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

描述（由申请方提供）：Na-Cl协同转运蛋白（NCC）在远曲小管的顶膜中表达，负责重吸收5%-10%的过滤Na和Cl。NCC是噻嗪类利尿剂的靶点，常用于治疗高血压、水肿和心力衰竭。NCC丰度受到以下因素的高度调节：饮食NaCl限制、醛固酮、雌激素和噻嗪治疗，此外，NCC在Gitelman综合征中突变，导致盐浪费、低钾血症和代谢紊乱，所有这些都证实了NCC丰度在调节Na平衡和血压中的稳态作用。我们最近测试的假设（在大鼠体内），高盐饮食引起的DCT NCC从顶端质膜（PM）的再分配到亚顶端细胞内膜池（IM）和盐限制有利于再分配到PM。应用密度梯度离心方法发现，NCC重新分配到高密度膜响应高盐饮食和低密度膜在盐限制。通过EM，NCC的顶下IM池是明显的，并且在IM与PM中的NCC标记的比率在高盐饮食中高于低盐饮食，其中大多数NCC都在PM中，支持我们的假设。我们的挑衅性的初步结果奠定了基础，解决调节NCC在体内亚细胞分布的分子机制。在本R21申请中，我们旨在充分证实NCC亚细胞分布受盐饮食慢性调节的假设，并继续检验NCC在PM与IM中的分布可受肾上腺素能刺激和/或急性高血压急性调节的假设。我们的研究将在确定以下方面取得新突破：NCC在响应肾上腺素能（抗利尿剂）和血压（利尿钠）信号中的作用、NCC的来源和目的地、途中的NCC相关蛋白。为了定义NCC蛋白-蛋白相互作用以及它们如何随着每个模型中明显的NCC重新分布而变化，我们需要产生试剂和方法来免疫沉淀NCC沿着相关蛋白以及沿着含有NCC的囊泡，用于随后的候选和蛋白质组学分析。这一系列研究旨在为NCC在体内的受管制的运输提供明确的证据，鉴定NCC运输的潜在介质，鉴定疾病中的NCC运输缺陷，并提出治疗性改变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.