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护一个独立的蛋白质组学和分子生物学为基础的计划，重点是调查事件，介导糖尿病肾病（DN）。DN是终末期肾病（ESRD）的主要原因。目前还没有完全预防进展为ESRD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DN的潜在治疗靶点的健康相关重要性。肾小管间质纤维化（TF）是由转化生长因子-β（TGF-β）介导的进行性DN的显著特征。Notch 4是一种迷人的膜结合转录调节因子，最近被认为与TGF信号传导和肾脏疾病有关。我们的初步研究结果表明，Notch 4激活抑制培养的肾小管细胞中的TGF-β信号传导，并显示与对照组相比，糖尿病小鼠肾小管细胞中Notch 4的表达降低。这提示了一种假说，即Notch 4活性的下调促进了TGF-β介导的DN效应。该项目将开发Notch 4缺陷型糖尿病小鼠。将进行肾病理生理学实验以描绘Notch 4在糖尿病介导的TF和TGF信号传导中的体内作用。预计这些动物也将为TF的研究提供更好的模型，因为目前的糖尿病动物模型都没有精确模拟糖尿病患者中观察到的TF。越来越多的证据表明TGF介导的上皮间质转化（EMT）在糖尿病相关TF中起关键作用。我们最近开发了TGF-介导的人肾小管细胞系EMT模型。我们还将开发葡萄糖和晚期糖基化终产物（AGE）介导的EMT的HK模型。我们将使用这些细胞模型结合主动Notch 4转染和RNAi介导的Notch 4敲低来剖析Notch 4在EMT中不同的信号传导和转录活性组分中的作用。最后，我们将使用最先进的蛋白质组学技术与Notch 4缺陷型糖尿病小鼠，以确定Notch 4介导的蛋白质表达和细胞器定位在DN。除了这些研究提供的分子见解外，在此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，以扩大其糖尿病相关计划并获得独立的资金。

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.