Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44106, USA.
Biochem J. 2010 Mar 15;427(1):105-12. doi: 10.1042/BJ20091373.
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.
肾功能随年龄增长而下降，线粒体也与之有关。本研究检测了来自 6 个月和 24 个月龄 Fischer 344 大鼠肾脏的分离线粒体的综合功能。使用极谱法测定完整线粒体的 OXPHOS（氧化磷酸化）和通透化线粒体中的细胞色素 c 氧化酶活性。使用分光光度法测定可溶性线粒体中 ETC（电子传递链）复合物的活性和细胞色素含量。使用蓝色非变性凝胶电泳评估呼吸复合物。通过免疫印迹法评估线粒体制剂中的细胞色素 c、Smac/ Diablo 和 VDAC（电压依赖性阴离子通道）。通过电子显微镜检查线粒体的形态。来自 24 个月龄动物的线粒体的 OXPHOS 与复合物 I、II、III 和 IV 以及脂肪酸底物降低了 15-25%。电子显微镜外观、ETC 复合物的活性以及单个复合物和超复合物的蛋白丰度均未改变。通过分光光度法测定和免疫印迹法证实，老年线粒体中细胞色素 c 的含量减少了 37%。用内源性细胞色素 c 进行的细胞色素 c 氧化酶活性的极谱测定表明，老年线粒体中的活性降低了 23%，用外源性细胞色素 c 的添加可纠正这种情况。Fischer 344 大鼠的肾脏线粒体 OXPHOS 随年龄增长而下降。线粒体细胞色素 c 含量降低是老年动物肾脏分离线粒体 OXPHOS 缺陷的主要因素。
