Gamma Interferon-inducible Protein 16 Regulates Hypoxia-induced Apolipoprotein L1 Expression in Human Podocytes

Richaundra K. Randle, Venkateswara Amara, and Waldemar Popik

Meharry Medical College, Nashville, TN Genetic risk variants (RVs) of Apolipoprotein L1 (APOL1) have been strongly associated with an increased risk of non-diabetic kidney diseases and kidney failure in African Americans. These RVs primarily affect podocytes, the crucial cells maintaining the kidney's filtration barrier. According to genome-wide association studies, environmental factors contribute significantly to this association. Recent studies show that hypoxia, or low oxygen tension, upregulates the expression of APOL1 in kidney podocytes. However, the underlying molecular mechanism still needs to be fully understood. The objective of this research is to identify molecular components that regulate APOL1 gene activation in response to hypoxia. To achieve this goal, we exposed human conditionally immortalized AB8/13 glomerular podocytes to roxadustat, a known inducer of hypoxia, or subjected to controlled hypoxic conditions (1% oxygen). We then analyzed changes in APOL1 expression using qPCR and immunoblotting. Our findings revealed that the DNA sensor IFI16 and hypoxia-associated transcription factor HIF-1α are critical components of hypoxia-induced APOL1 expression. We further confirmed the roles of IFI16 and HIF-1α by using siRNA-mediated knockdown and CRIPSR-Cas9 knockout assays. We identified four HIF-1α binding sites within the APOL1 promoter/enhancer sequence through ChIP-qPCR assays. We also determined the cellular localization and interactions between IFI16 and HIF-1α using subcellular fractionation, immunoprecipitation, and western blotting. Collectively, our results strongly indicate that, under hypoxic conditions, IFI16 and HIF-1α cooperate to promote APOL1 expression in human podocytes. These results are consistent with our hypothesis that hypoxic stress leads to upregulation of APOL1 RVs, further exacerbating glomerular podocyte damage. As there are currently no approved therapies specifically targeting APOL1-associated kidney disease, our research may present potential therapeutic targets. By preventing or reducing hypoxia driven expression of APOL1, we could potentially mitigate the pathogenesis of kidney disease in African Americans carrying APOL1 RVs. This project was supported, in part, by the School of Graduate Studies.