Differential Pathway Activation by APOL1 G1 and G2 Variants in Induced Pluripotent Stem Cell (iPSC)-Derived Endothelial Cells: Insights from Proteomic Analysis

Background: Apolipoprotein-L1 (APOL1) risk variants (RV), named G1 and G2, are associated with increased risk of chronic kidney disease (CKD) in people of African ancestry. Recent evidence has highlighted the role of APOL1 RVs as drivers of endothelial cell (EC) activation. However, the specific molecular mechanisms driving APOL1-mediated EC activation in humans and the differences between G1 and G2 variants remain unknown. Methods: We employed CRISPR Xential, a novel CRISPR/Cas9-based method to select and enrich for cells with two edited copies of a gene of interest, to engineer induced pluripotent stem cells (iPSCs) with inducible APOL1 expression. iPSCs were differentiated into ECs and treated with doxycycline for 24 or 48 hours to induce expression of APOL1 variants G0 (control), G1, and G2. We assessed EC viability and conducted global proteomics and bioinformatics analyses. Results: APOL1 induction reduced EC viability in a time-dependent manner, most significantly with G2 followed by G1 and G0. Proteomics analysis revealed more protein changes (119) after 24h in the G2 expressing ECs when compared with G1 (75) and G0 (6). After 48h, G2 and G1 showed similar amount of differentially regulated proteins, 154 and 175, respectively, whereas G0 expressing ECs presented 44. Among these proteins, IL8 and GLUT1 were upregulated in all conditions. Pathway enrichment analysis revealed inflammatory response and TNFa pathways to be common between G1 and G2 expressing ECs. Interestingly, after 48h, G1 expression led to changes in pathways related with EMT transition and hypoxia, whereas G2 enhanced pathways related with cell cycle and DNA replication. Conclusion: Our findings corroborate APOL1's role in EC activation and unveil distinct pathways influenced by G1 and G2 variants, underscoring their differential contribution to endothelial dysfunction. Funding: Commercial Support - AstraZeneca