Fine regulation of water reabsorption by the antidiuretic hormone [8-arginine]vasopressin (AVP) occurs in principal cells of the collecting duct and is largely dependent on regulation of the aquaporin-2 (AQP2) water channel. AVP-inducible long term AQP2 expression was investigated in immortalized mouse cortical collecting duct principal cells. Combined RNase protection assay, Western blot, and immunofluorescence analyses revealed that physiological concentrations of AVP added to the basal side, but not to the apical side, of cells grown on filters induced both AQP2 mRNA and apical protein expression. The stimulatory effect of AVP on AQP2 expression followed a V 2 receptor-dependent pathway because [deamino-8-D-arginine]vasopressin (dDAVP), a specific V 2 receptor agonist, produced the same effect as AVP, whereas the V 2 antagonist SR121463B antagonized action of both AVP and dDAVP. Moreover, forskolin and cyclic 8-bromo-AMP fully reproduced the effects of AVP on AQP2 expression. Analysis of protein degradation pathways showed that inhibition of proteasomal activity prevented synthesis of AVP-inducible AQP2 mRNA and protein. Once synthesized, AQP2 protein was quickly degraded, a process that involves both the proteasomal and lysosomal pathways. This is the first study that delineates induction and degradation mechanisms of AQP2 endogenously expressed by a renal collecting duct principal cell line.Kidneys are the major determinant of body water and electrolyte composition. Water reabsorption across the membranes of renal epithelial cells occurs through a complex process. Approximately 70 and 15% of the glomerular filtrate is reabsorbed in the proximal tubule and thin descending limb of Henle's loop, respectively. In contrast, the ascending limb of Henle's loop and the distal convoluted tubule are impermeable to water. These segments empty into the collecting duct (CD), 1 the chief site where tight regulation of water reabsorption occurs. In this segment, and in the connecting tubule of some species as well (1, 2), the excretion of electrolyte-free water is adjusted by principal cells under the control of the antidiuretic hormone [8-arginine]vasopressin (AVP) (3). Water movement across renal epithelial cells is facilitated by the presence of water channels of the aquaporin (AQP) protein family. Aquaporins exhibit a conserved homotetrameric organization, and the expression of different members of the aquaporin family is tissue-specific. AQP1 accounts for the transcellular selective water permeability of the proximal tubule and thin descending limb of Henle's loop (4, 5) and is constitutively expressed in the apical and basolateral membrane domains of both of these segments (6). Three aquaporins (AQP2, AQP3, and AQP4) are expressed in collecting duct principal cells where AVP regulates water reabsorption across the principal cell epithelium. AQP2 is located in subapical intracellular vesicles and in the apical plasma membrane (7, 8), whereas AQP3 and AQP4 are both located in the basolateral membrane (9, 10). Of all a...

In the renal collecting duct (CD) the major physiological role of aldosterone is to promote Na ؉ reabsorption. In addition, aldosterone may also influence CD water permeability elicited by vasopressin (AVP). We have previously shown that endogenous expression of the aquaporin-2 (AQP2) water channel in immortalized mouse cortical CD principal cells (mpkCCD C14 ) grown on filters is dramatically increased by administration of physiological concentrations of AVP. In the present study, we investigated the influence of aldosterone on AQP2 expression in mpkCCD C14 cells by RNase protection assay and Western blot analysis. Aldosterone reduced AQP2 mRNA and protein expression when administered together with AVP for short periods of time (<24 h). For longer periods of time, however, aldosterone increased AQP2 protein expression despite sustained low expression levels of AQP2 mRNA. Both events were dependent on mineralocorticoid receptor occupancy because they were both induced by a low concentration of aldosterone (10 ؊9 M) and were abolished by the mineralocorticoid receptor antagonist canrenoate. Inhibition of lysosomal AQP2 protein degradation increased AQP2 protein expression in AVP-treated cells, an effect that was potentiated by aldosterone. Finally, both aldosterone and actinomycin D delayed AQP2 protein decay following AVP washout, but in a non-cumulative manner. Taken together, our data suggest that aldosterone tightly modulates AQP2 protein expression in cultured mpkCCD C14 cells by increasing AQP2 protein turnover while maintaining low levels of AQP2 mRNA expression.Water permeability of the renal collecting duct (CD) 1 depends almost exclusively on the antidiuretic hormone [8-arginine]vasopressin (AVP), which exerts its action principally through tight regulation of aquaporin-2 (AQP2) expression. AQP2 belongs to the family of water channel proteins that facilitate osmotically driven water movement across cell membranes. At least six members of the AQP family (AQP1, AQP2, AQP3, AQP4, AQP6, and AQP7) are expressed in the kidney (1-4) and three of them, AQP2, AQP3, and AQP4, are expressed in CD principal cells (5-7). AVP increases CD water permeability by binding to the vasopressin V 2 -receptor located in the basolateral membrane of CD principal cells, an event that promotes AQP2 translocation from intracellular storage vesicles to the apical membrane (8). Water exits the cells through AQP3 and AQP4 water channels expressed in the basolateral membrane of CD cells. This process, induced by acute increases in AVP plasma concentration, occurs within minutes and is reversible. Declining levels of circulating AVP quickly lead to endocytotic retrieval of apical AQP2 and to reduced CD water permeability (8).Besides this rapid action, AVP also controls AQP2 expression over longer periods of time (from several hours to several days) (9). Accordingly, normal and AVP-deficient Brattleboro rats infused with AVP over a period of several days display increased levels of AQP2 expression (10, 13, 11). Conversely, animals admin...