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Abstract: Missense mutations in the WNK4 gene have been postulated to cause pseudohypoaldosteronism type II, an autosomal-dominant disorder characterized by hyperkalemia and hypertension. A previous study using Xenopus oocytes showed that wild-type WNK4 expression inhibited surface expression of renal K channel (ROMK) and that a disease-causing mutant further decreased the surface expression. The decreased surface expression of ROMK caused by mutant WNK4 was postulated to be a mechanism for decreased potassium secretion in distal nephrons that would presumably lead to hyperkalemia. To determine if the mutant WNK4 had such an inhibitory effect on the apical localization of ROMK in vivo, we generated transgenic mice using the CLCNKB gene promoter that expressed a mutant WNK4 (D564A) in distal nephrons. In contrast to the tight junction localization of wild-type WNK4 described previously, the mutant WNK4 was present in the cytoplasm in the distal tubules and in the apical membranes in the thick ascending limb of Henle’s loop. In both cell types, the apical localization of endogenous ROMK was not influenced by the co-expression of mutant WNK4. This result indicates that the mutant WNK4 does not have a dominant effect on the cellular localization of ROMK in vivo. [Copyright &y& Elsevier]

Abstract: Missense mutations in the WNK4 gene have been postulated to cause pseudohypoaldosteronism type II (PHAII), an autosomal-dominant disorder characterized by hyperkalemia and hypertension. Previous reports using Xenopus oocytes showed that wild-type WNK4 expression inhibited surface expression of the thiazide-sensitive NaCl cotransporter (NCC), while a disease-causing mutant lost the inhibitory effect on NCC surface expression. To determine if these changes observed in oocytes really occur in polarized epithelial cells, we generated stable MDCK II cell lines expressing NCC alone or NCC plus wild-type WNK4 or a disease-causing (D564A) WNK4. In contrast to the apical localization of NCC without co-expression of WNK4, immunofluorescence microscopy and biotin surface labeling revealed that this apical localization was equally decreased by both the wild-type and the mutant WNK4 expression. Apical localizations of two PHAII-unrelated apical transporters, sodium-independent amino acid transporter, BAT1 and bile salt export pump, Bsep, were also found to be decreased by both wild-type and mutant WNK4 expression. These results indicate that the regulation of NCC was not related to the disease-causing mutation and not restricted to the PHAII-related specific transporters. The regulation of intracellular localization of NCC by WNK4 might not be involved in the pathogenesis of PHAII. [Copyright &y& Elsevier]