Interplay of Renal Ca and Na Transport Pathways

描述(申请人提供)：近三分之一的美国成年人患有高血压，高血压是中风、心脏病发作和心力衰竭的主要风险因素。高血压的分子发病机制尚不清楚；然而，最近影响血压的遗传性疾病的发现突显了肾脏远端小管中对噻嗪敏感的Na-CI共转运体NCC和WNK蛋白激酶在血压调节中的重要作用。有趣的是，远端小管中的钠和钙转运途径是相反的；因此，远端小管中钙转运的增加减少了钠的转运，进而降低了血压。因此，详细了解钠和钙转运途径之间的相互作用在血压调节中是重要的。作为钙重吸收的守门人，TRPV5主要定位于远曲小管末段的顶膜，NCC和WNK4在此也有表达。WNK4促进TRPVS介导的钙转运，而对NCC的抑制作用则相反。此外，WNK4对TRPV5的增强作用被NCC剂量依赖性地阻断。此外，WNK4对TRPV5的增强作用被细胞内钙离子螯合作用所抵消。根据这些发现，我们推测WNK4通过增强TRPV5和抑制NCC来完整地调节Na和Ca的转运途径；WNK4的作用也受到TRPV5和NCC的调节。我们计划通过三个具体的目标来验证我们的假设：1)确定WNK4促进TRPVS介导的钙转运的机制；2)确定NCC调控WNK4的作用的机制；3)确定钙/钙调蛋白在调控WNK4介导的调控中的作用。这些研究将使用分子、生化和生理学方法进行，既有莱氏X.laevis卵母细胞，也有MDCK菌株I细胞。将建立稳定的MDCK I细胞系，以评估这些调节机制的生理意义。预计在该项目完成后，将通过运输蛋白与其共同调控因子WNK4之间的相互作用，对钙和钠转运途径的调控获得新的见解。随着从这些研究中获得的新知识，可能会开发一种新的策略，通过整合调控远端小管中的钠和钙通路来控制血压。

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.