Am J Physiol Cell Physiol 308: C779–C791, 2015. First published March 18, 2015; doi:10.1152/ajpcell.00065.2015.

Review

STEVE HEBERT LECTURESHIP OF THE EPITHELIAL TRANSPORT GROUP, 2013

Revisiting the NaCl cotransporter regulation by with-no-lysine kinases Silvana Bazúa-Valenti and Gerardo Gamba Molecular Physiology Unit, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán and Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, Mexico Submitted 9 March 2015; accepted in final form 13 March 2015

Bazúa-Valenti S, Gamba G. Revisiting the NaCl cotransporter regulation by with-no-lysine kinases. Am J Physiol Cell Physiol 308: C779 –C791, 2015. First published March 18, 2015; doi:10.1152/ajpcell.00065.2015.—The renal thiazidesensitive Na⫹-Cl⫺ cotransporter (NCC) is the salt transporter in the distal convoluted tubule. Its activity is fundamental for defining blood pressure levels. Decreased NCC activity is associated with salt-remediable arterial hypotension with hypokalemia (Gitelman disease), while increased activity results in salt-sensitive arterial hypertension with hyperkalemia (pseudohypoaldosteronism type II; PHAII). The discovery of four different genes causing PHAII revealed a complex multiprotein system that regulates the activity of NCC. Two genes encode for with-no-lysine (K) kinases WNK1 and WNK4, while two encode for kelch-like 3 (KLHL3) and cullin 3 (CUL3) proteins that form a RING type E3 ubiquitin ligase complex. Extensive research has shown that WNK1 and WNK4 are the targets for the KLHL3-CUL3 complex and that WNKs modulate the activity of NCC by means of intermediary Ste20-type kinases known as SPAK or OSR1. The understanding of the effect of WNKs on NCC is a complex issue, but recent evidence discussed in this review suggests that we could be reaching the end of the dark ages regarding this matter. WNK4; distal tubule; ion transport; hypertension; diuretics

to present the Steven Hebert Lecture in 2013 came as a very pleasant surprise since it gave me (G. Gamba) the opportunity to make a remembrance of one true scientist, with whom I had the privilege to be trained. I first met Steve at Harvard in the late 1980s when I was looking for a fellow position and he was an assistant professor of medicine. I was 27 and he was 42 years old. The day I met him in 1989 he explained to me his complicated tubular perfusion techniques, only to conclude by saying: “I am not going to do this anymore because next year I will pursue a transition from perfused tubules to expression cloning and molecular biology.” One year later, with the trust and patience that characterized him as a mentor, there he was, putting such a risky and ambitious enterprise in the hands of two clinicians (Kevin Ho from Ohio and myself from Mexico) with no experience at all in molecular biology, one of whom could not even speak good English at the time. In three years we identified at the molecular level the cDNA encoding the thiazide-sensitive Na⫹-Cl⫺ cotransporter (NCC) of the distal convoluted tubule (DCT) (21), the loop-diuretic sensitive Na⫹K⫹-2Cl⫺ cotransporter (NKCC2) (20) and the renal outer medulla potassium channel (ROMK) (28) of the thick ascending limb of Henle’s loop and the calcium-sensing receptor (CaSR) from bovine parathyroid (6). Steven Hebert was a true THE INVITATION OF THE EPITHELIAL TRANSPORT GROUP

Address for reprint requests and other correspondence: G. Gamba, Vasco de Quiroga, No. 15, Tlalpan 14000 Mexico City, Mexico (e-mail: gamba @biomedicas.unam.mx). http://www.ajpcell.org

visionary, with confidence enough to pursue areas of research that were considered high risk and controversial. He was always on the cutting edge. NCC cDNA was identified by expression cloning (Fig. 1) thanks to previous seminal works from Larry Renfro (59), David Ellison (17), and John Stokes (72) that revealed the functional characteristics of the cotransporter and the winter flounder urinary bladder as a unique source of mRNA for expression cloning. Following the predictions of the great Homer Smith, we then went from “fish to philosopher” to identify NCC and NKCC2 from mammalian sources using the flounder probe. Cloning NCC cDNA was the beginning of a new era in the distal nephron physiology that has revealed the key role of DCT in modulating arterial blood pressure, as well as salt and potassium metabolism. Gene Mutations Affecting NCC Activity In the year 2000, Melanie Cobb’s laboratory discovered a new family of serine/threonine kinases during their search for new kinases in the central nervous system (89). They named this new kinase with-no-lysine (K) 1 (WNK1) because it lacked a lysine residue within the subdomain II of the kinase domain, which is conserved in all serine/threonine kinases. Soon after the discovery of WNK1, three additional members of the family were identified and named WNK2, WNK3, and WNK4 (79). Without the seminal work of the Lifton group several months later, the discovery of this new family of kinases could have remained unnoticed in the field of ion

0363-6143/15 Copyright © 2015 the American Physiological Society

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Fig. 1. Steven Hebert and Gerardo Gamba at the Brigham and Women’s Hospital celebrating the day when the cDNA from winter flounder urinary bladder encoding the Na⫹-Cl⫺ cotransporter (NCC) was identified (circa 1992). The figures on the wall show the activity of NCC in oocytes induced by microinjection of cRNA in vitro transcribed from the identified cDNA.

transport physiology (85). The Lifton group demonstrated that mutations in the genes encoding WNK1 and WNK4 were associated with the development of a form of salt-sensitive hypertension affecting humans, known as pseudohypoaldosteronism type II (PHAII), familial hyperkalemic hypertension (FHHt), or Gordon’s syndrome (85). PHAII is an autosomal dominant disease featuring arterial hypertension accompanied by hyperkalemia, metabolic acidosis, and hypercalciuria and is characterized by a marked sensitivity to low doses of thiazidetype diuretics (44). The clinical picture of PHAII mirrors that of Gitelman’s syndrome, an autosomal recessive disease featuring salt-remediable hypotension, hypokalemia, metabolic alkalosis, and hypocalciuria. Gitelman’s syndrome is caused by

inactivating mutations of NCC (Table 1). Clinical evidence thus suggests that the PHAII phenotype mainly results from the increased activity of NCC by mutated WNKs (Table 1) (18, 66). Over the past 15 years, a tremendous amount of work has been dedicated to the comprehension of the physiological and pathophysiological effects of WNK kinases on this cotransporter. Although our understanding is far from complete, the recent work of many laboratories indicates that we could be reaching the end of the dark ages regarding this matter. Although WNK1 and WNK4 were associated with PHAII in 2001, it was known over the following decade that mutations in these two genes alone could not explain the occurrence of the disease in over 70% of all the affected families (26). This suspicion indicated that mutations in other genes were likely responsible for the development of a similar phenotype. Potential genomic regions were suggested (26, 42), although without enough power to precisely define which genes were involved. The identification of these genes was delayed for over a decade until the development of exome sequencing techniques. Then, two independent groups successfully applied these techniques and identified that mutations found in a protein known as kelch-like 3 (KLHL3) were causing either a dominant or a recessive form of PHAII (Table 1) (5, 81). One of these groups also identified mutations in another gene, known as cullin 3 (CUL3), in a severe form of PHAII that was caused mostly by de novo mutations (Table 1) (5). KLHL3 and CUL3 form a complex, leading to the ubiquitination of their target proteins, thereby marking them for degradation. As discussed below, we now know that the WNKs are targets of the KLHL3-CUL3 complex and that the disruption of this complex causes PHAII. Kelch3 and Cul3 Regulate WNK Ubiquitination and Degradation The ubiquitination process depends on three components: E1 ubiquitin-activating enzyme, E2 conjugating-enzyme, and E3 ubiquitin ligase (3, 8). Many types of E3 ligases exist, but the Cullin-RING ligases (CRLs) constitute the largest family. CUL3 acts as the scaffolding protein of subfamily CLR3; in other words, it brings together the substrate recognition protein with the rest of the ubiquitination machinery (22, 30). CUL3 binds to an adaptor protein of the BTB (Bric-a-brac, Tramtrack, and Broad complex)-containing family, which in

Table 1. Gene mutations affecting NCC activity Disease

NCC Activity

Gene

Effect

Reference

SLC12A3

There are over 100 different mutations in SLC12A3, most of them affecting either the structure or the trafficking of NCC. 1q31-42 Missense mutations in the acidic motif (E562K, D564A, Q565E) impair its degradation by KLHL3-CUL3, causing its overexpression. One mutation on the COOH-terminal domain (R1185C). Intronic deletions (intron 1) cause protein overexpression. Several missense mutations that impair KLHL3 binding to substrates (WNK1 and WNK4) or CUL3. These mutations result in overexpression of WNKs. Mutations produce a splice variant that lacks exon 9, resulting in a protein that degrades KLHL3. Probably a “gain of function” type of mutation.

(71, 78)

Gitelman

22

PHAIIA PHAIIB

1

? WNK4

PHAIIC PHAIID

1 111

WNK1 KLHL3

PHAIIE

1111

CUL3

?

NCC, Na⫹-Cl⫺ cotransporter; PHAII, pseudohypoaldosteronism type II; WNK, with-no-lysine kinase; KLHL3, kelch-like 3; CUL3, cullin 3. AJP-Cell Physiol • doi:10.1152/ajpcell.00065.2015 • www.ajpcell.org

(43) (85)

(85) (5, 40) (5)

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turn binds to the substrate protein being ubiquitylated (8). In the kidneys, CUL3 associates with KLHL3. CUL3 is expressed ubiquitously in the nephron, with higher expression in the proximal tubule, while KLHL3 is mainly expressed in the thick ascending limb of Henle’s loop, the DCT, and the collecting duct (5, 40, 45). KLHL3 harbors three major structural domains. The first two domains, the BTB and BACK (BTB and COOH-terminal kelch) domains, are responsible for the interaction with CUL3 (30). The third domain, the kelch domain, is located towards the COOH-terminal region and consists of six kelch repeats forming six blades arranged in a ␤-propeller conformation that is known to be important for substrate binding (5, 40, 70). In general, the PHAII-causing mutations in KLHL3 disrupt CUL3 binding or impair substrate binding, thereby abolishing the ubiquitination capacity of the CRL complex (30). After the demonstration that KLHL3 and CUL3 mutations cause PHAII, the researchers in the field focused on finding the substrates of this ubiquitination-promoting complex. Considering that the WNKs play a key role in regulating the activity of renal transporters (19, 66), it was hypothesized that KLHL3 and CUL3 could be targeting the WNK kinases. First, Ohta et al. (52) demonstrated that the KLHL3-CUL3 complex binds to and ubiquitinates WNK1, promoting its degradation. In addition, they showed that this phenomenon did not occur in the presence of a KLHL3 protein harboring PHAII-causing mutations. Furthermore, they showed that mutations in the WNK1 acidic domain, which were similar to the known WNK4 PHAII-causing mutations, prevented the KLHL3-WNK1 interaction. This result suggested that mutations in the acidic domain of WNK4 could also prevent the interaction of WNK4 with KLHL3. Indeed, three independent groups showed that KLHL3 interacted with and ubiquitinated WNK4 and that this phenomenon was prevented by PHAII-causing mutations in either WNK4 or KLHL3 (70, 84, 87). A direct mechanism of NCC regulation via ubiquitination has previously been described (1, 32), although this involves the HECT-type E3 ligase NEDD4-2 enzyme, rather than a CRL complex, such as KLHL3-CUL3. However, initial evidence supporting the direct interaction of KLHL3 with NCC also exists and suggests that the KLHL3-CUL3 CRL complex could modulate the integrity of the cotransporter directly (40). Further research investigating how the CUL3-KLHL3 complex directly regulates NCC is warranted to confirm this interesting perspective.

Evidence demonstrating that KLHL3 itself is a substrate of CUL3 and that KLHL3 ubiquitination leads to its degradation also exists (45, 52). CUL3 mutations result in the skipping of exon 9 (5), producing a truncated gain-of-function form of the protein that degrades KLHL3 at a much higher rate (45). In the absence of KLHL3, CUL3 cannot bind to and flag the WNKs for degradation, including both WNK4 and WNK1, leading to their overexpression. The severity of the PHAII phenotype supports the molecular observation that the KLHL3-CUL3 complex lies upstream of the WNKs. CUL3 mutations produce the most severe phenotypes and affect younger patients, followed in order of severity by recessive mutations in KLHL3, dominant mutations in KLHL3, WNK4 mutations and, finally, by WNK1 intronic mutations that produce the less severe clinical form (5). Overall, PHAII seems to result from increased WNK expression. For WNK1, the increase may be attributable to the elimination of repressive elements within the first intron of WNK1. For WNK4, it could be due to the missense mutations alleviating the kinase sensitivity to the ubiquitin ligase complex formed by KLHL3 and CUL3. Mutations in KLHL3 and CUL3 could lead to PHAII by decreasing the degradation rate of WNK1 and/or WNK4 (or WNK3). Thus, to clarify the pathophysiology of the disease it is critical to understand the effect of WNKs on NCC and other ion transport pathways in the kidney. Revisiting the Model of WNK-Mediated NCC Modulation: Evidence That WNK1 Activates NCC A straightforward explanation of the disease in PHAII patients due to mutations in WNK1 gene is that WNK1 may have a positive effect on NCC activity in a manner such that the elevated expression of WNK1 enhances NCC activation. However, the initial results of Yang et al. (90, 92) and Subramanya et al. (73) describing the role of WNK1 argue against this possibility (Table 2). These studies using Xenopus laevis oocytes provided results suggesting that WNK1 had no effect on NCC activity alone. However, WNK1 rescued the cotransporter activity after its reduction by WNK4, showing that WNK1 could prevent the negative effect of WNK4 on NCC (38, 86, 90). As discussed below, this finding in part gave rise to the working model shown in Fig. 2. It was shown later that activation of the cotransporters by WNK1 or WNK4 required

Table 2. Studies analyzing the effect of WNK1 on NCC Working Model

X. laevis oocytes X. laevis oocytes X. laevis oocytes X. laevis oocytes X. laevis oocytes Mice KS-WNK1 knockout mice WNK1⫹/FHHt mice X. laevis oocytes

Stimulus

Rat L-WNK1 Rat L-WNK1 and mouse WNK4 Rat-KS-WNK1 Rat-L-WNK1 Rat-KS-WNK1/l-WNK1 and WNK4 KS-WNK1 overexpression of fragment 1-253 KS-WNK1 Overexpression of L-WNK1 Human L-WNK1 variants

NCC Analysis

Effect

Reference

(90) (90)

Expression and phosphorylation

No effect WNK1 prevented the WNK4 negative effect No effect No effect KS-WNK1 prevents the WNK1 inhibition of WNK4 negative effect 22

Expression and phosphorylation Expression and phosphorylation

22 111

(27) (81)

Functional activity, surface expression, and phosphorylation

11

(12)

Functional activity Functional activity Functional activity Functional activity Functional activity

X. laevis, Xenopus laevis; KS-WNK1, kidney-specific WNK1; L-WNK1, long WNK1. AJP-Cell Physiol • doi:10.1152/ajpcell.00065.2015 • www.ajpcell.org

(73) (73) (73) (39)

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Fig. 2. Simplification of the initial model proposed for NCC regulation by with-nolysine kinases (WNKs). WNK4 has a negative effect on SPAK (Ste20-related proline alanine-rich kinase) and NCC; this can be prevented by the presence of WNK1. In contrast, WNK3 has a direct effect, activating SPAK and thus NCC. In accordance with this model, activation of NCC by increased expression of WNK1 could be the result of overcoming the WNK4 inhibition of NCC. OSR1, oxidative stress response 1.

the intermediate kinases, known as the SPAK/OSR1 Ste20type kinases (49, 61, 62, 82, 83). All WNK kinases activate the SPAK/OSR1 kinases that in turn phosphorylate the cotransporters. However, WNK1 was shown to be at least ten times more potent at activating SPAK/OSR1 than WNK4 (82). Therefore, the results describing the inhibition of NCC by WNK4 (described below) and the lack of effect of WNK1 on NCC activity contradicted the observations that both kinases activate SPAK/OSR1 and that WNK1 does so with a markedly greater potency. Recent studies have clarified this issue by confirming that WNK1 was a powerful NCC activator (Table 2). Indeed, Vidal-Petiot et al. (81) successfully generated a mouse model harboring an heterozygous deletion in the first intron of endogenous WNK1 that reproduces the human genetic condition observed in PHAII patients affected by an intronic deletion in the WNK1 gene (85). Accordingly, WNK1⫹/FHHt mice exhibit a twofold increase in WNK1 expression in the DCT, along with an increase in the expression and activation of NCC and a characteristic PHAII phenotype that includes hyperkalemia, hypertension, and metabolic acidosis. This phenotype confirmed that increasing WNK1 expression in mice results in overactivation of NCC and in the development of PHAII. In addition, knowing that the WNK1 gene gives rise to several different isoforms after alternative splicing of exons 9, 11, 12, and 26, the same group described a methodology to quantify the WNK1 protein isoforms lacking these exons (80). This analysis revealed that the variant lacking only exon 11 (WNK1-⌬11) was enriched in the kidneys and was responsible for most of the renal WNK1 expression. However, the previous studies addressing the effects of WNK1 on NCC were performed using the WNK1 variant lacking both exons 11 and 12 (90, 92). More recently, the effect of WNK1 on NCC was analyzed using the human WNK1-⌬11 isoform (12). These results consistently revealed that the WNK1-⌬11 isoform was a powerful NCC activator. In addition, the effect of the human WNK1-⌬11-12 isoform was also assessed and, surprisingly, this isoform was also able to promote a significant activation of the cotransporter, although with a lower potency. In the same

experiment, however, the rat WNK1-⌬11-12 isoform that was used in previous studies (90, 92), had no effect on NCC activity. Because of the dramatic difference between the effects of the rat and human WNK1-⌬11-12 isoforms on NCC activation, the rat WNK1 cDNA was fully sequenced and an unexpected mutation was identified. The glycine residue located at position 2120 was substituted by a serine. Site-directed mutagenesis experiments revealed that correcting this mutation in rat WNK1 turned the kinase into a NCC activator, while introducing the G2120S mutation in the human WNK1-⌬11 or WNK1 WNK1-⌬11-12 prevented WNK1 from activating NCC. Therefore, the originally reported lack of effect of WNK1 on NCC was likely due to the presence of an unexpected mutation in the WNK1 cDNA that was used in those studies. The recently described mechanism of WNK1 activation also provided four important observations (12). First, the positive effect of WNK1 on NCC depends on WNK1 catalytic activity because the catalytically inactive mutant WNK1-D321A no longer increases the cotransporter activity. Second, the effect of WNK1 on NCC is dependent on the WNK1-SPAK/OSR1 interaction because the WNK1-F316A mutant, bearing an amino acid substitution that eliminates the SPAK-binding site, cannot activate NCC (12). In fact, the WNK1-F316A mutant not only lost its ability to activate NCC but even completely inhibited NCC activity in oocytes. This suggests a dominantnegative effect of the WNK1-F316A mutant and that NCC activity depends on endogenous WNK1 activity in oocytes. The third observation is that the effect of WNK1 on NCC and the previously described effect of WNK3 on the cotransporter (63) require the presence of an intact HQ interaction motif in the WNKs. The HQ motif consists of a histidine and glutamic acid residue conserved among WNKs in the carboxyl terminal domain required for WNK to WNK interaction. Early work in the field suggested that the interaction between the WNK kinase isoforms could be an important mechanism regulating their function (37, 91, 92). More recently, Thastrup et al. (76) observed that the interaction between WNK isoforms occurred via their COOH-terminal domain. Alanine substitution of two

AJP-Cell Physiol • doi:10.1152/ajpcell.00065.2015 • www.ajpcell.org

Review NCC REGULATION BY WNKs

specific and conserved amino acid residues in all the WNKs (exemplified by His1145 and Gln1156 in human WNK4) prevented both homo- and heterodimerization between the isoforms. In this regard, the WNK1- or WNK3-HQ mutants are no longer able to stimulate NCC but do not affect its basal activity, suggesting that the WNK1-HQ-AA or the WNK3HQ-AA mutants also lose their ability to interact with endogenous WNK1 in oocytes (12). Finally, in addition to reducing the basal activity of NCC, WNK4 also reduced the positive effect of WNK1 and WNK3 on NCC. One plausible mechanism behind the inhibitory effect of WNK4 on NCC could reside in a dominant-negative effect of WNK4 on endogenous WNK1 activity in oocytes. Another possibility is that WNK4 may prevent the activation of endogenous OSR1 by endogenous WNK1, resulting in decreased NCC activity. As shown in the revisited model depicted in Fig. 3A, these observations suggest that, as opposed to what was previously suggested (Fig. 2), WNK4 acts upstream of WNK1. To prove this hypothesis, WNK1⫹/FHHt mice, which exhibit the PHAII phenotype and an increased phosphorylation level of NCC (81), were crossed with WNK4-null (WNK4⫺/⫺) mice, which exhibit a decreased expression of NCC and a Gitelman’s syndrome-like phenotype (9). It was reasoned that if WNK1 acted upstream of WNK4, as previously suggested (Fig. 2), the removal of WNK4 would rescue the PHAII phenotype in the WNK1⫹/FHHt background. In contrast, if WNK4 acted upstream of WNK1 (Fig. 3A), then the absence of WNK4 would have no effect on the PHAII phenotype in WNK1⫹/FHHt mice. The results showed that the phenotype of the WNK1⫹/FHHt-WNK4⫺/⫺ double-mutant mice was similar to that of the WNK1⫹/FHHt mice, including the increased activation of NCC (12). This finding provided in vivo evidence that WNK1 does not need WNK4 to modulate NCC activity. The negative effect of WNK4 can be prevented by the presence of angiotensin II (Fig. 3B)(10). This effect was

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demonstrated in Xenopus laevis oocytes, where the peptide hormone angiotensin II did not affect the basal level of NCC activity but exerted an inhibitory effect on WNK4-induced decrease of NCC activity (67). This phenomenon suggested that the previously reported positive effect of angiotensin II on NCC (77) was WNK4-dependent. In accordance with this hypothesis, angiotensin II induced SPAK and NCC phosphorylation in mDCT cells (67, 74). Conversely, in WNK4-null mice, the increased levels of SPAK/NCC phosphorylation in response to low-salt diet or angiotensin II infusion by subcutaneous minipump were not observed in the absence of WNK4 expression (9). In addition, Na et al. (51) presented evidence that WNK4 activity was sensitive to Ca2⫹ ions, with a maximal kinase activity at a Ca2⫹ concentration of ⬃1 ␮M. This finding further supported the possibility of an angiotensin II-mediated modulation of WNK4 activity. In addition, they showed that any of the PHAII-causing mutations found in the acidic motif affected the enzyme’s Ca2⫹ sensitivity, which could partly explain the mechanism by which mutated WNK4 induces the disease. Recently, another pathway explaining the angiotensin II-mediated modulation of WNK4 activity was discovered. Shibata et al. (69) observed that phosphorylation of KLHL3 on serine 433 prevented it from interacting with WNK4, thus impeding WNK4 ubiquitylation and degradation. Interestingly, serine 433 is a residue that is frequently mutated in families with PHAII, and the phosphorylation of this site is stimulated by angiotensin II via protein kinase C. Therefore, one mechanism for the angiotensin II-induced phosphorylation of NCC is apparently indirect and mediated by the prevention of WNK4 degradation. In addition to the full-length WNK1 splice variants mentioned above, a kidney specific-WNK1 (KS-WNK1) variant has been described (16). This variant is exclusively found in the kidney and lacks the first four exons of the WNK1 transcript, yielding a smaller protein with no kinase domain. The

Fig. 3. Revisited model for NCC regulation by WNKs. WNK4 lies upstream of WNK1 and WNK3. A: WNK1 and WNK3 are capable of activating SPAK and NCC. Thus, both kinases are positive activators of NCC. Interaction between monomers appears to be required since elimination of the HQ motif prevents the formation of homodimers and the positive effect of WNK1 or WNK3. The presence of WNK4 abrogates WNK1 or WNK3 effect on NCC. Since the negative effect of WNK4 requires the presence of the HQ motif in WNK4, it is likely that WNK4 inhibitor effect is due to dimer formation between WNK1 and WNK4 or WNK3 and WNK4, preventing the effect of these kinases on SPAK and NCC. B: observations suggest that the positive effect of angiotensin II on NCC requires the presence of WNK4. The possible mechanisms are that angiotensin II abrogates the dimer formation of WNK1 or WNK3 with WNK4, liberating them to activate NCC. Alternatively, angiotensin II could be activating the “inactive WNK4” not associated with WNK1 or WNK3, turning it into an active kinase that is able to interact with and activate SPAK and NCC. AJP-Cell Physiol • doi:10.1152/ajpcell.00065.2015 • www.ajpcell.org

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role of this variant is not clear at the moment but is thought to be implicated in the regulation of K⫹ secretion in the aldosterone-sensitive nephron. Further studies are needed to fully understand the role of KS-WNK1 and specifically, the molecular mechanisms by which this protein acts without a kinase activity. WNK3 Is a Powerful Activator of NCC WNK3 is the family member that has shown the most consistent and robust effect on NCC activity. WNK3 is expressed all along the nephron, and its coexpression with NCC or NKCC2 in Xenopus laevis oocytes resulted in a dramatic increase in the activity of these transporters (63). This positive effect was dependent on the kinase activity of WNK3. Indeed, the catalytically inactive WNK3-D295A mutant not only lost its positive regulatory effect on NCC but also became a powerful inhibitor. This effect suggests that, depending on their catalytic activity, which in turn depends on their autophosphorylation capability, the WNK kinases can act in two different modes, either as activators or as inhibitors. As shown in Table 3, all studies on WNK3 have consistently shown the robust activation of NCC. WNK3 is expressed in the brain and the kidneys as differently spliced variants due to the presence or absence of exons 18a or 18b, as well as of exon 22. One study showed that while the renal WNK3 isoforms activate NCC, the brain isoforms actually inhibit the cotransporter (24). However, another study reported that all WNK3 variants displayed the same effect on all members of the SLC12 family, including NCC (13). The WNK3-mediated activation of NCC requires the interaction with SPAK/OSR1 and is associated with an increased phosphorylation level of the cotransporter (56). Although WNK3 induces a dramatic activation of NCC, the elimination of WNK3 by knockout in mice generates no apparent consequences or obvious phenotype. Interestingly, however, the WNK3-null mice displayed an upregulation of WNK1 expression in the kidneys (46, 53). Because WNK1 and WNK3 exert the same effect on NCC (12), the upregulation of WNK1 in WNK3-null mice could be a compensatory response preventing the occurrence of a WNK3 deletion-related phenotype. This type of compensation is often observed in knockout animals. Therefore, it will be important to determine the effect of eliminating WNK3 using a conditional knockout strategy. In

conditional knockout mice, the sudden decrease in WNK3 expression might not be compensated by an overexpression of WNK1. The effect of WNK2 in all members of all SLC12 family, including NCC, is similar to that shown by WNK3 (65). However, by Western blot analysis, WNK2 seems to be expressed only in the central nervous system and the heart, so its interaction with NCC in the kidney is unlikely to occur. Effect of WNK4 on the NaCl cotransporter activity Soon after the discovery that mutations in the WNK1 and WNK4 genes were causing PHAII, the role of these kinases in NCC activity was assessed using the Xenopus laevis oocytes heterologous expression system. Two independent groups demonstrated that the coexpression of NCC with WNK4 resulted in a decreased NCC activity that was partly attributable to a reduction in NCC expression levels at the plasma membrane (86, 90). These studies provided evidence supporting that the PHAII-associated mutations in WNK4 were reducing its inhibitory effect on NCC. Accordingly, WNK4 was identified as an endogenous inhibitor of NCC under normal conditions, as depicted in the model proposed in Fig. 2. Furthermore, when mutated in PHAII patients, WNK4 lost its ability to inhibit NCC, thereby causing the disease by favoring an increase in NCC activity. However, as shown in Tables 4 and 5, several studies using similar strategies have reported over the years that WNK4 can act either as an NCC inhibitor or as an NCC activator. Evidence that WNK4 is an inhibitor of NCC. As discussed above, the initial work on oocytes suggested that WNK4 could inhibit the activity of NCC (Table 4). We observed that coinjection of NCC and WNK4 in Xenopus laevis oocytes resulted in a reduction of NCC activity and that this effect was not present using either the catalytically inactive form of WNK4 (WNK4-D321A) or a WNK4 construct containing one of the PHAII-associated mutations (WNK4-Q562E) (86). This result suggested that the inhibitory effect of WNK4 on NCC was dependent on the catalytic activity of the kinase, which was prevented by a PHAII-associated mutation. Yang et al. (90) simultaneously reported similar observations. In their study, three different PHAII-associated mutations were analyzed: WNK4-E559K, WNK4-D561A, and WNK4-Q562E.

Table 3. Studies that have analyzed the effect of WNK3 on NCC Working Model

Stimulus

NCC Analysis

X. X. X. X.

laevis laevis laevis laevis

oocytes oocytes oocytes oocytes

WT-human WNK3 WT-human WNK3 WT-human (fragment 421-1743) Human WNK3/Mouse WNK4 chimeras

Functional Functional Functional Functional

activity and surface expression activity and surface expression activity activity

X. X. X. X. X. X. X. X.

laevis laevis laevis laevis laevis laevis laevis laevis

oocytes oocytes oocytes oocytes oocytes oocytes oocytes oocytes

Human Human Human Human Human Human Human Human

Functional Functional Functional Functional Functional Functional Functional Functional

activity activity activity activity activity activity activity activity

“renal” WNK3 variant “brain” WNK3 variant WT-WNK3 “renal” WNK3 “brain” WNK3 WNK3 WNK3 WNK3

and and and and

phosphorylation phosphorylation phosphorylation phosphorylation

and phosphorylation

WT, wild type. AJP-Cell Physiol • doi:10.1152/ajpcell.00065.2015 • www.ajpcell.org

Effect

Reference

111 111 111 22 111 Depending on the chimeric construct used 111 22 111 111 111 111 111 111

(63) (91) (91) (68)

(24) (24) (56) (13) (13) (11) (35) (12)

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Table 4. Studies showing a negative effect of WNK4 on NCC Working Model

Stimulus

X. laevis oocytes

Mouse WT-WNK4

X. laevis oocytes

Mouse WNK4-Q562E

X. laevis oocytes

Mouse WT-WNK4

X. laevis oocytes X. laevis oocytes X. laevis oocytes X. laevis oocytes X. laevis oocytes X. laevis oocytes M1-Cells COS-7 Cells COS-7 Cells COS-7 Cells BAC mice

Mouse WNK4-Q562E Mouse WNK4-E559K Mouse WNK4-D561A Rat L-WNK1 WT-WNK4 (diverse fragments) WT-WNK4 WT-human WNK4 WT-human WNK4 Human WNK4-E562K Human WNK4-R1185C WT-WNK4 overexpression

BAC mice

NCC Analysis

Functional activity and surface expression Functional activity and surface expression Functional activity and surface expression Functional activity Functional activity Functional activity Functional activity Functional activity Functional activity Surface expression Surface expression Surface expression Surface expression DCT expression/physiological parameters

WNK4-Q562E overexpression

X. laevis oocytes X. laevis oocytes X. laevis oocytes X. laevis oocytes X. laevis oocytes COS-7 cells mDCT15 cells Wild-type mice mDCT cells mDCT cells Wild-type mice and mDCT15 cells X. laevis oocytes mDCT15 cells

Effect

Reference

22

(86)

No effect

(86)

22

(90)

2 22 22 No effect 22 22 22 22 No effect No effect 222 Gitelman-like phenotype

(90) (90) (90) (90) (92) (25) (7) (7) (7) (7) (36)

DCT expression/physiological parameters

111 PHAII-like phenotype

(36)

WT-WNK4 (445-1222) Mouse WT-WNK4 Mouse WT-WNK4

Functional activity Functional activity Functional activity

(91) (68) (67)

Mouse WT-WNK4 Human WNK4 variant WT-WNK4 WNK4 shRNA to knock down WNK4 Isoproterenol-induced decrease of WNK4 Overexpression of WT-WNK4 WNK4 knockdown with siRNA WNK4 increase by Per1 blockade Mouse WT-WNK4

Functional activity Functional activity Surface expression and half-like Functional activity and surface expression Expression and phosphorylation

22 22 22 Abrogated by ANG II 22 22 22 11

(23) (24) (95) (33)

11

(50)

22 22 22 22

(94) (94) (60) (12)

11

(34)

WNK4 shRNA to knock down WNK4

Protein Expression Protein expression mRNA expression Functional activity and phosphorylation Functional activity and surface expression

DCT, distal convoluted tubule.

They observed, however, that only WNK4-Q562E could partially prevent the inhibitory effect of WNK4 on NCC, while the other two mutations had no effect. This finding suggested that not all PHAII-associated mutations necessarily altered WNK4 activity at the level of its inhibitory effect on NCC. In addition, they also observed in this and a follow-up study (90, 92) that while WNK1 alone had no effect on NCC activity, the coinjection of NCC and WNK4 together with WNK1 could prevent the WNK4-induced inhibition of the cotransporter. Therefore, as discussed above, a model was proposed in which WNK4 acts as a NCC inhibitor in a manner that may be prevented by PHAII-associated mutations and WNK1 has no direct effect on NCC but acts as an inhibitor of WNK4 (Fig. 2). This model could explain the activation of NCC in patients with PHAIIassociated mutations in the WNK1 gene because, in these families, the deletion of a part of the intron 1 results in the elevated expression of an otherwise normal WNK1 protein. According to this model, an excess of WNK1 would activate NCC by alleviating WNK4-induced inhibition. The inhibitory effect of WNK4 on NCC was corroborated by a third group using Xenopus oocytes (25) and by a fourth group using mammalian transfected cells (7) (Table 4). In the latter

study using COS-7 cells, Cai et al. (7) observed that NCC expression levels at the plasma membrane were reduced by the presence of wild-type WNK4, but not by the PHAII-associated mutants of WNK4. Considering that the inhibitory effect of WNK4 on NCC observed in expression systems could have been an artifact of the high level of protein expression, it was desirable to corroborate this effect in other systems. In this regard, Ko et al. (33) took the advantage of the mDCT15 cell line that endogenously expresses both NCC and WNK4. They used specific shRNAs to reduce the expression of WNK4 by 68% and reported that following this reduction, NCC activity and cell surface expression were significantly increased by 92% and 117%, respectively. The fact that NCC activity and expression increased as a result of the reduction in endogenous WNK4 expression supported the previous observations showing that overexpression of WNK4 reduced NCC activity. The inhibitory effect of WNK4 on NCC was also corroborated using animal models (Table 4). Lalioti et al. (36) constructed two BAC transgenic mice presenting opposite modulations of WNK4 expression: one expressing four copies of the wild-type WNK4 gene (Tg-WNK4wt) and another expressing two copies of the wild-type WNK4 gene and two of the

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NCC REGULATION BY WNKs

PHAII-associated mutated WNK4 gene (Tg-WNK4PHAII). The Tg-WNK4wt mice developed low blood pressure and DCT hypoplasia, while the Tg-WNK4PHAII mice developed arterial hypertension with hyperkalemia and DCT hyperplasia. Thus, in these models, the overexpression of wild-type WNK4 resulted in a phenotype associated with reduced NCC activity, while the overexpression of the PHAII-associated mutant forms of WNK4 resulted in a PHAII-like phenotype with DCT hyperplasia and elevated NCC expression and activity. These observations support the hypothesis that WNK4 works as a molecular switch with distinct functional states. The fact that the Tg-WNK4PHAII mice harboring two wild-type WNK4 alleles developed PHAII eliminated the possibility that haploinsufficiency could be the mechanism explaining the PHAII disease in the case where mutations confer a loss-of-function effect. Instead, this result suggested that missense mutations in the acidic domain of WNK4 resulted in a gain-of-function effect. Another study using mouse models further confirmed the inhibitory role of WNK4 on NCC activity. Mu et al. (50) studied a mouse model of norepinephrine-induced salt-sensitive arterial hypertension (Table 4). They observed that the hypertension developed in response to high-salt diet was due to epigenetic changes in the glucocorticoid receptor gene after norepinephrine-induced activation of the renal ␤2-adrenergic receptor. Interestingly, WNK4 expression was reduced simultaneously with an increase in NCC expression and phosphorylation levels. The results obtained with this animal model were consistent with the idea that NCC activation was associated with a decrease in WNK4 expression, thereby supporting the model involving a negative effect of WNK4 on NCC function. In summary, studies in Xenopus laevis oocytes, two distinct mammalian cell lines (COS-7 and mDCT15), WNK4 BAC transgenic mice and wild-type mice treated with norepinephrine and high-salt diet are all consistent with the hypothesis that WNK4 behaves as an inhibitor of NCC activity. Evidence that WNK4 is an activator of NCC. Interestingly, some studies have suggested that WNK4 exerts a positive effect on NCC activity rather than a negative one (Table 5). In one study using Xenopus laevis oocytes, Wu et al. (87) observed that the coinjection of NCC and WNK4 cRNAs resulted in increased activity of NCC. One difference from previous studies is that, instead of using the rat or mouse WNK4 cRNA, these authors used human WNK4 cRNA. They also suggested that different conditions during the uptake experiments could explain this difference, although this was not further analyzed.

One study in mDCT cells showed that the calcineurin inhibitor cyclosporine induced an increase in WNK4 expression, which was associated with an increase in NCC expression (47) (Table 5). Some results using animal models also suggested that WNK4 acted as a positive activator of NCC (Table 5). Regarding calcineurin inhibitors, Hoorn et al. (29) presented evidence that tacrolimus-induced arterial hypertension was associated with increased WNK4 and WNK3 expression levels, together with an increased NCC phosphorylation level (T53-NCC) and SPAK expression level. In short, opposing studies showed that NCC expression and phosphorylation in wild-type mice were associated with either a decreased expression of WNK4 by norepinephrine (50) or an increased expression of WNK4 by tacrolimus (29). The results from two genetically engineered mouse models also suggested that WNK4 could activate NCC (Table 5). First, Castañeda-Bueno et al. (9) reported that WNK4-null mice exhibited a dramatic decrease in the expression and phosphorylation of NCC, accompanied by hypokalemia and metabolic alkalosis. In these mice, the blood pressure was normal despite increased plasma renin activity. The fact that the total absence of WNK4 resulted in the decreased expression of NCC and a phenotype resembling Gitelman’s disease suggested that WNK4 was an activator of NCC and that its absence resulted in a decreased expression and activity of the cotransporter. An alternative explanation that was not explored in this work is that the WNK4 knockout strategy could favor the expression of a shorter, truncated isoform of WNK4 that may act as a dominant-negative heterodimer partner with the other WNK isoforms to decrease NCC activity. The second model was developed by Wakabayashi et al. (84) using a similar strategy to that applied by Lalioti et al. (36), consisting of two wild-type WNK4 BAC transgenic mice. They generated two transgenic lines, one with a low copy number and another with a high copy number of WNK4, exhibiting an increased level of WNK4 expression by 1.7- and 9.1-fold relative to control mice, respectively. The increased expression and activity of SPAK/ OSR1 and NCC in these transgenic lines was accompanied by the development of a PHAII-like phenotype. In summary, there is evidence showing that wild-type WNK4 BAC transgenic mice can present either an inhibition of NCC and a Gitelman’s syndrome-like phenotype (36) or an activation of NCC with a PHAII-like phenotype (84). Taken together, these studies used similar strategies and experimental models, such as Xenopus laevis oocytes, cultured mammalian cells, as well as wild-type mice and transgenic

Table 5. Studies showing a positive effect of WNK4 on NCC Working Model

Stimulus

NCC Analysis

X. laevis oocytes

Mouse WT-WNK4

Functional activity

Wild-type mice mDCT cells WNK4 knockout mice

Increased WNK4 expression by tacrolimus Increased WNK4 expression by cyclosporine Knocking down WNK4

BAC mice X. laevis oocytes X. laevis oocytes

WT-WNK4 overexpression Human WT-WNK4 Human WT-WNK4 and low chloride hypotonic stress

NCC expression and phosphorylation NCC expression and phosphorylation NCC expression and phosphorylation, physiological parameters, response to low-salt diet or ANG II infusion NCC expression and phosphorylation Functional activity Functional activity and phosphorylation

AJP-Cell Physiol • doi:10.1152/ajpcell.00065.2015 • www.ajpcell.org

Effect

Reference

11 Only in the presence of ANG II 11 11 22

(67)

111 11 11

(84) (87) (2)

(29) (47) (9)

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NCC REGULATION BY WNKs

B

400

400 350

*

350 300 250 200

*

150 100

*

NCC activity (%)

A NCC activity (%)

mice, and they showed clear evidence for WNK4’s dual behavior towards NCC. Indeed, both inhibition and activation of NCC by WNK4 were reported, suggesting that when both states coexist, one will predominate depending on the specific physiological context. Moreover, it is possible that WNK4 inhibits NCC under a high-salt diet to promote natriuresis but activates NCC under a low-salt diet to maximize salt retention. Recent evidence on WNK4 regulation suggests that this is a reasonable hypothesis.

As discussed above, several lines of evidence suggest that WNK4 may behave as a molecular switch, either activating or inhibiting NCC depending on the physiological state of the organism and/or the cellular environment context at each particular moment. Therefore, the inhibitory or activating effects of WNK4 could coexist and be modulated at the intracellular level. It is known that the activity of all members of the electroneutral cation chloride cotransporter (SLC12) family are modulated by the intracellular chloride ion concentration ([Cl⫺]i) (14, 15, 41, 55, 58). This family is divided into two branches. The first is composed of three members (the Na⫹-K⫹-2Cl⫺ cotransporters NKCC1 and NKCC2 and the Na⫹-Cl⫺ cotransporter NCC) that couple sodium and chloride transport into the cell and are activated by phosphorylation of key residues in their NH2-terminal domain in response to the decrease in [Cl⫺]i (14, 55, 58). The other branch is composed of four members (the K⫹-Cl⫺ cotransporters, KCC1–KCC4) that couple potassium and chloride transport out of the cell and are inhibited by the phosphorylation of key residues in both the amino and carboxyl terminal domains in response to a decrease in [Cl⫺]i (48, 64). In short, a reduction of cell [Cl⫺]i results in the phosphorylation of SLC12 cotransporters, activating the sodium-coupled and inhibiting the potassium-coupled branch. Because these transporters move chloride into or out of the cell, their activities are key regulators of the [Cl⫺]i. Therefore, a classical feedback mechanism is established between SLC12 cotransporters activity and [Cl⫺]i, in which an increased [Cl⫺]i inhibits the influx of chloride in association with a dephosphorylation of the cotransporters, while a decrease in the [Cl⫺]i increases its influx, together with the phosphorylation of SLC12 members. Evidence from our group suggested that the signaling pathway linking the [Cl⫺]i and SLC12 phosphorylation could involve the activation/deactivation of WNK kinases (54, 58). As shown in Fig. 4, with the recent observation that WNK1 activates NCC, it became evident that exposing oocytes to low-chloride hypotonic stress (LCHS), which is known to be associated with a decreased [Cl⫺]i (2, 4, 31), potentiated the effect of WNK1 on NCC. However, the effect of WNK3, which is already dramatic in control conditions, remained unchanged by LCHS. Therefore, it was reasoned that WNKs could be modulated by [Cl⫺]i and present different affinities for chloride ions. In this scenario, the effect of WNK4 could also be modulated by [Cl⫺]i but with a greater affinity. This hypothesis resulted in the following affinity profile: WNK4 ⬎ WNK1 ⬎ WNK3. Supporting this idea, in the same series of experiments shown in Fig. 4, the absence or inhibitory effect of

WNK isoform

* *

250 200 150 100 50

50

The Effect of WNK4 on NCC Is Modulated by the Intracellular Chloride Concentration

*

300

0

-

W1

W3

W4

WNK isoform

0

-

W1

W3

W4

Fig. 4. Effect of WNK1, WNK3, and WNK4 on NCC activity in Xenopus laevis oocytes microinjected with NCC cRNA alone or together with each of the WNKs cRNA. A: uptake in control conditions in which intracellular chloride concentration ([Cl⫺]i) is ⬃45 mM (2). NCC activity is increased by WNK1 and WNK3 to 150% and 320%, respectively, while it is decreased by the presence of WNK4 (*P ⬍ 0.05 vs. black bar). B: oocytes from the same experiments were incubated in a low-chloride hypotonic stress medium for 16 h. This incubation reduces [Cl⫺]i to ⬃22 mM (2). In these conditions, WNK1 effect on NCC increased when compared with that observed in control conditions. The WNK3 remained unchanged. In contrast to WNK1 and WNK3, WNK4 turned into a positive activator of NCC. Thus, WNK1 and WNK4, but not the WNK3 effect on NCC, was modified by reduction of [Cl⫺]i (*P ⬍ 0.05 vs. black bar).

WNK4 on NCC activity in the control conditions was noticeably switched to an activating effect in the oocytes exposed to LCHS (2). The hypothesis that WNK4 activity towards NCC is modulated by [Cl⫺]i was further supported by the use of phospho-specific antibodies raised against the conserved WNK autophosphorylation sites (WNK1, S382; WNK3, S308; and WNK4, S335), which demonstrated that LCHS could modulate WNK1 and WNK4 autophosphorylation levels (2). In oocytes under control conditions, WNK4 autophosphorylation was absent, WNK1 was slightly autophosphorylated, and WNK3 was heavily autophosphorylated. LCHS increased WNK4 and WNK1 autophosphorylation, while the level of WNK3 autophosphorylation remained similarly strong in both conditions (2). Piala et al. (57) recently demonstrated that the kinase domain of long WNK1 (L-WNK1) harbors a chloride-binding site and that WNK1 autophosphorylation and activity were inhibited under high [Cl⫺]i when chloride was present on its binding site (with an IC50 of ⬃20 mM). Using crystallography techniques, this group identified two key leucine residues located in the DLG motif (L269 and L371) within the kinase domain that form the chloride-binding site. WNK kinases share a common structure with more than 80% identity in their kinase domain. Therefore, these DLG motif leucine residues are conserved among all WNKs, suggesting that the mechanism regulating the L-WNK1 chloride sensor function could be common among the WNKs. In this regard, it was recently demonstrated that mutating/substituting these leucine residues in WNK4 turned the kinase into a constitutively autophosphorylated NCC activator (2). In other words, in the absence of the L322 and L324 residues, WNK4 is no longer inhibited by [Cl⫺]i and acts as a constitutive NCC activator (2). From models in Figs. 3 and 5, it is possible that WNK4 inhibitory and activating effects on NCC could coexist and be modulated by [Cl⫺]i. In a high-chloride environment, WNK4 is not autophosphorylated, a condition in which it may interact with WNK1 and WNK3 and inhibit their activity, while in a low-

AJP-Cell Physiol • doi:10.1152/ajpcell.00065.2015 • www.ajpcell.org

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NCC REGULATION BY WNKs

chloride environment, autophosphorylation turns the kinase to an active mode, making it functional against SPAK and NCC, either as a homodimer or heterodimer with WNK1 and/or WNK3. The sensitivity of WNK4 to chloride may explain its dual effect in response to [Cl⫺]i, suggesting that the modulator of WNK4 activity has been identified. Interestingly, Terker et al. (75) recently demonstrated that the modulation of NCC phosphorylation in response to changes in extracellular potassium concentrations was associated with a potassium-dependent modulation of the membrane voltage in DCT cells, which in turn modulated the intracellular [Cl⫺]i. They provided evidence showing that when the extracellular potassium concentration was decreased, the resultant hyperpolarization of the DCT cells was associated with a chloride efflux decreasing the [Cl⫺]i and activating NCC in a WNK1-dependent manner. Similarly, Wang et al. (93) observed that the basolateral potassium conductance in the DCT determines the apical NCC expression and phosphorylation levels via a modulation of the [Cl⫺]i-dependent SPAK activity. This phenomenon explains the mechanism by which mutations in the basolateral K⫹ channel KCNJ10 result in NCC inhibition in response to intracellular chloride accumulation. Perspectives and Future Directions It is now clear that both WNK1 and WNK4 are able to activate NCC but that WNK4 is also able to specifically inhibit the cotransporter. The behavior of the kinases depends on the [Cl⫺]i modulating their autophosphorylation levels. However, because WNK1 and WNK4 activities are modulated by intracellular chloride concentrations, the increased expression of the kinases in PHAII does not appear to be sufficient to explain the development of the disease. Indeed, it is expected that the elevated kinase activity associated with the increased expression of the WNKs would be inhibited by the increase in intracellular chloride caused by NCC hyperactivation. This raises an interesting question: why does the increased WNK1 or WNK4 expression escape the intracellular chloride-mediated regulation? Could WNK4 missense mutations interfere with the capacity of the kinase to bind chloride, thereby

modifying its sensitivity to chloride? Further biochemical studies are required to study this possibility and to characterize the chloride sensitivity of different WNK isoforms. Additionally, the relative chloride sensitivity among the WNK isoforms could help us understand the specific roles of each WNK isoform in the different models and assess the correlation between their specific levels and sites of expression. A second important question concerns the mechanism behind the regulation of the whole WNK-SPAK-NCC complex by the chloride concentration. Does chloride binding to WNK1 or WNK4 affect the conformation of the WNK-SPAK-NCC complex? Alternatively, could it be that the complex is formed properly but that intracellular chloride elevation prevents the SPAK phosphorylation-dependent NCC phosphorylation? The latter potential mechanism could help explain the inhibiting vs. activating effect of WNK4. In a high-chloride environment, the complex may form but remain unphosphorylated, thereby hijacking and reducing SPAK-NCC activity. In contrast, in a low-chloride environment, the phosphorylation of these proteins would result in NCC activation. The modulation of WNKs by [Cl⫺]i may represent a central mechanism for the modulation of salt reabsorption in the DCT, as diverse physiological processes appear to converge on the [Cl⫺]i-dependent regulation of NCC. At least three key pieces of information have recently arisen. First, NCC regulation by extracellular potassium is due to modulations in the [Cl⫺]i (75). Second, mutations in the potassium channel KCNJ10 result in a Gitelman-like syndrome because of the increased [Cl⫺]i resulting from KCNJ10 inactivity (93). Third, NCC modulation by angiotensin II could be, at least in part, the consequence of a decrease in [Cl⫺]i considering that this peptide hormone can promote the opening of the basolateral membrane ion channels. Indeed, Wu et al. (88) provided evidence that angiotensin II promotes NKCC2 activation by stimulating chloride efflux through the opening of the basolateral CLC-KB channels. It will be interesting to determine whether a similar effect also regulates the DCT chloride channels. In summary, several recent findings have opened the path towards a better understanding of the signaling pathways

Fig. 5. Proposed model of [Cl⫺]i modulating WNK’s effects towards NCC. In a high [Cl⫺]i environment the effect of WNK1 or WNK4 on NCC is either reduced or prevented, depending on the [Cl⫺]i. Moreover, in these conditions, WNK1 and WNK4 autophosphorylation is inhibited by chloride binding to the kinase. Reported IC50 for chloride inhibition of WNK1 autophosphorylation is ⬃20 mM (57). Results shown in Fig. 4 suggest that WNK4 sensibility to chloride could be higher than that of WNK1 and WNK3. Thus, WNK4 inhibition is achieved with lower chloride concentrations than those needed to inhibit WNK1. WNK3 appears to be much less sensitive to [Cl⫺]i because, even in control conditions, the kinase is able to activate SPAK and NCC. The mutant WNK4-L322F construct, in which the key leucine residue of the chloride-binding site was eliminated, is no longer inhibited in control conditions (high [Cl⫺]i) and thus is a constitutive autophosphorylated activator of SPAK and NCC. Contrarily, wild-type WNK4 is inhibited in the same [Cl⫺]i. In a low [Cl⫺]i environment (right), WNK1 and WNK4 autophosphorylation is increased and thus activation of SPAK and NCC is increased. WNK3 remains similarly active as in control conditions.

AJP-Cell Physiol • doi:10.1152/ajpcell.00065.2015 • www.ajpcell.org

Review NCC REGULATION BY WNKs

regulating NCC activity, providing many interesting hypotheses to be tested in the upcoming years. GRANTS The work performed in G. Gamba’s laboratory is possible thanks to the support from the Consejo Nacional de Ciencia y Tecnología (Grants 165815, 188712, and 180075) and the Programa de Apoyo a Proyectos de Investigación e Innovación Tecnológica-Dirección General Asuntos del Personal Académico (PAPIIT-DGAPA Grant IN200513). SB-V was supported by a scholarship from Consejo Nacional de Ciencia y Tecnologíia (CONACyT)-Mexico and is a graduate student in the Biochemical Science Ph.D. program of the Universidad Nacional Autónoma de México. DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS S.B.-V. and G.G. prepared figures; S.B.-V. and G.G. drafted manuscript; S.B.-V. and G.G. edited and revised manuscript; S.B.-V. and G.G. approved final version of manuscript. REFERENCES 1. Arroyo JP, Lagnaz D, Ronzaud C, Vazquez N, Ko BS, Moddes L, Ruffieux-Daidie D, Hausel P, Koesters R, Yang B, Stokes JB, Hoover RS, Gamba G, Staub O. Nedd4-2 modulates renal Na⫹-Cl⫺ cotransporter via the aldosterone-SGK1-Nedd4-2 pathway. J Am Soc Nephrol 22: 1707–1719, 2011. 2. Bazua-Valenti S, Chavez-Canales M, Rojas-Vega L, Gonzalez-Rodriguez X, Vazquez N, Rodriguez-Gama A, Argaiz ER, Melo Z, Plata C, Ellison DH, Garcia-Valdes J, Hadchouel J, Gamba G. The effect of WNK4 on the Na⫹-Cl⫺ cotransporter is modulated by intracellular chloride. J Am Soc Nephrol doi: 10.1681/ASN.2014050470 [Epub ahead of print]. 3. Berndsen CE, Wolberger C. New insights into ubiquitin E3 ligase mechanism. Nat Struct Mol Biol 21: 301–307, 2014. 4. Bertram S, Cherubino F, Bossi E, Castagna M, Peres A. GABA reverse transport by the neuronal cotransporter GAT1: influence of internal chloride depletion. Am J Physiol Cell Physiol 301: C1064 –C1073, 2011. 5. Boyden LM, Choi M, Choate KA, Nelson-Williams CJ, Farhi A, Toka HR, Tikhonova IR, Bjornson R, Mane SM, Colussi G, Lebel M, Gordon RD, Semmekrot BA, Poujol A, Valimaki MJ, De Ferrari ME, Sanjad SA, Gutkin M, Karet FE, Tucci JR, Stockigt JR, KepplerNoreuil KM, Porter CC, Anand SK, Whiteford ML, Davis ID, Dewar SB, Bettinelli A, Fadrowski JJ, Belsha CW, Hunley TE, Nelson RD, Trachtman H, Cole TR, Pinsk M, Bockenhauer D, Shenoy M, Vaidyanathan P, Foreman JW, Rasoulpour M, Thameem F, Al Shahrouri HZ, Radhakrishnan J, Gharavi AG, Goilav B, Lifton RP. Mutations in kelch-like 3 and cullin 3 cause hypertension and electrolyte abnormalities. Nature 482: 98 –102, 2012. 6. Brown EM, Gamba G, Riccardi D, Lombardi M, Butters R, Kifor O, Sun A, Hediger MA, Lytton J, Hebert SC. Cloning and characterization of an extracellular Ca2⫹ sensing receptor from bovine parathyroid. Nature 366: 575–580, 1993. 7. Cai H, Cebotaru V, Wang YH, Zhang XM, Cebotaru L, Guggino SE, Guggino WB. WNK4 kinase regulates surface expression of the human sodium chloride cotransporter in mammalian cells. Kidney Int 69: 2162– 2170, 2006. 8. Canning P, Cooper CD, Krojer T, Murray JW, Pike AC, Chaikuad A, Keates T, Thangaratnarajah C, Hojzan V, Ayinampudi V, Marsden BD, Gileadi O, Knapp S, von Delft F, Bullock AN. Structural basis for Cul3 protein assembly with the BTB-Kelch family of E3 ubiquitin ligases. J Biol Chem 288: 7803–7814, 2013. 9. Castaneda-Bueno M, Cervantes-Perez LG, Vazquez N, Uribe N, Kantesaria S, Morla L, Bobadilla NA, Doucet A, Alessi DR, Gamba G. Activation of the renal Na⫹:Cl⫺ cotransporter by angiotensin II is a WNK4-dependent process. Proc Natl Acad Sci USA 109: 7929 –7934, 2012. 10. Castaneda-Bueno M, Gamba G. Mechanisms of sodium-chloride cotransporter modulation by angiotensin II. Curr Opin Nephrol Hypertens 21: 516 –522, 2012.

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11. Chavez-Canales M, Arroyo JP, Ko B, Vazquez N, Bautista R, Castaneda-Bueno M, Bobadilla NA, Hoover RS, Gamba G. Insulin increases the functional activity of the renal NaCl cotransporter. J Hypertens 31: 303–311, 2013. 12. Chavez-Canales M, Zhang C, Soukaseum C, Moreno E, PachecoAlvarez D, Vidal-Petiot E, Castaneda-Bueno M, Vazquez N, RojasVega L, Meermeier NP, Rogers S, Jeunemaitre X, Yang CL, Ellison DH, Gamba G, Hadchouel J. WNK-SPAK-NCC cascade revisited: WNK1 stimulates the activity of the Na-Cl cotransporter via SPAK, an effect antagonized by WNK4. Hypertension 64: 1047–1053, 2014. 13. Cruz-Rangel S, Melo Z, Vazquez N, Meade P, Bobadilla NA, Pasantes-Morales H, Gamba G, Mercado A. Similar effects of all WNK3 variants on SLC12 cotransporters. Am J Physiol Cell Physiol 301: C601– C608, 2011. 14. Darman RB, Forbush B. A regulatory locus of phosphorylation in the N terminus of the Na-K-Cl cotransporter, NKCC1. J Biol Chem 277: 37542– 37550, 2002. 15. de Los Heros P, Alessi DR, Gourlay R, Campbell DG, Deak M, Macartney TJ, Kahle KT, Zhang J. The WNK-regulated SPAK/OSR1 kinases directly phosphorylate and inhibit the K⫹-Cl⫺ co-transporters. Biochem J 458: 559 –573, 2014. 16. Delaloy C, Lu J, Houot AM, Disse-Nicodeme S, Gasc JM, Corvol P, Jeunemaitre X. Multiple promoters in the WNK1 gene: one controls expression of a kidney-specific kinase-defective isoform. Mol Cell Biol 23: 9208 –9221, 2003. 17. Ellison DH, Velazquez H, Wright FS. Thiazide-sensitive sodium chloride cotransport in early distal tubule. Am J Physiol Renal Fluid Electrolyte Physiol 253: F546 –F554, 1987. 18. Gamba G. Regulation of the renal Na⫹-Cl⫺ cotransporter by phosphorylation and ubiquitylation. Am J Physiol Renal Physiol 303: F1573– F1583, 2012. 19. Gamba G. Role of WNK kinases in regulating tubular salt and potassium transport and in the development of hypertension. Am J Physiol Renal Physiol 288: F245–F252, 2005. 20. Gamba G, Miyanoshita A, Lombardi M, Lytton J, Lee WS, Hediger MA, Hebert SC. Molecular cloning, primary structure and characterization of two members of the mammalian electroneutral sodium-(potassium)-chloride cotransporter family expressed in kidney. J Biol Chem 269: 17713–17722, 1994. 21. Gamba G, Saltzberg SN, Lombardi M, Miyanoshita A, Lytton J, Hediger MA, Brenner BM, Hebert SC. Primary structure and functional expression of a cDNA encoding the thiazide-sensitive, electroneutral sodium-chloride cotransporter. Proc Natl Acad Sci USA 90: 2749 –2753, 1993. 22. Genschik P, Sumara I, Lechner E. The emerging family of CULLIN3RING ubiquitin ligases (CRL3s): cellular functions and disease implications. EMBO J 32: 2307–2320, 2013. 23. Glover M, Mercier ZA, Figg N, O’Shaughnessy KM. The activity of the thiazide-sensitive Na(⫹)-Cl cotransporter is regulated by protein phosphatase PP4. Can J Physiol Pharmacol 88: 986 –995, 2010. 24. Glover M, Zuber AM, O’Shaughnessy KM. Renal and brain isoforms of WNK3 have opposite effects on NCCT expression. J Am Soc Nephrol 20: 1314 –1322, 2009. 25. Golbang AP, Cope G, Hamad A, Murthy M, Liu CH, Cuthbert AW, O’Shaughnessy KM. Regulation of the expression of the Na/Cl cotransporter by WNK4 and WNK1: evidence that accelerated dynamin-dependent endocytosis is not involved. Am J Physiol Renal Physiol 291: F1369 –F1376, 2006. 26. Hadchouel J, Delaloy C, Faure S, Achard JM, Jeunemaitre X. Familial hyperkalemic hypertension. J Am Soc Nephrol 17: 208 –217, 2006. 27. Hadchouel J, Soukaseum C, Busst C, Zhou XO, Baudrie V, Zurrer T, Cambillau M, Elghozi JL, Lifton RP, Loffing J, Jeunemaitre X. Decreased ENaC expression compensates the increased NCC activity following inactivation of the kidney-specific isoform of WNK1 and prevents hypertension. Proc Natl Acad Sci USA 107: 18109 –18114, 2010. 28. Ho K, Nichols CG, Lederer J, Lytton J, Vassilev PM, Kanazirska MV, Hebert SC. Cloning and expression of an inwardly rectifying ATPregulated potassium channel. Nature 362: 31–38, 1993. 29. Hoorn EJ, Walsh SB, McCormick JA, Furstenberg A, Yang CL, Roeschel T, Paliege A, Howie AJ, Conley J, Bachmann S, Unwin RJ, Ellison DH. The calcineurin inhibitor tacrolimus activates the renal sodium chloride cotransporter to cause hypertension. Nat Med 17: 1304 – 1309, 2011.

AJP-Cell Physiol • doi:10.1152/ajpcell.00065.2015 • www.ajpcell.org

Review C790

NCC REGULATION BY WNKs

30. Ji AX, Prive GG. Crystal structure of KLHL3 in complex with Cullin3. PLos One 8: e60445, 2013. 31. Keicher E, Meech R. Endogenous Na⫹-K⫹ (or NH4⫹)-2Cl⫺ cotransport in Rana oocytes; anomalous effect of external NH4⫹ on pHi. J Physiol 475: 45–57, 1994. 32. Ko B, Kamsteeg EJ, Cooke LL, Moddes LN, Deen PM, Hoover RS. RasGRP1 stimulation enhances ubiquitination and endocytosis of the sodium-chloride cotransporter. Am J Physiol Renal Physiol 299: F300 – F309, 2010. 33. Ko B, Mistry AC, Hanson LN, Mallick R, Cooke LL, Hack BK, Cunningham PN, Hoover RS. A new model of the distal convoluted tubule. Am J Physiol Renal Physiol 303: F700 –F710, 2012. 34. Ko B, Mistry A, Hanson L, Mallick R, Hoover RS. Mechanisms of angiotensin II stimulation of NCC are time-dependent in mDCT15 cells. Am J Physiol Renal Physiol 308: F720 –F727, 2015. 35. Lagnaz D, Arroyo JP, Chavez-Canales M, Vazquez N, Rizzo F, Spirli A, Debonneville A, Staub O, Gamba G. WNK3 abrogates the NEDD42-mediated inhibition of the renal Na⫹-Cl⫺ cotransporter. Am J Physiol Renal Physiol 307: F275–F286, 2014. 36. Lalioti MD, Zhang J, Volkman HM, Kahle KT, Hoffmann KE, Toka HR, Nelson-Williams C, Ellison DH, Flavell R, Booth CJ, Lu Y, Geller DS, Lifton RP. Wnk4 controls blood pressure and potassium homeostasis via regulation of mass and activity of the distal convoluted tubule. Nat Genet 38: 1124 –1132, 2006. 37. Lenertz LY, Lee BH, Min X, Xu BE, Wedin K, Earnest S, Goldsmith EJ, Cobb MH. Properties of WNK1 and implications for other family members. J Biol Chem 280: 26653–26658, 2005. 38. Lifton RP, Gharavi AG, Geller DS. Molecular mechanisms of human hypertension. Cell 104: 545–556, 2001. 39. Liu Z, Xie J, Wu T, Truong T, Auchus RJ, Huang CL. Downregulation of NCC and NKCC2 cotransporters by kidney-specific WNK1 revealed by gene disruption and transgenic mouse models. Hum Mol Genet 20: 855–866, 2011. 40. Louis-Dit-Picard H, Barc J, Trujillano D, Miserey-Lenkei S, BouatiaNaji N, Pylypenko O, Beaurain G, Bonnefond A, Sand O, Simian C, Vidal-Petiot E, Soukaseum C, Mandet C, Broux F, Chabre O, Delahousse M, Esnault V, Fiquet B, Houillier P, Bagnis CI, Koenig J, Konrad M, Landais P, Mourani C, Niaudet P, Probst V, Thauvin C, Unwin RJ, Soroka SD, Ehret G, Ossowski S, Caulfield M, Bruneval P, Estivill X, Froguel P, Hadchouel J, Schott JJ, Jeunemaitre X. KLHL3 mutations cause familial hyperkalemic hypertension by impairing ion transport in the distal nephron. Nat Genet 44: 456 –460, 2012. 41. Lytle C, Forbush B 3rd. Regulatory phosphorylation of the secretory Na-K-Cl cotransporter: modulation by cytoplasmic Cl. Am J Physiol Cell Physiol 270: C437–C448, 1996. 42. Mansfield TA, Simon DB, Farfel Z, Bia M, Tucci JR, Lebel M, Gutkin M, Vialettes B, Christofilis MA, Kauppinen-Makelin R, Mayan H, Risch N, Lifton RP. Multilocus linkage of familial hiperkalemia and hypertension, pseudohypoaldosternism type II, to chromosomes 1q31-42 and 17p11-q21. Nat Genet 16: 202–205, 1997. 43. Mansfield TA, Simon DB, Farfel Z, Bia M, Tucci JR, Lebel M, Gutkin M, Vialettes B, Christofilis MA, Kauppinen-Makelin R, Mayan H, Risch N, Lifton RP. Multilocus linkage of familial hyperkalaemia and hypertension, pseudohypoaldosteronism type II, to chromosomes 1q31-42 and 17p11-q21. Nat Genet 16: 202–205, 1997. 44. Mayan H, Vered I, Mouallem M, Tzadok-Witkon M, Pauzner R, Farfel Z. Pseudohypoaldosteronism type II: marked sensitivity to thiazides, hypercalciuria, normomagnesemia, and low bone mineral density. J Clin Endocrinol Metab 87: 3248 –3254, 2002. 45. McCormick JA, Yang CL, Zhang C, Davidge B, Blankenstein KI, Terker AS, Yarbrough B, Meermeier NP, Park HJ, McCully B, West M, Borschewski A, Himmerkus N, Bleich M, Bachmann S, Mutig K, Argaiz ER, Gamba G, Singer JD, Ellison DH. Hyperkalemic hypertension-associated cullin 3 promotes WNK signaling by degrading KLHL3. J Clin Invest 124: 4723–4736, 2014. 46. Mederle K, Mutig K, Paliege A, Carota I, Bachmann S, Castrop H, Oppermann M. Loss of Wnk3 is compensated for by ks-Wnk1 in the kidney of the mouse. Am J Physiol Renal Physiol 304: F1198 –F1209, 2013. 47. Melnikov S, Mayan H, Uchida S, Holtzman EJ, Farfel Z. Cyclosporine metabolic side effects: association with the WNK4 system. Eur J Clin Invest 41: 1113–1120, 2011. 48. Melo Z, de Los Heros P, Cruz-Rangel S, Vazquez N, Bobadilla NA, Pasantes-Morales H, Alessi DR, Mercado A, Gamba G. N-terminal

49.

50.

51.

52.

53.

54. 55.

56.

57.

58.

59. 60.

61.

62.

63.

64.

65.

66.

67.

serine dephosphorylation is required for KCC3 full activation by cell swelling. J Biol Chem 288: 31468 –31476, 2013. Moriguchi T, Urushiyama S, Hisamoto N, Iemura S, Uchida S, Natsume T, Matsumoto K, Shibuya H. WNK1 regulates phosphorylation of cation-chloride-coupled cotransporters via the STE20-related kinases, SPAK and OSR1. J Biol Chem 280: 42685–42693, 2005. Mu S, Shimosawa T, Ogura S, Wang H, Uetake Y, Kawakami-Mori F, Marumo T, Yatomi Y, Geller DS, Tanaka H, Fujita T. Epigenetic modulation of the renal beta-adrenergic-WNK4 pathway in salt-sensitive hypertension. Nat Med 17: 573–581, 2011. Na T, Wu G, Peng JB. Disease-causing mutations in the acidic motif of WNK4 impair the sensitivity of WNK4 kinase to calcium ions. Biochem Biophys Res Commun 419: 293–298, 2012. Ohta A, Schumacher FR, Mehellou Y, Johnson C, Knebel A, Macartney TJ, Wood NT, Alessi DR, Kurz T. The CUL3-KLHL3 E3 ligase complex mutated in Gordon’s hypertension syndrome interacts with and ubiquitylates WNK isoforms; disease-causing mutations in KLHL3 and WNK4 disrupt interaction. Biochem J 451: 111–122, 2013. Oi K, Sohara E, Rai T, Misawa M, Chiga M, Alessi D, Sasaki S, Uchida S. A minor role of WNK3 in regulating phosphorylation of renal NKCC2 and NCC co-transporters in vivo. Biol Open 1: 120 –127, 2012. Pacheco-Alvarez D, Gamba G. WNK3 is a putative chloride-sensing kinase. Cell Physiol Biochem 28: 1123–1134, 2011. Pacheco-Alvarez D, San Cristobal P, Meade P, Moreno E, Vazquez N, Munoz E, Diaz A, Juarez ME, Gimenez I, Gamba G. The Na-Cl cotransporter is activated and phosphorylated at the amino terminal domain upon intracellular chloride depletion. J Biol Chem 281: 28755– 28763, 2006. Pacheco-Alvarez D, Vazquez N, Castaneda-Bueno M, de los Heros P, Cortes-Gonzalez C, Moreno E, Meade P, Bobadilla NA, Gamba G. WNK3-SPAK interaction is required for the modulation of NCC and other members of the SLC12 family. Cell Physiol Biochem 29: 291–302, 2012. Piala AT, Moon TM, Akella R, He H, Cobb MH, Goldsmith EJ. Chloride sensing by WNK1 involves inhibition of autophosphorylation. Sci Signal 7: ra41, 2014. Ponce-Coria J, San Cristobal P, Kahle KT, Vazquez N, PachecoAlvarez D, De Los Heros P, Juarez P, Munoz E, Michel G, Bobadilla NA, Gimenez I, Lifton RP, Hebert SC, Gamba G. Regulation of NKCC2 by a chloride-sensing mechanism involving the WNK3 and SPAK kinases. Proc Natl Acad Sci USA 105: 8458 –8463, 2008. Renfro JL. Water and ion transport by the urinary bladder of the teleost Pseudopleuronectus americanus. Am J Physiol 228: 52–61, 1975. Richards J, Ko B, All S, Cheng KY, Hoover RS, Gumz ML. A role for the circadian clock protein Per1 in the regulation of the NaCl cotransporter (NCC) and the with-no-lysine kinase (WNK) cascade in mouse distal convoluted tubule cells. J Biol Chem 289: 11791–11806, 2014. Richardson C, Alessi DR. The regulation of salt transport and blood pressure by the WNK-SPAK/OSR1 signalling pathway. J Cell Sci 121: 3293–3304, 2008. Richardson C, Rafiqi FH, Karlsson HK, Moleleki N, Vandewalle A, Campbell DG, Morrice NA, Alessi DR. Activation of the thiazidesensitive Na⫹-Cl⫺ cotransporter by the WNK-regulated kinases SPAK and OSR1. J Cell Sci 121: 675–684, 2008. Rinehart J, Kahle KT, De Los Heros P, Vazquez N, Meade P, Wilson FH, Hebert SC, Gimenez I, Gamba G, Lifton RP. WNK3 kinase is a positive regulator of NKCC2 and NCC, renal cation-Cl⫺ cotransporters required for normal blood pressure homeostasis. Proc Natl Acad Sci USA 102: 16777–16782, 2005. Rinehart J, Maksimova YD, Tanis JE, Stone KL, Hodson CA, Zhang J, Risinger M, Pan W, Wu D, Colangelo CM, Forbush B, Joiner CH, Gulcicek EE, Gallagher PG, Lifton RP. Sites of regulated phosphorylation that control K-Cl cotransporter activity. Cell 138: 525–536, 2009. Rinehart J, Vazquez N, Kahle KT, Hodson CA, Ring AM, Gulcicek EE, Louvi A, Bobadilla NA, Gamba G, Lifton RP. WNK2 is a novel regulator of essential neuronal cation-chloride cotransporters. J Biol Chem 286: 30171–30180, 2011. San Cristobal P, De Los Heros P, Ponce-Coria J, Moreno E, Gamba G. WNK kinases, renal ion transport and hypertension. Am J Nephrol 28: 860 –870, 2008. San Cristobal P, Pacheco-Alvarez D, Richardson C, Ring AM, Vazquez N, Rafiqi FH, Chari D, Kahle KT, Leng Q, Bobadilla NA, Hebert SC, Alessi DR, Lifton RP, Gamba G. Angiotensin II signaling increases activity of the renal Na-Cl cotransporter through a WNK4-

AJP-Cell Physiol • doi:10.1152/ajpcell.00065.2015 • www.ajpcell.org

Review NCC REGULATION BY WNKs

68.

69.

70. 71.

72. 73. 74.

75.

76.

77.

78.

79. 80.

81.

SPAK-dependent pathway. Proc Natl Acad Sci USA 106: 4384 –4389, 2009. San Cristobal P, Ponce-Coria J, Vázquez N, Bobadilla NA, Gamba G. WNK3 and WNK4 amino-terminal domain defines their effect on the renal Na⫹-Cl⫺ cotransporter. Am J Physiol Renal Physiol 295: F1199 –F1206, 2008. Shibata S, Arroyo JP, Castaneda-Bueno M, Puthumana J, Zhang J, Uchida S, Stone KL, Lam TT, Lifton RP. Angiotensin II signaling via protein kinase C phosphorylates Kelch-like 3, preventing WNK4 degradation. Proc Natl Acad Sci USA 111: 15556 –15561, 2014. Shibata S, Zhang J, Puthumana J, Stone KL, Lifton RP. Kelch-like 3 and Cullin 3 regulate electrolyte homeostasis via ubiquitination and degradation of WNK4. Proc Natl Acad Sci USA 110: 7838 –7843, 2013. Simon DB, Nelson-Williams C, Johnson-Bia M, Ellison D, Karet FE, Morey-Molina A, Vaara I, Iwata F, Cushner HM, Koolen M, Gainza FJ, Gitelman HJ, Lifton RP. Gitelman’s variant of Bartter’s syndrome, inherited hypokalaemic alkalosis, is caused by mutations in the thiazidesensitive Na-Cl cotransporter. Nat Genet 12: 24 –30, 1996. Stokes JB, Lee I, D’Amico M. Sodium chloride absorption by the urinary bladder of the winter flounder. A thiazide-sensitive, electrically neutral transport system. J Clin Invest 74: 7–16, 1984. Subramanya AR, Yang CL, Zhu X, Ellison DH. Dominant-negative regulation of WNK1 by its kidney-specific kinase-defective isoform. Am J Physiol Renal Physiol 290: F619 –F624, 2006. Talati G, Ohta A, Rai T, Sohara E, Naito S, Vandewalle A, Sasaki S, Uchida S. Effect of angiotensin II on the WNK-OSR1/SPAK-NCC phosphorylation cascade in cultured mpkDCT cells and in vivo mouse kidney. Biochem Biophys Res Commun 393: 844 –848, 2010. Terker AS, Zhang C, McCormick JA, Lazelle RA, Zhang C, Meermeier NP, Siler DA, Park HJ, Fu Y, Cohen DM, Weinstein AM, Wang WH, Yang CL, Ellison DH. Potassium modulates electrolyte balance and blood pressure through effects on distal cell voltage and chloride. Cell Metab 21: 39 –50, 2015. Thastrup JO, Rafiqi FH, Vitari AC, Pozo-Guisado E, Deak M, Mehellou Y, Alessi DR. SPAK/OSR1 regulate NKCC1 and WNK activity: analysis of WNK isoform interactions and activation by T-loop transautophosphorylation. Biochem J 441: 325–337, 2012. van der Lubbe N, Lim CH, Fenton RA, Meima ME, Jan Danser AH, Zietse R, Hoorn EJ. Angiotensin II induces phosphorylation of the thiazide-sensitive sodium chloride cotransporter independent of aldosterone. Kidney Int 79: 66 –76, 2011. Vargas-Poussou R, Dahan K, Kahila D, Venisse A, Riveira-Munoz E, Debaix H, Grisart B, Bridoux F, Unwin R, Moulin B, Haymann JP, Vantyghem MC, Rigothier C, Dussol B, Godin M, Nivet H, Dubourg L, Tack I, Gimenez-Roqueplo AP, Houillier P, Blanchard A, Devuyst O, Jeunemaitre X. Spectrum of mutations in Gitelman syndrome. J Am Soc Nephrol 22: 693–703, 2011. Verissimo F, Jordan P. WNK kinases, a novel protein kinase subfamily in multi-cellular organisms. Oncogene 20: 5562–5569, 2001. Vidal-Petiot E, Cheval L, Faugeroux J, Malard T, Doucet A, Jeunemaitre X, Hadchouel J. A new methodology for quantification of alternatively spliced exons reveals a highly tissue-specific expression pattern of WNK1 isoforms. PLos One 7: e37751, 2012. Vidal-Petiot E, Elvira-Matelot E, Mutig K, Soukaseum C, Baudrie V, Wu S, Cheval L, Huc E, Cambillau M, Bachmann S, Doucet A,

82.

83.

84.

85.

86.

87. 88.

89.

90. 91.

92.

93.

94.

95.

C791

Jeunemaitre X, Hadchouel J. WNK1-related familial hyperkalemic hypertension results from an increased expression of L-WNK1 specifically in the distal nephron. Proc Natl Acad Sci USA 110: 14366 –14371, 2013. Vitari AC, Deak M, Morrice NA, Alessi DR. The WNK1 and WNK4 protein kinases that are mutated in Gordon’s hypertension syndrome, phosphorylate and active SPAK and OSR1 protein kinases. Biochem J 391: 17–24, 2005. Vitari AC, Thastrup J, Rafiqi FH, Deak M, Morrice NA, Karlsson HK, Alessi DR. Functional interactions of the SPAK/OSR1 kinases with their upstream activator WNK1 and downstream substrate NKCC1. Biochem J 397: 223–231, 2006. Wakabayashi M, Mori T, Isobe K, Sohara E, Susa K, Araki Y, Chiga M, Kikuchi E, Nomura N, Mori Y, Matsuo H, Murata T, Nomura S, Asano T, Kawaguchi H, Nonoyama S, Rai T, Sasaki S, Uchida S. Impaired KLHL3-mediated ubiquitination of WNK4 causes human hypertension. Cell Rep 3: 858 –868, 2013. Wilson FH, Disse-Nicodeme S, Choate KA, Ishikawa K, NelsonWilliams C, Desitter I, Gunel M, Milford DV, Lipkin GW, Achard JM, Feely MP, Dussol B, Berland Y, Unwin RJ, Mayan H, Simon DB, Farfel Z, Jeunemaitre X, Lifton RP. Human hypertension caused by mutations in WNK kinases. Science 293: 1107–1112, 2001. Wilson FH, Kahle KT, Sabath E, Lalioti MD, Rapson AK, Hoover RS, Hebert SC, Gamba G, Lifton RP. Molecular pathogenesis of inherited hypertension with hyperkalemia: the Na-Cl cotransporter is inhibited by wild-type but not mutant WNK4. Proc Natl Acad Sci USA 100: 680 –684, 2003. Wu G, Peng JB. Disease-causing mutations in KLHL3 impair its effect on WNK4 degradation. FEBS Lett 587: 1717–1722, 2013. Wu P, Wang M, Luan H, Li L, Wang L, Wang WH, Gu R. Angiotensin II stimulates basolateral 10-pS Cl channels in the thick ascending limb. Hypertension 61: 1211–1217, 2013. Xu B, English JM, Wilsbacher JL, Stippec S, Goldsmith EJ, Cobb MH. WNK1, a novel mammalian serine/threonine protein kinase lacking the catalytic lysine in subdomain II. J Biol Chem 275: 16795–16801, 2000. Yang CL, Angell J, Mitchell R, Ellison DH. WNK kinases regulate thiazide-sensitive Na-Cl cotransport. J Clin Invest 111: 1039 –1045, 2003. Yang CL, Zhu X, Ellison DH. The thiazide-sensitive Na-Cl cotransporter is regulated by a WNK kinase signaling complex. J Clin Invest 117: 3403–3411, 2007. Yang CL, Zhu X, Wang Z, Subramanya AR, Ellison DH. Mechanisms of WNK1 and WNK4 interaction in the regulation of thiazide-sensitive NaCl cotransport. J Clin Invest 115: 1379 –1387, 2005. Zhang C, Wang L, Zhang J, Su XT, Lin DH, Scholl UI, Giebisch G, Lifton RP, Wang WH. KCNJ10 determines the expression of the apical Na-Cl cotransporter (NCC) in the early distal convoluted tubule (DCT1). Proc Natl Acad Sci USA 111: 11864 –11869, 2014. Zhou B, Wang D, Feng X, Zhang Y, Wang Y, Zhuang J, Zhang X, Chen G, Delpire E, Gu D, Cai H. WNK4 inhibits NCC protein expression through MAPK ERK1/2 signaling pathway. Am J Physiol Renal Physiol 302: F533–F539, 2012. Zhou B, Zhuang J, Gu D, Wang H, Cebotaru L, Guggino WB, Cai H. WNK4 enhances the degradation of NCC through a sortilin-mediated lysosomal pathway. J Am Soc Nephrol 21: 82–92, 2010.

AJP-Cell Physiol • doi:10.1152/ajpcell.00065.2015 • www.ajpcell.org