Ameliorative effects of curcumin against renal injuries mediated by inducible nitric.pdf

Pulmonary, Gastrointestinal and Urogenital Pharmacology
Ameliorative effects of curcumin against renal injuries mediated by inducible nitric
oxide synthase and nuclear factor kappa B during gentamicin-induced toxicity in
Wistar rats
Ramar Manikandan a,
⁎, Manikandan Beulaja b
, Raman Thiagarajan c
, Asokan Priyadarsini b
,
Rajendran Saravanan a
, Munusamy Arumugam b
a
Department of Animal Health and Management, Alagappa University, Karaikudi- 630 003, India
b
Department of Zoology, University of Madras, Guindy Campus, Chennai- 600 025, India
c
Department of Biotechnology, School of Chemical and Biotechnology, SASTRA University, Thanjavur- 613401, India
a b s t r a c t
a r t i c l e i n f o
Article history:
Received 8 March 2011
Received in revised form 15 August 2011
Accepted 27 August 2011
Available online 10 September 2011
Keywords:
Gentamicin
Curcumin
Nitric oxide synthase
Nuclear factor kappa B
Oxidative stress
The ameliorative role of curcumin in attenuating gentamicin-induced nephrotoxicity has been reported ear-
lier however, the mechanism of action remains unclear. Gentamicin was injected intraperitoneally
(100 mg/kg body weight) once daily for 6 days. Curcumin was administered orally (200 mg/kg body weight)
once daily for 7, 15 and 30 days. Gentamicin-induced rats showed significant increase in the levels of kidney
markers and the activities of urinary marker enzymes, which was reversed upon curcumin treatment. A sig-
nificant increase in kidney lipid peroxidation (LPO) and decrease in activities of superoxide dismutase (SOD),
catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and reduced glutathione
(GSH) were observed in gentamicin-induced rats. Immunohistochemical, Western blot and RT-PCR studies
in gentamicin-induced rats also demonstrated an increase in the levels of inducible nitric oxide synthase
(iNOS) and nuclear factor-κB (NF-κB). All these effects induced by gentamicin were reduced upon treatment
with curcumin in a time dependent manner. To conclude, curcumin enhances antioxidants, and decreases
iNOS and NF-κB, thereby protecting the cells against oxidative stress induced by gentamicin.
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
Gentamicin, an aminoglycoside is commonly used as an antibiotic
for the treatment of serious infections caused by gram-negative bac-
teria, however, they are known to be nephrotoxic and ototoxic. In
some situations these side effects are so severe that the use of this
drug has to be discontinued. It has been estimated that up to 30% of
patients treated with aminoglycosides for more than 7 days show
some signs of nephrotoxicity (Mattew, 1992) and the specificity of
gentamicin-induced renal toxicity is related to its preferential accu-
mulation in the renal convoluted tubules and lysosomes (Nagai and
Takano, 2004).
The transcription factors belonging to nuclear factor-kappa B
(NF-κB)/Rel transcription family play a central role in inflammation
by its ability to activate proinflammatory genes (Baldwin, 1996).
NF-κB has been shown to be highly activated in many human inflam-
matory diseases and is well known to trigger proinflammatory cyto-
kines, chemokines, adhesion molecules, and inducible nitric oxide
synthase (iNOS) enzyme (Tak and Firestein, 2001). iNOS is the enzyme
responsible for inducible synthesis of nitric oxide, a free radical that
has been implicated to play vital roles in normal physiological processes
especially immunity. However, uncontrolled production of nitric
oxide has been demonstated to underlie pathophysiology of various
diseases including diabetes and nephrotoxicity (Kalayarasan et al.,
2009; Manikandan et al., 2011).
The mechanism of gentamicin-induced nephrotoxicity is not
completely known. However, studies have implicated reactive oxy-
gen species particularly superoxide anion radical in the pathophysiol-
ogy of gentamicin nephropathy (Cuzzocrea et al., 2002). It has been
demonstrated that gentamicin administration increases renal cortical
lipid peroxidation, nitric oxide generation and mitochondria hydro-
gen peroxide production (Karahan et al., 2005; Parlakpinar et al.,
2005; Yanagida et al., 2004; Yang et al., 1995). Abnormal production
of such molecules may damage macromolecules, induce cellular injury
and necrosis via several mechanisms including peroxidation, protein de-
naturation and DNA damage (Baliga et al., 1998; Parlakpinar et al.,
2005). The alteration in kidney functions induced by lipid peroxidation
is a proximal event in the injury cascade of gentamicin mediated neph-
rotoxicity (Karahan et al., 2005). Gentamicin also acts as an iron chelator
and iron-gentamicin complex is a potent catalyst of radical generation
European Journal of Pharmacology 670 (2011) 578–585
⁎ Corresponding author at: Department of Animal Health and Management, Alagappa
University, Karaikudi- 630 003, India. Tel.: +91 04565 225682.
E-mail address: manikandanramar@yahoo.co.in (R. Manikandan).
0014-2999/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejphar.2011.08.037
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmacology
journal homepage: www.elsevier.com/locate/ejphar

(Yanagida et al., 2004). Accordingly, the administration of compounds
with antioxidant activity has been successfully used to prevent or ame-
liorate gentamicin-induced nephrotoxicity (Cuzzocrea et al., 2002;
Karahan et al., 2005). However, none of these strategies were found
to be suitable/safe for clinical practice.
In the past few years, much interest has been laid on the role of
naturally occurring dietary substances for the control and manage-
ment of various chronic diseases, one such compound curcumin
has been used since ancient times for promoting human health. Cur-
cumin is a major yellow pigment in rhizomes of Curcuma longa Linn,
which is used widely as a spice and coloring agent in several foods as
well as cosmetics and drugs (Joe et al., 2004; Okada et al., 2001). Cur-
cumin has been reported to possess anti-inflammatory and antioxi-
dant properties with a potent ability to inhibit reactive oxygen
species formation (Biswas et al., 2005; Venkatesan et al., 2000). Cur-
cumin exhibited antioxidant activity in a renal cell line (Cohly et al.,
1998) and ameliorated ferric nitrilotriacetic acid (Fe-NTA) induced
renal oxidative stress in mice (Okada et al., 2001). Administration of cur-
cumin has also been reported to prevent renal lesions in streptozotocin-
induced diabetic rats (Suresh and Srinivasan, 1998).
A number of chemical compounds such as melatonin, a pineal hor-
mone (Sener et al., 2002), caffeic acid phenethyl ester (Parlakpinar
et al., 2005), chelerythrine, a protein kinase-C inhibitor (Parlakpinar
et al., 2005), garlic (Pedraza-Chaverri et al., 2000) and M40403, a low
molecular weight synthetic manganese containing superoxide dismu-
tase mimetic (Cuzzocrea et al., 2002) have been used to prevent
gentamicin-induced nephrotoxicity. However, the literature reporting
the mechanism involved in the ameliorative effects of plant-derived
substances on gentamicin-induced nephrotoxicity is scanty.
Therefore, in continuation of the search for a potential agent to
modulate the gentamicin mediated renal oxidative stress and dam-
ages, we have examined our hypothesis that curcumin is a potent an-
tioxidant and anti-inflammatory agent against gentamicin-induced
nephrotoxicity, in Wistar rats.
2. Materials and methods
2.1. Chemicals
Curcumin was purchased from Sigma Chemicals (St. Louis, MO,
USA). Gentamicin was procured from Ranbaxy laboratories, Mumbai,
India. Polyclonal anti-iNOS antibody and Rabbit polyclonal NF-κB
were obtained from BD Biosciences (San Jose, CA, USA). iNOS primer
and secondary antibody peroxide conjugated anti-rabbit IgG were
purchased from Bangalore Genei (Bangalore, India). All other chemi-
cals and reagents used were of the highest analytical grade commer-
cially available.
2.2. Animals
Male albino Wistar rats weighing between 150 g to 200 g were
procured from the National Institute of Nutrition (Hyderabad,
India). All experiments were approved by the Institutional Animal
Ethical Committee (IAEC), India, guidelines (IAEC 360/01/a/CPCSEA).
Rats were housed in an air-conditioned room at 22±10 °C with a
lighting schedule of 12 h light and 12 h dark. Rats were fed a balanced
commercial rat diet (Hindustan UniLever, Mumbai, India) and water
ad libitum.
2.3. Experimental design
The animals were randomly divided into five groups containing six
rats in each group. Gentamicin (Ranbaxy laboratories, Mumbai, India)
was injected intraperitoneally to animals at a dose of 100 mg/kg body
weight, for six consecutive days, which is well known to cause significant
nephrotoxicity in rats (Cuzzocrea et al., 2002). Curcumin (Sigma, St.
Louis, MO, USA) was administered orally to animals at a dose of
200 mg/kg body weight (Chuang et al., 2000).
1. Group I: animals administered with physiological saline alone
(control)
2. Group II: animals administered with gentamicin alone
3. Group III: animals administered with gentamicin and treated with
curcumin for 7 days
4. Group IV: animals administered with gentamicin and treated with
curcumin for 15 days
5. Group V: animals administered with gentamicin and treated with
curcumin for 30 days
After the last dose, all control and experimental animals were
immediately kept in individual metabolic cages to collect serum
for the estimation of renal function. The animals were sacrificed by
decapitation and the blood samples were drawn by cardiac puncture
and centrifuged to harvest the serum with which the renal function
assessment were analyzed. Kidney tissues were excised immediate-
ly, rinsed in ice-cold physiological saline, homogenized in 0.1 M
Tris–HCl buffer (pH 7.4) and the resultant tissue homogenate was
used for biochemical assays. Sections of the kidney were set aside
for histological, immunohistochemical, Western blot and RT-PCR
studies.
2.4. Renal function assessment
The levels of urea, uric acid, creatinine, blood urea nitrogen (BUN)
and glucose were assessed in the serum of control and experimental
animals using the methods of (Banday et al., 2008; Caraway, 1963;
Natelson et al., 1951; Owen et al., 1954; Sasaki and Matsui, 1972),
respectively.
2.5. Determination of lipid peroxidation
Lipid peroxidation was determined by the method of (Ohkawa et al.,
1979). The principle of this method being that malondialdehyde (MDA),
an end product of lipid peroxidation, reacts with thiobarbituric acid
(TBA) to form a pink chromogen. For this assay, 0.2 ml of 8.1% SDS,
1.5 ml of 20% acetic acid (pH 3.5) and 1.5 ml of 0.8% thiobarbituric acid
aqueous solution were added in succession in a reaction tube. To this re-
action mixture, 0.2 ml of the kidney homogenate was added, and the
mixture was then heated in boiling water for 60 min. After cooling to
room temperature, 5 ml of butanol: pyridine (15:1, v/v) solution was
added. The mixture was then centrifuged at 2236 x g for 15 min follow-
ing which the upper layer was separated, and the intensity of the result-
ing pink color was read at 532 nm. Tetramethoxypropane was used as an
external standard and the level of lipid peroxides was expressed as nmol
of MDA formed/g wet weight.
2.6. Antioxidant enzymes
Prior to biochemical analysis, kidneys of each group were homog-
enized in 10% 0.1 M Tris–HCl buffer (pH 7.2) and centrifuged at
12,879 x g for 30 min at 4 °C. The supernatant obtained was used for
the analysis of enzymatic as well as non-enzymatic antioxidants and
the amount of protein in each sample was estimated by (Lowry et
al., 1951).
2.6.1. Superoxide dismutase (SOD)
SOD activity was determined by the method of (Misra and
Fridovich, 1972). In this test, the degree of inhibition of pyrogallol
auto-oxidation by kidney homogenate supernatant was measured.
The change in absorbance was read at 470 nm against blank every
3 min on a spectrophotometer and the enzyme activity was
expressed as 50% inhibition of adrenaline auto oxidation/min.
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R. Manikandan et al. / European Journal of Pharmacology 670 (2011) 578–585

2.6.2. Catalase (CAT)
Catalase activity was determined by the method of Beers and Sizer
(1952). In this test, dichromatic acetic acid is reduced to chromic ac-
etate when heated in the presence of H2O2, with the formation of per-
chloric acid as an unstable intermediate. In the test, the green color
development was read at 590 nm against blank in a spectrophotome-
ter. The activity of catalase was expressed as μmole of H2O2 con-
sumed/mg protein/min.
2.6.3. Glutathione peroxidase (GPx)
The GPx activity was determined essentially as described by
(Rotruck et al., 1973). The rate of glutathione oxidation by H2O2, as
catalyzed by the GPx present in the supernatant is determined and
the color developed was read against a reagent blank at 412 nm in a
spectrophotometer. In the test, the enzyme activity was expressed
as μmole of glutathione oxidized/mg protein/min.
2.6.4. Glutathione-S-transferase (GST)
The GST activity was determined by the method of (Habig et al.,
1974). The conjugation of GSH with 1-chloro-2,4-dinitrobenzene
(CDNB), a hydrophilic substrate was observed spectrophotometrically
at 340 nm to measure the GST activity and the result was expressed in
conjugate/μmol of CDNB with GSH/min.
2.6.5. Reduced glutathione (GSH)
The GSH content was estimated by the method of (Moron et al.,
1979). The kidney homogenate was centrifuged at 2236 x g for 15 min
at 4 °C. To the resulting supernatant, 0.5 ml of 10% trichloroacetic acid
was added and centrifuged. The resulting protein-free supernatant
was allowed to react with 4 ml of 0.3 M Na2HPO4 (pH 8.0) and 0.5 ml
of 0.04% (w/v) 5, 5-dithiobis-2-nitrobenzoic acid. The absorbance of
the resulting yellow color was read spectrophotometrically at 412 nm
and the results were expressed as μmol of NADPH oxidized/min/mg.
2.7. Histological examinations
The kidney tissues of rats were fixed in buffered 10% formalin so-
lution for 24 h and embedded in a paraffin wax. Tissues were then
sectioned at 5-μm, stained with hematoxylin eosin (H & E). A semi-
quantitative evaluation of renal tissues was accomplished by scoring
the degree of severity according to the formerly published criteria
(Teixeira et al., 1982). For each renal section, whole slide was exam-
ined for parietal cell hyperplasia, tubular vacuolization and tubular
necrosis were observed under bright field using a Carl Zeiss Axioscop
microscope.
2.8. Immunohistochemistry
Immunohistochemistry was carried out by the method of
(Manikandan et al., 2009) on 5-μm paraffin-embedded tissue sections
on poly-L-lysine-coated glass slides. The tissue sections were deparaffi-
nized by placing the slides in an oven at 60 °C for 10 min and then
rinsed twice in xylene for 10 min each. The slides were then hydrated
in a graded ethanol series (100, 90, 70, 50, 30% for 10 min each) and
then finally in double-distilled water for 10 min. The sections were in-
cubated with 1% H2O2 in double-distilled water for 15 min at 22 °C, to
quench endogenous peroxidase activity. The sections were rinsed
with Tris–HCl containing 150 mM NaCl (pH 7.4) and blocked in block-
ing buffer: tris-buffered saline (TBS), 0.05% Tween, 5% non-fat dry
milk (NFDM) for 1 h at 22 °C. After washing with TBS containing
0.05% Tween 20, the sections were incubated with primary antibody,
anti-iNOS polyclonal rabbit antibody and rabbit polyclonal IgG to rat
NF-κB (BD Biosciences, San Jose, CA, USA) at a dilution of 1:500, over-
night at 4 °C. After incubation, the tissue sections were rinsed with
TBS containing 0.05% Tween 20 twice and incubated with secondary an-
tibody, goat anti-rabbit IgG-HRP conjugate (Bangalore Genei, Bangalore,
India), at a dilution of 1:3000, for 1 h at 4 °C. After another wash with
TBS containing 0.05% Tween 20, the immunoreactivity was developed
with 0.05% diaminobenzidine (DAB) and 0.01% H2O2 for 1–3 min and
the tissue sections were observed for brown color formation under
bright field using a Carl Zeiss Axioscop microscope.
2.9. Western blot analysis of iNOS and NF-κB
Kidney was homogenized in 135 mM NaCl, 20 mM tris, 2 mM
EDTA and 1 mM phenyl methyl sulfonyl fluoride (PMSF) (Sigma, St.
Louis, MO, USA) and the volume of buffer was 1 ml per 100 mg kidney
tissue. The homogenates were centrifuged (15 min, 8944 x g at 4 °C)
and the protein content of the supernatant was determined by Low-
ry's method with BSA as standard. Aliquots of supernatant (30 μg
total protein) were boiled for 5 min in sample buffer (0.2 M Tris–HCl
buffer, 10% glycerol, 2% SDS, 0.02% β-mercaptoethanol). Proteins
were separated by Tris–Glycine–SDS discontinuous 12% polyacryl-
amide gel electrophoresis, and electro blotted onto nitrocellulose
membrane (Amersham Biosciences, USA).Western transfer of the
proteins was performed at a constant current of 100 V for 90 min at
4 °C. The membrane was blocked with blocking buffer (TBS, 0.05%
Tween, 5% NFDM) for 1 h at 22 °C with constant shaking. The mem-
brane was once again rinsed four times for 5 min each with TBS con-
taining 0.05% Tween 20 and incubated with the primary antibody anti
iNOS polyclonal rabbit antibody and rabbit polyclonal IgG to rat
NF-κB (BD Biosciences, San Jose, CA, USA) at a dilution of 1:500, over-
night at 4 °C. After incubation, the membrane was rinsed four times
with TBS containing 0.05% Tween 20 for 5 min each and incubated
with the secondary antibody, goat anti-rabbit IgG-HRP conjugate
(Bangalore Genei, Bangalore, India) at a dilution of 1:3000, for 2 h at
4 °C. The membrane was developed with 0.05% diaminobenzidine
(Sigma, St. Louis, MO, USA) and 0.01% H2O2 for 3–5 min.
2.10. Reverse transcription-polymerase chain reaction (RTPCR)
Total RNA was extracted using trizol reagent (Sigma, St. Louis, MO,
USA). Oligo-dT primed first strand cDNA was prepared from kidney
RNA using AMV reverse transcriptase at 37 °C for 60 min. PCR was
performed with gene-specific primers using Taq DNA polymerase
(Bangalore Genei, Bangalore, India). The primers used for iNOS were
5′- GCCTCCCTCTGGAAAGA-3′ (Sense) and 5′-TCCATGCAGACAACCTT-
3′ (Antisense). The following cycling conditions were used: 120 s of ini-
tial denaturation at 94 °C followed by 30 cycles of 90 s at 94 °C, 60 s at
60 °C, 60 s at 72 °C, followed by 5 min at 72 °C. β-actin primer 5′-GTG
GCCGCTCTAGGCACCA-3′ and 5′- CGGTTGGCCTTAGGGTTCAGGGGGG-3′
were used as an internal control.
2.11. Statistical analysis
Quantitative data were reported as mean±S.D. and the statistical
significance of observed differences between the values in the different
groups were determined by Student's t-test where Pb0.05 and Pb0.001
was regarded as statistically significant.
3. Results
3.1. Renal function assessment
Urea, uric acid, creatinine, blood urea nitrogen (BUN) and glucose
levels were observed in serum. After gentamicin exposure group II
animals exhibited a significant increase in the levels of urea, uric
acid, creatinine, blood urea nitrogen (BUN) and glucose when com-
pared to control (group I) animals (Table 1). Interestingly, in animals
treated with curcumin for 7 days (group III) and 15 days (group IV),
there was a significant (Pb0.05) decrease in the levels of urea, uric
acid, creatinine, blood urea nitrogen and glucose when compared to
580 R. Manikandan et al. / European Journal of Pharmacology 670 (2011) 578–585

group II animals. Such a decrease was also seen in group V, where an-
imals were treated with curcumin for 30 days and showed a signifi-
cant (Pb0.001) decrease in the levels of urea, uric acid, creatinine,
blood urea nitrogen and glucose.
3.2. Levels of lipid peroxidation
Upon gentamicin administration (group II), LPO levels significantly
increased in the kidney of the animals. However, in group III & group
IV animals, treated with curcumin for 7 days and 15 days, respectively,
there was a significant (Pb0.001) decrease when compared to group II
animals. Such a decrease was observed in group V animals too
(Pb0.05; Fig. 1).
3.3. Antioxidant in rat kidney
3.3.1. Superoxide dismutase
SOD activity was measured in the kidney as it is a specific scavenger
of superoxide anion and gentamicin was earlier shown to induce O2
−
generation in kidney. The mean SOD activity in kidney of group II were
significantly reduced (Pb0.05). By contrast, in kidney belonging to
groups III (7 days) and IV (15 days), there was a significant (Pb0.001)
increase in the level of SOD activity when compared to group II animals.
However, in group V, animals treated with curcumin for 30 days, there
was a significant (Pb0.05) increase and the levels were equal to that ob-
served with control kidney (group I; Table 2).
3.3.2. Catalase
Catalase is a specific scavenger of hydrogen peroxide and catalase
activity was significantly reduced in gentamicin treated kidney
(group II; Pb0.05) when compared to control (group I). Interestingly,
levels of catalase in group III (7 days) and IV (15 days) animals were
significantly increased (Pb0.001) when compared to group II
animals. By contrast, animals treated with curcumin for 30 days
showed a significant (Pb0. 05) increase and the levels were recov-
ered to that of the control animals (Table 2).
3.3.3. Glutathione peroxidase
GPx is important for scavenging hydrogen peroxide and along with
catalase is a potent barrier against lipid peroxidation in the kidney. The
GPx level in kidney of group II were significantly decreased (Pb0.05),
whereas, GPx levels in group III (7 days) and IV (15 days) animals
were significantly increased (Pb0.001) when compared to group II ani-
mals. However, in group V, animals treated with curcumin for 30 days,
there was a significant increase (Pb0.05) which was equal to that ob-
served with control kidney of group I (Table 2).
3.3.4. Glutathione-S-transferase
GST, an important component involved in the recycling of glutathione
levels were found to be reduced in the kidney of group II (gentamicin
alone). Interestingly, GST levels in group III (7 days), IV (15 days) and V
(30 days) animals were significantly increased (Pb0.001; Pb0.05)
when compared to group II animals which was equal to that observed
with control kidney of group I (Table 2).
3.3.5. Reduced glutathione
Reduced glutathione is an important defense against free radical
mediated damage. The GSH activity in kidney of group II was signifi-
cantly (Pb0.05) lower than kidney of group I (untreated) and curcu-
min treatment led to (group III, IV and V) a significant (Pb0.001 and
Pb0.05) increase in the level of GSH as compared to that of group II
animals (Table 2).
3.4. Histological analysis
The kidney of control rats showed normal architecture of glomerulus
and tubules (Fig. 2A). Kidney of gentamicin-induced rats showed dam-
aged glomerular structure, tubular necrosis, tubular epithelial alteration,
apoptotic cells, and cellular proliferation with fibrosis, thickening of cap-
illary walls and atrophy of glomerular tuft in group II (Fig. 2B) animals.
These alterations were minimal in group III and IV (Fig. 2C and D). Ani-
mals treated with curcumin for longer period (group V: Fig. 2E), appar-
ently showed a normal architecture of glomeruli and tubules, similar to
the control group.
3.5. Immunohistochemical iNOS and NF-κB expression
The expression of iNOS and NF-κB in control and experimental
group of rats is shown in Figs. 3 and 4. Immunohistochemical analysis,
using a specific anti-iNOS and NF-κB antibody, showed positive stain-
ing in glomerular and tubular region in kidney of gentamicin-induced
rats (Figs. 3B and 4B). When animals were treated with curcumin for
different days like 7 days (group III; Figs. 3C and 4C), 15 days (group
IV; Figs. 3D and 4D) and 30 days (group V; Figs. 3E and 4E) the levels
of iNOS and NF-κB gradually decreased and in group V animals, the
Table 1
Effect of gentamicin and curcumin on kidney markers in control and experimental animals.
Experimental groups Urea (mg/dl) Uric acid (mg/dl) Creatinine (mg/dl) Blood urea nitrogen (mg/dl) Glucose (mg/dl)
Group I 35.86±2.57 3.42±0.37 1.32±0.12 18.12±1.28 52.12±4.23
Group II 71.91±4.84b
7.24±0.53b
2.84±0.15b
27.86±2.32b
62.86±4.98b
Group III 51.29±3.68a
5.13±0.24a
1.97±0.11a
23.75±2.01a
60.73±3.86a
Group IV 49.54±2.93a
4.62±0.27a
1.59±0.09a
20.87±1.85a
57.63±2.13a
Group V 40.97±2.48b
4.14±0.36b
1.41±0.09b
19.43±1.78b
54.33±2.33b
Each value represents the mean±S.D. of observation made on samples from four determinations from the same group. Statistical analysis was performed by the Student's t-test.
Alphabets in superscript indicate that the difference observed between the group II and group I or between group II, group III, group IV or group V are statistically significant at
b
Pb0.001 and a
Pb0.05.
Fig. 1. Quantitative analysis of malondialdehyde in the kidney of Wistar rats. Each value
represents the mean±S.D. of observation made on samples from four determinations
from the same group. Statistical analysis was performed by the Student's t-test. Asterisks
indicate that the difference observed between the group II and group I or between group
II, group III, group IV or group V are statistically significant at **Pb0.001 and *Pb0.05.
581

iNOS and NF-κB expression was found to be similar to that of control
(group I; Figs. 3A and 4A).
3.6. Expression of iNOS and NF-κB by Western blot
Kidney homogenate supernatant harvested from rats and main-
tained in Tris–HCl buffer were subjected to SDS-PAGE (12%) under
non-reducing conditions and processed for Western blot analysis,
using anti-iNOS and NF-κB antibody (Fig. 5). In the present study,
the animals injected with gentamicin alone (group II; Lane II)
revealed a higher level of iNOS and NF-κB when compared to control.
In animals treated with curcumin for 7 days (group III; Lane III) and
15 days (group IV; Lane IV), the expression of iNOS and NF-κB
showed a gradual decrease. However, in group V, animals treated
with curcumin for 30 days, iNOS and NF-κB expressions were found
to be similar to that of the control animals (group V; Lane V).
3.7. RT-PCR analysis of iNOS in the tissues (kidney)
The relative percentage of expression of candidate genes in gentami-
cin induced nephrotoxicity was analyzed by the expression of β-actin in
control animals as 100% (Fig. 6). iNOS expression in the kidney was
found to be elevated in group II animals (Lane II) compared to control
(group I; Lane I). In groups III (Lane III) and IV (Lane IV) animals the
iNOS expression was found to be gradually decreased. However, iNOS
expression in group V animals (Lane V) was found to be similar to
that of the control (Lane I).
4. Discussion
Aminoglycoside antibiotics are commonly used for the treatment
of severe gram negative bacterial infections (Parlakpinar et al.,
2003), but the most widely used drug in this category is gentamicin
(Reiter et al., 2002). Although this drug has proven its usefulness,
its nephrotoxicity effect limits its use widely. The exact mechanism
Fig. 2. Hematoxylin and eosin-stained kidney sections of Wistar rats. A: Physiological
saline-injected rat (group I). B: Animals administered with gentamicin alone (group
II). C: Animals administered with gentamicin and treated with curcumin for 7 days
(group III). D: Animals administered with gentamicin and treated with curcumin for
15 days (group IV). E: Animals administered with gentamicin and treated with curcu-
min for 30 days (group V).
Fig. 3. Immunohistochemistry of iNOS in kidney of Wistar rats. Bright ﬁeld photomi-
crographs show kidney sections from: (A) Physiological saline-injected rat (group I);
(B) Animals administered with gentamicin alone (group II); (C) Animals adminis-
tered with gentamicin and treated with curcumin for 7 days (group III); (D) Animals
administered with gentamicin and treated with curcumin for 15 days (group IV); (E)
Animals administered with gentamicin and treated with curcumin for 30 days (group
V). Kidney sections were preincubated with anti-iNOS polyclonal rabbit antibody
(1:500 dilution) and subsequently with goat anti-rabbit IgG-HRP conjugate (1:3000
dilution). The immunoreactivity was developed with 0.01% DAB and 2% H2O2.
Table 2
Effect of curcumin on the levels of glutathione and antioxidant enzymes of rats induced with gentamicin.
Enzymes analyzed (unit of activity) Group I Group II Group III Group IV Group V
Reduced glutathione (μmol NADPH oxidized/min/mg) 152.96±12.23 112.31±10.84b
139.29±11.32a
148.38±12.10a
147.36±13.23b
Superoxide dismutase (50% inhibition of adrenaline auto oxidation/min) 8.12±0.11 4.49±0.45b
5.73±0.28a
6.63±0.43a
8.10±0.12b
Catalase (μM H2O2 consumed/mg protein/min) 71.50±5.62 47.86±4.32b
58.03±4.13a
63.14±3.35a
68.49±3.43b
Glutathione peroxidase (μg GSH utilized/mg protein/min) 9.1±0.11 4.9±0.12b
6.12±0.27a
7.56±0.41a
8.5±0.43b
Glutathione-S-transferase (μmol of CDNB conjugated with GSH/min) 823.07±63.42 672.12±58.21b
687.17±56.34a
743.54±61.21a
792±61.82b
Each value represents the mean±S.D. of observation made on samples from four determinations from the same group. Statistical analysis was performed by the Student's t-test.
Alphabets in superscripts indicate that the difference observed between the group II and group I or between group II, group III, group IV or group V are statistically signiﬁcant at
b
Pb0.001 and a
Pb0.05.

by which gentamicin induces renal damage is unknown, however
several agents that scavenge or interfere with reactive oxygen species
production successfully ameliorate gentamicin- induced nephropathy
and renal failure (Ademuyiwa et al., 1990; Ali, 2002; Kacew and Bergeron,
1990; Sener et al., 2002). A potential therapeutic approach to protect or
reverse renal gentamicin damage would have a very important clinical
consequence in increasing the safety of the drug. Phenolic compounds
from dietary plants are known to be good scavengers of reactive oxygen
species. Thus in the present study, we assessed whether the nephrotoxic
effects caused by acute administration of gentamicin could be prevented
or ameliorated by treatment with curcumin, a herbal compound which
possesses a strong antioxidant property (Elisabeth and Rao, 1990). Sever-
al dosage schemes have been reported for gentamicin administration and
an intraperitoneal (i.p) dose of 100 mg/kg body weight, for 6 days, was
used which is an ideal dosage scheme reported to cause significant neph-
rotoxicity in rats (Cuzzocrea et al., 2002).
There are some experimental data suggesting that nephrotoxic
drugs can alter the levels of kidney markers, glutathione and other
antioxidant enzymes (Ozbek et al., 2000; Parlakpinar et al., 2003),
which are commonly used to monitor the development and extent
of renal tubular damage due to oxidative stress. The results of this
study shows that, gentamicin administration to wistar rats (group
II) produced a typical pattern of nephrotoxicity which was mani-
fested by marked increase in serum creatinine, blood urea nitrogen,
urea, uric acid and serum glucose levels. On the other hand, curcumin
administration showed a significant decrease in the levels of serum
creatinine, blood urea nitrogen, urea, uric acid and serum glucose. The
curative effect of curcumin on the kidney markers can be attributed to
its antioxidant property as it has been found that reactive oxygen spe-
cies may be involved in the impairment of glomerular filtration rate
(GRF) (Hughes et al., 1996). In the current study, gentamicin caused a
significant increase in the LPO levels, while GSH, GPx, SOD, catalase
and GST levels were reduced in the kidney tissue. Similar results were
also observed in one earlier study (Ozbek et al., 2000). Depletion of
renal GSH is one of the primary factors which permit lipid peroxidation,
suggested to be closely related to gentamicin-induced tissue damage.
Gentamicin nephropathy was associated with low activities of
Fig. 5. Effect of curcumin on iNOS and NF-κB protein expression in the kidney of Wistar
rats exposed to gentamicin. Lane I: Kidney protein from physiological saline-injected rat
(group I); Lane II: Kidney protein from animals administered with gentamicin alone
(group II); Lane III: Kidney protein from animals administered with gentamicin and trea-
ted with curcumin for 7 days (group III); Lane IV: Kidney protein from animals adminis-
tered with gentamicin and treated with curcumin for 15 days (group IV); Lane V:
Kidney protein from animals administered with gentamicin and treated with curcumin
for 30 days (group V).
Fig. 4. Immunohistochemistry of NF-κB in kidney of Wistar rats. Bright field photomicro-
graphs show kidney sections from: (A) Physiological saline-injected rat (group I); (B) An-
imals administered with gentamicin alone (group II); (C) Animals administered with
gentamicin and treated with curcumin for 7 days (group III); (D) Animals administered
with gentamicin and treated with curcumin for 15 days (group IV); (E) Animals adminis-
tered with gentamicin and treated with curcumin for 30 days (group V). Kidney sections
were preincubated with rabbit polyclonal IgG to rat NF-κB (1:500 dilution) and subse-
quently with goat anti-rabbit IgG-HRP conjugate (1:3000 dilution). The immunoreactivity
was developed with 0.01% DAB and 2% H2O2.
Fig. 6. Effect of curcumin on iNOS gene expression in the kidney of Wistar rats exposed to
gentamicin. Lane I: mRNA expression for iNOS in physiological saline-injected rat (group I);
Lane II: mRNA expression for iNOS in animals administered with gentamicin alone (group
II); Lane III: mRNA expression for iNOS in animals administered with gentamicin and trea-
ted with curcumin for 7 days (group III); Lane IV: mRNA expression for iNOS in animals ad-
ministered with gentamicin and treated with curcumin for 15 days (group IV); Lane V:
mRNA expression for iNOS in animals administered with gentamicin and treated with cur-
cumin for 30 days (group V).
583

antioxidant enzymes in renal cortex which could aggravate the oxida-
tive damage. Our results show that curcumin treatment significantly at-
tenuated the gentamicin mediated increase of LPO levels in kidney.
Furthermore, curcumin reversed the effect of gentamicin, by significantly
increasing the activities of GSH, GPx, SOD, catalase and GST content in the
kidney tissue. The apparent ameliorative effect might be due to the ability
of curcumin to neutralize the increase in free radicals caused by
gentamicin.
The histological studies of kidney from gentamicin treated rats
showed damaged glomerular structure, tubular necrosis, tubular epi-
thelial alteration, apoptotic cells, cellular proliferation with fibrosis,
thickening of capillary walls and atrophy of glomerular tuft. Similar
changes were also reported by (Al-Majed et al., 2002; Kumar et al.,
2000), and these alterations were found to be minimal in the animals
treated with curcumin for 7 and 15 days (group III and group IV). An-
imals treated with curcumin for a longer period i.e., 30 days (group V)
showed a normal architecture similar to the control animals, thus
showing its curative effect against gentamicin-induced tissue
damage.
To further understand the ameliorative role of curcumin in genta-
micin-induced nephrotoxicity, the expression of iNOS and NF-κB
were analyzed by immunohistochemical, Western blot and RT-PCR
studies. Nitric oxide (NO), which is a highly diffusible, short lived
free radical gas, had both physiological and pathological functions in
many mammalian tissues (Patel et al., 1999). Thus we speculate
that among various other radicals involved in the damage of kidney
cells, the involvement of iNOS would be more important and that
the blockade of iNOS could reduce gentamicin-induced nephrotoxici-
ty as reported earlier (Ghaznavi and Kadokhodaee, 2007). Reduction
of oxidative stress and a slight decrease in iNOS expression, as ob-
served in a time-dependent manner in curcumin treated animals,
may be responsible for the ameliorative effect of curcumin on genta-
micin-induced structural and functional alterations of kidney. NF-κB
is a highly conserved family of transcription factors that has a critical
role in mediating inflammation, apoptosis, and growth in chronic dis-
ease (Wardle, 2001). Activation of NF-κB, in response to oxidative
stress might play a role in gentamicin-induced nephrotoxicity by in-
ducing synthesis of inflammatory substances (cytokines, growth fac-
tors, adhesion molecules) that provoke kidney damage (Li and
Karin, 1999). Thus, blockade of NF-κB will be an effective approach
for the treatment of nephrotoxicity. Interestingly, in our study curcu-
min treatment led to a slight reduction in the expression of NF-κB
upon treatment for a longer period i.e. 30 days which further con-
firms its curative role against gentamicin-induced nephrotoxicity. It
is well known that curcumin prevents NF-κB activation and this effect
has been shown to be through the inhibition of IκBα phosphorylation
(Mandal et al., 2009). This inhibition of IkBα phosphorylation, pre-
vents its dissociation and ubiquitin-mediated degradation, and thus
keeps NF-κB from translocating into the nucleus.
In summary, we have confirmed that curcumin affords curative
role against nephrotoxicity induced by gentamicin exposure. Accord-
ing to our biochemical findings, which were supported by histopath-
ological, immunohistochemistry, Western blot and RT-PCR analysis,
administration of curcumin rescued the cells from the effects of gen-
tamicin. These findings indicate that curcumin administration may
reduce gentamicin-induced renal injury. Therefore, we propose that
curcumin might be a potential candidate agent against gentamicin-
induced nephrotoxicity via its antioxidant and anti-inflammatory
properties.
Acknowledgements
The authors thank the University Grants Commission (UGC), New
Delhi, India, for project funding in the form of UPE-HSP-33 and UGC-
SAP-DSA I.
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