Luciano Galizia

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.

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.

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.

ENaC Channels in Oocytes from Xenopus laevis and their Regulation by xShroom1 Protein

Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in X. oocytes and is required for the expression of amiloride sensitive sodium channels (ENaC). Oocytes were injected with α, β, and γ mENaC and xShroom1 sense or antisense oligonucleotides. We used voltage clamp techniques to study the amiloride-sensitive Na+ currents (INa(amil)). We observed a marked reduction in INa(amil) in oocytes co-injected with xShroom1 antisense. Oocytes expressing a DEG mutant β-mENaC subunit (β-S518K) with an open probability of 1 had enhanced INa(amil) although these currents were also reduced when co-injected with xShroom1 antisense. Addition of low concentration (20 ng/ml) of trypsin which activates the membrane-resident ENaC channels led to a slow increase in INa(amil) in oocytes with xShroom1 sense but had no effect on the currents in oocytes coinjected with ENaC and xShroom1 antisense. The same results were obtained with higher concentrations of trypsin (2 µg/ml) exposed during 2.5 min. In addition, fluorescence positive staining of plasma membrane in the oocytes expressing α, β and γ mENaC and xShroom1 sense were observed but not in oocytes coinjected with ENaC and xShroom1 antisense oligonucleotides. On this basis, we suggest that xShroom1-dependent ENaC inhibition may be through the number of channels inserted in the membrane.

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 (pHi) 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 pHi, as well as the receptors involved in these responses using a CCD cell line (RCCD1); pHi 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 RCCD1 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-/HCO3- exchanger. We conclude that in RCCD1 cells AVP selectively activates, via a complex of V1 and V2 receptor-mediated actions, different ion transporters linked to pHi regulation which might have physiological implications.

CFTR channel in oocytes from Xenopus laevis and its regulation by xShroom1 protein.

Shroom is a family of related proteins linked to the actin cytoskeleton. xShroom1 is constitutively expressed in Xenopus laevis oocytes, and it is required for the expression of the epithelial sodium channel (ENaC). As there is a close relationship between ENaC and the cystic fibrosis transmembrane regulator (CFTR), we examined the action of xShroom1 on CFTR expression and activity. Biotinylation was used to measure CFTR surface expression, and currents were registered with voltage clamp when stimulated with forskolin and 3-isobutyl-1-methylxanthine. Oocytes were coinjected with CFTR complementary RNAs (cRNAs) and xShroom1 sense or antisense oligonucleotides. We observed an increment in CFTR currents and CFTR surface expression in oocytes coinjected with CFTR and xShroom1 antisense oligonucleotides. MG-132, a proteasome inhibitor, did not prevent the increment in currents when xShroom1 was suppressed by antisense oligonucleotides. In addition, we inhibited the delivery of newly synthesized proteins to the plasma membrane with BFA and we found that the half-life of plasma membrane CFTR was prolonged when coinjected with the xShroom1 antisense oligonucleotides. Chloroquine, an inhibitor of the late endosome/lysosome, did not significantly increase CFTR currents when xShroom1 expression was inhibited. The higher expression of CFTR when xShroom1 is suppressed is in concordance with the functional studies suggesting that the suppression of the xShroom1 protein resulted in an increment in CFTR currents by promoting the increase of the half-life of CFTR in the plasma membrane. The role of xShroom1 in regulating CFTR expression could be relevant in the understanding of the channel malfunction in several diseases.

xshroom1 Regulates the Number of ENaC Channels Inserted in the Membrane of Oocytes From XENOPUS Laevis

Shroom is a family of proteins linked to the actin cytoskeleton. We studied its effect upon the currents through ENaC channels (INa amil) in oocytes (X. laevis) injected with α, β, and γ mENaC and xShroom1 sense or antisense oligonucleotides. We observed a strong reduction in INa amil with the xShroom1 antisense: inward conductances (Ginward) (−160 to 0 mV) were 36 ± 12 µS and 1.80 ±.50 µS with xShroom1 sense and antisense. Similar results were obtained in oocytes expressing a mutant β-mENaC subunit (β-S518K) with a open probability of 1 (Ginward 65 ± 10 µS and 1.80 ± 2.0 µS for oocytes with xShroom1 sense or xShroom1 antisense. The negative effects of xShroom1 antisense can not be reversed with forskolin which reduced the rate of ENaC retrieval: Ginward : 124 ± 27 µS and 7.0 ± 1.9 µS with xShroom1 sense or xShroom1 antisense. Trypsin in the range of ng/ml activates the membrane-resident ENaC channels (Bengrine et al.2007), being this effect dependent on activation of G-proteins. Addition of 20 ng/ml of trypsin led to a slow increase in INa amil with xShroom1 sense and it had no effect in most of the oocytes coinjected with ENaC and xShroom1 antisense (2 out of 20). Trypsin were without effects on the endogenous conductances. These data are consistent with the idea that the reduced INa amil when xShroom1 is blocked is most probably due to a lack of functional ENaC channels in the plasma membrane.Acknowledgements: ENaC cDNAs were provided by Dr M. Carattino (Pittsburgh, Pa). and the set for oocytes was a gift of Dr C. Peracchia (Rochester, NY). Supported by grants MO35 (UBA) and PICT 38181.