Download Protein kinase C- modulates mitochondrial function and active Na

Am J Physiol Renal Physiol 286: F307–F316, 2004.
First published October 21, 2003; 10.1152/ajprenal.00275.2003.
Protein kinase C-⑀ modulates mitochondrial function and
active Na⫹ transport after oxidant injury in renal cells
Grażyna Nowak, Diana Bakajsova, and Ginger L. Clifton
Department of Pharmaceutical Sciences, College of Pharmacy, University of Arkansas
for Medical Sciences, Little Rock, Arkansas 72205
Submitted 1 August 2003; accepted in final form 14 October 2003
a variety of drugs and toxicants
results in nephrotoxicity and acute renal failure (ARF). In
contrast to the heart or brain, the kidney has the potential for
complete recovery from ARF after toxicant- and ischemiareperfusion-induced injury. After the insult, surviving tubular
epithelial cells dedifferentiate, proliferate, and eventually replace the irreversibly injured tubular epithelial cells, thus
restoring tubular integrity and renal functions (28, 40). A
disruption of these processes contributes to prolonged renal
dysfunction. Renal proximal tubular cells (RPTC) are the
primary target of nephrotoxicants within the kidney, and RPTC
dysfunction is primarily responsible for the pathophysiological
and clinical presentations of ARF. Therefore, the repair of
RPTC functions allows for the return of renal functions after
ARF. Growth factors have been implicated in renal regeneration through promoting proliferation, differentiation, and the
recovery of mitochondrial function and active Na⫹ transport in
renal proximal tubules (10, 18, 19, 28, 29, 34, 36, 43). Recent
studies demonstrated that some extracellular matrix proteins
also play an important role in the promotion of the repair
processes in RPTC. Nony and colleagues (27) demonstrated
that collagen IV, but not collagen I, fibronectin, or laminin,
promotes the recovery of mitochondrial function and Na⫹/K⫹
activity in toxicant-injured RPTC. However, the exact cellular
mechanisms and pathways that mediate the repair of these
functions in RPTC remain unknown.
Protein kinase C (PKC) consists of a family of 11 phospholipid-dependent serine-threonine kinases classified by the requirement for Ca2⫹ and diacylglycerol for activation (26).
PKC is a key mediator of many diverse physiological and
pathological processes (11). PKC isozymes play a crucial role
in the regulation of major cellular functions, including proliferation, differentiation, motility, ion homeostasis, and transport
functions (11). PKC has also been implicated in the regulation
of cellular injury, apoptosis, cell survival, and the recovery
from injury (11).
PKC-⑀ is the diacylglycerol-dependent, Ca2⫹-independent
PKC isozyme that plays an important role in cell survival and
suppression of apoptosis in some cell types (11, 12). PKC-⑀ is
involved in ischemic injury in the heart and is a pivotal
signaling element in the cardioprotective mechanisms of ischemic preconditioning (23, 25). Studies in cardiac myocytes have
demonstrated that PKC-⑀ activation is required for protection
against ischemic injury (8, 9, 14, 16). The inhibition of PKC-⑀
activation and translocation results in the abolition of the
protection offered by ischemic preconditioning in the heart
(23). Although the exact mechanisms through which PKC-⑀
exerts its protective effects in the heart are not known at
present, these effects may be associated with the regulation of
ion homeostasis, including modulation of the mitochondrial
ATP-dependent K⫹ channel or plasma membrane Na⫹ channels. Furthermore, PKC-⑀ also mediates hydrogen peroxide/
hydroxyl radical-induced opening of mitochondrial ATP-dependent K⫹ channels and an inhibition of mitochondrial
GABA receptors in cardiomyocytes (44). Recent studies suggest that PKC-⑀ selectively inhibits the cardiac and neuronal
Na⫹ current (11, 42) and plays a role in the modulation of
Address for reprint requests and other correspondence: G. Nowak, Univ. of
Arkansas for Medical Sciences, Dept. of Pharmaceutical Sciences, 4301 W.
Markham St., Little Rock, AR 72205 (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
renal proximal tubular cells; recovery of physiological functions;
respiratory chain; adenosine 5⬘-triphosphate production; sodium-potassium adenosinetriphosphatase
EXPOSURE OF THE KIDNEY TO
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Nowak, Grażyna, Diana Bakajsova, and Ginger L. Clifton.
Protein kinase C-⑀ modulates mitochondrial function and active Na⫹
transport after oxidant injury in renal cells. Am J Physiol Renal
Physiol 286: F307–F316, 2004. First published October 21, 2003;
10.1152/ajprenal.00275.2003.—The aim of this study was to determine whether protein kinase C-⑀ (PKC-⑀) 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-⑀ was
activated three- to fivefold after injury. Active PKC-⑀ 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-⑀ translocation using a
peptide selective inhibitor, PKC-⑀V1–2, 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-⑀V1–2
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-⑀
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-⑀ is activated
and translocated to mitochondria; 2) PKC-⑀ activation decreases
mitochondrial respiration, electron transport rate, and ATP production
by reducing complex I-linked respiration; and 3) PKC-⑀ mediates
decreases in active Na⫹ transport and Na⫹-K⫹-ATPase activity.
These data show that PKC-⑀ activation after oxidant injury in RPTC
is involved in the decreases in mitochondrial function and active Na⫹
transport and that inhibition of PKC-⑀ activation promotes the repair
of these functions.
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PKC-⑀ MODULATES MITOCHONDRIAL FUNCTION AND NA⫹ TRANSPORT
MATERIALS AND METHODS
Materials. Female New Zealand White rabbits (2.0–2.5 kg) were
purchased from Myrtle’s Rabbitry (Thompson Station, TN). The cell
culture medium (a 50:50 mixture of DMEM and Ham’s F-12 nutrient
mix without phenol red, pyruvate, and glucose) was purchased from
MediaTech Cellgro (Herndon, VA). Myristoylated PKC-⑀V1–2 inhibitor (N-Myr-Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr) was supplied by
Biomol (Plymouth Meeting, PA). Protease inhibitors and the ATP
Bioluminescence Assay Kit HS II were obtained from Roche (Mannheim, Germany), and phosphatase inhibitor cocktails were from
Sigma (St. Louis, MO). Tris-glycine gels and nitrocellulose membranes were obtained from BioWhittaker Molecular Applications
(Rockland, ME) and Bio-Rad (Hercules, CA), respectively. PhosphoPKC-⑀ and PKC-⑀ antibodies were purchased from Upstate BiotechAJP-Renal Physiol • VOL
nology (Lake Placid, NY) and BD Transduction Laboratory (San
Diego, CA), respectively. Anti-mouse IgG coupled to horseradish
peroxidase was supplied by Kirkegaard & Perry Laboratory (Gaithersburg, MD) and Supersignal Chemiluminescent Substrate by Pierce
(Rockford, IL). The sources of the other reagents and cell culture
hormones have been described previously (30, 33, 35).
Isolation and culture of RPTC. 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 (35). 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/kgH2O). Human transferrin (5
␮g/ml), selenium (5 ng/ml), hydrocortisone (50 nM), bovine insulin
(10 nM), and L-ascorbic acid-2-phosphate (50 ␮M) were added to the
media immediately before daily media change (2 ml/dish).
TBHP treatment of the RPTC monolayer. RPTC monolayers
reached confluence within 6 days and were treated with TBHP (300
␮M, 45 min) on day 7 of culture. After TBHP exposure, the monolayer was washed with fresh, warm (37°C) medium and cultured for
an additional 4 days. Previously, we have shown that TBHP treatment
for this period of time induces ⬃24% cell death and loss from the
monolayer and sublethal injury to the remaining cells (32). In experiments using the selective inhibitor of PKC-⑀ translocation (PKC⑀V1–
2), RPTC were treated with 20 ␮M myristoylated PKC⑀V1–2 (NMyr-Glu-Ala-Val-Ser-Leu-Lys-Pro-Thr) for 1 h followed by TBHP
treatment. Next, PKC⑀V1–2 was added daily starting with the media
change immediately after TBHP exposure. RPTC samples were taken
at various time points after TBHP exposure for measurements of
mitochondrial functions, active Na⫹ transport, biochemical analyses,
and immunoblotting.
Oxygen consumption. RPTC monolayers were gently detached
from the dishes using a rubber policeman and transferred to the
oxygen consumption (QO2) measurement chamber. QO2 was measured
polarographically using a Clark-type electrode, as described previously (30, 32, 35). Basal QO2 was used as a marker of overall
mitochondrial function in RPTC. Uncoupled QO2 was used as a
marker of electron transfer rate and was measured in the presence of
carbonyl cyanide p-(trifluoro-methoxy)phenylhydrazone (2 ␮M).
Respiration with electron donors linked to the respiratory complex I,
II, or IV was measured after aspirating culture media and adding 2 ml
warm (37°C) sterile PBS containing 5 mM glutamate ⫹ 5 mM malate
(complex I), 10 mM succinate (complex II) ⫹ 0.1 ␮M rotenone, or 1
mM ascorbate ⫹ 1 mM N,N,N⬘,N⬘-tetramethyl-p-phenylenediamine
(complex IV).
Intracellular ATP content. Intracellular ATP content in RPTC was
measured by the luciferase method in freshly prepared cellular lysates
using an ATP Bioluminescence Assay Kit HS II (Roche) and following the manufacturer’s protocol.
ATP production rate. The assessment of state 3 respiration (the
maximum rate of ATP synthesis) was carried out by a modified
method of Borkan et al. (4). In brief, the culture media were aspirated
and replaced with 1 ml of a buffer solution resembling an intracellular
electrolyte milieu (in mM: 120 KCl, 5 KH2PO4, 10 HEPES, 1
MgSO4, and 2 EGTA, adjusted to pH 7.4 with KOH) containing
digitonin (0.1 mg/ml) and 5 mM glutamate ⫹ 5 mM malate or 10 mM
succinate as the substrates. The reaction was initiated by adding
excess ADP (2 mM final concentration) and was carried out for 5 min
at 37°C. Initial experiments determined that ATP production in these
conditions was linear for 10 min. The reaction was terminated by
adding an aliquot of ice-cold perchloric acid (3% final concentration),
and the suspension was snap-frozen in liquid nitrogen. After being
thawed, the suspension was spun down at 15,000 g for 1 min at 4°C.
The supernatant was neutralized to pH 7.5 and centrifuged again at
15,000 g for 10 min at 4°C. The final supernatant was analyzed for
ATP content using the ATP Bioluminescence Assay Kit HS II, as
described above. The initial pellet was assayed for protein content
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Ca2⫹-independent outward K⫹ channels on the plasma membrane (38).
Subcellular redistribution is an essential feature of PKC-⑀
activation. Translocation to specific subcellular compartments
is a critical step in the phosphorylation of target proteins and
PKC-⑀ signaling. The mitochondrion is thought to be an
important target for PKC-⑀ during cardiac ischemia-reperfusion injury. Ischemic preconditioning or phorbol ester induces the translocation of PKC-⑀ to the mitochondria followed
by opening of the mitochondrial ATP-sensitive K⫹ channel
(37, 45). Active PKC-⑀ also inhibits cytochrome c release from
mitochondria, the subsequent activation of caspases, and suppresses apoptosis (12). In cardiac mitochondria, PKC-⑀ forms
signaling modules with extracellular signal-regulated kinases
(ERKs) and, upon activation, phosphorylates mitochondrial
ERKs, which results in the phosphorylation and inactivation of
the pro-apoptotic protein Bad (12). Thus the activation of
PKC-⑀ in mitochondria is associated with suppression of cell
death and enhanced cell survival.
Although the evidence supports an important role of PKC-⑀
in the protection against cardiac injury, very little is known
about PKC-⑀ involvement in the physiological and pathological processes in the kidney. It is unknown whether toxicantinduced injury has any effect on renal PKC-⑀ and whether renal
PKC-⑀, and also the cardiac PKC-⑀, plays a protective role
during injury. It has been proposed that oxidative stress accompanying diabetes activates PKC-⑀ in the renal cortical
tubules (17). However, the role of PKC-⑀ in the renal dysfunction and repair after oxidant injury is completely unknown.
Previously, we have shown that tert-butylhydroperoxide
(TBHP) treatment induces ⬃24% cell death and loss from the
monolayer and sublethal injury to the remaining cells (32). The
sublethal injury induced by TBHP was associated with the
decrease in mitochondrial function and the consumption of an
oxidative substrate, intracellular ATP content, active Na⫹
transport, and Na⫹-dependent glucose uptake (32). These
RPTC functions are repaired over time without addition of any
exogenous factors. Our recent studies have shown that the
repair of RPTC functions, such as mitochondrial function and
active Na⫹ transport, after an oxidant-induced injury is mediated through PKC-dependent mechanisms (31). PKC-⑀ is one
of the major PKC isozymes present in RPTC. Therefore, the
aim of the present study was to examine whether 1) PKC-⑀
plays a role in mitochondrial dysfunction and decreases in
active Na⫹ transport and/or 2) PKC-⑀ mediates the repair of
mitochondrial function and active Na⫹ transport after oxidant
injury in RPTC.
PKC-⑀ MODULATES MITOCHONDRIAL FUNCTION AND NA⫹ TRANSPORT
AJP-Renal Physiol • VOL
(fatty acid free)] using a Dounce homogenizer and centrifuged at
1,000 g for 5 min at 4°C. The supernatant was collected and centrifuged at 15,000 g for 15 min at 4°C. The pellet containing RPTC
mitochondria was washed two times in the washing buffer (395 mM
sucrose, 10 mM HEPES, and 0.1 mM EGTA, adjusted to pH 7.4 with
KOH) and spun down again at 15,000 g for 15 min at 4°C. The final
mitochondrial pellet was resuspended in the Laemmli (21) sample
buffer and used for immunoblot analysis.
Immunoblotting. Immunoblot analysis was used to determine the
total and phosphorylated forms of PKC-⑀ in RPTC homogenates and
mitochondria. RPTC homogenates were lysed in the modified radioimmune precipitation assay buffer (50 mM Tris䡠HCl, 150 mM NaCl,
1 mM EGTA, 1% Triton X-100, 1 mM Na3VO4, 1 mM NaF, and the
protease and phosphatase inhibitor cocktails; pH 7.4), incubated on
ice for 10 min, and spun down at 100,000 g for 15 min at 4°C to pellet
the detergent-insoluble fraction; the supernatant was combined with
Laemmli sample buffer (21) and boiled as described previously (30).
Proteins were separated by SDS-PAGE and transferred electrophoretically to a nitrocellulose membrane. Blots were blocked for 1 h in
Tris-buffered saline buffer containing 0.5% casein and 0.1% Tween
20 (blocking buffer) and incubated overnight at 4°C in the presence of
anti-phospho-PKC-⑀ or anti-PKC-⑀ antibodies diluted in the blocking
buffer. After being washed, the membranes were incubated with
secondary IgGs coupled to horseradish peroxidase and washed again.
The supersignal chemiluminescent system was used for protein detection. Quantification of the results was performed using scanning
densitometry.
Protein assay. Protein concentration was determined using the
bicinchoninic acid assay with BSA as the standard.
Statistical analysis. Data are presented as means ⫾ SE and were
analyzed for significance by ANOVA. Multiple means were compared
using Fisher’s protected least-significant difference test with a level of
significance of P ⬍ 0.05. RPTC isolated from an individual rabbit
represented one experiment (n ⫽ 1) consisting of data obtained from
2 to 10 culture plates.
RESULTS
Activation of PKC-⑀ during RPTC repair after TBHP-induced injury. As shown in Fig. 1, the recovery of RPTC after
TBHP-induced injury was associated with the activation of
PKC-⑀. The protein levels of phosphorylated (active) PKC-⑀
increased at 1 h of the repair period and remained increased
during the 1st day of the recovery (Fig. 1A). The ratio of
phosphorylated PKC-⑀ to total PKC-⑀ protein in RPTC homogenates increased 2.5-, 4-, and 3-fold at 1, 2, and 4 h, respectively, after TBHP-induced injury (Fig. 1B). The ratio of
phosphorylated PKC-⑀ to total PKC-⑀ remained elevated until
24 h after TBHP injury. Phosphorylation of PKC-⑀ was associated with the translocation from the cytosol to the particulate
fraction of RPTC, which confirmed PKC-⑀ activation (Fig 1C).
The treatment of RPTC with the PKC-⑀ translocation inhibitor
(20 ␮M myristoylated PKC-⑀V1–2) prevented PKC-⑀ translocation to the membranous fraction of TBHP-injured RPTC,
which suggested that PKC-⑀V1–2 decreases PKC-⑀ activation
(Fig. 1D).
Mitochondrial PKC-⑀ during RPTC repair after TBHPinduced injury. Our previous report demonstrated that PKC-␣
is present in RPTC mitochondria (30). This is the first report
demonstrating the presence of PKC-⑀ in RPTC mitochondria.
The levels of PKC-⑀ and phosphorylated PKC-⑀ in RPTC
mitochondria decreased at 1 h after TBHP exposure but increased fourfold and sixfold at 4 and 6 h of the recovery period,
respectively (Fig. 2). On days 1, 2, and 4 after injury, the levels
of phosphorylated PKC-⑀ were similar in recovering RPTC and
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after solubilization in a buffer containing 100 mM Tris䡠HCl (pH 7.5),
150 mM NaCl, and 0.05% Triton X-100.
Mitochondrial membrane potential. Mitochondrial membrane potential (⌬⌿m) was assessed as described previously (30) using JC-1,
a cationic dye that exhibits potential-dependent accumulation and
formation of red fluorescent J-aggregates in mitochondria, which is
indicated by a fluorescence emission shift from green (525 nm) to red
(590 nm). At different time points of the recovery period, RPTC
monolayers were loaded with 10 ␮M JC-1 for 30 min at 37°C. After
being loaded, media were aspirated, and the monolayers were put on
ice, washed with ice-cold PBS, scraped off culture dishes, washed
again, and resuspended in PBS. Fluorescence was determined by flow
cytometry (FACSCalibur; BD Biosciences) using excitation by a
488-nm argon-ion laser. The JC-1 monomer (green) and the Jaggregates (red) were detected separately in FL1 (emission, 525 nm)
and FL2 (emission, 590 nm) channels, respectively. ⌬⌿m is presented
as the red-to-green fluorescence intensity ratio.
Rb⫹ uptake. Ouabain-dependent 86Rb⫹ uptake was used as a
cognate for measurement of ouabain-dependent K⫹ transport in
RPTC. Monolayers were incubated for 10 min at 37°C in the presence
of 10 mM HEPES, 140 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM
CaCl2, and 10 ␮M bumetamide (pH 7.4) and in the absence or
presence of 1 mM ouabain. This preincubation period allowed the
restoration of transmembrane ion gradients as well as bumetamide and
ouabain binding. Under these conditions, intracellular Na⫹ concentration is rate limiting for Na⫹-K⫹-ATPase-mediated Rb⫹ uptake.
The reaction was initiated by adding 86RbCl (1 ␮Ci; sp act 5.1
mCi/mg) and was carried out for 5 min. In the initial set of experiments, we determined that this reaction time was within the linear
86
Rb⫹ uptake range (data not shown). The uptake was terminated by
aspiration of the incubation buffer followed by extensive washing of
the monolayers with the ice-cold incubation buffer in which 5 mM
KCl was replaced by 5 mM RbCl. The cells were solubilized, and the
amount of 86RbCl associated with the monolayers was determined by
liquid scintillation spectrometry. Active Rb⫹ uptake was calculated as
the difference between total and ouabain-insensitive Rb⫹ uptake.
Na⫹-K⫹-ATPase activity. RPTC were scraped from the dishes,
resuspended in 5 mM HEPES buffer (pH 7.4), and lysed in 25 mM
imidazole buffer (pH 7.0) containing 0.065% SDS, 1% BSA, and
phosphatase inhibitor cocktail. After incubation for 10 min at room
temperature, 0.6 ml of 0.3% BSA in 25 mM imidazole buffer was
added to lower the SDS concentration, and aliquots were used for
measurement of Na⫹-K⫹-ATPase activity. Na⫹-K⫹-ATPase activity
was determined by measuring the difference between total ATPase
activity and ouabain-insensitive ATPase activity, as described previously (33).
Isolation of cytosolic and particulate fractions. RPTC samples
were harvested at various time points during recovery after TBHP
injury. Monolayers were washed with ice-cold PBS to remove all
nonviable cells, and the remaining cells were scraped from the dishes,
suspended in PBS, pelleted by centrifugation for 15 s in a microfuge,
and resuspended in ice-cold isolation buffer (20 mM Tris䡠HCl, pH
7.5, containing 10 mM MgCl2, 2 mM EGTA, 2 mM EDTA, 1 mM
NaF, 1 mM Na3VO4, 2 mM dithiothreitol, protease inhibitor cocktail,
and phosphatase inhibitor cocktail). After centrifugation at 1,000 g for
5 min to remove cell debris and nuclei, the supernatant was spun down
at 100,000 g for 30 min at 4°C. The supernatant resulting from the
ultracentrifugation represented the cytosolic fraction. The pellet was
resuspended in the isolation buffer containing 1% Triton X-100 and,
after 30 min incubation on ice, was centrifuged at 100,000 g for 30
min. The supernatant resulting from this ultracentrifugation represented the original particulate fraction. The cytosolic and particulate
fractions were combined with Laemmli sample buffer (21), boiled,
and used for immunoblotting.
Isolation of RPTC mitochondria. RPTC were homogenized in the
ice-cold isolation buffer [225 mM mannitol, 10 mM HEPES (adjusted
to pH 7.4 with KOH), 75 mM sucrose, 0.1 mM EGTA, and 0.1% BSA
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PKC-⑀ MODULATES MITOCHONDRIAL FUNCTION AND NA⫹ TRANSPORT
Fig. 1. A: protein levels of phosphorylated (P) protein kinase C (PKC)-⑀ and
total PKC-⑀ in renal proximal tubular cell (RPTC) homogenates during the
recovery after tert-butylhydroperoxide (TBHP)-induced injury. B: PKC-⑀ activation (the ratio of phosphorylated PKC-⑀ to total PKC-⑀). C: PKC-⑀ levels
in cytosolic and particulate fractions in RPTC during the recovery after
TBHP-induced injury. D: PKC-⑀ levels in cytosolic and particulate fractions in
RPTC grown in the presence of PKC-⑀ inhibitor, PKC-⑀V1–2, during and after
TBHP-induced injury. Blots in A are representative of 4 independent experiments. Results (quantified by densitometry) in B are averages ⫾ SE of 4
independent experiments (RPTC isolations). Blots in C and D are representative of 3 independent experiments. *P ⬍ 0.05, significantly different from 0 h
(controls).
QO2. Basal QO2 was used as a marker of the overall function
of RPTC mitochondria. At 4 h after TBHP exposure, basal QO2
in TBHP-injured RPTC was decreased to 42% of control levels
(Fig. 3). The inhibition of PKC-⑀ activation by PKC-⑀V1–2
reduced decreases in basal QO2 to 63% of controls (Fig. 3).
Basal QO2 in TBHP-injured RPTC recovered on day 4 after the
exposure regardless of the presence or absence of PKC-⑀V1–2
(Fig. 3).
Uncoupled QO2 was used as a marker of the mitochondrial
electron transfer rate and the integrity of respiratory complexes. Uncoupled QO2 in TBHP-treated RPTC decreased to
39% of control levels at 4 h after the injury (Fig. 4). The
inhibition of PKC-⑀ activation by PKC-⑀V1–2 reduced TBHPinduced decreases in uncoupled QO2 to 57% of controls at 4 h
of the recovery (Fig. 4). The inhibition of PKC-⑀ had no effect
on uncoupled QO2 in TBHP-treated RPTC on day 4 (Fig. 4).
in controls (Fig. 2, data not shown). These data demonstrate
that active (phosphorylated) PKC-⑀ is translocated to RPTC
mitochondria during the early recovery after TBHP-induced
injury.
Fig. 2. Protein levels of phosphorylated PKC-⑀ and total PKC-⑀ in RPTC
mitochondria during the recovery after TBHP-induced injury. Blots are representative of 3 independent experiments (RPTC isolations).
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Fig. 4. Uncoupled oxygen consumption (a marker for electron transfer rate)
during RPTC recovery after TBHP-induced injury. Results are averages ⫾ SE
of 8 independent experiments (RPTC isolations). Values with dissimilar
superscripts on a given day are significantly different (P ⬍ 0.05) from each
other.
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Fig. 3. Basal oxygen consumption (QO2, a marker for overall mitochondrial
function) during RPTC recovery after TBHP-induced injury. Results are
averages ⫾ SE of 8 independent experiments (RPTC isolations). Values with
dissimilar superscripts on a given day are significantly different (P ⬍ 0.05)
from each other.
PKC-⑀ MODULATES MITOCHONDRIAL FUNCTION AND NA⫹ TRANSPORT
Because the data suggested that the components of the
respiratory chain are the targets of PKC-⑀, we examined
whether respiration coupled to complexes I, II, or IV is under
the control of PKC-⑀. Complex I-coupled respiration (measured using glutamate and malate as electron donors) decreased
to 52% of controls at 4 h after TBHP-induced injury (Fig. 5A).
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In contrast, TBHP-induced injury had no effect on complex IIand complex IV-coupled respirations (Fig. 5, B and C). PKC⑀V1–2 had no effect on complex I-coupled respiration in
controls but diminished TBHP-induced decreases in site I-coupled respiration to 74% of those in control RPTC (Fig. 5A).
Furthermore, PKC-⑀V1–2 stimulated complex II-coupled respiration 1.5-fold in TBHP-injured RPTC, whereas it had no
effect in controls (Fig. 5B). Complex IV-linked respiration in
TBHP-injured RPTC at 4 h of the recovery period was stimulated 1.4-fold by the inhibition of PKC-⑀ activation but was
unaffected in controls (Fig. 5C).
These results demonstrate that PKC-⑀ activation plays a role
in mitochondrial dysfunction in TBHP-injured RPTC and that
the inhibition of PKC-⑀ activation protects against decreases in
mitochondrial respiration. Furthermore, these data show that
the electron transport chain is a target of PKC-⑀ during oxidant
injury and that the inhibition of PKC-⑀ activation in recovering
RPTC increases respiration through complexes I, II, and IV of
the respiratory chain.
ATP production. ATP synthesis is the fundamental function
of the mitochondria. The rate of ATP synthesis was measured
during state III (the maximal capacity of mitochondria to
generate ATP) in digitonin-permeabilized RPTC incubated in
the presence of ADP, and metabolic substrates linked to the
respiratory complex I (glutamate ⫹ malate) and the respiratory
complex II (succinate). The rate of ATP production in the
presence of electron donors linked to complex I was 43.9 ⫾ 4.1
nmol䡠mg protein⫺1 䡠min⫺1 in control RPTC. The early recovery period after TBHP exposure was associated with the
decrease in complex I-mediated ATP production to 59% of the
control levels (Fig. 6A). This decrease was prevented by the
inhibition of PKC-⑀ activation in TBHP-injured RPTC (Fig.
6A). Complex I-linked ATP synthesis in TBHP-injured RPTC
recovered on day 4 regardless of the presence or absence of
PKC-⑀V1–2. In contrast, ATP synthesis in the presence of
complex II-linked substrate (succinate) was not affected by
TBHP-induced injury, and PKC-⑀V1–2 had no effect on ATP
synthesis supported by succinate (Fig. 6B). These results demonstrate that PKC-⑀ mediates TBHP-induced decreases in ATP
production coupled to the oxidation of electron donors to
complex I.
Intracellular ATP levels. Intracellular ATP contents were
examined during RPTC recovery to determine whether the
inhibition of PKC-⑀ activation promotes intracellular ATP
levels in recovering RPTC after TBHP injury. ATP content in
control RPTC was 10.6 ⫾ 1.7 nmol/mg protein, and PKC⑀V1–2 had no effect on ATP levels in control RPTC (Fig. 7).
ATP content was diminished by 54% at 4 h of the recovery
after TBHP exposure, and the inhibition of PKC-⑀ activation
had no effect on the decreases in the intracellular ATP content
in TBHP-injured RPTC (Fig. 7). ATP content recovered on day
4 after TBHP injury regardless of the presence or absence of
PKC-⑀V1–2 (Fig. 7).
⌬⌿m. Mitochondrial respiration results in the generation of
proton and pH gradients across the inner mitochondrial membrane and produces the ⌬⌿m, which represents most of the
energy of the proton gradient. ⌬⌿m in RPTC was assessed by
the measurement of changes in the J-aggregate-to-JC-1 monomer ratio. The J-aggregate-to-JC-1 monomer ratio in control
RPTC was 1.15 ⫾ 0.31 and was not altered at 4 h or on day 4
after TBHP-induced injury (Fig. 8). Inhibition of PKC-⑀ acti-
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Fig. 5. Oxygen consumption coupled to complex I (A), complex II (B), and
complex IV (C) during RPTC recovery after TBHP-induced injury. Results are
averages ⫾ SE of 8 independent experiments (RPTC isolations). Values with
dissimilar superscripts on a given day are significantly different (P ⬍ 0.05)
from each other.
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PKC-⑀ MODULATES MITOCHONDRIAL FUNCTION AND NA⫹ TRANSPORT
Fig. 6. ATP production rate in the presence of electron donors linked to
complex I (A) and complex II (B) during RPTC recovery after TBHP-induced
injury. Results are averages ⫾ SE of 5 independent experiments (RPTC
isolations). Values with dissimilar superscripts on a given day are significantly
different (P ⬍ 0.05) from each other.
vation had no effect on ⌬⌿m in control or TBHP-injured RPTC
(Fig. 8). These data show that PKC-⑀ does not play any role in
the maintenance of the ⌬⌿m in RPTC.
Active Na⫹ transport. Active Na⫹ transport was used as a
marker of the basolateral membrane function and was assessed by
the measurement of ouabain-sensitive Rb⫹ uptake and the activity
of Na⫹-K⫹-ATPase. Ouabain-sensitive Rb⫹ uptake in control
RPTC was 36.3 ⫾ 4.8 nmol RbCl2䡠mg protein⫺1䡠min⫺1 and was
unaffected by PKC-⑀V1–2 (Fig. 9). At 4 h of the recovery period
after TBHP-induced injury, ouabain-sensitive Rb⫹ uptake was
decreased to 47% of levels in control RPTC (Fig. 9). However, in
the presence of PKC-⑀V1–2, ouabain-sensitive Rb⫹ uptake in
TBHP-treated RPTC was 67% of control levels (Fig. 9). Ouabainsensitive Rb⫹ uptake returned on day 4 after TBHP exposure
regardless of the presence or absence of PKC-⑀V1–2 (Fig. 9).
Na⫹-K⫹-ATPase activity was 290 ⫾ 25 mU䡠mg
protein⫺1 䡠min⫺1 in controls. TBHP-induced injury was associated with a 60% decrease in the Na⫹-K⫹-ATPase activity at
4 h of the recovery period (Fig. 10). On day 4 of the recovery
period, the Na⫹-K⫹-ATPase activity in TBHP-injured RPTC
was higher than at 4 h but still 29% lower than in controls (Fig.
10). Inhibition of PKC-⑀ translocation by PKC-⑀V1–2 had no
AJP-Renal Physiol • VOL
effect on the Na⫹-K⫹-ATPase activity in control RPTC but
prevented TBHP-induced decreases in the Na⫹-K⫹-ATPase
activity at 4 h after the injury and maintained the pump activity
throughout the 4-day recovery period (Fig. 10; data not
shown).
These data show that inhibition of PKC-⑀ activation in
TBHP-treated RPTC protects against the decreases in the
activity of Na⫹-K⫹-ATPase and active Na⫹ transport and
maintains this function during the recovery period.
DISCUSSION
PKC isozymes have been implicated in a variety of cellular
functions and physiological and pathophysiological responses
in different tissues. These responses include proliferation, mi-
Fig. 8. Mitochondrial membrane potential in RPTC during the recovery after
TBHP-induced injury. Results are expressed as the ratio of the aggregate-tomonomeric form of JC-1. Results are averages ⫾ SE of 5 independent
experiments (RPTC isolations). Values with dissimilar superscripts on a given
day are significantly different (P ⬍ 0.05) from each other.
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Fig. 7. Intracellular content of ATP in RPTC during the recovery after
TBHP-induced injury. Results are averages ⫾ SE of 4 independent experiments (RPTC isolations). Values with dissimilar superscripts on a given day
are significantly different (P ⬍ 0.05) from each other.
PKC-⑀ MODULATES MITOCHONDRIAL FUNCTION AND NA⫹ TRANSPORT
gration, permeability, contraction, secretion, injury, and apoptosis (11). It has been shown that PKC-⑀ plays an important
role in ischemic damage in the heart and that PKC-⑀ activation
is required for the protection of cardiac tissue from ischemiainduced cell death (16). The involvement of PKC-⑀ in cell
survival has been also demonstrated in cancer cells (12). The
protective effects of PKC-⑀ in cancer cells are the result of
dysregulation of one or more of the mitochondrial apoptotic
pathways responsible for caspase activation (12). PKC
isozymes have been also implicated in the renal recovery after
the ARF (1, 22) and in wound healing after mechanically
induced injury in renal tubular epithelial cells (39). The activation of PKC-␦, PKC-⑀, and PKC-␨, but not PKC-␣ or
PKC-␤, occurs during compensatory renal hypertrophy induced by unilateral nephrectomy (13). Our previous study has
shown that PKC plays an important role in the recovery of
mitochondrial and transport functions in RPTC after toxicantinduced injury (31). These data suggested that PKC-␣ and/or
PKC-⑀ are involved in the repair of RPTC functions after
sublethal injury induced by the oxidant, TBHP (31).
Our present results demonstrate that PKC-⑀ is activated
during the early recovery after oxidant injury as shown by both
the increases in phosphorylation and translocation of PKC-⑀
from the cytosolic to the particulate fraction of RPTC. These
data are consistent with recent reports showing that PKC-⑀ is
activated by oxidative stress induced by hyperglycemia in the
diabetic kidney (17). The activation of PKC-⑀ was most pronounced within a few hours after TBHP-induced injury, which
suggested that PKC-⑀ plays a role in the early repair process in
RPTC. PKC-⑀ activation subsided at 24 h after the injury and
was followed by the recovery of RPTC functions on day 4.
Elucidating the function of a specific PKC isozyme in the
presence of other PKCs requires selective tools to inhibit the
isozyme under study. Many current approaches include the use
of isozyme nonselective pharmacological inhibitors, preventing a meaningful interpretation of the results. Several years
ago, it was demonstrated that a translocation inhibitor peptide
(corresponding to the PKC-⑀-binding site for the specific reAJP-Renal Physiol • VOL
ceptor for activated C kinase, RACK) acts as a selective
competitor of PKC-⑀ translocation, binding, and function (14,
16). This inhibitor peptide (PKC-⑀V1–2) has been used to
demonstrate that the activation of PKC-⑀ mediates the protective effects of ischemic preconditioning in cardiac myocytes
(16).
In the present study, we used PKC-⑀V1–2 to block PKC-⑀
translocation during TBHP exposure and the recovery period
and to decrease PKC-⑀ function in regenerating RPTC. The use
of PKC-⑀V1–2 allowed us to show that PKC-⑀ mediates the
decreases in mitochondrial respiration and ATP production
after sublethal injury produced by the oxidant TBHP. Specifically, the inhibition of PKC-⑀ activation with PKC-⑀V1–2
diminished TBHP-induced decreases in basal and uncoupled
QO2 values. These data suggested that the respiratory chain is
the target for PKC-⑀. To look further into the mechanism of
these changes, we tested whether the complexes of the mitochondrial respiratory chain are the target(s) of PKC-⑀. Different oxidative substrates were used as electron donors to differentiate between respiration linked to the generation of
NADH and complex I, FADH2 production and complex II, or
complex IV. Electrons (reducing equivalents) from glutamate
and malate enter the electron transport chain mainly as NADH
at complex I. Electrons from succinate enter the electron
transport chain as FADH2 at complex II. Electrons from both
complexes I and II then feed into the Q cycle portion of
complex III. Interestingly, TBHP exposure decreased only
respiration linked to respiratory complex I but had no effect on
respiration linked to complexes II and IV. Because respiration
linked to complex II was not affected during TBHP exposure
and RPTC recovery, we conclude that the respiratory complex
III was not affected by sublethal injury induced by TBHP.
These data also suggest that the availability of ubiquinone,
which carries the electrons between complexes I or II and
complex III, was adequate to support the electron transport.
Therefore, we conclude that sublethal concentrations of TBHP
target components of the respiratory complex I or NADHproducing dehydrogenases upstream of complex I but have no
apparent effects on complex II and FADH2 production. The
Fig. 10. Na⫹-K⫹-ATPase activity in RPTC lysates during the recovery after
TBHP-induced injury. Results are averages ⫾ SE of 4 independent experiments (RPTC isolations). Values with dissimilar superscripts on a given day
are significantly different (P ⬍ 0.05) from each other.
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Fig. 9. Ouabain-sensitive 86Rb⫹ uptake (a marker of active Na⫹ transport) in
RPTC during the recovery after TBHP-induced injury. Results are averages ⫾
SE of 4 independent experiments (RPTC isolations). Values with dissimilar
superscripts on a given day are significantly different (P ⬍ 0.05) from each
other.
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PKC-⑀ MODULATES MITOCHONDRIAL FUNCTION AND NA⫹ TRANSPORT
AJP-Renal Physiol • VOL
Na⫹-K⫹-ATPase activity can be primarily controlled by PKC
through phosphorylation of the catalytic ␣-subunit of the pump
on serine and threonine residues (20, 24). In contrast to other
cell types, Na⫹-K⫹-ATPase activity in renal proximal tubules
is inhibited through phosphorylation by PKC (3, 20). Furthermore, the levels of intracellular Na⫹ modulate the effects of
PKC-mediated phosphorylation on Na⫹-K⫹-ATPase activity.
Increased intracellular Na⫹ concentrations are associated with
the inhibition of Na⫹-K⫹-ATPase activity in response to phosphorylation (15).
We assessed the function of Na⫹-K⫹-ATPase in the cell by
measuring the ouabain-sensitive 86Rb⫹ uptake and showed that
pump function is decreased early after TBHP exposure and
recovers on day 4 of the repair. Inhibition of PKC-⑀ activation
by PKC⑀V1–2 in TBHP-injured RPTC significantly reduced
the decreases in ouabain-sensitive 86Rb⫹ uptake during the
early recovery period. This suggested that the decrease in
active Na⫹ transport in RPTC after TBHP injury is dependent
on PKC-⑀ activation and that PKC-⑀ activation decreases
Na⫹-K⫹-ATPase function. The decrease in the pump function
of Na⫹-K⫹-ATPase could be the result of the decrease of
Na⫹-K⫹-ATPase catalytic activity or the reduced number of
pump molecules on the basolateral membrane. Our study
determined that the catalytic activity of Na⫹-K⫹-ATPase in
cell lysates is decreased in TBHP-injured RPTC but is maintained by treatment with PKC⑀V1–2. These results suggest that
the phosphorylation of Na⫹-K⫹-ATPase by PKC-⑀ decreases
the pump activity and show that the inhibition of PKC-⑀
activation during the early repair process maintains the catalytic activity of Na⫹-K⫹-ATPase. Therefore, our results suggest that Na⫹-K⫹-ATPase is an effector protein for PKC-⑀ in
TBHP-injured RPTC. Our data are in agreement with the study
by Buhagiar et al. (7), who demonstrated that PKC-⑀ regulates
the sarcolemmal Na⫹-K⫹ pump in cardiac myocytes. However, it is unknown, yet, whether PKC-⑀ phosphorylates Na⫹K⫹-ATPase directly or through activation of a downstream
kinase.
Thus our data show that oxidant-induced injury activates
PKC-⑀ in RPTC and that this activation mediates decreases in
mitochondrial function, specifically in complex I-mediated
respiration, active Na⫹ transport, and Na⫹-K⫹-ATPase activity. Therefore, the activation of PKC-⑀ in renal cells appears to
play a role distinct from the protective effects in the cardiac
tissue. In the heart, the activation of PKC-⑀ has been shown to
protect against injury, and it is thought that this protection is
through mitochondrial and/or transport mechanisms (2, 8, 9,
14, 37, 38, 42–45). In contrast, oxidant-induced activation of
PKC-⑀ in renal cells is detrimental to the respiratory and
transport functions, and the inhibition of PKC-⑀ activation
offers protection against the decrease in these functions. We
speculate that this apparent difference between cardiac and
renal responses to activation of PKC-⑀ may be the result of
different downstream targets of PKC-⑀ in cardiomyocytes and
RPTC, and phosphorylation of different mitochondrial enzymes that regulate oxidative metabolism or other proteins
essential for mitochondrial function. Furthermore, the effects
of PKC-⑀-mediated regulation of Na⫹-K⫹-ATPase, ion channels, and/or transporter proteins may be different in cardiomyocytes and RPTC, thus affecting ion homeostasis and cell
survival.
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lack of effects of TBHP on the ⌬⌿m at 4 h after the exposure
suggests that the integrity of the inner mitochondrial membrane
was not compromised and that mitochondrial NADH was not
lost because of leakage from the mitochondria.
Inhibition of PKC-⑀ activation with PKC⑀V1–2 reduced
decreases in the respiration linked to complex I at 4 h of the
recovery of TBHP-injured RPTC. This protective effect could
be the result of preserving the activities of NADH-producing
dehydrogenases in the tricarboxylic acid cycle or maintaining
the activity of complex I. The choice of respiratory substrates
used in this study precluded the involvement of dehydrogenases upstream of the tricarboxylic acid cycle. At the present
time, it is not clear whether complex I or the dehydrogenases
of the tricarboxylic acid cycle are the targets for PKC-⑀. It has
been shown, however, that the activities of aconitase and the
␣-ketoglutarate dehydrogenase complex are decreased by oxidative stress induced by H2O2 (41). The ␣-ketoglutarate dehydrogenase complex has been the most extensively studied
component of the tricarboxylic acid cycle, since it is a key
regulatory component of oxidative metabolism. It has been
shown that the ␣-ketoglutarate dehydrogenase complex is sensitive to some nephrotoxicants and heavy metals, including
zinc (5, 6, 41). Therefore, we speculate that, as a key regulatory
enzymatic complex of the oxidative metabolism, the ␣-ketoglutarate dehydrogenase is a very likely target of TBHP and
PKC-⑀ in our model. However, because the inhibition of
PKC-⑀ activation restored the respiration only partially, we
conclude that mechanisms other than PKC-⑀ contribute to the
decreases in complex I-linked respiration. Alternatively, the
TBHP-induced decline in complex I respiration is the result of
the decrease in activities of multiple enzymes of the tricarboxylic acid cycle and complex I, but PKC-⑀ targets only one of
them.
The decrease in QO2 in TBHP-injured RPTC was accompanied by a decline in glutamate- and malate-linked ATP production, whereas succinate-coupled ATP production remained
unaffected throughout the repair period. These results further
support the conclusion that the flow of electrons through
complex I but not complex II, III, or IV is decreased during the
early recovery of TBHP-injured RPTC. Furthermore, consistent with the promotion of respiration, blocking PKC-⑀ activation restored the glutamate- and malate-supported ATP production rate at 4 h of the recovery. Thus our results suggest that
the respiratory function and ATP synthesis in TBHP-injured
RPTC are, in part, under the control of PKC-⑀. However,
despite the increased ATP production rate at 4 h of the
recovery, the intracellular ATP content in TBHP-injured RPTC
was not restored in the presence of a PKC-⑀ inhibitor, which
suggested an increase in the ATP-consuming processes.
Active ion transport consumes a large portion of energy
(ATP) generated by renal proximal tubules. The proximal
tubule is the principal site of renal Na⫹ reabsorption, which
involves Na⫹-K⫹-ATPase, the ion pump using metabolic energy derived from ATP hydrolysis and generating the electrochemical gradient of Na⫹ and K⫹ across the plasma membrane. Na⫹-K⫹-ATPase is the main determinant of low cytosolic levels of Na⫹ and therefore plays a central role in the
regulation of cell volume, intracellular electrolyte content,
secondary active transmembrane transport of other ions, and
cellular uptake of organic compounds cotransported with sodium. It has been shown that, in proximal convoluted tubules,
PKC-⑀ MODULATES MITOCHONDRIAL FUNCTION AND NA⫹ TRANSPORT
In conclusion, PKC-⑀ plays a role in the early phase of the
recovery of mitochondrial function and active Na⫹ transport
after oxidant injury in RPTC. PKC-⑀ activation decreases
mitochondrial respiration, electron transport rate, and ATP
production by decreasing the electron flow through complex I.
The data also suggest that Na⫹-K⫹-ATPase is an effector
protein for active PKC-⑀, thereby establishing a molecular link
between oxidant injury and the decrease in active Na⫹ transport in RPTC. Thus the inhibition of PKC-⑀ activation early
after oxidant injury in renal proximal tubules represents a
means to promote the repair of mitochondrial function and
active Na⫹ transport.
ACKNOWLEDGMENTS
15.
16.
17.
18.
19.
20.
GRANTS
This work was supported by National Institute of Diabetes and Digestive
and Kidney Diseases Grant R01DK-59558.
21.
22.
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