Hypoxia-Induced Peroxisomal Stress Mediates Cytotoxicity of APOL1 Variants

Background: APOL1 variants (G1 and G2) are critical drivers of CKD in individuals of African ancestry. Not all individuals with APOL1 kidney risk variants (KRVs) develop kidney disease, consistent with the need for a "second hit" like interferon or hypoxia to initiate disease. The mechanisms that cause CKD under the influence of these triggers remain uncertain. This study aims to identify “second hit” trigger genes and pathways in order to develop therapeutic approaches to modify their function. Methods: A genome-wide siRNA and CRISPR screen on ~20,000 genes (3 targets/gene) was conducted in HEK293 cells expressing APOL1-G0, G1, and G2 under hypoxia (1%O2) using cytotoxicity as the readout, which identified peroxisomal genes. Individual targets were validated by siRNA knockdown. APOL1 localization to peroxisomes was analyzed using proximity biotinylation, peroxisome isolation and STORM imaging, and peroxisomal stress was measured by catalase activity in HEK293 cells and human podocytes. Candidate drug screening was performed in HEK293 cells. Peroxisomal localization of APOL1 in healthy human kidneys and FSGS was examined by PEX14 immunohistology. Glomerular peroxisomal protein expression (healthy kidneys n=4, FSGS n=12) was analyzed by mass spectrometry. Results: Hypoxia increased APOL1-G1 and G2 cytotoxicity in HEK293 cells and human podocytes. siRNA and CRISPR screens revealed that knockdown of genes involved in peroxisome biogenesis or Peroxins (PEX3, PEX5, PEX11, PEX13, PEX16, PEX19, FIS1) intensified APOL1 KRV-induced cytotoxicity under hypoxia. STORM imaging and fractionation studies showed translocation of APOL1s to peroxisomes under hypoxia. A C-terminal peroxisomal targeting signal facilitated this translocation, leading to increased catalase activity by APOL1 KRVs, indicating peroxisomal stress. Enhancing peroxisomal function genetically (PEX overexpression) or pharmacologically (PPAR-α agonists) alleviated hypoxia-induced APOL1 KRV cytotoxicity. APOL1 co-localized with peroxisomes in podocytes of healthy human kidneys, while Peroxin expression in glomeruli was reduced in FSGS. Conclusion: Peroxisomal genes are novel genetic modifiers of APOL1 nephropathy, protective against podocyte dysfunction caused by APOL1 KRVs under hypoxic conditions. Augmenting peroxisomal function represents a potential therapeutic strategy to ameliorate CKD associated with APOL1 KRVs. Funding: NIDDK Support