Protein kinase C-α interaction with iHSP70 in mitochondria promotes recovery of mitochondrial function after injury in renal proximal tubular cells

Abstract

This study determined the role of PKC-α and associated inducible heat shock protein 70 (iHSP70) in the repair of mitochondrial function in renal proximal tubular cells (RPTCs) after oxidant injury. Wild-type PKC-α (wtPKC-α) and an inactive PKC-α [dominant negative dn; PKC-α] mutant were overexpressed in primary cultures of RPTCs, and iHSP70 levels and RPTC regeneration were assessed after treatment with the oxidant tert-butylhydroperoxide (TBHP). TBHP exposure increased ROS production and induced RPTC death, which was prevented by ferrostatin and necrostatin-1 but not by cyclosporin A. Overexpression of wtPKC-α maintained mitochondrial levels of active PKC-α, reduced cell death, and accelerated proliferation without altering ROS production in TBHP-injured RPTCs. In contrast, dnPKC-α blocked proliferation and monolayer regeneration. Coimmunoprecipitation and proteomic analysis demonstrated an association between inactive, but not active, PKC-α and iHSP70 in mitochondria. Mitochondrial iHSP70 levels increased as levels of active PKC-α decreased after injury. Overexpression of dnPKC-α augmented, whereas overexpression of wtPKC-α abrogated, oxidant-induced increases in mitochondrial iHSP70 levels. iHSP70 overexpression 1) maintained mitochondrial levels of phosphorylated PKC-α, 2) improved the recovery of state 3 respiration and ATP content, 3) decreased RPTC death (an effect abrogated by cyclosporine A), and 4) accelerated proliferation after oxidant injury. In contrast, iHSP70 inhibition blocked the recovery of ATP content and exacerbated RPTC death. Inhibition of PKC-α in RPTC overexpressing iHSP70 blocked the protective effects of iHSP70. We conclude that active PKC-α maintains mitochondrial function and decreases cell death after oxidant injury. iHSP70 is recruited to mitochondria in response to PKC-α dephosphorylation and associates with and reactivates inactive PKC-α, which promotes the recovery of mitochondrial function, decreases RPTC death, and improves regeneration.

the pkc family is a family of 11 isozymes of serine/threonine protein kinases involved in regulating a variety of cellular processes, such as proliferation, differentiation, survival, migration, and invasion (1). Our previous study (26) demonstrated that nonselective activation of PKC improves the repair of mitochondrial functions in renal proximal tubular cells (RPTCs) after oxidant injury (26). PKC-α plays an essential role in cell survival in response to oxidative stress (40, 43) and is involved in the regulation of mitochondrial function in RPTCs injured by nephrotoxicants (27). The prosurvival functions of active PKC-α depend not only on its subcellular localization but also on the type of stress involved (13). Accumulation of active PKC-α in mitochondria occurs in response to PKC stimulation. PMA-induced activation and translocation of PKC-α to mitochondria alter mitochondrial membrane potential, decrease activity of complex I, and increase ROS generation in skeletal myocytes (47). On the other hand, inhibition of PKC-α by overexpression of the inactive mutant of PKC-α induces apoptosis (21).

PKC is involved in the expression of heat shock protein (HSP)70 (4, 5). HSPs are molecular chaperones that prevent abnormal protein folding and aggregation caused by different insults (53). The Hsp70 family contains multiple homologs ranging in size from 66 to 78 kDa. Overexpression of HSP70 protects against stress conditions such as heat shock, ischemia, hypoxia, and oxidative stress in a variety of cell types (49, 53). Two types of HSP70 are present in mammalian cells: HSP73, which is abundant and constitutively expressed, and inducible iHSP70, which is present at low levels in the absence of stress (50). The major stress-inducible HSP70s are highly homologous HSP70.1 and HSP70.2 (46). The cytoprotection offered by iHSP70 is associated with decreased ROS production and preservation of function of mitochondrial complexes, state 3 respiration, mitochondrial membrane potential, and ATP formation during stress (38, 46). Overexpression of HSP70 in intestinal epithelial cells prevents hypoxia-induced apoptosis initiated through the mitochondrial pathway. HSP70 targets mitochondria and protects their functions in cardiac ischemia (14). HSP70 inhibits apoptosis by increasing mitochondrial levels of the antiapoptotic protein Bcl-2 and preventing the accumulation of proapoptotic Bax in mitochondria (49, 53, 54). Overexpression of iHSP70 preserves cellular morphology and decreases cell death induced by H₂O₂ and cisplatin in LLC-PK1 cells and protects human proximal tubular cells against gentamicin-induced nephrotoxicity (15, 55).

Overexpression of PKC-α increases transcriptional activation of the HSP70 gene and protects against ischemia-reperfusion-induced injury in ventricular myocytes (3, 4). In contrast, inhibition of PKC-α decreases the expression of HSP70 and blocks the protective effect of heat shock pretreatment on TNF-α-induced apoptosis in hepatic epithelial cells (52). Exercise-induced myocardial protection against ischemic injury is mediated by PKC-α-induced HSP70 expression (23). Furthermore, HSP70 and PKC-α interact with each other in ventricular myocytes during HSP70-mediated protection against ischemia (3). The effect of PKC-α on HSP70 is not through phosphorylation, as the inhibition of PKC-α does alter HSP70 phosphorylation (23). Reintroduction of HSP70 in primary cultures of RPTCs obtained from HSP70 knockout mice prevents apoptosis after ATP depletion (49). Finally, pharmacological induction of HSP70 in vivo attenuates acute kidney injury after ischemia in wild-type mice but not in HSP70 knockout mice (49). However, the mechanisms and target proteins of protective actions of HSP70 against necrosis associated with the acute kidney injury are not known.

Our previous study (12) showed that primary cultures of RPTCs have the capacity to respond to stress conditions by synthesizing different HSPs, including HSP90 and HSP70, and general stress glycoproteins (GSPs), including GSP62, GSP50, and GSP38. Recently, we (35) have shown that PKC-α activation promotes the recovery of mitochondrial functions and RPTC survival after oxidant injury. However, the role of the mitochondrial PKC-α activation status in regulating mitochondrial levels of HSP70 in the kidney, particularly in RPTCs, is not known. Furthermore, it is not known whether mitochondrial PKC-α and HSP70 interact during RPTC injury and/or repair and whether such interaction plays a role in the recovery of mitochondrial function after injury in RPTCs. Therefore, the aim of the present study was to determine the relationship between the PKC-α activation status and iHSP70 levels in mitochondria of oxidant-injured RPTCs and the role of these two proteins in the recovery of mitochondrial function and RPTC survival and regeneration after oxidant injury. The second aim of the present study was to determine the mechanism of oxidant-induced RPTC death that is attenuated by HSP70.

MATERIALS AND METHODS

Animals and materials.

Female New Zealand White rabbits (2.0–2.5 kg) were purchased from Myrtle's Rabbitry (Thompson Station, TN). All animal procedures involved in this study were approved by the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences. Cell culture media (a 50:50 mixture of DMEM and Ham's F-12 nutrient mix without phenol red, pyruvate, and glucose) were purchased from MediaTech Cellgro (Herndon, VA). Phosphorylated PKC-α antibody was purchased from Upstate Biotechnology (Lake Placid, NY). Antibodies against the β-subunit of F₀F₁-ATPase and 5-(and 6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (carboxy-H₂DCFDA) were supplied by Molecular Probes (Life Technologies, Eugene, OR). VER-155008 and PKC-α antibody were supplied by Santa Cruz Biotechnology (Santa Cruz, CA). iHSP70 antibody was purchased from Enzo Life Sciences (Plymouth Meeting, PA). Cyclosporine A, ferrostatin, necrostatin-1, and Go-6976 were purchased from Sigma (St. Louis, MO), BioVision (Milpitas, CA), and EMD Millipore (Billerica, MA), respectively. The sources of the other reagents were as previously described (29, 33, 34).

Isolation and culture of RPTCs.

Renal proximal tubules were isolated from rabbit kidneys by the iron-oxide perfusion method and cultured in 35-mm culture dishes in improved conditions as previously described (33, 34). The culture medium was a 50:50 mixture of DMEM and Ham's F-12 nutrient mix without phenol red, pyruvate, and glucose supplemented with 15 mM NaHCO₃, 15 mM HEPES, and 6 mM lactate (pH 7.4, 295 mosmol/kg). Human transferrin (5 μg/ml), selenium (5 ng/ml), hydrocortisone (50 nM), bovine insulin (10 nM), and l-ascorbic acid-2-phosphate (50 μM) were added to the media immediately before the daily media change.

Adenoviral constructs and amplification.

The dominant negative (kinase dead, inactive) PKC-α mutant (dnPKC-α) was constructed by mutation at the ATP-binding site (replacement of lysine with arginine at position 368) (2). This mutation destroyed the construct's catalytic activity. Adenoviral vector encoding dnPKC-α was constructed by Dr. Trevor Biden (Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, NSW, Australia). Adenoviral vectors encoding wild-type PKC-α (wtPKC-α) and the shuttle plasmid were constructed as previously described (39, 45). Aliquots of both adenoviruses were generously provided by Dr. Alan Samarel (Loyola University Medical Center, Chicago, IL). Rat iHSP70 was constructed as previously described (24). Adenoviral vector encoding iHSP70 was constructed as previously described (25) by cloning the rat iHSP70 gene into the multiple cloning site of the E1 region of the adenoviral shuttle plasmid pACCMVpLpASR as previously described by Graham and Prevec (11). Adenoviruses were amplified in AD-293 cells and human embryonic kidney (HEK)-293 cells as we have previously described (42). Adenoviral particles were isolated and purified from HEK-293 lysates by centrifugation in a CsCl density gradient (7.5 and 8.3 M) at 175,500 g for 1 h. The multiplicity of infection (MOI) was determined by a viral dilution assay in HEK-293 cells grown in 96-well plates.

Overexpression of wild-type and inactive PKC-α and iHSP70.

All transfections were carried out in confluent quiescent cultures of RPTCs. Selective overexpression of wild-type and inactive PKC-α and iHSP70 was achieved by infecting RPTCs using adenoviral vectors encoding wtPKC-α (MOI: 75), dnPKC-α (MOI: 50), and iHSP70 (MOI: 280). Infection with adenoviral particles encoding the empty pShuttle vector was used as a control. Culture media were changed 24 and 48 h after infections of RPTCs with the respective PKC-α mutants, iHSP70, or empty pShuttle vector.

Oxidant treatment of the RPTC monolayer.

Confluent monolayers of RPTCs were treated with the model oxidant tert-butylhydroperoxide (TBHP; 350 μM) for 45 min. Controls were treated with the diluent (0.1% DMSO). After TBHP exposure, the monolayer was washed with fresh, warm (37°C) medium and cultured for an additional 4, 24, or 96 h. To assess the role of PKC-α in RPTC survival and recovery, RPTCs were transfected with wtPKC-α, dnPKC-α, and iHSP70 at 48 h before TBHP exposure.

Isolation of RPTC mitochondria.

RPTCs were homogenized and mitochondria were isolated as previously described (37, 41). Mitochondria resulting from this isolation were free of lysosomal and endoplasmic reticular contaminations and contained small amounts of peroxisomes (37, 41). The final mitochondrial pellet was resuspended in the lysis buffer used for immunoprecipitation or in Laemmli sample buffer if used for immunoblot analysis.

ROS/reactive nitrogen species generation.

carboxy-H₂DCFDA was used to assess oxidant generation in RPTCs as previously described (36, 37). Autofluorescence was measured in unstained RPTCs in each experiment. All results were corrected for autofluorescence and expressed as arbitrary fluorescence units per milligram of cellular protein.

Immunoprecipitation.

To identify proteins interacting with PKC-α, RPTCs were harvested in PBS and centrifuged at 200 g for 10 min, and pellets were homogenized in 500 μl RIPA buffer containing 50 mM Tris·HCl (pH 7.4), 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1 mM NaF, 1 mM sodium orthovanadate, and 1% Triton X-100 supplemented with protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN). Likewise, isolated mitochondria were lysed in RIPA buffer. Samples were centrifuged at 1,000 g for 10 min at 4°C, and the supernatant containing equal amounts of protein (500 μg) was used for immunoprecipitation. Supernatants were precleared using 20 μl of pure proteome protein G magnetic beads (Millipore, Billerica, MA) along with 1.0 μg of the appropriate nonimmune IgG. Precleared lysates were incubated with anti-PKC-α or iHSP70 antibodies or nonimmune IgG (5 μg) for 2 h at 4°C with gentle rotation. Immunoprecipitates were captured by gentle mixing with the magnetic beads for 1 h at 4°C. Bead-immunoprecipitate complexes were washed three times with washing buffer (PBS containing 0.1% Tween 20). Proteins were eluted from the complexes by a resuspension in elution buffer (1% SDS in PBS) and an incubation for 10 min with agitation at room temperature. Supernatants containing eluted proteins were mixed with Laemmli sample buffer, boiled, and used for immunoblot analysis.

Proteomic analysis.

To identify proteins interacting with PKC-α, bead-immunoprecipitate complexes were washed with PBS followed by a final wash with double deionized water. Protein complexes were eluted using buffer containing 2 M thiourea, 7 M urea, 4% CHAPS, and 30 mM Tris·HCl (pH 8.8). Eluates were used for proteomic analysis using two-dimensional differential in-gel electrophoresis performed at Applied Biomics (Hayward, CA). In brief, samples were covalently linked to green or red cyanine dye fluors and separated in the horizontal direction by isoelectric focusing (isoelectric focusing point: 3–10) followed by SDS-PAGE in the vertical direction (150–10 kDa). Image acquisition and in-gel analysis of protein fold changes were performed using DeCyder software (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). The gel was washed multiple times to remove staining dye and other chemicals interfering with mass spectrometry. Protein spots of interest were digested in gels at 37°C using trypsin digestion buffer. Digested peptide fragments were extracted from the gel, desalted, and identified by mass spectrometry (MS) analysis using matrix-assisted laser desorption ionization/time of flight. Protein identification was based on peptide fingerprint mass mapping (using MS data) and peptide fragmentation mapping (using MS/MS data). The MASCOT search engine was used to identify proteins from the primary sequence databases.

Cell proliferation assay.

To assess the effect of the PKC-α activation status on the regeneration of RPTC monolayers after oxidant injury, cell numbers were determined in RPTC cultures overexpressing wtPKC-α, dnPKC-α, or iHSP70 at different time points after TBHP injury. Briefly, the monolayer was washed twice with PBS, and cells were scraped using a rubber policeman and suspended in PBS. The cell suspension (10 μl) was applied to a slide, and the number of cells in each sample was determined in duplicates using the Countess Automated Cell Counter (Invitrogen, Grand Island, NY).

Immunoblot analysis.

Phosphorylation and levels of proteins of interest in RPTC lysates and mitochondria were assessed by immunoblot analysis as previously described (27).

Assessment of RPTC death.

RPTC apoptosis was evaluated by measuring phosphatidylserine externalization on the plasma membrane using the annexin V/propidium iodide-binding assay as previously described (31, 37). Cells positive for annexin V and negative for propidium iodide were considered apoptotic. Cells positive for propidium iodide and negative for annexin V were considered necrotic. In addition, cell lysis was assessed by measuring the release of lactate dehydrogenase (LDH) from RPTC monolayers into the culture media as previously described (32). Some monolayers were pretreated with cyclosporine A (1 and 5 μM), deferoxamine (1 mM), ferrostatin (10 μM), necrostatin-1 (10 μM), VER-155008 (5 μM), and Go-6976 (1 μM) before TBHP treatment and after TBHP removal and media replenishment. The final concentrations of these inhibitors were selected based concentration-dependent examination of minimum effective concentrations for each compound in RPTCs.

State 3 respiration.

State 3 and uncoupled O₂ consumptions were measured polarographically using a Clark-type electrode as previously described (29, 33, 34). State 3 respiration in mitochondria energized by electron donors to the respiratory complexes I (5 mM glutamate and 5 mM malate) and complex II (10 mM succinate and 0.1 μM rotenone) was measured in assay buffer containing digitonin (0.1 mg/ml) and respiratory substrates as previously described (30, 37). State 3 respiration was initiated by the addition of 0.4 mM ADP. State 4 respiration was measured after the addition of oligomycin (0.6 μg/ml) to RPTCs respiring at state 3.

Intracellular ATP content.

The intracellular ATP content in RPTCs was measured by the luciferase method in freshly prepared RPTC lysates using ATP Bioluminescence Assay Kit HS II (Roche, Mannheim, Germany) and the manufacturer's protocol, as previously described (27).

All results were normalized to cellular protein, which was measured by the bicinchoninic acid assay using BSA as the standard.

RESULTS

Oxidant injury induces transient activation of PKC-α in RPTCs.

To determine the effect of oxidant exposure on PKC-α activation status, we treated RPTC monolayers with the model oxidant TBHP, and the levels of phosphorylated (active) and total PKC-α proteins were determined. Phosphorylation of PKC-α in RPTCs occurred at the end of the TBHP exposure, lasted for the first 2 h of the recovery period, and decreased at 4 and 8 h after TBHP-induced injury (Fig. 1A, left). Total levels of PKC-α protein were decreased between 8 and 24 h and recovered at 48 h after injury (Fig. 1A, left). PKC-α phosphorylation in mitochondria increased at 4 h, decreased at 8 and 24 h, and recovered at 48 h after TBHP-induced injury. Total levels of PKC-α protein in mitochondria increased at 4 and 8 h, decreased at 24 h, and recovered at 48 h after TBHP exposure (Fig. 1A, right).

Fig. 1.

Protein levels of phosphorylated (p)PKC-α and total PKC-α in homogenates and mitochondria of noninfected renal proximal tubular cells (RPTCs; A) and RPTCs overexpressing adenoviral vector coding wild-type PKC-α (wtPKC-α; B) and inactive mutant dominant negative (dn)PKC-α (C) at different time points after tert-butylhydroperoxide (TBHP) exposure (0.35 mM, 45 min). Levels of actin and the β-subunit of F₀F₁-ATPase were used as loading controls for cell homogenates and mitochondria, respectively. D: ratio of p-PKC-α to total PKC-α in homogenates (left) and mitochondria (right) at different time points after TBHP exposure in RPTCs. Blots are representative of 3 independent experiments.

Overexpression of wtPKC-α in RPTCs prevented TBHP-induced decreases in PKC-α phosphorylation in RPTCs and mitochondria (Fig. 1B). In contrast, overexpression of dnPKC-α blocked the recovery of PKC-α phosphorylation and induced PKC-α degradation in both RPTC and mitochondria (Fig. 1C). These results demonstrate that an oxidant transiently activates PKC-α and that the phosphorylated PKC-α translocates to mitochondria. This is followed by the loss of PKC-α protein from injured mitochondria after the exposure. Overexpression of active PKC-α reduces, whereas overexpression of inactive PKC-α exacerbates, decreases in PKC-α levels in mitochondria after oxidant injury.

PKC-α activation decreases oxidant-induced RPTC death.

TBHP treatment increased RPTC lysis from 5% in controls to 27% and 41% at 4 and 24 h, respectively, after TBHP exposure (Fig. 2A). Overexpression of wtPKC-α, but not dnPKC-α, decreased RPTC lysis to 14% and 12% at 4 and 24 h, respectively, after TBHP exposure (Fig. 2A). TBHP exposure did not result in an increase in RPTC apoptosis (Fig. 2B). These results demonstrate that PKC-α activation decreases RPTC necrosis induced by an oxidant. To determine the mechanism of oxidant-induced RPTC necrosis, monolayers were treated with inhibitors of three different pathways of necrosis before exposure to TBHP. Pretreatment of RPTCs with cyclosporine A, an inhibitor of cyclophilin D, had no effect on oxidant-induced cell death at lower concentration (1 μM) and exacerbated necrosis at higher concentrations (Fig. 2C). Iron chelation by deferoxamine blocked TBHP-induced RPTC death (Fig. 2D). Likewise, inhibition of lipid peroxidation and ferroptosis by ferrostatin (10 μM) prevented oxidant-induced necrosis in RPTCs (Fig. 2D). Finally, necrostatin-1, an inhibitor of necroptosome formation and necroptosis, decreased RPTC lysis by 85% at the concentration of 10 μM and blocked RPTC death at the concentration of 50 μM (Fig. 2D). These results demonstrate that oxidant-induced RPTC necrosis is iron dependent and that its major mechanism is consistent with ferroptosis. In addition, necroptosome formation and necroptosis are additional mechanisms involved in RPTC necrosis. However, the opening of the mitochondrial permeability transition pore (MPTP) is not a major mechanism of oxidant-induced necrosis in RPTCs, which suggests that the mitochondrial permeability transition does not occur before RPTC death or is not a significant contributor to cell death in this model.

Fig. 2.

A and B: effects of activation and inhibition of PKC-α on the viability of RPTC monolayers after TBHP exposure (0.35 mM, 45 min). C: effects of blockade of the mitochondrial permeability pore (MPTP) on the viability of RPTC monolayers after TBHP exposure (0.35 mM, 45 min). CsA, cyclosporin A. D: effects of inhibition of ferroptosis (10 μM ferrostatin) and necroptosis (10 μM nectrostatin-1) on the viability of RPTC monolayers after TBHP exposure (0.35 mM, 45 min). Graphs show the percentage of propidium iodide (PI)-positive, annexin V-FITC-negative RPTCs (necrotic; A, C, and D) and annexin V-FITC-positive, PI-negative RPTCs (apoptotic; B) as determined by flow cytometry. Results are averages ± SE of 3–4 independent experiments (RPTC isolations). Values with different symbols (* and #) on a given day are significantly different (P < 0.05) from each other.

PKC-α activation improves RPTC regeneration after oxidant injury.

RPTC numbers decreased by 30% at 24 h and recovered by 96 h after TBHP-induced injury (Fig. 3A). Overexpression of wtPKC-α prevented the decreases in RPTC numbers after oxidant exposure (Fig. 3A). In contrast, overexpression of dnPKC-α decreased cell numbers in noninjured RPTCs and blocked RPTC proliferation and monolayer recovery after TBHP-induced injury (Fig. 3A). These data show that inhibition of PKC-α blocks RPTC proliferation and monolayer regeneration and that active PKC-α is critical for the promotion of RPTC viability and regeneration after oxidant injury.

Fig. 3.

Effects of activation and inhibition of PKC-α on monolayer cell numbers (A) and oxidant production (B) in TBHP-injured RPTCs. Results showing oxidant production are expressed as percentages of the control at the end (45 min) of TBHP (0.35 mM) exposure. Oxidant generation in controls was 87,218 ± 8,205 arbitrary fluorescence units/mg protein. Results are averages ± SE of 6 independent experiments (RPTC isolations). Values with different symbols (* and #) are significantly different (P < 0.05) from each other.

PKC-α inhibition attenuates oxidant production in RPTCs.

ROS production in RPTCs was increased 191% after TBHP exposure (Fig. 3B). Overexpression of wtPKC-α had no effect on ROS production in noninjured and TBHP-injured RPTCs. In contrast, overexpression of dnPKC-α decreased ROS generation in noninjured RPTCs and blocked increases in ROS production in TBHP-injured cells (Fig. 3B). Thus, our data suggest that PKC-α is involved in oxidant-induced ROS production, but blockade of ROS generation at the level of PKC-α activation does not protect RPTCs against injury and death.

PKC-α interacts with HSPs in RPTC mitochondria.

To test whether PKC-α interacts with other mitochondrial proteins, we immunopreciptated PKC-α from mitochondrial lysates and identified major proteins associated with PKC-α. Proteomic analysis of the PKC-α immunocomplexes identified several proteins that associate with PKC-α in mitochondria (Fig. 4A and Table 1). A major family of proteins present in these immunocomplexes was the HSP family, including HSP70, HSP90-β, cdc37 (cochaperone of HSP90), and HSP40 cochaperones of HSP70 (Table 1). These results were confirmed by immunoblot analysis of proteins present in PKC-α immunocomplexes, which revealed the association of PKC-α with iHSP70 (Fig. 4B). Reverse immunoprecipitation using iHSP70 antibody confirmed the association of iHSP70 with PKC-α (Fig. 4B). Therefore, we focused the study on the interaction of PKC-α with iHSP70 in mitochondria. Oxidant exposure resulted in a twofold increase in iHSP70 levels in PKC-α immunocomplexes (Fig. 4B, left). Overexpression of wtPKC-α in TBHP-injured RPTCs reduced mitochondrial iHSP70 association with PKC-α (Fig. 4, A, middle, and B, left). Overexpression of dnPKC-α resulted in a fivefold increase in HSP70 levels associated with PKC-α compared with nontransfected RPTCs (Fig. 4, A and B). These data show that inactive, but not active, PKC-α associates with HSP70 in RPTC mitochondria.

Fig. 4.

Proteins associated with PKC-α in RPTC mitochondria. A: two-dimensional differential in-gel electrophoresis images of major proteins immunoprecipitated from isolated mitochondria using PKC-α antibody. Left, nontransfected RPTCs (labeled green, Cy5) and RPTCs overexpressing dnPKC-α (labeled red, Cy3). Middle, nontransfected RPTCs (labeled green, Cy5) and RPTCs overexpressing wtPKC-α (labeled red, Cy3) at 4 h after TBHP exposure. Right, nontransfected RPTCs (labeled green, Cy5) and RPTCs overexpressing dnPKC-α at 4 h after TBHP exposure. Proteins were separated in the horizontal direction by isoelectric focusing point (pI) from pH 3 to 10 and vertically by molecular weight from 150 to 10 kDa. Proteins excised and eluted from the gel were identified by MS/MS analysis. Major proteins associated with PKC-α are labeled 1–7, and their identities are shown in Table 1. B: immunoblot analysis of proteins immunoprecipitated by PKC-α and inducible heat shock protein (iHSP)70 antibodies from mitochondria isolated from RPTCs at 4 h after TBHP exposure. Representative blots of 3 experiments are shown. IP, immunoprecipitation; WB, Western blot.

Table 1.

Mitochondrial proteins present in immunocomplexes with PKC-α in RPTCs

Spot	Top Ranked Protein Name	Accession No.	Protein Molecular Weight	Protein pI	Peptide Count	Protein Score
1	PKC α-type	gi 157786690	72,735.3	7.51	17	176
2	Heat shock cognate 71-kDa protein isoform 1	gi 5729877	70,854.2	5.37	25	498
3	HSP70.2-like isoform 1	gi 291395803	70,131	5.75	24	669
4	Heat shock 90-kDa protein 1, β	gi 291410975	84,371.5	4.93	27	602
5	Hsp90 cochaperone cdc37	gi 5901922	44,440	5.17	14	303
6	DnaJ subfamily A, member 2*	gi 114662436	57,451.9	8.63	7	50
7	DnaJ homolog subfamily A member 1†	gi 4504511	44,839.4	6.65	8	144

Proteins were identified using two-dimensional differential in-gel electrophoresis followed by mass spectrometry analysis using matrix-assisted laser desorption ionization/time of flight. Proteins are shown based on the numbering of gel spots shown in Fig. 4A. pI, isoelectric focusing point; HSP, heat shock protein.

^*HSP40 (cochaperone of HSP70).

^†Heat shock 40-kDa protein 4 (cochaperone of HSP70 suggested to play a role in protein import into mitochondria).

Oxidant injury increases levels of iHSP70 in RPTCs.

To determine whether iHSP70 levels are altered by oxidant injury, we analyzed protein levels of iHSP70 in RPTC homogenates and mitochondria at different time points after TBHP exposure. Figure 5A shows increased protein levels of iHSP70 in RPTC homogenates between 4 and 24 h and in mitochondria after TBHP exposure. Transient dephosphorylation of PKC-α preceded increases in iHSP70 levels. The highest levels of iHSP70 were observed at the time points when the levels of phosphorylated PKC-α were the lowest (Figs. 1A and 5A). The levels of iHSP70 in homogenates returned to control at 48 h after TBHP exposure, whereas the mitochondrial levels of iHSP70 were increased throughout the 96-h recovery period (Fig. 5A). These results show that TBHP-induced injury preferentially increases iHSP70 levels in mitochondria and suggest that mitochondria are an important site of action of iHSP70 in oxidant-injured RPTCs.

Fig. 5.

Protein levels of iHSP70 in homogenates and mitochondria of noninfected RPTCs (A) and RPTCs overexpressing wtPKC-α (B) and dnPKC-α (C) at different time points after TBHP exposure (0.35 mM, 45 min). Levels of actin and the β-subunit of F₀F₁-ATPase were used as loading controls for cell homogenates and mitochondria, respectively. D: ratio of iHSP70 levels to loading controls in homogenates (left) and mitochondria (right) at different time points after TBHP exposure in RPTCs. Blots are representative of 3 independent experiments.

Inhibition of PKC-α upregulates iHSP70 protein levels.

The results shown in Figs. 1 and 5 demonstrate that the TBHP-induced decreases in protein levels of phosphorylated PKC-α are associated with the increases in iHSP70 levels in RPTC homogenates and mitochondria. Therefore, we examined the relationship between the activation status of PKC-α and iHSP70 levels in injured RPTCs. Overexpression of wtPKC-α blocked the increases in iHSP70 levels in mitochondria of TBHP-injured RPTCs (Fig. 5B, right). In contrast, overexpression of dnPKC-α increased iHSP70 levels in homogenates and mitochondria of noninjured and TBHP-injured RPTCs (Fig. 5C). Thus, our data demonstrate that inhibition of PKC-α upregulates protein levels of iHSP70 in RPTCs and their mitochondria. Consequently, we tested whether iHSP70 overexpression alters the levels of phosphorylated PKC-α after TBHP-induced injury. The results shown in Fig. 6 demonstrate that iHSP70 overexpression increases the levels iHSP70 in cell lysates and mitochondria of noninjured and TBHP-injured RPTCs. Furthermore, overexpression of iHSP70 increases the levels of mitochondrial phosphorylated PKC-α and total PKC-α after oxidant injury. These data suggest that overexpression of iHSP70 is associated with increased levels of active PKC-α in mitochondria of injured RPTCs.

Fig. 6.

A: protein levels of iHSP70, p-PKC-α, and total PKC-α in homogenates and mitochondria isolated from nontransfected RPTCs and RPTCs overexpressing iHSP70 at 4 and 24 h after TBHP exposure (0.35 mM, 45 min). Levels of actin and the β-subunit of F₀F₁-ATPase were used as loading controls for cell homogenates and mitochondria, respectively. B: optical density ratio of p-PKC-α and total PKC-α versus the loading control (β-subunit of F₀F₁-ATPase) in mitochondria of TBHP-injured RPTCs at 24h after injury. Blots are representative of 3 independent experiments. *P < 0.05.

iHSP70 upregulation decreases oxidant-induced RPTC death.

Recently, we (37) have shown that overexpression of wtPKC-α increases the levels of phosphorylated PKC-α in RPTCs and their mitochondria and exerts protective effects on mitochondrial function and viability of oxidant-injured RPTCs. To determine whether upregulation of iHSP70 levels plays a role in mitochondrial function and RPTC survival and regeneration after oxidant injury, iHSP70 was overexpressed in RPTCs before TBHP exposure, and cell survival, monolayer regeneration, and repair of mitochondrial functions and energy status were assessed during the 4-day recovery period after TBHP exposure. The results shown in Fig. 7A demonstrate that overexpression of iHSP70 reduced RPTC death from 41% to 22% at 24 h after oxidant injury. iHSP70-offered protection was reversed by cyclosporine A, whereas necrostatin-1 did not have any effect on the protective actions of iHSP70 against oxidant-induced death (Fig. 7, A and B). Ferrostatin and deferoxamine further diminished necrosis in iHSP70-overexpressing RPTCs injured by the oxidant (Fig. 7B and data not shown). Neither TBHP nor iHSP70 overexpression had any effects on RPTC apoptosis (3.2 ± 0.6% vs. 2.7 ± 0.8% in control and TBHP-injured RPTCs, respectively; Fig. 7C). Thus, our data show that iHSP70 decreases RPTC necrosis caused by oxidant injury and that this protection is mediated through the MPTP and not downstream mechanisms of ferroptosis.

Fig. 7.

Effects of overexpression of iHSP70 and the MPTP inhibitor CsA (A and C), the inhibitor of ferroptosis ferrostatin (B and C), and the inhibitor of necroptosis necrostatin-1 (B and C) on RPTC viability at different time points after TBHP exposure (0.35 mM, 45 min). Graphs show the percentages of PI-positive, annexin V-FITC-negative RPTCs (necrotic; A and B) and annexin V-FITC-positive, PI-negative RPTCs (apoptotic; C) as determined by flow cytometry. Results are averages ± SE of 4 independent experiments (RPTC isolations). Values with different symbols (* and #) on a given day are significantly different (P < 0.05) from each other.

iHSP70 upregulation decreases cell loss and improves cell proliferation in RPTCs.

Cell numbers in TBHP-injured RPTC monolayers decreased 40% and did not recover by 96 h after oxidant injury (Fig. 8). Overexpression of iHSP70 did not reduce the decreases in RPTC numbers after TBHP exposure but promoted the full recovery of cell numbers after oxidant injury (Fig. 8). These data show that iHSP70 overexpression diminishes TBHP-induced cell loss and promotes RPTC proliferation after injury.

Fig. 8.

Effects of overexpression of iHSP70 on cell numbers in RPTC monolayers at different time points after TBHP exposure (0.35 mM, 45 min). Results are averages ± SE of 4 independent experiments (RPTC isolations). Values with different symbols (* and #) on a given day are significantly different (P < 0.05) from each other.

iHSP70 upregulation accelerates recovery of mitochondrial function after injury.

Complex I-coupled state 3 respiration in mitochondria energized with glutamate and malate decreased to 63% of controls after TBHP exposure and recovered at 96 h of the recovery period (Fig. 9A). State 3 respiration in mitochondria energized with succinate decreased to 79% of control at 4 h and also recovered at 96 h after TBHP exposure (Fig. 9B). Overexpression of iHSP70 reduced TBHP-induced decreases of state 3 respiration and accelerated its recovery in TBHP-injured RPTCs (Fig. 9, A and B). State 4 respiration was not changed in oxidant-injured RPTCs (data not shown). Due to decreases in state 3 respiration, the respiratory control ratio (RCR) decreased to 70% of controls (5.8 ± 0.3 vs. 4.1 ± 0.6 in controls and TBHP-injured RPTCs, respectively) at 4 h after TBHP exposure. Overexpression of iHSP70 had no effect on RCR at 4h after oxidant injury but restored RCR (4.8 ± 0.3 vs. 4.7 ± 0.6 in controls and injured RPTCs, respectively) at 24 h after TBHP exposure.

Fig. 9.

Effects of overexpression of iHSP70 on state 3 respiration coupled to the oxidation of electron donors through complex I (glutamate + malate; A), state 3 respiration coupled to the oxidation of electron donors through complex II (succinate + rotenone; B), oligomycin-sensitive respiration (C), and ATP content (D) in RPTCs at different time points after TBHP exposure (0.35 mM, 45 min). Results are averages ± SE of 3–6 independent experiments (RPTC isolations). Values with different symbols (* and #) are significantly different (P < 0.05) from each other.

Oligomycin is a specific inhibitor of F₀F₁-ATPase and blocks oxidative phosphorylation and ATP synthesis. Oligomycin-sensitive respiration represents the amount of O₂ consumed by oxidative phosphorylation, and it was used as an indirect measure of the function of F₀F₁-ATPase and RPTC capacity for oxidative phosphorylation. Oligomycin-sensitive respiration in RPTCs decreased to 70% of controls at 4 h and recovered within 96 h after TBHP-induced injury (Fig. 9C). Overexpression of iHSP70 improved oligomycin-sensitive respiration in TBHP-injured RPTCs (Fig. 9C). These data demonstrate that iHSP70 improves the mitochondrial capacity for respiration and oxidative phosphorylation and suggest that iHSP70 is involved in mechanisms maintaining mitochondrial coupling.

iHSP70 upregulation promotes recovery of ATP content after injury.

The ATP content was used to evaluate the energy status of RPTCs. The ATP content decreased by 43% at 4 h and recovered at 96 h after TBHP exposure (Fig. 10B). Overexpression of iHSP70 had no effect on ATP content in noninjured RPTCs, but it reduced the decreases in ATP content and promoted ATP recovery after oxidant exposure (Fig. 10B). In contrast, inhibition of HSP70 function using the selective HSP70 inhibitor VER-155008, an inhibitor that targets the ATPase-binding domain of HSP70, prevented iHSP70-mediated improvements in ATP levels in injured RPTCs (Fig. 10B). Inhibition of HSP70 had no effect on ATP levels in noninjured RPTCs (Fig. 9A).These data demonstrate that HSP70 overexpression promotes and HSP70 inhibition blocks the recovery of ATP content after oxidant injury in RPTCs.

Fig. 10.

Effects of overexpression and inhibition (5 μM VER-155008) of iHSP70 on oxidant production (A), ATP content (B), and cell lysis (C) in injured RPTCs at 24 h after TBHP injury. Oxidant generation data (A) are expressed as percentages of control at the end (45 min) of TBHP (0.35 mM) exposure. Oxidant generation in controls was 130,614 ± 20,731 arbitrary fluorescence units/mg protein. D: effects of inhibition of PKC (1 μM Go-6976) on RPTC lysis at 24 h after TBHP injury in RPTCs overexpressing iHSP70. Results are averages ± SE of 4 independent experiments (RPTC isolations). Values with different symbols (* and #) are significantly different (P < 0.05) from each other.

iHSP70 upregulation blocks TBHP-induced oxidant production.

TBHP exposure increased oxidant production in RPTC by twofold (Fig. 10A). Overexpression of iHSP70 in noninjured RPTCs decreased ROS production to 72% of controls (Fig. 10A). Furthermore, overexpression of iHSP70 blocked TBHP-induced increases in oxidant production in RPTCs (Fig. 10A).

Inhibition of PKC-α blocks the protective actions of iHSP70 against oxidant injury.

LDH release (used as a marker of RPTC lysis and death) increased to 44% at 24 h after TBHP exposure (Fig. 10, C and D). Overexpression of iHSP70 reduced LDH release to 22% in TBHP-injured RPTCs, whereas inhibition of iHSP70 by VER-155008 blocked the protective effects of iHSP70 overexpression in TBHP-injured RPTCs (Fig. 10C). Inhibition of PKC-α activity using Go-6976, a selective inhibitor that competes with ATP binding to the active site of classic PKC isozymes, did not have any effect on the viability of noninjured RPTCs and did not alter TBHP-induced necrosis (Fig. 10D). However, inhibition of PKC-α by Go-6976 abrogated the protective effects of iHSP70 overexpression in oxidant-injured RPTCs (Fig. 10D). These results demonstrate that the protective actions of HSP70 in mitochondria of oxidant-injured RPTCs are dependent on active PKC-α.

DISCUSSION

We (28) have previously shown that RPTCs regenerate and recover mitochondrial functions after sublethal injury induced by TBHP. Our previous reports (19, 27) have shown that PKC-α activation is involved in the regulation of mitochondrial function and cell death in RPTCs injured by the nephrotoxicants cisplatin and dichlorovinyl cysteine. Recently, we (25) have shown that the activation of PKC-α prevents mitochondrial hyperpolarization, dysfunction, and fragmentation, decreases cell death, and promotes the recovery of mitochondrial respiration and ATP content after oxidant injury in RPTCs. The results of the present study suggest that the maintenance of active PKC-α in mitochondria after oxidant injury is required for RPTC recovery and regeneration and that iHSP70 plays a supportive role in the maintenance of active PKC-α levels in mitochondria. Increased mitochondrial levels of phosphorylated (active) PKC-α in RPTC overexpressing wtPKC-α are associated with improved cell survival, proliferation, and monolayer regeneration after oxidant injury. In contrast, overexpression of inactive PKC-α block both proliferation and monolayer recovery after injury. Therefore, the results of our study suggest that active PKC-α is essential for improved mitochondrial function, survival, proliferation, and regeneration of RPTC after oxidant injury. The importance of PKC-α activation in renal cell survival has been previously demonstrated by Whelan and colleagues (51) in simian virus 40 immortalized COS-1 cells. Inhibition of PKC-α by overexpression of the inactive mutant of PKC-α caused loss of PKC function and induced apoptosis, whereas overexpression of wtPKC-α blocked apoptosis in COS-1 cells. Collectively, these reports show an essential role of PKC-α activation in renal cell survival after injury caused by an oxidant.

Proteomic analysis of mitochondrial proteins interacting with PKC-α focused the present study on a family of HSPs to determine whether they cooperate with PKC-α to improve mitochondrial function and RPTC survival and recovery after oxidant injury. Gao and Newton (8) demonstrated that HSP70 preferentially binds a hydrophobic residue preceding the unphosphorylated turn motif (Thr⁶⁴¹) of mature PKC that has become dephosphorylated and protects PKC from ubiquitination and degradation. Thus, HSP70 associates with PKC and prolongs the lifetime of active (phosphorylated) PKC. Coaxum and collaborators (4) have shown a close interaction between PKC-α and HSP70, which is independent of heat shock factor-1 activation and confers protection in cardiomyocytes injured by ischemia. The protection conferred by PKC-α in neonatal rat ventricular myocytes is mediated by the transcriptional induction of HSP70 (4). Overexpression of HSP72 is also protective against oxidative injury and cisplatin toxicity in RPTCs (15). However, these studies focused on the entire cellular pool of HSP70 and did not differentiate between cellular and mitochondrial pools of PKC-α and HSP70. Our data show that PKC-α activation increases the cellular pool of iHSP70 in RPTCs but that iHSP70 does not translocate to injured mitochondria if PKC-α is active. In contrast, inactive PKC-α increases both cellular and mitochondrial levels of iHSP70. In mitochondria, inactive PKC-α associates with several HSPs, including HSP70 and HSP90 and their cochaperones HSP40 and cdc37. Because the levels of active PKC-α decrease in mitochondria of oxidant-injured RPTCs, we hypothesize that this decrease promotes mitochondrial translocation of iHSP70 and binding to inactive PKC-α to protect it from unfolding and degradation until it is rephosphorylated and reactivated. Alternatively, iHSP70 translocation to mitochondria of injured RPTCs may occur in response to unfolding of PKC-α and the role of iHSP70 is to refold this protein kinase.

HSPs act as client proteins for many protein kinases, including Src, Akt, phosphoinositide-dependent kinase (PDK)-1 (the upstream kinase that phosphorylates PKC), and ErbB2-/HER2. HSP90 and HSP70 are recruited to protein kinases through interactions with cochaperones, which act as bridges between the HSP and its client kinase (10). Our results show that two HSP40 cochaperones of HSP70 and the cochaperone of HSP90 (cdc37) are associated with the HSP70-PKC-α complex in mitochondria of injured RPTCs and further support our hypothesis of a functional complex between iHSP70 and PKC-α and its protective role in injured mitochondria. The levels of these cochaperone proteins are greatly increased in mitochondria of RPTCs overexpressing inactive PKC-α and are nearly absent in noninjured and injured RPTCs overexpressing wtPKC-α, demonstrating that the activation status of PKC-α controls iHSP70 recruitment to mitochondria. It has been shown that the phosphorylation status of PKC at the COOH-terminus determines the affinity of binding between HSP70 and PKC-α (9). HSP70 binds to the COOH-terminus of mature unphosphorylated PKC and allows the rephosphorylation of PKC by its upstream kinase, PDK-1, thereby preventing the unfolding, aggregation, and degradation of PKC (8). Lack of iHSP70 binding to active PKC-α in mitochondria suggests that the affinity of this binding is lower than the binding of iHSP70 to inactive PKC-α or that the COOH-terminus of active PKC-α is inaccessible to HSP70 due to a conformational change induced by the phosphorylation of PKC-α.

PKC-α does not remain in mitochondria of oxidant-injured RPTCs in its inactive dephosphorylated form, suggesting that it is degraded if not rephosphorylated (Fig. 1). Overexpression of inactive PKC-α (dnPKC-α) increases the cellular pool of iHSP70 and recruits iHSP70 to mitochondria of noninjured RPTCs. Oxidant exposure and injury further augments the translocation of iHSP70 to mitochondria of RPTCs overexpressing dnPKC-α. In contrast, wtPKC-α overexpression increases the cellular pool of iHSP70 but does not recruit iHSP70 to mitochondria while protecting against mitochondrial dysfunction in oxidant-injured RPTCs. Our results suggest that the activation status of PKC-α is a major determinant of iHSP70 localization to mitochondria in noninjured and injured RPTCs. Overexpression of iHSP70 increased the cellular and mitochondrial levels of iHSP70 and maintained the levels of phosphorylated PKC-α in mitochondria of oxidant-injured RPTCs. This observation suggests that the formation of the iHSP70-PKC-α complex in mitochondria prevents the dephosphorylation and inactivation of mitochondrial PKC-α after oxidant exposure. The increases in the levels of phosphorylated PKC-α suggest that iHSP70 promotes PKC-α phosphorylation by the upstream kinase (possibly PDK-1) or prevents PKC-α dephosphorylation and, thus, its subsequent degradation. Therefore, we speculate that iHSP70 associates with unphosphorylated PKC-α in mitochondria to allow for PKC-α rephosphorylation and activation, which prevents the loss of this kinase from mitochondria.

We (35) have previously demonstrated that maintaining PKC-α in the active conformation is critical for RPTC survival after oxidant injury. Increasing the levels of active PKC-α in mitochondria improves mitochondrial respiration, oxidative phosphorylation, and ATP levels after TBHP-induced injury (35). Here, we show that iHSP70 overexpression increases the levels of phosphorylated PKC-α in mitochondria of TBHP-injured RPTCs, promotes the recovery of mitochondrial function and ATP levels, decreases cell death, and accelerates monolayer regeneration in injured RPTCs. iHSP70 translocation to mitochondria in response to oxidant exposure follows the dephosphorylation of mitochondrial PKC-α. Furthermore, mitochondrial levels of iHSP70 are the highest when PKC-α levels are the lowest. We hypothesize that the protective effect of iHSP70 is mediated through its interaction with PKC-α and that iHSP70 improves mitochondrial function and RPTC survival by maintaining the levels of active PKC-α in mitochondria. Our data suggest that iHSP70 plays a supportive role in the PKC-α-mediated protection and recovery of RPTCs and that the iHSP70 action is through the maintenance of PKC-α in its active state. Blockade of HSP70 function using VER-155008 inhibits the iHSP70-mediated recovery of ATP levels and cell survival in injured RPTCs overexpressing iHSP70. More importantly, inhibition of PKC-α in RPTCs overexpressing iHSP70 blocks the protective effect of iHSP70 against oxidant injury and cell death. This suggests that iHSP70 requires active PKC-α to promote mitochondrial function and survival in oxidant-injured RPTCs. Collectively, these data demonstrate that increased levels of active PKC-α in mitochondria are sufficient to maintain mitochondrial function and improved RPTC survival without the support of iHSP70, whereas iHSP70-mediated protection is dependent on functional PKC-α.

Our data show that multiple mechanisms of necrosis contribute to oxidant-induced RPTC death. Among the different pathways of necrosis (7), ferroptosis, the iron-dependent cell death that involves ROS formation and culminates in lipid peroxidation, is the predominant mechanism leading to mitochondrial fragmentation previously reported (35) and RPTC death and lysis. Iron chelation (deferoxamine) and inhibition of lipid peroxidation using ferrostatin block TBHP-induced cell death and lysis. Ferroptosis is distinct from other forms of necrosis because it does not involve bioenergetic failure (6). The fact that oxidant-induced RPTC death is preceded by mitochondrial dysfunction, fragmentation, and decreases in ATP content suggests that other pathways of necrosis also are activated by the oxidant in RPTCs. The fully formed necroptosome complex transduces the necroptotic signal into mitochondria, inducing their fragmentation and failure (17, 18, 44). Because an inhibitor of necroptosome formation, necrostatin-1, decreased oxidant-induced RPTC death, we conclude that necroptosis is another major mechanism of RPTC death. Our data suggest that necroptosis activated upstream of events leading to ferroptosis. In contrast, the MPTP opening is not a major mechanism of oxidant-induced RPTC death, and inhibition of the MPTP by cyclosporine A is not protective in this model. Because mitochondrial membrane hyperpolarization occurs shortly after TBHP exposure (35), it is likely that the MPTP remains closed until late after TBHP exposure and inhibitors of MPTP have no protective effect. However, our data suggest that a functional MPTP is necessary for HSP70-promoted cell survival and recovery of ATP levels. iHSP70-conferred protection is reversed by an inhibitor of MPTP, cyclosporine A, indicating that iHSP70 is involved in maintaining a functional MPTP, improved mitochondrial function, and increased ATP levels. iHSP70 does not appear to be involved in downstream events of ferroptosis or the formation of the necroptosome.

Previously, it has been shown that impaired oxidative phosphorylation and ATP depletion by the exposure of renal epithelial cells to the metabolic inhibitors sodium cyanide and 2-deoxy-d-glucose can be prevented by upregulation of HSP72 levels (16, 48). Increased HSP72 levels are associated with improved state 3 respiration and protection against mitochondrial depolarization (48). The improved recovery of oligomycin-sensitive respiration and ATP content by iHSP70 overexpression in our model suggests that iHSP70 is involved in promoting the recovery of oxidative phosphorylation and ATP production. This leads to the decrease in cell death and improved survival in RPTCs overexpressing iHSP70. Overexpression of dnPKC-α leads to the outcomes similar to those obtained by inhibition of HSP70 (VER-155008). Therefore, inactivation of PKC-α and inhibition of HSP70 have similar effects in oxidant-injured RPTCs.

The HSP70 family of proteins is also involved in protection against conditions that induce apoptosis in renal tubular cells. Induction of HSP72 by heat stress protects renal tubular cells against hypoxia-induced apoptosis through a PKC-dependent mechanism (22). Upregulation of HSP72 also inhibits tubular apoptosis caused by ureteric obstruction (20) and prevents apoptosis in renal epithelial cells by inhibiting activation of proapoptotic proteins (16). Because apoptosis was not induced by the oxidant in RPTCs, our report shows that HSP70 also plays a significant role in protecting against mitochondrial dysfunction and subsequent necrosis in oxidant-injured RPTCs.

We conclude that mitochondria are a target of iHSP70 and that an iHSP70 interaction with PKC-α in mitochondria improves oxidative phosphorylation, promotes the recovery of ATP levels, and decreases RPTC necrosis after oxidant injury in RPTCs. The protective actions of iHSP70 are through maintaining mitochondrial PKC-α in its active phosphorylated state and involve the maintenance of MPTP function, subsequent improvement of mitochondrial energetics, and a decrease in ROS production. These actions lead to reduced necrosis and improved proliferation and regeneration in oxidant-injured RPTCs.

GRANTS

This work was supported by National Institutes of Health (NIH) Grant 2-R01-DK-59558 (to G. Nowak). The University of Arkansas for Medical Sciences (UAMS) Translational Research Institute, supported by NIH Grant UL1-RR-029884, provided partial funding for the Flow Cytometry Core at UAMS.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: G.N. conception and design of research; G.N. and S.S. analyzed data; G.N. and S.S. interpreted results of experiments; G.N. prepared figures; G.N. edited and revised manuscript; G.N. and R.M. approved final version of manuscript; S.S. and R.M. performed experiments; S.S. drafted manuscript.