Papers by Valeria Rivarola
Journal of Cellular Biochemistry, Jan 5, 2017
AQP2-induced acceleration of renal cell proliferation involves the activation of a regulatory vol... more AQP2-induced acceleration of renal cell proliferation involves the activation of a regulatory volume increase mechanism dependent on NHE2 † Running head: NHE2-dependent RVI and AQP2-rised cell growth
Journal of cellular biochemistry, Jan 18, 2016
We have previously shown in renal cells that expression of the water channel Aquaporin 2 (AQP2) i... more We have previously shown in renal cells that expression of the water channel Aquaporin 2 (AQP2) increases the rate of cell proliferation by shortening the transit time through the S and G2 /M phases of the cell cycle. This acceleration is due, at least in part, to a down-regulation of regulatory volume decrease (RVD) mechanisms when volume needs to be increased in order to proceed into the S phase. We hypothesize that in order to increase cell volume, RVD mechanisms may be overtaken by regulatory volume increase mechanisms (RVI). In this study, we investigated if the isoform 2 of the Na(+) /H(+) exchanger (NHE2), the main ion transporter involved in RVI responses, contributed to the AQP2-increased renal cell proliferation. Three cortical collecting duct cell lines were used: WT-RCCD1 (not expressing AQPs), AQP2-RCCD1 (transfected with AQP2) and mpkCCDc14 (with inducible AQP2 expression). We here demonstrate, for the first time, that both NHE2 protein activity and expression was incr...
Los tubulos colectores corticales (CCD) del rinon de mamifero juegan un papel central en la regul... more Los tubulos colectores corticales (CCD) del rinon de mamifero juegan un papel central en la regulacion del transporte de agua-solutos y en el equilibrio acido-base del organismo. Si bien muchos estudios han sido realizados para tratar de entender los mecanismos implicados en estos procesos, los mismos no han sido completamente aclarados. En la presente tesis hemos utilizado a la linea celular RCCD1 (modelo de CCD) para intentar clarificar los mecanismos por los cuales se produce el movimiento de agua y la regulacion del pHi en este segmento del nefron. En lo referente al movimiento de agua observamos que la linea celular desarrolla importantes flujos en ausencia de fuerzas impulsoras osmoticas e hidrostaticas. Demostramos, ademas, que los mismos ocurririan por un mecanismo de cotransporte agua-soluto ya que la linea no expresa acuaporinas. Basalmente predominaria un flujo secretor asociado al movimiento de Cl-, HCO3- y K+. La hormona AVP estimularia, a “corto plazo”, una absorcion d...
Journal of Cellular Physiology, 2020
Increasing evidence indicates that aquaporins (AQPs) exert an influence in cell signaling by the ... more Increasing evidence indicates that aquaporins (AQPs) exert an influence in cell signaling by the interplay with the transient receptor potential vanilloid 4 (TRPV4) channel. We previously found that TRPV4 physically and functionally interacts with AQP2 in cortical collecting ducts (CCD) cells, favoring cell volume regulation and cell migration. Because TRPV4 was implicated in ATP release in several tissues, we investigated the possibility that TRPV4/AQP2 interaction influences ATP release in CCD cells. Using two CCD cell lines expressing or not AQP2, we measured extracellular ATP (ATPe) under TRPV4 activation and intracellular Ca2+ under ATP addition. We found that AQP2 is critical for the release of ATP induced by TRPV4 activation. This ATP release occurs by an exocytic and a conductive route. ATPe, in turn, stimulates purinergic receptors leading to ATPe‐induced ATP release by a Ca2+‐dependent mechanism. We propose that AQP2 by modulating Ca2+ and ATP differently could explain AQP2‐increased cell migration.
Journal of Cellular Physiology, 2019
Aquaporin-2 (AQP2) promotes renal cell migration by the modulation of integrin β1 trafficking and... more Aquaporin-2 (AQP2) promotes renal cell migration by the modulation of integrin β1 trafficking and the turnover of focal adhesions. The aim of this study was to investigate whether AQP2 also works in cooperation with Na + /H + exchanger isoform 1 (NHE1), another well-known protein involved in the regulation of cell migration. Our results showed that the lamellipodia of AQP2-expressing cells exhibit significantly smaller volumes and areas of focal adhesions and more alkaline intracellular pH due to increased NHE1 activity than AQP2-null cells. The blockage of AQP2, or its physically-associated calcium channel TRPV4, significantly reduced lamellipodia NHE1 activity. NHE1 blockage significantly reduced the rate of cell migration, the number of lamellipodia, and the assembly of F-actin only in AQP2expressing cells. Our data suggest that AQP2 modulates the activity of NHE1 through its calcium channel partner TRPV4, thereby determining pH-dependent actin polymerization, providing mechanical stability to delineate lamellipodia structure and defining the efficiency of cell migration.
Journal of cellular biochemistry, May 1, 2018
There is increasing evidence indicating that aquaporins (AQPs) exert an influence in cell signali... more There is increasing evidence indicating that aquaporins (AQPs) exert an influence in cell signaling by the interplay with the TRPV4 Ca channel. Ca release from intracellular stores and plasma membrane hyperpolarization due to opening of Ca -activated potassium channels (KCa) are events that have been proposed to take place downstream of TRPV4 activation. A major mechanism for Ca entry, activated after depletion of intracellular Ca stores and driven by electrochemical forces, is the store-operated Ca entry (SOCE). The consequences of the interplay between TRPV4 and AQPs on SOCE have not been yet investigated. The aim of our study was to test the hypothesis that AQP2 can modulate SOCE by facilitating the interaction of TRPV4 with KCa channels in renal cells. Using fluorescent probe techniques, we studied intracellular Ca concentration and membrane potential in response to activation of TRPV4 in two rat cortical collecting duct cell lines (RCCD ), one not expressing AQPs (WT-RCCD ) and...
Journal of cellular biochemistry, Aug 18, 2017
Neural activity alters osmotic gradients favoring cell swelling in retinal Müller cells. This swe... more Neural activity alters osmotic gradients favoring cell swelling in retinal Müller cells. This swelling is followed by a regulatory volume decrease (RVD), partially mediated by an efflux of KCl and water. The transient receptor potential channel 4 (TRPV4), a nonselective calcium channel, has been proposed as a candidate for mediating intracellular Ca(2+) elevation induced by swelling. We previously demonstrated in a human Müller cell line (MIO-M1) that RVD strongly depends on ion channel activation and, consequently, on membrane potential (Vm ). The aim of this study was to investigate if Ca(2+) influx via TRPV4 contributes to RVD by modifying intracellular Ca(2+) concentration and/or modulating Vm in MIO-M1 cells. Cell volume, intracellular Ca(2+) levels, and Vm changes were evaluated using fluorescent probes. Results showed that MIO-M1 cells express functional TRPV4 which determines the resting Vm associated with K(+) channels. Swelling-induced increases in Ca(2+) levels was due to...
Journal of Physiology and Biochemistry, 2019
We have previously shown in renal cells that expression of the water channel Aquaporin-2 increase... more We have previously shown in renal cells that expression of the water channel Aquaporin-2 increases cell proliferation by a regulatory volume mechanism involving Na + /H + exchanger isoform 2. Here, we investigated if Aquaporin-2 (AQP2) also modulates Na + /H + exchanger isoform 1-dependent cell proliferation. We use two AQP2-expressing cortical collecting duct models: one constitutive (WT or AQP2-transfected RCCD 1 cell line) and one inducible (control or vasopressin-induced mpkCCD c14 cell line). We found that Aquaporin-2 modifies Na + /H + exchanger isoform 1 (NHE1) contribution to cell proliferation. In Aquaporin-2-expressing cells, Na + /H + exchanger isoform 1 is anti-proliferative at physiological pH. In acid media, Na + / H + exchanger isoform 1 contribution turned from anti-proliferative to proliferative only in AQP2-expressing cells. We also found that, in AQP2-expressing cells, NHE1-dependent proliferation changes parallel changes in stress fiber levels: at pH 7.4, Na + /H + exchanger isoform 1 would favor stress fiber disassembly and, under acidosis, NHE1 would favor stress fiber assembly. Moreover, we found that Na + /H + exchanger-dependent effects on proliferation linked to Aquaporin-2 relied on Transient Receptor Potential Subfamily V calcium channel activity. In conclusion, our data show that, in collecting duct cells, the water channel Aquaporin-2 modulates NHE1-dependent cell proliferation. In AQP2-expressing cells, at physiological pH, the Na + /H + exchanger isoform 1 function is anti-proliferative and, at acidic pH, Na + /H + exchanger isoform 1 function is proliferative. We propose that Na + /H + exchanger isoform 1 modulates proliferation through an interplay with stress fiber formation. Keywords Aquaporin-2. Na/H exchanger 1. Acidosis. Cell proliferation Marina Mazzocchi and Gisela Di Giusto should be considered joint first authors. Key points • NHE1 activity in AQP2-expressing cells is anti-proliferative at pH = 7.4 and proliferative at pH = 7.0. • In the presence of AQP2, NHE1-dependent stress fiber formation modulates proliferation. • Interaction between AQP2-TRPV calcium channels modulates NHE1dependent proliferation.
PloS one, 2013
Mü ller cells are mainly involved in controlling extracellular homeostasis in the retina, where i... more Mü ller cells are mainly involved in controlling extracellular homeostasis in the retina, where intense neural activity alters ion concentrations and osmotic gradients, thus favoring cell swelling. This increase in cell volume is followed by a regulatory volume decrease response (RVD), which is known to be partially mediated by the activation of K + and anion channels. However, the precise mechanisms underlying osmotic swelling and subsequent cell volume regulation in Mü ller cells have been evaluated by only a few studies. Although the activation of ion channels during the RVD response may alter transmembrane potential (V m ), no studies have actually addressed this issue in Mü ller cells. The aim of the present work is to evaluate RVD using a retinal Mü ller cell line (MIO-M1) under different extracellular ionic conditions, and to study a possible association between RVD and changes in V m . Cell volume and V m changes were evaluated using fluorescent probe techniques and a mathematical model. Results show that cell swelling and subsequent RVD were accompanied by V m depolarization followed by repolarization. This response depended on the composition of extracellular media. Cells exposed to a hypoosmotic solution with reduced ionic strength underwent maximum RVD and had a larger repolarization. Both of these responses were reduced by K + or Cl 2 channel blockers. In contrast, cells facing a hypoosmotic solution with the same ionic strength as the isoosmotic solution showed a lower RVD and a smaller repolarization and were not affected by blockers. Together, experimental and simulated data led us to propose that the efficiency of the RVD process in Mü ller glia depends not only on the activation of ion channels, but is also strongly modulated by concurrent changes in the membrane potential. The relationship between ionic fluxes, changes in ion permeabilities and ion concentrations -all leading to changes in V m -define the success of RVD.
Journal of cellular biochemistry, 2012
We have previously demonstrated that in renal cortical collecting duct cells (RCCD1) the expressi... more We have previously demonstrated that in renal cortical collecting duct cells (RCCD1) the expression of the water channel Aquaporin 2 (AQP2) raises the rate of cell proliferation. In this study, we investigated the mechanisms involved in this process, focusing on the putative link between AQP2 expression, cell volume changes, and regulatory volume decrease activity (RVD). Two renal cell lines were used: WT-RCCD1 (not expressing aquaporins) and AQP2-RCCD1 (transfected with AQP2). Our results showed that when most RCCD1 cells are in the G1-phase (unsynchronized), the blockage of barium-sensitive K+ channels implicated in rapid RVD inhibits cell proliferation only in AQP2-RCCD1 cells. Though cells in the S-phase (synchronized) had a remarkable increase in size, this enhancement was higher and was accompanied by a significant down-regulation in the rapid RVD response only in AQP2-RCCD1 cells. This decrease in the RVD activity did not correlate with changes in AQP2 function or expression, demonstrating that AQP2—besides increasing water permeability—would play some other role. These observations together with evidence implying a cell-sizing mechanism that shortens the cell cycle of large cells, let us to propose that during nutrient uptake, in early G1, volume tends to increase but it may be efficiently regulated by an AQP2-dependent mechanism, inducing the rapid activation of RVD channels. This mechanism would be down-regulated when volume needs to be increased in order to proceed into the S-phase. Therefore, during cell cycle, a coordinated modulation of the RVD activity may contribute to accelerate proliferation of cells expressing AQP2. J. Cell. Biochem. 113: 3721–3729, 2012. © 2012 Wiley Periodicals, Inc.
Journal of neuroscience research, 2012
NMO-IgG autoantibody selectively binds to aquaporin-4 (AQP4), the most abundant water channel in ... more NMO-IgG autoantibody selectively binds to aquaporin-4 (AQP4), the most abundant water channel in the central nervous system and is now considered a useful serum biomarker of neuromyelitis optica (NMO). A series of clinical and pathological observations suggests that NMO-IgG may play a central role in NMO physiopathology. The current study evaluated, in well-differentiated astrocytes cultures, the consequences of NMO-IgG binding on the expression pattern of AQP4 and on plasma membrane water permeability. To avoid or to facilitate AQP4 down-regulation, cells were exposed to inactivated sera in two different situations (1 hr at 4°C or 12 hr at 37°C). AQP4 expression was detected by immunofluorescence studies using a polyclonal anti-AQP4 or a human anti-IgG antibody, and the water permeability coefficient was evaluated by a videomicroscopy technique. Our results showed that, at low temperatures, cell exposure to either control or NMO-IgG sera does not affect either AQP4 expression or plasma membrane water permeability, indicating that the simple binding of NMO-IgG does not affect the water channel's activity. However, at 37°C, long-term exposure to NMO-IgG induced a loss of human IgG signal from the plasma membrane along with M1-AQP4 isoform removal and a significant reduction of water permeability. These results suggest that binding of NMO-IgG to cell membranes expressing AQP4 is a specific mechanism that may account for at least part of the pathogenic process. © 2012 Wiley Priodicals, Inc.
Journal of cellular biochemistry, 2012
We have previously demonstrated that renal cortical collecting duct cells (RCCD1), responded to h... more We have previously demonstrated that renal cortical collecting duct cells (RCCD1), responded to hypotonic stress with a rapid activation of regulatory volume decrease (RVD) mechanisms. This process requires the presence of the water channel AQP2 and calcium influx, opening the question about the molecular identity of this calcium entry path. Since the calcium permeable nonselective cation channel TRPV4 plays a crucial role in the response to mechanical and osmotic perturbations in a wide range of cell types, the aim of this work was to test the hypothesis that the increase in intracellular calcium concentration and the subsequent rapid RVD, only observed in the presence of AQP2, could be due to a specific activation of TRPV4. We evaluated the expression and function of TRPV4 channels and their contribution to RVD in WT-RCCD1 (not expressing aquaporins) and in AQP2-RCCD1 (transfected with AQP2) cells. Our results demonstrated that both cell lines endogenously express functional TRPV4, however, a large activation of the channel by hypotonicity only occurs in cells that express AQP2. Blocking of TRPV4 by ruthenium red abolished calcium influx as well as RVD, identifying TRPV4 as a necessary component in volume regulation. Even more, this process is dependent on the translocation of TRPV4 to the plasma membrane. Our data provide evidence of a novel association between TRPV4 and AQP2 that is involved in the activation of TRPV4 by hypotonicity and regulation of cellular response to the osmotic stress, suggesting that both proteins are assembled in a signaling complex that responds to anisosmotic conditions. J. Cell. Biochem. 113: 580–589, 2012. © 2011 Wiley Periodicals, Inc.
Journal of Cellular Physiology, 2010
Collecting ducts (CD) not only constitute the final site for regulating urine concentration by in... more Collecting ducts (CD) not only constitute the final site for regulating urine concentration by increasing apical membrane Aquaporin-2 (AQP2) expression, but are also essential for the control of acid–base status. The aim of this work was to examine, in renal cells, the effects of chronic alkalosis on cell growth/death as well as to define whether AQP2 expression plays any role during this adaptation. Two CD cell lines were used: WT- (not expressing AQPs) and AQP2-RCCD1 (expressing apical AQP2). Our results showed that AQP2 expression per se accelerates cell proliferation by an increase in cell cycle progression. Chronic alkalosis induced, in both cells lines, a time-dependent reduction in cell growth. Even more, cell cycle movement, assessed by 5-bromodeoxyuridine pulse-chase and propidium iodide analyses, revealed a G2/M phase cell accumulation associated with longer S- and G2/M-transit times. This G2/M arrest is paralleled with changes consistent with apoptosis. All these effects appeared 24 h before and were always more pronounced in cells expressing AQP2. Moreover, in AQP2-expressing cells, part of the observed alkalosis cell growth decrease is explained by AQP2 protein down-regulation. We conclude that in CD cells alkalosis causes a reduction in cell growth by cell cycle delay that triggers apoptosis as an adaptive reaction to this environment stress. Since cell volume changes are prerequisite for the initiation of cell proliferation or apoptosis, we propose that AQP2 expression facilitates cell swelling or shrinkage leading to the activation of channels necessary to the control of these processes. J. Cell. Physiol. 224: 405–413, 2010. © 2010 Wiley-Liss, Inc.
Biology of The Cell, 2009
Background information. A major hallmark of apoptosis is cell shrinkage, termed apoptotic volume ... more Background information. A major hallmark of apoptosis is cell shrinkage, termed apoptotic volume decrease, due to the cellular outflow of potassium and chloride ions, followed by osmotically obliged water. In many cells, the ionic pathways triggered during the apoptotic volume decrease may be similar to that observed during a regulatory volume decrease response under hypotonic conditions. However, the pathways involved in water loss during apoptosis have been largely ignored. It was recently reported that in some systems this water movement is mediated via specific water channels (aquaporins). Nevertheless, it is important to identify whether this is a ubiquitous aspect of apoptosis as well as to define the mechanisms involved. The aim of the present work was to investigate the role of aquaporin-2 during apoptosis in renal-collecting duct cells. We evaluated the putative relationship between aquaporin-2 expression and the activation of the ionic pathways involved in the regulatory volume response.
Ajp: Renal Physiology, 2008
Galizia L, Flamenco MP, Rivarola V, Capurro C, Ford P. Role of AQP2 in activation of calcium entr... more Galizia L, Flamenco MP, Rivarola V, Capurro C, Ford P. Role of AQP2 in activation of calcium entry by hypotonicity: implications in cell volume regulation. We previously reported in a rat cortical collecting duct cell line (RCCD 1) that the presence of aquaporin 2 (AQP2) in the cell membrane is critical for the rapid activation of regulatory volume decrease mechanisms (RVD) (Ford et al. Biol Cell 97: 687-697, 2005). The aim of our present work was to investigate the signaling pathway that links AQP2 to this rapid RVD activation. Since it has been previously described that hypotonic conditions induce intracellular calcium ([Ca 2ϩ ]i) increases in different cell types, we tested the hypothesis that AQP2 could have a role in activation of calcium entry by hypotonicity and its implication in cell volume regulation. Using a fluorescent probe technique, we studied [Ca 2ϩ ]i and cell volume changes in response to a hypotonic shock in WT-RCCD 1 (not expressing aquaporins) and in AQP2-RCCD1 (transfected with AQP2) cells. We found that after a hypotonic shock only AQP2-RCCD 1 cells exhibit a substantial increase in [Ca 2ϩ ]i. This [Ca 2ϩ ]i increase is strongly dependent on extracellular Ca 2ϩ and is partially inhibited by thapsigargin (1 M) indicating that the rise in [Ca 2ϩ ]i reflects both influx from the extracellular medium and release from intracellular stores. Exposure of AQP2-RCCD 1 cells to 100 M gadolinium reduced the increase in [Ca 2ϩ ]i suggesting the involvement of a mechanosensitive calcium channel. Furthermore, exposure of cells to all of the above described conditions impaired rapid RVD. We conclude that the expression of AQP2 in the cell membrane is critical to produce the increase in [Ca 2ϩ ]i which is necessary to activate RVD in RCCD 1 cells. aquaporin 2; intracellular calcium; renal cells THE KIDNEY COLLECTING DUCT plays an important role in the process of urine concentration through a mechanism regulated by arginine vasopressin. This hormone induces an increase in osmotic water permeability (P f ) by triggering translocation and fusion of intracellular vesicles containing aquaporin 2 (AQP2) to the apical membrane of principal cells . In this condition, two-thirds of the hyposmotic luminal fluid entering the cortical collecting duct (CCD) is reabsorbed. Therefore, CCD cells are faced with both, changes in apical osmolarity and important volume flows which could result in cell volume increases. For this reason, potent volume regulatory mechanisms are needed to maintain cellular homeostasis and epithelial transport (29).
Comparative Biochemistry and Physiology A-molecular & Integrative Physiology, 2007
Cellular Physiology and Biochemistry, 2007
Arginine-vasopressin (AVP) has been proposed to be involved in the modulation of acid-base transp... more Arginine-vasopressin (AVP) has been proposed to be involved in the modulation of acid-base transporters; however, the nature of the mechanisms underlying AVP direct action on intracellular pH (pH i ) in the cortical collecting duct (CCD) is not yet clearly defined. The aim of the present study was to elucidate which are the proteins implicated in AVP modulation of pH i , as well as the receptors involved in these responses using a CCD cell line (RCCD 1 ); pH i was monitored with the fluorescent dye BCECF in basal conditions and after stimulation with basolateral 10 -8 M AVP. Specific V1-or V2-receptor antagonists were also used. RT-PCR studies demonstrated that RCCD 1 cells express V1a and V2 receptors. Functional studies showed that while V2-receptor activation induced a biphasic response (alkalinizationacidification), V1-receptor activation resulted in an intracellular acidification. The V2-mediated alkalinization phase involves the activation of basolateral NHE-1 isoform of the Na + /H + exchanger while in the acidification phase CFTR is probably implicated. On the other hand, V1-mediated acidification was due to activation of a Cl -/HCO 3 exchanger. We conclude that in RCCD 1 cells AVP selectively activates, via a complex of V1 and V2 receptor-mediated actions, different ion transporters linked to pH i regulation which might have physiological implications.
Journal of Membrane Biology, 2005
Transition from antidiuresis to diuresis exposes cortical collecting duct cells (CCD) to asymmetr... more Transition from antidiuresis to diuresis exposes cortical collecting duct cells (CCD) to asymmetrical changes in environment osmolality, inducing an osmotic stress, which activates numerous membrane-associated events. The aim of the present work was to investigate, either in the presence or not of AQP2, the transepithelial osmotic water permeability (P osm) following cell exposure to asymmetrical hyper- or hypotonic gradients. For this purpose, transepithelial net volume fluxes were recorded every minute in two CCD cell lines: one not expressing AQPs (WT-RCCD1) and another stably transfected with AQP2 (AQP2-RCCD1). Our results demonstrated that the rate of osmosis produced by a given hypotonic shock depends on the gradient direction (osmotic rectification) only in the presence of apical AQP2. In contrast, hypertonic shocks elicit P osm rectification independently of AQP2 expression, and this phenomenon may be linked to modulation of basolateral membrane permeability. No asymmetry in transepithelial resistance was observed under hypo- or hypertonicity, indicating that rectification cannot be attributed to a shunt through the tight junction path. We conclude that osmotic rectification may be explained in terms of dynamical changes in membrane permeability probably due to activation/incorporation of AQPs or transporters to the plasma membrane via some mechanism triggered by osmolality.
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Papers by Valeria Rivarola