WNK1 regulation of renal NaCl cotransport

描述（由申请人提供）：家族性高钾血症性高血压（FHHt，也称为II型假性醛固酮减少症或Gordon综合征）是一种血压和钾水平升高的疾病，在表型上是Gitelman综合征的“镜像”。FHHt是由丝氨酸-苏氨酸激酶WNK 1和WNK 4突变引起的，这两种蛋白在肾远曲小管（DCT）中高度表达。申办方先前已经证明WNK 1和WNK 4相互作用以调节噻嗪敏感性NaCl协同转运蛋白（NCC）。该建议集中在WNK 1上，由于WNK 1基因内含子1内的缺失，其转录在FHHt患者中上调。WNK 1经历组织特异性剪接，并且主要的肾同种型（kWNK 1）包含可能有缺陷的断裂激酶结构域。初步数据表明，kWNK 1可能通过抑制WNK 1的调节作用对NCC共转运产生显性负效应。本文提出的实验用于阐明WNK 1和kWNK 1在DCT中的功能作用。为此，我们将研究在非洲爪蟾卵母细胞表达系统的NCC共转运的kWNK 1的功能影响。我们将通过免疫沉淀研究和体外激酶测定来检测kWNK 1与WNK 1的相互作用。我们还提出了一系列的实验，用于确定顺式元件内的第一内含子的WNK 1基因，可能会被消融FHHt，导致上调全长WNK 1相对于kWNK 1。

DESCRIPTION (provided by applicant): Familial Hyperkalemic Hypertension (FHHt, also known as Type II Pseudohypoaldosteronism or Gordon's syndrome) is a disorder of elevated blood pressure and potassium levels, and is phenotypically the "mirror image" of Gitelman's syndrome. FHHt is caused by mutations in the serine-threonine kinases WNK1 and WNK4, proteins highly expressed in the renal distal convoluted tubule (DCT). The sponsor has shown previously that WNK1 and WNK4 interact to regulate the thiazide-sensitive NaCI cotransporter (NCC). This proposal focuses on WNK1, whose transcription is upregulated in patients with FHHt due to a deletion within intron 1 of the WNK1 gene. WNK1 undergoes tissue-specific splicing, and the predominant renal isoform (kWNK1), contains a fractured kinase domain which is probably defective. Preliminary data suggest that kWNK1 may exert a dominant-negative effect on NCC cotransport by inhibiting the regulatory effect of WNK1. The experiments proposed herein serve to clarify the functional role of WNK1 and kWNK1 in the DCT. To this end, we will study the functional effects of kWNK1 on NCC cotransport in a Xenopus oocyte expression system. We will test for kWNK1 interactions with WNK1 with immunoprecipitation studies and in vitro kinase assays. We also propose a series of experiments that serve to identify cis elements within the first intron of the WNK1 gene that may be ablated in FHHt, leading to the upregulation of full length WNK1 relative to kWNK1.