Experimental and computational analysis of mechanisms of mitochondrial-cellular ROS crosstalk in the kidney in salt-sensitive hypertension

盐敏感性高血压肾脏线粒体-细胞 ROS 串扰机制的实验和计算分析

• 批准号: 10529290
• 负责人: Allen W Cowley
• 金额: $ 60.83万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-01 至 2024-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10529290
• 关键词: ATP Synthesis Pathway; Adult; Affect; African American; Apical; Applications Grants; Asian; Bioenergetics; Blood Pressure; Blood Vessels; Cardiovascular Diseases; Cell membrane; Cell physiology; Cells; Cerebrovascular Disorders; Complex; Computer Analysis; Computer Models; Consumption; Dahl Hypertensive Rats; Data; Development; Diet; Disease; Environmental Risk Factor; Event; Excretory function; Exhibits; Foundations; Genetic; Genetic Engineering; Grant; Health; Heart; Human; Hydrogen Peroxide; Hypertension; Injury to Kidney; Kidney; Kidney Diseases; Kidney Failure; Knock-out; Laboratories; Limb structure; Measurement; Measures; Membrane; Membrane Potentials; Mitochondria; Mitochondrial Proteins; Modeling; Molecular; Morbidity - disease rate; NADH; NADPH Oxidase; Oxidation-Reduction; Oxidative Phosphorylation; Oxidative Stress; Oxygen; Pathologic; Patients; Play; Population; Process; Production; Publishing; Rattus; Reactive Oxygen Species; Resistance; Respiration; Risk Factors; Role; Signal Transduction; Sodium Chloride; Source; Testing; Thick; Time; Tubular formation; absorption; blood pressure reduction; effective therapy; experimental analysis; experimental study; feeding; high salt diet; hypertensive; mitochondrial dysfunction; mitochondrial membrane; mitochondrial metabolism; modifiable risk; mortality; novel; response; salt intake; salt sensitive; salt sensitive hypertension; sodium-potassium chloride cotransporter 2 protein; tissue injury; uptake

PROJECT SUMMARY Salt-sensitive hypertension is a significant health problem worldwide and there is a need to understand the underlying molecular mechanisms to enable more effective treatments. The proposed studies are based on a strong scientific foundation with experiments performed in our laboratories in Dahl salt-sensitive (SS) rats which mimic the human condition of the disease. We have demonstrated that this form of hypertension is associated with excess renal and vascular reactive oxygen species (ROS) production and reduced ability to excrete Na+. Excess reabsorption occurs in the renal medullary thick ascending limb (mTAL) leading to greater reabsorption of filtered Na+. Most relevant to this grant, SS rats exhibit a reduced ability to generate ATP through mitochondrial respiration in the mTAL, the tubular segment that is responsible for reabsorption of nearly 25% of the filtered Na+ of the kidney. In this region of the kidney, there exists high levels of oxidative stress (excess ROS production) emanating from both the mitochondria and cell membrane NADPH oxidases (NOX2 and NOX4). Two of the major gaps that remain in this field are first a lack of mechanistic studies of cellular/mitochondrial metabolism, and second, an absence of approaches to quantitatively evaluate the interdependence of the complex cellular processes. We hypothesize that a high salt diet which increases the delivery of Na+ to the mTAL of SS rats results in excess Na+ reabsorption and an increase of mTAL cytosolic [Na+] which stimulates mitochondrial ATP synthesis and ROS production which in turn stimulates membrane NOXs (ROS-ROS crosstalk and vicious cycle) leading to uncoupling of mitochondrial oxidative phosphorylation (OxPhos) and tissue injury. Aim 1 will utilize intact microdissected mTAL to test the hypothesis in SS rats that high salt diet increases cytosolic [Na+] thereby stimulating mitochondrial ROS production which in turn enhances greater uptake of Na+ into the cell and though ROS-ROS crosstalk of mitochondria and membrane NOX2 and NOX4 which amplifies total intracellular ROS production leading to OxPhos uncoupling. Contribution of membrane NOXs and mitochondrial ROS interactions will be determined using novel genetically engineered knockout strains SSNox4KO and SSp67/Nox4DKO rats. Aim 2 will determine the progression of the postulated bioenergetic events in isolated mitochondria of the kidney (both outer medulla and cortex) of high salt fed SS rats. Progressive alterations of mitochondrial bioenergetics and ROS production will be determined at four time points during the three weeks of high salt feeding. Aim 3 will utilize the measured data-driven computational modeling to provide a quantitative, integrated, and mechanistic framework that can predict the complex relationships existing between cellular oxygen utilization, energy production, and oxidative stress in the kidney during the development of salt-sensitive hypertension.

项目摘要 对盐敏感的高血压是全球重大的健康问题，有必要了解 基本的分子机制可以实现更有效的治疗方法。拟议的研究基于 强大的科学基础，在我们在达尔盐敏感（SS）大鼠的实验室中进行的实验，该实验 模仿该疾病的人类状况。我们已经证明了这种高血压相关的形式 过量的肾脏和血管活性氧（ROS）产生以及排泄Na+的能力降低。 过量的重吸收发生在肾脏厚度上升的肢体（mTAL）中，导致更大的重吸收 过滤的Na+。与这笔赠款最相关的是，SS大鼠通过线粒体表现出降低的ATP的能力 MTAL中的呼吸，导致近25％过滤的Na+重新吸收的管状段 肾脏。在肾脏的这个区域中，存在高水平的氧化应激（ROS产生过多） 由线粒体和细胞膜纳德氧化酶（NOX2和NOX4）发出。两个 在该领域中仍然存在的主要差距首先缺乏细胞/线粒体代谢的机械研究， 其次，缺乏定量评估复杂细胞相互依赖的方法 过程。我们假设高盐饮食将Na+递送到SS大鼠的MTAL 导致过量的Na+重吸收和刺激线粒体ATP的MTAL胞质[Na+]的增加 合成和ROS产生又刺激膜NOX（ROS-ROS串扰和恶性循环） 导致线粒体氧化磷酸化（OXPHOS）和组织损伤的解偶联。 AIM 1将使用 完整的微解剖MTAL测试SS大鼠的假设，高盐饮食会增加胞质[NA+] 刺激线粒体ROS的产生，从而增强了Na+对细胞的更大摄取 线粒体和膜NOX2和NOX4的ROS-ROS串扰，它们放大了总细胞内ROS 产生导致Oxphos解偶联。膜NOX和线粒体ROS相互作用的贡献 将使用新型的基因敲除菌株SSNOX4KO和SSP67/NOX4DKO大鼠确定。 AIM 2意志 确定在肾脏的孤立线粒体中假定的生物能事件的进展（均 高盐喂SS大鼠的外髓质和皮质）。线粒体生物能学和 在高盐喂养的三周内，将在四个时间点确定ROS的产生。目标3意志 利用测量数据驱动的计算建模来提供定量，集成和机械的 可以预测细胞氧利用，能量之间存在的复杂关系的框架 在盐敏感高血压发展过程中，肾脏的产生和氧化应激。