Sex differences in renal mitochondrial function of young Sprague Dawley rats

Abstract

Introduction Sex differences are implicated in many cardiovascular and renal pathologies; for instance, premenopausal females are typically less prone to renovascular damage than males. However, although mitochondrial bioenergetics per se is a well-known factor that can mediate the progression of the renal diseases, it is not known if there are sex-related dissimilarities in the performance of renal mitochondria prior to the development of any disease. The goal of this study was to test the differences in renal cortical and medullary mitochondrial function in healthy male versus female rats. Methods . Mitochondria were isolated from the kidneys collected from male and female Sprague Dawley (SD) rats procured from Charles River labs at 11 weeks of age. Mitochondrial membrane potential, superoxide and H2O2 levels were measured with luminescent or fluorescent dyes (TMRM, MCLA and Amplex Red) in mitochondria isolated from renal cortex and medulla. To measure the oxygen consumption rate (OCR) and mitochondrial calcium uptake, seahorse assay and spectrofluorimetry with CaGreen dye, respectively, were performed on isolated mitochondria. Lipid peroxide radical formation was detected using electron spin resonance spectroscopy (ESR) with in vivo spin trapping. The SOD activity and total antioxidant capacity were measured using commercially available kits. In order to analyze the expression levels of various mitochondria-related proteins, Western blot analysis was performed on snap-frozen isolated renal mitochondria. Results . Kidneys from SD male (SDM) and female rats (SDF) were divided into cortex (SDMC, FC) and medulla (SDMM, FM). We report higher membrane potential in SDFM compared to SDMM (p<0.001). H2O2 levels were elevated in both the SDFC and SDFM mitochondria compared to SDM (p<0.01), and GPX4 level was significantly increased the SDFM samples. Interestingly, superoxide production was increased in the medulla compared to the cortex for both SDM and SDF, while SOD2 expression was similar. Total SOD activity was increased in SDFC compared to all other groups (p<0.01). ESR showed similar lipid peroxide radical levels in all groups. Antioxidant capacity was reduced in SDFM tissues compared to other groups (p<0.05). Female mitochondria exhibited decreased OCR compared to males, and all OCR parameters were lower in medullary vs cortical mitochondria, independent of sex. Interestingly, calcium uptake was more active in the medulla vs cortex for both males and females, whereas the mitochondrial permeability transition pore (mPTP) opening was recorded earlier in females than males. Conclusions . We report differences in mitochondrial function in the cortex and medulla of young healthy male and female rats, primarily, in their ability to handle reactive oxygen species. The observed sex-related dissimilarities open new avenues of research aimed at establishing the mechanisms that may affect the predisposition of males and females to kidney disease development later in life.