Mechanism of Renal Cell Injury

肾细胞损伤机制

• 批准号: 8398924
• 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
• 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/8398924
• 关键词: 1-Phosphatidylinositol 3-Kinase; 2-tyrosine; 5' 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; public health relevance; receptor upregulation; research study; response; small hairpin RNA; urinary

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.

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，伴随着增殖和迁移。PDGFR增加导致系膜细胞活化的机制知之甚少。我们的研究结果提供了第一个证据，在系膜细胞PDGFR激活磷脂酰肌醇（PI）3激酶/Akt激酶调节增殖和迁移。此外，我们证明了PDGFR酪氨酸激酶活性的增加，导致PI 3激酶和Akt激酶活性在抗Thy-1诱导的系膜增生性肾小球肾炎大鼠。此外，非受体酪氨酸激酶c-Src调节Ras-GTdR，并与PI 3激酶结合，PI 3激酶调节Akt激酶活性。在这个建议中，使用系膜细胞培养和肾小球从大鼠抗Thy-1诱导的肾小球肾炎，我们将测试的假设，信号中继从c-Src PI 3激酶/Akt通过Ras调节系膜细胞活化的状态。在初步的数据中，我们发现PDGF刺激的Akt激酶激活I：B激酶1和2，导致NF：B激活。重要的是，我们提供了PDGF增加Akt激酶效应子Rheb（在脑中富集的Ras同源物）表达的第一个证据。我们推测Akt依赖的NF：B介导的Rheb表达调节系膜细胞活化。在第一个具体的目标，我们计划调查的分子机制的c-Src和Ras对PDGFR刺激PI 3激酶/Akt信号在系膜细胞和肾小球系膜增生性肾小球肾炎大鼠的作用。此外，c-Src和Ras之间的串扰将在系膜细胞活化中进行研究。在第二个具体目标中，将研究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I：B激酶同种型的磷酸化及其底物在抗Thy-1诱导的肾小球肾炎中的磷酸化。在具体的目标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。为了解决这些具体目标，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、突变酶的腺病毒介导的基因转移、si/shRNA表达和蛋白质的条件表达的技术。