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.