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

项目总结 盐敏感型高血压是世界范围内的一个重大健康问题，有必要了解 潜在的分子机制，以实现更有效的治疗。拟议的研究基于一项 在我们实验室对Dahl盐敏感(SS)大鼠进行的实验奠定了坚实的科学基础 模仿人类的疾病状况。我们已经证明，这种形式的高血压与 肾脏和血管产生过多的活性氧(ROS)，排泄Na+的能力降低。 肾髓质粗升支(MTAL)发生过度重吸收，导致更大的重吸收。 过滤后的钠离子。与此最相关的是，SS大鼠通过线粒体产生ATP的能力降低 MTAL中的呼吸作用，即负责重吸收近25%过滤后的Na+的管状部分 肾脏的组织结构。在肾脏的这一区域，存在高水平的氧化应激(过量的ROS产生)。 来自线粒体和细胞膜NADPH氧化酶(NOX2和NOX4)。其中两个 这一领域仍然存在的主要空白是首先缺乏对细胞/线粒体新陈代谢的机械性研究， 第二，缺乏定量评估复杂细胞相互依赖关系的方法 流程。我们推测，高盐饮食增加了Na+向SS大鼠的mTAL的输送 导致过量的Na+重吸收和mTAL胞浆[Na+]的增加，从而刺激线粒体ATP 合成和产生ROS，进而刺激膜NOx(ROS-ROS串扰和恶性循环) 导致线粒体氧化磷酸化(OxPhos)解偶联和组织损伤。目标1将利用 在SS大鼠中完整显微解剖mTAL以检验高盐饮食从而增加胞浆[Na+]的假设 刺激线粒体ROS的产生，进而促进细胞对Na+的更大吸收，尽管 线粒体和膜NOX2和NOX4的ROS-ROS串扰放大细胞内总ROS 生产导致OxPhos解偶联。膜NOx与线粒体ROS相互作用的贡献 将使用新型基因工程基因敲除株SSNox4KO和SSp67/Nox4DKO大鼠来确定。目标2将 确定肾脏分离线粒体中假定的生物能量事件的进展(两者 高盐饲养的SS大鼠的外髓和皮质)。线粒体生物能量学和进行性改变 ROS产量将在三周高盐饲养期间的四个时间点确定。目标3将 利用测量的数据驱动的计算建模来提供量化、集成和机械化的 可以预测细胞氧利用、能量利用之间存在的复杂关系的框架 在盐敏感型高血压的发展过程中，肾脏的产生和氧化应激。

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.