Pendrin Regulation in Mouse Kidney Primarily Is Chloride-Dependent
Marion Vallet*,,
Nicolas Picard*,,
Dominique Loffing-Cueni,
Marinos Fysekidis*,,
May Bloch-Faure,
Georges Deschênes,
Sylvie Breton||,
Pierre Meneton*,,
Johannes Loffing,
Peter S. Aronson¶,
Régine Chambrey*, and
Dominique Eladari*,,**
* INSERM U652, IFR58, Institut des Cordeliers; Université Paris-Descartes, Faculté de Médecine René Descartes; UMR 7134 CNRS-Université Pierre et Marie Curie; ** Département de Physiologie, Hôpital Necker-Enfant Malades, AP-HP, Paris, France; University of Fribourg, Department of Medicine, Unit of Anatomy, Fribourg, Switzerland; || Program in Membrane Biology, Massachusetts General Hospital, and Department of Medicine, Harvard Medical School, Boston, Massachusetts; and ¶ Department of Internal Medicine, Yale University School of Medicine, New Haven, Connecticut
Address correspondence to: Dr. Régine Chambrey or Dr. Dominique Eladari, INSERM U652, IFR58, Institut des Cordeliers, 15 rue de l’Ecole de médecine, 75006 Paris, France. Phone: +33-1-4441-3718; Fax: +33-1-4441-3717; E-mail: chambrey{at}ccr.jussieu.fr or eladari@ccr.jussieu.fr
Received for publication October 12, 2005.
Accepted for publication May 21, 2006.
Recent studies indicate that pendrin, an apical Cl–/HCO3–exchanger, mediates chloride reabsorption in the connectingtubule and the cortical collecting duct and therefore is involvedin extracellular fluid volume regulation. The purpose of thisstudy was to test whether pendrin is regulated in vivo primarilyby factors that are associated with changes in renal chloridetransport, by aldosterone, or by the combination of both determinants.For achievement of this goal, pendrin protein abundance wasstudied by semiquantitative immunoblotting in different mousemodels with altered aldosterone secretion or tubular chloridetransport, including NaCl loading, hydrochlorothiazide administration,NaCl co-transporter knockout mice, and mice with Liddle’smutation. The parallel regulation of the aldosterone-regulatedepithelial sodium channel (ENaC) was examined as a control forbiologic effects of aldosterone. Major changes in pendrin proteinexpression were found in experimental models that are associatedwith altered renal chloride transport, whereas no significantchanges were detected in pendrin protein abundance in modelswith altered aldosterone secretion. Moreover, in response tohydrochlorothiazide administration, pendrin was downregulateddespite a marked secondary hyperaldosteronism. In contrast,-ENaC was markedly upregulated, and the molecular weight ofa large fraction of -ENaC subunits was shifted from 85 to 70kD, consistent with previous results from rat models with elevatedplasma aldosterone levels. These results suggest that factorsthat are associated with changes in distal chloride deliverygovern pendrin expression in the connecting tubule and corticalcollecting duct.
Long-term BP regulation is remarkably linked to renal salt excretoryfunction. Normally, the kidney adapts urinary NaCl excretionto match exactly daily dietary NaCl intake. Among the differentnephron segments, the critical importance in vascular volumeand BP regulation of the aldosterone-sensitive distal nephron(ASDN), which consists of part of the distal convoluted tubule(DCT), the connecting tubule (CNT), and the collecting duct(CD), now is well established (1). In fact, during the pastdecade, mutations in genes that cause syndromes that are characterizedby hypertension or hypotension have been identified, and mostof these genes have turned out to be involved in the controlof Na+ absorption in the ASDN (for review, see reference [2]).
It is widely assumed that the pressor effect of dietary saltdepends primarily on Na+. However, several studies indicatethat Cl– is necessary for Na+ to exert its pressor effect(3–7), and primary alteration in distal Cl– transportmay have consequences on BP (8–10). Remarkably, in theCNT and the CD, Na+ reabsorption is not linked molecularly toCl– transport directly. Na+ reabsorption is activatedby the hormone aldosterone and is achieved through the apicalepithelial sodium channel (ENaC), which is expressed by CNTcells and principal cells of CD. Cl– transport does notoccur through principal cells but rather through the paracellularpathway or through the intercalated cells (11). In the B andnon-A non-B type intercalated cells, pendrin, an apical anionexchanger (12–14), is believed to play a key role in BPregulation, most likely by controlling distal nephron chloridereabsorption (15,16). Pendrin is upregulated by the injectionof deoxycorticosterone pivalate, a pharmacologic analogue ofaldosterone (16). However, we have demonstrated that pendrinalso can be regulated in response to changes in Cl– intakeby an aldosterone-independent mechanism (17). In vivo relevanceof both mechanisms remains unsettled, but this question is ofparticular importance. Therefore, the goal of our study wasto examine whether pendrin is regulated in vivo primarily bya chloride-dependent mechanism, by aldosterone, or by the combinationof both determinants.
To achieve this goal, we examined the protein expression ofpendrin in different mouse models with disturbed NaCl balancethat allow separate analysis of the effects of changes in distalchloride transport or in aldosterone secretion. We systematicallyexamined the parallel regulation of ENaC as a control. Our datademonstrate that Cl–-dependent pendrin regulation is ableto override the effects of aldosterone. These data also suggestthat pendrin might be an independent modulator of NaCl transportby the ASDN.
Animal Models and Treatments
All animals used in this study were treated in full compliancewith the French government animal welfare policy (agreementno. RA024647151FR).
NaCl Loading.
A treated group and a control group were handled in parallelduring a 6-d period. Each group consisted of seven adult C57BL/6male mice. Treated mice were given 0.28 M NaCl in the drinkingwater and were fed ad libitum with standard laboratory mousechow (AO3; Scientific Food and Engineering, Augy, France). Controlmice were fed ad libitum with standard laboratory mouse chowand had free access to distilled water for the same period oftime.
Mouse Model of Liddle Syndrome with Constitutive Activation of the Apical Sodium Channel ENaC.
Transgenic mice were generated by insertion of a stop codon(corresponding to residue R566 in human) into the mouse ENaCgene locus as described previously (18). Seven homozygous transgenicmice (ENaCL/L) and seven wild-type littermate controls (ENaCWT)were used for these series. All of the animals were outbredon a mixed 129/Ola-C57BL/6 genetic background. Animals were5 to 6 mo of age and weighed 28 to 30 g.
NaCl Co-Transporter (NCC) Knockout Mice.
Mice were generated by a standard gene-targeting technique asdescribed previously (19). Eight adult homozygous NCC-deficientmale mice (NCC–/–) and eight littermate wild-typemice (NCC+/+) were used for this series. All animals were inbredon the C57BL/6 genetic background (>10 generations of backcrossing).The animals were 7 to 8 mo of age and weighed 32 to 34 g.
Acute Hydrochlorothiazide Treatment
Hydrochlorothiazide (HCTZ; Sigma-Aldrich, St. Louis, MO), aspecific inhibitor of the NCC co-transporter of the DCT, wasgiven to seven adult C57BL/6 male mice for 48 h, at a dose of130 mg/kg body wt per d mixed with powdered standard laboratorymouse chow. The control group was composed of seven adult C57BL/6male mice and was fed with the same powdered food without HCTZ.The dose was determined by preliminary experiments in whichvarious doses were tested (65, 130, and 260 mg/kg body wt perd); 130 mg/kg body wt per d was found to be the minimal doserequired to detect a significant increase in renal Na+ excretionin metabolic studies (data not shown).
Physiologic Studies
Animals were housed in metabolic cages. They first were allowedto adapt for 5 d to the cages before the experimental period.Urine then was collected daily under light mineral oil for urinaryflow and electrolyte measurements. At the end of the experimentalperiod, animals were killed after anesthesia was induced byperitoneal injection of ketamine and xylazine (0.1 and 0.01mg/g body wt, respectively). Plasma was collected on heparin,and kidneys were removed. Urinary pH and Pco2 were measuredwith a pH/blood-gas analyzer (Radiometer ABL555; Copenhagen,Denmark). Serum and urine electrolytes and urine creatininewere measured with a Konelab 20i autoanalyzer (Thermo ElectronCorp., Eragny Parc, France). Blood pH, Pco2 and Po2 were measuredwith an AVL Compact 1 pH/blood-gas analyzer (AVL InstrumentsMédicaux, Eragny-sur-Oise, France). Plasma and urinaryaldosterone were measured by RIA (DPC Dade Behring, La Défense,France). Plasma renin activity was determined by RIA of angiotensinI generated by incubation of the plasma at pH 8.5 in the presenceof an excess of angiotensinogen as described previously (20).
Antibodies
Rabbit polyclonal antibody directed to C-terminal amino acids630 to 643 of mouse pendrin has been characterized (21,22).Affinity-purified rabbit polyclonal antibody against the B1subunit of the H+-ATPase was generated by S. Breton (MassachusettsGeneral Hospital, Charlestown, MA). This antibody was raisedagainst the last 13 amino acids of the C-terminal tail of therat ATP6V1B1 subunit isoform (C-QGAQQDPASDTAL). The peptidewas coupled to KLH via a cysteine positioned to its N-terminalend. Western blotting showed one single band in kidney extractsand complete inhibition of the signal after preincubation ofthe antibody with the B1 peptide (data not shown). Rabbit polyclonalantibodies directed to -ENaC (amino acids 46 to 68), -ENaC (aminoacids 617 to 638), and -ENaC (amino acids 629 to 650) were describedby Masilamani et al. (23). The ENaC antibodies were tested inpreliminary experiments to verify that the specificity was identicalto that of the antibodies from the original batch (data notshown).
Immunoblot Analyses
Membrane fraction preparation and immunoblotting proceduresfor comparing two sets of samples of renal cortical membraneswith regard to relative abundance of specific proteins weredescribed in detail previously (17,24). Coomassie blue–stainedpolyacrylamide gels were used to control equality of proteinloading for each series, as described in detail previously (17,24).The dilutions of primary antibodies used in this study wereas follows: Anti-pendrin, 1:30,000; anti–H+-ATPase, 1:30,000;anti–-ENaC, 1:3000; anti–-ENaC, 1:20,000; and anti–-ENaC,1:2000. Densitometric values were normalized to the mean forthe control group in a given experiment, which was defined as100%, and results were expressed as means ± SE.
Immunolabeling
Kidneys of vehicle and HCTZ-treated mice were fixed by vascularperfusion with 3% paraformaldehyde and 0.05% picric acid inPBS, frozen in liquid propane, and stored at –80°Cuntil use. Kidneys of NCC–/– and NCC+/+ mice weretaken from a previous experiment (25). Tissue was processedfor immunolabeling as described previously (25). Briefly, cryosections(5 µm thick) were incubated overnight at 4°C withanti-pendrin antibody (dilution 1:400). Binding sites of theprimary antibodies were detected with a Cy3-conjugated goatanti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove,PA) diluted 1:1000.
Morphometry
Kidney sections were immunostained for pendrin and were analyzedby an experienced investigator who was blinded to the treatmentand genotype of the mice. Using a Zeiss epifluorescence microscope,CNT and CCD profiles were randomly selected and the number ofintercalated cells with apical pendrin immunostaining was quantifiedand expressed as percentage of the total number of pendrin-positivecells analyzed. At least 200 pendrin-positive cells were evaluatedper animal.
Statistical Analyses
All data are presented as means ± SEM. Comparisons amonggroups were assessed by ANOVA or unpaired t test. Differenceswere considered significant at P < 0.05.
Mouse Pendrin Protein Abundance Was Decreased in Response to Long-Term NaCl Loading
We previously described that an increased oral Cl– loadreduces pendrin expression in kidneys of rats (17). Becauseit is possible that mice and rats differ with respect to mechanismsof NaCl transport regulation, we first tested whether we couldreproduce this effect in mice. In response to high NaCl intake,extracellular fluid volume (ECFV) was only moderately expandedin the treated mice, because there was no detectable changein plasma aldosterone concentration when compared with controlgroup values (361 ± 57 pM in the NaCl-loaded group versus467 ± 64 pM in the control group; P = 0.24). In the sameway, plasma renin activity tended to be lower in the treatedgroup, but this difference did not reach statistical significance(882 ± 286 ng angiotensin I/h per ml in treated miceversus 1452 ± 494 ng angiotensin I/h per ml in controls;P = 0.3). As previously found in rat, pendrin was significantlydownregulated (71 ± 3% in NaCl-loaded mice versus 100± 8% in controls; P = 0.006; Figure 1, A and B). -ENaCprotein abundance tended to be lower, but a statistically significantdifference was not demonstrated (74 ± 7% in NaCl-loadedmice versus 100 ± 11% in control group; P = 0.07; Figure 1, A and C).In contrast, as shown in Figure 1, A and D, the profile of -ENaCwas changed with a shift from the 70-kD "active" molecular form,which was decreased (23 ± 5% in NaCl-loaded mice versus100 ± 13% in control mice; P = 0.0001), toward the 85-kD"inactive" form (128 ± 2% in NaCl-loaded mice versus100 ± 3% in control mice; P < 0.0001). -ENaC proteinabundance was markedly upregulated in treated mice (184 ±10% in NaCl-loaded mice versus 100 ± 8% in control group;P < 0.0001; Figure 1, A and E). Importantly, this correspondedexactly to the opposite changes observed previously by Masilamaniet al. (26) in response to dietary NaCl restriction in the rat.
Figure 1. Effects of chronic NaCl loading on pendrin the protein abundance of the epithelial sodium channel (ENaC) subunits , , and in the mouse kidney cortex. (A) Immunoblots of membrane fractions from the cortex obtained from seven NaCl-loaded mice and seven control mice. Immunoblots were reacted with antibodies directed against pendrin and the three ENaC subunits. (B through E) Densitometric analyses of data showing the abundance of pendrin, -ENaC, -ENaC, and -ENaC, respectively, in response to chronic NaCl loading, expressed as percentage of control values. Values are means ± SEM. *Statistically significant difference versus controls (P < 0.05).
No Changes in Pendrin Protein Abundance Were Detectable in ENaCL/L Mice Despite a Marked Secondary Hypoaldosteronism
We next assessed the effect of ECFV expansion/low aldosteroneconcentration in the presence of unaltered chloride intake byanalyzing pendrin protein abundance in mice with Liddle’smutation. The phenotype of ENaCL/L mice was exactly as describedpreviously by Pradervand et al. (18,27). Despite their activatedNa+ transport in the CD, Liddle mice did not differ from theircontrols with respect to their body weight, acid–basestatus, or plasma electrolyte concentrations (Table 1). However,mutated mice exhibited an increase in ECFV as attested by adramatic decrease in urinary and plasma aldosterone concentrations(Table 1). Despite this secondary hypoaldosteronism, no changesin pendrin protein abundance were detectable in Liddle mice(95 ± 13% in ENaCL/L mice versus 100 ± 12% inENaCWT mice; P = 0.8; Figure 2, A and B). However, we cannotexclude the possibility that hypoaldosteronism secondary tothe Liddle mutation led to a small decrease in pendrin proteinabundance that was below the detection limit of our immunoblottingtechnique (<20% change). In contrast, both - and -ENaC subunitsclearly were downregulated in Liddle mice (66 ± 3% inENaCL/L mice versus 100 ± 5% in ENaCWT mice [P < 0.0001;Figure 2, A and C] and 38 ± 4% in ENaCL/L mice versus100 ± 8% in ENaCWT mice [P < 0.0001; Figure 2, A and D],for - and -ENaC, respectively). As expected, the -ENaC subunitwas not detectable in ENaCL/L mice because the mutation deletedthe C-terminal end of the protein that contains the epitopesthat are recognized by the -ENaC–specific antibody (Figure 2A).
Figure 2. Abundance of pendrin and -, -, and -ENaC proteins in kidney cortex of mice with Liddle’s mutation (ENaCL/L) and their wild-type controls (ENaCWT). (A) Immunoblots of membrane fractions from the cortex obtained from seven ENaCL/L mice and seven controls. Immunoblots were reacted with antibodies directed against pendrin and the three ENaC subunits. -ENaC subunit was not detectable in ENaCL/L mice because the mutation deleted the C-terminal end of the protein, which is recognized by the anti–-ENaC antibody. (B through D) Densitometric analyses showing the abundance of pendrin, -ENaC, and -ENaC, respectively, in ENaCL/L mice, expressed as percentage of control values. Values are means ± SEM. *Statistically significant difference versus ENaCWT mice (P < 0.05).
Pendrin Abundance Is Downregulated whereas ENaC Expression Is Stimulated in Response to Acute HCTZ Administration
To examine the effect of a secondary hyperaldosteronism as aresult of a renal loss of NaCl (i.e., without NaCl restriction)on pendrin protein expression, we next tested in wild-type micethe acute effect of HCTZ, a pharmacologic inhibitor of NCC,the NaCl transporter of the DCT. Physiologic data are shownin Tables 2 and 3. As expected, treated mice exhibited a markedincrease in urinary excretion of Na+ and Cl– after thefirst day of drug administration when compared with controlvalues. After 48 h of HCTZ treatment, the urinary excretionof Na+ and Cl– returned toward normal values, indicatingthat the mice compensated (Table 3). HCTZ-treated mice developeda marked decrease in ECFV, attested by a marked decrease inbody weight, and an increase in plasma protein, urea, and aldosteroneconcentrations (see Table 2). Unexpected, pendrin protein abundancewas not up- but downregulated in treated mice when comparedwith controls, as shown in Figure 3, A and B (67 ± 9%in HCTZ group versus 100 ± 11% in vehicle group; P =0.04). In contrast, -ENaC expression was increased (155 ±11% in HCTZ group versus 100 ± 5% in vehicle group; P< 0.001; Figure 3, A and C), -ENaC expression showed a shiftfrom the 85- to the 70-kD band (70 kD: 351 ± 23% in HCTZgroup versus 100 ± 20% in vehicle group [P < 0.0001];85 kD: 53 ± 4% in HCTZ group versus 100 ± 7% invehicle group [P < 0.0001]; Figure 3, A and D), and -ENaCexpression was decreased (47 ± 5% in HCTZ group versus100 ± 6% in vehicle group; P < 0.0001; Figure 3, A and E),consistent with previous results from rat models with elevatedplasma aldosterone levels (23,26).
Figure 3. Abundance of pendrin and -, -, and -ENaC proteins in the mouse kidney cortex in response to acute hydrochlorothiazide (HCTZ) administration. (A) Immunoblots of membrane fractions from the cortex obtained from seven mice that were treated for 48 h with HCTZ and seven control mice (vehicle). Immunoblots were reacted with antibodies directed against pendrin and the three ENaC subunits. (B through E) Densitometric analyses showing the abundance of pendrin, -ENaC, -ENaC, and -ENaC, respectively, in the HCTZ-treated mice, expressed as percentage of control values. Values are means ± SEM. *Statistically significant difference versus controls (P < 0.05).
Because recent data indicate that subcellular redistributionof pendrin is an important mechanism of pendrin regulation (15,16,21),the possibility exists that total protein abundance as assessedby Western blot on renal membrane fractions may not representthe physiologically active pool of pendrin. Therefore, we nextassessed whether HCTZ treatment induced a translocation of pendrinprotein to the apical membrane while total protein abundancewas decreased. However, Figure 4 shows that after 2 d of HCTZadministration, pendrin apical expression was markedly decreased,further supporting the conclusion that the functional pool ofpendrin is decreased in this situation.
Figure 4. Subcellular localization of pendrin in kidneys of control and HCTZ-treated wild-type mice. We performed immunofluorescence with rabbit antisera against pendrin on cryosections and then quantified pendrin-positive intercalated cells with apical pendrin immunostaining. In mice of both genotypes, the subcellular localization of pendrin is heterogeneous between cells. Cells with prominent apical and predominant intracellular localization of pendrin are intermingled in the stained tubular profiles (top). However, apical immunostaining is much more pronounced in the control than in the thiazide-treated mouse. Morphometry confirmed the less frequent apical localization of pendrin in intercalated cells of thiazide-treated mice (bottom).
Several Membrane Proteins from the Distal Nephron, Including Pendrin, Were Upregulated in NCC–/– Mice
The effect of HCTZ on pendrin protein that was observed in thepreceding series of experiments is paradoxic with respect tothe need for NaCl reclamation in response to blockade of theNaCl co-transporter of the DCT, NCC. Therefore, we next studiedthe regulation of pendrin in NCC–/– mice becausethese mice have the same defect in NaCl transport chronically.Moreover, in contrast to acute HCTZ treatment, this defect generallyis compensated in mice that ingest a NaCl-replete diet, presumablybecause of increased NaCl reabsorption in the downstream segmentsCNT and CD, where pendrin and ENaC are expressed (28). Physiologicdata from the NCC–/– and NCC+/+ mice that were usedin these studies are summarized in Table 4. As described previously(19), when fed a Na+-replete diet, NCC–/– mice werenormal with respect to their weight, plasma electrolyte concentrations,and acid–base balance. Figure 5, A and B, shows that pendrinprotein expression was markedly increased in the NCC–/–mice when compared with the NCC+/+ mice (260 ± 24% inNCC–/–versus 100 ± 8% in NCC+/+; P <0.0001). ENaC protein abundance also was increased in the NCC–/–mice (Figure 5), with an increase in the protein abundance ofall of the three ENaC subunits: -ENaC (162 ± 11% in NCC–/–versus 100 ± 9% in NCC+/+; P < 0.001; Figure 5, Aand C), -ENaC (183 ± 11% in NCC–/–versus100 ± 12% in NCC+/+; P < 0.001; Figure 5, A and E),and -ENaC (Figure 5, A and D). However, for -ENaC, the increaseconsisted of an increased expression of the two different 70-and 85-kD molecular forms of the protein (348 ± 34 and120 ± 5% in NCC–/–versus 100 ± 9and 100 ± 6% in NCC+/+; P < 0.0001 and P = 0.03 forthe 70- and 85-kD bands, respectively).
Figure 5. Abundance of pendrin and -, -, and -ENaC proteins in kidney cortex of NCC-null mice (NCC–/–) and their wild-type controls. (A) Immunoblots of membrane fractions from the cortex obtained from seven NCC–/– mice and seven controls. Immunoblots were reacted with antibodies directed against pendrin and the three ENaC subunits. (B through E) Densitometric analyses showing the abundance of pendrin, -ENaC, -ENaC, and -ENaC, respectively, in NCC–/– mice, expressed as percentage of control values. Values are means ± SEM. *Statistically significant difference versus controls (P < 0.05).
Loffing et al. (25) reported that the fractional cortical tubularvolume of CNT is approximately twice as high in NCC–/–than in NCC+/+ mice. This likely reflects a hypertrophy/hyperplasiaof the CNT in NCC knockout mice that contributes to compensationof the Na+ transport defect in the preceding DCT. To test whetherthe upregulation of the Cl– transporter pendrin that wasobserved in our experiments also was explained by the hypertrophy/hyperplasiaof the CNT and therefore to an increase in the number of pendrin-positiveintercalated cells, we assessed pendrin protein expression byimmunofluorescence studies on kidney sections from NCC–/–and NCC+/+ mice. Figure 6 shows that the number of pendrin-positivecells was markedly increased in NCC–/– mice whencompared with NCC+/+ mice, whereas the intensity of pendrinlabeling did not seem to be different. These results indicatedthat the upregulation of NaCl transporters from the CNT andCD was at least partly the consequence of tubular hypertrophy.This assumption was supported further by the fact that the proteinabundance of the intercalated cell–specific B1 subunitof the H+-ATPase was increased in magnitude similar to thatof pendrin in NCC–/– mice (317% ± 33 in NCC–/–versus 100% ± 12 in NCC+/+; P < 0.0001; Figure 7).However, although no obvious difference in subcellular localizationof pendrin was noted in this model (Figure 8A), careful morphometricquantification showed that the percentage of pendrin-positivecells with apical pendrin expression was increased slightly(Figure 8B).
Figure 6. Overviews on renal cortex of NCC+/+ and NCC–/– mice kept on a standard NaCl diet. Immunofluorescence with rabbit antisera against pendrin. The number of pendrin-positive tubular profiles and epithelial cells is increased drastically in the NCC–/– mice, which is consistent with the previously reported higher fractional cortical tubular volume of connecting tubules in NCC–/– mice when compared with NCC+/+ mice (25). Bar = approximately 100 µm.
Figure 7. B1 H+-ATPase protein abundance in kidney cortex of NCC–/– and NCC+/+ mice. (A) Immunoblots of membrane fractions from the cortex obtained from seven NCC–/– mice and seven controls. Immunoblots were reacted with anti-B1 subunit of the H+-ATPase antibody. (B) Densitometric analyses of data showing that the abundance of B1 subunit of the H+-ATPase, in NCC–/– mice, is increased compared with the control mice. Values are means ± SEM. *Statistically significant increase versus controls (P < 0.05).
Figure 8. Subcellular localization of pendrin in kidneys of NCC+/+ and NCC–/– mice on a standard NaCl diet. We performed immunofluorescence with rabbit antisera against pendrin on cryosections and then quantified pendrin-positive intercalated cells with apical pendrin immunostaining. In mice of both genotypes, the subcellular pendrin localization is heterogeneous between cells. Cells with prominent apical and predominant intracellular localization of pendrin are intermingled in the stained tubular profiles (top). Morphometry reveals that intercalated cells with apical pendrin immunostaining are slightly more frequent in NCC–/– mice than in NCC+/+ mice (bottom).
The CNT and the CD play a critical role in the fine-tuning ofNaCl and acid–base balance. In the CNT and the CD, unlikemost nephron segments, transepithelial reabsorption of Na+ andCl– occurs through two separate cellular pathways (11,29,30).The other unique feature of these nephron segments is that Cl–reabsorption is linked molecularly to bicarbonate secretion.Therefore, these distinct Na+ and Cl– pathways allow independentadaptation of NaCl and NaHCO3 reabsorption. However, Na+ reabsorption,which is under the control of the steroid hormone aldosterone,needs to be coordinated to Cl– reabsorption to affectECFV. Therefore, the aim of our study was to assess whetheraldosterone plays a major role in regulation of pendrin or whetherpendrin is regulated primarily by a Cl–-dependent mechanismthat is aldosterone independent. Our data confirm our previousobservation made in the rat that pendrin is strikingly dependenton factors that are associated with changes in distal Cl–delivery (17). In contrast, pendrin expression did not alwayscorrelate with circulating aldosterone level. Indeed, pendrinwas downregulated by an increase in NaCl intake even in theabsence of a detectable decrease of aldosterone secretion; conversely,in experiments with Liddle mice, we could not detect any significantchanges in pendrin protein abundance despite a marked decreasein aldosterone concentration. This could reflect that pendrinis sensitive mainly to an increase and not to a decrease inaldosterone levels, but the experiments shown in Figures 3 and4 clearly demonstrate that when distal chloride delivery wasincreased as a result of diminished NaCl reabsorption in theDCT, pendrin was downregulated despite a marked secondary hyperaldosteronism.This contrasts with the previous notion that aldosterone isa strong stimulatory factor of pendrin expression. Verlanderet al. (16) found that prolonged administration of the syntheticaldosterone analogue DOCP increases pendrin mRNA expressionand cell surface abundance of pendrin in intercalated cells.However, in the latter study, aldosterone was administered exogenouslyat high concentration (i.e., able to raise BP significantly).Therefore, it is possible that aldosterone when administratedin a supraphysiologic dose has a much more pronounced effecton pendrin. One other possibility, as noted by the authors themselves,is that the effect of DOCP was indirect. For example, chronicDOCP treatment leads to metabolic alkalosis and hypokalemia(16), two situations that have been shown to induce the translocationof pendrin from the cytosolic compartment to the apical membraneof the cells (21). Nevertheless, the data in Figures 3 and 4clearly suggest that in the physiologic range of aldosteroneconcentration, factors that are associated with changes in distalchloride delivery are the more important determinants regulatingpendrin.
The mechanism of Cl–-dependent regulation of pendrin remainsunsettled, but our data raise the possibility that it involvesthe capability to sense changes in the rate of urinary Cl–delivery or in luminal fluid Cl– concentration. In fact,we document adaptive changes in pendrin protein abundance inresponse to maneuvers that are expected to alter dramaticallydistal chloride delivery while plasma [Cl–] was unchanged.It is interesting that pendrin has been detected not only inmammals but also in the gill epithelium of a euryhaline elasmobranch,Dasyatis sabina (Atlantic stingray), in cells with a phenotypesimilar to that of B intercalated cells (31). In Dasyatis sabina,pendrin is believed to be an important mechanism for chloridereclamation required for the fish to adapt to fresh water (i.e.,to low [Cl–] conditions in the surrounding environment)(32). In contrast, increasing the salinity of the water markedlydecreased pendrin protein abundance (31). This observation supportsthe hypothesis that renal pendrin regulation in mice might bedue to changes in intratubular chloride concentration. However,because we did not measure luminal chloride delivery in theCNT and the CD in our various experimental conditions, it isnot possible from the data of this study to conclude firmlythat pendrin was regulated by changes in distal chloride deliveryor concentration per se. Moreover, several endocrine or paracrinefactors are expected to have been altered in parallel or bychanges in distal chloride delivery and might be variably involvedin pendrin regulation (e.g., vasopressin, bradykinin). Evidencefor additional complexity in the regulation of pendrin is thedisparity between the acute effect (48 h) of HCTZ to decreasegreatly pendrin expression and the upregulation of pendrin inNCC null mice. It should be emphasized that the latter modelinvolves a lifelong adaptation to the absence of NCC function,in contrast to the acute effect of HCTZ. Long-term changes inelectrolyte transport conditions may induce secondary morphologicchanges in the kidney. In fact, in the mouse model of NCC disruption,we found that the upregulation of pendrin was largely attributableto an increased prevalence of pendrin-positive intercalatedcells as a result of hypertrophy/hyperplasia of the CNT (seeFigures 6 and 8). Similar morphologic changes occur also inother models with increased ion transport activity in the ASDN,such as prolonged furosemide treatment (33–37), and mayexplain the reported upregulation of pendrin in response tothis diuretic (17). The signals underlying these chronic adaptationsare not known. Clearly, the disparity in pendrin regulationbetween the acute HCTZ model and the chronic NCC null modelindicates that factors other than acute changes in distal chloridedelivery must be involved in pendrin regulation.
Our results indicate that pendrin is regulated at the proteinlevel by factors that are associated with changes in distalchloride delivery or transport, whereas aldosterone in the physiologicrange plays a minor role. However, in response to situationsthat cause distal nephron hypertrophy and hyperplasia, suchas long-term inactivation of NCC, pendrin expression is upregulatedby several mechanisms, including hyperplasia of intercalatedcells. In this setting, the B and non-A non-B intercalated cellsmay participate in the renal compensatory mechanisms that limitNaCl loss by increasing pendrin-mediated Cl– reabsorption.
Acknowledgments
Support was provided to P.S.A. by National Institutes of Healthgrants DK33793 and DK17433, to S.B. by National Institutes ofHealth grant DK38452, to J.L. by Swiss National Science Foundationgrant 3200B0-105769/1, and to D.L.-C. by a Marie-Heim-VögtlinFellowship of the Swiss National Science Foundation.
We thank Dr. Edith Hummler and Prof. Bernard Rossier for providingthe Liddle mice and Dr. Gary E. Shull for providing the NCCknockout mice and for helpful discussions.
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
Meneton P, Loffing J, Warnock DG: Sodium and potassium handling by the aldosterone-sensitive distal nephron: The pivotal role of the distal and connecting tubule. Am J Physiol Renal Physiol 287: F593–F601, 2004[Abstract/Free Full Text]
Lifton RP, Gharavi AG, Geller DS: Molecular mechanisms of human hypertension. Cell 104: 545–556, 2001[CrossRef][Medline]
Kurtz TW, Morris RC Jr: Dietary chloride and bicarbonate as determinants of desoxycorticosterone hypertension. J Hypertens Suppl 2: S371–S373, 1984[Medline]
Kurtz TW, Al-Bander HA, Morris RC Jr: "Salt-sensitive" essential hypertension in men. Is the sodium ion alone important? N Engl J Med 317: 1043–1048, 1987[Abstract]
Schmidlin O, Tanaka M, Bollen AW, Yi SL, Morris RC Jr: Chloride-dominant salt sensitivity in the stroke-prone spontaneously hypertensive rat. Hypertension 45: 867–873, 2005[Abstract/Free Full Text]
Tanaka M, Schmidlin O, Yi SL, Bollen AW, Morris RC Jr: Genetically determined chloride-sensitive hypertension and stroke. Proc Natl Acad Sci U S A 94: 14748–14752, 1997[Abstract/Free Full Text]
Whitescarver SA, Ott CE, Jackson BA, Guthrie GP Jr, Kotchen TA: Salt-sensitive hypertension: Contribution of chloride. Science 223: 1430–1432, 1984[Abstract/Free Full Text]
Yamauchi K, Rai T, Kobayashi K, Sohara E, Suzuki T, Itoh T, Suda S, Hayama A, Sasaki S, Uchida S: Disease-causing mutant WNK4 increases paracellular chloride permeability and phosphorylates claudins. Proc Natl Acad Sci U S A 101: 4690–4694, 2004[Abstract/Free Full Text]
Kahle KT, Macgregor GG, Wilson FH, Van Hoek AN, Brown D, Ardito T, Kashgarian M, Giebisch G, Hebert SC, Boulpaep EL, Lifton RP: Paracellular Cl– permeability is regulated by WNK4 kinase: Insight into normal physiology and hypertension. Proc Natl Acad Sci U S A 101: 14877–14882, 2004[Abstract/Free Full Text]
Schambelan M, Sebastian A, Rector FC Jr: Mineralocorticoid-resistant renal hyperkalemia without salt wasting (type II pseudohypoaldosteronism): Role of increased renal chloride reabsorption. Kidney Int 19: 716–727, 1981[Medline]
Greger R: Chloride transport in thick ascending limb, distal convolution, and collecting duct. Annu Rev Physiol 50: 111–122, 1988[CrossRef][Medline]
Wall SM, Hassell KA, Royaux IE, Green ED, Chang JY, Shipley GL, Verlander JW: Localization of pendrin in mouse kidney. Am J Physiol Renal Physiol 284: F229–F241, 2003[Abstract/Free Full Text]
Kim YH, Kwon TH, Frische S, Kim J, Tisher CC, Madsen KM, Nielsen S: Immunocytochemical localization of pendrin in intercalated cell subtypes in rat and mouse kidney. Am J Physiol Renal Physiol 283: F744–F754, 2002[Abstract/Free Full Text]
Royaux IE, Wall SM, Karniski LP, Everett LA, Suzuki K, Knepper MA, Green ED: Pendrin, encoded by the Pendred syndrome gene, resides in the apical region of renal intercalated cells and mediates bicarbonate secretion. Proc Natl Acad Sci U S A 98: 4221–4226, 2001[Abstract/Free Full Text]
Wall SM, Kim YH, Stanley L, Glapion DM, Everett LA, Green ED, Verlander JW: NaCl restriction upregulates renal Slc26a4 through subcellular redistribution: Role in Cl– conservation. Hypertension 44: 982–987, 2004[Abstract/Free Full Text]
Verlander JW, Hassell KA, Royaux IE, Glapion DM, Wang ME, Everett LA, Green ED, Wall SM: Deoxycorticosterone upregulates PDS (Slc26a4) in mouse kidney: Role of pendrin in mineralocorticoid-induced hypertension. Hypertension 42: 356–362, 2003[Abstract/Free Full Text]
Quentin F, Chambrey R, Trinh-Trang-Tan MM, Fysekidis M, Cambillau M, Paillard M, Aronson PS, Eladari D: The Cl–/HCO3– exchanger pendrin in the rat kidney is regulated in response to chronic alterations in chloride balance. Am J Physiol Renal Physiol 287: F1179–F1188, 2004[Abstract/Free Full Text]
Pradervand S, Wang Q, Burnier M, Beermann F, Horisberger JD, Hummler E, Rossier BC: A mouse model for Liddle’s syndrome. J Am Soc Nephrol 10: 2527–2533, 1999[Abstract/Free Full Text]
Schultheis PJ, Lorenz JN, Meneton P, Nieman ML, Riddle TM, Flagella M, Duffy JJ, Doetschman T, Miller ML, Shull GE: Phenotype resembling Gitelman’s syndrome in mice lacking the apical Na+-Cl– cotransporter of the distal convoluted tubule. J Biol Chem 273: 29150–29155, 1998[Abstract/Free Full Text]
Menard J, Catt KJ: Measurement of renin activity, concentration and substrate in rat plasma by radioimmunoassay of angiotensin I. Endocrinology 90: 422–430, 1972[Medline]
Wagner CA, Finberg KE, Stehberger PA, Lifton RP, Giebisch GH, Aronson PS, Geibel JP: Regulation of the expression of the Cl–/anion exchanger pendrin in mouse kidney by acid-base status. Kidney Int 62: 2109–2117, 2002[CrossRef][Medline]
Knauf F, Yang CL, Thomson RB, Mentone SA, Giebisch G, Aronson PS: Identification of a chloride-formate exchanger expressed on the brush border membrane of renal proximal tubule cells. Proc Natl Acad Sci U S A 98: 9425–9430, 2001[Abstract/Free Full Text]
Masilamani S, Kim GH, Mitchell C, Wade JB, Knepper MA: Aldosterone-mediated regulation of ENaC alpha, beta, and gamma subunit proteins in rat kidney. J Clin Invest 104: R19–R23, 1999[Medline]
Quentin F, Eladari D, Frische S, Cambillau M, Nielsen S, Alper SL, Paillard M, Chambrey R: Regulation of the Cl–/HCO3– exchanger AE2 in rat thick ascending limb of Henle’s loop in response to changes in acid-base and sodium balance. J Am Soc Nephrol 15: 2988–2997, 2004[Abstract/Free Full Text]
Loffing J, Vallon V, Loffing-Cueni D, Aregger F, Richter K, Pietri L, Bloch-Faure M, Hoenderop JG, Shull GE, Meneton P, Kaissling B: Altered renal distal tubule structure and renal Na+ and Ca2+ handling in a mouse model for Gitelman’s syndrome. J Am Soc Nephrol 15: 2276–2288, 2004[Abstract/Free Full Text]
Masilamani S, Wang X, Kim GH, Brooks H, Nielsen J, Nielsen S, Nakamura K, Stokes JB, Knepper MA: Time course of renal Na-K-ATPase, NHE3, NKCC2, NCC, and ENaC abundance changes with dietary NaCl restriction. Am J Physiol Renal Physiol 283: F648–F657, 2002[Abstract/Free Full Text]
Pradervand S, Vandewalle A, Bens M, Gautschi I, Loffing J, Hummler E, Schild L, Rossier BC: Dysfunction of the epithelial sodium channel expressed in the kidney of a mouse model for Liddle syndrome. J Am Soc Nephrol 14: 2219–2228, 2003[Abstract/Free Full Text]
Brooks HL, Sorensen AM, Terris J, Schultheis PJ, Lorenz JN, Shull GE, Knepper MA: Profiling of renal tubule Na+ transporter abundances in NHE3 and NCC null mice using targeted proteomics. J Physiol 530: 359–366, 2001[Abstract/Free Full Text]
Schuster VL: Function and regulation of collecting duct intercalated cells. Annu Rev Physiol 55: 267–288, 1993[CrossRef][Medline]
Schuster VL, Stokes JB: Chloride transport by the cortical and outer medullary collecting duct. Am J Physiol 253: F203–F212, 1987
Piermarini PM, Verlander JW, Royaux IE, Evans DH: Pendrin immunoreactivity in the gill epithelium of a euryhaline elasmobranch. Am J Physiol Regul Integr Comp Physiol 283: R983–R992, 2002[Abstract/Free Full Text]
Evans DH, Piermarini PM, Choe KP: The multifunctional fish gill: Dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste. Physiol Rev 85: 97–177, 2005[Abstract/Free Full Text]
Stanton BA, Kaissling B: Regulation of renal ion transport and cell growth by sodium. Am J Physiol 257: F1–F10, 1989
Stanton B, Janzen A, Klein-Robbenhaar G, DeFronzo R, Giebisch G, Wade J: Ultrastructure of rat initial collecting tubule. Effect of adrenal corticosteroid treatment. J Clin Invest 75: 1327–1334, 1985[Medline]
Wade JB, Stanton BA, Field MJ, Kashgarian M, Giebisch G: Morphological and physiological responses to aldosterone: Time course and sodium dependence. Am J Physiol 259: F88–F94, 1990
Kaissling B, Stanton BA: Adaptation of distal tubule and collecting duct to increased sodium delivery. I. Ultrastructure. Am J Physiol 255: F1256–F1268, 1988
Loffing J, Le Hir M, Kaissling B: Modulation of salt transport rate affects DNA synthesis in vivo in rat renal tubules. Kidney Int 47: 1615–1623, 1995[Medline]
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Top
Abstract
Introduction
-ENaC was markedly upregulated, and the molecular weight of a large fraction of 
-ENaC subunits was shifted from 85 to 70 kD, consistent with previous results from rat models with elevated plasma aldosterone levels. These results suggest that factors that are associated with changes in distal chloride delivery govern pendrin expression in the connecting tubule and cortical collecting duct. 
ENaC gene locus as described previously (18). Seven homozygous transgenic mice (ENaCL/L) and seven wild-type littermate controls (ENaCWT) were used for these series. All of the animals were outbred on a mixed 129/Ola-C57BL/6 genetic background. Animals were 5 to 6 mo of age and weighed 28 to 30 g. 
