Dietary Electrolyte–Driven Responses in the Renal WNK Kinase Pathway In Vivo

Abstract

T he role of WNK1 and WNK4 in control of electrolyte balance and BP first became apparent with their mutation being associated with familial hyperkalemic hypertension (FHHt; also known as Gordon syndrome and pseudohypoaldosteronism type 2), a human autosomal dominant disorder that features hypertension, hyperkalemia, and acidosis that usually are hyperresponsive to thiazide diuretics (1,2). FHHt can be caused by intronic deletions in WNK1 or missense mutations in WNK4 (3). Mutations in either cause a broadly similar phenotype, suggesting that WNK1 and WNK4 function in a common pathway. Unlike most monogenic disorders that affect BP, which feature reciprocal Na ϩ and K ϩ (and/or H ϩ ) imbalances and share a relationship to the aldosterone pathway (4), FHHt features concurrent NaCl and K ϩ (and/or H ϩ ) retention (1,3,5). This unusual characteristic indicates the existence of a novel "WNK pathway" functioning in normal physiology, which may allow the "independent of aldosterone" regulation of K and Na balance (and extracellular volume) by the kidney, ultimately also maintaining BP within the normal range. The BP-regulatory role of this WNK pathway is conserved in evolution as WNK1ϩ/Ϫ mice are hypotensive (6).Previously, we and others demonstrated that a 5Ј-truncated kinase-deficient isoform (WNK1-S) predominates in kidney (7), this being conserved between human and mouse (7-9). Isoform-specific probes distinguished ubiquitous low-level expression of full-length WNK1-long (WNK1-L) from abundant WNK1-S expression in distal nephron. Recent Xenopus oocyte studies implicate WNK4 in inhibition of NaCl reabsorption by thiazide-sensitive Na ϩ Cl Ϫ co-transporter (NCC) (10,11) and/or Materials and Methods Animal TreatmentAll procedures were carried out under provisions of ethically approved licenses and involved adult, 25-to 30-g, male C57BL/6 mice (Charles River, Margate, UK). Modified electrolyte feeds for mice were obtained from Special Diet Services (Witham, UK). RNA ExtractionAt conclusion of treatments of mice, both kidneys were removed under terminal anesthetic, immediately frozen on dry ice, and stored at Ϫ80°C. Frozen kidneys were fragmented and immediately homogenized in TRIzol Reagent (Invitrogen, Paisley, UK), and total RNA was extracted following the manufacturer's guidelines. Real-Time PCRAssays used ABI PRISM 7900 relative quantification real-time methods (Applied Biosystems, Foster City, CA). PCR was performed in 384-well plates (AB Gene) and used 10-l reactions that contained 5.0 l of TaqMan Master Mix (Applied Biosystems), 200 nM of each primer, 5 nM of probe, and 4.5 l of template (1:40 dilution of cDNA synthesized as described previously [7]). PCR conditions involved 95°C for 10 min, then 40 cycles of 95°C for 15 s and 60°C for 60 s. Standard template dilution curves enabled target gene quantification and normalization to the endogenous control TATA-Box Binding Protein (TBP). All group values were calibrated to their control groups.Validation studies using mouse renal RNA established TBP as an excelle...

Regulation of WNK1 Expression by miR-192 and Aldosterone

“…mRNA expression of L-WNK1 and KS-WNK1 and protein ex- pression of L-WNK1 were measured in the kidney of 16-week-old C57BL/6N mice fed with a normal, low-sodium, or high sodium diet, submitted to a potassium load or a chronic aldosterone infusion for 10 days (Supplemental Table S2). In agreement with a previous study, 21 we confirmed that aldosterone and potassium load led respectively to a 1.6-and 1.5-fold increase in KS-WNK1 mRNA expression (Supplemental Figure S3) and that L-WNK1 mRNA expression is unaffected by the different challenges ( Figure 5A). At the protein level, L-WNK1 was not modified by changes in Na ϩ or K ϩ intake but was significantly increased by aldosterone infusion (1.9 Ϯ 0.17 versus 1 Ϯ 0.12 in control animals; Figure 5B).…”

“…؉ or K ؉ Intake and Aldosterone KS-WNK1 mRNA expression is stimulated by aldosterone, 21,22 whereas L-WNK1 expression is unaffected. We asked if L-WNK1 expression could be regulated at the post-transcriptional level.…”

“…This modest increase in L-WNK1 expression was comparable to the variation in WNK1 expression described in other studies. 21,28 The expression of KS-WNK1 protein could not be evaluated in the absence of specific antibody. These results suggest that L-WNK1 expression is regulated only at the post-transcriptional level by aldosterone.…”

Angiotensin II signaling via protein kinase C phosphorylates Kelch-like 3, preventing WNK4 degradation

“…Previous studies have demonstrated that both WNK4 and KLHL3 are present in the thick ascending limb of Henle (TAL), distal convoluted tubule (DCT), connecting tubule (CNT), and collecting duct (CD) (2,4,26,38). Levels of KLHL3 S433-P in nephrons of mice on a high-salt diet with and without AII infusion (five mice in each group) was compared by immunofluorescence microscopy.…”

Variations in the WNK1 gene modulates the effect of dietary intake of sodium and potassium on blood pressure determination

“…12 Furthermore, it has been proposed that the molecular switch for the splicing of L-WNK1 and KS-WNK1 is affected by the amount of dietary potassium intake. [19][20][21] In this study, we investigated whether the common variations in the WNK1 gene are potential contributors to individual variations in blood pressure. Furthermore, we analyzed the interactions between the common WNK1 variants and dietary potassium and sodium intake for determining inter-individual variations in blood pressure.…”