WNK4 kinase: from structure to physiology

Abstract

With no lysine kinase-4 (WNK4) belongs to a serine-threonine kinase family characterized by the atypical positioning of its catalytic lysine. Despite the fact that WNK4 has been found in many tissues, the majority of its study has revolved around its function in the kidney, specifically as a positive regulator of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule of the nephron. This is explained by the description of gain-of-function mutations in the gene encoding WNK4 that causes familial hyperkalemic hypertension. This disease is mainly driven by increased downstream activation of the Ste20/SPS1-related proline-alanine-rich kinase/oxidative stress responsive kinase-1-NCC pathway, which increases salt reabsorption in the distal convoluted tubule and indirectly impairs renal K⁺ secretion. Here, we review the large volume of information that has accumulated about different aspects of WNK4 function. We first review the knowledge on WNK4 structure and enumerate the functional domains and motifs that have been characterized. Then, we discuss WNK4 physiological functions based on the information obtained from in vitro studies and from a diverse set of genetically modified mouse models with altered WNK4 function. We then review in vitro and in vivo evidence on the different levels of regulation of WNK4. Finally, we go through the evidence that has suggested how different physiological conditions act through WNK4 to modulate NCC activity.

INTRODUCTION

The With No Lysine Family of Kinases

The with no lysine (WNK) family of kinases is a conserved group of serine/threonine kinases found in eukaryotic organisms (1). They were first reported in 2000 with the cloning of WNK1 from a rat cDNA library (2). Subsequent analysis of genomic sequences in the search for WNK1 paralogs identified additional family members including WNK2, WNK3, and WNK4 (3, 4). These proteins owe their name to the atypical positioning of its catalytic lysine (2). Whereas in most serine/threonine kinases this residue is located in subdomain II of the kinase domain, in the case of WNKs it is located in subdomain I. It has been speculated that this phenomenon is related to the Cl⁻-sensing ability of WNKs (5), as shall be explained below.

WNK kinases have been described to play a role in a variety of physiological and pathophysiological processes, such as cell volume regulation, modulation of transepithelial ion transport, and neurotransmission (6). Although several proteins have been shown to be modulated, directly or indirectly, by WNK kinases (7, 8), their best-characterized role involves their participation in a signaling pathway comprised of their direct substrates STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1) (9), which, in turn, act as serine/threonine kinases that phosphorylate and modulate cation-coupled Cl⁻ cotransporters (CCCs) of the SLC12 family (10).

In the past couple of decades, a large amount of information about the structure, regulation, and physiological functions of WNK kinases has accumulated (6, 7, 10, 11). Here, we review the current knowledge about one particular member of this family, WNK4, that plays a key role in kidney physiology.

WNK4

WNK4 is a highly conserved protein, as its orthologs in cartilaginous fishes show close to 50% identity with human WNK4 (hWNK4). In humans, the WNK4 gene is located in chromosome 17, whereas its mouse ortholog is located in chromosome 11. It is composed of 19 exons, and it was first described due to human disease-causing mutations found in 2001 by Richard Lifton’s group (4). Missense mutations in WNK4, as well as intronic deletions in WNK1, were found in patients with familial hyperkalemic hypertension (FHHt), also called pseudohypoaldosteronism type II (PHAII) or Gordon syndrome. Additionally, it has been described that loss-of-function mutations in WNK4 in Burmese cats are associated with feline hypokalemic periodic paralysis (12), which further suggests that WNK4 plays an important role in K⁺ homeostasis.

The fact that WNK4 mutations were the cause of FHHt prompted several groups around the world to study its relationship to the thiazide-sensitive renal Na⁺/Cl⁻ cotransporter (NCC), believed to be overactive and the cause of the electrolyte imbalance observed in FHHt (13). Several papers have shown that the presence of WNK4 in the distal convoluted tubule (DCT) is indispensable for the activity of NCC (14, 15). WNK4-mediated phosphorylation of the kinases SPAK and OSR1 increases their activity and allows them to phosphorylate and activate NCC (9). It was later found that mutations in the genes Kelch-like family member 3 (KLHL3) and cullin 3 (CUL3) (16, 17) also cause FHHt, which led to the finding that WNK4 protein abundance is negatively modulated by ubiquitin-mediated proteasome degradation promoted by the KLHL3-CUL3 E3 ubiquitin ligase complex (18–20).

Although the most studied role of WNK4 is the modulation of NCC in the distal nephron, several details are still not clear on the mechanisms that dictate the activity of the KLHL3/CUL3-WNK4-SPAK/OSR1-NCC pathway in response to different physiological states. Moreover, the roles of WNK4 outside the kidney are still obscure, and further investigation is required in this respect.

STRUCTURAL FEATURES OF WNK4

General Description

WNK4 is the smallest member of the mammalian WNK kinase family, with a molecular mass of ∼134 kDa. Roughly 35% of the primary sequence of WNK4 is predicted to have a secondary structure, whereas the remaining 65% is predicted to be disordered (Fig. 1A). Around one-half of those disordered regions contain low-complexity sequences. The low presence of globular domains extends to the other family members, being more notable in the largest member, WNK1, that doubles the size of WNK4. To what extent such disordered regions influence WNK kinase function remains unclear. Increasing evidence has demonstrated the key role of low-complexity sequences in driving proteins from a dispersed phase to a condensed, assembled state (21). Notably, numerous reports have shown that WNK-SPAK/OSR1 can form large cytoplasmic protein aggregates upon changes in potassium balance (22, 23).

Figure 1.

Conserved regions in with no lysine kinase 4 (WNK4). A: sequence conservation analysis was performed including the sequences of WNK1-4 kinases from the human, mouse, rat, zebrafish, pig, and clawed frog. Multiple sequence analysis was performed in Clustal Omega, and the conservation score was then calculated by https://compbio.cs.princeton.edu/conservation/score.html. The top graph shows the conservation score where three major conserved regions were identified. The first conserved region encompasses the kinase domain, the PF2a domain, and the acidic motif. The second and third conserved regions encompass the PF2b domain and the COOH-terminal coiled-coil domain (CT-CCD), respectively. The middle graph shows the conservation score observed along the WNK4 sequence in an analysis performed with the sequences of 27 WNK4 orthologs (including mammals, fishes, birds, reptiles, and amphibians). The analysis shows that, in addition to the domains and motifs that are conserved among different WNK kinases, a high degree of conservation was observed in the COOH-terminal segment, from the CT-CCD until the end of the protein. The bottom graph shows the results of the analysis of disordered protein regions. Many of these disordered regions overlap with low-complexity regions denoted by a green line. Low-complexity regions were predicted in http://smart.embl.de/. Disordered protein region probability was calculated in https://iupred2a.elte.hu/. B: amino acid sequence alignment of the COOH-terminal region of WNK4 of the human (KEGG entry 65266), rat (KEGG entry 287715), mouse (KEGG entry 69847), cat (KEGG entry 101100264), chicken (KEGG entry 777580), American alligator (KEGG entry 102565768), African clawed frog (KEGG entry 108701024), zebrafish (KEGG entry 100330953), and whale shark (KEGG entry 109912281). Numbers at the top represent the residue numbers of human WNK4. Different sites are indicated by arrows, such as critical residues for WNK-WNK binding located within the CT-CCD (153), familial hyperkalemic hypertension (FHHt) mutations (172, 188), calmodulin (CaM)-binding site (101), PKC/PKA/serum/glucocorticoid regulated kinase-1 (SGK1) phosphorylation sites (16, 124, 129), and a protein phosphatase-1 (PP1)-binding site (100). Alignment was generated in Clustal Omega (EMBL-EBI). KEGG, Kyoto Encyclopedia of Genes and Genomes.

Analysis of degree of conservation among different members of the WNK family shows sequence homology in three regions of the protein (Fig. 1A). These regions are also predicted to be globular domains by Globplot2 (http://globplot.embl.de/) and lie outside the predicted low-complexity regions shown in Fig. 1A. The first conserved region comprises the kinase domain, the first Pask-Fray 2-like (PF2-like) domain (initially described as the autoinhibitory domain) (24, 25), and the acidic domain (4). The second conserved region corresponds to the second PF2-like domain, termed PF2-like′ by Gagnon et al. (24, 25). The third conserved region includes the COOH-terminal coiled-coil domain (CT-CCD; Fig. 1B) (26).

Below, we describe the regions mentioned above as well as additional domains and motifs that are important for WNK4 function. We also integrate mechanistic insights derived from studies of other WNK kinases that may be applicable to WNK4 at the structural and functional levels.

Unique Features of the WNK Kinase Domain

The most conserved region among the WNK kinases is the kinase domain, with ∼90% sequence identity between WNK1, WNK2, and WNK3. The kinase domain of WNK4 appears to be the most divergent one, with an average 82% conservation score with respect to other WNKs. The WNK1 kinase domain crystal structure was first reported in 2004 and provided details for the mechanism of action of the WNK kinase family (27). Due to the high degree of homology, the same structural features may apply to the kinase domain of WNK4.

With a 25% identity to the kinase domains of other serine/threonine kinases, the kinase domain of WNK1 presents a similar overall fold with a dual-lobe architecture and 12 conserved subdomains (27). The COOH-terminal lobe has a standard general architecture. However, in the NH₂-terminal lobe, the β-sheet found in other kinases has an additional β-strand in WNK1 and is rolled up, forming a nearly complete β-barrel.

Regarding the active site, WNK kinases present several particular features. As stated above, one of them is the absence of the catalytic lysine in subdomain II. The crystal structure of WNK1 confirmed that Lys²³³ (Lys¹⁸⁶ in hWNK4) that emanates from β-strand 2 (subdomain I) is positioned in the active site, replacing the ATP-binding function of the catalytic lysine found in β-strand 3 (subdomain II) in other kinases. Another distinctive feature is the presence of a DLG motif in subdomain VII instead of the DFG motif found in most protein kinases. The aspartic acid residue of this motif is involved in the binding of Mg²⁺ that interacts with ATP’s β- and γ-phosphates and is important for catalysis. Interestingly, the leucine residue of the unique DLG motif of WNK kinases has been shown to be involved in the coordination of Cl⁻, whose binding stabilizes WNK1 in an inactive conformation (5). Substitution of this leucine residue with phenylalanine promotes higher levels of kinase autophosphorylation and prevents kinase inhibition at increasing NaCl concentrations. WNK kinases were long thought to be regulated, directly or indirectly, by intracellular Cl⁻ concentration, due to their ability to modulate the activation state of CCCs. The Cl⁻-sensing protein turned out to be the WNK kinase domain itself. In addition to Leu³⁶⁹ of the DLG motif, other residues involved in Cl⁻ coordination are Phe²⁸³, Leu²⁹⁹, and Leu³⁷¹, which establish hydrophobic interactions with the Cl⁻ ion (Fig. 2). The backbone amides of Gly³⁷⁰ and Leu³⁷¹ also establish hydrogen bonds with the Cl⁻ ion. Although at the structural level this site has only been described for WNK1, the observation that mutation of Leu³²² of hWNK4 (equivalent to Leu³⁶⁹ of WNK1) also decreases its sensitivity to inhibition by Cl⁻ suggests that this anion also binds to WNK4 in a structurally similar binding pocket (28).

Figure 2.

The with no lysine kinase (WNK) 4 kinase domain shares predicted structural features with WNK1. A: structural alignment of the WNK1 kinase domain (PDB 4Q2A) with the predicted structure of the kinase domain of WNK4 obtained from the sequence homology-based server http://raptorx.uchicago.edu/. B, inset: residues whose backbones amides and lateral chains are involved in the coordination of the Cl⁻ anion. Black labels indicate these amino acid residues in WNK1. Leu³²² in blue denotes the experimentally validated residue involved in Cl⁻ sensing in WNK4.

Finally, two residues, Ser³⁷⁸ and Ser³⁸², have been shown to be trans-autophosphorylated in the activation loop of WNK1 (also known as the T-loop). These correspond to Ser³³¹ and Ser³³⁵ in hWNK4 (29, 30). As with other kinases, phosphorylation of the activation loop promotes kinase activation because it induces the correct positioning of the catalytic loop and other structures within the active site (31). Phosphorylation at Ser³⁸² of WNK1 has been shown to increase with maneuvers that promote kinase activation like exposure to hyperosmotic stress (29, 30). Thus, evaluation of phosphorylation levels of this site has been used as a surrogate to evaluate kinase activity.

PF2-Like Domains

All WNK kinases share the presence of a PF2-like domain immediately after their kinase domain. In SPAK and OSR1, the best-known substrates of WNK kinases, two regulatory domains are found within their COOH-terminal region, which were named PF1 and PF2 (32). PASK was the name originally assigned to SPAK by the group that initially cloned it from the rat (33). Fray is the name of the Drosophila homolog (34). The PF2 domain in SPAK and OSR1 (also called CCT) can bind motifs with the consensus sequence Arg-Phe-Xxx-Val/Ile (RFxV/I) (35). These RFxV/I motifs are present in WNKs themselves and in CCCs, and they are essential to establish interactions with SPAK and OSR1 (36–39).

The first description of the PF2-like domain in WNK1 identified it as an autoinhibitory region. It was shown that the isolated domain had the ability to suppress the activity of WNK1’s kinase domain (29). Later, Gagnon and Delpire noticed that this domain was homologous to the PF2 domain of SPAK and OSR1 and thus coined the term PF2-like (Fig. 3A) (24, 40). The solution of its structure revealed that indeed it has a similar fold to that of the PF2 domain of SPAK and OSR1 (25, 35). It was also shown that this domain is able to bind RFxV/I-containing peptides with micromolar affinity (25), through similar interactions to those established between RFxV/I motifs and OSR1 (35). Gagnon and Delpire also identified another region in WNK kinases with homology to PF2 domains and referred to it as PF2-like′ (24). This region corresponds to the second conserved region among WNK kinases shown in Fig. 1A. For simplicity, we here refer to the PF2-like domains of WNK kinases as PF2a and PF2b.

Figure 3.

Sequence, structural, and functional analysis of the predicted PF2 domains in with no lysine kinase (WNK)4. A: sequence homology analysis guided by structural information of the PF2a domain of WNK1 (2LRU) reveals high degree of conservation with PF2 domains of all WNK members and oxidative stress responsive kinase-1 (OSR1). Arrows indicate the conservation of the residues involved in the recognition of the RFxV/I motifs. All sequences correspond to human proteins. B: structural alignment of the PF2a domain of WNK1 with the predicted structure of WNK4 PF2a and PF2b domains. Structural prediction was obtained by http://raptorx.uchicago.edu/. The predicted structures for both WNK4 PF2 domains show the groove where the RFxV/I motifs bind. R2 and F3 indicate the positions of the Arg and Phe residues of the GRFQVT peptide (93). C: representative Western blot of coimmunoprecipitation of human WNK4 and STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) coexpressed in human embryonic kidney (HEK)-293 cells. While the second RFxV/I motif found in mWNK4 (site 2, residues 1016-1019) drives the association of WNK4-SPAK, mutation of key residues within the PF2a (F476A,F478A) or PF2b (F703A,F705A) domains impacts the ability of WNK4 to optimally phosphorylate SPAK at Ser³⁷³. Similar results were observed in two independent experiments. D: the attributed function of WNK4’s PF2 domains remain to be discovered. To assess whether these domains are important for WNK multimerization, we used a FLAG-tagged full-length mouse WNK4 clone (FLAG-mWNK4 F.L.) and a FLAG-tagged truncated mWNK4 clone at residue 996 (FLAG-mWNK4 996X) that lacks the coiled-coil domain (CCD) as well as RFxV/I site 2. In this last clone, mutations of PF2 domains’ key residues were introduced individually or together (same mutations as those tested in C). E: by means of coimmunoprecipitation, we tested the interaction between FLAG-tagged WNK4 proteins and a HA-tagged mWNK4 full-length protein. We found that HA-mWNK4 F.L. interacted strongly with FLAG-mWNK4 F.L. (lane 2), but its ability to interact with mWNK4-996X decreased considerably (lane 3), probably due to lack of the COOH-terminal (CT)-CCD (153). However, mutation of the PF2 domains in FLAG-mWNK4-996X individually or together did not further decrease the interaction (lanes 4−6). This suggests that PF2 domains are not essential for multimerization of WNK4, and their function remains to be uncovered. Similar results were observed in two independent experiments.

Modeling of the PF2a and PF2b domains of WNK4 suggests that they can fold in a similar way to other PF2 domains whose structure has been described (Fig. 3B) (24). Through sequence alignment analysis of the WNK4 PF2 domains with those of WNK1 and OSR1, it is possible to note the conservation of several key residues (Fig. 3A). For OSR1’s PF2 domain, residues Asp⁴⁵⁹, Phe⁴⁵², and Ile⁴⁵⁰ drive the recognition of RFxV/I motifs within the groove formed by the β2-α1 interface (35). The same residues are observed to mediate interactions with the RFxV/I peptide in the WNK1-PF2a structure (corresponding to Asp⁵³¹, Phe⁵²⁴, and Ile⁵²² in WNK1) (25). Such residues are conserved in both PF2 domains of WNK4 as well (Fig. 3A), thus suggesting an inherent ability to recognize RFxV/I motifs.

The autoinhibitory role has been the only function attributed to the PF2a domain of WNK kinases, yet this function was proposed before it was identified as a PF2-like domain. More recently, we have shown that mutation of key residues within the PF2b domain of WNK4 impair its ability to phosphorylate SPAK (41), suggesting that it plays a positive role for kinase function. This effect is not due to impaired interaction with SPAK, as coimmunoprecipitation is still observed (Fig. 3C). Mutation of key residues within the PF2a domain of WNK4 also affect its ability to phosphorylate SPAK without impairing binding (Fig. 3C). Formation of homo- and heteromers among WNK monomers is key to their activity. For instance, it has been shown that mutation of two residues within the CT-CCD of WNKs prevents interaction between WNK monomers (26) and also prevents, for example, the ability of WNK1 to promote NCC activation in Xenopus laevis oocytes (42). Thus, we tested the hypothesis that PF2 domains in WNKs could also be important for mediating WNK-WNK interactions as WNK proteins contain one or more RFxV/I motifs. However, mutation of key residues within PF2a, PF2b, or both domains did not affect interaction between WNK4 monomers (Fig. 3, D and E).

RFxV/I Motifs

As mentioned above, the PF2 domains of SPAK and OSR1 have been shown to mediate interactions with RFxV/I motifs present in CCCs and WNK kinases (36–39). Regarding RFxV/I motifs in WNK kinases, five RFxV/I motifs are distributed along the sequence of WNK1, three can be found in WNK3, and only two are found in WNK2 and WNK4. In all cases, one of these motifs is located in the kinase domain, whereas the additional motifs are located within the COOH-terminal domain. In WNK3, mutation of each one of the three RFxV/I motifs has shown that absence of the motif located within the kinase domain impairs WNK3’s ability to activate NCC and Na⁺-K⁺-Cl⁻ cotransporters (NKCCs) and inhibit K⁺-Cl⁻ cotransporter 3 (KCC3) without impairing kinase activity. In contrast, mutation of the COOH-terminal RFxV/I motifs does not affect these functions (36). The kinase domain RFxV/I motif of WNK3 is conserved in WNK1 and WNK2 and localizes between α-helices 2 and 3. However, in WNK4, a glutamic acid replaces the valine in the last position of the RFxV/I motif. Instead, the kinase domain RFxV/I motif in WNK4 localizes before the α-helix 7 toward the end of the kinase domain. This motif is not conserved in other WNK members, as the phenylalanine is replaced by a tyrosine residue. In WNK4, this RFTI motif is conserved in mammals but changes to RYTI in reptiles, amphibians, birds, and fishes (Fig. 4A). We attempted to address the contributions of both RFxV/I motifs for WNK4’s ability to bind and phosphorylate SPAK. Transfection of WNK4 mutants for each of the RFxV/I motifs showed that, whereas the second motif is required for SPAK binding, the first is necessary for SPAK activation (Fig. 4B). Notably, the phenylalanine in the RFTI motif in WNK4 can be replaced by a tyrosine without affecting WNK4’s ability to phosphorylate SPAK (Fig. 4, C and D), consistent with the conservation seen in other vertebrates. This also suggests that this is not a bona fide SPAK-binding site, as the PF2 domain is unable to bind RYxV motifs (38).

Figure 4.

Sequence conservation and functional analysis of RFxV/I motifs in with no lysine kinase 4 (WNK4). A: multiple sequence alignment of WNK4 regions encompassing RFxV/I sites 1 and 2. Site 2 shows high degree of conservation, whereas site 1 is conserved in mammals but diverges in the second position (F changes to Y) in other classes. B: coimmunoprecipitation assay of STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and WNK4 proteins with mutated RFxV/I sites. SPAK and human (h)WNK4 clones were coexpressed in human embryonic kidney (HEK)-293 cells. Wild-type and mutant WNK4 proteins were immunoprecipitated, and their interaction with SPAK was assessed by Western blot. Appreciable interaction of SPAK with wild-type WNK4 and the WNK4 site 1 mutant (F421A) was observed. Nevertheless, only wild-type WNK4 was able to mediate SPAK phosphorylation at Ser³⁷³. Conversely, the WNK4 RFxV/I site 2 mutant (RF-1016,1017-AA) and the double mutant were unable to interact with SPAK. Similar results were observed in three independent experiments. C: the first RFXV/I motif in WNK4 is not a SPAK-binding site. Mutations affecting RFxV/I site 1 were introduced in a WNK4 clone that also carries the L321F mutation that affects the Cl⁻-binding site and thus impairs inhibition of kinase activity by Cl⁻. Expression of hWNK4-L321F in HEK-293 cells promoted SPAK Ser³⁷³ phosphorylation, whereas this effect was decreased with the F421A but not with F421Y mutation. As Tyr in this position would also affect binding to a PF2 domain (117), this suggests that RFxV/I site 1 is not a SPAK-binding site. Instead, it seems that the aromatic rings of Tyr and Phe establish key interactions that allow maintaining a functional structure. D: densitometric analysis of experiments corresponding to Fig. 1C (n = 3). *P < 0.05.

The observation that the second RFxV/I motif of WNK4 is a bona fide SPAK-binding site might help us to understand the pathophysiology of nonsense mutations in WNK4 found in Burmese cats, as mutant WNK4 in these animals lacks this SPAK-binding motif and therefore behaves as loss of function (12).

Acidic Motif

Most of the FHHt-causative missense mutations in WNK4 characterized so far lie within a 10-amino acid region highly enriched in negatively charged amino acids that is known as the acidic motif (4). One major breakthrough in understanding how WNK4 mutations caused FHHt came with the identification of mutations in KLHL3 and CUL3, which are components of an E3 ubiquitin ligase complex (16). Normally, WNK4 is targeted for degradation through ubiquitination in at least 15 sites. However, mutations found in the acidic motif of WNK4, which constitutes the binding site for KLHL3, prevent the association with the KLHL3-CUL3 E3 complex leading to protein accumulation (19, 20). The crystal structure of the KLHL3 Kelch domain together with the acidic domain of WNK4 provided definitive evidence on the role of the acidic motif (43).

Coiled-Coil Domains

WNK4 contains two coiled-coil domains (CCD). The first CCD lies immediately after the PF2a domain, whereas the second is located toward the COOH terminus (CT-CCD). These are regions that present sequence conservation in all WNK kinases (Fig. 1). The first CCD has not been described to exert any regulatory function yet. Thastrup and colleagues showed that the CT-CCD mediates the formation of homo- and heteromers between WNK kinases (26). Through an alanine-scanning mutagenesis analysis, it was possible to identify that residues His¹¹⁴⁵, Glu¹¹⁴⁸, and Gln¹¹⁵⁶ in hWNK4 are essential for the interaction with WNK1. These three residues have a high degree of conservation in all WNK kinases from different species, including WNK1 from Caenorhabditis elegans. It was also shown that WNKs can form high-molecular-mass complexes as detected by size exclusion chromatography, and this is in part dependent on the CT-CCD. The multimerization dependent on the CT-CCD is essential for the functional role of WNK4 over SPAK/OSR1-CCC pathway (42).

Conserved COOH-Terminal Region

From the end of the CT-CCD domain until the end of the protein there is a region with a length of ∼70 amino acids in WNK4 that is highly conserved in WNK4 orthologs across species of diverse vertebrate classes, including mammals, reptiles, birds, amphibians, and fishes (Fig. 1B). Within this region, several functional motifs are found. For instance, three phosphorylation sites that lie within RRxS motifs are present, whose modification has been shown to affect WNK4 function (see Regulatory Mechanisms below) (44–46). A calmodulin (CaM)-binding site has also been described in which the first RRxS motif is included. Interestingly, two dominant FHHt-causative mutations have been found within this region: K1169E and R1185C. Arg¹¹⁸⁵ also lies within the proposed CaM-binding site and just five residues upstream of the first COOH-terminal RRxS phosphorylation site (Ser¹¹⁹⁰ in hWNK4 and Ser¹¹⁶⁹ in mWNK4).

Phosphorylation of RRxS sites has been shown to promote WNK4-mediated SPAK phosphorylation (44), to promote WNK4-mediated NKCC2 activation (45), and to prevent the WNK4-mediated NCC inhibition that is observed on basal conditions in X. laevis oocytes (46). Deletion of the CaM-binding site has also been shown to promote WNK4-mediated NKCC2 activation (45). Thus, it has been proposed that this region of the protein may play a negative regulatory role on WNK4 kinase activity that may be relieved by phosphorylation of RRxS sites, FHHt mutations, or CaM binding in the presence of Ca²⁺.

Finally, a protein phosphatase-1 (PP1)-binding site has been identified within the last 12 residues of WNK4. Absence of this site promotes hyperphosphorylation and constitutive activation of WNK4 in HEK-293 cells (41). For more details regarding these observations, see Regulatory Mechanisms below.

Kidney-Specific Short Forms of WNK4

Our group first reported the presence of short versions of WNK4 specifically in lysates of kidney tissue (41). These short forms are not observed in other tissues, and they lack the COOH-terminal region of WNK4, which contains several regulatory motifs including the SPAK-binding motif (RFxV/I). Thus, according to in vitro experiments with truncated WNK4 mutants, these short forms are predicted to be unable to phosphorylate and activate SPAK.

The presence of short isoforms has been previously described for SPAK (47). These appear to be, at least in part, the product of a proteolytic event (48). In a similar manner, WNK4 short isoforms appear to originate from a proteolytic event mediated by a Zn²⁺-dependent metalloprotease (41). The cleavage site was shown to lie within the amino acids 740–781. The abundance of these WNK4 short forms is not modulated by changes in Na⁺ or K⁺ intake, maneuvers that are known to modulate NCC activity. Thus, further characterization is necessary to decipher their functional contribution to the regulation of renal electrolyte transport.

WNK4 IN PHYSIOLOGY AND PATHOPHYSIOLOGY

Expression Pattern

Initial analysis of the pattern of expression of WNK4 in humans indicated that this gene is highly expressed in the kidney, as determined by Northern blot (4), although it was also found in the colon and skin by RT-PCR (3). However, later RT-PCR assays in mouse tissues showed WNK4 expression in the kidney, testis, colon, heart, liver, brain, lung, and spleen (49). Knockout mouse-validated protein expression has been observed in the kidney, testis, lung, and brain (41).

In the kidney, immunostaining assays showed that WNK4 is present in podocytes, the cortical thick ascending limb of Henle’s loop (cTAL), the DCT, and cortical and medullary collecting ducts (50). Regarding subcellular localization, it was originally described that WNK4 localized to tight junctions of renal epithelial cells based on colocalization with zonula occludens 1 (ZO-1) protein (4). However, more recent immunostaining experiments performed with knockout-validated WNK4 antibodies have not confirmed this observation (23, 44, 50, 51). In the work by Ohno et al., strong staining was observed in the cytoplasmic subapical region of DCT, connecting tubule, and cortical thick ascending limb cells as well as principal cells of the cortical collecting duct. No colocalization was observed with ZO-1 in the DCT (50). In works by other authors, however, staining of kidney sections from mice maintained on standard conditions gives a very mild signal (most times indistinguishable from background signal) (23, 44, 51). However, in tissues from mice exposed to a low-K⁺ diet or volume depleted, conditions in which DCT Na⁺ reabsorption is stimulated, a strong punctuate cytoplasmic signal is observed in DCT cells (23, 44, 51). These structures have been termed “WNK bodies” as explained below in more detail.

Familial Hyperkalemic Hypertension

The DCT of the nephron participates in the regulation of electrolyte homeostasis, as it is involved in the renal handling of Na⁺, Cl⁻, Mg²⁺, and Ca²⁺. Specifically, the DCT mediates transcellular reabsorption of 5–10% of the filtered Na⁺ and Cl⁻ that cross the apical membrane via NCC (52). Interestingly, NCC also regulates K⁺ homeostasis, given that its activity indirectly modulates K⁺ secretion by principal cells in the aldosterone-sensitive distal nephron (ASDN) (53). This effect could be mediated by nephron remodeling (54). The importance of NCC in electrolyte and blood pressure homeostasis as well as acid-base balance became clear by the description of monogenic diseases affecting NCC activity, directly or indirectly, such as Gitelman syndrome and FHHt, respectively.

FHHt was initially described in 1970 (55). As the name indicates, it is a disease characterized by hypertension and hyperkalemia as well as hyperchloremic metabolic acidosis and normo- or hypercalciuria. Since the FHHt phenotype is opposite to the one observed in Gitelman syndrome [caused by loss-of-function mutations in the gene encoding NCC and characterized by hypokalemia, hypovolemia, metabolic alkalosis, and hypocalciuria (56)], and since it is corrected by a low dose of thiazide diuretics (specific inhibitors of NCC) (13, 57), it is thought that the main cause for the whole FHHt phenotype is NCC overactivation. This has been further supported by observations made in a transgenic mouse model where increased NCC activity is enough to promote the whole spectrum of FHHt abnormalities (54).

Despite NCC’s involvement in FHHt, no mutations in the gene encoding this protein have been described as a cause for FHHt. Instead, mutations in genes that regulate NCC function have been found in patients with FHHt (Table 1), such as WNK1, WNK4 (4, 59), KLHL3, and CUL3 (16, 17).

Table 1.

Genetic mutations found in patients with FHHt

Gene	Mutation	Mendelian Inheritance	Proposed Mechanism	Corresponding Mouse Model
WNK1	Deletions in intron 1 (4)	Autosomal dominant	Ectopic L-WNK1 expression specifically in the DCT (58)	Wnk1FHHt/+ (intron 1 deletion) (58)
	Acidic domain missense mutations: E631K, A634T, D635E, D635N, Q636E, Q636R (59)	Autosomal dominant	Decreased KS-WNK1 degradation in the DCT (59)	Wnk1delE631/+ (59)
WNK4	Acidic domain missense mutations: E562K, D564A, Q565E (4), D564H (60), P561L (61), and E560G (62)	Autosomal dominant	Decreased WNK4 degradation in the DCT (20)	Wnk4+/+/Q562E/Q562E (63); Wnk4D561A/+ (64)
	COOH-terminal missense mutations: R1185C (4) and K1169E (65)	Autosomal dominant	Disruption of the inhibitory domain, promoting increased WNK4 activity (45)	Not reported yet
KLHL3	Several missense mutations (16,17)	Autosomal dominant	Decreased WNK4/KS-WNK1 degradation in the DCT (66,67)	Klhl3R528H/+ (66);Klhl3M131V/+ (68)
	Several missense mutations (16,17) as well as nonsense mutations and splicing-altering mutations (16)	Autosomal recessive	Decreased WNK4/KS-WNK1 degradation in the DCT (69)	Klhl3-/- (69)
CUL3	Mutations in sites implicated in splicing of exon 9: intron 8 splice acceptor, intron 9 splice donor, putative intron 8 splice branch site, and a putative splice enhancer in exon 9 (12)	Autosomal dominant	Decreased WNK4/KS-WNK1 degradation in the DCT (70); impaired vascular relaxation through activation of the RhoA-ROCK pathway (71)	Cul3Δ403-459/+ (72);Cul3Het/Δ9 (70); pgk-Cul3Δ9 (71)

WNK, with no lysine kinase; KLH3, Kelch-like family member 3; CUL3, cullin 3; L-WNK1, full-length with WNK1; KS-WNK1 kidney-specific WNK1; DCT, distal convoluted tubule; ROCK, Rho kinase; FHHt, familial hyperkalemic hypertension.

FHHt due to mutations in WNK4 is an autosomal dominant trait with high penetrance. Most FHHt mutations within WNK4 are missense mutations that affect the acidic motif, such as E562K, D564A, Q565E (4), D564H (60), P561L (61), and E560G (62). As mentioned above, WNK4 mutations in the acidic motif disrupt its interaction with KLHL3 and prevent WNK4 degradation (18–20). A similar mechanism has been proposed to explain WNK4 activation by mutations in KLHL3 and CUL3, where WNK4 protein levels are increased in the DCT, driving downstream activation of SPAK/OSR1 and ultimately NCC (66, 70, 72).

Additionally, FHHt-causative mutations within the COOH-terminal region of WNK4 have also been described: K1169E (65) and R1185C (4). Although it is not clear how these particular mutations affect the WNK4-SPAK/OSR1-NCC pathway, a possibility is that these mutations may eliminate the inhibitory properties of the COOH terminus of WNK4 (41, 45, 73) toward its kinase activity and/or downstream signaling (see Structural Features above).

Murine Models With Altered WNK4 Function

To recapitulate the phenotype of patients with FHHt, Lalioti et al. generated a mouse strain harboring two transgenic copies of the FHHt mutant WNK4 Q562E, in addition to endogenous copies of WNK4 (Table 2) (63). These mice displayed high blood pressure levels, hyperkalemia, metabolic acidosis, and hypercalciuria. DCT hyperplasia was also reported. As expected, the phenotype of WNK4^{+/+/Q562E/Q562E} mice was completely reversed by genetic or pharmacological disruption of NCC, suggesting that FHHt is mainly driven by increased activity of this transporter in the DCT.

Table 2.

Genetically modified mouse models with altered WNK4 expression/function

Mouse Model	Mutation	Effect on Protein Function and/or Levels	Phenotype	Original Report
Transgenic WNK4+/+/Q562E/Q562E	Two additional copies of the WNK4 gene harboring FHHt mutation Q562E (equivalent to Q565E in human WNK4)	Higher WNK4 levels (19)	FHHt-like: increased blood pressure, hyperkalemia, metabolic acidosis, and hypercalciuria as well as DCT hyperplasia	(63)
WNK4D561A/+	D561A (equivalent to D564A in human WNK4)	Higher WNK4 levels, due to decreased degradation (20)	FHHt-like: increased blood pressure, hyperkalemia, hyperchloremic metabolic acidosis, higher NCC levels, and phosphorylation	(64)
WNK4 hypomorphic	Exon 7 deletion	Abnormal splicing between exons 6 and 9, with a protein lacking 14 kDa in the middle portion. WNK4ΔEx7-8 has decreased kinase activity	Gitelman-like: lower blood pressure and decreased OSR1 and NCC phosphorylation	(74)
WNK4-/-	Deletion of exon 1	Absent WNK4 expression	Gitelman-like: hypokalemia, hypochloremic metabolic alkalosis, hypomagnesemia, increased plasma renin activity; virtually absent NCC phosphorylation and lower NCC levels (both at mRNA and protein levels)	(14)
Transgenic (WNK4WT)	Additional copies of WNK4 (2 copies or 30 extra copies)	Higher WNK4 protein levels	FHHt-like: high blood pressure, hyperkalemia with hyperchloremic metabolic acidosis, higher SPAK, OSR1 and NCC expression. and phosphorylation	(20)
WNK4-/-	Deletion of exon 2	Absent WNK4 expression	Gitelman-like: lower blood pressure while on a low-NaCl diet and decreased SPAK and NCC expression and phosphorylation	(15)
WNK4-/-	Deletion of exons 1 and 2	Absent WNK4 expression	Gitelman-like: lower NCC expression and phosphorylation	(75)
WNK4L319F,L321F/L319F,L321F	L319F and L321F	Cl−-insensitive WNK4 and therefore, constitutively active kinase	FHHt-like: increased blood pressure, higher plasma K+ concentration, and hyperchloremic metabolic acidosis, with higher NCC expression and phosphorylation	(76)

WNK, with no lysine kinase; FHHt, familial hyperkalemic hypertension; DCT, distal convoluted tubule; NCC, NaCl cotransporter; OSR1, oxidative stress-responsive 1; SPAK, STE20/SPS1-related proline-alanine-rich protein kinase.

In a second FHHt mouse model, the D561A mutation was introduced in WNK4 (64). Heterozygous WNK4^+/D561A mice displayed higher blood pressure, hyperkalemia, hyperchloremia, and metabolic acidosis compared with WNK4^+/+ mice as well as increased total and phosphorylated levels of NCC and higher SPAK and OSR1 phosphorylation. Subsequent analysis also showed increased WNK4 protein levels in the kidneys of these mice (20). Accordingly, transgenic mice with additional copies of the WNK4 gene (either 2 or 30 extra copies) showed higher WNK4 protein levels, as expected, as well as increased downstream phosphorylation of SPAK/OSR1 and NCC, which caused hypertension, hyperkalemia, and metabolic acidosis (20). These observations showed that FHHt-causing mutations in WNK4 promote increased protein levels of WNK4 that are responsible for the FHHt phenotype.

As initial attempts to generate WNK4 knockout mice were not successful, hypomorphic WNK4 mice were generated by disrupting exon 7, which was expected to produce a COOH-terminally truncated WNK4 protein. However, splicing between exons 6 and 9 was observed, which had no frameshift, producing a WNK4 protein lacking the middle portion of WNK4, specifically, part of the PF2-like domain, the first CCD, and the acidic domain (74). Kinase assays showed that this mutant WNK4 immunoprecipitated from human embryonic kidney (HEK)-293 cells and mouse kidneys had a diminished kinase activity toward itself and SPAK. Hypomorphic WNK4 mice displayed mild hypotension as well as mildly decreased levels of phosphorylated (p)OSR1 and NCC. This was the first in vivo evidence that hinted at the positive modulation of NCC by WNK4. Right after FHHt mutations were described in WNK4, initial observations made in certain in vitro and in vivo models suggested that WNK4 had an inhibitory role on NCC activity that was reversed by FHHt mutations (63, 77, 78). However, these observations contrasted with the data later obtained by Vitari et al., showing that WNK4 can phosphorylate and activate SPAK and OSR1 in in vitro kinase assays (9), which, in turn, can phosphorylate and activate NCC (79).

Definitive confirmation of WNK4 as an activator of NCC came with the first report of a WNK4 knockout (−/−) mouse model generated by disruption of exon 1. These mice displayed a phenotype similar to that of Gitelman syndrome (14). Compared with their wild-type (WT) siblings, WNK4^−/− mice showed hypokalemia, hypochloremia, metabolic alkalosis, hypomagnesemia, and increased plasma renin activity. Hypokalemia was greatly accentuated by a low-K⁺ diet. WNK4^−/− mice had lower levels of NCC (both at transcript and protein levels) and virtually undetectable pNCC accompanied by a blunted response to thiazide diuretics. Increased response to amiloride suggested epithelial Na⁺ channel (ENaC) activation as a mechanism to compensate for NCC deficiency. Later, a second WNK4^−/− mouse model, with disruption of exon 2, was reported (15). These mice displayed a similar phenotype, showing a reduction of NCC expression and activity, with lower blood pressure levels while under a low-Na⁺ diet. Interestingly, these mice did not show hypokalemia in basal conditions, possibly due to an effect of the genetic background, sample collection, and/or differences of the diet. Notably, DCT morphological changes were observed in these mice, specifically tubule dilation and lower cell height. A third WNK4^−/− mouse model with exon 1 and 2 deletion showed similar findings with respect to total and pNCC levels as well as blunted thiazide response (75). These mice also did not show hypokalemia while under a control diet; however, plasma K⁺ concentration was lower while under a low-K⁺ diet compared with WT mice, further confirming that WNK4 plays an important role in the modulation of K⁺ homeostasis.

Even though WNK4 is expressed in a variety of organs, it is puzzling that WNK4^−/− mice have a relatively mild phenotype, with alterations of renal origin only. This might suggest that other WNK kinases might have the ability to compensate for the absence of WNK4 in other cells. Of note, although the expected Mendelian frequencies were observed among the progeny of reproductive crosses between WNK4^+/− mice, it was reported that WNK4^−/− mice, when crossed with each other, had a lower number of offspring (14). It is currently unknown whether this observation is related to a neurological or reproductive defect or both.

In 2019, a mouse model generated with CRISPR/Cas9 was reported where specific missense mutations were introduced in exon 3 of WNK4, causing the substitutions L319F and L321F in the WNK4 protein [Chen et al. (76)]. In vitro, these changes render WNK4 constitutively active, as it no longer has the ability to bind Cl⁻ (see Regulatory Mechanisms below). As expected, these mice showed a FHHt-like phenotype, with increased blood pressure and higher plasma K⁺ and Cl⁻ concentrations as well as metabolic acidosis, and higher levels of pSPAK/OSR1 and total and pNCC.

All of these studies in murine models have been instrumental in establishing WNK4 as a major positive regulator of SPAK/OSR1-NCC in the DCT in vivo, where this pathway is regulated by different mechanisms that will be detailed in later sections.

Interaction With Downstream Kinases SPAK and OSR1

As mentioned above, WNK4 promotes NCC activation by phosphorylating and activating the intermediate kinases SPAK and OSR1, which, in turn, phosphorylate NCC. The initial description of the participation of SPAK and OSR1 in this pathway came when Piechotta et al. (38) identified the interaction of both kinases with the NH₂ terminus of multiple CCCs, mediated by the RFxV/I motifs present in the latter. Later on, Dowd and Forbush showed in HEK-293 cells that overexpression of wild-type SPAK significantly increased NKCC1 activity, which correlated with increased cotransporter phosphorylation, and that kinase-inactive SPAK exerted dominant-negative effects on NKCC1 function, inhibiting activity and phosphorylation (80).

A couple of years later, a link was made between WNK and SPAK/OSR1 activity, when Vitari et al. (9) discovered that SPAK and OSR1 were among the proteins interacting with WNK1 and WNK4 and, through kinase assays, showed that the kinase domains of WNK1 and WNK4 were able to phosphorylate and increase the activity of SPAK and OSR1. Similar observations were made simultaneously by Moriguchi et al. (81). Two phosphorylation residues targeted by WNK kinases were found: Thr²³³ and Ser³⁷³ in hSPAK and Thr¹⁸⁵ and Ser³²⁵ in hOSR1. The first of these residues lies in the kinases’ T-loop and is critical for its activation. Meanwhile, the mutation of the second residue did not affect kinase activity (9), but its phosphorylation has been used as a readout of WNK activity toward SPAK/OSR1 (44, 64, 82, 83). A recent report has suggested that phosphorylation of this site might be involved in decreased SPAK degradation mediated by CUL4 (84).

In Vivo Models With Genetically Modified SPAK and OSR1 Reveal the Physiological Importance of These Kinases for the Modulation of NCC by WNK4

Several mouse models with gain- and loss-of-function mutations in SPAK or OSR1 have reinforced the concept that these kinases lie upstream to CCC phosphorylation. In addition, as explained below, these models have also been useful to demonstrate, for example, that SPAK and OSR1 serve as substrates of WNK4 in the DCT and that most of the function of WNK4 within this nephron segment depends on SPAK/OSR1 phosphorylation.

In 2010, Rafiqi et al. (85) reported that C57BL/6 mice carrying an inactive version of SPAK (SPAK^T243A/T243A mice) have diminished phosphorylation of NCC, NKCC1, and NKCC2 in the kidney. The decrease in NCC activity appears to predominate, as the mice display a Gitelman syndrome-like phenotype with hypocalciuria instead of the characteristic hypercalciuria observed in patients with Bartter syndrome who have dysfunctional NKCC2. Other Gitelman syndrome-like features observed were hypotension and hypokalemia (the latter was observed only under a low-Na⁺ diet) (85). A few years later, several groups reported the generation of SPAK^−/− mouse models, all of which also presented a Gitelman syndrome-like phenotype (47, 86, 87).

Attempts to generate a global OSR1 loss-of-function knockin or knockout mouse model have not been successful, as complete lack of function of this kinase is lethal during embryonic life (85, 87). However, heterozygous deletion of the kinase and kidney-specific knockout models have been studied. OSR1^+/− mice are hypotensive but have no differences in plasma or urinary electrolytes (87). Meanwhile, kidney-specific OSR1 knockout mice (KS-OSR1^−/−) are normotensive on a normal Na⁺ diet but display hypokalemia and lower urinary osmolality, reduced sensitivity to loop diuretics, along with hypercalciuria and diminishment of pNKCC2, with an increase in total NCC and pNCC (88). Thus, it was proposed that OSR1 activity is more relevant for NKCC2 regulation in the thick ascending limb of Henle’s loop, whereas in the DCT its absence can be compensated for by SPAK activity. In contrast, SPAK plays a more relevant role for NCC regulation in the DCT, where OSR1 cannot fully compensate for the absence of SPAK activity.

Nevertheless, further evidence has shown that OSR1 does play a role in NCC regulation and in the pathophysiology of FHHt caused by overexpression of WNK4. For instance, Chiga et al. (89) used WNK4^D561A/+ mice and crossbred them with SPAK^T243A/+ and OSR1^T185A/+ mice to analyze the effect of reducing or impairing SPAK and OSR1 activity on the FHHt phenotype. They observed that WNK4^D561A/+SPAK^T243A/T243A mice had an intermediate phenotype between wild-type and WNK4^D561A/+ mice (with systolic blood pressure, plasma K⁺ concentration levels, and pNCC levels lower than in WNK4^D561A/+ mice but higher than in wild-type mice). However, in WNK4^D561A/+SPAK^T243A/T243AOSR1^T185A/+ mice, systolic blood pressure, plasma K⁺ concentration levels, and pNCC levels were lower than in wild-type mice, showing that overactivation of both SPAK and OSR1 by WNK4 is responsible for the FHHt phenotype.

A similar strategy was performed with SPAK^−/− and KS-OSR1^−/− mice on a WNK4^D561A/+ background, where similar results were obtained (90). In this work, Chu et al. observed that WNK4^D561A/+SPAK^−/− mice had similar levels of NCC and pNCC to wild-type mice. Their systolic blood pressure and plasma electrolytes along with their thiazide-sensitive Na⁺ and Cl⁻ urinary excretion were normal. However, WNK4^D561A/+SPAK^−/− mice had higher levels of pNCC and NCC than SPAK^−/− mice, suggesting that most likely OSR1 was responsible for the observed phosphorylation. Indeed, pNCC and NCC were further reduced in triple mutants (WNK4^D561A/+SPAK^−/−KS-OSR1^−/−).

Additional evidence pointing to a relevant role for OSR1 in DCT physiology comes from observations made in mice in which NCC activity is stimulated by administration of a low-K⁺ diet. As discussed below in Role of Wnk4 in the Regulation of NCC by Physiological Stimuli, low K⁺ intake is one of the more potent stimuli for NCC activation, and WNK4 plays a key role in the signaling pathway that mediates this effect (75, 91). Interestingly, low K⁺-induced activation of NCC can be clearly observed in SPAK^T243A/T243A, SPAK^−/−, and KS-OSR1^−/− mice (91, 92), whereas activation is dramatically reduced in double-knockout mice (SPAK^−/−KS-OSR1^−/−) (92).

Finally, Grimm et al. used an elegant strategy to generate an in vivo model of gain of function of SPAK in the early DCT (54). These mice express constitutively active SPAK exclusively in parvalbumin-expressing cells, which in the kidney are restricted to DCT cells. All other cell types are effectively SPAK null. The mice were named CA-SPAK and were shown to develop the classic FHHt phenotype with hyperkalemia, hypertension, and acidosis as well as a lower fractional urinary excretion of K⁺, all of which resolved with thiazides. These findings correlated with a significant increase in NCC and pNCC and an increase in the area and length of the DCT1 at the expense of a reduction of these measurements in the CNT. Thus, this model demonstrated that SPAK hyperactivity in the DCT is sufficient to produce FHHt and that NCC hyperactivity is sufficient to drive morphological changes in the ASDN.

In summary, as can be noted, mouse models of SPAK gain or loss of function have many similarities in their renal phenotype with WNK4 gain- or loss-of-function mouse models, respectively, as is expected for proteins that are in the same signaling pathway. Taken together, all these findings strongly suggest that the main mechanism through which WNK4 mediates both the phosphorylation of NCC and pathogenesis of FHHt is through the phosphorylation and activation of SPAK and OSR1.

SPAK/OSR1-Independent Regulation of Phosphorylation of CCCs

The presence of PF2-like domains in WNK4 led to the in vitro finding of WNK4 being able to bind and directly phosphorylate NKCC1 and NKCC2 in the residues known to be targeted by SPAK/OSR1 when overexpressed in conjunction with Cab39/MO25 in X. laevis oocytes (93). Whether direct phosphorylation of CCCs by WNK4 is physiologically relevant in any in vivo condition remains unknown.

WNK4 Functions Not Involving NCC

In addition to the best-described role of WNK4 in the regulation of the SPAK/OSR1-NCC pathway, some works, mainly performed in in vitro systems, have shown that WNK4 can regulate other membrane transport proteins, many of which also participate in renal electrolyte handling (Fig. 5). Below, we make a brief mention of some of these findings.

Figure 5.

Proposed roles of with no lysine kinase 4 (WNK4) in the nephron. Works by different groups have suggested the participation of WNK4 in the regulation of multiple kidney transport proteins. A: in the thick ascending limb of the loop of Henle, WNK4 has been reported to positively regulate the activity of Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2) through the phosphorylation of STE20/SPS1-related proline-alanine-rich protein kinase (SPAK)/oxidative stress-responsive 1 (OSR1). Although a role for WNK4 in the regulation of renal outer medullary K⁺ channels (ROMK) has been postulated, it is unknown whether WNK4 specifically modulates this channel in the thick ascending limb. B: in the distal convoluted tubule (DCT), WNK4 is a positive regulator of NaCl cotransporter (NCC) through the phosphorylation of SPAK/OSR1. It also seems to play a role in Ca²⁺ handling through the positive regulation of transient receptor potential vanilloid 5 (TRPV5) channels. C: in the principal cells of the aldosterone-sensitive distal nephron (ASDN), WNK4 has been implicated as a negative regulator of electrogenic Na⁺ reabsorption and K⁺ secretion through epithelial Na⁺ channels (ENaC) and ROMK/large-conductance K⁺ (BK) channels, respectively. D: the Cl⁻/${\text{HCO}}_3^{-}$ antiporter pendrin has been postulated to be upregulated by WNK4, at least in the context of familial hyperkalemic hypertension (FHHt). No mechanisms are known for this phenomenon (figure created with Biorender.com).

The SLC12 Family of Cotransporters

CCCs of the SLC12 family participate in a wide range of physiological processes (6, 94), which include the regulation of epithelial transport, regulation of intracellular Cl⁻ concentration of excitable cells, regulation of cellular volume, etc. The seven well-characterized members of the SLC12 family (NCC, NKCC1, NKCC2, KCC1, KCC2, KCC3, and KCC4) are all subject to regulation by SPAK/OSR1-mediated phosphorylation (79, 80, 95, 96), and WNK4 is expressed in multiple tissues where different CCCs are expressed (3, 41, 97). Thus, it is possible that WNK4 could participate in the physiological regulation of CCCs other than NCC.

In vitro experiments have shown a possible role of WNK4 in the regulation of most members of the SLC12 family. For instance, it can increase the activity of NKCC1 and NKCC2 in X. laevis oocytes (45, 93, 98) and decrease the activity of KCCs (99). WNK4-induced phosphorylation of NKCC2 can also be observed in the HEK-293 cell system (100). However, only a couple of studies have analyzed the possible role of WNK4 in the regulation of these cotransporters in vivo.

For instance, a work by Terker et al. has recently suggested a role for WNK4 in NKCC2 regulation (100) (Fig. 5A). Given that WNK4^−/− mice present normocalciuria, Terker and coworkers hypothesized that this could be due to the additive dysfunction of NKCC2 and NCC, since the dysfunction of the former leads to hypercalciuria and dysfunction of the latter leads to hypocalciuria. They observed that WNK4^−/− mice have markedly decreased pNKCC2 levels in Western blot assays. However, recent findings have shown that, in mice bred on the C57BL/6 background (such as WNK4^−/− mice), the most frequently used antibodies targeting pNKCC2 (Thr⁹⁶/Thr¹⁰¹) mostly recognize NCC unspecifically in immunoblots (101). This is due to a genetic deletion of 15 bp encoding the highly conserved residues 96–100 of mouse (m)NKCC2 that prevents binding of the phosphoantibodies to NKCC2. Given this previously unknown fact, pNKCC2 levels in WNK4^−/− mice should be reassessed. However, a slight decrease in pNKCC2 was also observed by immunofluorescence in NKCC2-positive tubules by using antibodies directed against pThr⁹⁶/Thr¹⁰¹ and another phosphoacceptor site that is not affected by the deletion (pSer⁹¹).

Melo et al. also used WNK4^−/− mice on a low-Na⁺ diet to show that WNK4 was not required for the upregulation of KCC4 in this condition (102). Whether the absence of WNK4 leads to different levels of baseline total or pKCC4 was not examined.

Epithelial Na⁺ Channels

The description of FHHt-causative mutations in WNK4 and the renal origin of FHHt gave place to research focused not only on WNK4’s regulation of NCC but also of other transport proteins in the nephron whose dysregulation could potentially contribute to the observed phenotype. One of these proteins was ENaC. ENaC is a heterotrimeric amiloride-sensitive channel formed by three subunits (α, β, and γ) expressed in many tissues. Within the kidney, it is expressed in principal cells of the ASDN, where its activity drives Na⁺ reabsorption and K⁺ secretion (103, 104). Heterologous overexpression in X. laevis oocytes showed that coexpression of WNK4 with ENaC subunits led to a reduction of amiloride-sensitive currents (Fig. 5C). This was preserved when catalytically inactive versions of WNK4 were coexpressed (105). Other in vitro studies in distal tubular cells showed that WNK4 facilitates the internalization of ENaC independently of Nedd4-2 (106).

WNK4^−/− and WNK4 hypomorphic mice show an increase in ENaC function (14, 74), consistent with these inhibitory properties. However, it is unclear whether this is a direct consequence of the absence of WNK4 or an adaptation that occurs to compensate for NCC dysfunction. In the FHHt WNK4^{+/+/Q562E/Q562E} mouse model, in which WNK4 is overexpressed, an inverse phenomenon is observed: ENaC subunit levels and amiloride-sensitive currents are diminished in the kidneys of these animals (107). Interestingly, CA-SPAK mice also have a decreased abundance of ENaC subunits in total kidney lysates, which can be reversed with thiazides (54). This model strongly suggests that NCC overactivation is sufficient to produce a compensatory decrease in ENaC expression levels.

Renal Outer Medullary K⁺ Channels

Kir1.1, also known as the renal outer medullary K⁺ channel (ROMK), mediates both K⁺ recycling in the thick ascending limb of the loop of Henle and K⁺ secretion in the ASDN when coupled with the activity of ENaC (108).

Kahle et al. showed that WNK4 is an inhibitor of ROMK in X. laevis oocytes (109) (Fig. 5C). FHHt versions of WNK4 were more efficient inhibitors of ROMK in this system. This effect was also independent of the kinase activity of WNK4, was prevented by the phosphorylation of Ser¹¹⁶⁹ of WNK4 (105), and was shown to be mediated by intersectin, a scaffolding protein that participates in vesicle endocytosis (110). The phosphorylation of tyrosine residues in WNK4 (Tyr¹⁰⁹² and Tyr¹¹⁴³) by c-Src have been postulated to enhance ROMK inhibition by WNK4 (111, 112). WNK4^{+/+/Q562E/Q562E} animals have been recently found to possess lower activities of ROMK in patch-clamp, single-channel current experiments of their tubular cells (107), giving in vivo proof of the regulation of this channel by WNK4 in whole organisms. How much of a contribution is made to the FHHt phenotype by the direct dysregulation of ENaC and ROMK is not understood, since overactivity of WNK4 in the DCT is both necessary (63) and sufficient (54) to induce the phenotype in its totality.

Large-Conductance K⁺ or Maxi-K Channels

Large-conductance K⁺ (BK) channels are Ca²⁺-activated K⁺ channels involved in the regulation of neuronal excitability. They have also been described as flow-activated K⁺ channels that mediate K⁺ secretion in the ASDN (108).

WNK4 can inhibit BK channels in HEK-293 cells expressing α- and β-BK channel subunits (Fig. 5C). Unlike what has been observed for ENaC and ROMK, this inhibition is dependent on its kinase activity (113). Inhibition is achieved through a reduction in total and cell surface BK subunits through lysosomal degradation, as was shown by the rescue of the proteins with treatment with bafilomycin A1 or leupeptin. Wang et al. (114) found that the first CCD and first PF2-like domain of WNK4 (residues 1–584 of mWNK4) are necessary for its inhibition of BK channels. It is difficult to reconcile this knowledge with the recent finding of SPAK increasing BK channel activity (115), which is at odds with a WNK4 kinase-dependent inhibition of BK.

Claudins

Claudins are some of the main components of tight junctions, can facilitate paracellular epithelial transport of specific ions, and affect transepithelial resistance (116).

In mammalian cells expressing WNK4, claudin-1, -2, -3, and -4 have been coimmunoprecipitated with WNK4, and mutant WNK4 (D564A in hWNK4) showed an enhanced interaction with these tight-junction components. The presence of WNK4 also increased the phosphorylation of these claudins in different cell lines, with WNK4-D564A showing higher activity than wild-type WNK4 (117). Transepithelial Cl⁻ permeability was increased in cells expressing wild-type WNK4 and even more so in cells expressing FHHt mutants (49, 117). In these cell systems, WNK4 FHHt mutants may have been expressed at higher levels than wild-type WNK4. However, this was not assessed by the authors.

Tatum et al. later found that WNK4 colocalizes with claudin-7 in kidney tubules and coimmunoprecipitated both proteins from native tissues (Fig. 5C). Ser²⁰⁶ in claudin-7 was characterized as a putative WNK4 phosphoacceptor site. Its phosphorylation was determined to increase in the presence of wild-type WNK4 and to a greater extent with WNK4-Q562E, correlating with a decrease in transepithelial resistance (118). Conversely, immortalized collecting duct cells from mice lacking claudin-7 were shown to have increased transepithelial resistance. Interestingly, greater levels of WNK4 at the mRNA and protein levels were observed in these cells. This was speculated to occur as part of a compensatory mechanism to promote claudin-7 function (119).

Transient Receptor Potential Vanilloid Channels

The transient receptor potential (TRP) vanilloid (TRPV) subfamily of channels, which belongs to the TRP superfamily, is composed of a group of Ca²⁺-permeable channels that, in the kidney, can mediate Ca²⁺ reabsorption and have been postulated to play a role in osmolality and flow sensing (119a). In other cells, they play other important roles, such as heat and pain perception (119b).

Of all the TRPV channels, TRPV4 and TRPV5 have been shown to be modulated by WNK4. Regarding TRPV4, downregulation of the channel occurs when it is coexpressed with WNK4 in HEK-293 cells through a decrease in the surface expression of the channel, which cannot be activated by hypotonicity in the presence of this kinase (120).

Regarding TRPV5 (Fig. 5B), conflicting conclusions have been reached by different groups. In X. laevis oocytes, TRPV5 Ca²⁺ currents are stimulated in the presence of WNK4. A dose-dependent inhibition of WNK4 stimulation of TRPV5 occurs with increasing doses of NCC, which could possibly be mediated by an increase in intracellular Cl⁻ (121). FHHt mutants of WNK4 show similar activation of this channel. WNK4 might facilitate the forward trafficking of TRPV5 to the plasma membrane (121). On the other hand, negative regulation of TRPV5 by WNK4 has also been reported, with HEK-293 cells expressing WNK4 having an enhanced endocytosis of the channel (122). In vivo, lack of WNK4 causes a substantial reduction in TRPV5 measured by immunofluorescence. Furosemide-sensitive calciuria is enhanced in WNK4^−/− mice, which might be the consequence of a decrease in Ca²⁺ reabsorption proteins of the distal nephron, including TRPV5 (123). It must be noted that this finding goes in the opposite direction of the hypocalciuria normally observed with NCC downregulation or inhibition.

Cystic Fibrosis Transmembrane Conductance Regulator Channels

Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl⁻/${\text{HCO}}_3^{-}$-permeable channel that is expressed in many epithelial and glandular cells, where, in concert with the activity of NKCC1, it mediates Cl⁻ and water secretion (124). The regulation of CFTR by WNK4 is unclear. An initial report showed that, in X. laevis oocytes, WNK4 downregulated the presence of CFTR in the plasma membrane independently of its kinase activity in a dose-dependent manner (125). A more recent report in mammalian cells showed an enhancement of surface expression of CFTR by WNK4, also independently of kinase activity (126).

Pendrin

Pendrin is an anion antiporter involved in ear and thyroid physiology (127). In the kidney, it mediates Cl⁻/${\text{HCO}}_3^{-}$ exchange in β-intercalated cells and thus participates in ${\text{HCO}}_3^{-}$ secretion in the collecting duct. It has also been shown to mediate electroneutral NaCl reabsorption when its activity is coupled to Na⁺/Cl⁻/${\text{HCO}}_3^{-}$ transport mediated by the Na⁺-driven Cl⁻/${\text{HCO}}_3^{-}$ exchanger (128) and has been implicated in Na⁺ retention by aldosterone (129).

WNK4^{+/+/Q562E/Q562E} animals have been shown to have increased pendrin activity in their β-intercalated cells and increased β-intercalated cell mass (Fig. 5D) (130). The authors of that work argued that this protein could potentially contribute to metabolic acidosis and Na⁺ retention in FHHt, as deletion of pendrin in WNK4^{+/+/Q562E/Q562E} mice led to normalization of plasma ${\text{HCO}}_3^{-}$ and K⁺ and an increase in renin mRNA levels. It is unknown whether the pathophysiological dysregulation of pendrin observed in WNK4^{+/+/Q562E/Q562E} mice occurs in all models of FHHt and whether it is mediated directly by WNK4. The only published in vitro assay of pendrin function in the presence of WNK4 was made in X. laevis oocytes, where no effect was seen with coexpression of both proteins (97).

REGULATORY MECHANISMS OF WNK4 FUNCTION

WNK4 function is regulated at different levels. At the transcriptional level, no information is yet available about the regulation of WNK4 mRNA levels. Posttranslationally, however, a pathway for regulation of WNK4 protein levels has been well described. This pathway involves regulation of the WNK4 degradation rate by the CUL3-KLHL3 E3 ubiquitin ligase complex. In addition, kinase activity of WNK4 is also tightly regulated, and more than one mechanism appears to be involved (Fig. 6).

Figure 6.

Regulatory mechanisms of with no lysine kinase (WNK)4 function. In the distal convoluted tubule (DCT), WNK4 protein levels are regulated by the activity of the cullin 3 (CUL3)-Kelch-like family member 3 (KLHL3) E3 complex, which targets WNK4 for degradation by promoting its ubiquitylation at several sites (94, 136). KLHL3 binds to the acidic motif of WNK kinases through its propeller domain. Another level of regulation involves the modulation of WNK4 kinase activity. Several mechanisms have been described that participate in this regulation, which are the following. 1) Binding of Cl⁻ to a pocket within the active site of the kinase stabilizes an inactive conformation and prevents kinase autophosphorylation (4, 115). Intracellular Cl⁻ concentration levels are thus determinant on WNK4 activity. 2) WNK4 contains five phosphorylation sites within a RRxS motif, two located in the NH₂-terminal domain and three located in a COOH-terminal region. All five sites can be phosphorylated in vitro by PKC and PKA (16), and at least the three COOH-terminal sites can be phosphorylated by serum/glucocorticoid regulated kinase-1 (SGK1) (101, 124, 129). Phosphorylation levels of two of these sites (Ser⁶⁴ and Ser¹¹⁹⁶) correlate with levels of kinase activity [measured by its ability to autophosphorylate and to phosphorylate STE20/SPS1-related proline-alanine-rich protein kinase (SPAK)]. Phosphoablative mutations at Ser⁶⁴ and Ser¹¹⁹⁶ prevent WNK4-mediated phosphorylation of SPAK in response to cretain stimuli, like ANG II (16). 3) A protein phosphatase 1 (PP1)-binding site has been described close to the COOH-terminal end of the protein. Absence of this motif promotes WNK4 hyperphosphorylation and constitutive activation in cultured cells (100). 4) Kidney-specific (KS-)WNK1 interacts with WNK4 through the COOH-terminal coiled-coil domain (CT-CCD). Coexpression of KS-WNK1 in Xenopus laevis oocytes promotes WNK4 autophosphorylation and activation by a mechanism that is currently unknown (3). WNK1 mutations that produce amino acid substitutions within the acidic domain produce a mild familial hyperkalemic hypertension (FHHt) phenotype in humans and mice that has been speculated to be due to increased KS-WNK1-mediated activation of WNK4 (83) (figure created with Biorender.com).

Regulation of the WNK4 Degradation Rate by the CUL3-KLHL3 E3 Complex in the Kidney

Cullin-RING ligases (CRLs) are ubiquitin E3 enzymes that regulate the ubiquitylation of a wide variety of substrates. It has been estimated that ∼300 different CRL complexes may exist in humans (131), each of them with different substrate specificities that are determined by the cullin subtype and substrate adaptor molecule that make up the complex.

As mentioned above, mutations in the genes encoding for two proteins that are part of a CRL complex are responsible for the most severe forms of FHHt (16, 17). These are CUL3, the protein that forms the heterodimeric scaffold of the CRL complex, and KLHL3, the protein that acts as the substrate adaptor and that also binds to the NH₂-terminal domain of CUL3. Following the description of these mutations, it was rapidly identified that WNK kinases are substrates for the CUL3-KLHL3 E3 ligase complex. It was shown that, in vitro, KLHL3 can bind WNK1, WNK2, WNK3, and WNK4 (18–20), that the CUL3-KLHL3 E3 complex can promote ubiquitylation of WNK1 and WNK4 (18–20), and that this decreases WNK4 protein levels in cultured cells, suggesting that ubiquitylation promotes proteasomal degradation (19, 20). The observed binding, ubiquitylation, and degradation were impaired when tested with KLHL3 or WNK4 FHHt mutants. The acidic motif of WNK kinases, in which FHHt mutations in WNK4 are clustered, was shown to function as the binding site for KLHL3 (18).

In mutant mice carrying WNK4-FHHt mutations, increased renal levels of WNK4 were observed (19, 20), and in mutant mice carrying the KLHL3-R528H and KLHL3-M131V FHHt mutations, increased WNK4 and WNK1 renal expression levels were observed (66, 68). Given that KLHL3 expression in the kidney is largely restricted to the DCT (17, 132, 133), this upregulation is likely to exclusively occur within this nephron segment. Interestingly, it was shown that impaired activity of the CUL3-KLHL3 E3 complex (in KLHL3^−/− mice) increased WNK1 and WNK4 expression in the kidney but did not affect their expression levels in extrarenal tissues (134). Thus, in other tissues, WNK1 and WNK4 levels may not be subject to CRL-mediated regulation or, alternatively, they could be subject to regulation by a different CRL complex. A third possibility, given that KLHL3 expression is not kidney specific, is that KLHL3 absence may be compensated for by another KLHL protein. For instance, KLHL2 has been shown to be able to bind and promote ubiquitylation of WNK kinases (135). KLHL2^−/− mice were shown to display higher WNK4 expression levels in the renal medulla but not in the renal cortex. Expression levels of WNK kinases in extrarenal tissues were not reported.

Finally, it is interesting to note that the upregulation of SPAK and NCC activity (measured as phosphorylation) observed in KLHL3-R528H mice was completely prevented when these mice were crossed with WNK4^−/− mice, despite persistent WNK1 overexpression (67). Thus, the increased WNK1 levels were unable to compensate for WNK4 absence, and WNK4 seems to be key in the pathogenesis of FHHt caused by mutations in KLHL3. It remains to be determined whether the overexpressed WNK1 reported by Susa et al. corresponds to full-length WNK1 (L-WNK1) or the KS-WNK1 isoform. Interestingly, it has been recently shown that KS-WNK1 is much more sensitive to CUL3-KLHL3 E3-induced degradation than L-WNK1 (59).

All FHHt-causative mutations in CUL3 characterized so far cluster in sites implicated in splicing of exon 9 and cause the skipping of this exon. Thus, a protein with a 57-amino acid deletion is produced (CUL-Δ403–459) (16). It has been shown that expression of CUL3-Δ403–459 protein prevents ubiquitylation of WNK kinases even in the presence of wild-type CUL3 (72). CUL3^{+/Δ403–459} mice display higher levels of WNK4 protein, which drives downstream phosphorylation and activations of SPAK and NCC, causing FHHt. As CUL3-Δ403–459 promotes its autoubiquitylation and degradation and, thus, CUL3^{+/Δ403–459} mice have lower levels of CUL3 expression, it was initially proposed that haploinsufficiency was the cause for FHHt. However, it was later shown that Cul3^+/− (CUL3^Het) mice did not have a FHHt-like phenotype. In contrast, Cul3^Het mice that also carried a copy of a transgene that allowed inducible expression of CUL3-Δ403–459 (which were identified as CUL3^HET/Δ9 mice) did develop the FHHt phenotype. CUL3 protein levels were similarly decreased in both CUL3^Het and CUL3^HET/Δ9 mice. However, only CUL3^HET/Δ9 mice had higher WNK4, NCC, and pNCC protein levels (70). These results suggested that CUL3-Δ^403–459 protein has a dominant-negative effect on CUL3-KLHL3 E3-mediated degradation of WNK kinases, resulting in higher WNK4 levels and increased downstream activation of NCC.

FHHt-causative CUL3 mutations are responsible for the most severe form of FHHt (16). There is evidence that such severe phenotype may be due, at least in part, to additive effects of altered CUL3 function of renal electrolyte handling and vascular function (71). However, given that CUL3 expression seems to be ubiquitous, the absence of other manifestations of extrarenal origin are intriguing.

WNK4 Kinase Activity Is Sensitive to Intracellular Cl⁻ Levels

Modulation of the activity of CCCs by intracellular Cl⁻ levels is a well-known phenomenon supported by a large body of experimental evidence (136–139). Initial observations were made as far back as 1983, when John Russell described that intracellular Cl⁻ inhibited bumetanide-sensitive Na⁺-K⁺-Cl⁻ transport in the squid giant axon (140).

Later on, modulation of CCC activity by intracellular Cl⁻ concentration was shown to be related to their phosphorylation state (137, 141), and SPAK and OSR1 were shown to be the kinases responsible for such phosphorylation (38, 80). Thus, when WNK kinases were described as the upstream regulators of SPAK and OSR1 (9, 81), it was immediately tested and confirmed that WNK activity is upregulated by intracellular Cl⁻ depletion (81).

Direct modulation of WNK activity by Cl⁻ was not confirmed, however, until Piala and collaborators (5) described the crystallographic structure of the WNK1 kinase domain and the existence of a Cl⁻-binding site within the catalytic site of the protein. This site is structurally similar to the Cl⁻-binding sites found in Cl⁻ channels of the ClC family, with interactions to backbone amides and lateral chains of hydrophobic residues. It was shown that Cl⁻ binding stabilizes an inactive conformation of the kinase, preventing kinase autophosphorylation and activation. Mutation of one of the residues that makes up the Cl⁻-binding site (Leu³⁶⁹) decreased the inhibition of autophosphorylation observed at increasing concentrations of Cl⁻.

Bazua-Valenti et al. showed that the ability of WNK4 to activate NCC is also modulated by intracellular Cl⁻ concentration (28). This observation helped to resolve the long-lasting controversy of whether WNK4 acts as a positive or negative modulator of NCC. According to observations of Bazua-Valenti et al., in the X. laevis oocyte system, in which initial characterization of WNK4-regulatory activity of CCCs was performed, the intracellular Cl⁻ concentration is high, which renders WNK4 inactive. Thus, under basal conditions, WNK4 expression exerts an inhibitory effect on NCC activity, probably due to a dominant-negative effect over the endogenous WNK kinase or kinases (42). However, decreasing intracellular Cl⁻ concentrations by different maneuvers promoted WNK4 activation, measured as kinase autophosphorylation and through its ability to activate NCC (28). In addition, introduction of mutation L322F in hWNK4 (the residue equivalent to Leu³⁶⁹ in WNK1) rendered the kinase constitutively active.

Bazua-Valenti et al.’s work and a later work by Terker et al. suggested that WNK kinases, despite having very similar kinase domains, present different sensitivities to inhibition by Cl⁻. Interestingly, WNK4 has the more divergent kinase domain (with ∼80% identity with other WNKs, whereas the kinase domains of other WNKs have ∼90% identity among them) (6) and appears to be the family member that is more sensitive to inhibition by Cl⁻ (28, 83). It has been suggested that such high affinity of WNK4 for Cl⁻ is important for its physiological role in the regulation of NCC, given that intracellular Cl⁻ concentration in the DCT has been estimated to be rather low (142–144). Thus, the low intracellular Cl⁻ concentration of DCT cells allows WNK4 to remain active under basal conditions and allows rapid modulation of its activity in response to changes in intracellular Cl⁻ concentration. Definitive proof that modulation of WNK4 activity by intracellular Cl⁻ concentration is physiologically relevant, at least in the DCT, came with the description of a mouse model carrying the L319F/L321F-WNK4 mutation (L322F/L324F in hWNK4) (76). As mentioned above in WNK4 in Physiology and Pathophysiology, these mice present higher levels of NCC activity accompanied by the expected FHHt phenotype.

WNK4 Activity Is Modulated by Phosphorylation of Residues Located in Its Regulatory NH₂- and COOH-Terminal Domains

The initial description of a phosphorylation site located within the regulatory COOH-terminal domain of WNK4, whose modification affects WNK4 function, was made by Ring et al. (105). This site, Ser¹¹⁶⁹ (in mouse WNK4), was identified through a search for serum/glucocorticoid-regulated kinase-1 (SGK1) consensus phosphorylation motifs, as it was hypothesized that WNK4 may act as a transducer of aldosterone signaling in the distal nephron. In in vitro kinase assays, it was shown that SGK1 can indeed phosphorylate this WNK4 residue. Additionally, it was shown that introduction of the phosphomimetic mutation S1169D impaired the ability of WNK4 to inhibit ENaC and ROMK in X. laevis oocytes.

Later on, the works by Rozansky et al. and Na et al. described two additional phosphorylation sites within the COOH terminus of WNK4 that can be phosphorylated in vitro by SGK1: Ser¹¹⁸⁰ and Ser¹¹⁹⁶ in mouse WNK4 (Ser¹²⁰¹ and Ser¹²¹⁷ in hWNK4) (45, 46). As mentioned above (in Structural Features), these sites are clustered within a highly conserved region of WNK4 and are present from zebrafish to humans (44). Rozansky et al. showed that, in X. laevis oocytes, coexpression of SGK1 with WNK4 and NCC prevented WNK4 inhibitory activity on NCC and that the phosphomimetic mutant WNK4-S1169D/S1196D lost its ability to inhibit NCC (46). In addition, Na et al. showed, in the same expression system, that the phosphomimetic mutants S1169D/S1196D and S1169D/S1180D/S1196D had an enhanced positive effect on NKCC2 activity (45).

Several years later, we performed a comprehensive study of WNK4 phosphorylation sites by mass spectrometry analysis of WNK4 peptides generated from the immunoprecipitated protein from HEK-293 extracts (44). Eighteen phosphorylation sites were reproducibly identified, including the T-loop Ser³³² autophosphorylation site (Ser³³⁵ in hWNK4). We were particularly interested in five sites present within an RRxS motif, all of them located in segments of the protein that are highly conserved. These included, in addition to the Ser¹¹⁶⁹, Ser¹¹⁸⁰, and Ser¹¹⁹⁶ sites previously described as SGK1 targets, the Ser⁴⁷ and Ser⁶⁴ sites, located in the NH₂-terminal domain of the protein. Given that PKC and PKA show a preference for phosphorylation of sites within the RRxS sequence (145, 146), we explored the possibility that these sites might be targeted by these kinases. Indeed, we observed that pharmacological activation of PKC or PKA in HEK-293 cells promoted phosphorylation of all RRxS sites. The ability of PKC and PKA to phosphorylate these sites was also demonstrated in in vitro kinase assays. Importantly, phosphorylation of these sites was shown to promote WNK4 activation, measured by its ability to autophosphorylate and to phosphorylate SPAK. In particular, impaired phosphorylation of Ser⁶⁴ and Ser¹¹⁹⁶ had the greatest effect on WNK4 activity and kinase activity was completely abrogated in the S64A/S1196A double mutant. With the use of phosphospecific antibodies, we showed that these sites are phosphorylated in vivo in mouse kidney samples and that their phosphorylation levels increase in volume-depleted mice and in WNK4-FHHt mice. By immunofluorescent staining, the volume depletion-induced increase in phosphorylation at Ser⁶⁴ was observed to occur in the DCT.

Further studies will be necessary to determine whether these sites are mainly targeted in vivo by PKC, PKA, and/or SGK1. WNK4 phosphorylation by SGK1 was initially thought to occur in response to aldosterone stimulation (46, 105), since SGK1 expression is regulated by aldosterone (147). However, the sensitivity of DCT cells to aldosterone stimulation has recently been questioned (see Role of WNK4 in the Regulation of NCC by Physiological Stimuli) (148, 149). Thus, aldosterone-mediated regulation of WNK4 phosphorylation may only occur in the ASDN, which excludes the DCT. Within the DCT, phosphorylation of these sites may occur through aldosterone-independent regulated activity of SGK1 or through activity of PKC and/or PKA.

Possible Regulation of WNK4 Activity by Calmodulin Binding

Within the conserved region in which the COOH-terminal RRxS sites are located, Na et al. have additionally described the presence of a binding site for CaM (45). Their in vitro experiments demonstrated that CaM binds to the 1175–1194 segment of hWNK4 (1154–1173 of mWNK4), which presents a consensus CaM-binding site sequence, and that binding requires the presence of Ca²⁺. Deletion of the CaM-binding site in WNK4 prevented binding and also increased the ability of WNK4 to activate NKCC2 in X. laevis oocytes. On the basis of those observations, Na and coworkers proposed that this segment of the protein exerts a negative effect on WNK4 activity that may be relieved by binding to Ca²⁺/CaM or by RRxS phosphorylation, although demonstration that WNK4 activity increases in the presence of Ca²⁺/CaM is still missing. Indeed, Yang et al. have previously described a “negative regulatory signal region” within the last 47 amino acids of WNK4 (73).

Regulation of WNK4 Activity by PP1

Protein phosphatases target many components of the WNK-SPAK/OSR1-CCC pathway. In WNK4, there are two predicted PP1 sites with the consensus sequence KxVxF. Lin and colleagues described that PP1 binds to mouse WNK4 in amino acid regions 695–699 and 1211–1215, which both contain PP1-binding sites (150). In 2018, our group showed that the interaction between WNK4 and PP1 still occurred even in the absence of the two PP1-binding sites previously identified by Lin et al. (41). This suggested that other regions in WNK4 may contribute to the binding of PP1, or perhaps indirect binding through other endogenous WNKs was observed. However, mutation or deletion of the second PP1 motif near the COOH terminus of WNK4 resulted in a gain of function with increased WNK4 phosphorylation levels at the T-loop site Ser³³² (in mWNK4) and RRxS sites as well as increased WNK4-mediated phosphorylation of SPAK at Ser³⁷³. Accordingly, coimmunoprecipitation between PP1 and a shorter WNK4 COOH-terminal construct showed specific binding to the second PP1 motif, confirming it as a bona fide PP1-binding site that negatively modulates WNK4 phosphorylation and activity. PP1 α- and γ-isoforms, but not the β-isoform, were shown to promote dephosphorylation of WNK4 RRxS sites.

Interestingly, mutation of the first PP1 motif in WNK4 ablated the gain of function caused by the mutation of the second PP1 motif. Since this first PP1 motif lies within the predicted PF2b domain of WNK4 (see Structural Features), this raises the question of whether this mutation disrupts the function of the PF2 instead of preventing PP1 binding. Notably, this site is conserved in other WNKs, and it has been shown that it does not mediate PP1 binding in the case of WNK1 (151). In addition to PP1 sites, WNK4 also contains predicted PP2A and PP2B motifs, but none of them have been functionally described (http://elm.eu.org/).

Regulation of WNK4 Activity by Interaction With KS-WNK1

KS-WNK1 is a short isoform of WNK1 that is produced by transcriptional initiation at an upstream region regulated by an alternative promoter located within intron 4 of the WNK1 gene. Transcription from this promoter introduces into the transcript an alternative exon known as exon 4a. Thus, KS-WNK1 differs from L-WNK1 in that it lacks the segment encoded by exons 1−4 (that comprise most of the kinase domain) and contains a unique 30-residue segment in its NH₂ terminus encoded by exon 4a (152, 153). As the name indicates, this isoform is expressed only in the kidney, and its transcript levels are much higher in the DCT than in other nephron segments, although low levels have also been observed in the CNT (154).

Despite lacking a kinase domain, work from our group showed that KS-WNK1 can promote NCC activation when expressed in X. laevis oocytes (155). Such activation seems to be dependent on the interaction with an endogenous WNK kinase, as it is prevented by WNK463 (a specific inhibitor of WNK kinase activity). Coexpression of KS-WNK1 with WNK4 in the oocyte system promotes WNK4 autophosphorylation at Ser³³². The effect is not observed when a KS-WNK1 clone with mutations in the CT-CCD that prevent binding to WNK4 is expressed (26). Thus, interaction of WNK4 with KS-WNK1 within the DCT may be key to modulate WNK4 activity. Interestingly, it has recently been shown that missense mutations in the acidic domain of WNK1 produce a mild form of FHHt (59), and it was suggested that this phenotype is probably due to upregulation of KS-WNK1 protein expression, which then leads to increased activation of WNK4-SPAK/OSR1-NCC. This proposal is based on the following observations: 1) KS-WNK1 is more sensitive to CUL3-KLHL3 E3-induced degradation than L-WNK1 (59), and 2) KS-WNK1 is much more abundant in the DCT than L-WNK1. At the transcript level, the abundance of KS-WNK1 is 80 times higher than that of L-WNK1 (154). At the protein level, certain lines of evidence suggest that, in the DCT, L-WNK1 levels may be negligible. This is best highlighted by the effects of knockout of WNK4 in two different FHHt mouse models. In the KLHL3-R528H model, knockout of WNK4 completely abrogates NCC activity (measured by phosphorylation) (67). Western blots show that KLHL3-R528H mice have higher renal WNK1 levels. Although it was unclear to which isoform they correspond, it may be deduced that WNK1 upregulation in the DCT probably corresponds to KS-WNK1, as it does not compensate for the absence of WNK4. In contrast, in the WNK1-FHHt model (with an intronic deletion in WNK1 that promotes ectopic expression of L-WNK1 in the DCT), knockout of WNK4 does not prevent the pathway upregulation (42). Thus, in this model, the pathological ectopic expression of L-WNK1 appears to override the WNK4-mediated regulation of NCC. It is noteworthy that L-WNK1 is less sensitive to inhibition by Cl⁻ than WNK4 and thus may be constitutively active in intracellular Cl⁻ concentration levels of the DCT.

ROLE OF WNK4 IN THE REGULATION OF NCC BY PHYSIOLOGICAL STIMULI

NCC is regulated by multiple physiological stimuli (156). Given that, as explained above, WNK4 appears to be the major WNK kinase responsible for SPAK/OSR1 phosphorylation and activation in the DCT, this kinase plays a central role in many of the signaling pathways that transduce the extracellular signals that modulate NCC activity.

Extracellular K⁺ Concentration

Dietary K⁺ intake levels have been correlated with the level of NCC activity (assessed by its phosphorylation status). In rodent models, higher levels of NCC and pNCC are observed in animals on low-K⁺ diet (vs. control diet), and lower levels of NCC and pNCC are observed on high-K⁺ diet. Such modulation is triggered by subtle changes in extracellular K⁺ concentration that occur in response to the changes in dietary K⁺ content (51, 83, 157–159). Changes in extracellular K⁺ concentration affect the driving force for K⁺ movement through Kir4.1/Kir5.1 K⁺ channels expressed in the basolateral membrane of DCT cells (Fig. 7) (160, 161). Movement of K⁺ provokes changes in the membrane potential of DCT cells that, in turn, drive Cl⁻ fluxes through basolateral ClC-Kb channels (162, 163), ultimately affecting intracellular Cl⁻ concentration. These fluctuations in intracellular Cl⁻ concentration affect WNK4 activity and, thus, activity of the SPAK/OSR1-NCC pathway. Hence, a low-K⁺ diet promotes K⁺ efflux from DCT cells, membrane hyperpolarization, reduction of intracellular Cl⁻ concentration, and WNK4 activation; the opposite effects are produced by a high-K⁺ diet.

Figure 7.

Role of with no lysine kinase 4 (WNK4) in the regulation of NaCl cotransporter (NCC) by extracellular K⁺. In the face of hypokalemia, the extracellular K⁺ concentration ([K⁺]_e) gradient favors K⁺ exit from the distal convoluted tubule (DCT) cell through basolateral K⁺ channels formed by Kir4.1 and Kir5.1 subunits. This leads to membrane hyperpolarization, which, in turn, promotes exit of Cl⁻. The resulting decrease in intracellular Cl⁻ concentration ([Cl^-]_i) promotes WNK4 activation due to release of Cl⁻ from the Cl⁻-binding site, leading to increased STE20/SPS1-related proline-alanine-rich protein kinase (SPAK)/oxidative stress-responsive 1 (OSR1) and NCC phosphorylation and activation (152). An opposite mechanism may operate in hyperkalemia, although additional mechanisms are thought to participate in this setting. The decrease in [Cl⁻]_i induced by hypokalemia appears also to be responsible for the increase in WNK4 phosphorylation levels at RRxS sites that is observed in response to decreases in [K⁺]_e (99). The detailed mechanism is currently under investigation, but it may involve activation of PKC and/or PKA, given that these kinases can phosphorylate RRxS sites in vitro. In addition, phosphorylation of Kelch-like family member 3 (KLHL3) in the RRxS site located within the substate-binding propeller domain is also induced by decreases in [K⁺]_e (58). Given the similarity of this last mechanism to the regulation of WNK4 by phosphorylation at RRxS sites, it is possible that KLHL3-RRxS phosphorylation may also be secondary to [Cl⁻]_i depletion in the setting of hypokalemia (figure created with Biorender.com).

It has been proposed that among WNK kinases WNK4 is especially suited to participate in this mechanism, since, as mentioned above (see Regulatory Mechanisms), its sensitivity to inhibition by Cl⁻ is higher than that of other WNK kinases (28, 83). As DCT intracellular Cl⁻ concentration has been estimated to be rather low (142–144), it has been suggested that the other less sensitive WNK kinases would be constitutively active under these intracellular Cl⁻ concentration levels. The relevance that WNK4 plays in this regulatory mechanism was evidenced by the absence of upregulation of NCC phosphorylation in WNK4^−/− mice when placed on a low-K⁺ diet (75, 91). Consequently, mice developed severe hypokalemia. In addition, in mice expressing Cl⁻-insensitive WNK4 (harboring the L319F/L321F mutations), administration of a low-K⁺ diet did not promote NCC upregulation, and administration of an acute oral K⁺ load did not stimulate the NCC downregulation that was observed in wild-type controls, supporting the idea that the regulation of WNK4 activity by Cl⁻ is a key element in the signaling pathway for the regulation of NCC by extracellular K⁺ (76).

Besides the direct Cl⁻-sensing ability of WNK4, recent evidence from our group has shown that WNK4 phosphorylation at RRxS sites can be stimulated by a decrease in extracellular K⁺ concentration (164)_. This may be important to potentiate the activation of NCC. Interestingly, our data show that phosphorylation of these sites upon exposure to a decrease in extracellular K⁺ concentration is also secondary to the reduction in intracellular Cl⁻ concentration but is independent of WNK kinase activity. The molecular players that link intracellular Cl⁻ concentration depletion to WNK4 phosphorylation at RRxS sites are currently under investigation.

Finally, Ishizawa et al. have shown that CUL3-KLHL3 E3-induced degradation of WNK4 decreases in mice exposed to a low-K⁺ diet by a mechanism that involves KLHL3 phosphorylation at an RRxS site located within the substrate binding propeller domain (see Angiotensin II below) (165). Interestingly, such phosphorylation would prevent not only WNK4 degradation but also KS-WNK1 degradation (59). In 2018, Boyd-Shiwarski and colleagues coined the term “WNK bodies” to refer to WNK signaling complexes that are formed in the DCT under certain conditions (22). They also showed that KS-WNK1 expression is essential for the formation of WNK bodies, as these were absent in KS-WNK1^−/− mice. One of the conditions that have consistently been shown to induce the formation of WNK bodies is K⁺ deprivation (22, 51, 166). Thus, it is possible that KS-WNK1 induction under these conditions could promote the formation of these complexes. Interestingly, WNK bodies composed of WNK1 and SPAK/OSR1 are still formed in mice lacking WNK4; however, the phosphorylation of SPAK/OSR1 within the WNK bodies entirely depends on WNK4 (23). This evidence suggests that the localization of WNK4 to WNK bodies has functional relevance.

Aldosterone

Until relatively recently, NCC was thought to be a target of aldosterone. This idea was based on the observation that aldosterone infusion in rats increases NCC expression (167). The current view, however, is that aldosterone affects NCC activity indirectly, by affecting the levels of extracellular K⁺ concentration. That is, aldosterone promotes renal K⁺ secretion eventually leading to hypokalemia, and hypokalemia promotes NCC activation. This view is supported by the following observations. First, kidney-specific mineralocorticoid receptor (MR)-deficient mice have lower levels of NCC expression and activity that are reversed when hyperkalemia is corrected by a low-K⁺ diet (149). Second, in mice in which random deletion of MR occurs in only ∼20% of renal tubule cells (148), low-Na⁺ diet-induced upregulation of NCC and pNCC was observed in all cells irrespective of the absence or presence of MR. Third, 11β-hydroxysteroid dehydrogenase (11β-HSD) expression has been shown to be absent in the DCT of mice (168, 169), although this may vary between species (170, 171). This enzyme catalyzes the inactivation of glucocorticoids, thus preventing their binding to MRs. Cells that do not express 11β-HSD are aldosterone insensitive, because in these cells MR is mainly occupied by glucocorticoids that circulate in plasma at much higher levels than aldosterone. In contrast, in cells that express 11β-HSD, intracellular levels of glucocorticoids are much lower, and MR activity is mainly regulated by aldosterone.

All these pieces of evidence together have put into question the role that SGK1 plays on the regulation of the WNK4-SPAK/OSR1-NCC pathway. SGK1 expression is regulated by aldosterone in the ASDN, where phosphorylation of WNK4 by this enzyme could be important for the modulation of ENaC and ROMK. However, in the aldosterone-insensitive DCT, SGK1-mediated phosphorylation of WNK4 would have to be regulated by stimuli other than aldosterone. Alternatively, SGK1 target sites may be phosphorylated by another kinase. As mentioned above, we have observed that phosphorylation of RRxS sites (including Ser¹¹⁹⁶) increases when extracellular K⁺ concentration decreases (164). Thus, aldosterone-induced hypokalemia may promote NCC activation not only by decreasing Cl⁻ binding to the active site of WNK4 but also by promoting RRxS phosphorylation. Nevertheless, the kinase responsible for these phosphorylation events remains to be uncovered. It is unlikely, however, that SGK1 is involved, as the aldosterone-independent stimulation of SGK1 activity is triggered by hyperkalemia (172).

Angiotensin II

NCC expression and phosphorylation levels are modulated by dietary NaCl intake. Low-NaCl diets promote NCC activation and high-NaCl diets promote NCC inhibition (173–176). Aldosterone was initially thought to be behind such modulation, as low NaCl intake is a well-known stimulus for the activation of the renin-angiotensin-aldosterone system. However, as explained above, current evidence suggests that aldosterone does not regulate NCC directly.

Van der Lubbe et al. showed that NCC and pNCC levels are regulated by ANG II independently of aldosterone by observing a stimulatory effect of ANG II infusion on NCC and pNCC levels in adrenalectomized rats (177). Later on, we showed that WNK4 is a key element in the signaling pathway that transduces ANG II stimulation into NCC activation. In WNK4^−/− mice, ANG II infusion did not promote the increase in SPAK/OSR1 or NCC phosphorylation that was observed in their wild-type littermates.

Subsequent works described two different mechanisms by which WNK4 activity is regulated by ANG II (Fig. 8). First, Shibata et al. showed that stimulation of the ANG II type 1 (AT₁) receptor in HEK-293 cells (a Gα_q-coupled seven-transmembrane receptor) upregulates WNK4 levels by promoting PKC-mediated phosphorylation of KLHL3 within a site located in the substrate-binding propeller domain (178). Phosphorylation of this site prevents KLHL3 binding to WNK4 and thus prevents WNK4 degradation. In vivo, increases in WNK4 levels were reported in mice infused with ANG II. Second, stimulation of cells with ANG II can also promote PKC-mediated phosphorylation of WNK4 at RRxS sites. As discussed above, phosphorylation of these sites, especially Ser⁶⁴ and Ser¹¹⁹⁶, promotes an increase in WNK4 activity. In HEK-293 cells stimulated with ANG II, pSPAK levels increased only in the presence of WNK4. However, this effect was not observed in the presence of mutant WNK4 with phosphoablative mutations in Ser⁶⁴ and Ser¹¹⁹⁶. Phosphorylation levels of these sites were shown to increase in volume-depleted mice (44).

Figure 8.

Role of with no lysine kinase 4 (WNK4) in the regulation of NaCl cotransporter (NCC) by ANG II and by extracellular Ca²⁺. Stimulation of ANG II type 1 receptors (AT₁) or calcium-sensing receptor (CaSR) in human embryonic kidney (HEK)-293 cells promotes PKC activation that, in turn, phosphorylates RRxS sites in WNK4 and Kelch-like family member 3 (KLHL3) (5, 16, 135). KLHL3 phosphorylation prevents WNK4 degradation (135), and WNK4 phosphorylation promotes kinase activation (16). In mice, high circulating ANG II levels correlate with increased KLHL3 and WNK4 phosphorylation levels at RRXS sites as well as increased levels of WNK4 protein expression (16, 135). Higher levels of WNK4 expression and phosphorylation at an RRxS site are also observed in mice administered the calcimimetic R-568, which acts as a positive allosteric modulator of CaSR (5). On the basis of this evidence, our group has proposed that activation of ANG II receptors and CaSR receptors in distal convoluted tubule cells promotes NCC activation via the depicted pathway (figure created with Biorender.com).

More recently, NCC expression and phosphorylation were analyzed in AT₁ receptor knockout models (179, 180). No differences were observed between wild-type and AT₁^−/− mice. It remains to be determined whether this absence of effect is due to compensatory mechanisms that maintain NCC expression and activity in the absence of AT₁ receptor signaling or whether the reported ANG II effects on the DCT are independent of AT₁ receptor activity.

Calcium-Sensing Receptor

Stimulation of the calcium-sensing receptor (CaSR) in HEK-293 cells transfected with WNK4 and SPAK has been recently shown to promote SPAK-activating phosphorylation (82). This activation is WNK4 dependent and occurs through similar mechanisms to the those described above for ANG II-mediated regulation of WNK4 (Fig. 8). CaSR can be coupled to Gα_q proteins; thus, its stimulation can lead to activation of PKC. PKC then phosphorylates KLHL3 and WNK4, leading to increased levels of WNK4 expression and activity. It has been postulated that this mechanism is important for promoting NCC activation in circumstances in which increased extracelluar Ca²⁺ levels promote CaSR-mediated NKCC2 inhibition that leads to a decrease in Ca²⁺ but also NaCl reabsorption in the thick ascending limb of Henle’s loop. In this scenario, NCC activation would help to recover the NaCl to prevent urinary salt losses.

Distal Luminal NaCl Delivery

Several groups have reported that increased distal NaCl delivery to the DCT, induced by either loop diuretic administration or NaCl loading, promotes NCC activation and DCT hypertrophy (75, 173, 181, 182). In some of these models, loop diuretic administration produced a decrease in plasma K⁺ concentration; thus, it remains to be reassessed whether the observed effects on DCT were not due to changes in extracellular K⁺ concentration. In a more recent work, Yang et al. investigated such phenomena by inducing increased distal NaCl delivery in mice by subcutaneous injection of 0.5 mL of normal saline (with 0.1% KCl) for 3 days (75). With this maneuver, no changes in extracellular K⁺ concentration or renin levels occur. The amount of NaCl administered was low compared with that used in high-NaCl diet experiments in which NCC inhibition was observed. Interestingly, they found that their NaCl loading protocol promoted NCC activation (increased pNCC and thiazide sensitivity) in wild-type mice but also in WNK4^−/− mice. They proposed that activation of NCC by increased luminal NaCl delivery is independent of WNK4.

Other Hormonal Regulators of NCC Activity

Other hormones that have been shown to promote NCC activation include norepinephrine, insulin, fibroblast growth protein-23 (FGF23), prolactin, estrogens, and progesterone (180, 183–187). Insulin was also shown to promote SPAK and OSR1 phosphorylation (187) as well as estradiol and progesterone (186). Interestingly, Terker et al. reported that the effect of norepinephrine on NCC activation is dependent on OSR1 but not on SPAK activity (180). Regarding WNK4, Takahashi et al. showed that the insulin-induced activation of NCC is not observed in WNK4^−/− mice (15), suggesting that insulin promotes NCC activation via WNK4. In addition, work by Andrukhova et al. suggested that WNK4 is also involved in NCC activation induced by FGF23, given that they observed increased phosphorylation levels of WNK4 in renal cortex proteins extracted from mice treated with recombinant FGF23 (183).

CONCLUDING REMARKS

The study of the kinase WNK4 and its central role in renal physiology began with the identification of gain-of–function mutations causing inherited salt-sensitive hypertension in humans. Even though FHHt is a rare disease, that finding opened a field of research that had led to understanding several physiological and pathophysiological processes. It has now been established that the signaling pathway comprised of WNK4-SPAK/OSR1-NCC plays a pivotal role in the regulation of electrolyte homeostasis, such as Na⁺, Cl⁻, and K⁺ homeostasis, which have an impact on physiological parameters like blood pressure and extracellular K⁺ concentration levels. Additionally, these physiological parameters regulate the activity of WNK4 through different mechanisms, such as direct Cl⁻ binding, phosphorylation of different sites, and/or proteasomal degradation, forming negative feedback loops responsible for homeostatic balance. All this has been achieved in a record amount of time by the work of several groups of renal physiologists, increasingly collaborating at the international level. A lot of interesting discoveries are ahead of us in this exciting field.

GRANTS

Work in the researchers’ laboratories is possible due to the support of National Institute of Diabetes and Digestive and Kidney Diseases Grant DK51496 (to G.G.) and Consejo Nacional de Ciencia y Tecnología Mexico Grants A1-S-8290 and 101720 (to G.G. and M.C.B, respectively).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

A.R.M., A.R. and M.C. conceived and designed research; A.R.M. and A.R. performed experiments; A.R.M., A.R., G.G. and M.C. analyzed data; A.R.M., A.R., G.G. and M.C. interpreted results of experiments; A.R.M., A.R., H.C. and M.C. prepared figures; A.R.M., A.R., H.C., G.G. and M.C. drafted manuscript; A.R.M., A.R., H.C., G.G. and M.C. edited and revised manuscript; A.R.M., A.R., H.C., G.G. and M.C. approved final version of manuscript.