Mitochondria-mediated effects and therapeutic potential of Atrial Natriuretic Peptide in salt-sensitive hypertension

项目摘要 对于血压盐敏感性患者亚群，目前尚无特异性治疗方法 (BP)不幸的是，盐敏感性的分子机制仍然知之甚少。之一 盐敏感性（SS）高血压发展的主要机制涉及以下缺陷： 肾脏排泄盐的能力。由Nppa编码的心房利钠肽（ANP）是一种已知的激素， 促进盐的排泄和血压的降低，并且有临床数据表明， SS高血压的发展。在其他作用中，已知ANP（通过cGMP相关机制）是 有益于线粒体生物能量学和生物发生。然而，在知识方面存在差距， ANP对肾脏线粒体的影响，尤其是在SS高血压中。我们的试点研究表明， 在高盐攻击期间，Nppa-/-（ANP敲除）Dahl SS大鼠表现出加重的盐敏感性， 钠排泄减少，肾损伤加重，这与线粒体损伤有关， 功能障碍我们还发现，在Nppa-/-大鼠中， 与野生型对照相比，收集的细胞中上皮Na+通道（ENaC）的活性升高， 管道在野生型SS大鼠中，慢性ANP输注导致盐诱导的血压升高显著减弱 减轻了器官损伤 我们假设SS高血压患者ANP缺乏/对ANP敏感性降低是导致肾损害的原因。 线粒体功能障碍和相关的钠转运失衡。为了解决这个问题的核心假设， 在这个项目中，我们制定了三个具体目标：目标1。确定增加的ANP水平是否有益于 SS高血压患者的肾盐处理和心脏功能。目标二。确定肾cGMP水平是否低 由于缺乏ANP导致肾线粒体Ca 2+和活性氧（ROS）增加。 目标3。测试功能障碍性细胞破坏Ca 2+平衡和过量ROS产生的假设。 线粒体影响SS高血压肾钠处理。 我们产生了大量的证据来支持这些目标，建立了一个严格和全面的实验， 设计并建立了新的尖端技术来解决这个假设。我们招募了强壮的 合作的专业知识，并将实施整体动物研究和体内技术（血液）相结合 压力监测与药物输注、代谢研究和GFR测量）、电生理学（单次 通道和全细胞膜片钳的新鲜分离的肾单位和分离的线粒体），先进的 显微镜、线粒体荧光分光光度法和呼吸测定法以及常规分子生物学方法。的 成功完成拟议的研究将揭示盐敏感性的新的致病机制。

PROJECT SUMMARY There is no specific treatment available for the subpopulation of patients with salt sensitivity of blood pressure (BP); unfortunately, the molecular mechanisms underlying salt-sensitivity remain poorly understood. One of the major proposed mechanisms for the development of salt-sensitive (SS) hypertension involves a defect in the ability of the kidneys to excrete salt. Atrial Natriuretic Peptide (ANP) encoded by Nppa, is a hormone known to promote salt excretion and BP reduction, and there are clinical data implicating inherently low levels of ANP in the development of SS hypertension. Among other effects, ANP (via cGMP-related mechanisms) is known to be beneficial for mitochondrial bioenergetics and biogenesis. However, there is a gap in knowledge regarding the effects of ANP on mitochondria in the kidney, especially in SS hypertension. Our pilot studies demonstrated that during a high salt challenge Nppa-/- (ANP knockout) Dahl SS rats exhibit exacerbated salt-sensitivity, reduced sodium excretion, and aggravated kidney injury, which is associated with mitochondrial damage and dysfunction. We also showed that there is dysregulation of renal sodium transporters, in the Nppa-/- rats compared to wild-type controls, and the activity of the Epithelial Na+ Channel (ENaC) is elevated in the collecting ducts. Chronic ANP infusion in wild-type SS rats resulted in a dramatic attenuation of salt-induced BP increase and alleviated organ damage. We hypothesize that in SS hypertension ANP deficiency/reduced sensitivity to ANP is causative to renal mitochondrial dysfunction and associated sodium transport imbalance. To address the central hypothesis of this project, we developed three specific aims: Aim 1. Establish whether increased ANP levels are beneficial for renal salt handling and cardiac function in SS hypertension. Aim 2. Determine whether low renal cGMP level resulting from lack of ANP causes an increase in renal mitochondrial Ca2+ and reactive oxygen species (ROS). Aim 3. Test the hypothesis that disrupted Ca2+ balance and excessive ROS production by dysfunctional mitochondria affect renal sodium handling in SS hypertension. We generated abundant evidence to support these aims, created a rigorous and comprehensive experimental design and established novel cutting-edge techniques to address the hypothesis. We recruited strong collaborative expertise, and will implement a combination of whole-animal studies and in vivo techniques (blood pressure monitoring with drug infusion, metabolic studies and GFR measurements), electrophysiology (single channel and whole-cell patch-clamp of the freshly isolated nephrons and isolated mitochondria), advanced microscopy, mitochondrial spectrofluorimetry and respirometry, and routine molecular biology approaches. The successful completion of the proposed studies will unravel the novel causative mechanisms of salt-sensitivity.