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）： 高血糖和增加的转化生长因子-β（TGFβ）表达激活MTOR（雷帕霉素的机理靶标），这是促进肾小球和肾小管肥大和基质蛋白纤维蛋白纤连蛋白表达的关键节点。我们报告说，高葡萄糖和TGFβ激活了肾小球和近端管状上皮（PTE）细胞以及2型和1型糖尿病小鼠的肾脏中的MTOR复合物（C1和C2）。在这些糖尿病模型中，我们用雷帕霉素阻断了MTOR活性，显示出蛋白尿，肾肥大和基质扩张的减少。雷帕霉素对MTOR完全抑制可能是有害的，因为该激酶的活性是维持肾细胞稳态所必需的。此外，雷帕霉素会改变基因表达谱，从而导致不良反应。因此，需要替代治疗方法来阻断mTOR激酶。我们的数据表明，部署者的表达是一种利用检测器抑制以增加MTOR活性的组件可以识别出新的分子靶标，以阻止糖尿病肾脏并发症。在我们的初步数据中，我们 找到与EZH2表达增加（Zeste homolg 2的增强子）（Polycomb复制仪复合物2的催化亚基）相关的部署水平的降低，在赖氨酸-27处将组蛋白H3催化为阻断特定基因的转录。此外，我们显示了肾细胞中高葡萄糖和TGFβ的E3泛素连接酶βTRCP（含有蛋白质的蛋白质）的增强表达。 βTRCP靶向降解。此外，我们证明了高葡萄糖和TGFβ会增加microRNA，miR-181（a，b，c，d）和miR-221的表达。这些microRNA在1型和2型糖尿病小鼠的肾脏和目标部署中的下调也增加。在此提案中，使用培养的细胞和PTE细胞以及来自糖尿病OVE26和DB/DB小鼠的肾脏尖端，我们将测试以下假设：不适当的部署者下调下调有助于肾脏肥大和糖尿病肾脏病的基质扩张。探测潜在机制的规格，我们将定义抑制检测器的调节模块。在第一个特定目的中，我们将研究EZH2作为检测器，肥大，纤连蛋白和PAI-1（纤溶酶原激活剂抑制剂1）表达的转录抑制作用的候选者。 In the second aim, the role of βTrCP in detector protein degradation/mTORC1/C2 activation and its cross To address these aims, techniques including immunoblotting, immunoprecipitation, qRT-PCR, morphometry, immunohistochemistry, reporter transformation assays, chromatin immunoprecipitation assays and siRNA-mediated downregulation of proteins will be used.