Evaluation of mitochondrial function in a model of developmental programming of hypertension associated with transient neonatal oxygen exposure

EVALUATION OF MITOCHONDRIAL FUNCTION IN A MODEL OF DEVELOPMENTAL PROGRAMMING OF HYPERTENSION ASSOCIATED WITH TRANSIENT NEONATAL OXYGEN EXPOSURE Introduction: Rats exposed to oxygen (O2) as newborns suffer complications in adulthood, including: arterial hypertension, vascular dysfunction, nephropenia and indices of oxidative stress. Although the rats are born at term, their organ development is equivalent to that of a preterm fetus, allowing organs of interest such as the kidney to be compared to premature infants. Given that impaired nephrogenesis or reduced nephron numbers has been shown to promote the development of hypertension and mitochondrial dysfunction is associated with increased oxidative stress, we hypothesised that exposure to high oxygen concentrations in the neonatal period would significantly impact the expression and activity of key proteins involved in renal mitochondrial function and lead to an excessive production of reactive oxygen species by the mitochondria.  Methods: Sprague-Dawley rat pups were exposed to 80% O2 (Hyperoxic (H) group; O2 exposed) or 21% O2 (Control (Ctrl) group)  from day 3 to day 10 of life. At 4 and 16 weeks of age, kidneys were rapidly excised and the mitochondria isolated following a standard protocol; with a buffer containing 0.32 M sucrose and differential centrifugations. Expression of mitochondrial proteins was assessed by Western blot, whereas the release of hydrogen peroxide (H202), activities of key citric acid cycle enzymes and mitochondrial swelling were assessed by spectrophotometry. Results are expressed as the means ± SE. Both male and female offspring were studied. Results: In male H rats at 16 weeks of age (n=6), citrate synthase activity (internal standard and measure of relative mitochondrial abundance) was significantly increased (12.4 ± 8.4 vs 4.1 ± 0.5 μmole/mL/min), whereas aconitase activity (sensitive to ROS) was significantly decreased (0.11 ± 0.05 vs 0.20 ± 0.04 μmoles/min/mg mitochondria) and H202 release was significantly increased (7.0 ± 1.3 vs 5.4 ± 0.8 ρmoles/mg mitochondrial protein) compared to the controls (Ctrl, n=6 males and 4 females). The H group (vs Ctrl) also demonstrated a reduction in the expression of peroxiredoxin-3 (Prx3) (H 0.61±0.06 vs. Ctrl 0.78±0.02 relative units, -23%; p<0.05), a protein involved in the elimination of H202 and in the expression of cytochrome C oxidase (Complex IV) (H 1.02±0.04 vs. Ctrl 1.20±0.02 relative units, -15%; p<0.05), a protein in the mitochondrial respiratory chain, whereas the expression of uncoupling protein-2 (UCP2), a protein involved in dissipating the proton gradient, was significantly increased (H 1.05±0.02 vs. Ctrl 0.90±0.03 relative units, +17%; p<0.05). Female H rats (n=6) (vs Ctrl, n=6) at 16 weeks of age demonstrated a significant increase in aconitase activity (0.33±0.03 vs 0.17±0.02 μmoles/min/mg mitochondria), in the expression of ATP synthase β subunit (H 0.73±0.02 vs. Ctrl 0.59±0.02 relative units, +25%; p<0.05) (involved in ATP production) and in the expression of MnSOD (H 0.89±0.02 vs. Ctrl 0.74±0.03 relative units, +20%; p<0.05) (mitochondrial antioxidant involved in scavenging superoxide), whereas Prx3 expression was significantly reduced (H 1.1±0.07 vs. Ctrl 0.85±0.01 relative units, -24%; p<0.05 ).  In male H rats (vs Ctrl) at 4 weeks of age, the expression of Prx3 was significantly increased (H 0.72±0.03 vs. Ctrl 0.56±0.04 relative units, +31%; p<0.05). Female H rats (vs Ctrl) at 4 weeks of age demonstrated a significant increase in the expression of UCP2 (H 1.22±0.05 vs. Ctrl 1.03±0.04 relative units, +18%; p<0.05) and in the expression of MnSOD (H 1.36±0.01 vs. 1.19±0.06 relative units, +14%; p<0.05). Conclusion: The findings of this study demonstrate that transient oxygen exposure in the neonatal rat modifies protein expression, enzymatic activity and leads to indices of mitochondrial dysfunction (increase in ROS) in the adult kidney; these adverse changes in the mitochondria were more pronounced in adult males than in females. Overall, these findings,  suggest that impaired mitochondrial function is associated with and could play a role in the development of arterial hypertension, oxidative stress and cardiovascular disease associated with transient neonatal hyperoxic stress.  Keywords: Mitochondria, Hypertension, Kidneys, Developmental Programming, Oxygen, ROS

Keyphrases: Hypertension, ROS, Developmental programming, Mitochondria, Hypertension, Kidneys, Oxygen