Sodium-chloride co-transporter regulation in the kidney

描述(申请人提供)：钠氯共转运体(NCC)表达于远端曲管(DCT)的顶端质膜(APM)。抑制NCC可引起盐耗，降低血压，而刺激NCC可升高BP：WNK激酶突变可增加APM NCC和活性，失活WNK底物Spak激酶可降低NCC磷酸化(NCCP)和BP。肾素血管紧张素系统(RAS)通过AngiI-WNK4-SPAK依赖途径刺激NCC活性。我们提供了体内证据，在高盐饮食和ACE抑制时，NCC从APM重新分布到顶下细胞质小泡(SCV)，而在低盐饮食和AngII输注时，NCC从SCV重新分布到APM。我们现在发现，NCCP随着AngII治疗的增加而增加，而随着高盐饮食的增加而降低。血管紧张素转换酶I通过AT1R刺激NADPH氧化酶(NOx)，产生活性氧(ROS)。我们现在证明，在Angii治疗期间清除ROS可以阻止NCC贩运和NCCP。肾交感神经活动(RSNA)在高血压发病机制中也起主要作用。我们发现，RSNA和肾上腺素能激动剂都能刺激NCC向APM转运，并增加NCCP。这项建议的总体目标是确定负责对AngiI和/或RSNA反应的NCC进行整合调控的分子机制，以及饮食盐对这些途径的影响。我们的假设是，Angii(通过AT1R)和肾上腺素能激动剂(通过a1bAR)刺激ROS产生NOx，并激活Spak激酶，从而刺激APM和NCCP中NCC的积聚。我们推测食盐通过NOx独立地刺激ROS的产生。目的1.NADPH氧化酶和SPAK在血管紧张素Ⅱ刺激的NCC磷酸化和/或重分布到APM中起什么作用？这些影响是否受到食盐的影响？目的2.RSNA或肾上腺素能激动剂是否能刺激DCT NCC活性？NADPH氧化酶刺激和/或Spak磷酸化是必需的吗？食盐对这一规定有何影响？被安吉？方法：研究方法。这些AIMS将在急性或长期使用RAS、NADPH氧化酶、RSNA和改变的盐饮食的激动剂和抑制剂治疗的大鼠身上进行检验。我们将平行研究Spak、p47Phox和alpha1b肾上腺素受体的小鼠基因敲除模型，以确定这些调控途径或中间产物在NCC调控中的作用。NCC、NCCP、SPAK和SPAKp的分布将通过亚细胞分级和免疫印迹以及免疫荧光和免疫-EM来检测。将常规测量肾功能、氧化应激和血压，并使用噻嗪利尿剂试验测量NCC活性。这些目标的实现将建立主要的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的动态平衡设定点调节提供新的见解，并理想地指示基于抑制调节DCT NCC活性的多个通路的治疗顽固性高血压和/或水肿的治疗策略的开发。

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.