Drosophila nephrocytes as a model system to identify novel therapies for focal segmental glomerulosclerosis

局灶节段性肾小球硬化是一组异质性疾病，是肾病综合征和终末期肾病的常见原因。尽管在理解疾病病因学方面取得了进展，但治疗化合物的开发一直令人失望。使用整个动物的表型药物筛选可能会发现具有全新机制或过于复杂而无法通过传统筛选发现的疗法。果蝇模型已成功地用于人类药物发现，并且加兰细胞肾细胞被建立为肾病模型。我们提出了一种使用果蝇肾细胞的肾脏药物发现的体内模型。我们建议建立一种方法来评估肾细胞功能的幼虫在96孔板与含药物的液体食物（Aim 1）。示踪剂内吞作用反映肾细胞功能。我们提出了一个合适的体内试验，使用转基因动物，无处不在地表达两个荧光标记下的内源性启动子：绿色荧光蛋白，分泌到细胞外的空间，并受到内吞的肾细胞。解剖的肾细胞的绿色荧光强度可用作肾细胞功能的快速读出。第二种蛋白质是红色荧光mCherry，其保留在细胞内。因此，解剖的肾细胞的红色荧光与肾细胞功能无关，并且可以用作参考。将使用已建立的离体FITC-白蛋白试验作为确认的补充读数。然后，我们希望采用新系统来筛选FDA批准的药物库，这些药物在RhoGT 3活性失调的背景下（Aim 2）。这是局灶节段性肾小球硬化症的一个已被充分研究的发病机制，并且是相当大比例的遗传原因的基础。初步数据表明，RAC 1获得的功能和失去的功能的苍蝇直系同源物的ARHGAP 24诱导肾细胞中的类似表型，这是提示误效的狭缝隔膜蛋白。这导致肾细胞功能丧失。这些基因各自的功能丧失或获得将作为筛选的遗传背景。所有有效恢复肾细胞功能并已在果蝇中验证的药物将由合作者在永生化人类细胞系中使用足细胞迁移进行第二层筛选。使用FDA批准的化合物的已知靶标，这些化合物被证明是有效的，我们希望开发关于其作用机制的假设。在重点分析中，将在果蝇模型中探索潜在的生物学（Aim 3）。这可能会导致相关的新发现，并为小鼠和临床研究中的未来分析做好准备。在果蝇的两层分析中筛选FDA批准的药物，并在已建立的人类体外系统中进行验证，有望确定急需的局灶节段性肾小球硬化症的新治疗选择。

Focal segmental glomerulosclerosis is a heterogeneous group of disorders that are a common cause of nephrotic syndrome and end-stage renal disease. Despite advances in understanding disease etiologies, the development of therapeutic compounds has been disappointing. A phenotypic drug screen that uses whole animals may discover therapies that have an entirely novel mechanism or that are too complex to be discovered by conventional screening. The Drosophila model has been successfully used for human drug discovery and garland cell nephrocytes were established as a model for nephrotic renal disease. We propose an in vivo model for renal drug discovery using Drosophila nephrocytes. We suggest establishing an assay to evaluate nephrocyte function in larvae raised in 96-well plates with drug containing liquid food (Aim1). Tracer endocytosis reflects nephrocyte function. We propose a suitable in vivo assay using transgenic animals that ubiquitously express two fluorescent markers under endogenous promoters: EGFP that is secreted into the extracellular space and subject to endocytosis by nephrocytes. The green fluorescence intensity of dissected nephrocytes can serve as a rapid read-out of nephrocyte function. The second protein is red fluorescent mCherry that remains intracellularly. The red fluorescence of dissected nephrocytes thus is independent from nephrocyte function and can be applied as a reference. An established ex vivo FITC-albumin assay will be used as a complementary read-out for confirmation. Then we want to employ the new system to screen a library of FDA approved drugs in a background of dysregulated RhoGTPase activity (Aim2). This is a well-studied pathogenetic mechanism of focal segmental glomerulosclerosis und underlies a significant proportion of genetic causes. Preliminary data indicates that RAC1 gain-of-function and loss-of-function of the fly orthologue of ARHGAP24 induce a similar phenotype in nephrocytes that is suggestive for mistrafficking of slit diaphragm proteins. This results in a loss of nephrocyte function. The respective loss- or gain-of-function of these genes will serve as the genetic background for the screen. All drugs that are effective in restoring nephrocyte function and that have been validated in Drosophila will be analyzed in a second-tier screen using podocyte migration in an immortalized human cell line by a collaborator. Using the known targets of the FDA-approved compounds that proved to be effective, we want to develop hypotheses regarding their mechanism of action. In a focused analysis, the underlying biology will be explored in the Drosophila model (Aim3). This may lead to relevant novel findings and serves to prepare future analysis in mouse and clinical studies. Screening FDA approved drugs in a two-tiered analysis in Drosophila with validation in an established human in vitro system, holds the promise of identifying much needed novel therapeutic options for focal segmental glomerulosclerosis.