Rationale: Although sex differences in mitochondrial respiration have been documented in the heart, liver, and kidney, their consequences for cellular metabolism and substrate utilization remain poorly defined despite clear relevance in metabolic disease risk. Mitochondrial respiration and H 2 O 2 emission are key determinants of bioenergetic efficiency and oxidative stress, yet systematic sex-based comparisons between males and females are lacking. Because mitochondrial dysfunction contributes to heart, kidney, and liver disease and exacerbates ischemia-reperfusion injury in organ transplantation, identifying sex- and substrate-dependent differences is essential for understanding the underlying mechanisms. This study therefore examines sex-specific and substrate-dependent mitochondrial respiration and H 2 O 2 emission in the kidney cortex, heart, and liver to advance mechanistic insight and inform precision therapeutic strategies. Methods: Mitochondria were isolated from the kidney cortex, heart, and liver of adult Sprague-Dawley rats using the differential centrifugation method (n=5 per group; both sexes). To target different metabolic pathways of the TCA cycle and to assess the dependency of the electron transport chain (ETC) complexes, four substrate combinations, namely, pyruvate+malate (PM), glutamate+malate (GM), succinate (Suc), and palmitoyl-L-carnitine+malate (PCM), were used. A saturated dose of ADP was added to the respiring mitochondria to examine how various substrates affect mitochondrial oxygen consumption rate (OCR) and H 2 O 2 emission during oxidative phosphorylation. Mitochondrial OCR was assessed with a dual chamber Oroboros Oxygraph-2k instrument. H 2 O 2 emission was measured spectrofluorometrically with Amplex Red (AR) and Horseradish Peroxidase (HRP) in the respiration buffer, producing resorufin fluorescence. The non-H 2 O 2 -related resorufin signal was inhibited by phenylmethylsulfonyl fluoride (PMSF), a serine protease inhibitor. Results: Across all tissues, mitochondrial respiration displayed sex-specific differences. In the kidney cortex, males exhibited higher PM- and Suc- supported respiration than females, whereas no sex differences were observed in the heart. In the liver, females showed higher respiratory rates across all substrates. Similar sex- and substrate- dependent patterns were seen in H 2 O 2 emission. PMSF effectively inhibited the non-H 2 O 2 -mediated resorufin fluorescence in the cortex and liver, confirming assay specificity. Conclusion: These findings demonstrate that sex-dependent mitochondrial phenotypes are organ-specific and reflect differences in substrate utilization and metabolic regulation. Higher oxidative capacity and redox activity in male kidney mitochondria, contrasted with greater respiratory flux in female liver mitochondria, suggest that sex influences TCA cycle activity and redox balance in a tissue-dependent manner. Such distinctions may contribute to sex-specific vulnerability to metabolic, renal, and hepatic injury. This abstract was presented at the American Physiology Summit 2026 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.
Dave et al. (Fri,) studied this question.