利用小鼠模型研究WNK1在调控钠钾转运中的作用

WNK1 is a member of newly discovered serine-threonine kinases of which mutations causes a familial hypertension and hyperkalemia syndrome (FHH). WNK1 has at least two different isoforms, a ubiquitous full-length form (L-WNK1) and kidney-specific form (KS-WNK1) exclusively in the kidney. Studies using expression systems have shown that L-WNK1 activates Na-Cl cotransporter NCC, Na-K-2Cl cotransporter NKCC2 and the epithelial Na+ channel ENaC and inhibits renal K+ channel ROMK. KS-WNK1, by itself, has no effect on NCC, and ROMK, but antagonizes L-WNK1 regulation of NCC and ROMK. These results lead to the hypothesis that KS-WNK1 is a physiological antagonist of L-WNK1. In this hypothesis, the ratio of L-WNK1 over KS-WNK1 controls Na+ reabsorption and K+ secretion. An increase in the ratio causes Na+ and K+ retention and a decrease causing Na+ and K+ wasting. The goal of this proposal is to examine the hypothesis using genetic altered animal models. I propose the following three specific aims: Aim 1: To examine the in vivo physiological role of KS-WNK1 in the regulation of Na+ and K+ transport using transgenic mice overexpressing KS-WNK1 and KS-WNK1 knockout mice. Aim 2: To examine the in vivo physiological role of L-WNK1 in the regulation of Na+ and K+ transport using conditional L-WNK1 knockout mice. Aim 3: To test the hypothesis that KS-WNK1 regulates renal Na+ and K+ transport by antagonizing the effects of L-WNK1. These studies will provide new insights into the mechanism of FHH and may help development of WNK1 antagonists as potential therapeutic agents for the treatment of hypertension and hyperkalemia.

WNK1是新发现的丝氨酸/苏氨酸激酶家族成员, 其基因突变会导致家族性高血压高血钾(FHH)。WNK1至少有两种亚型, 广泛存在的全长型(L-WNK1)和肾脏特异型(KS-WNK)。我们通过体外功能研究首次发现L-WNK1调控肾脏钠钾离子转运, 而KS-WNK1则拮抗 L-WNK1的作用。我们推测KS-WNK1是L-WNK1的生理拮抗剂，而且L-WNK1对KS-WNK1的比率增加时，会使远端肾单位的钠离子重吸收增加，钾离子分泌减少，导致高血压和高血钾，这可能是WNK1突变导致FHH的一个新机制。为证实这个假说，我们建立KS-WNK1和L-WNK1转基因和基因敲除小鼠模型，研究KS-WNK1和L-WNK1在调节血压血钾平衡中新的调控路径以及KS-WNK1是否是L-WNK1的拮抗剂。此研究将从新的视角阐明FHH发生的分子机制，并为高血压和高血钾的防治提供新思路和新靶点。

WNK1是一类新的蛋白激酶，其基因突变会导致家族性高血压和高血钾 (FHH) ，前期工作发现其两个亚型全长型 (L-WNK1) 和肾脏特异型 (KS-WNK1) 在体外调控肾脏钠钾离子通道作用是互相拮抗。本课题通过构建L-WNK1和KS-WNK1的转基因和基因敲除小鼠模型来进一步研究L-WNk1和KS-WNK1 在调节血压血钾平衡中的体内作用以及KS-WNK1 是否是L-WNK1 的拮抗剂。我们首先构建L-WNK1转基因小鼠(L-WNK1-TG), 通过检测血压，血尿电解质和肾脏钠离子通道，发现L-WNK1-TG小鼠血压显著下降，在低钠情况下出现尿钠排泄增加，肾脏钠离子通道NCC和NKCC2表达量和功能都下降，而且上游调控的SPAK/OSR1表达也相应下降，表明L-WNK1可能通过WNK1-SPAK/OSR1-NCC/NKCC2激酶信号通路下调肾脏钠离子通道，进而引起血压下降。另外，我们又构建KS-WNK1 敲除小鼠(KS-WNK1-KO),发现KS-WNK1-KO血压增加，肾脏钠离子通道NCC/NKCC2和上游激酶SPAK/OSR1表达均上调，表明KS-WNK1也是通过KS-WNK1-SPAK/OSR1-NCC/NKCC2激酶信号通路上调肾脏钠离子通道，进而导致血压升高。L-WNK1和KS-WNK1小鼠交配验证它们是否拮抗的工作正在进行。综合上述结果，本研究为阐明家族性高血压和高血钾发生的分子机制奠定了坚实的工作基础。项目资助已发表核心论文1篇，另有已接受SCI论文1篇，待发表论文两篇。培养硕士生6名，其中1名已经取得硕士学位，今年两名硕士即将毕业，另三名硕士在读中。项目投入经费23万元，支出也近23万元，各项支出基本与预算相符。