Regulation of Na-CI cotransporter (NCC) subcellular distribution in DCT

描述（由申请人提供）：Na- ci共转运蛋白（NCC）表达于远曲小管顶端膜，负责过滤后5% -10%的Na和Cl的重吸收。NCC是噻嗪类利尿剂的目标，通常用于治疗高血压、水肿和心力衰竭。NCC丰度受以下因素的高度调控：饮食中限制NaCI、醛固酮、雌激素和噻嗪治疗，此外，NCC在Gitelman综合征中发生突变，导致盐消耗、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Na平衡和血压方面的稳态作用。我们最近在大鼠体内验证了一个假设，即高盐饮食会引起DCT NCC从根尖质膜（PM）向亚根尖胞内膜池（IM）重新分布，而盐限制有利于向PM重新分布。应用密度梯度离心方法发现，NCC在高盐饮食中重新分配到高密度膜，在盐限制期间重新分配到低密度膜。EM显示，NCC的亚根尖IM池明显，高盐饮食中NCC标记在IM和PM中的比例高于低盐饮食，其中大多数NCC都在PM中，支持我们的假设。我们令人振奋的初步结果为解决体内调节NCC亚细胞分布的分子机制奠定了基础。在这项R21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，并进一步验证NCC在PM和IM中的分布可由肾上腺素能刺激和/或急性高血压急性调节的假设。我们的研究将在确定：NCC在肾上腺素能（抗尿钠）和血压（利钠）信号响应中的作用、NCC的来源和目的地、NCC相关蛋白在途中的作用方面开辟新的领域。为了定义NCC蛋白之间的相互作用，以及它们如何随着每个模型中NCC的重新分布而变化，我们需要生成试剂和方法来免疫沉淀NCC以及相关蛋白和含有NCC的囊泡，用于随后的候选蛋白和蛋白质组学分析。这项研究旨在为体内NCC的调控贩运提供明确的证据，确定NCC贩运的潜在介质，确定NCC贩运在疾病中的缺陷，并提出在治疗上改变NCC的替代机制。

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.