Dual Influence of Aldosterone on AQP2 Expression in Cultured Renal Collecting Duct Principal Cells*

Udo Hasler {ddagger} §, David Mordasini {ddagger}, Matthieu Bianchi {ddagger}, Alain Vandewalle ¶, Eric Féraille {ddagger} || and Pierre-Yves Martin {ddagger} ||

From the {ddagger} Division of Nephrology, Fondation pour Recherches Médicales, 64 Avenue de la Roseraie, CH-1211, Genève 4, Switzerland, INSERM U478, Faculté de Médecine Xavier Bichat, Institut Fédératif de Recherche 02, BP416, F-75870 Paris Cedex 18, France

Received for publication, December 5, 2002 , and in revised form, March 26, 2003.
    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (109 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 mpkCCDC14 cells by increasing AQP2 protein turnover while maintaining low levels of AQP2 mRNA expression.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (14) and three of them, AQP2, AQP3, and AQP4, are expressed in CD principal cells (57). 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 (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 administered with V2-receptor antagonists display decreased levels of AQP2 expression (12). 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 (14), chronic renal failure (15), hypokalemia (16), hypercalcemia (17), lithium administration (18), and liver cirrhosis (19), whereas increased levels of AQP2 expression have been reported in pregnant rats despite normal levels of circulating AVP (20).

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 (21). 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 (2225). 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 (26), a situation that can be reproduced in adrenalectomized rats (27), and administration of the aldosterone antagonist spironolactone was found to increase dilute urine production in patients with severe congestive heart failure (28). 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 (29), AQP2 expression levels in hypovolemic rats with selective aldosterone deficiency were found to be increased in the inner medulla (30) but remained unchanged at the level of the whole kidney (31).

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 (32). 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 (32, 33). 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 (34). 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.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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) (32) 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 (TranswellTM, 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) (35) or with a polyclonal rabbit anti-mouse protein kinase A{alpha} 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 [GenBank] ) 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 [{alpha}-32P]UTP (Amersham Biosciences). Acidic ribosomal phosphoprotein P0 (EMBL accession number BC011106 [GenBank] ) 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 [{alpha}-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 x 105 cpm of AQP2 probe and 5 x 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (34). 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 109 M 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 106 M aldosterone (Fig. 1, A and B). In a second set of experiments, mpkCCDC14 cells were treated with either 109 M AVP alone or with both 109 M AVP and 106 M aldosterone, added simultaneously to the cell medium, for 3–48 h (Fig. 1, C and D). We used AVP at a concentration of 109 M to maximally stimulate AQP2 expression (34). Aldosterone was used at 106 M because this concentration maximally stimulates sodium transport in mpkCCDcl4 cells (32). 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 106 M aldosterone and then exposed to 109 M 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).



View larger version (37K):
[in this window]
[in a new window]
 
FIG. 1.
Aldosterone (Aldo) influences AQP2 protein expression in a time-dependent manner in mpkCCDC14 cells. Confluent mpkCCDC14 cells grown on filters were incubated at 37 °C in the absence (–) or presence (+) of 106 M aldosterone, administered 24 h after (A and B) or simultaneously with (C and D) 109 M AVP, for various periods of time. Total protein extracts (20 µg) were separated by 10% SDS-PAGE and AQP2 (A and C, upper panel) was detected by Western blot using a polyclonal anti-AQP2 antibody. As a loading control, protein kinase A{alpha} catalytic subunit (A and C, lower panel) was detected using a polyclonal anti-protein kinase A{alpha} catalytic subunit antibody. A and C, representative immunoblots are shown. For A, the strong signals illustrated in lanes 11 and 12 for AQP2 were underexposed as compared with those shown in lanes 1–10 to optimize visualization. B and D, densitometric quantification of AQP2 protein expressed as the ratio of optical density values measured for each experimental condition and the optical density measured at 48 (B) and 24 h (D) for AVP treatment in the absence of aldosterone. Bars are mean ± S.E. from five independent experiments. *, p < 0.05.

 

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 109 M aldosterone (a concentration corresponding to half-maximal MR occupancy) and 106 M 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 109 M AVP administered together with either 106 or 109 M 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 109 M AVP and 106 or 109 M 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, 106 M 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 109 M further adds to the physiological relevance of aldosterone action on AQP2 expression in cultured mpkCCDcl4 cells.



View larger version (34K):
[in this window]
[in a new window]
 
FIG. 2.
The effects of aldosterone (Aldo) on AVP-induced AQP2 long term expression are dependent on mineralocorticoid receptor occupancy in mpkCCDC14 cells. A and B, confluent mpkCCDC14 cells grown on filters were incubated at 37 °C in the presence of 109 M AVP administered simultaneously with or without 106 or 109 M aldosterone for either 9 h (left panel) or 48 h (right panel) prior to protein extraction and Western blot analysis as described in the legend of Fig. 1. A, a representative immunoblot is shown. B, densitometric quantification of AQP2 protein expressed as the ratio of optical density values measured for each experimental condition and the optical density measured at either 9 or 48 h AVP treatment in the absence of aldosterone. Bars are mean ± S.E. from four independent experiments. *, p < 0.05; **, p < 0.01. C and D, confluent mpkCCDC14 cells grown on filters were incubated at 37 °C in the presence of 109 M AVP administered simultaneously with or without 106 M aldosterone, 105 M canrenoate, or both aldosterone and canrenoate for either 9 (left panel) or 48 h (right panel) prior to protein extraction and Western blot analysis. C, a representative immunoblot is shown. D, densitometric quantification of AQP2 protein, expressed as the ratio of optical density values measured for each experimental condition and the optical density measured at either 9 or 48 h for AVP treatment in the presence of AVP alone. Bars are mean ± S.E. from three independent experiments. *, p < 0.05; **, p < 0.01.

 

The effect of aldosterone on AQP2 mRNA expression was next investigated by RNase protection assay (RPA). mRNA was extracted from cells treated with 109 M AVP alone or with both 109 M AVP and 106 M 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.



View larger version (29K):
[in this window]
[in a new window]
 
FIG. 3.
Aldosterone (Aldo) decreases AQP2 mRNA expression in mpkCCDC14 cells. Confluent mpkCCDC14 cells grown on filters were incubated at 37 °C in the presence of 109 M AVP administered simultaneously with (+) or without (–) 106 M aldosterone for various times prior to RNA extraction as described under "Experimental Procedures." RPA was performed with riboprobes for AQP2 mRNA (top panel) and for ribosomal phosphoprotein P0 (bottom panel) on 10 µg of total RNA. A, a representative RPA is shown. B, densitometric quantification of AQP2 mRNA. The ratio of optical density values of AQP2 mRNA and optical density values of ribosomal phosphoprotein P0 measured for each experimental condition were calculated. Results were expressed as the ratio of AQP2/P0 values obtained for each experimental condition and the AQP2/P0 value obtained after 24 h AVP treatment in the absence of aldosterone. Bars are mean ± S.E. from four independent experiments. *, p < 0.05.

 

We have previously shown that the increased expression levels of AQP2 mRNA and protein of mpkCCDcl4 cells incubated 24 h with 109 M 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 (34). Here, we investigated the effect of aldosterone under conditions of AVP chase by first incubating mpkCCDC14 cells 24 h with 109 M AVP and then for additional lengths of time (0.5–9 h) without or with 106 M 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 (34), 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 109 M AVP and then for an additional 9 h in the absence or presence of 106 M aldosterone following AVP washout (Fig. 4, C and D). As revealed by RPA analysis, whereas AQP2 mRNA expression of cells treated 9 h with 109 M AVP significantly decreased when the cells were co-incubated with 106 M 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.



View larger version (39K):
[in this window]
[in a new window]
 
FIG. 4.
Aldosterone increases AQP2 protein expression in AVP-pretreated mpkCCDC14 cells subjected to AVP chase. A and B, confluent mpkCCDC14 cells grown on filters were first incubated 24 h at 37 °C in the presence of 109 M AVP (lane 1) to induce expression of AQP2 protein and then for additional lengths of time (30 min to 9 h) in the absence of AVP (AVP chase) and in the absence (–, lanes 2–10) or presence (+, lanes 3–11) of 106 M aldosterone (Aldo) prior to protein extraction and Western blot analysis. A, a representative immunoblot is shown. B, densitometric quantification of AQP2 protein, expressed as the ratio of optical density values measured for each experimental condition and the optical density measured at 24 h AVP treatment in the absence of aldosterone. Bars are mean ± S.E. from five independent experiments. *, p < 0.05. C and D, RPA was performed with riboprobes for AQP2 mRNA (top panel) and for ribosomal phosphoprotein P0 (bottom panel) on 10 µg of total RNA extracted from cells that were not stimulated with AVP (lane 1), from cells that were stimulated 9 h with 109 M AVP (AVP pulse) in the absence (lane 2) or presence (lane 3) of 106 M aldosterone, and from 24-h AVP-pretreated cells subjected to 9 h of AVP chase in the absence (lane 4) or presence (lane 5) of 106 M aldosterone. C, a representative RPA is shown. D, densitometric quantification of AQP2 mRNA. The ratio of optical density values of AQP2 mRNA and optical density values of ribosomal phosphoprotein P0 measured for each experimental condition were calculated. Results were expressed as the ratio of AQP2/P0 values obtained for each experimental condition and the AQP2/P0 value obtained at 9 h AVP treatment in the absence of aldosterone. Bars are mean ± S.E. from four independent experiments as represented in C. *, p < 0.05.

 

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 (34). In the present study, we assessed the influence of aldosterone on AQP2 protein degradation in mpkCCDC14 cells by first incubating cells 24 h with 109 M AVP alone and then for an additional 9 h with AVP in the absence or presence of 106 M aldosterone and/or 104 M chloroquine, a weak base that increases lysosomal pH and thereby inhibits the proteolytic activity of lysosomal enzymes (Fig. 5, A and B). As previously reported, AQP2 protein content was increased in cells incubated with chloroquine as compared with that of cells incubated with AVP alone (Fig. 5, A, compare lanes 3 and 1, and B). Interestingly, whereas aldosterone decreased AQP2 protein expression when administered alone with AVP (Fig. 5, A, compare lanes 1 and 2, and B), it enhanced the rise in AQP2 protein content engendered by chloroquine (Fig. 5, A, compare lanes 3 and 4, and B). We next assessed whether these differences in AQP2 expression also occurred under conditions of AVP chase. This was performed by first incubating mpkCCDC14 cells 24 h with 109 M AVP alone and then for an additional 9 h without or with 106 M aldosterone and/or 104 M chloroquine following AVP washout (Fig. 5, C and D). Under these experimental conditions, the presence of either aldosterone or chloroquine increased AQP2 protein content as compared with that of cells subjected to 9 h of AVP chase in the absence of either drug (Fig. 5, C, compare lane 2 with lanes 3 and 4, and D). AQP2 protein content of cells subjected to 9 h of AVP chase in the presence of chloroquine was even higher than that of cells incubated 24 h with AVP alone (Fig. 5, C, compare lane 1 with lane 4, and D). This increase is most likely because of residual translational activity of AQP2 mRNA occurring during early periods of AVP chase. As observed in cells continuously subjected to AVP, the rise in AQP2 protein content in cells subjected to 9 h of AVP chase in the presence of chloroquine was even greater when chloroquine was co-administered with aldosterone (Fig. 5, C, compare lanes 4 and 5, and D). Taken together, these results suggest that AQP2 protein upregulation induced by aldosterone is associated with an increase of AQP2 protein turnover.



View larger version (29K):
[in this window]
[in a new window]
 
FIG. 5.
Aldosterone (Aldo) increases AQP2 protein turnover in mpkCCDC14 cells. A and B, confluent mpkCCDC14 cells grown on filters were first incubated 24 h at 37 °C with 109 M AVP and then for an additional 9 h with AVP alone (lane 1), or together with 106 M aldosterone (lane 2), 104 M chloroquine (lane 3), or both 106 M aldosterone and 104 M chloroquine (lane 4) prior to protein extraction and Western blot analysis. A, a representative immunoblot is shown. B, densitometric quantification of AQP2 protein, expressed as the ratio of optical density values measured for each experimental condition and the optical density measured at 33 h in the presence of AVP alone. Bars are mean ± S.E. from four independent experiments. *, p < 0.05. C and D, confluent mpkCCDC14 cells grown on filters were first incubated 24 h at 37 °C with 109 M AVP (lane 1) and then for an additional 9 h without AVP (AVP chase) (lane 2), or without AVP but with 106 M aldosterone (lane 3), 104 M chloroquine (lane 4), or both 106 M aldosterone and 104 M chloroquine (lane 5) prior to protein extraction and Western blot analysis. C, a representative immunoblot is shown. D, densitometric quantification of AQP2 protein, expressed as the ratio of optical density values measured for each experimental condition and the optical density measured after 9 h of AVP chase. Bars are mean ± S.E. from four independent experiments. *, p < 0.05.

 

Aldosterone May Increase AQP2 Expression by Reducing a Negative Feedback Pathway That Inhibits AQP2 mRNA Translation—The results presented so far indicate that aldosterone may influence AQP2 expression by simultaneously reducing AQP2 mRNA content and increasing AQP2 protein turnover in mpkCCDC14 cells, an event that should ultimately lead to upregulated AQP2 protein expression after long incubation periods. We assessed whether these events are dependent on transcriptional activity by measuring the effect of aldosterone on AQP2 mRNA and protein expression in mpkCCDC14 cells incubated with the classical transcriptional inhibitor actinomycin D.

For study of the effect of actinomycin D alone on AQP2 expression, cells were first incubated 24 h with 109 M AVP and then for additional lengths of time (0.5–9 h) with AVP alone or together with 5 x 106 M actinomycin D. Cells incubated with actinomycin D for 30 min displayed dramatically reduced levels of AQP2 protein expression as compared with that of cells treated with AVP alone for 24 h (Fig. 6, A, lanes 1 and 2, and B). The decrease in AQP2 protein content was transient because AQP2 protein content gradually increased in cells incubated with actinomycin D for longer periods of time (Fig. 6, A, lanes 3 and 4, and B). AQP2 protein content peaked after 9 h of incubation and reached a level that exceeded that of cells incubated in the presence of AVP alone (Fig. 6, A, compare lane 5 and lane 6, and B). The dramatic increase in AQP2 protein content observed in cells treated 9 h with actinomycin D was not associated with a decrease in AQP2 protein degradation because cells treated 9 h with both actinomycin D and chloroquine displayed a more than 2-fold increase in AQP2 protein expression as compared with that of cells treated with actinomycin D alone (Fig. 6, A, lane 7, and B). The very high levels of AQP2 content in cells treated with both actinomycin D and chloroquine may be readily explained by an elevated AQP2 protein turnover. RPA analysis performed on 24-h AVP-pretreated cells incubated for an additional 9 h with 5 x 106 M actinomycin D revealed that AQP2 protein expression was up-regulated (Fig. 6, A and B) despite a significant decrease of AQP2 mRNA expression (Fig. 6, C, compare lanes 2 and 3, and D). The time-dependent increase of AQP2 protein expression observed in mpkCCDC14 cells continuously subjected to actinomycin D strongly suggests the involvement of a feed-back mechanism dependent on transcriptional activity and synthesis of (a) regulatory protein(s) that continuously reduce(s) AQP2 mRNA translation.



View larger version (32K):
[in this window]
[in a new window]
 
FIG. 6.
AQP2 protein turnover is increased in mpkCCDC14 cells treated with actinomycin D. A and B, confluent mpkCCDC14 cells grown on filters were first incubated 24 h at 37 °C with 109 M AVP (lane 1), and then for various times with both AVP and 5 x 106 M actinomycin D (lanes 2–5), or for 9 h with either AVP alone (lane 6) or with both AVP and 104 M chloroquine (chloro) (lane 7) prior to protein extraction and Western blot analysis. A, a representative immunoblot is shown. The weaker signals illustrated in lanes 1–6 were overexposed as compared with that shown in lane 7 to optimize visualization. B, densitometric quantification of AQP2 protein, expressed as the ratio of optical density values measured for each experimental condition and the optical density measured after 24 h in the presence of AVP alone. Bars are mean ± S.E. from three independent experiments. *, p < 0.05. C and D, RPA was performed with riboprobes for AQP2 mRNA (top panel) and for ribosomal phosphoprotein P0 (bottom panel) on 10 µg of total RNA extracted from cells that were first stimulated 24 h with 109 M AVP (lane 1) and then for an additional 9 h with either AVP alone (lane 2) or with both AVP and 5 x 106 M actinomycin D (lane 3). C, a representative RPA is shown. D, densitometric quantification of AQP2 mRNA. The ratio of optical density values of AQP2 mRNA and optical density values of ribosomal phosphoprotein P0 measured for each experimental condition were calculated. Results were expressed as the ratio of AQP2/P0 values obtained for each experimental condition and the AQP2/P0 value obtained at 24 h treatment in the presence of AVP alone. Bars are mean ± S.E. from two independent experiments. *, p < 0.05.

 

We next compared the levels of AQP2 protein content in cells treated with actinomycin D alone or together with aldosterone to investigate the dependence of aldosterone action on cellular transcriptional activity. The down- and up-regulation of AQP2 protein induced by aldosterone when administered together with AVP for short incubation periods (≤24 h) and following AVP washout, respectively, were investigated by two separate protocols to better distinguish both actions from each other. Aldosterone-induced down-regulation of AQP2 protein was investigated by first incubating cells 24 h with 109 M AVP alone and then for an additional 9 h with AVP alone or together with 106 M aldosterone, 5 x 106 M actinomycin D, or both actinomycin D and aldosterone. Whereas the stimulatory effect of actinomycin D was marginally reduced when the cells were additionally treated with aldosterone (Fig. 7, A, compare lanes 4 and 5, and B) the extent of AQP2 protein down-regulation induced by aldosterone was much greater when aldosterone was administered alone (Fig. 7, A, compare lanes 2 and 3, and B). This suggests that aldosterone down-regulates AQP2 protein via a mechanism that depends on cellular transcriptional activity. Aldosterone-induced up-regulation of AQP2 protein was investigated by first incubating cells 24 h with 109 M AVP alone then for an additional 9 h in the absence of AVP and in the presence of 106 M aldosterone, 5 x 106 M actinomycin D, or both aldosterone and actinomycin D. As observed for cells continuously subjected to AVP, the presence of actinomycin D administered immediately following AVP washout increased AQP2 protein content as compared with that of cells subjected to9hofAVP chase alone (Fig. 7, C, compare lanes 2 and 4, and D). The high levels of AQP2 protein expression were not further increased in cells incubated with both actinomycin D and aldosterone (Fig. 7, C, compare lanes 4 and 5, and D). These findings suggest that AQP2 protein up-regulation induced by aldosterone depends on cellular transcriptional activity.



View larger version (33K):
[in this window]
[in a new window]
 
FIG. 7.
Down- and up-regulation of AQP2 protein induced by aldosterone (Aldo) are dependent on cellular transcriptional activity in mpkCCDC14 cells. A and B, confluent mpkCCDC14 cells grown on filters were first incubated 24 h at 37 °C with 109 M AVP (lane 1) and then for an additional 9 h with either AVP alone (lane 2), or together with 106 M aldosterone (lane 3), 5 x 106 M actinomycin D (lane 4), or both 106 M aldosterone and 106 M actinomycin D (lane 5) prior to protein extraction and Western blot analysis. A, a representative immunoblot is shown. B, densitometric quantification of AQP2 protein, expressed as the ratio of optical density values measured for each experimental condition and the optical density measured after 24 h in the presence of AVP. Bars are mean ± S.E. from three independent experiments as represented in A. *, p < 0.05. C and D, confluent mpkCCDC14 cells grown on filters were first incubated 24 h at 37 °C with 109 M AVP (lane 1) and then for an additional 9 h without AVP (lane 2). Three hours after AVP washout, cells were incubated for an additional 6 h with 106 M aldosterone (lane 3), 5 x 106 M actinomycin D (lane 4), or both 106 M aldosterone and 5 x 106 M actinomycin D (lane 5) prior to protein extraction and Western blot analysis. C, a representative immunoblot is shown. D, densitometric quantification of AQP2 protein, expressed as the ratio of optical density values measured for each experimental condition and the optical density measured after 24 h in the presence of AVP. Bars are mean ± S.E. from three independent experiments. *, p < 0.05.

 

The observation that actinomycin D increased AQP2 protein expression both in the absence and presence of AVP also suggests that AQP2 mRNA translation in mpkCCDC14 cells is under the inhibitory control of endogenously expressed repressor factors. Our results further indicate that aldosterone may up-regulate AQP2 protein content by repressing transcription of the same putative factor(s) that is (are) repressed by actinomycin D and that reduce AQP2 mRNA translation.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
We have previously shown that the low endogenous AQP2 expression levels of mpkCCDC14 cells grown on permeable filters in the absence of AVP can be rapidly up-regulated in response to physiological concentrations of AVP (34). In the present study we exploited this property by investigating the effect of aldosterone on AVP-induced AQP2 expression in mpkCCDC14 cells. Aldosterone was found to affect AQP2 expression in two distinct, MR-dependent manners. Cells treated with both aldosterone and AVP exhibited decreased AQP2 mRNA and protein expression after short (≤24 h) incubation periods as compared with cells treated with AVP alone. Conversely, AQP2 protein expression was increased in AVP-treated cells exposed to aldosterone for longer periods of time (48 h) or following AVP washout despite lower expression levels of AQP2 mRNA. These findings indicate that these opposed actions of aldosterone occur concurrently by mechanisms acting independently of each other. Indeed, whereas short term aldosterone-induced down-regulation of AQP2 protein most likely relies on reduced AQP2 mRNA expression, long term aldosterone-induced up-regulation of AQP2 protein may be explained by enhanced AQP2 mRNA translation.

RPA and Western blot analysis revealed that down-regulation of AQP2 mRNA and protein expression occurred in mpkCCDcl4 cells after short (≤24 h) incubation periods in the presence of aldosterone. In the absence of aldosterone, AQP2 mRNA and protein expression increased over time in a correlated manner in AVP-treated cells. This observation suggests that increased amounts of AQP2 mRNA are directly responsible for increased AQP2 protein content. Such coordinated control of AQP2 mRNA and protein expression would explain the reduced expression of AQP2 mRNA that occurs at early incubation times (≤24 h) in cells treated with aldosterone (see Figs. 1 and 3). Actinomycin D prevented aldosterone-induced down-regulation of AQP2 protein in AVP-pretreated cells (see Fig. 7, A and B), indicating that this effect promoted by aldosterone relies on the transcription of regulatory factor(s). Aldosterone may alter transcription of the AQP2 gene through modulation of the binding capacity of one or several regulatory factors that enhance or repress AQP2 gene transcription. Alternatively, aldosterone may reduce AQP2 mRNA half-life. Controlled degradation of mRNA plays a key role in the expression of specific genes and largely depends on the presence of cis-acting sequences that may be present throughout the transcript. Whereas certain cis-acting sequences modulate mRNA degradation others block mRNA decay as illustrated by amyloid precursor protein mRNA where binding of heterogeneous nuclear ribonucleoprotein C or nucleolin to specific sequences has been found to stabilize or degrade amyloid precursor protein mRNA, respectively (36, 37). The putative influence of aldosterone on AQP2 mRNA stability, however, possibly acting on trans-acting factors induced by AVP, remains to be confirmed.

Time course experiments revealed that long term (48 h) exposure of cells to aldosterone increased AVP-inducible AQP2 protein content (see Fig. 1). Aldosterone was also found to increase AQP2 protein abundance in AVP-pretreated cells 3–9 h following AVP washout (see Fig. 4). AQP2 protein up-regulation was not associated with increased AQP2 mRNA expression, indicating that aldosterone action is mediated by other cellular mechanisms that participate in increased expression of AQP2 protein. The aldosterone-inducible increase in AQP2 protein expression observed in this study using cultured mpkCCDcl4 cells is in agreement with results obtained in canrenoate-treated normal rats in which whole kidney AQP2 protein content was decreased by about 50% in the absence of a change in mRNA expression (29). In contrast, under conditions of clamped water intake Kwon et al. (31) detected a small decrease (20%) in whole kidney AQP2 protein content in aldosterone-deprived rats as compared with adrenalectomized rats supplemented with both dexamethasone and aldosterone (control), whereas under conditions of free water intake whole kidney AQP2 content remained unchanged. However, one might consider that aldosterone-deficient rats did not receive NaCl supplementation and were therefore severely hypovolemic as attested by the increase in serum creatine and urea levels reflecting probably prerenal acute renal failure. It is well established that under these conditions of aldosterone deficiency, circulating AVP levels are increased through hypovolemia-mediated non-osmotic AVP release (38), an event that largely contributes to drive water reabsorption (39, 40). Because increased AVP levels are associated with increased expression levels of AQP2 all along the collecting duct (9), it appears likely that aldosterone deficiency may have blunted the normal vasopressin response, i.e. an increase in whole kidney AQP2 content, indicating a modulatory role of aldosterone on AVP-dependent AQP2 expression. In addition, Ohara et al. (30) have recently shown that under the same conditions of aldosterone deficiency and hypovolemia, AQP2 expression levels are increased in the renal inner medulla. Because whole kidney AQP2 content is not, or at best, is slightly decreased in hypovolemic aldosterone-deprived rats, the increase in inner medullary AQP2 content implies a decrease in cortical and/or outer medullary AQP2 content. It is also worth mentioning that functional and morphological studies have demonstrated an axial gradient of aldosterone responsiveness along the aldosterone-sensitive distal nephron (23, 41), the response being strongest at initial portions of the aldosterone-sensitive distal nephron and progressively decreasing at more distal portions. In this respect, aldosterone action on AQP2 expression may vary considerably along the aldosterone-sensitive distal nephron. Altogether, in vitro and in vivo studies indicate that long term aldosterone reinforces the effect of AVP on AQP2 expression in collecting duct principal cells.

AQP2 protein up-regulation mediated by aldosterone found in the present study using cultured mpkCCDcl4 cells might be explained by decreased degradation and/or increased AQP2 protein synthesis. Our results show that when administered to AVP-pretreated cells together with chloroquine (which inhibits lysosomal AQP2 degradation (34)), aldosterone significantly increased AQP2 protein expression as compared with AVP-pretreated cells treated with chloroquine alone (see Fig. 5). Similar results were obtained under conditions of AVP chase. These findings can be readily explained by an increase in AQP2 protein turnover, i.e. synthesis and degradation, in response to aldosterone. In cells exposed to aldosterone for long periods of time or to conditions of AVP chase, the increase in AQP2 protein synthesis would dominate the increase in AQP2 protein degradation leading to a net accumulation of AQP2 protein. Our results also suggest that the mechanisms that lead to AQP2 up-regulation and those that mediate AQP2 down-regulation occur concurrently with each other shortly after addition of aldosterone to the cell medium. Indeed, down-regulated expression of AQP2 protein in AVP-pretreated cells challenged 9 h with aldosterone can be reversed when chloroquine is added to the cell medium together with aldosterone. Overall, our results indicate that increased AQP2 protein expression mediated by aldosterone is most likely because of an increase in AQP2 protein synthesis, an event that progressively overcomes the decreased expression of AQP2 mRNA.

The high tissue-specific expression of AQP2, restricted to principal cells of renal CD and of vas deferens (5, 9, 42), reflects the tight transcriptional regulation controlling AQP2 gene expression. Several cis-elements have been identified in the AQP2 promoter region (43, 44) and at least two of these represent negative cis-acting elements that may specifically bind negative trans-acting factors that repress AQP2 gene transcription (45). We have previously shown that the integrity of the proteasomal degradation pathway is mandatory for AVP-induced AQP2 expression in mpkCCDC14 cells (34). This observation suggests that AVP may induce proteasomal-mediated degradation of one or several transcription factors that negatively act on AQP2 expression. The results of the present study provide evidence that AQP2 protein expression is regulated at the level of AQP2 mRNA translation as well. Indeed, AQP2 protein expression in AVP-treated mpkCCDC14 cells is first reduced by inhibition of ongoing transcription by actinomycin D and then, in the continuous presence of actinomycin D, progressively increases to levels greater than those observed in cells treated with AVP alone despite a 2-fold reduction of AQP2 mRNA expression (see Fig. 6). In addition, actinomycin D transiently increases AQP2 protein expression under conditions of AVP chase (see Fig. 7). This response to transcriptional inhibition cannot be explained by a decrease in AQP2 protein degradation because AQP2 protein abundance was further increased by 50% when cells were co-incubated with both actinomycin D and chloroquine (see Fig. 6). Rather, inhibition of transcriptional activity most likely alleviated a negative feedback mechanism controlled by unidentified regulatory factors that reduce AQP2 mRNA translation. Our results further indicate that these regulatory factors, endogenously expressed in mpkCCDC14 cells, have short half-lives and are continuously transcribed. They may repress AQP2 mRNA translation either by direct binding to AQP2 mRNA and/or by altering the phosphorylation state of factors involved in AQP2 mRNA translation. Gene-specific regulation can be conferred by specialized mRNA-binding factors, as exemplified by aconitase and heterogeneous nuclear ribonucleoproteins K and E, which inhibit ferritin and 15-lipoxygenase mRNA translation, respectively (46, 47). In eukaryote cells, changes in the phosphorylation states of multiple components of the translational machinery, including initiation and elongation factors and ribosomal proteins, provide a second means to regulate mRNA translation (48). The phosphorylation states of these components modulate their ability to interact with one another and thus modulate their function. Insulin, for instance, has been shown to activate several components of the translational machinery including the translation initiation factors 4E and 4F as well as the elongation factor 2 (49). Whereas AQP2 protein content is transiently increased by the presence of either actinomycin D or aldosterone in mpkCCDCl4 cells subjected to an AVP chase this effect is not additive when both agents are simultaneously present (see Fig. 7). This indicates that the increased rate of AQP2 protein turnover induced by aldosterone is dependent on transcriptional activity and that aldosterone may increase AQP2 protein turnover by reducing negative feedback that represses AQP2 mRNA translation. Further work is needed to identify the regions of AQP2 mRNA and the corresponding mRNA binding factors that modulate AQP2 mRNA translation. The identification of factors participating in AVP and aldosterone regulation of AQP2 expression, which may include AVP-and aldosterone-induced or repressed factors previously identified by SAGE analysis (50), would not only improve our understanding of the mechanisms involved in the regulation of AQP2 mRNA and protein expression but may lead to the identification of unknown factors that mediate aldosterone action.

We also emphasize that while AQP2 mRNA expression returned to near baseline levels in mpkCCDcl4 cells subjected to 9 h of AVP chase, it was only reduced by 50% in AVP-pretreated cells incubated in the presence of actinomycin D for the same length of time (see Fig. 6). We cannot exclude the possibility that transcriptional activity is not entirely abolished by the concentration of actinomycin D used, although this would appear to be rather unlikely because AQP2 mRNA and protein expression are entirely abolished when AVP and actinomycin D are simultaneously added to the cell medium. Alternatively, actinomycin D may indirectly increase AQP2 mRNA half-life by increasing the rate of AQP2 mRNA translation. This effect would be consistent with several reports that have shown a correlation between mRNA translation and mRNA decay of specific genes (5154). Alternatively, putative factors may stabilize AQP2 mRNA in the continuous presence of AVP, an event that would occur independently of ongoing transcriptional activity, e.g. via phosphorylation of regulatory factors, and which would be abolished following removal of AVP from the cell medium. In this case, AQP2 mRNA stabilization would represent an additional means of AVP-mediated control of AQP2 expression.

In conclusion, the results of the present study indicate that, in addition to its classical stimulatory effect on sodium reabsorption, aldosterone may also modulate water reabsorption in collecting duct principal cells. We speculate that aldosterone may act as a physiological buffer of AVP action. Indeed, it may decrease the effects of acute variations in AVP secretion and may potentiate the effects of sustained increases in AVP secretion on water reabsorption by modulating AQP2 protein expression to prevent hypo-or hyperosmolar states. In addition, by reducing AQP2 mRNA content aldosterone may control AQP2 protein up-regulation more efficiently.


    FOOTNOTES
 
* This work was supported in part by Swiss National Science Foundation Grant 31-56504.99 (to P.-Y. M.), the Foundation Carlos et Elsie de Reuter (to P.-Y. M. and E. F.), the Department of Medecine of HUG (to P.-Y. M.), and INSERM. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

|| Both authors contributed equally to this work. Back

§ To whom correspondence should be addressed: Div. de Néphrologie, Fondation pour Recherches Médicales, 64 Avenue de la Roseraie, CH-1211, Genève 4, Switzerland. Tel.: 41-22-382-38-33; Fax: 41-22-347-59-79; E-mail: Udo.Hasler{at}medecine.unige.ch.

1 The abbreviations used are: CD, collecting duct; AQP, aquaporin; AVP, arginine-8 vasopressin; MR, mineralocorticoid receptor; RPA, RNase protection assay. Back



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Denker, B. M., Smith, B. L., Kuhajda, F. P., and Agre, P. (1988) J. Biol. Chem. 263, 15634–15642[Abstract/Free Full Text]
  2. Maunsbach, A. B., Marples, D., Chin, E., Ning, G., Bondy, C., Agre, P., and Nielsen, S. (1997) J. Am. Soc. Nephrol. 8, 358–360
  3. Yasui, M., Kwon, T. H., Knepper, M. A., Nielsen, S., and Agre, P. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 5808–5813[Abstract/Free Full Text]
  4. Nejsum, L. N., Elkjaer, M., Hager, H., Frokiaer, J., Kwon, T. H., and Nielsen, S. (2000) Biochem. Biophys. Res. Commun. 277, 164–170[CrossRef][Medline] [Order article via Infotrieve]
  5. Fushimi, K., Uchida, S., Hara, Y., Hirata, Y., Marumo, F., and Sasaki, S. (1993) Nature 361, 549–552[CrossRef][Medline] [Order article via Infotrieve]
  6. Ishibashi, K., Sasaki, S., Fushimi, K., Uchida, S., Kuwahara, M., Saito, H., Furukawa, T., Nakajima, K., Yamaguchi, Y., Gojobori, T., and Marumo, F. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 6269–6273[Abstract]
  7. Terris, J., Ecelbarger, C. A., Marples, D., Knepper, M. A., and Nielsen, S. (1995) Am. J. Physiol. 269, F775–F785[Medline] [Order article via Infotrieve]
  8. Nielsen, S., Chou, C. L., Marples, D., Christensen, E. I., Kishore, B. K., and Knepper, M. A. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1013–1017[Abstract]
  9. Nielsen, S., DiGiovanni, S. R., Christensen, E. I., Knepper, M. A., and Harris, H. W. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11663–11667[Abstract]
  10. Hayashi, M., Sasaki, S., Tsuganezawa, H., Monkawa, T., Kitajima, W., Konishi, K., Fushimi, K., Marumo, F., and Saruta, T. (1994) J. Clin. Invest. 94, 1778–1783[Medline] [Order article via Infotrieve]
  11. DiGiovanni, S. R., Nielsen, S., Christensen, E. I., and Knepper, M. A. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 8984–8988[Abstract]
  12. Marples, D., Christensen, B. M., Frokiaer, J., Knepper, M. A., and Nielsen, S. (1998) Am. J. Physiol. 275, F400–F409[Medline] [Order article via Infotrieve]
  13. Ecelbarger, C. A., Nielsen, S., Olson, B. R., Murase, T., Baker, E. A., Knepper, M. A., and Verbalis, J. G. (1997) J. Clin. Invest. 99, 1852–1863[Abstract/Free Full Text]
  14. Apostol, E., Ecelbarger, C. A., Terris, J., Bradford, A. D., Andrews, P., and Knepper, M. A. (1997) J. Am. Soc. Nephrol. 8, 15–24[Abstract]
  15. Kwon, T. H., Frokiaer, J., Knepper, M. A., and Nielsen, S. (1998) Am. J. Physiol. 275, F724–F741[Medline] [Order article via Infotrieve]
  16. Marples, D., Frokiaer, J., Dorup, J., Knepper, M. A., and Nielsen, S. (1996) J. Clin. Invest. 97, 1960–1968[Abstract/Free Full Text]
  17. Earm, J. H., Christensen, B. M., Frokiaer, J., Marples, D., Han, J. S., Knepper, M. A., and Nielsen, S. (1998) J. Am. Soc. Nephrol. 9, 2181–2193[Abstract]
  18. Marples, D., Christensen, S., Christensen, E. I., Ottosen, P. D., and Nielsen, S. (1995) J. Clin. Invest. 95, 1838–1845[Medline] [Order article via Infotrieve]
  19. Jonassen, T. E., Nielsen, S., Christensen, S., and Petersen, J. S. (1998) Am. J. Physiol. 275, F216–F225[Medline] [Order article via Infotrieve]
  20. Ohara, M., Martin, P.-Y., Xu, D. L., St. John, J., Pattison, T. A., Kim, J. K., and Schrier, R. W. (1998) J. Clin. Invest. 101, 1076–1083[Abstract/Free Full Text]
  21. Funder, J. F. (1998) Clin. Exp. Pharmacol. Physiol. Suppl. 25, S47–S50[Medline] [Order article via Infotrieve]
  22. MacDonald, P., MacKenzie, S., Ramage, L. E., Seckl, J. R., and Brown, R. W. (2000) J. Endocrinol. 165, 25–37[Abstract/Free Full Text]
  23. Loffing, J., Zecevic, M., Feraille, E., Kaissling, B., Asher, C., Rossier, B. C., Firestone, G. L., Pearce, D., and Verrey, F. (2001) Am. J. Physiol. 280, F675–F682
  24. Feraille, E., and Doucet, A. (2001) Physiol. Rev. 81, 345–418[Abstract/Free Full Text]
  25. Verrey, F., Hummler, E., Schild, L., and Rossier, B. (2000) in The Kidney, Physiology and Pathophysiology (Seldin, D. W., ed) 3rd Ed., pp. 1441–1471, Lippincott, Williams and Wilkins, Philadelphia, PA
  26. Wilson, D. M., and Sundermann, F. W. (1939) J. Clin. Invest. 18, 35–43
  27. Schwartz, M. J., and Kokko, J. P. (1980) J. Clin. Invest. 66, 234–242[Medline] [Order article via Infotrieve]
  28. van Vliet, A. A., Donker, A. J., Nauta, J. J., and Verheugt, F. W. (1993) Am. J. Cardiol. 71, 21A–28A[Medline] [Order article via Infotrieve]
  29. Jonassen, T. E., Promeneur, D., Christensen, S., Petersen, J. S., and Nielsen, S. (2000) Am. J. Physiol. 278, F246–F256
  30. Ohara, M., Cadnapaphornchai, M. A., Summer, S. N., Falk, S., Yang, J., Togawa, T., and Schrier, R. W. (2002) Biochem. Biophys. Res. Commun. 299, 285–290[CrossRef][Medline] [Order article via Infotrieve]
  31. Kwon, T-H., Nielsen, J., Masilamani, S., Hager, H., Knepper, M. A., Frokiaer, J., and Nielsen, S. (2002) Am. J. Physiol. 283, F1403–F1421
  32. Bens, M., Vallet, V., Cluzeaud, F., Pascual-Letallec, L., Kahn, A., RafestinOblin, M. E., Rossier, B. C., and Vandewalle, A. (1999) J. Am. Soc. Nephrol. 10, 923–934[Abstract/Free Full Text]
  33. Vandewalle, A., Bens, M., and Duong Van Huyen, J. P. (1999) Curr. Opin. Nephrol. Hypertens. 8, 581–587[CrossRef][Medline] [Order article via Infotrieve]
  34. Hasler, U., Mordasini, D., Bens, M., Bianchi, M., Cluzeaud, F., Rousselot, M., Vandewalle, A., Feraille, E., and Martin, P.-Y. (2002) J. Biol. Chem. 22, 10379–10386[CrossRef]
  35. Xu, D. L., Martin, P.-Y., Ohara, M., St. John, J., Pattison, T., Meng, X., Morris, K., Kim, J. K., and Schrier, R. W. (1997) J. Clin. Invest. 99, 1500–1505[Abstract/Free Full Text]
  36. Rajagopalan, L. E., Westmark, C. J., Jarzembowski, J. A., and Malter, J. S. (1998) Nucleic Acids Res. 26, 3418–3423[Abstract/Free Full Text]
  37. Westmark, C. J., and Malter, J. S. (2001) J. Biol. Chem. 276, 1119–1126[Abstract/Free Full Text]
  38. Boykin, J., De Torrente, A., Robertson, G. L., Erickson, A., and Schrier, R. W. (1979) Miner. Elecrolyte Metab. 2, 310–315
  39. Ufferman, R. C., and Schrier, R. W. (1972) J. Clin. Invest. 51, 1639–1646[Medline] [Order article via Infotrieve]
  40. Ishikawa, S. E., and Schrier, R. W. (1982) Kidney Int. 22, 587–593[Medline] [Order article via Infotrieve]
  41. Loffing, J., Pietri, L., Aregger, F., Bloch-Faure, M., Ziegler, U., Meneton, P., Rossier, B. C., and Kaissling, B. (2000) Am. J. Physiol. 279, F252–F258
  42. Nelson, R. D., Stricklett, P., Gustafson, C., Stevens, A., Ausiello, D., Brown, D., and Kohan, D. E. (1998) Am. J. Physiol. 275, C216–C226[Medline] [Order article via Infotrieve]
  43. Sasaki, S., Fushimi, K., Saito, H., Saito, F., Uchida, S., Ishibashi, K., Kuwahara, M., Ikeuchi, T., Inui, K., Nakajima, K., Watanabe, T. X., and Marumo, F. (1994) J. Clin. Invest. 93, 1250–1256[Medline] [Order article via Infotrieve]
  44. Uchida, S., Sasaki, S., Fushimi, K., and Marumo, F. (1994) J. Biol. Chem. 269, 23451–23455[Abstract/Free Full Text]
  45. Furuno, M., Uchida, S., Marumo, F., and Sasaki, S. (1996) Am. J. Physiol. 271, F854–F860[Medline] [Order article via Infotrieve]
  46. Hentze, M. W., Caughman, S. W., Rouault, T. A., Barriocanal, J. G., Dancis, A., Harford, J. B., and Klausner, R. D. (1987) Science 238, 1570–1573[Medline] [Order article via Infotrieve]
  47. Ostareck, D. H., Ostareck-Lederer, A., Shatsky, I. N., and Hentze, M. W. (2001) Cell 104, 281–290[CrossRef][Medline] [Order article via Infotrieve]
  48. Dever, T. E. (2002) Cell 108, 545–556[Medline] [Order article via Infotrieve]
  49. Proud, C. G., Wang, X., Patel, J. V., Campbell, L. E., Kleijn, M., Li, W., and Browne, G. J. (2001) Biochem. Soc. Trans. 29, 541–547[Medline] [Order article via Infotrieve]
  50. Robert-Nicoud, M., Flahaut, M., Elalouf, J. M., Nicod, M., Salinas, M., Bens, M., Doucet, A., Wincker, P., Artiguenave, F., Horisberger, J.-D., Vandewalle, A., Rossier, B. C., and Firsov, D. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 2712–2716[Abstract/Free Full Text]
  51. Jacobson, A., and Peltz, S. W. (1996) Annu. Rev. Biochem. 65, 693–739[CrossRef][Medline] [Order article via Infotrieve]
  52. Ross, J. (1995) Microbiol. Rev. 59, 423–450[Medline] [Order article via Infotrieve]
  53. Ruiz-Echevarria, M. J., Munshi, R., Tomback, J., Kinzy, T. G., and Peltz, S. W. (2001) J. Biol. Chem. 276, 30995–31003[Abstract/Free Full Text]
  54. Vilela, C., Ramirez, C. V., Linz, B., Rodrigues-Pousada, C., and McCarthy, J. E. (1999) EMBO J. 18, 3139–3152[Abstract/Free Full Text]