Endothelin Signaling and Actions in Renal Mesangium

在肾系膜中，内皮素-1（ET-1）通过过度收缩、增殖和细胞外基质（ECM） 累积导致肾小球硬化和肾衰竭。内皮素-1作用的分子机制 肾系膜的研究还不够。在目前的拨款申请中，我们的目标是证明新的ET-1 我们在培养的肾小球系膜细胞（GMC）中发现， 当ET-1产生增加和肾系膜功能障碍时，在体内肾小球疾病中的作用。 为了实现这些目标，我们已经产生了独特的大鼠品系，其中我们精确地修改了大鼠基因组， 工程化的锌指核酸酶（ZFN）与创新的体内敲入策略相结合。直到 最近，大鼠基因组的精确修饰是不可能的，但靶基因改变的产生 在近交系大鼠品系中使用ZFN已经成为该领域的主要突破之一， 研究人员利用大鼠进行生物医学研究的机会。在我们的初步研究中， 在GMC中发现了由ET-1刺激的新的信号通路，其涉及多单位的形成， 包括衔接蛋白p66 Shc的信号传导复合物。我们推测ET-1信号通过衔接蛋白 体内肾系膜中的p66 Shc有助于与肾功能异常相关的肾脏病理学。 肾系膜细胞在具体目标1中，我们将测试ET-1介导的经由p66 Shc的信号传导是否有助于 肾小球疾病中的肾损伤与ET-1产生增加和肾小球异常 功能我们将在大鼠中诱导抗Thy-1.1肾炎和高血压诱导的肾病， p66 Shc蛋白或表达引入突变的内源性p66 Shc。肾损伤的程度 评估。在具体目标2中，我们将使用来源于野生型和遗传修饰的大鼠品系的原代GMC 揭示p66 Shc信号在肾系膜中的分子机制。我们将检验这个假设， p66 Shc通过转录因子FOXO 3a失活促进GMC增殖并限制GMC 通过调节钙内流的收缩性。这些研究很重要，因为异常的GMC功能 在大多数患有高血压引起的肾病和肾小球硬化症的患者中检测到。的 阐明ET-1诱导的肾脏病理学机制将导致对这些机制的理解 潜在的增殖相关和氧化应激相关的肾小球疾病。

In renal mesangium endothelin-1 (ET-1) exerts excessive contraction, proliferation and extracellular matrix accumulation leading to glomerulosclerosis and kidney failure. The molecular mechanisms of ET-1 actions in renal mesangium are insufficiently studied. In the current grant application we aim to prove that novel ET-1 mediated signaling pathways, discovered by us in cultured glomerular mesangial cells (GMC), play principal role in glomerular diseases in vivo when ET-1 production is increased and renal mesangium is dysfunctional. To achieve these goals we have generated unique rat strains in which we precisely modified rat genome using engineered Zinc Finger Nucleases (ZFNs) in combination with innovative in vivo knock-in strategy. Until recently the precise modification of rat genome was not possible, but the generation of targeted gene changes using ZFNs in inbred rat strains has become one of the major breakthroughs in the field dramatically increasing opportunities of investigators in utilizing rats for biomedical research. In our preliminary studies we have discovered novel signaling pathway stimulated by ET-1 in GMC which involves the formation of multiunit signaling complex including adaptor protein p66 Shc. We hypothesize that ET-1 signaling via adaptor protein p66 Shc in renal mesangium in vivo is contributing to kidney pathologies associated with abnormal function of renal mesangial cells. In specific aim 1 we will test whether ET-1-mediated signaling via p66 Shc contributes to renal injury in glomerular diseases associated with enhanced ET-1 production and abnormal glomerular function. We will induce anti-Thy-1.1 nephritis and hypertension-induced nephropathy in rats which either lack p66 Shc protein or express endogenous p66 Shc with introduced mutations. The extent of renal injury will be assessed. In specific aim 2 we will use primary GMC derived from wild type and genetically modified rat strains to uncover the molecular mechanism of p66 Shc signaling in renal mesangium. We will test the hypothesis that p66 Shc promotes GMC proliferation via inactivation of transcription factor FOXO3a and restricts GMC contractility through regulation of calcium influx. These studies are important because abnormal GMC function is detected in the majority of patients with hypertension induced nephropathy and glomerulosclerosis. The elucidation of mechanisms of ET-1-induced renal pathologies will result in understanding of the mechanisms underlying proliferation-associated and oxidative stress related renal glomerular diseases.