Aldosterone and tight junctions: modulation of claudin-4 phosphorylation in renal collecting duct cells

Cathy Le Moellic,1 Sheerazed Boulkroun,1 Daniel González-Nunez,1 Isabelle Dublineau,2 Francoise Cluzeaud,1 Michel Fay,1 Marcel Blot-Chabaud,1,* and Nicolette Farman1,*

1Institut National de la Santé et de la Recherche Médicale Unité 478, Institut Fédératif de Recherches 02, Faculté de Médecine Xavier Bichat, Paris; and 2Laboratoire de Radiobiologie Digestive, Institut de Protection et de Sûreté Nucléaire, Fontenay aux Roses, France

Submitted 29 June 2005 ; accepted in final form 10 August 2005


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Aldosterone classically modulates Na transport in tight epithelia such as the renal collecting duct (CD) through the transcellular route, but it is not known whether the hormone could also affect paracellular permeability. Such permeability is controlled by tight junctions (TJ) that form a size- and charge-selective barrier. Among TJ proteins, claudin-4 has been highlighted as a key element to control paracellular charge selectivity. In RCCD2 CD cells grown on filters, we have identified novel early aldosterone effects on TJ. Endogenous claudin-4 abundance and cellular localization were unaltered by aldosterone. However, the hormone promoted rapid (within 15–20 min) and transient phosphorylation of endogenous claudin-4 on threonine residues, without affecting tyrosine or serine; this event was fully developed at 10 nM aldosterone and appeared specific for aldosterone (because it is not observed after dexamethasone treatment and it depends on mineralocorticoid receptor occupancy). Within the same delay, aldosterone also promoted an increased apical-to-basal passage of 125I (a substitute for 36Cl), whereas 22Na passage was unaffected; paracellular permeability to [3H]mannitol was also reduced. Later on (45 min), a fall in transepithelial resistance was observed. These data indicate that aldosterone modulates TJ properties in renal epithelial cells.

paracellular permeability; mannitol flux; occludin; iodine; sodium; kidney; RCCD2 cells


TIGHT JUNCTIONS (TJ) are highly specialized cell-cell adhesion structures that delineate apical and basolateral membrane domains (as reviewed in Refs. 1, 20, 32, 33). In epithelia, TJ play an important role as a paracellular seal that determines the permeability properties of the paracellular pathway, thus providing a physical barrier regulating ion and solute movements from one compartment to another. Evidence has been provided that TJ form a size- and ion-selective barrier; their permeability is modulated by various signals (1, 20, 32, 33). Transmembrane proteins of the TJ include occludin, claudins, and junction adhesion molecules, which interact with cytoplasmic proteins as those of the zonula occludens (ZO) family; this organization provides mechanisms allowing recruitment of other proteins of the cytoskeleton and regulatory proteins (1, 20, 32, 33). Such a complex architecture determines tissue-specific characteristics of TJ. It is now clear that claudins play a major role in determining the charge selectivity and conductance properties of the paracellular pathway, as highlighted in several excellent articles or reviews (1, 20, 28, 32, 33, 35, 40). At least 24 members of the claudin family have been identified in mammals; this gene family is conserved through evolution, including fish, Drosophila, and Caenorhabditis elegans (33). Overexpression of individual claudins in epithelial cells allowed determination (for some members of this family) of their intrinsic contribution to paracellular selectivity. It is considered that the overall properties of TJ depend on a combination of the cell background and the expressed claudins (5, 28, 34, 35). TJ permeability may be impaired in pathology, as demonstrated in cell or mouse models and in human diseases. Disorganization of TJ has been observed after several maneuvers such as ATP or Ca depletion or pharmacological disruption of F-actin (4, 12). Overexpression or suppression of several members of the claudin family in cultured cells (6, 34, 35, 40) or in mouse models (11) leads to changes in paracellular permeability. Mutations of claudin-16 (paracellin) have been identified in human familial hypercalciuric hypomagnesemia (27). With regard to hormonal modulations, it has been reported that EGF promotes selective changes in claudin-1 to -4 expression patterns and localization in detergent-insoluble fractions (28).

Properties of TJ vary among epithelia. Within the kidney, regulated ion and fluid transport depends on both transcellular and paracellular pathways. Distinct members of the claudin family are expressed along the nephron (17, 19, 41). The renal collecting duct is considered a tight epithelium [high transepithelial resistance (TER)], whereas other parts of the tubule are more leaky. Claudin-4 is specifically expressed in the collecting duct (together with claudin-3, -7, and -8). Among these claudins, several recent reports (5, 6, 34, 35) have established that claudin-4 is a major modulator of TJ, whereas less information is available for the other claudins. Claudin-4 overexpression in Madin-Darby canine kidney cells revealed its importance in the regulation of paracellular charge selectivity: it increases TER and reduces Na permeability, without affecting Cl permeability or mannitol flux (35). The first extracellular domain appears critical to determine its selectivity properties, and the cytoplasmic domains can influence the degree to which the extracellular domain affects paracellular properties (6). However, no information exists, to our knowledge, on the impact of claudin-4 phosphorylation on TJ function in epithelia.

This study questioned whether aldosterone could modulate the phosphorylation state of endogenous claudin-4 in renal collecting duct cells. In this view, it is of interest to note that claudin-4 can be phosphorylated in vitro (40) by the kinase With No Lysine Kinase (WNK)4, which is expressed in the collecting duct and is regulated by aldosterone (21). In addition, this study explored some aspects of early changes in TJ permeability properties after hormone exposure. In collecting duct cells, aldosterone binds to the mineralocorticoid receptor (MR), a transcription factor that regulates the synthesis of a cascade of proteins and signaling pathways leading to an increase in the number of active epithelial Na channels (ENaC) in the apical membrane (2, 10, 24, 37). It has been proposed that aldosterone could regulate both active transcellular Na transport and paracellular Cl reabsorption to maintain the electroneutrality of the transport (2, 9, 36). Paracellular permeability may be assessed by measurement of TER (29). Opposite results have been yielded after aldosterone addition in various model tissues, with observations of an increase, a decrease, or an absence of modifications of TER (3, 7, 13, 23), which may reflect their different patterns of claudin expression. In isolated, perfused cortical collecting ducts studied with the microelectrode technique, it was shown that in vivo chronic mineralocorticoid treatment leads to a decrease of the conductance of the paracellular pathway (25); however, the initial effects of the hormone have not been examined by such an approach. Herein we report the characterization of early effects of aldosterone (during the first hour) in the RCCD2 rat collecting duct cell line. This cell line exhibits high TER (2,000–5,000 {Omega}·cm2) and aldosterone-regulated Na transport (7). We found that aldosterone 1) does not modify the cellular pool or intracellular localization of endogenous occludin and claudin-4, 2) promotes selective phosphorylation of claudin-4 on threonine residues, and 3) reduces TER and increases apical-to-basal anion (125I) passage without modifying 22Na passage. In addition, the paracellular permeability to [3H]mannitol (a small, uncharged molecule) was also reduced. These changes occur relatively shortly (≤1 h) after hormone addition and involve a MR-specific genomic pathway. These data indicate that TJ represent a novel cellular target for aldosterone and point to mineralocorticoid-specific regulation of claudin-4 phosphorylation status as a new aldosterone-related event.


    MATERIALS AND METHODS
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 MATERIALS AND METHODS
 RESULTS
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RCCD2 cell culture. The rat cortical collecting duct cell line RCCD2 (7) was used between passages 5 and 20. The RCCD2 cells were grown at 37°C in a humidified incubator gassed with 5% CO2. Cells were cultured in a complete medium changed every other day (18) containing DMEM-Ham's F-12 (1:1), 14 mM NaHCO3, 2 mM glutamine, 50 nM dexamethasone, 50 nM sodium selenite, 5 µg/ml transferrin, 5 µg/ml insulin, 10 ng/ml EGF, 50 nM 3,5,3'-triiodothyronine, 100 U/ml penicillin-streptomycin, 2% fetal bovine serum (GIBCO), and 20 mM HEPES, pH 7.4. All experiments were performed with cells seeded on Transwell filters (Costar; 0.4-µm pore size) previously coated with rat tail collagen type I (Institut J. Boy, Reims, France). The medium bathing the apical surface of the RCCD2 cells was designated the "apical medium" (total volume 0.5 ml), and the medium bathing the basolateral surface of the monolayer was designated the "basal medium" (total volume 1.5 ml). To examine hormonal effects, the complete medium was replaced 16–18 h before experiments by minimum medium containing DMEM-Ham's F-12 (1:1), 14 mM NaHCO3, 2 mM glutamine, 100 U/ml penicillin-streptomycin, and 20 mM HEPES, pH 7.4.

Measurement of TER. The Millicell ERS (Millipore) apparatus was used to estimate changes in TER during the first hours after addition of aldosterone (10 nM) to RCCD2 cells grown on 12-mm-diameter Snapwell filters (Costar).

Measurement of apical-to-basal [3H]mannitol passage. RCCD2 cells were grown on 12-mm-diameter Transwell filters. Hormones [aldosterone, dexamethasone (Sigma)] or their solvent (ethanol 1:1,000) were added in the apical and basal medium, a tracer dose [50,000–100,000 counts per min (cpm)] of [3H]mannitol (17 Ci or 629 MBq/mmol; NEN) was added in the apical medium, and cells were kept at 37°C. Aliquots of basal medium were sampled at various time intervals (for time course experiments) or after 1-h incubation (for test of antagonists or inhibitors). Radioactivity accumulated in the basal medium was measured with a beta-scintillation counter (1217 Wallac; PerkinElmer).The total amount of [3H]mannitol recovered in the basal medium after 1 h represented at most 2–5% of the amount present in the apical medium. Apical-to-basal flux was expressed as moles per second. Apparent mannitol permeability (Pmann) was calculated from the equation:

Measurement of apical-to-basal 125I and 22Na passage. Because of security rules, it was not possible to perform 36Cl flux experiments. Instead, 125I was used as a substitute as previously described (9, 36). RCCD2 cells were grown on 12-mm-diameter Transwell filters as described for [3H]mannitol flux, 125I [5.106 cpm, 17.4 Ci/mg iodine (0.644 TBq/mg); Amersham, Orsay, France] was added to the apical medium together with diluent (control condition) or aldosterone (10 nM), and the cells were then incubated at 37°C. At various time intervals, aliquots of the basal medium were collected. Their radioactivity was counted with a gamma counter (Cobra II; Packard/PerkinElmer, Courtaboeuf, France). The same protocol was used to examine the basal accumulation of 22Na. One million counts per minute of 22Na (74 mBq/ml; Amersham) were added apically at time 0 on cells together with diluent (control condition) or aldosterone (10 nM). Aliquots of the basal medium were collected, and their radioactivity was measured with a Tri-carb 2300TR beta counter (PerkinElmer). Results are expressed as total radioactivity accumulated in the basal medium at each time point.

Immunofluorescence studies. The expression of occludin and claudin-4 was determined in the presence or absence of aldosterone (10 nM). The localization of occludin and claudin-4 was evaluated in cells seeded in 12-mm-diameter Transwell filters. Cells were fixed for 10 min with methanol at room temperature and incubated for 2 h at room temperature with rabbit polyclonal anti-occludin (1:50) or with mouse monoclonal anti-claudin-4 (1:50) (Zymed). After three rinses in PBS, cells were incubated with a secondary antibody associated with a fluorescent probe, cyanine Cy-3 (Jackson) or Alexa Fluor 568 (red) (Molecular Probes). In these experiments, the nucleus was stained with Sytox Green (Molecular Probes). Cells were examined by confocal microscopy (LSM 510; Zeiss).

Immunoprecipitation and Western blot experiments. Immunoprecipitation experiments were performed as described previously (18). Briefly, cells grown on 24-mm-diameter Transwell filters were treated or not with aldosterone or dexamethasone, washed in PBS with 1 mM sodium orthovanadate, scraped off the filters, and extracted with 200 µl of ice-cold RIPA buffer [in mM: 150 NaCl, 50 Tris·HCl (pH 7.4), 0.5 phenylmethylsulfonyl fluoride, 2.4 EDTA, and 1 sodium orthovanadate, with 1% Nonidet-40 (NP-40) and Sigma protease inhibitor cocktail (1:100)] for 30 min at 4°C. After centrifugation (12,000 g, 10 min, 4°C) to eliminate cell debris and nuclei, protein extracts were precleared with a Staphylococcus aureus slurry (Pansorbin; Calbiochem) before incubation overnight at 4°C under end-over-end rotation with the antibody directed against claudin-4 (Zymed; 1:40). Immunoprecipitates were then incubated with protein A-Sepharose beads (CL-4B; Pharmacia) at 4°C for 1 h. Beads were washed three times with 1 ml of high-salt buffer [500 mM NaCl, 1% NP-40, 50 mM Tris·HCl (pH 8.0)] and twice with 500 µl of low-salt buffer (20 mM Tris·HCl, pH 7.5) and resuspended in 40 µl of NuPage LDS sample buffer (Invitrogen). Samples of eluted immunoprecipitates were submitted to 15% NuPage SDS-polyacrylamide gel electrophoresis (Invitrogen) and transferred onto nitrocellulose membrane (Invitrogen). Phosphorylated residues were detected with mouse monoclonal anti-phosphoserine antibody (Sigma; 1:1,000), rabbit polyclonal anti-phosphothreonine antibody (Zymed; 1:500) or mouse monoclonal anti-phosphotyrosine antibody (Zymed; 1:500) coupled to peroxidase overnight at 4°C before detection with an ECL kit (Amersham). Nitrocellulose membranes were stripped, and Western blotting was done with the anti-claudin-4 mouse monoclonal antibody (Zymed; 1:1,000) followed by anti-mouse secondary antibody (Santa Cruz; 1:10,000) coupled to peroxidase and ECL. For Western blotting of occludin and claudin-4, the same protocol was used with the anti-occludin antibody (1:500), the anti-claudin-4 antibody (1:1,000), and a rabbit anti-{beta}-actin antibody (Santa Cruz; 1:200; 1 h at room temperature).

Hormones, inhibitors, and antagonists. Hormones [aldosterone, dexamethasone (Sigma) dissolved in 1:1,000 ethanol], antagonists, or inhibitors were added in both the apical and the basal media. The following inhibitors were added 1 h before aldosterone: cycloheximide (an inhibitor of protein synthesis; 10 µM in H2O), chelerythrine chloride (a PKC inhibitor; 100 nM in H2O), Gö-6976 (an inhibitor of PKC-{alpha}; 10 nM in DMSO 1:10,000). The MR antagonist spironolactone (10 µM in ethanol 1:1,000), or the glucocorticoid receptor (GR) antagonist RU-486 (10 µM in ethanol; 1:1,000) was added simultaneously to aldosterone (10 nM). In corresponding control conditions, diluent corresponding to that used for each inhibitor, antagonist, or hormone was added to the medium. RU-486 was a kind gift of Roussel-Uclaf.

Statistical analysis. Results are expressed as means ± SE. Differences between groups were tested using ANOVA and the Bonferroni test or an unpaired Student's t-test.


    RESULTS
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 MATERIALS AND METHODS
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Aldosterone does not modify expression and cellular localization of occludin and claudin-4. As a first step to investigate putative modifications of TJ by aldosterone, we examined the expression of some constituents of TJ. Attention was focused on two important TJ proteins, occludin and claudin-4, that were fully detectable in RCCD2 cells. Their amounts were not modified in RCCD2 cells grown on filters after aldosterone (10 nM) treatment, from 30 min to 5 h, as shown in Fig. 1A. Occludin immunofluorescence was strictly associated with the membranes, whereas claudin-4 was detectable at the level of the membranes and also in the cytoplasm to some extent. Their intracellular localization was not modified in the presence of aldosterone, at least during the first hour of exposure, as determined by confocal microscopy (Fig. 1B).



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Fig. 1. Lack of effect of aldosterone on occludin and claudin-4 expression and cellular localization in RCCD2 cells. A: Western blots of RCCD2 cells (grown on filters) treated with 10 nM aldosterone for 30 min to 5 h. Membranes were successively blotted with the anti-occludin or the anti-claudin-4 antibody and with the anti-{beta}-actin antibody. There was no change of occludin, claudin-4, or {beta}-actin expression over time. Similar results were observed in 3 independent experiments. B: RCCD2 cells grown on filters were treated for 15–45 min with 10 nM aldosterone, occludin or claudin-4 was immunodetected, and cells were examined by confocal microscopy. No consistent change in their localization was evidenced in 3 independent experiments. Bars: 10 µm.

 
Phosphorylation of endogenous claudin-4 is regulated by aldosterone. The phosphorylation status of several signaling molecules or constitutive proteins, including those of the TJ, exhibits changes on cell stimulation (8, 31). We examined the phosphorylation status of endogenous claudin-4 after aldosterone challenge. After incubation of RCCD2 cells with aldosterone (10 nM), endogenous claudin-4 was found to be phosphorylated on its threonine residues, whereas serine and tyrosine phosphorylation were unchanged, as detected by immunoprecipitation with an anti-claudin-4 antibody followed by Western blot with antibodies specific to phosphorylated serine, threonine, or tyrosine sites (Fig. 2A). In contrast, no modification of occludin phosphorylation on serine residues was evidenced after aldosterone treatment (not shown).



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Fig. 2. Time course of phosphorylation of claudin-4 in aldosterone-treated RCCD2 cells. A: RCCD2 cells grown on filters were incubated with aldosterone (10 nM) for various time intervals. Cell lysates were subjected to immunoprecipitation with the anti-claudin-4 antibody, followed by Western blot analysis with antibodies against phospho (P)-threonine, -serine, or -tyrosine (serial strippings of the same membrane). The membrane was also exposed to the anti-claudin-4 antibody to monitor gel loading in each lane. A representative experiment of aldosterone-dependent threonine phosphorylation of claudin-4 (while serine and tyrosine were unaffected) is shown. B: representative experiment showing the kinetics of threonine phosphorylation of claudin-4 after aldosterone treatment from 20 min to 4 h. The graph is a quantification of aldosterone-dependent threonine phosphorylation of claudin-4. Results are expressed as % increase in threonine phosphorylation (normalized to claudin-4 signal) in the presence of aldosterone compared with untreated cells (100%). Aldosterone induces threonine phosphorylation of claudin-4 within 15 min, and this effect is transient because it disappears after 1-h hormone treatment. Each point is the mean ± SE from 4 independent experiments (4–8 filters per time point). *P < 0.05, **P < 0.01, ***P < 0.001, any time point vs. 5 min (ANOVA and Bonferroni test).

 
Claudin-4 phosphorylation on threonine residues occurs within 15–20 min after hormone addition; important phosphorylation was observed 20–40 min after aldosterone addition and declined thereafter toward control values (Fig. 2B). This is a dose-dependent phenomenon, as shown in Fig. 3: full response was observed at 10 nM aldosterone, and no further increase was apparent at 10- and 100-fold higher concentrations. The apparent half-maximal concentration can be estimated to be ~1 nM, i.e., a physiological aldosterone concentration.



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Fig. 3. Dose dependence of aldosterone-induced threonine phosphorylation of claudin-4. RCCD2 cells grown on filters were incubated for 20 min with various aldosterone concentrations and processed as in Fig. 2: cell lysates were immunoprecipitated with the anti-claudin-4 antibody, followed by Western blot analysis with antibodies against phosphothreonine; the membrane was also exposed to the anti-claudin-4 antibody to monitor gel loading in each lane. Top: representative experiment showing aldosterone-dependent threonine phosphorylation of claudin-4. Bottom: quantification of 4 independent experiments. Results are expressed as % increase in threonine phosphorylation in the presence of aldosterone compared with untreated cells (100%). Threonine phosphorylation of claudin-4 increased with aldosterone concentration and was maximal at 10 nM. Each point is the mean ± SE from 6 filters. ***P < 0.001, each aldosterone concentration vs. 0.1 nM (ANOVA and Bonferroni test).

 
To examine whether this effect is mediated by the MR, similar experiments were performed with 10 nM aldosterone for 20 min in the presence or absence of a 1,000-fold excess of the MR antagonist spironolactone (Fig. 4A). Spironolactone suppressed the aldosterone-induced claudin-4 phosphorylation on threonine residues, indicating that it is mineralocorticoid specific. Because we previously showed (18) that in RCCD2 cells, PKC activity is rapidly (5–20 min) stimulated in the presence of aldosterone, this kinase seemed to be a good candidate for claudin-4 phosphorylation. However, inhibition of PKC by chelerythrine chloride or of PKC-{alpha} by Gö-6976 was ineffective in preventing claudin-4 phosphorylation (Fig. 4B), suggesting that it may depend on other kinases. Phosphorylation of claudin-4 did not occur in cells preincubated with cycloheximide and treated with aldosterone for 20 min (cycloheximide 112 ± 22%, cycloheximide + aldosterone 105 ± 10% of values observed with aldosterone alone; n = 3 filters in each group). This suggests that claudin-4 phosphorylation may require protein neosynthesis. Altogether, these data show that aldosterone induces selective threonine phosphorylation of claudin-4 as part of the early phase of hormone action.



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Fig. 4. Effect of inhibitors and antagonists on aldosterone-induced threonine phosphorylation of claudin-4. A: MR-specific antagonist spironolactone (Spiro, 10 µM) prevented the aldosterone-induced (10 nM, 20 min) phosphorylation of claudin-4 on threonine residues. B: aldosterone-induced (10 nM, 20 min) phosphorylation of claudin-4 was not affected when cells were preincubated with the PKC inhibitor chelerythrine chloride (CC, 100 nM) or the inhibitor of its {alpha}-isoform Gö-6976 (G, 10 nM). Each point is the mean ± SE from 3 filters. ***P < 0.001, aldosterone-treated vs. control cells (Student's t-test). C, cells incubated with diluents alone.

 
Early effects of aldosterone on TER and on apical-to-basolateral 125I and 22Na passage across RCCD2 cell monolayers. We examined whether the apical-to-basolateral permeability of RCCD2 cell monolayers to ions could be modified rapidly by aldosterone. As illustrated in Fig. 5B, accumulation of 125I in the basal medium increased with time after its apical addition to RCCD2 cell monolayers (control condition). In the presence of 10 nM aldosterone, the accumulation of 125I in the basal medium was clearly higher; the difference was apparent in 20 min, and no further modification occurred later. A clear difference between control and aldosterone-treated monolayers was also observed when the opposite flux (basal-to-apical 125I passage) was measured: at 30 min, apical accumulation of 125I was 1,067 ± 182 cpm under control conditions and 3,182 ± 874 cpm in the presence of aldosterone (means ± SE; n = 3 filters in each condition).



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Fig. 5. Effect of aldosterone on apical-to-basal 22Na and 125I passage and on transepithelial resistance (TER) in RCCD2 cells. RCCD2 cells grown on filters were incubated with aldosterone (Aldo, 10 nM) or diluent (control) and 22Na (A) or 125I (B) added in the apical medium; the radioactivity of the basal medium (50-µl aliquots) was measured at various time intervals. Results are expressed as counts per minute (cpm) accumulated in the basal medium. Basal accumulation of 125I was greater in aldosterone-treated cells than in control cells. In contrast, basal accumulation of 22Na was comparable. TER, measured with the Millicell apparatus, was reduced in aldosterone-treated cells compared with control cells (C). Each point is the mean ± SE from 6–9 filters. **P < 0.01, ***P < 0.001, aldosterone vs. control (ANOVA and Bonferroni test).

 
When 22Na was used (apical addition and basal collection), there was also a time-dependent accumulation of basal radioactivity; however, in this case, aldosterone did not modify it during the first hour (Fig. 5B). Later on (2 h), 22Na accumulation in the basal medium was enhanced in aldosterone-treated monolayers (not shown), consistent with the well-known delay of aldosterone-induced transepithelial Na transport. These results suggest that early events in aldosterone action in RCCD2 monolayers include an increase in anion permeability (presumably via the paracellular pathway) that does not coincide with Na passage but in fact precedes it.

It was previously shown that aldosterone reduces TER across RCCD2 monolayers (7); that observation was confirmed in this study, in which TER was estimated with the Millicell system in monolayers exposed to 10 nM aldosterone. As shown in Fig. 5C, initial resistance of the monolayers (4,500–5,000 {Omega}·cm2) was decreased by aldosterone at 45 min and thereafter.

[3H]mannitol flux is reduced in presence of aldosterone. To estimate another aspect of the paracellular pathway of RCCD2 cell monolayers, apical-to-basal flux of [3H]mannitol [a small uncharged molecule (Mr 182) that does not enter the cells] was measured in the presence or absence of aldosterone (10 nM). After addition of [3H]mannitol to the apical medium, its time-dependent accumulation was observed in the basal compartment (Fig. 6A). [3H]mannitol passage was ~15- to 20-fold higher through filters without cells (data not shown). As illustrated in Fig. 6A, aldosterone addition resulted in a reduced accumulation of [3H]mannitol in the basal chamber (compared with untreated cells). Comparison of 95% confidence interval bands derived from regression analysis shows that aldosterone-treated cells exhibited significantly lower mannitol passage as early as 20 min after hormone addition. Mannitol flux (in mol/s) in cells grown on 12-mm-diameter filters (1.13 cm2) was about two times lower in the presence than in the absence of aldosterone (control cells 24.6 x 10–18 mol/s, aldosterone 12.4 x 10–18 mol/s). Apparent Pmann was calculated to be 1.80 x 10–6 cm/s in control RCCD2 cells, i.e., values within the range of those reported in other cultured cell monolayers (30); in aldosterone-treated cells, [3H]mannitol permeability was about two times lower (0.91 x 10–6 cm/s). This result indicates that aldosterone elicits a reduction in paracellular pathway permeability to mannitol, which occurs rapidly after hormonal exposure.



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Fig. 6. Aldosterone-induced reduction in apical-to-basal [3H]mannitol passage in RCCD2 cells. RCCD2 cells grown on filters were incubated with aldosterone or diluent (control, C) on both sides of the monolayer, and tracer amounts of [3H]mannitol (50,000–100,000 cpm) were added to the apical medium. The radioactivity of the basal medium was measured in 50-µl aliquots; results are expressed as cpm accumulated in the basal medium or as their % variation compared with the control condition (without treatment). A: RCCD2 cells were incubated for various periods of time with 10 nM aldosterone or diluent and [3H]mannitol. Basal [3H]mannitol accumulation (in cpm) is shown on the y-axis. There is a linear increase in [3H]mannitol accumulation in the basal compartment over 3 h (y = 13.6x + 115.8); aldosterone reduced [3H]mannitol accumulation by a factor of 2 (y = 6.6x + 83.5). Each point is the mean ± SE from 3–8 filters. B: effect of aldosterone and dexamethasone (Dex): dose-dependent effect of aldosterone on [3H]mannitol basal accumulation (as % variation of control condition) was evidenced, with a significant reduction in [3H]mannitol flux at 10 nM (1 h) aldosterone and higher (*P < 0.05 between 10 nM and 1,000 nM). In contrast, dexamethasone (10 nM) was ineffective. Each point is the mean ± SE from 3–6 filters. C: MR-specific antagonist spironolactone (Spiro, 10 µM) prevented the aldosterone-induced (10 nM, 1 h) reduction in [3H]mannitol basal accumulation, whereas RU-486 (10 µM), an antagonist of the glucocorticoid receptor, did not block the effect. Each point is the mean ± SE from 3–6 filters. ***P < 0.001, aldosterone vs. control (Student's t-test).

 
Dose dependence and steroid specificity of the observed aldosterone-dependent reduction in apical-to-basal [3H]mannitol passage (in cpm accumulated in the basal compartment in 1 h) was examined after 1-h hormone exposure. As illustrated in Fig. 6B, aldosterone reduced mannitol flux in a dose-dependent manner: it was ineffective at 1 nM and progressively reduced mannitol flux at higher concentrations to yield 40% of control values at 1 µM hormone concentration. The glucocorticoid dexamethasone (10 nM) failed to reproduce the aldosterone effect. As a matter of fact, higher concentrations of dexamethasone had an effect opposite to that observed with aldosterone: apical-to-basal mannitol flux was enhanced by 183 ± 13% in the presence of 100 nM dexamethasone (P < 0.01; n = 6) and by 312 ± 56% at 1 µM dexamethasone (P < 0.001; n = 6). These data suggest that the reduction in paracellular permeability to [3H]mannitol is aldosterone specific and does not imply GR occupancy. To evaluate directly whether the MR or the GR are involved in the observed effect, antagonists (1,000-fold excess) of each receptor were tested. As shown in Fig. 6C, the MR-specific antagonist spironolactone (10 µM) fully prevented the 10 nM aldosterone-dependent reduction in paracellular mannitol passage. In contrast, the GR-specific antagonist RU-486 (10 µM) was without effect. This observation is indicative of a classic effect of aldosterone mediated by the MR. To evaluate whether the observed changes in mannitol flux could depend on protein synthesis, similar experiments were performed in the presence of the protein synthesis inhibitor cycloheximide (10 µM). Preincubation of cells with cycloheximide prevented the 10 nM aldosterone-induced reduction in mannitol permeability [cycloheximide 96 ± 4, cycloheximide + aldosterone 95 ± 1 (in % of values observed with aldosterone alone); n = 6 filters in each group]. Altogether, these data show that aldosterone reduces paracellular passage of mannitol through a mechanism that is highly specific for aldosterone; it requires MR occupancy and occurs rapidly after hormone addition.


    DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
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 DISCUSSION
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 REFERENCES
 
In epithelia with high resistance, contributions of TJ are of importance to allow fine-tuning of transepithelial ion movements (1, 20, 32, 33). Claudins play a key role in determining paracellular permeability properties, allowing dynamic regulation of TJ (33). In the renal collecting duct, electrogenic Na absorption is accompanied by transcellular K secretion (2). Aldosterone amplifies cation movements through upregulation of apical ENaC and basolateral Na-K-ATPase (10, 24, 37). In addition to transcellular ion transport, the contribution of the paracellular pathway may be important in pathophysiological situations, and limited information is available on its possible modulation by corticosteroid hormones. Glucocorticoid hormones stimulate TJ formation in 1–3 days, as documented in mammary gland epithelial cells by Firestone and colleagues (42). In kidney cells, it was proposed a long time ago that the steroid hormone aldosterone could affect paracellular permeability in addition to its well-established involvement in the control of transcellular Na transport in the distal parts of the nephron (2, 24).

Because of the pivotal role of claudin-4 in the regulation of paracellular charge selectivity, we have chosen to focus our experiments on claudin-4 and aldosterone in cortical collecting duct cells. To investigate the early effects of aldosterone, the RCCD2 rat renal collecting duct cell line was used as an aldosterone-sensitive cell model (7, 18). Within the first hour after aldosterone exposure, we did not find any detectable modification of endogenous claudin-4 expression level or cellular localization but rather changes in its phosphorylation: aldosterone promotes claudin-4 phosphorylation on threonine residues, whereas serine and tyrosine residues are unaffected. This event is part of the early response to the hormone because it occurs within 20 min and it depends on MR occupancy. Other claudins may be expressed in RCCD2 cells and phosphorylated after aldosterone treatment, but this hypothesis was not addressed in this study. Within 20 min, passage of 125I from the apical to the basal side of the filter was enhanced in aldosterone-treated cells; this passage occurred in the absence of changes in 22Na passage, indicating that the early increase in 125I accumulation in the basal compartment cannot be attributed to an increased electrical driving force for anion reabsorption secondary to Na movements. This observation is suggestive of a selective increase in anion permeability elicited by aldosterone, which was concomitant with claudin-4 phosphorylation; thereafter the difference between control and aldosterone-treated cells remained constant (no further increase in accumulation of radioactivity). In the renal collecting duct, it is estimated that Cl reabsorption occurs predominantly via the paracellular route (14, 26). Consistent with this notion, we have noted that apical-to-basal as well as basal-to-apical 125I passages were enhanced in the presence of aldosterone in RCCD2 cell monolayers; this observation is suggestive of the implication of the paracellular rather than the transcellular pathway. The observed change in anion passage is in agreement with the notion that claudin-4 is a regulator of the charge selectivity of the paracellular pathway (5, 6, 34, 35). Aldosterone reduced TER in RCCD2 cells, but this occurred later on, i.e., only 45 min after hormone addition as reported earlier (7), suggesting that it is not directly linked to claudin-4 phosphorylation. It can be proposed that changes in TER may reflect changes in ion permeability (Cl, HCO3, and K+). It was also observed that [3H]mannitol permeability was reduced in the early phase of aldosterone action. Changes in [3H]mannitol flux are often taken as an index of change in TJ permeability, even if the nature of these changes is poorly characterized with this tool (29). It may be related to the size of the permeability pore, which might be also modified by the hormone. Because claudin-4 phosphorylation may alter the charge selectivity of TJ rather than its size, changes in mannitol flux may not be directly related to claudin-4 phosphorylation.

Aldosterone-induced claudin-4 phosphorylation appears to be rapid and relatively transient (as expected for such protein modification); it is notable that mannitol and 125I accumulation were modified by aldosterone treatment at the same moment (20 min) and were not further altered later on. This was not the case for the changes in TER that occurred later. Claudin-4 phosphorylation may trigger (or participate in) a cascade of events leading to functional changes of the TJ. It could be proposed that claudin-4 phosphorylation is part of a signaling cascade that could modify cellular compartmentalization of other TJ proteins or their stability. It is also possible that the half-life of the TJ modifications is much longer than the phosphorylation event. Aldosterone-induced claudin-4 phosphorylation and reduction in [3H]mannitol permeability were sensitive to cycloheximide, suggestive of the need for protein neosynthesis. However, cycloheximide (added to cells 1 h before aldosterone) may also modify several cell parameters, precluding definite conclusions. Altogether, these observations indicate that aldosterone may be a regulator of TJ in RCCD2 cell monolayers.

A limitation of this study is that the link between aldosterone-induced claudin-4 phosphorylation and the observed changes in TJ properties has not been established. Indeed, comparison of the kinetics of the observed changes does not provide evidence for causative links. As a matter of fact, addressing specifically the functional consequences of claudin-4 phosphorylation in native (nontransfected) cells would be extremely difficult, because expression of threonine mutants would compete with endogenous claudin-4. Such a hint has already been underlined by Yamauchi et al. (40).

There are eight threonines within the rat claudin-4 coding sequence: four reside in the first extracellular loop, two reside in transmembrane domain 3, and two reside in the intracellular loop, one of which (T105) is conserved among rat, human, and mouse claudin-4. These two intracellular threonines might be those that are phosphorylated in the presence of aldosterone in RCCD2 cells. However, it is impossible to determine which threonine was specifically modified in this study, because we monitored the phosphorylation status of endogenous (not overexpressed) claudin-4.

Among the very large family of kinases, several kinases of the renal collecting duct may be responsible for claudin-4 phosphorylation, such as the serum- and glucocorticoid-activated kinase (sgk), the phosphatidylinositol 3-kinase (PI3-kinase), PKC, or the newly discovered WNKs (15, 18, 24, 37). The use of kinase inhibitors (when they exist) may not solve this question, because their specificity is often broad or unknown toward novel kinases such as the WNKs. Sgk, PI3-kinase, and PKC are regulated by corticosteroid hormones (18, 24, 37). Among them, PKC seemed interesting to consider, because its activity is upregulated by aldosterone in RCCD2 cells within 5–10 min (18), i.e., just before the observed increase in claudin-4 phosphorylation. However, we did not observe any effect of PKC inhibitors on aldosterone-induced claudin-4 phosphorylation. Other attractive candidates are the WNKs (16, 22). Claudin-4 can be phosphorylated by the kinase WNK4 (40). WNK4 is expressed in the distal nephron and appears to be a new regulator of ion transporters or channels such as thiazide-sensitive Na-Cl cotransporter or rat outer medullary K channel (14–16, 39). Mutations in the WNK4 gene have been identified in a Mendelian form of hypertension, accompanied by an increased Cl permeability of the distal nephron (38). Moreover, it has been shown that there is a correlation between increased Cl permeability and claudin phosphorylation induced by the disease-causing mutant WNK4 (40). WNK4 is thought to regulate paracellular ion permeability in the distal nephron, in particular the Cl shunt (14–16). WNK4 and claudin-4 interact directly (40), and it is tempting to propose that aldosterone might favor this interaction, thus promoting claudin-4 phosphorylation as reported in this study. Further studies are needed to test this hypothesis. Interestingly, WNK4 appears to be upregulated by long-term aldosterone treatment in the mouse kidney (21). Moreover, it was reported very recently that the kidney-specific WNK1 isoform is also induced by aldosterone as early as 30 min after hormonal exposure in a mouse collecting duct cell line transfected with the MR (22).

In conclusion, we report herein a series of data that designate the TJ as a novel target for aldosterone. We have shown that claudin-4 phosphorylation on threonine residues is an early event in aldosterone action. Its consequences on TJ properties remain to be elucidated. It can be proposed that claudin-4 may be interesting to consider as a candidate gene in patients with abnormal salt handling, with specific attention to its threonine residues.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This work was supported by Institut National de la Santé et de la Recherche Médicale. C. Le Moellic and S. Boulkroun were recipients of doctoral grants from the French Ministère de la Recherche and from the Fondation pour la Recherche Médicale. D. González-Nunez was supported by a Del Duca fellowship.


    ACKNOWLEDGMENTS
 
We acknowledge the participation of Severine Rousseau in some of the experiments as part of her master's degree training. We are indebted to Martine Muffat-Joly for help in statistical analyses. We thank D. Pelaprat and A. Gruaz for help in 125I flux experiments.

Present address of M. Blot-Chabaud: INSERM UMR 608, UFR de Pharmacie, 27 boulevard Jean Moulin, 13005 Marseille, France.


    FOOTNOTES
 

Address for reprint requests and other correspondence: N. Farman, INSERM U478, Faculté de Médecine Xavier Bichat, BP 416, 75870 Paris Cedex 18, France (e-mail: farman{at}bichat.inserm.fr)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

* M. Blot-Chabaud and N. Farman contributed equally to this work. Back


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 DISCUSSION
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