Pathogenesis of Diabetic Nephropathy

糖尿病肾病的发病机制

基本信息

批准号：
7383836
负责人：
Yashpal S. Kanwar
金额：
$ 30.34万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-02-15 至 2012-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7383836
关键词：
5&apos Flanking Region Affect Agonist Animal Model Antioxidants Apoptosis Attention Biochemical Biological Assay Biology Blood Vessels Cell Culture System Cell physiology Cells Characteristics Chimeric Proteins Co-Immunoprecipitations Complications of Diabetes Mellitus Cyclic AMP Cyclic AMP-Dependent Protein Kinases Development Diabetes Mellitus Diabetic Nephropathy Diabetic mouse Disease Dominant-Negative Mutation Employee Strikes Environment Epithelial Equilibrium Event Exhibits Extracellular Matrix Extracellular Matrix Proteins Figs - dietary Functional disorder GTP-Binding Proteins Gelshift Analysis Generations Genes Glucose Guanine Nucleotide Exchange Factors Guanosine Triphosphate HK2 gene Human Glandular Kallikrein 2 Hyperglycemia Injury Investigation Kidney Literature Mesenchymal Methods Minor Mitochondria Molecular Mus Neonatal Organ Culture Techniques Organogenesis Oxidants Oxidoreductase Pathogenesis Pathway interactions Phosphotransferases Precipitation Procedures Protein Biosynthesis Proteins Proto-Oncogenes Reactive Oxygen Species Regulatory Element Renal tubule structure Research Personnel Role Small Interfering RNA Streptozocin Stress System Time Transcriptional Activation Transcriptional Regulation Transfection Transgenic Mice Tubular formation Ubiquitin Up-Regulation Variant Yeasts activation product cell type chromatin immunoprecipitation db/db mouse defined contribution diabetic glycation inhibitor/antagonist interest interstitial nephrogenesis progenitor programs promoter research study small hairpin RNA subtraction hybridization transcription factor translocase vector yeast two hybrid system

项目摘要

DESCRIPTION (provided by applicant): Diabetic nephropathy is one of the major complications of diabetes, where cellular dysfunctions induced by hyperglycemia have certain degree of similarities in different compartments of the kidney, i.e., glomerular vs tubulo-interstitial or vascular. The cells affected may be derived from epithelial or mesenchymal progenitors; and at times hyperglycemia may induce a phenotypic change with epithelial-mesenchymal transformation. In target cells the high glucose activates various intracellular pathways that are similar except for minor variations. These pathways have been mainly studied in various cell types of the glomerulus, while the information available in the literature for the tubular compartment is limited. Intriguingly, our suppression subtraction analyses of kidneys of "neonatal" diabetic mice indicate that the majority of the differentially up- regulated genes are expressed in the renal tubules, e.g., GTP binding protein Rap1b, guanine nucleotide exchange factor Epac1 and ubiquitin fusion protein UbA52 and etc. The Rap1b was found to exert downstream effects leading to up-regulation of extracellular matrix (ECM) proteins. Interestingly, the co- expressed Epac1 happens to positively regulate the Rap1b activity. A recently described negative regulator of Rap1b is Cbl, a proto-oncogene. All the three molecules are modulated by high glucose ambience. Conceivably, both Cbl and Epac1 exert their effects on Rap1b to strike a balance in its activity to dampen the complications of diabetes related to ECM biology. To attest to this contention various experiments are proposed under the following specific aims: I. Modulation of Rap1b, Cbl and Epac1 expression in animal models of diabetes will be investigated and correlated with the disease activity. Attempts will be made to gauge the activity of Rap1b on the expression of tubulo-interstitial ECM proteins. II. Effect of high glucose ambience on their expression will be investigated in cell culture systems. Efforts will be devoted to tease out the modulation of Rap1b by Cbl/Epac1 under high glucose by employing various agonists, inhibitors, dominant constructs & shRNA/siRNA oligos. III. Mechanism(s) by which high glucose activate, directly or indirectly, Cbl and Epac1, will be investigated by carrying out promoter analysis, gel shift and chromatin immunoprecipitation assays. Role of glycative, oxidant/antioxidant and osmotic stresses in their activation will be investigated. IV. Other molecules that could activate Rap1b will be identified by using co-immuno- precipitation procedures and yeast-two-hybrid systems. V. Since Epac1 is up-regulated in the "neonatal" diabetic mice and exhibits differential expression during development, its modulation of nephrogenesis under high glucose ambience will be investigated. It is hoped that these studies would enhance our understanding of the "Renal Tubulo-interstitial ECM Pathobiology" relevant to diabetic nephropathy.

描述（申请人提供）：糖尿病肾病是糖尿病的主要并发症之一，其中由高血糖引起的细胞功能障碍在肾脏的不同室（即肾小球室与肾小管间质室或血管室）具有一定程度的相似性。受影响的细胞可能源自上皮或间质祖细胞；有时高血糖可能会引起上皮间质转化的表型变化。在靶细胞中，高葡萄糖激活各种细胞内途径，除了微小的变化外，这些途径相似。这些途径主要在肾小球的各种细胞类型中进行研究，而文献中有关管状室的信息有限。有趣的是，我们对“新生”糖尿病小鼠肾脏的抑制消减分析表明，大多数差异上调的基因在肾小管中表达，例如GTP结合蛋白Rap1b、鸟嘌呤核苷酸交换因子Epac1和泛素融合蛋白UbA52和研究发现 Rap1b 发挥下游效应，导致细胞外基质 (ECM) 蛋白上调。有趣的是，共表达的 Epac1 恰好正向调节 Rap1b 活性。最近描述的 Rap1b 负调节因子是 Cbl，一种原癌基因。所有这三个分子都受到高葡萄糖环境的调节。可以想象，Cbl 和 Epac1 都对 Rap1b 发挥作用，以平衡其活性，从而抑制与 ECM 生物学相关的糖尿病并发症。为了证明这一论点，根据以下具体目标提出了各种实验： I.将研究糖尿病动物模型中 Rap1b、Cbl 和 Epac1 表达的调节并将其与疾病活动相关联。我们将尝试测量 Rap1b 对肾小管间质 ECM 蛋白表达的活性。二.将在细胞培养系统中研究高葡萄糖环境对其表达的影响。我们将致力于通过使用各种激动剂、抑制剂、显性构建体和 shRNA/siRNA 寡核苷酸来阐明高葡萄糖条件下 Cbl/Epac1 对 Rap1b 的调节。三．将通过启动子分析、凝胶位移和染色质免疫沉淀测定来研究高葡萄糖直接或间接激活 Cbl 和 Epac1 的机制。将研究糖化、氧化剂/抗氧化剂和渗透应激在其激活中的作用。四．其他可以激活 Rap1b 的分子将通过使用免疫共沉淀程序和酵母双杂交系统来鉴定。 V.由于Epac1在“新生”糖尿病小鼠中表达上调，并且在发育过程中表现出差异表达，因此将研究其在高葡萄糖环境下对肾发生的调节。希望这些研究能够增进我们对与糖尿病肾病相关的“肾小管-间质ECM病理学”的理解。