![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
GASTROINTESTINAL, HEPATIC, PULMONARY, AND RENAL
Division of Nephrology, Department of Internal Medicine, Kawasaki Medical School, Kurashiki, Japan (M.S., N.K., S.F., H.H., T.T., T.N., T.S.); Department of Medicine and Clinical Science, Okayama University Graduate School of Medicine and Dentistry, Okayama, Japan (M.S., H.M.)
Received December 15, 2002; accepted March 13, 2003.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
).
Although effective, cisplatin is associated with many adverse drug reactions,
such as renal damage, gastrointestinal dysfunction, auditory toxicity, and
peripheral nerve toxicity (Cooley et al.,
1994). Nephrotoxicity in particular is a major complication and a
dose-limiting factor for cisplatin therapy
(Safirstein et al., 1986).
Administration of cisplatin is frequently associated with renal insufficiency
and tubular dysfunction. The possible involvement of peroxidative damage
caused by a reactive oxygen species (ROS) has been suggested in the
pathogenesis of cisplatin-induced renal failure
(Matsushima et al., 1998). In
particular, the hydroxyl radical is highly reactive among oxygen radicals.
Once excessive hydroxyl radicals are released, lipid peroxidation, which
causes changes in the fluidity and permeability of membranes, is induced
(Schmidley, 1990). Several
antioxidants and oxygen radical scavengers have been reported to be effective
in protection against these injuries (Rao
et al., 1999; Davis et al.,
2001; Hara et al.,
2001). The roles of hydroxyl radicals and other free radical
species in cisplatin nephrotoxicity have not been fully elucidated,
however.
The novel free radical scavenger, 3-methyl-1-phenyl-pyrazolin-5-one
(MCI-186; edarabone), has been shown to trap both hydroxyl radicals and
prevent iron-induced peroxidative injuries
(Murota et al., 1990). It has
also been shown to have protective effects against cerebral and myocardial
ischemia in rats. Pharmacological studies have suggested that the anticerebral
ischemic action of edarabone is related to its antioxidant action
(Watanabe et al., 1994a;
Wu et al., 2000). Oxidative
stress is also involved in the pathogenesis of glomerular and tubular injuries
in various acute renal diseases (Baud and
Ardaillou, 1986). Nevertheless, the usefulness of this compound in
acute renal disease has not been investigated.
The present study was designed to examine whether edarabone would prevent cisplatin-induced cytotoxicity in cultured renal epithelial cells. Furthermore, we investigated whether edarabone provided protection against cisplatin-induced deterioration of renal function and structural injury in vivo. Our results indicate that edarabone may be of value in preventing cisplatin-induced cytotoxicity and decline of renal function. The beneficial effect of edarabone relates to reducing the mitochondrial damage, ROS generation and lipid, protein, and nucleic acid oxidation, ultimately resulting in preservation of tubular cell structure.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
smooth muscle actin, and showed albumin reabsorption
(Takaya et al., 2003). Thus,
this cell line expressed proximal tubular phenotype. PTCs were cultured in
Dulbecco's modified Eagle's medium supplemented with 4 mM glutamine, 5% fetal
bovine serum, 100 U/ml penicillin, and 100 µg/ml streptomycin at 37°C
under a 95% room air/5% CO2 gas mixture on a 1% gelatin-coated
dish. Cultured cells from the 4th to 30th passages were used for the
experiments. Cell Viability Assays. Cell viability was examined by trypan blue exclusion for living cells and the tetrazolium salt (WST-1) degradation assay for mitochondrial viability. For trypan blue exclusion, cells were seeded in 12-well plates followed by exposure to medium containing phosphate-buffered saline or 20 µM cisplatin (Sigma-Aldrich Japan Co., Tokyo, Japan) for 24 h with or without edarabone (a gift from Mitsubishi Pharma Corp., Osaka, Japan). Nonadherent cells were removed, and adherent cells were harvested by trypsin-EDTA digestion and stained with 0.4% trypan blue for 5 min at 37°C. The number of cells excluding trypan blue were counted in a hemo-cytometer and expressed as a percentage of viable cells compared with vehicle-treated cells. To assess mitochondrial viability, cells were plated in 96-well plates and incubated with 20 µM cisplatin for 24 h. WST-1 measurement was performed according to the manufacturer's protocol (cell proliferation assay system; TaKaRa Biomedicals, Tokyo, Japan).
Measurement of Mitochondrial Membrane Potential
(
m) and Hydrogen Peroxide Production. To assess
changes in m, we used the potentiometric, fluorescent
dye
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine
iodide (JC-1; Molecular Probes, Inc., Eugene, OR)
(Di et al., 1995). We
determined changes in intracellular ROS levels by measuring the oxidative
conversion of cell-permeable 2',7'-dichlorofluorescein diacetate
(DCFH-DA; Molecular Probes, Inc.) to fluorescent dichlorofluorescein (DCF), by
confocal laser scanning microscopy or fluorescence-activated cell sorting
(FACS) (Mancini et al., 1998).
Cells were seeded in six-well plates followed by exposure to medium containing
phosphate-buffered saline or 20 µM cisplatin for 6 h with or without
edarabone. Cells were then incubated with 5 µg/ml JC-1 or 20 µM DCFH-DA
in medium for 15 min at 37°C in the dark. Both floating and adherent cells
were collected and pelleted by centrifugation at 500g for 5 min. In
each sample, a minimum of 10,000 cells was subjected to FACS using a FACS
Calibur (Nippon Becton Dickinson Co., Tokyo, Japan) and CellQuest software
(Nippon Becton Dickinson). Intracellular distribution of the dye was assessed
by confocal laser-scanning microscopy TCS-NT (Leica-Microsystems Co., Tokyo,
Japan).
In Vivo Studies
Animal Experiments. Male Wistar rats (300 ± 30 g b.wt.;
Charles River Japan, Inc., Kanagawa, Japan) were maintained on standard chow
and tap water ad libitum. The experimental protocol was approved in advance by
the Ethics Review Committee for Animal Experimentation of the Kawasaki Medical
School (Kurashiki, Japan). Rats were assigned to four groups as follows: group
1, saline-treated controls (n = 7); group 2, cisplatin-treated rats
(n = 7); group 3, rats treated with cisplatin plus edarabone at a
dose of 1 mg/kg (n = 7); and group 4, rats treated with cisplatin
plus edarabone at a dose of 5 mg/kg (n = 7). Under light ether
anesthesia, animals were injected intraperitoneally with 1.0 ml of saline or 5
mg/kg cisplatin (Matsushima et al.,
1998; Ramesh and Reeves,
2002). At the same time, 0.1 ml of saline or 1 or 5 mg/kg
edarabone was administered by a single intravenous injection
(Kawai et al., 1997). Animals
from each group were placed into metabolic cages for 24 h of urine collection
for measurement of N-acetyl-
-d-glucosaminidase (NAG)
excretion and creatinine clearance (Ccr) at 4 days after injection of
cisplatin or saline. After 5 days, animals were humanely killed using
pentobarbital anesthesia. Blood samples were obtained for measurement of blood
urea nitrogen (BUN) and creatinine, and the kidneys were dissected out.
Surgically removed kidney specimens were immediately frozen in liquid nitrogen
and unfixed cryostat sections (5-µm thick) were prepared for cytochrome
c oxidase staining. Another kidney cross-section was collected in 4%
neutral buffered paraformaldehyde for histopathological evaluation. The
remaining portions of the kidney were frozen in liquid nitrogen and stored at
-80°C for DNA and protein isolation.
Histopathology. The tissues collected in paraformaldehyde were processed, embedded in paraffin, sectioned (4-µm thick), and stained with H&E. A pathologist performed a semiquantitative analysis of the kidney sections in a blinded fashion. Changes observed were limited to the tubules, especially the proximal straight S3 portion, the main site of cisplatin toxicity, such as cell swelling, vacuolization, necrosis, and desquamation. Tubular lesions were graded as follows: 0, no damage; 1+, lesion areas <50%; 2+, lesion areas >50% with or without focal involvement of the S3 segment in the medullary rays; and 3+, lesion areas 100% with diffuse involvement of the medullary rays. The mean score for each group was calculated.
Cytochrome c Oxidase Staining. To confirm mitochondrial
activity in vivo, cytochrome c oxidase (COX) staining methods
(Seligman et al., 1968) were
used. The percentages of COX-positive areas, with the exception of the
glomerulus, were measured by using an image analyzer (MCID image analyzer;
Fuji Film, Tokyo, Japan). Ten consecutive fields from the renal cortex of each
rat were randomly selected and evaluated at a magnification of 100x.
Western Blot Analysis. Kidney samples were homogenized in lysis
buffer containing 0.25 M sucrose, 50 mM dithiothreitol, 3 mM HEPES (pH 7.9),
500 µM EGTA, 1 mM 4-(2-aminoethyl)-benzene-sulfonyl fluoride, 0.8 µM
aprotinin, 21 µM leupeptin, 36 µM bestatin, 15 µM pepstatin A, 14
µM (4-guanidino)butane, and 1% Triton X-100. After centrifugation
(8000g, 10 min, 4°C), the supernatants were used for Western
blotting. Protein concentrations were determined using a protein concentration
assay kit (QuantiPro BCA assay kit; Sigma-Aldrich). Western blot analysis for
oxidative protein was performed with the Oncor Oxyblot kit, according to the
manufacturer's protocol (Oncor, Gaithersburg, MD), as described previously
(Keller et al., 1993). For the
4-hydroxy-2-nonenal (HNE) immunoblot, proteins (10 µg) were separated in a
12.5% SDS-polyacrylamide gel and then transferred onto a polyvinylidene
diflouride membrane. The membranes were incubated for 1 h with anti-HNE
monoclonal antibody (1.0 µg/ml; Japan Institute for the Control of Aging,
Fukuroi, Shizuoka, Japan) followed by incubation with peroxidase-conjugated
goat anti-mouse IgG antibody (0.1 µg/ml) for 1 h. Bands were visualized
using the ECL-plus Western blotting detection system (Amersham Biosciences,
Inc., Tokyo, Japan).
Assessment of DNA Oxidative Injury. The amount of
8-hydroxy-deoxyguanosine (8-OHdG) in DNA was measured using a competitive
enzyme-linked immunosorbent assay kit (8-OHdG Check, Japan Institute for
Control of Aging) (Toyokuni et al.,
1997). Genomic DNA from the kidneys was extracted using DNAzol
reagent (Invitrogen Oriental Co., Tokyo, Japan). After hydrolyzing with DNA
nuclease and alkaline phosphatase, 10 µg of DNA were used for enzyme-linked
immunosorbent assay.
The number of apurinic/apyrimidinic (AP) sites in DNA was detected using a DNA damage quantification kit (Kumamoto Immuno-chemical Laboratory Co., Kumamoto, Japan). Purified DNA (1 µg) was incubated with N'-aminooxymethyl-carbonyl-hydrazino-d-biotin, which binds specifically to AP sites, for 1 h at 37°C. After fixing in 96-well plates overnight at room temperature, the plates were incubated with peroxidase-conjugated streptavidin for 1 h at 37°C. After color development with substrate solution, absorbances were measured at 660 nm.
Assessment of Apoptosis in Vivo. Apoptotic nuclei were detected with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method, using an apoptosis detection kit (in situ apoptosis detection kit; TaKaRa Biomedicals) according to the protocol specified by the manufacturer. The analysis was performed in a blinded fashion. Random areas were viewed at a magnification of x 100 and scored for the number of apoptotic nuclei present in cortical renal tubules. The mean number of stained cells in at least 20 random fields was expressed as the number of TUNEL-positive cells.
Statistical Analysis. All data are presented as mean ± S.E.M. The Student's t test was used for comparisons between two groups. Statistics were performed using StatView (SAS Institute, Inc., Cary, NC) on a Macintosh computer. Significance was defined as P < 0.05.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
|
m Change and Reactive Oxygen Species
Production. The m is an important indicator of
mitochondrial function and dysfunction. To assess mitochondrial damage induced
by cisplatin, we monitored changes in m using the
fluorescent dye JC-1. FACS analysis showed that high m
was maintained in control cells (68.9%)
(Fig. 2). After cisplatin
treatment, the rate of high m was decreased (5.4%), and
the rate of low m was increased (46.5%). By
coincubation with edarabone, the m was maintained at
high levels (47.1%), and the rate of low m was not
increased (1.3%).
|
To determine intracellular ROS generation induced by cisplatin and the oxygen radical elimination ability of edarabone, cisplatin-treated PTCs with or without edarabone were incubated with DCFH-DA, and oxidation of intracellular DCF was measured by confocal laser-scanning microscopy and FACS. No ROS production was evident in the control cells (Fig. 3), whereas enhanced ROS production was observed in cisplatin-treated cells. Edarabone treatment reduced ROS production in cisplatin-treated PTCs. FACS analysis showed that cisplatin caused a significant increase in ROS generation (1.4 versus 74.5%). Preincubation of PTCs with 10-4 M edarabone significantly reduced ROS levels (22.3%).
|
In Vivo Studies
Morphological Evaluation. Compared with controls at day 5, renal
tissue sections from the cisplatin group exhibited increased evidence of acute
structural damage, characterized by tubular necrosis, degeneration, casts, and
red blood cell extravasation (Fig.
4). These changes usually involved the entire S3 segments in the
outer stripe of the medulla zone. Semiquantitative assessment of the
histological lesions revealed a significantly higher score in
cisplatin-treated rats versus controls at day 5
(Table 1; 2.7 ± 0.2
versus 0.1 ± 0.1, P < 0.05). Edarabone treatment (5 mg/kg)
significantly lowered the semiquantitative score compared with cisplatin
treatment only (cisplatin + 5 mg/kg edarabone: 1.7 ± 0.3, P
< 0.05 versus the cisplatin group).
|
|
Assessment of Renal Function. Injection of cisplatin induced a significant increase in plasma BUN levels at day 5 (Table 2). Administration of edarabone significantly prevented the increase in BUN in cisplatin-treated animals. Creatinine levels also improved following edarabone treatment. Five days after cisplatin administration, a marked and significant decrease in Ccr was observed in the cisplatin treatment group versus controls (0.05 ± 0.01 ml/min/100 g b.wt. versus 0.93 ± 0.05 ml/min/100 g b.wt., P < 0.05). As shown in Table 2, Ccr was significantly higher in the edarabone-treated group compared with the cisplatin-treated group. Urinary NAG excretion, a marker of tubular damage, also increased in the cisplatin group but was significantly decreased by edarabone administration at a dose of 5 mg/kg (1.01 ± 0.15 versus 0.48 ± 0.07 IU/day, P < 0.05).
|
Assessment of Renal Mitochondrial Function. To investigate the involvement of mitochondrial function in the pathogenesis of cisplatin-induced renal dysfunction, we analyzed COX activity by histochemical COX staining (Fig. 5A). Histochemical staining for COX yielded an intense, fine granular pattern in the tubular cells in controls. Proximal tubular COX reactivity was markedly decreased in the cisplatin-treated compared with the control group. The edarabone-treated group exhibited equivalent COX reactivity to controls, however. The COX-positive area was significantly increased by edarabone treatment (Fig. 5B; cisplatin, 44.7%; cisplatin + edarabone 5 mg/kg, 79.6%).
|
Analysis of Oxidatively Modified Proteins, Lipids, and Nucleic Acids. To characterize the effects of edarabone on the level of protein oxidation in the cytosolic and mitochondrial fractions of the kidney, we measured levels of carbonyl groups in specific proteins by Western blot analysis. As shown in Fig. 6A, more carbonyl groups were present in cytosolic protein from the cisplatin-treated rats. Edarabone (5 mg/kg) reduced the protein oxidation by cisplatin in cytosol extracts. We also examined renal HNE content as an index of lipid peroxidation by Western blotting. The HNE content was increased by cisplatin treatment (Fig. 6B). The increase was completely prevented by treatment with 5 mg/kg edarabone. DNA damage caused by cisplatin-induced ROS was examined by investigating the 8-OHdG and AP sites in DNA. Cisplatin treatment resulted in a significant increase in 8-OHdG and the number of AP sites compared with controls (Table 3). The amount of these increases were reduced by edarabone treatment in a dose-dependent manner.
|
|
Quantification of Apoptosis in Vivo. The degree of apoptosis in the kidney was assessed using the TUNEL assay at day 5 after administration of cisplatin. As shown in Fig. 7A, significantly increased numbers of apoptotic nuclei were seen in the cisplatin group, but there were few apoptotic nuclei in the edarabone-treated group. Figure 7B shows the results of quantification of apoptotic nuclei after 5 days. The cisplatin-treated and cisplatin + edarabone-treated groups showed a 10.0- and 2.5-fold increase in apoptotic nuclei, respectively, over the saline-treated group.
|
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
),
hydrogen peroxide (H2O2), singlet oxygen
(1O2) or hydroxyl radical (·OH).
Superoxide anion is the most common oxygen radical and is a molecular species
generated in large amounts in the human body. Nevertheless, its reactivity is
very low such that it is not involved in modification of protein or lipid. In
contrast, the hydroxyl radical is one of the most reactive and aggressive
chemical species. Once excessive hydroxyl radicals are released, lipid
peroxidation, which causes changes in the fluidity and permeability of
membranes, is induced. No enzymes exist to eliminate the hydroxyl radical from
the cell. Edarabone, a free radical scavenger that traps hydroxyl radicals,
was developed for the potential treatment of cardiovascular disease,
cerebrovascular ischemia, and cerebral edema. This compound has been
registered in Japan for treatment of acute brain infarction. In the present
study, we demonstrated that edarabone treatment protected kidneys against
cisplatin-induced nephrotoxicity, both in vitro and in vivo. The present study
suggested that enhanced peroxidative damage caused by ROS, especially the
hydroxyl radical, might contribute to the pathogenesis of cisplatin-induced
acute renal failure.
Mitochondrial dysfunction is a central component of cisplatin
nephrotoxicity to proximal tubules in vivo
(Gordon and Gattone, 1986;
Davis et al., 2001) and in
vitro (Brady et al., 1993;
Zhang and Lindup, 1994). Renal
PTCs, which accumulate significantly greater amounts of cisplatin in vivo and
in vitro than other nephron segments
(Goldstein and Mayor, 1983),
undertake substance transport by consumption of large amounts of ATP. PTCs are
therefore sensitive to ATP production disturbances. Almost all ATP is produced
in mitochondria. Mitochondrial DNA does not bind histone protein, and thus, it
is readily susceptible to oxidative stress. Accumulation of oxidative damage
in mitochondrial DNA may affect mitochondrial respiratory function and cause
cellular dysfunction. In this study, we observed that mitochondria
dehydrogenation enzyme activity was deteriorated by cisplatin in PTCs and that
mitochondrial complex IV activity (determined by COX staining) was also
reduced. In vitro assays have revealed the generation of superoxide anion and
hydroxyl radical during interaction of cisplatin with DNA
(Masuda et al., 1994). We
found that treatment with edarabone, which has hydroxyl radical but not
superoxide anion scavenging activity, significantly ameliorated the
mitochondrial dysfunction induced by cisplatin both in vitro and in vivo.
These data indicate that hydroxyl radicals are important in the mechanism that
underlies cisplatin-induced renal injury.
Mitochondria normally generate small quantities of superoxide by
continuously converting 1 to 2% of consumed oxygen. Therefore, they are an
important source of ROS. Mitochondria are generally protected from oxidative
damage in several ways, including the activities of Mn-containing superoxide
dismutase and Se-containing glutathione peroxidase. Nevertheless, expression
of the superoxide dismutase and glutathione peroxidase genes is down-regulated
by cisplatin (Rao et al.,
1999; Huang et al.,
2001; Saad et al.,
2001) so that more ROS are generated. Furthermore,
cisplatin-induced mitochondrial injuries, especially inhibition of the
electron transfer system, produce large amounts of ROS
(Kruidering et al., 1997).
Namely, once mitochondria are impaired by cisplatin-induced ROS, more ROS are
generated, increasing the mitochondrial and cell damage. We demonstrated that
cisplatin-induced ROS generation in vitro by measuring the oxidative
conversion of DCFH-DA to DCF and found that edarabone could reduce
cisplatin-induced oxidation of intracellular DCF. The ROS detected by this
system was superoxide anion and hydrogen. The hydroxyl radical does not react
with DCFH-DA. Edarabone, which does not react with superoxide but scavenges
hydroxyl radicals, may therefore prevent ROS generation by protection of
mitochondria from hydroxyl radicals induced injury caused by cisplatin.
Although cells have developed various antioxidant defenses to protect
against ROS, free radicals that escape such defenses nonspecifically attack
and modify subcellular components, including proteins and free amino acids,
lipids and lipoproteins, nucleic acids, and connective tissue macromolecules.
These species may have an impact on cell activities such as membrane function,
metabolism, and gene expression, which results in further mitochondrial and
cell dysfunction. The oxidative proteins that have carbonyl groups at lysine,
arginine, proline, or threonine residues can modulate biochemical
characteristics of proteins, such as enzymatic activity, DNA binding
activities of transcription factors, and the susceptibility to proteolytic
degradation. We confirmed that many more carbonyl groups, which are formed in
the amino side chains of protein by metal catalyzed oxidation and are standard
markers of protein oxidation (Stadtman,
1992), were present in cisplatin-treated rats compared with
control rats. The radical scavenger edarabone could block cisplatin-induced
protein oxidation. This indicates that edarabone might protect against
cisplatin-induced functional disorders of renal metabolism.
It has been suggested that cisplatin-induced cytotoxicity may be due to
peroxidation of cell membranes (Montine
and Borch, 1988). The main content of cell membrane is lipid, and
thus lipid peroxidation in renal tissue might cause nephrotoxicity. Several
investigators have shown that cisplatin nephrotoxicity is associated with
lipid peroxidation in renal cortical slices and that antioxidants reverse
cisplatin-induced lipid peroxidation (Rao
et al., 1999; Saad et al.,
2001). The present study also demonstrated that cisplatin causes
lipid peroxidation. Edarabone was reported to inhibit iron-dependent lipid
peroxidation and to depress the lipo-oxygenase pathway
(Abe et al., 1988;
Watanabe et al., 1994a;
Watanabe and Egawa, 1994b).
Our data also indicate that edarabone is active in inhibiting lipid
peroxidation, suggesting that edarabone might protect against
cisplatin-induced renal functional disorders of membrane function.
Despite its toxic effects, cisplatin can induce DNA damage through the
generation of active oxygen species, such as the superoxide anion and hydroxyl
radicals (Crul et al., 1997).
Hydroxyl radicals have very strong oxidative power and, therefore, can injure
almost all bases and induce breakdown. It is known that 8-OHdG and AP site
increase as markers of oxidative DNA injury
(Lu et al., 2001). In this
study, we analyzed 8-OHdG and AP sites in extracted DNA from renal cortical
slices. Edarabone administration resulted in a suppression of 8-OHdG and AP
sites increase. We infer from this that oxidative damage to DNA was diminished
by edarabone treatment in cisplatin nephropathy, indicating that edarabone
might protect against cisplatin-induced disordered gene expression in the
kidneys.
Recent studies have shown that cisplatin causes apoptotic cell death in
renal tubule cells (Lieberthal et al.,
1996; Lau, 1999).
It has also been reported that activation of mitochondrial pathways were
important in apoptosis induced by cisplatin
(Davis et al., 2001;
Park et al., 2002). We
confirmed mitochondrial permeability transition by cisplatin exposure in
vitro. This led to release of cytochrome c, activation of caspase 9,
and entry into the execution phase of apoptosis
(Park et al., 2002). Edarabone
inhibited the cisplatin-induced decrease of m. It
should be noted that mitochondrial dysfunction could induce necrotic cell
death. Histological analysis of apoptotic and necrotic cell death in
prevention of cisplatin-induced renal dysfunction by edarabone is currently
under investigation in our laboratory.
Although edarabone did reduce the renal damage as evidenced on
histopathology, significant damage is still present. Another pathway, which is
not affected by edarabone, is thought to cause the cisplatin-induced
nephrotoxicity. The free radical scavenger, edarabone, can trap ·OH
only, so that another ROS such as
, or
H2O2 may cause renal damage. Recent findings suggest
that
can modulate nuclear factor-
B (NF-B) activation
(Schreck et al., 1991).
NF-B is a transcription factor implicated in the inducible regulation
of a wide range of genes involved in inflammatory and immune responses
(Baeuerle and Henkel, 1994;
Barnes and Karin, 1997).
Transcription of the intercellular adhesion molecule-1, inducible nitric oxide
synthase, and tumor necrosis factor- genes is regulated at the
NF-B site, and these genes have been shown to be up-regulated and to
participate in renal injury (Deng et al.,
2001; Ramesh and Reeves,
2002). So there is a possibility that these factors play a
significant role in cisplatin-induced renal damage. Another reason that
edarabone did not provide complete protection may be its short half-life.
Edarabone has a half-life of about 6 to 8 h. Because of this short half-life,
the cisplatin-induced nephropathy was not completely blocked by a single
intravenous injection of edarabone.
In conclusion, we have demonstrated in the present study that edarabone attenuated cisplatin-induced PTC damage in vitro and acute renal failure in vivo. In addition to its excellent antioxidant effects, the promising features of edarabone are that it is lipophilic, readily accessible to tissue, and thus an effective tissue level can be maintained with a single intravenous bolus injection. Our results also suggest that ROS induced by mitochondrial injuries contribute to renal damage after cisplatin treatment. ROS are involved in many other acute and chronic renal injuries, suggesting that edarabone treatment might be beneficial in various renal diseases.
|
Footnotes |
|---|
ABBREVIATIONS: ROS, reactive oxygen species; MCI-186,
3-methyl-1-phenyl-pyrazolin-5-one, edarabone; PTC, proximal tubular cell;
WST-1, tetrazolium salt; m, mitochondrial membrane
potential; JC-1,
5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl-carbocyanine
iodide; DCF, dichlorofluorescein; DCFH-DA,
2',7'-dichlorofluorescein diacetate; FACS, fluorescence-activated
cell sorting; NAG, N-acetyl--d-glucosaminidase; Ccr,
creatinine clearance; BUN, blood urea nitrogen; COX, cytochrome c
oxidase; HNE, 4-hydroxy-2-nonenal; 8-OHdG, 8-hydroxy-deoxyguanosine; AP,
apurinic/apyrimidinic; TUNEL, terminal deoxynucleotidyl transferase-mediated
dUTP nick end-labeling; NF-B, nuclear factor-B.
Address correspondence to: Dr. Minoru Satoh, Division of Nephrology, Department of Internal Medicine, Kawasaki Medical School, 577 Matsushima, Kurashiki, Okayama 701-0192, Japan. E-mail: satoh-minoru{at}mx1.tiki.ne.jp
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Abe K, Yuki S, and Kogure K (1988) Strong attenuation
of ischemic and postischemic brain edema in rats by a novel free radical
scavenger. Stroke 19:
480-485.
Baeuerle PA and Henkel T (1994) Function and
activation of NF-B in the immune system. Annu Rev
Immunol 12:
141-179.[Medline]
Barnes P and Karin M (1997) Nuclear factor-B: a
pivotal transcription factor in chronic inflammatory diseases. New
Engl J Med 336:
1066-1071.
Baud L and Ardaillou R (1986) Reactive oxygen species: production and role in the kidney. Am J Physiol 251: F765-F776.
Brady HR, Zeidel ML, Kone BC, Giebisch G, and Gullans SR
(1993) Differential actions of cisplatin on renal proximal tubule
and inner medullary collecting duct cells. J Pharmacol Exp
Ther 265:
1421-1428.
Cooley ME, Davis L, and Abrahm J (1994) Cisplatin: a clinical review. Part II. Nursing assessment and management of side effects of cisplatin. Cancer Nurs 17: 283-293.[Medline]
Crul M, Schellens JH, Beijnen JH, and Maliepaard M (1997) Cisplatin resistance and DNA repair. Cancer Treat Rev 23: 341-366.[CrossRef][Medline]
Davis CA, Nick HS, and Agarwal A (2001) Manganese
superoxide dismutase attenuates cisplatin-induced renal injury: importance of
superoxide. J Am Soc Nephrol
12:
2683-2690.
Deng J, Kohda Y, Chiao H, Wang Y, Hu X, Hewitt SM, Miyaji T, McLeroy P, Nibhanupudy B, Li S, and Star RA (2001) Interleukin-10 inhibits ischemic and cisplatin-induced acute renal injury. Kidney Int 60: 2118-2128.[CrossRef][Medline]
Di LF, Blank PS, Colonna R, Gambassi G, Silverman HS, Stern MD, and
Hansford RG (1995) Mitochondrial membrane potential in single
living adult rat cardiac myocytes exposed to anoxia or metabolic inhibition.
J Physiol (Lond) 486:
1-13.
Goldstein RS and Mayor GH (1983) The nephrotoxicity of cisplatin. Life Sci 32: 685-690.[CrossRef][Medline]
Gordon JA and Gattone Vh 2nd (1986) Mitochondrial alterations in cisplatin-induced acute renal failure. Am J Physiol 250: F991-F998.
Hara M, Yoshida M, Nishijima H, Yokosuka M, Iigo M, Ohtani KR, Shimada A, Hasegawa T, Akama Y, and Hirata K (2001) Melatonin, a pineal secretory product with antioxidant properties, protects against cisplatin-induced nephrotoxicity in rats. J Pineal Res 30: 129-138.[CrossRef][Medline]
Huang Q, Dunn R2, Jayadev S, DiSorbo O, Pack FD, Farr SB, Stoll RE,
and Blanchard KT (2001) Assessment of cisplatin-induced
nephrotoxicity by microarray technology. Toxicol Sci
63:
196-207.
Kawai H, Nakai H, Suga M, Yuki S, Watanabe T, and Saito K
(1997) Effects of a novel free radical scavenger, MCI-186, on
ischemic brain damage in the rat distal middle cerebral artery occlusion
model. J Pharmacol Exp Ther
281:
921-927.
Keller RJ, Halmes NC, Hinson JA, and Pumford NR (1993) Immunochemical detection of oxidized proteins. Chem Res Toxicol 6: 430-433.[CrossRef][Medline]
Kruidering M, van de Water B, Zhan Y, Baelde JJ, Heer E, Mulder GJ,
Stevens JL, and Nagelkerke JF (1997) Cisplatin-induced
nephrotoxicity in porcine proximal tubular cells: mitochondrial dysfunction by
inhibition of complexes I to IV of the respiratory chain. J
Pharmacol Exp Ther 280:
638-649.
Lau AH (1999) Apoptosis induced by cisplatin nephrotoxic injury. Kidney Int 56: 1295-1298.[CrossRef][Medline]
Lebwohl D and Canetta R (1998) Clinical development of platinum complexes in cancer therapy: an historical perspective and an update. Eur J Cancer 34: 1522-1534.
Lieberthal W, Triaca V, and Levine J (1996) Mechanisms of death induced by cisplatin in proximal tubular epithelial cells: apoptosis vs. necrosis. Am J Physiol 270: F700-F708.
Lu AL, Li X, Gu Y, Wright PM, and Chang DY (2001) Repair of oxidative DNA damage: mechanisms and functions. Cell Biochem Biophys 35: 141-170.[CrossRef][Medline]
Mancini M, Sedghinasab M, Knowlton K, Tam A, Hockenbery D, and Anderson BO (1998) Flow cytometric measurement of mitochondrial mass and function: a novel method for assessing chemoresistance. Ann Surg Oncol 5: 287-295.[Abstract]
Masuda H, Tanaka T, and Takahama U (1994) Cisplatin generates superoxide anion by interaction with DNA in a cell-free system. Biochem Biophys Res Commun 203: 1175-1180.[CrossRef][Medline]
Matsushima H, Yonemura K, Ohishi K, and Hishida A (1998) The role of oxygen free radicals in cisplatin-induced acute renal failure in rats. J Lab Clin Med 131: 518-526.[CrossRef][Medline]
Montine TJ and Borch RF (1988) Quiescent LLC-PK1 cells
as a model for cis- diamminedichloroplatinum (II) nephrotoxicity and
modulation by thiol rescue agents. Cancer Res
48:
6017-6024.
Murota S, Morita I, and Suda N (1990) The control of vascular endothelial cell injury. Ann N Y Acad Sci 598: 182-187.[Medline]
Park MS, De LM, and Devarajan P (2002) Cisplatin
induces apoptosis in LLC-PK1 cells via activation of mitochondrial pathways.
J Am Soc Nephrol 13:
858-865.
Ramesh G and Reeves WB (2002) TNF- mediates
chemokine and cytokine expression and renal injury in cisplatin
nephrotoxicity. J Clin Investig
110:
835-842.[CrossRef][Medline]
Rao M, Kumar MM, and Rao MA (1999) In vitro and in
vivo effects of phenolic antioxidants against cisplatin-induced
nephrotoxicity. J Biochem (Tokyo)
125:
383-390.
Saad SY, Najjar TA, Noreddin AM, and Al RA (2001) Effects of gemcitabine on cisplatin-induced nephrotoxicity in rats: schedule-dependent study. Pharmacol Res 43: 193-198.[CrossRef][Medline]
Safirstein R, Winston J, Goldstein M, Moel D, Dikman S, and Guttenplan J (1986) Cisplatin nephrotoxicity. Am J Kidney Dis 8: 356-367.[Medline]
Schmidley JW (1990) Free radicals in central nervous
system ischemia. Stroke
21:
1086-1090.
Schreck R, Rieber P, and Baeuerle PA (1991) Reactive
oxygen intermediates as apparently widely used messengers in the activation of
the NF-B transcription factor and HIV-1. EMBO (Eur Mol Biol
Organ) J 10:
2247-2258.[Medline]
Seligman AM, Karnovsky MJ, Wasserkrug HL, and Hanker JS
(1968) Nondroplet ultrastructural demonstration of cytochrome
oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine
(DAB). J Cell Biol 38:
1-14.
Stadtman ER (1992) Protein oxidation and aging.
Science (Wash DC) 257:
1220-1224.
Takaya K, Koya D, Isono M, Sugimoto T, Sugaya T, Kashiwagi A, and Haneda M (2003) Involvement of ERK pathway in albumin-induced MCP-1 expression in mouse proximal tubular cells. Am J Physiol Renal Physiol 294: F1037-F1045.
Toyokuni S, Tanaka T, Hattori Y, Nishiyama Y, Yoshida A, Uchida K, Hiai H, Ochi H, and Osawa T (1997) Quantitative immunohistochemical determination of 8-hydroxy-2'-deoxyguanosine by a monoclonal antibody N45.1: its application to ferric nitrilotriacetate-induced renal carcinogenesis model. Lab Investig 76: 365-374.[Medline]
Watanabe T and Egawa M (1994b) Effects of an
antistroke agent MCl-186 on cerebral arachidonate cascade. J
Pharmacol Exp Ther 271:
1624-1629.
Watanabe T, Yuki S, Egawa M, and Nishi H (1994a)
Protective effects of edarabone on cerebral ischemia possible involvement of
free radical scavenging and antioxidant actions. J Pharmacol Exp
Ther 268:
1597-1604.
Wu TW, Zeng LH, Wu J, and Fung KP (2000) Edarabone: further histochemical and biochemical evidence of neuroprotection. Life Sci 67: 2387-2392.[CrossRef][Medline]
Zhang JG and Lindup WE (1994) Cisplatin
nephrotoxicity: decreases in mitochondrial protein sulfhydryl concentration
and calcium uptake by mitochondria from rat renal cortical slices.
Biochem Pharmacol 47:
1127-1135.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  T. Arai, M. Nonogawa, K. Makino, N. Endo, H. Mori, T. Miyoshi, K. Yamashita, M. Sasada, M. Kakuyama, and K. Fukuda The Radical Scavenger Edaravone (3-Methyl-1-phenyl-2-pyrazolin-5-one) Reacts with a Pterin Derivative and Produces a Cytotoxic Substance That Induces Intracellular Reactive Oxygen Species Generation and Cell Death J. Pharmacol. Exp. Ther., February 1, 2008; 324(2): 529 - 538. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Prewett, D. S. Deevi, R. Bassi, F. Fan, L. M. Ellis, D. J. Hicklin, and J. R. Tonra Tumors Established with Cell Lines Selected for Oxaliplatin Resistance Respond to Oxaliplatin if Combined with Cetuximab Clin. Cancer Res., December 15, 2007; 13(24): 7432 - 7440. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Omori, Y. Shikata, K. Sarai, N. Watanabe, J. Wada, N. Goda, N. Kataoka, K. Shikata, and H. Makino Edaravone mimics sphingosine-1-phosphate-induced endothelial barrier enhancement in human microvascular endothelial cells Am J Physiol Cell Physiol, November 1, 2007; 293(5): C1523 - C1531. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. S. Razzaque Cisplatin nephropathy: is cytotoxicity avoidable? Nephrol. Dial. Transplant., August 1, 2007; 22(8): 2112 - 2116. [Full Text] [PDF]
|
|
|
|
 |
|
 C. Yang, V. Kaushal, S. V. Shah, and G. P. Kaushal Mcl-1 is downregulated in cisplatin-induced apoptosis, and proteasome inhibitors restore Mcl-1 and promote survival in renal tubular epithelial cells Am J Physiol Renal Physiol, June 1, 2007; 292(6): F1710 - F1717. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. J. Choi, Y. J. Piao, M. J. Lim, J. H. Kim, J. Ha, W. Choe, and S. S. Kim Overexpressed Cyclophilin A in Cancer Cells Renders Resistance to Hypoxia- and Cisplatin-Induced Cell Death Cancer Res., April 15, 2007; 67(8): 3654 - 3662. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Haruna, N. Kashihara, M. Satoh, N. Tomita, T. Namikoshi, T. Sasaki, T. Fujimori, P. Xie, and Y. S. Kanwar Amelioration of progressive renal injury by genetic manipulation of Klotho gene PNAS, February 13, 2007; 104(7): 2331 - 2336. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Kroemer, L. Galluzzi, and C. Brenner Mitochondrial Membrane Permeabilization in Cell Death Physiol Rev, January 1, 2007; 87(1): 99 - 163. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Zhuang, Y. Yan, R. A. Daubert, J. Han, and R. G. Schnellmann ERK promotes hydrogen peroxide-induced apoptosis through caspase-3 activation and inhibition of Akt in renal epithelial cells Am J Physiol Renal Physiol, January 1, 2007; 292(1): F440 - F447. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Seth, C. Yang, V. Kaushal, S. V. Shah, and G. P. Kaushal p53-dependent Caspase-2 Activation in Mitochondrial Release of Apoptosis-inducing Factor and Its Role in Renal Tubular Epithelial Cell Injury J. Biol. Chem., September 2, 2005; 280(35): 31230 - 31239. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Sunami, H. Sugiyama, D.-H. Wang, M. Kobayashi, Y. Maeshima, Y. Yamasaki, N. Masuoka, N. Ogawa, S. Kira, and H. Makino Acatalasemia sensitizes renal tubular epithelial cells to apoptosis and exacerbates renal fibrosis after unilateral ureteral obstruction Am J Physiol Renal Physiol, June 1, 2004; 286(6): F1030 - F1038. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|
 |
 |
 |
