Evaluation of Puromycin-Induced Podocyte Injury and Protective Effects of Voclosporin Using Induced Pluripotent Stem Cells from a Patient with Nephrotic Syndrome Harboring an INF2 Variant

Focal segmental glomerulosclerosis (FSGS) is a major cause of nephrotic syndrome and end-stage kidney disease (ESKD). Many cases are attributable to pathogenic variants in podocyte-related genes, such as inverted formin 2 ( INF2 ). However, no specific treatment exists for hereditary FSGS, and experimental platforms that faithfully model podocyte injury remain limited. Therefore, in this study, we developed an injury model by generating induced pluripotent stem cells (iPSCs) from a patient with INF2 -associated FSGS and differentiating them into kidney organoids. Podocytes were validated by immunofluorescence staining for podocyte-specific markers. We induced podocyte injury in this model using puromycin aminonucleoside (PAN) in a dose-dependent manner. Injury severity was quantified by measuring podocalyxin fluorescence intensity. Cyclosporine A (CsA) or voclosporin (VCS) was administered as a 1-h pretreatment before PAN exposure to evaluate their podocyte-protective effects. The kidney organoids exhibited well-defined podocyte marker expression, confirming successful differentiation. PAN exposure caused a significant and concentration-dependent reduction in podocalyxin fluorescence, indicating robust induction of podocyte injury in organoids harboring the INF2 variant. Pretreatment with CsA or VCS significantly attenuated PAN-induced podocyte injury and preserved podocyte marker expression. CsA and VCS reduced podocyte injury to similar extents. In conclusion, we established a patient-specific kidney organoid model of INF2 -associated FSGS that reliably recapitulated podocyte vulnerability to toxic injury. This platform demonstrated that calcineurin inhibitors, including the novel agent VCS, exert direct podocyte-protective effects in a genetic FSGS background and provide a practical system for mechanistic studies and therapeutic screening.