Clear cell Renal Cell Carcinoma (ccRCC) causes over 13,000 deaths each year, and about 20,000 new... more Clear cell Renal Cell Carcinoma (ccRCC) causes over 13,000 deaths each year, and about 20,000 new cases/year in Europe. In most cases, the causes are unknown and, most importantly, there are no reliable biomarkers for the diagnosis and prognosis of this disease. The search for sensitive biomarkers for early diagnosis and prognosis of clear cell Renal Cell Carcinoma (ccRCC) is currently a fast growing field. We carried out proteomics analysis of 93 urinary samples of healthy subjects (HS) and patients affected by ccRCC, prostate cancer (PCa) and chronic kidney disease (CKD), that was able to successfully distinguish each group. The most significant candidate biomarker was identified by mass spectrometry as Raf Kinase Inhibitor Protein (RKIP), a key regulator of cell signaling, already described in several cancer types as a metastasis suppressor. By combining ELISA, immunoblotting and tissue microarray, we demonstrated that, in ccRCC, urinary excretion of RKIP and its phosphorylated form (p-RKIP) reflected the tissue expression of these putative biomarkers. Baseline urinary RKIP, evaluated in an independent cohort of 56 ccRCC patients and 28 HS, successfully distinguished both groups and, most importantly, a cutoff value of 10 ng/mg/g Pr/uCr enabled a highly accurate prediction of Cancer-specific survival and Progression-free survival. Furthermore, p-RKIP was totally undetectable in both tissue and urine samples of ccRCC, showing a great potential for diagnostics purposes. Our data indicate that urinary RKIP encompasses both the unphosphorylated and the phosphorylated form and that their combined evaluation can help in the diagnosis and prognosis of ccRCC.
Protein phosphorylation is considered a key event in signal transduction. Peripheral blood mononu... more Protein phosphorylation is considered a key event in signal transduction. Peripheral blood mononuclear cells (PBMCs) are a critical component of the immune system. The analysis of PBMCs phosphoproteome might help elucidate the signaling pathways essential to their biological role in health, immunological diseases and cancer. Enrichment of phosphoproteins becomes a prerequisite for phosphoproteome analysis and conventionally requires a multi-step procedure and sophisticated equipments. In this study, we standardized 2D-PAGE phosphoproteome analysis of PBMCs and compared two phosphoprotein enrichment methods, lanthanum chloride precipitation and affinity micro-column. Further, the different specificity for PBMCs phosphorylated proteins of each method was investigated. PBMCs were isolated from fresh whole blood of ten healthy donors. PBMCs phosphoproteins were enriched either by phosphoprotein precipitation using lanthanum chloride, with an overall yield of 8.9 ± 4.7%, or by using an a...
Feature selection becomes a central task when 'signature' profiles specific to a pathological sta... more Feature selection becomes a central task when 'signature' profiles specific to a pathological status have to be extracted from high dimensional gene expression or proteomic data. In the present paper, we propose a feature selection method based on Singular Value Decomposition (SVD) and apply it to SELDI-TOF/MS proteomic data from a cohort of Type 2 Diabetics affected by Glomerulosclerosis and Membranous Nephropathy. We have selected a profile composed of 24 proteins that seems to be an effective signature for the pathology at hand, allowing to efficiently discriminate between the considered subtype of diabetes.
Objective: The intrarenal renin-angiotensin system (RAS) activation plays a pivotal role in immun... more Objective: The intrarenal renin-angiotensin system (RAS) activation plays a pivotal role in immunoglobulin A nephropathy (IgAN) pathogenesis, which is still largely undefined. Recently, vasopressin (AVP) has been advocated to contribute to the genesis and progression of chronic kidney diseases (CKD) directly, and indirectly, via RAS activation. Our aim is to explore the intrarenal activity of AVP, its relationship with RAS activity, as well as its modulation by therapies in IgAN. Design: In this observational study, we measured plasma copeptin, a surrogate marker of AVP, the urine excretion of aquaporin 2 (AQP2), a protein reflecting renal AVP action, and angiotensinogen (AGT), a parameter of renal RAS activation, and their relationship with renal function in 44 IgAN patients at the time of renal biopsy, without any drug therapy, and after 6-month treatment with ACEi or steroidCACEi. Twenty-one patients with other CKD and 40 healthy subjects were recruited as controls. Methods: ELISAs were used to measure all variables of interest. Results: At baseline, IgAN patients showed higher urinary levels of AQP2, compared with controls and patients with other CKD. Urinary AQP2 and AGT levels strongly correlated with the presence of arterial hypertension. SteroidsCACEi caused the decrease of all the variables examined. The fall of urinary AQP2 and AGT following drug treatments was associated with the decrease of daily proteinuria. Conclusion: Our findings would support the involvement of AVP-AQP2 axis, interacting with the RAS, in the progression of IgAN and candidate AQP2 as a possible novel marker of the disease.
Biomarkers discovery is one of the major goals in clinical proteomics. In fact, their measurement... more Biomarkers discovery is one of the major goals in clinical proteomics. In fact, their measurement in biological fluid is very important for an early diagnosis of disease. Urine can be collected no invasively in large amounts so that it provides a potential source of disease biomarkers. IgA nephropathy (IgAN) is the most common form of immune complex-mediated glomerulonephritis worldwide [1]. End-stage renal disease (ESRD) develops in about 30% of the patients. Early intervention and consequent therapy may prevent or at least delay the development of ESRD in these patients. Up to now, the diagnosis could only be achieved with a renal biopsy. An alternative approach is represented by the identification and comparison of urine proteins that can supply important information on the patient health status. The most established method is protein separation by two dimensional gel electrophoresis (2-DE) followed by in gel digestion and protein identification by mass spectrometry. In this field, mass spectrometry has played an increasingly significant role in the identification of proteins that can be carried out by their proteolysis followed by high resolution liquid chromatography separation and on-line detection of the different peptides by ESI-Ion trap mass spectrometry (ESI/IT/MS/MSn) or off-line detection by MALDI-TOF [2, 3]. Today 1D-and 2D-LC/ESI/IT/MS/MSn techniques represent a valid method for protein analysis since they allow automatic analyses and the possibility to optimize chromatographic condition in order to achieve optimal separation of proteins or peptides for a better collection of mass spectra. Moreover, the interpretation of ESI-MS/MS spectra is fundamental for the determination of peptide molecular weights and for the characterization of peptide structure by amino acid sequences essential to recognition of protein and for identification of post-translational modifications [4] performed by database searching or De Novo sequencing. In the present communication we report the study of different expression of urinary proteins in two subgroups of patients with IgA nephropathy. They were classified as responder (R) and non responder (NR) to ACE inhibitory therapies. Despite the therapy, NR patients progress towards renal failure while R patients do not. We investigated 5 IgAN NR male patients compared with 5 IgAN R male patients and 5 normal male individuals. Because large amounts of proteins in fluid such as albumin and globulin may hinder the detection of potential biomarker protein, all urine samples were depleted of albumin and IgG. Urine samples were analyzed by 2D-PAGE (two-dimensional electrophoretic analysis) coupled to nano-HPLC-ESI-ion trap-MS/MS.
In the post human genome era, several "omics" fields are emerging. Proteomics has experienced a r... more In the post human genome era, several "omics" fields are emerging. Proteomics has experienced a rapid growth in the recent past and has great potential for the future. Proteomic technologies are used with increasing frequency also in nephrology. The aim of this review is to examine the recent application of emerging proteomic technologies to the study of renal physiology and pathophysiology. We highlight the use in renal research of a number of available techniques including 2-dimensional gel electrophoresis, liquid chromatography/mass spectrometry, surface-enhanced laser desorption/ionization, and capillary electrophoresis/mass spectrometry. We examine the role, efficacy and diagnostic potential of the different proteomic approaches, focusing on current difficulties and potential solutions. The integrating role of bioinformatics and the need for standardized procedures for sample preservation and analysis and reporting of results are also discussed. Although the field is still in an embryonic stage, the knowledge gained up to now is important not only for a better understanding of renal physiology and pathophysiology, but also for the identification of disease markers and the development and follow-up of new therapies. This review gives an overview of proteomics, providing background information, outlining the scopes, highlighting the applications in nephrology, and reporting advantages and limitations. (G Ital Nefrol 2008; 25: 169-82)
Chronic renal insufficiency and/or proteinuria in type 2 diabetes may stem from chronic renal dis... more Chronic renal insufficiency and/or proteinuria in type 2 diabetes may stem from chronic renal diseases (CKD) other than classic diabetic nephropathy in more than one-third of patients. We interrogated urine proteomic profiles generated by surface-enhanced laser desorption/ionization-time of flight/mass spectrometry with the aim of isolating a set of biomarkers able to reliably identify biopsy-proven diabetic nephropathy and to establish a stringent correlation with the different patterns of renal injury.
Diabetic nephropathy (DN) has become the most frequent cause of chronic kidney disease worldwide ... more Diabetic nephropathy (DN) has become the most frequent cause of chronic kidney disease worldwide due to the constant increase of the incidence of type 2 diabetes mellitus in developed and developing countries. The understanding of the pathophysiological mechanisms of human diseases through a large-scale characterization of the protein content of a biological sample is the key feature of the proteomics approach to the study of human disease. We discuss the main results of over 10 years of tissue and urine proteomics studies applied to DN in order to understand how far we have come and how far we still have to go before obtaining a full comprehension of the molecular mechanisms involved in the pathogenesis of DN and identifying reliable biomarkers for accurate management of patients.
Clear cell Renal Cell Carcinoma (ccRCC) causes over 13,000 deaths each year, and about 20,000 new... more Clear cell Renal Cell Carcinoma (ccRCC) causes over 13,000 deaths each year, and about 20,000 new cases/year in Europe. In most cases, the causes are unknown and, most importantly, there are no reliable biomarkers for the diagnosis and prognosis of this disease. The search for sensitive biomarkers for early diagnosis and prognosis of clear cell Renal Cell Carcinoma (ccRCC) is currently a fast growing field. We carried out proteomics analysis of 93 urinary samples of healthy subjects (HS) and patients affected by ccRCC, prostate cancer (PCa) and chronic kidney disease (CKD), that was able to successfully distinguish each group. The most significant candidate biomarker was identified by mass spectrometry as Raf Kinase Inhibitor Protein (RKIP), a key regulator of cell signaling, already described in several cancer types as a metastasis suppressor. By combining ELISA, immunoblotting and tissue microarray, we demonstrated that, in ccRCC, urinary excretion of RKIP and its phosphorylated form (p-RKIP) reflected the tissue expression of these putative biomarkers. Baseline urinary RKIP, evaluated in an independent cohort of 56 ccRCC patients and 28 HS, successfully distinguished both groups and, most importantly, a cutoff value of 10 ng/mg/g Pr/uCr enabled a highly accurate prediction of Cancer-specific survival and Progression-free survival. Furthermore, p-RKIP was totally undetectable in both tissue and urine samples of ccRCC, showing a great potential for diagnostics purposes. Our data indicate that urinary RKIP encompasses both the unphosphorylated and the phosphorylated form and that their combined evaluation can help in the diagnosis and prognosis of ccRCC.
Protein phosphorylation is considered a key event in signal transduction. Peripheral blood mononu... more Protein phosphorylation is considered a key event in signal transduction. Peripheral blood mononuclear cells (PBMCs) are a critical component of the immune system. The analysis of PBMCs phosphoproteome might help elucidate the signaling pathways essential to their biological role in health, immunological diseases and cancer. Enrichment of phosphoproteins becomes a prerequisite for phosphoproteome analysis and conventionally requires a multi-step procedure and sophisticated equipments. In this study, we standardized 2D-PAGE phosphoproteome analysis of PBMCs and compared two phosphoprotein enrichment methods, lanthanum chloride precipitation and affinity micro-column. Further, the different specificity for PBMCs phosphorylated proteins of each method was investigated. PBMCs were isolated from fresh whole blood of ten healthy donors. PBMCs phosphoproteins were enriched either by phosphoprotein precipitation using lanthanum chloride, with an overall yield of 8.9 ± 4.7%, or by using an a...
Feature selection becomes a central task when 'signature' profiles specific to a pathological sta... more Feature selection becomes a central task when 'signature' profiles specific to a pathological status have to be extracted from high dimensional gene expression or proteomic data. In the present paper, we propose a feature selection method based on Singular Value Decomposition (SVD) and apply it to SELDI-TOF/MS proteomic data from a cohort of Type 2 Diabetics affected by Glomerulosclerosis and Membranous Nephropathy. We have selected a profile composed of 24 proteins that seems to be an effective signature for the pathology at hand, allowing to efficiently discriminate between the considered subtype of diabetes.
Objective: The intrarenal renin-angiotensin system (RAS) activation plays a pivotal role in immun... more Objective: The intrarenal renin-angiotensin system (RAS) activation plays a pivotal role in immunoglobulin A nephropathy (IgAN) pathogenesis, which is still largely undefined. Recently, vasopressin (AVP) has been advocated to contribute to the genesis and progression of chronic kidney diseases (CKD) directly, and indirectly, via RAS activation. Our aim is to explore the intrarenal activity of AVP, its relationship with RAS activity, as well as its modulation by therapies in IgAN. Design: In this observational study, we measured plasma copeptin, a surrogate marker of AVP, the urine excretion of aquaporin 2 (AQP2), a protein reflecting renal AVP action, and angiotensinogen (AGT), a parameter of renal RAS activation, and their relationship with renal function in 44 IgAN patients at the time of renal biopsy, without any drug therapy, and after 6-month treatment with ACEi or steroidCACEi. Twenty-one patients with other CKD and 40 healthy subjects were recruited as controls. Methods: ELISAs were used to measure all variables of interest. Results: At baseline, IgAN patients showed higher urinary levels of AQP2, compared with controls and patients with other CKD. Urinary AQP2 and AGT levels strongly correlated with the presence of arterial hypertension. SteroidsCACEi caused the decrease of all the variables examined. The fall of urinary AQP2 and AGT following drug treatments was associated with the decrease of daily proteinuria. Conclusion: Our findings would support the involvement of AVP-AQP2 axis, interacting with the RAS, in the progression of IgAN and candidate AQP2 as a possible novel marker of the disease.
Biomarkers discovery is one of the major goals in clinical proteomics. In fact, their measurement... more Biomarkers discovery is one of the major goals in clinical proteomics. In fact, their measurement in biological fluid is very important for an early diagnosis of disease. Urine can be collected no invasively in large amounts so that it provides a potential source of disease biomarkers. IgA nephropathy (IgAN) is the most common form of immune complex-mediated glomerulonephritis worldwide [1]. End-stage renal disease (ESRD) develops in about 30% of the patients. Early intervention and consequent therapy may prevent or at least delay the development of ESRD in these patients. Up to now, the diagnosis could only be achieved with a renal biopsy. An alternative approach is represented by the identification and comparison of urine proteins that can supply important information on the patient health status. The most established method is protein separation by two dimensional gel electrophoresis (2-DE) followed by in gel digestion and protein identification by mass spectrometry. In this field, mass spectrometry has played an increasingly significant role in the identification of proteins that can be carried out by their proteolysis followed by high resolution liquid chromatography separation and on-line detection of the different peptides by ESI-Ion trap mass spectrometry (ESI/IT/MS/MSn) or off-line detection by MALDI-TOF [2, 3]. Today 1D-and 2D-LC/ESI/IT/MS/MSn techniques represent a valid method for protein analysis since they allow automatic analyses and the possibility to optimize chromatographic condition in order to achieve optimal separation of proteins or peptides for a better collection of mass spectra. Moreover, the interpretation of ESI-MS/MS spectra is fundamental for the determination of peptide molecular weights and for the characterization of peptide structure by amino acid sequences essential to recognition of protein and for identification of post-translational modifications [4] performed by database searching or De Novo sequencing. In the present communication we report the study of different expression of urinary proteins in two subgroups of patients with IgA nephropathy. They were classified as responder (R) and non responder (NR) to ACE inhibitory therapies. Despite the therapy, NR patients progress towards renal failure while R patients do not. We investigated 5 IgAN NR male patients compared with 5 IgAN R male patients and 5 normal male individuals. Because large amounts of proteins in fluid such as albumin and globulin may hinder the detection of potential biomarker protein, all urine samples were depleted of albumin and IgG. Urine samples were analyzed by 2D-PAGE (two-dimensional electrophoretic analysis) coupled to nano-HPLC-ESI-ion trap-MS/MS.
In the post human genome era, several "omics" fields are emerging. Proteomics has experienced a r... more In the post human genome era, several "omics" fields are emerging. Proteomics has experienced a rapid growth in the recent past and has great potential for the future. Proteomic technologies are used with increasing frequency also in nephrology. The aim of this review is to examine the recent application of emerging proteomic technologies to the study of renal physiology and pathophysiology. We highlight the use in renal research of a number of available techniques including 2-dimensional gel electrophoresis, liquid chromatography/mass spectrometry, surface-enhanced laser desorption/ionization, and capillary electrophoresis/mass spectrometry. We examine the role, efficacy and diagnostic potential of the different proteomic approaches, focusing on current difficulties and potential solutions. The integrating role of bioinformatics and the need for standardized procedures for sample preservation and analysis and reporting of results are also discussed. Although the field is still in an embryonic stage, the knowledge gained up to now is important not only for a better understanding of renal physiology and pathophysiology, but also for the identification of disease markers and the development and follow-up of new therapies. This review gives an overview of proteomics, providing background information, outlining the scopes, highlighting the applications in nephrology, and reporting advantages and limitations. (G Ital Nefrol 2008; 25: 169-82)
Chronic renal insufficiency and/or proteinuria in type 2 diabetes may stem from chronic renal dis... more Chronic renal insufficiency and/or proteinuria in type 2 diabetes may stem from chronic renal diseases (CKD) other than classic diabetic nephropathy in more than one-third of patients. We interrogated urine proteomic profiles generated by surface-enhanced laser desorption/ionization-time of flight/mass spectrometry with the aim of isolating a set of biomarkers able to reliably identify biopsy-proven diabetic nephropathy and to establish a stringent correlation with the different patterns of renal injury.
Diabetic nephropathy (DN) has become the most frequent cause of chronic kidney disease worldwide ... more Diabetic nephropathy (DN) has become the most frequent cause of chronic kidney disease worldwide due to the constant increase of the incidence of type 2 diabetes mellitus in developed and developing countries. The understanding of the pathophysiological mechanisms of human diseases through a large-scale characterization of the protein content of a biological sample is the key feature of the proteomics approach to the study of human disease. We discuss the main results of over 10 years of tissue and urine proteomics studies applied to DN in order to understand how far we have come and how far we still have to go before obtaining a full comprehension of the molecular mechanisms involved in the pathogenesis of DN and identifying reliable biomarkers for accurate management of patients.
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Papers by M. Papale