Significance: Hypertension, one of the most common morbidity factors worldwide, is caused mostly by a deregulation of salt transport by the kidney, but underlying mechanisms remain elusive. The serine-threonine kinase With No lysine (K) 1 (WNK1) has been identified as a regulator of ion transport in the distal nephron, because its mutations cause Familial Hyperkalemic Hypertension (FHHt), a rare form of human hypertension. We generated a mouse model of WNK1-FHHt that fully recapitulates the disease and studied changes in expression of the Na ⁺ and K ⁺ transporters and channels involved in the maintenance of a correct blood pressure. Our study provides insights into the mechanisms underlying the pathogenesis of WNK1-FHHt and further corroborates the importance of WNK1 in ion homeostasis and blood pressure.

Mutations in WNK1 and WNK4 lead to familial hyperkalemic hypertension (FHHt). Because FHHt associates net positive Na ⁺ balance together with K ⁺ and H ⁺ renal retention, the identification of WNK1 and WNK4 led to a new paradigm to explain how aldosterone can promote either Na ⁺ reabsorption or K ⁺ secretion in a hypovolemic or hyperkalemic state, respectively. WNK1 gives rise to L-WNK1, an ubiquitous kinase, and KS-WNK1, a kinase-defective isoform expressed in the distal convoluted tubule. By inactivating KS-WNK1 in mice, we show here that this isoform is an important regulator of sodium transport. KS-WNK1 ^−/− mice display an increased activity of the Na-Cl cotransporter NCC, expressed specifically in the distal convoluted tubule, where it participates in the fine tuning of sodium reabsorption. Moreover, the expression of the ROMK and BKCa potassium channels was modified in KS-WNK1 ^−/− mice, indicating that KS-WNK1 is also a regulator of potassium transport in the distal nephron. Finally, we provide an alternative model for FHHt. Previous studies suggested that the activation of NCC plays a central role in the development of hypertension and hyperkalemia. Even though the increase in NCC activity in KS-WNK1 ^−/− mice was less pronounced than in mice overexpressing a mutant form of WNK4, our study suggests that the activation of Na–Cl cotransporter is not sufficient by itself to induce a hyperkalemic hypertension and that the deregulation of other channels, such as the Epithelial Na ⁺ channel (ENaC), is probably required.

The with-no-lysine (K) 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 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 Ste20 proline-alanine–rich kinase–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 toward NCC. In conclusion, WNK kinases form a network in which WNK4 associates with WNK1 and WNK3 to regulate NCC.

Mutations in the WNK1 gene, encoding a serine-threonine kinase of the WNK (With No lysine (K)) family, have been implicated in two rare human diseases, Familial Hyperkalemic Hypertension (FHHt) and Hereditary Sensory and Autonomic Neuropathy type 2 (HSAN2). Alternative promoters give rise to a ubiquitous isoform, L-WNK1, and a kidney-specific isoform, KS-WNK1. Several other isoforms are generated through alternative splicing of exons 9, 11 and 12 but their precise tissue distribution is not known. Two additional exons, 8b and HSN2, involved in HSAN2, are thought to be specifically expressed in the nervous system. The purpose of this study was to establish an exhaustive description of all WNK1 isoforms and to quantify their relative level of expression in a panel of human and mouse tissues and in mouse nephron segments. For the latter purpose, we developed a new methodology allowing the determination of the proportions of the different isoforms generated by alternative splicing. Our results evidenced a striking tissue-specific distribution of the different isoforms and the unexpected presence of exon HSN2 in many tissues other than the nervous system. We also found exon 26 to be alternatively spliced in human and identified two new exons, 26a and 26b, within intron 26, specifically expressed in nervous tissues both in humans and mice. WNK1 should therefore no longer be designated as a 28- but as a 32-exon gene, with 8 of them - 8b, HSN2, 9, 11, 12, 26, 26a and 26b - alternatively spliced in a tissue-specific manner. These tissue-specific isoforms must be considered when studying the different roles of this ubiquitous kinase.

WNK1 and WNK4 encode two members of the WNK serine-threonine kinase subfamily. Greater WNK1 expression associates with higher BP. A combination of promoters, enhancers, repressors, and insulators regulate WNK1 expression, but whether microRNAs also modulate WNK1 expression is unknown. Here, computational analysis revealed the presence of a target sequence for miR-192 and miR-215 at the same site in the 3′ untranslated region of the ubiquitous L- and the kidney-specific KS-WNK1. We functionally validated this target sequence by transient transfection and reporter assays. Although we observed expression of both miRs along the distal nephron, only miR-192 regulated endogenous WNK1 ex vivo. Furthermore, a potassium load, sodium depletion, and aldosterone infusion each significantly reduced miR-192 expression in the kidney. Taken together, these results suggest a miR-driven mechanism of gene regulation by aldosterone and a role for miR-192 in the regulation of sodium and potassium balance in the kidney.

Gain-of-function mutations in with no lysine (K) 1 (WNK1) and WNK4 genes are responsible for familial hyperkalemic hypertension (FHHt), a rare, inherited disorder characterized by arterial hypertension and hyperkalemia with metabolic acidosis. More recently, FHHt-causing mutations in the Kelch-like 3–Cullin 3 (KLHL3-CUL3) E3 ubiquitin ligase complex have shed light on the importance of WNK’s cellular degradation on renal ion transport. Using full exome sequencing for a 4-generation family and then targeted sequencing in other suspected cases, we have identified new missense variants in the WNK1 gene clustering in the short conserved acidic motif known to interact with the KLHL3-CUL3 ubiquitin complex. Affected subjects had an early onset of a hyperkalemic hyperchloremic phenotype, but normal blood pressure values”Functional experiments in Xenopus laevis oocytes and HEK293T cells demonstrated that these mutations strongly decrease the ubiquitination of the kidney-specific isoform KS-WNK1 by the KLHL3-CUL3 complex rather than the long ubiquitous catalytically active L-WNK1 isoform. A corresponding CRISPR/Cas9 engineered mouse model recapitulated both the clinical and biological phenotypes. Renal investigations showed increased activation of the Ste20 proline alanine–rich kinase–Na⁺-Cl^– cotransporter (SPAK-NCC) phosphorylation cascade, associated with impaired ROMK apical expression in the distal part of the renal tubule. Together, these new WNK1 genetic variants highlight the importance of the KS-WNK1 isoform abundance on potassium homeostasis.