Ceramides as Mediators of Mitochondrial Dysfunction Driving Kidney Injury

Abstract

Objective. Perturbations in proximal tubule mitochondrial function and lipid metabolism potentiate acute kidney injury and failed repair. Sphingolipids such as ceramides are recognized as lipotoxic drivers of metabolic dysfunction and cell death. We hypothesized that ceramides drive mitochondrial dysfunction and tubular insult and that lowering ceramides would prevent acute kidney injury in a mouse model of bilateral ischemia reperfusion injury (IRI). Methods. We examined acute effects of ceramide treatment on primary mouse proximal tubule epithelial cell (PTEC) mitochondrial oxygen consumption and mitochondrial supercomplex assembly by Seahorse respirometry and blue native PAGE, respectively. In vivo, we profiled accumulation of ceramides via HPLC-MS/MS in kidney cortex tissue of mice challenged with warm bilateral IRI. We lowered whole-body ceramides via Degs1 inducible global knockout ( Degs1iGKO) in male C57Bl/6J mice prior to challenge with IRI. Clinical, transcriptional, and histopathological renal outcomes were compared to sham-operated and Degs1fl/fl controls 24 hours after kidney reperfusion. Results. Treatment of PTECs with 5-50uM C2-ceramide resulted in acute and dose-dependent decreases in mitochondrial ATP production rates (15-65% decrease from baseline, respectively, p<0.05), as well as disruption of electron transport chain supercomplex structures (i.e., III2+IV and I+III2+IV). In mice, IRI led to a 25% increase in total cortex ceramides (sham: 995±110 vs IRI: 1239±185 pmol lipid/mg protein, p<0.05). Lipid species within the de novo ceramide synthesis pathway were elevated as early as 5 minutes after IRI (e.g., 34% increase in Cer(d18:0/16:0), sham: 2.38±0.27 vs 5 minutes: 3.20±0.81 pmol lipid/mg protein, p<0.05) and continued to accumulate throughout the progression of the AKI up to 24 hours after reperfusion (e.g., 122% increase in Cer(d18:0/16:0), sham: 2.38±0.27 vs 24 hours: 5.29±1.71 pmol lipid/mg protein, p<0.001). Cortex ceramides, particularly deleterious long-chain (e.g., Cer(d18:1/16:0)) species, correlated positively with kidney injury biomarkers (plasma creatinine, Pearson r=0.57; BUN, r=0.74; and cortex expression of Lcn2, r=0.60 , and Havcr1, r=0.62 , p<0.05). Degs1 global knockout decreased cortex ceramide levels by 56% (fl/fl: 995±110 vs KO: 435±174 pmol lipid/mg protein, p<0.001). Degs1iGKO animals had improved cortex histopathology, blood urea nitrogen (fl/fl: 81.02±2.13 vs KO: 49.82±6.34 mg/dL, p <0.001), plasma creatinine (fl/fl: 1.28±0.4 vs KO: 0.32±0.11 mg/dL, p<0.001), and cortex Lcn2 expression (fl/fl: 36.36±21.50 vs KO: 5.31±4.61, p<0.01) following IRI. Conclusions. Renal ceramides are implicated as drivers of kidney injury and mitochondrial dysfunction. Global Degs1 knockout prevented AKI induced by ischemia reperfusion injury. Thus, ceramide-lowering interventions may be an effective strategy to prevent AKI. This work was supported by funding from the National Institutes of Health [R01KD130296 (SAS, WLH), R01DK131609 (SAS), U01CA272529 (SAS), R44DK116450 (SAS), R01DK112826 (WLH), T32DK091317 (RJN), F31DK134088 (RJN)]. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.