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Alport syndrome is a hereditary chronic kidney disease, attributed to rare pathogenic variants in either of three collagen genes () with most localized in . Trimeric type IV Collagen α3α4α5 is essential for the glomerular basement membrane that forms the kidney filtration barrier. A means to functionally assess the many candidate variants and determine pathogenicity is urgently needed. We used , an established model for kidney disease, and identify as the functional homolog of human in the fly nephrocyte (equivalent of human podocyte). Fly nephrocytes deficient for showed an irregular and thickened basement membrane and significantly reduced nephrocyte filtration function. This phenotype was restored by expressing human reference (wildtype) , but not by carrying any of three established pathogenic patient-derived variants. We then screened seven additional patient variants; their ClinVar classification was either likely pathogenic or of uncertain significance. The findings support pathogenicity for four of these variants; the three others were found benign. Thus, demonstrating the effectiveness of this in vivo kidney platform in providing the urgently needed variant-level functional validation.