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The ClC-K2 Is Critical for Salt Handling in the Distal Nephron

† † † J. Christopher Hennings,* Olga Andrini, Nicolas Picard, Marc Paulais, Antje K. Huebner,* ‡ † † | Irma Karen Lopez Cayuqueo, § Yohan Bignon, Mathilde Keck, Nicolas Cornière, ‡ †† † David Böhm,* Thomas J. Jentsch,¶ Régine Chambrey, ** Jacques Teulon, ‡ ‡‡ Christian A. Hübner,* and Dominique Eladari **

*Institut für Humangenetik, University Hospital Jena, Friedrich-Schiller-Universität, Jena, Germany; †Centre National de la Recherche Scientifique Equipe de Recherche Labelisée 8228, Institut National de la Santé et de la Recherche Médicale Unité Mixte de Recherche en Santé 1138, Université Pierre et Marie Curie, Centre de Recherche des Cordeliers, Paris, France; ‡Institut National de la Santé et de la Recherche Médicale U970, Paris Cardiovascular Research Center, Paris, France; §Centro de Estudios Científicos, Valdivia, Chile; |Service de Néphrologie, Hôpital Felix Guyon, Centre Hospitalier Universitaire de la Réunion, St Denis, Ile de la Réunion, France; ¶Leibniz-Institut für Molekulare Pharmakologie and Max-Delbrück Centrum für Molekulare Medizin, Berlin, Germany; **Faculté de Médecine, Université Paris-Descartes, Paris, France; ††Centre National de la Recherche Scientifique, Paris, France; and ‡‡Département de Physiologie, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, Paris, France

ABSTRACT Chloride transport by the renal tubule is critical for blood pressure (BP), acid-base, and potassium homeostasis. Chloride uptake from the urinary fluid is mediated by various apical transporters, whereas basolateral chloride exit is thought to be mediated by ClC-Ka/K1 and ClC-Kb/K2, two chloride channels from the ClC family, or by KCl cotransporters from the SLC12 family. Nevertheless, the localization and role of ClC-K channels is not fully resolved. Because inactivating mutations in ClC-Kb/K2 cause , a disease that mimics the effects of the loop diuretic furosemide, ClC-Kb/K2 is assumed to have a critical role in salt handling by the thick ascending limb. To dissect the role of this channel in detail, we generated a mouse model with a targeted disruption of the murine ortholog ClC-K2. Mutant mice developed a Bartter syndrome phenotype, characterized by renal salt loss, marked hypokalemia, and metabolic alkalosis. Patch-clamp analysis of tubules isolated from knockout (KO) mice suggested that ClC-K2 is the main basolateral chloride channel in the thick ascending limb and in the aldosterone-sensitive distal nephron. Accordingly, ClC-K2 KO mice did not exhibit the natriuretic response to furosemide and exhibited a severely blunted response to thiazide. We conclude that ClC-Kb/K2 is critical for salt absorption not only by the thick ascending limb, but also by the distal convoluted tubule.

J Am Soc Nephrol 28: 209–217, 2017. doi: 10.1681/ASN.2016010085

Bartter syndrome (BS) is a group of renal tubulo- Received January 22, 2016. Accepted May 4, 2016. pathies with autosomal recessive inheritance. First J.C.H. and O.A. share first authorship. J.T., C.A.H., and D.E. share described by Frederic C. Bartter and Pacita Pronove, senior authorship and contributed equally to this work. this syndrome is characterized by marked secondary Published online ahead of print. Publication date available at hyperaldosteronism caused by renal salt-wasting www.jasn.org. with normal or low BP, hypokalemia, metabolic alkalosis, hypertrophy and hyperplasia of the juxta- Correspondence: Dr. Dominique Eladari, Paris Centre de Re- cherche Cardiovasculaire, Paris, France, or Dr. Christian A. 1 glomerular apparatus. Thesyndrometypically Hübner, Institut für Humangenetik, University Hospital Jena, presents during the neonatal period and is fre- Paris, France. Email: [email protected] or Christian. quently associated with hypercalciuria and [email protected] nephrocalcinosis. Copyright © 2016 by the American Society of Nephrology

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The free water clearance and distal fractional chloride (DCT), and in the basolateral membrane of both a-and reabsorption is low in patients affected by BS. Hence, it has been b-intercalated cells of the connecting tubule (CNT) and proposed that the primary pathogenic event leading to the the collecting duct (CD). Unexpectedly, ClC-K2 disruption syndrome is impaired NaCl absorption by the thick ascending completely abolished the natriuretic response to both hydro- limb (TAL) of the loop of Henle.2,3 Accordingly, genetic studies chlorothiazide and furosemide despite the presence of ClC-K1 revealed that BS is caused by inactivating mutations in one of in the mTAL. In accordance, ClC-K1 activity was barely at least four independent (numbered from type 1 to type detectable by patch-clamp experiments. Hence, our data in- 4BS):SLC12A1,4 KCNJ1,5 CLCNKB,6,7 and BSND.8 These dicate that chloride absorption all along the distal nephron genes encode for the Na+/K+/2Cl- cotransporter NKCC2, the from the TAL to the collecting duct depends on ClC-K2. ROMK, the chloride channel ClC-K2, and Barttin, an essential b-subunit for ClC-K1 and ClC-K2 Cl- channels,9 respectively. These findings suggest a transport RESULTS model, in which NaCl and K+ are taken up apically via NKCC2. Although K+ hastoberecycledacrosstheapical Disruption of the Clcnk2 Gene Results in a Severe membrane via ROMK to maintain a high luminal K+ concen- Phenotype with Early Lethality tration that is required for the sustained activity of NKCC2, To disrupt the Clcnk2 gene in mice, we flanked exons 5–10 NaCl leaves the cell basolaterally via the Na+/K+-ATPase and with loxP sites by homologous recombination (Supplemental ClC-Kb/Barttin. However, the expression pattern of each ClC- Figure 1A). A KO line was obtained by mating of the floxed K homolog has not been fully resolved because of the close line with a cre-Deleter mouse strain17 (Supplemental Figure 2/2 homology between ClC-K1 and -K2 and the lack of isoform 1, B–D). Homozygous (Clcnk2 )KOmicewerebornfrom specific antibodies. heterozygous matings at Mendelian ratio. At birth, homozy- Typically, NKCC2 and ROMK inactivation cause a “severe gous KO mice appeared indistinguishable from their wild-type +/+ +/2 form” of BS, which is characterized by a very early (even an- (WT; Clcnk2 ) and heterozygous (Clcnk2 ) littermates. 2/2 tenatal) onset, a marked salt-wasting phenotype, and pro- Subsequently Clcnk2 mice failed to thrive, became hypo- found hypokalemia and metabolic alkalosis, as described trophic (Supplemental Figure 2A), and exhibited early lethality initially by Bartter and Pronove. This clinical subtype of the (Supplemental Figure 2B). Compared with controls overall syndrome is often associated with polyhydramnios, failure to kidney size was reduced (Supplemental Figure 2C). Beginning 2/2 thrive, and severe hypercalciuria eventually leading to neph- at 2 weeks of age Clcnk2 mice developed hydronephrosis rocalcinosis. Because patients excrete high levels of PGE2 with (Supplemental Figure 2D). the urine and blockers of PGE2 synthesis significantly alleviate the symptoms, this variant is also called the “hyperprostaglan- ClC-K1 and ClC-K2 Immunolocalization in the Mouse dinuria syndrome”. Mutations in CLCNKB rather cause the Kidney “classic” BS, which is diagnosed later in life and characterized The localization of ClC-K channels was determined with an by a milder phenotype that lacks both hypercalciuria and antibody derived from guinea pig against the synthetic peptide nephrocalcinosis. Nevertheless, patients related to CLCNKB MEELVGLREGSSKKP, which corresponds to the N-terminal show a high phenotypic variability with clinical presentations end of the ClC-K2 . In immunofluorescence studies on ranging from very severe salt-losing nephropathy with marked Clcnk2+/+ kidney sections the guinea pig antibody reproduced hypokalemia to almost asymptomatic presentation.6 Some pa- the expression pattern as shown by Kobayashi et al. previ- tients with CLCNKB mutations exhibit a mild phenotype with ously18 (Figure 1, A and B). When the antibody was applied 2 2 moderate salt-wasting, hypocalciuria, and resistance to thia- on kidney sections from Clcnk2 / mice, the signal in the zide diuretics, i.e., typical features of Gitelman syndrome.10 cortical TAL (cTAL), the DCT, the CNT, and ICs was com- Gitelman syndrome is another salt-losing nephropathy, which pletely abolished (Figure 1, C–F). However, the staining per- is usually caused by inactivating mutations in the gene for the sisted in the mTAL and the tAL (Figure 1, G and H), indicating apical NaCl cotransporter NCC (SLC4A3) in the distal con- that ClC-K1 protein is likely to be expressed in the tAL as voluted tubule.6,11–15 Finally, mutations in the BSND gene described previously,18 but is also present in the mTAL. In lead to type 4 BS, the most severe form of BS with extreme overexpression studies the guinea pig antibody recognizes growth retardation, very severe salt-wasting, and sensorineu- both overexpressed ClC-K channels in HEK293 cells by West- ral deafness.16 ern blot and immunohistochemistry (Supplemental Figure 3). Here, we show that disruption of Clcnk2 in the mouse leads In parallel, we tested the rabbit R4 antibody described by to severe BS without hypercalciuria but with elevated levels of Kieferle et al.,19 another ClC-K1/K2 antibody, which is PGE2 in the urine. Using two different antibodies in our KO thought to cross react with both homologs, and compared 2/2 model, we demonstrate that ClC-K1 is expressed in the thin the staining pattern in Clcnk2+/+ and Clcnk2 mice. The ascending limbs and the medullary TALs (mTALs) of the loop signal distribution with the R4 antibody19 in kidney sections of Henle, whereas ClC-K2 is found in the medullary and cor- of Clcnk2+/+ mice was much more restricted compared with tical portion of the TAL, in the distal convoluted tubule the staining reported for rat.20 The basolateral staining in the

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A and B). To explain this finding, we de- termined the expression of ClC-K1 and ClC-K2 in the mouse kidney by quantita- tive RT-PCR. We found that ClC-K1 is roughly 25-fold less abundant than ClC- K2 in the adult mouse kidney (Supple- mental Figure 5A). The lower expression of ClC-K1 was further corroborated by Western blot analysis of whole kidney lysates 2/2 obtained from Clcnk2+/+ and Clcnk2 mice with the guinea pig and rabbit R4 an- tibodies, respectively (Supplemental Figure 5, B and C). We conclude that the ClC-K1 expression is below the detection limit in im- munofluorescence with the rabbit R4 antibody.

ClC-K2 Is a 10-pS Channel and Constitutes the Predominant Chloride Conductance in the Distal Nephron To verify the nature of the different chloride channels along the mouse nephron at the functional level, we performed patch-clamp experiments on several renal segments iso- 2 2 lated from Clcnk2+/+ or Clcnk2 / mice. Previous experiments have shown that the DCT and the intercalated cells of the CNT/ cortical collecting duct (CCD) are en- dowed with a 10-pS channel that we sug-

+/+ 2/2 gested to be mediated by ClC-K2 because Figure 1. Localization of ClC-K in Clcnk2 and Clcnk2 mice. The immunolocal- 21,22 2 2 of its ion selectivity and regulation. ization of ClC-K (red) on kidney sections from WT and Clcnk2 / mice using Additionally, the TAL and late DCT our new anti ClC-K antibody (A). Residual staining is detected in the thin ascending 2 2 limb (tAL) and mTAL in Clcnk2 / mouse kidney, consistent with a medullary ex- (DCT2) express another 10-pS chloride pression of ClC-K1. (B) Schematic representation of the staining along the mouse channel (showing contrasting anion selec- 2 2 – nephron obtained with this anti ClC-K antibody in WT and Clckn2 / mice. In the CNT tivity).23 25 The latter channel was called and CCD this antibody also labels intercalated cells as indicated by green dots. Lo- pseudo-cystic fibrosis transmembrane calization of ClC-K staining was ascertained using double labeling of ClC-K1 and 2 in conductance regulator (CFTR) because green and different tubular markers in red (C–H): NKCC2 for the cortical (C) and of its functional similarities to CFTR.24 medullary (G) TALs; NCC for the distal convoluted tubules (D); basolateral kidney In the early DCT (DCT1) and intercalated a anion-exchanger 1 (AE1) for -intercalated cells in the medulla (E); pendrin for cells of CNT/CCD only ClC-K2-like 10-pS b-intercalated cells (F). ClC-K staining is absent in the cortical TAL and the DCT, as 21,26 2/2 chloride channels are present. For this well as a-andb-intercalated cells in Clcnk2 mice. ClC-K staining is still present in - 2 2 reason, we first searched for Cl channels the mTAL and in the tAL in Clcnk2 / mice. A, scale bar = 200 mm; C, D, E, F, G, and H, in the DCT1 using a K+-rich (145 mM scale bar = 25 mm. KCl) pipette solution in the cell-attached configuration, in order to record simulta- TAL, DCT, and intercalated cells in the cortex (Supplemental neously K+ channels and Cl- channels.Asthispartofthe Figure 4, A and B) was consistent with that reported by renal tubule highly expresses Kir4.1/Kir5.1 potassium chan- Kobayashi et al. using their ClC-K1/2 antibody on kidney nels,27 this allowed us to discard silent patches (i.e., those 2 2 sections from Clcnk1 / mice.18 The staining extended to with no K+ channel activity) as formed by membrane vesi- mTALs in the outer medulla but was much less intense than cles. We recorded similar K+ channels from Clcnk2+/+ (n=9) 2 2 in the cortex. Importantly, the signal detected in the cortex and Clcnk2 / (n=13) DCT1 (Figure 2A). Although all DCT was fully abolished when the R4 antibody was applied on patches from Clcnk2+/+ mice showing K+ channel activity 2 2 kidney sections from our Clcnk2 / mice indicating that it expressed Cl- channels of 9.360.5 pS (Figure 2A, Table 1), is specific for ClC-K2 in the mouse (Supplemental Figure 4, no 9-pS chloride channels were recorded in DCT patches

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Table 1. Distribution of 10-pS Cl- channels in the DCT and 2 2 TAL of Clcnk2+/+ and Clcnk2 / mice Part of the Status FG,pS NPo Renal Tubule DCT [2] WT (9) 1 9.360.5 (9) 12.064.2 (9) DCT [3] KO (13) 0 NA NA CNT/CCD [4] WT (24) 0.70 10.960.6 (17) 34.569.6 (14) CNT/CCD [3] KO (10) 0 NA NA TAL [3] WT (14) 0.85 7.760.6 (12) 13.965.5 (10) TAL [2] KO (13) 0 NA NA [n], number of mice used in the experiments; (n), number of measurements. F, frequency for detecting channel activity; G, pS, unit conductance in 2 picosiemens; NPo, channel activity or normalized current due to 10-pS Cl channels; NA, not available.

2 2 from Clcnk2 / mice showing K+-channel activity (Figure 2A, Table 1). For intercalated cells of the CNT and CCD, we used NMDGCl in the pipette to decrease the frequent occurrence of cation channels in these cells. Seventy one percent of the patches formed on the intercalated cells from Clcnk2+/+ mice showed Cl- channels of 10.960.6 pS (n=17), whereas no such 2 2 channels were recorded in the CNT/CCD from Clcnk2 / n Figure 2. Channel current recordings in DCT and TAL patches mice ( =10) (Figure 2, Table 1). 2 2 +/+ 2/2 from Clcnk2+/+ and Clcnk2 / mice in the cell-attached config- In the cTAL of Clcnk2 and Clcnk2 mice (Figure 2B), we uration. (A) Distal convoluted tubule. Representative current re- consistently recorded channels of 7.760.6 pS (12 out of 14 cell- cordings from membrane patches formed on either a Clcnk2+/+ attached patches) from Clcnk2+/+ TALs but not in any of the 13 2 2 2 2 (WT) or Clcnk2 / DCT bathed with physiologic saline solution cell-attached patches from Clcnk2 / cTALs (see Table 1). (high-K+ solution in the pipette). The dotted line labeled with C In conclusion, the approximately 10-pS Cl- channel that we denotes the closed current level. At 276 mV holding potential, recorded in the DCT, TAL, and the intercalated cells of the + the dominating current is due to K channel activity (high am- CNT/CCD is ClC-K2, because this channel is totally absent plitudes of about 5 pA). At +84 mV holding potential, the activity Clcnk22/2 + from the renal tubules of mice. Unexpectedly, we of the K channel is no longer detected because this potential is 2/2 were unable to record pseudo-CFTR channels in Clcnk2 very close to the reversal potential for K+ currents. It is then possible to record accurately Cl- channels which show consider- TALs. This might be due to low occurrence under our exper- ably lower current amplitudes (about 0.7 pA at this voltage). K+ imental conditions. It could also indicate that this channel is channels and Cl- channels could be recorded in the same patches not spontaneously active in cell-attached patches. Because from Clcnk2+/+ DCTs (upper panel, left) whereas no Cl- channel 10-pS chloride channels having ClC-K-like anion selectivity 2 2 activity was recorded from Clcnk2 / DCTs, even though K+ sequence were not reported before, we performed anion sub- channel activity was present (lower panel, left). The arrows in- stitution experiments in the inside-out configuration to check dicate brief half-openings or closings of Cl- channel activity. that the two channels (ClC-K2 and pseudo-CFTR) were pre- Mean single-channel current (i)/voltage (Vc) relationship obtained sent in the cTAL (Supplemental Table 1). under the same condition are shown on the right. Each point is Paulais et al.22 described an approximately 40-pS Cl- chan- – the average of 3 9 determinations from nine patches. SEM is nel showing Cl- . Br- . NO - anion selectivity sequence in shown as error bars when larger than symbols. The K+ channel 3 the cTAL. This channel is most certainly formed by ClC-K1 conductances were similar for both genotypes (gK =38.8+/2 1.4 2 2 pS for Clcnk2+/+ versus 39.5 +/2 1.7 pS for Clcnk2 / ) as was the because a channel with similar properties was detected when +/+ mouse ClC-K1 was expressed in HEK293 cells.28 Because we number of channels per patch (NK =5.8+/2 0.7 for Clcnk2 2 2 versus 4.0 +/2 1.1 for Clcnk2 / ). (B) TAL. Left: Representative did not detect ClC-K1-like single channel currents in DCT 2/2 current recording from a membrane patch formed on Clcnk2+/+ patches from Clcnk2 mice and ClC-K1 was detected at 2 2 and Clcnk2 / TAL fragments bathed with physiologic saline solution (NMDGCl solution in the pipette). The dotted line la- beled with C denotes the closed current level. Holding potential: activity was recorded having a conductance of 7–10 pS. Right hand 86 mV. Clcnk2+/+: the two traces show the activity of 10-pS Cl- side: mean single-channel current (i)/voltage (Vc) relationship for channel under resting conditions (label 1) and when maximal the 10-pS Cl- channel recorded on Clcnk2+/+ TAL fragments. Each inhibition has been reached (label 2) after superfusing Na-acetate point is the average of 3–12 determinations from 14 patches. SEM 2 2 to acidify the intracellular compartment. Clcnk2 / : no channel is shown as error bars when larger than symbols.

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Clcnk2 Disruption Completely Abolishes Furosemide- Sensitive and Thiazide-Sensitive NaCl Transport Our preceding experiments support the hypothesis that ClC- K2 is the main basolateral chloride channel all along the distal nephron. We next assessed the impact of Clcnk2 disruption on chloride transport in the distal nephron in vivo.Wefirst tested whether ClC-K2 is the principal basolateral chloride channel in the TAL by assessing the effects of Clcnk2 disruption on the natriuretic response to furosemide, a diuretic that blocks NKCC2 and thereby abolishes NaCl absorption by the TAL. Injection of furosemide (Furo, 2 mg/kg body wt) intraperitoneally elicited a marked increase in urinary sodium excretion in Clcnk2+/+ 2 2 mice whereas the response was completely abolished in Clcnk2 / 2 2 mice (Figure 3A). Furthermore, TALs from Clcnk2 / mice appeared dilated with flattened epithelial cells when stained with NKCC2 (Figure 3B) and phosphorylated NKCC2 (pNKCC2, Figure 3C). Western blot analysis revealed highly variable NKCC2 and pNKCC2 abundance between individual mice and no significant differences between Clcnk2+/+ and 2 2 Clcnk2 / mice (Figure 3D). Clcnk2 disruption is expected to blunt the response to thiazides (HCTZ, 50 mg/kg body wt) by blocking NCC- dependentabsorptionintheDCTandbyblockingPds/ Ndcbe-mediated NaCl absorption by b-intercalated cells in the CNT and the CCD.29,30 Accordingly, the thiazide sensi- 2/2 tivity was markedly decreased in Clcnk2 mice (Figure 3E). 2 2 Figure 3. NKCC2 and NCC activities and localization in Clcnk2 / Similar to our observations in the TAL, the DCT also showed fl mice. Both furosemide (Furo, A) and hydrochlorothiazide (HCTZ, E) an abnormal morphology with marked attening of the cells, elicit significant natriuresis in Clcnk2+/+ mice 3 hours after in- tubule dilatation and reduced expression of the apical NaCl- traperitoneal injection. Furosemide induced natriuresis is abolished cotransporter NCC (Figure 3F). In contrast to NKCC2 and 2 2 in Clcnk2 / mice and HCTZ natriuretic effects strongly blunted. In pNKCC2, NCC and phosphorylated NCC (p-NCC) staining 2 2 the renal cortex of Clcnk2+/+ and Clcnk2 / mice, both NKCC2 (B) was clearly decreased (Figure 3, F–G). The reduced NCC ex- 2/2 and NCC (F) localize to the apical membrane of the TAL and the pression in Clcnk2 mice was confirmed by Western blot 2/2 DCT. In Clcnk2 mice, phospho-NKCC2 signals are reduced in analysis (Figure 3H). Similarly, we observed an almost com- ∞ some TAL ( in C panel) whereas signals in some TAL are normal plete absence of parvalbumin staining in kidney sections from (star in C panel) as compared with Clcnk2+/+ mice. NCC signals are also reduced. Decreased signal intensity is also observed for 2 2 phospho-NCC (G). The DCT in Clcnk2 / mice has abnormal flat- tened epithelium and dilatation of the lumen. Western blot for NKCC and pNKCC2 (D) and NCC and pNCC (H) on whole kidney 2 2 lysates from Clcnk2+/+ and Clcnk2 / mice. Statistical significance was analyzed by unpaired t test (D, H) or ANOVA with repeated measures (A, E). The effect of the pharmacological treatment rea- ches significant levels for both HCTZ and furosemide. Bonferroni multiple comparisons are presented in the figure and compare the 2 2 effect of the treatment for Clcnk2+/+ and Clcnk2 / mice *P,0.05; 2 2 ****P,0.001. Furo (Clcnk2+/+, n=9; Clcnk2 / , n=5); HCTZ 2 2 (Clcnk2+/+, n=10; Clcnk2 / , n=8). In panels B, C, F, and G, scale bar = 25 mm.

2 2 the same low frequency in Clcnk2+/+ and Clcnk2 / interca- 2 2 lated cells (20% for Clcnk2+/+ versus 10% for Clcnk2 / ) and 2 2 Clcnk2+/+ Clcnk2 / 2 2 TALs (14% for versus 8% for ), our results Figure 4. Blood parameters in Clcnk2+/+ and Clcnk2 / mice. indicate that there is no compensation for ClC-K2 by ClC-K1 Mice were fed a normal salt diet (0.3% Na+). Values are mean6 (as represented by the 40-pS channel) in the TAL, DCT, and SEM. Statistical significance was analyzed by unpaired t test. 2 2 CNT/CCD of ClC-K2 KO mice (Supplemental Table 2). **P,0.01; ***P,0.001; ****P,0.001 (Clcnk2+/+, n=7; Clcnk2 / , n=8).

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related patients usually present with a rela- tively mild phenotype. From previous work, it was proposed that ClC-K1 can to some extent compen- sate for the loss of ClC-K2 activity.8,9,22 Because ClC-K1 and ClC-K2 are thought to both be present in the TAL32,33 whereas only ClC-K2 is present in the DCT,21,32 this would explain why some patients with CLCNKB mutations rather present with a phenotype resembling Gitelman syndrome (characterized by impaired thiazide-sensitive absorption of NaCl by DCT cells) although others show Bartter syndrome (characterized by impaired 2 2 Figure 5. Urine measurements in Clcnk2+/+ and Clcnk2 / mice. Mice were fed a furosemide-sensitive absorption of NaCl normal salt (0.3% Na+) diet. Excretions of solutes were normalized to urine creatinine by TAL cells). Our patch-clamp analysis concentration. Values are mean6SEM. Statistical significance was analyzed by un- on isolated tubules suggests that ClC-K2 – paired t test. **P,0.01; ***P,0.001. corresponds with the 10-pS Cl channel previously described by us21,34 and medi- 2/2 Clcnk2 mice, indicating the atrophy and disappearance of ates most of the basolateral chloride conductance in the TAL the initial portion of the DCTas has already been described for and intercalated cells of the CNT/CCD. NCC KO mice31 (Supplemental Figure 6). In line with previous studies21,34 we found only one dom- inating 10-pS conductance in WT patches although we no- Disruption of Clcnk2 in the Mouse Causes a Severe ticed some brief half-level openings and closures. This may Hyperprostaglandin E Syndrome appear surprising because ClC-K channels are dimers with 2 2 Consistent with the biologic features of severe BS, Clcnk2 / two independently-gated pores controlled by a common mice exhibited a very strong metabolic alkalosis with partial gate. We never observed the characteristic “double-barreled” respiratory compensation as indicated by a high blood pH and activity as reported for ClC-0.35 In fact, ClC-K channels do - increased [HCO3 ]withhighpCO2 (Figure 4, Supplemental not have the glutamate amino acid in position 166 that is - 36 Table 3). The increase in plasma [HCO3 ] was accompanied responsible for protopore gating, suggesting that ClC-K gating by a decrease in plasma [Cl-]. Mutant mice also exhibited relies mostly on the common slow gate. For this reason, we hypokalemia of (3.3460.27 mM) compared with Clcnk2+/+ hypothesize that the 10-pS conductance represents the dimer mice (4.6260.18 mM). Mutant mice suffered from intense conductance, which could be proved by mutation of glutamate polyuria with low urine osmolality, but did not display 166 to create de novo protopore gating, as shown for rat37 and hypercalciuria as observed in some patients, whereas urinary mouse28 ClC-K1. Unfortunately, there has been no successful magnesium excretion was increased (Figure 5). recording of single-channel ClC-K2 currents until now.38 It is Because of the very severe phenotype we tested whether unlikely that the 10-pS conductance is mediated by ClC-K1/ClC- 2 2 Clcnk2 / mice have elevated PGE2 levels in the urine. Indeed, K2 heterodimers, because ClC-K1 has a much larger conduc- PGE2 excretion in mutant mice was increased approximately tance and is very infrequent in the setting of these experiments. 2 2 eight-fold compared with controls (Figure 5). Clcnk2 / mice This issue, however, would require direct investigation using also exhibited a hypertrophy of the juxtaglomerular apparatus ClC-K concateners because there are reports of functional and proliferation of renin-secreting cells as described1 (Figure hetero-dimeric ClC channels.39,40 6, A and B). Renin expressing cells even extended to interlob- In agreement with previous results ClC-K2 was the only ular arteries (Figure 6C). Accordingly, renin transcript abun- chloride channel detectable at the basolateral surface of 2 2 dance was approximately 50-fold higher in Clcnk2 / than in DCT cells.31 This nicely fits with our in vivo data that the re- Clcnk2+/+ mice (Figure 6D). sponse to furosemide and to hydrochlorothiazide is blunted in 2 2 Clcnk2 / mice. Our results indicate that ClC-K2 ablation is not compensated by ClC-K1 in the TAL and that ClC-K2 is the DISCUSSION main pathway for basolateral chloride exit in the TAL, the DCT, and the CNT/CCD. Here, we report the generation and characterization of a mouse The less severe phenotype in patients may be explained by model with a targeted disruption of Clcnk2. KO mice display some residual ClC-Kb activity whereas our gene targeting severe renal salt loss, volume depletion, marked hypokalemia, strategy leads to a complete ablation of ClC-K2. Though .50 and metabolic alkalosis. This is surprising because ClC-Kb CLCNKB mutations have been found in patients suffering

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KO mice. As observed in mice with Gitelman 2 2 syndrome, Clcnk2 / mice show a marked atrophy of the DCT1. Thus, the effects of TAL dysfunction on calcium homeostasis, which promotes hypercalciuria as observed in type 1 and type 2 Bartter syndrome, may be compensated by DCT atrophy and dys- function promoting hypocalciuria as ob- served in Gitelman syndrome. Our data further suggest that CLC-K2 mediates basolateral chloride exit in b-intercalated cells. We recently detected thepresenceofelectroneutralthiazide- sensitive NaCl absorption in b-intercalated cells.48 In these cells apical uptake of NaCl is mediated by the parallel operation of pendrin (Slc26a4) and the Na+-driven chloride/bicarbonate exchanger Ndcbe 2/2 Figure 6. Renin is stimulated in Clcnk2 mice. Renin immunostaining on kidney (Slc4a8),48 which is energized by the baso- +/+ 2/2 2/2 sections from Clcnk2 and Clcnk2 mice (A). In Clcnk2 mice, the strong lateral V-ATPase.29 Basolateral sodium exit stimulation of renin is accompanied by a hypertrophy of the juxtaglomerular apparatus occurs possibly via AE4 (Slc4a9), which ac- and a larger number of renin positive cells (B). Some renin positive cells are even cording to this model mediates Na-HCO found into the interlobular arteries (C, ia). Renin was analyzed by 3 2 2 29 quantitative RT-PCR (D) and is strongly stimulated in Clcnk2 / mice. Values are cotransport and ClC-K2. fl mean6SEM. Statistical significance was analyzed by unpaired t test. ****P,0.001. B, We detected ClC-K2 by immuno uores- scale bar = 30 mm; C, scale bar = 60 mm. cence at the basolateral surface of intercalated cells, including a-intercalated cells as de- from Bartter syndrome,41 only 20 mutations have been char- scribed previously.49 In the latter, basolateral chloride exit is crit- acterized functionally because of the difficulty to obtain robust ical to recycle the chloride that enters through the basolateral and reliable expression of ClC-K2 in heterologous systems. We anion exchanger AE1. If this recycling is impaired, AE1 activity is - have recently reviewed in-depth the different aspects of this expected to be limited by the resulting rise in [Cl ]i. This will problem,41 and it is noteworthy that among the 20 mutations decrease AE1 mediated basolateral bicarbonate extrusion and characterized, only 40% did not show any detectable activity in turn will block apical proton secretion via the V-ATPase. In whereas the other 60% exhibited significant chloride currents agreement with this model, disruption of the basolateral KCl (. 50% of WT channel activity).41 cotransporter KCC4 resulted in distal tubular acidosis in Interestingly, deletion of ClC-K2 leads to a marked down- mice.49 Because disruption of ClC-K2 causes a very profound regulation of NCC. Because our experiments identify ClC-K2 metabolic alkalosis, ClC-K2 appears to be dispensable for renal as the main (if not the only) pathway for basolateral chloride acidification. exit from DCT cells, whereas NCC is the apical entry pathway, In conclusion, our study and the previous studies from this suggests the presence of a coordinated regulation of apical Kobayashi et al.18,32 and from Matsumura et al.18,50 demon- and basolateral transporters. How renal cells are able to strate that ClC-K1 and ClC-K2 are the two basolateral renal achieve this crosstalk between both membrane domains chloride channels with complementary but not redundant remains unknown. However, recent studies have highlighted roles in the kidney. ClC-K2 is expressed all along the distal the critical role of intracellular chloride in the control of a nephron where it controls chloride absorption by the TAL, the cascade of kinases, like for example the with-no-lysine kinase DCT and the intercalated cells of the CD, whereas ClC-K1 is (WNK)-Ste20p-related proline- and alanine-rich kinase critical in the thin limb of the loop of Henle where it is re- (SPAK) pathway that controls NaCl transport by epithelial quired for the normal renal concentrating mechanism.50 42,43 - cells. For instance, a decrease in [Cl ]i is detected by the chloride-sensing kinase WNKs,43–45 which in turn promotes CONCISE METHODS the activation through an increase in the phosphorylation of the NaCl cotransporter NCC by SPAK.42,46,47 Because ClC-K2 Generation of Clcnk2 Deficient Mice and Balance provides the main chloride conductance in DCT cells it is Studies 2 2 likely that ClC-K2 disruption will lead to significant increase The generation of Clcnk2 deficient mice (Clcnk2 / ) is described in - in resting [Cl ]i, which is expected to block WNK activation, detail in the Supplemental Material. and thereby, to decrease NCC phosphorylation and expression For urine collection, mice were housed individually in metabolic which may explain the lack of hypercalciuria in patients and our cages (Tecniplast; Buguggiate, Varese, Italy) for a 24-hour period.

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Retro-orbital blood was sampled from isofluorane-anesthetized mice. sous égide de la Fondation pour la Recherche Médicale. N.P. is also Urine and blood were analyzed as described in the Supplemental Material. funded by grant ANR 15-CE14-0024-02/ConTarKiD from ANR. +/+ 2 2 DiureticinducednatriuresiswasassessedinClcnk2 andClcnk2 / littermates housed in metabolic cages using furosemide (2 mg/kg body wt, Furo) and hydrochlorothiazide (50 mg/kg body wt, HCTZ). After DISCLOSURES injection, urine was collected for 3 hours and urine sodium output was None. measured.

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