Discovering the intracellular mechanisms of the APOL1 protein-induced cell death

The markedly increased risk of chronic kidney disease (CKD) in the African-American population is attributed to 2 amino acid missense (G1) or deletion (G2) mutations in the human APOL1 protein which carries a membrane pore forming domain. These mutations confer cell injury and death of podocyte cells in the kidney, leading to development of CKD driven by its intracellular function. We found that tetracycline induction of APOL1 expression in HEK 293 cells promoted caspase-independent necrotic cell death, including cell swelling, cell detachment, and membrane permeabilization. TEM imaging revealed that severe mitochondrial damage was among the first phenotypes developing in response to mutant APOL1 variants, associated with reduction in mitochondrial membrane potential (MMP) as measured by TMRE staining. Treating cells with Cyclosporine A prior to APOL1 expression attenuated the drop in MMP, implicating an involvement of mitochondrial permeability transition pores. APOL1 was localized by immunostaining to the ER, possibly facing the cytosolic compartment. By deleting the membrane-addressing domain (MAD) of APOL1 its necrotic activity abolished, yet ER localization was retained, suggesting that this localization may be necessary but not sufficient to drive the necrotic function of APOL1. Deleting the C-terminal domain, where the mutations reside, also abolished the cellular damage caused by APOL1. Protein complementation assays suggest that APOL1 creates homo- and heterodimers with the WT and mutant variants.We propose that APOL1 is involved in ER-mitochondrial contact sites, possibly causing entry of Ca²⁺ to the mitochondria, leading to accumulation of Ca²⁺ in the mitochondrial compartment and the subsequent strong necrotic effects.