Acid-base homeostasis is critical for maintaining physiological functions. An acidic tumor microenvironment, driven by altered cellular metabolism, plays a pivotal role in tumor progression by fostering aggressive phenotypes, immune evasion, and resistance to therapy, often at the detriment of surrounding normal tissues. The Na⁺/H⁺ exchanger isoform 1 (NHE1) is a key regulator of intracellular pH and a critical factor in cancer cell survival and proliferation. This study aimed to evaluate the effect of mild alkaline treatment, combined with NHE1 inhibition, on cell viability in normal renal cells and clear cell renal cell carcinoma (ccRCC) cells. Our findings reveal that this therapeutic combination selectively induces cell death in ccRCC cells while sparing normal renal cells. Mechanistically, we demonstrate that NHE1 activity is higher in ccRCC cells than in normal cells. In our experimental model, mild alkaline treatment differentially affected NHE1 activity, stimulating it in normal cells but suppressing it in cancer cells. Furthermore, prolonged alkaline exposure alters the subcellular localization of NHE1 in the plasma membrane, with distinct patterns observed between normal and cancer cells. These results suggest that targeting NHE1 activity in conjunction with alkaline treatment represents a promising strategy for ccRCC treatment, providing a potential therapeutic avenue to exploit the differential pH regulation between cancerous and normal cells.

Müller cells are essential for retinal integrity and respond to injury by activating cell proliferation, migration and differentiation into a neuronal-like phenotype for tissue remodeling. Aquaporin-4 (AQP4), the most abundant water channel of the retina, contributes to water transport and the proliferation of Müller cells, but its role in the migration of these cells remains poorly understood. Because cell migration requires localized volume changes and cytoskeletal remodeling, and AQP4 has been linked to cytoskeleton dynamics, we investigated the contribution of AQP4 to Müller cell migration. MIO-M1 migrating Müller cells were exposed to TGN-020 (TGN, AQP4 inhibitor) or vehicle and we evaluated: cell migration (wound healing), AQP4, Actin and Vimentin (immunocytochemistry) and the kinetics of cell volume (videomicroscopy). Our results show that AQP4 inhibition decreased cell migration and water transport in the lamellipodia of Müller cells exposed to hypertonic media, without affecting cell volume regulation. In migrating cells, TGN also reduced the anisotropy of F-Actin fibers, which indicates their degree of organization, and changed Vimentin localization closer to the plasma membrane. Finally, the activation of the PI3K pathway regulates AQP4 expression in migrating cells. These findings identify, for the first time, a critical role of AQP4 in the migration of Müller cells, not only facilitating volume changes for lamellipodia formation but also the remodeling of the Actin and Vimentin cytoskeleton. Our results suggest the possibility of targeting AQP4 expression and/or function as a novel therapeutic strategy to prevent glial scar formation.

During the intense neuronal activity in the retina, Müller cells are exposed to a hypotonic environment and activate a regulatory volume decrease (RVD) response, which depends on Aquaporin-4 (AQP4) and the calcium channel Transient Receptor Potential Vanilloid 4 (TRPV4). It was reported that Osteopontin (OPN), a cytokine and component of the extracellular matrix (ECM), may modulate the RVD of Müller cells. In other cell types, OPN participates in cell survival and migration, which Müller cells undergo to maintain retinal homeostasis. Therefore, the aim of this work was to study the putative crosstalk of OPN with AQP4 and/or TRPV4 in the main functions of Müller cells: RVD, morphology maintenance and migration. We used a human Müller cell line (MIO-M1) exposed to OPN and evaluated cell volume and osmotic permeability (Pf) during an osmotic swelling, AQP4 expression, cell morphology and migration. We observed that OPN induced a reduced Pf and RVD by downregulating AQP4 expression, which was prevented by TRPV4 inhibition. OPN also induced significant changes in cell morphology with an increased number of cytoplasmic projections. Finally, OPN reduced the migration of Müller cells, being this effect dependent on TRPV4. We propose that OPN affects water permeability and cell volume regulation of Müller cells by activating TRPV4 to reduce AQP4 expression. This represents a novel mechanism of regulation of water permeability by the ECM in Müller cells. Additionally, OPN-induced changes in morphology and migration of Müller cells may have an impact on retinal physiology.

Aquaporin-4 (AQP4) is the target of the specific immunoglobulin G autoantibody (AQP4-IgG) produced in patients with neuromyelitis optica spectrum disorders (NMOSD). Previous studies demonstrated that AQP4-IgG binding to astrocytic AQP4 leads to cell-destructive lesions. However, the early physiopathological events in Müller cells in the retina are poorly understood. Here, we investigated the consequences of AQP4-IgG binding to AQP4 of Müller cells, previous to the inflammatory response, on two of AQP4's key functions, cell volume regulation response (RVD) and cell proliferation, a process closely associated with changes in cell volume. Experiments were performed in a human retinal Müller cell line (MIO-M1) exposed to complement-inactivated sera from healthy volunteers or AQP4-IgG positive NMOSD patients. We evaluated AQP4 expression (immunofluorescence and western blot), water permeability coefficient, RVD, intracellular calcium levels and membrane potential changes during hypotonic shock (fluorescence videomicroscopy) and cell proliferation (cell count and BrdU incorporation). Our results showed that AQP4-IgG binding to AQP4 induces its partial internalization, leading to the decrease of the plasma membrane water permeability, a reduction of swelling-induced increase of intracellular calcium levels and the impairment of RVD in Müller cells. The loss of AQP4 from the plasma membrane induced by AQP4-IgG positive sera delayed Müller cells' proliferation rate. We propose that Müller cell dysfunction after AQP4 removal from the plasma membrane by AQP4-IgG binding could be a non-inflammatory mechanism of retinal injury in vivo, altering cell volume homeostasis and cell proliferation and consequently, contributing to the physiopathology of NMOSD.

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 RCCD1 cell line) and one inducible (control or vasopressin-induced mpkCCDc14 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.

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 Ca(2+)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 Ca(2+)and ATP differently could explain AQP2-increased cell migration.

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 AQP2‐expressing 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.

There is increasing evidence indicating that aquaporins (AQPs) exert an influence in cell signaling by the interplay with the TRPV4 Ca2+ channel. Ca2+ release from intracellular stores and plasma membrane hyperpolarization due to opening of Ca2+ -activated potassium channels (KCa) are events that have been proposed to take place downstream of TRPV4 activation. A major mechanism for Ca2+ entry, activated after depletion of intracellular Ca2+ stores and driven by electrochemical forces, is the store-operated Ca2+ 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 Ca2+ concentration and membrane potential in response to activation of TRPV4 in two rat cortical collecting duct cell lines (RCCD1 ), one not expressing AQPs (WT-RCCD1 ) and the other transfected with AQP2 (AQP2-RCCD1 ). We found that AQP2 co-immunoprecipitates with TRPV4 and with the small-conductance potassium channel (SK3). We also showed that AQP2 is crucial for the activation of SK3 by TRPV4, leading to hyperpolarization of the plasma membrane. This seems to be relevant to modulate the magnitude of SOCE and is accompanied by TRPV4 translocation to the plasma membrane only in AQP2 expressing cells. These findings open the perspective to further investigate whether the interplay between different AQPs with TRPV4 and KCa channels can be an important mechanism to modulate SOCE with physiological relevance.

Neuronal activity in the retina generates osmotic gradients that lead to Müller cell swelling, followed by a regulatory volume decrease (RVD) response, partially due to the isoosmotic efflux of KCl and water. However, our previous studies in a human Müller cell line (MIO-M1) demonstrated that an important fraction of RVD may also involve the efflux of organic solutes. We also showed that RVD depends on the swelling-induced Ca2+ release from intracellular stores. Here we investigate the contribution of taurine (Tau) and glutamate (Glu), the most relevant amino acids in Müller cells, to RVD through the volume-regulated anion channel (VRAC), as well as their Ca2+ dependency in MIO-M1 cells. Swelling-induced [3H]Tau/[3H]Glu release was assessed by radiotracer assays and cell volume by fluorescence videomicroscopy. Results showed that cells exhibited an osmosensitive efflux of [3H]Tau and [3H]Glu (Tau > Glu) blunted by VRAC inhibitors 4-(2-butyl-6,7-dichloro-2-cyclopentylindan-1-on-5-yl)-oxybutyric acid and carbenoxolone reducing RVD. Only [3H]Tau efflux was mainly dependent on Ca2+ release from intracellular stores. RVD was unaffected in a Ca2+-free medium, probably due to Ca2+-independent Tau and Glu release, but was reduced by chelating intracellular Ca2+. The inhibition of phosphatidylinositol-3-kinase reduced [3H]Glu efflux but also the Ca2+-insensitive [3H]Tau fraction and decreased RVD, providing evidence of the relevance of this Ca2+-independent pathway. We propose that VRAC-mediated Tau and Glu release has a relevant role in RVD in Müller cells. The observed disparities in Ca2+ influence on amino acid release suggest the presence of VRAC isoforms that may differ in substrate selectivity and regulatory mechanisms, with important implications for retinal physiology. NEW & NOTEWORTHY The mechanisms for cell volume regulation in retinal Müller cells are still unknown. We show that swelling-induced taurine and glutamate release mediated by the volume-regulated anion channel (VRAC) largely contributes the to the regulatory volume decrease response in a human Müller cell line. Interestingly, the hypotonic-induced efflux of these amino acids exhibits disparities in Ca2+-dependent and -independent regulatory mechanisms, which strongly suggests that Müller cells may express different VRAC heteromers formed by the recently discovered leucine-rich repeat containing 8 (LRRC8) proteins.

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 Ca2+ 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 Ca2+ influx via TRPV4 contributes to RVD by modifying intracellular Ca2+ concentration and/or modulating Vm in MIO-M1 cells. Cell volume, intracellular Ca2+ 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 Ca2+ levels was due to both Ca2+ release from intracellular stores and Ca2+ influx by a pathway alternative to TRPV4. TRPV4 blockage affected swelling-induced biphasic response (depolarization-repolarization), suggesting its participation in modulating Vm changes during RVD. Agonist stimulation of Ca2+ influx via TRPV4 activated K+ channels hyperpolarizing Vm and accelerating RVD. We propose that TRPV4 forms a signaling complex with Ca2+ and/or voltage-dependent K+ channels to define resting Vm and Vm changes during RVD. TRPV4 involvement in RVD depends on the type of stimuli and/or degree of channel activation, leading to a maximum RVD response when Ca2+ influx overcomes a threshold and activates further signaling pathways in cell volume regulation. J. Cell. Biochem. 118: 2302-2313, 2017. © 2017 Wiley Periodicals, Inc.

ABSTRACTWe 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 were increased in AQP2‐expressing cells. NHE2 inhibition decreased cell proliferation and delayed cell cycle progression by slowing S and G2/M phases only if AQP2 was expressed. Finally, we observed that only in AQP2‐expressing cells a NHE2‐dependent RVI response was activated in the S phase. These observations suggest that the AQP2‐increased proliferation involves the activation of a regulatory volume increase mechanism dependent on NHE2. Therefore, we propose that the accelerated proliferation of AQP2‐expressing cells requires a coordinated modulation of the RVD/RVI activity that contributes to cell volume changes during cell cycle progression. J. Cell. Biochem. 118: 967–978, 2017. © 2016 Wiley Periodicals, Inc.

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.

We have previously demonstrated that renal cortical collecting duct cells (RCCD 1 ), 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‐RCCD 1 (not expressing aquaporins) and in AQP2‐RCCD 1 (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.

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.

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.

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. (C) 2010 Wiley-Liss, Inc.

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.Results. Apoptosis was induced by incubating cells with a hypertonic solution or with cycloheximide in two cortical collecting duct cell lines: one not expressing aquaporins and the other stably transfected with aquaporin-2. Typical features of apoptosis were evaluated with different approaches and the-water permeability was measured by fluorescence videomicroscopy. Our results show that the rate of apoptosis is significantly increased in aquaporin-2 cells and it is linked to the rapid activation of volume-regulatory potassium and chloride channels. Furthermore, the water permeability of cells expressing aquaporin-2 was strongly reduced during the apoptotic process and it occurs before DNA degradation.Conclusions. These results let us propose that under apoptotic stimulation aquaporin-2 would act as a sensor leading to a co-ordinated activation of specific ionic channels for potassium and chloride efflux, resulting in both more rapid cell shrinkage and more rapid achievement of adequate levels of ions necessary to activate the enzymatic apoptotic cascade.