Endothelin Signaling and Actions in Renal Mesangium

肾系膜中的内皮素信号传导和作用

• 批准号: 8735939
• 负责人: ANDREY SOROKIN
• 金额: $ 33.28万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-20 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8735939
• 关键词: Adaptor Signaling Protein; Adult; Apoptotic; Applications Grants; Area; Biomedical Research; Calcium; Capillary Endothelial Cell; Cell Cycle Regulation; Cell Proliferation; Cell physiology; Cells; Complex; Coupled; DBL Oncoprotein; Diabetic Nephropathy; Disease; Endothelial Cells; Endothelin; Endothelin Receptor; Endothelin-1; Engineering; Epithelial Cells; Excision; Extracellular Matrix; Extracellular Matrix Proteins; FOXO3A gene; Family; Fibrosis; Filtration; GTP-Binding Proteins; Gene Targeting; Generations; Genes; Glomerular Capillary; Glomerular Mesangial Cell; Goals; Human; Hypertension; Inbred Strains Rats; Injury; Kidney; Kidney Diseases; Kidney Failure; Knock-in Mouse; Knock-out; Longevity; Mediating; Modification; Molecular; Mutate; Mutation; Nephritis; Oxidative Stress; Parietal; Pathogenesis; Pathology; Patients; Peptides; Phagocytosis; Phosphorylation Site; Play; Production; Protein Isoforms; Rat Strains; Rattus; Regulation; Renal function; Renal glomerular disease; Research Personnel; Role; Signal Pathway; Signal Transduction; Src homology 2 domain-containing, transforming protein 1; Structure of glomerular mesangium; Testing; Urine; Zinc Fingers; age related; blood filtration; cell type; glomerular function; glomerulosclerosis; in vivo; innovation; member; mesangial cell; mutant; novel; nuclease; podocyte; protein protein interaction; public health relevance; rat genome; transcription factor

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.

描述(申请人提供)：在肾小球系膜，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功能衰竭。ET-1在肾小球系膜中作用的分子机制研究尚不充分。在目前的赠款申请中，我们的目标是证明我们在培养的肾小球系膜细胞(GMC)中发现的新的ET-1介导的信号通路在体内当ET-1产生增加和肾系膜功能障碍时在肾小球疾病中起主要作用。为了实现这些目标，我们培育了独特的大鼠品系，在这些品系中，我们使用工程锌指核酸酶(ZFN)结合创新的体内敲入策略精确地修改了大鼠基因组。直到最近，对大鼠基因组的精确修改是不可能的，但利用近交系大鼠品系中的ZFN产生靶向基因变化已经成为该领域的重大突破之一，极大地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刺激的GMC中新的信号通路，它涉及形成包括适配蛋白P66Shc在内的多单位信号复合体。我们假设ET-1信号通过肾小球系膜连接蛋白p66Shc在体内参与了与肾小球系膜细胞功能异常相关的肾脏病理改变。在特定的目标1中，我们将测试ET-1通过p66Shc介导的信号是否有助于与ET-1产生增加和肾小球功能异常相关的肾小球疾病的肾脏损伤。我们将在缺乏p66Shc蛋白或表达内源性p66Shc并引入突变的大鼠中诱导抗Thy-1.1肾炎和高血压肾病。将对肾脏损伤的程度进行评估。在具体目标2中，我们将使用来自野生型和转基因大鼠品系的原代GMC来揭示p66Shc信号在肾系膜中的分子机制。我们将验证p66 Shc通过失活转录因子FOXO3a促进GMC增殖，并通过调节钙内流限制GMC收缩的假设。这些研究很重要，因为在大多数高血压肾病和肾小球硬化患者中都检测到了GMC功能异常。阐明ET-1诱导的肾脏病理机制将有助于理解与增殖相关和氧化应激相关的肾小球疾病的机制。