Pharmacological Research 120 (2017) 10–22

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Pharmacological Research
journal homepage: www.elsevier.com/locate/yphrs

Vinpocetine reduces diclofenac-induced acute kidney injury through

inhibition of oxidative stress, apoptosis, cytokine production, and
NF-␬B activation in mice
Victor Fattori a , Sergio M. Borghi a , Carla F.S. Guazelli a , Andressa C. Giroldo a ,
Jefferson Crespigio b , Allan J.C. Bussmann c , Letícia Coelho-Silva a , Natasha G. Ludwig b ,
Tânia L. Mazzuco b , Rubia Casagrande d , Waldiceu A. Verri Jr. a,∗
a
Departamento de Ciências Patológicas, Centro de Ciências Biológicas, Universidade Estadual de Londrina, 86057-970 Londrina, Paraná, Brazil
b
Departamento de Medicina, Divisão de Endocrinologia, Centro de Ciências da Saúde, Universidade Estadual de Londrina, 86038-350 Londrina, Paraná,
Brazil
c
Laboratório de Anatomia Patológica, Centro de Ciências de Saúde, Universidade Estadual de Londrina, 86038-350 Londrina, Paraná, Brazil
d
Departamento de Ciências Farmacêuticas, Centro de Ciências da Saúde, Universidade Estadual de Londrina, 86038-350 Londrina, Paraná, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Acute kidney injury (AKI) represents a complex clinical condition associated with significant morbidity
Received 13 July 2016 and mortality. Approximately, 19–33% AKI episodes in hospitalized patients are related to drug-induced
Received in revised form nephrotoxicity. Although, considered safe, non-steroidal anti-inflammatory drugs such as diclofenac have
23 December 2016
received special attention in the past years due to the potential risk of renal damage. Vinpocetine is a
Accepted 28 December 2016
nootropic drug known to have anti-inflammatory properties. In this study, we investigated the effect and
Available online 14 March 2017
mechanisms of vinpocetine in a model of diclofenac-induced AKI. We observed that diclofenac increased
proteinuria and blood urea, creatinine, and oxidative stress levels 24 h after its administration. In renal tis-
Keywords:
Diclofenac
sue, diclofenac also increased oxidative stress and induced morphological changes consistent with renal
Acute kidney injury damage. Moreover, diclofenac induced kidney cells apoptosis, up-regulated proinflammatory cytokines,
NF-␬B and induced the activation of NF-␬B in renal tissue. On the other hand, vinpocetine reduced diclofenac-
Vinpocetine induced blood urea and creatinine. In the kidneys, vinpocetine inhibited diclofenac-induced oxidative
Proteinuria stress, morphological changes, apoptosis, cytokine production, and NF-␬B activation. To our knowledge,
Reactive oxygen species this is the first study demonstrating that diclofenac-induced AKI increases NF-␬B activation, and that vin-
pocetine reduces the nephrotoxic effects of diclofenac. Therefore, vinpocetine is a promising molecule
for the treatment of diclofenac-induced AKI.
© 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) represent the
most popular class of drugs currently prescribed and used world-
wide, reaching over than 70 million prescriptions each year in the
USA. This high number of prescription correlates with many cases
of NSAIDs intoxication in humans registered by The Association
of the Poison Control Centers’ National Poison Data System (AAPCC
NPDS) in the last years [1,2]. NSAIDs acute and chronic intoxications
∗ Corresponding author at: Department of Pathology, Londrina State University,
Rodovia Celso Garcia Cid Km 480 PR 445, CEP: 86.057-970, Postal Address 10.011,
Londrina, Paraná, Brazil. Tel.: +55 4333714979; fax: +55 4333714387.
E-mail addresses: waverri@uel.br, waldiceujr@yahoo.com.br (W.A. Verri Jr.).
are linked to increasing morbidity and mortality and have a direct
impact on public health costs [1,2]. NSAIDs are extremely efficient
as analgesic, anti-inflammatory, and antipyretic agents, and are
used routinely to treat varied forms of acute pain and inflammation
[2,3]. However, the indiscriminate use of NSAIDs represents a chal-
lenge to the public health due to their countless adverse effects.
Major adverse effects of NSAIDs include gastrointestinal damage
and acute kidney injury (AKI), and in more severe cases, increased
risk of serious cardiovascular dysfunctions [2,4]. The main mecha-
nisms of action of NSAIDs involve the inhibition of cyclooxygenase
enzymes isoforms 1 (COX-1) and 2 (COX-2), thereby preventing
the synthesis of their downstream effector prostaglandins and
thromboxane A2 [2]. The two main types of acute renal failure
induced by NSAIDs are hemodynamically-mediated nephritis and

http://dx.doi.org/10.1016/j.phrs.2016.12.039
1043-6618/© 2017 Elsevier Ltd. All rights reserved.
 V. Fattori et al. / Pharmacological Research 120 (2017) 10–22
acute interstitial nephritis, both associated with the low levels of
prostaglandins due to the COX inhibition by NSAIDs [2,5].
Diclofenac (2-[(2,6-diclorophenyl)amino]phenyl acetate), a
non-selective NSAID, is a phenyl acetic acid derivative that presents
analgesic, antipyretic, and anti-inflammatory activities [6]. In
chronic and acute pain conditions, diclofenac exhibits high effi-
® ®
cacy and good tolerability [3]. Commercial diclofenac (Voltaren )
is the most common NSAID prescribed for myalgia, osteoarthri-
tis, rheumatoid arthritis, and other rheumatic disorders [7–9].
Similarly to other NSAIDs, diclofenac adverse effects include
gastrointestinal disorders, liver injury, hypersensitive reactions,
hematotoxicity and AKI [6,10,11]. Prostaglandins maintain normal
glomerular filtration rate (GFR) and the inhibition of their synthe-
sis by diclofenac leads to abnormal renal function, the decline in
glomerular hydraulic pressure, and ultimately AKI [2]. The direct
effect of diclofenac-induced renal injury depends on targeting kid-
ney’s mitochondria leading to the production of reactive oxygen
species (ROS) causing DNA lesions and apoptosis [8,12]. This compi-
lation of data raises the concern about the cytotoxic effects related
to abuse and/or overdose of NSAIDs such as diclofenac.
Vinpocetine (ethyl apovincamine-22-oate) is a synthetic com-
pound belonging to the class of nootropic drugs. Vinpocetine is
a derivative of vincamine molecule, an alkaloid extracted from
the leaves of Vinca minor [13,14]. The clinical use of vinpocetine
includes the management of cerebrovascular disorders such as
stroke and cerebral hemorrhage as well as cognitive dysfunctions
[15,16]. Growing body of evidence also point out to the anti-
inflammatory effects of vinpocetine [13,14,17,18]. Vinpocetine acts
as an inhibitor of phosphodiesterase-1 (PDE-1) activity, regulating
the levels of cyclic adenosine monophosphate (cAMP) and cyclic
guanosine monophosphate (cGMP) [14]. Nevertheless, vinpocetine
inhibits NF-␬B activation and its downstream proinflammatory
mediators such as TNF-␣ and IL-1␤ [13,14,17,18] independently
of its PDE inhibition-related mechanisms [14,19]. Vinpocetine also
presents antioxidant effects that account for its anti-inflammatory
actions [13,17].
The mechanisms of diclofenac-induced AKI need further inves-
tigation for a better understanding of its physiopathology and
therapeutic profile. Glucocorticoids are one of the choices for AKI
management [20,21]. Considering that this class of drugs acts by
inhibiting NF-␬B by varied mechanisms [22], it is conceivable that
NF-␬B inhibitors could present beneficial effects in diclofenac-
induced AKI. Given that glucocorticoids treatment induces severe
side effects, this study investigated the efficacy of vinpocetine in
diclofenac-induced AKI.
2. Materials and methods
2.1. Animals
The experiments were performed on male one-month-old Swiss
mice, weighing between 20 and 25 g from Londrina State Uni-
versity, Paraná State, Brazil. Mice were housed in standard clear
plastic cages with free access to water and food, light/dark cycle of
12/12 h and controlled temperature. Mice were maintained in the
vivarium of the Department of General Pathology, Londrina State
University for at least two days before the experiments. Mice were
used only once and were acclimatized to the testing room at least
1 h before the experiments, which was conducted during the light
cycle. At the end of experiments described in the following items,
mice were anesthetized with isoflurane 3% (Abbot, Abbott Park, IL,
USA) by inhalation overdose, and terminally euthanized by cervical
dislocation followed by decapitation. The Londrina State Univer-
sity Ethics Committee on Animal Research and Welfare approved
animal care and handling procedures under the process number
11
27500.2014.40. All efforts were made to minimize the number of
animals used and their suffering.
2.2. Drugs
Materials were obtained from the sources as follows: sodium
diclofenac (Neutaren ) was purchased from Novartis Biociências
®
S. A. (São Paulo, SP, Brazil); vinpocetine (Vicog ) was purchased
from Marjan Indústria & Comércio Ltda. (São Paulo, SP, Brazil).
Sodium diclofenac and vinpocetine were diluted in saline imme-
diately before per oral (p.o.) administration.
2.3. Experimental design
In the first set of experiments, mice received toxic doses of
diclofenac (200 and 300 mg/kg, 100 ␮l, p.o.) administration and
24 h after, blood levels of urea, creatinine, superoxide anion, and
lipid peroxidation were measured to determine the adequate
nephrotoxic dose. Afterward, mice were treated with diclofenac
(200 mg/kg, 100 ␮l, p.o.) 30 min prior to the treatment with vin-
pocetine (0.3, 1, or 3 mg/kg, 100 ␮l, p.o.) or vehicle (sterile saline,
100 ␮l, p.o.). Twenty-four hours after diclofenac administration,
urea and creatinine blood levels were evaluated to determine the
best dose of vinpocetine. The dose of 3 mg/kg of vinpocetine was
selected for the following experiments: blood ALT and AST lev-
els; proteinuria; histopathological features; cytokine production
(ELISA); NF-␬B (immunohistochemistry and ELISA); COX-2 and c-
Myc immunohistochemistry. Diclofenac and vinpocetine doses, as
well as the time of samples collection, were based on previous stud-
ies [8,13,17]. All analyses in renal tissue collected described above
were performed using the entire organ.
2.4. Renal function tests
Blood samples were collected by cardiac puncture 24 h after
the diclofenac administration and added into microtubes contain-
ing anticoagulant (EDTA, 5000 IU/ml, Sigma Chemical Co., St. Louis,
MO, USA). The plasma was separated by centrifugation (200 × g,
10 min, and 4 ◦ C). Plasma samples were processed according to
the manufacturer’s instructions (Labtest Diagnóstico S.A., Lagoa
Santa, MG, Brazil) to evaluate urea and creatinine levels as indi-
cators of nephrotoxicity. Results are presented as milligram per
deciliter of plasma urea or creatinine. Urine was collected 24 h after
the diclofenac administration by the application of gentle pressure
over the bladder and added into microtubes for proteinuria analysis
using Lowry’s method.
2.5. Enzymatic markers of liver injury
Blood samples were collected by cardiac puncture 24 h after
diclofenac administration or 10 h after acetaminophen adminis-
tration (650 mg/kg, i.p.), and added into microtubes containing
anticoagulant (EDTA, 5000 IU/ml, Sigma Chemical Co., St. Louis, MO,
USA). The plasma was separated by centrifugation (200 × g, 10 min,
and 4 ◦ C). Plasma samples were processed according to the manu-
facturer’s instructions (Labtest Diagnóstico S.A., Lagoa Santa, MG,
Brazil) to evaluate ALT and AST levels as indicators of hepatotoxic-
ity. Results are presented as U/l of plasma ALT or AST.
2.6. Ferric-reducing ability potential and free radical scavenging
ability assays
The kidney was collected 24 h after the diclofenac adminis-
tration, immediately homogenized with 500 ␮l of 1.15% KCl and
centrifuged (10 min, 200 × g, and 4 ◦ C). The ability of the samples to
resist oxidative damage was determined as ferric-reducing ability
12 V. Fattori et al. / Pharmacological Research 120 (2017) 10–22
potential (FRAP) and free-radical scavenging ability (ABTS) assays
[23]. For the FRAP assay, 15 ␮l of supernatant was mixed with 45 ␮l
of deionized water and 200 ␮l of freshly prepared FRAP reagent
(Sigma Chemical Co., St. Louis, MO, USA). The reaction mixture was
incubated at 37 ◦ C for 30 min, and the absorbance was measured
at 595 nm. For the ABTS assay, the ABTS solution (Sigma Chemical
Co., St. Louis, MO, USA) was diluted with phosphate buffer saline at
pH 7.4 to an absorbance of 0.80 at 730 nm. Then, 200 ␮l of diluted
ABTS solution was mixed with 15 ␮l of the supernatant. After 6 min,
the absorbance was measured at 730 nm. The results were equated
against a Trolox standard curve (1.5–30 ␮mol/l, final concentra-
tions) (Sigma Chemical Co., St. Louis, MO, USA). The protein levels
in the samples were used for data normalization, and the results
are expressed as nmol of Trolox (Sigma Chemical Co., St. Louis, MO,
USA) equivalent per milligrams of protein in both assays.
2.7. Reduced glutathione (GSH) assay
The levels of GSH in renal tissue samples were determined using
a spectrophotometric method. Kidneys were collected 24 h after
the diclofenac administration and processed following a previously
described protocol [24]. The results were presented as mmols of
GSH per milligrams of protein (Sigma Chemical Co., St. Louis, MO,
USA).
2.8. Nitroblue tetrazolium reduction assay
For nitroblue tetrazolium (NBT) (Amresco, Solon, OH, USA)
reduction assay, blood and kidney tissue were collected 24 h after
the diclofenac administration as described previously [25]. Briefly,
the kidney was homogenized with 500 ␮l of 1.15% KCl, and aliquots
of 50 ␮l of homogenate were transferred to a 96-well plate, fol-
lowed by the addition of 100 ␮l of NBT solution, and maintained at
37 ◦ C in a warm bath for 5 min. For blood analysis, samples were
collected by cardiac puncture 24 h after the diclofenac administra-
tion, and added into microtubes containing anticoagulant (EDTA,
5000 IU/ml, Sigma Chemical Co., St. Louis, MO, USA). The plasma
was separated by centrifugation (200 × g, 10 min, and 4 ◦ C). The
supernatant was removed, and the formazan precipitated was then
solubilized by adding 120 ␮l of 2 M KOH (Sigma Chemical Co., St.
Louis, MO, USA) and 140 ␮l of dimethyl sulfoxide (DMSO, Sigma
Chemical Co., St. Louis, MO, USA). The optical density was measured
at 600 nm using a microplate spectrophotometer reader (Multi-
skan GO Microplate Spectrophotometer, Thermo Scientific, Vantaa,
Finland). The protein levels in the samples were used for data nor-
malization and the results were presented as NBT reduction (optical
density [OD] per milligram of protein or dl of plasma).
2.9. Lipid peroxidation assay
The levels of thiobarbituric acid (TBA)-reactive substances
(TBARS), primarily malondialdehyde (MDA), were quantified
spectrophotometrically using an adapted method as previously
described [26]. Tissue homogenate or plasma reacted with TBA
to form MDA-(TBA)2 complexes, and the proteins were pre-
cipitated with trichloroacetic acid. Samples were incubated for
15 min in a boiling water bath and transferred to an ice
bath. MDA, an intermediate product of lipid peroxidation, was
determined by the difference between absorbance at 535 and
572 nm using a microplate spectrophotometer reader (Multi-
skan GO Microplate Spectrophotometer, Thermo Scientific, Vantaa,
Finland). The results are presented as Lipid peroxidation (OD
A535-A572/mg of protein or dl of plasma).
2.10. Histopathological analysis
For renal histopathological analysis, kidneys were collected
24 h after diclofenac administration, fixed in 4% paraformalde-
hyde and dehydrated in a graded series of ethanol solutions for
paraffin embedding. Five ␮m sections of cortical and medullary
zones were prepared and stained with hematoxylin & eosin (H&E)
and periodic acid-Schiff (PAS). H&E and PAS stained sections were
examined at 40x magnification and scored by a pathologist using
light microscopy, and a semi-quantitative evaluation of renal tissue
damage was estimated in 10 high power fields (HPF) as described
previously with modifications [27]. The total histopathological
score was assayed considering morphological alterations in the:
1) glomerulus; 2) cortical and medullar loss of brush border; 3)
vacuolation; and 4) degeneration of epithelial tubular cells. The
degree of renal damage was classified on a scale of 0–3 (0: nor-
mal, 1: mild, 2: moderate, 3: severe). Results are presented as the
total histopathological score. The highest possible total score was
12.
2.11. Cytokine production
For cytokine production assessment, samples of renal tissue
were collected 24 h after diclofenac administration. The samples
were homogenized in 500 ␮l of the appropriate buffer containing
protease inhibitors. Cytokine levels were determined as described
previously by an enzyme-linked immunosorbent assay (ELISA)
using eBioscience kits (eBioscience, San Diego, CA, USA) accord-
ingly with manufacturer instructions. The results were expressed
as picograms (pg) of cytokine per 100 mg of tissue.
2.12. Immunohistochemistry
For immunohistochemical reaction, paraffin embedded kid-
ney sections at 4 ␮m were dewaxed, hydrated and heat-treated
in 1 mM EDTA buffer for antigenic unmasking on microwave at
95.8 ◦ C for 20 min. Sections were incubated overnight at room
temperature with rabbit polyclonal anti-NF-␬B p65 (Bioss, Sacra-
ment, CA, USA; catalog #bs-0456R, lot #999980W, 1:250), rabbit
polyclonal anti-c-Myc (Santa Cruz Biotechnology, Santa Cruz, CA,
USA; catalog #sc-40, 1:100. Prof. Isabelle Bourdeau from Univer-
sité de Montréal, Québec, Canada, gently donated this reagent),
or rabbit polyclonal anti-COX-2 (Bioss, Sacrament, CA, USA; cat-
alog #bs-0732R, 1:200. Prof. Isabelle Bourdeau from Université de
Montréal, Québec, Canada, gently donated this reagent) followed by
conjugation to the secondary antibody mouse/Rabbit ImmunoDe-
tector HRP/DAB (BioSB, Santa Barbara, CA, USA; catalog #BSB0005,
lot #0005GFI18). Immunohistochemically staining was performed
using the polymer-peroxidase based method, followed by develop-
ment with diaminobenzidine (Sigma Chemical Co., St. Louis, MO,
USA). A methodical negative control went through the first step of
the procedure (incubation with the vehicle instead of the primary
antibody). Histological slides were analyzed under the optic micro-
scope to identify areas that best represented NF-␬B p65, c-Myc, or
COX-2 immunostaining, whereas brownish color was considered
to be evidence of a positive expression. From each sample, pho-
tomicrographs of 800 × 600 pixels were obtained from 4x and 40x
magnification fields, using a Samsung camera (three images/slice),
adapted in the optic microscope. Digitally acquired images were
analyzed in the ImageJ 1.44 software for Windows (Java image
software in public domain: http://rsb.info.nih.gov/ij/), using the
threshold tool with color-based selection for positive staining. Rou-
tines for image analysis were defined in ImageJ macro language and
performed on RGB images without further treatment. The number
of pixels in the selected color range was divided by the total number
of pixels in each field. Four slices per sample of kidney tissue were
 V. Fattori et al. / Pharmacological Research 120 (2017) 10–22 13

Fig. 1. Diclofenac increases plasma levels of urea, creatinine, superoxide anion production, and lipid peroxidation. Mice received the stimulus with diclofenac (200 or
300 mg/kg, p.o.) and blood was collected after 24 h for the determination of plasma urea (A), creatinine (B), superoxide anion production (C), and lipid peroxidation levels
(D). Results are expressed as mean ± SEM, n = 6 mice per group per experiment, two independent experiments (* p < 0.05 vs. control group, one ANOVA followed by Tukey’s
post hoc).

analyzed and data were averaged. Results were expressed by the software GraphPad Prism 6.01 (GraphPad Software Inc., La Jolla,
relation between the immunopositive area fraction per total area CA, USA). Statistical differences were considered significant when
fraction as the percentage (%) of NF-␬B, c-Myc, or COX-2 staining. p < 0.05.
Control and experimental kidneys were processed under the same
conditions.
3. Results

2.13. NF-B activation
The determination of NF-␬B activation in renal tissue was
performed accordingly with manufacturer instructions. The kid-
ney was collected 24 h after diclofenac administration and
homogenized in ice-cold lysis buffer (Cell Signaling Technol-
ogy, Beverly, MA, USA). The homogenates were centrifuged
(16,100 g × 10 min × 4 ◦ C) and the supernatants used to assess the
levels of total and phosphorylated NF-␬B p65 subunit by ELISA
using PathScan kits #7836 and #7834, respectively (Cell Signal-
ing Technology, Beverly, MA, USA). The results are presented as the
sample ratio (phospho-p65/total-p65) measured at 450 nm.
2.14. Statistical analysis
3.1. Diclofenac induces kidney injury and increases blood
oxidative stress
First, the diclofenac dose capable of inducing kidney injury
was determined considering previously described doses [8]. Mice
received the stimulus with diclofenac (200 or 300 mg/kg, p.o.) and
a blood sample was collected after 24 h for the evaluation of urea,
creatinine, superoxide anion production, and lipid peroxidation
(Fig. 1). Diclofenac at 200 and 300 mg/kg elevated plasma urea
(Fig. 1A), creatinine (Fig. 1B), superoxide anion production (Fig. 1C),
and lipid peroxidation (Fig. 1D) when compared to control group.
Given there were no difference between the doses in these four
parameters, the diclofenac at 200 mg/kg was chosen for the fol-
lowing experiments.

Results are presented as mean ± standard error means (SEM)
of measurements made on six mice in each group per experi-
ment, except for those in the histopathological score (12 mice
in each group per experiment). Results are representative of two
independent experiments. Data were analyzed using one-way
ANOVA followed by Tukey’s post hoc and for categorical variables,
Kruskal–Wallis followed by Dunn’s post hoc, using the statistical
3.2. Vinpocetine reduces diclofenac-induced increase of plasma
urea and creatinine and proteinuria
Next, the vinpocetine dose capable of reducing diclofenac-
induced increase in urea and creatinine plasma levels, and
proteinuria was evaluated (Fig. 2). Mice received the stimulus with
diclofenac (200 mg/kg, p.o.) and after 30 min were treated with vin-
14 V. Fattori et al. / Pharmacological Research 120 (2017) 10–22

Fig. 2. Vinpocetine reduces diclofenac-induced increase in plasma urea and creatinine and proteinuria. Mice received the stimulus with diclofenac (200 mg/kg, p.o.) and after
30 min were treated with vinpocetine (0.3, 1, or 3 mg/kg, p.o.). Blood was collected 24 h after the stimulus for the determination of urea (A), creatinine (B), AST (C), and ALT
(D) plasma levels. Urine was collected 24 h after the stimulus for determination of proteinuria (C). Mice also received diclofenac (200 mg/kg, p.o,), or vinpocetine (3 mg/kg,
p.o.), or acetaminophen (650 mg/kg, i.p.) without further treatment, for evaluation of AST (C), and ALT (D) plasma levels. Results are expressed as mean ± SEM, n = 6 mice per
group per experiment, two independent experiments (* p < 0.05 vs. control group; # p < 0.05 vs. 0 mg/kg group, ** p < 0.05 vs. all groups, one ANOVA followed by Tukey’s post
hoc).

pocetine (0.3, 1, or 3 mg/kg, p.o.). Blood sample and urine were
collected 24 h after diclofenac administration. Diclofenac increased
urea and creatinine plasma levels, which were reduced by vinpoce-
tine treatment in a dose-dependent manner (Fig. 2A, B). The doses
of 1 and 3 mg/kg reduced urea plasma levels (IC50 0.965 mg/kg)
whereas only the dose of 3 mg/kg reduced creatinine plasma lev-
els (IC50 2.005 mg/kg) (Fig. 2B). The dose of 3 mg/kg reduced 71%
of creatinine levels. Therefore, vinpocetine at 3 mg/kg was chosen
for the following experiments. Diclofenac also increased protein-
uria, which was reduced in vinpocetine-treated mice (Fig. 2C).
Furthermore, diclofenac did not change kidney fresh weight (data
not shown). Vinpocetine (3 mg/kg, p.o.) alone did not induce any
changes in these markers (Fig. 2A–C).
3.3. Diclofenac does not induce liver injury
The next step was to evaluate whether diclofenac induces
injury of organs other than the kidney. Mice received the stimu-
lus with diclofenac (200 mg/kg, p.o.) and after 30 min were treated
with vinpocetine (3 mg/kg, p.o.). Mice were also stimulated with
diclofenac (200 mg/kg, p.o.) or acetaminophen (650 mg/kg, i.p.)
without further treatment. Twenty-four or 10 h after diclofenac
and acetaminophen stimulus, respectively, a blood sample was
collected for the evaluation of ALT and AST levels. Diclofenac did
not increase the levels of AST (Fig. 2D) and ALT (Fig. 2E), whereas
the known hepatotoxic drug acetaminophen increased both AST
(Fig. 2D) and ALT (Fig. 2E) levels. Vinpocetine (3 mg/kg, p.o.) alone
did not induce any changes in these markers (Fig. 2D, E).
3.4. Vinpocetine inhibits diclofenac-induced kidney oxidative
stress
The effect of vinpocetine in diclofenac-induced oxidative stress
was evaluated by the antioxidant defense status (FRAP, ABTS,
and GSH assays), superoxide anion production, and lipid per-
oxidation (Fig. 3). Mice received the stimulus with diclofenac
(200 mg/kg, p.o.) and after 30 min were treated with vinpoce-
tine (3 mg/kg, p.o.). The kidney was collected 24 h after diclofenac
administration. Diclofenac reduced antioxidant defenses in renal
tissue as observed by reductions in FRAP (Fig. 3A) and ABTS
assays (Fig. 3B), and GSH levels (Fig. 3C). Additionally, superox-
ide anion production (Fig. 3D), and lipid peroxidation (Fig. 3E)
were increased in diclofenac-stimulated animals. Treatment with
vinpocetine reverted diclofenac-induced depletion of antioxidant
defenses (Fig. 3A–C) as well as inhibited superoxide anion produc-
tion (Fig. 3D), and lipid peroxidation (Fig. 3E). Vinpocetine (3 mg/kg,
p.o.) alone did not induce any changes in these markers.
3.5. Vinpocetine reduces diclofenac-induced kidney
histopathological changes
Histopathological changes were evaluated using H&E (A–D, J–M)
and PAS (E–I, N–Q) staining. Mice received the stimulus with
diclofenac (200 mg/kg, p.o.) and after 30 min were treated with
vinpocetine (3 mg/kg, p.o.). The kidney was collected 24 h after
diclofenac administration. Fig. 4A, E shows kidney from control
group mice presenting intact glomeruli, intact proximal and distal
convoluted tubules in the cortical region. On the other hand, kid-
neys from animals that received diclofenac (Fig. 4C, G) presented
 V. Fattori et al. / Pharmacological Research 120 (2017) 10–22 15

Fig. 3. Vinpocetine inhibits diclofenac-induced kidney oxidative stress. Mice received the stimulus with diclofenac (200 mg/kg, p.o.) and after 30 min were treated with
vinpocetine (3 mg/kg, p.o.). The kidney was collected 24 h after the stimulus for the determination of FRAP (A), ABTS (B), GSH (C), superoxide anion production (D), and lipid
peroxidation levels (E). Results are expressed as mean ± SEM, n = 6 mice per group per experiment, two independent experiments (* p < 0.05 vs. control group; # p < 0.05 vs.
0 mg/kg group, one ANOVA followed by Tukey’s post hoc).

irregular shape glomeruli and Bowman’s capsule damage as noted
by glomeruli deformation (Fig. 4G) and glomeruli tip lesions (as
observed by the merge between the Bowman’s capsule and the
epithelial tubular cells) (Fig. 4I). Importantly, these changes were
reverted by vinpocetine (Fig. 4H, I). Moreover, diclofenac induced
tubular dilatation accompanied by flattened epithelium (Fig. 4C,
G) as well as loss of brush borders in the cortical proximal con-
voluted tubules (Fig. 4G). Vinpocetine-treated mice showed mild
tubular cell vacuolation, pronounced reduction in the glomeruli
damage, tubular dilatation, and loss of the brush border (Fig. 4D, H,
and I). Of note, diclofenac did not induce morphological changes in
the macula densa. Diclofenac also induced morphological changes
in the medullary portion of the kidney (such as loss of the brush
border), which were reduced by vinpocetine treatment (Fig. 4J–Q).
Fig. 4J, N show normal kidney medullary portion. Fig. 4R shows
the total histopathological score of experimental groups. Vinpoce-
tine (3 mg/kg, p.o.) alone did not induce histopathological changes
(Fig. 4B, F, K, O, and R).
3.6. Vinpocetine reduces diclofenac-induced kidney cytokine
production
The effect of vinpocetine on diclofenac-induced kidney cytokine
production was evaluated. Mice received the stimulus with
diclofenac (200 mg/kg, p.o.) and after 30 min were treated with
vinpocetine (3 mg/kg, p.o.). Kidney was collected 24 h after
diclofenac administration. Diclofenac increased the production of
IL-1␤ (Fig. 5A), IL-6 (Fig. 5B), IFN-␥ (Fig. 5C), IL-33 (Fig. 5D),
and TNF-␣ (Fig. 5E) in the kidney when compared to control
group. On the other hand, vinpocetine treatment was able to
reduce diclofenac-induced proinflammatory cytokine production
(Fig. 5A–E). Vinpocetine (3 mg/kg, p.o.) alone did not induce any
changes in cytokine levels.
3.7. Vinpocetine inhibits diclofenac-induced kidney NF-B
activation
Next, the role of NF-␬B in diclofenac-induced AKI was investi-
gated. NF-␬B activation was determined by immunohistochemistry
(NF-␬B p65 protein expression) (Fig. 6) and ELISA (phosphorylated-
p65/total-p65 ratio) (Fig. 7) assays. Mice received the stimulus
with diclofenac (200 mg/kg, p.o.) and after 30 min were treated
with vinpocetine (3 mg/kg, p.o.). The kidney was collected 24 h
after diclofenac administration. Diclofenac increased kidney NF-
␬B p65 immunohistochemical staining (Fig. 6C, G, and I) as well
as increased ELISA phosphorylated p65/total-p65 ratio (Fig. 7)
when compared to control group. Given the elevation in phospho-
rylated p65/total-p65 ratio, diclofenac not only increases NF-␬B
expression as well as increases its activation in renal tissue.
Vinpocetine treatment reduced diclofenac-induced NF-␬B p65
immunohistochemical staining (Fig. 6D, H, and I) and phosphory-
lated p65/total-p65 ELISA ratio (Fig. 7). Vinpocetine (3 mg/kg, p.o.)
alone did not induce any changes in these markers (Figs. 6B, F, and
I; and 7).
3.8. Vinpocetine inhibits diclofenac-induced kidney apoptosis
The effect of vinpocetine on diclofenac-induced apoptosis
was then investigated by immunohistochemistry (c-Myc protein
expression) (Fig. 8). Mice received the stimulus with diclofenac
(200 mg/kg, p.o.) and after 30 min were treated with vinpoce-
tine (3 mg/kg, p.o.). The kidney was collected 24 h after diclofenac
16 V. Fattori et al. / Pharmacological Research 120 (2017) 10–22

Fig. 4. Vinpocetine ameliorates diclofenac-induced kidney histopathological changes. Mice received the stimulus with diclofenac (200 mg/kg, p.o.) and after 30 min were
treated with vinpocetine (3 mg/kg, p.o.). The kidney was collected 24 h after the stimulus for the evaluation of histopathological changes using HE (A–D, J–M) and PAS (E–I,
N–Q) staining. Thick arrows indicate glomerulus, circle, and star indicate glomerulus deformation and tip lesion, respectively; thin arrows indicate loss of brush borders; arrow
heads indicate intact brush borders; crosses and asterisks indicate tubular dilatation and tubular cells vacuolation, respectively. PT: proximal convoluted tubules; DT: distal
convoluted tubules; MD: macula densa. Original magnifications 40x and 100x (only panel I). Total histopathological score for renal damage is presented in panel R. Results
are expressed as mean ± SEM, n = 12 mice per group per experiment, two independent experiments (* p < 0.05 vs. control group; # p < 0.05 vs. 0 mg/kg group, Kruskal–Wallis
followed by Dunn’s post hoc).

administration. Diclofenac increased kidney c-Myc immunohisto-
chemical staining (Fig. 8C, G, and I) when compared to control
group. Vinpocetine treatment reduced diclofenac-induced c-Myc
immunohistochemical staining (Fig. 8D, H, and I). Vinpocetine
(3 mg/kg, p.o.) alone did not induce any change in this marker
(Fig. 8B, F, and I).
3.9. Diclofenac does not change kidney COX-2 expression
Last, the effect of diclofenac in kidney COX-2 expression was
evaluated. Mice received the stimulus with diclofenac (200 mg/kg,
p.o.) and after 30 min were treated with vinpocetine (3 mg/kg,
p.o.). The kidney was collected 24 h after diclofenac administra-
tion. Neither diclofenac nor vinpocetine changed kidney COX-2
immunohistochemical staining (Fig. 9) when compared to control
group.
4. Discussion
In this study, vinpocetine treatment reduced diclofenac-induced
AKI as observed by inhibition of plasma urea and creatinine levels.
In addition, vinpocetine reduced diclofenac-induced histopatho-
logical changes (in cortical and medulla portions) and kidney
damage by a mechanism related to inhibition of cytokine pro-
duction, oxidative stress, NF-␬B activation, and apoptosis. To our
knowledge, these data show for the first time that diclofenac-
induced AKI increases NF-␬B activation, and that vinpocetine
reduces the nephrotoxic effects of diclofenac.
The nephrotoxic effect of diclofenac was previously demon-
strated in diverse experimental models using both in vitro and in
vivo analysis [8,12,28,29]. The attention of the scientific commu-
nity about diclofenac deleterious effects to renal system gained
strength after the publication of reports describing the threatening
effects of diclofenac to several species of wild vultures world-
wide [30–34]. For instance, diclofenac residues in the blood of
Indian vultures Gyps bengalensis correlates with the decline in
their population [30,33]. Other species of vultures Gyps endan-
gered in Africa are equally susceptible to the noxious effects of
diclofenac [32]. Accordingly, the decline in Gyps bengalensis vul-
tures populations in Nepal and India reduced after the withdrawal
of veterinary diclofenac from the market [31]. Regarding human
intoxication, diclofenac also induces nephrotoxicity in newborns
due to diclofenac ingestion by mothers [35], adults without other
risk factors than diclofenac administration [36,37], children [38],
and elderly patients [36,39,40]. In the first case, three newborns
presented renal failure and one of them died [35]. In addition,
ingestion of 2.5 g of diclofenac caused transient acute renal fail-
ure with recovery in 2 days [41], and a patient that used diclofenac
for chronic muscular pain and knee arthropathy presented acute
tubular injury due to medication [37]. In an environmental con-
 V. Fattori et al. / Pharmacological Research 120 (2017) 10–22 17

Fig. 5. Vinpocetine reduces diclofenac-induced kidney cytokine production. Mice received the stimulus with diclofenac (200 mg/kg, p.o.) and after 30 min were treated with
vinpocetine (3 mg/kg, p.o.). The kidney was collected 24 h after the stimulus to assess the tissue levels of IL-1␤ (A), IL-6 (B), IFN-␥ (C), IL-33 (D), and TNF-␣ (E). Results are
expressed as mean ± SEM, n = 6 mice per group per experiment, two independent experiments (* p < 0.05 vs. control group; # p < 0.05 vs. 0 mg/kg group, one ANOVA followed
by Tukey’s post hoc).

text, diclofenac that is eliminated in urine reaches affluent rivers
and causes kidney and liver injuries in fishes [42,43]. Therefore,
these set of data point out to diclofenac as a potential inductor of
AKI and raise concern about the ecologic impact of the ingestion of
diclofenac despite its clinical efficacy. It is also noteworthy to men-
tion that diclofenac is related to a series of severe complications
apart from AKI [44–46].
Clinical AKI is often diagnosed by an increase in blood urea
nitrogen and creatinine, which characterize a critical risk factor for
mortality in hospitalized and/or in intensive care unit patients [47].
Additionally, increase in blood oxidative stress is commonly found
in AKI [48,49]. In the present experimental condition, diclofenac
increased blood creatinine, urea, and oxidative stress levels 24 h
after the stimulus, which corroborates the toxic effect of this
molecule. Diclofenac induced deformation and formation of tip
lesions in the glomeruli, which are indicative of renal damage [50].
In addition, diclofenac induced tubular dilatation and loss of brush
border as observed by H&E and PAS staining data. Inhibition of
prostaglandin synthesis leads to the loss in glomeruli basic function
impairing GFR and renal metabolism [51,52]. In this regard, distur-
bances in GFR could be a possible mechanism of diclofenac-induced
AKI given that increased plasma urea and creatinine levels, and
oxidative stress (observed in this study) correlates to the decline
in GFR [51,52]. Tubular dilatation occurs as a result of increased
intracellular osmolality after autolysis shaping as a larger vesi-
cle or even as several smaller vesicles. In accordance with this
data, clear vacuoles in renal tubule epithelium are often located
in outer cortical tubules and commonly observed in the proximal
convoluted tubule epithelium [53,54]. Despite prostaglandin influ-
ence on GFR and NF-␬B induction of COX-2 expression in most
tissues, in this work COX-2 expression was not altered. A possi-
ble explanation is that kidney COX-2 expression is regulated by
the transcription factor NFAT rather than NF-␬B [55]. This regu-
lation occurs in a calcineurin-dependent mechanism, given that
kidney COX-2 expression was reduced by cyclosporin A (a cal-
cineurin/NFAT signaling inhibitor) treatment [55]. Under stressful
conditions, COX-2 expression increases mainly in the macula densa
[56]. Given that morphological changes were not observed in the
macula densa and that COX-2 expression is dependent on NFAT, but
not NF-␬B in the kidney, the inhibition of COX-2 expression might
not be a mechanism of diclofenac intoxication in this experimen-
tal conditions. Importantly, vinpocetine reduced histopathological
changes compared to the diclofenac-stimulated animals, indicating
that vinpocetine confers protection to the renal tissue exposed to
diclofenac nephrotoxic doses.
The elevated levels of ROS observed after diclofenac administra-
tion indicates that redox signaling also plays a key role in the kidney
damage [8]. In fact, in vitro data show that diclofenac induces oxida-
tive stress by affecting kidney mitochondrial complex I, leading to
a reduction of ATP formation [12], and consequently apoptosis [28]
and kidney damage. We observed that antioxidant defenses were
significantly reduced after diclofenac administration, reflected by
lower levels of FRAP and ABTS activity, and GSH levels. Super-
oxide anion and lipid peroxidation levels were also increased in
renal samples. Considering that ROS are linked to the develop-
ment and progression of AKI [51], strategies that target oxidative
metabolism may represent promising therapeutic approaches.
Vinpocetine reduces lipid peroxidation, nitrite production, and
restores GSH and total antioxidant defense in different models,
such as carrageenan and lipopolysaccharide-induced inflammation
[13,17], chronic cerebral hypoperfusion-induced vascular demen-
tia [57], and hepatic ischemia-reperfusion [58]. This mechanism
18 V. Fattori et al. / Pharmacological Research 120 (2017) 10–22

Fig. 6. Vinpocetine inhibits diclofenac-induced kidney NF-␬B p65 expression. Mice received the stimulus with diclofenac (200 mg/kg, p.o.) and after 30 min were treated
with vinpocetine (3 mg/kg, p.o.). The kidney was collected 24 h after the stimulus for NF-␬B p65 immunohistochemistry analysis (A–H). The average immunostaining was
quantified by image analysis (I). Original magnification 4x (A–D) and 40x (E–H). Results are expressed as mean ± SEM, n = 6 mice per group per experiment, two independent
experiments (* p < 0.05 vs. control group; # p < 0.05 vs. 0 mg/kg group, one ANOVA followed by Tukey’s post hoc).

accounts for the scavenging of hydroxyl radicals and other ROS
[58,59]. In fact, vinpocetine presented a similar antioxidant activ-
ity to curcumin in a model of ischemia-reperfusion liver injury
[58], a well-known antioxidant molecule [25]. Herein, we observed
that treatment with vinpocetine reduced oxidative stress in the
kidney corroborating its antioxidant properties. Given the close
relationship between ROS and GFR [51,52], the antioxidant effect of
vinpocetine may explain, at least in part, its renal protective effect.
In this work, diclofenac increased the production of the
classic proinflammatory cytokines IL-1␤, IL-6, IFN-␥, IL-33, and
TNF-␣. Considering that vinpocetine treatment reduced diclofenac-
induced cytokine production, and in vitro data demonstrated that
diclofenac increases NF-␬B activation in HCT116 cells [60], astro-
cytes [61], and microglia [62], the role of NF-␬B was investigated
in diclofenac-induced AKI. To our knowledge, this is the first report
demonstrating that diclofenac-induced AKI increases NF-␬B acti-
vation. NF-␬B modulates cytokine production, and therefore, the
increased levels of proinflammatory cytokines might depend on
diclofenac-induced kidney NF-␬B activation. This is consistent with
the diclofenac-induced kidney IL-6 mRNA expression [29] and hep-
atotoxic effects due to increasing IL-1␤, TNFRSF1A expression [63],
and TNF-␣, IFN-␥ production in vitro [64]. Although some reports
indicate that diclofenac induces in vivo liver injury [63,65,66], in
this experimental conditions, diclofenac did not induce hepato-
toxicity. This discrepancy might be attributed to differences in
mouse strain or experimental approaches, such as previous LPS
priming [65] or chronic treatment [66]. In addition, diclofenac
induces in vitro [12] and in vivo [8] kidney nuclear condensation and
fragmentation, which can also activate NF-␬B signaling pathway
and apoptosis [67]. Vinpocetine inhibited NF-␬B expression and
activation in renal samples, which lines up with other experimen-
tal models, such as in cerebral ischemia-reperfusion injury [18],
TNF-␣- [14], carrageenan- [17], and LPS-induced inflammation
[13]. Possible mechanisms by which vinpocetine inhibits NF-␬B
signaling pathway include targeting IKK [14] and inhibiting the
phosphorylation of the upstream enzyme AKT [19]. Vinpocetine
also inhibits PDE-1 [68] and PDE-4 [69]. However, the vinpocetine
inhibition of NF-␬B activation is PDE-independent [14,19]. This is
an important data given that higher levels of plasma proinflamma-
tory cytokines increase the risk of mortality in patients diagnosed
with AKI [70].
We next investigated the expression of the transcription factor
c-Myc, which is linked to ROS-, NF-␬B-, and p53-related apoptotic
mechanisms [71–73]. In fact, mice overexpressing c-Myc develop
polycystic kidney disease characterized by intense apoptosis mech-
anism [74]. Also, under stressful conditions, kidney tubular cells
undergo apoptosis in a mechanism dependent on the increases of
c-Myc and TNF-␣, and reduction of Bcl-2 [75]. In addition, in vitro
evidence demonstrated that the increased expression of c-Myc
is associated with the increase of oxidative stress in a model of
 V. Fattori et al. / Pharmacological Research 120 (2017) 10–22 19

chemical-induced nephrotoxicity in LLC-PK1 cells (renal tubular

cell lineage) [76]. In vitro data using Mantle cell lymphoma (MCL)
demonstrated that diclofenac increases p53 and caspase-3 expres-
sion, which ultimately lead to apoptosis [77]. Of note, diclofenac as
other NSAIDs have been used in cancer models due to their capac-
ity of inducing apoptosis [60,77]. In this work, diclofenac induced
kidney apoptosis, which was reduced by vinpocetine treatment,
as observed by c-Myc staining. In fact, vinpocetine reduces the
expression of the pro-apoptotic Bax and increases the expression
of the anti-apoptotic Blc-2, in addition to its antioxidant capacity
[78]. Therefore, these mechanisms may account for the reduced
expression of c-Myc in addition to the inhibition of NF-␬B and ROS.
Despite the two different techniques that were used for the NF-
␬B determination (ELISA and immunohistochemistry), mechanistic
studies are necessary to further advance in the investigation on
the role of NF-␬B and its inhibition by vinpocetine in diclofenac-
induced AKI. For instance, western blot analysis using kidney cells
lineage would allow separating the cytoplasmic and nuclear frac-
tions of NF-␬B p65 subunit to determine nuclear translocation. The
temporal demonstration of NF-␬B activation would contribute to
demonstrate whether this activation depends on the direct effect
Fig. 7. Vinpocetine inhibits diclofenac-induced kidney NF-␬B activation. Mice
of diclofenac or it is an event occurring after AKI-triggered ROS
received the stimulus with diclofenac (200 mg/kg, p.o.) and after 30 min were treated
production in vivo. Hence, studies using selective NF-␬B inhibitors with vinpocetine (3 mg/kg, p.o.). The kidney was collected for determination of NF-
would shed light on the role of NF-␬B in diclofenac-induced AKI. ␬B activation by measuring the tissue levels of total and phospho-NF-␬B p65 subunit
24 h after the stimulus. Results are expressed as mean ± SEM, n = 6 mice per group
per experiment, two independent experiments (* p < 0.05 vs. control group; # p < 0.05
vs. 0 mg/kg group, one ANOVA followed by Tukey’s post hoc).

Fig. 8. Vinpocetine inhibits diclofenac-induced kidney apoptosis. Mice received the stimulus with diclofenac (200 mg/kg, p.o.) and after 30 min were treated with vinpocetine
(3 mg/kg, p.o.). The kidney was collected 24 h after the stimulus for determination of c-Myc immunohistochemistry analysis (A–H). The average immunostaining was quantified
by image analysis (I). Original magnification 4x (A–D) and 40x (E–H). Results are mean ± SEM, n = 6 mice per group per experiment, two independent experiments (* p < 0.05
vs. control group; # p < 0.05 vs. 0 mg/kg group, one ANOVA followed by Tukey’s post hoc).
20 V. Fattori et al. / Pharmacological Research 120 (2017) 10–22

Fig. 9. Diclofenac does not change kidney COX-2 expression. Mice received the stimulus with diclofenac (200 mg/kg, p.o.) and after 30 min were treated with vinpocetine
(3 mg/kg, p.o.). The kidney was collected 24 h after the stimulus for determination of COX-2 immunohistochemistry analysis (A–H). The average immunostaining was
quantified by image analysis (I). Original magnification 4x (A–D) and 40x (EH). Results are mean ± SEM, n = 6 mice per group per experiment, two independent experiments
(* p < 0.05 vs. control group; # p < 0.05 vs. 0 mg/kg group, one ANOVA followed by Tukey’s post hoc).

5. Conclusions 2013/08216-2 (Center for Research in Inflammatory Disease -

We demonstrated that diclofenac induces AKI, which was char-
acterized by increased renal injury biomarkers and oxidative stress
plasma levels, in addition to increased tissue oxidative stress,
histopathological damages, proinflammatory cytokine production,
and apoptosis. Moreover, to the best of our knowledge, this is the
first report demonstrating that there is increase of NF-␬B acti-
vation in diclofenac-induced AKI. Also, it is worth to mention
that considering immunohistochemistry analysis (NF-␬B and c-
Myc proteins expression), diclofenac mainly targets kidney cortical
portion. Finally, vinpocetine treatment reduced kidney damage by
increasing total antioxidant system defense, and by reducing apo-
ptosis, NF-␬B activation, and downstream cytokine production.
Therefore, this work contributes to the understanding of diclofenac
nephrotoxic effects and reveals vinpocetine as a potential approach
to treat diclofenac-induced AKI.
Acknowledgments
This work was supported by Brazilian grants from Coorde-
nadoria de Aperfeiçoamento de Pessoal de Nível Superior (CAPES),
Conselho Nacional de Desenvolvimento Científico e Tecnológico
(CNPq), São Paulo Research Foundation under grant agreement
CRID), Ministério da Ciência, Tecnologia e Inovação (MCTI), Secre-
taria da Ciência, Tecnologia e Inovação (SETI), Fundação Araucária,
and Paraná State Government. SMB received post-doctoral fellow-
ships from CAPES and CNPq.
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