TFAM Deficiency Leads to STING-Mediated Podocyte Injury via Impaired Mitophagy in Diabetic Kidney Disease

Background: Mitochondrial dysfunction, innate immunity, and inflammation contribute to podocyte injury in diabetic kidney disease (DKD), the leading cause of end-stage kidney disease in the U.S. We recently showed that STING activation in glomeruli and podocytes in DKD is associated with increased apoptotic and autophagic podocyte death. TFAM deficiency is linked to mtDNA leakage and STING activation. TFAM facilitates autophagic mtDNA clearance by binding LC3 and directing mtDNA to lysosomes, suggesting impaired mtDNA autophagy may underlie disease. Damaged mitochondria are removed via PINK1/PARK2 or receptor-mediated (BNIP3) mitophagy. We hypothesize that TFAM deficiency drives DKD progression through suppressed mitophagy and autophagy, leading to mtDNA buildup and STING-mediated injury. Methods: In vitro, human podocytes with knockdown of STING (shSTING), TFAM (shTFAM) and scrambled control (shCTRL) were used to evaluate mitophagy using immunofluorescence approach and levels of cytosolic mtDNA using qRT-PCR. In vivo, we generated mice with podocyte specific Tfam deletion (pTFAMfl/fl) and induced diabetes using streptozotocin (STZ, 40 mg/kg). Diabetic db/db mice with TFAM overexpression were generated. Mice were characterized using urinary albumin-to-creatinine ratio (ACR), histological and serum analyses. scRNA-seq analysis of the kidneys was performed using fixation and barcoding kits by Parse Biosciences. Two-tailed t-test or One-Way ANOVA followed by Tukey’s post-test were used to detect statistical changes. Results: Our data show reduced BNIP3 expression in both clinical and experimental DKD, along with decreased TFAM and PINK1/PARK2 levels. In shTFAM podocytes, we observed reduced PINK1 phosphorylation, lower BNIP3, impaired mitophagy, decreased LC3B and p62, and increased apoptosis. STZ-induced diabetes led to more severe DKD in pTfamfl/fl mice, with mtDNA accumulation, elevated glomerular STING phosphorylation, and reduced PINK1 activity. scRNA-seq revealed decreased PINK1/PRKN, BNIP3, and autophagy gene expression in pTfamfl/fl podocytes. Conversely, TFAM overexpression in db/db mice improved DKD outcomes. Conclusion: In conclusion, these findings highlight critical role of TFAM in DKD progression. Targeting the TFAM/PINK1/BNIP3/STING pathway may lead to new treatment options for DKD. Funding: Private Foundation Support