Use of Zebrafish as a Model for Diabetic Nephropathy

描述(申请人提供)：基底膜增厚(BM)是 糖尿病的一种常见的继发性并发症，与 当肾小球骨髓(GBM)受到影响时，肾功能的异常。 虽然基底膜增厚的机制还不清楚，但它是 可见，GBM的正常生物合成和维持是一个重要的 肾功能方面。GBM增厚可以用问题来解释 在基质组分的合成、聚合和周转之间取得平衡。 这些过程都在一定程度上涉及细胞/细胞外基质的相互作用，并意味着 GBM的形成受到严格控制。要了解潜在的机制 在正常和病理条件下，基底膜的形成和维持 建议利用斑马鱼(ZF)作为模型系统。ZF将自己借给 这样的研究是因为它的肾脏系统发展迅速，而且生物体 可修改为细胞、分子和遗传方法。ZF肾脏系统 发展分两个阶段进行，包括： 1)在发育50小时后形成一过性前肾 由单个肾小球和2)通过3D形成的中肾组成 如哺乳动物所见，含有多个肾小球的周。不像 原肾，ZF中肾虽已有研究，但尚未得到很好的研究 需要与GBM的形成、成熟和周转相关的研究。这个 这项为期两年的R21(探索/发展助学金)的具体目标将集中在 关于建立ZF作为分析年度GBM形成和周转的模型 正常情况下的中肾和升高状态下的中肾 糖尿病患者观察到的血糖水平。具体目标包括：1) 确定在成熟过程中是否发生BM组分的异构体转换 ZF中肾小球，2)靶向表达的程序 肾小球基底膜成分及其相关的基质金属蛋白酶(MMP5) 转基因ZF的中肾，3)测定血糖水平是否升高 导致基底膜蛋白质糖基化，如果基底膜增厚 在啮齿动物肾脏模型中观察到的发生，以及4)实验的启动 利用上述GBM成分数据和转基因程序 开始检验ZF中肾中肾GBM增厚的假说 涉及到聚合和聚合之间的正常平衡的破坏 基质非酶糖化引起的基质组分周转 使它们不太容易被MMPs切割的成分。

DESCRIPTION (provided by applicant): A thickening in basement membranes (BM) is a common secondary complication of diabetes and is associated with abnormalities in kidney function when the glomerular BM (GBM) is affected. While the mechanisms underlying GBM thickening are not understood, it is evident that the normal biosynthesis and maintenance of GBM is an important aspect of kidney function. GBM thickening can be explained in terms of problems in the balance of matrix component synthesis, polymerization, and turnover. These processes all involve cell/ECM interactions to some degree and imply that GBM formation is tightly controlled. To understand the mechanisms underlying GBM formation and maintenance under normal and pathological conditions, we propose to utilize the zebrafish (ZF) as a model system. The ZF lends itself to such studies because its renal system develops rapidly and the organism is amendable to cellular, molecular, and genetic approaches. The ZF renal system develops in two stages including: 1) formation of a transient pronephros by 50 hrs of development that is composed of a single glomerulus and 2) formation of a mesonephros by the 3d week that contains multiple glomeruli as seen in mammals. Unlike the pronephros, the ZF mesonephric kidney has not been well studied although it is required for studies related to GBM formation, maturation, and turnover. The specific aims of this two-year R21 (Exploratory/Developmental Grant) will focus on establishing the ZF as a model for analysis of GBM formation and turnover in the mesonephric kidney under normal conditions and conditions of elevated glucose levels as observed in diabetes. The Specific Aims will include: 1) to determine if isoform switching of BM components occurs during maturation of the ZF mesonephric glomerulus, 2) development of procedures to target expression of GBM components and associated matrix metalloproteinases (MMP5) in the mesonephric kidney of transgenic ZF, 3) to determine if elevated glucose levels result in glycation of proteins in the GBM and if a thickening of the GBM occurs as observed in rodent kidney models, and 4) initiation of experiments using the above mentioned GBM compositional data and transgenic procedures to begin to test the hypothesis that 'GBM thickening in the ZF mesonphric kidney involves a disruption in the normal balance between the polymerization and turnover of matrix components due to non-enzymatic glycation of matrix components that makes them less susceptible to cleavage by MMPs."