Pathogenesis of Diabetic Nephropathy

描述（由申请人提供）：糖尿病肾病是糖尿病的主要并发症之一，其中由高血糖症诱导的细胞功能障碍在肾脏的不同隔室中具有一定程度的相似性，即，肾小球vs肾小管间质或血管。受影响的细胞可能来源于上皮或间充质祖细胞;有时高血糖可能诱导上皮-间充质转化的表型变化。在靶细胞中，高葡萄糖激活除了微小变化之外相似的各种细胞内途径。这些途径主要在肾小球的各种细胞类型中进行了研究，而文献中关于肾小管室的信息有限。有趣的是，我们对“新生”糖尿病小鼠肾脏的抑制减法分析表明，大多数差异上调的基因在肾小管中表达，例如，GTP结合蛋白Rap 1b、鸟嘌呤核苷酸交换因子Epac 1和泛素融合蛋白UbA 52等。Rap 1b具有下游效应，可上调细胞外基质（ECM）蛋白的表达。有趣的是，共表达的Epac 1恰好正向调节Rap 1b活性。最近描述的Rap 1b的负调节因子是Cbl，一种原癌基因。所有这三种分子都受到高糖环境的调节。可以想象，Cbl和Epac 1都对Rap 1b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，在以下具体目标下提出了各种实验：I.将研究糖尿病动物模型中Rap 1b、Cbl和Epac 1表达的调节，并将其与疾病活动性相关联。将尝试测量Rap 1b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糖环境对它们表达的影响。本论文将通过各种激动剂、抑制剂、优势构建体和shRNA/siRNA寡核苷酸来研究高糖条件下Cbl/Epac 1对Rap 1b的调控作用。三.高葡萄糖直接或间接激活Cbl和Epac 1的机制将通过进行启动子分析、凝胶迁移和染色质免疫沉淀试验进行研究。将研究抗氧化剂、氧化剂/抗氧化剂和渗透胁迫在其活化中的作用。四.其他可以激活Rap 1b的分子将通过使用共免疫沉淀程序和酵母双杂交系统来鉴定。V.由于Epac 1在“新生”糖尿病小鼠中上调，并在发育过程中表现出差异表达，因此将研究其在高葡萄糖环境下对肾发生的调节。我们希望这些研究将提高我们对糖尿病肾病相关的“肾小管间质ECM病理学”的理解。

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.