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

项目摘要 盐敏感性高血压是世界范围内的一个重大健康问题，需要了解盐敏感性高血压的发病机制。 潜在的分子机制，以实现更有效的治疗。拟议的研究是基于一个 在我们的实验室中对Dahl盐敏感（SS）大鼠进行的实验具有强大的科学基础， 模拟人类的疾病状况。我们已经证明，这种形式的高血压与 肾和血管活性氧（ROS）产生过多，Na+排泄能力降低。 过度重吸收发生在肾髓质粗升支（mTAL），导致更大的重吸收 过滤的Na+。与这项研究最相关的是，SS大鼠通过线粒体产生ATP的能力降低， mTAL中的呼吸，负责重吸收近25%的过滤Na+的小管段 肾脏的。在肾脏的这个区域，存在高水平的氧化应激（过量的ROS产生）。 来源于线粒体和细胞膜NADPH氧化酶（NOX 2和NOX 4）。两 该领域中存在的主要差距首先是缺乏细胞/线粒体代谢的机理研究， 其次，缺乏定量评估复杂细胞相互依赖性的方法 流程.我们假设高盐饮食增加了SS大鼠mTAL的Na+输送， 导致过量的Na+重吸收和mTAL胞质[Na +]增加，其刺激线粒体ATP 合成和ROS产生，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 完整的显微切割的mTAL来检验SS大鼠中的假设，即高盐饮食增加细胞溶质[Na +]， 刺激线粒体活性氧的产生，从而增强细胞对钠离子的吸收，尽管如此 线粒体和膜NOX2和NOX4的ROS-ROS串扰，其放大总细胞内ROS 产生，导致OxPhos解偶联。膜NOX和线粒体ROS相互作用的贡献 将使用新型基因工程敲除菌株SSNox4KO和SSp67/Nox4DKO大鼠测定。目标2将 确定假定的生物能量事件在分离的肾线粒体中的进展（两者都 外髓质和皮质）。线粒体生物能量学的进行性改变， 在三周高盐饲养期间的四个时间点测定ROS产生。目标3将 利用测量的数据驱动的计算建模，以提供一个定量的，综合的，和机械的 该框架可以预测细胞氧利用率、能量消耗和代谢之间存在的复杂关系。 盐敏感性高血压发展过程中肾脏的氧化应激。

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.