Interplay of Renal Ca and Na Transport Pathways

描述（由申请人提供）：高血压发生在近三分之一的美国成年人，是中风，心脏病发作和心力衰竭的主要危险因素。高血压的分子发病机制尚未得到很好的理解;然而，影响血压的遗传性疾病的最新发现突出了噻嗪敏感性Na-Cl协同转运蛋白NCC和肾脏远端小管中的WNK蛋白激酶在血压调节中的重要作用。有趣的是，钠和钙转运途径在远端小管是负相关的;因此，增加钙转运在远端小管减少钠转运，从而降低血压。因此，详细了解Na和Ca转运途径之间的相互作用在血压调节中是重要的。作为钙重吸收的看门人，TRPV 5主要定位于远曲小管晚期的顶膜，其中也表达NCC和WNK 4。WNK 4增强TRPVS介导的Ca转运，与其对NCC的抑制作用形成鲜明对比。此外，WNK 4对TRPV 5的增强作用被NCC剂量依赖性地阻断。此外，WNK 4对TRPV 5的增强作用被螯合细胞内Ca消除。基于这些发现，我们假设WNK 4通过增强TRPV 5和抑制NCC来整体调节Na和Ca转运途径; WNK 4的作用也受到TRPV 5和NCC的调节。我们计划通过追求三个具体目标来验证我们的假设：1）确定WNK 4增强TRPVS介导的Ca转运的机制; 2）确定NCC调节WNK 4作用的机制; 3）确定Ca/钙调蛋白在调节WNK 4介导的调节中的作用。这些研究将采用分子、生物化学和生理学的方法进行。Ileevis卵母细胞和MDCK株I细胞。将开发稳定的MDCK I细胞系以评估调节机制的生理意义。预计在该项目完成时，将通过转运蛋白及其共同调节因子WNK 4之间的相互作用获得对Ca和Na转运途径调节的新见解。随着从拟议的研究中获得的新知识，可能会开发一种通过对远端小管中Na和Ca通路的整体调节来控制血压的新策略。

DESCRIPTION (provided by applicant): Hypertension occurs in nearly one out of every three US adults and is a major risk factor for stroke, heart attack, and heart failure. The molecular pathogenesis of hypertension is not well understood; however, recent findings from genetic disorders affecting blood pressure highlight the important roles of the thiazide- sensitive Na-CI cotransporter NCC and the WNK protein kinases in the distal tubule of the kidney in blood pressure regulation. Intriguingly, Na and Ca transport pathways in the distal tubule are inversely related; therefore, an increase in Ca transport in the distal tubule decreases the Na transport and in turn lowers the blood pressure. Hence, a detailed understanding of the interplay between the Na and Ca transport pathways is important in blood pressure regulation. As a gatekeeper for Ca reabsorption, TRPV5 is prominently localized in the apical membrane of the late segment of the distal convoluted tubule, where NCC and WNK4 are also expressed. WNK4 enhances TRPVS-mediated Ca transport in striking contrast to its inhibitory effect on NCC. Furthermore, the enhancing effect of WNK4 on TRPV5 is dose-dependently blocked by NCC. In addition, the enhancing effect of WNK4 on TRPV5 is abolished by chelating intracellular Ca. Based on these findings we hypothesize that WNK4 integrally regulates Na and Ca transport pathways by enhancing TRPV5 and suppressing NCC; the actions of WNK4 are also modulated by TRPV5 and NCC. We plan to test our hypothesis by pursuing three specific aims: 1) determine the mechanism by which WNK4 enhances TRPVS-mediated Ca transport; 2) determine the mechanism by which NCC modulates the action of WNK4; and 3) determine the role of Ca/calmodulin in modulating WNK4-mediated regulation. The studies will be carried out using molecular, biochemical and physiological approaches with both X. laevis oocytes and MDCK strain I cells. Stable MDCK I cell lines will be developed to assess the physiological significance of the regulation mechanisms. It is expected that new insights into the regulation of Ca and Na transport pathways through the interplay between the transport proteins and their common regulator WNK4 will be obtained at the completion of the project. With new knowledge acquired from the proposed studies, it is likely a new strategy via integral regulation of Na and Ca pathways in the distal tubule could be developed for blood pressure control.