Mutations in the serine-threonine kinases WNK1 and WNK4 [with no lysine (K) at a key catalytic residue] cause pseudohypoaldosteronism type II (PHAII), a Mendelian disease featuring hypertension, hyperkalemia, hyperchloremia, and metabolic acidosis. Both kinases are expressed in the distal nephron, although the regulators and targets of WNK signaling cascades are unknown. The Cl ؊ dependence of PHAII phenotypes, their sensitivity to thiazide diuretics, and the observation that they constitute a ''mirror image'' of the phenotypes resulting from loss of function mutations in the thiazide-sensitive Na-Cl cotransporter (NCCT) suggest that PHAII may result from increased NCCT activity due to altered WNK signaling. To address this possibility, we measured NCCTmediated Na ؉ influx and membrane expression in the presence of wild-type and mutant WNK4 by heterologous expression in Xenopus oocytes. Wild-type WNK4 inhibits NCCT-mediated Na-influx by reducing membrane expression of the cotransporter ( 22 Nainflux reduced 50%, P < 1 ؋ 10 ؊9 , surface expression reduced 75%, P < 1 ؋ 10 ؊14 in the presence of WNK4). This inhibition depends on WNK4 kinase activity, because missense mutations that abrogate kinase function prevent this effect. PHAII-causing missense mutations, which are remote from the kinase domain, also prevent inhibition of NCCT activity, providing insight into the pathophysiology of the disorder. The specificity of this effect is indicated by the finding that WNK4 and the carboxyl terminus of NCCT coimmunoprecipitate when expressed in HEK 293T cells. Together, these findings demonstrate that WNK4 negatively regulates surface expression of NCCT and implicate loss of this regulation in the molecular pathogenesis of an inherited form of hypertension.protein serine-threonine kinases ͉ hypertension ͉ thiazide-sensitive Na-Cl cotransporter ͉ ion transport ͉ medical genetics H ypertension is the most common disease in industrialized societies, affecting Ͼ20% of the adult population and contributing to morbidity and mortality from stroke, myocardial infarction, renal failure, and congestive heart failure (1). Its pathogenesis is largely unknown, resulting in empiric pharmacologic therapy. In recent years, genetic approaches investigating rare Mendelian forms of high and low blood pressure have provided fundamental insight into mechanisms that contribute to blood pressure variation (2). These have demonstrated the causal role of inherited variation in renal salt homeostasis in blood pressure variation, with mutations in many genes known to play a role in mediating or regulating renal salt reabsorption resulting in altered blood pressure.Pseudohypoaldosteronism type II (PHAII; Online Mendelian Inheritance in Man database no. 145260) is an autosomal dominant disease featuring hypertension with hyperkalemia despite normal glomerular filtration rate; renal tubular acidosis is a variable associated finding. The clinical features of this disease are chloride dependent and are also corrected with thiazide diuretics, specific a...

Regulation of renal Naϩ transport is essential for controlling blood pressure, as well as Na ϩ and K ϩ homeostasis. Aldosterone stimulates Na ϩ reabsorption by the Na ϩ -Cl Ϫ cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na ϩ channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT 15 cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.

Angiotensin II (Ang II)-induced hypertension is associated with an increase in T cell production of interleukin 17A (IL-17A). Recently, we reported that IL-17A−/− mice exhibit blunted hypertension, preserved natriuresis in response to a saline challenge, and decreased renal sodium hydrogen exchanger 3 (NHE3) expression after 2 weeks of Ang II infusion compared to wild type (WT) mice. In the current study, we performed renal transporter profiling in mice deficient in IL-17A or the related isoform, IL-17F, after 4 weeks of Ang II infusion, a time when the blood pressure reduction in IL-17A−/− mice is most prominent. Deficiency of IL-17A abolished the activation of distal tubule transporters, specifically the sodium-chloride cotransporter (NCC) and the epithelial sodium channel (ENaC) and protected mice from glomerular and tubular injury. In human proximal tubule (HK-2) cells, IL-17A increased NHE3 expression through a serum and glucocorticoid regulated kinase 1 (SGK1) dependent pathway. In mouse distal convoluted tubule (mDCT15) cells, IL-17A increased NCC activity in an SGK1/Nedd4-2 dependent pathway. In both cell types, acute treatment with IL-17A induced phosphorylation of SGK1 at serine 78, and treatment with an SGK1 inhibitor blocked the effects of IL-17A on NHE3 and NCC. Interestingly, both HK-2 and mDCT15 cells produce endogenous IL-17A. IL17F had little or no effect on blood pressure or renal sodium transporter abundance. These studies provide a mechanistic link by which IL-17A modulates renal sodium transport and suggest that IL-17A inhibition may improve renal function in hypertension and other autoimmune disorders.

Abstract. The rat thiazide-sensitive Na-Cl cotransporter (rNCC) is expressed in the renal distal convoluted tubule and is the site of action of an important class of antihypertensive agents, the thiazide diuretics. The amino acid sequence contains two potential N-linked glycosylation consensus sites, N404 and N424. Either enzymatic deglycosylation or tunicamycin reduced the cotransporter to its core molecular weight (113 kD). Glycosylation site single mutants expressed in oocytes ran as thick bands at 115 kD, consistent with the highmannose glycoprotein. The double mutant produced the single thin 113-kD band seen in the deglycosylated cotransporter. Functional expression of cotransporters in Xenopus laevis oocytes revealed that the mutants displayed drastically decreased thiazide-sensitive 22 Na ϩ uptake compared with wild-type NCC. Analysis of enhanced green fluorescence protein (EGF-P)-tagged cotransporters demonstrated that this decrease in function is predominantly secondary to decreased surface expression. The elimination of glycosylation in the double mutant increased thiazide sensitivity by more than two orders of magnitude and also increased Cl Ϫ affinity. Thus, we have demonstrated that rNCC is N-glycosylated in vivo at two sites, that glycosylation is essential for efficient function and surface expression of the cotransporter, and that the elimination of glycosylation allows much greater access of thiazide diuretics to their binding site.