Small Molecule Inhibitors of APOL1 Reverse Dysfunctional Signaling Mediated by APOL1 Risk Variants in Immortalized Human Podocytes

Background: Two coding variants in the APOL1 gene (G1 and G2) increase risk for progressive kidney disease in individuals of African ancestry. APOL1 kidney disease (AKD) is in part driven by APOL1 toxicity in podocytes, leading to glomerular dysfunction and proteinuria. However, the molecular mechanisms by which APOL1 risk variants (RVs) drive podocyte injury in AKD are not completely understood. In this study, we examined the impact of APOL1 RV expression in immortalized human podocytes and evaluated whether small molecule APOL1 inhibitors could mitigate APOL1-induced changes. Methods: We generated doxycycline-inducible human immortalized podocyte cell lines expressing APOL1 RVs. Differentiated podocytes were treated with doxycycline to induce APOL1 expression, concurrently with either APOL1 inhibitor MZ-302 (a structural analog of MZE829) or MZ-303 (synthesized inaxaplin). Total RNA was extracted at timepoints preceding cytotoxicity and RNA was sequenced. Differential gene expression and pathway enrichment analyses were performed to assess the impact of APOL1 RV induction and the ability of APOL1 inhibitors to prevent these changes. In parallel, we performed cell painting assays to profile cellular phenotypes induced by APOL1 RVs and assess effects of small molecule APOL1 inhibitors. Results: APOL1 RV induction triggered widespread transcriptional alterations in immortalized podocytes, including upregulation of genes involved in inflammation, metabolism, and stress response pathways. These findings were corroborated by protein-level analyses. Treatment with MZ-302 or MZ-303 effectively prevented APOL1 RV-induced transcriptional changes and restored gene expression profiles to baseline levels. Consistent with these findings, cell painting analyses revealed that APOL1 RV-induced morphological perturbations were also mitigated by small molecule APOL1 inhibitors. Conclusion: These findings underscore the therapeutic potential of APOL1 small molecule inhibitors in mitigating APOL1-driven podocyte injury and provide mechanistic insights into podocyte dysfunction in AKD. Funding: Commercial Support - Maze Therapeutics Inc