It is widely recognized that the phenotype of familial hyperkalemic hypertension is mainly a consequence of increased activity of the renal Na + -Cl 2 cotransporter (NCC) because of altered regulation by with no-lysine-kinase 1 (WNK1) or WNK4. either by low chloride hypotonic stress or coinjection of oocytes with the solute carrier family 26 (anion exchanger)-member 9 (SLC26A9) cRNA, promoted WNK4 autophosphorylation and increased NCC-dependent Na + transport in a WNK4-dependent manner. Substitution of the leucine with phenylalanine at residue 322 of WNK4, homologous to the chloride-binding pocket in L-WNK1, converted WNK4 into a constitutively autophosphorylated kinase that activated NCC, even without chloride depletion. Elimination of the catalytic activity (D321A or D321K-K186D) or the autophosphorylation site (S335A) in mutant WNK4-L322F abrogated the positive effect on NCC. These observations suggest that WNK4 can exert differential effects on NCC, depending on the intracellular chloride concentration.

Objectives Insulin is recognized to increase renal salt reabsorption in the distal nephron and hyperinsulinemic states have been shown to be associated with increased expression of the renal NaCl cotransporter, NCC. However, the effect of insulin on NCC functional activity has not been reported. Methods Using a heterologous expression system of Xenopus laevis oocytes, a mouse distal convoluted cell line, mDCT15 cells, endogenously expressing NCC, and an ex vivo kidney perfusion technique, we assessed the effect of insulin on the activity and phosphorylation of NCC. The signaling pathway involved was analyzed. Results In Xenopus oocytes insulin increases the activity of NCC together with its phosphorylation at threonine residue 58. Activation of NCC by insulin was also observed in mDCT15 cells. Additionally, insulin increased the NCC phosphorylation in kidney under the ex vivo perfusion technique. In oocytes and mDCT15 cells, insulin effect on NCC was prevented with inhibitors of PI3K, mTORC2, and AKT1 kinases, but not by inhibitors of MAP or mTORC1 kinases, suggesting that PI3K-mTORC2-AKT1 is the intracellular pathway required. Additionally, activation of NCC by insulin was not affected by wild type or mutant versions of WNK1, WNK4, or SGK1, but it was no longer observed in the presence of wild type or the dominant negative, catalytically inactive WNK3, implicating this kinase in the process. Conclusion Insulin induces activation and phosphorylation of NCC. This effect could play an important role in arterial hypertension associated with hyperinsulinemic states, such as obesity, metabolic syndrome, or type 2 diabetes mellitus.

Using a SNP-based linkage approach, 7 suggestive linkage regions (maximum logarithm of odds [LOD] score = 1.8 in all linked regions) were identified in this panel (Supplemental Table 1B). In total, linked regions spanned 69 Mb and included 829 protein-coding genes. After filtering, WES performed in 1 unaffected and 2 affected individuals identified 71 possible disease-causing coding variants (Supplemental Table 1C). Four missense variants mapped to the linkage regions and were predicted in silico to be damaging. These variants were located in solute carrier family 30 member 7, a zinc transporter (SLC30A7), kinesin family member 11 (KIF11), tectonic family member 3 (TCTN3), and WNK1. The WNK1 variant (c.1905T>A; P.Asp635Glu) (Figure 1B) was located in exon 7 (ex7), which encodes the conserved acidic motif, previously shown to mediate the interaction with the substrate adaptor KLHL3 ( 14).Additional variants in the WNK1 acidic motif in other cases and kindreds. FHHt-causing WNK4 mutations are located in ex7 and ex17, encoding highly conserved acid and base motifs, respectively. Thus, we screened the homologous motifs of WNK1 encoded by ex7 and ex25, respectively, in 26 unrelated affected cases previously found as negative for the classical mutations in WNK4, KLHL3, CUL3, or the intron 1 deletion in WNK1. Direct sequencing identified 5 additional nonsynonymous heterozygous variants in ex7 in 8 unrelated subjects (Figure 2, A-C). The in silico pathogenicity of these variants is described in Supplemental Table 2. All were located within the acidic motif, between positions 631 and 636 of the L-WNK1 protein, and were predicted to be pathogenic. Four of the 6 missense variants were charge changing (E631K, D635N, Q636R, Q636E); 2 affected residues (D635, Q636) were also found mutated in the homologous acidic motif of WNK4 (D564 and Q565, Figure 2, C and D)Clinical and biochemical characteristics: hydrochlorothiazide-sensitive hyperkalemic acidosis without hypertension. Detailed clinical and biological characteristics of index cases are given in Table 1. The circumstances of discovery and the clinical symptoms of these index patients are detailed in Supplemental Table 3. In most of the cases, patients were asymptomatic and showed with no signs of hyperkalemia on an electrocardiogram. All displayed the electrolyte anomalies typical of FHHt, including marked hyperkalemia (median, 5.9 mmol/L; IQR, 5.3-6.3), hyperchloremia (median, 108 mmol/L; IQR, 106-110), and metabolic acidosis (total CO 2 , 20 mmol/L; IQR, 19-21) despite a normal glomerular filtration rate (GFR) (median creatinine, 58 μmol/L; IQR, 47-74). For the 7 index cases with prospective reliable clinical data, hyperkalemia and hyperchloremia were rapidly corrected with low doses of hydrochlorothiazide (HCTZ) (6.25 to 25 mg/d; Supplemental Figure 1). In comparison, an average drop in potassium of only 0.7 mmol/L was observed in normal, healthy subjects administered a much higher dose of HCTZ (50 mg for 3 weeks) (15). Compared with reference values (16), we also obser...

The With No lysine kinases WNK1 and WNK4 are key regulators of blood pressure. Their mutations lead to Familial Hyperkalemic Hypertension (FHHt), associated with an activation of the Na-Cl cotransporter, NCC. Although it is clear that WNK4 mutants activate NCC via SPAK (Ste20 Proline-Alanine rich Kinase), the mechanisms responsible for WNK1-related FHHt and alterations in NCC activity are not as clear. We tested whether WNK1 modulates NCC through WNK4, as predicted by some models, by crossing our recently developed WNK1-FHHt mice (WNK1+/FHHt) with WNK4−/− mice. Surprisingly, the activated NCC, hypertension, and hyperkalemia of WNK1+/FHHt mice remain in the absence of WNK4. We demonstrate that WNK1 powerfully stimulates NCC in a WNK4-independent and SPAK-dependent manner. Moreover, WNK4 decreases the WNK1 and WNK3-mediated activation of NCC. Finally, the formation of oligomers of WNK kinases through their C-terminal coiled-coil domain is essential for their activity towards NCC. In conclusion, WNK kinases form a network in which WNK4 associates with WNK1 and WNK3 to regulate NCC.

Familial hyperkalemic hypertension (FHHt) can be mainly attributed to increased activity of the renal Na:Cl cotransporter (NCC), which is caused by altered expression and regulation of the with-no-lysine (K) 1 (WNK1) or WNK4 kinases. The WNK1 gene gives rise to a kidney-specific isoform that lacks the kinase domain (KS-WNK1), the expression of which occurs primarily in the distal convoluted tubule. The role played by KS-WNK1 in the modulation of the WNK/STE20-proline-alanine rich kinase (SPAK)/NCC pathway remains elusive. In the present study, we assessed the effect of human KS-WNK1 on NCC activity and on the WNK4-SPAK pathway. Microinjection of oocytes with human KS-WNK1 cRNA induces remarkable activation and phosphorylation of SPAK and NCC. The effect of KS-WNK1 was abrogated by eliminating a WNK-WNK-interacting domain and by a specific WNK inhibitor, WNK463, indicating that the activation of SPAK/NCC by KS-WNK1 is due to interaction with another WNK kinase. Under control conditions in oocytes, the activating serine 335 of the WNK4 T loop is not phosphorylated. In contrast, this serine becomes phosphorylated when the intracellular chloride concentration ([Cl]) is reduced or when KS-WNK1 is coexpressed with WNK4. KS-WNK1-mediated activation of WNK4 is not due to a decrease of the [Cl]. Coimmunoprecipitation analysis revealed that KS-WNK1 and WNK4 interact with each other and that WNK4 becomes autophosphorylated at serine 335 when it is associated with KS-WNK1. Together, these observations suggest that WNK4 becomes active in the presence of KS-WNK1, despite a constant [Cl].

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The role of Cl − as an intracellular signaling ion has been increasingly recognized in recent years. One of the currently best described roles of Cl − in signaling is the modulation of the With-No-Lysine (K) (WNK) -STE20-Proline Alanine rich Kinase (SPAK)/Oxidative Stress Responsive Kinase 1 (OSR1) -Cation-Coupled Cl − Cotransporters (CCCs) cascade. Binding of a Cl − anion to the active site of WNK kinases directly modulates their activity, promoting their inhibition. WNK activation due to Cl − release from the binding site leads to phosphorylation and activation of SPAK/OSR1, which in turn phosphorylate the CCCs. Phosphorylation by WNKs-SPAK/OSR1 of the Na + -driven CCCs (mediating ions influx) promote their activation, whereas that of the K + -driven CCCs (mediating ions efflux) promote their inhibition. This results in net Cl − influx and feedback inhibition of WNK kinases. A wide variety of alterations to this pathway have been recognized as the cause of several human diseases, with manifestations in different systems. The understanding of WNK kinases as Cl − sensitive proteins has allowed us to better understand the mechanistic details of regulatory processes involved in diverse physiological phenomena that are reviewed here. These include cell volume regulation, potassium sensing and intracellular signaling in the renal distal convoluted tubule, and regulation of the neuronal response to the neurotransmitter GABA.

Background: The calcium sensing receptor (CaSR) in the distal convoluted tubule (DCT) activates the NaCl cotransporter (NCC). Glucose acts as a positive allosteric modulator of the CaSR. Under physiological conditions, no glucose is delivered to the DCT and fructose delivery depends on consumption. We hypothesized that glucose/fructose delivery to the DCT modulates the CaSR in a positive allosteric way, activating the WNK4-SPAK-NCC pathway and thus increasing salt retention. Methods: We evaluated the effect of glucose/fructose arrival to the distal nephron on the CaSR-WNK4-SPAK-NCC pathway using HEK-293 cells, C57BL/6 and WNK4-knockout mice, ex vivo perfused kidneys, and healthy humans. Results: HEK-293 cells exposed to glucose/fructose increased SPAK phosphorylation in a WNK4 and CaSR dependent manner. C57BL/6 mice exposed to fructose or a single dose of dapagliflozin to induce transient glycosuria showed increased activity of the WNK4-SPAK-NCC pathway. The calcilytic NPS2143 ameliorated this effect, which was not observed in WNK4-KO mice. C57BL/6 mice treated with fructose or dapagliflozin showed markedly increased natriuresis after thiazide challenge. Ex vivo rat kidney perfused with glucose above physiological threshold levels for proximal reabsorption showed increased NCC and SPAK phosphorylation. NPS2143 prevented this effect. In healthy volunteers cinacalcet administration, fructose intake, or a single dose of dapagliflozin increased SPAK and NCC phosphorylation in urinary extracellular vesicles. Conclusions: Glycosuria or fructosuria were associated with increased NCC, SPAK, and WNK4 phosphorylation in a CaSR-dependent manner.