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. 2003 Jan 21;100(2):680-4.
doi: 10.1073/pnas.242735399. Epub 2003 Jan 6.

Molecular pathogenesis of inherited hypertension with hyperkalemia: the Na-Cl cotransporter is inhibited by wild-type but not mutant WNK4

Frederick H Wilson  1 , Kristopher T Kahle, Ernesto Sabath, Maria D Lalioti, Alicia K Rapson, Robert S Hoover, Steven C Hebert, Gerardo Gamba, Richard P Lifton
Affiliations

Molecular pathogenesis of inherited hypertension with hyperkalemia: the Na-Cl cotransporter is inhibited by wild-type but not mutant WNK4

Frederick H Wilson et al. Proc Natl Acad Sci U S A. .

Abstract

Mutations in the serine-threonine kinases WNK1 and WNK4 [with no lysine (K) at a key catalytic residue] cause pseudohypoaldosteronism type II (PHAII), a Mendelian disease featuring hypertension, hyperkalemia, hyperchloremia, and metabolic acidosis. Both kinases are expressed in the distal nephron, although the regulators and targets of WNK signaling cascades are unknown. The Cl(-) dependence of PHAII phenotypes, their sensitivity to thiazide diuretics, and the observation that they constitute a "mirror image" of the phenotypes resulting from loss of function mutations in the thiazide-sensitive Na-Cl cotransporter (NCCT) suggest that PHAII may result from increased NCCT activity due to altered WNK signaling. To address this possibility, we measured NCCT-mediated Na(+) influx and membrane expression in the presence of wild-type and mutant WNK4 by heterologous expression in Xenopus oocytes. Wild-type WNK4 inhibits NCCT-mediated Na-influx by reducing membrane expression of the cotransporter ((22)Na-influx reduced 50%, P < 1 x 10(-9), surface expression reduced 75%, P < 1 x 10(-14) in the presence of WNK4). This inhibition depends on WNK4 kinase activity, because missense mutations that abrogate kinase function prevent this effect. PHAII-causing missense mutations, which are remote from the kinase domain, also prevent inhibition of NCCT activity, providing insight into the pathophysiology of the disorder. The specificity of this effect is indicated by the finding that WNK4 and the carboxyl terminus of NCCT coimmunoprecipitate when expressed in HEK 293T cells. Together, these findings demonstrate that WNK4 negatively regulates surface expression of NCCT and implicate loss of this regulation in the molecular pathogenesis of an inherited form of hypertension.

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Figures

Figure 1
Figure 1
Effect of WNK4 on 22Na influx mediated by NCCT in Xenopus oocytes. Oocytes were injected with cRNA encoding NCCT and wild-type or mutant WNK4; 22Na entry was measured as described in Methods. The injected cRNAs are indicated and the mean and standard error of 22Na influx is shown for each set of oocyte injections; for each set, the mean 22Na influx seen among oocytes injected with NCCT cRNA alone is expressed as 100%, and other injection series are expressed as a percentage of this value.
Figure 2
Figure 2
Effect of WNK4 on expression of NCCT in Xenopus oocytes. Confocal microscopy of Xenopus ooctyes expressing EGFP-tagged NCCT was performed in the presence and absence of expression of wild-type WNK4 as described in Methods. Representative examples of fluorescence seen in the absence of EGFP-NCCT (A), after expression of EGFP-NCCT alone (B), and after expression of EGFP-NCCT with wild-type WNK4 (C) are shown. The results demonstrate reduced expression of EGFP-NCCT at the cell surface after expression of WNK4.
Figure 3
Figure 3
Quantitation of expression of EGFP-NCCT in response to wild-type and mutant WNK4. Oocytes were injected with cRNA encoding EGFP-tagged NCCT and wild-type or mutant WNK4; green fluorescence was quantitated by confocal microscopy of oocytes as described in Methods. The injected cRNAs are indicated, and the mean and standard error of fluorescence is shown for each set of oocyte injections; as in Fig. 1, for each set of injections, the mean fluorescence seen among oocytes injected with NCCT cRNA alone is expressed as 100%, and other injection series are expressed as a percentage of this value.
Figure 4
Figure 4
Coimmunoprecipitation of WNK4 and C terminus of NCCT. (A) Full-length wild-type WNK4 tagged with the myc epitope and the C terminus of NCCT tagged with the V5 epitope were expressed in HEK 293T cells as described in Methods. Cellular extracts were prepared, and immunoprecipitation was performed with anti-V5 antibodies as described in Methods. The precipitated protein was subjected to SDS/PAGE on 4–15% gradient gels, transferred to membrane, and stained with anti-myc antibody. A 170-kDa protein corresponding to myc-tagged WNK4 is detected in immunoprecipitates from cells expressing both NCCT and WNK4, but not in cells transfected with only one or neither tagged protein. (B) The experimental protocol is as in A, except that the Q562E mutation found in PHAII kindred 13 has been introduced into WNK4; this mutant WNK4 is tagged with the HA epitope rather than myc, and staining is with anti-HA antibody. Mutant WNK4 is coprecipitated by anti-V5 antibody only when both tagged NCCT and WNK4 are transfected.
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