WNK1 regulation of renal NaCl cotransport

描述(申请人提供)：家族性高血钾高血压(FHHt，也称为II型假性低醛固酮血症或Gordon‘s综合征)是一种血压和钾水平升高的疾病，典型地是Gitelman’s综合征的“镜像”。FHHt是由丝氨酸苏氨酸激酶WNK1和WNK4突变引起的，这两种蛋白在肾脏远端曲管(DCT)中高表达。发起人先前已经证明，WNK1和WNK4相互作用，调节对噻嗪敏感的NaCI共转运蛋白(NCC)。这项建议关注的是WNK1，由于WNK1基因内含子1的缺失，其转录在FHHt患者中上调。WNK1经历组织特异性剪接，主要的肾脏亚型(KWNK1)包含一个断裂的可能是有缺陷的激酶结构域。初步数据表明，kWNK1可能通过抑制WNK1的调控作用而对NCC共转运起显性-负性作用。本文提出的实验有助于阐明WNK1和kWNK1在DCT中的功能作用。为此，我们将研究kWNK1在非洲爪哇卵母细胞表达系统中对NCC共转运的功能影响。我们将通过免疫沉淀研究和体外激酶试验来测试kWNK1与WNK1的相互作用。我们还提出了一系列实验，用于识别WNK1基因第一内含子中可能在FHHt中被消融的顺式元件，导致全长WNK1相对于kWNK1的上调。

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.