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• W1994587569 abstract "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 (mpkCCDC14) 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 mpkCCDC14 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 (10–9m) 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 mpkCCDC14 cells by increasing AQP2 protein turnover while maintaining low levels of AQP2 mRNA expression. 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 (mpkCCDC14) 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 mpkCCDC14 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 (10–9m) 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 mpkCCDC14 cells by increasing AQP2 protein turnover while maintaining low levels of AQP2 mRNA expression. Water permeability of the renal collecting duct (CD) 1The abbreviations used are: CD, collecting duct; AQP, aquaporin; AVP, arginine-8 vasopressin; MR, mineralocorticoid receptor; RPA, RNase protection assay.1The abbreviations used are: CD, collecting duct; AQP, aquaporin; AVP, arginine-8 vasopressin; MR, mineralocorticoid receptor; RPA, RNase protection assay. 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 (1Denker B.M. Smith B.L. Kuhajda F.P. Agre P. J. Biol. Chem. 1988; 263: 15634-15642Abstract Full Text PDF PubMed Google Scholar, 2Maunsbach A.B. Marples D. Chin E. Ning G. Bondy C. Agre P. Nielsen S. J. Am. Soc. Nephrol. 1997; 8: 358-360Google Scholar, 3Yasui M. Kwon T.H. Knepper M.A. Nielsen S. Agre P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5808-5813Crossref PubMed Scopus (216) Google Scholar, 4Nejsum L.N. Elkjaer M. Hager H. Frokiaer J. Kwon T.H. Nielsen S. Biochem. Biophys. Res. Commun. 2000; 277: 164-170Crossref PubMed Scopus (73) Google Scholar) and three of them, AQP2, AQP3, and AQP4, are expressed in CD principal cells (5Fushimi K. Uchida S. Hara Y. Hirata Y. Marumo F. Sasaki S. Nature. 1993; 361: 549-552Crossref PubMed Scopus (863) Google Scholar, 6Ishibashi K. Sasaki S. Fushimi K. Uchida S. Kuwahara M. Saito H. Furukawa T. Nakajima K. Yamaguchi Y. Gojobori T. Marumo F. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6269-6273Crossref PubMed Scopus (528) Google Scholar, 7Terris J. Ecelbarger C.A. Marples D. Knepper M.A. Nielsen S. Am. J. Physiol. 1995; 269: F775-F785Crossref PubMed Google Scholar). AVP increases CD water permeability by binding to the vasopressin V2-receptor located in the basolateral membrane of CD principal cells, an event that promotes AQP2 translocation from intracellular storage vesicles to the apical membrane (8Nielsen S. Chou C.L. Marples D. Christensen E.I. Kishore B.K. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1013-1017Crossref PubMed Scopus (877) Google Scholar). 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 (8Nielsen S. Chou C.L. Marples D. Christensen E.I. Kishore B.K. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1013-1017Crossref PubMed Scopus (877) Google Scholar). Besides this rapid action, AVP also controls AQP2 expression over longer periods of time (from several hours to several days) (9Nielsen S. DiGiovanni S.R. Christensen E.I. Knepper M.A. Harris H.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11663-11667Crossref PubMed Scopus (661) Google Scholar). Accordingly, normal and AVP-deficient Brattleboro rats infused with AVP over a period of several days display increased levels of AQP2 expression (10Hayashi M. Sasaki S. Tsuganezawa H. Monkawa T. Kitajima W. Konishi K. Fushimi K. Marumo F. Saruta T. J. Clin. Invest. 1994; 94: 1778-1783Crossref PubMed Scopus (168) Google Scholar, 13Ecelbarger C.A. Nielsen S. Olson B.R. Murase T. Baker E.A. Knepper M.A. Verbalis J.G. J. Clin. Invest. 1997; 99: 1852-1863Crossref PubMed Scopus (202) Google Scholar, 11DiGiovanni S.R. Nielsen S. Christensen E.I. Knepper M.A. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8984-8988Crossref PubMed Scopus (369) Google Scholar). Conversely, animals administered with V2-receptor antagonists display decreased levels of AQP2 expression (12Marples D. Christensen B.M. Frokiaer J. Knepper M.A. Nielsen S. Am. J. Physiol. 1998; 275: F400-F409PubMed Google Scholar). Several studies, however, have documented conditions under which AQP2 expression can be influenced by mechanisms other than AVP. Decreased levels of AQP2 expression that occur despite normal or even increased circulating AVP levels have been described in nephrotic syndrome (14Apostol E. Ecelbarger C.A. Terris J. Bradford A.D. Andrews P. Knepper M.A. J. Am. Soc. Nephrol. 1997; 8: 15-24PubMed Google Scholar), chronic renal failure (15Kwon T.H. Frokiaer J. Knepper M.A. Nielsen S. Am. J. Physiol. 1998; 275: F724-F741PubMed Google Scholar), hypokalemia (16Marples D. Frokiaer J. Dorup J. Knepper M.A. Nielsen S. J. Clin. Invest. 1996; 97: 1960-1968Crossref PubMed Scopus (253) Google Scholar), hypercalcemia (17Earm J.H. Christensen B.M. Frokiaer J. Marples D. Han J.S. Knepper M.A. Nielsen S. J. Am. Soc. Nephrol. 1998; 9: 2181-2193PubMed Google Scholar), lithium administration (18Marples D. Christensen S. Christensen E.I. Ottosen P.D. Nielsen S. J. Clin. Invest. 1995; 95: 1838-1845Crossref PubMed Scopus (391) Google Scholar), and liver cirrhosis (19Jonassen T.E. Nielsen S. Christensen S. Petersen J.S. Am. J. Physiol. 1998; 275: F216-F225Crossref PubMed Google Scholar), whereas increased levels of AQP2 expression have been reported in pregnant rats despite normal levels of circulating AVP (20Ohara M. Martin P.-Y. Xu D.L. St. John J. Pattison T.A. Kim J.K. Schrier R.W. J. Clin. Invest. 1998; 101: 1076-1083Crossref PubMed Scopus (104) Google Scholar). Several factors are expected to influence long term AQP2 expression. In the present study, we investigated the influence of aldosterone on AQP2 mRNA and protein expression. The major role of aldosterone is to promote unidirectional Na+ transport across a variety of epithelial tissues of high intercellular electrical resistance (21Funder J.F. Clin. Exp. Pharmacol. Physiol. Suppl. 1998; 25: S47-S50Crossref PubMed Scopus (23) Google Scholar). In the kidney, aldosterone promotes Na+ reabsorption principally by increasing whole cell abundance and cell membrane expression of apical epithelial Na+ channels and basolateral Na,K-ATPase units in the late portion of the distal tubule and in the collecting duct (22MacDonald P. MacKenzie S. Ramage L.E. Seckl J.R. Brown R.W. J. Endocrinol. 2000; 165: 25-37Crossref PubMed Scopus (34) Google Scholar, 23Loffing J. Zecevic M. Feraille E. Kaissling B. Asher C. Rossier B.C. Firestone G.L. Pearce D. Verrey F. Am. J. Physiol. 2001; 280: F675-F682Crossref PubMed Google Scholar, 24Feraille E. Doucet A. Physiol. Rev. 2001; 81: 345-418Crossref PubMed Scopus (427) Google Scholar, 25Verrey F. Hummler E. Schild L. Rossier B. Seldin D.W. The Kidney, Physiology and Pathophysiology. 3rd Ed. Lippincott, Williams and Wilkins, Philadelphia, PA2000: 1441-1471Google Scholar). Besides its role in controlled Na+ reabsorption, some pieces of evidence suggest that aldosterone influences water reabsorption as well. Patients with chronic adrenal insufficiency are unable to generate maximally concentrated urine (26Wilson D.M. Sundermann F.W. J. Clin. Invest. 1939; 18: 35-43Crossref PubMed Google Scholar), a situation that can be reproduced in adrenalectomized rats (27Schwartz M.J. Kokko J.P. J. Clin. Invest. 1980; 66: 234-242Crossref PubMed Scopus (55) Google Scholar), and administration of the aldosterone antagonist spironolactone was found to increase dilute urine production in patients with severe congestive heart failure (28van Vliet A.A. Donker A.J. Nauta J.J. Verheugt F.W. Am. J. Cardiol. 1993; 71 (A): 21A-228Abstract Full Text PDF PubMed Scopus (148) Google Scholar). The effect of aldosterone on AQP2 expression in animals has lead to apparently contrasting results. Whereas normal rats treated with the mineralocorticoid receptor antagonist canrenoate displayed increased urinary output and decreased renal AQP2 expression (29Jonassen T.E. Promeneur D. Christensen S. Petersen J.S. Nielsen S. Am. J. Physiol. 2000; 278: F246-F256Google Scholar), AQP2 expression levels in hypovolemic rats with selective aldosterone deficiency were found to be increased in the inner medulla (30Ohara M. Cadnapaphornchai M.A. Summer S.N. Falk S. Yang J. Togawa T. Schrier R.W. Biochem. Biophys. Res. Commun. 2002; 299: 285-290Crossref PubMed Scopus (31) Google Scholar) but remained unchanged at the level of the whole kidney (31Kwon T-H. Nielsen J. Masilamani S. Hager H. Knepper M.A. Frokiaer J. Nielsen S. Am. J. Physiol. 2002; 283: F1403-F1421Crossref PubMed Scopus (68) Google Scholar). The aim of the present study was to investigate the effect of aldosterone on long term AQP2 expression. To this end, we used the immortalized clonal collecting duct mpkCCDC14 cell line derived from microdissected cortical collecting ducts of a SVPK/Tag transgenic mouse (32Bens M. Vallet V. Cluzeaud F. Pascual-Letallec L. Kahn A. RafestinOblin M.E. Rossier B.C. Vandewalle A. J. Am. Soc. Nephrol. 1999; 10: 923-934Crossref PubMed Google Scholar). When grown on permeable filters, these cells form a tight epithelial monolayer that exhibits many major functional properties of CD principal cells including electrogenic Na+ transport stimulated by aldosterone and AVP (32Bens M. Vallet V. Cluzeaud F. Pascual-Letallec L. Kahn A. RafestinOblin M.E. Rossier B.C. Vandewalle A. J. Am. Soc. Nephrol. 1999; 10: 923-934Crossref PubMed Google Scholar, 33Vandewalle A. Bens M. Duong Van Huyen J.P. Curr. Opin. Nephrol. Hypertens. 1999; 8: 581-587Crossref PubMed Scopus (18) Google Scholar). In addition, we have recently shown that the low expression levels of endogenous AQP2 mRNA and protein in mpkCCDcl4 cells are rapidly up-regulated by physiological concentrations of AVP (34Hasler U. Mordasini D. Bens M. Bianchi M. Cluzeaud F. Rousselot M. Vandewalle A. Feraille E. Martin P.-Y. J. Biol. Chem. 2002; 22: 10379-10386Abstract Full Text Full Text PDF Scopus (150) Google Scholar). The results of the present study indicate that aldosterone significantly alters AQP2 whole cell protein content in cultured mpkCCDcl4 cells by modulating both AQP2 mRNA abundance and translational regulation. Cell Culture—mpkCCDC14 cells (passages 20–30) were grown in modified defined medium (DM: Dulbecco's modified Eagle's medium/Ham's F-12, 1:1 (v/v), 60 nm sodium selenate, 5 μg/ml transferrin, 2 mm glutamine, 50 nm dexamethasone, 1 nm triiodothyronine, 10 ng/ml epidermal growth factor, 5 μg/ml insulin, 20 mm d-glucose, 2% fetal calf serum, and 20 mm HEPES, pH 7.4) (32Bens M. Vallet V. Cluzeaud F. Pascual-Letallec L. Kahn A. RafestinOblin M.E. Rossier B.C. Vandewalle A. J. Am. Soc. Nephrol. 1999; 10: 923-934Crossref PubMed Google Scholar) at 37 °C in 5% CO2, 95% air atmosphere. The medium was changed every 2 days. Experiments were performed on confluent cells seeded on semi-permeable polycarbonate filters (Transwell™, 0.4 μm pore size, 1 cm2 growth area, Corning Costar, Cambridge, MA). Cells were grown in DM until confluence (day 6 after seeding), and then placed in serum-free, hormone-deprived DM 24 h before the experiments. Western Blot Analysis—After incubation in the presence of AVP and/or drugs, confluent cultured mpkCCDC14 cells grown on filters were incubated with AVP and without or with aldosterone and/or drugs to be tested. Cells were then rinsed twice with phosphate-buffered saline and then homogenized in 150 μl of ice-cold lysis buffer (20 mm Tris-HCl, 2 mm EGTA, 2 mm EDTA, 30 mm sodium fluoride, 30 mm Na4O7P2, 2 mm Na3VO4, 1 mm 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 μg/ml leupeptin, 4 μg/ml aprotinin, 1% Triton X-100, pH 7.4). Protein concentrations were measured by the BCA protein assay (Pierce). Equal amounts of protein from lysed samples were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilion-P, Millipore, Bedford, MA). Membranes were blocked by incubation with Tris-buffered saline (50 mm Tris, 150 mm NaCl) containing 0.2% (v/v) Nonidet P-40 and 5% (w/v) nonfat dry milk for 30 min at room temperature. Membranes were probed overnight at 4 °C with a polyclonal rabbit anti-rat AQP2 antibody (1:20000) (35Xu D.L. Martin P.-Y. Ohara M. St. John J. Pattison T. Meng X. Morris K. Kim J.K. Schrier R.W. J. Clin. Invest. 1997; 99: 1500-1505Crossref PubMed Scopus (273) Google Scholar) or with a polyclonal rabbit anti-mouse protein kinase Aα catalytic subunit antibody (1:2000, Santa Cruz Biotechnology, Santa Cruz, CA) washed three times with Tris-buffered saline containing 0.2% (v/v) Nonidet P-40, and then incubated with secondary horseradish peroxidase-conjugated goat anti-rabbit IgG (1:20000) (Transduction Laboratories, Lexington, KY) for 1 h at room temperature. The membranes were washed three times with Tris-buffered saline containing 0.2% (v/v) Nonidet P-40 and the antigen-antibody complexes were detected by the Super Signal Substrate method (Pierce). Identified protein bands were quantified using a video densitometer and ImageQuant software (Amersham Biosciences). RNase Protection Assay—Mouse genomic DNA was extracted from mpkCCDC14 cells using the DNeasy Tissue Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions. A mouse AQP2 cDNA (NCBI accession number NM_009699) fragment coding for the 81–279 nucleotide sequence was PCR amplified using sense and antisense primers containing EcoRI and XbaI restriction sites, respectively, and cloned into pCIneo (Promega, Madison, WI). Computer-assisted alignment sequence analyses confirmed the specificity of the cDNA fragment used. The sequence of the PCR-amplified fragment was checked by sequencing. The plasmid was then linearized with NheI restriction enzyme and 1 μg was used to produce antisense AQP2 transcripts (riboprobes) using T3 RNA polymerase in the presence of 50 μCi of [α-32P]UTP (Amersham Biosciences). Acidic ribosomal phosphoprotein P0 (EMBL accession number BC011106) antisense probe was used as an internal standard. A fragment coding for the 170–315-nucleotide sequence was PCR amplified using sense and antisense primers containing EcoRI and XbaI restriction sites, respectively, and cloned into pBluescript SK– (Stratagene, La Jolla, CA). Computer-assisted alignment sequence analyses confirmed the specificity of the cDNA fragment used, and the sequence of the PCR amplified fragment was checked by sequencing. The plasmid was then linearized with the XhoI restriction enzyme and 1 μg was used to produce antisense P0 riboprobes using T3 RNA polymerase in the presence of 5 μCi of [α-32P]UTP to avoid signal saturation because of the greater abundance of P0 rRNA as compared with AQP2 rRNA. Total RNA was extracted from cultured cells using the RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. Ten μg of riboprobe completed with 21 μg of yeast tRNA were used for hybridization with 2 × 105 cpm of AQP2 probe and 5 × 104 cpm of P0 probe. Yeast tRNA (25 μg) was used as a negative control. Hybridization was performed for 60 min at 70 °C followed by RNase A/T1 mixture digestion (Ambion, Austin, TX) for 30 min at 37 °C. The reaction was terminated by the addition of SDS and proteinase K. RNA duplexes were extracted with phenol/chloroform/isoamyl alcohol and precipitated with ammonium acetate/ethanol. Samples were then denatured in gel loading buffer at 95 °C for 5 min, run together with non-digested riboprobes on a 6% polyacrylamide sequencing gel, and autoradiographed. Identified fragments were quantified using a video densitometer and ImageQuant software (Amersham Biosciences). Statistics—Results are given as the mean ± S.E. from n independent experiments. Each experiment was performed on cultured cells from the same passage. Statistical differences were assessed using the Mann-Whitney U test or the Kruskal-Wallis test for comparison of two or more than two groups, respectively. A p < 0.05 was considered significant. Effects of Aldosterone on AVP-induced AQP2 Protein Expression in Mouse Collecting Duct Principal Cells—We have previously shown that the low levels of endogenous AQP2 mRNA and protein expression in untreated mpkCCDC14 cells grown on filters can be dramatically increased by addition of physiological concentrations of AVP to the basal medium (34Hasler U. Mordasini D. Bens M. Bianchi M. Cluzeaud F. Rousselot M. Vandewalle A. Feraille E. Martin P.-Y. J. Biol. Chem. 2002; 22: 10379-10386Abstract Full Text Full Text PDF Scopus (150) Google Scholar). In the present study, we examined the effects of aldosterone on AQP2 abundance in mpkCCDC14 cells treated with AVP. We first analyzed both short and long term aldosterone action by treating mpkCCDC14 cells with AVP alone or with both AVP and aldosterone for various lengths of time. Two different protocols were used for this study. In a first set of experiments, endogenous AQP2 expression was first increased by pretreating mpkCCDC14 cells with 10–9m AVP for 24 h after which time the cells were subjected to additional periods of incubation (3–48 h) in the continuous presence of AVP and in the presence or absence of 10–6m aldosterone (Fig. 1, A and B). In a second set of experiments, mpkCCDC14 cells were treated with either 10–9m AVP alone or with both 10–9m AVP and 10–6m aldosterone, added simultaneously to the cell medium, for 3–48 h (Fig. 1, C and D). We used AVP at a concentration of 10–9m to maximally stimulate AQP2 expression (34Hasler U. Mordasini D. Bens M. Bianchi M. Cluzeaud F. Rousselot M. Vandewalle A. Feraille E. Martin P.-Y. J. Biol. Chem. 2002; 22: 10379-10386Abstract Full Text Full Text PDF Scopus (150) Google Scholar). Aldosterone was used at 10–6m because this concentration maximally stimulates sodium transport in mpkCCDcl4 cells (32Bens M. Vallet V. Cluzeaud F. Pascual-Letallec L. Kahn A. RafestinOblin M.E. Rossier B.C. Vandewalle A. J. Am. Soc. Nephrol. 1999; 10: 923-934Crossref PubMed Google Scholar). Western blot analysis of AVP-treated mpkCCDC14 cells typically revealed a narrow 28-kDa band and a more diffuse band of about 35 kDa corresponding to the non-glycosylated and fully glycosylated forms of AQP2 protein, respectively. An additional faint band of about 31 kDa corresponding to the core-glycosylated form of AQP2 protein was also detected (Fig. 1, A and C). Although the amount of AQP2 protein increased over time in cells treated with AVP alone and in cells treated with both AVP and aldosterone, aldosterone was found to significantly alter AQP2 protein content when added 24 h after AVP and when added simultaneously with AVP. Indeed, AQP2 protein content significantly decreased shortly (≤24 h) following administration of aldosterone, as compared with AQP2 expression levels of cells treated with AVP alone (Fig. 1, A and C, lanes 1–10, and B and D). Longer periods (48 h) of aldosterone stimulation, however, significantly increased AQP2 protein expression as compared with that of cells treated with AVP alone (Fig. 1, A and C, lanes 11 and 12, and B and D). Both effects were mediated by aldosterone, i.e. down- and up-regulated abundance of AQP2 protein, could be reproduced in mpkCCDcl4 cells first pretreated 24 h with 10–6m aldosterone and then exposed to 10–9m AVP for additional lengths of time (3–48 h) (data not shown). Cells treated with aldosterone alone did not exhibit any change in AQP2 expression at any time (data not shown). These results indicate that aldosterone exerts a biphasic effect on AVP-induced AQP2 protein expression in mpkCCDcl4 cells: for short periods of time (≤24 h) aldosterone decreases AVP-elicited AQP2 protein expression whereas the steroid hormone enhances AQP2 expression for longer periods of time (>24 h). To investigate whether the short and long term effects of aldosterone on vasopressin-stimulated AQP2 protein expression were mediated through mineralocorticoid receptors (MR), we compared the effects of 10–9m aldosterone (a concentration corresponding to half-maximal MR occupancy) and 10–6m aldosterone (a concentration corresponding to half-maximal occupancy of both MR and glucocorticoid receptor). In addition, we tested the effect of canrenoate, a specific MR antagonist. The extent of decreased AQP2 protein expression was similar in confluent mpkCCDC14 cells treated with 10–9m AVP administered together with either 10–6 or 10–9m aldosterone for 9 h (Fig. 2, A, left panel, compare lanes 2 and 3, and B) whereas AQP2 protein expression of cells simultaneously treated with 10–9m AVP and 10–6 or 10–9m aldosterone for 48 h increased by 100 and 50%, respectively, as compared with that of cells treated with AVP alone (Fig. 2, A, right panel, compare lane 1 to lanes 2 and 3, and B). Moreover, 10–6m canrenoate, which had no effect on cells treated with AVP alone, abolished both aldosterone-dependent down- and up-regulation of AQP2 protein observed after 9 and 48 h of incubation, respectively (Fig. 2, C and D). These observations suggest that both effects of aldosterone on AQP2 protein expression are mediated by the MR. In addition, the observation that aldosterone induced AQP2 down- and up-regulation when administered at a concentration of 10–9m further adds to the physiological relevance of aldosterone action on AQP2 expression in cultured mpkCCDcl4 cells. The effect of aldosterone on AQP2 mRNA expression was next investigated by RNase protection assay (RPA). mRNA was extracted from cells treated with 10–9m AVP alone or with both 10–9m AVP and 10–6m aldosterone for 3–48 h. Signals corresponding to the protected fragments of the acidic ribosomal phosphoprotein P0 mRNA probe were visible in each tested condition at similar intensities (Fig. 3A, bottom panel) and were used as an internal standard to estimate the levels of AQP2 mRNA expression. A signal corresponding to protected fragments of the AQP2 mRNA probe was detected after only 3 h of AVP treatment in cells treated with AVP alone (Fig. 3A, top panel, lane 1) and gradually increased for longer incubation times (Fig. 3, A, lanes 3, 5, and 7, and B). The expression levels of AQP2 mRNA of cells treated with both AVP and aldosterone, however, were constantly lower than those of cells subjected to AVP alone over the entire length of time investigated (3–48 h) (Fig. 3, A, lanes 4, 6, and 8, and B). These results suggest that the increase of AQP2 protein expression elicited by aldosterone for long incubation periods (>24 h) in AVP-treated mpkCCDcl4 cells is not directly linked to an increase in AQP2 mRNA expression. We have previously shown that the increased expression levels of AQP2 mRNA and protein of mpkCCDcl4 cells incubated 24 h with 10–9m AVP return to near base-line levels when subjected to 24 h of AVP chase, i.e. an additional incubation period following AVP washout. Analysis of AQP2 protein expression under conditions of AVP chase revealed two distinct phases: an initial phase consisting of a sharp rise in AQP2 protein expression immediately following AVP removal from the medium and a second phase consisting of a gradual AQP2 protein decay (34Hasler U. Mordasini D. Bens M. Bianchi M. Cluzeaud F. Rousselot M. Vandewalle A. Feraille E. Martin P.-Y. J. Biol. Chem. 2002; 22: 10379-10386Abstract Full Text Full Text PDF Scopus (150) Google Scholar). Here, we investigated the effect of aldosterone under conditions of AVP chase by first incubating mpkCCDC14 cells 24 h with 10–9m AVP and then for additional lengths of time (0.5–9 h) without or with 10–6m aldosterone following AVP washout. Western blot analysis revealed that aldosterone significantly altered AQP2 protein expression in the absence of AVP (Fig. 4, A and B). As previously observed (34Hasler U. Mordasini D. Bens M. Bianchi M. Cluzeaud F. Rousselot M. Vandewalle A. Feraille E. Martin P.-Y. J. Biol. Chem. 2002; 22: 10379-10386Abstract Full Text Full Text PDF Scopus (150) Google Scholar), the amount of AQP2 protein measured in cells subjected to an AVP chase in the absence of aldosterone increased shortly following AVP washout (Fig. 4, A, compare lane 1 with lanes 2 and 4, and B) and then gradually decreased for longer periods of AVP chase (Fig. 4, A, lanes 6, 8, and 10, and B). AQP2 protein expression of cells treated with aldosterone also rapidly increased following AVP washout (≤1 h, Fig. 4, A, compare lane 1 with lanes 3 and 5, and B) but remained lower than that of aldosterone-deprived cells subjected to an AVP chase for the same period of time (Fig. 4, A, compare lanes 2 and 4 with lanes 3 and 5, and B). Three hours following AVP washout, however, AQP2 protein content of cells treated with aldosterone increased and reached significantly higher levels than those of aldosterone-deprived cells subjected to the same 3-h AVP chase period and those of aldosterone-treated cells subjected to 1 h of AVP chase (Fig. 4, A, compare lane 7 with lanes 5 and 6, and B). These high AQP2 protein expression levels gradually decreased for longer periods of AVP chase but remained higher than those of aldosterone-deprived cells subjected to the same conditions of AVP chase (Fig. 4A, compare lanes 9 and 11 to lanes 8 and 10, and B). AQP2 protein expression returned to baseline levels 24 h after AVP washout in both aldosterone-treated and aldosterone-deprived cells (data not shown). We next investigated AQP2 mRNA expression under conditions of AVP chase by first incubating cells 24 h with 10–9m AVP and then for an additional 9 h in the absence or presence of 10–6m aldosterone following AVP washout (Fig. 4, C and D). As revealed by RPA analysis, whereas AQP2 mRNA expression of cells treated 9 h with 10–9m AVP significantly decreased when the cells were co-incubated with 10–6m aldosterone (Fig. 4, C, lanes 2 and 3, and D), AQP2 mRNA expression of 24-h AVP-pretreated cells subjected to 9 h of AVP chase returned to baseline levels regardless of the presence or absence of aldosterone (Fig. 4, C, compare lanes 4 and 5 to lane 1, and D). These results indicate that the increase of AQP2 protein expression induced by aldosterone under conditions of AVP chase is not because of an increase in AQP2 mRNA expression. Effect of Aldosterone on AQP2 Protein Degradation in Mouse Collecting Duct Principal Cells—Our results show that aldosterone increases AQP2 protein content after long periods of incubation (48 h) and under conditions of AVP chase. This led us to investigate the role of protein degradation in the aldosterone-induced up-regulation of AQP2 protein. We have previously shown that inhibitors of the lysosomal protein degradation pathway enhance AQP2 protein expression in AVP-treated mpkCCDCl4 cells and reduce AQP2 degradation in cells subjected to an AVP chase (34Hasler U. Mordasini D. Bens M. Bianchi M. Cluzeaud F. Rousselot M. Vandewalle A. Feraille E. Martin P.-Y. J. Biol. Chem. 2002; 22: 10379-10386Abstract Full Text Full Text PDF Scopus (150) Google Scholar). In the present study, we assessed the i" @default.
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