Role of Rho Kinase in Diabetic Nephropathy

项目摘要 糖尿病肾病是美国终末期肾病（ESRD）的主要原因， 强调需要创新的治疗方法来防止其进展。我们有兴趣 了解糖尿病患者线粒体功能障碍的细胞和分子机制 环境，期望了解这些过程将揭示潜在的疾病机制 以及糖尿病肾病的治疗靶点。目前的建议是根据我们最近发表的 观察，表明线粒体断裂是必不可少的，促进线粒体功能障碍， 糖尿病环境中的足细胞详细了解线粒体分裂的机制， 肾脏仍然是不完整的，并且基于这些机制的治疗靶点不存在。因为 动力蛋白相关蛋白-1（Drp 1）在调节线粒体分裂中起着不可或缺的作用，我们关注于 研究Drp 1在线粒体断裂和糖尿病肾病进展中的作用。我们有 我们最近发表的观察结果表明，高葡萄糖会导致线粒体断裂， 促进Drp 1向线粒体的募集。在db/db糖尿病小鼠中缺失Drp 1可预防 线粒体分裂和改善晚期糖尿病肾病的组织学和生化特征。 重要的是，我们发现高糖环境通过磷酸化Drp 1的丝氨酸来触发线粒体分裂 600残留物。在这里，我们建议建立之间的串扰磷酸化的Drp 1和电子 转运复合物（ETC）作为线粒体ROS（mROS）的关键介质和潜在的治疗靶点 糖尿病肾病（DN）为了支持我们的假设，我们最近产生了一种新的糖尿病 敲入突变小鼠，其具有单个磷酸化缺陷（丝氨酸至丙氨酸）点突变， 内源性Drp 1等位基因中相应的S600位点（Drp 1 S600 A）。我们观察到糖尿病Drp 1 S600 A 小鼠表现出改善的DN的关键生化和组织学特征。评估Drp 1 S600的作用 接下来，我们将糖尿病Drp 1 S600 A小鼠与表达氧化还原-磷酸化的小鼠杂交， - 特异性地在线粒体基质中的灵敏的绿色荧光蛋白生物传感器（roGFP）（mt-roGFP），以及 观察到糖尿病小鼠中的Drp 1 S600 A突变导致糖尿病小鼠足细胞中的mROS减少， 动物这些发现提供了令人信服的初步证据，证明Drp 1在信号转导中的意想不到的作用。 调节mROS的级联反应，并代表了可能用于预防糖尿病的治疗靶点 肾病鉴于这些结果和本申请中提供的其他初步数据，本项目 将解决Drp 1磷酸化与线粒体ETC动态相互作用以增强 mROS通过由心磷脂激活调节的信号传导网络。这项研究的结果将 为线粒体形态学在糖尿病发展中的作用提供了重要的新见解， 肾病，并可能导致新的治疗目标，为未来的治疗糖尿病肾病。

Project Summary Diabetic nephropathy represents the primary cause of end stage renal disease (ESRD) in the US, underscoring the need for innovative therapies for preventing its progression. We are interested in understanding the cellular and molecular mechanisms that govern mitochondrial dysfunction in the diabetic milieu with the expectation that understanding of these processes will expose potential disease mechanisms and therapeutic targets in diabetic nephropathy. The present proposal is based on our recent published observation, indicating that mitochondrial fragmentation is essential for prompting mitochondrial dysfunction in podocytes in the diabetic milieu. A detailed understanding of mechanisms that govern mitochondrial fission in the kidney remains incomplete and therapeutic targets based on these mechanisms do not exist. Because dynamin-related protein-1 (Drp1) plays an integral part in regulating mitochondrial fission, we have focused on investigating the role of Drp1 in mitochondrial fragmentation and progression of diabetic nephropathy. We have been guided by our recent published observations that high glucose leads to mitochondrial fragmentation by promoting Drp1 recruitment to the mitochondria. Deletion of Drp1 in db/db diabetic mice prevented mitochondrial fission and improved histological and biochemical features of advanced diabetic kidney disease. Importantly, we found that high glucose milieu triggers mitochondrial fission by phosphorylating Drp1 at serine 600 residue. Here, we propose to establish the crosstalk between phosphorylation of Drp1 and electron transport complexes (ETC) as key mediators of mitochondrial ROS (mROS) and potential therapeutic targets in diabetic nephropathy (DN). In support of our hypothesis, we have recently generated a novel diabetic knockin mutant mouse harboring a single phosphorylation deficient (serine-to-alanine) point mutation at the corresponding S600 site in the endogenous Drp1 allele (Drp1S600A). We observed that diabetic Drp1S600A mice exhibited improved key biochemical and histological features of DN. To assess the role of Drp1S600 phosphorylation on mROS, We next crossed diabetic Drp1S600A mice with mice that express a redox- sensitive green fluorescent protein biosensor (roGFP) specifically in the mitochondrial matrix (mt-roGFP) and observed that Drp1S600A mutation in diabetic mice leads to reduced mROS in podocytes in live diabetic animals. These findings provide compelling initial evidence into the unexpected role of Drp1 in a signaling cascade that regulates mROS, and represents a therapeutic target that might be useful in preventing diabetic kidney disease. Given these results and additional preliminary data presented in this application, this project will address the hypothesis that Drp1 phosphorylation dynamically interact with mitochondrial ETC to enhance mROS though a signaling network that is regulated by cardiolipin activation. The results of this study will provide important new insights into the role of mitochondrial morphology in the development of diabetic nephropathy, and may lead to novel therapeutic targets for the future treatment of diabetic kidney disease.