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
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ABSTRACT |
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paracellular permeability; mannitol flux; occludin; iodine; sodium; kidney; RCCD2 cells
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 (2,0005,000 ·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.
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MATERIALS AND METHODS |
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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 [50,000100,000 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 25% 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:
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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--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-; 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.
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RESULTS |
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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 (4,5005,000 ·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 1018 mol/s, aldosterone 12.4 x 1018 mol/s). Apparent Pmann was calculated to be 1.80 x 106 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 106 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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DISCUSSION |
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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 510 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 (1416, 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 (1416). 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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GRANTS |
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ACKNOWLEDGMENTS |
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Present address of M. Blot-Chabaud: INSERM UMR 608, UFR de Pharmacie, 27 boulevard Jean Moulin, 13005 Marseille, France.
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FOOTNOTES |
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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.
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