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Risk-variant APOL1 expression in iPSC-derived macrophages increases cellular stress and inflammation

Esther Liu, Jennie Lin

DNA variants for the APOL1 gene have been linked to a higher risk of developing kidney and cardiovascular disease in individuals with recent African ancestry. APOL1 is expressed endogenously in not only kidney cells but also immune cells such as macrophages. Recent studies show that severe acute inflammatory illness such as COVID-19 increases the likelihood of individuals with the high-risk APOL1 genotype developing glomerulopathy, raising the question of the role of immune cells in APOL1-mediated disease. Therefore, we aim to understand the cellular stress and pro-inflammatory pathways increased by risk variant APOL1 expression in iPSC-derived macrophages (iPSDM).

nduced pluripotent stem cell (iPSC) expressing G0 and G1 variants of APOL1 were generated through CRISPR/Cas9. Macrophages (iPSDM) were differentiated from these iPSC lines. Expression of APOL1 in the cultured iPSDMs, was induced with IFNγ (20 ng/mL). We measured cytokine expression with ELISA. We measured metabolic respiration with the Seahorse Mito Stress Test (Agilent).

Upon stimulation with IFNγ, we observed a 4.6-fold decrease in TGF-β secretion (n=3, p<0.001) in G1 iPSDM compared to G0. When polarized to an M1-like phenotype with IFNγ and LPS, IL1β secretion was 1.4-fold increased (n=3, p<0.005) in G1 iPSDM compared to G0. G1 macrophages also had decreased levels of basal respiration compared to G0 (n=3, p=0.04). However, there was no observed difference in mitochondrial reactive oxygen species as measured by MitoSOX Red staining. Additionally, we observe a decrease in protein levels of LC3- II, an indicator of active autophagy, and a 4.4-fold increase of autophagy substrate P62 levels in G1 macrophages compared to G0.

The findings in our experiments show a significant increase in inflammatory cytokine expression in G1 macrophages and decreased levels of basal respiration and autophagy markers. These results suggest that APOL1 risk variant expression modulates macrophage functions which are relevant to kidney homeostasis and disease.