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Nephroprotective activities of rosmarinic acid against cisplatin-induced
kidney injury in mice
Robert Domitrovic
´
a,
⇑
, Iva Potoc
ˇnjak
b
,Z
ˇeljka Crnc
ˇevic
´-Orlic
´
c
, Marko Škoda
b
a
Department of Chemistry and Biochemistry, Medical Faculty, University of Rijeka, 51000 Rijeka, Croatia
b
Medical Faculty, University of Rijeka, 51000 Rijeka, Croatia
c
Department of Endocrinology, Clinical Hospital Rijeka, 51000 Rijeka, Croatia
article info
Article history:
Received 24 September 2013
Accepted 2 February 2014
Available online 8 February 2014
Keywords:
Rosmarinic acid
Cisplatin
Nephrotoxicity
Oxidative stress
Inflammation
Apoptosis
abstract
Rosmarinic acid (RA) is a natural phenolic compound with a broad range of applications, from food pre-
servatives to cosmetics. Increasing amounts of evidence suggests its beneficial effects against various
pathological conditions. The aim of this study was to investigate the therapeutic activity of rosmarinic
acid (RA) against cisplatin (CP)-induced nephrotoxicity. RA was administered by oral gavage at doses
of 1, 2 and 5 mg/kg for two successive days, 48 h after intraperitoneal CP injection (13 mg/kg). Twenty
four hours later, mice were sacrificed. Treatment with RA significantly ameliorated histopathological
changes and the increase in serum creatinine and blood urea nitrogen (BUN) induced by CP. Oxidative
stress induced by CP, evidenced by increased renal 4-hydroxynonenal (4-HNE), cytochrome P450 2E1
(CYP2E1) and heme oxygenase (HO-1) expression, was significantly reduced by RA administration. More-
over, RA inhibited the expression of nuclear factor-kappaB (NF-
j
B) and tumor necrosis factor-
a
(TNF-
a
),
indicating the inhibition of inflammation. Additionally, RA exhibited antiapoptotic activity through the
reduction of p53, phosphorylated p53 and active caspase-3 expression in the kidneys. These findings
show that RA ameliorates CP-induced oxidative stress, inflammation and apoptosis in the kidneys. The
nephroprotective activity of RA could be, at least in part, attributed to reduced CYP2E1 expression.
Ó2014 Elsevier Ltd. All rights reserved.
1. Introduction
Cisplatin (cis-diamminedichloroplatinum(II), CP) is an antitu-
mor drug commonly used in the treatment of testicular, ovarian,
bladder, cervical, esophageal, head and neck and small cell lung
cancer (Giaccone, 2009). Unfortunately, numerous side effects are
related to CP therapy, including ototoxicity, gastrointestinal toxic-
ity, myelosuppression, neurotoxicity and kidney injury (Miller
et al., 2010; Hartmann and Lipp, 2003). Kidneys represent the main
route of CP excretion, with proximal tubule cells as a primary site
of CP accumulation (Yao et al., 2007). Thus, nephrotoxicity is one of
the most serious dose-limiting side effects in CP chemotherapy.
The major mechanisms of CP-induced nephrotoxicity include tubu-
lar necrosis, oxidative stress, inflammation and apoptosis (Miller
et al., 2010).
Natural phenolics, such as hesperidin, rutin, silymarin and gan-
istein, were shown to ameliorate CP-mediated nephrotoxicity
(Sahu et al., 2013; Kang et al., 2011; Ninsontia et al., 2011;Sung
et al., 2008). Moreover, quercetin prevented the nephrotoxic
activity of CP without affecting its anti-tumor activity (Sanchez-
Gonzalez et al., 2011). These findings indicate that natural phenolic
compounds could be utilized as a nephroprotective agents against
CP-induced kidney injury.
Rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxy-
phenyllactic acid, is a widely occurring natural product with a
broad range of applications, from food preservatives to cosmetics
(Petersen and Simmonds, 2003). It possesses numerous biological
activities, including antioxidative (Zhang et al., 2010), anti-inflam-
matory (Chu et al., 2012), antiapoptotic (Lee et al., 2008), antitu-
mor (Venkatachalam et al., 2013), antialergic (Costa et al., 2012),
antibacterial (Moreno et al., 2006) and antiviral (Swarup et al.,
2007). The antioxidant activity of RA may be attributed to its
phenolic structure. Phenolic compounds can easily donate elec-
trons or hydrogen atoms to neutralize free radicals, whereas
resulting phenoxyl radicals could be enzymatically recycled to par-
ent phenolic (Sakihama et al., 2002). The antioxidant capacity of
phenolics seems greatly dependent on the number and even more
the position of hydroxyl groups. Hydroxyl groups in the ortho posi-
tion of the aromatic ring, such as in RA, can greatly enhance the
http://dx.doi.org/10.1016/j.fct.2014.02.002
0278-6915/Ó2014 Elsevier Ltd. All rights reserved.
⇑
Corresponding author. Address: Department of Chemistry and Biochemistry,
School of Medicine, University of Rijeka, B. Branchetta 20, 51000 Rijeka, Croatia.
Tel.: +385 51651135; fax: +385 51678895.
E-mail address: robert.domitrovic@medri.uniri.hr (R. Domitrovic
´).
Food and Chemical Toxicology 66 (2014) 321–328
Contents lists available at ScienceDirect
Food and Chemical Toxicology
journal homepage: www.elsevier.com/locate/foodchemtox
antioxidant capacity (Sroka and Cisowski, 2003). RA and luteolin
with four phenolic hydroxyl groups, including one catechol
structure, showed similar lipid peroxidation inhibition and free
radical scavenging activity in vitro (Özgen et al., 2011). Antioxidant
activity of RA makes this compound a good drug candidate for
treatment of oxidative stress-related pathological conditions. Pre-
viously, we showed that RA and luteolin ameliorated acute liver
damage in mice through the suppression of oxidative stress,
inflammation and fibrogenesis (Domitrovic
´et al., 2009, 2013). Re-
cently, Tavafi and Ahmadvand (2011) demonstrated that RA inhib-
its gentamicin-induced renal oxidative damage in rats. However,
the nephroprotective activity of RA against CP-induced kidney in-
jury has not been studied previously.
In the current study, we investigated the therapeutic activity of
RA against oxidative stress, inflammation and apoptosis induced
by administration of CP as a possible mechanisms of the nephro-
protective activity of RA.
2. Materials and methods
2.1. Chemicals
Rosmarinic acid (96%), cis-diamineplatinum(II) dichloride and dimethyl sulfox-
ide (DMSO) were purchased from Sigma–Aldrich (Steinheim, Germany). Diagnostic
kits for blood urea nitrogen (BUN) and creatinine were from Dijagnostika (Sisak,
Croatia). Radioimmunoprecipitation assay (RIPA) buffer (sc-24948) was purchased
from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Polyvinylidene difluoride
(PVDF) membrane and blocking reagent were obtained from Roche Diagnostics
GmbH (Mannheim, Germany). Mouse monoclonal antibodies to tumor necrosis
factor-alpha (TNF-
a
) (ab1793) and p53 (ab26), and rabbit polyclonal antibodies
to nuclear factor-kappa B (NF-
j
B) p65 (ab7970), heme oxygenase-1 (HO-1)
(ab13243), 4-hydroxynonenal (4-HNE) (ab46545) and cytochrome P450 2E1
(CYP2E1) (ab19140), HRP-conjugated anti-mouse IgG (ab97023) and HRP-conju-
gated anti-rabbit IgG (ab97051) for Western blotting were purchased from Abcam
(Cambridge, UK). Rabbit polyclonal antibodies to cleaved caspase-3 (Asp175,
#9661) and phospho-p53 (p-p53) (Ser15, #9284) were from Cell Signaling Technol-
ogy (Danvers, MA, USA). DAKO EnVision + System, Peroxidase/DAB kit with second-
ary antimouse/antirabbit antibodies (K500711) were from DAKO Corporation
(Carpinteria, CA, USA). Enhanced chemiluminescence (ECL) substrate was pur-
chased from Pierce Chemical Co. (Rockford, IL, USA). Anesthetic and analgesic
(Narketan 10 and Xylapan, respectively) were purchased from Vetoquinol (Bern,
Switzerland). All other chemicals were of the highest grade commercially available.
2.2. Animals
Male BALB/cN mice from our breeding colony, 14 week old, weighting 24-28 g,
were divided into 6 groups with 5 animals per group. Mice were fed a standard
rodent diet (pellet, type 4RF21 GLP, Mucedola, Italy), and water ad libitum. The
animals were maintained at 12 h light/dark cycle, at constant temperature
(20 ± 1 °C) and humidity (50 ± 5%). All experimental procedures were performed
in compliance with the Declaration of Helsinki and approved by the Ethical Com-
mittee of the Medical Faculty, University of Rijeka.
2.3. Experimental design
Group I (control group) received DMSO diluted with saline (5% DMSO, v/v) by
oral gavage. Group II was treated with RA (5 mg/kg) dissolved in the vehicle. Group
III received CP (13 mg/kg) dissolved in 5% DMSO (v/v) immediately before its
administration as a single intraperitoneal (ip) injection. Groups IV, V and VI were
treated with RA solution orally by gavage at doses of 1, 2 and 5 mg/kg, respectively,
for two consecutive days, two days after CP, whereas groups I, II and III received the
vehicle only. We minimized a possible effect of DMSO on cisplatin by reducing its
content. Additionally, controls received the same amount of DMSO as the CP- and
CP + RA-treated animals. RA or vehicle were given to mice after ip administration
of the combination of anesthetic and analgesic. We performed a preliminary inves-
tigation using RA in a dose range of 1–30 mg/kg with small groups of animals to
establish the suitable doses for usage in the main study. Several studies (Brahmi
et al., 2012; Sahu et al., 2011; Vijayan et al., 2007) demonstrated that pre-treatment
of animals with natural antioxidants was similarly or less renoprotective than post-
treatment. Since kidney damage develops gradually, we administered RA on day 2
and 3 following CP injection, targeting ongoing kidney injury. Twenty-four hours
after the last dose of RA or vehicle, mice were sacrificed. Previously, blood was
collected from retro-orbital sinus and serum was separated to determine serum
creatinine and blood urea nitrogen (BUN) concentration. The abdomen was open
and kidneys were removed. One kidney was frozen are later used for Western blot-
ting and other was immersed in buffered 4% paraformaldehyde solution for histo-
logical sections.
2.4. Serum markers of kidney damage
The level of serum markers of kidney function, BUN and creatinine, was mea-
sured by using a Bio-Tek EL808 Ultra Microplate Reader (BioTek Instruments,
Winooski, VT, USA) according to manufacturer’s instructions.
2.5. Histopathology
Paraformaldehyde-fixed tissues were processed as described previously (Dom-
itrovic
´et al., 2012). Histopathological changes in the kidneys were evaluated in
4
l
m thick deparaffinized sections stained by hematoxylin and eosin (HE). Tubular
damage was assessed by scoring tubular dilatation, necrosis, apoptosis and cast for-
mation in 10 different fields (Olympus BX51 microscope, Tokyo, Japan, 400origi-
nal magnification) in the corticomedullary junction of the kidneys (Leemans et al.,
2005). Histopathological changes were blindly scored by a pathologist on a 5-point
scale: 0 = no damage, 1 = 10% of the corticomedullary junction injured, 2 = 10–25%,
3 = 25–50%, 4 = 50–75%, 5 = more than 75%.
2.6. Immunohistochemistry
Immunohistochemical analysis on deparaffinized tissues sections was per-
formed using the primary antibodies against 4-HNE (1:1000), NF-
j
B p65
(1:1000), and cleaved caspase-3 (1:200), the secondary antimouse/antirabbit anti-
bodies and the DAKO EnVision kit, as described previously (Domitrovic
´et al.,
2011). Stained slides were analyzed by light microscopy (Olympus BX51, Tokyo,
Japan).
2.7. Western blot
Kidneys were lysed in RIPA buffer as described previously (Domitrovic
´et al.,
2011). Volume equivalents of 50
l
g of proteins were separated by 12% sodium
dodecyl sulfate polyacrylamide gel electrophoresis for 2.5 h at 4 °C under
reducing and denaturing conditions. Gels were blotted onto the PVDF membrane
for 45 min at room temperature, incubated in 1% blocking reagent for 1 h at
37 °C and incubated with the primary antibodies against CYP2E1 (1:5000),
HO-1 (1:2000), TNF-
a
(1:1000), p53 (1:1000) and p-p53 (1:1000) overnight at
4°C. The membranes were washed in Tris-buffered saline plus Tween 20 (TBST)
and incubated with the secondary antibodies for 1 h at 37 °C. Finally, the
membranes were exposed to ECL substrate, bands were detected and scanned
(Allianze 4.0, Cambridge, UK). The intensity of the bands was assayed by com-
puter image analysis software (NIH Image J software, available at http://rsb.info.
nih.gov/ij).
2.8. Statistical analysis
Data were analyzed using StatSoft STATISTICA version 12.0 software by Krus-
kal-Wallis test and post hoc comparisons were carried out with Dunn’s multiple
comparison test. Results of multiple comparisons tests were indicated by different
letters. Means with letters in common are not significantly different from each
other. Values in the text are means ± standard deviation (SD). Differences with
P< 0.05 were considered to be statistically significant.
3. Results
3.1. Kidney weight and serum markers of kidney damage
Both body weight and relative kidney weight of control and RA-
treated mice were similar (Table 1). CP-intoxication resulted in a
significant body weight reduction and increased relative kidney
weight. These changes were significantly ameliorated by RA. The
serum creatinine and BUN levels were significantly higher in CP-
treated animals when compared to control mice. Treatment with
RA significantly decreased the creatinine and BUN levels in a
dose-dependent manner.
3.2. Kidney histopathology
Normal tubular morphology was observed in cortical and
medullary regions of kidney in control mice (Fig. 1A) and mice
treated with RA (Fig. 1B). CP administration resulted in
322 R. Domitrovic
´et al./ Food and Chemical Toxicology 66 (2014) 321–328
histopathological changes in the corticomedullary junction of mice
kidneys, including tubular epithelial damage, tubular dilatation
and intratubular cast formation (Fig. 1C). Treatment with RA re-
sulted in a dose-dependent restoration of kidney morphology
(Fig. 1D and E), with only occasional degenerating tubules in
CP + RA5 treated mice (Fig. 1F).
3.3. Markers of oxidative stress
Oxidative stress, including lipid peroxidation, is a common fea-
ture of renal injury following cisplatin administration. The kidneys
of control mice and mice treated with RA were 4-HNE immunoneg-
ative (Fig. 2A and B), whereas CP administration induced strong
immunoreactivity toward this oxidative stress marker (Fig. 2C).
High 4-HNE immunopositivity was still present in mice receiving
the lowest dose of RA (Fig. 2D) but subsequently decreased by
higher doses (Fig. 2E, F and G). Similarly, administration of CP in-
creased CYP2E1 and HO-1 expression in mouse kidney lysates
(Fig. 2H) when compared to control mice and RA-treated animals.
CYP2E1 and HO-1 immunoreactivity decreased following RA
administration, with the expression level similar to control mice
by the highest RA dose.
Table 1
Body weight change, relative kidney weight and serum markers of kidney damage.
Body weight change (%) Relative kidney weight Creatinine (
l
mol/L) BUN (mmol/L)
Control +0.88 ± 0.7
a
8.17 ± 0.15
a
31.2 ± 0.9
a
16.3 ± 2.8
a
RA 5 mg/kg +0.16 ± 1.2
a
8.07 ± 0.38
a
33.2 ± 2.1
a
16.8 ± 1.7
a
CP 13 mg/kg 20.6 ± 3.1
b
9.35 ± 0.48
b
165.4 ± 14.7
b
89.6 ± 3.9
b
CP 13 mg/kg + RA 1 mg/kg 17.9 ± 1.8
b
9.26 ± 0.11
b
130.1 ± 9.7
c
69.2 ± 2.8
c
CP 13 mg/kg + RA 2 mg/kg 14.3 ± 2.2
c
8.56 ± 0.26
c
60.6 ± 10.5
d
29.2 ± 4.1
d
CP 13 mg/kg + RA 5 mg/kg 11.42 ± 1.7
c
8.04 ± 0.20
a
35.3 ± 4.6
a
14.8 ± 1.8
a
Mice were administered RA orally once daily for two consecutive, 48 h after intraperitoneal injection of cisplatin (CP). Animals were sacrificed 4 days after injection of CP.
Relative kidney weight is expressed as [(kidney weight/body weight) * 1000]. Each value represents the mean ± SD for 5 mice. Means within columns sharing the same letter
are not significantly different from each other (P<0.05).
Fig. 1. Representative histological abnormalities in the corticomedullary junction of mice kidneys. Mice treated with vehicle (A), RA 5 mg/kg (B), CP (C), CP + RA 1 mg/kg (D),
CP + RA 2 mg/kg (E) and CP + RA 5 mg/kg (F). Arrows show damaged tubular cells, stars show intratubular cast formation. Representative results from 5 similarly treated mice.
HE staining. Original magnification 400. Tubular injury score (G). Each value represents the mean ± SD for 5 mice. Means sharing the same letter are not significantly
different from each other (P< 0.05).
R. Domitrovic
´et al./ Food and Chemical Toxicology 66 (2014) 321–328 323
3.4. Inflammatory response
Immunohistochemical evaluation showed more intense expres-
sion of NF-
j
B p65 subunit in kidneys of mice exposed to CP
(Fig. 3C) when compared to controls (Fig. 3A) or RA-treated mice
(Fig. 3B). NF-
j
B p65 overexpression was noticed both in the
cytoplasm and the nuclei of tubular cells. Treatment with RA ame-
liorated NF-
j
B immunopositivity in a dose-dependent manner
(Fig. 3D, E, F and G). The lowest dose of RA reduced nuclear
NF-
j
B p65 immunopositivity, which diminished by higher doses
of RA, with concomitant reduction of the cytoplasmic staining
intensity. Western blot analysis revealed increased TNF-
a
expres-
sion in the kidneys of CP-treated mice when compared to control
and RA-treated mice (Fig. 3H).
3.5. Apoptosis
Apoptosis in the kidneys was determined by measuring the
expression of proapoptotic p53 protein and apoptotic executor
cleaved caspase-3. We subsequently determined p53 phosphoryla-
tion, which represents an important regulatory mechanism for
p53. As shown in Fig. 4A and B, the immunohistochemical analysis
revealed no detectable active caspase-3 staining in either cortical
or medullar structures of kidneys in control and RA-treated mice.
However, active caspase-3 staining was detected in the corticome-
dullary junction of CP-treated animals (Fig. 4C). The antiapoptotic
activity of RA was evident by dose-dependent reduction in cleaved
caspase-3 expression (Fig. 4D, E, F and G), which coincided with
decreased expression of p53 and p-p53 in the kidneys (Fig. 4H).
4. Discussion
CP is still a frequently used chemotherapeutic agent, despite of
its frequent adverse effects, including nephrotoxicity (Miller et al.,
2010). The toxic effects of CP occurs through increased oxidative
stress, tubulointerstitial inflammation and apoptosis (Sahu et al.,
2011). Therefore, combinatorial strategies which target multiple
mechanisms, including suppression of free radical production,
Fig. 2. 4-Hydroxynonenal (4-HNE) expression in the corticomedullary junction of mice kidneys. Mice treated with vehicle (A), RA 5 mg/kg (B), CP (C), CP + RA 1 mg/kg (D),
CP + RA 2 mg/kg (E) and CP + RA 5 mg/kg (F). Arrows show 4-HNE immunopositive cells. Representative results from 5 similarly treated mice. Immunohistochemistry
staining, original magnification 400. Measurement of the intensity of 4-HNE immunostaining (G). Western blot analysis of cytochrome P450 2E1 (CYP2E1) and heme
oxygenase-1 (HO-1) expression in mice kidney lysates (H). Each value represents the mean ± SD for 5 mice. Means sharing the same letter are not significantly different from
each other (P< 0.05).
324 R. Domitrovic
´et al./ Food and Chemical Toxicology 66 (2014) 321–328
anti-inflammatory and cytoprotective activities in the kidney, may
represent an optimal strategy for the prevention of CP-mediated
nephrotoxicity (Sahu et al., 2013). Based on antioxidant, anti-
inflammatory and antiapoptotic properties of RA, the present study
was undertaken to examine the protective effects of RA against CP-
induced kidney injury. The results of the current study showed that
RA may ameliorate tubular damage in CP-intoxicated animals,
which was achieved through the reduction of oxidative stress,
inflammation and apoptosis in the kidneys.
In the current study, CP administration resulted in a severe
nephropathy, accompanied by impaired histological features of
the kidneys. Body weight loss was used as a measure of CP-induced
toxicity (Leonetti et al., 2003). Increased creatinine and BUN in CP-
treated animals indicated reduction in the glomerular filtration
rate (Coca and Parikh, 2008). Similarly, increased relative kidney
weight was attributed to urine retention due to casts-induced
tubular obstruction (Schrier et al., 2004). However, post-treatment
with RA markedly ameliorated these pathological changes.
Oxidative stress plays a key role in the pathophysiology of
CP-mediated kidney injury (Chirino et al., 2008b; Matsushima
et al., 1998). CP induces the overproduction of free radicals,
including superoxide anion ðO
2
Þand hydroxyl radical ð
OHÞ(Shi-
no et al., 2003; Davis et al., 2001). Free radical scavengers prevent
acute renal failure through attenuation of tubular damage, en-
hanced regenerative response of tubular cells and preservation
of renal blood flow (Matsushima et al., 1998). As shown in
Fig. 2, renal oxidative stress induced by CP administration, dem-
onstrated as 4-HNE formation, was associated with kidney
parenchimal cells. This is in agreement with similar findings by
other authors, which demonstrated increased lipid peroxidation
in mice and rat kidneys and proximal tubular cells in vitro (Tanabe
et al., 2012; Pan et al., 2009; Chirino et al., 2008a). The increase in
4-HNE formation was subsequently decreased by RA, suggesting
the improvement in antioxidant defense system. Additionally,
RA ameliorated the increase in CYP2E1 expression. The catalytic
activity of CYP family of enzymes is associated with increased
production of free radicals and deterioration of antioxidant de-
fense (Kusirisin et al., 2009). Moreover, the interaction of CYP2E1
by CP is considered as the central pathway by which CP promotes
oxidative stress in kidneys (Liu and Baliga, 2003). In turn, CYP2E1
Fig. 3. Nuclear factor kappaB (NF-
j
B) cellular localization in the corticomedullary junction of mice kidneys. Mice treated with vehicle (A), RA 5 mg/kg (B), CP (C), CP + RA
1 mg/kg (D), CP + RA 2 mg/kg (E) and CP + RA 5 mg/kg (F). Arrows show NF
j
B immunopositive nuclei. Representative results from 5 similarly treated mice.
Immunohistochemistry staining, original magnification 400. Measurement of the intensity of NF-
j
B immunostaining (G). Western blot analysis of tumor necrosis
factor-alpha (TNF-
a
) expression in kidney lysates (H). Each value represents the mean ± SD for 5 mice. Each value represents the mean ± SD for 5 mice. Means sharing the
same letter are not significantly different from each other (P< 0.05).
R. Domitrovic
´et al./ Food and Chemical Toxicology 66 (2014) 321–328 325
inhibitors were shown to reduce free radical production and ame-
liorate tubular cell cytotoxicity (Liu et al., 2002). The increase in
expression of CYP2E1 in the kidneys of CP-treated animals ob-
served in this study is in agreement with similar findings by
Wu et al. (2011). Nevertheless, Liu and Baliga (2003) reported de-
creased levels of CYP2E1 in the kidneys of CP-treated mice, how-
ever, this could be attributed to a strain-specific differences in
renal expression of CYP isoforms (Muller et al., 2007). Notably,
the inhibition of renal CYP2E1 expression by RA agree with CYP
inhibiting properties of natural antioxidant compounds observed
in various cell types and tissues (Wu et al., 2011; Kimura et al.,
2010; Kusirisin et al., 2009; Wang et al., 2006). The increase in
CYP2E1 expression in the kidneys of CP-treated mice observed
in the current study was accompanied by the HO-1 overexpres-
sion, suggesting that the induction of CYP2E1 by CP resulted in
the mobilization of this cytoprotective enzyme. Nevertheless, RA
acted as an inhibitor of both CYP2E1 and HO-1 expression, sug-
gesting that the decrease in CYP2E1 expression plays an impor-
tant role in the nephroprotective activity of RA.
Inflammation plays an important role in the pathogenesis of CP
nephrotoxicity. It has been suggested that oxidative stress causes
local inflammation through up-regulation of pro-inflammatory
cytokines, including TNF-
a
(Hassan et al., 2012). Administration
of nephrotoxic doses of CP frequently results in the activation of
the NF-
j
B signaling pathway and increased expression of pro-
inflammatory mediators in renal parenchimal cells both in vivo
and in vitro (Jia et al., 2011; Pan et al., 2009; Ramesh and Reeves,
2004). Consequently, NF-
j
B inhibitors have shown protective
activity against CP-induced nephrotoxicity (Francescato et al.,
2007) and the suppression of pro-inflammatory mediators resulted
in reduced kidney tubular damage. Most recently, Amin et al.
(2012) showed that CP-mediated inflammation in rats was amelio-
rated by Ginkgo biloba extract through the inhibition of the NF-
j
B
pathway (Amin et al., 2012). Similarly, the inhibition of TNF-
a
by
salycilates resulted in decreased CP-mediated nephrotoxicity (Ra-
mesh and Reeves, 2004). In the current study, administration of
RA to CP-treated mice reduced NF-
j
B p65 nuclear translocation
in proximal tubular cells and expression of TNF-
a
, indicating the
Fig. 4. Cleaved caspase-3 expression in the corticomedullary junction of mice kidneys. Mice treated with vehicle (A), RA 5 mg/kg (B), CP (C), CP + RA 1 mg/kg (D),CP+RA
2 mg/kg (E) and CP + RA 5 mg/kg (F). Arrows show cleaved caspase-3 positive cells. Representative results from 5 similarly treated mice. Immunohistochemistry staining,
original magnification 400. Measurement of the intensity of cleaved caspase-3 immunostaining (G). Western blot analysis of p53 and p-p53 (Ser15) expression in kidney
lysates (H). Each value represents the mean ± SD for 5 mice. Each value represents the mean ± SD for 5 mice. Means sharing the same letter are not significantly different from
each other (P< 0.05).
326 R. Domitrovic
´et al./ Food and Chemical Toxicology 66 (2014) 321–328
anti-inflammatory effect of RA, which is in agreement with similar
findings in CCl
4
-induced liver injury (Domitrovic
´et al., 2013). We
hypothesize that inflammatory response is the result of oxidative
stress which occurs due to cisplatin metabolisation. The ability of
RA to abrogate the induction of the NF-
j
B pathway suggests its po-
tential use as an anti-inflammatory agent in CP-induced kidney
injury.
Reactive oxygen species were also suggested as an initiator of
apoptotic cell death triggered by CP (Kannan and Jain, 2000). Apop-
tosis of cancer cells is considered as the key mechanism of CP che-
motherapeutic activity (Bragado et al., 2007). Nevertheless, the
same mechanism seems to be involved in the development of kid-
ney injury. Thus, caspase-3, the executive cell death protease, has
been activated by CP treatment in renal proximal tubular cells
(Kaushal et al., 2001) and rat kidneys (Park et al., 2012). The tumor
suppressor p53, which plays an essential role in the induction of
apoptosis (Amaral et al., 2010; Shen and White, 2001), is rapidly
up-regulated following CP treatment and the inhibition of p53
can inhibit CP-induced apoptosis (Wei et al., 2007). p53 stability
and transcriptional activity are modulated by post-translational
modifications including phosphorylation (Sakaguchi et al., 1998).
Phosphorylation at N-terminal Ser15 resulted in reduced DNA re-
pair, enhanced DNA-binding activity and the induction of apopto-
sis (Offer et al., 2002). Previously, Pabla et al. (2008) and Jiang et al.
(2004) suggested a role for p-p53 (Ser15) in CP-induced renal cell
apoptosis in vitro and in vivo. Consistently with previous findings,
in the present study, the expression of p53, p-p53 (Ser15) and
cleaved caspase-3 increased in the kidneys of CP-treated mice. Or-
ally administered RA reduced the overexpression of p53, p-p53 and
cleaved caspase-3, suggesting the inhibition of tubular apoptosis.
In conclusion, the results of the current study suggest that the
suppression of oxidative stress, inflammatory response and apop-
totic cell death by RA could be the effective strategy for the treat-
ment of CP-induced kidney injury. However, controlled clinical
trials are necessary to confirm the therapeutic activity of RA in
the patients receiving CP chemotherapy.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Transparency Document
The Transparency Document associated with this article can be
found in the online version.
Acknowledgements
This research was supported by grants from Ministry of Science,
Education and Sport, Republic of Croatia (project 062-0000000-
3554).
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