Sex Differences in Renal Mitochondrial Respiration and H ₂ O ₂ Emission in Young Dahl Salt-Sensitive Rats

Introduction: Identifying andinvestigating sex differences in physiological processes and understanding the underlying regulatory mechanisms is crucial for personalized medicine. Oxidative stress is implicated in various animal models of human diseases including salt-sensitive (SS) hypertension as depicted in the Dahl SS rat model fed a high salt (HS) diet. The kidney is a key organ for blood pressure regulation and accurate measurements of renal mitochondrial H 2 O 2 emission (production – scavenging) is essential for understanding renal oxidative stress implicated in SS hypertension. In this study, we report sex differences in mitochondrial H 2 O 2 emission and the associated respiratory function (O 2 consumption) from the kidney cortex (Cx) and outer medulla (OM) of young Dahl SS rats fed a low salt (LS; 0.4% NaCl) diet. Methods: Freshly isolated mitochondria from the kidney Cx and OM of Dahl SS rats (male and female; 6-7 weeks old; fed a LS diet) were obtained by differential centrifugation method. Mitochondrial H 2 O 2 emission mediated by the complex II substrate succinate (state 2), ADP (state 3), and the uncoupler FCCP (state 5) was measured with a spectrofluorometer using amplex red (AR) and horseradish peroxidase (HRP) in the respiration buffer resulting in resorufin fluorescence with the non-H 2 O 2 -mediated resorufin fluorescence inhibited by PMSF (phenylmethylsulphonyl fluoride), a serine protease inhibitor. In addition, mitochondrial O 2 consumption was measured with the complex I-linked substrates pyruvate+malate (PM) and glutamate+malate (GM) and complex II-linked substrate succinate using an Oroboros Oxygraph-2k instrument. Results: Both kidney Cx and OM mitochondria exhibited HRP-independent (non-H 2 O 2 -mediated) resorufin fluorescence, albeit significantly higher in Cx samples. PMSF effectively inhibited this non-H 2 O 2 -mediated resorufin fluorescence. H 2 O 2 emission was higher in OM mitochondria in all respiratory states compared to Cx mitochondria in both sexes. Male rats were found to display higher rates of both succinate-induced (state 2) and ADP-inhibitable (state 3) H 2 O 2 emission in Cx mitochondria. In contrast, female rats exhibited higher rates of ADP-inhibitable (state 3) H 2 O 2 emission in OM mitochondria, with a trend towards increasing rates of succinate-induced (state 2) H 2 O 2 emission. Consistent with H 2 O 2 emission, male rats showed significantly higher respiration in Cx mitochondria as compared to female rats with different substrates in different respiratory states. Conclusions: This study provided novel quantitative data contributing to a deeper understanding of sex differences in the kidney Cx and OM mitochondrial respiration and H 2 O 2 emission of young Dahl SS rats. In addition, we found that careful consideration is necessary when measuring H 2 O 2 emission in renal Cx and OM mitochondria using the AR+HRP assay. The higher H 2 O 2 emission observed in the OM mitochondria of Dahl SS rats suggests its potential susceptibility to oxidative stress. The sex specific variations of mitochondrial respiration align with the known sex differences in Na + transporter distribution in the renal Cx and OM, suggesting a link between O 2 utilization, energy production, and Na + transport dependent on sex. NIH R01 HL151587 NIH P01 HL149620 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.