WNK kinases regulate thiazide-sensitive Na-Cl cotransport

Abstract

Pseudohypoaldosteronism type II (PHAII) is an autosomal dominant disorder of hyperkalemia and hypertension. Mutations in two members of the WNK kinase family, WNK1 and WNK4, cause the disease. WNK1 mutations are believed to increase WNK1 expression; the effect of WNK4 mutations remains unknown. The clinical phenotype of PHAII is opposite to Gitelman syndrome, a disease caused by dysfunction of the thiazide-sensitive Na-Cl cotransporter. We tested the hypothesis that WNK kinases regulate the mammalian thiazide-sensitive Na-Cl cotransporter (NCC). Mouse WNK4 was cloned and expressed in Xenopus oocytes with or without NCC. Coexpression with WNK4 suppressed NCC activity by more than 85%. This effect did not result from defects in NCC synthesis or processing, but was associated with an 85% reduction in NCC abundance at the plasma membrane. Unlike WNK4, WNK1 did not affect NCC activity directly. WNK1, however, completely prevented WNK4 inhibition of NCC. Some WNK4 mutations that cause PHAII retained NCC-inhibiting activity, but the Q562E WNK4 demonstrated diminished activity, suggesting that some PHAII mutations lead to loss of NCC inhibition. Gain-of-function WNK1 mutations would be expected to inhibit WNK4 activity, thereby activating NCC, contributing to the PHAII phenotype. Together, these results identify WNK kinases as a previously unrecognized sodium regulatory pathway of the distal nephron. This pathway likely contributes to normal and pathological blood pressure homeostasis.

Figure 1

(a) Effect of WNK4 on NCC-mediated ²²Na uptake, expressed as nmol/h per oocyte. Uptake rates ± SEM of oocytes injected with cRNA encoding NCC, WNK4, NCC + WNK4, NCC + an antisense WNK4 (AS WNK4), NCC + SGK (an unrelated kinase), and water are shown. WNK4 alone has no effect on uptake. Coexpression with WNK4 significantly reduces NCC-mediated Na uptake. Neither antisense WNK4 nor SGK affects NCC activity. *Uptake significantly different from NCC injection alone; P < 0.001. (b) Immunoblot of oocytes probed with anti-WNK4 antibody: oocytes were injected with water or with cRNA encoding WNK4, WNK4 + NCC, or NCC alone. Only oocytes injected with WNK4 cRNA express WNK4 protein. For NCC, WNK4, and NCC + WNK4, there were 12 repetitions of each experiment (15–30 oocytes per experiment). For NCC + antisense WNK4 and NCC + SGK, there were four experiments.Relative molecular weights are shown in kDa. HCTZ, hydrochlorothiazide.

Figure 2

(a) Immunoblot of immunoprecipitated oocyte membranes probed with anti-NCC antibody. Oocytes injected with NCC, NCC + WNK4, and NCC + WNK4 + WNK1 express the same amount of core-glycosylated protein. The ratio of mature glycosylated to core-glycosylated NCC is the same in all three groups. (b) Immunocytochemistry of oocytes stained with anti-NCC. Representative oocytes injected with NCC, NCC + WNK4, and NCC + WNK4 + WNK1 are shown. (c) Immunoblot of surface-biotinylated NCC. Only the mature glycosylated form of NCC was evident in this blot (data not shown). Coexpression of WNK4 with NCC reduced surface NCC expression by 85% (see Results). Coexpression with WNK1 and WNK4 restored surface NCC levels toward levels observed with NCC alone.

Figure 3

Effect of mutant WNK4 on NCC-mediated ²²Na uptake, expressed as nmol/h per oocyte. This figure compares rates of Na uptake (± SEM) of oocytes injected with cRNA encoding NCC and NCC + wild-type WNK4, E559K WNK4, D561A WNK4, and Q562E WNK4. Both wild-type and mutant WNK4 inhibited NCC activity (*P < 0.05 compared with NCC); Q562E reduced uptake significantly less than did wild-type WNK4 (^#P < 0.05 compared with NCC+ WNK4). There were six experiments (15–30 oocytes per experiment).

Figure 4

Effect of WNK1 on NCC-mediated ²²Na uptake, expressed as nmol/h per oocyte. This figure compares rates of Na uptake (± SEM) of oocytes injected with cRNA encoding NCC, WNK1, and NCC + WNK1, NCC + WNK4, and NCC + WNK4 + WNK1. *Uptake is significantly different (P < 0.001) from that of oocytes injected with NCC alone. There were seven experiments in each group (15–30 oocytes per experiment).