation of treatment effects; however, a
cut-off level for uAGT has not yet been established. Different quantification methods
for uAGT have been used in previous studies, rendering it difficult to compare past
results. Future studies might reveal a cut-off value that would enable us to use uAGT
in clinical practice.
In conclusion, a debate continues regarding whether uAGT indicates the impair-
ment of glomerular permeability (like proteinuria), tubulointerstitial injury, or the
activation of intrarenal RAS in various renal diseases. Therefore, additional research
is needed to determine the importance of uAGT.

Conditions
The above findings suggest that uAGT can be used as an index of intrarenal RAS
activation and thus may be a useful prognostic biomarker. In efforts to translate the
findings from the experimental studies to human subjects, various clinical studies
have demonstrated increased urinary AGT levels in hypertension, type 1 and type
2 diabetes mellitus, and several forms of chronic kidney diseases. However, no study
has described any effect of uAGT other than in renal diseases. Larger clinical studies
are needed in human subjects to determine if treatment with RAS inhibitors can
reduce uAGT levels, thus indicating reduction in intrarenal RAS activity.
The valid measurement method of uAGT is Elisa in samples. The development of
an ELISA kit for uAGT measurement would enable sensitive and specific quantifi-
cations as well as the evaluation of treatment effects; however, a cut-off level for
uAGT has not yet been established. Different quantification methods for uAGT have
been used in previous studies, rendering it difficult to compare past results. Future
studies might reveal a cut-off value that would enable us to use uAGT in clinical
practice.
Considering all the available data together, uAGT can be a potential biomarker of
treatment response with an ACEi or ARB or may be defining the severity of the renal
disease, but there is no evidence to use it as a screening marker for any renal disease.
Summary Points
• The kidney is a specific organ that contains and produces all of the RAS
components.
• The major source of intrarenal Ang II is AGT, and it is locally produced by
proximal tubular cells.
• Ang II plays an important role in maintaining hypertension and the development
of renal damage.
• uAGT, rather than urinary albumin, may be an earlier marker for diabetic
nephropathy.
• Treatment with an ACEi or AT1R blocker reduces blood pressure, urinary protein
excretion, and uAGT in both animal and human studies.
• uAGT is correlated with urinary protein excretion and hypertension.
• uAGT can be used to detect the severity of CKD.
• Many forms of renal diseases exhibit increased uAGT excretion, but there is still a
debate as to whether uAGT is an injury marker or a result of leakage from
damaged renal tissue.
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Flow Cytometry of Urinary Leukocytes
and Lymphocytes as a Biomarker of Renal 15
Disease
Philipp Enghard, Birgit Rudolph, Jan Klocke, and

Gabriela Riemekasten
Contents
Key Facts on Flow Cytometry of Urinary Immune Cells (For the Layperson) . . . . . . . . . . . . . . . . 329
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
Origin of the Urinary Leukocytes and Patterns of Associated Renal Injury . . . . . . . . . . . . . . . . . . . 331
General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333
Flow Cytometry as Biomarker for Specific Renal Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
IgA Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
Lupus Nephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334
ANCA-Associated Glomerulonephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 336
Renal Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 339
Urinary Cells as a “Window” into the Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 341
Potential Application to Prognosis, Other Diseases, or Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
P. Enghard (*)
Klinik mit Schwerpunkt Nephrologie und internistische Intensivmedizin, Charité Berlin, Berlin,
Germany
Klinik mit Schwerpunkt Rheumatologie und klinische Immunologie, Charité Berlin, Berlin,
Germany
e-mail: Philipp.Enghard@Charite.de; Enghard@Drfz.de
B. Rudolph
Institut f€ur Pathologie, Charité Berlin, Berlin, Germany
e-mail: Birgit.Rudolph@Charite.de
J. Klocke
Klinik mit Schwerpunkt Rheumatologie und klinische Immunologie Charité Berlin, Berlin,
Germany
e-mail: Jan.Klocke@Charite.de
G. Riemekasten
Rheumatologie, Universität L€
ubeck, L€
ubeck, Germany
e-mail: Gabriela.Riemekasten@Uksh.de

328 P. Enghard et al.
Abstract
Urine is normally nearly devoid of leukocytes. In various renal diseases however,
immune cells can be observed in the urine. Flow cytometry is the state-of-the-art
technique for analyzing the quantity and qualities of cells in suspension, making
this method an ideal approach for investigating urinary immune cells. Increased
amounts of urinary T cells and macrophages are routinely observed in inflamma-
tory renal disease. In contrast, noninflammatory renal syndromes are not associ-
ated with an increase in urinary immune cell counts. The composition and the
phenotype of the urinary immune cells are reminiscent of the infiltrating cells
observed in the respective kidney biopsies, indicating that the urinary immune
cells mirror local kidney inflammation. Therefore, they can be used as a “window
into the kidney” to investigate the local cellular pathogenesis of renal diseases.
Urinary T cells and macrophages have been probed as biomarkers in IgA nephri-
tis, lupus nephritis, ANCA-associated glomerulonephritis, and renal transplant
rejection, showing promising results in all entities.
Keywords
Anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis •
Flow cytometry as biomarker for renal diseases • IgA nephropathy • Lupus
nephritis (LN) • Renal transplantation • Urinary leukocyte origin
Abbreviations
ANCA Anti-neutrophil cytoplasmic antibody
Anti-HLA-DR Antibody staining HLA-DR MHCII molecules
Anti-URO-3 Antibody specific for tubular epithelial cells
BKV BK-virus, member of the polyomavirus family, can
cause infection of renal transplants
CCR7 Chemokine receptor of the CC family among others
expressed on naïve T cells
CD14 Marker to identify classical monocytes and macrophages
CD16 Surface molecule expressed by neutrophil granulocytes
and a subset of monocytes/macrophages
CD3 Identifies T cells which can be subdivided in CD3
+CD4+ T helper T cells and CD3+CD8+ T killer T
cells
CD4+ effector/memory Once naïve T cells encounter their cognate antigen,
T cells (EM T cells) they differentiate into effector/memory T cells
CD45RO Surface molecule expressed by memory T lymphocytes
CD54 ICAM-1 adhesion molecule
CD56 Marker expressed on NK T cells
CXCR3 Chemokine receptor of the CXC family binds CXCL9,
10, and 11. Primarily expressed on Th1 T cells and
mediates recruitment of these cells
15 Flow Cytometry of Urinary Leukocytes and Lymphocytes as a Biomarker. . . 329
DAPI Fluorescence stain that binds DNA visualizes cell

nucleuses in immunofluorescence
Fas-Ligand (Fas-L) Protein of the TNF family binding mediates apoptosis
SLEDAI Score reflecting the disease activity of SLE patients
LN Lupus nephritis
Key Facts on Flow Cytometry of Urinary Immune Cells (For

the Layperson)
• Normally, the urine is almost devoid of immune cells. In certain diseases, diverse
subsets of immune cells can be detected and quantified in the urine.
• Elevated amounts of immune cells in the urine reflect inflammation of the kidney.
• Immune cells in the urine can be used as a biomarker for various inflammatory
renal diseases, for diagnosis and follow-up.
• The immune cells in the urine offer the unique opportunity to directly investigate
the cellular components of the renal inflammation.
Definitions
ANCA-associated glomerulonephritis ANCA autoantibody-induced systemic

vasculitis affecting the kidneys and causing renal inflammation.
Flow cytometry Method for quantitative and qualitative analysis of large amounts
of suspended cells. Usually, cells are stained with fluorochrome-coupled antibodies,
which are detected using lasers and detectors for specific wavelengths.
Hematuria The presence of blood in the urine.
IgA nephropathy One of the most frequent forms of glomerulonephritis caused by

deposition of IgA in the mesangium.
Lupus nephritis (LN) Renal inflammation associated with the systemic autoim-
mune disease SLE.
Proliferative nephritis Renal disease associated with the proliferation of cells in

the glomeruli due to inflammation of the kidneys.
Proteinuria The presence of elevated amounts of protein in the urine.
Renal transplant rejection Immunologic reaction against the renal graft, a fre-
quent cause of graft failure.
SLE Systemic lupus erythematosus, a systemic autoimmune disease characterized

by the presence of anti-nuclear autoantibodies and inflammation of several potential
target organs.
Urine sediment Microscopic analysis of the insoluble contents of urine after

centrifugation.
Introduction
Simplified, the pillars of the classic laboratory workup of renal diseases consist of an
evaluation of the renal glomerular filtration rate (creatinine, cystatin C), assessment
of the function of the filtration barrier (proteinuria), and a microscopic analysis of the
urine sediment. Analysis of the sediment in particular holds clues to whether an
inflammatory kidney disease is present. However, it mainly relies on a semiquanti-
tative evaluation of unstained cells, is observer dependent, and does not have a high
sensitivity or specificity (Crop et al. 2010; Fogazzi et al. 2005).
A common observation in the pathology of inflammatory renal diseases is the
recruitment of inflammatory leukocytes and lymphocytes into the inflamed renal
tissue. Infiltration of immune cells can be present in the glomeruli and renal
interstitium. The invading cells consist mainly of macrophages and T cells and to
a lesser extent of NK cells, B cells, and plasma cells (Markovic-Lipkovski
et al. 1990). In large histologic studies, the interstitial infiltration of immune cells
was identified as being a main factor predicting the outcome in diverse renal diseases
(Bohle et al. 1992, 1994) (Table 1).
Table 1 Main flow cytometry findings in IgA nephropathy, lupus nephritis, ANCA-associated
glomerulonephritis, and renal transplantation
Summary of the main findings of flow cytometry in different renal diseases
IgA nephropathy Ratio of urinary CD3/CD14 distinguishes IgA nephropathy from other
forms of hematuria
Urinary CD3, CD4, and CD14 cells correlate with renal function and
crescents
Urinary macrophages correlate with disease activity
Lupus nephritis Urinary CD4 and CD8 identify patients with active, proliferative LN
Urinary T cells seem suitable to monitor treatment response
ANCA- Increase in urinary EM T cells in patients with active renal involvement
associated GN Decrease in urinary T cells correlates with remission
Renal transplant Increased amounts of urinary tubular epithelial cells indicate acute tubular
necrosis
Increased urinary T cells in patients with acute rejection
The presence of urinary HLA-DR+ cells and ratio of CD3+/URO+ >1 in
patients with acute rejection
Persistence of HLA-DR+ and CD3+ cells in the urine may indicate
subclinical rejection
Using specific antibodies for the detection of leukocyte and lymphocyte subsets,
T cells and macrophages can be detected in the urine using cytology or flow
cytometry. Flow cytometry is the state-of-the-art technique for analyzing and quan-
tifying cell suspensions in an easy and reliable form, making it a promising method
for the analysis of urinary leukocytes and lymphocytes. The first report of flow
cytometric analysis of urinary cells goes back to 1981 by W. Eisert et al. Flow
cytometry has further evolved ever since, and while most studies in the 1990s were
limited to the analysis of four parameters per analysis, modern studies were able to
simultaneously assess an ever-growing number of parameters.
The present chapter will review the current data on the use of urinary flow
cytometry in various kidney diseases. First, we will outline which patterns of kidney
injury are associated with certain leukocyte types in the urine; then, the application of
flow cytometry of urine cells will be discussed in specific renal diseases. Finally, the
application of urinary flow cytometry as a “window into the kidney” to analyze and
widen our understanding of renal diseases will be described. This chapter will focus on
advanced flow cytometry of urine leukocytes/lymphocytes in renal diseases. Related
topics, such as the automated flow cytometry of unlabelled urine cells and the usage of
urine flow cytometry to detect lower urinary tract malignancies, will not be discussed.
Origin of the Urinary Leukocytes and Patterns of Associated Renal

Injury
High numbers of T cells and macrophages can be observed in the urine of patients with
diverse renal diseases, such as anti-neutrophil cytoplasmic antibody (ANCA)-
associated rapid progressive glomerulonephritis, proliferative lupus nephritis (LN),
and IgA nephropathy, and renal transplant patients with acute rejection (see Fig. 1, e.
g., histology). However, the exact origin of these cells is not definitively established.
The phenotype of the cells in the urine is reminiscent of the intrarenal cells observed in
histology and differs from the phenotype of the cells in the peripheral blood, indicating
that the urine cells originate in the kidney and do not reflect a passive leak of blood cells
into the urine (Dolff et al. 2010, 2013; Enghard et al. 2009). Along that line, urinary
macrophages can be used for the evaluation of patients with hematuria: Patients with
glomerulonephritis (GN)-associated hematuria had high detectable numbers of macro-
phages and T cells in the urine. In contrast, patients with idiopathic renal hematuria,
patients with hereditary nephropathy including Alport’s disease, and patients with renal
stone-associated hematuria had no relevant amounts of T cells or macrophages in the
urine (Hotta et al. 1996; Hotta et al. 1999) (Fig. 2). Consequently it can be assumed that
the leukocytes/lymphocytes observed in the urine stem from the renal tissue.
In the kidney pathology of various renal diseases, invading T cells and macro-
phages can be detected in various sites, such as the glomeruli, the kidney
interstitium, and – in the case of tubulitis – invading the tubular epithelium itself.
Whether the detectable cells in the urine originate from a certain renal compart-
ment is presently unknown. The amount of urinary T cells and macrophages was
found to correlate well with the amount of glomerular crescents and the glomerular
Fig. 1 Examples for the kidney pathologies associated with the presence of elevated urinary T cells
and/or macrophages. (a) IgA nephropathy. (b) Proliferative lupus nephritis IV. (c) ANCA-
associated glomerulonephritis. (d) Acute renal transplant rejection
injury score (Deenitchina et al. 1999; Hotta et al. 1993), suggesting a glomerular
source of the urinary cells. Furthermore, the macrophages in urine and glomeruli
shared certain characteristics, such as CD16 expression (Hotta et al. 1999). The vast
majority of invading leukocytes/lymphocytes, however, is often observed in the renal
interstitium. Urinary T cells in lupus nephritis, for example, share many features of the
renal interstitial T cells, including subtype and chemokine-receptor expression, which
cannot be observed in the glomeruli (Dolff et al. 2010; Enghard et al. 2009). In renal
transplant patients, the amount of certain T cell subsets observed in the biopsy
correlated with the urinary numbers of these cells. The intrarenal T cells in renal
transplantation predominantly localize in the renal interstitium, providing further
evidence that the urinary cell may mirror inflammation in this compartment.
In line with the concept of the urinary immune cells origin, two patterns of renal
injury seem distinguishable according to the presence or absence of urinary T cells
and macrophages. In a wide variety of inflammatory proliferative forms of renal
disease, these cells can be observed in elevated numbers in the urine. In contrast,
various groups reported only negligible amounts of urinary lymphocytes/leukocytes
in less inflammatory renal diseases. In patients with nonproliferative nephrotic
syndromes, like minimal change nephropathy, membranous glomerulonephritis,
and focal segmental glomerulosclerosis, T cells and macrophages seem to be only
Fig. 2 Proposed flow chart to differentiate hematuria as proposed by Hotta et al. (Figure from
Hotta et al. 1993, 2000, with permission of the publishers)
present in low numbers in the urine (Deenitchina et al. 1999; Enghard et al. 2009,
2014; Hotta et al. 1994, 1999; Sakatsume et al. 2001). In most studies, these patients
have been included in small numbers as control groups. Nevertheless, the results
from various groups agree in the observation of only marginal numbers of urinary
inflammatory cells in nonproliferative forms of glomerulonephritis.
General Considerations
Using the immune cells in the urine as a biomarker for renal diseases has certain
advantages over a renal biopsy. Obviously analyzing urine is less invasive than is a
renal biopsy, and the urine analysis can be repeated as often as deemed necessary.
Importantly, the urinary cells may potentially be more sensitive to detect patholog-
ical changes: Many pathological alterations are not distributed evenly throughout the
renal tissue, but are patchy. Consequently, they can be missed by a random biopsy
sample from the kidney tissue. In contrast, it is reasonable to assume that the urinary
cells reflect the entire kidney and are therefore not at risk for sampling bias.
Given that urine is not a physiological place for leukocytes/lymphocytes, it seems
natural to assume that cell survival in this fluid is limited. In a seminal paper on cell
survival in urine, Stachowski et al. found that cell viability decreased with storage
time of the sample and high osmolarity; in contrast, high protein content in the urine
exerted a protective effect on cell survival. In line with this observation, a protocol
was developed adding 30 % fetal calf serum to the urine samples, which substan-
tially increased cell survival (Stachowski et al. 1998).
However, cell survival may not be very critical for analyzing and quantifying
immune cells in the urine, as these cells remain detectable even after cell death. Only
a minority of the published papers on urinary leukocytes/lymphocytes using flow
cytometry did use a live gate with special dyes for staining dead cells and excluding
them from analysis. All other studies did not exclude dead cells from their analysis,
which seems feasible as well.
Flow Cytometry as Biomarker for Specific Renal Diseases
IgA Nephropathy
IgA nephropathy (Morbus Berger) is among the most frequent forms of glomerulo-
nephritis worldwide. It is characterized by the accumulation of IgA deposits in the
glomerular mesangium. Hematuria is a common feature of patients with IgA
nephropathy, with severity ranging from minor hematuria to phases of gross hema-
turia. While many patients show only minor functional impairment, some patients
proceed to severe renal damage and renal insufficiency.
In the urine of patients with IgA nephropathy, CD3+ T cells and CD14+ macro-
phages can be observed, and the ratio of both allows for distinguishing IgA nephrop-
athy from other forms of hematuria, such as renal stones or hereditary hematuria (Hotta
et al. 1996). In IgA nephropathy, CD14+ macrophages usually outnumber the CD3 T
cells. Among the T cells, a slight predominance of CD8 T cells over CD4 T cells was
reported. Additionally, a considerable amount of CD56 NK cells can be found in the
urine, while CD20 B cells are almost absent (Hotta et al. 1994). The amount of CD3+ T
cells, CD4+ T cells, and CD14+ macrophages correlates with impaired renal function
(creatinine clearance) and histopathological changes, namely, interstitial cell infiltration
and glomerular crescents (Deenitchina et al. 1999; Hotta et al. 1994). The quantity of
macrophages also correlates with the activity of the disease, showing increased urinary
numbers during transient exacerbations, which normalize in remission. CD16, a marker
for inflammatory macrophages, is also detectable on the urinary macrophages, corre-
lating with acute exacerbations (Hotta et al. 1999) (Fig. 3).
A total of five papers from three independent research teams reported consistent data
on urinary T cells and macrophages as biomarkers, with good correlation with histology
and renal damage. Future studies, however, will have to prove that urinary flow
cytometry also helps predict the course of the disease and guide treatment decisions.
Lupus Nephritis
Lupus nephritis (LN) is one of the most common manifestations of SLE and is
associated with a poor prognosis (Mills 1994). Response to the standard therapeutic
regimes is still very unsatisfactory, with 45 % of all patients showing failure to achieve
remission during standard induction theraphy (Appel et al. 2009; Ginzler et al. 2005).
Biomarkers could help improve the prognosis of SLE and LN in two ways: first by
enabling a rapid diagnosis of LN, which is associated with better outcome (Faurschou
et al. 2006; Fiehn et al. 2003), and even more importantly by providing reliable tools to
monitor disease activity and treatment response, thus enabling clinicians to adjust
therapy toward a patient-tailored immunosuppressive treatment regime.
a
1,600
1,400
No. of cells / ml.urine
1,200
1,000
800
600
400
200
0
CD 14 CD 56 CD 3
b 5,000 r = 0.6853 c 3,000 r =0.6373

CD56+ cells / ml.urine
P<0.01 P<0.01

4,000 2,000
3,000 1,800
2,000 1,200
1,000 1,600
0 0
10 20 30 40 10 20 30 40
% crescents % crescents
Fig. 3 Urinary mononuclear cells in IgA nephropathy. (a) Number of urinary CD14+, CD56+, and
CD3+ cells in active crescentic (diagonal lines, columns on the left), inactive crescentic (finely
checkered, columns in the middle), and non-crescentic IgA nephropathy (grossly checkered,
columns on the right). (b) Correlation of the amount of CD14+ cells with the % of observed
crescents on the kidney biopsy. (c) Correlation of the amount of CD3+ cells with the % of observed
crescents (Figures from Hotta et al. 1993, with permission of the publishers)
In the course of LN, T cells and other leukocytes are recruited into the inflamed
kidney tissue and are thought to play a pivotal role in the mediation of tissue damage.
It was first observed in 1994 that some patients with SLE have relevant numbers of
macrophages in the urine (Hotta et al. 1994). However, it was not until 2009 that the
presence of urinary T cells was studied in larger cohorts of SLE patients. It was first
noted that urinary CD4+ T cells could be found in high numbers in patients with
active lupus nephritis. The amount of urinary CD4+ T cells correlates with the
disease activity and identifies patients with active lupus nephritis (Enghard
et al. 2009). Additionally, the urinary T cell counts decrease with therapy. Almost
in parallel, a second group reported similar findings for urinary CD8+ T cells as a
potential biomarker (Dolff et al. 2010).
In a large monocentric study, the findings of CD4+ T cells as a biomarker for lupus
nephritis were further consolidated. In a cohort of 147 SLE patients, the amount of
CD4+ T cells correlated with systemic and renal disease activity in patients with past
or present renal involvement (Enghard et al. 2014). In contrast, in patients without
renal involvement, hardly any T cells were observed in the urine, despite high levels of
disease activity. Using 800 CD4+ T cells as a cutoff, patients with active, proliferative
LN were identified with a high diagnostic precision (sensitivity 100 %, specificity
98 %, area under the Receiver Operating Characteristics (ROC) curve 0.9969).
Importantly, urinary T cells in terms of diagnosing proliferative LN performed better
than did classical biomarkers, such as proteinuria, the urine sediment, or creatinine
(Fig. 4a, b).
A group of 14 patients was monitored during the follow-up under treatment for
active lupus nephritis. The majority of patients showed a rapid normalization of their
urinary T cell counts as soon as 1–2 months after the initiation of treatment,
indicating a response to therapy. However, several patients showed persistent or
even increasing urinary CD4+ T cell counts under treatment, referred to as “non-
responders.” Interestingly, after the usual 6 months of induction treatment for lupus
nephritis, the responders presented a lower general disease activity and better
creatinine increment than did the “nonresponders” (Enghard et al. 2014) (Fig. 4c, d).
A total of 29 patients in this study were analyzed in parallel to a kidney biopsy,
the current gold standard for diagnosing lupus nephritis. All patients with prolifer-
ative lupus nephritis also presented with high urinary CD4+ T cell counts. In
contrast, patients with only minor abnormalities (class I) or nonproliferative forms
(class V) were not associated with increased urinary CD4+ T cells counts.
A second independent research team in a cohort of 46 SLE patients observed
similar results. In this study, urinary CD8 T cells (area under the ROC curve AUC
0.93) slightly outperformed urinary CD4 T cells (AUC 0.92) as a biomarker for
lupus nephritis. In patients with renal involvement, urinary T cells yielded a better
diagnostic precision than did creatinine or proteinuria. A subgroup of 16 SLE
patients was analyzed serially in parallel to treatment. After successful induction
of remission, a significant decrease in urinary CD4+ and CD8+ T cell numbers was
observed (Dolff et al. 2013).
ANCA-Associated Glomerulonephritis
Anti-neutrophil cytoplasmic antibody (ANCA)-associated glomerulonephritis is one

of the common manifestations of systemic ANCA vasculitis. It has an often
remitting-relapsing course, and exact determination of the inflammatory activity
can be challenging.
In a group of 102 patients with ANCA vasculitis, the amount of CD4+ effector/
memory T cells (EM T cells, defined as CD45RO+CCR7-CD3+CD4+) was quan-
tified in the peripheral blood and urine. In patients with active renal involvement, a
notable increase in urinary EM CD4+ T cells was observed (Abdulahad
et al. 2009). In parallel, the frequency of the same T cell subset decreased in the
blood, indicating compartmentalization of these cells from the blood into the
Fig. 4 Urinary T cells as a biomarker for proliferative lupus nephritis. (a) The amount of urinary
CD4+ T cells correlates with the SLEDAI in SLE patients with renal involvement ( filled circles). In
SLE patients without renal involvement, very few urinary CD4+ T cells are detectable, regardless of
disease activity (open boxes). (b) ROC curve showing the diagnostic performance of urinary CD4+
T cells for detecting proliferative/inflammatory LN in SLE patients. Using a cutoff of 800 CD4+ T
cells yielded a sensitivity of 100 % and a specificity of 98.0 %. (c) Follow-up under therapy:
Patients who normalized their urinary CD4+ T cell counts <800/100 ml within 6 months demon-
strated a significant reduction of their SLEDAI. In contrast, patients with persistent or increased
urinary CD4+ T cell numbers showed no treatment response regarding SLEDAI. (d) Patients with
normalized urinary CD4+ cells have significantly decreased creatinine levels than patients with
increase or persistence of these cells (6-month creatinine as % of initial creatinine) (Figures from
Enghard et al. 2014, with permission of the publishers)
inflamed kidneys. The amount of urinary EM CD4+ T cells differed significantly

between patients with active renal involvement and those in disease remission, and
patients without past or present renal involvement had hardly any EM CD4+ T in the
urine. Furthermore, the amount of urinary EM T cells decreased with successful
treatment in 12 patients assessed in the follow-up (Abdulahad et al. 2009) (Fig. 5).
A further case report on two patients with vasculitis-associated glomerulonephritis
(one patient with ANCAs, one without) reported that besides EM CD4+ T cells, naïve
CD4+ T cells and CD14+ monocytes/macrophages can also be observed in the urine
(Sakatsume et al. 2001).
338
450 100 30 1500 30 Patient 3 400 450 115

Patient 1 Patient 2 Patient 4
400 400 110
350 80 350 105
1250 300
300 20 20 300
60 100
250 250
1000 200 95
200 200
40 90
150 10 10 150
100 750 100 100 85
20
50 50 80
0 0 0 500 0 0 0 75
0 1 2 3 4 0 1 2 0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 9 10
80 Patient 5 600 55 Patient 6 400 45 Patient 7 165 30 Patient 8 110

70 50 40
500 45 160
60 40 300 35 155
400 35 30 20 100
50
30 25 150
40 300 200
25 20 145
30 200 20 15
15 140 10 90
20 100 10
100 10 135
10 5 5
0 0 0 0 0 130 0 80
0 1 2 3 4 5 6 7 8 9 10 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 0 1 2 3 4
Serum creatinine (μMol\L)
60 Patient 9 900 90 Patient 10 Patient 11 450

Patient 12
57.5 30 15 400
50 800 80 55.0
70 425 350
40 700 60 52.5 20 10
50
30 600 50.0 400 300
40
Urine CD4+TEM (cell/mL) / Urine creatinine (μMol/mL)

20 500 30 47.5 10 5
20 375 250
10 400 45.0
10
0 300 0 42.5 0 350 0 200
0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4
Weeks
Fig. 5 Follow-up of urinary effector/memory CD4+ T cells and creatinine in patients with ANCA-associated glomerulonephritis. The left axis shows EM CD4+
T cells (cell/ml) divided by urine creatinine (μmol/ml) (solid circles). The right axis displays serum creatinine (μmol/l) (open circles) (Figure from Abdulahad
et al. 2009, with permission of the publishers)
P. Enghard et al.
Renal Transplantation
Kidney transplantation is the preferred treatment for patients with terminal renal insuf-
ficiency. However, the need for immunosuppression and the risk of graft rejections need
to be kept in balance to enable graft survival and minimize the patient’s risk. Up until
now, there is no established biomarker for the fine-tuning of the immunosuppression or
the early, noninvasive detection of acute or chronic graft rejection (Lo et al. 2014).
Flow cytometry was used to analyze the cellular “profile” of the urine of renal
transplant patients as early as 1981. The first studies relied merely on the forward and
sideward scatter of the flow cytometer to identify cells according to their size and
granularity (Eisert et al. 1981; Gomez Jorge et al. 1991; Lee et al. 1992; Yu et al. 1999).
Using these parameters, cell debris, leukocytes, lymphocytes, and exfoliated tubular
epithelial cells can be differentiated. It was recognized that different urine signatures of
cell composition could be identified in patients with stable graft function, acute
rejection, acute tubular necrosis, and urinary tract infection. In particular, acute tubular
necrosis was identified by the presence of prominent numbers of tubular epithelial cells
with a high sensitivity (100 %) in two separate studies (Lee et al. 1992; Yu et al. 1999).
The diagnostic value of the cell scatter alone for identifying acute rejection was less
reliable, with a sensitivity of 63 % and specificity of 79 % (Yu et al. 1999).
In a large study analyzing 223 urinary samples from 127 renal transplant patients,
Nanni-Costa et al. used the scatters and specific fluorochrome-coupled antibodies for
the detection of different T cell subsets and NK cells (Nanni-Costa et al. 1992). They
confirmed different patterns for stable grafts, acute rejection, infection, and acute
tubular necrosis. Furthermore, they reported that patients with cyclosporine toxicity
might be identified by the presence of a large fraction of small cellular debris. During
acute rejection, urinary lymphocytes outnumbered the monocytes and consisted pre-
dominantly of the CD8+ cytotoxic subset. Large cellular debris (presumably epithelial
cells) was the dominating fraction in acute tubular necrosis, and neutrophils were
frequently found in urinary tract infections. Patients with stable graft function
presented only low cell numbers. Unfortunately, the study did not report receiver
operator curves of sensitivities and specificities of the respective cellular profiles
(Nanni-Costa et al. 1992). Another study from Brazil on 60 renal transplant patients
analyzed the frequency of CD3, CD4, CD8, HLA-Dr, Fas-L, ICAM, and CD25 using
flow cytometry of urine samples (Galante et al. 2006). Similar to other reports, CD3 T
cells were the largest urinary leukocyte population during acute rejection. The expres-
sion of Fas-L yielded the best results for diagnosing acute rejection with an area under
the ROC curve of 0.99, comparing acute rejection to patients with stable graft function
or pyelonephritis. Patients with chronic allograft dysfunction also showed elevated
Fas-L expression to some extent, which diminished the discriminatory power for
separating acute rejection from chronic allograft dysfunction (AUC 0.91). However,
the study analyzed only frequencies of cell subsets and not absolute cell numbers, and
including the absolute cell count may well increase the diagnostic utility.
A major contribution to the current knowledge of flow cytometry on urine cells in
renal transplantation comes from Isabel Roberti et al. over several reports. In a prospec-
tive double-blind study, 200 urine specimens from 40 renal transplant patients were
Fig. 6 Urinary flow cytometry in patients with renal transplant. Urinary cells observed in patients
with acute rejection (white columns), ischemic injury (gray columns), and other pathologies (black
columns). Samples were regarded as positive if the mean fluorescence significantly differed from
the control and at least 5 % of the cells stained with anti-HLA-DR or anti-CD3. Furthermore
samples with more than 500 HLA-DR positive events, samples with a CD3/URO3 ratio>1, with a
proportion of URO3 cells>3 %, and samples with more than 7500 events are displayed (Figure from
Roberti et al. 1995 with permission of the publishers)
analyzed. The presence of CD3+ T cells, URO+ tubular epithelial cells, and HLA-DR+
cells was analyzed using flow cytometry. These data were compared to the clinical
diagnosis of the patients, which was assigned retrospectively in a double-blind manner.
The presence of HLA-DR+ and the ratio of CD3+/URO+ cells differentiated patients
with acute rejection from other patients. A positive flow cytometry was defined as at least
5 % of all urinary cells staining with anti-HLA-Dr or Anti-CD3. Applying these criteria,
acute rejection was diagnosed with a sensitivity of 100 % and a specificity of 87.9 %
using flow cytometry, which was superior to the analysis of unstained cells using urine
cytology (Roberti et al. 1995) (Fig. 6). The good but non-perfect specificity was owed to
the observation that some of the patients with stable graft function also presented elevated
amounts of urinary HLA-DR+ and T cells. Interestingly, in a later study, the group
reported the 1-year follow-up data. The outcome of three patient groups was compared:
(1) patients without acute rejection and persistent low urine cells; (2) patients with early
acute rejection that had normalized their cells after antirejection therapy; and (3) patients
with stable function (no acute rejection), but with persistent positive urinary flow
cytometry. Strikingly, group 3 had a significant worse serum creatinine increment than
did group 1, indicating that detection of HLA-DR+ or T cells in the urine in the absence
of acute rejection may reflect subclinical rejection (Roberti et al. 1997a). In another study,
the same group reported an increase of HLA-DR, CD54, and CD3 expressing urinary
cells during rejection, while the presence of CD14+ monocytes was highly specific for
chronic rejection (Roberti et al. 1997b). Furthermore, the persistence of HLA-DR, CD14,
and CD54 expression on urinary T cells predicted the need for intensified treatment and
irreversible graft damage during acute rejection (Roberti and Reisman 2001).
A recent study of 35 renal transplant patients reported that CD4+ and CD8+
effector/memory T cells (EM T cells) and terminal differentiated T cells (TD T cells)
can also be observed in the urine. Their urinary counts correlated with the respective
amounts of CD4+/CD8+ EM and TD T cells in the respective renal biopsies. Urinary
numbers of the CD4+ and CD8+ EM and TD T cells differentiated between the
group with stable kidney function and the ones with allograft rejection. Patients with
BKV infection also showed increased numbers of urinary EM T cells. However, the
CD8+ EM and TD T cell numbers in the urine were significantly higher during
rejection compared to BKV nephropathy (van Doesum et al. 2014).
Urinary Cells as a “Window” into the Kidney
Besides their potential application as a biomarker for various renal diseases, the
analysis of urinary cells offers the opportunity to directly investigate different forms
of renal inflammation. As the urinary immune cells seem to mirror the inflammatory
process in the kidney, they offer a “window” into the kidney, to live and noninva-
sively observe the cellular pathogenesis or renal diseases.
Comparing the phenotype of T cells in the peripheral blood and urine, it was
demonstrated that mostly memory-effector T cells were recruited into the kidney and
found in the urine of renal transplant patients and those with IgA nephropathy,
ANCA-associated glomerulonephritis, and lupus nephritis (Abdulahad et al. 2009;
Dolff et al. 2010; Sakatsume et al. 2001; van Doesum et al. 2014). Interestingly, in
lupus nephritis and ANCA-associated glomerulonephritis, parallel to the observation
of EM T cells in the urine, a reciprocal decrease of the respective T cell subsets was
observed in the peripheral blood, suggesting migration of these cells from one
compartment to the other (Abdulahad et al. 2009; Dolff et al. 2010).
Presently, it is not known to what extent an antigen-specific T cell response
contributes to renal inflammation. A research paper on the T cell receptor repertoire
reported an enrichment of T cell receptors, indicating an antigen-driven response.
The same T cell receptor repertoire was detected in T cells in the urine, further
supporting the notion that the cells in the urine reflect the intrarenal immune
pathology (Hu et al. 2004).
Mainly using mouse models, several chemokine receptor pathways involved in
the recruitment of inflammatory cells into the renal tissue were identified. Although
these models have been informative, the observed results were, in part, contradic-
tory, and whether these models reflect the recruitment patterns in human patients
remains elusive. In human lupus nephritis, an enrichment of CXCR3-expressing T
cells in the kidneys was reported (Enghard et al. 2009; Segerer et al. 2004). CXCR3
is also expressed by the majority of urinary CD4+ T cells in patients with active
lupus nephritis. The frequency of urinary CXCR3-expressing T cells correlated with
disease activity in SLE patients with renal involvement. Importantly, compared to
the peripheral blood, CXCR3-expressing CD4+ T cells were significantly enriched
in the urine, suggesting participation of CXCR3 in mediating the migration of these
cells from the blood into the inflamed kidneys (Enghard et al. 2009) (Fig. 7).
b 100
solid line: CXCR3
on urinary CD4 T
cells
80
dotted line: CXCR3

on peripheral
% of the max.
60
blood CD4 T cells
40
20
0
100 101 102 103
CXCR3 APC
Fig. 7 CXCR3-expressing cells can be observed in the kidney and urine of patients with prolif-
erative lupus nephritis. (a) Immunofluorescence staining of a kidney biopsy from patients with
LN. Co-staining of CXCR3 (red), CXCL10 (green), and DAPI (blue) demonstrating co-localization
of CXCR3+ cells next to CXCL10 producers. (b) Histogram showing CXCR3 expression on CD3
+CD4+ T cells in the peripheral blood (red line) and urine (blue line) analyzed by flow cytometry.
While there are CXCR3 positive and negative CD4+ T cells in the blood, the urinary CD4+ T cell
uniformly expresses CXCR3 (Figures from Enghard et al. 2009, with permission of the publishers)
Potential Application to Prognosis, Other Diseases, or Conditions
The analysis of urinary immune cells by flow cytometry is presumably not only
applicable to the renal diseases described above, but renal diseases in general. Since
the urinary immune cell signature reflects the renal inflammation, it is reasonable to
assume that all kinds of renal diseases can be assessed for the extent of local kidney
inflammation, to facilitate diagnosis and enable monitoring under treatment.
Summary Points
• Urinary lymphocytes/leukocytes seem to directly reflect local renal inflammation.

• The presence of urinary T cells and/or macrophages yields promising results as a
biomarker for different renal diseases.
• These include IgA nephropathy, lupus nephritis, ANCA-associated glomerulone-
phritis, and renal transplant rejection.
• In these diseases, urinary T cells and/or macrophages can be used as a biomarker
for diagnosis and, presumably, for follow-up under therapeutic intervention.
• In contrast, noninflammatory renal diseases are not associated with increased
amounts of urinary immune cells.
• Additionally, flow cytometry of urinary immune cells offers an attractive tool for
investigating the cellular pathology of human renal diseases.
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Proteome of Human Urinary Exosomes
in Diabetic Nephropathy 16
Gloria Alvarez-Llamas and Irene Zubiri
Contents
Key Facts of Exosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348
Definitions of Words and Terms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 349
Introduction: Diabetic Kidney Disease (DKD) – Diagnosis, Treatment, and Challenges . . . . . 349
Urinary and Plasma Markers of DKD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 351
Exosomes: A Novel Source of Research in Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352
Exosomal Isolation from Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354
Characterization of the Isolated Exosomal Fraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356
Albumin Potential Interference in the Study of Kidney Diseases by Proteomics . . . . . . . . . . 357
Exosomal Markers of Diabetic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364
Abstract
Diabetic kidney disease (DKD) is the major complication in diabetic patients, the
leading cause of end-stage renal disease (ESRD), and main risk factor for cardio-
vascular disease (CVD). Its silent development, together with the lack of specific
and early accessible indicators of renal damage, often results in a late diagnosis
when kidney damage is irreversible. Omics approaches (genomics, proteomics,
metabolomics) account with the advantage of investigating the molecular milieu
as a whole, without preselection of potential targets. The complexity and wide range
of concentration levels of biological fluids as plasma, serum, or urine makes difficult
G. Alvarez-Llamas (*)
Department of Immunology, IIS-Fundación Jiménez Díaz, UAM, Madrid, Spain
e-mail: galvarez@fjd.es; gsllamas@yahoo.es
I. Zubiri
Queen Mary University of London, London, UK
e-mail: irenezubiri@gmail.com

348 G. Alvarez-Llamas and I. Zubiri
the discovery of novel markers of kidney disease progression, other than already
known high-abundance molecules (e.g., albumin). Exosomes are microvesicles
derived from kidney cells in contact with the urinary space with proven roles in
RNA and protein transfer and cell–cell communication. Exosomes may directly
reflect pathophysiological changes taking place in the damaged kidney, constituting
a feasible alternative to the invasive biopsy. Once released into urine or plasma,
exosomes can be isolated and thus represent a sub-proteome where molecular
messengers are enriched. This chapter overviews the current panorama in the
potential use of exosomes as a novel source of biomarkers able to improve DKD
current diagnosis, patients’ risk stratification, and prognosis prediction.
Keywords
Kidney disease • Diabetic nephropathy • Exosomes • Proteomics • Cardiovascular
disease • Urine • Plasma
Abbreviations
BP Blood pressure
CE Capillary electrophoresis
CVD Cardiovascular disease
DIGE Difference gel electrophoresis
DKD Diabetic kidney disease
GC Gas chromatography
iTRAQ Isobaric tags for relative and absolute quantitation
LC Liquid chromatography
MALDI Matrix-assisted laser desorption/ionization
MVB Multivesicular bodies
NTA Nanoparticle tracking analysis
RAS Renin–angiotensin system
SEC Size exclusion chromatography
SELDI-TOF Surface-enhanced laser desorption/ionization time-of-flight
TEM Transmission electron microscopy
THP Tamm–Horsfall protein
UAER Urinary albumin excretion rate
UC Ultracentrifugation
WB Western blotting
Key Facts of Exosomes
• Exosomes are 40–100 nm vesicles with density values in the range of

1.13–1.19 g/mL.
16 Proteome of Human Urinary Exosomes in Diabetic Nephropathy 349
• Exosomes derive from kidney cells in contact with the urinary space and have
proven roles in intercellular communication.
• Exosomes are direct messengers of what is happening in the kidney, both in acute
and chronic damage, and carry molecular markers of renal dysfunction and
structural injury.
• Several methodologies have been described for isolating exosomes from urine,
paying specific attention to the purity and recovery of the isolated fraction.
• In kidney disease patients, albumin can seriously interfere when being co-isolated
from urine with the exosomal fraction.
• A few exosomal markers of diabetic kidney disease and other renal disorders have
been found by proteomics approaches.
Definitions of Words and Terms
Albuminuria Abnormally increased amount of protein (albumin) detected in the urine.
Biomarker A characteristic (molecule) that is objectively measured and whose

levels are evaluated as an indicator of normal biological processes, pathogenic
processes, or pharmacological responses to therapeutic intervention.
Chronic kidney disease Progressive and permanent kidney damage, classified in

five different stages depending on severity of renal dysfunction.
Diabetic kidney disease Chronic renal disease affecting patients with type1 or
type2 diabetes.
End-stage renal disease Kidney failure which requires dialysis or kidney trans-
plantation. ESRD is the last and more severe stage in chronic kidney disease.
Exosome 40–100 nm microvesicles of endocytic origin secreted by most cell types.
Proteomics Study of the whole set of proteins present in a cell, organ, or biological
fluid in a certain moment.
Introduction: Diabetic Kidney Disease (DKD) – Diagnosis,

Treatment, and Challenges
Diabetes prevalence is globally increasing, and diabetes major complication is a renal

disease, commonly named diabetic nephropathy (DN) and more recently referred to
as diabetic kidney disease (DKD). Chronic kidney disease (CKD) is the major clinical
outcome of diabetic patients with DKD being the leading cause of end-stage renal
disease (ESRD) and a risk factor for cardiovascular disease (CVD), i.e., if diabetes is
present, ESRD patients mainly die from CVD. Unfortunately, the disease courses
silently, diagnosis is not straightforward, and kidney damage is irreversible. In many
Table 1 Clinical indicators of diabetic kidney disease (DKD) (Caramori et al. 2006; Jha
et al. 2014)
Initiators
Hyperglycemia
Genetics/epigenetics
Defining parameters
Albuminuria or ACR: normo (<30 mg/g), micro (30–300 mg/g), macro (300 mg/g)
GFR changes
Progression factors
Albumin
GFR changes
Glucose (HbA1C)
" BP
Lipids (cholesterol, triglycerides)
Uric acid
Novel indicators of kidney injury (pending confirmation)
Glomerular (adiponectin, ceruloplasmin, laminin)
Tubular (NGAL, KIM-1, α1- and β2-microglobulin, L-FABP, cystatin C)
Fibrosis (collagen type IV, TGF-β1-to-BMP-7 ratio)
Inflammation (TNF-α, TNFR1)
ACR albumin/creatinine ratio, GFR glomerular filtration rate, BP blood pressure
cases, initial diagnosis is not made by nephrologist, to whom the patient may be often
lately referred, resulting in an increase in mortality rates as no early management to
prevent disease progression has been attempted. Diagnosis is based on several clinical
manifestations with different interpretation depending on, e.g., patient with type 1 or
type 2 diabetes (Park 2014). Urinary albumin excretion rate (UAER or AER) and
rising blood pressure (BP) are the most commonly considered indicators together
with histological manifestations if biopsy material is available, which mainly happens
if atypical clinical course for diabetic nephropathy individuals is observed. In diabetic
patients, microalbuminuria is an indicator of nephropathy and a marker of vascular
damage and higher cardiovascular risk. Microalbuminuria reflects an abnormality in
glomerular capillary permeability to proteins and is also dependent on the tubular
capacity to reabsorb filtered albumin. Clinically, it is defined in the range 20–199 mg
albumin/g creatinine in males and 30–299 mg albumin/g creatinine in females.
Higher values are defined as macroalbuminuria or proteinuria and indicate a decline
in the renal function. AER as predictor for DKD accounts with several limitations.
It may be the case that healthy subjects with normal renal function show increased
AER or that diabetic patients with high risk of developing proteinuria are
normoalbuminuric in an early screening. In some cases, albuminuric patients revert
to normoalbuminuria in some years without treatment, and, in the opposite case,
normoalbuminuric individuals may develop DKD and progress to kidney failure
(Molitch et al. 2010; Kramer et al. 2003). Table 1 summarizes most commonly
used initiators, defining parameters, and progression factors of DKD.
Fig. 1 Kidney disease progression in diabetic patients. Type 1 or 2 diabetic patients may or may
not develop diabetic kidney disease (DKD). Novel markers able to predict individuals at risk for
DKD are pursued. Once renal function starts declining, early diagnosis is mandatory together with
the ability to predict whose patients will progress to end-stage renal disease (ESRD)
When facing individuals with diabetes, the main questions are as follows: Are they
going to develop DKD? And if so, will they progress to ESRD (Fig. 1)? Current risk
markers for DKD are albuminuria and decline in glomerular filtration rate (GFR) with
cutoff stratification values depending on age, serum uric acid, and serum soluble 1 and
2 TNF receptors, among others (MacIsaac et al. 2014), together with family history,
smoking habits, and ambulatory BP and lipids (Caramori et al. 2006; Gray and Cooper
2011). No cure is available. The best approach would be preventing microalbuminuria
development and CVD in diabetic patients, e.g., by means of tight BP control and
renin–angiotensin system (RAS) suppression (Ruilope et al. 2010) or attempting to stop
progression once DKD is diagnosed (Fernandez Fernandez et al. 2012). Precise glycemic
control, BP reduction, cholesterol management, and lifestyle improvement compose the
current therapy which should be defined as personalized and multitargeted (Bakris 2011).
Despite of all efforts, DKD prevalence remains stable (de Boer et al. 2011),
pointing to an urgent need of novel early markers of disease, markers of patient’s
risk, and predictors of patient’s prognosis once DKD is diagnosed in early stages.
Urinary and Plasma Markers of DKD
Limitations of current clinical makers of renal damage in diabetic patients prompt

further research aimed to discover novel indicators easily accessible (i.e., able to be
monitored in biological fluids as urine or serum/plasma). The ultimate goal is
twofold: (1) achieving early diagnosis of diabetic kidney disease, particularly in

those patients with apparent normal renal function (normoalbuminuric), and (2) suc-
cessful individual stratification of CV risk and renal damage progression.
Classical approaches focus to one or various molecules for which there is evidence
or proved connection with the disease under research. Preselection of potential targets
implies an enormous limitation in view of the complexity of the interactions and
underlying mechanisms operating in the cross talk among the different organs, tissues,
and cells. The advantage of the omics strategy is that no potential marker and no key
target are preselected, but all the protein and/or metabolite sets are investigated as a
whole in the search for significant differences. Thus, not only particular pathways or
responding molecules commonly measured in routine biochemical patient’s analysis
or previously discovered are being investigated but also those whose relationship with
the pathophysiological processes taking place is still unknown. A validation (confir-
mation) phase should then follow to further investigate candidate makers discovered,
to set valid conclusions for the clinical practice.
A systematic review of DKD markers is out of the scope of this chapter.
Representative studies are referred in Table 2, showing main urinary and plasma/
serum markers of DKD found by omics approaches mainly as responders to diabetic
condition itself, diabetic nephropathy, albuminuria development or progression, and
renal function decline over time or stable. Very different and complementary
approaches have been used, i.e., gas chromatography (GC) or liquid chromatography
(LC) or capillary electrophoresis (CE) coupled with mass spectrometry (GC-MS or
LC-MS or CE-MS), differential gel electrophoresis (DIGE) analysis, MALDI-TOF-
MS, SELDI-TOF-MS, and label-free or isotopic labeling (iTRAQ)-LC-MS/MS.
Exosomes: A Novel Source of Research in Kidney Disease
Urine exosomes are 40–100 nm vesicles coated with lipid bilayer membranes with
density values in the range of 1.13–1.19 g/mL, derived from all types of kidney cells
in contact with the urinary space, including renal tubule cells and podocytes.
Exosomes have proven roles in regulating immune response, antigen presentation,
RNA and protein transfer, and cell–cell interaction/signaling (Mathivanan
et al. 2010; Camussi et al. 2010; Van Balkom et al. 2011). These microvesicles
have an endosomal origin. They are formed by the fusion of multivesicular bodies
with the plasma membrane and release of their intraluminal vesicles, which are then
termed exosomes once in the extracellular space. Exosomes thus contain membrane
and cytosolic cellular proteins and are considered a mechanism of nonclassical
secretion of proteins, representing 3 % of the whole urine proteome. ExoCarta is a
protein, lipid, and RNA exosomal database providing with the contents of exosomes
which have been identified in multiple organisms, cells, and fluids.
The use of urinary exosomes as starting material for biomarker discovery has
shown to be advantageous. They constitute a sub-proteome of the whole urinary
proteome with minor complexity and reduced protein dynamic concentration range,
which represents a better alternative for detection of low-abundance proteins that
Table 2 Representative proteomics studies to approach diabetic nephropathy and discover novel
markers of disease
Biological Technical
Clinical groups source approach Main findings References
T1DM (n = 122): Plasma RPC18, C3f, apolipoprotein (Hansen
Normo, micro, macro peptidome wCX, C-I (markers of et al. 2010)
MALDI- DN)
TOF
T2DM (n = 6) Plasma PAGE+LC- Lumican, vasorin, (Ahn
Control (n = 6) glycoproteins MS/MS RBP4 et al. 2010)
T2DM (n = 90) Plasma and LC-MS/ Plasma: histidine, (Pena
Normo–micro, urine MS butenoylcarnitine et al. 2014)
micro–macro metabolome (T2DM vs. control)
HTN (n = 150) Urine: hexose,
glutamine, tyrosine
(risk predictors of
albuminuria
evolution)
DM Plasma GC-MS NEFAs, EFAs (Han
DN (n = 150) et al. 2011)
T2DM (n = 30) Plasma UPLC-MS/ Phospholipids (Zhu
DN (n = 52) MS PI C18:0/22:6 et al. 2011)
Control (n = 30) SM dC18:0/20:2
T1DM+micro Plasma LC- Kininogen (Merchant
(stable or declined renal peptides MALDI- et al. 2013)
function) TOF
DN (n = 66) Urine CE-MS Collagen fragments (Alkhalaf
T2DM (n = 82) peptides et al. 2010)
T2DM: normo (n = 43) Urine iTRAQ Alpha-1-antitrypsin (Jin
Micro (n = 43) Alpha-1-acid et al. 2012)
glycoprotein 1
Prostate stem cell
antigen
T1DM: normo (n = 52) Urine GC-MS, Metabolite panel (van der
(progressed (n = 26) or LC-MS Kloet
stable (n = 26)) et al. 2012)
T1DM (normo and macro) Urine LC-MS/ Vanin-1 (Fugmann
MS et al. 2011)
1. Control (n = 20), normo Urine SELDI- Ubiquitin (Papale
(n = 20), micro (n = 18) + TOF B2-microglobulin et al. 2010)
T2DM
2. DN (n = 65), T2DM
+ndCKD (n = 10), nDM
+CKD (n = 57)
T1DM+micro (normal Urine LC- α1(IV) collagen (Merchant
renal function): declined MALDI- α1(V) collagen et al. 2009)
renal function (n = 21) and TOF Tenascin-X
stable (n = 40) Inositol pentakis
phosphate
2-Kinase
(continued)
Table 2 (continued)
Biological Technical
Clinical groups source approach Main findings References
DM+albuminuria (n = 38) Urine SELDI- UbA52 (Dihazi
DM w/o albuminuria (n = TOF et al. 2007)
45)
noDM+albuminuria (n =
34)
Control (n = 45)
DM diabetes mellitus, T1DM type 1 diabetes mellitus, T2DM type 2 diabetes mellitus, DN diabetic
nephropathy, CKD chronic kidney disease, ndCKD nondiabetic CKD, HTN hypertension
otherwise could be masked by major proteins. As a consequence of their endocytic

origin, urinary exosomes contain proteins characteristic of every renal tubule epi-
thelial cell type and from the urinary collecting system, including proteins that are
characteristic of the membrane and cytoplasm of the cells in which they have been
generated. In particular, exosomes can be released in the kidney by cells as
podocytes, pass through the renal tubule, and either be untaken by recipient epithe-
lial cells of the collecting duct or influence them through secretion of their content
(Street et al. 2011). In this sense, more than a way of exocytic cell waste elimination,
exosomes should be considered as messengers, transferring information between
renal and nonrenal cells and carrying molecular markers of renal dysfunction and
structural injury (Salih 2014; Zhou et al. 2008). This role of exosomes as messengers
between cells and tissues gains particular importance in complex scenarios where
multi-organ cross talk takes place. That is the case of the cardiorenal syndrome,
defined (although not fully understood) by proved evidence that an acute/chronic
worsening of kidney function influences an acute/chronic cardiac dysfunction and
vice versa. In cardiovascular disease, exosomes have gained increasing interest
(Cosme et al. 2013) although their specific role in atherosclerosis development still
constitutes an underexplored field (Gonzalez-Calero et al. 2014).
Exosomal Isolation from Urine
Independent of the methodological approach to be used in the study of exosomal

molecular content, key aspects should be taken into account, which may strongly
influence the purity and recovery of the exosomal isolated fraction. Collection and
storage of urine samples influence in a high degree the quality of the recovered
exosomal fraction, and general guidelines have been published, including the use of
protease inhibitors at collection time, sample storage at 80 C, and extensive
vortexing of urine samples after thawing as mandatory steps (Zhou et al. 2006a).
Exosomal isolation (purification) from urine is not straightforward. High abundant
urinary proteins as Tamm–Horsfall protein (THP or uromodulin) and albumin when
renal function is compromised are co-isolated together with the exosomes. This
contamination source can be overcome using different methodological approaches,
as detailed below.
In the last decade, different methods have been proposed for the isolation of
exosomes from diverse biological fluids, and there is no consensus on the best
method to obtain a pure and well-characterized exosomal fraction from urine.
Despite the lack of agreement, most commonly used approaches are based on
(differential) ultracentrifugation (UC) (Pisitkun et al. 2004) (density gradient- or
cushion-based UC) (Raj et al. 2012) and based on the use of a nanomembrane
concentrator (Cheruvanky et al. 2007), immunoaffinity (Sun et al. 2012), or
microfluidic technology (He et al. 2014). There are also new commercial methods
such as the Total Exosome Isolation™ precipitation solution (Invitrogen),
immunobeads or immunoplates (HansaBioMed LLC), or ExoQuick™ precipitation
reagent suitable for the isolation of these microvesicles from urine, serum, and
plasma.
Differential centrifugation and UC isolation method is recommended by the
Human Kidney and Urine Proteome Project (http://www.hkupp.org/Exosome%
20Preparation.htm). Currently, this is the most frequently used methodology for
the isolation of exosomes from urine (Fig. 2). In brief, urine samples are centrifuged
at 17,000 g in order to remove the whole cells, large membrane fragments, and
debris and recover the supernatant, which is then ultracentrifuged (200,000 g, 1 h,
4 C). Exosomes are recovered in the pellet. Particular attention should be paid by
Fig. 2 Schematic view of isolation protocol of exosomes from urine. Ultracentrifugation based is
one of the most commonly used methodologies for isolating urinary exosomes. Serial centrifugation
steps and enrichment of the exosomal pellet by DTT treatment are applied to maximize purity and
recovery of the exosomal fraction. An extra albumin depletion step is recommended when analyz-
ing urine samples from kidney disease patients diagnosed with macroalbuminuria
the partial entrapment of exosomes by the polymerized THP network, thus reducing
exosomal recovery. This drawback can be overcome by treatment of the first
(low-speed centrifugation) pellet with reducing agents (e.g., DTT) and heat. Fol-
lowing centrifugation again, the supernatant is then collected and pulled together
with the supernatant obtained in the first low-speed centrifugation to proceed with
ultracentrifugation (Fernández-Llama et al. 2010). The final pellet can still contain
important amounts of THP polymers coprecipitating with the exosomes which can
be treated again with reducing agents and heat to dissolve the aggregates and
ultracentrifugated again to obtain a final clean exosome pellet (Gonzales
et al. 2010). Another option to minimize THP interference and further purify the
exosomal fraction is to perform extra steps of UC using sucrose gradient or 30 %
sucrose cushion.
Nanomembrane concentration is an alternative to ultracentrifugation, which is
time consuming and requires instrumentation not always available. This approach is
fast and simple and is based on the use of nanomembranes with a uniform pore size
of 13 mm. However, protein recovery is generally not uniform nor pure, and for
comparative proteomic analysis, this variation needs to be taken into account.
Exoquick ® is a commercial reagent designed for specific isolation of exosome by
precipitation, but obtaining enough exosomal recovery from control urine samples
and high purity of the isolated fraction from albuminuric samples is not guaranteed,
as urine-contaminating proteins can be co-isolated in proteinuric conditions
[unpublished data]. A modified Exoquick ® protocol has been described with
improved results (Alvarez et al. 2012). Six different protocols were compared
concluding that ultracentrifugation methods result in the purest exosomal protein
yield, and the fast and simple modified Exoquick ® protocol proved to be the most
effective alternative, particularly when analyzing exosomal mRNA and miRNA.
Exosomal isolations from plasma or secreted by B cell have been accomplished
by immunoaffinity based or by microfluidic isolation technology which separates
microvesicles as a function of diameter from heterogeneous populations of cancer-
cell-derived extracellular shed vesicles (Santana et al. 2014). These methods will be
probably adapted to the isolation of microvesicles from urine in the near future.
Characterization of the Isolated Exosomal Fraction
There are numerous techniques able to confirm the presence and purity of exosomes
obtained by any of the above-described isolation methods. Transmission electron
microscopy (TEM), Western blotting (WB), and most recently NanoSight technique
are the approaches most commonly used. TEM requires sample fixation with 4 %
paraformaldehyde to be later deposited on Formvar carbon-coated nickel grids and
stained with uranyl acetate to obtain images of the exosomes that can allow the user
to determine the size and shape (cup shaped) of these microvesicles under the
microscope. Immuno-electron microscopy (IEM) allows the immune detection and
direct imaging of exosomes. For WB characterization, specific well-known
exosomal proteins are detected. ALIX, TSG101, and clathrin are involved in the
maturation of MVB and are known to be present in the human urinary exosome
membrane. Exosomes are also rich in tetraspanins like CD9, CD63, and CD81 and
heat shock proteins like HSP60, HSP70, and HSPA5. All these specific markers are
independent from the origin of the exosomes and can be used to characterize
exosomes from urine as well as from other sources. In urine, exosomes originate
from podocytes and epithelial cells, and it is possible to detect the presence of
proteins that are segment specific such as aquaporin 2 (AQP2, collecting duct),
sodium proton exchanger 3 (NHE-3, proximal tubule), or podocalyxin (PODXL)
found in podocytes. Apart from WB, enzyme-linked immunosorbent assays
(ELISAs) or flow cytometry can be used for detection of specific exosomal markers.
Because of their small size, exosomes can only be analyzed in a flow cytometer after
linkage to larger particles of known size. Exosomes can be adsorbed to solid latex
microspheres, and microspheres/exosomes can be later incubated with specific
antibodies and analyzed on a flow cytometer (Benito-Martin et al. 2013). A novel
tool that has proven efficiency in the characterization of exosomes is the nanoparticle
tracking analysis (NTA) using the NanoSight which allows specific exosomes and
microvesicles in the range of 50–1,000 nm in liquid suspension to be directly and
individually visualized and counted in real time.
Albumin Potential Interference in the Study of Kidney Diseases by

Proteomics
The albuminuric condition may condition the purity of the exosomal isolated
fraction. Albumin overload in urine can represent an important problem when, e.
g., approaching a proteomics study of kidney diseases characterized by an abnor-
mally high content of this protein in urine (Martin-Lorenzo et al. 2014). Unspecific
co-isolation of albumin in the exosomal fraction may diminish reproducibility,
condition the robustness of the methodology with comparative purposes, and reduce
the possibility to detect low-abundance proteins, making more challenging the
comparison between healthy and disease condition. Total protein quantification of
exosomal fractions can vary substantially between control and disease samples,
resulting in a significantly higher total “exosomal protein content” in patient samples
due to albumin and thus causing underestimation of the low-abundance exosomal
proteins. Depletion of major soluble urine protein contaminants is therefore advis-
able to broaden our understanding of exosomal proteome changes apart from
albumin content in proteinuric kidney disease. An isolation methodology by serial
(ultra)centrifugation steps followed by depletion of the major proteins present in the
exosome fraction was described based on ProteoPrep ® Immunoaffinity Albumin &
IgG Depletion Kit (Sigma-Aldrich) originally developed for plasma samples but
adapted to urinary exosomal fraction. This method proved to be useful and simple,
allowing an increase up to 60 % in the number of identified proteins when using
LC-MS/MS techniques to investigate candidate exosomal markers of diabetic
nephropathy in human samples (Zubiri et al. 2013, 2014). The efficiency of isolation
methods in patients with nephrotic-range proteinuria was investigated by
comparison of three techniques: nanomembrane ultrafiltration, ultracentrifugation,

and ultracentrifugation followed by size exclusion chromatography (UC-SEC). They
demonstrate that highly abundant urinary proteins were still present in sufficient
quantity after ultrafiltration and ultracentrifugation and were able to overcome this
problem when using UC-SEC (Rood et al. 2010).
These two methods represent an improvement in the available exosomal isolation
methods, particularly challenging when dealing with nephropathy urine samples.
Exosomal Markers of Diabetic Kidney Disease
Since 2004 when the presence of exosomes in urine was reported (Sun et al. 2012), a
growing number of studies have been published aimed to the search of novel
biomarkers of disease in these microvesicles. Protein and RNA biomarker candi-
dates have been postulated for a variety of bladder, prostate, urinary tract diseases
and kidney diseases including DKD. In this specific context, several promising
biomarkers have been described in urinary exosomes from patients and animal
models (Table 3). The activity of dipeptidyl peptidase IV (DPP IV) in urine
microvesicles measured by ELISA positively correlated with the progression of
proteinuria in type 2 diabetic nephropathy patients, being a good candidate to
represent an early biomarker of renal damage before onset of albuminuria. Podocyte
injury contributes to the initiation and decline of kidney function in diabetic
nephropathy (Wolf et al. 2005), and podocyte apoptosis is an early mechanism
leading to diabetic nephropathy (Susztak et al. 2006). Measuring podocyte protein
expression changes in a noninvasive manner was possible after isolating urine
exosomes. Expression of Wilms’ tumor 1 (WT1) protein, a transcription factor and
podocyte marker, was measured in urine exosomes from 48 type 1 diabetic patients
and 25 healthy controls, showing for the first time a predominant expression of WT1
protein in urinary exosome in type 1 diabetic patients. This protein was not present in
healthy age-matched controls, and higher levels of this marker were found in
exosomes from patients with proteinuria. The strong correlation found between the
expression of WT1 and the increase in urine protein excretion suggests a consider-
able predictive value of this protein as an early biomarker of DN (Kalani et al. 2013).
Omics approaches account with the advantage of generating data which are not
individual, referred to a unique molecule (protein, metabolite), but global, describing
hundreds or thousands of compounds altered simultaneously in response to a certain
disease or stimulus. This is possible due to the ability of a wide range of available
techniques to characterize thousands of molecular species in each run, thus gener-
ating profiles or data sets which reflect the general situation of the sample (cell,
tissue, biopsy, serum, urine, etc.). Different methodological approaches currently
available can be applied to investigate the exosomal proteome, making the choice
mainly dependent on (1) the nature of the analytes to investigate (i.e., peptides,
proteins, metabolites, lipids); (2) the performance in terms of sensitivity, selectivity,
specificity, and throughput; and (3) the step in the, e.g., biomarker research pipeline
to approach (discovery or validation) which may require a targeted (e.g., SRM or
16
Table 3 Studies showing the potential of urinary exosomes in the search for biomarkers of kidney diseases
Disease Biomarker candidate Main technique Isolation method References
DKD miR-145 RT-QPCR UC (Barutta et al. 2013)
DKD AMBP LC-MS/MS UC (Wolf et al. 2005)
MLL3
VDAC1
DKD 13 mitochondrial metabolite GC-electron impact Volume exclusion (Sharma et al. 2013)
panels MS
DKD Xaa-Pro dipeptidase LC-MS/MS UC (Raimondo et al. 2013a)
Major urinary protein 1
Neprilysin
DKD DPP IV ELISA Immunoaffinity (Gonzales et al. 2010)
isolation
DKD WT1 Immunoblotting UC (Zhou et al. 2013)
Podocyte injury
CKD OPG Immunoblotting UC (Benito-Martin et al.
2013)
Proteome of Human Urinary Exosomes in Diabetic Nephropathy
ELISA
Kidney fibrosis mRNA of CD2AP RT-QPCR UC (Lv et al. 2014)
Cystinuria 38 protein panels IEF LC-MS/MS UC (Bourderioux et al. 2015)
AKI ATF3 RNA RT-QPCR UC (Chen et al. 2014)
(continued)
359
360
Table 3 (continued)
Disease Biomarker candidate Main technique Isolation method References
AKI ATF3 Immunoblotting UC (Zhou et al. 2008)
WT1 IHC
AKI Fetuin-A 2D-DIGE UC (Zhou et al. 2006b)
Renal carcinoma 10 protein panels LC-MS/MS UC (Raimondo et al. 2013b)
Renal transplantation NGAL Immunoblotting UC (Alvarez et al. 2013)
IgA nephropathy Aminopeptidase N vasorin LC-MS/MS UC (Moon et al. 2011b)
Thin basement membrane α-1-Antitrypsin, ceruloplasmin
nephropathy
Renal ischemia–reperfusion injury AQP1 Immunoblotting UC (Sonoda et al. 2009)
Bartter syndrome type I INKCC2 LC-MS/MS UC (Gonzales et al. 2009)
Immunoblotting
Autoimmune glomerulonephritis miR-26a RT-QPCR UC (Ichii et al. 2014)
Polycystic kidney disease PC1/TMEM2 PC2/TMEM2 LC-MS/MS Sucrose gradient (Hogan et al. 2015)
Obstructive nephropathy E-cadherin Immunoblotting UC (Trnka et al. 2012)
N-cadherin
TGFB
L1CAM
DKD diabetic kidney disease, AKI acute kidney injury, UC ultracentrifugation
G. Alvarez-Llamas and I. Zubiri
MRM) or a wider approach (e.g., label-free or (iTRAQ)-LC-MS/MS, CE-MS,

SELDI-TOF-MS). Proteomics analysis has been applied in the search for potential
markers of disease in isolated exosomes (Choi et al. 2013; Moon et al. 2011a;
Simpson et al. 2009). By label-free LC-MS/MS quantitative analysis of exosomes
isolated from urine of Zucker diabetic fatty (ZDF) rats as a model of type 2 DN,
286 proteins were identified and quantified. Confirmed by immunoblotting,
increased Xaa-Pro dipeptidase and decreased urinary protein 1 were shown. In a
similar study carried out in humans, spectral counting analysis revealed a total of
562 proteins identified (207 had been previously identified in urinary exosomes,
108 had been identified in exosomes from different origin, and 244 were identified as
exosomal proteins for the first time). Among those, a panel of 25 proteins signifi-
cantly changed in diabetic nephropathy. Confirmed by selected reaction monitoring
(SRM) mass spectrometry technique, three protein candidate markers of DN in
exosomes were postulated, alpha-1-microglobulin/bikunin precursor (AMBP),
voltage-dependent anion-selective channel protein 1 (VDAC1), and isoform 1 of
histone-lysine N-methyltransferase (MLL3), opening a new possibility to monitor
DN by analyzing urinary exosomes (Wolf et al. 2005).
The metabolome represents the downstream changes in the genome,
transcriptome, and proteome as a reflection of real-time processes occurring in living
organisms. Compared to more than ten million proteins in the proteome, a few
thousand metabolites present in an organism imply a considerable reduction in
complexity. Urine metabolomics is another important field for the study of diabetic
complications. By GC-MS 94 urine metabolites were quantified in cohorts of
patients with diabetes mellitus with and without kidney disease and in healthy
controls. Thirteen metabolites were found significantly reduced in the diabetic
nephropathy cohorts, related to the mitochondrial metabolism, indicating a suppres-
sion of mitochondrial activity in diabetic kidney disease. A consequence of this
dysregulation was also detectable in urine exosomes as they showed that urine
exosomes from patients contain a lower amount of mitochondrial DNA. This
founding was consistent with later gene expression measurements performed in
the kidney tissue, where a lower expression of PGC1α, a master regulator gene of
mitochondrial biogenesis, was observed.
The majority of the studies based in urine exosomes in the search of biomarkers
for DN are focused on the analysis of the proteomic composition of these
microvesicles in healthy and disease stages. The potential of the exosomal RNA as
source of kidney disease markers has also been reported (Miranda et al. 2010). RNA
present in urine tends to be easily degraded and can be originated in apoptotic or
necrotic cells not being representative of the transcriptional profile (Wang and Szeto
2007). RNAs protected by the lipid membrane of the exosomes are more stable and
can be recovered and analyzed through the isolation of the exosomal fraction. RNAs
contained in exosomes are produced in viable cells; thus they can provide a key
insight of the physiopathological processes taking place in the kidney (van Balkom
et al. 2011). Exosomes contain microRNA (miRNA), a class of small nonprotein-
encoding RNAs that regulate gene expression via suppression of target mRNAs.
miRNA expression was analyzed in urinary exosomes from type 1 diabetic patients
with and without diabetic nephropathy. Two hundred twenty-six miRNAs were
detected in the normoalbuminuric patient urinary exosomes, and 22 miRNAs
showed differential expression between normoalbuminuric and microalbuminuric
patients. In the validation phase, miR-145 was found enriched in urinary exosomes
from microalbuminuric patients, a glomerular marker of mesangial cells (Harvey
et al. 2008) induced by TGF-β1 in this cell type (Denby et al. 2011). The expression
of miR-145 was explored in both streptozotocin-induced diabetic mice and cultured
mesangial cells. An upregulation in miR-130a was observed in type 1 diabetic
patients. On the contrary, miR-155 and miR-424 were downregulated, and this effect
was observed specifically in those patients with incipient diabetic nephropathy. In
conclusion, miR-145 was identified as a new potential player in diabetic
glomerulopathy, and the feasibility of the study of urinary exosomal miRNA as a
source for candidate biomarker discovery in diabetic and other renal diseases was
demonstrated here.
The global results of these studies evidence the potential use of the urine
exosomes to monitor changes occurring in the kidney, opening an interesting
alternative to the invasive kidney biopsies used nowadays to diagnose patients and
follow progression. Further exosomal studies will follow to expand current knowl-
edge of underlying operating mechanisms in DKD, which ultimately end in the
discovery of novel therapeutic targets, and key molecules able to (a) diagnose
diabetic patients in asymptomatic stages, (b) predict who of them will or not further
progress to DKD or ESRD, and (c) stratify individual cardiovascular risk.

Conditions
This chapter shows the applicability of exosomes in the study of kidney diseases and
diabetic nephropathy in particular. The silent progression, asymptomatic at early
stages, and irreversible damage of kidney functionality prompt the application of
novel strategies in the search for novel markers, and exosomes arise as a powerful
underexplored source. Markers can be classified according to their utility in (1) “risk
assessment” (markers responding to disease susceptibility), (2) “screening” (markers
able to discriminate between healthy and asymptomatic diseases in large
populations), (3) “prognosis” (markers able to predict probable course of disease
or aggressiveness of therapy), (4) “stratification” (envisage responders and non-
responders to drug), and (5) “therapy monitoring” (indicators of the efficacy of
treatment once the responder status is established). Depending on the ultimate
goal, the experimental study should be carefully designed and patients’ cohorts,
properly matched with a healthy control group, carefully chosen (Fig. 3). Apart from
these general rules, all technical and methodological improvements focused to
efficiently isolate, characterize, and analyze the exosomal fraction from biological
fluids can be implemented to the study of this and other diseases. This is an open
Fig. 3 Schematic workflow. Exosomal proteome is a powerful tool in the search for novel markers
of disease. Isolated from urine, they constitute an enriched sub-proteome which directly reflects
changes taking place in the kidney
field of research, which, although not fully mature in methodology, already accounts
with proven applicability in the clinical proteomics field.
Summary Points
• Diabetic kidney disease is the major complication in diabetes and main risk factor
for cardiovascular disease.
• Diabetic kidney disease develops silently, it is asymptomatic at early stages, and it
is often diagnosed once the renal damage is irreversible.
• Proteomics arises as a powerful approach in the search for novel markers of
diabetic nephropathy, once the challenge of the proteome dynamic range in the
biological fluids is overcome.
• Exosomes are microvesicles released into urine which act as messengers of
changes taking place in the kidney.
• Exosomal isolation from biological fluid is challenging, and ultracentrifugation-
based method is one of the most efficient approaches. Particular care has to be
taken with co-isolation of albumin from urine of renal patients.
• Exosomes constitute a novel and enriched source of biomarkers of kidney
diseases and diabetic nephropathy, in particular.
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Renal Biomarkers N-Acetyl-Beta-D-
Glucosaminidase (NAG), Endothelin, 17
and Their Application
Serap Çuhadar and Tuna Semerci
Contents
Key Facts of Proximal Tubular Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 372
N-Acetyl-β-D-Glucosaminidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374
Assay Interferent Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377
Clinical Significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Endothelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 381
Endothelin Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382
Endothelin-Converting Enzyme . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383
Endothelin Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385
Clearance of ET-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
Detection Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 386
Physiological Functions of Endothelin (Table 3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387
Clinical Significance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 390
Abstract
The risk of acute kidney injury is rising due to aging in the world and with the
increased usage of therapeutic agents and diagnostic interventions which are
highly nephrotoxic. Kidneys, however, well tolerate with the injury, and with
the late rise in nitrogenous waste products such as creatinine and BUN in the
blood, the organ damage is underestimated. Biomarkers are being searched to find
S. Çuhadar (*)
Department of Medical Biochemistry, Ataturk Training and Research Hospital, Izmir, Turkey
e-mail: drcuhadar@gmail.com; sdcuhadar@yahoo.com
T. Semerci
Department of Medical Biochemistry, Giresun University, Giresun, Turkey
e-mail: tunasemerci@gmail.com

370 S. Çuhadar and T. Semerci
a more specific and sensitive one for kidney injury diagnosis like cardiac tropo-
nins (I and T) which is the gold standard in diagnosing acute coronary syndrome
nowadays replaced the total creatine kinase and CK-MB. However, there is still
no such a marker available to take place instead of creatinine. N-Acetyl-β-D-
glucosaminidase is a lysosomal enzyme that leaks into urine which is mainly
originated from the proximal tubular cells. This enzyme is defined as being more
specific and sensitive to renal tubular injury than creatinine especially with its
isoenzymes and when combined with other renal biomarkers, for example,
NGAL and Kim-1. N-Acetyl-β-D-glucosaminidase is stable in urine, and the
variation among individuals is minimal that the spot urine sample is adequate
for the assay practically with colorimetric and spectrophotometric methods with
its high reproducibility. Endothelins are paracrine hormones that stimulate myo-
cardial contraction and other smooth muscle contraction such as uterus, bronchus,
and stomach. They also promote vascular smooth muscle cell growth. Besides
they stimulate secretion in kidney, liver, and adrenals. Endothelins are implicated
in many pathophysiological conditions such as hypertension, myocardial
infarctus, subarachnoidal hemorrhage, and kidney failure.
Keywords
Creatinine ratio • Endothelin 1 • Endothelin-converting enzyme • Endothelin
receptor • Glomerular filtration rate • Proteinuria • Spot urine • Urinary N-
acetyl-β-D-glucosaminidase
Abbreviations
aa Amino acid
ANP Atrial natriuretic peptide
BNP Brain natriuretic peptide
cGMP Cyclic guanosine monophosphate
Cys C Cystatin C
ECE Endothelin-converting enzyme
ET Endothelin
ETA Endothelin receptor type A
ETB Endothelin receptor type B
GFR Glomerular filtration ratio
IL-1 Interleukin-1
IL-6 Interleukin-6
IP3 Inositol trisphosphate
LDL Low-density lipoprotein
NAG N-acetyl-β-D-glucosaminidase
NEP Neutral endopeptidase
17 Renal Biomarkers and Their Application 371
NO Nitric oxide
PKC Protein kinase C
RBP Retinol binding protein
SCr Serum creatinine
SRTX Sarafotoxin
TGF-B Transforming growth factor beta
Key Facts of Proximal Tubular Cells
• Nephrons are the main functional units of the kidney that their function is to
maintain the water, electrolyte, and acid–base balance and remove waste products
from the blood.
• Glomerulus and the Bowman’s capsule are the initial filtering parts of the
nephron.
• Proximal tubule is the part of the nephron that lies from Bowman’ s capsule to the
Henle loop.
• The apical surface of the proximal tubule epithelial cells is closely packed with
microvilli that gives its characteristic name brush border cell which increases the
surface area.
• N-Acetyl-β-D-glucosaminidase is the most active of the glycosidases found in the
lysosomes of the proximal renal tubule epithelial cells.
Definitions
Biomarker indicators of the normal physiological, pathological processes, or

response to pharmacological compounds or drugs that can be measured in biological
materials such as blood, body fluids, or tissues.
Acute kidney injury a deterioration of renal functions to maintain the fluid,

electrolyte, and acid–base homeostasis in an acute manner that results in nitrogenous
waste product retention in the blood.
Lysosome an intracellular cytoplasmic organelle that catabolizes the waste mate-

rials with its enzymes inside that works well at the acidic pH.
N-acetyl-β-D-glucosaminidase a lysosomal enzyme that is mostly abundant in

renal proximal tubular cells, and the function is to catalyze the hydrolysis of terminal
glucose residues in glycoproteins.
Ischemia-reperfusion ischemic injury occurs in a tissue with an interrupted blood

supply; reperfusion begins with the return of the circulation.
Hexosaminidase a term used to define both of the enzymes N-acetyl-β-D-

glucosaminidase and N-acetyl-β-D-galactosaminidase because of their similar
activities.
Endothelin-1 a localized hormone acts as a potent vasoconstrictor when stimulated

or administered intravenously.
Introduction
Kidneys are vital organs that tolerate well with the injury for a period with its
remaining undamaged nephrons instead which results in late response of functional
tests such as BUN, creatinine, and urine output. Because kidneys are target tissues
for toxic injuries by drugs such as aminoglycosides, chemotherapeutics, and chem-
ical toxins such as cadmium, lead, and contrast media, the aging population has high
rates of hospitalization, as a result of exposure to much more nephrotoxic agents
(Moriguchi et al. 2009; Leung et al. 2013; Uchino et al. 2005).
The disease prevention, attenuation of the severity, and proper treatment selection
require early identification. Biomarkers used for this purpose can be defined as the
indicators of the normal physiological and pathological processes or response to
pharmacological compounds or drugs that can be measured in biological materials
such as blood, body fluids, or tissues.
Renal biomarkers aid in the early treatment choices with the informational
advantage of differentiating the various types of kidney injuries such as prerenal
azotemia and ischemic or nephrotoxic injuries or the duration of kidney failure such
as acute, chronic, or acute-on-chronic. However, the current biomarkers are insen-
sitive to recognize injury early enough to aid in the prevention of nephrotoxic factors
in an acute manner.
The new biomarkers are being searched and studied in various patient groups to
find a more specific, sensitive, and earlier marker than creatinine, the gold standard,
used for decades for acute and chronic renal injury. As a valuable example, a novel
biomarker troponin acts as an indicator of myocardial injury as early as in a few
hours and acts as a reliable biologic parameter for diagnosis and therapeutic response
which is easily measured in a single tube of venous blood. The ideal renal biomarker
characteristics are listed in Table 1.
The urine itself is a useful product of the kidney which can be obtained easily
than serum or plasma which is appropriate for serial measurements. It contains
enzymes and protein release from injured tubular cells that can be early markers
for the tissue originated. As an example urine NGAL was superior to serum NGAL
for diagnosing AKI in children undergoing cardiopulmonary bypass (Mishra
et al. 2005).
Table 1 Ideal renal Noninvasive (study materials are blood or urine)

biomarker characteristics
Easily measured with good reproducibility
Inexpensive
Produces rapid results
Useful for early detection and monitoring
High sensitive (an early sign of organ injury)
High specific (typical of the organ damage)
Traceable among other laboratories
High sample stability
Minimum interferent factors (endogenous activators/inhibitors)
Monitors the effect of treatment
The protein or enzyme released from the segmental site of the injury, such as
glomerular, tubular or cytoplasmic, lysosomal, or membranous parts gives extensive
information. Urinary enzymes NAG, alanine aminopeptidase (AAP), α- and
π-glutathione S-transferase (GST), and lactate dehydrogenase (LDH) are commonly
used renal biomarkers. The injury to the renal tubular basal membrane sheds the
brush border enzymes of alkaline phosphatase (AP), gamma-glutamyl
transpeptidase (GGT), maltase, and leucine aminopeptidase into urine at increased
concentrations (Geus et al. 2012).
Low molecular weight urinary proteins such as α-1 and β2-microglobulin, neu-
trophil gelatinase-associated lipocalin (NGAL), and retinol-binding protein (RBP)
are found in urine at higher levels without kidney injury when filtration overload
surpasses tubular reabsorption capacity (Câmara et al. 2009) or with tubular injury.
The incidence of acute kidney injury (AKI) is rising due to increasing age and
severity of illnesses especially in critically ill patients although the annual inhospital
mortality rate seems to decline (Xue et al. 2006; Waikar et al. 2006). The incidence
of chronic kidney disease (CKD) is increasing primarily due to its major cause, the
type 2 diabetes. Sepsis and major surgery are other common predisposing factors for
acute and chronic renal diseases.
An abrupt decline in renal filtration function within 48 h is the recent definition of
AKI (KDIGO 2012) where the staging laboratory criteria are mainly an increase in
SCr and a reduction in urine output. Glomerular filtration rate (GFR) reduction is not
considered as reliable in acute stages (Wu and Parikh 2008). However, in chronic
renal failure, a small decline in the GFR is associated with a large increase in serum
creatinine level.
Serum creatinine (SCr), the current gold standard, has a limited predictive
performance for the early stages of acute renal injury (Waikar et al. 2009). It is
produced at a constant rate with nonenzymatic dehydration of muscle creatine, freely
filtered by the glomerulus, not reabsorbed by the tubules but secreted through the
tubules (Ferguson and Waikar 2012). SCr varies due to the age, gender, race, dietary
intake, muscle mass, muscle metabolism, strenuous exercise, volume overload, and
prerenal azotemia (Gupta et al. 2010). Because creatine is synthesized in the liver,
the SCr concentration in liver disease such as cirrhosis does not correlate well with
the kidney injury (Orlando et al. 2002).
The main function of the kidney is to maintain the homeostasis by glomerular
filtration, tubular reabsorption, and tubular excretion. Glomerular filtration rate
(GFR) is the best index to evaluate the overall kidney function (Ferguson and Waikar
2012). SCr is the best measuring method for the glomerular filtration estimation
(eGFR) in clinical practice. Consequently, the creatinine-based equations such as
modification of diet in renal disease equation (MDRD) or Cockcroft-Gault equation
are uncertain for GFR estimation due to the defined limitations (Bagshaw 2011).
Other defined gold standard for GFR assessment is inulin clearance which is
impractical especially in the acute setting.
SCr and cystatin C (Cys C) are markers of glomerular filtration in renal function
(Geus et al. 2012). Cys C is a cysteine protease inhibitor synthesized in all nucleated
cells. Due to its low molecular weight (~13 kDa), this molecule is freely filtered by
the glomerulus and nearly completely reabsorbed and catalyzed by proximal tubular
cells, but different from creatinine, not secreted by the renal tubules. This particu-
larity is an option used for GFR measurement, which is nowadays estimated as being
superior to the gold method, the creatinine measurement. Consequently, it better
evaluates GFR decline than SCr with a proportional rise in serum concentration
(Ferguson and Waikar 2012).
At early stages of kidney function decline, the tubular secretion of creatinine is
increased; hence, SCr level may be detected as unchanged that overestimates the
decrease in GFR estimation (Ferguson and Waikar 2012). Significant increase is seen
in 48–72 h of postinjury with a dysfunction of 30 % nephrons and more (Waikar
et al. 2009). Therefore, SCr is a late marker with poor specificity and sensitivity.
N-Acetyl-b-D-Glucosaminidase
N-Acetyl-β-D-glucosaminidase (NAG: EC 3.2.1.30) was purified from pig epididy-

mis in 1960 (Findlay and Levvy 1960) and from human spleen in 1967 (Robinson
and Stirling 1968). It is found in many tissues in the body; however, it is the most
active of the glycosidases found in the lysosomes of the proximal renal tubule
epithelial cells (see Fig. 1a, b) (Price 1992). The enzyme NAG catalyzes the
hydrolysis of terminal glucose residues in glycoproteins. The enzymes with a
molecular weight higher than 70 kDa cannot filter normally into the urine. Since it
is too large (140 kDa) to be filtered by the glomerulus freely, serum form of NAG is
not found in urine under physiological conditions. NAG is neither absorbed nor
secreted by renal tubules. Urine form of NAG exists in urine at elevated concentra-
tions with tubular cell damage due to nephrotoxic agents (drugs, contrast media,
Fig. 1 (a) Transmission electron microscopy image of proximal renal tubule in a control rat. Thin
arrows show vesicles of various sizes located in the apical cytoplasm. The thick arrow shows
lysosomes, short arrow shows the microvilli, and head of the arrow shows basal interdigitating
processes. (b) Transmission electron microscopy (Carl Zeiss Libra 120) image of two adjacent
proximal tubules in a control rat. The basal interdigitating processes (thin arrows), the mitochondria
among digitations (thick arrows), the apical surface of the cell shows the closely packed microvilli
(*), and vesicles of various sizes in the apical cytoplasm (short arrow) (*The images are supplied
kindly by Prof. Dr. Isıl Tekmen from her private archive. The images have not published anywhere.
Affiliation: Prof. Dr. Isıl Tekmen: Department of Histology and Embryology, Dokuz Eylul Univer-
sity Medical Faculty, Izmir Turkey)
cadmium, lead) and renal illnesses such as diabetes, obstructive uropathy, allograft
rejection, and hypertension being a useful early sign of tubular injury. Urinary NAG
is useful for the glomerular function detection in chronic kidney disease (Hong and
Lim 2012). Contrary to other urine biomarkers β2-microglobulin and
α1-microglobulin which are filtered through the glomeruli, NAG (coming only
from tubular cells) increase in urine reflects tubular dysfunction (Moriguchi
et al. 2009). Moreover, NAG is a very sensitive indicator of renal parenchymal
injury than creatinine due to its renal functional reserve.
It is important to note that in normal conditions NAG is excreted into urine at a
constant rate in small quantities as a result of normal exocytosis and leakage
processes. Even in the absence of renal injury, induced lysosomal activity may
cause an increase in urine NAG concentration (Bosomworth et al. 1999).
Isoenzymes: NAG has two main isoenzymes: “A” (acidic) and “B” (basic) which
are located mainly in human kidneys (Morita et al. 1998; Javed and Hussain 1992). The
functions of NAG enzyme are hydrolytic degradation of glycoproteins, mucopolysac-
charides, or glycolipids. Isoenzymes A and B have similar kinetic characteristics (Coma
et al. 1992). NAG A and B were also isolated from placenta (Numata et al. 1997).
Isoenzyme A is located in the soluble part of the lysosome and normally found in
urine due to exocytosis, where isoenzyme B could only be found in urine patholog-
ically due to the tubular damage as being a part of the lysosomal membrane (Gibey
et al. 1984). Normally isoenzyme A is dominant in urine in small quantities;
however in state of injury, isoenzyme B increases.
The minor isoenzymes of NAG are I1 and I2 forms (Numata et al. 1997). Inter-
mediate isoenzymes, I1 and I2, are found in normal sera, additionally I2 in the liver.
The intermediate I1 and I2 are described between the A and B peaks with lower
activities and are extracted mainly from human brain tissue. AS is serum and AT is
tissue form found in the liver, brain, spleen, and kidney (Ikonne and Ellis 1973).
During pregnancy, the urinary NAG activity increases due to the renal physio-
logical adaptation that might interfere the diagnostic value of NAG during renal
injury. Authors (Capodicasa et al. 2011) detected fluorometrically the prevalently
increased form of NAG as isoenzyme A. The intermediate isoenzyme “I” is the
placental form detected in sera (not urine) of pregnant women and is the small
fraction among other NAG isoenzymes. NAG isoenzyme I is an acidic variant of
isoenzyme B (Numata et al. 1997).
Isoenzymes differ in isoelectric points (pI), substrate specificity, and thermal
stability. The different isoelectric points (pI) of NAG A and NAG B (5.4 and 7.9,
respectively) allow the separation of these two isoenzymes by electrophoresis and
ion-exchange chromatography. Isoenzyme A has an acidic and B has a basic
isoelectric point. Isoenzyme isolation methods are ion-exchange chromatography,
HPLC, electrophoresis methods, ELISA, fluorometric methods (Mandić et al. 2005),
and a method that takes advantage of thermoinstability of NAG isoenzyme.
The heat stability of the two isoenzymes is different. The isoenzyme activities of
A and B are maximum at 50 C which abruptly decline from 50 C to 70 C (Nicot
et al. 1987). Isoenzyme A can be inactivated after heat treatment. By heating the
isoenzymes at 50–52 C for 1–3 h reduces the activity of isoenzyme A. NAG B is
more stable to heat and pH changes than isoenzyme A (Robinson and Stirling 1968).
NAG constitutes sialic acid residues which give its acidic property. Removing the
sialic acid groups transfers acidic form A to basic form B. Isoenzyme A has a greater
mobility toward the anode due to its acidic property (Robinson and Stirling 1968).
Isoenzyme A always exceeds isoenzyme B in normal serum and urine. Tissue
form of A is different from the serum form of isoenzyme A. In diabetic patients’
sera and urine, this situation does not change (Severinl et al. 1988). The interme-
diate form isoenzyme “I” is more evident in diabetics with microvascular
complications.
Numata et al. isolated NAG isoenzymes A and B with sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) method showing α and β chains.
Isoenzyme A dissociates into two chains of α and β and isoenzyme B into two or four
β subunits that can be dissociated into βa and βb subgroups. Probably isoenzyme “I”
can be dissociated into two β chains (Numata et al. 1997).
Detection method: NAG can be detected with enzymatic, colorimetric, fluoro-
metric, and ELISA methods. Enzymatic colorimetric assay method is based on
enzymatic cleavage of the colorimetric substrates: dichlorophenol sulfonephthalein,
p-Nitrophenyl N-Acetyl-β-D-Glucosaminide (PNP-NAG) (Horak et al. 1981), and

MCP-NAG (Noto et al. 1983) (sodio m-cresolsulfonphthaleinyl N-acetyl-β-D-
glucosaminide: MCP-NAG) where with substrate PNP-NAG detection sensitivity
was 0.15 U/L (Dalva et al. 2011). Detection wavelength differs (405, 505, 580) due
to the type of substrate (the formation of color) used. With the wavelength preferred,
no interference is expected in case of hemolysis or hyperbilirubinemia (Dalva
et al. 2011).
The first analytical method was fluorometric assay using 4-methylumbelliferyl-N-
acetyl-β-D-glucosaminide as a substrate (4-methylumbelliferyl-Glc NAc). Principle:
The rate of hydrolysis of nonfluorescent 4-methyllumberriferone derivative of N-
acetylglucosamine to fluorescent 4-methylumbelliferone is quantified by
spectrophotofluorometry. The fluorescent method is highly sensitive in determina-
tion of very low levels of NAG in urine diluted 20-to 50 fold to eliminate the
endogenous interferent substances and has been replaced with more practical spec-
trophotometric and colorimetric methods (Horak et al. 1981; Skálová 2005).
NAG activity can be expressed as U/g of creatinine for spot urine specimens to
minimize the dilutional or concentration effects. However, there is not a consensus
about the interpretation of urinary biomarker results as a normalized ratio to urinary
creatinine concentration. That might be affected by the urine creatinine excretion
variability where the decreased urine creatinine level might misinterpret the urine
biomarker level as lower than its exact value (Moriguchi et al. 2009; Hibi
et al. 2014). This easy calculation of the ratio of biomarker level to urinary creatinine
formula alternative to the gold standard excretion rate calculation of a urinary
biomarker is created assuming that creatinine excretion is constant. However, it
changes individually, according to the race, gender and muscle mass, etc., physio-
logically, and shows variations due to the tubular injury pathologically such as in
state of AKI. In contrary in an experimental study by Tonomura et al., the influence
of AKI on urinary NAG-to-creatinine ratio was examined. The urine creatinine
correction formula to urinary flow rate correction was compared in AKI conditions
for urinary enzyme NAG (Tonomura et al. 2011). According to that study, the
diagnostic accuracy of urinary creatinine correction for NAG displayed a higher
specificity and sensitivity (90.2 % and 72.2 %, respectively).
Assay Interferent Factors
Although urinary enzymes give information about the injury site where they are
originated, the interfering factors such as activators and inhibitors have to be taken
into consideration (Mueller et al. 1989). There exist low molecular weight endoge-
nous inhibitors in urine such as urea and ascorbic acid which may cause competitive
inhibition of NAG enzyme that leads to a loss in activity of nearly 6 % (Dalva
et al. 2011). At high concentrations, the endogenous urea in urine inhibits NAG A
and B isoenzyme activities (Bondiou et al. 1985). Gel filtration method is used to
separate the enzyme from inhibitors in urine before the analysis (Horak et al. 1981);
however, it is not declared as necessary (Dalva et al. 2011). NAG B is high in semen
Table 2 Advantages of N-acetyl-b-D-glucosaminidase assay in urine

Noninvasive
Diurnal variation of NAG is minimal to interfere with the assay results; therefore, untimed urine
specimen can be used
Detection in the site of injury
Sensitive marker for tubular damage
Excreted amount of the enzyme is correlated with the tubular injury
Allow early detection of renal injury
Identify the severity of renal injury
Stable in urine across to a range of pH and temperature
Urinary NAG is not gender dependent (Mohkam et al. 2008)
Easily detected by colorimetric or spectrophotometric assay
Reagent low cost
Reliability of measurements
(5800 μg/L), and contamination can interfere the results inevitably (Itoh et al. 1994).
The advantages of NAG assay in urine are listed in Table 2.
Specimens: Serum and urine are acceptable samples.
• Serum: The preferred blood sample is serum. Heparin has an interferent effect on
NAG activity that depresses activity by 50 % (Robinson and Stirling 1968).
• Urine: Untimed urine specimen can be used due to minimal diurnal variation of
NAG. The second morning urine or midstream urine is recommended instead of
the first morning urine. Second morning urine is the urine sample voided 2–4 h
after the first morning urine (Delanghe and Speeckaert 2014). First voided urine is
higher in epithelial cells, erythrocytes, leukocytes, and bacteria where gender
difference is higher than midstream or second morning urine.
Reference Intervals
Serum: Serum total NAG: Adult: 10–30 U/L
Higher in infants <1 year: 11–59 U/L (Oláh et al. 2004)
Serum NAG A isoenzyme: 56–76 % of total activity
Urine: Adult spot urine total NAG: 3.3–4.1 U/L (Horak et al. 1981)
1.0–4.6 U/g creatinine
Isoenzyme A: 80–90 % of total NAG, 10–20 % isoenzyme B (Morita
et al. 1998)
Isoenzyme B: 15.8–20 % of total NAG (Numata et al. 1997)
Sample stability: The stability of NAG isoenzymes is found as affected by the
pH. Lysosome is an organelle in which the interior side is at acidic pH
(4.5–5.0) where the enzymes are highly active. In cytoplasma, pH is slightly
basic (7.2), and lysosomal enzymes cannot destroy the cytoplasmic organ-
elles as they are pH sensitive and do not work well at basic pH. Therefore,
NAG is stable in acidic urine with its predominant A form, where B is the
active form in alkaline pH caused by urease-producing bacteria (Proteus,
Klebsiella, Serratia) or drugs (methotrexate, ranitidine, quinolone) (Morita

et al. 1998; Mandić et al. 2005). Under alkaline condition of about pH 8, the
enzyme activity of isoenzyme A is observed as inactivated where even at
pH 8 isoenzyme B maintained its activity as much as 84 % (Morita
et al. 1998).
To adjust urine to alkaline pH values leads to NAG denaturation irreversibly, so it
is not recommended (Mandić et al. 2005). As an example, in an experimental
study, the alkalinization decreased the NAG activity by about 30 % and later on
acidification of the urine decreased by an additional 12 %, irreversibly (Mandić
et al. 2005).
NAG is stable in urine for a few months stored at –80 C or –20 C and stable
up to five times of freeze-thaw cycles (Lockwood and Bosmann 1979).
Serum NAG A and B activities are found as stable up to 1 month of storage at
20 C. Second thaw caused a decrease in activity of ~33 % for both of the forms
(Coma et al. 1992).
Clinical Significance
The term “hexosaminidase” was first used by Robinson and Stirling in 1968 to
describe either the N-acetyl-β-D-glucosaminidase or N-acetyl-β-D-galactosaminidase
because of their similar activities (Robinson and Stirling 1968). The two major
isoenzymes liberate the terminal GlcNAc and GalNAc monomers from glycopro-
teins and glycosaminoglycans (so-called hexosaminidases). Isoenzyme A degrades
some glycolipids such as GM2 and GA2 gangliosides. Mutations in α or β chain result
in accumulation of undegraded metabolites in lysosomes and in circulation due to
inactivity of hydrolytic enzymes. Tay-Sachs disease is an autosomal recessive
condition, and the disease is a defect of hexosaminidase A activity which causes
ganglioside accumulation (Vidgoff et al. 1973; Sandhoff 1969; Okada and O’Brien
1969). In case of Tay-Sachs disease, serum and liver hexosaminidase A are absent,
but I1, I2, and B are present (Ikonne and Ellis 1973). Deficiency of both A and B
isoenzymes is named as Sandhoff variant of GM2 gangliosidosis (Sandhoff 1969).
Increased urinary activity of NAG not only indicates the renal tubular damage but
the increased lysosomal activity also (Bosomworth et al. 1999). In normal urine,
NAG B and I isoenzymes are present with a percentage of 10–20 % which are
concluded as more sensitive than total NAG (Numata et al. 1997). In pathological
urine of patients with renal diseases, the NAG showed significant increases and the
percentage of NAG B (Numata et al. 1997). Isoenzyme B increase was reported in
patients under therapy of aminoglycoside antibiotics (Gibey et al. 1984). In prema-
ture infants exposed to aminoglycoside antibiotics, urine NAG levels were found as
increased in the absence of a significant rise in SCr (Mcwilliam et al. 2012). In a
study (Assal et al. 2013), the urinary NAG activity was detected as the most sensitive
marker for early renal damage in type 2 diabetes than serum Cys C and urine NGAL.
NAG is an early warning of rejection of renal transplantation. Ischemia-reperfusion
injury begins in the proximal tubules as an earliest occurring factor damaging the
organ. For the early identification of the damage of the transplanted kidney, NAG is
used as a biomarker of the ischemia-reperfusion injury and acute rejection of the renal
allograft, because early damage of the tubulus is associated with rapidly processing
chronic allograft dysfunction. In their study, authors found NAG urine activity as
useful in the evaluation of early proximal tubule damage and predicting the long-term
function of the transplanted kidney (Kwiatkowska et al. 2014).
NAG excretion can also be found as increased due to the glomerular damage such
as in diabetic nephropathy (Gonzalez and Vincent 2012). In patients with vascular
complications, total NAG activity in both serum and urine was higher than in
diabetics without vascular complications, and the intermediate form was more
evident (Severinl et al. 1988).
In a study (Semerci et al. 2014), patients undergoing coronary angiography
demonstrated increased urinary excretion of NAG and high plasma endothelin-1 in
4 h of intervention although none of these patients developed contrast-induced
nephropathy. SCr increased slightly but insignificantly (from 0.71 to 0.79 mg/dL)
and serum BUN unchanged. This is an example that enzymuria reflected only slight
injury that is reversible and uncomplicated. In conclusion, monitoring patients at risk
with these sensitive biomarkers may detect the injury earlier than other late insen-
sitive standard clinical parameters of renal function.
Albumin is excreted into urine as a result of the changes in the glomerular
permselectivity barrier and intraglomerular pressure. The glomerular disease caused
proteinuria accompanied with high levels of urine NAG can be explained with
abnormal traffic of proteins thus caused intrinsic tubular toxicity. In cases of massive
proteinuria, the increase in tubular secretion of creatinine limits GFR estimation.
NAG is considered as a reliable marker of tubular toxicity of proteinuria in early
stages of nephrotic diseases (Bazzi et al. 2002). Urinary NAG is shown to be
increased significantly after surgery in patients suffering from acute kidney injury,
more sensitively than SCr (Jiang et al. 2013). In a study, a positive correlation of urine
NAG was observed with the level of the acute kidney injury (Liangos et al. 2007).
Urinary NAG provided a high sensitivity and specificity with a AUCROC 0.97
discriminating AKI in patients with urinary tract infection (Han et al. 2008).
Enzymes leak from proximal tubular cells into the tubular lumen. The urine
examination provides information about the site of the injury such as high molecular
proteinuria indicates glomerular injury; low molecular weight proteinuria, urinary
excretion of brush border antigens and urinary enzymes indicate proximal
convulated tubule injury (Sharma 2012). It is possible to enhance the sensitivity
and specificity in the diagnostic and therapeutic processes with the combination of
biomarkers. In many studies, enzymes or proteins; such as NAG, Kim-1 and NGAL,
combined according to the different sites of origination, glomerular or tubular, the
results were observed as more reliable (Han et al. 2009; Damman et al. 2011).
Urine NAG can specifically detect clinical outcome of renal tubular damage in
high-risk patients (Hiruma et al. 2014; Damman et al. 2011).
Serum NAG activity has not been investigated widely in clinical studies. How-
ever, elevated serum NAG activity was reported in HT, DM, hyperinsulinemia, and
renal disease (Inoue et al. 2000) and in chronic obstructive pulmonary disease and
myocardial infarction (Iqbal et al. 2003, 2005). Serum NAG activity is increased in
acute pancreatitis, as a marker of lysosomal dysfunction during the inflammatory
process (Milnerowicz et al. 2014).
Endothelin
Endothelin-1 (ET-1) is the most well-known peptide form of the endothelin family. It
was extracted from the endothelial cell cultures and was identified in 1988. It is
particularly known for its potent vasoconstrictor property as it has a long-lasting
activity for several hours when given intravenously (Yanagisawa et al. 1988a).
Endothelins are 21 amino acid peptides and have two disulfide bridges at the
positions of 1–15 and 3–11 cysteine amino acids. Amino acids of the N-terminal
end supply binding affinity to the receptor and some amino acids on the C-terminal
end cause to bind receptor (Sawamura et al. 1989).
Endothelin family consists of three members: endothelin-1 (ET-1), endothelin-2
(ET-2), and endothelin-3 (ET-3) (Inoue et al. 1989a). These members are encoded on
chromosomes 6, 1, and 20, respectively, in human (Arinami et al. 1991).
ET-2 and ET-3 differ from ET-1 by two and six various amino acids, respectively
(Yanagisawa et al. 1988b; Inoue et al. 1989a; Sawamura et al. 1989). ET-2 has a
tryptophan residue at position 6 which makes it most hydrophobic, and ET-3 has a
Lys7 residue instead of the hydrophobic Met7 which is the most polar endothelin
(Fig. 2) (Inoue et al. 1989a). Porcine and human endothelins are identical but the rat
endothelin has different six amino acid residues (Yanagisawa et al. 1988b).
Endothelin like toxins from the venom of the snake (Atractaspis engaddensis),
called sarafotoxins, has four isoforms: SRTX-a, SRTX-b, SRTX-c (S6c), and SRTX-
e (Yanagisawa and Masaki 1989). These 21 amino acid peptides are cardiotoxic that
induce coronary vasoconstriction (Nakajima et al. 1989). The vasoconstrictor activ-
ity of ET-1 is threefold of sarafotoxin S6b (Nakajima et al. 1989). Sarafotoxin 6c
(S6c) is used as a selective agonist of endothelin receptor type B (ETB) (Rubanyi
and Polokoff 1994).
Among the three types of endothelins, only the ET-1 is produced by endothelial
cells, but ET-2 and ET-3 cannot be obtained from endothelial cells of the vascular
tissue (Inoue et al. 1989a; Bloch et al. 1989; Yanagisawa and Masaki 1989).
Additionally, ET-1 mRNA is detected in vascular smooth muscle cells (VSMC)
and also in nonvascular cells such as the brain, lung, placenta, fetal lung, spleen,
pancreas, leukocytes and macrophages, endometrial cells, hepatocytes, Sertoli cells,
breast epithelial cells, fibroblasts, cardiomyocytes, tubular epithelial cells, kidney
mesangial cells and podocytes, etc. (Bloch et al. 1989; Thorin and Clozel 2010;
Kohan 1997; Bagnato et al. 2011). ET-2 is found in the kidney, intestinal tissues, and
heart, and ET-3 is in the brain, kidney, adrenal gland, intestinal tissues, fetal lung,
spleen, and pancreas (Itoh et al. 1989; Khimji and Rockey 2010; Plumpton
et al. 1993; Bloch et al. 1989; Sakurai et al. 1992; Arinami et al. 1991). Both the
ET-1 and ET-3 exist in the central nervous system (Shinmi et al. 1989). Especially in
the rat pituitary gland, ET-3 is higher than ET-1 (Matsumoto et al. 1989).
Endothelin-1
Ser Ser Cys Cys N

Ser
Leu
S-S
Met S-S
Cys His Leu Asp Ile

Asp Ile
Cys Val Phe Trp C
Lys Tyr
Glu
Endothelin-2
Ser Ser Cys Cys N

Ser
Trp
S-S
Leu S-S
Cys His Leu Asp Ile

Asp Ile
Cys Val Phe Trp C
Lys Tyr
Glu
Endothelin-3
Thr Phe Cys Cys N

Thr
Tyr
S-S
Lys S-S
Cys His Leu Asp Ile

Asp Ile
Cys Val Tyr Trp C
Lys Tyr
Glu
Fig. 2 Amino acid structures of endothelin-1, endothelin-2, and endothelin-3
Endothelin Synthesis
Endothelins act like local, paracrine/autocrine hormones as they are not stored but
released within minutes with a stimulus such as hypoxia, ischemia, or shear stress for
the regulation of the vasomotor tonus (Nakamura et al. 1990). The stimuli induce the
transcription of ET-1 mRNA, synthesis, and secretion. The half-life of mRNA is
~15–20 min (Inoue et al. 1989b).
Many stimuli including vasoactive hormones, growth factors, hypoxia, shear
stress, lipoproteins, free radicals, endotoxin, cyclosporine, and also endothelin
increase synthesis of ET-1. Endothelium-derived nitric oxide, nitrovasodilators,
natriuretic peptides, heparin, and prostaglandins which cause increases in
PEPTIDES
Cardiotrophin-1
HORMONES Endothelin PHYSICO-CHEMICAL
INHIBITING FACTORS Adrenaline Endotoxin STIMULI
OF ENDOTHELIN Angiotensin 2 TGF-B Shear stress (low)
Vasopressin IL-1 Free radicals STIMULATING FACTORS
Insulin IL-6 Hypoxia OF ENDOTHELIN
Shear stress (high) Cortisol Osmolarity
Prostaglandin E2 Homocysteine
BLOOD
Prostacyclin COMPONENTS
Nitric oxide Oxidised LDL
Heparin Calcium ions
ANP, BNP Glucose
Thrombin
Prepro ET-1
DNA XENOBIOTICS
cytoplasm
nucleus
A23187
Cyclosporin
Fig. 3 Stimulator and inhibitor factors of endothelin-1 synthesis. ANP atrial natriuretic peptide,
BNP brain natriuretic peptide, IL-1 interleukin-1, IL-6 interleukin-6, LDL low density lipoprotein,
TGF-B, transforming growth factor beta
intracellular level of cyclic guanosine monophosphate (cGMP) inhibit the synthesis

of endothelin (Fig. 3) (Haynes and Webb 1998; Attina et al. 2005; Kowalczyk
et al. 2015; Gray and Webb 1996; Saito et al. 1995).
Endothelins are synthesized by proteolysis of preproendothelins. Human
preproendothelin has 212 amino acid residues; the porcine preproendothelin has
203 amino acids (Itoh et al. 1988; Yanagisawa et al. 1988a). In the lumen of granular
endoplasmic reticulum, the preproendothelin leaves its signal peptide to occur
proendothelin-1 with 195 amino acids. Big endothelin-1, a 38 amino acid residue in
human but 39 amino acid in porcine, is formed with cleavage of proendothelin by an
enzyme named furin-like peptidase (Denault et al. 1995; Xu et al. 1994). A 21 amino
acid residue ET-1 is generated from big ET by endothelin-converting enzymes 1 and
2 (ECE-1 and ECE-2) by specific proteolytic processing between Trp21 and Val22
amino acids (Sawamura et al. 1989; Inoue et al. 1989a). Big ET-2 and ET-3 have
37 and 41 amino acid residues, respectively (Arinami et al. 1991; Opgenorth
et al. 1992). The synthesis pathways of ET-2 and ET-3 are similar to ET-1.
Endothelin-Converting Enzyme
In 1994 the amino acid sequence of ECE was found and called ECE-1 (Masaki 2004;
Xu et al. 1994; Schmidt et al. 1994). Later on the four isoforms of ECE-1 were
identified: ECE-1a, ECE-1b, ECE-1c, and ECE-1d. ECE-1a is found in intracellular
secretory vesicles in the endothelial cells. ECE-1b is also located in the intracellular
compartment close to the Golgi apparatus. ECE-1c and ECE-1d are located on the
cell surface (Masaki 2004). ECE-1 is mainly found in smooth muscle cells which
hydrolyzes bradykinin, substance P, and insulin also (Schmidt et al. 1994). ECE-2 is
ET-1 (1-31)
31aa ET-1 (1-21)
21aa
tissue
chymase ECE-1 or ECE-2
ET-1 (1-32)
32aa
Prepro ET-1 DNA

Big ET-1 non-ECE
nucleus (38aa) matrix
metalloproteinase
Prepro ET-1 mRNA
Furin-like
cytoplasm
enzyme
Prepro ET-1 Pro ET-1 vascular space
(212aa) (195aa)
Fig. 4 Endothelin-1 synthesis pathway
an intracellular enzyme that differs from ECE-1 by its pH sensitivity and is active at
acidic pH (pH 5.5) in contrast to neutral pH optimum of ECE-1. It subsists especially
in endothelial and neuronal cells (Emoto and Yanagisawa 1995). ECE-1 and ECE-2
are coded by chromosomes 1 and 3, respectively.
ECE-1 is a membrane-bound metalloproteinase and is similar to neutral endo-
peptidase 24.11 and Kell blood group protein structurally. This enzyme is inhibited
by the phosphoramidons, the metalloprotease inhibitors (Xu et al. 1994).
There exist also non-ECE pathways that convert big endothelin to various
endothelin forms with tissue chymases and matrix metalloproteinases (Nakano
et al. 1997; D’Orléans-Juste et al. 2003). When big endothelins are cleaved at the
Tyr31-Gly33 bond by human tissue chymases (e.g., mast cell chymases), 31-aa-
length endothelins (1–31) are formed. These products do not undergo any other
processes and have properties like endothelins (1–21). Among the (1–31)
endothelins, ET-2 (1–31) is the most potent vasoconstrictor, similar to ET-l (1–21),
and stronger than ET-2 (1–21) (Nakano et al. 1997; Oka et al. 2014). Endothelins
(1–32) are also formed by the matrix metalloproteinases (non-ECE metallopro-
teinases) (Fig. 4) (D’Orléans-Juste et al. 2003).
The vasoconstriction potency of ET-1 is 140-fold greater than big ET-1, and
preproendothelin-1, which is found in circulation and has no vasoconstrictor activity
(Rubanyi and Polokoff 1994). Although the contractile activity of big endothelin-1
in vascular beds is very low than ET-1, it is as potent as ET-1 in raising the blood
pressure in vivo as ECE-1 converts big ET-1 to active ET-1 rapidly (Sakurai
et al. 1992).
Endothelin Receptors
There are two kinds of receptors for endothelins which are called endothelin
receptor type A (ETA) and type B (ETB) and are coded by chromosomes 4 and
13, respectively (Arai et al. 1990; Sakurai et al. 1992). Endothelins present their
effects by these receptors (Sakurai et al. 1992). Both ETA and ETB receptors are
members of the heptahelical G-protein-coupled transmembrane receptors which
have seven hydrophobic membrane-spanning domains and a relatively long extra-
cellular N- terminal (Masaki 2004). The affinity order for ETA is ET-l ET-
2 >> ET-3 and ET-1 affinity is 100-fold of ET-3 (Lin et al. 1991). ETB affinity
is nearly equivalent for all three endothelins and sarafotoxins (Sakamoto
et al. 1991). Another receptor named ETC reported in amphibians has a greater
affinity for ET-3 and S6c than ET-1 and ET-2 (Masaki et al. 1994). The binding
affinity of ETA and ETB to ET-1 is equal (Davenport 2002), but the receptors’
intracytoplasmic C-termini are different, so the intracellular effect of ET-1 is
different (Nussdorfer et al. 1999).
ETA and ETB receptors are distinct from each other, both structurally and
functionally, and are widely distributed in the kidneys, lungs, adrenal gland, brain,
trigeminal nerve, fibroblasts, heart, cardiomyocytes, prostate, breast, and the female
reproductive tissue in addition to their main origin of vascular system (Karet and
Davenport 1996; Chichorro et al. 2010).
Type A receptors are located on vascular smooth muscle cells that cause vaso-
constriction and smooth muscle proliferation (Haynes and Webb 1994; Komuro
et al. 1988). Type B receptors are present on both endothelial and vascular smooth
muscle cells, especially on the endothelial cells (Davenport and Maguire 1994).
When ET-1 is bound to ETB receptor on smooth muscle cell, calcium ions increase
and vasoconstriction occurs (Chester and Yacoub 2014; Haynes et al. 1995).
ET-1 bound to an ETB receptor on endothelial cell causes synthesis of nitric oxide
(NO) and prostacyclin; consequently NO increases the cyclic guanosine
monophosphate (cGMP) levels in smooth muscle cells, thus leading to vasodilata-
tion (Chester and Yacoub 2014; Verhaar et al. 1998).
Binding of ET-1 to ETA receptor on smooth muscle cell leads to phospholipase C
activation that causes diacylglycerol (DAG) and inositol triphosphate (IP3) occur-
rence. IP3 causes a release of calcium ions from sarcoplasmic reticulum into the
cytosol. Also DAG causes an increase of protein kinase C (PKC) (Khimji and
Rockey 2010). Both pathways (calcium and PKC) cause vasoconstriction.
ET-1 shows its physiological effects with cell growth induction on endothelial
cells, smooth muscle cells, and astrocytes with its potent receptor ETA, whereas
ETB is suggested to be involved in cell apoptosis (Davenport 2002).
ECE-1 expression in endothelial cells is inhibited by activation of ETB receptors.
Pulmonary clearance of circulating ET-1 and reuptaking of ET-1 are mediated also
by ETB receptors (Davenport 2002).
ET-1 shows its proliferative effects through intracellular separate pathways by
increasing mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and
phosphatidylinositol 3-kinase (PI3K) (Chester and Yacoub 2014).
Clearance of ET-1
The circulating ET-1 is cleared from the plasma by ETB receptor followed by
lysosomal degradation for which the major site of clearance is the pulmonary
circulation with a minimum contribution of renal and splanchnic circulation
(Bremnes et al. 2000; Dupuis et al. 1996; Attina et al. 2005; Gasic et al. 1992).
Another way of degradation is the catabolism by extracellular neutral endopeptidase
24.11 (NEP, neprilysin) mainly found in the brush border vesicles of the renal
proximal tubules. NEP is a membrane-bound metallopeptidase with zinc. Although
ET-1 is cleared from the circulation approximately in 1–2 min, its biological effects
last longer (~60 min) (Abassi et al. 1992; de Nucci et al. 1988; Shah 2007).
Detection Method
ET-1 is at a very low concentration in plasma under physiological conditions being a

paracrine/autocrine hormone. It is detected in plasma with radioimmunosorbent
assay and enzyme-linked immunosorbent assay (ELISA) (Masaki 2004). In humans
ET-2 has not been detected yet in plasma (Haynes and Webb 1998).
The preferred blood sample is plasma with anticoagulant EDTA (2 mg/ml). The
centrifugation at 2000 g for 15 min at 4 C is recommended. Samples can be
stored at 25 C or 30 C until analysis (Miyauchi et al. 1991; Oka et al. 2014).
Up to date, for none of the endothelin family, no significant reference interval
study was performed with a large healthy population, to our knowledge. However,
there are studies performed with small groups. As an example in a study by Suzuki
et al., ET-1 level was found as 1.59
0.32 pg/ml (n = 24); however in another
study by Suzaki et al., ET-1 levels were found as 15.54
4.45 pg/ml (n = 5)
(Suzuki et al. 1989; Suzaki et al. 2003). These differences might be due to the
different study population, number of individuals, and different analysis methods
such as the difference of the column used for the purification of the antigens (Oka
et al. 2014).
Aging Difference
ET-1 showed to be increased with aging (Komatsumoto and Nara 1995) in healthy
women (young, middle-aged, and older women) from 1.02 to 2.9 pg/ml (Maeda
et al. 2003). However, in another study, no difference was observed with age (Oka
et al. 2014).
Gender Difference
There is not a consensus whether sex-related difference exists for ET-1. In a study
male ET-1 levels were found as higher than female (Miyauchi et al. 1992), but
another study regretted the difference (Oka et al. 2014).
Table 3 Functions of Regulation of the vascular tone and blood pressure

endothelin
Cell proliferation
Hormone production
Synthesis of vasoactive peptides
Bronchoconstrictor effect
Positive inotropic and chronotropic effect
Modulation of neurotransmission
Physiological Functions of Endothelin (Table 3)
Regulation of the Vascular Tone and Blood Pressure

ET-1 has a vasoconstrictor effect in vitro, and generally it increases blood pressure
in vivo (Yanagisawa et al. 1988a). The vasoconstrictor activities of ET-3 and
sarafotoxin S6b are about 1/60th and 1/3 of ET-1, respectively (Nakajima
et al. 1989). ET-1 increases the blood pressure by activating ETA receptors on
vascular smooth muscle cells and decreases the blood pressure by activating ETB
receptors on endothelial and renal cells (Shah 2007; Kohan et al. 2011). ET-1 leads to
vasodilatation by prostacyclin (Shah 2007). Also ET-1 has an effect on vascular tone
indirectly by sympathetic nervous system (Haynes and Webb 1994).
Cell Proliferation
Endothelin-1 has a proliferative effect on smooth muscle cells, mesangial cells and
fibroblasts, and cardiac myocytes. ET-1 has an increasing effect on growth-
promoting proto-oncogenes (c-fos and c-myc) and on growth factors (Bloch
et al. 1989; Haynes and Webb 1998).
Hormone Production
ET-1 stimulates atrial natriuretic factor secretion from atrial cardiocytes and stimulates
aldosterone synthesis from cortical zona glomerulosa cells and adrenaline secretion
from medullary chromaffin cells (Bloch et al. 1989; Haynes and Webb 1998).
Synthesis of Vasoactive Peptides

Endothelins activate cytokines like tumor necrosis factor (TNF), neutrophil chemo-
tactic factor, granulocyte-macrophage colony-stimulating factor, and interleukins
1, 6, and 8 (Bloch et al. 1989; Haynes and Webb 1998).
Both ET-1 and ET-3 stimulate pulmonary bronchoconstriction by thromboxane
(Levin 1995; Yanagisawa and Masaki 1989).
ET-1 has positive inotropic and chronotropic effects on myocardium. The activities
of ET-1(1–31) are similar to ET-1(1–21) which are vasoconstriction, neurotransmis-
sion, cell proliferation, and production of hormone cytokine (Yanagisawa and Masaki
1989). But its effects are more weaker than ET-1(1–21) (Oka et al. 2014).
Clinical Significance
Endothelin has a role on many illnesses such as ischemic heart diseases, myocardial
infarction, chronic heart failure, hypertension, atherosclerosis, pulmonary hyperten-
sion, chronic renal failure and cerebrovascular spasm, hypercholesterolemia, several
malignancies (like prostate, ovarial, renal, pulmonary, colorectal, cervical, breast,
bladder, and malign melanoma), sickle cell disease, Raynaud’s disease, and complex
regional pain syndrome, infectious diseases, and sepsis with its increased levels
(Shah 2007; Masaki 2004; Levin 1995).
Endothelin and Kidney

ET-1 is the mainly potent isoform in the kidney and is produced by glomerular
epithelial and mesangial cells, vasa recta, and arcuate arteries, renal tubular, and
medullary collecting duct tubule cells (Eid et al. 1994; Karet and Davenport 1996).
ET-1 regulates renal and intrarenal blood flow, sodium and water homeostasis, and
acid–base balance. ET-1 stimulates HCO3 reabsorption and H+ secretion along the
collecting duct with ETA receptors.
Renal and vascular ET-1 act independently with each other. In urine, only kidney-
originated ET-1 exists so the urinary ET-1 excretion is independent of its plasma
form (Serneri et al. 1995; Dhaun et al. 2006; Abassi et al. 1992). ET-1 is catabolized
by neutral endopeptidase that is found in the brush border vesicles of the renal
proximal tubules.
The exogenously administered ET-1 mainly affects renal vasculature, as they are
more sensitive than other vascular beds. The effect is seen as vasoconstriction of the
afferent and efferent arterioles (esp. the afferent arteriole) and consequently a
reduction in glomerular filtration rate (Rabelink et al. 1994; Loutzenhiser
et al. 1990; Schildroth et al. 2011). With the increased water and sodium retention,
blood pressure increases. In cases of renal function reduction, plasma ET-1 levels
were found as increased (Zoccali et al. 1995).
In experimental studies, ETB receptors, both renal and extrarenal, played protec-
tive roles against hypertension (Ohkita et al. 2005). In state of hypertension,
increased vascular ET system activity was observed in experimental studies
performed with ET receptor antagonists (Cardillo et al. 1999). In a study, chronic
blockade of the ETB receptor increased blood pressure (Pollock and Pollock 2001).
It is suggested that renal ET-1 production and function might be so altered that
inappropriate volume homeostasis is contributed in the pathophysiology of hyper-
tension (Dhaun et al. 2006).
Renal cortical ET-1 causes hypertension by increasing renal vascular resistance,
and glomerular ETA activation increases inflammation through enhanced production
of inflammatory factors. The released factors by the inflammatory cells release a
number of factors within the kidney that cause vasoconstriction and increase in
sodium reabsorption resulting in increased blood pressure (Speed and Pollock 2013).
Therefore, ET-1 antagonists are being searched for the arterial hypertension
treatment.
ET-1 has pro-inflammatory, promitogenic, and profibrotic actions in the kidney that
might take part in the hypertensive effect of ET-1. A study demonstrated that ET-1
directly increased glomerular proteinuria with increasing the permeability to albumin
and renal inflammation via ETA receptor activation in vitro (Saleh et al. 2010).
ET and Chronic Kidney Disease

ET-1 is suggested to be a major factor in chronic kidney disease which contributes to
hypertension, proteinuria, and renal inflammation (Dhaun et al. 2011). In chronic
renal failure, both the circulating (Koyama et al. 1989) and urine (Dhaun et al. 2009)
ET-1 levels were found as increased. The increased ET-1 causes the morphological
and functional alterations characteristic for chronic renal failure (Richter 2006).
Summary Points
• This chapter reviews the biomarkers N-acetyl-β-D-glucosaminidase and

endothelin and their significance for renal disorders.
• The insensitivity of creatinine leads to a new standardized biomarker research.
• N-Acetyl-β-D-glucosaminidase is a lysosomal enzyme mainly found in the prox-
imal tubular cells, and the urine NAG has a diagnostic value for tubular injury.
• Endothelin-1 (ET-1) is the most well-known peptide form of the endothelin
family which was extracted from the endothelial cell cultures and named in 1988.
• Serum creatinine is the gold standard used widely to estimate the glomerular
filtration rate (GFR) since no biomarker was identified instead of that endogenous
compound up to date.
• Serum creatinine concentrations vary among individuals with age, race, gender,
muscle mass, and muscle metabolism.
• Both the physiological (children, pregnancy) and pathological (cirrhosis, muscle
wasting, volume overload) conditions may underestimate the acute renal injury as
the defined cutoffs.
• N-Acetyl-β-D-glucosaminidase is the most commonly used renal biomarker with
the advantages of minimal diurnal variation, minimal variability among individ-
uals, and easy quantification with untimed urine that gives information of the site
of the injury since urine N-acetyl-β-D-glucosaminidase originates from the prox-
imal tubular cells.
• Ischemia-reperfusion injury begins in the proximal tubules as an earliest occur-
ring factor damaging the organ, and N-acetyl-β-D-glucosaminidase is used for
early identification of the transplanted kidney injury.
• The exogenously administered ET-1 mainly affects renal vasculature. The effect
is seen as vasoconstriction of afferent and efferent arterioles, thus a reduction in
glomerular filtration rate.
• Endothelins act like local, paracrine/autocrine hormones and are not stored, but
released within minutes with a stimulus such as hypoxia, ischemia, or shear stress
for the regulation of the vasomotor tonus.
• Only endothelin-1 is produced by endothelial cells, but endothelin-2 and

endothelin-3 cannot be obtained from endothelial cells of the vascular tissue.
• ETA receptor activation within the blood vessels, detected on the smooth muscle,
modulates vasoconstriction. ETB receptors, mainly found on the vascular endo-
thelium, modulate vasodilatation with prostacyclin and nitric oxide.
• ET-1 has pro-inflammatory, promitogenic, and profibrotic actions in the kidney.
• ET-1 is suggested to be a major factor in chronic kidney disease which contributes
to hypertension, proteinuria, and renal inflammation.
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Methylated Arginines as Biomarkers
in Renal Disease 18
Arduino A. Mangoni, Angelo Zinellu, Salvatore Sotgia,
Andrew Rowland, and Ciriaco Carru
Contents
Key Facts of Methylated Arginines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 399
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
Synthesis, Transport, and Metabolism of Methylated Arginines . . . . . . . . . . . . . . . . . . . . . . . . . . . 400
Determination of Methylated Arginines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 402
Pathophysiological Effects of Methylated Arginines in the Cardiovascular System . . . . . . 407
Methylated Arginines and Other Cardiovascular Risk Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . 408
Methylated Arginines and Adverse Clinical Outcomes in Patients with Renal Disease . . . 410
Generalizability of Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418
Methylated Arginines and Other Disease States . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 418
Applications in Clinical Practice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
Future Research Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 419
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 420
Abstract
The endogenous methylated analogues of the amino acid arginine, asymmetric
dimethylarginine, symmetric dimethylarginine, and monomethylarginine are gen-
erated following the degradation of proteins containing methylated arginine
residues. Research conducted over the last 25 years has convincingly demon-
strated that methylated arginines, in particular asymmetric dimethylarginine and
monomethylarginine, play a key role in vascular homeostasis by inhibiting the
A.A. Mangoni (*) • A. Rowland

Department of Clinical Pharmacology, School of Medicine, Flinders University, Adelaide, Australia
e-mail: arduino.mangoni@flinders.edu.au; andrew.rowland@flinders.edu.au
A. Zinellu • S. Sotgia • C. Carru
Department of Biomedical Sciences, University of Sassari, Sassari, Italy
e-mail: azinellu@uniss.it; ssotgia@uniss.it; carru@uniss.it

398 A.A. Mangoni et al.
activity of the three isoforms of nitric oxide synthase, endothelial, neuronal, and
inducible, with consequent reduced synthesis of nitric oxide. The increased
synthesis and/or reduced catabolism or clearance of methylated arginines might
contribute to the onset and progression of endothelial dysfunction, vascular
damage, and atherosclerosis through relatively high local exposure in critical
organs and tissues. The initial evidence of a detrimental effect of methylated
arginines on vascular homeostasis originated from studies in patients with either
chronic kidney disease or end-stage renal disease. As the kidney represents a
major elimination route, it is not surprising that the plasma/serum concentrations
of asymmetric dimethylarginine, symmetric dimethylarginine, and monomethy-
larginine are significantly increased in patients with renal disease. Methylated
arginine concentrations have been shown to independently predict adverse clin-
ical outcomes, e.g., cardiovascular morbidity and mortality and all-cause mortal-
ity, both in chronic kidney disease and in end-stage renal disease patients.
This review discusses the synthesis, transport, and elimination of methylated
arginines in physiological conditions and their main pathophysiological effects in
the cardiovascular system. It also discusses the role of methylated arginines as
predictors of adverse outcomes in patients with renal disease and their potential
clinical use as biomarkers.
Keywords
Methylated arginines • Chronic kidney disease • End-stage renal disease • Out-
comes • Endothelium • Nitric oxide • Dialysis
Abbreviations
ADMA Asymmetric dimethylarginine
AQC 6-Aminoquinolyl-N-hydroxysuccinimidyl carbamate
CE Capillary electrophoresis
DAH Dialysis-associated hypotension
DMA Dimethylamine
eNOS Endothelial nitric oxide synthase
FITC Fluorescein isothiocyanate
FLR Fluorescence
GC Gas chromatography
HD Hemodialysis
HILIC Hydrophilic interaction liquid chromatography
HMA Homoarginine
HPLC High-performance liquid chromatography
LCMS Liquid chromatography-mass spectrometry
18 Methylated Arginines as Biomarkers in Renal Disease 399
LIF Laser-induced fluorescence

MI Myocardial infarction
MMA Monomethylarginine
MRM Multiple reaction monitoring
NDA Naphthalene-2,3-dicarboxaldehyde
NO Nitric oxide
NOS Nitric oxide synthase
OPA O-phthaldialdehyde
PFP Pentafluoropropionyl
SDMA Symmetric dimethylarginine
UPLC Ultra-performance liquid chromatography
Key Facts of Methylated Arginines
• Methylated arginines are arginine analogues containing one or two methyl

groups.
• The methylation of arginine residues in proteins is mediated by a family of
enzymes known as protein arginine methyltransferases.
• Methylated arginines are released into the cytoplasm following protein degradation.
• Methylated arginines in the cytoplasm are either metabolized by the enzyme
dimethylarginine dimethylaminohydrolase or transported into the extracellular
space by means of a cationic transporter.
• Circulating plasma methylated arginines are either eliminated by the kidney or
catabolized by dimethylarginine dimethylaminohydrolase.
• Different analytical methods have been developed to measure methylated argi-
nine concentrations from plasma and other biological fluids.
• Reference ranges for methylated arginines concentrations have been published
for healthy subjects. In this group, methylated arginines concentrations are
primarily dependent on age and renal function.
Definitions
Cardiovascular events Cardiovascular events include a number of cardiovascular

complications as clinical manifestations of the onset and progression of atheroscle-
rosis, e.g., myocardial infarction, stroke, and peripheral arterial occlusive disease.
Dialysis Dialysis is treatment strategy consisting in the removal of excess water,

toxins, and other substances from the blood or other body compartments. It is used
primarily to artificially replace the function of the kidney in patients with renal disease.
Methylated arginines Methylated arginines are a group of endogenous arginine

analogues generated by the methylation of arginine residues in protein and subse-
quent degradation of such proteins.
Nitric oxide Nitric oxide is an endogenous molecule that plays a key role in the
physiological regulation of vascular tone, regional blood flow, and blood pressure in
health and in disease states.
Renal disease Renal disease is a condition where the capacity of the kidney to
eliminate water as well as toxic compounds is impaired. Different degrees of renal
impairment predict the risk of complications and death.
Introduction
Cardiovascular disease (CVD) represents the main cause of morbidity and mortality
in either chronic kidney disease (CKD) or end-stage renal disease (ESRD). At least
two factors may explain the association between CVD and renal disease: (1) several
CVD risk factors, particularly hypertension and diabetes, increase the risk of CKD
and ESRD onset and progression, and (2) a good amount of clinical evidence has
demonstrated that both CKD and ESRD independently increase the risk of CVD
events, e.g., myocardial infarction (MI) and stroke (Gansevoort et al. 2013).
A number of functional and structural abnormalities of various components of the
cardiovascular system, generally grouped under the term “cardiovascular
remodeling,” have been demonstrated in CKD/ESRD patients. They include endo-
thelial dysfunction with impaired endothelium-dependent vasodilatation, increased
intima-media thickness, arterial stiffening, and left ventricular hypertrophy
(Gansevoort et al. 2013). Alterations in vascular homeostasis, mainly secondary to
impaired synthesis of the endogenous vasodilator nitric oxide (NO) by the enzyme
isoform endothelial NO synthase (eNOS), play a key role in this context (Napoli
et al. 2006). Over the last 25 years or so, experimental and clinical studies have
demonstrated the biological and pathophysiological relevance of endogenous meth-
ylated arginine analogues in modulating NO synthesis and vascular homeostasis. A
significant number of human studies addressing this issue have been conducted in
patients with CKD/ESRD.
Synthesis, Transport, and Metabolism of Methylated Arginines
Three methylated arginine analogues have been identified in mammalian cells:

asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and
monomethylarginine (MMA) (Vallance and Leiper 2004) (Fig. 1). ADMA, SDMA,
and MMA are synthesized following the methylation of arginine residues in proteins
by protein arginine methyltransferases (PRMTs). PRMTs use S-adenosylmethionine
as methyl group donor (Vallance and Leiper 2004).
Following protein degradation, ADMA, SDMA, and MMA are released into the
cytoplasm (Vallance and Leiper 2004). Intracellular ADMA and MMA, but not
SDMA, are further metabolized to citrulline and dimethylamine by the enzyme
dimethylarginine dimethylaminohydrolase (DDAH) (Fig. 2). DDAH is present in
two isoforms, DDAH-1 and DDAH-2 (Vallance and Leiper 2004). DDAH-1 has
CH3 CH3 CH3 CH3 CH3
NH NH2 NH N NH NH NH NH
C C C C
NH NH NH NH
CH2 CH2 CH2 CH2
CH2 CH2 CH2 CH2
CH2 CH2 CH2 CH2
CH CH CH CH
NH2 COOH NH2 COOH NH2 COOH NH2 COOH
Arginine ADMA SDMA MMA
Fig. 1 Chemical structure of the amino acid arginine and the methylated arginines asymmetric
dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and monomethylarginine (MMA)
Protein arginine residues
PRMT (transmethylation)
Proteolysis
ADMA SDMA
Protein methylated arginine
MMA
residues
AH
DD
Citrulline
dimethylamine
Intracellular
CAT
Extracellular
ADMA, SDMA, MMA
Fig. 2 Pathways of intracellular synthesis, metabolism, and transport of the methylated arginines
asymmetric dimethylarginine (ADMA), symmetric dimethylarginine (SDMA), and monomethy-
larginine (MMA). PRMT protein arginine methyltransferases, DDAH dimethylarginine dimethyla-
minohydrolase, CAT cation transporter
been identified primarily in the liver, and the kidney, and brain (Vallance and Leiper
2004). By contrast, DDAH-2 is predominantly expressed in the endothelium, heart,
placenta, and kidney (Vallance and Leiper 2004). ADMA and MMA metabolism by
DDAH involves a nucleophilic attack on the guanidine carbon by a cysteine held in
an activated state in the tertiary structure of the enzyme (Vallance and Leiper 2004).
There is evidence that increased NO concentrations nitrosate DDAH, inhibiting its
activity (Vallance and Leiper 2004). This suggests a negative feedback system
whereby increased NO can inhibit further NO synthesis through increased concen-
trations of ADMA and MMA.
Although ADMA and MMA are extensively metabolized in the cytoplasm, a
fraction is transported, together with SDMA, across the cell membrane into the
extracellular space and the systemic circulation via the y+ cationic amino acid
transport system (Vallance and Leiper 2004) (Fig. 2). The fraction of ADMA and
MMA that escapes intracellular metabolism is either metabolized in the liver or in
the kidney through DDAH or eliminated unchanged by the kidney (Vallance and
Leiper 2004). SDMA is not metabolized by DDAH. Instead, it undergoes renal
clearance, although there is some evidence of additional uptake by the liver
(Vallance and Leiper 2004). Therefore, it is not surprising that renal and/or liver
disease states are characterized by a significant increase in plasma concentrations of
methylated arginines (Vallance and Leiper 2004).
Determination of Methylated Arginines
ADMA is the methylated arginine most studied in human studies. Table 1 describes
the methods used for its measurement in human plasma, including concentrations in
apparently healthy subjects. The majority of methods utilize liquid chromatography
(LC) with separation of analytes achieved on either reverse phase (C18) or hydro-
philic interaction (HILIC) analytical columns and detection of analytes by either
ultraviolet (UV), fluorescence (FLR), or mass spectrometry (MS) (MacAllister
et al. 1996; Anderstam et al. 1997; Marescau et al. 1997; Pettersson et al. 1997; Pi
et al. 2000; Teerlink et al. 2002; Marra et al. 2003; Heresztyn et al. 2004; Zhang and
Kaye 2004; Sotgia et al. 2008; Blackwell et al. 2009; Jones et al. 2010; Ivanova
et al. 2010; Vishwanathan et al. 2000; Martens-Lobenhoffer and Bode-Boger 2003,
2006, 2012; Martens-Lobenhoffer et al. 2004; Huang et al. 2004; Schwedhelm
et al. 2007; Bishop et al. 2007; D’Apolito et al. 2008; Gervasoni et al. 2011; Yi
et al. 2011; Davids et al. 2012; Hui et al. 2012; El-Khoury et al. 2012; Servillo
et al. 2013; Di Gangi et al, 2010). To a lesser extent, various other technologies such
as capillary electrophoresis (CE) (Zinellu et al. 2007a, 2011; Causse et al. 2000; Linz
et al. 2012; Linz and Lunte 2013; Desiderio et al. 2010), gas chromatography
(GC) (Tsikas et al. 2003; Albsmeier et al. 2004), and enzyme-linked immunosorbent
assay (ELISA) (Schulze et al. 2004) have also been reported. Many of these methods
can simultaneously measure also arginine, SDMA, and MMA concentrations. How-
ever, the simultaneous quantification of arginine and its methylated metabolites in
biological fluids raises several analytical issues: (a) plasma concentrations of ADMA
Table 1 Determination of ADMA in human plasma. Description of the analytical methods

developed to measure asymmetric dimethylarginine (ADMA) in human plasma including informa-
tion on method of detection, reported ADMA concentrations, and detection limit
Plasma
ADMA Detection
References Apparatus Detection Derivative concentration limit
(MacAllister HPLC UV None 0.39
0.05 ND
et al. 1996) (n = 9)
(Anderstam HPLC FLR AQC 0.36
0.08 ND
et al. 1997) (n = 7)
(Marescau et al. 1997) HPLC FLR OPA 0.41
0.09 ND
(n = 66)a
(Pettersson et al. 1997) HPLC FLR OPA 0.58
0.02 0.015
(n = 10)
(Pi et al. 2000) HPLC FLR OPA 0.30
0.05 0.015
(n = 7) (LOQ)
(Teerlink et al. 2002) HPLC FLR OPA 0.42
0.06 0.01
(n = 53) (LOQ)
(Marra et al. 2003) HPLC FLR NDA 0.38–1.30 0.01
(range,
n = 50)
(Heresztyn et al. 2004) HPLC FLR AQC 0.44
0.08 0.1
(n = 12)
(Zhang and Kaye HPLC FLR OPA 0.76
0.12 0.0015
2004) (n = 35) (LOQ)
(Sotgia et al. 2008) HPLC FLR Ninhydrin 0.58
ND 0.004
(n = 50)
(Blackwell et al. 2009) HPLC FLR OPA 0.46
0.08 0.001
(n = 70)
(Jones et al. 2010) HPLC FLR AQC 0.45
0.07 0.20
(n = 30) (LOQ)
(Ivanova et al. 2010) HPLC FLR OPA 0.36–0.63 ND
(range,
n = 225)
(Vishwanathan HPLC MS/MS None 0.12
0.05 0.005
et al. 2000) (n = 20)
(Martens-Lobenhoffer HPLC MS OPA 0.45
0.13 0.20
and Bode-Boger 2003) (n = 15)a (LOQ)
(Martens-Lobenhoffer HPLC MS OPA 0.36
0.07 0.15
et al. 2004) (n = 47) (LOQ)
(Huang et al. 2004) HPLC MS None 0.48
0.07 0.010
(n = 40)
(Martens-Lobenhoffer HPLC MS/MS None 0.37
0.06 0.02
and Bode-Boger 2006) (n = 14)
(Schwedhelm HPLC MS 1-butanol 0.46
0.09 0.0005
et al. 2007) (n = 85)
(Bishop et al. 2007) HPLC MS/MS None 0.66
0.12 0.06
(n = 15)
(continued)
Table 1 (continued)
Plasma
ADMA Detection
References Apparatus Detection Derivative concentration limit
(D’Apolito et al. 2008) HPLC MS/MS None 0.57
0.11 ND
(n = 30)
(Gervasoni et al. 2011) HPLC MS/MS None 0.46
0.07 0.005
(n = 103)a
(Yi et al. 2011) HPLC MS 1-butanol 0.46
0.17 0.08
(n = 21) (LOQ)
(Davids et al. 2012) HPLC MS/MS 1-butanol 0.47
0.06 0.0004
(n = 100) (LOQ)
(Martens-Lobenhoffer HPLC MS/MS None 0.45
0.06 0.003
and Bode-Boger 2012) (n = 10)
(Hui et al. 2012) HPLC MS/MS NDA 0.30
011 0.0026
(n = 123)
(El-Khoury et al. 2012) HPLC MS/MS None 0.36–0.67 ND
(range,
n = 51)
(Servillo et al. 2013) HPLC MS/MS None 0.67
0.04 0.06
(n = 12) (LOQ)
(Di Gangi et al. 2010) UPLC MS/MS 1-butanol 0.56
0.10 0.02
(n = 20)
(Zinellu et al. 2007b) CE UV None ND (n = 77) 0.03
(Zinellu et al. 2011) CE UV None 0.46
0.11 0.01
(n = 50)
(Causse et al. 2000) CE LIF FICT 0.34
0.02 0.05
(n = 5)
(Linz et al. 2012) CE LIF NDA ND 0.02
(Linz and Lunte 2013) CE LIF NDA 0.37
0.05b 0.005
(Desiderio et al. 2010) CE MS/MS None 0.42
0.05 0.02
(n = 1)
(Tsikas et al. 2003) GC MS/MS PFP 0.39
0.06 0.0001
(n = 12)
(Albsmeier et al. 2004) GC MS PFP 0.60
0.08 0.0004
(10)
(Schulze et al. 2004) ELISA UV None 0.65
0.13 0.05c
(n = 10)
Concentrations, limits of detection (LOD), and limits of quantification (LOQ) are given in μmol/L
AQC 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate, CE capillary electrophoresis, ELISA
enzyme-linked immunosorbent assay, FITC fluorescein isothiocyanate, FLR fluorescence, GC gas
chromatography, LIF laser-induced fluorescence, MS mass spectrometry, ND not declared, NDA
naphthalene-2,3-dicarboxaldehyde, OPA o-phthaldialdehyde, PFP pentafluoropropionyl, UPLC
ultra-performance liquid chromatography
a
Values from serum
b
Pooled serum
c
Analytical sensitivity
and SDMA are, in fact, two orders of magnitude lower than those of arginine and an
order of magnitude higher when compared to the concentration of MMA; (b) the
discrimination between the chemically related compounds ADMA and its stereoiso-
mer SDMA, which is produced in equivalent quantities, and MMA and
homoarginine (HMA) represents a primary analytical challenge; and (c) arginine,
ADMA, SDMA, and MMA are lacking of intrinsic chromophores, making direct
detection by optical-based devices (UV or FLR) particularly challenging; however,
the latter is overcome by the use of MS. For these reasons, assays of high specificity
and sensitivity are desirable, and sample cleanup by solid-phase extraction is often
required prior to analysis.
Underivatized ADMA has been successfully measured by HPLC-CE with UV
detector (MacAllister et al. 1996; Zinellu et al. 2007b, 2011), high-performance
liquid chromatography-mass spectrometry (HPLC/MS) (Huang et al. 2004), and
HPLC-CE/MS/MS (Vishwanathan et al. 2000; Martens-Lobenhoffer and Bode-
Boger 2006, 2012; Bishop et al. 2007; D’Apolito et al. 2008; Gervasoni
et al. 2011; El-Khoury et al. 2012; Servillo et al. 2013; Zinellu et al. 2007a, 2011;
Desiderio et al. 2010) detection. By CE, detection of underivatized analytes may also
be accomplished by a pre-column sample concentration (Zinellu et al. 2007b) or by
online sample concentration using the field-amplified sample injection (FASI) tech-
nique (Zinellu et al. 2011; Desiderio et al. 2010). Commonly, however, a pre-column
derivatization is performed before the analysis (Anderstam et al. 1997; Marescau
et al. 1997; Pettersson et al. 1997; Pi et al. 2000; Teerlink et al. 2002; Marra
et al. 2003; Heresztyn et al. 2004; Zhang and Kaye 2004; Sotgia et al. 2008;
Blackwell et al. 2009; Jones et al. 2010; Ivanova et al. 2010; Martens-Lobenhoffer
and Bode-Boger 2003; Martens-Lobenhoffer et al. 2004; Schwedhelm et al. 2007; Yi
et al. 2011; Davids et al. 2012; Hui et al. 2012; Di Gangi et al. 2010; Causse
et al. 2000; Linz et al. 2012; Linz and Lunte 2013; Tsikas et al. 2003; Albsmeier
et al. 2004). Ortho-phthaldialdehyde (OPA) has been widely used for fluorescence
application in this context (Marescau et al. 1997; Pettersson et al. 1997; Pi
et al. 2000; Teerlink et al. 2002; Blackwell et al. 2009; Ivanova et al. 2010). A
major drawback of OPA is the instability of derivatives due to rapid decomposition,
thus requiring online derivatization. More stable derivatives, even at room temper-
ature, are obtained by using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate
(ACQ) as derivatization agent (Anderstam et al. 1997; Heresztyn et al. 2004;
Jones et al. 2010). Other fluorescence probes used especially in CE/laser-induced
fluorescence (LIF)-based methods are fluorescein isothiocyanate (FITC) (Causse
et al. 2000) and naphthalene-2,3-dicarboxaldehyde (NDA) (Linz et al. 2012; Linz
and Lunte 2013). Ninhydrin is also used for fluorescence detection. Unlike other
derivatization reagents, it shows reaction specificity toward ADMA (Sotgia
et al. 2008). An ELISA assay for the specific analysis of ADMA is also available
(Schulze et al. 2004).
Mass spectrometric detection hyphenated with HPLC (Vishwanathan et al. 2000;
Martens-Lobenhoffer and Bode-Boger 2003, 2006, 2012; Martens-Lobenhoffer
et al. 2004; Huang et al. 2004; Schwedhelm et al. 2007; Bishop et al. 2007; D’Apolito
et al. 2008; Gervasoni et al. 2011; Yi et al. 2011; Davids et al. 2012; Hui et al. 2012;
El-Khoury et al. 2012; Servillo et al. 2013), UPLC (Di Gangi et al. 2010), CE
(Desiderio et al. 2010) or Gas Chromatography (GC) (Tsikas et al. 2003; Albsmeier
et al. 2004) is increasingly used. Quantification is carried out by either MS (Martens-
Lobenhoffer and Bode-Boger 2003; Martens-Lobenhoffer et al. 2004; Huang
et al. 2004; Schwedhelm et al. 2007; Yi et al. 2011) or MS/MS (Vishwanathan
et al. 2000; Martens-Lobenhoffer and Bode-Boger 2006, 2012; Bishop et al. 2007;
D’Apolito et al. 2008; Gervasoni et al. 2011; Davids et al. 2012; Hui et al. 2012;
El-Khoury et al. 2012; Servillo et al. 2013). Detection can be performed either for
underivatized arginines (Vishwanathan et al. 2000; Huang et al. 2004; Martens-
Lobenhoffer and Bode-Boger 2006, 2012; Bishop et al. 2007; D’Apolito et al. 2008;
Gervasoni et al. 2011; El-Khoury et al. 2012; Servillo et al. 2013) or after derivatiza-
tion with OPA (Martens-Lobenhoffer and Bode-Boger 2003; Martens-Lobenhoffer
et al. 2004), NDA (Hui et al. 2012) or 1-butanol (Schwedhelm et al. 2007; Yi
et al. 2011; Davids et al. 2012; Di Gangi et al. 2010).
As described previously, the major limitation of LC-based approaches used to
quantify methylated arginines has been the requirement to achieve substantial chro-
matographic separation of these chemically similar compounds. However, the chal-
lenges associated with achieving chromatographic separation of ADMA and SDMA
have been overcome, to some degree, in recent years through the use of HILIC
chromatography or the in situ generation of unique analytes (mass fragments) for
these compounds, using triple-quadrupole MS with multiple reaction monitoring
(MRM) and collision-induced dissociation of underivatized analytes (Martens-
Lobenhoffer and Bode-Boger 2012). The application of this approach to other chem-
ically related arginine metabolites (MMA and HMA) remains to be considered. While
HILIC chromatography offers significant advantages for the separation of chemically
related highly polar compounds, such as arginines, the logistical and technical chal-
lenges associated with this type of chromatography continue to limit its application. It
is likely that the different approaches utilized to overcome these technical challenges
account for the large inter-assay variability in reported plasma ADMA concentrations.
Measurements of ADMA concentrations in human urine (Marescau et al. 1997;
Pi et al. 2000; Teerlink et al. 2002; Martens-Lobenhoffer and Bode-Boger 2003,
2006; Servillo et al. 2013; Tsikas et al. 2003; Gopu et al. 2013; Markowski
et al. 2007), cerebrospinal fluid (Martens-Lobenhoffer et al. 2007), exhaled breath
condensate (Di Gangi et al. 2012), and human vascular endothelial cells (Davids
et al. 2012; Shin et al. 2011) have been also reported. Moreover, evaluations of
methylated arginines in animal plasma (Schwedhelm et al. 2007; Chae et al. 2011;
Nonaka et al. 2005; Tsunoda et al. 2005) or tissues both as free (Ueno et al. 1992;
Nonaka et al. 2006) or total form (free plus protein incorporated) (Teerlink 2005)
have been described. A specific method for the evaluation of protein-incorporated
arginine, ADMA, and SDMA by capillary electrophoresis UV detection has been
recently reported (Zinellu et al. 2007a).
Pathophysiological Effects of Methylated Arginines

in the Cardiovascular System
As previously described, NO is a pivotal endogenous modulator of vascular and

cardiovascular homeostasis both in health and in disease states (Napoli et al. 2006).
Its key role in mediating endothelium-dependent vasodilation, vascular smooth
muscle relaxation, regional blood flow, and blood pressure control has been exten-
sively characterized both in vitro and in vivo (Napoli et al. 2006). Research
conducted over the last 10–15 years has demonstrated additional effects of NO on
prevention of atherosclerosis onset and progression, e.g., reduction of arterial stiff-
ness and intima-media thickness, anti-inflammatory effects, reduced platelet activity,
and reduced cardiac remodeling (Napoli et al. 2006). Therefore, factors leading to an
impaired NO synthesis are likely to exert detrimental effects to cardiovascular
homeostasis, favoring pro-atherosclerotic processes (Fig. 3) (Napoli et al. 2006).
Fig. 3 Putative
pathophysiological effects by
ADMA
which the methylated SDMA
arginines asymmetric MMA
dimethylarginine (ADMA),
symmetric dimethylarginine
(SDMA), and
monomethylarginine (MMA)
modulate the onset and
progression of altered Nitric oxide synthesis –
vascular homeostasis, Endothelium-dependent vasodilation –
atherosclerosis, and Vascular smooth muscle cell contractility +
thrombosis
Vascular smooth muscle cell proliferation +
Intima-media-thickness +
Vasoconstriction +
Arterial stiffness +
Blood pressure +
Peripheral vascular resistance +
Left ventricular mass +
Local inflammation +
Oxidative stress +
Monocyte adhesion +
LDL oxidation +
Impaired vascular homeostasis

Atherosclerosis
Thrombosis
A large number of experimental and human studies have investigated the effects of
methylated arginines on established markers of cardiovascular homeostasis and
remodeling, e.g., endothelial function, intima-media thickness, arterial stiffness, periph-
eral vascular resistance, blood pressure, and left ventricular hypertrophy (Fig. 3).
Endothelial Function
Studies have shown that the plasma concentrations of ADMA and MMA are
independently associated with impaired endothelial function and endothelium-
dependent vasodilation (Boger et al. 1998). Although SDMA does not directly
inhibit eNOS activity, recent studies have reported independent associations between
SDMA concentrations and impaired endothelial function. The latter might be medi-
ated by indirect NO synthesis inhibition secondary to reduced arginine availability
(Memon et al. 2013).
Intima-Media Thickness
Plasma ADMA, SDMA, and MMA concentrations have shown positive and inde-
pendent associations with carotid intima-media thickness in patients with CKD or
ESRD (Zoccali et al. 2002a; Nanayakkara et al. 2005).
Arterial Stiffness
An acute increase in arterial stiffness after MMA administration has been demon-
strated both in animal and in human studies (Wilkinson et al. 2002). Population
studies investigating associations between methylated arginine concentrations and
measures of arterial stiffness have provided conflicting results. Some studies showed
independent associations between ADMA concentrations and arterial stiffness
(Schutte et al. 2010), whereas other studies did not (Chirinos et al. 2008).
Blood Pressure and Peripheral Vascular Resistance

Intravenous administration of ADMA and MMA has been shown to acutely increase
blood pressure and renal, mesenteric, and peripheral vascular resistance in animal
models (Gardiner et al. 1993). By contrast, SDMA administration was not associated
with significant changes in blood pressure (Veldink et al. 2013). Increased plasma
ADMA and SDMA, but not MMA, concentrations have been observed in hyper-
tensive subjects (Wang et al. 2009).
Left Ventricular Hypertrophy

Significant associations between serum ADMA concentrations and echocardio-
graphic measures of left ventricular hypertrophy have been demonstrated in CKD
patients as well as ESRD patients (Shi et al. 2010; Zoccali et al. 2002b).
Methylated Arginines and Other Cardiovascular Risk Biomarkers
From a pathophysiological point of view, it seems unlikely that a single biomarker,

or group of biomarkers, can fully account for the various molecular and biochemical
processes responsible for the onset and progression of atherosclerosis and its various
clinical manifestations. In this context, there is good evidence of associations
between methylated arginines and other established cardiovascular biomarkers,
e.g., homocysteine, C-reactive protein (CRP), low-density lipoprotein (LDL)
cholesterol concentrations, and insulin resistance. Notably, these biomarkers are
also significantly raised in CKD/ESRD patients and independently predict adverse
outcomes in this population.
Homocysteine
Positive associations between homocysteine, ADMA, and SDMA concentrations
have been demonstrated in human studies (Meinitzer et al. 2011). These associations
can be explained by a number of factors: (1) PRMTs utilize S-adenosylmethionine,
an intermediate in the methionine-homocysteine pathway, as a methyl donor for the
methylation of arginine residues in proteins (Vallance and Leiper 2004); and
(2) homocysteine inhibits DDAH activity in vitro (Wadham and Mangoni 2009).
CRP
Human studies have shown positive associations between ADMA and CRP concen-
trations in CKD patients, although other studies have not reported a significant
association (Zoccali et al. 2002a; Nanayakkara et al. 2005). Positive associations
between SDMA and CRP have been reported in hyperuricemia (Tenderenda-
Banasiuk et al. 2013). The inhibitory effect of pro-oxidative pathways on DDAH
activity might explain the positive associations between ADMA, but not SDMA, and
CRP (Wadham and Mangoni 2009).
Low-Density Lipoprotein (LDL) Cholesterol

Positive associations between ADMA and plasma LDL cholesterol concentrations
have been reported in ESRD patients undergoing peritoneal dialysis (PD) (Gocmen
et al. 2008). By contrast, no significant correlation between ADMA and LDL
cholesterol was observed in patients with ischemic heart disease and in patients
with polycystic ovary syndrome (Meinitzer et al. 2007). Positive associations
between SDMA and plasma LDL cholesterol concentrations have been reported in
patients with thyroid dysfunction (Arikan et al. 2007). Similarly to CRP, it is possible
that oxidized-LDL-induced pro-oxidative pathways might reduce DDAH activity
with consequent accumulation of ADMA, but not SDMA.
Insulin Resistance
There is good evidence of a strong, positive association between ADMA concentrations
and markers of insulin resistance in healthy subjects and in patients with rheumatoid
arthritis (Stuhlinger et al. 2002). By contrast, no relationship between ADMA and
insulin resistance was demonstrated in young patients with metabolic syndrome (Garcia
et al. 2007). Ethnic differences might explain, at least in part, the discrepancy in
the results (Reimann et al. 2007). Plasma ADMA, but not SDMA, concentrations
have been also shown to predict worsening of insulin resistance over time in
patients with ischemic heart disease (Surdacki et al. 2013). Hyperglycemia-induced
prooxidant pathways might reduce DDAH activity (Wadham and Mangoni 2009).
This phenomenon, similarly to CRP and LDL cholesterol, might explain the positive
associations between ADMA and insulin resistance.
Methylated Arginines and Adverse Clinical Outcomes in Patients

with Renal Disease
Several studies in CKD, ESRD, and post-kidney transplant patients have investi-
gated the predictive capacity and/or association between plasma methylated arginine
concentrations and adverse clinical outcomes (Table 2).
Dialysis-Associated Hypotension (DAH)

Two relatively small studies have investigated the association between methylated
arginines and DAH (Bergamini et al. 2004; Mangoni et al. 2008). In one study, both
pre-HD and post-HD ADMA, but not SDMA, concentrations were significantly
higher in DAH versus non-DAH patients (Bergamini et al. 2004) (Table 2). In
another study, pre-HD and HD-induced changes in SDMA, but not ADMA, con-
centrations independently predicted DAH (Mangoni et al. 2008). The limited avail-
able evidence suggests that higher concentrations of methylated arginines pre-HD,
and/or their acute HD-induced reduction, might increase the risk of DAH through
reduced inhibition of NO synthesis, with consequent vasodilatation and reduced
blood pressure. Further research is needed to corroborate this hypothesis in larger
study populations, including ESRD patients on PD.
Progression to ESRD
Three studies have investigated the impact of methylated arginines on the progression
to ESRD, HD, or PD, in patients with CKD (Ravani et al. 2005; Lajer et al. 2008;
Levin et al. 2014). Two studies demonstrated an independent effect of plasma ADMA
concentrations in predicting a further deterioration of renal function (Ravani
et al. 2005; Lajer et al. 2008). No associations between SDMA and progression to
ESRD were observed in one of these studies (Lajer et al. 2008). In another larger
study, ADMA did not independently predict progression to ESRD (Levin et al. 2014).
Notably, the duration of follow-up in this study was relatively short, 12 months,
compared with the first two studies, 27 months and 11.3 years, respectively.
Cardiovascular Events and Mortality

The impact of methylated arginines on the risk of fatal/nonfatal cardiovascular
events, e.g., myocardial infarction (MI), stroke, and peripheral arterial occlusive
events, in CKD/ESRD has been extensively studied. Eight studies demonstrated that
higher plasma ADMA concentrations significantly predict cardiovascular events in
this population (Lajer et al. 2008; Zoccali et al. 2001; Mallamaci et al. 2004, 2005;
Testa et al. 2005; Young et al. 2009; Tripepi et al. 2011; Ignjatovic et al. 2013).
Furthermore, two studies demonstrated an interaction effect between ADMA and
other biomarkers, i.e., eNOS genetic polymorphism, C-reactive protein, and brain
natriuretic peptide (Mallamaci et al. 2005; Testa et al. 2005). As a result of this
interaction, the predictive capacity was significantly improved when the biomarkers
were considered together, rather than separately. By contrast, one study did not
report significant associations (Zoccali et al. 2004), whereas another study did report
an inverse association between plasma ADMA concentrations and risk of cardio-
vascular events (Busch et al. 2006). Although the results of the study from Busch
et al. are intriguing, a mixed cohort of CKD, ESRD, and renal transplant patients was
considered in the analysis. This is potentially relevant as there is evidence that
ADMA and SDMA plasma concentrations significantly decrease in renal transplant
patients, particularly in the early post-transplant phase (Claes et al. 2014). Finally,
three studies have investigated whether SDMA plasma concentrations predict car-
diovascular events. However, none of them reported significant associations (Lajer
et al. 2008; Zoccali et al. 2001; Ignjatovic et al. 2013).
All-Cause Mortality
Eleven studies have investigated the predictive role of ADMA on all-cause mortality,
either as separate or composite end-point, in CKD/ESRD patients. Positive indepen-
dent associations between higher plasma ADMA concentrations and risk of all-cause
mortality were reported in ten studies (Ravani et al. 2005; Levin et al. 2014; Zoccali
et al. 2001; Mallamaci et al. 2004, 2005; Young et al. 2009; Tripepi et al. 2011;
Ignjatovic et al. 2013; Aucella et al. 2009; Lu et al. 2011). An interaction between
ADMA, C-reactive protein, and brain natriuretic peptide was also shown to signif-
icantly enhance the predictive capacity of each biomarker alone (Mallamaci
et al. 2005). By contrast, one study reported no significant associations between
ADMA and all-cause mortality (Lajer et al. 2008). Similarly to the studies on
adverse cardiovascular outcomes, four studies failed to report significant associa-
tions between plasma SDMA concentrations and all-cause mortality (Lajer
et al. 2008; Zoccali et al. 2001; Aucella et al. 2009).
Clinical Outcomes in Renal Transplant Patients

Two studies have specifically investigated the predictive role of methylated arginines
in renal transplant patients. A small study reported a significant association between
plasma ADMA concentrations and risk of acute rejection (Esposito et al. 2009).
Another larger study with a relatively long follow-up showed that plasma ADMA
concentrations independently predicted a number of adverse outcomes, including
graft failure, fatal and nonfatal cardiovascular events, and all-cause mortality
(Abedini et al. 2010).

Conditions
The published evidence supports the role of methylated arginines, particularly

ADMA, in predicting adverse clinical outcomes in CKD and ESRD patients. Higher
plasma ADMA concentrations were associated with an increased risk of
412
Table 2 Prospective studies on methylated arginines, morbidity, and mortality in renal disease. Studies investigating the capacity of methylated arginine
concentrations to predict morbidity and mortality in different cohorts of patients with renal disease. Information regarding the clinical and demographic
characteristics of the patient population, the type of methylated arginines measured, the duration of follow-up, the type of end-points assessed, and a summary of
the results is provided for each study
Methylated
arginines
References Population (determination) Follow-up End-points Results
(Zoccali ESRD on HD ADMA and 33.4
14.6 months CV events# HR (95 % CI) per 1 μmol/L increasea
et al. 2001) SDMA (HPLC)
N = 225 All-cause mortality* ADMA 1.17 (1.04–1.33)#
Age 59.9
15.1 years SDMA 1.00 (0.88–1.14)#
ADMA 1.26 (1.11–1.41)*
SDMA 1.06 (0.94–1.18)*
(Mallamaci ESRD on HD ADMA 42.3 months (range CV events# HR (95 % CI) per 1 μmol/L increaseb
et al. 2004) N = 224 (HPLC) 0.2–70.5) All-cause mortality* ADMA 1.19 (1.08–1.32)#
Age not provided for ADMA 1.22 (1.11–1.35)*
total population
(Zoccali ESRD on HD or PD ADMA 41
22 months CV events HR (95 % CI) per 1 μmol/L increase c
et al. 2004) N = 254 (HPLC) ADMA 1.06 (0.96–1.16)
Age not provided for
total population
(Bergamini ESRD on HD ADMA – DAH ADMA higher in DAH versus non-DAH
et al. 2004) patients (6.23
1.04
vs. 3.62
0.62 μmol/L, P < 0.05)
N = 20 SDMA SDMA not significantly different between
DAH and non-DAH patients
Age not provided for (HPLC) (7.21
1.02 vs. 6.30
0.72 μmol/L)
total population P < 0.05)
A.A. Mangoni et al.
18
(Mallamaci ESRD on HD or PD ADMA 34

16 months CV mortality# Predictive value addedd
et al. 2005) N = 246 (HPLC) All-cause mortality* ADMA: +3.3 %#
Age 60.2
15.3 years CRP + ADMA: +8.4 %#
CRP + ADMA + BNP: +10.5 %#
Predictive value addede
ADMA: +5.6 %*
CRP + ADMA: +9.0 %*
CRP + ADMA + BNP: +11.6 %*
(Ravani CKD ADMA 27.0 months (range Progression to HR (95 % CI) per 0.1 μmol/L increasef
et al. 2005) N = 131 (ELISA) 3.4–36.0) ESRD or all-cause ADMA 1.20 (1.07–1.35)
Age 71.1
11.0 years mortality
(Testa ESRD on HD or PD ADMA 42
22 months CV mortality HR (95 % CI) per 1.0 μmol/L increaseg
et al. 2005) N = 261 (HPLC) ADMA 1.20 (1.07–1.34)
Age not provided for HR (95 % CI) of eNOS T allele 1 +
total population ADMA >75th percentile versus eNOS T
allele 1 + ADMA <75th percentile
2.71 (1.38–5.35)
Methylated Arginines as Biomarkers in Renal Disease
(Busch CKD + ESRD on HD ADMA Median 24 months CV events# ADMA highest versus lowest quartile
et al. 2006) + post-kidney (range 1–52) 0.27 (0.10–0.71)h#
transplant
N = 200 SDMA Initiation or
Age 57.6
13.0 years (HPLC) re-initiation of HD* HR (95 % CI) per 1.0 μmol/L increasei
SDMA 1.63 (1.15–2.32)*
(continued)
413
414
Table 2 (continued)
Methylated
arginines
(Lajer T1DN ADMA 11.3 years (range Fatal and nonfatal HR (95 % CI) above versus below
et al. 2008) 0.0–12.9) CV events# medianl
N = 397 SDMA Decline in GFR* ADMA 2.05 (1.31–3.20)#
Age 42.1
10.5 years (HPLC) Progression to SDMA NS#
ESRD^
All- cause ADMA 0.9 (0.0–1.7)*
mortality SDMA NS*
ADMA 1.85 (0.99–3.46)^
SDMA NS^
ADMA NS
SDMA NS
(Mangoni ESRD on HD ADMA – DAH OR (95 % CI) per 0.10 μmol/L increasem
et al. 2008) N = 52 SDMA SDMA 1.31 (1.04–1.65)
Age 64.4
13.4 years (LCMS)
(Young CKD ADMA 9.5 years (range CV mortality# HR (95 % CI) per 0.25 μmol/L increasen
et al. 2009) N = 821 (ELISA) 0.2–11.6) All-cause mortality* ADMA 1.19 (1.00–1.42)#
Age 52
12 years ADMA 1.09 (0.99–1.26)*
(Aucella ESRD on HD or PD ADMA 56
28 months All-cause mortality HR (95 % CI) per 1.0 μmol/L increaseo
et al. 2009) N = 288 SDMA ADMA 1.92 (1.16–3.16)
Age 58
16 years (HPLC) SDMA NS
(Esposito Post-kidney transplant ADMA 6 months Acute rejection ADMA associated with acute rejection in
et al. 2009) N = 41 (ELISA) multivariate analysis (P < 0.05, variables
not provided)
A.A. Mangoni et al.
Age 49.9
12.9 years
18
(Abedini Post-kidney transplant ADMA 5–6 years GFDSC# HR (95 % CI)p

et al. 2010) N = 1,847 (HPLC) MACE* ADMA 2.78 (1.22–5.82)#
Age 30–75 years CV mortality or ADMA 2.61 (1.03–6.61)*
nonfatal MI^
Stroke ADMA 4.90 (1.70–14.10)^
All-cause mortality& ADMA 7.63 (2.52–23.13)
ADMA 4.87 (2.12–11.18)&
(Lu et al. 2011) CKD ADMA 2.9
1.2 years All-cause mortality, HR (95 % CI) per 0.1 μmol/L increaseq
nonfatal MI or stroke#
N = 298 (HPLC) All-cause mortality* ADMA 1.37 (1.09–1.73)#
Age 73
10 years ADMA 1.25 (0.94–1.66)*
(Tripepi ESRD on HD ADMA 156 months Fatal and nonfatal HR (95 % CI) per 1.0 μmol/L increaser
et al. 2011) CV events#
N = 225 (HPLC) All-cause mortality* ADMA 1.18 (1.07–1.30)#
Age 60
15 years ADMA 1.22 (1.12–1.34)*
(Ignjatovic ESRD on HD ADMA 3 years CV mortality# HR (95 % CI) per 1.0 μmol/L increases
et al. 2013) N = 153 SDMA All-cause mortality* ADMA 1.51 (0.99–2.28)#
Age not provided for (HPLC) SDMA 0.21 (0.03–1.48)#

total population ADMA 1.71 (1.32–2.21)*
SDMA 1.41 (0.56–3.56)*
(continued)
415
Table 2 (continued)
416
Methylated
arginines
(Levin CKD ADMA 12 months Progression to renal HR (95 % CI) per 0.11 μmol/L increaset
et al. 2014) replacement
therapy#
N = 2,544 (ELISA) All-cause mortality* ADMA 1.03 (0.88–1.20)#
Age 68.1
12.7 years ADMA 1.13 (1.01–1.29)*
CKD chronic kidney disease, ESRD end-stage renal disease, ADMA asymmetric dimethylarginine, SDMA symmetric dimethylarginine, CRP C-reactive protein,
BNP brain natriuretic peptide, HD hemodialysis, PD peritoneal dialysis, CV cardiovascular, HPLC high-performance liquid chromatography, HR hazard ratio,
CI confidence interval, DAH dialysis-associated hypotension, ELISA enzyme-linked immunosorbent assay, eNOS endothelial nitric oxide synthase, T1DN type
1 diabetic nephropathy, GFR glomerular filtration rate, LCMS liquid chromatography-mass spectrometry, NS nonsignificant, GFDSC graft failure or doubling of
serum creatinine, MACE major adverse cardiovascular events, MI myocardial infarction
a
Adjusted for arginine, SDMA, age, sex, previous cardiovascular events, systolic blood pressure, smoking, diabetes, total cholesterol, LDL cholesterol,
fibrinogen, C-reactive protein, homocysteine, hemoglobin, calcium phosphate product, albumin, duration of dialysis, fractional urea clearance
b
Adjusted for age, sex, smoking, diabetes, previous cardiovascular events, cholesterol, blood pressure, antihypertensive treatment, fractional urea clearance,
duration of dialysis, hemoglobin, albumin, calcium phosphate product, C-reactive protein, homocysteine, plasma norepinephrine
c
Adjusted for age, ejection fraction (or endocardial fractional shortening or mid-wall fractional shortening), previous cardiovascular events, smoking, dialysis
modality, antihypertensive treatment, circumferential end-systolic stress, systolic blood pressure, diabetes, albumin, C-reactive protein
d
Basic model including age, gender, smoking, diabetes, comorbidity score
e
Basic model including age, gender, smoking, diabetes, albumin, dialysis modality, comorbidity score
f
Adjusted for homocysteine, hemoglobin, proteinuria, glomerular filtration rate
g
Adjusted for endothelial nitric oxide polymorphisms, age, gender, smoking, previous cardiovascular events, diabetes, pulse pressure, albumin, C-reactive protein
h
Adjusted for hemodialysis, diabetes, age, body mass index, history of cardiovascular disease, albumin, C-reactive protein, hemoglobin, creatinine
i
Adjusted for renal transplant, age, hypertension, albumin, C-reactive protein, hemoglobin, creatinine, LDL cholesterol
l
Adjusted for sex, age, glycated hemoglobin, systolic blood pressure, glomerular filtration rate, cholesterol, smoking, previous cardiovascular events,
antihypertensive treatment
m
Adjusted for age, gender, baseline systolic blood pressure, hypertension, diabetes
n
Adjusted for age, gender, race, randomization assignment, history of cardiovascular disease, diabetes, smoking, diastolic blood pressure, LDL cholesterol, HDL
cholesterol, C-reactive protein, body mass index, glomerular filtration rate, proteinuria, cause of kidney disease
A.A. Mangoni et al.
o
18
Adjusted for diabetes, albumin, age, treatment modality, previous cardiovascular events, gender, duration of dialysis, hemoglobin, antihypertensive treatment,
smoking, homocysteine, systolic blood pressure, cholesterol
p
Adjusted for age, gender, systolic blood pressure, previous coronary artery disease, diabetes, LDL cholesterol, smoking, creatinine
q
Adjusted for age, gender, hypercholesterolemia, diabetes, glomerular filtration rate, hypertension, smoking
r
Adjusted for age, gender, smoking, diabetes, cholesterol, systolic blood pressure, cardiovascular comorbidities, antihypertensive treatment, duration of dialysis,
albumin, hemoglobin, calcium phosphate product, homocysteine, C-reactive protein, interleukin-6
s
Adjusted for age, gender, smoking, C-reactive protein, serum amyloid, albumin
t
Adjusted for age, gender, history of cardiovascular disease, glomerular filtration rate, serum phosphate, albumin
*
all-cause mortality, initiation of HD, decline in GFR, or MACE
#
CV events, CV mortality, GFDSC, all-cause mortality, nonfatal MI or stroke, progression to renal replacement therapy
^
progression to ESRD, CV mortality or nonfatal MI

all-cause mortality
&
all-cause mortality
417
cardiovascular morbidity and mortality and all-cause mortality in studies with

different population characteristics, renal disease etiology, and follow-up. The pre-
dictive capacity of ADMA in these studies was accounted for a number of clinical,
demographic, and biochemical variables associated per se with adverse outcomes in
this patient group. Therefore, plasma ADMA concentrations can independently
predict risk in renal disease patients. Some studies have also demonstrated that the
inclusion of other biomarkers, e.g., C-reactive protein, in ADMA-based statistical
models can further enhance risk prediction. By contrast, published studies failed to
demonstrate a predictive role for SDMA, except for one study demonstrating
independent associations with DAH and another study showing an increased risk
of undergoing HD in a mixed cohort of CKD, ESRD, and renal transplant patients
(Mangoni et al. 2008; Busch et al. 2006). Virtually no evidence is available on the
predictive role of MMA in renal disease patients.
Generalizability of Findings
Several potential issues might limit the applicability of the findings of published
studies on the prognostic role of methylated arginines in CKD/ESRD patients. First,
as previously discussed, the concentrations of methylated arginines reported in these
studies significantly depend on the technique used for their determination. Second,
the majority of studies recruited either middle-aged or older patients <80 years
(Table 2). As both CKD and ESRD affect pediatric patients and adolescents as well
as patients 80 years, further clinical studies are required to ascertain whether
plasma methylated arginines, particularly ADMA, have similar predictive capacity
in these age groups. Of note, age is a significant determinant of plasma methylated
arginine concentrations (Schwedhelm et al. 2009). Third, the available evidence has
been largely derived from white Caucasian populations. Recent studies have
highlighted significant differences in the concentrations of methylated arginines
and other arginine metabolites between different ethnic groups (Sydow
et al. 2010). This suggests an interaction between genetic polymorphisms and
environmental factors in modulating arginine metabolic pathways. Fourth, although
a limited number of studies failed to demonstrate a significant impact of SDMA in
CKD/ESRD patients, there is increasing evidence of its predictive role in other
related disease states (Meinitzer et al. 2011). Further larger studies in renal disease
patients should better clarify the role of SDMA, and MMA, in risk stratification.
Methylated Arginines and Other Disease States
There is increasing evidence of the predictive capacity of methylated arginines in

other disease states, particularly those related to the pathophysiology of atheroscle-
rosis and thrombosis. ADMA and SDMA have been associated with adverse out-
comes in patients with type 2 diabetes (Krzyzanowska et al. 2007) and ischemic
heart disease (Meinitzer et al. 2011).
Applications in Clinical Practice
For its routine use in clinical practice, a biomarker should possess several key
characteristics, e.g., (1) it should be easily measurable at population level by
means of robust and relatively inexpensive techniques; (2) it should be indepen-
dently associated with the risk of disease onset, progression, or outcomes in a
predictable fashion; and (3) it should be modifiable by means of pharmacological
and/or non-pharmacological interventions. The measurement of methylated argi-
nines plasma/serum concentrations can be reliably accomplished by means of HPLC
and LCMS methods. Although the determination of methylated arginines is cur-
rently limited to research, a widespread use in the clinical setting might be justified
on the base of cost-effectiveness. The latter would require further confirmation of the
role of methylated arginines in specific disease states as well as their capacity to
significantly enhance risk stratification and reclassification. Both in vitro and in vivo
studies have investigated the pharmacological modulation of methylated arginine
concentrations, particularly ADMA. There is evidence that some lipid-lowering
agents, angiotensin receptor blockers, and beta-blockers can enhance or restore
DDAH activity in experimental studies (Wadham and Mangoni 2009). Both
angiotensin-converting enzyme inhibitors and angiotensin receptor blockers have
been shown to reduce plasma ADMA concentrations in human studies (Delles
et al. 2002). However, other studies, also investigating the effects of beta-blockers,
have not confirmed these findings (Kelly et al. 2012). As both angiotensin-
converting enzyme inhibitors and angiotensin receptor blockers are the cornerstone
of management in patients with renal disease, further research is warranted to
establish whether their renoprotective effects are mediated by their modulatory
effects on methylated arginine concentrations.
Future Research Directions
Although there have been significant advances in knowledge regarding the patho-
physiological and prognostic role of methylated arginines in renal disease, the
available evidence has also identified important knowledge gaps. The latter should
encourage further investigations in this area. For example, relatively little is known
on the role of methylated arginines in predicting further deterioration in renal
function, particularly in CKD patients, and the risk of acute and post-acute renal
transplant rejection. The availability of data from published studies on the effect size
in both CKD and ESRD patients should allow a robust power sample size calculation
as well as the selection of appropriate clinical, demographic, and biochemical
confounders for multivariate analyses, in such further studies.
Additional in vitro and in vivo studies should investigate the interaction between
ADMA, MMA, and markers of NO synthesis. Although it is well established that
ADMA and MMA are competitive inhibitors of NOS enzymes, positive associations
between ADMA, MMA, and nitrite/nitrate concentrations have been reported in
ESRD (Bouteldja et al. 2013). This supports the concept of NO-mediated inhibition
of DDAH activity to prevent excessive NO synthesis, primarily by the inducible

NOS isoform iNOS, in the presence of a pro-inflammatory environment, such as
CKD or ESRD.
Another opportunity for further research is the assessment of the potential
enhancement of predictive capacity using combined versus single biomarkers.
Two studies have already provided evidence that adding either specific genetic
polymorphisms or biochemical surrogate markers can significant increase the pre-
dictive capacity of ADMA-based statistical models (Mallamaci et al. 2005; Testa
et al. 2005). The increasing availability of analytical platforms able to perform
combined measurements of one or more biochemical pathways from the same
biological sample offer a number of opportunities in this context.
Summary Points
• This chapter focuses on the biology and the pathophysiology of the endogenous
arginine analogues asymmetric dimethylarginine, symmetric dimethylarginine,
and monomethylarginine and their role in renal disease.
• Asymmetric dimethylarginine, symmetric dimethylarginine, and monomethy-
larginine inhibit nitric oxide synthesis either directly or indirectly.
• Experimental and human studies have shown that the kidney is an important
elimination site of methylated arginines.
• Plasma asymmetric dimethylarginine, symmetric dimethylarginine, and
monomethylarginine concentrations are increased in patients with different
degrees of renal disease.
• Plasma concentrations of methylated arginines, particularly asymmetric
dimethylarginine, independently predict the risk of adverse cardiovascular events
and all-cause mortality in patients with renal disease.
Acknowledgments A Visiting Professorship granted to Professor Mangoni by the Department of

Biomedical Sciences, University of Sassari (Italy), facilitated this work.
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Albuminuria as a Biomarker of the Renal
Disease 19
Visnja Lezaic
Contents
Key Facts of Kidney Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 428
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 429
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 431
Mechanisms of Albuminuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432
Values and Categories of Albuminuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 432
Methods of Albuminuria Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 434
Screening for Chronic Kidney Disease in the General Population . . . . . . . . . . . . . . . . . . . . . . . . . 436
Albuminuria as a Cardiovascular Disease Marker . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439
Albuminuria and Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 439
Link Between Albuminuria and Increased Cardiovascular and Renal Risk . . . . . . . . . . . . . . . 439
Albuminuria as a Target for Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
Not Resolved Questions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 440
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 441
Abstract
Albuminuria is an early and sensitive marker of kidney damage in diabetic
patients, a good predictor of kidney outcome and cardiovascular disease.
Screening for albuminuria is important to identify individuals at risk for renal
outcome, i.e., developing end-stage renal disease, acute kidney injury, and pro-
gressive chronic kidney disease as well as cardiovascular disease and all-cause
mortality in both general and high-risk population (diabetes, cardiovascular
disease, hypertension, and older patients). Also, it is the most widely used clinical
marker of diabetic nephropathy. The terminology of “early diabetic nephropathy”
indicated diabetic subjects with albuminuria. In this early phase of diabetic
V. Lezaic (*)
Faculty of medicine, University of Belgrade, Belgrade, Serbia
e-mail: mf.bg@med.bg.ac.rs; visnjalezaic@gmail.com

428 V. Lezaic
nephropathy, the glomerular filtration rate is usually well preserved. In patients with
several cardiovascular risks, albuminuria is predicting outcome, even below the
level to be taken as normal. It seems that the pathophysiologic mechanisms linking
albuminuria to cardiovascular and renal risk are generalized loss of vascular endo-
thelial function in organs. Furthermore, albuminuria can be used as a target for
treatment for primary and secondary prevention of renal and cardiovascular disease
development. ACE inhibition in subjects with nondiabetic albuminuria may prevent
future cardiovascular events. Measurement of albuminuria can help to determine
whether or not the patient with hypertension should be treated, how aggressively
they should be treated, and what medications we should treat them with.
Keywords
Albuminuria • Healthy subject • Diabetic nephropathy • Screening of chronic
kidney disease • Prediction of renal progression • Cardiovascular mortality •
Target for treatment • Hypertension
Abbreviations
ACR Albumin-to-creatinine ratio
RAAS Renin–angiotensin–aldosterone system
RRT Renal replacement therapy
UAER = UAE Urinary albumin excretion rate
Key Facts of Kidney Function
• Healthy kidneys regulate the body’s fluid levels and filter wastes and toxins from
the blood into the urine. Also, they release a hormone that regulates blood
pressure, activate vitamin D to maintain healthy bones, release the hormone
that directs production of red blood cells, and keep blood minerals in balance
(sodium, phosphorus, potassium).
• Patients at risk for kidney disease should have simple blood and urine tests to
check if their kidneys are working properly. These include diabetes, high blood
pressure, family history of kidney failure and being age 60 or older, kidney
stones, smoking, obesity, and cardiovascular disease.
• The glomerular filtration rate is a measure of kidney work to remove wastes from
the blood. To assess the glomerular filtration rate is necessary to determine the
concentration of creatinine in the serum. Reduction of glomerular filtration rate
and increased serum creatinine show renal failure.
• The presence of high albumin concentration in urine indicates renal damage most
probably at an early stage. The presence of high protein in the urine may indicate
the kidney disease.
19 Albuminuria as a Biomarker of the Renal Disease 429
• Part of routine health screening is urinalysis. Urine sample is collected from the
patient in a specimen cup (about 50 ml). It is evaluated by its physical appearance
(color, cloudiness, odor, clarity); macroscopic, chemical, and molecular proper-
ties; or microscopic assessment.
• Urine is used to diagnose a urinary tract or kidney infection, to evaluate causes of
kidney failure, and to screen for progression of some chronic conditions such as
diabetes mellitus and high blood pressure (hypertension).
• Patients with decreased glomerular filtration rate or proteinuria should be evalu-
ated to determine its cause(s).
Definitions
Acute kidney injury (AKI) Acute kidney injury (AKI) is the sharp loss of kidney
function, resulting in the retention of urea and other nitrogenous waste products and
in the dysregulation of extracellular volume and electrolytes.
Albumin-to-creatinine ratio (ACR) Expression of albuminuria from the ratio of

albumin to creatinine in urine, determined in individual sample of urine.
Angiotensin-converting enzyme inhibitors Are drugs that block the production of

angiotensin II. The latter is a hormone that circulates in the blood and has many
effects on the cardiovascular system; angiotensin II is a strong vasoconstrictor.
Angiotensin II receptor antagonists Are the drugs that modulate the renin–angio-
tensin–aldosterone system by blocking angiotensin II receptors.
Chronic kidney disease (CKD) Chronic kidney disease, also called chronic kid-
ney failure, describes the gradual loss of kidney function. Its stages are based on the
patient’s level of glomerular filtration rate (GFR) which is a measure of filtering
capacity of the glomeruli.
Diabetic nephropathy Is a slow progressive kidney disease that occurs in patients

with diabetes.
End-stage renal disease (ESRD) Means glomerular filtration rate below 15 ml/
min/1.73 m2.
Estimated glomerular filtration rate (eGFR) Describes how much fluid filtered
through the kidney. It is usually measured with creatinine clearance rate: it is the
volume of plasma that is cleared of creatinine per unit time. Nowadays, glomerular
filtration rate is estimated using different formulas based on creatinine or other
markers such as cystatin C.
430 V. Lezaic
Framingham score for assessment of cardiovascular risk It gives an estimate of

the probability that a person will develop cardiovascular disease within a specified
amount of time, usually 10–30 years. It also indicates who is most likely to benefit
from prevention.
Glomeruli and tubule Both represent part of the nephron, the basic structural and
functional unit in the kidney.
Immunochemistry assays (Immunonephelometry, immunoturbidimetry, radioim-

munoassay, enzyme-linked immunosorbent assay) Are based on the interaction of
the urinary albumin with anti-albumin antibodies in the reagents.
Insulin resistance A decreased sensitivity to the action of insulin. Conditions in

which the increased amount of insulin is inadequate to induce normal insulin
responses in insulin-sensitive tissues (liver, skeletal muscles, adipose tissues).
Likelihood ratios Tell us how much we should shift our suspicion for a particular
test result. The “positive likelihood ratio” (LR+) tells us how much to increase the
probability of disease if the test is positive, while the “negative likelihood ratio”
(LR) tells us how much to decrease it if the test is negative.
Nonsteroidal anti-inflammatory drugs It is a group of medication with antipy-

retic, analgesic, and anti-inflammatory effects.
Progression of chronic kidney disease Worsening of kidney function, usually

slow, which is determined by measuring glomerular filtration rate and albuminuria
and/or proteinuria.
Renal replacement therapy (RRT) Refers to the three ways of replacing the lost
kidney function: dialysis (hemo- or peritoneal) and kidney transplant.
Renin–angiotensin–aldosterone system Is a hormone system that regulates blood

pressure and water balance in the body.
Sensitivity vs. specificity Sensitivity (the true positive rate) measures the propor-
tion of actual positives which are correctly identified (the percentage of sick
people who are correctly identified as having the condition). Specificity (the true
negative rate) measures the proportion of negatives which are correctly identified
(the percentage of healthy people who are correctly identified as not having the
condition).
Type 1 diabetes mellitus It is a type of glucose control disturbance that results from
the autoimmune destruction of insulin-producing cells in the pancreas. There is
absolute lack of insulin. Type 2 diabetes mellitus It is characterized by hyperglyce-
mia due to insulin resistance and relative lack of insulin.
Urinary albumin excretion (UAE) The presence of albumin in the urine as a

consequence of high permeable glomerular membrane and inhibition of tubular cell
reabsorption.
Introduction
Albuminuria refers to abnormal loss of albumin in the urine. Albumin is one fraction of
plasma protein found in the urine in normal subjects but in larger quantity in patients
with kidney disease. Although the measurement of urinary proteins has been a standard
tool for nephrologists to diagnose kidney diseases for more than two centuries, the
introduction of novel methods in 1980s enabled the measurement of small quantities of
albumin in the urine. This triggered a series of investigations showing that increased
urinary albumin excretion was an early and sensitive marker of diabetic nephropathy
(Viberti 1982; Parving 1982), and an independent risk factor for cardiovascular disease
in patients with hypertension and diabetes, cardiovascular and peripheral vascular
disease (Parving 2001; Heart Outcomes Prevention Evaluation (HOPE) Study Inves-
tigators 2000) and in the general population (Hillege 2002; Romundstad 2003).
All these statements derived from the screening studies, which have shown
that there are signs of kidney disease in apparently healthy individuals (Kiberd
2006; Levey 2007). Thus renal disease became epidemic worldwide. The overall
prevalence of albuminuria in the general population has been reported to be 7.2 %
in the PREVEND cohort from the Netherlands (n = 40,548) (Hillege 2002) and
10.7 % in the more racially diverse United States National Health and Nutrition
Examination Survey (NHANES III) study (n = 15,939) (Mattix 2002). About
8 % of adults have microalbuminuria (30–300 mg of albumin per 24 h), and 1 %
have macroalbuminuria (i.e., excretion of more than 300 mg of albumin per 24 h).
Furthermore, albuminuria was detected in one of every three persons with
diabetes, one of every seven persons with high blood pressure but no diabetes,
and one of every six persons older than 60 years (Collins 2009; Levey 2009).
Also, assessment of urinary albumin excretion rather than plasma creatinine or
estimated glomerular filtration rate (eGFR) seems the utmost tool for recognizing
the chronic kidney disease especially in the early stages (Brantsma 2008; de Jong
2008; van der Velde 2009). Finally, in the majority of guidelines, albuminuria as
a marker of kidney damage joined the glomerular filtration rate (GFR) as a
measure of kidney function for definition of chronic kidney disease stages in
order to evaluate chronic kidney disease (CKD) (National Kidney Foundation
2002; NICE clinical guideline 73 http://guidance.nice.org.uk/cg73, American
Diabetes Association 2014).
In this review, the etiology and physiologic mechanisms of albuminuria in renal
disease are highlighted. Also, clinical impact of albuminuria as a factor in renal
disease compromises and establishes the link toward cardiovascular diseases, dia-
betes, and hypertension is shown. Therapeutic possibilities influencing on albumin-
uria and consequences of this are shown, too. Finally, a few dilemmas will be set up
as a proposal for further investigation.
432 V. Lezaic
Mechanisms of Albuminuria
Mechanisms underlying increased urinary albumin excretion are complex. Plasma

albumin filtered in the glomeruli is considered to be the major source of urinary
albumin. Most albumin passing (intact-unprocessed albumin) through the glomer-
ular membrane is reabsorbed by proximal tubular cells (known as the retrieval
pathway). The small amount of albumin not taken up by this pathway is destined for
excretion through the degradation pathway in tubular cell lysosomes. Thus, normal
protein excretion in humans is now recognized to involve the excretion of 1–3 g/day
of albumin-derived fragments in combination with less than 25 mg/day of intact
albumin (Greive 2001). In pathological states, the glomeruli may become increas-
ingly permeable to circulating albumin due to disturbances in endothelial cell
function, basement membrane abnormalities, or podocyte disorders. In addition,
inhibition in proximal tubule reabsorption of filtered albumin can also contribute to
albuminuria (Russo 2009).
Data suggest that the excreted albumin itself and/or bound ligands, such as fatty
acids, initiate a series of events that eventually leads to fibrosis (Chen 2000; Thomas
2002; Arici 2002). In vitro studies have shown that albumin induces changes in tubular
cells stimulating the expression of inflammatory and fibrogenic mediators (Wang
1997; Tang 2003; Zoja 1995; Yard 2001). Although inflammation appears to be an
important pathogenic factor in a number of renal diseases characterized by albumin-
uria, the possible pathogenic role of albumin itself is not yet fully elucidated in humans.
Values and Categories of Albuminuria
Previously, the presence of albumin in the urine is called microalbuminuria. This

name was changed in albuminuria, because this is not a small molecule of plasma
albumin, but a low quantity of albumin present in the urine that could not be detected
by standard dipstick test. The current definition of proteinuria, albuminuria, and
microalbuminuria is shown in Table 1. Lately, albuminuria is categorized into the
following stages: normal or <30 mg albumin/g creatinine (<3.4 mg/mmol), moder-
ately increased (formerly called microalbuminuria) from 30 to 299 mg/g (3.4–34.0 mg/
mmol), and severely increased (formerly called macroalbuminuria or nephrotic range)
at 300 mg/g (>34.0 mg/mmol) (Levey UpToDate last visit 2015) (Table 2).
Urinary albumin excretion (UAE) can be measured in the urine collected during
24 h or overnight (8–12 h). As UAE changes throughout the day, the amount of
albumin excreted over 24 h has been considered the “gold standard.” Nevertheless,
Table 1 The terminology and the difference between albuminuria and microalbuminuria
Proteinuria Abnormal excretion of protein by the kidney including any or all proteins
excreted
Albuminuria Abnormal excretion rate of albumin
Microalbuminuria Abnormally increased excretion rate of albumin in the urine in the range of
30–299 mg/g creatinine
Table 2 Categories of albuminuria

UAER Overnight ACR ACR
Categories mg/24 h UAER μg/min mg/mmol mg/g
Normal <30 <20 <3 <30
Male: <2.5 <17
Female: <3.5 <25
Moderately increased (previous 30–300 20–200 3–30 30–300
name microalbuminuria)
Severe increased (previous name >300 >200 >30 >300
macroalbuminuria)
Derived from KDIGO (2012) Clinical Practice Guideline for the Evaluation and Management of
Chronic Kidney Disease 2013; Joint Specialty Committee of the Royal College of Physicians of
London and the British Renal Association (2005, 20–3; Mattix 2002; and The CARI guidelines.
Caring for Australasians with Renal Impairment. Sydney: Australian and New Zealand Society of
Nephrology http://www.cari.org.au/guidelines.php
Levin 2008 http://www.cmaj.ca/cgi/data/179/11/1154/DC1
UAER urinary albumin excretion rate, ACR albumin-to-creatinine ratio
24-h urine collection is a cumbersome procedure. In daily practice albumin can be

determined in any urine sample: first morning urine specimens or random spot urine
collections. In such cases, its excretion can be estimated by measurement of the
albumin alone or albumin-to-creatinine ratio (ACR). Comparisons among all albu-
minuria measurements and 24-h albuminuria showed that albuminuria from first
morning voids is a more reliable alternative to 24-h urinary albumin excretion than
spot urine samples for diagnosis of albuminuria and to monitor it over time. If it is
decided to collect the first morning sample, then measurement of the ACR is to be
preferred over urinary albumin concentration alone (Witte 2009; Jafar 2007).
The cutoff value that traditionally indicates the presence of albuminuria is considered
to be albumin excretion equal or greater than 30 mg/24 h. For the diagnosis of kidney
disease, guidelines suggest diverse values as the limit of normal albuminuria (Table 2).
Some of them recommend different threshold values depending on the individual’s sex,
so albuminuria is defined as an ACR greater than 2.5 g/mmol creatinine for men and
3.5 g/mmol creatinine for women (Joint Speciality Committee of the Royal College of
Physicians of London and the British Renal Association. Guidelines for identification,
management and referral of adults with chronic kidney disease. London: Department of
Health for England 2005;20–3. http://www.rcplondon.ac.uk/pubs/books/kidney/), or
17 mg/g creatinine for men and 25 mg/g creatinine for women (Mattix 2002), while a
threshold value for albuminuria of 30 mg/g (3.4 mg/mmol) regardless of the sex of the
patients is recommended by others (National Kidney Foundation 2002 http://www.
kidney.org/professionals/KDOQI/guidelines_ckd/toc.htm
The CARI guidelines. Caring for Australasians with Renal Impairment. Sydney:
Australian and New Zealand Society of Nephrology n.d.. http://www.cari.org.au/
guidelines.php Levin 2008 http://www.cmaj.ca/cgi/data/179/11/1154/DC1). Indi-
vidual laboratories express the ACR in milligrams of albumin per gram of creatinine,
while others use albumin in milligrams per millimole of creatinine, so specified limit
values are expressed in several units.
434 V. Lezaic
There are some limitations that must be considered to maximize the reliability of
the ACR. Many conditions can cause a false-positive value for albuminuria. Thus,
urine samples should not be collected during: marked acute hyperglycemia; urinary
tract infection; marked hypertension; congestive cardiac failure; heavy exercise (due
to increased protein catabolism and altered renal circulation); fever, immediately
after surgery or after an acute fluid overload; and contamination with seminal or
menstrual fluid (which contains more albumin). Also, the value of the ACR depends
on the rate of urinary creatinine excretion that, in turn, reflects interindividual
differences in muscle mass. Therefore, persons with low muscular mass often have
the moderate ACR elevation used to define albuminuria in the absence of a true
elevation in absolute albuminuria. That contributes to the difference in values for
albuminuria between men and women (Joint Speciality Committee of the Royal
College of Physicians of London and the British Renal Association. Guidelines for
identification, management and referral of adults with chronic kidney disease.
London: Department of Health for England 2005, 20–3. http://www.rcplondon.ac.
uk/pubs/books/kidney/, Mattix 2002).
Furthermore, due to variability in urinary albumin excretion, two out of three
specimens collected within a 3- to 6-month period should be abnormal before
considering a patient to have developed increased UAE or to have exhibited
progression in albuminuria.
Methods of Albuminuria Assessment
Collection of the urine sample is very important when albuminuria is measured, as

many factors can alter the value and errors may occur due to inadequate aseptic
precautions or improper storage and handling. After collection it is preferable to
measure on the same day, but if urine albumin is not estimated immediately, then the
urine can be stored at 4 C. Specimens are stable for at least 2 weeks at 4 C and
5 months at 70 C. Freezing samples may decrease the albumin result but mixing
immediately before assay eliminates this effect (David et al. 1999).
There are two types of assay used for assessment of albuminuria: colorimetric test
strips (semiquantitative) and immunochemistry-based assays (quantitative) (Table 3).
Traditionally, detection of albuminuria starts with the dipstick test. These test strips
have been used in some countries for screening program of kidney damage (Iseki
Table 3 Methods of albuminuria assessment

Semiquantitative colorimetric assays
Dipstick test: Micral microalbumin urine test strip, CLINITEK Microalbumin
Quantitative immunochemistry-based assays
Immunoturbidimetry
Nephelometry
Radioimmunoassay (RIA)
Chemiluminescent immunoassay (CLIA)
Enzyme-linked immunosorbent assay (ELISA)
Derived from Martin (2011)
1996; Ležaić 2011). Some disadvantages are recognized. The test is semiquantitative
and insensitive for reliable detection of albumin in concentration ranges around
300 mg/day. Furthermore, concentrated urines may give a color change in the positive
range of a reagent strip device even though protein loss remains normal and vice
versa. False-positive results may occur if the urine is alkalinized (e.g., due to urinary
tract infection) or in the presence of quaternary ammonium compounds that alter the
urine pH. The performance of reagent strips is operator dependent (Rumley 2000) and
affected by the presence of colored compounds such as bilirubin and certain drugs
(e.g., ciprofloxacin, quinine, and chloroquine) (Scotti da Silva-Colombeli 2007).
Immunochemical assays (immunonephelometry, immunoturbidimetry, radioim-
munoassay, enzyme-linked immunosorbent assay) are based on the interaction of
urinary albumin with anti-albumin antibodies in the reagents. With these methods it
is possible to detect very small concentrations of albumin in the urine. Each method
has advantages and disadvantages, and the choice depends on local experience and
technical support. All methods have similar precision, sensitivity, and range. Cur-
rently urinary albumin is predominantly measured in diagnostic laboratories using
immunoturbidimetric assays (Martin 2011). Radioimmunoassay (RIA) and chemi-
luminescent immunoassay (CLIA) are highly sensitive, specific, and reproducible
methods. Disadvantages are unavailability, the cost factor, proper infrastructure
needed, and radioactive hazards (Agarwal 2002).
Measuring albumin in the urine is complex, especially as multiple species of
intact albumin (immunoreactive albumin and immuno-unreactive albumin) and
albumin-derived fragments have been reported (Comper 2003). Albumin fragments
are not detected by conventional immunoassays commonly used to measure urinary
albumin. At present there is no diagnostic tool available to measure albumin
fragments in urine routinely.

Conditions
Albuminuria (ACR more than 3 mg/mmol) is a marker for kidney disease in

apparently healthy subjects as well as in patients with different comorbidities such
as diabetes, hypertension, or obesity and is widely used in clinical practice (Chronic
kidney disease: early identification and management of chronic kidney disease in
adults in primary and secondary care. NICE guidelines [CG182] Published date: July
2014). It may precede the appearance of type 2 diabetes mellitus, being present in the
insulin resistance syndrome. Also albuminuria is considered to be a marker of
cardiovascular risk in the general population (Mykkanen 1998; MacIsaac 2004;
Thomas 2011). In addition, albuminuria has predictive significance, i.e., the higher
the albuminuria the higher the risk for mortality (cardiovascular and all cause),
progression of chronic kidney disease (CKD), and end-stage renal disease (ESRD)
independent of eGFR as a measure of kidney function. No apparent threshold value
was found in the general population and a population at high risk for kidney disease
(Matsushita 2010; Gansevoort 2011; van der Velde 2011). All this derives from the
results of large studies conducted over the last three decades.
436 V. Lezaic
Screening for Chronic Kidney Disease in the General Population
Screening for chronic kidney disease in the general population is usually performed
using eGFR and albuminuria. Recent data have shown a positive correlation between
UAE and rate of decline in eGFR, i.e., patients with higher levels of UAE had a more
rapid decline in eGFR, especially beyond a UAE of above 150 mg/24 h. The average
rate of renal function decline in the UAE category above 300 mg/24 h was four times
more rapid than that for UAE less than 15 mg/24 h (van der Velde 2009). In Table 4
the predictive cutoff albuminuria values for renal replacement therapy (RRT) start
during 6 years of follow-up was shown. During follow-up a UAE concentration
above 20 mg/L identified individuals with the risk of RRT with 58 % sensitivity and
92 % specificity. The likelihood ratio of a positive test result showed that an
individual at risk, i.e., with a history of hypertension or diabetes and a UAC
>20 mg/L, has 24.3 times the previous odds to start RRT during follow-up; for a
UAC >200 mg/L, this odds to start RRT is 118.7. These data suggest that screening
for albuminuria might be effective to identify individuals at risk for developing
ESRD (van der Velde 2009).
Albuminuria is a risk factor not only for ESRD but also acute kidney injury and
progressive CKD in both general and high-risk populations (Gansevoort 2011).
Hazard ratios for renal outcomes at different ACR presented in Table 5 show
similarity between general population and high-risk cohorts. The patterns for
ESRD were less steep in the high-risk cohorts compared with the general population,
whereas the patterns for acute kidney injury and progressive CKD were similar in the
general population cohorts and high-risk cohorts. These associations are indepen-
dent of other cardiovascular risk factors (Gansevoort 2011) (Table 6).
Lower eGFR and higher albuminuria were each independently associated with
mortality and ESRD as it was shown in the meta-analysis of 13 cohorts, including
21,688 individuals selected because of CKD (Astor et al. 2011). This risk increased
progressively with every higher level of albuminuria: an eightfold higher ACR was
Table 4 Various cutoff values of albumin excretion rate in a single first morning void urine to
identify individuals who started RRT during follow-up
AER category mg/L Sensitivity % Specificity % PPV % NPV % LR+ LR
General population
>20 58 92 0.8 99.9 7.43 0.46
>200 36 99 5.7 99.9 54.44 0.65
With known HTA/DM
>20 44 98 2.6 99.9 24.3 0.57
>200 31 99 11.6 99.9 118.7 0.69
Age >55 years
>20 44 96 1.3 99.9 12.1 0.58
>200 27 99 7.1 99.9 68.9 0.74
Derived from van der Velde (2009)
AER albuminuria excretion rate, LR+ likelihood ratio of a positive test, LR likelihood ratio of a
negative test, NPV negative predictive value, PPV positive predictive value
HTA arterial hypertension, DM diabetes mellitus
Table 5 Pooled hazard ratios for renal outcomes, i.e., end-stage renal disease, acute kidney injury,
and progressive chronic kidney disease by albuminuria categories for the general and high-risk
population
Albumin-to-creatinine ratio Albumin-to-creatinine ratio
30–299 mg/g 300 mg/g
General population
ESRD 12 (7.9–18.1) 72.1 (64.3–121)
AKI 2.5 (1.7–3.7) 6.0 (4.5–8.0)
Progressive CKD 3.1 (2.5–3.8) 11.2 (5.8–21.5)
High-risk patients
ESRD 4.3 (2.6–7.1) 38.1 (15.6–93.5)
AKI 2.7 (2.2–3.4) 7.4 (5.5–9.8)
Progressive CKD 2.2 (1.9–2.7) 9.9 (6.7–14.5)
Derived from Gansevoort (2011)
Pooled adjusted hazard ratios (95 % confidence interval) for ESRD and acute kidney injury and
pooled adjusted odds ratios (95 % confidence interval) for progressive CKD
ESRD end-stage renal disease, AKI acute kidney injury, CKD chronic kidney disease
Table 6 Adjusted hazard ratio (95 % confidence interval) for mortality, by albuminuria category
HR 95 % confidence interval
Albumin-to-creatinine ratio, mg/g
30–299 1.5 1.28–1.75
300–999 1.85 1.08–3.16
>1,000 2.7 1.74–4.26
Estimated glomerular filtration rate, ml/min/1.73 m2
30–44 1.35 1.23–1.49
15–29 2.25 1.81–2.79
<15 3.74 2.69–5.20
Derived from Astor et al. (2011)
Adjusted for age, sex, race, previous cardiovascular disease, smoking status, diabetes mellitus,
systolic blood pressure, and serum total cholesterol concentration
associated with an estimated 40 % higher risk of death and an estimated threefold

higher risk of ESRD after adjustment for eGFR (Astor et al. 2011).
For evaluation of chronic kidney disease, albuminuria as a marker of kidney
damage is combined with eGFR as a measure of kidney function. Both markers were
used in the classification of chronic kidney disease (CKD) (National Kidney Foun-
dation 2002; NICE clinical guideline 73 http://guidance.nice.org.uk/cg73, American
Diabetes Association. Standards of Medical Care in Diabetes 2014). That is why
regular screening for albuminuria is of utmost importance, especially at stages 1 and
2 of CKD, where therapeutic interventions may be of benefit to prevent or delay the
development of renal injury.
Albuminuria is the most widely used clinical marker of diabetic nephropathy and
has been recognized as a predictor of progression to ESRD in both type 1 and type
2 diabetes (Perkins 2010; Adler 2003). It is one of the earliest markers of diabetic
438 V. Lezaic
Categories of albuminuria
< 30 30-300 >300
0
Rate of change of eGFR
–1
–2
ml/min/1.73m 2
–3
–4
–5
–6
–7
Fig. 1 Comparison of the rate of the change in estimated glomerular filtration rate (eGFR)
monitored at least 5 years, adjusted for age and baseline eGFR in diabetic women and men, and
classified according to the average urinary albumin excretion at baseline. White (women) and gray
(men) bars; *p < 0.001 versus normal albuminuria value (Derived from Babazono (2009))
nephropathy that precede severely increased albuminuria (>300 mg/24 h). The
impact of elevated UAE in predicting future renal function loss in subjects with
diabetes was emphasized in the 1980s (Mogensen 1986; Parving 1982). During
these years, the terminology of “early diabetic nephropathy” was introduced to
indicate diabetic subjects with albuminuria. In this early phase of diabetic nephrop-
athy, eGFR is usually well preserved. Early nephropathy contrasts with the later
phase of overt nephropathy in which albuminuria increases into overt proteinuria and
the eGFR falls below 60 ml/min finally progressing to the level of ESRD. Docu-
mentation of the early phase of kidney damage with albuminuria but still normal
eGFR has helped us to understand better the impact of albuminuria during the loss of
renal function in diabetes. Moreover, it led to the demonstration that early interven-
tion by interfering in the renin–angiotensin–aldosterone system (RAAS) slows the
progression of nephropathy in diabetic patients (Parving 2001; Ruggenenti 2004).
The changes between these albuminuria states represent a hallmark of disease
progression or regression (American Diabetes Association 2014). More recent
studies have shown that an increase in albuminuria, even within the range that is
currently considered normal, indicates higher renal risk (Babazono et al. 2009;
Gansevoort 2011). In patients with type 2 diabetes monitored for at least 5 years,
higher albuminuria at baseline was associated with a faster decline in renal function
(Fig. 1), according to the data of Babazono T and coworkers’ study (Babazono
2009). Importantly, although within the normal range, it was calculated that albu-
minuria of 10 mg/g in women or 5 mg/g in men was associated with a
significantly greater rate of renal function decline (Babazono 2009). Not only the
albuminuria level itself but also changes in albuminuria (ACR within 30–300 mg/g)
over time predict renal or cardiovascular risk changes. In patients with type 2 diabetes
and ACR 30–300 mg/g, those subjects in whom albuminuria decreased by more than
50 % over 2-year follow-up had a subsequent renal function decline of 1.8 ml/min per
year. In contrast, in subjects without a 50 % reduction in albuminuria, long-term renal
function decline was significantly greater, being 3.1 ml/min per year (Araki 2007).
During the 1990s, there was growing evidence that albuminuria is important for
progressive reduction of renal function in nondiabetic renal disease. The findings
from large clinical trials showed the beneficial effect of lowering albuminuria by
using agents that interfere with the RAAS to prevent progression to ESRD (The
Gruppo Italiano di Studi Epidemiologici in Nefrologia (GISEN) Group 1997; Jafar
2003). This focused attention on treatment of renal patients for albuminuria and not
only for high blood pressure (De Jong 1999).
Albuminuria as a Cardiovascular Disease Marker
Some evidence suggested that high albuminuria is associated with cardiovascular

risk. Three large studies conducted in the general population showed that high
albuminuria predicts cardiovascular risk (Hillege 2001; Romundstad 2003; Yuyun
2004). The predictive power of albuminuria on cardiovascular risk is independent of
other known cardiovascular risks. Hence the idea that albuminuria should be com-
bined with Framingham scores for assessment of cardiovascular risk is suggested as
a primary prevention strategy with higher efficiency (Asselbergs 2004b). In patients
with several cardiovascular risk factors, albuminuria predicts outcome, even below
the level taken as normal (Mann 2004).
Albuminuria and Hypertension
Albuminuria may not only be the consequence of but it may also precede the
development of hypertension (Forman 2008; Scheven 2013). Albuminuria is found
in 11–40 % of persons with hypertension, the prevalence increasing with age and
the duration of hypertension (Rossa 2000; Özyilmaz 2013). According to the
PREVEND study results, in subjects with elevated albuminuria and newly discov-
ered hypertension or hypercholesterolemia, the cardiovascular risk exceeded by
20 % the risk in normoalbuminuric hypertensive patients. Thus, detecting albumin-
uria might also help the clinician decide when to initiate antihypertensive therapy. In
addition, identification of target organ damage is an indication for treatment in
patients with lower blood pressure (European Society of Hypertension-European
Society of Cardiology guidelines for the management of arterial hypertension 2003).
Link Between Albuminuria and Increased Cardiovascular

and Renal Risk
The pathophysiological mechanisms linking albuminuria to cardiovascular and renal

risk are still not fully understood, but much evidence appears to connect any level of
440 V. Lezaic
UAE to generalized loss of vascular endothelial function in many organs including

kidneys (Solbu 2009; Foster 2008).
Albuminuria as a Target for Treatment
Albuminuria can be used as a target for treatment for primary and secondary
prevention of renal and cardiovascular disease development. Previous studies
showed that the degree of albuminuria reduction was associated with a more
beneficial renal outcome in long-term follow-up (Apperloo 1994; Rossing 1994).
Therefore, several measures were introduced in clinical practice in order to reduce
albuminuria, such as dietary protein restriction (El Nahas 1984), nonsteroidal anti-
inflammatory drugs (Vriesendorp 1986), angiotensin-converting enzyme inhibitors,
and angiotensin II receptor antagonists (Gansevoort 1994). Thus far, no trials have
shown that lowering albuminuria in the early phase (i.e., microalbuminuria with a
GFR >60 ml/min, namely, stage 1 or 2 CKD) slows the progressive decline of renal
function. On the other side, there is some evidence that ACE inhibition in subjects
with nondiabetic albuminuria may prevent future cardiovascular events (Asselbergs
2004). Also, measurement of albuminuria can help to determine whether or not a
patient with hypertension should be treated, how aggressive should the therapy be,
and what medications should be used (European Society of Hypertension-European
Society of Cardiology guidelines for the management of arterial hypertension 2003).
Not Resolved Questions
Although the strong association between age and kidney function is well established,
the clinical significance of CKD in older asymptomatic people remains disputable.
In addition, it is unclear what portion of this decline is due to the higher prevalence of
risk factors for kidney disease at older ages, such as hypertension, diabetes, and
vascular disease. It seems to be worth investigating:
• The cutoff value of albuminuria that contribute to the accurate diagnosis of CKD
in the elderly
• The introduction of standardized laboratory tests that will clearly separate the
whole molecule, antibodies’ recognizable molecules, and fragments of albumin in
the urine
Summary Points
• This chapter focuses on albuminuria: definition, classification, importance in

renal disease detection, prognostic significance, mechanism of occurrence, and
methods of estimation.
• Currently, albuminuria categorizes into the following stages: normal, less than
30 mg/g (<3.4 mg/mmol); moderately increased, 30–299 mg/g (3.4–34.0 mg/
mmol); and severely increased albuminuria, 300 mg/g (>34.0 mg/mmol).
• Albuminuria is a marker for kidney disease in apparently healthy subjects and in
patients with different comorbidities.
• Screening for albuminuria is important to identify individuals at risk for devel-
oping end-stage renal disease, acute kidney injury, and progressive chronic
kidney disease, as well as cardiovascular disease and all-cause mortality in both
general and high-risk population (diabetes, cardiovascular disease, hypertension,
and older patients).
• Albuminuria is the most widely used clinical marker of early stage of diabetic
nephropathy when the glomerular filtration rate (GFR) is usually well preserved.
• The predictive power of albuminuria on cardiovascular risk is independent to
other known cardiovascular risks.
• Albuminuria can be used as a target for treatment for primary and secondary
prevention of renal and cardiovascular disease development.
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Serum Cystatin C as a Biomarker
20
Serap Çuhadar
Contents
Key Facts of Renal Biomarker Cystatin C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 446
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447
Cystatin Superfamily . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 447
Structure of Cys C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 449
Assay Interferent Factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Reference Range . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Cys C and Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Cys C and Pregnancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
Factors Affecting Cys C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
Advantages of Cys C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
Disadvantages of Cys C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
Cys C and GFR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
Cys C and Cirrhosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
Cys C and Malignancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
Amyloid Angiopathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
Abstract
Cystatin C is a low molecular weight cationic protein produced by all nucleated cells
which is a potent cysteine protease inhibitor. Its plasma concentration is proportional
with glomerular filtration as it is synthesized at a constant rate, freely filtered through
the glomerulus, and largely reabsorbed and catabolized in the proximal renal tubule
with no tubular secretion which makes it ideal for GFR estimation. This protein has a
S. Çuhadar (*)
Department of Medical Biochemistry, Ataturk Training and Research Hospital, Izmir, Turkey
e-mail: drcuhadar@gmail.com; sdcuhadar@yahoo.com

446 S. Çuhadar
capability to detect early renal failure as it gives reliable GFR estimation at the
critical level of 60 ml/min/1.73 m2. Though cystatin C is superior to serum creati-
nine, non-standardization and several clinical situations such as thyroid dysfunction
and the use of high doses of glucocorticoid limit its acceptance as a GFR marker to
replace creatinine, the current endogenous gold standard biomarker.
Keywords
Creatinine • Cystatin C • Cystatins • Cysteine proteases • Glomerular filtration
rate • Kidney diseases • Renal functions
Abbreviations
Ahsg Alpha 2HS glycoprotein
CRES Cystatin-related epididymal spermatogenic
CRPs Androgen-regulated cystatin-related proteins
CSF Cerebrospinal fluid
Cys C Cystatin C
HCHWA-1 Hereditary cerebral hemorrhage with amyloidosis of Icelandic type
HMWK High molecular weight kininogen
HRG Histidine-rich glycoprotein
LMWK Low molecular weight kininogen
MDRD Modification of diet in renal disease
Key Facts of Renal Biomarker Cystatin C
• Kidney filtration function is tested with serum creatinine-based equations named

as estimated glomerular filtration rate (eGFR).
• Creatinine is affected by nonrenal factors: age, gender, muscle mass, volume
status, and analytical interferences such as bilirubin, ketone, and protein.
• Cystatin C is an endogenous protein synthesized in all nucleated cells that its
plasma level is proportional with renal filtration capacity.
• Cystatin C is catabolized in the tubules of the kidney, that its high concentration in
urine reflects tubular injury.
• In contrary to serum creatinine, cystatin C is less effected from volume status and
not affected from hepatic dysfunctions which make it more reliable in the state of
cirrhosis.
Definitions
Cathepsin Lysosomal enzyme responsible for terminal protein degradation
Cystatin A low molecular weight protein that inhibits reversibly the lysosomal
enzyme cysteine proteases
20 Serum Cystatin C as a Biomarker 447
GFR A test to determine the filtration function of the kidney
Housekeeping gene A type of gene that indicates a stable production rate
Introduction
Cystatin C (Cys C), originally called as gamma-trace and post-gamma-globulin, is a

non-glycosylated low molecular weight protein found in many body fluids and
secretions. In 1961, it was isolated as a cerebrospinal fluid-specific protein in
human and named as gamma-trace protein (γ-CSF) (Clausen 1961). In 1981, the
complete amino acid sequence of human cystatin C was determined by Grubb and
Löfberg (Grubb and Löfberg 1982). The amino acid sequence of Cys C was the first
sequence to be determined among cystatins. Later on the sequence similarity of the
isolated human protein and chicken egg white cystatin was shown, and Cys C was
identified as an inhibitor of cysteine proteases (Turk et al. 1983).
The cystatins inhibit the cysteine peptidases, the papain-like proteases (cathep-
sins), classified as clan C1, and some also inhibit the asparaginyl endopeptidase/
legumain as clan C13 enzymes, and parasite proteases like cruzipain, where they
appear to provide protective functions. Also cystatins have a role in the defense
mechanism against microbial infections (Magister and Kos 2013; Turk et al. 2000;
Abrahamson et al. 2003). Known as the tightest binding inhibitor of lysosomal and
extracellular cysteine proteases, Cys C inhibits papain, cathepsin L, and cathepsin S
in pM range. Cys C neutralizes the protease activity strongly and rapidly (Turk and
Turk 2008).
Lysosomal cysteine proteases, generally known as cathepsins (clan C1), are the
papain family that are responsible for terminal protein degradation in the lysosomes
(Magister and Kos 2013; Turk et al. 2000). Also they may be secreted to degrade
extracellular matrix components (Turk and Turk 2008). The increased cysteine
protease activity has been shown to be related to a variety of pathopysiological
conditions such as bone resorption, chronic inflammation (rheumatoid arthritis,
bronchiectasis), cancer progression and metastasis, viral and parasitic infections,
and septic shock (Turk and Turk 2008; Ni et al. 1997). Likewise, failure in the
function of the protease inhibition results in neurodegeneration, cardiovascular
diseases, osteoporosis, arthritis, and cancer. In atherosclerotic lesions,
overexpression of cathepsins was found (Dinic et al. 2014).
Cystatin Superfamily
Cystatins, a superfamily of cysteine protease inhibitors, are comprised of 12 different

inhibitors. They provide protective functions for uncontrolled proteolysis and tissue
damage which are found in a variety of human fluids and secretions (Turk
et al. 2002).
448 S. Çuhadar
Table 1 The human cystatin superfamily classification

Family 1 (stefins) Family 2 (cystatins) Family 3 (kininogens)
Intracellular Extracellular/transcellular Intravascular
Stefin A Cystatin C LMW kininogen
Stefin B Cystatin D HMW kininogen
Cystatin E/M
Cystatin F
Cystatin G
Cystatin S
Cystatin SA
Cystatin SN
The cystatin superfamily consists of three types classified on the basis of the
presence of one, two, or three copies of cystatin-like segments and the presence or
absence of disulfide bond (Table 1). The members of the cystatin superfamily are
reversible competitive inhibitors of cysteine proteases (Rashid et al. 2006). In
addition to the family 1, 2, and 3 cystatins, the proteins containing cystatin domains
but lacking inhibitory activities are also the members of the cystatin superfamily.
Family 1 cystatins (stefins): Stefin A (also named cystatin A, acid cysteine
protease inhibitor, epidermal SH-protease inhibitor) and stefin B (also named
cystatin B, neutral cysteine protease inhibitor) are intracellular cystatins (Magister
and Kos 2013). These single-chain proteins lack disulfide bonds and carbohydrate
side chains and are composed of 98 amino acid residues with 11,175 Da and
11,006 Da, respectively (Machleidt et al. 1983; Ochieng and Chaudhuri 2010).
The structure of stefin molecule consists of a five-stranded antiparallel β-sheet
wrapped around a five-turn α-helix with an additional C-terminal strand (Stubbs
et al. 1990).
Stefin A and B are potent inhibitors of papain and cathepsins L, S, and H. Their
genes do not encode signal peptides (Ni et al. 1997). These intracellular cytoplasmic
proteins of many types of cells have been detected in extracellular fluids as well (Kos
and Schweiger 2002).
Family 2 cystatins (cystatins): Cys C, D, E/M, F, G, S, SA, and SN are type
2 extracellular and/or transcellular proteins distributed in body fluids at high con-
centrations with molecular masses of 13–14 kDa. Cys C, D, E/M, F, G, S, SA, and
SN are encoded by genes located on the chromosome 20 (Abrahamson et al. 1990).
Some members of the family 2 are glycosylated (Ochieng and Chaudhuri 2010).
They all contain characteristic intrachain disulfide bonds toward the C-terminal
unlike stefins. S type (S, SA, and SN) were first isolated from human saliva
(Magister and Kos 2013; Isemura et al. 1984). Cys D, S, SN, and SA, mainly
found in saliva, are poorer inhibitors of cysteine proteases than Cys C which is the
most abundant human cystatin that strongly inhibits clan C1 and clan C13 (Magister
and Kos 2013). Human Cys D, present in tears and saliva, strongly inhibits cathepsin
H and S and weakly neutralizes cathepsin L, but not cathepsin B (Balbín et al. 1994).
The target proteases of cystatin E/M are the papain-like cysteine proteases
including cathepsin B, L, and V (Cheng et al. 2006). Legumain, the asparaginyl
endopeptidase, is mostly inhibited by its potent inhibitor cystatin E/M (Abrahamson
et al. 2003).
Cys C is a major local regulator of extracellular proteolytic activity that inhibits
cysteine proteases belonging to the papain (C1) and legumain (C13) families and
especially inhibits the cathepsin B, H, L, and S. Cys C controls the activity of
cathepsins which have elastolytic and collagenolytic activities that contributes to
atherosclerotic process (Dinic et al. 2014). The high activity of legumain and
cathepsins has been shown to ease the invasion of tumor cells (Briggs et al. 2010).
It has been suggested that in breast cancer, the loss of cystatin E/M activity leaded an
increase in tumor cell growth and metastasis (Ni et al. 1997).
Family 3 cystatins (kininogens): Intravascular low molecular weight kininogen
(LMWK; MW 50e80 kDa) and high molecular weight kininogen (HMWK; MW
120 kDa) are large extracellular proteins comprised of about 335 amino acid residues
that contain three family 2-like domains. These glycosylated forms of human
kininogens have additional disulfide bonds and differ in length of the C-terminal
regions. Structurally both HMWK and LMWK are composed of a light chain, a
heavy chain connected by disulfide bridges, and the kinin segment. The light chains
of both kininogens are different; however, the heavy chain and the kinin segment
have identical amino acid sequences (Kellermann et al. 1986). LMWK binds papain
and cathepsin L and S; HMWK binds papain, cruzipain, and cathepsin S.
CRES (cystatin-related epididymal spermatogenic) proteins: cystatin-like pro-
teins named as cystatin-related epididymal-specific proteins were firstly found in
mouse epididymis. Though structural homology was shown with cystatins, CRES
proteins have no inhibitory effect on cysteine proteases papain and cathepsin B
(Cornwall and Hsia 2003).
Other cystatin-like proteins, which lack cysteine protease inhibitory properties,
are fetuin A, alpha 2HS glycoprotein (ahsg), histidine-rich glycoprotein (HRG), and
androgen-regulated cystatin-related proteins (CRPs), testatin, and cystatin T. Testatin
and cystatin T, specifically expressed in the testis, have similar sequence with family
2 cystatins (Ochieng and Chaudhuri 2010; Eriksson et al. 2002).
Structure of Cys C
The structure of the Cys C is a composition of five antiparallel β-sheets wrapped

around a central helix with the disulfide bonds of between residues 73 and 83 and
between residues 97 and 117. The molecular mass of Cys C is 13,343 Da
450 S. Çuhadar
Cys C in body fluids, mg/L

CSF 5,8
Plasma 0,96
Urine 0,095
Saliva 1,8
Seminal plasma 51
Amniotic fluid 1
Tears 2,4
Milk 3,4
Fig. 1 Cystatin C concentrations (mg/L) in the body fluids
(nonhydroxylated) and 13,359 Da (hydroxylated proline residue at position 3). The

isoelectric point of Cys C is 9.3 and thus positively charged in all body fluids (Filler
et al. 2005).
Cys C is composed of 120 aa residues encoded by the “housekeeping type” CST3
gene located in the short arm of chromosome 20 at p.11.2 (Grubb and Löfberg 1982;
Abrahamson et al. 1990). The protein is synthesized as a 146 aa pro-protein with a
26-residue hydrophobic signal peptide by all nucleated cells (Abrahamson
et al. 1987). With the cleavage of the signal peptide, mature Cys C is released into
the bloodstream in a short time. The N-terminal amino acid residue is the part of the
Cys C molecule that has a high binding affinity to papain.
The amino acid sequence of the single polypeptide chain of human Cys C is
SSPGK PPRLV GGPMD ASVEE EGVRR ALDFA VGEYN KASND MYHSR
ALQVV RARKQ IVAGV NYFLD VELGR TTCTK TQPNL DNCPF HDQPH
LKRKA FCSFQ IYAVP WQGTM TLSKS TCQDA (Grubb and Löfberg 1982).
The concentrations of Cys C in human biological fluids are figured (Fig. 1).
Cystatin C is found at high concentrations in the body fluids, particularly in
cerebrospinal fluid and seminal plasma (Filler et al. 2005; Magister and Kos
2013). The CSF concentration is five times more than in blood plasma which is
the dominant cysteine proteinase inhibitor supplied mainly by the choroid plexus
(Cole et al. 1989).
Detection method: PETIA (particle-enhanced turbidimetric immunoassay) and
PENIA (particle-enhanced nephelometric immunoassay). Both methods make pos-
sible to measure routinely and rapidly, but the higher cost compared with SCr
constraints its use routinely.
Specimens: Serum, plasma (EDTA, lithium heparin).
Assay Interferent Factors
Hemoglobin8 g/L, triglycerides 23 mmol/L, bilirubin 488 μL, and rheumatoid
factor 2,000 kIU/L did not show any significant interference analyzing with
nephelometric and turbidimetric methods (Finney et al. 1997; Delanaye et al. 2008).
Stability of cystatin C: Cys C is stable up to 48 h in the whole blood. Stored
plasma samples are stable up to 4 years at 80 C (Hoek et al. 2003).
Reference Range
Cys C production rate is constant throughout the ages of 1–50 years and increases
significantly above the age of 50–60 in both gender which might be due to the
physiological aging of the renal function (Finney et al. 2000; Galteau et al. 2001).
Plasma
<1 year: 0.59–1.97 mg/L (Finney et al. 2000)
1–50 years: 0.53–0.92 mg/L
>50 years: 0.58–1.02 mg/L
In premature infants, reference ranges are higher than adults (1.10–2.06 mg/L)
(Schwartz and Work 2009).
Urine: 0.03–0.18 mg/L (Conti et al. 2005).
Cys C cannot be detected in urine under physiological conditions; however, in
state of tubular injury, urine Cys C may become a measurable level, a potential
biomarker for AKI (Slocum et al. 2012).
Cys C and Children
In pediatric population, Cys C is more advantageous than serum creatinine, espe-

cially under 4 years of age due to their lower muscle mass. Cys C levels are higher in
the first weeks of life until 1 year of age, and the production rate is constant till
50–60 years unlike serum creatinine which increases until the early years of adoles-
cence, due to muscle mass gain (Finney et al. 2000).
Cys C and Pregnancy
During the pregnancy, the serum Cys C concentrations were found as differed
significantly with gestational age. In the first trimester, Cys C values were detected
as higher than the second trimester and were increased in the third trimester which is
452 S. Çuhadar
at the highest value after delivery. It was considered as a reliable GFR marker in
pregnancy instead of serum creatinine that is unreliable (Babay et al. 2005).
Factors Affecting Cys C
Cystatins are involved in a number of immunomodulatory functions; it was shown to

be associated in the pathophysiology of multiple sclerosis and Alzheimer’ s disease
(Bollengier 1987; Levy et al. 2001) During the inflammatory processes, Cys C
release has been found as downregulated, contributing to increased cysteine protease
activities in the macrophage microenvironment (Chapman et al. 1990) However,
contrary to earlier suggestions, no significant effect of systemic inflammation on
plasma Cys C concentrations was detected in a recent study by Grubb et al. (2011).
Cys C levels were found as affected by visceral obesity and insulin resistance
(Ognibene et al. 2006). In fact, visceral obesity and insulin resistance are frequently
associated with GFR increase and lower Cys C concentrations. Thyroid hormones
increase Cys C levels; thus, hypothyroidism has been associated with lower Cys C
concentrations and hyperthyroidism with higher Cys C concentrations (Schmid
et al. 2012; Fricker et al. 2003).
It has been shown that moderate or high doses of glucocorticoids increase the Cys
C production (Risch et al. 2001).
Advantages of Cys C
Cys C is a promising marker due to its proven satisfactory criteria, thus gaining
popularity. Cys C has a short half-life (1.5 h) compared with serum creatinine (4 h
with normal GFR) (Sjostrom et al. 2004). Serum creatinine is distributed in whole
body water, whereas Cys C is distributed in extracellular part. Therefore Cys C rises
more rapidly than serum creatinine and advantageous for early contrast-induced
renal injury detection (Briguori et al. 2010). Less than 10 % increase in Cys C at 24 h
can be a reliable marker for ruling out contrast-induced acute renal injury, and more
than 10 % increase in serum Cys C at 24 h is an independent predictor of 1-year
major adverse events such as death and dialysis (Briguori et al. 2010).
Although there are some conflicting data regarding greater intraindividual vari-
ability of Cys C than serum creatinine, in a recent study intraindividual variability of
Cys C has been confirmed as similar to serum creatinine that Cys C seems as
accurate as SCr for longitudinal patient follow-up (Delanaye et al. 2008).
Interindividual variations of Cys C and creatinine were found as similar about
15.1 % and 14.4 %, respectively (Reinhard et al. 2009).
Cys C plasma concentration is inversely correlated with GFR as it is only
catabolized in the kidney. Its renal clearance cannot be measured. Urine Cys C can
Table 2 Concentrations Blood plasma 0.96, 0.57–1.79

of human cystatin c in
Cerebrospinal fluid 5.8, 3.2–12.5
body fluids of healthy
adults (mg/L; mean and Urine 0.095, 0.033–0.29
range) (Filler et al. 2005) Saliva 1.8, 0.36–4.8
Seminal plasma 51.0, 41.2–61.8
Amniotic fluid 1.0, 0.8–1.4
Tears 2.4, 1.3–7.4
Milk 3.4, 2.2–3.9
only be detected in case of renal proximal tubular impairment, which is more specific
than serum Cys C (Koyner et al. 2008). Rapid testing is available with commercial
automated assay procedures.
Disadvantages of Cys C
The test is relatively expensive; in daily routine, a large volume of testing requires
more evidences clinically and careful consideration.
Cys C levels are lower in the hypothyroid and higher in hyperthyroid state. Very
high doses of glucocorticoids increase the Cys C production, whereas low or
medium doses of glucocorticoids decrease the Cys C production. Some evidence
suggests that inflammation, osteoporosis, and diabetes affect the cystatin levels. In a
study authors found 8.5 % higher levels of Cys C in patients with diabetes mellitus.
However, that effect might be due to the interaction with proteinuria and Cys C. As
in a study with type 1 diabetic patients with normal renal function without protein-
uria, no influence of diabetes was found on the relationship between Cys C and GFR
(Hofstra et al. 2009). The advantages and disadvantages are summarized in Table 2.
Cys C and GFR
GFR estimation is a widely used measurement protocol instead of the invasive

methods based on exogenously injection of substances such as inulin,
125-
Iothalamate, iohexol, 51Cr-EDTA which are traumatic for patients, especially
for pediatric population. Moreover some techniques imply exposure to radiation.
Creatinine is produced by the muscle from creatine; thus, the muscle mass affects
its concentration. Besides serum creatinine differs by age, gender, rhabdomyolysis,
and dietary meat (Ochieng and Chaudhuri 2010). Moreover creatinine is secreted by
renal tubules in a varied amount, and drugs may influence the tubular secretion of
creatinine (Grubb et al. 2012). Therefore a new endogenous biomarker is being
searched for GFR estimation that Cys C seems ideal in this aspect (Table 3).
454 S. Çuhadar
Table 3 Advantages and disadvantages of cystatin C measurement

Advantages of Cys C
Less effected from nonrenal factors: gender, ethnicity (Caucasian, Afro-American and Asian), and
muscle mass
Cys C production rate is constant from 1 year of age to 50 years
Cys C secretion does not have a circadian rhythm (Larsson et al. 2008)
The absence of a circadian rhythm for Cys C allows its quantification in a single urine sample
(Conti et al. 2005)
Interindividual variation of Cyc C is about 15.1 % (Reinhard et al. 2009), intraindividual variation
4.5 % (Delanaye et al. 2008; Bandaranayake et al. 2007)
Short half-life (1.5 h) and extracellular distribution cause a rapid rise in serum concentration that is
an advantage over serum creatinine
Less effected from volume status
Rapid and precise testing is available with automated methods
Sensitive to small changes in GFR
Reliable in liver disease
More convenient in the pediatric population
Specific to proximal renal tubule injury; only small amounts of Cys C can be found in urine
(tenfold lower than in plasma), and increased concentrations in single-void urine samples directly
reflect tubular damage (Conti et al. 2005)
Disadvantages of Cys C
Non-standardized (lack of international standard)
Expensive
Levels altered by thyroid dysfunctions
High doses of corticosteroids increase Cys C production
Malignancy increases serum concentrations
To overcome the creatinine-based limitations, several formulas have been devel-

oped and some parameters have been added to the GFR prediction equations
(Cockcroft and Gault 1976; Levey et al. 1999). However, neither Cockcroft and
Gault equation that produces GFR values in ml/min nor the modification of diet in
renal disease (MDRD) equation that gives results as ml/min (1.73 m2)1 is suitable
for children or for adults especially at eGFR >90 ml/min (1.73 m2)1 level. Instead,
Schwartz equation or Counahan-Barratt equation is preferred for children (Schwartz
et al. 1976; Counahan et al. 1976; Grubb et al. 2005).
Cys C is present in many biological fluids at higher concentrations, and its low
molecular weight and positive net charge facilitate its glomerular filtration. Its
plasma concentration is proportional with glomerular filtration as it is produced
constantly by all nucleated cells. Cys C is later largely reabsorbed and catabolized in
the proximal renal tubule with no tubular secretion which makes it ideal for GFR
estimation (Grubb et al. 1985, 1992). Cys C has been proposed as an alternative
marker of renal function because of its possible advantages over serum creatinine
(Dharnidharka et al. 2002; Grubb et al. 2005). This protein has a capability to detect
early renal failure as it gives reliable GFR estimation at the critical level of 60 ml/
min/1.73 m2 (Bargnoux et al. 2012). Grubb et al. concluded that instead of
Table 4 Ideal GFR marker characteristics (Ochieng and Chaudhuri 2010; Swan 1997)
Endogenously produced at a constant rate
Filtered and excreted only by the kidney
Unaffected by nonrenal factors such as age, gender, weight, nutrition, disease state, etc.
Freely filtered through the glomerulus
Neither reabsorbed nor secreted by the renal tubules
Undergo no extrarenal elimination
Stable in urine for further analysis
creatinine, Cys C-based prediction equation for GFR estimation that uses only serum
concentration and a prepubertal factor (GFR [ml/min1.(1.73 m2)1] = 84.69 cys
c (mg/L)1.680 1.384 (if <14 years)) might replace both the MDRD prediction
equation for adults and the Schwartz and Counahan-Barratt prediction equations for
children. In that formula, a prepubertal factor added to compensate the prepubertal
concentrations that is higher than the older individuals where 13–14 years of age
limit represents the start of the puberty (Grubb et al. 2005). Ideal GFR marker
characteristics are listed in Table 4.
The gold standard exogenous marker 51Cr-EDTA has been compared with Cys C
to estimate GFR; this endogenous marker gave excellent correlation with GFR
(Simonsen et al. 1985). Cys C is more sensitive to small changes in the so-called
creatinine-blind GFR (40–70 ml/min) (Schwartz and Work 2009). It was reported
that in children, the serum Cys C is better correlated with GFR than serum creatinine
(Grubb et al. 2005).
There are equations recommended to estimate GFR from Cys C concentration:
(A) Grubb’s equation : GFRGrubb ¼ 84:69 Cystatin C1:680 ð1:384 if child < 14
yearsÞ (Grubb et al. 2005).
(B) Larsson’s equation:GFRLarsson ¼99:43CystatinC1:5837 ;cystatinC ismeasured
inmg=L (Larsson et al. 2004).
The problem that restricts the widespread use of Cys C is the lack of standard-
ization. Though recently a certified reference material (ERM-DA471/IFCC) has
been released, the different assay systems, differences in the established reference
intervals for different populations are the factors that affect the reliability of Cys
C-based equations inevitably (Grubb et al. 2005). Besides the use of large doses of
corticosteroids, thyroid dysfunction reduces the performance of Cys C-based equa-
tions. In those cases the diagnostic performance of creatinine-based equation is more
reliable.
The use of Cys C in combination with SCr to calculate eGFR strengthens the risk
classification of chronic kidney disease. The risk of death was found as increased
below the threshold of ~85 ml/min/1.73 m2 when using both Cys C and creatinine-
based eGFR (Shlipak et al. 2013). It has been concluded that Cys C is more sensitive
to small changes in GFR than serum creatinine in contrast-induced acute kidney
injury (Briguori et al. 2010).
456 S. Çuhadar
Although Cys C-based equations offer significant advantages over creatinine-

based equations, Cys C cannot replace creatinine as it also has some limitations
mentioned above and not perfectly tested in some clinical situations. Consequently
Cys C-based equations cannot replace gold standard methods (Grubb et al. 2005;
Andersen et al. 2009).
Cys C and Cirrhosis
In patients with liver cirrhosis, renal dysfunction is associated with poor prognosis.
In these patients, SCr may be influenced directly by nonrenal factors such as protein-
calorie malnutrition, muscle wasting, and increased tubular secretion, and impaired
liver function caused reduced creatinine production. And indirectly, hemodynamic
changes will affect serum creatinine concentrations. In cirrhosis, GFR estimation
with creatinine has been shown to overestimate the true GFR by up to 200 %
(Sherman et al. 2003); consequently the renal failure is greatly underestimated.
Inulin clearance was considered as the gold standard in cirrhosis (Caregaro
et al. 1994). However, this method is very cumbersome to perform in clinical
practice. Moreover, urine collection is difficult to execute in clinical practice because
of urinary losses and incomplete urinary bladder emptying. 51CR-EDTA and 99TC-
DPTA are other measurement techniques that imply exposure to radiation. Con-
versely, Cys C is independent of gender, age, and muscle mass, and not influenced
by serum bilirubin, inflammation, or malignancy. Accordingly, Cys C has been
proposed as a specific marker of GFR and an early indicator of impaired renal
function in patients with cirrhosis (Ćulafić et al. 2014; Ustundag et al. 2007).
Cys C and Malignancy
Cys C is shown to be increased in malignancies in some studies irrespective of

kidney function. High concentrations of cathepsin B and H are detected in the sera of
patients with colorectal cancer and malign melanoma which are associated with
increased serum Cys C (Kos et al. 1998). Imbalance between cysteine proteinases
and cystatins is associated with tumoral cell metastasis that is considered to facilitate
tumor cell invasion and metastasis. Though high levels of Cys C may inhibit
cathepsin activities, increased levels of Cys C was found to be associated with
poor prognosis (Kos et al. 2000). Increase in the inhibitory functions of cystatin
could lead to a harmful impairment of the antitumor response of cysteine cathepsins
(Magister and Kos 2013).
Cysteine proteases take part in tumoral metastasis that involves local invasion and
angiogenesis. Cathepsin B, detected to be present on the surface of the tumor cell
(Mai et al. 2000), plays a key role in tumoral cell invasion (Ochieng and Chaudhuri
2010) which is particularly inhibited by Cys C. Though increases in Cys C concen-
tration were determined in malignant diseases such as colorectal cancer or mela-
noma, in those studies, GFR measurement was not performed with a reference
technique (Kos et al. 1997, 1998). In myeloma, serum Cys C was found as more
sensitive than SCr for GFR estimation that detected moderate GFR reductions.
Additionally, Cys C also correlated with advanced disease and provided important
information for prognosis (Terpos et al. 2009; N€uckel et al. 2012).
Amyloid Angiopathy
Cys C is a protein with amyloidogenic properties that aggregates in the brain arteries
of elderly people with amyloid angiopathy. A more severe disease massive amy-
loidosis is associated with the L68Q mutant of human Cys C that leads to death in
young adults with massive cerebral hemorrhage. This autosomal dominant disorder,
associated with mutation in the Cys C gene CST3, is known as Icelandic type. A
point mutation that replaces leucine amino acid with glutamine at position codon
68 of the Cys C gene is a disorder known as hereditary cerebral hemorrhage with
amyloidosis of Icelandic type (HCHWA-1) (Ghiso et al. 1986; Levy et al. 1989;
Revesz et al. 2009). This less stable deposit of mutant Cys C (ACys) aggregates
mainly in the brain arteries to form plaques called amyloid deposits that impairs the
elasticity of the arterial wall by replacing the muscle fibers and elastic fibers (Pezzini
et al. 2009).
Summary Points
• This chapter reviews cystatin C and its significance for renal function.
• Cystatins are cysteine protease inhibitors where they appear to provide protective
functions.
• Cystatin C is a protein synthesized by all nucleated cells and secreted shortly after
its synthesis and not affected by nonrenal factors.
• Cystatin C is freely filtered through the glomerulus and almost completely
catabolized by tubules that are an advantage for GFR estimation.
• Low molecular weight and positive net charge of Cys C facilitate its glomerular
filtration. The plasma concentration is proportional with glomerular filtration as it
is produced constantly.
• Cystatin C is an endogenous GFR marker that can be easily detected in serum
with automated methods.
• Urine concentration is very low (tenfold lower than plasma concentration), and
increased concentrations in urine samples directly reflect tubular damage.
• Cys C is a protein with amyloidogenic properties that aggregates in the brain
arteries of elderly people with amyloid angiopathy.
• Lysosomal cysteine proteases, generally known as cathepsins (clan C1), are the
papain family that are responsible for terminal protein degradation in the
lysosomes.
458 S. Çuhadar
• The cystatin superfamily consists of three types classified on the basis of the
presence of one, two, or three copies of cystatin-like segments and the presence or
absence of disulfide bond.
• Cystatin C is found at high concentrations in the body fluids, particularly in the
cerebrospinal fluid and seminal plasma.
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Homocysteinemia as a Biomarker in Kidney
Disease 21
Velibor Čabarkapa and Mirjana Đerić
Contents
Key Facts of Homocysteine as a Biomarker in Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467
The Structure and Origin of Homocysteine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467
The Metabolism of Homocysteine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468
The Regulation of Homocysteine Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
Methods for the Determination of Homocysteine and Reference Values and Factors
Affecting the Level of Homocysteine in the Blood . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
The Causes of Hyperhomocysteinemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473
The Biological Effects and Mechanisms of the Toxicity of Hyperhomocysteinemia . . . . . . . . . 474
Homocysteine and the Renal Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476
Homocysteine in Patients with Terminal Renal Failure and in Transplanted Patients . . . . . . . . . 479
Diabetes Mellitus, Diabetic Nephropathy, and Homocysteine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480
Homocysteine in the Nephrotic Syndrome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481
Homocysteine and Vascular Disease in Patients with Chronic Kidney Disease . . . . . . . . . . . . . . . 482
The Potential Use of Homocysteine in the Diagnostics and Monitoring of
Renal Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484
Potential Applications of Homocysteine in Other Diseases or Conditions . . . . . . . . . . . . . . . . . . . . 485
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 486
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 487
Abstract
Homocysteine is an amino acid containing sulfur in the form of the thiol group.
Homocysteine is a metabolite of the essential amino acid methionine. First,
methionine is converted to S-adenosylmethionine, which is then converted to
S-adenosylhomocysteine, wherein transmethylation processes take place.
V. Čabarkapa (*) • M. Đerić

Department of Pathophysiology, Medical Faculty Novi Sad, University of Novi Sad, Novi Sad,
Serbia
e-mail: veliborcabarkapa@gmail.com; lulica@uns.ac.rs; mirjanadjeric@gmail.com

464 V. Čabarkapa and M. Đerić
Homocysteine finally emerges from S-adenosylhomocysteine. It then enters

either the transsulfuration pathway when converted to cystathionine or the
remethylation pathway when converted back into methionine in the presence of
the 5-methyltetrahydrofolate reductase, cystathionine β-synthase, and methionine
synthase and with the participation of vitamin B12 and folic acid. Therefore, the
decreased activities of these enzymes, as well as the deficit of the vitamin B12 and
folic acid, are frequent causes of hyperhomocysteinemia. In addition, chronic
kidney disease and the use of certain drugs are also very important factors that
cause hyperhomocysteinemia.
In the bloodstream, homocysteine is found in one of two forms, the bound and
the free form, which together makes up the total homocysteine. The most suitable
sample for determining the circulating levels of total homocysteine is plasma.
Enzymatic and immunochemical methods are most commonly used in routine
clinical practice. Reference values of the total homocysteine vary among different
countries and even among different laboratories as a result of the use of different
methods for determining the levels of total homocysteine but also the different
lifestyles of the residence.
The plasma total homocysteine level is positively correlated with the serum
creatinine values, and it is in a high inverse correlation with the glomerular
filtration rate, so it could be used as a marker of renal hypofunction. In addition,
the kidneys possess enzymes involved in metabolic pathways. That is why
chronic kidney disease is one of the most common causes of hyperhomocys-
teinemia. The level of homocysteine in patients with chronic kidney disease can
be elevated even in the initial stages. Hyperhomocysteinemia is present in most
patients with glomerular filtration rate <60 ml/min/1.73 m2, with the highest
values in patients on chronic dialysis treatment. Hyperhomocysteinemia occurs in
50–60 % of patients with transplanted kidneys. On the other hand, hyperhomo-
cysteinemia is an important factor that contributes to the structural damage of the
kidney, both glomerular and tubular, along with damage to the interstitial tissue.
Therefore, when there are hyperhomocysteinemia and absence of the deficiency
of the vitamin B12 and folic acid, it is necessary to examine the renal function.
Homocysteine is a vascular toxin, and it is one of the risk factors for the
development of coronary heart disease, cerebrovascular disease, and peripheral
artery disease. Also, it has a role in the development of venous thrombosis.
Therefore, in patients with chronic kidney disease, the plasma concentration of
homocysteine is a significant determinant of the risk of morbidity and mortality
from cardiovascular disease. The measurement of homocysteine in patients with
renal dysfunction is necessary for making decisions about therapeutic treatment,
especially if the patient has not yet had signs of development of cardiovascular
disease.
In addition, it is assumed that homocysteine has a role in the pathogenesis of
other pathological conditions, such as neurodegenerative diseases, dementia,
cognitive disorders, osteoporosis, epigenetic changes, hypertension, as well as
the stimulation of an immune response.
21 Homocysteinemia as a Biomarker in Kidney Disease 465
Keywords
Homocysteine (Hcy) • Vascular disease in patients with chronic kidney disease •
Determination • Diabetes mellitus and diabetic nephropathy • Diagnostics and
monitoring of renal dysfunction • Metabolism • Nephrotic syndrome • Potential
applications • Regulation • Renal function • Structure and origin • Terminal renal
failure and in transplanted patients • Hyperhomocysteinemia • Biological effects
and mechanisms • Causes • Nephrotic syndrome • Homocysteine
Abbreviations
5-MTHF Methyltetrahydrofolate
5-MTHFR Methyltetrahydrofolate reductase
apo A-I Apolipoprotein A-I
ATP Adenosine triphosphate
bHcy Bound homocysteine
BHL Bleomycin hydrolase
BHMT Betaine-homocysteine methyltransferase
CBS Cystathionine β-synthase
CMIS Chronic malnutrition-inflammation state
CSE Cystathionine γ-lyase
DM Diabetes mellitus
fHcy Free homocysteine
H2S Hydrogen sulfide
Hcy Homocysteine
HDL High-density lipoprotein
HHcy Hyperhomocysteinemia
HTL Homocysteine thiolactone
IL Interleukin
Km Michaelis constant
MAT Methionine adenosyltransferase
MCP-1 Monocyte chemoattractant protein-1
Met Methionine
MIP-2 Macrophage inflammatory protein-2
MMP Matrix metalloproteinase
MS Methionine synthase
NAC N-acetylcysteine
NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells

NO Nitric oxide
NS Syndroma nephroticum
oxLDL Oxidized low-density lipoprotein
PON1 Paraoxonase-1
ROS Reactive oxygen species
SAH S-adenosylhomocysteine
SAM S-adenosylmethionine
TAFI Thrombin activatable fibrinolysis inhibitor
TGF-β1 Transforming growth factor β1
tHcy Total homocysteine
t-PA Tissue plasminogen activator
tRNA Transfer ribonucleic acid
VSMC Vascular smooth muscle cells
Key Facts of Homocysteine as a Biomarker in Kidney Disease
• The kidneys are a major regulator of the circulating levels of homocysteine (Hcy),
as they take part in its excretion and metabolism.
• In patients with chronic kidney disease (CKD), the glomerular filtration rate
(GFR) is the main determinant of the total Hcy levels. Hyperhomocysteinemia
(HHcy) is present in renal transplant patients as well.
• Hemodialysis treatment leads to a transient reduction in the levels of tHcy.
• HHcy in patients with diabetes mellitus mainly occurs with the development of
diabetic nephropathy.
• HHcy contributes to the progression of renal dysfunction. Therefore, in all
patients with HHcy and without deficit of folic acid and vitamin B12, it is
necessary to evaluate the functional status of the kidneys.
• HHcy in CKD may contribute to the development of vascular comorbidity and
comortality.
• In patients with CKD, the determination of the tHcy levels has significance in the
assessment of the risk for the morbidity and mortality due to CVD, then in the
assessment of the progression of renal dysfunction, the estimation of the total
mortality in CKD patients with malnutrition-inflammation syndrome, as well as
for making a decision regarding the therapeutic treatment.
Definitions
Homocysteinylation The reaction in which the Hcy in the form of homocysteine

thiolactone binds to amino groups of amino acids, leading to a dysfunction of the
proteins.
Hyperhomocysteinemia Increasing levels of total homocysteine (tHcy), which

implies an increase in the level of the free (fHcy) and bound Hcy (bHcy).
Hypomethylation Reduction in the volume of the transmethylation reactions in

hyperhomocysteinemia (HHcy), due to the inhibition of the methyltransferase by the
increased levels of S-adenosylhomocysteine (SAH).
Remethylation The conversion of Hcy into methionine by the enzyme methionine

synthase, the folic acid as a donor of the methyl groups, and of vitamin B12 as a
cofactor.
Transmethylation The reaction of the methyl group transfer from the donor to the
acceptor. This reaction takes place during the conversion of methionine into Hcy,
and it is dependent on the intermediates S-adenosylmethionine (SAM) and SAH.
Transsulfuration Reaction to the conversion of Hcy into cystathionine with the

participation of vitamin B6. Cystathionine, after being converted into various com-
pounds, is excreted by the kidneys, which are the pathways for eliminating Hcy from
the body.
Introduction
Homocysteine (Hcy) was discovered in the fourth decade of the twentieth century.
However, only when its role in the pathogenesis of vascular diseases was realized, in
the last 30 years, has it received greater attention. In addition, an increasing number
of scientific papers indicate a possible causal relationship between hyperhomocys-
teinemia (HHcy) and other pathological conditions.
The kidneys play an important role in the metabolic homeostasis of Hcy, because
they participate in Hcy metabolism, and on the other hand, they are involved in Hcy
excretion. Hyperhomocysteinemia is almost an inevitable phenomenon in patients
with kidney disease, particularly chronic.
The Structure and Origin of Homocysteine
Homocysteine is a nonessential amino acid which contains sulfur in the form of thiol
(-SH) group (Fig. 1). Hcy is produced in all human cells, as a product of the
intracellular metabolism of the essential amino acid methionine (Met). When Hcy
production is beyond the scope of its intracellular metabolic processing, Hcy
released from the cells is increased which might lead to the development of HHcy.
Fig. 1 Homocysteine – the

structure. Homocysteine is an
amino acid which contains
sulfur in the form of -SH
group
Hcy is eliminated from the bloodstream in a number of ways, the most important
being the reuptake by cells and excretion in the urine.
Hcy is present in two forms in the bloodstream. Approximately 70–80 % of Hcy
is in the bound form (bHcy), by disulfide bond to plasma proteins, mainly albumin.
About 10–30 % is in the free form (fHcy), which can be in one of the following
forms: the reduced (1–2 % of the total Hcy (tHcy)) or the oxidized form, as a
disulfide that is bounded by disulfide bond to cysteine or another Hcy molecule
(homocysteine) (Nekrassova et al. 2003; Medina et al. 2001).
The Metabolism of Homocysteine
Homocysteine is an intermediate metabolite of the Met. The process begins with the
submission of an adenosyl group from the adenosine triphosphate (ATP) to a sulfur
atom of Met, with the participation of the methionine adenosyltransferase (MAT),
which results in the conversion of Met into S-adenosylmethionine (SAM). SAM is
the donor in most transfer reactions of the methyl group (transmethylation) (Fig. 2).
Most of the methyl groups originating from SAM bind with guanidinoacetic acid
and in that form is used for the synthesis of creatine (about 70 %). The rest of the
methyl group acceptors and their methylation products are deoxyribonucleic acids
(DNA) adenine or cytosine (DNA-N-methyladenine-DNA or 5-methylcytosine),
Fig. 2 The main metabolic pathways of homocysteine (Hcy). Hcy is the product of methionine
(Met) metabolism. First, the amino acid is converted into S-adenosylmethionine (SAM) in the
presence of the methionine adenosyltransferase (MAT). Then SAM is converted into
S-adenosylhomocysteine (SAH) by the methyltransferase, and the released methyl group is submit-
ted to various acceptors (transmethylation). From SAH by the SAH hydrolase, Hcy is generated.
Hcy can then be reconverted to Met by the methionine synthase (MS), or it can be converted into
cystathionine in the presence of the cystathionine β-synthase (CBS), which is excreted in the urine
after subsequent conversion. In conditions of hyperhomocysteinemia, Hcy can be converted into
SAH by the adenosylhomocysteinase
transfer ribonucleic acid (tRNA) bases (tRNA-methylated bases), L-arginine (asym-

metric dimethylarginine (ADMA)), etc. After submitting a methyl group to an
acceptor, with the participation of methyltransferase, SAM is converted into
S-adenosylhomocysteine (SAH). Hcy is generated from SAH, under the influence
of SAH hydrolase; and thereafter Hcy is the substrate for the synthesis of
cystathionine and then cysteine (transsulfuration of Hcy) and resynthesis of Met
(remethylation). However, in conditions of HHcy, in the presence of adenosine, Hcy
can be converted to SAH, which disrupts the SAM/SAH ratio. The concentration of
SAH is an important determinant of the methyltransferase activity in tissues; it acts
as an inhibitor of these enzymes as they interact with higher affinity with SAH than
with SAM. Therefore, in conditions of HHcy, due to an increase in the concentration
of SAH, there is a decrease in the intensity of the transmethylation processes, or so to
say, it leads to hypomethylation.
In healthy individuals, the Hcy clearance from plasma is constant, and the
transsulfuration pathway is the only way to eliminate Hcy from the body. Within
this pathway, in reactions that are dependent on pyridoxal phosphate, and with the
participation of the cystathionine β-synthase (CBS), Hcy is converted into
cystathionine, which is then converted into cysteine, under the influence of
cistationase (Fig. 3). The kidneys, liver, intestine, and pancreas possess both
enzymes of the transsulfuration pathway (Hirsch et al. 2002). Activation of these
two enzymes is induced, among other factors, by Met originating from food, which
potentiates the clearance of Hcy. Interruption of dietary protein rich with Met leads
to favoring remethylation pathway. Remethylation can occur with the participation
of the betaine-homocysteine methyltransferase, which happens almost exclusively in
the liver and has a small role in the metabolism of Hcy, also with the participation of
MS or 5-methyltetrahydrofolate-homocysteine methyltransferase, when Hcy
receives a methyl group from the monocarbonyl pool, and the
5-methyltetrahydrofolate (5-MTHF) is used as a donor of the methyl group. Vitamin
B12 is required as a cofactor in this reaction of transmethylation.
The biological functions of Hcy are as follows: it is the main substance for the
synthesis of cystathionine, cysteine, and other metabolites of the transsulfuration
pathway; it participates in the conservation of Met; it participates in the catabolism of
choline, as a receptor of the methyl group from betaine, which is a product of the
choline degradation process; and it serves as a substrate for the recycling of tissue
folate (Finkelstein and Martin 2000; Wu et al. 2012).
The Regulation of Homocysteine Metabolism
The level of Hcy in the plasma depends on the balance between the release of Hcy
from the cells into the bloodstream and its elimination from the plasma. The
mechanisms involved in regulating the metabolism of Hcy include difference in
the affinity of enzymes to the substrate that determines in which pathway Hcy will be
included, as well as the influence of SAM, SAH, and 5-MTHF. In addition, the Hcy
Fig. 3 The regulation of homocysteine (Hcy) metabolism. After the intake of food rich in proteins,
the methionine (Met) is converted to S-adenosylmethionine (SAM), which is converted into
S-adenosylhomocysteine (SAH), by the reaction of transmethylation. SAH is also the inhibitor of
this reaction. Hcy is generated through the conversion of SAH, and then Hcy can be converted again
into Met through the reaction of remethylation that depends on the presence of folate and vitamin
B12. Hcy can also be converted to Met in a reaction with betaine. Additionally, Hcy is converted into
cystathionine and cysteine by transsulfuration reactions which depend on vitamin B6, which
potentiated by SAM and SAH. Transsulfuration pathway is the only way of eliminating Hcy.
SAM is an inhibitor of the folic acid cycle, the conversion of Met into SAM, as well as the
conversion of Hcy to Met via betaine. SAH is an inhibitor of the transmethylation processes, and
that is why hypomethylation occurs in the case of decrease in the SAM/SAH ratio; dUTP
deoxyuridine triphosphate, dTMP deoxythymidine monophosphate, A acceptor, DHF
dihydrofolate, THF tetrahydrofolate
metabolism is affected by the oxidative state in the cell and by the distribution of
enzymes in the body.
The enzymes can be divided into two groups: those which participate in the
conservation of Met (they have a low Michaelis constant (Km) value), and enzymes
that are involved in the catabolism of Met (which have a higher Km value). Whether
Hcy will go through transsulfuration or remethylation pathway depends on the Km
enzymes value versus Hcy. Km value for CBS is about 100 times higher than the Km
value of the methylase, which means that the affinity of CBS is significantly lower
than the affinity of the methylase. That is why under physiological conditions, the
remethylation pathway is favored. Activation of the transsulfuration pathway of Hcy
occurs only when there is a maximum capacity utilization of the methylation
pathways of Hcy. Apart from CBS, cistationase and MAT – the III form – have a
high Km value (Finkelstein and Martin 2000; Ramakrishnan et al. 2006; Finkelstein
2007). MAT forms I and II, MS, and betaine-homocysteine methyltransferase
(BHMT) have a low Km value. While CBS is active in the oxidative form, oxidation
inactivates MS, and that is how oxidative stress favors the transsulfuration pathway
of Hcy, generating cysteine or glutathione. Therefore, in conditions of oxidative
stress, Hcy release from the cells is reduced (Durand et al. 2001).
Intermediate metabolites of Met, primarily SAM and SAH, are significant regu-
latory factors of Hcy metabolism. An increased concentration of SAM inhibits the
activity of MTHFR and the synthesis of 5-MTHF, as well as the activity of BHMT,
all of which have a purpose to reduce converting Hcy into Met. Simultaneously, it
also leads to the activation of CBS, favoring the transsulfuration pathway. Further-
more, SAM inhibits its own synthesis by acting as an inhibitor to the forms I and II of
MAT (extrahepatic forms) and activating the form III of MAT (the hepatic form).
Also, SAM activates some methyltransferases and potentiates the transmethylation
pathway. The ultimate effect of SAM is to increase the catabolism of Hcy and to
prevent its accumulation. Adequate folate intake provides sufficient amounts of
5-MTHF, which inhibit the alternative transmethylation pathways. The final effect
is an increase in the level of SAM which consequently favors the clearance of Hcy
(Wu et al. 2012). On the other hand, a high level of SAM leads to the inhibition of
5,10-MTHF conversion into 5-MTHF. In case of the deficiency of folates, which are
essential for the proper flow of the Hcy remethylation process, there is a deficit of
SAM, whose synthesis is dependent on the Hcy remethylation, especially in condi-
tions of insufficient intake of Met. Consequently, there is a decreased activity of CBS
and HHcy is induced. In addition, a reduction of 5-MTHF contributes to the increase
of SAH or Hcy, because it leads to a reduced inhibition of some methyltransferase
enzymes which induces alternative transmethylation pathways.
Methods for the Determination of Homocysteine and Reference

Values and Factors Affecting the Level of Homocysteine
in the Blood
The main indications for determining homocysteinemia are diagnostics on

homocystinuria, examination of the deficiency of vitamin B12 and folic acid, and
the assessment of cardiovascular risk (Refsum et al. 2004). In determining the levels
of tHcy, it is necessary to determine the level of folates and vitamin B12.
The most suitable sample for determining the circulating levels of Hcy is potas-
sium EDTA or lithium heparin plasma. Citrate as an anticoagulant is not
recommended because it has a dilution effect and lowers the tHcy levels by
5–15 % in the individual samples (Refsum et al. 2004; Rasmussen and Møller
2000). After blood sampling, it is necessary to immediately put the test tube on
ice, in order to prevent an increase in the concentration of Hcy due to its synthesis
and release from erythrocytes. The separation of plasma or serum is preferably
carried out after 30–60 min but no longer than 6 h. The concentration of tHcy in
serum samples is about 5–10 % higher than in plasma because the coagulation of the
serum is slower on ice (Rasmussen and Møller 2000). Because samples should not
contain fibrin, red blood cells, or particles, special caution is needed in patients
receiving anticoagulant or thrombolytic therapy. The stability of Hcy in plasma or
serum is 4 days at 20–25 C, 14 days at 2–8 C, and at least 12 months at 20 C.
Available routine methods usually allow us to determine the levels of tHcy, and
the first step usually involves treating the sample of plasma or serum with a reducing
agent and converting all forms of Hcy into the reduced form whose level is then
measured. The methods used to determine levels of tHcy levels are chromatographic
methods, gas chromatography-mass spectrometry, capillary electrophoresis with
fluorescence detection, as well as enzymatic and immunochemical methods
(Rafii et al. 2009). The development of the immunochemical and enzymatic methods
has facilitated the determination of tHcy levels in the general clinical practice. The
most commonly used methods in this group are the fluorescence polarization
immunoassay and the chemiluminescent immunoassay (Ramakrishnan et al. 2006).
Increasingly there is a need to identify other forms of Hcy. For example, it has
been found that the reduced form of Hcy has the strongest toxic effects on blood
vessels (Chambers et al. 2001). However, determining the levels of the fHcy with the
most current methods is not suitable for routine clinical practice (Refsum et al. 2004;
Nekrassova et al. 2003).
The reference values for tHcy vary among different countries and even among
different laboratories. The use of different methods for determining the level of tHcy
and people’s different life habits (especially folate intake habits, or the use of fruits
and vegetables in general diet, then smoking, and consumption of coffee and tea) are
most likely the cause for that. The use of coffee, as well as smoking, was positively
correlated, while the consumption of tea was negatively correlated with the level of
tHcy (Golbahar et al. 2004; Grubben et al. 2000). In smokers, vitamin B6 interacts
with carbon monoxide from cigarette smoke, which leads to a disruption in the
transsulfuration pathway; also, nicotine disrupts the methylation processes and
affects the activity of MS (Bergmark et al. 1997). In premenopausal women, the
circulating tHcy level was slightly lower than that in men, which is mainly attributed
to the effect of estrogen which leads to a decrease in tHcy levels probably by
influencing the catabolism of Met, as well as the possible influence of estrogen on
the level of folates (Refsum et al. 2004; Stanger et al. 2003; Zappacosta et al. 2013;
Stanislawska-Sachadyn et al. 2008). During pregnancy, there is a reduction in
homocysteinemia, due to hemodilution, reduced albumin concentration in the
plasma, and because of the renal hyperfiltration (Walker et al. 1999). The levels of
tHcy increase along with the body aging, and the values practically double from
childhood to old age (Refsum et al. 2004); also, in older people the values of tHcy
are 2–5 μmol/L higher than in people of middle age, who have the same level of
creatinine in the serum (Elshorbagy et al. 2007). Possible causes of this trend are
decrease in renal clearance with aging, reduction in the CBS activity, reduced intake
of folate, and the intracellular functional deficiency of vitamin B12, as well as the use
of different drugs (Refsum et al. 2004; Elshorbagy et al. 2007). In the supine
position, homocysteinemia is about 10 % lower than in the sitting position, probably
due to the reduction of plasma albumin concentration in this position. In addition,

6–8 h after a meal rich in proteins, an increase may occur in the plasma levels of tHcy
by 10–15 % (Nurk et al. 2001). This may be the cause of the daily individual
differences of the tHcy levels which are lower in the morning than in the second
half of the day.
In most European countries, the upper limit for the reference value of tHcy does
not exceed 15 μmol/L (Refsum et al. 2004; Nekrassova et al. 2003). In addition, in a
significant number of laboratories, the upper limit for the reference value is
12 μmol/L (Stanger et al. 2003; Finkelstein and Martin 2000). It is considered that
it would be best to establish the limit values of tHcy in relation to the intake of food
enriched with folates, the intake of beverages, and smoking, so that in a population
with favorable living habits, this limit should be 12 μmol/L, while in a population
with unhealthy lifestyle, the upper limit should be 15 μmol/L (Refsum et al. 2004).
The given values are related to individuals aged 15–65 years old. For the elderly with
adequate intake of folates, tHcy upper limit should be set at 15 μmol/L or 20 μmol/L
in patients with poor intake of folates. Hcy level above 15 μmol/L is present in
approximately 5 % of the general population and in about 50 % of patients who
suffered a stroke or have a cardiovascular disease (CVD) (Guthikonda and Haynes
2006). In any case, tHcy levels up to 30 μmol/L are considered as a moderate HHcy;
values between 30 and 100 μmol/L are considered as an intermediate HHcy, while
values above 100 μmol/L are considered as an extreme or severe HHcy (Stanger
et al. 2003).
However, since HHcy is associated with an increased cardiovascular risk, the
question is whether there is a need for establishing upper limits for “normal”
homocysteinemia. Namely, tHcy concentration below 10 μmol/L is considered to
be “ideal” when it comes to low risk of CVD (Dhonukshe-Rutten et al. 2009).
Moreover, when reducing the tHcy level by 3 μmol/L, it leads to a reduction in the
risk for ischemic heart disease by 16 %, deep vein thrombosis by 25 %, and stroke by
24 % (Wald et al. 2002). It has been established that in countries in which the average
values of tHcy are closer to “ideal,” there is lower incidence of vascular disease
(Golbahar 2004).
The Causes of Hyperhomocysteinemia
HHcy implies an increase in the level of fHcy and bHcy. HHcy is caused by different
factors. The most common factors are genetic factors, nutritional factors, the use of
drugs, and renal disease.
5-MTHFR and CBS deficits are the most common enzyme deficiencies that lead
to HHcy and homocystinuria (increased Hcy excretion in urine). So far, 60 different
mutations have been described in patients with 5-MTHFR deficiency (Iacobazzi
et al. 2014), which is characterized by a decrease in SAM/SAH ratio and impair-
ments in the methylation processes. The most common mutations that are associated
with reduced activity of 5-MTHFR are C677T and A1298C. C677T mutation leads
to the manifestation of a thermo-sensitive form of 5-MTHFR, which is frequently
associated with coronary artery disease. In heterozygotes, the activity of 5-MTHFR

is reduced by 40 %, while in monozygotes, it is reduced by more than 70 %
(Iacobazzi et al. 2014). However, there is an interaction between the genotype and
the nutritional status. In homozygotes for the thermo-sensitive form of 5-MTHFR,
an adequate folate intake may lead to the maintenance of homocysteinemia within
the reference range (McLean et al. 2004). The deficiency of CBS, another very
important enzyme involved in the metabolism of Hcy, particularly in the homozy-
gous form, can be the cause of severe HHcy.
A vitamin B12 and folic acid deficit is one of the most common causes of HHcy,
and on the other side, HHcy can be a very sensitive marker of the intracellular
deficiency of these vitamins (Ramakrishnan et al. 2006). Vitamin B12 is a cofactor
for MS, and folic acid participates in the donation of a methyl group to Hcy in the
process of remethylation. Because of the impairment in the remethylation process
caused by the deficiency of these vitamins, Hcy conversion into Met is reduced, as
well as the synthesis of SAM, and due to HHcy there is an increase in the reversible
synthesis of the SAH, leading to a disorder in the methylation processes. Apart from
malnutrition, deficiency of these vitamins may also occur due to their malabsorption
or increased consumption, or it may be caused by the use of some medications.
Those who have an increased risk for deficiency of these vitamins are vegetarians or
people with reduced intake of fresh fruits and vegetables, then the elderly, persons
with malignancies or bowel diseases, pregnant women, and alcoholics (Stanger
et al. 2003). In order to reduce the cardiovascular risk and achieve the “ideal” values
of homocysteinemia (<10 μmol/L), it is necessary to establish an adequate folate
(>15 nmol/L) and vitamin B12 status (>350 pmol/L) (Dhonukshe-Rutten
et al. 2009).
Various medications have an influence on cofactors in the metabolic pathways of
Hcy. HHcy is caused by folate metabolic antagonists (methotrexate, trimethoprim,
antiepileptics), hypolipemics which are antagonists of vitamin B6 (niacin), inhibitors
of vitamin B12 and folic acid absorption (colestipol/cholestyramine), as well as those
which lead to renal damage (fibrates), then drugs that reduce the absorption of
vitamin B12 (metformin and omeprazole); cyclosporine A, probably due to its effects
on the renal function; isoniazid as an antagonist of B6 vitamin; and others.
The effect of renal function on the Hcy metabolic homeostasis will be discussed
in detail hereinafter.
The Biological Effects and Mechanisms of the Toxicity

of Hyperhomocysteinemia
Given the high reactivity of Hcy in biological systems, in HHcy, this amino acid can
be converted to various metabolites such as Hcy-thiolactone (HTL), S-nitroso-Hcy,
Hcy-containing disulfides, adenosylhomocysteine, etc., which take a part in various
pathophysiological processes.
In the plasma, Hcy is constantly subjected to oxidation to form homocysteine,

Hcy-mixed disulfide bonds, and thiolated proteins. During the oxidation of -SH
groups, free radicals are generated, such as hydrogen peroxide (H2O2) and superox-
ide anion (O2-), which exert adverse effects on lipids and proteins in the cell
membrane, thus resulting in endothelial dysfunction (Medina et al. 2001). They
also react with nitric oxide (NO) and reduce the bioavailability of this potent
vasodilator and inhibitor of platelet adhesion (Fischer et al. 2003). In addition,
HHcy leads to a decreased activity of enzymes involved in the catabolism of
ADMA, primarily the dimethylarginine dimethylaminohydrolase. This causes an
increase in the level of ADMA, which is an inhibitor of NO synthase, thus resulting
in a reduced NO production (Sen et al. 2014).
In the milieu of HHcy, especially in situations where the transsulfuration and
remethylation pathways are impaired, for example, in conditions of CBS or
5-MTHFR deficiency, the production of highly reactive HTL is induced
(Ramakrishnan et al. 2006). The level of HTL in plasma is up to 0.3 % of tHcy
(Chwatko and Jakubowski 2005), and one of the indicators of an increased
production of HTL is an elevated Hcy/Met ratio. In the form of HTL, Hcy reacts
with the -NH2 group of the amino acids, especially lysine. This so-called process of
N-homocysteinylation of proteins, which is specific for Hcy, leads to structural
changes in the proteins which may ultimately result in their denaturation. In
addition, as a result of the homocysteinylation, modification occurs on lipoproteins,
hemoglobin, and other plasma proteins. The N-homocysteinylation of fibrinogen
leads to an increased thrombogenesis (Karolczak and Olas 2009); homocystei-
nylation of the high-density lipoprotein (HDL) reduces their defensive role (Ferretti
et al. 2003), while homocysteinylation of the endothelial NO synthase (eNOS)
causes a decrease in the NO production (Sen et al. 2014). Also, as a result of protein
homocysteinylation, vascular damage may occur due to the fact that these modified
proteins accumulate on the surface of the blood vessel wall. Then they are identi-
fied directly by macrophages, or they can first be recognized by the anti-Hcy
antibodies with which they form antigen-antibody complexes that are then recog-
nized by macrophages. In both cases the result is phagocytosis with the consequent
destruction of endothelial cells (Jakubowski 1999). The enzymes involved in the
organism’s defense from proteins’ homocysteinylation are the paraoxonase, which
is a component of the HDL, and the bleomycin hydrolase (BHL) (Zimny
et al. 2006). Both enzymes lead to HTL hydrolysis.
Due to the increased level of SAH, which is an inhibitor of the
methyltransferases, in HHcy, there is a hypomethylation of the DNA, RNA, and
other substances. Hypomethylation is associated with vascular damage, malignant
diseases, the aging process, and other pathological conditions (Perez et al. 2007).
Other effects are being considered as well, such as genotoxicity, endoplasmic
reticulum stress, unfolded protein response, stimulation of the immune response,
activation of the metalloproteinases, mitochondrial and telomerase damage, stimu-
lation of the vascular smooth muscle cell proliferation (VSMC), etc. (Perła-Kajàn
et al. 2007; Jakubowski 2007; Jacobsen et al. 2005; Perez et al. 2007).
However, there is still a dilemma whether Hcy is only a biomarker of a certain

pathological condition or an active participant in the processes that lead to these
pathological conditions.
Homocysteine and the Renal Function
The processes of filtration, reabsorption, degradation, and urinary excretion of amino

acids take place in the kidneys, and that is why the kidneys have an important role in
the amino acids’ homeostasis (Garibotto et al. 2010). The kidneys are, in addition to
the liver, the main places for eliminating Hcy from the plasma. The kidneys
eliminate about 70 % of plasma Hcy, so it is not surprising that kidney failure is
one of the most common causes of HHcy.
In patients with chronic kidney disease (CKD), HHcy is most prominent in
end-stage renal disease (ESRD) patients, i.e., patients on dialysis (85–100 %), while
in patients with CKD who are not on dialysis, HHcy is present in 36–89 % of them
(in most cases, between 12 and 30 μmol/L) (Čabarkapa 2007; Jardine et al. 2012).
Even when a slight reduction occurs in the glomerular filtration rate (GFR), it results in
an increase in the Hcy level, if compared to the level before the reduction in GFR,
which may actually be within the reference range (Refsum et al. 2004). Hcy level
>12 μmol/L is present in about 10 % of CKD patients with GFR >60 ml/min/1.73 m2,
in about 90 % of CKD patients with GFR <60 ml/min/1.73 m2, and in patients on
dialysis (Čabarkapa et al. 2007). The plasma tHcy level is in positive correlation with
the plasma creatinine level and in an inverse correlation with the GFR (R = 0.71,
p < 0.001) (Čabarkapa et al. 2012) (Graph 1). Therefore, tHcy level could be used as a
marker of renal hypofunction, particularly in patients with GFR <60 ml/min/1.73 m2.
Only a small fraction of tHcy is subjected to glomerular filtration, and it is that
which is not bound to proteins (fHcy). Most of the filtered Hcy is reabsorbed in the
tubules, and only a small portion of it is eliminated in the urine, about 6–10 μmol per
day (Van Guldener and Stehouwer 2003). Arginine and lysine inhibit the Hcy
reabsorption, because they share the same path of reabsorption with Hcy.
Since the amount of Hcy that is normally excreted in the urine is relatively small,
it is assumed that the main factor that leads to HHcy in renal insufficiency is a
disorder in the intrarenal cellular metabolism. The kidneys are a place of intense Hcy
metabolism (Sen et al. 2014). They play an active role in the remethylation and
transsulfuration metabolic pathways of Hcy, because they have the enzymes (CSB,
MS, and BHMT) involved in the mentioned metabolic pathways. After entering the
cell, Hcy is reduced and becomes accessible for action of these enzymes. Therefore,
to maintain the levels of tHcy within the limits of the reference range, in addition to
the preserved functional ability of the kidneys, a preserved function and level of the
enzymes involved in Hcy metabolism is essential.
In CKD, it is most likely that the remethylation pathway is one that is primarily
disturbed (i.e., the metabolic clearance of Hcy), while the transsulfuration metabolic
180
R=-0.71, p<0.001
160
140
120
GFR (ml/min/1.73 m2)
100
80
60
40
20
0
0 5 10 15 20 25 30 35
Hcy (μmol/L)
Graph 1 The correlation between the glomerular filtration rate (GFR) and the plasma levels of
total homocysteine (tHcy). A statistically significant inverse correlation was determined (correlation
coefficient R = 0.71, p < 0.001) in a sample of 142 chronic kidney disease patients (100 diabetics
and 42 patients without diabetes) (Čabarkapa et al. 2007; Čabarkapa 2011)
pathway of Hcy is preserved in the initial stages, but as the renal damage progresses,
there is a consequent reduction in the functioning of that metabolic pathway as well
(de Koning and Hu 2010). The kidneys are also the main routes for the removal of
SAH from the blood, which indicate their very important role in the regulation of the
transmethylation reactions in the body (Garibotto et al. 2009). In CKD, especially in
ESRD, due to increasing levels of SAH and the consequent reduction in the
SAM/SAH ratio in the plasma and erythrocytes, as well as delays in the
methyltransferase activity, there is a reduction in the extent of transmethylation.
The altered expression of certain genes, altered cell differentiation, changes in the
properties and structure of cell membranes, damaged chemotaxis and phagocytosis,
reduced repair of damaged proteins, and reduced synthesis of neurotransmitters are
all just some of the consequences of hypomethylation in these patients. Moreover,
hypomethylation in CKD/ESRD patients is a very important factor that contributes
to the development of vascular diseases, which are the leading causes of mortality in
these patients. Besides that, particularly in the pronounced reduction in the func-
tional abilities of the kidneys, in addition to the renal, the extrarenal metabolism of
Hcy is damaged as well. This is due to presence of uremic toxins that inhibit the
intracellular metabolism of Hcy, which leads to an increased Hcy release from the
cells (de Koning and Hu 2010; Elshorbagy et al. 2007), as well as due to the
disrupted hepatic metabolism in chronic renal failure and the presence of inhibitors
of various enzymes in the circulation. Although the liver has a significant capacity
for remethylation and transsulfuration of Hcy, it still fails to remove the excess of
Hcy in patients with a significant reduction in the GFR, especially in ESRD.
Apart from the fact that the decrease in renal function leads to HHcy, HHcy itself
can contribute to the damage in the kidney structures, the glomerulus, the tubules,
and the corresponding interstitium (Mao et al. 2014). The mechanisms that may be
involved in the pathogenesis of the glomerular damage are changes in renal hemo-
dynamics, endoplasmic reticulum stress, hypomethylation, homocysteinilation, apo-
ptosis of the mesangial cells, and increase in the levels of ADMA, which reduce NO
production and lead to a consequent endothelial dysfunction (Finocchiaro and
Zoccali 2005). In addition, HHcy causes oxidative stress, which may play a role in
the damage of kidney structures.
Also, one of the mechanisms by which HHcy leads to the progression of the renal
dysfunction is an induction of the inflammatory molecules in the mesangial cells, such
as monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory
protein-2 (MIP-2) (Sen et al. 2009, 2010). Cytokines and chemokines contribute to
the recruitment of monocytes and their deposition in the wall of the glomerular blood
vessels, which leads to deposition of extracellular matrix and ultimately to the
occurrence of glomerulosclerosis (Sen et al. 2010), a decline in renal function and
development of chronic renal failure. The result is the expression of the matrix
metalloproteinases in the kidneys (MMPs) (Tan and Liu 2012), and HHcy contributes
to their activation. Increased activity of MMP-2 and MMP-9 leads to the dysregulation
of the synthesis and degradation of the extracellular matrix (Sen et al. 2014) which
results in the development of glomerular-tubulointerstitial fibrosis.
HHcy also stimulates a hydrogen sulfide (H2S), which is one of the molecules that
play an essential role in the regulation of the inflammatory response. Hcy is a
precursor in the synthesis of H2S. However, the inhibition of the cystathionine
γ-lyase (CSE) occurs in HHcy. CSE participates in the transsulfuration pathway
and catalysis of H2S formation, thus its inhibition leading to a decreased production
of H2S in the mesangial cells. This could be one of the most important mechanisms
by which HHcy contributes to kidney damage and the eventual progression of the
renal dysfunction in patients with CKD. H2S somewhat inhibits the production of
MCP-1 and MIP-2, which is stimulated by HHcy, thus preventing renal damage
associated with HHcy (Sen et al. 2009) and contributing to the reparation of lesions
caused by HHcy. Also, the renoprotective effect of H2S is reflected in the regulation
of the extracellular matrix remodeling and inhibition of the renal glomerular fibrosis
(Sen et al. 2010). In addition to the direct renoprotective effects, H2S also exhibits an
antioxidant, vasodilator, and antihypertensive effects. A reduction in the level of this
substance contributes to the pathogenesis of neurodegenerative diseases, hyperten-
sion, inflammation, and atherosclerosis (Sen et al. 2014). Due to its antioxidant and
vasodilator characteristics which oppose the harmful effects of HHcy, H2S could be a
very useful therapeutic agent in patients with HHcy.
Homocysteine in Patients with Terminal Renal Failure

and in Transplanted Patients
HHcy is almost an inevitable companion of patients on dialysis. The type of dialysis

affects the plasma tHcy levels. HHcy is more common and more pronounced in
patients on hemodialysis (approximately 90 %) than those on peritoneal dialysis
(approximately 70 %). Plasma levels of Hcy in ESRD patients are typically two to
three times higher than in the healthy population and range, in most cases, between
16 and 100 μmol/L, with a median of 30 μmol/L (Perna et al. 2007). The hemodi-
alysis treatment leads to a reduction in tHcy levels by 30–40 %, but the Hcy levels do
not usually return back to reference range. In addition, the tHcy level increases back
rapidly after the hemodialysis treatment and within 24 h reaches the predialysis
level. Hcy bound to proteins cannot be removed by hemodialysis, only the fHcy,
which makes about 20 % of the tHcy. That is why the amount of Hcy that could be
eliminated by dialysis treatment is very small when compared to the influx of Hcy in
the plasma. However, uremic toxins are efficiently removed by hemodialysis treat-
ment which contributes to the reduction of tHcy levels in ESRD patients, especially
when using the superflux dialysis, which significantly reduces the level of Hcy (Van
Guldener and Stehouwer 2003). In patients on peritoneal dialysis, the intensity of
removing excess Hcy via dialysate is low, due to the predominance of Hcy fraction
which is bound to proteins and the possible reabsorption of Hcy from the dialysate,
as well as the application of dialysis solutions containing amino acids including Met.
The extent of Hcy removal significantly depends on its predialysis level in the
bloodstream; the higher the level of Hcy in the plasma is associated with, the higher
the percentage of reduction of the homocysteinemia. Therefore, in these patients, it
would be preferable to put the emphasis on the reparation of the Hcy metabolic
pathways, which can be achieved by using folates.
If there is a lowering of levels of Hcy with ESRD patients with malnutrition-
inflammation syndrome (CMIS), it indicates disturbed nutritional status, and it is
associated with increased mortality in these patients (Perna et al. 2007; Ingrosso and
Perna 2009; Kalantar-Zadeh et al. 2004; Marcus et al. 2007).
HHcy appears in about 50–60 % of patients with a transplanted kidney with a
median of 16–19 μmol/L (Einollahi et al. 2011; Sengoegle et al. 2003), even though
it would be expected that the transplantation leads to a significant reduction in the
levels of tHcy. Immediately after transplantation, the tHcy level is reduced averagely
by about one-seventh of levels before transplantation, although almost a third of
patients experience an increase in the levels of tHcy. In addition to the reduced renal
function and the impaired vitamin status, HHcy in transplant patients is also induced
by the immunosuppressive therapy, especially cyclosporine, as well as increased

expression of the transforming growth factor beta 1 (TGF-β1). This cytokine stim-
ulates the release of Hcy from the cells thus leading to HHcy in these patients.
Diabetes Mellitus, Diabetic Nephropathy, and Homocysteine
HHcy is not characteristic for patients with diabetes mellitus (DM) until it comes to
the development of diabetic nephropathy (DN). In patients with DM, tHcy level can
be increased, decreased, or within the reference range. For patients with DM and
without DN, it is characteristic to have lower plasma concentrations of tHcy com-
pared to people who are not suffering from diabetes or renal disease (Čabarkapa
2007). The cause for this is the activation of the transsulfuration pathway enzymes in
conditions of chronic hyperglycemia, and a contributory factor is the glomerular
hyperfiltration. However, when DN is developed, even in the initial stages, it is
characterized by the appearance of microalbuminuria (urinary albumin excretion of
30–300 mg/day), and especially after the manifestation of macroalbuminuria (uri-
nary albumin excretion >300 mg/day) and with the decrease in the GFR, impaired
renal function is the main factor that affects the level of tHcy and that leads to the
appearance of HHcy (Čabarkapa et al. 2012; Mao et al. 2014) (Graph 2). In addition
to the high inverse correlation between tHcy and the GFR, there is also a significant
correlation between albuminuria and homocysteinemia. The level of tHcy is signif-
icantly higher in diabetic patients with macroalbuminuria than in diabetic patients
with microalbuminuria and normoalbuminuria (Graph 2), which confirms the sig-
nificant connection between the functional status of the kidneys and the
homocysteinemia. Given the fact that albuminuria is also a risk factor for the
development of cardiovascular disease (CVD), the simultaneous presence of HHcy
and pathologic albuminuria significantly increases this risk. In addition, insulin
therapy may increase the level of tHcy, because of the inhibition of the CBS activity.
Although the underlying disease and DN lead to an increased inflammatory
response, Hcy has a contributory role as well, because HHcy is associated with
stimulation of the inflammatory response. It is most likely that the increase in the
inflammatory response is one of the major factors that lead to the development and
progression of the DN (Wada and Makino 2013). The level of oxidized low-density
lipoprotein (oxLDL) is higher in diabetic patients with HHcy than in patients with
tHcy levels within the reference range. Oxidized LDL may have a role in the
progression of the renal dysfunction in diabetic patients, because it stimulates the
production of collagen in the mesangial cells. In fact, patients with DM and tHcy
levels >10 μmol/L have a significantly higher risk of progression of the renal disease
(OR 3.4, p < 0.001) (Čabarkapa 2007). Therefore, the homocysteinemia may serve
as a predictor of the development of DN (Mao et al. 2014), as well as the progression
of the renal failure in patients with DN.
HHcy may contribute to the development and progression of microvascular
complications in DM due to the fact that Hcy exerts toxic effects on the blood
vessels, mainly in its free, reduced form, whose level is increased in the bloodstream
160
tHcy (μmol/L): p<0.001; GFR (ml/min/1.73 m2): p<0.001
140
Hcy (μmol/L)
GFR (ml/min/1.73 m2)
120
100
80
60
40
20
0
macroalbuminuric microalbuminuric normoalbuminuric
Diabetic patients
Graph 2 The level of total homocysteine (tHcy) in macroalbuminuric (n = 34), microalbuminuric

(n = 46), and normoalbuminuric (n = 52) diabetic patients. Glomerular filtration rate (GFR) and
tHcy levels are significantly different between all groups of patients ( p < 0.001), wherein the lowest
GFR is characteristic for the macroalbuminuric patients and consequently the highest tHcy
(Čabarkapa et al. 2012; Čabarkapa 2011)
of patients with DM. The cause of this increase is the condition of chronic hyper-
glycemia, which is associated with the nonenzymatic glycation of albumin, which
results in a decrease in the Hcy fraction that is bound to albumin, and which
increases its free fraction. HHcy, combined with the mutation of MTHFRC667T,
is a predictive factor for the development of microvascular complications in type
2 diabetes, especially in DN (Mtiraoui et al. 2007).
Homocysteine in the Nephrotic Syndrome
The tHcy level in the nephrotic syndrome (NS) can be normal, increased, or even
decreased, and the level of tHcy does not correlate with the proteinuria, so the GFR is
the main determinant of the tHcy level. Increased tHcy level in the NS is a
contributing risk factor for thrombosis of the blood vessels.
Homocysteine and Vascular Disease in Patients with Chronic

Kidney Disease
The progressive reduction in the functional ability of the kidneys in patients with
CKD is accompanied by numerous complications, including CVD which is the
leading cause of mortality (Garibotto et al. 2010). CKD patients with GFR
<60 ml/min/1.73 m2 have more than a tenfold increased risk, while CKD patients
with GFR 60–90 ml/min/1.73 m2 have twice the risk of developing a CVD when
compared with the healthy population (Francis et al. 2004). In fact, more than 50 %
of CKD patients die of CVD even before starting dialysis. In about one-third of the
patients with CKD, the incidence of cardiovascular complications cannot be linked
to any of the traditional risk factors, so Hcy could be that connection.
Hcy is an independent risk factor for the development of coronary heart disease,
cerebrovascular disease, and peripheral arterial disease. Increasing the levels of Hcy
by 5 μmol/L increases the risk of developing a coronary heart disease to the same
extent as the increase in total cholesterol level by 0.5 mmol/L (Di Minno et al. 2010).
Pro-atherogenic effect of the Hcy is manifested already in the mild to moderate
increase in the plasma concentration of Hcy (Sarwar et al. 2007). An increase in the
plasma level of tHcy by 3 μmol/L increases the risk for coronary artery disease by
10 %, and for cerebral infarction by 20 % (Nakao et al. 2014), and a reduction in the
level of Hcy up to 25 % leads to a reduction in the risk for myocardial infarction by
20 % (Cheng 2013). In ESRD patients, an increase of the tHcy level by 5 μmol/L
leads to an increase in the mortality rate by 7 % (Heinz et al. 2009), and a reduction
in the level of Hcy level in patients on dialysis by 10 μmol/L leads to a reduction in
the risk for cardiovascular events by 20 % (Zoccali et al. 2000).
The mechanisms linking HHcy and vascular damage are the endothelial dysfunc-
tion due to a decreased NO bioavailability, the proliferation of VSMC and their
impaired function, the accelerated degradation of elastin and the imbalance in the
elastin/collagen relations with the consequent reduction in the blood vessel elasticity,
the metalloproteinase activation, the H2S inhibition, the interleukin-8 (IL-8) stimu-
lation, the stimulation of the oxidative transformation of LDL, the increased adhe-
sion of platelets, and the inhibited synthesis of apolipoprotein A-I (apo A-I)
(Doronzo et al. 2010; Sen et al. 2010; Cheng 2013). HHcy leads to the thickening
of the intima media of blood vessels (Sarwar et al. 2007). Due to the activation of the
nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) via reactive
oxygen species (ROS), HHcy leads to the proliferation of the VSMC, which is
accompanied by an increased production of collagen and VSMC calcification. Hcy is
also an important marker of the oxidative stress in CKD and ESRD patients
(Wu et al. 2012). During the oxidation of Hcy into HTL and homocysteine, these
newly created substances mediate in the oxidative damage of the endothelial cells of
blood vessels, as well as in the oxidative modification of LDL, which leads to the
formation of foam cells that release inflammatory cytokines and ROS
(Wu et al. 2012).
Despite that, in CKD patients, there is a reduced activity of the paraoxonase
1 enzyme (PON1). PON1 in the bloodstream is bound to the HDL, and it plays a
role in the protection of LDL from oxidation (in the presence of apo A-I) and from
the detrimental effect of HTL. Also, PON1 is important for protecting proteins
from N-homocysteinylation which would lead to their damage and may play a role
in the occurrence of CVD. In addition, the process of protein homocysteinylation
contributes to the development of glomerulosclerosis which in turn leads to
impairment in the renal homeostasis (Suszynska-Zajczyk et al. 2014). Therefore,
this enzyme represents a link between the HDL and the Hcy metabolism. In CKD,
there is an increased clearance of apo A-I from the circulation, which lowers the
HDL levels and consequently reduces the PON1 activity. Given the cardioprotective
role of PON1, the existence of a reduction of its activity is associated with an
increased risk for CVD and with an increase in the mortality rate (Suszynska-
Zajczyk et al. 2014). The use of the recombinant human erythropoietin, which is
primarily used in patients on dialysis in the treatment of anemia, leads to an increase
in the PON1 activity, which certainly has a favorable effect on the risk for CVD.
In addition to HHcy, increased levels of SAH and the consequent DNA
hypomethylation are typical for patients with CKD, especially in those with vascular
complications (Stenvinkel et al. 2007). DNA hypomethylation is associated with the
development of atherosclerosis, and it is in correlation with the level of the circu-
lating Hcy, not only with HHcy (Ingrosso and Perna 2009). In hypomethylation, due
to the weakened repair process of damaged proteins, there is a consequent cell
damage which favors the formation of atherosclerotic lesions.
Furthermore, Hcy may be considered as an inflammatory marker in patients with
CKD, as it contributes to the induction of inflammation due to the increased
oxidative stress, NF-κB activation, and the inhibition of the H2S production in
cases of HHcy (Wu et al. 2012). In patients on hemodialysis, in the absence of the
chronic malnutrition-inflammation state (CMIS), characteristic is the existence of
HHcy, and Hcy is an independent risk factor for CVD. However, in the presence of
CMIS, the lower Hcy level reflects the disturbed nutritional status, especially the
reduction of the amino acid pool and hypoalbuminemia. Lower levels of Hcy are
associated with an increased mortality rate in these patients, independent from other
CVD risk factors (Ingrosso and Perna 2009; Kalantar-Zadeh et al. 2004; Marcus
et al. 2007).
The intake of folates in patients with CKD may lead to improvements in the
transmethylation and remethylation pathways, or so to say, it may lead to the
reduction of hypomethylation. However, due to the permanent impairment of the
transsulfuration pathway, the normalization of Hcy levels, especially in ESRD
patients, is very difficult. In patients with ESRD, the MTHFR genotype may affect
the response of these patients to folate substitution. The hemodialysis patients with
MTHFR677TT require higher doses of folic acid than the patients with CC or CT
genotype, to achieve reduction in the tHcy levels (Wu et al. 2012). Also, diabetic
patients with ESRD are less responsive to the treatment with folates and require
higher doses (15 mg/day) than the ESRD patients without diabetes (5 mg/day)
(Wu et al. 2012). In addition to the folate substitution in the attempt to restore the
remethylation pathway, for an improved regulation of the intracellular metabolism of
Hcy, it is required to use 500 mg of methylcobalamin.
Despite clear evidence of the association between HHcy and CVD, the questions
whether Hcy is a marker or a risk factor for cardiovascular complications, and
whether therapeutic lowering of the Hcy level using folates, B12, and other vitamins
leads to a significant reduction in the risk for CVD, still remain open. The HOPE
(Jamison et al. 2007) and the RCT study (Mann et al. 2008) showed that the use of
folic acid, vitamin B6, and B12 in patients with HHcy in different stages of CKD,
including the ESRD patients, did not show satisfactory effects in reducing the
cardiovascular complications and in improving the rate of survival in these patients,
even though there has been a reduction in the level of Hcy. A meta-analysis, from
2012 (Pan et al. 2012), showed that the therapeutic lowering of Hcy level does not
reduce the rate of the repeated cardiovascular events and stroke and the rate of total
mortality in CKD patients. When the clinical manifestations of atherosclerosis are
already present or there is a confirmation of a positive history of vascular diseases,
treatment with vitamins of the B group could even have adverse effects and thus
nullify the beneficial effects of lowering the Hcy levels (Cheng 2013). The use of
folic acid could induce pro-atherogenic changes in the endothelial cells of the blood
vessels, and the use of a combination of folates and vitamin B12 and the consequent
stimulation of the DNA synthesis could stimulate the cells’ proliferation and
neointimal proliferation. In patients with DN, the application of high doses of
vitamin B may even lead to a reduction in the GFR and increase the rate of vascular
complications (Wu et al. 2012). Although the therapeutic lowering of the Hcy level
has not led to the expected favorable results in the secondary prevention of vascular
events, it also does not have to be a factor that excludes the causal relationship
between HHcy and CVD. Unlike in secondary prevention, in the primary preven-
tion, when there are still no significant signs of atherosclerotic lesions, lowering the
tHcy could have beneficial effects.
The poor efficiency of the folic acid and B vitamins in lowering the levels of Hcy
in ESRD patients can be partly explained by the Hcy trait to bind with high affinity to
proteins in the circulation. Therefore, new therapeutic measures are aimed at liber-
ating the Hcy from its protein complexes, because the fHcy can be removed from the
circulation by hemodialysis thus achieving a significant reduction in the tHcy level.
Agents that are used for that are the N-acetylcysteine (NAC) and thiol-containing
drug sodium 2-mercaptoethanesulfonate (Mesna) (Scholze et al. 2004; Urquhart
et al. 2007).
The Potential Use of Homocysteine in the Diagnostics

and Monitoring of Renal Dysfunction
Because of the significant inverse correlation between Hcy levels and GFR, Hcy
could be used as a marker of renal hypofunction. Therefore, in all patients with
HHcy, unless they have a folic acid or vitamin B12 deficiency, it is necessary to carry
out an assessment of the functional status of the kidneys.
In patients with CKD, the determination of tHcy levels would be significant in the
assessment of the overall risk for morbidity and mortality from CVD, followed by
the assessment of progression of renal dysfunction, and Hcy level is a prognostic

factor for overall mortality assessment in CKD patients with CMIS.
The measurement of Hcy in patients with renal dysfunction is also necessary for
making a decision about the necessary therapeutic treatment, especially if the patient
has not yet suffered from the development of CVD.
Potential Applications of Homocysteine in Other Diseases or

Conditions
First of all, Hcy is a significant marker of vitamin B12 and folic acid deficiency. Hcy,
i.e., HHcy, may be also an important risk factor for the development of some
neurodegenerative diseases, osteoporosis, epigenetic changes, and arterial hyperten-
sion and may have a role in the stimulation of the immune response. In addition, the
screening for HHcy is a significant test for assessing the risk for developing venous
thrombosis.
Neurotoxic effects of HHcy are the results of hypomethylation of proteins and/or
genes and the increased production of ROS. Hyperhomocysteinemia stimulates the
inflammation, and it is well known that inflammation, which is almost mandatory
present in many neurodegenerative diseases, contributes significantly to the devel-
opment and progression of disease.
Due to the induction of hypomethylation, especially of the DNA, HHcy is
associated with genetic instability and malignant diseases. Hypomethylation induced
by HHcy plays also a part in the aging processes (Perez et al. 2007). In addition,
HHcy leads to increased oxidative stress, which leads to the damage of proteins.
Due to the activation of osteoclasts, the stimulation of bone resorption, and the
disturbance of collagen cross-links, as well as the induction of pre-osteoblastic cell
apoptosis, HHcy is a risk factor for developing osteoporosis and for the occurrence
of bone fractures (Herrmann et al. 2007). Since that the thyroid hormones influence
the activity of the enzymes involved in the processes of remethylation, particularly in
the liver, hypothyroidism is characterized by the presence of HHcy; on the other
hand, in hyperthyroidism, the level of Hcy is reduced.
The fact that HHcy inhibits the production of H2S, which in turn leads to
vasoconstriction, an increased arterial stiffness and stimulation of the inflammatory
response, HHcy may be associated with the pathogenesis of hypertension. Increased
plasma concentrations of Hcy by 5 μmol/L was associated with an increase in
systolic blood pressure by 0.7–1.2 mmHg and the diastolic blood pressure by
0.5–0.7 mmHg, regardless of the vitamin status and the functional status of the
kidneys (Stehouwer and van Guldener 2003).
HHcy has a role in the development of arterial and venous thrombosis. It leads to
changes in the platelet function, reduces the bioavailability of NO, and increases
expression and activity of coagulation factors II, V, and VIII; it increases the activity
of thrombin activatable fibrinolysis inhibitor (TAFI). It also leads to a decrease in the
levels of protein C and antithrombin III deficiency, reduces the expression of
thrombomodulin, and reduces the binding sites for the activator of the tissue
Chronic
kidney
Cardiovascular disease Drugs
disease
Hypertensio
artherialis Ageing
HHcy
Pregnancy Malignancy
complications
Neurodegenerative Osteoporosis
disease
Lifestyle and
Genetics
vitamin status
Fig. 4 The most common causes of hyperhomocysteinemia (HHcy) (blue arrows) and the condi-
tion in whose pathogenesis of HHcy (red arrows) has an important role. The most common factors
that can cause HHcy are genetic factors, nutritional factors, the use of certain medications, and
chronic kidney disease, and, the conditions in whose pathogenesis of HHcy has an important role
are some neurodegenerative diseases, osteoporosis, arterial hypertension, cardiovascular disease,
malignancy, pregnancy complications, and aging
plasminogen (t-PA). The fibrin clot that is formed in the milieu of HHcy is more
resistant to fibrinolysis (Di Minno et al. 2010).
Because of hypervolemia and glomerular hyperfiltration, Hcy level is lower
during pregnancy than in the general population. However, a disorder of metabolism
of Hcy and an increase in its plasma values during pregnancy are associated with an
increased incidence of preeclampsia, placental abruption, and recurrent pregnancy
loss in the mother, as well as neural tube defects in the fetus. These complications
can be overcome by the use of folates and other dietetic supplements, especially
omega-3 fatty acids (Kulkarni et al. 2011) (Fig. 4).
Summary Points
• This chapter is focused on the importance of homocysteine as a marker in renal

diseases.
• Homocysteine is synthesized from methionine and metabolized with the partic-
ipation of vitamin B12 and folic acid.
• The concentrations of total homocysteine (free and bound) are routinely mea-
sured in the plasma, and the reference values are still not precisely determined.
• The increase in the plasma concentrations of homocysteine is caused by genetic

factors, nutritional factors, the use of certain medications, and chronic kidney
disease.
• Homocysteine is a significant risk factor for the development of numerous
pathological conditions, especially cardiovascular disease.
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Fetal Beta2-Microglobulin as a Biomarker
of Kidney Disease 22
Gilles Grangé, Marie Clémence Leguy, Vassilis Tsatsaris, and
Jean Guibourdenche
Contents
Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 492
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 493
Physiology and Analysis of Beta-2 Microglobulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 494
Fetal Kidney and Urinary Tract: Normal Development and Pathological Situations . . . . . . 499
Assessment of Fetal Renal Function: Usefulness of β2-Microglobulin . . . . . . . . . . . . . . . . . . . . 505
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 509
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510
Abstract
Ultrasound diagnosis of fetal kidney and urinary tract anomalies is generally
performed at 18–20 WG, showing renal agenesis, cystic kidneys, and urinary
tract enlargement. Prognosis of the renal function and its postnatal outcome relies
on the US follow-up of the amniotic fluid volume and renal parenchyma. In case
of lower urinary tract obstruction, bilateral hydronephrosis, and nephropathies,
fetal puncture may be useful to analyze β2-microglobulin. It is a small protein
which circulates in a free and stable soluble form in biological fluids and can be
G. Grangé • V. Tsatsaris
CHU Cochin, AP-HP, Maternité Port Royal, Paris, France
e-mail: gilles.grange@cch.aphp.fr; vassilis.tsatsaris@cch.aphp.fr
M.C. Leguy
CHU Cochin, AP-HP, Biologie hormonale, Paris, France
e-mail: marie-clemence.leguy@cch.aphp.fr
J. Guibourdenche (*)
CHU Cochin, AP-HP, Biologie hormonale, INSERM U1139, Physiologie, Faculté de Pharmacie,
Université Paris Descartes, Paris, France
e-mail: jean.guibourdenche@cch.aphp.fr; jean.guibourdenche@parisdescartes.fr

492 G. Grangé et al.
measured using different immunoassay. As β2-microglobulin is entirely filtered

and reabsorbed and degraded by the nephron, serum levels rise when glomerular
filtration is impaired, while urinary levels increase when tubular reabsorption is
affected. Viral fetal infections may also increase serum levels, activating the
lymphocyte turnover and thus β2-microglobulin release. An increase in β2-
microglobulin in serum (above 5–6 mg/L) and to some extent in urine (above
4–5) constitutes an accurate marker to predict a postnatal renal failure in case of
urinary tract obstruction and bilateral hydronephrosis. In urines, measurement of
Na and Ca if possible has to be associated. In contrast to uropathies, measurement
of β2-microglobulin is poorly informative in nephropathies. However, discrepan-
cies remain due to the difficulty in defining a good postnatal outcome and
choosing of a reliable cutoff value and to the fact that nephrogenesis is a dynamic
complex process in which injury is not entirely reflected by β2-microglobulin.
Keywords
Beta-2 microglobulin • Human fetus • Kidney • Microproteins • Nephropathies •
Prognosis • Renal function • Serum • Urine • Uropathies
Abbreviations
β2-m Beta-2 microglobulin
Ca Calcium
Da Dalton
HLA Human leukocyte antigens
LuTO Lower urinary tract obstruction
MCDK Multicystic dysplastic kidney disease
MHC-I Class I major histocompatibility complex
Na Sodium
NK Natural killer cells
NMR Nuclear magnetic resonance spectroscopy
NSAIDs Nonsteroidal antiinflammatory drugs
PUV Posterior urethral valve
US Ultrasound
WG Weeks of gestation
Key Facts
• Human nephrogenesis occurs in the renal parenchyma from 5 to 35 WG and leads

to the formation of functional nephrons in terms of glomerular filtration and
tubular secretion and reabsorption.
22 Fetal Beta2-Microglobulin as a Biomarker of Kidney Disease 493
• It can be alterated in case of lower urinary tract obstruction, bilateral

hydronephrosis, and some nephropathies.
• Finding specific and sensitive markers of the renal parenchyma injury is still a
challenge.
• Serum β2-m is an index of glomerular filtration, while urinary β2-m reflects
tubular reabsorption.
• β2-m levels is the most reliable biochemical marker to predict postnatal outcome
when fetal ultrasound findings are doubtful.
• There may be a poor prognosis of the postnatal renal function if β2-m levels
increase in the urine or serum.
• However, there is still a gray zone in which prognosis is difficult to establish.
Definitions
The nephron is the functional unit of the kidney. It consists in the glomerulus
responsible for the plasma filtration and the tubules where secretion and reabsorption
take place, leading to the definitive urine which is thus an ultrafitra of the plasma.
Renal failure can be defined as impaired plasma clearance by the nephron. It can be
functional or organic and rapidly lethal if there is no treatment requiring dialysis and
kidney transplantation.
Nephropathies are purely kidney diseases affecting the development of the kidney
(agenesis, hypoplasia), its location (ectopia, abnormal rotation), or the
nephrogenesis and the differentiation of the renal parenchyma (defective
corticomedullary differentiation, parenchymal dysplasia, cysts).
Uropathies are urinary tract disease which can lead to an alteration of the
renal parenchyma and kidney functions due to urinary impaired excretion and reflux.
Introduction
Thanks to advances in the field of ultrasound scanning, the diagnosis of fetal renal
and urinary tract anomalies has widely improved. However, the assessment of fetal
renal functions and the prognosis of their postnatal outcome (i.e., renal failure) are
still a matter of challenge. Thus, fetal sampling can help to analyze biochemical
markers reflecting the renal parenchyma injury. Several studies pointed out the
interest of β2-microglobulin, a small protein that is entirely filtered and reabsorbed
and degraded by the nephron. We aim in this chapter to make an overview of the past
and the current knowledge on β2-microglobulin as a biomarker of fetal kidney
disease.
Physiology and Analysis of Beta-2 Microglobulin
Structure, Synthesis, and Function

Human beta-2 microglobulin (β2-m) was first identified in urines from patients with
chronic kidney dysfunction at the end of the 1960s (Berggård and Bearn 1968). It is a
highly conserved protein among species (Grooves and Greenberg 1982). Human β2-
m is a small monomeric protein of 11,800 Da which is devoid of carbohydrate and
has a pH of 5.7. It is made up of 100 residues with a seven-stranded β-sandwich fold
typical of the Ig superfamily and a single inter-sheet disulfide bond between cysteine
residues 25 and 80 in β-strands B and F. β2-m displays partial homology with
immunoglobulin constant region domains and particularly the CH III domain (Trinh
et al. 2002). It is encoded by a single gene on chromosome 15 which is expressed in
numerous tissues in fetus and adult (Cejka et al. 1975; G€ussow et al. 1987). It is
synthesized and expressed on the surface of nucleated cells. Lymphocytes release β2-
m during cell division and after stimulation. Indeed, different cytokines (e.g.,
interferons) can stimulate β2-m synthesis in hepatocytes and T lymphocytes (Vraetz
et al. 1999). On the cell surface, β2-m forms the small invariable light chain of the
class I major histocompatibility complex (MHC-I) bound to the heavy chain (also
called the α-chain) extracellular region (Grey et al. 1973). Its expression is regulated
in a coordinated manner with that of MHC-I antigens (also referred to as human
leukocyte antigens [HLA]) It binds non-covalently to these antigens which form the
α-chain of the MHC-I. β2-m interacts with and stabilizes the tertiary structure of the
MHC-I α-chain to present antigens (e.g., antigens present on cancer cells) to
cytotoxic T lymphocytes (CD8+) and NK cells (Yokoyama 1998; Hill et al. 2003).
It is probable that β2-m not only reflects but also modulates cell turnover, i.e.,
proliferation, apoptosis, and the immune response, particularly in the setting of
cancer. No receptor for β2-m has yet been identified, and it acts via different
pathways: cytokines, growth factors or hormones and their receptors, and different
kinases (cAMP-dependent protein kinase A, phosphatidylinositol 3-kinase, etc.)
(Rowley et al. 1995; Zhao et al. 2013; Nomura et al. 2014).
Release, Excretion, and Catabolism

β2-m is principally a cell membrane protein. However, because it is non-covalently
associated with MHC-I antigens and not directly attached to the cell membrane, β2-m
can be released from a cell in a free soluble form. Such forms of β2-m are detectable in
many body fluids, such as blood, urine, and amniotic fluid but also in cerebrospinal fluid,
seminal fluid, pleural fluid, and ascites (Berggård et al. 1968; Cejka et al. 1975; Puolakka
et al. 1982; Lutz et al. 1991) (Table 1). Its rate of production in normal adult subjects is
quite constant at around 0.13 mg/h.kg with relatively stable serum levels of 1–3 mg/L,
while it is excreted in urine at low levels of less than 0.5 mg/L. Levels are not age or
gender dependent (Filler et al. 2002). Catabolism occurs in the kidney. β2-m is a low-
molecular-weight protein (40,000 Da) like α1-acid glycoprotein, retinol-binding
protein, and α1-microglobulin (Regeniter et al. 2009). It is thus totally filtered through
Table 1 Levels of b2-m in different human body fluids. b2-m was measured in body fluids from
healthy children, adults, and mothers using different assays and antibodies. Fetal fluids were also
analyzed in fetus devoided of any renal impairment at the US or with no neonatal renal failure
β2-m (mg/L) Range Reference
Non pregnant
Serum 1.4 1–2.3 Ikezumi et al. 2013
3.09 2.88–3.3 Filler et al. 2002
Urine <0.5 Davey and Gosling 1982
Cerebrospinal fluid 1 0.6–2 Lutz et al. 1991
Pregnancy
Maternal blood 1.72
0.42 Ferreira et al. 1991
1.83
0.48 Nolte et al. 1991
1.2
0.3 Jauniaux et al. 1998
1.6
0.51 J Guibourdenche, ANR Perinat
Collection, personnal data
Coelomic fluid 4.5
Amniotic fluid 6.7 1–24.7 Burghard et al. 1988
3.5
5.1
2.1 12–1.2 Oliveira et al. 2002
Fetal blood 3.6 0–5.8 Berry et al. 1995
3.4 2–4.9 Tassis et al. 1996
2.93 0.64–7.94 Cobet et al. 1996
3.6
4.28 2.95–6.61 Bökenhamp et al. 2001
3.2 1.5–4.7 Dommergues et al. 2000
3.7 3.1–4.8 Fabbri et al. 2011
3.4
1.5 J Guibourdenche, ANR Perinat
Fetal urines 1.22 0.41–2.04 Muller et al. 1993
0.96 0–3.3 Muller et al. 1996
1.4 0.1–6.8 Spaggiari et al. 2012
1.6 <4 J Guibourdenche, ANR Perinat
Fetal ovarian cysts 3.1 2–4.1 Lecarpentier et al. 2012
Over intra abdominal cysts 7.3 0.1–41 Lecarpentier et al. 2012
Cord blood 2.78
0.84 Ferreira et al. 1991
3.3 2.1–4.5 Nolte et al. 1991
the glomeruli, reabsorbed at 99.9 %, and then degraded by lysosomes in the proximal
tubules (Fig. 1). Around 5 μg/h of the protein is excreted in the final urine. The plasma
disappearance curve of 125I-β2-m displays a rapid turnover (t1/2 = 2.1 h; range:
1.1–2.8 h in normal healthy patients) (Karlsson et al. 1980). Its levels in blood reflect
the rates of both nucleated cell turnover and glomerular filtration, whereas in urine they
MHC
nucleated cells tubular reabsorption
and degradation
nephron
blood
definitive urine
glomerular filtration
bladder
≠ b2-m in serum
Impaired filtration ≠ b2-m in urines

(alteration of glomeruli membrane,
decrease in nephrons number) Impaired tubular reabsorption
Increase nucleated cells turn over
(infections, cancers)
β2-m α-chain
Fig. 1 Renal physiology and β2-microglobulin in the human nephron. β2-m is mainly a nucleated
cell membrane protein associated with MHC α-chain, which is also released in free soluble form.
This form is entirely filtered through the glomeruli and reabsorbed and degraded at 99.9 % in the
proximal tubules. Serum β2-m levels rise mainly when glomerular filtration is impaired, while
urinary β2-m levels increase when tubular reabsorption is affected
are indicative of tubular reabsorption and degradation. Soluble β2-m can aggregate and
polymerize into insoluble fibrils in pathological situations such as chronic renal failure
and acidic conditions (Vincent et al. 1994). β2-m is the major protein component in
amyloid plaques and forms filamentous structures in the joints and connective tissue.
Samples collected from dialysis-related amyloidosis patients exhibit such fibrils which
are mostly formed by full-length β2-m together with a few additional proteolytic split
products (Rosano et al. 2005).
Sampling and Assay of b2-microglobulin in Human Fluids

In serum, β2-m circulates physiologically as a free monomer protein. In pathological
situations, dimeric or insoluble forms may occur. Truncated forms have also been
characterized in pathological conditions, resulting in enzymatic cleavage in the
N-terminal part. Under acidic conditions (pH<5–6), which may affect the urine in
pathological situations, β2-m is spontaneously unstable and in vitro may form
Table 2 Urinary proteins in renal disease. Troubles within the glomeruli filtration and the tubular
reabsorption can result in different types of proteinuria. Diagnosis can be made by measuring the
excreted urinary proteins or performing an electrophoresis of urinary proteins. Tubular proteinuria
is characterized by an increased excretion of low-molecular-weight proteins (40,000 Da) such as
b2-m, whereas glomerular proteinuria is associated with the excretion of high-molecular-weight
proteins (66,000 Da)
Protein Molecular mass (Da) Normal urine concentration mg/L Proteinuria
IgG 150,000 <10 Glomerular
Transferrin 79,550 <2.5 Glomerular
Albumin 66,460 <30 Mixed
α1-microglobulin 33,000 <12 Tubular
Retinol-binding 20,960 <0.5 Tubular
protein
β2-microglobulin 11,800 <0.3 Tubular
amyloid-like fibrils (Davey and Gosling 1982; Vincent et al. 1994). The stability of
β2-m in the urine decreases when the temperature rises, so urine specimens should be
alkalinized if necessary and stored at +4 C before being rapidly frozen if not
arranged quickly. Froze and thaw cycles should be avoided even if the molecule
seems quite stable (Juraschek et al. 2012).
β2-m levels can be measured by different immunoassays using a variety of
antibodies and detection systems (e.g., immunochemiluminescence, immunotur-
bidimetry) (Lutz et al. 1991; Terrier et al. 2004). These assays mainly recognize
the monomer protein in serum, plasma, or urine. Most assays are correlated but with
differences fluctuating from 2 % to 35 %, which may influence cutoff values. In
addition, some assays present matrix effects so they cannot be applied to all
biological fluids. There is no significant difference between plasma and serum levels.
Assay values can range from 0 to 16 mg/L, depending on the type of sample.
Clinical Usefulness in Children and Adults

Diseases affecting glomerular filtration and tubular reabsorption may result in
different types of proteinuria (Peterson et al. 1969; Gorriz and Martinez-Castelao
2012) (Table 2, Fig. 2). Important diagnostic information can be obtained by
determining excreted urinary proteins in order to locate both the site and extent of
renal injury. The pathological excretion of high-molecular-weight proteins, i.e.,
IgG, transferrin (66,000 Da), reflects an alteration of glomerular filtration.
The increased excretion of low-molecular-weight proteins (40,000 Da) such
as β2-m is an index of tubular proteinuria indicative of impaired tubular
reabsorption.
In serum, any elevation of β2-m levels may result from either increased
production and release due to a greater nucleated cell turnover or impaired renal
filtration and clearance (Fig. 1). However, in the latter case, creatinine is still the
most available indicator used for the diagnosis and follow-up of renal
Fig. 2 Electrophoresis of standard

fetal urinary proteins (1: mixte
of standards; 2: amniotic fluid;
3: b2-m standard; 4: RBP
a
standard; 5: a1-m standard; 6:
albumin standard; 7: TRF
standard; 8: IgG standard).
Fetal urines (a) and protein
standards (b) were applied for b2-m
sodium dodecyl sulfate
polyacrylamide gel
electrophoresis followed by
an argentic detection (b). This
allowed the separation of
those proteins according to
their apparent molecular size:
low-molecular-weight
proteins (β2-m; α1m; RBP),
albumin, high-molecular-
weight proteins (IgG; TRF),
β2-m β2-microglobulin, α1m
α1microglobulin, RBP
retinol-binding globulin, IgG
1 2 3 4 6 7 8
immunoglobulin G, TRF
transferrin
b
b2-m
RBP
a1-m
migration
albumin
TRF
IgG
insufficiency in a daily practice in children and adults (Ikezumi et al. 2013;

Juraschek et al. 2013; Reichel 2014). An increase in β2-m may provide informa-
tion on the progression of infection and the lymphoid response, in a setting such as
HIV infection (Fabbri et al. 2011; Chiltra et al. 2011). It may also be useful to the
prognosis and treatment of tumors such as lymphoma, reflecting an advanced
disease stage, tumor mass, and its malignant environment (Changhoon et al. 2014;
Nomura et al. 2014).
-bilateral renal agenesis cortex

Nephropathies:
-cystic dysplasia
serum b2-m
-polycystic kidney medulla
poorly informative
pyelectasia Kidney
calyces
cystic dysplasia
poor corticomedullary renal pelvis

differenciation
ureteropelvic junction
hydronephrosis
Uropathies
hydro uretro nephrosis ureter
b2-m increase enlarge ureter Urinary
in serum or urines
track
ureterovesical
junction
megacystis
cystis
uretra
3B: Pathology 3A: Physiology
Fig. 3 Normal and pathological structures of the fetal kidney and the urinary tract. This is a
schematic representation of the normal kidney and normal urinary tract (a) and the most frequent
pathological findings discovered at the US (b)
Fetal Kidney and Urinary Tract: Normal Development

and Pathological Situations
The placenta ensures clearance of the fetal compartment, while the fetal kidneys and
excretory system develop during pregnancy (Fig. 3a).
Anatomical Development
Fetal kidneys develop from three successive embryonic structures of mesodermal
origin. The intermediate mesoderm gives rise to the nephrogenic cord which in turn
forms the pronephros (3–5 WG) that is never functional and will regress; the
mesonephros (5–12 WG), a transient structure which can produce urine; and the
metanephros (as from 6 WG) which will lead to the definitive kidney (Wolff and
Winyard 2002; Dressler 2009). This results from an interaction between the ureteric
bud which will lead to the urinary excretory tract and the nephrogenic blastema that
will become the nephrons. The metanephros migrates from the pelvis to the lumbar
region and then rotates. It produces urine as from 10 WG through the establishment
of glomerular filtration. Nephrogenesis is a complex process that combines cell
growth and differentiation. It occurs from 5 to 35 WG, with an intense phase
between 18 and 32 WG. It occurs centrifugally, with newly formed nephrons
being located in the peripheral cortex. This results in the typical US appearance
with renal corticomedullary differentiation (Fig. 4a). At birth, each fetal kidney
contains around one million nephrons. Nephrogenesis is induced by penetration of
the ureteric bud into the nephrogenic blastema. Renal vesicles form and develop into
S-shaped tubules. The end of these S tubules opposite the ureteric bud will
Fig. 4 (continued)
Fig. 4 Ultrasound scanning of the fetal kidney and the urinary tract in normal and pathological
situations (Gilles Grangé, personal data). (a) Normal appearance of fetal kidney at the end of
pregnancy. Its outline is bumpy and corticomedullary differentiation is clearly visible. (b)
Multicystic fetal kidneys with oligohydramnios. The kidney carries numerous irregular cysts on
the periphery with a loss of corticomedullary differentiation and hyperechogenic appearance. (c)
Renal dysplasia and oligohydramnios within a Meckel syndrome. The kidneys are large with a
hyperechogenic appearance of the parenchyma due to renal dysplasia. It should look for associated
encephalocele. (d) Obstructive megaureter. The ureter appears as a dilated tortuous fluid-filled
tubular structure located between the bladder and the renal pelvis. (e) Severe megacystis at the first
trimester. The urinary bladder is enlarged, full of urines, and occupies most of the fetal abdomen
differentiate into the glomerulus. The remainder of the tubule extends and differen-
tiates into proximal and distal convoluted tubules and the loops of Henle, connecting
to the collecting ducts. The ureter arises from the lower ureteric bud which grows
toward the nephrogenic blastema and undergoes repeated dichotomous branching to
form the collecting system (i.e., pelvis, calyces, collecting ducts). Branching of
the collecting system is completed by 20 WG. The bladder derives mainly from
the cloacal endoderm. At 6 WG, the cloaca divides into two separate chambers: the
anorectal chamber and the urogenital sinus. The latter will form the major part of
the bladder and the urethra.
Renal Function Development

Morphological development of the kidney and urinary tract can be followed by
fetal ultrasound scanning (US) from the end of the first trimester and is associated
with functional maturation (Quigley 2012). Glomerular filtration is likely to be
initiated at around 9–10 WG and then rapidly increases with gestational age,
reflecting the rise in the number of nephrons up to 35 WG. This filtration is the
first step in the formation of urine which becomes the principal source of amniotic
fluid from around 20 WG. From the second half of gestation, amniotic fluid volume
reflects fetal diuresis so that evaluation of its volume by US constitutes a reliable
index of renal function (Shackelford et al. 1992; Yamamoto et al. 2007). Its
biochemical analysis, but preferentially that of fetal urine or blood whenever
possible, can provide valuable information on functional development and renal
maturation. Amniotic fluid is vital for lung and skeletal development. Decreased
amniotic fluid volume, i.e., oligohydramnios and anhydramnios, causes lung
hypoplasia and soft tissue deformities when it occurs at an early stage of pregnancy
affecting the canalicular phase (16–25 WG) of lung development. At birth, the
glomerular filtration rate is low but increases rapidly in line with postnatal hemo-
dynamic changes. Fetal creatinine is cleared by the placenta. At birth, serum
creatinine levels are equal to those of the mother, at around 70 μmol/L, and then
fall during the first week of life (Nolte et al. 1991). Biochemical analysis of
amniotic fluid (Burghard et al. 1988; Oliveira et al. 2002) and mainly fetal urine
(Nicolaides et al. 1992; Muller et al. 1996) has suggested that glomerular filtration
is functional as from 20 WG due to the decreasing low levels of total proteins in
amniotic fluid and urine (Fig. 5). Tubular functions, i.e., secretion and
reabsorption, are initiated later at around 12–14 WG leading to hypotonic fetal
urine relative to plasma, maturing in late pregnancy. In utero, sodium reabsorption
is low and glucose reabsorption is also observed at around 22 WG with an initially
low threshold, increasing during pregnancy. The absence of glucose and phospho-
rus after 20 WG and the increase in calcium in urines are in favor of the maturation
of tubular reabsorption and secretion. The fetal kidney seems able to acidify the
urine and to reabsorb bicarbonate.
Different regulatory systems have been identified in the fetus and develop
gradually during pregnancy. Arginine vasopressin is present but the fetal kidney
only becomes sensitive to it toward the end of pregnancy. The renin-angiotensin
system (RAS) is also expressed early and is independent of the maternal RAS
(Schutz et al. 1996). Renin does not cross the placental barrier. The fetus produces
renin and angiotensin II at higher levels than in the mother; they are involved in
regulating the fetal circulation and in kidney development. Several prostaglandins
are also produced by the kidney and are involved in modulating the fetal RAS.
Fig. 5 Fetal β2-m levels b2-m

during pregnancy. β2-m can be (mg/L)
measured in amniotic fluid
since the end of the first 7
trimester and in fetal urine and
blood in the second half of 6
pregnancy. Levels are higher amniotic fluid
in amniotic fluid compared to
5 fœtal urines
fetal urine and blood. They
decreased significantly during
the third trimester of 4 fœtal blood
pregnancy (Muller et al. 1996;
Burghard et al. 1988). In cord 3
blood, levels were increased
in preterms compared to full-
term neonates (Nolte 2
et al. 1991)
1
1 2 3 cord blood
trimester
Renal Impairment in the Fetus

Fetal renal impairment may have several causes; they may be renal or extrarenal or
endogenous or exogenous (Vanderheyden et al. 2003; Fig. 3b). Some can be detected
by US scanning but it may be difficult to pronounce upon renal functionality and a
future prognosis. As exogenous causes, drugs or medicines taken by the mother may
cross the placenta and impair fetal renal function, leading to a reduction in amniotic
fluid volume. Angiotensin-converting enzyme inhibitors (ACE inhibitors) can directly
disrupt the fetal RAS which can impair both prenatal and postnatal renal functions.
Renal tubular dysgenesis is often noted which may have long-term consequences such
as renal failure and hypertension (Plazanet et al. 2014). Nonsteroidal antiinflammatory
drugs (NSAIDs) can decrease prostaglandin synthesis by cyclooxygenase inducing
transient or irreversible renal impairment (Phadke et al. 2012).
Fetal Nonrenal Causes

Intrauterine growth restriction (IUGR) of placental origin may be associated with
chronic hypoxemia inducing the redistribution of blood flow to vital organs, i.e., the
brain and the heart, at the expense of others such as the kidneys. This may disturb the
physiological increase in the number of glomeruli and their functional maturation
and regulation. Fetuses may therefore develop oligohydramnios and be at risk of
decreased glomerular filtration rates during childhood (Bacchetta et al. 2009). In
twin-twin transfusion syndrome, a major complication of monochorionic twin preg-
nancies, the donor twin may develop reduced renal perfusion. This upregulates the
production of renin and the secretion of angiotensin II which can be transferred to the
recipient twin, thus activating its own RAS (Mahieu-Caputo et al. 2005).
Renal Causes: Nephropathies

Nephropathy can result from abnormal kidney development. Except in a familial
context where previous index cases are confirmed, they may be discovered fortu-
itously by ultrasound scanning (Avni and Hall 2006).
Bilateral renal agenesis can be detected in the case of oligohydramnios or
anhydramnios as the absence of both kidneys, non-visualization of the bladder, or
a failure to visualize the renal arteries using color Doppler US. It is less common than
unilateral agenesis but has a fatal outcome. It is sporadic and usually isolated but
may sometimes form part of a genetic syndrome or abnormal development sequence
(e.g., Fraser syndrome). When unilateral, the bladder can be seen and the amniotic
fluid volume is normal.
Ultrasound scanning is able to detect the abnormal presence of cysts in the kidney
which may indicate a diagnosis of multicystic kidneys or cystic dysplasia (Fig. 4b).
Multicystic dysplastic kidney disease (MCDK) results from non-coordinated devel-
opment of the metanephros and the branching ureteric bud. It usually affects only
one of the two kidneys which becomes enlarged and sometimes deformed, with an
abnormal renal parenchyma and the presence of irregular cysts. When isolated the
prognosis is good and the amniotic fluid volume is normal. The prognosis depends
on the state of the contralateral kidney, which is generally normal or hypertrophic.
The presence of two enlarged and echogenic kidneys with numerous small
cortical cysts is suggestive of infantile polycystic kidney disease (PKD) also referred
to as autosomal recessive cystic kidney disease. It can also be part of few syndromes
as Meckel (Fig. 4c). The prognosis is variable but usually fatal if this condition
appears early in pregnancy, leading to oligohydramnios. Among the rare fetuses
which survive, it often results in chronic renal failure.
Fetal Excretory Tract Abnormalities: Uropathies

Uropathies essentially arise from the obstruction and enlargement of the urinary tract
(Figs. 3a, 4d). This leads to cystic renal dysplasia if a complete obstruction occurs
at an early stage. If the obstruction is intermittent, or develops later in the second
half of pregnancy, long-standing hydronephrosis can cause renal cystic dysplasia,
the severity of which depends on both the degree and duration of the obstruction
(Morris 2008; Longpre et al. 2012).
On an US, dilatation of the renal pelvis (i.e., pyelectasis) or of the calyces may be
indicative of impaired urinary flow. Hydronephrosis refers to dilatation of the renal
pelvis which may be transient and resolve at birth. Unilateral hydronephrosis is more
common than its bilateral counterpart. It can result from an abnormal ureteropelvic
or ureterovesical junction or from urethral obstruction. In the latter case, the obstruc-
tion is bilateral and involves some or all of the urinary tract. The prognosis for upper
urinary tract obstruction is relatively good if it is found in isolation. By contrast,
obstruction in the lower urinary tract (LuTO) may be associated with cystic renal
dysplasia, oligohydramnios, or pulmonary hypoplasia, with a variable prognosis.
Hydroureteronephrosis combines hydronephrosis and an enlarged ureter, which is
common in the case of megacystis. This enlargement may be moderate or severe or
unilateral or bilateral.
Urethral obstruction can result in an enlarged bladder (megacystis) which can be

detected from the end of the first trimester (Fig. 4e). Posterior urethral valve (PUV) is
the principal cause in male fetuses, associating oligohydramnios, a large thick-
walled bladder, and different degrees of bilateral hydronephrosis and hydroureter.
The prognosis is poor for fetuses diagnosed antenatally when an oligohydramnios is
present. In rare cases, it may be associated with normal or increased amniotic fluid
volume, suggestive of megacystis microcolon intestinal hypoperistalsis syndrome.
Megacystis may also be observed in the context of prune belly syndrome.
Assessment of Fetal Renal Function: Usefulness of b2-Microglobulin
The assessment of fetal renal function is based on ultrasound findings which help to
diagnose and monitor nephropathies and uropathies and can be indicative of renal
impairment. Depending on the gestational age, the US should provide detail on
amniotic fluid volume, kidney size and appearance, renal parenchyma,
corticomedullary differentiation, renal dysplasia (presence of cortical cysts and
echogenicity of the renal parenchyma), collecting system, and bladder aspect and
size (Fig. 4). Poor prognostic factors include dilatation of the upper tract, increased
bladder wall thickness, oligohydramnios, echogenic renal cortex, and cysts, partic-
ularly before 24 weeks (Winyard and Chitty 2008; Morris 2008; Longpre
et al. 2012). In certain cases, the biochemical analysis of fetal fluids can be a useful
adjunct to ultrasound findings.
Amniotic Fluid
The analysis of amniotic fluid has been suggested because fetal urine is the principal
source of amniotic fluid as from 20 WG. β2-m is detectable in amniotic fluid, and its
concentration rises after 10 WG when glomerular filtration becomes operational,
picks at around 20–24 WG and then falls (Burghard et al. 1988; Gulbis et al. 1996;
Jauniaux et al. 1998; Oliveira et al. 2002) (Table 1, Fig. 5). The reduction in its
concentration during the third trimester is likely to be related to the maturation of
tubular function. However, because oligohydramnios is often present in the event of
impaired renal function, an amniotic puncture is rarely performed. Furthermore,
because the origin of β2-m in amniotic fluid is not unequivocal, its measurement is of
little value (Kim et al. 2012).
Fetal Blood
The biochemical assay of fetal blood enables the evaluation of glomerular filtration.
Serum creatinine cannot be used because the molecule is very small so it can easily
cross the placenta and be cleared by the mother. Analyses of low-molecular-weight
proteins such as α1-microglobulin and β2-m have been proposed because they
cannot cross the placenta and are entirely filtered and reabsorbed by the nephron
(Nolte et al. 1991). Levels in fetal blood are quite stable around 3–4 mg/l with a trend
to decrease at the end of pregnancy (Table 1, Fig. 5). There is no correlation between
cord blood and maternal blood so that the assessment of maternal blood is of no
Table 3 Cutoff values for fetal serum b2-m in obstructive uropathies. b2-m was measured using
different assays in the serum of fetuses affected by LuTO. Cutoff values were established to
distinguish between LuTO leading to termination of pregnancy or neonatal renal failure, from
LuTO associated with normal neonatal renal function or no renal failure in the first year of life
Fetal serum β2m (mg/L)
5.6 Berry et al. 1995
4.1 Cobet et al. 1996
4.9 Tassis et al. 1997
5 Dommergues et al. 2000
5.6 Bökenhamp et al. 2001
5 Spaggiari et al. 2012
5 Nguyen et al. 2013
interest in case of fetal nephropathies or uropathies. Elevation of these low-

molecular-weight proteins in fetal blood is therefore likely to reflect a reduction in
the number of nephrons and/or an alteration of the glomerular filtration. The first
observation was based on the relationship between serum β2-m levels and neonatal
renal function in 15 affected fetuses with urinary tract or renal malformation,
compared with 64 “unaffected” fetuses (Berry et al. 1995). By applying a cutoff
value of 5.6 mg/L, elevated β2-m levels were found to be both sensitive (80.0 %) and
specific (98.6 %) in predicting a poor outcome (termination of pregnancy or renal
failure at birth). These findings were confirmed by subsequent studies which never-
theless noted an overlap in β2-m levels between the controls and fetuses with urinary
tract anomalies of bad prognosis (Tassis et al. 1997; Nicolini and Spelzini 2001).
Other micro-proteins such as α1-microglobulin had little (Cobet et al. 1996;
Bökenhamp et al. 2001; Nguyen et al. 2013). α1-microglobulin is likely to be
more independent of the gestational age than β2-m which tends to decrease at the
end of pregnancy. Cystatin C could be more specific than β2-m which remains more
sensitive in predicting impaired renal function. A significant correlation has been
found between α1-microglobulin and β2-m and cystatin C. Other studies have
confirmed the usefulness of a cutoff value at around 4–6 mg/L for β2-m (Table 3).
It is now clear that fetal serum β2-m levels rise markedly above 5 mg/L in the context
of bilateral renal agenesis (median: 9.5 mg/L) and uropathies ending in terminal
renal failure after 1 year of life (i.e., serum creatinine >50 μmol/L) (median
5.3–7.4 mg/L) (Muller et al. 2004; Nguyen et al. 2013). In newborns, fetal β2-m is
higher compared to young children with a decrease which is age but not gender
dependent, while serum creatinine increases (Ikezumi et al. 2013). All survivors with
normal postnatal creatinine values (50 μmol/L) had fetal β2-m levels <5 mg/L
(Dommergues et al. 2000). The same team confirmed that with this cutoff value,
elevated serum β2-m levels were still sensitive in the event of obstructive uropathy
complicated by urinary ascites (Spaggiari et al. 2013a). The potential usefulness of
sequential measurement of fetal serum β2-m to enable improvements to the course of
the disease or its treatment has not been clearly established, especially when
compared to the potential additional risk to the fetus resulting from the invasiveness
blood sample collection (Craparo et al. 2007). Indeed, data regarding sampling
intervals are still arbitrary and based on US follow-up.
Very few studies have focused specifically on hypoplastic kidney in which fetal
serum β2-m is likely to be increased (8.3 mg/L, n = 7; Muller et al. 2004). Using the
cutoff value of 5 mg/L, this team showed that β2-m has displayed a positive
predictive value of 87.1 % (n = 31) in the case of hypoplasia in the second half of
pregnancy, identical to those of amniotic fluid volume evaluation (Spaggiari 2013).
β2-m increases with the severity of the oligohydramnios. In the context of cystic
dysplasia, amniotic fluid volume has the same prognostic value as β2-m, which was
elevated (8.8 mg/L, n = 9, Muller et al. 2004) but lack sensitivity. In the case of
nephropathies, the sensitivity and specificity of β2-m have been shown to be poor to
predict renal function, with an important overlap with normal ranges in contrast to
uropathies (Muller et al. 2004; Nguyen et al. 2013). Thus, a normal serum β2-m
value cannot exclude postnatal renal failure. In fetuses exposed to RAS blockers
who present signs of renal impairment, β2-m values 5 mg/L are as reliable as
persistent severe oligohydramnios in predicting poor postnatal renal function during
the first 18 months of life (Spaggiari et al. 2012).
In addition, as fetal serum β2-m may increase in case of viral infection, the interest
of the sampling should be discussed facing the US findings in such a situation
(Fabbri et al. 2011). Results should then be cautiously interpreted.
Fetal Urine
Although amniotic fluid volume depends on fetal diuresis, its composition does not
reflect strictly fetal renal function because its composition fluctuates during preg-
nancy, depending on renal and fecal excretion, respiratory secretion, and placental
exchange. Any contamination of the urinary sample by amniotic fluid enables any
analyses and interpretation (Spaggiari 2013). Fetal urine production has been
documented as early as 10–12 WG. An increase in β2-m urine excretion is a good
indicator of tubular proteinuria resulting from changes in tubular reabsorption
(Fig. 2). The determination of fetal urine parameters has been proposed since the
1980s in the case of uropathies because its collection may lower the pressure in the
urinary tract and enables biochemical analysis (Cromblebome et al. 1990). Different
technologies such as high-field proton nuclear magnetic resonance spectroscopy
(NMR) have shown that LuTO may be associated with glycosuria, aminoaciduria,
and organic aciduria (Foxall et al. 1995). The levels of biochemical fetal urine
compounds fluctuate during pregnancy. Different publications concluded that cur-
rent evidence is insufficient to pronounce upon the clinical usefulness of analyzing
fetal urine alone (Clark et al. 2003; Morris 2008). However, it confirmed that two
tests provided more accurate pointers: Ca >95th percentile for gestational age and
Na >95th percentile for gestational age. Threshold values of urinary Na >100 meq/
L and Ca >2 mmol/L are widely accepted as being predictive of a poor outcome,
with variable sensitivity and specificity (Nicolini et al. 1992). The usefulness of
measuring β2-m in urine was highlighted in 1993 (Lipitz et al. 1993). “Normal”
ranges for several urinary markers in a significant cohort of patients were published
in 1996 (Muller et al. 1996) (Table 1, Fig. 5). Forty-one fetuses presenting with
dyplasia
moderate lesions
8.8 mg/L
normal renal parenchyma
normal renal parenchyma
4.4 mg/L
fœtal serum b2m

1.5 mg/L
Fig. 6 Histological section of the fetal kidney in relation with β2-m levels in LuTO (Luton et al.
2013 with the permission of J Guibourdenche). The fetal kidneys were removed just after termina-
tion of pregnancy. After formalin fixation, 5 μ medio-sagittal paraffin sections were stained with
hematein-eosin-safran for a histological evaluation of the renal parenchyma, i.e., the blastema, the
number of mature glomeruli, the mesangial glomerular fibrosis, the cortical tubules density, and
primitive ducts medullar fibrosis
bilateral obstructive uropathy but no oligohydramnios who subsequently displayed

normal postnatal renal function at age 1–2 years (i.e., serum creatinine <50 μmol/L)
were used as controls. A drop in urinary β2-m levels as from 20WG was correlated
with a reduction in Na values. The normal β2-m median value was 0.96
1.2 mg/L
and levels higher than 4–6 mg/L were predictive of tubular injury. Urinary β2-m is
both specific (0.83) and sensitive (0.80) in predicting elevated serum creatinine at the
age of 2 years. Urinary glucose, calcium, phosphorus, ammonium, and total proteins
were specific but lacked sensitivity in the context of obstructive uropathies (Muller
et al. 1993). Urinary Na and β2-m predicting poor prognosis are correlated with the
severity of the renal injury but to a smaller extent compared to serum β2-m (Daïkha-
Dahmane et al. 1997; Luton et al. 2013) (Fig. 6). Fetal urinary β2-m, but not Na, is
correlated with postnatal clearance at ages 10–17 years, thus helping to predict the
long-term outcome of bilateral uropathies. However, several limitations regarding
the use of urine parameters are often discussed. First of all, cutoff values should be
analyzed more accurately with respect to gestational age because urinary β2-m levels
decrease physiologically and are largely independent of renal function before
20–22 WG. Secondly, it is not always possible to puncture the calyces, and most
samples are collected from the bladder. Unfortunately, urine from the bladder reflects
the renal function of both kidneys. It does not allow to state on the unaffected kidney
on which the prognosis is dependent in the event of urinary tract obstruction.
In addition, the first urine void does not accurately reflect renal function, as the
urine may have been present in the bladder for some time which could influence its
composition. Sequential sampling has been proposed to improve diagnosis and
prenatal management, such as vesicoamniotic shunting, but such procedures are
invasive and do not significantly improve outcome (Biard et al. 2005)
Conclusion
Antenatal investigations to determine fetal renal function and predict postnatal renal
outcome present different degrees of accuracy. Ultrasound findings and their evolu-
tion during pregnancy can be set against biochemical results. Biochemical analysis
of amniotic fluid is of little value, and the benefits of performing an invasive
procedure to collect fetal urine or blood are still a matter of debate. Its indication
is limited to isolated cases of LuTO, bilateral hydronephrosis, and nephropathies. β2-
m is a low-molecular-weight protein that is entirely filtered by the glomeruli and
reabsorbed and degraded by the renal tubules. High β2-m levels in serum are not
unequivocal but mainly reflect a reduction in the number of glomeruli or their
impaired capacity for filtration. They are well correlated to the severity of renal
parenchymal injury. Their increase in urine is due to impaired reabsorption but the
accuracy of this assay is questionable because of the heterogeneity of samples and its
variable correlation with parenchymal injury. The results obtained in fetuses
suspected of being affected may overlap those of normal fetuses because of the
difficulties encountered in establishing normal fetal ranges and defining a good
postnatal renal outcome. This may be due to the fact that there is no linear relation
between β2-m levels and the number of nephrons that remain functional and main-
tain normal β2-m levels. To date, β2-m especially in fetal serum has been shown to be
the most reliable biochemical marker for the prediction of postnatal impairment
when fetal US follow-up is doubtful. However, because β2-m does not constitute a
gold standard, it is likely that new and more reliable biomarkers may emerge in the
future (Taranta-Janusz et al. 2014).
Summary Points
• We aim to update the current knowledge on fetal β2-microglobulin in the human

kidney and urinary tract diseases.
• Renal and urinary tract anomalies can be identified during a routine fetal ultra-
sound scan at 18–20 WG.
• Once diagnosed, the prognosis is dependent on monitoring the amniotic fluid

volume and renal parenchyma.
• Biochemical markers partially reflect the renal parenchyma injury and can
improved the prognosis of the renal function.
• Serum β2-m levels rise when glomerular filtration is impaired, while urinary β2-m
levels increase when tubular reabsorption is affected.
• β2-m determinations reflect renal function at the time of sampling.
• Its measurements in serum and urine can be useful in the context of lower urinary
tract obstruction, bilateral hydronephrosis, and sometimes in nephropathies.
• Discrepancies remain because of the difficulty in defining a good postnatal
outcome, the heterogeneity of the cases studied, the lack of prenatal reference
values, the invasive collection procedures, and the assay to be used.
• However, by applying a cutoff value around 5 mg/L, β2-m constitutes an accurate
marker to predict postnatal renal function that is stable and easy to measure
especially in fetal serum.
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Proteinuric Biomarkers in Chronic Kidney
Disease 23
Claudio Bazzi and Omran Bakoush
Contents
Key Facts of Proteinuria in CKD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518
Determinants of Proteinuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518
Kidney Damage Consequent to Proteinuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
Tubulointerstitial Compartment Damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519
Glomerular Damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521
Measurement of Proteinuria and Risk Stratification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 521
Albuminuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 522
Diabetic Kidney Disease (DKD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 522
Glomerulonephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 524
Studies of Patient Cohorts Including Different Types of GN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 524
Idiopathic Membranous Nephropathy (IMN) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 524
Primary Focal Segmental Glomerulosclerosis (FSGS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 526
IgA Nephropathy (IgAN) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 527
Crescentic IgA Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528
Membranoproliferative Glomerulonephritis (MPGN) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528
ANCA-Associated Vasculitis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 528
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 529
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 530
C. Bazzi (*)
D’Amico Foundation for Renal Disease Research, Milan, Italy
e-mail: claudio.bazzi@alice.it
O. Bakoush
Department of Nephrology, Lund University, Lund, Sweden
Department of Internal Medicine, UAE University, Al Ain, United Arab Emirates
e-mail: Omran.Bakoush@uaeu.ac.ae; Omran.Bakoush@med.lu.se

516 C. Bazzi and O. Bakoush
Abstract
Risk stratification of patients with chronic kidney disease is crucial for early
identification of patients at risk of disease progression and for timely initiation of
treatments to prevent progression and the associated cardiovascular morbidity and
mortality. Proteinuria and its components have been widely used for diagnosis and
staging system of chronic kidney diseases. Albuminuria is used for diagnosis of
kidney disease in the general population and kidney involvement in systemic
diseases such as diabetes. However, albuminuria is not the most powerful predictor
of disease outcome, and the urinary excretion of proteins larger than albumin such as
IgM and IgG shows a higher predictive value: IgM-uria could be helpful for kidney
and cardiovascular risk assessment of diabetic patients, and IgG-uria could be
helpful for risk assessment of glomerulonephritis patients. This chapter presents a
concise review of the clinical value of proteinuria profile for prediction of functional
outcome and responsiveness to treatments in patients with chronic kidney disease.
Keywords
Proteinuric patterns • CKD • Diabetic nephropathy • Glomerulonephritis •
Progression • Remission • Treatment responsiveness
Abbreviations
ACEi Angiotensin-converting enzyme inhibitors
α2m/C α2-macroglobulin/urinary creatinine ratio
Albumin/C Urinary albumin/urinary creatinine ratio
ANCA Antineutrophil cytoplasmic antibody
Anti-PLA2R Anti-phospholipase A2 receptor antibody
α1m α1-microglobulin
β2m β2-microglobulin
β-NAG N-acetyl-β-D-glucosaminidase
CSA Cyclosporine A
CYC Cyclophosphamide
CV Cardiovascular
DKD Diabetic kidney disease
ESKD End-stage kidney disease
FE Fractional excretion
FE α1m Fractional excretion of α1-microglobulin
FE IgG Fractional excretion of immunoglobulin G
GBM Glomerular basement membrane
GCW Glomerular capillary wall
GFB Glomerular filtration barrier
GGS Global glomerular sclerosis
23 Proteinuric Biomarkers in Chronic Kidney Disease 517
HR Hazard ratio
hsCRP High-sensitive C-reactive protein
IgA Immunoglobulin A
IgAN IgA nephropathy
IgM Immunoglobulin M
IMN Idiopathic membranous nephropathy
MCD Minimal change disease
MPGN Membranoproliferative glomerulonephritis
MW Molecular weight
NS Nephrotic syndrome
PTECs Proximal tubular epithelial cells
ROC analysis Receiver-operating characteristic analysis
RTX Rituximab
sCr Serum creatinine
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SG Surviving glomeruli: glomeruli without global sclerosis
SI IgG/transferrin selectivity index
TID Tubulointerstitial damage
TGF-β Transforming growth factor β
Key Facts of Proteinuria in CKD
• Proteinuria is the presence in urine of amount of proteins exceeding the normal

value (150 mg/24 h).
• Methods of measurement of proteinuria:
– Total proteinuria measured in 24-h urine collection
– Protein/creatinine ratio: ratio between total or single proteins and urinary
creatinine expressed as g or mmol in morning urine sample
– Fractional excretion of single proteins: clearance of protein related to creati-
nine clearance
• Determinants of proteinuria in CKD: increased loss of proteins across damaged
glomerular wall (glomerular filtration barrier, GFB) and reduced reabsorption by
proximal tubular epithelial cells (PTECs).
• Proteinuric pattern: the presence in urine of proteins with different molecular
weight (MW) may allow a classification of proteinuria: glomerular (only middle
and high MW proteins); tubular, low MW proteins and trace amount of albumin;
and mixed, the presence of high, middle, and low MW proteins.
• Clinical significance of proteinuria: proteinuria is a marker of disease severity
(defined nephrotic when it exceeds 3.5 g/24 h); persistent proteinuria is itself
responsible of further renal damage at tubulointerstitial and glomerular level
triggering a vicious circle of nephron loss and worsening renal function.
• Risk stratification by amount and type of proteinuria: in each CKD the variable
amount and type of proteinuria between patients allow a risk stratification iden-
tifying patients with high probability of progression to renal failure and patients
with low probability of progression.
• Responsiveness to treatments: the ability of risk stratification to predict renal
outcome and in some CKD to identify responsiveness to treatments may be useful
to guide decisions on the start and type of treatment.
Introduction
Patients with chronic kidney disease (CKD) frequently progress to end-stage kidney
disease (ESKD) or succumb to cardiovascular disease. This is a costly worldwide
public health problem (Jha et al. 2012). In the USA and Europe, about 10 % of adults
suffer from CKD, mostly due to diabetes mellitus (DM), hypertension, and glomer-
ulonephritis (Levey et al. 2009; Eckardt et al. 2013). In clinical practice, proteinuria
is not only a biomarker for the diagnosis and severity of CKD but is also associated
with increased risk of progression to ESKD, ischemic heart disease, heart failure,
cerebrovascular insults, and cardiovascular death (Liu et al. 2003; Jafar et al. 2009).
This association exists even after adjustment for other traditional atherosclerotic risk
factors (diabetes, hypertension, dyslipidemia, and smoking) and nontraditional risk
factors, such as elevated plasma levels of C-reactive protein (Arnlov et al. 2005;
Stehouwer and Smulders 2006).
Determinants of Proteinuria
The glomerular filtration barrier (GFB) normally does not allow the passage of high
molecular weight (MW) proteins such as IgG (150 kDa), α2-macroglobulin
(720 kDa), and IgM (900 kDa), while the low MW proteins [α1-microglobulin
(α1m, 31.8 kDa), β2-microglobulin (β2m, 11.8 kDa), and retinol-binding protein
(20 kDa)] are freely filtered and almost completely reabsorbed by proximal tubular
epithelial cells (PTECs). The middle MW protein albumin (67 kDa) passes through
GFB in small amounts (26 g/day) and is also almost completely reabsorbed by
PTECs. The main known mediators of protein reabsorption by PTECs are megalin
(Srorm et al. 2013), cubilin (Amsellem et al. 2010), amnionless, neonatal Fc receptor
(FcRn) (Rath et al. 2013), and CD36 (Baines et al. 2012).
The pathologically damaged GFB allows the passage of increased amounts of
middle and high MW proteins according to the type and severity of damage
(D’Amico and Bazzi 2003a). Maintenance of GFB integrity depends on structural
and functional interactions among its three components: the fenestrated endothelium
and its cell surface layer, the glomerular basement membrane (GBM), and the
visceral epithelial cell layer (podocytes) (Haraldsson et al. 2008; Lowik
et al. 2009; Toblli et al. 2012; Menon et al. 2012). The endothelial cell layer is
characterized by fenestrae of 50–100 nm in size covered by negatively charged
proteoglycans (glycocalyx) that contribute to size and charge perm-selectivity and

restrict the passage of the negatively charged albumin. The GBM consists of type IV
collagen and laminin along with sulfated proteoglycans (Miner 2011). Podocytes
and their slit diaphragms contribute to the size selectivity of the barrier. The
molecular structure of podocytes has been elucidated mainly in familiar and exper-
imental forms of focal segmental glomerulosclerosis (Gbadegesin et al. 2011). Sev-
eral molecules are responsible for maintenance of podocyte integrity: nephrin,
podocin, alpha-actinin 4, CD2-associated protein, transient receptor potential cation
channel type 6, Wilms’ tumor 1, phospholipase C epsilon-1, and lamininβ2. Alter-
ation of the GFB is characterized by the opening of nonselective channels (pores)
that allow high and middle MW proteins to escape into the urinary space.
The molecular machinery for reabsorption cannot handle the elevated tubular
load of proteins passing through the damaged GFB. This leads to increased urinary
excretion of albumin and high MW proteins. Also, the urinary excretion of LMW
proteins is increased (tubular proteinuria) due to the competition for tubular
reabsorption between high, middle, and low MW proteins and due to the functional
and structural alteration of PTECs (Baines and Brunskill 2011). The proteinuria and
the functional/structural alterations in PTECs are also responsible for the increased
urinary excretion of the lysosomal enzyme N-acetyl-β-D-glucosaminidase (β-NAG),
a reliable marker of tubular damage (Bazzi et al. 2002; D’Amico and Bazzi 2003b).
Thus, the quantity and composition of proteinuria in final urine depend on a complex
interplay between filtration and reabsorption.
Kidney Damage Consequent to Proteinuria
Proteinuria is strongly associated with the risk of progression to ESKD not only as
marker of the degree of structural alterations of GFB and PTECs but also because
proteinuria itself is responsible of further renal damage at different levels.
Tubulointerstitial Compartment Damage
Regardless of the underlying cause, various types of proteinuric CKD may share a
common pathogenetic mechanism of kidney disease progression. Brenner and
coworkers, using the “remnant kidney” model, proposed a hypothesis for the
mechanism of kidney disease progression. Brenner hypothesized that nephron loss
increases glomerular hydraulic pressure and stretches the glomerular capillary wall
(GCW), impairing its selectivity and increasing the protein content of the glomerular
filtrate (Olson et al. 1982). PTECs persistently exposed to urinary proteins increase
their expression of chemokines, cytokines, growth factors, and vasoactive molecules
and activate complement, which together induce the infiltration of interstitial inflam-
matory cells (Ruggenenti 2012a; Cravedi and Remuzzi 2013) (Fig. 1). This leads to
renal scarring and initiation of a vicious circle of further nephron loss and worsening
renal function. Several studies showed a strong correlation between some proteinuria
Glomerular damage
Plasma proteins
Tubular cells
MHC TNF-a PDGF

ICAM-1class II CD40 RANTES IL-6 Transdifferentiation
ET-1 FGF-1
antigens MCP1 into fibroblast
GFG-2
Osteopontin IL-8
TGF-b1
CD40L
IL-1
IL-2 TNF-a
Interstitium IFN-g PDGF

TGF-b1
T-lymphocyte Macrophage
TFG-b1
IL-4
TNF-a TGF-b1
IFN-g Fibroblast Myofibroblast
Synthesis
Degradation
Extracellular
Matrix proteins
Fig. 1 Molecular and cellular features associated with tubulointerstitial compartment damage
(From Jorge Toblli et al., Int J Nephrol, 2012 with permission. Courtesy of Dr. Jorge Toblli)
components and the extent of tubulointerstitial damage (TID) evaluated histologi-

cally or by proteinuric markers of TID. It has been demonstrated that the extent of
TID depends not only on the amount but also on the type of proteinuria, as it is
significantly associated with increased excretion of high MW proteins such as IgG
(Bazzi et al. 2000). Lowering capillary pressure by RAS inhibition reduces protein-
uria and the decline in kidney function, as has been demonstrated in several
experimental and clinical studies in diabetic and nondiabetic patients (van der
Meer et al. 2010). Another important determinant of TID is interstitial hypoxia
caused by reduction of blood flow in the interstitial compartment due to vasocon-
striction of efferent arterioles due to local activation of the renin-angiotensin system,
increased synthesis of endothelin, and reduced synthesis of nitric oxide. Global
glomerular sclerosis and interstitial fibrosis reduce the number of peritubular capil-
laries and increase the distance between capillaries and tubular cells further
compromising the reabsorption of proteins by PTECs (Nangaku 2004, 2006;
Nangaku et al. 2007).
Glomerular Damage
Proteinuria is associated not only with TID but also with glomerular damage.
Excessive protein load on podocytes induces overexpression of TGF-β (Loeffler
and Wolf 2014; Ghayur and Margetts 2013), which may lead to GBM thickening,
podocyte apoptosis and detachment, differentiation of mesangial cells into
myofibroblasts, increased extracellular matrix production, and dysregulation of the
balance between extracellular matrix deposition and breakdown with consequent
development of glomerulosclerosis.
Thus the amount and persistence of proteinuria is one of the main factors respon-
sible for progressive kidney damage and worsening renal function. In the last two
decades, several studies evaluated the value of urinary excretion of high and low
molecular weight (MW) proteins for predicting disease outcome, responsiveness to
treatment, and performing risk stratification aimed at improving clinical practice.
Measurement of Proteinuria and Risk Stratification
Critical to reducing the kidney disease burden of ESKD is early diagnosis and timely
initiation of treatments to prevent disease progression (Levey et al. 2009). Dipstick is a
semiquantitative method used mainly for preliminary diagnostic assessment of
suspected kidney disease (White et al. 2011; Samal and Linder 2013). Twenty-four-
hour proteinuria had been widely measured in the past, but is now rarely used because
urine collection is cumbersome and frequently incorrect. The protein/creatinine ratio in
the first or second morning urine sample, which is highly correlated with 24-h protein-
uria (Ruggenenti et al. 1998), is at present the most frequently used method for
measurement of proteinuria (Mc Taggart et al. 2012). Fractional excretion (FE) of
single proteins in relation to creatinine clearance is a better proteinuric marker com-
bining two predictors of kidney function decline: increased protein excretion and GFR,
a marker of functioning nephron mass (Park and Kim 2011).
Depending on the type of kidney disease, different agents are used to prevent
kidney disease progression. They are mainly immunosuppressive (IS) agents for
glomerulonephritis (GN) patients and RAS inhibitors for diabetic and GN
patients. Unfortunately, the response to treatment is heterogeneous in CKD
patients for both unknown and known reasons, including incomplete knowledge
of the etiology and pathogenetic mechanisms of lesions and the different clinical
features at presentation. Thus, it is of paramount importance in the management
of CKD to identify the risk factors that can accurately predict the functional
outcome (Halbesma et al. 2011; Tangri et al. 2013) and can be used for risk
stratification in order to assess drug responsiveness and develop more targeted

treatment. Risk stratification could also be useful for the planning of controlled
trials to avoid the confounding effect of including different percentages of low-
and high-risk patients in the trial arms. Knowledge of the pathogenetic mecha-
nisms of kidney damage has greatly increased in the last decades. Genetic and
molecular studies have elucidated several damage mechanisms at the glomerular,
tubular, interstitial, and vascular levels, but assessment of these markers is often
limited to research laboratories and is frequently rather expensive and not avail-
able in the clinical practice. Conversely, laboratory devices for measuring the
components of proteinuria, such as nephelometry and turbidimetry, are available
in most hospital laboratories.
Here, we review the potential use of different urine proteins, such as albuminuria,
IgG-uria, and IgM-uria, for diagnosis, risk stratification, and prediction of disease
outcome in CKD patients.
Albuminuria
Measurement of urinary albumin excretion in the general population can identify

patients at risk of development of kidney failure (Astor et al. 2011). A low eGFR and
albuminuria are associated with adverse kidney outcomes (Gansevoort et al. 2011).
Values of eGFR <60 mL/min/1.73 m2 and albumin/creatinine ratio 10 mg/g could
predict a high risk for cardiovascular (CV) mortality in the general population
(Matsushita et al. 2010). Mild albuminuria may progress to heavier albuminuria,
so its follow-up is indicated. Although albuminuria is important for early diagnosis
of kidney diseases, not all glomerulonephritis patients with albuminuria progress to
kidney failure, and not all diabetic patients with kidney disease develop albuminuria
(Cohen-Bucay and Viswanathan 2012). Up to 30 % of diabetic patients with diabetic
kidney disease (DKD) have normal urine albumin excretion despite the decline in
kidney function and progressive vascular endothelial damage (Marshall 2014).
Many recent longitudinal epidemiological studies of patients with diabetes and
glomerulonephritis have pointed out the inferiority of albuminuria compared to
other high MW proteins such as IgG and IgM for prediction of kidney disease
outcome (Bakoush et al. 2001a, b, 2003, 2006).
Diabetic Kidney Disease (DKD)
The pathophysiological mechanisms of DKD could differ between type 1 and type
2 diabetes (Fioretto et al. 2008; Bakoush et al. 2002; Steinke and Maurer 2008). In
type 1 diabetes, hyperglycemia could be the main mechanism for DKD, whereas in
type 2 diabetes, the metabolic syndrome (obesity, hypertension, and dyslipidemia) and
hyperglycemia are major contributors to DKD (Altemtam et al. 2012) (Fig. 2). In
DKD, urinary excretion of albumin is used to differentiate normo-albuminuria
(<30 mg/g uC) from microalbuminuria (30 and <300 mg/g uC) and
- RAS activation
- Oxidative stress
Diabetes,Hypertension, - Inflammation
Hyperglycaemia, - Fibrosis stimulation
Dyslipidaemia, etc. - Decrease capillary
bloodflow
Vascular endothelial
damage and
atherosclerosis
Ischemic glomerular
Coronary artery disease Cerebrovascular disease
disease
- Acute myocardial
Ischemic and Increase number of
infarction
- Angina haemorrhagic stroke shunt-pathways
- Heart failure
- Arrhythmia
IgM-uria
Fig. 2 A proposed mechanism for association of IgM-uria with cardiovascular disease
macroalbuminuria (300 mg/g uC) (Cohen-Bucay and Viswanathan 2012). In recent

years it has been found that high values of normo-albuminuria are also associated with
DKD progression and cardiovascular morbidity and mortality. Consequently, it has
been suggested that the category of microalbuminuria should be eliminated
(Ruggenenti and Remuzzi 2006). However, irrespective of the level of albuminuria,
increased urine IgM excretion in patients with type 1 or type 2 diabetes was associated
with significantly lower kidney and patient survival. The presence of IgM-uria
increased the risk of kidney and cardiovascular death threefolds (Tofik et al. 2009)
in patients with micro- or macroalbuminuria (Fig. 2). IgM-uria was found to be
associated with a risk of the occurrence of subsequent cardiovascular events in patients
with coronary artery disease with acute chest pain (Tofik et al. 2013). Markers of
general inflammation, such as pro-inflammatory cytokines and high-sensitive C-reac-

tive protein (hsCRP), are also associated with cardiovascular disease (Kaptoge
et al. 2010; Tedgui and Mallat 2006). However, recent studies on large cohorts raised
doubts about the clinical value of these markers in the estimation of cardiovascular risk
in nondiabetic and diabetic patients (Schottker et al. 2013; Kaptoge et al. 2012).
Although the strength of the association between IgM-uria and the degree of
atherosclerosis should be studied, the presence of IgM-uria might reflect severe size-
selectivity dysfunction of the GFB, secondary to generalized atherosclerosis and
systemic inflammation. Atherosclerosis is associated with elevated glomerular vas-
cular wall resistance, resulting in glomerular ischemia and a markedly increased
population of highly unselective glomerular shunt pathways (Bakoush et al. 2004,
2006; Rippe et al. 2006).
Glomerulonephritis
More than 50 years after the introduction of kidney biopsy and identification of
several types of primary and secondary GN, the etiology and pathogenesis of GN are
still largely unknown. There is no standard first-line treatment that is effective in all
patients of each type of GN. Several clinical, functional, laboratory, and histologic
features have been evaluated to assess disease severity and predict functional
outcome and responsiveness to treatment.
Studies of Patient Cohorts Including Different Types of GN
In 89 patients with nephrotic syndrome [9 minimal change disease (MCD), 29 pri-

mary focal segmental glomerulosclerosis (FSGS), 51 idiopathic membranous
nephropathy (IMN)] (Bazzi et al. 2000), the outcome was predicted by a combina-
tion of IgG/transferrin selectivity index and the TID marker fractional excretion of
α1-microglobulin (FE α1m). In 45 patients with selectivity index >0.10 and FE α1m
above a cutoff value, the remission rate was 6 %, and progression rate was 69 %. In
the 30 patients who were treated with steroids alone or in combination with cyclo-
phosphamide (CYC), the remission rate was 75 % in low-risk patients, and 10 % in
high-risk patients. In a cohort of 97 patients with various types of GN but without
nephrotic syndrome (NS) (Mc Quarrie et al. 2011), fractional excretion of IgG and
albumin was a good predictor of kidney disease progression and was more accurate
than several other proteinuric markers. In a cohort of 189 patients with various types
of GN (Tofik et al. 2011), urinary IgG/creatinine ratio of more than 5 mg/mmol was
associated with the highest rate of kidney disease progression to ESKD.
Idiopathic Membranous Nephropathy (IMN)
In a study of 57 patients with IMN and NS (Branten et al. 2005), renal survival varied
significantly with urinary β2m (< vs 0.5 μg/min) and urinary IgG (< vs 250 mg/
24 h). The authors concluded that excretion of β2m and IgG accurately predicts renal
Table 1 Predictive value of progression and remission by fractional excretion of IgG alone (IMN,
FSGS, MPGN) or divided by percentage of glomeruli without global sclerosis (SG surviving
glomeruli) (IgAN and crescentic IgAN)
No. of Proteinuric
Diagnosis Reference patients biomarkers Progression Remission
IMN Bazzi et al. 84 FE IgG <0.020 3 % vs. 43 % 90 % vs.
2014 vs. ≥0.020 P = 0.003 24 %
P < 0.001
Untreated FE IgG <0.020 12 % vs. 0 % 94 % vs.
n. 35 vs. p: ns 85 %
treated p: ns
with ST + FE IgG ≥0.020 72 % vs. 43 % 0 % vs. 36 %
CYC n. 37 P = 0.014 P = 0.025
FSGS Bazzi et al. 38 FE IgG <0.112 0 % vs. 75 % 77 % vs.
2013 vs. ≥0.112 P < 0.0001 25 %
P = 0.016
FE IgG <0.112 0 % vs. 89 % 83 % vs.
and α2m/C P < 0.0001 11 %
<4.79 vs. FE P = 0.008
IgG ≥0.112
and α2m/C ≥
4.79
IgAN Bazzi et al. 68 ACEi − FE IgG/SG < 2 % vs. 87 %
2012 vs. ≥0.00010 P < 0.0001
64 ACEi + FE IgG/SG < 2 % vs. 36 %
vs. ≥ 0.00010 P < 0.0001
Crescentic Bazzi et al. 34 FE IgG/SG < 0 % vs. 89 %
IgAN 2009 vs. ≥0.00034 P < 0.0001
23 treated FE IgG/SG 0 % vs. 100 %
steroids + <0.00034 + P < 0.0001
cycloph sCr vs. <1.74
treated mg/dL vs. FE
IgG/SG ≥
0.00034 + sCr
≥ 1.74
MPGN Bazzi et al. 18 FE IgG < vs. 9 % vs. 86 %
2002 ≥0.180 P = 0.002
outcome and can be used to guide decisions on the start of immunosuppressive

treatment. In a subsequent study on 129 patients with IMN and NS, the same group
(van der Brand et al. 2011) confirmed the predictive value of the progression of
urinary excretion of the tubular markers α1-microglobulin and β2-microglobulin. In
IMN with NS, the efficacy of various IS agents remains controversial, as evidenced in
the last meta-analysis published in 2013 (Chen et al. 2013). In a study of 86 patients
with IMN and NS (Bazzi et al. 2014), the proteinuric marker with the highest
sensitivity and specificity for progression was FE IgG (cutoff, 0.020). Kidney sur-
vival and remission rate were significantly higher in low- (FE IgG <0.020) versus
high-risk patients (FE IgG 0.020) (Table 1, Fig. 3). Comparison of 35 untreated
patients with 37 patients treated with steroids and cyclophosphamide (CYC)
Fig. 3 Kidney survival and remission rate in 84 patients with IMN and NS according to FE IgG <
vs 0.020
according to a published protocol (Ponticelli et al. 1998) showed that in untreated

low-risk patients, progression is very low (12 %), and spontaneous remission is very
high (94 %). In high-risk patients, treatment reduced progression from 72 % to 43 %
and increased the remission rate from 0 % to 36 % (Table 1). Rituximab (RTX), a new
and promising immunosuppressive agent for treatment of patients with IMN with NS,
induced complete or partial remission in 65 of 100 treated patients: in 32 patients
RTX was used as rescue treatment because of previous unresponsivity to various
immunosuppressive or anti-proteinuric agents (Ruggenenti et al. 2012b). In a study of
20 patients with IMN and NS (Irazabal et al. 2013), fractional excretion of IgG and
α1m was associated with the response to rituximab at 12 months, but not at 24 months
(overall remission rate was 89 % at 24 months). In these patients, a decrease in anti-
PLA2R antibodies preceded the decline in proteinuria, but baseline anti-PLA2R
levels did not correlate with the response to therapy. Thus, in IMN patients, FE IgG
may be used to guide the treatment: conservative treatment is recommended for
low-risk patients, unless severe NS is present with risk of complications. Immuno-
suppressive agents or rituximab therapy of high-risk patients should be started soon
after diagnosis to avoid the inefficacy of late treatment, when kidney disease damage
may become irreversible.
Primary Focal Segmental Glomerulosclerosis (FSGS)
Very few studies evaluated the predictive value of proteinuric patterns in primary
focal segmental glomerulosclerosis (Bazzi et al. 2003; Deegens and Wetzels 2007).
In the largest and more recent study on 38 patients with primary FSGS and NS
(Bazzi et al. 2013), FE IgG and α2-macroglobulin/creatinine ratio showed the
Fig 4 Probability of ESRD and remission in 38 patients with FSGS and NS according to FE IgG
and α2-macroglobulin/Cr < vs respective cutoffs
highest sensitivity and specificity for progression to ESKD or remission (Table 1,

Fig. 4). In the low-risk group with a combination of FE IgG <0.112 and α2m/C
<4.79, 89 % of the patients had remission as first event and 78% of them had
sustained remission after 104
54 months. In 22 % of these cases, remission was
obtained with steroids alone and in 61 % with steroids in combination with CYC;
17 % were unresponsive to steroids and CYC and remitted after treatment with other
agents (mycophenolate mofetil, pentoxifylline, cyclosporine A). In the high-risk
group (FE IgG 0.112 and α2m/C 4.79), 89 % of the patients progressed to
ESKD after 32
35 months; one patient was treated with steroids alone and eight
were treated with steroids and CYC. Other markers (urinary protein/creatinine ratio,
eGFR, FE α1m, segmental sclerosis, global sclerosis, and TID score) did not predict
remission and had a lower predictive value of ESKD. The best predictor for ESKD
was α2m/creatinine ratio and for disease remission it was FE IgG. The disease
outcome predicted by these proteinuric markers is more accurate than that of the
old long-term predictor of favorable outcome that was steroid-induced remission
(about 30 %). These proteinuric biomarkers could be useful for individualizing
treatment of patients with FSGS with NS.
IgA Nephropathy (IgAN)
In the last two decades, several laboratory methods for evaluating different features of
proteinuria have been tested for their ability to predict outcome in IgAN (SDS-PAGE
pattern, selectivity index, and proteomic analysis) (Woo et al. 1991, 1997; Haubitz
et al. 2005). In a recent study of 132 patients with non-crescentic IgA nephropathy
(Bazzi et al. 2012), global glomerular sclerosis (GGS) varied from 0 % to more than
50 %. To assess glomerular loss and tubular load of proteins more precisely, the
proteinuric markers were divided by the percentage of glomeruli without GGS,

conventionally referred to as “surviving glomeruli” (SG). The fractional excretion of
IgG and α1m and 24-h proteinuria were, respectively, 40-, 13-, and ninefold higher in
patients with <50 % of SG than in those with 100 % of SG. These results validate the
experimentally observed association between nephron loss and increased proteinuria.
FE IgG/SG proved to be the best predictor of kidney disease progression to kidney
failure (Table 1). In patients with FE IgG/SG above the cutoff, treatment with ACE
inhibitors reduced kidney disease progression from 87.5 % to 36 %. In patients with
FE IgG/SG below the cutoff, the increase in eGFR over time was significantly higher
in ACEi-treated patients than in untreated patients.
Crescentic IgA Nephropathy
In a study of 37 patients with crescentic IgA nephropathy (Bazzi et al. 2009) (cellular
crescents in 19
15 % of glomeruli), fractional excretion of IgG was strongly
correlated with baseline sCr (r = 0.749), percentage of global glomerular sclerosis
(GGS) (r = 0.536), cellular crescents (r = 0.414), and TID score (r = 0.725). The
values of proteinuric markers were divided by the percentage of glomeruli without
GGS, conventionally defined as “surviving glomeruli” (SG). In 34 patients FE
IgG/SG below or above a cutoff level assessed by ROC analysis was the most
powerful predictor of kidney disease progression to kidney failure: 5 % vs. 83 %.
In 23 patients treated with steroids and CYC soon after kidney biopsy, FE IgG/SG
in combination with serum creatinine, both below and above their respective cutoffs,
predicted kidney disease progression in 0 % versus 100 % of patients (Table 1).
These data suggest that high-risk patients should be treated with alternative agents.
Membranoproliferative Glomerulonephritis (MPGN)
Only one study evaluated the predictive value of proteinuric markers in MPGN.
Bazzi et al. (Abstract, J Am Soc Nephrol 2002; 13: SA-P905; Int. Congress Am. Soc.
Nephrol. 2002) studied a cohort of 30 patients with membranoproliferative glomer-
ulonephritis (type I, n = 23, type II n = 1, type III n = 4, fibrillary type n = 2). They
reported that FE IgG <0.180 predicted progression in 9 % of 18 patients with NS,
whereas FE IgG 0.180 predicted progression in 86 % of them.
ANCA-Associated Vasculitis
In 83 patients with ANCA-associated renal vasculitis (Bakoush et al. 2006), IgM

excretion was a strong predictor of ESKD, and in addition to age, it predicted the risk
of cardiovascular death. IgM-uria reflects a markedly increased population of shunts
and large defects in the GFB and thereby is a good indicator of the severity of
glomerular damage. Serum creatinine is a late kidney biomarker and is associated
with cardiovascular mortality only in cases with severe kidney impairment. In that
study, IgM-uria was able to predict the risk in patients with relatively good kidney
function and thus was a better predictor of disease outcome.
Summary
In CKD patients, the first physiopathological alteration is the dysfunction of the

GFB, which allows passage of proteins normally absent in urine. Later, proteinuria
itself causes further kidney damage in the glomerular and tubulointerstitial compart-
ments. The characteristics of the overall proteinuric pattern thus depend on the
interplay of glomerular and tubulointerstitial damage. The urinary proteins more
frequently evaluated for prediction of kidney disease progression are albumin, IgG,
IgM, α2-macroglobulin, and low MW proteins. Albuminuria in combination with
low eGFR predicts the risk of adverse kidney outcomes, cardiovascular diseases, and
death. In diabetes, IgM-uria is associated with progression of DKD and risk of
cardiovascular events. In nine studies collectively covering 674 patients with glo-
merulonephritis (GN), IgG-uria (expressed as IgG/creatinine ratio or more fre-
quently as fractional excretion) seems to be the best predictor of disease outcome.
In non-crescentic IgA nephropathy, FE IgG predicts responsiveness to ACE inhib-
itors. In IMN, treatment of FE IgG high-risk patients with steroids and CYC reduced
the progression rate from 72 % to 43 % and increased the remission rate from 0 % to
36 %. In primary FSGS, 89 % of FE IgG and α2m/creatinine ratio high-risk patients
were unresponsive to treatment with steroids and CYC, and 78 % of low-risk
patients had sustained remission. In crescentic IgA nephropathy, none of the patients
assessed as high risk according to FE IgG and sCr responded to steroids and CYC.
So, in glomerulonephritis the urinary excretion of IgG is not only a strong predictor
of functional outcome but also a valuable predictor of responsiveness to some
treatments. We recommend evaluation of proteinuric patterns at diagnosis of kidney
disease because of its low cost and its potential influence on outcome through early
identification and management of patients at high risk of kidney disease progression.
Summary Points
• The frequent progression of CKD to end-stage kidney failure and cardiovascular

morbidity and mortality is a costly worldwide public health problem.
• Risk stratification of patients with CKD disease is crucial for early identification
of patients at risk of kidney disease progression and cardiovascular morbidity and
mortality and for timely initiation of preventive treatments.
• This chapter focuses on the usefulness of proteinuric patterns for prediction of the
functional outcome (progression, remission) and responsiveness of CKD to some
treatments.
• The glomerular filtration barrier (GFB) normally does not allow the passage of
high molecular weight (MW) proteins (IgG, IgM, α2-macroglobulin); low
amounts of the middle MW albumin pass through and are almost completely
reabsorbed by proximal tubular epithelial cells (PTECs); low MW proteins (α1-
microglobulin, β2-microglobulin, retinol-binding protein) pass through freely and
are almost completely reabsorbed by PTECs.
• Proteinuria is dependent on alteration of glomerular filtration barrier and func-
tional or structural alteration of PTECs; the urinary protein amount and compo-
sition measured in final urine are consequent to complex interplay between
filtration and reabsorption.
• Measurement of albuminuria is useful for preliminary diagnosis of suspected
kidney disease.
• However, albuminuria is not as strong predictor of kidney disease progression as
the urinary excretion of proteins larger than albumin such as IgM and IgG.
• IgM-uria could be helpful for the assessment of kidney and cardiovascular disease
risk in diabetic patients.
• IgG-uria could be helpful for assessment of risk in glomerulonephritis. Fractional
excretion of IgG, a marker that combines two predictors of kidney function
decline, is a much better predictor of the functional outcome and responsiveness
of primary glomerulonephritis to treatment.
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Apelin and Copeptin as Biomarkers
of Kidney Disease 24
Antonio Lacquaniti, Valeria Chirico, Valeria Cernaro, Rosaria Lupica,
Antonio David, and Michele Buemi
Contents
Key Facts of Glomerular Filtration Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 537
Key Facts of Vasopressin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 537
Key Facts of Autosomal Dominant Polycystic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 537
Key Facts of End-Stage Renal Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538
Key Facts of Aquaretics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 538
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 539
Physiologic Point of View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 541
Pathological Involvement of Copeptin and Apelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 543
Diabetes Mellitus and Kidney Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544
Autosomal Dominant Polycystic Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547
End-Stage Renal Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548
Potential Applications to Prognosis, Other Diseases, or Conditions . . . . . . . . . . . . . . . . . . . . . . . 548
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554
A. Lacquaniti (*) • V. Cernaro • R. Lupica • M. Buemi

Department of Clinical and Experimental Medicine, University Hospital “G. Martino”, Messina,
Italy
e-mail: ant.lacq@gmail.com; alacquaniti@unime.it; valecern82@gmail.com;
lupicarosaria@alice.it; buemim@unime.it
V. Chirico
Department of Pediatrics, University Hospital “G. Martino”, Messina, Italy
e-mail: valeriachirico@hotmail.it
A. David
Department of Neuroscience and Anesthesiology, University Hospital “G. Martino”, Messina, Italy
e-mail: davida@unime.it

536 A. Lacquaniti et al.
Abstract
The need for faster diagnosis, more accurate prognostic assessment, and treat-
ment decisions in renal diseases has led to the investigations of new biomarkers.
Arginine vasopressin (AVP) is one of the main hormones of the hypothalamic–
pituitary–adrenal axis, and it is mainly stimulated by hyperosmolarity.
AVP plays deleterious renal effects, inducing hypertension, glomerular
hyperfiltration, albuminuria, and glomerulosclerosis, whereas its inhibition, by
drinking water or by V2 antagonism, led to a renoprotection. The direct mea-
surement of AVP in humans is problematic, due to its bond with platelets and its
instability in isolated plasma.
However, copeptin, the C-terminal portion of the AVP precursor and released
into the circulation from the posterior pituitary gland in equimolar amounts with
AVP, represents a measurable substitute. In many studies copeptin mimed AVP
levels and its behavior, modified by plasma osmolality, stress, and various disease
states, revealing some of the various physiologic and pathophysiologic conditions
associated with increased or decreased AVP.
Apelin, belonging to a signaling pathway implicated in the regulation of
cardiovascular function and fluid homeostasis, is highly expressed in hypotha-
lamic nuclei, is co-localized with AVP, and exerts an active diuretic action,
counteracting the effects of vasopressin.
In this chapter, we examine the diagnostic and prognostic role of copeptin and
apelin, reporting the last data about their physiological functions, their receptor
interactions, and their functions in nephrologic field.
Conclusion: Copeptin and apelin represent prognostic marker for nephropathic
patients and predictive factors for progression to end-stage renal disease. Their
measurement may be included in a “renal” work-up for patients at high risk, and
their evaluation may be helpful also to predict a therapy response. However,
further studies are needed in order to make these results more reliable and
applicable in larger populations.
Keywords
Apelin • Copeptin • Vasopressin • APJ signaling • Aquaretic • Tolvaptan • CKD
progression • Kidney biomarkers • ADPKD
Abbreviations
ADH Antidiuretic hormone
ADPKD Autosomal dominant polycystic kidney disease
Ang II Angiotensin II
APJ Angiotensin I receptor-related protein J receptor
AQP Aquaporin
AVP Arginine vasopressin
DM Type 2 diabetes mellitus
24 Apelin and Copeptin as Biomarkers of Kidney Disease 537

HD Hemodialysis
HIF Hypoxia-inducible factor
MI Myocardial infarction
SIADH Syndrome of inappropriate antidiuretic hormone
TKV Total kidney volume
VR Vasopressin receptor
UAE Urinary albumin excretion
Key Facts of Glomerular Filtration Rate
• Glomerular filtration rate (GFR) describes the flow rate of filtered fluid through
the kidney.
• GFR is the best test to measure the level of kidney function and determine the
stage of kidney disease.
• According to the National Kidney Foundation, normal results range from 90 to
120 mL/min/1.73 m2.
• There are recommended equations for estimating GFR from serum creatinine,
such as the Modification of Diet in Renal Disease (MDRD) Study equation and
the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation.
• If GFR estimates are likely inaccurate, a measured GFR is an important
confirmatory test.
Key Facts of Vasopressin
• Arginine vasopressin (AVP) is a peptide hormone formed in the hypothalamus

and then transported via axons to, and released from, the posterior pituitary into
the blood.
• AVP has two principle sites of action: the kidney and blood vessels.
• At renal level, AVP regulates extracellular fluid volume acting on the collecting
ducts via V2 receptors and leading to decreased urine formation.
• For this reason it is also called antidiuretic hormone.
• A secondary function of AVP is exerted in vessels, binding V1 receptors on the
vascular smooth muscle and causing vasoconstriction.
Key Facts of Autosomal Dominant Polycystic Kidney Disease
• Autosomal dominant polycystic kidney disease (ADPKD) is one of the most

common forms of polycystic kidney disease.
• It is a genetic disorder characterized by the growth of numerous cysts in both

kidneys.
• The cysts are filled with fluid, and their progressive expansion slowly replaces
much of the normal mass of the kidneys, reducing kidney function and leading to
kidney failure.
• When ADPKD causes kidneys to fail, the patient requires dialysis or kidney
transplantation.
• ADPKD can be diagnosed using ultrasound, computed tomography scan, or
magnetic resonance imaging studies of the kidneys.
• A genetic test can detect mutations in the PKD1 and PKD2 genes.
Key Facts of End-Stage Renal Disease
• End-stage renal disease (ESRD) occurs when kidney function is permanently lost.
• According to the National Kidney Foundation classification, it is defined by a
glomerular filtration rate <15 mil/min.
• Most cases of ESRD are caused by diabetes or high blood pressure.
• Dialysis or a kidney transplant is necessary to live.
• Possible complications of ESRD include heart and blood vessel problems, mal-
nutrition, or anemia.
Key Facts of Aquaretics
• They are drugs that selectively bind vasopressin receptors in the renal collecting
duct promoting excretion of solute-free water.
• These drugs are also called vaptans.
• Tolvaptan is an orally active selective V2 receptor antagonist approved for the
treatment of the syndrome of inappropriate antidiuretic hormone secretion.
• Tolvaptan may rapidly increase the level of sodium in the blood.
• Tolvaptan would become the first pharmaceutical therapy in Europe for patients
with autosomal polycystic kidney disease.
Definitions
Biomarker Biomarker is an objectively measured and evaluated indicator of nor-

mal biological processes, pathogenic processes, or pharmacological responses to a
therapeutic intervention.
Brain natriuretic peptide BNP is a substance secreted from the ventricles or lower
chambers of the heart in response to changes in pressure that occur when heart failure
develops and worsens.
Cellular receptor A receptor is a structure on the surface of a cell (or inside a cell)
that selectively recognizes and binds with specific molecules, producing a specific
effect in the cell.
Hemodialysis Hemodialysis is the most common method used to treat advanced

and permanent kidney failure. This medical procedure is based on the removal of
fluids and waste products from the blood, correcting electrolyte imbalances. This is
accomplished using a machine and a dialyzer, also referred to as an “artificial kidney.”
Peritoneal dialysis Peritoneal dialysis represents another renal replacement ther-

apy, using, as a filter, the lining of the abdominal cavity (peritoneal membrane) and a
solution (dialysate), infused through a specific catheter.
Renin–angiotensin–aldosterone system The renin–angiotensin–aldosterone sys-

tem (RAAS) is a signaling pathway responsible for regulating the body’s blood
pressure. As the name implies, there are three important components to this system:
(1) renin, (2) angiotensin, and (3) aldosterone.
Test sensitivity It evaluates the probability that a test result will be positive when
the event is present.
Test specificity It measures the probability that a test result will be negative when
the event is not present.
Total kidney volume Total kidney volume (TKV) growth is considered the best
surrogate marker predicting the decline of renal function in autosomal dominant
polycystic kidney disease. This datum is obtained by magnetic resonance imaging
scans with an optimized T1-weighted acquisition protocol without gadolinium-
based contrast agents.
Introduction
Kidney disease is characterized by progressive destruction of the renal parenchyma

and loss of functional nephrons, leading to chronic renal failure. In clinical practice,
the diagnosis is dependent on levels of unreliable biomarkers, such as blood urea,
nitrogen, and serum creatinine, characterized by low sensitivity and specificity. In
fact, the time relationship between changes in serum creatinine and concomitant
changes in glomerular filtration rate (GFR) does not allow accurately estimating
timing of injury and severity of renal dysfunction (Lacquaniti et al. 2013a).
Moreover, creatinine levels are affected by several confounding factors, such as diet
and muscle mass, and, despite the use of weighting for age, gender, and race in the GFR
estimating equations, this can result in misclassification of patients (Tangri et al. 2011).
Arginine vasopressin (AVP), also known as antidiuretic hormone (ADH), plays
an important role in the regulation of volume status. It is secreted into the blood if
dehydration (increase in plasma osmolality) or volume loss occur, acting through a

water reabsorption in the nephron tubules by binding to the AVP V2 receptor
(VR) (Birnbaumer 2000).
In various experimental models, including rodent models of diabetes, it has been
shown that AVP infusion induces hypertension, glomerular hyperfiltration, albumin-
uria, and glomerulosclerosis, playing deleterious renal effects (Bankir et al. 2001),
whereas its inhibition, by drinking water or by V2 antagonism, led to a decrease in
proteinuria in rats with renal failure (Okada et al. 1994). The direct measurement of
vasopressin in humans is problematic. More than 90 % in the circulation is bound to
platelets, it is unstable in isolated plasma, and most assays have relatively limited
sensitivity (Preibisz et al. 1983).
Recently, copeptin, the C-terminal portion of the precursor of vasopressin and
stoichiometrically secreted with it, becomes a reliable marker and a useful substitute
to detect circulating concentration in clinical routine. Moreover, several studies in
healthy subjects demonstrated a close relation between plasma copeptin, AVP
concentrations, and a wide range of osmolalities (Szinnai et al. 2007; Balanescu
et al. 2011).
However, the interpretation of copeptin levels must take into account potential
confounding factors, such as gender, renal impairment, and the fluid status of the
subject. Hence, a single reference range for normal copeptin will not be valid
considering the need to adjust for the independent effects of gender and renal
function (Bhandari et al. 2009).
Whereas water retention in the kidney is known to be an active phenomenon,
controlled by AVP, water excretion is not a passive process, as a result of AVP
release blockade, because this event is also controlled by a diuretic peptide, called
apelin.
It belongs to a signaling pathway which is implicated in the regulation of
cardiovascular function and fluid homeostasis. It is highly expressed in the supra-
optic and paraventricular hypothalamic nuclei, co-localized with AVP in a subset of
magnocellular neurons (Fig. 1).
Moreover, water deprivation, increasing systemic AVP release and causing
depletion of hypothalamic AVP stores, decreases plasma apelin concentrations,
indicating that AVP and apelin are conversely regulated to prevent additional
water loss at kidney level (Llorens-Cortes and Moos 2012).
Until now, efforts to retard (or even prevent) progression of chronic kidney
disease (CKD) have mainly focused on reducing protein consumption and lowering
blood pressure and renin–angiotensin system (RAS) blockade. The outcomes from
these approaches, however, remain far from satisfactory, and additional strategies are
therefore welcome.
The issues of optimal water intake and vasopressin receptor antagonism may
therefore represent novel frontiers in the effort to slow progression of renal pathologies.
In this chapter, we examine the diagnostic and prognostic role of copeptin and
apelin, reporting the last data about their physiological functions, their receptor
interactions, and their functions in nephrologic field.
Fig. 1 Vasopressin/copeptin and apelin release and their systemic effects
Physiologic Point of View
Copeptin physiologically contributes to the correct structural formation of AVP prior

to release into the circulation. The measurement of copeptin appears to be a clinically
relevant method for reliably assessing AVP plasma concentrations, which cannot be
determined in routine practice. This would be particularly helpful in diseases where
disturbances of the vasopressinergic system contribute to or are the immediate result
of the pathogenesis. The kinetics of copeptin, however, has not been well explored,
and it is not yet known whether copeptin-specific receptors exist nor whether
copeptin exerts biological functions.
Target organs and cells perceive hormonal stimuli mediated by AVP through three
distinct AVP receptors, V1a, V1b, and V2, all of which belong to a family of
heptahelical guanine nucleotide-binding protein-coupled receptors (Jard 1998).
In the kidney, AVP exerts its antidiuretic effect by regulating sodium and water
transport via the V2 receptor, which is expressed in the basolateral membrane of the
thick ascending limb of Henle’s loop, distal tubules, and collecting ducts. Through
the V2 receptors, AVP stimulates the Gs protein and adenylate cyclase to increase
intracellular cAMP, stimulating the translocation of aquaporin (AQP)-2 and the
amiloride-sensitive ENaC in the principal cells of the collecting duct and thereby
increasing water reabsorption (Imbert et al. 1975).
Fig. 2 Copeptin and apelin generation and maturation
The discovery of apelin constitutes an important advance in both fundamental

research and clinical medicine. Experimental data have shown that apelin has
diuretic effects via its central and renal actions: by inhibiting the phasic activity of
vasopressinergic neurons and systemic AVP secretion and by its direct effect on the
renal microcirculation and probably tubular function. Besides its diuretic action,
when injected into the blood stream, apelin decreases blood pressure and increases
the contractile force of the myocardium while decreasing pre- and post-load, actions
opposing those of vasopressin and angiotensin II. These data confirm that this new
circulating vasoactive (neuro)peptide could play a crucial role in maintaining water
and electrolyte balance and cardiovascular functions (Galanth et al. 2012).
It is very difficult to mention a standard normal level of apelin due to the presence
of various forms of apelin (apelin-12, apelin-13, apelin-18, apelin-36) in the circu-
lation (Fig. 2).
Besides this factor, different kits for measurement and the cross-reaction between
the types of apelin lead to various levels mentioned in the literature. Malyszko
reported apelin-36 levels as 84.0
9.26 pg/ml, whereas mean apelin-12 level was
reported as 304 pg/ml, both in healthy populations (Karadag et al. 2014).
The angiotensin I receptor-related protein J (APJ) receptor was first identified as an
orphan G-protein-coupled receptor, with the closest identity to the angiotensin II (Ang II)
receptor, type AT1a. In the ensuing years, the receptor was de-orphanized when its
cognate ligand, apelin, was isolated from bovine stomach extracts (Tatemoto et al. 1998).
Fig. 3 Copeptin and apelin signaling pathways
The inner strip of the outer medulla in kidneys contains the highest level of APJ
mRNA. Additionally, the collecting duct also contains apelin receptor, suggesting
that apelin might act as an aquaretic peptide in this segment (Hus-Citharel
et al. 2008).
Moreover, apelin and its receptor can be found extensively in the heart, vascular
endothelium, lungs, mammary glands, and central nervous system (Kawamata
et al. 2001) (Fig. 3).
Pathological Involvement of Copeptin and Apelin
Animal models and human studies strongly suggest a role for vasopressin, via V2
receptors, in the progression of various forms of CKD and in salt-sensitive hyper-
tension. Recent insights also point to the possible role of vasopressin in albuminuria
and diabetic nephropathy, via V2-receptor-mediated actions. These results raise the
possibility that, in addition to blood-pressure lowering, renin–angiotensin system
blockade, and other treatments, inhibiting the V2-receptor-mediated actions of
vasopressin, might provide further therapeutic benefit for these patients. Whereas
the effect of vasopressin in autosomal dominant polycystic kidney disease (ADPKD)
is relatively straightforward and mainly dependent on V2 receptors, its role in other
forms of CKD is more complex (Table 1).
Diabetes Mellitus and Kidney Dysfunction
Type 2 diabetes mellitus (DM) is a major cause of kidney and cardiovascular

diseases, and screening for albuminuria and proteinuria is recommended for strati-
fication of risk of these comorbidities.
Table 1 Apelin and copeptin roles in renal diseases

Future
Disease Apelin Copeptin Conclusion prospects References
Acute Not Serum In critically ill patients, Early Meyer
kidney available "" an elevation of plasma diagnostic et al. (2008)
disease data in copeptin is strongly marker
humans associated with
development of AKI
Apelin-13 treatment Late Chen
protects against renal prognostic et al. (2015)
ischemia/reperfusion factor
(I/R) injury in rats Further
inhibiting apelin
inflammatory factors, analyses in
TGF-β1, and apoptosis human
studies
Apelin as a
potential
therapy
Chronic Serum Serum Copeptin levels are Prognostic Li
kidney " "" significantly high in the marker et al. (2013)
disease CKD group and closely
related to GFR and
atherosclerosis markers
Elevated apelin in Malyszko
CKD patients may be et al. (2008)
due to impaired renal
clearance and
inflammation
Diabetic Serum Serum High copeptin level is Prognostic Velho
nephropathy ## "" strongly associated marker et al. (2013)
with the risk of severe
renal outcomes, with a
direct effect on urinary
albumin excretion
Patients with high Silva
apelin levels have et al. (2013)
better survival rates
than patients with
lower levels of apelin,
with a decreased risk of
cardiovascular
mortality
(continued)
Table 1 (continued)
Future
ADPKD Serum Serum High copeptin levels Diagnostic Nakajima A
## "" are independently markers et al. Clin
associated with an Exp
increase in TKV and a Nephrol.
decrease in GFR 2015
Low serum apelin Prognostic Boertien
levels in ADPKD markers et al. (2012)
patients represent an Marker of Lacquaniti
independent predictor therapy et al. (2013)
of renal disease response
progression and renal Apelin as a
replacement therapy potential
therapy
ESRD/ Serum Serum AVP in ESRD patients Prognostic Fenske
dialysis ## "" with little or no residual marker et al. (2011)
urine output exerts its
functions primarily via
the V1a and V1b
receptors
There is a strong and Further Codognotto
independent studies are et al. (2007)
association of required
increased copeptin
levels with stroke,
cardiovascular events,
sudden death, and
all-cause mortality
AKI acute kidney injury, TFG tumor growth factor, CKD chronic kidney disease, GFR glomerular
filtration rate, TKV total kidney volume, ADPKD autosomal dominant polycystic kidney disease,
AVP arginine vasopressin, ESRD end-stage renal disease
Despite improvements in glycemic and blood pressure control and the efficacy of
RAS blockade for proteinuria reduction, diabetic nephropathy is the most frequent
cause of end-stage renal disease in developed countries (Lacquaniti et al. 2013b).
However, as shown in recent randomized trials, even with optimal treatment, up
to 20 % of patients with diabetes and proteinuria develop end-stage renal disease
(ESRD) within a 3-year follow-up (Brenner et al. 2001).
In various experimental models, it has been shown that AVP infusion induces
glomerular hyperfiltration and albuminuria (Bankir et al. 2001).
While high AVP levels may be beneficial in the short term by limiting the amount
of water required for the excretion of the high osmolar load presented by glucosuria,
in the long term, it might cause adverse outcomes by aggravating hyperglycemia and
its sequelae, such as diabetic nephropathy. In this context, it is also of interest that
copeptin concentrations are significantly associated with hypertension, abdominal
obesity, inflammation, and the metabolic syndrome, suggesting that increased
activity of the AVP system may be “a unifying factor” in the metabolic syndrome
(Enhörning et al. 2011).
It was in fact demonstrated that high copeptin concentration was strongly asso-
ciated with the risk of severe renal outcomes (doubling of plasma creatinine con-
centration and/or end-stage renal disease) in DM patients, independently of relevant
covariates such as age, duration of diabetes, blood pressure and baseline levels of
glycated hemoglobin, urinary albumin excretion (UAE), and eGFR (Velho
et al. 2013).
Moreover, baseline copeptin levels were significantly associated with high
albumin-to-creatinine ratio (ACR) and low eGFR, but only in DM patients who
were not treated with RAS inhibitors. A possible explanation is that the deleterious
effect of AVP (measured as copeptin) is mediated at least in part via the
renin–angiotensin system, explaining the significant interaction between RAS inhib-
itor use and copeptin for the association with eGFR (Boertien et al. 2013a).
The association between copeptin and UAE was independent of systemic blood
pressure, indicating that other mechanisms of action may be important. Urinary
osmolality was independently associated with UAE, but less strong than the associ-
ation between copeptin and UAE, suggesting that besides the antidiuretic effect of
vasopressin, other effects (e.g., pressor effects) may also be involved in the relation-
ship of copeptin with UAE (Enhörning et al. 2013).
In a recent population study (Prevention of Renal and Vascular End-Stage
Disease [PREVEND]), including adults with and without kidney dysfunction, the
association between copeptin and microalbuminuria was confirmed, highlighting a
direct effect of AVP on urinary albumin excretion. In particular, microalbuminuria
was about two times more prevalent in individuals with copeptin plasma levels in the
highest quintile (>10.5 pmol/L) compared with individuals with copeptin levels in
the lowest two quintiles (<5.3 pmol/L).
In the same study, high levels of copeptin were associated with low renal
function, hypothesizing that copeptin caused renal function decline or that subjects
with low renal function were less sensitive to the actions of copeptin/vasopressin
(Meijer et al. 2010).
Several studies have shown an association between apelin levels and overt
diabetes.
It was demonstrated that apelin, as an endocrine or paracrine peptide, mediated
angiogenesis, facilitates abnormal vessel formation, and increased permeability in
diabetic glomeruli, assuming a crucial role in the pathogenesis and progression of
diabetic nephropathy (Zhang et al. 2013).
Moreover, Erdem demonstrated that plasma apelin was lower in newly diagnosed
and untreated DM patients than healthy controls (Erdem et al. 2008).
The role of apelin in diabetes pathogenesis was also confirmed by Soriguer who
showed the association between apelin levels, glucose concentrations, and insulin
sensitivity (Soriguer et al. 2009).
An association of apelin with mortality and hospitalization events, as well as its
relationship with renal function and cardiovascular risk factors, was assessed in a
homogeneous population of DM patients with a diagnosis of mild-to-moderate

CKD. In particular, patients with higher apelin levels tended to have better survival
rates than patients with lower levels of apelin, with a decreased risk of cardiovascular
mortality (Silva et al. 2013).
However, further studies with a larger sample size are needed to investigate the
relationship between these biomarkers, DM, and its comorbidities.
Autosomal Dominant Polycystic Kidney Disease
GFR is recognized as a poor predictive marker of renal function decline in ADPKD

patients, as glomerular hyperfiltration in functioning nephrons is able to compensate
for the ongoing loss of renal tissue. In early stages, the change in total kidney volume
(TKV) still appears to be the most sensitive marker of disease progression
(Grantham et al. 2006).
AVP, having a detrimental role in the pathogenesis of ADPKD, is already
elevated in the early stages of the disease, as a compensatory mechanism to maintain
fluid balance and plasma osmolality within the normal range, in patients character-
ized by a urinary concentrating defect. In subjects with ADPKD, copeptin is
associated with a decline in kidney function and need for renal replacement therapy,
independently of confounding parameters, such as age, gender, or baseline GFR
(Boertien et al. 2012).
Moreover, high baseline copeptin levels were independently associated with an
increase in TKV and a decrease in GFR, representing a biomarker easy to measure
that may help to predict outcome in ADPKD (Boertien et al. 2013b).
Furthermore, a cross-sectional study involving 102 ADPKD patients confirmed
these data, revealing that copeptin levels were associated with various markers of
disease severity, such as albuminuria, GFR, renal blood flow, and TKV (Meijer
et al. 2011).
The rise in vasopressin (measured as copeptin) precedes a decline in GFR.
Consequently, lowering vasopressin can lead to renoprotection. To lower vasopres-
sin concentration, one of the options is to achieve ample hydration.
Plasma osmolality and plasma copeptin level decreased, in fact, after high water
intake with a positive correlation between plasma copeptin and TKV slope, suppos-
ing that increase urine volume by drinking relatively solute-free water ameliorates
cyst growth via AVP reduction (Higashihara et al. 2014).
Another way to suppress the effect of vasopressin is to block the V2 receptor in
the kidney with medication. This option has been tested in animal experiments and
humans, revealing that vasopressin antagonism through tolvaptan prevented cyst
growth and kidney function decline (Torres et al. 2011, 2012).
Whereas several studies evaluated the role of copeptin in cystic kidney disease,
only one prospective study demonstrated that low serum apelin levels in ADPKD
patients represented an independent predictor of renal disease progression and renal
replacement therapy (Lacquaniti et al. 2013c).
End-Stage Renal Disease
Dialysis patients are at a very high risk of death (about 20 % per year). Thus, the
identification of new risk factors with the potential of applying intervention strate-
gies to improve outcome in the future is of utmost importance (Buemi et al. 2008).
Several biomarkers have consistently been shown to be closely associated with
patient mortality and improve risk prediction provided by clinical variables alone
(Lacquaniti et al. 2009, 2011).
In this context, the search for new biomarkers reflecting different facts of cardio-
vascular risk is still ongoing.
AVP in ESRD patients with little or no residual urine output can probably not
efficiently act via V2 receptors, but exerts its functions primarily via the V1a and
V1b receptors. Thus, an increase in cardiovascular risk and all-cause mortality in
ESRD patients might be partly linked to the predominant activation of V1a and V1b
receptor function (Teitelbaum and McGuinness 1995). It was demonstrated a strong
and independent association of increased copeptin levels with stroke, cardiovascular
events, sudden death, and all-cause mortality in hemodialysis patients, but not with
myocardial infarction or death caused by chronic heart failure (Fenske et al. 2011).
This discrepancy could be related to the cohort and statistical power since the
study comprised 1,241 solely diabetic patients reaching the end point by 37–49 %.
The increased copeptin levels in hemodialysis patients most likely reflect accu-
mulation, indicating an impaired clearance of this low molecular weight protein
(Artunc et al. 2014).
On the other hand, modern dialysis practices such as high-flux dialyzers or
hemodiafiltration can remove significant amounts of low molecular weight proteins,
such as β2-microglobulin.
The literature data about apelin levels in ESRD is limited, reporting lower values
in dialysis patients compared with the general population.
Apelin level was found to be lower in uremic patients with dilated cardiomyop-
athy than in non-uremic counterparts, demonstrating that uremia decreases apelin
levels irrespective of the degree of heart failure (Codognotto et al. 2007).
There is no data about the clearance of apelin during hemodialysis (HD) or perito-
neal dialysis (PD) in the literature, but, due to its molecular weight, it is expected to be
filtered through the glomerulus but not through low-flux hemodialysis membranes; so
dialysis clearance cannot be the reason of lower levels in HD patients, whereas it could
happen in PD patients, due to larger pore size of the peritoneal membrane.
Moreover, AVP/apelin balance changes with plasmatic osmolality variations and
a close relationship with arterial hypo- and hypertension induced by hemodialysis
were also demonstrated (Cernaro et al. 2012).
Kidney disease detection, whether acute or chronic, is based mainly on serum

creatinine, which is considered to delay prompt diagnosis and management, thus
increasing substantially patient morbidity and mortality and prolonged hospitaliza-

tion. Several biomarkers have been evaluated as early prognostic markers, but they
are still far from being implicated into clinical practice. Hence, more sensitive and
specific biomarkers are needed to enable us recognize individuals at increased risk
for progression of CKD to ESRD and occurrence of cardiovascular complications.
It has been demonstrated that apelin and copeptin could represent valid bio-
markers for various types of kidney diseases, such as diabetic nephropathy or
ADPKD, and may serve as a future target molecule potentially useful for under-
standing disease processes or innovative therapeutic strategies, due to their role in
body fluid homeostasis and in the regulation of the cardiovascular system, reflecting
the dialog between the heart and kidney (Table 2).
In fact, Voors demonstrated that copeptin was a strong marker for mortality and
morbidity in patients with heart failure after acute myocardial infarction (MI) in a
subset of 224 patients of the OPTIMAAL study (Optimal Trial in Myocardial
Infarction with Angiotensin II Antagonist Losartan). In addition, the predictive
value of copeptin was even stronger than brain natriuretic peptide (BNP) and
NT-proBNP (Voors et al. 2009).
Atrium is known as one of the major sources of apelin production, and during
heart failure the apelin level in atria falls. Thus, its level in plasma drops conse-
quently (Foldes et al. 2003; Basile et al. 2014).
Kadoglou confirmed this trend in patients with unstable angina and acute MI,
revealing significantly lower level of apelin compared to patients with asymptomatic
coronary artery disease (Kadoglou et al. 2010).
In addition to the possibility of application of apelin as a heart failure biomarker,
it also plays direct biological roles, including vasodilatory with hypotensive
response and a positive cardiac inotropic action, opposing its effects to angiotensin
II on vascular tone, blood pressure, and fluid homoeostasis (Brame et al. 2015).
It was in fact demonstrated, in vivo studies, that apelin injection resulted in an
improvement in cardiac function, as well as a reduction in cardiac loading (Ashley
et al. 2005; Chandrasekaran et al. 2008).
Starting from these assumptions, it will be plausible that an optimum dose of
apelin will improve both systolic and diastolic functions of the heart and thus could
have beneficial effects on the failing hearts in patients with chronic renal diseases
(Berry et al. 2004).
Carcinogenesis could represent another potential field of application for apelin,
due to its marked properties to regulate angiogenesis, both in vitro and in vivo,
stimulating endothelial cell proliferation and migration.
Apelin is in fact upregulated during tumor angiogenesis, and its overexpression
was demonstrated to increase in vivo tumor growth. The molecular mechanisms
which regulate its expression are still largely unknown, but it has been reported that
hypoxia-inducible factor (HIF)-1 regulates apelin synthesis (Bolignano et al. 2010).
It was demonstrated in humans that apelin represents a prognostic factor for
survival and a predictive factor for cancer disease progression, becoming a bio-
marker of enhanced angiogenesis and closely related to increased metastatic capac-
ity. The inhibition of apelin signaling may be a promising target of therapies for
Table 2 Apelin and copeptin in cardiac diseases

Future
Acute Serum Serum Copeptin is Precocious Gu et al. (2011)
myocardial ## "" elevated in the diagnostic
infarction early hours after the marker
onset of an
ST-elevation AMI
when CK-MB and
cTnT are still low
Apelin values are Late Kadoglou
lower in patients prognostic et al. (2010)
with unstable factor
angina and AMI Marker of
than patients with therapy
asymptomatic response
coronary artery Apelin as a
disease potential
therapy
Chronic Serum Serum Copeptin is a Prognostic Voors
heart ## "" strong marker for marker et al. (2009)
failure mortality and
morbidity in
patients with HF
The predictive Marker of Chandrasekaran
value of copeptin is therapy et al. (2008)
even stronger than response
BNP
Apelin is reduced Apelin as a
in patients with potential
heart failure and therapy
upregulated
following
favorable left
ventricular
remodeling
Atrial Serum Serum High copeptin Prognostic Smith
fibrillation # " levels are associated marker et al. (2010)
with AF and a
higher risk of CVE
Low plasma apelin Marker of Falcone
levels before therapy et al. (2010)
external electrical response
cardioversion are Further
an independent studies are
prognostic factor required
for arrhythmia
recurrence in
patients with AF
treated with anti-
arrhythmic drugs
(continued)
Table 2 (continued)
Future
Heart Not Serum Copeptin Prognostic Malyszko
transplant available " independently marker et al. (2010)
data predicts outcomes Further Przybylowski
of heart transplant studies are et al. (2010)
patients, and it is required,
closely related to involving
kidney function apelinergic
and intraventricular system
septal thickness
AMI acute myocardial infarction, CK-MB creatine kinase–myocardial band, cTnT cardiac
troponin T, HF heart failure, BNP brain natriuretic peptide, AF atrial fibrillation, CVE cardiovas-
cular events
cancer treatment, with a new class of anti-angiogenesis drugs. Furthermore, consid-

ering its vasopressin antagonist role, apelin could be a new drug to treat the
syndrome of inappropriate antidiuretic hormone (SIADH)-related hyponatremia,
often involving oncologic patients (Lacquaniti et al. 2015).
Table 3 resumed the potential diagnostic and prognostic roles of copeptin and
apelin in various diseases.
Conclusion
These findings are of great importance toward understanding the pathophysiological

mechanisms involving several types of kidney diseases, as well as the role of
vasopressin, copeptin, and apelin as diagnostic and prognostic biomarkers.
Copeptin and apelin represent in fact prognostic marker for these patients and
predictive factors for progression to ESRD. Their measurement may be included in a
“renal” work-up for patients at high risk, and their evaluation may be helpful also to
predict a therapy response, especially in the immediate future, when the aquaretic
tolvaptan, selective V2 receptor antagonist, will be available for treating patients
with ADPKD.
However, further studies are needed in order to make these results more reliable
and applicable in larger populations.
Summary Points
• Arginine vasopressin plays deleterious renal effects, inducing hypertension, albu-

minuria, and glomerulosclerosis, whereas its inhibition led to renoprotection.
• This chapter focuses on apelin and copeptin, two biomarkers closely related to
vasopressin system.
Table 3 Apelin and copeptin in different disorders behind cardiorenal pathologies

Future
Sepsis Serum Serum Copeptin is positively Precocious Jochberger
and " "" correlated with diagnostic et al. (2009)
septic APACHE II score and marker
shock inflammatory markers
in patients with sepsis,
reflecting disease
severity
Serum Apelin level was Late Gad
significantly high in prognostic et al. (2014)
adult patients and factor
neonates with sepsis Marker of
and septic shock therapy
response
Diabetes Not Serum Without prior thirsting, Early Timper K
insipidus available "" a single baseline diagnostic et al. J Clin
data copeptin level marker Endocrinol
>21.4 pmol/L Metab. 2015
differentiated jc20144507
nephrogenic DI from
other etiologies with
100 % sensitivity and
specificity, rendering
water deprivation
testing unnecessary in
such cases
In patients undergoing Prognostic Winzeler B
pituitary procedures, marker et al. J Clin
low copeptin levels Endocrinol
reflect postoperative DI, Metab. 2015
while high levels jc20144527
virtually exclude it
SIADH Serum Serum High copeptin levels Diagnostic Fenske
" " reflect the abnormal markers et al. (2014)
AVP secretion in the
case of SIADH
High apelin Marker of Blanchard
concentrations and therapy et al. (2013)
apelin-to-copeptin response
ratios characterize Further
SIADH patients studies are
required
(continued)
Table 3 (continued)
Future
Liver Serum Serum Copeptin Prognostic Moreno
cirrhosis and "" concentrations markers et al. (2013)
histology increased along with the
" severity of liver disease,
predicting 1-year
mortality or liver
transplantation
Apelin is expressed in Marker of Farid
the cirrhotic liver, and it therapy et al. (2014)
is involved in the response
disease progression. It Apelin as a
may be implicated in potential
multiple facets in the therapy
fibrosis–carcinoma axis target
Cancer Serum Serum High apelin and Precocious Belting
" " copeptin levels could be diagnostic et al. (2012)
found in oncologic biomarker
patients because of their
vasoactive properties
Apelin behaves as a Prognostic Lacquaniti
potent activator of marker et al. (2015)
tumor neoangiogenesis, Apelin as a
representing a strong potential
and independent risk therapy
marker for cancer target
progression Further
studies are
required
APACHE II Acute Physiology and Chronic Health Evaluation II, DI diabetes insipidus, SIADH
syndrome of inappropriate antidiuretic hormone
• Copeptin represents the C-terminal portion of the precursor of vasopressin, it is

stoichiometrically secreted with it, and it is detectable through a reliable and
sensitive analytical method.
• Apelin, highly expressed in the supraoptic and paraventricular hypothalamic
nuclei and co-localized with vasopressin in a subset of magnocellular neurons,
exerts an active diuretic action, counteracting the effects of vasopressin.
• High copeptin levels, closely associated with marker of renal dysfunction, such as
microalbuminuria and high albumin-to-creatinine ratio, were strongly associated
with the risk of severe renal outcomes in diabetic patients.
• Low apelin values were related with mortality and hospitalization events in diabetic
patients, with a strict relationship with renal function and cardiovascular risk factors.
• Vasopressin plays a critical role in the pathophysiology of the autosomal domi-
nant polycystic kidney disease (ADPKD).
• In fact, copeptin levels are elevated in ADPKD patients, and it is associated with
various markers of disease severity.
• Furthermore, apelin represents an independent predictor of renal disease progres-
sion in ADPKD patients, providing predictive information about the patient’s risk
for renal replacement therapy.
• In patients with end-stage renal disease, treated by hemodialysis or peritoneal
dialysis, apelin and copeptin represent new risk factors, closely associated with
cardiovascular events and patient mortality.
• Behind nephrologic application, apelin and copeptin play diagnostic and prognostic
roles in cardiologic and oncologic pathologies, providing a target pathway poten-
tially useful for understanding disease processes or innovative therapeutic strategies.
• However, further larger prospective studies are necessary to confirm these poten-
tial applications of copeptin and apelin and to ascertain their eventual relevance as
a parameter for monitoring the development and the progression of renal diseases.
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BLyS and APRIL Cytokines as Biomarkers
of Kidney Diseases 25
Natavudh Townamchai, Wannarat Pongpirul,
Asada Leelahavanichakul, and Yingyos Avihingsanon
Contents
Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559
Key Facts of BLyS and APRIL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559
Key Facts of Lupus Nephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
Key Facts of Kidney Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561
Unmet Needs for AKD and CKD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561
N. Townamchai
Division of Nephrology, Department of Medicine, Faculty of Medicine, Chulalongkorn University
and King Chulalongkorn Memorial Hospital, Bangkok, Thailand
e-mail: ntownamchai@gmail.com
W. Pongpirul
Department of Medicine, Bamrasnaradura Infectious Diseases Institute, Ministry of Public Health,
Nonthaburi, Thailand
e-mail: awannarat@yahoo.com
A. Leelahavanichakul
Division of Immunology, Department of Microbiology, Faculty of Medicine, Chulalongkorn
University, Bangkok, Thailand
Center of Excellence in Immunology and Immune-Mediated Diseases, Faculty of Medicine,
Chulalongkorn University, Bangkok, Thailand
e-mail: a_leelahavanit@yahoo.com
Y. Avihingsanon (*)
Center of Excellence in Immunology and Immune-Mediated Diseases, Faculty of Medicine,
Chulalongkorn University, Bangkok, Thailand
e-mail: yingyos.a@gmail.com

558 N. Townamchai et al.
Biology of BLyS and APRIL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562

Current Biomarkers Used to Diagnose and Prognose Kidney Diseases . . . . . . . . . . . . . . . . . . . 564
BLyS and APRIL as a Therapeutic Biomarker to Guide the Treatment for Kidney
Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565
Other Roles of BLyS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565
Factors that Affect the BLyS Level During Kidney Transplantation . . . . . . . . . . . . . . . . . . . . . . . 568
Levels of BLyS Pretransplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568
BLyS Levels Posttransplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569
Immunoregulation of BLyS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570
BLyS as a Biomarker for Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572
Abstract
Kidney damages mediated by the immune system have been reported for glo-
merular diseases, tubulointerstitial nephritis, and rejection of the allograft
posttransplantation. B-lymphocytes have an important role in the pathogenesis
for these kidney diseases. There are increasing evidences that TNF-like ligands
which regulate B-cell homeostasis such as B-lymphocyte stimulator (BLyS) and a
proliferation-inducing ligand (APRIL) can be used to detect kidney diseases as
well as acute rejection of the kidney allograft. This chapter will review these two
ligands, BLyS and APRIL, and its promising prospect in detecting glomerular
diseases and acute rejection of the kidney allograft.
BLyS and APRIL are found in the plasma and membrane proteins of macro-
phages, monocytes, and dendritic cells. Both can activate the proliferation of
B-cells and plasma cells. They are expressed on tissues of the lymph node, bone
marrow, synovium, and kidney. Even though both cytokines share a common
receptor on the B-cells, however, their roles in B-cell activation are significantly
different. When BLyS is activated, this will result in severe B-cell hyperplasia in
BLyS-transgenic mice. In contrast, when APRIL is activated, there is no effect on
the homeostasis of the lymphoid in APRIL-transgenic mice.
BLyS was initially discovered to be a potential therapeutic target for lupus
patients. Later, it was shown that both BLyS and APRIL were somehow associ-
ated with lupus nephritis. Both cytokines are expressed on the infiltrating mono-
cytes and/or macrophages of patients with lupus nephritis. It remains unknown
whether the blockade of both cytokines can be used to treat the lupus disease.
However, serum APRIL has been shown to be a useful biomarker in detecting
lupus nephritis in patients with a poor prognosis.
In kidney transplantation, B-cell is an important factor for the long-term
survival of the kidney allograft. High levels of BLyS or APRIL can activate
alloreactive B-cells by inducing antibodies against the donor antigens (donor-
specific antibody, DSA) and result in rejection of the allograft. Aside from that,
BLyS is an important growth factor for IL-10-producing B-cells known as the
25 BLyS and APRIL Cytokines as Biomarkers of Kidney Diseases 559
regulatory B-cells. It has been shown that BLyS from the mice can increase a
proportion of regulatory B-cells ex vivo. These evidences indicate that BLyS
plays a major role in the activation and regulation of the immune system affecting
the success and/or outcome of the kidney transplantation.
Keywords
Biomarker • BLyS • BAFF • APRIL • Kidney disease • SLE • Kidney transplan-
tation • B-cell
Abbreviations
ABMR Antibody-mediated rejection
AKD Acute kidney disease
APRIL A proliferation-inducing ligand
BAFF B-cell-activating factor
BAFF-R B-cell-activating factor receptor
BCMA B-cell maturation antigen
BCR B-cell receptor
BLyS B-lymphocyte stimulator
CD Cluster of differentiation
CR Complement receptor
Cr Creatinine
DSA Donor-specific antibody
HLA Human leukocyte antigen
IF/TA Interstitial fibrosis/tubular atrophy
IL Interleukin
NSTDA National Science and Technology Development Agency
PLA2R M-type phospholipase A2 receptor
suPAR Soluble urokinase-type plasminogen activator receptor
TACI Transmembrane activator and calcium modulator and cyclophilin
ligand interactor
Key Facts
Key Facts of BLyS and APRIL
• BLyS is required for the early stages of B-cell development, whereas APRIL is
needed much later for the survival of the plasma cell.
• BlyS and APRIL are mainly produced by innate immune cells such as macro-
phages, monocytes, and B-cells.
• BLyS and APRIL contribute to the development of the immune-mediated kidney

disease (i.e., SLE) and rejection of the kidney allograft.
• BLyS and APRIL are new biomarkers that can be used to detect active B-cell-
mediated kidney disease and antibody-mediated rejection in kidney
transplantation.
• BLyS and APRIL are markers for B-cell homeostasis required by both the
regulatory and allo-/autoreactive B-cells for their survival.
• Current B-cell target therapy such as belimumab, atacicept, or rituximab can
affect the serum levels of BLyS and APRIL.
Key Facts of Lupus Nephritis
• An autoimmune kidney disease that typically affected young adult female.

• A leading cause of secondary glomerular diseases worldwide.
• Typical symptoms and signs are leg edema and foamy urine.
• Autoreactive B-lymphocyte cells play a pivotal role in the mechanism of the
disease.
• Kidney transplantation is the best treatment of end-stage kidney disease.

• The important barrier of kidney transplantation is alloreactive cellular immunity.
• Biomarkers are required to monitor for an immune response to kidney allograft
so-called “rejection.”
Definitions
Antibody-mediated rejection (ABMR) Allograft rejection due mainly to anti-

HLA antibody.
Anti-HLA Alloantibody to HLA antigen. Patients sensitive to self-antigen will

have high levels of anti-HLA.
Autoreactive B-cell B-cells that can differentiate into plasma cells and produce
antibody to the self-antigen.
Donor-specific antibody (DSA) Anti-HLA that is specific to the donor HLA.
Glomerulonephritis Glomerular disease is caused by the inflammation of the

glomeruli and has the following clinical presentations of proteinuria, red blood
cells/casts in the urine, and elevated levels of the serum creatinine.
IL-10-producing B-cell Regulatory B-cell with immunoregulatory function can

inhibit inflammation by controlling the production of IL-10. Regulatory B-cell plays
an important role in the long-term survival of the allograft post-kidney transplantation.
Immune-mediated kidney disease Kidney disease caused by the immune system

(e.g., lupus nephritis, membranoproliferative glomerulonephritis (MPGN), membra-
nous nephropathy).
Immunoregulation The immune mechanism which regulatory cells counterbal-

ance an inflammation. Those cells include regulatory T-lymphocyte and regulatory
B-lymphocyte cells.
Renal vasculitis Kidney disease is caused by the inflammation of mainly small

vessels (e.g., Wegener granulomatosis and microscopic polyangiitis).
Introduction
Unmet Needs for AKD and CKD
The kidney is one of the most vulnerable organs that can be damaged by several
factors. The damages of the kidney function can appear early or much later during
the course of the destruction of the kidney and tend to result in end-stage kidney
disease. Kidney diseases are simply classified according to its severity which can
be either acute or chronic. A 3-month observational period can distinguish between
an acute kidney disease (AKD) and the chronic kidney disease (CKD). An
impaired kidney function is defined as having a glomerular filtration rate (GFR)
less than 60 mL/min/1.73 m2. The gold standard for determining GFR is via the
inulin clearance or radioisotope nuclear clearance (Stevens and Levey 2009).
However, the most commonly used kidney function test is the serum creatinine
or creatinine clearance. Both tests have long been used in the medical field despite the
fact that they are not sensitive enough for detecting early stages of the kidney disease.
Also, creatinine is not involved in any part of the processes that causes damage to the
kidney so it is not recommended to solely use it to diagnose kidney injury (Shemesh
et al. 1985; Yurasov et al. 2005; Meyer-Bahlburg et al. 2008). Thus a more accurate
diagnostic test that can specifically detect each type of kidney disease is needed.
This is possible if mechanisms involved in various kidney diseases are targeted. It
has been shown that the B-lymphocyte is a major player in causing immune-
mediated kidney diseases such as glomerulonephritis, renal vasculitis, or rejection
of the kidney allograft (Yurasov et al. 2005; Parsons et al. 2009; Redfield et al. 2011;
Wilde et al. 2013). Therefore further investigations of the B-cells have dramatically
improved the performances of the diagnostic and prognostic tests for immune-
mediated kidney diseases as well as provide better targets for therapy. A perfect
example of this can be seen with the treatment for systemic lupus erythematosus
(SLE) and nephritis. Because the B-lymphocytes and plasma cells are involved in the
development of SLE and nephritis (Odendahl et al. 2000; Arce et al. 2001; Liu
et al. 2011), hence, the only way to counteract that would be by blocking the specific
autoreactive B-lymphocytes which have proved to be quite helpful (Sfikakis
et al. 2005; Anolik et al. 2007). But it should be mentioned that none of the targeted
B-cell treatments have been recognized or approved by the regulatory health agency
to treat the disease. Many clinical trials failed to show the benefit of targeted B-cell
therapy which can partly be explained by their poor inclusion criteria (Merrill
et al. 2010; Furie et al. 2011; Rovin et al. 2012). This can be rectified by stratifying
the SLE patients based on their B-cell markers. This technique will allow the
detection of a subgroup of SLE patients that will benefit from the targeted therapy.
Therefore it is recommended that serum B-cell cytokine levels such as
B-lymphocyte stimulator (BLyS) or a proliferation-inducing ligand (APRIL) be
used to identify groups of people who will benefit from the B-cell therapy.
Biology of BLyS and APRIL
BLyS and APRIL do not directly destroy the kidney but enhance the activities of the
B-cells and plasma cells resulting in the immune-mediated kidney disease. The
cytokines are associated with the development of autoreactive B-cell in autoimmune
diseases. One of the autoreactive B-cells is the B2 cells which come from the bone
marrow and are highly dependent on BLyS and APRIL to survive. The other
autoreactive B-cell is the B1 cell or innate immune response B-cell which is less
dependent on BLyS and APRIL because it is originated from the fetal liver, the
peritoneum, and the mucosa. In general, harmful autoreactive B-cell is controlled by the
bone marrow and spleen. During the development of the B-cell, these processes are
neutralized by apoptosis or changes in the morphological structure of the B-cell
receptor (BCR). Thus the affinity of BCR (BCR engagement) and availability of
survival cytokines such as BLyS and APRIL are important for the survival of the B-cell.
It should be noted that APRIL plays a different role compared to that of BLyS
during the autoimmune reactivation process (Fig. 1). While BLyS is required earlier
during the development of the B-cell, in contrast, the APRIL cytokine is needed much
later post-development for the survival of the plasma cell (Mackay and Schneider
2009). The plasma cells of APRIL-deficient mice mature normally but have a poorer
survival rate and impaired switching of the isotype class (Dillon et al. 2006; Belnoue
et al. 2008; Benson et al. 2008). The functions between BLyS and APRIL are different
even though they use the same receptor. BLyS can bind to any of the three receptors at
a time: B-cell-activating factor receptor (BAFF-R), transmembrane activator and
calcium modulator and cyclophilin ligand interactor (TACI) receptor, and B-cell
maturation antigen (BCMA) receptor. Among the three receptors, APRIL can bind
to TACI and BCMA. The only receptor that is not shared between BLyS and APRIL is
the BAFF-R which is expressed on the immature B-cell. BAFF-R is important for the
survival of the B-cell and upon its activation produces a large volume of cells that can
synthesize proteins and translate mRNAs but cannot undergo cell division (Mackay
and Schneider 2009). Unlike BAFF-R, TACI and BCMA are expressed on mature
Fig. 1 The interactions between BAFF/BLyS-APRIL and cellular receptors. BAFF appears in the
early stages of B-cell differentiation. The survival of the mature B-cell (plasma cell) is highly
dependent on APRIL
B-cells and important for switching of the immunoglobulin isotype class as well as the
survival of the plasma cell. When BLyS is attached to either TACI or BCMA, it will
cause the B-cell to mature because its affinity for BAFF-R is stronger than TACI and
BCMA. On the other hand, when APRIL is attached to TACI or BCMA, this will help
increase the life-span of the plasma cell because its affinity for BCMA is very strong
(Marsters et al. 2000; Day et al. 2005).
Notable characteristics and key features of BLyS and APRIL were derived from
the mouse models so are worthy to be mentioned here (Fig. 1):
1. Animal models for BAFF-R

In BAFF-R-deficient mice, BAFF-R is expressed initially on immature B-cells
and strongly on follicular and marginal zone B-cells (Meyer-Bahlburg et al. 2008;
Hsu et al. 2002; Stadanlick et al. 2008). The expression of BAFF-R increases
when the BCR is activated. Upon the activation of BAFF-R, increased levels of
CD21 (complement receptor 2, CR2) and an additional signal released by the C3d
receptor will help activate B-cells (Gorelik et al. 2004). BAFF –/– mice (BAFF-
deficient mice) and transgenic A/WySnJ BAFF-R mice (mutated BLyS rendered
to be nonfunctional) have similar characteristics such as impaired maturation of
the B-cell, low levels of immunoglobulin, and weaker B-cell immune responses
(Mackay et al. 2003; Shulga-Morskaya et al. 2004; Mackay and Leung 2006).
When there is an overexpression of BAFF-R activity, this will induce B-cell
hyperplasia and phenotypic characteristics of the autoimmune diseases (i.e.,
glomerulonephritis and destruction of the salivary gland) (Mackay et al. 2005;
Groom et al. 2007).
2. Animal models for APRIL

APRIL –/– mice (APRIL-deficient mice) are able to form a normal germinal
center formation, but its plasma cells have a shorter life-span (Dillon et al. 2006;
Belnoue et al. 2008; Benson et al. 2008). On the other hand, the life-span of B1
cell in the transgenic APRIL mice shows a longer life-span and possibly develops
a neoplasia (Dillon et al. 2006). These data support the importance of APRIL as
an important survival factor.
3. Animal models for BCMA and TACI
BCMA, a receptor for BLyS and APRIL, is expressed on B-cells of the germinal
center, memory B-cells, and plasma cells (Gras et al. 1995; Darce et al. 2007;
Hildebrand et al. 2010). BCMA –/– mice (BCMA-deficient mice) have a normal
B-cell response and B-cell development, but its plasma cells have a shorter life-
span (Xu and Lam 2001; O’Connor et al. 2004).
TACI is expressed on both mature B-cells and plasma cells. TACI can be
activated only by the oligomeric or membrane-bound form of either BLyS or
APRIL (Bossen et al. 2008). TACI –/– mice (TACI-deficient mice) have exces-
sive amount of B-cells, B-cell hyperplasias, overproduction of autoantibodies,
and an extensive presence of B-cell lymphomas (Mackay and Schneider 2008).
Current Biomarkers Used to Diagnose and Prognose Kidney Diseases
Physicians or nephrologists usually require certain laboratory tests for diagnosing or

prognosing the kidney diseases. For example, serum creatinine has been used to
determine the kidney function but its interpretations are quite limiting. A patient with
a subtle kidney disease or focal histological changes may have a normal level of the
serum creatinine or kidney function. Hence, the measurement of serum creatinine
level alone cannot detect any significant changes of the kidney function (Doolan
et al. 1962; Levey 1990; van Acker et al. 1992).
Another biomarker used to diagnose glomerular disease is the use of albuminuria.
One of its limitations is that it is unable to differentiate the scars of the kidney from
the presence of active disease. High levels of albuminuria are associated with having
an active disease which requires higher doses of immunosuppressant such as steroids
(Yamagata et al. 1996). This can ultimately harm the patient unnecessarily if the
wrong diagnosis is made.
Other biomarkers such as urine cells or sediments can also be used to detect
glomerular diseases. These cells can be easily seen with a simple microscope but it
requires a skilled medical technician. However, the red blood cells or white
blood cells from the urine can appear during an infection of the urinary bladder,
tumor, or inflammation making it difficult to correctly diagnose for glomerular
diseases. In addition, the urine cells can also be found in the kidney stones, the
ureter, or the area up to the kidney so it cannot be used to specifically detect for
glomerular diseases.
However, the gold standard for diagnosing glomerular diseases is the kidney
biopsy because histopathology can provide the basic pathology of the glomerular
disease and the area of the damaged tissues. Unfortunately its invasive procedure can
result in various complications that can range from mild hematuria to serious
bleeding. Yet like the other biomarkers previously discussed, one of its limitations
is that it is a cross-sectional observation so it is difficult to predict the course of
glomerular disease. Furthermore, if the condition of the patient becomes worse or
resistant to treatment, another biopsy will be required. Many clinicians try to avoid
repeating the procedure and most of the patients will also refuse to have it done again.
As a result of this, there is an urgent need for noninvasive techniques utilizing
biomarkers that can clinically and accurately detect kidney diseases. An ideal
biomarker should be accurate, reproducible, and not invasive. Such a biomarker
has not been discovered but there are many promising biomarker candidates. The
most interesting biomarkers are from soluble proteins that are involved in the
mechanisms of the glomerular diseases such as lupus nephritis’ anti-DNA antibody
(Cortes-Hernandez et al. 2004; Forger et al. 2004; Alba et al. 2003), membranous
nephropathy’s anti-M-type phospholipase A2 receptor (anti-PLA2R) antibody (Beck
et al. 2009), or focal segmental glomerulosclerosis’ soluble urokinase-type plasmin-
ogen activator receptor (suPAR) (Wei et al. 2012; Li et al. 2014).
BLyS and APRIL as a Therapeutic Biomarker to Guide the Treatment

for Kidney Diseases
The biomarkers that are of interest are BLyS and APRIL because both are involved
in the mechanisms of SLE and lupus kidney disease. Serum BLyS level is associated
with SLE disease activity and complement levels (Table 1, Fig. 2). Hence the
blockage of BLyS can attenuate the disease activity (Morimoto et al. 2007; Vincent
et al. 2014). In contrast, the level of serum APRIL is associated with lupus nephritis
(Table 1, Fig. 2). When the cutoff level for serum APRIL was used at 3.6 ng/mL, it
proved to be very useful in predicting resistance to treatment (Treamtrakanpon
et al. 2012; Eilertsen and Nossent 2014) as well as difficult-to-treat lupus nephritis
patients. High levels of serum APRIL are indicative of active nephritis and associ-
ated with resistance to steroids and cyclophosphamide therapy. The expression of
APRIL in the kidney tissue can identify refractory nephritis (Treamtrakanpon
et al. 2012). Based on these information, a new schema using BLyS and APRIL
has been proposed to guide the treatment for kidney diseases (Fig. 3).
Other Roles of BLyS
One of the factors in having successful kidney transplantation is dependent on the

use of immunosuppressant agents at the right dosage. Overdose of these agents can
result in infections and cancers, whereas underusage can result in the loss of the
allograft. Currently, therapeutic drug monitoring of the immunosuppressive drug is
the standard of care for managing kidney transplantation. However, the pharmaco-
kinetic monitoring of the drug cannot predict the future biological changes of the
Table 1 Serum levels of BLyS and APRIL in SLE patients

BLyS APRIL
Spearman Spearman
r p-value r p-value
R_SLEDAI 0.255 0.057 Duration of disease 0.353 0.017*
Anti-dsDNA 0.362 0.090 R_SLEDAI 0.219 0.148
C3 0.560 0.002* Uprotein 0.438 0.002*
C4 0.621 0.002* WBC 0.267 0.061
CH50 0.568 0.003* PMN 0.262 0.006
WBC 0.485 <0.001* Activity score 0.335 0.017*
PMN 0.402 <0.001*
Lymphocyte 0.417 0.002*
Prednisolone (dose) 0.543 <0.001*
Mycophenolate (dose) 0.369 0.007*
ACEI (dose) 0.272 0.051
BLyS at 6 months 0.580 0.005*
apart
Serum BLyS and APRIL levels are associated with different stages of systemic lupus erythematosus
(SLE) and lupus nephritis. Serum APRIL is associated with kidney activity of lupus nephritis (i.e.,
urine protein, active score of renal pathology). In contrast, serum BAFF is associated with the
activity indices of systemic lupus disease (i.e., serum complement levels, total leukocyte count, and
renal systemic lupus erythematosus disease activity index (R_SLEDAI))
kidney allograft. Hence a better understanding of the allograft rejection and regula-
tion of the immune system can provide newer methods to appropriately monitor the
outcome of the kidney transplantation (Fig. 4).
It has been known for a long time that the immunosuppression of the T-cell
contributes to the rejection of the allograft. This information is used to prevent the
rejection of the kidney allograft. Over the years, there have been many new advances
in the field of T-cell immunology and immunosuppression. Despite this, only short-
term graft outcomes have improved. To date, long-term graft outcome and the
condition of the patient have not changed (Kasiske et al. 2005). The reason for
this may be due to the involvement of other parts of the immune system. Recent
studies highlighted the role of the humoral response, such as the B-cells, plasma
cells, and anti-HLA, as the key factor in providing a successful long-term outcome of
the kidney allograft (Everly et al. 2013; Wiebe and Nickerson 2013). Previous
murine cardiac allograft model showed that the BLyS-deficient mice have a longer
allograft survival rate compared to the wild type (Ye et al. 2004). In humans, the
presence of BLyS in the recipients’ kidney allograft was associated with acute
rejection. The interstitial fibrosis/tubular atrophy (IF/TA) of the graft can be detected
when stained with C4d (Xu et al. 2009). These studies confirm the role of BLyS in
transplantation. BLyS has been proposed to be used as a noninvasive monitoring
biomarker for both pre- and post-kidney transplantation. Clinical trials manipulating
the levels of BLyS by anti-BLyS agents are still ongoing.
Fig. 2 An association of serum APRIL and pathology of the lupus nephritis. For patients with SLE,
a level of APRIL more than 3.6 ng/mL is associated with active renal pathology which consists of
endocapillary proliferation, fibrinoid necrosis, infiltrations of the polymorphonuclear (PMN) cells,
and depositions of hyaline in the glomerular capillaries
Fig. 3 Biomarker-guided strategy. Biomarker-guided strategy can provide early diagnosis and appro-
priate treatment for the kidney diseases. The early stages of the kidney diseases can be detected by
serum levels of BLyS and APRIL or urine cytokines. However, the subclinical pathology occurs at the
late stage so an invasive tissue biopsy is needed. An alternative noninvasive method currently utilizes
the serum creatinine to diagnose the late stage of kidney disease. The prognosis of the kidney disease
becomes more accurate when the biomarkers are used together to diagnose and prognose the disease
Fig. 4 Mechanism of antibody-mediated kidney allograft rejection. Recipient’s antigen-presenting

cells recognize donor antigen. The antigen-presenting cells bring antigens to regional lymph nodes.
Alloreactive T-cells recognize donor antigen. The alloreactive immune response is activated.
B-cells differentiate to plasma cells and produce antibodies against donor HLA. The anti-HLA
antibodies, produced by plasma cells, continuously injure the kidney graft
Factors that Affect the BLyS Level During Kidney Transplantation
The production of BLyS by innate immune cells and consumption of BLyS by

various B-cells at different stages of development can affect the levels of BLyS.
High levels of BLyS indicate an increased production and/or decreased consumption
of BLyS by B-cells (Fig. 5). Besides this, other important factors that affect the BLyS
levels after a kidney transplantation are the characteristics of the recipients and the
duration posttransplantation.
Levels of BLyS Pretransplantation
There are various degrees of sensitized patients that can affect the pretransplant
BLyS level. Patients with high titer of anti-HLA antibodies also have high BLyS
levels, but this has not been positively correlated with the rejection rate posttrans-
plantation (Snanoudj et al. 2014). However, there are a subgroup of patients who
have a higher pre-desensitization or pretransplant BLyS level which was positively
correlated with the amount of posttransplant antibody-mediated rejection (ABMR)
(Banham et al. 2013). These findings indicate that the recipient with high
pretransplant BLyS levels will need a more intensive pretransplant desensitization
protocol and immunosuppressive regimen. In addition, anti-BLyS treatment may be
beneficial for these groups of kidney transplant recipients.
Fig. 5 Factors associated with B-lymphocyte stimulator (BLyS) factor. B-lymphocyte stimulator
(BLyS) factor is the key factor for the development of plasma cells and production of antibodies.
The innate immune responses enhance BLyS levels, whereas immunosuppressive drugs inhibit the
production of BLyS. Immunosuppressive drugs currently used to inhibit the activity of BlyS are
belimumab and atacicept. Other immunosuppressive drugs such as rituximab, bortezomib, and
alemtuzumab are used to suppress the antibody production of B-cells/plasma cells
BLyS Levels Posttransplantation
Thibault-Espitia A et al. assessed the BLyS level in 143 recipients posttrans-

plantation and showed that there was an association between the high levels of
BLyS and the development of the donor-specific antibody (DSA) (Thibault-Espitia
et al. 2012). However, the trial was a cross-sectional study and did not mention the
use of any immunosuppressant agent such as rituximab which can also alter the
BLyS level. Moreover, the study did not show the BLyS level prior to transplanta-
tion. In another study, recipients who received antirejection therapy such as
rituximab had a significant peak of BLyS levels at 3 months posttreatment (Zarkhin
et al. 2009). The high BLyS levels were positively correlated with more than
6 months of B-cell depletion after therapy. These results confirm that the B-cells
can lower the levels of BLyS by consuming it. On the other hand, patients treated
with a different immunosuppressant agent such as alemtuzumab had a higher rate of
ABMR and excessive amount of serum BLyS (Bloom et al. 2009). The BLyS levels
from the alemtuzumab-treated patients did not correlate with the amount of CD20
B-cells. This can be explained by the effects of alemtuzumab which has an activity
against B-cells as well as other innate immune cells. On the other hand, data from a
well-designed, longitudinal study of BLyS levels in pediatric recipients found no
correlation between BLyS levels and the development of de novo DSA. Therefore
the monitoring of BLyS could not be used to predict the development of DSA and
chronic ABMR in the pediatric recipients (Comoli et al. 2015). However, the
recipients with and without de novo DSA have similar BLyS kinetics. The BLyS
levels are seen to gradually increase in the first 12 months after transplantation and
eventually reach a plateau indicating the presence of early pan-activated B-cells in
both unsensitized and previously sensitized patients. Hence BLyS may contribute to
the rejection or tolerance of the kidney allograft.
Immunoregulation of BLyS
The immune system has a mechanism to counterbalance all types of immune

responses. When the immune system is activated, there are regulatory systems that
counterbalance the inflammatory immune response. There are evidences that regu-
latory B-cells can influence the levels of BLyS. This regulatory function is of
concern especially during the anti-BLyS therapy. ELISPOT data showed that the
regulatory IL-10-producing B-cells were significantly abundant when B-cells were
cocultured with BLyS. Mice treated with BLyS also had an increased amount of
IL-10-producing B-cells (Yang et al. 2010). The regulatory role of BLyS was later
confirmed in a placebo-controlled, double-blind phase 2 trial of atacicept, a BLyS
signal inhibitor. This study was conducted in patients with multiple sclerosis whose
clinical activity peaked and relapsed after receiving atacicept (Kappos et al. 2014).
As a result of this, the manipulation of the BLyS system with an inhibitor is a double-
edged sword that can affect both the activated B-cells and regulatory B-cells.
BLyS as a Biomarker for Transplantation
To date, BLyS can significantly impact the outcome of the kidney transplantation. The
concerns to use BLyS as a biomarker for the kidney transplantation are discussed here.
First, the production of BLyS from the innate immune system is counterbalanced by
the B-cells’ consumption of BLyS. To further complicate the matter, high levels of
BLyS are not always associated with B-cell activation, but a direct inhibition of the
B-cells can increase the BLyS level by reducing the consumption of BLyS. Second,
there are evidences that BLyS can regulate the immune system. BLyS can activate
B-cells, plasma cells, and regulatory B-cells of the immune system. Certain, favorable
conditions of the BLyS level can induce tolerance posttransplantation.
Thus additional studies of the BLyS levels in different, specific conditions are
needed to rule out the potential effects of the confounding factors and assess the
benefits of using this noninvasive method to monitor the outcome of the transplan-
tation. Since there is no single best biomarker, hence, the monitoring of BLyS should
be done with other biomarkers and interpreted by correlating the results to the
clinical data. A longitudinal follow-up of the BLyS level may yield future potential
benefits when monitoring the immune system posttransplantation.

Conditions
BLyS and APRIL may in the future be used to diagnose various B-cell-/plasma cell-
related diseases such as multiple myeloma, mixed cryoglobulinemia, and other
autoimmune diseases including Sjögren’s syndrome and multiple sclerosis. The
level of serum BLyS may be useful in predicting the activity and severity of the
disease. Additional studies looking at serial monitorings of BLyS are warranted for
future treatment guidelines.
For example, the combined use of BLyS levels and donor-specific antibody
(DSA) may enhance the accuracy to detect antibody-mediated rejection of the
graft. DSA alone can only detect ABMR in one-third of the patients
(Wu et al. 2013). So a combination of its use with BLyS may be useful for future
diagnostic tests of solid organ transplantation.
As for APRIL, its other uses may be its ability to prognose various B-cell-
mediated diseases. The reason for this is based on the data obtained from
Treamtrakanpon et al. who showed that a high serum APRIL level was associated
with treatment failure and early loss of kidney function (Treamtrakanpon
et al. 2012). There are evidences that have shown APRIL’s ability to accurately
prognose lupus kidney disease. Hence APRIL is considered a good prognostic
biomarker for lupus kidney disease. Thus the use of APRIL should also be beneficial
for other diseases caused by memory and/or plasma cells.
Conclusions
Biomarkers to detect kidney diseases are an unmet medical need. Noninvasive tests
can be repetitively used to safely monitor the injury of the kidney. Levels of B-cell-
activating cytokines may be potential candidates for detecting immune-mediated
kidney injury.
Summary Points
• BLyS and APRIL are key cytokines of B-cell growth and maturation.
• The effects of the innate immune system and homeostasis of the B-cells affect the
serum levels of BLyS and APRIL.
• B-cell markers such as BLyS and APRIL cytokines are good examples of bio-
markers that can be used to prognose lupus kidney disease and outcome of the
kidney transplantation.
• Data interpretations of BLyS biomarkers should be done per patient together with
clinical data and other biomarkers to increase the accuracy of the prognosis for
patients with kidney diseases or those who have had a kidney transplantation.
• BLyS biomarkers can be used to diagnose, prognose, and help guide the treatment
for B-cell-associated kidney disease particularly lupus nephritis and kidney
transplantation.
Acknowledgment This work is supported by the National Science and Technology Development
Agency (NSTDA), Thailand (P-13-00505).
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Erythrocyte Glutathione Transferase
as a Biomarker in Kidney Health 26
and Disease
Alessio Bocedi, Annalisa Noce, Raffaele Fabrini, Nicola Di Daniele,

Francesco Galli, and Giorgio Ricci
Contents
Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580
Biomarkers of Stress Response in Kidney Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581
Molecular and Kinetic Properties of e-GST . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584
Activity Determination of e-GST and Its Correlation with the Protein Expression . . . . . . . . . . . 584
Overexpression of e-GST in Uremic Patients Under Hemodialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . 585
e-GST in CKD Patients Under Conservative (Pre-dialysis) Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . 588
Environmental and Endogenous Factors Affecting e-GST Levels in Healthy
Subjects and in Non-uremic Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
Overexpression of e-GST in Kidney-Transplanted Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591
Future Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 595
A. Bocedi • R. Fabrini • G. Ricci (*)

Department of Chemical Sciences and Technologies, University of Rome “Tor Vergata”, Rome,
Italy
e-mail: bocedi@uniroma3.it; raffaele.fabrini@uniroma2.it; riccig@uniroma2.it
A. Noce • N. Di Daniele
Department of Systems Medicine, Nephrology and Hypertension Unit, University of Rome “Tor
Vergata”, Rome, Italy
e-mail: annalisa.noce@libero.it; didaniele@med.uniroma2.it
F. Galli
Department of Pharmaceutical Sciences, University of Perugia, Perugia, Italy
e-mail: francesco.galli@unipg.it

578 A. Bocedi et al.
Abstract
Erythrocyte glutathione transferase (e-GST), an enzyme involved in the detoxi-
fication of endogenous and exogenous toxic compounds, is overexpressed in
chronic kidney disease (CKD) patients. e-GST transcription increases linearly
along the stages of kidney disease progression, and a positive correlation with the
extent of hyperhomocysteinemia, a characteristic defect of thiol metabolism in
CKD, has consistently been demonstrated. e-GST expression varied in the com-
parison between patients treated with pure diffusive and convective dialytic
procedures, and preliminary data demonstrate the persistence of increased
e-GST levels in kidney transplanted patients.
Analogously with the use of glycated hemoglobin in the retrospective moni-
toring of blood glucose in diabetic patients, e-GST may serve as an indicator over
a temporal span of a few weeks of the systemic exposure to uremic toxins, which
could be particularly important in verifying the efficacy of depurative techniques
and devices. Applications as prognostic marker of toxicological processes and
comorbidity in CKD as well as in transplanted patients are worth of further
clinical investigation.
Keywords
Biomarker • Erythrocyte glutathione transferase • Maintenance hemodialysis •
Blood toxicity • Chronic kidney disease • Kidney transplantation
Abbreviations
ADQI Acute dialysis quality initiative
AhR Aryl hydrocarbon receptor
ARE Antioxidant-responsive element
ASK1 Apoptosis signal-regulating kinase 1
Bax BCL-2-associated X protein
C/EBPβ CAATT/enhancer-binding protein
CAR Constitutive androstane receptor
e-CAT Erythrocyte catalase
e-GST Erythrocyte glutathione transferase
EpRE Electrophile-responsive element
GSH Glutathione
GSTP1-1 Glutathione transferase class P1-1
Hcy L-homocysteine
HD Hemodialysis
HDF Hemodiafiltration
IL-6, IL-8; IL-18 Interleukin-6, -8, -18
K/DOQI Kidney disease outcomes quality initiative
26 Erythrocyte Glutathione Transferase as a Biomarker in Kidney Health. . . 579
KDIGO Kidney disease improving global outcomes

KIM-1 Kidney injury molecules
L-FABP LTYPE fatty acid-binding protein
MHD Maintenance hemodialysis
NHE3 Sodium-hydrogen exchanger 3
Nrf2 NF-E2-related factor 2
PINI Prognostic inflammatory and nutrition index
PPARγ Peroxisome proliferator-activated receptor-γ
Prxd-6 Peroxiredoxin-6
PXR Pregnane X receptor
RBC Red blood cell
T2DM Type 2 diabetes mellitus
TNF-α Tumor necrosis factor-alpha
TRAF2 TNF-receptor associated factor 2
Key Facts
e-GST is involved in the cell detoxification against many toxic compounds.

e-GST can be used as a prognostic biomarker and monitoring tool of human
conditions associated with increased exposure to endogeneous toxins or xenobi-
otics and oxidative stress.
e-GST overexpression is part of the stress adaption response of bone marrow and
blood and thus is a natural defense mechanism against endogenous and exoge-
nous stressors (such as uremic toxins, xenobiotics, pollutants, etc.).
e-GST activity may be determined by a rapid, easy, and unexpensive spectrophoto-
metric procedure.
The evauation of e-GST overexpression can be used to assess systemic toxicity with
analogy to glycated hemoglobin used in clinical monitoring of blood glucose.
Definitions
Biomarker Natural component (or substance) used to indicate biological events

such as a specific disease or the response to a therapy.
Chronic kidney disease Known as chronic renal disease which consists in a

progressive loss in renal function.
Dialysis Technology accomplished by a filtration device used in biology and

medicine to depurate fluids and solutions such as the same human blood.
e-GST Enzyme expressed in red blood cells devoted to toxic compound

detoxifications.
Glomerular filtration rate The glomerular filtration rate is a test to measure the
level of kidney function and to determine the stage of kidney disease.
Glutathione Also referred as reduced glutathione (GSH) is a tripeptide containing

glutamic acid, cysteine, and glycine, which serves as a reducing agent in many
biochemical reactions.
Glycated hemoglobin Glycated hemoglobin is a particular form of hemoglobin

synthesized via a nonenzymatic glycation pathway during its exposure to plasma
glucose.
Homocysteine A modified amino acid that often increases above normal concen-
trations in chronic kidney disease patients.
Kt/Vurea A mathematical model that takes into account the urea clearance in a
single hemodialysis session to quantify the dose and thus the adequacy of dialysis
therapy.
Systemic sclerosis Also known as scleroderma is a chronic connective tissue

disease generally classified as one of the autoimmune rheumatic diseases.
Introduction
Acute kidney injury (AKI) and chronic kidney disease (CKD) are conditions asso-
ciated with poor prognosis and high impact on healthcare systems. Achieving timely
and appropriate diagnosis and therapeutic prescription in these patient populations
has major impact on morbidity and mortality indices.
Unluckily, available diagnostic and prognostic tools cannot fulfill these goals.
Routine laboratory protocols identify macroscopic defects of glomerular filtration
and tubular reabsorption, but cannot provide a timely appropriate diagnosis of the
complex series of metabolic and toxicologial aspects of the renal disease, and conse-
quently their prognostic value is limited or null. Actually, many of these protocols
focus on water balance and small- to middle-size molecule removal, thus completely
missing several of the toxicological aspects associated with formation and retention of
uremic solutes. Compelling clinical research has revealed that biological and toxico-
logical effects of uremic solutes strongly depend on their reactivity with components
of cells and body fluids (Piroddi et al. 2013). Secondary products of this abnormal
chemistry, such as lipid-derived reactive carbonyls and protein adducts, are bioactive
compounds themselves that produce increasing waves of scavenging and inflamma-
tory responses of tissues. During kidney disease progression, such an adverse chem-
istry sustains mechanisms of stress and toxicity that spread from renal tissue, in the
earliest phases of the disease, to a systemic level. The consequent chronic-

degenerative lesions of tissues further sustain mechanisms of toxicity and the abnor-
mal signaling of uremic tissues. All these aspects of the uremic stress response are
disregarded in the clinical investigation and management of AKI and CKD patients.
Even worst the performance of current laboratory protocols in monitoring the
efficacy of depurative dialysis procedures remains a simple weighing of solute and
water exchanges throughout dialyzer membranes.
Biomarkers of Stress Response in Kidney Disease
Classically, the assessment of glomerular function is based on the measurement of

creatinine clearance (Ronco et al. 2010). However, creatinine cannot be considered
an ideal biomarker. This should be an endogenous substance that circulates at almost
stable concentrations and completely filtered by the kidney and excreted in the urine
without being reabsorbed or excreted from the renal tubule. In this respect, the
creatinine clearance test has a number of limitations. In fact, creatinine is completely
filtered by the glomerulus, but it is also in part reabsorbed and especially excreted by
the renal tubule. Moreover, circulating levels of creatinine are influenced by age and
body mass, and dietary habits.
Creatinine tests cannot be applied in the course of AKI, since increased values of
creatinine occur in presence of a significant, often irreversible, tissue damage. Again,
the large functional reserve of the kidney implies that serum creatinine significantly
changes only when more than 50 % of the renal function is compromised (Ronco
et al. 2008; Cruz et al. 2009). As a consequence biomarkers of kidney injury more
specific and sensitive than creatinine are needed (Devarajan 2010a; Parikh et al. 2010).
For this purpose, other biomarkers have been proposed as clinically useful in
diagnostic protocols of AKI syndrome, such as cystatin C, KIM-1 (kidney injury
molecule-1), NGAL (neutrophil gelatinase-associated lipocalin), NHE3 (sodium-
hydrogen exchanger 3), some cytokines (IL-6, IL-8; IL-18), the depolymerizing
factor of the actin-actin complex (actin-actin-depolymerizing F lipocalin), L-FABP
(LTYPE fatty-acid-binding protein), Netrin-1, and chemokine derived from keratin
(keratin-derived chemokine) (Ronco et al. 2008, 2010). However, clinical validation
in AKI has been provided and is available in the literature only for cystatin C and
NGAL (Devarajan 2010b; Parikh et al. 2010), and reliable unbiased laboratory
protocols are not available for the routine clinical assessment of other biomarkers.
Finally, most recent guidelines published by the acute dialysis quality initiative
(ADQI) (Ronco et al. 2010) reported that only NGAL and cystatin C could be
integrated in clinical practice protocols in the near future.
However, cystatin C should be considered an index of kidney function rather than
of tubular damage. Recent clinical trials suggest that NGAL is a reliable diagnostic
and prognostic biomarker of AKI (Devarajan 2010b; Vaidya et al. 2008; Haase
et al. 2009). ADQI guidelines recommend the use of NGAL assay in patients with
suspected AKI syndrome, being this an early and reliable marker of renal damage,
both in animal models and humans (Ronco et al. 2010).
However, increased circulating levels of NGAL may result from damage or

impairment/activity of other organs or tissues, such as cells of the immune system.
As far as CKD concerns, the introduction in 2002 of a classification system based
on estimated glomerular filtration rate (eGFR) produced a compelling need for
accurate methods to estimate GFR. As a consequence, the Modification of Diet in
Renal Disease (MDRD) estimating equation was adopted worldwide (K/DOQI
2004; Levey et al. 2006; KDIGO 2013; Fraser et al. 2015).
The more recently developed Chronic Kidney Disease Epidemiology Collabora-
tion (CKD-EPI) serum creatinine equation has been shown to improve accuracy of
eGFR estimation over the MDRD equation also improving prediction models for
mortality risk and progression rate to end-stage renal disease (ESRD) (Levey
et al. 2009; Matsushita et al. 2012). Subsequently, in 2012, Kidney Disease Improv-
ing Global Outcomes (KDIGO) recommendations advocate the routine use of the
CKD-EPI equation for reporting eGFR levels (KDIGO 2013).
As previously stated, serum creatinine levels are influenced by factors such as diet
and muscle mass, and, despite the use of weighting for age, gender, and race in the
eGFR estimating equations, this can result in patient misclassification (Stevens
et al. 2007, 2010). Serum cystatin C is less influenced by these factors, which suggests
a valid alternative for estimating GFR (Tangri et al. 2011; Baxmann et al. 2008).
Moreover, no one of the proposed biomarkers may provide any level of informa-
tion on extrarenal events of toxicity associated with the uremic condition and the
onset and progression of different aspects of CKD comorbidity.
Therefore, the lack of specific biomarkers of the efficiency of the kidney organ as
well as of dialysis therapy in depurating the organism from different types of
circulating toxins is a major clinical limitation. The ideal biomarker should provide
such clinical information in temporal spans not limited to a single day or dialysis
session, thereby representing a sort of long-term and versatile biosensor of uremic
toxicity. In this context, erythrocyte glutathione transferase (e-GST), an enzyme
compartmentalized in the red cells, and then non-dialyzable, seems to be an inter-
esting long-term probe either to assess kidney function or the adequacy of depurative
therapies. GSTs represent a superfamily of ubiquitous enzymes devoted to cell
protection by promoting the conjugation of glutathione with toxins of very different
shapes and chemical nature (Armstrong 1997; Hayes et al. 2005; Awasthi
et al. 1994). Furthermore, GSTs may act like ligandins by binding and sequestering
a variety of small or large toxic compounds and peptides. Protein-protein interaction
properties are also emerging as an important role of GSTP1-1, which appear to
control signaling pathways and transcriptional responses of cells (Bartolini and Galli
2016). The apoptotic signaling of Jun-kinase (Wang et al. 2001) and Bax (Kampranis
et al. 2000) is under the influence of this interaction. GSTs also modulate calcium
channels decreasing the apoptotic mobilization of calcium ions (Dulhunty
et al. 2001). Other relevant protein interactions of GSTP in the apoptotic pathway
include TNF-α and TNF-receptor associated factor 2 (TRAF2) and downstream the
apoptosis signal-regulating kinase 1 (ASK1) (Wu et al. 2006); also the activity of
Prxd-6, a 1-Cys membrane peroxidase, is controlled in a redox-dependent manner by
the interaction with GSTP protein, and recent evidence has been obtained on the
Fig. 1 X-ray crystallographic

structure of e-GST (GSTP1-
1). Three-dimensional
structure of glutathione
transferase P1-1 (i.e.,
erythrocyte glutathione
transferase) from PDB code:
6gss (Oakley et al. 1997). The
two monomers are in green
and red ribbons. Glutathione
molecule is also reported
according to atom type
(Picture was drawn by means
of UCSF Chimera (Pettersen
et al. 2004))
existence of a GSTP-dependent feedback of Nrf2 transcription factor activity that

may share the same molecular mechanism (Bartolini et al. 2015). The interleukin 6
activated STAT3 transcription factor is another node of the regulatory interactome of
GSTP (Bartolini and Galli 2016).
GSTs have been found to bind the dinitrosyl-glutathionyl-iron complex, a natural
derivative of nitric oxide with toxic properties, which becomes harmless when bound
to GST (De Maria et al. 2003; Bocedi et al. 2013). Other functions include selenium-
independent peroxidase activity with some organic peroxides (Bartling et al. 1993).
Human cytosolic GSTs are dimeric proteins classified into seven different gene-
independent classes termed alpha, mu, pi, theta, omega, sigma, and zeta. Human
glutathione transferase P1-1 (hGSTP1-1) is an homodimeric intracellular protein of
about 46 kDa expressed in different organs and cell types (Sheehan et al. 2001). The
GSTP1-1 is the most abundant form of intra-erythrocyte transferase representing 95 %
of an entire GST pool (Awasthi and Singh 1984). Its X-ray structure is reported in
Fig. 1.
The expression of GSTP gene varies depending on the activity of different
trascription factors (Bartolini and Galli 2016; Higgins and Hayes 2011). Besides
classical transcriptional activators such as some anticancer drugs and other xenobi-
otics, GSTP gene is modulated by the levels of endogenous activators. These
include toxins produced by the functional defect of depurative organs such as liver
and kidney (Carmagnol et al. 1981). In CKD patients, this hyperactivity of GSTP
gene was observed also in blood leukocytes and was proposed to represent an attempt
of the cell detoxification machinery to set a defense response against the systemic
toxicity of the uremic condition (Mimic-Oka et al. 1992; Galli et al. 2014). GSTP is, in
fact, one of the most effective components within the family of phase II genes
associated with the detoxification function and redox signaling of glutathione.
This chapter collects the available information in literature that suggests how
e-GST investigation can represent a very simple and inexpensive laboratory tool in
clinical nephrology. This could monitor toxicological aspects of uremia associated

with the transcriptional activation of detoxification and stress-response genes that
share their regulation with that of e-GST. Similarly to glycated hemoglobin analysis
in the long-term monitoring of blood glucose, the e-GST induction response pro-
vides a span coverage of several weeks in the retrospective evaluation of molecular
effects of uremic toxicity, i.e., that deriving from red blood cell (RBC) life span.
Molecular and Kinetic Properties of e-GST
e-GST has been extensively characterized in terms of molecular and kinetic proper-
ties. It was identified as GSTP1-1, an acidic GST isoenzyme belonging to the P1
class GST that is also present in the human spleen, skin, and brain. It is a dimeric
protein composed by two identical subunits of about 23 kDa (Armstrong 1997).
Each subunit has an active site competent for glutathione (GSH) binding (G-site) and
a second one able to accommodate different toxic compounds (H-site) which are
conjugated to GSH (Armstrong 1997). e-GST, as well as other GST isoenzymes,
may act also like a ligandin, binding toxic compounds and facilitating their elimi-
nation (Armstrong 1997). In blood, this enzyme is exclusively present in erythro-
cyte, where its concentration in healthy subjects is about 5.6 U/g Hb (Dessì et al.
2012). Serum does not contain detectable levels of e-GST. The presence of other
GST isoenzymes (in particular of the alpha-class isoenzyme) in sufficient amount to
be detected with a simple spectrophotometer has been reported in a single study, but
recent reexamination established that this result is merely artifactual (Fabrini
et al. 2012a).
In healthy subjects, e-GST activity levels remain virtually constant throughout
childhood and adult life (Strange et al. 1980), but upon transcriptional stimulation,
the enzyme protein can be up-regulated several folds over baseline non-stimulated
levels. GSTs contain in the promoter region the consensus sequences “antioxidant-
responsive element” (ARE) and “electrophile-responsive element” (EpRE), as well
as other sequences with an equivalent or synergistic function downstream of
transcriptional factors such as PXR, CAR, AhR, Nrf2, PPARγ, and C/EBP β
(Higgins and Hayes 2011). Such a molecular redundancy and the presence of
transregulation mechanism between the transcriptional factors that control GST
expression demonstrate the importance of this family of genes in cellular homeosta-
sis and particularly in the stress adaption response to electrophilic substances.
Activity Determination of e-GST and Its Correlation

with the Protein Expression
e-GST activity can be easily determined using a conventional spectrophotometric

apparatus. Using 1-chloro-2,4-dinitobenzene as co-substrate, the rate of conjugation
with GSH can be followed at 340 nm (extinction coefficient of the GSH-conjugation
complex = 9,600 M1 cm1) (Habig and Jakoby 1981). The pH optimum for this
enzyme is 7.5, but the assay is usually performed at pH 6.5 because at higher pH
values the spontaneous reaction becomes too fast. In early studies, e-GST activity
was determined after erythrocytes purification (Carmagnol et al. 1981; Mimic-Oka
et al. 1992; Galli et al. 1999). More recent papers described a simplified procedure
which uses only four microliters of hemolyzed total blood (Dessì et al. 2012).
This assay, which is accurate and specific for e-GST since this enzyme is not
detectable in serum, is very simple, unexpensive, and rapid (about 1 min per sample).
A major advancement for the application of this assay in high-throughput routine
protocols has been the development of an automatic procedure that performs hun-
dreds of determinations in a short time (Dessì et al. 2012).
However, accuracy of GST assay in blood and the relationship between the
enzyme activity and protein expression investigated in some of the earliest studies
on CKD patients (Galli et al. 1999) can be biased by the interference of some
relevant factors. First, inactive forms of GSTP1-1 could be present in total blood
being this a redox-sensitive form of GST. In fact the isoenzyme shows two reactive
cysteines (Cys47 and Cys101) that can form an intramolecular disulfide bridge after
exposure to conditions of oxidative stress to produce a completely inactive form of
this enzyme protein (Ricci et al. 1991). However, the oxidized inactive form can be
efficiently restored to active by the reducing activity of cellular thiols. Accordingly,
the studies on purified e-GST of healthy or nephropathic patients suggested that, if
present, inactive forms of e-GST in blood are negligible (Dessì et al. 2012), and
immunoblotting experiments were in line with this finding (Galli et al. 1999). In
conclusion, the increased e-GST activity of CKD patients is directly related with the
levels of protein expressed in the circulating RBCs.
Other important aspects in assay performance deal with sample storage and
processing. e-GST is a very stable enzyme when total blood samples are stored at
4 C (100 % activity recovered after 7 days), but storage at temperatures 0 C must
be avoided since this may lead to irreversible inactivation (Dessì et al. 2012). Simple
storage precautions are thus sufficient to handle samples in large multicenter trials or
screening programs.
Overexpression of e-GST in Uremic Patients Under Hemodialysis
Early in the 1980s, Carmagnol and coworkers first reported of increased e-GST
levels in pediatric hyperbilirubinemia and in CKD patients (Carmagnol et al. 1981).
After a decade, Mimic-Oka and coworkers (Mimic-Oka et al. 1992) confirmed this
finding reporting of increased GST and GSH levels in RBC and leukocytes of CKD
patients either in pre-dialysis conservative therapy or hemodialysis treatment. Only a
few years later, Galli and coworkers (Galli et al. 1999) first demonstrated that the
increased e-GST activity of CKD patients is the consequence of an increased
expression rather than a kinetic modulation of the enzyme protein. In this larger
observational study carried out on 118 subjects, e-GST overexpression was found to
be a highly prevalent trait in the different subpopulations of dialysis patients (72 % of
Fig. 2 e-GST activity in

healthy subjects and
pre-dialysis and dialysis
patients. e-GST activity is
reported as U/gHb. Healthy
subjects, control group;
pre-dialysis patients,CKD
stage I–IV; dialysis patients,
MHD patients (Modified from
Dessì et al. 2012)
patients on standard three times/week bicarbonate hemodialysis and 57 % of peri-

toneal dialysis patients get over the mean + 2SD cutoff levels of healthy subjects).
The study also suggested that e-GST overexpression is not associated with surrogate
markers of oxidative stress and was not influenced by the increased content of RBC
vitamin E produced after treatment with vitamin E-modified hemodialyzers. In the
same manner, even the response to erythropoietin therapy apparently did not influ-
ence e-GST levels, and preliminary experiments in that study suggested that high-
molecular-weight or protein-bound toxins could play some role in GST
overexpression. In the same study, only a few subjects in pre-dialysis phase were
assessed, and a lower prevalence of GST overexpression than in dialysis patients was
reported (approx. 20 %).
A later study (Dessì et al. 2012), performed with an automated procedure of
analysis on total blood of 62 maintenance hemodialysis patients and 80 healthy
controls, further confirmed the increased levels of e-GST activity in MHD patients
(5.8
0.4 vs 10.2
0.4 U/g Hb, respectively) (Fig. 2) and first described the linear
correlation between enzyme activity and plasma levels of homocysteine (Hcy) (r2 =
0.64, P < 0.0001) (Fig. 3), a modified amino acid that increases above normal
concentrations in more than 90 % of CKD patients. No other significant correlations
were found when e-GST activity was matched with hemoglobin, transferrin, blood
iron, and markers of systemic inflammation and renal function such as alpha-1 acid
glycoprotein and high-sensitive C-reactive protein, beta-2 microglobulin, and the
malnutrition-inflammation index PINI. This automated procedure of analysis was
Fig. 3 Correlation between plasma homocysteine levels (mM) and e-GST in MHD patients.
Correlation between plasma Hcy levels (μM) and e-GST activity (U/g Hb) (Modified from Dessì
et al. 2012)
validated and is now available to further explore this phase-II-dependent response of

the uremic syndrome at the clinical level (Dessì et al. 2012).
A later study (Noce et al. 2012) verified the potential of e-GST as a biomarker
complementary to the Kt/Vurea parameter to assess the dose and adequacy of
dialysis techniques, i.e., standard bicarbonate hemodialysis (HD) and online
hemodiafiltration (HDF). In this respect, e-GST may assess adequacy not as a
one-spot evaluation procedure performed during a single session of dialysis, as it
occurs for Kt/Vurea, but rather as an indicator of depurative efficiency of a series of
treatments accomplished during a span of few weeks. In this study the increased
e-GST activity of CKD patients was confirmed in 103 patients that were compared
with 82 healthy controls (9.0
0.4 vs 5.8
0.4 U/g Hb, respectively) (Fig. 4). Then
of this patient population, 44 patients on HD and 59 patients on HDF were compared
and e-GST activity was found to be significantly lower in HDF than HD subgroup
(8.2
0.4 vs. 10.0
0.4 U/g Hb, respectively). Single-pool Kt/Vurea and total
weekly Kt/Vurea were higher in HDF than in HD (1.5
0.1 vs. 1.3
0.1, and 4.6
0.1 vs. 3.9

0.2), but no significant correlation was found between e-GST activity
and Kt/Vurea data (Noce et al. 2012). The findings in this study were consistent with
the use of e-GST as a biomarker of long-term depurative efficacy of dialysis
treatments, which is worth of further clinical investigation.
Finally a recent retrospective study (Galli et al. 2014) of a population of 98 HD
patients investigated for plasma Hcy and blood thiol status between 1999 and 2004
demonstrated that a daily (2 h) dialysis schedule (daily HD or DHD) (n = 28) can
lead to a better correction of the uremic retention solute Hcy than a standard three
times/week protocol of HD (SHD) (n = 70). Levels of Cys and Cys-Gly measured
Fig. 4 e-GST activity in

uremic patients under
hemodialysis. e-GST activity
in all the uremic patients in
standard bicarbonate
hemodialysis (HD group), in
online hemodiafiltration
therapy (HDF group), and in
the control group. *P <
0.0001 e-GST activity in total
uremic patients vs the control
group. **P < 0.0001 e-GST
activity in the HD group vs the
HDF group (Modified from
Noce et al. 2012)
in plasma were also influenced, and according to past observation (Dessì

et al. 2012), the levels of hyperhomocysteinemia correlated with e-GST levels as
well as with plasma GSH. According with the findings obtained with the compar-
ison between diffusive and convective methods (Noce et al. 2012), these findings
point to a better depurative function of high-efficiency dialysis procedures,
such as frequent dialysis. Uremic retention solutes could be among the toxic
components that may interfere with thiol metabolism and redox balance of CKD
patients, some of which may interfere with e-GST transcription in the bone marrow.
These solutes await further investigation for molecular identification and better
removal by more efficient dialysis therapies.
e-GST in CKD Patients Under Conservative (Pre-dialysis) Therapy
When e-GST activity was investigated for the first time in pre-dialysis patients
(Mimic-Oka et al. 1992), a linear negative correlation was found between the
enzyme activity and the levels of creatinine clearance, a measure of renal function
directly reflecting GFR values. A more extensive and recent study (Dessì et al. 2012)
examined e-GST activity in 72 pre-dialysis patients, and a significant increase of
e-GST activity was also reported with a positive correlation with disease severity
weighted according to the four stages of “Kidney Disease Outcomes Quality Initia-
tive” classification (7.4
0.5, 8
1, 9.5
0.6, 12
1 U/g Hb, respectively)
(Fig. 2). In these patients, e-GST activity did not correlate with conventional markers
of either acute phase or chronic inflammation and kidney disease, although these
markers also increased according with the severity of the uremic disease (Table 1).
An extension of this study correlated the levels of e-GST, Hcy, and erythrocyte
catalase (e-CAT) in 328 type-2 diabetes mellitus patients (T2DM), of which 61 were
non-nephropathic patients and 267 were also CKD patients under conservative
pre-dialysis therapy. Average e-GST activity was significantly higher in all T2DM
patients compared to the control group (7.9
0.3 vs. 5.8
0.4 U/g Hb) (Fig. 5), and
enhanced activity levels were also observed in all the four subgroups of CKD
diabetic patients divided according with the K/DOQI guidelines (Noce
et al. 2014). Mean Hcy levels in diabetic patients were higher than in healthy
subjects (33.4
1.2 vs. 13.6
0.8 μM), and Hcy increased according with the
stage of CKD. As expected from previous studies (Noce et al. 2012), a significant
correlation was found between e-GST and Hcy levels (Noce et al. 2014). These
findings demonstrated that e-GST transcriptional activation is a specific response to,
and thus an early biomarker of the uremic condition.
Environmental and Endogenous Factors Affecting e-GST Levels

in Healthy Subjects and in Non-uremic Patients
Two different studies showed that, besides CKD, e-GST overexpression can be the
consequence of other conditions, such as the exposure to environmental pollutants or in
the case of the autoimmune disease known as scleroderma or systemic sclerosis (SSc).
Also in this latter disease the exposure to toxins of unknown origin and nature is
proposed to play a major pathogenic role. SSc is characterized by endothelial
dysfunction and fibrosis of the skin and internal organs (Fabrini et al. 2013), and a
renal involvement is frequent in these patients. Interestingly, e-GST is highly
overexpressed in SSc patients (n = 102) reaching a mean value of 13 U/g Hb, i.e.,
more than two times higher than normal healthy levels (5.8 U/g Hb) (Fig. 6a, b)
(Fabrini et al. 2013). Enzyme levels in these patients correlated (r2 = 0.49, P <
0.0001) with the Medsger DSS and DAI Valentini indices that quantify the severity
and activity of the disease. Interestingly, e-GST levels of SSc patients were not
influenced by the presence of kidney damage or by other defects of specific organs
taken separately. e-GST hyper-expression in this condition appears thus to be linked
with the exposure to putative toxins that cause the disease and not to the autoimmune
disease per se, to the damage of specific organs, or to other consequences of the
disease that may also include oxidative stress.
A study on about 500 healthy volunteers living in eight distinct areas at or near
the Sacco river valley, a region of the Frosinone district (Lazio, Italy) well known for
its environmental pollution (Table 2) (Fabrini et al. 2012b), proposed a role for
e-GST evaluation as a biomarker of environmental pollution hazard. Subjects of six
590
Table 1 Main clinical features and laboratory findings in 72 pre-dialysis patients divided into four subgroups according to K/DOQI stage (stage I to IV) CKD,
62 ESRD patients on MHD, and 80 healthy subjects (control group) (Modified from Dessì et al. 2012)
Control group Stage I (CKD) Stage II (CKD) Stage III (CKD) Stage IV (CKD) ESRD on MHD
e-GST (U/g Hb) 5.8
0.4 7.4
0.5 8
1 9.5
0.6 12
1 10.2
0.4
hs-CRP (mg/l) 1.2
0.7 3
1 3.4
0.4 4.0
0.8 7
2 7
1
GFR (ml/min)a 118
2 109
3 77
2 42
2 20
1 <4.7b
PINI 0.5
0.1 0.6
0.3 1.0
0.6 0.5
0.1 1.3
0.4 2
1
Alpha- 1 acid glycoprotein (g/l) 0.70
0.03 0.90
0.06 0.95
0.04 0.92
0.06 1.21
0.09 1.24
0.05
Beta-2 microglobulin (mg/l) 0.90
0.08 1.84
0.09 2.2
0.1 5.6
0.5 12
1 37
2
Mean age (years) 46.1 45.8 51.1 64.4 62.3 58.0
Data are expressed as mean
SEM
a
GFR was calculated on the basis of MDRD equation
b
Only two MHD patients showed a residual renal function while 60 were totally anuretic
A. Bocedi et al.
Fig. 5 E-GST activity in

diabetic CKD patients. e-GST
activity in diabetic patients
non-CKD group (CKD 0) and
all CKD stages (CKD I–IV)
compared to the control group
(healthy subjects) (Modified
from Noce et al. 2014)
different areas of that region showed 18–44 % increased levels of e-GST when
compared to 400 volunteers living in the Rome hinterland, and the highest GST
levels were observed in the areas of higher risk of pollution (Fig. 7). Oxidation-
dependent changes of GST activity were not observed in the blood specimens of the
exposed populations (Fabrini et al. 2012b).
Overexpression of e-GST in Kidney-Transplanted Patients
Preliminary data about e-GST activity in renal-transplanted patients are available

(Bocedi and Ricci unpublished data) and surprisingly demonstrated a remarkable
enzyme overexpression in these patients (n = 80) (Fig. 8a) with levels of activity
similar to those found in stage IV CKD patients. Only slightly different e-GST levels
were observed between patients receiving organs from living donors and cadavers.
No correlation has been found between e-GST levels and the clinical parameters
used to assess kidney function in these patients, except for eGFR. Also different
immunosuppressive therapies seem to not influence the e-GST expression. These
results, if further confirmed, may suggest the use of e-GST as an indicator of the
Fig. 6 Correlation of e-GST

with DSS and DAI in
sclerodermic patients.
a Correlation of e-GST
activity with the Medsger
disease severity scale (DSS) in
all sclerodermic patients (r2 =
0.49; P < 0.0001).
b Correlation of e-GST
activity with the Valentini
scleroderma disease activity
index (DAI) in all
sclerodermic patients (r2 =
0.49; P < 0.0001) (Modified
from Fabrini et al. 2013)
subclinical impairment of the transplanted kidneys. Interestingly, oxidized albumin,

used as a long-term biomarker of oxidative stress, demonstrated the presence of an
oxidative challenge in the transplanted patients and particularly in those receiving
postmortem-explanted kidneys (Fig. 8b).
Future Perspectives
The use of e-GST as a CKD biomarker is promising and worth of further clinical
validation. Reference values of e-GST levels in different ethnic groups and
populations as well as in different regions and habitats (i.e., urban, mountain,
country, coastal, etc.) need to be determined.
Table 2 Geographic features of selected areas (Modified from Fabrini et al. 2012b)
Territorial
Selected areas in the extension
Frosinone district (Km2) Geographic features
Area 1 25 Nearby confluence of the Sacco and Liri rivers
Area 2 10 Close to the Liri river
Area 3 90 After confluence of the Sacco and Liri rivers
Area 4 40 Near the Sacco river – presence of industrial site
Area 5 40 Liri river flows through the area – presence of
regularized landfill and compost sites
Area 6 30 Close to important industrial site
Area 7 60 Sacco river flows through the area
Area 8 40 Close to Sacco and Liri rivers – presence of
incineration plant
Fig. 7 e-GST activity in the

Sacco river valley and its
relative increase compared to
the Rome area. Cumulative
(men and women) e-GST
activity. Continuous dotted
line is the reference value for
the Rome area (Modified from
Fabrini et al. 2012b)
e-GST will become an even more promising biomarker of kidney disease if

personal reference values could be obtained along the different ages and situations of
life, which may be attained by screening protocols in childhood and youth. Only a few
microliters of total blood obtainable by means of a simple pinprick on a finger are
needed to perform an e-GST assay according with the procedure described in this
chapter, and the development of an automatic analysis procedure makes this a rela-
tively feasible and cheap task even for large populations of subjects.
Fig. 8 e-GST activity in transplanted patients and oxidized albumin in serum of transplanted
patients. a Preliminary e-GST levels in 80 kidney-transplanted patients compared to healthy sub-
jects and CKD (stage IV) and MHD patients. b Oxidized serum albumin in kidney-transplanted
patients compared to healthy subjects (Bocedi and Ricci unpublished results)
The study of e-GST levels in the different moments and context of life of a single
subject and the comparison with other subjects of the same area will eliminate major
biases for diagnostic and prognostic application of this biomarker.

Conditions
e-GST analysis represents a reliable diagnostic and prognostic tool of nephropathic

diseases. Other potential applications in other uncommon situations associated with
increased exposure to endogenous or environmental stressors or toxins have been
identified, such as the chronic exposition to environmental pollutants in urban areas
or working places. Given that all mammalians express e-GST with very similar
molecular properties, enzyme investigation could be used also to broader protocols
of environmental, veterinary and, food safety monitoring.
Summary Points
• e-GST is an inducible enzyme involved in cellular detoxification and redox

homeostasis of blood and systemic compartments.
• e-GST overexpression is highly prevalent in chronic kidney disease and is also
observed in other human conditions such as hyperbilirubinemia and systemic
sclerosis.
• e-GST overexpression appears very early in CKD and the extent of this transcrip-
tional response follows that of disease severity, thus pointing to a role of this
enzyme as a reliable and sensitive biomarker of uremic toxicity.
• e-GST overexpression in CKD can be used to monitor the depurative efficiency
and the dose of dialysis methods and devices. High-efficiency dialysis therapies
can decrease, at least in part, e-GST levels in maintenance HD patients.
• e-GST is also overexpressed in kidney-transplanted subjects that may further
demonstrate the sensitivity of this gene to even a partial (subclinical) defect of
kidney function.
• e-GST expression represents a circulating biosensor useful in the retrospective assess-
ment of the systemic exposure to electrophilic toxicants and also to other cellular
stressors. Retrospection spans through the 3 months of the erythrocyte life span.
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Plasma Growth Arrest-Specific Protein
6 (Gas6) as a Biomarker of Renal Diseases 27
Aybala Erek Toprak
Contents
Key Facts of Enzyme-Linked Immunosorbent Assay (ELISA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 601
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 602
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603
Gas6 Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 603
Gas6 in Cellular Actions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 605
Gas6-Receptor Interrelations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
Gas6/TAM System in Vasculature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 607
Gas6 in Renal Damage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609
Gas6 Stimulates Mesangial-Cell Proliferation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 609
Gas6 Induces Acute and Chronic Renal Nephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 610
Gas6 in Chronic Allograft Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 611
Gas6 in Diabetic Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 612
Gas6 in Human Chronic Renal Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
Gas6 in Human Inflammatory Renal Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
Gas6 in IgA Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 614
Gas6 Effect on Renin-Angiotensin-Aldosterone System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615
Gas6 in Renal Cell Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 616
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 618
Abstract
Gas6 (growth arrest-specific 6) is a unique protein belonging to the family of
plasma vitamin K-dependent proteins and a ligand for the tyrosine kinase family
(Tyro3, Axl, Mer). Gas6 bears a great structural resemblance to the anticoagulant
A.E. Toprak (*)

Göztepe Training and Research Hospital, Medical Biochemistry Lab, Istanbul Medeniyet
University School of Medicine, Kadıkoy/Istanbul, Turkey
e-mail: aybalaerek@yahoo.com; serhantoprak@hotmail.com

600 A.E. Toprak
protein S. Interestingly, in spite of the presence of a g-carboxyglutamic acid

domain in its structure, Gas6 has not been discored to show any function in the
coagulation cascade. Gas6 has been reported to participate in cell proliferation,
apoptosis, chemotaxis, leukocyte migration, sequestration, platelet aggregation,
vascular homeostasis, and inflammation. In addition, it plays a role in the activa-
tion of various kinds of cells, including platelets and endothelial and vascular
smooth-muscle cells (VSMC). After it was demonstrated that Gas6 induces
proliferation of VSMC and serum-starved NIH 3T3 (mouse embryonic fibroblast
cell lines) fibroblasts, mitogenic effects of Gas6 were found in cultured mesangial
cells. As a result, it has been reported to take part in various pathophysiological
processes, for instance, atherosclerosis, renal diseases, cancer, and thrombosis.
The main subject of the present review is the role Gas6 plays in renal diseases.
Keywords
Chronic allograft injury • Diabetic nephropathy • Gas6 • Gas6 (growth arrest-
specific 6) • Acute and chronic renal nephritis induction • Cellular actions •
Human chronic renal failure • Human inflammatory renal diseases • IgA nephrop-
athy • Mesangial-cell proliferation stimulation • Receptor interrelations • Renal
cell carcinoma • Renal damage • Renin-angiotensin-aldosterone system • TAM
system in vasculature • Mitogenic activity
Abbreviations
Ang Angiotensin
CR Chronic rejection
DOCA Deoxycorticosterone acetate
EC Endothelial cells
ECM Extracellular matrix
Gas6 Growth arrest-specific gene protein 6
Gla g-Carboxyglutamic acid
GO Glycoxidized
HD Hemodialysis
IMT Intima-media thickening
MHC Myosin heavy chain
MMCs Mouse mesangial cells
MR Mineralocorticoid receptor
NADPH Nicotinamide adenine dinucleotide phosphate
NIH 3T3 Mouse embryonic fibroblast cell lines
NK Natural killer
NTN Nephrotoxic nephritis
27 Plasma Growth Arrest-Specific Protein 6 (Gas6) as a Biomarker of. . . 601
NTS Nephrotoxic serum

PDGF Platelet-derived growth factor
PI3K Phosphatidylinositol-3-kinase
RAAS Renin-angiotensin-aldosterone system
RCC Renal cell carcinoma
RTK Receptor tyrosine kinase
RT-PCR Reverse transcription polymerase chain reaction
SHGB Sex hormone-binding globulin
STAT Signal transducers and activators of transcription
STZ Streptozotocin
TAM receptors Tyro3, Axl, and Mer receptors
TGF-β Transforming growth factor beta
VKD Vitamin K-dependent
VSMC Vascular smooth-muscle cells
Key Facts of Enzyme-Linked Immunosorbent Assay (ELISA)
• The enzyme-linked immunosorbent assay (ELISA) is a technique that usually

uses antibodies and color change to determine an antigen or uses antigens and
color change to identify an antibody in a liquid sample.
• If an antigen is a subject for detection, sample is applied to a solid phase which is
pre-coated microstrip plates with specific capture antibodies and antigens
are attached to capture antibodies. After incubation, the plates are washed with
a mild detergent solution to remove any nonspecifically bound or unbound
material. Then, a further specific detection antibody is applied over the solid
phase so it can bind to the antigen which is binded to capture antibody. This
detection antibody is linked to an enzyme, and, after an incubation, second
washing step proceeds. Finally the enzyme’s substrate is added. The further
reaction causes producing of a detectable signal, generally a color change in the
substrate. After stop solution is added, the optical density is read with an ELISA
plate reader (Fig. 3).
• Capture antibody, substance which is wanted to detect and detecting antibody
binds each other, respectively, and is called “sandwich” ELISA because of the
sandwich-like formation and it is the most common form.
• Unlike other spectrophotometric lab assay formats where the same reaction well
(e.g., a cuvette) can be reused after washing, ELISA plates are not reusable
because they are coated with antibody or antigen.
• Enzyme/substrate reaction is short term so microstrip plate must be read as soon
as possible (up to 20 min).
• Eligible for analyzing a wide variety of test parameters, and quick (approximately
86 tests in 3–4 h application are the advantages of ELISA method. And also
antigens of very low concentration level able to be detected if capture antibody is
very specific to antigen.
602 A.E. Toprak
Definitions
Angiotensin II It causes blood vessels to constrict (vasoconstriction) and high

blood pressure. Also it triggers the release of the water-retaining hormone vasopres-
sin from the pituitary gland and afterward secretion of aldosterone, adrenaline, and
noradrenaline in the adrenal gland.
Chronic kidney disease Chronic kidney disease comprises conditions that

damage kidneys and reduce their capacity to keep our body healthy, being present
for at least 3 months. Both lower GFR and greater levels of albuminuria are
independently related to cardiovascular events, mortality, and the rate of end-stage
renal disease. If kidney disease gets worse, wastes can accumulate and become
high levels in blood and some complications may develop like high blood
pressure, anemia (low blood count), poor nutritional health, weak bones, and
nerve damage.
Glomerular filtration rate (GFR) GFR is the gold test to determine level of
kidney function which describes the flow rate of filtered fluid through the kidney
and identifies the stage of renal disease. In clinical practice, serum creatinine
level-based estimates of creatinine clearance are used to quantify GFR. A widespread
used formula for estimate of creatinine clearance is the Cockcroft-Gault formula. It
can be calculated from the blood creatinine test and patient’s age, gender, and body
size. GFR indicates patient’s stage of kidney disease and is used while planning
treatment.
Renin-angiotensin-aldosterone system RAAS is a hormone signaling system

responsible for the regulation of fluid balance and blood pressure in our body.
When blood pressure is low or certain nerve impulses occur by stressful situations,
the kidneys release an enzyme into the circulation called renin. Plasma renin then
triggers the conversion of angiotensinogen (released from the liver) to angiotensin
I. This is converted by another enzyme, the angiotensin-converting enzyme (ACE)
(found in the lungs), into angiotensin II.
Reverse transcription polymerase chain reaction (RT-PCR) RT-PCR is a kind of

the polymerase chain reaction (PCR). This assay is usually used in molecular
biology and genetics to determine RNA expression. RT-PCR enables creation
of complementary DNA (cDNA) transcripts from RNA and identifies gene
expression.
Vitamin K-dependent (VKD) proteins VKD proteins require the presence of

vitamin K to become biologically active via carboxylation. Commonly known
VKD proteins are the factors II, VII, IX, and X in coagulation system. Other well-
known VKD proteins in anticoagulation system are protein C and protein
S. Osteocalcin, Gas6, matrix Gla protein, proline-rich Gla proteins 1,2, and trans-
membrane Gla proteins 3,4 are the VKD proteins which are less popular.
Introduction
Expansion of mesangial cells is shown in many kinds of glomerular disease and

usually is linked with matrix enlargement, contributing to progression to glomerulo-
sclerosis (Doi 2001).
It has been pointed out that several growth factors and cytokines cause mesangial-
cell proliferation: platelet-derived growth factor, basic fibroblast growth factor,
transforming growth factor beta 1, angiotensin (Ang) II, vascular endothelial growth
factor, interleukin-6 (Donate-Correa et al. 2015), Smad1 (Mima et al. 2008), and
growth arrest-specific 6 (Gas6) (Yanagita 2004). Gas6, a newly discovered vitamin
K-dependent (VKD) protein, is a ligand for TAM receptors Tyro3, Axl, and Mer.
Gas6/TAM signaling plays a part in chemotaxis and phagocytosis (Fridell
et al. 1998); cell migration, proliferation, and adhesion (McCloskey 1997); cell
survival and mitogenic activities (Goruppi et al. 1996); and hematopoiesis and
inflammation (Angelillo-Scherrer et al. 2005; Tjwa et al. 2008).
In 1988, Gas6 was determined by screening genes whose expression was
upregulated in embryonic mouse NIH 3T3 fibroblasts. The name of the protein
was derived from the property of cells whose growth was arrested (Schneider
et al. 1988). The gene was sequenced 5 years later (Manfioletti et al. 1993) and
named growth arrest-specific gene 6; it has been shown to share 44 % similarity with
protein S, a further VKD protein encoded by the PROS1 gene in humans. The best
determined role of protein S is its function in the anticoagulation pathway, where it
acts as a cofactor to protein C in the inhibition of factors Va and VIIIa. To the best of
our knowledge, Gas6 functions are limited to TAM-receptor activation.
Both Gas6 and its receptor Axl are expressed in the kidneys, principally in
intraglomerular mesangial cells. Mesangial-cell proliferation, a hallmark of glomer-
ular sclerosis, has been stimulated by Gas6/Axl. Put-down of mesangial-cell prolif-
eration with either warfarin, an inhibitor of gamma-carboxylation, or an extracellular
portion of Axl suggests that Gas6 plays a role in glomerular disease (Yanagita 2004).
Gas6 appears to exhibit an important role in a wide variety of cell types and
generally is associated with specifications of injury, inflammation, and repair. This
chapter focuses on the role of Gas6 in kidney diseases.
Gas6 Structure
Gas6 is a 75 kDa molecular weight multimodular protein and containing several

posttranscriptional modifications. The main one is gamma (γ)-carboxylation of the
N-terminal domain (the Gla module). The Gla module contains 11–12
γ-carboxylated glutamyl residues. Glutamyl residues are modified to
γ-carboxylated glutamyl residues by VKD carboxylase enzyme posttranslationally.
Carboxylation allows VKD clotting factors to bind to membrane phospholipids on
platelets that induce the coagulation pathway. In the absence of carboxylation, the
coagulation process becomes defective, yet it has not been shown that Gas6 has a
direct effect on the coagulation cascade (Huang et al. 2003).
604 A.E. Toprak
Fig. 1 Structure of Gas6. Gas6 consists of N-terminal Gla domain, a loop region with a disulfide
bridge, four EGF-like domains, and 2 C-terminal LamG-like subdomains containing the SHBG
domain. EGF epidermal growth factor, SHBG sex hormone-binding globulin
The Gla domain contributes to VKD proteins the capability of binding anionic
phospholipids at the cell surface. In the remaining cells, situated in the inner leaflet of
the plasma membrane are anionic phospholipids. At the cell surface, the demonstra-
tion of anionic phospholipids is a characteristic of cell injury, activation, and
apoptosis. In situations similar to these, such lipids maintain a docking surface on
which VKD proteins bind and their activity takes place. As a result, Gla domains aim
Gas6 to apoptotic or activated cells that occur in a wide range of pathologies
(Laurance et al. 2012).
Provided by a disulfide bridge, a loop follows the Gla domain. The Gas6 loop
does not appear to be susceptible to the performance of serine proteases, in contrast
with the protein S loop. In the event of protein S, this loop is necessary since it
interacts with activated protein C and is inhibited by thrombin (He et al. 1998). Four
epidermal growth-factor modules, two of which including calcium-binding consen-
sus sequences, followed by a sex hormone-binding globulin (SHBG)-like module,
compose the principal framework of Gas6 after the loop. This module includes two
submodules which have a framework similar to the globular modules of laminin A
(LamG). It is likely to observe such kind of globular structure in proteins since they
interact with steroids, heparin sulfates, or integrins (Fig. 1).
Although there is remarkable structural similarity between protein S and Gas6,
Gas6 tissue expressions for Gas6 and PROS 1 definitely are different. Most plasma
VKD factors have a gene expression restricted to the liver, especially factor IX and
factor X. Expression of PROS1 is not so specific to the liver, and comparable levels
of transcript can be found in the kidney, lungs, gonads, and endothelial cells. Gas6 is
unique among the genes of the family, because Gas6 expression in the liver is minor
compared with expression in other tissues (Nakano et al. 1995). Gas6 is substantially
expressed and has been shown in the lungs, heart, kidneys, intestines, and terminally
differentiated cells, as well as in endothelial cells within the capillaries, vascular
smooth-muscle cells (VSMCs), bone marrow, and monocytes (Avanzi et al. 1997).
Several enzyme-linked immunosorbent assay (ELISA) methods have been
developed to quantify Gas6, and its presence in plasma has been demonstrated
(Balogh et al. 2005; Borgel et al. 2006; Clauser 2007; Alciato et al. 2008; Ekman
et al. 2010; Erek-Toprak et al. 2014). Gas6 has been found in human plasma in
concentrations (15–63 ng/ml) that are much lower than the other VKD proteins of
plasma.
Gas6 in Cellular Actions
The function of Gas6 has been debated since its discovery. In reality, Gas6 was
identified as a growth factor in several varieties of cells, particularly cultured cells
derived from serum (Nakano et al. 1995; Li et al. 1996; Goruppi et al. 1996). Growth
factors are primary influences on cell adhesion, differentiation, migration, duplication,
and survival. These influences are mediated by interaction with specific cell-membrane
receptors, most of them with intrinsic protein-tyrosine kinase activity; thus they are
known as receptor tyrosine kinases (RTKs) (Schlessinger and UIIrich 1992).
Binding the extracellular portion of its cognate polypeptide ligand causes dimer-
ization or oligomerization of the receptor, transducing the signal to the intracellular
portion of the molecule and triggering tyrosine kinase activity, which results in
tyrosine phosphorylation of the receptor. RTKs are classified according to their
extracellular domain, which generally is composed of a modular structure. Members
of RTK subgroups often bind common or similar ligands.
A crucial discovery in the understanding of Gas6 biology was the discovery that
it was a ligand of a previously orphan RTK, Axl. Axl is part of a three-member family
of receptors comprising Axl, Tyro3, and MerTK, which had been cloned by various
groups and given various names: Axl (ARK, Tyro7, Ufo), Tyro3 (Sky, Rse, Brt, Dtk,
Tif, Etk2, Rek), and MerTK (Mer, c-Eyk, Nyk, Tyro12). The architecture of these
receptors is preserved, and it is constituted by two immunoglobulin domains
sequenced by two fibronectin type III (Fig. 2). This RTK family is named TAM
after the first letter of the names of its three components (Tyro3, Axl, and MerTK).
Beginning with the first studies of Gas6 action on cultured cells, the antiapoptotic
effect of Gas6 has been found in many cells, including NIH 3T3 fibroblasts, VSMCs,
chondrocytes, endothelial cells, neurons, oligodendrocytes, epithelial cells, and
several types of cancer cells. Investigations have shown that serum deprivation,
TNF-α, changes in pH, oxidative stress, soluble phospholipase, and the amyloid
peptide cause Gas6’s antiapoptotic effects (Bellido Martin and Frutos 2008).
Mitogenic activity is another research subject that shows the cellular effects of
Gas6. Mitogenic effects have been described in VSMCs, cardiac fibroblasts,
Schwann cells, and mesangial cells (Nakano et al. 1995; Stenhoff et al. 2004; Li
et al. 1996; Yanagita et al. 2001b).
Another cellular action of Gas6 is induction of phagocytosis by macrophages and
other phagocytic cells. This induction has been shown to be specific to apoptotic cell
removal or phosphatidylserine-containing liposomes, imitating the situation in the cel-
lular membranes of apoptotic cells (Ishimoto et al. 2000; Hall et al. 2001; Wu et al. 2005).
Some studies have demonstrated Gas6-induced chemotaxis of VSMCs (Fridell
et al. 1998). In certain tumor types, migration and invasiveness correlate with
606 A.E. Toprak
Fig. 2 Structure of TAM

receptors. TAM family
receptors consist of 2 Ig-like
modules, 2 FN III domains, a
TM, and a TK domain. FN III
fibronectin type III-like, TAM
Tyro3, Axl, and Mer, TK
tyrosine kinase, TM
transmembrane domain
expression of Gas6 receptors, especially in Axl (Shieh et al. 2005), and Gas6
subsidiary signaling via Axl leads to invasiveness of glioma cells in animal models
(Vajkoczy et al. 2006).
A further cellular action of Gas6 is cell activation and differentiation. It has been
reported that natural killer (NK) cells express Axl, Mer, and Tyro3, and all three
TAM receptors are essential for normal differentiation and maturation of NK cells
(Caraux et al. 2006). In addition, it has been demonstrated that Gas6 stimulation
downregulates expression of inflammatory cytokines (Correll et al. 2004).
Furthermore, induction of differentiation by Gas6 has been reported in the
maturation process of adipocytes (Maquoi et al. 2005). Collett et al. (2003) have
shown that Gas6 activity modulates differentiation models on vessel-wall cells,
including inhibiting osteogenic differentiation of pericytes. Ming et al. (2001)
have shown the differentiation of VSMCs into foam cells. These properties
could play a role in cellular homeostasis of vasculature after injury (Ruan and
Kazlauskas 2012).
Gas6-Receptor Interrelations
As mentioned, Gas6 is the ligand for the TAM-receptor family, which consists of
Axl, Tyro3, and Mer. The affinity sequence for Gas6 is Axl, Tyro3, and Mer, from
highest to lowest . The extracellular domain of these receptors contains two Ig-like
domains, which are features of adhesion molecules, sequenced by two fibronectin
type III-like figures. The cytoplasmic last segment contains a tyrosine kinase domain
(Fig. 2). Discovered to a product of a transforming gene in T-cell leukemia cells, this
gene was given the name “Axl,” which originates from the Greek “anexelekto,”
meaning uncontrolled, on the basis of initial observations of its function. Axl is a
140-kDa protein expressed omnipresent (Laurance et al. 2012; Nagata et al. 1996).
With its ability to transform, Gas6’s intracellular domain can activate tumors in
mice independently of its ligand. Furthermore, it seems to be overexpressed in many
human cancers (Wu et al. 2014). In addition to the role it plays in the development of
cancer, Axl takes part in cell adhesion due to its extracellular domain, free from its
tyrosine kinase domain (Bellosta et al. 1995).
Tyro3 and Mer were determined by a number of researchers in 1994. Tyro3 is
expressed mainly in the brain and central nervous system, whereas Mer is expressed
in monocytic-cell generation. In contrast to Axl, there is not so much data concerning
possible roles of Tyeo3 and Mer in cancer. However, recently it has been found that
knocking down Gas6 and Mer restricts the proliferation of myeloma cell lines, while
downregulation of Axl or Tyro has no effect (Waizenegger et al. 2015).
From the first studies on Gas6, numerous researchers have pointed out that
following Gas6/Axl binding, phosphatidylinositol-3-kinases (PI3K) is activated in
many cell types, including endothelial cells (EC), VSMC, chrondrocytes, fibroblasts,
neurons, oligodendrocytes, and a number of cancer cells (Goruppi et al. 1999; Allen
et al. 1999; Hasanbasic et al. 2004; Wu et al. 2014). PI3K/Akt activation is essential
for Gas6’s antiapoptotic role. The cell-survival effect is blocked by pharmacological
inhibitors of the PI3K pathway (Goruppi et al. 1999). Activation of Akt causes to
inactivation of Bad, a proapoptotic mediator, and to an enhancement in the
antiapoptotic protein Bcl-2 by an NFkB-dependent mechanism (Hasanbasic
et al. 2004). Several isoforms of PI3K have been shown to interact with Axl and
potentially could cause discrete signaling pathways from the receptor (Hafizi
et al. 2002). In this context, it is interesting to note that C1-TEN, a protein phos-
phatase similar to the PI3K/Akt/PKB inhibitor PTEN, has been found to interact
with Axl, although the potential regulatory role of the Axl/C1-TEN interaction has
not been elucidated (Hafizi et al. 2002).
Gas6/TAM System in Vasculature
Evidence from several in vivo and in vitro models has suggested that Gas6 plays an
important role in vascular biology. In early studies, Gas6 was demonstrated in
cloning a growth-inducing factor of VSMCs, and this was the initial relationship
between Gas6 and the biology of vascular vessels (Nakano et al. 1995). Shortly
608 A.E. Toprak
thereafter, it was shown that Gas6 and Axl proteins were increased by vascular injury
in rat carotid arteries. In VSMC culture, Axl expression and stimulation were
potentialized by thrombin and Ang II (Melaragno et al. 1998). In addition, it was
shown that Axl is a redox-sensitive receptor activated by H2O2 in cultured VSMC
and intact aorta VSMC (Konishi et al. 2004). More recently, a redox-induced
interaction between Axl and glutathiolated nonmuscle myosin heavy chain
(MHC)-IIB in VSMCs has been found. MHC-IIB is involved in directed cell
migration. The Axl-MHC-IIB relationship occurs via inducing VSMCs with both
reactive oxygen species and Gas6 (Cavet et al. 2010).
Another study has reported the effect of glucose levels on Axl signal transduction
in VSMCs. It demonstrated Gas6-Axl signal alteration by glucose by modulating
interaction of Axl with special signaling proteins. Gas6-Axl stimulation
increased ERK1/2 and PI3K-Akt-mTOR activation in high and low glucose con-
centrations, respectively, showing that 140-kDa Axl could be responsible for ERK1/
2 activation, whereas the 114-kDa Axl is responsible for Akt activation (Cavet
et al. 2008).
In addition, a function for Gas6/Axl has been described in a reproducible mouse
model of flow-dependent vascular remodeling that resembles human intima-media
thickening (IMT). It found that in Axl knockout mice, IMT of the carotid was
decreased by rising cell apoptosis and changing vascular inflammation. A major
finding of this research was that the Gas6/Axl pathway adjusted the performance of
some types of cells in the vessel wall and seemed to behave in an autocrine/paracrine
manner. Another finding was that remodeled carotids from Axl/ mice had
importantly changed inflammatory responses in comparison with Axl+/+ mice.
Specific changes, such as relatively more monocytes than VSMC, diminished
macrophages and neutrophils in the intima and augmented neutrophils in the adven-
titia. There was an important retardation of inflammatory response in Axl/ mice
during vascular remodeling, which could be attributed to apoptosis and/or a failure to
complete phagocytosis brought about by impaired macrophage function (Korshunov
et al. 2006). A similar situation was demonstrated in atherosclerosis of Gas6
knockout animals, which had a lower content of lymphocytes and macrophages
(Lutgens et al. 2000).
Furthermore, it has been shown that Gas6/Axl interactions play a role in vascular
calcifications. Regulation of Axl signaling is a crucial event in matrix mineralization
and pericyte differentiation. After it had been demonstrated that Axl and its ligand,
Gas6, are expressed in bovine retinal pericytes, research found that expression was
downregulated in post-confluent cultures containing mineralized nodules. Research
suggested that the ability to modulate the Axl signaling pathway in vivo may lead to
a novel therapeutic target to stop or prevent ectopic calcification (Collet et al. 2003).
This effect is supported by several reports that implicate the Gas6/TAM system in
osteoclast function (Kawaguchi et al. 2004). More recently, it has been shown that
Axl inhibits induction of calcification of cultured VSMCs (Collett et al. 2007).
In another study, statins were shown to prevent apoptosis of aortic smooth-muscle
cells by renovating the Gas6/Axl pathway, eventually inhibiting calcification (Son
et al. 2006). In this context, the link between genetic variants in Gas6 and stroke
could be due to the influence of Gas6 on vascular calcification, a process obviously

associated with risk of stroke (Munoz et al. 2007).
A different effect of Gas6 on vascular biology is its role on the endothelium,
which has been suggested from its antiapoptotic effect in culture (Goruppi 1997;
Hasanbasic et al. 2004). Furthermore, addition of Gas6 has been shown to inhibit
adhesion of granulocytes to endothelial cells (Avanzi et al. 1998).
Platelets are leading members of the vascular system, mainly providing primary
hemostasis, and mediating inflammatory responses and cellular interactivities. The
first proof suggesting a function for TAM receptors in platelet physiology originated
from researches of Gas6-deleted mice. It was shown that Gas6/ mice avoided
thrombosis and presented defective platelet aggregation. Moreover, reverse tran-
scription polymerase chain reaction (RT-PCR) study represented that platelets
express Tyro3, Axl, and Mer (Angelillo-Scherrer et al. 2001). A follow-up study
showed that all three receptors are necessary for normal platelet aggregation
(Angelillo-Scherrer et al. 2005). Furthermore, soluble Mer (Mer-Fc) inhibits platelet
aggregation in vitro and protects against collagen/epinephrine-stimulated thrombosis
in vivo (Sather et al. 2007). In additionally, mice with two or three TAM receptors
deleted exhibited more critical impairment of platelet function than single-deleted
mice (Wang et al. 2007). More recently, it was found that Gas6 promotes and
increases sequestration of circulatory platelets and leukocytes in a P-selectin-depen-
dent fashion (Tjwa et al. 2008).
Even though it has been reported that Gas6 is present in rat (İshimato et al. 2000)
and mouse platelets, Balogh et al. (2005) reported that using immunoblotting,
combined IP immunoblotting and a highly sensitive and specific ELISA for Gas6
did not enable detection of Gas6 in washed human platelets. The previously pro-
posed role of Gas6 in platelet aggregation may be due to Gas6 coming from the
circulation.
Gas6 in Renal Damage
In many types of glomerular disease, too much proliferation of mesangial cells

greatly affects glomerular structure and function. Regardless of the source of injury,
an impairment in the control of mesangial-cell proliferation seems to play a vital role
in developing progressive renal injury and glomerulosclerosis (Doi 2001).
Gas6 Stimulates Mesangial-Cell Proliferation
Gas6 contributes to renal diseases mainly by stimulating mesangial-cell prolifera-

tion. After it was shown that Gas6 induces proliferation of VSMC and serum-starved
NIH 3T3 fibroblasts (Nakano et al. 1995; Goruppi et al. 1999), the mitogenic effect
of Gas6 was found in mesangial-cell culture (Yanagita et al. 1999). Additionally,
Yanagita et al. exhibited that the conditioned medium of serum-starved mesangial
cells prepared in the presence of warfarin did not stimulate proliferation of mesangial
610 A.E. Toprak
cells. It was concluded that Gas6 is an autocrine growth factor for mesangial cells,
and warfarin restrains mesangial-cell proliferation by decreasing production of
active Gas6 as a consequence of inhibiting the VKD γ-carboxylation of its Gla
domain.
Additional studies have pointed out that STAT3, a member of the signal trans-
ducer and activator of transcription (STAT) protein family, is a main signaling
molecule involved in Gas6-mediated mesangial-cell proliferation in vitro and
in vivo. STAT proteins, which are latent transcription factors, are activated via
phosphorylation (Yanagita et al. 2001b). Activated STAT proteins cause transloca-
tion to the nucleus and induce STAT-specific transcription. Activation of STAT
proteins is linked to regulation of cell differentiation and growth. It has been
shown that phosphorylation of STAT proteins is directly proportional to increasing
proliferation of mesangial cells. In addition, administering warfarin or Axl-Fc
restricts the Gas6-specific pathway by phosphorylating STAT3 in glomeruli and by
mesangial-cell proliferation (Yanagita et al. 2001b).
Gas6 Induces Acute and Chronic Renal Nephritis
Another effect of Gas6 related to renal damage was shown in an acute rat model of
glomerulonephritis (GN) stimulated by injecting anti-Thy1.1 antibody. Expression
of Gas6 and Axl was increased significantly in Thy1 GN in correlation with the
degree of mesangial-cell proliferation. Double immunostaining indicated that most
parts of Gas6 and Axl were localized together in mesangial cells. Thus, it seems
logical that Gas6 is secreted from mesangial cells in vivo to induce the mesangial-
cell surface receptor, Axl. Treating rats with Thy1 GN with low doses of warfarin
and Axl-ECD can greatly inhibit mesangial-cell proliferation and extracellular
matrix accumulation and can reduce the degree of glomerular injury in vivo in a
dose-related way. Furthermore, it has been reported that blocking the Gas6/Axl
pathway with warfarin or Axl-Fc reduced expression of platelet-derived growth
factor B (PDGF-B) in Thy1 GN, indicating that the Gas6/Axl pathway can affect
PDGF-B production in vivo. These findings show that the Gas6/Axl pathway plays
an important role in progression of glomerular diseases by modulating expression of
other growth factors in vivo (Yanagita et al. 2001a).
Investigating the function of Gas6 in stimulated nephrotoxic nephritis (NTN)
contributes further insight into understanding whether Gas6 is involved in chronic
renal injury. Early studies with Thy1 GN were important as a means of demonstrat-
ing the role of Gas6 in human diseases, but they did not provide sufficient proof
about progressive kidney diseases leading to chronic renal failure in human subjects,
because Thy1 GN is self-limited and calms spontaneously. NTN is a model of a
progressive form of glomerulonephritis in which glomerular hypercellularity, cres-
cent lesion formation, and glomerular sclerosis occur via inflammatory-cell infiltra-
tion and glomerular proliferation (Topham et al. 1999). NTN is stimulated by
injecting preimmunized mice with heterologous nephrotoxic serum (NTS, a sheep
anti-rat glomerular antiserum) that has reactivity to different glomerular cell and
basal membrane antigens. Gas6/ mice have been used to research the role of
Gas6 in NTN. In Gas6/ mice, decreased phosphorylation of STAT3, remarkable
reduction of proteinuria, less glomerular cell proliferation, less crescent formation,
and decreased mortality were found compared with Gas6+/+ mice. Furthermore,
administering recombinant Gas6 to NTS-treated Gas6/ mice induced massive
proteinuria, glomerular cell proliferation, and glomerulosclerosis compared to the
responses observed in wild-type NTN mice. These data indicate that Gas6 is
involved in progression of chronic kidney damage (Yanagita et al. 2002).
Apart from receptor Axl, another member of TAM family, Mer, has been exam-
ined in experiments on glomerulonephritis. The crucial role of Mer in clearing
apoptotic cells in the immune system and its function in lessening immune responses
by modulating cytokine production has caused a great deal of interest in this
molecule in the field of autoimmunity. Mer-deficient mice develop a lupus-like
autoimmune syndrome, possibly resulting from Mer-mediated inflammatory
response with impaired clearing of apoptotic cells (Lemke and Rothlin 2008).
More recently, Shao et al. (2010) used NTS-mediated kidney disease, which is a
mouse model of GN. In this model, the severity of injury correlates directly with the
dose of antibody injected. Histological changes stimulated by inflammatory injury
exhibit apoptosis linked subtly with degrees of abnormalities. The study detected
high levels of Mer protein in the kidney and more precise localized expression of
renal Mer in mesangial and endothelial cells within the glomerulus, and the study
found that Mer was upregulated during experimental GN. Interestingly, Mer knock-
out mice were much more prone to NTS-nephritis than were the wild type. Severe
renal damage was observed within 3 days of NTS injection in Mer knockout mice
but not in wild-type controls. In addition, it was found that early-onset renal damage
in Mer/ mice was associated with increased inflammatory cytokines, massive
amounts of apoptotic cells, and abundant infiltration of neutrophils. It seems Mer
plays a protective role in development of NTS-mediated nephritis, in contrast with
the reported role of Axl in worsening renal disease.
Thus, Mer and Axl, two receptor kinases that belong to the same subfamily,
possibly play contrasting roles in glomerular inflammation. Their opposing roles
in vivo may depend on their relative expression, which is far greater for Mer in the
basal state and in availability of ligands. Further studies will be required to clarify
how capillary-rich organs, such as the kidneys, are protected via Mer and to clarify
the obviously complicated relationship between Axl and Mer in renal pathology.
Gas6 in Chronic Allograft Injury
Chronic allograft injury still is a contributory factor to late kidney-graft loss, despite
improvements in immunosuppressive drugs and a reduction in acute T-cell-mediated
rejection (Zhang et al. 2015). Chronic rejection is responsible for more than half of
graft failures in surviving recipients after renal transplantation (Paul 1995). Typical
pathological features of chronic rejection are cellular proliferation and fibrosis in
glomeruli, tubules, and the media layer of arterioles. Immunological and
612 A.E. Toprak
nonimmunological mechanisms may play roles in tissue remodeling by helping local

production of growth factors that affect cell migration and proliferation.
Yin et al. (2001) investigated expression of Gas6 in a rat model of
chronic rejection (CR) of kidney transplant and found that Gas6 expression accel-
erates significantly above already-high basal levels during development of CR. The
Gas6 response has a significant peak at 4 weeks – meaning during the initial stages of
CR – in the allograft compared with expression in the isograft. Transforming growth
factor beta (TGF-β) remains elevated at all time points tested up to 24 weeks post
transplant. This suggests that Gas6 plays a role in the initial stages of CR develop-
ment. Moreover, the same researchers later investigated expression of Gas6 and
its receptors in human renal allografts that were demonstrating either acute
rejection, chronic rejection, acute tubular necrosis, or calcineurin inhibitor toxicity.
They found that expression of Gas6 increased in the acute tubular necrosis and CR
groups, whereas Axl expression was upregulated in acute tubular necrosis. This
shows that Gas6 expression positively correlates with expression of a CR marker,
α-smooth muscle actin. These findings suggest the possibility that Gas6 plays a
part not only in the process of kidney allograft dysfunction but also in CR
(Yin et al. 2002).
Gas6 in Diabetic Nephropathy
More than 387 million people worldwide suffer from diabetes mellitus, and it is the
main factor in chronic kidney disease, responsible for up to 45 % of end-stage kidney
disease (www.idf.org/diabetesatlas/6e/Update20124). Given this, lightening the
molecular mechanism of diabetic nephropathy (DN) is crucial.
The main characteristics of the initial phase of DN are mesangial-cell expansion,
glomerular hypertrophy, increasing glomerular filtration rate (GFR), accumulation of
extracellular matrix (ECM), and albuminuria, which frequently progress to
glomerulosclerosis and impairment of renal function if untreated. Much comprehen-
sive research has been done to identify molecules involved in mesangial broadening
and glomerular hypertrophy.
Nagai et al. (2003) used streptozotocin (STZ)-induced diabetic rats and reported
12 weeks after injection that they exhibited characteristics of the initial phase of DN,
such as glomerular hypertrophy, increased glomerular filtration rate, and albumin-
uria. Gas6 and Axl expression in the glomeruli of these rats was augmented, and
application of low-dose warfarin to STZ-induced rats diminished GFR increase,
albuminuria, and glomerular hypertrophy. In addition, in STZ-treated Gas6/
mice, less mesangial and glomerular hypertrophy was found compared to identically
treated Gas6+/+ mice. These findings showed that the Gas6/Axl pathway may play a
crucial part in the initial phase of DN in vivo.
To research the signaling molecules that contribute to mesangial hypertrophy and
find how Gas6 is involved in DN, Nagai et al. (2005) studied the Akt/mTOR
pathway involved in mesangial-cell hypertrophy and examined a major mechanism
responsible for glomerular hypertrophy in the pathway’s downstream signaling
molecules, p70 S6 kinase and 4EBP-1, in vivo and in vitro. The in vivo part of the
study analyzed the glomeruli of STZ-treated Gas6 knockout mice and found activa-
tion of Akt and 4E-BP-1, induction of p27, and localization of phosphorylated Akt in
mesangial cells of diabetic glomeruli. Furthermore, STZ-treated Gas6/ mice
showed less phosphorylated Akt-positive areas than did STZ-treated, +/+ mice.
The in vitro part of the study examined the influence of high glucose on Gas6/Axl,
the role of Gas6/Axl in high glucose-stimulated mesangial hypertrophy, and the role
of the Akt/mTOR pathway in Gas6-stimulated mesangial hypertrophy. It concluded
that Gas6 can be stimulated by high glucose and is able to stimulate mesangial
hypertrophy in an Akt-dependent fashion. The Akt/mTOR pathway is induced
primarily in mesangial cells when mesangial and glomerular hypertrophy are deter-
mined in the initial phase of diabetic DN in vivo. Gas6 may be one of the molecules
contributing to activation of this pathway.
Low-density lipoprotein (LDL) has been associated with diabetic microvascular
complications and is modified by enhanced oxidation, glycation, or glycoxidation.
Modified LDL levels are increased abundantly in diabetic patients, despite good
glycemic control (Lyons 1993). Modified LDL leads to development of glomerular
injury in diabetes by increasing transforming growth factor (TGF)-b1 expression,
which plays a key role in proliferation of mesangial cells in diabetes. More recently,
Kim et al. (2015) examined the relationship between glycoxidized (GO)-LDL
and Axl/Gas6 signaling pathways in DN and studied glomerular mouse mesangial
cells (MMCs) in vitro to show a GO-LDL-induced gene expression profile. They
reported that Axl gene and protein expression was significantly increased by
GO-LDL in MMCs. In addition, they found that Axl expression was increased in
MMCs cultured under diabetic conditions. Furthermore, Gas6 stimulated TGF-b1
secretion, and this TGF-b1secretion stimulated Axl expression. It seems GO-LDL
can increase Axl expression by Gas6-induced TGF-b1 upregulation. The study con-
cluded that Axl/Gas6 signaling may be a new therapeutic aim for renal diseases induced
by GO-LDL.
There are not many studies investigating Gas6 effects in human DN. The first, by
Erek-Toprak et al. (2014), showed that in patients with type 2 diabetes, plasma Gas6
levels were significantly higher in patients with albuminuria than in patients with
normoalbuminuria. The plasma Gas6 levels between subgroups of type 2 diabetes
were checked and found to be lower in the group with microalbuminuria than in the
group with macroalbuminuria. There was a significant correlation between albumin-
uria and plasma Gas6 levels and between Gas6 and HbA1c.. The study concluded
that there seems to be an association between plasma Gas6 levels and albuminuria in
patients with type 2 diabetes.
In this context, more recently, Erkoc et al. (2015) suggested that the Gas6 intron
8 c.834+7G>A gene polymorphism might be a risk factor for DN in type 2 diabetic
individuals and compared frequency of Gas6 intron 8 c.834+7G>A gene polymor-
phism between type 2 diabetic patients with DN and non-diabetic individuals. No
correlation between Gas6 intron 8 c.834+7G>A gene polymorphism and DN in type
2 diabetic individuals was found. Interestingly, the study found that Gas6 intron
8 c.834+7G>A polymorphism was associated with diabetic retinopathy among DN
614 A.E. Toprak
patients. Further studies with more subjects are needed to investigate the genetic
basis of type 2 DN and its relationship to the Gas6 gene.
Gas6 in Human Chronic Renal Failure
In the only research on human chronic kidney disease (CKD), Lee et al. (2012)
studied all stages of human CKD in hemodialysis (HD) patients (23 % of them
having diabetes) and reported increased levels of Gas6 in HD and CKD patients
compared with control subjects. In addition, this study found that Gas6 increased
significantly as the stages of CKD advanced. Furthermore, patients who had been on
dialysis longer had higher Gas6 levels. The study concluded that dysregulation of the
Gas6 protein could represent a novel inflammatory pathway contributing to human
vascular disease in renal failure.
Gas6 in Human Inflammatory Renal Diseases
One of the first studies to investigate Gas6 effects in human renal diseases was
conducted by Fiebeler et al. (2004). They gathered human renal specimens from
26 patients with IgA nephritis, lupus nephritis, diffuse immune complex glomeru-
lonephritis, antineutrophil cytoplasmic antibody-associated glomerulonephritis, and
acute rejection and examined whether Axl/Gas6 expression is influenced by nico-
tinamide adenine dinucleotide phosphate (NADPH) oxidase in vitro. The study
found that Gas6 and Axl immunofluorescence were barely detectable in normal
kidneys, whereas in specimens with diseases, Axl was abundantly expressed in the
small vessel media, distal tubules, glomeruli, and collecting ducts. Similarly, Gas6
was upregulated in all segments of the renal tubules, small vessel intima and media,
and glomeruli. Gas6 and Axl upregulation was a noticeable but nonspecific finding
in these renal diseases. Cultured rat VSMCs and immortalized human mesangial
cells were induced with Ang II for 6 or 18 h. Western blot and confocal microscopy
showed Ang II-dependent Gas6 and Axl expression. The study reported that the Ang
II-induced Gas6 and Axl expression may be dependent on NADPH-oxidase and
concluded that Gas6 and Axl are important molecules in human renal diseases and
seem to be potential targets for therapy.
Gas6 in IgA Nephropathy
Human IgA nephropathy (IgAN) is a form of glomerulonephritis which is the most

common worldwide. However, pathological demonstrations of IgAN are extensive
and can range from mild mesangial hypercellularity to a rapidly progressive glo-
merulonephritis with fulminant crescents and endocapillary proliferation. The results
of IgAN vary hugely (Tumlin et al. 2007). Therefore, IgAN might be divided into
different subgroups according to etiology, histopathology, or clinical manifestations.
In this respect, expression of Gas6 and its receptors were investigated by using
biopsy-proven IgAN cases to see whether Gas6 and its receptors were participated in
the progression of IgAN by analyzing the link between expression of Gas6 and
clinicopathological features (Nagai et al. 2013). In this study, Gas6 was upregulated
in either podocytes or endothelial/mesangial cells. In IgAN, Axl was in endothelial/
mesangial cells while Tyro3 was the receptor for Gas6 in podocytes. The study
reported that Gas6 expression in podocytes correlated with several prognostic
factors, for instance, mesangial proliferation and proteinuria, and was inversely
associated with p27 expression. Gas6 was involved in human IgAN, via Tyro3 and
Axl in podocytes and endothelial/mesangial cells, respectively (Yanagita et al. 2001a).
However, in human IgAN, Gas6 upregulation was observed mainly in podocytes,
while endothelial/mesangial-dominant expression was seen in few patients. The study
concluded that the expression pattern can be a marker to classify IgAN and therapeutic
effects and prognosis should reevaluated according to the pattern.
Gas6 Effect on Renin-Angiotensin-Aldosterone System
The renin-angiotensin-aldosterone system (RAAS) is a widely known regulator of

blood pressure (BP) and plays a main role in tissue perfusion, extracellular volume
and determining target-organ damage. It controls fluid and electrolyte balance via
regulated influences on the heart, blood vessels, and kidneys. RAAS activation can
cause hypertension, atherosclerosis, and cardiac and renal failure, which can be
prevented by blocking Ang II action. Ang II exerts its vasoconstrictor effect mainly
on the postglomerular arterioles by accelerating glomerular hydraulic pressure and
ultrafiltration of plasma proteins, influences that may be involved in onset
and progression of chronic kidney damage. In addition, Ang II may directly con-
tribute to accelerating renal damage by maintaining cell growth, fibrosis, and inflam-
mation. Ang II is an effective, direct stimulus of aldosterone (Ald) synthesis. Ald
signaling is mediated via the mineralocorticoid receptor (MR), and MR blocking has
a renoprotective function and may slow or even stop progression of chronic nephrop-
athies and reduce cardiovascular and renal morbidity (Atlas 2007). Nevertheless,
MR-regulated pathways and signal molecules are incompletely understood.
To explore whether Gas6 plays a role in RAAS, Park et al. used an Ald-induced
target-organ damage experimental model. They reported that Gas6 expression was
upregulated in a highly activated RAAS rat model. MR blocking decreased this
upregulation and prevented target-organ damage. In addition, VSMCs were tested
in vitro and it was found that Ald induced Gas6 expression without the presence
of Ang II. Furthermore, the effects of deoxycorticosterone acetate (DOCA) on
Gas6/ mice were explored, and it was observed that the gene-deleted mice
were protected from cardiac hypertrophy, inflammation, and fibrosis and had
improved renal function with reduced albuminuria, renal fibrosis, and fibronectin
deposition compared to wild-type mice. This protection was independent of BP
reduction. The study concluded that Gas6 seems to play a role in mineralocorticoid
receptor-mediated target-organ damage.
616 A.E. Toprak
More recently, Batchu et al. (2013) reported the differences in immune-specific

mechanisms regulated by Axl during initial versus late phases of salt-dependent
hypertension. They found that expression of Axl in hematopoietic cells is crucial to
kidney pathology in the initial phase of salt-dependent hypertension. In addition, in
both hematopoietic and nonhematopoietic cell lines, Axl contributes to the late phase
of hypertension.
Gas6 in Renal Cell Carcinoma
Renal cell carcinoma (RCC) is the most common cancer of the adult kidney and
represents 2–3 % of all human cancers. Incidence of RCC peaks at 60–70 years of
age. Unfortunately, the outcome of metastatic RCC has a dismal prognosis, and
mortality remains very high (Jonasch et al. 2014).
Because no markers are currently recommended to assess the risk of progressive
disease, there have been great efforts to find novel markers. Taking into account that
Axl is overexpressed in several cancers, Gustafsson et al. (2009a) conducted a study
to determine expression of Axl and its ligand, Gas6, in various RCC types in
308 patients. They reported that Axl and Gas6 expression in RCC are associated
with tumor advancement and patient survival. In particular, low-tumor Axl mRNA
levels independently correlated with improved survival.
Another study carried out by the same authors and published the same year used a
cell-based RCC model system to explore the complex role of Gas6 and Axl in RCC.
This study demonstrated inhibition of migration and survival in RCC 786-O cells as
a result of Gas6 signaling through the Axl RTK (Gustafsson et al. 2009b). To date,
Axl has been reported as increasing oncogenic effects in most cancers in which it has
been studied (Wu et al. 2014). However, in line with the results of Gustaffson et al.,
Gas6 has been reported to have contrasting effects, inhibiting vascular endothelial
growth factor A (VEGFR-A)-driven migration, specifically through activating Axl
RTK (Gallicchio et al. 2005). So, the literature implies that the biology of Gas6 and
Axl is complex and probably context-specific. Perhaps in RCC, where high Gas6
expression correlates with improved prognosis (Gustafsson et al. 2009a), Gas6 can
be protective and can retard cancer progression by mechanisms such as decreasing
migratory potential and decreased survival.
Conclusion
The Gas6 signaling pathway is highly regulated in several pathological conditions.

Gas6/Axl-dependent signaling plays a crucial role in cell survival, aggregation,
migration, and growth through multiple downstream pathways.
Further studies are needed to identify the molecules participating in the Gas6/Axl
pathway. Understanding the potential role and effects of the Gas6/Axl signaling
pathway may help affect the course of the future therapies by aiming this pathway in
patients with renal diseases.
Fig. 3 ELISA for the quantitation of an antigen in a biological sample
The levels of Gas6 in circulation have been studied in a variety of renal diseases, and
generally it has been detected increased comparing with controls. The serum or
plasma concentrations or expression of Gas6 is also determined in diabetes (Folli
et al. 2011), obesity, insulin resistance, atherosclerosis, acute coronary syndrome,
cardiovascular diseases, hypertension, coronary artery bypass grafting, stroke, rheu-
matoid arthritis (Kim et al. 2014), systemic lupus erythematosus, Behcet disease,
multiple sclerosis, sepsis, preeclampsia, and psoriasis. All studies demonstrate that
Gas6/Axl signaling alters and may be associated with prognosis and treatment of the
disease.
In different cancer types including ovarian, endometrial, gastric, thyroid, renal
clear cell carcinoma (Gustafsson et al. 2009a), and glioblastoma tumors, Gas6
expression is accelerated. Also in several primary human cancers, including lung
adenocarcinoma (Shieh et al. 2005), brain cell tumor (Vajkoczy et al. 2006), ocular
melanoma (Van Ginkel et al. 2004), pancreatic cancer (Wu et al. 2014) leukemia,
gastric cancer, colon cancer, breast cancer, ovarian cancer, and glioblastoma, Axl is
found overexpressed (Laurance et al. 2012). Furthermore AXL and GAS6 activated
in cancers have been correlated with short survival time, poor prognosis, stimulation
of accelerated invasiveness/metastasis, and drug resistance.
618 A.E. Toprak
The literature supports GAS6 /AXL as a new candidate of treatment target in a

variety of cancers as well as in different renal diseases and several other diseases.
However most of the researches was in vitro and the number of in vivo studies is not
so much especially in human. Future perspectives of more comprehensive researches
will highlight in detail the role of Gas6 in diagnosis, follow-up, and treatment of
diseases (Fig. 3).
Summary Points
• Gas6 (growth arrest-specific gene protein 6) is structurally linked to proteins from

the family of plasma vitamin K-dependent ones.
• Gas6 is a ligand for Tyro3, Axl, and Mer (TAM receptors), and these receptors are
members of a large family, receptor tyrosine kinases.
• Gas6/TAM signaling plays a role in chemotaxis; phagocytosis; cell migration,
proliferation, adhesion, survival, and mitogenic activities; hematopoiesis; and
inflammation.
• Gas6 plays a crucial role in several renal diseases.
• Uncovering the possible function and effects of the Gas6/Axl pathway may
contribute forthcoming therapies which intend this pathway in patients with
renal diseases.
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Soluble Urokinase Receptor as a Biomarker
in Kidney Disease 28
Takehiko Wada
Contents
Key Facts of Focal Segmental Glomerulosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 624
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 625
Potential Implication of Circulating Permeability Factors in FSGS . . . . . . . . . . . . . . . . . . . . . . . . . . . 626
Searching for a Circulating Permeability Factor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 628
suPAR as a Potential Circulating Permeability Factor in FSGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 629
Is suPAR a Valid Diagnostic Biomarker for Primary FSGS? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 630
Association Between Serum suPAR and Renal Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 633
suPAR as a Biomarker Predicting Clinical Course . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 634
Issues Concerning the Measurement of suPAR Levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 635
Urinary suPAR as a Biomarker for FSGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 638
suPAR as a Causative Factor for Primary FSGS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 638
suPAR and Antineutrophil Cytoplasmic Antibodies-Associated Glomerulonephritis . . . . . . . . . 639
suPAR and Lupus Nephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 640
suPAR and Diabetic Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641
suPAR and IgA Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 641
Potential Applications to Prognosis, Other Disease, or Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 642
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 642
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 643
Abstract
Urokinase receptor (uPAR), a glycosylphosphatidylinositol (GPI)-anchored pro-
tein, engages in multiple protein-protein interactions and various biological
functions. Its soluble form, soluble uPAR (suPAR), has been linked to various
T. Wada (*)
Division of Nephrology, Endocrinology and Metabolism, Tokai University School of Medicine,
Isehara, Japan
e-mail: twada-tky@umin.ac.jp; twada@tsc.u-tokai.ac.jp

624 T. Wada
diseases and conditions including focal segmental glomerulosclerosis (FSGS). It

has been long speculated that a circulating permeability factor should be impli-
cated in the pathogenesis of the disease because a substantial portion of
the patients experience recurrence shortly after renal transplantation. In 2011,
Reiser and colleagues suggested that suPAR might be not only a potential
pathogenic permeability factor but also a diagnostic biomarker. Following
this report, renal researchers worldwide have analyzed the validity of suPAR
as a diagnostic biomarker for primary FSGS and posttransplant FSGS. Further-
more, the utility of suPAR as a biomarker in other renal diseases including
diabetic nephropathy and immunoglobulin A (IgA) nephrosis has been also
suggested.
In this chapter, a comprehensive review of suPAR as a diagnostic biomarker
and as a predictive biomarker in kidney diseases is provided.
Keywords
Antineutrophil cytoplasmic antibody-associated glomerulonephritis
(ANCA-GN) • Biomarker suPAR (see soluble urokinase receptor (suPAR)) •
Cardiotrophin-like cytokine 1 (CLC-1) • C-reactive protein (CRP) • Cytokine
receptor-like factor 1 (CRLF-1) • Diabetic nephropathy • Enzyme-linked immu-
nosorbent assay (ELISA) • Focal segmental glomerulosclerosis (FSGS) • Circu-
lating permeability factors • End-stage renal disease • In vitro experiments •
Posttransplant recurrence • Primary nephrotic syndrome • ROC analysis • Immu-
noglobulin A nephropathy (IgAN) • Lupus nephritis • Minimal change disease
(MCD) • Nephrotic Syndrome Study Network (NEPTUNE) • Receiver operating
characteristic (ROC) analysis • Soluble urokinase receptor (suPAR) • Diabetic
nephropathy • Systemic lupus erythematosus (SLE) • Systemic Lupus
Erythematosus Disease Activity Index (SLEDAI) • Urokinase receptor
(uPAR) • Monoclonal blocking antibody
Key Facts of Focal Segmental Glomerulosclerosis
• Focal segmental glomerulosclerosis (FSGS) was first described in 1957.

• FSGS was originally a histopathological term that describes scarring which
occurred only in segments in some of glomeruli.
• FSGS is one of the major glomerular diseases that cause nephrotic syndrome.
• A variety of underlying mechanism of FSGS have been suggested, and podocyte
injury is considered to be a common cause of FSGS.
• A large proportion of cases show resistance to treatment and the renal prognosis is
generally poor.
• Renal transplantation is a treatment option for FSGS patients developing renal
failure; however, the rate of posttransplant recurrence is substantially high
(20–50 %).
28 Soluble Urokinase Receptor as a Biomarker in Kidney Disease 625
Definitions
Enzyme-linked immunosorbent assay (ELISA) ELISA is a plate-based assay

technique designed for detecting and quantifying peptides, proteins, antibodies, etc.
In quantitative sandwich immunoassay technique employed in the assay for uPAR, an
antibody specific for uPAR has been precoated onto a microplate. Samples and controls
are pipetted into the wells, and uPAR is bound by the immobilized antibody. After
washing away unbound substances, an enzyme-linked polyclonal antibody specific for
uPAR is added to the wells. Following a washing process, a substrate solution is added,
and the intensity of the developed color is measured.
Galactose affinity chromatography Affinity chromatography is a separation

method based on a specific interaction between an immobilized ligand and its
binding partner. In search for a circulating permeability factor, Savin and colleagues
postulated that the factor might have a lectin-like interaction with sugar of the
podocyte glycocalyx. Therefore, they used galactose-agarose beads to isolate a
substance which could interact with galactose.
Glycosylphosphatidylinositol (GPI) anchor GPI anchor is a posttranslational mod-

ification that links modified protein in the outer leaflet of the plasma membrane.
Podocyte The podocyte is one of the resident cells in the glomerulus. Podocytes are
located at the outer aspect of capillary tuft. A podocyte is considered to be a
terminally differentiated cell, and it comprises a large cell body, major processes,
and foot processes. The cells play an important role in filtration barrier system.
Receiver operating characteristic (ROC) analysis An ROC graph is a technique

for visualizing, organizing, and selecting classifiers based on their performance. The
curve is created by plotting the true positive rate against the false-positive rate at
various threshold settings. An analysis using ROC curve (ROC analysis), which is
useful to select possibly optimal models, has been extended for use in visualizing
and analyzing the behavior of diagnostic systems.
Introduction
The urokinase receptor (also known as urokinase-type plasminogen activator recep-

tor: uPAR) is a critical regulator of extracellular matrix (ECM) proteolysis, cell-ECM
interactions, and cell signaling. The human uPAR cDNA encodes a polypeptide of
335 amino acids, including an N-terminal 22-residue secretion signal peptide and a
C-terminal segment of 30 amino acids that is removable with the attachment of a
glycosylphosphatidylinositol (GPI) anchor.
uPAR is a protein with three Ly-6 and uPAR (LU) domains (D1, D2, and D3) that
is a proteinase receptor for urokinase. It is also involved in non-proteolytic pathways
626 T. Wada
αvβ3 integrin
α β
D1
D2
Vitronectin
D3
Fig. 1 Urokinase receptor and β3-integrin signaling. Urokinase receptor (uPAR) activates β3-integrin
outside-in signaling. Vitronectin, which binds to uPAR and β3-integrins, is required for the activity of
this pathway. The activation of β3-integrin signaling stimulates cell motility and invasion in murine
podocytes
by forming signaling complexes with other transmembrane proteins, including

integrins, caveolin, and G-protein-coupled receptors. uPAR is expressed in mono-
cytes (Estreicher et al. 1990), neutrophils (Plesner et al. 1994), activated T cells
(Nykjaer et al. 1994), endothelial cells (Barnathan et al. 1990), keratinocytes
(Grondahl-Hansen et al. 1988), fibroblasts (Plow et al. 1986), smooth muscle cells
(Reuning et al. 1994), megakaryocytes (Wohn et al. 1997), and tumor cells (Thuno
et al. 2009). In the kidney, its expression has been detected in tubular epithelial cells
(Florquin et al. 2001) and podocytes (Wei et al. 2008). The coordination of extra-
cellular matrix proteolysis and cell signaling by uPAR underlies its biological
function in cell migration, proliferation, and survival (Smith and Marshall 2010).
Because uPAR is attached to the plasma membrane with a GPI anchor and lacks both
transmembrane and intracellular domains, it requires transmembrane co-receptors
such as integrins and vitronectin (Fig. 1). Importantly, uPAR can be released from
the plasma membrane by the cleavage of the GPI anchor to become soluble. This
form is called suPAR (soluble urokinase receptor, or soluble urokinase-type plas-
minogen activator receptor, Fig. 2). Recently, suPAR has attracted much attention
from nephrologists. Here, suPAR as a biomarker of kidney diseases, mainly focal
segmental glomerulosclerosis (FSGS), will be reviewed.
Potential Implication of Circulating Permeability Factors in FSGS
FSGS is a group of clinicopathological syndromes sharing a common glomerular

lesion. It is estimated that primary FSGS accounts for approximately 40 % of
primary nephrotic syndrome cases in adults worldwide, and the estimated incidence
D1
D1
D2
D1
D3 D2
linker region
D2
D3
D3
GPI anchor
Fig. 2 The structure of urokinase receptor (uPAR) and the mechanism of soluble uPAR (suPAR)
formation. Urokinase receptor (uPAR) is expressed on various cell types and is attached to the
plasma membrane with a glycosylphosphatidylinositol (GPI) anchor. The cleavage of a GPI anchor
results in the release of its soluble form, soluble urokinase receptor (suPAR). Both suPAR and uPAR
contain three Ly-6 and uPAR (LU) domains, designated D1–D3, connected by short linker regions.
The cleavage of the linker regions results in the shorter suPAR fragments
is approximately seven per one million (Kitiyakara et al. 2003). The patients with
primary FSGS typically show a similar clinical presentation as minimal change
disease (MCD), including heavy proteinuria with abrupt onset, severe
hypoalbuminemia, and marked peripheral edema. Many cases are refractory to
treatment by steroids and/or immunosuppressants, resulting in progressive renal
impairment. This disorder is one of the most common primary glomerular diseases
that cause end-stage kidney disease (ESRD) and has a prevalence of approximately
4 % in the USA. Although various factors including viruses, drugs, and genetic
mutations have been identified as causes of FSGS, approximately 80 % of cases are
idiopathic (D’Agati et al. 2011).
Primary FSGS has long been attributed to a putative circulating permeability
factor for several reasons. First, disease recurrence after initial renal transplantation
occurs in 20–50 % of recipients with primary FSGS. The recurrence rate may exceed
80 % in patients with a history of allograft loss due to recurrence (Vincenti and
Ghiggeri 2005). Some recipients experience disease recurrence within hours after
transplantation. Moreover, several lines of evidence have shown that patients with
recurrent primary FSGS might have a substantial reduction in proteinuria after
plasmapheresis (Davenport 2001; Matalon et al. 2001). Second, the plasma or
plasma fractions from patients with FSGS can cause proteinuria in rats (Zimmerman
1984; Sharma et al. 2002; Avila-Casado Mdel et al. 2004). Third, sera from some
628 T. Wada
FSGS patients increased the permeability to albumin in glomeruli isolated from rats
(Savin et al. 1996). Fourth, an infant born to a mother with FSGS had heavy,
transient proteinuria, suggesting that a circulating permeability factor may be trans-
mitted from the mother to her infant and may be responsible for the development of
proteinuria (Kemper et al. 2001).
Furthermore, an interesting case of renal retransplantation was reported in 2012
(Gallon et al. 2012). A 27-year-old patient with ESRD due to primary FSGS received
a kidney transplant from his healthy 24-year-old sister. Despite repeated plasmaphe-
resis during his perioperative period, heavy proteinuria developed on the second
post-operation day, and his renal function progressively declined. Allograft biopsy
on day 6 revealed disease recurrence. On posttransplantation day 14, the allograft
was removed and retransplanted to another patient, who was a 66-year-old man with
ESRD due to type 2 diabetes mellitus. Immediately after the retransplantation, the
allograft regained function and proteinuria improved from 25 to 1.2 g/24 h. More-
over, allograft biopsy on the post-retransplantation days 8 and 25 showed a reversal
of the histopathologic lesions. In addition, the authors reported that the recipient
continued to have excellent allograft function and mild proteinuria (0.27 g per 24 h)
at 8 months after the retransplantation. This clinical course of “serial renal trans-
plantation” strongly suggests that a circulating permeability factor is involved in the
pathogenesis of the primary FSGS of the first recipient.
Searching for a Circulating Permeability Factor
Based on the observations described above, identification of a circulating perme-

ability factor in FSGS has been a top priority for renal researchers. Thus far,
cardiotrophin-like cytokine 1 (CLC-1) and anti-CD40 antibody have been suggested
as potential circulating factors that can impair the glomerular filtration barrier and
cause FSGS. CLC-1 is a member of interleukin-6 family and was found in serum
from patients with active FSGS. In addition, several lines of evidence have
suggested that CLC-1 might be a circulating factor associated with primary FSGS.
Virginia Savin and her colleagues found CLC-1 in the active fraction from galactose
affinity chromatography. They found that CLC-1 mimicked the effects of FSGS
plasma on the permeability to albumin and decreased nephrin expression in glomer-
uli and cultured podocytes. A monoclonal antibody against CLC-1 blocked the effect
of FSGS sera on albumin permeability (McCarthy et al. 2010), and recombinant
human CLC-1 increased the albumin permeability of isolated rat glomeruli (Sharma
et al. 2015). This effect was inhibited by a heterodimer composed of CLC-1 and
co-secreted molecule cytokine receptor-like factor 1 (CRLF-1) (Sharma et al. 2015).
It was suggested that Janus kinase 2 (JAK2)/signal transducer and activator of
transcription 3 (STAT3) signaling might be involved in the effects of CLC-1.
However, the precise roles of CLC-1 in primary FSGS remain to be determined.
Recently, Delville et al. described a potential circulating antibody that can
contribute to FSGS disease pathogenesis (Delville et al. 2014). They evaluated
pathogenic antibodies in posttransplant recurrent FSGS utilizing serum samples
from 64 patients with and without recurrent FSGS and 34 non-FSGS control
patients. They screened approximately 9,000 antigens in pre-transplant sera and
selected ten antibodies targeting glomerular antigens. Among these, anti-CD40
antibody had the best correlation with the risk of posttransplant recurrent FSGS.
Interestingly, although the binding capacity of anti-CD40 antibody to its antigen was
low in the enzyme-linked immunosorbent assay (ELISA), a peptide microarray scan
revealed that the immunogenicity of the CD40 protein in the two β-strand regions
was specifically altered in the recurrent FSGS sera, suggesting that a perturbation in
the conformation of the CD40 protein might cause posttransplant recurrence of
FSGS. Furthermore, anti-CD40 antibodies purified from recurrent FSGS patients
caused injury in human cultured podocytes, and this injury was ameliorated with a
monoclonal blocking antibody against uPAR or with cycloRGDfv, a small molecule
that blocks αvβ3 integrin activity. The injection of anti-CD40 antibodies purified
from recurrent FSGS sera into wild-type mice caused a mild but significant increase
in albuminuria during the first 8 days after injection, and albuminuria was markedly
enhanced in the presence of suPAR. In contrast, no effect was observed in CD40-
deficient mice or wild-type mice injected with blocking antibody to CD40. Based on
these results, the authors suggested that the combination of anti-CD40 antibody and
suPAR might contribute to glomerular injury in mice.
Taken together, CLC-1 and anti-CD40 antibody are potential candidates that
functionally cause proteinuria and glomerulosclerosis through podocyte injury.
However, to date, no data are available on the potential application of these mole-
cules as diagnostic biomarkers for FSGS.
suPAR as a Potential Circulating Permeability Factor in FSGS
After a long search by many researchers, Wei et al. published an article reporting that
suPAR might be a potential circulating permeability factor (Wei et al. 2011). Prior to
this report, they had suggested that uPAR expressed in podocytes might have a
deleterious effect on podocyte integrity. Utilizing uPAR-deficient mice and cultured
cells, they demonstrated that uPAR activation led to foot process effacement and
proteinuria through a mechanism that included the activation of αvβ3 integrin and
the small GTPases cdc42 and Rac1 (Wei et al. 2008).
Following the study on uPAR, they focused on its soluble form, suPAR. They
reported that the suPAR serum levels were elevated in 70 % of FSGS patients, and
the suPAR levels in FSGS patients were significantly higher than those in patients
with MCD (either in relapse or in remission), membranous nephropathy (MN), and
preeclampsia or in healthy control subjects. They also reported that serum suPAR
levels in patients with recurrent FSGS were significantly higher than those in patients
with primary FSGS or nonrecurrent FSGS (Wei et al. 2011). In posttransplant
recipients 1 year after transplantation, the patients who developed recurrent FSGS
had had significantly higher levels of suPAR compared with those who did not (Wei
et al. 2011).
630 T. Wada
This study also demonstrated that circulating suPAR activated podocyte β3

integrin and suggested that suPAR might play a causal role in primary FSGS. This
indicates that suPAR might not only be a biomarker but also a pathogenic perme-
ability factor for primary FSGS. This article caused considerable excitement in the
field. Moreover, this group reported elevated serum suPAR levels in two different
FSGS cohorts compared with healthy control subjects (Wei et al. 2012). They
measured the serum suPAR concentration in 70 patients from the North America-
based FSGS clinical trial (CT) and 94 patients from PodoNet, the Europe-based
consortium studying steroid-resistant nephrotic syndrome. While only 6 % of age-
and gender-matched control subjects showed serum suPAR levels higher than
3,000 pg/mL, elevated suPAR levels were observed in 84.3 % and 55.3 % of patients
with FSGS patients in the CT and PodoNet cohorts, respectively. Their multiple
regression analysis demonstrated that lower suPAR levels were associated with
higher estimated glomerular filtration rate (eGFR), male gender, and treatment
with mycophenolate mofetil. Huang et al. measured plasma suPAR in 74 patients
with primary FSGS and demonstrated that their plasma suPAR levels were signifi-
cantly higher than those of patients with MCD and MN and normal subjects,
although they could not discriminate secondary FSGS from primary FSGS (Huang
et al. 2013). On the basis of these promising data, renal researchers directed their
attention to serum/plasma suPAR levels as a potential diagnostic biomarker
for FSGS.
Is suPAR a Valid Diagnostic Biomarker for Primary FSGS?
Following the promising data reported by Wei and colleagues, several clinical
studies were conducted worldwide to validate serum suPAR levels for the diagnosis
of primary FSGS. However, data obtained from those different cohorts called the
exciting hypothesis in question.
Maas and colleagues were the first to refute this hypothesis (Maas et al. 2012,
2013) by showing no difference in serum suPAR concentrations between idiopathic
FSGS, secondary FSGS, and MCD in their small cohort. Later, this group reported the
suPAR levels in 54 patients with biopsy-proven idiopathic FSGS as well as
476 non-FSGS patients (Meijers et al. 2014). In this study, they found that the
serum suPAR levels and eGFR were negatively correlated and that the suPAR levels
in idiopathic FSGS overlapped with those in non-FSGS controls. Taken together, they
concluded that suPAR is not a clinical biomarker for FSGS (Meijers et al. 2014). A
Japanese group performed a multicenter cross-sectional cohort study for patients with
primary glomerular diseases, including FSGS (Wada et al. 2014). Serum suPAR
concentrations in 69 patients with biopsy-proven primary glomerular diseases
(38 patients with primary FSGS, 11 with MCD, 11 with IgA nephropathy, nine with
MN) were measured. A reverse relationship between renal function and suPAR levels
for the entire group of patients was detected (Fig. 3). Among the patients with normal
renal function (eGFR >60 mL/min/1.73 m2), the suPAR levels could not discriminate
primary FSGS from other glomerular diseases or even healthy controls (Fig. 4).
8000
6000
suPAR (pg/ml)
4000
2000
0
FSGS MCD IgAN MN
suPAR FSGS MCD IgAN MN

Mean 2723.0 2260.6 2055.0 3110.2
S.D. 671.2 577.2 678.1 630.9
Fig. 3 Serum levels of the soluble urokinase receptor (suPAR) in patients with primary glomerular
diseases without impaired renal function. Japanese patients with primary glomerular diseases whose
estimated glomerular filtration rate (eGFR) was 60 ml/min/1.73 m2 or higher were analyzed for their
serum suPAR levels. One-way analysis of variance (ANOVA) revealed that there were significant
differences in suPAR levels among the disease groups. However, no significant difference was
detected in a multiple comparison (From Wada et al. 2014). Abbreviations: FSGS focal segmental
glomerulonephritis, MCD minimal change disease, IgAN immunoglobulin A nephrosis, MN mem-
branous nephropathy. The unit for suPAR concentration is pg/mL
8000
6000
suPAR (pg/ml)
4000
2000
0
0 50 100 150
2
eGFR (mL/min/1.73 m )
Fig. 4 The correlation between soluble urokinase receptor (suPAR) and the estimated glomerular
filtration rate (eGFR). Serum suPAR levels were significantly and inversely correlated with the
eGFR (Pearson’s correlation coefficient test, R2 = 0.242, P < 0.0001) in a cohort of Japanese
patients with primary glomerular diseases. Open diamonds indicate focal segmental glomerulo-
sclerosis (FSGS) patients. Filled diamonds indicate patients with minimal change disease, IgA
nephropathy, or membranous nephropathy (From Wada et al. 2014)
632 T. Wada
1.0
0.8
Sensitivity
0.6
0.4
0.2
0.0
0.0 0.2 0.4 0.6 0.8 1.0
1 - Specificity
Fig. 5 Receiver operating characteristic (ROC) analysis of the serum soluble urokinase receptor
(suPAR) in Japanese patients with focal segmental glomerulosclerosis (FSGS). ROC analysis in
patients with FSGS or minimal change disease (MCD) from Wada et al. (2014). Solid line, ROC
curve for patients with FSGS or MCD whose estimated glomerular filtration rate (eGFR) was 60 ml/
min/1.73 m2 or higher. The area under the ROC curve (AUC-ROC) was 0.684
0.114 (95 %
confidence interval (CI): 0.461–0.907, P = 0.13). Dotted line, ROC curve for the nephrotic patients
with FSGS or MCD whose eGFR was 60 ml/min/1.73 m2 or higher. The AUC-ROC was
0.621
0.093 (95 % CI: 0.438–0.803, p = 0.21)
In this study, receiver operating characteristic (ROC) analysis was performed to

determine whether the serum suPAR level is a valid diagnostic biomarker that can
discriminate FSGS from MCD. As a result, the area under the ROC curve
(AUC-ROC) was only 0.684
0.114 (95 % confidence interval: 0.461–0.907,
p = 0.13), which suggests that suPAR cannot be used to differentiate FSGS from
MCD (Fig. 5).
There are also several reports on pediatric cohorts. Bock and colleagues described
that suPAR levels were higher in children with nonglomerular kidney diseases
compared with FSGS (Bock et al. 2013). Interestingly, female patients with heavy
proteinuria had lower suPAR levels than those without proteinuria. Moreover,
posttransplantation patients with either FSGS or non-FSGS had similar suPAR levels
as before transplantation, independent of proteinuria, race, or sex. On the basis of
those data, they also concluded that serum suPAR is unlikely the leading cause of
childhood idiopathic FSGS (Bock et al. 2013). Sinha et al. measured serum suPAR
levels prospectively in an Indian cohort of 469 children with renal disease, including
steroid-resistant (n = 237), steroid-sensitive (n = 138), and congenital nephrotic
syndrome (n = 9) and other proteinuric kidney diseases (n = 85), with samples
from control children (n = 85) (Sinha et al. 2014). Similar percentages of patients in
each group had elevated serum suPAR levels (>3,000 pg/mL). While approximately
half of the children with proteinuric renal disease had elevated suPAR levels, there
were no significant differences between the different histopathological disease
groups. The serum suPAR levels were inversely correlated with eGFR as in the
studies described above and directly correlated with C-reactive protein (CRP).
Furthermore, suPAR levels did not change significantly after therapy or during
remission (Sinha et al. 2014). Harita et al. evaluated serum suPAR levels in Japanese
pediatric patients with FSGS (n = 20), steroid-sensitive nephrotic syndrome (SSNS:
n = 26), chronic glomerulonephritis (CGN: n = 24), and nonglomerular kidney
disease (n = 24) (Harita et al. 2014). They reported that serum suPAR levels were
significantly higher in patients with FSGS than in patients with SSNS or CGN but
not higher compared with patients with nonglomerular kidney diseases. Notably,
FSGS patients had lower eGFR compared with patients with SSNS or CGN.
Therefore, higher suPAR levels in FSGS patients could be attributable to lower
renal function. In this study, serum suPAR levels were negatively correlated with
eGFR. Interestingly, serum suPAR levels in four patients who underwent renal
transplantation decreased after transplantation. However, the same tendency was
observed in three transplant recipients with nonglomerular kidney diseases, indicat-
ing that the decrease in suPAR levels after kidney transplantation is not disease-
specific and that the reduction in suPAR might be due to improved renal function.
The authors also observed that suPAR levels were not significantly high at the acute
phase of posttransplant FSGS recurrence, even in patients who responded well to
plasmapheresis. These results suggest that elevated suPAR levels are attributed
mainly to impaired renal function.
Association Between Serum suPAR and Renal Function
As described above, most of the studies for both adult and pediatric cohorts
showed that the suPAR levels in serum or plasma negatively correlated with
renal function. In addition to those studies, several studies that tested a relatively
large number of patients have been reported. Taniguchi et al. reported that this was
the case in a Japanese cohort of 476 patients with chronic kidney disease (CKD),
irrespective of underlying kidney diseases (Taniguchi et al. 2014). They also found
that suPAR levels were associated with the decline rate of renal function. A recent
report on 241 patients from the prospective, longitudinal, multicenter observa-
tional cohort of the Nephrotic Syndrome Study Network (NEPTUNE) described
that serum suPAR levels at baseline were inversely correlated with eGFR (Spinale
et al. 2015). In contrast, the initial report by Wei et al. (2011) did not
provide information on the renal function of the patients studied. In their subse-
quent study on the CT cohort, they reported data from a multiple regression
analysis showing that suPAR was negatively associated with eGFR at baseline
(Wei et al. 2012). Although Li et al. reported that serum suPAR levels in
FSGS patients were significantly higher than those in patients with MCD or MN,
renal function in FSGS patients was significantly lower than in the other
disease groups and the control subject group. Therefore, we cannot exclude the
possibility that there were some biases in terms of renal function in this study
(Li et al. 2014).
634 T. Wada
These results confirmed that serum suPAR levels are inversely associated with
renal function. Although the behavior of suPAR in the kidney is currently unclear,
given that its molecular size is small (20–50 kDa, depending on the degree of
glycosylation and proteolytic cleavage), the molecule is likely to be filtered through
the glomerular slit diaphragm. Therefore, it is possible that the decline in suPAR
excretion with decreased GFR may cause increased serum levels.
suPAR as a Biomarker Predicting Clinical Course
As described above, most of the clinical studies on suPAR and primary glomerular
diseases concluded that serum suPAR levels are not a valid diagnostic biomarker for
FSGS. However, several lines of evidence have suggested that suPAR might be valid
as a biomarker predicting clinical course.
Wei et al. reported in their article on CT and PodoNet cohorts that there was a
positive association between the relative reduction of suPAR after 26 weeks of
treatment and the reduction of proteinuria, with higher odds for complete remission
in the CT cohort (Wei et al. 2012). Huang et al. described the relationship between
the change in plasma suPAR level and clinical outcome during follow-up with a
mean duration of 78.0 (IQR 22.0–119.4) weeks. Although plasma suPAR levels did
not differ among the patient groups with treatment response (complete remission,
partial remission, and treatment failure), the suPAR level of patients with complete
remission decreased significantly, whereas patients with partial remission showed no
significant change. Furthermore, the treatment failure group showed a significant
increase in plasma suPAR levels (Huang et al. 2013).
Fujimoto et al. investigated the change in suPAR levels at baseline and at
2 months after treatment induction with the responsiveness to treatment. The
serum suPAR levels in MCD patients significantly decreased during the 2-month
treatment period, while those in the patients with FSGS or MN did not change
significantly. As for suPAR levels in the non-intractable nephrotic syndrome group
and intractable nephrotic syndrome group, they found significant suPAR decreases
in patients with non-intractable nephrotic syndrome, whereas patients with intracta-
ble nephrotic syndrome showed significant suPAR increases. These results suggest
that the change in suPAR during the first 2 months of treatment might be associated
with clinical outcome (Fujimoto et al. 2015). Taniguchi et al. reported that serum
suPAR levels at baseline were significantly associated with the decline rate of renal
function during the first year and during the first 2 years of follow-up in their CKD
cohort (Taniguchi et al. 2014). Li et al. performed a prospective study utilizing the
Nanjing cohort to evaluate the relationship between serum suPAR levels and steroid
responsiveness. They found that patients who were sensitive to steroids had signif-
icantly higher suPAR levels than patients who were resistant to steroids. For
evaluation of the predictive value of baseline suPAR concentration on steroid
responsiveness, they defined a cutoff value (3,400 pg/mL) based on their ROC
analysis. As a result, at the 6-month follow-up in 84 patients with FSGS, suPAR
levels were significantly decreased in those with high suPAR (≧3,400 pg/mL), while
suPAR levels did not change in the low-suPAR group even though they exhibited
reduced proteinuria. In addition, a higher proportion of patients with higher suPAR
were more likely to reach complete remission compared with patients with low
suPAR concentrations (Li et al. 2014). In contrast, Meijers et al. reported that there
was no difference in serum suPAR levels between steroid-resistant FSGS patients
(n = 15) and steroid-sensitive FSGS patients (n = 23) in their Nijmegen cohort
(Meijers et al. 2014). This discrepancy might be explained by the exclusion of
patients with low eGFR (<40 ml/min/1.73 m2) in the study by Li et al. Based on
these studies, suPAR might be useful as a diagnostic marker in patients with
reasonably preserved kidney function (Meijers and Sprangers 2014). Further studies
will be needed to settle this controversy (Table 1).
It is also controversial whether serum suPAR levels are associated with the risk
of posttransplant recurrence of FSGS. Wei et al. reported that subjects with
recurrent FSGS sustained higher serum suPAR levels over the course of 1 year
compared to those in which no posttransplant recurrence occurred, although no
difference in posttransplant eGFR was observed between subjects with and without
recurrent FSGS. Moreover, plasmapheresis significantly reduced serum suPAR
levels in FSGS patients, and β3-integrin activity evaluated with AP5 staining was
lower in human cultured podocytes incubated with pheresed sera. They also
reported the effects of plasmapheresis on clinical outcome in patients with
posttransplant recurrent FSGS. While the serum suPAR levels in two patients
who reached a clinical remission fell below 2,000 pg/mL, those in two patients
who failed to reach a remission despite plasmapheresis remained elevated (Wei
et al. 2011). In contrast, Harita et al. demonstrated that serum or plasma suPAR
levels at posttransplant recurrence in the acute phase were not significantly high
in their pediatric cohort, suggesting that posttransplant recurrence of FSGS was
not caused by elevated suPAR levels (Harita et al. 2014). Their data also suggested
that serum or plasma suPAR levels did not have predictive value for recurrent
FSGS.
Issues Concerning the Measurement of suPAR Levels
The current ELISA system provided by R&D systems, which has been employed in
virtually all of the studies examining the potential ability of suPAR as a diagnostic
biomarker, measures all of its fragments. As discussed above, suPAR consists of
three LU domains and can be cleaved to smaller fragments, a D1 fragment and a
D2-D3 fragment (Fig. 2). Reiser’s group suggested that the D2-D3 fragment of
suPAR, which was specifically detected in sera from patients with FSGS, might
contribute to FSGS through the activation of αvβ3-integrin signaling (unpublished
data). However, to date, a system that can measure a specific suPAR fragment has not
been developed. That type of ELISA system would facilitate the evaluation of the
potential role of suPAR as a diagnostic biomarker. Moreover, because the glycosyl-
ation of suPAR molecules might be associated with their activity, the relationship
between the glycosylation and activity of suPAR should be investigated.
636
Table 1 List of the studies investigating the validity of serum/plasma suPAR levels as a diagnostic biomarker for primary focal segmental glomerulosclerosis
(FSGS)
Correlation
suPAR in FSGS with renal
n (FSGS) Age (pg/mL) Diseases for comparison (n) function Reference Notes
23 19 (12–44) Unknown MCD (25) MN (16) preeclampsia (7) Unknown Wei Sera from 54 transplant
et al. 2011 FSGS patients were also
evaluated
70 Unknown 4,588
203 C (150) Yes (multiple Wei CT cohort
regression et al. 2012
analysis)
94 Unknown 3,497
195 C (150) Unknown Wei PodoNet cohort
et al. 2012
11 Unknown 2,392 (median) MCD (7) secondary FSGS (5) Yes Maas
et al. 2012
74 29 (13–84) 2,923 MCD (14) MN (29) secondary FSGS Yes (only in Huang
(2,205–4,360) (56) C (56) FSGS patients) et al. 2013
20 12.1
5.0 2,487 Non-FSGS glomerular diseases (24), Yes Bock
(2,191–3,351) nonglomerular CKD (26), C (29) et al. 2013
44 47 (33–60) 3,772 FSGS remission (10) non-FSGS CKD Yes Meijers
(2,622–4,422) (476) et al. 2014
126 9.4
4.8 3,316.1
101.2 Steroid-resistant MCD (142) steroid- Yes Sinha eGFR >30 mL/min/1.73 m2
sensitive nephrotic syndrome (151) et al. 2014 only
proteinuric CKD (85)
16 55.6
16.3 2,723
6,71.2 MCD (11) MN (9) IgAN (11) C (17) Yes Wada eGFR >60 mL/min/1.73 m2
et al. 2014 only
T. Wada
28
20 13.1
5.2 2,837
1,266 SSNS (26) CGN (24) nonglomerular Yes Harita
kidney disease (24) et al. 2014
109 28
14 3,512 MCD (20) MN (22) Yes (univariate Li
(2,232–4,231) model only) et al. 2014
95 36 (17–52) 3,178 MCD (62) MN (52) IgAN (32) Yes Spinale NEPTUNE cohort
(2,681–3,763) et al. 2015
8 48.0 3,212 MCD (12) MN (15) MPGN (2) ANCA- Yes Fujimoto
(29.0–68.0) (2,769–4,196) GN (13) et al. 2015
FSGS focal segmental glomerulosclerosis, MCD minimal change disease, MN membranous nephropathy, C control, CKD chronic kidney disease, MPGN
membranoproliferative glomerulonephritis, ANCA-GN antineutrophil cytoplasmic antibodies-associated glomerulonephritis, IgAN immunoglobulin A nephrop-
athy, SSNS steroid-sensitive nephrotic syndrome, CGN chronic glomerulonephritis
Continuous variables are presented as median (interquartile range) or mean
standard deviation
Soluble Urokinase Receptor as a Biomarker in Kidney Disease
637
638 T. Wada
Urinary suPAR as a Biomarker for FSGS
As described above, the results from most of the clinical studies conducted so far
suggested that the serum suPAR concentration was not a valid diagnostic biomarker
for FSGS. In contrast, urinary suPAR levels might be a more promising parameter as
a biomarker, and several clinical studies have been conducted to evaluate the value
of urinary suPAR as a diagnostic tool. Several study groups have described positive
correlations between serum or plasma suPAR and urinary suPAR. Huang
et al. analyzed urinary suPAR levels in patients with several different glomerular
diseases in a Chinese cohort. In their study, urinary suPAR levels were significantly
correlated with plasma suPAR levels in primary FSGS patients but not in MCD or
MN patients (Huang et al. 2014). Fujimoto et al. showed that urinary suPAR levels
correlated with serum suPAR levels in patients with primary nephrotic syndrome due
to FSGS, MCD, MN, or membranoproliferative glomerulonephritis (MPGN) in a
Japanese cohort (Fujimoto et al. 2015). The significant positive correlation was also
observed in a NEPTUNE cohort (Spinale et al. 2015). However, in an Indian
pediatric cohort, urinary suPAR levels were not significantly correlated with serum
suPAR levels (Sinha et al. 2014).
Urinary suPAR levels consistently correlated with urinary protein excretion,
although the patients with glomerular diseases other than primary FSGS failed to
show positive associations in a Chinese cohort. Huang et al. found that urinary
suPAR levels positively correlated with urinary protein excretion in primary FSGS
patients but not in MCD, MN, or secondary FSGS patients. The urinary suPAR
levels in primary FSGS patients were significantly higher than those in patients with
MCD, MN, and secondary FSGS and normal subjects (Huang et al. 2014).
suPAR as a Causative Factor for Primary FSGS
Aside from the role of suPAR as a diagnostic biomarker, suPAR in FSGS patient sera
could play a causative role in primary FSGS. Wei et al. used immunoprecipitation to
demonstrate that suPAR could interact with β3-integrin. Their in vitro experiments
revealed that sera from recurrent FSGS patients with high levels of suPAR strongly
induced the AP5 signal, indicating that β3-integrin signaling was activated. Activa-
tion of podocyte β3-integrin signaling was also shown in human biopsy specimens
from patients with primary or recurrent FSGS. Furthermore, they demonstrated that
suPAR caused proteinuria and FSGS utilizing three different mouse models, includ-
ing uPAR-knockout mice injected with recombinant suPAR, hybrid-transplant mice
modeling endogenous suPAR release, and genetically engineered wild-type mice
that drive the expression of a suPAR plasmid in the skin (Wei et al. 2011).
In contrast, Spinale et al. could not reproduce the pathogenic effects of suPAR in
their recent study (Spinale et al. 2015). They utilized wild-type mice injected with
uPAR/Fc chimera as an acute model. However, they did not observe an increase in
urinary protein excretion at 12 or 24 h after the injection, although serum levels of
suPAR showed a 12-fold increase at 4 h, and a nearly 6-fold increase in suPAR
persisted at 24 h. As a chronic model, they generated an inducible transgenic mouse

that can express suPAR in its liver. Although serum suPAR concentration increased
approximately twofold by day 13 and nearly threefold at day 44, urinary protein
excretion was not detected when the experiment was terminated. These results
suggest that the upregulation of suPAR in the circulation might not be pathogenic.
In addition, Cathelin et al. also investigated the effects of forced increases in suPAR
levels in mice. In that experiment, they failed to induce podocyte injury or protein-
uria by the injection of monomeric mouse uPAR produced in eukaryotic S2 cells or
uPAR/Fc chimera, even though glomerular suPAR deposits were observed (Cathelin
et al. 2014). As discussed above, the studies on the pathogenesis of suPAR used
different uPAR/suPAR molecules. Monomeric three-domain mouse suPAR was
produced in Drosophila melanogaster Schneider 2 cells and used in the study by
Cathelin and colleagues (2014). This monomeric mouse suPAR was structurally well
characterized (Gardsvoll and Ploug 2007). The recombinant uPAR/Fc chimera
protein produced in mouse NS0 cells can be structurally considered a circulating
molecule, and elevated levels of suPAR were detected by ELISA assay in the
injected mice (Spinale et al. 2015). The administration of this molecule induced
significant proteinuria in Plaur-deficient mice (Wei et al. 2011) but not in wild-type
mice (Spinale et al. 2015; Cathelin et al. 2014). Delville et al. used recombinant
human suPAR in their study. Their animal study revealed that proteinuria induced by
anti-CD40 antibody was exacerbated in the presence of this human suPAR.
Wei et al. used a mouse suPAR cDNA clone (IMAGE cDNA clone 3158012) to
evaluate the effects of chronic suPAR overexpression. This clone, which contains the
coding sequence for the D1 and D2 domains, was delivered into mouse skin by
in vivo electroporation. This clone contains a retained intron 4 that results in a
frameshift mutation at the site corresponding to amino acid residue 133 within the
second uPAR domain and premature termination of translation within the uPAR D2
domain (Fig. 2). On the basis of its predicted structure, some have questioned
whether this cDNA could produce a properly folded and stable protein. As
discussed, different experimental settings have yielded conflicting results, and the
causative roles of suPAR in proteinuria remain elusive.
suPAR and Antineutrophil Cytoplasmic Antibodies-Associated

Glomerulonephritis
As described above, it remains controversial whether serum suPAR levels are a valid
diagnostic biomarker for FSGS. Among the other renal diseases, antineutrophil
cytoplasmic antibodies-associated glomerulonephritis (ANCA-GN) has been linked
to suPAR. Wada et al. compared serum suPAR concentrations in five patients with
ANCA-GN with those in five patients with primary glomerular diseases matched for
age and eGFR. The suPAR concentrations in the ANCA-GN group were signifi-
cantly higher than in the control group (6,791.3
1,513.0 pg/mL vs. 3,727.5
8,182 pg/mL, P = 0.01). Fujimoto et al. also measured serum suPAR levels in
myeloperoxidase (MPO)-ANCA-GN patients (n = 13). In that study, they found
640 T. Wada
that serum suPAR levels before immunosuppressive therapy were significantly

correlated with clinical severity determined by age, serum creatinine level, serum
CRP level, and coexistence of pulmonary lesions (Fujimoto et al. 2015). They also
reported that serum suPAR and CRP levels were significantly correlated in the
ANCA-GN group. Given that previous studies suggested that elevated serum
suPAR levels were associated with nonspecific inflammation (Backes et al. 2012;
Koch et al. 2011; Yilmaz et al. 2011), the high levels of suPAR observed in ANCA-
GN patients might be attributable to inflammation in this disease setting.
suPAR and Lupus Nephritis
A Chinese group recently reported plasma suPAR levels in patients with lupus
nephritis (Qin et al. 2015). They compared plasma suPAR levels among the lupus
nephritis group, the nonrenal systemic lupus erythematosus (SLE) group (SLE
patients without renal involvement), and the control group. The plasma suPAR
levels in the lupus nephritis group were significantly higher than those in the
nonrenal SLE group or in the control group. When they compared the plasma
suPAR levels longitudinally in 14 patients with lupus nephritis between the active
and remission phases, the average plasma suPAR levels in remission decreased
significantly compared with the self-controls in the active phases. As with FSGS
described above, plasma suPAR levels in patients with lupus nephritis negatively
correlated with eGFR. When the subjects were limited to the lupus nephritis patients
whose eGFR was normal (>60 mL/min/1.73 m2), plasma suPAR levels in the
patients with lupus nephritis were still higher than those in the nonrenal SLE patients
or in the control subjects. Interestingly, plasma suPAR levels positively correlated
with the clinical disease activity assessed by Systemic Lupus Erythematosus Disease
Activity Index (SLEDAI). In terms of histopathological findings, within lupus
nephritis, no significant difference in plasma suPAR levels was observed among
the histological classes according to the International Society of Nephrology/Renal
Pathology Society (ISN/RPS) 2003 classification. However, they described signifi-
cant correlations between plasma suPAR levels and renal activity indices scores,
endocapillary hypercellularity, cellular crescents, interstitial inflammation, renal
chronic indices scores, tubular atrophy, and interstitial fibrosis. In their longitudinal
study, patients with lupus nephritis were followed up for an average duration of
38 months. Their univariate analysis of renal prognosis showed that plasma suPAR
level was a significant risk factor ( p = 0.01, HR 6.326, 95 % CI 1.466–27.298),
while their multivariate analysis failed to detect a significant association ( p = 0.61,
HR 1.091, 95 % CI 0.790–1.507). They additionally described a group of patients
with higher suPAR levels (>3,443.4 pg/mL) that presented with worse renal out-
come, although they did not show clinical and histopathological profiles in these two
groups, and it is unclear if there were any confounders (Qin et al. 2015).
A Hungarian group reported that SLE patients had significantly higher levels of
serum suPAR (n = 89, median 4,580 pg/mL, IQR: 3,720–6,300) compared with
healthy controls (n = 29, median 2,800, IQR: 2,060–3,420). Their ROC analysis
revealed that the cutoff value of suPAR to discriminate between patients with high
disease activity (SLEDAI >8) and those with moderate disease activity or in remission
(SLEDAI ≦8) was 5,700 pg/mL. However, they did not show the data of the suPAR
levels specifically in the patients with lupus nephritis (Toldi et al. 2012).
Enocsson et al. recently described the association of suPAR levels with organ
damage in patients with SLE (Enocsson et al. 2013). In their cohort, serum creati-
nine, CRP, erythrocyte sediment rate (ESR), C4, and urinary albumin as well as
leukocyte, erythrocyte, and platelet counts were significantly associated with serum
suPAR levels. No significant association between suPAR levels and the disease
activity defined as either the SLE disease activity index 2000 (SLEDAI-2K) or the
physician’s global assessment (PGA) was detected. However, a significant positive
association was found between suPAR levels and organ damage. The group found
that renal, ocular, neuropsychiatric, skin, and peripheral vascular organ damages had
a significant positive association with serum suPAR levels. Renal injury had the most
pronounced impact on suPAR levels.
suPAR and Diabetic Nephropathy
Several lines of evidence have indicated that serum or plasma suPAR levels may be
associated with diabetic complications. To date, most of the evidence is on diabetic
macrovascular complications such as atherosclerosis (Olson et al. 2010), cardiovascu-
lar disease (Persson et al. 2012, 2014; Sehestedt et al. 2011), and stroke (Persson
et al. 2014). Microinflammation in atherosclerosis might be associated with elevated
suPAR levels. Recently, Theilade et al. described the results from their single-center
cross-sectional study on type 1 diabetes patients (Theilade et al. 2015). In terms of renal
complications, they found a stepwise increase in median suPAR levels in patients with
normo-, micro-, and macroalbuminuria. Using ROC analyses, the authors found that
suPAR was a better predictor of albuminuria than age, systolic blood pressure, or CRP.
However, further large-scale prospective studies will be needed to determine if serum
suPAR concentration is a biomarker of diabetic nephropathy.
suPAR and IgA Nephropathy
Immunoglobulin A nephropathy (IgAN), characterized by IgA deposition in the

glomerular mesangium, is the most common form of idiopathic glomerulonephritis
leading to chronic kidney disease. Segmental glomerulosclerosis, one of the variables
in the Oxford classification of IgAN, is similar to the histopathological feature observed
in FSGS and is associated with poor prognosis. A group from China recently described
their data on plasma suPAR in 569 IgAN patients (Zhao et al. 2015). Plasma suPAR
levels were similar between patients with and without segmental glomerulosclerosis.
However, suPAR plasma levels positively correlated with proteinuria and negatively
correlated with eGFR. Even after the data were adjusted for eGFR, plasma suPAR
remained positively correlated with proteinuria. The plasma suPAR levels in IgAN
642 T. Wada
patients and in primary FSGS patients with nephrotic syndrome were not significantly
different. In that study, plasma suPAR levels in patients with extensive effacement of
podocyte foot processes were significantly higher than those with segmental efface-
ment on the basis of comparable eGFR (Zhao et al. 2015). Wada et al. also reported that
the serum suPAR levels in IgAN patients were not different from those in patients with
other glomerulopathies in the cohort of patients with normal renal function (Fig. 3,
Wada et al. 2014). Taken together, in IgAN patients, plasma or serum suPAR levels are
again associated with renal function, and they are likely to be dependent upon the
severity of glomerular damage.
Potential Applications to Prognosis, Other Disease, or Conditions
As discussed in the section “suPAR as a Biomarker Predicting Clinical Course,” the

change in serum/plasma suPAR levels might be associated with treatment response
in patients with nephrotic syndrome; however, conflicting results have been
reported, and the predictive value for treatment response remains controversial. A
biomarker that can predict posttransplant FSGS recurrence would be extremely
valuable for FSGS patients. However, it is still undetermined if suPAR levels are
predictive in posttransplant recurrence. Taken together, the value of suPAR levels as
a prognostic biomarker in renal diseases remains elusive.
Meanwhile, the elevated serum levels of suPAR have been reported under several
different disease conditions such as sepsis (Backes et al. 2012), liver cirrhosis
(Berres et al. 2012), rheumatic arthritis (Slot et al. 1999), and malignancies (Mazar
et al. 1999). However, suPAR does not appear to be superior to other biomarkers
(e.g., CRP and procalcitonin for sepsis) (Donadello et al. 2012). Recently, the value
of suPAR as a biomarker for cardiovascular disease (CVD) has gotten substantial
attention (Hodges et al. 2015). In the Danish MONICA cohort, elevated levels of
suPAR were associated with the incidence of CVD, type 2 diabetes mellitus, cancer,
and mortality (Eugen-Olsen et al. 2010). In this population, suPAR predicted CVD
with HR 1.7 (95 % CI 1.1–2.8, p = 0.027) for women and HR 2.1 (95 % CI 1.4–3.2,
p < 0.001) for men (Lyngbæk et al. 2013). Meijers et al. conducted a prospective
observational study of 476 patients with mild-to-moderate CKD (mean follow up
period; 57 months). Higher suPAR levels were significantly associated with overall
mortality (univariate HR 5.35) and cardiovascular events (univariate HR 5.06), and
the multivariate analysis also demonstrated that higher suPAR levels were associated
with cardiovascular events (Meijers et al. 2015).
Conclusion
As discussed above, although suPAR was once viewed as a promising diagnostic

biomarker for primary FSGS, studies from several different groups have questioned
its validity. Most clinical studies have revealed that serum or plasma suPAR levels
are negatively correlated with renal function. Serum or plasma suPAR levels
measured with the currently available ELISA kit are likely to reflect the patients’
renal function, inflammation, and some other clinical conditions and are not valid
diagnostic biomarkers for primary glomerular diseases. Further research will be
necessary to determine if a certain form of suPAR (i.e., shorter fragments of
suPAR or hypoglycosylated suPAR) could be a valid biomarker. It also remains
unclear whether suPAR could be a pathogenic factor to induce podocyte injury,
resulting in glomerulosclerosis.
Summary Points
• The discovery of a diagnostic biomarker for focal segmental glomerulosclerosis

(FSGS) has been awaited because differentiating between FSGS and minimal
change disease (MCD) is difficult, especially in the early phase of the disease.
• Clinical findings have suggested that circulating permeability factor(s) might be
implicated in the pathogenesis of idiopathic or recurrent FSGS.
• Soluble urokinase receptor (suPAR) was described as a potential diagnostic
biomarker for primary FSGS and posttransplant recurrent FSGS and as a patho-
genic factor that causes podocyte injury through the activation of β3-integrin
signaling.
• Most studies have not supported the initial data showing that suPAR could be
used as a diagnostic biomarker for FSGS and reported that serum/plasma suPAR
levels were inversely correlated with renal function.
• Serum/plasma suPAR levels might be a valid biomarker to predict the patients’
clinical course.
• The ELISA system currently used in virtually all of the studies on suPAR levels
measured all forms of suPAR, which might mask the differences in shorter
fragments or glycosylation of suPAR.
• Several studies have suggested that the suPAR urinary level might be a better
biomarker than the serum/plasma levels.
• Data from animal experiments have also questioned the causative roles of suPAR
in FSGS.
• Clinical data on the validity of suPAR as a diagnostic marker for other renal
diseases have yielded some positive data. However, we cannot exclude the
possibility that there might be confounding factors such as renal dysfunction
and inflammation.
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Salivary Urea Nitrogen as a Biomarker
for Renal Dysfunction 29
Viviane Calice-Silva, Jochen G. Raimann, Wen Wu,
Roberto Pecoits-Filho, Peter Kotanko, and Nathan Levin
Contents
Key Facts of Saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 649
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 651
Saliva Physiology, Properties, and Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 653
Urea Measurements in the Saliva . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 656
Dipstick Methods to Measure Saliva Urea Nitrogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 658
Limitations of SUN Assessments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 660
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 661
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 662
Abstract
Kidney disease is highly prevalent all over the world in acute and chronic forms.
Patients with kidney dysfunction have a higher morbidity and mortality especially
due to cardiovascular events. Biomarkers nowadays available for the assessment
of renal function such as serum creatinine, cystatin C, and blood urea nitrogen are
mostly laboratory dependent. Many studies have been conducted following the
aim to determine biomarkers of kidney disease allowing early diagnosis, faster
treatment initiation, and consequent better outcomes. However, in many places
V. Calice-Silva (*) • R. Pecoits-Filho

School of Medicine, Pontifícia Universidade Católica do Paraná, Curitiba, PR, Brazil
e-mail: vivicalice@gmail.com; vivicalice@hotmail.com; r.pecoits@pucpr.br
J.G. Raimann • P. Kotanko • N. Levin
Renal Research Institute, New York, NY, USA
e-mail: jochen.raimann@rriny.com; peter.kotanko@rriny.com; nwlevin3@gmail.com
W. Wu
Integrated Biomedical Technology, Elkhart, IN, USA
e-mail: ibtbiomed@earthlink.net

648 V. Calice-Silva et al.
worldwide there are a large number of patients affected by acute and chronic
kidney disease without receiving adequate diagnosis or treatment. The presence
of urea in the saliva was firstly described in 1841 and has since then been
repeatedly studied as an indicator of renal function. While it can be measured
by laboratory methods, a real advantage is found in it as a bedside methodology
(e.g., as a dipstick), which allows early and cost-effective diagnosis in areas with
limited resources. The aim is to review current evidence of the saliva as a general
biomarker and more specifically salivary urea nitrogen as a noninvasive and
useful biomarker of decreased kidney function.
Keywords
Kidney disease • Renal biomarkers • Salivary renal biomarkers • Blood urea
nitrogen • Saliva • Salivary urea nitrogen and renal disease
Abbreviations
BUN Blood urea nitrogen
Ca+2 Calcium
Cl Chloride
CO(NH2)2 Urea
CO2 Carbon dioxide
DHEA Dehydroepiandrosterone
H2O Water
HCO3 Bicarbonate
HIV Human immunodeficiency virus
K+ Potassium
KIM -1 Kidney injury molecule -1
Mg+2 Magnesium
MUC5B Mucin 5B
MUC7 Mucin 7
Na+ Sodium
NH3 Ammonia
NH4 Ammonium ion
OH Hydroxyl ion
ROC Receiver operating characteristic
29 Salivary Urea Nitrogen as a Biomarker for Renal Dysfunction 649
SUN Salivary urea nitrogen

UN Urea nitrogen
Key Facts of Saliva
• Saliva is an important fluid to maintain the mouth homeostasis with many

diagnosis properties and has been widely studied since many years ago because
of the many potential biomarkers it contains.
• The oral fluid composition varies in relation to the type of gland where it is
produced. Mainly (90 %) of this fluid originates from three pairs of major salivary
glands (parotid, sublingual, and submandibular) and from a larger number of
minor salivary glands.
• There are many medications and systemic disease that can interfere in the saliva
production causing dry mouth, which is known as the medical term xerostomy.
• Many substances measured in the blood can be assessed in the saliva such as urea
nitrogen, which can, to some extent, be seen as a biomarker relating to kidney
function.
• In 1841, Wright et al. first described the presence of urea in the saliva. At that time
it was thought the saliva could be used to increase the excretion of urea in patients
with kidney disease.
Definitions
Blood urea nitrogen (BUN) It is produced by the liver and is a waste product of
protein digestion. It is an indicator of renal function. The normal ranges are 6–20 mg/
dL. High-protein intake, liver diseases, dehydration, kidney disease, and gastroin-
testinal bleeding can cause an increase in BUN levels.
Creatinine It is an end product of muscle catabolism, derived from the phospho-

creatine in the muscle. Its generation can be increased by creatine intake in meat and
supplements. Creatinine is freely filtered, secreted by tubular cells, and eliminated
via extrarenal. It is easily measured in the blood and urine at a low cost.
Electrolytes These are substances that ionize when diluted in some specific sol-
vents. Salts, acids, bases, and gases can be dissolved and became some specific ions
that are very important to the organism homeostasis. Some examples of electrolytes
are the ions: sodium, chloride, potassium, magnesium, calcium, phosphate, hydro-
gen, ammonium, and many others.
IL-18 Interleukin-18 is a cytokine that is produced by macrophages and other cells.

It can be measured in the urine due to its expression in the proximal tubule
promoting tubule cell apoptosis and necrosis after an ischemic kidney injury. It
has been proposed as an early biomarker of kidney dysfunction.
Kidney disease Some degree of impairment in renal function that can happen
suddenly (acute kidney disease) or after many years of permanent damage (chronic
kidney disease). It is diagnosed through the assessment of glomerular filtration rate
and presence of proteins in the urine without other causes, with or without structural
kidney damage detected by biopsy.
KIM-1 The kidney injury molecule-1 is a type 1 transmembrane protein, with an

immunoglobulin and mucin domain. Its expression is upregulated in the proximal
tubule in the post-ischemic kidney event, because it has been proposed as an early
biomarker measured in the urine in patients with acute renal failure.
Negative predictive value It is a method that evaluates, according the prevalence

of a specific disease or condition, the diagnostic performance of a test or statistical
measurement. It represents the event when the test makes a negative prediction and
the subject has a negative result under the gold standard method.
NGAL The neutrophil gelatinase-associated lipocalin is a protein identified as a

biomarker for early diagnosis of kidney injury and can be measured in the plasma
and urine.
Positive predictive value It is a method that evaluates, according the prevalence of

a specific disease or condition, the diagnostic performance of a test or statistical
measurement. It represents the event when the test makes a positive prediction and
the subject has a positive result under the gold standard method.
Receiver operating characteristic It is a graphical representation of the true

positive rate (sensitivity) and false negative rate (one-specificity) for a specific
diagnostic test.
Renal replacement therapy It is the treatment proposed to substitute the blood

filtration when the kidneys do not have the capability to do this anymore. The
options are hemodialysis (HD) and its variations (daily HD, home HD, continuous
HD, and others), peritoneal dialysis, and kidney transplantation. Each modality has
specific indication and contraindication, and the moment to start the therapy varies
from different literatures with a consensus to start as soon as the glomerular filtration
rate is lower than 10–15 ml/min/1.73 m2 or when the patients have uremic symptoms
or uncontrolled hypervolemia.
Saliva urea nitrogen It is the blood urea nitrogen that achieves the salivary fluid
through diffusion mechanisms and can be measured by laboratory devices and
salivary strips.
Sensitivity It is a statistical measure that considers the proportion of positive results

which are correctly identified as positive events, for example, when an individual has a
positive test for some disease that he/she is a carrier. It is calculated by the number of true
positive tests divided by the sum of true positive and false negative tests.
Spearman’s rank correlation It is a nonparametric statistical method to assess the

relationship between two variables.
Specificity It is a statistical measure that considers the proportion of negative results

which are correctly identified as negative events, for example, when an individual
has a negative test for some disease that he/she is not a carrier. It is calculated by the
number of true negative tests divided by the sum of true negative and false positive
tests.
Introduction
Renal disease is highly prevalent worldwide (KDIGO 2012, 2013). Kidney dysfunc-
tion can happen suddenly, named a pathophysiologic process termed acute kidney
injury (AKI) that often occurs as a consequence of comorbidities such as sepsis,
hypovolemic shock, drug toxicity, and urinary tract obstruction, among others
(KDIGO 2012). Alternatively renal function can also decrease in a slower fashion
due to long-standing kidney injury as a consequence of chronic conditions such as
diabetes mellitus, arterial hypertension, autoimmune glomerular diseases, or also
prolonged AKI. The latter is named chronic kidney disease (CKD) (KDIGO 2013).
Both entities, acute and chronic kidney disease, are classified according to the
degree of kidney dysfunction ranging from mild to severe impairment (KDIGO
2012, 2013). Over the decades many biomarkers have been proposed for an accurate
and reliable assessment of kidney function. At this point glomerular filtration rate
(GFR) is considered the most accurate measure of renal function. This parameter is
determined by the clearance of a specific substance over a 24-h period and requires
urine output collection over the entire time. Normalization to body surface area has
been proposed to improve the accuracy of the estimation (Inker and Levey 2014).
Most commonly used clearances are those of creatinine (90–120 ml/min/1.73 m2),
urea (40–65 ml/min/1.73 m2), and inulin (100–130 ml/min/1.73 m2) (Inker and
Perrone 2014).
Creatinine is the waste product of muscle catabolism, derived from the metabolism
of phosphocreatine in the muscle. It is primarily excreted by the kidneys, where it is
freely filtered, secreted by tubular cells, and in addition eliminated via extrarenal such
as via intestinal after degradation by intestinal flora (Levey et al. 1988). Creatinine
generation can be increased by creatine intake in meat or dietary supplements; it is
easily measured in the blood and urine at a considerably low cost; however, it is
quantified through laboratory-dependent technique (Inker and Levey 2014).
As mentioned above urea nitrogen (UN) is another marker utilized to assess renal
function. It is produced by the liver and is a waste product of protein digestion and
cellular decomposition. It is freely filtered by the glomerulus and passively
reabsorbed in the proximal and distal tubules. Besides renal dysfunction, high-protein
intake, volume depletion, and gastrointestinal bleeding can also cause an increase in
UN levels (Dossetor 1966). Creatinine and urea were widely used jointly to quantify
renal function, once creatinine could overestimate and urea, on the other hand, could
underestimate GFR due to the mechanisms of secretion and reabsorption; both
together could estimate GFR more accurately (Inker and Levey 2014).
Despite the low applicability in clinical practices, inulin clearance is considered
the reference method to quantify kidney function due to its biochemical properties.
This polysaccharide is metabolized by the bacterial flora in the colon and is
completely excreted by the kidneys. It is not secreted or reabsorbed in any portion
of the nephron allowing GFR to be calculated. It is determined by continuous
intravenous infusion with blood and urine sample collections (Robson et al. 1949).
In clinical routine where time and cost effectiveness are of greatest importance,
kidney function is often evaluated by the estimation of the GFR (eGFR) using the
serum concentration of endogenous filtration biomarkers such as creatinine, urea,
and cystatin C (Earley et al. 2012). Demographic characteristics are included in these
estimating equations to provide more reliable results (Cockcroft and Gault 1976;
Levey et al. 1999; Schwartz and Work 2009). One example of these equations
widely used to estimate eGRF is the Modification of Diet in Renal Disease
(MDRD) study equation with standardized creatinine expressed as per below
(Levey et al. 1999):

eGFR mL=min=1:73 m2 ¼ 175 SCr1:154 age0:203 0:742½if female
1:210 ½if black
New biomarkers for kidney damage have been proposed in the last years such as
KIM-1, NGAL, and IL-18, with the aim of providing early diagnosis of tubular
injury due to acute hemodynamic changes leading to acute kidney injury (Wasung
et al. 2014; Devarajan and Murray 2014). Despite their excellent diagnostic perfor-
mance, they are very costly, laboratory dependent and are not useful to evaluate
chronic kidney damage.
Renal disease often goes undiagnosed, and many patients are not even aware of
the risk factor causing it (Sumaili et al. 2009). Partly as a consequence, the incidence
and prevalence of CKD and AKI are increasing in both developed and developing
countries. According to the scarce data available in developing countries, renal
disease most often is a de novo occurrence affecting young adults in their productive
age (Tao Li et al. 2013). In contrast, in the developed world, AKI and CKD mainly
affect older patients and those with many comorbidities. Nevertheless, in both
worlds kidney diseases are responsible for a large number of deaths (Sumaili
et al. 2009; Tao Li et al. 2013; El Nahas 2005).
The lack of medical infrastructure, particularly of laboratories, is one of
the most important reasons for the limited capacity of health workers to diagnose
CKD and AKI in many countries. Other contributory factors include late referral
to a nephrologist or hospital, limited renal replacement therapy, especially in
developing countries, where in addition to a lower level of awareness, the
scarcity of financial resources may affect prevention, diagnosis, and treatment

(El Nahas 2005; Barsoum 2006). It was hypothesized that a cheap diagnostic
tool used in the most remote areas would allow to increase diagnosis and, if an
adequate treatment is available, could affect outcomes in these patients.
For a long time, saliva has been proposed as a fluid with diagnostic qualities for
various purposes including diagnosis and monitoring of infectious diseases (HIV,
hepatitis C, and others) and other systemic diseases such as neoplasms and renal
diseases, assessment of hormonal dysfunctions, performance of genetic tests, mea-
surement of pharmaceutical drug levels, and for the purpose of forensic analyses
(Nagler et al. 2002; Malamud 2011; Vining and McGinley 1982; Aps and Martens
2005). Saliva allows the measurement of many different components present in the
blood. These include potassium, sodium, uric acid, phosphate, and many others
(Nagler et al. 2002; Lima et al. 2010; Chiappin et al. 2007) (Table 1).
Most interestingly for the herein reviewed purpose, UN can also be assessed
through saliva, making this fluid possibly a valuable biomarker for the evaluation of
kidney function. This suggests that the use of saliva for diagnosis of oral and
systemic diseases could be the marker called for above, which allows the diagnosis
of a reduction kidney function and may aid to improve outcomes of kidney injury in
the developing world and areas with limited medical resources.
Saliva Physiology, Properties, and Function
Salivary glands are developed through a sensible embryonic process. The parotid
gland originates from the oral ectoderm and mesenchyme. The embryogenic origin
of the submandibular and sublingual glands is similar (Carlson 2000). Innervation of
salivary glands by the parasympathetic and sympathetic branches of the autonomic
nervous system is essential to secretion and tissue homeostasis of these glands
(Martin and Burgen 1962). According to the stimuli the saliva secreted will have
different compositions in the different glands (Aps and Martens 2005; Humphrey
and Williamson 2001; Proctor and Carpenter 2007) (Table 2).
Salivary glands are composed of two main cell types: the acini, which produce the
saliva, and the ductal cells (intercalated, striated, and excretory), which modify and
transport the saliva to the mouth (Malamud 2011). The secretion of saliva involves
the active secretion of sodium and chloride ions by the acinar cells into the ductal
lumen of the gland after appropriate neural stimulation. Water derived from the
blood system passes around via tight junctions and through/via aquaporin channels
to the acinar cells to form saliva, which is isotonic with respect to the serum. In the
parotid and submandibular glands, the sodium is mostly recovered by the striated
ducts, which are impermeable to water (Amerongen 2008; Mandel 1989). This
process changes the saliva from isotonic, as is secreted by the acini, to a hypotonic
fluid, which allows salt detection in much lower levels. This fluid composition can
change from resting to stimulated saliva and according the time of the day due to the
circadian rhythm of these glands (Lima et al. 2010; Humphrey and Williamson 2001;
Tiwari 2011).
Table 1 Substances found in the saliva and blood

Inorganic compounds (mmol/l) (Aps
and Martens 2005; Chiappin Whole unstimulated
et al. 2007) Blood values salivary range
Na+ mmol/L 145 5
K+ mmol/L 4 22
Cl mmol/L 120 15
Ca+2 mmol/L 2.2 1–4
HCO3 mmol/L 25 5
Mg+2 mmol/L 1.2 0.2
NH3 mmol/L 0.03 6
Organic compounds (Nagler et al.
2002)
Uric acid mg/dL 4.4
0.83 2.7
1.67
Bilirubin mg/dL 0.5
0.24 0.03
0.07
BUN mg/dL 18.08
8.5 20.7
11.8
Creatinine mg/dL 1.0
0.10 0.07
0.03
Glucose mg/dL 97.33
10.4 0.67
0.14
Cholesterol mg/dL 193.5
27.9 0.42
1.04
Hormones (Chiappin et al. 2007;
Patel et al. 2004; Hansen et al. 2003;
Lu et al. 1999; Chatterton et al. 2005;
Low 2011)
Cortisol 140–700 mmol/l 3.6–35.1 nmol/l
Testosterone 300–1,000 ng/dL (men) 140.3
154.15 pg/ml
DHEA-s 20–640 μg/dL (according 291.21
294.81 pg/
age and gender) ml
Progesterone 5–20 ng/ml (women Luteal phase: 436
mid-cycle) 34 pmol/l
<1 ng/ml (women Follicular phase: 22.1
pre-ovulation)
2.7 pmol/l
Estradiol 30–400 pg/ml Luteal phase: 20.6
(premenopausal) 0.4 pmol/l

Aldosterone <20 ng/ml (adults) 138–475 pmol/l
This table shows some examples of substances present in the blood and saliva and their respective
values in each fluid according the reference used
Na+ sodium, K+ potassium, Cl chloride, Ca+2 calcium, HCO3 bicarbonate, Mg+2 magnesium,
NH3 ammonia, BUN blood urea nitrogen, DHEA dehydroepiandrosterone
Saliva has several functions: digestion (presence of α-amylase), facilitation of

taste perception, buffer and lubrication capacity, defense against microorganisms,
and assists in teeth mineralization (Amerongen 2008; Tiwari 2011; Carpenter 2013).
The oral fluid composition varies in relationship to the serous or mucous gland
component, and it mainly (90 %) originates from three pairs of major salivary glands
(parotid, sublingual, and submandibular) and from a larger number of minor salivary
glands (Von Ebner and Blandin-N€uhm glands) (Amerongen 2008). The major
Table 2 Characteristic differences in the saliva produced according to different nervous stimuli
and consequent saliva aspect
Alpha-adrenergic Cholinergic
Parameter Beta-adrenergic stimulation stimulation stimulation
Volume Low Low High
Viscosity High Low Low
Protein High High Low
concentration
Mucin Very high Low Very low
concentration
Consequence Viscous saliva rich in Low-volume saliva, not Watery and high-
proteins and mucins with foamy or viscous, rich volume saliva rich in
foamy appearance in proteins electrolytes
The characteristics of the saliva produced by each gland vary according to the central stimulation
these glands receive. In this table the fluid characteristics are shown in agreement with the type of
stimuli. It is important to be aware that the salivary glands receive more than one type of central
stimuli (beta, alpha-adrenergic, and/or cholinergic) and can produce different types of fluid each
time according to the more prominent stimuli (Adapted from Aps and Martens (2005)- with the
permission of Elsevier)
contributions for the whole saliva production during resting conditions are subman-
dibular, sublingual, and parotid glands, respectively. Under mechanical and chemical
stimulation, parotid and submandibular glands are the most important ones to the
saliva secretion. Non-glandular saliva sources are responsible for the other 10 % of
the saliva composition and are represented by the gingival crevicular fluid, oral
epithelial cells, nasopharyngeal discharge, food debris, bacteria, and their products
(Nagler et al. 2002; Malamud 2011).
The principal compounder of saliva is water (99 %), while others are inorganic,
organic nonprotein, protein, hormone, and lipid molecules (Table 1). The parotid
gland is essentially a serous gland since it does not produce mucin. The parotid gland
fluid composes of water, bicarbonate, and a large number of proteins. The main
proteins secreted by the parotid glands are amylase (20 %), phosphoproteins
(statherin – 7 %), and proline-rich proteins (60 %); these two last groups of proteins
are responsible for the constitution of the protein pellicle on dental surfaces which
keep the saliva supersaturated in calcium and phosphate which helps in tooth
mineralization. On the other hand, submandibular and sublingual glands are mixed
serous-mucous glands and are responsible, associated with the minor glands, for
salivary mucin secretions (MUC5B and MUC7) that have important protective
functions such as avoiding infection, inflammation, and mechanical injuries in
both hard and soft tissues (Humphrey and Williamson 2001; Carpenter 2013;
Tabak 1995). An amount of 0.5–1.5 ml of the whole saliva is produced per day.
The normal pH of saliva ranges between 5.3 (low flow) and 7.8 (peak flow). The
major glands contribute most of the volume secretion and electrolyte contents, and
the minor glands contribute little volume secretion and most of the blood group
components (Mandel 1989; Garrett et al. 1998).
The saliva and plasma exchange substances through a thin layer of epithelial cells
separating the salivary ducts from the systemic circulation. The clearance of these
compounds from the plasma into the saliva may involve several processes such as
ultrafiltration (through gap junctions between cells of secretory units involving
molecules with <1,900 Da: water, ions, and hormones such as catecholamine and
steroids), transudation of plasma (e.g., albumin and other proteins), and selective
transport through cellular membranes by passive diffusion of lipophilic molecules or
by active transport through protein channels (Chiappin et al. 2007; Garrett
et al. 1998; Marini and Cabassi 2002).
While these complex relationships and dynamics suggest many factors involved
in the composition of the saliva, it has been, as mentioned above, repeatedly
proposed as a body fluid with diagnostic qualities, which is easily accessible.
Urea Measurements in the Saliva
Generally the accuracy with which any random biomarker can be measured in the
saliva mainly depends on the biochemical nature of the marker, the source and type
of sample being taken, and the mechanism by which the marker enters the oral cavity
(Humphrey and Williamson 2001). Saliva collection is an easy, noninvasive method,
not expensive and already in use in many areas, particularly genetics and forensics
(Vining and McGinley 1982; Aps and Martens 2005).
Salivary urea nitrogen (SUN) was first described by Wright in 1841 in a case
study of a patient who developed a uterine tumor, consequently ascites and a decline
in urine output. He believed that once the patient could not eliminate the urea
through the urine, her organism developed compensatory mechanisms to eliminate
urea through ascites and the saliva avoiding the neurological manifestations of this
excretion product accumulation (Wright 1841).
Urea reaches the saliva by diffusion transports and, associated with bicarbonate,
is responsible for the buffer capacity of this fluid (Humphrey and Williamson 2001;
Amerongen 2008; Mandel 1989). In 1922 Hench and colleagues described a positive
relationship between the salivary and blood urea levels, especially in patients with
urea retention. The determination of urea for this study was made by the urease
method (Hench and Aldrich 1922). Hench also found, in patients with urea retention,
that the combined urea and ammonia nitrogen in the saliva was in average 80 % of
the blood urea nitrogen, and that was always increased with an increase in BUN
levels. The authors suggested that salivary urea nitrogen could precede or replace
BUN determination. Salivary stimulation was proposed as a treatment for urea
retention when patients had a low urine output and could not eliminate BUN through
urine anymore (Hench and Aldrich 1922).
Later in 1923, Hench proposed a salivary urea nitrogen index and showed a
distinct and continuous parallelism of the SUN and BUN values. After salivary
stimulation with paraffin, two samples were collected, and SUN was determined
using a mercury titration method modified from the one used by Friedlander in 1921
to measure urea in the urine. In this study Hench showed correlation between BUN
and SUN in healthy and kidney disease patients. The salivary urea index, defined as
the number of milliliters (ml) of 5 % solution of mercuric chloride used for each
100 ml of saliva, increased directly with the degree of urea retention as indicated by
BUN. The SUN index had a sensitivity of 91 % for urea retention detection. He
concluded that the SUN index could be a substitute for BUN determination (Hench
and Aldrich 1923).
Barnett et al. in 1929 studied the influence of saliva stimulation in the SUN
results. Many previous studies had shown a wide variation in the SUN results with
stimulation by chewing paraffin, ranging from 50 % to 130 % in different publica-
tions (Hench and Aldrich 1923; Barnett and 1929). In this study, samples were
collected with and without stimulation; the whole saliva and parotid saliva were used
to perform the measurements. Stimulation was conducted using solutions of tartaric
acid (0.05–0.5 %). The results were consistent with the previous studies, showing
SUN and BUN correlating significantly when SUN was measured with and without
stimulation. SUN measured in the unstimulated saliva had a better agreement with
BUN when measured in both whole saliva and parotid salivary samples. On average
SUN was 7.2 mg lower when the saliva flow was increased by stimulation (Barnett
and Bramkamp 1929).
Later, studies were conducted to analyze SUN in specific salivary glands such as
the parotid (Shannon and Prigmore 1961a, b; Shannon et al. 1977). Mean parotid
urea concentrations were reported to range from 73 % to 95 % of serum levels.
Forland et al. in 1964 proposed that SUN measured in the saliva collected from
parotid glands could be used to monitoring hemodialysis patients (Forland
et al. 1964). In Forland’s study Urea was determined in both fluids with an auto
analyzer with the use of a previously described modification of the Ormsby method
(Ormsy 1942). Saliva was stimulated by a chewing gum or with a hard candy. The
correlation coefficient of mean values for BUN and SUN monitored during hemo-
dialysis was 0.99 despite rapid changes in BUN and SUN levels during treatment
(Forland et al. 1964).
The first study using a test-strip method to measure SUN was done by Akai
et al. in 1983 when a dipstick was created to assess it. The urease-containing (urease-
bromocresol-green method) test strip and an automatic reflectance spectrometer
were used to conduct these analyses. Forty-four CKD patients at different disease
stages and 12 healthy patients serving as a control group were studied. SUN and
BUN results changed according the temperature and the reaction time of 15 min,
increasing their values with higher temperatures and prolonged time exposure. A
saliva/serum ratio of approximately 1.03 was found with this method, and the degree
of correlation was similar in both populations (Akai et al. 1983; Okuda et al. 1980).
Some years later, 1987, Sein et al. published another study where significant
correlation coefficients (r = 0.74 and r = 0.99) between SUN and BUN in unstimulated
whole saliva were found in 56 healthy subjects and in 50 patients undergoing hemo-
dialysis, respectively. Serum and saliva urea nitrogen were determined by the urease/
glutamate method using Roche reagent kits in a Cobas-Bio centrifugal analyzer (Sein
and Arumainayagam 1987; Shull and Cerins-Overbey 1982).
Table 3 Outline of the saliva urea nitrogen chemical reaction

Salivary urea nitrogen cleavage reaction
COðNH2 Þ2 þ H2 O ! 2 NH3 þ CO2
Urease
ðUreaÞ
NH3 þ H2 O ! NH4 þ þ OH
pH Indicator + OH- pH Indicator

(Yellow) (Green/Blue)
In the urea cleavage-process urea in contact with water is cleaved by urease enzyme and becomes
ammonia and carbon dioxide. Ammonia in contact with water becomes the ion ammonium and
hydroxyl. The release of hydroxyl ions will alter the pH which leads to changes in the test-pad color
reflecting the salivary urea nitrogen result.
CO(NH2)2 urea, H2O water, NH3 ammonia, NH4+ ion ammonium, OH ion hydroxyl
In 2007 a dipstick was developed to measure SUN in the bedside setting. This
tool is the first non-laboratory-dependent resource that has been applied in research
settings in patients with acute and chronic renal disease and presented a good
agreement with BUN levels (AUC ROC of 0.90 (95 % CI 0.85–0.95) for CKD –
68 patients and AUC ROC of 0.91; (95 % CI 0.80–1.0) for AKI – 44 patients)
(Raimann et al. 2011; Calice-Silva et al. 2014).
Another study performed by Cardoso et al. in 2009 found a significant correlation
between SUN and BUN (r = 0.91, p < 0.001) in both 78 healthy subjects and
154 CKD patients. The SUN had a sensitivity and specificity of 100 % in both
groups in this study. The unstimulated whole saliva collected in the morning in
fasting conditions was evaluated. A serum enzymatic colorimetric assay (urea color
2R, Wiener Laboratory, Argentina) was adapted for the SUN measurements (Car-
doso et al. 2009).
In the last few years, other studies in this field were performed analyzing different
techniques to measure SUN, comparing saliva stimulation versus non-stimulation
and whole saliva versus saliva collected from specific glands, in healthy and CKD
patients, but all of them utilized laboratory-dependent methods (Peng et al. 2013;
Zuniga et al. 2012).
Dipstick Methods to Measure Saliva Urea Nitrogen
The aforementioned work by Akai in 1983 studied a dry strip method using a
chromatographic method which required an automatic reflectance spectrometer
(Akai et al. 1983). A more recently developed dipstick method developed by
Integrated Biomedical Technology, IN, USA, allows the assessment of SUN, with-
out additional devices.
For the employment of this method, 50 μL of liquid saliva are transferred to the
test pad of the colorimetric SUN dipstick (Integrated Biomedical Technology, IN).
The SUN in the saliva is cleaved by urease present in the test pad into ammonia and
hydroxyl ions which change the pH and consequently the test-pad color (Table 3).
Table 4 Saliva urea nitrogen test-pad number and respective SUN ranges
SUN test-pad number (SUN range, mg/dL) Color of the test pad
1 (5–14)
2 (15–24)
3 (25–34)
4 (35–54)
5 (55–74)
6 (75)
The SUN (saliva urea nitrogen) results are divided in six ranges from the lower to the higher levels
and are represented by the colors and test-pad numbers showed above. Test-pad result 1 refers to
SUN between 5 and 14 mg/dL, test-pad 2 (15–24 mg/dL), test-pad 3 (25–34 mg/dL), test-pad
4 (35–54 mg/dL), test-pad 5 (55–74 mg/dL), and test pad 6 (75 mg/dL). The greater the test-pad
number the worse the kidney function
In 1 min the result can be read and the test-pad color compared to six standardized
color fields indicating SUN concentrations of 5–14 (color pad #1), 15–24 (#2), 25–34
(#3), 35–54 (#4), 55–74 (#5), and 75 (#6) mg/dL, respectively (Table 4). This strip
showed a good agreement between SUN and BUN at different concentrations.
Raimann et al. (2011) published a study where 68 CKD patients in the stages 1 to
5D were studied. The elevated BUN was diagnosed using this SUN dipstick;
unstimulated whole saliva was collected to perform the measurements. SUN and
BUN were correlated significantly (RS = 0.63; p < 0.01). Elevated BUN was
diagnosed by SUN determination with an AUC ROC curve of 0.90 (95 % CI
0.85–0.95) and an interobserver coefficient of variation of 4.9 % at SUN levels
>50 mg/dL and within-sample reproducibility of 90 % (Raimann et al. 2011).
The same tool was also applied in the AKI setting. Forty-four patients suffering
from acute renal disease were analyzed in this cross-sectional study performed by
Calice-Silva et al. (2014). In this group of patients, the SUN dipstick had a sensitivity
of 0.91 and specificity of 0.79 to diagnose AKIN 3 (AUC ROC 0.91–95 % CI
0.80–1.0), the worse degree of AKI, and when the most part of the patients will need
some kind of renal replacement therapy (RRT – hemodialysis or peritoneal dialysis).
These findings make this tool useful to screen patients with suspected renal disease
in areas with limited resources (Calice-Silva et al. 2014).
Despite promising results SUN and BUN do not have a good agreement at the
lower levels of BUN. The delicate balance of strip buffer composition relative to the
saliva pH and buffer capacity has been put forward as possible causes of inaccurate
measurements at low levels. Further developments to increase accuracy at lower
levels of BUN are under investigation at this time. Furthermore, more studies are
needed to better understand the applicability of this tool in more diverse population,
such as healthy patients, children, patients using multiple medications, and those
suffering from others disorders potentially interfering with the accuracy of the
method.
Limitations of SUN Assessments
Some biological factors may alter the BUN and SUN relationship, in particular
urease-producing oral bacterial flora, which may give rise to falsely low SUN values
(Burne and Chen 2000); this, however, does not affect the agreement at higher levels
of SUN. Another important limitation is the circadian rhythm that salivary glands
have, potentially leading a different secretion rate of some saliva compounder
according the time of the measurement in relation to the time of the day. This
suggests that the agreement between SUN and BUN may differ at different times
of the day (Dawes 1972; Dawes and Ong 1973; Ferguson et al. 1973).
Many are the mechanisms that lead to a diminished saliva production. Both
systemic diseases such as Sjögren syndrome and the use of multiple medications
such as antihypertensive, antihistaminic, antidepressive, anticonvulsants, analgesics,
and many others can interfere in the salivary flow and buffer capacity and cause
changes in the salivary pH that possibly could result in inaccurate SUN measure-
ments (Marques et al. 2014; Thomson et al. 1999). Regardless, this method has very
important strengths: most importantly, the tool enables to assess a marker to a certain
extent relating to kidney function quickly, noninvasively, and independently of a
laboratory directly at bedside (Raimann et al. 2011; Calice-Silva et al. 2014).
New developments of the dipsticks are currently being investigated and promise
to increase the precision of SUN strip. The main improvement is an additional
control pad which contains the same compounds as the main test pad excluding
the enzyme urease in order to evaluate the pH and buffer capacity of saliva, which, as
mentioned above, may be one of the causes of inaccuracies.
This auxiliary test pad will show darker greenish/bluish color if there is an
elevated pH (higher than 7.0) or some alteration in the buffer capacity in this fluid.
Consideration of such a discoloration when comparing the color of the main test pad
against the color code on the container allows an adjustment of the measurement or
even the replication of this to confirmate it. This improvement aims to analyze SUN
more accurately, particularly at lower levels where false negative results have been
observed and also will help to increase the usefulness of the dipstick in cases where
the SUN may be possibly low (e.g., for the screening of patients with only mild
impairments of kidney function).
Potential Applications to Prognosis, Other Diseases,

or Conditions
Renal disease leads to a high burden on society and health systems. Diagnosis and
even more treatment are expensive. In many areas people do not have access to either
due to high costs, available resources, and insurance coverage in different healthcare
systems (Grassmann et al. 2005; Jha et al. 2013). Depending on the underlying
cause, the disease can, despite often treatable and reversible when diagnosed early,
be fatal in a short period of time. For those suffering from slow-progressing renal
disease, early diagnosis may also be of benefit and allow, at the very least, dietary
interventions or other affordable therapeutic options (Tao Li et al. 2013).
Despite a remarkable scarcity of data, it may be assumed that particularly in

remote areas with limited medical infrastructure, there are still a large number of
patients suffering from CKD or AKI without access to renal replacement therapy
resulting in a fatal outcome as a consequence of uremia, hyperkalemia, or fluid
overload. Even with the increment in dialysis facilities numbers and healthcare
coverage all over the world, early diagnosis remains a challenge. As one of the
most important examples, it needs mentioning that in developing countries many
children die daily due to AKI secondary to infectious diseases (Rosa-Diez
et al. 2014; De Vecchi et al. 1999; Schieppati et al. 2014; Cerda et al. 2008). It is
of note that such an occurrence may be entirely avoided by adequate treatment, in the
best case only requiring adequate volume replacement or a short regimen of perito-
neal dialysis (Callegari et al. 2013).
For those with chronic disease, the situation is more complex since maintenance
RRT requires more resources and is more difficult to successfully implement.
However, early diagnosis may be of help to diagnose the condition early and
allow preventive measures to slow the disease progression. Global screening pro-
grams for renal disease, as already proposed in the past, early detection, and
prevention of progressive renal disease may be seen as the keys for a successful
improvement in patients care and survival (Sumaili et al. 2009; Barsoum 2006).
Another important application could be in the setting of disasters where quick
decisions based on easy-to-use methods are the key to improve outcomes and curtail
fatalities. This has been repeatedly observed, hypothesized, and even shown in vivo
during latest events (Vanholder et al. 2010; Naicker 2010).
SUN could also be an important biomarker for the monitoring of the treatment
regimen for those maintained in home dialysis therapies such as home hemodialysis
and peritoneal dialysis. It could be aid physicians, family members, and the patient
himself or herself to control their treatment. This however needs to first be studied in
a real-life setting.
Overall, early diagnosis may not only save lives but may also preserve years of
productivity for those affected (Tao Li et al. 2013; Cerda et al. 2008). Regular
screening of the population may allow the initiation of prevention programs and
the early diagnosis for many suffering from undetected and undiagnosed renal
disease. The combination of these with patients education in terms of diet and risk
factors may possible an important improvement of public health (El Nahas 2005).
Measurement of SUN with low-cost, noninvasively, and accurately may be of
benefit to improve overall outcomes and should thus be considered as a marker for
the diagnosis of deteriorating renal function.
Summary Points
• Renal disease can be manifested acutely or chronically and is responsible for an

increased mortality risk in patients suffering from this illness. According to the
degree of dysfunction, the patient may need renal replacement therapy
represented mainly by hemodialysis, peritoneal dialysis, and kidney transplanta-
tion. Around 8–10 % of the worldwide population has some degree of renal
disease. Renal disease has a high morbidity and mortality, especially due to
cardiovascular disease.
• Glomerular filtration rate is the principal method used to assess kidney function.
Another form to assess kidney function is measuring some specific substances
such as creatinine, urea, and cystatin C in the blood and urine.
• Saliva is also a fluid with diagnostic properties that has been studied since many
years. Many substances measured in the blood can be assessed in the saliva such
as urea nitrogen, which can, to some extent, be seen as a biomarker relating to
kidney function. Salivary urea nitrogen (SUN) was first described in 1841 by
Wright et al. A good agreement between the values of urea nitrogen in the blood
and saliva has been shown in several studies.
• A recently developed salivary dipstick allows the measurement of SUN giving
physicians the opportunity to diagnose renal disease at bedside. This may result in
earlier treatment initiation and possible improved outcomes. Particularly for
developing countries and medically isolated areas, this tool could be of great
benefit. It may also be used for triage in the setting of natural disaster with the
need of quick diagnosis and early treatment.
• Salivary urea nitrogen dipstick is a promising noninvasive, bedside tool to screen
patients with suspected renal disease, in areas with limited health resources. SUN
dipstick has higher diagnosis applicability in those patients with higher levels of BUN.
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Part III
Specific Diseases and Conditions
Chronic Allograft Damage Index (CADI)
as a Biomarker in Kidney Transplantation 30
Ilkka Helanterä, Fernanda Ortiz, and Petri Koskinen
Contents
Key Facts of Kidney Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 670
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 671
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 671
Development of Chronic Allograft Damage Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 675
Description of CADI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 675
Correlation of CADI with Prognosis and Use of CADI as Surrogate
Marker in Clinical Trials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 676
CADI as a Surrogate of Graft Function in Basic Science . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 679
Comparison of CADI to Individual Histopathological Lesions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 680
CADI from Donor Biopsies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 680
Progression of CADI as a Biomarker of Graft Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 681
CADI in Pediatric Kidney Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 682
Reproducibility of CADI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 682
CADI in Helping Clinical Decision-Making . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683
Limitations of CADI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 683
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 684
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 685
Abstract
Although the outcome of patients after kidney transplantation has improved
markedly during the past decades, chronic scarring and loss of function of the
allograft along time are a common problem. Histopathological changes precede
I. Helanterä (*)
Transplantation and Liver Surgery, Helsinki University Hospital, Helsinki, Finland
e-mail: Ilkka.helantera@helsinki.fi; Ilkka.helantera@hus.fi
F. Ortiz • P. Koskinen
Department of Medicine, Division of Nephrology, Helsinki University Hospital, Helsinki, Finland
e-mail: fernanda.ortiz@hus.fi; petri.koskinen@hus.fi

670 I. Helanterä et al.
the loss of function in the transplant, and chronic allograft damage index (CADI)
was introduced in the early 1990, with the purpose of numerically classifying
pathological lesions in transplanted kidneys. CADI is a sum score of six histo-
pathological lesions commonly seen in biopsies taken from transplanted kidneys
that correlate with the function and outcome of the graft. CADI includes intersti-
tial inflammation, tubular atrophy, interstitial fibrosis, arterial fibrointimal thick-
ening, glomerular mesangial matrix increase, and the percentage of globally
sclerosed glomeruli. CADI score, analyzed from either donor biopsies taken
before transplantation or from protocol biopsies taken after transplantation,
correlates with graft function and later outcome and can be used as a surrogate
marker in clinical trials. CADI has also been extensively studied in experimental
models of kidney transplantation. In addition, CADI can be useful in clinical
decision-making, as it gives a simple numeric score of the extent of chronic injury
in the kidney allograft.
Keywords
Kidney transplantation • Pathology • Protocol biopsy • Donor biopsy • Outcome
Abbreviations
CADI Chronic allograft damage index
CAN Chronic allograft nephropathy
CsA Cyclosporine A
MP Methylprednisolone
MMF Mycophenolate mofetil
• Kidney transplantation is the treatment of choice in patients with end-stage renal

disease, and compared to maintenance dialysis treatment, it is very cost-effective
and improves quality of life and survival.
• Annually more than 70,000 kidney transplantations are performed globally, of
which approximately 40 % are from living donors and 60 % from donors after
brain death or cardiac death.
• The average survival of a transplanted kidney is approximately between 10 and
20 years, depending on the patient and donor characteristics.
• Life-long immunosuppressive treatment is needed to prevent rejection of the
transplant, and maintenance immunosuppression is usually a combination of
two to three drugs. Immunosuppression is associated with increased risk of
infections and malignancies, and in addition current drugs are associated with
several adverse metabolic effects.
• The most common reason for the loss of a kidney transplant is premature death of
the patient with a functioning graft, mostly to cardiovascular causes of
malignancies.
• Chronic scarring of the transplanted kidney is an unsolved problem, leading to
gradual loss of the transplant function and return to dialysis.
30 Chronic Allograft Damage Index (CADI) as a Biomarker in Kidney. . . 671
• Multiple causes lead to similar histopathological picture seen in kidney transplant

biopsies, consisting of scarring and chronic inflammation.
• Histopathological changes precede loss of function in the transplants, and histo-
pathological scoring of the lesions of well-functioning grafts gives estimates of
the prognosis of the allograft and can be used as surrogate markers in clinical
studies.
Definitions
Chronic allograft nephropathy A term referring to chronic scarring of the kidney

allograft. It was originally introduced to include the multifactorial etiology of long-
term allograft injury, including immunological and nonimmunological causes, but
has later been abandoned from classifications due to the nondiagnostic and very
unspecific nature of the term.
Chronic rejection Chronic immunological injury to the transplanted kidney. During

the time chronic allograft damage index was developed, chronic rejection was used
commonly to describe the unspecific long-term scarring of the kidney allograft, but
later histopathological classifications have specified chronic rejection as its own entity,
defined as evidence of certain characteristics of chronic inflammation in the biopsies.
Donor biopsy A core needle biopsy taken from a transplanted organ before the
transplant operation, e.g., taken from a brain-dead donor during the explantation
operation of the organ.
Protocol biopsy A core needle biopsy taken from the (kidney) allograft at a
predefined time point after transplantation during stable graft function. The opposite
of a protocol biopsy is an indication biopsy, which is taken due to graft dysfunction
or signs of injury in the graft.
Introduction
Kidney transplantation is an excellent treatment for end-stage renal disease for

patients who are evaluated suitable both for the operation and for long-term exposure
to immunosuppressive drugs. The most common reason for the loss of a kidney
transplant is premature death of the patient with a functioning graft, mostly due to
cardiovascular causes or malignancies. However, chronic scarring of the
transplanted kidney is an unsolved problem, leading to gradual loss of the transplant
function and return to dialysis. Multiple causes of injury to the transplanted kidney
all lead to similar histopathological picture seen in kidney transplant biopsies,
consisting of scarring and chronic inflammation.
Despite advances in molecular diagnostics and development of several potential
biomarkers from blood or urine, histopathological evaluation of the kidney from a
core needle biopsy remains the gold standard for diagnosing kidney diseases.
In addition to native kidneys, biopsies are very important in assessing the well-being
of a transplanted kidney, especially as it is well known that histopathological
changes precede the loss of function in the transplants. The procedure of taking a
biopsy from a kidney transplant with an automated biopsy device under ultrasound
guidance is very safe, and serious complications occur very rarely. Two main studies
provided knowledge about the safety on kidney graft biopsies performed either by
indication or by protocol. The first study reported an incidence of 0.4 % for major
complications after reviewing 2127 protocol biopsies (Furness et al. 2003a). In the
second study including 1171 protocol biopsies and 499 indication biopsies, only 1 %
of major complications were reported in either group (Schwarz et al. 2005). There-
fore, many transplant centers have adopted the policy of taking protocol biopsies
from the stable well-functioning transplanted kidney at a predefined time point after
transplantation. Protocol biopsies are taken on one hand to detect underlying pathol-
ogy despite normal graft function (most importantly subclinical rejection) (Rush
et al. 1998), but on the other hand histopathology provides important information
about the prognosis of the transplanted kidney. Protocol biopsies are also increas-
ingly used as surrogate markers of graft prognosis in the context of randomized
controlled trials, but a concrete benefit in the routine follow-up of kidney transplant
patients is yet to be fully demonstrated. Therefore, graft function is usually moni-
tored by serum creatinine, estimated glomerular filtration rate, and proteinuria,
whereas a kidney biopsy is indicated to ascertain the cause of graft dysfunction.
The potential benefit of recognizing unexpected pathological patterns in well-
functioning kidneys early enough may have an impact on graft survival (Bosmans
et al. 2008).
The most commonly used classification of renal allograft histopathology is the
Banff classification. The Banff classification was created in a meeting held in Banff,
Canada, in August 1991 and published in 1993 (Solez et al. 1993). The Banff
Working Classification of Renal Allograft Pathology is a schema developed to
standardize the interpretation of kidney allograft pathology across frontiers. Subse-
quent meetings have been held every other year to refine the classification. The
modifications to the Banff schema made in 1997 synthesized two different classifi-
cation systems: Banff ’95 and the classification developed by CCTT (Collaborative
Clinical Trials in Transplantation) (Racusen et al. 1999). In Banff classification,
individual histopathological lesions are scored separately semiquantitatively (from
0 to 3), and certain diagnostic categories are defined. Banff ’97 is still the most
commonly used classification system in the literature of kidney transplant pathology.
Details of the Banff ’97 classification are shown in Table 1. The earliest versions of
the Banff classification emphasized the diagnosis of acute rejection, mainly because
this was the most common problem in those days. Chronic allograft nephropathy
(CAN) was graded into three categories – mild, moderate, or severe – without an
attempt to determine the pathophysiological mechanisms involved in this entity. The
improvements in immunosuppressive regimens dramatically reduced the incidence
of acute rejection, exposing the problem of chronic allograft damage. The elucida-
tion of the role of donor-specific antibodies, suggested by C4d staining, caused a
revolutionary modification of the Banff classification and led to the omission of the
Table 1 Description of the Banff 1997 classification for the pathology of a kidney allograft
(Racusen et al. 1999)
1. Normal
2. Antibody-mediated rejection, demonstrated to be due, at least in part, to antidonor antibody
A. Immediate (hyperacute)
B. Delayed (accelerated acute)
3. Borderline changes suspicious for acute rejection. This category is used when no intimal
arteritis is present, but there are foci of mild tubulitis (1–4 mononuclear cells/tubular cross section)
and at least i1
4. Acute/active rejection
Type Histopathological findings
(grade)
IA Cases with significant interstitial infiltration (>25 % of parenchyma affected) and
foci of moderate tubulitis (>4 mononuclear cells/tubular cross section or group of
10 tubular cells)
IB Cases with significant interstitial infiltration (>25 % of parenchyma affected) and
foci of severe tubulitis (>10 mononuclear cells/tubular cross section or group of
10 tubular cells)
II A Cases with mild to moderate intimal arteritis (v1)
II B Cases with severe intimal arteritis comprising >25 % of the luminal area (v2)
III Cases with transmural arteritis and/or arterial fibrinoid change and necrosis of
medial smooth muscle cells (v3 with accompanying lymphocytic inflammation)
5. Chronic/sclerosing allograft nephropathy
Grade Histopathological findings
I (Mild) Mild interstitial fibrosis and tubular atrophy without (a) or with (b) specific
changes suggesting chronic rejection
II Moderate interstitial fibrosis and tubular atrophy (a) or (b)
(Moderate)
III (Severe) Severe interstitial fibrosis and tubular atrophy and tubular loss (a) or (b)
6. Other Changes considered not to be due to rejection
general term “chronic allograft nephropathy” (Solez et al. 2007) (Table 2). In an
attempt to discriminate the underlying processes, the present recommendation dis-
tinguishes acute and chronic antibody-mediated rejection from acute and chronic
T-cell-mediated rejection. Unspecific interstitial fibrosis and tubular atrophy (IFTA)
are now a separate category, graded from mild to moderate and severe.
Although the Banff classification is an excellent diagnostic tool for pathologists
for assessment of histopathological lesions and helps clinicians in interpreting the
findings, there are limitations for the use of Banff classification in research and
correlation of histopathological findings to later outcomes, which derive especially
from the fact that every lesion is assessed separately. There is a real need for “score”
of different histopathological lesions to evaluate the total damage in the allograft for
both research purposes and for clinicians.
Chronic allograft damage index (CADI) was developed in the early 1990s to
provide better tools for both research and clinical use in evaluating the severity of
chronic changes in the kidney allograft. The terminology has changed many times
Table 2 Update of the Banff classification in 2007, which includes the addition of antibody-
mediated rejection and new concepts of chronic active T-cell-mediated rejection (Solez et al. 2007)
1. Normal
2. Antibody-mediated rejection
Due to documented anti-donor antibody
Acute antibody-mediated rejection
Type (grade)
I. ATN-like – C4d+, minimal inflammation
II. Capillary margination and/or thromboses, C4d+
III. Arterial – v3, C4d+
Chronic active antibody-mediated rejection
Glomerular double contours and/or peritubular capillary basement membrane multilayering
and/or interstitial fibrosis/tubular atrophy and/or fibrous intimal thickening in arteries, C4d+
3. Borderline changes: “suspicious” for acute T-cell-mediated rejection
No intimal arteritis is present, but there are foci of tubulitis (t1, t2, or t3 with i0 or i1) although
the i2 t2 threshold for rejection diagnosis is not met
4. T-cell-mediated rejection
Acute T-cell-mediated rejection
Type (grade)
IA. Cases with significant interstitial infiltration (>25 % of parenchyma affected, i2 or i3)
and foci of moderate tubulitis (t2)
IB. Cases with significant interstitial infiltration (>25 % of parenchyma affected, i2 or i3)
and foci of severe tubulitis (t3)
IIA. Cases with mild to moderate intimal arteritis (v1)
IIB. Cases with severe intimal arteritis comprising >25 % of the luminal area (v2)
III. Cases with “transmural” arteritis and/or arterial fibrinoid change and necrosis of medial
smooth muscle cells with accompanying lymphocytic inflammation (v3)
Chronic active T-cell-mediated rejection
“Chronic allograft arteriopathy” (arterial intimal fibrosis with mononuclear cell infiltration in
fibrosis, formation of neointima)
5. Interstitial fibrosis and tubular atrophy, no evidence of any specific etiology
Grade
I. Mild interstitial fibrosis and tubular atrophy (<25 % of cortical area)
II. Moderate interstitial fibrosis and tubular atrophy (26–50 % of cortical area)
III. Severe interstitial fibrosis and tubular atrophy/loss (>50 % of cortical area)
6. Other:
Changes not considered to be due to rejection – acute and/or chronic
during the past decades, and in the 1990s “chronic rejection” was the most widely
used term for chronic failure of kidney allografts (Hayry et al. 1993). In the early
2000s “chronic allograft nephropathy” dominated the literature as the description of
the unspecific scarring and failing of allografts (Paul 1999; Nankivell et al. 2003). In
recent literature, progress has been made to try to define the specific cause of chronic
scarring in the transplanted kidney and to try to avoid the use of nonspecific
nondiagnostic terms such as chronic allograft nephropathy (Solez et al. 2007).
Chronic rejection still exists in the current classification, but is only one subgroup
of reasons for scarring of the graft, and should include signs of continuous immu-
nological damage to the graft. CADI was developed before Banff classification was
presented, but these classification systems are not competing, rather more
complementing each other. The purpose of this review is to describe the develop-
ment of CADI and to describe how CADI can be employed both for clinical and
research use.
Development of Chronic Allograft Damage Index
The development of CADI dates to the late 1980s to the early 1990s, when the
concept of protocol biopsies was getting more popular within the transplant com-
munity. Research group in Helsinki lead by Prof. Pekka Häyry was interested in
finding out whether histopathological changes in well-functioning kidney allografts
may predict later outcome and to what extent they correlate to graft function and later
chronic rejection. Key focus was on chronic rejection, as defined by that time (Häyry
et al. 1993). The original paper in which CADI was introduced described a patient
population participating in randomized controlled trial of immunosuppressive drugs.
In this trial, triple-drug therapy with cyclosporine (CsA), methylprednisolone (MP),
and azathioprine (AZA) was compared to any combination of two of these drugs. In
this study, a protocol core needle biopsy was taken from all patients at 2 years after
transplantation, and the patient population consisted of 89 patients with adequate
biopsy material. In the first paper, 35 individual histopathological parameters were
scored semiquantitatively from 0 to 3 (Isoniemi et al. 1992). From these parameters,
the most common findings were correlated with graft function and donor age. The
individual parameters, which were the best predictors of graft function, were selected
to the CADI scoring system (Tables 3 and 4). In CADI, all individual parameters are
scored from 0 to 3. A confirmation of the usefulness of CADI was published 2 years
later, when from the same initial patient population, CADI score at 2 years was
correlated to graft function and outcome at 6 years. CADI correlated well with graft
function 4 years after the biopsy, and CADI also predicted graft failure (chronic
rejection) at 6 years (Isoniemi et al. 1994).
Description of CADI
CADI results from the sum of the following six histopathological parameter scores:
interstitial inflammation, tubular atrophy, interstitial fibrosis, arterial fibrointimal
thickening, glomerular mesangial matrix increase, and the percentage of globally
sclerosed glomeruli. The CADI score ranges from 0 to a maximum of 18. Details on
individual scoring are depicted in Table 3 and Fig. 1. In the modern application of
CADI, the individual parameters scored from 0 to 3 according to Banff ’97 classi-
fication, except for the percentage of globally sclerosed glomeruli, which is not
included in the Banff classification (0, no globally sclerosed glomeruli; 1, <15 %;
2, 16–50 %; and 3, >50 % globally sclerosed glomeruli). In the original CADI
Table 3 Description of the chronic allograft damage index, which is a sum score of six parameters
of chronic lesions in kidney allograft pathology
Inflammation (Focal/diffuse)
0 No or trivial inflammation
1 Up to 25 % of parenchyma inflamed
2 26–50 % of parenchyma inflamed
3 >50 % of parenchyma inflamed
Interstitial fibrosis (Focal/diffuse/subcapsular)
0 No fibrosis
1 Up to 25 % of the interstitium affected
2 26–50 % of the interstitium affected
3 >50 % of the interstitium affected
Tubular atrophy
0 No tubular atrophy
1 Tubular atrophy up to 15 % of proximal tubules
2 Tubular atrophy in 16–30 % of proximal tubules
3 Tubular atrophy in more than 31 % of proximal tubules
Mesangial matrix increase
0 No mesangial matrix increase
1 Up to 25 % of non-sclerotic glomeruli affected (at least moderate)
2 26–50 % of non-sclerotic glomeruli affected (at least moderate)
3 >50 % of non-sclerotic glomeruli affected (at least moderate)
Intimal Changes at least seen in 1 artery or 3 arterioles
proliferation
0 No intimal thickness
1 Intimal thickness up to 25 % of the remaining lumen
2 Intimal thickness 26–50 % of the remaining lumen
3 Intimal thickness >50 % of the remaining lumen
Sclerosis (Increase in extracellular matrix, sclerotic areas positive stained with
PAS)
0 No changes
1 <15 % of glomeruli affected
2 16–50 % of glomeruli affected
3 >50 % of glomeruli affected
papers, the Banff ’97 classification was not available, and some of the individual
lesion scores differed slightly. Minor differences in the grading of chronic lesions
between CADI and Banff are detailed in Table 5.
Correlation of CADI with Prognosis and Use of CADI as Surrogate

Marker in Clinical Trials
Already the first studies presenting CADI described one of the key strengths of
CADI, which is correlation of CADI to later graft function and outcome (Isoniemi
Table 4 Parameters evaluated in the histological specimens of renal grafts in the development
phase of chronic allograft damage index (CADI)
Interstitium
Lymphocytes Hemorrhage
Macrophages Fibrosis
Pyroninophilic cells Inflammation
Neutrophils Edema duplication
Eosinophils Fibrin deposits
Glomeruli
Mesangial cell proliferation Number of glomeruli
Capillary basement membrane thickening Mesangial matrix increase
Capillary thrombosis Capillary basement membrane
Glomerular inflammation Bowman capsular thickening
Glomerular necrosis Glomerular sclerosis
Tubuli
Epithelial vacuolation isometric Epithelial swelling
Epithelial atrophy Epithelial vacuolation anisometric
Casts Necrosis
Dilatation Inflammation
Basement membrane thickening
Vessels
Endothelial proliferation Endothelial swelling
Inflammation Intimal proliferation
Obliteration Sclerosis
In red, the components selected for CADI
et al. 1994). After that, several other studies have correlated CADI with later
outcome with similar results (Yilmaz et al. 2003). In two multicenter randomized
controlled trials of mycophenolate in kidney transplantation, US trial and
Tricontinental trial (Sollinger 1995; The Tricontinental Mycophenolate Mofetil
Renal Transplantation Study Group 1996; Mathew 1998; US Renal Transplant
Mycophenolate Mofetil Study Group 1999), protocol core needle biopsies were
performed in certain centers participating in the studies, and the biopsy slides were
sent for centralized blinded reading using CADI score. Altogether 621 protocol
biopsies were analyzed from these studies and scored with CADI. Of the biopsies,
111 were taken at baseline, 302 at 12 months, and 206 at 36 months after transplan-
tation. In multivariable models CADI score at 1 year was significantly and indepen-
dently associated with 3-year graft survival (OR 1.6). For every unit increase in
CADI score, the odds ratio of a graft loss or death increased by almost 50 % (Yilmaz
et al. 2003). Clinical factors that independently correlated with elevated CADI at
1 year were donor age, acute rejection during the first posttransplant year, and CMV
infections. In addition to the linear association of CADI and graft survival, CADI
can be categorized as low (<2), elevated (2–4), and high (>4). In this study, none
of the patients with low CADI lost their graft, whereas 4.6 % of patients with
elevated CADI lost a graft and 16.7 % of patients with high CADI lost their graft
Fig. 1 Examples of rising CADI score in kidney transplant biopsies. Photomicrographs of four
kidney biopsies showing a rising CADI score. (a) A 6-month protocol biopsy with normal
histology, score: 0. (b) A 6-month protocol biopsy, score: 2, comprised of chronic vasculopathy
(cv 1, not shown in the photomicrograph) and glomerulosclerosis (gs 1). (c) A 6-month protocol
biopsy, score: 4, comprised of interstitial fibrosis (ci 1), tubular atrophy (ct 1), mesangial matrix
increase (mm 1), and glomerulosclerosis (gs 1). (d) A 12-month protocol biopsy, score: 8, comprised
of interstitial fibrosis (ci 1), tubular atrophy (ct 1), inflammation (i 1), glomerulosclerosis (gs 2), and
severe chronic vasculopathy (cv 3). Masson’s trichrome. Magnification 100
(Yilmaz et al. 2003). As the authors of this study conclude, the good predictive value
of CADI suggests that CADI can be used not only as surrogate marker in prevention
trials but can also be used to identify the patient cohort at risk for intervention trials.
After this study, several other studies have also described the predictive power of
CADI in predicting later graft function or outcome (Helanterä et al. 2007), and also
later randomized controlled trials have used CADI as a surrogate end point (Mota
et al. 2004). Previously acute rejection and graft survival were mostly used as end
points in clinical trials of immunosuppressive drugs. However, in the current era, in
which graft survival is on average between 10 and 20 years and acute rejection is
seen in only approximately 10 % of patients, reliable surrogate markers are crucial
for clinical trials to get results in a timely manner and to avoid very expensive and
long trials. Therefore, CADI provides an excellent biomarker and surrogate marker
for clinical trials for immunosuppressive drugs. In addition, CADI as a linear
Table 5 Minor differences in the grading of chronic lesions between CADI and Banff (Racusen
et al. 1999)
CADI Banff
Inflammation Same grading
Interstitial fibrosis (Focal/diffuse/subcapsular)
0 No fibrosis Up to 5 %
1 Up to 25 % affected 6–25 %
2 Same grading
3 Same grading
Tubular atrophy
0 Same grading
1 Tubular atrophy up to 15 % of proximal tubules Up to 25 %
2 Tubular atrophy in 16–30 % of proximal tubules 26–50 %
3 Tubular atrophy in more than 31 % of proximal tubules Over
Mesangial matrix Same grading
increase
Intimal Same grading
proliferation
Sclerosis (Increase in extracellular matrix, sclerotic areas positive Not graded
stained with PAS)
0 No changes
1 <15 % of glomeruli affected
2 16–50 % of glomeruli affected
3 >50 % of glomeruli affected
numeric score is very practical in different types of studies analyzing correlation of

different variables with graft histopathology (Ortiz et al. 2005; Helanterä et al. 2007,
2010).
CADI as a Surrogate of Graft Function in Basic Science
The use of CADI expands to animal experimentation. Using a rat model for studying
chronic rejection, CADI was useful to assess immunosuppressive drugs that may
prevent the development of theses lesions (Malmstrom et al. 2008; Rintala
et al. 2008; Palin et al. 2013). Similarly CADI was used to show the association
between cytomegalovirus infections and chronic kidney transplant rejection
(Lautenschlager et al. 1997; Kloover et al. 2000).
In human trials the detection of plasminogen activator inhibitor-1 in the serum of
renal transplanted patients showed the potential to predict CADI scores (Chang
et al. 2009). Also in a study focused on microarrays, the CADI score combined to
certain biomarkers from the NLR family predicted the graft function at 1 year
posttransplantation (Perco et al. 2009). In another research focused on the develop-
ment of chronic changes possibly attributed to CsA toxicity, CADI score was not
correlated to transforming growth factor-β in protocol biopsies from patients under

low-calcineurin inhibitor regimen (Ortiz et al. 2013), suggesting only a minor role
for CsA nephrotoxicity in the development of chronic changes in the kidney graft.
Comparison of CADI to Individual Histopathological Lesions
There are several studies analyzing the association of single histopathological

lesions with outcome. Both chronic scarring (interstitial fibrosis and tubular atrophy)
and inflammation have been associated with inferior outcome (Seron et al. 1997;
Mengel et al. 2007), and also several studies show an association with transplant
glomerulopathy (duplication of glomerular capillary basement membranes) and
inferior outcome (Husain and Sis 2013). However, whether single lesions are more
powerful predictors of outcome compared to composite scores has not been studied.
A few studies have addressed this question indirectly. In one analysis (Yilmaz
et al. 2007), the CADI score or individual histopathological lesions from protocol
biopsies taken at 1 or 3 years after transplantation were correlated with clinical
parameters. Donor age, acute rejection, recipient age, and cold ischemia time
correlated better with CADI and to lesser extent with individual histopathological
lesions. Only about 60 % of the variation in histopathological score was explained
by clinical factors, supporting the use of CADI score in risk evaluation in protocol
biopsies taken after transplantation. In an analysis of donor biopsies (Anglicheau
et al. 2008), CADI score was compared with clinical risk factors and a clinicopath-
ological composite scoring in an attempt to find the best predictor of later graft
function in marginal donors. In this study, CADI analyzed from donor biopsies was
associated with low renal function after transplantation, as was also glomerulo-
sclerosis or arteriolar hyalinosis as single lesions. However, best prediction of low
renal function after transplantation was achieved with a composite score of serum
creatinine, donor hypertension, and glomerulosclerosis.
CADI from Donor Biopsies
The mere use of clinical scores while assessing kidney quality before transplantation
has not been predictors of subsequent graft function. Thus, the inclusion of histo-
logical analysis in the donor evaluation has become crucial, especially in the last
years when marginal donors are more frequently accepted. CADI has been first
utilized to evaluate donor biopsies in 1999 (Lehtonen et al. 1999). The authors
detailed the nature of the lesions observed in implant biopsies and correlated the
scores with subsequent graft function. The mean CADI score was 0.74, pointing the
good quality of the kidneys. Although histopathological changes were uncommon,
organs from donors older than 40 years of age had more frequent anisometric
vacuolization, interstitial fibrosis, vascular hyalinosis, glomerular sclerosis, and
tubular basement membrane thickening. CADI was useful to quantitate the lesions in
implant biopsies, but these abnormalities did not show an impact on the incidence of
delayed graft function.
More recently, a study focused on marginal donors (Anglicheau et al. 2008)
described that the factors associated with low glomerular filtration rate at 1 year
posttransplant were glomerulosclerosis, arteriolar hyalinosis, the Pirani clinical
score, and CADI. Other authors have also proposed the use of composite risk scores
when evaluating donor kidney quality. Kahu et al. published the results of the
follow-up of 481 donor biopsies and the paired recipients. They observed that >3
CADI score in donor biopsies was associated with worse graft function and survival.
In this study CADI was used also in follow-up biopsies to assess the development of
histological lesions in the first year after transplantation and its impact on prognosis
(Kahu et al. 2011).
Progression of CADI as a Biomarker of Graft Prognosis
As previously described, histological lesions could be already observed in implant

biopsies. It is of outstanding importance to follow up both the progression of these
lesions and the generation of new pathological findings across time. CADI was used
for this purpose in a study involving 83 kidney transplant patients (Ortiz et al. 2005).
The authors concluded that delta CADI (defined as the difference in CADI scores at
6 months and implant biopsies) was affected by glomerulosclerosis in donor biopsies
and serum creatinine concentration at hospital discharge. The delta-CADI concept
allowed to investigate the factors related to this progression. In this study the risk for
renal allograft damage progression was increased not only by donor histology but
also by nonimmunologic factors, such as hypertension and dyslipidemia. In a similar
fashion Yango et al. studied the progression of histological lesions in the graft from
implantation to 6 months after transplantation, focusing on the impact of immuno-
suppression regimen on the progression. The authors found the use of CADI score as
a suitable tool to evaluate the progression, even when the graft function was stable
(Yango et al. 2008). In a similar fashion Nainani et al. used CADI to quantify the
progression of the histological lesions in sequential biopsies obtained from patients
under different immunosuppressive regimens and interestingly concluded that ste-
roid discontinuation did not affect the progression in CADI score in patients free of
acute rejection (Nainani et al. 2012).
Histological lesions increase with time. CADI allows also the quantification of
theses lesions in later biopsies and the possible effects of changes in immunosup-
pression made later on. A few studies have focused on the switch from calcineurin
inhibitor to sirolimus, and the effects were evaluated in follow-up biopsies scored
with CADI (Mota et al. 2004; Ruiz et al. 2003). In both investigations the effect of
the change in the immunosuppressive regimen was properly evaluated with CADI,
resulting in lower scores in biopsies from patients on sirolimus.
CADI in Pediatric Kidney Transplantation
The progression of histopathological lesions in children after kidney transplan-

tation has also been studied using CADI and serial protocol biopsies taken 1.5,
3, 5, and 7 years after transplantation, from 51 children transplanted under the age
of 5 years (Qvist et al. 2000). However, in this study a modified version of CADI
was used. Due mostly to very mild histopathological changes, all the individual
parameters were scored with a scale of 0–6, and a sum of these scores was
calculated (CADI range 0–36). This taken into account, the CADI scores were
overall very low; at 1.5 years, mean CADI was 2.5 and at 7 years only 3.5 (Qvist
et al. 2000), suggesting that the progression of histopathological lesions in small
children with well-functioning grafts may be slower compared to adults.
Concerning the individual parameters, interstitial inflammation tended to
decrease with time, whereas interstitial fibrosis tended to increase over time.
The most prominent finding was the progression of glomerular sclerosis. At
1.5 years, 3 % of the glomeruli were totally sclerosed, whereas 7 years after
transplantation 36 % of the glomeruli were totally sclerosed. CADI correlated
with graft function at the time of biopsy and also predicted later graft function,
whereas the individual histological parameters did not predict graft function
(Qvist et al. 2000). Especially in this pediatric material with very mild histopath-
ological lesions, CADI may provide better prognostic tools compared to individ-
ual histopathological lesions. Clinical parameters, which were associated with
higher CADI, were previous acute rejection episodes and kidney from a deceased
donor (vs. living donor) (Qvist et al. 2000).
Reproducibility of CADI
A common problem with histopathological classification systems is the poor

interobserver reproducibility, especially in quantifying the severity of lesions. This
holds true also for kidney transplant pathology. There are several studies showing
that quantifying lesions in the kidney allograft biopsy based on Banff classification
differs widely between pathologists (Furness and Taub 2001, Furness et al. 2003b).
When kappa values were calculated for the level of interobserver agreement (0 = no
agreement; 1 = perfect agreement), all evaluated lesions had very poor reproduc-
ibility with kappa values differing between 0.1 and 0.4 (Furness and Taub 2001,
Furness et al. 2003b). Even feedback systems applied in these multicenter studies did
not significantly improve the interobserver variability (Furness et al. 2003b). These
same limitations apply for when individual histopathological parameters are scored
for CADI. However, in the pivotal studies of CADI score, an excellent interobserver
agreement was reached with the two pathologists who were involved in the devel-
opment of CADI; the correlation coefficient between the two pathologists was as
high as 0.85 (Yilmaz et al. 2003). This suggests that reproducibility is possible with
CADI, especially with devoted pathologists working in the same center. However,
due to the large interobserver variability, comparison of CADI between different

centers must be interpreted with caution.
CADI in Helping Clinical Decision-Making
Although CADI was developed primarily as a tool for research, it may also be very
useful in helping clinical practice. All kidney transplant biopsies in the authors’
institution have been classified according to CADI already for the past 10 years. A
numeric score of all the individual parameters and the sum score (CADI) are
provided by local pathologists from all transplant biopsies and also from donor
biopsies. The numeric table gives a quick overview of the severity of chronic
changes and makes comparison of the change in the parameters compared to
previous biopsies very easy. Also CADI score can be very illustrative and useful
when the findings of the biopsy are interpreted to the patient. We recommend taking
CADI as a part of routine clinical practice when interpreting kidney transplant
biopsies.
Limitations of CADI
In addition to the general problem of reproducibility of pathological lesions in

kidney transplant biopsies, several limitations exist in the CADI scoring system,
and critique has been pointed for certain characteristics of CADI. One point of
criticism is that CADI mixes chronic (fibrosis, tubular atrophy, glomerular sclerosis)
and active lesions (inflammation). It is, however, well known that various degrees of
inflammation are commonly seen in biopsies of stable kidney allografts and that this
inflammation is one of the most important predictors of graft outcome (Mengel
et al. 2007) regardless of the degree of inflammation. Therefore, strong arguments
exist for including inflammation in CADI.
Another point is that mesangial matrix increase included in the CADI is not very
strongly correlated with outcome and is not linked to any particular pathogenic
process. Also, this variable is not very reproducible.
Another problem with CADI is that it is simply a sum score of all the parameters,
whereas ideally based on their predictive value each parameter should be given
individual weight in the score. In addition, some of the components are strongly
correlated with each other (such as tubular atrophy and interstitial fibrosis) and are
not very independent variables. The weight of this component is thus doubled in the
current score.
All of these points of criticism are indeed adequate. However, one of the
advantages of CADI is its simplicity, and calculating a weighted score would
probably increase the predictive power in clinical trials, but would on the other
hand complicate the interpretation of this score in clinical use. The current advan-
tages and applications of CADI score are summarized in Figure 2.
Fig. 2 Applications for chronic allograft damage index (CADI). Summary of the applications of
chronic allograft damage index (CADI) as a biomarker in kidney transplantation

Conditions
As CADI is developed specifically for kidney transplant pathology, it cannot there-

fore be applied directly to other transplanted organs or diseases. The chronic scarring
in different transplanted organs differs in the cell types and tissues involved, and
therefore same principles are not applicable in other organs. However, composite
scoring systems could be helpful in quantifying the extent of chronic changes also in
other transplanted organs, but must be developed specifically to each organ system.
As described above, one of the main advantages of CADI as a biomarker in
kidney transplantation is its excellent correlation with graft prognosis, and it is also
one of the key reasons why this composite scoring system was in fact developed.
Linear correlation of CADI with graft function and prognosis makes it an excellent
surrogate marker for clinical and experimental studies.
Summary Points
• Chronic allograft damage index (CADI) was introduced in the early 1990s, with
the purpose of numerically classifying pathological lesions in transplanted
kidneys.
• CADI is a sum score of six histopathological lesions commonly seen in biopsies
taken from transplanted kidneys that correlate with the function and outcome of
the graft.
• CADI includes interstitial inflammation, tubular atrophy, interstitial fibrosis,

arterial fibrointimal thickening, glomerular mesangial matrix increase, and the
percentage of globally sclerosed glomeruli.
• CADI score, analyzed from either donor biopsies taken before transplantation or
from protocol biopsies taken after transplantation, correlates with graft function
and later outcome and can be used as a surrogate marker in clinical trials.
• CADI can also be useful in clinical decision-making, as it gives a simple numeric
score of the extent of chronic injury in the kidney allograft.
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Biomarkers for Kidney Injury in Cystic
Fibrosis 31
Kevin J. Downes and Stuart L. Goldstein
Contents
Key Facts of Cystic Fibrosis and Kidney Injury . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 691
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 692
Kidney Injury Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 693
The Impact of Kidney Injury in Cystic Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 694
Causes of Acute Kidney Injury in Cystic Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 696
Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 696
NSAIDs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698
Other Nephrotoxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 698
Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 699
Stones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 699
Markers of Kidney Injury in Cystic Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 700
Serum Creatinine and Estimated GFR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 700
Aminoglycoside Clearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 701
N-Acetyl-β-D-glucosaminidase (NAG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 705
Urinary Albumin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 706
Cystatin C (CysC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 707
Beta-2-Microglobulin (β2M) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 708
Retinol-Binding Protein (RBP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 710
Neutrophil Gelatinase-Associated Lipocalin (NGAL) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 710
Kidney Injury Molecule-1 (KIM-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 711
Other Urinary Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712
K.J. Downes (*)

Department of Pediatrics, Perelman School of Medicine, The University of Pennsylvania,
Philadelphia, PA, USA
Division of Infectious Diseases, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA
e-mail: downeskj@email.chop.edu; kjdownes@yahoo.com
S.L. Goldstein
Division of Nephrology and Hypertension, Center for Acute Care Nephrology, Cincinnati
Children’s Hospital Medical Center, Cincinnati, OH, USA
e-mail: Stuart.Goldstein@cchmc.org

690 K.J. Downes and S.L. Goldstein
Biomarkers and the Future of Kidney Injury in Cystic Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 712

Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 713
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 714
Abstract
Cystic fibrosis (CF) is an autosomal recessive disease affecting sodium and
chloride transport predominantly in the lungs and digestive system. Patients with
this disease develop recurrent and chronic respiratory tract infections often neces-
sitating repeated and aggressive antimicrobial therapy. Patients also commonly
develop pancreatic insufficiency, diabetes, malabsorption, and liver problems. The
role of the CF gene mutation in the kidney is not well described. However, patients
with CF are at risk for development of acute kidney injury (AKI) due to receipt of
nephrotoxic medications, particularly antibiotics, as well as long-term renal dam-
age from long-standing diabetes and repeated nephrotoxin exposures. The tradi-
tional marker of kidney injury and function, serum creatinine, is an unreliable
marker of kidney function and injury in patients with CF and low muscle mass: it
often overestimates true kidney function and does not detect kidney injury until a
significant number of nephrons have been affected. Novel biomarkers, such as
cystatin C (CysC), retinol-binding protein (RBP), kidney injury molecule-1
(KIM-1), and neutrophil gelatinase-associated lipocalin (NGAL) have been stud-
ied preliminarily in this population but hold significant promise for early detection
of AKI, risk stratification, and prognosis. With advances in medical therapies
leading to substantial improvement in lifetime survival among CF patients, the
long-term ramifications of the disease and its treatments on the kidney are becom-
ing apparent. Methods to improve detection of kidney injury and risk of develop-
ment of chronic kidney disease in patients with CF are paramount. This chapter
reviews the available literature on the causes and impact of AKI in patients with
CF, as well as the strengths, limitations, and potential uses of traditional and novel
biomarkers of kidney injury in this population.
Keywords
Acute kidney injury • Aminoglycosides • Chronic kidney disease • Cystic
fibrosis • Urinary biomarkers
Abbreviations
AG Aminoglycoside
Β2M Beta-2-microglobulin
CCl Creatinine clearance
CF Cystic fibrosis
CFRD Cystic fibrosis-related diabetes
31 Biomarkers for Kidney Injury in Cystic Fibrosis 691

CysC Cystatin C
eCCl Estimated creatinine clearance
IV Intravenous
L-FABP Liver-type fatty acid-binding protein
NAG N-Acetyl-β-D-glucosaminidase
NSAIDs Nonsteroidal anti-inflammatory drugs
RBP Retinol-binding protein
TDM Therapeutic drug monitoring
UCr Urine creatinine
Key Facts of Cystic Fibrosis and Kidney Injury
• Cystic fibrosis (CF) is an autosomal recessive genetic disease.

• A defect in the cystic fibrosis transmembrane conductance receptor (CFTR) leads
to impaired sodium and chloride transport across epithelial cells.
• As a result of abnormal electrolyte transport, patients develop thick mucus which
predisposes them to chronic and recurrent pulmonary infections, intestinal mal-
absorption, and pancreatic insufficiency.
• The impact of CFTR on kidney disease in CF has not been fully elucidated.
• The majority of kidney injury in this population stems from secondary insults
from nephrotoxin administration (antibiotics, immunosuppressive medications),
formation of renal stones, and CF-related diabetes (CFRD).
• As survival for patients with CF increases, so do opportunities for kidney injury
and the likelihood of development of chronic kidney disease (CKD).
• The traditional marker of kidney injury and function, serum creatinine, is often
inaccurate in patients with CF and decreased muscle mass.
• Novel serum and urine biomarkers hold great promise for the early detection and
prognostication of acute kidney injury in patients with CF and other high-risk
populations.
Definitions
Acute kidney injury (AKI) An acute, reversible decline in kidney function

manifested by an increase in serum creatinine (SCr) combined with an inability of
the kidney to regulate fluid and electrolytes appropriately.
Albuminuria The presence of albumin, a plasma protein, in the urine; this often
reflects damage to the glomerulus (the filter) of the kidney.
Aminoglycoside A class of antibiotic used to treat certain bacterial infections.
Biomarker A molecule (protein, enzyme, etc.) found in the body (blood, urine,
tissue, etc.) that signifies the presence of a disease.
Chronic kidney disease (CKD) A progressive and often irreversible loss of kidney
function.
Creatinine A by-product of muscle breakdown; its concentration in the serum is

often reflective of kidney function because it is nearly exclusively cleared by the
kidney. Elevated serum creatinine concentrations reflect impaired kidney function.
Creatinine clearance (CCl) Describes how much creatinine is removed from the
body by the kidney; used as a proxy for kidney function and glomerular
filtration rate.
Drug clearance A measure of the amount of drug eliminated from the serum in a
given amount of time.
Glomerular filtration rate (GFR) A measure of kidney function describing the

rate of fluid filtered by the kidney.
Nephrotoxin A drug or medically administered substance (i.e., intravenous con-

trast) which can have injurious effects on the kidney.
Introduction
Cystic fibrosis (CF) is an autosomal recessive genetic disease that predisposes

individuals to chronic and recurrent pulmonary infections, intestinal malabsorption,
and pancreatic insufficiency. A defect in the cystic fibrosis transmembrane conduc-
tance receptor (CFTR) leads to impaired sodium and chloride transport across
epithelial cells and is the underlying cause of the clinical manifestations of CF. In
the human kidney, CFTR is present predominantly in the proximal and distal tubules
(Crawford et al. 1991), but the impact of CFTR on kidney disease in CF has not been
fully elucidated. Despite the presence of CFTR in the kidney, primary kidney
diseases are relatively rare in CF patients. Thus, the majority of kidney injury in
this population stems from secondary insults from nephrotoxin administration,
formation of renal stones, and CF-related diabetes (CFRD). As survival for patients
with CF increases, so do opportunities for kidney injury and the likelihood of
development of chronic kidney disease (CKD). Improved methods for early detec-
tion of kidney injury are needed.
This chapter reviews the causes and methods of detection of acute kidney injury
(AKI) in CF patients, with a focus on nephrotoxin-associated kidney injury, which is
the most prevalent and potentially modifiable cause of kidney injury in this population.
The impact of AKI in CF patients and the potential roles of novel urinary and serum
kidney injury biomarkers in AKI detection are also discussed. Laboratory methods of
biomarker measurement are not reviewed and can be found elsewhere in this book.
Kidney Injury Definitions
Acute kidney injury, formerly denoted acute renal failure, is defined as an acute,
reversible increase in serum creatinine (SCr) and nitrogenous waste products com-
bined with an inability of the kidney to regulate fluid and electrolytes appropriately
(Andreoli 2009). Traditionally, the method for monitoring kidney injury (acute or
chronic) has been through surveillance of SCr and blood urea nitrogen (BUN)
measurements and monitoring urine output. Serum creatinine can be monitored
directly or used to estimate the glomerular filtration rate (GFR) or creatinine
clearance (CCl) through a variety of derived formulae. A number of AKI definitions
have been developed based on the magnitude of SCr or estimated creatinine clear-
ance (eCCl) changes and/or a reduction in urine output. Figure 1 displays the AKI
GFR Criteria* Urine Output Criteria

Increased SCreat x1.5 or UO<.5ml/Kg/h
GFR decrease>25% x 6 hr
Risk
High
Sensitivity
Increased SCreat x2 UO<.5ml/Kg/h
or GFR decrease>50%
Injury x 12 hr
Increased SCreat x3 UO<.3ml/Kg/h

ria
GFR decrease 75% x 24 hr or

Oligu
Failure OR SCreat≥4mg/dl Anuria x 12 hrs

Acute rise≥0.5mg/dl
High
Specificity
Loss Presistent ARF** = complete loss
of kidney function > 4 weeks
End Stage Kidney Disease

ESKD (> 3 months)
Fig. 1 RIFLE classification scheme for acute renal failure (ARF). The classification system
includes separate criteria for creatinine and urine output (UO). A patient can fulfill the criteria
through changes in serum creatinine (SCreat) or changes in UO or both. Abbreviations: ARF acute
renal failure, GFR glomerular filtration rate (#2004; licensee BioMed Central Ltd. This is an Open
Access article. http://ccforum.com/content/8/4/R204. Reproduced with permission from Bellomo
et al. 2004)
classification scheme of the RIFLE criteria (Bellomo et al. 2004), which is one of the
most frequently employed AKI definitions for adults. This classification system
demonstrates how SCr-, GFR-, and urine output-based criteria relate and can be
applied to identify patients with AKI. Most AKI definitions have been studied and
validated in specific patient populations. However, there is no universally accepted
and validated definition for AKI specific to patients with CF. As will be discussed
below, SCr-based definitions of AKI may be inadequate in patients with CF due to
the limitations and inaccuracies of SCr in this population.
Similar to AKI, chronic kidney disease (CKD) encompasses a number of stages
which reflect the degree of kidney dysfunction. Chronic kidney disease typically
reflects sustained (3 months) and significant (GFR <60 mL/min/1.73 m2) impair-
ment (K/DOQI 2002). A reduction in kidney function to this degree, referred to as
stage 3 CKD, generally equates to a loss of half of normal adult kidney function.
Persistent kidney damage, manifest as structural or function abnormalities, can also
meet the CKD definition.
The Impact of Kidney Injury in Cystic Fibrosis
The incidence of kidney injury in CF may be significantly higher than in the non-CF
population. Bertenshaw and colleagues estimated the incidence of acute renal failure
in individuals with CF to be between 4.6 and 10.1 cases per 10,000 per year, more
than 100 times the average rate of kidney injury in children and three to eight times
that of adults (Bertenshaw et al. 2007). This survey study identified patients by
physician report and defined acute renal failure as, “raised plasma creatinine for
age.” Despite the use of this broad definition, the majority of subjects included in the
study (54 %) had severe renal impairment requiring dialysis, making it likely that
many patients with modest changes in SCr, which would constitute AKI by recently
validated definitions, were not reported. Therefore, this study may actually under-
estimate the true incidence of AKI in the CF population.
Acute kidney injury has both short- and long-term ramifications, and even small
changes in SCr of 0.3–0.5 mg/dL are independently associated with worse out-
comes, including mortality, in children and adults (Moffett and Goldstein 2011;
Chertow et al. 2005). Meanwhile, children and adults who sustain AKI are also at
increased risk for long-term mortality and the development of chronic kidney disease
(Askenazi et al. 2006; Wehbe et al. 2011), including AKI resulting from exposure to
nephrotoxic medications (Menon et al. 2014). Data on the impact of AKI episodes in
CF patients are more limited. Among adults with CF, episodes of AKI correlate with
some markers of chronic kidney impairment (Florescu et al. 2012). This cohort study
followed 113 adult patients in the United States with CF for up to 8.5 years. While
there were no significant changes in BUN ( p = 0.92) or SCr ( p = 0.2) among the
entire study population, individuals who experienced an episode of AKI had a
significant increase in BUN (P = 0.002) and a nearly significant increase in SCr
(P = 0.056) at the end of follow-up. Only the use of inhaled colistin correlated with
AKI episodes ( p = 0.03).
Unfortunately, there are limited additional data about the long-term outcomes of
CF patients in relation to episodes of AKI specifically. Yet, there is mounting
evidence that patients with CF sustain repeated renal insults over time from a variety
of etiologies. As survival for patients with CF increases, the risk of developing CKD
rises. Quon et al. estimated that the age-adjusted prevalence of CKD is about twice
that of the United States general population (Quon et al. 2011). A strong association
has been described between patients CFRD requiring insulin and the development of
CKD (Quon et al. 2011). Meanwhile, others have noted an association between
repeated intravenous (IV) aminoglycoside (AG) use and long-term renal impairment
(Al-Aloul et al. 2005a). Ultimately, the development of CKD is more likely to result
from repeated or chronic insults than progression of primary kidney disease.
Children with CF may also develop chronic renal dysfunction. In a retrospective
analysis of children with CF who had GFR measured by 99mTc-DTPA at a single
center, Prestidge et al. found that 6 % (4/63) had evidence of stage 2 CKD (GFR
<90 mL/min/1.73 m2, persistent abnormalities in urinary sediment, abnormal renal
imaging): one child with reduced GFR and three others with persistent microscopic
hematuria (Prestidge et al. 2011). Although the rate of renal impairment in this study
was low, the authors observed that 40–56 % of children had evidence of glomerular
hyperfiltration depending on the definition used (GFR >2 standard deviations for
age or >90th percentile for age). In diabetic adults without CF, glomerular
hyperfiltration precedes the development of albuminuria and subsequent GFR
decline (Moriya et al. 2012) and it is possible, as the authors suggest, that glomerular
hyperfiltration in CF patients similarly portends subsequent kidney function decline,
although this has not been established.
The impact of repeated nephrotoxic insults in the CF population becomes most
apparent in patients undergoing lung transplantation. In a large cohort study of
pediatric lung transplant recipients, patients with CF had more rapid decline in
kidney function following lung transplant than other transplant recipients (Hmiel
et al. 2005). In this study, Kaplan-Meier analyses determined that older age at
transplant and diagnosis of CF were both associated with loss of renal function
over time. Broekroelofs similarly found that rate of renal function loss was greatest
among lung transplant recipients who also had CF (10 mL/min/year, range: 14 to
6 mL/min/year) compared to others (Broekroelofs et al. 2000). Meanwhile, using
data from the CF Foundation Patient Registry, Quon determined that the 2-year risk
of post-lung transplant kidney dysfunction among CF patients was 35 % (95 % CI:
32–39 %) and the risk increased substantially with increasing age (Quon et al. 2012).
There are a number of potential reasons why patients with CF are at high risk for
development of renal impairment following lung transplantation: receipt of repeated
and chronic nephrotoxic medications prior to and following transplantation
(calcineurin inhibitors, antibiotics), diabetic nephropathy, and the presence of kidney
stones. Renal reserve may be impaired heading into transplant, because of the
numerous insults sustained prior to the procedure, and compounded by infection,
receipt of additional antibiotic courses, and advanced diabetic disease afterward
(Hmiel et al. 2005). With the high risk of development of CKD for patients with
CF undergoing lung transplantation, it is paramount to identify means to reduce
insults to the kidney prior to transplant.
Causes of Acute Kidney Injury in Cystic Fibrosis
There are a number of potential causes of AKI in patients with CF including toxins,
intrinsic renal disease, obstruction, and other insults. In this population, the most
likely inciting factor is the use of nephrotoxic medications. Individuals with CF have
frequent lung infections which contribute to a decline in lung function over time, and
aggressive antimicrobial therapy is used in both the acute and long-term manage-
ment of CF lung disease. Unfortunately, a number of antibiotics commonly admin-
istered in the CF population, most notably aminoglycosides and colistin, have
nephrotoxic effects. Nonsteroidal anti-inflammatory drugs (NSAIDs) are also fre-
quently used in CF to mitigate lung inflammation but can have detrimental effects on
the kidney. Other nephrotoxic medications such as antihypertensive medications and
immunosuppressants, although less commonly given, diabetic nephropathy, renal
stones, and other causes may also contribute to kidney injury in this population; each
of these potential causes will be discussed in further detail below.
Antibiotics
Aminoglycosides are a commonly used class of antibiotics in CF because of their

activity against Gram-negative bacteria, particularly Pseudomonas aeruginosa, the
most common pathogen infecting the lungs of patients with CF. These are
concentration-dependent antibiotics, meaning that bacterial killing is optimized at
high concentrations. To take advantage of this property, AGs are typically administered
in high doses once daily to maximize killing and allow sufficient time for clearance of
the medication prior to re-dosing. However, nephrotoxicity is a known side effect of
AGs and the cellular mechanisms leading to toxicity are complex. Aminoglycoside
nephrotoxicity results from accumulation of drug within proximal tubule cells leading
to cytotoxicity, apoptosis, and cell death (Rougier et al. 2004). After glomerular
filtration, a portion of the drug binds to an endocytic receptor, megalin, located on
the apical surface of the proximal tubule epithelial cell and is endocytosed (Moestrup
et al. 1995). Expression of megalin is directly related to the degree of drug accumula-
tion as it is the principle receptor for AG uptake in the kidney (Schmitz et al. 2002), as
well as a number of other important ligands. Following endocytosis, the drug accumu-
lates within lysosomes, causes damage to membrane phospholipids and is released into
the cytosol where cellular toxicity occurs (Servais et al. 2005). Tubule cell damage and
apoptosis then lead to a reduction in the glomerular filtration rate (GFR) and impaired
kidney function (Lopez-Novoa et al. 2011).
Most reports of AKI in patients with CF have implicated AG as the cause
(Bertenshaw et al. 2007; Al-Aloul et al. 2005b; Drew et al. 2003; Kennedy
et al. 2005; Smyth et al. 2008). In a survey of CF centers in the United Kingdom,
an AG was administered prior to onset of AKI in 88 % of cases reported (Bertenshaw
et al. 2007). An additional risk factor such as dehydration, underlying renal disease,
or long-term receipt of a nephrotoxic drug also often accompanies cases of
aminoglycoside-associated AKI (Smyth et al. 2008). Yet, the true incidence of
AG-associated AKI in the CF population had not been known. This is because
detection of AKI is reliant upon systematic SCr measurement, which is rarely
performed. Among children with CF, AKI rates during AG courses of up to 20 %
have been described when SCr is monitored daily (Downes et al. 2014).
But, detection of AKI is significantly impacted by the frequency of SCr measure-
ment. Nonsystematic SCr measurement, which is common practice, and dependence
upon a suboptimal marker (SCr) lead to an underestimation of the actual incidence
of AKI.
Similar to AG, the antibiotic colistin has broad Gram-negative activity and is used
often in patients with CF to treat more resistant pathogens. It is a polymyxin
antibiotic whose nephrotoxic potential is recognized but not well understood. The
mechanism of nephrotoxicity is thought to be similar to the mechanism by which the
drug exhibits its antibacterial activity: increasing cell membrane permeability (Lewis
and Lewis 2004). Damage to renal tubule cells leads to acute tubular necrosis,
mitochondrial dysfunction, and impaired kidney function (Dai et al. 2014). Histor-
ically, colistin was associated with nephrotoxicity in up to 50 % of recipients
(Falagas and Kasiakou 2006). However, newer formulations of the drug, careful
monitoring of patients, and avoidance of coadministration with other known
nephrotoxins have led to a reduction in reported toxicity.
The nephrotoxic potential of colistin in patients with CF is conflicting. In a
retrospective review of 52 patients receiving 135 courses of colistin at a single
center, Ledson and colleagues found that there was no change in renal function
following receipt of the drug among the 122 evaluable courses (Ledson et al. 1998);
colistin was used in combination with other drugs in 85 % of courses in this study.
Meanwhile, in a randomized trial of IV colistin monotherapy versus combination
antipseudomonal therapy (Conway et al. 1997), recipients of monotherapy did not
demonstrate a change in creatinine clearance after 12 days, while those who received
IV colistin in combination with another non-AG antibiotic had a significant decline
by day 12 (day 1 = 109 mL/min vs. day 12 = 91 mL/min, p <0.01). Nevertheless,
reports of kidney injury with colistin are less prevalent than with IV aminoglycosides
in CF, perhaps owing to the different frequency of use of these agents.
There have been no studies directly comparing the development of kidney injury
from AG vs. colistin in CF patients. But, receipt of repeated courses of nephrotoxic
antibiotics may be associated with long-term renal damage even in the absence of
detected SCr-based AKI. In a prospective study of 80 CF outpatients with Pseudo-
monas aeruginosa, Al-Aloul and colleagues found a strong correlation between IV
AG use and diminishing kidney function (r = 0.32, P = 0.0055) as determined by
measured creatinine clearance (Al-Aloul et al. 2005a). Figure 2 displays the inverse
correlation between kidney function and number of courses of IV nephrotoxic antibi-
otics found in this study. The association between decreased renal function and
repeated antimicrobial exposure was not significant for regimens including IV colistin
with a beta-lactam antibiotic (r = 0.02, p = 0.83). However, there was a significant
association between renal function decline and receipt of IV aminoglycosides with a
beta-lactam antibiotic (r = 0.35, p = 0.0018); the effect was more pronounced when
an IV aminoglycoside was coadministered with colistin (r = 0.51, p <0.001).
Fig. 2 Correlation between renal function (mCCL) and lifetime use of IV nephrotoxic antibiotics
(courses containing aminoglycosides and/or colistin). R = 0.65, P <0.00001. Abbreviations: mCCL
measured creatinine clearance (Reproduced with permission from Al-Aloul et al. 2005. #2004
John Wiley & Sons, Inc)
NSAIDs
Beyond the treatment of pain, nonsteroidal anti-inflammatory drugs (NSAIDs) may

have a role in the treatment of chronic lung inflammation in CF patients. However,
NSAIDs alter kidney function through their effects on prostaglandins (Weir 2002)
and in the setting of altered renal blood flow (dehydration, shock, etc.) may com-
promise renal perfusion and lead to kidney injury. Despite their potential adverse
effects, NSAIDs have rarely been reported to have significant nephrotoxic effects in
patients with CF. In a single-center retrospective study of patients on chronic
ibuprofen, 50 % patients had to discontinue the therapy due to adverse events
(Fennell et al. 2007) but only one (2 %) discontinued the drug due to renal toxicity.
Similarly, Lahiri et al. reported that high-dose ibuprofen was not associated with
increased non-creatinine biomarkers of kidney injury (Lahiri et al. 2014); these
biomarkers will be discussed in further detail below.
Other Nephrotoxins
A number of other medications administered in patients with CF may be toxic to the

kidneys, as with other groups of patients. Lung transplantation is an important option
for CF patients with severe and end-stage lung disease. Immunosuppressant medi-
cations such as the calcineurin inhibitors cyclosporine and tacrolimus have important
therapeutic roles in transplant recipients but can lead to rapid decline in kidney
function following transplant (Quon et al. 2012; Hmiel et al. 2005; Broekroelofs
et al. 2000). These medications contribute to kidney injury and a reduction in GFR
by causing vasoconstriction of afferent and efferent glomerular arterioles (Lanese
and Conger 1993). Their nephrotoxic effects may be compounded by years of prior
nephrotoxin receipt in CF patients and may unmask decreased renal reserve (Hmiel
et al. 2005). Antihypertensives such as loop diuretics and angiotensin-converting
enzyme inhibitors may alter renal hemodynamics causing kidney injury (Ferguson
et al. 2008). And antimicrobials such as acyclovir and amphotericin have their roles
in CF care for the treatment of herpes virus and fungal infection, respectively, but
may also be nephrotoxic.
Diabetes
In 2012, nearly 20 % of individuals with CF had cystic fibrosis-related diabetes

(CFRD) with more than a third of adults being affected (Cystic fibrosis foundation
patient registry 2012 annual data report 2013). Similar to type I and type II diabetes
in patients without CF, CFRD causes long-term kidney damage. In a study using
registry data from Germany and Austria (Konrad et al. 2013), the rate of diabetic
nephropathy (defined as the presence of microalbuminuria) was similar among
adults with CFRD (25.2 %) compared to non-CF patients with type I (17.2 %) or
type II diabetes (24.7 %, p-values not reported) after adjustment for demographics.
Using the US CF Registry data from 2001 to 2008, Quon determined that CFRD
requiring insulin therapy was a significant risk factor for the development of stage
3 CKD as defined by an estimated GFR <60 mL/min/1.73 m2 (Quon et al. 2011).
Considering that this study was a retrospective analysis of registry data with reliance
on eGFR for detection of CKD, this may actually underestimate the impact of CFRD
on CKD development. CFRD requiring insulin therapy is also a significant risk
factor for renal dysfunction following lung transplant (HR 1.30; 95 % CI, 1.02–1.67)
(Quon et al. 2012). While the microvascular complications of CFRD can lead to
chronic renal insufficiency, it is unknown whether CFRD also contributes to epi-
sodes of acute kidney injury or compounds the nephrotoxic effects of antibiotics in
the CF population.
Stones
Patients with CF are at higher risk for nephrocalcinosis compared to the general
population which can contribute to an impairment of kidney function. In a single-
center cohort study of pediatric CF patients, the prevalence of risk factors for stone
formation was high – hyperoxaluria (N = 58/83, 78 %), hypocitraturia (57/76,
75 %), hypercalciuria (16/87, 18 %), and hyperuricuria (15/83, 18 %) (Andrieux
et al. 2010). However, no patients had symptomatic kidney stone formation and only
2 % had stones diagnosed by ultrasonography. Some studies have reported a
prevalence of nephrolithiasis as high as 21 % (Terribile et al. 2006). Microscopic
nephrocalcinosis may also be a frequent occurrence in CF patients, and the increased
sodium secretion that occurs in CF may lead to dehydration and low urine volumes,
further compounding the risk for stone formation. Repeated exposure to antimicro-
bials may alter gut flora and decrease the Oxalobacter formigenes, which leads to
reduced degradation of oxalate and an increased risk of hyperoxaluria and stone

formation (Sidhu et al. 1998).
Markers of Kidney Injury in Cystic Fibrosis
With the myriad of potential causes of kidney injury in patients with CF, the long-
term ramifications of repeated and chronic renal insults on kidney function is
becoming more apparent as patients with this disease live longer. Traditional
markers of kidney function and injury such as serum creatinine are inadequate.
And, there is a need for accurate and sensitive markers of kidney injury and
incorporation of these markers into routine CF care. Yet, the ideal biomarkers for
AKI detection among patients with CF have not been established, and further
research is urgently needed.
Novel biomarkers for kidney injury may be clinically useful for early detection of
AKI. These biomarkers, of which several have been identified and will be discussed
below, are more sensitive than traditional SCr measurements in detecting kidney
injury directly and are under investigation for their utility in risk stratification and
prognostication in AKI (Parikh et al. 2005). Increased levels have been associated
with poor clinical outcomes irrespective of SCr measurements (Haase et al. 2011;
Parikh et al. 2005). Serum biomarkers often reflect abnormal kidney function
(impaired GFR), while urinary biomarkers may reflect kidney injury and/or function
(compromised reabsorption). Depending on the process, urinary biomarkers also
have the potential to signify specific sections of the nephron that are affected. This
type of specificity could be useful to elucidate the mechanism of underlying injury.
However, the determination of the optimal biomarkers for use in specific clinical
settings has not been established.
The following sections will discuss the utility of traditional and novel biomarkers
for detection of kidney injury in CF. Many of the biomarkers reviewed in this section
are described in more detail in other chapters in this book. Therefore, this section will
focus primarily on these biomarkers in CF patients specifically. Of note, few studies
explicitly examine the role of biomarkers in AKI detection in CF patients. The
majority of studies attempt to establish the relationship between biomarkers of
interest and GFR. This section summarizes available data and, when applicable,
provides guidance as to how kidney injury biomarkers can be used or studied in the
future to improve AKI detection in patients with CF.
Serum Creatinine and Estimated GFR
The glomerular filtration rate is widely considered the best measure of kidney
function. Direct measurement of GFR, however, is difficult, often costly, and
frequently impractical in the hospitalized setting. Serum creatinine is the traditional
biomarker most often used for simple assessment of kidney function as well as
detection of kidney injury. Creatinine, a product of muscle breakdown, undergoes
glomerular filtration and is excreted in the urine. Because there is minimal extrarenal
clearance of creatinine in healthy individuals, renal creatinine clearance is used as a
surrogate for kidney function (Perrone et al. 1992). Twenty-four hour urine collec-
tion allows for calculation of CCl but is methodologically tedious and prone to error.
Therefore, serum creatinine values are used to estimate CCl and GFR through
implementation of a number of derived formulae. These formulae tend to be most
reliable in the setting of stable kidney function and thus stable SCr. And the validity
of these formulas is based on the assumptions that SCr is completely filtered and that
the rate of production equals the rate of renal excretion (Perrone et al. 1992). In the
setting of unstable renal function or ongoing kidney injury, unfortunately, these
criteria are not always met.
Changes in SCr values are nonspecific, often delayed, and do not directly reflect
cellular kidney injury. A demonstrable change in SCr is not detected until significant
renal mass, roughly 70–80 %, has been affected (Pfaller and Gstraunthaler 1998).
Creatinine is formed as a result of muscle breakdown which makes it an additionally
problematic marker of kidney function and injury in patients with CF, who fre-
quently have reduced muscle mass compared to healthy patients. Because of this,
SCr-based formulae for estimating GFR are often inaccurate and typically underes-
timate renal impairment in patients with CF (Al-Aloul et al. 2007). Al-Aloul
compared measured CCl from timed urine collections with ten SCr-based formulae
used to estimate CCl in 74 adult CF patients and 29 healthy, age- and BMI-matched
control subjects (Al-Aloul et al. 2007) and concluded that all formulae for estimating
CCl were unreliable in CF patients. The correlation between estimated and measured
creatinine clearance ranged from 0.55 to 0.7 with a bias of 9.1 to 22.9 depending
on the equation used. The formulae were also less accurate in CF patients than in
healthy controls. Additionally, the two equations most commonly used clinically for
estimating CCl, the Cockcroft-Gault formula (Cockcroft and Gault 1976) and the
abbreviated MDRD equation (Rule et al. 2004), grossly overestimated renal function
in adult CF patients with reduced CCl (<80 mL/min, Table 1) in their study
population.
Although traditional monitoring for kidney injury involves SCr measurement, the
sensitivity of this parameter is poor. While more accurate methods of estimation of
kidney function are available, such as 24-h urine creatinine collection or nuclear
GFR studies, they are typically used to assess GFR at a single time point. These
approaches are generally not practical in the hospitalized setting and cannot gener-
ally be used for monitoring or early detection of AKI due to the methodological rigor
involved in their use.
Aminoglycoside Clearance
Therapeutic drug monitoring (TDM) is used to try to improve efficacy and minimize
toxicity from aminoglycosides. The goal of TDM is to maximize effectiveness and
safety by determining patient-specific doses and dosing intervals through drug level
monitoring. Trough levels are typically monitored to assure adequate clearance of
702
Table 1 Comparison of mCCl and eCCl (CGF and abbreviated MDRD [four variable]) in renally impaired CF patients (mCCl <80 mL/min)
Mean mCCl eCCl Mean eCCl % eCCl within 33 % of 95 % CI for Limits of
N (SD) formula (SD) mCCl Bias bias agreement
mCCl 35 63.5 (12.3) CGF 81.7 (18.1) 60 % 18.3 13.6–23.0 9.7–46.3
<80 aMDRD 79.3 (20.2) 65 % 15.8 10.5–22.0 17.5–50.1
mCCl 39 100.7 (13.8) CGF 102.3 (16.6) 95 % 1.6 3.5–6.7 30.4–33.6
80 aMDRD 95.1 (16.1) 84 % 5.6 11.1–0.1 39.6–28.4
Total 74 83.1 (22.9) CGF 92.6 (20.1) 78 % 9.7 5.5–13.5 24.9–43.9
aMDRD 88 (19.7) 77 % 4.9 0.3–9.5 35.3–45.1
Reproduced with permission from Al-Aloul et al. (2007). Elsevier #2006
aMDRD abbreviated Modification of Diet in Renal Disease formula, CI confidence interval, CGF Cockcroft-Gault formula, eCCl estimated creatinine clearance,
mCCl measured creatinine clearance, N number, SD standard deviation
K.J. Downes and S.L. Goldstein
the drug and elevated serum trough concentrations most closely relate to nephrotox-
icity (Bertino et al. 1993). Both sophisticated and simple methods have been
developed to determine an individual patient’s pharmacokinetic parameters (volume
of distribution, elimination rate constant, total body clearance) based on serum drug
concentrations (Tod et al. 2001). Although there is variability in monitoring practices
between CF centers, TDM is almost universally used.
In theory, AG clearance should be directly related to kidney function since these
drugs are almost exclusively eliminated via glomerular filtration. Unfortunately, the
correlation between GFR and actual drug clearance in CF patients is variable. Some
studies (Town et al. 1996) describe a significant correlation between the measured
creatinine clearance and tobramycin total body clearance (r = 0.52, p = 0.02). Other
more recent studies (Soulsby et al. 2010), however, observed that tobramycin
clearance correlates poorly with measured GFR in both adults (r = 0.1, p = 0.71)
and children (r = 0.25, p = 0.19) with CF. Figure 3 displays Bland-Altman plots
comparing GFR estimated via tobramycin clearance and measured GFR.
Aminoglycoside clearance is more reflective of kidney function than of kidney
injury. Changes in drug clearance over time may suggest that an individual has
sustained injury, but the extent of kidney injury needed to result in clinically
significant alterations in drug clearance has not been described. Theoretically,
longitudinal monitoring of tobramycin clearance could be used to identify chronic
renal insufficiency. However, to our knowledge, no studies have demonstrated
decreased AG clearance as a marker of CKD in patients with CF.
In a recent population pharmacokinetic study, Alghanem observed that AG
clearance appears to be stable over time in patients with CF despite receipt of
multiple courses of therapy over several years (Alghanem et al. 2013). The authors
evaluated 1,075 aminoglycoside courses in 166 patients aged 14–66 years; subjects
received as many as 28 courses over a 15 year period. There were no significant
changes in kidney function (based on eCCl) over time, and only a single patient had
moderate renal impairment (eCCL 45–58 mL/min). Additionally, there was little
change in aminoglycoside clearance from one course to the next (between-occasion
variability = 11 %), and no significant trends were detected in AG clearance over
time based on the number of prior aminoglycoside courses. The authors concluded
that in the population of CF patients they studied, there was no decline in AG
clearance over time.
These findings contrast with data from other studies suggesting an increased
prevalence of kidney dysfunction over time in patients with CF (Quon et al. 2011;
Al-Aloul et al. 2005a). The discrepancy may lie in the variable relationship between
AG clearance and eGFR in CF patients. Creatinine clearance had only a weak
relationship to AG clearance in the Alghanem study which likely reflects the
unreliable nature of SCr-based equations for estimating kidney function in CF
patients. Additionally, Alghanem and colleagues relied on drug levels drawn within
72 h of the start of therapy in the majority (83 %) of cases. It is possible that patients
with mild/moderate underlying renal dysfunction do not demonstrate impaired drug
clearance early in antibiotic courses. The mechanism of AG toxicity is dependent
upon accumulation of drug in proximal tubule cells, and decreased AG clearance
Tobramycin clearance in adults (n=20)

100 +1.96 SD
80 89.8
60
GFR - tob clearance
40
Mean
20 27.1
-20
-1.96 SD
-40 -35.7
-60
60 80 100 120 140
AVERAGE of GFR and tob clearances
Tobramycin clearance in children (n=24)

200
150 +1.96 SD
137.8
GFR - Tob Cl
100
Mean
50 59.6
0
-1.96 SD
-18.6
-50
90 100 110 120 130 140 150 160
AVERAGE of GFR and Tob Cl
Fig. 3 Bland and Altman analysis for differences between tobramycin clearance and measured
GFR in mL/min/1.73 m2 in adults (a) and children (b). The x-axis represents the average GFR and
the y-axis represents the difference between the measured GFR and tobramycin clearance
(Reproduced with permission from Soulsby et al. 2010. #2010 Elsevier)
may not manifest until a sufficient amount/duration of drug has been administered.
Serial AG level measurements would be needed to determine when individuals pass
the cutoff that leads to impaired kidney function, but the frequency of measurement
needed to accurately capture this may be impractical. Ultimately, the inconsistent
relationships between AG clearance and measured and estimated CCl make it
difficult to rely on drug clearance as a marker of kidney injury.
N-Acetyl-b-D-glucosaminidase (NAG)
In the setting of kidney injury, enzymes located within tubular epithelial cells may be
released into the urine. N-Acetyl-β-D-glucosaminidase (NAG) is one such enzyme.
NAG is a proximal tubule lysosomal enzyme and detection in the urine increases in
the setting of a number of causes such as nephrotoxic injury and diabetic nephrop-
athy (Skalova 2005). Although not specific to a particular mechanism of injury, it is a
sensitive marker of renal tubular injury and has been studied in a variety of patient
populations. Typically, NAG is expressed as a ratio with urinary creatinine to
account for biologic variability.
In patients with CF, NAG has been primarily studied in the context of
nephrotoxin receipt, in particular aminoglycosides, and is a highly sensitive and
specific marker of tubular injury in this population (Godson et al. 1988). Urinary
levels of NAG increase significantly during courses of IV gentamicin (Godson
et al. 1988), amikacin (Halacova et al. 2008), and tobramycin (Steinkamp
et al. 1986; Glass et al. 2005; Etherington et al. 2007; Master et al. 2001;
Riethmueller et al. 2009; Smyth et al. 2005). Elevated levels of NAG can even be
observed during courses of inhaled tobramycin (Guy et al. 2010).
Most studies of NAG have confirmed that urinary concentrations increase despite
stable serum creatinine. Glass studied 22 children with normal GFR and stable SCr
receiving IV tobramycin three times daily for 14 days (Glass et al. 2005). NAG
significantly increased following completion of therapy compared to measurements
obtained prior to the start of the drug ( p <0.0001). Steinkamp studied 14 subjects
before, during, and after receipt of 10 mg/kg/day of IV tobramycin with azlocillin
and observed a six- to tenfold increase in urinary NAG during therapy (Steinkamp
et al. 1986). Meanwhile, Etherington measured urinary NAG in 88 patients receiving
IV tobramycin or colistin on days 1, 14, and at first clinic follow-up (Etherington
et al. 2007). Although there were no changes in SCr, a 3.5-fold increase in urinary
NAG occurred between day 1 and 14, and NAG excretion was higher in subjects
receiving tobramycin compared to colistin (day 14 median NAG ratio, 2.24 vs. 0.98,
p <0.001), suggesting an increased risk of tubular toxicity from tobramycin. Addi-
tionally, NAG was higher at each time point of the study for subjects with CFRD.
Studies of urinary NAG in CF patients also provide some evidence that tubular
injury from nephrotoxic antibiotics may be sustained. In the study by Glass, urinary
NAG levels remained higher than pretreatment levels at 4 weeks after the course
( p <0.001) (Glass et al. 2005). Meanwhile, Etherington observed that the majority
(80 %) of patients who received multiple courses of treatment during the study
period had day 1 NAG levels that were significantly higher in subsequent courses
( p < 0.001) (Etherington et al. 2007). And, almost half (46 %) of patients had an
elevated NAG level at their clinic follow-up visit. Although NAG values returned to
normal during follow-up assessments in the study by Steinkamp, the study was small
(N = 14), and follow-up measurements were obtained at 4–120 days after treatment
cessation (Steinkamp et al. 1986).
Urinary NAG has also been used to monitor for nephrotoxicity in several studies
comparing different dosing regimens of aminoglycosides (Master et al. 2001;
Riethmueller et al. 2009; Smyth et al. 2005). Two of these studies showed lower
NAG values with once-daily administration (Master et al. 2001; Smyth et al. 2005),
providing evidence for the improved safety of once-daily dosing. Although NAG
levels were similar between the once- and thrice-daily tobramycin groups in a
randomized trial, significant rises in urinary NAG develop during AG therapy
despite stable serum creatinine (Riethmueller et al. 2009).
Urinary NAG is a sensitive marker of proximal tubule kidney injury receiving
nephrotoxic medications, and several studies in CF patients have observed increasing
and often elevated values over the course of AG therapy without a demonstrable
change in serum creatinine. This corroborates that NAG is a more sensitive marker of
tubular injury than SCr during aminoglycoside courses and that it may have a role in
detection of subclinical kidney injury in patients receiving these medications. How-
ever, longitudinal studies are needed which seek to elucidate the relationship between
episodes of kidney injury detected by changes in NAG and long-term renal outcomes.
Urinary Albumin
The presence of albuminuria may result from glomerular damage due to increased
permeability or may signal proximal tubule dysfunction and decreased reabsorption
of the protein (Vaidya et al. 2008). Albumin is freely filtered by the glomerulus, and
urinary detection can be increased in the setting of non-pathologic conditions which
cause proteinuria, such as dehydration and vigorous exercise. Microalbuminuria,
defined as 30–300 mg/L (Vaidya et al. 2008), is often present in patients with
diabetes including CF patients with CFRD. In a large cohort study of CF children
and adults, CFRD contributed to a sevenfold increased odds (95 % CI: 2.5–20.0, p =
0.0002) of persistent microscopic albuminuria: 10.7 % of patients with CFRD versus
1.6 % of CF patients without CFRD (Lind-Ayres et al. 2011). Transient
microalbuminuria was present in a similar percentage of patients with and without
CFRD in this study, which was comparable to the rate found in the general popu-
lation (6–7 %).
Other factors aside from diabetes may also contribute to the development of
albuminuria in CF patients. Lind-Ayres found that a significantly higher percentage
(40 %) of patients who had undergone lung transplant, all of whom either had CFRD
or glucose intolerance, had persistent microscopic albuminuria. Meanwhile, in a
small cohort study assessing proteinuria via 24-h urine collection, CF genotype was
the only factor associated with the presence of high (>150 mg/day, N = 6/22)
vs. low (<150 mg/day, N = 16/22) proteinuria (Cemlyn-Jones and Gamboa 2009).
The multifactorial nature of albuminuria and its high prevalence in the CF popula-
tion limits the utility of albuminuria as a marker of acute kidney injury. Persistent
detection of microalbuminuria heralds the onset of nephropathy in patients with
CFRD. But, the predictive capability of a single measurement is limited. Whether
albuminuria can be used as a reliable marker of acute kidney injury is yet to be
determined. Additional studies which provide data on serial urine albumin measure-
ments would be needed to determine its potential role for it in AKI detection.
Table 2 Results for correlations between measured GFR and estimated renal function (95 %
confidence interval reported in brackets)
Test R value (adults) R value (children)
Cystatin C 0.64 [0.43–0.78] 0.61 [0.39–0.76]
P = 0.0059 P = 0.0011
Cockcroft and Gault equation (adults) or 0.51 [0.36–0.75] 0.60 [0.34–0.74]
Schwartz (children) P = 0.0252 P = 0.0015
Tobramycin clearance 0.10 [0.83–0.52] 0.25 [0.04–0.50]
P = 0.7117 P = 0.1920
aMDRD equation (adults) or updated 0.29 [0.17–0.65] 0.60 [0.28–0.80]
Schwartz (children) P = 0.2145 P = 0.001
Reproduced with permission from Soulsby et al. (2010), #2010 Elsevier
Cystatin C (CysC)
Cystatin C is a low molecular weight protein that is freely filtered by the glomerulus
and reabsorbed by proximal tubule cells via megalin-assisted endocytosis (Kaseda
et al. 2007). It does not undergo tubular secretion, and its function is to serve as an
extracellular inhibitor of cysteine proteases. Injury to proximal tubule cells leads to
increased excretion in the urine, while elevated plasma levels are reflective of
impaired glomerular filtration. Therefore, it may be a useful marker of proximal
tubule injury as well as a functional marker of impaired GFR. Numerous studies have
examined its role as a serum marker of GFR in a variety of populations, including CF.
Soulsby compared the correlation between measured GFR, using a radioisotope
technique, and eGFR using SCr-based equations, serum CysC levels, and
tobramycin clearance in adults and children with CF (Soulsby et al. 2010). The
results for the correlations are shown in Table 2. CysC had the strongest correlation
with measured GFR for both adults and children. However, CysC had no advantage
over SCr-based equations for detecting the 4/47 patients with impaired kidney
function (measured GFR <90 mL/min/1.73 m2). The sensitivity and specificity of
CysC for detection of decreased GFR was 100 % and 85.7 %, respectively. There
was very poor correlation between tobramycin clearance and measured GFR (R =
0.1 and 0.25 for adults and children, respectively), and the correlation between
tobramycin clearance and CysC was not reported. Given the low prevalence of
renal dysfunction in this population, the authors concluded that CysC offers no
benefit over SCr-based estimates of GFR.
This study contrasts with an earlier study by Beringer which demonstrated
superiority of CysC to SCr-based methods for GFR estimation in both CF patients
and healthy, age-matched controls (Beringer et al. 2009). GFR estimates based on a
CysC-based equation (GFR = 100/CysC – 14 Tidman et al. 2008) provided greater
precision in both the CF and control populations. And, for those with CF, the CysC-
based equation demonstrated a significantly higher AUC for the prediction of
impaired kidney function (measured GFR <90 mL/min/1.73 m2) compared with
the Cockcroft-Gault equation (AUC 0.928 vs. 0.556, p = 0.005) and aMDRD
equation (AUC 0.928 vs. 0.539, p = 0.003).
Serum CysC has also shown utility during AG courses in CF patients. Halacova
measured serum CysC, CysC clearance, creatinine clearance, urinary NAG, and
amikacin clearance in 71 patients receiving intermittent infusion amikacin therapy
for 12 days (Halacova et al. 2008). Serum CysC levels increased throughout
amikacin treatment (P <0.001, Dunnett’s multiple comparisons test) and 80 % of
patients demonstrated CysC levels above the normal range. Consequently, the
estimated GFR using a CysC-based equation demonstrated a significant decline
over the course of amikacin treatment. Conversely, there were no significant changes
in creatinine clearance or serum creatinine from day 0 compared with day
12, although serum creatinine was above the normal range in 45 % (N = 32) of
patients on day 12 of therapy. Figure 4 shows a comparison of the creatinine
clearance and CysC clearance values over the course of amikacin in this study.
The GFR estimated by SCr was significantly higher than that predicted by CysC:
1.76
0.02 versus 1.18
0.04 mL/s/1.73 m2 (105.6
1.2 vs. 70.8
2.4 mL/min/
1.73 m2), p <0.0001. ROC analyses demonstrated that serum CysC and CysC
clearance were better predictors of amikacin clearance than creatinine clearance:
AUC values of the ratios of amikacin clearance to creatinine clearance, CysC, and
CysC clearance on day 12 compared to day 0 were 0.51, 0.92, and 0.84, respectively.
Urinary CysC may also be beneficial for AKI monitoring during
aminoglycosides, which are reabsorbed in the proximal tubule via the same
endocytic receptor, megalin, as CysC. In a rat model, significant changes in urinary
CysC could be detected as early as day 1 of gentamicin therapy (Hoffmann
et al. 2010). However, to our knowledge, this urinary biomarker has not been studied
in patients with CF. It holds promise for the noninvasive detection of AKI during
aminoglycoside courses and warrants investigation.
Beta-2-Microglobulin (b2M)
Beta-2-microglobulin is another low molecular weight protein which, similar to

CysC, is filtered by the glomerulus and reabsorbed in the proximal tubule. It is the
light chain of the major histocompatibility class I molecule and, unlike CysC, is not a
useful serum biomarker due to its expression on the cell surface of all nucleated cells
(Ferguson et al. 2008). Its role as a kidney injury biomarker is limited to detection in
the urine which increases in the setting of impaired proximal tubule function. In a rat
model of nephrotoxicity (Sasaki et al. 2011), β2M increased on day 1 of gentamicin,
and the rate of increase was higher than other urinary biomarkers (CysC, NGAL,
NAG).
Data on the role of β2M in patients with CF are limited. In a prospective case-
control study evaluating the role of serum β2M as a marker of lung inflammation,
Kearns et al. measured β2M in CF outpatients (N = 12), CF patients receiving
antibiotics for a pulmonary exacerbation (N = 6), and healthy controls (N = 10)
(Kearns et al. 1989). Serum β2M values were significantly lower in healthy controls
than both CF groups ( p <0.05), but there were no differences in urinary values of
β2M between healthy controls and patients with CF. Meanwhile, in a randomized
3·0 ns
Creatinine clearance (m/s/1·73 m2)
2·5 P < 0·01 P < 0·001
1·97 ± 0·06 1·88 ± 0·05 1·89 ± 0·06

2·0
1·65 ± 0·05 1·62 ± 0·05 1·54 ± 0·06
1·5
1·0
0·5
0·0
A B C D E F
Days
P < 0·001
P < 0·05
1·64 ± 0·08
Cystatin C clearance Grubb
2·0
1·54 + 0·09
(ml/s/1.73m2)
1·5
1·16 + 0·07 1·04 + 0·08
0·90 + 0·08
1·0 0·77 + 0·07
0·5
0·0
A B C D E F
Days
Fig. 4 Comparison of creatinine clearance (a) and cystatin C clearance (b) during amikacin
therapy in CF patients. Data are expressed as column bars (mean
SEM). A: Day before start of
amikacin treatment (day 0). B–F 3rd, 5th, 7th, 10th, and 12th day of amikacin treatment
(Reproduced with permission from Halacova et al. 2008. #2008 Blackwell Publishing Ltd)
controlled trial of once- versus thrice-daily IV tobramycin, combined with

ceftazidime, urinary β2M was higher in the thrice-daily group (0.87
0.5 mg/L
vs. 0.18
0.2 mg/L, P <0.01), although values were in the normal range for both
groups (Vic et al. 1998). The study population was small (N = 22) but SCr was
unchanged over the course of therapy in both groups. Unfortunately, this data
combined with the fact that β2M is unstable in acidic urine make it an impractical
kidney injury biomarker in the clinical setting.
Retinol-Binding Protein (RBP)
Retinol-binding protein (RBP) is a hepatically synthesized chaperone for vitamin A

transport to tissues. It is freely filtered by the glomerulus and reabsorbed in the
proximal tubule via megalin (Christensen et al. 1999). RBP is a sensitive marker of
tubule dysfunction and can be detected in the urine soon after nephrotoxin exposure
(Ferguson et al. 2008). This biomarker has been studied preliminarily in CF patients.
Glass measured urinary RBP and NAG in 22 children with CF receiving a 14-day
course of IV tobramycin (Glass et al. 2005). Measurements were performed imme-
diately prior to and following therapy, as well as 4 weeks following completion of
therapy. Urinary NAG (P <0.001) and RBP (P = 0.03) both increased over the
course of therapy. Unlike NAG, however, urinary RBP returned to pretreatment
levels at 4-week follow-up. Interestingly, pretreatment levels of RBP were elevated
in 18 of 21 subjects with prior AG receipt vs. zero for NAG. However, there were no
differences in RBP following therapy based on the number of prior aminoglycoside
courses.
Given that RBP is a chaperone for vitamin A transport, serum levels may be
affected by nutritional status or the presence of liver disease in CF patients. In fact,
patients with CF have lower plasma RBP levels than age- and sex-matched controls
(Mrugacz et al. 2005) which could be a result of underlying pancreatic insufficiency
associated with this disease. In this context, the findings by Glass that urinary RBP
both: (a) increased during therapy and (b) was higher at baseline among subjects
with prior AG receipt are particularly interesting. These data suggest that RBP may
be a highly sensitive marker for both acute and chronic tubular dysfunction in the CF
population. Additional studies assessing the relationship between aminoglycoside
receipt and RBP levels should be performed, and the time course of elevation of RBP
during aminoglycosides needs to be elucidated.
Neutrophil Gelatinase-Associated Lipocalin (NGAL)
Neutrophil gelatinase-associated lipocalin (NGAL) is a protein involved in iron

homeostasis whose expression is upregulated in the setting of ischemia or infection.
In humans, increased urinary levels can be detected following a variety of kidney
insults and becomes elevated prior to SCr-based AKI develops (Gaspari et al. 2010;
Hirsch et al. 2007). Increased levels have been associated with poor clinical out-
comes irrespective of SCr changes (Haase et al. 2011; Singer et al. 2011). Urinary
NGAL concentrations may reflect dysfunction in glomerular filtration, impaired
proximal tubule reabsorption, and increased production by distal nephrons,
depending on the mechanism of injury (Kuwabara et al. 2009). Similar to RBP
and cystatin C, urinary excretion is increased in the setting of nephrotoxic proximal

tubule injury (Kuwabara et al. 2009).
The role of NGAL as a marker of kidney injury in CF has not been defined. In a
study evaluating serum NGAL (Zughaier et al. 2013), values were higher in patients
with CF compared to healthy controls (P <0.001) yet similar when compared among
CF patients with stable disease vs. those experiencing a pulmonary exacerbation.
Meanwhile, when peripheral monocytes of both CF and healthy patients were
infected with Pseudomonas aeruginosa, NGAL secretion increased. This raises the
possibility that serum NGAL varies in response to lung infection and whether this
influences its utility as a kidney injury urinary biomarker has been debated (Nazareth
and Walshaw 2013). Nevertheless, the relationship between serum and urinary
concentrations in the setting of proximal tubule injury, as would result from
aminoglycoside exposure, has not been explored in CF patients. Animal studies
show a time-dependent increase in urinary NGAL following administration of
gentamicin (Zhou et al. 2014) suggesting that it may be a useful marker to determine
the optimal duration of aminoglycoside therapy in CF patients. Additionally, the
trajectory of NGAL values over the course of therapy may be a good indicator of
drug accumulation given the saturability of megalin-facilitated endocytosis.
Kidney Injury Molecule-1 (KIM-1)
Kidney injury molecule-1 (KIM-1) is a transmembrane protein located predomi-

nantly (90 %) in the proximal tubule (Zhou et al. 2008; Chiusolo et al. 2010). Its
gene expression, and subsequent protein detection in the urine, is increased in the
setting of both ischemia and toxin administration, specifically gentamicin (Zhou
et al. 2008; Chen et al. 2010). Changes in KIM-1 precede rises in SCr following
ischemic and toxic insults (Tu et al. 2014; Torregrosa et al. 2015), making it a useful
biomarker of AKI development and severity.
As with many biomarkers of kidney injury, KIM-1 has not been extensively
studied in the CF population. Urinary KIM-1, NAG, and protein were studied in
52 children and young adults to determine the impact of high-dose ibuprofen use on
kidney biomarkers (Lahiri et al. 2014). Half of subjects were on high-dose ibuprofen
therapy, and there were no differences in mean biomarker concentrations between
the groups: KIM-1 = 0.306
0.28 ng/mg creatinine in subjects on ibuprofen
vs. 0.381
0.28 ng/mg creatinine in subjects not receiving the medication (P =
0.34). The authors did observe a correlation between KIM-1 values and lifetime AG
exposure (r = 0.35, P = 0.012), although other factors which could influence this
association (age, recent AG exposure, receipt of oral antibiotics, etc.) were not
explored. Of note, this was a small study conducted in relatively healthy CF patients
for whom urinary biomarkers were measured at only a single point in time. There-
fore, results may not be reflective of the long-term effects of ibuprofen on the kidney.
Longitudinal studies are needed in patients with CF to define its role in detection
of AKI.
Other Urinary Biomarkers
There are a number of other novel AKI biomarkers that have been studied in non-CF
populations. Interleukin-18 (IL-18), liver-type fatty acid-binding protein (L-FABP),
and clusterin, for example, may have a role in AKI detection or prognostication. But
studies are needed in patients with CF to determine their utility in this population.
Biomarkers and the Future of Kidney Injury in Cystic Fibrosis
Kidney injury is becoming an increasingly recognized issue in patients with CF. The
untoward effects of repeated antibiotic courses, CF-related diabetes, and other insults
on the kidney lead to an increased risk of development of chronic kidney disease in
this population (Al-Aloul et al. 2005a; Wehbe et al. 2012; Quon et al. 2011). As
lifetime survival in patients with CF increases, the long-term ramifications of this
disease and the associated treatments on renal health are becoming more evident. It
becomes paramount, therefore, to identify and employ strategies which seek to
characterize those at highest risk for AKI, improve detection of kidney injury, and
mitigate its long-term risks.
While diabetes may provide the highest threat to long-term kidney function, the
major risk factor for development of AKI in CF patients is the receipt of nephrotoxic
antibiotics such as aminoglycosides. There are no well-recognized methods for
decreasing the toxic effects of these antibiotics, aside from early discontinuation of
therapy. Unfortunately, avoidance of these medications is not a feasible or advisable
approach considering the impact of recurrent and chronic lung infections in this
population. While there is general agreement that monitoring of aminoglycoside
drug levels and renal function is necessary, there is a lack of consistency regarding
the optimal monitoring strategy to both identify and prevent AKI. Traditional
monitoring for kidney injury via measurement of serum creatinine is suboptimal.
Urinary biomarkers are highly sensitive for kidney injury. In general, however,
they have not been extensively studied in patients with CF, and their role in
monitoring for kidney injury in these patients has not been defined. There are a
number of avenues for future research in this field. In theory, biomarkers could be
used to risk stratify CF patients prior to start of nephrotoxic therapy and indicate
specific patients in whom alternative antibiotics should be considered. An improved
correlation between biomarkers and aminoglycoside pharmacokinetic parameters
(AUC, clearance) may allow for noninvasive and rapid monitoring of renal drug
handling. Biomarkers may detect AKI earlier than with SCr and promote implemen-
tation of nephro-protective strategies in patients receiving nephrotoxins. Alterna-
tively, they could be used to monitor the impact of interventions which seek to
reduce kidney injury.
The majority of studies conducted in CF patients have focused on determining
which biomarker is most reflective of kidney function in this population. While
important, the overall utility of these novel biomarkers lies in their ability to improve
the safety of nephrotoxic medications, promote earlier detection of AKI, and identify
patients at higher risk for poor short- and long-term outcomes. Ultimately,
biomarker-driven trials will be needed prior to their implementation in the clinical
setting.

Conditions
A number of urinary and serum biomarkers have been identified which are highly
sensitive for the detection of kidney injury. These biomarkers increase in the serum
or urine following a variety of insults such as nephrotoxin receipt, ischemia, and
sepsis. Although these biomarkers have not been extensively studied in the CF
population, they show promise for improving the earlier detection of AKI compared
to traditional biomarkers in a variety of clinical scenarios. Urinary biomarkers also
have prognostic implications and have been linked to poor outcomes irrespective of
serum creatinine. These biomarkers could be used clinically to identify patients at
highest risk for morbidity and mortality. Urinary and serum biomarkers are applica-
ble in a variety of settings where patients are at risk for kidney injury: critical illness,
patients receiving nephrotoxins, cardiac bypass, and other. Since the mechanism and
severity of kidney injury relates to release/expression/excretion of these biomarkers,
they should have utility in multiple patient populations at risk for or sustaining
kidney injury.
Summary Points
• Cystic fibrosis (CF) is a genetic disease that predisposes patients to recurrent and
chronic respiratory tract infections.
• Patients with CF are at risk for development of acute kidney injury (AKI) due to
receipt of nephrotoxic medications, particularly antibiotics, as well as long-term
renal damage from long-standing diabetes and repeated nephrotoxin exposures.
• With advances in medical therapies leading to substantial improvement in life-
time survival among CF patients, the long-term ramifications of the disease and
its treatments on the kidney may become apparent.
• Chronic kidney disease is prevalent in patients with CF, and means to detect and
mitigate kidney insults are important to stem long-term deleterious effects.
• The traditional marker of kidney injury and function, serum creatinine, is
unreliable in patients with CF as it often overestimates true kidney function and
does not detect kidney injury until a significant number of nephrons have been
affected.
• Biomarkers, such as cystatin C (CysC), retinol-binding protein (RBP), kidney
injury molecule-1 (KIM-1), neutrophil gelatinase-associated lipocalin (NGAL),
and others, hold significant promise for early detection of AKI, risk stratification,
and prognosis.
• Additional research is urgently needed to explore and define the potential roles of
these novel biomarkers in detection of both acute kidney injury and chronic
kidney disease in the CF population.
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Biomarkers in IgA Nephropathy
32
Contents
Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 721
Key Facts of IgA Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 721
Key Facts of Uromodulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 722
Key Facts of Immunosuppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 722
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 722
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 723
Diagnostic Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 724
Urine Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 724
Serum Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 728
Prognostic Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 729
Urine Kidney Injury Molecule-1 (KIM-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 730
Fractional Excretion of IgG (FE IgG) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 731
Soluble CD89 (sCD89) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 731
Urinary Angiotensinogen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 732
Complement Components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 732
Inflammatory Cytokines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 733
Markers of Oxidative Stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 734
Biomarker Correlation with Histologic Findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 735
Podocalyxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736
TGF-β and IL-6 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736
Thioredoxin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737
Soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737
Urine Secretory IgA (sIgA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737
Proteomic Analysis of Urine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 737
Biomarkers of Response to Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 738
M. Nafar • S. Samavat (*)

Department of Nephrology, Labbafinejad Medical Center, Shahid Beheshti University of Medical
Sciences, Tehran, Iran
Chronic Kidney Disease Research Center (CKDRC), Shahid Beheshti University of Medical
e-mail: nafar@sbmu.ac.ir; m.nafar.md@gmail.com; shsamavat@gmail.com;
sh_samavat@yahoo.com

Biomarker Response to Blockade of the Renin–Angiotensin System . . . . . . . . . . . . . . . . . . . . . 739

Biomarker Response to Fish Oil . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 740
Biomarker Response to Tonsillectomy
Steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 741
Biomarker Response to Immunosuppressive Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 742
Potential Applications to Prognosis, Other Diseases, or Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . 743
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 743
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 744
Abstract
IgA nephropathy (IgAN) is the most common glomerulonephritis worldwide,
diagnosis of which is dependent on kidney biopsy with its invasive nature.
Besides diagnosis, histologic lesions are predictors of prognosis and response
to therapy. In addition some clinical and laboratory factors predict the risk of
progression to end-stage renal disease, such as hypertension, baseline serum
creatinine, and time-averaged proteinuria. Biomarkers are measurable sub-
stances that are indicators of specific physiologic or pathologic process. Serum
and urine are easily accessible and perfect biofluids. This review is focused on
recently discovered biomarkers with diagnostic, prognostic, histologic, and
response prediction capacities. Yet none of the described biomarkers could be
used instead of biopsy. A long road is ahead to reach the optimal biomarker
profile with bedside utility.
Keywords
IgA nephropathy • Diagnostic biomarkers • Prognostic biomarkers • Response to
treatment • Proteomics • Metabolomics
Abbreviations
2D PAGE Two-dimensional polyacrylamide gel electrophoresis
2DE Two-dimensional gel electrophoresis
AOPPs Advanced oxidation protein products
ARB Angiotensin receptor blocker
AUC Area under curve
BAFFs B-cell-activating factors
BMP-1 Bone morphogenetic protein-1
EGF Epidermal growth factor
FE IgG Fractional excretion of IgG
FOXP3 Forkhead box P3
Gd-IgA1 Galactose-deficient IgA1
GS Glomerular sclerosis
IF/TA Interstitial fibrosis and tubular atrophy
32 Biomarkers in IgA Nephropathy 721

IL Interleukin
IL-2R IL-2 receptor
INF-γ Interferon-γ
LC-MS/MS Liquid chromatography/tandem mass spectrometry
LG3 Laminin G-like 3
MAC Membrane attack complex
MALDI-TOF-MS Matrix-assisted laser desorption/ionization time-
of-flight mass spectrometry
MBL Mannose-binding lectin
MCP-1 Monocyte chemoattractant protein-1
miRNAs MicroRNAs
mRNA Messenger RNA
MVBs Multivesicular bodies
Nano-HPLC-ESI-MS/MS Nano-high-performance liquid chromatography–e-
lectrospray ionization–tandem mass spectrometry
NFκB Nuclear factor κB
RANTES Regulated upon activation normal T cell expressed
and secreted
SELDI Surface-enhanced laser desorption/ionization
sIgA Secretory IgA
STAT-3 Signal transducer and activator of transcription 3
sTfR Soluble transferrin receptor
sVCAM-1 Soluble vascular cell adhesion molecule-1
TAL Thick ascending limb
TBMN Thin basement membrane nephropathy
TECs Tubular epithelial cells
TGF-β Transforming growth factor-β
TLR-9 Toll-like receptor-9
TMAO Trimethylamine N-oxide
TNF-α Tumor necrosis factor-α
Treg Regulatory T cell
Key Facts
Key Facts of IgA Nephropathy
• IgA nephropathy (IgAN) is glomerulopathy diagnosed by dominant or codom-

inant deposition of IgA antibodies in mesangial areas.
• Jean Berger first described IgAN in 1968.

• The fundamental step is aberrantly galactosylated IgA1 (Gd-IgA1).
• Antibody formation against Gd-IgA1 leads to immune complex formation and
mesangial deposition of these complexes.
• Genetic factors are involved in the pathogenesis of IgAN.
• The mesangial deposition results in macrophage infiltration and tubulointerstitial
inflammation, podocyte activation, and further tubular damage.
• The choice of treatment is dictated by the risk of progression. The higher is the
risk of progression, the more aggressive is the immunosuppression.
Key Facts of Uromodulin
• Uromodulin precursor contains 640 amino acids.

• The precursor is processed and becomes mature in thick ascending limb (TAL)
of Henle’s loop and polymerized upon secretion.
• Uromodulin regulates water and electrolyte transport in TAL. It increases the
expression of apical potassium channel.
• It has a role in prevention of urinary tract infection.
• It plays a role in innate immunity and acts as a chemoattractant.
• Mutations in uromodulin gene lead to decreased urinary level of uromodulin and
result in interstitial inflammation and fibrosis.
Key Facts of Immunosuppression
• Treatment approach in IgAN depends on the risk of progression.

• The risk of progression is estimated based on the amount of proteinuria, renal
function, and the presence of hypertension on presentation.
• Those at intermediate or high risk of progression are suitable for immunosup-
pressive therapy.
• Those with proliferative disease in the kidney biopsy might have better response
to immunosuppressive drugs.
• Corticosteroids, mycophenolate mofetil (MMF), cyclophosphamide, and azathi-
oprine are all studied in IgAN.
Definitions
Immunonephelometric assay Immunonephelometric assay is the technique to

measure plasma protein level based on the amount of turbidity and scattering of
light caused by small particles in the sample.
ELISA The enzyme-linked immunosorbent assay is based on antigen and anti-

body interaction and enzyme-induced color changes in substrate. Antigens are
attached into wells in a plate. Then an antibody that can bind to the antigen and
is linked to an enzyme is added. The next step is the addition of substrate. The
reaction causes color change in the substrate, and the intensity of the color signal is
indicative of the amount of antigen present.
MicroRNAs MicroRNAs (miRNAs) are 21–23-nucleotide noncoding RNAs that

regulate posttranscriptional gene expression by binding to target mRNA leading to
either the degradation of mRNA or inhibition of their transcription.
Metabolomics Metabolomics comprehensively identify the metabolites in a

biofluid and help to understand the cellular pathways and therefore the pathogenesis
of diseases. Metabolome is a collection of metabolites a physiologic process had
left behind. Each tissue has its specific metabolome, but analyzing urine or plasma
metabolomes results in more general data, which is not tissue specific.
Proteomics Proteomics is the analysis of the whole protein content of a biofluid.

The changes in the proteomes are caused by differences in synthesis or modifica-
tions during the course of biologic or pathologic processes. These modifications can
be used as specific markers of the process.
Oxidative stress Oxidative stress is an imbalance between pro-oxidant processes

and antioxidant defense mechanisms.
Introduction
IgA nephropathy (IgAN) is known as the most common glomerulonephritis in the

world. It is characterized by predominant or codominant mesangial deposition of
IgA. Light microscopic findings vary from near-normal to crescentic glomerulone-
phritis. The spectrum of clinical findings is as wide as histologic ones: from
asymptomatic hematuria to rapidly progressive glomerulonephritis. This made
IgAN a diagnosis based on pathology.
The exact pathogenesis of IgAN is not still completely clear, but abnormally
glycosylated polymeric IgA1 (Gd-IgA1) and autoantibodies against it are present
in the mesangial deposits and the circulation. The origin of this aberrantly
glycosylated IgA1 might be the production of mucosal IgA in the bone marrow
(mis-homing). Microbial pathogens activate B cells by binding to TLR-9 and
induce class switching and antibody production. The immune complexes containing
Gd-IgA1 then bind to transferrin receptor (CD71) on mesangial cells and lead to in
situ cytokine production and complement activation and renal injury.
Once known as a benign glomerulonephritis, IgAN is now one of the causes of
end-stage renal disease (ESRD). Approximately 20–40 % of IgAN patients will
reach ESRD within 20 years after diagnosis. Thus, timely and noninvasive diagno-
sis, better understanding of pathogenesis and predictors of clinical course, and
response to therapy may help to change patient’s destiny (Boyd et al. 2012;
Wyatt and Julian 2013; Kim et al. 2012).
Diagnostic Biomarkers
The diagnosis of IgAN is based on kidney biopsy, an invasive and inconvenient

procedure with minor risk of bleeding, need for transfusion, and even nephrectomy.
In order to develop a noninvasive way, various serum and urine biomarkers were
assessed.
Urine Biomarkers
Urine is an accessible and noninvasive biofluid, and its composition reflects the
pathogenetic changes in the kidney. Besides filtered proteins, urine contains various
cytokines and “fingerprints” of immunologic processes and complements system
activation. Although there is no single well-documented urinary marker with
approved clinical use, several candidate markers have been studied.
Uromodulin
First identified in the early 1950s, uromodulin is a glycoprotein that is exclusively
found in the thick ascending limb (TAL) of Henle’s loop. It is synthesized in the
form of 640 amino acid precursor. Uromodulin maturation occurs in the endoplas-
mic reticulum by glycosylphosphatidylinositol anchoring and N-glycosylation.
After modification in Golgi, it undergoes some proteolytic modifications, which
makes it ready for polymerization and urinary excretion. It has different biologic
functions, including protection against urinary tract infection, prevention of renal
stone formation, and is involved in innate immunity. It binds to IgG, C1q, and
TNF-α and, on the other hand, has the capability to activate monocytes and
neutrophils (Rampoldi et al. 2011).
In an effort to identify a diagnostic biomarker for IgAN, proteomic techniques
based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF-MS) were utilized on urine samples of 32 IgAN patients, and a
uromodulin fragment, urinary peptide with m/z 1913.14, was identified. The decreased
intensity of this peptide was reported to be diagnostic. It was also demonstrated that
urine peptide pattern can discriminate IgAN and healthy controls with 100 % sensi-
tivity and specificity. The reported sensitivity and specificity for this peptide pattern to
distinguish IgAN from other glomerulopathies were 85.7 % and 76.5 %, respectively
(Wu et al. 2010). Following this study, Obara et al. (2012) evaluated urinary
IgA–uromodulin complex as a diagnostic marker for IgAN. The ELISA for
IgA–uromodulin complex was applied on urine samples of IgAN, disease controls,
and healthy controls. They calculated the cutoff of 0.705 from the ROC curve, which
was able to distinguish healthy controls from those with kidney disease (including
IgAN) with 90.5 % sensitivity, 90.0 % specificity, and 90.4 % diagnosis efficiency.
With the cutoff of 2.45, IgA–uromodulin complex discriminated IgAN from other
kidney diseases (sensitivity of 92.9 % and specificity of 57.4 %). The results indicated
that IgA–uromodulin complex could differentiate active but not inactive form of IgAN
from other renal diseases with hematuria or proteinuria except lupus nephritis. These
data suggested IgA–uromodulin complex as a marker of active IgAN.
Soluble Transferrin Receptor (sTfR) or CD71

Aberrantly glycosylated IgA1 immune complexes interact with cell surface IgA1
receptor, TfR or CD71. An increased mesangial expression of CD71 was reported
in IgAN. Binding of IgA immune complex leads to mesangial proliferation,
proinflammatory cytokine production (IL-1, IL-6, and TNF-α), and complement
activation (Monteiro 2005).
sTfR (molecular mass 84,910 Da) lacks the first 100 amino acids of TfR and is
upregulated in IgAN, which is proposed to be eliminated in urine. In order to assess
the urinary sTfR as a diagnostic marker in IgAN, fixed-time immunonephelometry
assay was utilized on urine samples of 71 patients with biopsy-proven IgAN.
Urinary sTfR/creatinine ratio was significantly higher in patients with active
IgAN in comparison to those with inactive disease. Although urinary sTfR was
also detected in other morphological types of glomerulopathy, the level was much
lower (>5 μg/L vs. 2 μg/L) (Delanghe et al. 2013). Thus according to this study,
urinary sTfR could be used as a diagnostic marker for active IgAN.
Interleukin 6 (IL-6)
Cytokines play a main role in clinical and pathological manifestations of IgAN.
Produced by activated monocytes and mesangial cells, IL-6 leads to mesangial cell
proliferation and has a role in fibrogenic process. On the other hand, IL-6 is a
plasma cell differentiating factor and by binding to its gp130 receptor and activa-
tion of STAT-3 pathway stimulates Gd-IgA1 production (Reily et al. 2014). Thus,
urinary IL-6 (uIL-6) can serve as a potential diagnostic biomarker in IgAN.
Elevated levels of uIL-6 were shown in IgAN patients compared with healthy
controls at the time of biopsy (Stangou et al. 2009). Of course, in an earlier study,
this upregulated uIL-6 also has been shown in IgAN patients but it was not disease
specific (Hrvacevic et al. 1996).
Furthermore, uIL-6 and its ratio to epidermal growth factor (EGF) were reported
to be related to disease progression in IgAN (Harada et al. 2002; Ranieri
et al. 1996). Recently, in a study in children with asymptomatic hematuria, authors
demonstrated higher uIL-6 activity with IgAN. Despite sample size limitations,
they suggested uIL-6 as a screening tool in children with hematuria and as a guide
to perform kidney biopsy (Kanemoto et al. 2014).
Complement Components
Histologic evaluations of IgAN reveal the footprint of complement activation.
There are evidences of C3, properdin, and mannose-binding lectin (MBL) within
mesangial deposits, which show alternative and lectin pathway activation. There-
fore, complement proteins might be markers of diagnosis and prognosis. In a
study, urinary level of properdin and membrane attack complex (MAC) was
significantly higher in IgAN patients than healthy controls, but due to lack of
disease controls, this could not be supposed as disease-specific marker. Moreover,

there was an incremental pattern in urine MAC and factor H with increasing
disease activity, which makes them prognostic rather than diagnostic markers
(Onda et al. 2011). In a study on urine samples of 162 IgAN patients, urine MBL
levels were significantly higher than the healthy controls (Liu et al. 2012). These
studies point to the fact that aside from the pathogenetic role of complement system,
the markers of complement system activity could guide diagnosis and follow-up of
disease activity.
Laminin G-Like 3 (LG3)

Laminin G-like 3 (LG3) is a product of perlecan cleavage. Perlecan is a proteo-
glycan and major component of extracellular matrix and basement membranes.
Proteoglycans play a main role in charge selectivity of filtration barrier. Data
suggested that perlecan gene expression is negatively correlated with albuminuria
(Ebefors et al. 2011). Cleavage of perlecan by bone morphogenetic protein-1
(BMP-1)/Tolloid-like proteases results in LG3 fragment formation which has
anti-angiogenic activity. Urinary excretion of LG3 fragment has also been reported
to be significantly reduced in IgAN patients. Evaluation of urine proteome pattern
by MALDI-TOF-MS/MS and Western blotting showed lower excretion of LG3 in
IgAN patients in comparison to healthy controls and patients with chronic kidney
disease (Rocchetti et al. 2013). Moreover, two studies suggested LG3 urinary level as
a possible marker of severity both clinical and histological. They reported an inverse
correlation between renal function and urine LG3 (Rocchetti et al. 2013; Surin
et al. 2013).
Exosomes and Vesicles

Multivesicular bodies (MVBs) contain cellular and cytoplasmic compartments and
exosomes. MVBs fuse with the membrane, and exosomes are secreted to the
urinary space. Therefore, exosomes are reflection of biological processes in the
kidney and are not affected by filtered proteins or uromodulin (Santucci
et al. 2013).
Moon et al. (2011) reported 4 exosomal proteins out of 1,877 urinary exosome
proteins, analyzed by LC-MS/MS, to be of diagnostic value in early IgAN. These
proteins were aminopeptidase N, vasorin precursor, α-1-antitrypsin, and cerulo-
plasmin and were able to differentiate IgAN from thin basement membrane
nephropathy (TBMN) and healthy controls. Urinary excretion of aminopeptidase
N and vasorin precursor was higher in TMBN patients and controls than in those
with IgAN. On the contrary, the amount of α-1-antitrypsin and ceruloplasmin was
higher in the IgAN group than in normal group and TBMN. Increased amount of
α-1-antitrypsin might be due to hematuria. Ceruloplasmin level probably
increased due to inflammation as an acute phase reactant. Assessing the area
under curve of these markers, ceruloplasmin was the best marker to discriminate
IgAN from TBMN.
MicroRNAs
MicroRNAs (miRNAs) are 21–23-nucleotide noncoding RNAs that regulate post-
transcriptional gene expression by binding to target mRNA leading to either the
degradation of mRNA or inhibition of their transcription. Recent data suggested
that changes in parenchymal, serum, or urine miRNAs be responsible in pathogen-
esis of diseases including IgAN. miRNAs in biofluids such as urine and serum could
be used for better understanding of pathogenesis and diagnosis and predicting the
clinical course of IgAN (Szeto and Li 2014).
In urine miRNAs can be detected by conventional real-time quantitative PCR.
Increased urinary expression of miRNA-146a and miRNA-155 has shown in IgAN
patients compared with healthy subjects. These miRNAs have immunoregulatory
capacity. miR-146a level was inversely related to proinflammatory cytokine (IL-1β,
IL-6, and TNF-α) levels and directly related to RANTES. Urinary level of miR-155
was also related with suppression of IL-1β and TNF. miR-155 was positively
correlated with FOXP3, and this finding suggested a role for Tregs in development
of IgAN (Wang et al. 2011). In another study the same group demonstrated a
significantly lower urinary miR-29b and miR-29c, but higher miR-93 levels in
IgAN patients than controls. This miRNA profile is mostly related to TGF-β
signaling pathway, renal fibrosis, and glomerular scarring (Wang et al. 2012).
Comparing IgAN with diabetic glomerulosclerosis and hypertensive
nephrosclerosis, upregulated miR-17 in IgAN could be a potential marker for
diagnosis (Szeto et al. 2012). Large population studies and better technical
approaches are needed for assessment of this miRNA function and introduction
of diagnostic as well as prognostic markers of disease.
Urine Metabolomics
Metabolomics comprehensively identify the metabolites in a biofluid and help to
understand the cellular pathways and therefore the pathogenesis of diseases. These
metabolites could also be used as diagnostic and prognostic biomarkers (Zhao and
Lin 2014).
Nuclear magnetic resonance (NMR) spectroscopy is one of the proper tech-
niques to study these metabolic fingerprints. Compared with healthy subjects, urine
of IgAN had elevated levels of creatinine, trimethylamine N-oxide (TMAO),
acetate, and betaine and lower levels of hippurate, citrate, and lactate. Del Coco
et al. showed a connection between betaine and TNF-α and suggested increased
clearance of betaine and therefore lower plasma betaine as a pathogenic event that
led to increased TNF-α levels (Del Coco et al. 2012).
Urinary Proteome
In 2005, Park et al. reported a urinary protein map of IgAN by the means of 2D gel
electrophoresis (2DE) for the first time. They described under-excretion of 35 pro-
teins in IgAN in comparison to healthy controls and suggested that they might be
involved in the pathogenesis of the disorder (Park et al. 2006). Furthermore,
Table 1 Differentially expressed urine proteins in IgAN and healthy subjects

Protein Fold
ID Protein name Biological process change
GP2 Pancreatic secretory Antigen transcytosis by M cells in mucosa- 3.7
granule membrane major associated lymphoid tissue
glycoprotein
VASN Vasorin Inhibitor of TGF-β signaling 3.7
EGF Epidermal growth factor Innate immune response/positive 3.1
regulation of cell proliferation
CLM9 CMRF35-like molecule 9 Immunity 2.9
PCDH1 Protocadherin-1 Cell–cell signaling +8.9
UTER Uteroglobin Regulation of inflammatory response +3.5
DPP4 Dipeptidyl peptidase-4 Positive regulation of cell proliferation/T- +3.5
cell activation
SLAF5 SLAM family member Blood coagulation/leukocyte migration +2.7
5/CD84
NHLC3 NHL repeat-containing It may be involved in a variety of enzymatic +1.9
protein 3 processes, including protein modification
through ubiquitination
Differentially expressed proteins in urine samples of 13 patients with IgAN in comparison to eight
healthy subjects. The biologic processes in which these proteins are involved and the pattern of
changes are demonstrated
utilizing 2DE, absence of increased alpha-1-microglobulin in urine samples of

IgAN patients is reported as a marker of disease (Yokota et al. 2007).
Recently, in an attempt to identify a diagnostic urinary proteome profile, urine
samples of IgAN patients were analyzed by the means of liquid chromatography/
tandem mass spectrometry (nLC-MS/MS). Differentially expressed proteins were
evaluated, and a diagnostic panel was suggested, which was decreased urinary
expression of GP2 (pancreatic secretory granule membrane major glycoprotein),
vasorin, EGF, and CLM9 (CMRF35-like molecule 9) and increased urinary expres-
sion of protocadherin, uteroglobin, DDPIV (dipeptidyl peptidase-4), NHLC3 (NHL
repeat-containing protein 3), and SLAF5 (SLAM family member 5/CD84)
(Samavat et al. 2014). These proteins and their proposed biologic role are demon-
strated in Table 1.
Serum Biomarkers
Galactose-Deficient IgA1 (Gd-IgA1) and Gd-IgA1-Containing Immune

Complexes
Aberrantly glycosylated IgA1 is the key point in the pathogenesis of IgAN. The
Gd-IgA1 acts as a new antigen and stimulates autoantibody production and immune
complex formation, mostly Gd-IgA1-specific IgG and IgA. In a study on large
cohort of IgAN, CKD controls, and healthy volunteers, the prevalence of elevated
serum levels of IgA, Gd-IgA1, and glycan-specific IgG and IgA was evaluated by
the means of ELISA. Among these markers, Gd-IgA1-specific IgG had the best
performance in diagnosis of IgAN with 89 % sensitivity and 92 % specificity. The
marker discriminated IgAN from healthy controls and those with nonimmune
kidney diseases (Yanagawa et al. 2014).
Although the elevated serum levels of galactose-deficient IgA1 alone are insuf-
ficient for diagnosis, it could be used as a predicting factor for progression of IgAN
(Zhao et al. 2012).
Serum BAFF
B cells play a central role in the pathogenesis of IgAN. Pathogen-associated
molecular patterns bind to TLR-9 and activate B cells and induce class switching
and mucosal IgA production (Boyd et al. 2012). B-cell-activating factor (BAFF) is
a cytokine that belongs to the tumor necrosis factor (TNF) ligand family and
participates in class switching (Mecklenbrauker et al. 2004).
The role of BAFF and TLR-9 was evaluated in IgAN patients, and serum levels
of BAFF and TLR-9 mRNA were elevated in IgAN patients compared with healthy
controls and patients with mild glomerular abnormalities. Serum IgA1 level, IgA
deposition in mesangium, and TLR-9 protein expression were positively correlated
with serum BAFF levels (Li et al. 2014). In another study, Xin et al. also reported a
twofold elevation in serum BAFF level in IgAN compared with the healthy controls
and demonstrated a correlation between serum BAFF and renal function tests
(eGFR). Elevated serum BAFF level (>1.47 ng/ml) was mostly correlated with
mesangial hypercellularity, segmental glomerulosclerosis, and interstitial fibrosis
and tubular atrophy (IF/TA) (Xi et al. 2013).
Metabolomics Studies
Nuclear magnetic resonance (NMR) spectroscopy is an analytic technique to study
the metabolome. In a study, serum samples of 35 IgAN patients and 23 healthy
controls were evaluated by NMR, and multivariate pattern recognition analysis was
performed. Compared with healthy controls, higher levels of lactate, myo-inositol,
phenylalanine, and L6, L5, and L3 lipids were detected in serum of IgAN patients.
Lower serum levels of β-glucose, α-glucose, valine, tyrosine, phosphocholine,
lysine, isoleucine, glycerophosphocholine, glycine, glutamine, glutamate, alanine,
acetate, 3-hydroxybutyrate, and 1-methylhistidine were reported in IgAN patients.
Further analysis showed high sensitivity and specificity for diagnosis of IgAN (Sui
et al. 2012).
Despite the recent developments, achieving a diagnostic profile is a long road
to hit.
Prognostic Biomarkers
IgAN is a common glomerular disease with a significant risk to progress toward

ESRD. Various clinical and pathologic features have been described as prognostic
factors. Male gender, hypertension, elevated serum creatinine at diagnosis, and
Table 2 Clinical and histologic prognostic factors in IgAN

Risk factor Reference
Male gender Nachman et al. 2012
Older age Nachman et al. 2012
Obesity Floege and Feehally 2013
Smoking Floege and Feehally 2013
Sustained hypertension (>140/90)a Floege and Feehally 2013
Persistent hematuria Nachman et al. 2012
Persistent proteinuria (proteinuria of >1 g per day)a Nachman et al. 2012
Impaired renal function at diagnosisa Moriyama et al. 2014
Tubular atrophy/interstitial fibrosis Lee et al. 2014
Higher uric acid Moriyama et al. 2014
a
The KDIGO guideline for glomerulonephritis currently divides patients according to their risk
profile based on proteinuria, blood pressure, and eGFR at the time of diagnosis and during follow-
up: low risk (normal eGFR, no hypertension, urine protein <0.5 g/day), intermediate risk (pro-
teinuria >0.5–1 g/day
reduced eGFR
hypertension), high risk (rapid loss of eGFR)
persistent proteinuria are most studied clinical characteristics, and glomerular

sclerosis, tubular atrophy and interstitial fibrosis, endocapillary hypercellularity,
and crescent formation are pathologic features predicting progression (Peters
et al. 2011).
These prognostic factors do not have high sensitivity and specificity, and there is
a need for more accurate, less invasive, and reproducible ones. The clinical and
histologic prognostic factors in IgAN are listed in Table 2.
Urine Kidney Injury Molecule-1 (KIM-1)
KIM-1 is a glycoprotein with immunoglobulin and mucin domain that is expressed

on injured proximal tubule cells. The metaloproteinases cleave the ectodomain of
shed KIM-1. The ectodomain (90 kDa) is proposed to have a proinflammatory role
and cause macrophage recruitment and inflammation following tubular injury.
Thus, it is involved in both regenerative and fibrosing processes (Peters
et al. 2011; Xu et al. 2011). Urinary KIM-1 level has been evaluated as a predictor
of renal outcome in several studies.
Urinary KIM-1 has been shown to be an independent predictor of ESRD along
with serum creatinine in IgAN patients with an AUC of 0.86 %. It kept its predictive
value even in the subgroup of patients with serum creatinine less than 1.5 mg/dl
(Peters et al. 2011).
Of note, the relation between pathologic findings and urinary KIM-1 was diverse.
Urinary KIM-1 excretion was not correlated with tubulointerstitial score (Peters
et al. 2011). In a study by Xu et al., urinary KIM-1 was correlated with pathologic
findings of mesangial proliferation, crescent formation, glomerular sclerosis, and
interstitial fibrosis, but after applying multivariate analysis, no significant correla-

tion was found with any of these pathologic parameters. They also demonstrated a
worse renal outcome in a group of patients with urinary KIM-1 higher than 4.17 ng/
mg urinary creatinine after more than 12 months of follow-up (Xu et al. 2011).
In a more recent study, increased urinary KIM-1 was reported in IgAN patients
compared with healthy subjects, which was not correlated with estimated GFR. In
the contrary to the previous studies, urinary KIM-1 was not correlated with initial
serum creatinine, which might be due to milder disease. They reported urinary
KIM-1 level as a marker of tubulointerstitial injury even in patients with mild renal
dysfunction and serum creatinine less 2 mg/dl (Lee et al. 2014).
It seems that urinary KIM-1 level could be used as a marker of renal survival if a
large study with a long-term follow-up is conducted.
Fractional Excretion of IgG (FE IgG)
Persistent proteinuria is one of the predictors of outcome in IgAN (Glassock

2008). Data have shown that proteins with high molecular weight (>100 kDa)
were associated with decline in kidney function (Mackinnon et al. 2003). IgG is
one of the high molecular weight proteins, and its fractional excretion has been
reported to be related with progression of renal failure. In a study including
34 IgAN patients, FE IgG with AUC of 0.96 predicted the outcome of reaching
stage 5 of CKD. FE IgG with a cutoff of 0.029 had 88.9 % sensitivity and 88 %
specificity in predicting the primary outcome of decline in GFR. In the whole
cohort, patients with higher FE IgG had 37.1-fold higher risk of disease progres-
sion (McQuarrie et al. 2011).
Claudio Bazzi et al. analyzed the relationship of FE IgG with histologic findings,
renal outcome, and response to therapy in 37 patients with crescentic IgAN. Apart
from FE IgG, the ratio of FE IgG to percentage of glomerular sclerosis (FE IgG/GS)
was also evaluated as a predicting factor of progression. FE IgG/GS had 91 %
sensitivity and 92 % specificity in predicting progression with an AUC of 0.901,
and FE IgG/GS was 16-fold higher in progressors. In a group treated with cyclo-
phosphamide and steroids, FE IgG/GS in combination with serum creatinine of
more than 1.74 mg/dl had the highest value in prediction of response to therapy.
They reported no response to therapy in 89 % of those with FE IgG/GS of more than
0.0034 (Bazzi et al. 2009a).
Soluble CD89 (sCD89)
CD89 (FcαRI) is an IgA-specific Fc-binding receptor, which is expressed on

macrophages, neutrophils, eosinophils, dendritic cells, and Kupffer cells, but not
mesangial cells. Binding of IgA to membrane-bound receptor leads to phagocytosis
and inflammatory cytokine release. The soluble form of CD89 (sCD89) might have
a role in IgA immune complex formation in IgAN. sCD89 30 kDa isoform makes
sCD89–polymeric IgA (sCD89–pIgA) complexes both in healthy subjects and
those with glomerular disorders (IgAN or non-IgAN causes) (Boyd and Barratt
2010). Vuong et al. reported decreased serum levels of sCD89–pIgA in IgAN
patients with progressive disease (doubling of serum creatinine during at least
1-year follow-up or reaching stage 5 of chronic kidney disease). No correlation
has been observed between sCD89–pIgA and disease progression in non-IgAN
glomerular disorders. Two different explanations for this finding could be
suggested, one is the possibility of deposition of complexes in mesangium and
the other is the protective role of shed sCD89–pIgA complexes which prevents anti-
IgA1 autoantibodies binding to undergalactosylated IgA1 (Vuong et al. 2010).
Urinary Angiotensinogen
The renin–angiotensin system (RAS) has been long recognized to have pathophys-
iologic role in renal diseases. Apart from the systemic angiotensin II, intrarenal RAS
activation has been proposed to be involved in pathogenesis of glomerular injury and
progression toward end-stage renal disease (Kobori et al. 2007). Urinary
angiotensinogen has been demonstrated as a reflection of intrarenal RAS activation
in several studies (Xu et al. 2014; Kobori et al. 2002). In order to assess the intrarenal
RAS activity in IgAN patients, urinary angiotensinogen (UAGT) level in 52 IgAN
subjects was evaluated and compared with that of healthy controls and patients with
minimal glomerular abnormality (MGA). UAGT/Ucr was not significantly different
between healthy controls and patients with MGA. But UAGT/Ucr was significantly
higher in the IgAN group. Data showed a significant increase in tissue expression of
angiotensinogen mRNA and angiotensin II by immunostaining (Nishiyama
et al. 2011). In a study, UAGT level was correlated with urine protein-to-creatinine
ratio and serum creatinine. IgAN patients with UAGT >100 ng/mgCr had higher
serum creatinine and worse renal function after therapy than those with UAGT
<100 ng/mgCr. These data suggested UAGT as a predictor of outcome in IgAN
(Kim et al. 2011).
Complement Components
Complement system activation, both alternative and lectin pathway, has been
shown in IgAN by demonstrating mesangial deposition of complement components
such as mannose-binding lectin (MBL), C3, C4d, and C5b-9 (Wada and Nangaku
2013). Increased serum levels of C3a (footprint of alternative and lectin pathway
activation) and C4a (footprint of lectin pathway activation) have been reported in
IgAN (Abou-Ragheb et al. 1992).
In a study on 162 IgAN patients, urinary MBL level was reported higher in IgAN
than healthy controls. The patients were divided into three groups based on their
Lee’s pathologic class, with Lee-I or Lee-II in the first group, Lee-III in the second,
and Lee-IV or Lee-V in the third group. Patients in the third group had the highest
urinary MBL level. Urinary MBL was in positive correlation with serum creatinine
and the degree of proteinuria and severity of pathologic changes (mesangial
hypercellularity, endocapillary proliferation, tubular atrophy/interstitial fibrosis,
glomerular sclerosis) (Liu et al. 2012).
In a study aimed to explore serum biomarkers in IgAN, serum proteome was
analyzed by SELDI system. The pathologic glomerular patterns were scored.
Compared to the healthy controls, 93 proteins were differently expressed in
IgAN. Among these proteins, the one with protein signal at 8,592 m/z was recog-
nized to be C4a desArg by Western blotting. C4a desArg level evaluated by ELISA
was positively correlated with mesangial hypercellularity that acts as a prognostic
factor. Thus, C4a desArg could be used as potential biomarker associated with
severe glomerular injury (Sogabe et al. 2013).
Studies have shown elevated serum IgA and/or C3 to differentiate IgAN with
verity of histologic severity. In addition, Zhang et al. found that the higher serum
IgA and the lower serum C3 level are (thus, increased IgA/C3 ratio) in IgAN
patients, the more is the chance of progression of kidney injury. IgA/C3 ratio at a
cutoff of 3.32 had a sensitivity coefficient of 71.43 % and a specificity coefficient of
68.88 %. Those with serum IgA/C3 3.32 had lower GFR after mean follow-up
duration of 36 months and have reached the primary outcome of 50 % decline in
GFR or the need for renal replacement therapy (Zhang et al. 2013).
Inflammatory Cytokines
The pathogenetic role of T helper2 (Th2) has been described in IgAN. Th2 cells upon
activation produce IL-2 and express IL-2 receptor (IL-2R) and can induce B-cell
class switching and IgA production. The aberrantly galactosylated IgA1 complexes
deposit in the mesangium and stimulate mesangial cells. Activated mesangial cells
secrete proinflammatory cytokines such as TNF-α, IL-1, IL-6, MCP-1, and PDGF.
These cytokines activate tubular epithelial cells (TECs). Activated TECs in turn
produce IL-18, which modulates macrophage activity through NFκB pathway. The
abovementioned mediators could be used as a prognostic marker since interstitial
inflammation and fibrosis are key prognostic indicators in IgAN (Lundberg
et al. 2012; Stangou et al. 2013; Torres et al. 2008; Shi et al. 2012).
IL-2 and soluble IL-2R (sIL-2R) have been long shown to be elevated in IgAN
(Schena et al. 1989; Parera et al. 1992). In a cross-sectional study on 194 patients
with IgAN, sIL-2R was analyzed by Luminex system. sIL-2R level of more than
153.1 pg/ml was associated with progression to either CKD stage 5 or a 50 %
decline of eGFR during follow-up or a 30 % decline of eGFR in 5 years of follow-
up. This predictive value was independent of baseline serum creatinine or the
amount of albuminuria during the follow-up period. Baseline sIL-2R level was
also demonstrated to be predictive of the rate of GFR decrement and was correlated
with degree of tubulointerstitial fibrosis (>25 % fibrosis) (Lundberg et al. 2012).
Upregulated urinary cytokines might be a marker of disease activity and
mesangial cell proliferation. Studied by Stangou et al., urinary levels of IL-1β,
MCP-1, IL-17, INF-γ, and IL-6 were correlated with endocapillary proliferation in
pathologic specimen. Baseline serum creatinine level was correlated with IL-1β,
MCP-1, and IL-2. Among the mentioned cytokines, urinary IL-6 was predictive of
renal outcomes (Stangou et al. 2013).
Expression of MCP-1 in the mesangium induces interstitial macrophage infil-
tration and profibrotic growth factor production. On the other side, tubular epithe-
lial cells produce epidermal growth factor to counteract with tissue fibrosis in renal
tubular injury. Thus, decreased level of EGF and increased level of MCP-1 might
cause progressive interstitial damage and fibrosis. In patients with IgAN, decreased
urinary EGF/MCP-1 has been reported, in patients with the lowest ratio (less than
8.9) having the most severe pathologic lesions. Of note is detrimental effect of low
EGF/MCP-1 ratio on renal survival. Those with EGF/MCP-1 ratio <8.9 had 36 %
renal survival after 84 months of follow-up. At the cutoff of 23.2, EGF/MCP-1 ratio
had 88.9 % sensitivity and 86.4 % specificity for prediction of serum creatinine
doubling or development of ESRD (Torres et al. 2008).
Elevated serum IL-18, produced by activated TECs, is positively correlated
with the amount of proteinuria and serum creatinine level and negatively corre-
lated with eGFR and serum albumin. Serum IL-18 with a cutoff value of
323.69 pg/ml has 94.7 % and 78.6 % sensitivity and specificity, respectively, in
predicting tubulointerstitial injury. Serum IL-18, tubulointerstitial injury, and
serum creatinine at diagnosis were independent predictors of composite end
point of creatinine doubling or renal replacement therapy or death of any cause
(Shi et al. 2012).
Markers of Oxidative Stress
Oxidative stress is a state of imbalance between oxidative activity and antioxidant

defense. The state of oxidative stress as reduction in superoxide dismutase, catalase,
and glutathione peroxidase activity and increased advanced oxidation protein
products (AOPPs) has been demonstrated in different studies. IgAN patients had
higher levels of AOPPs than healthy controls, and this high level of oxidative
marker was directly correlated with the amount of proteinuria at diagnosis and
time-averaged proteinuria during the course of follow-up. AOPP level above
100.7 μmol/L also had sensitivity and specificity of about 75 % in predicting
progressive eGFR loss. The increased proteinuria and the decline in eGFR might
be related to the toxic effects of AOPPs on podocytes and increased production of
TGF-β (Camilla et al. 2011). Therefore, diminution of oxidative stress could lead to
attenuation of rate of decline in eGFR and slowing the progression of disease.
Table 3 Different histologic classifications of IgAN

Histologic
classifications Advantages Disadvantages
The Lee Simple for routine use Qualitative description, less
classification reproducibility
Take into account the severity of Not scoring chronic injury (IF/TA)
crescent formation Not scoring endocapillary proliferation
as an individual marker
The Haas Simple for routine use Not scoring endocapillary proliferation
classification as an individual marker
Not scoring crescent formation as an
individual marker
Combining markers of disease activity
(hypercellularity) and chronic injury in
same categories
The Oxford Individually scoring the More complex than the others
classification pathologic changes (active or
(MEST) chronic lesions)
Quantitative and reproducible Not taking crescents into account
METS: mesangial hypercellularity (M), endocapillary proliferation (E), tubular atrophy/interstitial
fibrosis (T), segmental glomerulosclerosis (S)
Even with all these well-conducted studies, drawing a solid conclusion from the
information on the prognostic marker in IgAN is too soon. Large cohort studies may
lead to more precise results.
Biomarker Correlation with Histologic Findings
Histologic lesions in IgAN have a wide spectrum, from minimal glomerular

abnormality, mesangial hypercellularity, endocapillary and extracapillary
hypercellularity, segmental glomerulosclerosis, tubular atrophy, and interstitial
fibrosis. Each of these findings may indicate a distinct prognostic or therapeutic
implication. The last three histologic features are indicators of chronic lesions and
are reported to be related to renal outcome. The proliferative lesions may predict
response to immunosuppressive therapies. In order to minimize interobserver
variations in pathology reports, various classification systems have been developed,
including the Lee classification, the Haas classification, and the Oxford classifica-
tion (Roberts 2014). All these classifications have limitation in predicting progno-
sis, when compared with clinical prognostic factors. The advantages and
disadvantages of each are demonstrated in Table 3.
All of these pathologic classifications are invasive and need kidney biopsy.
Several groups had studied correlation of different biomarkers with the pathologic
findings. Some of the biomarkers will be reviewed here; the others have been
summarized in Table 4.
Table 4 Biomarkers correlated with histologic lesions

Histologic lesion Biomarker Reference
Mesangial hypercellularity Urine MBL Liu et al. 2012
C4a desArg Sogabe et al. 2013
Endocapillary proliferation Urine MBL Liu et al. 2012
Crescent formation FE IgG Bazzi et al. 2009
Tubulointerstitial damage Serum uric acid Zhou et al. 2014
sIL-2R Lundberg et al. 2012
Serum IL-18 Shi et al. 2012
FE IgG, FE α1m Bazzi et al. 2009
Urine α1m and β2m Peters et al. 2011
Urine EGF, IL-6, MCP-1 Stangou et al. 2009
Urine MBL Liu et al. 2012
Glomerular sclerosis miRNA-429 Wang et al. 2010
Urine IL-6 Stangou et al. 2009
Urine MBL Liu et al. 2012
α1m α1-microglobulin, β2m β2-microglobulin, EGF epidermal growth factor, FE α1m fractional
excretion of α1-microglobulin, FE IgG fractional excretion of IgG, IL-6 interleukin 6, IL-18
interleukin 18, MBL mannose-binding lectin, miRNA-429 microRNA-429, MCP-1 monocyte
chemoattractant protein-1, sIL-2R soluble interleukin 2 receptor
Podocalyxin
Podocalyxin is an apical membrane protein of podocytes. Its extracellular portion

consists of a juxtamembrane stalk and four different domains: O-glycosylated,
sialylated, N-glycosylated, and a globular domain. The intracellular domain of
podocalyxin interacts with Na+/H+ exchanger regulatory factors 1 and 2, ezrin,
and actin cytoskeleton of podocyte. It has a critical role in the development of
glomeruli and slit diaphragm. Urine level of podocalyxin is related to the degree of
podocyte injury in IgAN. In a study in adult IgAN patients, a significant correlation
has been reported between the urinary level of this protein and the severity of acute
extracapillary abnormalities (Sekulic and Sekulic 2013). This marker can be used to
predict the renal lesions and possibly the prognosis.
TGF-b and IL-6
In a study aimed to evaluate response to treatment with steroids, urinary TGF-β and
IL-6 levels were evaluated before and after treatment in IgAN patients. The
baseline urinary TGF-β level was significantly higher in patients with mesangial
hypercellularity, mesangial expansion, and crescent formation, and urinary IL-6
level was directly related to glomerulosclerosis (Kalliakmani et al. 2011). There is a
study that showed a significant correlation between chronicity of IgAN and
glomerulosclerosis (Stangou et al. 2009). Conversely, urine IL-6 has been corre-
lated with endocapillary proliferation along with other proinflammatory cytokines
such as IL-1β, MCP-1, IL-17, and INF-γ (Stangou et al. 2013), and the pediatric
patients’ urine IL-6 level was positively correlated with mesangial hypercellularity,
crescent formation, and endocapillary proliferation (Kanemoto et al. 2014). TGF-β
and IL-6 as markers of disease severity and pathologic findings should be evaluated
in large-scale study.
Thioredoxin
Thioredoxin is a redox-active protein and a marker of oxidative stress that has been
evaluated in IgAN patients (Nosaki et al. 2012). In this study serum level of
thioredoxin above 40 ng/ml was significantly associated with mesangial cell pro-
liferation. Although it was not specific for IgAN and it was also seen in other causes
of mesangial proliferation such as lupus nephritis, it still is a valuable marker that
needs to be proven in larger studies.
Soluble Vascular Cell Adhesion Molecule-1 (sVCAM-1)
sVCAM-1 is known as a marker of endothelial injury. Following endothelial

damage and activation, sVCAM-1 is cleaved from the membrane-binding domain
and released into the circulation. Severe pathologic finding of crescent formation is
the consequence of endothelial injury. sVCAM-1 was related to active crescentic
lesion rather than chronic glomerular lesions. Those with tubular atrophy and
interstitial fibrosis score (T in Oxford classification) had significantly higher
sVCAM-1 (Zhu et al. 2013).
Urine Secretory IgA (sIgA)
The sIgA has been shown to have a role in pathogenesis of IgAN, and increased
urinary levels of it were evident in patients when compared with healthy controls.
Data suggested that this increased level is associated with increased scores of
mesangial proliferation, crescent formation, and higher score of tubular atrophy
and interstitial fibrosis. The higher the urine sIgA was, the more severe the
pathologic findings were (Tan et al. 2009). Thus, this marker might be used as a
predictor of pathologic changes in IgAN.
Proteomic Analysis of Urine
In a proteomic analysis of urine samples of 13 IgAN patients using liquid chroma-

tography/tandem mass spectrometry (nLC-MS/MS), Kalantari et al. reported a
Table 5 Biomarker expression and their correlation with eGFR

Protein Up-/ Correlation with
ID Protein name downregulation eGFR
AFAM Afamin (alpha-albumin) # Positive
A2GL1 Leucine-rich alpha-2 # Positive
glycoprotein
CERU Ceruloplasmin # Positive
AMBP Alpha-1-microgolbulin " Negative
HEMO Hemopexin " Negative
APOA1 Apolipoprotein A-I " Negative
CO3 Complement C3 " Negative
VTDB Vitamin D-binding protein " Negative
APOA4 Apolipoprotein A-IV " Negative
B2MG Beta-2-microglobulin " Negative
RET4 Retinol-binding protein 4 " Negative
All the markers significantly correlate with eGFR with a p value of <0.006
panel of biomarkers that can differentiate various pathological subclasses based on

Oxford parameters specially endocapillary proliferation. These candidate bio-
markers were afamin, leucine-rich alpha-2-glycoprotein, ceruloplasmin, alpha-1-
microgolbulin, hemopexin, apolipoprotein A-I, complement C3, vitamin D-binding
protein, beta-2-microglobulin, and retinol-binding protein 4. Changes in urinary
expression of these markers were correlated with eGFR, as shown in Table 5
(Kalantari et al. 2013).
It seems that a single marker might be insufficient to differentiate pathologic
changes and predict disease course. A panel of biomarkers, serum, or urine might be
helpful.
Biomarkers of Response to Therapy
Better understanding of pathogenesis of IgAN helps targeted therapy. Floege has

proposed five pathogenetic steps. The first step is overproduction of Gd-IgA1,
which is thought to be from tonsillar origin. Therefore tonsillectomy is considered
as a therapeutic option, although the results on the course of IgAN are inconsistent.
The next step is antibody formation against Gd-IgA1 (autoimmunity). To address
this step, steroids and immunosuppressive therapy come to action. Deposition of the
immune complexes in mesangium and binding to mesangial IgA receptors (CD71)
induce inflammation and complement activation. Removal of glomerular IgA,
blocking the receptor, and prevention of complement activation are the proposed
approaches to target third and fourth steps of the pathogenesis. The last two steps
are cellular damage and growth factor (such as platelet-derived growth factor,
TGF-β) and profibrotic mechanism activation (Floege 2011).
Table 6 Biomarkers as predictors of response to therapy

Treatment Biomarkers Reference
RAS blockade Urine kininogen, ITIH4, transthyretin Rocchetti et al. 2008
Urinary KIM-1 Seo et al. 2013
FE IgG Bazzi et al. 2009
Fish oil Serum oxylipins Zivkovic et al. 2012
Tonsillectomy
steroids Serum Gd-IgA1 Nakata et al. 2014
Changes in GalNAc content of IgA1 Iwantani et al. 2012
IgA–uromodulin complex Obara et al. 2012
Serum thioredoxin Nosaki et al. 2012
Immunosuppressive therapy Serum IL-18 Shi et al. 2012
Urinary MBL Liu et al. 2012
FE IgG/GS Bazzi et al. 2009
FE IgG fractional excretion of IgG, FE IgG/GS FE IgG to glomerulosclerosis ratio, GalNAc
N-acetylgalactosamine, Gd-IgA1 galactose-deficient IgA1, ITIH4 inter-α-trypsin-inhibitor heavy
chain H4 precursor, KIM-1 kidney injury molecule-1, MBL mannose-binding lectin, RAS
renin–angiotensin system
Apart from novel anti-growth factor therapy, angiotensin-converting enzyme

inhibitors (ACEi) and angiotensin receptor blockers (ARBs) are the mainstay of
therapy.
According to the 2012 Kidney Disease: Improving Global Outcomes (KDIGO)
guidelines, in those with persistent proteinuria after 3–6 months of treatment with
ACEi or ARBs and estimated GFR of more than 50 cm3/min per 1.73 m2, treatment
with fish oil or steroids is recommended. Immunosuppressive drugs such as cyclo-
phosphamide, azathioprine, or mycophenolate mofetil are reserved for those with
rapidly progressive disease (KDIGO 2012).
Biomarkers can be used as a guide to select patients for specific treatment,
follow the response, and predict recurrences (Table 6).
Biomarker Response to Blockade of the Renin–Angiotensin System
In 2008, Rocchetti et al. evaluated response to treatment with ACEi in IgAN

patients. Responder patients were defined as more than 50 % reduction in protein-
uria and stable GFR after 6 months of treatment with ACEi. They utilized 2D
PAGE and nano-HPLC-ESI-MS/MS to analyze the urinary proteome in patients.
Patients who responded to treatment had a significantly higher urine kininogen
level at the baseline compared to the nonresponders. Additionally, there was a
significant increase in urine level of kininogen after treatment in the responder
group, but did not reach the normal values. They also reported decreased level of
inter-α-trypsin-inhibitor heavy chain H4 precursor (ITIH4, 35 kDa fragment) and
increased level of transthyretin in nonresponders when compared with responders
(Rocchetti et al. 2008).
Urinary KIM-1 has been introduced as a marker of renal survival. The changes in
its level have also been evaluated as a marker of response. Urinary KIM-1 was
measured at diagnosis and after about 2 years of treatment with different regimens
including a low-salt diet, blood pressure control, pharmacotherapy with angiotensin
receptor blockers or angiotensin-converting enzyme inhibitors, and if necessary
immunosuppressives (in 13.5 % of patients). The urinary KIM-1/creatinine ratio
was decreased to 0.26 [0.12–0.65] ng/mg from the baseline of 1.16 [0.51–1.83]
ng/mg after mean follow-up period of 23.56
5.08 months. In about half of the
patients, KIM-1 levels became normal. This study suggested KIM-1 as a valuable
predictor of response to therapy, but larger study on patients with more severe renal
dysfunction is needed to verify it (Seo et al. 2013).
Fractional excretion of IgG (FE IgG) is known as a marker of changes in size
selectivity and, as it was mentioned previously, a marker of disease progression in
IgAN. Bazzi et al. evaluated the clinical, laboratory, and histologic risk factors of
disease progression in two groups of IgAN patients (baseline serum creatinine less
than 3 mg/dl and no immunosuppressive therapy) treated with or without ACEi. The
risk factors were blood pressure, 24-hour proteinuria, FE IgG, fractional excretion of
α1-microglobulin, glomerular sclerosis, and tubulointerstitial damage. Progression
was defined as primary end points of end-stage renal disease or serum creatinine
doubling or secondary end point of 25 % increase in serum creatinine during follow-
up. The cutoff point for progression was calculated for each risk factor with highest
sensitivity and specificity. ACE inhibitors were renoprotective in cases with baseline
risk factors above the defined cutoff. In these patients, treatment with ACEi reduced
the progression rate significantly. Although different players are involved in ACEi
renoprotection, FE IgG with the cutoff value of 0.006 was the most powerful
predictor of renoprotection in this study (Bazzi et al. 2009).
Biomarker Response to Fish Oil
Fish oil (3.3 g/day) is one of the recommended treatments in KDIGO guideline for
patients with more than 1 g/day proteinuria and no response to ACEi after
3–6 months (KDIGO 2012). Studies reported different outcomes of using fish oil.
Apart from differences in patient population, drug dosage, and composition,
fatty acid metabolism variances might be the cause of inconsistency in response to
therapy. Fish oil is rich in ω-3 fatty acids (FAs). FA metabolism leads to production
of oxylipins. Oxylipins are produced by enzymatic and nonenzymatic oxidation of
FAs. Enzymatic oxidation involves the cyclooxygenase (COX), lipoxygenase
(LOX), and cytochrome P450 (CYP) pathways. Oxylipins play an important role
in different signaling pathways, such as coagulation cascade, vasodilation, and
cellular immunity and autoimmunity. Analyzing oxylipin profile in IgAN patients
with diverse response to therapy may help predicting fish oil responsiveness.
LC-MS/MS was used to identify oxylipin profile in serum of IgAN patients under
treatment with fish oil. When responders (those with at least 25 % decrease in
proteinuria) were compared with nonresponders, the major difference was among
arachidonic acid and linoleic acid metabolites in the LOX pathways. Lower levels
of hydroxyeicosatetraenoic acids (HETEs) and the leukotriene B4 (LTB4) were
associated with kidney function improvement. Fish oil can result in reduction of
these metabolites (Zivkovic et al. 2012).
Biomarker Response to Tonsillectomy 6 Steroids
Serum galactose-deficient IgA1 (Gd-IgA1) has the leading role in the pathogenesis
of IgAN, thus following its serum level during the course of treatment might be
helpful. In a study, changes in serum level of Gd-IgA1 with treatment were
evaluated. Patients were treated with tonsillectomy followed by steroids if not
responding to tonsillectomy alone. Clinical response was defined as remission of
hematuria and protein/creatinine ratio less than 0.15 g/g. Twenty-two out of
37 patients responded to tonsillectomy alone, 13 patients responded to tonsillec-
tomy and steroids, and 2 responded to neither. The changes in serum Gd-IgA1 was
parallel to the clinical response, declined in those with remission in proteinuria and
hematuria. Thus, apart from its pathogenetic role, serum Gd-Iga1 could be used as a
marker of clinical response (Nakata et al. 2014).
Aberrant O-glycosylation of IgA1 including hyposialylation, reduced galactose
(Gal) number, and reduced N-acetylgalactosamine (GalNAc) number in the hinge
region is the cornerstone of IgAN. The variations in glycosylation pattern after
tonsillectomy and steroid therapy and the molar content of GalNAc and galactose in
the hinge region were evaluated as a marker of response. There was a significant
increase in GalNAc in patients in remission but the level was still lower than
healthy controls. Therefore, quantitative changes in GalNAc content of IgA1,
analyzed by MALDI-TOF-MS, might be a new marker of therapeutic response
(Iwantani et al. 2012).
As previously mentioned, elevated IgA–uromodulin complex level could be a
diagnostic marker of active IgAN. Thus, it could be used as a criterion for selection
of patients in early active phase to be treated with tonsillectomy and steroid (Obara
et al. 2012).
Data have suggested that oxidative stress enhances the nephrotoxicity of aber-
rantly galactosylated IgA1. It is proposed that improvement in oxidative stress in
response to either steroids or tonsillectomy might be related to the clinical response.
Thioredoxin is a redox-active protein, which was used as a marker of oxidative
stress in patients with IgAN. Serum and urine levels of thioredoxin were measured
with ELISA before and after tonsillectomy. Serum thioredoxin level in IgAN
patients was higher at baseline compared with healthy controls and was signifi-
cantly decreased after tonsillectomy but did not reach the levels in controls (Nosaki
et al. 2012).
Most of the abovementioned studies have included small number of patients, and
a large clinical trial is needed to draw a net conclusion.
Biomarker Response to Immunosuppressive Therapy
Most of the studies, which reported a biomarker as predictor of response to

immunosuppressive therapy, were single centered with small number of cases
short follow-up. Beyond all, they were mostly designed for outcomes of progres-
sion and prognosis.
KDIGO recommends steroids in IgAN patients with nephrotic-range proteinuria
and in patients with GFR of more than 50 ml/min/1.73 m2 and proteinuria of more
than 1 g/day after 3–6 months of treatment with RAS blockers (KDIGO 2012).
In order to study disease activity after remission of proteinuria with steroids,
urine IL-6 and TGF-β were measured in 21 patients with IgAN at diagnosis and at
the end of treatment. Following treatment despite a significant reduction in pro-
teinuria, the urinary level of these factors remains high, which might represent
ongoing inflammation (Kalliakmani et al. 2011). Serial measurements of factors
and longer follow-up might help to define a tool for clinical course monitoring and
patient selection for steroid therapy. In a different study, baseline serum level of
IL-18 predicted the response to steroid therapy. The higher the serum IL-18 was,
the more was the probability of the nonresponder to therapy. This correlation might
be due to association of this marker with tubulointerstitial damage (Shi et al. 2012).
When steroids were used in combination with azathioprine in subjects
nonresponsive to RAS blocker and fish oil, affected urinary markers of inflamma-
tion including IL-6 were suppressed (Stangou et al. 2013).
Liu et al. in a study aimed to evaluate the value of urinary MBL in prognosis
and progression of IgAN included patients at diagnosis who have been followed for
286
59 months, and they were treated either with non-immunosuppressive
therapy (ACEi, ARB, fish oil, antiplatelet) or steroids alone or in combination with
cytostatics, cyclosporin A, and mycophenolate mofetil. In both groups
(non-immunosuppressive vs. immunosuppressive), patients who had reached
remission had lower baseline urinary MBL level (Liu et al. 2012). Thus baseline
urinary MBL could serve as a predictor of response rather than a surrogate of
response to therapy.
The pathologic finding of crescentic glomerulonephritis with rapidly progressive
renal failure is one of the grave clinical features of IgAN. Different therapeutic
approaches have been employed to treat this condition including combined treat-
ment with steroids and cyclophosphamide. Thirty-seven crescentic IgAN patients
were investigated. The relationship between the fractional excretion of IgG
(FE IgG) and histologic lesions and response to therapy was analyzed. FE IgG to
glomerulosclerosis ratio (FE IgG/GS) and serum creatinine in combination were the
most accurate predictors of responsiveness to therapy. Patients with FE IgG/SG
0.00034 and sCr 1.74 mg/dl were at 100 % risk of progression even with
treatment. So it could be used in patient selection for immunosuppression therapy
(Bazzi et al. 2009).
According to the evidences, FE IgG/SG is the best predictor of response to
treatment with RAS blocker and steroid and cyclophosphamide. Further multicen-
ter large-scale trails are needed to evaluate predicting markers of response.
Although great evolutions have occurred in protein isolation techniques, valida-

tion of these biomarkers in large population studies and bringing them from bench
to beside are at their infancy.

Conditions
Urine is a biofluid, which can be easily collected in a noninvasive procedure. It is

representative of local processes in the kidney and least affected by systemic
processes. It could be examined repeatedly during the course of disease. Thus, it
seems to be a perfect sample for biomarker evaluation in glomerular disease.
Moreover, according to available studies, a panel of biomarkers rather than a single
biomarker seems to be more accurate and practical. Accordingly, proteomics and
metabolomics studies might be of more help in the path to design diagnostic,
prognostic, and predictive biomarkers, as they are representative of active ongoing
processes in the kidney. In that way, light would be shed on pathogenetic pathways
too, and this would help for targeted therapy.
In order to extract a panel of biomarkers in different fields of glomerular
diseases, it is practical to find a proteomics or metabolomics bank with various
samples from patients with different glomerular diseases (minimal change disease,
focal segmental glomerulosclerosis, membranous nephropathy, etc.). This will help
to define disease-specific biomarker profile and little by little will fade the need to
perform kidney biopsy in order to make a diagnosis or predict the outcome of the
glomerulopathy.
Summary Points
• This chapter focuses on novel biomarkers in IgA nephropathy.

• IgA nephropathy is one of the most common glomerulonephritis worldwide,
diagnosis of which is dependent on renal biopsy.
• Different clinical and pathological factors influence the outcome and response to
therapy.
• As an easily accessible biofluid sample, urine protein and metabolite profile is
the most studied markers in IgAN.
• In a search for noninvasive biomarkers for diagnosis of IgA nephropathy,
various proteomics methods (2DE, MALDI-TOF-MS, LC-MS/MS, SELDI-
MS/MS) or metabolomics (NMR) were used.
• Uromodulin, sTfR, IL-6/EGF, and complement components have been repeat-
edly reported as diagnostic urinary markers.
• Analysis of urine exosomes using LC-MS/MS, evaluation of urine metabolomics
by the means of NMR spectroscopy, or urine proteome study by nLC-MS/MS
are new approaches for noninvasive diagnosis of IgAN. These data also could
help clarifying the pathogenesis of disease.
• Numerous studies have reported urinary KIM-1, FE IgG, and inflammatory

cytokines as predictor of renal outcome in IgAN.
• Various markers are proposed as predictor of response to immunosuppressive
therapy. The most widely studied is FE IgG.
• A wide range of studies have conducted and various markers have been intro-
duced, but large cohort studies are still needed to reach the goal of noninvasive
diagnostic, pathogenetic, and prognostic markers in IgAN.
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MicroRNAs as Biomarkers of Diabetic
Nephropathy 33
Aaron D. McClelland and Phillip Kantharidis
Contents
Key Facts of Diabetic Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 750
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 751
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752
Need for Better Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 752
miRNA in Biofluids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754
miRNA in Renal Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 754
Circulating miRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 755
Urinary miRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 758
Exosomal miRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 760
Diagnostic and Prognostic Profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 762
Notable Differences in Profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 765
Potential Applications for Other Nephropathies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 770
Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 773
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 774
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 775
Abstract
Over the course of the past decade, miRNAs (microRNA) have established
themselves as important players in many aspects of biology, not least of all in
disease pathology. Indeed, microRNA (miRNA) dysregulation has been demon-
strated in numerous diseases and in almost all tissues with a number of them
displaying promise as therapeutic targets. In recent years, the presence of miRNA
in various biofluids, including blood and urine, has been well documented.
Importantly, there have been a number of studies demonstrating that miRNA
profiles in these biofluids undergo distinct shifts in both the levels of particular
A.D. McClelland • P. Kantharidis (*)

JDRF Danielle Alberti Memorial Centre for Diabetes Complications, Diabetes Domain, Baker IDI
Heart and Diabetes Institute, Melbourne, VIC, Australia
e-mail: Aaron.McClelland@bakeridi.edu.au; Phillip.Kantharidis@bakeridi.edu.au

750 A.D. McClelland and P. Kantharidis
miRNA species and also which specific miRNA is present. This has sparked
interest in their potential as noninvasive biomarkers for disease. Interestingly, the
vast majority of these miRNAs have no current known role in their respective
diseases. Furthermore, recent discoveries of exosome-bound miRNA being
excreted from cells into both the urine and blood have sparked further interest
in the field. Here, we review the current literature regarding clinical investigation
of miRNAs as diagnostic and prognostic markers for diabetic nephropathy.
Specifically, we discuss those studies utilizing miRNA profiles in blood, urine,
and also exosomes, their importance to the field miRNA biomarker research, and
any potential issues arising from these studies.
Keywords
Diabetic nephropathy • Diabetes • MicroRNA • Biomarkers • Urine • Plasma •
Serum • Exosome
Abbreviations
BMI Body mass index
DGS Diabetic glomerulosclerosis
FPG Fasting plasma glucose
HTN Hypertensive nephropathy
MCN Minimal change nephropathy
MGN Membranous nephropathy
miRNA/miR Microribonucleic acid
mRNA Messenger ribonucleic acid
OGGT Oral glucose tolerance test
qRT-PCR Quantitative real-time polymerase chain reaction
SNP Single nucleotide polymorphism
T1D Type 1 diabetes
T2D Type 2 diabetes
UAER Urinary albumin excretion rate
Key Facts of Diabetic Nephropathy
• A significant proportion of both type 1 and type 2 diabetics develop diabetic

nephropathy.
33 MicroRNAs as Biomarkers of Diabetic Nephropathy 751
• The condition is progressive and involves renal fibrosis and destruction of tissue
architecture, microvascular damage, and loss of function of the glomerulus, the
filtration unit of the kidney.
• Diabetic nephropathy is the leading cause of end-stage renal disease which results
in eventual renal failure and therefore requirement of either renal transplantation
or hemodialysis.
• Prognosis for those with end-stage renal failure is poor and is further worsened by
concomitant cardiovascular complications.
• There is a reciprocal relationship between cardiovascular complications and
diabetic nephropathy meaning those with one complication are at higher risk of
developing the other.
• Diabetic nephropathy can progress for 5–10 years without any adverse physio-
logical manifestation and therefore is often somewhat advanced at the time of
diagnosis.
• There are no therapeutic compounds available that effectively prevent the devel-
opment or progression of diabetic nephropathy.
• With the exception of cardiovascular complications, there are no means to
accurately predict those that will develop diabetic nephropathy.
Definitions
Biomarker Any biological substance (protein, DNA/RNA sequence, metabolite)

which is measurable and enables diagnosis or prognosis of a disease.
Diabetic nephropathy A degenerative complication of T1D and T2D ultimately

leading to end-stage renal disease.
Exosome 50–100-nm lipid vesicles excreted directly from the cell plasma mem-
brane and are transported in the blood or urine.
Gene array Silica chips containing oligonucleotide tags designed to capture spe-
cific mRNA sequences.
Glomerulosclerosis An aspect of diabetic nephropathy which entails glomerular

hypertrophy, microvascular endothelial dysfunction, and loss of podocytes.
High-throughput sequencing A technology which sequences every tagged RNA

molecule in a given sample.
MicroRNA 22–25 nucleotide noncoding RNA sequences posttranscriptionally

regulate protein production.
MicroRNA family A group of miRNA which contain identical seed regions which
allow them to target mRNA for posttranscriptional regulation.
mRNA A sequence that is transcribed from the genome and translated into protein
by ribosomes.
Plasma Blood preparation with blood cells removed by centrifugation.
qRT-PCR panel Fixed arrays of specific mRNA or miRNA preloaded on com-

mercial qRT-PCR plates.
Serum Blood preparation with cells removed by clotting followed by

centrifugation.
Tubulointerstitial fibrosis Deposition and accumulation of extracellular matrix

proteins in interstitial space of the kidney.
Introduction
MicroRNA research has flourished since the initial discovery of their presence in
humans. Recent years have seen numerous volumes published on miRNA and their
relevance to specific areas of transcriptional and medical biology. This chapter aims
to highlight the developments in a forefront region of miRNA research, miRNA
biomarkers. Specifically, the detection of miRNA in biofluids including urine and
plasma will be discussed. These topics will be focused on their relevance to the early
diagnosis of diabetic nephropathy and also the identification of those at risk of
progressive diabetic renal disease. Consideration will also be given to potential
benefit of this field to nondiabetic nephropathies.
Need for Better Biomarkers
Clinicians are recommended to test T2D patients at the time of diagnosis and every
year after this for urinary protein exceeding 30–300 mg/24 h thus indicating
impaired renal function as defined by microalbuminuria. While this test is well
established and a rather accurate measure of renal and, in particular, glomerular
health, the test fails to identify those at risk of DN, and therefore no action can be
taken to prevent its onset. Moreover, tissue and cellular damage incurred during DN
is progressive and currently irreversible (Dronavalli et al. 2008). There is a pertinent
need to improve evaluation of both renal damage and also identification of diabetic
subjects at risk of developing DN.
A number of experimental markers of renal damage are reported in the literature
or are available to clinicians but are generally either cumbersome or have known
inaccuracies. Creatinine clearance has a tendency to over- or underestimate renal
function in both healthy and diseased subjects (Wuyts et al. 2003). Furthermore,
there are several formulae for calculating creatinine clearance with each method
accounting for different physiological parameters and possessing differing accura-
cies. Direct GFR measurement by way of monitoring clearance of infused inulin is
both cumbersome and prone to inaccuracies (Hsu and Bansal 2011). However, inulin
clearance is considered a more accurate, earlier marker for renal damage than
albuminuria or creatinine clearance alone. However, there are issues with inter-lab
consistency of results due largely to management of this rigorous test.
There are numerous proteins or metabolites that indicate potential to be used as
predictive markers to identify those at risk of going on to develop DN. The presence
of a number of podocyte proteins can indicate podocyte damage and stress, the
presence and concentration of a number of immunoglobulin G isoforms can both
predict and diagnose progression of glomerular damage, and KIM-1, a marker of
tubular epithelial cell proliferation, can indicate tubular damage (Moresco
et al. 2013; Wang et al. 2013a). There are many other markers reported in the
literature that demonstrate effectiveness in diagnosing DN or identifying those at
risk of development. However, a number of these markers may not be truly specific
to DN and may indicate other forms of renal disease as is the case with transferring
and primary glomerulonephritis (Wang et al. 2013a). Additionally, many of these
markers are expensive to analyze and possess inter-assay variability due to testing
methods and protein stability.
Thus, the need for highly stable, cost-effective, and reproducible markers of DN
and pre-DN subjects is present. MicroRNA is well poised to fill this need.
MicroRNA is highly stable at room temperature (Mall et al. 2013), their
dysregulation is often cell/tissue specific (Babak et al. 2004), and their expression
profiles can indicate DN or the potential for developing DN (Zampetaki et al. 2010).
Furthermore, targeted miRNA analysis is comparatively cheap, reproducible, and
well suited to high-throughput qRT-PCR analysis such as that found in large
diagnostic labs. Finally, isolation of these novel biomarkers from either urine or
plasma is relatively straightforward and quite forgiving compared to numerous other
protein- or metabolite-type biomarkers, both established and experimental (Fig. 1).
Fig. 1 Depiction of the various samples obtainable for miRNA analysis from both blood and urine
and the methods by which they may be analyzed
miRNA in Biofluids
Biofluids, in particular plasma, are attractive for the diagnosis of a number of pathol-
ogies as it may carry indications of disease in any region of the body given that the
appropriate markers are observed. While this concept holds true for well-defined
conditions such as cancer, where miRNA research has flourished, the same cannot be
said for more complex diseases such as type 2 diabetes (Chen et al. 2008). The
multifactorial nature of diabetes results in damage to an array of tissues, and as such
a single biomarker is unlikely to provide sufficient information on the state of disease.
Accordingly, miRNA, which often possesses tissue- or cell-type-specific profiles, may
provide an excellent alternative to traditional clinical biomarkers (Babak et al. 2004).
miRNA is remarkably stable in plasma as their ~22-nucleotide structure is both
mechanically and thermodynamically sound. This inherent stability is further enhanced
by being paired with protective chaperones such as Ago2 or being encased in small
lipid vesicles such as exosomes (Creemers et al. 2012). While the regulatory mecha-
nisms of exosomal and extracellular miRNA are currently unclear, current research
indicates that the profile of the miRNA populations is both cell specific and disease
specific. These plasma-borne miRNAs have also been demonstrated to be taken up by
nonhost cells and exert regulatory effects on the target cell translational processes
in vitro (Momen-Heravi et al. 2014). miRNA may therefore play a role in communi-
cation between tissues in both homeostatic and pathological processes and thus further
enhance the prospect of miRNA biomarker therapeutics.
While it is true that plasma represents the whole body miRNA secretosome, urine
more specifically represents that of the kidney and its components. The cells of the
kidney secrete exosomal and protein-bound miRNA into the urinary stream and are
equally as stable as those in plasma (Mall et al. 2013). Analysis of urinary miRNA has
the potential to specifically identify the health of the diabetic kidney and may also
provide a measure of the likelihood that a patient will progress to diabetic nephropathy.
miRNA in Renal Disease
miRNAs have rapidly proven themselves as important regulators in a vast array of

tissues in addition to playing roles in development and homeostasis (Sayed and
Abdellatif 2011; Guo et al. 2014). The kidney is no exception, with much work
published in regard to a great number of nephropathies. In particular, renal carcino-
mas (Nakada et al. 2008), PKD (Wessely and Tran 2012), IgAN (Szeto and Li 2014),
and, importantly, DN (McClelland and Kantharidis 2014) have all been found to be
mediated in part by a growing number of miRNAs.
A small group of miRNAs have been repeatedly shown to be involved in
glomerulosclerotic and tubulointerstitial injury. These major renal pathologies are
predominately mediated by the resident cells of the glomerular and tubular structures
of the kidney, respectively (Schena and Gesualdo 2005). Surprisingly, in the diabetic
milieu, these cells exhibit similar changes in cellular physiology such as increased
extracellular matrix production and concomitant reduction on ECM turnover
proteins, increased growth factor production and secretion, cellular hypertrophy, loss
of cell-cell contacts, and disturbances to basement membranes. The processes have
been shown to be dependent upon dysregulation of a central set of miRNAs. This list
includes the miR-29 and miR-200 families, miR-192/215 and miR-21 (McClelland
et al. 2014). There have been a considerable number of miRNAs added to this list in
recent years and this list continues to grow. Some of these, such as the miR-30 and
let-7 families, appear in biofluids alongside miRNA of no known relevance to DN
(Zhou et al. 2013). The presence of these miRNAs suggests potential to be effective
biomarkers for the detection and diagnosis of DN.
Despite the state of knowledge regarding miRNA and DN, there have been
surprisingly few studies specifically investigating their use as biomarkers for dia-
betic kidney disease, though recent years have seen increasing interest in the field. A
number of studies have demonstrated the validity of biofluid miRNA profiles in not
only diagnosing DN but also identifying those at risk of developing DN. These
studies clearly authenticate the validity of miRNA as potential biomarkers in the
diagnosis of DN and those at risk of developing DN. Validation of these miRNA
biomarker profiles has obvious implications for early detection and subsequently the
timely interventions required to prevent or attenuate development and progression of
an otherwise degenerative condition.
Circulating miRNA
Circulating miRNAs have been the subject of a number of recent studies. These
studies have focused on both diagnosis of diabetes in general and also of DN. A
number of studies have also sought to identify miRNA in plasma or serum which
may differentially diagnose various stages of diabetic nephropathy.
The seminal paper on serum miRNA signatures in disease was conducted by
Chen et al. (2008). The authors performed Solexa sequencing on serum and blood
cell fractions from subjects with lung cancer, colorectal cancer, and diabetes and
healthy controls. Remarkable similarity in the miRNA profiles from both plasma and
cellular fractions between each of the diseased groups was reported. Specifically,
23 miRNAs were detected in the serum of those with lung cancer or diabetes that
were not detected in the sera of healthy controls. Conversely, there were 16 miRNAs
detected in healthy sera that were not detected in that from diseased subjects. Of
particular interest, there were 39 miRNAs uniquely detected in sera from lung cancer
patient serum compared to diabetic samples and with three unique to diabetic sera.
The study also provided compelling evidence that miRNAs are differentially
expressed in serum and are therefore suitable biomarkers for the noninvasive deter-
mination of disease states. Although there were 69 miRNAs detected in both serum
and cellular fractions of lung cancer patients, there were further 28 and 63 unique
miRNAs detected in normal and diseased serum, respectively. Furthermore, when
miRNA profiles were compared within the lung cancer group, there were 57 common
miRNAs yet 76 and 15 unique miRNAs in plasma and cellular fractions, respectively.
Likewise, there were 84 common miRNAs between plasma and cellular fraction from
diabetic subjects and 17 and 27 unique miRNAs in these fractions, respectively. This
study provided the catalyst for research into biofluid-borne miRNAs and their role as
both signaling molecules and as clinical biomarkers themselves.
A large prospective study by Zampetaki et al. followed over 800 subjects for
15 years and revealed significant changes in five miRNAs in subjects who developed
DM over the course of the study (Zampetaki et al. 2010). Additionally, 8 of 13 miRNAs
analyzed were dysregulated in those with DM at the onset of the study. However, a
number of these, namely, miR-197, miR-28-3p, and miR-150, were not reported in the
Chen study though this may be an artifact of analysis platforms and their relative
sensitivities or, more importantly, disease-specific expression (Chen et al. 2008). Alter-
natively, as Zampetaki utilized plasma miRNA as opposed to serum miRNA, these
discrepancies may lie in sample preparation. The authors also reported that miR-126
secretion was decreased in high glucose-treated endothelial cells. These changes also
correlate with onset and diagnosis of peripheral vascular disease in the study cohort.
Due to the role of endothelial dysfunction in the glomerular aspects of DN,
decreases in miR-126 in urine may prove to be an effective marker for glomerular
injury (Karalliedde and Gnudi 2011). However, miR-126 is elevated in the urine of
subjects with DN compared to non-nephropathic diabetics or healthy controls (Liu
et al. 2014). In further contrast to the study by Zampetaki, these investigators found
no difference in urinary miR-126 between healthy controls and DM subjects without
DN. Considering the in vitro data from the Zampetaki study, it is likely that
decreased circulating miR-126 is likely more indicative of cardiovascular risk/
disease rather than DN specifically. However, given clear links between cardiovas-
cular disease and DN, the expression of this miRNA remains important (Van Buren
and Toto 2011). Regardless, circulating miR-126 has been recently supported as a
suitable biomarker for detecting those that are susceptible to DM (Zhang et al. 2013).
A study by Kong et al. also sought to identify a miRNA signature in serum from
patients with and without diabetes using seven T2D-related miRNAs (Kong
et al. 2011). All seven miRNAs were found to be elevated in diabetic patients
compared to those who were deemed to be at risk of diabetes due to excessive
BMI or family history. Interestingly, these miRNAs, with the exception of miR-375,
were detected at similar levels in at-risk individual as those with clinically defined
prediabetes. The panel of miRNAs was also used to correctly classify diabetic and
nondiabetic patients in a blind test. Although the study excluded patients with known
nephropathy, it is interesting to note that these miRNAs, with the exception of miR-9
and miR-375, have been implicated in renal pathology or have been identified in
urine (Wang et al. 2012a Li et al. 2013; Shi et al. 2013; He et al. 2014; Huang
et al. 2014). A number of these miRNAs were also not detected in the Chen study;
however, this may result from differences in microarrays compared to Solexa
platforms and more specifically the libraries used to generate the arrays.
miR-135a has been implicated in the development of diabetic nephropathy
through targeting of TRPC1 (He et al. 2014). miR-135a was elevated in both plasma
and renal biopsy material from subjects with diabetic nephropathy compared to
healthy controls and those with DM thus providing a potential plasma biomarker
for DN. The authors also reported changes in a number of miRNAs in those with DM
compared to controls with these changes being magnified in those with DN. Of
particular interest in this study, a number of miRNAs were reported as upregulated,
namely, miR-15a, miR-21, and miR-126. These miRNAs were reported as being
downregulated in subjects in the Zampetaki study (Zampetaki et al. 2010). These
discrepancies may result from differences in the ethnicity of the patient cohorts
employed. Furthermore, as He et al. included subjects with DN, these differences
may represent a shift in circulating miRNA during development of DN thus provid-
ing an indication of disease progression in any given individual. Supporting this
notion, miR-34a was increased in diabetic serum in the Kong cohort which excluded
diabetics with no apparent renal disease while being increased in DN subjects in the
He cohort (Kong et al. 2011; He et al. 2014).
Single nucleotide polymorphisms are a popular target for studies attempting to
identify genetic elements responsible for disease, particularly in those employing
genome-wide association analyses. Interestingly, Zhou et al. reported potentially
important genetic variation in the promoter region of let-7a-2, a member of the highly
conserved let-7 family (Roush and Slack 2008; Zhou et al. 2013). The authors analyzed
both miRNA and genomic material from plasma of over 260 Han Chinese, a cohort
constituting subjects with DN, diabetics without DN, and also control subjects. A
number of let-7 family members were more than twofold downregulated in those with
DN compared to diabetics without DN. The authors also reported 22 miRNAs with
dysregulated expression between DN subjects and diabetics with no DN. No data was
presented for either group compared to controls with the exception of let-7a which was
reported as being ~80 % upregulated in DM subjects compared to control. Interestingly,
the expression of this miRNA was reduced below control levels in those with DN. This
again provides a clear example of a specific miRNA whose expression levels may
noninvasively indicate the state of disease in diabetic subjects.
The let-7 family are reported to have roles in diabetic renal fibrosis with their
downregulation leading to derepression of TGFβRI and subsequently fibrotic sig-
naling (Brennan et al. 2013; Wang et al. 2014). This downregulation may result from
defective genomic regulatory units. Zhou et al. analyzed three SNPs in the promoter
region of let-7a and reported that 50 % of DM subjects’ genomes contained the CT
SNP compared to only 40 % of healthy controls (Zhou et al. 2013). This association
increased to 66 % in those that had progressed to DN. Both the TT and the CC
phenotype alone presented no correlation with disease state. Of great importance was
that 82 % of DM subjects possessed the CC/TT phenotype compared to only 68 % in
controls. DN subjects were only slightly higher at 85 %. These findings, both
miRNA expression profiles and SNP incidence, provide excellent measures for the
diagnosis of DN and also the detection of those at risk of developing DM and
identification of those that will progress to DN.
Finally, miR-199a has been detected at elevated levels in serum of those with T2D
compared to healthy subjects (Yan et al. 2014). The authors confirmed GLUT4 as a
target of miR-199a through in vitro gene knockdown and replacement experiments
and demonstrated that miR-199a targeting of GLUT4 resulted in modulation of
glucose uptake in L6 skeletal muscle cells. These findings have obvious implications
to the development and progression of DN. Increased circulating miR-199a may
potentially be taken up by metabolic tissues thus decreasing GLUT4 expression and

concordantly decreasing insulin sensitivity (Michael et al. 2001). The resultant
increase in serum glucose has ramifications for the kidney, a tissue whose functional
components are composed of cells well documented to be sensitive to chronic
hyperglycemic conditions (Brownlee 2001). Clinical investigation of this miRNA in
reference to diabetic nephropathy would be of value to the progression of miRNA
biomarker research.
Urinary miRNA
Urine is particularly suited for determination of renal health. The cells that comprise
the nephron are highly specialized, and each serves a specific function in both the
filtration of plasma and the recovery and exclusion of particular metabolites (Smith
1951). As such, the resulting urinary product contains a number of biomolecules
which indicate function or dysfunction of various parts of the nephron. miRNA is no
exception and the quantity and type of miRNA present may indicate adverse
physiology in any of these cells. This urinary miRNA may be detected both in
urinary sediment and urinary exosomes which represent shed apoptotic or damaged
cells and actively secreted miRNA, respectively (Szeto et al. 2012; Barutta
et al. 2013). These features, combined with the relative ease of urinary miRNA
purification and stability in conjunction with noninvasive availability of clinical
samples, have led to a considerable number of studies in recent years analyzing
miRNA from diabetic urine.
A comparative study by Szeto et al. analyzed miRNA in urinary sediments from
subjects with IgAN, HTN, and DGS (Szeto et al. 2012). The authors reported
significantly less miR-15 in urinary sediments from those with DGS compared to
those with IgAN and HTN. There were also decreased levels of miR-21, miR-17,
and miR-216a though these differences were not significant. Importantly, expression
levels of a number of miRNAs correlated with indicators of renal function and
damage including proteinuria, eGFR, glomerulosclerosis, and tubulointerstitial
fibrosis. The rate of eGFR decline was inversely correlated with both miR-21 and
miR-216a levels. miR-21 has been implicated in fibrotic signaling in proximal tubule
epithelial cells and mesangial cells, while miR-216a has a role in glomerulosclerosis
(Kato et al. 2010; Dey et al. 2011). Their level in urinary sediments therefore
provides important information about the health of the nephron.
The miR-29 family is also implicated in renal fibrosis through regulation of ECM
proteins such as collagen (Peng et al. 2013). All three members of this fibrotic family
of miRNA were analyzed in urinary supernatant from DM patients with and without
abnormal renal function as defined by urinary albumin concentration. Although all
three miRNAs were detected in urinary supernatant, only miR-29a was found to be
increased those with albuminuria compared to those without. Although miR-29a
levels were positively correlated with UAER, no correlation was found with other
indicators of renal function including urea, cystatin, β2-microglobulin levels, creati-

nine clearance, or eGFR. It is interesting to note that miR-29a, miR-29b, and miR-29c
are downregulated in renal tissues from experimental diabetes models which in part
leads to increased ECM expression (Wang et al. 2012a). Increased expression in the
urine is therefore unexpected. Regardless, these findings by Peng et al. are in line with
those of Kong et al. who reported increased circulating miR-29a (Kong et al. 2011).
DN progresses through a number of stages typically marked by the presence and
level of albuminuria (Brownlee 2001). This clinical presentation has its origins in the
underlying physiology of the nephron and, in particular, the glomerulus. As such,
urinary miRNA profiles may differ between patients exhibiting varying degrees of
disease. Argyropoulos et al. attempted to ascertain these potential differences by
analyzing miRNA expression levels in total urine from subjects with T1D who failed
to develop DN over a 20-year period, those who developed DN, and those that
presented intermittent or persistent microalbuminuria (Argyropoulos et al. 2013).
The authors reported high-level dysregulation of a number of miRNAs between
albuminuric and non-albuminuric subjects. Unique signatures were also reported for
those with intermittent vs. persistent microalbuminuria and also for those that went on
to develop DN over the course of the study compared to those T1D subjects that did
not develop DN. Although the panels of miRNA presented in each of the comparisons
are largely unique, it should be noted that these very differences highlight the
possibility of highly specific miRNA signatures which exist at various stages of DN.
In addition to the unique profiles reported in each group, there are two miRNAs
which were reported in multiple comparisons, namely, miR-221-3p and miR-323b-5p
(Argyropoulos et al. 2013). Specifically, miR-221-3p was downregulated in those with
microalbuminuria compared to those without microalbuminuria and was also
decreased in those that developed DN. Conversely, miR-323b-5p was decreased in
those with manifest microalbuminuria when compared to those without albuminuria.
Interestingly, this miRNA was increased in those with persistent microalbuminuria
compared to those with intermittent microalbuminuria indicating that the level of this
miRNA may indicate the duration of disease. Although neither of these miRNAs have
reported functions in diabetic nephropathy, pathway analysis reveals that miR-221-3p
targets a number of genes involved in cell cycle regulation, protein synthesis, and
PI3K signaling pathway and is therefore an attractive target for future research (http://
diana.cslab.ece.ntua.gr/). Likewise, miR-323b-5p targets multiple genes in proximal
tubule-specific pathways and pathways important for tubular function and will also be
of interest to basic research (http://diana.cslab.ece.ntua.gr/).
Upon summation of the literature at the time, Yang et al. proposed a miRNA
signature for urine analysis in those with DN (Yang et al. 2013). The authors
highlight that increased levels of miR-377, miR-192, miR-216/217, and miR-144
in addition to decreased miR-21 and miR-375 may be most indicative of renal health
in diabetic patients. This hypothesis was based on previously reported urinary
analysis of these miRNAs in addition to their reported roles in cellular physiology.
Although there was no clinical study to support this notion, the paper provides a
good platform for future urinary miRNA biomarker work.
Exosomal miRNA
Exosomes are 50–100-nm lipid microvesicles that are extruded from the cellular
plasma membrane (Raposo and Stoorvogel 2013). Their role in cellular biology and
the modes by which they are regulated are relatively unknown. In recent years,
evidence has emerged indicating that exosomes may play a fundamental role in cellular
and physiology (Camussi et al. 2010). These inferences come from the observation that
proteomic and RNA profiles of microvesicles differ significantly to that of their host
cells (Koppers-Lalic et al. 2014). Importantly, miRNA profiles also differ significantly
suggesting that miRNA may be trafficked between cells and tissues as a means of
communication between cells and tissue (Xiao et al. 2012). Indeed, in vitro and in vivo
experiments have demonstrated that exosome-bound miRNA may be taken up by
non-donor cells and exert posttranscriptional regulatory control in a manner identical to
miRNA transcribed from the target cells’ genome (Rana et al. 2013).
Although their presence in, and secretion to, biofluids is likely required for proper
physiological function, the isotype and quantity of these molecules vary with
differing stages of disease and are therefore important for diagnosis (Barutta
et al. 2013; Lv et al. 2013a). Importantly, over-/underexpression of specific
miRNA species in biofluids may prime individuals for development of disease by
modulating key signaling pathways in target cell types.
Exosomes are secreted from cells in both homeostatic and pathological conditions
and are relatively stable (Raposo and Stoorvogel 2013; Ge et al. 2014). As such,
exosomal miRNA provides an attractive target for clinical biomarker evaluation.
Their purification is relatively simple when compared to blood-borne miRNA, and
sample collection is completely noninvasive and may be collected at any time
(Cheng et al. 2014). Despite the great advantages to be had in the use of exosomal
miRNA for the diagnosis of diabetes and more specifically DN, their potential has
gone largely unappreciated. There are a great number of reviews discussing the
advantages of this approach yet surprisingly few research publications investigating
their potential; however, this field is quickly gaining traction.
Progress has recently been bolstered with a technical study by Cheng et al. which
determined the efficiency of a number of exosomal isolation methods along with
analysis of RNA profiles of exosomes from each method (Cheng et al. 2014). The
authors demonstrated that a simple, on-column protocol provided small RNA of
sufficient quantity and quality to allow high-throughput massively parallel sequenc-
ing. Additionally, the on-column system by Norgen required much shorter
processing times (1.5 h vs. 4–4.5 h) and also much less sample volumes (5–10 mL
vs. >20 mL), characteristics well suited to diagnostic labs where time, equipment,
and samples may be limited. Although high-throughput sequencing is currently not
employed in most diagnostic labs, that the authors were able to employ this analysis
clearly demonstrates that the RNA obtained is more than suitable for more conven-
tional qRT-PCR applications. It should also be noted that high-throughput sequenc-
ing is largely an exploratory technique and as such will not be required once a
diagnostic profile has been established.
The study has provided an excellent platform from which future studies may be
launched. Following the removal of low-read signals, a total of 66 abundant
miRNAs were detected in exosomes isolated with the Norgen kit and 166 miRNAs
from ultracentrifugation-based protocols (Cheng et al. 2014). The vast majority of
these miRNAs were detected across all subjects in the study thus validating not only
consistency in the isolation protocols but also consistency in miRNA expression in
healthy individuals. This study therefore provides an important reference for which
future studies may compare samples from diseased individuals.
In a study utilizing a cohort containing subjects with DN, FSGS, and IgAN, Lv
et al. isolated exosomes by ultracentrifugation from cleared urine (Lv et al. 2013).
They reported increasing levels of miR-29c and miR-200c between each of the
groups with DN subjects having the lowest expression and IgAN subjects presenting
the highest. None of these changes reached statistical significance nor were there any
control subjects in the study cohort. Nonetheless, this study demonstrated that
exosomal miRNA expression profiles may shift with differing renal pathologies
highlighting that microvesicle-bound miRNA may prove to be effective for deter-
mining disease state and possibly progression. The authors also reported remarkable
stability of exosomal RNA indicating that exosomes provide a highly stable source
of miRNA for biomarker analysis (Lv et al. 2013).
In a follow-up report, Lv et al. again reported changes in miR-29c and miR-200c
levels in urinary exosomes (Lv et al. 2013). Here, the study cohort comprised those
with DN, FSG, IgAN, membranous nephropathy, and mesangial proliferative
glomerulosclerosis and healthy controls as defined by UAER. Unfortunately, all
pathological groups were pooled in the reported data with no delineation or com-
parisons drawn between the different groups. Regardless, all three members of the
miR-29 family, in addition to miR-200a/b/c, were significantly downregulated in the
urinary exosome fractions from the CKD group compared to controls
(Lv et al. 2013). Interestingly, when comparing CKD samples based on fibrotic
scoring, they found that miR-29a/c and miR-200b/c were significantly higher in
those with mild fibrosis compared to those with moderate to severe fibrosis.
Although no comparisons were drawn directly between fibrotic and control subjects,
it is interesting to speculate, due to the established roles of these miRNAs in fibrotic
signaling, that these miRNAs continue to decrease as fibrosis progresses (Wang
et al. 2012a; Xiong et al. 2012). Indeed, there was a significant correlation between
tubulointerstitial fibrosis and miR-29c levels with the remaining miRNAs showing
nonsignificant correlations to fibrotic score.
Barutta et al. analyzed miRNA content of urinary exosomes from those with
incipient DN as marked by microalbuminuria and T1D without DN and healthy
controls (Barutta et al. 2013). qRT-PCR panel analysis revealed 22 dysregulated
miRNAs in microalbuminuric and normoalbuminuric subjects compared to healthy
controls. Regrettably, this list was not published. However, the authors did report
upregulation of miR-145 and miR-130a in microalbuminuric exosomes compared to
both T1D and control samples. The expression of these miRNAs was also increased
in T1D subjects compared to controls indicating that these miRNAs may be suitable
Table 1 miRNA which may be prognostic markers for development of DM

miRNA Change Source Prognosis References
miR-126 Down Plasma Marker for risk of developing DM (Guo et al. 2014)
miR-15a Down Plasma Marker for risk of developing DM (Guo et al. 2014)
miR-223 Down Plasma Marker for risk of developing DM (Guo et al. 2014)
miR-28-3p Up Plasma Marker for risk of developing DM (Guo et al. 2014)
miR-29b Down Plasma Marker for risk of developing DM (Guo et al. 2014)
to track progression of renal damage in those with T1D, at least to the stage of
microalbuminuria. Conversely, miR-155 and miR-424 were decreased between
groups with microalbuminuria subjects expressing the lowest levels of these
miRNA species and healthy controls the highest. Again these expression profiles
may be utilized for tracking renal health in those with T1D.
Diagnostic and Prognostic Profiles
A number of the above studies have identified miRNAs which have potential to be
used as either prognostic or diagnostic biomarkers (Tables 1 and 2). Of the studies
reviewed here, that by Zampetaki holds special significance. This prospective study
followed over 800 individuals to the completion of the 15-year study (Zampetaki
et al. 2010). Importantly, 19 subjects developed T2D over the final 10 years of the
study. These individuals were found to have altered expression of a number of
miRNAs at the outset of the study therefore providing a potential miRNA signature
for identification of individuals that may develop diabetes in the near future (Table 1).
However, this profile finds little support in subsequent studies (Table 3). Indeed,
miR-15a, miR-126, and miR-29b are reported as being upregulated (Kong
et al. 2011; Argyropoulos et al. 2013; Peng et al. 2013; He et al. 2014). Regardless,
the size, design, and length of the study, especially in comparison to other studies
reviewed here, add credit to these findings, and as such, subsequent follow-up
studies of a similar nature which will either support or refute the findings of
Zampetaki are of great importance.
Urinary miRNA profiles were also identified by Argyropoulos who utilized
samples from 40 subjects from the Pittsburgh Epidemiology of Diabetes Complica-
tions study which entailed a >20-year follow-up period (Argyropoulos et al. 2013).
The selected cohort contained four groups of ten subjects each comprising those with
T1D that did not develop DN, those who developed DN, those who displayed
intermittent microalbuminuria, and those with persistent microalbuminuria. This
study resulted in three panels of miRNA which potentially identify
microalbuminuric T1D subjects when compared to non-albuminuric T1D subjects
and differentiate between intermittent and persistent microalbuminuria and T1D
subjects with overt DN compared to those without (Table 2). Surprisingly, the vast
majority of miRNAs identified in these panels were not reported at significant levels
Table 2 miRNAs which are differentially expressed in various biofluids and display promise as
diagnostic markers for various stages of DM and, more specifically, DN
miRNA Change Source Disease/stage References
let-628-5p Up Urine Differentiates intermittent from (Rana
persistent microalbuminuria et al. 2013)
miR-1224-3p Up Urine Marks overt diabetic nephropathy (Rana
et al. 2013)
miR-124a Up Serum Marks progression from pre-DM to (Karalliedde
T2D and Gnudi
2011)
miR-126 Up Serum Changes mark the presence of DM (Koppers-Lalic
and increase with progression of DN et al. 2014)
miR-135a Up Serum Changes mark the presence of DM (Koppers-Lalic
et al. 2013)
T2D and Gnudi
2011)
miR-15a Up Serum Changes mark the presence of DM (Koppers-Lalic
miR-17-5p Up Urine Differentiates intermittent from (Rana
miR-188-3p Down Urine Marks microalbuminuria (Rana
et al. 2013)
miR-1912 Up Urine Marks overt diabetic nephropathy (Rana
et al. 2013)
miR-1913 Up Urine Marks microalbuminuria (Rana
et al. 2013)
miR-194-1 Down Serum Changes mark the presence of DM (Koppers-Lalic
miR-205 Down Serum Changes mark the presence of DM (Koppers-Lalic
miR-214-3p Up Urine Marks microalbuminuria (Rana
et al. 2013)
miR-215 Down Serum Changes mark the presence of DM (Koppers-Lalic
et al. 2013)
miR-221-3p Down Urine Marks overt diabetic nephropathy (Rana
et al. 2013)
miR-222-3p Up Urine Differentiates intermittent from (Rana
(continued)
Table 2 (continued)
T2D and Gnudi
2011)
miR-29a Up Urine Differentiates intermittent from (Nakamura
miR-29b-1-5p Up Urine Marks overt diabetic nephropathy (Rana
et al. 2013)
miR-29c Up Urine Differentiates intermittent from (Nakamura
miR-30b Up Serum Changes mark the presence of DM (Koppers-Lalic
miR-30d Up Serum Marks progression from pre-DM to (Karalliedde
T2D and Gnudi
2011)
miR-323b-5p Down Urine Marks microalbuminuria (Rana
et al. 2013)
miR-323b-5p Up Urine Differentiates intermittent from (Rana
T2D and Gnudi
2011)
miR-34a Down Serum Changes mark the presence of DM (Koppers-Lalic
et al. 2013)
miR-373-5p Up Urine Marks microalbuminuria (Rana
et al. 2013)
miR-373-5p Down Urine Differentiates intermittent from (Rana
miR-375 Up Serum Marks progression from pre-DM to (Karalliedde
T2D and Gnudi
2011)
et al. 2013)
et al. 2013)
miR-433 Up Urine Differentiates intermittent from (Rana
et al. 2013)
miR-520h Down Urine Differentiates intermittent from (Rana
et al. 2013)
(continued)
Table 2 (continued)
et al. 2013)
miR-589-5p Down Urine Differentiates intermittent from (Rana
et al. 2013)
et al. 2013)
et al. 2013)
miR-9 Up Serum Marks progression from pre-DM to (Karalliedde
T2D and Gnudi
2011)
miR-92a-3p Down Urine Differentiates intermittent from (Rana
miR-92b Up Urine Marks microalbuminuria (Rana
et al. 2013)
in any of the studies reviewed here. This may merely be the result of T1D pathology
compared to T2D (Table 3).
Conversely, these discrepancies may originate from differences in the miRNA
libraries used in either high-throughput sequencing, qRT-PCR arrays, or gene arrays
which are based on the miRBase database. This is especially likely when one
considers that from miRBase 10, which the Chen and Zhou studies utilized, to
miRBase 18, which Argyropoulos aligned their data sets to, there have been
379 miRNA records modified, 1217 miRNAs added, and 18 miRNAs deleted
from the miRBase libraries (http://www.mirbasetracker.org/). It is also important to
note that studies using commercial qRT-PCR arrays and gene arrays may be missing
a considerable proportion of miRNA as commercial products are rarely updated as
often as miRBase.
Notable Differences in Profiles
As previously discussed, urine and urine-derived exosomes are particularly well

suited to diagnosis of renal health as they are rich in nephron miRNA as compared to
plasma which may contain miRNA from any number of tissues (Lorenzen and Thum
2012). It is therefore pertinent that the miRNA from these distinct sources be
compared in order to arrive at a consensus for what may be viable markers for
diabetic nephropathy and its various stages. However, despite the large number of
miRNAs reported among the above studies, few miRNAs have been reported to exist
at significant levels in more than one biofluid, an observation which may find clarity
Table 3 Comparison of miRNA reported to be differentially expressed in various biofluids in the

reviewed DN- and DM-related studies
Exosomes
miRNA Serum/plasma (references) Urine (references) (references)
let-628-5p – Up (Rana –
et al. 2013)
let-7 Down (Glowacki et al. 2013) –– –
miR-1224-3p – Up (Rana –
et al. 2013)
miR-124a Up (Karalliedde and Gnudi 2011) – –
miR-126 Down (Guo et al. 2014), up – –
(Koppers-Lalic et al. 2014)
miR-130a – – Up (Momen-
Heravi
et al. 2014)
miR-135a Up (Koppers-Lalic et al. 2014) – –
et al. 2013)
miR-145 – – Up (Momen-
Heravi
et al. 2014)
miR-146a Up (Karalliedde and Gnudi 2011) – –
miR-150 Downa (Guo et al. 2014) – –
miR-155 – – Down (Momen-
Heravi
et al. 2014)
miR-15a Down (Guo et al. 2014), up Down (Michael –
(Koppers-Lalic et al. 2014) et al. 2001)
miR-17 Up (Koppers-Lalic et al. 2014) Up (Rana –
et al. 2013)
miR-188-3p – Down (Rana –
et al. 2013)
(Glowacki et al. 2013)
miR-1912 – Up (Rana –
et al. 2013)
et al. 2013)
miR-1915 Up (Glowacki et al. 2013) – –
miR-192 Up (Koppers-Lalic et al. 2014) – –
miR-194-1 Down (Koppers-Lalic et al. 2014) – –
miR-197 Down (Guo et al. 2014) – –
miR-200 – – Down (Szeto and
Li 2014; Wang
et al. 2011)
miR-205 Down (Koppers-Lalic et al. 2014) – –
miR-20b Down (Guo et al. 2014) – –
(continued)
Table 3 (continued)
Exosomes
(Koppers-Lalic et al. 2014)
et al. 2013)
miR-215 Down (Koppers-Lalic et al. 2014) – –
et al. 2013)
et al. 2013)
miR-26a Down (Glowacki et al. 2013) – –
miR-28-3p Up (Guo et al. 2014) – –
miR-29b Down (Guo et al. 2014), up Up (Nakamura Down (Szeto and
(Karalliedde and Gnudi 2011) et al. 2000), up Li 2014; Wang
(Rana et al. 2013) et al. 2011)
miR-30b Up (Karalliedde and Gnudi 2011, – –
Koppers-Lalic et al. 2014,
Glowacki et al. 2013)
miR-320b Downa (Guo et al. 2014), up – –
miR-323b-5p – Down (Rana –
et al. 2013)
miR-323b-5p – Up (Rana –
et al. 2013)
miR-34a Up (Karalliedde and Gnudi 2011), – –
down (Koppers-Lalic et al. 2014)
et al. 2013)
miR-363 Down (Glowacki et al. 2013) – –
miR-373-5p – Up/down (Rana –
et al. 2013)
miR-375 Up (Karalliedde and Gnudi 2011) – –
miR-377 Up (Koppers-Lalic et al. 2014) – –
miR-3940-5p Up (Glowacki et al. 2013) – –
miR-424 – – Down (Momen-
Heravi
et al. 2014)
et al. 2013)
et al. 2013)
(continued)
Table 3 (continued)
Exosomes
et al. 2013)
miR-4707-5p Up (Glowacki et al. 2013) – –
et al. 2013)
miR-486-5p Downa (Guo et al. 2014), up – –
miR-520h – Down (Rana –
et al. 2013)
et al. 2013)
mir-552 – Up (Rana –
et al. 2013)
et al. 2013)
et al. 2013)
et al. 2013)
et al. 2013)
miR-9 Up (Karalliedde and Gnudi 2011) – –
miR-92a-3p – Down (Rana –
et al. 2013)
miR-92b – Up (Rana –
et al. 2013)
a
Reported as a nonsignificant trend
b
miRNAs of the same family are listed as a single miRNA species
when considering the potential source of miRNAs in these biofluids (Table 3).
Exceptions to this observation are miR-15a and the miR-29 family.
miR-15a was reported by Zampetaki to be downregulated in serum, while He
reported this miRNA to be upregulated (Table 3; Zampetaki et al. 2010; He
et al. 2014). This difference may lie in the analysis of serum versus plasma as
serum preparation requires the clotting of blood before generation of a cleared
supernatant (Luque-Garcia and Neubert 2007). The clotting process required to
produce serum may induce release of miRNA either in miRNA-protein complexes
or in exosomes which are not cleared by the low-speed centrifugation required to
remove the cellular fraction (Hunter et al. 2008; Duttagupta et al. 2011; Wang
et al. 2012c). This highlights the need for standardization of sample preparation
for biofluid miRNA studies. Although the Szeto data supports that of Zampetaki, it
must be noted that the Szeto study did not draw comparisons to control samples but
to nondiabetic forms of nephropathy, namely, IgAN and HTN (Szeto et al. 2012).
While this is important, control samples are required in future studies to draw
conclusion about the validity of changes in miRNA as being indicative of disease,
particularly DN.
There is little congruency in the changes of miR-29 family members across the
various sample sources (Table 3). For example, miR-29b was reported to be
downregulated in serum by Zampetaki and in urinary exosomes by LV, while
Argyropoulos reported upregulation in urine (Zampetaki et al. 2010; Argyropoulos
et al. 2013; Lv et al. 2013). However, it should be noted that Argyropoulos utilized
uncleared urine for miRNA analysis and samples were also obtained from type
1 diabetics, while all other studies reviewed here have utilized samples from type
2 diabetics. Furthermore, uncleared urine may contain considerable cellular material
especially in the case of those with advanced nephropathy (Detrisac et al. 1983;
Nakamura et al. 2000). It is therefore important to remove this cellular sediment,
however small, to avoid occlusion of any obtained data. Furthermore, as highlighted
by Szeto, various nephropathies produce differing miRNA profiles (Szeto
et al. 2012). This gains particular importance with the Lv study which grouped
subjects with DN, FSGS, IgAN, and membranous nephropathy into a single “CKD”
group (Lv et al. 2013). Although the data reported by Lv is supported by a number of
experimental studies, it is important to report data from individual phenotypes to
enable proper identification of disease-specific biomarkers.
There are further differences in changes of particular miRNA reported in serum
studies including miR-191, miR-21, the miR-320 family, miR-34a, and miR-486-5p
(Table 3). The prospective study by Zampetaki reported decreased levels of miR-191
in those that went on to develop T2D during the course of the 20-year study
(Zampetaki et al. 2010). Conversely, Zhou reported that miR-191 was increased in
T2D subjects that had progressed to DN compared to those that had not (Zhou
et al. 2013). However, the authors did not provide comparisons to control samples,
and RNA extraction was performed on whole blood which has obvious implications
for the observed miRNA profile. Given the differences in design study and sample
preparation, it is difficult to speculate which directional change in miR-191 is most
representative of disease state, and therefore further studies are required to clarify
this. miR-21 was reported as downregulated in those that developed DM in the
Zampetaki study which is contrary to experimental data for this miRNA in both
in vitro and in vivo models of diabetic nephropathy (Zampetaki et al. 2010; Denby
et al. 2011; Chau et al. 2012; Dey et al. 2012). On the other hand, He et al. found that
miR-21 is increased in those with DM compared to controls with further increases
seen in those with DN compared to those without DN, a finding supported by
Glowacki et al. who observed increased circulating miR-21 in renal allograft sub-
jects (Glowacki et al. 2013; He et al. 2014).
There is a marked difference in the level of miR-34a between the Kong and He
studies (Table 3). Kong reported that miR-34a is decreased in serum of those with
DM and is further decreased in DN subjects (Kong et al. 2011). Conversely, He
reported that miR-34a was increased in T2D subjects compared to those with
prediabetes as defined by OGGT/FPG and those susceptible to T2D
(He et al. 2014). This difference is unlikely to be a result of cohort ethnicity as
both studies were conducted with Chinese populations. Furthermore, both studies
utilized patient serum for their analysis. As with many miRNA biomarker studies,
the cohorts enlisted were much smaller than what is typically seen in most other
cohort-based studies. The cause of this is generally twofold. Healthy control biopsy
material is generally difficult to obtain due to the invasive nature of renal biopsy
collection and therefore limits the size and number of studies. Another major
consideration is the cost involved in performing analysis of large numbers of
samples. As this field is still largely exploratory, it requires utilization of gene arrays
or high-throughput sequencing platforms which are costly to run and also require
specialist knowledge to assemble and analyze the large amounts of data obtained.
Potential Applications for Other Nephropathies
There have been a number of studies concerning miRNA biomarkers in a wide array
of nondiabetic nephropathies (Table 4). Of the studies previously reviewed here, that
by Szeto demonstrated differential levels of urinary miRNA species in HTN, DGS,
and IgAN (Szeto et al. 2012). Specifically, miR-17 was uniquely upregulated in
urine from those with IgAN while there was a nonsignificant increase in the levels of
both miR-21 and miR-216a in HTN urine samples. There have been a considerable
number of studies into miRNA biomarkers for various nephropathies in urine and
plasma, a selection of which will be discussed here.
A study by Neal et al. sought to measure miRNA levels in plasma and urine from
subjects in various stages of nondiabetic CKD (Neal et al. 2011). The authors found
that not only did the total concentration of miRNA decrease as disease worsened but
that the levels of a number of circulating miRNAs were inversely correlated with
renal function. Furthermore, a number of these miRNAs were found to be
downregulated in various groups compared to controls. Of the miRNAs analyzed,
only miR-638 displayed changes in detectable levels contrary to those reported
previously in diabetic nephropathy albeit from a different source (Argyropoulos
et al. 2013). Interestingly, the levels of these miRNAs were largely unchanged in
urine from the various study groups indicating that the changes in plasma miRNA
may be originating distal to the kidney and are likely the result of increased blood
toxicity due to impaired renal function.
In another study comparing various nephropathies, Wang et al. compared miRNA
levels in urinary sediment from subjects with DGS, MCN, FSGS, and MGN
(Wang et al. 2013b). Considering the studies’ use of urinary sediment rather than
clarified urine, not surprisingly, the miRNAs reported to be most dysregulated are
those that have been well studied in regard to renal cell dysfunction and fibrotic
signaling, namely, miR-29a, miR-192, and miR-200a/c (Chung et al. 2010; Wang
et al. 2011, 2012c). With the exception of miR-29a and miR-200a, these miRNAs
were found to be downregulated across all groups compared to controls.
Table 4 Summary of miRNA reported to be dysregulated in various nephropathies outside of DN

miRNA Change Nephropathy Reported comparison References
miR-223 Up PKD Differential diagnosis of PKD to (Ben-Dov et
non-PKD CKD al. 2014)
miR-199a Up PKD Differential diagnosis of PKD to (Ben-Dov et
miR-199b Up PKD Differential diagnosis of PKD to (Ben-Dov et
miR-146a Up Lupus Identify lupus nephritis over control (Wang
nephritis et al. 2012b)
miR-155 Up Lupus Identify lupus nephritis over control (Wang
nephritis et al. 2012b)
miR-15 Down DGS Differential expression in DGS to (Michael
HTN and IgAN et al. 2001)
miR-17 Up IgAN Differential expression in IgAN to (Michael
DGS and HTN et al. 2001)
miR-216a Up HTN Differential expression in HTN to (Michael
DGS and IgAN et al. 2001)
miR-210 Down Nondiabetic Differential expression in stage 3/4 (Zampetaki
CKD CKD, ESRD, and controls et al. 2010)
miR-638 Down Nondiabetic Differential expression in stage (Zampetaki
CKD 4 CKD and ESRD to controls et al. 2010)
miR-192 Down DGS Differential expression compared to (Zampetaki
MCN/FSGS, MGN, and controls et al. 2010)
miR-192 Up MCN/FSGS, Differential expression compared to (Zampetaki
MGN DGS and controls et al. 2010)
miR-200c Down DGS, MGN Differential expression compared to (Zampetaki
MCN/FSGS and controls et al. 2010)
miR-200c Up MCN/FSGS Differential expression compared to (Zampetaki
DGS, MGN, and controls et al. 2010)
miR-638 Down DGS, Indicates abnormal renal pathology (Zampetaki
MCN/FGSG, et al. 2010)
MGN
Additionally, in support of the Neal study, miR-638 was decreased in all groups
compared to controls (Neal et al. 2011). In support of miRNA biomarkers acting as a
means for differential diagnosis, levels of various miRNAs were correlated with
different clinical parameters between the studied groups (Table 5). Furthermore,
differential correlations were also seen between miRNA levels and histological
Table 5 Summary of correlations between miRNA levels in various nephropathies and clinical/
histological markers
miRNA Proteinuria eGFR GS TIF
miR-21 – DN – DN
miR-29a – – – DN, MGN
miR-29b – DNa – DN
miR-29c – – – –
miR-122 – – DN DN
miR-141 – MCN/FSGS – –
miR-150 – DN MGN –
miR-184 – MCN/FSGS – MGN
miR-192 DN MCN/FSGS – MGN
miR-198 DNa – – –
miR-200a – – – –
miR-200b – MCN/FSGS – –
miR-200c – MCN/FSGS DNa –
miR-205 – MCN/FSGS MGNa –
miR-375 – MCN/FSGS – MGN
miR-429 – – DN –
miR-638 DNa DN – –
GS glomerulosclerosis, TIF tubulointerstitial fibrosis
Nonsignificant correlations ( p <0.06), adapted from Wang et al. (2013)
a
parameters between the various groups. These correlations clearly illustrate both the
complexity and redundancy of miRNA signaling systems. By their very nature,
dysregulation of any number of miRNA can ultimately result in similar outcomes.
However, given that miRNA is regulated as any other gene, their profiles can hint at
the source of the insult therefore aiding in differential diagnosis.
Changes in urinary miRNA in PKD have also been reported in comparison to
various stages of non-PKD CKD providing a further measure of differential diag-
nostic capability (Ben-Dov et al. 2014). This study compared urinary sediment,
urinary exosomes, and various cultured cell lines including primary proximal tubule
cells. Urine samples were obtained from males and females within each group, and
all samples were analyzed by high-throughput sequencing. This study produced a
massive amount of data, all of which is available online. The authors highlight that
there were increased levels of miR-223 and miR-199a/b in urinary sediments of
those with PKD compared to non-PKD CKD. Interestingly, miR-223 was increased
in DM plasma though, as already demonstrated, there can be vast differences in
miRNA profiles pending the source of the sample, both in regard to biofluids and
disease state of the donor (Zampetaki et al. 2010).
Lupus nephritis is a transient complication of systemic lupus erythematosus
which may be caused by a number of distinct glomerular phenotypes (Weening
et al. 2004). Furthermore, not all lupus patients will develop some form of nephritis,
and a single patient may display differing phenotypes throughout their lifetime.
Although lupus nephritis is generally easily treated, each phenotype requires spe-
cialized regimens, and therefore effective diagnosis without repeated biopsies is
required (Szeto 2014). Although the various phenotypes have not been studied in
regard to miRNA, Wang et al. reported that miR-145 and miR-155 were both
increased in urinary sediment of lupus nephritis subjects compared to controls
(Wang et al. 2012b). Furthermore, miR-145 levels correlated with GFR and
miR-155 with the level of proteinuria and also with SLEDAI score, an index used
in clinical studies which demonstrate lupus activity.
These examples are but a few of the many studies demonstrating the effectiveness of
utilizing miRNA as biomarkers for disease. However, as with many of those published
in the field of diabetic nephropathy, many of these have utilized small cohorts of
patients. The reasons for this are the same as those for reviewed studies concerning
DN, cost and sample availability. Regardless, it is obvious that miRNAs have a
promising future as diagnostic and prognostic tools given enough time and research
interest.
Concluding Remarks
This chapter has sought to highlight the current knowledge regarding the use of
miRNA in the diagnosis and detection of those with DN and also those at risk of
developing DN. It should be apparent that, although there has been a considerable
amount of work conducted in the field, there is still much to be done. Although the
above studies have clearly demonstrated that miRNAs show great potential as
clinical biomarkers, there remain a number of diverse challenges to be overcome
before miRNA can be effectively utilized in the clinic.
The most basic of these is technical congruence. The need for experimental
standardization is particularly highlighted by the Cheng study which demonstrated
differences in miRNA profiles from exosomes isolated through utilization of differ-
ent commercially available kits (Cheng et al. 2014). Furthermore, purification of
miRNA from blood and urine needs to be standardized due to the presence of whole
cells and cellular debris which unequivocally contain miRNA profiles which differ
to that of free or exosome-bound miRNA. Additionally, cellular fractions of plasma
and urine undoubtedly contain RNases which are free to degrade miRNA upon cell
lysis during miRNA isolation. These factors likely contribute to intra-study differ-
ences and clearly necessitate standardization of an inherently sensitive assay.
Another major concern in current miRNA biomarker studies is the cohort size in
the bulk of studies. While most clinical studies regarding pharmaceutical interven-
tion or protein-based biomarkers typically involve hundreds of subjects, miRNA
studies are typically restricted to no more than 20 subjects with many being limited
to fewer than 10 subjects per group. While it is easy to account this to sample
availability, one needs to keep in mind that miRNA biomarkers are typically sought
in noninvasive, freely available biofluids. For effective analysis of miRNA profiles
to be enabled, high-throughput sequencing methods must be employed which are
costly and require specialist knowledge. This cost can be offset by the use of
conventional qRT-PCR analysis at the cost of analytical depth. It is therefore

important that governments and other funding bodies recognize the costs involved
in establishing a putative miRNA profile which may be used in a clinical setting.
While this cost will initially undoubtedly be high, it will be greatly offset by greater
clinical management of those at risk of, and those with, DN.
In addition to the cost of sample analysis, there are added costs of prospective
studies such as those conducted by Zampetaki. Prospective studies are required to
enable development of miRNA profiles which may identify individuals at risk of
diabetes and, more specifically, DN. These types of studies provide the opportunity
to establish profiles which indicate the various stages of DN progression. However,
the cost of following large cohorts of patients is largely prohibitive to a developing
field of research. The desired outcomes of prospective studies also necessitate
multiple high-throughput sequencing of plasma/urine/exosomes from numerous
individuals over time which dramatically add to an already large project cost.
Again, if these costs can be met, we will see a shift in clinical diagnostic paradigms
which would be rapidly seen as reduction in costs incurred to the public health
sector.
There is a great need for improved diagnosis of DN and also those diabetics at risk
of developing DN. This need is generated both by a desire to improve patient quality
of life and also to reduce financial burden on public health systems associated with
ongoing cost of care. Furthermore, given the poor prognosis for those that progress
to end-stage renal disease coupled with a lack of organ donation, the need for
establishment of improved diagnostic protocols becomes much more evident. Estab-
lishment of these profiles may also lead to discovery of suitable miRNA targets for
therapeutic intervention in both extant and developing DN.
miRNA research is an exceedingly young field considering their discovery in
humans almost 15 years ago. It is exceptional that we are already beginning to see
the potential for miRNAs as noninvasive biomarkers for DN and other nephropa-
thies. However, we must learn to crawl before we can walk and walk before we can
run, although it seems that we have taken our first steps toward our goals of
improved prognostic and diagnostic tools. With continued funding, protocol stan-
dardization, and sample availability, there will be great progress in filling a pertinent
need in a burgeoning public health sector. For those involved in miRNA research,
these are exciting times with each day bringing us one step closer to establishing
biomarker profiles.
Summary Points
• Blood contains miRNA as either free, protein-bound complexes or packaged into

exosomes and can indicate dysfunction of a number of tissues.
• Various components of the nephron secrete miRNA into the urinary stream, and
therefore urinary miRNA provides an excellent marker for renal health.
• miRNA is dysregulated in diabetic nephropathy, and the profile of secreted
miRNA, both in serum/plasma and urine, is also dysregulated.
• Circulating miRNA and those excreted in urine are highly stable and easily
extracted therefore providing a source of noninvasive biomarkers for diabetic
nephropathy.
• Potential miRNA profiles, from both urine and plasma/serum, have been identi-
fied which can differentially diagnose specific stages of diabetic nephropathy
including the onset of microalbuminuria.
• Prediction of those that will develop type 2 diabetes or type 2 diabetic subjects
that will progress to diabetic nephropathy is also possible.
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Biomarkers in Focal Segmental
Glomerulosclerosis 34
Mohsen Nafar and Shiva Kalantari
Contents
Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781
Key Facts of TGF-ß Pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 781
Key Facts of Notch Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782
Key Facts of Wnt Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 782
Key Facts of NF-κB Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783
Key Facts of TWEAK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 783
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 784
Diagnostic Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787
Protein Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 787
Ribonucleic Acid Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 792
Metabolite Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 794
Prognostic Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795
WT1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 795
Afamin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797
AMBP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 797
Osteopontin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 798
Uncommon Prognostic Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 798
M. Nafar
Department of Nephrology, Labbafinejad Medical Center, Shahid Beheshti University of Medical
e-mail: nafar@sbmu.ac.ir; m.nafar.md@gmail.com
S. Kalantari (*)
Department of Basic Science, Faculty of Paramedical Sciences, Shahid Beheshti University of
e-mail: shiva.kalantari@sbmu.ac.ir; shivakalantari_81@yahoo.com

780 M. Nafar and S. Kalantari
Candidates for Both Diagnosis and Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 800

Predictive Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 801
Apolipoprotein A-I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 801
Alpha 1-B Glycoprotein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 802
Uncommon Predictive Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 802
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 803
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 805
Abstract
Focal segmental glomerulosclerosis (FSGS) is a podocyte-related disease and
one of the common causes of idiopathic glomerulonephritis. The pathogenesis of
FSGS is not well understood and still under study; however, some clues have
been found regarding implications of critical signaling pathways. Diagnosis of
FSGS like other glomerulopathies is based on renal biopsy. Although biopsy
considered a gold standard in nephrology world for diagnosis, it is invasive and
not always possible to be performed. Discovery of the biomolecules which are
easily measurable, noninvasive, specific, and sensitive and their changes reflect
the type and stage of the disease can simplify the diagnosis. These biomolecules
are referred to as “biomarkers” and can be complementary to biopsy for faster
and more accurate diagnosis. The advances in biomedicine fields and nascency
of the high-throughput platforms such as proteomics, transcriptomics,
metabolomics, and other related “omics” have brought the novel way of detec-
tion biomarkers in various diseases. In this review, we focus on the recently
presented biomarkers for diagnosis, prognosis, and prediction of the responsive-
ness to drugs for FSGS detected by different methods. However, most of the
current biomarkers are still under more examination; they will be dependable
complementary of traditional diagnostic methods in the future.
Keywords
Focal segmental glomerulosclerosis • Diagnostic biomarker • Prognostic bio-
marker • Predictive biomarker • Proteomics • Metabolite biomarker •
Ribonucleic acid biomarker • Podocyte
Abbreviations
2DE Two-dimensional electrophoresis
ADR Adriamycin
ARB Angiotensin receptor blocker
CLCF1 Cardiotrophin-like cytokine factor 1
ConA Concanavalin A
EMT Epithelial–mesenchymal transition
FDR False discovery rate
FN Fibronectin
34 Biomarkers in Focal Segmental Glomerulosclerosis 781
GBM Glomerular basement membrane

GC-MS Gas chromatography mass spectrometry
GPI Glycosylphosphatidylinositol
HLA Human leukocyte antigen
IEF Isoelectrofocusing
IGFBP Insulin-like growth factor-binding protein
INS Idiopathic nephrotic syndrome
LC Liquid chromatography
LN Lupus nephritis
MALDI-TOF Matrix-assisted laser desorption/ionization time of flight
MCNS Minimal change nephrotic syndrome
MCP-1 Monocyte chemotactic protein-1
MDA Malondialdehyde
MMF Mycophenolate mofetil
MMP9 Matrix metallopeptidase 9
MN Membranous nephropathy
NOS Not otherwise specified
RT-qPCR Real-time quantitative polymerase chain reaction
SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis
SELDI-TOF Surface-enhanced laser desorption/ionization time of flight
SRM Single reaction monitoring
SRNS Steroid-resistant nephrotic syndrome
SSNS Steroid-sensitive nephrotic syndrome
suPAR Soluble urokinase plasminogen activator receptor
TGF-ß Transforming growth factor-ß
TWEAK TNF-like weak inducer of apoptosis
UPAR Urokinase plasminogen activator receptor
Key Facts
Key Facts of TGF-ß Pathway
• TGF-ß signaling regulates a variety of cellular processes in embryonic and

mature cells such as: apoptosis, cell proliferation, and differentiation.
• This pathway is a conserved signaling pathway from invertebrates to higher
vertebrates.
• The signaling is begun by binding TGF-ß ligands to receptors on the cell surface,
activation of phosphorylation cascade of Smad proteins, translocation of the
Smad complexes into the nucleus, and regulation of the target genes accompa-
nied with other nuclear factors.
• TGF-ß is able to trigger extracellular component production in glomeruli and

hence is involved in pathogenesis of glomerular diseases including FSGS.
• Collagens (such as collagen type III), fibronectin, and proteoglycans are some of
the synthesized ECM proteins in damaged glomeruli.
• Induction of ECM component production leads to the increase of permeability of
glomeruli which results in proteinuria.
Key Facts of Notch Signaling
• Notch signaling is initiated by binding the ligands to the notch receptors which
switch on a series of proteolytic cleavage steps of notch. The final product of
these proteolytic steps is a transcription factor that can induce the target genes.
• The target genes of activated notch protein during the signaling pathway are Hes
and Hey transcription factors that are responsible for negative regulation of
tissue-specific differentiation.
• Notch signaling is important for developing the glomeruli and differentiation of
podocytes.
• Continued activity of notch signaling in mature podocytes can be harmful and
damage these cells.
Key Facts of Wnt Signaling
• Wnt signaling is reported for the first time in 1987 during the identification of an
oncogene named int1. This gene is homolog to wingless gene in Drosophila.
Therefore, the name “Wnt” comes from “int” and “wingless.”
• It is divided into two signaling pathways: canonical and noncanonical. The
noncanonical is further divided into two pathways: planar cell polarity pathway
and Wnt/calcium pathway.
• Canonical is considered as a pathway which is dependent to ß-catenin, and
noncanonical is considered as a pathway which is independent to ß-catenin.
• Each of the signaling pathways is triggered by different kinds of receptors. For
example, canonical pathway is triggered by Wnt1, Wnt3a, and Wnt8, while
noncanonical pathway is triggered by Wnt5a and Wnt11.
• Wnt proteins are secreted glycoproteins that act through frizzled family
receptors.
• Some of the Wnt protein functions include: cell migration, cell differentiation,
and cell proliferation.
• Canonical pathway can be inhibited by negative feedback of one of its target
genes named DKK1.
• Activation of Wnt-ß-catenin pathway through integrin-linked kinase in the
podocytes has been reported recently as one of the mechanisms involved
in FSGS.
Key Facts of NF-kB Signaling
• NF-κB is the abbreviation of nuclear factor kappa-light-chain enhancer of

activated B cells.
• This protein is a transcription factor that has a critical role in regulation of both
innate and adaptive immune systems.
• This family of DNA-binding protein has five members including: RelA,
NF-κB1, NF-κB2, cRel, and RelB.
• Activation of NF-κB signaling may affect podocyte cytoskeleton which ends up
to impaired glomerular permeability and leaking proteins into urine.
• Repression of NF-κB signaling is one of the mechanisms of glucocorticoid
therapy.
Key Facts of TWEAK
• TWEAK is the abbreviation of tumor necrosis factor (TNF)-like weak inducer of

apoptosis.
• TWEAK is a type of cytokine that binds to its receptor Fnt14 (FGF-inducible
molecule 14) and stimulates proinflammatory responses, angiogenesis, and cell
growth/death.
• It is involved in some inflammatory and autoimmune diseases including lupus
nephritis (LN).
• Fnt14 in the kidney is located in podocytes, tubular, and mesangial cells.
Definitions
Podocyte A type of highly differentiated cells in the glomerulus which composed

glomerular filtration barrier besides glomerular basement membrane and endothe-
lial cells.
End-stage renal disease The last stage of progression of kidney disease that renal
function is lost and dialysis or renal transplantation is recommended.
Circulating permeability factors Circulating proteins that cause abnormal per-

meability of glomeruli to the plasma proteins. These factors might be related to
dysfunction of T cells.
Extracellular matrix A collection of secreted molecules from the cells is mostly

composed of fibrous proteins and glycosaminoglycans which structurally and
biochemically support the cells. Cell-to-cell communication, cell adhesion, and
regulating the cell dynamics are some of the functions of extracellular matrix.
Glomerular filtration rate A calculated factor which can serve as a test for
evaluation of the kidney function or judgment about the progression of kidney
disease. This factor describes the rate of filtered plasma which passes through the
kidney.
MALDI-TOF technique One of the proteomic techniques for identification of

proteins extracted from different sources of samples using mass spectrometer. The
extracted proteome is mixed with a kind of matrix and ionized by laser and
delivered to an analyzer named “time of flight” which is connected to a detector.
A user can identify the molecules (e.g., proteins) using these experimental spectra
that are matched with theoretical spectra available in the database.
Single reaction monitoring technique This is a mass spectrometric-based tech-

nique for determining the absolute abundance of a molecule (e.g., protein) in the
sample. The abundance of the molecule is determined by a calibration curve of a
known molecule.
SELDI-TOF technique One of the proteomic techniques for profiling the prote-
ome of different types of samples using mass spectrometer. This technique does not
need sample preparation procedure and may serve as a diagnostic tool. Low
resolution and lack of reproducibility are some of the limitations of this technique.
Introduction
Focal segmental glomerulosclerosis (FSGS) is a podocyte-derived glomerular

disease described for the first time by Arnold Rich in 1959 (Rich 1959). This
clinical-pathological syndrome has multiple etiologies and pathogenic mecha-
nisms, and therefore it might be a combination of several pathological conditions
and not a single disease (Falk et al. 2000). The incidence of FSGS has been
increased in the past two decades and expected to be increasing since the proportion
of patients with primary FSGS has been raised recently and reached to approxi-
mately 25 % of adult nephropathies (Falk et al. 2000).
In fact the clinical features of FSGS are the features of a nephrotic syndrome and
include: peripheral edema, marked proteinuria (with or without nephrotic range),
hypertension, hypoalbuminemia, hyperlipidemia, and progressive loss of renal
function. It may be found either as primary or secondary forms. Primary FSGS
sometimes has no identifiable cause or known etiology (idiopathic) and linked to
genetic mutations in podocyte-specific proteins (e.g., membrane and podocyte slit
diaphragm proteins), while secondary FSGS may occur in response to previous
glomerular injury, glomerular hypertension, hypertrophy, drug toxicity (e.g.,
pamidronate, adriamycin), some malignancies or viral infections (e.g., HIV infec-
tion), obesity, and reduced renal mass. FSGS as one of the most common causes of
primary glomerular disease in children and adults may progress to end-stage renal
disease (ESRD) with a relatively high risk.
Pathological features of FSGS on light microscopy are recognized by sclerotic

lesions that affect some of the glomeruli (focal) and a portion of the glomerular tuft
(segmental). Accumulation of foam cells in glomerular capillary, swelling of epithelial
cells, collapse of capillaries, and expansion of extracellular matrix lead to hyalinosis
that also may be observed in microscopic section (Falk et al. 2000). Diffuse efface-
ment of foot process in podocytes is a significant finding on electron microscopy.
Based on these pathological features, five histological variants were described:
perihilar variant, cellular variant, tip variant, collapsing variant, and FSGS not
otherwise specified (NOS) (Deegens et al. 2008).
It is suggested that there is a relationship between the frequency of these
variants, race, and ethnicity. Accordingly, collapsing and cellular variants are
dominant in African American population, while they are relatively rare in Indian
and Dutch population (Deegens et al. 2008). In contrast tip variant is more frequent
in whites (Chun et al. 2004). In general, the frequency of FSGS variants according
to published literature is different. Accordingly NOS is the most frequent (up to
72.5 %) and collapsing is the least frequent (up to 24 %) variant.
These variants are also different in prognosis. In general, collapsing variant is
known to have the worst renal survival rate and hence has worse prognosis
compared to other variants, while the tip variant has clinical features similar to
that of minimal change disease (MCD) and responsive to steroid therapy, hence
considered to have good prognosis. Cellular variant is also known to have an
intermediate prognosis between the two variants.
The pathogenesis of FSGS is poorly understood. However, FSGS is known to be
caused by an abnormality in podocytes (highly differential epithelial cells of the
glomerulus).
Transforming growth factor-ß (TGF-ß) is the possible mediator of lesions in
different renal injuries including FSGS via induction of podocyte injury through
apoptosis, proliferation, and epithelial–mesenchymal transition (EMT) and
increased production of extracellular matrix proteins including collagen (Lee 2013).
The role of mutation in specific genes is also known to be involved in patho-
genesis of FSGS. Some of these genes are podocin (NPHS2), nephrin (NPHS1),
actinin-4 (ACTN-4), CD2-associated protein (CD2AP), WT1 transient receptor
potential cation 6 (TRPC6), and phospholipase C (PLCE1/NPHS3) (Woroniecki
and Kopp 2007).
Injury of epithelial and endothelial cells as a consequence of compensatory
capillary hypertension after loss of nephrons as well as mesangial transformation
is another hypothesis for FSGS pathogenesis (Falk et al. 2000). Lipid metabolic
disorders, abnormalities of the coagulation pathway, and alterations in T cell
function also may play important role in FSGS. Association of some specific
HLA types is known to be involved in familial and even nonfamilial forms of
FSGS. Accordingly, people who have these alleles of HLA are more prone to
FSGS: HLA-DR4, HLA-B12, HLA-DRw8, HLA-DRw5, HLA-B8, HLA-DR3
and HLA-DR7, and HLA-DR4303 and HLA-Bw53 (Falk et al. 2000).
There are also some evidence regarding involvement of Wnt/Ctnnb1 pathway
and notch signaling in podocytes of patients and animal models with FSGS. TGF-ß
is believed to act upstream of both pathways (Kato and Susztak 2012).
There are also several reports regarding the possible role of permeability factors
in FSGS pathogenesis via an effect on podocyte-specific proteins (nephrin and
podocin), phosphorylation of cellular proteins in the podocyte, activity of serine
proteases, and activity of integrin-like kinase that may lead to detachment of
podocyte from GBM (Sharma et al. 2004; Hattori et al. 2008; Carraro et al. 2004).
Treatment of FSGS follows two major goals: complete remission of proteinuria
and preservation of renal function. However, complete remission might not occur in
all cases because of heterogeneity of the disease and different responsiveness to
therapeutic strategies. Initial immunosuppressive therapy with corticosteroids
including prednisone, prednisolone, and methylprednisolone is the first line of
treatment for idiopathic nephrotic syndrome; however, a proportion of patients
might not respond to this therapy. The mechanism of action of corticosteroids in
FSGS still has some ambiguities and has not been completely elucidated.
Calcineurin inhibitors such as cyclosporine A and tacrolimus may be used as initial
therapy, especially in patients at increased risk for corticosteroids toxicity (Kato
and Susztak 2012). Angiotensin-converting enzyme (ACE) inhibitors or angioten-
sin II receptor blockers (ARBs), rosiglitazone (a peroxisome proliferator-activated
receptor-γ), antiproliferative agents such as mycophenolate mofetil (MMF),
adalimumab (a human monoclonal antibody), abatacept, and rituximab
(a chimeric monoclonal antibody) are the other possible treatments which could
be used for FSGS.
It is worthy to note that FSGS patients may respond to these therapies (especially
to steroid drugs) differently. Since prediction of responsiveness of FSGS patients to
steroid drugs is almost impossible at presentation, the steroid-resistant patients
might be imposed to side effects of an ineffective therapy and lose the time for
appropriate therapy. In spite of some attempts for discovery of the biomarkers
responsible for different responses, prediction of responsiveness before medication
is still impossible.
In addition, diagnosis of FSGS underlies on kidney biopsy. As FSGS variants
show different responsiveness to therapies, recognition of variant could be helpful
to choose the therapeutic strategy. Furthermore, some investigators have argued
that achievement of complete or partial remission of nephrotic syndrome (Hattori
et al. 2008) and quality of the responsiveness to therapies could be a better predictor
of outcome in FSGS than the histological variant. Biomarker discovery researches
are promising in this regard to extricate nephrologists from invasive biopsy and
bewilderment in choosing the best responsive and least risky therapy. Proteomics
and metabolomics as well as transcriptomics, the major platforms in biomarker
discovery, are valuable for differentiating between variants and also for diagnosis
and therefore reducing the rate of inefficient therapies; however, such these studies
are few.
In this review we focus on different types of biomarkers reported in the literature
for diagnosis and prognosis purposes as well as prediction of the responsiveness to
therapy for FSGS.

Conditions
However, FSGS is a well-characterized glomerular disease with defined features

(e.g., podocyte injury and histological patterns); its hallmarks are common with
other glomerular diseases which end up to nephrotic syndrome. A few of candidate
biomarkers that were reported in the literature and were named in the present
review such as alpha-1-antitrypsin, beta-2-microglobulin, and transferrin were
detected in other glomerular diseases as well (including IgA nephropathy, mem-
branous glomerulonephritis, and diabetic nephropathy). Detection of these
nonspecific candidates may let to provide a rational for this hypothesis: “however,
all the glomerular diseases have distinct specific mechanism; they are
interconnected with each other in some parts which lead to represent the same
symptoms including proteinuria and swelling.” Therefore, if we could recognize
this shared pattern of pathogenesis and sketching a therapy for that, a broad
spectrum of renal disease may be treated. This is not an unattainable dream.
Mapping of disease network for each glomerular disease using available bio-
markers and available information in the databases and comparing them via
advanced bioinformatic methods is one of the possible ways to make this dream
come true. Several attempts have been started to map a network for some renal
diseases recently that are still in progress.
Owing to this viewpoint and application, the nonspecific candidate biomarkers
should be considered as valuable as specific candidates. On the other hand, the
relative or absolute abundance of these nonspecific biomarkers might be quite
different in glomerular diseases which could be applied in the quantitative diag-
nostic protein arrays as a “panel of diagnostic/prognostic biomarkers” in the future.
Diagnostic Biomarkers
Protein Biomarkers
This section focuses on protein candidates for discrimination of FSGS from other
types of glomerular diseases or normal subjects reported in the literature (see
Table 1). The other kinds of biomarkers including mRNAs and small metabolites
have been discussed in the sections “Ribonucleic Acid Biomarkers” and “Metab-
olite Biomarkers.”
suPAR
suPAR stands for soluble urokinase plasminogen activator receptor, a circulating
form of uPAR, which is a membrane glycosylphosphatidylinositol (GPI)-anchored
protein in numerous types of cells including podocytes (Jefferson and Shankland
2013). suPAR is produced through proteolytic cleavage of uPAR in the linker
Table 1 Diagnostic protein biomarker candidates for FSGS

Protein
ID Protein name Source Analytical technique Reference
suPAR Soluble Serum/ ELISA kit/1DE-LC-MS/ Wei et al. 2011, 2012;
urokinase urine/ MS Huang et al. 2014;
plasminogen cultured Matsumoto et al. 2010
activator receptor podocyte
CD80 T lymphocyte Serum/ ELISA kit Cara-Fuentes et al. 2013
activation urine
antigen CD80
NGAL Neutrophil Serum/ Enzyme immunoassay Chehade et al. 2013;
gelatinase- urine Proletov et al. 2013
associated
lipocalin
Cys C Cystatin C Serum/ Enzyme immunoassay Proletov et al. 2013
urine
IGFBP- Insulin-like Urine – Worthmann et al. 2010
1 growth factor-
binding protein 1
IGFBP- Insulin-like Urine – Worthmann et al. 2010
3 growth factor-
binding protein 3
IGFBP- Insulin-like Cultured 1DE-LC-MS/MS Matsumoto et al. 2010
rP1 growth factor- podocytes
binding protein-
related protein-1
IBP7 Insulin-like Urine Nano-LC-MS/MS Nafar et al. 2014
growth factor-
binding protein 7
A1AT1 Alpha-1- Urine Nano-LC-MS/MS, Nafar et al. 2014;
antitrypsin 2DE-MALDI-TOF, Candiano et al. 2006;
magnetic bead peptide Navarro-Muñoz
enrichment-MALDI-TOF et al. 2012; Pérez
et al. 2014
Different protein biomarkers detected in urine, serum, or cultured cells which were discriminated
FSGS subjects from normal subjects or other glomerular diseases and hence have diagnostic value
region between domains DI and DII and released in the circulation in the form of
different fragments (Smith and Marshall 2010) and hence categorized as a circu-
lating permeability factor.
It is believed that suPAR acts through binding to and activating beta 3 integrin
signaling in podocytes. Two members of the integrin family (α3β1 and αvβ3) have a
major role in podocyte structure and function in which their aberrant activity via
binding to suPAR may lead to foot process effacement followed by proteinuria
(Wei et al. 2011). Contribution of suPAR-mediated signaling in FSGS is suggested
by Wei and colleagues in 2011 (Wei et al. 2011).
The role of circulating permeability factors in pathogenesis of FSGS has been
discussed in the literature and postulated to the following reasons: (a) rapid recur of
primary FSGS after kidney transplantation in adults and children (30 % and 50 %,
respectively) and delayed relapse using plasmapheresis before transplantation,
(b) occurrence of proteinuria in rats after injection of plasma fractions of patients
with FSGS, (c) increment of albumin permeability in an ex vivo model of isolated
glomerulus after addition of sera of FSGS patients, and (d) transmission of FSGS
from mother to fetus (McCarthy et al. 2010). Therefore, one can hypothesize that
suPAR as a circulating permeability factor which probably stems from injured
podocytes might be detected in serum or urine of FSGS patients and has diagnostic
value. Several research groups have tested this hypothesis from 2011 up to now
(Wei et al. 2011; McCarthy et al. 2010; Meijers et al. 2014). Wei and colleagues for
the first time reported elevated levels of serum suPAR in FSGS subjects compare
with other glomerular diseases (Wei et al. 2011). They reported elevated serum
suPAR level in native and recurrent FSGS cases and then verified this report in two
different cohorts composed of the FSGS Clinical Trial and the PodoNet European
FSGS consortium (Wei et al. 2011, 2012). Versus the fans of serum suPAR
diagnostic value in FSGS, Bock and Meijers believe that serum suPAR cannot
discriminate FSGS patients from non-FSGS patients or healthy controls (Bock
et al. 2013; Meijers et al. 2014).
Jefferson has reviewed the limitations of the studies which demonstrated elevated
levels of serum suPAR as follows: (1) Lack of enough sensitivity (only a part of
FSGS subjects in each group had elevated suPAR level). This might be due to
heterogeneity of the studied population or confounding factors that were not con-
sidered. (2) Sampling in different courses of disease in which levels of suPAR might
be variable. (3) Association of FSGS with other conditions which might result in
increased suPAR level such as inflammatory disorders, atherosclerotic disease,
myocardial infarction, systemic lupus erythematosus, and some of the cancers
(Jefferson and Shankland 2013). Although suPAR was detected in some studies as
a differential molecule in FSGS compared with healthy condition or other glomer-
ular diseases, being not specific to FSGS (it showed elevated level also in membra-
nous nephropathy (Wei et al. 2011)), inconsistent and irreproducible results in other
cohorts (Bock et al. 2013; Meijers et al. 2014) bring the value of suPAR as a valid
biomarker under the question. While suPAR level in serum is controversial, there are
no conflicting results in measuring urinary levels of suPAR in FSGS up to now.
Urinary suPAR was measured in a robust and well-designed study using ELISA
kit in 62 patients with primary FSGS (Huang et al. 2014). suPAR level is, then,
compared with minimal change disease (MCD), membranous nephropathy (MN),
secondary FSGS, and normal subjects. Subsequently, the effect of urinary suPAR
on activating β3 integrin was investigated in cultured podocytes. The conclusion
was interesting: urinary suPAR level is specifically increased in primary FSGS
(especially in cellular variant) and β3 integrin signaling is induced by urinary
suPAR level in human podocytes (Huang et al. 2014).
Taken together, suPAR is implied in pathogenesis of podocyte disorders such as
FSGS, and future investigations on its molecular mechanism will definitely help to
better insight of the disease. Although suPAR has pathogenic value, its diagnostic
value is still unclear. We suggest that measuring urinary suPAR might be more
promising in biomarker discovery of FSGS compared with serum suPAR which has
a heterogeneous population of fragments.
CD80
CD80 also known as B7-1 is a transmembrane protein which is mostly expressed on
the surface of B lymphocytes and antigen-presenting cells (Henry et al. 1999).
Expression of CD80 on podocytes in some patients with primary FSGS suggests the
contribution and relationship between this protein and FSGS (Reiser and Mundel
2004). Remodeling of actin cytoskeleton and modulation of components of the slit
diaphragm due to expression of CD80 on podocytes under stress conditions might
justify its contribution in the pathogenesis of FSGS (Reiser and Mundel 2004;
Reiser et al. 2004).
Recently, Cara-Fuentes demonstrated CD80 as a differential biomarker
between genetic form and primary FSGS and thus considered it as a diagnostic
urinary biomarker accompanied with serum suPAR (Cara-Fuentes et al. 2013). In
another study by the same group, CD80 is represented as a differential biomarker
between MCD and FSGS (Cara-Fuentes et al. 2014). The latter has been reviewed
by Davin in a letter in Pediatric Nephrology journal recently (Davin 2014). Devin
believes in bias in their conclusion regarding pathophysiological role of CD80 in
MCD patients and not in FSGS and linked this bias to higher number of values
considered in MCD compared with in FSGS. On the other hand, their claim was
discordant with the report of Yu et al., based on positive staining for B7-1 (CD80)
along peripheral capillary walls in biopsy slides of patients with primary FSGS
(two of three) and MCD (three of five). In general, Yu et al. showed clear
involvement of CD80 at least in some cases in the pathogenesis of FSGS and
suggested CD80 as a treatment biomarker for FSGS and a target for abatacept
(Yu et al. 2013).
Albeit there is conflict about the pathophysiological role of CD80 in FSGS in the
mentioned studies, diagnostic value of CD80 cannot be excluded. Evaluating CD80
is suggested with other high-resolution techniques and not only by commercially
available kits and in larger cohorts.
uNGAL
Neutrophil gelatinase-associated lipocalin (NGAL), also called lipocalin 2 or
siderocalin or 24p3, is a member of lipocalin superfamily (Flower 1996). NGAL
has several functions including participation in innate immune system (Goetz
et al. 2002), activation or repression of iron-responsive genes (Yang et al. 2002),
and induction of apoptosis through deprivation of trophic factors (Devireddy
et al. 2005). This protein was detected originally in form of bound with MMP9 in
human activated neutrophils (Kjeldsen et al. 1993). High expression of NGAL in
human kidney in response to injury and correlation of its serum and urinary level
with the severity of renal injury have been reported in various studies, and, hence, it
is considered as a promising biomarker in clinical nephrology. NGAL, specially its
urinary isoform (uNGAL), is mostly referred to as a sensitive biomarker for
progression of acute kidney injury (Makris et al. 2009). Diagnostic value of this
protein has been demonstrated for FSGS recently. Chehade et al. showed uNGAL
could significantly discriminate between healthy children and those with FSGS
( p = 0.007) and between children with MCD and those with FSGS ( p = 0.01)
(Chehade et al. 2013). Proletov et al. also measured uNGAL and Cyc C in
104 patients with primary glomerulopathies including FSGS. Urinary Cys C and
NGAL excretion in their study correlated with the degree of glomerulosclerosis and
proteinuria and the reduced glomerular filtration rate (GFR) regardless of the
method of its determination (Proletov et al. 2013). According to Korzeniecka-
Kozerska’s findings, MMP-9/NGAL ratio may serve as differentiation marker
between minimal change nephrotic syndrome (MCNS) and FSGS in nephrotic
children (Korzeniecka-Kozerska et al. 2013). Although Kozerska’s study had
some limitations, it was well designed. The limitations were small group of patients
who were under treatment at enrollment, lack of biopsy for MCNS group, and use
nonparametric statistics to analyze the data. The positive point of their study was
subdividing the cohort into two groups and subjected them to examination twice
(before treatment and after regression of proteinuria) which shows a wise study
design.
Although there are not enough definitive studies regarding the role played by this
molecule in the pathogenesis of kidney diseases and specially FSGS, this seems to
be a promising diagnostic marker of FSGS besides its well-known potential value
for monitoring the progression of chronic kidney diseases.
IGFBPs
Circulating growth factors that are filtered from the bloodstream and pass the
glomerular filtration barrier influenced glomeruli and seem to be important candi-
dates for renal disease pathogenesis as well as diagnosis. Several members of
insulin-like growth factor-binding protein family with the gene name of IGFPB
have been demonstrated as a diagnostic urinary marker for FSGS. Urinary excretion
of IGFBP-1 and IGFBP-3 was reported by Worthmann and Peters as discriminating
proteins between FSGS and MCD patients (Worthmann et al. 2010). They could
find a relationship between the local expression of IGFBPs in podocytes as well as
endothelial cells and the pathogenesis of glomerular disease. The induction of
podocyte expression of these proteins by TGF-beta and bradykinin in this study
suggested the involvement of TGF-beta signaling in the pathogenesis of FSGS. A
recent proteomic study on podocyte cultured cells and mouse model of FSGS
validated the role of IGFBP-rP1 in pathogenesis of podocyte disorders like FSGS
(Matsumoto et al. 2010) and strengthened the probability of nomination of IGFBP
family as biomarker. A recent data regarding noninvasive urinary diagnostic bio-
markers for FSGS also revealed another member of insulin-like growth factor-
binding proteins, namely, IBP7 (Nafar et al. 2014). Despite the small cohort of this
study, detection of IBP7 and other suggested biomarkers in this study using a high-
resolution mass spectrometry technique and high quality of multivariate statistical
analysis were valuable.
Alpha-1-antitrypsin
Alpha-1-antitrypsin (A1AT), also referred to as SERPINA1, is a member of a
family of serine protease inhibitors. A1AT inhibits trypsin, chymotrypsin, and
plasminogen activator irreversibly. Serpins have several functions in the cell,
including regulation of homeostasis, cellular survival, and blood clotting
(Normandin et al. 2010). Urinary A1AT or at least some fragments of this protein
in patients with FSGS have been reported by several studies. Candiano et al. used
proteomic technique (two-dimensional electrophoresis followed by MALDI-TOF
MS) to detect specific pattern of A1AT fragmentation in association with albumin
in the urine of subject with nephrotic syndrome (FSGS, MCD, and MGN)
(Candiano et al. 2006). Their findings were confirmed by Western blotting.
Navarro-Muñoz et al. also reported two major peptides belonging to uromodulin
and A1AT that distinguished between proliferative forms of glomerular kidney
disease and nonproliferative forms (Navarro-Muñoz et al. 2012). According to their
findings, nonproliferative forms correlated with higher A1AT peptide levels of
which focal segmental glomerulosclerosis was linked more closely to high levels
of the m/z 1945 peptide (one of the peptides of A1AT) than minimal change
disease. They used magnetic bead peptide enrichment, MALDI-TOF MS analysis,
and ClinProTools v2.0 to select differential peptides. The same group recently
published another paper regarding urine peptide profiling of FSGS and MCD
patients. Alpha-1-antitrypsin, in their study, showed lower peak area (unlike
uromodulin with higher peak area) in FSGS patients compared with MCD patients
and hence could be suggested as a potential diagnostic urinary marker for FSGS
(Pérez et al. 2014). Elevated urinary excretion of A1AT is also reported in a
proteomic study on FSGS and healthy controls using nano-LC-MS/MS technique
(Nafar et al. 2014).
To sum up detection of A1AT by several advanced proteomic techniques in
FSGS is enough to candidate this protein as a potential diagnostic biomarker;
however, it does not seem to be highly specific.
Ribonucleic Acid Biomarkers
Fn14 and MCP-1 mRNA, miR-192, miR-205 and miRNA-186, miR-10a, and
miR-30d and miR-200c are recent ribonucleic acid biomarkers reported for FSG
(see Table 2). A comprehensive transcriptomic study on cultured podocytes and
two distinct populations of patients with FSGS showed upregulation of Fn14 and
monocyte chemotactic protein-1 (MCP-1) mRNA in glomeruli from patients with
focal segmental glomerulosclerosis (Sanchez-Niño et al. 2013). Expression of
both transcripts was also correlated. This finding also was confirmed in a second
focal segmental glomerulosclerosis cohort. As NF-κB inhibitor parthenolide
could prevent the activation of NF-κB signaling mediated by TWEAK activation
and the following increase of MCP-1 mRNA and protein, a regulatory role was
suggested for TWEAK receptor Fn14 in mediating glomerular inflammation. A
significant difference observed in the urinary mRNA expression of MCP-1 (and a
Table 2 Diagnostic ribonucleic acid candidate biomarkers

Name of Analytical
transcript Name of expressed protein Source technique Reference
Fn14 Fibroblast growth factor- Cultured RT-PCR Sanchez-Niño
mRNA inducible immediate-early podocyte et al. 2013
response protein 14
MCP-1 Monocyte chemotactic protein-1 Cultured RT-PCR Sanchez-Niño
mRNA podocyte/ et al. 2013; Szeto
urine et al. 2005
miR- – Serum RT-qPCR Cai et al. 2013
192,
miR-205
miR-186 – Serum RT-qPCR Zhang et al.
2014a
miR- – Urine RT-qPCR Wang et al. 2013
200c
miR-10a, – Urine RT-qPCR Wang et al. 2012
miR-30d
mRNA or microRNA diagnostic biomarkers detected in different sources by transcriptomic
techniques
few other target genes) between disease groups (including IgA nephropathy and
glomerulosclerotic patients) and controls in the other transcriptomic study is
performed by real-time polymerase chain reaction (RT-PCR) (Szeto
et al. 2005). In this report urinary MCP-1 expression correlated with the degree
of glomerulosclerosis.
MicroRNAs (miRNAs, miRs) are involved in most physiological, developmen-
tal, and pathological processes and hence scientists devoted much attempts to
investigate miRNAs recently. miR-192 and miR-205 are expressed preferentially
in the renal cortex and believed to have a role in renal diseases. Serum levels of
these two miRNAs in FSGS, MCD, and healthy control subjects have been evalu-
ated by RT-qPCR technique (Cai et al. 2013). Accordingly patients with FSGS had
higher serum levels of miR-192 and miR-205 than those with MCD. Positive
correlation between the levels of miR-192 and miR-205 and proteinuria is also
observed in FSGS patients.
Plasma miR-186 was proposed by Zhang et al. (2014a) as a biomarker for FSGS
with nephrotic proteinuria (Zhang 2014a). They applied quantitative reverse
transcription–polymerase chain reaction analysis for studying the plasma miRNAs
in FSGS and healthy subjects.
Wang and colleagues in two distinct studies reported urinary miR-10a and
miR-30d as differential candidate biomarkers for FSGS and healthy kidney donors
and urinary miR-200c as differential between FSGS or MCD patients and those
with other causes of nephrotic syndrome (Wang et al. 2012, 2013).
Since miRNAs are relatively stable and many of them can be found in extracel-
lular fluid such as plasma, serum, and urine in addition to tissues and cells, we
believe that they can be ideal source of biomarkers for disease. In this regard,
miRNAs beside protein biomarkers are promising in diagnosis of glomerular

diseases such as FSGS.
Metabolite Biomarkers
Metabolomics is defined as “the quantitative measurement of the dynamic

multiparametric metabolic response of living systems to pathophysiological stimuli
or genetic modification” (Nicholson et al. 1999). A vast variety of molecules are
considered as metabolites such as: lipids, sugars, amino asides, and small peptides.
The major techniques for detection and investigation of these molecules are nuclear
magnetic resonance (NMR), gas chromatography coupled with mass spectrometry
(GC-MS), and liquid chromatography coupled with mass spectrometry. Serum and
urinary metabolites are appropriate candidates for noninvasive diagnosis and could
shed light on the pathogenesis of the diseases and especially renal diseases.
In this regard, Rosendale et al. analyzed urinary metabolites using NMR tech-
nique on patients with membranous lupus nephritis (LN), proliferative lupus
nephritis, and FSGS (Romick-Rosendale et al. 2011). They suggested hippurate
as a perfect discriminator for distinguishing between class V LN and FSGS patients
with 100 % specificity, 100 % sensitivity, and 100 % accuracy.
Later, Hao et al. performed a comprehensive study on urinary metabolite bio-
markers differentiating FSGS from other glomerular diseases with the same tech-
nique (Hao et al. 2013).
In FSGS patients they detected elevated urinary levels of glucose,
dimethylamine, and trimethylamine compared with healthy controls and
decreased level of pyruvate, valine, hippurate, isoleucine, phenylacetylglycine,
citrate, tyrosine, 3-methylhistidine, and β-hydroxyisovalerate. Additionally,
FSGS patients had lower urine N-methylnicotinamide levels compared with
other glomerulopathies. The advantages of this study are (1) a relatively enough
number of glomerular diseases as control disease, (2) enough clinical data with
consideration of exclusion criteria, and (3) strong statistical analysis with valida-
tion using permutation tests.
In a recent study, urinary proteins and metabolites from pediatric idiopathic
nephrotic syndrome (INS) are profiled via label-free mass spectrometry technique
(Sedic et al. 2014). Hydroxyphenylacetate and uridine showed upregulation, while
glutamine and phenylalanine levels showed downregulation in INS subjects. The
data showed involvement of oxidative stress in pathogenesis of nephrotic syn-
drome. However, they revealed involvement of some specific metabolites for the
first time; the study had some limitations including a relatively small cohort and
lack of specification of nephrotic syndrome.
Serum and urinary malondialdehyde (MDA), an end product of lipid peroxida-
tion, are suggested as a diagnostic biomarker discriminating FSGS from MCD
patients by two research groups during 2005 and 2010 (Kuo et al. 2005; Nezhad
et al. 2010). Since MDA is an important and known biomarker of oxidative stress,
these findings emphasize the involvement of oxidative stress-induced lipid
Table 3 Diagnostic metabolite candidate biomarkers

Analytical
Metabolite name Source technique Reference
Hippurate Urine NMR Romick-
Rosendale et al.
2011
Glucose, dimethylamine, Urine NMR Hao et al. 2013
trimethylamine, pyruvate, valine,
hippurate, isoleucine,
phenylacetylglycine, citrate, tyrosine,
3-methylhistidine, β-hydroxyisovalerate,
N-methylnicotinamide
Hydroxyphenylacetate, uridine, Urine Label-free mass Sedic et al. 2014
glutamine, phenylalanine spectrometry
Malondialdehyde Serum/ Immunostaining Kuo et al. 2005;
urine/ Nezhad
glomeruli et al. 2010
Metabolites detected in urine, serum, or tissue of FSGS subjects using metabolomics techniques
for diagnosis purposes
peroxidation in FSGS pathogenesis. Metabolite candidate biomarkers which are

discussed above have been tabulated in Table 3.
Prognostic Biomarkers
We focus here on the important protein biomarkers reported in the literature.

There is no adequate evidence regarding ribonucleic acid or metabolite bio-
markers for monitoring the disease progression. Future analysis using powerful
high-throughput techniques that are complement of proteomics such as microarray
in transcriptomic platform and NMR or GC-MS in metabolomics platform may
clarify new candidates for prognosis of FSGS. Protein candidate biomarkers which
are presented here have been tabulated in Table 4.
WT1
WT1, a transcription factor belonging to zinc finger superfamily, is associated with

podocytes and essential for normal podocyte function (Zhou et al. 2008). It has a
crucial role in development of kidney (maturation of podocytes) and genital system
and regulates its target genes by binding to both DNA and messenger RNA
(mRNA). Its mutation is associated with some diseases including Denys–Drash
syndrome (mutation in exon 8 or 9), Frasier syndrome (mutation in intron 9), and
WAGR syndrome (a deletion in WT1) (Niaudet and Gubler 2006; Fischbach
et al. 2005). All of these mentioned syndromes are associated with FSGS. Since
one of the target genes of this transcription factor is TGF-ß (Niaudet and Gubler
Table 4 Prognostic candidate biomarkers

Protein
ID Protein name Source Analytical technique Reference
WT1 Wilms tumor Urinary exosome/ Western blot Zhou et al. 2013
protein podocyte-like cell
line
AFAM Afamin Urine LC-MS/MS, Zhao et al. 2014;
concanavalin A Kalantari et al.
(ConA) enrichment 2014a
AMBP α1- Urine Nano-LC-MS/MS Zhao et al. 2014;
Microglobulin Kalantari et al.
2014a
OPN Osteopontin Glomeruli, urine RT-PCR, Western Shui et al. 2007
blot,
immunohistochemistry
Protein biomarkers useful for monitoring the disease severity
2006), one can hypothesize that one of the mechanisms of its involvement in
glomerular diseases especially those with podocyte disorders could be via TGF-ß
signaling. The relationship between TGF-ß and WT1 was described for the first
time in a study in 2011 on human podocyte cell line treated with TGF-ß1 and
kidneys in Alb/TGF-beta1-transgenic mice (Sakairi et al. 2011). Their findings
showed TGF-beta1 reduced WT1 expression. It appears that there is a complex
and mutual relationship between WT1 and TGF-ß and they can regulate each other.
In the latter study, also WT1 is introduced as a potential useful marker of early
podocyte injury.
Urinary exosomal WT1 is showed by another study to be a prognostic marker
for monitoring the progression of podocyte injury in animal models as well as a
diagnostic biomarker in FSGS patients (Zhou et al. 2008). Their findings showed
the increased WT1 very early in the first and third day after puromycin injection,
before albuminuria was detectable, and continued elevation at 7th day to 14th day
in PAN-treated rats. Later, another study by the same author revealed urinary
exosomal WT1 as a predictor of the onset of disease earlier than proteinuria in a
mouse model of collapsing glomerulopathy (Zhou et al. 2013). Their findings also
indicated the potential differentiating power of urinary exosomal WT1 in
distinguishing active FSGS from active steroid-sensitive nephrotic syndrome
(SSNS). WT1 in these studies was not detected in healthy subjects.
As several studies show the involvement of WT1 in FSGS and podocyte injury
(elevated level with the increase of podocyte injury), it could be a useful candidate
for diagnosis as well as prognosis. We suggest more investigations on the level of
changes in a cohort consist of patients with well (lower proteinuria or higher GFR)
and worse (higher proteinuria or lower GFR) prognosis using high-resolution
techniques like single-reaction monitoring (SRM).
Afamin
The glycoprotein afamin, a member of the albumin gene family, is a vitamin

E-binding protein which plays a crucial role in oxidative stress-related anti-apoptotic
cellular processes (Heiser et al. 2002). It has been detected in kidney diseases and
described as a candidate biomarker; nonetheless, very little is known about its
physiological or pathological function. However, there is only a single report up to
now regarding afamin as a prognostic biomarker for FSGS and involvement in disease
progression; we brought it here because of reliable and valid data as well as the value
of this study in terms of discovery of noninvasive biomarkers in urine. Proteomic
technique such as LC-MS/MS (triple TOF mass spectrometer) accompanied by
Western blot and concanavalin A (ConA) enrichment of urinary proteins are state-
of-the-art techniques which authors applied (Zhao et al. 2014). They reported a 1.16-
fold change in the rat urine on the third day after ADR injection with a continuous
elevation of up to 5.63-fold on day 23 after the ADR injection. Despite this fact that
afamin is an orthologous protein in human and also detection of this protein in a pilot
study on FSGS patients used high-resolution proteomic tools (Kalantari et al. 2014a),
further studies on human samples in a large cohort are essential.
AMBP
α1-Microglobulin (AMBP), a 27–33 kDa glycoprotein, is synthesized by the liver

and is a known marker for tubular proteinuria. It can pass freely through filtration
barrier in glomerulus and reabsorbed by the proximal tubule (Devarajan et al. 2010).
Some of the possible roles of α1-microglobulin are immunoprotective/anti-
inflammatory role, inhibition of IL-1-beta, and monocyte free radical production
in a complex form with collagen (Santin and Cannas 1999). Nevertheless, the full
biological function of this protein is not clear. It has been reported previously as a
prognostic marker for nephrological disorders such as membranous nephropathy and
diabetic nephropathy (Ponticelli and Passerini 2010; Marczewski et al. 1996) as well
as a diagnostic marker for urological disorders (Everaert et al. 2000). AMBP was
detected for the first time as a prognostic biomarker in the urine samples from FSGS
patients with different prognoses using proteomic tools (Kalantari et al. 2014a).
Soon later another research group reported AMBP as a useful candidate for early
detection of FSGS, as it decreased during the first 7 days after ADR injection to the
rat models (Zhao et al. 2014). They verified the findings using Western blot.
Although changes of AMBP in both studies were detected during different phases
of FSGS in the urine samples from rat models and human subjects, the trend was
opposite. This protein with animal source showed a decrease during the early phase
of ADR-induced FSGS; however, it indicated an increase with the ratio of 4.39 from
the human source. Since both studies applied powerful analytical tools, the judgment
is tough and further analysis with a large sample size may ensure us about the trend
of changes of AMBP during the disease course.
Osteopontin
Osteopontin (OPN) is an acidic 70 kD glycoprotein that is classified as an extra-

cellular matrix (ECM) protein. Various cell types can secrete OPN in the normal
conditions including vascular smooth muscle cells, lymphocytes, macrophages,
epithelial cells, cells of tubular system, and also podocyte cells in the pathogenic
conditions (Schordan et al. 2013).
Overexpression of OPN is a well-known marker for tubulointerstitial diseases,
FSGS, and diabetic nephropathy (Shui et al. 2007; Susztak et al. 2004). OPN
mechanisms of action in glomerular injury have been investigated in several studies
(Shui et al. 2007; Lorenzen et al. 2008; Endlich et al. 2002; Nakamura et al. 2005;
Teramoto et al. 2005). It is suggested that it induces the expression of urokinase-
type plasminogen activator (uPA), MMP-2, and MMP-9 through the NF-κB path-
way which results in increased podocyte motility and proteinuria (Lorenzen
et al. 2008). Contribution of OPN in F-actin reorganization involved in podocyte
foot process effacement (Endlich et al. 2002), recruiting macrophage toward the
compromised glomeruli (Shui et al. 2007), and binding of OPN to CD44 which
induces the loosening of cell-matrix adhesion and cellular crescent formation are
other possible mechanisms (Nakamura et al. 2005; Teramoto et al. 2005). However,
some of the investigators believe that OPN is essential for preventing early damage
of podocytes rather than promoting glomerulosclerosis or recruitment of macro-
phages (Schordan et al. 2013). Therefore, there is still controversy regarding
inflammatory role of OPN in developing FSGS and its proinflammatory role in
preventing podocyte injury.
Besides several attempts to elucidate the pathophysiological role of OPN, a well-
designed study by Shui and colleagues (Shui et al. 2007) showed its potential
capacity as a biomarker of disease progression in FSGS. They applied RT-PCR
and Western blot to evaluate OPN in isolated glomeruli and urine from
FSGS-induced rat models and immunohistochemistry technique to assess the
OPN expression in epithelial lesions and macrophage infiltration around the glo-
meruli. They found that OPN mRNA and protein correlate with sclerosis and may
be helpful in the prognosis of FSGS. The only weak point of their study was the lack
of comparison of the results with human subjects.
Uncommon Prognostic Biomarkers
Serotransferrin, kininogen-1, fibronectin (FN), Rab23, and annexin A1 are some of

the well-defined prognostic biomarkers in FSGS-induced rat models that are not
validated in human subjects (Zhao et al. 2014; Shui et al. 2006, 2008) (see Table 5).
Serotransferrin and kininogen were detected in the urine of rat models by two
independent research groups that both applied proteomic techniques for identifica-
tion (LC-MS/MS and two-dimensional electrophoresis followed by MALDI-TOF-
MS). Other candidates that had dynamic changes in the urine of FSGS-induced rat
Table 5 Uncommon prognostic candidate biomarkers

Protein name Source Analytical technique Reference
Kininogen, kallikrein, glutathione Urine 2DE-MALDI-TOF Shui
S-transferase, apoptosis-inducing factor-2, et al. 2008
annexin A1, E-cadherin, collagen fragment,
ECM protein 1, cerberus, tomoregulin
fragment, ADAM 32
Ceruloplasmin, cadherin-2, fetuin-B, beta- Urine LC-MS/MS Zhao
2-microglobulin, alpha-1-antiproteinase, et al. 2014
alpha-2-HS-glycoprotein
Fibronectin Serum, Immunoassay, Shui
tissue immunohistochemistry, et al. 2006
Western blot
Rab23 Serum, Western blot, RT-PCR, Huang
urine, immunohistochemistry et al. 2009
tissue
Annexin A1 Urine, 2DE-MALDI-TOF , Shui
tissue immunohistochemistry et al. 2008;
Ka et al. 2014
Ribonuclease 2, CD59, prostaglandin-H2 Urine Nano-LC-MS/MS Kalantari
D-isomerase, beta-2-microglobulin, et al. 2014a
extracellular sulfatase 2, corticosteroid-
binding globulin, matrix-remodeling-
associated protein 8, collagen alpha-1
(VI) chain, actin, and haptoglobin
Prognostic candidates detected in different sources which are reported in single study or only in
animal models without validation in human subjects
models and are identified by 2DE-MALDI-TOF technique were kallikrein and

kininogen precursor which caused hemodynamic problems, glutathione
S-transferase, apoptosis-inducing factor-2 (AIF-2), annexin A1 which is involved
in raised oxidative stress and apoptosis, E-cadherin which causes epithelial cell
damage, collagen fragment, ECM protein 1, cerberus, tomoregulin fragment, and
ADAM 32 which affects glomerular sclerosis (Shui et al. 2008). The urinary
candidate proteins with dynamic changes during FSGS induction identified using
LC-MS/MS were ceruloplasmin, cadherin-2, fetuin-B, and beta-2-microglobulin.
The following proteins, identified using LC-MS/MS, are found to be orthologous
with human proteins and verified by Western blot: alpha-1-antiproteinase, alpha-2-
HS-glycoprotein, as well as afamin and AMBP which are mentioned earlier (Zhao
et al. 2014). Fibronectin (FN) is suggested by Shui’s research group in 2006 (Shui
et al. 2006). They measured FN in the serum and urine using immunoassay tech-
nique and assayed in the tissue using Western blot, real-time PCR, and immunohis-
tochemistry. As the increased trend of FN was first detected in the serum, then in the
glomeruli and ultimately in the urine, one hypothesized that elevation of serum FN
may contribute to FN deposition in glomeruli and final release to the urine. Verifi-
cation of this valuable finding with human subjects has not been performed yet.
Rab23 was introduced in 2009 as a candidate biomarker that indicates the

severity of FSGS in mice models (Huang et al. 2009). It was evaluated in the
serum, urine, and mice kidneys by Western blot and monitored up to 20 days after
FSGS induction using ADR injection to the mice models. Real-time PCR and
immunohistochemistry are also applied to investigate its expression. The elevations
of Rab23 were observed in the urine and glomeruli (mesangial cells) but not in the
serum. The possible role of elevated Rab23 in FSGS state might be suppression of
hedgehog signaling and/or influence collagen synthesis.
Urinary annexin A1 was detected in two independent studies on adriamycin-
induced glomerulopathy mice models (Shui et al. 2008; Ka et al. 2014). However,
in one of them, it was also measured in the urine of FSGS patients and other
glomerulopathy human subjects; it was not specific for FSGS and hence we
considered it as uncommon prognostic marker. Annexin A1 was also evaluated in
the glomeruli by immunohistochemistry technique. The authors suggested that
urinary ANXA1 comes from damaged and/or apoptotic renal tissues.
Prognostic urinary biomarker candidates recently detected in FSGS patients
using nano-LC-MS/MS technique that are not confirmed by complementary tech-
niques or in a larger population are ribonuclease 2, CD59, prostaglandin-H2
D-isomerase, beta-2-microglobulin, extracellular sulfatase 2, corticosteroid-
binding globulin, matrix-remodeling-associated protein 8, collagen alpha-1
(VI) chain, actin, and haptoglobin (Kalantari et al. 2014a).
Because all of these candidates are detected in one single experiment without
validation or only in animal models, we brought them under the title of “uncommon
prognostic biomarkers.”
Candidates for Both Diagnosis and Prognosis
Several biomarker candidates may serve as both diagnostic and prognostic includ-
ing: urinary NGAL, urinary suPAR, and WT1.
These kinds of biomarkers appear to be more important and worth for more
investigation and validation due to their capability of determining the type and
severity of kidney damage simultaneously in a single test in the future clinical
world.
uNGAL had a decrease in children with FSGS (in remission) in a 1-year follow-
up study. Its level correlated negatively with creatinine clearance and correlated
positively with both reduction of GFR and proteinuria (Youssef and El-Shal 2012).
It should be noted that the elevated level of uNGAL compared with healthy subjects
could be used as a diagnostic marker, while its decreased level is important to
monitor the prognosis of FSGS.
In a comprehensive study on evaluating the urinary suPAR changes among
FSGS patients and other nephropathies and normal controls, urinary suPAR could
discriminate FSGS from other conditions as well as FSGS with complete remission
after therapy (Huang et al. 2014). Accordingly, a decreasing trend was observed in
the median of urinary suPAR level in eight patients with complete remission but not
partial remission after 80 weeks follow-up.
Several studies on exosomal WT1 on mice models as well as human subjects
verified its value as a candidate biomarker for diagnosis and prognosis (Zhou
et al. 2008, 2013; Orloff et al. 2005).
Predictive Biomarkers
Predictive biomarkers mostly refer to biomolecules that have different amounts in

patients who are responder or nonresponder to a selective drug. In addition, these
kinds of biomolecules can predict the state of relapse or remission before initiation
therapy or predict the recurrence of FSGS after renal transplantation. Although
predictive biomarkers are very applicable in saving time for choosing the appro-
priate therapy and preventing the side effects of noneffective therapy, the number of
studies on these kinds of biomarkers for FSGS is very few. The noninvasive
biomarkers detected in urine or serum have special value for prediction and most
of the studies carried on these specimens. Because there are few overlaps between
these studies and there is no validated biomarker for responsiveness or recurrence
prediction, most of the following mentioned biomarkers may be considered as
“uncommon predictive biomarkers.” However, some of them might have diagnostic
or prognostic value as well which were explained in sections “Diagnostic Bio-
markers” and “Prognostic Biomarkers.”
Apolipoprotein A-I
Apolipoprotein A-I (APOA-I) was detected in two independent studies on urinary

predictive biomarkers on responder and nonresponder FSGS patients to steroid
therapy and relapsing and non-relapsing FSGS patients (Lopez-Hellin et al. 2013;
Kalantari et al. 2014b). Proteomic tools were applied in both studies.
Two-dimensional electrophoresis followed by MALDI-TOF analysis showed a
form of apolipoprotein A-I named APOA-Ib exclusively in the urine of relapsing
FSGS. This study suggested the capacity of APOA-Ib for prediction of relapse
before the clinical manifestation. This finding was verified using Western blot in the
same patients as well as independent group of patients (Lopez-Hellin et al. 2013).
Kalantari et al. applied nano-LC-MS/MS technique and detected 3.15-fold change
of APOA-I in the urine of steroid-sensitive compared to steroid-resistant FSGS
patients. The false discovery rate (FDR) <1 % in label-free quantification and
introducing a predictive model with 100 % accuracy using multivariate statistical
analysis for differentiating steroid-sensitive and steroid-resistant group are the
strength aspects of this study (Kalantari et al. 2014b). They also reported 20 other
candidates besides APOA-I as urinary predictive biomarkers for FSGS based on
multivariate predictive model: PGRP2, ACTG, FBLN3, PGBM, AACT, IGHG1,

TRFE, YIPF3, A1AG2, THBG, A1BG, A2GL, ANAG, S10A9, CUBN, TITIN,
AMPN, CLUS, IPSP, and MXRA8.
It is not well understood whether APOA-I is a consequence of relapsing FSGS or
is related to its pathogenesis or how it is related to responsiveness, but there are
some evidence about involvement of HDL or its components (including APOA-I
and APOA-II) in FSGS. One possible hypothesis is the different sclerotic potential
of SSNS patients in comparison with SRNS due to different excretions of APOA-I
(Kalantari et al. 2014b).
Alpha 1-B Glycoprotein
Alpha 1-B glycoprotein (A1BG) is a member of the immunoglobulin superfamily

with an unknown function. Detection of this protein in two independent studies on
urine proteome profile of FSGS patients with different responsiveness enhances the
possibility of being a specific biomarker of A1BG for differentiating SSNS from
SRNS. Upregulation of this protein in the urine of steroid-resistant FSGS patients
was consistent in both reports. Piyaphanee and colleagues identified an 11-fold
upregulated 13.8 kDa fragment of α 1-B glycoprotein (A1BG) in urine of SRNS
patients using SELDI-TOF and verified their finding by Western blot (Piyaphanee
et al. 2011). A 1.2-fold upregulation of this protein is also detected recently using
nano-LC-MS/MS (Kalantari et al. 2014b). It has worth to be validated in a large
sample size in the future studies.
Uncommon Predictive Biomarkers
A pilot study in 1997 showed excretion of some nonspecific proteins with homog-
enous anionic charge (albumin and transferrin) in the urine of steroid-resistant
nephrotic syndrome (SRNS) patients as well as some proteins with heterogeneity
of electrical charge (IgG, beta2-microglobulin, and lysozyme) in FGS (Ramjee
et al. 1997).
Few years later in 2006, SELDI-TOF technique was used to classify SSNS and
SRNS patients, and beta-2-microglobulin was reported as a biomarker associated
with SRNS (Khurana et al. 2006). Beta-2-microglobulin is not a specific biomarker
and known as a common excreted protein in renal failure. In addition the low
resolution and low reproducibility of SELDI-MS technique diminish the reliability
of the results. Some special fragments of albumin and/or of alpha1-antitrypsin in
the urine of a group of nephrotic syndrome (SSNS and SRNS) were reported in the
same year (Candiano et al. 2006). In the later study, the versatile techniques
including 2D-E followed by MALDI-TOF and Western blot were applied.
Haptoglobin was reported as a serum predictive biomarker for SSNS patients
(Wen et al. 2012). A relatively high number of subjects (n = 146), detection, and
identification of biomarkers using proteomic tools (2D-E/MALDI-TOF) and
verification using two antibody-based techniques (Western blot and ELISA) were
the privileges of this study. However, haptoglobin was not reported in the other
similar study on SSNS and SRNS.
Cardiotrophin-like cytokine factor 1 (CLCF1), one of the permeability circulat-
ing factors, was reported as a predictive candidate for recurrence of FSGS when its
level can reach to 100 times higher than normal subjects (Savin et al. 2008).
Besides these candidates which were reported in single studies are not further
evaluated; few other candidates for predicting responsiveness to steroid drugs are
also reported which have diagnostic value as well including suPAR (Li et al. 2014),
urinary miR-30a-5p (Zhang et al. 2014b), and uNGAL (Bennett et al. 2012).
Uncommon predictive candidates have been tabulated in Table 6.
To sum up, it is worthy to note that the majority of studies on predictive
biomarkers performed by proteomic tools and most of the specimens were urine.
This indicates the prominence of high-throughput techniques such as SELDI-TOF,
LC-MS/MS, and 2D-E in clinical researches and excellence of urine as a source of
noninvasive biomarkers. Further studies for validation of these noninvasive bio-
marker candidates in the future for clinical usage will be promising.
Summary Points
• This review focuses on different candidate biomarkers detected by proteomics

(2DE-MALDI-TOF-MS, LC-MS/MS, SELDI-MS/MS), metabolomics (NMR,
MS), transcriptomic (RT-PCR) tools, and other immune-based assay techniques
(such as Western blot and ELISA).
• Some of the candidates that have been reported by different research groups
through independent studies and detected by different techniques have been
discussed under a separate subheading and also explained its pathogenic impor-
tance. The other candidates which have been reported only by one group or
detected in animal models without validation in human subjects are discussed
under the title of “uncommon biomarkers.”
• suPAR, NGAL, and WT1 proteins which have been reported in numerous
researches have special value for both diagnosis and prognosis of FSGS.
• High-throughput platforms especially proteomics, transcriptomics, and
metabolomics besides the recent advances in mass spectrometry have shown
exciting results in biomarker discovery fields and more confident identification
of biomarkers.
• As urine is in direct contact with the injured kidney, its collection is noninvasive
and less complex than serum and could be the best choice for biomarker
discovery of glomerular diseases including FSGS. In addition some of the
urinary biomarkers such as suPAR are less controversial among the scientists
than their serum isoforms.
• Although lots of attempts have been devoted for identification of biomarkers
which are specific and sensitive enough for FSGS, there is still not a consensus
on the single ideal biomarker. Therefore, using a panel of biomarkers as a
Table 6 Uncommon predictive candidate biomarkers

Analytical
Protein ID Protein/miRNA name Source technique Reference
APOA1, PGRP2, ACTG, Apolipoprotein A-I , Urine LC-MS/MS Kalantari
FBLN3, PGBM, AACT, N-acetylmuramoyl-L- et al.
IGHG1, TRFE, YIPF3, alanine amidase, gamma- 2014b
A1AG2, THBG, A1BG, actin, EGF-containing
A2GL, ANAG, S10A9, fibulin-like extracellular
CUBN, TITIN, AMPN, matrix protein
CLUS, IPSP, MXRA8 1, basement membrane-
specific heparan sulfate
proteoglycan core protein,
alpha-1-
antichymotrypsin, Ig
gamma-1 chain C region,
transferrin, killer lineage
protein 1, alpha-1-acid
glycoprotein 2, thyroxine-
binding globulin, alpha-
1B-glycoprotein, leucine-
rich alpha-2-glycoprotein,
alpha-N-
acetylglucosaminidase,
calgranulin-B, cubilin,
connectin,
aminopeptidase N,
clusterin, plasma serine
protease inhibitor, matrix-
remodeling-associated
protein 8
IgG, B2MG, LYZ Immunoglobulin G, Urine 2DE Ramjee
beta2-microglobulin, and et al. 1997
lysozyme
B2MG Beta-2-microglobulin Urine SELDI- Khurana
TOF et al. 2006
ALB, A1AT Albumin, alpha1- Urine 2DE- Candiano
antitrypsin MALDI- et al. 2006
TOF
HPT Haptoglobin Serum 2D-E/ Wen
MALDI- et al. 2012
TOF
suPAR Soluble urokinase Serum ELISA Li
plasminogen activator et al. 2014
receptor
– miR-30a-5p Urine Probe-based Zhang
quantitative et al.
RT-PCR 2014b
NGAL Neutrophil gelatinase- Urine ELISA Bennett
associated lipocalin et al. 2012
Candidates detected mostly by proteomic techniques are reported in single study or in animal
models which are useful for predicting responsiveness to steroid therapy
diagnostic array can serve to be more reliable, efficient, and practical in the
future clinical world.
• Besides the mentioned high-throughput platforms which are also part of systems
biology, the other areas of this science including bioinformatics and network
science are able to bright the dark sides of mechanism and pathogenesis of the
diseases and suggest the possible candidate biomarkers hypothetically to be
tested and evaluated in the wet lab.
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Biomarkers of Renal Microthrombosis
in Lupus Nephritis 35
María Galindo-Izquierdo, Elena Gonzalo-Gil, Oscar Toldos, and
José Luis Pablos-Álvarez
Contents
Key Facts of Renal Biopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 812
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 813
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 814
Histological Patterns of Renal Vascular Lesions in Patients with Lupus Nephritis . . . . . . . . . . . 815
Immunohistochemistry Detection of Specific Markers of Microthrombosis . . . . . . . . . . . . . . . . . . 821
Serological Biomarkers of Vascular Involvement in Patients with LN . . . . . . . . . . . . . . . . . . . . . . . . 825
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 826
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 827
Abstract
The involvement of the kidneys, or lupus nephritis, develops in 30–50 % of
patients with systemic lupus erythematosus and is a major cause of morbidity and
mortality. Although some clinical variables have prognostic value, histological
information obtained from biopsies continues to be indispensable for classifica-
tion and outcome prediction, including the status of renal vascular lesions. In this
chapter, we describe main histological patterns of vasculopathies by using routine
methods for light microscopy, electron microscopy, and direct immunofluores-
cence. Lastly, we report more sensitive and specific methods for immunohisto-
chemistry detection of platelet aggregates. These procedures could help to better
identify vascular involvement in patients with lupus nephritis.
M. Galindo-Izquierdo (*) • E. Gonzalo-Gil • J.L. Pablos-Álvarez

Rheumatology Department, Hospital Universitario 12 de Octubre, Madrid, Spain
e-mail: mgalindo@h12o.es; mgali69@gmail.com; elenagonzalo_bio@hotmail.com;
jlpablos@h12o.es
O. Toldos
Pathology Department, Hospital Universitario 12 de Octubre, Madrid, Spain
e-mail: oscar-toldos@hotmail.com

812 M. Galindo-Izquierdo et al.
Keywords
Lupus nephritis • Systemic lupus erythematosus • Thrombosis • Inflammation •
Immunohistochemistry markers • Antiphospholipid antibodies • Platelet aggre-
gates • Complement
Abbreviations
aCL antibodies Anticardiolipin antibodies
ADAMTS-13 Disintegrin and metalloproteinase with a thrombospondin type
1 motif, member 13
ANXA5 Annexin A5
aPL antibodies Antiphospholipid antibodies
APS Antiphospholipid syndrome
APSN APS nephropathy
CEC Circulating endothelial cell
CV Cardiovascular
DIF Direct immunofluorescence
EM Electron microscopy
GP Glycoproteins
H&E Hematoxylin and eosin
HUS Hemolytic uremic syndrome
IC Immunocomplex
IF Immunofluorescence
IHC Immunohistochemistry
LA Lupus anticoagulant
LM Light microscopy
LN Lupus nephritis
MHA Microangiopathic hemolytic anemia
PAS Periodic acid-Schiff
RAS Renal artery stenosis
sTM Soluble thrombomodulin
TMA Thrombotic microangiopathy
TTP Thrombotic thrombocytopenic purpura
VCAM-1 Vascular cell adhesion molecule-1
VWFCP Von Willebrand factor-cleaving protease
Key Facts of Renal Biopsy
• The inflammation and involvement of kidneys in patients with lupus is known as

lupus nephritis.
• Renal biopsy is necessary in patients with lupus and evidence of renal disease
because the clinical presentation may not accurately reflect the histologic findings.
35 Biomarkers of Renal Microthrombosis in Lupus Nephritis 813
• There are various types of lupus-associated renal involvement although

immunocomplex-mediated glomerular diseases are the commonest.
• The status of renal vascular lesions in lupus nephritis is also important as their
presence can adversely affect the prognosis of the renal disease.
• In clinical practice, light microscopy, electron microscopy, and direct immuno-
fluorescence are used to detect vascular renal lesions.
• Immunohistochemistry technique is able to detect thrombus formation from their
earliest phases of the disease.
Definitions
Systemic lupus erythematosus Chronic inflammatory disease of unknown etiol-

ogy that can affect the skin, joints, kidneys, lungs, nervous system, serous mem-
branes, and/or other organs of the body, associated with immunologic abnormalities
such as the production of autoantibodies.
Lupus nephritis Inflammatory disease affecting kidneys in patients with lupus,

most commonly as an immunocomplex-mediated glomerular disease, which is
usually differentiated with a renal biopsy.
Antiphospholipid syndrome Disease defined by the occurrence of venous or

arterial thrombosis or of specific pregnancy morbidity, in the presence of laboratory
evidence of antiphospholipid antibodies. These antibodies include lupus anticoagu-
lant and/or anticardiolipin antibodies (IgG and/or IgM) and/or anti-beta-2-glycopro-
tein-I (IgG and/or IgM).
Immunocomplex Derived from complex interactions between antibody, antigens,

complement, and various receptors as a part of adaptive immunity.
Microthrombi Small thrombus located in a capillary or other small blood

vessels.
Thrombotic microangiopathy Is a descriptive name for the histologic presence of

thrombi in the glomeruli and arterioles. Definition by electron microscopy includes
the presence of subendothelial widening of the glomerular capillary wall due to the
deposition of fibrin-like material.
Platelet glycoprotein The membrane glycoproteins of human platelets act as

receptors that mediate two important functions, adhesion to the subendothelial
matrix and platelet-platelet cohesion or aggregation.
Annexin A5 Protein that binds to phospholipid bilayers, forming two-dimensional

crystals and blocking the phospholipids from availability for coagulation enzyme
reactions.
Complement factor C4d C4d is a split product of C4 activation, without a

biological function, that is considered as “a footprint” of antibody-mediated tissue
injury.
Circulating endothelial cells Endothelial progenitor cells described in several

pathologic conditions that have in common the presence of vascular injury.
Thrombomodulin Is a cell surface glycoprotein which is widely expressed in a

variety of cell types and acts as a cofactor for thrombin binding that mediates protein
C activation and inhibits thrombin activity.
Angiopoietin-2 Regulating factor of angiogenesis that exerts context-dependent

effects on EC by binding the endothelial-specific receptor tyrosine kinase
2 (TIE2). Angiopoietin-2 acts as a negative regulator of ANG-1/TIE2 signaling
during angiogenesis.
Vascular cell adhesion molecule-1 (VCAM-1) Or cell surface adhesion

molecule involved in the recruitment of leukocytes to endothelial cells on arterial
walls.
Immunofluorescence microscopy Technique that utilizes fluorescent-labeled anti-

bodies to detect specific target antigens.
Electron microscopy Microscope that produces an electronically magnified image

of a specimen with a greater resolving power than a light-powered optical microscope.
Introduction
Systemic lupus erythematosus (SLE) is a multisystem disease affecting many

organs. The involvement of the kidneys, or lupus nephritis (LN), with proteinuria
and hypertension as its most prominent features, develops in 30–50 % of patients
with SLE (Cameron 1999) and is a major cause of morbidity and mortality
(Faurschou et al. 2010). There are many variations in the prevalence and course of
SLE-associated renal disease, and several factors, both clinical and demographic,
have been shown to influence the outcome (Mok 2005). Most renal abnormalities
emerge within 3–5 years after SLE diagnosis (Seligman et al. 2002). Although
eventually up to one-third of SLE patients will develop elevated serum creatinine,
evidence of significantly impaired renal function is an uncommon early manifesta-
tion. The standard clinical practice is to perform a renal biopsy if clinical or analytic
parameters suggest renal involvement. Although some clinical variables, such as
elevation of serum creatinine or persistent elevations of blood pressure, have prog-
nostic value, histological information obtained from biopsies continues to be indis-
pensable for classification and outcome prediction (Esdaile et al. 1991; Austin
et al. 1995). Clinical findings alone can underestimate the true incidence of renal
involvement as some patients have significant pathological abnormalities without

relevant clinical signs of renal involvement (silent LN) (Gonzalez-Crespo
et al. 1996).
The renal biopsy is an invaluable method in the diagnosis of patients with renal
disease. This procedure allows for an accurate diagnosis and gives information about
the outcome and prognosis of renal disease, and it is a helpful tool in order to choose
the best treatment.
Klemperer and his colleagues (Klemperer et al. 1984) were the first to describe
the light microscopic (LM) renal pathology of SLE glomerulonephritis (GN) with
cellular proliferation, wire loops, hematoxylin bodies, and fibrin thrombi in autop-
sies from untreated patients. There are various types of SLE-associated renal
involvement although immunocomplex (IC)-mediated glomerular diseases are the
commonest (Balow and Austin 1988). In addition to GN, the status of renal vascular
lesions in LN is also important because their presence can adversely affect the
prognosis of the renal disease (Banfi et al. 1991; Appel et al. 1994). Several studies
report more frequent hypertension, fibrosis, and worse outcomes.
Histological Patterns of Renal Vascular Lesions in Patients

with Lupus Nephritis
Renal vascular lesions in patients with SLE diagnosis may be inflammatory, throm-
botic, or secondary to a podocytopathy. In vasculitic lesions, immune deposits are
found in nearly every case of LN in the mesangium and less consistently in the
capillary wall. Glomerular fibrin thrombi are a histologic feature of proliferative LN
(Kant et al. 1981), and in the presence of severe glomerular inflammation, thrombus
formation can result from endothelial activation or damage with activation of the
coagulation system. Thrombi also occur without other signs of glomerular inflam-
mation in the thrombotic thrombocytopenic purpura (TTP)-like lesion, a rare com-
plication of SLE (Hamasaki et al. 2003). Autoantibody against ADAMTS-13
(a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member
13), also known as von Willebrand factor-cleaving protease (VWFCP), is a common
cause of acquired von Willebrand factor protease deficiency in patients with TTP.
Although immunoglobulin G anti-ADAMTS-13 antibody is unusual in patients with
SLE (Mannucci et al. 2003; Rieger et al. 2005), the association remains a potential
cause of thrombosis. The formation of large multimers of von Willebrand factor
leads to platelet aggregation and thrombosis (Gungor et al. 2001). Glomerular
thrombosis also occurs in SLE in the absence of severe inflammation, suggesting
that several additional pathogenic mechanisms could be implied. Lupus anticoagu-
lant (LA) or antiphospholipid (aPL) antibodies are present in a significant proportion
of patients with SLE, and these autoantibodies, directed against the phospholipid
elements in the intrinsic clotting cascade, may arise by mechanisms analogous to
antinuclear antibodies (Nzerue et al. 2002). Patients with diagnosis of
antiphospholipid syndrome (APS) may develop thrombosis in the arteries, veins,
and glomerular capillaries.
In some SLE patients, nephrotic syndrome may be also due to a podocytopathy

with a non-immunocomplex-mediated injury to the glomerular podocyte, rather than
immunocomplex-mediated glomerular injury.
In clinical practice, we use the following three methods to detect vascular renal
lesions: LM, electron microscopy (EM), and direct immunofluorescence (DIF). The
core for LM must be fixed and embedded in paraffin. Among the variety of fixatives
that are recommended for the LM study, Zenker’s fixative provides a good architec-
tural and cytological detail. Hematoxylin and eosin (H&E), periodic acid-Schiff
(PAS), Masson trichrome, and methenamine silver impregnation are the routine
employed stains.
Nowadays, EM is an essential tool in the study of renal disease, as some disorders
are still defined according to EM findings. After its fixation in glutaraldehyde, the
specimen is embedded in an epoxy resin, to make a plastic-embedded tissue block,
from which we get 1 μm thick section and we stain it with toluidine blue. Toluidine
blue gives great information about the histology of the kidney and helps to choose
the better place to do the thin section from EM study.
Finally, DIF is the best technique to detect immune deposits in renal parenchyma
by using sera anti-IgG, anti-IgA, anti-IgM, anti-C1q, anti-C3, anti-C4, and anti-
fibrinogen. For DIF study, renal tissue must be frozen in liquid nitrogen.
In the 1990s, Appel and Descombes published two extensive reviews where they
described main histological subtypes of vascular lesions in patients with LN (Appel
et al. 1994; Descombes et al. 1997). Lesions were categorized into the following
types:
1. Lupus vasculopathy or noninflammatory necrotizing vascular lesion, defined as

the presence of hyaline thrombi occluding the glomerular capillary and/or the
arteriolar lumen. These hyaline thrombi are eosinophilic PAS positive and are not
accompanied by inflammatory changes of the vascular wall (Fig. 1a,b). By using
DIF microscopy, we can detect high content of immunoglobulins and comple-
ment complexes and fibrin. By EM, these deposits are electron dense and discrete
and often have a granular texture, although occasionally a fingerprint or tactoid
substructure is seen (Fig. 1c). They are most commonly located below an intact
vascular endothelium or within the basement membranes surrounding the medial
myocytes. Rarely, perivascular (adventitial) deposits are present. Such vascular
deposits are commonly found with active glomerular proliferative forms of
LN. They may be overlooked in biopsies because of their focal distribution and
because greater attention is usually directed to the glomerular pathology. This
type of lupus vasculopathy must be differentiated from hyalinosis, frequently
associated with hypertension and arteriosclerosis, that commonly accompanies
the more chronic and inactive forms of LN.
2. Thrombotic microangiopathy (TMA) is characterized by the extensive presence
of thrombi in the arteries, arterioles, and glomerular capillaries, mainly containing
fibrin, fragmented red blood cells, and leukocytes into the vascular lumen
(Fig. 2a, b). Sometimes, plasmatic components infiltrate the vessel wall. This
type of vasculopathy may involve the renal vessels of lupus patients with TTP or
Fig. 1 Lupus vasculopathy or noninflammatory necrotizing vascular lesion. Renal tissue from a
patient with lupus nephritis and noninflammatory necrotizing vasculopathy. (a, b) Hyalinization
with vascular wall replaced by hyaline and eosinophilic PAS-positive material without inflamma-
tory changes detected by hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining
(black arrows). Fibrinoid necrosis is also observed (b, arrow head). Original magnification: 200.
(c) Electron microscopy with dense subendothelial deposits in a glomerular capillary (Image from
“image collection” of the Spanish Rheumatology Society and courtesy of Dr. S. Castañeda)
Fig. 2 Thrombotic microangiopathy. Detection of microthrombi (black arrows) in glomerular

capillaries by H&E (a) and Masson trichrome (b) staining. Original magnification: 400
APS or may occur without a recognizable thrombotic systemic process. Most

commonly, it affects preglomerular arterioles and small interlobular arteries.
Histologically, it appears identical to the vascular changes seen in non-lupus
patients with hemolytic uremic syndrome (HUS), TTP, malignant hypertension,
scleroderma, and other thrombotic microangiopathies (Tsai 2012). In the acute
phase, there is marked luminal narrowing or total occlusion by intraluminal,
subendothelial, or medial accumulation of eosinophilic, fuchsinophilic material
with staining properties of fibrin, invariably associated with endothelial swelling,
denudation, and sometimes fragmented and/or hemolyzed erythrocytes. It is
distinguished from noninflammatory vasculopathy by the predominance of fibrin
and the absence of discrete immune deposits on immunofluorescence (IF) and
EM. In the chronic phase, mucoid edema of the intima and/or “onion skin” type of
intimal fibroplasia may occur.
Microangiopathic hemolytic anemia (MHA), and specifically the presence of
peripheral blood schistocytes, is a hallmark of TMA. Hu et al. described that
MHA is present in 60 % of the patients with LN and TMA. Therefore, the
detection of serum markers of MHA could be valuable for determining whether
LN is complicated with TMA or not (Hu et al. 2010). They also described the
presence of increased levels of circulating endothelial cells (CECs) in peripheral
blood and soluble thrombomodulin (sTM) in patients with LN complicated by
TMA (Yao et al. 2008a).
3. Vasculitis defined by the presence of fibrinoid necrosis of the arterial wall
accompanied by an infiltration of inflammatory cells. This is the least frequent
renal vascular lesion encountered in SLE. Its morphologic appearance is identical
to that of microscopic polyangiitis. The affected vessels are usually intralobular
small- and medium-sized arteries. There is prominent inflammatory cell infiltra-
tion of the arterial wall by neutrophils and mononuclear leukocytes, affecting the
vessel eccentrically and circumferentially (Fig. 3). Acute mural inflammation is
accompanied by fibrinoid necrosis, usually most severe in the intima and to a
lesser extent in the media. IF discloses fibrin-related antigen sometimes associ-
ated with less intense staining for immunoglobulin and/or complement fractions.
Although there are few EM descriptions, they have showed no electron-dense
deposits of the immune type, being common the presence of fibrin deposition.
4. Fibrous intimal thickening and arteriosclerosis, considered nonspecific sclerotic
vascular lesions and defined as fibrous thickening of the intima without necrosis,
proliferation, or thrombosis and subendothelial hyaline deposits.
5. Uncomplicating vascular immune deposits, characterized by the presence of
granular deposits of immunoglobulins and complement between the smooth
muscle cells of the vascular wall or the intimal basement membrane. These
deposits may appear either isolated or associated with one of the other preceding
vascular lesions.
In patients with APS, renal manifestations include renal artery stenosis (RAS)
and/or renovascular hypertension, renal infarction, APS nephropathy (APSN), renal
vein thrombosis, and increased allograft vascular thrombosis.
Fig. 3 Vasculitis.
Inflammatory involvement of
glomerular capillaries
detected by H&E staining.
Fibrinoid necrosis of the
arterial wall accompanied by
polymorphonuclear cell
infiltration. Original
magnification: 400
Specifically, APSN is characterized by vascular involvement associated with

hypertension, acute and/or chronic renal failure, and low-grade proteinuria. APSN
vascular lesions may be acute (TMA) and/or chronic (arteriosclerosis, arterial fibrous
intimal hyperplasia (FIH), tubular thyroidization, arteriolar occlusions, and focal
cortical atrophy (FCA)) (Nochy et al. 1999). On repeated kidney biopsies, we can
observe a progression of the acute lesions to chronic proliferative, obstructive, and
fibrotic forms (Tektonidou et al. 2004). Biopsy-proven renal vascular lesions are
statistically more related to LA than with anticardiolipin (aCL) antibodies, and their
presence and severity represent independent risk factors to develop hypertension,
elevated serum creatinine, and increased interstitial fibrosis (Fakhouri et al. 2003).
Acute APSN vascular lesions and TMA are identical to those previously reported
in this text. Arteriosclerosis is characterized by fibrous intimal thickening and
arteriolar hyalinosis that reduces the vascular lumen of arcuate and interlobular
arteries.
FIH is different from that observed as a consequence of aging arteriosclerosis,
because cellular proliferation is more intense. The myofibroblastic intimal cellular
proliferation leads to intimal thickening, with tortuosity of interlobular arteries and
their branches. The media shows two different patterns, proliferative with hypertro-
phic myocytes or, alternatively, atrophic and fibrous. Areas of fibrous thickening of
intima (Fig. 4a) or recanalyzing thrombosis often reduce the lumen (Fig. 4b,c).
Fibrocellular and fibrous arterial and arteriolar occlusions are often present in
small interstitial arteries.
FCA involves superficial zones of the subcapsular cortex, associated with a
depression of the contour of the renal capsule. With these areas involved by FCA,
the glomeruli can appear small and sclerotic or, in contrast, pseudocystic and
voluminous, often clustered in groups, sometimes in the same biopsy. This kind of
Fig. 4 Chronic lesions of antiphospholipid syndrome-associated nephropathy. (a) Fibrous intimal

hyperplasia and thickening detected by Masson trichrome (black arrows). Original magnification:
200. (b, c) Recanalyzing thrombi with reduced lumen (black arrows) by H&E (b) and Masson
trichrome (c) staining. Original magnification: 2200 and 400, respectively. (d) Thyroidization of
renal parenchyma (black arrow), tubular atrophy, and interstitial fibrosis (arrow head) by H&E.
Original magnification: 200
lesion is considered to be typical of APSN and leads to dense interstitial fibrosis,

with thyroidization and tubular atrophy (Fig. 4d). Very often, FCA associates with
vascular lesions of FIH. Immunofluorescence can reveal fibrin and more incon-
stantly C3 and IgM deposits in the vessels showing thickening, and, sometimes,
renin deposits are observed in the juxtaglomerular apparatus. Tubular thyroidization
is characterized by tubular atrophy, with eosinophilic casts, resembling thyroid
tissue, often in the deep cortex or medulla.
Finally, both primary APS and aPL-positive patients with SLE nephritis are prone
to develop thrombosis of the renal veins and inferior vena cava, associated with
nephrotic-range proteinuria, especially in those with LA positivity.
APSN may occur independently of lesions attributable to lupus because its
presence is not correlated to the WHO class of the lupus glomerulopathy (Daugas
et al. 2002).
Immunohistochemistry Detection of Specific Markers

of Microthrombosis
Current observation of renal vascular lesions mainly depends on renal biopsy

examination, and biomarkers for clinical dynamic estimation of patients are greatly
needed. The classification and clinicopathological correlations of vascular renal
lesions are limited by two factors. Firstly, the sensitivity of routine histology to
detect microthrombosis is limited by its relatively low presence and the small size of
tissue samples obtained by renal biopsy. Moreover, vascular lesions include acute
thrombosis but also less specific chronic vascular lesions such as arteriolar sclerosis
or hyalinosis (Tektonidou et al. 2004).
Besides routine histological methods, recent data suggest that immunohistochem-
istry (IHC) detection of platelet aggregates on paraffin-embedded kidney sections
increases the sensitivity to detect microthrombosis.
CD61 and CD41 are epitopes of the alpha-IIb-beta-3 integrin chain that is present
in platelets, in microparticles arising from platelet activation, or in membrane
fragments that are detectable by IHC in formalin-fixed tissues. Both markers provide
a sensitive method to identify intravascular platelet aggregates. In fact, IHC studies
focusing on cardiac and renal allograft rejection have shown the specific stain of
small deposits of CD61/CD41 platelets to detect thrombus formation from their
earliest phases (Arbustini et al. 2000; Meehan et al. 2003; Wierzbicki et al. 2006). In
contrast, in mural layers or old thrombotic material, CD61/CD41 immunoreactivity
is no longer detectable (Arbustini et al. 2000). Recently, it has been demonstrated
that the detection of intravascular platelet microthrombi CD61+ analyzed by IHC is
a more sensitive and specific marker for aPL-related microthrombi than histological
criteria to detect the acute microthrombosis in patients with LN (Galindo et al. 2009)
(Fig. 5a, b). In contrast, histological chronic vascular occlusions previously included
as APS-associated kidney lesions were often negative by CD61 IHC. The presence
of CD61+ aggregates was significantly associated with aPL and not to aging or
cardiovascular risk factors. However, in our series, APS histological lesions were not
associated with aPL but to an older age and to cardiovascular risk factors associated
with arteriosclerotic disease. We also did not detect nonspecific immunostaining
either in normal vessels of healthy kidney control sections or in extravascular
structures of TMA control or SLE kidney sections, confirming the anti-CD61
antibody specificity.
Although CD41+ detection was found in both glomerular and extraglomerular
sections, this marker is less specific and sensitive than CD61+ in patients with LN
(Fig. 5c, d).
In cardiac allograft rejection, the amount of fibrin progressively increases with the
increase of thrombus size. Fibrin is the only identifiable thrombus component in old
mural thrombi embedded within the intimal lesions. Recent occlusive thrombi
immunoreact with anti-CD41a and anti-CD61 and with anti-fibrin antibodies,
whereas organized occlusive thrombi exclusively immunoreact with anti-fibrin
Fig. 5 Immunohistochemical detection of platelet microthrombi lesions, macrophagic infiltration,

and activated complement deposition in kidney biopsies. CD61 (a, b), CD41 (c, d), CD68 (e, f), and
C4d (g, h) antigens were detected with immunoperoxidase (brown) in serial sections of SLE
nephritis tissues (d–l). SLE glomerular (a, c, e, g) or extraglomerular (b, d, f, h) markers are
shown. Sections were counterstained with hematoxylin. Original magnification: 400. One of the
marked areas containing microthrombi is shown in inset with higher magnification (630)
Fig. 6 Immunohistochemical detection of fibrinogen. Fibrinogen antigen is detected with

immunoperoxidase (brown) in SLE nephritis tissue. SLE glomerular (a), extraglomerular (b), and
a thrombus (c) marker are shown. Sections were counterstained with hematoxylin. Original
magnification: 400
antibodies (Arbustini et al. 2000). However, in renal tissue from patients with LN,
immunoreaction with anti-fibrin antibodies leads to a diffuse and nonspecific pattern.
This pattern of staining does not allow differentiating vascular lesions from glomer-
ular and interstitial fibrosis (Fig. 6).
To identify the nature of the vascular intimal proliferation in patients with APS,
Nochy et al. have used a monoclonal mouse antihuman muscle-specific actin
antibody (HHF35) (Nochy et al. 1999). This antibody recognizes alpha and
gamma smooth muscle actin on paraffin sections from smooth and striated muscle,
stained using streptavidin-biotin method. It identifies FIH where the intima is
thickened, primarily by an intense myofibroblastic intimal cellular proliferation.
Whether microthrombotic lesions are a consequence of renal inflammation or
independently contribute to renal damage is unclear (Descombes et al. 1997; Nochy
et al. 1999; Tektonidou et al. 2004). Glomerular and interstitial macrophagic accu-
mulation is one of the individual variables that better correlates with clinical
parameters and renal activity in SLE, and it is a feature of the most aggressive
forms of human GN (Yang et al. 1998; Gonzalo et al. 2012). These cells, together
with dendritic cells, are the major source of inflammatory cytokines, and their
interaction with resident T cells may amplify renal inflammation. In patients with
LN, it has been confirmed that detection of intravascular microthrombi CD61+
associates with intra- and extraglomerular infiltration of CD68+ macrophages
(Gonzalo et al. 2012), suggesting the importance of macrophagic infiltration as
marker of SLE and renal activity in proliferative LN (Fig. 5e, f).
On the other hand, many studies have shown that complement activation may
play an important role in thrombotic events. An association between the presence of
GN and complement activation measured as glomerular C3 staining has been
described. Complement-derived inflammatory mediators or anaphylatoxins such as
C3a, C4a, and C5a increase vascular permeability, activate platelets and neutrophils,
and promote the release of cytokines with induction of systemic inflammation and
coagulation. Studies on murine models have highlighted how complement activation
is essential for aPL-induced pregnancy morbidity, suggesting that tissue injury in
APS may be caused by a complement-mediated inflammatory process, rather than by
thrombosis alone (Salmon et al. 2007). In humans, a few studies also point to
complement activation in aPL-mediated thrombosis (Distelmaier et al. 2009). C4d
is a sensitive marker for the classical pathway of complement activation. In SLE
patients, C4d deposition may be considered as a highly specific indicator of throm-
botic and vascular complications (Navratil et al. 2006), and it has been suggested that
intensity of staining correlates with the extent of IC deposition (Kim and Jeong
2003). Furthermore, immunodetection of glomerular C4d deposition in renal biopsy
from LN patients could be a convenient method of identifying individuals at risk of
TMA and vascular pathology associated with the presence of aPL (Cohen
et al. 2008) and even with the absence of aPL antibodies. However, because both
processes may be associated with proliferative LN and higher activity indexes, this
may be an indirect association (Distelmaier et al. 2009). However, in patients with
LN, no direct relationship has been demonstrated between C4d deposition and
microthrombosis. Instead, C4d deposition correlates with the intensity of macro-
phagic infiltration, which in turn was associated with microthrombosis (Gonzalo
et al. 2012) (Fig. 5g, h).
Annexin A5 (ANXA5) participates in the inhibition of blood coagulation by
competing with prothrombin for phosphatidylserine binding sites. Also, ANXA5
inhibits the activity of phospholipase A1, and it has been proposed a role in large
vessels as one of the mechanisms by which aPL might contribute to the increased
risk of atherothrombosis in patients with SLE. This could be explained by causing
decreased ANXA5 binding to endothelium in systemic circulation (Cederholm
and Frostegard 2005). Other authors have revealed colocalization of ANXA1 with
IgG2 in glomeruli of patients with LN by IHC techniques (Bruschi et al. 2014).
In this sense, glomerular ANXA2, essential in the coagulation by recruiting plas-
minogen and tissue plasminogen activator, has been associated with IgG and C3
deposits in active LN and thrombosis (Yung et al. 2010). This could indicate the
importance of ANX as glomerular target antigens in renal biopsy samples from
patients with LN.
Serological Biomarkers of Vascular Involvement in Patients

with LN
The presence of CEC has been suggested as a potential and useful marker for
vasculopathy in patients with LN (Yao et al. 2008a). Actually, circulatory levels of
thrombomodulin and vascular cell adhesion molecule-1 (VCAM-1) can be useful
biomarkers of renal vascular in LN patients (Yao et al. 2008b). The increase of
VCAM-1 levels suggests a state of endothelial cell activation or endothelial damage.
Angiopoietin-2 activates CEC and increases vascular inflammation. Although an
association between angiopoietin-2 and proliferative and nonproliferative lesions in
LN has not been demonstrated, it may be used as a biomarker of disease activity and
renal involvement in SLE patients (El-Banawy et al. 2012).
Other authors found that endothelial damage measured as CEC number was
markedly elevated in patients with TMA and vasculitis (Erdbruegger et al. 2006).
Even it has been suggested that the activated phenotype of CEC might be capable of
further potentiating vascular injury by the production of inflammatory and
pro-thrombotic immune mediators (Clancy et al. 2001).

Conditions
The sensitivity of routine histological methods to detect microthrombosis in patients

with LN and/or aPL is very low. The identification of intravascular platelet aggre-
gates CD61+ by IHC is more sensitive and specific to detect acute microthrombosis
but not chronic occlusive vascular lesions. Microthrombi detected with IHC may
have a lower impact on renal function and outcome than do larger histological
microthrombi. Further longitudinal prospective studies, including patients with LN
and repeated biopsies after induction treatment, will help us to better define the real
significance of this kind of histological and IHC lesions. Consequently, we will be
able to better define correlation between histopathological and IHC findings, with
serum and urinary data of renal function, the presence of aPL antibodies, and the
degree of response to treatment.
CD61+ microthrombi were firstly described in heart transplant recipients with
allograft vascular disease, the major cause of late graft failure. In allograft vascular
disease, coronary thrombosis is a frequent and major complication. Whereas recent
thin mural thrombi are mostly constituted of CD41- and CD61-positive platelets, the
amount of fibrin increases with the increase of thrombus thickness and is the major
component of occlusive thrombi. Fibrin is the only identifiable thrombus component
in old mural thrombi embedded within the intimal lesions (Arbustini et al. 2000).
Similar findings have been reported in renal acute humoral rejection, where CD61
has been able to detect intracapillary platelet activation in specimens without
thrombi detectable by LM (Meehan et al. 2003).
C4d, a sensitive marker for the classical pathway of complement activation, has
demonstrated to be a good marker of humoral rejection in renal transplant biopsy
samples (Collins et al. 1999).
Most centers involved in the management of transplant recipients have incorpo-
rated routine C4d staining in diagnostic pathology evaluation of all renal, heart, and
pancreas allograft biopsies. A solid base for regular C4d staining of biopsied
allograft tissue is now established for heart transplantation and pancreas transplan-
tation (Crespo-Leiro et al. 2005; de Kort et al. 2010). For other transplanted organs
such as the lung, the usage of C4d staining is still controversial (Magro et al. 2003).
In liver and short bowel transplantation, C4d seems to have no additional diagnostic
value (Neil and Hubscher 2010).
Cohen et al. have demonstrated that complicated pregnancies of patients with
SLE and APS share several pathophysiological aspects with antibody-mediated
rejection. They describe that placental C4d is detectable in the majority of SLE
and APS cases (>60 %) in a diffuse staining pattern at the fetal-maternal interface,
whereas in normal pregnancies C4d is always negative. Excessive placental C4d
relates to impaired fetal outcome due to fetal loss or due to prematurity in the setting
of preeclampsia (Cohen et al. 2011).
Glomerular C4d deposition has been also found in some patients with
antineutrophil autoantibody-negative pauci-immune glomerulonephritis, whereas
in the antineutrophil autoantibody-positive patients, it is absent (Xing et al. 2010).
In the setting of thrombotic microangiopathies, independently of the underlying
disease, performing a C4d stain might help clinicians understand the mechanisms of
renal microvascular thrombosis. A positive C4d stain could indicate that comple-
ment is involved and could even guide future treatment, for instance, with comple-
ment inhibitors. However, this needs further basic study, and its clinical utility must
await trials of complement inhibitory therapies.
Summary Points
• The major cause of morbidity and mortality in patients with systemic lupus
erythematosus is lupus nephritis.
• Renal vascular lesions in patients with systemic lupus erythematosus may be
inflammatory, thrombotic, or secondary to a podocytopathy and adversely affect
the prognosis of the renal disease.
• Main histological subtypes of vascular lesions in patients with lupus nephritis
include lupus vasculopathy, thrombotic microangiopathy, vasculitis, fibrous inti-
mal thickening, and arteriosclerosis.
• Patients with antiphospholipid syndrome, in addition to acute vascular lesions or
thrombotic microangiopathy, may develop chronic vasculopathies as arterioscle-
rosis, arterial fibrous intimal hyperplasia, tubular thyroidization, arteriolar occlu-
sions, and focal cortical atrophy.
• The sensitivity of routine histology to detect microthrombosis is limited by its

relatively low presence and the small size of tissue samples obtained by renal
biopsy.
• Detection of intravascular platelet microthrombi CD61+ by immunohistochem-
istry is a more sensitive and specific marker for antiphospholipid antibody-related
acute microthrombi than the routine methods.
• Detection of intravascular microthrombi CD61+ is associated with intra- and
extraglomerular infiltration of CD68+ macrophages.
• C4d deposition in patients with systemic lupus erythematosus may be considered
as a highly specific indicator of thrombotic and vascular complications.
• Serological markers as circulatory levels of thrombomodulin and vascular cell
adhesion molecule-1 can be useful biomarkers of renal vascular disease in LN
patients.
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Planar Antibody Arrays for Biomarkers
in Nephritis 36
Christer Wingren
Contents
Key Facts of Planar Antibody Arrays and Nephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 832
Definition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 832
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 833
Antibody Micro- and Nanoarray: Basic Technological Concepts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 834
Resistin as a Potential Marker in Lupus Nephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 840
Plasma Profiling Reveals Candidate Biomarker for Renal Impairment . . . . . . . . . . . . . . . . . . . . . . . . 842
Biomarker of Renal Pathology Chronicity in Lupus Nephritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 842
Planar Antibody Arrays for Biomarker Discovery in Lupus Nephritis . . . . . . . . . . . . . . . . . . . . . . . . 843
Planar Antibody Microarrays: Biomarker Discovery in Systemic Lupus Nephritis . . . . . . . . . . . 843
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 844
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 844
Abstract
Affinity proteomics, represented by planar antibody arrays, is an established
methodology for high-throughput disease proteomics. The technology can be
used to generate multiplexed protein expression profiles of even crude proteomes.
The antibodies are deposited one by one in an ordered pattern, an array, onto a
planar, solid support, where they will act as specific catcher molecules. Next, the
sample is added, and any specifically bound proteins are detected and quantify
using mainly fluorescence as sensing technology. The observed binding pattern is
then converted into a high-resolution protein expression map, or protein atlas,
C. Wingren (*)
Department of Immunotechnology and CREATE Health, Lund University, Lund, Sweden
e-mail: christer.wingren@immun.lth.se

832 C. Wingren
outlining the composition of the sample at the molecular level. Using state-of-the-
art bioinformatics, candidate biomarker signatures are identified. Hence, the
technology platforms provide unique opportunities for, e.g., biomarker discovery,
disease diagnostics, monitoring, and evidence-based therapy selection, setting the
stage for personalized medicine. Nephritis is inflammation of the kidney, a focal
or diffuse proliferative or destructive disease, for which new panels of high-
performing, blood-based biomarkers could have a clinical impact. In this chapter,
we will describe the design and development of planar antibody microarrays for
biomarker discovery and illustrate their use for delineating disease-associated
biomarkers in nephritis.
Keywords
Recombinant antibodies • Antibody arrays • Protein expression profiling •
Disease proteomics • Nephritis • SLE • Biomarkers
Abbreviations
GPS Global proteome survey
scFv Single-chain fragment variable
TXP Triple-X Proteomics
Key Facts of Planar Antibody Arrays and Nephritis
• Planar antibody arrays are miniaturized assays for multiplexed profiling of pro-
teins in even crude samples, such as serum.
• Antibody arrays rely on the specific, sensitive, and selective binding properties of
the arrayed antibodies for capture of the corresponding proteins (antigens).
• Planar antibody arrays can be used for protein expression profiling, resulting in
biomarker discovery.
• Nephritis is a chronic or acute inflammatory condition of the kidneys.
• Nephritis-associated serum and urine biomarkers can provide the clinicians with
actionable information (e.g., diagnosis and monitoring).
Definition
Array A miniaturized, ordered pattern of, e.g., dispensed antibodies.
Biomarker A measurable indicator of some biological state, condition, or disease.
Clinical proteomics A branch of proteomics, involving the application of proteo-

mic technologies on clinical samples.
36 Planar Antibody Arrays for Biomarkers in Nephritis 833
Microarray An array with micro-sized spot features.
Nanoarray An array with nano-sized spot features.
Nephritis An acute or chronic inflammatory condition of the kidneys.
Planar arrays Arrays printed on planar surfaces.
Proteome All proteins in a given sample, cellular system, or organism, at a given

time point.
Proteomics Large-scale comprehensive study of all proteins (the proteome) in

sample.
scFv The smallest fragment of an intact antibody containing the antigen-

binding site.
Introduction
Nephritis is a chronic or acute inflammatory condition of the kidneys, involving the

glomerulus, tubule, or interstitial tissue. The disease is due to a variety of causes,
including kidney disease, infection, and autoimmune disease, and the treatment
depends on the cause. In many cases, the damage is reversible when the cause is
identified and removed, but can in severe cases progress to renal failure and fibrosis.
Data indicates that this condition could be the ninth highest cause of death in humans
across the world. There are several different types of nephritis, such as acute
nephritis, chronic nephritis, glomerulonephritis, interstitial nephritis, pyelonephritis,
autoimmune nephritis, and lupus nephritis.
Lupus nephritis is caused by systemic lupus erythematosus (SLE) and one of the
most serious complications that can result from SLE (D’Cruz et al. 2007; Herbst
et al. 2012; Mok 2010; Rovin et al. 2007). Data indicates that 35 % of the patients
display signs of nephritis at the time of lupus diagnosis, and about 40–60 % of the
patients will show kidney involvement during the course of this chronic autoimmune
connective tissue disease. If not diagnosed and treated early enough, kidney nephri-
tis could result in severe condition and even death. The clinical manifestations of
SLE vary among the patients, and the signs and symptoms evolve over time and
overlap with those of other autoimmune diseases, why SLE is often misdiagnosed
and/or overlooked (Liu et al. 2010; Merrill 2005; Manzi 2009). In fact, SLE is often
referred to as the “invisible disease.” Hence, high-performing blood- and/or urine-
based biomarkers would thus have a significant clinical impact, providing the
clinicians with actionable information. However, deciphering disease-associated
biomarker panels in crude samples, such as serum or plasma, has proven to be
technologically very challenging.
834 C. Wingren
Proteomics is the large-scale comprehensive study of all proteins in a given

sample, cellular system, or organism, defined as the proteome. Clinical proteomics
is a branch of proteomics, involving the application of proteomic technologies on
clinical samples, such as blood. The aim is to decipher disease-associated bio-
markers for, e.g., diagnosis, prognosis, classification, and therapeutic prediction, as
well as for screening and/or monitoring how well the patient responds to a given
treatment. In addition, and most importantly, the traditional approach of searching
for a single, unique biomarker as the solution to an unmet clinical need (e.g.,
diagnosis) has been replaced by the concept of defining multiplexed biomarker
panels. Such biomarker panels have been validated to provide a much more selec-
tive, specific, and robust disease classifier (Cordero et al. 2008; Hanash et al. 2008;
Mischak et al. 2007; Borrebaeck and Wingren 2007) and will become the golden
standard to aim for. In this process, the need for multiplexed, high-performing (e.g.,
resolution, specificity, sensitivity, and reproducibility) protein bioassays capable of
handling also crude, complex samples (e.g., non-fractionated plasma) has become
evident (Hanash 2003; Hanash et al. 2008). The challenging analytical nature of a
proteome is well illustrated by plasma, containing thousands of individual proteins,
ranging in concentration over more than nine orders of magnitude. This has been a
major driving force in the development of a new line of proteomic technologies,
denoted affinity proteomics, mainly represented by antibody microarrays
(Borrebaeck and Wingren 2009a, 2011). The antibody microarray-based technology
has rapidly evolved from early proof-of-concept setups to multiplexed, high-
performing protein bioassays and today constitutes a key established approach
within clinical proteomics at frontline laboratories (Borrebaeck and Wingren 2009a).
In 2000, the first set of papers was published, reporting focused efforts toward
developing antibody microarrays (Haab et al. 2001; MacBeath and Schreiber 2000).
In these publications, low-density (<10) antibody microarrays were generated by
printing polyclonal and/or monoclonal antibodies one by one. The basic concepts of
the antibody microarrays were demonstrated, but the work also highlighted some of
the technical challenges that would have to be addressed and resolved before the
technology would become an established proteomic approach. During the last
15 years, major efforts have therefore been launched to develop the technology
further. As a result, a set of high-performing antibody micro- and nanoarray tech-
nology platforms are now at hand, providing novel opportunities for large-scale
protein expression profiling of high- and low-abundant targets in crude,
non-fractionated proteomes, such as serum (Borrebaeck and Wingren 2009a).
Antibody Micro- and Nanoarray: Basic Technological Concepts
An antibody array is a specific form of protein array that relies on the specific
binding property of the antibody. More specifically, the antibodies are printed one by
one onto a solid support in an ordered pattern, an array, where they are exploited as
capture molecules, or probes, for the corresponding antigens, with the aim of
detecting and quantifying the levels of the target proteins in the sample at hand
Protein expression
Dispensing of antibodies Labelling of sample profile, or protein
one-by-one map, of the sample
Sample
• Planar antibody arrays

• One antibody per spot
Array Data
Antibodies Assay Array use
production processing
• Monoclonal Protein expression profiling

• Polyclonal
• Diagnostics
• Recombiant
Detection of • Prognostics
specifically • Classification
bound proteins • Disease monitoring
• etc
Labeled sample added
Fig. 1 Schematic illustration of planar antibody microarray setup
(Borrebaeck and Wingren 2009a; Wingren and Borrebaeck 2009) (Fig. 1). Produc-
ing such miniaturized, high-density arrays based on antibodies with a broad range of
specificities enables the simultaneous screening of many protein targets, while
consuming minute (μL range) amount of reagents. When the antibody microarray
has been produced, the assay is run like a conventional immunoassay (e.g., ELISA).
The observed signal intensities are then transformed into a protein expression map,
or detailed protein atlas, revealing the composition of the sample at a molecular
level. In other words, the antibody array technology provides unique opportunities
for performing protein expression profiling of crude, non-fractionated proteomes
that will enhance our fundamental knowledge of biological processes in both disease
and health (Borrebaeck and Wingren 2007, 2009b; Haab 2006; Hartmann
et al. 2009; Kingsmore 2006; Wingren and Borrebaeck 2006).
The current concept of generating miniaturized antibody arrays, ranging in size
from mm2 to cm2, is based on either printing (pL scale or less) (Borrebaeck and
Wingren 2007; MacBeath 2002; Wingren and Borrebaeck 2006), self-addressing
(Wacker and Niemeyer 2004; Wacker et al. 2004), or self-assembling
(He et al. 2008a, b; He and Taussig 2001; Ramachandran et al. 2004, 2008) small
amounts (fmol range) of individual antibodies with the desired specificity onto a
solid support (Fig. 2). Direct printing is by far the most commonly used approach
and is based on using various dispensing methodologies, with non-contact ink-jet
printers dominating the scene (Borrebaeck and Wingren 2007; Wingren and
Borrebaeck 2007). The purified probes are printed one by one in the pL scale,
generating ~150 μm sized spot features, depending on the printing buffer and the
surface properties of the solid support. Self-addressing is a new method for
836 C. Wingren
1. Dispensing 2. Self-addressing 3. Self-assembly

- ink-jet technology - Antibody-nucleotide conjugate - Antibodies expressed directly on the
- Antibodies printed one-by-one - Nucleotide-tag acts as zipcode array in their unique spot using
- ~300 pL/drop - Complementary nucleotide on cell-free expression methods.
the array (“home”)
- Antibodies find their way to their
unique spot (“home”) on their own DNA coding for
the antibody
antibody
Zipcode-tag
Complementary Cell-free expression / spot

zipcode-tag
Fig. 2 Three main ways of producing planar recombinant antibody arrays
potentially producing truly high-density arrays, but still in its exploratory phase
(Wacker and Niemeyer 2004; Wacker et al. 2004). In this approach, each individual
antibody is tagged with a unique zip code tag, a short stretch of DNA. When added to
the array in bulk, the antibodies will find their way on their own to their unique home
(spot) on the array, composed of complementary DNA. Self-assembling antibody
microarrays is also a new, exploratory approach to potentially generate high-density
antibody arrays (He et al. 2008b). In this setup, the antibodies are produced directly
on the chip in their unique position, using cell-free protein expression
(He et al. 2008a, b; He and Taussig 2001; Ramachandran et al. 2004, 2008).
The size of the individually printed spots determines whether the array is denoted
a microarray (spot diameter (Ø) in the μm range) or a nanoarray (Ø in the nm range)
(Wingren and Borrebaeck 2007). Regarding antibody microarrays, arrays with an
overall footprint of < 1 cm2, based on 18 103 μm2 (diameter (Ø) of ~150 μm)
sized spots at a density of 2,000 spots/cm2, have mainly been produced and
applied (Hoheisel et al. 2013; Borrebaeck and Wingren 2009a; Kingsmore 2006;
Sanchez-Carbayo 2010). Further, the multiplexity, i.e., the number of antibodies
with different specificities per array, has been in the range of <900 different anti-
bodies/array. Adopting ink-jet-based printers to produce the arrays, the antibodies
have been sequentially spotted in parallel (1 to 4 antibodies at a time), and the
multiplexity has been achieved by washing the nozzles and loading them with new
antibodies.
In the case of nanoarrays, conceptual protein (antibody) nanoarrays displaying
truly miniaturized (spot size; <0.8 μm2, Ø < 1 μm) and high-density (spot density;
>100,000 spots/cm2) features have been designed and produced (Nettikadan
et al. 2006; Lee et al. 2010; Hoff et al. 2004; Backmann et al. 2005; Arntz
et al. 2003; Zheng et al. 2005; Ellmark et al. 2009; Ghatnekar-Nilsson et al. 2007;
Bruckbauer et al. 2004; Tran et al. 2010) (for review see Wingren and Borrebaeck
2007). Despite the success, these nanoarray designs have been shown to be associ-
ated with three key technical bottlenecks. First, the production methodologies at
Microarrays (submicro)Arrays Nanoarrays

Spot diameter ~150 μm 10 μm < 1 μm
Spot area 18x103μm2 78.5 μm2 < 0.8 μm2
Spot density ≤ 200 spots/cm2 ≤ 250,000 spots/cm2 >>100,000 spots/cm2
A. B. Dip-pen nanolitography-based printer.

Nanoprinter
etc
Inkjet printer A. Microcantilever-based surface

patterning tool
B. Dip-pen nanolitography-based
printer
Fig. 3 Three main types of planar antibody arrays (with respect to size of the spots)
hand are currently limited to producing only 1-plex arrays (i.e., arrays composed of
multiple spots of a single antibody), or in rare cases <5-plex designs (Wingren and
Borrebaeck 2007; Nettikadan et al. 2006; Lee et al. 2002, 2010; Hoff et al. 2004;
Backmann et al. 2005; Arntz et al. 2003; Zheng et al. 2005; Ellmark et al. 2009;
Ghatnekar-Nilsson et al. 2007; Bruckbauer et al. 2004; Tran et al. 2010; Berthet-
Duroure et al. 2008; Meister et al. 2004). Second, reducing the spot size < 1μm will
lead to impaired rather than improved assay performance (e.g., sensitivity) (Ekins
1998). Third, hardware for sensitive (such as fluorescence-based) sensing of high-
density nanoarrays remains to be established (Wingren and Borrebaeck 2007).
However, the density and multiplexity of antibody arrays are essential for large-
scale protein expression profiling endeavors. In order to meet this demand without
having to further develop the technologically challenging antibody nanoarray
designs, miniaturized arrays based on submicron-sized (Ø 10 μm) rather than
nano-sized (Ø < 1 μm) spot features have surfaced (Fig. 3) (Irvine et al. 2011;
Jang et al. 2010; Lynch et al. 2004; Nettikadan et al. 2006; Petersson et al. 2014b).
Using a nanoarrayer, based on dip-pen technology, the first 12- and 48-plex planar
recombinant antibody arrays, based on 78.5 um2 (Ø 10 μm) sized spots at a density
of 38,000 spots/cm2, interfaced with a fluorescent-based readout were recently
produced (Petersson et al. 2014b, c). Importantly, their use for biomarker discovery
in serum was also outlined, using systemic lupus erythematosus as showcase
(Petersson et al. 2014c). Interestingly, adopting a microcantilever-based surface
patterning tool, it was recently demonstrated that 16-plex recombinant antibody
arrays, based on miniaturized spot features (78.5 um2, Ø 10 μm) at a 7- to
125-times increased spot density (250,000 spots/cm2 vs. 38,000 spots/cm2
(Petersson et al. 2014b) or 2,000 spots/cm2 (Borrebaeck and Wingren 2009a)),
interfaced with a fluorescent-based readout could be produced (Petersson
et al. 2014a). The feasibility of this conceptual array platform for serum protein
profiling was also indicated (Petersson et al. 2014a).
In order to achieve high density, access to numerous renewable antibodies is a
must. By using large antibody libraries, with, e.g., 1010 members (Söderlind
et al. 2000), as a probe source, renewable antibodies displaying “any” specificity
838 C. Wingren
TTEDAVLHHEAR
Identify peptide motifs common to
1. Proteome a limited (1-100) number proteins
(e.g. HEAR) LNHVAAYRWHEAR
3. Affinity
2. Trypsination TTEDAVLHHEAR proteomics SDFITYEWHHEAR
SDFITYEWHHEAR
LNHVAAYRWSEAR
LNHVAAYRWHEAR
HAERRTAGSSSLESR Enrichment of motif-carrying
peptides using motif-specific
antibodies
TTEDAVLHHEAR
Discovery mode profiling
4. MS-MS SDFITYEWHHEAR 5. Bioinformatics 6. Applications
100
200 CIMS antibodes
LNHVAAYRWHEAR
Original wt protein 50 proteins / CIMS antibody
- Identified 1 x10 4 proteins coverage
%
- Quantified
Specie independent
0 m/z
Fig. 4 Schematic illustration of the global proteome survey (GPS) setup, designed for global
proteome profiling
can readily be selected and included on the arrays. The logistics behind large-scale
selections could potentially constitute a logistical bottleneck. If so, two similar
concepts were recently presented, demonstrating one solution to how to use a smaller
set of antibodies while still targeting numerous proteins. The two concepts denoted
Triple-X Proteomics (Poetz et al. 2009) (TXP) and global proteome survey (Wingren
et al. 2009) (GPS) (Fig. 4) are based on the same fundamental principle and is based
on combining antibody arrays with mass spectrometry. Briefly, antibodies are
generated against short peptide motifs, four to six amino acid residues long, each
motif being shared among 2 to 100 different proteins. These motif-specific anti-
bodies could then be used to target motif-containing peptides. From a practical point
of view, the proteome is first digested (e.g., trypsinated), and the peptide-specific
antibodies are then used to specifically capture and enrich motif-containing peptides.
Next, the motif-containing peptides are detected, identified, and quantified using
tandem mass spectrometry, thereby allowing us to backtrack the original proteins in
a quantitative manner. By using 200 such motif-specific antibodies, each targeting a
motif shared among 50 unique proteins, would thus enable us to target about half the
non-redundant proteome. As an example, a recent study showed that about 1400
tissue proteins could be profiled in a quantitative manner using only nine such motif-
specific antibodies (Olsson et al. 2013).
Planar antibody arrays, printed on (microscope) slides (16 subarrays/slides;
made of plastic, glass, or polymer) or on the bottom of ELISA plates, are the
dominating format, although bead-based arrays, or arrays in solution, have also
been manufactured (Borrebaeck and Wingren 2009a).
The assay is run like a traditional immunoassay (~4 h assay time), but consuming
only μL scale volumes of the samples. It should be noted that crude, non-fractionated
proteomes, such as serum, plasma, urine, cell lysates, and tissue extracts, can, in
contrast to many competing proteomic technologies, be directly used without having
to pre-fractionate the sample (Belov et al. 2001, 2003; Campbell et al. 2006; Dexlin
et al. 2006; Mischak et al. 2007; Ingvarsson et al. 2007; Haab 2003; Wingren
et al. 2007; Haab et al. 2001; Wingren and Borrebaeck 2009).
In a majority of cases, the samples are labeled with a fluorescent dye, either
directly or indirectly, and interfaced with a fluorescent-based readout (Kusnezow
et al. 2007; Wingren and Borrebaeck 2008; Wingren et al. 2007). A dynamic range
of at least four orders of magnitude and an assay sensitivity in the pM range can be
obtained, thus allowing low-abundant (pg/ml) analytes to be targeted in crude
proteomes. By quantifying the signal intensity of each spot in the array, the array
images are transformed into protein expression profiles, outlining the protein com-
position of the sample. State-of-the-art bioinformatics is then applied in order to
identify any disease-associated biomarker panels that can be explored and exploited
for, e.g., diagnosis, prognosis, and classification (Borrebaeck and Wingren 2009a, b;
Wingren and Borrebaeck 2009).

Conditions
To date, planar antibody microarrays have been used for protein expression profiling
of almost any kind of crude sample format, such as plasma and serum, with the aim
of deciphering disease-associated biomarker signatures (for review see, e.g.,
Borrebaeck and Wingren 2007; 2009b; 2009a; Haab 2005; Haab 2006; Hartmann
et al. 2009; Kingsmore 2006; Griffiths et al. 2005; Wingren and Borrebaeck 2009).
The design of the applications ranges from small proof-of-concept studies to large
semi-global protein expression profiling efforts. Reviewing the antibody array field,
from early to recent applications, shows that the technology can be used in, but not
limited to, the following areas: (1) autoimmunity, (2) allergy, (3) bladder proteomics,
(4) cell proteomics, (5) drug abuse, (6) glycomics, (7) heart proteomics, (8) heredi-
tary disease, (9) inflammatory conditions/infections, (10) liver proteomics, (11) lung
proteomics, (12) medical microbiology, (13) neurology/psychiatry, (14) obstetrics/
gynecology, (15) oncoproteomics, (16) periodontology, (17) phosphoproteomics,
(18) protein expression, and (19) protein signaling (Table 1).
Cancer is by far the most targeted disease using this technology, and several
publications have demonstrated the potential of the antibody microarray methodol-
ogy for pin-pointing cancer-associated biomarkers for, e.g., diagnosis, prognosis,
classification, predicting the risk for relapse, and evidence-based therapy selection,
as illustrated by a few selected representative references (Sanchez-Carbayo 2010;
Alhamdani et al. 2012; Wingren et al. 2012; Hoheisel et al. 2013). While planar
antibody arrays have been frequently applied within the field of cancer, nephritis has
so far only been addressed in a limited set of studies. Below, we have outlined the
840 C. Wingren
Table 1 General overview of planar antibody array-based applications and area of use
A. Area of use (example disease, biological process) B. Applications
A1. Autoimmunity Systemic lupus erythematosus B1. Protein expression
profiling
A2. Allergy Cytokine profiling B2. (Early) Diagnosis
A3. Bladder proteomics Smooth muscle hypertrophy B3. Differential diagnosis
A4. Cell proteomics Blood phenotyping B4. Classification
A5. Drug abuse Screening B5. Phenotyping
A6. Glycomics Pancreatic cancer B6. Evidence-based
therapy selection
A7. Heart proteomics Myocardial infarction B7. Predicting the risk for
relapse
A8. Hereditary disease Cystic fibrosis B8. Drug abuse screening
A9. Inflammatory Atherosclerosis, obesity B9. Bacterial detection/
conditions/infections profiling
A10. Liver proteomics APAP-induced liver disease B10. Bacterial toxin
detection
A11. Lung proteomics Chromium(VI) treatment
A12. Medical microbiology Detection of bacteria/toxin
A13. Neurology/psychiatry Cerebral palsy
A14. Obstetrics/gynecology Preeclampsia
A15. Oncoproteomics Pancreatic cancer, breast cancer,
lymphomas
A16. Periodontology Model system
A17. Phosphoproteomics Lung cancer
A18. Protein expression Posttranslational profiling
A19. Protein signaling Various model systems
findings of some of those applications by selecting a set of representative publica-

tions (Table 2).
Resistin as a Potential Marker in Lupus Nephritis
In this study, the authors used commercially available planar antibody arrays to
discover candidate biomarkers in serum and urine of patients suffering from SLE and
lupus nephritis (Hutcheson et al. 2015). The hypothesis was to explore whether
serum and urine levels of adipokines could act as biomarkers for lupus nephritis. In
previous work, adipokines have been associated with SLE and cardiovascular
disease. Based on the antibody array work, 15 adipokines, adiponectin, leptin, and
resistin were selected. Next, ELISA was applied in an attempt to validate the
biomarkers. Compared to matched controls, the results showed that the expression
Table 2 Overview of the selected applications, using (planar) antibody arrays for protein expres-
sion profiling and biomarker discovery in nephritis, used as representative examples
Antibody array
Study (aim/target disease/ (design/
reference) antibodies) Key finding(s)
1. Serum and urine protein Commercially Resistin was indicated as a potential
profiling for biomarker discovery available planar biomarker in lupus nephritis
2. SLE and lupus nephritis antibody arrays
3. Hutcheson et al. 2015 Mono-/
polyclonal
antibodies
1. Plasma protein profiling for In-house Fibulin was outlined as a candidate
biomarker discovery designed bead- biomarker for renal impairment, in
2. Glomerulonephritis, diabetic based arrays particular for glomerulonephritis
nephropathy, obstructive uropathy, Polyclonal
and analgesic abuse antibodies
3. Neiman et al. 2011
1. Urine profiling for biomarker Commercially Angiostatin was outlined as a
discovery available planar candidate urinary biomarker of renal
2. Lupus nephritis antibody arrays disease in SLE
3. Wu et al. 2013 Mono-/
polyclonal
antibodies
1. Design of miniaturized planar In-house First generation of miniaturized
antibody arrays and serum protein designed planar antibody arrays. Three serum
profiling for biomarker discovery miniaturized, biomarkers associated with SLE were
2. SLE planar arrays detected
3. Petersson et al. 2014c Recombinant
single-chain Fv
antibodies
1. Serum protein profiling for In-house Multiple serum biomarker signatures
biomarker discovery designed for diagnosis, classification, and
2. SLE (including lupus nephritis) miniaturized, prognosis of SLE. SLE and systemic
and systemic sclerosis planar arrays sclerosis could be differentiated
3. Carlsson et al. 2011 Recombinant
single-chain Fv
antibodies
levels of adiponectin and resistin were increased in both serum and urine, while
leptin was increased in lupus nephritis. Further, the levels of resistin in serum, but
not in urine, were found to correlate with renal dysfunction in lupus nephritis. Taken
together, resistin might thus prove useful as a biomarker of renal dysfunction in
patients with lupus nephritis. Additional work targeting additional, independent
patient cohorts will, however, be required to validate the data and to preferentially
extend this single biomarker into a multiplex marker panel to increase the anticipated
assay performance (specificity, sensitivity, and selectivity).
842 C. Wingren
Plasma Profiling Reveals Candidate Biomarker for Renal

Impairment
The ability to detect early signs of kidney toxicity and to monitor progression of
disease represents essential unmet clinical needs. Spurred by this, the authors
applied an in-house designed antibody suspension bead array to perform plasma
protein expression profiling targeting four types of kidney disorders, including
glomerulonephritis, diabetic nephropathy, obstructive uropathy, and analgesic
abuse (Neiman et al. 2011). To this end, 129 polyclonal antibodies, targeting
94 unique proteins, were used to produce the bead-based array. In total, 200 clinical
plasma samples, including renal-associated cases and controls, were profiled.
Significantly higher expression levels were observed for 1 of 94 proteins, fibulin-
1, in glomerulonephritis patients compared to all of the other patient cohorts,
indicating a potential for differential diagnosis. Most importantly, using three
different antibodies directed toward three separate, non-overlapping epitopes on
fibulin-1 showed similar expression levels, further supporting the data. In addition,
Western blot analysis of selected plasma samples confirmed the observations.
Next, a novel, independent patient cohort, including glomerulonephritis and con-
trols, was applied in an attempt to validate the findings in the discovery cohort. The
data confirmed the indications, outlining fibulin-1 as a potential indicator to
monitor kidney damage or kidney malfunction. The performance of the biomarker
might be even further improved by finding additional markers, in the end resulting
in a multiplexed panel.
Biomarker of Renal Pathology Chronicity in Lupus Nephritis
In this study, the authors used a commercially available, multiplexed antibody

microarray to perform protein expression profiling of about 280 proteins in urine
targeting lupus nephritis (Wu et al. 2013). The data indicated elevated levels of urine
angiostatin. Angiostatin has been shown to have modulatory function in inflamma-
tion and angiogenesis. Using ELISA, the increased levels of urinary angiostatin were
then validated in an independent cohort of SLE patients. Next, the authors investi-
gated whether the levels of angiostatin also reflected the SLE disease activity.
Indeed, the results showed that higher levels were observed in active SLE versus
inactive SLE. In fact, the patients with the most severe form of SLE were found to
have the highest levels of urinary angiostatin. The biomarker might also be used to
differentiate SLE patients with active SLE versus inactive SLE, as illustrated by
receiver operating curve analysis resulting in an area under the curve of 0.90. Finally,
when analyzing lupus nephritis patients, urine-angiostatin levels were found to
correlate with renal pathology chronicity index, but not with the activity index.
Hence, angiostatin surfaced as a novel, candidate noninvasive biomarker of renal
disease in SLE. Further studies will be required in order to validate these promising
findings, targeting novel, independent patient cohorts.
Planar Antibody Arrays for Biomarker Discovery in Lupus

Nephritis
In this exploratory work, the authors first developed and designed a 48-plex mini-
aturized recombinant single-chain Fv antibody array platform (Petersson
et al. 2014c). In more detail, individual spot features with a diameter of 10 μm and
an area of 78.5 μm2 were printed at a density of 38,000 spots per cm2 using dip-pen
nanolithography. The setup was interfaced with a high-resolution scanner for
fluorescence-based sensing. The performance and applicability of the in-house
designed planar antibody arrays were demonstrated by performing protein expres-
sion profiling of lupus nephritis. To this end, the observed serum profiles of lupus
nephritis (n = 45) versus healthy controls (n = 30) were compared, and differentially
expressed proteins were defined. The results showed that differentially expressed
serum levels of three proteins in lupus nephritis versus healthy controls were
detected, including complement protein C1q (downregulated), interleukin
6 (upregulated), and low-density lipoprotein (upregulated). Of note, these data
supported previous findings, based on using conventional recombinant antibody
microarrays (Carlsson et al. 2011). Taken together, the data outlined that planar
recombinant antibody arrays could be used to define lupus nephritis-associated
serum biomarkers, while consuming minute amount of sample (<1 single drop of
serum).
Planar Antibody Microarrays: Biomarker Discovery in Systemic

Lupus Nephritis
In this discovery study, the authors used in-house designed 135-plex recombinant
single-chain Fv antibody microarrays to perform protein expression profiling of
systemic sclerosis, systemic lupus nephritis, and healthy controls (Carlsson
et al. 2011). The 135 antibodies were directed against 60 different proteins,
including mainly immunoregulatory proteins. The hypothesis was to explore
(parts of) the immune system as an early, specific, and sensitive sensor for disease.
The results showed that several candidate SLE-associated multiplexed serum
biomarker panels were successfully deciphered, reflecting disease (with impact
on diagnosis), disease severity (enabling phenotyping), and disease activity (indi-
cating ability to detect, monitor, and potentially even predict flares). In addition,
biomarker panels differentiating SLE and systemic sclerosis were detected, and the
observed differences increased with severity of SLE. Hence, the study demon-
strated that molecular portraits of systemic lupus nephritis (and systemic sclerosis)
could be extracted from a crude serum sample. Of note, the assay was performed
while consuming less than a single drop of serum, and low-abundant biomarkers
(pg/ml) could readily be detected. In the end, the disease-associated marker panels
might also enhance our fundamental understanding of these complex autoimmune
diseases. Of note, the authors have a set of additional manuscripts in the pipeline,
further validating the candidate serum biomarker signature for diagnosis and
844 C. Wingren
outlining additional marker panels for classification and prognosis (Wingren et al.,
unpublished observations).
Summary Points
• This chapter focuses on the design of planar antibody arrays for protein expres-
sion profiling and biomarker discovery in nephritis.
• Planar antibody arrays have been developed for biomarker discovery in clinical
proteomics.
• Miniaturized planar antibody arrays can be used to perform multiplexed protein
expression profiling, targeting crude proteomes.
• Planar antibody arrays have been successfully used for biomarker discovery in
nephritis.
• Nephritis-associated urine, serum, or plasma biomarkers have been deciphered
using planar antibody arrays.
• Multiplexed biomarker panels can be deciphered in a single drop of blood, or less,
using planar antibody arrays.
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Malondialdehyde as a Biomarker in Kidney
Transplantation 37
Isabel Fonseca
Contents
Key Facts of Kidney Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 851
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 851
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 852
Oxidative Stress, Reactive Oxygen Species and Antioxidants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 853
Lipid Damage by Reactive Oxygen Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 855
Malondialdehyde . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 857
Malondialdehyde and the Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 860
Biomarkers of Oxidative Stress in Kidney Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 861
Malondialdehyde and Kidney Transplantation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 864
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 869
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 869
Abstract
Delayed graft function (DGF) is a clinical diagnosis that describes dysfunction of
the kidney allograft immediately after kidney transplantation, which as a detri-
mental impact on kidney graft survival. In recent years, there is a considerable
interest in identifying biomarkers indicative of early graft dysfunction and pre-
dictive of allograft survival. A variety of markers have been proposed as candi-
dates to signal graft dysfunction earlier. Of these markers, malondialdehyde
(MDA) appear to have a clinical relevance, given the results obtained in prelim-
inary clinical studies and/or its biological properties that are directly related with
its toxicity towards several cells and tissues.
I. Fonseca (*)
Department of Nephrology and Kidney Transplantation, Centro Hospitalar do Porto, Hospital de
Santo António, Porto, Portugal
e-mail: isabelf27@gmail.com; ifonseca.defi@chporto.min-saude.pt

850 I. Fonseca
Almost 30 % of DGF following kidney transplantation is attributable to

ischemia reperfusion injury, which is one of the most important non-specific
and non-immunologic factor that affect early and long-term allograft function.
The production of excessive quantities of reactive oxygen species (ROS) is an
important mechanism of ischemia-reperfusion injury and lipid peroxidation is one
of the most widespread hypotheses of ROS-mediated cell injury. Lipid oxidation
gives rise to several aldehydes, including MDA, which are good indicators of
nephrotoxicity and tissue damage. Malondialdehyde is a reliable diagnostic
biomarker of initial graft injury and accurately detect graft dysfunction earlier
than serum creatinine.
Moreover, levels of MDA in plasma within the first week after reperfusion of
the graft predicts long-term graft outcome. As in every other domain in medicine,
in organ transplantation early diagnosis and timely intervention will improve
outcomes. Malondialdehyde is, therefore, more than a promising marker, but an
alarm of cellular and tissue damage. And the approach of using MDA as a trigger
to initiate and monitor immunosuppressive therapies, and as a safety biomarker
when using potentially nephrotoxic agents, is also promising.
Keywords
Oxidative stress • Malondialdehyde • Lipid peroxidation • Kidney transplanta-
tion • Ischemia-reperfusion injury
Abbreviations
4-HNE Dyhidroxynonel
AGEs Advanced glycation end products
AOPP Advanced oxidation protein products
GPx Glutathione peroxidase
GR Glutathione reductase
GSH Glutathione
GSSG Oxidized glutathione
H2O2 Hydrogen peroxide
HOO• Hydroperoxyl radical
LOOHs Lipid hydroperoxides
MDA Malondialdehyde
MHC Major histocompatibility complex
NADPH Reduced nicotinamide adenine dinucleotide phosphate
O2• Superoxide
OH• Hydroxyl radical
OLOO• Epoxy-allylic peroxyl radicals
PUFAs Polyunsaturated fatty acids
37 Malondialdehyde as a Biomarker in Kidney Transplantation 851
SOD Superoxide dismutase

TBARS Thiobarbituric acid reactive substances assay
• Kidney transplantation is the treatment of choice for almost all cases of renal
failure due to better quality of life and survival than for chronic dialysis.
• The short-term outcome of renal transplantation has improved substantially in the
past 20 years, however long-term survival has not paralleled that improvement.
• Delayed graft function is the most common complication in the immediate post-
transplantation period mainly in deceased renal allografts, almost invariably in
the non-heart beating and in some live donor transplants.
• Kidney DGF represents acute kidney injury in the immediate postoperative
period after transplantation and continues to pose a significant challenge in
kidney transplantation
• Several studies have found associations between DGF and increased risk for acute
rejection, chronic allograft dysfunction, and worse graft survival.
• The association between DGF and worse outcomes has led to increased efforts to
better understand the mechanisms of ischemia-reperfusion injury and to develop
interventions to reduce its occurrence and impact.
Definitions
Antioxidants Antioxidants are substances, endogenous or exogenous, that, when

present at low levels compared to an oxidizable compound (e.g. lipids), delay or
inhibit oxidation of the substrate and the inherent oxidative damage.
Cold ischemia time Cold ischemia time is defined as the time between the chilling
of a tissue or organ after its blood supply has been reduced or cut off and the time in
which the tissue or organ reach physiological temperature after restoration of blood
supply during implantation procedures.
Delayed graft function (DGF) Delayed graft function (DGF) represents renal
failure persisting after transplantation and is usually defined as requirement for
dialysis in the first week of transplantation (although several other definitions are
used due to the difficulty of defining DGF precisely). Prolonged cold ischemia time
is usually associated with a greater incidence of DGF, which as a detrimental impact
on kidney graft survival.
Free radicals Free radicals are defined as molecules or molecular fragments

containing one or more unpaired electron in atomic or molecular orbits, which
gives a considerable reactivity to the free radical.
852 I. Fonseca
Ischemia Ischemia refers to reduction or cessation of arterial blood flow with

immediate oxygen deprivation of cells.
Ischemia-reperfusion injury Ischemia-reperfusion injury refers to cellular/tissue

damage that results damage that results from a period of ischemia that is followed by
the reestablishment of the blood supply. The absence of blood and nutrients from
blood creates a condition in which the restoration of circulation results in inflam-
mation and oxidative damage.
Lipid peroxidation Lipid peroxidation is the metabolic process in which ROS

attack and induce oxidative deterioration of lipids, especially polyunsaturated fatty
acids (PUFAs), affecting cell membrane structure and function and causing tissue
damage.
Malondialdehyde (MDA) Malondialdehyde (MDA) is a highly reactive three-

carbon dialdehyde produced as an endproduct of lipid peroxidation, being consid-
ered a good marker of oxidative stress and of tissue damage.
Oxidative stress Oxidative stress can be defined as an imbalance favouring the

oxidant (reactive oxygen species) generation over the ability of antioxidant mecha-
nisms for detoxifying the reactive intermediates or for repairing the resulting damage
that leads to macromolecular damage and dysfunction.
Pro-oxidants Pro-oxidants are species that causes or promotes oxidative stress,

either by generating reactive oxygen species or by inhibiting antioxidant systems.
Depending on the circumstances, a compound may exhibit pro- or antioxidant
activity. Examples: polyphenols, thiols, α-tocopherol.
Reactive oxygen species (ROS) Reactive oxygen species (ROS) are highly reac-
tive metabolites of molecular oxygen (O2), such as oxygen-derived free radicals
(superoxide, hydroxyl radical, nitric oxide) and non-radical oxygen derivatives of
high reactivity (singlet oxygen, hydrogen peroxide, peroxynitrite, hypochlorite).
ROS are constantly produced endogenously and under physiological conditions,
ROS production and scavenging capacity are balanced. At high doses ROS become
deleterious, exhibiting pathophysiological actions.
Introduction
Cellular damage caused by restoring the blood supply to previously viable ische-
mic tissues is defined as ischemia-reperfusion injury. This phenomenon is inevita-
ble in organ transplantation. In kidney transplantation, decreased blood supply is
associated with flow deviation from cortex to medulla, which preserves the oxy-
genation of the metabolically vulnerable medulla at the cost of cortical perfusion
and glomerular filtration (Woolfson et al. 1994). Sensitivity to oxygen deprivation,
or ischemia, has been demonstrated in both the proximal tubules (Shanley

et al. 1986) and their thick ascending limbs (Brezis et al. 1985). Thus, tubuloin-
terstitial damage begins soon after transplantation due to ischemia-reperfusion
injury. The resolution of this process is critical to obtain a more successful outcome
for the kidney, because ischemia-reperfusion has been identified as a major risk
factor for the development of delayed graft function (DGF), acute rejection and
long-term chronic graft dysfunction (Woolfson et al. 1994; Koo et al. 1998;
Kosieradzki and Rowinski 2008).
A number of mechanisms have been proposed to mediate the injury caused by the
ischemia that occurs during the organ transfer from the donor to the recipient and the
reperfusion once the vascular anastomosis in the recipient is complete. The most
frequently cited mechanisms of ischemia-reperfusion damage include intracellular
calcium overload, endothelial and microvascular dysfunction, leukocyte-
endothelium interactions, altered kidney metabolism and perhaps most importantly
the formation of oxygen free radicals (Kosieradzki and Rowinski 2008; Gulec 2011).
Oxidative Stress, Reactive Oxygen Species and Antioxidants
Oxygen free radicals or, more generally, reactive oxygen species (ROS) are products
of normal cellular metabolism that exist in all aerobic cell, and their level is
controlled by a balance between pro-oxidants and antioxidants. The average person
has approximately 10,000–20,000 free radicals attacking each cell in his body every
day (Halliwell and Gutteridge 1986; Valko et al. 2004). These well-known ROS,
such as superoxide (O2•), hydrogen peroxide (H2O2) and the hydroxyl radical
(OH•), are derived from oxygen and are formed as intermediates in reduction-
oxidation processes. They represent the most important class of reactive species
generated in living systems (Valko et al. 2004; Small et al. 2012).
One to 3 % of inspired molecular oxygen (O2) is converted to superoxide (O2•)
rather than being reduced to water. Superoxide is the most common of the ROS and a
precursor to other ROS generated in cells. In fact, the formation of this oxygen free
radical leads to a cascade of other ROS. Superoxide has an unpaired electron that
makes it highly reactive and renders it unstable and short-lived as an oxidising agent.
Because of its charge, the superoxide anion is poorly permeable to cell membrane
and remains mostly within the mitochondrial matrix, although it can also cross cell
membranes via anion channels (Burton and Jauniaux 2011; Small et al. 2012). It can
undergo several chemical reactions depending on the amount generated and the
proximity to other radicals and enzymes. This free radical anion is a powerful
precursor of hydrogen peroxide (H2O2), which is lipid soluble and freely crosses
the cell membrane (Burton and Jauniaux 2011). Although cellular hydrogen perox-
ide is stable, it has the potential to interact with a variety of substrates and cause
damage, particularly in the presence of the reduced metal ion Fe2+. This interaction
leads to the breakdown of H2O2 and the formation of the most highly reactive, toxic
and damaging of the free radicals produced during normal metabolism and/or by
exogenous sources, the hydroxyl radical (OH•) (Valko et al. 2004; Small et al. 2012).
854 I. Fonseca
This small, highly mobile, water-soluble, and short-lived molecule can be produced
from molecular oxygen (O2) in cell metabolism and under a variety of stress
conditions.
A cell produces approximately 50 hydroxyl radicals every second. In a full day,
each cell can generate four million hydroxyl radicals, which can either be neutralised
or attack biomolecules (Ayala et al. 2014). Mitochondria constantly metabolize
oxygen thereby producing ROS as a by-product. The estimated levels of ROS within
the mitochondria are five to tenfold higher than other cytosolic and nuclear com-
partments because they are mostly generated by the mitochondria during oxidative
phosphorylation (energy generation) and by the activation of cellular enzymes,
including NADPH oxidase, cyclooxygenase, nitric oxide synthase and xanthine
oxidase (Valko et al. 2004; Small et al. 2012). Under physiological conditions,
ROS are produced specifically to serve in essential biological functions and play a
physiological role in cellular responses to noxia, such as in defence against infec-
tious agents and in the function of a number of cellular signalling systems (Rahman
2007). Under these conditions, ROS are the by-products of normal metabolic
processes, and the rates of free radical production and elimination are similar,
leading to a steady state that is presumably tolerated by the cell. In other cases,
mostly pathological, ROS production exceeds the cellular antioxidant capacity and
can be deleterious to biological systems, which causes oxidative stress. Traditionally,
oxidative stress is defined as an imbalance between oxidant generation and antiox-
idant mechanisms for detoxifying the reactive intermediates or for repairing the
resulting damage, which can lead to macromolecular damage and dysfunction
(Halliwell and Gutteridge 1986; Rahman 2007).
The antioxidant defence mechanisms can be divided into two major
groups: (i) endogenous, which are primarily enzymes, such as superoxide dismutase
(SOD), catalase, glutathione reductase (GR) and glutathione peroxidase (GPx),
(ii) and small molecules, which are primarily exogenous and act as free radical
scavengers, such as reduced glutathione (GSH), vitamins A, C, and E, carotenoids
and polyphenol (Valko et al. 2004; Lu 2009; Burton and Jauniaux 2011).
In aerobic organisms the antioxidant enzymes are widely involved in scavenging
free radicals. The reactions catalysed by these defence enzymes are briefly presented
in (Fig. 1). Superoxide dismutases, namely copper/zinc-SOD (CuZn-SOD) and
manganese-SOD (Mn-SOD), play a key role in the oxidative stress process because
Fig. 1 Brief description of

the reactions catalysed by the SOD GPx
main antioxidant defence
enzymes. SOD Superoxide 2O2·- + 2 H+ H2O2 H2O + O2
dismutase, GPx Glutathione
peroxidase +
O2
Catalase
they accelerate the transformation of the highly reactive superoxide anion (O2•) into
hydrogen peroxide (H2O2), a much more stable ROS. Hydrogen peroxide is then
converted enzymatically into oxygen and water by catalase and glutathione perox-
idase (Gutteridge 1995; Small et al. 2012). Hydrogen peroxide is not a free radical
because it does not have an unpaired electron, but it is a ROS and must be promptly
removed by catalase. In many tissues, catalase activity, which is largely localised to
peroxisomes, is very low and frequently not available for decomposition of hydro-
gen peroxide (H2O2). Therefore, in most tissues, hydrogen peroxide is deactivated
with GPx. Thus, high SOD activity, which results in increased H2O2 production,
must be accompanied by increased GPx and/or catalase activity to limit injury. If this
does not happen, the excess hydrogen peroxide can interact with superoxide in the
presence of transition metal-containing molecules and yield a hydroxyl radical (HO•)
(Haber-Weiss reaction), which is one of the most deleterious and potent oxidising
agents known (Gutteridge 1995; Small et al. 2012). Glutathione peroxidase provides
an effective mechanism against cytosolic injury because it reduces peroxides and
hydroxyl radicals into nontoxic forms using GSH (Burton and Jauniaux 2011). Thus,
the activity of GPx depends on the presence of reduced GSH as a hydrogen donor,
which plays a critical role as antioxidant. In fact, GSH plays a critical role as an
antioxidant because it participates in a large number of detoxifying reactions by
forming glutathione disulphide. Glutathione disulphide is converted back to GSH by
the action of glutathione reductase at the expense of NADPH, forming a redox cycle
(Lu 2009; Burton and Jauniaux 2011).
When these antioxidant mechanisms cannot offset the generation of free radicals,
oxidative stress-associated tissue injury occurs. Because free radicals are compounds
highly unstable and reactive with a half-lives of only seconds, it is difficult to
measure their levels directly, which makes understanding in vivo oxidative damage
challenging. Oxyradical-modified lipids, proteins, carbohydrates and nucleic acids
remain stable from hours to weeks, which makes them ideal surrogate markers of
oxidative stress (Locatelli et al. 2003). Moreover, defects or low activity in the
antioxidant defence system lead to the impaired clearance of ROS, which can also be
used as indirect oxidative markers. A list of the common oxidative markers is found
in (Table 1; Gutteridge 1995; Locatelli et al. 2003; Valko et al. 2004; Rahman 2007;
Burton and Jauniaux 2011).
Lipid Damage by Reactive Oxygen Species
Reactive oxygen species may cause tissue injury via several mechanisms. Because
they are potent oxidising and reducing agents, ROS directly damage cellular mem-
branes and modify biological molecules, such as lipids, proteins, and nucleic acids.
Of the many biological targets of oxidative stress, lipids are the most affected (Del
Rio et al. 2005). Unsaturated phospholipids, glycolipids, and cholesterol in cell
membranes and other organised systems are prominent targets of oxidant attack
and lead to a process known as lipid peroxidation (Girotti 1985; Romero et al. 1998;
Ayala et al. 2014). Lipid peroxidation is the most important source of free radical-
856 I. Fonseca
Table 1 Biomarkers of oxidative stress

Oxidants
1. Protein oxidation products
Protein carbonyls
Advanced oxidation protein products (AOPP)
Carboxymethyllysine and pentosidine
Pentosidine
2. Carbohydrate oxidation products
Advanced glycation end products (AGEs)
3. Lipid peroxidation products
Malondialdehyde
F2-isoprostane
4-Hydroxynonenal
Acrolein
Thiobarbituric acid‐reactive substances
4. DNA oxidation products
8-hydroxy-20 deoxyguanosine (8-OHdG)
8-hydroxyguanosine (8-oxoG)
Prooxidants
5. Prooxidant enzymes
Xanthine oxidase
NADPH oxidase
Antioxidants
6. Non-enzymatic antioxidants (low molecular weight antioxidants)
Antioxidant vitamins (from diet or pharmacological supplements)
Ascorbic acid (vitamin C)
Tocopherol (vitamin E)
Retinol (vitamin A)
Carotenoids
Innate compound
Glutathione (GSH)
7. Antioxidant enzymes
Superoxide dismutase
Catalase
Glutathione peroxidase
GSH/GSSG ratio in erythrocyte
NADPH Reduced nicotinamide adenine dinucleotide phosphate, GSH Glutathione, GSSG Oxidized
glutathione, DNA deoxyribonucleic acid, AOPP Advanced oxidation protein products, AGEs
Advanced glycation end products
mediated injury that directly damages cellular membranes and yields a number of
secondary products responsible for extensive cellular damage (Romero et al. 1998).
The two most prevalent ROS that can profoundly affect lipids are the hydroxyl
(OH•) and hydroperoxyl (HOO•) radicals. Polyunsaturated fatty acids (PUFAs) of
the membrane phospholipids are the most susceptible molecules to attack by these
free radicals. Damage to PUFAs initiates self-propagating chain reactions that

undergo peroxidation and form lipid hydroperoxides (LOOHs) and a variety of
decomposition products, such as aldehydes or isoprostanes. In general, PUFAs are
more susceptible to oxidation as the number of double bonds increase. Of the various
biologically relevant PUFAs, long-chain n-3 fatty acids, such as eicosapentaenoic
acid (20:5n-3) and docosahexaenoic acid (22:6n-3), oxidise more readily than the
less saturated fatty acids, such as linoleic acid (18:2n-6) (Girotti 1985).
Lipid peroxidation is naturally present in small amounts in the body because of
the effect of ROS. However, because lipid peroxidation is a self-propagating chain-
reaction, the initial oxidation of only a few lipid molecules can result in significant
tissue damage. Any free radical with sufficient energy to extract a hydrogen atom
from a methylene carbon (-CH2-) of an unsaturated fatty acid can initiate lipid
peroxidation, which results in a carbon lipid radical (L•). The free-radical chain
reaction of lipid peroxidation propagates until two free radicals annihilate each other
to terminate the chain (Gutteridge 1995). The lipid carbon radical (L•) reacts with
molecular oxygen to generate a reactive lipid peroxyl radical (LOO•), which may
then extract a hydrogen from a nearby unsaturated fatty acid to form the non-radical
intermediate lipid hydroperoxides (LOOHs). Thus, LOOHs are the primary products
of fatty acid peroxidation and may undergo iron-mediated electron reduction and
oxygenation to yield epoxy-allylic peroxyl radicals (OLOO•), which trigger the
chain reaction of lipid peroxidation (Girotti 1985).
Under normal physiological conditions, these lipid hydroperoxides (LOOHs) pro-
duced in the course of normal conditions are completely inactivated by cellular and
extracellular defence mechanisms. In certain pathological conditions, increased
ROS-initiated peroxidation reactions and/or the depletion of antioxidant defence sys-
tems lead to the accumulation of lipid hydroperoxides (LOOHs). Accumulated LOOHs
tend to perturb membrane structure/function and alterations in membrane integrity,
fluidity, and permeability can occur, which results in cellular and tissue damage (Girotti
1985). The destruction of membrane lipids and the end-products from lipid peroxida-
tion reactions are especially dangerous to the viability of cells and several tissues.
Therefore, lipid oxidation is now known to be a crucial step in the pathogenesis of
several disease states (Gutteridge 1995; Agarwal et al. 2004; Rahman 2007). Three of
the major targets of the lipid peroxidation process are the brain, the kidney and the liver.
Thus, the toxicity of lipid peroxidation products in mammals generally involves
neurotoxicity, nephrotoxicity and hepatotoxicity (Repetto et al. 2012). The detection
and measurement of lipid peroxidationis the evidence most frequently cited to support
the involvement of free-radical reactions in diseases; thus, lipid peroxidation may be an
excellent marker of tissue damage (Gutteridge 1995).
Malondialdehyde
Many lipid peroxidation products are formed during ROS attacks on the double
bounds PUFAs, including lipid hydroperoxides (LOOHs) and conjugated dienes, the
primary products of lipid peroxidation. A great number of aldehydes are among the
858 I. Fonseca
secondary products formed during lipid peroxidation, such as malondialdehyde

(MDA) and 4-dyhidroxynonel (Fig. 2), as well as the prostaglandin F2-like com-
pounds (F2-isoprostanes), the gold standard in lipid peroxidation studies (Janicka
et al. 2010). All of these by-products can serve as biomarkers of lipid peroxidation.
Choosing a convenient marker for evaluating the importance of lipid peroxidation
in vivo is difficult because of the analytical problems of specificity and sensitivity.
Isoprostanes are the most specific markers of lipid peroxidation but they are also
most difficult to measure (Janicka et al. 2010). Thus, MDA is the principal and the
most studied product of lipid peroxidation (Romero et al. 1998).
Malondialdehyde is a highly toxic and reactive bifunctional molecule that reacts
with DNA and forms adducts with deoxyguanosine and deoxyadenosine. It can also
modify RNA, proteins and other biomolecules (Siddique et al. 2012). The main source
of MDA in biological samples is the peroxidation of PUFAs with two or more
methylene-interrupted double bonds. Thus, MDA is widely used as a reliable bio-
marker for estimating the tissue damage from ROS and lipid peroxidation (Del Rio
et al. 2005). In recent years, oxidative stress has been implicated in various diseases,
and elevated serum or plasma MDA levels has been noted in different pathological
conditions, including airway diseases, cancer, diabetes, renal disease, neurodegener-
ative disorders, cardiovascular diseases, among others (Table 2; Pucheu et al. 1995;
Keith et al. 1998; Templar et al. 1999; Gonenc et al. 2001; Dib et al. 2002;
Kolanjiappan et al. 2002; Dut et al. 2008; Bandeira et al. 2012; Antus et al. 2014).
Historically, the thiobarbituric acid reactive substances assay (TBARS) was the
common method used to estimate MDA levels. However, this assay is notoriously
Oxidative damage
ROS Tissue injury
Targets:
DNA
Proteins
Lipids
Lipid MDA
Peroxidaon 4-HNE Isoprostanes
LOOHs Reactive
Aldehydes
Oxidation
Fig. 2 Lipid peroxidation and MDA production. ROS Reactive oxygen species, PUFAs Polyun-
saturated fatty acids, LOOHs Lipid hydroperoxides, MDA Malondialdehyde, 4-HNE
Dyhidroxynonel
Table 2 Malondialdehyde in some pathological conditions

Author Disease or condition Conclusions
(Pucheu Myocardial infarction MDA is a good marker of reperfusion-related radical
et al. 1995) stress after thrombolysis, which might represent a
simple and reliable test of reperfusion efficacy
following thrombolysis, and it might enable one to
test the effect of various antioxidant therapies
associated with thrombolytic treatment
(Keith Congestive heart failure A significant increase in MDA levels in patients with
et al. 1998) congestive heart failure was related to the functional
severity of heart failure, with the highest levels being
observed in patients in functional class III and
IV. Increased MDA levels can be a objective marker
of prognosis in congestive heart failure
(Gonenc Breast and lung cancer MDA, a marker of lipid peroxidation in patients with
et al. 2001) breast and lung cancer
(Dib Neurodegenerative Plasma MDA levels are elevated in several
et al. 2002) diseases neurodegenerative disorders, namely amyotrophic
lateral sclerosis, Alzheimer’s disease, Parkinson’s
disease and Huntington’s disease and there are
indications that these levels correlate with clinical
state. MDA can be considered as a marker for the
evolution of these diseases
(Templar Chronic kidney disease Lipid peroxidation in patients with chronic renal
et al. 1999) and hemodialysis failure is further exacerbated by hemodialysis, as
evidenced by significant increased MDA levels. In
the non-dialysis group, patients with
glomerulonephritis presented the higher MDA
levels, suggesting the possible involvement of ROS
in the pathophysiology of glomerular disease
(Dut Asthma Asthma is associated with an extremely powerful
et al. 2008) oxidative stress, expressed by higher levels of MDA
(Bandeira Diabetes mellitus Increased level of MDA in diabetics suggests that
et al. 2012) peroxidative injury may be involved in the
development of diabetic complications
(Antus Chronic obstructive MDA useful marker for monitoring exacerbation-
et al. 2014) pulmonary disease associated oxidative stress in patients with acute
(COPD) exacerbation of COPD
MDA Malondialdehyde, ROS Reactive oxygen species, COPD Chronic obstructive pulmonary
disease
nonspecific because it also reacts with saturated and unsaturated non-functional

aldehydes, carbohydrates and prostaglandins, which has led to controversy over its
use for quantifying MDA from in vivo samples. The poor assay specificity associ-
ated with TBARS may lead to an overestimation of the levels of MDA in human
plasma and other biological tissues and fluids, and this, in turn, may limit the
likelihood of detecting the true differences in the levels of lipid peroxidation in
clinical studies (Siddique et al. 2012; Moselhy et al. 2013; Ayala et al. 2014). Several
technologies for determining free and total MDA, such as high-performance liquid
860 I. Fonseca
chromatography (HPLC) techniques, and several derivatisation-based strategies,

have been developed during the last decade. These new assays have improved
specificity and sensitivity of MDA determination. Consequently, due to the higher
accuracy in the detection of the degree of lipid peroxidation and the indirect level of
the free oxygen radicals, HPLC is currently recommended as the preferred MDA
assay for human plasma samples. This is a remarkable advance in biomedical
research community because MDA is becoming a very relevant molecule and a
promising biomarker of tissue damage (Siddique et al. 2012; Moselhy et al. 2013;
Ayala et al. 2014).
Malondialdehyde and the Kidney
Oxidative stress causes tissue damage by different mechanisms including lipid per-
oxidation, DNA damage, and protein modification. All of these processes have been
implicated in the pathogenesis of several systemic diseases, including kidney disease.
The kidney is very vulnerable to ROS damage because renal lipids are composed of an
abundance of long-chain PUFAs. Though free radicals can attack many critical
biological molecules, such as DNA and cellular proteins, the high content of unsat-
urated lipids marks lipid peroxidation as the central feature of oxidant injury in the
kidney (Rodrigo and Rivera 2002; Chung and Perrella 2004; Ozbek 2012).
Chronic kidney disease is a pro-oxidant state and the degree of intracellular and
extracellular oxidative stress is related to the severity of renal failure (Massy and
Nguyen-Khoa 2002; Small et al. 2012). Formation of ROS is evident in many areas
of the kidney, predominantly in the renal cortices. The renal medulla, however, is
susceptible to hypoxia, but with less ROS production under physiological and
pathological conditions (Bedard and Krause 2007). The structural characteristics
of chronic kidney disease include increased tubular atrophy, interstitial fibrosis,
glomerulosclerosis, renal vasculopathy and reduced renal regenerative capability.
These characteristics may be caused, at least in part, by the gradual loss of renal
energy through the development of mitochondrial dysfunction and resultant increase
of oxidative stress (Small et al. 2012). Some studies report that ROS are involved in
the pathogenic mechanisms of conditions such as glomerulosclerosis, renal tubular
damage and tubulointerstitial fibrosis (Shah 1995; Rodrigo and Rivera 2002;
Agarwal et al. 2004; Raju et al. 2013).
It has also been observed that free radical-induced lipid peroxidation tissue
damage plays a significant role in the pathogenesis of various renal diseases (Raju
et al. 2013). Elevated levels of MDA and decreased antioxidant activity have been
reported in patients with renal disease, which is a reflection of the increased
oxidative stress in these patients. This oxidative imbalance is exacerbated by dialysis
procedure and by severe chronic inflammation (Boaz et al. 1999; Templar
et al. 1999; Oberg et al. 2004; De Vecchi et al. 2009; Raju et al. 2013). Urinary
MDA is considered to be a reliable indicator of enhanced lipid peroxidation in renal
tubules and is thought to be directly proportional to renal damage in non-dialysis
kidney patients (Draper et al. 1986). Although there are few studies in this field,
MDA levels seem to depend on type of renal disease; it is generally higher in patients
with glomerular-associated reduced plasma clearance (Templar et al. 1999). Patients
with chronic kidney disease on regular haemodialysis exhibit higher MDA levels
than healthy controls. It has been suggested that enhanced lipid peroxidation might
result from free radical activity generated by complement activation and the release
of cytokines during the dialysis procedure. This was mostly attributed to the
exposure of blood to bio-incompatible dialysis membranes and the diffusion of
hydrophilic compounds to the dialysate and the influx of endotoxin from the
dialysate (Templar et al. 1999; Locatelli et al. 2003). Increased lipid peroxidation,
especially in haemodialysis patients, also results in excess consumption of antiox-
idant enzymes such as SOD. The loss of antioxidants, such as zinc and copper that
act as cofactors of SOD activity, in the dialysate fluid also contribute to the further
decrease in antioxidant activity of these patents (Locatelli et al. 2003).
Biomarkers of Oxidative Stress in Kidney Transplantation
There is substantial literature on oxidative stress and renal disease, but there is a lack
of data on oxidative stress in kidney transplantation. Restoring kidney function after
transplantation can lead to an improvement in oxidative stress, but some studies
demonstrate increased systemic biomarkers of oxidative stress in kidney transplant
recipients, (Simic-Ogrizovic et al. 1998; Campise et al. 2003) specifically in the
early post-transplant phase (Zahmatkesh et al. 2010; Ardalan et al. 2013) and,
thereafter, coexisting with chronic allograft tubular atrophy/interstitial fibrosis
(Simic-Ogrizovic et al. 1998; Djamali 2007).
The first line of cellular defence against oxidative injury includes the antioxidant
enzymes catalase, SOD, and GPx. There are conflicting results in the literature on the
activities of antioxidant enzymes in kidney transplant patients. Glutathione com-
pounds and SOD have been reported to increase (Whitin et al. 1998; Zachara
et al. 2004), decrease (Campise et al. 2003), or not change (Vostalova et al. 2012)
following renal transplantation. Whitin and colleagues reported a rapid increase in
plasma GPx activity after transplantation (Whitin et al. 1998). Plasma GPx activity
was two times higher 3 days after transplantation in adult patients who received a
kidney transplant from a related donor and rapidly increased over the first 2 weeks
post-transplant in adult recipients from a deceased-donor and in paediatric patients
undergoing kidney transplantation from related donors (Whitin et al. 1998). Zachara
et al. demonstrated that plasma GPx activity increases rapidly 3 days after renal
transplantation and doubles 2 weeks later (Zachara et al. 2004). Both of these studies
suggested that monitoring plasma GPx might be a useful biomarker for monitoring
the transplanted kidney function and a valuable tool for the postoperative detection
of early graft dysfunction and/or DGF (Whitin et al. 1998; Zachara et al. 2004).
In kidney transplantation, some processes including immunologic disorders of
the kidneys, ischemic insults, and nephrotoxic drugs, lead to oxidative stress,
subsequent renal injury and graft dysfunction (Chung and Perrella 2004). In the
perioperative period, all solid organs used for transplantation undergo varying
862 I. Fonseca
degrees of ischemic damage and reperfusion injury after retrieval, storage, and
transplantation into the recipient (Koo et al. 1998). Ischemic changes start at the
time of a donor’s brain death. Specifically, brain death is associated with generalised
ischemia due to a hyperactivity of the sympathetic system, which aims to maintain
the cerebral perfusion pressure. Subsequently, free radical-mediated injury induces
the generation and release of proinflammatory cytokines and activates the innate
immune response. The immune response against a transplanted organ may not solely
involve a major histocompatibility complex (MHC)-specific alloimmune response,
but instead, an immediate nonspecific inflammatory response caused by ischemia-
reperfusion injury (Koo et al. 1998). It has been suggested that all of these changes
(the early innate response and the ischemic tissue damage) play roles in the devel-
opment of adaptive responses, which may in turn lead to early graft dysfunction
and/or acute kidney rejection (Kosieradzki and Rowinski 2008). Hypothermic
storage before transplantation add ischemic tissue damage to the organ and increase
the susceptibility to injury upon reperfusion, which is the final stage of ischemic
injury. The reperfusion injury is considered the effector phase of ischemic injury and
develops hours or days after the initial insult. The whole process has been described
as ischemia-reperfusion injury and it has a profound influence on not only the early
but also the late function of a transplanted kidney (Koo et al. 1998; Kosieradzki and
Rowinski 2008).
In the kidney, once the ischemia-reperfusion injury occurs, it causes a highly
complex cascade of events (Fig. 3; Koo et al. 1998; Kosieradzki and Rowinski 2008;
Gulec 2011). During ischemia, hypoxia is rapidly induced and followed by a sudden
drop in intracellular levels of ATP and a rapid increase in ROS production, including
superoxide (Edelstein et al. 1997; Dagher 2000; Lee et al. 2005). The kidney is
highly energetic and, therefore, relies heavily on aerobic metabolism for the pro-
duction of ATP by oxidative phosphorylation. The reduction of molecular oxygen
along the electron transport chain within the mitochondria is vital for renal cellular
function, and the interruption of blood supply results in an ischemic injury
(Small et al. 2012). The tubulointerstitium is a section of the kidney that includes
the tubules, which comprise approximately 80 % of the kidney volume, and the
compartment of the kidney bounded by the vasculature and nephrons. This compo-
nent of the kidney performs vital functions and is particularly vulnerable to oxygen
depletion. Thus, renal cells and the endothelial and tubular epithelial cells have high
energy requirements and are highly susceptible to injury in situations of relative
oxygen and nutrient deprivation (Khand et al. 2002; Kim et al. 2002; Maenpaa
et al. 2008; Nilakantan et al. 2008). In fact, the absence of blood supply causes a state
in which the restoration of circulation results in inflammation and oxidative damage
through the induction of oxidative stress (Sydykov 2009).
Although crucial for tissue survival, oxygen can be injurious during the reperfu-
sion of previously ischemic organ. Molecular oxygen, when reintroduced into a
previously ischemic kidney, undergoes sequential reduction steps, leading to the
generation of oxygen free radicals, which is a key process in the development of
reperfusion injury (Ponticelli 2014). Even thought the restoration of circulation
(reperfusion) is essential for the recovery of normal cellular function and the
Fig. 3 Brief description and sequence of events in ischemia-reperfusion injury. ATP Adenosine
triphosphate, ROS Reactive oxygen species
prevention of irreversible tissue injury, the reperfusion itself may initiate a series of
pathophysiological alterations that can augment the tissue injury produced by
ischemia alone (Sydykov 2009). The reperfusion of the ischemic kidney further
worsens the state of oxidation by promoting the additional release of free radicals. In
an study performed in animal model, Bolli and colleagues showed that potent
oxidant radicals, such as the superoxide anion, hydroxyl radical, and peroxynitrite,
are produced within the first few minutes of reflow and play a critical role in the
development of reperfusion injury (Bolli et al. 1989). Hence, ROS are generated
during both the ischemia phase and the reperfusion phase (Ponticelli 2014).
In kidney transplantation, not only do the ischemic and reperfusion periods
required for the organ preservation and implantation procedures induce oxidative
stress, but placing the kidney into an immune milieu can also act as an adjuvant for
oxidative damage. The warm ischemia after the kidney vessels are clamped and the
cold ischemia after refrigeration also reduce the oxygen and nutrients supply to the
tissues. A wide range of protective substances, such as antioxidant enzymes, may
protect the transplanted organ by limiting the production of ROS and the damage
from oxidative stress following ischemia-reperfusion injury of the kidney graft. The
kidney has naturally occurring antioxidant enzymes to counteract the effects of
oxygen free radicals. Superoxide dismutase catalyses the conversion of superoxide
864 I. Fonseca
to the harmless hydrogen peroxide. Glutathione works in a similar manner, but it can
also act on organic peroxides (Deneke and Fanburg 1989; Weinberg 1992; Lu 2009).
The protective abilities of these scavengers are overwhelmed when oxygen free
radical concentrations exceed the reducing capabilities of the scavengers (Granger
1988). There is some evidence that during the early phase of ATP depletion, which
typically occurs in ischemia, manganese superoxide dismutase (MnSOD), a major
mitochondrial antioxidant that eliminates superoxide, is inactivated (Parajuli and
MacMillan-Crow 2013). Some studies performed on heart transplantation (animal
model), have revealed a decrease in the antioxidant protein levels and a sequential
loss in MnSOD, catalase and GPx activity in acutely rejecting cardiac allografts
(Nilakantan et al. 2005a, b). To the best of our knowledge, there are no similar
studies in humans.
Malondialdehyde and Kidney Transplantation
Delayed graft function (DGF) is a clinical diagnosis that describes dysfunction of the
kidney allograft immediately after kidney transplantation, usually related to ischemic
damage to the graft. The rate of DGF after kidney transplantation varies from 2 % to
50 %, depending on the definition used and the centre’s transplant protocols. It is one
of the most important risk factors for both acute rejection and impaired renal function
at 1 year post-transplant. The has been a large amount of research devoted to the
search for DGF predictors or biomarkers that would allow for an early assessment of
allograft function and prediction of 1-year prognosis and long-term graft survival
(Koo et al. 1998; Yarlagadda et al. 2008, 2009; Ponticelli 2014).
A range of factors could lead to DGF such as organ procurement, donor charac-
teristics, prolonged ischemia time, recipient factors, renal toxicity, and ureteral
obstruction, among others (Schroppel and Legendre 2014). Almost 30 % of DGF
following kidney transplantation is attributable to ischemia reperfusion injury, which
is one of the most important non-specific and non-immunologic factor that affect
early and long-term allograft function (Gulec 2011). The production of excessive
quantities of ROS is an important mechanism of ischemia-reperfusion injury. As
previously discussed, these ROS cause tissue injury through lipid peroxidation and
the activation of endothelial cells, resulting in functional and structural cell damage
(Li and Jackson 2002; Eltzschig and Collard 2004; Dolegowska et al. 2012). Thus,
the importance of oxidative stress in kidney transplantation is highlighted by the
observation that increased oxidative stress exists in the presence of ischemia-reper-
fusion injury and possibly other risk factors for kidney graft dysfunction. Measure-
ment of the circulating levels of oxidative stress markers are useful along the whole
spectrum of the graft injury process and may be used for precise evaluation of
oxidative stress status of these patients in vivo. Moreover, oxidative markers could
be used for screening and risk assessment and may have may have a predictive value
for early graft function and long-term graft function.
Because of considerable amount of long-chain PUFAs in the composition of renal
lipids, lipid peroxidation is one of the most widespread hypotheses of ROS-mediated
cell injury (Ozbek 2012). Despite the controversy regarding whether lipid peroxi-
dation is the cause or the result of injury, increased lipid peroxidation is observed in
ischemia-reperfusion injury. Malondialdehyde is the principal by-product of PUFA
peroxidation and can be an indicator of tissue damage, thus it would reflect the
ischemia-reperfusion stress of the graft (Del Rio et al. 2005). Moreover, a recent
study in animal model revealed that clearance and kidney levels of MDA could
predict cyclosporine-induced nephrotoxicity (Sereno et al. 2015). Oxidants and
antioxidants can be biomarkers of graft dysfunction with diagnostic accuracy, not
only in the early post-transplant period but also in the longer-term. Increased plasma
and intragraft levels of MDA and decreased antioxidant activity were found in
kidney allografts with chronic tubular atrophy/interstitial fibrosis, which suggest
the possibility of early detection even when graft dysfunction is undetectable with
serum creatinine (Simic-Ogrizovic et al. 1998; Djamali et al. 2005; Djamali 2007).
Very few studies have investigated MDA levels in kidney transplantation as a
marker of lipid peroxidation and as a biomarker of ischemia-reperfusion injury
(Pincemail et al. 1993; Davenport et al. 1995; Fonseca et al. 2014). Although
these studies have shown elevated levels of MDA in plasma after graft reperfusion,
the potential correlations of MDA with subsequent graft function were examined in a
recent study (Fonseca et al. 2014). This study was done in 40 kidney recipients from
deceased and living donors and the mean levels of MDA were higher in the deceased
donor recipients at all measured time points. However, the mean difference was only
statistical significant on the second and fourth days (Fig. 4). Tubular and vascular
damage in the donor organ during cold ischemia before transplantation is associated
with subsequent ischemia-reperfusion injury and DGF. Thus, the kidneys from
deceased donors are more susceptible to ischemia and reperfusion damages than
from living donor, leading to higher production of ROS and subsequent lipid
peroxidation. However, in this study the mean differences in MDA levels between
deceased- and living-donors were not statistical significant on most of the days. In
addition, cold ischemia time was not correlated with MDA values. More research is
needed in this area to clarify these findings.
As the MDA content is an indicator of ischemia-reperfusion damage and as renal
ischemia-reperfusion injury occurring after kidney transplantation contributes to
kidney dysfunction and it is the most common cause of DGF, it seems rational that
MDA levels could be a considered a marker of graft injury, and then a predictive
biomarker of DGF. And effectively, the author observed that recipients who devel-
oped DGF presented increased MDA levels during the first week after kidney
transplantation, which appear to reflect the postischemic tissue damage of DGF
kidneys. Compared to pre-transplant, these patients presented higher MDA levels at
8–12-h following kidney transplantation, in contrast to recipients with prompt graft
function whose MDA levels continuously decreased throughout the week (Fig. 5).
Moreover, MDA on day-1 accurately predicted the need for dialysis within the first
week (AUC = 0.90), with a diagnostic performance higher than serum creatinine
(AUC = 0.73) and similar to that of cystatin C (AUC = 0.91), which is considered a
marker with greater sensitivity for the detection of impaired renal function. In
addition, the independent association of high levels of plasma MDA with poorer
866 I. Fonseca
Donor
.50 Living Donor
Deceased Donor
.40 *
Mean MDA ( micromol /L)
*
.30
.20
.10
.00
Pre- Day 1 Day 2 Day 4 Day 7
transplant
Time
Fig. 4 Longitudinal changes of the pre-transplant and 1 week post-transplant circulating levels of
MDA regarding donor status. Evolution of mean values of MDA with 95 % confidence intervals
according to donor status; measurements were performed preoperatively (pre-transplant), and then
at first (day-1), second (day-2), fourth (day-4) and seventh (day-7) days after kidney transplantation.
*Statistical significant (P < 0.05)
1-year allograft function was also reported (Fonseca et al. 2014). This suggests that
the products of oxygen free radical damage can be measured during kidney trans-
plantation, and that they may have an adverse effect on early and long-term graft
function.
A wide range of protective substances, such as antioxidant enzymes, may
potentially exert a protective influence by limiting the production of ROS and
the damage of oxidative stress following an ischemia-reperfusion injury of the
kidney graft. The antioxidant activity was also evaluated in this study. Compared
with healthy controls, kidney patients had significantly higher levels of SOD and
GR prior to kidney transplantation, which were, most likely, in response to the
increased oxidative stress in end-stage renal disease patients. However, no signif-
icant changes were observed following kidney transplantation, even when strati-
fied by graft function. Kidney DGF is a manifestation of acute kidney injury; thus,
for an existing or acute injury, it is natural for higher quantities of ROS to be
released. Under normal conditions, endogenous antioxidant enzymes neutralise
these radicals and the enzyme activity subsequently decreases. Another possibility
is that the capacity of the antioxidant defence system increases as a response to a
DGF
No Yes
*
.50 *
*
.40 *
Mean MDA (micromol/L)
.30
.20
.10
.00
Pre- Day1 Day2 Day4 Day7
transplant
Time
Fig. 5 Longitudinal changes of the pre-transplant and 1 week post-transplant circulating levels of
MDA regarding graft function. Evolution of mean values of MDA with 95 % confidence intervals
according to the development of delayed graft function (DGF) or not; measurements were
performed preoperatively (pre-transplant), and then at first (day-1), second (day-2), fourth (day 4)
and seventh (day-7) days after kidney transplantation. *Statistical significant (P < 0.05)
higher production of oxidant radicals. Because neither of these two situations

occurred, it was hypothesised that in the first week after kidney transplantation,
the antioxidant defence system does not effectively respond to the higher levels of
oxidative stress detected in DGF.
Oxidative stress is believed to be a common pathway that leads to both
immunological and nonimmunological stress in the setting of kidney transplan-
tation and to the development or progression of chronic allograft nephropathy.
Our understanding of oxidative stress has significantly advanced in the last
decade, but these experimentally derived ideas have yet to be fully integrated
into clinical practice. Current hypotheses of renal oxidative stress caused by
ischemia-reperfusion injury include the direct impact of free radicals; and the
ability of ischemia-reperfusion generate ROS, which are able to start lipid
peroxidation either directly or by exhausting antioxidant defence substances. It
is also possible that, similar to MnSOD, the major enzymes of the antioxidant
system become inactivated during the early phase of ATP depletion in ischemia
868 I. Fonseca
(Parajuli and MacMillan-Crow 2013). Maybe, one way of combating this

increased oxidative stress and the following damages would be to increase
antioxidant defences, namely through the supplementation of non-enzymatic
antioxidant molecules.
Reliable biomarkers enabling early discrimination of DGF in kidney transplantation

are lacking, which impairs timely therapeutic interventions. There are many new
approaches devoted to the search for new DGF biomarkers that would allow for early
determination of allograft dysfunction and of prognoses for 1-year and long-term
graft survival. Elevated MDA levels reflected kidney dysfunction and predicted the
need for renal replacement therapy within the first week following kidney transplan-
tation with more precisions and efficiency than monitoring serum creatinine, which
is a promising finding because it will possibly enable to target any intervention to
those patients who will benefit most.
With the understanding of the mechanisms of the pathophysiology of kidney
ischemia-reperfusion having evolved, many possible interventions suggest them-
selves. Oxidative stress is one of the most important components of the ischemia-
reperfusion process, which is an inevitable phenomenon in kidney transplantation
(Paller 1992; Kim et al. 2009; Hariharan et al. 2011; Dolegowska et al. 2012).
Given the important role that ROS play in ischemia-reperfusion injury, it is not
surprising that there is growing interest in the possibility that supplementation with
nutritional antioxidants will provide renoprotection. In other organs, such as the
myocardium and cerebrum, some research using animal models reveals that the
free radical-associated ischemia-reperfusion injury can be prevented or reduced by
a pre- or post-insult treatment with nutritional antioxidants such as vitamin E,
selenium, vitamin C, and beta carotene (Yoshida 1989; Poltronieri et al. 1992;
Coombes et al. 2000). Although animal studies suggest that dietary supplementa-
tion with antioxidants provides protection against ischemia-reperfusion induced
injury, it is unclear if antioxidant supplementation can provide the same protection
in humans and specifically in kidney transplantation against ischemia-reperfusion
injury. Moreover, the discovery of novel biomarkers that are able to predict the
clinical response to antioxidant interventions is a crucial challenge to overcome to
allow for the personalization and timely intervention of kidney transplant thera-
pies. Will MDA be able to evaluate the effectiveness of antioxidant therapy?
Although researchers already have gained substantial insight into the mechanisms
and consequences of ischemia-reperfusion injury and tissue damage induced by
oxidative stress, there is also almost a complete absence of studies which address
the MDA molecule and kidney transplantation. Thus, additional studies are
required to further clarify the ability of MDA to monitor the progression
(or regression) of the graft damage after ischemia-reperfusion injury, particularly
in patients who experienced DGF.
Summary Points
• The production of excessive quantities of ROS is an important mechanism of

reperfusion injury. In kidney transplantation, oxygen free radicals are the most
likely agents responsible for initiating the damage associated with reperfusion
injury.
• Oxidative stress, and specifically lipid peroxidation, is a vicious cycle: the disease
causes increased lipid peroxidation, which is then responsible for toxicity and
more cellular and tissue damage
• Lipid peroxidation is a well-established mechanism of cellular injury and is used
as an indicator of oxidative stress in cells and tissues. Lipid peroxides, derived
from PUFAs, are unstable and decompose to form a complex series of com-
pounds. These include reactive carbonyl compounds, which is the most abundant
is MDA. Therefore, measurement of MDA is widely used as a biomarker of lipid
peroxidation.
• Lipid peroxidation and MDA content are good indicators of ischemia-reperfusion
damage.
• Malondialdehyde is a reliable diagnostic biomarker of initial graft injury and
accurately detect graft dysfunction earlier than serum creatinine.
• Malondialdehyde is a predictor of dialysis requirement during the first week post-
transplantation and of kidney graft function within the first year post-
transplantation
• The endogenous antioxidant defence system does not effectively respond to the
higher levels of oxidative stress detected in DGF.
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Utility of Neutrophil Gelatinase-Associated
Lipocalin in Kidney Transplantation: 38
Detailed Review
Juan C. Ramirez-Sandoval, William Herrington, and

Luis E. Morales-Buenrostro
Contents
Key Facts of Urinary Neutrophil Gelatinase and Graft Loss in Kidney Transplant . . . . . . . . . . . 876
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 877
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 877
Utility of NGAL Assessment During the Early Posttransplantation Period . . . . . . . . . . . . . . . . . . . 879
Early Diagnosis of Delayed Graft Function (Fig. 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 880
Prognosis of Graft Loss Months or Years After Transplantation (Fig. 3) . . . . . . . . . . . . . . . . . . 883
Utility of NGAL Assessment After AKI in Late Posttransplantation Period (Fig. 4) . . . . . . . . . 887
Utility of NGAL Assessment During KTR Surveillance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 888
Utility of NGAL in Differentiating Etiology of Graft Failure (Fig. 5) . . . . . . . . . . . . . . . . . . . . . . . . . 888
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 889
Potential Applications to Prognosis, Other Disease, or Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 890
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 891
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 891
Abstract
Neutrophil gelatinase-associated lipocalin (NGAL) is a protein expressed by
kidney tubular cells in response to ischemia but may also be an early indicator
of immunological rejection, calcineurin inhibitor toxicity, obstructive nephropa-
thy, subclinical tubulitis, or infection. Although there is currently no evidence to
support the routine serial measurement of blood or urinary NGAL to detect
subclinical acute tubular injury, NGAL has the potential to provide useful infor-
mation to those that care for kidney transplant recipients (KTRs). First, high
J.C. Ramirez-Sandoval • L.E. Morales-Buenrostro (*)

Department of Nephrology and Mineral Metabolism, National Institute of Medical Sciences and
Nutrition Salvador Zubirán, D.F., Mexico
e-mail: juancarlosramirezsandoval@yahoo.com; luis_buenrostro@yahoo.com
W. Herrington
Oxford Kidney Unit, Oxford University Hospitals NHS Trust, Churchill Hospital, Oxford, UK
e-mail: will.herrington@ouh.nhs.uk

876 J.C. Ramirez-Sandoval et al.
urinary or serum NGAL concentrations shortly after transplantation are a predic-

tor of delayed graft function and are associated with reduced graft function at
1 year. Secondly, among KTRs with previously stable graft function who then
suffer acute graft dysfunction, a high urinary NGAL predicts graft loss at 1 year.
If further refined, diagnostic tests based on NGAL levels may provide future
useful clinical tools.
Keywords
NGAL • Biomarkers • Kidney transplantation • Acute kidney injury • Delayed
graft function • Immunological rejection • Graft survival • Early diagnosis •
Sensitivity and specificity • ROC curve
Abbreviations
KTR Kidney transplant recipient
ROC Receiver-operating characteristic curve
Se Sensitivity
sNGAL Serum neutrophil gelatinase-associated lipocalin
Sp Specificity
uNGAL Urinary neutrophil gelatinase-associated lipocalin
Key Facts of Urinary Neutrophil Gelatinase and Graft Loss

in Kidney Transplant
• Kidney transplantation replaces normal kidney function and should (in most
situations) be regarded as the optimum treatment of end-stage kidney disease.
• Patients who are fit for transplantation survive longer and have a better quality
of life.
• According to 2009 and 2005 SRTR National Report and 2002 UNOS report,
kidney graft survival for adult recipients after 10 years of transplant is 51 % and
68 % for deceased and living donor, respectively.
• Living donor transplantations had better graft and KTR survival than deceased
transplantation, probably explained by less ischemia-reperfusion damage to graft
and closer major histocompatibility complex (MHC) matching before surgery.
• Deceased donor transplantation is the most common form of transplant in devel-
oped countries, but there is a significant and growing organ shortfall.
• Retrieving of kidneys from deceased donors is exposed to ischemia, between
circulatory arrest and the start of cold storage (warm ischemia) and during the
time that graft is on cold storage before transplantation (cold ischemia).
38 Utility of Neutrophil Gelatinase-Associated Lipocalin in Kidney. . . 877
• Current means of graft evaluation after transplantation, particularly using creat-

inine, show multiple flows, especially when the patient is in an unstable state.
• A biomarker identifying the quality of donor kidneys at the time of donor
evaluation would therefore be very useful.
• For now, the ideal noninvasive biomarker of graft dysfunction does not exist.
Definitions
Area under receiver operating characteristic curve (AUC/ROC) A curve plots

the true positives (sensitivity) vs false positives (1 specificity) at each “cutoff”
value for any diagnostic test that uses a continuous variable. An area under curve of
100 % (1.0) defines a perfect test. An area value of 0.5 indicates no discriminative
value.
Calcineurin inhibitors Drugs that suppress the immune system by inhibiting

interleukin production in T cells and are associated with acute or chronic graft
dysfunction when blood concentrations are in high levels (i.e., tacrolimus or
cyclosporine).
Delayed graft function Normally, kidney transplant recipients have progressive

diuresis and gradual decrease in serum creatinine levels in the following hours after
kidney transplant surgery. If dialysis is required within 7 days after transplantation, it
is classified as delayed graft function.
Immunological rejection Adaptive immune response against the graft caused by

cellular and/or antibody recognition of proteins perceived as foreign by the recipient.
Estimated glomerular filtration rate Calculation based generally in serum or

urinary creatinine that describes the milliliters of blood filtered through kidneys in
1 min. It depends from several factors including age, gender, and race, among others.
As a rule, its value in young people must be higher than 90 mL/min/1.73 m2.
Introduction
In appropriately selected patients with end-stage kidney disease, kidney transplan-

tation offers improved long-term survival when compared to continuing dialysis
(Suthanthiran and Strom 1994).
Nevertheless, those caring for kidney transplant recipient (KTRs) still face many
challenges. First, even in countries with well-developed kidney services, the number
of patients on dialysis eligible for a kidney transplant exceeds supply of donor
organs (Pussell et al. 2012). Secondly, although in recent years there have been
significant gains in the prevention of immunological graft loss during the first year
after transplantation, there has been little to no improvement in long-term graft
survival (Meier-kriesche et al. 2004a). Thirdly, despite apparently increasingly

effective treatment of acute rejection, in about two-fifths of cases, graft function
does not return to its pre-rejection baseline, which is associated with an incremental
increase in death-censored graft loss (Meier-Kriesche et al. 2004b). Lastly, the
currently available, inexpensive, noninvasive diagnostic tests (such as measurement
of serum creatinine) are poor predictors of early histopathological changes associ-
ated with rejection (Yilmaz et al. 2007). Therefore, in addition to increasing the
donor pool, novel early diagnostic and prognostic biomarkers are needed to detect
those at risk of future graft injury and loss.
Several proteins that are shed by (or are failed to be resorbed by) injured kidney
tubule epithelial cells have been described and may have the potential to identify
ongoing kidney injury and perhaps even distinguish the etiology of the injury (Tesch
2010).
Neutrophil gelatinase-associated lipocalin (NGAL), also known as lipocalin-2, is
a 25 kDa single disulfide-bridged polypeptide covalently bound to a gelatinase
(Kjeldsen et al. 1993). As has been discussed in other chapter of this book, NGAL
has several forms. The dimeric form of NGAL is expressed in neutrophils and
functions as part of the innate immune system by sequestering iron siderophores
(Flo et al. 2004). These processes reduce iron availability, limiting bacterial prolif-
eration (Goetz et al. 2002; Paragas et al. 2012).
The dimeric form of NGAL is detectable in the urine of those with urinary tract
infections (Mårtensson et al. 2012) but is also induced by aseptic injury. It is
proposed that, in addition to bacteriostasis, NGAL may, by inhibiting apoptosis
and enhancing cellular proliferation/differentiation (Kashiwagi et al. 2014; Schmidt-
Ott et al. 2007), also have a reparatory role in injured epithelial tubular cells.
NGAL is secreted in low levels from several cell types and, due to its size and
charge, is freely filtered through the glomerulus. Filtered NGAL is then almost
completely resorbed by healthy tubular cells. The average urinary NGAL
(uNGAL) concentration in healthy adults is also about 20 ng/mL (Kuwabara
et al. 2009). Similar average concentrations of NGAL (unadjusted for urine concen-
tration) have been observed in KTRs with stable good graft function. In a sample of
seven stable KTR with an estimated glomerular filtration rate of 96 mL/min/1.73 m2
and a normal graft biopsy, median uNGAL concentration was 5 (interquartile range
1–7) ng/mL or, corrected for grams of urinary creatinine, 0.212 (interquartile range
55–289) ng/mg creatinine. These biomarker values are similar to those obtained
from healthy donors (Ramirez-Sandoval et al. 2014).
Lower creatinine excretion in the setting of acute kidney injury may amplify a
tubular injury biomarker signal, thereby increasing its clinical utility. However, one
report demonstrated marked variability in creatinine excretion across and within
individuals in the setting of AKI or kidney transplantation (Lebkowska et al. 2009).
Response to acute kidney injury in non-KTRs is an early rise in uNGAL and serum
NGAL (sNGAL) concentration, especially in its monomeric form (Mårtensson
et al. 2012). Some of the increased uNGAL represents impaired proximal tubular
resorption, but the main source of the raised sNGAL and uNGAL is secretion from the
distal nephron (Singer et al. 2013). This rapid change in NGAL concentration is
Fig. 1 NGAL is released from ischemic kidney tubules and may serve as an early diagnostic
biomarker better than serum creatinine. Raised urinary or serum NGAL levels are useful for early
diagnosis of kidney injury
detectable far in advance of changes in serum creatinine (Fig. 1) (Devarajan 2010;

Barrera-Chimal and Bobadilla 2012).
Serum creatinine is also an imperfect prognostic biomarker of long-term graft
survival in KTRs, as although a raised creatinine at 1 year (or decline in creatinine
between 6 months and 1 year from transplantation) predicts those that are likely to
progress to graft loss (i.e., the test is highly sensitive), a large proportion of those
who go on to have graft failure have a normal creatinine at 1 year (i.e., the test has
poor specificity) (Kaplan et al. 2003).
Although the gold standard assay to certify NGAL concentration is immunoblot
based (which is a time-consuming and impractical assay for routine clinical use), an
ELISA, a chemiluminescent microparticle immunoassay (ARCHITECT ®, Abbott),
and a point-of-care fluorescence-based immunoassay (Triage ®, Biosite) are all
available and suited for clinical use (with acceptable correlation with ELISA)
(Hollmen et al. 2014). In this chapter, the potential for NGAL as a biomarker for
clinical use in KTR is considered.
Utility of NGAL Assessment During the Early Posttransplantation

Period
Delayed graft function (DGF) is commonly defined as the necessity for dialysis
within the first week after kidney transplantation (Mallon et al. 2013). DGF occurs
less frequently in grafts from living donor (4–10 %) compared with deceased donor
(5–50 %) (Irish et al. 2003; Troppmann et al. 1995; Sharif and Borrows 2013). This
complication is associated with an increased risk of late graft loss, acute
immunological rejection, and KTR death even after recovery of delayed graft
function (Yarlagadda et al. 2009; Tapiawala et al. 2010).
The principal causes of delayed graft function are derived from donor ischemic
injury prior to retrieval or during preservation and recipient-derived ischemia-reper-
fusion injury after implantation (Siedlecki et al. 2011).
In kidney biopsies taken approximately 1 h after reperfusion, NGAL expression
is observed in proximal and distal tubular epithelial cells. Intensity of NGAL
staining is greatest in deceased donor kidneys and is strongly correlated with cold
ischemic time (R = 0.86) and peak postoperative creatinine (Mishra et al. 2006).
Measurement of uNGAL and/or sNGAL in the immediate posttransplant period
would be expected to predict DGF.
Early Diagnosis of Delayed Graft Function (Fig. 2)
Indeed, a systematic review of 1,079 KTR from 14 studies has demonstrated

that a uNGAL or sNGAL measurement 6–12 h after transplantation is highly
predictive of DGF. Aggregation of results suggested that a “raised” NGAL concen-
tration was both sensitive (82 %) and specific (82 %) for predicting DGF (aggregated
area under receiver operating characteristic curve [AUC/ROC] was 0.87) (Haase
et al. 2009).
Ten of the twelve studies that have investigated the role of uNGAL posttrans-
plantation have found that it predicts DGF (these studies are summarized in Table 1).
However, although in theory urinary NGAL might be expected to be more specific
for kidney injury than sNGAL (as NGAL can be secreted into circulation from
other organs), this hypothesis is not borne out in practice due to the high frequency
of oliguria and anuria postoperatively (between 12 % and 38 % of KTRs of
deceased donors are anuric in the postoperative period) (Pajek et al. 2014;
Fonseca et al. 2013). Furthermore, differences in glomerular filtration of sNGAL
derived from extrarenal tissues and urine from residual functioning native kidneys
Fig. 2 NGAL is a short-term

prognosis biomarker in kidney
transplantation. The serum or
urinary NGAL levels 6–12 h
after transplantation predict
delayed graft function
38
Table 1 Predictive capacity for delayed graft function of urinary NGAL

Number of Optimal threshold value
DGFa or SGFb/ Hours after AUC/ (sensitivity, specificity) and
First author Year total KTR transplantation Test NGAL ROC comments
Hollmen 2011a, 66/176 Before surgery Chemiluminescent microparticle 0.595 4 ng/mL (Se 77 %, Sp 28 %)
b immunoassay (ARCHITECT, Abbott 20 ng/mL (Se 35 %, Sp 79 %)
Diagnostics)
Hall 2011 34a/91 6h ELISA (Antibody Shop, Gentofte, 0.81 350 ng/mL (Se 77 %, Sp 74 %)
24 h Denmark) 0.82
Hollmen 2011a, 66a/176 24 h Chemiluminescent microparticle 0.75 560 ng/mL (Se 68 %, Sp 73 %)
b immunoassay (ARCHITECT, Abbott
Diagnostics)
Salamzadeh 2012 11/68 24 h ELISA kits were utilized for Not 3.77 ng/mL in group with DGF vs
measuring urine NGAL (Antibody reported 1.82 ng/mL
Shop, Gentofte, Denmark)
Rostami 2013 31c/64 2h ELISA test kit (Antibody Shop, 0.71 204 ng/mL (Se 72 %, Sp 67 %)
24 h Gentofte, Denmark) 0.68 77 ng/mL (Se 72 %, Sp 64 %)
Fonseca 2013 18/40 8–12 h after Chemiluminescent microparticle 0.88 286 ng/mL (Se 100 %, Sp 76 %)
transplant immunoassay (ARCHITECT, Abbott
48 h after Diagnostics) 0.96
transplant
Utility of Neutrophil Gelatinase-Associated Lipocalin in Kidney. . .
Choi 2013 14b/69 48 h ELISA (BioPorto Diagnostics, 0.778 uNGAL 254.7 ng/mg UCr
Gentofte, Denmark) (Se 85.7 %, Sp 66.7 %)
Field 2014a, 49a/182 Before surgery ELISA using Luminex (R&D systems, No uNGAL was similar in donors
b Minneapolis, MN, USA) differences giving immediate or aberrant
functioning kidneys (92.17 ng/mL
versus 92.63 ng/mL, respectively)
(continued)
881
882
Table 1 (continued)
Number of Optimal threshold value
DGFa or SGFb/ Hours after AUC/ (sensitivity, specificity) and
First author Year total KTR transplantation Test NGAL ROC comments
Qurashi 2014 11/67 6h Chemiluminescent microparticle – DGF had 950
750 ng/mL vs
immunoassay (ARCHITECT, Abbott 375
387 ng/mL in group without
Diagnostics) DGF
Buemi 2014 20a/97 6h Automated two-site sandwich Not uNGAL no differences compared in
24 h immunoassay (Abbott Laboratories, reported KTR with DGF versus KTR
48 h Abbott Park, IL) and fluorescence without DGF
immunoassay Triage NGAL test
(Biosite–Inverness Medical, Waltham,
MA)
Pajek 2014 40b/71 24 h ELISA (BioPorto Diagnostics, 0.82 33.1 μg/mmol UCr (Se 68 %, Sp
Gentofte, Denmark) 93 %)
Pianta 2015 23/81 4h ELISA (R&D DuoSets, R&D 0.77
systems, Minneapolis, MN)
KTR kidney transplant recipients, DGF delayed graft function, AUC/ROC area under receiver operating characteristic curve, Se sensitivity, Sp specificity, UCr
urinary creatinine
a
DGF was defined as the need for at least one dialysis course in the first 7 days following transplantation
b
SGF was defined as a serum creatinine (Scr) reduction ratio (difference between Scr at 0 h and the Scr on day 7 divided by Scr at 0 h) less than 0.75
c
Includes DGF and defined as Cr level more than 1.5 mg/dL on the second postoperative day
J.C. Ramirez-Sandoval et al.
may modify the uNGAL signal from graft tubules. As an example of relationship
between uNGAL and sNGAL, Buemi et al. has demonstrated that uNGAL concen-
trations may be half that of sNGAL at 6 h posttransplant. Median values of sNGAL
6 h after transplant were 620 ng/mL and 538 ng/mL in KTR who received living and
deceased donor kidneys, respectively, while the corresponding uNGAL measure-
ments were 92.5 ng/mL and 212.3 ng/mL, respectively (Buemi et al. 2014).
Moreover, evidence to date suggests that uNGAL does not improve the predic-
tion of DGF when urine output and urinary creatinine excretion are already known.
Pajek et al. found that a urinary creatinine excretion rate of less than 0.56 mmol/h
10 h posttransplant predicted DGF and slow graft function with 94 % sensitivity
and 84 % specificity (AUC/ROC of 0.90, 95 % confidence interval
[CI] 0.80–0.96), while a uNGAL-to-creatinine ratio >33.1 mcg/mmol had 68 %
sensitivity and 93 % specificity and similar AUC/ROC of 0.82, 95 % CI 0.70–0.91
(Pajek et al. 2014).
Nine of the ten studies have found that sNGAL predicts DGF (Table 2). sNGAL
measurement is easier than uNGAL and negatively correlates with urine output.
Hollmen et al. reported that the mean posttransplant sNGAL was 822 ng/mL if the
patient is anuric, 697 ng/ml if the diuresis was between 100 and 1,000 ml in 24 h,
and 561 ng/ml if the diuresis was more than 1,000 ml in 24 h; a day 1 post-op
sNGAL concentration of over 426 mg/mL is a predictor of DGF with sensitivity of
over 90 % and specificity of 83 % (AUC/ROC 0.91, 95 % CI 0.86–0.96). sNGAL
was also an independent predictor of DGF even when other factors had been taken
account of (including urine output and donor characteristics) (Hollmen
et al. 2014).
There are several difficulties with interpreting the studies of NGAL: notable
limitations include variability in the performance of NGAL assays, heterogeneity
in DGF definition, heterogeneous kidney transplant patient populations, and the lack
of uniformly applicable cutoff values (see Tables 1 and 2). Nevertheless, the current
data suggest that sNGAL may offer additional diagnostic information with regard to
which kidney transplants are likely to develop delayed graft function.
Prognosis of Graft Loss Months or Years After Transplantation

(Fig. 3)
It has also been suggested that sNGAL concentration measured at 24 and 48 h after
transplantation predicts long-term prognosis after transplantation. Four studies have
tested this hypothesis (Table 3).
Interestingly, after excluding those KTR with DGF, uNGAL predicts different
long-term outcomes in patients with early normal graft function. Fonseca et al. found
that uNGAL measured on the fourth and seventh days postsurgery was indepen-
dently associated with 1-year graft function, independent of donor characteristics,
acute rejection episodes, and rehospitalizations (Fonseca et al. 2013). Similarly, Choi
et al. found that a uNGAL-to-creatinine ratio cutoff value of 153 ng/mgCr had a
sensitivity and specificity of 95 % and 65 %, respectively (AUC/ROC 0.83), for
884
Table 2 Predictive capacity for delayed graft function of serum/plasma NGAL

Number of DGFa
or SGF***/total Hours after ROC/ Optimal threshold value
Author Year KTR transplantation Test NGAL AUC (sensitivity, specificity)
Parikh (include 2006 10/60 24 h ELISA (HYB211–01B, 0.90 450 ng/mg UCr (Se 90 %, Sp
adults and children) Antibody Shop) 48 %); 1,000 ng/mg UCr
(Se 90 % Sp 83 %)
Kusaka 2008 7/67 24 Chemiluminescent microparticle 0.99 500 ng/mL (Se 91 %, Sp 97 %)
immunoassay (ARCHITECT,
Abbott Diagnostics)
Lebkowska 2009 4/41 24 h ELISA (Antibody Shop, Not Four KTR with DGF did not
Gentofte, Denmark) reported show a fall in sNGAL
Hall 2011 26/78 0, 24, 48 ELISA (Antibody Shop) No sNGAL was not different
differences between KTR with DGF and
others
Bataille 2011 15/41 24 Triage meters (NGAL test 0.97 400 ng/mL (Se 93.3 %, Sp
Triage; Biosite Inc., Inverness 88.5 %)
Medical, San Diego, CA)
Lee 2012 14/59 24 h ELISA (BioPorto Diagnostics, 0.86 411.4 ng/mL (Se 57 %, Sp 98 %)
Gentofte, Denmark)
Rahimzadeh 2012 2b/27 24 h ELISA (?) 0.95 174 ng/mL (Se 100 %, Sp
(include only 95.5 %)
children <18 years)
J.C. Ramirez-Sandoval et al.
38
Buemi 2014 20/97 6h Automated two-site sandwich 0.73

24 h immunoassay (Abbott 0.80
48 h Laboratories, Abbott Park, IL) 0.85
and fluorescence immunoassay
Triage NGAL test
(Biosite–Inverness Medical,
Waltham, MA)
Hollmen 2014 66/176 24 h ELISA kit (BioPorto 0.85 423 ng/mL (Se 87 %, Sp 77 %)
Diagnostics, Gentofte,
Denmark) and point-of-care
(POC) fluorescence
immunoassay NGAL kit and
device (Triage Biosite, San
Diego, California, USA)
Pajek 2014 40b/71 24 h ELISA (BioPorto Diagnostics, 0.82 33.1 μg/mmol UCr (Se 68 %, Sp
Gentofte, Denmark) 93 %)
KTR kidney transplant recipients, DGF delayed graft function (the need for hemodialysis procedure in the first week), SGF slow graft function (less than 70 %
reduction of serum creatinine in the first week), AUC/ROC area under receiver operating characteristic curve, Se sensitivity, Sp specificity, UCr urinary
creatinine
a
DGF was defined as the need for at least one dialysis course in the first 7 days following transplantation
b
DGF or SGF
Utility of Neutrophil Gelatinase-Associated Lipocalin in Kidney. . .
885
Fig. 3 NGAL might be a long-term prognosis biomarker in kidney transplantation. In some

studies, the serum or urinary NGAL levels assessed the first week after transplantation predict
long-term graft function or graft loss at 1 year
Table 3 Predictive capacity for long-term graft prognosis of urinary NGAL assessed early after
transplantation
Assessment of
Author Year Outcome NGAL Comments
Hall 2011 eGFR at 3 months after At time 0, 1st No association
transplantation day and 2nd
day after
transplant
Hollmen 2011a, Graft survival after 1 year Before donor No differences
b between donor with serum operation between groups
NGAL 214 ng/mL vs donors
with NGAL <214 ng/mL
Choi 2013 eGFR <60 mL/min/1.73 m2 at 2nd day after AUC/ROC of 0.832
1 year after transplantation transplant Cutoff value
152.9 ng/mL
(Se 95 %, Sp 65 %)
Fonseca 2013 Multivariate analysis for 4th day after Regression
prediction of graft function transplant coefficient adjusted
Ln uNGAL at 4th
day = 0.067
7th day after Regression
transplant coefficient adjusted
Ln uNGAL at 7th
day = 0.0138
Urinary NGAL was
associated with
1-year serum
creatinine
Yang 2014 eGFR <60 mL/min/1.73 m2 at At time 0, 2nd No association
2 years after transplantation day and 6th
day
eGFR estimated glomerular filtration rate, AUC/ROC area under receiver operating characteristic
curve
predicting “poor 1-year outcome” (defined as a eGFR <60 mL/min/1.73 m2) in

62 KTR (Choi et al. 2013).
Interestingly, raised sNGAL measured 2 days after transplant surgery was not
associated graft function 3 months after transplant (Hall et al. 2010, 2011), raising
the hypothesis that uNGAL is a more useful prognostic for subclinical tubular
ischemic injury postsurgery as significant extrarenal NGAL secretion in the recovery
phase from surgery may mask any increased kidney secretion of NGAL into
circulating blood.
Utility of NGAL Assessment After AKI in Late Posttransplantation

Period (Fig. 4)
KTR have an increased risk of severe AKI which carries with it high risk of graft
failure (Nakamura et al. 2012). Paradoxically, AKI might be more frequent in KTRs
with higher eGFR (Mehrotra et al. 2012). uNGAL concentrations during AKI are an
independent predictor of graft loss. In a study of 67 KTRs with AKI occurring on
average nearly 3 years since successful transplantation, uNGAL predicted graft loss
with sensitivity and specificity of 84 % and 91 %, respectively (AUC/ROC 0.89,
95 % CI 0.81–0.97), using a cutoff value of 210 ng/mL (Fig. 6) (Ramirez-Sandoval
et al. 2014). Similar urinary NGAL cutoffs predict the development of AKI or need
for renal replacement therapy initiation/death in non-KTR populations at high risk of
AKI (Haase et al. 2009).
Fig. 4 NGAL might be a long-term prognosis biomarker after acute graft injury. In kidney
transplant recipients with stable graft function, NGAL assessed during an episode of acute kidney
injury is useful to predict graft loss
Utility of NGAL Assessment During KTR Surveillance
The available evidence suggests that there is no utility in routinely measuring

NGAL during routine KTR surveillance. Kaufeld et al. compared protocol trans-
plant biopsies with uNGAL results over 6 months in 140 KTR. Median urinary
NGAL-to-creatinine ratios did not discriminate KTRs with the presence or absence
of acute tubular injury on biopsy. NGAL-to-creatinine ratios were also not useful at
discriminating those with serial evidence of acute tubular injury (Kaufeld
et al. 2012).
However, there is some evidence among high-risk KTRs that NGAL surveillance
may have a use. Field et al. studied sNGAL in 94 highly sensitized patients
following HLA-incompatible kidney transplantation, among who 44 experienced a
rejection episode within 30 days. A sNGAL concentration 6,865 ng/mL at day
1 postsurgery had a sensitivity of 73 % and a specificity of 60 % for predicting
immunological rejection (AUC 0.67) (Field et al. 2014a).
Utility of NGAL in Differentiating Etiology of Graft Failure (Fig. 5)
uNGAL’s potential to distinguish rejection from other causes of AKI in KTRs was
hypothesized in data from 44 KTR with AKI. Compared to 138 stable allografts,
uNGAL was significantly higher in the 35 individuals with non-rejection AKI
(median uNGAL concentration 8 [IQR 4–17] ng/mL compared to 59 [IQR
33–136] ng/mL). Moreover, compared to non-rejection AKI, nine KTRs with
Fig. 5 NGAL and long-term graft loss after acute kidney injury. This survival graft curve shows
that kidney transplant recipients with acute graft dysfunction and urinary NGAL higher than
163 ng/mL at diagnosis have a worst prognosis of graft survival compared with lower levels
Fig. 6 Utility of NGAL for differential diagnosis of acute kidney injury etiology in kidney
transplant recipients. At this time, NGAL seems not to be a useful biomarker for etiology diagnosis
in acute graft injury, especially to identifying immunological rejection
biopsy-proven acute rejection had substantially elevated uNGAL (median

339 [165–499] ng/mL). A cutoff of 100 ng/mL represented 100 % sensitivity and
93 % sensitivity (AUC of ROC 0.98) (Heyne et al. 2012). However, these promising
results have not been replicated in other studies. Ramirez-Sandoval et al. did not find
utility in uNGAL when 20 KTR with immunological rejection were compared with
47 non-immunological causes of AKI. uNGAL-to-creatinine ratio with a cutoff point
of 59 ng/mg had 60 % sensitivity and 58 % specificity at differentiating rejection
from other causes of AKI (AUC/ROC 0.75, 95 % CI 0.61–0.88) (Ramirez-Sandoval
et al. 2014). Furthermore in a study of KTRs by Zhang et al., no significant
difference was observed between uNGAL measurements in 41 KTR with biopsy-
confirmed acute rejection, 29 with biopsy-proven acute tubular necrosis, and 15 sta-
ble allograft controls (Zhang et al. 2013) (Fig. 6).
Table 4 summarizes other studies that have assessed NGAL as a biomarker to
differentiate calcineurin inhibitor toxicity, obstructive nephropathy, or subclinical
tubulitis. Overall, the current evidence suggests that measuring NGAL, at least in
isolation, does not help distinguish etiology of acute graft dysfunction.
Conclusions
NGAL is a promising biomarker of graft injury and may have clinical utility in
predicting early and long-term prognosis after kidney transplantation, for long-term
prognosis after an acute kidney injury and, possibly, for early diagnosis of graft
dysfunction. Appropriate randomized clinical trials on important outcomes in grafts
and recipients comparing the use of NGAL versus the current standard of clinical
Table 4 Different causes of urinary NGAL elevation in graft dysfunction

Cause Author/year Example
Reperfusion See Table 1 See Table 1
injury
Calcineurin Wasilewska A group of children who receive cyclosporine and uNGAL
inhibitors et al. (2010) were determined before and 3, 6, and 12 months after
transplantation. uNGAL increase from basal 4.74 to 15.6 ng/
mL (uNGAL/uCr 4.2–18.3)
Tsuchimoto Twenty liver transplant recipients developed AKI by
et al. (2014) tacrolimus (n = 31). The AUC/ROC of urinary NGAL was
0.876
Obstructive Lucarelli Not study in kidney transplant but evidence in other kidney
nephropathy et al. (2014) diseases
IgA Ding Not study in kidney transplant but evidence in other kidney
nephropathy et al. (2007) diseases
Urinary tract Kaufeld Urinary tract infection has a linear correlation with urinary
infection et al. (2012) NGAL
BK virus Rau One report did not find significant difference in plasma
nephropathy et al. (2013) NGAL expression between BKV+ patients and BKV
negative patients
AUC/ROC area under receiver operating characteristic curve, UCr urinary creatinine
care are required (with economic assessments) in order to promote more widespread
use of NGAL in kidney transplantation.
Changes in NGAL concentration however are not specific for a particular mech-
anism of kidney injury, and therefore, NGAL will not replace the need of graft
biopsy in order to distinguish different etiologies of graft dysfunction.
Potential Applications to Prognosis, Other Disease, or Conditions
Evidence-Based Applications of NGAL

• Early diagnosis of delayed graft function
• Prognostic tools to predict graft loss after acute kidney injury
Possible Applications of NGAL

• Screening of patients with increased risk of graft dysfunction in order to early
therapeutic interventions.
• Assisting clinical decision-making in the management of transplant-related com-
plications or during the follow-up of kidney transplant recipients.
• The ability of monomeric kidney-derived NGAL to detect specific graft damage
needs to be further explored.
Summary Points
• Urine and serum NGAL concentration in kidney transplant recipients with normal
graft function appear to be similar to the concentration observed in health
individuals.
• Studies of NGAL in kidney transplant recipients have focused on its utility for
predicting short- and long-term graft failure.
Utility of NGAL in Transplantation

• Higher sNGAL or uNGAL levels 6–72 h after transplantation predict delayed
graft function (i.e., the necessity for dialysis within the first week after kidney
transplantation). For example, uNGAL concentrations >150 ng/mL assessed a
few hours after transplantation predict reduced graft function at 1 year.
• In kidney transplant recipients with acute graft dysfunction and previously stable
graft function, uNGAL concentrations >210 ng/mL predict graft loss.
Nonutility of NGAL in Kidney Transplantation

• Routine serial measurements of NGAL in grafts with stable function do not
identify subclinical acute tubular injury.
• Measurement of NGAL during acute kidney injury does not distinguish etiology
of the cause of graft dysfunction and may rise because of ischemic injury,
calcineurin inhibitors toxicity, obstructive nephropathy, subclinical tubulitis, or
infection.
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miR-210 as a Biomarker in Renal Carcinoma
39
Hideto Iwamoto, Mitsuhiko Osaki, Masashi Honda,
Takehiro Sejima, Atsushi Takenaka, and Futoshi Okada
Contents
Key Facts of Diagnosis and Management of Renal Cell Carcinoma (RCC) . . . . . . . . . . . . . . . . . . 897
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 897
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 898
miR-210 Investigation in RCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 900
miR-210 as a Diagnostic Biomarker in RCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 900
miR-210 as a Prognostic Biomarker in RCC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 903
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 907
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 908
Abstract
Renal cell carcinoma (RCC) is the most common urological neoplasm of the
kidney in adults. The worldwide incidence and mortality rate is in the order of
270,000 cases and 120,000 deaths, respectively. Despite the fact that the diag-
nostic modalities and therapeutic techniques for RCC continue to improve, the
overall incidence and mortality of RCC has increased in the last 20 years. The
5-year survival rate is approximately 98 % for stage I disease and approximately
50 % for stage III or higher disease. These data underscore the importance of early
detection and adequate prediction of prognosis of RCC.
H. Iwamoto • M. Honda • T. Sejima • A. Takenaka

Division of Urology, Faculty of Medicine, Tottori University, Yonago, Tottori, Japan
e-mail: gakkoura@med.tottori-u.ac.jp; honda@med.tottori-u.ac.jp; sejimat@med.tottori-u.ac.jp;
atake@med.tottori-u.ac.jp
M. Osaki (*) • F. Okada
Division of Pathological Biochemistry, Faculty of Medicine, Tottori University, Yonago, Tottori,
Japan
e-mail: osamitsu@med.tottori-u.ac.jp; osamitsu@grape.med.tottori-u.ac.jp;
fuokada@med.tottori-u.ac.jp

896 H. Iwamoto et al.
However, both early detection and adequate prediction are often difficult in the
clinical setting. Renal tumor in the early stage is often asymptomatic and
non-palpable, and it is impossible to correctly distinguish between RCC and
BRT, even with the full use of diagnostic modalities such as imaging examina-
tions, and determination of the adequate frequency of radiological imaging
examinations as a screen for tumor recurrence is confusing. These problems are
caused by the lack of an accurate biomarker for diagnosis and prognosis in RCC.
Recent studies suggest that tissue microRNAs (miRNAs), which are non-
protein-coding small RNAs, are involved in carcinogenesis and cancer progres-
sion. Further studies suggest that microRNAs are highly stable and abundant in
the serum, urine, and other body fluids. Therefore, both tissue miRNAs and
circulating miRNAs might be good biomarkers for diagnostic and prognostic
considerations in a variety of cancers.
Of all the miRNAs, miR-210, a well-known hypoxia-inducible miRNA, is one
of the best studied miRNA in various cancers. Some research groups have
reported that miR-210 was induced under hypoxic conditions via hypoxia-
inducible factors (HIFs) in various cancer cell lines. In the case of RCC, it is
well known that HIF1α and HIF2α accumulate in clear cell carcinoma (ccRCC),
which is the largest subtype of RCC, as a result of the deficiency of the von
Hippel-Lindau (VHL) tumor suppressor gene. Therefore, a number of research
groups have attempted to determine the relationship between miR-210 expression
and the VHL-HIF pathway and to use miR-210 as a new biomarker in ccRCC.
Regarding the possibility of using miR-210 as a diagnostic biomarker in RCC,
two studies have reported that serum miR-210 expression was significantly higher
in ccRCC patients than in healthy controls. These studies found no significant
correlation between serum miR-210 levels and clinicopathological parameters of
ccRCC patients. However, there has been no report regarding the utility of urine
miR-210 as a diagnostic biomarker in RCC, and there has been no study of the
difference between miR-210 expression levels of RCC and benign renal tumor.
Regarding the use of miR-210 as a prognostic biomarker in RCC, some studies
have demonstrated that high miR-210 expression in ccRCC tissue was statistically
related to poor prognosis. On the other hand, several other studies have demonstrated
precisely the opposite results regarding miR-210 levels and prognosis. Thus, there is
no consensus regarding the utility of miR-210 as a prognostic biomarker. Further
studies with a larger number of patients are warranted to validate these results.
Keywords
Renal cell carcinoma • MicroRNA-210 (miR-210) • Circulating miRNA in body
fluids • Hypoxia-inducible factors (HIFs) • Von Hippel-Lindau tumor suppressor
gene (VHL tumor suppressor gene) • Diagnostic biomarker • Prognostic
biomarker
Abbreviations
BRT Benign renal tumor
CAIX Carbonic anhydrase IX
39 miR-210 as a Biomarker in Renal Carcinoma 897
ccRCC Clear cell carcinoma

chRCC Chromophobe RCC
DFS Disease-free survival
HCs Healthy controls
HIFs Hypoxia-inducible factors
ISCU1/2 Iron-sulfur cluster assembly protein
MIBC Muscle-invasive bladder cancer
miRNAs MicroRNAs
NMIBC Non-muscle-invasive bladder cancer
OS Overall survival
pRCC Papillary RCC
VHL Von Hippel-Lindau
Key Facts of Diagnosis and Management of Renal Cell

Carcinoma (RCC)
• Diagnosis of RCC is generally performed based on imaging examination, includ-

ing abdominal ultrasonography, computed tomography, magnetic resonance
imaging, and so on.
• Since histopathological diagnosis represented by tissue biopsy is not basically
performed to avoid dissemination of cancer cell, it is impossible to distinguish
between RCC and BRT correctly.
• Most patients with renal tumor have been applied to surgical treatment
represented by nephrectomy and partial nephrectomy.
• It is known that approximately 20 % of small renal tumors less than 4 cm are BRT.
• Surgical treatment is associated with a risk of deterioration in renal function, even
if partial nephrectomy is performed to spare normal renal parenchyma.
• It is known that RCC is basically characterized by low response rate to chemo-
therapy, radiation therapy, and so on.
• In recent years, although efficacy of targeted therapy for progressive RCC such as
metastatic RCC has been reported, the response rate is still definitive.
• To this day, progressive RCC indicates a poor prognosis.
• At this time, there are no accurate biomarkers for diagnosis and prognosis in RCC.
Definitions
TNM stage The TNM staging is one of the most popular systems used in cancer
staging. The TNM system is based on the size and/or extensity of the primary tumor
(T), the size and/or number of regional lymph nodes metastasis (N), and the
existence of distant metastasis (M). For many cancers, TNM combinations corre-
spond to one of five stages. Criteria for stages differ for different types of cancer. For
example, bladder cancer T3N0M0 is stage III, whereas colon cancer T3N0M0 is
stage II. This system is useful for estimating a prognosis of cancer patients and helps
the doctor plan the appropriate treatment.
Exosome Exosomes are membrane-bound particles, 50 to 90 nm, formed via

invagination of the early endosome and released upon fusion of late endosome
with plasma membrane.
RNase RNase is a general term for enzyme that catalyzes the degradation of RNA
into smaller components. RNase is also contained in the body fluid such as blood,
urine, saliva and so on.
Hypoxia-inducible factors (HIFs) HIF is a protein which has been shown to

control mitochondrial function and is essential for this repression of mitochondrial
respiration during hypoxia. HIFs are known familiarly as a master regulator of the
cellular hypoxic response.
Von Hippel-Lindau (VHL) tumor suppressor gene VHL gene is located on the
short arm of chromosome 3. Deficiency of VHL gene leads to HIF accumulation;
this accumulation induces upregulation of their transcriptional target genes, such as
vascular endothelial growth factor (VEGF) and glucose transporter type 1 (GLUT1),
thus promoting angiogenesis and metabolic changes associated with carcinogenesis.
Since VHL gene deficiency is observed in 70 % or more of ccRCCs, it is suggested
that its deficiency occurs in the early stage of carcinogenesis in ccRCC.
Carbonic anhydrase IX (CA IX) Carbonic anhydrase catalyzes the reversible

hydration of carbon dioxide to carbonic acid and plays a role in pH regulation. To
date, 14 human isoforms have been reported. CA IX is strongly induced by hypoxia
in a range of tumor cell lines.
Iron-sulfur cluster assembly protein (ISCU1/2) Prosthetic groups are critical for
electron transport and mitochondrial oxidation-reduction reactions. ISCU1/2 facili-
tates the assembly of iron-sulfur clusters.
Introduction
Renal cell carcinoma (RCC) is the most common urological neoplasm of the kidney
in adults, representing over 90 % of primary renal neoplasms and accounting for
approximately 3–5 % of all adult malignancies in western countries. In Japan, the
results of a 2002 survey revealed that the crude incidence rates of RCC were 8.2 and
3.6 per 100,000 population for men and women, respectively (Marumo et al. 2007).
In the United States, 58,000 new cases were diagnosed in 2010, with approximately
13,000 deaths (Jemal et al. 2010). The worldwide incidence and mortality rate is in
the order of 270,000 cases and 120,000 deaths, respectively (Ferlay et al. 2010).
Moreover, RCC represents a spectrum of histologic subtypes that are
morphologically and cytogenetically distinct. The most common subtype is clear cell
RCC (ccRCC), which accounts for approximately 75–80 % of cases. Other subtypes
include papillary RCC (pRCC) (10–15 %), chromophobe RCC (chRCC) (5 %), and
collecting duct carcinoma (Meloni-Ehrig 2002). It is known that these different
histological subtypes of RCC vary in their clinical courses and their prognosis and
different clinical strategies have been developed for their management. Oncocytoma,
papillary adenoma, mesonephric adenoma, and angiomyolipoma are the main
benign renal tumors (BRT).
Despite the fact that the diagnostic modalities and therapeutic techniques for
RCC continue to improve, the overall incidence and mortality of RCC has
increased in the last 20 years (Hollingsworth et al. 2006). At the time of initial
diagnosis, approximately 60 % of the patients have a localized carcinoma and
nearly 20 % are identified with regional or distant metastases (Howlader
et al. 2009). Approximately 30 % of RCC cases develop metastatic disease
following surgery with curative intent. It is known that RCC is basically charac-
terized by low response rate to chemotherapy, radiation therapy, and so
on. Therefore, the 5-year survival rate is approximately 98 % for stage I disease
and approximately 50 % for stage III or higher disease (Devita et al. 2008).
These data underscore the importance of early detection of renal tumor before
metastasis and of adequate prediction of tumor recurrence and distant metastases
after surgery.
However, both early detection and adequate prediction are often difficult in the
clinical setting. Renal tumor in the early stage is often asymptomatic and
non-palpable. Recent data show that approximately 70–80 % of renal tumors are
detected incidentally as a result of scanning for another medical problem. It is
impossible to correctly distinguish between RCC and BRT, even with the full use
of diagnostic modalities such as imaging examinations, abdominal ultrasonogra-
phy, computed tomography, and magnetic resonance imaging. Additionally, scan-
ning for tumor recurrence and distant metastases must rely mainly on radiological
imaging examinations in the outpatient clinic after surgery. However, determina-
tion of the adequate frequency of radiological imaging examinations is confused
by differences in factors including T stage and tumor histopathology (Sejima
et al. 2013). These problems are caused by the lack of an accurate biomarker for
diagnosis and prognosis in RCC. Therefore, identification of novel biomarkers is
urgently needed.
Recent studies demonstrated that microRNAs (miRNAs), which are non-protein-
coding small RNAs, are involved in carcinogenesis, cancer progression, and metas-
tasis. MicroRNAs are approximately 22 nucleotides in length and regulate gene
expression at the posttranscriptional level by binding to the untranslated region
(30 UTR) of target mRNAs, leading to translational inhibition and/or mRNA degra-
dation (Slaby et al. 2010). Specific expression profiles of miRNAs in tissue have
been reported in a variety of cancers, including in RCC (Volinia et al. 2006). Early
studies suggested that miRNAs were strictly intracellular molecules. However, more
recent studies have reported that miRNAs are highly stable and abundant in the
serum, urine, and other body fluids, which is likely due to exosome protection of
miRNAs against degradation by RNase (Mitchell et al. 2008; Chim et al. 2008).
Interestingly, serum miRNA levels are similar in men and women and do not vary
with patient age (Hunter et al. 2008). Thus, both tissue miRNAs and circulating
miRNAs might be good biomarkers for diagnostic and prognostic considerations in a
variety of cancers. Indeed, several studies have demonstrated that specific tissue and
circulating miRNAs were valuable for distinguishing patients with cancer from
healthy controls (HCs).
miR-210 Investigation in RCC
Of all of the miRNAs, miR-210, a well-known hypoxia-inducible miRNA, is one of

the best studied miRNA in cancer. MiR-210 is upregulated in various types of
human cancer (Camps et al. 2008; Ho et al. 2010), suggesting that it has an important
role in carcinogenesis. Previous studies reported that miR-210 is induced under
hypoxic conditions via hypoxia-inducible factors (HIFs) in various cancer cell lines
and that miR-210 is an important factor in cell proliferation, mitochondrial respira-
tion, DNA repair, vascular biology, and angiogenesis in various types of cancer
(Jung et al. 2012; Chan et al. 2012).
In the case of RCC, most of the studies have investigated miR-210 expression in
clear cell carcinoma (ccRCC), which is the largest subtype of RCC. It is well known
that HIF1α and HIF2α accumulate in ccRCC as a result of abrogated ubiquitin-
mediated degradation due to loss or deficiency of the von Hippel-Lindau (VHL)
tumor suppressor gene (Eble et al. 2004). Therefore, a number of research groups
have attempted to determine the relationship between miR-210 expression and the
VHL-HIF pathway in RCC. Nakada et al. (2011) demonstrated that miR-210
expression clearly correlated with the accumulation of HIF1α under normoxia as
well as under hypoxia, suggesting that miR-210 upregulation in ccRCC was most
likely due to the accumulation of HIF1α. They further confirmed that restoration of
VHL expression in a VHL-deficient cell line led to the degradation of HIF1α and
suppressed the expression of miR-210, suggesting that miR-210 expression is
regulated via the VHL-HIF1α pathway. Neal et al. (2010) reported that miR-210
levels showed a correlation with a HIF-regulated mRNA, carbonic anhydrase IX
(CAIX), and VHL mutation or promoter methylation. They also reported that the
inverse correlation between miR-210 levels and iron-sulfur cluster assembly protein
(ISCU1/2) provides support for the hypothesis that ISCU1/2 is a target of miR-210
and that it may contribute to the anaerobic respiration seen in renal tumors. Along
with these studies, research aimed at using miR-210 as a new biomarker in RCC,
especially in ccRCC, has also been advancing.
miR-210 as a Diagnostic Biomarker in RCC
Circulating miRNA is suitable for analysis as a diagnostic biomarker. In particular,

serum and urinary miRNAs have been frequently used for this analysis.
Previous studies have described the potential use of serum miRNA as a diagnostic
biomarker for various cancers (e.g., miR-29a and miR-92 in colorectal cancer
(Huang et al. 2010); miR-195 in breast cancer (Heneghan 2010); miR-17-5p,
miR-21, miR-106a, and miR-106b in gastric cancer (Tsujiura et al. 2010); and
miR-141 and miR-26a in prostate cancer (Mitchell 2008; Mahn et al. 2011)).
These two studies have investigated the utility of serum miR-210 as a diagnostic
biomarker for RCC. Zhao et al. (2013) reported that tissue miR-210 levels in
33 ccRCC patients were significantly higher in tumor tissue than in adjacent
non-tumoral renal parenchyma tissue (P = 0.004) (Fig. 1). Serum miR-210 levels
were also significantly higher in 68 ccRCC patients than in 42 HCs (P < 0.001)
(AUC, 0.87; sensitivity, 81.0 %; specificity, 79.4 %) (Fig. 2). Furthermore, they
confirmed that serum miR-210 was significantly elevated in patients with TNM stage
I ccRCC as well as in patients with other stages compared with HCs, although there
was no difference between the different stages and serum miR-210 levels in patients
with ccRCC that was decreased 1 week after surgical resection of the tumor.
Iwamoto et al. (2014) demonstrated a similar result; tissue miR-210 levels in
34 ccRCC patients were significantly higher in tumor tissue than in adjacent
non-tumoral renal parenchyma (P < 0.001). In 31 cases (92 %), the miR-210
level in tumor tissues was increased by > twofold when compared with that in
normal tissues. Serum miR-210 levels were also significantly higher in ccRCC
patients than in 24 HCs (P = 0.001) (AUC, 0.77; sensitivity, 65 %; specificity,
83 %). Moreover, there was no significant correlation between serum miR-210 levels
and age, gender, tumor size, or the existence of metastasis at diagnosis (Fig. 3). The
results of these two studies suggest that at least some of the serum miR-210 arises
from release from the primary renal tumors and that upregulation of serum miR-210
may occur in the early stage of ccRCC. There have been a few studies regarding the
utility of serum miRNAs as a diagnostic biomarker in RCC using a similar study
methodology (e.g., miR-1233, miR-378, miR-451, and miR-378; refer to the next
section).
Some studies using urinary miRNA as a diagnostic biomarker in other cancers
have been published (e.g., miR-143, miR-222, miR-452, miR-145, and miR-200a in
bladder cancer; refer to the next section). Lorenzen et al. (2011) reported that urinary
miR-210 levels identified patients with acute T-cell rejection and predicted long-term
kidney function in renal allograft recipients. However, there have been no reports of
the availability of urinary miR-210 as a diagnostic biomarker in RCC.
In clinical practice, it is also important to distinguish the subtypes of renal tumor,
in particular, to distinguish between RCC and BRT in order to avoid invasive
overtreatment. Munari et al. (2014) analyzed the miRNA expression profile of a
set of 15 tissue samples of clear cell papillary renal cell carcinoma (ccpRCC) and
evaluated similarities and differences between ccRCC and pRCC. They reported that
miR-210 was upregulated in both ccpRCC and ccRCC compared with normal tissue.
As an alternative approach, although it was not a study using miR-210 alone,
Fridman et al. (2010) used expression levels of six microRNAs in tissue samples
including 17 ccRCC, 20 pRCC, 13 chRCC, and 21 oncocytoma, to demonstrate a
two-step decision-tree classifier that could distinguish four common subtypes of
1000.00 P = 0.0001
miR-210 expression (normalized)

100.00
10.00
1.00
1
Normal (n=42) cRCC (n=68)
Fig. 1 Serum miR-210 level in ccRCC patients and HCs. Serum miR-210 level was signifi-
cantly higher in the ccRCC patients than in HCs. The lines inside the boxes represent the median
value (Data are from Zhao et al. (2013), with permission from the Publishers)
Fig. 2 Receiver operating miR -210

characteristic (ROC) curve 1.0
analysis using serum miR-
210 for discriminating
ccRCC patients. Serum 0.8
miR-210 yielded an AUC (the
areas under the ROC curve) of
0.874 (95 % Cl: 0.806–0.941)
with a sensitivity of 81.0 % 0.6
Sensitivity
and a specificity of 79.4 % for

discrimination between the
ccRCC patients and HCs
(Data are from Zhao 0.4
et al. (2013), with permission
from the Publishers)
0.2
AUC = 0.874
0.0
0.0 0.2 0.4 0.6 0.8 1.0
1 - Specificity
renal tumor (Fig. 4). The first step used expression levels of miR-210 and miR-221 to
distinguish between the two pairs of subtypes: ccRCC and pRCC vs. oncocytoma
and chRCC. The second step used either miR-200c with miR-139-5p to distinguish
oncocytoma from chRCC or miR-31 with miR-126 to distinguish ccRCC from
pRCC. Using this classifier, these four subtypes of RCC could be distinguished
5,000000E-3 P=0.067
Serum miR-210 levels (normalized)

4,000000E-3
5
3,000000E-3
2,000000E-3
1,000000E-3
0,000000E0
No matastasis Metastasis present

Metastasis at diagnosis
Fig. 3 Analysis of the correlation between serum miR-210 level and existence of metastasis at
diagnosis. There was no significant association between serum miR-210 level and existence of
metastasis at diagnosis (Data are from Iwamoto et al. (2014), with permission from the Publishers)
more than 90 % of the time. Youssef et al. (2011) also developed a classifier that
could distinguish the different RCC subtypes using multiple miRNAs from 70 tissue
samples, although miR-210 was not included in their analysis. Although the diag-
nostic accuracy of their analysis was very high, the use of multiple miRNAs was
somewhat confusing. These studies were innovative. However, preoperative diag-
nosis using tissue miRNAs is difficult. There have been no similar studies using
circulating miRNAs to distinguish the different RCC or BRT subtypes.
Studies regarding the significance of miR-210 as a diagnostic biomarker in RCC
are summarized as follows. It is clear that miR-210 levels are upregulated in tissue
and serum in the early stage of ccRCC. However, some problems remain to be
solved in preparation for clinical application. Examples of such problems are the
problem of organ specificity, i.e., distinguishing between ccRCC and other organ
cancers, and the problem of cancer specificity, i.e., distinguishing between ccRCC
and BRT. Moreover, the use of urine samples as a less invasive sample for analysis of
biomarkers will be expected in the future.
miR-210 as a Prognostic Biomarker in RCC
A number of studies have reported the effect of miR-210 on the prognosis of

different cancers, including breast cancer (Camps et al. 2008), epithelial ovarian
cancer (Giannakakis et al. 2008), diffuse large B-cell lymphoma (Lawrie et al. 2008),
lung cancers (Duncavage et al. 2009), and pancreatic adenocarcinomas
Kidney Tumor
Oncocytoma/ Conventional/
Chromophobe Papillary
C D
Oncocytoma Chromophobe Conventional Papillary
Fig. 4 Two-step decision-tree classifier to distinguish four common subtypes of renal tumor.
First, samples are classified into either the oncocytoma/chromophobe pair or the conventional/
papillary pair, using expression levels of hsa-miR-210 and hsa-miR-221 (B). In the second step,
oncocytoma is differentiated from chromophobe using expression levels of hsa-miR-200c and
hsa-miR-139-5p (C), and conventional is differentiated from papillary using expression levels of
hsa-miR-31 and hsa-miR-126 (D) (Data are from Fridman et al. (2010), with permission from the
Publishers)
(Ho et al. 2010). Most of these studies found upregulation of miR-210 levels in
cancer tissues or blood of cancer patients compared with the levels in normal tissues
or blood of HCs, which was related to a poor survival outcome (Camps et al. 2008;
Duncavage et al. 2009; Gee et al. 2010; Ho et al. 2010; Rothe et al. 2011; Toyama
et al. 2012; Qiu et al. 2013).
In the case of RCC, several studies that investigated the correlation between
miR-210 levels and the prognosis of patients with RCC have been published
(Table 1). Neal et al. (2010) found a significant inverse correlation between
miR-210 expression in the tissue samples of 31 clear cell RCC patients (Fig. 5)
and 5-year overall survival (r = 0.481, P = 0.006). Samaan et al. (2015) analyzed
tissue samples of 276 primary clear cell RCC patients and confirmed that patients
with high miR-210 expression had a statistically higher chance of disease recurrence
(hazard ratio, 1.82; P = 0.018) and shorter overall survival (HR, 2.46; P = 0.014).
However, in multivariate analysis, miR-210 lost its statistically significant associa-
tion with shorter disease-free survival and overall survival after adjusting for tumor
size and for tumor, node, and metastasis stage. These results are in agreement with
most of the previous studies in other cancers.
On the other hand, Wotschofsky et al. (2013) suggested that there was no
significant difference between miR-210 expression in tissue samples of primary
RCC and that of metastatic RCC and that miR-210 expression was not statisti-
cally related to recurrence-free survival. Iwamoto et al. (2014) reported similar
results using serum samples. However, McCormick et al. (2013) demonstrated
Table 1 Summary table of studies regarding miR-210 as a biomarker. This table indicates
specimen and intended purpose of miR-210 as a biomarker for each study
Authors Year Specimen Purpose
Zhao 2013 Serum Early diagnosis of ccRCC
Iwamoto 2014 Serum Early diagnosis of ccRCC
Lorenzen 2011 Urine Prediction of acute rejection and kidney function in renal
allograft recipients
Munari 2014 Tissue Differential diagnosis of RCC subtypes
Fridman 2010 Tissue Differential diagnosis of RCC subtypes
Youssef 2011 Tissue Differential diagnosis of RCC subtypes
Neal 2010 Tissue Prediction of poor prognosis in ccRCC
Samaan 2015 Tissue Prediction of poor prognosis in ccRCC
Wotschofsky 2013 Tissue Prediction of poor prognosis in ccRCC
McCormick 2013 Tissue Prediction of good prognosis in ccRCC
miR-210
Normalised Expression Ratio
1.2
1.0
0.8
0.6
0.4
0.2
0 2 4 6 8 10 12
Survival (years)
Fig. 5 Analysis of the correlation between tissue miR-210 level and patient survival. A
significant correlation was found between miR-210 level and 5-year overall survival
(r = 0.481, P = 0.006) (Data are from Neal et al. (2010), with permission from the Publishers)
precisely the opposite results; high miR-210 expression in clear cell RCCs was
associated with lower stage and grade, and high miR-210 expression was asso-
ciated with improved survival post nephrectomy, compared with medium and low
levels of miR-210. As an explanation for these results, they suggested that,
although VHL inactivation in ccRCC should lead to constitutive HIF activation
and miR-210 upregulation, further loss of cell differentiation coupled with
ongoing mutations may manifest with reduction in expression of genes such as

miR-210 and reduced miR-210 expression reflects a shift from HIF-1 to HIF-2
predominance.
Those studies regarding the significance of miR-210 as a prognostic biomarker
in RCC are summarized as follows. Unfortunately, at the present time, there does
not appear to be a consensus as to whether miR-210 expression is associated with
good prognosis or poor prognosis. Further studies with a larger number of patients’
clinical specimens are warranted to validate correctly these results.

Conditions
A few studies have investigated the utility of serum miRNAs as a diagnostic

biomarker for RCC. Wulfken et al. (2011) were the first to report that the serum
miR-1233 level was increased in RCC patients. They found miR-1233 levels were
increased in 84 patients with RCC from a multicenter cohort (AUC, 0.588; sensi-
tivity, 77.4 %; specificity, 37.6 %). They included in this investigation 13 samples
from patients with angiomyolipoma or oncocytoma whose serum miR-1233 levels
were similar to those of patients with RCC. Redova et al. (2012) demonstrated that
serum miR-378 and miR-451 were potential biomarkers for RCC. When the utility
of miR-378 plus miR-451 as a biomarker was evaluated in an independent cohort of
90 patients with RCC and 35 HCs, the combination of serum miR-378 and miR-451
enabled identification of RCC with a relatively high accuracy rate (AUC, 0.86;
sensitivity, 81 %; specificity, 83 %). Hauser et al. (2012) confirmed that serum
miR-378 was significantly increased in ccRCC (25 patients, P = 0.006), but they
did not detect a difference in the level of this biomarker when they compared
117 patients with RCC versus 123 HCs. Comparison of these miRNAs with
miR-210 in terms of diagnostic accuracy indicated that miR-210 had the highest
diagnostic accuracy as a single miRNA marker.
Many studies have reported the significance of miR-210 as a diagnostic and
prognostic biomarker in different cancers, especially in breast cancer. Jung
et al. (2012) reported that high expression levels of plasma miR-210 before
trastuzumab-based neoadjuvant chemotherapy were associated with resistance to
treatment in 29 patients (P = 0.035) and that expression levels of plasma miR-210
were significantly lower in postoperative samples than in preoperative samples of
39 patients (P = 0.029). Camps et al. (2008) studied 219 early breast cancer patients
with long-term follow-up and reported that miR-210 expression levels showed an
inverse correlation with disease-free survival (DFS) and overall survival (OS), which
was significant in both univariate and multivariate analyses (DFS, p = 0.003 and
OS, P < 0.001, respectively). Toyama et al. (2012) demonstrated a similar relation-
ship between high miR-210 levels and poor prognosis of breast cancer in 161 Asian
patients. They found that triple-negative breast cancer patients with low miR-210
expression experienced significantly better disease-free and overall survival than
those with high miR-210 expression (P = 0.02 and P = 0.05, respectively).
Furthermore, higher expression of miR-210 in triple-negative breast cancer patients

was an independent prognostic factor, indicating a worse prognosis than lower
miR-210 expression.
Additionally, the utility of urinary miRNA as a diagnostic and prognostic bio-
marker has been reported in other cancers, especially in bladder cancer. Yun
et al. (2012) demonstrated that the levels of miR-145 were significantly decreased
in 138 non-muscle-invasive bladder cancer (NMIBC) patients and 69 muscle-
invasive bladder cancer (MIBC) patients compared to 144 healthy controls
(P < 0.001). Also, MIBC patients had significantly lower miR-145 levels than
NMIBC patients (P = 0.036). Furthermore, they confirmed in multivariate analysis
that miR-200a was an independent predictor of NMIBC recurrence (P = 0.013).
Puerta-Gil et al. (2012) also reported that urinary miR-452 (AUC, 0.848) and
miR-222 (AUC, 0.718) provided high accuracies for bladder cancer diagnosis. In
the case of RCC, only one study has been carried out that focused on urinary
miRNAs. Brandenstein et al. (2012) found that urinary miR-15a levels showed a
marked decrease postoperatively in ten ccRCC patients (P < 0.005) and that there
was significant upregulation of miR-15a levels in the urine of 10 ccRCC patients
compared to 35 patients with other medical conditions including conditions such as
oncocytoma, urothelial carcinoma, colon carcinoma, and urinary bladder infection
(Fig. 6). Even though there are a number of studies that have reported an association
between miRNAs and RCC in tissue and serum samples, the number of studies that
have analyzed urinary miRNAs is extremely small. This small number of reports
indicates that some unconventional mechanism may make it difficult to detect
miRNAs associated with RCC in urine samples. However, urine is the most mini-
mally invasive sample in clinical examination. Therefore, if urinary miR-210 can be
used as a biomarker in RCC, it will be of practical value.
Summary Points
• This chapter focuses on the significance of miR-210 as a diagnostic and prog-

nostic biomarker in RCC.
• miR-210, a well-known hypoxia-inducible miRNA, is one of the best studied
miRNAs in cancer.
• miR-210 is upregulated in various types of human malignancies, suggesting it has
an important role in carcinogenesis.
• In the case of RCC, it is well known that HIF1α and HIF2α accumulate in ccRCC
as a result of a deficiency of the VHL tumor suppressor gene.
• A number of research groups have attempted to reveal the relationship between
miR-210 expression and the VHL-HIF pathway.
• Research regarding the utility of miR-210 as a new biomarker in RCC, especially
in ccRCC, has also been advancing.
• In terms of its use as a diagnostic biomarker, previous studies have found that serum
miR-210 levels were significantly higher in ccRCC patients than in HCs.
Fig. 6 Box plot analyses from the urine of patients with different disease entities in the
urogenital tract. Carcinoma of the urogenital tract and other tumors, such as colon cancer and
hepatic cell carcinoma, failed to show increased miRNA 15a levels. Furthermore, inflammatory
conditions were equally incapable of increasing miRNA 15a levels. The increased expression of
miRNA 15a in patients’ urine with RCCs compared with all other collected urine samples was
significant, as indicated by asterisks (Data are from von Brandenstein et al. (2012), with permission
from the Publishers)
• No studies of the investigation of the utility of urine miR-210 as a diagnostic

biomarker have been published.
• In terms of its use as a prognostic biomarker, some studies have suggested that high
miR-210 expression in ccRCC tissue is statistically related to poor prognosis.
• Several studies have demonstrated precisely the opposite results, i.e., that low
miR-210 levels correlate with RCC prognosis.
• A consensus has not yet been reached regarding the significance of miR-210 as a
prognostic biomarker.
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Matrix Metalloproteinase-2 (MMP-2)
and Plasminogen Activator Inhibitor-1 40
(PAI-1) in Peritoneal Dialysis: Biological
Implications and Clinical Utility
Deirisa Lopes Barreto and Raymond T. Krediet
Contents
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 913
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 913
Structure and Biological Functions of MMP-2 and PAI-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 916
Cellular and Peritoneal Tissue Expression of MMP-2 and PAI-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 918
Systemic Levels of MMP-2 and PAI-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 920
Potential Applications to Prognosis, Other Diseases, or Conditions . . . . . . . . . . . . . . . . . . . . . . . 921
Peritoneal Effluent MMP-2 and PAI-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 922
Appearance of MMP-2 and PAI-1 in the Peritoneal Cavity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 922
Associations with PD Duration, Peritoneal Transport Parameters, and Peritoneal Effluent
Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 924
Effect of PD Solutions on Levels of MMP-2 and PAI-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 924
Peritoneal Effluent Levels of MMP-2 and PAI-1 During PD-Related Complications . . . . . 925
Clinical Relevance of Peritoneal Effluent MMP-2 and PAI-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 926
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 926
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 927
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 927
Abstract
Peritoneal dialysis (PD) represents an important renal replacement therapy for
end-stage renal disease patients. Besides several advantages, long-term PD ther-
apy is associated with functional and anatomical modifications that may lead to
peritoneal membrane dysfunction. The clinical presentation of these complica-
tions is not always reflected in underlying morphological abnormalities. Unfor-
tunately, it is not feasible to gain insight into intraperitoneal events that affect the
structure of the peritoneal membrane without invasive procedures. The peritoneal
D. Lopes Barreto (*) • R.T. Krediet

Division of Nephrology, Room A3-274, Academic Medical Center – University of Amsterdam,
Amsterdam, The Netherlands
e-mail: D.LopesBarreto@amc.uva.nl; R.T.Krediet@amc.uva.nl

912 D. Lopes Barreto and R.T. Krediet
effluent after drainage is the most clinically relevant specimen in PD. As proxies,
peritoneal effluent biomarkers could be utilized as noninvasive tools for moni-
toring and diagnostic purposes. To date, no biomarkers have yet been identified to
assess the degree of tissue remodeling and fibrosis in chronic PD patients.
In this chapter a review is given on matrix metalloproteinase-2 (MMP-2) and
plasminogen activator inhibitor-1 (PAI-1) as biomarkers for the clinical practice of
PD therapy. MMP-2 is a 72 kDa gelatinase that is involved in tissue remodeling.
PAI-1 is a 50 kDa member of the serine protease inhibitors (SERPINs) that inhibits
fibrinolysis and proteolysis. Peritoneal effluent MMP-2 and PAI-1 have recently
been gauged as biomarkers for peritoneal membrane alterations. Their biological
functions during physiological and pathological conditions are discussed as well
as the expression in cells and tissue. This is followed by a brief synopsis of the
current literature on circulatory MMP-2 and PAI-1 in PD therapy. Investigations
on the levels of peritoneal effluent MMP-2 and PAI-1 are discussed more exten-
sively with respect to their appearance in the peritoneal cavity, associations with
parameters of peritoneal transport, and potential clinical applications. Finally, the
clinical relevance of effluent MMP-2 and PAI-1 will be addressed.
Keywords
Adhesions • Biomarker • Effluent biomarker • Fibrosis • Matrix
metalloproteinase-2 • Peritoneal dialysis • Peritoneal effluent • Peritoneal mem-
brane dysfunction • Plasminogen activator inhibitor-1 • Tissue remodeling
Abbreviations
CA125 Cancer antigen 125
EMT Epithelial-to-mesenchymal transition
EPS Encapsulation peritoneal sclerosis
HD Hemodialysis
IL-6 Interleukin-6
kDa Kilodalton
MMP-2 Matrix metalloproteinase-2
MTAC Mass transfer area coefficients
PAI-1 Plasminogen activator inhibitor-1
SERPINs Serine protease inhibitors
TGF-β1 Transforming growth factor-beta1
TIMP Tissue inhibitor of metalloproteinases
TNF-α Tumor necrosis factor-α
t-PA Tissue plasminogen activator
UFF Ultrafiltration failure
u-PA Urokinase plasminogen activator
40 MMP-2 and PAI-1 in Peritoneal Dialysis 913
Definitions
Encapsulation peritoneal sclerosis (EPS) EPS is the most severe complication of

PD therapy that is accompanied by high mortality rates and severe morbidity.
Anatomically, EPS is characterized by a dense cocoon of fibrous tissue covering
the abdomen.
Epithelial-to-mesenchymal transition (EMT) Transdifferentiation of mesothelial

cell into mesenchymal cell phenotype. EMT is a short-term consequence of PD
therapy due to the continuous exposure of PD solutions to the mesothelium.
Free water transport (FWT) Aquaporin-1 mediated transport of water, without

dissolved solutes and electrolytes. FWT is one of the peritoneal transport parameter
that becomes impaired in long-term PD patients.
Mass transfer area coefficients (MTAC) A peritoneal transport parameter of small

solutes that reflects the peritoneal vascular surface area. In general, the MTAC of low
molecular weight solutes increase with duration of PD therapy.
Peritoneal function test A standardized peritoneal test to assess the function status
of the peritoneal membrane. Several forms of peritoneal function tests are available;
however, all are characterized by a predefined dwell time with or without interme-
diate sampling of the peritoneal effluent. Depending on the duration and complexity
of the test, more peritoneal transport parameters are gained.
Peritoneal membrane The peritoneal membrane is described to be a semiperme-

able membrane that consists of three main layers. Firstly, the mesothelial cell layer is
encountered, followed by the interstitium in which the peritoneal capillaries are
imbedded. Long-term and continuous exposure of PD solutions to this membrane
may lead to functional and morphological alterations.
Introduction
For the past five decades, peritoneal dialysis (PD) has been used as a well-
established renal replacement therapy for end-stage renal disease patients
(Table 1). Other than hemodialysis (HD), PD is an intracorporeal dialysis system
where the peritoneum is responsible for the removal of excess fluid and toxic waste
products from the circulation into the peritoneal cavity. The peritoneum is a contin-
uous membrane that consists of mesothelial cells and underlying tissues, lining the
anterior abdominal wall (parietal peritoneum) and the intraperitoneal organs (vis-
ceral peritoneum). The peritoneal cavity is the space between these two opposing
surfaces (Fig. 1). A permanent catheter is inserted into the peritoneal cavity through
which 1.5–2.5 l of a hyperosmolar dialysis solution can be infused per dialysis
Table 1 Key facts of peritoneal dialysis

The invention of continuous ambulatory PD occurred in 1976 by Popovich and Moncrief.
However, PD was already applied in patients, but solely suitable as a treatment option for acute
renal failure since the 1940s. In the following decades, the technique has been optimized by
undertaking prevention measures for PD-related peritonitis and exit-site infections. Additionally,
novel dialysis solutions were introduced and referred as the more biocompatible PD solutions. All
these precautions resulted in a diminished rate of PD therapy discontinuation
For almost half of a century, PD therapy belongs to one of the renal replacement therapy that is
offered to end-stage renal disease patients. The proportion of PD patients in the worldwide dialysis
population covers 11 %. The mortality rate remains high, but the 5-year patient survival has
improved from 29 % to 41 %. In many countries, PD therapy is presented as the primary dialysis
modality choice, as there is a survival benefit over hemodialysis in the first 3 years. Nevertheless, a
global decrease in the proportion of PD patient is observed except for developing countries
In PD therapy the peritoneum serves as a biological dialysis membrane. Through this
semipermeable serous membrane, toxins and excess fluid are removed from the circulation into
the peritoneal cavity. For proper adequacy, three to five daily exchanges of PD solutions take place
varying in dose and dwell duration. The efficacy of PD therapy is measured by means of
peritoneal function tests. These tests provide insight into the magnitude of small solute removal,
fluid transport, and ultrafiltration capacity
Chronic exposure to PD solutions may affect the peritoneal membrane negatively. Especially, in
patients who are treated with PD for more than 2 years. The observed modifications are reflected
by the function peritoneal transport parameters. Moreover, effluent biomarkers as proxies are
capable to reveal anatomical alterations. However, attempts are being made to reduce or delay the
development of peritoneal membrane dysfunction
PD peritoneal dialysis
session. Several exchanges are performed throughout the day or night with drainage
of the peritoneal dialysate after prespecified time intervals.
In the worldwide dialysis population, the prevalence of PD therapy approximates
11 % (Jain et al. 2012). Paradoxically, PD is underutilized as a primary dialysis therapy
despite its initial survival benefit over a 3-year period when compared with incident
hemodialysis patients (van de Luijtgaarden et al. 2011; Weinhandl et al. 2010). More-
over, a European registry indicated prolongation of this survival benefit for up to 5 years
after commencing dialysis treatment (van de Luijtgaarden 2014). Discontinuation of PD
therapy occurs because of catheter-related complications, psychosocial features, perito-
neal membrane dysfunction, or other clinical factors. On a continuum (Fig. 2), peritoneal
membrane dysfunction may be partitioned into two time frames, namely, short-term and
long-term consequences of PD therapy. Peritoneal membrane dysfunction is the cause of
modifications that may arise on a functional as well as anatomical level. Functionally,
early discontinuation is merely due to inherent ultrafiltration failure, i.e., a rapid
dissipation of the osmotic gradient that is observed in approximately 15 % of incident
PD patients. The global identification strategy of ultrafiltration failure (UFF) is by
performing a 4-h peritoneal function test with a 3.86 % or 4.25 % glucose-containing
dialysis solution. The ultrafiltration volume is thereafter measured from the drained
dialysate, and when this amount is below the threshold of 400 ml, the diagnosis of UFF
is made (Mujais et al. 2000). The actual or acquired peritoneal membrane dysfunction is
usually observed after more than 2 years of PD therapy (Kolesnyk et al. 2010). At this
stage, anatomical modifications, such as neoangiogenesis, are becoming more overt as
Peritoneal capillary
Microvilli
Basement
membrane
Internal Abdo
organs -minal
wall
Lymph vessel
Resident
fibroblast
Mesothelial
cell
Visceral peritoneum Peritoneal cavity Parietal peritoneum
Fig. 1 Anatomy of the peritoneum. The peritoneum lines the inner abdominal wall and external
surface of the internal organs in a continuous manner with the peritoneal cavity as space between the
facing surfaces. The peritoneal membrane consists of three main layers: mesothelium, interstitium,
and peritoneal capillaries
reflected by some parameters of peritoneal membrane transport. In the initial phase of

PD therapy, the peritoneal membrane experiences low-grade inflammation due to the
instillation of the nonbiological PD solutions. Epithelial-to-mesenchymal transition
(EMT) has also been detected in the first 2 years of PD therapy (Del Peso
et al. 2008). Further alterations in the anatomy of the peritoneal membrane comprise
loss of mesothelial cell mass and neoangiogenesis, and after a treatment period of more
than 4 years, adhesions or extensive fibrosis may be detected. However, these structural
modifications are difficult to assess, as peritoneal biopsies are required which may lead
to discontinuation of PD therapy. Moreover, classical imaging techniques have shown to
be insufficient in the timely detection of anatomical alterations. Nevertheless, as PD
therapy requires drainage of the peritoneal cavity in order to discard toxins and excess
fluid, the drained dialysate, i.e., peritoneal effluent, is an important diagnostic tool,
because it contains a large proportion of proteins and substances that are readily
available for analysis. Therefore, the peritoneal effluent should be regarded as the
most clinically relevant diagnostic specimen in PD. Over the years several candidate
effluent biomarkers have been investigated (Lopes Barreto and Krediet 2013). The
purpose of these effluent biomarkers is to gain insight into pathophysiological events
within the peritoneal cavity and indirectly assess morphological alterations without
Fig. 2 Continuum PD therapy: Possible modifications and consequences. Depending on the

duration of PD therapy, several functional and morphological peritoneal membrane alterations are
induced. However, the degree of peritoneal impairment is patient specific, and therefore, not all PD
patients encounter and develop certain PD-related complications
causing harm to the peritoneal membrane. More importantly, acknowledged effluent

biomarkers are expected to aid in early detection of severe complications or to determine
the right time/moment to discontinue PD treatment.
The anatomical alterations that mainly affect the peritoneal membrane are the
increase in peritoneal capillaries, loss of mesothelial cells, and tissue remodeling and
fibrosis. Surrogate markers have been identified to monitor the increase of peritoneal
capillaries and loss of mesothelial cells, like vascular endothelial growth factor
(VEGF) and cancer antigen 125 (CA125), respectively. Unfortunately, no markers
have yet been identified or acknowledged to monitor the degree of peritoneal adhe-
sions and fibrosis. However, matrix metalloproteinase-2 (MMP-2) and plasminogen
activator inhibitor-1 (PAI-1) have been promoted recently as emerging effluent bio-
markers for peritoneal tissue remodeling and fibrosis. Besides their biological func-
tions and implications, this chapter will focus on PD-related investigations of systemic
as well as peritoneal effluent MMP-2 and PAI-1. Furthermore, their potential clinical
applications and utility as effluent biomarkers in PD will be discussed.
Structure and Biological Functions of MMP-2 and PAI-1
MMPs have been associated with several physiological and pathological processes
including cell differentiation, migration, angiogenesis, apoptosis, and growth. How-
ever, their main function remains the degradation of the extracellular matrix (ECM)
components. Primarily MMPs are synthesized as latent zymogens and their activa-
tion is contingent upon the cellular environment (Nagase et al. 1991; Khasigov
et al. 2001). This zinc-dependent matrix-degrading endopeptidase family is further-
more subdivided into five main classes: interstitial collagenases, stromelysins,
metalloelastases, membrane-type MMPs, and gelatinases. MMP-2 (molecular
weight 72 kDa) is classified into the latter one and is therefore also referred as
gelatinase A. The common trait of MMPs is a pro-peptide domain, which structures
the inactive form for the majority of matrixins. The distinctive characteristic of
MMP-2 is that it also exhibits a sequence of fibronectin type II-resembling modules
in the catalytic domain for collagen- and gelatin-binding purposes (Bode et al. 1999).
MMP-2 is the only gelatinase that is constitutively secreted by mesothelial cells on the
apical side (Martin et al. 2000; Marshall et al. 1993). Initially, the expression of its gene,
which is located on human chromosome 16q13, is regulated by various cytokines such
as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) (Borden and Heller 1997;
Kossakowska et al. 1999). Furthermore, in vitro experiments in endothelial, mesenchy-
mal, and vascular smooth muscle cells suggest tissue-specific regulation of MMP-2
(Zucker et al. 1995; Kaizuka et al. 1999; Galis et al. 1997). Other regulators of MMP-2
function encompass oxidative stress and a decreased bioavailability of nitric oxide
(Wang et al. 2005). MMP-2 susceptibility to oxidative modification diminishes its
activity, potentially leading to an altered turnover of the extracellular matrix (Mattana
et al. 1998). Membrane-type MMPs, TIMP-2, and plasmin and serine proteases are a few
enzymes through which pro-MMP-2 may be activated. MMP-2 substrates cover a broad
range of ECM components including collagens, elastin, fibronectin, gelatins, and laminin
(Klein and Bischoff 2011). Overall, evidence has been found for MMP-2 in angiogen-
esis, cell growth and migration, invasion, and metastasis (Chang and Werb 2001).
PAI-1 is a single-chain glycoprotein that is a member of the serine protease
inhibitors (SERPINs) with a molecular weight of 50 kDa. The encoding gene is
located at chromosome 7 and approximates 12.2 kilobases. In general, SERPINs are
known to control proteolytic pathways and to date over 36 human SERPINs are
acknowledged (Law et al. 2006). In comparison to the other SERPINs, PAI-1 is
exceptional as it appears in four distinct conformations: (1) an active form that
interacts with plasminogen activator, (2) a nonreactive latent form, (3) a
non-inhibitory cleaved substrate, and (4) a cleaved form. Due to these entities,
PAI-1 is able to respond rapidly in changing microenvironments. Hence, the stability
of PAI-1 is affected merely by cellular acidity and thermal dynamics. The active
form is considered to be the least stable conformation and the cleaved form as the
most stable (Sancho et al. 1995). At neutral pH the half-life of active PAI-1 is nearly
2 h at 37 C (Lindahl et al. 1989). The latent form may be converted into the active
form as well by negatively charged phospholipids or denaturants (Lambers
et al. 1987; Hekman and Loskutoff 1985). However, PAI-1 in plasma or extracellular
matrix is stabilized when bound to vitronectin. In this conformation the activity of
PAI-1 decays with a 12-fold increased half-life (Lijnen 2005; Mimuro et al. 1987).
The main sites of production are endothelium, mesothelium, and vascular smooth
muscle cells (Loskutoff et al. 1989; Holmdahl et al. 1997). The enzymatic activities
of tissue plasminogen activator (t-PA) and urokinase plasminogen activator (u-PA)
Insults peritoneal membrane:

- Uremic environment
- Exposure PD solution
Release cytokines
1. Low grade inflammation Increase vascular permeability Plasminogen
t-PA
– Plasmin
– Fibrinolysis and
2. Oxidative stress u-PA
proteolysis
+
3. Activation local host defence Loss mesothelial cell mass PAI-1
mechanism of mesothelial cells and integrity mesothelium
MMP-2 Pro-
Epithelial-to-mesenchymal Growth factors TGF-β1
MMP-2
transition
– +
Extracellular matrix Extracellular matrix –

accumulation and degradation
fibrosis
Tissue remodelling
Extracellular matrix
synthesis
Fig. 3 Schematic diagram of MMP-2 and PAI-1 pathways. Depicted in this figure are the
factors and mediators of MMP-2 and PAI-1. Both markers encompass pleiotropic entities, which
may result in excessive deposition of extracellular matrix constituents and fibrosis when impaired
are mainly restricted by PAI-1. For this reason PAI-1 is regarded as the predominant
inhibitor of fibrinolysis. Additionally, PAI-1 interacts with integrins and ECM
components (Dellas and Loskutoff 2005). PAI-1 release occurs through cytokines,
growth and inflammatory factors, and hormones. At a transcriptional level,
transforming growth factor-β1 (TGF-β1) is a major regulator of PAI-1, and in
cultured human peritoneal mesothelial cells, PAI-1 antigen was increased after
stimulation with TGF-β1 (Rougier et al. 1998). The biological processes to which
PAI-1 has been related are angiogenesis, cell migration, fibrosis, inflammation,
metastasis, and tissue remodeling (Dellas and Loskutoff 2005). The development
of abundant interstitial fibrosis and peritoneal membrane thickening is likely a
consequence of long-term PD therapy. A disbalance in proteolytic activity may
result in accumulation of ECM in basement membranes and interstitium. It is here
where MMP-2 and PAI-1 are intertwined due to their individual pleiotropic charac-
teristics. Figure 3 is a schematic diagram in which the pathways are depicted.
Cellular and Peritoneal Tissue Expression of MMP-2 and PAI-1
The efficacy of PD therapy relies on the integrity of the peritoneal membrane through
which the exchange of fluid and solutes occurs. However, anatomical and morpho-
logical studies describing consecutive peritoneal tissue modifications in PD patients
are seldom performed, as the procedure forms a direct threat to the continuation of PD
therapy. Even though the mesothelium is not considered as a prominent barrier for
peritoneal solute transport, it is an important constituent as it is the source from which
several substances are secreted and excreted including MMP-2 and PAI-1. Unfortu-
nately, the mesothelium is also difficult to assess due to its susceptibility to artifactual
damage during a peritoneal biopsy. From the abovementioned reasons, it can be
deduced that in vitro experiments using human peritoneal mesothelial cell lines or
in vivo rodent models are valuable proxies despite discordant results on some aspects
when compared with the dialyzed human peritoneal membrane condition.
So far, no histological studies have been performed to examine MMP-2 in human
peritoneal tissue. However, in a chronic PD rodent model, the degree of fibrosis was
correlated with effluent levels of MMP-2 (Lopes Barreto 2013). MMP-2 has been
investigated in harvested arteries from hemodialysis and PD kidney transplant
recipients. The densitometric measurements indicated a higher level of MMP-2
activity and expression in these previously dialyzed patients, compared to chronic
renal failure patients or healthy living kidney donors, suggestive for altered vascular
remodeling (Chung et al. 2009). More recently, immunohistochemical analysis of
renal allograft biopsies from patients with chronic humoral rejection revealed the
presence of MMP-2 in podocytes, whereas this was less common or absent in
patients experiencing other renal complications (Wong et al. 2010). Involvement
of MMP-2 in EMT of peritoneal mesothelial cells has been demonstrated. During
TGF-β1-induced EMT, MMP-2 gene and protein expression was upregulated, coin-
ciding with demolition of the basement membrane (Margetts et al. 2005). Addition-
ally, in human peritoneal mesothelial cells stimulated by >1 U/mL thrombin, the
activity of MMP-2 indicated a maximum reduction of 56 % in cell supernatants.
Moreover, the effect of thrombin was associated with a decline in MMP-2 produc-
tion and an enhanced tissue inhibitor of metalloproteinases (TIMP)-1 synthesis
(Haslinger et al. 2000). If prolonged, a potential consequence as a result of this
loss in homeostasis is peritoneal tissue remodeling where the decrease in MMP-2
leads to accumulation of ECM constituents.
The association between glucose-induced PAI-1 activation contributes to the
interest of in vitro experiments investigating the effect of glucose in PD solutions
on PAI-1 expression. For this purpose, isolated human peritoneal mesothelial cell
has been exposed to conventional, biocompatible, and icodextrin (a glucose
polymer)-based PD solutions varying in pH, glucose concentration, or duration of
exposure. Common findings comprise elevated PAI-1 antigen levels for PD solutions
high in glucose (>2.5 %) versus low-glucose PD solutions (<1.5 %). Icodextrin-
based PD fluids indicated a consistently lower PAI-1 activity. Additionally, a time-
dependent increase in PAI-1 release has been detected (Katsutani et al. 2007; Mandl-
Weber et al. 2001a; Breborowicz et al. 1997). Regulators of PAI-1 activity have also
received great attention. In vitro, the expression of PAI-1 is enhanced by TGF-β1 and
thrombin, but no significant impact seems to be present for hyaluronan (Rougier
et al. 1998; Mandl-Weber et al. 1999; Sitter et al. 2003). Depending on the strain,
bacterial peritonitis augments mesothelial PAI-1 synthesis, hence diminishing the
intraperitoneal fibrinolytic activity (Mandl-Weber et al. 2001b).
The formation of adhesions is a physiological response to peritoneal tissue repair.

The capacity to restore peritoneal insults in a controlled manner is therefore highly
dependent on the balance between fibrinogenesis and fibrinolysis. As a consequence,
PAI-1 has been studied extensively in patients undergoing laparotomy due to
peritoneal inflammation or trauma, or elective surgery. Nevertheless, studies on the
peritoneal distribution and localization of PAI-1 have not yet been performed in
patients receiving PD therapy. When comparing homogenates from normal perito-
neal tissue to an inflamed peritoneum, it was clear that the latter exhibited elevated
levels of PAI-1 (Vipond et al. 1990). The authors stated that a decrease in functional
fibrinolytic activity under inflammatory conditions is mediated by PAI-1. Later, the
expression of PAI-1 in the human peritoneum was localized. Immunohistochemical
analyses revealed that PAI-1 is present in the mesothelium and capillary vascular
walls of the peritoneum. However, in inflamed peritoneal tissue, PAI-1 is extensively
distributed in the submesothelium and partly co-localized with immunoreactivity of
macrophages (Holmdahl et al. 1997).
Systemic Levels of MMP-2 and PAI-1
As the critical importance of tissue remodeling and fibrosis in PD is centered at the

integrity of the peritoneal membrane, a limited number of PD study groups have
investigated circulatory MMP-2 and PAI-1. Nevertheless, these investigations have
related the systemic levels of these markers to major causes of morbidity and
mortality in PD such as cardiovascular risk and inflammation. The reported systemic
levels of MMP-2 and PAI-1 in PD patients are summarized in Table 2.
In a cross-sectional study, similar serum values between HD and PD patients, and
between pre-end-stage renal disease patients and transplant patients, were found for
MMP-2. The findings from this study indicated inferior MMP-2 levels of dialysis
patients suggesting a dialysis-related suppression of MMP-2 activity (Preston et al.
2002). Within the PD population, the median serum level of MMP-2 approximates
243 ng/mL when measured the first year after commencing PD therapy in incident
PD patients (Lopes Barreto et al. 2013). These serum levels are in line with a second
cross-sectional study, which additionally reported MMP-2 levels of healthy controls
without diabetes mellitus, hypertension, and renal or vascular diseases (Pawlak et al.
2010). Moreover, in the same study, it was demonstrated that PD patients with a
previous diagnosis of cardiovascular diseases exhibit a significantly augmented
serum level of MMP-2 with concentrations ranging from 200 to 625 ng/mL.
However, it is ambiguous whether elevated serum MMP-2 resides in the causal
pathway or reflects a consequence of cardiovascular diseases in PD patients.
Normal values of PAI-1 activity and antigen have been found in PD patients (Irish
1997; Kim et al. 1997). In PD patients with atherosclerosis, serum PAI-1 antigen is
increased and averages 25 ng/mL, which points toward an impaired fibrinolytic activity
(Kim et al. 1997). Comparative investigations on the effect of PAI-1 activity in chronic
renal failure and dialysis patients have also been executed. Throughout the subdivided
study population, a significantly lower activity of PAI-1 appeared to be present for HD
Table 2 Systemic levels of MMP-2 and PAI-1

Serum level in study
population (ng/mL)
Sample Detection Healthy PD
Author and year size method controls patients
MMP-2
Preston et al. (2002) 21 ELISA 542.0 850.0
Pawlak et al. (2010) 56 ELISA 192.4 258.2
Lopes Barreto 86 ELISA – 242.9
et al. (2013)
PAI-1
Kim et al. (1997) 82 ELISA 18.0 17.0
Pawlak et al. (2006) 34 ELISA 24.5 13.5
Arikan et al. (2009) 46 ELISA – 50.4
Lopes Barreto 86 ELISA – 20.9
et al. (2013)
Systemic levels of the biomarkers are expressed as average of study population
PD peritoneal dialysis, MMP-2 matrix metalloproteinase-2, ELISA enzyme-linked immunosorbent
assay, PAI-1 plasminogen activator inhibitor-1
patients, compared to patients with low and high proteinuria, and continuous ambulatory
PD patients (Irish 1997). The opposite was found in a study focused at assessing the risk
of cardiovascular diseases between HD and PD patients (Tomura et al. 1996).
Conflicting evidence encompasses PAI-1 antigen as well, where enhanced or impaired
fibrinolysis is described (Pawlak et al. 2006). In summary, data on the impact of PAI-1
activity between dialysis modalities are inconclusive and likely to be attributed to the
circadian rhythm of PAI-1 that peaks in the morning (Angleton et al. 1989) and analytical
procedures. Recently, a cohort study with a minimal follow-up duration of 5 years
reported a worse survival rate for all-cause and cardiovascular mortality in prevalent PD
patients with PAI-1 plasma levels exceeding 41 ng/mL (Arikan et al. 2009).
MMP-2 has been associated with arterial stiffness (Yasmin et al. 2005). Additionally,
in patients with an acute coronary syndrome, MMP-2 was considered as a predictive
factor for all-cause mortality (Dhillon et al. 2010). In renal diseases elevated serum
and plasma levels of MMP-2 have been related to progressive glomerulosclerosis.
Systemic levels of MMP-2 in chronic kidney disease patients are negatively corre-
lated with kidney function as measured by the estimated glomerular filtration rate
(eGFR) and increase linearly with the degree of proteinuria (Nagano et al. 2009).
Moreover, a recent finding indicated that plasma MMP-2 may be an independent
predictor of rapid decline in eGFR, accompanied by a hazard ratio of 2.47 for
progression of kidney disease in patients with MMP-2 levels above >861 ng/mL
(Hsu et al. 2013).
As a consequence of PAI-1 main function, i.e., an inhibitor of fibrinolysis, the

primary use in clinical practice is usually to detect deficiencies in the fibrinolytic
system. Elevated plasma levels of PAI-1 are therefore merely associated with
coronary diseases. Renal cell carcinomas stain positive for cytoplasmatic PAI-1
(Zubac et al. 2010). Furthermore, in a large multiethnic cohort, patients with an
eGFR <60 mL/min/1.73 m2 had 6.5 % higher levels of PAI-1 in comparison to those
with better kidney function. Additionally, PAI-1 was associated with eGFR by
cystatin C calculations, but not with creatinine-based eGFR (Dubin et al. 2011).
PAI-1 has been proposed as a potential therapeutic target in renal fibrogenesis
(Rerolle et al. 2000).
Peritoneal Effluent MMP-2 and PAI-1
The identification of effluent biomarkers in PD is merely from a hypothesis-driven

perspective rather than a discovery-based approach. The exact sequence of patho-
physiological events with the peritoneal cavity is unknown. However, since the
inception of PD therapy, numerous study groups have revealed molecular mecha-
nisms indicating short- and long-term peritoneal membrane alterations. From these
data, abundant peritoneal adhesions and fibrosis are considered as final events in the
continuum, which sometimes ends with the clinical presentation of encapsulating
peritoneal sclerosis (EPS). EPS is a rare (prevalence 3–7 %), but devastating
complication of PD therapy that is high in morbidity and mortality. Several attempts
have been made to timely identify PD patients at risk. However, no early detection
strategy is yet available.
The lead time of peritoneal tissue remodeling and interstitial fibrosis encompasses
years of chronic exposure to PD solutions. The biological functions of MMP-2 and
PAI-1, including remodeling of the ECM and prevention of excessive collagen and
fibrin depositions, led to investigations on their presence, potential significance, and
local effects of impaired homeostasis in PD therapy. Additionally, these markers
have been related to peritoneal transport parameters, PD-related peritonitis, and
effects of various PD solutions.
Appearance of MMP-2 and PAI-1 in the Peritoneal Cavity
Peritoneal effluent MMP-2 has been studied in incident as well as prevalent PD

patients. These studies reported average values of effluent MMP-2 and PAI-1,
revealing an overall increasing tendency in a time-dependent manner (Table 3).
Preferably, effluent levels are presented in appearance rates, which are an adjusted
value that is corrected for the drained effluent volume. However, this can only be
done when a marker has demonstrated to increase linearly during a predefined dwell
time. PAI-1 is known to increase linearly during a 4-h dwell regardless of the PD
Table 3 Peritoneal effluent levels of MMP-2 and PAI-1

Study Sample Detection PD duration Effluent level
Author and year design size method (months)a (ng/mL)b
MMP-2
Nishina Cohort 13 Zymography 7–137 126a
et al. (2004)
Hirahara Cross- 444 ELISA 13–92 167
et al. (2007) sectional
Minami Cross- 16 EIA 10–97 319
Hirahara Cross- 215 ELISA 22–69 167
Lopes Barreto Cohort 86 ELISA 0.5–12 21.4c
et al. (2013)
Cho et al. (2016) Cohort 178 ECL 0–3 34.2c
PAI-1
Selgas Cross- 20 ELISA 6–121 7.3
Goedde Cross- 16 ELISA Unknown 2.6
Opatrný et al. Cross- 31 ELISA 4–35 1.45
(1998) sectional
Lopes Barreto Cohort 86 ELISA 0.5–12 0.9c
et al. (2013)
PD peritoneal dialysis, MMP-2 matrix metalloproteinase-2, ECL electrochemiluminescence immu-
noassay, EIA enzyme immunoassay, ELISA enzyme-linked immunosorbent assay, PAI-1 plasmin-
ogen activator inhibitor-1. Peritoneal effluent levels of the biomarkers expressed as average of study
population
a
Range PD duration study population
b
Biomarker levels represent baseline measurement of study
c
Median biomarker level
solution glucose concentration (Lin et al. 1995; Opatrný et al. 1998). The release
pattern of MMP-2 has not yet been determined.
One of the main criteria for an effluent biomarker is the presence of local
peritoneal production. This is established by computing a peritoneal transport line
based on dialysate to plasma ratios of β2-microglobulin, albumin, IgG, and α2-
macroglobulin when plotted against their molecular weight. By interpolation of the
candidate marker, a positive discrepancy is attributed to local production. In the case
of MMP-2, local production accounts for 90 % of the effluent concentration.
Intraperitoneal release of PAI-1 has been established in both pediatric and adult
PD patients (de Boer et al. 1999; Selgas et al. 1992; Lopes Barreto et al. 2013).
Currently, the biological variability of effluent MMP-2 and PAI-1 between and
within PD patients is not known.
Associations with PD Duration, Peritoneal Transport Parameters,

and Peritoneal Effluent Markers
With regard to PD therapy duration, transversal analyses report positive relationships

with MMP-2 and PAI-1 (Lopes Barreto et al. 2013; Hirahara et al. 2007). However,
time-trend analyses of MMP-2 report conflicting results. A possible reason could be
the difference in follow-up duration (Lopes Barreto et al. 2013; Cho et al. 2016). In
contrast, time courses of PAI-1 indicate a significant elevation in effluent levels with
duration of PD therapy alongside an explained variance of 5 % in cross-sectional
analyses.
Peritoneal transport parameters reflect the functional integrity of the peritoneal
membrane and are necessary in order to determine the efficacy of PD therapy. The
functional parameters are derived from standardized peritoneal membrane function
tests, such as the standard peritoneal permeability analysis (SPA), and include
components of solute and fluid transport. The net ultrafiltration, mass transfer area
coefficients (MTAC) of small solutes, and free water transport are the most essential
parameters. High levels of MMP-2 and PAI-1 are accompanied by high MTAC of
creatinine and low free water transport. Also dialysate to plasma ratios of creatinine
and the maximal initial dialysate sodium dip have indicated moderate to strong
correlation coefficients with both effluent markers (Hirahara et al. 2007, 2011; Lopes
Barreto 2013). A relationship between net ultrafiltration and MMP-2 or PAI-1 is
absent (Lopes Barreto 2013).
Besides correlating peritoneal transport parameters with MMP-2 and PAI-1,
linear regression analyses have also been executed with other peritoneal effluent
markers. Research findings suggested no or less active participation of the mesothe-
lium in the production of MMP-2 or PAI-1 in long-term incident PD patients. Even
though moderate, the total variance of 22 % and 32 % for, respectively, MMP-2 and
PAI-1 can be explained by differences in IL-6 levels (Lopes Barreto 2013). This is in
line with pathophysiological findings reporting on inflammatory conditions and loss
in mesothelial cell function preceding fibrotic processes.
Effect of PD Solutions on Levels of MMP-2 and PAI-1
With the introduction of novel PD solutions, the aim has also been to objectify
the effect of conventional dialysis solutions high in glucose versus the more
biocompatible PD solutions in terms of peritoneal membrane integrity. However,
these studies have only been performed with established effluent biomarkers and
currently with effluent MMP-2 as well. Unfortunately, effluent PAI-1 has not yet
been incorporated. The first study addressing this question was performed a
decade ago in a number of 13 prevalent PD patients with a follow-up duration
of 21 months. The authors concluded that MMP-2 levels were decreased in
patients treated with the more biocompatible PD solutions (Nishina et al. 2004).
A second study, characterized by a cross-sectional design, compared a glucose-
based PD solution with icodextrin. The study population consisted of 16 prevalent
PD patients who received both solutions in an 8-h overnight dwell divided over
2 days. The dialysate exchange with icodextrin had significantly higher levels of
MMP-2 in comparison to the 2.27 % glucose-based dwell (Minami 2007). More
recently, the BalANZ trial evaluated the influence of conventional dialysis solu-
tion on the time course of effluent MMP-2 versus neutral pH PD solutions low in
glucose degradation products. Solely incident PD patients with archived effluent
specimens were included in the study (n = 178). Their findings indicated
increasing levels of effluent MMP-2, irrespective of the type of PD solution
(Cho et al. 2016).
Peritoneal Effluent Levels of MMP-2 and PAI-1 During PD-Related

Complications
Even though peritonitis has a cure rate of more than 88 %, it still remains a frequently
encountered complication in PD (van Esch et al. 2014). In general it is advocated to
measure peritoneal effluent biomarkers after resolution of a peritonitis episode as the
laboratory results in the acute phase are usually elevated. Nevertheless, the behavior of
effluent markers has been investigated right before the onset of peritonitis as well as
during a peritonitis episode. During inflammation the vascular permeability is increased
and the fibrinolytic activity is altered (Sitter et al. 1995). Measurements of effluent
PAI-1 on PD-related peritonitis have been performed in adult as well as pediatric PD
patients. Still, no data is available describing MMP-2 in pediatric PD-related peritonitis.
In pediatric PD patients, the role of the fibrinolytic system has been investigated
as well. It indicated that stable pediatric and adult PD patients have similar concen-
trations of effluent PAI-1 (Lin et al. 1995; Goedde 1997). To the same extent both
populations have significantly elevated effluent PAI-1 levels when experiencing a
peritonitis episode (Goedde 1997; de Boer et al. 1999). Concerning effluent MMP-2,
median values approximate 250 ng/mL and 160 ng/mL for, respectively, PD patients
with and without infectious peritonitis when measured by gelatin zymography
(Hirahara et al. 2007). However, contrary results are found in the activity of
MMP-2 where no differences were detected between peritonitis-free PD patients
and those with peritonitis. Moreover, an alteration in effluent MMP-2 activity
between the onset and recovery of peritonitis was absent (Fukudome et al. 2001).
Frequent peritonitis episodes are believed to be one of the major risk factors for
the development of EPS. Since effluent MMP-2 and PAI-1 are recently gauged to
reflect the degree of intraperitoneal fibrosis and due to the rarity of EPS, current
literature relating these candidate markers to EPS is limited to two investigations: a
single- and multicenter study. A Japanese multicenter study aimed to assess the
potential of MMP-2 as indicator of the progression to EPS. For this purpose, the
authors stratified adult PD patients with peritoneal injury into four categories,
including patients identified as EPS cases, and added a control group. Subsequently,
they measured MMP-2 by means of an enzyme-linked immunosorbent assay. The
cross-sectional analysis demonstrated elevated levels of effluent MMP-2 in those
with overt peritoneal damage (Hirahara et al. 2007). The objective of the single-
center study was to describe time trends and to evaluate the clinical validity of
effluent MMP-2 and PAI-1 in 4 years preceding EPS diagnosis. This study followed
a nested case-control design including 10 patients diagnosed with EPS and 34 long-
term controls. The same detection method as in the multicenter study was used for
both effluent markers. Interestingly, the time course of MMP-2 illustrated no differ-
ence between the two groups, whereas EPS patients exhibited persistent elevated
levels of PAI-1 with an increasing tendency when compared with long-term PD
patients. In addition, diagnostic accuracy measures indicated the potential for efflu-
ent PAI-1 to detect preclinical EPS. The discriminative capacity for effluent MMP-2
was restricted to 1 year prior to the diagnosis of EPS (Lopes Barreto et al. 2014).
Clinical Relevance of Peritoneal Effluent MMP-2 and PAI-1
From the preliminary literature of MMP-2 in EPS follows that MMP-2 is unlikely to
be significant as a biomarker that can be used for the early detection of EPS.
However, as all physiological and pathological processes require remodeling of
the ECM for cell migration, a significant application for MMP-2 in short-term
consequences of PD should not be ruled out. This is supported by its involvement
in EMT illustrating that MMP-2 is a necessary component during the transdiffer-
entiation of mesothelial cells. For this reason it would be of interest to assess the
association of effluent MMP-2 with PD technique failure due to peritoneal mem-
brane dysfunction.
The potential application of effluent PAI-1 resides more in the purpose of a
screening or diagnostic tool for EPS. The capacity of effluent PAI-1 to discriminate
between long-term PD patients and those who will develop EPS is not of neglectable
magnitude. A histological study on the distribution and localization of PAI-1 in
peritoneal tissue within the PD population would be of great relevance. Furthermore,
such explorations would have the ability to objectify the degree of concordance
between the actual measured effluent levels of PAI-1 and its expression in peritoneal
tissue. Effluent PAI-1 could also be utilized as an effluent biomarker to monitor
progression of PD therapy, as the development of interstitial fibrosis is process of
large lead time.
Conclusions
Intraperitoneal events that occur as a consequence of PD therapy are multifactorial

processes. The use of effluent biomarkers would facilitate a noninvasive strategy to
monitor PD therapy and support the early identification of PD-related outcomes.
This chapter has given insight into the biological implications and clinical utility of
MMP-2 and PAI-1 as biomarkers in PD. It is obvious that beforehand implementa-
tion in routine patient care external validation is necessary with respect to their
clinical validity and utility. Nevertheless, a panel of effluent biomarkers including
MMP-2 or PAI-1 in conjunction with a routine peritoneal function test should be

utilized for optimal chronic PD patient care.
Summary Points
• This chapter focuses on the biological implications and clinical utility of matrix
metalloproteinase-2 (MMP-2) and plasminogen activator inhibitor-1 (PAI-1) in
peritoneal dialysis (PD) therapy.
• The peritoneal effluent is the most clinically relevant specimen in PD.
• MMP-2 is a 72 kDa gelatinase that is involved in tissue remodeling.
• PAI-1 is a 50 kDa member of the serine protease inhibitors (SERPINs) that
inhibits fibrinolysis and proteolysis.
• Peritoneal effluent MMP-2 and PAI-1 have recently been gauged as biomarkers
for peritoneal membrane alterations.
• Current literature is limited with respect to the assessment of MMP-2 and PAI-1
clinical validity and utility.
• Incorporating peritoneal effluent biomarkers in routine PD patient care, alongside
peritoneal function tests, will lead to a more personalized medicine.
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Part IV
Molecular, Cellular, and Histological Variables
Assessing Fibrosis in Kidney Biopsies
41
Behtash Ghazi Nezami and Alton B. Farris
Contents
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 935
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 936
Mechanisms of Renal Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 937
Interstitial Fibrosis Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 941
Fibrosis Patterns in Biopsies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 941
Staining Techniques and Light Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 942
Computer-Based Morphometric Study . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 946
Molecular Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 947
Proteomic Approaches, Including Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 947
Fibrosis Measurement and Methodologies: Challenges and Promises . . . . . . . . . . . . . . . . . . . . . . . . . 948
Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 949
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 949
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 949
Abstract
Renal interstitial fibrosis and tubular atrophy (IFTA) is one of the primary end
points of kidney injury, and accurate IFTA quantitation in biopsy samples is
crucial in establishing the diagnosis and assessing disease severity. Therefore,
knowing the basic procedures in the preparation of biopsy for IFTA and specific
staining techniques available for IFTA is pivotal to the pathologists’ practice. This
B.G. Nezami
Department of Digestive Diseases, Department of Medicine, Emory University School of Medicine,
Atlanta, GA, USA
Department of Pathology and Laboratory Medicine, Emory University Hospital, Atlanta, GA, USA
e-mail: bnezami@emory.edu
A.B. Farris (*)
Department of Pathology and Laboratory Medicine, Emory University Hospital, Atlanta, GA, USA
e-mail: abfarri@emory.edu

934 B.G. Nezami and A.B. Farris
chapter reviews the mechanisms of IFTA pathogenesis pertinent to biopsy eval-

uation and the common techniques used to evaluate biopsies for IFTA. The
challenges facing IFTA evaluation in biopsies and recent technical developments
in this field are discussed.
Keywords
Kidney/renal fibrosis • Epithelial–mesenchymal transition • Myofibroblast •
Morphometry • Masson’s trichrome stain • Picrosirius red stain
Abbreviations
BMP Bone morphogenic protein
CCR-2 C-C chemokine receptor type 2
DC Dendritic cell
EMR-1 EGF-like module-containing mucin-like hormone receptor-like 1
EMT Epithelial-mesenchymal transition
EndoMT Endothelial-mesenchymal transition
ERKs Extracellular-signal-regulated kinases
FGF-2 Fibroblast growth factor 2
FSP-1 Fibroblast-specific protein 1
GSK Glycogen synthase kinase
HGF Hepatocyte growth factor
HIF Hypoxia-induced factor
IF Interstitial fibrosis
IFTA Interstitial fibrosis/tubular atrophy
IHC Immunohistochemical
ILK Integrin-linked kinase
JAK/STAT Janus kinase/signal transducer and activator of transcription
LTBP Latent TGF-β binding protein
miRNA MicroRNA
NF-κB Nuclear factor κB
PAPMS Pathogen-associated molecular patterns
PKC Protein kinase C
SMA Smooth muscle actin
TA Tubular atrophy
TEC Tubular epithelial cell
TGF-β Transforming growth factor-beta
TIMP Tissue inhibitors of metalloproteinases
41 Assessing Fibrosis in Kidney Biopsies 935
TLR Toll-like receptor

tPA Tissue plasminogen activator
TSP-1 Thrombospondin-1
tTG Tissue transglutaminase
USAG-1 Uterine sensitization-associated gene 1 (also known as sclerostin
domain-containing protein 1)
Definitions
Birefringent Birefringence in the context of this article refers to colors that are
somewhat iridescent or anomalous (Howie et al. 2008). This anomalous quality is
imparted to tissue when viewed under polarized light due to a particular interaction
of the polarized light with the tissue or a combination of the stain’s interaction with
the tissue. For example, a Sirius red stain of fibrotic tissue, particularly the collagen
in this fibrotic tissue, has a yellowish birefringence when viewed under polarized
light.
Computerized morphometry Tissue is imaged and then analyzed with computer-

ized programs called algorithms to obtain quantitative measures of different param-
eters. For example, a positive pixel count algorithm tuned to detect the color of
fibrotic tissue can be used to quantitate fibrotic areas.
Endothelial/mesenchymal transition (EMT) The change in endothelial pheno-

type during renal injury leading to fibrosis, in which endothelial cells lose their
specific markers and acquire the mesenchymal phenotype, becoming more invasive
with increased migratory abilities.
Epithelial/mesenchymal phenotype (EMP) The putative phenotypic change of

epithelial cells leading to the loss of their cell polarity and adhesion surface proteins
and gain of migratory properties to become mesenchymal stem cells.
Extracellular matrix (ECM) Connective tissue material that essentially serves as

the framework for tissue and that is not contained within cells.
Fibrosis This term refers to the deposition of excess fibrous tissue within an organ
and is derived from the Latin fibra, meaning “fiber,” and the Greek -osis, meaning a
condition or state [derived from Dictionary.com].
Genomics The analysis of the genetic state of a biologic specimen or set of biologic
specimens. Genomics is one of the main “-omics” disciplines in which the suffix “-ics”
derived from the Latin “-ica” and Greek “-ika” [derived from Dictionary.com].
IFTA Parenchymal contraction in which intervening tubulointerstitial parenchyma

between obsolescent glomeruli have been lost and occupied by fibrotic matrix
through interstitial fibrosis (IF), which is usually accompanied by tubular
atrophy (TA).
microRNA Short single-stranded RNA molecules that regulate a wide range of

genes posttranscriptionally by targeting mRNAs and are stable in tissues for
extending periods of time.
Proteomics Large-scale study of structures and functions of proteins using protein

purification techniques and mass spectrometry. Referring to proteome as the entire
set of proteins of an organism or tissue. This is an example of another “-omics”
discipline.
Sclerosis Refers to hardening or stiffening of tissue or organ, often due to a

pathologic growth of fibrous tissue or chronic inflammation [derived from Dictio-
nary.com]. The word is derived from the Greek sclerosis, which means “hardening.”
Introduction
Interstitial extracellular matrix (ECM) accumulation is common to many chronic

kidney diseases and contributes to loss of kidney function. After renal damage, a
constellation of intra- and extrarenal cells are activated to limit the extent of tissue
damage and start the healing process. Interstitial fibrosis is the result of such
processes (Table 1), when a complete regeneration of the renal tissue cannot be
achieved. Interstitial fibrosis (IF) mostly refers to the excessive and pathological
deposition of ECM, which is often accompanied by tubular atrophy (TA) and is
collectively termed IFTA (Liu 2006; Boor et al. 2010; Zeisberg and Neilson 2010;
Table 1 Key facts of renal fibrosis pathogenesis

A variety of cells contribute to interstitial fibrosis, including fibroblasts, dendritic cells,
lymphocytes, and monocyte/macrophages
Tubular epithelial cells possibly contribute to fibrosis as they are damaged and undergo a putative
epithelial-mesenchymal transition (EMT)/phenotype (EMP). Endothelial cells also possibly
contribute to fibrosis through an endothelial to mesenchymal transition (EndoMT). However,
despite evidence for these processes, some question EMT and EndoMT
Cellular damage is thought to contribute to the release of molecular mediators, many of which are
pro-fibrotic, including tumor growth factor beta (TGF-β), a major fibrogenic and inflammatory
cytokine. Other growth factors include bone morphogenic protein (BMP), platelet-derived growth
factor (PDGF), and hepatocyte growth factor (HGF)
Fibrosis ultimately occurs through the deposition of extracellular matrix (ECM), including
collagen, proteoglycans, and admixed ECM molecules such as fibronectin, biglycan, decorin,
tissue transglutaminase (tTG), matrix metalloproteinase (MMP), tissue plasminogen activator
(tPA), and laminin
This table lists the key facts of the cellular and molecular contributors to renal fibrosis
Farris and Colvin 2012; Farris and Alpers 2014). TA is characterized by the presence
of small dilated and thin tubules with pale cytoplasm and thickened and irregularly
contoured basement membranes. TA probably has distinct mechanisms than IF,
related to blood flow restriction, glomerular filtration rate (GFR), or loss of tubular
continuity. Many studies show a reciprocal correlation between kidney function and
the IF extent. IF has been shown to predict the outcome of renal allograft (Choi
et al. 2005; Farris et al. 2010; Meas-Yedid et al. 2011) and native kidney diseases
such as IgA nephropathy (Working Group of the International IgA Nephropathy
Network and the Renal Pathology Society et al. 2009).
Renal biopsies play major role in clinical diagnosis of IFTA. However, there is
still controversy over the best qualitative and quantitative techniques for IFTA
(Farris et al. 2014). Accurate IFTA measurement is required in numerous applica-
tions, including in comparison of renal allograft protocol biopsies, helping in the
determination of prognosis in glomerular disease (e.g., IgA nephropathy and lupus
nephritis), and pharmaceutical studies on therapeutic inhibition of IF (Liu 2006;
Vilayur and Harris 2009). Furthermore, as stem cell therapy is gaining attention in
renal fibrosis research, the need for establishing accurate and valid assessment
techniques for fibrosis is more pronounced (Reinders et al. 2014; Choi et al. 2015).
Mechanisms of Renal Fibrosis
Normal renal interstitium consists of sparse cells, mainly fibroblasts and dendritic
cells (DCs), embedded in ECM network. During and after renal injury (infection,
ischemia, diabetes, allograft rejection, etc.), a constellation of mechanisms are
activated to protect the damaged tissue and speed up regeneration (such as reepithe-
lialization, epithelial barrier repair, regeneration after vascular injury, construction of
the ECM skeleton, and recovering from mesangial damage). A variety of cells
(Fig. 1) and molecular mechanisms (Fig. 2) contribute to IF.
IF is most likely driven by a lack of highly differentiated cells, which are replaced
by scarring connective tissue. Tumor growth factor beta (TGF-β) is a major
fibrogenic and inflammatory cytokine, produced by damaged native and inflamma-
tory cells (Farris and Colvin 2012; Friedman et al. 2013). Tubular epithelial cells
unable to regenerate get arrested in the G2/M phase and produce TGF-β (Yang
et al. 2010), which causes augmented deposition of ECM proteins and renal fibrosis
(Bottinger 2007). TGF-β-inducible integrins (e.g., αVβ6) ultimately act through
integrin-linked kinases (ILK) and other mediators to produce collagen, contributing
to the production of the ECM (Farris and Colvin 2012). ECM production is also
affected by other systemic physiologic states, such as the renin/angiotensin system
(Naito et al. 2010).
ECM accumulation usually occurs prior to chronic kidney disease (CKD) and
contains sulfated and non-sulfated proteoglycans and glycosaminoglycans involved
in IF and other major molecules such as types I and III collagen, fibronectin,
biglycan, decorin (Boor et al. 2010; Zeisberg and Neilson 2010), tissue transglu-
taminase (tTG) (Huang et al. 2009), matrix metalloproteinase (MMP) (Wang
Fig. 1 Cells contributing to interstitial fibrosis: A variety of cells contribute to interstitial fibrosis,
including renal vasculature, tubular cells, and inflammatory cells such as lymphocytes, monocyte/
macrophages, mast cells, and dendritic cells. The renal tubules undergo changes to acquire
epithelial–mesenchymal phenotype (EMP) and may undergo the process of epithelial–me-
senchymal transition (EMT). The endothelium is possibly involved in a process of endothelial–-
mesenchymal transition (EndoMT). Inflammatory cells have important role in both the processes of
EMT/EMP and EndoMT. Fibrocytes and pericytes can transform to fibroblasts and myofibroblasts.
Fibroblasts/mesenchymal cells mediate the production of fibrosis and extracellular matrix (ECM)
deposition and also may undergo a transition to a myofibroblastic phenotype through paracrine
signaling from inflammatory cells, further increasing ECM deposition and fibrosis
et al. 2010), tissue plasminogen activator (tPA) (Yang et al. 2002), and laminin
(Abrass et al. 2010). Proteoglycans fill the majority of renal extracellular interstitial
space and act as a reservoir of pro-fibrotic growth factors, such as the latent forms of
TGF-β or fibroblast growth factor 2 (FGF-2). Fibronectin and thrombospondin-1
(TSP-1) are adhesive glycoproteins involved in IF. Fibronectin accumulation is one
of the first events during renal fibrosis (Eddy 1996).
Other mediators important in IF include Smads, bone morphogenic proteins
(BMPs), particularly BMP-7, sclerostin domain-containing protein 1 (also known
as uterine sensitization-associated gene 1 (USAG-1)), protein kinase C (PKC),
extracellular-signal-regulated kinases (ERKs) (Sun et al. 2014), platelet-derived
growth factor (PDGF), PDGF-β (Wilkinson et al. 2009), hepatocyte growth factor
(HGF), the Janus kinase/signal transducer and activator of transcription (JAK/STAT)
pathway, fibrinogen (Sorensen et al. 2011), and possibly toll-like receptors (TLRs).
Smads act on ILK, stimulating glycogen synthase kinase (GSK) to produce
β-catenin, which traverses into the nucleus to induce transcription that ultimately
leads to fibrosis (Farris and Colvin 2012).
Fig. 2 Key molecular mechanisms contributing to interstitial fibrosis: A variety of molecular

mechanisms contribute to interstitial fibrosis. Transforming growth factor (TGF-β) is released
from activated inflammatory and damaged mesangial cells through interactions with matrix
metalloproteinases (MMPs), plasmin, integrin, angiotensin, tissue plasminogen activator (tPA),
and tissue transglutaminase (tTG). When it is released from inhibition by latent TGF-β-binding
protein (LTBP) and latency-associated peptide (LAP), TGF-β binds the transforming growth factor
receptor (TGF-R), activating intracellular signals such as the Smads, jagged/notch, Akt, Bcl-2, and
NF-κB pathways. These lead to nuclear transcription, ultimately culminating in collagen and ECM
production. In epithelial cells it may lead to epithelial-mesenchymal transition (EMT). Smads also
increase fibrosis via integrin-linked kinase (ILK). ILK acts through glycogen synthase kinase
(GSK) to produce β-catenin, which traverses into the nucleus to induce transcription. Integrins
can also act through ILK in a similar manner. Bone morphogenic proteins (BMPs) generally restrict
fibrosis. BMPs bind to their receptor (BMPR) to inhibit Smads, a process inhibited by uterine
sensitization-associated gene 1 (USAG-1) and platelet-derived growth factor (PDGF)
Renal fibroblasts are the major constituent cells in ECM and are in charge of
producing excessive collagen (Boor et al. 2010). Thrombospondin-1 stimulates
fibroblast proliferation and migration in CKD and is correlated with the degree of
tubulointerstitial fibrosis in rat models of renal fibrosis (Mason and Wahab 2003).
Fibroblasts stain for vimentin (intermediate filament protein) and stain weakly for
alpha smooth muscle actin (α-SMA). Activated fibroblasts stain for fibroblast-
specific protein 1 (FSP-1). However, there are not any completely specific markers
for fibroblasts that are widely utilized, and this makes the study of fibroblasts quite
difficult (Xia et al. 2013; Farris and Alpers 2014).
Myofibroblasts secrete collagen and glycosaminoglycans and are activated by
various mechanisms such as paracrine signals derived from lymphocytes and mac-
rophages, autocrine factors, and pathogen-associated molecular patterns (PAMPS).
Myofibroblasts have multiple potential origins with candidates being fibroblasts,
fibrocytes, pericytes, and epithelial or endothelial cells (Humphreys et al. 2010).
These cells share features with smooth muscle cells, including the expression of
α-SMA, and contain vimentin, fibronectin, and S100A4 (also known as FSP-1) (Lin
et al. 2008). Pericytes contribute to vascular reconstruction via tissue inhibitors of
metalloproteinases (TIMPs) and ADAMTS1 (Schrimpf et al. 2012). Fibrocytes are
derived from peripheral blood leukocytes (Pilling et al. 2009) producing ECM and
expressing both hematopoietic (e.g., CD45) and stromal cell markers (e.g., type I
collagen). Recent in vitro studies suggest that fibrocytes develop outside the kidney
independent of infiltrating monocytes and rely on CCR2 for migration into target
organs (Reich et al. 2013).
Endothelial cells and tubular epithelial cells (TECs) participate in the recruitment
of circulating leukocyte populations and facilitating the inflammatory response in the
injured kidney. TECs play major role in recruiting macrophages and lymphocytes
via NF-κB and pro-inflammatory chemokines (Mezzano et al. 2004). Epithelial and
endothelial cells increase the IF through a process of differentiation to
myofibroblasts, in which they undergo a phenotypic conversion termed epithelial–-
mesenchymal or endothelial–mesenchymal transition (EMT/EndoMT) (LeBleu
et al. 2013). In this process they lose their markers, such as E-cadherin, and acquire
mesenchymal markers, such as vimentin and α-SMA (Zeisberg and Neilson 2010;
Friedman et al. 2013). These markers can be used to determine EMT process in the
tissue (Wang et al. 2015). However, many experts have questioned this migration
feature of EMT (Kriz et al. 2011). Endothelial cells can suppress inflammation via
endothelial nitric oxide synthase (eNOS) production in injured sites. VEGF allevi-
ates fibrosis (Lian et al. 2011), and hypoxia promotes fibrosis through multiple
mediators including hypoxia-inducible factor-1α (HIF-1α) (Higgins et al. 2008).
A wide range of mononuclear inflammatory cells can be identified in normal renal
interstitium, including DCs, macrophages, and lymphocytes (T cells, B cells, and
natural killer cells) (Paust et al. 2011). Lymphocytes play a variety of roles in the
development of IFTA (Tapmeier et al. 2010). Type 2 T-helper cells (TH2) produce
mostly pro-fibrotic cytokines, inducing differentiation of fibrocytes, triggering mac-
rophage recruitment and inflammatory response (Liu et al. 2012), and in contrast,
type 1 T-helper cells (TH1) inhibit differentiation of fibrocytes (Niedermeier
et al. 2009). Microarray analysis of renal allografts has shown increased T cell and
natural killer gene sets in IFTA development (Scian et al. 2011). High T cell and
macrophage but not B cell infiltration is associated with low IL-10 expression, which
confers susceptibility to IFTA (Khan et al. 2010).
Monocyte/macrophages are heterogeneous, consisting of both infiltrating and
resident cells (Anders and Ryu 2011). Renal DCs seem to mediate the recruitment
of other cell types. During exposure to bacteria, DCs generate chemokines to attract
effector cells, such as neutrophils (Rogers et al. 2014). Macrophages or DCs can be
identified by F4/80 (also known as EGF-like module-containing mucin-like hor-
mone receptor-like 1 (EMR1), CD11b+ (integrin αM), and the DC marker CD11c+
(integrin αx). However, these markers are not specific (Rogers et al. 2014). The
degree of macrophage infiltration correlates with both the severity of damage and
extent of IF (Eardley et al. 2008). Some subsets of bone marrow-derived monocytes
such as CD11b+ cells may attenuate fibrosis (Semedo et al. 2010).
Mast cells express immune-related surface receptors and store inflammatory
cytokines, which give them the ability to immediately release pro-fibrosis mediators
such as TGF-β and MMPs (Snelgrove et al. 2011). There is a correlation between the
accumulation of mast cells and the degree of renal interstitial fibrosis (Mack and
Rosenkranz 2009).
Interstitial Fibrosis Assessment
Interstitial fibrosis is typically assessed in a qualitative and a quantitative manner. A

variety of methods can be used (Fig. 3) and are discussed below. Briefly, microscopy
is one of the primary methods of this assessment since it assists in the qualitative
measurement of fibrosis, and it also allows quantitation of the degree of fibrosis.
Furthermore, molecular methods are becoming more widespread and will also likely
aid in both qualitative and quantitative assessment of fibrosis (Table 2).
Fibrosis Patterns in Biopsies
It is important to note that depending on the pathology there are different patterns of
IFTA. Table 3 highlights the features of IFTA in different pathologic conditions.
Despite these associations, there is often an essentially nonspecific pattern of
fibrosis in renal biopsies of patients with chronic kidney disease, including diffuse
or patchy fine IF surrounding tubules, which can be either normal or atrophic. This
is associated with either diffuse or focal disease of glomeruli, tubules, or vessels
(Farris and Colvin 2012; Farris and Alpers 2014; Haas et al. 2014). It is important
to note that even though the clinical emphasis is usually put on cortical IF,
medullary IF is likely happening in parallel to cortical IF and epithelial loss (Farris
et al. 2013).
Fig. 3 Methods for interstitial fibrosis assessment: Various methods are available for assessing
interstitial fibrosis. Renal biopsy samples are routinely prepared for light, immunofluorescent, and
electron microscopic studies. For clinical evaluation of renal fibrosis, H&E, trichrome, Periodic
acid–Schiff (PAS), picrosirius red, and collagen III immunohistochemical (IHC) stainings are
commonly used. Other methods have been used for research only purposes to study renal fibrosis,
such as electron microscopy, microRNA analysis, or transcriptomics or proteomics. However, these
methods are not yet used for routine clinical evaluation of renal fibrosis
Staining Techniques and Light Microscopy
The preferred approach in staining for IFTA is using multiple complementary

techniques to confirm the degree of fibrosis, because each technique has shortcom-
ings and advantages. The routine techniques to obtain sample and assess renal
fibrosis and tubular atrophy may be different between laboratories. Hence, a gener-
ally accepted algorithm is usually applied in each laboratory (Agarwal et al. 2013).
In the assessment of IFTA, paraffin embedded kidney sections are often stained
for hematoxylin and eosin (H&E), trichrome, periodic acid–Schiff (PAS), and
methenamine silver. Other methods such as picrosirius red or immunohistochemistry
for collagen (particularly collagen types I and III) or SMA can give more information
depending on the specimen. Table 4 summarizes the characteristics, advantages, and
disadvantages of currently popular stains for IFTA in routine clinical diagnosis and
research. Trichrome highlights fibrosis by color, typically with a blue or green hue,
depending on the trichrome method employed (e.g., Mallory’s, Masson’s, etc.). For
quantitation, visual assessment of slides is the standard practice at many institutions
(Moreso et al. 2001). Picrosirius red stain is also a commonly used method to detect
Table 2 Key facts of renal fibrosis assessment

Biopsies are often performed to assess interstitial fibrosis, which is often accompanied by tubular
atrophy (IFTA)
A variety of stains can be used to highlight interstitial fibrosis, including trichrome, Sirius red, and
collagen III immunohistochemistry
Assessment of histologic stains can be combined with image analysis to provide an objective
method for IFTA assessment
Numerous IFTA assessment methods have shown relationships with renal function measures (e.g.,
glomerular filtration rate). In this sense, IFTA assessment can act as a biomarker of renal disease
Molecular methods such as transcriptional profiling and proteomic approaches have also been
used to assess renal fibrosis
Knowing the degree of fibrosis and its relationship to genomic, proteomic, and other -omic data
helps an investigator go from an understanding of the disease state (“-osis”) to understanding on a
variety of -omic levels
This table lists the key facts of renal fibrosis assessment
Table 3 Interstitial fibrosis patterns in different pathologic conditions

Etiology Fibrosis pattern
Aging Subcapsular fibrosis due to marginal supply of the peripheral cortex
Often accompanied by thickening of capillary loop basement membrane
Secondary to the ischemia associated with peritubular capillary injury
Pyelonephritis Diffuse fibrosis with severe loss of tubules
In acute phase is associated with inflammatory infiltrates
Relative preservation of the glomerular structure
Infarct Broad with loss of tubules
Calcineurin Patchy and striped corresponding with TA
inhibitor Hyaline arteriopathy and glomerulosclerosis
Preferential involvement of medullary rays – toxic injury to discrete
segments
Chronic allograft Loss of peritubular capillaries in parallel to IF
injury Diminished supply of nutrients to the tubulointerstitium
Chronic IFTA with relative glomerular sparing, atubular glomeruli, dilated tubules,
obstruction and intratubular Tamm–Horsfall protein casts with extravasation into the
interstitium
Diabetic Diffuse and homogeneous
nephropathy
Interstitial fibrosis and tubular atrophy (IFTA) occurs in specific patterns depending on the etiology
of the IFTA, as shown above
fibrosis in tissues (Fig. 5). Picrosirius red is typically considered to be specific for
collagen types I and III under polarized light in which the collagen has a birefringent
yellow hue (Sund et al. 2004). Movat’s pentachrome stain allows the assessment of
collagen, proteoglycan, and elastic tissue content with a single-staining procedure.
It imparts different colors to collagen fibers, glycosaminoglycans, elastic fiber, fibrin,
nuclei, and muscle. There have been modifications for this staining to increase
Table 4 Staining techniques

Method Description Advantages Disadvantages
Hematoxylin Performed for routine Routinely performed by Does not provide
and eosin diagnosis most laboratories on much contrast for
(H&E) nearly every specimen tissue with IF
Periodic Imparts a pinkish hue to Routinely performed by Does not provide
acid–Schiff glomerular and tubular many laboratories on much contrast for
(PAS) basement membranes renal biopsies tissue with IF
Relatively inexpensive
Useful for assessing TA
Useful for tubulitis
Masson’s Imparts a blue or green Routinely performed by Reproducibility
trichrome hue to ECM, including many laboratories on sometimes limited
collagen renal biopsies
Relatively inexpensive Possibly less sensitive
Useful for assessing in mild fibrosis
extracellular matrix Sensitive to the length
of formalin fixation
Picrosirius Stains ECM, Specific for collagen type Not widely used
red particularly collagen I and III
Examined with High signal-to-noise Subject to polarized
polarized and ratio vs. unpolarized
unpolarized light measurement
discrepancies
Polarization color may Computerized image Dependent on
reflect the extent of analysis possible operator performance
cross-linking and the Shown correlates with Time consuming and
age of fibrosis (fine the interstitial volume expensive
vs. large bundles) or fraction of the cortex. Difficult to
density of fibers standardize, lacks
cross institutional
validation
Cannot differentiate
between collagen type
Movat’s Five-colored stain, Collagen content, Not routinely
pentachrome imparting different proteoglycan content, performed on renal
colors to collagen fibers, and elastic tissue content biopsies and may not
glycosaminoglycans, with a single-staining be available in all
elastic fibers, fibrin, procedure laboratories
nuclei, and muscle Reveals early and subtle
changes in the
interrelationships of
many of the tissues
(continued)
Table 4 (continued)
Method Description Advantages Disadvantages
Collagen IHC utilizing antibody High signal-to-noise Not widely available
type III IHC for collagen III ratio
Computerized image Difficult to
analysis possible standardize
Dependent on
operator’s
performance
α –SMA IHC IHC utilizing antibody Interstitial SMA can be May not be present in
for smooth muscle actin detected in some cases of all types of IFTA
(SMA) IFTA
This table describes stains used for the assessment of interstitial fibrosis (IF) and tubular atrophy
(TA). Most of these stains are considered to be either “special” histochemical stains or immuno-
histochemistry (IHC). For the assessment of IF, stains are typically considered to be useful if they
highlight extracellular matrix (ECM) or collagen in particular
Fig. 4 Morphologic approaches for interstitial fibrosis assessment: Interstitial fibrosis can be
approached in two basic ways by pathologists. (1) The fibrosis percentage taken as the percentage
of tissue occupied by fibrosis excluding tubules and glomeruli and healthy islands of tissue,
(2) assessing the percentage of the tissue that is abnormal
consistency and reliability and decrease preparation time (Doello 2014). Immuno-
histochemistry (IHC) staining can be used to identify particular protein or cell
population in the biopsy sample, such as collagen I, III, and IV, smooth muscle
actin (e.g., α-SMA) (Choi et al. 2015), Smad7, E-cadherin (Liu et al. 2013), and
CD11c+ cells (Kruger et al. 2004). Among the wide range of IHC stains available,
collagen III (Satoh et al. 2001) is most often used to assess fibrosis (Fig. 5).
Computer-Based Morphometric Study
Morphometry techniques use computerized image analysis to measure the surface

area affected by fibrosis. These computer-based methods include morphometry of
slides stained with trichrome (Farris et al. 2011, 2014; Meas-Yedid et al. 2011),
picrosirius red (Sund et al. 2004; Lattouf et al. 2014), and collagen III IHC (Farris
et al. 2011, 2014; Farris and Colvin 2012) or a combination of staining techniques
and automated analysis (Street et al. 2014). Analysis in some of these studies has
shown correlation with GFR (Farris et al. 2011, 2014; Meas-Yedid et al. 2011);
however, as shown in the studies by Farris et al. (2011), this may not improve upon
the assessment made by the unaided human eye (Farris et al. 2011). As computerized
scanners gain more widespread use in research and clinical settings, it is possible that
computerized morphometry will integrate more in clinical standard practice. Com-
monly used software programs include NIH ImageJ (available at http://imagej.nih.
Fig. 5 Commonly used stains to assess renal fibrosis are shown: Fibrosis is visualized by different
colors in each staining: Blue in trichrome, light pink/purple in PAS (vs. dark pink/purple for
basement membranes), dark brown in collagen III immunohistochemistry, and red in picrosirius
red stain. A glomerulus can be seen in the center of the images, and an area of fibrosis can be seen to
the left of the glomerulus in each of the images (all at an original magnification of 200)
gov/ij/), Fiji (available at http://fiji.sc/Fiji), Icy (available at http://icy.

bioimageanalysis.org/), and proprietary programs that are stand-alone and/or are
provided by microscope manufacturers (e.g., Aperio [Leica], Olympus,
NIS-Elements [Nikon], MetaMorph, etc.). Whole slide scanners allow scanning of
slides, often in an automated batch mode, which further facilitates the ease of this
analysis (Farris et al. 2011, 2014).
Molecular Methods
A variety of molecular pathways can be probed through transcriptional profiling of

gene expression (Maluf et al. 2008; Bunnag et al. 2009; Scian et al. 2011). Many
approaches involve examining genes from set pathways together to determine which
pathways predominate in IFTA, showing, for example, the importance of chronic
inflammation in a study that examined immune response, cell-to-cell interaction, and
inflammation pathways (Maluf et al. 2008). Other studies show the importance of
specific derangements in the development of IFTA, showing that tissue injury and
dedifferentiation are linked with functional deterioration (Bunnag et al. 2009). Older
studies have focused on the analysis of DNA and RNA; however, newer studies have
evaluated microRNAs (miRNAs), short single-stranded RNA molecules that regu-
late wide ranges of genes posttranscriptionally by targeting mRNAs (Zununi Vahed
et al. 2014). MicroRNAs are stable in tissues for extended periods of time. So far a
few miRNAs are the center of clinical attention, which may find implications in renal
fibrosis assessment, especially in renal allograft tissue (Zununi Vahed et al. 2014;
Chung and Lan 2015) with some miRNAs being pro-fibrotic and upregulated in
renal fibrotic tissue and some miRNAs showing a decrease in IFTA (miR-107,
211, 204, 324, and 30a-3p) (Chung and Lan 2015).
Proteomic Approaches, Including Mass Spectrometry
There are a growing number of studies on fibrotic pathophysiological mechanisms

using proteomic approaches in cell cultures, animal models, and human tissues
(Klein et al. 2011; Prunotto et al. 2011). Many proteomic methods utilize techniques
for protein fractionation and mass spectrometry (Konvalinka et al. 2010; Sethi
et al. 2013). Proteomic methods can potentially be used on a variety of specimens,
including urine, to provide noninvasive assessment of renal fibrosis. Current prote-
omic work has mainly explored in vitro systems. Databases, specific for normal and
pathological human kidney proteomes, are available including normal glomeruli and
medulla (e.g., http://www.hkupp.org/ and http://www.proteinatlas.org/
humanproteome/kidney) (Habuka et al. 2014). However, despite the recent technical
advancements and studies, proteomic biomarkers have not yet found direct impli-
cations in clinic (Konvalinka et al. 2010; Sethi et al. 2013).
Current techniques have specific shortcomings when it comes to IFTA evalua-
tion as the whole kidney proteomic analysis does not provide accurate information
regarding localization (Walker et al. 2004), and, therefore, each kidney compart-
ment should be separated prior to proteomic analysis (such as with laser capture
microdissection). Still, the minute amount of available tissue severely limits
exploration.
Fibrosis Measurement and Methodologies: Challenges

and Promises
The measurement of IF has limitations including sampling issues and variable

quality and quantity of the IF. Overall, there is no consensus regarding the best
way to assess IF (Farris et al. 2014; Haas et al. 2014). Unfortunately, there is a lack of
consistency in measuring fibrosis among pathologists, which stems from different
conceptual ways of considering the percentage of fibrosis. Some pathologists con-
sider IF percentage as the percent of overall tissue occupied by fibrous matrix,
whereas others consider the area containing both fibrotic matrix and intact glomeruli
and tubular structures. This is depicted in the Fig. 4. This discrepancy was
highlighted in an interobserver variability study among numerous pathologists in
multiple countries by Furness et al. (2003), and a recent study prompted by the Banff
Allograft Pathology Conference also showed a great deal of variability in fibrosis
assessment among pathologists (Farris et al. 2014). Discrepancies among patholo-
gists involve the threshold of matrix needed in order to identify a region as being
involved by fibrosis. Therefore, special effort should be made to overcome this
caveat by constant education and unifying the definition.

Conditions
Fibrosis assessment is important in the kidney and also other organs because it
provides a surrogate marker of chronic injury. IFTA assessment is important in the
kidney in particular because it shows correlations with renal function (Farris
et al. 2014; Farris and Alpers 2014). Some investigators distinguish conventional
fibrosis from “sclerosis” since sclerosis may represent a late “hardened” stage at
which fibrosis is more chronic in nature and thus be pathologically distinct. It is
likely that future method refinements will help recognize later stages of fibrosis and
also identify stages at which fibrosis may be reversible (Farris and Colvin 2012;
Farris and Alpers 2014), and intervention may be possible at earlier stages of fibrosis
(Friedman et al. 2013). Furthermore, fibrosis assessment is useful in a variety of
other diseases including pulmonary fibrosis, liver fibrosis (cirrhosis), nephrogenic
systemic fibrosis, systemic sclerosis, wound healing, and cancer (Farris and Colvin
2012; Friedman et al. 2013; Farris and Alpers 2014; Rybinski et al. 2014). Therefore,
although this review has focused mostly on the kidney, it is likely that the kidney can
serve as a window to view other organs and disease processes and thus improve the
lives for patients (Friedman et al. 2013).
Conclusion
IFTA is brought about through complex molecular mechanisms of renal injury.

Fibrosis can be assessed in a number of ways. Efforts to improve these methods
could lead to improvements in the surrogate measures provided by pathologists,
making pathologic assessment a better biomarker of renal disease, and molecular
approaches including transcriptomics and proteomics will further complement these
assessment methods. Assessing fibrosis, a type of -osis or state of the biologic
system, can eventually help provide an -omic measure that can be correlated with
other -omic measures (e.g., genomics/transcriptomics, proteomics, metabolomics,
etc.). Going from the assessment of the -osis can eventually lead to better assessment
of -omics. In this sense, fibrosis assessment can act as a biomarker. Ultimately, it is
hoped that better assessment will facilitate the development of methods to ameliorate
fibrosis and improve patient outcomes (Friedman et al. 2013).
Summary Points
• This chapter focuses on renal fibrosis, which is the pathologic accumulation of

ECM proteins following tissue injury.
• IF is accompanied by TA, and these are collectively referred to as interstitial
fibrosis and tubular atrophy (IFTA).
• IFTA is shown to have prognostic value in chronic kidney diseases.
• Renal biopsy can be used to diagnose and quantitate IFTA and further prognos-
ticate renal function.
• A variety of staining techniques are useful in assessing the severity of IF,
including trichrome, picrosirius red, and collagen III immunohistochemistry.
• Molecular methods such as transcriptional profiling and proteomics can provide
surrogate measures of IFTA.
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Next-Generation Sequencing (NGS)
in Biomarker Discovery and Applications 42
in Nephrology
Imari Mimura and Masaomi Nangaku
Contents
Key Facts of ChIP-seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 957
Key Facts of RNA-seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 957
Key Facts of RIP-seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 958
Key Facts of 3C and Hi-C Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 958
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 959
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 959
ChIP-seq for Genome-Wide Analysis in Renal Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 960
Applications of ChIP-seq in Kidney Tissue Samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 960
Identification of Targets for Nuclear Hormone Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 960
ChIP-seq Analysis in the Developmental Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 961
ChIP-seq Applications for Metabolic Memory and Legacy Effect . . . . . . . . . . . . . . . . . . . . . . . . . 961
Technical Evolution of ChIP-seq for In Vivo Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 962
Traditional Method for ChIP-seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 962
PAT-ChIP-seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 963
RNA-seq and Applications for Kidney Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 964
Technical Method for RNA-seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 964
Applications of RNA-seq for Kidney Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 965
Quartz-seq for Identifying a Single-Cell RNA-seq Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 966
New Methods for Revealing RNA-Binding Proteins, RIP-seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 966
Technical Method for RIP-seq . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 966
Application of RIP-seq in the Research Fields . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 967
High-Resolution Circular Chromosome Conformation Capture Technology (3C, 4C,
5C, and Hi-C) Methods for Identifying Genome-Wide Chromosome Conformations . . . . . . . . 969
Technical Method of 3C, 4C, 5C, and Hi-C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 969
Applications of 3C or Hi-C in Research Fields Including Nephrology . . . . . . . . . . . . . . . . . . . . 971
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 972
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 972
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 973
I. Mimura • M. Nangaku (*)

Division of Nephrology and Endocrinology, The University of Tokyo, Tokyo, Japan
e-mail: imimura-tky@umin.ac.jp; mnangaku-tky@umin.ac.jp; mnangaku@gmail.com

956 I. Mimura and M. Nangaku
Abstract
Next-generation sequencing (NGS) has been rapidly evolved in these 10 years.
The practical use of high-throughput sequencers makes it possible to identify the
localization of epigenetic modifications in detail. Recent technologies including
ChIP-seq and RNA-seq allowed demonstration of protein-DNA bindings or
splicing variants even with a small number of cells. These technologies have
great potential in a wide range of renal diseases because only a few amounts of
human renal biopsy samples can be harvested. In addition these techniques have
been adapted to a variety of tissues in different model organs. PAT-ChIP (pathol-
ogy tissue-ChIP)-seq protocol on freshly isolated mouse embryonic kidneys can
be used for in vivo analysis of transcriptional factor recruitment on chromatin.
RIP-seq can be used to analyze the RNA-binding proteins on genome-wide scale.
Chromosome conformation capture (3C) assay makes tremendous progress into
Hi-C which can detect genome-wide long interactions on chromosomes which
have cell-type specificity. It is important to catch up with the speed of technical
development and make use of these tools in order to understand the epigenetic
mechanisms systematically.
Keywords
ChIP-seq • RNA-seq • RIP-seq • Chromosome conformation capture assay •
Epigenetics
Abbreviations
3C Chromosome conformation capture
4C-seq Circular chromosome conformation capture
AGO2 Argonaute 2
AR Androgen receptor
ChIP-seq Chromatin immunoprecipitation-sequencing
DCCT/EDIC Diabetes Control and Complications Trial/Epidemiology of Dia-
betes Interventions and Complications
DCR2 Dicer 2
Dpc Day post-coitum
Ezh2 Enhancer of zeste 2 polycomb repressive complex 2 subunit
FFPE Formalin-fixed paraffin-embedded
GLUT3 Glucose transporter 3
H3K27me3 Lysine 27 trimethylation
H3K4m3 Lysine 4 trimethylation
H3K9me3 Lysine 9 trimethylation
Hi-C High-resolution circular chromosome conformation capture
HIF-1 Hypoxia-inducible factor-1
HK-2 Human kidney-2
HUVECs Human umbilical vein endothelial cells
KDM3A Lysine-specific demethylase 3A
KLF1 Kruppel-like factor
42 Next-Generation Sequencing (NGS) in Biomarker Discovery and. . . 957
mDCT Murine distal convoluted tubular epithelial cell line

MM Metanephric mesenchyme
MR Mineralocorticoid receptor
PAT-ChIP Pathology tissue-ChIP
piRNA PIWI-interacting RNA
PRC2 Polycomb repressive complex 2
pre-miRNAs Precursor miRNAs
pri-miRNAs Primary miRNAs
RBP RNA-binding protein
RISC RNA-induced silencing complex
SLC2A3 Solute carrier family 2A3
Suv39h1 Suppressor of variegation 3–9 homolog 1
VSMC Vascular smooth muscle cells
Key Facts of ChIP-seq
• Recently the number of genome-wide analysis such as ChIP-seq has increased

because of their technological progress and a reduction in costs.
• ChIP-seq can be used in order to analyze the mechanisms of kidney disease and
development of the kidney.
• Long-term effects such as metabolic memory and legacy effect may be mediated
by epigenetic changes, and ChIP-seq is a powerful tool to study epigenetic
modifications.
• Advanced technology of ChIP-seq called PAT-ChIP makes it possible to analyze
the kidney tissue samples for in vivo analysis.
• The improved PAT-ChIP protocol coupled with laser capture microdissection can
be used for discovery and validation of novel epigenetic factors in human samples.
Key Facts of RNA-seq
• RNA-seq is used for showing genome-wide gene expressions and gene variants.
• RNA-seq data for 234 renal clear cell carcinoma patients clarified that both gene
and isoform expression signatures are useful for distinguishing cancer stages and
that they help to identify advanced stage cancers, predict clinical outcome, and
present a comprehensive view of cancer development and progression.
• Advanced technique for RNA-seq is a single-cell RNA-seq, which can profile
cell-to-cell variability on a genomic scale.
• Making use of this technique, we can identify the difference between tubuloin-
terstitial cells and fibrotic cells or mesangial cell, endothelial cells, and podocytes
in the glomerulus.
• RNA-seq using total mouse kidneys at E11.5 and E12.5 as well as the renal
vesicles at P4 identified a large number of genes with partially degraded
noncoding RNA. It was also found that single cells at early developmental times
often expressed genes related to several developmental pathways, providing
powerful evidence that initial organogenesis involves a process of multilineage
priming.
Key Facts of RIP-seq
• RIP-seq can analyze the RNA-binding proteins on genome-wide scale.

• RIP-seq using AGO2 antibody demonstrated that AGO2 is strongly enriched in
small RNAs that encompass the promoter regions and other regions on both the
sense and antisense DNA strands.
• A new method of RIP-seq in combination with northern blotting identified
various types of small RNAs associated with the BmAgo2 protein as well as
miRNAs and piRNAs.
• It can examine the miRNA-mRNA associations under specific conditions and
find novel putative miRNA targets.
• RIPSeeker, a free open-source for de novo RIP peak predictions, demonstrates
superior sensitivity and specificity in discriminating high-confidence peaks.
Key Facts of 3C and Hi-C Assay
• Chromosome conformation capture (3C) assay has recently been developed in

order to clarify a conformational proximity between promoter and a distal
enhancer.
• A study of human genome identified thousands of significant long-range looping
interactions between gene promoters and distal loci, revealing that gene pro-
moters engage with distal elements through looping.
• We performed ChIP-seq of HIF-1 (hypoxia-inducible factor-1) and showed that
HIF-1 and KDM3A (lysine-specific demethylase 2A3) cooperatively upregulate
the downstream target gene, SLC2A3 (solute carrier family 2A3), via removal of
suppressive histone marks and chromosome conformational change under
hypoxia.
• In response to glucose, 4C-seq identified the contact in human islets between
insulin (INS) promoter and the calcium-activated chloride channel ANO1 gene.
The contact was strengthened and the expression of ANO1 increased. It is
demonstrated that networks of long-range physical contacts are important to the
regulation of insulin metabolism.
• 3C-based techniques have evolved into Hi-C (high-resolution circular chromo-
some conformation capture assay) at a rapid speed and make it possible to analyze
the long-range interactions on multiple chromosomes.

Conditions
We can use NGS to analyze the genome-wide sequencing of patients with diseases
and find novel epigenetic factors which are associated with the diseases not only in
nephrology but also in other fields. In addition we can analyze and compare the
relationships between survival data and genome sequencing. We can predict the
prognosis of diseases using the data of NGS coupled with survival data in the
future.
Definitions
ChIP-seq Genome-wide sequencing analysis of chromatin immunoprecipitation

using high-throughput analyzers.
Hi-C High-resolution circular chromosome conformation capture based on 3C

assay.
Legacy Effects The intensive treatment of blood glucose leads to delaying pro-
gression of diabetic nephropathy.
Metabolic Memory Early metabolic control affects a beneficial and long-term

influence on the clinical outcome of diabetic complications.
PAT-ChIP Pathology tissue-ChIP methods to extract and immunoprecipitate chro-

matin from paraffin-embedded samples.
Quartz-seq New technique which can detect different cell types and different cell
cycle phases of a single-cell type.
RIP-seq Genome-wide sequencing analysis of RNA-binding proteins.
RNA-seq Genome-wide sequencing analysis of total RNA or mRNA.
Introduction
Progression of high-throughput genome sequencers in the early years of the twenty-

first century and the determination of human genome in 2003 promotes the genome-
wide analysis of many kinds of species. These results clarify that the expressions of
genome information are affected intricately not only by the sequences of bases but
also by methylation of DNA, modifications of histones which consist of chromatin in
the nucleus, and small nucleic acids such as microRNA (Tsukada et al. 2006).
The practical use of high-throughput sequencers recently makes it possible to

identify the localization of epigenetic modifications in detail. Increasing number of
recent papers has demonstrated the roles of epigenetic mechanisms for embryogen-
esis, cell differentiation, genome imprinting, inactivation of X chromosome, and
aging (Ware et al. 2009). Epigenetic changes are accumulated as a memory in a cell,
and epigenetic memory results in the cancer, congenital disorders, and adult-onset
diseases (Mimura et al. 2011, 2013). However, molecular mechanisms of transcrip-
tional factors and epigenetic modifications still remain to be solved.
ChIP-seq for Genome-Wide Analysis in Renal Diseases
Applications of ChIP-seq in Kidney Tissue Samples
ChIP-seq has been widely used in fields related to nephrology as shown below. Sun
et al. have performed ChIP-seq experiments using RNA polymerase II antibody in
five adult mouse tissues including kidney (Gupta et al. 2010; Sun et al. 2011). They
clarified that 6,384 promoters are tissue specific among 12,270 novel promoters.
ChIP-seq analysis of RNA polymerase II made it possible to identify the novel
tissue-specific promoters.
The kidney is composed of various different types of cells, and cell type-specific
analysis is desirable. Recent technologies allowed demonstration of protein-DNA
bindings using ChIP-seq with a small number of cells (Furey 2012). This technology
can be used for a wide range of renal diseases.
Identification of Targets for Nuclear Hormone Receptors
Recently the number of genome-wide analysis such as ChIP-seq has increased

because of their rapid progress and prevalence. AR (androgen receptor) binds
male sex steroids and other mediators and induces physiological androgen
actions. Pihlajamaa et al. performed ChIP-seq analysis of AR-binding sites in
murine kidney and epididymis (Pihlajamaa et al. 2014). They found two novel
collaborating factors for AR signaling in vivo, Hnf4α (hepatocyte nuclear factor
4α) in the kidney and AP-2α (activating enhancer-binding protein 2α) in the
epididymis. They clarified that these factors are constitutively bound to chroma-
tin and guide AR to specific genome loci on hormone exposure. Ueda et al. also
demonstrated the genome-wide distribution of MR (mineralocorticoid receptor)
using mDCT (murine distal convoluted tubular epithelial cell line) (Ueda
et al. 2014). MR is a member of nuclear receptor family proteins and contributes
to fluid homeostasis in the kidney. They performed ChIP-seq and microarray
analysis using DNA and mRNA samples of mDCT cells overexpressing
3FLAG-hMR after treatment with aldosterone. Twenty-five genes were indi-
cated as the candidate targets of MR by ChIP-seq and microarrays. Four genes
such as Sgk1, Rasl12, Tns1, and Tsc22d3 were validated as the direct targets of
MR by ChIP-qPCR.
ChIP-seq Analysis in the Developmental Kidney
Li et al. analyzed the genome-wide binding sites of the p53 gene in the embryonic
kidney (Li et al. 2014). They found that the p53-regulated transcriptome is largely
composed of genes regulating developmental, morphogenesis, and metabolic path-
ways. They demonstrated that 78 % of p53 peaks in the developing kidney lie within
the proximal promoters of annotated genes and that 25 % of the differentially
expressed p53-bound genes are present in nephron progenitors and nascent neph-
rons, including components of Fgf, Wnt, Bmp, and Notch pathways. They showed
the comprehensive analysis of the p53 transcriptome and cistrome in a developing
mammalian organ for the first time.
The metanephric mesenchyme (MM) is known to give rise to nephrons. The MM is
composed of uninduced (Six2, high; Lhx1, low) and induced (Wnt stimulated, Six2,
low; Lhx1, high) cells. McLaughlin et al. performed ChIP-seq analysis using
uninduced (mK3) and induced (mK4) metanephric mesenchyme (McLaughlin
et al. 2013). mK3 cells express genes characteristic of early mesenchyme, not epithe-
lial progenitor genes, while mK4 cells resemble induced MM undergoing epithelial
conversion. ChIP-seq revealed that H3K4me3 active region peaks are enriched in
metabolic genes and that H3K27me3 peaks, histone repressive marks, are enriched
mesenchyme and epithelial cell fate commitment genes. They further demonstrated
that one of histone methyltransferases, G9a, occupies the promoter region of Six2 in
induced cells. Stimulation of Wnt signaling in uninduced cells provokes an active
chromatin state (H3K4m3 (lysine 4 trimethylation), high; H3K27me3 (lysine
27 trimethylation), low), recruitment of β-catenin, and loss of prebound Ezh2 (one
of histone methyltransferases). As shown above, ChIP-seq analysis revealed that the
chromatin signature correlates strongly with their gene expression states.
Renal hypoplasia is a congenital reduction in nephron number. It is a predisposing
factor for chronic kidney disease and hypertension. Saifudeen et al. examined whether
p53 and Pax2 cooperate in nephrogenesis by using mice with germ line p53 deletion
(Saifudeen et al. 2012). Mice or humans with germ line heterozygous mutations in
Pax2 exhibit renal hypoplasia. They performed ChIP-seq of p53 and clarified that peaks
of p53 occupancy in chromatin regions of the Pax2 promoter and gene in embryonic
kidneys. They also demonstrated that p53 binding to Pax2 gene is significantly more
enriched in Pax2-expressing than non-expressing MM cells. They suggested that the
cross talk between p53 and Pax2 may provide a promotion of nephrogenesis.
ChIP-seq Applications for Metabolic Memory and Legacy Effect
Epidemiological and clinical data support the idea that early metabolic control
affects a beneficial and long-term influence on the clinical outcome of diabetic
complications. This phenomenon has recently been called as “metabolic memory.”

In addition, large clinical studies showed that the intensive treatment of blood
glucose leads to delaying progression of diabetic nephropathy (Writing Team for
the Diabetes, Complications Trial/Epidemiology of Diabetes et al. 2003; Holman
et al. 2008). It is called “legacy effect.” Strict control for blood glucose levels in type
1 diabetes patients was effective in inhibition of microalbuminuria and microvascu-
lar complications in DCCT/EDIC study (Writing Team for the Diabetes, Complica-
tions Trial/Epidemiology of Diabetes et al. 2003). Patients with type 2 diabetes
mellitus who receive intensive glucose therapy also had a lower risk of microvas-
cular complications (Holman et al. 2008). In addition, de Boer et al. demonstrated
that the albumin excretion rate came out to be controlled and that GFR remained in
the normal range with type 1 diabetes mellitus patients during DCCT/EDIC study
through 10 years of follow-up (de Boer et al. 2014).
It is natural to presume that this long-term effect such as metabolic memory and
legacy effect may be mediated by epigenetic changes and that deep sequencing such
as ChIP-seq is a powerful tool to study epigenetic modifications. To support this
point of view, ChIP-seq analysis demonstrated hyperglycemia-mediated induction of
genes through modulation of acetylated H3K9/K14 (Pirola et al. 2011). They
performed ChIP-seq of H3K9/K14 and CpG methylation assays followed by mas-
sive parallel sequencing (CpG-seq) using primary cultured vascular cells. They
compared the ChIP-seq and CpG-seq and analyzed them and clarified that modula-
tion of acetylated H3K9/K14 is inversely correlated with methyl-CpG content.
Villeneuve et al. demonstrated that H3K9me3 (lysine 9 trimethylation) plays an
important role in metabolic memory (Villeneuve et al. 2008). H3K9me3 levels were
decreased at the promoters of inflammatory genes in cultured db/db vascular smooth
muscle cells (VSMC) relative to control db/+ cells. One of the H3K9me3
transmethylferases, Suv39h1 (suppressor of variegation 3–9 homolog 1), was also
reduced in db/db VSMC and overexpression of SUV39H1 in db/db VSMC reversed
diabetic phenotype. These results demonstrate the protective roles for H3K9me3 and
Suv39h1 against the pre-activated state of diabetic VSMC. The mechanism of
metabolic memory needs further research using deep sequencing.
Technical Evolution of ChIP-seq for In Vivo Analysis
Traditional Method for ChIP-seq
The traditional method for ChIP-seq is indicated in Fig. 1 (Mimura et al. 2014). First,
we cross-link cultured cells in the dishes by using paraformaldehyde (Step 1). Next,
the cell lysate is homogenized and sonicated into fragments. The antibody is attached
with sepharose beads (Step 2). Then we immunoprecipitate the cell lysate and the
antibody with beads (Step 3). Then, we reverse cross-linking at 65 C incubation.
And DNA is purified by ethanol precipitation (Step 4). Finally, we use immunopre-
cipitated DNA for high-throughput sequencing (Step 5).
Step1
Paraformaldehyde fixation (10-20min)
Step2 Cell Lysis Antibody Beads
Step3 Immunoprecipitation
Step4 DNA Purification
Step5 High-throughput sequencers
Fig. 1 ChIP-seq methods. Step 1: First we cross-link cultured cells in the dishes by using
paraformaldehyde for 10–20 min. Step2: The cell lysis is homogenized and sonicated into frag-
ments. The antibody is attached with sepharose beads. Step3: We immunoprecipitate the cell lysis
and antibody with beads for overnight at 4 C. Step 4: After we reverse cross-linking at 65 C
incubation, DNA is purified by ethanol precipitation. Step5: Immunoprecipitated DNA can be used
for high-throughput sequencers
PAT-ChIP-seq
Recently this technique has been adapted to a variety of tissues in different model
organs. Heliot et al. demonstrated the ChIP protocol on freshly isolated mouse
embryonic kidneys for in vivo analysis of transcription factor recruitment on chro-
matin (Heliot and Cereghini 2012). Fanelli et al. developed a methodology called
PAT-ChIP (pathology tissue-ChIP) to extract and immunoprecipitate chromatin from
paraffin-embedded patient samples (Fanelli et al. 2010). The brief protocol of
PAT-ChIP is shown in Fig. 2. First, we perform deparaffinization and rehydration
of formalin-fixed paraffin-embedded (FFPE) samples (Step 1). After we homogenize
the cell lysate, MNase is used for the digestion of cell lysis buffer (Step 2). We
sonicate the cell lysate into fragments by using sonicator (Step 3). The antibody is
needed to react with sepharose beads. Then, we immunoprecipitate the cell lysate
with antibody for overnight (Step 4). Finally, we purify DNA fragment after
reversing cross-linking (Step 5). They showed that PAT-ChIP can be coupled with
high-throughput sequencing (PAT-ChIP-seq) for the genome-wide analysis of dis-
tinct chromatin modifications (Fanelli et al. 2011). They developed the new method
for PAT-ChIP-seq coupled with laser capture microdissection (Amatori et al. 2014).
Step1 Deparaffination and rehydration of FFPE (formalin-fixed paraffin-embedded) samples
Step2 Homogenization and Mnase digestion

Mnase
Antibody Beads
Step3 Sonication
Step4 Immunoprecipitation
Step5 DNA purification
Fig. 2 PAT-ChIP methods. Step 1: We perform deparaffination and rehydration of FFPE (formalin-
fixed paraffin-embedded) samples. Step 2: After we homogenize the cell lysis, MNase is used for
the digestion of cell lysis buffer. Step 3: We sonicate the cell lysis into fragments by using sonicator.
The antibody is needed to react with sepharose beads. Step 4: We immunoprecipitate the cell lysis
with antibody for overnight. Step 5: We purify DNA fragment after reversing cross-linking
The improved PAT-ChIP protocol can be used for discovery and validation of novel
epigenetic factors in human samples.
RNA-seq and Applications for Kidney Diseases
Technical Method for RNA-seq
As shown in Fig. 3, RNA samples for RNA sequencing are prepared as total RNA or
mRNA (Step 1). The cDNA library is constructed using reverse transcription (Step
2). Adaptors are attached to both ends of cDNA library (Step 3). One fragment is
sequenced with or without amplification in a high-throughput sequencer to obtain
short sequences from one (single end) or both ends (pair end). The resulting reads are
aligned to a reference genome or reference transcripts to produce a genome-scale
transcription map (Step 4). As shown in step 4, longer reads or pair-end reads reveal
connectivity between multiple exons. RNA-seq is useful for revealing the complex
Step1 Total RNA or mRNA

AAAAAA
Step2 Reverse transcribed cDNA

AAAAAA
TTTTTT
Step3 Ligate sequence adaptors

Forward primer
Reverse primer
Step4 Sequence reads Exonic reads

Junction reads
Poly A end reads
exon1 exon2 exon3
AAAAAA
AAAAAA
Fig. 3 RNA-seq methods. Step 1: RNA samples for RNA sequences are prepared for total RNA or
mRNA. Adaptors need to be added on both sides of them with poly (A). Step 2: The cDNA library
is constructed using reverse transcription. Step 3: RNA is converted to a library of cDNA fragments
with adaptors attached to one or both ends. Step 4: One fragment is sequenced with or without
amplification in high-throughput sequencers to obtain short sequences from one (single end) or both
ends (pair end). The resulting reads are aligned to a reference genome or reference transcripts to
produce a genome-scale transcription map
transcriptomes (Yae et al. 2012). In addition, RNA-seq can also demonstrate

sequence variations in the transcribed regions (Mimura et al. 2014).
Applications of RNA-seq for Kidney Diseases
RNA-seq is used for showing genome-wide gene expressions and gene variants.
Liu et al. analyzed gene expressions and isoform levels of RNA-seq data for
234 renal clear cell carcinoma patients (Liu et al. 2013). They found that both
gene and isoform expression signatures are useful for distinguishing cancer stages
and that they help to identify advanced stage cancers, predict clinical outcome, and
present a comprehensive view of cancer development and progression. Brunskill
and Potter performed RNA-seq using cap mesenchyme progenitors and renal
vesicles, identified hundreds of novel splice patterns and new genes, and clarified
the RNA processing complexities of the Hox clusters (Brunskill and Steven Potter
2012).
Tallack et al. identified novel target genes of KLF1 (Kruppel-like factor) by

RNA-seq from erythroid tissue (Tallack et al. 2012). They clarified two novel long
noncoding RNAs that are dynamically expressed during erythroid differentiation.
These results suggest that we can find novel functional molecules in the kidney using
RNA-seq in vivo. RNA-seq was also helpful in detecting a new fusion of chromo-
somes from individuals diagnosed with Ewing sarcoma. Pierron discovered a novel
fusion between BCOR (encoding the BCL6 corepressor) and CCNB3 (encoding the
testis-specific cyclin B3) on the X chromosome (Pierron et al. 2012). Sung found
that recurrent HBV integration occurred at the TERT, MLL4, and CCNE1 genes,
which showed upregulated expression, and that this was observed more frequently
by RNA-seq of tumors (Sung et al. 2012). Thiagarajan et al. performed RNA-seq and
compared the results with preexisting microarray datasets using 15.5 dpc (day post-
coitum) embryonic mouse kidney (Thiagarajan et al. 2011). The resolution of
RNA-seq provides the basis for a transition from classical gene-centric models of
kidney development.
Quartz-seq for Identifying a Single-Cell RNA-seq Method
A new technique named Quartz-seq has been recently developed (Sasagawa

et al. 2013). It is a single-cell RNA-seq method. Sasagawa et al. developed the
new technique which can detect different cell types and different cell cycle phases of
a single-cell type. Making use of this technique, we can identify the difference
between tubulointerstitial cells and fibrotic cells or mesangial cell, endothelial
cells, and podocytes in the glomerulus. Brunskill et al. created an atlas of gene
expression patterns in the developing kidney by using a single-cell RNA-seq
strategy (Brunskill et al. 2014). They performed RNA-seq using total mouse kidneys
at E11.5 and E12.5 as well as the renal vesicles at P4. They identify a large number
of genes with partially degraded noncoding RNA. They also found that single cells
at early developmental times often expressed genes related to several developmental
pathways, providing powerful evidence that initial organogenesis involves a process
of multilineage priming. As shown in this paper, a single-cell RNA-seq must be a
breakthrough in order to analyze the expression of specific types of kidney cells
because human samples can be harvested with a very small amount, leading to a
limitation of analyzing the data.
New Methods for Revealing RNA-Binding Proteins, RIP-seq
Technical Method for RIP-seq
RIP-seq is firstly developed to capture the polycomb repressive complex 2 (PRC2)

transcriptome and identify a genome-wide pool of more than 9,000 PRC2-
interacting RNAs in embryonic stem cells in 2010 (Zhao et al. 2010). They demon-
strated the direct RNA-protein interaction via Ezh2 (enhancer of zeste 2 polycomb
repressive complex 2 subunit) subunit and identified Gtl2 RNA as a PRC2 cofactor.
The method is shown in Fig. 4. To construct RIP-seq libraries, cell nuclei are
isolated, and nuclear lysates are prepared with DNAse. Cell lysate is incubated
with RBP (RNA-binding protein) antibody or control IgG with agarose beads
(Step 1). RNA-protein complexes are immunoprecipitated with beads (Step 2) and
RNA are extracted using RNA purification processes (Step 3). Purified RNAs are
used for high-throughput sequencers using the same procedures of RNA-seq (Step 4)
(Jayaseelan et al. 2014).
Application of RIP-seq in the Research Fields
Small RNAs such as miRNA, siRNA, and piRNA have been known to play roles as
a silencer of mRNA or retrotransposons, which is called “RNA silencing.” piRNA
(PIWI-interacting RNA) comes mainly from retrotransposon and binds to a subset of
PIWI proteins. piRNA functions as a guide molecular for targeted RNA which has
complementary sequences. A set of Argonaute proteins binds to small RNAs and
plays important roles in RNA silencing. Argonaute protein is one of RNA-binding
Step1 Cell Lysis

RBP (RNA-binding protein)
mRNA
Beads Antibody for RBP
Immunoprecipitation
Step2
Precipitate specific mRNA with RBP
Step3 Purification of mRNA
Step4 RNA-seq procedures
Fig. 4 RIP-seq methods. Step 1: Cell nuclei are isolated, nuclear lysates are prepared with DNAse.
Cell lysate is incubated with RBP (RNA-binding protein) antibody or control IgG with agarose
beads Step 2: RNA-protein complexes are immunoprecipitated with beads. Step 3: RNA are
extracted using RNA purification processes. Step 4: Purified RNA are used for high-throughput
sequencers using the same procedures of RNA-seq
proteins, which have PIWI domains. miRNAs are processed as shown in Fig. 5.
miRNAs are transcribed by RNA polymerase II (Pol II) into the primary miRNAs
(pri-miRNAs). Pri-miRNAs are processed by RNase III Drosha into precursor
miRNAs (pre-miRNAs). Pre-miRNAs are exported by the nuclear export factor,
Exportin 5, into the cytoplasm. In the cytoplasm the pre-miRNAs are processed by
Dicer, another RNase III, into mature miRNAs. The miRNA strand is incorporated
into the RISC (RNA-induced silencing complex) and target complementary mRNA.
Targeted mRNA receives degradation or translational repression.
Because RIP-seq is a recently developed technique, there are a few papers using
RIP-seq. We do not have any report of RIP-seq in the field of the kidney so far.
Cernilogar et al. performed RIP-seq using AGO2 antibody and demonstrated that
AGO2 is strongly enriched in small RNAs that encompass the promoter regions and
other regions of heat shock and other genetic loci on both the sense and antisense
DNA strands (Cernilogar et al. 2011). They showed that DCR2 (Dicer 2) and AGO2
are globally associated with transcriptionally active loci and have a pivotal role in
shaping the transcriptome by controlling the processivity of RNA polymerase II.
Transcription
Pri-miRNA
Drosha
Processing
Pre-miRNA
Nucleus
Exportin
Cytoplasm
¸ Translational repression
¸ mRNA degradation
Dicer
processing
AGO2 RISC
RISC
AAAAA
miRNA Target mRNA
AGO2: Argonaute 2
RISC: RNA induced silencing complex
Fig. 5 miRNAs processing in the nucleus and the cytoplasm. miRNAs are transcribed by RNA
polymerase II (Pol II) into the primary miRNAs (pri-miRNAs). Pri-miRNAs are processed by
RNase III Drosha into precursor miRNAs (pre-miRNAs). Pre-miRNAs are exported by the nuclear
export factor, Exportin 5, into the cytoplasm. In the cytoplasm the pre-miRNAs are processed by
Dicer, another RNase III, into mature miRNAs. The miRNA strand is incorporated into the RISC
(RNA-induced silencing complex) and target complementary mRNA. Targeted mRNA receives
degradation or translational repression
Lu et al. developed a multi-targeting RIP-seq strategy to identify Sm-associated

RNAs from Drosophila ovaries and cultured human cells (Lu et al. 2014). Sm
proteins are multimeric RNA-binding factors, and they discovered three major
categories of Sm-associated transcripts: small nuclear (sn) RNAs, small Cajal
body (sca) RNAs, and mRNAs. They also showed both ubiquitous and tissue-
specific interactions. Nie et al. developed a new method of RIP-seq in combination
with northern blotting and identified various types of small RNAs associated with
the BmAgo2 protein as well as miRNAs and piRNAs (Nie et al. 2013). Kanematsu
et al. screened for miRNA-mRNA associations using RIP-seq in colon cancer cell
lines (Kanematsu et al. 2014). They also examined the miRNA-mRNA associations
under hypoxic condition which included several well-characterized cancer-related
genes as novel putative miRNA targets.
Meier et al. performed RIP-seq by using HEK293T cells with stable ectopic
expression of miR-155 (Meier et al. 2013). They found 100 AGO2-associated
mRNAs in miR-155-expressing cells and indicated that these targets were either
regulated by mRNA decay or by translational repression. Li et al. developed
RIPSeeker, a free open-source for de novo RIP peak predictions (Li et al. 2013).
RIPSeeker demonstrates superior sensitivity and specificity in discriminating high-
confidence peaks.
High-Resolution Circular Chromosome Conformation Capture

Technology (3C, 4C, 5C, and Hi-C) Methods for Identifying
Genome-Wide Chromosome Conformations
Technical Method of 3C, 4C, 5C, and Hi-C
Chromosome conformation capture (3C) assay has recently been developed to

clarify a conformational proximity between promoter and a distal enhancer. Dekker
developed the 3C assay to detect the frequency of interaction between any two
genomic loci (Dekker et al. 2002). As shown in Fig. 6, firstly intact nuclei are
isolated, and cells are subjected to formaldehyde fixation, which cross-links proteins
to other proteins and to DNA (Step 1). For quantification of cross-linking frequen-
cies, cross-linked DNA is digested with a restriction enzyme (Step 2). Then cross-
linked DNA is subjected to ligation at very low concentration (Step 3). Cross-linking
is reversed and individual ligation products are detected and quantified by the
quantitative PCR (3C) using locus-specific primers or DNA microarray (4C) (Step
4). In Table 1, 3C-based methods were summarized. 3C or 4C generates single
interaction profiles for individual loci (Dekker et al. 2013). While 3C typically yields
a long-range interaction of a selected gene promoter, 4C generates a genome-wide
interaction profile for a single locus. 5C and Hi-C methods are not anchored on a
single locus of interest but instead generate matrices of interaction frequencies as
two-dimensional heat maps with genomic positions along the two axes. 5C com-
bines 3C with hybrid capture approaches to identify millions of interactions in
parallel between two large sets of loci. Hi-C method is genome-wide adaptation of
Step1 Step2 Step3 Step4

Formaldehyde Restriction
Fixation Ligation Detection by
Enzyme Cut
qPCR ( C) or
Reverse DNA microarray
Digestion crosslink (C )
Restriction enzyme
cut sites
Fig. 6 3C or 4C methods. Step 1: Intact nuclei are isolated and cells are subjected to formaldehyde
fixation, which cross-links proteins to other proteins and to DNA. Step 2: For quantification of
cross-linking frequencies, cross-linked DNA is digested with a restriction enzyme. Step 3: Cross-
linked DNA is subjected to ligation. Step 4: Cross-linking is reversed and individual ligation
products are detected and quantified by the quantitative PCR (3C) using locus-specific primers or
DNA microarray (4C)
Table 1 Summary of 3C-based methods

Interactions Detection methods Image
3C One-by-one PCR or sequencing Gene Promoter
Interaction
frequency
Distance from prm

4C One-by-all Inverse PCR sequencing anchor
Numbers
of reads
5C Many-by-many Sequencing 5C map
Refseq Genes
Hi-C All-by-all Sequencing Hi-C interaction map
Chromosome
Interaction
enrichment
While 3C typically yields a long-range interaction of a selected gene promoter, 4C generates a

genome-wide interaction profile for a single locus. 5C and Hi-C methods are not anchored on a
single locus of interest but instead generate matrices of interaction frequencies as two-dimensional
heat maps with genomic positions along the two axes
3C and includes a unique step in which the DNA ends are filled in with biotinylated
nucleotides after restriction digestion. Hi-C could provide all-by-all genome-wide
interaction map.
Applications of 3C or Hi-C in Research Fields Including Nephrology
Because chromosome conformation capture assays have recently been developed,

there are few papers using these techniques in kidney research fields. In this
paragraph, we will check papers of other fields. A study of human genome identified
thousands of significant long-range looping interactions between gene promoters
and distal loci, revealing that gene promoters engage with distal elements through
looping (Sanyal et al. 2012). Many of the looping are cell type-specific interactions
between active gene promoters and distal elements. They also demonstrated that
relationships between genes and regulatory elements are far from exclusive: genes
can interact with multiple distal elements and that elements can interact with multiple
genes.
We demonstrated that the chromosome conformation can change under hypoxic
condition via HIF-1 (hypoxia-inducible factor-1) and one of histone demethylases,
KDM3A (lysine-specific demethylase 3A), using HUVECs (human umbilical vein
endothelial cells) by 3C assay (Mimura et al. 2012). We performed ChIP-seq of
HIF-1 and clarified that HIF-1 binds to not only promoter regions but also enhancer
regions. We showed that HIF-1 and KDM3A cooperatively upregulate the down-
stream target gene, SLC2A3 (solute carrier family 2A3; also known as GLUT3,
glucose transporter 3), via removal of suppressive histone marks and chromosome
conformational change. We also performed 3C assay under normoxic condition with
knockdown of HIF-1 by siRNA and clarified that HIF-1 contributes to the loop
formation under normoxia on the loci of SLC2A3.
Xu et al. performed 4C-seq (circular chromatin conformation capture) to identify
a physical contact in human islets between insulin (INS) promoter and the calcium-
activated chloride channel ANO1 gene (Xu et al. 2014). In response to glucose, this
contact was strengthened and the expression of ANO1 increased. They demonstrated
that networks of long-range physical contacts are important to the regulation of
insulin metabolism. Bhattacharya et al. demonstrated that chromatin-chromatin
interactions exist between upstream regulatory elements and the Lmo2 (Lim domain
only 2) promoter in erythroid cells using 3C experiments (Bhattacharya et al. 2012).
Lmo2 encodes a transcriptional cofactor critical for the development of hematopoi-
etic stem cells. They also showed that the interactions are absent from the kidney
where Lmo2 is transcribed at 12-fold lower levels. Binding of CTCF (CCCTC-
binding factor) and cohesin which support chromatin loops was found within the
distal regions of Lmo2 and proximal promoter of Lmo2, suggesting that these
intergenic transcripts play an important role in regulating Lmo2.
Kim et al. performed 3C assays using in vivo kidney samples (Kim et al. 2012).
Induction of HO-1 (heme oxygenase-1) is a beneficial response to tissue injury
including acute kidney injury. They confirmed that transcription factors such as
USF1/2, Jun B, Sp1, and CTCF were found to associate with regulatory regions of
the human HO-1 in the kidney following rhabdomyolysis by 3C in the formation of
chromatin looping in vivo. They also demonstrated hemin-inducible chromatin
looping between the intronic enhancer and the promoter region using HK-2
(human kidney-2), a renal epithelial cell line (Deshane et al. 2010). They also
showed that Sp1 small interfering RNA and mithramycin A, a Sp1 binding site
inhibitor, resulted in loss of the loop formation between the intronic enhancer and the
distal HO-1 promoter. These results also demonstrated molecular interactions that
underlie human HO-1 regulation in HK-2.
Cohesin and CTCF are known to be required for long-range interactions in
eukaryotic genomes (Lee and Iyer 2012). However, how local chromatin interactions
govern higher-order folding of chromatin fibers and the function of cohesion remain
poorly understood. Mizuguchi et al. performed Hi-C analysis to explore the high-
resolution organization of Schizosaccharomyces pombe genome (Mizuguchi
et al. 2014). They revealed that heterochromatin mediates chromatin fiber compac-
tion at centromeres and promotes prominent inter-arm interactions within
centromere-proximal regions.
Conclusions
Genome-wide analysis using high-throughput sequencing has become more avail-

able in various kinds of research fields because of their technological progress and a
reduction in costs. ChIP-seq, RNA-seq, RIP-seq, and chromosome conformation
capture (3C) techniques are very useful tools for analyzing and clarifying the
epigenetic molecular mechanisms. Advanced technology of ChIP-seq makes it
possible to analyze the kidney tissue samples for in vivo analysis. Advanced
technique for RNA-seq, a single-cell RNA-seq, can profile cell-to-cell variability
on a genomic scale. RIP-seq can examine the miRNA-mRNA associations under
specific conditions. 3C-based techniques have evolved into Hi-C at a rapid speed and
make it possible to analyze the long-range interactions on multiple chromosomes.
We make use of these tools to identify new biomarkers and to detect novel epigenetic
factors which play important roles in progression of kidney diseases.
Summary Points
• Genome-wide analysis using high-throughput sequencing has become more

available in research fields including nephrology.
• ChIP-seq, RNA-seq, RIP-seq, and chromosome conformation capture techniques
are very useful tools for analyzing and clarifying the epigenetic molecular
mechanisms.
• PAT-ChIP (pathology tissue-ChIP)-seq protocol on freshly isolated mouse embry-

onic kidneys can be used for in vivo analysis of transcriptional factor recruitment
on chromatin.
• Advanced technique for RNA-seq is a single-cell RNA-seq, which can profile
cell-to-cell variability on a genomic scale.
• RIP-seq can be used to analyze the RNA-binding proteins on genome-wide scale.
• 3C-based techniques have evolved into Hi-C at a rapid speed and make it possible
to analyze the long-range interactions on multiple chromosomes.
• We make use of these tools to identify new biomarkers and to detect novel
epigenetic factors which play important roles in progression of kidney diseases.
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Cell Cycle Arrest Biomarkers in Kidney
Disease 43
Kianoush Kashani, Erin N. Frazee, and John A. Kellum
Contents
Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 979
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 979
Cell Cycle in Renal Tubular Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 980
Relationship Between the Cell Cycle and AKI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 981
Cell Cycle Arrest Urinary Biomarkers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 981
IGFBP7 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 982
TIMP-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 982
Mechanism of Action of Cell Cycle Arrest Biomarkers in AKI . . . . . . . . . . . . . . . . . . . . . . . . . . . 983
Applications of Cell Cycle Arrest Biomarkers for AKI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 984
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 986
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 986
Abstract
Acute kidney injury remains one of the most common and deadly complications
of critical illness. Early recognition of this syndrome potentially allows more
efficient prophylactic and potentially therapeutic options. The functional bio-
markers of kidney injury are very insensitive to the changes of kidney function
K. Kashani
Division of Nephrology and Hypertension, Department of Medicine, Mayo Clinic, Rochester, MN,
USA
Division of Pulmonary and Critical Care Medicine, Department of Medicine, Mayo Clinic,
Rochester, MN, USA
E.N. Frazee
Hospital Pharmacy Services, Mayo Clinic, Rochester, MN, USA
J.A. Kellum (*)
The Center for Critical Care Nephrology, CRISMA, Department of Critical Care Medicine,
University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
e-mail: kellumja@ccm.upmc.edu

978 K. Kashani et al.
early in the course of AKI and also nonspecific to the etiology of kidney damage.
The critical need for novel biomarkers of AKI resulted in a significant number of
efforts which concluded discovery and validation of several new AKI bio-
markers. The most recent and indeed the most specific biomarkers of kidney
stress are recently discovered and validated and currently approved by the Food
and Drug Administration (FDA) for identification of AKI high-risk individuals
among ICU patients. These biomarkers, i.e., insulin growth factor-binding pro-
tein-7 (IGFBP7) and tissue inhibitor metalloproteinases-2 (TIMP-2), are able to
identify high-risk patients in ICU, 12 h before the functional biomarkers are able
to detect AKI. These proteins are involved in the pathophysiology and natural
history of AKI by halting the progression of the cell cycle following injury during
the G1- to S-phase transition. In this review, we will describe the role of the cell
cycle during AKI and the relationship between the cell cycle arrest and maladap-
tive recovery of the kidney following an injury. Then we focus on cell cycle arrest
biomarkers and their relationship with AKI, their physiological roles, and finally
potential clinical applications.
Keywords
Tissue inhibitor metalloproteinases-2 (TIMP-2) • Insulin-like growth factor bind-
ing protein-7 (IGFBP-7) • Cell cycle arrest • Biomarker • Acute kidney injury
Abbreviations
βFGF β fibroblast growth factor
CDK Cyclin-dependent protein kinase
DAMP Damage-associated molecular pattern
DDR DNA Damage Response
EGF Epithelial growth factor
FDA Food and Drug Administration
G1 Gap 1
G2 Gap 2
ICU Intensive care unit
IGFBP7 Insulin-like Growth Factor Binding Protein-7
ITG α3 β1 Integrin α3/β1
KDIGO Kidney Disease Improving Global Outcomes
KIM-1 Kidney injury molecule -1
L-FABP Liver fatty acid binding protein
M Mitosis
MMP Metalloproteinases
NGF Nerve growth factor
PAMP Pathogen-associated molecular pattern
PCNA Proliferating cell nuclear antigen
43 Cell Cycle Arrest Biomarkers in Kidney Disease 979

S Synthesis
SA-β-gal Senescence-associated galactosidase
TIMP-2 Tissue Inhibitor Metalloproteinases-2
TGF-β Transforming growth factor- β
Key Facts
• AKI pathophysiology is very complex. Cell cycle arrest is a part of the patho-
genesis of AKI.
• Biomarkers of cell cycle arrest can predict moderate to severe AKI at least 12 h
before its clinical presentation.
• There are two cutoffs defined for [TIMP-2] [IGFBP7], a high-sensitivity cutoff at
0.3 and a high-specificity cutoff at 2 (ng/ml)2/1,000.
Introduction
Acute kidney injury (AKI) is one of the most common complications of hospital and
intensive care unit (ICU) admissions and it is associated with significant clinical
consequences. In a large population-based epidemiological study, the incidence of
AKI was found to be 1,811 cases per million persons (Ali et al. 2007). Among all
hospitalized patients and specifically those in the ICU, the incidences of AKI rise
dramatically to 20 % and 67 %, respectively (Uchino et al. 2006; Ostermann and
Chang 2007; Hoste et al. 2006). In addition, AKI carries an independent risk of
mortality and transition to both chronic kidney disease and end-stage kidney disease
(Chawla et al. 1361; Joannidis n.d.; Uchino et al. 2010).
Despite the gravity of AKI as a critical illness complication, very few therapeutic
interventions have been tested in humans, the majority of which have been found to
be ineffective (Aydin et al. 2007). A plausible explanation for the lack of success
with previously tested therapies could relate to an inability to successfully identify
patients at high risk for AKI early in their disease course when they most likely
benefit from prophylactic or therapeutic strategies. Apart from risk stratification
tools to identify high-risk patients, sensitive and early biomarkers of kidney stress
or injury could play significant roles in this regard. Early identification and inter-
vention is likely to result in a significant improvement in the outcomes of patients
with AKI, a goal that has as yet been difficult to achieve (Himmelfarb et al. 2008).
In the past decade, the number of studies to discover and validate novel bio-
markers of AKI has significantly grown. Kidney injury molecule-1 (KIM-1), neu-
trophil gelatinase-associated lipocalin (NGAL), urinary interleukin-18 (IL-18), and
liver fatty acid-binding protein (L-FABP) are among these new discoveries
(Ichimura and Mou 2008; Mishra et al. 2005; Siew et al. 1497; Yokoyama
et al. 2009). These biomarkers are able to predict AKI development and its
consequences. However, despite the seemingly excellent progress in this field, a

recent systematic review concluded that these biomarkers are only effective in early
recognition of AKI in a well-defined timed injury in the pediatric population. In adult
patients with multiple comorbid conditions and nebulous time course of injury,
however, these biomarkers were found to be significantly less robust for early
recognition of AKI (Vanmassenhove et al. 2012).
Recently, a new group of biomarkers associated with the cell cycle arrest were
found to be predictive of AKI in a heterogenous cohort of critically ill adults and
validation studies confirmed these findings (Kashani et al. 2013; Bihorac
et al. 2014). In this chapter, we will review the physiology of the cell cycle, its
relationship with AKI, and the implications for new biomarkers in clinical practice.
Cell Cycle in Renal Tubular Cells
Under normal physiologic conditions, the majority of renal tubular epithelial cells
are in a quiescent [Gap 0 (G0)] state (Shankland et al. 2000). After AKI, as part of
the normal repair mechanisms, tubular cells enter the active cell cycle to replace any
necrotic or apoptotic cells or any other gaps in the epithelial barrier due to detached
cells. The first step of the active cell cycle is interphase, wherein cells prepare for
mitosis (M). The interphase includes three stages: gap 1 (G1), synthesis (S), and
finally gap 2 (G2). During G1 or the growth phase, cell biosynthetic activities
increase significantly. Cells gather supplements required for replication of
deoxyribonucleic acid (DNA) content, including proteins and organelles such as
ribosomes and mitochondria. In the S phase, all DNA content of the cell is replicated.
Cells continue their growth during the G2 phase and then pass into mitosis. Mitosis
itself includes four phases (prophase for condensation of chromatin to chromosomes,
metaphase for alignment of chromosomes at the equator of the cell, anaphase for
splitting sister chromosomes to the opposite pole of the cell, and finally telophase for
formation of two daughter cells) (Temple and Raff 1986; Peters 1998; Karp 2005).
During the cell cycle, eukaryotic cells undergo vigorous self-examination to
ensure the fidelity of their DNA content. This process happens during at least
three well-recognized checkpoints. The first cell cycle checkpoint, also called
restriction point at G1/S, happens immediately before cells enter the S phase.
Cyclin-dependent protein kinase (CDK) inhibitors, including P21, P16, and P53,
halt the progression of the cell cycle from the G1 to S phase by inhibiting the CDK
complexes (CyclD-CDK4 and CyclE-CDK2) (Chkhotua et al. 2006; Melk
et al. 2005; Price et al. 2004; Tanaka et al. 2005). The second well-known checkpoint
is immediately before the beginning of mitosis at the G2 phase. There is evidence
that the inability to transition from G2 to M promotes a fibrogenic, “maladaptive”
recovery after AKI (Yang et al. 2010). Finally, the third checkpoint is located at the
metaphase to assess the tension in bipolar attachments among chromosomes.
If at any of these checkpoints, cells fail to proceed to the next step, they encounter
one of these outcomes: (1) transient arrest, repair, and return to cell cycle;
(2) defective repair typically leading to apoptosis or cell senescence; and (3) direct
apoptosis when the damage is very severe (Price et al. 2009).
Relationship Between the Cell Cycle and AKI
The pathogenesis of AKI is extremely complex. A combination of injuries to

endothelial and epithelial cells in the setting of inflammation leads to the clinical
presentation of AKI (Sharfuddin and Molitoris 2011). One of the proposed mecha-
nisms involved in the evolution of AKI is derangements in the cell cycle and its
arrest. In response to DNA damage, the DDR (DNA-damage response network) and
P53 are activated to determine the ultimate fate of an injured cell. During cell cycle
arrest, the cells assess the extent of damage and need for repair.
In a murine model of septic AKI caused by cecal ligation and puncture, G1 cell
cycle arrest was found to play an essential role in the development of AKI (Yang
et al. 2009). Flow cytometry of the DNA content of kidney cells showed the number
of cells in G1 significantly increased within 6 h of insult, while the amount of cells in
the S phase decreased in this time frame. The increased G1 to S cell ratio continued
for at least 24 h, and it was only after 72 h that the number of S cells increased
significantly and clinical improvement of AKI occurred. In this experience, the
authors noted a significant upregulation of P53 and P21 during the first 24 h [the
cell cycle arrest indicators], CDK [the S phase indicators] after the first 24 h, and
finally retinoblastoma protein [a proliferation indicator] 72 h from the original insult.
In another murine model of AKI induced by clamping renal arteries, investigators
noted changes of injury, G/S transition, and differentiation markers in the S3
segment of proximal tubules happen in series (Witzgall et al. 1994). These investi-
gators found an initial increase in clusterin [injury marker], followed by
up-regulation of proliferating cell nuclear antigen (PCNA) [G1 to S transition
marker], and finally vimentin [differentiation marker] during the first 120 h of initial
insult. In other models of AKI including cisplatin exposure, ischemia reperfusion,
and ureteral obstruction, rapid induction of P21 in proximal and distal tubular cells is
demonstrated (Megyesi et al. 1996). Knowing that P21 is heavily involved with the
G1/S cell cycle arrest would suggest that the cell cycle can be initiated very early in
the course of injury, and as an integrated part of the process, G1/S cell cycle arrest
happens in the initial steps of the cell cycle if certain conditions are met (e.g.,
damage, ongoing stress).
Cell Cycle Arrest Urinary Biomarkers
In recent discovery and validation studies, two cell cycle arrest biomarkers of AKI,
insulin growth factor-binding protein-7 (IGFBP7), and tissue inhibitor
metalloproteinases-2 (TIMP-2) showed high performance in prediction of AKI
before its clinical presentation (Kashani et al. 2013; Bihorac et al. 2014). In this
section we describe characteristics of these two proteins and their relationship with
the cell cycle arrest during AKI.
IGFBP7
IGFBP7 is a 27-kDA protein from the 16-member IGFBP superfamily and it is

expressed in renal epithelial cells (Degeorges et al. 2000; Matsumoto et al. 2010).
IGFBP7 is known to weakly bind IGF-1 and IGF-2, and this bond is weaker than
other IGFBP family members 1–6. It also binds insulin with over 500-fold greater
affinity than other family members (Burger et al. 2005). Its expression is induced by
P53, DNA injury induced by retinoic acid, transforming growth factor- β (TGF-β),
glucocorticoids, or reactive oxygen species (ROS) (Burger et al. 2005; Pereira
et al. 1999; Swisshelm et al. 1995). IGFBP7 regulates tissue availability of
insulin-like growth factors, stimulates cell adhesion (Nagakubo et al. 2003), and
promotes cell repair. IGFBP7 is induced in renal microvasculature after ischemia
(Usui et al. 2002) and is involved in cell senescence (Acosta et al. 2008; Cichowski
and Hahn 2008; Vicencio et al. 2008). It also plays a role in the epithelial cell cycle
arrest including M12 prostate cells (Sprenger et al. 2002). Given the involvement of
IGFBP7 in senescence, proliferation, and the cell cycle arrest, the potential exists
that it may also be an early marker of cellular (DNA) damage.
The role of IGFBP7, particularly during AKI, is of interest. In an in vitro
experiment with human melanoma cell lines, it was noted that increasing content
of recombinant IGFBP7 in the culture environment is associated with a dose-
dependent decrease in cell proliferation and also increased apoptosis (Wajapeyee
et al. 2008). In another in vitro experiment with MCF-7 breast cancer cells, inves-
tigators noted transfecting cells with IGFBP7 or adding it to the culture media
induced senescent phenotypes, including decreased cell proliferation, increased
G1/S cell cycle arrest cells, altered cell morphology, and increased senescence-
associated galactosidase (SA-β-gal) activity. The observed growth stoppage was
associated with increased expression of the CDK inhibitor P21 and dephosphoryla-
tion of the retinoblastoma protein. This phenomenon was partially reversed by P21
knockdown in MCF-7 cells, while P53 knockdown did not influence the growth
inhibition and cellular senescence (Zuo et al. 2012).
TIMP-2
Tissue inhibitor metalloproteinases-2 is a 21-kDA protein from the four-member

TIMP family. This protein is expressed in melanoma cells and renal tubular cells
(e.g., in polycystic kidney disease). TIMP-2 irreversibly inactivates metallopro-
teinases (MMP) by binding to their catalytic zinc cofactor. TIMP-2 acts on
MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-13,
MMP-14, MMP-15, MMP-16, and MMP-19 and is also required for activation of
MMP-2 (Goldberg et al. 1989; Stetler-Stevenson et al. 1989). TIMP-2 expression is
influenced by a number of cytokines and chemokines and is upregulated by signals
that control proliferation (βFGF and EGF) and differentiation (retinoic acid and
NGF) (Jaworski and Pérez-Martínez 2006). Additionally, TIMP-2 has
metalloproteinase-independent cell-signaling activities. Studies have shown that
TIMP-2 binds to human endothelial cells through integrin α3/β1 (ITG α3 β1),
which results in G1 cell cycle arrest and inhibition of proliferation (Seo
et al. 2006; Stetler-Stevenson 2008; Chang et al. 2006; Henriet et al. 1999; Perez-
Martinez and Jaworski 2005). Similarly, binding of TIMP-2 through ITG α3 β1
mediates suppression of FGF2 or VEGF and induces endothelial cell proliferation
in vitro and angiogenesis in vivo (Bourboulia et al. 2011; Seo et al. 2003, 2008,
2011). ITG α3 β1 regulates kidney epithelial cell responses to TGF-β which indicates
the ITG-α3-β1-specific mechanisms described for endothelial cells could occur in
renal epithelium (Kim et al. 2009). TIMP-2 excretion is induced by ROS, differen-
tiation signals (retinoic acid), and proliferation signals (EGF). During kidney injury
TIMP-2 is involved in a variety of events including factors involved in innate
immunity such as structural changes influencing leukocyte transmigration from the
capillaries to areas of injury in the renal tubule (Opdenakker et al. 2001; Stefanidakis
et al. 2006), changes in endothelial permeability (Catania et al. 2007), and modula-
tion of the inflammatory response (Manicone and McGuire 2008; Garton
et al. 2006). It is also involved in later events that occur with more serious injury
such as apoptosis (cell death) (Ii et al. 2006a; Mannello et al. 2005), loss of cell-cell
adhesion, and sloughing of tubular epithelial cells (Catania et al. 2007; Manicone
and McGuire 2008; Ii et al. 2006b). Seo et al., in an in vitro investigation of the effect
of TIMP-2 on human microvascular endothelial cell proliferation, observed that
TIMP-2, via a mechanism independent of MMP inhibition, decreased endothelial
cell proliferation (Seo et al. 2003).
Mechanism of Action of Cell Cycle Arrest Biomarkers in AKI
As outlined above, both of the candidate biomarkers are excreted during DNA damage
and are involved with the G1/S cell cycle arrest. During early phases of AKI, induced
by ROS, pathogen-associated molecular pattern (PAMP), and damage-associated
molecular pattern (DAMP) molecules, renal epithelial cells enter the cell cycle and
then stop immediately before the S phase. This arrest is highly regulated and several
proteins are involved. IGFBP7 and TIMP-2 as part of this machinery are expressed
during tubular epithelial cell injury. They appear to act as autocrine signals but also in
a paracrine fashion alerting neighboring epithelial cells. P53 and P21 expression is
directly induced by IGFBP7 and P27 by TIMP-2. The cell cycle promotion effect of
CDK complexes (CyclD-CDK4 and CyclE-CDK2) is directly blocked by P-proteins
which results in the initiation of a transient G1/S cell cycle arrest.
Applications of Cell Cycle Arrest Biomarkers for AKI
In recent years the role of cell cycle arrest biomarkers in the early detection of AKI
has been validated (Kashani et al. 2013; Bihorac et al. 2014). In the discovery phase,
all patients with known AKI at the time of enrollment were excluded. Then, in a
protocolized process, more than 340 proteins were measured in multiple samples
collected at predefined time points (Kashani et al. 2013; Bihorac et al. 2014). These
candidate proteins were selected based on the current knowledge of AKI pathophys-
iology and its related processes including inflammation, apoptosis, necrosis, endo-
thelial injury, cell-cell and cell-matrix adhesion, cytoprotection, oxidative processes,
and the cell cycle. Among all of these potential biomarkers, two molecules related to
the cell cycle arrest exhibited superior performance in the early detection of AKI.
Hereafter, the clinical validation phase involved a large-scale multicenter study of
728 patients from 35 medical centers in North America and Europe (Kashani
et al. 2013). These patients were critically ill adults more than 21 years of age who
were admitted to the ICU. Patients with Kidney Disease: Improving Global Out-
comes (KDIGO) stage 2 or 3 AKI were excluded during the screening process. This
study found that cell cycle arrest biomarkers performed better in the early detection
of this syndrome compared to previously described AKI biomarkers such as KIM-1
and NGAL (Kashani et al. 2013). During this study investigators noted that in
surgical patients the best individual marker was IGFBP7 while TIMP-2 performed
better in patients with sepsis. The product of these two markers ([TIMP-2]
[IGFBP7]) was selected as a biomarker panel for AKI risk stratification.
In the follow-up validation study, investigators enrolled 420 patients within the
first 24 h of admission to the ICU (Bihorac et al. 2014). Twenty three centers in the
USA participated in this study. Investigators excluded patients who had KDIGO
stage 2 or 3 AKI at the screening phase. Diagnosis of AKI within 12 h of enrollment
adjudicated by a clinical adjudication committee was used as the primary endpoint.
Investigators used the highly sensitive threshold of 0.3 (ng/ml)2/1,000 for [TIMP-2]
[IGFBP7]. In this study the performance of a clinical model to predict AKI was only
fair [the receiving operating characteristic area under curve (AUC) was only 0.70
(95 % CI, 0.63–0.76)]. The performance of the model was significantly enhanced
when the urinary [TIMP-2] [IGFBP7] was added to the model (AUC increased to
0.86 (95 % CI, 0.80–0.90)) (Bihorac et al. 2014).
Cutoffs for [TIMP-2] [IGFBP7] for clinical use were validated in another follow-
up investigation. In this study 154 patients from six sites in the USA were enrolled
(Hoste et al. 2014). Unlike the other two studies that used a central laboratory and
ELISA for measurement of [TIMP-2] [IGFBP7], each site used the commercial
platform (NephroCheck ®) to measure the urinary [TIMP-2] [IGFBP7] at the local
level. Eligibility criteria for enrollment were similar to the earlier studies. Two
previously determined thresholds from the original validation study (0.3 and
2 (ng/ml)2/1,000) were validated as the sensitivity and specificity cutoffs, respec-
tively. Investigators found a sensitivity of 89 % for the 0.3 (ng/ml)2/1,000 cutoff and
a specificity of 90 % for the 2 (ng/ml)2/1,000 as the cutoff. Therefore, authors
concluded the lower cutoff could be used for screening and risk stratification
Fig. 1 Clinical application of cell cycle arrest biomarkers. Identifying patients who are at high risk
or very high risk for development of AKI is very important. This figure delineates how identification
of these patients could be in alignment with the Kidney Disease: Improving Global Outcomes
(KDIGO) guidelines for AKI (Summary of Recommendation Statements 2012)
processes while the higher cutoff could be used to identify patients at very high
likelihood of developing AKI (Hoste et al. 2014) (Fig. 1). A subsequent analysis of
data from the Sapphire trial revealed that these cutoffs were able to accurately predict
9-month death or dialysis in ICU patients developing AKI (Koyner et al. 2013).
Further studies have revealed that in patients who underwent cardiac surgery,
serial urinary samples of [TIMP-2] [IGFBP7] were predictive of postoperative AKI
(Meersch et al. 2014). This cohort included 50 patients, 52 % of whom developed
AKI (including stage 1). The authors reported that the maximum urinary [TIMP-2]
[IGFBP7] concentration achieved within the first 24 h after cardiopulmonary bypass
was predictive of AKI with an AUC of 0.84 – sensitivity of 92 % and specificity of
81 % at a cutoff of 0.5 (ng/ml)2/1,000 (Meersch et al. 2014).
In September 2014, the US Food and Drug Administration (FDA) approved the
marketing of [TIMP-2] [IGFBP7] to assess a patient’s risk for developing AKI (FDA
& FDA 2014). This milestone not only allows the clinical use of these biomarkers
for early detection of patients with AKI but may also facilitate enrollment in
interventional studies designed to prevent or treat this deadly syndrome (Endre
et al. 2014). Indeed, this approval announcement is the “beginning of a new era”
(Endre et al. 2014).
Importantly, while cell cycle arrest clearly has a “dark side” and is associated with
development of AKI and long-term adverse outcomes, there is a “light side” as well.
Cell cycle arrest can protect cells from the disastrous consequences of entering cell
division with damaged DNA or insufficient bioenergetic resources during injury or
stress. Whether we can use the light side to help prevent AKI remains to be seen, but
there is already evidence that cell cycle arrest biomarkers such as IGFBP7 and
TIMP-2 are indicators of both sides of this complex physiology.
Summary Points
In conclusion, AKI is a deadly syndrome that affects millions of patients around

the world. Damaged renal epithelial cells enter the cell cycle shortly after the
injury. There is a cell cycle arrest immediately prior to the S phase, and IGFBP7
and TIMP-2 are among the mediators of this process. These two proteins are
sensitive and specific markers for the prediction of AKI and have now been validated
in several large-scale investigations. [TIMP-2] [IGFBP7] is currently FDA approved
for clinical use to allow accurate assessment for the risk of developing AKI.
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Integrin-Linked Kinase (ILK) Expression
as a Biomarker in Cancer of the Kidney 44
Miriam de Fatima Brasil Engelman and Gustavo Gonçalves
Engelman
Contents
Key Facts: Activation of the Phosphoinositide 3-Kinase (PI3K)-Akt-Signaling Pathway
and Integrin-Linked Kinase in Clear-Cell Renal Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 993
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 994
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 995
ILK and Tumorigenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 998
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1008
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1009
Abstract
Integrin-linked kinase (ILK) is a serine/threonine protein kinase implicated in cell-
cycle control via integration of integrins with the extracellular matrix. Integrin-
linked kinase overexpression promotes anchorage-independent growth and may
induce tumorigenesis and invasion. Integrin-linked kinase suppresses anoikis,
suggesting that it has a role in oncogenic transformation, particularly in the process
of metastasis. Due to its effects on the cell cycle, apoptosis, cell adherence, and in
the breakdown of collagen and cellular mobility, integrin-linked kinase has been the
subject of numerous studies, particularly in the field of oncology. ILK expression
and activity are increased in several human cancers, including prostate, colon,
stomach, ovary, malignant melanomas, Ewing’s sarcoma, primitive
M.d.F.B. Engelman (*)

Department of Pathology, Faculdade de Ciências da Saúde Dr. José Antônio Garcia Coutinho,
Universidade do Vale do Sapucaí (UNIVÁS), Pouso Alegre, Minas Gerais, Brazil
e-mail: mi.engelman@uol.com.br
G.G. Engelman
Faculdade de Medicina Universidade José do Rosário Vellano (UNIFENAS), Alfenas,
Minas Gerais, Brazil
e-mail: gustavo_engelmann@hotmail.com

992 M.d.F.B. Engelman and G.G. Engelman
neuroectodermal tumor, non-small-cell lung cancer, bladder cell carcinoma, basal

cell carcinoma, squamous cell/adenosquamous carcinomas, adenocarcinoma of the
gallbladder, malignant pleural mesothelioma, chondrosarcoma, and pancreatic
cancer. Renal cell carcinoma is a tumor derived from epithelial cells of the renal
tubules and represents 80–85 % of all primary malignant tumors of the kidney and
2–3 % of all cancers in adults. Among renal cell carcinomas, clear renal cell
carcinomas are the most frequent, accounting for 70–80 % of cases. These neo-
plasms may be family associated or, in the majority of cases (95 %), sporadic. Both
types are related to loss of function of the VHL gene, which acts as a tumor
suppressor. Clear renal cell carcinomas at identical stages and pathological grades
may exhibit distinct biological behavior, and consequently prognosis markers are
urgently needed. In clear renal cell carcinoma, integrin-linked kinase immunoex-
pression is related to loss of intercellular adhesion and degree of differentiation, and
it is positively correlated with the proliferation index, renal capsule and renal vein
invasion, tumor size, and Robson stage. Integrin-linked kinase may be essential for
invasion and metastasis in renal cell carcinoma. Integrin-linked kinase may also act
through the phosphoinositide 3-kinase (PI3K)-Akt pathway to promote cell sur-
vival in human renal cell carcinoma. Integrin-linked kinase may act as a
phosphoinositide-dependent kinase (PDK)-2 by facilitating Akt phosphorylation
at S473. Altogether, results reviewed here indicate that the PI3K/ILK/Akt axis is a
promising target for therapeutic intervention in renal cell carcinoma and that
integrin-linked kinase expression might be used in clinical practice as an important
predictive and prognostic tumor marker for treatment customization.
Keywords
Immunohistochemistry • Biomarker • Carcinoma • Renal cell • Integrin-linked
kinase • Human
Abbreviations
ADRP Adipose differentiation related protein
AKT/PKB Protein kinase B
AP1 Activator protein 1
BCC Basal cell carcinoma
CCRC Clear cell renal carcinoma
COX-2 Cyclooxygenase-2
CRC Renal cell carcinoma
FA Focal adhesion
FAK Focal adhesion kinase
FFA Free fatty acid
FKHR Forkhead transcription factor
GPR40 G protein-coupled receptor 40
GPR40 G protein-coupled receptors
GSK-3 Glycogen synthase kinase
44 Integrin-Linked Kinase (ILK) Expression as a Biomarker in Cancer of. . . 993
HFI 1α Hypoxia-inducible factor-1alpha

HFI 2α Hypoxia-inducible factor-1alpha
IGF-1 Growth factor 1 insulin
IHC Immunostaining
ILK Integrin-linked kinase
ILKAP ILK phosphatase associated
MMP9 Metalloproteinase-9
mRNA Messenger ribonucleic acid
mTOR Mammalian target of rapamycin
MUC 1 Mucin1
NF-κB Nuclear factor kappa B
NSCLC Non-small cell lung cancer
PanIN Pancreatic intraepithelial neoplasm
PAS Periodic acid-Schiff
PCR Polymerase chain reaction
PDGF PLATELET derived growth factor
PDK-1 PI 3-kinase-dependent kinase
PI3K 3 kinase fosfatilinositol
PTEN Phosphatase (fosfatilinositol of 3, 4, 5-triphosphate) and tensin
homolog
Pthrp Parathyroid hormone-related protein
RNA Ribonucleic acid
TGF-α Transforming growth factor α
TMA Tissue microarray
VLH Von Hippel-Lindau
α-NAC Alpha-chain of the nascent polypeptide-associated complex
α-Pix PAK (p21-activated kinase)-interacting exchange factor
α-SMA α-smooth muscle actin
Key Facts: Activation of the Phosphoinositide 3-Kinase (PI3K)-Akt-

Signaling Pathway and Integrin-Linked Kinase in Clear-Cell Renal
Carcinoma
• The immunohistochemical expression of integrin-linked kinase in clear-cell renal

carcinomas correlates with loss of intercellular adhesion, degree of differentia-
tion, cell proliferation index, invasion of the renal capsule and renal vein, tumor
size, and Robson stage. ILK may be essential for invasion and metastasis in RCC.
• The phosphoinositide 3-kinase (PI3K)-Akt-signaling pathway is involved in
many cellular processes including proliferation, death, migration, and angiogenesis.
• Once recruited to the plasma membrane through the activity of PI3K, Akt is
activated by phosphorylation at two sites: T308 in the kinase domain by PDK-1
and S473 in the regulatory tail by PDK-2.
• Integrin-linked kinase phosphorylates PKB/Akt at the Ser473 residue, leading to

evasion of apoptosis by inhibition of caspase-3 activation or activation of nuclear
factor қ B (NF-қB). Activation of this factor may also stimulate cyclooxygenase-
2 (COX-2), which is implicated in tumor progression by stimulating angiogenesis
and invasion.
• Integrin-linked kinase promotes cell survival in human renal cell carcinomas.
PTHrP activates integrin-linked kinase, which acts as a phosphoinositide-
dependent kinase (PDK)-2 in facilitating Akt phosphorylation at S473.
• Free-fatty acids are associated with the development of renal cell carcinomas,
through the activation of GPR40/ILK/Akt, revealing a new mechanism for the
correlation between metabolic disorders and renal carcinoma.
Definitions
Anoikis From the ancient Greek, which means homelessness. It is the apoptosis of
normal epithelial cells in response to detachment from their extracellular matrix.
Epithelial–mesenchymal transition (EMT) Mechanism by which cancer cells

acquire the invasive and stem-like traits necessary for distant metastasis.
GPR40, also known as free-fatty-acid receptor 1 (FFA1) A class A G-protein-

coupled receptor for medium- and long-chain free-fatty acids that may be involved in
the metabolic regulation of insulin secretion.
Integrin Proteic transmembrane receptors which interact and connect cell to cell
and their extracellular matrix.
Oncogenes Proto-oncogenes are genes that normally help cells grow. When a
proto-oncogene mutates or there are too many copies of it, it becomes a gene that
can become permanently turned on or activated when it is not supposed to be. When
this happens, the cell grows out of control, which can lead to cancer and this gene is
called an oncogene.
Parathyroid hormone-related protein (PTHrP) A cytokine-like polyprotein

expressed in fetal and adult tissues and a key regulator of cellular calcium transport
and smooth muscle cell contractility, as well as a control factor in cell proliferation,
development, and differentiation.
Predictive marker A biomarker that predicts the likelihood of benefit from a

specific clinical intervention or the differential outcomes of two or more interven-
tions or treatment.
Prognostic marker A biomarker that assesses a patient’s overall outcome, such as

the probability of cancer recurrence after standard treatment.
Tumor suppressor genes Normal genes that slow down cell division, repair DNA
mistakes, or tell cells when to die (apoptosis or programmed cell death). When tumor
suppressor genes don’t work properly, cells can grow out of control, which can lead
to cancer.
Von Hippel–Lindau (VHL) disease Autosomal dominantly inherited tumor syn-

drome, caused by mutations of the von Hippel–Lindau tumor suppressor (VHL)
gene. It is characterized by frequent development of specific types of tumors in
selective organs such as: retinal hemangioblastomas, cerebellar and spinal
hemangioblastomas, renal clear-cell carcinomas and cysts, epididymal and broad
ligament cystadenomas, pancreatic cysts, microcystic serous adenomas, neuroendo-
crine tumors, and pheochromocytomas.
Introduction
Cell interaction with the extracellular matrix (ECM) regulates fundamental processes
such as cell shape, motility, growth, survival, differentiation, and gene expression
through integrin-mediated signal transduction. Integrin-linked kinase (ILK) was
isolated approximately 20 years ago in a yeast two-hybrid screen using the cyto-
plasmic tail of integrin β as bait (Hannigan et al. 1996). This serine/threonine kinase
interacts with the β-integrin cytoplasmic domain in focal adhesions, where it func-
tions as a scaffolding protein in the formation of multiprotein complexes connecting
integrins to the actin cytoskeleton and modulating intracellular signaling pathways
originated by those connections (McDonald et al. 2008).
ILK comprises three structurally distinct domains. The N-terminus consists of five
ankyrin repeats that mediate the interaction with PINCH, a family of LIM-domain-
only proteins that consists of two members, PINCH-1 and PINCH-2. Both PINCH
proteins contain five LIM domains, the first of which binds ILK (Tu et al. 1999;
Chiswell et al. 2008). The pleckstrin homology (PH)-like domain of ILK has been
shown to bind phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3)
(Delcommenne et al. 1998; Pasquali et al. 2007). The C-terminal kinase-like domain
binds several adaptor proteins including α-parvin, also known as actopaxin or
CH-ILKBP, β-parvin, also known as affixin, and γ-parvin (Nikolopoulos and Turner
2000; Olski et al. 2001; Tu et al. 2001; Yamaji et al. 2001; Chu et al. 2006) (Fig. 1).
The ILK/PINCH/parvin (IPP) complex represents a central constituent of adhe-
sion sites that contain β1 and β3 integrin, from where it regulates multiple cellular
processes (Wickström et al. 2010). ILK mediates the phosphorylation of a variety of
intracellular substrates, including protein kinase B (PKB/Akt) and glycogen
synthase kinase-3 (GSK-3). However, ILK lacks key catalytic residues, and thus
Fig. 1 ILK functional domains and interactions. ILK is localized in focal adhesions, where it forms
multiprotein complexes with several proteins that are involved in cytoskeletal dynamics and cell-
signaling cascades. The N-terminal ankyrin repeats of ILK interact directly with several key
proteins, including PINCH and ILKAP. The central PH-like domain of ILK binds to PtdIns(3,4,5)
P3 and is required for PI3K-dependent activation of ILK. The C-terminal kinase-like domain of ILK
interacts with integrins, as well as with several actin-binding adaptor proteins, including α-parvin,
β-parvin, and paxillin
its ability to function as a “true” kinase was questioned (Qin and Wu 2012). Gain-
and loss-of-function strategies have shown that overexpression and/or constitutive
activation of ILK results in oncogenic transformation and progression to invasive
and metastatic phenotypes in human tumors (Persad and Dedhar 2003).
Usually, ILK is overexpressed in human malignancies, and it correlates with
tumor stage and grade (Tables 1 and 2). Moreover, ILK overexpression predicts poor
patient survival in several types of cancers. Because of ILK roles in the cell cycle,
apoptosis, cellular adhesion, collagen degradation, angiogenesis, and cell motility,
numerous studies have documented it as a potentially tumorigenic molecule (Fig. 2).
Among RCCs, clear renal cell carcinomas (CRCC) are the most frequent,
accounting for 70–80 % of cases. These neoplasms may be family associated or,
in the majority of cases (95 %), sporadic. Both cases are related to a loss of function
of the VHL gene, which acts as a tumor suppressor. CRCCs at identical stages and
pathological grades may exhibit distinct biological behavior. Therefore, prognosis
markers are urgently needed.
Table 1 ILK expression in human malignancies

Malignancy Reference ILK expression
Medulloblastoma Chung ILK expression in 100 % off cells
et al. 1998
Primitive Chung ILK expression in 100 % off cells
neuroectodermal et al. 1998
tumor
Ewing’s sarcoma Chung ILK expression in 100 % off cells
et al. 1998
Prostate carcinoma Graff ILK expression increased with tumor progression and
et al. 2001 correlated with poor prognosis
Ovarian tumor Ahmed ILK expression increased with tumor progression.
et al. 2003 Normal epithelium negative
Colon carcinoma Marotta ILK overexpression in malignant acini compared with
et al. 2003 normal crypts
Gastric carcinoma Ito et al. 2003 ILK expression associated with tumor thickness and
nodal metastasis
Melanoma Dai ILK expression associated with tumor thickness and
et al. 2003 correlated with poor prognosis
Anaplastic thyroid Younes ILK overexpression in tumor tissue, but not in normal
carcinoma et al. 2005 thyroid tissue
Lung carcinoma, Takanami ILK expression correlated with tumor invasion, grade,
non-small cell 2005 stage, and poor prognosis
Colon carcinoma Bravou ILK expression correlated with tumor invasion, grade,
et al. 2006 and stage. Overexpression in metastasis
Lung carcinoma, Okamura ILK overexpression in a subset of tumor associated with
non-small cell et al. 2007 poor prognosis
Hepatocellular Peroukides ILK overexpression in liver oncogenesis and hepatic
carcinomas et al. 2008 cirrhosis correlates with activation of Akt
Squamous Goulioumis ILK expression nuclear associated with p-Akt
laryngeal et al. 2008 expression
carcinomas
Lung carcinoma, Watzka ILK overexpression and strong pAkt staining that are
non-small cell et al. 2010 mutually associated with poor prognosis
Basal cell Papanikolaou ILK overexpression in 100 % of cases and correlated
carcinoma et al. 2010 with tumor invasion
Pancreatic Schaeffer ILK expression elevated in tumors but minimal in
carcinoma et al. 2010 pancreatic intraepithelial neoplasm lesions
Bladder cell Matsui ILK expression correlates to the invasiveness of cancer
carcinoma et al. 2011
Clear-cell renal Engelman ILK expression correlated with tumor invasion, grade,
carcinoma et al. 2013 increase in proliferation index, loss of intercellular
adhesion, and stage
Renal cell Han ILK expression regulates vimentin and E-cadherin
carcinoma et al. 2015 expression by regulating the EMT-related transcription
factors Snail and Zeb1
ILK is overexpressed in several human malignancies
(Republished with permission of Company of Biologists Ltd., from Journal of Cell Science, Paul C.
McDonald, Andrew B. Fielding, Shoukat Dedharauthor, 121, 2008; permission conveyed through
Copyright Clearance Center, Inc.)
Table 2 ILK expression in human tumor progression

ILK overexpression
Association Tumor Reference
Histological grade Prostatic carcinoma Graff et al. 2001
Ovarian tumor Ahmed et al. 2003
Colonic carcinoma Bravou et al. 2006
Small-cell pulmonary carcinoma Takanami 2005
Clear-cell renal carcinoma Engelman et al. 2013
Increase in proliferation Prostatic carcinoma Graff et al. 2001
Apoptosis evasion Anaplastic thyroid cancer Younes et al. 2005
Pancreatic adenocarcinoma Duxbury et al. 2005
Clear-cell renal carcinoma Sourbier 2007
Apoptosis evasion Clear-cell renal carcinoma Engelman et al., 2013
Invasion and stage Colon cancer Bravou et al.2006
Melanoma Dai et al. 2003
Non-small-cell lung cancer Takanami 2005
Gastric carcinoma Ito et al. 2003
Basal cell carcinoma Papanikolaou et al. 2010
Pancreatic carcinoma Schaeffer et al. 2010
Bladder cell carcinoma Matsui et al. 2011
Renal cell carcinoma Han et al. 2015
ILK is overexpressed in several human malignancies, and it correlates with histological grade,
increase in proliferation, apoptosis evasion, invasion, and tumor stage
In this chapter we discuss the potential of ILK as a biomarker for these kidney
tumors.
ILK and Tumorigenesis
Overexpression of ILK promotes anchorage-independent growth and can induce

tumorigenesis and invasiveness. In normal epithelial cells, loss of integrin–ECM
interaction induces a form of apoptosis called anoikis. Work by Attwell et al. (2000)
has shown that ILK suppresses anoikis and might therefore play an important role in
oncogenic transformation, particularly in metastasis (Attwell et al. 2000). ILK is
overexpressed in Ewing’s sarcoma, primitive neuroectodermal tumors, and medul-
loblastomas (Chung et al. 1998). ILK also takes part in chemically induced tumors in
mice, probably via alteration of the PKB/Akt pathway (Segrelles et al. 2002).
Numerous studies have related the development and progression of different
malignancies to ILK overexpression and ILK-mediated activation of important
signaling pathways involved in neoplastic transformation such as PI3K, PKB/Akt,
and GSK-3β. In colon cancer, β-catenin activation, decreased immunoexpression of
Fig. 2 Overview of the intracellular signaling pathways regulated by ILK. ILK activation promotes
processes such as motility and contractility, survival, EMT, invasion, proliferation, and angiogen-
esis that are crucial for the progression of malignant PIP3, PtdIns(3,4,5)P3; NAC, nascent
polypeptide-associated complex and coactivator; and HIF1 hypoxia-inducible factor 1 (Republished
with permission of Company of Biologists Ltd., from Journal of Cell Science, Paul C. McDonald,
Andrew B. Fielding, Shoukat Dedharauthor, 121, 2008; permission conveyed through Copyright
Clearance Center, Inc.)
E-cadherin, and activation of Akt correlate with increased expression of ILK and
tumor progression (Bravou et al. 2006). Specifically in colon adenocarcinomas, ILK
has been associated with overexpression of phosphorylated GSK-3β and nuclear
translocation of β-catenin (Marotta et al. 2003). In gliomas, the growth factor Cyr61
activates ILK, thus triggering the phosphorylation of GSK-3β and downstream
activation of the β-catenin signaling pathway, β-TCF/Lef-1, and activation of Akt
phosphorylation with Pik3 and antiapoptotic protein Bad (Xie et al. 2004). The
evaluation of 118 samples of non-small-cell lung cancers showed strong ILK
cytoplasmic expression, strong integrin β1 membranous staining, and strong phos-
phorylated Akt (pAkt) cytoplasmic staining and provided evidence that ILK, integrin
β1, and pAkt are mutually associated with poor prognosis (Okamura et al. 2007).
In liver oncogenesis and hepatic cirrhosis, ILK overexpression correlates with
Akt activation but not with other conventional ILK targets. The expression levels of
ILK, β-catenin, E-cadherin, and pAkt were evaluated by immunohistochemistry in
69 human hepatocellular carcinomas (HCC) and adjacent normal and cirrhotic liver
parenchyma. ILK and pAkt immunostaining was observed in 100 % and 79.7 % of
HCCs, respectively, and their protein levels correlated significantly with each other.
Activation of β-catenin and downregulation of E-cadherin were frequently observed
in HCC, but they were not related to ILK expression (Peroukides et al. 2008).
A study of 97 invasive laryngeal squamous cell carcinomas showed that ILK
overexpression correlates with activation of Akt but not with downregulation of
E-cadherin or activation of β-catenin. Activated Akt seems to characterize well-
differentiated tumors, while loss of E-cadherin and activation of β-catenin correlated
with high-grade carcinomas (Goulioumis et al. 2008).
Moreover, tumors with an inactive PTEN gene, such as prostate carcinomas
(Persad et al. 2000) and glioblastomas (Obara et al. 2004), had upregulated ILK.
Other work has shown that ILKAP, a protein phosphatase that inhibits both ILK and
PTEN, also mediates the inhibition of GSK-3β and has an important role in
suppressing carcinogenesis (Kumar et al. 2004).
Patients with familial adenomatous polyposis (FAP), a condition that precedes
colon cancer, showed impaired regulation of ILK (Marotta et al. 2001). More
recently, work by the same group suggested that disturbances in ILK signaling
represent an early event in the development of colon cancer. ILK was overexpressed
in the crypts of both primary and metastatic lesions. In functional tests, high ILK
activity coincided with changes in the target molecule GSK3 β. Finally, the authors
showed that in colon carcinomas, activation of β-catenin, downregulation of
E-cadherin, and activation of the Akt-FKHR pathway were all significantly corre-
lated with ILK expression and tumor progression (Marotta et al. 2003).
ILK expression also inversely correlates with the degree of histological differen-
tiation in several human malignancies. High-grade prostate carcinomas display
higher levels of ILK immunoexpression compared to low-grade carcinomas (Graff
et al. 2001). In 53 cases of ovarian tumors, intensity of immunohistochemical
staining for ILK directly correlates with tumor grade. An ovarian tumor cell line
expressed high ILK levels, as observed by Western blotting, whereas immortalized
cells derived from normal ovarian tissue displayed low basal expression of ILK
(Ahmed et al. 2003).
ILK expression is also significantly correlated with histological grade in non-
small-cell lung carcinomas (Takanami 2005; Okamura et al. 2007; Watzka
et al. 2010). Bravo et al. (2006), studying 125 primary colon carcinomas, found
ILK expression in 98.4 % of tumors and reported strong correlation between
immunoprotein levels and degree of histological differentiation.
Elevated ILK immunoexpression in pancreatic carcinomas correlates with the
expression of Snail, which suppresses E-cadherin. In 23 of 25 carcinoma cases, ILK
displayed extensive positivity (>50 %). On the other hand, pancreatic intraepithelial
neoplastic (PanIN) lesions stained minimally for Snail and ILK (Schaeffer
et al. 2010).
Many studies encompassing various types of cancers suggest the involvement of
ILK in metastatic spread. For example, 4 out of 5 gastric carcinoma cell lines and
22 out of 35 (63 %) microdissected tumor samples of primary gastric carcinomas
expressed ILK mRNA. In this study, strong expression of ILK protein significantly
correlated with deep invasion of tumor cells into the gastric wall and presence of
nodal metastasis (Ito et al. 2003).
Immunohistochemical ILK expression significantly correlates with human mela-
noma thickness. Biopsies of melanomas overexpressed ILK protein in 0 %, 22 %,
33 %, and 63 % of the individual samples when tumors measured 0.75, 0.76–1.50,
1.51–3.0, and >3.0 mm in thickness, respectively. Similarly, 83 % of tumors with
lymph node invasion strongly expressed ILK in comparison to only 18 % of tumors
without lymph node invasion (Dai et al. 2003).
Increased ILK immunoexpression was also significantly associated with lymph
node metastasis and tumor stage in 134 cases of non-small-cell lung carcinoma. The
study suggests that increased ILK expression is associated with poor prognosis in
patients with non-small-cell lung carcinoma (Okamura et al. 2007).
Bravo et al. (2006) also showed a correlation of immunohistochemical ILK
expression with local invasion and metastasis in colon carcinomas. All metastatic
tumors showed ILK immunohistochemical expression. Moreover, the intensity of
immunoreaction was higher in metastatic tumors. However, there was no direct
correlation between protein overexpression and depth of wall invasion or stage of
the disease.
In human basal cell carcinomas (BCC), ILK expression also correlates with
epithelial–mesenchymal transition markers and tumor invasion. Histological sec-
tions of 100 human BCCs were evaluated by immunohistochemistry for the expres-
sion of ILK, E-cadherin, Snail, β-catenin, and alpha-smooth muscle actin (alpha-
SMA). ILK overexpression was observed in 100 % of the sections and expression
levels strongly correlated with tumor invasion and infiltrative BCCs. There was a
significant correlation between ILK expression and all ECM markers investigated
(Papanikolaou et al. 2010).
Results from a tissue microarray (TMA) performed with human bladder cell
carcinomas showed that ILK expression correlated with invasiveness. This study
suggests that invasive bladder cancers overexpress ILK, which plays an important
role in the epithelial–mesenchymal transition (EMT) of bladder cancer via control of
E-cadherin and MMP-9 expression (Matsui et al. 2011).
Another study indicated that ILK expression in 45 human clear-cell renal carci-
nomas (CRCC) correlated with the loss of intercellular adhesion, loss of differenti-
ation and increased cell proliferation, renal capsule and renal vein invasion, tumor
size, and Robson stage (Engelman et al. 2013). ILK immunoexpression directly
correlated with CRCC, paving the way for the potential development of new
molecular therapies targeting specific pharmacologic inhibitors of the ILK pathway.
The phosphoinositide 3-kinase (PI3K)-Akt-signaling pathway is constitutively
activated in human CRCC independently of VHL expression, and it plays an
essential role in CRCC progression through inhibition of tumor cell apoptosis
(Sourbier et al. 2006). PI3K constitutes a family of enzymes involved in monitoring
of cell growth, proliferation, motility, adhesion, survival, and angiogenesis. Binding
of a number of growth factors to membrane receptor tyrosine kinases (EGFR –
epidermal growth factor receptor, c-kit, and INS-1 – insulin receptor 1) initiates
signaling via the PI3K pathway. Growth factors include insulin-like growth factor,
epidermal growth factor, fibroblast growth factor, interleukin 3 and 6, and vascular
endothelial growth factor (Clark et al. 2002; Meier et al. 1997). The ligand–receptor
interaction determines the conversion of phosphatidylinositol 4,5-2P (PIP2) into
phosphatidylinositol 3,4,5-3P (PIP3), which relays growth and survival signals by
recruiting Akt, also known as protein kinase B, and phosphoinositide-dependent
kinase (PDK). Cytoplasmic Akt is activated in the cell membrane by phosphoryla-
tion at two independent positions with the involvement of PDK1 and mTOR. Once
activated, the mTOR complex (mTORC) acts through its downstream effectors to
stimulate protein synthesis and entrance into G1 phase as well as to control proteins
that regulate apoptosis (Hay 2005).
At the renal level, the PI3K phosphorylation product PIP3 recruits cytoplasmic
Akt to the membrane. Once activated, Akt inhibits apoptosis by phosphorylation and
inactivation of the proapoptotic proteins procaspase-9, apoptosis signal-regulating
kinase-1 (ASK1), and BAD, a member of the bcl-2 family. PI3K inhibits GSK-3β,
which normally phosphorylates and induces degradation of cell-cycle control protein
cyclin D1 and of transcription factors that promote proliferation such as c-myc,
β-catenin, c-Jun, and Notch (Cojocaru et al. 2015). The PI3K–Akt pathway is
constitutively activated in various human cancers where it plays a critical role in
tumor progression and in tumor resistance to therapies (Hanada et al. 2004).
Parathyroid hormone-related protein (PTHrP) is a cytokine-like polyprotein that
is normally expressed throughout the body where it plays a variety of roles,
including regulation of cellular growth, differentiation, and death. PTHrP was
initially identified as the factor responsible for the paraneoplastic syndrome humoral
hypercalcemia of malignancy (Martin et al. 1997; Philbrick et al. 1996). PTHrP
represents an essential growth factor for CRCC and a target for the von
Hippel–Lindau (VHL) tumor suppressor gene. In fact, pVHL suppresses PTHrP
expression at both the mRNA and protein levels (Massfelder et al. 2004). PTHrP-
mediated inhibition of tumor cell apoptosis is crucial for human RCC growth
(Sourbier et al. 2006).
The PI3K/ILK/Akt/NF-κB axis provides a promising target for therapeutic inter-
vention in CRCC. PTHrP is one of the main factors involved in the constitutive
activation of the PI3K-Akt-signaling pathway in human RCC, regardless of VHL
expression. PTHrP induces phosphorylation of Akt at S473 but not at T308. Trans-
fections with ILK constructs and RNA interference provide evidence that ILK is
involved in human RCC cell survival. PTHrP activates ILK, which then acts as a
phosphoinositide-dependent kinase (PDK)-2 in facilitating phosphorylation of Akt
at S473. NF-κB is the downstream Akt target regulated by PTHrP (Agouni
et al. 2007).
Induced overexpression of ILK also leads to RCC progression via free-fatty-acid
(FFA)-mediated activation of the GPR40/ILK/Akt pathway, revealing a novel mech-
anism for the correlation between metabolic disturbances and renal carcinomas (Liu
et al. 2013). Oleic acid is an n-9 monounsaturated fatty acid that activates G-protein-
coupled receptors, which, in turn, lead to ERK1/2 phosphorylation and cancer cell
proliferation in breast cancer. High concentration levels of oleic acid have been used
to imitate the effects of abnormal levels of FFAs on tumor growth in human RCC
786-O cells (Liu et al. 2013). Results indicated that oleic acid stimulates 786-O cell
viability and delayed apoptosis, both in a concentration-dependent manner. Western
blot analysis revealed that oleic acid treatment upregulated ILK expression in a
concentration-dependent manner. Overexpression of ILK was found to increase the
phosphorylation of Akt on Ser-473, and, in addition, when siRNA was used to target
and knock down ILK, the effects of oleic acid on 786-O cell growth were weakened
and the expression of ILK was suppressed.
Recent investigation regarding the role of ILK in cancer progression and metas-
tasis in RCC showed that ILK may be essential for invasion and metastasis and that it
regulates vimentin and E-cadherin expression by regulating the EMT-related tran-
scription factors Snail and Zeb1 (Han et al. 2015). ILK is expressed at a low level in
normal cells and low-stage RCC cells and is highly expressed in advanced and
metastatic cells. In Caki-1, a metastatic RCC cell, both ILK and its downstream
EMT-related effectors are highly expressed. However, ILK knockdown suppressed
the formation of stress fibers and focal adhesions and impeded phenotypic EMT
markers, including cell migration and invasion. In vivo knockdown of ILK
suppressed the progression, invasion, and metastasis of primary RCC in nude mice
by downregulation of EMT markers. Overexpression of ILK increased tumor cell
migration and invasion.
Tumor growth requires the conjunction of several factors, including increased
replication potential, anchor-independent growth capacity, resistance to apoptosis,
angiogenesis, adjacent tissue invasion, and metastasis. When disturbed or
overexpressed, ILK promotes all of these factors. Currently, ILK is considered a
potential prognosis marker for the following human malignancies: melanoma
(Lu et al. 2013), non-small-cell lung cancer (Posch et al. 2014; Watzka et al. 2011;
Okamura et al. 2007), colorectal cancer (Li et al. 2013b), squamous cell/
adenosquamous carcinomas and adenocarcinoma of the gallbladder
(Li et al. 2013a), malignant pleural mesothelioma (Watzka et al. 2010; Schramm
et al. 2010), chondrosarcoma (Papachristou et al. 2008), and pancreatic cancer
(Sawai et al. 2006) (Table 3).

Conditions
RCC represents almost 3 % of malignant tumors in adult humans and most of the
neoplasms arising from the kidney. RCC is characterized by a lack of early warning
signs as well as by diverse and variable clinical manifestations, thus more than 30 %
of patients have advanced-stage RCC at diagnosis (Koul et al. 2011). Only 10 % of
individuals with RCC present with the classic triad of hematuria, pain, and a flank
mass. RCC has the highest mortality rate of the genitourinary cancers, as more than a
third of patients with RCC will die from the disease. Novel and well-established
approaches for the early detection and management of renal cancer are therefore
extremely important.
Table 3 ILK as a prognostic marker in human cancer

Type of cancer Clinical significance Detection Reference
Melanoma Concomitant JWA and ILK expression is IHC Lu et al. 2013
closely correlated with survival
Non-small-cell Increased sILK is associated with adverse Serum ILK Posch
lung cancer survival quantified by et al. 2014
ELISA
Non-small-cell ILK is an adverse prognostic factor IHC Watzka
lung cancer et al. 2010
Non-small-cell ILK and Akt are mutually associated with IHC Okamura
lung cancer poor prognosis and are independent et al. 2007
prognostic factors
Colorectal ILK overexpression is associated with IHC, PCR, Li
cancer tumor progression and poor prognosis and Western et al. 2013b
blotting
Carcinomas of Positive ILK and PRDX1 expressions are IHC Li
the gallbladder closely related to the progression and poor et al. 2013a
prognosis
Chondrosarcoma ILK might serve as biological marker that IHC Papachristou
could accurately predict a high-grade et al. 2008
tumor
Pancreatic Association between strong expression of IHC Sawai
cancer ILK and poor prognosis et al. 2006
ILK has prognostic significance in several types of human tumors
The incidence of RCC directly correlates with the existence of genetic factors
such as von Hippel–Lindau disease, hereditary papillary renal cancer, and tuberous
sclerosis (Delahunt 2009). Other suggested risk factors include cigarette smoking,
obesity, diuretic use, high-protein diets, hypertension, kidney transplantation, and
HIV infection as well as exposure to petroleum-derived products, chlorinated sol-
vents, cadmium, lead, asbestos, and ionizing radiation (Cairns 2011).
RCC comprises a heterogeneous group of epithelial tumors with variable clinical
outcomes. Genetic hallmarks identify the various histological subtypes: clear-cell
RCC also called conventional RCC, papillary RCC, chromophobe RCC, collecting
duct RCC, and unclassified forms. Clear-cell renal cell carcinoma (CRCC) repre-
sents the most common histological type (80 %) and originates in renal proximal
tubular epithelium (Cairns 2011).
In 95 % of the cases, CRCC occurs without an obvious cause, but it may also
result from genetic factors such as von Hippel–Lindau disease. In both cases,
biallelic inactivating mutations of the VHL tumor suppressor gene occur (Kaelin
2007; Arjumand and Sultana 2012). The presence of germ-line mutations of the
VHL gene has been ascertained in 100 % of the cases of hereditary CRCC and in
two-thirds of sporadic CRCC cases. The VHL gene is inactivated by point muta-
tions, deletions, or hypermethylation of gene promoters (Li et al. 2007; Lianjie
et al. 2007).
Historically, CRCC prognosis has relied on clinicopathologic variables, such as

pathological stage, histological grade (Fuhrman nuclear grade), presence of
sarcomatoid differentiation, histological type, vascular invasion, and presence of
necrosis. However, carcinomas at identical stages and pathological grades may have
different biological behaviors.
At present, no markers segregate between aggressive and indolent behaviors.
Prognostic information regarding long-term CRCC outcome for an individual
patient is based primarily upon pathologic data obtained from definitive surgical
resection. Markers that can be detected through noninvasive methods and still
indicate the malignancy of small renal masses or predict growth rates are urgently
needed (Cairns 2011).
In recent years, many studies have focused on the molecular basis of RCC. The
discovery of reliable biomarkers in RCC could have an important impact on diag-
nosis, prognosis, and prediction of therapeutic benefit. To date, most biomarker
research has centered on by-products of the VHL pathway including VHL muta-
tions, vascular endothelial growth factor ligands and receptors, HIF, and carbonic
anhydrase IX. Elucidation of the VHL/HIF pathway has led to the successful
evaluation and regulatory approval of agents targeting the VEGF and mTOR axes.
Multiple research reports provide solid evidence for the implication of PI3K/Akt/
mTOR in CRCC carcinogenesis (Cojocaru et al. 2015). Other prognostic factors
have little consistency, thus they are not routinely used. The most commonly studied
include DNA ploidy, which strongly correlates with the Fuhrman grade, cell prolif-
eration by flow cytometry, Ki-67 or PCNA, CD44 expression, mucin1 (MUC 1), and
insulin growth factor 1 (IGF-1). The p53 mutation seems to correlate with poor
survival and metastatic disease in patients with early-stage disease.
Several potential RCC biomarkers have been identified, although none has
progressed beyond the discovery phase. Some of the most promising RCC markers
are proteins such as the B7-H1 factor, insulin-like growth factor II, and mRNA-
binding protein 3, which display strong independent prognosis but have not been
validated. Several studies have shown that C-reactive protein, an easily measured
routine marker, is produced by RCC cells and has significant prognostic value for
metastasis and mortality (Saito and Kihara 2011), but it was not further evaluated.
The best-known genetic abnormalities associated with CRCC include loss of
chromosome 3p (70–80 %) and gain of chromosome 5q (50–60 %). It is believed
that loss-of-function mutations in the remaining allele and VHL represent early
events in CRCC carcinogenesis, albeit not sufficient to result in tumor evolution.
Antiangiogenic therapies targeting vascular endothelial growth factor (VEGF) have
benefited patients with advanced RCC (Singer et al. 2011), but results are generally
thought to be cytostatic and do not cure patients. These therapies effectively inhibit
tumor progression through deprivation of oxygen and nutrition from the tumor
microenvironment but cannot block metastasis of CRCC cells.
However, these treatments do not target tumor cells directly, leaving room for
disease progression (Virtanen and Lehto 2004). A recent large-scale analysis
suggested that CRCCs have substantial genetic heterogeneity that involves genes
implicated in the regulation of methylation in 15 % of cases, confirming the
Table 4 ILK expression in RCC

Reference ILK expression
Sourbier (2007) ILK overexpression correlates with activation of Akt and induces cell
survival
Engelman ILK expression correlated with tumor invasion, increase in proliferation
et al. (2013) index, grade, loss of intercellular adhesion, and stage
Liu et al. (2013) ILK expression associated with p-Akt expression
Han et al. (2015) ILK expression regulates vimentin and E-cadherin expression by regulating
the EMT-related transcription factors Snail and Zeb1
ILK expression is correlated with tumor progression and activation of Akt in RCC
importance of epigenetic modification and truncating mutations of PRMB1 in 41 %

of cases (Audenet et al. 2012).
The implication of multiple genes suggests the existence of genetically distinct
CRCC subgroups, similarly to what has been shown for breast cancer. In fact, CRCC
studies established that these tumors may also be segregated based on gene expres-
sion profiles, and this effort provides important independent information. Work
focused on the genetic profile of tumors separated CRCC into two distinct subtypes,
named CCA and CCB. CCA tumors overexpress genes associated with hypoxia,
angiogenesis, and fatty acid metabolism and have a favorable prognosis compared to
CCB tumors. The latter group overexpresses a more aggressive panel of genes
associated with epithelial–mesenchymal transition, cell cycle, and wound healing
(Brannon et al. 2012). Interestingly, VHL profiles are similar in the two groups.
ILK represents a potential prognostic marker for CRCC as a consequence of its
tumorigenic properties as well as recent evidence of its immunoexpression in various
forms of human malignancies (Table 4). Increased ILK immunoreactivity in the
primary tumor is an adverse prognostic factor in a variety of preclinical and clinical
models of human cancer. Despite the small number of studies focusing on the role of
ILK in renal carcinogenesis, existing results provide relevant data and may have
important prognostic and therapeutic implications.
In fact, ILK immunoreactivity correlates with tumor severity in CRCC
(Engelman et al. 2013), and ILK is essential for invasion and metastasis of RCC
both in vitro and in vivo (Han et al. 2015). In the latter study, the RCC cell line from
a metastatic lesion showed the highest expression of ILK and displayed higher
expression of EMT-related transcriptional factors Snail and Zeb1. This finding
suggests that ILK immunoreactivity might be related to the metastatic potential of
RCC cells, which would point to ILK as a potential prognostic marker for CRCC.
Two recent studies have provided clear descriptions of the route of CRCC tumor
progression. There is strong evidence for the participation of ILK in CRCC carci-
nogenesis, involving the signaling pathway PI3K-Akt (Agouni et al. 2007; Liu
et al. 2013). ILK phosphorylates PKB/Akt at the Ser473 residue, leading to evasion
of apoptosis by inhibition of caspase-3 activation or activation of nuclear factor қ B
(NF-B қ). Activation of this factor may also stimulate cyclooxygenase-2 (COX-2),
which is implicated in tumor progression by stimulating angiogenesis and invasion.
Moreover, the PI3K-Akt-signaling pathway intersects with the signaling cascade
Fig. 3 Schematic representation of activation of the Akt/PKB pathway in human CRCC. The VHL
gene is mutated in a majority of sporadic CRCCs. As a result of mutation, the VHL protein cannot
target and degrade hypoxia-inducible factors (HIF) 1α and 2α. Excess HIF causes increased
transcription of downstream genes, such as vascular endothelial growth factor (VEGF), platelet-
derived growth factor (PDGF), and transforming growth factor alpha (TGF-α). The PI3K-Akt-
signaling pathway intersects with the signaling cascade triggered by VHL inactivation via
AKT-mediated induction of HIF-1α through mTOR. PTHrP or free-fatty acids activate NF-κB
through stimulation of ILK, one of the kinases with PDK-2 activity in human CRCC
triggered by VHL inactivation, as shown in Fig. 3, provided that Akt can upregulate
HIF-1α expression by increasing mTOR-mediated protein translation (Chou
et al. 2015). The same authors describe a novel regulatory feedback loop in which
hypoxia induces ILK expression through an HIF-1α-dependent mechanism and ILK,
in turn, stimulates HIF-1α expression through cell-type- and cell-context-dependent
pathways. The ILK-HIF-1α regulatory loop could underlie the maintenance of high
HIF-1α expression levels and the promotion of EMT under hypoxic conditions. The
small-molecule ILK inhibitor T315 can disrupt this regulatory loop in vivo and
suppress xenograft tumor growth, thereby providing proof of concept that targeting
ILK represents an effective strategy to block HIF-1α expression and aggressive
phenotypes in cancer cells.
Both VHL inactivation and ILK-mediated activation of the Akt pathway result in
HIF-1α overexpression, which promotes hyperangiogenesis in CRCC. Antiangiogenic
therapies do not prevent CRCC metastases, and ILK is essential for RCC invasiveness
and metastases (Han et al. 2015). Thus, the PI3K/ILK/Akt axis provides a promising
target, and ILK could be a predictive marker indicating the activity in this pathway in a
group of patients and guiding therapy toward ILK inhibition.
Notably, ILK expression can be measured at the protein level by immunohisto-
chemistry of histological CRCC samples, a low-cost and routinely available tech-
nique. Alternatively, serum ILK (sILK) could be measured in CRCC patients. This
possibility is supported by the fact that ILK has been detected in the serum of non-
small-cell lung cancer (NSCLC) patients. Moreover, sILK was preoperatively quan-
tified by ELISA in 50 newly diagnosed NSCLC patients. After surgery, patients
received follow-up examinations for a median interval of 2.5 years. Mean sILK was
2.3 times more elevated in the 16 patients who died as compared to the 34 patients
who survived (Posch et al. 2014).
Recently published data on urinary bladder cancer show that methylation markers
can predict the progression of early lesions to muscle-invasive bladder tumors with a
high degree of accuracy, suggesting that these markers can be used to diagnose
bladder cancer (Costa et al. 2011; Kandimalla et al. 2012). Similarly, tumor markers
such as urinary ILK may, in the near future, be used in CRCC allowing early
detection of disease.
Although the causes for ILK overexpression in various tumors have not been
fully unveiled, data reviewed here suggest that ILK may be involved in various
pathogenic mechanisms of human malignancies. It has become increasingly clear
that a complex network of protein–protein interactions controls the ILK-mediated
loss of cell adhesion, migration, growth, cell-cycle progression, and survival. On the
other hand, much like oncoproteins that stimulate cell proliferation during embry-
onic development, ILK plays essential roles during embryogenesis. Could a new
super-oncogene code for ILK?
Summary Points
• Integrin-linked kinase is a serine/threonine kinase implicated in cell-cycle control

via integration of integrins with the extracellular matrix.
• Integrin-linked kinase overexpression promotes anchorage-independent growth
and may induce tumorigenesis and invasion. Integrin-linked kinase suppresses
anoikis, suggesting an important role for it in oncogenic transformation, partic-
ularly in the process of metastasis.
• Integrin-linked kinase expression and activity increase in several human cancers,
such as prostate, colon, stomach, ovary, malignant melanomas, Ewing’s sarcoma,
primitive neuroectodermal tumor, non-small-cell lung cancer, bladder cell carci-
noma, basal cell carcinoma, squamous cell/adenosquamous carcinomas, adeno-
carcinoma of the gallbladder, malignant pleural mesothelioma, chondrosarcoma,
and pancreatic cancer.
• Renal cell carcinomas are tumors derived from epithelial cells of the renal tubules
and represent 80–85 % of all primary malignant tumors of the kidney and 2–3 %
of all cancers in adults. The most common renal cell carcinoma forms are clear-
cell renal carcinoma, papillary, and chromophobe.
• Clear-cell renal carcinomas represent the most frequent form of renal cell cancer,
accounting for 70–80 % of cases. These neoplasms may be family associated or,
in a majority of cases (95 %), sporadic. In both cases, tumors are related to loss of
function of the VHL gene, a tumor suppressor gene.
• Clear-cell renal carcinomas at identical stages and similar pathological grades
may exhibit distinct biological behavior. Therefore, other markers are needed for
prognostic evaluation.
• In 2013, Integrin-linked kinase immunoexpression was evaluated in a tissue
microarray of 45 human clear-cell renal carcinomas. Results suggested that
integrin-linked kinase immunoexpression is related to the loss of intercellular
adhesion and with the degree of differentiation. It also is positively correlated
with the proliferation index, invasion of the renal capsule and renal vein, tumor
size, and Robson stage.
• Recent investigation has shown that integrin-linked kinase may be essential for
renal cell carcinoma progression and metastasis, and that it regulates vimentin and
E-cadherin expression by regulating the extracellular matrix-related transcription
factors Snail and Zeb1.
• Integrin-linked kinase affects cell survival in human renal cell carcinomas
through the PI3K-Akt-signaling pathway. PTHrP activates ILK, which acts as a
PDK-2 in facilitating Akt phosphorylation at S473. These results indicate that the
PI3K/ILK/Akt/NF-κB axis may provide a target for therapeutic intervention in
clear-cell renal carcinomas.
• In 2013, free-fatty acids, through the activation of GPR40/ILK/Akt, were asso-
ciated with renal cell carcinoma. This finding revealed a new mechanism for the
correlation between metabolic disorders and renal carcinoma.
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Molecular Biomarkers and Treatments
for Renal Cell Carcinoma 45
Juan Chipollini, Martin J.P. Hennig, and Vinata B. Lokeshwar
Contents
Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1017
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1017
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1017
Systemic Therapy for Renal Cell Carcinoma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1020
Markers in Targeted Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1022
Carbonic Anhydrase IX (CAIX) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1022
VHL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1022
VEGF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1024
Circulating Endothelial Cells (CECs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1024
Circulating Tumor Cells (CTCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1025
Neutrophil Gelatinase-Associated Lipocalin (NGAL) and Matrix Metalloproteinase-9
(MMP-9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1025
MicroRNA (miRNA) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1026
mTOR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1026
Other RNA-Related Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1026
Inflammatory Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1027
Polymorphisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1027
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1028
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1028
J. Chipollini
Department of Urology, University of Miami Miller School of Medicine, Miami, FL, USA
e-mail: juan.chipollini@jhsmiami.org; jchipollini@med.miami.edu
M.J.P. Hennig
Department of Urology, University of L€
ubeck, L€
ubeck, Germany
e-mail: mjp.hennig@gmail.com
V.B. Lokeshwar (*)
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Georgia Regents
University, Augusta, GA, USA
e-mail: vlokeshwar@gru.edu

1016 J. Chipollini et al.
Abstract
Recent advances in the biological understanding of renal cell carcinoma (RCC)
have led to the identification of several molecular biomarkers with prognostic and
therapeutic value. Clinical trials have proven the effectiveness of targeted thera-
pies for advanced RCC and a growing body of work is underway to provide
molecular biomarkers for response of these new therapies which act primarily
against the vascular endothelial growth factor or mammalian target of rapamycin
pathways. Finding optimal biomarkers will help tailor targeted therapies
according to tumor and patient innate molecular factors and provide the best
individualized treatments that can prolong survival while minimizing adverse
side effects.
Keywords
Renal cell carcinoma • Molecular biomarkers • Prognostic markers • Vascular
endothelial growth factor • Tyrosine kinase inhibitor • Mammalian target of
rapamycin inhibitor
Abbreviations
ccRCC Clear cell renal cell carcinoma
CECs Circulating endothelial cells
CEPCs Circulating endothelial progenitor cells
CTCs Circulating tumor cells
ECOG Eastern Cooperative Oncology Group
FDA Food and Drug Administration
HIF Hypoxia-inducible factor
IFN Interferon
ILN Interleukin
LDH Lactate dehydrogenase level
LLN Lower limit of normal
Metastatic RCC Metastatic renal cell carcinoma
miRNA MicroRNA
mTOR Mammalian target of rapamycin
OS Overall survival
PFS Progression-free survival
PI3K Phosphatidylinositol 3-kinase
RECIST Response evaluation criteria in solid tumors
SAA Serum amyloid A
SEER Surveillance, Epidemiology, and End Results
ULN Upper limit of normal
45 Molecular Biomarkers and Treatments for Renal Cell Carcinoma 1017

VEGFR VEGF receptor(s)
VHL Von Hippel-Lindau
Key Facts
• Renal cell carcinoma (RCC) is one of the major genitourinary cancers. RCCs are
often found incidentally and surgically curable if still localized. Once RCC spreads
(metastasizes), it becomes a deadly disease. There are different types of RCC,
which have different invasive and biological behaviors. In order to effectively treat
these types, it is important to understand their differences, which are linked to
tumor-related or tumor-produced by-products, so-called biomarkers. Biomarkers
are used to help clinicians to choose the best treatment for patients and different
tumor types. They can be useful to improve diagnostics or to monitor the effect of
treatment. This chapter takes a closer look and gives an overview over many
different biomarkers important for treatment and diagnostics.
Definitions
Prognostic marker A biomarker that predicts disease progression, treatment

response, or mortality (disease specific or overall).
Renal cell carcinoma Renal cell carcinoma (RCC) is an adenocarcinoma of the

renal parenchyma.
Von Hippel-Lindau (VHL) Tumor suppressor gene frequently deleted in kidney

cancer
Introduction
In 2014, an estimated 63,920 new kidney and renal pelvis cancers will be diagnosed
in the USA, and approximately 13,860 will die of this disease (Siegel et al. 2014).
Renal cell carcinoma (RCC) is an adenocarcinoma of the renal parenchyma and
accounts for more than 80 % of kidney cancer in adults (King et al. 2014). Associ-
ated risk factors for RCC are cigarette smoking, obesity, and hypertension, and its
highest incidence and death rates are found among American Indians and Alaska
Natives, which may be due in part to high rates of obesity and smoking in these
populations.
There are several subtypes of RCC based on histopathological and genetic
characteristics. The most common histology is clear cell followed by papillary,
chromophobe, oncocytic, and rare types such as collecting duct and medullary
Type Incidence Mutations

o Clear Cell o 65 - 70% o VHL gene
o Papillary 1 o 5 - 7.5% o C-Met oncogene
o Papillary 2 o 5 - 7.5% o Fumarate

hydratase
Fig. 1 Incidence of RCC and genetic associations
carcinomas. Both hereditary and sporadic types have been associated with mutation
in the short arm of chromosome 3, with implication in tumor suppressor genes (i.e.,
VHL) or oncogenes (i.e., c-Met). Many of the hereditary types are associated with
familial syndromes such as Birt-Hogg-Dubé, hereditary papillary renal carcinoma,
and von Hippel-Lindau disease (see Fig. 1).
Surgical extirpation has been the mainstay of treatment for patients who present
with stage I–III RCC. For those who present late with advanced and metastatic
disease (stage IV), the overall clinical course of RCC varies; approximately 50 % of
patients survive less than 1 year and 10 % survive for more than 5 years (Yuen 2009).
Chemotherapy has been shown to be ineffective in the treatment of this disease, and
until recently, the only effective treatment for metastatic RCC (mRCC) was
cytokine-based immunotherapy with interferon (IFN)-α or interleukin (IL)-2.
Besides anatomic and histologic characteristics, others have evaluated various
clinical factors for prognostic purposes. Motzer and associates evaluated the rela-
tionship between pretreatment clinical features and survival in 670 patients enrolled
in phase II and phase III clinical trials of chemotherapy or immunotherapy at the
Memorial Sloan Kettering Cancer Center (MSKCC) in order to create a multivariate
model that can predict survival. Prognostic factors associated with shorter survival
were low Karnofsky performance status (KPS) (<80 %), lactate dehydrogenase level
(LDH) >1.5 the upper limit of normal (ULN), hemoglobin level < the lower limit
of normal (LLN), high corrected serum calcium level (>10 mg/dL), and absence of
nephrectomy. The median time to death in patients with 0 risk factors (favorable
risk), 1 or 2 risk factors (intermediate risk), and 3 risk factors (poor risk) were
20 months, 10 months, and 4 months, respectively (Motzer et al. 2004).
The MSKCC model was modified in 2002 and was restricted to 400 patients who
received IFN-α. Five variables were used as risk factors for short survival: low KPS,
high LDH, low serum hemoglobin, high corrected serum calcium, and time from
initial RCC diagnosis to start of IFN-α therapy of less than 1 year. The median time
to death for patients deemed favorable, intermediate, and poor risk was 30 months,
14 months, and 5 month, respectively (Motzer et al. 2002). Fortunately, advances in
the understanding of molecular and genetic factors of RCC have led to the emer-
gence of newer targeted therapeutic agents for its treatment. According to the
Surveillance, Epidemiology, and End Results (SEER) cancer registry, advanced
Anatomic Histologic Clinical

o Tumor size o Fuhrman grade o Performance
o Metastasis o Sarcomatoid status
o Venous features o Anemia
involvement o Microvascular o Hypercalcemia
o Lymph node Invasion o Lactate
status o Tumor necrosis dehydrogenase
Fig. 2 Anatomic, histologic, and clinical prognostic factors in RCC
Fig. 3 Treatments for RCC
RCC cases (regional and distant disease) in the targeted therapy era had a statistically
significant improvement in overall survival (OS) than the pre-targeted therapy group
(P < 0.001) (Vaishampayan et al. 2014) (see Fig. 2).
The treatment of advanced RCC has evolved significantly following the identi-
fication of the von Hippel-Lindau (VHL) gene and the subsequent development of
antiangiogenic therapies. There are currently eight US Food and Drug Administra-
tion (FDA)-approved agents available for the treatment of mRCC. Five of these
agents target either the vascular endothelial growth factor (VEGF) or its receptors
(VEGFR), two inhibit activity of the mammalian target of rapamycin (mTOR), and
one is a recombinant form of the endogenous cytokine IL-2. Each of these agents
provides clinical benefit to a subset of patients, and the overall outlook for patients
with mRCC is better today than it was 10 years ago (see Fig. 3).
There are still many unmet needs when it comes to treating mRCC. More
specifically, there are no validated systems that predict those patients at risk for
early metastatic disease that may benefit from some type of adjuvant therapy. In
addition, there is no clear mechanism to identify nonresponders as well as those

patients more susceptible to certain drug toxicities. For years, molecular biomarkers
from serum, tissue, or urine have been acknowledged as potential prognostic and
predictive adjuncts in the treatment of mRCC. In this review, we provide an update
on current investigations of the most well-known molecular biomarkers associated
with RCC.
Systemic Therapy for Renal Cell Carcinoma
The immune system has long been believed to have an important role in the
development of renal cancer. Reports of complete remissions have been described
in the literature, although it is estimated that the true incidence of spontaneous
regression is less than 1 %. Clear cell RCC (ccRCC) is the most common subtype,
occurring in approximately 80 % of patients. It is characterized by the inactivation of
the von Hippel-Lindau (VHL) tumor suppressor gene located on chromosome 3p25
and encodes for proteins in charge of proteolysis of hypoxia-inducible factors
(HIFs). Disruption of VHL results in production of defective VHL protein and
accumulation of HIF, which leads to the transcription of several hypoxia-inducible
genes such as vascular endothelial growth factor (VEGF), platelet-derived growth
factor (PDGF), epidermal growth factor receptor (EGFR), erythropoietin, and others
(Choueiri 2008; Bardos and Ashcroft 2004).
Overproduction of these angiogenic factors promotes tumor growth and progres-
sion after binding with its corresponding receptors. VEGF functions by promoting
the proliferation and migration of endothelial cells, stimulating vessel formation, and
inhibiting apoptosis. The crucial role of VEGF in RCC growth and metastasis has led
to the development of FDA-approved small molecule VEGF inhibitors such as
sunitinib, sorafenib, and axitinib, that target the VEGF and PDGF receptors, and
also of a recombinant human monoclonal anti-VEGF antibody, bevacizumab. All of
these agents have been shown to improve clinical outcomes in patients with clear cell
mRCC, although sunitinib is superior to sorafenib (Larkin et al. 2015).
The targeting of the mTOR pathway has also been proven effective in the
treatment of mRCC, although its mechanism of action is not completely understood.
Along with phosphatidylinositol 3-kinase (PI3K) and Akt, its signaling regulates cell
growth, metabolism, proliferation, and motility (Hudes 2009). The relevance of
PI3K/Akt/mTOR pathway led to the development of the new therapies such as
temsirolimus which was shown to prolong OS and progression-free survival (PFS)
compared to IFN alone in poor-risk patients (Hudes et al. 2007). Another mTOR
inhibitor, everolimus, was approved after its phase III trial was halted early once
interim analysis showed significant improvement in PFS for patients who had failed
previous VEGF-targeted therapy (Motzer et al. 2008). Table 1 summarizes current
therapies that target the VHL-VEGF and PI3k/Akt/mTOR pathways.
45
Table 1 FDA-approved targeted agents for the treatment of mRCC

Name Class Pivotal study Primary endpoint HR (95 % CI) Common side effects
Sunitinib TKI Motzer PFS: 11 vs 5 months (INF-α) 0.42 (0.32–0.54) Diarrhea, N/V, HTN, hand-foot
et al. (2007) p < 0.001 syndrome
Sorafenib TKI Escudier OS: 17.8 vs 14.3 months (placebo) 0.78 (0.62–0.97) Diarrhea, HTN, hand-foot
et al. (2009) p < 0.05a syndrome, rash/desquamation
Pazopanib TKI Sternberg PFS: 9.2 vs 4.2 months (placebo) 0.46 (0.34–0.62) Diarrhea, HTN, hair color
et al. (2010) p < 0.0001 changes, N/V
Axitinib TKI Rini PFS: 6.7 vs 4.7 months (sorafenib) 0.665 Diarrhea, HTN, fatigue
et al. (2011) (0.544–0.812)
p < 0.0001
Bevacizumab VEGF Escudier OS: 23.3 (bevacizumab + IFN) vs 0.91 (0.76–1.10) Fatigue, asthenia, proteinuria,
monoclonal et al. (2010) 21.3 months (IFN + placebo) p = 0.336 HTN
antibody
Temsirolimus mTOR inh Hudes OS: 10.9 (alone) vs 7.3 (IFN) vs 0.73 (0.58–0.92) Rash, edema, hyperglycemia,
et al. (2007) 8.4 months (both) p = 0.008 hyperlipidemia
Everolimus mTOR inh Motzer PFS: 4 vs 1.9 months (placebo) 0.30 (0.22–0.40) Stomatitis, rash, fatigue,
Molecular Biomarkers and Treatments for Renal Cell Carcinoma
et al. (2008) p < 0.0001 pneumonitis

HTN hypertension, mo months, mTOR inh mammalian target of rapamycin inhibitor, N/V nausea or vomiting, OS overall survival, PFS progression-free
survival, TKI tyrosine kinase inhibitor, VEGF vascular endothelial growth factor
a
Analysis after crossover from placebo
1021
Markers in Targeted Therapy
Efficacy of markers is challenging due to the molecular heterogeneity in advanced

RCC. These molecular differences can account for the mixed results and treatment
responses of targeted therapies. Several groups have confirmed that molecular
markers may predict tumor behavior, in addition to response to treatment. Molecular
markers could become helpful adjuncts in deciding particular therapies and dosage
for optimal treatment of patients. Table 2 summarizes selected prognostic markers in
advanced RCC.
Carbonic Anhydrase IX (CAIX)
Tissue-based biomarkers have been used to predict progression and survival out-
comes. CAIX is a well-known immunohistochemical marker in the diagnosis of
RCC. It is a cell surface enzyme that is overexpressed in upward of 90 % of ccRCC
cases, and its expression is regulated by HIF-1α and the inactivation of VHL (Sun
et al. 2011). A retrospective study by Bui et al. showed that decreased CAIX levels
are associated with poor survival in advanced RCC. Low CAIX (85 %) staining
was an independent poor prognostic factor for survival in mRCC ( p < 0.001), and
overall expression of CAIX decreased with development of metastasis (Bui
et al. 2003). In a recent meta-analysis of 2611 patients, low CAIX expression was
associated with poor disease-specific survival (DSS) (HR = 1.89, 95 %CI:
1.20–2.98), unfavorable PFS (HR = 2.62, 95 %CI: 1.14–6.05), and worse OS
(HR = 2.03, 95 %CI: 1.28–3.21). Furthermore, low CAIX expression was signifi-
cantly associated with lymph node metastases (OR = 0.31, 95 %CI: 0.15–0.62) and
higher tumor grade (OR = 0.41, 95 % CI: 0.31–0.5; (Zhao et al. 2014)). In general, a
high expression of CAIX appears to be a favorable prognostic marker, and it may
even contribute to improved response rates for IL-2 therapy (Atkins et al. 2005).
VHL
ccRCC is characterized by VHL inactivation through biallelic gene loss due to a dual
hit in most patients (Garcia-Donas et al. 2013). It has been proposed that
VHL-inactivated tumors are more susceptible to VEGF-targeted therapies (Choueiri
2008). It is inactivated in almost all patients with VHL syndrome and in approxi-
mately 70 % of sporadic ccRCC (Yao et al. 2002) VHL mutations (intragenic or
hypermethylation) were strongly associated with better cancer-free survival and
cancer-specific survival for 134 patients with stage I–III ccRCC ( p = 0.024 and
0.023, respectively; (Yao et al. 2002)).
Table 2 Selected prognostic markers in advanced RCC

Author n Specimen Study Marker Therapy Endpoint
Bui 321 Tissue RT CAIX n/a DSS
et al. (2003)
Choueiri 123 Tissue RT VHL (LOF Sunitinib, RR
et al. (2008) mutation) sorafenib,
axitinib,
bevacizumab
Rini 43 Tissue RT VHL (mutation Bevacizumab TTP
et al. (2006) or methylation) + IFN-α
Escudier 900 Serum RCT VEGF Sorafenib OS, RR
et al. (2009)
Harmon 30 Serum RCT VEGF-A, Sunitinib or PFS, OS
et al. (2014) VEGF-C, IFN-α
sVEGFR-3, IL-8
Deprimo 63 Serum PT VEGF, Sunitinib RR
et al. (2007) sVEGFR-2,
PIGF, sVEGFR-3
Porta 85 Serum RT NGAL, VEGF Sunitinib PFS
et al. (2010)
Steffens 1,161 Serum RT CRP n/a CSS
et al. (2012)
Armstrong 404 Serum RT LDH Temsirolimus OS
et al. (2012) or IFN-α
Garcia- 101 Serum, PT VEGFR3 Sunitinib PFS,
Donas saliva polymorphisms toxicity
et al. (2011) (rs307826,
rs307821),
CYP3A5*1
Xu 397 Serum PT Polymorphisms Pazopanib PFS, RR
et al. (2011) in IL-8 (2767TT/-
251TT), HIF1A
(1790G), NR1ǀ2
(-25385TT),
VEGF-A
(-1498CC)
Lambrechts 110 Serum RCT Polymorphism in IFNα-2a + PFS, OS
et al. (2012) VEGFR1 bevacizumab
(rs799341/ or placebo
rs9554316,
rs9513070)
CAIX carbonic anhydrase IX, CSS cancer-specific survival, DSS disease-specific survival, IL-8
interleukin-8, IFN-α interferon alpha, LOF loss of function, OS overall survival, PIGF placenta
growth factor, PFS progression-free survival, PT prospective, RCT randomized control trial, RR
response rate, RT retrospective, sVEGFR soluble vascular endothelial growth factor receptor, TTP
time to tumor progression, VHL von Hippel-Lindau protein
A retrospective analysis of temsirolimus-treated patients observed no difference

in outcome for tumors harboring VHL mutations. CAIX was also studied but found
no correlation with objective response or clinical benefit (Cho et al. 2007). Another
study of patients treated with sunitinib, sorafenib, axitinib, or bevacizumab at the
Cleveland Clinic and University of California, San Francisco, showed VHL inacti-
vation had a Response Evaluation Criteria in Solid Tumors (RECIST)-defined
response rate of 41 % vs 31 % for patients with wild-type VHL ( p = 0.34).
However, PFS and OS did not correlate with VHL status (Choueiri et al. 2008).
VEGF
VEGF is a glycoprotein that plays a critical role in the induction of endothelial cell
division and migration by interacting with the transmembrane tyrosine kinase
receptors VEGFR-1 and VEGFR-2 selectively expressed on vascular endothelial
cells (Rini et al. 2005). Another VEGF-receptor family member is VEGFR-3, which
is mainly expressed in lymphatic, endothelial cells (Iljin et al. 2001). Porta
et al. reported VEGF baseline values were significant predictors of PFS for patients
treated with sunitinib on multivariate analysis (RR = 1.96, 95 %CI: 1.47–2.45;
(Porta et al. 2010)). The use of VEGF as a prognostic biomarker was also evaluated
in the TARGET study of 900 patients randomly assigned to sorafenib versus
placebo. Baseline VEGF levels correlated with Eastern Cooperative Oncology
Group (ECOG) PS, MSKCC score, PFS, and OS in univariate and multivariate
analyses. Results suggested that patients in the 75th percentile of VEGF level may
experience greater benefit from sorafenib (HR = 0.27, 95 % CI: 0.15–0.460) than
those with low VEGF (HR = 0.58, 95 %CI: 0.43–0.78; (Escudier et al. 2009)). An
update demonstrated elevated VEGF levels correlated with shorter PFS in placebo-
treated patients on univariate analysis (HR = 1.645, 95 % CI: 1.19–2.28) but failed
to show as an independent prognostic factor on multivariate analysis (Pena
et al. 2010). No links have been found so far regarding the relation of VEGFR-3
and RCC. A study by Virman et al. could only confirm that a low expression of
CD31 was significantly associated with poorer survival but failed to show any
association toward VEGFR-3 (Virman et al. 2015).
Circulating Endothelial Cells (CECs)
A growing body of evidence shows that CECs and circulating endothelial progenitor
cells (CEPCs) contribute to tumor angiogenesis. RCC shows a high expression of
VEGF, which plays a role in the mobilization, recruitment, homing, and incorpora-
tion of endothelial progenitor cells for tumor vasculogenesis during tumor progres-
sion (Ding et al. 2008). An analysis of 53 patients showed the preoperative CEPC
level correlated with serum VEGF level in patients with RCC (r = 0.710,
p < 0.001). The mean CEPC level in patients with RCC was significantly higher
than in patients with benign renal tumors and healthy controls (0.281 % vs 0.073 %
and 0.076 %, respectively; (Yang et al. 2012)). Recently, Gu and associates were able
to isolate and culture CEPCs from patients with RCC and found CEPC levels
significantly higher than control group (0.276 % vs 0.086 %, p < 0.001). Further-
more, earlier emergence of CEPC colonies (6.72 vs 14.67 days), higher number of
colonies (10.06 vs 1.83), and higher cell culture success rate (87.8 % vs 40 %; each
p < 0.001) were also noted in RCC patients (Gu et al. 2015). Given that CECs
decrease after nephrectomy in patients with localized RCC, CECs may be a potential
marker of progression and recurrence, although the difficulty in quantifying them
reliably has decreased the enthusiasm for their use a biomarker.
Circulating Tumor Cells (CTCs)
Although there has been great interest in CTCs as biomarkers in RCC, the detection
of RCC cells in the blood has been hindered by the absence of markers that are
specific enough for RCC cells against the background of hematological cells
(Hernandez-Yanez et al. 2012). The only commercially available FDA-approved
platform for the enumeration of CTCs is the CellSearch™ assay which relies on the
expression of epithelial cell adhesion molecule (EpCAM) and cytokeratins for
cellular detection (Mego et al. 2011). Although this system has been useful for
detection of CTCs in metastatic breast, colorectal, and prostate cancer patients, its
use has been limited in RCC due to the lack of EpCAM positivity of RCC cells.
Other filtration-based technologies have been developed to allow for antigen-
independent isolation of CTCs based on size and cytomorphological features in
comparison to hematological cells (Vona et al. 2000). Using such methods,
El-Heliebi et al. aimed to identify CTCs in the blood of 30 RCC patients; nonethe-
less, they were unable to distinguish epithelial from endothelial non-hematologic
cells based on morphological criteria alone (El-Heliebi et al. 2013).
Neutrophil Gelatinase-Associated Lipocalin (NGAL) and Matrix

Metalloproteinase-9 (MMP-9)
NGAL was identified as 25 kDa protein covalently linked to MMP-9 in neutrophils.

NGAL protects MMP-9 from degradation, thereby preserving the activity of MMP-9
which has been shown to play a critical role in tumor cell invasion and metastasis in
many cancers (Yan et al. 2001). NGAL, along with VEGF, showed significant
prediction of PFS in 85 patients treated with sunitinib. NGAL level above threshold
resulted in increased risk of progression (RR = 1.86, 95 % CI: 1.42–3.019) and
decreased PFS compared to those below the NGAL threshold level (3.4 vs
8.2 months, p = 0.03; (Porta et al. 2010)). The role of MMP-9 and other metallopro-
teinases has been investigated in RCC progression. Kugler et al. found that MMP-2
and MMP-9 are elevated in RCC tumor tissue with more expression seen in
advanced tumors (Kugler et al. 1998). One study of paraffin-embedded tissue
samples from 232 patients revealed MMP-9 had a high correlation with systemic
symptoms (OR = 1.02, 95 %CI: 0.36–2.62; (Kawata et al. 2006)). Although there is
tissue evidence, other studies have failed to find any correlation of MMP-2 and
MMP-9 activity in serum and urine (Di Carlo 2012).
MicroRNA (miRNA)
miRNAs are endogenous ~23 nt RNAs that have gene-regulatory roles by pairing to
the mRNAs of protein-coding genes to direct their posttranscriptional repression.
Due to having crucial impact on the regulation of cell growth, evasion of apoptosis,
angiogenesis, tissue invasion, and metastasis, miRNAs have become a focus of
intensive investigation throughout recent years (Bartel 2009). Expression analyses
have shown that a variety of miRNAs are either up- or downregulated in RCC when
compared to normal kidney tissue. Patients with RCC have higher levels of circu-
lating miRNA-221 and miRNA-222 when compared to healthy individuals. Fur-
thermore, even higher levels of these miRNAs were observed in mRCC patients
when compared to nonmetastatic patients (Teixeria et. al. 2014). Signatures of
certain combinations of miRNAs, e.g., miRNA-451, miRNA-221, miRNA-30a,
miRNA-10b, and miRNA-29a, may also be useful in differentiating between meta-
static and nonmetastatic RCC (Heinzelmann et. al. 2011). However, it remains to
be determined whether changes in the expression of specific miRNAs that
were reported in different studies are either the reason or the result of RCC
(Li et. al. 2015).
mTOR
As described above, the targeting of mTOR provides one of the best approaches for
clinicians to effectively treat mRCC. Predictors for success of this treatment option
are still under investigation. In 2015 Bodnar et al. showed that for histological grade
1 and 2 tumors, increased LDH level before treatment and the PIK3CA gene variant
rs6443624 were predictors of treatment-related effects for everolimus (Bodner et. al.
2015).
Other RNA-Related Markers
Due to the advances in genomic profiling and easy to use laboratory techniques, such
as RT-(q)-PCR, it has become easier to measure the expression of genes and, thereby,
to identify up- and downregulated genes in RCC patients. For example, CD1d is a
member of the CD1 family of glycoproteins that is expressed on the surface of
various human antigen-presenting cells and is involved in the presentation of lipid
antigens to T cells. CD1d expression was found to significantly correlate with stage,
grade, higher relapse rates, poor cancer specific, and OS (Chong et. al. 2015).
Inflammatory Markers
Studies have investigated the role of inflammatory proteins and cytokines in RCC.
Serum amyloid A (SAA) and C-reactive protein (CRP) have been identified as
potential biomarkers for RCC. Fischer et al. analyzed blood samples of 115 patients
with localized or advanced RCC. Preoperative values of IL-6, CRP, and SAA were
significantly higher in advanced versus localized RCC cases. SAA had the best
diagnostic accuracy with sensitivity of 78 % and specificity of 82 % compared to
CRP (69 % and 82 %) and IL-6 (44 % and 94 %; (Fischer et al. 2012)). In another
study of 422 RCC patients, SAA showed prognostic significance with higher levels
in advanced stage disease (i.e., T3-M1; (Mittal et al. 2012)). Large studies have
correlated high serum preoperative CRP levels as a significant prognostic factor for
unfavorable clinical outcome (Steffens et al. 2012; de Martino et al. 2013; Johnson
et al. 2010). A preoperative CRP level of >10 mg/l indicates a 2.5-fold increased risk
of death from RCC when compared to a preoperative CRP level of 4 mg/l (Steffens
et al. 2012).
LDH has long been recognized as a prognostic biomarker as part of the MSKCC
risk classification model for mRCC (Motzer et al. 2004). LDH is an enzyme
involved in anaerobic glycolysis and is regulated by the PI3K/Akt/mTOR pathway
(Armstrong et al. 2012). A phase III trial evaluated pre-and posttreatment LDH
levels in 404 poor-risk patients treated with temsirolimus or IFN-α. Patients with
LDH >1 ULN indicated a hazard ratio for death of 2.8 with a median survival
time of 6.9 months for patients treated with temsirolimus versus 4.2 months median
survival for those treated with IFN-α (Armstrong et al. 2012). This study validated
that a higher baseline LDH is a predictor of OS in patients treated with mTOR
inhibitors.
Polymorphisms
Genetic factors have been associated with deficiencies of certain therapies according
to their targeted pathway. Various VEGF polymorphisms have been found as risk
factors for renal cancer (Bruyere et al. 2010). The association between VEGF single
nucleotide polymorphisms (SNPs) and sunitinib has been the most widely studied
among the anti-VEGF therapies. In an observational, prospective study of
101 patients, sunitinib tolerability and RECIST response were assessed in correlation
with 16 polymorphisms of nine genes involved in sunitinib pharmacokinetics. Two
VEGFR3 polymorphisms (rs307826 and rs307821) and CYP3A5 (rs776746) were
associated with reduced PFS (HR = 3.57 and HR = 3.31) and increased toxicity
(HR = 3.75), respectively. Three additional polymorphisms in ABCB1, ABCG2,
and VEGFR2 (rs1128503, rs2231142, and rs1870377) showed a tendency for worse
response although not significant (Garcia-Donas et al. 2011). Other studies that
investigated tolerability of sunitinib in RCC patients found an increased risk of
leukopenia associated with CYP1A1 (OR = 6.24), FLT3 (OR = 2.8), the NR1ǀ3
haplotype (OR = 1.74), mucosal inflammation with CYP1A1 (OR = 4.03), and
hand-foot syndrome with ABCB1 haplotype (OR = 2.56; (van Erp et al. 2009)).
Using data from the recent phase III COMPARZ trial of pazopanib- vs sunitinib-
treated patients, two IL8 SNPs (rs1126647 and rs4073) showed significant associ-
ation with poor OS outcomes ( p 0.05); rs1126647 also showed association with
OS in another independent dataset of sunitinib-treated patients ( p 0.05)
(Xu et al. 2015). In the AVOREN bevacizumab trial, VEGFR1 SNP rs7993418, as
well as rs9554316 (full linkage) and rs9513070, correlated with PFS (HR = 1.81
and 1.68, respectively), but only rs7933418 remained significant after covariate
adjustment (Lambrechts et al. 2012).

Conditions
Greater understanding of the biological heterogeneity of RCC has led to the discov-
ery of a number of biomarkers with prognostic and therapeutic value. These prog-
nostic biomarkers show an opportunity to individualize treatment therapies based on
patient and tumor molecular background and require further external validation
before they can be incorporated in routine clinical practice. Eventually, markers
can help the health-care community guide available targeted therapies toward
improving prognostication, risk stratification, and survival outcomes for patients
with advanced RCC and even cut down on costs for the health-care system.
Summary Points
• Clinical course and subtypes of kidney cancer

• Treatment strategies for renal cell carcinoma
• Biomarkers for metastasis
• Biomarkers for treatment
• Biomarkers for clinical decision making
Acknowledgments Grant Support: 5R01CA176691-02; Martin J. P. Hennig is a fellow of the

Biomedical Exchange Program and receives funding from the German Academic Exchange Service
(DAAD).
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M-Type Phospholipase A2 Receptor
as a Biomarker in Kidney Disease 46
Elion Hoxha and Rolf A.K. Stahl
Contents
Key Facts of Membranous Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1034
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1034
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1035
Pathogenesis of Primary and Secondary Membranous Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . 1036
First Antigens Discovered in Human Membranous Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1036
Clinical Course and Treatment of Membranous Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1037
The Discovery of PLA2R Antibodies in Membranous Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . 1038
The Role of PLA2R Antibodies Differentiating Primary from Secondary Membranous
Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1040
THSD7A Is Another Target Antigen in Membranous Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . 1041
The Role of PLA2R Antibodies in the Clinical Management of Patients with Membranous
Nephropathy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1042
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1044
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1045
Abstract
The identification of the M-type phospholipase A2 receptor as a major antigen in
membranous nephropathy is leading to a paradigm shift in our understanding of the
pathomechanisms involved in this disease as well as the clinical management of
these patients. On the one side, the autoimmune nature of the disease, in which
circulating autoantibodies bind to podocytic antigens, is confirmed, and on the
other side, a whole new area of research on this condition has opened. Genome-
wide association studies show an association of membranous nephropathy with
PLA2R and HLA loci, and major antibody-binding epitope regions have been
identified on the PLA2R. These findings might enable us in the future to better
E. Hoxha • R.A.K. Stahl (*)

University Medical Center Hamburg-Eppendorf, Hamburg, Germany
e-mail: ehoxha@uke.de; rstahl@uke.de

1034 E. Hoxha and R.A.K. Stahl
characterize the processes leading to the development of these antibodies, thus

leading to new treatment options. At the same time, PLA2R antibody findings are
playing an increasing role in the differential diagnosis of primary from secondary
membranous nephropathy and treatment management helping to better adapt
therapy to the risk profile and disease activity of an individual patient and are a
perfect example of how experimental research can lead to individualized medicine.
Keywords
Membranous nephropathy • PLA2R antibodies • Differential diagnosis • Treat-
ment • Prognosis
Abbreviations
ACTH Adrenocorticotropic hormone
PLA2R M-type phospholipase A2 receptor
THSD7A Thrombospondin type-1 domain-containing 7A
Key Facts of Membranous Nephropathy
Primary membranous nephropathy is an autoimmune disease, caused by binding of

circulating autoantibodies on antigen(s) on the podocytes.
The clinical course of primary membranous nephropathy is very variable ranging
from spontaneous remission of disease in approximately one-third of patients to
development of end-stage renal disease in another third of patients.
Considering the high rate of spontaneous remissions, the severe potential adverse
effects of an immunosuppressive therapy, and the lack of prospective biomarkers
to characterize the risk for disease progression, the decision which patients should
be treated with immunosuppressants is challenging.
PLA2R and, less frequently, THSD7A are target antigens in primary membranous
nephropathy.
There is increasing evidence that PLA2R antibody levels are reliable biomarkers in
primary membranous nephropathy.
Definitions
Immune deposits Immune deposits can be detected by electron microscopy in the

kidney tissue of patients with membranous nephropathy. They are localized in the
subepithelial space and consist of antibodies against a target epitope, which is
localized on the podocyte, antigen, and molecules of the complement system.
In situ immune-complex formation In situ immune-complex formation is pre-

sumably a key step leading to the development of membranous nephropathy. In this
regard, in situ formation describes the binding of circulating antibodies to an
46 M-Type Phospholipase A2 Receptor as a Biomarker in Kidney Disease 1035
endogenous podocytic antigen; thus, the antibody-antigen complexes are formed

directly at the subepithelial space, where they can be detected in renal biopsies.
Nephrotic syndrome Nephrotic syndrome is a kidney disorder characterized by

loss of large amounts of protein (>3.5 g/1.73 m2 body surface area per day) in the
urine, low levels of serum albumin, high levels of cholesterol in the blood and
edema. A nephrotic syndrome is caused by a number of diseases such as diabetic
nephropathy, amyloidosis, or kidney-specific diseases such as membranous
nephropathy, focal segmental glomerulosclerosis, minimal change disease, etc.
Spontaneous remission Spontaneous remission of proteinuria in patients with

membranous nephropathy is a frequent event. A remission of proteinuria is usually
defined as a decrease of proteinuria by at least 50 % to a level of <3.5 g per day
(partial remission) or <0.3 g per day (complete remission). The remission of
proteinuria is considered spontaneous when the patient did not receive any immu-
nosuppressive medication, but was treated with supportive therapy only.
Subepithelial space Subepithelial space is the area between the glomerular base-
ment membrane and the podocytes in the kidney.
Introduction
Membranous nephropathy is a common cause of nephrotic syndrome in adults

(Ponticelli and Glassock 2014). At presentation, patients usually show a normal or
only slightly impaired renal function. The clinical hallmark of the disease is the large
proteinuria and the nephrotic syndrome (Ponticelli and Glassock 2014). Often
patients also show an arterial hypertension. The diagnosis is made by renal biopsy
(Fig. 1), which typically shows (a) no pronounced glomerular infiltration of inflam-
matory cells, while the glomerular capillary wall might appear thickened by light
microscopy, (b) a granular positivity for immunoglobulins along the glomerular
basement membrane by immunohistochemistry or immunofluorescence, and
Fig. 1 PAS staining, immunohistochemical staining for IgG, and transmission electron microscopy
of a renal biopsy from a patient with membranous nephropathy with kind permission of Prof. T. Wiech
(c) electron-dense immune deposits strictly on the epithelial side of the glomerular
basement membrane. In most cases membranous nephropathy is an autoimmune
disease restricted to the kidney, where autoantibodies against podocytic antigens
lead to the formation of the subepithelial immune deposits (Glassock 2012). For the
last 50 years, great efforts have been made to identify the podocytic antigen(s) and
the corresponding autoantibodies responsible for the development of this disease,
leading to the discovery of the M-type phospholipase A2 receptor (PLA2R) as a
major antigen in membranous nephropathy (Beck et al. 2009).
Pathogenesis of Primary and Secondary Membranous

Nephropathy
In some cases membranous nephropathy is clinically presented in the context of a

systemic disease, e.g., systemic lupus erythematosus, malignant diseases, and chronic
active viral infection (e.g., hepatitis B), or use of certain medications (e.g., nonsteroidal
anti-inflammatory drugs, gold, penicillamine) (Glassock 2013). These cases make
approximately 20 % of all cases of membranous nephropathy. In the absence of detailed
knowledge on the pathophysiological processes leading to membranous nephropathy,
all cases, in which no secondary cause of disease could be identified, were considered to
be primary or idiopathic. For a long time, it has been postulated that immune processes
play a central role in the development of primary membranous nephropathy. Most of
the evidence derives from experimental work on the Heymann nephritis, a rat model of
primary membranous nephropathy (Heymann et al. 1959). It was shown that circulating
autoantibodies to antigens on the podocytes lead to an in-situ immune-complex
formation, complement activation, and proteinuria (Beck and Salant 2014). While
these findings were very important for more in-depth investigation and better under-
standing of the development of membranous nephropathy, the responsible podocytic
antigen in Heymann nephritis, megalin, is not found in humans (Ronco and Debiec
2011). Thus, it was postulated that primary membranous nephropathy is an autoim-
mune disease, caused by binding of circulating autoantibodies on antigen(s) on the
podocytes; however, the responsible antigen remained elusive for years to come.
First Antigens Discovered in Human Membranous Nephropathy
The first direct evidence that in situ immune-complex formation is responsible for
development of primary membranous nephropathy in humans came in 2002 (Debiec
et al. 2002). A newborn from a woman genetically deficient for neutral endopepti-
dase developed membranous nephropathy due to the fetomaternal alloimmunization
of the mother to neutral endopeptidase in a previous pregnancy. Transplacental
passage of the autoantibodies to neutral endopeptidase leads to the development of
membranous nephropathy in the newborn. These cases of membranous nephropathy
are very rare, and neutral endopeptidase was not found to be the target antigen in
membranous nephropathy in adults. However, these findings were important,
showing that binding of circulating autoantibodies to a podocytic antigen leads to the

development of membranous nephropathy (Debiec et al. 2002). Over the course of
years, other mechanisms were attributed to the formation of immune deposits and
development of membranous nephropathy. In children it was shown that circulating
autoantibodies against bovine serum albumin bind to the antigen, which is natively
not expressed on the podocytes, but rather “planted” in the first months or years of
life (Debiec et al. 2011). These children develop membranous nephropathy, which
usually resolves without need of specific treatment. This form of early-childhood
membranous nephropathy has not been described in adults, and serum bovine
albumin was not the target antigen in membranous nephropathy in adults. Some
evidence exists that circulating immune complexes can lead to development of
immune deposits; however, one would expect immune deposits deriving from
circulating immune complexes to also be present in the mesangium and the endo-
thelial side of the glomerular basement membrane, which is not the case in mem-
branous nephropathy (Fujigaki et al. 1993). Thus, it is not known which role (if any)
such processes might play in human membranous nephropathy.
Clinical Course and Treatment of Membranous Nephropathy
The clinical course of primary membranous nephropathy ranges from spontaneous

remission of disease in approximately one-third of patients to development of
end-stage renal disease in another third of patients (Schieppati et al. 1993). The
remaining patients have a persisting proteinuria but a stable renal function. Patients
with spontaneous remission have an excellent prognosis and don’t need specific
treatment. On the other side, patients who don’t have a remission of disease have a
very unfavorable prognosis (Polanco et al. 2010). Different immunosuppressants
have been shown to be effective in patients with primary membranous nephropathy
(Waldman and Austin III 2012). However, considering the high rate of spontaneous
remissions and the severe potential adverse effects of an immunosuppressive ther-
apy, the decision which patients should be treated is challenging. Different models
have been proposed in order to define the patient’s risk for disease progression
(Branten et al. 2005; Cattran et al. 1997; van den Brand et al. 2012). Proteinuria and
serum creatinine play a central role in these models; however, without knowledge on
the pathophysiological and immunological processes leading to disease progress, no
conclusive decision could be made which patients should receive immunosuppres-
sion. This is due to a number of factors: (i) spontaneous remission may take more
than 1 year to happen, (ii) patients with very high proteinuria can still have a
spontaneous remission, (iii) patients with low proteinuria may develop nephrotic
proteinuria, or other factors, and there were no markers which would specifically
differentiate these patients prospectively (Polanco et al. 2010; Hladunewich
et al. 2009). Therefore, clinical management of patients with membranous nephrop-
athy relied on short-term and midterm clinical observation, reacting to eventual
disease progress (decrease of renal function), failure of disease recovery (persistence
of proteinuria), or severe uncontrollable symptoms (severe edema, not reacting to

supportive treatment).
Treatment guidelines recommend a supportive treatment of all patients with
primary membranous nephropathy for at least 6 months, consisting of inhibitors of
the renin-angiotensin axis, diuretics, lipid-lowering drugs, and, depending on the
individual thromboembolic risk of a patient, anticoagulants. Patients with no remis-
sion of proteinuria and decrease in renal function and who are at a high risk for
disease progression should then be treated with immunosuppressive drugs (KDIGO
2012). Alkylating agents have been shown to be effective in inducing remission of
proteinuria and preserve renal function (Hofstra and Wetzels 2010). For these agents
the most long-term data on treatment of membranous nephropathy are available
(Ponticelli et al. 1992, 1998; Jha et al. 2007). Cyclophosphamide seems to be as
efficacious as chlorambucil, however, with fewer side effects (Ponticelli et al. 1998).
Calcineurin inhibitors are also effective in inducing remission of proteinuria in
patients with membranous nephropathy; however, high relapse rates upon cessation
of treatment have been reported (Cattran et al. 2001, 1995; Praga et al. 2007; Caro
et al. 2014). At the same time, use of alkylating agents might be associated with a
better preserved renal function, especially in patients with impaired renal function
(Howman et al. 2013). Rituximab has been shown to be efficacious in inducing
remission of proteinuria; however, long-term data on renal function are still lacking
(Ruggenenti et al. 2012; Fervenza et al. 2010). Most importantly, up to now, there are
no randomized, controlled studies comparing the effectiveness and safety of
rituximab to the other treatment options. The use of glucocorticoids alone is not
recommended in patients with membranous nephropathy (Ponticelli et al. 1992).
Other immunosuppressants such as mycophenolate mofetil, ACTH, etc., are also
used for treatment of membranous nephropathy; however, there are not sufficient
data on their long-term efficacy (Chan et al. 2007; Dussol et al. 2008; Ponticelli
et al. 2006; Bomback et al. 2012).
The Discovery of PLA2R Antibodies in Membranous Nephropathy
In 2009, a pivotal work from Beck et al. identified the PLA2R as a major target antigen
in patients with primary membranous nephropathy (Beck et al. 2009). Autoantibodies
against PLA2R are found in the blood of patients with primary membranous nephrop-
athy (Fig. 2), but not in healthy controls, in patients with secondary membranous
nephropathy or other glomerular diseases (Hoxha et al. 2011). PLA2R antibodies were
found to be detectable in patients with active disease or with a relapse; however, they
were negative in patients with stable remission of disease (Hofstra et al. 2011). The
fact that the PLA2R antibodies co-localize with IgG4 in, and were eluted from the
immune deposits of a kidney with membranous nephropathy, makes a direct patho-
genetic role of these antibodies more plausible (Beck et al. 2009). This is further
highlighted by the finding that a patient who was on dialysis because of membranous
nephropathy had a very rapid relapse of membranous nephropathy after renal trans-
plantation and was positive for PLA2R antibodies in the serum (Stahl et al. 2010). The
Membranous nephropathy Membranous nephropathy

PLA2R antibody positive PLA2R antibody negative
Tumor associated Lupus erythematosus Drug (NSAID) associated

membranous nephropathy type V membranous nephropathy
Minimal change disease lgA nephropathy ANCA-associated

glomerulonephritis
Fig. 2 Detection of circulating PLA2R antibodies by an immunofluorescence test. Serum samples

were diluted 1:10 prior to measurement. No PLA2R antibodies can be detected in the serum of
patients with secondary forms of membranous nephropathy or other glomerular diseases
formal pathogenesis of membranous nephropathy, positive for PLA2R antibodies,

however, still has to be verified in animal models. A major still unanswered question
is how and why PLA2R antibodies develop. Genome-wide association studies have
identified several SNP mutations in the PLA2R and HLA genes which were associated
with the incidence of membranous nephropathy, PLA2R antibody positivity, and
enhanced PLA2R staining of renal biopsy (Stanescu et al. 2011; Lv et al. 2013).
Whether these genetic factors play a role or are associated with clinical parameters,
disease progression or outcome remains to be seen. The description of major antigenic
epitope sites on the PLA2R from several groups might further help understand the
pathogenesis of disease and perhaps lead to new prognostic markers and therapeutic
targets (Fresquet et al. 2015; Kao et al. 2015).
Since 2009 different methods have been established to easily and reliably mea-
sure PLA2R antibodies in the serum, thus allowing a number of studies to establish
the role of PLA2R antibodies as biomarkers for diagnosis, disease activity, treatment
success, and prognosis in membranous nephropathy (Hoxha et al. 2011; Dähnrich
et al. 2013; Hofstra et al. 2012; Behnert et al. 2014).
The Role of PLA2R Antibodies Differentiating Primary from

Secondary Membranous Nephropathy
Many studies have reported on the role of PLA2R antibodies to differentiate primary
form secondary membranous nephropathy (Qin et al. 2011; Hoxha et al. 2011, 2012;
Dähnrich et al. 2013). Since the pathogenesis, treatment, and prognosis of primary
membranous nephropathy are different from that of secondary membranous
nephropathy, differentiating the two forms of disease is very important. In primary
membranous nephropathy, the dominant immunoglobulin in the immune deposits is
usually IgG4, while in the secondary forms of disease, the other immunoglobulin
subtypes are more dominant (Ohtani et al. 2004). However, neither the histological
patterns nor the clinical findings can reliably differentiate primary from secondary
membranous nephropathy, since the coexistence of membranous nephropathy and a
potential secondary cause does not prove the secondary pathogenesis of membra-
nous nephropathy, but might be coincidental. PLA2R antibodies can be detected in
the serum of approximately 70 % of patients with primary membranous nephropathy
(Beck et al. 2009). Since PLA2R antibodies can disappear from the blood (sponta-
neously or under immunosuppressive therapy), a negative PLA2R antibody test does
not rule out the possibility that the patient has PLA2R-associated disease (Beck
et al. 2011; Hoxha et al. 2014c). In these cases, histological finding on the PLA2R
expression in renal biopsy can be helpful. Patients with PLA2R antibodies in the
serum show a markedly enhanced glomerular PLA2R expression (Fig. 3), which is
not seen in renal tissue of patients with other glomerular diseases or with secondary
Fig. 3 Immunohistochemical staining of PLA2R in renal biopsies from a patient with primary
membranous nephropathy and detectable PLA2R antibodies in the serum, a patient with secondary
membranous nephropathy (PLA2R antibody negative), and a normal kidney with kind permission
of Prof. T. Wiech
membranous nephropathy (Hoxha et al. 2012; Svoboda et al. 2013). The enhanced
staining for PLA2R does not seem to be caused by an increase in the glomerular
PLA2R production (Hoxha et al. 2012). Future studies will show if these findings are
due to conformational changes on the PLA2R or increased PLA2R deposition. Until
then, one can only speculate on the mechanisms leading to these findings. An
eventual conformational change on the PLA2R could be induced by PLA2R antibody
binding, but it could also be a preexisting condition, leading to the development of
PLA2R antibodies. An increased PLA2R deposition may be a result of podocytic
antigen shedding and/or antigen-antibody captured in the subepithelial space.
Patients who are negative for PLA2R antibodies in the serum, but have an
enhanced PLA2R staining in renal biopsy, might have an immunological remission
of disease, or PLA2R antibody levels in these patients are so low that the antibodies
are captured in the kidney and elude detection in the blood (Debiec and Ronco
2011). Thus, the combination of PLA2R antibody measurement in blood and PLA2R
staining of renal tissue can be used to differentiate primary from secondary mem-
branous nephropathy.
It is important to note that there have been reports of patients with PLA2R
antibody-positive membranous nephropathy, in whom hepatitis B, lupus
erythematosus, sarcoidosis, or malignancies have been diagnosed (Qin et al. 2011;
Larsen et al. 2013; Knehtl et al. 2011). In these cases the two diseases might have
coincidentally developed independently of each other, or by a yet unknown mech-
anism, the secondary cause might have led to the development of PLA2R antibodies.
While the former is more likely to be true, further research is needed to fully
understand the development of membranous nephropathy in these cases. Of partic-
ular importance is the question on the incidence of malignancies in PLA2R antibody-
positive patients, whether it is lower than in PLA2R antibody-negative patients or
perhaps same as in the general population. This is of utmost importance, since
membranous nephropathy characteristically develops in patients older than
50 years, who have a higher risk for developing malignancies.
THSD7A Is Another Target Antigen in Membranous Nephropathy
In a considerable number of patients who are negative for PLA2R antibodies in the
serum and show no enhanced staining for PLA2R in renal biopsy, no secondary
cause of membranous nephropathy can be found (Hoxha et al. 2012). There are a
number of factors which may play a role in this finding: (i) these patients might have
a secondary cause of membranous nephropathy, which could not be detected;
(ii) antibodies against podocytic antigens other than the PLA2R might lead to the
development of membranous nephropathy. This was shown to be the case for
thrombospondin type-1 domain-containing 7A (THSD7A), a podocytic membrane
protein, against which antibodies are found in the blood of patients with membra-
nous nephropathy (Tomas et al. 2014). THSD7A antibodies co-localize with IgG4 in
glomeruli and were isolated from immune deposits of a kidney from a THSD7A
antibody-positive patient with membranous nephropathy. THSD7A antibodies and
Fig. 4 In primary membranous nephropathy, PLA2R and THSD7A have been identified as
potential antigens. However, there are patients who are negative for PLA2R antibodies and
THSD7A antibodies in the serum, don’t show an enhanced PLA2R or THSD7A staining in the
renal biopsy, and also don’t have any known secondary cause of membranous nephropathy. In these
patients, the search for further antigens or clinical causes that might lead to the development of
membranous nephropathy continues
PLA2R antibodies seem to be exclusive to each other, since until now no cases of
THSD7A antibody-positive patients are reported, who are also positive for PLA2R
antibodies (Tomas et al. 2014). More work is needed to clinically characterize
THSD7A antibody-positive patients and analyze if there are differences in the
clinical characteristics of THSD7A antibody-positive from PLA2R antibody-
positive membranous nephropathy. Of particular importance for future research
work is the understanding of the pathophysiological mechanisms leading to the
development of membranous nephropathy in patients who are negative for PLA2R
and THSD7A but don’t show any known secondary cause of disease (Fig. 4).
There have been other antibodies found in the serum of patients with membra-
nous nephropathy; however, the characterization of the role of these antibodies for
the development of membranous nephropathy is still lacking (Murtas et al. 2012).
Further studies are needed to analyze whether these antibodies play a direct role in
the pathogenesis of membranous nephropathy or they develop as a consequence of
the podocytic damage upon formation of immune deposits by binding of PLA2R
antibodies on the podocytes and activation of the complement system.
The Role of PLA2R Antibodies in the Clinical Management

of Patients with Membranous Nephropathy
PLA2R antibody levels in the serum are associated with the clinical activity of
disease (Hofstra et al. 2011). This was shown for patients who had a spontaneous
remission of disease, as well as for patients treated with immunosuppressants,
where a decrease of PLA2R antibody levels preceded a decrease in proteinuria
(Hoxha et al. 2014c). There have been conflicting data whether PLA2R antibody
levels and proteinuria correlate at a defined time point, with retrospective studies
showing a correlation, which was not confirmed in prospective analyses (Hofstra
et al. 2012; Hoxha et al. 2012, 2014c). The fact that changes in PLA2R antibody
levels precede changes in proteinuria by months is an explanation why no direct
correlation of PLA2R antibody levels and proteinuria at a defined time point was
found (Hoxha et al. 2014c; Beck et al. 2011). At the same time, proteinuria depends
not only on the immunological disease activity but also on other factors such as
glomerular sclerosis, concomitant diseases (e.g., arterial hypertension, diabetes),
etc. The precise relationship between PLA2R antibody levels and proteinuria at a
defined time remains to be verified in animal models of PLA2R antibody-positive
membranous nephropathy.
Upon initiation of an immunosuppressive treatment, PLA2R antibody levels
decrease more rapidly than proteinuria, perhaps because of the time glomerular
reparatory processes need to effect proteinuria (Beck and Salant 2010; Hoxha
et al. 2014c). Experimental work on animal models will further elucidate the
mechanisms which may play a role in these processes. At the same time, patients
with higher PLA2R antibody levels at the time of diagnosis were less prone and
needed longer time to achieve a remission of proteinuria (Hofstra et al. 2012, 2014c).
Preliminary data indicate that the effect of different immunosuppressive drugs on the
PLA2R antibody levels is not different, suggesting that PLA2R antibody levels may
serve as a target for treatments and the efficacy of any immunosuppressive agent
may depend on its capability to lower PLA2R antibody levels (Hoxha et al. 2014c).
However, randomized, controlled clinical trials are needed to prove which immuno-
suppressive drugs have the best efficacy and safety profile in PLA2R antibody-
positive membranous nephropathy. At the same time, experimental work on animal
models will help us better understand how immunosuppressive drugs influence
disease activity and if there is a direct podocytic effect, in addition to the immuno-
modulatory effect, of drugs such as cyclosporine A and rituximab in PLA2R
antibody-positive membranous nephropathy.
In patients with low proteinuria, high PLA2R antibody levels are associated with
the risk for developing a nephrotic syndrome (Hoxha et al. 2014b). If it can be
confirmed that relapse of proteinuria in patients with membranous nephropathy is
preceded by an increase in PLA2R antibody levels or is associated with persistence
of PLA2R antibodies as suggested by a study (Bech et al. 2014), treatment of patients
might be adapted accordingly. If the high relapse rate of proteinuria upon cessation
of an immunosuppressive treatment is due to persisting PLA2R antibodies in the
blood, than another immunosuppressant, a higher treatment dosage or a longer
treatment time might be recommended. In other patients, if PLA2R antibodies
disappear very quickly, some immunosuppressive therapy might be safely saved,
thus resulting in less adverse events.
One of the most important findings was that patients with high PLA2R antibody
levels are at higher risk for progression of renal function impairment (Kanigicherla
et al. 2013; Hoxha et al. 2014a). This finding might lead to further changes in the
way treatment decisions are made in patients with membranous nephropathy. Future
clinical studies will shed more light into these findings. If they are confirmed, the
therapeutic strategy of membranous nephropathy might profoundly change, and
PLA2R antibody levels might take a central role when deciding which patient should
receive what treatment and when, leading to a more personalized treatment approach
in this disease.
The discovery of the PLA2R antibodies has led to a major burst in the clinical and
experimental research of membranous nephropathy and has already substantially
improved the diagnosis and the clinical management of patients with membranous
nephropathy.

Conditions
PLA2R antibody levels are associated with disease activity in membranous nephrop-
athy and may predict the prognosis of these patients. This is shown by the fact that
higher PLA2R antibody levels are associated with a lower chance to reach a
remission of proteinuria and, perhaps, more importantly with the risk of renal
function deterioration. Therefore, PLA2R antibody levels could be helpful when
making treatment decisions. Experimental animal models of PLA2R-associated
membranous nephropathy are needed to further analyze whether PLA2R antibody
levels lead to a worse prognosis in membranous nephropathy because of the
prolonged high proteinuria in these patients or if higher PLA2R antibody levels
have a direct glomerular effect. When we better understand how reduction or
disappearance of PLA2R antibodies leads to remission of proteinuria and mainte-
nance of renal function, treatment of membranous nephropathy might focus on these
antibodies.
Summary Points
• The PLA2R is the major target antigen in primary membranous nephropathy.

• The combination of PLA2R antibody measurement in blood and PLA2R staining
of renal tissue can be used to differentiate primary from secondary membranous
nephropathy.
• PLA2R antibody levels in the serum are associated with the clinical activity of
disease, and a decrease of PLA2R antibody levels precedes a decrease in
proteinuria.
• Patients with higher PLA2R antibody levels at the time of diagnosis are less prone
to have a remission of proteinuria and need longer time to achieve a remission of
proteinuria.
• In patients with low proteinuria, high PLA2R antibody levels are associated with
the risk for developing a nephrotic syndrome.
• Patients with high PLA2R antibody levels are at higher risk for progression of
renal function impairment.
• PLA2R antibody levels may serve as a target for treatments, and the efficacy of
any immunosuppressive agent may depend on its capability to lower PLA2R
antibody levels.
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Part V
Functional and Structural Variables
Ultrasound Elastography in Kidney Disease
47
Fuat Ozkan, Cemil Goya, Sema Yildiz, Mahmut Duymus,
Mehmet Sait Menzilcioglu, Serhat Avcu, and Mehmet Fatih Inci
Contents
Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1053
Key Facts of Quasi-static Elastography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1053
Key Facts of Dynamic Elastography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1053
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1053
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1054
US Elastography Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1055
Quasi-Static Elastography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1055
Dynamic Elastography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1056
Viscoelasticity of the Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1060
Influence of Renal Characteristics on Elasticity Values . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1060
Normal Renal Elasticity Values and Reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1062
Measurement of Renal Fibrosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1063
Preclinical Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1064
Evaluation of Renal Transplants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1064
Evaluation of Renal Mass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1066
Elastographic Properties of End-Stage Renal Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1066
Metabolic Diseases of the Kidney . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1069
Evaluation of Renal Damage in Urinary Tract Abnormality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1070
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1072
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1072
F. Ozkan (*) • S. Yildiz • M. Duymus • M.S. Menzilcioglu • S. Avcu

Department of Radiology, Faculty of Medicine, Gazi University, Ankara, Turkey
e-mail: drfozkan@yahoo.com; drfozkan@gmail.com; drsemayildiz@yahoo.com;
mahmutduymush@yahoo.com; dr.m.sait@hotmail.com; serhatavcu@hotmail.com
C. Goya
Department of Radiology, Faculty of Medicine, Dicle University, Diyarbakir, Turkey
e-mail: cegoya1@yahoo.com
M.F. Inci
Department of Radiology, Izmir Katip Celebi University, School of Medicine, Izmir, Turkey
e-mail: drfatihinci@gmail.com

1052 F. Ozkan et al.
Abstract
Ultrasound elastography is a relatively new imaging modality that can measure
tissue elasticity quantitatively by using different techniques. With the develop-
ment of technology, kidney elasticity measurements can be performed accurately
and quickly using ultrasound. Nowadays, kidney stiffness is used not only in
fibrosis but also in the characterization of focal lesions, staging of metabolic
kidney damage including diabetes mellitus, gout disease, etc.., and evaluating
renal damage in urinary tract abnormality. However, kidney is a unique tissue and
has a complex internal architecture. Therefore intrinsic or extrinsic factors such as
perfusion, urinary pressure, anisotropy, depth of kidney, and hydronephrosis may
affect the viscoelasticity of the kidney.
Elastography is a useful, quick, noninvasive complementary method in the
diagnosis of kidney diseases but needs specific training and optimization techniques
as well as acknowledging technical and pathological factors which may influence it.
Keywords
Elastography • Renal-acoustic radiation force imaging • Real-time elastography •
Transient elastography-supersonic shear wave imaging • Renal transplant
Abbreviations
1D One-dimensional
2D Two-dimensional
3D Three-dimensional
ARFI Acoustic radiation force imaging
CDUS Color Doppler ultrasonography
CT Computed tomography
IF/TA Interstitial fibrosis and tubular atrophy
kPa Kilopascal
L-NAME Nω-nitro-L-arginine methyl ester hydrochloride
MRI Magnetic resonance imaging
ROI Region-of-interest
RTE Real-time elastography
SDUV Shear wave dispersion ultrasound vibrometry
SSI Supersonic shear wave imaging
SWV Shear wave velocity
Tc-99m DMSA Technetium-99m dimercaptosuccinic acid
TE Transient elastography
US Ultrasonography
VTI Virtual touch imaging
VTQ Virtual touch quantification
VUR Vesicoureteral reflux
β2-MG β2-microglobulin
47 Ultrasound Elastography in Kidney Disease 1053
Key Facts
Key Facts of Quasi-static Elastography
• The principle of compression-based elastography (quasi-static) is based on the

relationship between differences in tissue deformability and its elastic properties.
• Mechanical pressure (transducer pressure exerted by operator, carotid pulses,
breathing movements, muscular contractions) on a tissue produces a minor
axial deformability in the hard tissue (incompressible) and high deformability
in the soft areas (compressible).
• Quasi-static elastography software evaluates in real time this tissue deformability
by evaluating the sound wave frequency alteration data before and after the
application of a compressive force.
• The different stiffness of tissues is measured and represented as a color-scale
image. Hard areas are represented as blue, soft areas as red, and firm areas as an
intermediate consistency in green.
• Strain ratio, semiquantitative estimation of elasticity, compares a lesion’s elastic-
ity to elasticity of surrounding normal tissue, providing a numeric outcome by
using elastograms.
Key Facts of Dynamic Elastography
• The methods are classified according to applied force.

• In ARFI imaging, short-duration acoustic “push pulses” generate shear waves
perpendicular to the main ultrasound beam; detection pulses are generated
simultaneously.
• The shear wave speed can be expressed as numeric values in m/s by VTQ in ARFI
imaging.
• TE evaluates shear wave speed no forming an image because it uses a surface
mechanical force rather than acoustic radiation force.
• In SSI, multiple focal spots are created in depth along a same longitudinal axis,
generating a Mach cone acting as a shear wave source. The measurement of the
velocity of this plane in each point of the image in real time provide a 2D
quantitative elasticity map by using the ultrafast imaging technique.
Definitions
Elasticity Elasticity is the feature of objects to retrieve their original shape and size
after the stress is stopped.
Strain Strain (ε) is the outcome of subjecting stress to an elastic object. It describes
alteration in shape size of an elastic object as a result of stress. Longitudinal strain,
like compression, gives rise to change in length of an object. Shear strain gives rise to
changes in angles of an object. Strain has no units.
Stress Stress (σ) is defined as power producing deformation (elongation, compres-

sion, torsion). Unit of stress is pascal or pounds per square inch (psi). (pascal =
newton/m2). Although both stress and pressure have same units, stress mostly can
be a vector and is applied in a specified direction, whereas pressure effects equally in
all directions.
Viscoelasticity Biological tissues exhibit both viscous and elastic properties. This
means that, at low strain rates, because molecular structural “flow” can occur, there is
a time delay between application of force and displacement. However, this does not
have time to become at high strain rates. Therefore, for shear wave methods, the use
of higher vibration frequencies will give rise to higher values of wave speed.
Viscosity Viscosity is the quantity of resistance of a fluid when it subjects shear

stress or tensile (compression or stretching) stress.
Introduction
To date, renal imaging has primarily been based on morphologic evaluation of the
kidney parenchyma, excretory system, and vascularity using ultrasonography, color
Doppler ultrasonography (CDUS), computed tomography (CT), and magnetic res-
onance imaging (MRI). Moreover, functional parameters could easily be studied
with MRI such as perfusion, filtration, and diffusion measurements. Since radiolog-
ical evaluation of structural differences between normal parenchyma and patholog-
ical tissues remains a challenge, elastography has been accepted as an attractive
technique firstly having been established in the liver (Bavu et al. 2011; Castera
et al. 2008; Palmeri et al. 2011).
Elastography is a noninvasive imaging technique of stiffness or elasticity of the
tissue via measuring movement or distortion of the tissue in response to a slight
pressure (Singla et al. 2013). Although clinical practice of ultrasonography
(US) elastography in liver tissue alters related to fibrosis or steatosis, yet little validation
has been done to estimate its potential role in renal tissue changes (Grenier et al. 2013).
Progressive changes of extracellular matrix causing progression of fibrosis are
associated with decreased functional alteration in kidneys, as in the liver. This
process, called chronic kidney disease (CKD), is mostly encountered in developed
countries, particularly depending on diabetes and hypertension-related nephropa-
thies (El Nahas 2005). It leads to end-stage renal failure, with high morbidity and
mortality and raised health costs. Most types of kidney diseases may progress to
CKD, with progressive fibrotic process affecting first either glomeruli (glomerulo-
sclerosis) or the interstitial space (interstitial fibrosis), depending on the early-stage
nephropathy (Chatziantoniou et al. 2004; Ricardo et al. 2008). Likewise, the devel-
opment of interstitial fibrosis and tubular atrophy (IF/TA), formerly named chronic
allograft nephropathy (Nankivell et al. 2003; Stegall et al. 2011), is the main reason
of renal allograft failure in renal transplantation.
Renal biopsy is a valuable procedure for the diagnosis of renal disease, and it can
help to guide treatment and state prognosis, especially with the evaluation of the
degree of fibrosis or inflammation in the assessment of allograft rejection (Urban
et al. 2013). However, the biopsy procedure can be complicated with small and large
hematomas, gross hematuria, arteriovenous fistula, and even death (Maya and Allon
2009; Urban et al. 2013). Moreover, the biopsy is inherently prone to sampling errors
due to the small volume of tissue extracted (Urban et al. 2013). Therefore, more
efficient diagnostic strategies are emerging to evaluate the renal pathologies in vivo
and noninvasively. These new approaches are expected to require defining and
confirming adequate imaging biomarkers, with their intrinsic variability and
interoperator reproducibility before clinical practice. Recently, diffusion-weighted
MRI has been suggested to assess the parenchymal fibrosis via evaluating the
restriction of water molecule motion (Togao et al. 2010; Warner et al. 2011) but it
has no specificity for the fibrosis. Besides, US elastography is shown to be a
promising technique in the evaluation of stiffness with first encouraging results of
hepatic evaluation (Grenier et al. 2013). However, kidneys have a complex intrarenal
architecture compared with liver, having two anatomical compartments, cortex and
medulla, a high vascularity, and a function of urinary excretion.
The purpose of this chapter is to represent the main results, advantages, and
limitations of US elastography technique in the evaluation of several kidney diseases
and to present the new horizons of US elastography application in renal imaging.
US Elastography Techniques
The elastography techniques can be classified into two main categories: the quasi-
static technique and the dynamic technique. These two categories do not obtain the
same knowledge on mechanical parameters, so the difference between them is quite
important. Although dynamic elastography yields a quantitative image or only one
quantitative value of elasticity, quasi-static elastography yields a strain image of the
investigated tissue and demonstrates qualitative knowledge in proportional to the
elasticity (Grenier et al. 2013).
Quasi-Static Elastography
This is also called by manufacturers as “real-time elastography” (RTE) and is the first
elastography technique, developed by Ophir et al. in the beginning of the 1990s
(Ophir et al. 1991). It is an easily applicable and widespread technique (Grenier
et al. 2013) based on a quasi-static distortion of a tissue due to a stress (σ). A stress
(σ) induces a strain (ε), depending on the degree of tissue elasticity. The Young
modulus E is identified by the ratio between the stress and the strain (Eq. 1):
a move probe
σ
b Measure depth c Display as a
shifts strain image
shift
Imaging
plane
Fig. 1 Principle of quasi-static elastography. The probe is placed at the surface of the investigated
organ and small compression is applied. Images are acquired before and after compression (a). By
taking small windows within the image of raw US signals, a displacement shift in depth is recovered
(b). Then, by calculating the derivative of the displacement, the final strain image is obtained and
displayed (c) (From Grenier et al. 2013, with permission)
E ¼ σ=ε (1)
In this technique, a stress is applied to the tissue (through the ultrasonic probe
itself) and observed by ultrasound imaging. The strain induced by the propagation of
the stress is evaluated by comparing data before and after stress, and we estimate the
displacement (Fig. 1). Though the stress (S) is not measurable, the resulting map
does not display the Young modulus. This system provides parametric maps that
differentiate rigid and soft tissues, and these maps may be in color and/or gray scale
depending on the constructor.
In this technique, the tissue strain is reliant on the amount of applied compression.
Therefore, this makes the technique operator-dependent. Although this technique is
known as a qualitative imaging, nowadays semiquantitative measurement can be
made with evaluation of strain ratio, which is the ratio of strain between the lesion
and the adjacent normal tissue.
This technique seems useful for superficial organs, mainly in the evaluation of
breast or thyroid, due to the limitation of operator-dependent strain (Grenier
et al. 2013). Though it is accepted as non-applicable for renal pathologies because
of the deep location of kidneys, a recent study has reported a quantitative evaluation
of tissue elasticity from deep soft tissue which could be achievable with modified
form of this technique (Baghani et al. 2012).
Dynamic Elastography
This technique is based on the propagation of mechanical or elastic waves in the

body (Grenier et al. 2013). In a quasi-incompressible medium such as the human
body consisted with 75 % of water, which is an incompressible material, the Young
modulus is directly related to the speed (VS) of a kind of elastic waves, the shear
waves (Grenier et al. 2013), and the tissue density (ρ) is assumed to be constant and
equal to 1,000 kg m3 (Eq. 2):
E ¼ 3ρV2 S (2)
Dynamic elastography can state a quantitative value of the Young modulus by
estimating the speed of the shear waves by both generating a shear wave within
the tissues using an external source (mechanical vibration and impulse or acoustic
radiation force) and using one-dimensional (1D) or two-dimensional (2D) US to
follow their propagation (Grenier et al. 2013).
Fibroscan Transient Elastography

It was originally called the “shear elasticity probe” and was the first quantitative
elastography method available on the market (Grenier et al. 2013). It was
manufactured at the Langevin Institute at the end of the 1990s (Sandrin
et al. 2002, 2003). This system is based on a mechanical pulse made by a mechanical
impulse or vibrating “punch” which generates a transient shear wave (pulse) that
propagates longitudinally through the skin. The velocity and amplitude of the shear
wave are calculated in a region-of-interest. The velocity (V) is converted into
kilopascal (kPa), and it reflects the tissue stiffness. This is not an imaging-guided
system and the probe is positioned randomly in the skin surface. The measurements
are obtained at a depth between 20 and 60 mm (Grenier et al. 2013).
The Fibroscan transient elastography (TE) system is also inappropriate for the
kidneys: (1) first, there is no B-mode control, and as the sample volume is fixed at a
certain depth and size, it is extremely difficult and hazardous to position the sample
volume on the renal cortical parenchyma, which is located at a variable depth
(Grenier et al. 2013). A manual adjustment could be considered in the transplanted
kidney, due to its superficial localization. Nevertheless it is hazardous without real-
time US control, and this technique would require a variable amount of pressure
applied on the probe, which would change the values of elasticity measurements
(Grenier et al. 2013); (2) second, the mechanical wave should be applied on a
rigid surface, such as the rib cage, to avoid compression effects of the probe,
which is not able to occur for the kidney. Despite these limitations, Fibroscan
transient elastography was used to evaluate the elasticity in three recent series of
transplanted kidneys (Arndt et al. 2010; Sommerer et al. 2013; Lukenda et al. 2014).
Sommerer et al. have reported that body mass index, skin-transplant distance, and
perirenal or intrarenal fluid collection were important parameters of successful
measurement of kidney stiffness by this technique. The same authors revealed that
heterogeneous renal morphology and factors mentioned above may affect measur-
ability of elasticity negatively. More technical modifications were needed to better
assess the kidney.
ARFI = Acoustic Radiation Force Imaging

In this technique which was initially developed by Siemens (ARFI Virtual Touch™),
estimation of stiffness of deep tissues can be ensured with short-term acoustic forces
Fig. 2 Principle of acoustic radiation force imaging (ARFI). A radiation force is created from a
focal spot within the organ by focusing US, creating a local shear wave source that is propagated
perpendicularly to the US axis (a). On a few transducers close to the focal spot, the displacements of
the shear wave are recovered, allowing a 1D measurement of shear elasticity in the region-of-
interest (ROI) (b) (From Grenier et al. 2013, with permission)
named as pushing pulses. When USs are focused for a long time (hundreds of μs
vs. 1–10 μs for classical US B-mode), a force, known as US radiation pressure, is
generated at the focal spot, and this acoustic force is used to deform the tissue
(Grenier et al. 2013). Tissue comes back to the original position after the force
application and meanwhile generates shear waves which spread away from the focal
excitation point as a local vibration (Singla et al. 2013). The velocity of the shear
waves depends on the tissue stiffness and is quantitatively measured by software in
an evaluated area and expressed as m/s (Virtual Touch Quantification, Siemens
Healthcare, Mountain View, CA). The Virtual Touch Quantification (VTQ) is a
noninvasive and painless examination and can obtain multiple measurements on a
small box located at the chosen depth within organs. Though US devices use 1D
technique for the elasticity measurement, 2D US image (B-mode) is preferred to
position the estimation window at the needed location within the organ (Grenier
et al. 2013) (Fig. 2).
The inability of this technique results from underestimation of tissues at a depth
of more than 8–10 cm due to weakening of the radiation forces at greater depths
(Singla et al. 2013).
Supersonic Shear Wave Imaging

Acoustic radiation force (US radiation pressure) generating a Mach cone, and US
ultrafast imaging are the two basic models of Supersonic shear wave imaging (SSI)
Fig. 3 Principle of supersonic shear wave imaging (SSI). Multiple focal spots are created in depth
along the same longitudinal axis, generating a Mach cone acting as a shear wave source (a). By
using an ultrafast imaging mode, the propagating shear wave is caught in the entire field of imaging,
allowing the reconstruction of a 2D elasticity map (b) (From Grenier et al. 2013, with permission)
technique (Bercoff et al. 2004; Grenier et al. 2013). Various repeated focalized US
spots at different depths (typically four to five spots) are used to evaluate a wide area
at the same time (Grenier et al. 2013). Multiply located shear waves form a conical
shear wave front like a Mach cone of an aircraft with supersonic speed (Singla
et al. 2013) (Fig. 3). An ultrafast scanner with 5,000 frames per second capability is
needed for the imaging of this shear wave propagation (Singla et al. 2013). This
ultrafast scanner allows the formation of real-time images by achieving the entire
shear wave field in less than 30 ms, and fast average processing of acquired images
to increase robustness without stroboscoping the acquisition several times to obtain a
whole shear wave field (Grenier et al. 2013). In conclusion, a real-time dimensional
quantitative mapping is obtained, and low-frequency curved arrays are used for
native and deep transplanted kidneys and high-frequency linear arrays for superficial
transplanted kidneys for examination with SSI technique (Grenier et al. 2013).
The main limitation of this technique is certain depth of tissue is required for
shear wave penetration. Therefore evaluation of superficial structures can be
difficult.
Shear Wave Dispersion Ultrasound Vibrometry

Shear wave dispersion ultrasound vibrometry (SDUV) has a focused ultrasound
beam to form harmonic shear waves or impulse multi-frequency shear waves that
spread out from the central vibration point (Amador et al. 2011). Dispersion of shear
wave is a feature that means the measured speed of shear waves vary depending on
the frequency in a viscoelastic medium and may specify viscoelastic properties of the
tissue quantitatively.
Viscoelasticity of the Kidney
In recent years, several previous studies have reported on the usefulness of

elastographic methods focusing on the elastic properties of the tissue, not the
viscosity (Scola et al. 2012). However, it has been claimed that if viscosity is not
regarded, measurements of stiffness can be biased, and the results may be higher
than their real values (Amador et al. 2011).
The kidney can be modeled as a viscoelastic material due to its both elastic and
viscous physical characteristics (Amador et al. 2011). For an accurate diagnosis,
single evaluation of shear elasticity is not enough for a correct diagnosis despite the
correlation of stiffness with pathology (Deffieux et al. 2009). Therefore it is essential
to measure some mechanical parameters such as shear viscosity for accurate char-
acterization of the kidney. SSI with specialized hardware can form maps of stiffness
and viscosity as distinct from TE and MR elastography that only evaluate the elastic
part, not the viscous part of the tissue response (Amador et al. 2011).
SDUV uses an intermittent pulse sequence suitable with ultrasound scanners on
the market and can measure the tissue viscosity and elasticity quantitatively
(Amador et al. 2011).
Influence of Renal Characteristics on Elasticity Values
Tissue stiffness is characterized by its acquired intrinsic mechanical characteristics

(e.g., fibrosis) and hemodynamic factors of turgor (Korsmo et al. 2013).
The sensitivity of the elastography to mechanical parameters causes the measure-
ment of renal shear wave velocities to be made with caution (Grenier et al. 2013).
There are some intrinsic factors of kidneys such as anisotropy of renal architecture,
compressibility of renal transplants, high degree of renal vascularity, and possible
increase of urinary pressure. It is important to assess the variation of these parameters
to increase the reproducibility of the technique and to decrease the intrinsic variabil-
ity of the measurements (Grenier et al. 2013).
A manual compression by the US probe may be possible during scanning of renal
transplants which are more superficially located in the iliac fossa whereas native
kidneys are bedded quite deeply and hardly compressible. The same phenomenon is
valid for elastographic evaluation of the liver parenchyma with higher values on the left
lobe obtained using a compliant epigastric window, than on the right lobe, using a rigid
intercostal window. Syversveen et al. (2012) evaluated the effect of probe compression
on elasticity values of transplanted kidneys, and they calibrated the compression forces
with ARFI on the renal cortex of 31 kidney transplants by a mechanical device. They
claimed that there were highly significant differences in mean shear wave velocity
between five different compression levels (Syversveen et al. 2012).
In the kidney Henle’s loops and vasa recta within medulla and the collecting
ducts within cortex and medulla are parallel and directed from the capsule to the
papilla within each renal segment. Therefore, the intrinsic architecture of the renal
parenchyma is accepted as anisotropic, and (Grenier et al. 2013) the degree of
anisotropy has been assessed to be around 15 % in the cortex and 30 % in the
medulla with MR evaluation (Ries et al. 2001). It has been claimed that when
emission of the US beam is sent parallel to renal microstructures, the shear wave
propagates perpendicular to these, and this causes multiple vascular and tubular
interfaces, decreases the speed of shear wave propagation, and results in lower
elasticity values (Grenier et al. 2013). Conversely, when emission of the US beam
is sent perpendicular to these structures, higher elasticity values are obtained
(Grenier et al. 2013) (Fig. 4). It has been also claimed that the mean variation of
the Young modulus was 10.5 % in the outer cortex, 29.7 % in the inner cortex, and
31.8 % in the medulla due to anisotropy in normal conditions (Gennisson
et al. 2012). Therefore, a clear identification of sampled renal segments and their
orientation according to the US beam are mandatory when performing renal US
elastography, and it is important to position the main axis of US probe as parallel as
possible to the main axis of pyramids to reduce the effect of anisotropy in order to
quantify the shear wave velocity.
The kidney is a highly vascularized organ, receiving approximately 20 % of
cardiac output, and this vascularity may significantly affect in vivo elasticity mea-
surements (Warner et al. 2011; Asano et al. 2014). It has been reported that reduction
of blood flow may affect shear wave velocity values more than tissue fibrosis in
patients with CKD (Asano et al. 2014). It has been shown with MR elastography that
cortical and medullary stiffness gradually decreased ~30 % and ~20 %, respectively,
during reduction in renal blood flow (Warner et al. 2011). Similarly, a significant
decrease in elasticity affecting mainly cortex was described after ligation of the renal
artery (Gennisson et al. 2012). Medullary stiffness is less affected from the alter-
ations in renal perfusion since medullary blood flow has less dependence on
hydrostatic pressure and renal vascular resistance, contributing around 90 % of
RBF, exists generally in cortical microvessels (Warner et al. 2011). Conversely, a
giant increase in renal elasticity was observed after ligation of the renal vein in an
animal study (Gennisson et al. 2012).
Urinary obstruction, especially acute and complete forms, may increase the
intrarenal pressure. Gennisson et al.’s pig study defined that elasticity values were
linearly affected with the severity of urinary obstruction (Gennisson et al. 2012).
Consequently, before remarking increased elasticity measurements, urinary obstruc-
tion will have to be ruled out, and especially in renal transplant patients, it should be
noted to the degree of bladder filling due to the shortness of the ureter and its
denervation; eventually a filled bladder may affect the pyelocaliceal system (Grenier
et al. 2013).
Fig. 4 Effect of anisotropy. Elasticity map of a pig kidney showing higher values in the lower pole
than on the anterior aspect (a). Macroscopic image of a lower half of a pig kidney showing the
corticomedullary differentiation and, superimposed, schematic representation of 2 renal segments:
one shows a predominant vertical anisotropy in the direction of US beam and the other a horizontal
anisotropy perpendicular to the direction of US beam (red arrows illustrate the direction of anisot-
ropy). Axis of propagation of the shear wave (black arrows) is perpendicular to the oriented renal
structures in the first case and parallel in the second (b) (From Grenier et al. 2013, with permission)
Normal Renal Elasticity Values and Reproducibility
It is important to know “normal” kidney elasticity before interpretation of the results

obtained from patients with kidney pathology, and there are many factors that
influence the renal shear wave speed: age, gender, overweight/obesity, and measure-
ment depth (Bota et al. 2014). In the first study with SSI technique (Arda et al. 2011),
normal cortical elasticity values were defined as 5.2
2.9 (1–13) kPa and 4.9
2.9
(1–26) kPa in men and women, respectively. They stated that there were no signif-
icant difference in elasticity measurements between sexes and no significant positive
or negative correlation between age and elasticity of the renal cortex.
In a study by Guo et al. (2013), a negative correlation between shear wave
velocity (SWV) of renal parenchyma evaluated by ARFI and age and significantly
different SWV values between men and women (2.0660.48 m/s vs. 2.260.52 m/s,
respectively) was declared in contrast to Arda et al.’s study (Grenier et al. 2013).
Bota et al. also reported that kidney shear wave speed values measured by ARFI
elastography were decreasing with age, and values were significantly lower in men
than in women in patients between 31–50 and 51–65 years of age (Bota et al. 2014).
It was previously reported that mean SWVs in normal adult kidneys were
2.24–2.37 m/s, with no significant difference between the right kidney and the left
kidney (Goertz et al. 2011; Sohn et al. 2014).
In a study evaluating age-related changes in kidney stiffness in healthy children
by ARFI technique with an average age of 8.1
4.7 years (Lee et al. 2013), it was
stated that the mean SWVs were 2.19 m/s for the right kidney and 2.33 m/s for
the left kidney. It has been declared that SWV of kidneys changed with aging in
pediatric population with the most notable increase less than 5 years of age (Lee
et al. 2013).
Interobserver variability has been accepted as a major limitation of elastography.
Though some authors claimed that elastography was an operator-dependent exam
and higher intra- and interobserver variabilities were stated as inevitable (Yoon
et al. 2011; Ozkan et al. 2013), many other studies reported higher interobserver
agreement especially in RTE (Merino et al. 2011; Orlacchio et al. 2014). These
contradictory results may be due to the different elastography techniques and
softwares preferred.
Different results were reported in interobserver agreement of transient
elastography, ARFI, and SSI techniques. Although previous studies reported low
reproducibility of all these techniques, recent studies reported good reproducibility
especially in RTE and ARFI techniques by using extra dedicated software or gained
experience.
Measurement of Renal Fibrosis
Increased extracellular matrix synthesis, with excessive fibrillary collagens in

the glomerular, interstitial, and vascular compartments, may progressively cause
the end-stage renal failure (El Nahas 2005; Grenier et al. 2013). These pathophys-
iological pathways have been well investigated and numerous therapeutic inter-
ventions should be made to prevent or favor regression of fibrosis in
experimental models (Chatziantoniou et al. 2004; Boffa et al. 2003). Therefore,
improvement of new noninvasive methods for definition and quantification of
fibrosis is emerging.
Preclinical Studies
The first study evaluating US elastography in an experimental model reported the

influence of renal anisotropy and vascular and urinary pressure on elasticity values in
six pig kidneys by ultrasonic supersonic shear wave elastography (Gennisson
et al. 2012). Second study used SSI to detect kidney cortex elasticity changes and
tried to predict histopathological development of fibrosis in a rat model of
glomerulosclerosis induced by Nω-nitro-L-arginine methyl ester hydrochloride
(L-NAME) administration (Derieppe et al. 2012). It was shown that increased
cortical elasticity values were correlated with the degree of renal dysfunction, but
no correlation was found between the global histological score and the severity of
cortical stiffness (Derieppe et al. 2012).
Evaluation of Renal Transplants
The natural history of IF/TA in transplanted kidneys has been well studied through the
protocol biopsies. The early phase, which generally occurs during the first years of
post-transplantation, is characterized by fibrogenesis and the occurrence of tubuloin-
terstitial damage due to immunologic phenomena; the late phase is characterized by
the worsening of parenchymal lesions (irreversible IF, TA, arteriolar hyalinosis) and
glomerular sclerosis leading to graft lost (Nankivell et al. 2003; Stegall et al. 2011).
Protocol biopsies are the only reliable tool for diagnosis of IF/TA due to lacking of
noninvasive markers of abovementioned pathological changes. However, the role of
renal biopsy protocol is controversial in renal transplantation due to its invasive nature
and highly cost (Orlacchio et al. 2014). Recently, ultrasonographic elasticity imaging
has been emerging to evaluate the viscoelastic properties of renal tissue in transplan-
tation patients (Urban et al. 2013; Amador et al. 2012) (Fig. 5). There are several
studies about elasticity properties of renal transplants reporting controversial informa-
tion using different elastographic systems (Table 1).
Arndt et al. (2010) found a high positive correlation between renal stiffness and
the degree of internal fibrosis in renal transplants by using transient elastography
(Fibroscan). Sommerer et al. have measured significant higher renal stiffness in renal
transplants with histologically verified advanced fibrosis on a large cohort of
transplanted patients by transient elastography (Sommerer et al. 2013). They have
declared that the sensitivity and specificity of transient elastography detecting renal
allograft fibrosis were 54 % and 73 %, respectively. Stock et al. (2011) stated that an
average increase of shear wave velocities of ARFI technique was seen in 15 % of
transplants with pathologically proven acute rejection. However, Syversveen
et al. (2011) reported that there was no correlation between shear wave velocities
and the presence and/or severity of fibrosis by TE technique. Both TE and ARFI
techniques were used to detect the tissue fibrosis, but they were not directly
comparable because of their dissimilar technologies (although ARFI used short
duration, high-intensity acoustic pulses to produce shear waves, TE used vibrations)
Fig. 5 Virtual Touch Quantification (VTQ) measurement of renal allograft rejection. Acoustic
radiation force impulse quantification measurement in the 47-year-old man transplanted kidney is
performed. Shear wave velocity = 3.29 m/s shows renal allograft rejection
Table 1 Several studies performed on renal transplants with different elastography techniques
Authors Technique Results
Arndt TE Correlation between stiffness and fibrosis (Pearson r=0.67;
et al. (2010) p=0.002; R2=0.45)
Stiffness difference according to Banff score
Syversveen ARFI No significant difference in stiffness according to Banff score
et al. (2011)
Stock ARFI Moderate correlation between stiffness and fibrosis (r = 0.468,
et al. (2011) p = 0.026)
Grenier SSI No correlation (excepted with total Banff score)
et al. (2012)
Orlacchio RTE High correlation between stiffness and fibrosis according to
et al. (2014) Banff score (r = 0.52, p < 0.05)
Gao RTE Strong correlation between stiffness and fibrosis according to
et al. (2013) Banff score ( p < 0.05)
Lukenda TE High correlation between stiffness and fibrosis according to
et al. (2014) Banff score (r = 0.727; p = 0.0001)
Cui ARFI Significant difference between the stiffness values of the
et al. (2014) nonfibrosis group and mild-moderate fibrosis group (p < 0.01)
(Orlacchio et al. 2014). TE is a quasi-static elastography method, and its measure-

ments are qualitative or semiquantitative.
Gao et al. have introduced a novel technique for assessing the severity of cortical
fibrosis in renal transplants by applying the corticomedullary strain ratio as a quantitative
marker (Gao et al. 2013). In this technique, two-dimensional speckle-tracking software

was used to make offline analysis of cortical and medullary strain produced by external
compression by the ultrasound probe (Gao et al. 2013). Then, corticomedullary strain
ratio (cortical normalized strain/medullary normalized strain; normalized strain =
developed strain/applied strain [deformation from the abdominal wall to the pelvic
muscles]) was calculated (Gao et al. 2013). They concluded that measuring the renal
strain by a strong speckle-tracking technique, analyzing the internal tissue deformation
stimulated by external compression, might have advantages over other methods based
on shear wave velocity (Gao et al. 2013).
Grenier et al. evaluated 49 consecutive kidney transplant recipients by using SSI
scheduled for renal biopsy (Grenier et al. 2012), and none of each individual score of
the semiquantitative Banff classification was correlated with the measurement of
cortical stiffness. Moreover, cortical stiffness was correlated with neither the level of
IF measured by quantitative image analysis nor the scoring and grading of IF/TA. These
results may be due to impact of other parameters on parenchymal stiffness besides the
IF. Nevertheless, in a study measuring viscoelastic properties of renal transplants with
varied biopsy scores by using some post-processing software applications (Urban
et al. 2013), the measurements of shear modulus in transplanted kidneys were found
to be positively correlated with increased Banff scores. Though the measurement of
viscoelasticity in renal transplants may be feasible, it is needed to be studied in large
cohorts to evaluate its prognostic and diagnostic value (Urban et al. 2013).
Evaluation of Renal Mass
There are only a few studies investigating the renal masses. The first study about this
topic reported a series of 15 cases (2 pseudotumors, 2 hemorrhagic cysts, 8 renal cell
carcinomas, 1 chromophobe cell carcinoma, 2 tubulo-papillary carcinomas), by
ARFI technique, and the elastographic values of renal masses were between 1.61
and 3.97 m/s, without distinction among different types (Clevert et al. 2009). The
second study found a difference between the ratios of angiomyolipomas to adjacent
parenchyma and renal cell carcinoma to adjacent parenchyma by using strain
imaging (Tan et al. 2013). The third study evaluated shear wave velocities of the
tumors by ARFI quantification, and the authors claimed that there were differences
in the shear wave velocities between the SWV values of benign renal lesions
including hematomas and malignant renal lesions (Goya et al. 2014a) (Figs. 6, 7,
8, and 9). More experience is necessary to evaluate the potential role of elastography
in separating benign and malignant tumors.
Elastographic Properties of End-Stage Renal Disease
CKD is an important health problem in developed countries, particularly in the

context of diabetes and hypertension-related nephropathies. It is important to diag-
nose the disease in early phase, because the earlier treatment is efficient in slowing
Fig. 6 Virtual Touch Quantification (VTQ) measurement of renal angiomyolipoma. VTQ mea-
surement of a renal angiomyolipoma in anterior upper pole of the left kidney of a 47-year-old male
patient
Fig. 7 Virtual Touch Quantification (VTQ) measurement of papillary renal cell carcinoma. VTQ
measurement of a histopathologically confirmed papillary renal cell carcinoma in the upper pole of
the right kidney of a 51-year-old female patient
the progression to kidney failure irrespective of the reason (K/DOQI clinical practice
guidelines 2002). However, conventional signs of CKD, such as serum creatinine,
proteinuria, and urea nitrogen, are insensible and may cause loss of time to diagnose
(Guo et al. 2013; Levey et al. 2009). Guo et al. have used ARFI elastography to
determine changes in elasticity in chronic renal disease, and they found that the
Fig. 8 Virtual Touch Quantification (VTQ) measurement of hydatid cyst. VTQ measurement of a
histopathologically confirmed hydatid cyst in the upper pole of the right kidney of a 32-year-old
male patient
Fig. 9 Wilms’ tumor. VTQ measurement (a) and eSie Touch Elasticity Imaging (b) of a Wilms’
tumor in a 5-year-old male patient
Fig. 10 eSie Touch Elasticity Imaging of the chronic kidney disease. Acoustic radiation force
impulse (ARFI) imaging in a 45-year-old man with chronic kidney disease due to hypertension
shows decreased stiffness (yellow-green-red areas) in atrophic kidney
SWV was significantly higher in control group than each stage of CKD patients
(Fig. 10).
However, Wang et al. stated that SWV did not correlate with any pathological
marks of fibrosis, and ARFI could not predict the dissimilar stages of CKD in a study
evaluating ARFI technique in CKD with common etiology of IgA nephropathy
(Wang et al. 2014).
The contradictory results of these studies may arise from complex intrarenal
architecture that affects the viscoelasticity of the kidney. Further studies are
warranted to elucidate the sources of the variability.
Metabolic Diseases of the Kidney
Primary gout is a metabolic disease characterized with increased blood uric acid and
tissue injury depending on a long-term purine metabolism disorder (Tian et al. 2014).
Renal involvement may be seen as acute and chronic renal insufficiency due to
chronic interstitial nephritis and uric acid stones (Tian et al. 2014). In a study
evaluating the value of VTQ technology combined with urinary β2-microglobulin
(β2-MG) measurement, a small-molecule immunoglobulin increases in urine in early
phase of renal damage (Fassett et al. 2011), in the early diagnosis of gouty kidney
(Tian et al. 2014). In this study, patients with kidney damage had significantly
increased renal parenchyma and sinus shear wave velocities, and urinary β2-MG
level was positively linearly correlated with the parenchymal shear wave velocities.
Diabetes mellitus is the leading cause of end-stage renal disease (ESRD), and
diabetic nephropathy is one of the most significant complications that is directly
related to progression of the disease (Yu et al. 2014). Laboratory examinations such
as albumin-to-creatinine ratio, blood β2-MG, and urine β2-MG are indicators that
appear before renal diabetic involvement (Yu et al. 2014). It has been stated that there
was significant difference in SWV of the renal cortex between the patients with
microalbuminuria and macroalbuminuria groups and normal population
(Yu et al. 2014), and the renal cortical SWVs were positively correlated with the
albumin-to-creatinine ratio.
Goya et al. found that ARFI imaging was able to distinguish diabetic nephropathy
stages (except for stage 5 in the right and except for stages 4–5 in the left kidneys) by
VTQ technique, and they stated a correlation between the SWV and estimated
glomerular filtration rate (eGFR), serum urea nitrogen, urinary protein, and creati-
nine (Goya et al. 2015).
Evaluation of Renal Damage in Urinary Tract Abnormality
There are only a few studies evaluating renal pathologies in children with urinary
tract abnormalities. Bruno et al. reported that the kidneys in pediatric patients with
vesicoureteral reflux (grade III) had significantly higher SWV values compared
with healthy kidneys by ARFI technique (Bruno et al. 2013). The authors claimed
that ARFI might ensure reliable information about the severity of renal damage and
might be useful in the diagnostic workup in children with chronic reflux disease
(Bruno et al. 2013). In a study evaluating hydronephrotic kidney stiffness in
30 children (range, 0–23 months), a significant difference in the median
SWVs between normal kidneys (1.75 m/s) and high-grade hydronephrotic kidneys
(2.02 m/s) was also stated by using ARFI technique (Sohn et al. 2014). These results
were compatible with decreased elasticity and increased stiffness in high-grade
hydronephrotic kidneys (Sohn et al. 2014). In a comprehensive study about detec-
tion of the renal damage with ARFI technique as a complementary tool to
technetium-99m dimercaptosuccinic acid (Tc-99m DMSA) scintigraphy, high-
grade (grade V–IV) refluxing kidneys had the lowest SWV values, while
non-refluxing kidneys had the highest values, and severely damaged kidneys had
the lowest SWV values (Goya et al. 2014b) (Fig. 11). Goya et al. considered that
other parameters such as vascularization, anisotropy, external pressure, and
hydronephrosis might be more effective in renal elasticity than fibrosis (Goya
et al. 2014b). It has been also claimed that ARFI measurements could not differen-
tiate the reason of the stiffness such as tissue fibrosis and edema (Sohn et al. 2014).
Therefore, further researches with larger patient population and pathologic corre-
lation are needed.
In summary, ultrasound elastography is a more favorable imaging modality than
others including magnetic resonance elastography. Firstly, ultrasound is less
expensive and easier to use and especially capable of ensuring quantitative mea-
surements of tissue both viscosity and elasticity with new techniques. Therefore it
will likely become the most widely used modality for clinical elasticity estimation
and imaging. However, kidney has unique biomechanical properties including
viscosity and anisotropy due to complex intrarenal architecture. This means that
changeability of measurements in the renal parenchyma is an actual concern.
Further studies have to be made to obtain more experience with preclinical models
Fig. 11 eSie Touch Elasticity Imaging of the grade V vesicoureteral reflux (VUR). Technetium-
99m dimercaptosuccinic acid (Tc-99m DMSA) summed image shows right atrophic kidney in a
5-year-old boy with grade V VUR (a). Acoustic radiation force impulse (ARFI) imaging shows a
small kidney with no corticomedullary differentiation and entirely decreased stiffness as yellow-
green-red areas (b) (Images taken by a high-frequency probe)
and to clarify better the known/unknown sources of variability and the histopath-
ological causes of elasticity changes in patient cohorts, with pathological
correlation.
The innovation of elastography is giving rise to new avenues for prognosis, diag-
nosis, and therapy. This is especially welcome in the renal disease because of an
increasing incidence of chronic kidney disease worldwide with its high morbidity
and mortality. Technological advances in ultrasound probe, for example, three-
dimensional (3D) and combining with new elastographic techniques may offer
advantages in diagnosis and treatment of diseases (Treece et al. 2008). 3D evaluation
is easy and feasible and provides post-processing evaluation in different planes.
Combining these techniques may allow not only volumetric evaluation which could
be important in order to plan invasive treatment but also better visualization of

features of the organ/lesion (type of vascularization).
Ultrafast imaging technique which is used to track the wave in SSI leads to many
groundbreaking applications of ultrasound (Tanter and Fink 2014). This technique
may detect slow flow in very small vessels, which allows functional imaging
(fUltrasound). Moreover, it may pave the way to molecular imaging owing to higher
contrast and non-disruption of microbubbles.
The unique combination of these properties with elastography may provide a
multiparametric biomarker which will not necessitate histopathological techniques
for prognosis, diagnosis, and therapy in kidney diseases.
Summary Points
• This chapter mainly focuses on recent developments in the field of renal ultra-
sound elastography, especially its use in kidney diseases.
• Ultrasound elastography is a new imaging modality that can measure stiffness of
the tissue qualitative/quantitatively.
• The most common elastography techniques for clinical use are quasi-static
(compression-based) and dynamic elastography.
• Kidney is a unique tissue and has complex internal architecture. Therefore
intrinsic or extrinsic factors such as perfusion, urinary pressure, anisotropy,
depth of kidney, and hydronephrosis may affect viscoelasticity of the kidney.
• Elastography can be considered as a potential diagnostic biomarker in kidney
diseases; however, it needs specific training and optimization techniques as well
as acknowledging technical and pathological factors which may influence it.
Acknowledgments Figures 1–4 are reprinted from, Ultrasound Clinics, 8/4, Grenier N, Gennisson
J-L, Cornelis F, et al, Ultrasound elastography of the kidney, 551–64, 2013, with permission from
Elsevier (license number: 3446661223710).
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Aortic Pulse Wave Velocity as a Biomarker
in Chronic Dialysis Patients 48
Petar Avramovski and Aleksandar Sikole
Contents
Key Facts of Arteriosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1079
Key Facts of Doppler Effect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1079
Key Facts of Aortic Pulse Wave Velocity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1080
Key Facts of Hemodialysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1080
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1080
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1081
Document Map . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1083
Pulse Wave Velocity Estimation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1083
PWV Measured by Doppler . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1084
Arterial Stiffness and Hemodialysis Duration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1085
Progression of Arterial Stiffness . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1088
Arterial Stiffness and Cardiovascular Mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1090
Estimation of Cut-Off Point . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1092
Predictors of Cardiovascular Survival . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1092
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1097
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1097
Abstract
Cardiovascular mortality is considered the main cause of death in patients
receiving dialysis and is 10–20 times higher in such patients than in the general
population. A high percentage of all cardiovascular mortality diseases are
P. Avramovski (*)
Department of Internal Medicine, Clinical Hospital “D-r Trifun Panovski”, Bitola, Macedonia
e-mail: avramovski@gmail.com; dravramovski@hotmail.com
A. Sikole
Medical Faculty “Ss. Cyril and Methodius University” Skopje, University Clinic of Nephrology,
Skopje, Republic of Macedonia
e-mail: asikole@hotmail.com; asikole@gmail.com

1078 P. Avramovski and A. Sikole
associated with stiffening of the arteries, a direct consequence of atherosclerosis.

Increased central arterial stiffening is a hallmark of the aging process and
consequence of many disease states, such as diabetes, atherosclerosis, and
chronic renal compromise. Accelerated arteriosclerosis is a major risk to long-
term survivors on maintenance hemodialysis.
Measuring of the pulse wave velocity provides useful information regarding
the mechanical properties of the arterial tree and can be used to assess the stiffness
and endothelial function. From all the different methods to assess arterial stiff-
ness, carotid to femoral pulse wave velocity has emerged as the gold standard
method. Two Doppler waves are recorded transcutaneously at the base of the neck
for the right common carotid artery and over the right common femoral artery.
After that, the Doppler waves are identified and their time delay diversity is
measured simultaneously with electrocardiography. Time delay (transition time,
ΔT) is the time from the R wave to the foot of the carotid or femoral Doppler
waveform.
There is a high prevalence of increased pulse wave velocity in a relatively
young hemodialysis patient population. Vascular stiffening likely begins much
earlier and progresses more rapidly in hemodialysis patients. Accelerated arterio-
sclerosis is a major risk to long-term survivors on maintenance hemodialysis. The
pulse wave velocity measured at baseline was markedly higher in chronic hemo-
dialysis patients than in general population patients, with a greater than twofold
higher annual increase. In the general population group, only factors associated
with the progression of arterial stiffness in the elderly were evident (traditional
risk factors), but in chronic kidney disease patients, arterial stiffness (i.e. pulse
wave velocity) is accelerated due to synergism between age and traditional risk
factors plus factors related to renal comorbidity (nontraditional risk factors).
Patients with end stage renal disease face a particularly high risk of cardiovascular
disease and total mortality. It is now known that pulse wave velocity, C-reactive
protein and serum albumin are strongly and independently predictive of outcome
in chronic hemodialysis patients. Whether enhanced arterial stiffness is a risk
factor contributing to the development of cardiovascular disease or a marker of
established cardiovascular disease is a matter of debate. The pulse wave velocity
is a strong independent predictor of over-all and cardiovascular mortality with
high-level performance values, assessed by simple, indirect, reproducible and
noninvasive evaluation of regional arterial stiffness.
Keywords
Pulse wave velocity • Stiffness • Clinical biomarker • Chronic dialysis • Doppler •
Cardiovascular mortality • Traditional risks factors • Nontraditional risk factors
Abbreviations
C Incisura
CCA Common carotid artery
CFA Common femoral artery
CHP Chronic dialysis patients
48 Aortic Pulse Wave Velocity as a Biomarker in Chronic Dialysis Patients 1079

CV Cardiovascular
D Dicrotic wave
DD Dialysis duration
ECG Electrocardiogram
GPP General population patients
P Percussion wave
PWV Pulse wave velocity
S Starting point
T Tidal wave
TT Transit time
ΔD Relative change in vascular diameter
ΔS Vascular cross sectional surface area
ΔT Time delay, time diversity
Key Facts of Arteriosclerosis
• Increased arterial stiffness is the result of atherosclerosis, vascular calcification

and changes in collagen/elastin ratio content in the vessel wall.
• Stiffening and thickening of the arterial walls are two important components of
atherosclerosis.
• Arterial stiffness is a cause of premature return of reflected waves in late systole,
increasing central pulse pressure and the load on the ventricle.
• Accelerated atherosclerosis in renal disease is also driven by diffuse calcifications
in the arterial media without much inflammation, producing a histological picture
quite different from calcifications in complex atherosclerotic plaque.
• Accelerated arteriosclerosis is a major risk to long-term survivors on maintenance
hemodialysis and the most frequent cause of cardiovascular morbidity in patients
with end stage renal disease.
Key Facts of Doppler Effect
• The Doppler ultrasound, measuring the changes in ultrasound waves, can actually
measure how fast or slow blood is moving, which can indicate a circulatory
problem.
• Doppler ultrasound imaging can also be used to identify atherosclerotic plaque
buildup in the blood vessels, narrowed or blocked arteries.
• The most widely used method of evaluating arterial stiffness is Doppler ultra-
sound that measures aortic pulse wave velocity in the area running from the
common carotid artery to the common femoral artery.
Key Facts of Aortic Pulse Wave Velocity
• Pulse wave velocity is a measure of arterial stiffness, or the rate at which pressure
wave (not blood) moves down the vessel.
• In aging and arteriosclerosis, central elastic arteries become stiffer, diastolic
pressure decreases while systolic and pulse pressures are augmented due to
increased pulse wave velocity.
• By measuring the pulse wave velocity it is possible to noninvasively assess
stiffness and age of the arteries and thus the risk of cardiovascular events with
fatal ending.
Key Facts of Hemodialysis
• Hemodialysis is the default therapy for patients in end stage renal disease.
• Hemodialysis is a method that is used to achieve the extracorporeal removal of
waste products such as urea, creatinine and free water from the blood when the
kidneys are in end stage renal disease.
• In chronic hemodialysis patients, increased arterial stiffness has recently become
intensively investigated as a major novel cardiovascular risk factor.
• Dialysis patients have rigid blood vessels in which stiffening starts earlier and are
more pronounced by accelerated aging of blood vessels compared with patients
from the general population.
Definitions
Arterial stiffness Arterial stiffness is a term that describes rigidity of the arterial
walls and the reduced capability of arteries to expand and contract according
pressure changes.
Arteriosclerosis It is a broad term that describes hardening and elasticity loss of the
inner and middle layers of the artery wall.
Atherosclerosis This is a condition where the arteries become stiffer and narrowed
due to progressive thickening and hardening of their walls from waxy plaques on the
inner lining.
Chronic Renal Insufficiency It is a stage based on reduced filtering capacity of the

kidneys so that they are no longer capable to remove fluids and wastes from the body
or of maintaining the adequate level of certain kidney-regulated chemical in the
bloodstream.
Doppler ultrasound This is a noninvasive ultrasound test that is used to estimate

blood flow through blood vessels based of series of generated pulses that is trans-
mitted and then reflected from blood cells to detect movement of blood.
Foot of the wave This is the terminal point of diastolic spectral waveform that
occurs at the beginning of the first ascent after diastole.
Hemodialysis Hemodialysis is a renal replacement therapy method in renal failure

disease that is used to achieve the extracorporeal removal of waste products and free
water from the blood.
Pulse Wave Velocity Measures the rate of pressure wave propagation across the
vessel. Pressure wave is generated during blood flows through the vessels of
circulatory system.
Introduction
The pulse wave is a physiological phenomenon that propagates through the arteries
due to the reciprocal transformation between the kinetic energy of a segment of the
expelled blood volume and the potential energy of a stretched segment of the
resilient vascular wall. The pulse wave analysis provides useful information regard-
ing the mechanical properties of the arterial tree and can be used to assess the
stiffness and endothelial function. Arterial stiffness is a general term that collectively
describes distensiblility, compliance, and elastic modulus of the arterial vascular
system. These properties are not homogeneous along the arterial tree, muscular and
elastic vessels differ. The elastic properties of arteries vary along the arterial tree,
with more elastic proximal arteries and stiffer distal arteries. The velocity of prop-
agation of the pulse wave increases with decreased arterial distensibility. Moreover,
wave reflections, which amplify the pressure wave, are generated at the level of
peripheral arterial bifurcations and smaller muscular arteries (Laurent et al. 2006).
In recent years, great emphasis has been placed on the role of arterial stiffness in
the development of CV diseases. Indeed, the assessment of arterial stiffness is
increasingly used in the clinical assessment of patients. Increased central arterial
stiffening is a hallmark of the aging process and the consequence of many disease
states, such as diabetes, atherosclerosis, and chronic renal compromise. The most
consistent and well-reported changes are luminal enlargement with wall thickening
(remodeling) and reduction of elastic properties (stiffening) at the level of large
elastic arteries, namely arteriosclerosis (Izzo and Shykoff 2001). Arteriosclerosis
refers to reduced arterial compliance due to increased fibrosis, loss of elasticity, and
vessel wall calcification affecting the media of large and middle-sized arteries. In
dialysis patients, both atherosclerosis (affecting mainly the intima of the arteries) and
arteriosclerosis (affecting predominantly the media of large and middle-sized arteries
diffusely) is prominent (Kanbay 2010).
Arterial stiffness describes the reduced capability of an artery to expand and

contract in response to pressure changes. Parameters that describe vessel stiffness
include compliance and distensibility. The consequence of reduced compliance/
distensibility is an increased propagation velocity of the pressure pulse along the
arterial tree, called pulse wave velocity (PWV). PWV inversely correlates with
arterial distensibility and relative arterial compliance (Cecelja and Chowienczyk
2012).
Mechanical behaviour of large arteries is extremely complex and provides
serious difficulties, both on the theoretical and technical aspects. Indeed, arteries
have marked anisotropy, exhibit non-linear visco-elastic properties, and have
powerful adaptive mechanisms (Nichols and O’Rourke 2005). An understanding
of the basic principles of haemodynamics and generating pulse wave, require
integrated knowledge of physicists, physiologists, biologists and medical doctors.
Earlier physicists such as Young (1808), Poiseuille (1840), and Korteweg (1878)
established hydraulic and elastic theory. Important contributions to the analysis of
the pressure wave and PWV were made by physiologist Marey (1860) and Mac-
kenzie (1902) by developing and experimenting with various types of sphygmo-
graphs. It is impossible to extrapolate segmental arterial stiffness to the whole
arterial tree, because no single arterial segment has identical viscoelastic proper-
ties. Despite these obstacles, simple parameters derived either from the Windkessel
model or based on the arterial wave propagation that have been developed. Otto
Frank (1899) originally used the principle of conservation of mass to quantify the
Windkessel model of the arterial system. According to this description, the large
conductance arteries distend to accommodate blood ejected from the heart during
systole and recoil to propel blood through the small resistance vessels in diastole
(Sagawa K et al. 1990).
Total arterial compliance (Ctot), the sum of all arterial compliances in the system,
determines the ability of the arterial system to store blood, whereas the total
peripheral resistance (Rtot), the average input pressure divided by the average flow,
determines the ability of the arterial system to resist blood flow. According to this
description, Ctot is higher in young subjects, allowing the arterial system to accom-
modate an entire stroke volume without generating much pulse pressure (i.e.,
difference between systolic and diastolic pressures). However, with an increase in
arterial stiffness with age, Ctot decreases and ejection creates a larger pulse pressure
(Quick et al. 2006).
Arterial stiffening increases in patients with chronic renal insufficiency, and aortic
PWV, a marker of stiffening, is a strong independent predictor of mortality in this
population.
In uremic patients, elasticity and digestibility of collagen and other extracellular
matrix proteins are reduced because of reactions with methylglyoxal and other
reactive carbonyl compounds, which are increased. Intima-medial thickening occurs
in response to increased wall stress from hypertension. Arterial stiffening in renal
disease is also driven by diffuse calcifications in the arterial media without much
inflammation, producing a histological picture quite different from calcifications in
complex atherosclerotic plaque (Goldsmith et al. 2004).
Document Map
While we mention three ways for PWV measuring, attention is kept on the stiffness
measuring using Doppler ultrasound. Foot-to-foot Doppler estimating method is
explained by time delay of the signal acquired by the carotid and femoral arteries
with synchronous ECG monitoring. It is a method of PWV calculating based on the
carotid to femoral signals time delay using a standard equation of speed. In the
following text, an associative correlation between hemodialysis duration and arterial
stiffness expressed by PWV through coefficient of determination and scatter diagram is
presented. We emphasize the associated interaction of traditional and dialysis-specific
factors in the impact of increasing stiffness of the arteries in patients on dialysis.
Estimation of PWV progression have concluded that dialysis patients have rigid
blood vessels with more pronounced stiffening by accelerated aging of blood vessels
compared with GPPs. To distinguish the patients who survived and who did not
survived, we used discrimination ability of a model, by assessing cut-off point of PWV.
Both groups (survived or not survived) formed by the PWV cut-off point value
present statistically significant difference regarding survival. A plot of the Kaplan-
Meier estimate of the survival function indicates significantly higher CV mortality
observed in patients with PWV 11.8 m/s. PWV as an independent predictor for CV
outcome is assessed by Cox-regression model analysis.
Pulse Wave Velocity Estimation
Arterial stiffness, estimated by aortic PWV, is an independent predictor of CV

mortality and morbidity. However, the clinical applicability of these measurements
and the elaboration of PWV reference values are difficult due to differences between
the various devices used Salvi et al. (2008)). Arterial stiffness is assessed noninva-
sively by PWV measurement, that is, the velocity of the pulse wave to travel a given
distance between two sites of the arterial system.
There are three different non-invasive techniques to measure arterial stiffness:
1. Measuring of PWV expressed through time delay ΔT [ms].

2. Measuring of the relative change in vascular diameter ΔD = D1/D2, or relative
change in vascular cross sectional surface area ΔS = S1/S2 during PWV pressure
propagation.
3. Registration and analysis of different segments from arterial pressure waveforms
(S, P, T, C and D) and calculating indexes: ejection elastic index, dicrotic
dilatation index and dicrotic elastic index (Fig. 1).
Using the first technique, PWV is estimated from foot-to-foot transit time in the
aorta and path length measurement. This technique offers a simple, reproducible,
cheap, and noninvasive evaluation of segmental aortic stiffness.
Transit times are assessed as the time difference between two characteristic points
on carotid and femoral waveforms. The characteristic points chosen are dependent
Fig. 1 Arterial pressure waveforms in systole and diastole. Graphic variation of pressure changes
throughout one full heart cycle. This diagram can be used for classification of the arterial elasticity.
There are several specific points on the pressure diagram that reflect appropriate hemodynamic
events in the specific phases of the cardiac cycle: S (starting point) – blood discharging after aortic
valve is open; P (percussion wave) – linearly increasing of arterial wall by left ventricular (LV)
ejection; T (tidal wave) – reflected wave from the distal small arteries; C (Incisura) – end-point of
systolic phase, aortic valve is closed now and D (dicrotic wave) – reflective blood pressure of aorta
that crash into aortic valve
on the type of waveform (flow, pressure, or diameter distension) and the algorithm
used for its detection. The two most popular algorithms are: (I) the intersecting
tangent algorithm (Sphygmocor ® system and for manual identification) and (II) the
point of maximal upstroke during systole (as used in the Complior ® system).
Different algorithms applied on the same waveforms can lead to differences in
measured PWV values of 5–15 % (Millasseau et al. 2005).
PWV Measured by Doppler
Carotid and femoral waves are analyzed by a General Electric Logiq pro 5 Doppler
ultrasound machine. Although it is not possible to analyze the carotid and femoral
waves simultaneously, they can be normalized separately with the electrocardiogram
(ECG) (gatting).
Three parameters needed for foot-to-foot PWV calculating are obtained by Doppler
ultrasound with a linear array (10 MHZ) probe synchronized with ECG during 2-s
minimum sliding window: T1 – time delay from “R – wave” of ECG to foot of the
Common Carotid Artery (CCA) wave, T2 – time delay from “R – wave” of ECG to foot
of the Common Femoral Artery (CFA) wave and distance D measured from sternal
notch (CCA) to the groin (CFA). The foot of the wave is defined at the end of diastole,
when the steep rise of the systolic waveform begins. Path length (distance D) was
defined by direct anthropometric measurement of the distance between suprasternal
Fig. 2 Doppler of carotid artery – time delay measurement by CCA Doppler synchronized with
ECG. Picture shows the basic principle of time delay estimation from heart beat to the emergence of
carotid flow wave. Doppler of right common carotid artery obtains grey spectral wave and the green
curve below it is obtained by synchronous recording of electrocardiography. The distance deter-
mined by two calipers mark, presented by two “red cross” is calculated as 50.0 ms time delay (TM).
This time delay period is needed for the pulse wave to arrive from the heart to the carotid artery
notch (fossa jugularis sternalis) and groin. Each of the three consequent recordings
involved two or three cardiac cycles. To find the transit time (TT) we measured the time
from the R wave of ECG to the foot of the waveform using digital calipers (Fig. 2).
It is now known that the measurement of carotid-femoral PWV is calculated by
dividing the distance D by the ΔT, so-called TT (Transit Time). Time diversity ΔT is
calculated by the time differences T1 and T2 yielding the time delay: ΔT = T2T1.
The speed of pulse wave (V) is calculated by standard equation for the speed: V
(m/s) = S (m)/ΔT (s). Hence, PWV = D/Δt (m/s).
Arterial Stiffness and Hemodialysis Duration
While traditional risk factors predominated in the general population, in chronic

dialysis patients (CHP), nontraditional risk factors play an increasingly important
role, being perhaps dominant in end-stage renal disease (ESRD) patients. Recently,
many studies have focused on newly discovered nontraditional risk factors, such as
vitamin D deficiency, CRP, fibrinogen, hyperhomocysteinemia, high plasma

norepinefrin, accumulation of the endogenous inhibitor of the nitric oxide synthase
asymmetric dimethylarginine, extracellular volume overload, hyperphosphatemia,
and oxidant stress as a link between traditional and other nontraditional risk factors
in CHPs. Nontraditional risk factors are more prevalent in ESRD patients compared
to the general population (Zoccali et al. 2005). These include specific factors like
uremia, infection, biocompatibility of dialysis membranes, hyperhomocysteinemia,
acidosis, and hyperphosphatemia. Parallel testing and comparing the two groups (the
hemodialysis and the general population) are providing an important data for the
influence of traditional risk factors for atherosclerosis in the general population and
the combined impact of traditional and dialysis-specific risk factors in patients
undergoing dialysis (Avramovski et al. 2013).
Coefficient of determination R2 (0.3723) is showing that 37.23 % from the total
variability is explained with the linear relation between PWV and dialysis duration
(DD) or that 37.23 % from PWV is dependent on the DD. Only 37.23 % from the
changes in PWV are a result of the DD value changes and the rest 62.77 % from the
total variability between them are not explained (62.77 % of aortic PWV are
dependent on other factors, which are not covered with the regression model).
This model is used as criterion for best regression equation choice, so the greater
its value is, the better the model of approximation will be (Table 1).
The regression parameter bo = 9.7797 is showing the expected theoretical value
of aortic PWV in case if DD would have a value equal to zero. This parameter also
shows the point of the y-axis (dependent variable axis, aortic PWV) through which
the regression line passes across. The regression parameter b1 = 0.2914 signifies that
at each increase of one unit (year) in DD, aortic PWV score increases for 0.2914 m/s.
The equation of simple linear regression y59:7797þ0:2914X shows the average
coordination of aortic PWV and DD variations. With this equation, we get the
evaluated (theoretical) aortic PWV values in opposition to its empirical values.
A Fig. 3 shows a scatter plot of aortic PWV and DD. There is a positive
association between these variables. The data from each one of 80 examined patients
is displayed as a collection of colored points (blue circles) determining the cross-
sectional point of “x” axis – DD value, with “y” axis – PWV value. Each point has
the value of one variable determining the position on the horizontal axis and the
value of the other variable determining the position on the vertical axis. Linear
regression lines computed by data acquired from different aortic PWV patient’s
status dependent on aortic stiffness are plotted and shown by different colors and line
styles (light blue solid line, red dashed line and dark blue solid line). Linear
regression line plotted with dark blue solid line shows a positive correlation between
aortic PWV and DD. The 95 % confidence interval is presented by red dashed line
and prediction interval is presented by light blue solid line.
The high CV mortality rate in dialysis population has become a major issue and
it’s not simply related to the increased acceptance of elderly subjects. A recent cross-
sectional haemodialysis study found that while traditional coronary risk factors may
apply to this population, other factors including the uraemic milieu and the
haemodialysis procedure itself were probably contributory (Cheung et al. 2000).
48
Table 1 Linear regression analysis of aortic PWV and dialysis duration. This table shows the results of relationship between a scalar dependent variable
(PWV) and explanatory variable (DD) obtained by linear regression. The high value of the coefficient of determination (R2 = 0.3723) and the low value of the
coefficient of statistical significance ( p < 0.0001) shows that there is a strong positive correlation between PWV and DD. This means that 37.23 % from the total
variability is explained with the linear relation between PWV and DD or that 37.23 % from PWV is dependent of the DD
Regression
Dependent Y Pulse wave velocity, m/s
Independent X Dialysis duration, years
Sample size 80
Coefficient of determination R2 0.3723
Residual standard deviation 1.9666
Regression equation
y ¼ 9:7797 þ 0:2914 X
Parameter Coefficient Std. error 95 % CI t P
Intercept b0 9.7797 0.3216 9.1395–10.4199 30.4118 <0.0001
Slope b1 0.2914 0.04285 0.2061–0.3767 6.901 <0.0001
PWV pulse wave velocity, DD dialysis duration, Std. Error standard error, CI confidence interval
Aortic Pulse Wave Velocity as a Biomarker in Chronic Dialysis Patients
1087
Fig. 3 Linear regression scatter plot of aortic PWV and dialysis duration. A Fig. 3 shows the results
from linear regression analysis between pulse wave velocity (PWV) and dialysis duration (DD)
presented as scatter plot, a graph of plotted points that shows the relationship between two sets of
data. Linear regression line plotted with dark blue solid line shows a positive correlation between
aortic PWV and DD, the 95 % confidence interval is presented by red dashed line and prediction
interval is presented by light blue solid line. Abbreviations: PWV pulse wave velocity, DD dialysis
duration
Haemodialysis causes numerous changes including abnormal complement activa-

tion with disordered leukocyte-endothelial interactions, the release of plasma factors
including tumour necrosis factor-alpha and reactive oxygen species (Himmelfarb
et al. 2002; Schroder et al. 2001). These processes cause vascular oxidative stress
and consecutive elevation of PWV in latter period as result of the vascular stiffness
increase. Because of this, the impact of nontraditional risk factors and the above-
mentioned factors, nontraditional and specific dialysis factors are conditioning
progressive yearly increase of artery stiffness in dialysis patients or PWV increase
of average 0,291 m/s in every single year spent in dialysis.
Progression of Arterial Stiffness
Aortic stiffness is associated with increased CV mortality in patients with chronic

kidney disease (CKD). Traditional CV risk factors may play some role in the
progression of aortic stiffness before development of advanced CKD, and that the
enhanced rates of progression of aortic stiffness in CKD patients on dialysis are
probably determined by more specific CKD-related risk factors such as advanced-
glycation end products (AGEs) (Utescu et al. 2013). Modification of vascular
extracellular matrix by advanced AGEs may result in progression of vascular
stiffness. Because of higher exposure to glucose and uremic toxins, patients on
dialysis have higher tissue levels of AGEs, increased vascular stiffness, and
Fig. 4 Pronounced progression of arterial stiffness in chronic hemodialysis patient (CHP) com-
pared to the general population patient (GPP) group. This box whisker diagram shows comparative
results from PWV progression in 36-month follow-up period among CHPs and GPPs. ΔPWV in
both groups is presented as a difference between PWV in the baseline and after 36 months. Its mean
value and SD is 63.95
18.373 cm/s for CHP and 27.28
28.519 cm/s for GPP. It is obvious that
there is a faster and pronounced progression of PWV in CHP than in the GPP group. The results of
mean, range, 75th percentiles, median and 25th percentiles, test statistics, difference and two-tailed
probability of P are presented in this figure, too (Figure courtesy of Korean Journal of Internal
Medicine (KJIM 2013; 28: Fig. 4, p 469). The figure is published with permission from the KJIM
and copyrights are reserved). Abbreviations: PWV pulse wave velocity, CHP chronic hemodialysis
patients, GPP general population patients
enhanced central augmentation pressure compared to general population patients

(GPP). However, the rate of progression of arterial stiffness and the role of CV risk
factors in the progression of arterial stiffness are not enough established in longitu-
dinal prospective studies. Although age is one of the most important determinants of
CV risk, large artery stiffness is also a key independent predictor of CV mortality
(London et al. 2001).
It is now known that progression of arterial stiffness in CHPs compared to the
GPPs is pronounced. It was evaluated in longitudinal prospective study in 3 years
period (Avramovski et al. 2013). The progression of PWV in CHP during the
36-month period (mean difference 0.6395
0.18373 m/s, p < 0.001) compared to
the progression of PWV in GPP during the same period (mean difference 0.2728
0.28519 m/s, p < 0.001) is pronounced (Fig. 4).

Estimated and compared patients from the control group did not include a young
healthy population. The control group consisted of participants from the general
population who were not spared from the normal process of atherosclerosis, aging,
and osteoporosis. The patients in this group had functioning kidneys, to exclude the
influence of renal comorbidity. There is a high prevalence of increased PWV in a
relatively young hemodialysis patient population. Vascular stiffening likely begins
much earlier and progresses more rapidly in hemodialysis patients ( p < 0.001). The
PWV value measured at baseline was markedly higher (24 %) in CHP than in GPP,
with a greater than twofold higher annual increase. In the GPP group, only factors
associated with the progression of arterial stiffness in the elderly were evident
(traditional risk factors), but in CKD patients, arterial stiffness (i.e., PWV) is
accelerated due to synergism between age and traditional risk factors plus factors
related to renal comorbidity (nontraditional risk factors).
The marked increase in aortic stiffness with aging and little change in peripheral
arterial stiffness results in a reversal of the gradient of arterial stiffness from the
youthful pattern of a compliant proximal aorta, which was evident in individuals
aged <50 years, to a pattern of greater aortic stiffness in older participants (Fantin
et al. 2007). The progression of blood vessel aging is significantly greater in dialysis
patients. In this population, the chronological age is greater than biological age,
expressed through the increased arterial stiffness (Fig. 5).
It is now known that progression of PWV over a 36-month period, and the
significant difference between the CHP and GPP groups, suggest that arterial
stiffening has progressed further in dialysis patients compared to the general popu-
lation, which suggests a significant distinction in the aging and stiffness of their
arteries, and so thus the biological age of both populations (despite almost identical
chronological age: 59.3
11.8 vs. 59.7
11.9 years).
GPPs have an increased vascular stiffness; this is associated with traditional risk
factors and urea, hemoglobin, albumin, CRP, and glucose levels. Nontraditional risk
factors, or uremia-related specific factorssuch as anemia (hemoglobin), inflammation
(CRP), hypoalbuminemia, and abnormal lipoproteins-might play a role in the accel-
erated progression of arterial stiffness only in CHPs (Avramovski et al. 2013).
Fortunately, arterial stiffening can be monitored by a simple noninvasive method,
measuring the PWV, which enables evaluation of the risk of CVr events.
Arterial Stiffness and Cardiovascular Mortality
A high percentage of all CV diseases are associated with stiffening of the arteries, a
direct consequence of atherosclerosis. Increased arterial stiffness is the result of
many contributing factors, such as atherosclerosis, vascular calcification and
changes in collagen/elastin ratio content in the vessel wall. The increase in artery
wall stiffness is noticeable from the beginning of the arteriosclerosis process, before
anatomical changes and clinical manifestations are observed. Atherosclerosis is the
most frequent cause of CV morbidity in patients with ESRD. Patients with ESRD
face a particularly high risk of CV disease and total mortality (Zaccali et al. 2003).
Fig. 5 Values of 3-year PWV follow-up period in CHP (baseline and after 36 months). The results
from the PWV progression in CHPs during 36-months follow-up period are presented in Fig. 5. The
mean value of 11.18
2.29 m/s is compared with the mean value of 11.82
2.34 m/s after 3-years.
Box plots, notched box plots and lines are presenting results of mean, range, 75th percentiles,
median and 25th percentiles of PWV in CHPs. Abbreviations: PWV pulse wave velocity, CHP
chronic hemodialysis patients, 36 mon 36-months follow-up period
Accelerated arteriosclerosis is a major risk to long-term survivors on maintenance

hemodialysis (Safar et al. 2002). Myocardial infarction and cerebrovascular events
occupy an important place in the mortality of these patients. The CV mortality rate of
CHPs is approximately 20 times higher than that of the general population, and the
cerebrovascular death rate is nearly 10 times higher.
The arterial system in ESRD patients undergoes structural remodeling very
similar to changes with aging, and is characterized by diffuse dilation, hypertrophy
and stiffening of the aorta and major arteries. In comparison with nonuremic
patients, the intima-media thickness of major central arteries is increased in ESRD
patients (London et al. 1997). There are large-scale cohort studies, which provide
evidence that high PWV as a noninvasive marker for arterial stiffness is a useful
predictive marker for CV events in subjects with CKD, hypertension and diabetes.
Since epidemiological and clinical studies have shown that damage of large arteries
is a major contributory factor to the high CV morbidity and mortality of patients with
ESRD, such a population is particularly appropriate to analyze the impact of arterial
stiffness on mortality (Blacher et al. 1999, 2003). PWV is a strong independent
predictor of overall and CV mortality in a population of ESRD patients undergoing
hemodialysis but (Guérin et al . 2001) have recently showed that arterial stiffness is
not only a risk factor contributing to the development of CV disease but is also a
marker of established more advanced, less reversible arterial changes.
Avramovski et al. (2014) in 36-month follow-up period, comparing the PWV
results in survived (11.26
2.37 m/s) and nonsurvived ESRD patients (13.13
1.70 m/s) got significantly ( p < 0.001) higher PWV in deceased patients. PWV in
deceased patients from CV disease is more pronounced, it is equal to 13.7
1.24 m/s
( p < 0.001). At first sight, it is not very big difference, only about two and a half
meters. But, if we know the fact, that an increase of aortic PWV by 1 m/s corresponds
to an age, sex and risk factor adjusted, risk increases for 14 %, 15 % and 15 % in total
CV events, CV mortality and all-cause mortality, respectively, the above mentioned
fact is not for underestimation. An increase in aortic PWV by 1 SD (standard
deviation) was associated with respective increases of 47 %, 47 % and 42 %
(Vlachopoulos et al. 2010b).
Estimation of Cut-Off Point
The most relevant way of structuring the comparison groups of ESRD, in order to
obtain the statistical significance between them is grouping by cut-off PWV value.
The PWV cut-off point value for ESRD patients where the sensitivity and specificity
are highest (94.1 % and 61.4 %, respectively) is 11.8 m/s. Avramovski et al. found
that the PWV cut-off point of 11.8 m/s is predictive of increased mortality in ESRD
patients, especially for CV mortality. However, different studies have determined
different cut-off points of PWV that is predictive of increased overall and CV
mortality. The cut-off point of 12 m/s or greater was chosen based upon a study
demonstrating this to be the level associated with clinically significant negative
prognosis in patients with ESRD (Covic et al. 2005). Based on receiver operating
characteristics (ROC) curve analysis mean PWV levels in CHPs show an optimal
cut-off point at 12.0 m/s, while mean PWV levels in GPPs show an optimal cut-off
point at 9.6 m/s/ (Boutouyrie 2010). The role of age in presenting normal, reference
and cut-off point values needs careful consideration. As for blood pressure, it is not
immediately clear whether normality should be defined according to age. It is now
known that considering the PWV of 11.8 m/s as a relevant cut-off point speed, for
all-cause and especially for CV mortality prediction generates two different sub-
groups of ESRD patients. There are statistically significant differences between
those subgroups ( p < 0.001) according to PWV value (13.65
1.32 vs. 9.76
1.29 for each one).
Predictors of Cardiovascular Survival
Vertical drop in a plot of the Kaplan-Meier indicates an event as series of horizontal

steps of declining magnitude approaching the true survival function in CHPs
(Fig. 6). Considering the fact that the mean value of PWV is 8.3 m/s (mean age
Fig. 6 Kaplan-Meier survival time according different cut-off value of PWV and CV events. This
figure presents survival probability in CHPs according different PWV (above or below the cut-off
velocity): PWV 11.8 m/s (red staircase line) and PWV <11.8 m/s (blue staircase line). Every vertical
drop in a plot of the Kaplan-Meier indicates a CV event as series of horizontal steps of declining
magnitude approaching the true survival function in CHPs during 36-months follow-up period.
Abbreviations: PWV pulse wave velocity, CV cardiovascular, CHP chronic hemodialysis patients
61.0 years) in the general population (Inoue et al. 2009) the majority of patients with
ESRD could be considered to have increased arterial stiffness and elevated PWV
equal to 12.50 m/s (mean age 59.3) as results of accelerated atherosclerosis
(Avramovski et al. 2014). It is now known that there is a more than fourfold
increased relative risk for lethal outcomes (all-causes mortality) in subgroups with
more stiffened arteries (PWV 11.8 m/s, P = 0.0037). Relative risk for exposed
groups according to CV lethal outcomes is a about 14-fold increased risk in sub-
groups with more stiffened arteries (PWV 11.8 m/s, P < 0.0080).
Avramovski hypothesized that there is no differences in survival in both subgroups
of patients on dialysis just below and above the cut-off point (11.8 m/s). Comparative
results of the two curves [(logrank), x2 = 13,1001; degree of freedom (DF) = 1;
significance (P) = 0,0003; relative risk = 0.1744; 95 % CI = (0.0767–0.3965)]
indicate significantly higher CV mortality in patients with PWV above cut-off point
(PWV 11.8 m/s). With threshold of 0.95 or security risk error of 0.05, Avramovski
rejected the null hypothesis and concluded that there is a statistically high significant
difference (significance is very large) in both subgroups of dialysis patients with
different PWV, regarding survival. Survival time according to different cut-off value
of PWV dependent on CV events is presented in Fig. 6.
Are the traditional risks factors for atherosclerosis sufficient alone to describe
high prevalence of CV disease in this condition? The traditional risk factors for
Fig. 7 Cox-regression
survival analysis (predictors
of CV outcome). The results
from Cox-regression analysis
of CV survival according to
PWV as an independent
predictor for the CV outcome
in CHPs: regression
coefficient b = 0.357, p value
= 0.0005, hazard ratio
coefficient Exp (b) = 1.429
and 95 % CI of Exp (b) =
1.169–1.745 are presented in
Fig. 7. Red staircase line
indicates CV survival
probability in CHPs in
36-months follow-up period.
Every vertical drop in a plot
means one fatal CV event.
Abbreviations: CV
cardiovascular, CHPs chronic
hemodialysis patients, PWV
pulse wave velocity, CI
confidence interval
atherosclerosis (age, elevated blood pressure, smoking status, low levels of HDL
cholesterol, high levels of LDL cholesterol and triglycerides, obesities and diabetes)
interacting to initiate atherosclerosis and promote the development of CV disease
have enhanced our ability to assess risk in individual patients. In addition, under-
standing of new, so-called novel risk factors (CRP, homocysteine, plasma fibrinogen,
interleukin-10, impaired glucose tolerance and metabolic syndrome) and when these
are included along with the classic risk factors in assessing the global risk profile,
may improve ability to predict future risk precisely. In uremic patients, traditional
risk factors are added to specific, disease-related (inflammation and malnutrition)
and treatment-related risk factors (incompatibility of dialysis membrane and dialysis
adequacy) (Fruchart et al. 2004).
Using Mantel – Cox- regression analysis (proportional hazards regression) of CV
survival in hemodialysis patients, the potential predictors of events ending with
death we’re analyzed. Assessments (regression coefficient [b], hazard ratio coefficient
Exp [b], p value, and 95 % CI [confidence interval] of Exp [b]) of independent
predictors for CVoutcome after Cox-regression model analysis are presented in Fig. 7.
According to the Cox-regression analysis, the significant covariates retained by
the model (backward stepwise) are only PWV, CRP and albumin. Covariates with
positive regression coefficients (b), PWV (0.357) and CRP (0.083) are predictors of
the CV events. They indicate decreased hazard and increased survival time. Albu-
min, as covariate with negative regression coefficient (b) (0.1881), indicates
decreased hazard and increased survival time. The predictor PWV has an Exp
(b) hazard ratio coefficient of 1.429. The HR increases by 1.429 (42.9 %) with
each unit increase in PWV. Foremost biomarker in predicting CV risk is PWV with
more expressed statistical significance ( p < 0.0001) than statistical significance of
other covariates (CRP, p < 0.001; albumin, p < 0.003).
Aortic PWV may represent a surrogate end point, which may in fact indicate in
which patients the traditional CV risk factors translate into real risk. Summary
comparative results from meta-analysis of the predictive value of aortic stiffness
(carotid-femoral PWV) for all-cause and CV events are presented by Vlachopoulos
et al.: HR: 1.63 for CV and 1.61 for all-cause mortality; HR: 1.44 for CV and 1.35 for
all-cause mortality; HR: 1.20 for CV and 1.14 for all-cause mortality. Considering
earlier before mentioned arguments, it remains to explain whether PWV as the main
determinant of arterial stiffness, has some independent predictive value for the
overall and CV – mortality. Several pathophysiological mechanisms may explain
the association between increasing PWV and CV-mortality. Increased stiffness of the
arteries is the cause of premature return of reflected waves in late systole, resulting in
increased central pulse pressure and further ventricular overload. It reduces ejection
fraction and increases the myocardial oxygenation demand.
The estimation of PWV as an indicator of artery stiffness has never been ascertained
as a CV risk marker. Recently, many studies have confirmed its importance that
aortic PWV is strongly associated with the presence and extent of atherosclerosis and
constitutes a forceful marker and predictor of CV risk in hypertensive patients,
diabetes, chronic kidney disease, rheumatoid arthritis, degenerative disease and
many other diseases. PWV as a biomarker of disease is a predictor of coronary heart
disease and stroke in a population-based study among apparently healthy subjects
(Mattace-Raso et al. 2006), and provides additional predictive value above CV risk
factors, measures of atherosclerosis, stiffness and pulse pressure. Viscoelastic prop-
erties of large arteries play an essential role in CV hemodynamics, especially in
systolic blood pressure determination.
Large artery damage is a major contributing factor to the elevated CV morbidity and
mortality observed in CV risk factors such as hypertension. Rich qualitative and
quantitative information about the large arteries (stiffness, distensibility, pulsatility,
compliance) is easily obtained by Doppler determination of PWV. Reduced arterial
distensibility contributes to a disproportionate increase in systolic pressure and an
increase in arterial pulsatility that is associated with an increase in CV morbidity and
mortality. A number of longitudinal studies among hypertensive patients report the
effects of elevated PWV as an independent predictor of cerebrovascular diseases and
all-cause mortality. The relative risk of stroke mortality is 1.7 for PWVelevation of 4 m/
s and that of all-cause mortality is 2.1 for PWV elevation of 5 m/s (Laurent et al. 2003).
Patients with type 2 diabetes have increased stiffness of central elastic arteries.
However, whether peripheral muscular artery stiffness is equally affected by the
disease remains sparsely examined. Diabetes is a predictor of central artery stiffness,

and glucose is a determinant of peripheral artery stiffness (Zhang et al. 2011).
Hyperhomocystinaemia is associated with macro and microangiopathic diabetic
complications. Vitamin B12 is significantly associated with homocystein concentra-
tions and is identified as a marginally independent correlate of PWV in diabetic
patients in the absence of folate deficiency. Elevated homocystein and reduced
vitamin B12 have a key role in the development of atherogenesis in diabetic patients
(Shargorodsky et al. 2009). Mortality risk doubled in subjects with diabetes (hazard
ratio 2.34, 95 % CI 1.5–3.74) and in those with glucose intolerance (2.12, 95 % CI
1.11–4.0) compared with controls (Cruickshank et al. 2002).
Rheumatoid arthritis (RA) is a chronic, systemic, inflammatory disease, which
is associated with increased CV risk that is not explained by traditional CV risk
factors but may be due in part to increased aortic stiffness, an independent predictor
of CV mortality (Mäki-Petäjä et al. 2006). The involvement of the CV system in the
course of inflammatory lesion and connective tissue diseases may result in serious
morbidity and mortality. PWV as an indicator of arterial distensibility predicts CV
risk in RA patients. Decreased dilatation capacity leads to a reduction in arterial
blood pressure and flow dynamics and impairment in coronary perfusion. Aortic
PWV increases by only +6 % per decade in healthy individuals, which suggests that
subjects with RA have arteries 20 years older than chronological control subjects.
Contrary to findings by Klocke et al. (2003), there is not a significant difference in
PWV between the RA and control groups.
Systemic inflammation may contribute to the increased incidence of CV disease
in RA, because inflammation is known to play a pivotal role in the pathogenesis of
CV disease (Libby 2002). All these changes in the CV system are in line with
increased CV morbidity and mortality in RA patients. The CV mortality risk in
patients with RA is approximately equal to the CV risk in patients with diabetes. In a
prospective study, the 3-year incidence rate of fatal and nonfatal CV events was
9.0 % in RA patients and 4.3 % in the general population. Compared with the
non-diabetic population, non-diabetic patients with RA and those with type 2 diabe-
tes had comparable hazard ratios, 2.16 and 2.04 respectively (Peters et al. 2009). It is
very likely that the use of common risk calculators (e.g., Framingham, SCORE) will
underestimate the CV risk in RA patients.
Effective control of inflammation reduces the CV risk in patients with RA as it
improves arterial stiffness and endothelial function. The measurement of PWV gives
data values about the current state of blood vessels inflammation and assesses to the
risk of CV disease in RA patients. This diagnostic method for assessment of arterial
stiffness is useful not only to assess vascular features of blood vessels (rigidity,
elastance, compliance, distensibility) but can assess the effectiveness of certain drugs
in the course of therapy in certain diseases.
Conventional and noninvasive tools for evaluating atherosclerosis have been
recently developed and are currently in use. The use of PWV has received increasing
attention as a non-invasive method to measure vascular injury (Khoshdel
et al. 2007). The first use of PWV was reported in 1922 in a study examining an
association between age and arterial stiffness (Bramwell and Hill 1922). During the
1960s, as new techniques were developed to assess pulse wave and pressure, reports
on PWV increased. At the time, PWV measurement procedures were complicated
and not suitable for diagnostic purposes. The development of ultrasound Doppler
technique made a big step forward, so now measuring the PWV has become a
relatively simple method for fast, inexpensive, accurate and routine assessment of
arterial stiffness. Following its commercialization in 1999, many reports on PWV
have been published.
Here, we have indicated the potential value of using PWV, which is a convenient,
inexpensive, and noninvasive test to identify vascular injury and predict vascular
disease. The European Society of Hypertension (ESH) and the European Society of
Cardiology (ESC) have added PWV measurement as an early index of large artery
stiffening in the “2007 Guideline for the Management of Arterial Hypertension”
(Mancia et al. 2007).
Summary Points
• This chapter focuses on arterial stiffness, which describes the reduced capability
of an artery to expand and contract in response to pressure changes.
• Increased central arterial stiffening is a hallmark of the aging process and the
consequence of many disease states, such diabetes, atherosclerosis, and chronic
renal compromise.
• The pulse wave velocity is a physiological phenomenon that is used to assess the
stiffness of large vessels, measuring the speed of pressure wave propagation, not
the displacement of the blood.
• While traditional risk factors predominated in the general population,
nontraditional risk factors (uremia, infection, biocompatibility of dialysis mem-
branes, acidosis, etc. . .) play an increasingly important role, being perhaps
dominant in end-stage renal disease patients.
• The larger stiffness of the blood vessels in patients on hemodialysis, which occurs
earlier and progresses rapidly, increases the speed of the pulse wave and the
number of cardiovascular events.
• Aortic pulse wave velocity may represent a surrogate end point, which may in fact
indicate in which patients the traditional cardiovascular risk factors translate into
real risk.
• The salient finding of this chapter is that the pulse wave velocity was a strong
independent predictor of cardiovascular mortality with high-level performance
values, assessed by simple, indirect, reproducible, and noninvasive evaluation of
regional arterial stiffness in chronic dialysis patients.
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Renal Arterial Resistance Index
49
Massimo Iacoviello, Valeria Antoncecchi, Marta Leone, and
Marco Matteo Ciccone
Contents
Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1102
Key Facts of Cardiorenal Syndrome (CRS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1102
Key Facts of Doppler Ultrasonography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1103
Key Facts of Heart Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1104
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1104
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1106
Renal Circulation and Renal Arterial Resistances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1107
Doppler Evaluation of Arterial Resistances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1109
Factors Influencing RRI . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1111
Potential Applications to Prognosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1114
Arterial Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1114
Diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1114
Renal Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1114
Heart Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1115
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1117
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1117
Abstract
The kidney is a profusely vascularized organ which, unlike other organs, does not
regulate renal blood flow (RBF) mainly by oxygen demand. Reflex (myogenic effect
and tubular-glomerular feedback) and neurohormonal mechanisms modulate RBF and
M. Iacoviello (*)
Cardiology Unit, Cardiothoracic Department, University Hospital Policlinico Consorziale of Bari,
Bari, Italy
e-mail: massimo.iacoviello@policlinico.ba.it
V. Antoncecchi • M. Leone • M.M. Ciccone
Cardiology Unit, School of Cardiology, Department of Emergency and Organ Transplantation,
University of Bari, Bari, Italy
e-mail: valeriaantoncecchi@libero.it; martaleo84@yahoo.it; marcomatteo.ciccone@uniba.it

1102 M. Iacoviello et al.
renal resistances by regulating the tone of afferent and efferent arterioles as well as that
of the major resistance vessels in the kidneys. Arterial renal resistances are also
influenced by a number of other pathophysiological factors, such as increased arterial
stiffness, arterial atherosclerosis, renal parenchymal abnormalities, and intra-abdominal
and central venous pressure. Finally, renal resistances can be permanently increased if a
microvascular disease and vascular rarefaction occur as a result of vasoconstriction-
related ischemia, endothelial dysfunction, and inflammatory cytokine activity.
In this setting, a parameter reflecting renal resistances could offer a useful tool
to better characterize renal disease and the risk of progression. This chapter
focuses on renal resistance index (RRI), a parameter obtained by pulsed Doppler
which reflects renal arterial resistances. The possible clinical usefulness of RRI
has been demonstrated in studies which show the pathophysiological correlates
and prognostic role in predicting a greater risk of chronic kidney disease progres-
sion and cardiovascular events.
Keywords
Renal resistance index • Renal circulation • Chronic kidney disease • Cardiovas-
cular diseases • Heart failure • Worsening of renal failure
Abbreviations
CHF Chronic heart failure
CVP Central venous pressure
HF Heart failure
HFPEF Heart failure with preserve ejection fraction
HFREF Heart failure with reduced ejection fraction
NO Nitric oxide
RAAS Renin-angiotensin-aldosterone system
RAS Renal artery stenosis
RBF Renal blood flow
RI Resistance index
RRI Renal resistance index
WRF Worsening of renal function
Key Facts
Key Facts of Cardiorenal Syndrome (CRS)
• Kidney and heart diseases share many pathophysiological mechanisms such as

tissue hypoperfusion, venous congestion, inflammatory status, and neurohor-
monal activation.
49 Renal Arterial Resistance Index 1103
• Acute or chronic dysfunction of one of these two organs can lead to functional
worsening of the other. Cardiorenal syndrome is the condition in which the heart
and kidney can negatively affect each other.
• The Consensus Conference of the Acute Dialysis Quality Group divided this
syndrome into five subtypes.
• Types 1 and 2 (cardiorenal syndrome): acute heart failure causing acute kidney
injury (AKI) and chronic heart failure leading to a progressive renal dysfunction,
respectively.
• Types 3 and 4 (renocardial syndrome): acute kidney dysfunction causing acute
heart failure and chronic kidney disease leading to a worsening of cardiac
function, respectively.
• Type 5: systemic diseases involving both the kidney and heart.
• Therefore, in patients with heart failure, an accurate evaluation of renal
function is important, and vice versa; therefore, close collaboration between
nephrologists and cardiologists is necessary in order to improve patient
prognosis.
Key Facts of Doppler Ultrasonography
• Doppler ultrasonography is a noninvasive technique that uses high-

frequency sound waves (ultrasound) to estimate blood flow through blood
vessels.
• During examination the operator uses a transducer that sends and
receives ultrasounds. The sound waves are reflected by blood cells, and their
frequency is modified (increase or decrease) by the movement of red cells
(Doppler effect).
• The Doppler shift is the difference in frequency between the reflected
ultrasound and the initial ultrasound and is correlated with the velocity of the
blood cells.
• A computer receives and processes this information and gives a spectrum or an
image that represents the blood flow.
• Three main Doppler techniques are used: continuous wave, pulsed wave, and
color Doppler.
• Continuous wave Doppler: the transducer continuously emits trains of ultra-
sounds along a line and continuously receives reflected signals. Continuous
wave Doppler ultrasound is unable to determine the specific location of velocities
within the beam.
• Pulsed wave Doppler: the transducer emits a train of ultrasound to a certain depth
and then captures the reflected ultrasounds. The computer then calculates the flow
velocity at that point.
• Color Doppler: the principle is the same as for pulsed wave Doppler, but
analyzes an area of small sample volumes. Based on the velocities
calculated in that area, the computer builds a color image that represents the
blood flow.
Key Facts of Heart Failure
• Heart failure represents one of the main causes of morbidity and mortality in
industrialized countries. The prevalence ranges from 0.4 % to 2.0 % in the
European population. The incidence increases with age.
• It is a condition in which the heart is not able to pump an adequate supply of blood
to satisfy the body’s needs (oxygen and nutrients).
• Heart dysfunction can involve the left or right ventricle or both. However, the left
ventricle is usually affected first.
• There are two different mechanisms underlying left heart dysfunction: the heart is
not able to contract with enough force (systolic dysfunction), or the ventricle
becomes stiff and develops resistance to filling.
• Heart failure is defined as acute if signs and symptoms arise suddenly and require
immediate medical treatment. It is defined as chronic if the patient is in a stable clinical
condition but has, or has had, signs and symptoms of heart failure for some time.
• Causes of heart failure are coronary artery disease; myocardial infarction; valvu-
lar, pericardium, endocardium, or heart muscle disease; congenital heart disease;
severe lung disease; diabetes; abnormalities of heart rhythm; etc.
• Possible symptoms are shortness of breath (dyspnea) during activity or at rest,
tiredness, and palpitations. Possible signs are high jugular venous pressure,
pulmonary crepitations and displaced apical impulse, and excess fluid in body
tissues (edema) with swelling of feet, ankles, legs, or abdomen.
• Heart failure treatment includes lifestyle changes, drugs (angiotensin-
converting enzyme inhibitors, angiotensin II receptor blockers, beta-blockers,
diuretics, vasodilators, antiplatelet agents, and anticoagulants), implantable defibrilla-
tors to prevent arrhythmic complications, left ventricular assist devices (a mechanical
device that helps the heart to pump blood), and, ultimately, heart transplantation.
Definitions
Arteriolosclerosis This is a disease of the small blood vessels characterized by a

thickening and hardening of the arteriole wall. It can be due to concentric, smooth
muscle wall hypertrophy or the buildup of hyaline material. It reduces blood flow
and determines tissue ischemia.
Central venous pressure (CVP) This is the pressure in the thoracic vena cava near
the heart; therefore, it is an estimate of pressure in the right atrium. It can be
measured invasively using a central venous catheter, but noninvasive evaluation is
also possible. The most common method used is to evaluate the diameter of the vena
cava and its changes during inspiration.
Ejection fraction This is the percentage of blood ejected by the heart with each
beat. It is an indicator of heart contractility. Mathematically, it is the difference
between the end-diastolic volume and the end-systolic volume divided by the
end-diastolic volume. Normal values are 55–70 %.
Glomerular filtration rate (GFR) This is considered the best index to assess renal
function. It measures kidney filtration capacity using renal clearance of an exoge-
nous marker (inulina or I-iothalamate) or more often an endogenous marker, i.e.,
serum creatinine. GFR is generally estimated on the basis of serum creatinine levels.
Glomerulosclerosis This is a disease characterized by scarring and hardening of

glomeruli. If only a part of glomeruli are affected, then it is defined as focal
segmental glomerulosclerosis. It can be idiopathic or due to known causes (HIV,
obesity, diabetes, drugs, lupus, etc).
Heart failure with preserved ejection fraction (HFpEF) This term is used to
define patients with signs and symptoms of heart failure but who have a normal or
mildly reduced ejection fraction, no left ventricle dilatation, abnormalities of cardiac
structure, and/or diastolic dysfunction. HFpEF has a higher prevalence among the
elderly, females, and patients with hypertension.
Heart failure with reduced ejection fraction (HFrEF) This term is used to define
patients with signs and symptoms of heart failure but who have a reduced ejection
fraction. The most frequent cause is coronary artery disease. Other possible causes
are myocarditis, alcohol abuse, chemotherapy, idiopathic forms, etc.
Pulse arterial pressure This is the difference between the systolic pressure and the
diastolic pressure. It represents the maximal change in aortic pressure during systole.
It depends on the compliance of the aorta and on cardiac output.
Pulse wave velocity This is the best index of aortic stiffness. It is usually measured
by determining the propagation time of the pulse pressure from the carotid to femoral
artery. Different methods are used in order for measurement, among which the most
common are mechanical methods requiring specific devices. However, it may also
be easily measured using high reproducible, noninvasive ultrasound methods. A
threshold of >10 m/s has been suggested by a recent expert consensus statement as
an index of an altered arterial distensibility in hypertensive patients.
Renal blood flow (RBF) This is the amount of blood that passes through the kidney
in a specific time unit. Mathematically, it is the difference between the aortic pressure
and renal venous pressure, divided by renal vascular resistance. The normal value is
1,200 ml/min.
Worsening renal function (WRF) In patients affected by cardiovascular diseases,

this is generally defined as an increase in serum creatinine of >0.3 mg/dl and/or
>25 % between two time points. It has been shown to be associated with a worse
prognosis, hospitalizations for heart failure and higher mortality.
Introduction
The kidney is a profusely vascularized organ, and, unlike other organs, renal blood
flow (RBF) is not mainly influenced by oxygen demand but is determined by reflex
(myogenic effect and tubular-glomerular feedback) and neurohormonal mechanisms
that regulate the tone of afferent and efferent arterioles as well as of the major
resistance vessels in the kidneys (Braam et al. 2012). Moreover, arterial renal
resistances are also influenced by a number of other factors, such as an increased
arterial stiffness, arterial atherosclerosis, renal parenchymal relevant pathophysio-
logical abnormalities, and increased central venous pressure. These factors can cause
a permanent increase in vascular resistance due to microvascular remodeling and
capillary rarefaction caused by vasoconstriction-related ischemia, endothelial dys-
function, and the production of inflammatory cytokines and fibrosis (Chade 2013).
Therefore, a parameter that provides information on functional and permanent
changes in RBF by reflecting abnormalities in renal arterial resistances could offer
an incremental value to better characterize renal function.
This potential use is even more obvious when considering the limitations of the
parameters currently used to assess renal function. The estimation of renal function is
generally based on the calculation of glomerular filtration rate (GFR) by serum
creatinine levels (Stevens et al. 2006). While creatinine serum levels and estimated
GFR represent the cornerstone in the evaluation of renal function and its worsening,
they are limited by several factors, such as between-person and within-person
variability, age, diet, gender and body mass, the active creatinine tubular secretion
(Damman et al. 2012), drug interference, and the loss of muscle mass frequent in the
end stages of systemic diseases (Smilde et al. 2006). Finally, it is worth noting that
the kidneys use only part of their filtering capacity. A normal GFR does not exclude
an impairment in filtration capacity; in other words, a normal GFR in kidneys with a
reduced renal reserve which increases the filtration capacity of residual nephrons
(Bosch et al. 1995) could be observed. Due to the limitations of creatinine, new
biomarkers have been proposed to detect early glomerular dysfunction (cystatin C)
or identify tubular damage (neutrophil gelatinase-associated lipocalin, N-acetyl-
beta-glucosaminidase, and kidney injury molecule) preceding the drop in GFR.
In this clinical setting, a parameter reflecting RBF alterations could be useful
because, by detecting abnormalities, patients at a higher risk of renal disease onset
and/or its progression could be better identified (Fig. 1). This more accurate evalu-
ation of renal function could be relevant not only for the treatment of chronic kidney
disease (CKD) patients, but also for a better management of patients affected by
cardiovascular diseases.
Over the last few years, more attention has been focused on the link between the
kidney and the heart. These two organs are characterized by many common patho-
physiological mechanisms which can negatively affect each other, and, conse-
quently, the term “cardiorenal syndrome” has recently been introduced. This term
indicates a condition characterized by an acute or chronic dysfunction of one of the
two organs which may induce the acute or chronic dysfunction of the other (Ronco
et al. 2008).
Fig. 1 Changes in renal blood flow preceding the drop in glomerular filtration rate. The
pathophysiological background of possible clinical usefulness of renal resistances (Modified by
Damman et al. 2010). The glomerular filtration rate drops when the kidneys are no longer able to
compensate for the loss of nephrons by enhancing the filtration of those remaining. Renal resistance
index, as well as microalbuminuria and tubular dysfunction biomarkers, could provide early
detection of functional and structural renal changes preceding the fall in GFR. GFR glomerular
filtration rate, RBF renal blood flow, WRF worsening of renal function
In patients affected by chronic cardiovascular diseases and especially in those

with heart failure (HF), CKD as well as worsening renal function (WRF), i.e., type
2 cardiorenal syndrome, represents a clinical condition associated with a poor
prognosis (Hillege et al. 2000; Smith et al. 2006; Damman et al. 2009).
The aim of this chapter is to focus on the possible clinical role of renal arterial
resistances in patients with renal and cardiovascular diseases.
Renal Circulation and Renal Arterial Resistances
Twenty-two percent of cardiac output is directed to the kidney. It has a terminal-type

circulation, without anastomosis (Fig. 2). At the level of the hilum branch, the renal arteries
branch out into the segmental, arcuate, and interlobular arteries. The afferent arterioles
derive from the interlobular arteries leading into the glomerular capillaries that coalesce to
form the efferent arterioles. Efferent arterioles give rise to a second capillary network, i.e.,
peritubular capillaries that are followed by the venous system (Chade 2013).
In order to control arterial pressure and water and sodium reabsorption, reflex
mechanisms (such as myogenic effect and tubular-glomerular feedback) and neuro-
hormonal mechanisms (sympathetic system, renin-angiotensin-aldosterone system,
Fig. 2 Anatomy of renal circulation. The renal arterial and vein anatomy is shown
endothelin, nitric oxide, prostaglandins, bradykinin, and natriuretic peptides) mod-

ulate the arteriolar tone, thus affecting renal resistance.
The myogenic effect is a fast mechanism consisting in the vasoconstriction of
kidney small vessels when wall tension increases. The tubuloglomerular feedback is
a slower mechanism of vasoconstriction of the afferent arteriole that occurs when an
increase in GFR produces a rise in NaCl concentration, which is then sensed by the
macula densa, and a vasoconstrictor mediator (probably adenosine) is secreted. Of
the neurohormonal mediators, the most important are catecholamines and the renin-
angiotensin-aldosterone system (RAAS) which determine vasoconstriction and an
increased reabsorption of water and sodium (Braam et al. 2012).
The RBF self-regulation maintains the renal perfusion constant for arterial pres-
sure changes between 70 and 180 mmHg. The abovementioned mechanisms regu-
late RBF and GFR in order to keep them constant when there are fluctuations in renal
perfusion pressure both in physiological situations and pathological conditions.
A depletion or an overstimulation of these systems, for example, in heart failure,
could determine an increase in intrarenal resistances and a dissociation between
cardiac output and RBF. This is the consequence of a disproportional decrease in
renal perfusion pressure and RBF in reduced cardiac output (Braam et al. 2012).
The arterial renal resistances are also influenced by a number of other relevant
pathophysiological factors such as increased arterial stiffness, arterial atherosclero-
sis, renal parenchymal abnormalities, and increased central venous pressure (Fig. 3).
Moreover, endothelin, nitric oxide (NO), prostaglandins, bradykinin, and natriuretic
peptides are also involved in the regulation of arteriolar tone.
Among these, the decreased availability of NO seems to play a pivotal role in
determining abnormalities in renal circulation. A reduction, mediated by oxidative
stress (reactive oxygen species, ROS), promotes vasoconstriction. Moreover,
Fig. 3 Factors influencing renal circulation. The factors influencing renal perfusion and renal
resistances are summarized. The myogenic reflex and the tubular-glomerular feedback regulate the
tone of afferent and efferent arterioles. However, arterial renal resistances are also influenced by a
number of other pathophysiological factors, such as neurohormonal mechanisms, arterial stiffness,
arterial atherosclerosis, renal parenchymal abnormalities, and intra-abdominal and central venous
pressure. CVP central venous pressure, IAP intra-abdominal pressure, RAAS renin-angiotensin-
aldosterone system, SNS sympathetic nervous system
ROS-mediated inflammation further reduces NO availability thus perpetuating a

vicious circle leading to permanent changes in renal vasculature.
In fact, the ischemia related to endothelial dysfunction and the inflammatory cyto-
kines can cause fibrosis, vascular remodeling, and rarefaction (Chade 2013). This latter
condition is generally due to changes in renal microcirculation, which are also defined as
microvascular disease, and is a frequent feature of CKD. When it occurs, a permanent
increase in renal resistances and a further decline in renal function can be observed.
Doppler Evaluation of Arterial Resistances
Arterial pulsed Doppler has been proposed as a useful tool to estimate the arterial
resistance of an organ. The parameter generally used is the Doppler resistive index
(RI), i.e., a measure obtained from pulsed wave Doppler velocity curves of the
peripheral arteries according to the Pourcelot equation ([peak systolic velocity –
end-diastolic velocity]/peak systolic velocity) (Pourcelot 1974).
The first studies on RI were aimed at evaluating its role in the assessment of the
grade of stenosis of internal carotid artery lesions (Pourcelot 1974). Several studies
have subsequently suggested the possible usefulness of RI also in the evaluation of

other vascular beds, among which the kidney.
Assessment of renal resistance index. The examination of kidney and renal
arteries is usually performed by an anterior approach by means of a convex ultra-
sound probe. The patient should be adequately prepared to avoid intestinal gases.
Nonetheless, it is possible to evaluate the renal resistance index (RRI) at the end
of an echocardiogram also using the echocardiographic probe (Ciccone et al. 2014).
With the patient in a lateral or sitting position, the scan images of the kidney are
obtained from a posterior approach. The use of Color or Power Doppler helps to
localize the vessels.
RRI slightly decreases from the hilum toward the renal cortex. Usually, pulsed
Doppler volume sample is placed at the level of the segmental arteries. It is necessary
to achieve the best alignment between the ultrasound beam and flow direction to
record Doppler velocities maximizing waveforms gain and size, to obtain peak
systolic velocity and end-diastolic velocity (Fig. 4). Three to five reproducible
waveforms should be evaluated.
In healthy adult subjects, the RRI mean value is around 60 with nonsignificant
differences between the two kidneys (Pontremoli et al. 1999). The index is affected
by extreme bradycardia and tachycardia and pathologically increases with severe
aortic valve insufficiency and decreases with severe aortic valve stenosis. It is less
reliable during arrhythmias (Krumme et al. 2007).
An RRI greater than 70 is generally considered abnormal in adults, while it could
physiologically exceed this cutoff in the first years of life (Tublin et al. 2003).
According to a recent meta-analysis (Lubas et al. 2014), measures taken by expert
staff showed an intraobserver variability ranging from 2.07 % to 5.1 % and an
interobserver variability from 3.61 % to 6.2 %. Therefore, overall RRI has a good
reproducibility and repeatability
RRI and renal resistances. The relationship between RRI and renal resistance is
not actually linear. RRI changes have been evaluated in an ex vivo system in which
vascular compliance and resistance could have been separately modified (Bude
et al. 1999). The study authors concluded that RRI is influenced by a combination
of compliance and resistance. For fixed compliance, the RRI increases with increas-
ing resistance.
In vivo conditions are even more complicated than this because many factors play
a role in perfusion pressure regulation. Also, considering that the majority of
published studies have enrolled nonhomogeneous study populations, this preamble
explains why the literature on RRI sometimes produced discordant results that have
caused some skepticism about the routine use of this parameter in clinical practice.
Renal artery stenosis and RRI. Besides the exploration of the entire renal artery,
also the examination of the intrarenal arteries is extremely important in the diagnosis
of renal artery stenosis (RAS) (Fig. 5). The RRI, together with the acceleration time
index, is in fact an indirect parameter of stenosis. In the presence of a significant
RAS, reduction in blood flow and pulsatility occur leading to a tardus-parvus pattern
Doppler waveform characterized by a longer time needed to achieve the peak
systolic velocity. In addition, the vasodilatation of the distal capillary bed produces
Fig. 4 Renal resistance index calculation. Calculation of the renal resistance index. The renal
arterial Doppler was performed using a 4 MHz probe with the patient in the sitting position and
using a posterior approach to visualize the kidney. The course of the segmental arteries is visualized
by color Doppler flow, and, at the middle tract level of the best one visualized, pulsed wave Doppler
is performed. Peak systolic velocity and end-diastolic velocity are used to calculate the renal arterial
resistance index according to Pourcelot formula
a reduced peak systolic velocity and a relatively increased end-diastolic velocity,

reducing overall the poststenotic RRI.
Zeller and Schwerck have proved, in different studies, the accuracy of a side-to-side
difference in an RRI >5 to detect RAS, while an AT >100 ms was found to be reliable
for the diagnosis, with an 89 % sensitivity and a 91 % specificity (Zeller et al. 2008).
Factors Influencing RRI
In the absence of a significant renal artery stenosis, RRI is influenced by a number of

physiological and pathophysiological factors able to modify arterial and venous RBF.
Aging and arterial stiffness. As previously mentioned, RRI could be influenced
by changes in arterial compliance and resistance (Bude et al. 1999).
Fig. 5 Patterns of renal resistance index. Examples of possible alterations of the renal Doppler
pattern. The figure shows a normal subject at the top, a subject with increased renal resistances in
the middle, and a subject with renal arterial stenosis at the bottom
Consequently, RRI is influenced by all physiological and pathophysiological

factors which change arterial compliance and resistance. It has been widely demon-
strated that RRI positively correlates with age, pulse pressure, pulse wave velocity
(Schwenger et al. 2006; Lubas et al. 2014; Ohta et al. 2005), and ambulatory arterial
stiffness index (Ratto et al. 2006), which are all markers of reduced arterial
compliance.
In a population of renal transplanted patients, RRI was correlated to the recipi-
ent’s ankle-brachial blood pressure index (Heine et al. 2005) and to subject’s age,
rather than the age of the grafted kidney (Krumme et al. 1997).
All cardiovascular risk factors which can modify arterial stiffness, such as
diabetes and essential hypertension, are also able to modify RRI. RRI has been
found to be significantly correlated to daytime systolic blood pressure variability
(Kawai et al. 2012). Similarly, RRI was also independently associated with blood
glucose level (Otha et al. 2008) and insulin resistance (Afsar et al. 2010). These two
risk factors affect the RRI not only by modifying arterial stiffness but also by
determining parenchymal renal lesion and/or promoting oxidative stress.
Parenchymal renal diseases. Platt et al. (1990) first showed the utility of RRI to
differentiate between isolated glomerular disease and vascular or interstitial disease
as assessed by renal biopsies. Patients with glomerulopathies had normal RI values
(mean value: 58), whereas the others had markedly elevated RI values (mean values
are 87 in vascular alterations and 75 in interstitial fibrosis).
Numerous studies have subsequently confirmed the association of RRI with

tubulointerstitial injury (Bigè et al. 2012; Sugiura et al. 2004; Boddi et al. 2005)
and arteriolosclerosis. In particular, one found that RRI was correlated to renal
involvement in patients with progressive sclerosis (Aikimbaev et al. 2001).
It is probable that parenchymal diseases, involving any parenchymal component,
produce a scarring process that, in turn, leads to a reduction in the intrarenal vessels
area and to an increase in interstitial pressure, which decreases vascular compliance
and increases resistance to blood flow.
Endothelial dysfunction. As previously mentioned, endothelial dysfunction and
the related NO imbalance play a key role in the vasomotor nephropathy. Intrarenal
NO regulates glomerular hemodynamics, tubular transport, and tubuloglomerular
feedback (Majid et al. 2001). The net result is increased renal and glomerular
perfusion, natriuresis, and diuresis.
Changes in NO availability could influence RRI by modifying both afferent and
efferent arteriole tone as well as renal medullary blood flow. Moreover, the ischemia
related to reduced NO and the related increase in inflammatory cytokines can cause
fibrosis, vascular remodeling, and rarefaction (Chade 2013).
Although these are potential effects, there are limited data about the relationship
between RRI and endothelial dysfunction (Bruno et al. 2011).
Intra-abdominal and central venous pressure. Another important determinant of
renal resistances is renal venous pressure. Its changes influence RBF more than
changes in arterial blood pressure. It has been demonstrated experimentally that RBF
decreases when renal venous pressure increases (Winton 1931).
The mechanisms influencing renal venous pressure include the obstruction of
renal vein (thrombosis, neoplastic mass, inferior vena cava syndrome), an increase in
abdominal pressure (ascites and hemorrhage), and a rise in central venous pressure
(CVP) (HF and pulmonary hypertension) (Braam et al. 2012).
An increased renal venous pressure causes a decrease in arteriovenous gradient
with a consequent reduction in RBF. Moreover, it determines a rise in efferent
arterioles and end glomerular capillary pressures, thus inducing a decrease in net
filtration pressure and in GFR and an increase in arterial renal resistances (Jessup and
Costanzo 2009). Finally, besides the effects on gradients of renal vasculature, an
increased renal venous pressure causes a rise in interstitial pressure. This is due to the
low compliance of interstitium and to the tight capsule of the kidney (Braam
et al. 2012).
As a consequence, in the clinical setting of heart failure, venous congestion is the
other determinant mechanism responsible for the decrease in renal perfusion
together with reduced cardiac output. This has recently been supported by a study
evaluating a series of CHF outpatients in which a high CVP was independently
associated with higher RRI values (Ciccone et al. 2014).
This is even more interesting considering that both in patients with acute and
chronic heart failure (CHF) an increased CVP has been found to be associated with a
WRF and a progression of cardiorenal syndrome (Damman et al. 2007; Winton
1931; Mullens et al. 2009; Iacoviello (a) et al. 2013).
Potential Applications to Prognosis
Arterial Hypertension
Several data show that a high RRI in hypertensive patients is also associated to
organ-damage markers such as left ventricular hypertrophy and carotid intima-media
thickness (Florczak et al. 2009; Kawai et al. 2012; Parolini et al. 2009; Otha
et al. 2008; Doi et al. 2012).
Independently from the presence of overt nephropathy, hypertensive patients
show higher vascular resistance and a higher RRI (Raff U et al. 2010).
In 426 hypertensive patients, Doi and coll. (2012) demonstrated that RRI >73 for
males and >72 for females was predictive of a combined end point of cardiovascular
and renal events (i.e., all-cause death, myocardial infarction, stroke, congestive heart
failure requiring hospitalization, aortic dissection, and end-stage renal failure requir-
ing regular hemodialysis).
Although RAAS inhibitors seem to decrease RRI values, showing a
renoprotective effect, in some studies the stratification of the population on the
basis of the use of these drugs did not alter the prognostic results (Sugiura and
wada 2009; Doi et al. 2012).
Diabetes
As in arterial hypertension, diabetes is a systemic disease with a multiorgan target.

Diabetic nephropathy is characterized by nodular and diffuse forms of intercapillary
glomerulosclerosis, afferent and efferent glomerular arteriolar hyalinization, tubular
atrophy, and interstitial fibrosis (Ohta et al. 2005), although glomerulosclerosis is the
histological hallmark of the disease.
It has been demonstrated that in diabetic patients, high RRI values are signifi-
cantly correlated to the observation of microvascular diabetic complications, includ-
ing nephropathy, retinopathy, or sensory neuropathy (Liu et al. 2012). Moreover, in
patients with renal diseases, RRI is higher in diabetic nephropathy than in chronic
glomerulonephritis and/or nephrosclerosis (Ohta et al. 2005). Finally, higher RRI in
diabetic patients is also associated with left ventricular diastolic dysfunction
(MacIsaac et al. 2008), thus highlighting that type 2 diabetes leads to multiorgan
damage (including simultaneous injury to the heart and kidneys).
In advanced CKD (eGFR <30 ml/min/1.73 m2), however, the RRI difference
between diabetic and nondiabetic patients loses statistical significance (Kawai
et al. 2011), thus suggesting the prevalent relevance of the advanced renal disease
in affecting the value of the parameter.
Renal Diseases
More data are available for RRI in renal diseases.

Chronic kidney disease. In this clinical setting, several studies have shown that the RRI
assessed at enrollment is predictive of WRF (Radermacher et al. 2002; Ikee et al. 2005;
Splendiani et al. 2002; Sugiura et al. 2009; Parolini et al. 2009; Hanamura et al. 2012).
Splendiani et al. (2002) demonstrated a positive correlation between the initial
value of RRI and the percentage of serum creatinine variation.
Radermacher and coll. (2002) found that RRI >80 can identify patients with a
worse prognosis. In a multivariate regression analysis, only proteinuria and RRI
were independent predictors of progressive renal dysfunction. In a study conducted
by Hanamura and coll. in patients with CKD (Hanamura et al. 2012), a RRI >70 was
an independent risk factor for WRF, and it was considered a marker of organ
dysfunction, histological damage, and renal prognosis and a possible determinant
for steroid therapy.
Bigè and coll. (2012) studied 58 patients affected by CKD, defined according to
the KDOQI definition, and undergoing renal biopsy. In this series, a RRI >70 was
predictive of a reduction in renal function and identified patients at higher risk of
end-stage renal disease independently from GFR baseline values.
Also the studies conducted on renal transplanted patients showed very interesting
conclusions. A RRI >80 was predictive of allograft failure. However, it did not
permit identification of a specific cause of failure, but could raise the suspicion of
vascular complications associated with transplantation, such as arteriovenous fistula
or vein thrombosis. What is more important, however, is that the higher values of
RRI were associated with patient deaths, also in cases of functioning grafts
(Radermacher et al. 2003).
Acute kidney injury. Very recent studies suggest the usefulness of RRI in
predicting acute kidney injury (AKI) in intensive care unit patients (namely, patients
with polytrauma and sepsis or patients who underwent cardiac surgery) (Schnell
et al. 2012; Bossard et al. 2011; Dewitte et al. 2012). Moreover, (Darmon et al. 2011)
found even higher values of RRI in patients with persistent AKI.
Renal artery stenosis. Radermacher and coll. (2001) suggested RRI as a marker to
predict survival after therapeutic intervention (angioplasty or surgery) for RAS.
Patients with a RRI >80 showed no improvement in renal function, hypertension,
or renal survival even after a successful procedure. Two subsequent studies (Yuksel
et al. 2012; Cianci et al. 2010) confirmed similar results while another disproved
them (Zeller et al. 2008).
Heart Failure
Renal impairment in CHF patients is very common and is associated with higher
morbidity and mortality (Hillege et al. 2000; Smith et al. 2006; Damman et al. 2009).
Therefore, its accurate characterization plays a key role in the management of these
patients. The role of RRI in the clinical setting of CHF is a topic of interest since,
reflecting kidney vascular and parenchymal abnormalities (Fig. 6), it adds informa-
tion about renal impairment and the outcome of these patients (Ennezat et al. 2011;
Ciccone et al. 2014).
Fig. 6 Pathophysiology of renal resistance index in cardiorenal syndrome. The pathophysio-

logical factors influencing the renal resistances and the consequent prognostic impact on cardiorenal
syndrome are summarized. The combination of hemodynamic and neurohormonal factors leads to
functional and structural changes responsible for the increase in renal resistance index. This further
promotes the activation of neurohormonal systems as well as sodium and water retention, thus
favoring the progression of cardiorenal syndrome
The first association between high RRI and worse outcome has been demon-
strated in a series of patients affected by HF with preserved ejection fraction
(HFpEF) (Ennezat et al. 2011). Subsequently, this association has been found in a
series of CHF outpatients mainly affected by HF with reduced ejection fraction
(HFrEF). In this series, RRI was an independent predictor of both a composite end
point reflecting HF progression (i.e., death hospitalization due to HF worsening)
(Ciccone et al. 2014) and mortality for all causes (Monitillo et al. 2014).
Also in CHF patients, the different pathophysiological conditions underlying the
increase in renal vascular resistance and, as a consequence, in RRI can explain the
relevant prognostic information carried by this parameter. This is confirmed by the
independent association that has been found in CHF patients between RRI and the
variables reflecting the presence of atherosclerosis and/or an increased arterial
stiffness (i.e., age, diabetes, and pulse pressure) as well as an increased CVP.
This pathophysiological background underlying RRI values can also explain its
incremental value when added to GFR. A high RRI could identify patients at higher
risk of HF progression both in patients with GFR above and below 60 ml/
min*1.73 m2. On the other hand, patients with low RRI were characterized by a
similar risk of events also when dichotomized according to the presence or not of
reduced GFR (Ciccone et al. 2014).
But it is also worth noting that RRI is not only associated with HF progression but
also with WRF in CHF outpatients. In fact a RRI >70 is independently associated
with a 1-year increase in creatinine >0.3 mg/dl (Citarelli et al. 2014).
Finally, in CHF outpatients, RRI was also demonstrated to be independently
associated with high doses of loop diuretics as well as with their increase during a
midterm follow-up (Iacoviello (b) et al. 2015). The increased intrarenal resistance
could, in fact, cause a reduction in filtration pressure and lead to a reduced delivery
of diuretic molecules at the level of Henle’s loop and a reduced response (Paul 2002).
As a result, an increased RRI can allow the detection of patients with an altered
diuretic dose-response curve who may develop diuretic resistance.
These data suggest that this parameter could be used together with GFR,in the
clinical setting of CHF in order to obtain a better characterization of kidney function.
This is further strengthened by the fact that RRI evaluation has been found to be
easy, fast, and highly reproducible (Ciccone et al. 2014).
Summary Points
• Renal resistances play a key role in renal function as well as in CKD onset and
progression. It has been shown that RRI, a Doppler-derived parameter, reflects
renal arterial resistances and is a noninvasive evaluation tool.
• An altered RRI reflects the many factors which can modify renal resistances, such as
increased arterial stiffness, arterial atherosclerosis, oxidative stress, endothelial dysfunc-
tion, renal parenchymal abnormalities, renal microvascular disease and vascular rare-
faction, intra-abdominal and central venous pressure, and neurohormonal activation.
• In patients with renal diseases, RRI is not a marker for a specific disease because it
increases in different clinical conditions. However, it certainly could be an index
to indicate renal disease progression as it reflects the abnormalities of both the
systemic and local vascular bed.
• In renal diseases, an increased RRI is associated with a higher risk of CKD
progression and a worse outcome.
• In cardiovascular diseases, RRI seems particularly relevant in patients with
arterial hypertension and in those with CHF. In the latter group, an increased
RRI has been found to be independently associated both with HF progression and
mortality. Moreover, it offers incremental prognostic information when added to
GFR and is able to detect patients prone to developing WRF.
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Pulmonary Pressure as a Novel Prognostic
Biomarker in Renal Patients 50
Davide Bolignano, Francesco Mattace-Raso, Eric J. Sijbrands,
Anna Pisano, and Giuseppe Coppolino
Contents
Key Facts of Pulmonary Circulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1123
Key Facts of Pulmonary Hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1123
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1124
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1124
Pulmonary Circulation and Pulmonary Pressure Assessment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1125
Diagnosing High PP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1126
Significance of High PP in the General Population and in Renal Patients . . . . . . . . . . . . . . . . . . . . 1129
Risk Factors of High PP in Renal Patients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1131
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1138
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1138
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1138
Abstract
Renal patients are notoriously at high risk for cardiovascular complications, but
such risk is not fully explained by traditional and chronic kidney disease (CKD)-
related risk factors. New prognostic biomarkers are therefore needed to refine
outcome prediction in this population. High pulmonary pressure (PP; also known
as pulmonary hypertension) is remarkably prevalent among persons with CKD,
D. Bolignano (*) • A. Pisano

e-mail: davide.bolignano@gmail.com; pisanoanna@hotmail.it
F. Mattace-Raso • E.J. Sijbrands
Department of Internal Medicine, Erasmus University Medical Center, Rotterdam, The Netherlands
e-mail: f.mattaceraso@erasmusmc.nl; e.sijbrands@erasmusmc.nl
G. Coppolino
e-mail: gcoppolino@hotmail.it

1122 D. Bolignano et al.
particularly in hemodialysis patients. High PP is a powerful and independent

predictor of death in the general population and in subjects with heart or lung
diseases. In renal patients, there is now evidence showing that PP may hold the
same prognostic utility. High PP predicts adverse cardiovascular outcomes in
dialysis and pre-dialysis populations. In kidney transplant recipients, high PP is
associated with worse renal outcomes. In this chapter, we will focus on high PP in
the CKD population, spanning from the main techniques for assessing PP to the
pathophysiology of pulmonary hypertension (PH) in renal patients. Prognostic
implications of PH in CKD patients for risk stratification and therapeutic man-
agement will also be discussed.
Keywords
Pulmonary pressure • Pulmonary hypertension • Chronic kidney disease •
End-stage kidney disease • Dialysis • Cardiovascular risk
Abbreviations
APAH Associated forms of pulmonary arterial hypertension
ARBs Angiotensin receptor blockers
AVF Arteriovenous fistula
CKD-5D Chronic kidney disease stage 5 on dialysis
CKD-ND Chronic kidney disease not on dialysis
COPD Chronic obstructive pulmonary disease
CV Cardiovascular
E/e0 Early trans-mitral flow velocity [E]/early mitral annular tissue
velocity [e0 ]
EGD Early graft dysfunction
ePP Estimated pulmonary pressure
ERS European Respiratory Society
ESC European Society of Cardiology
ESKD End-stage kidney disease
FPAH Familial pulmonary arterial hypertension
HD Hemodialysis
HIV Human immunodeficiency virus
ILD Interstitial lung disease
IPAH Idiopathic pulmonary arterial hypertension
KDOQI Kidney Disease Outcomes Quality Initiative
LV Left ventricular
mPP Measured pulmonary pressure
NO Nitric oxide
NYHA New York Heart Association
PAH Pulmonary arterial hypertension
50 Pulmonary Pressure as a Novel Prognostic Biomarker in Renal Patients 1123
PAWP Pulmonary artery wedge pressure

PH Pulmonary hypertension
PP Pulmonary pressure
PVR Pulmonary vascular resistance
RAP Right atrial pressure
RHC Right heart catheterization
Vmax Maximum tricuspidal jet velocity
WHO World Health Organization
Key Facts of Pulmonary Circulation
• The pulmonary circulation is a low-resistance, low-impedance, high-capacitance,

and high-flow circuit.
• This circuit has hemodynamic and structural characteristics that are deeply
different from those of the systemic circulation.
• Pressure values in the pulmonary arteries are roughly one fourth to one sixth of
those of the systemic circulation.
• Medial thickening of the major branches of the pulmonary artery is lower than
that of systemic arteries, being comparable to the structure of large veins.
• The normal pulmonary circulation consists of highly compliant vessels and a vast
capillary network with large recruitment capability.
Key Facts of Pulmonary Hypertension
• Pulmonary hypertension (PH) is a condition characterized by a pathological

increase in pulmonary artery pressure (PP).
• Such increase may result from an increase in pulmonary artery resistances (“pre-
capillary” forms) or by a passive congestion of the venous side of the pulmonary
circulation (“post-capillary” forms) that is usually secondary to left heart dysfunction.
• PH may arise during several lung or systemic diseases and, particularly, in the
presence of left heart dysfunction. In rare cases, PH manifests as a primary disease.
• The overall, estimated prevalence of asymptomatic PH in the general population
ranges from 5 % to 10 %. Precapillary forms are much more rare with an
estimated prevalence of about 15 cases per million and an annual incidence of
about 2–3 per million.
• Echocardiography may be useful as a screening test to identify patients at risk
of PH.
• Invasive maneuvers (right heart catheterization) are needed for a clear-cut diag-
nosis of PH, for the etiological framework, and for an optimal therapeutic
planning (vasoreactivity test).
• Prognosis and response to treatment are highly variable, being mostly dependent
on the type of PH.
• Subjects with pre-capillary PH (particularly primary forms) are usually scarcely

responsive to treatments and may need lung transplant.
Definitions
Arteriovenous fistula (AVF) Artificial vascular shunt surgically created by

connecting a peripheral artery and a vein in the elbow or in the wrist of patients
that need to start chronic hemodialysis treatment. AVFs serve as a high-flow vascular
access for performing the dialysis procedure.
Chronic kidney disease (CKD) A pathological condition characterized by a

reduced renal function with consequent alterations in fluid regulation, blood pressure
control, waste product elimination, and metabolic alterations. CKD may progress,
more or less rapidly, to end-stages.
End-stage kidney disease (ESKD) The final stage of CKD, also known as terminal
uremia. Residual renal function is not anymore sufficient to control the body
homeostasis so that patients need to start in due course chronic hemodialysis
treatment or be transplanted.
Hemodialysis (HD) Chronic or acute therapy for replacing renal function. ESKD
patients usually undergo chronic hemodialysis thrice a week. Each HD session lasts
3.5–4 h. During the HD session, fluid and electrolyte excess and waste products are
removed from the blood circulation.
Peritoneal dialysis (PD) Alternative technique to hemodialysis consisting of using

the patient’s own peritoneal membrane as an exchange surface for balancing fluid,
electrolyte, and waste homeostasis. The peritoneal cavity is accessed via a perma-
nent catheter placed in the patient’s abdomen that is linked to an external machine.
Exchanges are induced and regulated according to diffusion and convection princi-
ples by fluid bags with established content.
Right heart catheterization (RHC) Invasive maneuver consisting of reaching the

right side of the heart via a small catheter inserted from a peripheral vein (e.g.,
femoral, subclavian, or jugular). This procedure allows diagnostic measurements
(e.g., right heart pressures, pulmonary artery pressure) as well as interventional
procedures.
Introduction
The incidence of chronic kidney disease (CKD) and end-stage kidney disease
(ESKD) is on the rise. Currently, it has been estimated that over 50 million people
are affected by CKD and over 2 million persons need chronic dialysis for ESKD
(Eggers 2011). This amount is expected to increase by 60 % by 2020 (Gilbertson).

CKD ranks now as one of the main risk factors for cardiovascular (CV) mortality and
morbidity with a substantial impact on health resources. In the USA, annual costs
attributable to manage CV complications of CKD patients rank from 1,700 (KDOQI
CKD stage 1) to 12,700 (CKD stage 4) dollars (Honeycutt et al. 2013). Although a
large percentage of patients with CKD have traditional CV risk factors such as
diabetes, hypertension, and lipid abnormalities, interventions targeting these factors
have failed to significantly decrease CV mortality and morbidity. Similarly, normal-
ization of other nontraditional risk factors peculiar of CKD including anemia,
microalbuminuria, inflammation, oxidative stress, and altered mineral metabolism
was not totally effective in improving event-free survival.
High pulmonary pressure (PP) has recently emerged as a novel CV risk factor in
the general population. In a surveillance from 1980 to 2002 (Hyduk et al. 2005), the
Centers for Disease Control and Prevention identified increasing rates of hospitali-
zation associated with high PP and stable death rates ranging from 5.2 to 5.4 per
100.000 persons. Conversely, during the last decade, an increasing trend in mortality
was documented with an estimated age-adjusted death rate of 4.5–12.3 per 100.000
(George et al. 2014). High PP rarely presents as an idiopathic condition, being more
frequently associated with systemic, cardiac, or lung disorders which may affect the
pulmonary vascular circuit.
There is now accruing evidence indicating that masked, non-symptomatic high
PP is exceedingly prevalent in CKD persons, particularly in ESKD patients on
chronic renal replacement therapy (Bolignano et al. 2013). Several underlying
conditions, such as volume overload, the presence of high-flow arteriovenous
fistulas, breath disorders, and sympathetic hyperactivation have been postulated to
explain such a high frequency. Nevertheless, PP is emerging as a novel, important
prognostic biomarker in the renal population. In fact, the presence of high PP in
CKD is generally associated with poor outcomes, spanning from increased mortality
to higher rate of CV events and delayed graft function in renal transplanted patients
(Bolignano et al. 2013). The evaluation of PP might therefore represent an addi-
tional, helpful tool for risk assessment and risk stratification of renal patients.
Pulmonary Circulation and Pulmonary Pressure Assessment
Less known to nephrologists, who are more familiar with the systemic circulation,
the pulmonary circulation is a delicate and exclusive low-resistance, low-impedance,
high-capacitance, and high-flow circuit. Under physiological conditions, pressure
levels in the pulmonary arteries are roughly one fourth to one sixth of those normally
found in the systemic circulation (Naeije 2013). Medial thickening of the major
pulmonary arteries is notably lower than that of systemic arteries, being more similar
to the structure of large veins. The normal pulmonary circulation therefore consists
of highly compliant pulmonary arteries and a vast capillary network with large
recruitment capability which is able to accommodate large increases in blood flow
without significant increases in PP, e.g., during sustained exercise or in the presence
of left-to-right congenital intra-cardiac shunts.
How can PP be assessed in clinical practice? The gold standard of measurement is
represented by right heart catheterization (RHC), an invasive procedure which
consists in reaching the right heart with a catheter inserted via a peripheral vein
(Badesch et al. 2009). The catheter can be moved until the right atrium or even
further, reaching the right ventricle and the main pulmonary artery branches. As long
as the catheter proceeds through the right heart to the pulmonary circulation, this gets
characteristic pressure responses, very similar to a sequence of spikes, peaking at
about 20–25 mmHg. When in the distal branch of the pulmonary artery, values of the
measured PP of about 14
3 mmHg are considered as normal. The assessment of
the so-called pulmonary artery wedge pressure (PAWP), that is, the pressure mea-
sured by wedging a Swan-Ganz catheter with an inflated balloon into a small
pulmonary arterial branch, may give additional information, particularly in the
presence of pathological PP values (see below). In fact, PAWP allows to assess the
pulmonary vascular resistance (PVR), expressed as the ratio between the difference
of mean PP and PAWP and the cardiac output. RHC is crucial for assessing PP, but as
it is an invasive procedure, it may be associated with an increased risk of dangerous
complications. Therefore, with some exceptions, cardiac catheterization in daily
practice is usually not considered as the first-line approach to evaluate PP. In most
cases, PP is firstly estimated by non-invasive procedures, like a simple transthoracic
Doppler echocardiography. PP estimation with such technique is based on the
eventual finding of tricuspidal regurgitation, a phenomenon that can be minimally
present also in apparently healthy subjects. If tricuspidal regurgitation is present, the
echocardiography instrument can automatically assess the maximum tricuspidal jet
velocity (Vmax). This parameter is important to finally estimate the PP (ePP)
according to the so-called Bernoulli’s equation as the product of the square of the
Vmax by 4 (4x Vmax2) (Rudski et al. 2010). However, this equation often gives a
too much rough estimate of ePP that can be further refined by implementing also
information about the estimated right atrial pressure (RAP) in the “modified”
Bernoulli’s equation (4x Vmax2+ RAP) (Yock and Popp 1984). RAP is supposed
to range from 10 to 30 mmHg in case of absence or presence of relevant inferior vena
cava collapse.
Diagnosing High PP
A recent joint guideline (Galie et al. 2009) made by the European Society of
Cardiology (ESC) and the European Respiratory Society (ERS) has established the
main criteria for the definition of “pathological” PP values, finally making clearness
in a very controversial and debated topic.
High, pathological PP (a condition also known as pulmonary hypertension (PH))
is defined by a documented increase in the measured PP (mPP) 25 mmHg at rest.
However, although not diagnostic, ePP values 35–49 mmHg (roughly
corresponding to Vmax values of 2.8–3.4 m/s) can be considered suggestive of PH
“Per-Capillary” “Post-Capillary”
Primary increase in pulmonary “Passive” congestion of the venous side
artery resistance Capillary barrier PWP>15 mmHg
PWP£15 mmHg PVR<3 W.U.
PVR>3 W.U.
Pulmonary Arterial
High PP associated to
hypertension (PAH)
left heart disorders
idiopathic, familiar or
associated to connective Left heart systolic dysfunction,
diseases, HIV, drug or Left heart diastolic dysfunction,
toxins, portal hypertension, left-sided valve disease (mitral
pulmonary veno-occlusive and/or aortic)
disease
High PP of unclear or
multifactorial etiology
chronic thromboembolism
Obstruction of pulmonary arterial
vessels by emboli, tumors or foreign
bodies
lung diseases
COPD, ILS, sleep-apnea,fibrosis
Fig. 1 Differential diagnosis of high pulmonary pressure in relation to the anatomic site of disease.
COPD chronic obstructive pulmonary disease, ILS interstitial lung syndrome, PAWP pulmonary
artery wedge pressure, PP pulmonary pressure, PVR pulmonary vascular resistance
while values 50 mmHg are strongly indicative of the true presence of PH. Echocar-
diography estimation can then be useful as screening test for selecting patients who
deserve more invasive exams for a clear-cut diagnosis of pathological PP. Even
though it is of foremost importance to be sure about the true diagnosis/presence of
pathological PP, it is also important to identify the underlying cause of such
alteration.
As briefly alluded to before, mPP or ePP values alone are not sufficient to make a
differential diagnosis of PH and additional parameters, such as PAWP and PVR, are
required.
According to the ESC-ERS guideline, which has recently been endorsed by a
WHO document, different types of PH exist, each one with its peculiar natural
history and clinical approach. The WHO classification (McGlothlin 2012) of the
different types of PH mostly looks at the pathogenesis and the anatomic location of
the primary alteration responsible of increased PP (Fig. 1).
As mentioned, the pulmonary circulation consists of an arterial side that mostly
recalls the characteristics of systemic veins in terms of compliance and sectional
structure and a venous side bringing the oxygenated blood back to the heart and,
from there, to the systemic circulation. The capillary barrier, that is, ideally in the
middle between the two sides, can be useful to distinguish pathological conditions
Estimated PP
35-49 mmHg ≥50 mmHg

PAWP≤15 mmHg Pre-capillary high
and
PVR≥3 W.U. PP
Suggestive of Diagnostic of
‘truly’ high PP ‘truly’ high PP
PAWP>15 mmHg
and Post-capillary high
PVR<3 W.U. PP
mPP≥25
mmHg RHC needed for differential
diagnosis of high PP
PP measurement
by RHC needed
for diagnosis
Fig. 2 Diagnostic algorithm for approaching patients with suspected high pulmonary pressure.
mPP measured pulmonary pressure, PAWP pulmonary artery wedge pressure, PP pulmonary
pressure, PVR pulmonary vascular resistance, RHC right heart catheterization
characterized by a primary increase in pulmonary artery resistances, the so-called

“pre-capillary” pulmonary hypertension, from those mostly secondary to a passive
venous congestion (“post-capillary”). PAWP and PVR can be helpful in discrimi-
nating pre-capillary from post-capillary forms of high PP. In pre-capillary forms,
PAWP is low (<15 mmHg) while PVR is expectedly high (>3 Woods units);
conversely, in post-capillary forms, PAWP values are increased (>15 mmHg) with
usually low PVR (<3 Woods units) (Badesch et al. 2009). Pre-capillary forms of PH
include the so-called pulmonary arterial hypertension (PAH) which encompasses
idiopathic (IPAH), familial (FPAH), and forms associated (APAH) to congenital
heart disease, connective tissue diseases, drugs and toxins, HIV infection, portal
hypertension, or pulmonary veno-occlusive disease. In addition, pre-capillary PH
can arise as a consequence of chronic thromboembolism such as in the presence of
obstruction of pulmonary arterial vessels (proximal or distal) by thromboemboli,
tumors, or foreign bodies. Post-capillary forms are most frequently found as asso-
ciated to left heart disorders such as systolic or diastolic dysfunction or valve
diseases (mitral and/or aortic).
High PP can be found also in the presence of other conditions primarily affecting
the lungs (e.g., chronic obstructive pulmonary disease (COPD), interstitial lung
disease (ILD), sleep apnea) or as manifestation of several systemic diseases
(McGlothlin 2012). These two latter cases can present either with a prevalent pre-
capillary or post-capillary involvement so that a proper differential diagnosis based
on PAWP and PVR is not always possible. Figure 2 depicts a diagnostic algorithm
that can be useful for approaching patients with suspected pathological PP. As said,
the first approach would be to perform a non-invasive assessment of PP by echo-
cardiography. ePP of 35–49 mmHg or Vmax 2.8–3.4 m/s are suggestive of PH but
would require RHC for the final diagnosis. ePP>50 mmHg or Vmax >3.4 m/s might
be considered diagnostic of PH, but RHC is needed for further characterization of

PH (pre- or post-capillary) on the basis of PAWP and PVR values.
Significance of High PP in the General Population and in Renal

Patients
What is the epidemiological impact of elevated PP? In other words, what do studies
and registries report about the penetrance and diffusion of this condition? Recently,
evidence is accruing showing that high PP is much more prevalent in the general
population than expected (Simonneau et al. 2009). This condition might remain
undetected because of the absence of symptoms in the early phases and suspected
only when clinical signs of right ventricular dysfunction (dyspnea, fatigue, non-
productive cough, angina pectoris, syncope, peripheral edema, and, rarely, hemop-
tysis) are manifested (Badesch et al. 2009). In the Olmsted County study (Lam
et al. 2009), a general population study conducted in a random sample of the same
county, the prevalence of high PP defined by a Doppler-derived PP >35 mmHg was
about 5 % in individuals older than 45 years. Most cases of high PP detected in this
population were secondary to concomitant left heart disorders, and the presence of
high PP was predicted by diastolic dysfunction as measured by the E/e0 (early trans-
mitral flow velocity [E]/early mitral annular tissue velocity [e0 ]) ratio and by the
presence of systemic hypertension and high pulse pressure.
In a survey on 4.579 patients undergoing echocardiographic examinations
(Strange et al. 2012), the prevalence of high PP (>40 mmHg) was 10.5 %. Among
the 483 cases with elevated PP, 78.7 % had left heart disease, 9.7 % had lung
diseases, 4.2 % had primary pulmonary artery hypertension, and 0.6 % had pulmo-
nary thromboembolism. In another study (Ghio et al. 2001), the prevalence of PH in
patients with chronic heart failure increased with the progression of NYHA class. Up
to 60 % of patients with severe LV systolic dysfunction and up to 70 % of patients
with isolated LV diastolic dysfunction had pathologically high PP.
Pre-capillary forms of PH are less frequent with an annual incidence of about 2–3
per million and an estimated prevalence of about 15 cases per million (Simonneau
et al. 2009). Adult females are almost three times more likely to present with PAH
than males. In children, the presence of PAH is equally split along gender lines. But
what happens if we refer specifically to renal patients? Do things change in this
particular population?
Although epidemiological data are scarce and sparse and mainly based on
retrospective studies, high PP appears to be exceedingly prevalent among renal
patients and not only confined to connective tissue and systemic diseases (Fig. 3).
Among pre-dialysis patients, the prevalence of PH is about two to eight times
higher than in the general population, ranging from 9 % to 39 % (Bolignano
et al. 2013). PH prevalence is higher in the dialysis population (CKD-5D) than in
CKD-ND patients. With regard to dialysis modality, the prevalence of PH is lower in
patients on peritoneal dialysis (from 0 % to 42 %) than in hemodialysis patients
(from 18.8 % to 68.8 %) (Bolignano et al. 2013). Unfortunately, there is a lack of
70
60
50
Prevalence of high ePP (%)
40
30
20
10 Estimated prevalence
of high ePP in the GP
0
CKD-1-4 CKD-5ND CKD-5D
Fig. 3 Reported prevalence of high estimated pulmonary pressure (ePP) in renal patients. Each dot
represents a single prevalence reported in a different study cohort (see text). Gray dots indicate
prevalence of high ePP in CKD stage 1–4 populations; blue dots in CKD stage-5 not on dialysis;
purple dots in CKD stage-5 on chronic hemodialysis treatment; azure dots in CKD stage-5 on
chronic peritoneal dialysis treatment. Correspondent lines indicate the median (calculated) value of
high ePP prevalence in a given CKD class. ePP estimated pulmonary pressure, CKD 1–4 chronic
kidney disease stage 1–4, CKD 5ND chronic kidney disease stage 5 not on dialysis, CKD 5D
chronic kidney disease stage 5 on dialysis, GP general population
uniformity among studies with respect to the ePP cut-offs considered as “patholog-
ical” (ranging from 25 to 45 mmHg). Such a variability in the diagnostic criteria
explains the wide range of PH prevalence reported in CKD patients and hampers the
possibility to perform rough comparisons between studies and to provide reliable
overall estimates of the frequency of high PP among renal patients.
But what kind of PH do renal patients have? Understanding the type of PH would
be useful to better understand the pathophysiology of this condition and, eventually,
to plan also the best treatment in such patients. Unfortunately, as stressed before, the
only way to characterize the nature/origin of high PP is to perform RHC to measure
PP but also to assess PAWP and PVR.
In the only study measuring PP by RHC (Pabst et al. 2012), PH was present in
81 % of HD and 71 % of pre-dialysis patients. The prevalence of (pre-capillary) high
PP was 6 % in CKD stage 4–5 patients and 13 % in HD patients, and the prevalence
of post-capillary PH was 71 % and 65 %. These observations, although partly biased
by the strict inclusion criteria of the study population (all subjects underwent RHC
for characterization of an unexplained dyspnea after exclusion of other potential

causes), seem to indicate that high PP is mostly post-capillary also in the renal
population.
Risk Factors of High PP in Renal Patients
As alluded to before, post-capillary high PP is likely to depend on the presence of LV

disorders. Heart dysfunction is prevalent in the renal population, particularly in chronic
hemodialysis patients. This condition can trigger volume overload but can be aggra-
vated itself by fluid excess, therefore generating a vicious circle that can contribute to
pulmonary congestion and high PP. Nevertheless, other risk factors/conditions may
trigger and/or exacerbate PH in renal patients (Fig. 4). Arteriovenous fistula (AVF) is
known to induce a decrease in systemic vascular resistances, which increase venous
return and cardiac output to maintain proper blood flow to peripheral tissues. These
hemodynamic effects might increase pulmonary blood flow and set the stage for high
PP. Accordingly, high PP are more prevalent among HD than PD patients or CKD
patients not on dialysis; furthermore, PP rises in strict parallelism with AVF creation
(Abassi et al. 2006) and tends to worsen overtime in the HD population (Havlucu
et al. 2007; Fabbian et al. 2011). However, evidence that renal transplantation may
revert to normal PP in patients who still have a functioning AVF (Nakhoul et al. 2005)
indicates that other risk factors are involved in the genesis of high PP in CKD.
Endothelial dysfunction is a major determinant of PH in the general population
Chronic Kidney Disease
Volume overload
Pulmonary circuit
LV Disorders congestion Chronic hypoxia Sleep disordered
Heart failure breathing
High sympathetic activation
Lower NO and
Hypoxia aggravation increased ADMA Vasular and endothelial
Anaemia dysfunction
Vascular stiffness
Hemodialysis Neutrophil activation Background co-morbidities

(AV-fistula, (COPD, diabetes, hypertension,
bio-incompatibility) Increased PBF Lung microvascular lupus and collagen diseases,)
tone alterations
Increased pulmonary
pressure
Fig. 4 Main pathophysiological mechanisms leading to an increase in pulmonary pressure in renal

patients. ADMA asymmetric dimethylarginine, AV fistula arteriovenous fistula, COPD chronic
obstructive pulmonary disease, LV left ventricular, NO nitric oxide, PBF pulmonary blood flow
(Giaid 1998) and is exceedingly frequent in renal patients (Zoccali 2007). Plasma
levels of the powerful, endothelial-derived, vasodilator nitric oxide (NO) are more
reduced in HD patients with higher PP (Yigla et al. 2004). Furthermore, asymmetric
dimethylarginine (ADMA), an endogenous inhibitor of NO synthase which is copi-
ously synthesized at lung level (Arrigoni et al. 2003), has been strongly involved in
experimental (Sasaki et al. 2007) and in primary forms (Kielstein et al. 2005) of PH
and accumulates in subjects with renal function impairment (Zoccali et al. 2001).
Interestingly, ADMA is increased in patients with sleep breathing disorders (Barcelo
et al. 2009). Sleep breathing disorders, particularly sleep apnea, are highly pervasive in
both pre-dialysis (Sakaguchi et al. 2011) and dialysis patients (Zoccali et al. 2002), and
nocturnal hypoxemia by sleep apnea is a strong trigger of high PP by enhancing
sympathetic activation (Ward and McMurtry 2009; Sica et al. 2000).
Chronic exposure of blood to dialysis membranes causes reversible neutrophil
sequestration in the lung and neutrophil activation (Craddock et al. 1977) which may
contribute to microvascular lung disease and PP increase in HD patients (Kiykim
et al. 2010). The control of microvascular tone in the lung might also be affected by
other diseases, such as diabetes and connective, liver, infectious, and hematologic
diseases. Severe anemia is a recognized cardiovascular risk factor in renal patients and
its impact on the cardiovascular system includes direct effects to the pulmonary circu-
lation. Indeed, anemia could worsen PH by aggravating hypoxia (Buemi et al. 2007).
As a consequence of the overall dysfunction in mineral metabolism, arterial
rigidity is increased in renal patients and calcium deposits have been found even
in the pulmonary artery of CKD patients (Nitta et al. 2003). These findings are in line
with observations in the general population showing that stiffening of the pulmonary
artery is significantly correlated to high PP (Lam et al. 2009).

Conditions
What is the prognostic impact of high PP? Do we have to fear the presence of PH in
our patients?
A large US survey (Hyduk et al. 2005), which collected data on PH at the
community level over a 22-year period (1980–2002), documented a stable death
rate in patients with pathologically high PP, ranging from 5.2 to 5.4 per 100.000.
Conversely, over the last 10 years, an increasing trend in mortality was documented
with an estimated age-adjusted death rate of 4.5–12.3 per 100.000 (George
et al. 2014). In addition, high PP was also associated with steadily increasing rates
of hospitalizations (Hyduk et al. 2005). With this background in mind, one would
easily argue that this trend simply reflects that one of “common” cardiovascular or
pulmonary diseases; in this view, PH would be nothing more than a simple compli-
cation of traditional heart or lung disorders. However, a milestone study on PH in
cardiopathic subjects has promptly rejected this hypothesis (Kjaergaard et al. 2007).
The authors studied over 400 patients with known or presumed heart failure. After
echocardiographic assessment of PP and LV ejection fraction, patients were followed
for up to 5.5 years. Patients presenting with a substantial increase in PP

(>39 mmHg) had a significantly increased mortality in the long term (5 years) and
even in the short term (1 year) with respect to those with lower PP values. Interest-
ingly, these observations remained unaffected if patients were stratified according to
the presence of a conserved or impaired LV function (50 % or >50 %). Multiple
Cox analyses apportioned a 9 % increase in mortality per each 5 mmHg increase in
PP, independent of age and presence of chronic lung disease, heart failure, and
impaired renal function. These solid findings indicate that the prognostic impact of
high PP is not influenced by traditional risk factors, including heart dysfunction.
Similar observations have been extended to other populations, such as patients with
primary PH or PH mostly secondary to chronic lung disease (Lau et al. 2014).
What happens in renal patients? Does high PP hold the same prognostic power
even in this particular, high-risk population? A study in 2003 (Yigla et al. 2003)
tested for the first time this possibility in a cohort of chronic hemodialysis patients.
Indeed, at unadjusted Kaplan-Meier survival curves, patients with ePP 35 mmHg
showed poorer survival with respect to those with lower ePP over a 5-year follow-
up. These preliminary observations prompted the authors to carry out an observa-
tional study (Yigla et al. 2009) on a wider cohort of 127 ESKD patients initiating
chronic renal replacement therapy. Patients’ survival was assessed over a longer
follow-up period (7 years), and subjects were stratified according to the absence of
PH (ePP <45 mmHg) or to the presence of prevalent PH (ePP >45 mmHg already
present before starting HD treatment) or incident PH (ePP >45 mmHg developed
after starting HD). Overall, the presence of high PP conferred a higher risk of death
(hazard ratio 2.1 and 3.6 for incident and prevalent PH, respectively), and patients
already having abnormal PP values before starting dialysis had poorer survival as
compared to those developing PH after dialysis initiation. Furthermore, the prog-
nostic power of high PP was independent of gender, age at onset of HD, and
presence of diabetes mellitus, left ventricular dysfunction, and clinically relevant
valve disease. In this study, each 10 mmHg increase in ePP was associated with a
fully adjusted increased risk of mortality of 50 %.
Similar findings were reported in another cohort of 278 patients on chronic renal
replacement therapy who were followed for 24 months (Li et al. 2013). In this series,
the prevalence of ePP >35 mmHg was as high as 35 %. Subjects with baseline PH
had a hazard ratio of cardiovascular death of 2.36 with respect to those with
“normal” ePP. This increased risk was fully independent of several potential con-
founders, such as gender, age at HD onset and duration of HD, pre-dialysis diastolic
blood pressure, serum phosphorus, urea levels, presence of diabetes, systolic dys-
function, and history of CV events. Of note, the presence of high ePP was also a
powerful predictor of non-fatal cardiovascular events (hazard ratio 2.27), even after
adjustment for the above-mentioned risk factors plus medication with ACEi or ARB,
dialysis adequacy, and C-reactive protein.
In another study of 288 chronic HD patients (Agarwal 2012), high ePP
(>35 mmHg) was found in 38 % of subjects and its presence predicted by left atrial
dysfunction, urea removal ratio, and use of vitamin D receptor activators. During
follow-up (median 2.15 years), patients with higher ePP had significantly worsen
cumulative hazard estimates of cardiovascular fatal events (53 %, crude mortality

rate 168.9/1,000 patient-years vs. 22 %, crude mortality rate 52.5/1,000 patient-
years). After adjustment for race, age, vascular access, serum albumin, and history of
CV disease, the presence of ePP >35 mmHg still conveyed a higher risk of death
(hazard ratio 2.17) in this population.
Another study (Ramasubbu et al. 2010) found abnormal ePP values (defined as
Vmax >2.5 m/s) in 42/90 HD patients (47 %). Subjects were followed over
12 months to assess all-cause mortality and hospitalizations. There was a statistically
significant parallelism among severity of PH (defined as low, Vmax <2.5 m/s; mild,
Vmax 2.6–2.9 m/s; or severe, Vmax >3 m/s) and mortality trend, but no differences
were found in all-cause hospitalizations.
Recently, the prognostic impact of PP was extended also to persons with early
CKD (KDOQI stages 2–4) (Bolignano et al. 2015). In a series of 468 subjects with
early renal impairment, the estimated prevalence of PH according to an ePP cut-off of
35 mmHg was 23 % and the presence of high ePP was predicted by age and left atrial
volume. Patients were followed over time (median 4 years) to assess the incidence of
a combined endpoint including cardiovascular death, acute decompensated heart
failure, coronary artery disease events, and cerebrovascular and peripheral artery
events. In a Cox multivariate model adjusting for age, eGFR, hemoglobin, left atrial
volume, left ventricular mass, and presence of diabetes mellitus and background CV
disease, high ePP predicted a high risk for the combined cardiovascular endpoint
(hazard ratio 1.75). Thus, PH is remarkably prevalent also in patients with
non-advanced CKD and holds its prognostic capacity with respect to adverse CV
outcomes independently of classical and CKD-specific risk factors.
Accruing evidence indicates that the prognostic utility of PP might be extended
also to outcomes of kidney transplant recipients. In a retrospective, 3-year observa-
tional study (Zlotnick et al. 2010), 55 patients undergoing renal transplantation were
studied with respect to the incidence of early graft dysfunction (EGD), defined either
as delayed or slow graft function. The percentage of patients developing EGD was
higher within the subgroup of patients with high ePP (>35 mmHg). Furthermore,
this increased risk (odds ratio 15.0) was fully independent from other traditional risk
factors such as age of recipient, history of coronary artery disease, left ventricular
ejection fraction, pre-transplant HD, presence of functional AVF, age of donor, and
cold ischemia time. Another retrospective study (Issa et al. 2008) analyzed the
clinical course of 215 transplant candidates. The authors stratified these subjects
into three different ePP categories (<35 mmHg, 35–59 mmHg, and >60 mmHg).
Despite most of them having ePP apparently falling within normal values, more than
one third presented with frankly pathological values. Roughly 10 % of the
study cohort had even seriously high ePP (>60 mmHg). The severity of ePP was
directly correlated to dialysis vintage, and the presence of a severe PH (ePP
>50 mmHg) was a significant predictor of death after transplantation even after
adjustment for age, reduced left ventricular ejection fraction, serum albumin, and
delayed graft function.
Table 1 summarizes the main findings from prognostic studies of high PP in renal
patients.
50
Table 1 Main prognostic studies of high PP in renal patients

Studies looking at mortality and CV outcomes
High ePP
Study/year Country Population prevalence ePP cut-off Follow-up Results
Yigla Israel 58 HD 39.7 % HD 35 mmHg >5 years At unadjusted Kaplan-Meier survival curves, patients with
et al. 2003 5 PD 0 % PD ePP 35 mmHg had poorer survival with respect to those
with lower ePP
Yigla Israel 127 HD 29.1 % 45 mmHg 7 years High PP conferred a higher risk of death (HR 2.1 and 3.6
et al. 2009 for incident and prevalent PH, respectively) with a fully
adjusted HR of mortality of 1.5 (1.2–1.9) for each
10 mmHg increase in ePP; ( p = 0.0007)
Li et al. 2013 China 278 HD 64.7 % 30 mmHg 2 years Subjects with baseline high PP had a HR of CV death of
2.36 with respect to those with normal ePP. High ePP was
also an independent predictor of non-fatal CV events
(HR 2.27)
Agarwal 2012 USA 288 HD 38 % 35 mmHg 2.15 years The presence of ePP >35 mmHg conveyed a higher risk of
(median) death (HR 2.17) after adjustment for race, age, vascular
access, serum albumin, and history of CV disease
Ramasubbu USA 90 HD 47 % Vmax >1 year Statistically significant parallelism between severity of PH
et al. 2010 2.5 m/s (defined as low, Vmax <2.5 m/s; mild, Vmax 2.6–2.9 m/s;
or severe, Vmax >3 m/s) and mortality trend but no
differences in all-cause hospitalizations
Pulmonary Pressure as a Novel Prognostic Biomarker in Renal Patients
Bolignano Italy/ 468 CKD 23 % 35 mmHg 4 years In a Cox multivariate model adjusting for age, eGFR,
et al. 2015 Germany (median) hemoglobin, left atrial volume, LVM, and presence of
diabetes mellitus and background CV disease, high ePP
predicted a high risk for a combined CV endpoint
(HR 1.75)
(continued)
1135
1136
Table 1 (continued)
Studies looking at mortality and CV outcomes
High ePP
Study/year Country Population prevalence ePP cut-off Follow-up Results
Studies looking at renal outcomes in kidney transplant recipients
Zlotnick USA 55 HD 38 % 35 mmHg 3 years Higher percentage of patients developing EGD within the
et al. 2010 subgroup with high ePP (>35 mmHg) with a fully
adjusted OR of 15.0
Issa et al. 2008 USA 215 32 % 35 mmHg 2.5 years The presence of a severe PH (ePP >50 mmHg) was a
CKD/HD/ significant predictor of death after transplantation, after
PD adjustment for age, reduced left ventricular ejection
fraction, serum albumin, and delayed graft function
CKD chronic kidney disease, CV cardiovascular, EGD early graft dysfunction, eGFR estimated glomerular filtration rate, ePP estimated pulmonary pressure,
HD hemodialysis, HR hazard ratio, LVM left ventricular mass, OR odds ratio, PD peritoneal dialysis, PH pulmonary hypertension, PP pulmonary pressure,
Vmax maximum tricuspidal jet velocity
D. Bolignano et al.
Right heart catheterization to

Improve LV-
dysfunction and confirm Pulmonary hypertension
sleep apnea diagnosis and assess severity
Volume overload PH associated to left heart dysfunction

correction by HD Supportive treatment: Anticoagulation
+/− Diuretics +/− Oxygen +/− Digoxin
intensification or PD
High-flow AVFs Acute vasoreactivity test

identification Positive Negative
and treatment
Oral CCBs Thromboembolic PH

PH associated to Heart or
Lung diseases
No Epoprostenol or Treprostinil
Sustained response ERAs or PDE-5 Is (oral) (IV)
Epoprostenol or Treprostinil (IV) iloprost (inhaled)
iloprost (inhaled) ERAs or PDE-5 is (Oral)
Treprostinil (SC) Trerpostinil (SC)
Continue CCBs No response

No response
Combination therapy
ERA+PDE-5 Is + prostanoids
Carefully consider Atrial septostomy
tratment on a Lung transplant
risk/benefit basis
Investigational protocols
Fig. 5 Potential therapeutic approach to high pulmonary pressure in renal patients. AVFs arterio-
venous fistulas, CCBs calcium-channel blockers, ERAs endothelin-receptor antagonists, HD hemo-
dialysis, IV intravenous, LV left ventricular, PDE-5 Is phosphodiesterase-5 inhibitors, PD peritoneal
dialysis, PH pulmonary hypertension, SC subcutaneous
Although few doubts exist on the prognostic usefulness of PP in this population,

one question still remains open: is pathologically high PP also as a modifiable factor
that deserves correction? Unfortunately, to date there are no specific studies of PH
treatment in CKD patients. In the absence of such evidence, one can refer to the
therapeutic algorithm proposed by current guidelines on PH (Galie et al. 2009)
(Fig. 5). In the general population, the first step is basically to confirm the presence
of high PP and to assess the severity and possible type of PH by right heart
catheterization. The second step would be to tailor the best treatment to the patient
according to the (invasive) vasoreactivity test and the individual response to drug.
Nevertheless, given the peculiar characteristics of renal patients, is there any addi-
tional approach that could be particularly useful in the CKD setting? Of course, the
ideal solution would be to encourage kidney transplantation since accumulating
(although not univocal) evidence indicates that PP may revert to normal values
after receiving a renal transplant. Yet, as briefly alluded to before, a higher risk of
death may persist in kidney transplant recipients with previous PH even after
normalization of PP (Issa et al. 2008). Volume overload correction by ultrafiltration
intensification or peritoneal dialysis might improve LV dysfunction and sleep apnea.
The identification and surgical treatment of very high flow AVF would probably be
helpful as well. Finally, when a pharmacological approach is needed, this should be
always weighted on a risk/benefit balance, considering that most drugs for PH
treatment can be dangerous in patients with impaired renal function or on dialysis.
Conclusions
Evaluation of pulmonary pressure might represent an additional prognostic tool for

risk stratification of renal patients. Pulmonary hypertension is highly prevalent in
CKD patients, particularly in stage 5 patients on chronic HD. PH in CKD is a
potentially reversible process because it may regress after kidney transplantation.
However, in dialysis patients with established PH, the excess risk for death may
persist after kidney transplantation. Accruing evidence indicates that high PP pre-
dicts a high risk of death, particularly in ESKD patients, and worse outcomes in
kidney transplant recipients. Large prospective studies adopting well-standardized
criteria of PP measurement, such as right heart catheterization, are needed to clarify
the exact risk of PH in persons with CKD.
Summary Points
• This chapter focuses on pulmonary pressure (PP), that is, the pressure in the
arterial side of the pulmonary circulation, as a novel prognostic biomarker in
nephrology.
• In renal patients, PP assessment is now receiving growing attention because high
PP (also known as pulmonary hypertension (PH)) is exceedingly prevalent in
such population.
• Several factors have been called into question to explain high PP in renal patients,
including left heart dysfunction, volume overload, breath disorders, and the
presence of high-flow arteriovenous fistulas.
• High PP portends a risk excess for mortality and adverse outcomes in the general
population that is fully independent of traditional heart and lung risk factors.
• Similar observations have been reported in renal patients, particularly in dialysis
patients.
• In addition, PH predicts adverse renal outcomes in kidney transplant recipients.
• Future studies are eagerly awaited to clarify the exact risk of high PP in renal
patients and to demonstrate whether normalization of PP translates into better
outcomes.
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Estimation of Glomerular Filtration Rate
51
Antonín Jabor, Janka Franeková, and Lenka Hošková
Contents
Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1145
Glomerular Filtration Rate: Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1145
Assessment of Glomerular Filtration Rate: Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1146
Creatinine and Cystatin C in the Assessment of GFR: Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . 1146
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1146
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1147
Historical Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1147
Physiology of Glomerular Filtration Rate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1148
Creatinine, Cystatin C, and Urea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1149
Creatinine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1149
Cystatin C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1151
Urea . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1154
Biological and Analytical Variability of GFR Measures and Related Tests . . . . . . . . . . . . . . . 1155
Estimation Methods of GFR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1155
The Principle of Renal Clearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1155
Creatinine Clearance and the Historical Cockcroft and Gault Equation . . . . . . . . . . . . . . . . . . . 1157
eGFR Based on Cystatin C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1158
Urea Clearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1158
Mean of Urea Clearance and Creatinine Clearance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1159
MDRD Formula . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1159
CKD-EPI Equations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1160
Lund-Malmö Equations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1160
Estimation of GFR in Children . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1163
A. Jabor (*) • J. Franeková

Department of Laboratory Methods, Institute for Clinical and Experimental Medicine, CZ, Prague,
Czech Republic
e-mail: anja@medicon.cz; jafa@ikem.cz
L. Hošková
Cardiology Clinic, Institute for Clinical and Experimental Medicine, CZ, Prague, Czech Republic
e-mail: lenka.hoskova@ikem.cz

1144 A. Jabor et al.
Interpretation of eGFR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1165

Specific Problems with Cystatin C Measurement and Interpretation . . . . . . . . . . . . . . . . . . . . . . 1165
GFR and Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1165
Glomerular Filtration Rate and Body Surface Area . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1166
Specific Clinical Situations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1167
Chronic Kidney Disease (CKD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1167
Diabetes Mellitus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1168
AKI and Intensive Care . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1169
Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1171
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1172
Abstract
We describe the principles of estimating glomerular filtration rate (GFR) with an
emphasis on the role of creatinine and cystatin C in the calculation of estimated
GFR (eGFR). We present a list of selected equations for eGFR calculation
together with a critical evaluation of their role in the diagnostics, classification,
and monitoring of kidney function.
History: The first part briefly describes the history of GFR assessment.
Pathophysiology: The second part deals with the physiology of GFR, the
Starling equation, and the regulation of GFR.
Creatinine, cystatin C, and urea: The third part describes the pathophysiology,
analytical details on the measurements, interferences, reference intervals, and
important features of creatinine, cystatin C, and urea, together with data on
intraindividual and interindividual variability of these compounds, desirable
accuracy, and precision as well as data on real analytical quality, indexes of
individuality, and reference change values.
Estimation of GFR: The fourth part describes the principle of renal clearance,
creatinine clearance and the Cockcroft and Gault equation, eGFR based on
cystatin C, urea clearance and the mean of urea and creatinine clearance, the
MDRD formula, the CKD-EPI equations, the Lund-Malmö equations, and equa-
tions used in children. Comparison of inulin clearance with other equations for
eGFR is also given.
Interpretation: The fifth part deals with the weaker aspects and sources of
errors that may occur when using creatinine and cystatin C in the estimation of
GFR. The relation of age and body surface area to GFR is also described.
Clinical use of eGFR: The sixth part describes the importance of eGFR in the
classification of chronic kidney disease, diabetes mellitus, and acute kidney
injury, with an emphasis on recommendations in recent guidelines.
Keywords
Glomerular filtration rate • Chronic kidney disease • CKD-EPI equation • Creat-
inine • Cystatin C • Urea
51 Estimation of Glomerular Filtration Rate 1145
Abbreviations
ADA American Diabetes Association
ADQI Acute Dialysis Quality Initiative
AKIN Acute Kidney Injury Network
BMI Body mass index
BSA Body surface area
CCr Clearance of creatinine
CKD-EPI Chronic Kidney Disease Epidemiology Collaboration
ID-MS Isotope dilution mass spectrometry
IFCC International Federation of Clinical Chemistry and Laboratory
Medicine
LBM Lean body mass
MDRD Modification of Diet in Renal Diseases
NDF Net driving force (net filtration pressure)
NIST SRM National Institute of Standards and Technology, Standard Refer-
ence Material
PCR Protein-to-creatinine ratio
RCV Reference change value, critical difference
RIFLE Risk, Injury, Failure, Loss of function, End-stage kidney disease
Key Facts
Glomerular Filtration Rate: Key Facts
• Doctors require information on two principal kidney properties: function of

glomeruli and function of tubuli.
• Glomerular filtration rate (GFR) is a measure of the filtration ability of the kidney.
• Glomerular filtration rate can be measured, or – much more frequently – esti-
mated (eGFR).
• Doctors use eGFR for diagnostics and staging of kidney diseases according to
internationally accepted guidelines.
Assessment of Glomerular Filtration Rate: Key Facts
• The gold standard for direct measurement of GFR is inulin clearance. Other
slightly less precise but acceptable methods also include iohexol or iothalamate
clearance.
• Direct measurement of GFR is impractical and time consuming; therefore, GFR is
estimated from the concentration of serum biomarkers.
• Two serum biomarkers are used: creatinine and cystatin C, where the higher the
creatinine or cystatin C, the lower the GFR.
• Laboratories provide physicians with information on eGFR by means of calcula-
tions based on serum creatinine and/or serum cystatin C.
• Reference ranges for estimated GFR (eGFR) are not specified, because of the
necessity to evaluate kidney function in the context of the clinical condition of the
patient; however, values lower than 90 mL/min per 1.73 m2 (1.5 mL/s per
1.73 m2) are considered decreased.
Creatinine and Cystatin C in the Assessment of GFR: Key Facts
• Creatinine and cystatin C are endogenous compounds eliminated by the

kidneys, mainly via glomerular filtration. Both are widely used in medical
laboratories.
• There are a number of internationally accepted equations for eGFR calculation.
• Creatinine and cystatin C have some disadvantages: creatinine is influenced by a
high-protein diet, muscle mass changes, and muscle catabolism; tubular secretion
of creatinine can occur; some methods interfere with body substances and drugs;
cystatin C is influenced by corticosteroids, in diabetes mellitus and diseases of the
thyroid gland.
Definitions
CKD-EPI equations The recommended equations for estimating glomerular filtra-

tion rate, based on serum creatinine (the 2009 CKD-EPI creatinine equation), serum
cystatin C (the 2012 CKD-EPI cystatin C equation), or both (the 2012 CKD-EPI
cystatin C and creatinine equations).
Cockcroft-Gault equation Not recommended. An obsolete formula for estimating

glomerular filtration rate, based on sex, weight, and serum creatinine.
Creatinine A metabolic product of muscle creatine, produced by the body at a

constant rate. Elimination is influenced by kidney glomerular function, and
increased concentration in blood is found in patients with decreased glomerular
filtration rate.
Creatinine clearance Not recommended. A method of estimating glomerular

filtration rate, based on the measurement of creatinine concentration in serum and
urine together with the collection of urine during a specified time interval. Creatinine
clearance has been replaced by more modern equations based on serum concentra-
tions of creatinine and/or cystatin C.
Cystatin C A protein inhibitor of cysteine proteases, produced by all nucleated

cells at a constant rate. Elimination is influenced by kidney filtration, and increased
concentration in blood is found in patients with decreased glomerular filtration rate.
In contrast to creatinine, a very low amount of cystatin C is excreted by the kidney,
because of tubular reabsorption of this compound.
Glomerular filtration rate The volume of plasma ultrafiltrate (primary urine)

generated by the kidneys per unit of time. Values are expressed in ml/min (or ml/s)
per standardized body surface area (1.73 m2).
MDRD equation Not recommended. A formula that was used to estimate glomer-
ular filtration rate between 1999 and 2009.
Urea A metabolic product of body proteins, synthesized by the liver. Urea repre-
sents the main route of nitrogen elimination from the body. Increased concentration
in the blood occurs during hypercatabolism of proteins or kidney failure.
Introduction
Estimation of glomerular filtration rate (GFR) is a basic method of assessing kidney

function. Decreased GFR, present for more than 3 months, is one of the important
diagnostic criteria for chronic kidney disease (CKD). Furthermore, CKD is classified
based on the GFR category (KDIGO 2012 Guideline 2013). Gold-standard methods
(renal or plasma inulin clearances) are precise, but time consuming. Creatinine has
been used as a surrogate marker in GFR assessment for years, but physicians are
more interested in function (GFR) than concentration (creatinine in plasma). There-
fore, the reliable conversion of plasma biomarker concentration to eGFR is used for
practical reasons. Among the available biomarkers, creatinine and cystatin C are
mostly recommended to calculate eGFR, provided they are traceable to international
calibration and suitable equations are used. Due to the plethora of eGFR equations, it
is necessary to develop practical guidelines for international application and support
their use in clinical practice.
Historical Background
Max Eduard Jaffé published his paper on the estimation of creatinine in 1886 (Jaffé
1886). Many modifications appeared because of the interference of what became
known as Jaffé-positive substances. However, the Jaffé reaction with alkaline
picrate is used even today, and enzymatic determination of creatinine is only

slowly becoming the main method for the determination of creatinine in routine
clinical laboratories. Inulin clearance was described in 1935 by Shannon and Smith
(Shannon and Smith 1935), and the recent KDIGO 2012 Guideline (KDIGO 2012
Guideline 2013) recognizes inulin clearance as a gold standard of GFR assessment.
The Cockcroft and Gault equation was published in 1976 (Cockcroft and
Gault 1976). This equation was derived from samples of 249 patients aged
18–92. The MDRD equation was published by Andrew Levey (Levey et al.1999),
and the same author published the CKD-EPI equation in 2009 (Levey et al. 2009).
The best and most recent recommendation describing the use of creatinine,
cystatin C, and estimations of GFR was published in 2013 in the “KDIGO 2012
Clinical Practice Guideline for the Evaluation and Management of Chronic Kidney
Disease.”
Physiology of Glomerular Filtration Rate
Approximately 80 % of plasma volume entering afferent renal arterioles passes to

efferent arterioles, and the remaining 20 % of plasma volume is filtered. Filtration is
illustrated by the Starling equation.
The general Starling equation describes the rate of fluid through the capillary
wall:
JV ¼ KF A½ðPC PT Þ δðπC πT Þ
where
JV = rate of fluid
KF = capillary filtration constant
A = area for fluid exchange
PC and PT = capillary and tissue hydrostatic pressure
πC and πT = capillary and tissue oncotic pressure
δ = coefficient describing possible transfer of proteins
A simplified equation describing net filtration pressure (or net driving force,
NDF) in the glomeruli is as follows:
NDF ¼ ðPC PT Þ ðπC πT Þ
where
PC = hydrostatic pressure of plasma (blood pressure)

PT = fluid pressure in the Bowman’s capsule
πC = oncotic pressure of plasma proteins
πT = oncotic pressure of plasma ultrafiltrate in the Bowman’s capsule
Filtration appears where NDF > 0. Filtration is influenced by the filtration area
(e.g., 50 % in patients with one kidney), the quality of the glomerular membrane
(changed in acidemia or in kidney disease), and the ability of various proteins to
enter the Bowman’s capsule (molecular weight, shape of the molecule, isoelectric
point of the protein). Approximately 120 mL/min (~170 L per day) of ultrafiltrate is
generated every day, while about 1 mL/min (~1.5 L per day) of urine is excreted
daily. This means that 99 % of the ultrafiltrate is reabsorbed (i.e., the fractional
excretion of water is about 1 %). Glomerular filtration rate (GFR) is regulated by
myogenic stretch and tubuloglomerular feedback. Tubuloglomerular feedback rep-
resents the autoregulation system, where GFR is regulated according to tubular
urine flow. The aim of the system is to decrease loss of water and ions in situations
with increased glomerular filtration rate. In case of increased GFR, the delivery of
the chloride ion to the thick ascending limb of the loop of Henle is increased, which
results in the constriction of afferent arterioles and the decrease of GFR. Sympa-
thetic regulation, angiotensin II, prostaglandins, and natriuretic peptides contribute
to the regulation of GFR.
Creatinine, Cystatin C, and Urea
Creatinine
Pathophysiology
Muscle creatine is converted to creatinine by a spontaneous, nonenzymatic reaction
at a constant rate; however, ingested meat is a source of creatine and hence of
creatinine. Creatinine is eliminated by glomerular filtration, but proximal secretion
of creatinine exists; proximal secretion of creatinine increases with decreasing GFR.
Also, a small amount of creatinine is reabsorbed by tubuli.
Analytical Remarks
International calibration is based on NIST SRM 967 (human liquid serum, traceable
to the ID-MS (isotope dilution mass spectrometry) reference method). Two groups of
methods are used throughout the world: the group of Jaffé methods with alkaline
picrate and the group of enzymatic methods. Two key enzymes are used: creatinine
deaminase (creatinine iminohydrolase, EC 3.5.4.21) and creatininase (creatinine
amidohydrolase, EC 3.5.2.10). Other methods, e.g., HPLC, high-performance liquid
chromatography, are not used routinely. The reference method is ID-MS.
Interferences
The Jaffé reaction is specific not only to creatinine, but many compounds – known as
“Jaffé-positive chromogens” – can falsely increase concentration of creatinine, when
measured in plasma or serum. Among them, glucose, protein, fatty acids, acetone,
acetoacetate, pyruvate, and cephalosporins are the most important compounds. The
measurement in urine is less influenced by these compounds. Other substances can
falsely decrease creatinine concentration, e.g., ascorbic acid and bilirubin.
0,3
dU-creatinine (mmol/d per kg of body weight)
0,2
0,1
0,0
15 16 17 18 19 20 21 22 23
BMI (kg/m2; displayed values above 16 only)
Fig. 1 The relationship between BMI and creatinine output over 24 h calculated per kilo-
gram of the total body weight in adolescents (Based on data published by Remer 2002). As can
be seen from the Figure, urinary output (dU) of creatinine (recalculated per kg of the body weight) is
constant over a wide range of body mass index (BMI). Two outliers were excluded. Circle = boys,
square = girls
Enzymatic methods can be influenced by bilirubin, hemoglobin, monoclonal

immunoglobulin M, and some drugs, e.g., dobutamine.
Reference Intervals
Reference intervals are based on the abovementioned international calibration.
Reference intervals for plasma (serum) creatinine are always different for both sexes
due to the difference in muscle mass. Reference intervals for plasma (serum) are
64–104 μmol/L (0.72–1.18 mg/dL) in men and 49–90 μmol/L (0.55–1.02 mg/dL) in
women.
Output of Creatinine
In order to predict the output of creatinine, a great number of equations for estimat-
ing glomerular filtration rate (eGFR), e.g., the Cockcroft-Gault equation, are based
on the patient’s weight. Therefore, this equation fails in individuals with fluid
retention or obesity. Output of creatinine calculated per kilogram of the body weight
is expected to be constant in adults without fluid retention, excessive amount of body
fat, or muscle atrophies. Figure 1 shows body mass index (BMI) 16–22 kg/m2,
where creatinine output calculated per kilogram of the body weight is practically
independent of BMI. Output of creatinine ranges from 0.15 to 0.18 mmol/day/kg of
the body weight (median 0.17 mmol/day/kg of body weight, 10th percentile 0.13,
90th percentile 0.20 mmol/day/kg of body weight).
If a constant amount of creatinine is excreted from one kilogram of body mass

(except for individuals with muscle atrophies, fluid retention, or excessive obesity), we
may then check the output of creatinine in adults in comparison to body mass. Table 1
indicates the reference limits of daily creatinine excretion in the urine according to
ARUP’s Laboratory Test Directory (http://ltd.aruplab.com/Tests/Pub/0020473).
If values are recalculated based on body weight, i.e., we want to calculate the
body weight in relation to the amount of excreted creatinine (a coefficient of
0.17 mmol of creatinine based on one kilogram of the total body weight per day
was used), unrealistic values are obtained in some cases. Maximum creatinine output
is derived from the maximum possible GFR (150 mL/min, i.e., 2.5 mL/s). When
diuresis is 1,500 mL/day, plasmatic concentration of creatinine is 90 μmol/L, and
concentration of creatinine in the urine is 13 mmol/L, then creatinine clearance is
2.508 mL/s and creatinine output is 19.5 mmol/day (Sch€uck, 2012, personal com-
munication). Nevertheless, the maximum possible output of creatinine does not
represent the reference limit. Therefore, we recommend the following reference
ranges for creatinine output in the urine over 24 h:
Men 10–16 mmol/day (1.13–1.81 g/day)

Women 8–14 mmol/day (0.90–1.58 g/day)
Note: If the coefficient of 0.17 mmol/day per kilogram of the body weight is used,
the corresponding weights are 59–94 kg in men and 47–82 kg in women.
Ratios of Urinary Compounds to Creatinine

Ratios of urinary compounds to creatinine are becoming increasingly popular as
tools suitable for evaluating substances eliminated through the urine, based on the
principle that the concentration of creatinine reflects diuresis (Fig. 2). It is obvious
that the reliability of the estimation of diuresis from creatinine concentration
decreases with decreasing creatinine concentration.
The most used ratios are PCR (protein-to-creatinine ratio) and ACR (albumin-to-
creatinine ratio). As can be seen from Fig. 2, lower concentrations of urinary
creatinine are rather poor predictors of urine volume. On the other hand, interpreting
ratios is practical and much better than evaluation of concentration alone. However,
timed urine samples can provide the physician with more accurate protein and
albumin excretion values.
Cystatin C
Pathophysiology
Cystatin C (cystatin 3, gamma-trace protein) is a member of cystatin superfamily II,
gen CST3, locus 20p11.2. Cystatin C is a basic (isoelectric point, pI, 9.3) protein
composed of a single polypeptide chain of 120 amino acid residues with a molecular
weight of 13.4 kDa (13,359 g/mol). Cystatin C is synthesized at a constant rate by all
nucleated cells. The synthesis is not influenced by inflammation, catabolism, or diet.
1152
Table 1 The reference limits of creatinine output indicated in the literature (ARUP’s Laboratory Test Directory)
Men (mmol/day) Women (mmol/day) Men (g/day) Women (g/day)
Age Lower limit Upper limit Lower limit Upper limit Lower limit Upper limit Lower limit Upper limit
3–8 years 1.2 6.2 1.2 6.2 0.14 0.70 0.14 0.70
9–12 years 2.7 11.5 2.7 11.5 0.31 1.30 0.31 1.30
13–17 years 4.4 20.3 3.5 14.1 0.50 2.30 0.40 1.60
18–50 years 8.8 22.1 6.2 14.1 1.00 2.50 0.70 1.60
51–80 years 7.1 18.6 4.4 12.4 0.80 2.10 0.50 1.40
>81 years 5.3 17.7 3.5 11.5 0.60 2.00 0.40 1.30
Daily output of creatinine increases with increasing muscle mass. However, the upper limit of creatinine daily output is unrealistic. For example, the upper limit
of creatinine in the group of men aged 18–50 years (22.1 mmol/day) corresponds to 130 kg of the body weight, when a factor of 0.170 mmol/kg of the body
weight is used. The error is due to an incorrect collection of urine during experiments when defining reference ranges
A. Jabor et al.
Fig. 2 The relationship between diuresis and concentration of creatinine in the urine.
Creatinine is excreted at a constant rate. Therefore, concentration of creatinine in the urine decreases
with increasing volume of urine. However, kidney function influences the balance between serum
and urine creatinine. Higher concentrations of creatinine in a lower volume of urine have an
acceptable low error estimate. On the other hand, lower concentrations of creatinine in the urine
may represent different volumes of urine, and the estimation of urinary volume is imprecise. For
example, concentration of creatinine of 20 mmol/L means that the volume of urine is between
approximately 500 and 1,000 mL/day. In contrast, concentration of creatinine of about 5 mmol/L
means that the volume of urine is between 1,200 and 3,500 mL/day for different kidney functions.
Nevertheless, ratios of urinary compounds to creatinine are better estimates for interpretation
purposes than concentrations of these compounds without any correction
Measurable concentrations of cystatin C are found in the plasma (about 1 mg/L),

urine (0.2 mg/L), cerebrospinal fluid (3–14 mg/L), seminal plasma (40–60 mg/L),
milk (2 mg/L), saliva (0.4–5 mg/L), tears (1 mg/L), and amniotic (1 mg/L) and
synovial fluid (2 mg/L). With regard to molecular weight and pI, cystatin C is freely
filtered and completely reabsorbed and degraded in the proximal tubuli. Therefore,
the plasma concentration of cystatin C indirectly reflects glomerular filtration rate,
and increased urine concentration reflects tubular injury.
Analytical Remarks
International calibration is based on CRM ERM-DA471/IFCC. Immunoassay is a
dominant principle of cystatin C measurement.
The Use of Cystatin C in Nephrology

The measurement of cystatin C is recommended in situations where eGFR based on
serum creatinine is less accurate and also for confirmation of CKD in adult patients
with mildly to moderately decreased eGFR (based on creatinine), i.e., 45–59 mL/min
per 1.73 m2 (G3a) (KDIGO 2012 Guideline 2013). As cited from KDIGO 2012
Guideline: “1.4.3.2: We suggest using additional tests (such as cystatin C or a

clearance measurement) for confirmatory testing in specific circumstances where
eGFR based on serum creatinine is less accurate. (2B).”
Cystatin C, Cardiovascular Mortality, and Morbidity

Shlipak (Shlipak et al. 2005) published a relation between increased concentrations
of cystatin and the annual rate of death due to all causes and also death due to
cardiovascular causes. While the relation between cystatin C and the rate of death
was almost exponential (the higher the level of cystatin C, the higher the hazard
ratio), association of creatinine categories and mortality revealed a J-shaped curve.
One important result was that within each quintile of creatinine concentration, higher
quintiles of cystatin C were associated with increased risk of mortality due to all
causes. Cystatin C was further assessed as a cardiovascular risk factor (Taglieri
et al. 2009; Woitas et al. 2013) and is classified as a cardiorenal biomarker
(Chowdhury et al. 2013).
Reference Intervals
Reference intervals based on the abovementioned international calibration are
0.31–0.99 mg/L in men and 0.4–0.99 mg/L in women.
Urea
Pathophysiology
Urea is an end product of protein catabolism, synthesized in the liver. About 90 % of urea
is excreted by the kidneys. Free filtration in the glomeruli is followed by several stages of
passive tubular transport, which results in the reentering of the plasma compartment.
Clearance of urea is lower than true GFR. Impaired kidney function, dehydration,
increased protein catabolism (including catabolism of protein in gastrointestinal bleed-
ing), a high-protein diet, administration of cortisol, and obstruction of the urinary tract
are the main reasons for increased urea concentration in plasma. Creatinine rises more
slowly than urea in prerenal failure or in cases of decreased renal blood flow.
Analytical Remarks
Older nonenzymatic methods of urea determination are used in exceptional cases,
and the majority of laboratories use the enzymatic method with urease (EC 3.5.1.5)
and glutamate dehydrogenase (EC 1.4.1.3). International calibration is based on
NIST SRM 912a.
Reference Intervals
Increased concentrations of urea are found in older populations and in men. Different
reference values can be found in the literature. Reference intervals in men up to
50 years of age are 3.2–7.4 mmol/L and 3.0–9.2 mmol/L in men above 50 years of
age. The intervals are 2.5–6.7 mmol/L in women up to 50 years of age and
3.5–7.2 mmol/L in women above 50 years of age.
Output of Urea
Output of urea is influenced by protein catabolism. The normal concentration of urea
in the urine is 250–433 mmol/L. If daily volume of urine is about 1,350 mL/day,
daily production of urea is calculated at 340–585 mmol/day.
Biological and Analytical Variability of GFR Measures and Related

Tests
Intraindividual (variability within a subject) and interindividual (variability between

subjects) biological variabilities are important characteristics required for accurate
interpretation of laboratory tests. Desirable precision, maximum tolerable bias, and
total allowable error are calculated according to biological variability, and they
define the requirements for laboratory tests. The index of individuality is calculated
as a ratio of intraindividual biological variability and analytical variability. Reason-
able analytical variability is expressed as a coefficient of variability (CVa, given in
%) and calculated from intermediate precision (long-term analytical variability
assessed over a period of several weeks) in the clinical laboratory. The reference
change value (RCV, or critical difference, given in %) is calculated from
intraindividual variability (CVi) and analytical variability (CVa) as
1=2
RCV ¼ 2:77 CVa2 þ CVi2
and expresses changes (in %) in the concentration of the analyte, which represents a
significant departure from the basal value. The absolute RCV is calculated for the
selected basal value. Required parameters for accurate interpretation of laboratory
tests are given in Table 2.
Estimation Methods of GFR
The Principle of Renal Clearance
Renal clearance is defined as “the volume of plasma from which the substance is
completely cleared by the kidneys per unit of time” (Burtis 2006). For a substance
with specific properties (stable rate of synthesis, stable plasma concentration, freely
filtered, no influence of renal tubuli in terms of reabsorption, secretion, synthesis, or
catabolism), the amount of the filtered substance is the same as the amount of the
excreted substance:
GFR PSubst ¼ USubst V
where GFR is the glomerular filtration rate,

1156
Table 2 Required parameters for accurate interpretation of laboratory tests (S/P = serum or plasma, dU = output per day)
S/P creatinine dU creatinine S/P cystatin C S/P urea dU urea
Intraindividual 6.0 % 11.0 % 4.6 % 12.3 % 17.4 %
biological variability
Interindividual 14.7 % 23.0 % 13.0 % 18.3 % 25.4 %
biological variability
Desirable precision 3.0 % 5.5 % 2.3 % 6.2 % 8.7 %
Maximum tolerable bias 4.0 % 6.4 % 3.5 % 5.5 % 7.7 %
Total allowable error 8.9 % 15.4 % 7.2 % 15.7 % 22.1 %
Index of individuality 0.41 0.48 0.35 0.67 0.69
Reasonable CVa 2.0 % 2.7 % 2.0 % 3.4 % 2.6 %
Reference change value 17.5 % 31.4 % 13.9 % 35.3 % 48.7 %
(%)
Reference change value// 17.5 μmol/L// 3.77 mmol/d// 0.14 mg/L// 3.54 mmol/L// 171 mmol/d//
for baseline value 100 μmol/L 12 mmol/d 1 mg/L 10 mmol/L 350 mmol/d
Intraindividual variability corresponds to the fluctuation of the concentration “within” a person, while interindividual variability corresponds to the difference
“between” persons. Analytical quality must reflect the parameters of biological variability: the lower the biological variability, the better analytical performance
required. Analytical quality derived from biological parameters is represented by desirable imprecision, maximum tolerable bias, and total allowable error. It is
essential to use the reference change value in order to properly interpret two consecutive measurements and to achieve a significant difference: the higher the
biological (CVi) and analytical (CVa) variability, the higher the difference needed between the two consecutive values to be significant
A. Jabor et al.
PSubst and USubst are plasma and urine concentrations of that substance,
respectively, and V is the volume of urine per unit of time.
Then we have
GFR ¼ ðUSubst VÞ=PSubst
The problem consists in the properties of an “ideal” substance: creatinine is far from
ideal due to proximal tubular secretion; inulin, 51Cr-EDTA, and iohexol display
extrarenal clearance; the use of 51Cr-EDTA, 125I-iothalamate, and 99mTc-DTPA is
connected with the risk of ionizing radiation; other markers are neither sensitive nor
specific for the assessment of GFR (beta-2-microglobulin, alpha-1-microglobulin,
urea, and retinol-binding protein).
Creatinine Clearance and the Historical Cockcroft and Gault Equation
Creatinine is supposed to be filtrated freely by the glomeruli and excreted in the urine
without any tubular secretion or reabsorption. Unfortunately, this is not the case
since creatinine is secreted in the proximal tubuli and this secretion increases with
decreasing GFR. Renal clearance of creatinine (CCr) is calculated by the following
simple equation:
eGFR CCr ¼ ðUCreat VÞ=SCreat (1)
where UCreat is the concentration of creatinine in the urine, V is the volume of urine
per unit of time, and SCreat is the plasma (serum) concentration of creatinine.
Volume is expressed in mL, time in minutes (SI units: in seconds), and concentra-
tions of creatinine must be in the same units (both urine and plasma in mg/dL, or
both urine and plasma in mmol/L). The product (UCreat * V) in Eq. 1 represents the
rate of creatinine elimination in the urine, e.g., in g/day (SI: mmol/day). Creatinine is
supposed to be synthesized at a constant rate, where the amount of creatinine
produced daily is a function of body muscle mass. Because of the correlation
between the muscle mass and body weight, one can assume that the rate of creatinine
elimination can be deduced from
eGFR CCr ¼ X=SCreat (2)
where X represents the amount of creatinine excreted per day. For example, here is
the Cockcroft and Gault formula
eGFR ¼ ½ð140 AgeÞ Weight=ð48, 8 SCreatÞ F (3)
where Age is in years, Weight is in kg, SCreat is in μmol/L, factor F is 1.0 for men
and 0.85 for women, and eGFR is in mL/s.
Cockcroft and Gault derived this formula in 1976 based on data from 249 healthy
volunteers. This formula is not recommended any more. However, there have been
instances where this formula has been used among clinical pharmacists; also, some
pharmacokinetic programs still use the obsolete equation. A similar principle is used
in other equations, where the estimation of creatinine output is calculated from body
measurements (height in children, weight in adults, etc.).
eGFR Based on Cystatin C
There are many equations based on the assumption that cystatin C is produced at a
constant rate:
eGFR ¼ X=SCystC
where X is an estimate of the daily output of cystatin C.

However, certain extrarenal clearance of cystatin C exists, and therefore, new
equations are based on the regression between cystatin C and the “gold” standard.
In 2005, Grubb derived a cystatin C-based equation for use in adults and children
(Grubb 2005):
eGFR ¼ 84:69 SCystC1:680 1:384ðif < 14yearsÞ
or with gender factor
eGFR ¼ 87:62 SCystC1:693 1:376ðif < 14yearsÞ 0:940ðif femaleÞ
Results are in mL/min per 1.73 m2.

A review of other cystatin C-based equations in children was published by
Andersen (Andersen et al. 2009) and Filler (Filler et al. 2012).
The CKD-EPI formula based on cystatin C was published in 2013 (KDIGO 2012
Guideline 2013). It should be noted, however, that Grubb published a new cystatin
C-based equation in 2014:

eGFR ¼ 130 SCystC1:3069 Age0:117 7 mL=min per 1:73m2
This equation disregards sex and race and can be used as an assay-independent
calculation (Grubb 2014).
Urea Clearance
When compared with GFR measured by inulin clearance, urea clearance underesti-
mates GFR, while creatinine clearance overestimates GFR. Therefore, an equation
was derived as a mean of urea and creatinine clearance (listed in the European Best
Practice Guidelines for Haemodialysis (ERA-EDTA 2002)).
Mean of Urea Clearance and Creatinine Clearance
This equation is based on the assumption that urea clearance underestimates “true”
GFR and creatinine clearance (due to the proximal secretion of creatinine, increased
with decreasing GFR) overestimates GFR:
eGFR ¼ V=ð2 tÞ ðUUrea=SUrea þ UCreat=SCreatÞ 1:73=BSA
V = volume of urine in mL per time

t = time in minutes or seconds
UUrea and UCreat = urinary concentrations of urea and creatinine in mmol/L
SUrea nad SCreat = serum concentrations of urea and creatinine in mmol/L,
BSA = body surface area in m2
eGFR is then in mL=min per 1:73m2 or mL=s per 1:73m2 :
MDRD Formula
MDRD (Modification of Diet in Renal Diseases) was published by Levey (Levey

et al. 1999). Three equations were derived for chronic kidney diseases, the first based
on age, gender, and serum creatinine (and race); the second on age, gender, serum
creatinine, and serum urea (and race); and the third on age, gender, serum creatinine,
serum urea, and serum albumin (and race). The equations were derived from a
sample of 1,070 patients and validated using a sample of 558 patients with chronic
kidney disease. Renal clearance of 125I-iothalamate was selected as the “gold”
standard; creatinine was measured using a kinetic alkaline picrate method. The
first equation was enhanced by improvements in international creatinine standardi-
zation: the “new” MDRD formula (called the “175 MDRD equation”) replaced
previous versions (Levey et al. 2007):

eGFRðMDRDÞ ¼ 175 SCreat1:154 Age0:203 F
where SCreat is in mg/dL, Age is in years, and F is 1.0 for men and 0.742 for women.
Another factor is used for race (1,210 if African American). eGFR (MDRD) is in
mL/min per 1.73 m2.
For the SI unit, the MDRD equation is
h i
eGFRðMDRDÞ ¼ 2:9167 ðSCreat 0:0113Þ1:154 Age0:203 F
where SCreat is in μmol/L, Age is in years, and the other factor as above. eGFR
(MDRD) is in mL/s per 1.73 m2.
CKD-EPI Equations
The CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) equation was

published by Levey (Levey et al. 2009), and the authors recommend using this equation
instead of the older MDRD equation. The 2009 CKD-EPI is based on creatinine in
serum, derived from a sample of 8,254 participants and validated using a data set of
3,896 participants. Iothalamate clearance was selected as the “gold” standard.
The KDIGO CKD 2012 Guideline strictly recommends using CKD-EPI equations.
The use of other creatinine-based equations is possible only when improved accuracy is
proved, compared with the 2009 CKD-EPI creatinine equation. When recording eGFR,
the equation used should be specified (KDIGO 2012 Guideline 2013).
There are three recently recommended CKD-EPI equations:
• 2009 CKD-EPI creatinine-based equation

• 2012 CKD-EPI cystatin C-based equation
• 2012 CKD-EPI cystatin C- and creatinine-based equation
These equations are described in Table 3.
Lund-Malmö Equations
These equations were derived from a population of Swedish Caucasians (derivation

set from Lund, N = 436; validation set from Malmö, N = 414); clearance of
iohexol was used as the “gold” standard. The entire cohort of 850 patients was
within an age interval of 26–85 years; plasma creatinine was 45–545 μmol/L, and
iohexol clearance was 9–121 mL/min per 1.73 m2 (Björk et al. 2007). All equations
(Table 4) used creatinine, age in years, and sex; adjustment to lean body mass can be
applied. Lean body mass (LBM) can be calculated according to James (see Björk
et al. 2007) as follows:
Women : LBM ¼ 1:07 Weight 148 ðWeight=HeightÞ2
Men : LBM ¼ 1:1 Weight 120 ðWeight=HeightÞ2
where Weight is in kg, Height in cm, and LBM is in kg.

One of the advantages of the Lund-Malmö equations lies in the fact that the
equation estimates GFR using a broad age range and predicts GFR in patients with
“normal” serum creatinine. A further advantage is its derivation from a European
population. Björk published a revised version of the Lund-Malmö equations in 2011
(Björk et al. 2011). Nyman published a better performance of the revised Lund-
Malmö eGFR than MDRD or CKD-EPI in a population of 2,847 adults (Nyman
et al. 2014). However, CKD-EPI is now the standard method of GFR estimation.
Table 3 CKD-EPI creatinine equations
51
Type of equation Sex Condition Equation

2009 CKD-EPI creatinine equations Women SCreat 2:4 ðSCreat=61:9Þ0:329 0:993Age ð 1:159 if blackÞ
62 μmol/L
SCreat 2:4 ðSCreat=61:9Þ1:209 0:993Age ð 1:159 if blackÞ
>62 μmol/L
Men SCreat 2:35 ðSCreat=79:6Þ0:411 0:993Age ð 1:159 if blackÞ
80 μmol/L
SCreat 2:35 ðSCreat=79:6Þ1:209 0:993Age ð 1:159 if blackÞ
>80 μmol/L
2012 CKD-EPI cystatin C equation Women and SCystC 2:217 ðSCystC=0:8Þ0:499 0:996Age ð 0:932 in womenÞ
men 0.8 mg/L
SCystC 2:217 ðSCystC=0:8Þ1:328 0:996Age ð 0:932 in womenÞ
>0.8 mg/L
Estimation of Glomerular Filtration Rate
2012 CKD-EPI creatinine-cystatin C Women SCreat 2:17 ðSCreat=61:9Þ0:248 ðSCystC=0:8Þ0:375 0:995Age ð 1:08 if blackÞ
equations 62 μmol/L
SCystC
0.8 mg/L
SCreat 2:17 ðSCreat=61:9Þ0:248 ðSCystC=0:8Þ0:711 0:995Age ð 1:08 if blackÞ
62 μmol/L
SCystC
>0.8 mg/L
>62 μmol/L
SCystC
0.8 mg/L
>62 μmol/L
SCystC
0.8 mg/L
1161
(continued)
1162
Table 3 (continued)
Type of equation Sex Condition Equation
Men SCreat 2:25 ðSCreat=79:6Þ0:207 ðSCystC=0:8Þ0:375 0:995Age ð 1:08 if blackÞ
80 μmol/L
SCystC
0.8 mg/L
80 μmol/L
SCystC
>0.8 mg/L
>80 μmol/L
SCystC
0.8 mg/L
>80 μmol/L
SCystC
>0.8 mg/L
Creatinine should be in μmol/L, age in years, and cystatin C in mg/L. Results are in mL/s per 1.73 m2
SCreat is given in μmol/L; therefore, the original factors used for creatinine should be recalculated. Example: 0:7 88:4 ¼ 61:88 ¼ 61:9
Note: CKD- EPI equations are not intended for use in children
A. Jabor et al.
Table 4 Lund-Malmö equations

Type of
equation Condition Equation
Lund-Malmö SCreat eðð4:620:0112SCreatÞ0:0124Ageþ0:339ðlnðAgeÞÞ0:226SexÞ =60
equation <150 μmol/L
without SCreat eðð8:17þ0:0005SCreat1:07lnðSCreatÞÞ0:0124Ageþ0:339ðlnðAgeÞÞ0:226SexÞ =60
correction to 150 μmol/L
LBM
Lund-Malmö SCreat eðð4:950:011SCreatÞ0:00587Ageþ0:00977LBMÞ =60
equation with <150 μmol/L
correction to SCreat eðð8:58þ0:0005SCreat1:08lnðSCreatÞÞ0:00587Ageþ0:00977LBMÞ =60
LBM 150 μmol/L
Creatinine should be in μmol/L and age in years. Results are in mL/s per 1.73 m2. Sex is 0 for men and
1 for women. For LBM calculation, see the section “Lund-Malmö Equations”
Estimation of GFR in Children
The Schwartz formula (Schwartz et al. 1976) has been used for years, based on
serum creatinine and body height:
eGFR ¼ 41:3 ðHeight=SCreatÞ
where Height is in meters and SCreat is in mg/dL.

This equation was recommended in the KDIGO 2012 Guideline as the “bedside”
Schwartz formula.
In 2009, Schwartz published a new formula based on height, serum creatinine,
cystatin C, and BUN (Schwartz et al. 2009):
eGFR ¼ 39:1 ½ðHeight=SCreatÞ0:516 1:8=SCystC0, 294 30=BUNðmg=dLÞ0:169

1:099ðif maleÞ Height=1:40, 188
where Height is in meters, SCrea in mg/dL, SCystC in mg/L, and BUN in mg/dL.
Schwartz used plasma iohexol clearance as the “gold” standard. Other
equation is
eGFR ¼ 40:7 ðHeight=SCreatÞ0:64 ð30=BUNÞ0:202
where Height is in meters, SCreat and BUN are in mg/dL, and results are in mL/min
per 1.73 m2. This equation was recommended in the 2012 KDIGO CKD (KDIGO
2012 Guideline 2013).
However, Nyman tested the use of the Lund-Malmö equation in children and
found this equation performed well in 85 pediatric Caucasian patients, aged
0.3–17 years. The Lund-Malmö equation performed better when not corrected for
lean body mass (Nyman et al. 2008).
Table 5 Measured and calculated values

Variable Value Unit Value Unit
Serum creatinine 0.1 mmol/L 1.13 mg/dL
(100) (μmol/L)
Serum urea 5.0 mmol/L 5.0 mmol/L
Serum cystatin C 1.1 mg/L 1.1 mg/L
Urine creatinine 7 mmol/L 0.79 g
Urine urea 250 mmol/L 250 mmol/L
BSA 1.94 m2 1.94 m2
Clearance of creatinine 1.08 mL/s per 64.9 mL/min per
1.73 m2 1.73 m2
Clearance of urea 0.77 mL/s per 46.4 mL/min per
1.73 m2 1.73 m2
Mean of urea and creatinine clearance 0.93 mL/s per 55.7 mL/min per
1.73 m2 1.73 m2
eGFR (2009 CKD-EPI creatinine) 1.254 mL/s per 75.2 mL/min per
1.73 m2 1.73 m2
eGFR (2012 CKD-EPI cystatin C) 1.189 mL/s per 71.3 mL/min per
1.73 m2 1.73 m2
eGFR (2012 CKD-EPI 1.217 mL/s per 73.0 mL/min per
creatinine + cystatin C) 1.73 m2 1.73 m2
eGFR (MDRD) 1.14 mL/s per 68.6 mL/min per
1.73 m2 1.73 m2
The differences among various GFR estimations are natural and caused by using different
populations when deriving the respective equations. Some equations perform well during the
interval of “normal” GFR values, and others perform better in pathology
Example: Male, white, 50 years, 24-h collection of urine (1,440 min.), volume of
collected urine 1,500 mL/day. Weight 75 kg, height 180 cm. Measured and calcu-
lated values are given in Table 5.
The use of an exogenous filtration marker is recommended in situations where
more accurate GFR values are needed to make better treatment decisions. Inulin
clearance is recognized as the “gold” standard for GFR assessment (KDIGO 2012
Guideline 2013). There are two principles of inulin clearance measurement: “renal”
clearance of inulin (continuous infusion of inulin with measurement of plasma and
urine inulin concentrations under steady-state conditions, where timed collection of
urine is necessary) and “plasma” clearance of inulin (measurement of several plasma
inulin concentrations after an intravenous bolus of inulin). There are several models
of “plasma” clearance of inulin (simple exponential analysis, area under curve
calculation, the Jung model using early and late plasma concentrations, the
biexponential model, etc.).
Table 6 shows our own experience with plasma inulin clearance, based on the
Jung model (Jung et al. 1991). Results of other equations are compared.
Table 6 Comparison of “plasma” inulin clearance (the Jung model of calculation) with other
eGFR equations
Absolute
bias Relative Correlation Percentage
30 %
eGFR (mL/min) bias (%) coefficient (accuracy)
2009 CKD-EPI creatinine 5.1 2.0 0.504 80
Clearance of creatinine 9.7 14.7 0.380 46
“Plasma” clearance of inulin 10.5 14.0 0.959 87
(monoexponential model)
2012 CKD-EPI cystatin C 7.4 9.1 0.627 73
MDRD equation 11.8 10.9 0.461 71
2012 CKD-EPI cystatin 7.5 7.6 0.602 76
C + creatinine
The table shows the “gold” standard (inulin clearance) compared with various equations for
estimating GFR. As can be seen, the creatinine clearance and MDRD equations perform poorly
while the CKD-EPI equation performs better. The different mathematical inulin clearance model
performs well for obvious reasons (calculation is based on the same variables)
Interpretation of eGFR
Specific Problems with Cystatin C Measurement and Interpretation
Sources of errors in GFR estimation using creatinine and cystatin C (KDIGO 2012
Guideline, modified) are shown in Table 7. Synthesis, tubular processes, and
extrarenal elimination are listed as “non-GFR variables that differ from derivation
sets of patients” in KDIGO 2012 Guideline.
GFR and Age
GFR decreases with age; however, values below 60 mL/min per 1.73 m2 (1 mL/s per
1.73 m2) are rather unusual in individuals without renal pathology. Mathew (Mathew
2007) used data from Sikaris (personal communication) to elucidate this relation-
ship. More than 97.5 % of the mixed population (more than 300,000 examinations in
a large private pathology, exclusive of creatinine outliers for each decade) have an
eGFR above 60 mL/min per 1.73 m2 (1 mL/s per 1.73 m2) at ages 57 years, and
80 % of the mixed population have an eGFR above 60 mL/min per 1.73 m2 (1 mL/s
per 1.73 m2) at ages 80 years. Similar data are available in the KDIGO 2012
Guideline, where inulin clearance is above 60 mL/min per 1.73 m2 in older healthy
men and women.
Table 7 Factors influencing the interpretation of creatinine, cystatin C, and eGFR. According to
the KDIGO 2012 Guideline, modified
Source of
error Creatinine Cystatin C
Non-steady AKI AKI
state
Synthesis Increased with great muscle mass, Increased by administration of
high-protein diet, creatine corticosteroids
supplements, ingestion of cooked
meat, muscle hypercatabolism,
African American race
Decreased in malnutrition, marasmus, Diseases of thyroid glands
muscle-wasting diseases, amputations, Diabetes, adiposity
liver disease, vegetarian diet
Tubular Drug-induced inhibition of tubular
processes secretion (trimethoprim, cimetidine,
fenofibrate)
Extrarenal Decreased after dialysis Increased by severe decrease in GFR
elimination
Higher GFR Higher biological variability in Higher biological variability in
non-GFR determinants relative to non-GFR determinants relative to
GFR, higher measurement error GFR, higher measurement error
Interferences Jaffé method Heterophile antibodies
Decreased: ascorbic acid, bilirubin,
hemoglobin (neonates)
Increased: acetone, acetoacetate,
albumin, cephalosporins (cefoxitin),
glucose, methyldopa pyruvate,
trimethoprim, uric acid
Enzymatic method
Decreased: dobutamine, dopamine,a
bilirubin increased: calcium dobesilate
The important part of the interpretation lies in the fact that both creatinine and cystatin C can be
influenced by a variety of processes and compounds (both of exogenous and endogenous origin).
Creatinine is influenced mainly by changes in muscle mass while cystatin C is influenced by diseases.
Both creatinine and cystatin C are also influenced by interference. The “classic” Jaffé reaction with
alkaline picrate for measuring creatinine produces a lot of interference. Enzymatic measurement of
creatinine and immunoanalytic methods for measuring cystatin C can also be influenced by interference
a
Note: Dobutamine and dopamine only interfere with supratherapeutic concentrations, e.g., con-
tamination of the sample
Glomerular Filtration Rate and Body Surface Area
Results of all “new” equations for eGFR are always expressed in standardized
format, i.e., in mL/min per 1.73 m2 (or mL/s per 1.73 m2). However, for drug dosing
it is necessary to recalculate the standardized results here to reflect the actual
filtration of the patient (in mL/min or mL/s).
The following simple equation is used:
GFR ¼ eGFR=1:73 BSA
where BSA (in m2) is calculated according to the DuBois and DuBois formula
(DuBois and DuBois 1916)
BSA ¼ 0:007184 Weight0:425 Heigth0:725
where Weight is in kg and Height is in cm. GFR is then in mL/min or mL/s.
Specific Clinical Situations
Chronic Kidney Disease (CKD)
“Chronic kidney disease (CKD) is defined as abnormalities of kidney structure or

function, present for more than 3 months, with implications for health and CKD is
classified based on cause, GFR category, and albuminuria category (CGA)”
(KDIGO 2012 Guideline 2013). This completely new definition reflects the need
for both a more “clinical” definition and a more precise “laboratory” classification.
GFR categories together with recommended terms are listed in Table 8.
Lower values of eGFR correlate with worse prognoses: the age-standardized
rate of hospitalization, rate of death, and rate of cardiovascular events (per
100 person-years) increase almost exponentially with decreasing eGFR (Levey
et al. 2006).
Table 8 GFR categories in CKD (KDIGO 2012 Guideline 2013)

GFR GFR (mL/min per GFR (mL/s per
category 1.73 m2) 1.73 m2) Terms
G1 1.50 90 Normal or high
G2 1.00–1.49 60–89 Mildly decreaseda
G3a 0.75–0.99 45–59 Mildly to moderately
decreased
G3b 0.50–0.74 30–44 Moderately to severely
decreased
G4 0.25–0.49 15–29 Severely decreased
G5 <0.25 <15 Kidney failure
The importance of the KDIGO 2012 Guideline for chronic kidney disease lies in the combination of
the GFR and albuminuria categories for staging and prognostication. Terminology is also clearly
recommended for each GFR category. GFR categories G1 and G2 do not fulfill the criteria for CKD
unless evidence of kidney damage is present
a
Note: Relative to a young adult level
Diabetes Mellitus
Pathophysiology and the Development of Diabetic Nephropathy

In type 1 diabetes mellitus (T1DM), five stages of diabetic nephropathy can be
distinguished:
(a) Early hypertrophy and hyperfunction, lasting anywhere from months to years. At
this stage, eGFR is increased of about 20–40 %, and hyperfiltration is supposed
to be a risk factor.
(b) Clinically latent stage of renal impairment, where eGFR can be increased, but
the basal membrane is thickened and mesangial expansion can be found.
(c) Incipient nephropathy, with positive albuminuria; eGFR is sometimes increased,
but a thickened basal membrane and mesangial expansion is more common.
(d) Manifest nephropathy with proteinuria and decreased eGFR; GFR decreases at a
rate of 1.2 mL/min per year (0.2 mL/s per year).
(e) Chronic kidney disease with severely decreased GFR or kidney failure.
Only 25 % of patients with type 2 diabetes (T2DM) mellitus have similar renal
impairment, as is common in T1DM, frequently as a result of decompensated
diabetes mellitus. About 40 % of patients with T2DM display slight or moderate
histological findings with borderline renal function. About 35 % display tubuloin-
tersticial changes or glomerulosclerosis.
Guidelines
Recently American Diabetes Association Standards of Medical Care in Diabetes
(ADA 2016) describes consequences of decreased GFR in the following chapters:
Cardiovascular Disease and Risk Management and Microvascular Complications
and Foot Care. Estimated GFR should be used as a screening tool at least once a year
in patients with type 1 diabetes mellitus lasting five or more years and in all patients
with type 2 diabetes mellitus. Serum creatinine with MDRD formula or preferably
CKD-EPI formula is recommended for eGFR assessment of diabetes mellitus. The
five stages of CKD are defined in the 2016 ADA Standards of Medical Care in
Diabetes (Table 9). In contrast to the KDIGO 2012 Guideline, stage 3a and 3b are not
distinguished here.
Both eGFR and albuminuria (and serum/plasma potassium) are measured
together. Screening based on albuminuria alone is not sufficient. It should be
stressed, however, that albuminuria is measured repeatedly due to its high biological
variability. The new term “increased urinary albumin excretion” can be used only if
two of three urine specimens collected within a 3- to 6-month period contain
30 mg/g creatinine (3.0 g/mol creatinine). The use of the term
“microalbuminuria” (30–299 mg/g creatinine, 3–29.9 g/mol creatinine) should no
longer be used; similarly, the term “macroalbuminuria” (or “clinical albuminuria”,
>299 mg/g creatinine or 30 g/mol creatinine) is also inappropriate.
The ADA Standards (ADA 2016) recommends actions for the management of
CKD. Patients in stages 4 and 5 should always be referred to a nephrologist, as well
Table 9 Stages of CKD from the ADA Standards of Medical Care in Diabetes 2016
GFR GFR mL/s
mL/min per per Comparison with KDIGO
Stage Description 1.73 m2 1.73 m2 2012
1 Kidney damage with 90 1.5 Normal or high in KDIGO
normal or increased 2012 without remarks on
GFR damage
2 Kidney damage with 60–89 1–1.49 Same in KDIGO 2012,
mildly decreased GFR without remarks on damage
3 Moderately decreased 30–59 0.50–0.99 Mildly to severely decreased
GFR GFR
4 Severely decreased 15–29 0.25–0.49 Same in KDIGO 2012
GFR
5 Kidney failure <15 or <0.25 or Dialysis is not mentioned in
dialysis dialysis KDIGO 2012
The American Diabetes Association uses a similar GFR classification to the KDIGO Guideline. In
contrast to the KDIGO 2012 Guideline, stages 3a and 3b are not distinguished here
as patients with eGFR 45–60 mL/min per 1.73 m2 (0.75–1.0 mL/s per 1.73 m2) and
suspect of nondiabetic kidney disease. More frequent monitoring of an expanded set
of laboratory tests is recommended in patients with eGFR 30–44 mL/min per
1.73 m2 (0.5–0.74 mL/s per 1.73 m2).
Estimated GFR (or creatinine measurement) should also be monitored (together
with potassium levels in serum/plasma) in diabetic patients using ACE inhibitors,
angiotensin receptor blockers, loop diuretics, hydrochlorothiazide, or
chlorthalidone. Hydrochlorothiazide and chlorthalidone should be avoided with
eGFR levels under 30 mL/min per 1.73 m2 (0.5 mL/s per 1.73 m2).
AKI and Intensive Care
Definition and Staging of Acute Kidney Injury

Acute kidney injury (AKI) is present when
• Serum creatinine increases by 26.5 μmol/L or more (0.3 mg/dL and more) within
48 h, or
• Serum creatinine increases by at least 50 % above the baseline (at least 1.5 times
above the baseline) within 7 days, or
• The volume of urine is lower than 0.5 mL/kg per hour for 6 h (oliguria).
There are two staging systems – the older RIFLE system (Risk, Injury, Failure,
Loss of function, End-stage kidney disease) and the newer AKIN system (Acute
Kidney Injury Network) (Ronco 2013). Comparison of the RIFLE and AKIN
systems is given in Table 10. Both systems can be used, but a fraction of the patients
classified in the respective stages is not equal.
Table 10 Comparison of the RIFLE and AKIN systems

Urine Urine
RIFLE SCreat/GFR output AKIN output
staging criteria criteria staging SCreat criteria criteria
Risk Increased SCreat Urine Stage Increased Urine
>50 % or GFR output 1 SCreat > +50 % to output
decreases >25 % <0.5 mL/ +100 % (i.e., <0.5 mL/
kg per 1.5–2.0-fold from kg per hour
hours the baseline) within 6 h
within 6 h or > increase of
26.5 μmol/L
(0.3 mg/dL)
Injury Increased SCreat Urine Stage Increased SCreat Urine
>100 % or GFR output 2 +101 % to +200 % output
decreases >50 % <0.5 mL/ (i.e., 2.01–3.0-fold <0.5 mL/
kg per hour from the baseline) kg per hour
for more for more
than 12 h than 12 h
Failure Increased SCreat Urine Stage Increased SCreat Urine
>200 % or GFR output 3 +200 % and more output
decreases >75 % <0.3 mL/ (more than 3.0-fold <0.3 mL/
or SCreat kg per hour from the baseline) or kg per hour
>354 μmol/L for 24 h or SCreat higher than for 24 h or
(4 mg/dL) (with anuria for 354 μmol/L (4.0 mg/ anuria for
acute rise of 12 h dL) with an acute 12 h
44 μmol/L or more increase of at least
(0.5 mg/dL and 44 μmol/L (0.5 mg/
more) dL)
Staging of AKI is based on changes of serum creatinine and urine volume (similar in both the
RIFLE and AKIN classifications) or GFR changes (RIFLE only). Loss of function and end-stage
renal disease (ESRD) do not have a parallel stage in the AKIN system and were removed from the
staging system in 2007 (Mehta et al. 2007). Patients receiving renal replacement therapy (RRT) are
considered to meet the criteria for Stage 3 in the AKIN system (irrespective of the stage before RRT)
Several methods for GFR estimation are discussed among intensivists with strong
emphasis on frequent monitoring of plasma (serum) creatinine; however, creatinine
clearance based on a 2-h collection of urine is also admitted, and iohexol or
iothalamate is classified as acceptable and realistic by the ADQI group. GFR can
be used for staging of acute kidney injury as part of the RIFLE (but not the AKIN)
classification.
Monitoring of creatinine concentration is an essential tool for GFR assessment in
intensive care. Plasma (serum) creatinine is not a precise predictor of GFR
under acute circumstances. On the other hand, dynamic changes in creatinine
concentrations are similar to the dynamic changes in GFR. In cases of
unknown basal creatinine concentration, it is possible to assess creatinine concen-
tration using the CKD-EPI equation. This principle was recommended for the
MDRD equation by Bellomo et al. (2004). They used “basal” GFR of 75 mL/min
per 1.73 m2 (1.25 mL/s per 1.73 m2). Table 11 is based on the 2009 CKD-EPI
Table 11 Estimated baseline creatinine from the 2009 CKD-EPI equation for “basal” GFR of
75 mL/min per 1.73 m2 (1.25 mL/min per 1.73 m2) (According to Bellomo et al. 2004)
Male baseline
Age creatinine Male baseline Female baseline Female baseline
(years) (mg/dL) creatinine (μmol/L) creatinine (mg/dL) creatinine (μmol/L)
20 1.35 119 1.06 94
30 1.28 113 1.01 89
40 1.20 106 0.95 84
50 1.13 100 0.89 79
60 1.07 95 0.85 75
70 1.01 89 0.80 71
80 0.95 84 0.76 67
Changes in serum creatinine are used for the diagnosis and staging of acute kidney injury. In acute
settings, physicians do not know the basal value of serum creatinine prior to the development of
acute kidney injury. Because of the relation between age, creatinine, and GFR, basal creatinine can
be estimated from the expected GFR before injury (here it is at least 75 mL/min (1.25 mL/s) per
1.73 m2 of body surface area)
creatinine equation with “basal” GFR of 75 mL/min per 1.73 m2 (1.25 mL/s per
1.73 m2).

Conditions
The relation between eGFR and prognosis is given in the KDIGO 2012 Guideline for
CKD. Four variables should be taken into account: cause of CKD, GFR category
(G1–G5), albuminuria category (A1–A3), and complications (other risk factors,
comorbidities). eGFR is used to diagnose and monitor other diseases and conditions:
increased GFR can be expected during pregnancy, with a high-protein diet and in
conditions where there is increased output of osmotically active substances (osmotic
diuresis, e.g., after mannitol, or in hyperglycemic situations). For extrarenal reasons,
decreased eGFR can occur during and after cases of dehydration, e.g., hemorrhagic
shock.
Summary
• Estimated glomerular filtration rate (eGFR) is used for the diagnostics, staging,
and monitoring of kidney function.
• Estimation is based on serum creatinine and/or cystatin C concentration.
• Methods of determining creatinine and cystatin C should be traceable to interna-
tional calibrators; enzymatic determination of creatinine is preferred to the Jaffé
(alkaline picrate) method.
• The gold-standard method for GFR assessment (inulin clearance) is preferred to

other methods (125I-iothalamate, iohexol).
• The CKD-EPI equations (Chronic Kidney Disease – Epidemiology Collabora-
tion) are recent equations for calculating eGFR.
• Three CKD-EPI equations are recommended: the 2009 CKD-EPI creatinine,
2012 CKD-EPI cystatin C, and 2012 CKD-EPI cystatin C and creatinine equa-
tions, respectively.
• The MDRD equation or Cockcroft-Gault formula should not be used for eGFR
assessment.
• The KDIGO 2012 Guidelines (both for chronic kidney disease and acute
kidney injury) and ADQI recommendations (Acute Dialysis Quality Initiative)
provide suggestions for health care that positively impact on patient benefit and
safety.
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Red Blood Cell Distribution Width: Useful
Predictor for Treatment Response 52
in Primary Glomerular Diseases
Kenan Turgutalp, Simge Bardak, Serap Demir, and Ahmet Kıykım
Contents
Key Facts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1176
Definitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1177
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1177
Pathogenesis of Primary Glomerular Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1178
Prognostic Factors in Primary Glomerular Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1180
What Is RDW? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1182
Clinical Importance of Increased RDW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1183
RDW in Patients with Proteinuria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185
Potential Role of RDW in Primary Glomerular Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185
Summary Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1188
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1188
Abstract
Red blood cell distribution width (RDW) shows the variation in size of erythro-
cytes in the circulation and it is obtained easily by automated blood cell counters.
RDW is associated with systemic inflammation. Clinical or subclinical inflam-
mation is usually present in pathogenesis of primary glomerular diseases (PGDs).
Control of many of PGDs can be achieved by immune modulator agents and this
supports the underlying systemic inflammatory process. Treatment response
cannot be achieved in some patients. There is neither a standard dose nor a
standard dose reduction protocol for immunosuppressive treatment in PGDs. In
this aspect, use of predictive biomarkers for treatment response can protect
patients from side effects of unnecessary immunosuppressive drugs. New
K. Turgutalp (*) • S. Bardak • S. Demir • A. Kıykım

Division of Nephrology, Department of Internal Medicine, School of Medicine, Mersin University,
Mersin, Turkey
e-mail: k.turgutalp@hotmail.com; kenanturgutalp@gmail.com; bardaksimge@gmail.com;
serapbas@yahoo.com; ahmetkiykim@gmail.com; aakiykim@yahoo.com

1176 K. Turgutalp et al.
biomarkers are essential in prediction of treatment response and prognosis of

PGDs. So, studies have been focused to investigate potential clinical importance
of RDW in prediction of prognosis and clinical outcome of PGDs. RDW is
assessed as part of the complete blood count which is a quite cheap test, and
therefore, it may be a useful novel marker for evaluation of treatment response in
nephrotic syndrome due to PGDs.
Keywords
Biomarker • Immunosuppressive drugs • Nephrotic syndrome • Primary glomer-
ular diseases • Red blood cell distribution width • Treatment response
Abbreviations
Anti-PLA2R Phospholipase A2 receptor
FSP1+ Fibroblast-specific protein 1-positive
IgM Immunoglobulin M
L FABP L fatty-acid-binding protein
MG Membranous glomerulopathy
MPGN Type 1 membranoproliferative glomerulonephritis
MPV Mean platelet volume
NAG N-acetyl-beta-glucosaminidase
NHANES National health and nutrition examination survey
NS Nephrotic syndrome
PGDs Primary glomerular diseases
RBC Red blood cell
RDW Red blood cell distribution width
SOCS Suppressors of cytokine signaling
α1M α1-microglobulin
β2 M β2-microglobulin
Key Facts
• NS due to PGDs is an immunoinflammatory condition.

• Clinical course of PGDs varies widely from complete remission to end-stage
renal disease.
• Control of many of PGDs can be achieved by conservative treatment and immune
modulator agents.
• Predictive markers of therapeutic response are useful especially in determining
the treatment plan and follow-up of PGDs
• Use of predictive biomarkers for treatment response can protect patients from the
side effects of unnecessary immunosuppressive drugs.
52 Red Blood Cell Distribution Width: Useful Predictor for Treatment. . . 1177
• Most of classical predictors are weak, unpractical in use, and also they are quite
expensive, so their use is limited in most centers.
• RDW may be associated with systemic inflammation and may predict prognosis
and clinical outcome of PGDs
Definitions
Antibodies An immunoglobulin, a specialized immune protein, produced due to

the introduction of an antigen into the body which possesses the significant capa-
bility to combine with the antigen that triggers its production. The production of
antibodies is a major function of the immune system and is carried out by a type of
white blood cell called a B cell (B lymphocyte).
Biomarkers A predictor that can be used to measure the presence or progress of a

disease or the effects of treatment. For example, prostate-specific antigen is a
biomarker for prostate cancer.
End-stage renal disease End-stage renal disease is the stage of kidney impairment
which is irreversible, cannot be controlled by conservative management alone, and
requires dialysis or kidney transplantation to maintain life.
Inflammation A protective tissue response of the organism to injury which serves

to remove injurious insult and to mediate the healing process.
Primary glomerular diseases Primary glomerular diseases consist of a group of

disorders characterized by pathologic alterations in normal glomerular structure and
function, independent of systemic disease courses. This distinction is important
because the clinical presentation and pathologic findings of glomerulopathy secondary
to systemic diseases may mimic primary glomerular disorders, yet the correct diagnosis
of the underlying systemic disease may significantly alter the treatment of the patient.
Proteinuria The presence of abnormally large amounts of protein in the urine,

usually resulting from kidney diseases, but sometimes results from fever, excessive
exercise, and other abnormal conditions. It is often defined as an amount in excess of
300 mg per day.
Introduction
Clinical course of primary glomerular diseases (PGDs) varies widely from complete
remission to end-stage renal disease. Besides conservative treatment, clinicians need
to use immunosuppressive drugs which have serious side effects. However, unfor-
tunately only some of the patients respond to the treatment. For this reason, some
patients may use these therapeutic agents though treatment response would never be
achieved and they face serious side effects of the treatment regimen.
Table 1 Universal characteristics of any biomarker. These features of new biomarkers are essential
for diagnosis, prediction of treatment response, and prognosis in patients with primary glomerular
disease (Data are from Biomarkers Definitions Working Group (2001))
They should be noninvasive, easily measured, and inexpensive
They should be from readily available sources, such as blood or urine
They should have a high sensitivity, allowing early detection, and no overlap in values between
diseased patients and healthy individuals
They should have a high specificity, being greatly upregulated (or downregulated) specifically in
the diseased samples and unaffected by comorbid conditions
Biomarker levels should vary rapidly in response to treatment
Biomarker levels should aid in risk stratification and possess prognostic value in terms of real
outcomes
Biomarkers should be biologically plausible and provide insight into the underlying disease
mechanism
Renal biopsy is still the gold standard method for diagnosis of glomerular
diseases. However, it is not only an invasive procedure which cannot be performed
in every clinic, but it also results in important complications, and pathologic
evaluation may be time-consuming. Although serial renal biopsies might improve
treatment, they are often difficult to justify because of the risk, discomfort, and
expense. New methods are needed to identify the cause of renal diseases and
prognosis without a biopsy. On the other hand, various biomarkers which have
been used for years in order to investigate the etiology and progression rate of
glomerular diseases are available. However, their low selectivity and specificity
limit their use.
The National Institutes of Health Biomarkers Definitions Working Group has
defined a biological marker (biomarker) as “A characteristic that is objectively
measured and evaluated as an indicator of normal biological processes, pathogenic
processes, or pharmacologic responses to a therapeutic intervention” (Biomarkers
Definitions Working Group 2001). Universal characteristics for any ideal biomarker
are listed in Table 1 (Biomarkers Definitions Working Group 2001). New bio-
markers with such these features are essential in diagnosis, prediction of treatment
response, and prognosis of PGDs. In this section, we discussed markers which have
been still used to predict treatment response in PGDs like minimal change disease
(MCD), focal segmental glomerulosclerosis (FSGS), membranous glomerulopathy
(MG), type 1 membranoproliferative glomerulonephritis (MPGN) and IgA nephrop-
athy (IgAN). We focused on red blood cell distribution width (RDW) which we
thought can predict treatment response in PGDs.
Pathogenesis of Primary Glomerular Diseases
Proteinuria due to glomerular diseases was first described 200 years ago by Richard
Bright (1836). During this period, besides the developments in histopathologic
evaluation, many investigations about underlying etiologic factors, genetic studies,
studies on experimental models, studies about mediators effecting the development

of tissue injury, and studies investigating the role of immune response were all held
to understand more about glomerular injury. Although many studies focusing to
identify nephritogenic factors were carried out in last 50 years, we still do not know
exactly what the precipitating factors are and how can we prevent development of
PGDs. Understanding of immune response caused by environmental factors (espe-
cially infections, drugs etc.) and genetic features can allow development of new
biomarkers for PGDs.
Nowadays, it has been suggested that nephrotic syndrome (NS) due to PGDs is an
immunoinflammatory condition (Camici 2007). Many PGDs are associated with
positive staining for immunoglobulins, often with components of the complement
system, and with the presence of immune complexes. Infiltration by inflammatory
cells is largely determined by the site of immune complexes. Some of the authors
thought that specific or nonspecific infections which can be acute or chronic may
play a role in PGDs’ pathogenesis (Couser and Johnson 2014). Glomerular diseases
can develop as a consequence of infection with organisms releasing super antigens
that cause a polyclonal activation of B cells. The main organism responsible for
super antigen-associated glomerular injury is Staphylococcus aureus. Once an
immune response is initiated, the local injury may lead to the release of additional
antigens that may further increase the immune response. Normal immune response is
a reaction only against the pathogen. As deletion of self-reactive T cells which arise
during development of thymus occurs in adulthood, autoimmune response is not
observed (central tolerance). If self-reactive T and B cells are not deleted for any
reason and pass into the systemic circulation, mechanisms may be activated to detect
and either delete or suppress them (peripheral tolerance). Loss of tolerance for
various reasons (genetic susceptibility and problems, T-regulator cell dysfunctions,
antigenic similarity, persistence of self-reactive cells, structural changes in epitopes,
increase in number of similar epitopes, development of antibodies and immune
complexes) can initiate autoimmune injury (Couser and Johnson 2014). Generally,
this process occurs silently and remains subclinical.
Twenty thousand five hundred of 1,000,000 microbial derived genes in humans
are inherited and pass to future generations (Pillai 2013). Microorganisms were
suggested to trigger autoimmune mechanism in type 1 diabetes mellitus and auto-
immune arthritis (Couser and Johnson 2014). Sufficient amount of data supporting
the role of infectious agents in development of PGDs is actually present in literature
(post-streptococcal glomerulonephritis, etc.). Systemic inflammatory response due
to infectious agents play an important role in the pathogenesis of PGDs MCD,
FSGS, MG, type 1 MPGN, and IgAN (Couser and Johnson 2014). Furthermore,
antibody-mediated or immune complex-related glomerular injuries due to microor-
ganisms are evident in IgAN, MG, and type 1 MPGN (Couser and Johnson 2014).
On the other hand, there are non-antibody-mediated injury mechanisms modu-
lated by microorganisms such as problems in T-cell regulation in MCD and problems
in parts of immune system like complement pathway in C3 nephropathies (Couser
and Johnson 2014). In contrast to the immune complex mechanisms, certain glo-
merular diseases develop primarily through cell-mediated immunity. A direct role
for T cells in mediating glomerular injury and crescent formation has been shown in
some experimental crescentic nephritis (Atkins et al. 1996).
All these processes lead to a clinical or subclinical inflammation in PGDs.
Besides control of many of these diseases can be achieved by immune modulator
agents, and this supports the underlying systemic inflammatory process (Camici
2007). So, studies have been focused to investigate potential clinical importance of
RDW which is directly associated with systemic inflammation in prediction of
prognosis and clinical outcome of PGDs (Turgutalp et al. 2014).
Prognostic Factors in Primary Glomerular Diseases
There are many factors affecting the prognosis and outcome of PGDs. Predictive
markers of therapeutic response are useful especially in determining the treatment
plan and follow-up of PGDs.
Prognosis of MG and FSGS are usually better in women than men probably due
to lower proteinuria and blood pressure. However, women lose the advantage at
higher levels of proteinuria (Cattran et al. 2008). Older age is one of the other poor
prognostic parameters in MG. However, some studies declared that prognosis is
similar in elderly and young idiopathic MG patients. Indeed, elderly patients have
higher risk for infection due to immunosuppressive drugs (Yamaguchi et al. 2014).
Impaired renal function, higher blood pressure, and higher proteinuria are asso-
ciated with worse prognosis in membranous nephropathy (Troyanov et al. 2006).
Basal level of proteinuria is important in predicting the prognosis of MG (Caro
et al. 2014). Titer of the antibodies against the M-type phospholipase A2 receptor
(anti-PLA2R) is also correlated with the clinical activity and the response to immu-
nosuppressive treatment in patients with MG (Segarra-Medrano et al. 2014). Deple-
tion of anti-PLA2R autoantibodies can predict proteinuria response in MG (Beck
et al. 2011). It has been documented that the amount of low-, medium-, and high-
molecular-weight urinary proteins may be related with the rate of response to the
treatment in patients with MG (Irazabal et al. 2013). Selective urinary biomarkers
such as α1-microglobulin (α1M), β2-microglobulin (β2M), immunoglobulin G
(IgG), and M (IgM) are used to predict both the outcome and the treatment response
in MG (Irazabal et al. 2013). Urinary complement levels, N-acetyl-beta-
glucosaminidase (NAG) and L fatty-acid-binding protein (L FABP), are some of
the other predictive urinary markers for progression of MG. High urinary concen-
tration of C5b9, a complement activation marker, is associated with poor prognosis
(Brenchley et al. 1992). NAG, a lysosomal enzyme of proximal tubular cells, is a
marker of tubular cell injury like β2M. β2M was found superior in predicting
progression of renal disease when compared with NAG (Hofstra et al. 2008).
These markers can be measured by noninvasive methods and can be repeated during
the follow-up. However, their efficacy is still controversial. Particular histologic
findings like tubulointerstitial damage, glomerular, and vascular sclerosis are also
associated with poor prognosis in MG (Troyanov et al. 2006). Detection of specific
protein expression in renal biopsy specimen may help to predict the rate of
progression. Interstitial alpha smooth muscle actin staining (Badid et al. 1999) and
interstitial infiltration of CD68-positive cells with MCP-1/CCR2 expression are
associated with progression to end-stage renal disease among patients with MG
(Yoshimoto et al. 2004).
Severity of proteinuria, elevated serum creatinine, and black race are clinical risk
factors for poor outcome in FSGS. Collapsing variant and tubulointerstitial fibrosis
are histopathologic features of poor prognosis (Appel and D’Agati 2015). Urinary
fractional excretion of IgG, and α 2-macroglobulin/creatinine ratio were
documented as powerful predictors of outcome and responsiveness to steroids
and cyclophosphamide (Bazzi et al. 2013). Low β2M may suggest a good prognosis
without immunosuppressive therapy in FSGS (Deegens and Wetzels 2007).
Urinary-retinol binding protein, a marker of proximal tubular dysfunction, was
studied in patients with FSGS, MCD, or mesangial proliferative glomerulonephritis
and higher levels were correlated with treatment unresponsiveness (Mastroianni
et al. 2000). Urinary NAG may be an indicator of relapse in both FSGS and MCD
(Dillon et al. 1998).
Elevated serum creatinine concentration at presentation, higher blood pressure,
and persistent and severe proteinuria are signs of poor prognosis in IgAN. Reduced
proteinuria is associated with better renal function (Shimizu et al. 2009). Urinary
β2M levels are correlated with renal function and proteinuria in IgAN (Shin
et al. 2014). Urinary excretion of low molecular weight proteins is not superior to
total proteinuria and serum creatinine in predicting prognosis in patients with IgAN
(Peters et al. 2009). Higher IgA/C3 ratio is correlated with worse prognosis (Ishiguro
et al. 2002). Urinary interleukin 6 (IL 6) has prognostic value in patients with IgAN
and was found higher in progressors (Harada et al. 2002). The ratio of epidermal
growth factor (EGF) to monocyte chemotactic peptide 1 (MCP 1) in the urine was
also used as a prognostic marker for patients with IgAN (Torres et al. 2008).
Glomerular changes with segmental glomerulosclerosis, crescents, mesangial
hypercellularity, and tubulointerstitial changes are poor histological parameters for
IgAN (Shin et al. 2014). As the glomerular changes are related to hypertension,
serum creatinine levels, and proteinuria levels, glomerular grading system may be
useful in predicting the prognosis of IgAN (Shin et al. 2014). Renal biopsy can also
help distinguishing crescentic variant of IgAN which has a worse prognosis than
other noncrescentic variants. In crescentic IgAN, ratio of fractional excretion of IgG
to surviving glomeruli could inform us about progression. Its predictive value was
found higher when it was evaluated with serum creatinine (Bazzi et al. 2009). Renal
biopsy requirement was its disadvantage. On the other hand, immunohistochemical
evaluation is useful in prediction of outcome. Positive staining of GMP-17, a protein
found on cytotoxic T lymphocytes, is associated with progression of IgAN (van Es
et al. 2008). Fibroblast-specific protein 1-positive (FSP1+) fibroblasts are associated
closely with the interstitial fibrosis and tubulointerstitial fibrosis correlates with renal
survival; therefore, a number of (FSP1+) cells may predict response to the treatment
with corticosteroids (Harada et al. 2008). Smoking, hyperuricemia, gross obesity,
long duration of preceding symptoms, and increasing age are the other poor prog-
nostic factors (Feehally and Floege 2015).
Elevated serum creatinine, nephrotic range proteinuria, severe hypertension, and

crescents (50 %) or marked interstitial fibrosis on biopsy are the risk factors for
progression of MPGN. Patients with nephritic syndrome and high C1q staining in
glomerular deposits are poor prognostic parameters for MPGN (Schena and Alpers
2015).
1-β glycoprotein fragmentation was documented as a powerful urinary biomarker
for steroid resistance in pediatric NS (Piyaphanee et al. 2011), whereas NPHS2 allele
combination was reported as another marker of steroid resistance in patients with NS
(Santín et al. 2011). Serum IgG level and IgG/IgM ratio were found to be signifi-
cantly lower in children who had corticosteroid-resistant NS (Roy et al. 2009).
Plasma levels of suppressors of cytokine signaling (SOCS) 3 and SOCS 5 might
predict initial resistance to steroids in NS patients (Ostalska-Nowicka et al. 2011).
Fractional excretion of magnesium was found to be correlated with various renal
histopathologic findings in children and it was reported that it might be an early
predictor of clinical outcome in steroid-resistant NS (Rumana et al. 2014). Mean
platelet volume (MPV) was found higher on admission in therapy-resistant patients.
In the same study it was documented that MPV levels were significantly correlated
with the proteinuria level during a yearly follow-up (Kocyigit et al. 2013).
Most of the predictors are weak, impractical in use, and also they are quite
expensive, so their use is limited in most centers. Ultimately, some diseases are
likely to require the use of combinations of markers in useful clinical assays.
Immunosuppressive drugs are the mainstay in the treatment of patients with
PGDs who are unresponsive to conservative treatment. On the other hand, it is
well known that immunosuppressive drugs have many short- and long-term side
effects like increase in malignant diseases, metabolic complications, sleep and mood
disorders, gastrointestinal discomfort, hypertension, and life-threatening infections.
Immunosuppressive drugs should be given in appropriate doses only for an appro-
priate duration to reduce these side effects. There is neither a standard dose nor a
standard dose reduction protocol in PGDs. In this aspect, use of predictive bio-
markers for treatment response can protect patients from side effects of unnecessary
immunosuppressive drugs (Turgutalp et al. 2014). Studies have been focused to
investigate potential clinical importance of RDW which is directly associated with
systemic inflammation in prediction of prognosis and clinical outcome of PGDs
(Turgutalp et al. 2014).
What Is RDW?
RDW shows the variation in size of erythrocytes in the circulation. The first
quantitative assessments of variation in red blood cell diameter measurements in
fixed stained peripheral blood smear were reported by Price-Jones in normal indi-
viduals and the patients with anemia (Price-Jones 1910). He postulated that differ-
ence in size and range variation might be diagnostically useful, but his method was
tedious, was time-consuming, and had some limitations. Currently, RDW value is
obtained easily by automated blood cell counters.
Higher RDW values indicate higher difference in size of circulating erythrocytes

(Simel et al. 1988). RDW is a semi-quantitative statistical value which is routinely
obtained in a complete blood cell count. Automated cell counters estimate red blood
cell (RBC) volume cell by cell, sampling millions of RBCs in the process. It is a
simple, cheap, and easily repeatable parameter. RDW is calculated with the follow-
ing formula:
RDW ¼ ðStandard deviation of MCV=mean MCVÞ 100
Normal RDW values are 11.6–14.6 % in adults (Vajpayee et al. 2011). If RDW is
measured in EDTA anticoagulated blood sample instead of citrated blood, false high
results can be obtained. Prolonged storage of the specimen at room temperature may
result in decrease in RDW values. Except such this condition, there is not any
clinical situation in which RDW values are below normal. Therefore RDW value
is either in normal range or high.
Clinical Importance of Increased RDW
It has been used in differential diagnosis of anemia for many years. Deficiency of
iron, folate, or vitamin B12 can lead to higher RDW values, whereas RDW is in
normal range in anemia with homogeneous structure in erythrocytes like thalassemia
syndromes. However, the finding of an increased RDW is not specific for any one
abnormality. On the other hand, a normal RDW does not mean that the main
population of red cells is normal.
Furthermore, elevated RDW value has been associated with poor prognosis in an
increasingly large number of non-hematological clinical conditions, particularly in
disorders with underlying inflammatory processes. Systemic inflammatory process
and oxidative stress can affect iron metabolism negatively, can suppress erythrocyte
maturation, can shorten erythrocyte survival, can lead to early escape of reticulo-
cytes into the blood circulation, and therefore can increase the RDW values
(Spiropoulos et al. 2010; Pierce and Larson 2005; Ghaffari 2008). RDW has a
positive, independent linear correlation with acute phase reactants like C-reactive
protein (CRP) and IL-6 (Lippi et al. 2009; Veeranna et al. 2013). For this reason, the
value of RDW started to be investigated in detail in clinical disorders other than
hematologic problems. RDW was firstly evaluated and found increased in diseases
with underlying inflammatory mechanisms (hearth failure, inflammatory bowel
disease, malnutrition, etc.) (Song et al. 2012; Ozcan et al. 2013; Huang
et al. 2014). Furthermore, meta-analysis reports showed that RDW could predict
mortality and morbidity in disorders with underlying inflammatory processes like
hearth failure (Yu et al. 2011).
Recent studies have documented that circulating erythrocytes participate in
coagulopathy and development of atherosclerotic vascular disease (Yu et al. 2011).
It was suggested that erythrocyte deformability is reduced in people with high RDW
values (Patel et al. 2013), therefore blood flow slows, blood viscosity increases, and
Table 2 Clinical Hematological

conditions for which
Multiple myeloma
predictive value of RDW
was tested. RDW is a Hairy cell leukemia
predictive marker for Lymphoma
prognosis in many different Cardiovascular
clinical situations Acute coronary syndrome
Stable angina pectoris
Acute/chronic heart failure
Stroke
Peripheral artery disease
Pulmonary embolism
Eisenmenger syndrome
Connective tissue diseases
Rheumatoid arthritis
Systemic lupus erythematosus
Takayasu’s arteritis
Systemic sclerosis
Malignancies
Breast cancers
Prostate cancer
Pancreatic cancer
Malignant mesothelioma
Lung cancer
Miscellaneous
Nonalcoholic steatohepatitis/hepatic fibrosis
Hip fracture
Crohn’s disease
Diabetes complications
Acute pancreatitis
Critically ill patients
Preeclampsia
Trauma patients
Sarcoidosis
this can increase aggregation and can lead to arterial and venous thrombosis
(Rezende et al. 2014). RDW is also a predictor for acute coronary syndrome in
general population without cardiovascular disease (Skjelbakken et al. 2014). These
findings suggested subclinical inflammatory process as the leading factor for “out-
of-sight” or undefined clinical problems in these people. Besides, meta-analysis
reports documented that RDW could predict acute coronary syndrome in patients
with documented coronary artery disease (Su et al. 2014). Some clinical conditions
that have investigated the predictive value of RDW are listed in Table 2. Absence of
randomized controlled trials is the limited side of these reports.
These findings support the importance of RDW as a predictive marker for

prognosis in many different clinical situations. However, underlying mechanisms
of this important relationship is not clear enough.
RDW in Patients with Proteinuria
As both RDW and microalbuminuria are found to be related with cardiovascular

morbidity and mortality, it can be hypothesized that a link between RDW and
microalbuminuria may exist (Arbel et al. 2014; Schmieder et al. 2014; Deveci
et al. 2010). The cross-sectional analysis of the National Health and Nutrition
Examination Survey (NHANES) data from 1999 to 2006 showed that RDW
increases significantly as the degree of the urinary albumin ratio increases (Afonso
et al. 2011). The same study also showed that the risk of microalbuminuria increases
as RDW values increase, which is independent of the potential comorbidities such as
hypertension, diabetes, and obesity. It was suggested that common interactions
between inflammation, oxidative stress, endothelial dysfunction, and neurohumoral
overactivity may explain the association between RDW and microalbuminuria
(Afonso et al. 2011). However, in another study by Mathew, RDW was shown to
be unrelated with microalbuminuria among patients with diabetes mellitus and
hypertension (Mathew et al. 2014).
Relation between RDW and preeclampsia, which is a disorder associated with
proteinuria, is controversial. It was suggested that RDW level is correlated with both
the presence and the severity of preeclampsia; however, in another study, it was
found that there is no association between RDW and preeclampsia (Abdullahi
et al. 2014; Keskin et al. 2013).
So, there may be a relation between RDW and disorders associated with
microalbuminuria or proteinuria. But, there are very few studies and further studies
should be performed to display whether there is a direct correlation between RDW
and proteinuria or it is the common characteristics of the disorders associated with
increased RDW and microalbuminuria/proteinuria, such as inflammation, oxidative
stress, or endothelial dysfunction.
Potential Role of RDW in Primary Glomerular Diseases
There are insufficient data on the potential role of RDW in PGDs. Only one study
was carried out to investigate the importance of RDW in PGDs. In this study it was
hypothesized that higher RDW levels can represent resistance to the treatment of
PGDs. It was designed to show the predictive value of RDW, a simple, inexpensive
biomarker, for the treatment response in patients with PGDs. Then, a prospective
study including 176 patients with NS due to biopsy-proven PGDs (MCD, n = 42;
MG, n = 47; FSGS, n = 41; type 1 MPGN, n = 46) was performed (Turgutalp
et al. 2014). Patients were grouped according to their response to the treatment. All
subjects had been followed for 12 months. Group 1 was composed of 55 patients
17,8%
18
13,4% 14,8% 17,8%
16
14 11,9% 12,5%
12 Mean RDW value
RDW %
10 during new diagnose

8
6 Mean RDW value at the
4 12th months of
2 treatment
0
remission partially resistance
group remission group
group
Fig. 1 Mean RDW values of all the groups and their comparisons both before and at the end of the
treatment. While baseline mean RDW value was low in group 1 patients, it was higher in group
2 and group 3 subjects. RDW values when evaluated before treatment in all groups, the highest
mean RDW level was found in group 3 patients ( p < 0.05), the lowest mean RDW level was found
in group 1 patients ( p < 0.05). In group 1 and group 2, there was a significant decrease in mean
RDW levels after treatment comparing with baseline levels ( p < 0.05). In group 3 patients, there
was no decrease in RDW levels after treatment comparing with first admission levels ( p > 0.05)
(Data are from Turgutalp et al. (2014))
with complete remission whereas group 2 was composed of 53 patients with partial
remission and group 3 was composed of 68 patients who were resistant to therapy at
the end of treatment (12th month). Mean RDW values of the all groups and their
comparisons both before and at the end of the treatment are shown in Fig. 1.
According to the treatment response, mean RDW values of patients with different
types of PGDs are shown in Table 3. Statistically significant difference was not
detected between histological types of PGDs (Turgutalp et al. 2014). Additionally,
they reported that sensitivity (ability to determine of treatment sensitive patients) and
specificity (ability to determine of treatment resistance patients) of RDW was 98.2 %
and 81.8 %, respectively.
In the same study, most of the patients with complete remission had a baseline
RDW value 14 % (90 %), and most of the patients who were resistant to the
treatment had a baseline RDW value >15 % (86.1 %). Similarly, most of the patients
with partially remission had baseline RDW values between 14.1 % and 15 %
(78.7 %) (Fig. 2) (Turgutalp et al. 2014) (Table 4).
These results suggested that serum RDW level may be a useful predictive
biomarker for estimating the response to the therapy and may reflect increased
inflammatory response in NS. RDW level can be seen as a part of the complete
blood count which is a cheap test and therefore it may be a cost-effective novel
marker for evaluation of treatment response in NS due to PGDs. However, it should
Table 3 Mean RDW values (%) in patients with different types of PGDs according to response to
the therapy
Groups MCD MG FSGS MPGN p**
Remission (1) 11.82
0.21 11.80
0.20 12.15
0.19 12.14
0.15 NS
Partial remission (2) 12.3
1.3 12.4
1.3 12.6
1.4 12.7
1.5 NS
Resistant (3) 17.7
2.0 17.7
2.1 17.8
2.1 17.9
2.2 NS
p* <0.05 <0.05 <0.05 <0.05
Mean RDW value in patients with different types of PGDs according to response to the therapy.
Statistically significance was not detected between histological types (for all p < 0.05) (Data are
from Turgutalp et al. (2014))
RDW red cell distribution width, MCD minimal change disease, MGN membranous nephropathy,
FSGS focal segmental glomerulosclerosis, MPGN membranoproliferative glomerulonephritis
(Multiple comparisons: Dunn test was applied for variables which showed non-normal distribution)
p* Difference between group 1, 2, and -3 in same histopathological type of PGDs, p** difference
between histopathological types of PGDs in the same group
90.0%
86.1%
90.0% 78.7%
80.0%
70.0%
60.0%
50.0% RDW≤14%
40.0%
RDW 14.1-15%
30.0% 17.3%
11.3% RDW>15%
20.0% 4.0%
6.0% 4.0%
10.0% 2.6%
0.0%
Remission Partially Resistance
group Remission group
group
Fig. 2 Proportion of response to treatment according to initial RDW value. Most of the patients
with complete remission had a baseline RDW value 14 % (n = 45, 90 %) ( p < 0.001, Kendal Tau:
0.86), and most of the patients who were resistant to the treatment had a baseline RDW value
>15 % (n = 68, 86.1 %) ( p < 0.001, Kendal Tau: 0.87). Similarly, most of the patients with
partial remission had baseline RDW values between 14.1 % and 15 % (n = 37, 78.7 %) ( p < 0.001,
Kendal Tau: 0.85) (Data are from Turgutalp et al. 2014)
be considered that RDW value may also increase due to the other comorbid
conditions. Therefore it may be confusing whether the increase in RDW value is
associated with PGDs or comorbid conditions. Cutoff values for RDW may be
useful in differential diagnosis. Therefore new studies may be designed to assess
these cutoff values. Further research is also required to clarify the association
between the factors that have a role in the pathogenesis of different histopathological
types of PGDs and RDW (Turgutalp et al. 2014).
Table 4 Determinants of proteinuria levels in the entire study population as evaluated by

ANCOVA analysis. The ANCOVA results showed that RDW was a strong predictor of proteinuria
independent of age, albumin, CRP values. The coefficient of determination (R2) shows that RDW as
an independent variable predicts 65.3 % of the changes in proteinuria by regression analysis. Both
the coefficients of the regression equation (slope and intercept) are significant at p < 0.001 level.
The independent variable RDW is a significant predictor of the change in proteinuria
Source Mean square F value p
Age 167,404.965 0.056 >0.05
Albumin 2,460,952.409 0.826 >0.05
RDW 746,972,702.509 250.628 <0.001
hs-CRP 349,886.563 0.117 >0.05
eGFR 279,617.394 0.094 >0.05
RDW red cell distribution width, hs-CRP high sensitive C-reactive protein, eGFR estimated
glomerular filtration rate (Data are from Turgutalp et al. (2014))
Summary Points
• This chapter focuses on RDW which may be a novel biomarker predicting the
response to the therapy in patients with PGDs.
• RDW shows the variation in size of erythrocytes in the circulation and it is
obtained easily by automated blood cell counters.
• Studies have been focused to investigate potential clinical importance of RDW
which is directly associated with systemic inflammation in prediction of progno-
sis and clinical outcome of PGDs.
• RDW level can be seen as a part of the complete blood count.
• RDW is a cheap test and therefore it may be a cost-effective novel marker for
evaluation of treatment response in NS due to PGDs.
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Part VI
Resources
Recommended Resources on Biomarkers
in Kidney Disease 53
Rajkumar Rajendram, Vinood B. Patel, and Victor R. Preedy
Contents
Key Points . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1196
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1196
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1203
Abstract
Biomarkers of kidney disease are substances that identify damage to the renal
tract and may reflect renal function. They may be released from the kidney or
result from a specific response to damage to the renal tract or changes in renal
function.
Serum creatinine is currently the most widely used marker of kidney disease
and renal function in clinical practice. However as it is affected by several non-
renal factors it is unreliable and so better biomarkers are urgently required.
Several potentially relevant novel biomarkers of kidney disease have been
discovered through omic technologies such as genomics, and proteomics. These
R. Rajendram (*)
Division of Diabetes and Nutritional Sciences, Faculty of Life Sciences and Medicine, King’s
College London, London, UK
Department of Anaesthesia and Intensive Care, Stoke Mandeville Hospital, Aylesbury, UK
e-mail: rajkumarrajendram@hotmail.com; rajkumarrajendram@doctors.org.uk
V.B. Patel
Department of Biomedical Sciences, Faculty of Science & Technology, University of Westminster,
London, UK
e-mail: V.B.Patel@westminster.ac.uk
V.R. Preedy
Department of Nutrition and Dietetics, Division of Diabetes and Nutritional Sciences, Faculty of
e-mail: biomarkers2@publicationeditor.org.uk

1196 R. Rajendram et al.
novel biomarkers could be used to predict the risk of kidney disease, diagnose
renal disease after an acute event, suggest the likely outcome (prognosis) in the
absence of treatment, and predict the likely response to treatment. Keeping up-to-
date with the rapid pace of the developments in this field is difficult. When
attempting to do so it is difficult to know which of the myriad of available
resources are reliable. To assist our colleagues we have therefore written this
chapter which includes tables containing reliable, up-to-date resources. The
experts who assisted with the compilation of these tables of resources are
acknowledged.
Keywords
Biomarkers • Kidney Disease • Evidence • Resources • Books • Journals •
Regulatory bodies • Professional societies
Key Points
• Biomarkers are of significant value in modern nephrology.

• Biomarkers are used in screening for kidney disease.
• Biomarkers are used in risk stratification, diagnosis, prognostication, directing
initial therapy, monitoring response to treatment, and guiding the choice of further
treatments.
• This chapter lists the most up-to-date resources on the regulatory bodies, journals,
books, professional bodies, and websites that are relevant to an evidence-based
approach to the use of biomarkers of renal disease.
Introduction
As defined at the beginning of this millennium, biomarkers are objective measures

that indicate normal biological processes, pathogenic processes, or pharmacologic
responses to a health care intervention (Atkinson et al. 2001). Biomarkers have
significant scientific and clinical value in kidney diseases which are common causes
of morbidity and mortality (Brown et al. 2015; Findlay et al. 2015; Panocchia
et al. 2016).
A renal disease biomarker is a substance that indicates the presence of kidney
disease. It could be released from the kidney or result from a specific response to
kidney disease. Ideally, such a biomarker could be assayed in noninvasively col-
lected fluids (e.g., urine, blood or serum).
Serum creatinine is currently the most commonly used marker of renal function in
routine clinical practice. It is used to estimate the glomerular filtration rate (GFR),
but is unfortunately an unreliable indicator of kidney function, for many reasons. For
53 Recommended Resources on Biomarkers in Kidney Disease 1197
Table 1 Regulatory bodies and organizations. This table lists the regulatory bodies and orga-
nizations involved with various aspects of biomarkers
Biomarkers Consortium biomarkersconsortium.org
Biomarker, Imaging and Quality of Life cancer.gov/aboutnci/organization/ccct/funding/
Studies Funding Program, National BIQSFP
Cancer Institute, USA.
Biomarker Qualification Program US fda.gov/Drugs/DevelopmentApprovalProcess/Dru
Food and drug administration gDevelopmentToolsQualificationProgram/u
cm284076.htm
Canadian Coordinating Office for Health www.ccohta.ca
Technology Assessment
Centers for Disease Control and www.cdc.gov/globalhealth/countries/egypt
Prevention (CDC)
CKD Biomarkers Consortium www.ckdbiomarkersconsortium.org/
European Medicines Agency ema.europa.eu/ema/index.jsp?curl=pages/special_
topics/general/general_content_000349.jsp
George Institute for Global Health www.georgeinstitute.org/publications/biomarkers-
in-kidney-fibrosis-are-they-useful
International Federation of Clinical www.ifcc.org
Chemistry and laboratory Medicine
(IFCC)
KIDNEY, Swiss National Centre of www.nccr-kidney.ch/index.php?nav=43GEN
Competence in Research Exclusives
Medicines and Healthcare products mhra.gov.uk
Regulatory Agency (MHRA)
National Center for Environmental www.epa.gov/ncer/rfa/2006/2006_star_pbk_mode
Research ling.html
National Institutes of Health www.nlm.nih.gov/medlineplus/ency/anatomyvide
os/000023.htm
National Research Council Canada www.nrc-cnrc.gc.ca/eng/transparency/access_info
rmation/infosource.html
U.S. Food and Drug Administration www.fda.gov/Drugs/DevelopmentApprovalProce
ss/DrugDevelopmentToolsQualificationProgram/u
cm437988.htm
example, serum creatinine is affected by several nonrenal factors such as age,

gender, muscle mass, and hydration.
In acute kidney injury (AKI), serum creatinine does not reflect the actual decrease
in the GFR and takes several hours or days to reach a new steady state. In fact, serum
creatinine may not increase until over half of the patient’s renal function is lost
because there is a significant renal reserve.
As a result of the limitations of serum creatinine, several possible biomarkers of
kidney disease have been studied over the past decade. These include urinary and
serum proteins, molecules, and, most recently, microRNAs. The driver for this line
Table 2 Professional societies. This table lists the professional societies involved with biomarkers
and/or kidney disease
American Association for Clinical www.aacc.org
Chemistry
American Society of Nephrology (ASN) www.asn-online.org
Association for Clinical Biochemistry and www.acb.org.uk
Laboratory Medicine
Australasian Association of Clinical www.linkedin.com/company/australasian-
Biochemists association-of-clinical-biochemists
Australian and New Zealand Society of www.nephrology.edu.au
Nephrology (ANZSN)
Biomarkers Consortium www.biomarkersconsortium.org
Carriel Institute for Medical Research www.carriel.org
Deutsche Gesellschaft f€ur Nephrologie www.dgfn.eu
European Renal Association – European www.era-edta.org
Dialysis and Transplant Association
European Society of Endocrinology (ESE) www.ese-hormones.org
High Blood Pressure Research Council of www.hbprca.com.au
Australia
Hypertension, Dialysis and Clinical www.hdan.com
Nephrology
Institute of Biomedical Science www.ibms.org
International Society of Hypertension ish-world.com
International Society of Nephrology www.theisn.org
Japanese Society for Biomedical Mass www.jsbms.jp/english
Spectrometry
Mass Spectrometry Society of Japan www.mssj.jp/en/index.html
National Kidney Foundation www.kidney.org
Sociedad Portugesa de Nefrolagia www.spnefro.pt
Société Française de Biologie Clinique www.sfbc.asso.fr
(SFBC)
Société française d’endocrinologie (SFE) www.sfendocrino.org
Society of Medical Biochemists of Serbia www.dmbj.org.rs
Society of Nephrologists of Serbia www.udruzenjenefrologa.com
Viapath – Clinical Biochemistry www.viapath.co.uk/departments-and-laboratories/
Laboratory at King’s College Hospital clinical-biochemistry-laboratory-at-kings
of investigation is that prompt specific intervention may prevent permanent renal

damage if AKI is detected at any early stage.
These novel biomarkers could be used to predict the risk of kidney disease,
diagnose renal disease after an acute event, suggest the likely outcome (prognosis)
in the absence of treatment, and predict the likely response to treatment. Thus, there
are four main potential uses for biomarkers in renal disease:
Table 3 Journals publishing on kidney disease. This table lists the top 25 journals publishing
original research and review articles related to kidney disease. The list was generated from SCOPUS
(www.scopus.com) using general descriptors. The journals are listed in descending order of the total
number of articles published in the past 5 years. Of course, different indexing terms or different
databases will produce different lists so this is a general guide only. For example, journals associated
with biomarker discovery will produce a different list (see Table 4)
Plos One
Nephrology Dialysis Transplantation
Transplantation Proceedings
Kidney International
American Journal of Kidney Diseases
Journal of Urology
Transplantation
American Journal of Physiology Renal Physiology
Pediatric Nephrology
Urology
American Journal of Transplantation
Renal Failure
BMJ Case Reports
Nature Reviews Nephrology
International Urology and Nephrology
Clinical and Experimental Nephrology
Saudi Journal of Kidney Diseases and Transplantation an Official Publication of the Saudi Center
for Organ Transplantation Saudi Arabia
Nephrology
Journal of Endourology
BMC Nephrology
Clinical Nephrology
BJU International
New England Journal of Medicine
American Journal of Nephrology
International Journal of Cardiology
Risk stratification
Diagnosis
Prognostication
Monitoring response to treatment
Several potentially relevant renal biomarkers have been discovered through omic
technologies such as genomics and proteomics. The use of emerging high-
throughput technologies to integrate biomarkers into clinical practice will allow
“personalization” of disease management in the future.
Table 4 Journals publishing on kidney disease and biomarkers. This table lists the top
25 journals publishing original research and review articles related to kidney disease and bio-
markers. The list was generated from SCOPUS (www.scopus.com) using general descriptors. The
journals are listed in descending order of the total number of articles published in the past 5 years.
Of course, different indexing terms or different databases will produce different lists so this is a
general guide only
Plos One
Nephrology Dialysis Transplantation
Kidney International
Transplantation Proceedings
Pediatric Nephrology
Renal Failure
Transplantation
American Journal of Physiology Renal Physiology
American Journal of Kidney Diseases
Nephrology
BMC Nephrology
Journal of Nephrology
Saudi Journal of Kidney Diseases and Transplantation an Official Publication of the Saudi Center
for Organ Transplantation Saudi Arabia
Clinical Nephrology
Clinical and Experimental Nephrology
International Urology and Nephrology
Journal of Urology
American Journal of Nephrology
Tumor Biology
Nature Reviews Nephrology
Critical Care
Nephron Clinical Practice
American Journal of Transplantation
Clinica Chimica Acta
International Journal of Cardiology
Biomarker discovery in relation to kidney disease is also important for several

reasons other than the patient’s physical domains. Kidney diseases impact on
quality-of-life measures (Brown et al. 2015; Griva et al. 2016; Moreira
et al. 2015). Furthermore, one needs to consider the global impact of kidney diseases
(Jha et al. 2013; Mills et al. 2015). Kidney diseases are estimated to have a global
prevalence of 8–16 % and are driven by risk factors such as obesity and hyperten-
sion, though other less well-defined factors may also contribute (Weaver et al. 2015).
Kidney diseases are ranked within the top 20 causes of all global deaths. There are
about half a billion people with chronic kidney disease alone (Mills et al. 2015)
though less precise figures have been obtained for acute kidney disease. The Lancet
highlighted the importance of kidney disease as being on par with diabetes in terms
Table 5 Relevant books on biomarkers. This table lists books on biomarkers

Aptamers in Bioanalysis. Mascini M. Wiley-Interscience, 2009, USA.
Aptamer Handbook: Functional Oligonucleotides and Their Applications. Klussmann S (editor).
Wiley-VCH, 2006, Germany.
Biomarkers in Bone Disease. Preedy VR, Patel VB. Springer, 2017, Netherlands.
Biomarkers in Cardiovascular Diseases. Tousoulis D, Stefanadis C. CRC Press, 2013, UK.
Biomarkers for CKD: Glomerular Filtration and Progression of Kidney Disease (CD). Dalton
R. American Association of Clinical Chemists, 2012, USA.
Biomarkers in Kidney Disease. Edelstein CL, Elsevier, 2010, USA.
Biomarkers in Renal Disease. Rosner MH, Okusa M. Nova Publisher, 2008, USA.
Handbook of Biomarkers. Kewal KJ. Lippincott, 2010, USA.
Biomarker Guide. Peters KE, Walters CC, Moldowan JM. Cambridge University Press, 2010,
USA.
Role of Novel Biomarkers in Chronic Kidney Disease: Renalase. Desir GV. Springer, 2010, Italy.
Table 6 Relevant books on kidney disease. This table lists books on kidney disease
Brenner and Rector’s the Kidney. Brenner BM. Saunders, 2008, Philadelphia.
Comprehensive Clinical Nephrology. Johnson RJ, Feehally J, Flaege J, Elsevier, 2014, USA
Diagnostic Pathology: Kidney Diseases, 2nd Edition. Colvin R, Chang A. Elsevier, 2015, USA
Kidney Disease and Laboratory Medicine. Lamb E, Delaney M. ACB Venture Publications 2009,
UK.
Pathophysiology. McCance KL, Brashers VL, Rote NS. Mosby Elsevier, 2010, USA.
Uremic Toxins. Niwa T (Editor). John Wiley & Sons, 2012, USA
Urinalysis and Body Fluids, 6th Edition. Strasinger SK, Di Lorenzo MS. Elsevier, 2011, USA
of burden (Anon 2013). Other areas of the causes and impact of kidney-related
disease are highlighted in this book.
It is now difficult even for experienced scientists and clinicians to remain up-to-
date. For those new to the field, it is difficult to know which of the myriad of
available sources are reliable. To assist colleagues who are interested in understand-
ing more about biomarkers of kidney disease, we have therefore produced tables
containing reliable, up-to-date resources in this chapter. The experts who assisted
with the compilation of these tables of resources are acknowledged below.
Examples of the definitions, measurement, and applications of biomarkers can be
found in this book and also via the recommended resources in the tables below.
Tables 1, 2, 3, 4, 5, 6, 7, and 8 list the most up-to-date information on the
regulatory bodies (Table 1), professional bodies (Table 2), journals on cardiovascular
disease (Table 3), journals on biomarkers (Table 4), books on biomarkers (Table 5),
books on cardiovascular disease (Table 6), emerging techniques and platforms
(Table 7), and websites (Table 8) that are relevant to an evidence-based use of
biomarkers in cardiovascular disease.
Table 7 Sources and resources for emerging techniques and platforms. This table lists some
emerging sources, resource platforms in biomarker discovery, and application
Biobanking and Biomolecular bbmri.eu
Resources Research Infrastructure
Clinical Center of Vojvodina, Novi www.kcv.rs/rs
Sad, Serbia
Eurolab www.eurolab.org
GEN Exclusives www.genengnews.com/gen-articles/biomarker-discover
y-methods-evolving/4097/
Human genome variation society www.hgvs.org
Fondation PremUP www.premup.org
Intech, open science, open minds www.intechopen.com/books/latest-research-into-qualit
y-control/quality-control-of-biomarkers-from-the-samp
les-to-data-interpretation
Laboratory for emerging Opto.brown.edu
technologies
MediBEACON www.medibeacon.com/products/nephrology/renal-func
tion-system/
Medical Faculty, Novi Sad, Serbia www.mf.uns.ac.rs
NanoINK, Nano biodiscovery www.marketwired.com/press-release/nanoink-platform-
proven-reproducibly-detect-protein-biomarkers-from-
dried-blood-spot-1562611.htm
Quintiles, American Heart & www.quintiles.com/library/white-papers/biomarkers-re
Quintiles cent-advances-in-their-application-to-the-treatment-of-
hematologic-malignancies
University of Zurich Progenetix progenetix.org/cgi-bin/pgHome.cgi
database
Table 8 Relevant internet resources. This table lists some Internet resources on biomarkers and
kidney disease
Acute Dialysis Quality Initiative www.adqi.org
Biomarkers Test (BMT) www.biomarkers.it
Biomed Central (BMC) biomarkerres.org
Biomarkers
Broad Institute www.broadinstitute.org/scientific-community/science/platf
orms/proteomics/biomarkers
Insightomics – Biomarkers www.insightomics.com/biomarker-discovery/?gclid=CJvJ
discovery 9–Sp8gCFYUIwwodnNQIwQ
Kidney Health Australia www.kidney.org.au
Medscape www.medscape.com
Mercer’s institute for successful www.misa.ie/research/clinical/biomarkapd
aging (MISA)
Military Medical Academy, www.vma.mod.gov.rs
Belgrade, Serbia
News medical www.news-medical.net/health/What-is-a-Biomarker.aspx
Acknowledgment We would like to thank the following authors for contributing to the develop-
ment of this resource.
Anderson K, Avramovski P, Bashford G, Čabarkapa V, Carnero A, Clarke S, Çuhadar S, De
Rossi A, Diakos C, Gad M, Guedes-Marques M, Jabor A, Kobayashi T, Mangoni A, Nistal JF, Noh
D-Y, Schraml PH, Simeoni M, Stahl RAK, Staikou C, Takenaka S, Tomai F, Wonshik H, Yi L
References
Anon. The global issue of kidney disease. Lancet. 2013;382(9887):101.
Atkinson AJ, Colburn WA, DeGruttola VG, DeMets DL, Downing GJ, Hoth DF, Oates JA, Peck
CC, Schooley RT, Spilker BA, Woodcock J, Zeger SL, NCI-FDA Biomarkers Definitions
Working Group. Biomarkers and surrogate endpoints: preferred definitions and conceptual
framework. Clin Pharm Ther. 2001;69:89–95.
Brown MA, Collett GK, Josland EA, Foote C, Li Q, Brennan FP. CKD in elderly patients managed
without dialysis: survival, symptoms, and quality of life. Clin J Am Soc Nephrol. 2015;10
(2):260–8.
Findlay M, Donaldson K, Robertson S, Almond A, Flynn R, Isles C. Chronic kidney disease rather
than illness severity predicts medium- to long-term mortality and renal outcome after acute
kidney injury. Nephrol Dial Transplant. 2015;30(4):594–8.
Griva K, Goh CS, Kang WCA, Yu ZL, Chan MC, Wu SY, Krishnasamy T, Foo M. Quality of life
and emotional distress in patients and burden in caregivers: a comparison between assisted
peritoneal dialysis and self-care peritoneal dialysis. Qual Life Res. 2016;25(2):373–84.
Jha V, Garcia-Garcia G, Iseki K, Li Z, Naicker S, Plattner B, Saran R, Wang AYM, Yang C-W.
Chronic kidney disease: global dimension and perspectives. Lancet. 2013;382(9888):260–72.
Mills KT, Xu Y, Zhang W, Bundy JD, Chen C-S, Kelly TN, Chen J, He J. A systematic analysis of
worldwide population-based data on the global burden of chronic kidney disease in 2010.
Kidney Int. 2015;88(5):950–7.
Moreira JM, Bouissou Morais Soares CM, Teixeira AL, Simoes e Silva AC, Kummer AM. Anxiety,
depression, resilience and quality of life in children and adolescents with pre-dialysis chronic
kidney disease. Pediatr Nephrol. 2015;30(12):2153–62.
Panocchia N, Tazza L, Di SE, Liberatori M, Vulpio C, Giungi S, Lucani G, Antocicco M, Bossola
M. Mortality in hospitalized chronic kidney disease patients starting unplanned urgent
haemodialysis. Nephrology. 2016;21(1):62–7.
Weaver VM, Fadrowski JJ, Jaar BG. Global dimensions of chronic kidney disease of unknown
etiology (CKDu): a modern era environmental and/or occupational nephropathy? BMC
Nephrol. 2015;16(1):1–8.
Index
A Aminoglycosides, 696–697, 701, 705,

Acoustic radiation force imaging (ARFI), 708, 710, 712
1057–1058, 1063, 1064, 1066, 1069, Angiotensin II, 304–308, 311
1070 Angiotensinogen (AGT), 304, 305, 309
Acute kidney injury (AKI), 87, 91–101, Anoikis, 998
210, 693, 694 Antibody arrays, nephritis. See Nephritis,
cystatin C, 31 antibody arrays
in cystic fibrosis, causes of, 696–700 Antineutrophil cytoplasmic antibodies-
definition, 29 associated glomerulonephritis
fibroblast growth factor-2, 34 (ANCA-GN), 337, 639–640
incidence of, 29 Antiphospholipid antibodies (aPL), 815, 825
interleukin-18, 32–33 Apelin, 540, 542, 544, 547–553
kidney injury molecule-1, 33 APJ signalling, 543
neutrophil gelatinase-associated lipocalin, Apoptosis signal regulating kinase-1 (ASK1),
32 1002
NGAL and, 210–213 A proliferation-inducing ligand (APRIL)
urinary epidermal growth factor, 34 animal models for, 564
urine metabolomic profiling, 34–35 BCMA, 564
Acute kidney injury (AKI), CKD patients and cellular receptors, 563
cause of, 164 and pathology, 566
peri-operative AKI, 158–159 plasma cells of, 562
post-operative AKI, 155, 156, 158 serum, high levels of, 565
renal function, recovery of, 143–144 in systemic lupus erythematosus patients,
SORO-ESRD, 145–146 566
Acute rejection, 6–10, 12–19, 21 TACI, 564
Adhesions, 915–916, 920, 922 Aquaretic, 543, 551
ADPKD. See Autosomal dominant polycystic Assay, creatinine. See Creatinine assays
kidney disease (ADPKD) Autosomal dominant polycystic kidney disease
Albuminuria (ADPKD), 543, 547, 549
assessment of, 434–435
cardiovascular disease marker, 439
definition, 431 B
and hypertension, 439 Basal cell carcinomas (BCC), 1001
mechanisms of, 432 B-cell, 562–564, 569–571
prevalence of, 431 Beta-2 microglobulin (β2m)
renal risk, 439–440 amniotic fluid, 505
screening for chronic kidney disease, clinical usefulness, 497–498
436–439 fetal blood, 505–507
target for treatment, 440 foetal kidney and urinary tract, 499–505

Methods, Discoveries and Applications, DOI 10.1007/978-94-007-7699-9
1206 Index
Beta-2 microglobulin (β2m) (cont.) roles of, 565–566

foetal urine, 507–509 in systemic lupus erythematosus patients, 566
release, excretion and catabolism, 494–496
sampling and assay of, 496–497
structure, synthesis and function, 494 C
Biomarker(s), 56, 58, 61–62, 64–65, 76, 78–79, Cardiopulmonary bypass (CPB), 88, 91–92,
210, 213, 222, 223, 581–584, 587, 590, 94, 98
594, 915, 922, 926, 1177–1178, 1182, Cardiotrophin-like cytokine, 1 (CLC-1), 628
1186 Cardiovascular diseases (CVD), 186, 187,
AKI (see Acute kidney injury (AKI)) 189, 192, 349, 354, 1106–1107
APRIL (see A proliferation-inducing ligand Cardiovascular mortality, 435
(APRIL)) arterial stiffness and, 1091–1097
allograft rejection, 7–19 Cardiovascular risk, 1132
blood, 15–19 Cell cycle
BLyS (see B lymphocyte stimulator (BLyS)) vs. AKI, 981
urine, 7–15 applications, 984–986
books on, 1201 arrest urinary biomarkers, 981–983
internet resources on, 1202 clinical application, 985
journals, 1200 in renal tubular cells, 980–981
microRNA (see Micro ribonucleic acid Chemical markers, 179–194
(miRNA/miR)) albuminuria, 186–189
nephritis (see Nephritis) bilirubin, 182
professional societies, 1198 blood, 180–181
regulatory bodies and organisations, 1197 electrolytes, 192–193
of renal disease, chemokines glucose, 184–186
see Chemokines ketones, 183
sources, 1202 nitrites and leukocyte esterase, 183–184
suPAR (see Soluble urokinase receptor pH, 181–182
(suPAR)) protein, 189–192
of tolerance, 19 uric acid, 192
uses for, 1198 urobilinogen, 182
Blood pressure, 303, 305–307, 310–311 Chemokines
Blood toxicity, 580, 582–583 CCL2/MCP-1, 235
Blood urea nitrogen, 656–659 CCL5/RANTES, 237
BLyS. See B lymphocyte stimulator (BLyS) CXCL8/IL-8, 234–235
BOLD MRI, 89–91, 100 in glomerular diseases, 238
Books leukocyte trafficking, 233
on biomarkers, 1201 mRNA, 235
on kidney disease, 1201 nephrotic syndrome, 234
B lymphocyte stimulator (BLyS) tubular epithelial cells, 233
BCMA, 563 ureteropelvic junction obstruction, 240–242
blockage of, 565 vesicoureteral reflux, 242–243
and cellular receptors, 563 ChIP-seq
donor-specific antibody, 571 genome-wide analysis, 960–962
factors, 569 methods, 964–965
features of, 563 in vivo analysis, 962–964
immunoregulation of, 570 Chromosome conformation capture assays, 971
innate immune cells and consumption of, Chronic allograft damage index (CADI)
568 advantages of, 684
monitoring of, 570 clinical decision-making, 683
post-transplantation, 566–570 clinical trials, 676–679
pre-transplantation, 566 development of, 675
production of, 570 donor biopsies, 680–681
Index 1207
individual histopathological lesions, 680 reference intervals, 1150

limitations of, 683 urinary compounds, ratios of, 1151
microarrays, 679 Creatinine assays
pediatric kidney transplantation, 682 clinical application, 295–296
progression of, 681 creatinine clearance, 275–277
rat model, 679 cystatin C, 289–295
reproducibility of, 682–683 GFR (see Glomerular filtration rate (GFR))
Chronic allograft injury, 611–612 IDMS traceable assays, 281–289
Chronic dialysis, 1085, 1097 urinary creatinine measurements, 277–281
Chronic kidney disease (CKD), 87–90, Creatinine ratio, 377
210, 214–215, 236, 238, 240, 243, Cystatin C (Cys C), 447, 1158
311, 320, 400, 580–581, 584–586, advantages of, 452–453
588–590, 692, 694, 712, 1106, 1114, amyloid angiopathy, 457
1124, 1167 analytical remarks, 1153
AKI (see Acute kidney injury (AKI), CKD assay interferent factors, 451
patients) cardiovascular mortality and morbidity, 1154
carotid-intima media thickness, 408 and children, 451
CKD-ESRD progression, 153–155 and cirrhosis, 456
clinical outcomes in, 411 detection method, 450
CRP concentrations in, 409 disadvantages of, 453
left ventricular hypertrophy, 408 factors, 452
LORFFAB, impact of, 155–156 and GFR, 453–456
metabolomics (see Metabolomics, CKD) kininogens, 449
NGAL and, 214–221 and malignancy, 456–457
prediction and prognostication, 163 measurement and interpretation, 1165
progression, 543, 549 in nephrology, 1153–1154
proteinuric biomarkers (see Proteinuric pathophysiology, 1154
patterns) and pregnancy, 451–452
sickle cell nephropathy, 162–163 reference intervals, 1154
stage III, 152 reference range, 451
stage IV, 152 stability of, 451
stage V, 151–152 stefins, 448
Chronic Kidney Disease Epidemiology Cysteine proteases, 447, 449, 456
Collaboration (CKD-EPI) equation, Cystic fibrosis (CF), 692
1148, 1160, 1171 acute kidney injury, causes of, 696–700
Chronic renal failure (CRF), 251–252, 254, 262 biomarkers, 712–713
Circulating miRNA in body fluids, 900, 903 kidney injury in, 694–695
Clear renal cell carcinomas (CRCC), markers of kidney injury, 700–712
996, 1001–1002, 1004–1008 Cytokine receptor-like factor, 1 (CRLF-1), 628
Clearance, 273–275, 296
Clinical biomarker, 1095
Complement, 818, 824, 826 D
Conclusions, 199 Diabetes, 754, 756, 760
Congenital anomalies of the kidney and urinary Diabetic kidney disease (DKD), 522–524
tract (CAKUT), 239, 240, 243 Diabetic nephropathy (DN), 349, 357,
Copeptin, 540–541, 543–553 361–362, 429, 437, 641
C-reactive protein (CRP), 640 Gas6, 612–614
Creatinine, 452–453 microRNAs see Micro ribonucleic acid
analytical remarks, 1149 (miRNA/miR)
clearance of creatinine, 1157 Diagnosis, AKI. See Acute kidney injury (AKI)
interferences, 1149–1150 Diagnostic biomarkers, 724–729, 787–795
output of, 1149–1152 miR-210, 900–906
pathophysiology, 1149 Dialysis, 409, 410, 1124, 1129, 1132–1133
1208 Index
Differential diagnosis, 1040 Epigenetics, 959–960, 962–963, 972

Discovery, 56, 58, 61–62, 64 Epithelial-to-mesenchymal transition (EMT),
Disease proteomics. See Proteomics 938–940, 1001, 1003, 1006–1007
Donor biopsy, 680–681 Erythrocyte glutathione transferase (e-GST),
Doppler, 1083, 1097 582
pulse wave velocity measurement, 1084–1085 Estimated GFR (eGFR). See Glomerular
filtration rate (GFR)
Evidence, 1201
E Exosomes, 754, 760–762, 765
Early diagnosis, 880–883 isolation from urine, 354–356
Effluent biomarker, 56–58, 60–61, 64, 915, research in kidney disease, 352–358
923, 924
Elastography, 1054
ARFI, 1057–1058 F
elasticity values, renal characteristics on, Familial adenomatous polyposis (FAP), 1000
1060–1062 Fibrosis, 915, 916, 919, 922
end stage renal disease, 1066–1069 Flow cytometry, as biomarker for renal
Fibroscan-transient elastography, 1057 diseases, 334–341
metabolic diseases, 1069–1070 Focal segmental glomerulosclerosis (FSGS),
normal renal elasticity values and 630, 784–787, 789–794, 796
reproducibility, 1062–1063 circulating permeability factors in, 626–628
quasi-static elastography, 1055–1056 CLC-1, 628
renal fibrosis, measurement of, 1063–1066 post-transplant recurrence of, 635
renal mass, evaluation of, 1066 primary nephrotic syndrome, 638
shear wave dispersion ultrasound ROC analysis, 632, 634
vibrometry, 1059–1060 in vitro experiments, 638
supersonic shear wave imaging, 1058–1059 Free-fatty-acid (FFA), 1002
urinary tract abnormality, renal damage,
1070–1071
viscoelasticity, 1060 G
End stage renal disease (ESRD), 145–146, Genomic(s), 16–18, 58, 61, 63–64
153–155 Glomerular diseases, 238–239
Endothelin converting enzyme, 383–384 Glomerular filtration rate (GFR), 373, 388,
Endothelin-1 (ET-1), 380–381 452–457
clearance, 386 acute kidney injury and intensive care,
clinical significance, 388–389 1169–1171
converting enzyme, 383–384 and age, 1165–1166
detection method, 386 biological and analytical variability of, 1155
physiological functions, 387 and body surface area, 1166–1167
receptors, 385 in children, 1163–1165
stimulator and inhibitor factors, 383 CKD-EPI equations, 1160
synthesis pathway, 384 Cockcroft-Gault equation, 1157–1158
synthesis, 382–383 creatinine, 1149–1155
Endothelin receptor, 385 creatinine clearance, 275
Endothelium, 400, 402, 407–408 CysC, 290, 1153–1155
End-stage kidney disease (ESKD), 1124 development of, 277
End-stage renal disease (ESRD), 400 diabetes mellitus, 1168–1171
clinical outcomes in, 411 eGFR, 275–277
peritoneal dialysis, 409 estimation of, 284
progression to, 410 history, 1147–1148
Enzymatic assay, 277, 280, 287 Lund-Malmö equations, 1160
Enzyme-linked immunosorbent assay (ELISA), maturational GFR changes, 275
629, 635, 639 MDRD formula, 1159
Index 1209
physiology of, 1148–1149 and vascular disease in patients with chronic

renal clearance, 1155, 1157 kidney disease, 482–484
urea, 1154–1155 Human chronic renal failure, Gas6, 614
Glomerulonephritis (GN), 123–127, 522 Human foetus
ANCA-associated vasculitis, 528–529 amniotic fluid, 505
idiopathic membranous nephropathy, 524–526 anatomical development, 499–502
IgA nephropathy, 528 fetal non renal causes, 503
membrano-proliferative glomerulonephritis, foetal blood, 505–507
529 foetal urine, 507–509
patient cohorts, 525 nephropathies, 504
primary focal segmental glomerulosclerosis, renal function development, 502
526–527 uropathies, 504–505
Graft survival, 877, 879, 888 Human hepatocellular carcinomas (HCC), 1000
Growth arrest-specific, 6 (Gas6), 603 Human inflammatory renal diseases, Gas6, 614
acute and chronic renal nephritis induction, Hyperhomocysteinemia
610–611 biological effects and mechanisms, 474–476
cellular actions, 605–606 causes, 473–474
chronic allograft injury, 611–612 Hypertension, 439
diabetic nephropathy, 612–614 Hypoxia-inducible factors (HIFs), 900, 905
human chronic renal failure, 613
human inflammatory renal diseases, 613
IgA nephropathy, 614–615 I
mesangial-cell proliferation stimulation, IgA nephropathy (IgAN), 334, 641–642
609–610 biomarkers response to therapy
receptor interrelations, 607 (see Response to treatment, IgAN)
renal cell carcinoma, 616 exosomes and vesicles, 726
renal damage, 609–616 fractional excretion of IgG, 731
renin-angiotensin-aldosterone system, effect Gas6, 614–615
on, 615–616 Gd-IgA1-containing immune complexes,
structure, 603–605 728–729
TAM system in vasculature, 607–609 histologic evaluations of, 725
inflammatory cytokines, 733–734
Interleukin-6, 725, 736
H laminin G-like 3, 726
Healthy subject, 434 metabolomics studies, 729
Heart failure (HF), 1107–1108, 1112–1117 microRNAs, 727
Hematuria, 176, 179–180, 187, 195 oxidative stress, 734–735
Hemodialysis, 251–252, 255–256, podocalyxin, 736
259, 261–263 proteomic analysis of urine, 737–738
Homocysteine (Hcy) serum BAFF, 729
determination, 471–473 soluble CD89, 731–732
diabetes mellitus and diabetic nephropathy, soluble vascular cell adhesion molecule
480–481 1, 737
in diagnostics and monitoring of renal sTfR/CD71, 725
dysfunction, 484–485 TGF-β, 736–737
metabolism, 468–469 thioredoxin, 737
nephrotic syndrome, 481 urinary angiotensinogen, 732
potential applications, 485–486 urinary MBL level, 733
regulation, 469–471 urinary proteome, 727–728
renal function, 476–479 urine kidney injury molecule-1, 730–731
structure and origin, 467–468 urine metabolomics, 727
terminal renal failure and in transplanted urine secretory IgA, 737
patients, 476–479 uromodulin, 724–725
1210 Index
IGFBP7, 982 APRIL (see A proliferation-inducing ligand

Immunoglobulin A nephropathy (IgAN). (APRIL))
See IgA nephropathy BLyS (see B lymphocyte stimulator (BLyS))
Immunohistochemistry markers, 821–824 books on, 1201
Immunological rejection, 877, 888, 889 chronic, 349, 561
Immunosuppressive drugs, 1180, 1182 diabetic, 349–351, 358–362
Infants, creatinine assays. See Creatinine assays diagnosis and prognosis, 564–565
Inflammation, 815, 818, 823, 824 exosomes, research in, 352–358
Integrin linked kinase (ILK) internet resources on, 1202
Akt-FKHR pathway, 1000 journals, 1199–1200
β-catenin activation, 998 mechanisms, 561
BCC, 1001 prevalence, 1200
CRCC (see Clear renal cell carcinomas professional societies, 1198
(CRCC)) Kidney, foetal beta-2 microglobulin. See Beta-2
Cyr61, 999 microglobulin (β2m)
cytoplasmic expression, 999 Kidney physiology, 114, 117–120
FAP, 1000 Kidney/renal fibrosis
FFAs, 1002 assessment, 941
high-grade prostate carcinomas, 1000 computer based morphometric study,
human malignancies, 996 946–947
laryngeal squamous cell carcinomas, 1000 measurement and methodologies, 948
liver oncogenesis and hepatic cirrhosis, 999 mechanisms of, 937–941
lymph node metastasis, 1001 molecular methods, 947
non-small cell lung carcinomas, 1000 patterns in biopsies, 941
oncogenic transformation, 996 proteomic approaches, mass spectrometry,
ovarian tumor cell, 1000 947–948
pancreatic carcinomas, 1000 staining techniques and light microscopy,
pleckstrin homology (PH)-like domain, 995 942–946
PTHrP, 1002 Kidney transplantation, 6, 127–130, 566, 568,
tissue microarray, 1001 570, 591–592
and VHL inactivation, 1006, 1007 biomarkers, 19
Intensive care, 101 CADI (see Chronic allograft damage index
Ischemia-reperfusion injury, 852, 862–865, 867 (CADI))
Isotope dilution mass spectrometry (IDMS), MDA (see Malondialdehyde (MDA))
276, 296
enzymatic quantification methods, 286
impact of, 281–286 L
interchangeability of, 280 Late onset renal failure from angiotensin
Scr standardization program, 287 blockade (LORFFAB), 155–156
Lipid peroxidation, 855–860
Lupus nephritis (LN), 334–336, 640–641,
J 814–820
Jaffe method, 278, 284, 286
Journals
biomarkers, 1200 M
kidney disease, 1199, 1200 Maintenance hemodialysis, 586
Malondialdehyde (MDA)
in biological samples, 858
K high-performance liquid chromatography,
Kidney biomarkers, 539, 547 859–860
Kidney diseases, 175, 186, 188, 199, 455, 650, lipid peroxidation, 852, 855–861, 864, 867
651, 657 pathological conditions, 859
acute kidney disease, 561 plasma and intragraft levels of, 865
Index 1211
pre-transplant and post-transplant progression to ESRD, 410

circulating levels, 865, 866 synthesis, transport and metabolism of,
thiobarbituric acid reactive substances 400–402
assay, 858 Micro ribonucleic acid (miRNA/miR)
urinary MDA, 860 in biofluids, 754
Mammalian target of rapamycin (mTOR), 1019 diagnostic and prognostic profiles, 762–765
Masson’s trichrome stain, 942, 944 exosomes, 760–762
Mass spectrometry (MS), 71–76, 78, 79 and genomic material, 757
Matrix metalloproteinase-2 (MMP-2) let-7 family, 757
arterial stiffness, 921 lung cancer patient, 755
cellular and peritoneal tissue expression, miR-9, 756
918–920 miR-15a, 768
dialysis-related suppression of, 920 miR-17, 770
estimated glomerular filtration rate, 921 miR-21, 769
peritoneal effluent, 922–926 miR-29, 768
serum and plasma levels of, 921 miR-34a, 757, 769
structure and biological functions of, miR-126, 756
916–918 miR-145, 773
Maturational changes, 275, 278, 282, 296 miR-155, 773
Membranous nephropathy miR-199a, 757
clinical course and treatment of, 1037–1038 miR-375, 756
pathogenesis of, 1036 miR-638, 771
PLA2R antibodies (see M-type plasma and cellular fraction, 755
phospholipase A2 receptor (PLA2R) in PKD, 772
antibodies) in renal disease, 754–755
THSD7A, 1041–1042 urinary miRNA, 758–759
transplacental passage, 1036 Microproteins, 506
Metabolic acidosis, 47–50 MicroRNAs (miRNAs). See also Micro
Metabolite biomarkers, 794–795 ribonucleic acid (miRNA/miR)
Metabolomics, 58, 61, 63–64, 727, 729, 743 animal models, 132–133
Metabolomics, CKD, 72 antagonists, 131–132
mass spectrometry, 73–76 biogenesis of, 112–114
non-target analysis, 76–78 in blood and urine, 130–131
target analysis, 78–79 blood flow and oxygen supply, 117–119
Methylated arginines C3-glomerulopathy, 125
all-cause mortality, 411 crescentic glomerulonephritis, 125
arterial stiffness, 408 detection of, 114–117
blood pressure and peripheral vascular glomerular filtration, 119
resistance, 408 HIV associated nephropathy, 127
cardiovascular events and mortality, human diseases, 133
410–411 IgA nephropathy, 123–124
clinical outcomes, 411 interstitial osmolarity, 119–120
clinical practice, 419 in kidney transplantation, 127–130
CRP, 409 membranous glomerulonephrits, 125
dialysis-associated hypotension, 410 minimal change disease and focal segmental
endothelial function, 408 glomerulosclerosis, 124
evaluations of, 406 polycystic kidney disease, 120–122
homocysteine, 409 systemic lupus erythematosus, 125–127
insulin resistance, 409–410 tubular reabsorption and excretion, 119
intima media thickness, 408 urine, 12
LC-based approaches, 406 MicroRNA-210 (miR-210), 903
left ventricular hypertrophy, 408 diagnostic biomarker, 900–903
low-density lipoprotein cholesterol, 409 prognostic biomarker in RCC, 903–906
1212 Index
Microscopic markers, 194 O

casts, 196 Outcomes, 411, 673, 675, 676, 680
cells, 196 Oxidative stress, 855
crystals, 196 circulating levels of, 864
erythrocytes, 195 definition, 854
leucocytes, 195 evaluation of, 864
lipids, 196 immunological and nonimmunological
organisms, 197 stress, 867
Minimal change disease (MCD), 627, 630, ischemia-reperfusion process, 868
632, 634 in kidney transplantation, 861–864
Mitogenic activity, 605 superoxide dismutases, 854
Molecular biomarkers, 1020 tissue damage, 860
Morphometry, 946–947
M-type phospholipase A2 receptor (PLA2R)
antibodies P
clinical management of, 1042–1044 Pancreatic intraepithelial neoplastic (PanIN)
histological finding, 1040 lesions, 1000
and HLA genes, 1039 Parathyroid hormone-related protein (PTHrP),
IgG4, 1038 994, 1002
renal biopsy, 1041 Pathology, 672, 682
role of, 1040–1041 Peritoneal dialysate, 56, 58–59, 61
and THSD7A antibodies, 1041 Peritoneal dialysis (PD), 56
Multivariate statistics, 79 MMP-2 (see Matrix metalloproteinase-2
Myofibroblast, 937, 940 (MMP-2))
morbidity and mortality in, 920
PAI-1 (see Plasminogen activator inhibitor-
N 1 (PAI-1))
N-acetyl-β-D-glucosaminidase, urinary, 374–381 prevalence of, 914
Neonatal period, 30 short-term and long-term consequences of,
Nephritis, antibody arrays 914
cancer, 839–840 Peritoneal effluent, 56–65, 915, 922–926
micro-and nanoarray, 834–839 Peritoneal membrane dysfunction, 914, 926
protein expression profiling, 839 Phosphoinositide, 3-kinase (PI3K)-Akt-
recombinant antibody, 843 signaling pathway, 1001
renal impairment, 842 Physical markers, 176
renal pathology, biomarker of, 842 color, 177
resistin, 840–841 odor, 177
systemic lupus nephritis, 843–844 relative density, 178–179
Nephropathies, 504 turbidity or clarity, 177–178
Nephrotic syndrome (NS), 1179, 1182 Picrosirius red stain, 941, 946
homocysteine, 481 Plasma, 352, 356, 753–756, 762, 765, 772, 773
Nephrotic Syndrome Study Network Plasminogen activator inhibitor-1 (PAI-1)
(NEPTUNE), 633 cellular and peritoneal tissue expression,
Neutrophil gelatinase-associated lipocalin 918–920
(NGAL), 208 effect of, 921
and acute kidney injury, 210–213 estimated glomerular filtration rate, 921
and autosomal dominant polycystic kidney normal values of, 920–921
disease, 213–214 peritoneal effluent, 922–926
Newborn structure and biological functions of,
AKI see Acute kidney injury (AKI) 916–918
creatinine assays see Creatinine assays Platelet aggregates, 821, 825
Nitric oxide (NO), 400, 402, 407, 410, 419 Podocyte, 785, 787, 789, 791, 795, 798
Nontraditional risk factors, 1085–1086, 1090 Polycystic kidney disease (PKD), 120–122
Index 1213
Potential applications to prognosis, other Real-time elastography (RTE). See Quasi-static

diseases or conditions, 199–200 elastography
Prediction of renal progression, 437 Receiver operating characteristic (ROC)
Predictive biomarkers, 801–803 analysis, 632, 634, 640
Preterm neonate, 277, 290, 297 Recombinant antibodies, 837, 843
Primary glomerular diseases (PGDs), 1177 Red blood cell distribution width,
pathogenesis, 1178–1180 1178, 1180
prognostic factors, 1180–1182 clinical importance, 1183–1185
Professional societies, 1198 description, 1182
Prognosis, 498, 504, 508, 1037, 1040, 1044 in patients with proteinuria, 1185
Prognostic biomarkers, 729–735, 795–801 Regulatory bodies, 1197
miR-210, 903–906 Remission, 525, 529
Prognostic marker, 1022, 1023 Renal biomarkers, 651–653
Progression, 521, 525, 526, 529 Renal cell carcinoma (RCC), 898, 903,
Protein expression profiling, 834, 837, 1002–1003, 1005–1006, 1017
839, 842–843 Gas6, 616
Proteinuria, 176, 180, 186, 189–192, 196, 380, systemic therapy, 1020
389 Renal circulation
Proteinuric patterns anatomy, 1108
albuminuria, 522 factors influencing, 1109
diabetic kidney disease, 522–524 and renal arterial resistances,
glomerular basement membrane, 518 1107–1109
glomerular damage, 522 Renal damage, Gas6, 609–616
glomerular filtration barrier, 518 Renal failure, worsening of, 1106
glomerulonephritis, 524–529 Renal functions, 453, 454, 456, 457,
measurement and risk stratification, 502, 505–509
521–522 Renal imaging, US elastography.
tubulo-interstitial compartment damage, See Elastography
519–521 Renal impairment, 252, 255, 260
Proteomics, 58, 61–62, 357–358, 737–738, Renal replacement therapy (RRT), 143, 145,
743, 786, 795, 797, 798, 801, 150, 153, 155, 156
834, 838 Renal resistance index, 1107, 1116
urine, 12–15 assessment, 1110
Protocol biopsy, 672, 675, 677, 680 calculation, 1111
Pulmonary hypertension (PH), 1126–1127, pathophysiology, 1116
1137 patterns, 1112
Pulmonary pressure (PP) Renal transplantation, 210, 339–341
assessment, 1125–1126 Renal transplants, 1060, 1064–1066
diagnosis, 1126–1129 Renin-angiotensin-aldosterone system, Gas6,
renal patients, 1128–1131 615–616
Pulse wave velocity (PWV), 1082 Renin-angiotensin system (RAS),
estimation, 1083–1084 303, 305–306
measurement by Doppler, 1084–1085 Renoprevention, 158, 163–165
Resources, biomarkers. See Biomarkers
Response to treatment, IgAN
Q fish oil, 740–741
Quasi-static elastography, 1055–1056 immunosuppressive therapy,
742–743
renin–angiotensin system, 739–740
R tonsillectomy and steroids, 741
Random spot urine, 175, 193, 199 Ribonucleic acid biomarkers,
RDW. See Red blood cell distribution width 792–794
(RDW) RIP-seq, 966–969
1214 Index
RNA-seq Systemic Lupus Erythematosus Disease

and applications, kidney diseases, Activity Index (SLEDAI), 640
964–966 Systemic lupus erythematosus (SLE), 561, 565,
methods, 965 640, 814, 816, 818, 820, 822–825,
ROC curve, 877, 880 833, 841–843
S T
Saliva, 653 Target for treatment, 440
Salivary renal biomarkers, 653, 656–657, 660 Term neonate, 277, 290
Salivary urea nitrogen (SUN) and renal disease, The specimen for analysis, 176–177
656–659 Thrombosis, 815, 818–820, 824–826
physiology, properties and function, Tissue inhibitor metaloproteinases-2 (TIMP-2),
653–656 982–983
urea measurements, 656–658 Tissue remodeling, 920, 922
Screening for chronic kidney disease, Tolerance, 7, 8
436–439 biomarkers, 19–22
Sediment, 180, 194, 195 Tolvaptan, 547, 551
Sensitivity and specificity, 887 Traditional risks factors, 1093
Sepsis, 87, 93–94 Transient elastography (TE), 1057
Serum, 496–497, 505, 507, 509, 755–757, Transplant
768, 769 kidney, 255, 256, 264
Serum creatinine. See Serum creatinine renal, 256, 259, 262
trajectories Treatment, 1037–1038, 1043, 1044
Serum creatinine trajectories Treatment response, 1177–1178, 1180, 1182,
autosomal dominant polycystic kidney 1185, 1186
disease, 147–149 Treatment responsiveness, 522, 524
CKD (see Chronic kidney disease (CKD)) Tumor markers, 251, 260, 262, 263
HIV-associated nephropathy, 160–162 Tyrosine kinase inhibitor, 1024
LORFFAB, 155–156
prognostication and management, 165
sickle cell disease nephropathy, 162–163 U
Serum osmolal gap, 43, 46–50 Ultrasound elastography, kidney diseases.
SLE. See Systemic lupus erythematosus (SLE) See Elastography
Soluble urokinase receptor (suPAR) Urea
and antineutrophil cytoplasmic antibodies- analytical remarks, 1154
associated glomerulonephritis, output of, 1155
639–640 pathophysiology, 1154
clinical course, 634–635 reference intervals, 1154
and diabetic nephropathy, 641 urea clearance, 1158
FSGS, 629–633, 638–639 Urinalysis, 175–177, 190
and IgA nephropathy, 641–642 Urinary angiotensinogen (uAGT), 305–320
and lupus nephritis, 640–641 Urinary biomarkers, 175, 700, 712
measurement of, 635 Urinary leukocytes origin, 331–333
and renal function, 633–634 Urinary N-acetyl-β-D-glucosaminidase,
Spot urine, 377 374–381
Stiffness, 1081–1083, 1085–1092, Urinary oxygen tension, 98–99, 101
1095, 1096 Urinary protein, 307, 309, 311, 312, 316, 319
Supersonic shear wave imaging (SSI), Urine, 352, 507–509, 756, 758–759,
1058–1059 765, 770–771
Syndrome of rapid onset end stage renal disease exosomes isolation from, 354
(SORO-ESRD), 145–146, 153 Urine osmolal gap, 44, 46, 47, 49–50
Index 1215
Urokinase receptor (uPAR), 625 Vasopressin, 540, 541, 543, 546,

monoclonal blocking antibody, 629 547, 551
structure of, 627 von Hippel-Lindau tumor suppressor gene
Uropathies, 504–505 (VHL tumor suppressor gene),
900, 1002, 1004
V
Vascular endothelial growth factor (VEGF), W
1005, 1019, 1020 Worsening renal function (WRF), 1107

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Biomarkers in Disease:

Methods, Discoveries and Applications

1. General Methods in Biomarker Research and Their Applications

More information about this series at http://www.springer.com/series/13842

With 208 Figures and 142 Tables

ISBN 978-94-007-7698-2 ISBN 978-94-007-7699-9 (eBook)

Library of Congress Control Number: 2016939598

# Springer Science+Business Media Dordrecht 2016

Printed on acid-free paper

This Springer imprint is published by Springer Nature

In the present volume, Biomarkers in Kidney Disease, we have sections on

While the Editors recognize the difﬁculties in assigning particular chapters

April 2015 Victor R. Preedy

• Key Facts (areas of focus explained for the lay person)

The material in Potential Applications to Prognosis, Other Diseases, or Con-

April 2015 Victor R. Preedy

Part I General Aspects .................................... 1

1 Biomarkers in Kidney Transplantation ..................... 3

10 Overview of Neutrophil Gelatinase-Associated Lipocalin (NGAL)

Part II Circulating and Body Fluid Biomarkers . . . . . . . . . . . . . . . . 269

13 Creatinine Assays in Early Infancy: How to Aim for

22 Fetal Beta2-Microglobulin as a Biomarker of Kidney Disease . . . . 491

Part III Specific Diseases and Conditions .................... 667

30 Chronic Allograft Damage Index (CADI) as a Biomarker in

35 Biomarkers of Renal Microthrombosis in Lupus Nephritis . . . . . . 811

36 Planar Antibody Arrays for Biomarkers in Nephritis . . . . . . . . . . 831

37 Malondialdehyde as a Biomarker in Kidney Transplantation . . . . 849

38 Utility of Neutrophil Gelatinase-Associated Lipocalin in

39 miR-210 as a Biomarker in Renal Carcinoma . . . . . . . . . . . . . . . . 895

40 Matrix Metalloproteinase-2 (MMP-2) and Plasminogen Activator

Part IV Molecular, Cellular, and Histological Variables . . . . . . . . . 931

41 Assessing Fibrosis in Kidney Biopsies . . . . . . . . . . . . . . . . . . . . . . . 933

42 Next-Generation Sequencing (NGS) in Biomarker Discovery

43 Cell Cycle Arrest Biomarkers in Kidney Disease . . . . . . . . . . . . . . 977

44 Integrin-Linked Kinase (ILK) Expression as a Biomarker in

45 Molecular Biomarkers and Treatments for Renal

46 M-Type Phospholipase A2 Receptor as a Biomarker in

Part V Functional and Structural Variables .................. 1049

47 Ultrasound Elastography in Kidney Disease . . . . . . . . . . . . . . . . . 1051

Part VI Resources ....................................... 1193

53 Recommended Resources on Biomarkers in Kidney Disease . . . . . 1195

Dr. Vinood B. Patel B.Sc., Ph.D., FRSC is currently a

Victor R. Preedy B.Sc., Ph.D., D.Sc., FRSB, FRSH, FRIPHH, FRSPH,

Alessio Bocedi Department of Chemical Sciences and Technologies, University of

Nicola Di Daniele Department of Systems Medicine, Nephrology and Hyperten-

María Galindo-Izquierdo Rheumatology Department, Hospital Universitario

University of Athens, Medical School, Aretaieio Hospital, Department of Neonatol-

Massimo Iacoviello Cardiology Unit, Cardiothoracic Department, University

Hideto Iwamoto Division of Urology, Faculty of Medicine, Tottori University,

Antonín Jabor Department of Laboratory Methods, Institute for Clinical and

Jan Klocke Klinik mit Schwerpunkt Rheumatologie und klinische Immunologie,

Peter Kotanko Renal Research Institute, New York, NY, USA

Antonio Lacquaniti Department of Clinical and Experimental Medicine,

Mehmet Sait Menzilcioglu Department of Radiology, Faculty of Medicine, Gazi

José Luis Pablos-Álvarez Rheumatology Department, Hospital Universitario

Shiva Samavat Department of Nephrology, Labbaﬁnejad Medical Center, Shahid

Chiara Summaria Nephrology and Dialysis Unit, University Hospital of Catan-

M. Nafar • S. Samavat (*)

# Springer Science+Business Media Dordrecht 2016 3

(a) Calculated serum osmolality (mOsmol/kgH2O) = 2 [Na+] + [glucose]/18 +

(a) Calculated urine osmolality (mOsmol/kgH2O) = 2 {[Na+] + [K+]} +