e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 392404

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journal homepage: www.elsevier.com/locate/etap

Nephroprotective effect of vanillic acid against

cisplatin induced nephrotoxicity in wistar rats:
A biochemical and molecular study
Ganapathy Sindhu , Emayavaramban Nishanthi, Ramalingam Sharmila
Department of Biochemistry and Biotechnology, Annamalai University, Annamalai Nagar 608 002, India

a r t i c l e

i n f o

a b s t r a c t

Article history:

Cisplatin is one of the extensively used anticancer drugs against various cancers. Dosage

Received 9 August 2014

dependent nephrotoxicity is the major problem in cisplatin chemotherapy. Cisplatin

Received in revised form

induced nephrotoxicity results in the depletion of renal antioxidant defence system. Our

10 December 2014

present study is aimed to investigate the nephroprotective effect of vanilic acid to against cis-

Accepted 14 December 2014

platin induced nephrotoxicity in male wistar rats. Elevated levels of serum creatinine, blood

Available online 22 December 2014

urea nitrogen, serum uric acid and reduced antioxidant status were observed as indicatives of nephrotoxicity in cisplatin (7 mg/kg bw) alone administered rats. Animals which are

Keywords:

pre-treated with vanillic acid (50 mg/kg and 100 mg/kg) restored the elevated levels of renal

Cisplatin

function markers and reduced antioxidant status to near normalcy when compared to cis-

Vanillic acid

platin alone treated animals. Cisplatin induced lipid peroxidation was markedly reduced by

Nephroprotective

oral administration of vanillic acid at a high dose. The ndings in the present study suggest

TNF-

that vanillic acid is a potential antioxidant that reduce cisplatin nephrotoxicity and can be

NF-B

as a combinatorial regimen in cancer chemotherapy.

Antioxidants

1.

Introduction

The kidney is a major organ in the human body carrying

out the essential functions like clearance of metabolic waste
products, control of uid volume status, maintenance of
electrolyte and acidbase balance, and endocrinal function.
Renal tubular damage is a well-known renal complication
induced by anticancer drugs (Kakihara et al., 2003). cisDichlorodiaminoplatinum (II), cisplatin is one of the most
widely used antineoplastic drug against testicular, bladder,
ovarian, lung, head and neck tumours. In spite its effectiveness as an antitumour drug, various side effects has been

2014 Elsevier B.V. All rights reserved.

reported, especially nephrotoxicity, has restricted its clinical use. It was reported that 30% of patients suffered from
renal disorders after cisplatin treatment (Faubel et al., 2007;
Arany and Sarstein, 2003; Ramesh and Reeves, 2002). Cisplatin causes tubular injury through multiple mechanisms
like, hypoxia, oxidative stress, inammation, and apoptosis.
Cisplatin-associated cytotoxicity and generation of reactive
oxygen species (ROS) leads to failure of antioxidant defence
that results in damage to cellular macromolecules such as proteins and nucleic acids. Furthermore, the nephrotoxic effect
of cisplatin is dose limiting (Sultana et al., 2012), and is
manifested by imbalances in kidney function markers and
electrolytes. Cisplatin-induced nephrotoxicity is a complex

Corresponding author at: Department of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalai Nagar
608 002, Tamil Nadu, India. Tel.: +91 9788083335; fax: +91 4144 238 141.
E-mail address: ganapathysindhu@gmail.com (G. Sindhu).

http://dx.doi.org/10.1016/j.etap.2014.12.008
1382-6689/ 2014 Elsevier B.V. All rights reserved.

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 392404

process that comprises of acute cytotoxic effects on tubular

epithelial cells, resulting in loss of tubular epithelial cells by
necrosis and apoptosis, followed by inammatory cell inltration and bro proliferative changes (Yao et al., 2007).
Strong evidence indicates the involvement of inammatory mechanisms in the pathogenesis of acute kidney
injury. There are multiple lines of evidence suggesting the
release of pro-inammatory cytokines like tumour necrosis factor- (TNF-), recruitment of inammatory cells,
such as macrophages and leukocytes (Ramesh and Reeves,
2002; Faubel et al., 2007) caused mitochondrial dysfunction
(Rodrigues et al., 2009) in the pathogenesis of cisplatininduced nephrotoxicity. NF-B signalling pathway plays a
crucial role in inammatory as well as immune responses.
Besides, NF-B is a redox sensitive transcriptional factor, regulates expression of various inammatory cytokines, adhesion
molecules, and chemokines (Cichocki et al., 2010).
TNF- is an important key element in the inammatory
pathway is activated in kidney by cisplatin. Obstruction of
TNF- expression prevents the activation of cytokine network and provides protection against cisplatin nephrotoxicity.
In addition to proinammatory molecules, renal parenchymal cells can synthesize a variety of cytoprotective factors in
response to tissue injury which may inhibit the ongoing cell
injury and/or facilitate renal remodelling after initial tubular injury (Wang et al., 2008; Goodman et al., 2007; Ogawa
et al., 2001). Recent studies suggest that certain cytokines,
such as IL-4, IL-10, IL-13 and TGF-, may protect kidney
against tissue injury, but the source of these cytokines
and their role in renal injury is largely unknown. IL-10 is
an anti-inammatory cytokine inhibiting the production of
pro-inammatory cytokines and chemokines and activates
immune cells. IL-10 ameliorates tissue injury in different
pathophysiological conditions including kidney injury in several models of kidney diseases (Jiangping et al., 2001; Ronald
et al., 2010).
Various synthetic and natural entities are screened for
nephroprotector activity against cisplatin-induced nephrotoxicity, where treatment carried out with plant derived
compounds. Phenolic compounds form a substantial part
of plant food in which phenolic acids have received much
attention because of their role in the prevention of many
human diseases particularly atherosclerosis and cancer and
promote health benets due to their antioxidant property
(Mattila and Kumpulainen, 2002). Vanillic acid (4-hydroxy3-methoxybenzoic acid) is a phenolic derivative from edible
plants and fruits, and has antibacterial (Rai and Maurya,
1996), antimicrobial (Delaquis et al., 2005), anti-larial (Varma
et al., 1993) and hepatoprotective properties (Singh et al.,
2005). Further, it is used in traditional Chinese medicine for
diabetes, haemorrhagic inammation, hypertension, ulcers,
and fever. Tsuda et al. (1994) reported that vanillic acid
exhibits chemopreventive effect in experimentally induced
carcinogenesis. Vanillic acid is a signicant antioxidant
because of the presence of the carboxyl group (Chou et al.,
2010) and possess antihypertensive activity in L-NAMEinduced hypertensive rats (Kumar et al., 2011). Vanillic acid
showed inhibitory effect on inammatory mediators by
suppressing NF-B in lipopolysaccharide-stimulated mouse
(Kim et al., 2011). Stanely Mainzen Prince et al. (2011) studied

393

the protective effect of vanillic acid due to its free radical

scavenging, antioxidant and anti-inammatory properties
in isoproterenol induced cardiotoxic rats. Gitzinger et al.
(2012) have recommended that vanillic acid is licensed as
food additive may facilitate its approval by governmental agencies for applications in future biopharmaceutical
manufacturing scenarios as well as in gene- and cell-based
therapies. The present study is aimed to explore the possible
mechanism(s) for the nephroprotective effect of vanillic acid
on cisplatin-induced nephrotoxicity.

2.

Materials and methods

2.1.

Drugs and chemicals

Vanillic acid, cisplatin and oligonucleotide primers were purchased from SigmaAldrich Chemical Pvt. Ltd. (St. Louis, MO,
USA). All other chemicals used were of analytical grade,
purchased from Hi-media Laboratories Pvt. Ltd., Mumbai,
India.

2.2.

Animals

Healthy adult male albino rats (810 weeks old; weighing

120180 g) of Wistar strain, were purchased from the National
Institute of Nutrition, Hyderabad, India, and were maintained
in the Central Animal House, Department of Experimental Medicine, Rajah Muthaiah Medical College and Hospital,
Annamalai University, Annamalai Nagar, India. The animals
were housed in polypropylene cages and were provided with
standard pellet diet (Amrut Laboratory Animal Feed Mysore
Feeds Limited, Bangalore, Karnataka, India) and water ad
libitum. The animals were maintained under controlled conditions of temperature (23 2 C) and humidity (6570%) with
a 12 h light/dark cycle. The animal treatment and protocol
employed was approved by the Institutional Animal Ethics
Committee (Registration number 160/1999/CPCSEA; Proposal
No. 976: dated 07.02.2013), Annamalai University. The animals were cared in accordance with the Guide for the
care and use of laboratory animals and committee for the
purpose of control and supervision on experimental animals.

2.3.

Experimental design

A total of 30 Male Wistar rats were randomized into ve

groups including control and experimental of 6 rats in each
group. Group I rats served as the vehicle control (0.9% saline).
Group II rats were treated with cisplatin (7 mg/kg bw i.p.,
injection on 19th, 20th and 21st day) alone. Group III and
IV rats were orally administered with vanillic acid (50 mg/kg;
100 mg/kg, bw, po, respectively) started from day 1 and continued daily for 21 days and were injected intraperitonially with
cisplatin (7 mg/kg bw on 19th, 20th and 21st day). Group V rats
were orally administered with vanillic acid (100 mg/kg) alone
throughout the experimental period. All the animals were
sacriced by cervical dislocation at the end of experimental
period.

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2.4.

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Biochemical analysis

Biochemical studies were conducted on blood, liver and kidney of the control and experimental group. Blood samples
were collected in heparinized tubes for plasma (separated
by centrifugation at 1000 g for 15 min) and in a dry test
tube and allowed to coagulate at ambient temperature for
30 min for serum (separated by centrifugation at 2000 g
for 10 min). Liver and kidney tissue samples from animals
were washed with ice-cold saline and homogenized using
the appropriate buffer [thiobarbituric acid reactive substances
(TBARS), 0.025 mol/l TrisHCl buffer, pH 7.5; superoxide dismutase (SOD), 0.025 mol/l sodium pyrophosphate buffer, pH 8.3;
catalase (CAT), 0.01 mol/l phosphate buffer, pH 7.0; glutathione
peroxidase (GPx) and reduced glutathione (GSH), 0.4 mol/l
phosphate buffer, pH 7.0] in an all-glass homogenizer with a
Teon pestle.
Aliquots from these preparations (blood and tissues) were
used for the lipid peroxidation TBARS (Fraga et al., 1988), SOD
(Kakkar et al., 1984), CAT (Sinha, 1972), GPx (Rotruck et al.,
1973) and GSH (Ellman, 1959). Blood urea was estimated by
the diacetyl monoxime method (Natelson, 1957). Serum creatinine was estimated by the alkaline picrate method (Bonsnes
and Taussky, 1945) and Uric acid in plasma was estimated by
the method of Brown (1945).

2.5.
PCR detection of TNF-, NF-B and IL-2 and IL-10
genes expression
Total RNA was extracted from renal tissue by the guanidium
thiocyanate method. RNA content and purity were measured
by Nano spectrophotometer (Implen, Germany) at 260280 nm.
RT-PCR was performed using a thermal cycler (Eppendorf
Mastercycler Personal) with extracted RNA for detection of
TNF-, NF-B and IL-2 and IL-10 genes. -Actin expression
was determined as loading control for each sample. The
primer sequences are listed in Table 1. The primer design
was adapted from previous literatures (Deepa and Anuradha,
2011; Ikezumi et al., 2000). For amplication of the target
genes, reverse transcription and PCR were run in two separate steps. Briey, the rst strand (DNA was made by mixing
2 g of total RNA with oligo DT primer (0.5 g/l), dNTP mix
(10 mM each, 2 l) and 2 l DEPC water in a 0.2 ml microfuge
tube. After incubation for 5 min at 65 C, add 1 l reverse transcriptase (1.5 U) in 5 M-MuLV (Moloney-Murine Leukaemia
virus) reverse transcription buffer (4 l) and RNase inhibitor
solution (4 U, 0.5 l), incubate at 42 C for 90 min to reversely
transcribe the RNA. The reaction was terminated by heating
at 85 C for 5 min and then cooled to 4 C. Reactions were
run in a total volume of 50 l, including 25 l PCR reactions
Mix (RED Taq-Ready Mix), 2 l of each primer at 10 M concentrations, 5 l of the previously reverse transcribed cDNA
template and 16 l of DEPC water. All reactions were run in
triplicates. All PCR products were run on 1.5% agarose stained
with ethidium bromide and visualized by UV transilluminator.
Semi-quantitative determination of PCR products was performed using the gel documentation system (UV Tech, Genie).
Relative expression of each studied gene (R) was calculated
using the following formula, R densitometrical units of each
studied gene/densitometrical units of -actin.

2.6.

Histopathological examination

Histopathological investigations were performed on liver and

kidney tissues of the control and experimental animals in
each group. Tissues were xed in 10% buffered formalin and
routinely processed and embedded with parafn; 23 m sections were cut in a rotary microtome, xed on glass slides, and
stained with haematoxylin and eosin.

2.7.

Statistical analysis

Statistical analysis was performed using one-way analysis

of variance (ANOVA), followed by Duncans multiple range
test (DMRT) using SPSS version 12.0 for windows (SPSS Inc.,
Chicago, Illinois, USA; http://www.spss.com). Values are represented as mean SD and p < 0.05 was considered statistically
signicant.

3.

Results

3.1.

Effect of vanillic acid on renal function parameters

Fig. 1 shows the levels of serum creatinine and urea and

uric acid in plasma of control and experimental rats in each
group. Rats treated with cisplatin alone (Group II) showed
increase in serum creatinine and urea and uric acid levels in
plasma, when compared to control rats. Normalcy in the levels of serum creatinine, and urea and uric acid in plasma was
observed in Groups (III and IV) when treated with cisplatin.
Rats treated with vanillic acid alone (Group V) showed no signicant difference in levels of creatinine, urea, and uric acid
when compared to control rats (Group I).

3.2.

Effect of vanillic acid on oxidative stress

Fig. 2 shows the concentration of TBARS in plasma, liver and

kidney of control and experimental rats in each group. There
was increase in TBARS level in the plasma, liver and kidney tissue of rats treated with cisplatin alone (Group II). Oral
administration of vanillic acid to rats in Groups III and IV
keep the concentration of TBARS in near normal range when
treated with cisplatin. No signicant difference in TBARS levels was noticed in rats treated with vanillic acid alone (Group
V) as compared to control rats (Group I).

3.3.

Effect of vanillic acid on antioxidant status

Figs. 35 shows the status of enzymatic (SOD, CAT, GPx) and

non-enzymatic (GSH) antioxidants in the plasma, kidney and
liver tissues of control and experimental rats in each group.
Decrease in enzymatic and non-enzymatic antioxidant status
was observed in rats treated with cisplatin alone (Group II).
Normal values in the SOD, CAT, GPx and GSH were observed
in Groups III and IV rats when treated with cisplatin. No signicant differences in antioxidants status were noticed in rats
treated with vanillic acid alone (Group V) as compared to control rats (Group I).

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Table 1 Primer sequences are used in RT-PCR.

S. no.

PCR product

Primer sequences

Annealing temperature

Size (bp)

TNF-

Sense: 5 -CTCGAGTGACAAGCCCGTAG-3
Antisense: 5 -TTGACCTCAGCGCTGAGCAG-3

52 C

386

NF-B

Sense: 5 -AAGATCAATGGCTACACGGG-3
Antisense: 5 -CCTCAATGTCTTCTTTCTGC-3

60 C

493

IL-10

Sense: 5 -CCAAGCCTTATCGGAAATGA-3
Antisense: 5 -AGGGGAGAAATCGATGACAG-3

55 C

346

IL-2

Sense: 5 -GCGCACCCACTTCAAGCCCT-3
Antisense: 5 -CCACCACAGTTGCTGGCTCA-3

63 C

351

-Actin

Sense: 5 -TGTGATGGTGGGAATGGGTCAG-3
Antisense: 5 -TTTGATGTCACGCACGATTTCTC-3

58 C

213

3.4.

Effect of vanillic acid on renal inammation

The TNF-, NF-B, IL-2 and IL-10 mRNA expression patterns

of control and experimental rats are depicted in Figs. 69. Cisplatin induced signicant increase in the expression of TNF-,
NF-B and IL-2 genes and reduced IL-10 gene expression compared to control (p < 0.05). Vanillic acid pretreatment increased
the IL-10 gene expression and reduced IL-2, NF-B and TNF-
genes expression. Similar pattern of TNF-, NF-B, IL-2 and
IL-10 mRNA expression was observed in control rats and rats
treated with vanillic acid alone.

3.5.

Histopathology

Histopathological studies of kidney of Groups I and V rats

showed normal glomerulus and tubules with regular morphology (Fig. 10A and E). Group II rats showed degenerating tubular
structures with vacuolization and loss of tubular architecture
and mononuclear inltration in lumen (Fig. 10 B). Administration of vanillic acid for 21 days at the dose of 50 mg/kg (Group

III) showed normal glomerulus, presence of mild degenerating

tubules with vacuolated cells (Fig. 10C). However, high dose of
vanillic acid (100 mg/kg) for 21 days (Group IV) revealed mostly
normal kidney morphology with occasional tubular necrosis
(Fig. 10D).
Histological studies on liver showed that cisplatin alone
treated rats (Group II) revealed vacuolization of cytoplasm,
nuclear fragmentation and also increased necrotic hepatocytes (Fig. 11B). Fig. 11C and D revealed reduction in necrotic
hepatic cells was observed in cisplatin + vanillic acid treated
rats (Groups III and IV). Rats administered with vanillic acid
alone (Group V) and control (Group I) showed normal cellular
architecture (Fig. 11A and E).

4.

Discussion

Metabolism and excretion of exogenously administered therapeutic and diagnostic agents are the major functions of
kidneys. The capacity of the kidney to concentrate urine

Fig. 1 Status of kidney function markers serum creatinine, plasma urea and uric acid in control and experimental rats in
each group. Values are given as mean SD from six rats in each group. Values not sharing a common superscript differ
signicantly at p < 0.05 (DMRT). Cis cisplatin, VA vanillic acid.

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Fig. 2 Status of plasma, kidney and liver TBARS in control and experimental rats in each group. Values are given as
mean SD from six rats in each group. Values not sharing a common superscript differ signicantly at p < 0.05 (DMRT). Cis
cisplatin, VA vanillic acid.

Fig. 3 Status of enzymatic and non-enzymatic antioxidants in plasma of control and experimental rats in each group. UA
the amount of enzymes required to inhibit 50% nitroblue-tetrazolium (NBT) reduction. Values are given as mean SD from
six rats in each group. Values not sharing a common superscript differ signicantly at p < 0.05 (DMRT). Cis cisplatin, VA
vanillic acid.

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397

Fig. 4 Status of enzymatic and non-enzymatic antioxidants in kidney of control and experimental rats in each group.
Values are given as mean SD from six rats in each group. Values not sharing a common superscript differ signicantly at
p < 0.05 (DMRT). Cis cisplatin, VA vanillic acid.

Fig. 5 Status of enzymatic and non-enzymatic antioxidants in liver of control and experimental rats in each group. Values
are given as mean SD from six rats in each group. Values not sharing a common superscript differ signicantly at p < 0.05
(DMRT). Cis cisplatin, VA vanillic acid.

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Fig. 6 mRNA expression patterns of TNF- and

quantitative analysis of the expression by densitometry in
the kidney of control and experimental rats in each group.
Data are expressed as relative intensity and are means SD
(n = 6) after normalization with that of -actin. Values not
sharing a common superscript differ signicantly at p < 0.05
(DMRT).

can result in increased localized accumulations of toxicants

and initiate renal dysfunction. Renal tubular damage is a
well-known renal complication induced by anticancer drugs
(Kakihara et al., 2003). Cisplatin causes tubular injury through
multiple mechanisms, including hypoxia, oxidative stress,
inammation, and apoptosis. Furthermore, signicant interactions among these various pathways may occur in cisplatin
injury. Several agents were co-administered to prevent cisplatin induced nephrotoxicity. However, their effects on the
anticancer potential of cisplatin were not studied in most
cases. Vanillic acid is one such plant derived medicinal compound widely used in traditional medicine to treat urinary
problems. Vanillic acid, a strong antioxidant has been associated with a variety of pharmacologic activities such as
suppressor of potent snake venom, cell apoptosis in Neuro2A cells, and immune-mediated liver inammation in mice
(Huang et al., 2008; Gitzinger et al., 2012). It has been reported
to inhibit the mutagenesis induced by chemical and physical
mutagens in various models (Shaughnessy et al., 2001). It has
been showed that vanillic acid increased apoptosis in nonsmall lung cancer NCI-H460 cell line and might be useful for
as an anti-cancer drug (Del Rio et al., 2013). It also showed
chemopreventive effect in chemical carcinogenesis models in
the rat (Tsuda et al., 1994). It has been reported that vanillic
acid displayed inhibited DNA-dependent protein kinase and

Fig. 7 mRNA expression pattern of NF-B and quantitative

analysis of the expression by densitometry in the kidney of
control and experimental rats in each group. Data are
expressed as relative intensity and are means SD (n = 6)
after normalization with that of -actin. Values not sharing
a common superscript differ signicantly at p < 0.05 (DMRT).

enhanced sensitivity of cancer cells to cisplatin (Durant and

Karran, 2003). In the present study, we demonstrated the possible mechanisms involved in the nephroprotective effect of
vanillic acid on cisplatin-induced nephrotoxicity.
The histopathology of tissue sections suggested that the
cisplatin alone treated group had encountered vast histological damages as evidenced by the presence of glomerular
congestion, tubular degeneration, vacuolization and necrosis
in the proximal tubular epithelial cells and the presence of
tubular cast. Inammatory cells were also seen in kidney section from the cisplatin group. Oral pretreatment with vanillic
acid both doses were found to reduce such changes in kidney
histology induced by cisplatin. Present results suggested
that vanillic acid suppressed renal toxicity occurring during
cisplatin induced renal toxicity, as evidenced by predominant
protection of renal histology in kidney tissue of pretreated rats.
A large number of phytoconstituents from medicinal plants
have been shown to be regulators of renal function (Mohana
Lakshmi et al., 2012). BUN, Serum creatinine and uric acid
are the most sensitive markers of nephrotoxicity employed in
the diagnosis of renal damage. Creatinine is a waste product
formed in muscle from the high energy storage compounds
as creatine phosphate. The amount of creatinine produced
is fairly constant and is primarily a function of muscle
mans. Creatinine is removed from plasma by glomerular ltration and then excreted in urine without any appreciable

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399

Fig. 8 mRNA expression pattern of IL-10 and quantitative

analysis of the expression by densitometry in the kidney of
control and experimental rats in each group. Data are
expressed as relative intensity and are means SD (n = 6)
after normalization with that of -actin. Values not sharing
a common superscript differ signicantly at p < 0.05 (DMRT).

Fig. 9 mRNA expression pattern of IL-2 and quantitative

analysis of the expression by densitometry in the kidney of
control and experimental rats in each group. Data are
expressed as relative intensity and are means SD (n = 6)
after normalization with that of -actin. Values not sharing
a common superscript differ signicantly at p < 0.05 (DMRT).

reabsorption by the tubules (Negishi et al., 2007). Creatinine

is used to assess renal function however; serum creatinine
levels do not start to rise until renal function has decreased
by at least 50%. An elevated serum creatinine level occurs
due to obstruction of urinary tract. Many a times serum
urea/creatinine ratio is used for assessment of kidney function and disease diagnosis. Profound studies reported that
the levels of renal function markers signicantly elevated during cisplatin induced nephrotoxicity (Sung et al., 2008). It has
been reported that administration of vanillic acid modied the
serum urea, uric acid and creatinine levels towards normal
which was altered during acetaminophenone induced hepatotoxicity (Deepa Mol and Raja, 2010). Our results are in line with
these ndings. Since cisplatin can be dened as possessing
nephrotoxic potential, blocking of this process corresponds to
prevention/protection of nephrotoxicity. Such increased levels
of these nephrotoxicity markers in the serum might be due to
the renal membrane damage during cisplatin administration.
This is indicative of the onset of renal cellular damage, kidney
dysfunction and alteration in the membrane permeability. In
contrast, rats administered with vanillic acid showed lower
BUN and serum creatinine levels as compared with the control rats. This is an indicator of the possible nephroprotective
efcacy offered by vanillic acid against cisplatin.
Reactive oxygen species (ROS) directly act on cell components, including lipids, proteins, and DNA, and destroy their

structure. ROS are produced via the xanthinexanthine oxidase system, mitochondria, and NADPH oxidase in cells. In
the presence of cisplatin, ROS are produced through all these
pathways and are implicated in the pathogenesis of acute
cisplatin-induced renal injury (Kawai et al., 2006). Profound
studies showed that the pathogenesis of cisplatin induced
acute renal toxicity actively involves oxidative stress (Yilmaz
et al., 2004; Sultana et al., 2012). Biological membranes contain
a large amount of polyunsaturated fatty acids, which are particularly susceptible to peroxidative attacks to produce lipid
peroxides. Lipid peroxides have been used as a sensitive indicator of oxidant induced cell injury (Khan et al., 2011). Our
study corroborates this: there was an increase in lipid peroxidation in the cisplatin treated group as compared with the
control group and there was dose dependent inhibition of LPO
by vanillic acid administration. The present results signifying
that vanillic acid can be a potent scavenger of free radicals produced during cisplatin toxicity thereby protecting the kidney
from free radical damage. The results indicated that vanillic
acid rendered signicant preventive effect against cisplatininduced nephrotoxicity.
Oxidative stress was characterized by increased lipid
peroxidation and/or altered non-enzymatic and enzymatic
antioxidant systems due to enzymatic inactivation during
cisplatin injury (Hawkins et al., 2001). The defense provided by antioxidant systems is crucial for the survival of

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Fig. 10 Histopathological changes in kidney tissue. (A, E) Control and vanillic acid alone treated animals showing normal
glomerulus and renal tubules. (B) Cisplatin alone treated animals showing degenerative changes within the glomerulus and
in tubular cells. (C) Vanillic acid 50 mg/kg and cisplatin treated animal showing moderate degeneration of luminal cells of
tubular structure. (D) Vanillic acid 100 mg/kg and cisplatin treated animals showing the glomerulus and the tubular
structures with mild degenerative changes.

organisms. Detoxication of ROS in the cell is provided by both

enzymatic and non-enzymatic systems which constitute the
antioxidant defence systems. These antioxidants play a role
in delaying, intercepting, or preventing oxidative reactions
catalyzed by free radicals (McCord, 2000). GSH is the major
antioxidative tripeptide in the cell and plays pivotal role in
the detoxication of toxicants including chemotherapeutic
drugs, metabolism of nutrients and regulation of various
pathways to maintain cellular homeostasis (Wu et al., 2004)
and also serves as a substrate for GST and GPx. GPx in
association with its cosubstrate reduced glutathione protects
the cell from oxidative damage by catalyzing the reduction
of hydro peroxides (Husain et al., 1998). SOD constitutes an
important link in the biological defense mechanism through
dismutation of endogenous cytotoxic superoxide radicals to
H2 O2 . CAT is a hemoprotein which catalyses the reduction of
hydrogen peroxide to molecular oxygen and H2 O and protects

the tissue from highly reactive hydroxyl radicals. Previous

studies suggested that antioxidant enzymes are inhibited by
cisplatin, and renal activities of superoxide dismutase, glutathione peroxidase and catalase are signicantly decreased
(Sahreen et al., 2010; Husain et al., 1998).
During cisplatin administration, platinum sulfhydryl
group complexes formed are taken up by renal cells and
stabilized by intracellular GSH which leads to depletion of
antioxidant system. Thus GSH depletion results in increased
toxicity of cisplatin by undergoing rapid transformation of
platinum complexes to reactive metabolites GSH depletion
also results in lipid peroxidation and this seems to be the
prime factor that permits lipid peroxidation and impaired
antioxidant enzyme activities (Wu et al., 2004). The decreased
SOD activity is insufcient to scavenge the superoxide anion
produced during the normal metabolic process. The superoxide anion can cause initiation and progression of lipid

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401

Fig. 11 Histopathological changes in liver tissue. (A, E) Control and vanillic acid alone treated animals respectively,
showing normal architecture. (B) Cisplatin alone treated animals showing necrotic cells, lobular architecture of liver and
pronounced dilatation of sinusoids and destruction of the hepatocytes. (C) Vanillic acid 50 mg/kg and cisplatin treated
animals showing reductions in necrotic hepatic cells. (D) Vanillic acid 100 mg/kg and cisplatin treated animals showing mild
destruction of the hepatocytes.

peroxidation. The depletion in the activity of CAT, as seen in

the cisplatin treated group compared to the control group,
results in decreased ability to scavenge toxic hydrogen peroxide, further contributing to oxidative stress (Sahreen et al.,
2010). Our results are supported by previous studies which
elucidate the potency of cisplatin to inhibit the antioxidant
armoury. Erdem et al. (2012) has been evaluated the possible
ameliorative activity of vanillic acid on MMC-induced genotoxic effects and they were found possibly contributing to
this activity is the hydroxyl group, which places vanillic acid
in the category of phenolic antioxidants. These observations
support our hypothesis that the mechanism of nephrotoxicity
is related to free radical generation and the nephroprotection
offered by vanillic acid is due to antioxidant defense system.
Cisplatin induces a series of inammatory changes that
mediate renal injury. Recent evidences indicate that inammation has an important role in the pathogenesis of cisplatin

induced renal injury (Ueki et al., 2013; Sahu et al., 2013).

Cisplatin causes direct tubular injury through multiple mechanisms. Cisplatin increases degradation of IB and increases
NF-B binding activity. These events lead to the enhanced
renal expression of TNF-. For example, TNF- induces apoptosis, produces ROS, and coordinates the activity of a network
of cytokines that all contribute to cellular injury. Apoptosis
is an important mode of cell death in cisplatin nephrotoxicity (Arjumand et al., 2011). Engagement of a cell surface
receptor with extracellular TNF- activates apoptosis in renal
tubule. Ramesh and Reeves (2002) showed that TNF- may
play an important role in inammation and apoptosis in renal
tubular epithelial cells in cisplatin nephrotoxicity models. NFB activation is pivotal in the expression of proinammatory
cytokines like TNF- and other mediators involved in acute
inammatory responses and other conditions associated with
increased ROS generation. Acute inammatory responses

402

e n v i r o n m e n t a l t o x i c o l o g y a n d p h a r m a c o l o g y 3 9 ( 2 0 1 5 ) 392404

have driven via induction of NF-B and TNF- (Collins et al.,

1995). We found that mRNA levels for NF-B and TNF- were
increased in the kidney tissue after cisplatin injection. The
present study reveals that both NF-B and TNF- expression
were abrogated substantially by the prophylactic treatment
of vanillic acid, thus reducing inammatory response implicated in the acute renal toxicity caused by the cisplatin. In
the present study, we showed that the cytokine levels were
lowered after the administration of vanillic acid in cisplatin
treated rats. Oral administration of vanillic acid exhibited
protective mechanism which may include decreased expression of pro apoptotic TNF- and by reducing apoptosis. These
results are consistent with previous observations. Vanillic
acid, a phenolic compound that possess o-methoxy group,
covalently modify sulfhydryl groups by oxidation and alkylation, and the modication might be responsible for the
inhibition of the NF-B dependent process. Therefore, it is
possible that the immunomodulatory effect of vanillic acid
is mediated by inhibiting the NF-B dependent process. The
role of inammation in the pathogenesis of renal diseases of
various origins, are ischaemic and toxic kidney injury. Renal
cells and resident leukocytes, in response to ischaemic or toxic
insults, secrete a wide range of chemokines and cytokines
(Huang et al., 2010; Zhang et al., 2008; Huang et al., 2007).
These mediators of inammation up-regulate the expression
of adhesion molecules and attract different populations of
leukocytes that include neutrophils, macrophages, T cells,
NK cells and dendritic cells, which may further exacerbate
injury by producing (Ascon et al., 2006; Furuichi et al., 2003;
Ramesh and Reeves, 2002). Concurrent with the induction of
a stress activated inammatory response, many agents with
anti-inammatory properties (e.g., IL-10) are produced that
may prevent tissue injury, or help in tissue repair/remodelling
subsequent to injury in different organs and tissues, including the kidneys (Bamboat et al., 2010). IL-10 inhibits the
production of pro-inammatory cytokines and chemokines
and the activation of immune cells. IL-10 ameliorates tissue injury in different pathophysiological conditions IL-10 is
a multifunctional anti-inammatory cytokine that has been
reported to attenuate different renal pathologies (Kitching
et al., 2002). Several interleukins, including IL-2, IL-4, IL-5 and
IL-10, shave been shown to induce IL-2R expression in inammatory processes and viral infections. Among the cytokines,
interleukin-2 (IL2) is the most important for stimulating T-cell
proliferation. When a T-lymphocyte is activated by antigenpresenting cells, it produces and secretes IL2 (Ronald et al.,
2010). In the present study demonstrated the nephroprotective potential of vanillic acid in cisplatin nephrotoxicity. Oral
administration of vanillic acid 50 mg/kg and 100 mg/kg body
weight down regulated the expression of inammatory markers (TNF-, NF-B and IL-2). Also vanillic acid enhanced the
expression of IL-10. These results indicated that vanillic acid
exhibit protective effect on cisplatin-induced renal injury.

5.

Conclusion

The present study concludes that the nephroprotective effect

of vanillic acid might be due to its antioxidative and antiinammatory potential during cisplatin induced nephrotoxicity

and it may nd use as an adjunct in the cancer chemotherapy in which cisplatin is used as rst line drug. Therefore,
the development of a combination therapy strategy is needed
to further validate the prophylaxis and suggestive treatment of cisplatin-induced nephrotoxicity in both in vivo
and in vitro experimental models. Further, the underlying
suggested mechanism(s) and whether this nephroprotective
effect interferes with the cytotoxic effect of cisplatin on cancer
cells need to be investigated in future.

Conict of interest
The authors declare that there are no conicts of interest.

Transparency document
The Transparency document associated with this article can
be found in the online version.

Acknowledgement
The authors are thankful to the authorities of Annamalai University for providing all facilities to carry out this work.

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