Regulation of Na-CI cotransporter (NCC) subcellular

描述(由申请人提供)：Na-CI共转运体(NCC)表达于远端曲管的顶膜，负责5%-10%过滤后的钠和氯的重吸收。NCC是经常用于治疗高血压、浮肿和心力衰竭的噻嗪类利尿剂的靶标。NCC的丰度受到以下因素的高度调控：饮食中的NaCI限制、醛固酮、雌激素和噻嗪治疗，此外，NCC在Gitelman综合征中发生突变，导致盐耗、低钾血症和代谢性碱中毒，所有这些都证实了NCC丰度在调节钠平衡和血压方面的动态平衡作用。我们最近(在大鼠体内)测试了一种假说，即高盐饮食引起DCT NCC从根尖质膜(PM)重新分布到根尖下细胞内膜池(IM)，而盐限制有利于重新分布到PM应用密度梯度离心法发现，NCC在高盐饮食下重新分布到高密度膜上，在盐限制期间重新分布到低密度膜上。免疫组织化学显示NCC的亚尖下IM池明显，高盐饮食IM/PM的NCC标记比率高于低盐饮食，低盐饮食NCC大部分位于PM，支持我们的假说。我们具有挑衅性的初步结果为研究体内调控NCC亚细胞分布的分子机制奠定了基础。在R21的应用中，我们的目的是充分证实NCC亚细胞分布受盐饮食长期调节的假设，并进一步检验PM和IM中NCC分布可以受到肾上腺素能刺激和/或急性高血压的强烈调节的假说。我们的研究将在以下方面开辟新的领域：NCC在响应肾上腺素(抗心钠药)和血压(钠尿)信号中的作用，NCC的来源和目的地，NCC相关蛋白的途径。为了确定NCC蛋白与蛋白质之间的相互作用以及它们如何随着NCC在每个模型中的表观再分布而变化，我们需要开发试剂和方法来免疫沉淀NCC及其相关蛋白和含有NCC的囊泡，用于后续的候选和蛋白质组学分析。这项调查旨在为活体内受管制的NCC贩运提供确凿证据，确定NCC贩运的潜在调解人，确定NCC贩运在疾病中的缺陷，并提出替代机制来改变NCC的治疗。

DESCRIPTION (provided by applicant): The Na-CI cotransporter (NCC) is expressed in the apical membrane of the distal convoluted tubule and is responsible for the reabsorption of 5% -10% of filtered Na and Cl. NCC is the target of the thiazide diuretics prescribed very frequently for treatment of hypertension, edema and heart failure. NCC abundance is highly regulated by: dietary NaCI restriction, aldosterone, estrogen and thiazide treatment, additionally, NCC is mutated in Gitelman's syndrome leading to salt wasting, hypokalemia and metabolic alkalosis, all confirming a homeostatic role of NCC abundance in regulating Na balance and blood pressure. We recently tested the hypothesis (in rats in vivo) that high salt diet provokes redistribution of DCT NCC from apical plasma membrane (PM) to sub-apical intracellular membrane pools (IM) and that salt restriction favors redistribution to the PM. Applying a density gradient centrifugation approach discovered that NCC redistributes to higher density membranes in response to high salt diet and to lower density membranes during salt restriction. By EM, sub-apical IM pools of NCC were evident and the ratio of NCC labeling in the IM to PM was higher in high salt diet than low salt diet where most all the NCC is in the PM, supporting our hypothesis. Our provocative preliminary results set the groundwork for addressing the molecular mechanisms regulating NCC subcellular distribution in vivo. In this R21 application we aim to fully confirm the hypothesis that NCC subcellular distribution is chronically regulated by salt diet and proceed to test the hypothesis that NCC distribution in PM vs. IM can be acutely regulated by adrenergic stimulation and/or acute hypertension. Our studies will break new ground in determining: the role of NCC in responding to adrenergic (antinatriuretic) and blood pressure (natriuretic) signals, the source and destination of NCC, the NCC associated proteins en route. In order to define NCC protein-protein interactions and how they change with apparent NCC redistribution in each model we need to generate reagents and methods to immunoprecipitate NCC along with associated proteins and along with NCC containing vesicles for subsequent candidate and proteomic analyses. This line of investigation aims to provide definitive evidence for regulated trafficking of NCC in vivo, identify potential mediators of the NCC trafficking, identify NCC trafficking defects in disease, and suggest alternative mechanisms to alter NCC therapeutically.