Metabolic Reprogramming by Deletion of Mitochondria Citrate Carrier in Kidney Tubules Ameliorated Vancomycin-induced Acute Tubular Injury Through Upregulation of GPX4

The mitochondrial citrate carrier (CIC, also known as the citrate carrier SLC25A1), is a l mitochondrial transporter mediating the bidirectional transport of citrate across mitochondrial membrane. Vancomycin and gentamycin-induced acute tubular injury (V+G-ATI) is a severe complication affecting hospitalized or critically ill patients, increasing the risk of long-term renal failure and the need for hemodialysis. However, the exact pathophysiological mechanisms underlying V+G-ATI remain poorly understood. We hypothesize: that metabolic reprogramming by inhibiting mitochondrial citrate transport will ameliorate AKI induced by VA, offering a potential pathway for therapeutic intervention. Method and Results: Adeno-associated virus (AAV) serotype 9 vector encoding Cre recombinase driven by KSP promoter (AAV9-Ksp-Cre) was injected intraperitoneally in 4 to 6-month-old CIC flox/flox mice to induce CIC deletion in kidney tubules. Approximately 2 weeks after the CIC deletion (KO), we induced ATI by daily intraperitoneal injections of vancomycin + gentamycin for two consecutive days. Kidney tissues were harvested at 48 hours for histological and proteomics analysis. Shotgun proteomic profiling was conducted using the label-free Data Independent Analysis method. The in vitro studies using HK-2 (human proximal tubular epithelial cell line) were performed to examine oxidative stress by MitoSOX Red, DCFDA, and BODIPY lipid peroxidation sensors. The V+G induced extensive ATI, characterized by vacuolation, attenuation of tubular epithelial cells, tubular dilation, and/or cell death. Quantitative pathological analysis revealed that ~50% of tubules were injured in wild-type mice 48 hours after V+G. Tubular-specific CIC deletion significantly protected kidney tubules from V+G-induced ATI. To obtain mechanistic insight, we performed quantitative proteomics. KEGG pathway analysis highlights necroptosis, lysosome, ferroptosis, and NF-KB pathways in V+G-induced ATI. Many of these were attenuated by CIC KO, particularly the ferroptosis pathway. Specifically, GPX4, a key regulator of ferroptosis, was decreased in V+G-ATI but was significantly upregulated in CIC knockout, shown by proteomic and confirmed by immunoblot. V+G-ATI significantly altered levels of Acsl4 and Tfrc (markers of ferroptosis), and these were significantly ameliorated by Ksp-CIC deletion. In HK2 cells, V+G increases mitochondrial ROS (MitoSOX), H2O2 (DCFDA), and lipid peroxidation. Interestingly, Iron blue stain showed an accumulation of iron in the injured tubules of V+G-ATI in WT mice, and these were attenuated in CIC KO, supporting the protective role of CIC in mitigating ferroptosis-associated damage. As CIC KO inhibits citrate transfer from mitochondria to cytosol, it is expected to enhance the TCA cycle, increasing several TCA intermediates, including alpha-ketoglutarate (AKG). AKG is well known to exert an anti-oxidative effect, such as by enhancing the endogenous glutathione peroxidase pathway. To examine the roles of these downstream metabolic pathways, we administered AKG or glutathione (GSH), simultaneously with V+G. Our data showed that either AKG or GSH attenuated V+G-ATI, further highlighting the critical metabolic interplay in drug-induced AKI. Conclusion: Metabolic reprogramming by tubular-specific CIC knockout mitigated ferroptosis by upregulating GPX4, which reduced mitochondrial ROS, lipid peroxidation, iron accumulation, hence protecting against V+G-induced kidney tubular ferroptosis/injury. Administration of AKG or GSH recapitulated the nephroprotective effects of CIC KO. Our findings suggest the potential of metabolic treatment to prevent antibiotics-induced AKI. NIH R01DK133118 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.