Pathogenesis of Diabetic Nephropathy

糖尿病肾病的发病机制

• 批准号: 7208448
• 负责人: Yashpal S. Kanwar
• 金额: $ 30.96万
• 依托单位: NORTHWESTERN UNIVERSITY AT CHICAGO
• 依托单位国家: 美国
• 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/7208448
• 关键词: 5' 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; Kidney Diseases; 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

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，其中高血糖引起的细胞功能障碍在肾脏的不同腔室，即肾小球与小管间质或血管间质有一定的相似性。受影响的细胞可能来源于上皮或间充质祖细胞；有时高血糖可引起表型改变，伴有上皮-间质转化。在靶细胞中，高葡萄糖激活了各种相似的细胞内通路，除了微小的差异。这些途径主要在肾小球的各种细胞类型中进行研究，而文献中关于小管室的信息有限。有趣的是，我们对“新生儿”糖尿病小鼠肾脏的抑制减法分析表明，大多数差异上调基因在肾小管中表达，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等。Rap1b被发现发挥下游作用，导致细胞外基质（ECM）蛋白的上调。有趣的是，共表达的Epac1恰好正调控Rap1b的活性。最近描述的Rap1b的负调控因子是Cbl，一种原癌基因。这三种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Rap1b的活性，抑制与ECM生物学相关的糖尿病并发症。为了证明这一观点，我们提出了各种实验，具体目的如下：1 .研究糖尿病动物模型中Rap1b、Cbl和Epac1表达的调节及其与疾病活动的相关性。我们将尝试测定Rap1b对小管间质ECM蛋白表达的活性。2。高糖环境对其表达的影响将在细胞培养系统中进行研究。我们将通过使用各种激动剂、抑制剂、优势结构体和shRNA/siRNA寡核苷酸，努力理清Cbl/Epac1在高糖条件下对Rap1b的调节。3。高糖直接或间接激活Cbl和Epac1的机制将通过启动子分析、凝胶转移和染色质免疫沉淀试验进行研究。糖化、氧化/抗氧化和渗透胁迫在其活化中的作用将被研究。其他可以激活Rap1b的分子将通过使用共免疫沉淀程序和酵母双杂交系统进行鉴定。5 .由于Epac1在“新生儿”糖尿病小鼠中上调，并在发育过程中表现出差异表达，因此将研究其在高糖环境下对肾发生的调节。希望这些研究能够加深我们对与糖尿病肾病相关的“肾小管-间质ECM病理生物学”的认识。