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)： 人和实验性系膜增生性肾小球肾炎的一个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，并伴随着增殖和迁移。PDGFR增加导致系膜细胞活化的机制尚不清楚。我们的结果首次证明了在系膜细胞中，PDGFR激活的磷脂酰肌醇(PI)3激酶/Akt激酶调节细胞的增殖和迁移。此外，在抗Thy-1诱导的系膜增生性肾小球肾炎中，我们发现PDGFR酪氨酸激酶活性增加，导致PI3和Akt激酶活性增加。此外，非受体酪氨酸激酶c-Src调节Ras-GTP酶，并与调节Akt激酶活性的PI3激酶结合。在本研究中，我们将利用抗Thy-1诱导的肾炎大鼠肾小球系膜细胞和肾小球系膜细胞，验证c-Src信号通过RAS传递到PI3K/Akt调节系膜细胞激活状态的假说。在初步数据中，我们发现PDGF刺激的Akt激酶激活了I：B激酶1和2，导致了核因子：B的激活。重要的是，我们首次提供了PDGF增加Akt激酶效应器Rheb(脑中富含RAS同源基因)表达的证据。我们推测，Akt依赖的NF：B介导的Rheb的表达调节系膜细胞的激活。在第一个特定目标中，我们计划研究c-Src和RAS在PDGFR刺激的系膜细胞和系膜增生性肾炎大鼠肾小球PI3K/Akt信号转导中的作用机制。此外，c-Src和RAS之间的串扰将在系膜细胞激活中进行研究。在第二个特定目标中，将研究I：B激酶1和2以及核因子：B在系膜细胞增殖和迁移中的作用。在抗Thy-1诱导的肾小球肾炎中，将研究I：B激酶亚型的磷酸化及其底物的磷酸化。在具体目标3中，我们将研究PI3K/Akt信号及其上游调控因子Ras和c-Src在Rheb表达中的作用。将研究核因子：B对Rheb表达的贡献。为了解决这些特定的目的，将使用免疫组织化学/免疫荧光、免疫沉淀、免疫印迹、报告基因转染、电泳迁移率改变分析、染色质免疫沉淀、腺病毒介导的突变酶基因转移、si/shRNA表达和蛋白质的条件表达等技术。