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中，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不清楚；然而，最近来自影响血压的遗传疾病的研究结果强调了噻嗪敏感的Na-CI共转运体NCC和肾远端小管中的WNK蛋白激酶在血压调节中的重要作用。有趣的是，Na和Ca在远端小管中的运输途径呈负相关；因此，远端小管钙转运的增加会减少钠转运，从而降低血压。因此，详细了解钠钙转运途径之间的相互作用对血压调节很重要。TRPV5作为Ca重吸收的守门人，主要定位于远曲小管末段的顶膜，NCC和WNK4也在此表达。WNK4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，WNK4对TRPV5的增强作用被NCC剂量依赖性阻断。此外，WNK4对TRPV5的增强作用通过螯合细胞内Ca而被消除。基于这些发现，我们假设WNK4通过增强TRPV5和抑制NCC来整体调节Na和Ca的转运途径；WNK4的作用也受TRPV5和NCC的调控。我们计划通过三个具体目标来验证我们的假设：1)确定WNK4增强trpvs介导的钙转运的机制；2)确定NCC调控WNK4的作用机制；3)确定Ca/calmodulin在wnk4介导的调节中的作用。该研究将采用分子、生化和生理学方法，用X. laevis卵母细胞和MDCK株I细胞进行。将开发稳定的MDCK I细胞系来评估调节机制的生理意义。预计在项目完成后，通过转运蛋白与其共同调节因子WNK4之间的相互作用，将获得对Ca和Na转运途径调控的新见解。随着从这些研究中获得的新知识，很可能通过对远端小管中Na和Ca通路的整体调节来开发一种新的血压控制策略。

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.