APOL1 Induction Increases Cytosolic Calcium in 293 Cells but Not HeLa or Isogenic Induced Pluripotent Stem Cell (iPSC)-Derived Podocytes

Background: Coding variants in APOL1 are associated with a spectrum of kidney diseases, but the biology underlying this genetic association remains uncertain. The channel function of APOL1 imparts its trypanolytic activity and in vitro models demonstrate APOL1 can insert into lipid bilayers conferring a non-selective cation transport without variant-dependent differences. Recently overexpression of APOL1 kidney disease variants in cell culture models of 293 cells were shown to increase intracellular calcium. However, these model systems rely on APOL1 overexpression that may lead to aberrant APOL1 trafficking and cytotoxic phenotypes that may not reflect the pathogenesis of human APOL1 kidney disease. Our data suggests that APOL1-mediated cytosolic calcium elevations are cell line dependent. Methods: We utilized isogenic induced pluripotent stem cell (iPSC) with APOL1-G0 and gene edited to G1 or G2, and differentiated to podocytes using a differentiation protocol developed in our lab. We also used tetracycline regulated G0, G1 or G2 in HeLa or 293 cells. We performed surface biotinylation with streptavidin pulldowns, SYTOX microplate viability assays and fura-2 microplate calcium assays after 6h (293), 24h (HeLa and iPSC) and 72h (iPSC) of tetracycline or IFN-γ induced APOL1 expression. Results: In 293 cells APOL1 localizes to the plasma membrane after 6h of induced expression and is associated with an increase in intracellular calcium in a variant independent manner (p <0.05 Kruskal-Wallis). In HeLa cells after 24 hours of APOL1 induction APOL1 does not localize to the plasma membrane and there is no APOL1 mediated cell death (p >0.05 Kruskal-Wallis). Similarly, iPSC-derived podocytes do not have APOL1 localization at the plasma membrane even after 72h of IFN-γ induction and there is no variant-dependent differences in cell viability or intracellular calcium at 24h, 48h or 72h (p >0.05 Kruskal-Wallis). Conclusion: Our data indicates that overexpression of APOL1 in 293 cells results in plasma membrane insertion and variant independent intracellular calcium elevations and cell death. On the other hand, tetracycline induced APOL1 induction in HeLa cells and IFN-γ APOL1 induction in isogenic iPSC derived podocytes do not have APOL1 plasma membrane localization, intracellular calcium differences or cytotoxicity. Funding: NIDDK Support, Private Foundation Support