Mass Spectrometry Analysis of Notch4 Networks in Diabetes-Related Tubule Injury

描述(由申请人提供)： 候选人的总体职业目标是建立和维持一个独立的、以蛋白质组和分子生物学为基础的计划，专注于研究与糖尿病肾病(DN)有关的事件。糖尿病肾病是终末期肾病的主要病因。目前还没有完全防止进展为终末期肾病的治疗方法。这强调了发现新的调节事件作为诊断、预防和治疗糖尿病肾病的潜在治疗靶点与健康相关的重要性。肾小管间质纤维化(TF)是由转化生长因子-β(TGF-β)介导的进展性糖尿病肾病的一个显著特征。Notch4是一种引人入胜的膜结合转录调节因子，最近被认为与转化生长因子信号转导和肾脏疾病有关。我们的初步发现表明，与对照组相比，Notch4的激活抑制了培养的肾小管上皮细胞中的转化生长因子-β信号，并显示了Notch4在糖尿病小鼠肾小管上皮细胞中的表达减少。这提示了一种假说，即下调Notch4活性促进了转化生长因子-β介导的糖尿病肾病效应。该项目将培育Notch4基因缺陷的糖尿病小鼠。肾脏病理生理学实验将描述Notch4在糖尿病介导的转铁蛋白和转化生长因子信号转导中的体内作用。预计这些动物也将为转铁蛋白的研究提供一个更好的模型，因为目前的糖尿病动物模型都不能准确地模拟糖尿病人中观察到的转铁蛋白。越来越多的证据表明，转化生长因子介导的上皮细胞向间充质转化(EMT)在糖尿病相关的转移性纤维化中起着关键作用。我们最近建立了转化生长因子介导的人肾小管上皮细胞系EMT模型。我们还将开发葡萄糖和晚期糖基化终末产物(AGE)介导的EMT的香港模型。我们将使用这些细胞模型，结合活跃的Notch4转染和RNAi介导的Notch4敲除，来剖析Notch4在EMT中不同信号和转录活性成分中的作用。最后，我们将使用最先进的蛋白质组学技术对Notch4缺陷的糖尿病小鼠进行研究，以确定Notch4介导的蛋白质在糖尿病肾病中的表达和细胞器的定位。除了这些研究提供的分子洞察力外，在此次职业发展奖期间获得的动物模型和病理生理学培训将为候选人提供宝贵的资源，以扩大他的糖尿病相关项目并获得独立水平的资助。

DESCRIPTION (provided by applicant): The Candidate's overall career goal is to establish and maintain an independent proteomic and molecular biology-based program focused on investigation of events that mediate diabetic nephropathy (DN). DN is the leading cause of end-stage renal disease (ESRD). There are no current treatments that completely prevent progression to ESRD. This emphasizes the health-related importance for discovery of novel regulatory events as potential therapeutic targets for the diagnosis, prevention and treatment of DN. Tubulointerstitial fibrosis (TF) is a prominent feature of progressive DN that is mediated by transforming growth factor-beta (TGF-beta). Notch4 is a fascinating membrane-bound transcription regulator that was recently implicated in TGF signaling and renal disease. Our preliminary findings indicate that Notch4 activation inhibits TGF-beta signaling in cultured renal tubular cells and show decreased expression of Notch4 in tubular cells from diabetic mice as compared with controls. This suggests a hypothesis that down-regulation of Notch4 activity promotes the TGF-beta-mediated effects in DN. This project will develop Notch4-deficient diabetic mice. Renal pathophysiology experiments will be performed to delineate the in vivo role of Notch4 in diabetes-mediated TF and TGF signaling. It is anticipated that these animals will also provide a better model for the study of TF, as none of the current diabetic animal models exactly mimic the TF observed in diabetic humans. Accumulating evidence suggests a key role of TGF-mediated epithelial to mesenchymal transition (EMT) in diabetes-associated TF. We have recently developed models for TGF-mediated EMT in human kidney (HK) tubule cell lines. We will also develop HK models for glucose and advanced glycation end product (AGE)-mediated EMT. We will use these cellular models in combination with active Notch4 transfection and RNAi-mediated Notch4 knockdown to dissect the role of Notch4 in different components of signaling and transcription activity in EMT. Lastly, we will use state-of-the-art proteomic techniques with the Notch4-deficient diabetic mice to define Notch4-mediated protein expression and organelle localization in DN. In addition to molecular insight provided by these studies, the animal model and pathophysiology training obtained during this Career Development Award will provide valuable resources for the Candidate to expand his diabetes-related program and obtain independent-level funding.