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. © 2012 Wiley Periodicals, Inc.     

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. © 2010 Wiley‐Liss, Inc.     

Arginine-vasopressin modulates intracellular pH via V1 and V2 receptors in renal collecting duct cells.

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 (alkalinization-acidification), 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.     

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. 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.