Abstract
Kidney function declines with advancing age and mitochondria have been implicated. In the present study we have examined the integrated function of mitochondria isolated from kidneys of 6- and 24-month-old Fischer 344 rats. OXPHOS (oxidative phosphorylation) of intact mitochondria and cytochrome c oxidase activity in permeabilized mitochondria were determined with polarographic assays. The activities of the ETC (electron transport chain) complexes and the cytochrome content in solubilized mitochondria were measured using spectrophotometric methods. The respiratory complexes were evaluated with blue native gel electrophoresis. Mitochondrial preparations were evaluated by immunoblotting for cytochrome c, Smac/Diablo and VDAC (voltage-dependent anion channel). Mitochondrial morphology was examined by electron microscopy. OXPHOS of mitochondria isolated from 24-month-old animals was decreased 15-25% with complexes I, II, III and IV, and fatty acid substrates. The electron microscopic appearance of mitochondria, the activity of the ETC complexes and the protein abundance of individual complexes and supercomplexes were unchanged. The content of cytochrome c was decreased by 37% in aged mitochondria, as determined by spectrophotometric methods and confirmed with immunoblotting. Polarographic determination of cytochrome c oxidase activity with endogenous cytochrome c demonstrated a 23% reduction in aged mitochondria, which was corrected with the addition of exogenous cytochrome c. Renal mitochondrial OXPHOS decreased with aging in the Fischer 344 rat. Decreased mitochondrial cytochrome c content is a major factor contributing to the OXPHOS defect of mitochondria isolated from kidneys of elderly animals.