Regulation of Podocyte Function By Angiotensin II

描述(申请人提供)：血管紧张素II(AngII)对足细胞功能的调节：肾小球上皮细胞(足细胞)是肾小球滤过屏障的重要组成部分。足细胞损伤与蛋白尿和进行性肾功能丧失有关。大量证据表明，肾小球损伤至少部分是由血管紧张素Ⅱ通过1型(AT1)和2型(AT2)受体亚型介导的。在肾小球内，血管紧张素Ⅱ受体表达于足细胞、内皮细胞和系膜细胞。足细胞同时表达AT1和AT2受体。这种受体系统的激活可能调节足细胞的功能，在疾病状态下，可能会促进足细胞的损伤。在许多细胞类型和动物模型中，Angii的促损伤作用是由AT1受体介导的，而AT2受体可能是负调控的。在这方面，我们的初步数据表明，ATL诱导的Gqα亚单位(Galphaq)的激活在介导Angii在体外和体内的损伤效应中起着关键作用。基于这一初步工作，我们假设：AT1受体在肾小球足细胞中具有促进损伤的作用，这种作用主要是通过Galphaq依赖的途径介导的，并受AT2受体的相反作用的调节。在拟议的研究中，我们将围绕三个具体目标来研究这一假说。首先，我们将利用药理学和分子生物学技术确定AT1和AT2依赖的信号通路，这些信号通路调控培养的足细胞的增殖、蛋白质合成和凋亡。基于我们的初步结果，在第二个特定目标#2中，我们将通过利用小鼠newitin启动子在足细胞中表达具有结构性活性的Galphaq(Galphaq&gt；L)来确定Galphaq偶联通路在体内促进足细胞损伤中的作用。最后，我们将确定AT1受体在促进肾小球损伤中的作用，尤其是足细胞，以及足细胞AT2受体在调节AT1和Galphaq依赖性肾损伤中的作用。在这些研究中，我们将使用转基因技术在肾小球足细胞上特异性地过度表达AT1或AT2受体。在确定转基因对未被操纵的小鼠的影响后，我们将使用我们的转基因动物来研究如下引起的肾脏损伤的严重程度：1.输注AngII；2.足细胞AT_2受体和Galphaq&gt；L的共表达；以及3.足细胞损伤。这些研究应该为足细胞Angii受体在疾病状态下促进肾小球损伤中的作用提供新的、重要的见解。了解调节肾小球疾病过程的生化机制可能为制定治疗决策提供理论基础，并可能导致开发治疗肾小球疾病的新策略。

DESCRIPTION (provided by applicant): Regulation of podocyte function by angiotensin II (ANGII): Glomerular epithelial cells (podocytes) are a critical component of the glomerular filtration barrier. Podocyte damage is associated with proteinuria and progressive loss of kidney function. A large body of evidence indicates that glomerular damage is mediated, at least in part, by ANGII through type 1 (AT1) and type 2 (AT2) receptor subtypes. Within the glomerulus, ANGII receptors are expressed on podocytes,endothelial cells and mesangial cells. Podocytes express both AT1 and AT2 receptors. Activation of this receptor system presumably regulates podocyte function and, in disease states, may promote podocyte injury. In many cell types as well as animal models, the injury promoting effects of ANGII are mediated by AT1 receptors and may be negatively modulated by AT2 receptors. In this regard, our preliminary data suggest that ATl-induced activation of the Gq alpha-subunit (Galphaq) plays a key role in mediating the damaging effects of ANGII both in vitro and in vivo. Based on this preliminary work, we hypothesized that: AT1 receptors have injury promoting effects in glomerular podocytes that are largely mediated through Galphaq-dependent pathways and are modulated by the opposing actions of AT2 receptors. In the proposed studies, we will investigate this hypothesis focusing on three specific aims. First, we will identify AT1- and AT2-dependent signaling pathways that regulate proliferation, protein synthesis, and apoptosis in cultured podocytes using pharmacological and molecular biological techniques. Based on our preliminary results, in second specific aim #2, we will define the role of Galphaq-coupled pathways in promoting podocyte injury in vivo by expressing a constitutively active form of Galphaq (GalphaqQ>L)in podocytes using the mouse nephrin promoter. Lastly, we will determine the role AT1 receptors in promoting glomerular injury specifically in podocytes as well as the role of podocyte AT2 receptors in modulating both AT1- and Galphaq-dependentrenal damage. In these studies, we will use transgenic techniques to overexpress either AT1 or AT2 receptors specifically on glomerular podocytes. After determining the effect of the transgenes in unmanipulated mice, we will use our transgenic animals to investigate the severity of renal damage induced by: 1. infusion of ANGII, 2. co-expression of AT2 receptors and GalphaqQ>L in podocytes, and 3. podocyte injury using a model developed in our laboratory. These studies should provide new, important insights into the role of podocyte ANGII receptors in promoting glomerular injury in disease states. Understanding the biochemical mechanisms that regulate glomerular disease processes may provide a rationale strategy for making therapeutic decisions and could lead to the development of novel strategies for treating glomerular diseases.