Mechanism of Renal Cell Injury

肾细胞损伤机制

基本信息

批准号：
8043246
负责人：
GOUTAM GHOSH CHOUDHURY
金额：
--
依托单位：
SOUTH TEXAS VETERANS HEALTH CARE SYSTEM
依托单位国家：
美国
项目类别：
财政年份：
2011
资助国家：
美国
起止时间：
2011-07-01 至 2015-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8043246
关键词：
1-Phosphatidylinositol 3-Kinase 2-tyrosine 5&apos Flanking Region Address Adenoviruses Albumins Antibodies Biological Assay Cell Proliferation Cell physiology Cells Creatinine Creatinine clearance measurement DNA DNA biosynthesis Data Disease Disease Progression Dominant-Negative Mutation Drug Design Electrophoretic Mobility Shift Assay Elements End stage renal failure Enzymes Excretory function Experimental Models Firefly Luciferases Gene Transfer Genes Genetic Transcription Glomerulonephritis Growth Factor Guanosine Triphosphate Phosphohydrolases Hour Human IkappaB kinase Immunoblot Analysis Immunoblotting Immunofluorescence Immunologic Immunohistochemistry Immunoprecipitation Isoenzymes Kidney Mediating Messenger RNA Modality Modeling Molecular Molecular Mechanisms of Action Monomeric GTP-Binding Proteins Pathogenesis Pathology Phosphorylation Phosphotransferases Plasmids Platelet-Derived Growth Factor Platelet-Derived Growth Factor Receptor Platelet-Derived Growth Factor beta Receptor Play Population Process Proliferating Cell Nuclear Antigen Proliferative Glomerulonephritis Protein Tyrosine Kinase Proteins Ras homolog enriched in brain Rattus Regulation Renal function Reporter Role SRC gene SU 6656 Sequence Analysis Signal Pathway Signal Transduction Signal Transduction Pathway Staining method Stains Structure of glomerular mesangium Techniques Testing Therapeutic Therapeutic Effect Transcription Initiation Site Transfection Veterans anti-Thy-1 cell injury cell motility chromatin immunoprecipitation cytokine design in vivo inhibitor/antagonist kidney cell mesangial cell migration mutant p65 promoter protein expression protein-tyrosine kinase c-src receptor upregulation research study response small hairpin RNA urinary

项目摘要

DESCRIPTION (provided by applicant): A major manifestation of mesangioproliferative glomerulonephritis in human and experimental model is increased expression of platelet-derived growth factor receptor-2 (PDGFR) in mesangial cells with concomitant proliferation and migration. The mechanism by which increased PDGFR results in the activation of mesangial cells is poorly understood. Our results provide first evidence that in mesangial cells PDGFR-activated phosphatidylinositol (PI) 3 kinase/Akt kinase regulates proliferation and migration. Furthermore, we demonstrate an increase in PDGFR tyrosine kinase activity, resulting in PI 3 kinase and Akt kinase activities in anti-Thy-1-induced mesangioproliferative glomerulonephritis in the rat. Moreover, the non-receptor tyrosine kinase c-Src regulates Ras-GTPase and associates with PI 3 kinase, which regulates Akt kinase activity. In this proposal, using mesangial cells in culture and glomeruli from rats with anti-Thy-1-induced glomerulonephritis, we will test the hypothesis that signal relay from c-Src to PI 3 kinase/Akt through Ras regulates the state of mesangial cell activation. In the preliminary data, we show that PDGF-stimulated Akt kinase activates I:B kinases 1 and 2, resulting in NF:B activation. Importantly, we provide the first evidence that PDGF increases expression of Akt kinase effector Rheb (Ras homolog enriched in brain). We hypothesize that Akt-dependent NF:B-mediated expression of Rheb regulates mesangial cell activation. In the first specific aim, we plan to investigate the molecular mechanisms of action of c-Src and Ras on PDGFR-stimulated PI 3 kinase/Akt signaling in mesangial cells and in glomeruli of rats with mesangioproliferative glomerulonephritis. Also a cross-talk between c-Src and Ras will be studied in mesangial cell activation. In the second specific aim, the roles of I:B kinases 1 and 2 and NF:B in mesangial cell proliferation and migration will be studied. Phosphorylation of I:B kinase isotypes and phosphorylation of their substrates in anti-Thy-1-induced glomerulonephritis will be investigated. In the specific aim 3, role of PI 3 kinase/Akt signaling and its upstream regulators Ras and c-Src in expression of Rheb will be examined. Contribution of NF:B to the Rheb expression will be investigated. To address these specific aims, techniques including immunohistochemistry/immunofluorescence, immunoprecipitation, immunoblotting, reporter transfection assays, electrophoretic mobility shift assays, chromatin immunoprecipitation asays, adenovirus-mediated gene transfer of mutant enzymes, si/shRNA expression and conditional expression of proteins will be used. PUBLIC HEALTH RELEVANCE: Glomerulonephritis is the third most common case of end stage renal disease in the population at large and in veterans. Many forms of proliferative glomerulonephritis are associated with mesangial cell proliferation. Platelet-derived growth factor-mediated signal transduction pathways contribute to the pathology of the disease. The mechanism of the signaling pathways will be studied using mesangial cells in culture and in a rat model of mesangioproliferative glomerulonephritis. The results obtained from the experiments described in the proposal will help designing therapeutic modalities targeting many common forms of proliferative glomerulonephritis.

描述（由申请人提供）：人和实验模型中系膜增生性肾小球肾炎的主要表现是系膜细胞中血小板衍生生长因子受体2（PDGFR）的表达增加，并伴有增殖和迁移。 PDGFR 增加导致系膜细胞激活的机制尚不清楚。我们的结果提供了第一个证据，证明在系膜细胞中，PDGFR 激活的磷脂酰肌醇 (PI) 3 激酶/Akt 激酶调节增殖和迁移。此外，我们证明PDGFR酪氨酸激酶活性增加，导致大鼠抗Thy-1诱导的系膜增生性肾小球肾炎中PI 3激酶和Akt激酶活性增加。此外，非受体酪氨酸激酶 c-Src 调节 Ras-GTPase 并与调节 Akt 激酶活性的 PI 3 激酶相关。在本提案中，使用培养物中的系膜细胞和抗 Thy-1 诱导的肾小球肾炎大鼠的肾小球，我们将测试以下假设：信号从 c-Src 通过 Ras 传递到 PI 3 激酶/Akt 调节系膜细胞激活的状态。在初步数据中，我们表明 PDGF 刺激的 Akt 激酶激活 I:B 激酶 1 和 2，从而导致 NF:B 激活。重要的是，我们提供了第一个证据表明 PDGF 增加了 Akt 激酶效应器 Rheb（大脑中富集的 Ras 同源物）的表达。我们假设 Akt 依赖性 NF:B 介导的 Rheb 表达调节系膜细胞活化。在第一个具体目标中，我们计划研究 c-Src 和 Ras 对系膜增生性肾炎大鼠肾小球系膜细胞和肾小球中 PDGFR 刺激的 PI 3 激酶/Akt 信号传导的分子机制。还将研究系膜细胞激活中 c-Src 和 Ras 之间的串扰。第二个具体目标是研究 I:B 激酶 1 和 2 以及 NF:B 在系膜细胞增殖和迁移中的作用。将研究抗 Thy-1 诱导的肾小球肾炎中 I:B 激酶同种型的磷酸化及其底物的磷酸化。在具体目标 3 中，将检查 PI 3 激酶/Akt 信号传导及其上游调节因子 Ras 和 c-Src 在 Rheb 表达中的作用。将研究 NF:B 对 Rheb 表达的贡献。为了实现这些特定目标，将使用包括免疫组织化学/免疫荧光、免疫沉淀、免疫印迹、报告基因转染测定、电泳迁移率变动测定、染色质免疫沉淀测定、腺病毒介导的突变酶基因转移、si/shRNA表达和蛋白质条件表达等技术。公共卫生相关性：肾小球肾炎是普通人群和退伍军人中第三常见的终末期肾病病例。许多形式的增殖性肾小球肾炎与系膜细胞增殖有关。血小板衍生生长因子介导的信号转导途径有助于该疾病的病理学。将使用培养的系膜细胞和系膜增生性肾小球肾炎的大鼠模型来研究信号传导途径的机制。该提案中描述的实验获得的结果将有助于设计针对许多常见形式的增殖性肾小球肾炎的治疗方式。