Regulation of NCC by WNK kinases in kidney cells. In wild-type mice (left panel), WNK kinases send a positive signal to OSR1 and SPAK kinases by phosphorylation, and activated OSR1 and SPAK phosphorylate and activate NCC. Red arrows indicate positive regulation of NCC. NCC has been shown to be negatively regulated by wild-type WNK4 when assayed in Xenopus oocytes, which is independent of the WNK-OSR1/SPAK cascade. Blue arrows indicate negative regulation of NCC. In Wnk4^D561A/+ mice (middle panel), the PHAII-causing D561A mutation in a single WNK4 allele increases WNK kinase activity towards OSR1 and SPAK. Whether the increased WNK kinase activity is due to an increase in kinase activity of mutant WNK4 itself, or other mechanisms involving other WNK kinases, remains to be determined. The activated OSR1 and SPAK then activate NCC by phosphorylation. The disease-causing WNK4 mutants reportedly lack the inhibitory effect of wild-type WNK4 on NCC, as indicated by the dotted light blue arrow. Both mechanisms might work in parallel to activate NCC in PHAII. However, PHAII phenotypes can be restored only by the partial interruption of the signal from WNK kinases to OSR1 and SPAK in Wnk4^D561A/+Spak^T243A/+Osr1^T185A/+ triple knock-in mice (right panel). Because the level of NCC phosphorylation in wild-type mice and Wnk4^D561A/+Spak^T243A/+Osr1^T185A/+ triple knock-in mice is almost equal (Fig. 2), the triple knock-in mice should still show some PHAII-like phenotypes if the inhibitory power of wild-type WNK4 (dark blue arrow) is substantial. However, these mice showed normal phenotypes (Fig. 3), clearly suggesting that the inhibitory effect of wild-type WNK4 might not be as strong in in vivo kidney cells as in the Xenopus oocyte expression system.