Sodium-chloride co-transporter regulation in the kidney

描述（由申请人提供）：Na+Cl-共转运蛋白（NCC）在远曲小管（DCT）的尖质膜（APM）中表达。NCC抑制可引起盐消耗，降低BP，而NCC刺激可提高BP: WNK激酶突变可增加APM NCC及其活性，WNK底物SPAK激酶失活可降低NCC磷酸化（NCCp）和BP。肾素血管紧张素系统（RAS）通过angi - wnk4 - spak依赖通路刺激NCC活性。我们提供的体内证据表明，在高盐饮食和ACE抑制期间，NCC从APM重新分布到根尖下细胞质囊泡（SCV），在低盐饮食和AngII输注期间，NCC从SCV重新分布到APM。我们现在表明，NCCp随着AngII治疗而增加，而高盐饮食则降低。AngII通过AT1R刺激NADPH氧化酶（Nox），产生活性氧（ROS）。我们现在表明，在AngII治疗期间，ROS清除可阻断NCC的贩运和NCCp。肾交感神经活动（RSNA）在高血压发病中也起主要作用。我们发现RSNA和肾上腺素能激动剂都刺激NCC向APM转运并增加NCCp。本研究的总体目标是确定NCC对AngII和/或RSNA的综合调控的分子机制，以及膳食NaCl对这些途径的影响。我们的假设是AngII（通过AT1R）和肾上腺素能激动剂（通过a1bAR）刺激Nox生成ROS并激活SPAK激酶，从而刺激APM和NCCp中NCC的积累。我们假设膳食盐通过Nox独立刺激ROS生成。目的1。NADPH氧化酶和SPAK在AngII刺激的NCC磷酸化和/或再分配到APM中的作用是什么？这些效果会受到饮食盐的影响吗？目标2。RSNA或肾上腺素能激动剂是否刺激DCT NCC活性？NADPH氧化酶刺激和/或SPAK磷酸化是必需的吗？饮食中的盐对这种调节有何影响？AngII ?方法。这些目标将在急性或慢性用RAS、NADPH氧化酶、RSNA激动剂和抑制剂和改变盐饮食治疗的大鼠中进行检验。SPAK、p47phox和alpha1b肾上腺受体敲除小鼠模型将被平行检查，以确定这些调节途径或中间体在NCC调节中的作用。NCC、NCCp、SPAK和SPAKp的分布将通过亚细胞分离、免疫印迹、免疫荧光和免疫电镜检测。常规测量肾功能、氧化应激和血压，并使用噻嗪类利尿剂试验测量NCC活性。实现这些目标将建立主要血压调节信号对DCT NCC细胞分布、NCCp和活性的综合影响，为DCT调节ECFV和BP的稳态设定点调节提供新的见解，并在理想情况下，为开发基于抑制调节DCT NCC活性的多种途径的治疗顽固性高血压和/或水肿的治疗策略提供指导。

DESCRIPTION (provided by applicant): The Na+Cl- cotransporter (NCC) is expressed in the apical plasma membrane (APM) of the distal convoluted tubule (DCT). NCC inhibition provokes salt wasting and can lower BP, while NCC stimulation can raise BP: WNK kinases mutations increase APM NCC and activity, inactivating the WNK substrate SPAK kinase reduces NCC phosphorylation (NCCp) and BP. The renin angiotensin system (RAS) stimulates NCC activity via an AngII-WNK4-SPAK dependent pathway. We provided in vivo evidence that NCC redistributes out of the APM into subapical cytoplasmic vesicles (SCV) during high NaCl diet and ACE inhibition and redistributes into the APM from SCV during low NaCl diet and AngII infusion. We now show that NCCp increases with AngII treatment and decreases with high salt diet. AngII via AT1R stimulates NADPH oxidase (Nox), generating reactive oxygen species (ROS). We now show that ROS scavenging during AngII treatment blocks NCC trafficking and NCCp. Renal sympathetic nerve activity (RSNA) also plays a primary role in hypertension pathogenesis. We show that both RSNA and adrenergic agonists stimulate NCC trafficking to APM and increase NCCp. The overall aim of this proposal is to determine the molecular mechanisms responsible for integrated regulation of NCC in response to AngII and/or RSNA and the influence of dietary NaCl on these pathways. Our hypothesis is that AngII (via AT1R) and adrenergic agonists (via a1bAR) stimulate Nox generation of ROS and activates SPAK kinase which stimulates NCC accumulation in APM and NCCp. We postulate that dietary salt independently stimulates ROS generation via Nox. Aim 1. What is the role of NADPH oxidase and SPAK in AngII stimulated NCC phosphorylation and/or redistribution to APM? Are these effects influenced by dietary salt? Aim 2. Do RSNA or adrenergic agonists stimulate DCT NCC activity? Are NADPH oxidase stimulation and/or SPAK phosphorylation requisite? How is this regulation affected by dietary salt? By AngII? Methods. The aims will be examined in rats treated acutely or chronically with agonists and inhibitors of RAS, NADPH oxidase, RSNA and altered salt diets. Mouse knockout models of SPAK, p47phox, and alpha1b adrenoreceptors will be examined in parallel to define the roles of these regulatory pathways or intermediates in NCC regulation. Distribution of NCC, NCCp, SPAK and SPAKp will be examined by both subcellular fractionation and immunoblots and immunofluorescence and immuno-EM. Renal function, oxidative stress and BP will be measured routinely and NCC activity measured using a thiazide diuretic test. Accomplishing the aims will establish integrated effects of major BP regulating signals on DCT NCC cellular distribution, NCCp and activity, providing novel insights into homeostatic set point regulation of ECFV and BP by the DCT and, ideally, indicating strategies for the development of therapies to treat resistant hypertension and/or edema based on inhibiting multiple pathways that regulate DCT NCC activity.