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）：在人类和实验模型中，肾上管增生性肾小球肾炎的主要表现是增加了血小板衍生的生长因子受体-2（PDGFR）在伴有增殖和迁移时的血小板衍生生长因子受体-2（PDGFR）。众所周知，增加了PDGFR导致肾小球细胞激活的机制。我们的结果提供了首先证据表明，在膜细胞中，PDGFR激活的磷脂酰肌醇（PI）3激酶/Akt激酶可调节增殖和迁移。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诱导的大鼠抗血管造影的肾小球肾炎中。此外，非受体酪氨酸激酶C-SRC调节Ras-GTPase并与PI 3激酶相关，该激酶调节Akt激酶活性。在该建议中，使用抗THY-1诱导的肾小球肾炎的大鼠的培养和肾小球中的肾小球细胞，我们将测试以下假说，即通过RAS通过RAS信号继电器通过RAS通过RAS来调节膜细胞活化状态。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激酶效应子Rheb的表达（RAS富含大脑的RAS同源物）。我们假设依赖AKT的NF：B介导的Rheb的表达调节肾小球细胞的激活。在第一个具体目的中，我们计划研究c-SRC和RAS对PDGFR刺激的PI 3激酶/AKT信号的分子机制，并在伴有大鼠的肾小球可促肾上腺素肾炎中和大鼠的肾小球中的分子机制。同样将研究C-SRC和RAS之间的串扰，并在膜细胞激活中进行研究。在第二个特定目的中，将研究I：B激酶1和2和NF：B的作用在肾小球细胞增殖和迁移中。 I：B激酶同型的磷酸化和抗THY-1诱导的肾小球肾炎中底物的磷酸化。在特定目标3中，将检查PI 3激酶/AKT信号传导及其上游调节剂RAS和C-SRC在RHEB表达中的作用。 NF：B对RHEB表达的贡献将进行研究。为了解决这些具体目的，包括免疫组织化学/免疫荧光，免疫沉淀，免疫印迹，报告基因转染测定法，电泳迁移率转移测定法，染色质免疫沉淀ASASES，腺病毒介导的基因介导的基因转移突变的enzymes，si/shrynna表达蛋白质和条件表达蛋白质的基因转移。公共卫生相关性：肾小球肾炎是总体和退伍军人人口中第三大常见的末期肾脏疾病病例。许多形式的增殖性肾小球肾炎与肾小球细胞增殖有关。血小板衍生的生长因子介导的信号转导途径有助于该疾病的病理。信号通路的机制将在培养物中和大鼠肾小球肾小球肾炎的大鼠模型中研究。从提案中描述的实验中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。