Renal Failure, 2013; 35(2): 268–274

Copyright © Informa Healthcare USA, Inc.

ISSN 0886-022X print/1525-6049 online
DOI: 10.3109/0886022X.2012.743859

LABORATORY STUDY

Pomegranate Extract Attenuates Gentamicin-Induced Nephrotoxicity

in Rats by Reducing Oxidative Stress

Mustafa Cekmen1, Alper Otunctemur2, Emin Ozbek2, Suleyman Sami Cakir2, Murat Dursun2,
Emre Can Polat3, Adnan Somay4 and Nurver Ozbay4
1
Department of Biochemistry, Kocaeli University, Kocaeli, Turkey; 2Department of Urology, Okmeydani Training and Research
Hospital, Istanbul, Turkey; 3Department of Urology, Balikligol Government Hospital, Sanlıurfa, Turkey; 4Department of
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Pathology, Fatih Sultan Mehmet Training and Research Hospital, Istanbul, Turkey

Abstract
Nephrotoxicity is a major complication of gentamicin (GEN), which is widely used in the treatment of severe Gram-
negative infections. Reactive oxygen species are important mediators of GEN-induced nephrotoxicity. Because of the
strong antioxidant properties of pomegranate extract (PE), we evaluated the protective effect of PE against GEN-induced
nephrotoxicity. Thirty-two adult male rats were randomly divided into four equal groups: (1) controls; (2) treated with GEN
for 14 consecutive days (100 mg/kg/day); (3) treated with GEN plus distilled water; and (4) treated with GEN plus PE (100
For personal use only.

μL). After 15 days, the rats were killed and their kidneys were taken, and blood analysis was performed. Tubular necrosis
and interstitial fibrosis scores were determined histopathologically; and biochemically, nitric oxide (NO), malondialdehyde
(MDA), and reduced glutathione (GSH) levels in kidneys were determined. Urea, creatinine, Naþ, and Kþ levels were
investigated in the blood analysis. Statistical analyses were made by the chi-square test and analysis of variance. Serum
urea and creatinine levels were significantly higher in rats treated with GEN alone than rats in the control and the GEN þ
PE-treated groups. The GSH level in renal tissue of only GEN-treated rats was significantly lower than those in the control
group, and administration of PE to GEN-treated rats significantly increased the level of GSH. The group that was given
GEN and PE had significantly lower MDA levels in kidney cortex tissue than those given GEN alone. There was no
significant difference of NO levels between the groups. In rats treated with GEN þ PE, despite the presence of mild tubular
degeneration and tubular necrosis is less severe, and glomeruli maintained a better morphology when compared with the
GEN-treated group. We think that PE prevents kidney damage by decreasing oxidative stress in kidney.

Keywords: gentamicin, pomegranate extract, nephrotoxicity, nitric oxide, reactive oxygen species

INTRODUCTION aminoglycosides are excreted in the urine correspond to

Gentamicin (GEN) is an antibiotic of aminoglycoside
group and is widely used in the treatment of Gram-
negative infections. A major complication of this drug is
nephrotoxicity, and it has been estimated that approxi-
mately 10–20% of cases are treated with aminoglycoside
therapy.1 Despite the introduction of less nephrotoxic
antibiotics against Gram-negative microorganism, it is
still used because of its low cost and efficacy against
resistant beta-lactam positive microorganisms.2 GEN is
not metabolized in the body but is essentially eliminated
by glomerular filtration and partially reabsorbed by prox-
imal tubular cells.3 No conclusive evidence exists of
tubular secretion of GEN, and consequently, most
filtrates.4 The specificity of GEN for renal toxicity is
apparently related to its preferential accumulation in
the renal proximal convoluted tubules, reaching a con-
centration of 5–50 times higher than plasma in the
tubular renal cell.5
It has been demonstrated that nephrotoxicity induced
by GEN is characterized by direct tubular necrosis,
which is localized mainly in the proximal tubules. The
exact mechanisms of GEN-induced nephrotoxicity still
remain unclear. However, several studies demonstrated
that reactive oxygen species (ROS) may be important in
GEN-induced nephrotoxicity.6 Moreover, GEN has also
been shown to enhance the generation of ROS. Lipid
peroxidation (LPO) mediated by ROS has been

Address correspondence to Alper Otunctemur, Department of Urology, Okmeydani Research & Education Hospital, Sisli, 34384 Istanbul,
Turkey. Tel.: þ90 212 314 5500; Fax: þ90 212 314 5503; E-mail: alperotunctemur@yahoo.com
Received 28 July 2012; Revised 18 October 2012; Accepted 23 October 2012

268

suggested as a causative agent of cell death in different
pathological states including various models of renal dis-
eases.7,8 Besides their direct damaging effects on tissues,
ROS seem to trigger the accumulation of leukocytes in
the tissue involved, and thus cause tissue injury indirectly
through activated neutrophils. It has been shown that
activated neutrophils secrete enzymes such as myeloper-
oxidase, elastase, and proteases and liberate oxygen radi-
cals.9 On the other hand, in vivo and in vitro studies have
shown that the scavengers of reactive oxygen metabolites
are protective in GEN-induced renal failure.10,11
Pomegranate extract (PE) and its derivatives have been
used for centuries to confer health benefits in a number of
inflammatory diseases. Edible parts of pomegranate fruit
represent 52% of total fruit weight, comprising 78% juice
and 22% seeds.12 Fresh juice is rich in vitamin C, and
polyphenolic compounds such as anthocyanins, punicala-
gin, and ellagic and gallic acids.13,14 PE is a rich source of
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potent polyphenolic, flavonoid antioxidants (anthocya-
nins). The soluble polyphenol content in PE varies within
the limits of 0.2%–1.0% depending on the variety and
includes mainly anthocyanins that have been shown to
possess antiatherogenic properties. Anthocyanins were
shown to be effective inhibitors of LPO, production of
nitric oxide (NO), and inducible nitric oxide synthase
(iNOS) activity in different model systems.15,16
For personal use only.
Pomegranate has become more popular because of the
attribution of important physiological properties, such as
anticancer,17,18 cholesterol-lowering, cardioprotective,19
and so on. Many investigators have reported that pome-
granate and its derivatives have free radical scavenger and
potent antioxidant activity.20–22 It has also been shown
that pomegranate can suppress nuclear factor kappa B
(NF-κB) activation through a novel mechanism in vas-
cular endothelial cells.23
As a result of this information, in this study we investi-
gated the possible inhibitory effects of pomegranate against
the GEN-induced oxidative stress and renal injury in rat
models. The nephroprotective effect of PE is previously
established in many studies, however, to our knowledge
this is the first study in literature concerning the protective
role of pomegranate against GEN nephrotoxicity.
MATERIALS AND METDODS
Drugs
GEN was purchased from Bilim Pharmaceuticals
(Istanbul, Turkey), and PE (100% pure, pasteurized
pomegranate juice, 250 mL; Elite Natural Beverage Co.,
Ankara, Turkey) was purchased from a local store. GEN
was dissolved in saline and injected intraperitoneally. PE
was dissolved in distilled water and administered via naso-
gastric gavage. The average of 2.5 mL diluted PE contains
100-μL PE, which is equivalent to 2.8 μmol of total poly-
phenol per day.
Animals
Adult male Wistar albino rats (200–250 g) were housed in
clean plastic cages in a temperature and humidity-
© 2013 Informa Healthcare USA, Inc.
Pomegranate Extract Attenuates Gentamicin Nephrotoxicity in Rats 269
controlled facility with a constant 12 h light/dark cycle
with free access to food and water. The use of animals
and the experimental protocol were approved by the
Institutional Animal Care and Use Committee, and ani-
mals were treated in accordance with the Guide for the
Care and Use of Laboratory Animals of Research Council.
Treatment and Experimental Design
After a quarantine period of 7 days, 32 rats were randomly
divided into four equal groups, each consisting of eight
animals as follows: (1) controls; (2) intraperitoneally
injected with GEN for 14 consecutive days (100 mg/kg/
day); (3) treated with GEN plus distilled water via naso-
gastric gavage for 14 days; (4) treated with GEN plus PE
(100 μ/L) for 14 days. Rats were treated for 14 days. After
15 days, rats were killed and their kidneys were taken, and
blood analysis was performed. Tubular necrosis and inter-
stitial fibrosis scores were determined histopathologically
in a part of kidneys; NO, malondialdehyde (MDA), and
reduced glutathione (GSH) levels were determined in the
other part of kidneys. Urea, creatinine, Naþ, and Kþ levels
were investigated in a blood analysis.
Sample Collection and Biochemical Assays
Twenty-four hours after the administration of last doses of
GEN and PG, on the 15th day, the rats were anesthetized
by intraperitoneal injection of ketamine and were sacrificed.
Twenty-four-hour urine collections were obtained in stan-
dard metabolic cages a day before the rats were killed.
Renal cortical tissues were separated into two parts for
biochemical analysis and light microscopic examination.
Blood samples were also obtained by cardiac puncture to
assess the serum levels of urea, creatinine, Naþ, and Kþ
concentrations. The tissues were shock-frozen in liquid
nitrogen and were kept in 80 C. In the frozen tissues,
MDA, end product of LPO, reduced GSH, nonenzymatic
antioxidant, and total nitrite (NOx), a stable product of
NO, were evaluated biochemically as a means of oxidative
stress.
Renal impairment was assessed by serum urea and
creatinine levels, as well as by the kidney histology.
Serum urea and creatinine levels were determined
using an autoanalyzer (SYNCHRON LX20, Beckman
Coulter Inc., Ireland) by using commercial Becman
Coulter diagnostic kits. Kidney tissue (300 mg) was
homogenized in ice-cold tamponade containing
150 mM KCl for determination of MDA. MDA levels
were assayed for products of LPO. MDA referred to as
thiobarbituric acid reactive substance was measured with
thiobarbituric acid at 532 nm using a spectrofluorom-
eter, as described previously.24 GSH was determined by
the spectrophotometric method, which was based on the
use of Ellman’s reagent.25
NOx was quantified by the Griess reaction26 after
incubating the supernatant with nitrate reductase from
Escherichia coli to convert nitrate (NO3) to nitrite (NO2).
Griess reagent (1 mL—1% sulfanilamide, 0.1%
 270 M. Cekmen et al.
naphthyl-ethylenediamine hydrochloride, and 2.5%
phosphoric acid; Sigma Chemical Co., St. Louis, MO,
USA) was then added to 1 mL of supernatant. The
absorbance was read at 545 nm after 30-min incubation.
The absorbance was compared with the standard graph
of NaNO2, obtained from the reduction of NaNO3
(1–100 lmol/L). The accuracy of the assay was checked
in two ways; the interassay and intraassay coefficients of
variation were 7.52% and 4.61%, respectively. To check
conversion of nitrate to nitrite (recovery rate), known
amounts of nitrate were added to control plasma sam-
ples; these samples were deproteinized and reduced as
above.
Histopathological Examinations
Histopathological evaluation of the kidney tissues was
done. Paraffin-embedded specimens were cut into
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6-μm thickness and stained with Hematoxylin–Eosin
stain for light microscopic examination using a conven-
tional protocol27 (Olympus BH-2, Olympus, Tokyo,
Japan). A semiquantitative evaluation of renal tissues
was accomplished by scoring the degree of severity
according to previously published criteria.28 All sections
of the kidney samples were examined for parietal cell
hyperplasia, tubular vacuolization, and tubular necrosis.
Briefly, minimum of 50 proximal tubules associated with
For personal use only.
50 glomeruli were examined for each slide, and an aver-
age score was obtained. Severity of lesion was graded
from 0 to 3 according to the percentage of tubular invol-
vement. Slides were examined and assigned for severity
of changes using scores on scale of none (0), mild (1),
moderate (2), and severe (3) damages, in which (0)
denotes no change; grade (1) changes affecting <25%
tubular damage (mild); grade (2) changes affecting 25%–
50% of tubules (moderate); grade (3) changes affecting
>50% of tubules (severe).
To evaluate kidney fibrosis, the specimens obtained
from kidney were embedded in paraffin, sectioned at
6-μm sections, and stained with Masson’s trichrome.
Specimens were scored after painting with, in brief, ()
no fibrosis, (þ) fibrosis in <25% of total kidney tissue
(mild), (þþ) fibrosis in 25%–50% of total kidney tissue
(moderate), (þþþ) fibrosis in >50% of total kidney tis-
sue (serious).29
Table 1. Effects of GEN alone and its combination with PE on plasma urea, creatinine, Naþ, Kþ, and 24-h urine volume levels in rats.
Parameters Control
Urea (mg/dL) 34  8.1
Creatinine (mg/dL) 0.44  0.1
Naþ (mmol/L) 138.5  1.4
Kþ (mmol/L) 3.9  0.2
24-h Urine volume (mL) 8.8  1.1
Notes: Values are expressed as mean  SD for eight rats in each group.
Groups: control, GEN-treated, GEN þ vehicle-treated, and GEN þ PE-treated.
a
Significantly different from the control group.
b
Significantly different from the GEN-treated group (p < 0.001).
Abbreviations: NO, nitric oxide; MDA, malondialdehyde; GSH, reduced glutathione; GEN, gentamicin.
Statistical Analyses
Results of all groups were shown as mean
values  standard deviation (SD). Statistical analyses of
the histopathological evaluation of the groups were carried
out by the chi-square test, and biochemical data were
analyzed by the one-way analysis of variance (ANOVA).
The significance between two groups was determined by
the Dunnett’s multiple comparison test, and p < 0.05 was
accepted as statistically significant value.
RESULTS
No deaths or remarkable signs of external toxicity were
observed in the groups of rats given GEN either alone or
combination with PE. The biochemical and histopatho-
logical results were similar for the control and PE groups,
and we decided to consider them without distinction and
report only the control group.
Urinary Volume
The 24-h urine volume in the GEN-treated group was
significantly higher than in the group control (p < 0.01),
indicating the presence of GEN-induced polyuria,
whereas in the group treated with GEN þ PE, it was
not different from that of the group control, pointing
out the protective role of PE against acute tubular necro-
sis (Table 1).
Biochemical Variables in Plasma and Tissue
Naþ and Kþ concentrations among four groups were
similar. Serum urea and creatinine levels were signifi-
cantly higher in rats treated with GEN alone than rats
in the control and GEN þ PE-treated groups
(p < 0.01). Administration of PE to the GEN-treated
rats caused decrease in serum urea and creatinine
levels (Table 1).
The GSH level in renal tissue of only GEN-treated rats
was significantly lower than those in the control group
(p < 0.05), and administration of PE to GEN-treated rats
significantly increased the level of GSH (p < 0.05; Table 2).
The group that was given GEN and PE had signifi-
cantly lower MDA levels in kidney cortex tissue than
those given GEN alone. There was no significant differ-
ence for any levels between the groups (Table 2).
GEN GEN þ vehicle GEN þ PE
109  14.9 a
105  14.4 45  10.8b
2.1  0.5a 1.99  0.5 0.8  0.3b
140.3  2.7 139.1  2.4 138.8  1.6
4.2  0.5 4.1  0.4 4.1  0.3
21.8  4.5a 20.3  3.4 9.6  1.7b
Renal Failure
 Pomegranate Extract Attenuates Gentamicin Nephrotoxicity in Rats 271

Table 2. Effects of PE on NO, MDA, and GSH levels of rat kidney.

Parameters Control GEN GEN þ vehicle GEN þ PE

NO (nmol/g wet tissue) 30.3  9.3 47.2  7.3 a

44.5  5.8 43.7  11.9
MDA (nmol/g wet tissue) 2.6  0.7 4.1  1.1a 3.9  1.1 2.2  0.9b
GSH (umol/g wet tissue) 2.3  0.8 1.4  0.7a 1.3  0.6 2.4  0.4b
Notes: Values are expressed as mean  SD for eight rats in each group.
a
Significantly different from the control group.
b
Significantly different from the GEN-treated group (p < 0.05).
Abbreviations: NO, nitric oxide; MDA, malondialdehyde; GSH, reduced glutathione; GEN, gentamicin.

Results of Histopathological Examinations
The histopathological examination of kidney showed no
pathologic findings in the control group (Figure 1A). In
rats treated with GEN and GEN þ vehicle, there were
mild and severe tubular necrosis, tubular degeneration,
and epithelial vacuolization in the proximal tubules, and
parietal cell hyperplasia compared with the control group
(Figure 1B and C). In rats treated with GEN þ PE,
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mild and moderate fibrosis in kidney specimen (Table 4;
Figure 2A and D).
DISCUSSION
The kidneys are easily susceptible to damage from drugs
because of larger perfusion and accumulation of excreted
compounds that occur in renal tubular cells during

despite the presence of mild tubular degeneration and
epithelial vacuolization in the proximal tubules, parietal
cell hyperplasia and tubular necrosis are less severe, and
glomeruli maintained a better morphology when com-
pared with the GEN group (Figure 1D). These changes
are summarized in Table 3.
After staining with Masson’s trichrome, no statistical
difference was found between the groups in kidney fibro-
For personal use only.
absorption and secretion. Aminoglycoside is an antibio-
tic whose clinical use is limited by its nephrotoxicity.
GEN is a widely used aminoglycoside antibiotic and
has been shown to cause marked histological damage in
particular to renal proximal convoluted tubules,30,31
resulting in swelling, vacuolization, and necrosis of
epithelial cells and accumulation of myelin-like bodies.32
Furthermore, the results of many studies have shown

sis scores. However, in GEN-treated rats, there was more that altered concentrations of various biochemical

(A) (B)

(C) (D)

Figure 1. (A) Normal tubules and glomeruli in kidney cortex, staining with H&E 100 (control group). (B) Severe tubular necrosis, tubular
degeneration, and epithelial vacuolization in the proximal tubules, staining with H&E 100 (GEN-treated group). (C) Moderate tubular
necrosis, tubular degeneration, and epithelial vacuolization in the proximal tubules, staining with H&E 100 (GEN þ vehicle-treated group).
(D) Mild epithelial vacuolization in the proximal tubules and normal glomeruli, staining with H&E 100 (GEN þ PE-treated group).

© 2013 Informa Healthcare USA, Inc.

 272 M. Cekmen et al.
Table 3. Semiquantitative analysis of tubular necrosis, tubular vacuolization, parietal cell hyperplasia in the control, GEN-, GEN þ vehicle-,
and GEN þ PE-treated rats.
Tubular necrosis
Hyperplasia n 0 1 2
Control 8 8 0 0
GENa 8 0 2 5
GEN þ vehicle 8 1 2 4
GEN þ PEb 8 4 3 1
Notes: Score 0, no degeneration; 1, mild degeneration; 2, moderate degeneration; 3, severe degeneration.
a
Statistical significant difference from the control group.
b
Statistical significant difference from the GEN-treated group (p < 0.05).
Table 4. Analysis of kidney fibrosis in the control, GEN-,
GEN þ vehicle-, and GEN þ PE-treated rats.
n () (þ) (þþ)
Control 8 0 0 0
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GEN 8 3 4 1
GEN þ vehicle 8 4 4 0
GEN þ PE 8 6 2 0
Notes: Score (), no fibrosis; (þ), mild fibrosis; (þþ), moderate
fibrosis; (þþþ), serious fibrosis.
No statistical difference between the groups (p > 0.05).
indicators of oxidative stress in kidney tissue are due to
GEN.33 Because of the obvious responsibility of ROS in
For personal use only.
GEN-induced renal damage, several antioxidant agents
have been used to prevent GEN nephrotoxicity.34 PE is
rich in antioxidants of the polyphenolic class, which
includes tannins and anthocyanins. These antioxidants
are more potent, on a molar basis, than many other anti-
oxidants, including vitamins C and E, coenzyme Q-10,
and lipoic acid. The antioxidant levels in PE were found to
be higher than that in other natural juices, such as blue-
berry, cranberry, and orange, as well as in red wine.15
Pomegranate has become more popular because of the
attribution of important physiological properties, such as
anticancer, cholesterol-lowering, and cardioprotective.
This study demonstrated ameliorative effects of PE, a
phenolic antioxidant, on GEN-induced nephrotoxicity,
in line with the consideration that oxygen-free radicals
are important mediators of GEN-induced acute renal
failure. The understanding of aminoglycoside nephro-
toxicity is clinically important; such nephrotoxicity is
typically associated with anoliguric acute renal failure,
that is, azotemia in the presence of 1–2 L/day urine out-
put. In this study, the 24-h urine volume in the GEN
group was significantly higher than in the control group,
indicating the presence of GEN-induced polyuria,
whereas in the GEN þ PE-treated group, it was not
different from that of the control group, suggesting the
protective role of PE against acute tubular necrosis. In
addition, increased serum urea and creatinine levels in
GEN-treated rats reflect the renal damage. In contrast to
the previous studies, serum Kþ levels were similar
between the GEN-induced nephrotoxicity and the con-
trol groups. The final studies show that Kþ levels
Tubular vacuolization Parietal cell
3 0 1 2 3 0 1 2 3
0 7 1 0 0 8 0 0 0
1 0 2 4 2 0 2 5 1
1 1 2 3 2 0 3 4 1
0 1 6 1 0 3 4 1 0
were not increased in rats with GEN-induced nephro-
toxicity.35–37 Administration of PE protects the kidney
function from GEN as indicated by preventing an
(þþþ)
increase in serum urea and creatinine levels. PE restores
0 the renal function by preserving the structural integrity of
0 renal cells against GEN challenge, evidenced by signifi-
0
cantly preventing an increase in the levels of serum crea-
0
tinine and urea.
Since brush border membrane (BBM) and other intra-
cellular organelles such as mitochondria and lysosomes
are known GEN target,38,39 the structural/functional
integrity was assessed by the status of their respective
biomarker enzymes. So, here, we measured the MDA,
GSH, and NO, as a means of oxidative stress. Our find-
ings corroborate those of the earlier studies demonstrat-
ing that an enhanced endogenous oxidative stress has a
major role in the severity of GEN-induced acute renal
failure.40,41 MDA, a stable lipid hydroperoxide, provides
an index of the LPO in biological tissues.42 In this study,
we found increased MDA levels in the GEN-treated
group, and as a protective effect of PE, lower MDA levels
were found in the group determined by GEN þ PE. The
GSH antioxidant system is considered as the most notable
cellular protective mechanism. GSH has a very important
role in protecting against oxygen-free radical damage by
providing reducing equivalents for several mechanism, as
well as scavenging hydroxyl radicals and singlet oxygen.
Its depletion is a common consequence of increased for-
mation of ROS5 like GEN-induced nephrotoxicity. In the
group treated with GEN þ PE, we found increased GSH
levels. However, our study has shown that PE does not
affect NO levels protectively in contrast to some previous
studies with different antioxidant agents.3,43 So, it may
enhance LPO by a different way. These findings strongly
indicate that PE is important in protecting the kidney from
GEN-induced injury through improvement in oxidant
status.
GEN treatment provokes acute tubular necrosis and
acute renal failure in about 30% of high-risk patients.6
Animal models of aminoglycoside nephrotoxicity also
present residual areas of interstitial fibrosis in the renal
cortex and progressive tubular injury.44,45 After staining
with Masson’s trichrome, no statistical difference was
found between the groups in kidney fibrosis scores.
However, in GEN-treated rats, there was more mild
Renal Failure
 Pomegranate Extract Attenuates Gentamicin Nephrotoxicity in Rats 273

(A) (B)
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For personal use only.

(C) (D)

Figure 2. (A) No fibrosis in the control group, staining with Masson’s trichrome 100. (B) Mild fibrosis in interstitium, staining with
Masson’s trichrome 400 (GEN-treated group). (C) Mild fibrosis in interstitium, staining with Masson’s trichrome 200 (GEN þ vehicle-
treated group). (D) No fibrosis in GEN þ PE-treated group, staining with Masson’s trichrome 100.

and moderate fibrosis in kidney specimen as previous
studies. In this study, the histopathological examination
of kidneys showed severe and extensive damage in GEN-
treated rats which have tubular necrosis and edema. This
could be due to the formation of highly reactive radicals as
a consequence of oxidative stress caused by GEN. The
kidneys of the control group showed normal histological
features, but the GEN-treated group revealed more exten-
sive and marked tubular necrosis. On the other hand, the
tubules from rats of the GEN þ PE-treated group were
nearly normal in histological appearance except for a
slight desquamation and atrophy of the tubular epithelial
cells. Similar changes were also reported by some studies,
which demonstrated structural changes in renal tissue of
GEN-treated animals and its reversal by various
agents.3,46,47
In conclusion, the data indicated that GEN-induced
nephrotoxicity might be related to oxidative damage.
The results obtained in this study suggested that PE has
an overall protective effect against GEN-induced nephro-
toxicity in rat model. The observed protective effects can
be attributed to the antioxidant properties of PE. So, PE is
a highly free-radical scavenger agent and offers protection
against GEN-induced acute renal failure.
Declaration of interest: The authors report no con-
flicts of interest. The authors alone are responsible for the
content and writing of this article.
REFERENCES
[1] Humes HD, Weinberg JM. Toxic nephropathies. In: Brenner
BM, Rector FC, eds. The Kidney. Philadelphia: WB Saunders
Co.; 1986:1491–1532.
[2] Edson RS, Terrell CL. The aminoglycosides. Mayo Clin Proc.
1999;74:519–528.
[3] Karadeniz A, Yildirim A, Simsek N, Kalkan Y, Celebi F.
Spirulina platensis protects against gentamicin-induced nephro-
toxicity in rats. Phytother Res. 2008;22:1506–1510.

© 2013 Informa Healthcare USA, Inc.

 274 M. Cekmen et al.
[4] De la Cruz Rodriguez LC, Araujo CR, Posleman SE, Rey MR.
Attenuation of gentamicin-induced nephrotoxicity: trimetazi-
dine versus N-acetyl cysteine. J Appl Toxicol. 2010;30:343–353.
[5] Abdel-Raheem IT, Abdel-Ghany AA, Mohamed GA . Protective
effect of quercetin against gentamicin-induced nephrotoxicity in
rats. Biol Pharm Bull. 2009;32:61–67.
[6] Cuzzocrea S, Mazzon E, Dugo L, et al. A role for superoxide in
gentamicin-mediated nephropathy in rats. Eur J Pharmacol.
2002;450:67–76.
[7] Grune T, Sommerburg O, Petras T, et al. Postanoxic formation
of aldehydic lipid peroxidation products in human renal tubular
cells. Free Radic Biol Med. 1995;18:21–27.
[8] Baliga R, Ueda N, Walker PD, et al. Oxidant mechanisms in
toxic acute renal failure. Drug Metab Rev. 1999;31:971–997.
[9] Winterbourn CC, Pichorner H, Kettle AJ. Myeloperoxidase-
dependent generation of a tyrosine peroxide by neutrophils.
Arch Biochem Biophys. 1999;338:15–21.
[10] Fryer MJ. Vitamin E may slow kidney failure owing to oxidative
stress. Redox Rep. 1997;3:259–261.
[11] Pedraza-Chaverrıí J, Maldonado PD, Medina-Campos ON,
et al. Garlic ameliorates gentamicin nephrotoxicity: relation to
Ren Fail Downloaded from informahealthcare.com by Nyu Medical Center on 11/03/14
antioxidant enzymes. Free Radic Biol Med. 2000;29:602–611.
[12] Tsuda T, Horio F, Cyanidin OT. 3-O-beta-d-glucoside sup-
presses nitric oxide production during a zymosan treatment in
rats. J Nutr Sci Vitaminol (Tokyo). 2002;48:305–310.
[13] Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys.
1959;82:70–77.
[14] de Nigris F, Williams-Ignarro S, Lerman LO, et al. Beneficial
effects of pomegranate juice on oxidation-sensitive genes and
endothelial nitric oxide synthase activity at sites of perturbed
shear stress. Proc Natl Acad Sci USA. 2005;102:4896–4901.
For personal use only.
[15] Aviram M, Dornfeld L, Kaplan M, et al. Pomegranate juice
flavonoids inhibit low-density lipoprotein oxidation and cardio-
vascular diseases: studies in atherosclerotic mice and in humans.
Drugs Exp Clin Res. 2002;28:49–62.
[16] Tsuda T, Horio F, Osawa T. Cyanidin 3-O-beta-d-glucoside
suppresses nitric oxide production during a zymosan treatment
in rats. J Nutr Sci Vitaminol (Tokyo). 2002;48:305–310.
[17] Kaplan M, Hayek T, Raz A, et al. Pomegranate juice supple-
mentation to atherosclerotic mice reduces macrophage lipid
peroxidation, cellular cholesterol accumulation and develop-
ment of atherosclerosis. J Nutr. 2001;131:2082–2089.
[18] Houghton DC, Plamp CE III, DeFehr JM, Bennett WM, Porter
G, Gilbert D. Gentamicin and tobramicin nephrotoxicity. A
morphologic and functional comparison in the rat. Am J
Pathol. 1978;93:137–152.
[19] Buege JA, Aust SD. Microsomal lipid peroxidation. Methods
Enzymol 1978;52:302–310.
[20] Chen WC, Chen HY, Wu HC, Wu MC, Hsu CD, Tsai FJ.
Vascular endothelial growth factor gene polymorphism is asso-
ciated with calcium oxalate stone disease. Urol Res. 2003;31:
218–222.
[21] Esen T, Akinci M, Aytekin Y, Altug T, Hekim N. The effects of
citrate, polyphosphates and pridoxin on intratubular crystalliza-
tion in hyperoxaluric rats. Turk J Urol. 1990;16:365–370.
[22] Selvam R. Calcium oxalate stone disease: role of lipid peroxida-
tion and antioxidants. Urol Res. 2002;30:35–47.
[23] Itoh Y, Yasui T, Okada A, Tozawa K, Hayashi Y, Kohri K.
Preventive effects of green tea on renal stone formation and the
role of oxidative stress in nephrolithiasis. J Urol. 2005;173
(1):271–275.
[24] Villegas I, Martín AR, Toma W, et al. Rosiglitazone, an agonist
of peroxisome proliferator-activated receptor c, protects against
gastric ischemia–reperfusion damage in rats: role of oxygen free
radicals generation. Eur J Pharmacol. 2004;505:195–203.
[25] Wasowicz W, Neve J, Peretz A. Optimized steps in fluorometric
determination thiobarbituric acid-reactive substances in serum:
importance of extraction pH and influence sample preservation
and storage. Clin Chem. 1993;39:2522–2528.
[26] Beutler E. Glutathione in Red Blood Cell Metabolism. A Manual of
Biochemical Methods. New York: Grune and Stratton;
1984:112–114.
[27] Sun Y, Oberley LW, Li Y. A simple method for clinical assay of
superoxide dismutase. Clin Chem. 1988;34:497–500.
[28] Allen CT. Laboratory methods in histochemistry. In: Prophet
EB, Mills B, Arrington JB, Sobin LH, eds. American Registry of
Pathology. 1st ed. Washington DC: Armed Forces Institute of
Pathology; 1992:53.
[29] Ayyıldız A, Nuhoğlu B, Gülerkaya B, et al. Effect of intraurethral
mitomycin C on healing and fibrosis in rats with experimentally
induced urethral stricture. Int J Urol. 2004;11:1122–1126.
[30] Humes HD, Connor RPO. Aminoglycoside nephrotoxicity. In:
Schrier RW, Gottschalk CW, eds. Diseases of the Kidney. Vol. 2,
no. 4. Boston, MA: Little Brown; 1988:1229–1273.
[31] Abdel-Gayoum AA, Ali BH, Abdel-Razig AA, Bashir AA,
Ghywarsha K. Effect of gentamicin-induced nephrotoxicity on
some carbohydrate metabolic pathways in the rat renal cortex.
Arch Toxicol. 1994;68:643–647.
[32] Ali BH, Bashir AA. Effect of fish oil treatment on gentamicin
nephrotoxicity in rats. Anal Nutr Metab. 1994;38:336–339.
[33] Humes HD. Aminoglycoside nephrotoxicity. Kidney Int.
1988;33:900–911.
[34] Simmons CF Jr, Bogusky RT, Humes HD. Inhibitory effects of
gentamicin on renal mitochondrial oxidative phosphorylation.
J Pharmacol Exp Ther. 1980;214:709–715.
[35] Silan C, Uzun O, Comunoglu NU, Gokcen S, Bedirhan S,
Cengiz M. Gentamicin-induced nephrotoxicity in rats amelio-
rated and healing effects of resveratrol. Biol Pharm Bull.
2007;30:79–83.
[36] Cronin RE, Thompson JR . Role of potassium in the pathogenesis
of acute renal failure. Miner Electrolyte Metab. 1991;17:100–105.
[37] Said MM. The protective effect of eugenol against gentamicin-
induced nephrotoxicity and oxidative damage in rat kidney.
Fundam Clin Pharmacol. 2011; 25:708–716.
[38] Farooq N, Priyamvada S, Khan F, Yusufi ANK. Time depen-
dent effect of gentamicin on enzymes of carbohydrate metabo-
lism and terminal digestion in rat intestine. Hum Exp Toxicol.
2007;26:587–593.
[39] Banday AA, Farooq N, Priyamvada S, Yusufi ANK, Khan F.
Time dependent effects of gentamicin on the enzymes of carbo-
hydrate metabolism, brush border membrane and oxidative
stress in rat kidney tissues. Life Sci. 2008;82(9):450–459.
[40] Martinez-Salgado C, Lopez-Hernandez FJ, Lopez-Novoa JM.
Glomerular nephrotoxicity of aminoglycosides. Toxicol Appl
Pharmacol. 2007;223:86–98.
[41] Walker PD, Shah SV. Gentamicin enhanced production of
hydrogen peroxide by renal cortical mitochondria. Am J
Physiol. 1987;253:C495–C499.
[42] Kuhad A, Tirkey N, Pilkhwal S, et al. Effect of Spirulina, a blue
green algae, on gentamicin-induced oxidative stress and renal
dysfunction in rats. Fundam Clin Pharmacol. 2006;202:121–128.
[43] Ozbek E, Cekmen M, Ilbey YÖ, et al. Atorvastatin prevents
gentamicin-induced renal damage in rats through the inhibition
of p38-MAPK and NF-kB pathways. Renal Fail. 2009;31:
382–392.
[44] Houghton DC, English J, Bennett WM. Chronic tubulointerstitial
nephritis and renal insufficiency associated with longterm “sub-
therapeutic” gentamicin. J Lab Clin Med. 1988;112: 694–703.
[45] Fuhr J, Kaczmarczyk J, Kruttgen CD. A simple colorimetric
method of inulin determination in renal clearance studies on
metabolically normal subjects and diabetics. Klin Wochenschr.
1955;33:729–730.
[46] Kumar KV, Shifow AA, Naidu MU, et al. Carvedilol: a beta
blocker with antioxidant property protects against gentamicin-
induced nephrotoxicity in rats. Life Sci. 2000;66:2603–2611.
[47] Nakakuki M, Yamasaki F, Shinkawa T, et al. Protective effect of
human ulinastatin against gentamicin-induced acute renal
failure in rats. Can J Phys Pharmacol. 1996;74:104–111.
Renal Failure