Sodium-chloride co-transporter regulation in the kidney

描述（由申请方提供）：Na+Cl-协同转运蛋白（NCC）在远曲小管（DCT）的顶端质膜（APM）中表达。NCC抑制引起盐消耗并可降低BP，而NCC刺激可升高BP：WNK激酶突变增加APM NCC和活性，失活WNK底物SPAK激酶降低NCC磷酸化（NCCP）和BP。肾素血管紧张素系统（RAS）通过AngII-WNK 4-SPAK依赖性途径刺激NCC活性。我们提供了在体内的证据表明，NCC重新分配的APM到亚顶端细胞质囊泡（SCV）在高NaCl饮食和ACE抑制和重新分配到APM从SCV在低NaCl饮食和血管紧张素II输注。我们现在表明，NCCp随着AngII治疗而增加，随着高盐饮食而减少。AngII通过AT 1 R刺激NADPH氧化酶（Nox），产生活性氧（ROS）。我们现在表明，在AngII治疗期间清除ROS阻断了NCC运输和NCCp。肾交感神经活动（RSNA）也在高血压发病机制中起主要作用。我们发现，RSNA和肾上腺素能激动剂刺激NCC贩运到APM和增加NCCp。本提案的总体目标是确定负责响应于AngII和/或RSNA的NCC的综合调节的分子机制以及饮食NaCl对这些途径的影响。我们的假设是AngII（通过AT 1 R）和肾上腺素能激动剂（通过a1 bAR）刺激ROS的Nox产生并激活SPAK激酶，SPAK激酶刺激NCC在APM和NCCP中的积累。我们假设，饮食中的盐独立地刺激ROS的产生，通过氮氧化物。目标1. NADPH氧化酶和SPAK在AngII刺激的NCC磷酸化和/或再分布至APM中的作用是什么？这些影响是否受饮食盐的影响？目标2. RSNA或肾上腺素能激动剂刺激DCT NCC活性吗？NADPH氧化酶刺激和/或SPAK磷酸化是必需的吗？这一规定如何受到膳食盐的影响？被AngII？方法.将在急性或慢性接受RAS、NADPH氧化酶、RSNA激动剂和抑制剂和改变盐饮食的大鼠中检查目的。SPAK、p47 phox和alpha 1b肾上腺素受体的小鼠敲除模型将被平行检查，以确定这些调节途径或中间体在NCC调节中的作用。将通过亚细胞分级分离和免疫印迹以及免疫荧光和免疫EM检查NCC、NCCp、SPAK和SPAKp的分布。将常规测量肾功能、氧化应激和BP，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BP调节信号对DCT NCC细胞分布、NCCP和活性的综合影响，为DCT对ECFV和BP的稳态设定点调节提供新的见解，并且理想地，指示基于抑制调节DCT NCC活性的多种途径来治疗顽固性高血压和/或水肿的疗法的开发策略。 公共卫生相关性：肾脏肾单位的一个短区域称为远曲小管，通过钠-氯化物协同转运蛋白（NCC）仅重吸收5-10%的递送至肾脏的盐，但该区域似乎是血压（BP）的关键决定因素，也是靶向治疗以降低BP的关键区域。超过25%的人群患有高血压，其中很大一部分人群对目前的治疗方法有抵抗力。我们的目标是确定主要的BP调节信号，即激素，神经系统和饮食中的盐，如何影响远端小管中的NCC活性，并确定这些信号是如何同时整合的。这些结果将提供对肾脏如何设置BP的深入了解，并且理想地，指示基于抑制调节NCC活性的多个途径的联合疗法的开发策略，以治疗顽固性高血压和/或水肿。

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. PUBLIC HEALTH RELEVANCE: A short region of the kidney nephron known as the distal convoluted tubule reabsorbs only 5-10% of the salt delivered to the kidney, via the sodium-chloride cotransporter (NCC), yet this region appears to be a key determinant of blood pressure (BP), and also a key region to target therapies to lower BP. More than 25% of the population has high BP and a significant fraction of this population is resistant to current therapies. We aim to define how the major BP regulating signals, namely, hormones, nervous system and dietary salt, affect the NCC activity in the distal tubule, and determine how these signals are simultaneously integrated. The results will provide insight into how BP is set by the kidney and, ideally, indicate strategies for the development of combination therapies to treat resistant hypertension and/or edema based on inhibiting multiple pathways that regulate NCC activity.