Nowak Grazyna, Bakajsova Diana, Clifton Ginger L
Dept. of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
Am J Physiol Renal Physiol. 2004 Feb;286(2):F307-16. doi: 10.1152/ajprenal.00275.2003. Epub 2003 Oct 21.
The aim of this study was to determine whether protein kinase C-epsilon (PKC-epsilon) is involved in the repair of mitochondrial function and/or active Na+ transport after oxidant injury in renal proximal tubular cells (RPTC). Sublethal injury was produced in primary cultures of RPTC using tert-butylhydroperoxide (TBHP), and the recovery of functions was examined. PKC-epsilon was activated three- to fivefold after injury. Active PKC-epsilon translocated to the mitochondria. Basal oxygen consumption (Qo2), uncoupled Qo2, and ATP production decreased 58, 60, and 41%, respectively, at 4 h and recovered by day 4 after injury. At 4 h, complex I-coupled respiration decreased 50% but complex II- and IV-coupled respirations were unchanged. Inhibition of PKC-epsilon translocation using a peptide selective inhibitor, PKC-epsilonV1-2, reduced decreases in basal and uncoupled Qo2 values and increased complex I-linked respiration in TBHP-injured RPTC at 4 h of recovery. Furthermore, PKC-epsilonV1-2 prevented decreases in ATP production in injured RPTC. Na+-K+-ATPase activity and ouabain-sensitive 86Rb+ uptake were decreased by 60 and 53%, respectively, at 4 h of recovery. Inhibition of PKC-epsilon activation prevented a decline in Na+-K+-ATPase activity and reduced decreases in ouabain-sensitive 86Rb+ uptake. We conclude that during early repair after oxidant injury in RPTC 1) PKC-epsilon is activated and translocated to mitochondria; 2) PKC-epsilon activation decreases mitochondrial respiration, electron transport rate, and ATP production by reducing complex I-linked respiration; and 3) PKC-epsilon mediates decreases in active Na+ transport and Na+-K+-ATPase activity. These data show that PKC-epsilon activation after oxidant injury in RPTC is involved in the decreases in mitochondrial function and active Na+ transport and that inhibition of PKC-epsilon activation promotes the repair of these functions.
本研究的目的是确定蛋白激酶C-ε（PKC-ε）是否参与肾近端小管细胞（RPTC）氧化损伤后线粒体功能的修复和/或活性Na⁺转运。使用叔丁基过氧化氢（TBHP）对RPTC原代培养物造成亚致死性损伤，并检测功能的恢复情况。损伤后PKC-ε被激活3至5倍。活性PKC-ε转位至线粒体。基础氧消耗（Qo2）、解偶联Qo2和ATP生成在损伤后4小时分别下降了58%、60%和41%，并在损伤后第4天恢复。在4小时时，复合体I偶联呼吸下降了50%，但复合体II和IV偶联呼吸未改变。使用肽选择性抑制剂PKC-εV1-2抑制PKC-ε转位，可减少恢复4小时时TBHP损伤的RPTC中基础和解偶联Qo2值的下降，并增加复合体I相关呼吸。此外，PKC-εV1-2可防止损伤的RPTC中ATP生成的下降。在恢复4小时时，Na⁺-K⁺-ATP酶活性和哇巴因敏感的⁸⁶Rb⁺摄取分别下降了60%和53%。抑制PKC-ε激活可防止Na⁺-K⁺-ATP酶活性下降，并减少哇巴因敏感的⁸⁶Rb⁺摄取的下降。我们得出结论，在RPTC氧化损伤后的早期修复过程中：1）PKC-ε被激活并转位至线粒体；2）PKC-ε激活通过降低复合体I相关呼吸来减少线粒体呼吸、电子传递速率和ATP生成；3）PKC-ε介导活性Na⁺转运和Na⁺-K⁺-ATP酶活性的下降。这些数据表明，RPTC氧化损伤后PKC-ε的激活与线粒体功能和活性Na⁺转运的下降有关，并且抑制PKC-ε激活可促进这些功能的修复。
