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Overexpression of in Intercalated Cells Produces Chloride-Sensitive Hypertension

† ‡ †| Thibaut Jacques,* Nicolas Picard,* R. Lance Miller,§ Kent A. Riemondy,§ Pascal Houillier,* † † ‡ ‡ Fabien Sohet,* Suresh K. Ramakrishnan,* Cara J. Büsst,* Maximilien Jayat,* †| †† ‡‡ Nicolas Cornière,* Hatim Hassan,** Peter S. Aronson, Jean Christopher Hennings, ‡‡ ‡ ‡| Christian A. Hübner, Raoul D. Nelson,§ Régine Chambrey,* and Dominique Eladari*

*Faculté de Médecine, Université Paris-Descartes, Paris, France; †Institut National de la Santé et de la Recherche Médicale Unité Mixte re Recherche 872, Centre de Recherche des Cordeliers, Paris, France; ‡Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche 970, Centre de Recherche Paris Cardiovasculaire, Paris, France; §Department of Pediatrics, Division of Nephrology, School of Medicine, University of Utah, Salt Lake City, Utah; |Hopital Européen Georges Pompidou, Département de Physiologie, Assistance Publique-Hopitaux de Paris, Paris, France; **Section of Nephrology, Department of Medicine, University of Chicago, Chicago, Illinois; ††Department of Internal Medicine, Section of Nephrology, Yale University School of Medicine, New Haven, Connecticut; and ‡‡Institute of Human Genetics, Jena University Hospital, Jena, Germany

ABSTRACT Inherited and acquired disorders that enhance the activity of transporters mediating renal tubular Na+ reabsorption are well established causes of hypertension. It is unclear, however, whether primary activa- tion of an Na+-independent chloride transporter in the kidney can also play a pathogenic role in this disease. Here, mice overexpressing the chloride transporter pendrin in intercalated cells of the distal nephron (TgB1-hPDS mice) displayed increased renal absorption of chloride. Compared with normal mice, these transgenic mice exhibited a delayed increase in urinary NaCl and ultimately, developed hyperten- sion when exposed to a high-salt diet. Administering the same sodium intake as NaHCO3 instead of NaCl did not significantly alter BP, indicating that the hypertension in the transgenic mice was chloride-sensitive. Moreover, excessive chloride absorption by pendrin drove parallel absorption of sodium through the epithelial sodium channel ENaC and the sodium-driven chloride/bicarbonate exchanger (Ndcbe), despite an appropriate downregulation of these sodium transporters in response to the expanded vascular volume and hypertension. In summary, chloride transport in the distal nephron can play a primary role in driving NaCl transport in this part of the kidney, and a primary abnormality in renal chloride transport can provoke arterial hypertension. Thus, we conclude that the chloride/bicarbonate exchanger pendrin plays a major role in controlling net NaCl absorption, thereby influencing BP under conditions of high salt intake.

J Am Soc Nephrol 24: 1104–1113, 2013. doi: 10.1681/ASN.2012080787

Hypertension is one of the most common human diseases in industrialized countries. Among the different environmental and genetic factors predis- Received August 9, 2012. Accepted March 6, 2013. posing individuals to hypertension, chronic expo- T.J. and N.P. contributed equally to this work. sure to high dietary Na+ intake has emerged as one Published online ahead of print. Publication date available at of the most important risk factors. However, the www.jasn.org. molecular mechanisms underlying salt-sensitive hypertension are still poorly understood in the ma- Correspondence: Prof. Dominique Eladari, Institut National de la Santé et de la Recherche Médicale (INSERM) Unité Mixte de jority of patients. Although it is widely assumed Recherche (UMR) 970, Centre de Recherche Paris Cardiovascu- that all pressor effects of dietary NaCl depend on laire (PARCC), 56 Rue Leblanc, F-75015, Paris, France. Email: its Na+ content, several studies have drawn atten- [email protected] tion to the fact that the sodium has to be in the form Copyright © 2013 by the American Society of Nephrology

1104 ISSN : 1046-6673/2407-1104 JAmSocNephrol24: 1104–1113, 2013 www.jasn.org BASIC RESEARCH of NaCl to produce an increase in BP. In 1929, Berghoff and collecting duct (CCD; detailed methods of the transgene con- Geraci1 reported that BP increased in seven individuals on a struction and genotyping can be found in Supplemental Ma- high NaCl intake but not a high NaHCO3 intake, an observa- terial and Supplemental Table 1). The transgene includes the tion subsequently confirmed by Morgan.2 In humans or ani- 59-flanking region of the ATP6V1B1 gene extending to but mals with salt-sensitive hypertension, selective dietary loading excluding the endogenous translational start codon, the hu- 2 of Na+ without Cl has repeatedly failed to induce an increase man SLC26A4 cDNA with its own translational start site, and in BP.3–7 Likewise, in hypertensive and normotensive subjects, the SV40 late region polyadenylation signal, and it is referred substitution of dietary NaCl with equimolar NaHCO3 leads to as B1-hPDS (Figure 1A). The rationale for using the human to a reduction of BP, further suggesting a modulating effect of instead of the mouse cDNA is to be able to distinguish the dietary chloride on BP.8,9 Surprisingly, although it is widely expression of the transgene from the expression of the en- accepted that excessive sodium reabsorption can lead to hy- dogenous pendrin gene. Approximately 110 live pups were pertension, the potential role of primary activation of renal analyzed for the presence of the B1-hPDS transgene by per- chloride transport in the pathogenesis of hypertension is forming PCR analysis of tail DNA (Figure 1B), and 63 pups poorly understood. were found to be positive for transgene integration. However, 2 It was recently shown that Cl absorption in the collecting only one founder transmitted the transgene in a Mendelian duct occurs through intercalated cells and requires the pres- fashion and therefore, was used to generate animals for this 2 2 10,11 ence of the Cl /HCO3 exchanger pendrin (Pds/Slc26a4). study. The mice that were used in the present study were F6. We subsequently showed that the functional coupling of Mendelian transmission of the transgene and Southern blot + 2 2 pendrin together with the Na -dependent Cl /HCO3 exchanger analyses (Supplemental Figure 2) were compatible with a sin- (Ndcbe/Slc4a8) results in electroneutral, thiazide-sensitive gle site of integration. Figure 1C shows that high levels of the NaCl absorption, and hence, mimicks the NaCl human pendrin transcript were detected by RT-PCR in the (NCC) of the distal convoluted tubule.12 Although the impor- renal cortex and medulla of transgenic B1-hPDS (TgB1-hPDS) tance of this system in vascular volume regulation is suspected mice, whereas it was not present in the wild-type littermates. based on the observation that pendrin disruption impairs nor- We previously reported that, beyond expression in renal in- mal renal NaCl balance11 or protects against mineralocorti- tercalated cells, our ATP6V1B1 promoter fragment was able to coid-induced hypertension,13 its potential pathogenic role drive expression of EGFP in epididymis, uterus, small and as a primary determinant of hypertension is unknown. large intestines, and nonciliated airway epithelial cells15 and Thus, to investigate the potential primary role of enhanced Cre-recombinase in the brain.14 Thus, we tested expression of pendrin activity in the pathogenesis of salt-sensitive hyperten- the transgene in these different tissues (Figure 1D). No hPDS sion, we have created a mouse model overexpressing pendrin transcript was detected in tissues isolated from wild-type in the intercalated cells of the aldosterone-sensitive distal mice, whereas in tissues isolated from TgB1-hPDS mice, hPDS nephron. We show that pendrin overexpression results in in- transcript was detected at high levels in uterus and epididymis. creased chloride absorption that confers on mice markedly hPDS transcript was also detectable in lungs and brain, but no salt-sensitive elevation of BP.Interestingly, the pressor effect of significant level of hPDS transcript was detected in small or high salt intake was strictly chloride-dependent and occurred large intestine or liver. despite appropriate downregulation of the sodium transporters We next examined the expression of the hPDS protein by in the aldosterone-sensitive distal nephron. We conclude from immunohistochemistry. However, all the different antipendrin these experiments that a primary abnormality of renal chloride antibodies available to us recognize only the rodent orthologs reabsorption can lead to NaCl-sensitive hypertension. of pendrin. Thus, a new rabbit polyclonal antibody was raised against the synthetic peptide APGGRSEPPQLPEYS (corre- sponding to amino acids 3–18 of the N-terminal end of human RESULTS pendrin protein) (Supplemental Material). Preliminary ex- periments showed that, although this antibody was recogniz- Generation of a Mouse Model Overexpressing Pendrin ing human pendrin, it also crossreacted with the murine in the Intercalated Cells ortholog (Supplemental Figure 3). We used this antibody to A 6.9-kb fragment corresponding to the human V-ATPase B1- localize both endogenous (murine) and transgenic (human) subunit gene (ATP6V1B1 gene; GenBank accession no. pendrin on kidney sections from TgB1-hPDS and control mice NM_039350) has been successfully used to drive expression (Figure 1, E and F). As previously shown by others,16 pendrin of both enhanced green fluorescent protein (EGFP) and the expression in wild-type mice was detected exclusively in b-or Cre-recombinase (Cre) into renal intercalated cells.14,15 Im- non-a,non-b intercalated cells, which were identified by the portantly, preliminary experiments revealed that the promoter coexpression of the V H+-ATPase but not the anion exchanger is likely to be insensitive to a high-salt diet (Supplemental 1 (AE1). By contrast, pendrin staining was also detected in Figure 1). We used this promoter fragment to drive the a-intercalated cells (V H+-ATPase and AE1-positive) of the expression of human pendrin (hPDS/SLC26A4) cDNA in in- renal cortex and medulla in TgB1-hPDS mice. No staining was tercalated cells of the connecting tubule and the cortical detected in principle cells of the collecting duct or the

J Am Soc Nephrol 24: 1104–1113, 2013 A Mouse Model of Chloride-Sensitive Hypertension 1105 BASIC RESEARCH www.jasn.org

Figure 1. Generation and characterization of transgenic mouse model overexpressing human pendrin in the renal intercalated cells. (A) Schematic of the transgene (B1-hPDS) containing ;6.9 kb from the 59-flanking region of the ATP6V1B1 gene extending to but ex- cluding the endogenous translational start codon, the human SLC26A4 cDNA (with its own translational start site; indicated by arrow), and the SV40 late region polyadenylation signal. Positions of the primers used for genotyping or RNA expression (quantitative PCR) are shown. (B) Normal mouse DNA spiked with 0, 1, 10, 102,103,104,105,and106 copies per cell equivalent of the purified transgene DNA (left) was amplified by PCR using primers specific for the hB1-EGFP transgene. PCR genotyping (right) of tail DNA from different pups (TgB1-hPDS-1 to -4); in this example, mice 1 and 4 are hemizygous for the B1-hPDS transgene, whereas mice 2 and 3 do not carry the B1-hPDS transgene, (i.e., wild-type littermates). The intensity of the PCR product from mice 1 and 4 compared with normal DNA spiked with variable amounts of the purified transgene indicates that the transgenics have an almost 1000 copies per cell equivalent of the transgene. (C) Quantitative RT-PCR analyses of the human pendrin transcript in the renal cortex and medulla from TgB1-hPDS and

1106 Journal of the American Society of Nephrology J Am Soc Nephrol 24: 1104–1113, 2013 www.jasn.org BASIC RESEARCH connecting tubule. Ectopic expression of hPDS in a-intercalated tubule, which is not accessible to the microperfusion. Thus, cells was expected, because our B1 promoter fragment was to assess the effects of the transgene on ENaC activity, we next shown to drive expression of EGFP and Cre in both intercalated tested whether ENaC-dependent Na+ absorption and K+ se- cell subtypes.14,15 Noteworthy, the expression of the transgene cretion are inhibited in TgB1-hPDS mice by monitoring the had no particular impact on AE1 or the V H+-ATPase cellular or effects of an acute injection of amiloride (1.45 mg/kg body subcellular localization. wt) on urinary excretion of Na+ and K+. As shown in Figure 2, In our first B1-EGFP transgenic mouse model, we noted E and F, amiloride injection increased urine Na+ excretion and that EGFP expression was slightly stronger in the medulla than decreased K+ excretion similarly in the TgB1-hPDS mice and in the cortex.15 In the present study, the transgene expression wild-type littermates. Taken together, these results indicate also looked stronger in the renal medulla than in the cortex that, although the B1-hPDS transgene promotes electroneu- (Figure 1, E and F). However, immunohistochemistry is not a tral NaCl absorption, it does not inhibit ENaC. good technique for quantitation, and we were not able to check the level of expression of the trangene by Western Increased Electroneutral NaCl Absorption in the Distal blot, because our antibody was not suitable for this technique. Nephron Does Not Lead to High Serum K+ Thus, we next checked pendrin activity in the renal cortex by Concentration or Metabolic Acidosis but Provokes measuring intracellular pHi changes consecutive to manipu- Chloride-Sensitive Hypertension 2 lation of luminal Cl concentration in intercalated cells in Phenotypical analyses of TgB1-hPDS mice and wild-type litter- cortical collecting ducts isolated from TgB1-hPDS and control mates are summarized in Supplemental Table 2. Increased 2 2 mice. Basolateral Cl /HCO3 activity was not assessed in electrogenic chloride absorption in the CCD, also known 2 2 these experiments. Figure 1G shows that apical Cl /HCO3 as a chloride shunt, has been proposed to decrease the trans- exchange activity was much higher in intercalated cells from epithelial voltage; thereby, it impairs tubular K+ secretion and TgB1-hPDS transgenic mice than wild-type littermates, showing provokes metabolic acidosis and hyperkalemia.17,18 However, that our transgenic strategy for overexpressing pendrin is ef- TgB1-hPDS mice exhibit net NaCl absorption in the CCD, but in fective in cortical intercalated cells. the absence of any Vte, they did not exhibit high serum K+ concentration. Another question was whether overexpression Pendrin Overexpression Stimulates Chloride of pendrin can favor the development of acidosis. In fact, Absorption by the Renal Tubule we initially anticipated that ectopic expression of hPDS in To determine whether the B1-hPDS transgene expression a-intercalated cells would short circuit acid secretion and promotes excessive chloride absorption, cortical collecting thereby, lead to distal tubular acidosis. Thus, to determine ducts were isolated from either TgB1-hPDS transgenics or wild- whether TgB1-hPDS mice display hyperchloremic metabolic ac- type littermates. All animals were fed a normal salt diet (i.e., idosis, we performed blood gas analyses in TgB1-hPDS mice and 0.3% Na+ administered as NaCl). Transepithelial fluxes of Na+ wild-type littermates. These analyses showed that both TgB1-hPDS 2 + (JNa), Cl (JCl), and K (JK) and transepithelial voltage (Vte) mice and wild-type littermates fed a normal salt diet had normal were measured as described previously.12 Figure 2, A and B blood acid–base parameters and did not display an alkaline urine shows that, although no transport activity was detectable in pH, indicating the absence of significant bicarbonaturia. CCDs isolated from control mice, tubules from TgB1-hPDS Because the effects of the transgene might have been, at mice exhibited net NaCl absorption. The magnitudes of Na+ least partly, masked by some compensatory mechanisms, the 2 and Cl fluxes were very similar to those magnitudes observed same analyses were again performed after feeding the mice in CCDs isolated from mice maintained on a low-salt diet for 2 weeks with a high-salt (3% Na+ administrated as NaCl) (figure 1 in ref. 12). CCDs from TgB1-hPDS mice did not diet. This maneuver is expected to increase urinary chloride + 2 2 develop a net negative Vte and did not secrete K (Figure 2, delivery to the CCD and hence, accelerate Cl /HCO3 ex- C and D). The latter findings suggested that epithelial Na+ change through the transgene. However, as shown in Supple- channel (ENaC) activity is impaired as a consequence of ex- mental Table 2, serum K+ concentration and acid–base status cessive electroneutral NaCl absorption. However, under nor- of TgB1-hPDS mice remained normal and identical to those mal conditions, ENaC is mostly active in the connecting levels observed in wild-type littermates. wild-type (WT) mice. (D) Quantitative RT-PCR analyses of the human pendrin transcript in different tissue. (E) Colocalization studies of pendrin (red) and AE1 (blue) on mouse kidney sections reveal the presence of transgenic hPDS in a-intercalated cells (arrows) in TgB1-hPDS but not WT mice. (F) Colocalization studies of pendrin (red), AE1 (blue), and the H+-ATPase reveal the absence of pendrin staining in principle cells and a normal pattern of expression of AE1 and the . CCD, cortical collecting duct; OMCD, outer medullary 2 2 B1-hPDS collecting duct. (G) Apical Cl /HCO3 exchange activity in intercalated cells of CCD isolated from either Tg or WT mice. Traces are 2 2 + the average of pHi changes recorded when luminal Cl is removed and then readded in the presence of extracellular HCO3 (25 mM) and Na - 2 2 2 free solutions. The initial rate of intracellular Cl -dependent alcalinization (right), reflecting apical Cl /HCO3 exchange, is much higher in TgB1-hPDS than WT mice. Statistical significance is assessed by two-tailed unpaired t test; n=3tubulesineachgroup.***P,0.001 versus control group.

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2 diet (3% Na+), urinary excretion of both Na+ and Cl dra- matically increased in both groups. However, these adaptive changes were significantly delayed in TgB1-hPDS mice com- pared with wild-type littermates. Urinary aldosterone was not different between genotypes in animals fed a normal salt diet, and they decreased similarly to nearly undetectable levels when animals were fed a high-salt diet (Figure 3C). The high- salt diet led to a significant rise in systolic BP in TgB1-hPDS mice, whereas the wild-type littermates experienced no change in BP (Figure 3D). Almost the same values of BP were obtained when recorded by radiotelemetry (Supplemen- tal Figure 4); accordingly, all subsequent BP measurements were performed using the tail-cuff method. Monitoring of heart rate detected a significant decrease in heart rate in both genotypes fed a high-salt diet but did not reveal any difference between genotype (Supplemental Figure 4). We recently described that pendrin works in tandem with Ndcbe to mediate electroneutral and thiazide-sensitive NaCl absorption.12 Pech et al.19 reported that pendrin is required for normal ENaC activity.19 Thus, to determine whether pen- drin overexpression secondarily drives Na+ absorption by Ndcbe, ENaC, or both molecules, we tested the effects of acute injections of either amiloride (1.45 mg/kg) or hydrochlorothi- azide (50 mg/kg). These experiments were performed in mice fed a high-salt diet, because the hypertensive phenotype observed in the TgB1-hPDS mice was only apparent in this situation. Figure 4 shows that the response to amiloride was increased in TgB1-hPDS mice compared with wild-type litter- mates, indicating that ENaC activity is increased in this model; Figure 2. Characterization of ion transport activities in the aldosterone- the response to hydrochlorothiazide (HCTZ) was also greater B1-hPDS sensitive distal nephron in TgB1-hPDS and WT mice. Analyses of Na+, in Tg mice. The NCC and pendrin/Ndcbe transporters 2 Cl ,andK+ transepithelial fluxes and transepithelial voltage in are targeted by HCTZ in vivo.12 However, it is unlikely that CCDs isolated from TgB1-hPDS and WT mice fed a normal salt diet. higher sensitivity to HCTZ in the TgB1-hPDS mice reflects in- + 2 (A) JNa is the rate of Na absorption. (B) JCl is the rate of Cl creased activity of NCC, because we directly observed the pres- absorption. (C) Vte is the transepithelial voltage. (D) JK is the rate ence of electroneutral NaCl absorption in the collecting duct + fi of K secretion. Statistical signi cance is assessed by two-tailed (Figure 2), a nephron segment devoid of NCC. Moreover, in , unpaired t test; n=7 in each group. *P 0.05 versus control group. our model, expression of human pendrin is driven by the B1 Effect of amiloride injection on (E) urinary Na+ excretion and (F) + B1-hPDS promoter; it is not expressed in the distal convoluted tubule urinary K excretion in Tg and WT mice. Mice kept under normal Na+ diet (0.3% Na+ in food) were subcutaneously injected but exclusively expressed in the connecting tubule and collect- with amiloride (1.45 mg/kg body wt). Urines from TgB1-hPDS and ing duct, which is evident from two previous studies using the WT mice were collected over 2 days starting at 9 AM and ending exact same promoter to drive expression of either EGFP or 2 at 3 PM the next day. Vehicle was injected on day 1, whereas Cre.14,15 Thus, these results indicate that Cl absorption in the amiloride was injected at 9 AM on the second day (black bars). distal nephron is paralleled by Na+ absorption occurring by Excreted ion concentrations (millimolar) were normalized to the either ENaC or Ndcbe. urinary concentration of creatinine (millimolar) to minimize the Pech et al.19 have recently reported that extracellular bicar- effects of incomplete urine sampling over such short periods. bonate stimulates ENaC activity by increasing b-andg-ENaC 6 Values are the mean SEM from seven determinations in each protein abundance and more importantly, promoting fi group. Statistical signi cance was tested versus time controls by g-ENaC proteolytic cleavage, a process associated with an in- paired t test. *P,0.05, **P,0.01. crease in the channel activity.20 Conversely, pendrin knockout mice exhibit decreased ENaC activity associated with de- Wenext testedthe effectsof pendrin overexpression onrenal creased b-andg-ENaC activity and decreased g-ENaC pro- adaptation to a high-salt diet. Figure 3, A and B shows that teolytic activation. In the experiments shown in Figure 4, we animals from both genotypes exhibited the same systolic BP detected increased ENaC activity in TgB1-hPDS mice fed a high- 2 and urinary excretion of Na+ and Cl when fed a normal salt salt diet; therefore, we next assessed the profile of expression of diet (0.3% Na+). When animals were switched to a high-salt different sodium transporters, including ENaC, along the

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protein expression. Possible mechanisms include changes in transporter surface ex- pression, phosphorylation, activation state, and/or driving force. Tofurthershow thecriticalroleofchloride inthepathogenesisofsalt-sensitivehyperten- sion in this model, we examined the effects of chloride on BP by administering NaHCO3 instead of NaCl as previously described.4,7 Contrasting with the marked pressor effects of high NaCl intake observed in the preced- ing experiments, high NaHCO3 intake did not alter systolic BP in the TgB1-hPDS mice or wild-type littermates, which confirms that the abnormal renal handling of chloride is the primary cause of salt-sensitive hyper- tension in TgB1-hPDS mice (Figure 6).

DISCUSSION

In most human populations, sodium is almost invariably ingested as a chloride salt, and sodium ion has no pressor effects when chloride is substituted with another anion like sulfate or bicarbonate.3,8,9 The lat- Figure 3. Effects of dietary NaCl loading in TgB1-hPDS mice. Time course of renal 2 ter observation has led several groups to con- excretion of (A) Na+ and (B) Cl in TgB1-hPDS and WT mice fed a normal salt diet and then switched to a high NaCl diet. (C) Urine aldosterone concentration was measured clude that salt-sensitive hypertension is 3,4,7 under a normal salt diet or after 2 or 6 days of high NaCl diet. Data are presented as chloride-dependent. However, renal the mean 6 SEM; n=7 for each genotype. Statistical significance versus WT was as- transporters that have been shown to sessed by unpaired t test. *P,0.05, **P,0.01. (D) Systolic BP measured by tail-cuff have a clear impact on BP, like the Na/K/ under normal salt diet (0.3% Na+/0.8% NaCl) or after 2 weeks of high-salt (3% Na+/8% 2Cl cotransporter type 2 or NCC,21 trans- NaCl) diet. Data are presented as the mean 6 SEM; n=10 for each genotype. The port equimolar amounts of sodium and difference in systolic BP was assessed by ANOVA followed by Bonferroni’sposthoc chloride. In the collecting duct, it was test. **P,0.01 versus normal salt diet. thought until recently that chloride is mostly absorbed passively because of the lumen- negative transepithelial potential difference nephron. Unexpectedly, expression of NCC, p(T55)NCC, that results from ENaC-dependent electrogenic absorption of ENaC (both a-andg-subunits), and Ndcbe assessed by im- Na+.22,23 As a consequence, most efforts have been made to munoblotting of membrane fractions isolated from the renal dissect the mechanisms accounting for renal sodium absorption cortex was decreased in TgB1-hPDS mice compared with wild- and its regulation, whereas the pathogenic effects of abnormal type littermates (Figure 5 and Supplemental Table 3). This renal chloride transport have not been studied as extensively. downregulation of sodium transporters in the aldosterone- Here, we show that overexpression of pendrin leads to sensitive distal nephron in TgB1-hPDS mice is expected in chloride-dependent hypertension. Because the trangene hPDS response to the hypoaldosteronism and the salt-sensitive hy- was detectable by PCR in the brain, we cannot rule out the pertension observed under these conditions. The proteolytic possibility that overexpression of pendrin in the central product of g-ENaC subunit was also undetectable, which was nervous system did also alter central regulation of BP.However, expected in salt-loaded animals. Taken together, these results the expression of hPDS in the brain was marginal compared indicate that excessive chloride absorption by the B1-hPDS with the kidney. We also did not detect differences in heart rate transgene is able to accelerate Na+ absorption through ENaC between TgB1-hPDS and control mice that might have revealed and Ndcbe, although their expression levels as assayed by im- some dysregulation of the autonomous nervous system. Im- munoblotting were downregulated. This result suggests that portantly, we show that renal overexpression of pendrin drives primary activation of chloride reabsorption by pendrin stim- excessive renal chloride reclamation, which in turn, is respon- ulates Na reabsorption through these transporters sufficiently sible for chloride-sensitive hypertension. Moreover, we show to overcome the observed downregulation at the level of that, despite normal downregulation of all the different

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Figure 4. Effects of amiloride and hydrochlorothiazide injections in TgB1-hPDS and WT mice fed a high-salt diet. Urinary (A) Na+ and (B) K+ excretion after amiloride injection in TgB1-hPDS and WT mice. Urinary (C) Na+ and (D) K+ excretion after HCTZ injection in TgB1-hPDS and WT mice. Mice on a high-salt diet (3% Na+/8% NaCl in food) for 2 weeks were subcutaneously injected with amiloride (1.45 mg/kg body wt), HCTZ (50 mg/kg body wt), or B1-hPDS vehicle. Urines from Tg and WT mice were collected over 2 Figure 5. Protein abundance of critical sodium transporters along fi days from 9 AM to 3 PM; the rst-day injections were in vehicle the nephron in TgB1-hPDS and WT mice fed a high-salt diet. (A) only (white bars), whereas the diuretics were injected at 9 AM on Immunoblots of membrane fractions from the renal cortex ex- + the following day (black bars). The concentration of excreted Na tracted from seven TgB1-hPDS and seven WT mice fed a high-salt + and K (millimolar) were normalized to the urinary concentration diet for 2 weeks. Each lane was loaded with 10–15 mg protein. of creatinine (millimolar) to minimize the effects of incomplete Immunoblots were probed with the different primary antibodies 6 urine sampling over such short periods. Values are the mean as indicated. (B) Densitometric analyses of data were expressed SEM from seven determinations in each group. Statistical signif- as percentages of control (i.e., percent of control). Mean control , icance was tested versus time controls by paired t test. *P 0.05, values and SEMs for each respective immunoblot are provided in , **P 0.01. Supplemental Table 3. Bars represent mean 6 SEM. Statistical significance is assessed by two-tailed unpaired t test; n=7 in each group. *P,0.05, **P,0.01 versus controls. sodium transporters in the aldosterone-sensitive distal neph- ron, excessive chloride absorption by transgenic pendrin is still able to drive parallel sodium absorption. This important changes in chloride transport can drive NaCl absorption also finding challenges the classic paradigm assuming that sodium has important therapeutic implications, because our results sug- transport is always primary and secondarily drives chloride as gest that pendrin might be a very interesting target to modulate the accompanying anion. Rather, these studies support the NaCl absorption by the distal nephron; pendrin blockers could, possibility that a primary increase in chloride transport can therefore, represent a new class of antihypertensive compounds. serve as the primary event driving NaCl absorption in the This possibility is further supported by a recent study showing distal nephron. It was previously shown that mice with pen- that double deletion of NCC and pendrin leads to massive so- drin disruption do not increase vascular volume and do not dium wasting, hypovolemia, and hypotension.24 raise BP in response to administration of deoxycorticosterone From the study of two independent mouse models of pivalate,13 a mineralocorticoid that is expected to maximally pseudohypoaldosteronism type II,25,26 it has been concluded stimulate sodium absorption by ENaC. We now show that a that increased electroneutral NaCl absorption by NCC is the primary increase in pendrin activity can cause hypertension, main mechanism leading to this syndrome. Functional cou- even when aldosterone is decreased. The fact that primary pling of pendrin and Ndcbe mediates NCC-like activity.12

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and Use of Laboratory Animals (National Institutes of Health publication No.93–23, revised 1985).

Metabolic Studies All experimentswere performedusingage- andsex-matchedTg(B1-hPDS) mice and wild-type littermate (3–5 months). For urine collection, mice were housed in metabolic cages (Techniplast, France). Mice were given deionized water ad libitum and pair-fed with standard laboratory chow containing 0.3% sodium (INRA, France). They were allowed to adapt for 3–5 days in the cages. At steady state, urine collection was performed daily under mineral oil in the urine collector for electrolyte measurements. Mice were then switched to a high-salt diet (3% Na+),

either as NaCl or NaHCO3 (INRA, France). After the switch, urine was collected every 24 hours. All biochemical and hormonal analyses were performed using standard methods as detailed in Supplemental Material.

B1-hPDS Figure 6. Effect of NaHCO3 loading on systolic BP in Tg BP Measurements in Conscious Mice and WT mice. Systolic BP measured by tail-cuff on normal salt + Mice were fed normal or high-salt diet for at least 2 weeks. Systolic BP diet (0.3% Na+) or after 2 weeks of high-salt (3% Na adminis- was measured using a computerized tail-cuff system after 2 weeks of trated as NaHCO ) diet. Data are presented as the mean 6 SEM; 3 daily training, which has been described elsewhere.29 Then, at least 10 n=8 for each genotype. The difference in systolic BP was tested measurements were performed every day for at least 7 consecutive by ANOVA. days. Only the last 4 days were kept for analyses. If the variability of the measurements made in a single day exceeded the SD by more than Moreover, this transport system is expressed in the nephron 20%, the day was discarded and replaced by an additional day of segments where ENaC-dependent K+ and H+ secretion occurs. measurement. Moreover, to ensure that it was the case in our study, Thus, we hypothesized that upregulation of this transport sys- BPs were also measured by radiotelemetry in mice fed either a normal tem might decrease ENaC activity and hence, might impair K+ or high-salt diet in a pilot experiment (Supplemental Material). Sim- and H+ secretion even more than NCC upregulation. However, ilar values were obtained using both techniques (compare Figure 4 our present results clearly indicate that this hypothesis is not with Supplemental Figure 3). the case. The absence of effects on and acid–base homeostasis cannot be attributed to a failure of our transgenic Immunofluorescence Studies and Immunoblot strategy, because we clearly show that the B1-hPDS transgene Analyses 2 2 was able to increase apical Cl /HCO3 exchange activity in the Kidney sections were triple labeled with a rabbit polyclonal human cortical collecting duct. Moreover, we were able to show that pendrin antibody (diluted 1:200), a guinea pig anti-AE130 (diluted the transgene accounts for excessive renal NaCl absorption, 1:5000), and a chicken anti-Atp6v1e1,31 which detects the V H+-ATPase and our TgB1-hPDS mice displayed salt-sensitive hypertension. (diluted 1:500), using the standard technique as previously described in Nevertheless, our results are in line with two recent studies that detail.32 For Western blot analyses, 10–60 mg membrane-enriched pro- reported that increased NCC activity is not sufficient to cause tein fraction were separated on reducing 7.5% SDS polyacrylamide gels. Gordon’s syndrome.27,28 Protein loading was assessed on gels ran in parallel and stained with In summary, our study solidifies the concept of chloride- Coomassie blue.33 Blots were probed with anti a-ENaC (dilution sensitivehypertension byshowingthat a primaryenhancement 1:10,000), anti g-ENaC (1:30,000),34 anti-Ndcbe (1:250),12 anti- of renal chloride transport by overexpression of pendrin can NHE3 the Na/H exchanger type 3 (1:1000),35 anti-NKCC2 the Na/K/ lead to salt-sensitive hypertension. Thus, we conclude that the 2Cl cotransporter type 2 (1:5000),35 anti-NCC (1:50,000),36 and anti- chloride/bicarbonate exchanger pendrin has a major role in NCC phospho-Thr55 antibody (residues 41–60 of human NCC phos- controlling net NaCl absorption and hence, BP in the setting of phorylated at Thr55, HPSHLTHSSTFCMRpTFGYNT, S908B; 1:275).37 high salt intake. Proteins were detected by chemiluminescence (ECL Kit; Amersham Biosciences). Antibodies against a-andg-subunits of ENaC were gen- erously donated by J. Loffing (University of Zurich, Zurich, Switzer- CONCISE METHODS land). Antibody against a-ENaC was raised against the N terminus of mouse a-ENaC (MLDHTRAPELNLDLDLDVSNC). Animals All the experimental procedures have been reviewed and approved by RT-PCR the local ethics committee from the University Pierre et Marie Curie, Total RNA was extracted using the RNeasy Kit (Qiagen). Reverse and they were performed in accordance with the Guide for the Care transcriptionwasperformed byusing afirst-strand cDNA synthesis kit

J Am Soc Nephrol 24: 1104–1113, 2013 A Mouse Model of Chloride-Sensitive Hypertension 1111 BASIC RESEARCH www.jasn.org for RT-PCR (Roche Diagnostics). Real-time PCR was performed on a 7. Whitescarver SA, Ott CE, Jackson BA, Guthrie GP Jr, Kotchen TA: Salt- LightCycler (Roche Diagnostics) with a LightCycler 480 SYBR Green I sensitive hypertension: Contribution of chloride. Science 223: 1430– Master qPCR Kit (Roche Diagnostics). No amplification was observed 1432, 1984 fi 8. Schorr U, Distler A, Sharma AM: Effect of sodium chloride- and sodium in the absence of reverse transcription, which con rmed that samples bicarbonate-rich mineral water on blood pressure and metabolic pa- were free of genomic DNA. Primer sequences and a methodology for rameters in elderly normotensive individuals: A randomized double- the PCR amplification are provided in detail in Supplemental Mate- blind crossover trial. J Hypertens 14: 131–135, 1996 rial. Results are expressed as the mean 6 SEM from six mice. 9. Luft FC, Zemel MB, Sowers JA, Fineberg NS, Weinberger MH: Sodium Transgene expression in different organs was assessed in one male bicarbonate and sodium chloride: Effects on blood pressure and electrolyte homeostasis in normal and hypertensive man. J Hypertens mouse and one female mouse as described in Figure 1D. 8: 663–670, 1990 10. Pech V, Kim YH, Weinstein AM, Everett LA, Pham TD, Wall SM: An- In Vitro Microperfusion of Isolated Collecting Ducts giotensin II increases chloride absorption in the cortical collecting duct CCDs were isolated and microperfused in vitro as described by Burg in mice through a pendrin-dependent mechanism. Am J Physiol Renal et al.38 Intracellular pH was monitored by using the pH-sensitive dye Physiol 292: F914–F920, 2007 BCECF.12 [Na+], [K+], and [creatinine] measurements were per- 11. Wall SM, Kim YH, Stanley L, Glapion DM, Everett LA, Green ED, Verlander JW: NaCl restriction upregulates renal Slc26a4 through 12 formed by HPLC ; transepithelial voltage (Vte) was measured con- subcellular redistribution: Role in Cl- conservation. Hypertension 44: – tinuously between Ag-AgCl electrodes connected to 0.15 M NaCl agar 982–987, 2004 bridges inserted in the perfusion pipette and bathing solutions as de- 12. Leviel F, Hübner CA, Houillier P, Morla L, El Moghrabi S, Brideau G, 12 + + scribed previously to calculate the rate of K (JK) and Na (JNa) Hassan H, Parker MD, Kurth I, Kougioumtzes A, Sinning A, Pech V, transepithelial transport, which is described in detail in Supplemental Riemondy KA, Miller RL, Hummler E, Shull GE, Aronson PS, Doucet A, Wall SM, Chambrey R, Eladari D: The Na+-dependent chloride- Material. bicarbonate exchanger SLC4A8 mediates an electroneutral Na+ re- absorption process in the renal cortical collecting ducts of mice. J Clin Invest 120: 1627–1635, 2010 13. Verlander JW, Hassell KA, Royaux IE, Glapion DM, Wang ME, Everett ACKNOWLEDGMENTS LA, Green ED, Wall SM: Deoxycorticosterone upregulates PDS (Slc26a4) in mouse kidney: Role of pendrin in mineralocorticoid-induced R.C. and D.E. are funded by Institut National de la Santé et de la hypertension. Hypertension 42: 356–362, 2003 Recherche Médicale, Centre National de la Recherche Scientifique, 14. Miller RL, Lucero OM, Riemondy KA, Baumgartner BK, Brown D, Breton S, Nelson RD: The V-ATPase B1-subunit promoter drives expression of the Transatlantic Network for Hypertension from the Fondation Cre recombinase in intercalated cells of the kidney. Kidney Int 75: 435– Leducq, Grant subvention de recherche 2010 AMGEN from the So- 439, 2009 ciété de Néphrologie (to R.C.), Grant subvention de recherche 2010 15. Miller RL, Zhang P, Smith M, Beaulieu V, Paunescu TG, Brown D, Breton from the association pour l’information et la recherche sur les mal- S, Nelson RD: V-ATPase B1-subunit promoter drives expression of adies rénales génétiques AIRG (to R.C.), and Grant HYPERCLO EGFP in intercalated cells of kidney, clear cells of epididymis and airway – programm BLANC 2010-R10164DD from l’Agence Nationale de la cells of lung in transgenic mice. Am J Physiol Cell Physiol 288: C1134 C1144, 2005 Recherche (to D.E.). 16. Kim YH, Kwon TH, Frische S, Kim J, Tisher CC, Madsen KM, Nielsen S: Immunocytochemical localization of pendrin in intercalated cell sub- types in rat and mouse kidney. Am J Physiol Renal Physiol 283: F744– DISCLOSURES F754, 2002 None. 17. Schambelan M, Sebastian A, Rector FC Jr: Mineralocorticoid-resistant renal hyperkalemia without salt wasting (type II pseudohypoaldoster- onism): Role of increased renal chloride reabsorption. Kidney Int 19: 716–727, 1981 REFERENCES 18. Take C, Ikeda K, Kurasawa T, Kurokawa K: Increased chloride re- absorption as an inherited renal tubular defect in familial type II pseu- 1. Berghoff RSGeraci AS: The influence of sodium chloride on blood dohypoaldosteronism. NEnglJMed324: 472–476, 1991 pressure. Intern Med J 56: 395–397, 1929 19. Pech V, Pham TD, Hong S, Weinstein AM, Spencer KB, Duke BJ, Walp 2. Morgan TO: The effect of potassium and bicarbonate ions on the rise in E, Kim YH, Sutliff RL, Bao HF, Eaton DC, Wall SM: Pendrin modulates blood pressure caused by sodium chloride. Clin Sci 63: 407s–409s, ENaC function by changing luminal HCO3-. J Am Soc Nephrol 21: 1982 1928–1941, 2010 3. Kurtz TW, Al-Bander HA, Morris RC Jr: “Salt-sensitive” essential hy- 20. Carattino MD, Hughey RP, Kleyman TR: Proteolytic processing of the pertension in men. Is the sodium ion alone important? NEnglJMed epithelial sodium channel gamma subunit has a dominant role in 317: 1043–1048, 1987 channel activation. 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24. Soleimani M, Barone S, Xu J, Shull GE, Siddiqui F, Zahedi K, Amlal H: 32. Eladari D, Cheval L, Quentin F, Bertrand O, Mouro I, Cherif-Zahar B, Double knockout of pendrin and Na-Cl cotransporter (NCC) causes Cartron JP, Paillard M, Doucet A, Chambrey R: Expression of RhCG, a severe salt wasting, volume depletion, and renal failure. Proc Natl Acad new putative NH(3)/NH(4)(+) transporter, along the rat nephron. JAm Sci U S A 109: 13368–13373, 2012 Soc Nephrol 13: 1999–2008, 2002 25. Lalioti MD, Zhang J, Volkman HM, Kahle KT, Hoffmann KE, Toka HR, 33. Quentin F, Chambrey R, Trinh-Trang-Tan MM, Fysekidis M, Cambillau Nelson-Williams C, Ellison DH, Flavell R, Booth CJ, Lu Y, Geller DS, M, Paillard M, Aronson PS, Eladari D: The Cl-/HCO3- exchanger pen- Lifton RP: Wnk4 controls blood pressure and potassium homeostasis drin in the rat kidney is regulated in response to chronic alterations in via regulation of mass and activity of the distal convoluted tubule. Nat chloride balance. Am J Physiol Renal Physiol 287: F1179–F1188, 2004 Genet 38: 1124–1132, 2006 34. Wagner CA, Loffing-Cueni D, Yan Q, Schulz N, Fakitsas P, Carrel M, 26. Yang SS, Morimoto T, Rai T, Chiga M, Sohara E, Ohno M, Uchida K, Lin Wang T, Verrey F, Geibel JP, Giebisch G, Hebert SC, Loffing J: Mouse SH, Moriguchi T, Shibuya H, Kondo Y, Sasaki S, Uchida S: Molecular model of type II Bartter’s syndrome. II. Altered expression of renal so- pathogenesis of pseudohypoaldosteronism type II: Generation and dium- and water-transporting proteins. Am J Physiol Renal Physiol 294: analysis of a Wnk4(D561A/+) knockin mouse model. Cell Metab 5: 331– F1373–F1380, 2008 344, 2007 35. Kim GH, Ecelbarger C, Knepper MA, Packer RK: Regulation of thick 27. Hadchouel J, Soukaseum C, Büsst C, Zhou XO, Baudrie V, Zürrer T, ascending limb ion transporter abundance in response to altered acid/ Cambillau M, Elghozi JL, Lifton RP, Loffing J, Jeunemaitre X: Decreased base intake. J Am Soc Nephrol 10: 935–942, 1999 ENaC expression compensates the increased NCC activity following 36. Schmitt R, Ellison DH, Farman N, Rossier BC, Reilly RF, Reeves WB, inactivation of the kidney-specific isoform of WNK1 and prevents hy- Oberbäumer I, Tapp R, Bachmann S: Developmental expression of pertension. Proc Natl Acad Sci U S A 107: 18109–18114, 2010 sodium entry pathways in rat nephron. Am J Physiol 276: F367–F381, 28. McCormick JA, Nelson JH, Yang CL, Curry JN, Ellison DH: Over- 1999 expression of the sodium chloride cotransporter is not sufficient to cause 37. Richardson C, Rafiqi FH, Karlsson HK, Moleleki N, Vandewalle A, familial hyperkalemic hypertension. Hypertension 58: 888–894, 2011 Campbell DG, Morrice NA, Alessi DR: Activation of the thiazide-sen- 29. Ito M, Oliverio MI, Mannon PJ, Best CF, Maeda N, Smithies O, Coffman sitive Na+-Cl- cotransporter by the WNK-regulated SPAK and TM: Regulation of blood pressure by the type 1A angiotensin II re- OSR1. J Cell Sci 121: 675–684, 2008 ceptor gene. Proc Natl Acad Sci U S A 92: 3521–3525, 1995 38. Burg M, Grantham J, Abramow M, Orloff J: Preparation and study of 30. Stehberger PA, Shmukler BE, Stuart-Tilley AK, Peters LL, Alper SL, fragments of single rabbit nephrons. Am J Physiol 210: 1293–1298, Wagner CA: Distal renal tubular acidosis in mice lacking the AE1 (band3) 1966 Cl-/HCO3- exchanger (slc4a1). J Am Soc Nephrol 18: 1408–1418, 2007 31. Breton S, Wiederhold T, Marshansky V, Nsumu NN, Ramesh V, Brown D: The B1 subunit of the H+ATPase is a PDZ domain-binding protein. Colocalization with NHE-RF in renal B-intercalated cells. J Biol Chem This article contains supplemental material online at http://jasn.asnjournals. 275: 18219–18224, 2000 org/lookup/suppl/doi:10.1681/ASN.2012080787/-/DCSupplemental.

J Am Soc Nephrol 24: 1104–1113, 2013 A Mouse Model of Chloride-Sensitive Hypertension 1113 Supplemental Informations

1- Supplemental methods Generation of the Tg(B1-hPDS) transgenics: The human SLC26A4 cDNA was ligated into the pBluescript cloning vector containing 6.9kbp of the human ATP6V1B1 promoter1, 2. The SV40 late region polyadenylation signal was cloned downstream of the cDNA. The transgene includes the 5'-flanking region of the ATP6V1B1 gene extending to but excluding the endogenous translational start codon, the human SLC26A4 cDNA, with its own translational start site, and the SV40 late region polyadenylation signal, and is referred as B1-hPDS. Transgene integrity was confirmed by restriction digest and bidirectional sequencing of ligation sites. To prepare for injection, the transgene was linearized from the vector by SalI and NotI digestion, followed by gel purification using an electroelution method and then concentrated using ElutipD columns (Whatman). The transgene was then further concentrated by ethanol precipitation and resuspended in low EDTA injection buffer (10mM tris 0.1mM EDTA). Transgene integrity was confirmed by restriction digest and bidirectional sequencing of ligation sites. Transgenic mice were created by the University of Utah transgenic mouse core facility using standard procedures as described1, 2. Genotyping demonstrated that 63 pups were positive for transgene integration. One founder transmitted the transgene in a Mendelian fashion and was subsequently used for all the experiments described. The mice that were used in the present study were F6. Mendelian transmission of the transgene and southern blot analyses (Suppl. Fig S1) were compatible with a single site of integration. The transgenic founder was crossed with wild- type C57BL/6 x CBA F1 mice and the resulting hemizygous offspring were analyzed. The control mice consisted of sex-matched wild type littermates.

Genotyping: Mouse genomic DNA was prepared from tail tissue by standard methods. To detect the B1-hPDS transgene, PCR primers were designed to amplify across the hPDS cDNA and SV40 Polyadenylation signal sequence to provide a PCR product of 345 bp (Forward primer: 5’- AGAGGGTCAAGGTTCCATTTTAG-3’; Reverse primer: 5’- CAAACCACAACTAGAATGCAGTG-3’). PCR consisted in denaturation 95°C for 7min followed by 40 cycles of amplification (30 sec at 95°C, 30 sec at 57°C, 30 sec at 72°C) and 5 min at 72°C.

RNA extraction and reverse transcription: Animals were killed and kidneys were harvested and rapidly frozen in liquid nitrogen. Snap-frozen kidneys (six kidneys for each condition) were homogenized in RLT-Buffer (Qiagen, Basel, Switzerland) supplemented with ß-mercaptoethanol to a final concentration of 1%. Total RNA was extracted from 200 µl aliquots of each homogenized sample using the RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. Quality and concentration of the isolated RNA preparations were analyzed by the ND-1000 spectrophotometer (NanoDrop Technologies). Total RNA samples were stored at –80°C. Each RNA sample was diluted to 100 ng/µl and 3 µl used as a template for reverse transcription using the Bioline cDNA synthesis kit (BioLine, Randolph, MA). For reverse transcription, 1 µg of RNA template were diluted in a 10-µl reaction mix that contained (final

concentrations) RT buffer (1X), oligo (dT)18 (2.5 µM), RNase inhibitor (0.5 U/µl), the Bioline reverse transcriptase (2.5 U/µl), dNTP mix (0.5 µM each), and RNase-free water.

Real-time quantitative PCR: Primers were designed using Primer Express software from Applied Biosystems for GAPDH (Forward 5’- GCACAGTCAAGGCCGAGAAT-3’; Reverse 5’- GCCTTCTCCATGGTGGTGAA-3’), human pendrin (Forward 5’- AAATCTCAAGAGGGTCAAGGTTC-3’; Reverse 5’- ACATCAAGTTCTTCTTCCGTCAG-3’), mouse Atp6v1b1 (Forward 5’- ATCAATGTGCTCCCATCCCTCT-3’; Reverse 5’- AATGCGCTTCAGCATCTCTTTC-3’). Primers were chosen to produce amplicons ≤150 bp that spanned intron-exon boundaries as to exclude amplifying genomic DNA. The specificity of all primers was first tested on mRNA derived from kidney. Real-time PCR was performed using a 5 ng of cDNA quantity on a LightCycler (Roche Diagnostics, Meylan, France) with LightCycler 480 SYBR Green I Master qPCR kit (Roche Diagnostics, Meylan, France). Briefly, 2µl (2.5 ng/µl) cDNA, 0.5 µl of each primer (25 µM), 2 µl RNAse free water, 5 µl SYBR Green Master mix (Roche Applied Biosystems, Meylan, France); for a final volume of 10 µl. Reaction conditions were as follows: 95oC for 10 minutes followed by 45 cycles of 95oC for 20 seconds, 62oC for 20 seconds, and 72°C for 20 seconds. All reactions were run in duplicate. A negative control was amplified in the absence of the reverse transcriptase. Cycle thresholds were recorded at within the linear range of fluorescence intensity, which was set at 0.06. Gene expression was normalized to that glyceraldehyde 3-phosphate dehydrogenease (GAPDH). Relative expression ratios were calculated as R=2(Ct(GAPDH)-Ct(test gene)), where Ct represents the cycle number at the threshold 0.06.

Southern Blot analyses of Genomic DNA: A 894 bp fragment of the hPDS coding sequence was excised by EcoRI and served as a probe template. The probe was radiolabelled with 32P-dCTP (Hartmann Analytik, Braunschweig, Germany) and purified with Illustra ProbeQuant G-50 columns (GE Healthcare, Waukesha, WI, USA). Phenol-Chloroform purified genomic DNA (5 µg) from wild-type and transgenic B1-hPDS mice was digested with either BamHI or EcoRI (Thermo, Waltham, MA, USA), respectively, and separated on 0.8% agarose gels. Digested genomic DNA was blotted on Hybond-XL membranes (GE Healthcare, Waukesha, WI, USA) and hybridized with radiolabelled probes following standard protocols.

Generation and characterization of a anti human pendrin antibody: The synthetic peptide (APGGRSEPPQLPEYS) corresponding to amino-acids 3 to 18 of the N-terminal end of human pendrin protein was synthesized by the DB-BioRun Laboratory (Nantes, France), coupled to keyhole limpet hemocyanin with the Imject Maleimide Activated Immunogen Conjugation Kit (Pierce), and used to immunize rabbits to generate polyclonal antiserum (DB- BioRun Laboratory, Nantes, France). Supplemental Figure 2 shows that analyses of mouse and human kidney sections labeled with this new antibody revealed exactly the same pattern of labeling in both species that was consistent with known pendrin expression. Pre-immune serum did not yield any labeling, and all staining was abolished when the antiserum was pre- incubated with the immunizing peptide. Moreover, when the antiserum was used to stain kidney sections obtained from mice with pendrin disruption3 no signal was detected which confirmed that this antiserum is specific for pendrin. (Supplemental Figure 1).

Biochemical and hormonal measurements: Urine creatinine (enzymatic method) was measured with a Konelab 20i auto-analyzer (Thermo Electron Corporation, Eragny Parc, France). Urinary chloride was measured with a DL 55 titrator (Mettler Toledo, Viroflay, France). Urinary Na+ and K+ were measured by flame photometry (IL943, Instruments Laboratory, Lexington, MA). Urine aldosterone was measured by RIA (DPC Dade Behring, La Défense, France). Blood was collected by tail incision on mice anesthetized by peritoneal injection of a mixture (0.1ml/g body weight) of ketamine (Imalgene®, Rhône Mérieux, Lyon, France; 10%) and xylazine (Rompun®, Bayer AG, Leverkusen, Germany; 5%) and [Na+], [K+], hematocrit and [Cl-] were measured with an ABL 77 pH/blood-gas analyzer (Radiometer, Copenhagen, Denmark). Blood gases analyses were performed by retro- orbitary punction on awake animals and pH, PCO2, and PO2 were measured with an ABL 77 pH/blood-gas analyzer (Radiometer, Copenhagen, Denmark). Blood bicarbonate concentration was calculated by the autoanalyzer from the measured values using the Henderson-Hasselbach equation.

Measurements of blood pressure by radiotelemetry : Animals were anaesthetized with pentobarbital (50 mg/kg) by intraperitoneal (IP) injection. Telemetric devices were implanted in 6 wild-type and 6 TgB1- hPDS according to the standard protocols. Briefly, catheters were inserted into the right carotid artery and attached to a radiotransmitter (Physio Tel HD-X11; Data Sciences International) located on the back. Mice were allowed to recover for two to three days at which time they were placed in metabolic cages (Hatteras Instruments, Cary, NC) and permitted to acclimate to the cages and the gel diet for an additional five days. Blood pressure was continuously recorded using a telemetry receiver (Physio Tel DSI receiver; Data Sciences International).

In vitro microperfusion of mouse CCDs: Kidneys were removed and cut into 1 to 2 mm coronal slices that were transferred into a chilled dissection medium containing (mM): 118 NaCl, 25 NaHCO3, 2.0 K2HPO4, 1.2 MgSO4, 2.0 calcium lactate, 1.0 sodium citrate, 5.5 glucose, and 12 creatinine, pH 7.4,

and gassed with 95% O2-5% CO2. CCD segments were isolated from cortico- medullary rays under a dissecting microscope with a sharpened forceps. Because CCDs are highly heterogeneous, relatively short segments (0.45-0.6 mm) were dissected to maximize the reproducibility of the isolation procedure. In vitro microperfusion was performed as described by Burg et al. 4. Briefly, isolated CCDs were rapidly transferred to a 1.2 ml temperature- and environmentally-controlled chamber, mounted on an inverted microscope, and perfused and bathed initially at room temperature with dissection solution. The specimen chamber was continuously suffused with 95% O2-5% CO2 to maintain pH at 7.4. Once secure, the inner perfusion pipette was advanced and the tubule was opened with a slight positive pressure. The opposite end of the tubule was then pulled into a holding collection pipette. In the holding collection pipette, 2 to 3 cm of water-saturated mineral oil contributed to maintain the tubule open at a low flow rate of perfusion. The perfusing and collecting end of the segment was sealed into a guard pipette using Dow- Corning 200 dielectric fluid (Dow Corning Corp., Midland, MI). The tubules were then warmed to 37°C and equilibrated for 20 minutes while the collection rate was adjusted to a rate of 1-4 nl/min. The length of each segment was measured using an eyepiece micrometer. Because CCDs from mice are frequently unstable and collapse, measurements were conducted during the first 90 minutes of perfusion. Usually, collections from 4 periods of 15 minutes were performed in which 25 to 30 nanoliters of fluid were collected. The volume of the collections was determined under water-saturated mineral oil with calibrated volumetric pipettes. 20 nanoliters were required for [Na+], [K+] and [creatinine] measurements. Transepithelial voltage (Vte) was measured continuously using Ag-AgCl electrodes connected to 0.15 M NaCl-agar bridges inserted in the perfusion pipette and bathing solutions. The initial value after rewarming of the tubule was noted. Values of each period were averaged.

Measurements of Na+, K+ and creatinine concentrations with high- pressure liquid chromatography: Cation concentrations were measured as previously described5. The Dionex-500 system (Dionex DX-500, Dionex Corp., Voisins le Bretonneux, France) consisted of an AS50 autosampler, a GP50 gradient pump, an ED40 electrochemical detector (Na+ and K+), and an AD20 UV absorbance detector 220 nm (creatinine). The signal-to-noise ratio of the conductivity measurement was enhanced by employing a cation self- regenerating suppressor (CSRS-ultra, 4mm) that was set in the autosuppression recycle mode. The HPLC column consisted of a Dionex IonPac CS12 column (4 x 250 mm), equipped with a guard column CG12A, (4 x 50 mm). The mobile phase consisted of 18mM methanesulfonic acid. Tubular fluid, perfusion solution, and standard solutions were drawn under mineral oil with a calibrated pipette (about 20 nl) and transferred to a vial

containing 39 µl to the mobile phase of the HPLC with LiNO3 as an internal standard. Peaks of each measured analyte (Na+, K+, Creatinine) were adjusted with the value of the Li+ internal standard to limit the variations due to automatic injection. In each run of experiments, perfusion and bath solutions were tested in 4 or 5 replicates and the reproducibility of the measure was evaluated: Coefficient of variation (CV) < 0.10 for K+ determination, and CV <0.05 for creatinine determination. In addition, a calibration curve for each analyte was tested with correlation coefficients >0.98.

Measurement of fluid absorption: Creatinine was used as the volume marker, and therefore was added to the perfusion solutions (both perfusate and bath) at a concentration of 12 mM. The rate of fluid absorption (Jv) was calculated as Jv = (Vperf – Vcoll)/L, with Vperf = Crcoll/Crperf x Vcoll.

Crcoll and Crperf are the concentrations of creatinine in the collected fluid and perfusate, respectively. Vcoll is the collection rate at the end of the tubule. L is the length of the tubule.

Calculation of the rate of absorption of Na and K: For each collection, Na+ flux (JNa) and K+ flux (JK) where calculated and reported to the length of the tubule: JNa =[( [Na]perf x Vperf )– ([Na]coll x Vcoll )] /L JK =[( [K]perf x Vperf )– ([K]coll x Vcoll )] /L Therefore, positive values indicate net absorption, whereas negative values indicate net secretion of the ion. For each tubule, the mean of the 4 collection periods was used.

Intracellular pH measurements on isolated microperfused tubules During intracellular pH measurement experiments, the average tubule length exposed to bath fluid was limited to 300 – 350 µm in order to prevent motion of the tubule. Two solutions were used, differing in their content in chloride. The composition of the solutions were as follows ; chloride-containing solution was

composed of (in mM) 119 NMDG-Cl, 23 NMDG-HCO3, 2 K2HPO4, 1.5 CaCl2,

1.2 MgSO4, 10 HEPES, and 5.5 D-glucose ; chloride-free solution was composed of (in mM) 119 NMDG-gluconate, 23 NMDG-HCO3, 2 K2HPO4, 7.5

Ca-gluconate, 1.2 MgSO4, 10 HEPES, and 5.5 D-glucose. All solutions was

adjusted to pH 7.40 and continuously bubbled with 95% O2/5% CO2. At the beginning of all experiments, tubules were bathed and perfused with the Cl-containing solution. To identify principal and intercalated cells, we labeled the apical membrane of intercalated cells by adding fluorescent peanut lectin (PNA,Vector Labs) to the luminal perfusate for 5 minutes and observed which cells were fluorescent. Intracellular pH in CCD cells was assessed with imaging-based, dual excitation-wavelength fluorescence microscopy with use of the fluorescent probe 2',7'-Bis- (2-Carboxyethyl)-5- (And-6)- carboxyfluorescein (BCECF, Molecular probes). Tubules were loaded for ~20 min at room temperature with 5x10-6mol/L of the acetoxymethyl ester of BCECF added to the peritubular fluid. Loading was continued until the fluorescence intensity at 440 nm excitation wavelength was at least one order of magnitude higher than background fluorescence. The loading solution was then washed out by initiation of bath flow and the tubule was equilibrated with dye-free solution for 5-10 minutes. Bath solution was delivered at a rate of 20 ml/min and warmed to 37±0.5°C by water jacket immediately upstream to the chamber. During the fluorescence recording, the Cl-containing solution was delivered to the perfusion pipette via a chamber under an inert gas (N2 pressure around 1 bar) connected through a manual 6-way valve. With this system, opening of the valve instantaneously activated flow of one solution. The majority of the fluid delivery to the pipette exited the rear of the pipette system through a drain port at a rate of 4 ml/min. This method resulted in a smooth and complete exchange of the luminal solution in less than 3 to 4 s as measured by the time necessary for appearance of colored dye at the tip of the perfusion pipette. After 3-minutes recording, luminal fluid was instantaneously (at the rate of 4 ml/min in the draining) replaced by the corresponding Cl-free solution for 3 minutes. Finally, the luminal solution was changed again for the Cl-containing solution. Intracellular dye was excited alternatively every 2 seconds at 440 and 500 nm with a light-emitting diode (Optoled, Cairn Research, Faversham, UK). Emitted light was collected through a dichroïc mirror, passed through a 530 nm filter and focused onto a EM-CCD camera (iXon, Andor Technology, Belfast, Ireland) connected to a computer. The measured light intensities were digitized with 14-bit precision (16384 grey level scale) for further analysis. For each tubule, 3-4 intercalated cells were analyzed and the mean grey level was measured with the Andor IQ software (Andor Technology, Belfast, Ireland). Background fluorescence was subtracted from fluorescence intensity to obtain intensity of intracellular fluorescence. Intracellular dye was calibrated at the end of each experiment using the high [K+]-nigericin technique. Tubules were perfused and bathed with a HEPES- buffered, 95-mM K+ solution containing 10 µM of the K+/H+ exchanger nigericin. Four different calibration solutions, titrated to pH 6.9, 7.3, 7.5 or 7.8 were used.

References 1. Miller, RL, Lucero, OM, Riemondy, KA, Baumgartner, BK, Brown, D, Breton, S, Nelson, RD: The V-ATPase B1-subunit promoter drives expression of Cre recombinase in intercalated cells of the kidney. Kidney international, 75: 435-439, 2009. 2. Miller, RL, Zhang, P, Smith, M, Beaulieu, V, Paunescu, TG, Brown, D, Breton, S, Nelson, RD: V-ATPase B1-subunit promoter drives expression of EGFP in intercalated cells of kidney, clear cells of epididymis and airway cells of lung in transgenic mice. Am J Physiol Cell Physiol, 288: C1134-1144, 2005.

3. Choi, BY, Kim, HM, Ito, T, Lee, KY, Li, X, Monahan, K, Wen, Y, Wilson, E, Kurima, K, Saunders, TL, Petralia, RS, Wangemann, P, Friedman, TB, Griffith, AJ: Mouse model of enlarged vestibular aqueducts defines temporal requirement of Slc26a4 expression for hearing acquisition. J Clin Invest, 121: 4516-4525, 2011.

4. Burg, M, Grantham, J, Abramow, M, Orloff, J: Preparation and study of fragments of single rabbit nephrons. Am J Physiol, 210: 1293-1298, 1966.

5. Leviel, F, Hubner, CA, Houillier, P, Morla, L, El Moghrabi, S, Brideau, G, Hassan, H, Parker, MD, Kurth, I, Kougioumtzes, A, Sinning, A, Pech, V, Riemondy, KA, Miller, RL, Hummler, E, Shull, GE, Aronson, PS, Doucet, A, Wall, SM, Chambrey, R, Eladari, D: The Na+-dependent chloride-bicarbonate exchanger SLC4A8 mediates an electroneutral Na+ reabsorption process in the renal cortical collecting ducts of mice. J Clin Invest, 120: 1627-1635, 2010.

2- Supplemental figures

Supplemental Figure S1. Effects of high salt diet on Atp6v1b1 mRNA. Wild type C57BL/6 mice were fed either a normal salt (0.3% Na+ as NaCl salt) or high salt diet (3% Na+) for two weeks. RT-PCR analysis were performed using the primers shown in suppl. table S1. Results were normalized to GAPDH as described above in the method section. Results are the mean ± S.E., n = 6 in both groups. The absence of statistical difference was tested by unpaired Student’s t test.

Supplemental Figure S2. Southern Blot analyses of Genomic DNA from WT or TgB1-hPDS mice. Genomic DNA from wildtype (Wt) and transgenic mice (Tg) was digested with either BamHI or EcoRI as indicated above. Digestion with BamHI which does not cut within the transgenic construct, revealed a single band of strong intensity supportive of a single integration site. An additional genomic digest with EcoRI, which also excises the probe template, resulted in a single band corresponding to the size of the probe. Binding of the probe to fragments which include the endogenous murine Pds sequence are marked by asterisks.

Supplemental Figure S3. Characterization of a new anti human pendrin antibody. Low magnification (A) and high magnification (B) of mouse kidney sections, and high magnification (C, D) of human kidney sections labeled with the rabbit anti human pendrin antibody (red labeling), plasmic membranes were counterstained with FITC-conjugated phalloidin, and nuclei were counterstained with DAPI. The pattern of labeling was identical in human and mouse kidney, and was consistent with the known localization of pendrin in β- ICs. Staining of mouse kidney sections (E), was completely abolished when the antibody was pre-incubated with the immunizing peptide in excess (F), and was absent when pre-immune serum was used instead of the immune serum (G). Staining was also absent in sections obtained from mice with pendrin disruption (Pds-/-) (I) compared to WT mice (H) and stained using same procedure than in panel A and B demonstrating the specificity of the signal.

Supplemental Figure S4: Systolic blood pressure and heart rate measured in WT and TgB1-hPDS mice by radio-telemetry. Mice were fed either normal (0.3% Na+ as NaCl) or high salt (3% Na+ as NaCl) for two weeks. Data are means ± S.E. of data from 5 independent mice in each group. Data were analyzed by ANOVA followed by bonferroni post hoc-test when appropriate. ** indicate p<0.01 vs. Normal salt diet.

3. Supplemental Tables

Supplemental Table 1 : Primers used in this study Forward primers Reverse Primers Mouse 5’-GCACAGTCAAGGCCGAGAAT-3’ 5’-GCCTTCTCCATGGTGGTGAA-3’ Gapdh primers B1- 5’-AGAGGGTCAAGGTTCCATTTTAG-3’ 5’- hPds CAAACCACAACTAGAATGCAGTG- (genotyping) 3’ human PDS 5’-AAATCTCAAGAGGGTCAAGGTTC-3’ 5’- ACATCAAGTTCTTCTTCCGTCAG- 3’ Mouse 5’-ATCAATGTGCTCCCATCCCTCT-3’ 5’-AATGCGCTTCAGCATCTCTTT-3’ Atp6v1b1

Supplemental Table 2 : Physiological data from TgB1-hPDS and WT mice fed either a normal salt or a high salt diet for two weeks 0.8% NaCl 8%NaCl (0.3% Na+) (3% Na+) TgB1-hPDS WT TgB1-hPDS WT [Na+], mM 148 ± 1 149 ± 1 154 ± 0.6 154 ± 1.4 Plasma [K+], mM 4.15 ± 0.19 4.03 ± 0.11 3.60 ± 0.25 3.76 ± 0.15 [Cl-], mM 117 ± 1.3 116 ± 0.9 119 ± 0.4 118 ± 0.4 Ht, % 41 ± 1.02 42 ± 0.77 42 ± 0.6 44 ± 1.2 pH 7.24 ± 0.01 7.23 ± 0.02 7.25 ± 0.01 7.28 ± 0.01 - blood [HCO3 ], mM 21.3 ± 0.8 21.5 ± 0.8 22.0 ± 0.5 22.3 ± 0.3 pCO2, mmHg 51 ± 1.0 54 ± 1.8 52 ± 1.5 50 ± 0.9 urine pH 5.76 ± 0.07 5.83 ± 0.06 6.22 ± 0.05 6.13 ± 0.03

Data are means ± S.E. of data obtained from 8 TgB1-hPDS and 8 WT mice. Both diet conditions are presented in same table but are from two independent series and statistical significance was only assessed vs. WT fed same diet, using a two-tailed unpaired Student’s t-test.

Supplemental Table 3 : Effect of a high salt diet on different renal transporters protein abundance. Densitometric analyzes of immunoblots of membrane fractions isolated from the renal cortex of TgB1-hPDS mice or WT mice. All animals were pair-fed a high salt diet (3%Na+ as NaCl) for one week.

WT KO p value NHE3 100 ± 9.9 75.5 ± 12 0.19 NKCC2 100 ± 5.4 109.5 ± 6.7 0.29 NCC 100 ± 8.1 58.0 ± 8.2 0.003* pNCC 100 ± 16.5 58.0 ± 8.0 0.041* αENaC 90kDa 100 ± 6.5 75.0 ± 5.3 0.012* αENaC 30kDa 100 ± 9.1 55.3 ± 5.7 0.001* γENaC 100 ± 12.8 64.3 ± 7.3 0.032* mPds 100 ± 7.0 86.7 ± 11.9 0.33 B1-H-ATPase 100 ± 6.2 93.1 ± 11.9 0.45 Ndcbe 100 ± 3.9 61.4 ± 7.2 0.0005*

Data are mean ± S.E and are expressed as a percentage of control values. Data obtained from 7 TgB1-hPDS mice and 7 WT mice. statistical significance was determined by a two-tails unpaired Student’s t-test, a p value < 0.05 is considered as significant and labeled with *.