With-no-lysine (WNK) kinases are highly expressed along the mammalian distal nephron. Mutations in either WNK1 or WNK4 cause familial hyperkalemic hypertension (FHHt), suggesting that the protein products converge on a final common pathway. We showed previously that WNK4 downregulates thiazide-sensitive NaCl cotransporter (NCC) activity, an effect suppressed by WNK1. Here we investigated the mechanisms by which WNK1 and WNK4 interact to regulate ion transport. We report that WNK1 suppresses the WNK4 effect on NCC activity and associates with WNK4 in a protein complex involving the kinase domains. Although a kinase-dead WNK1 also associates with WNK4, it fails to suppress WNK4-mediated NCC inhibition; the WNK1 kinase domain alone, however, is not sufficient to block the WNK4 effect. The carboxyterminal 222 amino acids of WNK4 are sufficient to inhibit NCC, but this fragment is not blocked by WNK1. Instead, WNK1 inhibition requires an intact WNK4 kinase domain, the region that binds to WNK1. In summary, these data show that: (a) the WNK4 carboxyl terminus mediates NCC suppression, (b) the WNK1 kinase domain interacts with the WNK4 kinase domain, and (c) WNK1 inhibition of WNK4 is dependent on WNK1 catalytic activity and an intact WNK1 protein. These findings provide insight into the complex interrelationships between WNK1 and WNK4 and provide a molecular basis for FHHt. IntroductionFamilial hyperkalemic hypertension (FHHt; also known as pseudohypoaldosteronism type II; Online Mendelian Inheritance in Man reference number #145260) is an autosomal dominant disease characterized by hypertension, hyperkalemia, and sensitivity to thiazide diuretics. Wilson and colleagues (1) reported that mutations in 2 genes encoding homologous proteins, WNK1 (PRKWNK1) and WNK4 (PRKWNK4), can cause FHHt. The with-no-lysine (WNK) kinases are novel serine/threonine kinases that were named because they lack lysine at a location previously thought to be essential for kinase activity (2). Both WNK1 and WNK4 are highly expressed in the kidney. The FHHt-causing WNK1 mutations are deletions within the first intron. These mutations increase WNK1 expression in leukocytes and were postulated to be gain-of-function mutations (1). Recently, heterozygous WNK1-deficient mice were shown to exhibit lower blood pressure than wild-type controls (3), supporting the hypothesis that WNK1 exerts a positive effect on blood pressure. The WNK4 mutations that cause FHHt are discrete missense mutations in 2 areas of the coding region (1). These mutations cause phenotypic features similar to those that result from WNK1 intron mutations (1), but the mechanisms involved may be different.We showed previously that WNK4 inhibits the thiazide-sensitive NaCl cotransporter (NCC, gene symbol SLC12A3) when expressed in Xenopus oocytes (4); similar results were obtained by others (5).

Pseudohypoaldosteronism type II (PHAII) is an autosomal dominant disorder of hyperkalemia and hypertension. Mutations in two members of the WNK kinase family, WNK1 and WNK4, cause the disease. WNK1 mutations are believed to increase WNK1 expression; the effect of WNK4 mutations remains unknown. The clinical phenotype of PHAII is opposite to Gitelman syndrome, a disease caused by dysfunction of the thiazide-sensitive Na-Cl cotransporter. We tested the hypothesis that WNK kinases regulate the mammalian thiazide-sensitive Na-Cl cotransporter (NCC). Mouse WNK4 was cloned and expressed in Xenopus oocytes with or without NCC. Coexpression with WNK4 suppressed NCC activity by more than 85%. This effect did not result from defects in NCC synthesis or processing, but was associated with an 85% reduction in NCC abundance at the plasma membrane. Unlike WNK4, WNK1 did not affect NCC activity directly. WNK1, however, completely prevented WNK4 inhibition of NCC. Some WNK4 mutations that cause PHAII retained NCC-inhibiting activity, but the Q562E WNK4 demonstrated diminished activity, suggesting that some PHAII mutations lead to loss of NCC inhibition. Gain-of-function WNK1 mutations would be expected to inhibit WNK4 activity, thereby activating NCC, contributing to the PHAII phenotype. Together, these results identify WNK kinases as a previously unrecognized sodium regulatory pathway of the distal nephron. This pathway likely contributes to normal and pathological blood pressure homeostasis

We investigated the activity of thiazide-sensitive sodium-chloride cotransporter (NCC) in experimental metabolic syndrome (MS) and the role of insulin in NCC activation. Renal responses to NCC inhibitor hydrochlorothiazide (HCTZ), as a measure of NCC activity in vivo were studied in 12-week old Zucker obese rats (ZO), a model of MS, and in lean control animals (ZL), together with renal NCC expression, and molecular markers of NCC activity, such as localization and phosphorylation. Effects of insulin were further studied in mammalian cell lines with inducible and endogenous expression of this molecule. ZO rats displayed marked hyperinsulinemia, but no differences in plasma aldosterone as compared to ZL. In ZO, natriuretic and diuretic responses to NCC inhibition with HCTZ were enhanced compared with ZL, and associated with a decrease in blood pressure (BP). ZO rats displayed enhanced Thr53 NCC phosphorylation and predominant membrane localization of both total and phosphorylated NCC, together with a different profile in expression of SPAK isoforms, and lower expression of WNK4. In vitro, insulin induced NCC phosphorylation, which was blocked by PI3 kinase inhibitor. Insulin-induced reduction in WNK4 expression was also observed, but delayed compared with the time course of NCC phosphorylation. In summary, we report increased NCC activity in hyperinsulinemic rodents in conjunction with SPAK expression profile consistent with NCC activation and reduced WNK4 as well as an ability of insulin to induce NCC stimulatory phosphorylation in vitro. Together, these findings indicate that hyperinsulinemia is an important driving force of NCC activity in MS with possible consequences for BP regulation.

With no lysine kinase 4 (WNK4) is essential to activate the thiazide-sensitive NaCl cotransporter (NCC) along the distal convoluted tubule, an effect central to the phenotype of familial hyperkalemic hypertension. Although effects on potassium and sodium channels along the connecting and collecting tubules have also been documented, WNK4 is typically believed to have little role in modulating sodium chloride reabsorption along the thick ascending limb of the loop of Henle. Yet wnk4 mice (knockout mice lacking WNK4) do not demonstrate the hypocalciuria typical of pure distal convoluted tubule dysfunction. Here, we tested the hypothesis that WNK4 also modulates bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) function along the thick ascending limb. We confirmed that w nk4 mice are hypokalemic and waste sodium chloride, but are also normocalciuric. Results from Western blots suggested that the phosphorylated forms of both NCC and NKCC2 were in lower abundance in wnk4 mice than in controls. This finding was confirmed by immunofluorescence microscopy. Although the initial response to furosemide was similar in wnk4 mice and controls, the response was lower in the knockout mice when reabsorption along the distal convoluted tubule was inhibited. Using HEK293 cells, we showed that WNK4 increases the abundance of phosphorylated NKCC2. More supporting evidence that WNK4 may modulate NKCC2 emerges from a mouse model of WNK4-mediated familial hyperkalemic hypertension in which more phosphorylated NKCC2 is present than in controls. These data indicate that WNK4, in addition to modulating NCC, also modulates NKCC2, contributing to its physiological function in vivo.

Background: Mutations in the ubiquitin ligase scaffold protein Cullin 3 (CUL3) cause the disease Familial Hyperkalemic Hypertension (FHHt). In the kidney, mutant CUL3 (CUL3-Δ9) increases abundance of With-No-Lysine [K] Kinase 4 (WNK4), inappropriately activating Sterile 20/SPS-1-related proline/alanine-rich kinase (SPAK), which then phosphorylates and hyperactivates the Na+-Cl- cotransporter (NCC). The precise mechanism by which CUL3-Δ9 causes FHHt has been unclear. We tested the hypothesis that reduced abundances of CUL3 and of Kelch-like 3 (KLHL3), the CUL3 substrate adaptor for WNK4, are mechanistically important. Since JAB1, an enzyme that inhibits CUL3 activity by removing the ubiquitin-like protein NEDD8, cannot interact with CUL3-Δ9, we also determined whether Jab1 disruption mimicked the effects of CUL3-Δ9 expression. Methods: We used an inducible renal tubule-specific system to generate several mouse models expressing CUL3-Δ9, mice heterozygous for both CUL3 and KLHL3 (Cul3+/−/Klhl3+/−), and mice with short-term Jab1 disruption (to avoid renal injury associated with long-term disruption). Results: Renal KLHL3 was higher in Cul3−/− mice, but lower in Cul3−/−/Δ9 mice and in the Cul3+/−/Δ9 FHHt model, suggesting KLHL3 is a target for both WT and mutant CUL3. Cul3+/−/Klhl3+/− mice displayed increased WNK4-SPAK activation and phospho-NCC abundance, and an FHHt-like phenotype with increased plasma [K+] and salt-sensitive blood pressure. Short-term Jab1 disruption in mice lowered abundances of CUL3 and KLHL3, and increased abundances of WNK4 and phospho-NCC. Conclusions:Jab1-/- mice and Cul3+/−/Klhl3+/− mice recapitulated the effects of CUL3-Δ9 expression on WNK4-SPAK-NCC. Our data suggest that degradation of both KLHL3 and CUL3 plays a central mechanistic role in CUL3-Δ9-mediated FHHt.