Univ. of Arkansas for Medical Sciences, Dept. of Pharmaceutical Sciences, 4301 West Markham St., Little Rock, AR 72205, USA.
Am J Physiol Renal Physiol. 2012 Aug 15;303(4):F515-26. doi: 10.1152/ajprenal.00072.2012. Epub 2012 Jun 6.
We demonstrated that nonselective PKC activation promotes mitochondrial function in renal proximal tubular cells (RPTC) following toxicant injury. However, the specific PKC isozyme mediating this effect is unknown. This study investigated the role of PKC-α in the recovery of mitochondrial functions in oxidant-injured RPTC. Wild-type PKC-α (wtPKC-α) and inactive PKC-α mutants were overexpressed in RPTC to selectively increase or block PKC-α activation. Oxidant (tert-butyl hydroperoxidel; TBHP) exposure activated PKC-α in RPTC but decreased PKC-α levels in mitochondria following treatment. Uncoupled and state 3 respirations and activities of complexes I and IV in TBHP-injured cells decreased to 55, 44, 49, and 65% of controls, respectively. F(0)F(1)-ATPase activity and ATP content in injured RPTC decreased to 59 and 60% of controls, respectively. Oxidant exposure increased reactive oxygen species (ROS) production by 210% and induced mitochondrial fragmentation and 52% RPTC lysis. Overexpressing wtPKC-α did not block TBHP-induced ROS production but improved respiration and complex I activity, restored complex IV and F(0)F(1)-ATPase activities, promoted recovery of ATP content, blocked mitochondrial fragmentation, and reduced RPTC lysis to 14%. In contrast, inhibiting PKC-α 1) induced mitochondrial hyperpolarization and fragmentation; 2) blocked increases in ROS production; 3) prevented recovery of respiratory complexes and F(0)F(1)-ATPase activities, respiration, and ATP content; and 4) exacerbated TBHP-induced RPTC lysis. We conclude that 1) activation of PKC-α prevents mitochondrial hyperpolarization and fragmentation, decreases cell death, and promotes recovery of mitochondrial respiration and ATP content following oxidant injury in RPTC; and 2) respiratory complexes I and IV and F(0)F(1)-ATPase are targets of active PKC-α.
我们证明，非选择性蛋白激酶 C（PKC）激活可促进肾近端小管细胞（RPTC）在毒物损伤后的线粒体功能。然而，介导这种效应的特定 PKC 同工酶尚不清楚。本研究探讨了 PKC-α 在氧化应激损伤的 RPTC 中线粒体功能恢复中的作用。野生型 PKC-α（wtPKC-α）和失活的 PKC-α 突变体在 RPTC 中过表达，以选择性地增加或阻断 PKC-α 激活。氧化应激（叔丁基过氧氢；TBHP）暴露激活了 RPTC 中的 PKC-α，但处理后线粒体中的 PKC-α 水平下降。TBHP 损伤细胞的解偶联和状态 3 呼吸以及复合物 I 和 IV 的活性分别下降至对照的 55%、44%、49%和 65%。损伤的 RPTC 中的 F(0)F(1)-ATP 酶活性和 ATP 含量分别下降至对照的 59%和 60%。氧化应激使活性氧（ROS）的产生增加了 210%，并诱导线粒体碎片化和 52%的 RPTC 裂解。过表达 wtPKC-α 并未阻断 TBHP 诱导的 ROS 产生，但改善了呼吸和复合物 I 活性，恢复了复合物 IV 和 F(0)F(1)-ATP 酶的活性，促进了 ATP 含量的恢复，阻断了线粒体碎片化，并将 RPTC 裂解减少至 14%。相比之下，抑制 PKC-α：1）诱导线粒体超极化和碎片化；2）阻断 ROS 产生的增加；3）阻止呼吸复合物和 F(0)F(1)-ATP 酶、呼吸和 ATP 含量的恢复；4）加剧 TBHP 诱导的 RPTC 裂解。我们得出结论：1）PKC-α 的激活可防止线粒体超极化和碎片化，减少细胞死亡，并促进 RPTC 中氧化应激损伤后线粒体呼吸和 ATP 含量的恢复；2）呼吸复合物 I 和 IV 以及 F(0)F(1)-ATP 酶是活性 PKC-α 的靶标。
