Claudia Capurro

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.

Distribution of mRNA encoding the FA-CHIP water channel in amphibian tissues: Effects of salt adaptation

A water channel, the frog aquaporin-CHIP (FA-CHIP) was recently cloned from Rana esculenta urinary bladder. The 28.9 kDa encoded protein shows 78.8%, 77.4%, 42.4% and 35.6% identity with rat CHIP28, human CHIP28, rat WCH-CD and γ-TIP, other members of the new transmembrane water channel family (Aquaporin-CHIP). We have now studied membranes from different frog (R. esculenta) organs employing semiquantitative PCR using FA-CHIP specific primers and an internal standard to quantify the PCR products. The FA-CHIP mRNA was abundantly expressed in the frog urinary bladder, skin, lung and gall bladder, while a lower expression was detected in the colon, liver and oviduct. FA-CHIP mRNA was not detected in the frog kidney, erythrocytes and brain but its expression was observed in the toad (Bufo arenarum) urinary bladder and skin, showing that FA-CHIP is probably a general amphibian water channel. Salt acclimation is known to increase the water permeability of frog and toad epithelia. We have now observed that salt acclimation for 1, 3, 4 or 5 days markedly increased skin and urinary bladder FA-CHIP mRNA expression. It is generally accepted that water permeability is controlled in these tissues by the rate of water channel transfer from subapical vesicles (aggrephores) to the apical membrane. Our results indicate that water permeability is also regulated at the level of the FA-CHIP transcription.

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.

Uroguanylin Regulates Net Fluid Secretion via the NHE2 Isoform of the Na+/H+ Exchanger in an Intestinal Cellular Model

Uroguanylin (UGN) has been proposed as a key regulator of salt and water intestinal transport. Uroguanylin activates cell-surface guanylate cyclase C receptor (GC-C) and modulates cellular function via cyclic GMP (cGMP), thus increasing electrolyte and net water secretion. It has been suggested that the action of UGN could involve the Na+/H+ exchanger, but the actual contribution of this transporter still remains unclear. The objective of our study was to investigate the putative effects of UGN on some members of the Na+/H+ exchanger family (NHEs), as well as to clarify its consequences on transepithelial fluid flow in T84 cells. In order to do so, transepithelial fluid flow (Jv) was studied by optic techniques and intracellular pH (pHi) was measured with a fluorescence method. Results showed that NHE2 is found at the apical membrane and has a major role in Na+ absorption; NHE1 and NHE4 are localized at the basolateral membrane with a house-keeping role in steady state pHi. In the assayed conditions, cell exposure to apical UGN increases net secretory Jv, without changing short-circuit currents nor transepithelial resistance, and reduces NHE2 activity. Therefore, at physiological pH, the effect on net Jv was produced mainly by a reduction in normal Na+ absorption through NHE2, rather than by the stimulation of electrolyte secretion. Our study shows that the effect of UGN on pHi is GC-C/cGMP-mediated and enhanced by sildenafil, thus involving PDE5 enzyme. Additionally, cell exposure to apical UGN results in intracellular alkalinization, probably due to indirect effects on basolateral NHE1 and NHE4, which have a major role in pHi 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.

Water permeability properties of the ovarian oocytes from Bufo arenarum and Xenopus laevis : a comparative study

The water permeability properties of ovarian oocytes from Xenopus laevis and Bufo arenarum, a toad species found in the Buenos Aires region, were studied. We report that: (i) the water osmotic permeability (Pf, cm/sec × 10−4) was significantly higher in Bufo (6°C=12.3±2.4; 18°C = 20.8±4.8) than in Xenopus oocytes (6°C=5.3±0.3; 18°C=6.2±1.6). The corresponding water diffusion permeability values (Pd, cm/sec × 10−4) were: Xenopus = 2.3±0.3 (6°C) and 4.8±0.7 (18°C); Bufo=2.7±0.4 (6°C) and 6.0 ±0.5 (18°C). (ii) Amphotericin B increased the Pf and Pd values. The observed ΔPfΔPd ratio was not significantly different from the expected results (n=3), after amphotericin B incorporation in both species. This means that the influence of unstirred layers and other potential artifactual compounds did not significantly affect our experimental results, (iii) Preincubation with gramicidin during 12 hr induced a clear increase in the oocyte volume. After that, a hypotonic shock only slightly increased the oocyte volume. Conversely, a hypertonic challenge induced a volume change significantly higher than the one observed in control conditions, (iv) Mercury ions did not affect the osmotic permeability in Xenopus oocytes but clearly inhibited, in a reversible way, the osmotic permeability in oocytes from B. arenarum. (v) Mercury ions did not reduce Pd values in either species, (vi) The ΔPfΔPd values calculated from the differences observed in these parameters between both species were 11.9±5.1 at 18°C and 15.5±2.4 at 6°C. These numbers are similar to those previously reported in the case of membranes having water channels. From these results, we propose that water channels are present in the ovarian oocyte from B. arenarum but not in the ovarian oocyte from X. laevis.