Pathogenesis of 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，高血糖引起的细胞功能障碍在肾脏的不同部分有一定程度的相似之处，即肾小球与肾小管间质或血管。受影响的细胞可能来自上皮祖细胞或间充质祖细胞；有时高血糖可诱导上皮间充质转化为表型改变。在靶细胞中，高糖激活了各种细胞内途径，除了微小的差异外，这些途径都是相似的。这些途径主要在肾小球的不同细胞类型中进行研究，而文献中关于肾小管间隔区的信息有限。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、鸟核苷酸交换因子Epac1和泛素融合蛋白UbA52等。有趣的是，共表达的Epac1恰好正向调节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糖环境的调节。可以想象，Cb1和Epac1都对Rap1b发挥作用，以达到其活性的平衡，以减少与ECM生物学相关的糖尿病并发症。为了证明这一论点，在以下特定目的下提出了各种实验：1.将研究糖尿病动物模型中Rap1b、Cb1和Epac1的表达调节并与疾病活动性相关。将尝试测量Rap1b在肾小管间质ECM蛋白表达中的活性。高糖环境对它们表达的影响将在细胞培养系统中进行研究。将致力于通过使用各种激动剂、抑制剂、优势结构&shRNA/siRNA寡核苷酸来梳理Cb1/Epac1在高糖条件下对Rap1b的调控。高糖直接或间接激活Cb1和Epac1的机制(S)将通过启动子分析、凝胶漂移和染色质免疫沉淀实验来研究。糖化、氧化剂/抗氧化剂和渗透胁迫在其激活中的作用将被研究。可以激活Rap1b的其他分子将通过免疫共沉淀法和酵母双杂交系统进行鉴定。由于Epac1在“新生”糖尿病小鼠中表达上调，并在发育过程中表现出差异表达，因此我们将研究它在高糖环境下对肾脏形成的调控。希望这些研究能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。

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.