Use of Zebrafish as a Model for Diabetic Nephropathy

描述（由申请人提供）：基底膜（BM）增厚， 糖尿病常见的继发性并发症， 当肾小球BM（GBM）受到影响时，肾功能异常。 虽然GBM增厚的机制尚不清楚，但它是 很明显，GBM的正常生物合成和维持是一个重要的 肾功能方面。GBM增厚可以解释为 在基质组分合成、聚合和周转的平衡中。 这些过程都在一定程度上涉及细胞/ECM相互作用，并意味着 GBM的形成受到严格控制。为了了解这些现象背后的机制， 在正常和病理条件下GBM的形成和维持，我们 建议利用斑马鱼（ZF）作为模型系统。ZF有助于 这种研究，因为它的肾脏系统发展迅速， 适用于细胞、分子和遗传学方法。ZF肾脏系统 发展分为两个阶段，包括： 1)通过50小时的发育形成短暂的前肾， 由单个肾小球组成，2）由3d形成中肾 哺乳动物中含有多个肾小球的一周。不像 尽管ZF中肾肾在前肾，但尚未得到很好的研究， 这是与GBM形成、成熟和周转相关的研究所必需的。的 这项为期两年的R21（探索/发展赠款）的具体目标将集中在 建立ZF作为分析GBM形成和周转的模型， 中肾在正常条件下和升高条件下 糖尿病患者的血糖水平。具体目标包括：1） 确定BM组分的同种型转换是否发生在 ZF中肾肾小球，2）开发靶向表达 GBM组分和相关的基质金属蛋白酶（MMP 5）在 转基因ZF的中肾，3）以确定是否升高的葡萄糖水平 导致GBM中蛋白质的糖化，并且如果GBM的增厚 如在啮齿动物肾脏模型中观察到的那样发生，以及4）实验开始 使用上述GBM组成数据和转基因程序， 开始检验ZF中肾GBM增厚的假设 涉及聚合和聚合之间的正常平衡的破坏， 由于基质的非酶糖化导致基质组分的转换 这使得它们不易被MMPs切割。"

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."