Nowak Grazyna, Takacsova-Bakajsova Diana, Megyesi Judit
Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, Arkansas; and
Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, Arkansas; and.
Am J Physiol Renal Physiol. 2017 Jan 1;312(1):F109-F120. doi: 10.1152/ajprenal.00115.2016. Epub 2016 Oct 19.
Previously, we documented that activation of protein kinase C-ε (PKC-ε) mediates mitochondrial dysfunction in cultured renal proximal tubule cells (RPTC). This study tested whether deletion of PKC-ε decreases dysfunction of renal cortical mitochondria and improves kidney function after renal ischemia. PKC-ε levels in mitochondria of ischemic kidneys increased 24 h after ischemia. Complex I- and complex II-coupled state 3 respirations were reduced 44 and 27%, respectively, in wild-type (WT) but unchanged and increased in PKC-ε-deficient (KO) mice after ischemia. Respiratory control ratio coupled to glutamate/malate oxidation decreased 50% in WT but not in KO mice. Activities of complexes I, III, and IV were decreased 59, 89, and 61%, respectively, in WT but not in KO ischemic kidneys. Proteomics revealed increases in levels of ATP synthase (α-subunit), complexes I and III, cytochrome oxidase, α-ketoglutarate dehydrogenase, and thioredoxin-dependent peroxide reductase after ischemia in KO but not in WT animals. PKC-ε deletion prevented ischemia-induced increases in oxidant production. Plasma creatinine levels increased 12-fold in WT and 3-fold in KO ischemic mice. PKC-ε deletion reduced tubular necrosis, brush border loss, and distal segment damage in ischemic kidneys. PKC-ε activation in hypoxic RPTC in primary culture exacerbated, whereas PKC-ε inhibition reduced, decreases in: 1) complex I- and complex II-coupled state 3 respirations and 2) activities of complexes I, III, and IV. We conclude that PKC-ε activation mediates 1) dysfunction of complexes I and III of the respiratory chain, 2) oxidant production, 3) morphological damage to the kidney, and 4) decreases in renal functions after ischemia.
此前,我们记录到蛋白激酶C-ε(PKC-ε)的激活介导了培养的肾近端小管细胞(RPTC)中的线粒体功能障碍。本研究检测了PKC-ε基因敲除是否能减轻肾缺血后肾皮质线粒体功能障碍并改善肾功能。缺血后24小时,缺血肾脏线粒体中的PKC-ε水平升高。野生型(WT)小鼠缺血后,复合体I和复合体II偶联的状态3呼吸分别降低了44%和27%,而PKC-ε基因敲除(KO)小鼠缺血后该指标未发生变化且有所升高。与谷氨酸/苹果酸氧化偶联的呼吸控制率在WT小鼠中降低了50%,而在KO小鼠中未降低。WT小鼠缺血肾脏中复合体I、III和IV的活性分别降低了59%、89%和61%,而KO小鼠缺血肾脏中这些复合体的活性未降低。蛋白质组学分析显示,缺血后KO小鼠而非WT小鼠体内ATP合酶(α亚基)、复合体I和III、细胞色素氧化酶、α-酮戊二酸脱氢酶以及硫氧还蛋白依赖性过氧化物还原酶的水平升高。PKC-ε基因敲除可防止缺血诱导的氧化剂生成增加。WT缺血小鼠的血浆肌酐水平升高了12倍,KO缺血小鼠升高了3倍。PKC-ε基因敲除减少了缺血肾脏中的肾小管坏死、刷状缘丧失和远端节段损伤。在原代培养的缺氧RPTC中,PKC-ε的激活加剧了,而PKC-ε的抑制减轻了以下指标的降低:1)复合体I和复合体II偶联的状态3呼吸;2)复合体I、III和IV的活性。我们得出结论,PKC-ε的激活介导了1)呼吸链复合体I和III的功能障碍;2)氧化剂生成;3)肾脏的形态学损伤;4)缺血后肾功能的降低。