Valeria Rivarola

Functional interaction between AQP2 and TRPV4 in renal cells

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.

Volume regulation in cortical collecting duct cells: role of AQP2

Background information. The renal CCD (cortical collecting duct) plays a role in final volume and concentration of urine by a process that is regulated by the antidiuretic hormone, [arginine]vasopressin. This hormone induces an increase in water permeability due to the translocation of AQP2 (aquaporin 2) from the intracellular vesicles to the apical membrane of principal cells. During the transition from antidiuresis to diuresis, CCD cells are exposed to changes in environmental osmolality, and cell‐volume regulation may be especially important for the maintenance of intracellular homoeostasis. Despite its importance, cell‐volume regulation in CCD cells has not been widely investigated. Moreover, no studies have been carried out till date to evaluate the putative role of AQPs during this process in renal cells.

Differential Role of Na+/H+ Exchange Isoforms NHE-1 and NHE-2 in a Rat Cortical Collecting Duct Cell Line

The Na+/H+ exchanger (NHE) constitutes a gene family containing several isoforms that display different membrane localization and are involved in specialized functions. Although basolateral NHE-1 activity was described in the cortical collecting duct (CCD), the localization and function of other NHE isoforms is not yet clear, This study examines the expression, localization, and regulation of NHE isoforms in a rat cortical collecting duct cell line (RCCD1) that has previously been shown to be a good model of CCD cells. Present studies demonstrate the presence of NHE-1 and NHE-2 isoforms, but not NHE-3 and NHE-4, in RCCD1 cells. Cell monolayers, grown on permeable filters, were placed on special holders allowing independent access to apical and basolateral compartments. Intracellular pH (pHi) regulation was spectrofluorometrically studied in basal conditions and after stimulation by NH4Cl acid load or by a hyperosmotic shock. In order to differentiate the roles of NHE-1 and NHE-2, we have used HOE-694, an inhibitor more selective for NHE-1 than for NHE-2. The results obtained strongly suggest that NHE-1 and NHE-2 are expressed in the basolateral membrane but that they have different roles: NHE-1 is responsible for pHi recovery after an acid load and NHE-2 is mainly involved in steady-state pHi and cell volume regulation.

Role of AQP2 during apoptosis in cortical collecting duct cells

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.

Neuromyelitis Optica Immunoglobulin G present in sera from neuromyelitis optica patients affects aquaporin-4 expression and water permeability of the astrocyte plasma membrane

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 channels 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.

Aquaporin 2-increased renal cell proliferation is associated with cell volume regulation†

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.

Cell volume regulation in cultured human retinal Müller cells is associated with changes in transmembrane potential.

Müller 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 Müller cells have been evaluated by only a few studies. Although the activation of ion channels during the RVD response may alter transmembrane potential (Vm), no studies have actually addressed this issue in Müller cells. The aim of the present work is to evaluate RVD using a retinal Müller cell line (MIO-M1) under different extracellular ionic conditions, and to study a possible association between RVD and changes in Vm. Cell volume and Vm changes were evaluated using fluorescent probe techniques and a mathematical model. Results show that cell swelling and subsequent RVD were accompanied by Vm 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− 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 Müller 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 Vm– define the success of RVD.

Adaptation to alkalosis induces cell cycle delay and apoptosis in cortical collecting duct cells: role of Aquaporin-2.

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.

Asymmetry in the osmotic response of a rat cortical collecting duct cell line: role of aquaporin-2.

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 (Posm) 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 Posm 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.

Functional and Molecular Adaptation of Cl-/HCO3- Exchanger to Chronic Alkaline Media in RenalCells

The Cl-/HCO3- exchanger (AE) is one of the mechanisms that cells have developed to adjust pH Despite its importance, the role of AE isoforms in controlling steady-state pH during alkalosis has not been widely investigated. In the present study, we have evaluated whether conditions simulating acute and chronic metabolic alkalosis affected the transport activity and protein levels of Cl-/HCO3- exchangers in a rat cortical collecting duct cell line (RCCD1). pHi was monitored using the fluorescent dye BCECF in monolayers grown on permeable supports. Anion exchanger function was assessed by the response of pHi to acute chloride removal. RT-PCR and immunoblot assays were also performed. Our results showed that RCCD1 cells express two members of the anion exchanger gene family: AE2 and AE4. Functional studies demonstrated that while in acute alkalosis pHi became alkaline and was not regulated, after 48 h adaptation; steady-state pHi reached a value similar to the physiological one. Chronic treated cells also resulted in a 3-fold rise in Cl-/HCO3- exchange activity together with a 2.2-fold increase in AE2, but not AE4, protein abundance. We conclude that RCCD1 cells can adapt to chronic extracellular alkalosis reestablishing its steady-state pHi and that AE2 would play a key role in cell homeostasis.