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

局灶性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方面取得了进展，但治疗化合物的发展一直令人失望。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治疗方法。果蝇模型已成功应用于人类药物研发，花环细胞肾细胞被建立为肾病肾病模型。我们提出了一种利用果蝇肾细胞发现肾脏药物的体内模型。我们建议建立一种评价用含药液体食物(Aim1)培养在96孔板中的幼虫肾细胞功能的方法。示踪内吞反应反映肾细胞功能。我们建议使用在内源性启动子下普遍表达两种荧光标记的转基因动物进行体内检测：EGFP分泌到细胞外空间，并受到肾细胞的内吞作用。解剖肾细胞的绿色荧光强度可作为肾细胞功能的快速读数。第二种蛋白质是红色荧光mCherry，它留在细胞内。因此，解剖肾细胞的红色荧光与肾细胞功能无关，可作为参考。已建立的体外FITC-白蛋白检测将作为补充读数用于确认。然后，我们希望使用新系统在RhoGTPase活性失调的背景下筛选FDA批准的药物库(AIM2)。这是局灶性节段性肾小球硬化的一种已被充分研究的发病机制，也是相当大比例的遗传原因的基础。初步数据表明，ARHGAP24的苍蝇同源基因RAC1的功能获得和功能丧失在肾细胞中诱导了类似的表型，这提示缝隙隔膜蛋白的错误洗脱。这会导致肾细胞功能的丧失。这些基因各自的功能丧失或获得将作为筛查的遗传背景。所有在恢复肾细胞功能方面有效并已在果蝇中得到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的第二级筛查进行分析。利用FDA批准的已被证明有效的化合物的已知靶点，我们想要开发关于它们的作用机制的假设。在重点分析中，将在果蝇模型(Aim3)中探索潜在的生物学。这可能会导致相关的新发现，并为未来在小鼠和临床研究中的分析做准备。在果蝇体内进行两级分析筛选FDA批准的药物，并在已建立的人类体外系统中进行验证，有望确定局灶性节段性肾小球硬化急需的新治疗方案。

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.