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

• Applicant: Privatdozent Dr. Tobias Hermle
• Subject Area: Nephrology
• Term: from 2018 to 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 407240209

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.

• DFG Programme: Research Grants