Am J Physiol Renal Physiol 303: F1264F1274, 2012.

First published August 29, 2012; doi:10.1152/ajprenal.00227.2012.

Translational Physiology

Epicatechin limits renal injury by mitochondrial protection in cisplatin

nephropathy
Katsuyuki Tanabe,1 Yoshifuru Tamura,1 Miguel A. Lanaspa,1 Makoto Miyazaki,1 Norihiko Suzuki,2
Waichi Sato,2 Yohei Maeshima,3 George F. Schreiner,4 Francisco J. Villarreal,5 Richard J. Johnson,1
and Takahiko Nakagawa1,6
1

Division of Renal Diseases and Hypertension, University of Colorado Denver, Aurora, Colorado; 2Division of Nephrology,
Nagoya University, Nagoya, Japan; 3Department of Medicine and Clinical Science, Okayama University Graduate School,
Okayama, Japan; 4Cardero Therapeutics, Incorporated, Menlo Park, California; 5Department of Cardiology, University
of California, San Diego, California; and 6TMK Project, Medical Innovation Center, Kyoto University Graduate School
of Medicine, Kyoto, Japan
Submitted 18 April 2012; accepted in final form 28 August 2012

Tanabe K, Tamura Y, Lanaspa MA, Miyazaki M, Suzuki N,

Sato W, Maeshima Y, Schreiner GF, Villarreal FJ, Johnson RJ,
Nakagawa T. Epicatechin limits renal injury by mitochondrial
protection in cisplatin nephropathy. Am J Physiol Renal Physiol 303: F1264 F1274, 2012. First published August 29, 2012;
doi:10.1152/ajprenal.00227.2012.Cisplatin nephropathy can be
regarded as a mitochondrial disease. Intervention to halt such deleterious injury is under investigation. Recently, the flavanol ()-epicatechin emerges as a novel compound to protect the cardiovascular
system, owing in part to mitochondrial protection. Here, we have
hypothesized that epicatechin prevents the progression of cisplatininduced kidney injury by protecting mitochondria. Epicatechin was
administered 8 h after cisplatin injury was induced in the mouse
kidney. Cisplatin significantly induced renal dysfunction and tubular
injury along with an increase in oxidative stress. Mitochondrial
damages were also evident as a decrease in loss of mitochondrial mass
with a reduction in the oxidative phosphorylation complexes and low
levels of MnSOD. The renal damages and mitochondrial injuries were
significantly prevented by epicatechin treatment. Consistent with
these observations, an in vitro study using cultured mouse proximal
tubular cells demonstrated that cisplatin-induced mitochondrial injury,
as revealed by a decrease in mitochondrial succinate dehydrogenase
activity, an induction of cytochrome c release, mitochondrial fragmentation, and a reduction in complex IV protein, was prevented by
epicatechin. Such a protective effect of epicatechin might be attributed
to decreased oxidative stress and reduced ERK activity. Finally, we
confirmed that epicatechin did not perturb the anticancer effect of
cisplatin in HeLa cells. In conclusion, epicatechin exhibits protective
effects due in part to its ability to prevent the progression of mitochondrial injury in mouse cisplatin nephropathy. Epicatechin may be
a novel option to treat renal disorders associated with mitochondrial
dysfunction.
AKI; cocoa; complex IV; dark chocolate; MnSOD
CISPLATIN IS ONE OF THE MOST widely used chemotherapeutic
agents in patients with cancer of the testes, head, neck, ovary,
cervix, lung, and other organs. Cisplatin is particularly beneficial in testicular cancer, where the cure rate exceeds 90% and
is nearly 100% for early stages (7, 34, 38). However, side
effects of cisplatin, especially in the kidney, limit its use.
Specifically, tubular epithelial cells in the kidney are one of the

Address for reprint requests and other correspondence: T. Nakagawa, TMK

Project, Medical Innovation Center, Kyoto Univ. Graduate School of Medicine, 53 Shogoin Kawaharacho, Sakyo-ku, Kyoto, Japan 606-8507 (e-mail:
nakagawt@gmail.com).
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major targets for cisplatin (2, 30). Many research groups have
been intensively investigating how cisplatin injures tubular
cells to find a way to protect the kidney.
One mechanism could be by impairing mitochondrial function. Previous studies demonstrated that cisplatin induces activation of the Bcl2-family protein Bax, which forms pores on
the mitochondrial outer membrane (24, 40). The p53/PUMA-
pathway is also activated by cisplatin and leads to mitochondrial membrane damage (19, 20). Recent reports have suggested that cisplatin also directly binds mitochondrial DNA
and the voltage-dependent anion channel in cancer cells (43).
The injury to the mitochondrial membrane causes a loss of
membrane potential and an impaired function in mitochondrial
oxidative phosphorylation (22, 23). Similarly, cisplatin-induced apoptosis could be caused by a release of cytochrome c
from mitochondria as a consequence of an increase in mitochondrial membrane permeability (5). This anticancer agent
also impairs mitochondrial structure, leading to mitochondrial
fragmentation (5).
()-Epicatechin is a flavonoid (a subclass of polyphenols)
and is predominantly contained in cacao. Therefore, cocoa
powder or dark chocolate is typically rich in epicatechin (8,
13). Our attention to the favorable effect of cocoa-derived
flavonoid was generated from an epidemiological observation
in the Kuna Indians, a native population living on islands off
the coast of Panama (16, 17). The island-living Kuna belong to
one of the few cultures that are protected against age-related
cardiovascular disease, including an increase in blood pressure
and a decline in kidney function (16, 17). Interestingly, this
native population regularly consumes significant amounts of
cocoa drink (16). For those Kuna who moved to mainland
Panama, where the homemade cocoa was not available, the
protection from cardiovascular disease was lost (16).
Recently, Villarreals research group (28, 31, 32, 36, 41, 42)
has examined the effects of epicatechin in the setting of
cardiovascular disease. They showed that the beneficial effect
of epicatechin could be due in part to protection of mitochondria (36). Huttemann et al. (18) also recently reported that
epicatechin could maintain exercise-induced mitochondrial capacity in the hindlimb muscles of mice even after discontinuation of exercise.
Given evidence that cisplatin induces mitochondrial injury
in tubular cells, we tested the hypothesis that epicatechin might
alleviate cisplatin-induced renal damage by protecting mitochondria.

1931-857X/12 Copyright 2012 the American Physiological Society

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Translational Physiology
EPICATECHIN LIMITS MITOCHONDRIAL DAMAGE IN CISPLATIN AKI
METHODS

Experimental protocols. All animal experiments were performed in

accordance with the Animal Care and Use Committee of the University of Colorado. Male C57BL/6J mice (Jackson Laboratory, Bar
Harbor, ME) at 8 wk of age were used in the present study. The mice
were fed a standard pellet laboratory chow ad libitum. Mice were
divided into four subgroups: 1) a control (saline) group, 2) an
epicatechin-treated control group, 3) a cisplatin group, and 4) an
epicatechin-treated cisplatin group (n 8 12 for each group). Cisplatin [cis-diamminedichloro-platinum (II); Aldrich Chemical, Milwaukee, WI] was dissolved in normal (0.9%) saline at a concentration
of 1 mg/ml immediately before administration. Mice were given 30
mg/kg of cisplatin or normal saline (as a vehicle control) intraperitoneally to induce acute kidney injury (AKI). As previously reported
(12), this model shows overt renal dysfunction and severe tubular
injury on day 3 after cisplatin injection. First of all, ()-epicatechin
(Sigma-Aldrich, St. Louis, MO) was prepared in normal saline every
time before injection, and the dosage of 1 mg/kg was given intraperitoneally twice a day until the day before death. Epicatechin treatment
started 8 h after cisplatin injection (Fig. 1A). All the mice were
euthanized 72 h after the injection of cisplatin, and blood samples and
kidney tissues were obtained.
Renal function. Blood urea nitrogen (BUN) was measured using an
Alfa Wassermann VetACE autoanalyzer (Schiaparelli Biosystems).
Serum creatinine concentration was analyzed with HPLC-MS/MS
(Applied Biosystems 3200 Qtrap). Creatinine and [2H3]creatinine
were detected in multiple reaction monitoring mode, monitoring the
transitions of the m/z from 114 to 44.2 and from 117 to 47.2,
respectively.
Histological analysis. Formalin-fixed, paraffin-embedded sections
(3 m) were stained with the periodic acid-Schiff reagent (PAS) for
light microscopy. On coronal sections of the kidney, acute tubular
injuries were evaluated and quantified as the acute tubular necrosis
(ATN) score, which was determined by the percentage of tubules
displaying detachment of epithelium, loss of brush border, or cast
formation, as follows: 0 none, 1 10%, 2 10 25%, 3
26 50%, 4 5175%, and 5 75%. Ten fields on each section at

Cisplatin

Epicatechin

day 0

day 1

day 2

day 3

With/without
Epicatechin

With/without
Cisplatin

-1

Sacrifice

24 hours

Fig. 1. Experimental designs for in vivo and in vitro study. A: experimental

design for mouse study. Eight hours after cisplatin injection, epicatechin
treatment was started every 12 h by day 3. B: experimental design for cell
culture study. After incubation of mouse proximal tubular cells with 100
M cisplatin for 1 h, cisplatin-containing culture medium was removed and
replaced with new medium with/without epicatechin and incubated for
4 24 h.

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200 magnification were examined. Kidney sections were observed

by two investigators in a blinded manner.
Apoptosis. For the detection of apoptotic cells in the kidney,
terminal uridine deoxynucleotidyl transferase-mediated dUTP nickend labeling (TUNEL) staining was performed on frozen section (10
m) using a TACS 2 TdT-Blue Label In Situ Apoptosis Detection Kit
(Trevigen, Gaithersburg, MD) according to the manufacturers protocol. Digital images were obtained with a Scan Scope CS Digital Slide
Scanner (Aperio, Vista, CA) at 200 magnification. Ten fields were
examined to determine the average number of TUNEL-positive nuclei
in each section.
Immunohistochemistry. Formalin-fixed, paraffin-embedded sections
(3 m) were used for immunohistochemistry as previously described
(35). The following antibodies were used as primary antibodies: 1) rabbit
anti-4-hydroxynonenal (4-HNE) antibody (Abcam, Cambridge, MA);
and 2) rabbit anti-nitrotyrosine antibody (Chemicon International, Temecula, CA). Briefly, after deparaffinization, the sections were treated
with 3% H2O2 for 10 min to inactivate endogenous peroxidase activity.
The sections were treated with 10 mM citrate buffer (pH 6.0) for 30 min
in a steamer for antigen retrieval. After incubation with a background
sniper (Biocare Medical, Concord, CA) for 15 min, sections were
incubated with primary antibodies overnight at 4C. Then, the sections
were incubated with Mach2 rabbit horseradish peroxidase (HRP) polymer (Biocare Medical). Diaminobenzidine (DAB) was used as a chromogen. Slides were counterstained with hematoxylin. Digital images
were obtained with a Scan Scope CS Digital Slide Scanner (Aperio) at
200 magnification.
Mitochondrial DNA content. Total DNA was extracted from kidney
tissue and purified using DNeasy Blood and Tissue Kit (Qiagen,
Chatsworth, CA). For the detection of cytochrome c oxidase subunit
(COX)-II, a mitochondrial gene and UCP-2 (nuclear gene encoding
mitochondrial protein), the following oligonucleotide primers were
used: COX-II, 5=- TTTTCAGGCTTCACCCTAGATGA-3= (forward)
and 5=-GAAGAATGTTATGTTTACTCCTACGAATATG-3= (reverse);
and UCP-2, 5=-GCGTTCTGGGTACCATCCTAAC-3= (forward) and
5=-GCGACCAGCCCATTGTAGA-3= (reverse). Diluted DNA was
added to SYBR Green JumpStart (Sigma-Aldrich) with the specific
primer to make reaction mixes. The PCR program was optimized and
performed as denaturation at 95C for 3 min followed by 50 cycles of
amplification (COX-II, 95C for 30 s, 55C for 30 s, and 72C for 1
min; and UCP-2, 95C for 30 s, 57C for 30 s, and 72C for 1 min,
respectively) using the MyiQ Single-Color Real-Time PCR Detection
System (Bio-Rad). The amount of COX-II PCR products was normalized with UCP-2 to determine the ratio for mitochondrial DNA/
genomic DNA.
Cell culture. Conditionally immortalized mouse proximal tubular
cells (TKPTS) (10) were cultured with DMEM/F-12 50/50 medium
(Mediatech, Manassas, VA) supplemented with 5% FBS, penicillin
(100 U/l), streptomycin (100 g/l), and SITE-liquid media supplement (Sigma-Aldrich) at 37C. HeLa cells were cultured with MEM
(Mediatech) supplemented with 10% FBS, penicillin (100 U/l), and
streptomycin (100 g/l). At 90% confluence, the cells were stimulated
by 100 M cisplatin in complete medium for 1 hour at 37C before the
cisplatin-containing medium was removed. Then, the cells were cultured
in complete medium with or without 1 107, 5 107, or 1 106
M epicatechin, or 1 mM tempol (Enzo Life Science, Plymouth Meeting,
MA) for appropriate time in each experiment (Fig. 1B).
Cell survival assay. To determine the number of live cells, a
CyQUANT NF Cell Proliferation Assay Kit (Invitrogen), which can
measure nuclear DNA content via fluorescent dye binding, was used.
Mouse proximal tubular cells or HeLa cells were seeded in 96-well
plates at 7,500 or 1,500 cells/well and allowed to adherent at 37C
overnight. One hour after stimulation by 100 M cisplatin, this
medium was replaced with new medium containing various concentrations of epicatechin. Cells were incubated for 4 or 24 h before the
culture medium was replaced with fluorescent dye-containing HBSS,
and incubated for 1 h at 37C. Fluorescent intensity was measured

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EPICATECHIN LIMITS MITOCHONDRIAL DAMAGE IN CISPLATIN AKI

using a Synergy 2 Multi-Mode Microplate Reader (Bio-Tek, Winooski, VT) with excitation at 485 nm and emission detection at 528
nm. A strongly positive correlation was shown between this fluorescence intensity and the real number of cells on 96-well plates (R2
0.98, P 0.05). Cell number was expressed as an estimate (cells/
well) using this trendline equation.
Succinate dehydrogenase activity. A conventional MTT assay was
used to assess the activity of succinate dehydrogenase, complex II as
a mitochondrial oxidative phosphorylation protein (6). Mouse proximal tubular cells were stimulated in the same way as in the abovenoted cell survival assay. The cells were treated with epicatechin in
100 l of complete medium for 4 h after the cisplatin-containing
medium was removed. Then, 20 l of thiazolyl blue tetrazolium
bromide (Sigma-Aldrich) dissolved in PBS at 5 mg/ml was added to
each well, and the plate was incubated for 3 h at 37C. Then, culture
medium was replaced with MTT solvent (4 mM HCl and 0.1% NP-40
in isopropanol), and cells were agitated on an orbital shaker for 15 min
in the dark. Absorbance was read at 590 nm with a reference filter of
620 nm. Data were expressed as values relative to control.
Western blotting. Kidney cortex tissues were homogenized in RIPA
buffer at 4C. Mitochondria were isolated from kidney tissues using a
Mitochondria Isolation Kit for Tissue (Thermo Scientific, Waltham,
MA) and lysed in RIPA buffer at 4C. Cultured cell lysate was
prepared in cell lysis buffer (Cell Signaling, Danvers, MA). Samples
were processed for SDS-PAGE and electrotransferred onto a nitrocellulose membrane. After being blocked with 5% nonfat dry milk in
1 TBS containing 0.1% Tween 20 for 1 h, membranes were
incubated overnight with the following antibodies at 4C: 1) mouse
anti-nitrotyrosine antibody (Chemicon International); 2) mouse antibovine Complex I antibody (Abcam); 3) rabbit anti-human CYC1
(component of Complex III antibody, Abcam); 4) rabbit anti-human
COX-IV antibody (Abcam); 5) mouse anti-rat MnSOD antibody
(Enzo Life Science); 6) mouse monoclonal -actin antibody (SigmaAldrich); 7) rabbit anti-human phospho-p53 (Ser15) antibody (Cell
Signaling); 8) mouse monoclonal anti-p53 antibody (Cell Signaling);
9) rabbit anti-human phospho-cyclin-dependent kinase 2 (CDK2;
Thr160 antibody, Cell Signaling); 10) rabbit monoclonal anti-CDK2
antibody (Cell Signaling); 11) mouse monoclonal anti-p-ERK antibody (Santa Cruz Biotechnology, Santa Cruz, CA); and 12) rabbit
anti-human ERK1 antibody (Santa Cruz Biotechnology). After incubation with an HRP-labeled anti-rabbit or anti-mouse IgG antibody
(Cell Signaling) for 1 h, signals were detected using Immune Star
HRP (Bio-Rad, Hercules, CA). The membranes were reprobed with a
rabbit anti--actin antibody (Sigma-Aldrich) or COX-IV antibody to
serve as controls for equal loading. The density of each band was
determined using National Institutes of Health Image software and
expressed relative to the density of the corresponding band of -actin
or COX-IV, with an arbitrary unit value of 1 assigned to the normal
control.
Cytochrome c release. The cytoplasmic fraction was separated
from the mitochondrial fraction in mouse proximal tubular cells using
a Mitochondrial Isolation Kit for Cultured Cells (Thermo Scientific).
Released cytochrome c in cytoplasm from mitochondria was detected
by Western blotting with a mouse monoclonal anti-cytochrome c
antibody (BD Biosciences, San Jose, CA) as a primary antibody. The
membranes were reprobed with a mouse monoclonal anti--tubulin
antibody (Sigma-Aldrich) to serve as a control for equal loading.
Mitochondrial membrane potential. Mitochondrial membrane potential was evaluated using a JC-1 Mitochondrial Potential Sensor
(Invitrogen). In this assay, potential dependent accumulation of JC-1
causes the fluorescence to shift from green to red. On 96-well plates,
adherent mouse proximal tubular cells were treated with 100 M
cisplatin for 1 h and 106 M epicatechin for 8 h. The cells were then
incubated with medium containing JC-1 at 1:2,000 for 30 min at 37C.
Fluorescent intensity was measured using a Synergy 2 Multi-Mode
Microplate Reader with excitation at 485 nm and emission at 528 nm

for green and excitation at 530 nm and emission at 590 nm for red.
Data were expressed as ratio of red to green fluorescence.
Oxidative stress detection assay. An Image-iT LIVE Green Reactive Oxygen Species Detection Kit (Invitrogen) was used for the
detection of increased oxidative stress in vitro. Adherent mouse
proximal tubular cells seeded on 96-well plates were treated with
cisplatin for 1 h. Then, the cells were incubated with medium
containing 106 M epicatechin or 1 mM tempol for 4 h. Culture
medium was replaced with 25 M carboxy-H2DCFDA-containing
HBSS and incubated for 1 h at 37C. Cells were washed with HBSS
two times, and then fluorescent intensity was measured using a
Synergy 2 Multi-Mode Microplate Reader with excitation at 485 nm
and emission at 528 nm. Data were expressed as values relative to
control.
Mitochondrial imaging in vitro. To obtain mitochondrial images
in vitro, mitochondria on unfixed murine proximal tubular cells were
stained with Mitotracker Mitochondrion-Selective Probes (Invitrogen). After incubation in the complete medium with or without 106
M epicatechin or 1 mM tempol, the cells were incubated with 500 nM
Mitotracker in PBS at 37C for 45 min. After washes with PBS three
times, the cells were observed by a laser-scanning confocal microscope (LSM 510 META, Carl Zeiss Microimaging, Thornwood, NY)
to obtain images. Fragmented mitochondria looked punctate and
rounded, whereas normal mitochondria showed a threadlike structure.
Cells with mitochondrial fragmentation were defined as those containing a majority (70%) of fragmented mitochondria as in a
previous report (4), and they were counted to determine the percentage in 30 35 cells/sample.
Statistical analysis. All values are expressed as means SD.
Statistical analysis was performed with one-way ANOVA using
Tukeys method to compare the groups. A level of P 0.05 was
considered a statistically significant difference.
RESULTS

Renal function. Cisplatin significantly increased serum creatinine and BUN on day 3 compared with vehicle (saline)
treatment while these elevations were significantly blocked by
epicatechin treatment (Fig. 2, A and B).
Tubular injury. Tubular epithelial cells are the primary site
for cisplatin injury. Hence, we examined tubular injury including detachment of epithelium, loss of brush border, or cast
formation. Compared with control, epicatechin treatment had
no effects on tubular epithelial cells (Fig. 2, C and D). In
contrast, cisplatin markedly caused tubular injury while such
injuries were partially ameliorated by epicatechin treatment.
Similarly, the cisplatin group had a significantly higher ATN
score (Fig. 2, EG), suggesting this anticancer drug caused
widespread severe injury. However, such tubular damage was
alleviated by epicatechin. In addition, TUNEL staining was
performed to detect apoptotic cells. The cisplatin group significantly exhibited a higher number of apoptotic cells compared
with the control group and epicatechin group (Fig. 2, HL).
However, epicatechin partially but significantly blocked the
formation of apoptosis (Fig. 2, K and L).
Oxidative stress. Oxidative stress is a major factor causing
renal injury in response to cisplatin. We examined the level of
oxidative stress using two markers, 4-hydroxynonenal (4HNE) and nitrotyrosine (NT) (Fig. 3, AF). By immunohistochemistry, both markers were highly expressed in the kidney
treated by cisplatin compared with the control. In particular,
damaged tubules showed strong signals for both markers (Fig.
3, B and E). However, treatment with epicatechin reduced
these signals (Fig. 3, C and F). These data suggest that cisplatin

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Translational Physiology
EPICATECHIN LIMITS MITOCHONDRIAL DAMAGE IN CISPLATIN AKI
P <0.001

3
2

P <0.05

300
200
100

1
0

Control

Epicatechin

Cisplatin

Control

Cisplatin
P <0.05

5
ATN score

P <0.001

400

Epicatechin

P <0.05

BUN (mg/dL)

Serum creatinine (mg/dL)

4
3
2
1

0
Epicatechin

Control

TUNEL positive cell number (/field)

L
H

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Cisplatin

50

P <0.01

40
30

Fig. 2. Effect of epicatechin on renal function and histology in mouse cisplatin nephropathy at death. The level of serum creatinine (A) and blood urea nitrogen (BUN; B)
are shown. Periodic acid-Schiff (PAS) staining demonstrates the representative histological features in the sham group (C), sham
group with epicatechin treatment (D), cisplatin group (E), and cisplatin with epicatechin
group (F). Quantification of acute tubular
necrosis (ATN) score from these groups is
shown (G) as well as terminal uridine deoxynucleotidyl transferase-mediated dUTP nickend labeling (TUNEL) staining in the sham
group (H), sham with epicatechin group (I),
cisplatin group (J), and cisplatin with epicatechin group (K). Blue dot indicates positive
cells, while red color indicates nuclear staining. A quantification of TUNEL-positive
cells from these groups is also shown (G).
Bar 50 m.

20
10
0

Epicatechin

+
Control

induces oxidative stress in damaged tubules while such stress

was significantly blocked by epicatechin treatment. This finding was next confirmed by Western blotting for NT using the
mouse kidney. NT expression was significantly induced by
cisplatin whereas such induction was significantly blocked by
epicatechin (Fig. 3G). Since mitochondria are a source of
oxidative stress, we also isolated mitochondria to examine the
NT level. As shown in Fig. 3H, cisplatin treatment resulted in
higher levels of NT in isolated mitochondria while it was
significantly reduced by epicatechin in this model.
Mitochondrial effect of epicatechin in the mouse kidney.
Since we found that epicatechin reduced oxidative stress in
mitochondria in this model, we further examined the mitochondrial effect of epicatechin. First, we examined mitochondrial

Cisplatin

DNA content, which is supposed to correspond to mitochondrial number. Consistent with mitochondrial oxidative stress,
cisplatin significantly reduced mitochondrial DNA whereas
epicatechin significantly blocked such a reduction (Fig. 4A).
Similarly, the protein expression of mitochondrial oxidative
phosphorylation complexes and MnSOD was significantly reduced by cisplatin while such a reduction was ameliorated by
epicatechin (Fig. 4, BD).
Mitochondrial effect of epicatechin in cultured mouse proximal tubular cells:. To confirm the benefit of epicatechin on
mitochondria, we next performed an in vitro study using mouse
proximal tubular cells (TKPTS) cells. In this experiment, we
tested the effect of epicatechin after cells had been treated with
100 M cisplatin for 1 h. First, we examined the activity of

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EPICATECHIN LIMITS MITOCHONDRIAL DAMAGE IN CISPLATIN AKI

NT

4-HNE

H
NT

NT

-actin

COX-IV

Epicatechin

Control

2
1

+
Cisplatin

P <0.01

6
NT/COX-IV ratio

NT/-actin ratio

+
Control

P <0.01

0
Epicatechin

Epicatechin

Cisplatin

P <0.01

P <0.01

Control

Cisplatin

Epicatechin

Control

Cisplatin

Fig. 3. Effect of epicatechin on oxidative stress in renal cortex and mitochondria. Immunohistochemistry for 4-hydroxynonenal (4-HNE; AC) and nitrotyrosine
(NT; DF) in the saline group (A and D), cisplatin group (B and E), and cisplatin with epicatechin group (C and F). Immunoblot with renal cortex for NT and
its quantification (G). as well as the immunoblot with mitochondria isolated from renal cortex for NT and its quantification are also shown (H). COX, cytochrome
c oxidase. Bar 50 m.

succinate dehydrogenase in complex II of the mitochondrial

phosphorylation complex. A MTT reduction, as an index of
this enzyme activity, was significantly low in the cells without
epicatechin compared with control cells at 8 h. In contrast,
epicatechin-treated cells exhibited significantly higher level of
MTT reduction compared with cells without epicatechin, suggesting that succinate dehydrogenase was functionally activated by epicatechin (Fig. 5A). Because MTT reduction is
also associated with cell number, we determined whether
the higher level of reduced MTT could be due to an increase

in cell number. To address this issue, we examined DNA

content in the culture cell dish as a surrogate of cell number.
As shown in Fig. 5B, we did not find any difference in DNA
content, suggesting that cisplatin as well as subsequent
epicatechin treatment did not alter cell number at this time
point.
We next examined whether epicatechin protects the mitochondrial membrane from cisplatin injury. Cisplatin caused a
marked release of cytochrome c from mitochondria to cytoplasm while epicatechin treatment prevented such injury at

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EPICATECHIN LIMITS MITOCHONDRIAL DAMAGE IN CISPLATIN AKI

mtDNA / gDNA ratio

(COXII / UCP2)

P <0.01

1.5

P <0.05

COX-I

1
COX-III
0.5

-actin

Epicatechin

0
Epicatechin

Control

+
Control

+
Cisplatin

Cisplatin

COX-IV

MnSOD

-actin

-actin

Control

COX-IV/-actin ratio

1.5

Cisplatin

P <0.05

0.5

Control

Cisplatin

Control

Cisplatin
P <0.01

1.5

P <0.01

0
Epicatechin

Epicatechin

MnSOD/-actin ratio

Epicatechin

P <0.05

0.5

0
Epicatechin

Control

Cisplatin

Fig. 4. Effect of epicatechin on mitochondria in mouse cisplatin nephropathy at death. Mitochondrial DNA content corrected by genomic DNA in the renal cortex
is shown (A). Immunoblotting for complex I and III in the renal cortex (B) and immunoblotting for complex IV (C) and MnSOD (D) and their quantifications
are also shown.

24 h (Fig. 5C). Similarly, cisplatin induced a significant loss of

mitochondrial membrane potential, as shown in the JC-1 mitochondrial potential sensor assay, and epicatechin also suppressed this alteration (Fig. 5D).
To determine mechanisms by which epicatechin protects
mitochondria from cisplatin injury, three representative signalings were examined. Phosphorylation of p53, which has been
recently implicated in cisplatin-induced mitochondrial membrane damage, was markedly accelerated by cisplatin treatment
whereas epicatechin did not affect it (Fig. 5E). Similarly,
activation of CDK2 caused by cisplatin was not inhibited by
epicatechin (Fig. 5F). In contrast, phosphorylation of ERK
MAPK, which is a key pathway involved in cisplatin nephropathy, was prevented by epicatechin (Fig. 5G).
Antioxidative effect of epicatechin might be a key mechanism
for mitochondrial protection. As shown in the in vivo experiments, oxidative stress was found to be induced by cisplatin

treatment in proximal tubular cells, suggesting that oxidative

stress might be a key mediator of cisplatin injury in mitochondria.
To confirm this hypothesis, we examined whether blocking oxidative stress with the potent antioxidant tempol could prevent
mitochondrial injury in response to cisplatin. As expected, an
increased in oxidative stress was significantly prevented by tempol as potently as epicatechin (Fig. 6A). Furthermore, a cisplatinmediated reduction in complex IV protein expression was also
ameliorated by epicatechin as well as tempol at 24 h (Fig. 6B).
Finally, we examined the effect of epicatechin in mitochondrial
structure using a laser-scanning confocal microscope. The typical
reticulotubular appearance of mitochondria in healthy TKPTS
cells (Fig. 6C) had disintegrated into multiple small rounded
organelles in response to cisplatin at 8 h (Fig. 6D). However,
epicatechin treatment prevented these structural changes in mitochondria (Fig. 6, E and G). Interestingly, tempol showed similar
protection on mitochondrial structural changes (Fig. 6, F and G),

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EPICATECHIN LIMITS MITOCHONDRIAL DAMAGE IN CISPLATIN AKI

MTT relative absorbance

1.5

Estimated cell number (x103)

A
P <0.01
P <0.05

0.5

0
Epicatechin 0
(M)
Control

10-7

5x10-7

10-6

Cisplatin (100M)

10
8
6
4
2
0

Epicatechin 0
(M)
Control

10-6

5x10-7

Cisplatin (100M)

cyto c
Relative red/green
fluorescence ratio

1.5

-tubulin

Epicatechin

Control

10-7

+
Cisplatin

P <0.05

0.5

p-p53

Epicatechin

p53

Control

Epicatechin

Control

Cisplatin

F
p-CDK2

Cisplatin

CDK2

P <0.01

3
p-p53 / p53 ratio

P <0.01

Epicatechin
2

Control

+
Cisplatin

G
p-ERK

ERK
0
Epicatechin

Epicatechin

Control

Cisplatin

Control

+
Cisplatin

Fig. 5. Effect of epicatechin on mitochondria in mouse cultured proximal tubular cells. The activity of succinate dehydrogenase is measured by a reduction of
MTT to formazan at 8 h (A). In the same condition, DNA content was measured (B). Released cytochrome c into cytoplasmic fraction separated from
mitochondrial fraction was detected by Western blotting (C). Loss of mitochondrial membrane potential was evaluated by JC-1 Mitochondrial Potential
Sensor assay (D). Data are expressed as the ratio of red to green fluorescence. Western blots for p53 and phosphorylated p53 and its quantification are
shown (E). Western blots for cyclin-dependent kinase 2 (CDK2) and phosphorylated (activated) CDK2 (F) and ERK and phosphorylated-ERK (G) are
also shown.

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Translational Physiology
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EPICATECHIN LIMITS MITOCHONDRIAL DAMAGE IN CISPLATIN AKI

Relative fluorescence

P <0.01

3
P <0.01

P <0.05

COX-IV
2

-actin
1
Epicatechin

Tempol

+
-

Control

+
-

Control

Cisplatin

Cisplatin

G
%cells with mitochondrial
fragmentation

Epicatechin Tempol
-

P <0.05

80

P <0.01

P <0.05

60
40
20
0

Epicatechin Tempol
-

Control

+
-

Cisplatin

Fig. 6. Effects of epicatechin on oxidative stress and mitochondrial fragmentation. The increased production of reactive oxygen species (ROS) in cisplatin-treated
mouse proximal tubular cells was detected by the reaction with carboxy-H2DCFDA (A). Immunoblotting for complex IV protein is shown (B). Increased ROS
and the reduced complex IV level caused by cisplatin (100 M) were prevented by both epicatechin (106 M) and the antioxidant tempol (1 mM). Images by
using a laser confocal microscope show mitochondrial structure detected by Mitotracker (green) staining on control (C), cisplatin (100 M) alone (D)-,
cisplatin106 M epicatechin (E)-, and cisplatin1 mM tempol (F)-treated mouse proximal tubular cells. Mitochondria in control conditionally immortalized
mouse proximal tubular cells (TKPTS) exhibit a reticulotubular appearance (white arrow in C). Cisplatin fragmented them into multiple small rounded organelles
(mitochondrial fragmentation; white arrow in D), whereby subsequent 106 M epicatechin improved such structural perturbation at 8 h (E) as well as did 1 mM
tempol (F). The percentage of cells with mitochondrial fragmentation is shown (G).

suggesting these morphological alterations of mitochondria were

at least partially mediated by oxidative stress.
Anticancer effect of cisplatin is not disturbed by epicatechin
in HeLa cells. It is clinically important that supplemental
treatment should not block the anticancer effect of cisplatin,
but should selectively protect the kidney. Hence, we next
examined whether epicatechin could disturb cell toxicity in
HeLa cells, which are derived from a patient with cervical
adenocarcinoma. In the TKPTS cells, cisplatin significantly
reduced DNA content whereas epicatechin (106 M) prevented
such toxicity of this anticancer agent at 24 h (Fig. 7A). In
contrast, epicatechin failed to protect HeLa cells from cisplatin
toxicity in cell number (Fig. 7B).
DISCUSSION

Consistent with previous studies, we found that cisplatincaused renal dysfunction and morphological changes were

associated with mitochondrial injury in the mouse kidney.

These findings was also confirmed by using immortalized
mouse proximal tubular epithelial cells in which we documented that cisplatin caused mitochondrial dysfunction as
revealed by a reduction in protein expression of the mitochondrial oxidative phosphorylation complex and MnSOD. Here,
we tested whether epicatechin prevents such renal injury because of mitochondrial protection. In particular, we examined
whether epicatechin administration after pretreatment with
cisplatin could prevent cisplatin-induced renal injury. Our
primary finding is that epicatechin exhibited protective effects,
and such protection was likely owing to an ability of epicatechin to protect mitochondria. In fact, both in vivo and in vitro
studies demonstrated that cisplatin-mediated injuries, including a reduction in mitochondrial protein, its functional
perturbation, and morphological alterations, were significantly prevented by epicatechin. Importantly, epicatechin

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Translational Physiology
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Estimated cell number (x103)
in TKPTS

EPICATECHIN LIMITS MITOCHONDRIAL DAMAGE IN CISPLATIN AKI

10

P <0.01

P <0.05

6
4
2
0

Epicatechin 0
(M)
Control
Estimated cell number (x103)
in HeLa cell

10-7

5x10-7

10-6

Cisplatin (100M)

P <0.01

4
3
2
1
0

Epicatechin 0
(M)
Control

10-7

5x10-7

10-6

Cisplatin (100M)

Fig. 7. Anticancer effect of cisplatin is not disturbed by epicatechin in the

HeLa cells. The anticancer activity of cisplatin is examined by cell number,
which corresponds to survived cell number at 24 h. Cisplatin significantly
reduced cell number in both TKPTS cells (A) and HeLa cells (B). However,
epicatechin treatment prevented a loss of cell number in TKPTS at 24 h, but
not in HeLa cells.

selectively protect tubular epithelial cells, but not cancer

(HeLa) cells.
Recently, the cardiovascular benefits of epicatechin have
been shown in several studies. Villarreals research group (41)
has performed a series of studies and found that epicatechin
potently ameliorated myocardial injury in the setting of myocardial ischemia-reperfusion injury in rats. Subsequent studies
using rats also documented that myocardial infarction caused
by permanent coronary occlusion was also significantly alleviated by this compound (42). In turn, clinical studies have
indicated that the underlying mechanisms for epicatechin benefits could be endothelial protection since epicatechin is capable of inducing vasorelaxation as well as activation of endothelial nitric oxide synthase (14, 33). Recently, epicatechin was
also shown to improve exercise performance due to an increase
in capacity for muscle aerobic metabolism (18, 28). While
several mechanisms may be involved, a study in skeletal
muscle documented that a potential mechanism is likely a
stimulation of mitochondrial biogenesis (28, 36).
To confirm mitochondrial protection by epicatechin, cultured mouse proximal tubular cells were stimulated by cisplatin

for 1 h, cisplatin was removed, and the medium was replaced

with new conditioned medium with/without epicatechin. This
protocol was designed to test whether epicatechin could be
protective after injection of cisplatin injury. In this experiment,
mitochondrial function, as revealed by oxidative phosphorylation protein expression as well as its activity, remained low at
8 h but was rescued by epicatechin treatment. Similarly, other
markers of mitochondrial injury, including a release of cytochrome c from mitochondria and loss of mitochondrial membrane potential, were also prevented by epicatechin treatment.
These data suggest that epicatechin protects mitochondria from
cisplatin injury.
Cisplatin injury was found to be regulated by several pathways. The activation of p53, a proapoptotic protein, was
initiated as a consequence of DNA damage in response to
cisplatin, and it occurs before a release of cytochrome c,
indicating that activation of p53 could be upstream of mitochondrial injury (9, 19). Similarly, CDK2, a cell cycle protein,
seems to be activated by cisplatin and mediates cell injury
independently of the p53 pathway (26). However, the current
study documented that epicatechin had no effects on these
pathways. Another pathway involved in this injury could be the
MAPK pathway, which regulates a variety of cell responses,
including proliferation, differentiation, and apoptosis (29). Interestingly, this pathway seems to be involved both upstream
and downstream of mitochondrial injury. In fact, pharmaceutical inhibition of ERK was shown to be capable of preventing
cytochrome c release and Bax activation (21) whereas mitochondria-induced oxidative stress could cause ERK activation
(39). In the current study, we documented that epicatechin
potently blocked the activation of ERK, thereby providing a
potential mechanism for the beneficial effect of epicatechin.
We also found that cisplatin causes mitochondrial fragmentation as well as a reduction in mitochondrial number, and such
findings are consistent with previous papers (5). Given the fact
that mitochondria are a major source of oxidative stress, it is
intuitively assumed that mitochondrial dysfunction could result
in a reduction of reactive oxygen species (ROS) production.
However, as opposed to such an assumption, fragmented mitochondria are found to be not bioenergically functional (3).
Moreover, blocking oxidative phosphorylation with either
pharmaceutical maneuvers or molecular manipulation can accelerate ROS production (11, 25, 37). Hence, an increase in
mitochondrial oxidative stress may be due in part to cisplatininduced mitochondrial injury. In contrast, we also found that
blocking oxidative stress with tempol potently inhibited mitochondrial injury, suggesting that oxidative stress may also
drive mitochondrial injury under cisplatin stimulation. Consistent with our results, a recent study has demonstrated that a
reduction in mitochondrial-specific oxidative stress ameliorated kidney injury in a mouse cisplatin nephropathy model
(27). Since mitochondrial dysfunction is usually tightly connected with oxidative stress, it seems likely that ROS can be a
cause and a consequence of mitochondrial injury.
While we found that epicatechin exhibits antioxidative effects as potently as tempol, it remains unclear as to how this
compound blocks ROS production. One potential mechanism
may be by enhancing mitochondrial antioxidative function by
activating MnSOD and glutathione (1). Epicatechin can also
block NADPH oxidase activity in the model of cyclosporine
nephropathy (15). Taking this together, we now speculate that

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Translational Physiology
EPICATECHIN LIMITS MITOCHONDRIAL DAMAGE IN CISPLATIN AKI

epicatechin may have several pathways for blocking oxidative

stress, and these pathways should be explored in a future study.
In conclusion, we demonstrated that epicatechin is protective in mouse cisplatin nephropathy even if treatment starts
after cisplatin injury had occurred in tubular cells. Mechanisms
are likely that epicatechin exhibits mitochondrial protection.
Given the fact that epicatechin is a derivative of cocoa, it might
be safe for us to use this compound in clinical nephrology
studies.

13.
14.

15.

ACKNOWLEDGMENTS
We thank Dr. Francisco Villarreal (University of California, San Diego) for
a great discussion.
GRANTS

16.

17.

This study is also supported by a Japan Heart Foundation/Bayer Yakuhin

Research Grant (K. Tanabe).
18.

DISCLOSURES
G. Schreiner is a member of Cardero, Inc., which is developing epicatechin
as a treatment for various disorders. R. J. Johnson and T. Nakagawa have stock
in Cardero, Inc. All other investigators declare no conflicts of interest.

19.

AUTHOR CONTRIBUTIONS

20.

Author contributions: K.T. and T.N. provided conception and design of

research; K.T., Y.T., M.A.L., M.M., N.S., W.S., and T.N. performed experiments; K.T., M.A.L., M.M., and T.N. analyzed data; K.T., Y.M., G.F.S., R.J.J.,
and T.N. interpreted results of experiments; K.T. prepared figures; K.T. and
T.N. drafted manuscript; K.T., F.J.V., and R.J.J. edited and revised manuscript;
K.T., Y.T., M.A.L., M.M., N.S., W.S., Y.M., G.F.S., F.J.V., R.J.J., and T.N.
approved final version of manuscript.
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