Endothelin Signaling and Actions in Renal Mesangium

描述（由申请人提供）：在肾系膜中，内皮素-1（ET-1）过度收缩、增殖和细胞外基质积聚，导致肾小球硬化和肾衰竭。目前对ET-1在肾系膜中作用的分子机制的研究还不够深入。在目前的资助申请中，我们的目的是证明，新的ET-1介导的信号通路，我们发现在培养的肾小球系膜细胞（GMC），发挥主要作用，在肾小球疾病在体内时，ET-1的生产增加，肾系膜功能障碍。为了实现这些目标，我们已经产生了独特的大鼠品系，其中我们使用工程化的锌指核酸酶（ZFN）结合创新的体内敲入策略精确地修饰了大鼠基因组。直到最近，大鼠基因组的精确修饰是不可能的，但是在近交系大鼠品系中使用ZFN产生靶向基因变化已经成为该领域的重大突破之一，这大大增加了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，我们发现了ET-1刺激GMC形成包括衔接蛋白p66 Shc在内的多单位信号复合物的新的信号通路。我们推测ET-1信号通过衔接蛋白p66 Shc在肾系膜在体内是有助于肾脏病理与肾系膜细胞功能异常。在具体的目标1中，我们将测试是否ET-1介导的信号通过p66 Shc有助于肾损伤肾小球疾病与增强ET-1的生产和异常肾小球功能。我们将在缺乏p66 Shc蛋白或表达引入突变的内源性p66 Shc的大鼠中诱导抗Thy-1.1肾炎和高血压诱导的肾病。将评估肾损伤的程度。在具体目标2中，我们将使用来自野生型和遗传修饰的大鼠品系的原代GMC来揭示肾系膜中p66 Shc信号传导的分子机制。我们将检验p66 Shc通过转录因子FOXO 3a失活促进GMC增殖和通过调节钙内流限制GMC收缩性的假设。这些研究是重要的，因为在大多数高血压肾病和肾小球硬化患者中检测到GMC功能异常。阐明ET-1诱导的肾脏病变的机制将导致对增殖相关和氧化应激相关肾小球疾病的机制的理解。

DESCRIPTION (provided by applicant): 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 studie 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.