Metabolic Reprogramming Induced by Renal Tubule-Specific Mitochondrial Citrate Carrier Deletion Mitigates Vancomycin-Induced Acute Tubular Injury by Preserving GPX4

Background: The mitochondrial citrate carrier (CIC) is a mitochondrial transporter mediating the bidirectional transport of citrate across the mitochondrial membrane. CIC is implicated in various human diseases characterized by mitochondrial oxidative damage. Vancomycin and gentamycin-induced acute tubular injury (V+G-ATI) is a severe complication affecting hospitalized patients. The detailed mechanisms underlying V+G-ATI remain unclear, and effective therapeutic interventions are lacking. Methods: We injected 2 daily Vancomycin and Gentamicin, and collected kidneys at 48 hours for wild type and CIC KSP-knock out mice, followed by pathology, proteomics, metabolomics and C13 metabolic flux analysis. HK2 cells were used to elucidate the underlying protective mechanisms of CIC knock-down in tubular cells. Results: The V+G-ATI induced up to 50% of tubular injury. CIC knockout substantially protected kidney tubules from V+G-ATI. Quantitative proteomic and KEGG pathway analysis highlights necroptosis, lysosome, ferroptosis and NF-κB pathway. Specifically, GPX4, a key regulator of ferroptosis, was decreased in V+G-ATI but was significantly upregulated in CIC knockout mice, by both proteomic analysis and Western blot. V+G-ATI significantly altered levels of Acsl4 and Tfrc, additional markers of ferroptosis, and these were significantly ameliorated by Ksp-CIC deletion. In HK2 cells, V+G increases mitochondrial ROS, H2O2, and lipid peroxidation. As CIC KO inhibits citrate transfer from mitochondria to cytosol, it enhances the TCA cycle 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. Either AKG or GSH attenuated V+G-ATI. In HK2 cells, knocking down the second transporters (SLC25A11 Oxoglutarate or SLC25A18 Glutamate) attenuated the protective effect of CIC knock-down, suggesting that the efflux of these intermediates is required. Conclusion: Metabolic reprogramming by CIC KO protected against V+G-ATI and attenuated ferroptosis through enhancement of endogenous GSH pathway, which requires the efflux of ketoglutarate / glutamate from mitochondria. Funding: NIDDK Support