View: Evolving Concepts in Potassium Homeostasis and Hypokalemia

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Renal Tubular Ubiquitin-Protein Ligase NEDD4-2 Is Required for Renal Adaptation during Long-Term Potassium Depletion

† † ‡ † Lama Al-Qusairi,* Denis Basquin,* Ankita Roy, Renuga Devi Rajaram,* Marc P. Maillard,§ ‡ † Arohan R. Subramanya, and Olivier Staub*

*Department of Pharmacology and Toxicology, University of Lausanne, Lausanne, Switzerland; †National Centre of Competence in Research “Kidney.ch”, Zurich, Switzerland; ‡Department of Medicine, University of Pittsburgh School of Medicine and VA Pittsburgh Healthcare System, Pittsburgh, Pennsylvania; and §Service of Nephrology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland

ABSTRACT Adaptation of the organism to potassium (K+)deficiency requires precise coordination among organs involved in K+ homeostasis, including muscle, liver, and kidney. How the latter performs functional and molecular changes to ensure K+ retention is not well understood. Here, we investigatedtheroleofubiquitin-protein ligase NEDD4-2, which 2 negatively regulates the epithelial sodium channel (ENaC), Na+/Cl cotransporter (NCC), and with no-lysine-kinase 1 (WNK1). After dietary K+ restriction for 2 weeks, compared with control littermates, inducible renal tubular NEDD4-2 Pax8/LC1 knockout (Nedd4L ) mice exhibited severe hypokalemia and urinary K+ wasting. Notably, expression of the ROMK K+ channel did not change in the distal convoluted tubule and decreased slightly in the cortical/medullary collecting duct, whereas BK channel abundance increased in principal cells of the connecting tubule/collecting ducts. Pax8/LC1 However, K+ restriction also enhanced ENaC expression in Nedd4L mice, and treatment with the ENaC inhibitor, benzamil, reversed excessive K+ wasting. Moreover, K+ restriction increased WNK1 and WNK4 expression Pax8/LC1 and enhanced SPAK-mediated NCC phosphorylation in Nedd4L mice,withnochangeintotalNCC.We proposeamechanisminwhichNEDD4-2deficiency exacerbates hypokalemia during dietary K+ restriction primarily through direct upregulation of ENaC, whereas increased BK channel expression has a less significant role. These changes outweigh the compensatory antikaliuretic effects of diminished ROMK expression, increased NCC phosphorylation, and enhanced WNK pathway activity in the distal convoluted tubule. Thus, NEDD4-2 has a crucial role in K+ conservation through direct and indirect effects on ENaC, distal nephron K+ channels, and WNK signaling.

J Am Soc Nephrol 28: 2431–2442, 2017. doi: https://doi.org/10.1681/ASN.2016070732

Organisms handle potassium (K+)deﬁciency by acti- delivered to the CNT/CD, which promotes electrogenic vating mechanisms aimed at conserving K+ and estab- Na+ reabsorption via epithelial sodium channel (ENaC) lishing normokalemia.1,2 Hypokalemia may result from renal and extrarenal disorders and is a side effect Received July 8, 2016. Accepted February 1, 2017. of many drug therapies.3–5 The renal response to K+ deﬁciency occurs in the distal nephron, where K+ se- A.R.S. and O.S. contributed equally to this work. + cretion via principal cells is diminished and K reab- Published online ahead of print. Publication date available at sorption in intercalated cells is activated, allowing the www.jasn.org. + decrease of K excretion to near zero.1,2,6 Genetic, phys- Correspondence: Prof. Olivier Staub, Department of Pharmacology iologic, and pathophysiologic evidences indicate that and Toxicology, University of Lausanne, Rue de Bugnon 27, 1011 + Lausanne, Switzerland, or Prof. Arohan R. Subramanya, Department of net urinary K excretion results from an interplay be- Medicine, Renal-Electrolyte Division, University of Pittsburgh School of tween different ion transport processes in the ASDN. Medicine, S828A Scaife Hall, 3550 Terrace Street, Pittsburgh, PA 2 Indeed, the Na+/Cl cotransporter (NCC) in the distal 15261. E-mail: [email protected] or [email protected] + convoluted tubule (DCT) controls how much Na is Copyright © 2017 by the American Society of Nephrology

J Am Soc Nephrol 28: 2431–2442, 2017 ISSN : 1046-6673/2808-2431 2431 BASIC RESEARCH www.jasn.org and K+ excretion via ROMK and BK channels.7–9 The regulation mice were initially kept under high K+ diet (HKD) for 5 days, of these proteins involves both aldosterone-dependent and followed by another 5 days of low K+ diet (LKD). During this -independent mechanisms.8,10–14 Aldosterone-dependent K+ se- maneuver, no difference in body weight, food/water con- cretion predominates during hyperkalemia, when aldosterone sumption, and urine volume was observed between genotypes is elevated. This is supported by observations that deletion of (Figure 1, A–D). As expected, HKD increased and LKD de- aldosterone synthase, the mineralocorticoid receptor (MR), the creased plasma aldosterone levels in all mice (Figure 1E). HKD aldosterone-induced kinase SGK1, or aENaC all result in hyper- induced kaliuresis, which reached a maximum after 2 days kalemia.10,12,15–18 It is well accepted that under such hyperkalemic (Figure 1H). Upon switching to LKD, K+ excretion declined conditions, SGK1 is strongly expressed and phosphorylates the dramatically (Figure 1H), and Nedd4LPax8/LC1 mice tended to ubiquitin-protein ligase NEDD4-2, thereby interfering with preserve less K+ (Figure 1I). Plasma K+ and Na+ at 4 days of downregulation of ENaC and the WNK/SPAK/NCC path- LKD were similar in both genotypes (Figure 1, F and G). Sim- way.17,19–21 On the other hand, it is less well understood what ilar to recent observations by Walter et al.,23 urinary Na+ mechanisms are active during K+ restriction, when the body has excretion transiently increased under LKD in both groups to preserve K+. Under such low aldosterone conditions, the aldo- (Supplemental Figure 1A) but was recovered after 2 weeks sterone/MR/SGK1 regulatory axis is switched off. Nevertheless, (Figure 2D). This transient LKD-induced natriuresis is most NCC is stimulated because of the aldosterone-independent effect likely because of diminished aldosterone and reduction of of hypokalemia.11,22,23 One can reasonably assume that mecha- ENaC activity,33,34 and diminution of NKCC2 expression/ nisms that interfere with K+ secretion are important as well, and activity attributed to a reduction in filtered K+.35–38 These that such inhibitory mechanisms might involve NEDD4-2. observations suggest that the kidney is able to conserve K+ Although NEDD4-2 is best characterized as an ENaC in- independently of NEDD4-2 action in the short term. hibitor,24–32 itwasrecentlyshownthatitsroleismorecomplex. Pax8/LC1 Both in vitro,aswellasinNEDD4-2knockout(Nedd4LPax8/LC1) Nedd4L Mice Display a Defect in K+ Retention mice given a high Na+ diet (HSD), NEDD4-2 acts as a NCC in- under Prolonged K+ Deficiency hibitor,20,21 whereas it affects ENaC to a lesser degree. These effects As mutant mice showed a tendency to K+ loss, we extended were observed under HSD, a maneuver that suppresses endogenous the LKD treatment to 2 weeks, expecting total body K+ to be aldosterone production, thereby maximizing NEDD4-2 activity. depleted, inducing frank hypokalemia. Indeed, under these However, it is not known how regulation of these proteins by conditions both groups developed hypokalemia. However, NEDD4-2 is coordinated during K+ restriction, a qualitatively dif- in the Nedd4LPax8/LC1 mice, the hypokalemia was more se- ferent stress that also suppresses aldosterone. If the effect of NEDD4- vere (Figure 2A) and was accompanied by increased urinary 2 on NCC predominates under K+ restriction, then the increased K+ excretion (Figure 2C). Mutant mice exhibited slight hy- NCC activity seen with NEDD4-2 deletion would result in dimin- pernatremia and a tendency toward decreased Na+ excre- ished Na+ delivery to the CNT/CCD, minimizing the hypokalemic tion (Figure 2, B and D). Because both aldosterone and effect of K+ deficiency. In contrast, if NEDD4-2–dependent inhibi- renin expression were decreased in Nedd4LPax8/LC1 mice tion of ENaC outweighs its effect on NCC, NEDD4-2 deletion compared with controls (Figure 2, E and F), hypervolemia would be expected to increase voltage-dependent distal K+ excre- islikelytheprimarycausefortherelativediscrepancyin tion, exacerbating the hypokalemia. Our findings in nephron- aldosterone. We note the surprisingly high aldosterone specific Nedd4LPax8/LC1 mice20 clearly demonstrate that the latter is levels measured in control mice as already previously re- the case, particularly when dietary K+ is restricted over the long term. ported for standard diet (1350 pg/ml),20 which appears to Thus, taken together with prior observations, our data indicate that be inherent to the model used. No differences in body the primary regulatory target of NEDD4-2 action may change de- weight, food and water intake, urine volume (Supplemen- pending on the type of physiologic stimulus that suppresses aldoste- tal Table 1), or urine osmolality (Supplemental Figure 1B) rone secretion; under conditions of volume expansion caused by were observed. dietary Na+ loading, NEDD4-2 primarily regulates NCC,20 whereas These findings suggest that NEDD4-2 is regulated by al- under conditions where hypokalemia is induced by K+ restriction, terations in dietary K+. Toevaluate this further, we subjected NEDD4-2 primarily regulates ENaC. These observations reveal a control mice either to LKD for 2 weeks or HKD for 2 days previously unrecognized role for NEDD4-2 as an essential suppressor andanalyzedNEDD4-2expressionandphosphorylation of tubular ENaC activity when dietary K+ is scarce. levels in total kidney lysates. After 2 weeks of LKD, NEDD4-2 was less phosphorylated at S222 and S328 by ap- proximately 75% and 65%, respectively, compared with RESULTS HKD, with no significant change in total NEDD4-2 (Supple- mental Figure 2, A and B). However, immunofluorescence The Early Adaptation to K+ Deficiency Is Independent further showed that total NEDD4-2 is upregulated in the of Renal Tubular NEDD4-2 CNT/CD under LKD (Supplemental Figure 2, C and D). To evaluate how NEDD4-2 regulates renal tubular transport This effect was masked in Western blots, likely because of processes under varying K+ diets, control and Nedd4LPax8/LC1 expression of NEDD4-2 elsewhere in the kidney.39

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B). In contrast, immunoﬂuorescence staining indicated that ROMK membrane localization was less abundant in the CNT/CD by about 20% (Figure 3, C and E), whereas no difference was observed in the DCT (Figure 3, C and D). These data suggest that CNT/CD ROMK expression is altered in mutant mice; however, this alteration does not explain the observed K+ wasting.

Pax8/LC1 The Hypokalemia in Nedd4L Mice Is Caused by Increased ENaC Activity We assessed ENaC expression by Western blot and observed increased full-length (but not cleaved) aENaC, and full-length gENaC in mutant mice (Figure 4, A and B). The cleaved form of gENaC was not detectable, likely because ENaC was not sufficiently expressed under LKD conditions. Immunofluorescence analyses suggested a 30% increase in the intracellular staining of aENaC (Figure 4, C and E) and a tendency toward elevated gENaC (Figure 4, D and E). No obvious differences were observed for bENaC staining, consistent with the immunoblotting data (Sup- plemental Figure 3B). ENaC mRNAs were similar in both genotypes (Supplemental Figure 3A). To analyze ENaC activity, we treated mice with a single dose (1 mg/kg fi Figure 1. Metabolic parameters of Nedd4LPax8/LC1 mice challenged by HKD and LKD. body wt) of benzamil, a speci cENaCin- 40 (A–D) Body weight (A), food intake/body weight (B), water intake/body weight (C), and hibitor. Benzamil treatment increased urine volume (D). No significant difference was observed between control and mutant urine volume to the same extent in control mice in the measured parameters (eight controls and seven Nedd4LPax8/LC1 mice). and mutant mice (Supplemental Figure (E–G) Plasma aldosterone (E), plasma K+ (F), and plasma Na+ (G) levels after 2 days of 3C) but induced a significantly stronger na- HKD (ten controls and seven Nedd4LPax8/LC1 mice) and after 4 days of LKD (five triuresis (145%) in Nedd4LPax8/LC1 mice Pax8/LC1 controls and four Nedd4L mice). Note the sharp reduction in aldosterone (Figure 4F). Interestingly, intraperitoneal levels in response to LKD. No difference was observed between control and mutant + + + benzamil injection completely reversed ex- mice in plasma aldosterone, Na ,andK levels. (H and I) Urinary K excretion per day cessive K+ wasting in mutant mice (Figure under normal diet and during 5 days of HKD and 5 days of LKD; (I) is a magnification of 4G). In contrast, we did not detect an anti- the dotted box in (H), indicating K+ excretion under LKD starting from day 2 until day 5. Mutant mice seem to be less able to reduce their urinary K+ excretion than control kaliuretic effect of benzamil treatment in littermates. The difference was not statistically significant (eight controls and seven control mice, indicating that ENaC-mediated + + Nedd4LPax8/LC1 mice). HK, day of high K+ diet; LK, day of low K+ diet; ND, normal diet. Na reabsorption was not linked to K excretion under these hypokalemic conditions. Taken together, these data suggest that the exacerbated hypokalemia observed Decreased ROMK Membrane Localization in Nedd4L in Nedd4LPax8/LC1 versus control mice is caused by ENaC over- Pax8/LC1 Mice activation. Previously, Nedd4LPax8/LC1 mice challenged by HSD for 10 days exhibited enhanced abundance and apical localization The WNK/SPAK/NCC Pathway Is Enhanced in Pax8/LC1 of ROMK in the distal nephron.20 We therefore analyzed Nedd4L Mice ROMK expression and membrane abundance in control and Previous work showed that Nedd4LPax8/LC1 mice fed a HSD/ mutant mice after 2 weeks of LKD. Western blot analyses normal K+ diet exhibited an increase in WNK1 protein abun- showed no difference in ROMK expression (Figure 3, A and dance, resulting in enhanced SPAK-mediated NCC

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Because WNK1 and WNK4 are well known to regulate NCC via phosphorylation by Ste20- and SPS1-related proline alanine-rich kinase (SPAK), we investigated how LKD affects SPAK-mediated regulation of NCC in control and Nedd4LPax8/LC1 mice. Western blot analysis revealed increased phosphorylation of SPAK at S373 in mutant mice (Figure 6, A and C). SPAK phosphorylation at this site has previously been reported to be a signature of WNK-mediated activation.19,41 Consistent with this obser- vation, the SPAK T-loop activation site (T233) exhibited stronger phosphorylation in the DCT, with no apparent change in total SPAK expression or localization (Figure 6, D and E). This correlated with increased NCC phosphorylation, whereas total NCC expression and membrane localization were not different (Figure 6, B, C, E, and F). A single injection of thiazide Figure 2. Nedd4LPax8/LC1 mice develop a K+ wasting phenotype after 2 weeks of LKD. (A and B) Plasma K+ and Na+ in control (white) and Nedd4LPax8/LC1 (gray) mice resulted in similar natriuresis in both ge- after 2 weeks of LKD. The renal suppression of NEDD4-2 results in severe hypokalemia notypes (Figure 6G), whereas thiazide- and a slight increase in plasma [Na+](n= 12 controls and five Nedd4LPax8/LC1 mice). (C sensitive kaliuresis was greater in mutant and D) Analysis of 24-hour urinary K+ and Na+ excretion reveal elevated K+ excretion versus control mice (Figure 6H), indicat- in mutant mice and concomitant but not significant Na+ retention (six mice in each ing that NCC cannot be the cause of K+ group). (E and F) Plasma aldosterone (n=12 controls and five Nedd4LPax8/LC1 mice) wasting and hypokalemia observed in and renin expression (n= five control and five Nedd4LPax8/LC1 mice) are decreased in mutant mice. Altogether, our data suggest Pax8/LC1 Nedd4L mice as compared with controls. *P,0.05; **P,0.01. that the WNK/SPAK/NCC pathway is activated in mutant mice, functioning as a phosphorylation.19 We therefore wondered if NEDD4-2 compensatory mechanism that attenuates the excessive urinary deletion affects WNK1 regulation under hypokalemic condi- K+ wasting associated with NEDD4-2 deletion. tions. Accordingly, we analyzed WNK1 abundance and cellular localization using a C-terminal antibody specificfor BK Channels Are Increased in Principal Cells of the Pax8/LC1 NEDD4-2–sensitive isoforms19 andfoundincreasedWNK1 CNT/CD in Nedd4L Mice protein levels in mutant mice (Figure 5, A and B). Further- Because WNK1 is a positive regulator of BK channels,42,43 we more, immunofluorescence analyses showed that WNK1 also assessed BK expression by Western blot, and found that exhibited a differential segment-specificpatterninthedistal the channel-forming BKa and BKb1(butnotBKb4) subunits nephron. Specifically, WNK1 accumulated in intracellular were increased in total kidney extracts (Figure 7, A and B). puncta in the DCT (Figure 5C). In the DCT of Nedd4LPax8/LC1 Immunostaining showed that BKa subunits were detectable in mice, WNK1 puncta were larger and more intense than in the CNT/CD despite active hypokalemia, and their expression control littermates (Figure 5C). In addition, WNK1 protein was enhanced by about 30% in the CNT/CCD of Nedd4LPax8/LC1 expression was enhanced at the apical membrane of both cor- mice (Figure 7, C and D). Interestingly, the increased BKa abun- tical (Figure 5E) and medullary CNT/CD in Nedd4LPax8/LC1 dance in Nedd4LPax8/LC1 mice appeared to be restricted to prin- mice (Figure 5F). Interestingly, the enhanced WNK1 signal cipal cells, as demonstrated by AQP2 costaining (Figure 7C). in the CNT/CD was primarily localized in principal cells, as These findings indicate that under hypokalemic conditions, shown by AQP2 costaining (Figure 5, E and F). In addition, the K+ loss in Nedd4LPax8/LC1 mice correlates with enhanced total WNK4 expression was enhanced in mutant mice relative expression of BK channels in principal cells, suggesting that to controls during long-term K+ restriction (Figure 5, A and B), enhanced BK-mediated K+ secretion may contribute to the pro- and similarly to WNK1, WNK4 puncta were more prominent found hypokalemia observed in these mice. in the DCT of mutant mice (Figure 5D). These data indicate that the protein abundance of WNK1 and WNK4 is increased in Evaluation of Epithelial Hypertrophy in the CNT/CD of Pax8/LC1 Nedd4LPax8/LC1 versus control mice under severe hypokalemic Nedd4L Mice under LKD conditions, and that there is a difference in subcellular localiza- Rats fed with LKD for 2 weeks had been previously shown to tion in different nephron segments. exhibit distal nephron hypertrophy and diabetes insipidus,

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150 CNT/CD cells per genotype and detected a tendency toward epithelial hypertrophy in mutant versus control mice, although the difference was not statistically signiﬁcant (Supplemental Figure 4C). These observations suggest that distal tubular morphology and concentrating functions are not different under LKD between control and Nedd4LPax8/LC1 mice.

DISCUSSION

NEDD4-2 is a well appreciated master regulator of distal nephron Na+ reabsorption and BP,47–49 but its role in regulating these processes during changes in K+ balance is not well understood. Here, we show that Nedd4LPax8/LC1 mice exhibit severe urinary K+ wasting, exacerbating the hypokalemia caused by chronic dietary K+ deficiency. This severe hypokalemia was associated with increased ENaC expression and activity, increased BK channel abundance, enhanced NCC phosphorylation, and decreased ROMK apical localization in the CNT/CD. Thus, the hypokalemic phenotype observed in these mice under chronic LKD seems to be driven by increased ENaC and BK channel expression. Not surprisingly, our data show that HKD-inducedkaliuresisispreservedin Nedd4LPax8/LC1 mice. Under such conditions, NEDD4-2 activity is low because of inhibition by aldosterone and SGK1.17,50 Hence, the knockout of NEDD4-2 in K+ -loaded mice would have minimal effects on K+ balance relative to K+-loaded controls. This contrasts with the inactivation of stim- Figure 3. ROMK apical localization is decreased in the distal nephron of Nedd4LPax8/LC1 ulatory actors in the pathway, such as aldo- mice under LKD. (A) Western blot analysis of ROMK from control and Nedd4LPax8/LC1 sterone synthase, MR, SGK1, and aENaC, all mice after 2 weeks of LKD; the band at 50 kDa is nonspecific.17 (B) Protein quantifi- of which cause hyperkalemia after genetic cation of (A) showing similar expression levels of ROMK in both genotypes (seven deletion.10,12,15–18 Taking this into consider- fl mice in each group). (C) ROMK uorescence intensity in DCT and CNT/CCD segments ation, we investigated what would happen (from D and E) from control and Nedd4LPax8/LC1 mice. Shown are the ratios of cortical to Nedd4LPax8/LC1 mice when restricting K+ ROMK labeling over the surface area of NCC or AQP2-expressing cells, respectively. *P,0.05. (D and E) Costaining of ROMK (green) with NCC (red) (D) and AQP2 (red) (E) supply, a condition that should be associated from control and Nedd4LPax8/LC1 mice. ROMK level is significantly decreased in the with high NEDD4-2 activity. Would overac- CNT/CCD (C and E) segments of Nedd4LPax8/LC1 mice, the decrease of ROMK in the tivation of NCC (as shown in Ronzaud + DCT is not statistically significant (C and D) (four mice in each group). Fg, fully gly- et al.20) lead to a reduction of Na delivery cosylated; Ng, nonglycosylated ROMK; Pg, partially glycosylated. to downstream segments, and consequently balance the K+ excretion? Or would NEDD4-2 deletion lead to increased ENaC evidenced by decreased AQP2 expression.44–46 We therefore activity, enhanced K+ excretion, and hypokalemia, as is the case assessed AQP2 expression and found no difference between in Liddle syndrome51,52?Ourfindings suggest that the second genotypes (Supplemental Figure 4, A and B). Moreover, to scenario predominates, namely that NEDD4-2 suppression of evaluate distal hypertrophy, we quantified the size of at least ENaC is most active under K+-restricted conditions.

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interactions has largely been explored within the context of Liddle syndrome or signaling pathways that block NEDD4-2 activity. However, a rationale explaining why NEDD4-2 was selected during evolution to tonically inhibit ENaC activity when aldosterone levels are low has been lacking. This study suggests that the inhibition of ENaC by NEDD4-2 is crucial under conditions where dietary K+ is scarce, to limit excessive K+ wasting and prevent life-threatening hypokalemia. Our biochemical analyses show an upregulation of primarily intracellular aENaC. Accumulation of intracellular bENaC and gENaC was observed in the same model under dietary Na+ loading,20 and it was suggested by an accompanying editorial by Ellison that NEDD4-2 may not control directly the cell surface expression, but intracellular degradation.55 Our data support such a model. Additionally, they suggest that under chronic K+ depletion, NEDD4-2–dependent regulation of ENaC may act independently of factors that regulate its proteolytic cleavage. Intrigu- ingly, ENaC regulation seems to be diet- dependent, although both HSD and LKD result in reduced circulating aldosterone. When mice were kept under HSD, intracellular bENaC and gENaC were increased, but aENaC (including aENaC cleavage) was decreased because of low aldosterone,20 and no increase in amiloride sensitivity was observed. In contrast, LKD results in accumulation of intracellular aENaC, a slight increase in gENaC, and no alteration of bENaC, together with increased benzamil sensitivity. It is well known that ENaC has a high conductance, Figure 4. ENaC expression and activity are increased in Nedd4LPax8/LC1 mice after 2 and only a low number of channels per cell weeks of LKD. (A and B) Western blot analysis of ENaC in control and Nedd4LPax8/LC1 areexpectedtobeactiveattheplasma mice after 2 weeks of LKD and protein quantification showing a significant increase in membrane; consequently, the biochemical the full-length aENaC and gENaC subunits (seven mice in each group). (C and D) or histochemical detection of ENaC may Costaining of aENaC (C) and gENaC (D) (both in green) and AQP2 (in red) in control not be sensitive enough to detect subtle Pax8/LC1 and Nedd4L mice. (E) Mean of aENaC and gENaC fluorescence intensity in CNT/ changes at the membrane. This may explain CCD segments showing a significant increase in aENaC (but not gENaC) abundance in Pax8/LC1 that alterations related to ENaC activation Nedd4L mice compared with controls (four mice in each group). (F) Benzamil (cleavage of either aENaC or gENaC) are significantly increased Na+ excretion compared with vehicle (DMSO) in both genotypes, not observable under the LKD condi- and the loss of Na+ in the benzamil-treated Nedd4LPax8/LC1 mice is significantly higher 56,57 than that of control mice. (G) Benzamil treatment efficiently prevents the K+ wasting tions. Similar data regarding ENaC abun- observed in Nedd4LPax8/LC1 mice (seven mice in each group). *P,0.05; **P,0.01 dance have been reported in other germline models of Nedd4L inactivation.24–26 Numerous prior reports have shown that NEDD4-2 interacts Recently, it was shown that SGK1/NEDD4-2 regulates with and ubiquitylates ENaC, reducing channel cell surface expres- WNK1.19 Similarly, we found that WNK1 is increased in the sion.24–26,28,32,53,54 The physiologic relevance of ENaC/NEDD4-2 DCT and the CNT/CD of Nedd4LPax8/LC1 mice under LKD. In

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addition, we observed a net augmentation in WNK signaling, as both WNK1 and WNK4 were increased, and phosphorylation of the WNK effectors SPAK/OSR1 was enhanced. Moreover, both WNK1 and WNK4 exhibited a punctate pattern in the DCT, a finding that was previously reported in instances where WNK kinases are active.11,58 By contrast, in the CNT/ CD, WNK1 localized to the apical membrane, which was particularly evident in mutant mice. This segment-specificsub- cellular distribution may reflect a difference in WNK1 interactors or regulatory networks, and supports extensive litera- ture implicating WNK1 as a regulator of ion transport processes in the CNT/CD that influence K+ balance.59 Wealso show that NCCphosphorylation is enhanced in Nedd4LPax8/LC1. This correlates with plasma K+ and the observed increase in DCT WNK/SPAK pathway activity in mutant mice. Consistent with increased NCC activity in the mutant model, thiazide-mediated inhibition of NCC exacerbated urinary K+ losses. This suggests that the high NCC activity may, in part, be a compensatory response during active hypokalemia to limit distal Na+ delivery. In this regard, recent studies have suggested that NCC phosphorylation status is tightly linked to the concentration of extracellular K+.11,60 Specifically, hypokalemia has been shown to be a potent NCC activator. The effect is believed to be because of changes in the basolateral membrane voltage of the DCT, which becomes hyperpolarized during hypokalemia, resulting in enhanced chloride efflux and activation of WNK kinases.11 The experiments presented here did not dissociate the effect of NEDD4-2 from the role of hypokalemia in WNK regulation. However, it is notable that under high NaCl/normal K+ dietary conditions, Nedd4LPax8/LC1 mice exhibit increased WNK1 protein abundance relative to controls, despite no difference in plasma K+

Figure 5. WNK1 and WNK4 are upregulated in Nedd4LPax8/LC1-deficient mice. (A and are more prominent in the DCT of mutant B) Western blot analysis of WNK1 and WNK4 in control and Nedd4LPax8/LC1 mice after versus control mice. (E and F) Costaining of 2 weeks of LKD, and protein quantification showing a significant increase in the two WNK1 (green) and AQP2 (red) showing an WNK proteins in Nedd4LPax8/LC1 mice (* indicates a nonspecific band; seven controls increase in WNK1 apical localization in the and five Nedd4LPax8/LC1 mice). (C and D) Costaining of NCC (red) with WNK1 (C) and CNT/CCD (E) (arrows) and MCD (F) (arrows) of WNK4 (D) (both in green) in control and Nedd4LPax8/LC1 mice. WNK1 and WNK4 foci mutant mice. *P,0.05; *P,0.01

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reduced NEDD4-2–mediated WNK degradation and hypokalemia. In addition, our data revealed that Nedd4LPax8/LC1 mice exhibit an increase in BK protein, and a decrease in ROMK apical localization in the CNT/CD. Recently, Liu et al. and Webb et al. both found that WNK1 activates BK channels in vitro, through the regulation of protein expression and trafficking.42,43 Because WNK1 protein abundance was increased in the CNT and CD in Nedd4LPax/LC1 mice, it seems plausible that the increase in BK expression is WNK1-dependent. However, the increased expression of BK subunits did not appear to be the predominant cause of K+ wasting in Nedd4LPax8/LC1 mice, as the defective kaliuresis was fully corrected by benzamil administration, suggesting that the increased K+ losses are primarily ENaC-dependent in etiol- ogy. The decreased ROMK expression might be explained by enhanced WNK signaling because L-WNK1 and WNK4 are well known inhibitors of ROMK, as evidenced in various in vitro and in vivo systems.61–64 On the other hand, ROMK wasmoreabundantinthenormokalemic Nedd4LPax8/LC1 mice under HSD conditions,20 a condition also associated with enhanced WNK1/4 expression.19 Thus, the observed changes in ROMK cannot be fully explained by a regulatory effect of the WNK signaling pathway. Other molecular components of the ROMK regulatory network, or perhaps a WNK- Figure 6. NCC and SPAK phosphorylation is increased in Nedd4LPax8/LC1 mice after 2 independent effect of plasma [K+]itself, weeks of LKD. (A and B) Western blot analysis of SPAK (A) and NCC (B) phosphory- may predominate under the conditions Pax8/LC1 fi lation in control and Nedd4L mice. (C) Protein quanti cation from (A) and (B) studied here. fi showing a signi cant increase in S373 phosphorylated SPAK and NCC phosphorylation at In summary, this study demonstrates several residues including T53, T91, or the combination of T43, T53, and T58 (3P-NCC), with + fi fi Pax8/LC1 that K -restricted mice lacking renal tubu- no modi cation of total NCC (seven controls and ve to seven Nedd4L mice). (D) + Immunostaining of total SPAK in control and Nedd4LPax8/LC1 mice showing comparable level lar NEDD4-2 exhibit severe urinary K and localization of the protein in the cortex of both genotypes. (E) Costaining of NCC (green) wasting and hypokalemia, and describes and T233 phosphorylated SPAK (red) in control and Nedd4LPax8/LC1 mice.Anincreasein how NEDD4-2 orchestrates the regulation SPAK phosphorylation was observed in the DCT of mutant mice. (F) Immunostaining of total of its different targets in order to ensure (upper panel) and T53 phosphorylated NCC (lower panel) in control and Nedd4LPax8/LC1 mice adequate K+ handling (Figure 8). The pri- after 2 weeks of LKD. Increased NCC phosphorylation is observed in Nedd4LPax8/LC1 mice, mary defect caused by NEDD4-2 deletion with similar apical localization of the transporter in both genotypes. (G and H) Thiazide is the impaired downregulation of ENaC, + Pax8/LC1 treatment induced an equivalent increase in Na excretionincontrolandNedd4L which subsequently increases the driving + mice compared with vehicle (DMSO) (G), whereas K excretion was exacerbated in mutant force for K+ secretion. Our findings suggest , , mice by thiazide treatment (H) (seven mice in each group). *P 0.05; **P 0.01. that the tonic inhibition of ENaC by NEDD4-2 may be an evolutionarily con- levels.19,20 On the basis of these results, we propose that the served mechanism that allows the kidney to conserve K+ stimulatory effect of NEDD4-2 ablation on WNK activity and prevent life-threatening hypokalemia when access to under LKD conditions is because of a combined effect of dietary K+ is limited.

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separated according to the manufacturer’s instructions. Mice were then humanely eutha- nized by cervical dislocation. Experimental pro- tocols were designed with respect to the Swiss Animal Welfare Act and approved by the veter- inary administration of the Canton of Vaud, Switzerland.

Metabolic Cages, Diuretic Treatment, and Urine and Plasma Analyses After 2 days of adaptation in metabolic cages, data related to body weight and food and water intake were registered, and 24-hour urine samples were collected. Urine analysis (Na+,K+, creatinine, and urea) was performed by the Lab- oratory of Clinical Chemistry at the Lausanne Hospital (CHUV) using a Modular Analytics System (Roche Diagnostics). For diuretics treatment, control and mutant mice were intra- peritoneally injected with vehicle containing benzamil (1 mg/kg body wt) and thiazide (20 mg/kg body wt), and urine samples were collected 3 hours after injection. Diuretic treat- Figure 7. BK channels are upregulated in CNT/CCD principal cells of Nedd4LPax8/LC1 ment was applied during the light cycle (when mice. (A and B) Western blot analysis of BK in control and Nedd4LPax8/LC1 mice after 2 the aldosterone level was minimal) to avoid any weeks of LKD, and protein quantification revealing a significant increase in BKa and BKb1 expression (seven mice of each genotype). (C) Costaining of BKa (green) and overlap between aldosterone effect and the ef- + AQP2 (red) in control and Nedd4LPax8/LC1 mice. (D) Quantification of BKa fluores- fect induced by drug treatment. Plasma Na and + cence intensity in the CNT/CCD segments. BKa labeling was significantly increased in K levels were measured with a flame photom- the principal cells of mutant CNT/CCD segments. *P,0.05; **P,0.01. eter (Cole-Palmer Instrument). Plasma aldosterone measurements was performed at the CONCISE METHODS Service of Nephrology of the CHUV using the radioimmunoassay kit ALDO-RIACT, according to the manufacturer’sinstructions. Handling and Induction of Renal Tubule–Specific Pax8 LC1 Nedd4L / Mice Kidney Lysates Preparation and Western Blot Analyses fl fl Inducible renal tubule–specific Nedd4L ox/ ox/Pax8-rTA/LC1 knock- Frozen tissues were homogenized using buffer containing Tris/HCl out (Nedd4LPax8/LC1) mice were generated as described previously.20 50 mM at pH 7.5, EDTA 1mM, EGTA 1 mM, sucrose 0.27 M, NaF Mice were housed in a temperature-controlled room (19°C–22°C) 50 mM, and Na-pyrophosphate 5 mM in addition to protease inhib- with a 12:12-hour light/dark cycle. To induce the gene deletion, mice itors purchased from Roche (Complete, no. 11836145001). Protein aged 21–24 days were treated with doxycycline (2 mg/ml in 2% su- homogenates were then centrifuged at 10,0003g for 10 minutes at crose in drinking water) for 12 days. Genotype was identified by PCR 4°C, supernatants were collected, and protein concentration was performed on ear biopsies, Nedd4L gene deletion was analyzed by measured using the BradFord method (no. UPF86420; Uptima). PCR performed on genomic DNA preparation from kidneys. For ROMK detection, an additional ultracentrifugation step at 100,0003g for 1 hour was performed for membrane enrichment. Dietary Manipulation Proteins were separated by SDS-PAGE and transferred to nitrocellu- Control (Nedd4LPax8 or Nedd4LLC1)andmutant(Nedd4LPax8/LC1) lose membranes using a wet transfer apparatus (Bio-Rad). Mem- doxycycline-treated mice were fed a standard or control diet (0.3% K+; branes were blocked with 5% nonfat milk in TBS and 0.1% Tween 0.2% Na+; Ssniff Spezialitäten GmbH), LKD (,0.03% K+;0.2%Na+; 20 for 40 minutes and incubated with primary antibodies overnight at Ssniff Spezialitäten GmbH), or HKD (5% K+;0.2%Na+; Ssniff Spezia- 4°C. Secondary antibodies were applied for 2 hours at room temper- litäten GmbH) for the periods indicated in the related results. Mice were ature. A list of the antibodies used in the study is available in Sup- given free access to food and water during the period of experiments. plemental Table 2. Western blot data were quantified using ImageJ software. Plasma and Organs Collection Mice were anesthetized by ketamine/xylazine intraperitoneal injec- Immunofluorescence tion; blood was collected by exsanguination from the retro-orbital Mice were anesthetized by ketamine/xylazine intraperitoneal injec- plexus in SARSTEDT heparin-containing microtubes and plasma was tion. Cardiac perfusions of PBS followed by 4% PFA were performed

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Figure 8. Model of NEDD4-2 action in K+ conservation in the CNT/CCD. Under K+ restriction, the aldosterone/MR/SGK1 regulatory axis is switched off, resulting in maximal NEDD4-2 activity. In controls, the active NEDD4-2 primarily downregulates ENaC. WNK1 is also downregulated, resulting in limited BK channel activity, Moreover, hypokalemia causes WNK4 activation, and stimulation of SPAK and NCC to limit distal Na+ delivery to ENaC and voltage-dependent K+ excretion via ROMK. Collectively, these transport processes act synergistically to promote K+ conservation. The deregulation of this process in Nedd4LPax8/LC1 mice results in K+ wasting, primarily because of the release of ENaC from tonic NEDD4-2 inhibition. WNK1 is also increased, causing elevated BK expression. The severe hypokalemia caused by enhanced K+ wasting further stimulates WNK1/WNK4, SPAK and NCC, which, together with reduced ROMK expression, partially compensates for the K+ wasting induced by NEDD4-2 deletion. to fix the kidneys. Before freezing, fixed kidneys were kept in 30% the ImageJ user guide.66,67 The mean size of 150 cells from control sucrose in PBS solution at 4°C overnight. Immunostaining was per- and 200 cells from mutant mice was calculated. formed on 5 mm cryosections using primary antibodies applied overnight at 4°C. Secondary antibodies were applied for 2 hours at room RNA Extraction and Quantitative RT-PCR temperature. The antibodies and dilutions used are listed in Supple- Total RNA was purified from total kidney using TRIzol reagent mental Table 2. DNA was counterstained with 0.1 mg/ml DAPI. Sam- (Ambion) and precipitated by isopropyl alcohol. cDNA was synthe- ples were mounted in Dako immunofluorescence medium, and im- tized from 2 to 5 mg total RNA using Superscript II reverse transcrip- aged by fluorescence microscopy using either a Zeiss Axiovision tion (Invitrogen) and random hexamers. TaqMan Gene Expression (v4.8) or Zeiss Axioscan Z1 microscope. Entire kidney sections con- Assays (Applied Biosystems) were used to analyze gene expression. taining at least 200 segments of interest were quantified using ImageJ Primers/probes were Scann1a (aENaC, Mm00803386_m1; Applied software. Integrated density and corrected total cell fluorescence were Biosystems), Scann1b (bENaC; Mm00441215_m1), Scann1c assessed for all analyzed signals.65 To evaluate tubular hypertrophy in (gENaC; Mm00441228_m1), Ren1 (Renin1; Mm02342889_g1), the distal nephron, CNT/CD were determined as AQP2-expressing Gapdh (GAPDH; Mm99999915_g1). segments. Kidney sections were imaged with the 340 objective of a Zeiss Axiovision microscope. The surface area of each individual cell Statistical Analyses of at least ten CNT/CD segments in each condition were analyzed Data were statistically analyzed using an unpaired paired t test. Values using ImageJ ROI manager and measurement tools, as described in were considered significant when P#0.05. Data are represented as

2440 Journal of the American Society of Nephrology J Am Soc Nephrol 28: 2431–2442, 2017 www.jasn.org BASIC RESEARCH means6SEM. The significance of the metabolic parameters of the and indirect mineralocorticoid effects determine distal salt transport. J experimental groups under different diets were analyzed using two- Am Soc Nephrol 27: 2436–2445, 2016 13. Sorensen MV, Grossmann S, Roesinger M, Gresko N, Todkar AP, way ANOVA followed by Bonferroni multiple comparisons tests. Val- fi fi fi , Barmettler G, Ziegler U, Odermatt A, Lof ng-Cueni D, Lof ng J: Rapid ues were considered signi cant when a 0.05. dephosphorylation of the renal sodium chloride cotransporter in response to oral potassium intake in mice. Kidney Int 83: 811–824, 2013 14. Rengarajan S, Lee DH, Oh YT, Delpire E, Youn JH, McDonough AA: ACKNOWLEDGMENTS Increasing plasma [K+] by intravenous potassium infusion reduces NCC phosphorylation and drives kaliuresis and natriuresis. Am J Physiol We thank Matteo Stifanelli, Andrée Tedjani, and Marianne Sidhom Renal Physiol 306: F1059–F1068, 2014 for technical help. 15. Canonica J, Sergi C, Maillard M, Klusonova P, Odermatt A, Koesters R, fi fi This work was supported by the Swiss National Science Foundation Lof ng-Cueni D, Lof ng J, Rossier B, Frateschi S, Hummler E: Adult nephron-specificMR-deficient mice develop a severe renal PHA-1 grant no. 310030_159735 (to O.S.), the National Centre of Competence in phenotype. Pflugers Arch 468: 895–908, 2016 “ ” Research Swiss Kidney.ch (to O.S.), networking support by the European 16. Huang DY, Wulff P, Völkl H, Loffing J, Richter K, Kuhl D, Lang F, Vallon Cooperation in Science and Technology (COST) Action Aldosterone and V: Impaired regulation of renal K+ elimination in the sgk1-knockout Mineralocorticoid Receptor: Pathophysiology, clinical implication, and mouse. J Am Soc Nephrol 15: 885–891, 2004 therapeutic innovations (ADMIRE) BM1301 (to O.S.), Novartis 17. Al-Qusairi L, Basquin D, Roy A, Stifanelli M, Rajaram RD, Debonneville A, Nita I, Maillard M, Loffing J, Subramanya AR, Staub O: Renal tubular Foundation for medical biological research (to O.S.), and National In- SGK1 deficiency causes impaired K+ excretion via loss of regulation of stitutes of Health grant R01DK098145 (to A.R.S.). L.A.-Q. was supported NEDD4-2/WNK1 and ENaC. Am J Physiol Renal Physiol 311: F330– by a fellowship of the Marie Curie cofunding International Fellowship F342, 2016 Program on Integrative Kidney Physiology and Pathophysiology. 18. Perrier R, Boscardin E, Malsure S, Sergi C, Maillard MP, Loffing J, Loffing-Cueni D, Sørensen MV, Koesters R, Rossier BC, Frateschi S, Hummler E: Severe salt-losing syndrome and hyperkalemia induced by adult nephron-specific knockout of the epithelial sodium channel DISCLOSURES a-subunit. J Am Soc Nephrol 27: 2309–2318, 2016 None. 19. 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Legends of supplementary figures:

Figure S1: Urinary Na+ excretion and urine osmolality of control and mutant mice

(A) Urinary Na+ excretion under normal (ND), high K (HKD) and low K (LKD) diets showing no significant difference between both groups (n: 8 control and 7 KO mice). (B) Urine osmolality after 2 weeks of LKD. No significant difference between control and mutant animals was observed (n: 6 mice in each group).

Figure S2: Dietary potassium controls NEDD4-2 phosphorylation and total expression

(A) WB analysis of NEDD4-2 phosphorylation and total expression in mice fed either with high

K+ diet (HKD) for 2 days or with low K+ diet (LKD) for 2 weeks. (B) Protein quantification from (A) showing significant decrease of NEDD4-2 phosphorylation at S222 and S328 under

LKD with no change in total NEDD4-2 expression (n: 6 mice in each group; **: p value <

0.01). (C, D) Co-immunostaining of total NEDD4-2 (green) and AQP2 (red) in WT animals fed with HKD and LKD (C) and quantification of NEDD4-2 fluorescence intensity in the

CNT/CCD segments (D). NEDD4-2 abundance was significantly increased in CNT/CCD cells in mice fed with LKD (n: 2 mice in each group; *: p value < 0.05).

Figure S3: ENaC expression and activity in Nedd4LPax8/LC1 mice after 2 weeks of LKD.

(A) Q-RT-PCR analysis (TaqMan) showing normal levels of αENaC (Scnn1a), βENaC

(Scnn1b) and γENaC (Scnn1g) mRNA in Nedd4LPax8/LC1 mice compared to control after 2 weeks of LKD (n: 6 control and 5 KO animals). (B) Immunostaining of βENaC in control and

Nedd4L-KO mice showing a similar pattern in both genotypes. (C) Benzamil treatment results

NEDD4-2 in K+ handling

in a comparable increase of urine volume excreted by control and KO mice (n: 7 animals in each group).

Figure S4: AQP2 expression and CNT/CD cell size in control versus mutant mice.

(A) WB analyses showing AQP2 protein in control and mutant mice. Fg: Fully glycosylated,

Ng: Non glycosylated (B) Quantification of A, showing no significant difference between both groups (n: 5 animals per group). (C) Histogram illustrating mean cell size in the CNT/CD of control and Nedd4LPax8/LC1 mice (n= 150 control and 200 mutant cells), showing no significant difference between groups.

Figure S1 Figure S2

A HKD LKD kDa B HKD LKD 1.2 pS222 150 N4-2 100 1

pS328 150 0.8 N4-2 100 0.6

150 ** Total N4-2 0.4 Protein levels 100 **

(relative to GAPDH) 0.2

GAPDH 37 0 pS222 N4-2 pS328 N4-2 Total N4-2

C AQP2 NEDD4-2 Merge D

160 * DCT DCT 140 120 100 80 2 days HKD CNT/CCD 60 50μm 40 20 in CNT/CCD segments (%) N4-2 fluorescence intensity 0 HKD LKD CNT/ CCD DCT 2 weeks LKD DCT Figure S3

A C Control Nedd4LPAX8/LC1 1.6 0.6 1.4 0.5 1.2 1 0.4 0.8 0.3 0.6 0.2 0.4 Urine volume (ml) 0.1

Fold changes in mRNA 0.2 levels (relative to GAPDH ) 0 0 Scnn1a Scnn1b Scnn1g DMSO Benzamil

B Control Nedd4LPAX8/LC1 β ENaC

50μm Figure S4 Normal diet (0.3 %) Low K+ diet (<0.03%) WT KO WT KO Body weight (g) 24.10 +/- 0.21 22.62 +/- 0.66 21.0 +/-0.6 19.8 +/-0.5

Food intake (g/g BW.24h) 0.16 +/- 0.01 0.17 +/-0.01 0.13 +/- 0.002 0.11 +/-0.01

Water intake (ml/g BW.24h) 0.20 +/- 0.01 0.20 +/- 0.01 0.55 +/-0.04 0.63 +/- 0.08

Urine volume (ml.24h) 1.57 +/- 0.14 1.94 +/- 0.21 5.81 +/- 0.73 5.24 +/-0.87

Table S1: Body weight, food and water intake and urine volume are not changed between control and mutant mice after 2 weeks of LKD Primary antibodies Host Dilution for WB Dilution for IF References Total AQP2 Goat 1/1000 Santa Cruz Biotechnology (C-17):sc-9882 Total AQP2 Rabbit 1/1000 Wagner CA., AJP-renal Physiol 2008 Total NCC Rabbit 1/500 1/500 Sorensen MV., Kidney international 2013 Total NCC Guinea-Pig 1/500 Kindly provided by Johannes Loffing pT53 NCC Rabbit 1/500 1/500 Sorensen MV., Kidney international 2013 pT58 NCC Rabbit 1/500 Sorensen MV., Kidney international 2013 pT91 NCC Sheep 1/700 Richardson C., J Cell Science 2008 3pNCC S(45-55-60) Sheep 1/700 Kindly provided by Dario Alessi pT233 SPAK Sheep 1/100 Richardson C., J Cell Science 2008 pS373 SPAK Rabbit 1/1000 Millipore (072273) Total SPAK Rabbit 1/100 Millipore (072271) WNK1 (Ex12) Rabbit 1/100 1/50 RoyA., JCI 2015 WNK4 Mouse 1/50 abcam (52847) WNK4 Rabbit 1/1000 Novus Biologicals (NB600-284) αENaC Rabbit 1/500 1/500 Sorensen MV., Kidney international 2013 βENaC Rabbit 1/500 1/500 Wagner CA., AJP-renal Physiol 2008 γENaC Rabbit 1/500 1/500 Wagner CA., AJP-renal Physiol 2008 pS222 NEDD4-2 Sheep 1/600 Faresse N, AJP-renal Physiol 2012 pS328 NEDD4-2 Rabbit 1/500 Flores S., JASN 2005 Total NEDD4-2 Rabbit 1/1000 1/50 Kamynina E., FASEB J 2001 ROMK Guinea-Pig 1/200 1/50 Al-Qusairi L., AJP-renal physiol 2016 Maxi K α Rabbit 1/500 1/100 Alomone labs (APC-151) Maxi K β1 Rabbit 1/1000 abcam (3587) Maxi K β4 Rabbit 1/1000 Alomone labs (APC-061) GAPDH Mouse 1/5000 Millipore (MAB374) Actin Mouse 1/1000 Sigma-Aldrich (A5316) Tubulin Mouse 1/1000 Sigma-Aldrich (T5201)

Secondary antibodies Host Dilution for WB Dilution for IF References Goat, AlexaFluor® 555 Donkey 1/500 Invitrogen (A-21432) Guinea-Pig, AlexaFluor® 546 Goat 1/500 Invitrogen (A-11074) Mouse, AlexaFluor® 555 Goat 1/500 Invitrogen (A-21422) Rabbit, AlexaFluor® 488 Goat 1/500 Invitrogen (A-11034) Rabbit, AlexaFluor® 488 Donkey 1/500 Invitrogen (A-21206) Sheep, AlexaFluor® 546 Donkey 1/500 Invitrogen (A-21098) Jackson ImmunoReasearch (706-035- Guinea-Pig, HRP Donkey 1/10000 148) Mouse, HRP Sheep 1/10000 GE Healthcare UK Limited (NA931V) Rabbit, HRP Donkey 1/10000 GE Healthcare UK Limited (NA934V) Sheep, HRP Rabbit 1/10000 Milipore (12-342) Table S2: List of the antibodies used in this study References for Table S2:

1. Wagner, CA, Loffing-Cueni, D, Yan, Q, Schulz, N, Fakitsas, P, Carrel, M, Wang, T, Verrey, F, Geibel, JP, Giebisch, G, Hebert, SC, Loffing, J: Mouse model of type II Bartter's syndrome. II. Altered expression of renal sodium- and water-transporting proteins. Am J Physiol Renal Physiol, 294: F1373-1380, 2008. 2. Sorensen, MV, Grossmann, S, Roesinger, M, Gresko, N, Todkar, AP, Barmettler, G, Ziegler, U, Odermatt, A, Loffing-Cueni, D, Loffing, J: Rapid dephosphorylation of the renal sodium chloride cotransporter in response to oral potassium intake in mice. Kidney Int, 83: 811-824, 2013. 3. Richardson, C, Rafiqi, FH, Karlsson, HK, Moleleki, N, Vandewalle, A, Campbell, DG, Morrice, NA, Alessi, DR: Activation of the thiazide-sensitive Na+-Cl- cotransporter by the WNK-regulated kinases SPAK and OSR1. J Cell Sci, 121: 675-684, 2008. 4. Roy, A, Al-Qusairi, L, Donnelly, BF, Ronzaud, C, Marciszyn, AL, Gong, F, Chang, YP, Butterworth, MB, Pastor-Soler, NM, Hallows, KR, Staub, O, Subramanya, AR: Alternatively spliced proline- rich cassettes link WNK1 to aldosterone action. J Clin Invest, 125: 3433-3448, 2015. 5. Faresse, N, Lagnaz, D, Debonneville, A, Ismailji, A, Maillard, M, Fejes-Toth, G, Naray-Fejes-Toth, A, Staub, O: Inducible kidney-specific Sgk1 knockout mice show a salt-losing phenotype. Am J Physiol Renal Physiol, 302: F977-985, 2012. 6. Flores, SY, Loffing-Cueni, D, Kamynina, E, Daidie, D, Gerbex, C, Chabanel, S, Dudler, J, Loffing, J, Staub, O: Aldosterone-Induced Serum and Glucocorticoid-Induced Kinase 1 Expression Is Accompanied by Nedd4-2 Phosphorylation and Increased Na+ Transport in Cortical Collecting Duct Cells. J Am Soc Nephrol, 16: 2279-2287, 2005. 7. Kamynina, E, Debonneville, C, Bens, M, Vandewalle, A, Staub, O: A novel mouse Nedd4 protein suppresses the activity of the epithelial Na+ channel. FASEB J, 15: 204-214, 2001. 8. Al-Qusairi, L, Basquin, D, Roy, A, Stifanelli, M, Rajaram, RD, Debonneville, A, Nita, I, Maillard, M, Loffing, J, Subramanya, AR, Staub, O: Renal tubular SGK1 deficiency causes impaired K+ excretion via loss of regulation of NEDD4-2/WNK1 and ENaC. Am J Physiol Renal Physiol, 311: F330-342, 2016.