KCNJ10 determines the expression of the apical Na-Clcotransporter (NCC) in the early distal convolutedtubule (DCT1)Chengbiao Zhanga,b,1, Lijun Wangb,1, Junhui Zhangc, Xiao-Tong Sub, Dao-Hong Linb, Ute I. Schollc, Gerhard Giebischd,2,Richard P. Liftonc, and Wen-Hui Wangb,2

aJiangsu Province Key Laboratory of Anesthesiology, Xuzhou Medical College, Xuzhou 221002, China; bDepartment of Pharmacology, New York MedicalCollege, Valhalla, NY 10595; and cDepartment of Genetics, Howard Hughes Medical Institute, and dDepartment of Cellular and Molecular Physiology,Yale University School of Medicine, New Haven, CT 06510

Contributed by Gerhard Giebisch, June 25, 2014 (sent for review June 3, 2014)

The renal phenotype induced by loss-of-function mutations of in-wardly rectifying potassium channel (Kir), Kcnj10 (Kir4.1), includes saltwasting, hypomagnesemia, metabolic alkalosis and hypokalemia.However, the mechanism by which Kir.4.1 mutations cause thetubulopathy is not completely understood. Here we demonstratethat Kcnj10 is a main contributor to the basolateral K conductance inthe early distal convoluted tubule (DCT1) and determines the ex-pression of the apical Na-Cl cotransporter (NCC) in the DCT. Immu-nostaining demonstrated Kcnj10 and Kcnj16 were expressed in thebasolateral membrane of DCT, and patch-clamp studies detected a40-pS K channel in the basolateral membrane of the DCT1 of p8/p10wild-type Kcnj10+/+ mice (WT). This 40-pS K channel is absent inhomozygous Kcnj10−/− (knockout) mice. The disruption of Kcnj10 al-most completely eliminated the basolateral K conductance and de-creased the negativity of the cell membrane potential in DCT1.Moreover, the lack of Kcnj10 decreased the basolateral Cl conduc-tance, inhibited the expression of Ste20-related proline–alanine-richkinase and diminished the apical NCC expression in DCT. We concludethat Kcnj10 plays a dominant role in determining the basolateral Kconductance and membrane potential of DCT1 and that the basolat-eral K channel activity in the DCT determines the apical NCC expres-sion possibly through a Ste20-related proline–alanine-rich kinase-dependent mechanism.

SeSAME/EAST syndrome | Kir.5.1 | SPAK | WNK

The distal convoluted tubule (DCT) of the kidney is re-sponsible for the absorption of 5% of the filtered NaCl load

and plays an important role in the reabsorption of Ca2+ and Mg2+

(1–3). The DCT has been functionally divided into early DCT(DCT1) and late DCT (DCT2). Whereas Na-Cl cotransporter(NCC) expression was detected throughout DCT, epithelial Nachannel and renal outer medullary K channel have been shownto be expressed in DCT2 (4−6). In DCT1, Na enters the cellthrough the apical Na-Cl cotransporter (NCC) and leaves thebasolateral membrane via the Na-K-ATPase while Cl leaves thecells through basolateral Cl channels or the KCl cotransporter(4, 7, 8). The K channels in the basolateral membrane participatein generating the cell membrane potential and are also re-sponsible for K recycling, which is important for sustaining theNa-K-ATPase activity (9, 10). It is well established that the mainbasolateral K channel in the DCT is a 40-pS inwardly rectifyingK channel which is a heterotetramer composed of Kcnj10 andKcnj16 (11−15). In humans, loss-of-function mutations in theKCNJ10 gene have been shown to cause epilepsy, ataxia, sen-sorineural deafness and tubulopathy (SeSAME/EAST) syn-drome (16–18). The renal manifestation of the disease isa reminiscence of Gitelman’s syndrome characterized by saltwasting, hypomagnesemia, metabolic alkalosis, and hypokalemia.This suggests that the membrane transport in the DCT is com-promised by KCNJ10 mutations. However, the molecularmechanism by which KCNJ10 channel activity modulates the

membrane transport in the DCT is not completely understood.Studies performed in Kcnj16 knockout mice demonstrated thatthe disruption of Kcnj16 did not recapitulate the phenotypes ofSeSAME/EAST syndrome except for hypokalemia (19). More-over, the basolateral K conductance of the DCT was increased inKcnj16 knockout mice, indicating that Kir4.1 could compensatefor the loss of Kir5.1 and sustains the transport in the DCT.Thus, it is essential to conduct the study in Kcnj10 knockout miceto understand the role of Kcnj10 in regulating the membranetransport in DCT. However, as described previously (20, 21), thegrowth of Kcnj10−/− mice was significantly retarded at 7 d (p7)after birth, and the body weight of the knockout mice was sig-nificantly lower than that of heterozygous (het) and WT litter-mates (Fig. S1). Moreover, homozygous Kcnj10−/− mice were notable to survive more than 2 wk after birth. Thus, the present studywas carried out in p7/p10 neonatal littermates. The first aim of thepresent study is to examine whether Kir4.1 is essential for thebasolateral K conductance in DCT1 and for generating the cellmembrane potential. The second aim of the study is to examinewhether the disruption of basolateral K channel activity impairsthe activity of the apical NCC and the basolateral Cl channel inthe DCT.

Significance

Loss of function mutations in the potassium channel KCNJ10causes a salt-wasting syndrome. The phenotype resemblesGitelman syndrome, which results from loss of sodium chloridetransport along the distal convoluted tubule (DCT), but themechanisms involved are not clear. Here, we perform experi-ments in the kidney from Kcnj10 knockout mice and demon-strate that Kcnj10 is a main contributor to the basolateralpotassium conductance in the DCT and that the potassiumchannel activity regulates the expression of ste20-related pro-line–alanine-rich kinase (SPAK) and determines Na-Cl cotrans-porter (NCC) expression. Our study suggests the possibility thatthe regulation of basolateral Kcnj10 activity in the DCT may bethe first step in response to a variety of physiological stimuli forinitiating SPAK-WNK-dependent modulation of NCC expressionin the kidney.

Author contributions: C.Z., L.W., G.G., R.P.L., andW.-H.W. designed research; C.Z., L.W., J.Z.,X.-T.S., D.-H.L., U.I.S., and W.-H.W. performed research; J.Z. contributed new reagents/analytic tools; C.Z., L.W., J.Z., X.-T.S., D.-H.L., U.I.S., G.G., R.P.L., and W.-H.W. analyzed data;and W.-H.W. wrote the paper.

The authors declare no conflict of interest.1C.Z. and L.W. contributed equally to this work.2To whom correspondence may be addressed. Email: wenhui_wang@nymc.edu orgerhard.giebisch@yale.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1411705111/-/DCSupplemental.

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ResultsWe first used the fluorescence microscope to examine the ex-pression of Kcnj10 in the kidney of 4-wk-old and p8 WT mouse.We confirmed the previous report that Kcnj10 is expressed in thebasolateral membrane of DCT, connecting tubule (CNT), andinitial cortical collecting duct (CCD) of both adult and p8 mousekidneys (22). Because the present study is focused on the DCT,we only presented the results regarding the expression of Kcnj10in DCT. Fig. S1 is immunostaining images demonstrating thatKcnj10 is highly expressed in the cortex region of WT mice but isabsent in the knockout mice. Fig. 1A is an image showing Kcnj10immunostaining in the kidney of a 4-wk-old WT mouse whileFig. 1B shows immunostaining of parvalbumin, a Ca2+ bindingprotein that is exclusively expressed in the DCT (23). Fig. 1 Cand D shows merged images of Kcnj10 and parvalbumin at a lowand high magnification, respectively. It is apparent that Kcnj10staining is extensively located in the basolateral membrane of theDCT. Kcnj10 has been shown to interact with Kcnj16 to forma 40-pS inwardly rectifying K channel in the basolateral mem-brane of mouse DCT1 (11, 19). Thus, we used the patch-clamptechnique to examine whether this 40-pS K channel was alsopresent in the basolateral membrane of DCT1 of p7/p10 mice.Fig. S2 shows the kidney size and isolated DCT from both WTand Kcnj10 knockout mice. It is apparent that the isolated DCTsin the Kcnj10−/− mice had a similar length and appearance tothose of WT mice. Fig. 2A is a typical channel recording showingthe 40-pS K channel activity in the basolateral membrane ofDCT1 of a p8 WT mouse. We detected the 40-pS K channels in23 patches from all 32 experiments and the mean channel openprobability was 0.5 ± 0.05 (n = 10). Thus, the probability offinding the 40-pS K channel and the channel open probability inDCT1 of p7/p10 WT mice was similar to that in adult mice (15).However, the probability of finding the 40-pS K channel de-creased in Kcnj10+/− mice (9 patches from all 22 patches) andwas zero from all 32 experiments in homozygous Kcnj10−/− mice.Moreover, we failed to detect any K channel activity in these 32

patches in the basolateral membrane of DCT1 of the knockoutmice. Thus, the results strongly suggest that Kcnj10 is absolutelyrequired for forming the 40-pS K channel in the DCT1 and thatthis 40-pS K channel should play a dominant role in generatingthe basolateral K conductance in the DCT1.The notion that Kcnj10 is an indispensable component for the

basolateral K conductance is further confirmed by the experi-ments in which we used the perforated whole-cell recording tomeasure the K currents in the DCT1. Two unique features ofDCT1 cell make it possible to assess the basolateral K conduc-tance by measuring the whole-cell K currents: (i) there is noapical K channel in the DCT1 (15) and (ii) the DCT1 cells lackthe cell coupling as evidenced by the fact that carboxyfluresceindye injected into the clamped cells did not spread into neigh-boring cells (Fig. S3). Fig. 2B is a typical recording demon-strating the whole-cell currents of DCT1 of WT mice withsymmetrical 140 mM KCl in the bath and in the pipette. Fromthe inspection of Fig. 2B, it is apparent that adding 1 mM Ba2+

and 10 μΜ 5-Nitro-2-(3-phenylpropylamino) benzoic acid (NPPB)almost completely abolished both inward and outward currents.This suggests that the Ba2+-sensitive K currents and NPPB-sensitive Cl currents are the main membrane conductance inDCT1. The mean K and Cl currents of DCT1 at −60 mVwere −2650 ±190 pA/cell (n = 10) and −1670 ± 140pA/cell (n =10), respectively. However, the Ba2+-sensitive K currents de-creased in the DCT1 cells of heterozygous Kcnj10+/− and werelargely abolished in homozygous Kcnj10−/− mice. Fig. 3A is awhole-cell recording showing that K currents in Kcnj10+/− micewere reduced to −1500 ± 90 pA (n = 11) whereas they were only−280 ± 60 pA/per cell at −60 mV (n = 11) in Kcnj10−/− mice. Incontrast, K currents in their corresponding WT littermates were2850 ± 180 pA per cell (n = 5). This finding strongly indicates thatKcnj10 plays a key role in determining the basolateral K con-ductance in the DCT1.Because the basolateral K conductance participates in gener-

ating the cell membrane potential, a decrease in the K conduc-tance induced by the disruption of Kcnj10 is expected to reduce

Fig. 1. Kcnj10 is expressed in the basolateral membrane of the DCT. Theexperiments were performed in the kidney slice obtained from a 4-wk-oldWT mouse. (A) Kcnj10 immunostaining in the mouse kidney with lowmagnification. (B) Parvalbumin immunostaining in the mouse kidney withlow magnification. Fluorescence microscope image showing the mergedstaining of Kcnj10 (red) and parvalbumin (green) with the low magnification(C) and a high magnification (D).

Fig. 2. Kcnj10 forms a 40-pS K channel in the basolateral membrane ofDCT1. (A) A patch-clamp recording shows the 40-pS K channel activity in thebasolateral membrane of DCT1. The experiments were performed in a cell-attached patch of DCT1 in the p8 WT mice. The DCT was bathed in a 140 mMNaCl+5 mM KCl solution, and the pipette solution contains 140 mM KCl. Theholding potentials are indicated on the top of each trace, and the channelclosed levels are labeled by “C.” (B) (Upper) A whole-cell recording showingthe Ba2+-sensitive K currents and NPPB-sensitive Cl currents in a DCT1 cell ofp8 WT mouse. Ba2+ (1 mM) and NPPB (10 μΜ) were directly added to thebath. (Lower) The bar graph summarizes the results from 10 measurementsat −60 mV on DCT1 of p7/p10 mice. The K and Cl currents were measuredwith the perforated whole-cell recording with 140 mM KCl in the pipetteand in the bath.

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the negativity of DCT1 membrane potential. This possibility wasexamined by measuring the K reversal potential (an index of thecell membrane potential) with 140 mM Na/5 mM K in the bathand 140 mM K in the pipette. Fig. 3B is a typical recordingshowing the currents measured in the DCT1 cells, which wereclamped from −100 mV to 100 mV with a ramp protocol. The Kreversal potential was −65 ± 2 mV (n = 8) in the DCT1 of WTmice but it depolarized to −39 ± 3 mV (n = 8) in the homozy-gous Kcnj10−/− mice. The K reversal potential in DCT1 ofKcnj10+/− mice was also lower (−57 ± 3 mV, n = 4) than that inWT. Thus, the results strongly indicate that Kcnj10 plays a keyrole in generating the negative membrane potential in DCT1.Also, our finding suggests that Kcnj16 alone was not able to forma functional K channel in the basolateral membrane in theDCT1. This notion was further suggested by immunocytochem-ical experiments in which the Kcnj16 immunostaining was ex-amined with fluorescence microscope in the WT and Kcnj10−/−

mice, respectively. The positive Kcnj16 staining was detected inthe DCT, CNT, and CCD of both WT and knockout mice. Fig.4A is an immunostaining image showing that Kcnj16 is expressedin the parvalbumin-positive DCT. Moreover, Kcnj16 immunos-taining is highly located in the basolateral membrane of the DCTin WT mice (Fig. 4B). The disruption of Kcnj10 did not affect theoverall expression of Kcnj16 in parvalbumin-positive DCT andother distal nephron (Fig. 4C). However, it largely abolished themembrane staining of Kcnj16 not only in the DCT but also in theparvalbumin-negative tubules, presumably in the CNT or CCD(Fig. 4D). Thus, our results further confirmed the previousfinding that homotetramer of Kcnj16 alone was not able to forma functional K channel and are also in consistence with the ob-servation that the basolateral K conductance was largely abol-ished in Kcnj10−/− mice despite of the presence of Kcnj16 (24).The basolateral K channel plays a key role in providing the

driving force for Cl exit across the basolateral membranethrough Cl channels. We speculate that the lack of the baso-lateral K channel may also inhibit the Cl channel. Thus, we usedthe whole-cell recording to examine NPPB-sensitive Cl currentsin WT and homozygous mice. Fig. 5A is a recording showingNPPB-sensitive Cl currents in WT and homozygous Kcnj10−/−

mice. It is apparent that Cl currents in the DCT1 of Kcnj10−/−

mice (−460 ± 100 pA per cell at −60 mV, n = 8) were signifi-cantly lower than those of WT mice (−1750 ± 180 pA per cell at−60 mV, n = 8). Thus, the lack of Kcnj10 decreased Cl channelactivity in DCT1. A decrease in the basolateral Cl conductanceand basolateral membrane potential is expected to increase theintracellular Cl concentration in the DCT. A high intracellular Clconcentration has been shown to inhibit with-no-lysine kinase(WNK) autophosphorylation and its kinase activity and it alsoimpairs WNK-ste20-related proline–alanine-rich kinase (SPAK)interaction (25, 26). Therefore, we next examined the possibilitythat the disruption of Kcnj10 may suppress the SPAK expression

Fig. 3. The disruption of Kir4.1 abolished the baso-lateral K conductance and caused a depolarization inDCT1. (A) (Left) A whole-cell recording showing Ba2+-sensitive K currents in DCT1 cells of p8/p10 WT, het-erozygous (het), and knockout (KO) mice. (Right) Abar graph summarizes the results measured at−60 mV. The K currents were measured with perfo-rated whole-cell recording with symmetrical 140 mMKCl in the bath and in the pipette. (B) (Left) A whole-cell recording showing K reversal potential in DCT1cells of p8/p10 WT and knockout mice. (Right) Themean value of K reversal potential from eightmeasurements is summarized in a bar graph. Formeasurement of K reversal potential, the bath so-lution contains 140 mM NaCl+ 5 mM KCl while thepipette solution has 140 mM KCl.

Fig. 4. The disruption of Kcnj10 affects the membrane expression ofKcnj16. Immunostaining of Kir5.1 (Kcnj16) (red) and parvalbumin (green) inp8 Kcnj10+/+ (A and B) and p8 Kcnj10−/− mice (C and D). A and B are mergedimages showing Kir5.1 and parvalbumin staining with low magnificationwhile C and D show the double staining with a high magnification. Arrowindicates the intracellular staining of Kcnj16 in the knockout mice.

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by examining the expression of SPAK in WT, heterozygous, andknockout mice. Fig. 5B is a Western blot showing that the ex-pression of the full-length SPAK was significantly decreased inboth Kcnj10+/− (52 ± 6% of the control value) and Kcnj10−/−

mice (26 ± 4% of the control value) in comparison with those inWT mice (n = 4). Thus, the inhibition of the basolateral Kconductance decreased full-length SPAK expression. Becausefull-length SPAK stimulates NCC activity through the phos-phorylation (27–29), it is conceivable that the disruption ofKcnj10-induced inhibition of SPAK should suppress the NCCactivity. Moreover, because Kcnj10 is the major type of Kchannel contributing to the basolateral K recycling, loss-of-function of Kcnj10 is expected to jeopardize K recycling acrossthe basolateral membrane that is important to sustain the Na-K-ATPase activity and the transepithelial transport (30). There-fore, we examined the NCC expression in the DCT usingimmunostaining and Western blot. Fig. 6 A and B showsimmunostaining images of NCC with a low magnification in WTmice and in homozygous Kcnj10−/− mice, respectively. It is ap-parent that the intensity of NCC immunostaining in Kcnj10−/−

mice was lower than those of WT mice. The low expressionof NCC in Kcnj10−/− mice was also confirmed by the Westernblot analysis, demonstrating that the total NCC expression inKcnj10−/− mice was only 28 ± 5% (n = 6) of the control value(Fig. 6 C and D). The notion that Kcnj10 expression affectedNCC expression was also suggested by examining the expressionof NCC in Kcnj10+/− mice. Western blot analysis demonstratedthat the total renal NCC expression in Kcnj10+/− mice was alsosignificantly reduced to 45 ± 8% (n = 4) of the control value butwas higher than those of Kcnj10−/− mice (26 ± 5% of the control)(Fig. 7A). Fluorescence images further demonstrated that NCCstaining was mainly localized in the apical membrane of WTmice (Fig. 7B) and also in Kcnj10+/− mice (Fig. 7C). However,not only was the total expression of NCC low in homozygousKcnj10−/− mice, but also the intensity of NCC immunostaining inthe apical membrane of DCT was decreased, and it was diffusedinto the intracellular space (Fig. 7 D and E). Thus, the results

strongly suggest that the basolateral K channel activity plays animportant role in the regulation of total NCC expression inthe DCT.

DiscussionThe first finding of the present study is that Kir.4.1 (Kcnj10) isa major contributor to the basolateral K conductance in theDCT1. Previous studies using single tubule RT-PCR, Westernblot analysis, and immunocytochemistry demonstrated thatKir4.1, Kir.5.1, Kir7.1, and KCNQ1, a voltage-gated K channel,were expressed in the DCT (31–33). Moreover, Kir7.1 proteinwas detected as early as in p7 mice, and it was also expressed inthe basolateral membrane of DCT (32). However, the observa-tion that the basolateral K conductance in the DCT1 was largelyabolished in Kcnj10−/− mice strongly indicates that Kir4.1 playsa dominant role in determining the basolateral K conductance.This conclusion was also supported by the finding that the dis-ruption of Kcnj10 expression shifted the K reversal potential toa less negative value. Thus, although K channels other thanKir4.1 and Kir.5.1 are expressed in the basolateral membrane ofDCT1, they play a minor role in determining the basolateralK conductance. However, because our experiments were per-formed in p7/p10 mice, we could not exclude the possibility thatK channels other than Kir.4.1 may increase their contributions tothe basolateral K conductance in DCT1 of the adult kidney.However, even if this occurs, it is unlikely that they would exceedthe Kcnj10 because no K channels other than the 40-pS Kchannel were detected in the DCT1 from adult mice in over 300patches (15).It is well established that Kir.4.1 and Kir.5.1 form a hetero-

tetramer 40-pS K channel in the DCT1 (11). This finding isconfirmed by our present observation that the 40-pS K channelwas completely absent in the basolateral membrane of DCT1 ofthe knockout mice. Although Kir5.1 (Kcnj16) was still expressedin the DCT and nephrons other than DCT in the knockout mice,no membrane staining in the basolateral membrane was visible inKcnj10−/− mice. Moreover, the K channel activity was completelyabsent in the basolateral membrane of DCT1 of knockout mice,suggesting that Kcnj16 was not able to form homotetramer aloneor heterotetramer with other inwardly rectifying K channel suchas Kir.7.1. In contrast, Kcnj10 was able to form a 20-pS

Fig. 5. The disruption of Kcnj10 decreased Cl conductance and SPAK ex-pression in DCT. (A) (Upper) A recording showing NPPB-sensitive Cl currentsin DCT1 of WT and knockout mice. The measurements were carried out withperforated whole-cell recording with symmetrical 140 mM KCl in the bathand pipette. (Lower) The results from eight experiments measured at -60 mVare summarized in a bar graph. (B) (Upper) A Western blot showing theexpression of full-length SPAK in the kidney from WT, heterozygous, andhomozygous Kcnj10−/− mice. (Lower) Results from four experiments aresummarized in a bar graph.

Fig. 6. The disruption of Kcnj10 inhibits NCC expression. Fluorescence mi-croscope image with a low magnification shows overall NCC immunostain-ing in (A) p8 Kcnj10+/+ and (B) p8 Kcnj10−/− mice. (C) A Western blot showingthe NCC expression in Kcnj10+/+ and Kcnj10−/− mice. (D) A bar graph sum-marizes results of experiments similar as those shown in C.

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homotetramer in the basolateral membrane of DCT in Kcnj16−/−

mice (19). Therefore, the present study suggests that Kcnj10 isessential for the basolateral K conductance and the cell mem-brane potential in the DCT1.The second finding is that the full-length SPAK expression

decreased in the kidneys of both heterozygous and knockoutmice. We speculate that the membrane potential or basolateralK conductance may play an important role in the regulation ofSPAK expression because the expression level of SPAK wasclosely correlated with the negativity of the membrane potentialin the DCT1 of WT, heterozygous, and knockout mice. However,it is not known whether the cell membrane potential directly orthe intracellular Cl concentration indirectly regulates SPAK ex-pression. The basolateral K channels participate in generatingthe membrane potential and K recycling across the basolateralmembranes (9). Because the membrane potential provides thedriving force for the Cl exit across the basolateral membranethrough Cl channels, a depolarization is expected to decrease Clexit thereby increasing the intracellular Cl concentration. Theobservation that the basolateral Cl conductance was significantlydecreased in Kcnj10−/− mice in comparison with those of WTmice supports the notion that the transcellular Cl transport inthe DCT1 was compromised after the disruption of Kcnj10.Thus, a depolarization of the cell membrane potential with di-minished Cl conductance is expected to cause an increase inintracellular Cl concentration in the DCT1, which has beenshown to inhibit WNK autophosphorylation and WNK-SPAKinteraction, thereby inhibiting the SPAK activity (25, 26).A large body of studies has demonstrated that WNK and

SPAK interactions play a key role in the regulation of NCCphosphorylation and activity (34–39). It has been reported thata decrease in NCC phosphorylation leads to enhancing theubiquitination and internalization of NCC (40). Thus, a decreasein the full-length SPAK expression in Kcnj10 knockout mice isexpected to decrease NCC phosphorylation, thereby decreasingNCC expression in the DCT. The role of Kcnj10 in determiningNCC expression was indicated by observations that the expres-sion of NCC in Kcnj10−/− and Kcnj10+/− mice was significantlydecreased in comparison with those of WT, although heterozy-gous mice had as normal growth and life span as those of WT.Relevant to our finding is the report that the lack of SPAK ac-tivity decreased NCC expression in SPAK knockout mice (41).Thus, the results suggest that the basolateral K channel activityor the cell membrane potential plays a role in the regulation ofNCC expression possibly by SPAK-dependent pathways. The

lack of SPAK has been reported to mimic the effect of the de-letion of NCC on the epithelial structure, significantly decreasingthe DCT volume (29, 42). Thus, it is conceivable that a decreasein DCT volume may be also involved in decreasing NCC ex-pression in Kcnj10 knockout mice.Fig. 8 is a scheme illustrating the mechanism by which the

disruption of Kcnj10 decreases NCC expression. We hypothesizethat the inhibition of the basolateral K conductance decreases Clexit across the basolateral membrane, thereby suppressing SPAKactivity in the DCT. A decrease in SPAK expression inhibits theNCC expression. Thus, our data suggest that the loss-of-functionmutation of Kcnj10-induced salt wasting is the result of decreasingNCC expression in the DCT, thereby increasing Na delivery to theconnecting tubule and cortical collecting duct. An enhanced Naabsorption in the distal nephron causes excessive K wasting,thereby causing hypokalemia and metabolic alkalosis. In addition,a depolarization in the DCT reduces the driving force for trans-cellular Mg2+ absorption in the DCT and leads to hypomagne-semia (43). We conclude that Kcnj10 plays a predominant role indetermining the basolateral K conductance in the DCT1 and thatthe disruption of Kcnj10 decreases the NCC expression.

MethodsAnimals. Kcnj10−/−, Kcnj10+/−, and Kcnj10+/+ mice were obtained throughmating Kcnj10+/− mice, which were kindly provided by Paulo Kofuji at Uni-versity of Minnesota (Minneapolis) to R. Lifton (20, 21). Primers used forgenotyping were the following: Kcnj10, forward 5′-TGG ACG ACC TTC ATTGAC ATG CAA TGG-3′ and reverse 5′-CTT TCA AGG GGC TGG TCT CAT CTACCA CAT -3′ (20). Although Kcnj10−/− mice were viable and had no obviousabnormality at birth in comparison with their WT littermates, they diedwithin 2 wk (21). Thus, we carried out the experiments using 7- to 10-d-oldhomozygous Kcnj10−/−, heterozygous Kcnj10+/−, and Kcnj10+/+ mice. Theanimal-using protocol was approved by independent Institutional AnimalCare and Use Committee at both Yale University and New YorkMedical College.

Preparation of DCT1. The experiments were performed on DCT1, a nephronsegment immediately adjacent to the glomerulus (Fig. S2B). The methodsfor dissecting DCT were described previously (15). Moreover, the appear-ance of DCT1 isolated from Kcnj10−/− mice was similar to those in WT mice(Fig. S2B). The isolated DCT was transferred onto a 5 mm × 5 mm coverglass coated with polylysine (Sigma) to immobilize the tubule, and thecover glass was placed in a chamber mounted on an inverted microscope(Nikon). The DCT was superfused with Hepes-buffered NaCl solution

Fig. 7. NCC expression is decreased in heterozygous and knockout mice. (A)(Upper) A Western blot showing the NCC expression in WT, heterozygous,and Kcnj10−/− mice. (Lower) Results from four experiments are summarizedin a bar graph. Double immunostaining images show NCC expression (red) inparvalbumin-positive (green) DCT cell of p8 WT (B), p8 heterozygous (C), andp8 homozygous Kcnj10−/− mice (D and E).

Fig. 8. A cell scheme illustrating the mechanism by which the basolateral Kchannel activity regulates the apical NCC expression in the DCT1. The solidand dotted lines, respectively, mean an enhanced or a diminished signaling.

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containing: 140 mM NaCl, 5 mM KCl, 1.8 mM MgCl2, 1.8 mM CaCl2, and 10mM Hepes (pH = 7.4) at room temperature for the single-channel patchesand for the measurement of K reversal potential.

Electrophysiology. The methods for the single-channel patch-clampexperiments, whole-cell recording, and measurement of K reversalpotentials were described previously and also discussed in the supplementalmaterial and ref. 15.

Immunostaining. Mice were anesthetized with ketamine (100 mg/kg) andXylazine (10mg/kg) and the abdomens cut open for perfusion of kidneys with2 mL PBS containing heparin (40 unit/mL) followed by 20 mL of 4% (vol/vol)paraformaldehyde. After perfusion, the kidneys were removed and sub-jected to postfixation with 4% (vol/vol) paraformaldehyde for 12 h. Thekidneys were dehydrated and cut into 8- to 10-μM slices with Leica1900cryostat (Leica). The tissue slices were dried at 42 °C for 1 h. The slides werewashed with 1× PBS for 15 min, and permeablized with 0.3% Triton dis-solved in 1× PBS buffer containing 1% BSA and 0.1% lysine (pH = 7.4) for15 min. Kidney slices were blocked with 2% (vol/vol) horse serum for 30 minat room temperature and then incubated with primary antibodies (Kir.4.1,parvalbumin, Kir5.1 and NCC) for 12 h at 4 °C. Slides were thoroughlywashed with 1× PBS and followed by addition of second antibodies mixturein 0.4% Triton dissolved in 1× PBS for 2 h at room temperature.

Preparation of Protein Samples and Western Blot. The tissue of renal cortexwas homogenized in an ice-cold solution containing 250 mM sucrose, 50 mMTris·HCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1% protease and phos-phatase inhibitor mixtures (Sigma) titrated to pH 7.6. After homogenization,the protein concentration was measured using the DC Protein Assay Kit (Bio-Rad). The proteins were separated by electrophoresis on 4–15% (vol/vol)SDS-polyacrylamide gels and transferred to nitrocellulose membrane. Themembranes were blocked with LI-COR blocking buffer (PBS). An Odysseyinfrared imaging system (LI-COR) was used to scan the membrane at awavelength of 680 nM or 800 nM.

Experimental Materials and Statistics. Kir5.1 and parvalbumin antibodies werepurchased from Santa Cruz. We obtained Kir4.1 antibody from Millipore.Antibodies for NCC and SPAKwere a gift fromRobert Hoover (EmoryUniversity,Atlanta, GA), who used this antibody in his previous publication (44), and fromJ. McCormick and D. Ellison (Oregon Health & Science University, Portland, OR).The 5-Nitro-2-(3-phenylpropylamino)benzoic Acid (NPPB) was purchased fromSigma. The data are presented as mean ± SEM. We used the paired Student ttest or one-way analysis of variance to determine the statistical significance.

ACKNOWLEDGMENTS. The authors thank Drs. Robert Hoover, David Ellison,and James McCormick for their generosity in providing NCC and SPAKantibodies. The work is supported by National Institutes of Health GrantRO1 DK54983.

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Zhang et al. PNAS | August 12, 2014 | vol. 111 | no. 32 | 11869

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