Mechanism of Renal Cell Injury

肾细胞损伤机制

• 批准号: 9269454
• 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 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9269454
• 关键词: 20 year old; 3' Untranslated Regions; Accounting; Address; Adverse effects; Affect; Albuminuria; Biological Assay; Catalytic Domain; Cells; Chronic Disease; Chronic Kidney Failure; Complex; Complications of Diabetes Mellitus; Data; Deposition; Development; Diabetes Mellitus; Diabetic Nephropathy; Diabetic mouse; Disease; Down-Regulation; EZH2 gene; Elements; End stage renal failure; Enhancers; Epigenetic Process; Epithelial Cells; FRAP1 gene; Family; Fibronectins; Fibrosis; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Glucose; Histone H3; Homeostasis; Homologous Gene; Hyperglycemia; Hypertrophy; Immunoblotting; Immunohistochemistry; Immunoprecipitation; Injury; Insulin-Dependent Diabetes Mellitus; Kidney; Kidney Diseases; Lysine; Mediating; Messenger RNA; MicroRNAs; Modeling; Molecular Target; Non-Insulin-Dependent Diabetes Mellitus; Pathologic; Patients; Pattern; Pharmacology; Phosphotransferases; Plasmids; Plasminogen Activator Inhibitor 1; Polycomb; Population; Process; Proteins; Quantitative Reverse Transcriptase PCR; Reagent; Renal Tissue; Renal function; Reporter; Reporting; Repression; Rodent; Role; Signal Transduction; Signaling Protein; Sirolimus; Small Interfering RNA; Specificity; Techniques; Testing; Therapeutic; Transducin; Transfection; Transforming Growth Factors; Tubular formation; Ubiquitination; Veterans; age group; beta-Transducin Repeat-Containing Proteins; cell injury; chromatin immunoprecipitation; db/db mouse; demographics; diabetic; effective therapy; falls; inhibitor/antagonist; kidney cell; mTOR inhibition; morphometry; mouse model; novel; protein degradation; protein expression; public health relevance; regenerative; response; sensor; therapeutic target; type I diabetic; ubiquitin-protein ligase

 DESCRIPTION (provided by applicant): Hyperglycemia and increased transforming growth factor-β (TGFβ) expression activate mTOR (mechanistic target of rapamycin), which acts as a key node to promote glomerular and tubular hypertrophy and matrix protein fibronectin expression. We have reported that high glucose and TGFβ activate both mTOR complexes (C1 and C2) in mesangial and proximal tubular epithelial (PTE) cells and in kidneys of type 2 and type 1 diabetic mice. Blocking mTOR activity with rapamycin, we showed reduction in albuminuria, renal hypertrophy and matrix expansion in these models of diabetes. Complete inhibition of mTOR by rapamycin can be detrimental as the activity of this kinase is required for maintaining renal cell homeostasis. Also, rapamycin changes gene expression profile, which causes adverse effects. Therefore, alternative therapeutic approach is necessary to block mTOR kinase. Our data demonstrate that the expression of deptor, a component of both mTOR complexes, and inhibitor of their kinase activities, is reduced in kidneys of diabetic rodents and in mesangial and PTE cells treated with high glucose or TGFβ. Exploiting how deptor is suppressed to increase mTOR activity can identify novel molecular targets to block diabetic renal complications. In our preliminary data, we find reduction in deptor levels concomitant with increased expression of EZH2 (enhancer of zeste homolg 2), the catalytic subunit of the polycomb repressor complex 2, which trimethylates histone H3 at lysine- 27 to block transcription of a specific gene. Moreover, we show enhanced expression of the E3 ubiquitin ligase βTrCP (transducin repeat containing protein) by high glucose and TGFβ in renal cells. βTrCP targets deptor for degradation. Furthermore, we demonstrate high glucose and TGFβ increase the expression of a family of microRNA, miR-181 (a,b,c,d), and miR-221. These microRNAs are also increased in kidneys of type 1 and type 2 diabetic mice and target deptor for downregulation. In this proposal, using cultured mesangial and PTE cells and renal tissues from diabetic OVE26 and db/db mice, we will test the hypothesis that inappropriate deptor downregulation contributes to renal hypertrophy and matrix expansion in diabetic kidney disease. Probing the specificities of underlying mechanisms, we will define the regulatory modules of deptor suppression. In the first specific aim, we will investigate EZH2 as a candidate for transcriptional suppression of deptor, hypertrophy and, fibronectin and PAI-1 (plasminogen activator inhibitor-1) expression. In the second aim, the role of βTrCP in deptor protein degradation/mTORC1/C2 activation and its cross talk with TGFβ signaling in forcing mesangial and PTE cell hypertrophy and, fibronectin and PAI-1 expression will be examined. In specific aim 3, we will investigate the contribution of miR-181 family and miR-221 to hypertrophy and, fibronectin and PAI-1 expression in response to high glucose and TGFβ in mesangial and PTE cells and in diabetic mice kidneys. To address these aims, techniques including immunoblotting, immunoprecipitation, qRT-PCR, morphometry, immunohistochemistry, reporter transfection assays, chromatin immunoprecipitation assays and siRNA-mediated downregulation of proteins will be used.

 描述(由申请人提供)： 高血糖和转化生长因子-β(转化生长因子-β)的表达增加激活了雷帕霉素的机械靶点mTOR，它是促进肾小球和肾小管肥大及基质蛋白纤维连接蛋白表达的关键节点。我们已经报道，高糖和转化生长因子β激活系膜和近端小管上皮细胞以及2型和1型糖尿病小鼠肾脏中的mTOR复合体(C1和C2)。用雷帕霉素阻断mTOR活性，我们发现在这些糖尿病模型中，蛋白尿减少，肾脏肥大和基质扩张。雷帕霉素完全抑制mTOR可能是有害的，因为这种激酶的活性是维持肾细胞动态平衡所必需的。此外，雷帕霉素会改变基因表达谱，从而导致不良反应。因此，有必要采用替代治疗方法来阻断mTOR激酶。我们的数据表明，在糖尿病啮齿动物的肾脏以及在高糖或转化生长因子β处理的肾小球系膜细胞和PTE细胞中，两种mTOR复合体的组成成分Deptor及其激酶活性抑制物Deptor的表达减少。研究Deptor是如何被抑制以增加mTOR活性的，可以发现阻断糖尿病肾脏并发症的新的分子靶点。在我们的初步数据中，我们 Deptor水平的降低伴随着EZH2(Zust Homolg 2的增强子)的表达增加，EZH2是多梳抑制物复合体2的催化亚单位，它使组蛋白H3在赖氨酸-27处三甲基化，以阻止特定基因的转录。此外，我们还发现高糖和ββ促进了E3泛素连接酶TCPTrCP(转导重复序列蛋白)在肾细胞中的表达。βTrCP以降解依赖物为目标。此外，我们还证明了高糖和转化生长因子β增加了一个microRNA家族miR-181(a，b，c，d)和miR-221的表达。这些microRNAs也在1型和2型糖尿病小鼠的肾脏中增加，并作为下调的靶标。在这项研究中，利用培养的糖尿病OVE26和db/db小鼠的肾小球系膜细胞和PTE细胞和肾组织，我们将检验这一假说，即在糖尿病肾病中，不适当的Deptor下调导致肾脏肥大和基质扩张。为了探索潜在机制的特殊性，我们将定义Detor抑制的调节模块。在第一个特定目标中，我们将研究EZH2作为一个候选转录抑制因子，抑制Deptor、肥大以及纤维连接蛋白和PAI-1(纤溶酶原激活物抑制物-1)的表达。第二个目的是研究转化生长因子TrCP在蛋白降解/β/C2激活中的作用及其与转化生长因子β信号通路在促进系膜细胞和PTE细胞肥大以及纤维连接蛋白和纤连蛋白-1表达中的相互作用。在特定的目标3中，我们将研究miR-181家族和miR-221在高糖和转化生长因子β诱导的系膜细胞、PTE细胞和糖尿病小鼠肾脏肥大、纤维连接蛋白和PAI-1表达中的作用。为了达到这些目的，将使用免疫印迹、免疫沉淀、qRT-PCR、形态计量学、免疫组织化学、报告基因转染法、染色质免疫沉淀法和siRNA介导的蛋白质下调等技术。