Differential roles of WNK4 in regulation of NCC in vivo

The Na ⁺ -Cl ^- cotransporter (NCC) in distal convoluted tubule (DCT) plays important roles in renal NaCl reabsorption. The current hypothesis for the mechanism of regulation of NCC focuses on WNK4 and intracellular Cl ⁺ concentration ([Cl ^- ] _i ). WNK kinases bind Cl ⁺ , and Cl ^- binding decreases the catalytic activity. It is believed that hypokalemia under low K ⁺ intake decreases [Cl ^- ] _i to activate WNK4, which thereby phosphorylates and stimulates NCC through activation of SPAK. However, increased NCC activity and apical NaCl entry would mitigate the fall in [Cl ^- ] _i . Whether [Cl ^- ] _i in DCT under low-K ⁺ diet is sufficiently low to activate WNK4 is unknown. Furthermore, increased luminal NaCl delivery also stimulates NCC and causes upregulation of the transporter. Unlike low K ⁺ intake, increased luminal NaCl delivery would tend to increase [Cl ^- ] _i . Thus we investigated the role of WNK4 and [Cl ^- ]i in regulating NCC. We generated Wnk4-knockout mice and examined regulation of NCC by low K ⁺ intake and by increased luminal NaCl delivery in knockout (KO) and wild-type mice. Wnk4-KO mice have marked reduction in the abundance, phosphorylation, and functional activity of NCC vs. wild type. Low K ⁺ intake increases NCC phosphorylation and functional activity in wild-type mice, but not in Wnk4-KO mice. Increased luminal NaCl delivery similarly upregulates NCC, which, contrary to low K ⁺ intake, is not abolished in Wnk4-KO mice. The results reveal that modulation of WNK4 activity by [Cl ^- ] _i is not the sole mechanism for regulating NCC. Increased luminal NaCl delivery upregulates NCC via yet unknown mechanism(s) that may override inhibition of WNK4 by high [Cl ^- ] _i .