The renal Na ؉ :Cl ؊ cotransporter rNCC is mutated in human disease, is the therapeutic target of thiazide-type diuretics, and is clearly involved in arterial blood pressure regulation. rNCC belongs to an electroneutral cation-coupled chloride cotransporter family (SLC12A) that has two major branches with inverse physiological functions and regulation: sodium-driven cotransporters (NCC and NKCC1/2) that mediate cellular Cl ؊ influx are activated by phosphorylation, whereas potassium-driven cotransporters (KCCs) that mediate cellular Cl ؊ efflux are activated by dephosphorylation. A cluster of three threonine residues at the amino-terminal domain has been implicated in the regulation of NKCC1/2 by intracellular chloride, cell volume, vasopressin, and WNK/STE-20 kinases. Nothing is known, however, about rNCC regulatory mechanisms. By using rNCC heterologous expression in Xenopus laevis oocytes, here we show that two independent intracellular chloride-depleting strategies increased rNCC activity by 3-fold. The effect of both strategies was synergistic and dose-dependent. Confocal microscopy of enhanced green fluorescent protein-tagged rNCC showed no changes in rNCC cell surface expression, whereas immunoblot analysis, using the R5-anti-NKCC1-phosphoantibody, revealed increased phosphorylation of rNCC amino-terminal domain threonine residues Thr 53 and Thr 58 . Elimination of these threonines together with serine residue Ser 71 completely prevented rNCC response to intracellular chloride depletion. We conclude that rNCC is activated by a mechanism that involves amino-terminal domain phosphorylation.The renal Na ϩ :Cl Ϫ cotransporter (NCC 4 or TSC, gene symbol SLC12A3, locus identification number 6559) that is expressed at the apical membrane of the mammalian distal convoluted tubule represents the major salt transport pathway in this segment of the nephron (1-4). Its essential role in preserving the extracellular fluid volume and blood pressure has been established by the identification of inactivating mutations of the SLC12A3 gene as the cause of Gitelman's disease (5, 6), an inherited disorder featuring arterial hypotension, renal salt wasting, hypokalemic metabolic alkalosis, hypocalciuria, and hypomagnesemia. In addition, a defect in NCC regulation by serine/threonine kinases WNK1 and WNK4 has been implicated in the pathogenesis of a salt-dependent form of human hypertension known as pseudohypoaldosteronism type II (PHAII) (7,8), which features marked sensitivity to hydrochlorothiazide and a clinical picture that is a mirror image of Gitelman's disease (9). NCC is the pharmacological target of thiazide-type diuretics that are currently recommended by the Joint National Committee VII for the detection, evaluation, and treatment of high blood pressure as the first line treatment of arterial hypertension either as the unique drug or in combination with other antihypertensive agents (10). Despite the importance of NCC for cardiovascular and renal physiology, pharmacology, and pathophysiology, little is known about t...

Modulation of Na+-Cl− cotransporter (NCC) activity is essential to adjust K+ excretion in the face of changes in dietary K+ intake. We used previously characterized genetic mouse models to assess the role of Ste20-related proline-alanine-rich kinase (SPAK) and with-no-lysine kinase (WNK)4 in the modulation of NCC by K+ diets. SPAK knockin and WNK4 knockout mice were placed on normal-, low-, or high-K+-citrate diets for 4 days. The low-K+ diet decreased and high-K+ diet increased plasma aldosterone levels, but both diets were associated with increased phosphorylation of NCC (phospho-NCC, Thr44/Thr48/Thr53) and phosphorylation of SPAK/oxidative stress responsive kinase 1 (phospho-SPAK/OSR1, Ser383/Ser325). The effect of the low-K+ diet on SPAK phosphorylation persisted in WNK4 knockout and SPAK knockin mice, whereas the effects of ANG II on NCC and SPAK were lost in both mouse colonies. This suggests that for NCC activation by ANG II, integrity of the WNK4/SPAK pathway is required, whereas for the low-K+ diet, SPAK phosphorylation occurred despite the absence of WNK4, suggesting the involvement of another WNK (WNK1 or WNK3). Additionally, because NCC activation also occurred in SPAK knockin mice, it is possible that loss of SPAK was compensated by OSR1. The positive effect of the high-K+ diet was observed when the accompanying anion was citrate, whereas the high-KCl diet reduced NCC phosphorylation. However, the effect of the high-K+-citrate diet was aldosterone dependent, and neither metabolic alkalosis induced by bicarbonate, nor citrate administration in the absence of K+ increased NCC phosphorylation, suggesting that it was not due to citrate-induced metabolic alkalosis. Thus, the accompanying anion might modulate the NCC response to the high-K+ diet.