Research Article

Amelioration of cisplatin-induced nephrotoxicity by statins

Rajesh A. Maheshwari, Girish U. Sailor, Lalji Patel1, R. Balaraman

ABSTRACT
Objectives: This study aimed to investigate the protective effect of simvastatin (SIM) and
rosuvastatin (RST) on cisplatin (CIS)-induced nephrotoxicity.
Materials and Methods: Adult female Wistar rats were divided into six groups: Control
group (Group 1) received 0.5% sodium carboxy methyl cellulose, group 2 and group 3
Department of Pharmacy, received SIM and RST for 10 days, respectively, and group 4 was injected single dose of
Sumandeep Vidyapeeth, Piparia,
CIS (7 mg/kg, i.p.). Group 5 and 6 were treated with SIM (10 mg/kg, p.o.) and RST (10 mg/
Vadodara, 1Department of
kg, p.o.) for 10 days, respectively. All groups received cisplatin on the 5th day of treatment.
Pharmacology and Toxicology,
Zydus Research Centre, Renal function tests like serum creatinine, urea, BUN, albumin, calcium, uric acid and
Ahmedabad, Gujarat, India magnesium, serum lipids, and markers of oxidative stress such as renal malondialdehyde
(MDA) level and superoxide dismutase (SOD) and catalase (CAT) activities were measured.
Received: 04-10-2012 All tissues were investigated for histopathological changes.
Revised: 19-12-2012 Result: CIS reduced the renal function, which was reflected with significant increase in
Accepted: 23-04-2013 serum urea, BUN, serum creatinine, uric acid and also significant decrease serum calcium,
magnesium, albumin levels. In addition, cisplatin caused renal tubular damage with a
Correspondence to: higher MDA level, depletion of SOD and CAT activity, and elevation of serum lipids.
Mr. Rajesh Maheshwari, SIM or RST ameliorate CIS induced renal damage due to improvement in renal function,
E-mail: rajpharma2007@gmail.com oxidative stress, suppression of serum lipids, and histological alteration.
Conclusions: This finding suggests that simvastatin and rosuvastatin may have a protective
effect against cisplatin-induced kidney damage via amelioration of lipid peroxidation as
well as due to improvement of renal function, and lipid-lowering effects.

KEY WORDS: Cisplatin, nephrotoxicity, rosuvastatin, simvastatin

Introduction
Many studies have assessed the effect of various substances
to neutralize the nephrotoxic action of cisplatin. Statins are widely
used clinically for lowering hypercholesterolemia because of
their inhibitory effect on 3-hydroxy-3-methylglutaryl coenzyme A
(HMG-CoA) reductase, the enzyme that catalyzes the rate-limiting
step of the cholesterol synthesis in the liver and other tissues.[1]
Statins have beneficial changes in other lipid fractions, along
with significant effects on numerous measures of inflammation,
immunity, oxidative stress, thrombosis, and vascular and
renal function.[2-8] Pleiotropic effects of statins have also been
implicated in reductions in heart failure.[9] Cis-dichlorodiammine
platinum (II) or cisplatin (CIS) is currently among the most widely
used agents in the chemotherapy of cancer.[10] Limitation to its
greater efficacy is due to its nephrotoxicity. Acute and chronic
nephrotoxicity of CIS occur in man and animals especially after
repeated administration.[11] The pathogenesis of renal damage
caused by CIS is due to an increase in lipid peroxide levels and
causes generation of reactive oxygen metabolites (ROM) and
inhibits the activity of antioxidant enzymes in renal tissue.[12,13]
The present study was undertaken to investigate the protective
effect of simvastatin (SIM) and rosuvastatin (RST) in CIS-induced
nephrotoxicity through pleiotropic effect.
Materials and Methods

Drugs and Chemicals

Access this article online
Quick Response Code:
Rosuvastatin and simvastatin were obtained from Zydus
Website: www.ijp-online.com Cadila, Ahmedabad, India. Cisplatin injection was obtained from
DOI: 10.4103/0253-7613.115016 VHB limited, Mumbai, India. All other chemicals and reagents
used in the study were of analytical grade.
Experimental Animals
The experimental protocol was approved by the Institutional
Animal Ethics Committee for the Purpose of Control and

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 Maheshwari, et al.: Renoprotective effect by statins

Supervision of Experiments on Animals (CPCSEA). The
experiment was carried out on healthy adult female Wistar
rats weighing 200-250 g. Rats were housed in polypropylene
cages, maintained under standardized condition (12-h light/
dark cycle, 24°C, 35 to 60% humidity) and allowed free access
to diet (Nav Maharashtra Oil Mills Pvt. Ltd., Pune) and purified
drinking water ad libitium.
Induction of cisplatin-induced kidney damage
The rats were divided into six groups of six animals each.
Group 1 animal received 0.5% sodium CMC orally for 10 days.
Group 2 and 3 received SIM (10 mg/kg, p.o.) and RST (10 mg/kg,
p.o.) for 10 days, respectively. Group 4 was injected single dose
of CIS (7 mg/kg, i.p.). Group 5 and 6 were treated with SIM (10
mg/kg, p.o) and RST (10 mg/kg, p.o) for 10 days, respectively.
All groups received cisplatin on the 5th day of treatment.[14]
At the end of treatment on 10th day, blood samples were
withdrawn from retro-orbital plexus under light ether anesthesia
without any anti-coagulant and allowed for 10 minutes to clot
at room temperature. It was centrifuged at 2500 rpm for 20
minutes for serum separation. The serum obtained was kept
at 4ºC until used. Studies have been carried out to determine
kidney function markers such as urea, BUN, creatinine, albumin,
calcium and magnesium and uric acid and serum lipid levels
like total cholesterol, LDL-cholesterol and triglycerides were
estimated from serum sample using standard diagnostic kit
(SPAN Diagnostics and Crest Biosystems, India).
Estimation of biomarkers of Oxidative stress
Kidney was removed and kept in cold conditions (precooked
in inverted Petridis on ice). The tissues were cross chopped
with surgical scalpel into fine slices in chilled 0.25 M sucrose,
quickly blotted on a filter paper. The tissues were minced and
homogenized in 10 mM Tris-HCl buffer, pH 7.4 (10 %w/v) with 25
strokes of tight Teflon pestle of glass homogenizer at a speed of
2500 rpm. The clear supernatant was used for oxidative stress
markers assays. The levels of malondialdehyde (MDA) formation
and the activities of endogenous antioxidant enzymes such as
catalase (CAT) and superoxide dismutase (SOD) were estimated
by the method of Slater and Sawyer[15] Hugo Aebi as given by
Hugo[16] and Mishra and Fridovich,[17] respectively.
Histopathology
For light microscopic evaluation, kidney tissues of each
group were fixed in 10% phosphate buffered formalin. Paraffin-
embedded specimens were cut into 6 mm-thick sections and
stained with hematoxylin and eosin (H&E). The kidney tissues
were examined under a light microscope (Olympus Bioxl) for
the presence of tubular changes by an observer blinded to the
animal treatment group.
Statistical Analysis
All of the data are expressed as mean ± SEM. Statistical
significance between more than two groups was tested using
one-way ANOVA followed by the Bonferroni multiple comparisons
test. Calculations were done using Graphpad Prism software.
The significance level was set at P < 0.05 for all tests.
Results
Effects of statins on CIS-induced kidney dysfunction
Administration of single injection of CIS (7 mg/kg, i.p.)
caused a marked reduction in renal function, as characterized by
significant (P < 0.001) increase in serum urea, BUN, creatinine,
uric acid and a significant (P < 0.001) decrease serum calcium,
magnesium, albumin levels when compared to control group
[Table 1]. Thus, these data indicate that a single intraperitoneally
injection of 7 mg/kg CIS impairs kidney functions.
The treatment with SIM showed significant decrease in
BUN (P < 0.01), urea (P < 0.01), and creatinine (P < 0.05)
and a significant increase in albumin (P < 0.01) as compared
to CIS-treated group. The treatment with RST showed highly
significant decrease in BUN (P < 0.001), urea (P < 0.001),
creatinine (P < 0.01) and significant increase in albumin (P <
0.001) as compared to CIS-treated group.
Treatment with SIM showed a significant increase in
calcium (P < 0.001) as well as magnesium (P < 0.05) level
and a significant decrease in uric acid (P < 0.01) level as
compared to CIS-treated group, and the treatment with RST
showed a significant increase in calcium (P < 0.001) as well
as magnesium (P < 0.01) level and a significant decrease in
uric acid (P < 0.01) level as compared to CIS-treated group.
CIS treatment significantly increased serum total cholesterol,
LDL-cholesterol, and TG levels compared with control group (P
< 0.001). RST or SIM alone did not affect serum lipids levels,
but co-administration of SIM and RST in cisplatin-treated rats
significantly suppressed serum total cholesterol (P < 0.001),
LDL-cholesterol (P < 0.001), and TG (P < 0.01) levels as
compared to CIS-treated alone [Table 2].

Table 1:
Effect of simvastatin and rosuvastatin treatment (10 mg/kg/day p.o. for 10 days) on kidney function tests of control and cisplatin-
injected rats

Groups Serum Urea BUN (mg/dl) Serum Serum Albumin Serum Serum SerumUric
(mg/dl) Creatinine (gm/dl) Calcium Magnesium Acid (mg/dl)
(mg/dl) (mg/dl) (mEq /l)
Cont 39.15 ± 1.29 18.28 ± 0.60 0.66 ± 0.046 3.41 ± 0.44 9.79 ± 0.28 3.14 ± 0.28 2.63 ± 0.23
SIM Cont 40.09 ± 1.67 18.72 ± 0.78 0.73 ± 0.071 3.51 ± 0.29 9.62 ± 0.31 2.79 ± 0.20 2.46 ± 0.23
RST Cont 39.87 ± 1.22 18.62 ± 0.57 0.68 ± 0.054 3.54 ± 0.20 9.77 ± 0.36 2.75 ± 0.25 2.76 ± 0.23
CIS 108.6 ± 6.62### 50.73 ± 3.09### 2.13 ± 0.18### 1.47 ± 0.11### 5.95 ± 0.31### 1.66 ± 0.20### 4.82 ±0.38###
SIM + CIS 89.87 ± 2.53 ** 41.74 ± 1.18 ** 1.64 ± 0.11 * 3.02 ± 0.10 ** 7.81 ± 0.18*** 2.68 ± 0.17* 3.29 ± 0.27**
RST + CIS 64.19 ± 1.89 *** 29.97 ± 0.88 *** 1.53 ± 0.07 ** 3.18 ± 0.11 *** 8.69 ± 1.16*** 2.88 ± 0.11** 3.21 ± 0.14**
Values are expressed as mean ± SEM for six animals in the group. #P < 0.05, ## P < 0.01, ###P < 0.001 considered statistically significant as compared to control group; * P <
0.05, ** P < 0.01, ***P < 0.001 considered statistically significant as compared to cisplatin group.

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 Maheshwari, et al.: Renoprotective effect by statins

Table 2:
Effect of simvastatin and rosuvastatin treatment (10 mg/kg/day p.o. for 10 days) on serum levels of lipid on control and cisplatin-
injected rats

Groups Total cholesterol(mg/dl) LDL-cholesterol(mg/dl) Triglyceride(mg/dl)

Cont 65.37 ± 3.55 15 ± 1.71 116.5 ± 2.18
SIM Cont 63.17 ± 2.38 15.17 ± 2.00 109.5 ± 3.73
RST Cont 62 ± 2.70 16.67 ± 1.38 111.8 ± 2.07
CIS 92.6 ± 1.88### 35.33 ± 1.40### 139.8 ± 4.2###
SIM + CIS 77 ± 1.71*** 25.33 ± 0.84*** 95.67 ± 2.77**
RST + CIS 74.5 ± 3.93*** 23.83 ± 1.24*** 98.17 ± 3.12**
Values are expressed as mean ± SEM for six animals in the group. #P < 0.05, ##P < 0.01, ###P < 0.001 considered statistically significant as compared to control group; * P < 0.05,
** P < 0.01, ***P < 0.001 considered statistically significant as compared to cisplatin group.

Effect statins on markers of oxidative stress Figure 1: Effect of simvastatin and rosuvastatin on biomarkers of
Oxidative stress increases in CIS-treated group as compared oxidative stress: (a) MDA, (b) SOD, and (c) CAT activity, #P < 0.05, ##
P < 0.01, ###P < 0.001 vs. control; * P < 0.05, ** P < 0.01, ***P < 0.001
to control group. The treatment with SIM and RST showed a vs. cisplatin
significantly reduced oxidative stress level.
The treatment with SIM showed a significant increase in
SOD (P < 0.001), CAT (P < 0.001) activities and a significant
decrease in MDA (P < 0.01) levels as compared to CIS-treated
group [Figure 1]. The treatment with RST showed a significant
increase in SOD (P < 0.01), CAT (P < 0.001) activities and
significant decrease in MDA (P < 0.001) level as compared to
CIS-treated group [Figure 1].
Histopathological changes
The architecture of the kidney was disturbed with CIS
administration as compared to normal structural features of
control animal. CIS administration caused a severe tubular
necrosis, vaculation, and tubular dilation. SIM and RST a
treatment showed amelioration of tubular damage induced
by CIS while SIM and RST alone did not have any effect on
morphology of kidney [Figure 2]. These results indicated that
treatment with SIM and RST afforded protection against CIS-
induced nephrotoxicity.
Discussion
The results demonstrate that daily SIM and RST treatment
markedly ameliorate CIS-induced kidney damage as shown in
microscopic examination and biochemical parameters. CIS
is one of the widely used cytotoxic agents in the treatment
of several forms of cancers. In spite of its clinical usefulness,
there are constraints in using this drug as it causes
nephrotoxicity and neurotoxicity. Other less frequent toxic
b
effects like hepatotoxicity, which are generally observed after
administration of high doses of CIS, can also alter the clinical
situation in patients.[18-21] The mechanism of nephrotoxicity due
to cisplatin might be due to the release of free radicals.[22] In
the present study, CIS injection produced severe degeneration
in glomeruli, proximal, and distal tubules. Tubular swelling and
necrosis are in line with earlier studies, which show similar
findings.[23,24] The renal function tests such as BUN, serum urea,
serum creatinine, and serum uric acid were elevated and serum
albumin, calcium, and magnesium levels were lowered in CIS-
injected animals as compared to control group. It is probable
that increased reactive oxygen species (ROS) production in the
renal tissue may be responsible for this damage of the organ
as reflected by the change in the levels of MDA and activities of c

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 Maheshwari, et al.: Renoprotective effect by statins
Figure 2: Light microscopy of kidney tissues from rats (HE stained
kidney sections). (A) Control group, (B) Simvastatin group, (C)
Rosuvastatin, (D) Cisplatin. Shows severe glomerular degeneration
(arrow) and degeneration in tubular cells (asterisks), (E) Cisplatin +
Simvastatin, (F) Cisplatin + Rosuvastatin
SOD and catalase in the study. Another player in the induction
of these changes is the depletion of thiol groups in kidney.
Both these effects, in concert, led to the development of CIS-
induced nephrotoxicity. DNA fragmentation, tubular necrosis
as well as tubular apoptosis have also been implicated in
nephrotoxicity induced by CIS.[25] It is evident from prior clinical
data that CIS administration results in elevation in serum urea
and creatinine levels as a result of nephrotoxicity.[26,27] Statins
represent a class of anti-hyperlipidemic drugs that have a lot of
pleiotropic effects. In the present study, pre-treatment with RST
and SIM was able to counteract CIS-mediated renal damage.
Treatment with these statins improved functional nephrotoxicity
indices. Several studies have shown a renoprotective effect
of statins in different animal models.[28] Renal tissue lesions
induced by CIS were significantly improved by SIM and RST.
This is evident from histopathological findings and by an
improvement in serum creatinine, urea, and BUN values.
Since ROS generation mediates CIS nephrotoxicity, it may be
presumed that antioxidant effect of statins may be protecting
these conditions. SIM, in particular, reverses oxidant-antioxidant
imbalance and has good hydroxyl radical scavenging activity.[29]
RST also shows a dose-dependent antioxidant effect. These
effects are primarily mediated by up-regulation of antioxidant
defense protein heme oxygenase-1(HO-1).[30] The antioxidant
effect of SIM and RST plays a significant role in amelioration
of CIS-induced renal damage, but the lipid-lowering effect of
statins may also be involved in this mechanism. RST and SIM
treatment decreased serum cholesterol, LDL-cholesterol, and
TG level in CIS-treated rats as compared to CIS treated alone.
Therefore, it is possible that lipid accumulation on the renal
tubule might be prevented, thereby showing renoprotective
effects. The major renoprotective effect of both, RST and SIM
is, therefore, mediated by normalization of ROS production.
Conclusion
These results indicate that RST and SIM improve biochemical
and histological alterations induced by CIS. However, RST
shows more significant renoprotective effect than SIM. These
renoprotective effects are beyond the lipid-lowering effects of
statins. Mechanisms of this renoprotective effect mainly include
amelioration of lipid peroxidation induced by CIS as well as
activation of defense mechanisms.
Acknowledgement
We acknowledge our Head of the Department Dr. A. K. Seth for his
co-operation and encouragement for publishing this research paper.
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Cite this article as: Maheshwari RA, Sailor GU, Patel L, Balaraman R.
Amelioration of cisplatin-induced nephrotoxicity by statins. Indian J Pharmacol
2013;45:354-8.
Source of Support: This research received no specific grant from
any funding agency in the public, commercial, or not-for-profit sectors.
Conflict of Interest: No.
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