Previously, we showed that oxidant exposure in renal proximal tubular cells (RPTC) induces mitochondrial dysfunction mediated by PKC-epsilon. This study examined the role of ERK1/2 in mitochondrial dysfunction induced by oxidant injury and whether PKC-epsilon mediates its effects on mitochondrial function through the Raf-MEK1/2-ERK1/2 pathway. Sublethal injury produced by tert-butylhydroperoxide (TBHP) resulted in three- to fivefold increase in phosphorylation of ERK1/2 and p38 but not JNK. This was followed by decreases in basal and uncoupled respirations (41%), state 3 respiration and ATP production coupled to complex I (46%), and complex I activity (42%). Oxidant exposure decreased aconitase activity 30% but not pyruvate, alpha-ketoglutarate, and malate dehydrogenase activities. Inhibition of ERK1/2 restored basal and state 3 respirations, DeltaPsi(m), ATP production, and complex I activity but not aconitase activity. In contrast, activation of ERK1/2 by expression of constitutively active MEK1 suppressed basal, uncoupled, and state 3 respirations in noninjured RPTC to the levels observed in TBHP-injured RPTC. MEK1/2 inhibition did not change Akt or p38 phosphorylation, demonstrating that the protective effect of MEK1/2 inhibitor was not due to activation of Akt or inhibition of p38 pathway. Inhibition of PKC-epsilon did not block TBHP-induced ERK1/2 phosphorylation in whole RPTC or in mitochondria. We conclude that 1) oxidant-induced activation of ERK1/2 but not p38 or JNK reduces mitochondrial respiration and ATP production by decreasing complex I activity and substrate oxidation through complex I, 2) citric acid cycle dehydrogenases are not under control of the ERK1/2 pathway in oxidant-injured RPTC, 3) the protective effects of ERK1/2 inhibition are not due to activation of Akt, and 4) ERK1/2 and PKC-epsilon mediate oxidant-induced mitochondrial dysfunction through independent pathways.

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.

We previously reported that mitochondrial function, intracellular ATP levels, and complex I activity are decreased in renal proximal tubular cells (RPTC) after oxidant (tert-butyl hydroperoxide; TBHP)-induced injury. This study examined the hypothesis that succinate supplementation decreases mitochondrial dysfunction, ameliorates energy deficits, and increases viability in TBHP-injured RPTC. Basal and uncoupled respirations in injured RPTC decreased 33 and 35%, respectively, but remained unchanged in injured RPTC supplemented with 10 mM succinate (electron donor to respiratory complex II). State 3 respiration supported by electron donors to complex I decreased 40% in injured RPTC but improved significantly by succinate supplements. The activity of mitochondrial complex I in TBHPinjured RPTC decreased 48%, whereas complex II activity remained unchanged. Succinate supplementation prevented decreases in complex I activity. ATP levels decreased 43% in injured RPTC but were maintained in injured cells supplemented with succinate. Lipid peroxidation increased 19-fold in injured RPTC but only 9-fold in injured cells supplemented with succinate. Exposure of primary cultures of RPTC to TBHP produced 24% cell injury and lysis but no apoptosis. In contrast, no cell lysis was found in RPTC supplemented with succinate. We conclude that mitochondrial dysfunction and energy deficits in oxidant-injured RPTC are ameliorated by succinate, and we propose that succinate supplementation may prove therapeutically valuable. Succinate 1) uses an alternate pathway of mitochondrial energy metabolism, 2) improves activity of complex I and oxidation of substrates through complex I, and 3) decreases oxidative stress and cell lysis in oxidant-injured RPTC.The major source of energy for metabolic processes and active transport in renal proximal tubular cells (RPTC) is oxidative metabolism, and ATP levels in RPTC are maintained almost entirely by oxidative phosphorylation. Therefore, decreases in ATP levels are the initiating factor in the development of both lethal and sublethal RPTC injury, and the degree of ATP decline determines the severity of injury (Bonventre and Weinberg, 2003). RPTC are the major target of ischemic and nephrotoxic injury within the kidney due to their dependence on oxidative phosphorylation and their limited capacity for generating ATP in glycolysis (Bagnasco et al., 1985;Bonventre and Weinberg, 2003). Ischemia and toxicants impair oxidative phosphorylation in RPTC both in vivo and in vitro and result in ATP depletion, leading to the loss of ion homeostasis, opening of the glycine-sensitive plasma membrane death channels/pores, and necrotic cell death (Miller et al., 1994;Dong et al., 1998;Chen et al., 2001;Bonventre and Weinberg, 2003).Structural and functional alterations of mitochondria are important pathogenic factors underlying injury induced by ischemia, hypoxia, and toxicants in many cell types, including RPTC