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RENIN-MINERALOCORTICOIDS-ANF-ADH |
INSERM, U-478, IFR02, Faculté de Médecine Xavier Bichat (C.E.D., S.V., N.F., M.L.), 75870 Paris, France; and Klinik für Nephrologie und Rheumatologie, Heinrich Heine Universität (C.E.D., P.J.H., B.G.), 40225 Dusseldorf, Germany
Address all correspondence and requests for reprints to: Dr. Marc Lombès, INSERM, U-478, IFR02, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, BP 416, 75870 Paris Cedex 18, France. E-mail: . mlombes{at}bichat.inserm.fr
Abstract
Renal transplant recipients treated with cyclosporine A (CsA) and FK506 (tacrolimus) develop signs of hypoaldosteronism despite normal plasma aldosterone levels, suggesting a relative resistance of the distal nephron to aldosterone action. To examine the effects of immunosuppressants on human MR (hMR) function, we established the M cell model, renal tubular cells stably transfected with hMR. Upon CsA and FK506 administration, hMR mRNA levels and aldosterone binding in M cells remained unchanged (maximum number of sites, 
80 fmol/mg protein; Kd = 1 nM). Aldosterone-dependent intracellular localization of green fluorescent protein-hMR was not affected by immunosuppressants. A major impact of CsA or FK506 on the multidrug resistance gene product in cellular accumulation of aldosterone was also excluded. In contrast, aldosterone-stimulated hMR transcriptional activity was reduced to 53 ± 11.2% (P < 0.03) after pretreatment of M cells for 3 d with CsA and to 71 ± 9.6% (P < 0.05) after pretreatment with FK506. These effects were both time and concentration dependent (IC50 of CsA, 10-6 M; IC50 of FK506, 10-5 M) and needed at least 2 d to develop. Such an inhibitory effect does not depend on the N-terminal part of hMR, as CsA also reduced transcriptional activity of a 1–453 deletion mutant of hMR. Our results demonstrate that immunosuppressants inhibit hMR transcriptional activity without affecting hMR expression, aldosterone binding properties, and hMR nucleocytoplasmic trafficking. They suggest that ion transport alterations in renal graft recipients are in part induced by impaired hMR function.
TRANSPLANT PATIENTS receiving treatment with the immunosuppressive agents cyclosporine A (CsA) or FK506 (tacrolimus) frequently exhibit electrolyte alterations such as hyperkalemia and metabolic acidosis (1, 2, 3). These symptoms clinically resemble features of hypoaldosteronism despite normal plasma aldosterone levels in these patients (4, 5) and have been directly attributed to the administration of immunosuppressants (4, 6). Plasma inactive renin, a crucial step in the renin-angiotensin-aldosterone system, is elevated in these patients (5). These findings suggest a disturbance within the renin-angiotensin-aldosterone system that leads to signs of impaired aldosterone action. CsA and FK506 are known to induce nephrotoxicity by stimulating interstitial fibrosis and altering tubular function. They exert various effects on ion exchanging systems of the distal nephron, for example, inhibition of the Na+/K+-adenosine triphosphatase and the Na+/K+/2Cl- cotransporter (7, 8), thereby playing a crucial role in the development of hyperkalemia by altering potassium excretion and reabsorption. Interestingly, these electrolyte alterations are reversible upon administration of fludrocortisone (1), a finding also in favor of a potentially impaired aldosterone function. We therefore hypothesized that CsA and FK506 induce a relative resistance of distal tubular cells to the action of aldosterone, leading to impaired electrolyte balance in transplant patients.
Aldosterone is a key hormone involved in the regulation of ion and water homeostasis by stimulating ion transport in the distal nephron (9). Its action is mediated by the MR, which is a nuclear receptor belonging to the superfamily of ligand-regulated transcription factors. After hormone binding, the aldosterone-MR complex undergoes a conformational change (10). During this process molecular chaperones bound to the MR are liberated, and the complex is translocated to the nucleus (11). Some of these molecular chaperones, such as heat shock protein 90 (hsp90), are capable of binding proteins called immunophilins. These proteins are known to complex immunosuppressants; for instance, Cyp40 binds CsA, or FKBP51 and FKBP52 bind FK506 (12, 13). The activated MR complex usually binds in the form of a dimer to target DNA sequences (14), thereby modulating transcription of hormone-dependent genes. The interactions between immunophilins and nuclear receptors suggest a role of immunosuppressants in the signaling pathway of steroid hormones, as shown previously for the GR and PR (15, 16).
The aim of the present study was to investigate the influence of immunosuppressive agents on human MR (hMR) properties in terms of mRNA expression, aldosterone binding, subcellular localization, and transcriptional activity. We used renal tubular cells stably transfected with wild-type or N-terminal-truncated hMR. We demonstrate that CsA and FK506 significantly reduce aldosterone-mediated transcriptional activity of hMR without affecting hMR expression levels or aldosterone binding to the receptor. This impairment of the mineralocorticoid signaling pathway may account for the symptoms of secondary pseudohypoaldosteronism observed in renal transplant patients.
Materials and Methods
Cell culture
Rabbit kidney tubule cells, RC.SV3 (17), were maintained in DMEM-Ham’s F-12 supplemented with 2% charcoal-stripped FCS (Life Technologies, Inc., Cergy Pontoise, France), 100 IU/ml penicillin, 100 µg/ml streptomycin, 2 mM glutamine, 5 µg/ml insulin, 50 nM dexamethasone, 5 µg/ml transferrin, 20 mM HEPES, and 5 nM sodium selenite in a humidified atmosphere with 5% CO2. M cells, which are RC.SV3 cells stably transfected with an expression vector of hMR (pcDNA3-hMR) (18), were grown in 200 µg/ml geneticin G418 (Life Technologies, Inc.). All products for cell culture were obtained from Life Technologies, Inc.
Transfection procedures
Before transfection, cells were preincubated with different concentrations of CsA (Novartis Pharma GmbH, Wehr, Germany) or FK506 (Fujisawa, Germany) for various periods of time as indicated. Six hours before transfection, cells were seeded in six-well culture dishes using medium without dexamethasone. For RC.SV3 cells, the pcDNA3-hMR plasmid, the pSV-ß-galactosidase plasmid encoding for ß-galactosidase used as internal control for cellular viability and transfection efficiency, and the pF31-luciferase plasmid containing the mouse mammary tumor virus (MMTV) promoter driving hMR-dependent luciferase gene expression were transiently transfected into the cells by the calcium phosphate precipitation method. M cells were only transfected with ß- galactosidase and pF31-luciferase plasmids. Sixteen hours later, cells were treated with various concentrations of aldosterone and/or CsA and FK506 as indicated. After 24 h, cells were lysed for enzymatic assays. Results were normalized for transfection efficiency and expressed as the ratio of luciferase activity over ß-galactosidase activity in arbitrary units. These cotransfection assays minimize possible toxic effects of immunosuppressants on our cell system.
Generation of mutated hMR stable transfectants
To generate the N454 plasmid, pcDNA3-hMR plasmid was digested by BamHI, the XmaIII-BamHI fragment of hMR was removed, and the plasmid was religated. This plasmid encodes for a truncated hMR lacking the 1–453 first N-terminal amino acids of the receptor. Stable transfectants were generated using the calcium phosphate precipitation method. Clones were selected with geneticin-containing medium as previously described (18).
Luciferase and ß-galactosidase assays
Luciferase activity was determined as previously described (19). After washing twice with ice-cold PBS, transfected cells were lysed at 4 C in lysis buffer. Supernatant (100 µl) was mixed with 100 µl luciferine reagent (0.07 mg/ml; Sigma, St. Louis, MO), and luciferase activity was determined by a luminometer (E.G.&G. Berthold, Nashua, NJ). For ß- galactosidase assay, 100 µl supernatant were used as previously described (19), and the OD of the solution was determined at a wavelength of 420 nm with a spectrophotometer (Hitachi, Hialeah, FL).
Steroid binding assays
M cells grown in steroid-free medium for 24 h were frozen under liquid nitrogen, ground in a mortar, and homogenized at 4 C in TEGW buffer [20 mM Tris-HCl, 1 mM EDTA, 10% glycerol (vol/vol), and 20 mM sodium tungstate, pH 7.4, at 25 C] with a Teflon-glass potter (10 strokes), followed by a centrifugation at 12,000 x g at 4 C for 30 min. The resulting supernatant was considered the cytosolic fraction. Increasing concentrations of [3H]aldosterone (Amersham Pharmacia Biotech, Little Chalfont, UK; SA, 1.92 TBq/mmol) were added to 180 µl cytosol. After 4 h at 4 C, total radioactivity was determined by counting with a ß-counter (Wallace, Pharmacia Biotech, Piscataway, NJ). Subsequently, bound and unbound steroids were separated by dextran-charcoal treatment; the cytosolic fraction was incubated for 5 min with a dextran-charcoal mixture (4% Norit A and 0.4% dextran T70 in TEGW buffer) and centrifuged at 10,000 x g for 3 min, 4 C. Bound steroid was determined by counting the radioactivity of the supernatant. The bound as a function of the unbound was analyzed by a computer method as previously described (20), and the maximum number of sites, the dissociation constant (Kd), and the constant of the nonspecific binding (ß) were calculated. The protein content of the cytosol was determined by the Bradford method (21).
Binding of [3H]aldosterone in a whole cell assay
Cells were grown in steroid-free medium 24 h before binding studies, washed twice with serum-free medium, and incubated for 1 h at 37 C with 10-8 M [3H]aldosterone alone or in the presence of CsA and FK506 and in combination with a 1000-fold excess of unlabeled steroid to determine specific and nonspecific binding of [3H]aldosterone. Subsequently, cells were washed with PBS twice, and ice-cold ethanol was added for steroid extraction. Radioactivity was determined with a ß-counter. After counting of a representative sample of cells, results were expressed as number of binding sites per 104 cells.
Generation of probes for multidrug resistance gene product (MDR) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by RT-PCR
Total RNA was isolated from M cells and mouse kidney using the TRIzol reagent (Life Technologies, Inc.). Two micrograms of total RNA were reverse transcribed with 200 U reverse transcriptase (SuperScript II, Life Technologies, Inc.). GAPDH and MDR cDNAs were then amplified for 30 cycles (95 C for 45 sec, 57 or 52 C for 45 sec, and 72 C for 45 sec) in a total volume of 25 µl containing 1x PCR buffer (50 mM KCl and 20 mM Tris-HCl, pH 8.4), 200 µM dNTP, 1.5 mM MgCl2, 10 pmol sense and antisense primers, and 0.25 U Taq polymerase (Life Technologies, Inc.). The following primers were used for amplification of mouse MDR: sense, 5'-GGCATTTACTTCAAACTTGTCAT-3'; antisense, 5'-TCGCTTGGTGAGGATCTCTCC-3', and for the amplification of rabbit GAPDH: sense, 5'-CTAGCGCGTCCCCGAGACACGAT-3'; and antisense, 5'-AACTTGCCGTGGGTGGAATCATA-3'. MDR- and GAPDH-derived amplicons were purified from a 2% gel using the PCR Purification Kit Protocol (QIAGEN, Courtaboeuf, France). These cDNA were thereafter subcloned into the pGEM-T easy cloning plasmid (Promega Corp., Madison, WI). cDNA for the MDR was 501 bp long; cDNA for GAPDH was 182 bp long.
Subcellular localization of hMR chimera
The human collecting duct cell line H5 (22), provided by P. Ronco (INSERM, U-489; Tenon Hospital, Paris, France) was stably transfected with pcDNA3-hMR-enhanced green fluorescent protein (EGFP), an expression vector plasmid for a chimera protein consisting of hMR and EGFP, a plasmid provided by M. E. Oblin (INSERM, U-478, Paris, France). Clones were then selected with 200 µg/ml geneticin G418 (Life Technologies, Inc.). The transcriptional activity of hMR on MMTV- luciferase reporter assays and in vivo aldosterone binding were used to select clone 20, which expresses the highest levels of functional hMR. Clone 20 cells, pretreated, or not, for 3 d with 10-6 M CsA or FK506, were grown on glass slides and incubated with 10 nM aldosterone for various periods of time (0–30 min). Cells were rinsed with PBS at 4 C and fixed with 2% paraformaldehyde in PBS for 15 min at room temperature, followed by dehydration with 100% ethanol for 5 min. The fluorescence of EGFP was observed directly on a Leica Corp. (Rueil-Malmaison, France) inverted DM IRB microscope using an N2.1 filter (515–560 nm band-pass filter).
Northern blot analysis
Fifteen micrograms of total RNA were analyzed by Northern blotting as previously described (23). 
-32P-Labeled probes were synthesized by random priming (Megaprime, Amersham Pharmacia Biotech) of cDNA encoding for hMR, MDR, and GAPDH. Membranes were subjected to autoradiography and specific hybridization signals (hMR, 3.0 kb; MDR, 4.3 kb; GAPDH, 1.3 kb) were quantified using a Packard InstantImager (Downers Grove, IL). Results are expressed in arbitrary units corresponding to the ratio of specific counts for hMR or MDR vs. GAPDH signal.
Statistical analysis
Statistical analysis was performed using t test for unpaired comparisons (InStat, version 2.01, GraphPad Software, Inc., San Diego, CA). P < 0.05 was considered significant.
Results
CsA and FK506 do not modify hMR mRNA expression level or aldosterone binding to hMR in M cells
To examine the influence of immunosuppressive agents on hMR function, we established M cells, a model of rabbit renal tubular RC.SV3 cells (17) in which hMR expression vector was stably transfected, because no human cell line expressing functional hMR is currently available. Using a MMTV-luciferase reporter gene assay, we showed that RC.SV3 cells, in the absence of transfected hMR, were unable to generate a specific aldosterone response (Fig. 1A
). When the hMR plasmid was transiently transfected into the parental cells, a concentration-dependent stimulation of transcriptional activity of hMR was detected with an EC50 for aldosterone of 10-11 M. A concentration-dependent increase in transcriptional response was also observed in M cells that were stably transfected with hMR (Fig. 1B), indicating that these cells express functional MR. However, compared with transiently transfected hMR, a slightly increased EC50 at 10-10 M was obtained, indicating that M cells were somehow less sensitive to aldosterone. These findings are supported by other studies of steroid receptors (24, 25, 26) in which a leftward shift of concentration-response curves was also observed on transient vs. stable transfection.
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). Northern blot analysis revealed no significant modification in steady state levels of hMR mRNA, suggesting that hMR gene transcription and presumably mRNA stability in M cells were not measurably affected by either drug. Furthermore, we examined [3H]aldosterone binding to cytosolic fractions of M cells (Table 1). Scatchard plot analysis of [3H]aldosterone binding to the receptor in the presence or absence of 10-6 M CsA or FK506 revealed no major difference in aldosterone binding parameters (number of binding sites, Kd, and ß), indicating that these immunosuppressants do not modify binding of aldosterone to hMR. We also pretreated M cells with 10-6 M CsA or FK506 for 6 d and then prepared cytosolic fractions. Here again no difference in aldosterone binding parameters to hMR under these pretreatment conditions was observed (Fig. 3).
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), in accordance with previous studies (11, 27, 28). Similarly, the EGFP-hMR protein remained almost exclusively in the cytoplasmic compartment in the absence of aldosterone when the cells were treated for 3 d with CsA or FK506, (Fig. 4, D and G). A 5-min exposure to aldosterone induced a rapid nuclear translocation of hMR (Fig. 4B) that was almost exclusively located in the nucleus after 30 min (Fig. 4C). The aldosterone-dependent nuclear translocation of EGFP-hMR was not altered upon immunosuppressive treatment. In addition, determination of [3H]aldosterone partitioning in intact cells (nuclear and cytoplasmic compartments) revealed no major difference between treated or untreated cells, suggesting the lack of a significant modification in intracellular hMR trafficking upon immunosuppressive exposure (data not shown).
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). No difference between treated or untreated cells was observed, leading to the assumption that CsA and FK506 did not modify the number of specific aldosterone-binding sites reflected by the measured aldosterone uptake. These results suggest that the alteration of MDR mRNA expression levels by CsA does not account for the observed impairment of aldosterone responsiveness because whole cell binding of aldosterone remains unchanged.
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, simultaneous treatment of M cells with aldosterone and CsA or FK506 did not significantly alter ED50, which remained at 10-10 M, providing additional support for the lack of change in hMR affinity for aldosterone upon CsA or FK506 treatment. As biological alterations in CsA- or FK506-treated patients were generally observed after several weeks of treatment, we exposed M cells with immunosuppressants for several days before transfection of the reporter plasmid. Pretreatment with CsA or FK506 for 3 d resulted in a considerable decrease in hMR transcriptional activity (Fig. 6B). CsA reduced transcriptional activity to 53 ± 11.2% (P < 0.03) at 10-8 M aldosterone. The inhibitory effect of FK506 was less pronounced, decreasing transcriptional activity to only 71 ± 9.6% (P < 0.05) compared with that in untreated cells (Fig. 6C). The concentration of immunosuppressants needed for inhibiting the transcriptional activity of hMR was determined by incubating M cells with increasing concentrations of CsA or FK506 for 3–5 d (Fig. 7A). Our results indicate that for CsA the inhibitory effect became evident at a concentration of 10-6 M and was even more pronounced at 5 x 10-6 M [residual activity, 53 ± 11.2% (P < 0.03) and 34 ± 5.7% of untreated cells (P < 0.007), respectively]. We failed to cultivate M cells in medium supplemented with 10-5 M CsA, reflecting its toxic effects on cellular growth and survival. For FK506, a significant reduction in hMR transcriptional activity was also observed at 10-6 M (71 ± 9.6%; P < 0.05), although it was weaker and reached 55 ± 6.9% (P < 0.007) of untreated cells at 10-5 M. Both immunosuppressants exerted an inhibitory effect on hMR transcriptional activity in a time-dependent manner (Fig. 7B). The effect of CsA compared with FK506 was more pronounced and became significant after 2 d of pretreatment, whereas the effects of FK506 were not observed before 3 d. At d 7, aldosterone-mediated transcriptional activity was reduced to 49 ± 9.3% (P < 0.03) by CsA and to 67 ± 5.5% (P < 0.03) by FK506. These results indicate that CsA is more potent than FK506 in reducing the aldosterone-mediated transcriptional activity of hMR in our cell system.
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). We examined whether the N-terminal region was required for the inhibitory effect of CsA. RC.SV3 cells were stably transfected with a deletion mutant of hMR lacking N-terminal amino acids 1–453 (Fig. 8A). N454 cells were pretreated for 3 d with 10-6 M CsA and stimulated by 10-8 M aldosterone. The deleted mutant hMR was still able to trans-activate the luciferase reporter gene in a ligand-dependent manner, although its relative activity was less pronounced than that of the wild-type receptor (Fig. 8B). Compared with the 47% inhibition observed in M cells, CsA decreased aldosterone-mediated transcriptional activity by 72 ± 6.2% (P < 0.001) in N454 cells, indicating that the effect of CsA in N454 cells was more pronounced than that in M cells. These results demonstrate that inhibition of transcriptional activity of hMR by CsA does not require most of the N-terminal domain of hMR.
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The present study aimed at examining the pathophysiological mechanisms responsible for the partial resistance to aldosterone action observed in kidney transplant recipients treated with CsA or FK506. Both immunosuppressive drugs are known to bind to specific intracellular proteins called immunophilins, which form a complex with molecular chaperones such as hsp90 bound to the MR in its nonfunctional state (13). Immunophilins have been reported to be involved in the stability of the nontransformed receptor complex (33, 34), in the trafficking of the receptor between the cytoplasm and the nucleus (35), and in the alteration of DNA binding. Therefore, CsA and FK506 may play a role in receptor conformation and in its intracellular localization, posttranslational modification, and transcriptional activity (36). It has been shown that both immunosuppressants exert inhibiting or stimulating effects on steroid receptor function. On the one hand, both immunosuppressants reduced corticosterone-mediated transcriptional activity in mouse fibroblasts (37), and FK506, but not CsA, inhibited steroid-induced transcriptional activity of GR and PR in human breast cancer T47D cells (33). The mechanism of this inhibition is unclear, but it might be due to a regulatory influence of immunophilins on steroid receptor function. On the other hand, CsA and FK506 stimulate transcriptional activity of the GR by inducing intracellular steroid accumulation in mouse fibroblasts (15), because both substances are potent inhibitors of a steroid efflux pump, P-gp (38). This protein is a product of the multidrug resistance gene and a member of the ATP-binding cassette family. Similar stimulatory effects have been documented for the PR, with FK506 potentiating progesterone-induced transcription in a yeast model by P-gp inhibition (16).
In the present study we investigated the influence of immunosuppressants on hMR transcriptional activity. We focused our studies on a renal tubular cell line (RC.SV3) stably transfected with hMR (M cells) because these cells represent a biologically relevant system, in that presumably hMR levels are lower than those reached in transiently transfected cells. Furthermore, constitutively expressed hMR is likely to be more sensitive to intracellular cross-talk of a wide variety of transcriptional factors. The question of whether studies on steroid receptor function carried out with transiently expressed receptors are representative of what would occur in vivo has not yet been answered. In our model we demonstrate that both immunosuppressants significantly decreased the transcriptional activity of hMR. Interestingly, this effect needed at least 2 d to develop and was less pronounced with FK506, consistent with previous studies that demonstrated a weaker functional disturbance of FK506 (39). This is an interesting finding, because it is well known that after aldosterone stimulation hMR will translocate to the nucleus within 30 min. Indeed, our results using nucleo-cytoplasmic partition of radiolabeled aldosterone and subcellular localization of EGFP-hMR protein chimera showed that aldosterone-dependent nuclear translocation of hMR was not significantly altered upon immunosuppressive treatment. In our cell system the observed transcriptional modification takes much longer to develop, indicating that de novo protein synthesis might be involved in the process.
It is known that steroid receptors contain at least two distinct activating functions, one located in the N-terminal domain of the receptor (AF1) and another in the C-terminal region (AF2) that is ligand dependent. An N-terminal deletion mutant of hMR stably transfected into M cells (N454) subsequently treated with immunosuppressants also showed this inhibition of hMR transcriptional activity described above. Our findings indicate that most of the N-terminal domain of hMR was not required for establishing this inhibitory effect. Taken together, these results suggest that the inhibitory effects of immunosuppressants on the trans-activating function of hMR involve the AF2 domain of the receptor and presumably lead to an altered interaction with cofactors.
In our study we used concentrations of immunosuppressants generally higher than those reported for basal trough levels in immunosuppressive therapy (10-6 M CsA and FK506 in vitro compared with basal trough levels of 2 x 10-7 M CsA and 10-8 M FK506 in vivo). However, it has to be taken into account that serum levels in vivo generally range between 10-8–10-6 M. Nonetheless, the observed effects of immunosuppressants on hMR transcriptional activity are specific effects on steroid receptor signaling, because we always cotransfected an internal control (the pSV-ß-galactosidase plasmid) accounting for transfection efficacy and cellular viability.
The decreased transcriptional activity observed was not due to a modification of hMR mRNA expression levels in M cells. However, we could not exclude that in renal transplant patients expression of hMR and its isoforms (40, 41) might be altered by CsA and FK506. More importantly, no decrease in aldosterone binding to hMR by CsA or FK506 was detected in Scatchard analysis. There was no direct inhibitory effect of aldosterone binding to hMR on either simultaneous administration of immunosuppressants together with aldosterone or long-term treatment of cells with CsA or FK506. As adequate MR conformation requires its interaction with hsp90 (13), also known to complex with immunophilins, it is unlikely that CsA or FK506 modified the heterooligomeric structure of hMR, because both immunosuppressants were unable to impede aldosterone binding to hMR in M cells.
CsA and FK506 inhibit a potent transporter for drugs and hormones, P-gp (38). It has been shown recently that aldosterone transport from the adrenal gland to the extracellular compartment is mediated by P-gp (42). Therefore, P-gp inhibition by CsA or FK506 should increase intracellular aldosterone levels. According to our results, the intracellular aldosterone concentration remained constant despite treatment of cells with immunosuppressants. Furthermore, CsA and FK506 have been shown to also be capable of inducing P-gp expression, thereby reducing intracellular steroid levels (29, 30). These effects might establish a balance between steroid influx and efflux, presumably leaving intracellular steroid levels unchanged. We were able to show a slight increase in P-gp mRNA levels by CsA, but not by FK506, treatment, confirming the results of Hauser et al. (43). This increase in P-gp mRNA levels by CsA may lead to an increase in aldosterone extrusion from the cells. However, we were unable to demonstrate any modification in cellular aldosterone uptake in whole cell assays, indicating that despite P-gp induction, a new balance of transmembraneous aldosterone trafficking is established. Therefore, this effect is probably not responsible for the observed inhibition of hMR transcriptional activity. Whether other steroid transport systems, such as multidrug resistance related protein or LEM1 (44, 45), are also implied in the transport of aldosterone remains to be determined.
Both CsA and FK506, by complexing with their respective cytoplasmic binding proteins, cyclophilin (Cyp40) and FKBP12, are known to inhibit calcineurin, a calcium/calmodulin-dependent protein serine/threonine phosphatase (46), thereby affecting the phosphorylation status of various intracellular proteins. It could be hypothesized that CsA and FK506 might also modify the hMR phosphorylation level. Changes in receptor function by alteration of phosphorylation status have been described for GR, PR, and AR (47, 48, 49). Massaad et al. (18) found a modulation of hMR function by PKA, although this effect was thought to occur via the phosphorylation of cofactors and not via direct phosphorylation of the receptor protein itself. The slow kinetics of inhibitory effects of immunosuppressants are not in favor of a rapid change in phosphorylation status of hMR itself or of cofactors involved in aldosterone-dependent trans-activation.
Aldosterone is known to modulate a variety of genes, for example, the amiloride-sensitive epithelial sodium channel ENaC (50), serum- and glucocorticoid-regulated kinase (51), and other as yet unidentified genes. These genes might also be the targets of repressed hMR transcriptional activity by immunosuppressants, thereby modifying mineralocorticoid-dependent genes and altering cellular electrolyte transport mechanisms. This does not exclude the possibility of CsA and FK506 directly modulating the expression or function of sodium and potassium channels or transporters (52, 53).
Our study suggests that aldosterone resistance in kidney transplant patients is at least in part due to an impaired transcriptional activity of hMR, thereby contributing to hyperkalemia and metabolic acidosis observed in transplant patients. These findings open an interesting therapeutic perspective on whether transplant patients might benefit from mineralocorticoid agonist treatment. Our results also bring some new insights into the mineralocorticoid signaling pathway and novel aspects in the mechanism of action of immunosuppressive agents.
Acknowledgments
We thank Novartis and Fujisawa Laboratories for the gifts of CsA and FK506 immunosuppressants, and Dr. P. Ronco (INSERM, U-489) for RC.SV3 and H5 cells. We also thank Dr. M. E. Oblin (INSERM, U-478) for the gift of EGFP-hMR plasmid. Dr. M. C. Zennaro (INSERM, U-478) is gratefully acknowledged for her critical reading of the manuscript and helpful discussions.
Footnotes
1 Recipient of a fellowship from the Fondation pour la Recherche Médicale. 
Abbreviations: AF, Activating function; CsA, cyclosporine A; EGFP, enhanced green fluorescent protein; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hMR, human MR; hsp90, heat shock protein 90; MDR, multidrug resistance gene product; MMTV, mouse mammary tumor virus; P-gp, P-glycoprotein.
Received November 19, 2001.
Accepted for publication February 1, 2002.
References
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) or with pF31-luciferase and pSV-ß-galactosidase alone (RC.SV3 - hMR; 
) and subsequently treated with increasing concentrations of aldosterone for 24 h. C on the x-axis indicates the control condition in which no aldosterone was added. B, M cells, stably transfected with a plasmid containing the full-length coding sequence of hMR, were also transiently transfected with pF31-luciferase and pSV-ß-galactosidase. Sixteen hours after transfection, cells were incubated with increasing concentrations of aldosterone for 24 h. C indicates the control condition in which no aldosterone was added. After cell lysis, luciferase and ß-galactosidase activities were measured. Results are calculated as the ratio of luciferase activity and ß-galactosidase activity and are expressed as a percentage of relative transcriptional activity of RC.SV3 cells or M cells stimulated by 10-8 M aldosterone (100%). They represent the mean ± SEM of four different experiments performed in triplicate.
) or in the presence of 10-6 M CsA (
) or 10-6 M FK506 (
) for only 24 h. C on the x-axis indicates the control condition in which no aldosterone was added. Luciferase and ß-galactosidase activities were measured. Results are calculated as the ratio of luciferase activity to ß-galactosidase activity and are expressed as the percentage of relative transcriptional activity of M cells stimulated by 10-8 M aldosterone alone (100%). They represent the mean ± SEM of four different experiments performed in triplicate. No significant difference was observed in the presence of CsA or FK506. M cells were preincubated 3 d before transient transfection with 10-6 M CsA (B, 
), or they were preincubated 1–7 d before transient transfection with 10-6 M CsA (B, 