With no lysine kinase 4 (WNK4) is essential to activate the thiazide-sensitive 
NaCl cotransporter (NCC) along the distal convoluted tubule, an effect central 
to the phenotype of familial hyperkalemic hypertension. Although effects on 
potassium and sodium channels along the connecting and collecting tubules have 
also been documented, WNK4 is typically believed to have little role in 
modulating sodium chloride reabsorption along the thick ascending limb of the 
loop of Henle. Yet wnk4-/- mice (knockout mice lacking WNK4) do not demonstrate 
the hypocalciuria typical of pure distal convoluted tubule dysfunction. Here, we 
tested the hypothesis that WNK4 also modulates bumetanide-sensitive Na-K-2Cl 
cotransporter (NKCC2) function along the thick ascending limb. We confirmed that 
w nk4-/- mice are hypokalemic and waste sodium chloride, but are also 
normocalciuric. Results from Western blots suggested that the phosphorylated 
forms of both NCC and NKCC2 were in lower abundance in wnk4-/- mice than in 
controls. This finding was confirmed by immunofluorescence microscopy. Although 
the initial response to furosemide was similar in wnk4-/- mice and controls, the 
response was lower in the knockout mice when reabsorption along the distal 
convoluted tubule was inhibited. Using HEK293 cells, we showed that WNK4 
increases the abundance of phosphorylated NKCC2. More supporting evidence that 
WNK4 may modulate NKCC2 emerges from a mouse model of WNK4-mediated familial 
hyperkalemic hypertension in which more phosphorylated NKCC2 is present than in 
controls. These data indicate that WNK4, in addition to modulating NCC, also 
modulates NKCC2, contributing to its physiological function in vivo.