Deletion of protein kinase C-ε attenuates mitochondrial dysfunction and ameliorates ischemic renal injury.

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.

Protein kinase C-α interaction with F0F1-ATPase promotes F0F1-ATPase activity and reduces energy deficits in injured renal cells.

We showed previously that active PKC-α maintains F0F1-ATPase activity, whereas inactive PKC-α mutant (dnPKC-α) blocks recovery of F0F1-ATPase activity after injury in renal proximal tubules (RPTC). This study tested whether mitochondrial PKC-α interacts with and phosphorylates F0F1-ATPase. Wild-type PKC-α (wtPKC-α) and dnPKC-α were overexpressed in RPTC to increase their mitochondrial levels, and RPTC were exposed to oxidant or hypoxia. Mitochondrial levels of the γ-subunit, but not the α- and ß-subunits, were decreased by injury, an event associated with 54% inhibition of F0F1-ATPase activity. Overexpressing wtPKC-α blocked decreases in γ-subunit levels, maintained F0F1-ATPase activity, and improved ATP levels after injury. Deletion of PKC-α decreased levels of α-, ß-, and γ-subunits, decreased F0F1-ATPase activity, and hindered the recovery of ATP content after RPTC injury. Mitochondrial PKC-α co-immunoprecipitated with α-, ß-, and γ-subunits of F0F1-ATPase. The association of PKC-α with these subunits decreased in injured RPTC overexpressing dnPKC-α. Immunocapture of F0F1-ATPase and immunoblotting with phospho(Ser) PKC substrate antibody identified phosphorylation of serine in the PKC consensus site on the α- or ß- and γ-subunits. Overexpressing wtPKC-α increased phosphorylation and protein levels, whereas deletion of PKC-α decreased protein levels of α-, ß-, and γ-subunits of F0F1-ATPase in RPTC. Phosphoproteomics revealed phosphorylation of Ser(146) on the γ subunit in response to wtPKC-α overexpression. We concluded that active PKC-α 1) prevents injury-induced decreases in levels of γ subunit of F0F1-ATPase, 2) interacts with α-, ß-, and γ-subunits leading to increases in their phosphorylation, and 3) promotes the recovery of F0F1-ATPase activity and ATP content after injury in RPTC.

Assessment of mitochondrial functions and cell viability in renal cells overexpressing protein kinase C isozymes.

The protein kinase C (PKC) family of isozymes is involved in numerous physiological and pathological processes. Our recent data demonstrate that PKC regulates mitochondrial function and cellular energy status. Numerous reports demonstrated that the activation of PKC-a and PKC-ε improves mitochondrial function in the ischemic heart and mediates cardioprotection. In contrast, we have demonstrated that PKC-α and PKC-ε are involved in nephrotoxicant-induced mitochondrial dysfunction and cell death in kidney cells. Therefore, the goal of this study was to develop an in vitro model of renal cells maintaining active mitochondrial functions in which PKC isozymes could be selectively activated or inhibited to determine their role in regulation of oxidative phosphorylation and cell survival. Primary cultures of renal proximal tubular cells (RPTC) were cultured in improved conditions resulting in mitochondrial respiration and activity of mitochondrial enzymes similar to those in RPTC in vivo. Because traditional transfection techniques (Lipofectamine, electroporation) are inefficient in primary cultures and have adverse effects on mitochondrial function, PKC-ε mutant cDNAs were delivered to RPTC through adenoviral vectors. This approach results in transfection of over 90% cultured RPTC. Here, we present methods for assessing the role of PKC-ε in: 1. regulation of mitochondrial morphology and functions associated with ATP synthesis, and 2. survival of RPTC in primary culture. PKC-ε is activated by overexpressing the constitutively active PKC-ε mutant. PKC-ε is inhibited by overexpressing the inactive mutant of PKC-ε. Mitochondrial function is assessed by examining respiration, integrity of the respiratory chain, activities of respiratory complexes and F0F1-ATPase, ATP production rate, and ATP content. Respiration is assessed in digitonin-permeabilized RPTC as state 3 (maximum respiration in the presence of excess substrates and ADP) and uncoupled respirations. Integrity of the respiratory chain is assessed by measuring activities of all four complexes of the respiratory chain in isolated mitochondria. Capacity of oxidative phosphorylation is evaluated by measuring the mitochondrial membrane potential, ATP production rate, and activity of F0F1-ATPase. Energy status of RPTC is assessed by determining the intracellular ATP content. Mitochondrial morphology in live cells is visualized using MitoTracker Red 580, a fluorescent dye that specifically accumulates in mitochondria, and live monolayers are examined under a fluorescent microscope. RPTC viability is assessed using annexin V/propidium iodide staining followed by flow cytometry to determine apoptosis and oncosis. These methods allow for a selective activation/inhibition of individual PKC isozymes to assess their role in cellular functions in a variety of physiological and pathological conditions that can be reproduced in in vitro.

Protein kinase C-α activation promotes recovery of mitochondrial function and cell survival following oxidant injury in renal cells.

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.

γ-Tocotrienol protects against mitochondrial dysfunction and renal cell death.

Oxidative stress is a major mechanism of a variety of renal diseases. Tocopherols and tocotrienols are well known antioxidants. This study aimed to determine whether γ-tocotrienol (GT3) protects against mitochondrial dysfunction and renal proximal tubular cell (RPTC) injury caused by oxidants. Primary cultures of RPTCs were injured by using tert-butyl hydroperoxide (TBHP) in the absence and presence of GT3 or α-tocopherol (AT). Reactive oxygen species (ROS) production increased 300% in TBHP-injured RPTCs. State 3 respiration, oligomycin-sensitive respiration, and respiratory control ratio (RCR) decreased 50, 63, and 47%, respectively. The number of RPTCs with polarized mitochondria decreased 54%. F0F1-ATPase activity and ATP content decreased 31 and 65%, respectively. Cell lysis increased from 3% in controls to 26 and 52% at 4 and 24 h, respectively, after TBHP exposure. GT3 blocked ROS production, ameliorated decreases in state 3 and oligomycin-sensitive respirations and F0F1-ATPase activity, and maintained RCR and mitochondrial membrane potential (ΔΨ(m)) in injured RPTCs. GT3 maintained ATP content, blocked RPTC lysis at 4 h, and reduced it to 13% at 24 h after injury. Treatment with equivalent concentrations of AT did not block ROS production and cell lysis and moderately improved mitochondrial respiration and coupling. This is the first report demonstrating the protective effects of GT3 against RPTC injury by: 1) decreasing production of ROS, 2) improving mitochondrial respiration, coupling, ΔΨ(m), and F0F1-ATPase function, 3) maintaining ATP levels, and 4) preventing RPTC lysis. Our data suggest that GT3 is superior to AT in protecting RPTCs against oxidant injury and may prove therapeutically valuable for preventing renal injury associated with oxidative stress.

Protein kinase C-epsilon activation induces mitochondrial dysfunction and fragmentation in renal proximal tubules.

PKC-ε activation mediates protection from ischemia-reperfusion injury in the myocardium. Mitochondria are a subcellular target of these protective mechanisms of PKC-ε. Previously, we have shown that PKC-ε activation is involved in mitochondrial dysfunction in oxidant-injured renal proximal tubular cells (RPTC; Nowak G, Bakajsova D, Clifton GL Am J Physiol Renal Physiol 286: F307-F316, 2004). The goal of this study was to examine the role of PKC-ε activation in mitochondrial dysfunction and to identify mitochondrial targets of PKC-ε in RPTC. The constitutively active and inactive mutants of PKC-ε were overexpressed in primary cultures of RPTC using the adenoviral technique. Increases in active PKC-ε levels were accompanied by PKC-ε translocation to mitochondria. Sustained PKC-ε activation resulted in decreases in state 3 respiration, electron transport rate, ATP production, ATP content, and activities of complexes I and IV and F(0)F(1)-ATPase. Furthermore, PKC-ε activation increased mitochondrial membrane potential and oxidant production and induced mitochondrial fragmentation and RPTC death. Accumulation of the dynamin-related protein in mitochondria preceded mitochondrial fragmentation. Antioxidants blocked PKC-ε-induced increases in the oxidant production but did not prevent mitochondrial fragmentation and cell death. The inactive PKC-ε mutant had no effect on mitochondrial functions, morphology, oxidant production, and RPTC viability. We conclude that active PKC-ε targets complexes I and IV and F(0)F(1)-ATPase in RPTC. PKC-ε activation mediates mitochondrial dysfunction, hyperpolarization, and fragmentation. It also induces oxidant generation and cell death, but oxidative stress is not the mechanism of RPTC death. These results show that in contrast to protective effects of PKC-ε activation in cardiomyocytes, sustained PKC-ε activation is detrimental to mitochondrial function and viability in RPTC.

Succinate ameliorates energy deficits and prevents dysfunction of complex I in injured renal proximal tubular cells.

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 TBHP-injured 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.

Activation of ERK1/2 pathway mediates oxidant-induced decreases in mitochondrial function in renal cells.

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.

Protein kinase C-epsilon modulates mitochondrial function and active Na+ transport after oxidant injury in renal cells.

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.