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。我们感兴趣的是 了解糖尿病患者线粒体功能障碍的细胞和分子机制 希望对这些过程的了解将揭示潜在的疾病机制 糖尿病肾病的治疗靶点。目前的建议是基于我们最近出版的 观察表明，线粒体片段化是促使线粒体功能障碍的关键 糖尿病环境中的足细胞。对线粒体分裂调控机制的详细了解 肾脏仍然不完整，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(Dynamin-Related Protein-1，drp1)在调控线粒体分裂中起着不可或缺的作用，我们主要关注的是 探讨Drp1在线粒体断裂和糖尿病肾病进展中的作用。我们有 受到我们最近发表的观察结果的指导，即高糖通过以下方式导致线粒体碎裂 促进线粒体的Drp1募集。阻止db/db糖尿病小鼠中Drp1的缺失 晚期糖尿病肾病的线粒体分裂和改善的组织学和生化特征。 重要的是，我们发现高糖环境通过使丝氨酸上的drp1磷酸化来触发线粒体的分裂。 600个残留物。在这里，我们建议在drp1的磷酸化和电子之间建立串扰。 转运复合体(ETC)作为线粒体ROS(MRO)的关键介体和潜在的治疗靶点 在糖尿病肾病(DN)中。为了支持我们的假设，我们最近产生了一种新的糖尿病 Knockin突变小鼠携带单个磷酸化缺陷(丝氨酸到丙氨酸)突变 内源性Drp1等位基因(Drp1S600A)中对应的S600位点。我们观察到糖尿病患者的Drp1S600A 小鼠表现出改善了糖尿病肾病的关键生化和组织学特征。评估Drp1S600的作用 在MRO上的磷酸化，我们接下来将糖尿病Drp1S600A小鼠与表达氧化还原的小鼠进行杂交- 线粒体基质特异性绿色荧光蛋白敏感生物传感器(mt-roGFP)和 观察到糖尿病小鼠中Drp1S600A突变导致活体糖尿病小鼠足细胞MRO减少 动物。这些发现为Drp1在信号转导中出人意料的作用提供了令人信服的初步证据 调节MRO的级联反应，代表了可能对预防糖尿病有用的治疗靶点 肾脏疾病。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DRp1磷酸化与线粒体等动态相互作用的假设，以增强 MRO通过一个受心磷脂激活调节的信号网络。这项研究的结果将 为线粒体形态在糖尿病发展中的作用提供了重要的新见解 并可能为未来糖尿病肾病的治疗带来新的治疗靶点。

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.