Acute insulin stimulation induces phosphorylation of the Na-Cl cotransporter in cultured distal mpkDCT cells and mouse kidney

The NaCl cotransporter (NCC) is essential for sodium reabsorption at the distal convoluted tubules (DCT), and its phosphorylation increases its transport activity and apical membrane localization. Although insulin has been reported to increase sodium reabsorption in the kidney, the linkage between insulin and NCC phosphorylation has not yet been investigated. This study examined whether insulin regulates NCC phosphorylation. In cultured mpkDCT cells, insulin increased phosphorylation of STE20/SPS1-related proline-alanine-rich kinase (SPAK) and NCC in a dose-dependent manner. This insulin-induced phosphorylation of NCC was suppressed in WNK4 and SPAK knockdown cells. In addition, Ly294002, a PI3K inhibitor, decreased the insulin effect on SPAK and NCC phosphorylation, indicating that insulin induces phosphorylation of SPAK and NCC through PI3K and WNK4 in mpkDCT cells. Moreover, acute insulin administration to mice increased phosphorylation of oxidative stress-responsive kinase-1 (OSR1), SPAK and NCC in the kidney. Time-course experiments in mpkDCT cells and mice suggested that SPAK is upstream of NCC in this insulin-induced NCC phosphorylation mechanism, which was confirmed by the lack of insulin-induced NCC phosphorylation in SPAK knockout mice. Moreover, insulin administration to WNK4 hypomorphic mice did not increase phosphorylation of OSR1, SPAK and NCC in the kidney, suggesting that WNK4 is also involved in the insulin-induced OSR1, SPAK and NCC phosphorylation mechanism in vivo. The present results demonstrated that insulin is a potent regulator of NCC phosphorylation in the kidney, and that WNK4 and SPAK are involved in this mechanism of NCC phosphorylation by insulin.     

Disease-causing mutations in the acidic motif of WNK4 impair the sensitivity of WNK4 kinase to calcium ions

WNK4 is a serine/threonine protein kinase that is involved in pseudohypoaldosteronism type II (PHAII), a Mendelian form disorder featuring hypertension and hyperkalemia. Most of the PHAII-causing mutations are clustered in an acidic motif rich in negatively charged residues. It is unclear, however, whether these mutations affect the kinase activity in any way. In this study, we isolated kinase domain of WNK4 produced by Escherichia coli, and demonstrated its ability to phosphorylate the oxidative stress-responsive kinase-1 (OSR1) and the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC) in vitro. Threonine 48 was identified as the WNK4 phosphorylation site at mouse NCC. The phospho-mimicking T48D mutant of mouse NCC increased its protein abundance and Na(+) uptake, and also enhanced the phosphorylation at the N-terminal region of NCC by OSR1. When the acidic motif was included in the WNK4 kinase construct, the kinase activity of WNK4 exhibited sensitivity to Ca(2+) ions with the highest activity at Ca(2+) concentration around 1 μM using kinase-inactive OSR1 as a substrate. All tested PHAII-causing mutations at the acidic motif exhibited impaired Ca(2+) sensitivity. Our results suggest that these PHAII-causing mutations disrupt a Ca(2+)-sensing mechanism around the acidic motif necessary for the regulation of WNK4 kinase activity by Ca(2+) ions.     

WNK4 is the major WNK positively regulating NCC in the mouse kidney

By analysing the pathogenesis of a hereditary hypertensive disease, PHAII (pseudohypoaldosteronism type II), we previously discovered that WNK (with-no-lysine kinase)–OSR1/SPAK (oxidative stress-responsive 1/Ste20-like proline/alanine-rich kinase) cascade regulates NCC (Na–Cl co-transporter) in the DCT (distal convoluted tubules) of the kidney. However, the role of WNK4 in the regulation of NCC remains controversial. To address this, we generated and analysed WNK4^−/− mice. Although a moderate decrease in SPAK phosphorylation and a marked increase in WNK1 expression were evident in the kidneys of WNK4^−/− mice, the amount of phosphorylated and total NCC decreased to almost undetectable levels, indicating that WNK4 is the major WNK positively regulating NCC, and that WNK1 cannot compensate for WNK4 deficiency in the DCT. Insulin- and low-potassium diet-induced NCC phosphorylation were abolished in WNK4^−/− mice, establishing that both signals to NCC were mediated by WNK4. As shown previously, a high-salt diet decreases phosphorylated and total NCC in WNK4^+/+ mice via AngII (angiotensin II) and aldosterone suppression. This was not ameliorated by WNK4 knock out, excluding the negative regulation of WNK4 on NCC postulated to be active in the absence of AngII stimulation. Thus, WNK4 is the major positive regulator of NCC in the kidneys.     

Regulation of with‐no‐lysine kinase signaling by Kelch‐like proteins

In 2001, with‐no‐lysine (WNK) kinases were identified as the genes responsible for the human hereditary hypertensive disease pseudohypoaldosteronism type II (PHAII). It took a further 6 years to clarify that WNK kinases participate in a signaling cascade with oxidative stress‐responsive gene 1 (OSR1), Ste20‐related proline‐alanine‐rich kinase (SPAK), and thiazide‐sensitive NaCl cotransporter (NCC) in the kidney and the constitutive activation of this signaling cascade is the molecular basis of PHAII. Since this discovery, the WNK–OSR1/SPAK–NCC signaling cascade has been shown to be involved not only in PHAII but also in the regulation of blood pressure under normal and pathogenic conditions, such as hyperinsulinemia. However, the molecular mechanisms of WNK kinase regulation by dietary and hormonal factors and by PHAII‐causing mutations remain poorly understood. In 2012, two additional genes responsible for PHAII, Kelch‐like 3 (KLHL3) and Cullin3, were identified. At the time of their discovery, the molecular mechanisms underlying the interaction between these genes and their involvement in PHAII were unknown. Here we review the pathophysiological roles of the WNK signaling cascade clarified to date and introduce a new mechanism of WNK kinase regulation by KLHL3 and Cullin3, which provides insight on previously unknown mechanisms of WNK kinase regulation.     

SPAK/OSR1 regulate NKCC1 and WNK activity: analysis of WNK isoform interactions and activation by T-loop trans-autophosphorylation

Mutations in the WNK [with no lysine (K) kinase] family instigate hypertension and pain perception disorders. Of the four WNK isoforms, much of the focus has been on WNK1, which is activated in response to osmotic stress by phosphorylation of its T-loop residue (Ser382). WNK isoforms phosphorylate and activate the related SPAK (SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase 1) protein kinases. In the present study, we first describe the generation of double-knockin ES (embryonic stem) cells, where SPAK and OSR1 cannot be activated by WNK1. We establish that NKCC1 (Na+/K+/2Cl- co-transporter 1), a proposed target of the WNK pathway, is not phosphorylated or activated in a knockin that is deficient in SPAK/OSR1 activity. We also observe that activity of WNK1 and WNK3 are markedly elevated in the knockin cells, demonstrating that SPAK/OSR1 significantly influences WNK activity. Phosphorylation of another regulatory serine residue, Ser1261, in WNK1 is unaffected in knockin cells, indicating that this is not phosphorylated by SPAK/OSR1. We show that WNK isoforms interact via a C-terminal CCD (coiled-coil domain) and identify point mutations of conserved residues within this domain that ablate the ability of WNK isoforms to interact. Employing these mutants, we demonstrate that interaction of WNK isoforms is not essential for their T-loop phosphorylation and activation, at least for overexpressed WNK isoforms. Moreover, we finally establish that full-length WNK1, WNK2 and WNK3, but not WNK4, are capable of directly phosphorylating Ser382 of WNK1 in vitro. This supports the notion that T-loop phosphorylation of WNK isoforms is controlled by trans-autophosphorylation. These results provide novel insights into the WNK signal transduction pathway and provide genetic evidence confirming the essential role that SPAK/OSR1 play in controlling NKCC1 function. They also reveal a role in which the downstream SPAK/OSR1 enzymes markedly influence the activity of the upstream WNK activators. The knockin ES cells lacking SPAK/OSR1 activity will be useful in validating new targets of the WNK signalling pathway.     

WNK4 inhibits NCC protein expression through MAPK ERK1/2 signaling pathway

WNK [with no lysine (K)] kinase is a subfamily of serine/threonine kinases. Mutations in two members of this family (WNK1 and WNK4) cause pseudohypoaldosteronism type II featuring hypertension, hyperkalemia, and metabolic acidosis. WNK1 and WNK4 were shown to regulate sodium chloride cotransporter (NCC) activity through phosphorylating SPAK and OSR1. Previous studies including ours have also shown that WNK4 inhibits NCC function and its protein expression. A recent study reported that a phorbol ester inhibits NCC function via activation of extracellular signal-regulated kinase (ERK) 1/2 kinase. In the current study, we investigated whether WNK4 affects NCC via the MAPK ERK1/2 signaling pathway. We found that WNK4 increased ERK1/2 phosphorylation in a dose-dependent manner in mouse distal convoluted tubule (mDCT) cells, whereas WNK4 mutants with the PHA II mutations (E562K and R1185C) lost the ability to increase the ERK1/2 phosphorylation. Hypertonicity significantly increased ERK1/2 phosphorylation in mDCT cells. Knock-down of WNK4 expression by siRNA resulted in a decrease of ERK1/2 phosphorylation. We further showed that WNK4 knock-down significantly increases the cell surface and total NCC protein expressions and ERK1/2 knock-down also significantly increases cell surface and total NCC expression. These data suggest that WNK4 inhibits NCC through activating the MAPK ERK1/2 signaling pathway.     

Regulation of apical localization of the thiazide-sensitive NaCl cotransporter by WNK4 in polarized epithelial cells

Missense mutations in the WNK4 gene have been postulated to cause pseudohypoaldosteronism type II (PHAII), an autosomal-dominant disorder characterized by hyperkalemia and hypertension. Previous reports using Xenopus oocytes showed that wild-type WNK4 expression inhibited surface expression of the thiazide-sensitive NaCl cotransporter (NCC), while a disease-causing mutant lost the inhibitory effect on NCC surface expression. To determine if these changes observed in oocytes really occur in polarized epithelial cells, we generated stable MDCK II cell lines expressing NCC alone or NCC plus wild-type WNK4 or a disease-causing (D564A) WNK4. In contrast to the apical localization of NCC without co-expression of WNK4, immunofluorescence microscopy and biotin surface labeling revealed that this apical localization was equally decreased by both the wild-type and the mutant WNK4 expression. Apical localizations of two PHAII-unrelated apical transporters, sodium-independent amino acid transporter, BAT1 and bile salt export pump, Bsep, were also found to be decreased by both wild-type and mutant WNK4 expression. These results indicate that the regulation of NCC was not related to the disease-causing mutation and not restricted to the PHAII-related specific transporters. The regulation of intracellular localization of NCC by WNK4 might not be involved in the pathogenesis of PHAII.     

WNK1 regulates phosphorylation of cation-chloride-coupled cotransporters via the STE20-related kinases, SPAK and OSR1

The WNK1 and WNK4 genes have been found to be mutated in some patients with hyperkalemia and hypertension caused by pseudohypoaldosteronism type II. The clue to the pathophysiology of pseudohypoaldosteronism type II was its striking therapeutic response to thiazide diuretics, which are known to block the sodium chloride cotransporter (NCC). Although this suggests a role for WNK1 in hypertension, the precise molecular mechanisms are largely unknown. Here we have shown that WNK1 phosphorylates and regulates the STE20-related kinases, Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress response 1 (OSR1). WNK1 was observed to phosphorylate the evolutionary conserved serine residue located outside the kinase domains of SPAK and OSR1, and mutation of the OSR1 serine residue caused enhanced OSR1 kinase activity. In addition, hypotonic stress was shown to activate SPAK and OSR1 and induce phosphorylation of the conserved OSR1 serine residue, suggesting that WNK1 may be an activator of the SPAK and OSR1 kinases. Moreover, SPAK and OSR1 were found to directly phosphorylate the N-terminal regulatory regions of cation-chloride-coupled cotransporters including NKCC1, NKCC2, and NCC. Phosphorylation of NCC was induced by hypotonic stress in cells. These results suggested that WNK1 and SPAK/OSR1 mediate the hypotonic stress signaling pathway to the transporters and may provide insights into the mechanisms by which WNK1 regulates ion balance.     

Overexpression of human WNK1 increases paracellular chloride permeability and phosphorylation of claudin-4 in MDCKII cells

Pseudohypoaldosteronism type II (PHAII), an autosomal dominant disorder characterized by hypertension, hyperkalemia, and hyperchloremic acidosis, is reportedly due to mutations in WNK1 and WNK4 kinase genes. However, the pathogenesis of the disease remains unknown. Mutations in the WNK1 gene are the deletions in the first intron, which reportedly increases WNK1 mRNA expression. Thus, we generated WNK1 over-expressing stable cell lines using MDCKII cells to model the distal nephron of PHAII patients. Using these cell lines, we investigated whether increased WNK1 expression might affect paracellular chloride permeability and claudin phosphorylation, since we previously observed an increase in both with a disease-causing mutant WNK4. WNK1 expression in MDCKII cells increased chloride permeability two to threefold. Co-expression of wild-type WNK4 did not further increase WNK1-enhanced chloride permeability. WNK1 expression also induced phosphorylation of endogenous claudin-4 in MDCKII cells, as well as over-expressed claudin-4. Combined, these results suggest that increased WNK1 expression has the same effect on chloride permeability and claudin phosphorylation as the mutant WNK4. Thus, increased chloride shunt may be involved in the pathogenesis of PHAII caused by WNK1 mutations.     

WNK3-SPAK interaction is required for the modulation of NCC and other members of the SLC12 family

The serine/threonine with no lysine kinase 3 (WNK3) modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC) to promote Cl(-) influx and prevent Cl(-) efflux, thus fitting the profile for a putative "Cl(-)-sensing kinase". The Ste20-type kinases, SPAK/OSR1, become phosphorylated in response to reduction in intracellular chloride concentration and regulate the activity of NKCC1. Several studies have now shown that WNKs function upstream of SPAK/OSR1. This study was designed to analyze the role of WNK3-SPAK interaction in the regulation of CCCs with particular emphasis on NCC. In this study we used the functional expression system of Xenopus laevis oocytes to show that different SPAK binding sites in WNK3 ((241, 872, 1336)RFxV) are required for the kinase to have effects on CCCs. WNK3-F1337A no longer activated NKCC2, but the effects on NCC, NKCC1, and KCC4 were preserved. In contrast, the effects of WNK3 on these cotransporters were prevented in WNK3-F242A. The elimination of F873 had no consequence on WNK3 effects. WNK3 promoted NCC phosphorylation at threonine 58, even in the absence of the unique SPAK binding site of NCC, but this effect was abolished in the mutant WNK3-F242A. Thus, our data support the hypothesis that the effects of WNK3 upon NCC and other CCCs require the interaction and activation of the SPAK kinase. The effect is dependent on one of the three binding sites for SPAK that are present in WNK3, but not on the SPAK binding sites on the CCCs, which suggests that WNK3 is capable of binding both SPAK and CCCs to promote their phosphorylation.     

Effect of angiotensin II on the WNK-OSR1/SPAK-NCC phosphorylation cascade in cultured mpkDCT cells and in vivo mouse kidney

In our recent study using Wnk4^D561A/+ knockin mice, we determined that the WNK-OSR1/SPAK-NaCl cotransporter (NCC) phosphorylation cascade is important for regulating NCC function in vivo. Phosphorylation of NCC was necessary for its plasma membrane localization. Previously, angiotensin II infusion was shown to increase apical membrane expression of NCC in rats. Therefore, we investigated whether angiotensin II was an upstream regulator for the WNK-OSR1/SPAK-NCC cascade in cultured cells and in vivo kidney. In mpkDCT cells, the phosphorylation of OSR1 and NCC was increased 30 min after the addition of angiotensin II (10^-9-10⁻⁷ M) but returned to baseline after 18 h. In mice, a 5-min infusion of angiotensin II (5 ng/g/min) increased NCC phosphorylation in the kidney at 30 min and 2 h after the injection but returned to baseline 24 h later. This increase was inhibited by angiotensin II receptor blocker (valsartan) but not by aldosterone receptor blocker (eplerenone). Ten-day infusions of angiotensin II (720 ng/day) also increased phosphorylation of OSR1 and NCC in the mouse kidney, and both valsartan and eplerenone inhibited the increased phosphorylation. Although angiotensin II is identified as an upstream regulator for the WNK-OSR1/SPAK-NCC cascade in vivo, aldosterone appears to be the major regulator of this signal cascade in the long-term regulation by angiotensin II.     

A Novel Ste20-related Proline/Alanine-rich Kinase (SPAK)-independent Pathway Involving Calcium-binding Protein 39 (Cab39) and Serine Threonine Kinase with No Lysine Member 4 (WNK4) in the Activation of Na-K-Cl Cotransporters

Na⁺-dependent chloride cotransporters (NKCC1, NKCC2, and NCC) are activated by phosphorylation to play critical roles in diverse physiological responses, including renal salt balance, hearing, epithelial fluid secretion, and volume regulation. Serine threonine kinase WNK4 (With No K = lysine member 4) and members of the Ste20 kinase family, namely SPAK and OSR1 (Ste20-related proline/alanine-rich kinase, Oxidative stress-responsive kinase) govern phosphorylation. According to present understanding, WNK4 phosphorylates key residues within SPAK/OSR1 leading to kinase activation, allowing SPAK/OSR1 to bind to and phosphorylate NKCC1, NKCC2, and NCC. Recently, the calcium-binding protein 39 (Cab39) has emerged as a binding partner and enhancer of SPAK/OSR1 activity, facilitating kinase autoactivation and promoting phosphorylation of the cotransporters. In the present study, we provide evidence showing that Cab39 differentially interacts with WNK4 and SPAK/OSR1 to switch the classic two kinase cascade into a signal kinase transduction mechanism. We found that WNK4 in association with Cab39 activates NKCC1 in a SPAK/OSR1-independent manner. We discovered that WNK4 possesses a domain that bears close resemblance to the SPAK/OSR1 C-terminal CCT/PF2 domain, which is required for physical interaction between the Ste20 kinases and the Na⁺-driven chloride cotransporters. Modeling, yeast two-hybrid, and functional data reveal that this PF2-like domain located downstream of the catalytic domain in WNK4 promotes the direct interaction between the kinase and NKCC1. We conclude that in addition to SPAK and OSR1, WNK4 is able to anchor itself to the N-terminal domain of NKCC1 and to promote cotransporter activation.     

OSR1-sensitive regulation of Na+/H+ exchanger activity in dendritic cells

The oxidative stress-responsive kinase 1 (OSR1) is activated by WNK (with no K kinases) and in turn stimulates the thiazide-sensitive Na-Cl cotransporter (NCC) and the furosemide-sensitive Na-K-2Cl cotransporter (NKCC), thus contributing to transport and cell volume regulation. Little is known about extrarenal functions of OSR1. The present study analyzed the impact of decreased OSR1 activity on the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. DCs were cultured from bone marrow of heterozygous WNK-resistant OSR1 knockin mice (osr(KI)) and wild-type mice (osr(WT)). Cell volume was estimated from forward scatter in FACS analysis, ROS production from 2',7'-dichlorodihydrofluorescein-diacetate fluorescence, cytosolic pH (pH(i)) from 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein fluorescence, and Na(+)/H(+) exchanger activity from Na(+)-dependent realkalinization following ammonium pulse and migration utilizing transwell chambers. DCs expressed WNK1, WNK3, NCC, NKCC1, and OSR1. Phosphorylated NKCC1 was reduced in osr(KI) DCs. Cell volume and pH(i) were similar in osr(KI) and osr(WT) DCs, but Na(+)/H(+) exchanger activity and ROS production were higher in osr(KI) than in osr(WT) DCs. Before LPS treatment, migration was similar in osr(KI) and osr(WT) DCs. LPS (1 μg/ml), however, increased migration of osr(WT) DCs but not of osr(KI) DCs. Na(+)/H(+) exchanger 1 inhibitor cariporide (10 μM) decreased cell volume, intracellular reactive oxygen species (ROS) formation, Na(+)/H(+) exchanger activity, and pH(i) to a greater extent in osr(KI) than in osr(WT) DCs. LPS increased cell volume, Na(+)/H(+) exchanger activity, and ROS formation in osr(WT) DCs but not in osr(KI) DCs and blunted the difference between osr(KI) and osr(WT) DCs. Na(+)/H(+) exchanger activity in osr(WT) DCs was increased by the NKCC1 inhibitor furosemide (100 nM) to values similar to those in osr(KI) DCs. Oxidative stress (10 μM tert-butyl-hydroperoxide) increased Na(+)/H(+) exchanger activity in osr(WT) DCs but not in osr(KI) DCs and reversed the difference between genotypes. Cariporide virtually abrogated Na(+)/H(+) exchanger activity in both genotypes and blunted LPS-induced cell swelling and ROS formation in osr(WT) mice. In conclusion, partial OSR1 deficiency influences Na(+)/H(+) exchanger activity, ROS formation, and migration of dendritic cells.     

SPAK Isoforms and OSR1 Regulate Sodium-Chloride Co-transporters in a Nephron-specific Manner

STE20/SPS-1-related proline-alanine-rich protein kinase (SPAK) and oxidative stress-related kinase (OSR1) activate the potassium-dependent sodium-chloride co-transporter, NKCC2, and thiazide-sensitive sodium-chloride cotransporter, NCC, in vitro, and both co-localize with a kinase regulatory molecule, Cab39/MO25α, at the apical membrane of the thick ascending limb (TAL) and distal convoluted tubule (DCT). Yet genetic ablation of SPAK in mice causes a selective loss of NCC function, whereas NKCC2 becomes hyperphosphorylated. Here, we explore the underlying mechanisms in wild-type and SPAK-null mice. Unlike in the DCT, OSR1 remains at the TAL apical membrane of KO mice where it is accompanied by an increase in the active, phosphorylated form of AMP-activated kinase. We found an alterative SPAK isoform (putative SPAK2 form), which modestly inhibits co-transporter activity in vitro, is more abundant in the medulla than the cortex. Thus, enhanced NKCC2 phosphorylation in the SPAK knock-out may be explained by removal of inhibitory SPAK2, sustained activity of OSR1, and activation of other kinases. By contrast, the OSR1/SPAK/M025α signaling apparatus is disrupted in the DCT. OSR1 becomes largely inactive and displaced from M025α and NCC at the apical membrane, and redistributes to dense punctate structures, containing WNK1, within the cytoplasm. These changes are paralleled by a decrease in NCC phosphorylation and a decrease in the mass of the distal convoluted tubule, exclusive to DCT1. As a result of the dependent nature of OSR1 on SPAK in the DCT, NCC is unable to be activated. Consequently, SPAK^−/− mice are highly sensitive to dietary salt restriction, displaying prolonged negative sodium balance and hypotension.     

Up-regulation of the clusterin gene after proteotoxic stress: implication of HSF1-HSF2 heterocomplexes

The SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase-1) kinases interact and phosphorylate NKCC1 (Na+-K+-2Cl- co-transporter-1), leading to its activation. Recent studies indicated that SPAK and OSR1 are phosphorylated and activated by the WNK1 [with no K (lysine) protein kinase-1] and WNK4, genes mutated in humans affected by Gordon's hypertension syndrome. In the present study, we have identified three residues in NKCC1 (Thr175/Thr179/Thr184 in shark or Thr203/Thr207/Thr212 in human) that are phosphorylated by SPAK and OSR1, and have developed a peptide substrate, CATCHtide (cation chloride co-transporter peptide substrate), to assess SPAK and OSR1 activity. Exposure of HEK-293 (human embryonic kidney) cells to osmotic stress, which leads to phosphorylation and activation of NKCC1, increased phosphorylation of NKCC1 at the sites targeted by SPAK/OSR1. The residues on NKCC1, phosphorylated by SPAK/OSR1, are conserved in other cation co-transporters, such as the Na+-Cl- co-transporter, the target of thiazide drugs that lower blood pressure in humans with Gordon's syndrome. Furthermore, we characterize the properties of a 92-residue CCT (conserved C-terminal) domain on SPAK and OSR1 that interacts with an RFXV (Arg-Phe-Xaa-Val) motif present in the substrate NKCC1 and its activators WNK1/WNK4. A peptide containing the RFXV motif interacts with nanomolar affinity with the CCT domains of SPAK/OSR1 and can be utilized to affinity-purify SPAK and OSR1 from cell extracts. Mutation of the arginine, phenylalanine or valine residue within this peptide abolishes binding to SPAK/OSR1. We have identified specific residues within the CCT domain that are required for interaction with the RFXV motif and have demonstrated that mutation of these in OSR1 inhibited phosphorylation of NKCC1, but not of CATCHtide which does not possess an RFXV motif. We establish that an intact CCT domain is required for WNK1 to efficiently phosphorylate and activate OSR1. These data establish that the CCT domain functions as a multipurpose docking site, enabling SPAK/OSR1 to interact with substrates (NKCC1) and activators (WNK1/WNK4).     

Immunolocalization of WNK4 in mouse kidney

Initial reports claim that WNK4 localization is mainly at intercellular junctions of distal convoluted tubules (DCT) and cortical collecting ducts (CCD) in the kidney. However, we recently clarified the major targets of WNK4 kinase to be the OSR1/SPAK kinases and the Na–Cl co-transporter (NCC), an apical membrane protein in the DCT, thus raising the question of whether the cellular localization of WNK4 is at intercellular junctions. In this study, we re-evaluate the intrarenal and intracellular immunolocalization of WNK4 in the mouse kidney using a newly generated anti-WNK4 antibody. By performing double immunofluorescence of WNK4 with several nephron-segment-specific markers, we have found that WNK4 is present in podocytes in glomeruli, the cortical thick ascending limb of Henle’s loop including macula densa, and the medullary collecting ducts (MCD), in addition to the previously identified nephron segments, i.e., DCT and CCD. These results are consistent with the finding that WNK4 constitutes a kinase cascade with OSR1/SPAK and NCC in the DCT, and highlights a novel role for WNK4 in nephron segments newly identified as being WNK4-positive in this study.     

A SPAK isoform switch modulates renal salt transport and blood pressure

The renal thick ascending limb (TAL) and distal convoluted tubule (DCT) play central roles in salt homeostasis and blood pressure regulation. An emerging model suggests that bumetanide- and thiazide-sensitive NaCl transporters (NKCC2 and NCC) along these segments are phosphorylated and activated by WNK kinases, via SPAK and OSR1. Here, we show that a kidney-specific SPAK isoform, which lacks the kinase domain, inhibits phosphorylation of NCC and NKCC2 by full-length SPAK in?vitro. Kidney-specific SPAK is highly expressed along the TAL, whereas full-length SPAK is more highly expressed along the DCT. As predicted from the differential expression, SPAK knockout in animals has divergent effects along TAL and DCT, with increased phosphorylated NKCC2 along TAL and decreased phosphorylated NCC along DCT. In mice, extracellular fluid volume depletion shifts SPAK isoform abundance to favor NaCl retention along both segments, indicating that a SPAK isoform switch modulates sodium avidity along the distal nephron.     

WNK3 is a putative chloride-sensing kinase

The with-no-lysine kinase 3 (WNK3) is a serine/threonine kinase that modulates the activity of the electroneutral cation-coupled chloride cotransporters (CCC). Using the Xenopus laevis oocyte heterologous expression system, it has been shown that WNK3 activates the Na(+)-coupled chloride cotransporters NKCC1, NKCC2, and NCC and inhibits the K(+)-coupled chloride cotransporters KCC1 through KCC4. Interestingly, the effect of catalytically inactive WNK3 is opposite to that of wild type WNK3: inactive WNK3 inhibits NKCCs and activates KCCs. In doing so, wild type and catalytically inactive WNK3 bypass the tonicity requirement for activation/inhibition of the cotransporter. Thus, WNK3 modulation of the electroneutral cotransporters promotes Cl(-) influx and prevents Cl(-) efflux, thus fitting the profile for a putative "Cl(-)-sensing kinase". Other kinases that potentially have these properties are the Ste20-type kinases, SPAK/OSR1, which become phosphorylated in response to reductions in intracellular chloride concentration and regulate the activity of NKCC1. It has been demonstrated that WNKs lie upstream of SPAK/OSR1 and that the activity of these kinases is activated by phosphorylation of threonines in the T-loop by WNKs. It is possible that a protein phosphatase is also involved in the WNK3 effects on its associated cotransporters because activation of KCCs and inhibition of NKCCs by inactive WNK3 can be prevented by known inhibitors of protein phosphatases, such as calyculin A and cyclosporine, suggesting that a protein phosphatase is also involved in the protein complex.     

Decrease of WNK4 ubiquitination by disease-causing mutations of KLHL3 through different molecular mechanisms

Recently, we demonstrated that WNK4 is a substrate for KLHL3–Cullin3 (CUL3) E3 ubiquitin ligase complexes and that impaired WNK4 ubiquitination is a common mechanism for pseudohypoaldosteronism type II (PHAII) caused by WNK4, KLHL3, and CUL3 mutations. Among the various KLHL3 mutations that cause PHAII, we demonstrated that the R528H mutation in the Kelch domain decreased the binding to WNK4, thereby causing less ubiquitination and increased intracellular levels of WNK4. However, the pathogenic mechanisms of PHAII caused by other KLHL3 mutants remain to be determined. In this study, we examined the pathogenic effects of three PHAII-causing mutations in different KLHL3 domains; the protein levels of these mutants significantly differed when they were transiently expressed in HEK293T cells. In particular, S410L expression was low even with increased plasmid expression. The cycloheximide chase assay revealed that an S410L mutation in the Kelch domain significantly decreased the intracellular stability. Mutations in E85A in the BTB domain and C164F in the BACK domain decreased the binding to CUL3, and S410L as well as R528H demonstrated less binding to WNK4. In vitro and in vivo assays revealed that these mutants decreased the ubiquitination and increased the intracellular levels of WNK4 compared with wild-type KLHL3. Therefore, the KLHL3 mutants causing PHAII investigated in this study exhibited less ability to ubiquitinate WNK4 because of KLHL3’s low stability and/or decreased binding to CUL3 or WNK4.