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Protective effect of naringenin against gentamicin-induced nephrotoxicity in rats Amr A. Fouad a,∗,1 , Waleed H. Albuali b , Ahmed Zahran c,2 , Wafaey Gomaa d,3 a
Department of Biomedical Sciences, Pharmacology Division, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia b Department of Pediatrics, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia c Department of Internal Medicine, Nephrology Division, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia d Department of Pathology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
a r t i c l e
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a b s t r a c t
Article history:
The protective effect of naringenin, a flavonoid compound isolated from citrus fruits,
Received 15 March 2014
was investigated against nephrotoxicity induced by gentamicin (80 mg kg−1 /day, i.p., for
Received in revised form
eight days) in rats. Naringenin treatment (50 mg kg−1 /day, p.o.) was administered for eight
19 July 2014
days, starting on the same day of gentamicin administration. Gentamicin caused signif-
Accepted 24 July 2014
icant elevations of serum creatinine, and kidney tissue levels of malondialdehyde, nitric
Available online 2 August 2014
oxide, and interleukin-8, and a significant decrease in renal glutathione peroxidase activity. Naringenin treatment significantly ameliorated the changes in the measured biochemical
Keywords:
parameters resulted from gentamicin administration. Also, naringenin markedly attenuated
Acute kidney injury
the histopathological renal tissue injury observed with gentamicin. Immunohistochem-
Gentamicin
ical examinations showed that naringenin significantly reduced the gentamicin-induced
Naringenin
expression of kidney injury molecule-1, vascular endothelial growth factor, inducible nitric oxide synthase, and caspase-9, and increased survivin expression in the kidney tissue. It
Rats
was concluded that naringenin, through its antioxidant and anti-inflammatory effects, may represent a therapeutic option to protect against gentamicin nephrotoxicity. © 2014 Elsevier B.V. All rights reserved.
1.
Introduction
Gentamicin is an aminoglycoside antibiotic commonly used for treatment of serious and life-threatening aerobic Gramnegative bacterial infections. Its rapid bactericidal action and
low incidence of bacterial resistance have made it a firstline drug in a variety of clinical situations (Barza et al., 1996). However, nephrotoxicity which is the major adverse effect limits the clinical usefulness of gentamicin. Gentamicin nephrotoxicity accounts for 10–15% of all cases of acute renal failure, and about 30% of gentamicin-treated
∗ Corresponding author at: Department of Biomedical Sciences, Pharmacology Division, College of Medicine, King Faisal University, Al-Ahsa 31982, Saudi Arabia. Tel.: +966 501776517. E-mail addresses: [email protected], [email protected] (A.A. Fouad). 1 Primary address: Department of Pharmacology, Faculty of Medicine, Minia University, El-Minia, Egypt. 2 Primary address: Department of Internal Medicine, Nephrology Division, Faculty of Medicine, Menofia University, Egypt. 3 Primary address: Department of Pathology, Faculty of Medicine, Minia University, El-Minia, Egypt. http://dx.doi.org/10.1016/j.etap.2014.07.015 1382-6689/© 2014 Elsevier B.V. All rights reserved.
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experiments. The research protocol was approved by the Scientific Research Ethics Committee, King Faisal University, and the experimental procedures were carried out in accordance with international guidelines for care and use of laboratory animals (Institute for Laboratory Animal Research, 2011). The rats were randomly divided into four groups as follows: - First (control) group (n = 6) received a daily i.p. injection of 0.9% NaCl for eight days. - Second group (n = 8) received gentamicin (80 mg kg−1 /day, i.p., for eight days) and 0.5% carboxymethyl cellulose, p.o., for eight days starting on the same day of gentamicin administration. - Third group (n = 8) received gentamicin and naringenin (50 mg kg−1 /day, p.o.), for eight days starting on the same day of gentamicin administration. - Fourth group (n = 6) received only naringenin for eight days without induction of gentamicin nephrotoxicity.
(A)
1.5
Serum creatinine level (mg/dl)
1.0
0.5
N A R
C on tr eh ol ic le + G EN N A R + G EN
0.0
V
(B) 500
IL-8 (pg/10 0 mg tissue)
300
200
Male Sprague-Dawley rats, weighing 200–220 g, were obtained from College of Medicine, King Faisal University. The animals were kept at standard housing facilities including 24 ± 1 ◦ C, 45 ± 5% humidity and 12 h light/dark cycle. They were supplied with standard laboratory chow and water ad libitum, and left to acclimatize for one week before the
G EN N A R
+
G EN
l
0
eh ic le +
Animals and treatments
V
2.2.
100
C
Gentamicin sulfate and naringenin powders were purchased from Sigma–Aldrich Chemical Co., USA. Gentamicin sulfate was dissolved in 0.9% NaCl, and naringenin was prepared in 0.5% carboxymethyl cellulose. The doses of gentamicin and naringenin used in the present work were selected based on our preliminary experiments and in accordance with previous reports (Gnanasoundari and Pari, 2006; Patel Manali et al., 2011; Renugadevi and Prabu, 2009).
NO (nmol/100 mg tissue)
tr o
Drugs
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on
2.1.
Materials and methods
Kidney tissue levels
2.
MDA (nmol/g tissue) GPx (U/g tissue)
N A R
patients show signs of nephrotoxicity (Mattew, 1992; Shifow et al., 2000). Gentamicin-induced nephrotoxicity is characterized by morphological alterations including destruction, necrosis and apoptosis of kidney cells which eventually lead to acute kidney injury (AKI) and dysfunction (Selby et al., 2009). The precise mechanism of gentamicin nephrotoxicity is not well elucidated. However, increased generation of reactive oxygen species (ROS), increased lipid peroxidation, depletion of antioxidant defenses, and increased production of inflammatory cytokines and chemokines seem to play an important role (Martinez-Salgado et al., 2007; Parlakpinar et al., 2005; Priyamvada et al., 2008). Oxidative stress leads to activation of inflammatory cascades, nitrosative tissue stress, and up-regulation of caspase family of proteases with subsequent apoptotic cell death (SanchezGonzalez et al., 2011). Previous studies revealed the protective effect of antioxidants and anti-inflammatory agents against AKI induced by gentamicin (Anandan and Subramanian, 2012; Feyissa et al., 2013; Lee et al., 2012; Stojiljkovic et al., 2012). Naringenin (4 ,5,7-trihydroxy flavonone) is a plant bioflavonoid found mainly in citrus fruits. Previous studies demonstrated that naringenin significantly ameliorated oxidative and inflammatory tissue injuries in different experimental models (Arul and Subramanian, 2013; Esmaeili and Alilou, 2014; Mershiba et al., 2013; Ortiz-Andrade et al., 2008; Ramprasath et al., 2014). Also, previous studies showed that naringenin treatment significantly protected against cisplatin nephrotoxicity in rats (Badarya et al., 2005), cadmiuminduced oxidative renal dysfunction in rats (Renugadevi and Prabu, 2009), and carbon tetrachloride-induced acute nephrotoxicity in mice (Hermenean et al., 2013). Therefore, naringenin has the potential to protect against AKI induced by gentamicin, and to the best of our knowledge, there is no other investigations have been carried out on the protective effect of naringenin against gentamicin-induced nephrotoxicity.
Fig. 1 – Effects of naringenin (NAR) on: (A) serum creatinine level; (B) kidney tissue levels of malondialdehyde (MDA), nitric oxide (NO), interleukin-8 (IL-8), and glutathione peroxidase (GPx) activity of rats exposed to gentamicin (GEN) nephrotoxicity. Data are mean ± S.E.M., *P < 0.05 vs. control group, • P < 0.05 vs. vehicle plus gentamicin group.
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2.3. Sample preparation and biochemical measurements The rats were euthanized 24 h following the last gentamicin administration by i.p. injection of thiopental (100 mg kg−1 ). Blood samples were collected through a puncture in the left ventricle, and centrifuged for 10 min at 2430 × g to obtain clear sera which were stored at −20 ◦ C. Subsequently, serum creatinine level was measured by an enzymatic assay method (Roche Diagnostics GmbH, Germany) using a Hitachi automatic analyzer (Hitachi Co. Ltd., Tokyo, Japan). The kidneys were dissected out from each animal and their fresh weight was recorded. The renal cortex of the right kidney obtained from each animal was separated, kept at −80 ◦ C and subsequently homogenized in cold potassium phosphate buffer (0.05 M, pH 7.4). The homogenates were centrifuged at 2430 × g for 10 min at 4 ◦ C. Colorimetric assay kits were used according to the instructions of the manufacturers for determination of malondialdehyde (MDA), as an indicator of lipid peroxidation, and glutathione peroxidase (GPx) activity (Biodiagnostic, Egypt), and nitric oxide (NO) level (Cayman Chemical Company, USA). Also, kidney tissue level of interleukin-8 (IL8) was assessed using rat IL-8 ELISA kit as indicated by the manufacturer (MyBioSource, Inc., USA).
2.4.
Histopathological examination
The left kidneys were fixed in 10% formalin solution and then dehydrated in ascending grades of alcohol and embedded in paraffin. Sections at 4 m-thickness were taken, stained with hematoxylin and eosin (H&E) and examined under light microscope by a pathologist unaware of the treatment protocol. Also, renal tubular necrosis scoring was assessed using a semi-quantitative scale of Ramesh and Reeves, 2005 (0 = normal; 1 = less than 10%; 2 = 10–25%; 3 = 25–75%; 4 = more than 75%).
2.5.
Immunohistochemical examinations
Sections at 4 m-thickness prepared from different animal groups were deparaffinised, rehydrated, and endogenous peroxidase activity was blocked with 3% H2 O2 in methanol. Sections were pre-treated in citrate buffer (pH 6.0) in a microwave. Sections were incubated at room temperature with rabbit polyclonal antibodies specific for the rat targets. The antibodies used were anti-kidney injury molecule-1 (KIM-1), anti-vascular endothelial growth factor (VEGF), and anti-inducible nitric oxide synthase (iNOS), anti-survivin, and anti-caspase-9 antibodies (Thermo Scientific, USA, dilution
Fig. 2 – Rat kidney photomicrographs from: (A, 200×) control group showing normal kidney histology; (B, 200× and C, 400×) vehicle plus gentamicin group showing marked renal tubular necrosis, tubular dilatation, vacuolization (C, white arrow), desquamation of epithelial lining, and cast formation inside the tubular lumen (black arrows); (D, 200×) naringenin plus gentamicin group showing a histological picture similar to that of the control group with minimal tubular damage, (E) renal tubular necrosis scoring, Data are mean ± S.E.M., *P < 0.05 vs. control group, • P < 0.05 vs. vehicle plus gentamicin group, NAR = naringenin, GEN = gentamicin.
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1:200). The sections were incubated with biotinylated goat anti-polyvalent, then with streptavidin peroxidase and finally with diaminobenzedine plus chromogen. Slides were counterstained with hematoxylin. The slides were visualized under light microscope and the extent of cell immunopositivity was assessed. The area (m2 ) occupied by the immunopositive cells was measured in five separate microscopic fields in each slide using a digital imaging software program (cellSens, Olympus Corporation, USA), and the mean area for each slide was obtained, then the mean ± S.E.M. was calculated for each group. The same procedures were repeated using normal rabbit serum instead of the primary antibodies to obtain negative control and indicate the specificity of the used antibodies (Rasmussen, 2009).
2.6.
Statistics
All values are expressed as mean ± S.E.M. The results were analyzed by one-way analysis of variance (ANOVA) followed
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by Tukey test for multiple comparisons using SPSS for Windows (version 18) and P < 0.05 was selected as the criterion of significant difference.
3.
Results
Significant elevations of serum creatinine, and kidney tissue levels of MDA, NO, and IL-8, and a significant decrease in renal GPx activity were detected in rats received gentamicin (80 mg kg−1 /day, i.p., for eight days) as compared to the control group (Fig. 1). However, naringenin-treated rats showed significant reductions of serum creatinine, and renal MDA, NO, and IL-8, and a significant increase in renal GPx activity as compared to the vehicle plus gentamicin group (Fig. 1A and B). Histopathological examination shows that gentamicin caused widespread necrosis with dilatation, vacuolar degeneration, epithelial desquamation and intraluminal cast
Fig. 3 – Immunohistochemical staining (200×) of kidney injury molecule-1 (KIM-1) in rat kidney from: (A) control group showing no expression of KIM-1; (B) vehicle plus gentamicin group showing a significant increase in KIM-1 immunoreactivity in the renal tubular cells; (C) naringenin plus gentamicin group demonstrating a significant reduction in KIM-1 immunostaining. Brown color indicates KIM-1 positivity; (D) area of immunopositivity (m2 ), data are mean ± S.E.M., *P < 0.05 vs. control group, • P < 0.05 vs. vehicle plus gentamicin group, ND = non-detectable, NAR = naringenin, GEN = gentamicin. (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)
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formation mainly in the proximal convoluted tubules. However, naringenin treatment markedly attenuated the gentamicin-induced renal tissue injury with a histopathological picture comparable to that of the control group. In addition, naringenin caused a significant reduction in the gentamicininduced renal tubular necrosis score (Fig. 2). Significant increases in the expression of KIM-1, VEGF, iNOS, and caspase-9, and a significant decrease in survivin expression in the cells of proximal tubules were observed in rats in the vehicle plus gentamicin group as compared to the control animals. Naringenin plus gentamicin group showed significant decreases in the expression of KIM-1, VEGF, iNOS, and caspase-9, and a significant increase in survivin expression in the cells of proximal tubules as compared to the vehicle plus gentamicin group (Figs. 3–7). The slides from the vehicle plus gentamicin group which were incubated with normal rabbit serum instead of the primary antibodies showed no positive staining indicating the specificity of the used antibodies (figures not shown).
4.
Discussion
The present study showed that naringenin significantly downregulated oxidative, nitrosative, inflammatory, and apoptotic biomarkers in the serum and kidney tissue of rats exposed to gentamicin-induced AKI. It was demonstrated that oxidative stress, increased generation of ROS, depletion of antioxidant defenses, and increased lipid peroxidation in renal tissue play a crucial role in AKI induced by gentamicin (Aygün et al., 2012; Manikandan et al., 2011). It also induces a cascade of inflammatory reactions with increased production of inflammatory cytokines and chemokines (Dam et al., 2012; Kalayarasan et al., 2009). Gentamicin also induces iNOS leading to increased production of NO which interacts with superoxide to generate the potent cytotoxic agent, peroxynitrite (Lee et al., 2012; Otunctemur et al., 2013). The major effect of peroxynitrite is the nitration of cellular proteins leading to nitrosative stress and tissue injury (Negrette-Guzmán et al., 2013). Moreover,
Fig. 4 – Immunohistochemical staining (200×) of vascular endothelial growth factor (VEGF) in rat kidney from: (A) control group showing normal expression of VEGF; (B) vehicle plus gentamicin group showing a significant increase in VEGF immunoreactivity in the renal tubular cells; (C) naringenin plus gentamicin group demonstrating a significant reduction in VEGF immunostaining. Brown color indicates VEGF positivity; (D) area of immunopositivity (m2 ), data are mean ± S.E.M., *P < 0.05 vs. control group, • P < 0.05 vs. vehicle plus gentamicin group, NAR = naringenin, GEN = gentamicin. (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)
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Fig. 5 – Immunohistochemical staining (200×) of inducible nitric oxide synthase (iNOS) in rat kidney from: (A) control group showing no expression of iNOS; (B) vehicle plus gentamicin showing a significant increase in iNOS immunoreactivity in the renal tubular cells; (C) naringenin plus gentamicin group showing a significant reduction in iNOS immunostaining. Brown color indicates iNOS positivity; (D) area of immunopositivity (m2 ), data are mean ± S.E.M., *P < 0.05 vs. control group, • P < 0.05 vs. vehicle plus gentamicin group, ND = non-detectable, NAR = naringenin, GEN = gentamicin. (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)
increased renal expression of VEGF was reported in animals upon exposure to various nephrotoxic agents including gentamicin, and in patients with AKI (Hoffmann et al., 2010; Mohamed et al., 2013). VEGF enhances angiogenesis, endothelial proliferation, cell migration, and vascular permeability. Increased ROS generation in AKI leads to up-regulation of VEGF causing NO overproduction which increases peroxynitrite formation and nitrosative stress (El-Remessy et al., 2004; Feliers et al., 2006). KIM-1, the novel biomarker for kidney injury, is induced by various nephrotoxic agents or ischemia. KIM-1 is a cell membrane glycoprotein acts as a receptor for apoptotic-cell phagocytosis which is up-regulated in the renal tubular cells in cases of kidney injury (Ichimura et al., 2012; Martensson et al., 2012). It is considered as an excellent indicator in nephrotoxicity due to its marked expression in injured kidney tissue, but not in intact kidneys (Bonventre, 2009). In addition, IL-8 is
an endothelial-derived chemochine involved in recruitment of neutrophils to the injured organs including kidneys. IL-8 is also important for neutrophil chemotaxis and oxidative burst. Previous studies demonstrated that IL-8 level was elevated in cases of AKI associated with renal allograft ischemia (Kwon et al., 2003), cardiopulmonary bypass surgery (Liangos et al., 2009), and liver transplantation (Sirota et al., 2013). Therefore, IL-8 is also considered a novel biomarker for AKI. The results of the present work revealed that naringenin significantly reduced IL-8 level and KIM-1 expression in kidney tissue of rats exposed to gentamicin nephrotoxicity, and to the best of our knowledge, this is the first study which demonstrated the effects of naringenin on IL-8 and renal KIM-1 expression in AKI. Naringenin is the major bioactive flavonoid compound derived from citrus fruits. Naringenin exerts marked antioxidant activity, scavenges ROS, suppresses lipid peroxidation,
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Fig. 6 – Immunohistochemical staining (200×) of survivin in rat kidney from: (A) control group showing that survivin is normally expressed; (B) vehicle plus gentamicin group showing a significant decrease in survivin immunoreactivity in the renal tubular cells; (C) naringenin plus gentamicin group showing a significant increase in survivin immunostaining. Brown color indicates survivin positivity; (D) area of immunopositivity (m2 ), data are mean ± S.E.M., *P < 0.05 vs. control group, • P < 0.05 vs. vehicle plus gentamicin group, NAR = naringenin, GEN = gentamicin. (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)
and maintains the antioxidant defense mechanisms (AlRejaie et al., 2013; Mershiba et al., 2013). Also, naringenin inhibits iNOS activity and therefore prevents nitrosative tissue stress (Annadurai et al., 2013; Jayaraman et al., 2012), and reduces the release of inflammatory cytokines and chemokines (Lim et al., 2013). This in accordance with the present results which revealed that naringenin significantly suppressed lipid peroxidation and nitrosative stress, maintained antioxidant defenses, and reduced the renal tissue expression of VEGF and iNOS in rats received gentamicin. Survivin is a new member of the inhibitor-of-apoptosis proteins and is considered the most powerful antiapoptotic gene in vivo. Upon apoptotic cell death stimulation, survivin is released from the mitochondria, binds to the X-linked inhibitor of apoptosis protein and synergizes its inhibitory effect on caspase-9 activation (Cheung et al., 2013). Survivin also suppresses cell apoptosis by inhibiting Fas/Fas ligand
stimulation, and by preventing cytchrome c release from the mitochondria (Hossain et al., 2012; Ryan et al., 2009). Also, the results of present work revealed that naringenin significantly inhibited the gentamicin-induced expression of caspase-9 and renal cell apoptosis. In agreement with present study, previous studies showed that gentamicin resulted in activation of caspase-9 and apoptotic renal cell death (Chen et al., 2011; Negrette-Guzmán et al., 2013). Therefore it could be stated that naringenin exerts its antiapoptotic activity via induction of survivin protein, and inhibition of caspase family of proteases, and to the best of our knowledge, this is the first study which shows the effect of gentamicin and naringenin on survivin expression in the kidney. Also, the antioxidant and anti-inflammatory activities may be responsible for the antiapoptotic effect of naringenin. The results of the present study indicate that naringenin provided a significant protective effect against gentamicin
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Fig. 7 – Immunohistochemical staining (200×) of caspase-9 in rat kidney from: (A) control group showing no expression of caspase-9; (B) vehicle plus gentamicin group showing a significant increase in caspase-9 immunoreactivity in the renal tubular cells; (C) naringenin plus gentamicin group showing a significant reduction in caspase-9 immunostaining. Brown color indicates caspase-9 positivity; (D) area of immunopositivity (m2 ), data are mean ± S.E.M., * P < 0.05 vs. control group, • P < 0.05 vs. vehicle plus gentamicin group, ND = non-detectable, NAR = naringenin, GEN = gentamicin. (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)
nephrotoxicity in rats. The antioxidant and anti-inflammatory activities can be considered the main factors responsible for the nephroprotective effect of naringenin. Therefore, naringenin may represent a potential therapeutic option to prevent gentamicin-induced renal injury and dysfunction, which is a major and dose-limiting clinical problem.
Transparency document The Transparency document associated with this article can be found in the online version.
Acknowledgements Funding None.
The teamwork of the present study is greatly thankful to the Deanship of Scientific Research, King Faisal University for the sincere motivation and support.
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Conflict of interest There is no conflict of interest.
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kidney biomarker panel in rat models of acute and subchronic nephrotoxicity. Toxicology 277, 49–58. Hossain, M.M., Banik, N.L., Ray, S.K., 2012. Survivin knockdown increased anti-cancer effects of (-)-epigallocatechin-3-gallate in human malignant neuroblastoma SK-N-BE2 and SH-SY5Y cells. Exp. Cell Res. 318, 1597–1610. Ichimura, T., Brooks, C.R., Bonventre, J.V., 2012. Kim-1/Tim-1 and immune cells: shifting sands. Kidney Int. 81, 809–811. Institute for Laboratory Animal Research, 2011. Guide for the Care and Use of Laboratory Animals, 8th ed. National Academies Press, Washington, DC. Jayaraman, J., Jesudoss, V.A., Menon, V.P., Namasivayam, N., 2012. Anti-inflammatory role of naringenin in rats with ethanol induced liver injury. Toxicol. Mech. Methods 22, 568–576. Kalayarasan, S., Prabhu, P.N., Sriram, N., Manikandan, R., Arumugam, M., Sudhandiran, G., 2009. Diallyl sulfide enhances antioxidants and inhibits inflammation through the activation of Nrf2 against gentamicin-induced nephrotoxicity in Wistar rats. Eur. J. Pharmacol. 606, 162–171. Kwon, O., Molitoris, B.A., Pescovitz, M., Kelly, K.J., 2003. Urinary actin, interleukin-6, and interleukin-8 may predict sustained art after ischemic injury in renal allografts. Am. J. Kidney Dis. 41, 1074–1087. Lee, I.C., Kim, S.H., Lee, S.M., Baek, H.S., Moon, C., Kim, S.H., Park, S.C., Kim, H.C., Kim, J.C., 2012. Melatonin attenuates gentamicin-induced nephrotoxicity and oxidative stress in rats. Arch. Toxicol. 86, 1527–1536. Liangos, O., Kolyada, A., Tighiouart, H., Perianayagam, M.C., Wald, R., Jaber, B.L., 2009. Interleukin-8 and acute kidney injury following cardiopulmonary bypass: a prospective cohort study. Nephron Clin. Pract. 113, c148–c154. Lim, R., Barker, G., Wall, C.A., Lappas, M., 2013. Dietary phytophenols curcumin, naringenin and apigenin reduce infection-induced inflammatory and contractile pathways in human placenta, foetal membranes and myometrium. Mol. Hum. Reprod. 19, 451–462. Manikandan, R., Beulaja, M., Thiagarajan, R., Priyadarsini, A., Saravanan, R., Arumugam, M., 2011. Ameliorative effects of curcumin against renal injuries mediated by inducible nitric oxide synthase and nuclear factor kappa B during gentamicin-induced toxicity in Wistar rats. Eur. J. Pharmacol. 670, 578–585. Martensson, J., Martling, C.R., Bell, M., 2012. Novel biomarkers of acute kidney injury and failure: clinical applicability. Br. J. Anaesth. 109, 843–850. Martinez-Salgado, C., Lopez-Hernandez, F.J., Lopez-Novoa, J.M., 2007. Glomerular nephrotoxicity of aminoglycosides. Toxicol. Appl. Pharmacol. 223, 86–98. Mattew, T.H., 1992. Drug-induced renal disease. Med. J. Aust. 156, 724–728. Mershiba, S.D., Dassprakash, M.V., Saraswathy, S.D., 2013. Protective effect of naringenin on hepatic and renal dysfunction and oxidative stress in arsenic intoxicated rats. Mol. Biol. Rep. 40, 3681–3691. Mohamed, H.E., El-Swefy, S.E., Mohamed, R.H., Ghanim, A.M., 2013. Effect of erythropoietin therapy on the progression of cisplatin induced renal injury in rats. Exp. Toxicol. Pathol. 65, 197–203. Negrette-Guzmán, M., Huerta-Yepez, S., Medina-Campos, O.N., Zatarain-Barrón, Z.L., Hernández-Pando, R., Torres, I., Tapia, E., Pedraza-Chaverri, J., 2013. Sulforaphane attenuates gentamicin-induced nephrotoxicity: role of mitochondrial protection. Evid. Based Complement. Alternat. Med. 2013, 135314. Ortiz-Andrade, R.R., Sanchez-Salgado, J.C., Navarrete-Vazquez, G., Webster, S.P., Binnie, M., Garcia-Jimenez, S., Leon-Rivera, I., Cigarroa-Vazquez, P., Villalobos-Molina, R., Estrada-Soto, S.,
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2008. Antidiabetic and toxicological evaluations of naringenin in normoglycaemic and NIDDM rat models and its implications on extra-pancreatic glucose regulation. Diabetes Obes. Metab. 10, 1097–1104. Otunctemur, A., Ozbek, E., Cekmen, M., Cakir, S.S., Dursun, M., Polat, E.C., Somay, A., Ozbay, N., 2013. Protective effect of montelukast which is cysteinyl-leukotriene receptor antagonist on gentamicin-induced nephrotoxicity and oxidative damage in rat kidney. Ren. Fail. 35, 403–410. Parlakpinar, H., Tasdemir, S., Polat, A., Bay-Karabulut, A., Vardi, N., Ucar, M., Acet, A., 2005. Protective role of caffeic acid phenethyl ester (cape) on gentamicin-induced acute renal toxicity in rats. Toxicology 207, 169–177. Patel Manali, B., Deshpande, S., Shah, G., 2011. Evaluation of efficacy of vitamin E and N-acetyl cysteine in gentamicin-induced nephrotoxicity in rats. Ren. Fail. 33, 341–347. Priyamvada, S., Priyadarshini, M., Arivarasu, N.A., Farooq, N., Khan, S., Khan, S.A., 2008. Studies on the protective effect of dietary fish oil on gentamicin-induced nephrotoxicity and oxidative damage in rat kidney. Prostaglandins Leukot. Essent. Fatty Acids 78, 369–381. Ramesh, G., Reeves, W.B., 2005. p38 MAP kinase inhibition ameliorates cisplatin nephrotoxicity in mice. Am. J. Physiol. Renal Physiol. 289, F166–F174. Ramprasath, T., Senthamizharasi, M., Vasudevan, V., Sasikumar, S., Yuvaraj, S., Selvam, G.S., 2014. Naringenin confers protection against oxidative stress through upregulation of
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Available online at www.sciencedirect.com
ScienceDirect journal homepage: www.elsevier.com/locate/etap
Protective effect of naringenin against gentamicin-induced nephrotoxicity in rats Amr A. Fouad a,∗,1 , Waleed H. Albuali b , Ahmed Zahran c,2 , Wafaey Gomaa d,3 a
Department of Biomedical Sciences, Pharmacology Division, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia b Department of Pediatrics, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia c Department of Internal Medicine, Nephrology Division, College of Medicine, King Faisal University, Al-Ahsa, Saudi Arabia d Department of Pathology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia
a r t i c l e
i n f o
a b s t r a c t
Article history:
The protective effect of naringenin, a flavonoid compound isolated from citrus fruits,
Received 15 March 2014
was investigated against nephrotoxicity induced by gentamicin (80 mg kg−1 /day, i.p., for
Received in revised form
eight days) in rats. Naringenin treatment (50 mg kg−1 /day, p.o.) was administered for eight
19 July 2014
days, starting on the same day of gentamicin administration. Gentamicin caused signif-
Accepted 24 July 2014
icant elevations of serum creatinine, and kidney tissue levels of malondialdehyde, nitric
Available online 2 August 2014
oxide, and interleukin-8, and a significant decrease in renal glutathione peroxidase activity. Naringenin treatment significantly ameliorated the changes in the measured biochemical
Keywords:
parameters resulted from gentamicin administration. Also, naringenin markedly attenuated
Acute kidney injury
the histopathological renal tissue injury observed with gentamicin. Immunohistochem-
Gentamicin
ical examinations showed that naringenin significantly reduced the gentamicin-induced
Naringenin
expression of kidney injury molecule-1, vascular endothelial growth factor, inducible nitric oxide synthase, and caspase-9, and increased survivin expression in the kidney tissue. It
Rats
was concluded that naringenin, through its antioxidant and anti-inflammatory effects, may represent a therapeutic option to protect against gentamicin nephrotoxicity. © 2014 Elsevier B.V. All rights reserved.
1.
Introduction
Gentamicin is an aminoglycoside antibiotic commonly used for treatment of serious and life-threatening aerobic Gramnegative bacterial infections. Its rapid bactericidal action and
low incidence of bacterial resistance have made it a firstline drug in a variety of clinical situations (Barza et al., 1996). However, nephrotoxicity which is the major adverse effect limits the clinical usefulness of gentamicin. Gentamicin nephrotoxicity accounts for 10–15% of all cases of acute renal failure, and about 30% of gentamicin-treated
∗ Corresponding author at: Department of Biomedical Sciences, Pharmacology Division, College of Medicine, King Faisal University, Al-Ahsa 31982, Saudi Arabia. Tel.: +966 501776517. E-mail addresses: [email protected], [email protected] (A.A. Fouad). 1 Primary address: Department of Pharmacology, Faculty of Medicine, Minia University, El-Minia, Egypt. 2 Primary address: Department of Internal Medicine, Nephrology Division, Faculty of Medicine, Menofia University, Egypt. 3 Primary address: Department of Pathology, Faculty of Medicine, Minia University, El-Minia, Egypt. http://dx.doi.org/10.1016/j.etap.2014.07.015 1382-6689/© 2014 Elsevier B.V. All rights reserved.
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experiments. The research protocol was approved by the Scientific Research Ethics Committee, King Faisal University, and the experimental procedures were carried out in accordance with international guidelines for care and use of laboratory animals (Institute for Laboratory Animal Research, 2011). The rats were randomly divided into four groups as follows: - First (control) group (n = 6) received a daily i.p. injection of 0.9% NaCl for eight days. - Second group (n = 8) received gentamicin (80 mg kg−1 /day, i.p., for eight days) and 0.5% carboxymethyl cellulose, p.o., for eight days starting on the same day of gentamicin administration. - Third group (n = 8) received gentamicin and naringenin (50 mg kg−1 /day, p.o.), for eight days starting on the same day of gentamicin administration. - Fourth group (n = 6) received only naringenin for eight days without induction of gentamicin nephrotoxicity.
(A)
1.5
Serum creatinine level (mg/dl)
1.0
0.5
N A R
C on tr eh ol ic le + G EN N A R + G EN
0.0
V
(B) 500
IL-8 (pg/10 0 mg tissue)
300
200
Male Sprague-Dawley rats, weighing 200–220 g, were obtained from College of Medicine, King Faisal University. The animals were kept at standard housing facilities including 24 ± 1 ◦ C, 45 ± 5% humidity and 12 h light/dark cycle. They were supplied with standard laboratory chow and water ad libitum, and left to acclimatize for one week before the
G EN N A R
+
G EN
l
0
eh ic le +
Animals and treatments
V
2.2.
100
C
Gentamicin sulfate and naringenin powders were purchased from Sigma–Aldrich Chemical Co., USA. Gentamicin sulfate was dissolved in 0.9% NaCl, and naringenin was prepared in 0.5% carboxymethyl cellulose. The doses of gentamicin and naringenin used in the present work were selected based on our preliminary experiments and in accordance with previous reports (Gnanasoundari and Pari, 2006; Patel Manali et al., 2011; Renugadevi and Prabu, 2009).
NO (nmol/100 mg tissue)
tr o
Drugs
400
on
2.1.
Materials and methods
Kidney tissue levels
2.
MDA (nmol/g tissue) GPx (U/g tissue)
N A R
patients show signs of nephrotoxicity (Mattew, 1992; Shifow et al., 2000). Gentamicin-induced nephrotoxicity is characterized by morphological alterations including destruction, necrosis and apoptosis of kidney cells which eventually lead to acute kidney injury (AKI) and dysfunction (Selby et al., 2009). The precise mechanism of gentamicin nephrotoxicity is not well elucidated. However, increased generation of reactive oxygen species (ROS), increased lipid peroxidation, depletion of antioxidant defenses, and increased production of inflammatory cytokines and chemokines seem to play an important role (Martinez-Salgado et al., 2007; Parlakpinar et al., 2005; Priyamvada et al., 2008). Oxidative stress leads to activation of inflammatory cascades, nitrosative tissue stress, and up-regulation of caspase family of proteases with subsequent apoptotic cell death (SanchezGonzalez et al., 2011). Previous studies revealed the protective effect of antioxidants and anti-inflammatory agents against AKI induced by gentamicin (Anandan and Subramanian, 2012; Feyissa et al., 2013; Lee et al., 2012; Stojiljkovic et al., 2012). Naringenin (4 ,5,7-trihydroxy flavonone) is a plant bioflavonoid found mainly in citrus fruits. Previous studies demonstrated that naringenin significantly ameliorated oxidative and inflammatory tissue injuries in different experimental models (Arul and Subramanian, 2013; Esmaeili and Alilou, 2014; Mershiba et al., 2013; Ortiz-Andrade et al., 2008; Ramprasath et al., 2014). Also, previous studies showed that naringenin treatment significantly protected against cisplatin nephrotoxicity in rats (Badarya et al., 2005), cadmiuminduced oxidative renal dysfunction in rats (Renugadevi and Prabu, 2009), and carbon tetrachloride-induced acute nephrotoxicity in mice (Hermenean et al., 2013). Therefore, naringenin has the potential to protect against AKI induced by gentamicin, and to the best of our knowledge, there is no other investigations have been carried out on the protective effect of naringenin against gentamicin-induced nephrotoxicity.
Fig. 1 – Effects of naringenin (NAR) on: (A) serum creatinine level; (B) kidney tissue levels of malondialdehyde (MDA), nitric oxide (NO), interleukin-8 (IL-8), and glutathione peroxidase (GPx) activity of rats exposed to gentamicin (GEN) nephrotoxicity. Data are mean ± S.E.M., *P < 0.05 vs. control group, • P < 0.05 vs. vehicle plus gentamicin group.
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2.3. Sample preparation and biochemical measurements The rats were euthanized 24 h following the last gentamicin administration by i.p. injection of thiopental (100 mg kg−1 ). Blood samples were collected through a puncture in the left ventricle, and centrifuged for 10 min at 2430 × g to obtain clear sera which were stored at −20 ◦ C. Subsequently, serum creatinine level was measured by an enzymatic assay method (Roche Diagnostics GmbH, Germany) using a Hitachi automatic analyzer (Hitachi Co. Ltd., Tokyo, Japan). The kidneys were dissected out from each animal and their fresh weight was recorded. The renal cortex of the right kidney obtained from each animal was separated, kept at −80 ◦ C and subsequently homogenized in cold potassium phosphate buffer (0.05 M, pH 7.4). The homogenates were centrifuged at 2430 × g for 10 min at 4 ◦ C. Colorimetric assay kits were used according to the instructions of the manufacturers for determination of malondialdehyde (MDA), as an indicator of lipid peroxidation, and glutathione peroxidase (GPx) activity (Biodiagnostic, Egypt), and nitric oxide (NO) level (Cayman Chemical Company, USA). Also, kidney tissue level of interleukin-8 (IL8) was assessed using rat IL-8 ELISA kit as indicated by the manufacturer (MyBioSource, Inc., USA).
2.4.
Histopathological examination
The left kidneys were fixed in 10% formalin solution and then dehydrated in ascending grades of alcohol and embedded in paraffin. Sections at 4 m-thickness were taken, stained with hematoxylin and eosin (H&E) and examined under light microscope by a pathologist unaware of the treatment protocol. Also, renal tubular necrosis scoring was assessed using a semi-quantitative scale of Ramesh and Reeves, 2005 (0 = normal; 1 = less than 10%; 2 = 10–25%; 3 = 25–75%; 4 = more than 75%).
2.5.
Immunohistochemical examinations
Sections at 4 m-thickness prepared from different animal groups were deparaffinised, rehydrated, and endogenous peroxidase activity was blocked with 3% H2 O2 in methanol. Sections were pre-treated in citrate buffer (pH 6.0) in a microwave. Sections were incubated at room temperature with rabbit polyclonal antibodies specific for the rat targets. The antibodies used were anti-kidney injury molecule-1 (KIM-1), anti-vascular endothelial growth factor (VEGF), and anti-inducible nitric oxide synthase (iNOS), anti-survivin, and anti-caspase-9 antibodies (Thermo Scientific, USA, dilution
Fig. 2 – Rat kidney photomicrographs from: (A, 200×) control group showing normal kidney histology; (B, 200× and C, 400×) vehicle plus gentamicin group showing marked renal tubular necrosis, tubular dilatation, vacuolization (C, white arrow), desquamation of epithelial lining, and cast formation inside the tubular lumen (black arrows); (D, 200×) naringenin plus gentamicin group showing a histological picture similar to that of the control group with minimal tubular damage, (E) renal tubular necrosis scoring, Data are mean ± S.E.M., *P < 0.05 vs. control group, • P < 0.05 vs. vehicle plus gentamicin group, NAR = naringenin, GEN = gentamicin.
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1:200). The sections were incubated with biotinylated goat anti-polyvalent, then with streptavidin peroxidase and finally with diaminobenzedine plus chromogen. Slides were counterstained with hematoxylin. The slides were visualized under light microscope and the extent of cell immunopositivity was assessed. The area (m2 ) occupied by the immunopositive cells was measured in five separate microscopic fields in each slide using a digital imaging software program (cellSens, Olympus Corporation, USA), and the mean area for each slide was obtained, then the mean ± S.E.M. was calculated for each group. The same procedures were repeated using normal rabbit serum instead of the primary antibodies to obtain negative control and indicate the specificity of the used antibodies (Rasmussen, 2009).
2.6.
Statistics
All values are expressed as mean ± S.E.M. The results were analyzed by one-way analysis of variance (ANOVA) followed
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by Tukey test for multiple comparisons using SPSS for Windows (version 18) and P < 0.05 was selected as the criterion of significant difference.
3.
Results
Significant elevations of serum creatinine, and kidney tissue levels of MDA, NO, and IL-8, and a significant decrease in renal GPx activity were detected in rats received gentamicin (80 mg kg−1 /day, i.p., for eight days) as compared to the control group (Fig. 1). However, naringenin-treated rats showed significant reductions of serum creatinine, and renal MDA, NO, and IL-8, and a significant increase in renal GPx activity as compared to the vehicle plus gentamicin group (Fig. 1A and B). Histopathological examination shows that gentamicin caused widespread necrosis with dilatation, vacuolar degeneration, epithelial desquamation and intraluminal cast
Fig. 3 – Immunohistochemical staining (200×) of kidney injury molecule-1 (KIM-1) in rat kidney from: (A) control group showing no expression of KIM-1; (B) vehicle plus gentamicin group showing a significant increase in KIM-1 immunoreactivity in the renal tubular cells; (C) naringenin plus gentamicin group demonstrating a significant reduction in KIM-1 immunostaining. Brown color indicates KIM-1 positivity; (D) area of immunopositivity (m2 ), data are mean ± S.E.M., *P < 0.05 vs. control group, • P < 0.05 vs. vehicle plus gentamicin group, ND = non-detectable, NAR = naringenin, GEN = gentamicin. (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)
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formation mainly in the proximal convoluted tubules. However, naringenin treatment markedly attenuated the gentamicin-induced renal tissue injury with a histopathological picture comparable to that of the control group. In addition, naringenin caused a significant reduction in the gentamicininduced renal tubular necrosis score (Fig. 2). Significant increases in the expression of KIM-1, VEGF, iNOS, and caspase-9, and a significant decrease in survivin expression in the cells of proximal tubules were observed in rats in the vehicle plus gentamicin group as compared to the control animals. Naringenin plus gentamicin group showed significant decreases in the expression of KIM-1, VEGF, iNOS, and caspase-9, and a significant increase in survivin expression in the cells of proximal tubules as compared to the vehicle plus gentamicin group (Figs. 3–7). The slides from the vehicle plus gentamicin group which were incubated with normal rabbit serum instead of the primary antibodies showed no positive staining indicating the specificity of the used antibodies (figures not shown).
4.
Discussion
The present study showed that naringenin significantly downregulated oxidative, nitrosative, inflammatory, and apoptotic biomarkers in the serum and kidney tissue of rats exposed to gentamicin-induced AKI. It was demonstrated that oxidative stress, increased generation of ROS, depletion of antioxidant defenses, and increased lipid peroxidation in renal tissue play a crucial role in AKI induced by gentamicin (Aygün et al., 2012; Manikandan et al., 2011). It also induces a cascade of inflammatory reactions with increased production of inflammatory cytokines and chemokines (Dam et al., 2012; Kalayarasan et al., 2009). Gentamicin also induces iNOS leading to increased production of NO which interacts with superoxide to generate the potent cytotoxic agent, peroxynitrite (Lee et al., 2012; Otunctemur et al., 2013). The major effect of peroxynitrite is the nitration of cellular proteins leading to nitrosative stress and tissue injury (Negrette-Guzmán et al., 2013). Moreover,
Fig. 4 – Immunohistochemical staining (200×) of vascular endothelial growth factor (VEGF) in rat kidney from: (A) control group showing normal expression of VEGF; (B) vehicle plus gentamicin group showing a significant increase in VEGF immunoreactivity in the renal tubular cells; (C) naringenin plus gentamicin group demonstrating a significant reduction in VEGF immunostaining. Brown color indicates VEGF positivity; (D) area of immunopositivity (m2 ), data are mean ± S.E.M., *P < 0.05 vs. control group, • P < 0.05 vs. vehicle plus gentamicin group, NAR = naringenin, GEN = gentamicin. (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)
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Fig. 5 – Immunohistochemical staining (200×) of inducible nitric oxide synthase (iNOS) in rat kidney from: (A) control group showing no expression of iNOS; (B) vehicle plus gentamicin showing a significant increase in iNOS immunoreactivity in the renal tubular cells; (C) naringenin plus gentamicin group showing a significant reduction in iNOS immunostaining. Brown color indicates iNOS positivity; (D) area of immunopositivity (m2 ), data are mean ± S.E.M., *P < 0.05 vs. control group, • P < 0.05 vs. vehicle plus gentamicin group, ND = non-detectable, NAR = naringenin, GEN = gentamicin. (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)
increased renal expression of VEGF was reported in animals upon exposure to various nephrotoxic agents including gentamicin, and in patients with AKI (Hoffmann et al., 2010; Mohamed et al., 2013). VEGF enhances angiogenesis, endothelial proliferation, cell migration, and vascular permeability. Increased ROS generation in AKI leads to up-regulation of VEGF causing NO overproduction which increases peroxynitrite formation and nitrosative stress (El-Remessy et al., 2004; Feliers et al., 2006). KIM-1, the novel biomarker for kidney injury, is induced by various nephrotoxic agents or ischemia. KIM-1 is a cell membrane glycoprotein acts as a receptor for apoptotic-cell phagocytosis which is up-regulated in the renal tubular cells in cases of kidney injury (Ichimura et al., 2012; Martensson et al., 2012). It is considered as an excellent indicator in nephrotoxicity due to its marked expression in injured kidney tissue, but not in intact kidneys (Bonventre, 2009). In addition, IL-8 is
an endothelial-derived chemochine involved in recruitment of neutrophils to the injured organs including kidneys. IL-8 is also important for neutrophil chemotaxis and oxidative burst. Previous studies demonstrated that IL-8 level was elevated in cases of AKI associated with renal allograft ischemia (Kwon et al., 2003), cardiopulmonary bypass surgery (Liangos et al., 2009), and liver transplantation (Sirota et al., 2013). Therefore, IL-8 is also considered a novel biomarker for AKI. The results of the present work revealed that naringenin significantly reduced IL-8 level and KIM-1 expression in kidney tissue of rats exposed to gentamicin nephrotoxicity, and to the best of our knowledge, this is the first study which demonstrated the effects of naringenin on IL-8 and renal KIM-1 expression in AKI. Naringenin is the major bioactive flavonoid compound derived from citrus fruits. Naringenin exerts marked antioxidant activity, scavenges ROS, suppresses lipid peroxidation,
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Fig. 6 – Immunohistochemical staining (200×) of survivin in rat kidney from: (A) control group showing that survivin is normally expressed; (B) vehicle plus gentamicin group showing a significant decrease in survivin immunoreactivity in the renal tubular cells; (C) naringenin plus gentamicin group showing a significant increase in survivin immunostaining. Brown color indicates survivin positivity; (D) area of immunopositivity (m2 ), data are mean ± S.E.M., *P < 0.05 vs. control group, • P < 0.05 vs. vehicle plus gentamicin group, NAR = naringenin, GEN = gentamicin. (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)
and maintains the antioxidant defense mechanisms (AlRejaie et al., 2013; Mershiba et al., 2013). Also, naringenin inhibits iNOS activity and therefore prevents nitrosative tissue stress (Annadurai et al., 2013; Jayaraman et al., 2012), and reduces the release of inflammatory cytokines and chemokines (Lim et al., 2013). This in accordance with the present results which revealed that naringenin significantly suppressed lipid peroxidation and nitrosative stress, maintained antioxidant defenses, and reduced the renal tissue expression of VEGF and iNOS in rats received gentamicin. Survivin is a new member of the inhibitor-of-apoptosis proteins and is considered the most powerful antiapoptotic gene in vivo. Upon apoptotic cell death stimulation, survivin is released from the mitochondria, binds to the X-linked inhibitor of apoptosis protein and synergizes its inhibitory effect on caspase-9 activation (Cheung et al., 2013). Survivin also suppresses cell apoptosis by inhibiting Fas/Fas ligand
stimulation, and by preventing cytchrome c release from the mitochondria (Hossain et al., 2012; Ryan et al., 2009). Also, the results of present work revealed that naringenin significantly inhibited the gentamicin-induced expression of caspase-9 and renal cell apoptosis. In agreement with present study, previous studies showed that gentamicin resulted in activation of caspase-9 and apoptotic renal cell death (Chen et al., 2011; Negrette-Guzmán et al., 2013). Therefore it could be stated that naringenin exerts its antiapoptotic activity via induction of survivin protein, and inhibition of caspase family of proteases, and to the best of our knowledge, this is the first study which shows the effect of gentamicin and naringenin on survivin expression in the kidney. Also, the antioxidant and anti-inflammatory activities may be responsible for the antiapoptotic effect of naringenin. The results of the present study indicate that naringenin provided a significant protective effect against gentamicin
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 8 ( 2 0 1 4 ) 420–429
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Fig. 7 – Immunohistochemical staining (200×) of caspase-9 in rat kidney from: (A) control group showing no expression of caspase-9; (B) vehicle plus gentamicin group showing a significant increase in caspase-9 immunoreactivity in the renal tubular cells; (C) naringenin plus gentamicin group showing a significant reduction in caspase-9 immunostaining. Brown color indicates caspase-9 positivity; (D) area of immunopositivity (m2 ), data are mean ± S.E.M., * P < 0.05 vs. control group, • P < 0.05 vs. vehicle plus gentamicin group, ND = non-detectable, NAR = naringenin, GEN = gentamicin. (For interpretation of the references to color in the text, the reader is referred to the web version of the article.)
nephrotoxicity in rats. The antioxidant and anti-inflammatory activities can be considered the main factors responsible for the nephroprotective effect of naringenin. Therefore, naringenin may represent a potential therapeutic option to prevent gentamicin-induced renal injury and dysfunction, which is a major and dose-limiting clinical problem.
Transparency document The Transparency document associated with this article can be found in the online version.
Acknowledgements Funding None.
The teamwork of the present study is greatly thankful to the Deanship of Scientific Research, King Faisal University for the sincere motivation and support.
references
Conflict of interest There is no conflict of interest.
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