Toxicology and Applied Pharmacology 277 (2014) 8–20

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Naringin ameliorates gentamicin-induced nephrotoxicity and associated mitochondrial dysfunction, apoptosis and inflammation in rats: Possible mechanism of nephroprotection Bidya Dhar Sahu a, Srujana Tatireddy b, Meghana Koneru a, Roshan M. Borkar c, Jerald Mahesh Kumar d, Madhusudana Kuncha a, Srinivas R. c, Shyam Sunder R. e, Ramakrishna Sistla a,⁎ a

Medicinal Chemistry and Pharmacology Division, Indian Institute of Chemical Technology (IICT), Hyderabad 500 007, India National Institute of Pharmaceutical Education and Research (NIPER), Hyderabad 500 037, India c National Centre for Mass Spectrometry, Indian Institute of Chemical Technology (IICT), Hyderabad 500 007, India d CSIR-Centre for Cellular and Molecular Biology (CCMB), Hyderabad 500 007, India e Faculty of Pharmacy, Osmania University, Hyderabad 500 007, India b

a r t i c l e

i n f o

Article history: Received 2 December 2013 Revised 24 February 2014 Accepted 28 February 2014 Available online 14 March 2014 Keywords: Gentamicin-induced nephrotoxicity Naringin Apoptosis Inflammation Mitochondrial dysfunction

a b s t r a c t Gentamicin-induced nephrotoxicity has been well documented, although its underlying mechanisms and preventive strategies remain to be investigated. The present study was designed to investigate the protective effect of naringin, a bioflavonoid, on gentamicin-induced nephrotoxicity and to elucidate the potential mechanism. Serum specific renal function parameters (blood urea nitrogen and creatinine) and histopathology of kidney tissues were evaluated to assess the gentamicin-induced nephrotoxicity. Renal oxidative stress (lipid peroxidation, protein carbonylation, enzymatic and non-enzymatic antioxidants), inflammatory (NF-kB [p65], TNF-α, IL-6 and MPO) and apoptotic (caspase 3, caspase 9, Bax, Bcl-2, p53 and DNA fragmentation) markers were also evaluated. Significant decrease in mitochondrial NADH dehydrogenase, succinate dehydrogenase, cytochrome c oxidase and mitochondrial redox activity indicated the gentamicin-induced mitochondrial dysfunction. Naringin (100 mg/kg) treatment along with gentamicin restored the mitochondrial function and increased the renal endogenous antioxidant status. Gentamicin induced increased renal inflammatory cytokines (TNF-α and IL-6), nuclear protein expression of NF-κB (p65) and NF-κB-DNA binding activity and myeloperoxidase (MPO) activity were significantly decreased upon naringin treatment. In addition, naringin treatment significantly decreased the amount of cleaved caspase 3, Bax, and p53 protein expression and increased the Bcl-2 protein expression. Naringin treatment also ameliorated the extent of histologic injury and reduced inflammatory infiltration in renal tubules. U-HPLS-MS data revealed that naringin co-administration along with gentamicin did not alter the renal uptake and/or accumulation of gentamicin in kidney tissues. These findings suggest that naringin treatment attenuates renal dysfunction and structural damage through the reduction of oxidative stress, mitochondrial dysfunction, inflammation and apoptosis in the kidney. © 2014 Elsevier Inc. All rights reserved.

Introduction Gentamicin (GM) is probably one of the most commonly used aminoglycoside antibiotics for the treatment of serious and life-threatening infections caused by Gram-negative aerobes (Negrette-Guzman et al., 2013). Despite its beneficial effects, low cost and low levels of resistance, serious complications like nephrotoxicity and ototoxicity are dose-limiting factors in the use of aminoglycosides (Ali et al., 2011; Sun et al., 2013). It has been reported that aminoglycoside antibiotics

⁎ Corresponding author. Fax: +91 40 27193189. E-mail address: [email protected] (R. Sistla).

http://dx.doi.org/10.1016/j.taap.2014.02.022 0041-008X/© 2014 Elsevier Inc. All rights reserved.

induce a dose-dependent nephrotoxicity in 10–25% of therapeutic courses; despite rigorous monitoring of serum drug concentrations and adequate fluid volume control (Martínez-Salgado et al., 2007). Although several studies have been undertaken to investigate the mechanisms underlying these unwanted side effects, the mechanism of nephrotoxicity induced by gentamicin is not completely known and remains to be studied further. Experimental evidence suggests the role of reactive oxygen/nitrogen species, in association with increased lipid peroxide formation and decreased activity of antioxidant enzymes in gentamicin-induced nephrotoxicity (Balakumar et al., 2010; Lee et al., 2012). Recent studies have also postulated that renal inflammation, which is characterized by infiltration of inflammatory cells such as monocytes/macrophages and subsequent release of pro-inflammatory cytokines and activation of NF-κB in

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response to oxidative stress, is involved in this process (Bae et al., 2013; Kalayarasan et al., 2009). Furthermore, apoptosis/necrosis of renal tubular epithelial cells (Juan et al., 2007; Lee et al., 2012; Sue et al., 2009), mitochondrial dysfunction (Morales et al., 2010; Negrette-Guzman et al., 2013; Servais et al., 2005) and activation of renal matrix metalloproteinases (Romero et al., 2009) are also involved in gentamicin-induced nephrotoxicity. Flavonoids occur ubiquitously in the plant kingdom and are common components of the human diet. In recent years, the use of naringin had received considerable attention as dietary antioxidant. Naringin (4′, 5, 7-trihydroxy flavanone 7-rhamnoglucoside) is a major and active flavanone glycoside found in grape fruits and other related citrus species (Singh et al., 2004). Numerous reports have documented the protective actions of naringin in various models of oxidative stress due to its direct free radical scavenger activity and its indirect antioxidant properties (Amudha and Pari, 2011; Badary et al., 2005; Singh et al., 2004). It is also reported that naringin up-regulates the gene expression of superoxide dismutase, catalase and glutathione peroxidase (Jeon et al., 2001). Experimental data further showed the hypolipidemic (Jeon et al., 2004), anticancer (Marchand et al., 2000; So et al., 1996), anti-inflammatory (Gopinath and Sudhandiran, 2012; Nie et al., 2012), cardioprotective (Rajadurai and Prince, 2009), antimutagenic (Higashimoto et al., 1998) and antimicrobial (Kim et al., 1998) properties of naringin. To the best of our knowledge, the effect of naringin against gentamicin-induced nephrotoxicity has not been studied. Based on these finding, we hypothesized that combining naringin with gentamicin would be a novel strategy to protect the kidney from gentamicin induced renal side effects. Therefore, the present study is initiated with an aim to evaluate the protective effect and the potential mechanisms of naringin against nephrotoxicity following gentamicin administration in rats.

Materials and methods Drugs and chemicals Gentamicin sulfate was obtained from Abbott Healthcare Pvt Ltd, India. Naringin (purity: ≥ 90% from citrus fruit), reduced glutathione (GSH), glutathione oxidized (GSSG), catalase, glutathione reductase, glutathione peroxidase, 2-thiobarbituric acid (TBA), superoxide dismutase assay kit, caspase 3 fluorimetric assay kit, caspase 9 substrate colorimetric, cytochrome c oxidase assay kit, o-dianisidine, Bradford reagent, 5, 5-dithio-bis (2-nitrobenzoic acid) (DTNB) were obtained from Sigma-Aldrich Co., St Louis, MO, USA. Antibodies against NF-kB (p65), cleaved caspase-3, Bax, Bcl-2, p53, lamin B1, β-actin and HRPconjugated secondary antibody were purchased from Cell Signaling Technology (Boston, MA). NE-PER nuclear and cytoplasmic extraction kit was obtained from Pierce Biotechnology, Rockford, IL, USA. NF-κB (p65) transcription factor assay kit was obtained from Cayman Chemical Company, Ann Arbor, MI. Rat TNF-α and IL-6 ELISA (Ready-SET-Go) kits were obtained from eBioscience, USA. All other chemicals were of analytical grade.

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Experimental design Rats were randomly divided into five groups consisting of eight rats in each and were treated as follows: Group I: Vehicle control (Control); rats were orally treated with gum acacia (2%) and an intraperitoneal (i.p.) injection of normal saline daily for a period of 7 consecutive days. Group II: Naringin control (Nar); rats were orally treated with naringin (100 mg/kg body weight) and an intraperitoneal (i.p.) injection of normal saline daily for a period of 7 consecutive days. Group III: Gentamicin control (GM); rats were orally treated with gum acacia (2%) and an intraperitoneal (i.p.) injection of gentamicin (120 mg/kg body weight) daily for a period of 7 consecutive days. Group IV: GN50; rats were treated daily with both naringin (50 mg/kg body weight, orally) and gentamicin (120 mg/kg body weight, intraperitoneally) at an interval of 1 h for a period of 7 consecutive days; Group V: GN100; rats were treated daily with both naringin (100 mg/kg body weight, orally) and gentamicin (120 mg/kg body weight, intraperitoneally) at an interval of 1 h for a period of 7 consecutive days. Because of very low aqueous solubility, naringin (purity ≥ 90% from citrus fruit, Sigma-Aldrich Co., St Louis, MO, USA), was suspended in 2% gum acacia. The dose of gentamicin (120 mg/kg, i.p.) was selected based on our pilot study (data not shown) and the doses of naringin (50 mg/kg and 100 mg/kg) were selected based on reported literature, in which naringin did not produce any detectable toxicity to experimental animals (Li et al., 2013). At the end of the study, whole blood samples were collected through retro-orbital plexus to obtain serum for the quantification of serum specific renal function parameters (blood urea nitrogen and creatinine). Body weights of all animals were recorded and the animals were humanely euthanized using carbon dioxide gas in an air-tight chamber. Kidney tissues were harvested, fatty and conjunctive tissue layer were removed, rinsed in normal saline and stored in − 80 °C freezer for further biochemical and immunoblot studies. Preparation of kidney tissues A known weight of the kidney tissue (right side) was homogenized in phosphate buffer saline (PBS, 50 mM, pH 7.4) containing 1% protease inhibitor cocktail (Sigma-Aldrich) to give a 10% homogenate suspension. A part of the homogenate was centrifuged at 14,000 rpm for 1 h, at 4 °C and the supernatant obtained was used for the estimation of various biochemical parameters. The protein contents were measured by using Bradford reagent (Sigma-Aldrich) against bovine serum albumin (BSA) as standard. To another part of the homogenate, an equal quantity of 10% trichloroacetic acid (TCA) was added, mixed properly and centrifuged at 5000 rpm for 15 min at 4 °C. The supernatant obtained was used for estimation of thiobarbituric acid reactive substance (TBARS) and vitamin C and the pellet obtained was used for estimation of protein carbonyl content.

Experimental animals

Biochemical analysis

Male Sprague–Dawley rats weighing between 180 and 200 g were obtained from National Institute of Nutrition (NIN), Hyderabad, India. Animals were housed in a central facility under controlled conditions (12 h light schedule, temperature at 22 ± 2 °C). Food and water were provided ad libitum. The experiments involved with animals were conducted according to the ethical norms of the CPCSEA, Government of India and after obtaining approval from the Institutional Animal Ethics Committee (IAEC) of the institute.

Non-enzymatic antioxidants Reduced glutathione was measured using DTNB [5, 5′ dithiobis-(2nitrobenzoic acid)]. This reagent reacts with the \SH groups to produce a yellow colored complex which has peak absorbance at 412 nm (Ellman, 1959). GSH was determined from a standard curve produced using commercially available standard GSH (Sigma-Aldrich). Levels of GSH were expressed as μg/g tissue. Kidney tissue vitamin C level from different experimental groups was estimated by using 2, 4-dinitrophenyl

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hydrazine reagent (2, 4-DNPH and 4% thiourea in 9 N H2SO4) (Omaye et al., 1979). The content of vitamin C was determined from a standard curve produced using commercially available standard vitamin C (Sigma-Aldrich) and was expressed as mg/g tissue. Enzymatic antioxidants The catalase activity was measured by the method of Aebi (1974), and the rate of decomposition of H2O2 was followed at 240 nm and the activity was expressed as U/mg protein. Total superoxide dismutase (SOD) activity in kidney tissues of different experimental groups was determined by using SOD assay kit (Sigma-Aldrich Co., St. Louis, MO, USA) and was expressed as percentage of activity while the vehicle control group was taken as 100%. Glutathione S-transferase (GST) activity was measured spectrophotometrically at 340 nm by mixing suitable amount of enzyme with 1 mm CDNB (1-chloro-2, 4-dinitrobenzene) and 1 mm GSH as described earlier by Habig et al. (1974). The GST activity was calculated at molar extinction coefficient of 0.0096 μM−1 cm−1 and was expressed as nmol of CDNB conjugated/min/ml. Glutathione reductase (GR) activity was measured as described by Carlberg and Mannervik (1975) by using 2 mm oxidized glutathione (GSSH) and 2 mm NADPH. The GR activity was calculated at molar extinction coefficient of 6.22 mM−1 cm−1 and was expressed as U/mg protein. Glutathione peroxidase activity was determined spectrophotometrically by employing 30 U glutathione reductase, 10 mM GSH, 2.5 mM NADPH and potassium phosphate buffer containing EDTA and sodium azide (0.25 M, pH 7). The enzyme activity was determined by measuring the disappearance of NADPH at 340 nm and was expressed as U/mg protein (Paglia and Valentine, 1967). Lipid peroxidation and protein carbonyl content Lipid peroxidation was determined in the kidney tissue of different experimental groups by the measurement of the thiobarbituric acid reactive substances (TBARS) as described earlier (Ohkawa et al., 1979). Similarly, protein carbonyl content in the kidney homogenate was determined as previously described, by reaction with DNPH and HCl and finally with guanidine hydrochloride (Dalle-Donne et al., 2003). Isolation of renal mitochondria Mitochondria were isolated from kidney tissues as described by Johnson and Lardy (1967). Briefly, a part of the kidney tissue (left side) from all experimental animals was homogenized in isolation buffer containing 10 mM Tris, 250 mM Sucrose, 1 mM EGTA, pH 7.2 at 4 °C using a Teflon Potter homogenizer to get 1:10 w/v homogenate. The homogenate was centrifuged at 600 g for 10 min to remove cellular debris and the resultant supernatant obtained was centrifuged at 10,000 g for 10 min at 4 °C. The mitochondrial pellet obtained was washed once with the same medium and another time with medium without EGTA and the same centrifugation procedure being used. The final pellets were resuspended in the storage buffer containing 10 mM Tris and 250 mM Sucrose to give a protein concentration of 1–2 mg/ml. Protein content was determined using Bradford reagent (Sigma-Aldrich) against bovine serum albumin (BSA) as standard. Mitochondrial enzyme activity assays NADH dehydrogenase activity assay NADH dehydrogenase (NDH) activity was measured spectrophotometrically by the method as described earlier (King and Howard, 1967). The method involves catalytic oxidation of NADH to NAD + with subsequent reduction of cytochrome C. Briefly, to reaction mixture containing 0.2 M glycylglycine buffer pH 8.5, 6 mM NADH in 2 mM glycylglycine buffer and 10.5 mM cytochrome C, mitochondrial sample from different experimental groups containing 100 μg protein was added and the change in optical density at 550 nm was

recorded for 180 s at 15 s intervals. NADH dehydrogenase activity was expressed as percentage activity where normal control group was taken as 100%. Succinate dehydrogenase activity assay Mitochondrial succinate dehydrogenase (SDH) activity in different experimental groups was measured spectrophotometrically according to the method that involves oxidation of succinate by an artificial electron acceptor, potassium ferricyanide, as described earlier (King, 1967). Briefly, to the reaction mixture containing 0.2 M phosphate buffer pH 7.8, 1% bovine serum albumin (BSA), 0.6 M succinic acid and 0.03 M potassium ferricyanide, mitochondrial sample containing 100 μg protein was added and the change in optical density at 420 nm was recorded for 120 s at 15 s intervals. Succinate dehydrogenase activity was expressed as percentage activity where normal control group was taken as 100%. Mitochondrial redox activity assay MTT [3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide] assay is a measure of viable cells. Mitochondrial redox activity was measured based on the reduction of MTT by hydrogenase activity in functionally intact mitochondria. The MTT reduction rate was used to assess the activity of the mitochondrial respiratory chain in isolated mitochondria by the method described earlier (Liu et al., 1997). Briefly, 100 μl mitochondrial samples were incubated with 10 μl MTT (5 mg/ml 50 mM phosphate buffer saline (PBS, pH 7.4) for 3 h at 37 °C. The blue formazan crystals thus formed were solubilized with dimethyl sulfoxide (DMSO) and optical density was recorded at 580 nm. Mitochondrial redox activity was expressed as percentage activity where normal control group was taken as 100%. Cytochrome c oxidase activity assay Cytochrome c oxidase (COX) activity was determined using cytochrome c oxidase assay kit (Sigma-Aldrich Co., St. Louis, MO, USA) according to the manufacturer's specifications. The assay is based on the decrease in absorbance at 550 nm of ferrocytochrome c caused by its oxidation to ferricytochrome c by cytochrome c oxidase. First, cytochrome c was reduced with dithiothreitol and then reoxidized by the cytochrome c oxidase. The difference in extinction coefficients between reduced and oxidized cytochrome c is 21.84 at 550 nm and was expressed as U/ml. DNA extraction and DNA fragmentation assay Genomic DNA was isolated from kidney tissues of normal as well as experimental rats by phenol/chloroform/isoamyl alcohol (PCI; 25:24:1, v/v/v, pH 8.0) method and was then electrophoresed (Bio-Rad, Gel Electrophoresis Unit) on 1.5% agarose gel in presence of 0.5 μg/ml ethidium bromide. The fragmented DNA (DNA ladder) was visualized by UV light using UV transilluminator (Bio Doc-It, Imaging system). Caspase (3 and 9) activity assay Caspase-3 activity in kidney tissue homogenate was measured by using fluorimetric assay kit (Fluorimetric caspase 3 assay kit) from Sigma-Aldrich St Louis, MO, USA at excitation and emission wavelengths 360 nm and 460 nm, respectively. Similarly, caspase-9 activity was assayed in kidney tissue homogenate of different experimental groups by colorimetric detection of p-nitroanilide at 405 nm after cleavage from a labeled substrate, acetyl-Leu-Glu-His-Asp-p-nitroanalide (Ac-LEHD-pNa) (Sigma-Aldrich St Louis, MO, USA). Changes in caspase activities in kidney tissue samples of gentamicin control group were calculated against the mean value of caspase activity in normal control tissue and expressed as fold increase over normal control. Assay of pro-inflammatory cytokines TNF-α and IL-6 concentrations in the renal tissue samples were analyzed using commercially available kits from ebiosciences, USA.

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The principle of assay was sandwich ELISA and limit of detection was 4 and 8 pg/ml for TNF-α and IL-6 respectively. Absorbance was taken at 450 nm. The protein level of supernatant was estimated and the TNF-α and IL-6 levels were expressed as pg/mg of protein. Myeloperoxidase activity assay Tissue-associated myeloperoxidase (MPO) activity is frequently utilized to estimate tissue neutrophil infiltration in inflamed tissues. Kidney tissue myeloperoxidase activity was assayed as a marker of neutrophil infiltration according to the method of Xia and Zweier (1997). The kidney tissue (left side) samples (100 mg) were homogenized in ice-cold potassium phosphate buffer (50 mM-K 2HPO4 , pH6) containing hexadecyltrimethylammonium bromide (0.5%, w/v) to get 10% homogenate. The homogenate was allowed for three freeze and thaw cycles and then centrifuged at 41,400 g for 10 min at 4 °C. MPO activity was assessed by measuring the H2O2-dependent oxidation of o-dianisidine. One unit of enzyme activity was defined as the amount of MPO present per gram of tissue weight that caused a change in absorbance of 1·0/min at 460 nm.

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binding site. After washing, samples were incubated by addition of specific primary antibody directed against NF-κB (p65). A secondary antibody conjugated to horseradish peroxidase (HRP) was added to provide a sensitive colorimetric readout at 450 nm.

Histopathology For the light microscopic investigations, kidney tissues (N = 3/group) from different experimental groups were fixed in 10% buffered formalin and processed routinely for embedding in paraffin. At least two sections of approximately 5 μm-thick, from each kidney tissue and a total of 6 histological slides (both left and right kidney) from each experimental group were prepared and stained with hematoxylin and eosin (H and E). The extent of kidney damage at the cortex region was assessed by examining the magnitude of renal proximal tubular degeneration, tubular necrosis and infiltration of inflammatory cells in a high power field (objective lens: 20 ×; magnification: 200 ×) under a light microscope (NIKON 800 photomicroscope).

Preparation of nuclear and total protein extracts Frozen renal tissue cortices (a fragment of kidney tissue of right side) from different experimental groups were homogenized in ice-cold RIPA buffer (Pierce Biotechnology, Rockford, IL, USA) containing 1% protease inhibitor cocktail (Sigma-Aldrich) to get total protein extracts. After being centrifuged at 12,000 g for 20 min at 4 °C, the supernatant was collected and used for analysis of cleaved caspase-3, p53, Bax and Bcl2 expression. Similarly, nuclear extracts were prepared by using NEPER nuclear and cytoplasmic extraction kit (Pierce Biotechnology, Rockford, IL, USA) containing 1% protease inhibitor cocktail (Sigma-Aldrich) according to the manufacturer's protocol and used for analysis of NF-κB (p65) protein expression as well as NF-κB-DNA binding assay. The protein contents were measured by using biscinchonic acid (BCA) kit (Pierce Biotechnology, Rockford, IL, USA) against bovine serum albumin (BSA) as standard. Western blot analysis Samples (40 μg/lane) were loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred to polyvinylidine difluoride (PVDF) membranes. The membranes were blocked with 3% BSA for 1 h and incubated with primary antibodies overnight at 4 °C against cleaved caspase-3 (rabbit monoclonal 1:1000, Cell Signaling Technology), p53 (mouse monoclonal 1:1000, Cell Signaling Technology), Bax (rabbit monoclonal 1:1000, Cell Signaling Technology), Bcl-2 (rabbit monoclonal 1:1000, Cell Signaling Technology) and NF-κB (p65) (rabbit monoclonal 1:1000, Cell Signaling Technology) followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (1:3000) for 1 h and visualization with the enhanced chemiluminescence system (Pierce Biotechnology, Rockford, IL, USA). β-actin was used as the loading control for total proteins and Lamin B1 was used as the internal control of nuclear proteins. Densitometric analysis of immunoblots was performed with the Image J software (NIH).

Estimation of gentamicin in kidney tissues U-HPLC-MS method was developed and validated for quantification of gentamicin in kidney tissues. The sample preparation for determination of gentamicin in kidney tissue was done by slightly modifying the method reported by Heller et al. (2005). In brief, kidney homogenate (200 μl), internal standard neomycin (10 μl, 10 ng/ml), water (140 μl) and 30% trichloroacetic acid (40 μl) were mixed together. Then the mixture was vortexed and centrifuged at 20,000 g for 15 min. The supernatant obtained was filtered using disposable PVDF syringe filters (0.2 μm pore size) in HPLC vials and injected into the U-HPLC-MS instrument. Analysis was carried out with the U-HPLC instrument (Thermo Scientific Accela, Germany) equipped with a quaternary pump, a de-gasser, a diode-array detector, an auto sampler and a column compartment. Mass spectrometric detection was carried out with an Orbitrap mass analyzer (Exactive Thermo Scientific, Germany) equipped with an electrospray ionization (ESI) source. The data acquisition was done using Xcalibur software. The typical operating source conditions for MS scan in positive ion ESI mode were optimized as follows: sheath gas flow rate 30; aux gas flow rate 10; spray voltage 4.50 kV; capillary temperature 330 °C; capillary voltage 72.50 V; tube lens voltage 160.0 V; skimmer voltage 16.00 V and heater temperature 250 °C. For full scan MS mode, the mass range was set at m/z 100–1000. All the spectra were recorded under identical experimental conditions and scan rate of 4.9 scans/s. Gentamicin concentration was determined by using Aquasil C-18 column (50 mm × 2.1 mm I.D., particle size 3 μm, Thermo Scientific), C18 HQ105 (10 × 2.1 mm) guard column and mobile phase comprising a mixture of acetonitrile: 0.1% trifluoroacetic acid (90:10). The other conditions were, flow rate 0.2 ml/min, column temperature 25 °C and injection volume 10 μl. The concentration of gentamicin in kidney tissues was expressed as ng/mg of kidney tissue.

Analysis of NF-κB (p65) activation by ELISA

Statistical analysis

The activation of NF-κB (p65) was assessed by an enzymatic immunoassay. The nuclear extract samples were used for analysis of NF-κB DNA-binding using the NF-κB (p65) transcription factor ELISA assay kit (Cayman Chemical Company, Ann Arbor, MI) according to the manufacturer's instructions. Briefly, nuclear extracts were incubated in the oligonucleotide-coated wells where the oligonucleotide sequence contains the NF-κB response element consensus-

Statistical analysis was carried out using the Graph Pad Prism, version 5.0 software. To test for statistically significant differences among various groups for a given parameter, one way analysis of variance (ANOVA) with Dunnett's multiple comparison procedure was used. All data were expressed as the mean and standard deviation (mean ± SD) or as percent activity compared to control group. Differences were considered statistically significant when p b 0.05.

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Results

Effect of naringin on gentamicin-induced renal tubular damage

Effect of naringin on gentamicin-induced renal dysfunction and associated oxidative stress

Light microscopic examination of kidney tissue in vehicle control (Fig. 2A) and naringin control (Fig. 2B) rats showed normal glomerular and tubular structures. Kidney section from gentamicin treated rats displayed tubular degeneration (white arrow), massive necrosis and foci of inflammation containing infiltration of inflammatory cells (yellow arrow) and the tubular lumen filled with pink color inflammatory fluid accumulation (asterisk) in proximal tubules (Fig. 2C). Kidney tissue sections from naringin (50 mg/kg) along with gentamicin treated rats showed moderate tubular degeneration (white arrow), and decreased necrosis and pink color inflammatory fluid accumulation in proximal tubules with mild infiltration of inflammatory cells when compared to gentamicin treated control rats (Fig. 2D). Photomicrographs of kidney sections of naringin (100 mg/kg) along with gentamicin treated rats showed apparently normal histoarchitecture with mild tubular degeneration without any inflammatory infiltration when compared to gentamicin treated control rats (Fig. 2E).

The levels of serum specific renal function parameters such as BUN (Fig. 1A) and creatinine (Fig. 1B) were significantly (p b 0.001) increased in gentamicin per se administered rats compared to vehicle control rats. Co-administration of naringin at two doses (50 and 100 mg/kg) to gentamicin administered rats significantly (p b 0.01) decreased the serum BUN and creatinine levels compared to gentamicin administered control rats. Naringin did not show dose dependent inhibition of BUN and creatinine in experimental groups. The differences in serum BUN and creatinine levels in naringin per se group are insignificant (p N 0.05) when compared to those of vehicle control rats. Gentamicin administration showed a significant (p b 0.001) increase in kidney to body weight ratios when compared to vehicle control group. The addition of naringin treatment (50 and 100 mg/kg) along with gentamicin significantly (p b 0.05) attenuated the increase in kidney to body weight ratios when compared to gentamicin alone treated rats (Fig. 1C). Gentamicin administration produced a significant (p b 0.001: GSH, GPx, GR, CAT and vitamin C; p b 0.01: GST and SOD) decrease in kidney tissue antioxidants when compared to vehicle control rats. Addition of naringin treatment at a dose of 100 mg/kg body weight to gentamicin significantly restored the levels of antioxidants (p b 0.001: GSH, GPx, GR, CAT and vitamin C; p b 0.01: GST; p b 0.05: SOD) when compared to gentamicin alone group. Though naringin treatment at the dose of 50 mg/kg body weight did not show significant change in GSH, GST, SOD and vitamin C levels, a significant (p b 0.01) increase in GPx, GR and CAT activities were observed when compared to gentamicin per se administered rats (Table 1).

Effect of naringin on gentamicin-induced renal lipid peroxidation and protein oxidation Gentamicin induced a significant (p b 0.05: for TBARS; p b 0.01: for carbonyl content) increase in kidney tissue thiobarbituric acid reactive substance (TBARS) (Fig. 3A) as an index of lipid peroxidation and protein carbonyl content (Fig. 3B) as an index of protein oxidation when compared to vehicle control rats. Animals treated with naringin at both doses along with gentamicin significantly abolished the increase in TBARS and carbonyl levels (50 mg/kg naringin: p b 0.01; 100 mg/kg naringin: p b 0.001, respectively) when compared to gentamicin per se administered rats.

Fig. 1. Effect of naringin on gentamicin-induced changes in serum specific renal function parameters, (A) Blood urea nitrogen (BUN) and (B) serum creatinine (Cr). (C) Effect of naringin on gentamicin-induced changes in kidney to body weight ratio. Values are the means ± SD (n = 8) with Control, group of animals treated with gum acacia (2%) along with normal saline (i.p.) daily for 7 consecutive days; Nar, group of animals treated with both naringin (100 mg/kg, orally) and normal saline (i.p.) daily for 7 consecutive days; GM, group of animals treated with gentamicin (120 mg/kg, i.p.) daily for 7 consecutive days; GN50, group of animals treated with both naringin (50 mg/kg, orally) and gentamicin (120 mg/kg, i.p.) daily for 7 consecutive days; GN100, group of animals treated with both naringin (100 mg/kg, orally) and gentamicin (120 mg/kg, i.p.) daily for 7 consecutive days. ⁎⁎⁎p b 0.001 vs. vehicle control group, ⁎p b 0.05 and ⁎⁎p b 0.01 vs. GM control group.

B.D. Sahu et al. / Toxicology and Applied Pharmacology 277 (2014) 8–20

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Table 1 Effect of naringin on gentamicin (GM) induced changes in renal GSH, GPx, GR, GST, SOD, CAT and vitamin C levels. Parameters estimated GSH levels (μg/g of tissue) GPx activity (U/mg protein) GR activity (U/mg protein) GST activity (nmols of CDNB conjugated/min/ml) SOD activity (% of control) CAT activity (U/mg protein) Vitamin C levels (mg/g tissue)

Control

GM

GN50

GN100

83.61 ± 15.22

58.57 ± 3.18$

75.52 ± 6.85

95.93 ± 12.5⁎⁎⁎

23.27 ± 1.78

15.87 ± 1.71#

19.39 ± 1.29⁎⁎

23.63 ± 0.86⁎⁎⁎

30.70 ± 3.33

13.94 ± 3.10#

26.04 ± 8.93⁎⁎

28.07 ± 3.93⁎⁎⁎

19.19 ± 4.09

11.88 ± 0.51$

15.86 ± 2.50

19.38 ± 2.28⁎⁎

100.00 ± 2.47

77.07 ± 5.21$

89.02 ± 10.60

95.39 ± 5.11⁎

11.14 ± 0.96

8.04 ± 1.14#

9.67 ± 0.44⁎

12.26 ± 1.21⁎⁎⁎

0.66 ± 0.08

0.45 ± 0.09#

0.56 ± 0.06

0.68 ± 0.09⁎⁎⁎

All data were expressed as mean ± SD, N = 8. Control, vehicle control rats treated with 2% gum acacia along with normal saline for 7consecutive days; GM, gentamicin control rats treated with 2% gum acacia (orally) along with gentamicin (120 mg/kg, i.p.) for 7 consecutive days; GN50, 50 mg/kg of naringin (orally) with GM (120 mg/kg, i.p.) for 7 consecutive days; GN100, 100 mg/kg of naringin (orally) with GM (120 mg/kg, i.p.) for 7 consecutive days; GSH, reduced glutathione; GPx, glutathione peroxidase; GR, glutathione reductase; GST, glutathione S-transferase; SOD, superoxide dismutase; CAT, catalase; CDNB, 1-chloro-2, 4-dinitrobenzene. $ p b 0.01. # p b 0.001 vs control. ⁎ p b 0.05. ⁎⁎ p b 0.01. ⁎⁎⁎ p b 0.001 vs GM control.

Effect of naringin on gentamicin-induced mitochondrial dysfunction The activities of NADH dehydrogenase (NDH) (Fig. 4A), succinate dehydrogenase (SDH) (Fig. 4B), mitochondrial redox activity (MTT) (Fig. 4C) and cytochrome c oxidase (COX) (Fig. 4D) were significantly

(p b 0.05: for NDH, SDH and COX; p b 0.001: for MTT) decreased in mitochondria isolated from gentamicin alone treated rats when compared with vehicle control rats. Naringin treatment (100 mg/kg) significantly (p b 0.05: for NDH and SDH; p b 0.01: for MTT and COX) restored mitochondrial enzyme NDH, SDH and COX activities and improved

Fig. 2. Representative photomicrographs of kidney sections from rats treated with naringin and/or gentamicin (H and E staining). Light microscopic examination (×20 objective lens) of kidney tissue in vehicle control (Fig. 2A, scale bar: 50 μm) and naringin treated control rats (Fig. 2B, scale bar: 50 μm) showing normal glomerular and tubular structures. Kidney sections of gentamicin treated rats showing tubular degeneration (white arrow), massive necrosis and foci of inflammation containing infiltration of inflammatory cells (yellow arrow) and the tubular lumen filled with pink color fluid accumulation (asterisk) (Fig. 2C, scale bar: 50 μm). Kidney tissue sections from naringin (50 mg/kg) along with gentamicin treated rats showing moderate tubular degeneration (white arrow), decreased necrosis and pink color fluid accumulation in proximal tubules with mild infiltration of inflammatory cells (yellow arrow) (Fig. 2D, scale bar: 50 μm). In contrast, photomicrographs of kidney sections of naringin (100 mg/kg) treated rats showing apparently normal histo-architecture with mild tubular degeneration without any inflammatory infiltration (Fig. 2E, scale bar: 50 μm). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. Effect of naringin on gentamicin-induced changes in lipid peroxidation and protein oxidation markers in kidney tissues of rats. (A) Thiobarbituric acid reactive substance (TBARS), (B) protein carbonyl content. Values are the means ± SD (n = 8). Control, group of animals treated with gum acacia (2%) along with normal saline (i.p.) daily for 7 consecutive days; GM, group of animals treated with gentamicin (120 mg/kg, i.p.) daily for 7 consecutive days; GN50, group of animals treated with both naringin (50 mg/kg, orally) and gentamicin (120 mg/kg, i.p.) daily for 7 consecutive days; GN100, group of animals treated with both naringin (100 mg/kg, orally) and gentamicin (120 mg/kg, i.p.) daily for 7 consecutive days. ⁎p b 0.05 and $p b 0.001 vs. vehicle control group, ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001 vs. GM control group.

mitochondrial redox activity (MTT) when compared to gentamicin alone treated rats. Naringin, at 50 mg/kg, significantly (p b 0.05) improved mitochondrial redox activity (MTT), but did not produce any significant effect on mitochondrial NDH, SDH and COX activities. Effect of naringin on gentamicin-induced renal inflammation Gentamicin administration produced a significant (p b 0.001) elevation of TNF-α (Fig. 5A) and IL-6 (Fig. 5B) levels when compared to vehicle control rats. Naringin treatment (100 mg/kg) along with gentamicin significantly (p b 0.01) decreased the TNF-α levels when compared with gentamicin control, whereas naringin, at 50 mg/kg, did not produce any significant change to TNF-α levels. However, IL-6 levels were significantly (p b 0.001) decreased in naringin treatment at both doses (50 mg/kg and 100 mg/kg) compared to gentamicin alone treated rats. Myeloperoxidase (MPO) activity was significantly (p b 0.001) increased in the kidney tissues of gentamicin alone treated

rats when compared to vehicle control rats and this increase was significantly (p b 0.01, at 50 mg/kg and p b 0.001, at 100 mg/kg naringin) decreased in naringin plus gentamicin treated rats (Fig. 5C). In addition, gentamicin significantly increased the nuclear translocation of p65 subunit of NF-κB (Fig. 5D) and NF-κB-DNA binding activity (Fig. 5E) compared to vehicle control rats. Naringin treatment at 100 mg/kg significantly reduced both nuclear NF-κB (p65) protein expression and DNA-binding activity when compared to gentamicin alone treated rats. Effect of naringin on gentamicin-induced renal apoptosis Gentamicin administration significantly increased the renal protein expression of Bax, p53 and cleaved caspase 3 and caspases (3 and 9) activities and significantly (p b 0.05) decreased the Bcl-2 protein expression when compared to vehicle control rats. Gel electrophoresis analysis of DNA extracted from whole kidney tissues of rats exhibited

Fig. 4. Effect of naringin on gentamicin-induced changes in mitochondrial enzyme activities. (A) NADH dehydrogenase, (B) succinate dehydrogenase, (C) mitochondrial redox activity (MTT assay), and (D) cytochrome C oxidase activity. Values are the means ± SD (n = 8). Control, group of animals treated with gum acacia (2%) along with normal saline (i.p.) daily for 7 consecutive days; GM, group of animals treated with gentamicin (120 mg/kg, i.p.) daily for 7 consecutive days; GN50, group of animals treated with both naringin (50 mg/kg, orally) and gentamicin (120 mg/kg, i.p.) daily for 7 consecutive days; GN100, group of animals treated with both naringin (100 mg/kg, orally) and gentamicin (120 mg/kg, i.p) daily for 7 consecutive days. $p b 0.05 and ⁎⁎⁎p b 0.001 vs. vehicle control group, ⁎p b 0.05 and ⁎⁎p b 0.01 vs. GM control group.

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Fig. 5. Effect of naringin on gentamicin-induced changes in inflammatory markers in kidney tissues of rats. (A) Tumor necrosis factor-α (TNF-α), (B) interleukin-6 (IL-6) and (C) myeloperoxidase (MPO) activity. Values are the means ± SD (n = 8). (D) Immunoblot analysis of nuclear NF-κB (p65) expression in the kidney tissue of rats treated with naringin and/or gentamicin. Lamin B1 expression was used as a loading control. Representative bar diagram showing quantitative relative levels of NF-κB (p65) protein for vehicle, GM and GM and naringin treated rats. Values are the means ± SD (n = 3). (E) Nuclear NF-κB (p65) DNA-binding activity was determined by using NF-κB p65) transcription factor ELISA assay kit. Values are the means ± SD (n = 6). Control, group of animals treated with gum acacia (2%) along with normal saline (i.p.) daily for 7 consecutive days; GM, group of animals treated with gentamicin (120 mg/kg, i.p.) daily for 7 consecutive days; GN50, group of animals treated with both naringin (50 mg/kg, orally) and gentamicin (120 mg/kg, i.p.) daily for 7 consecutive days; GN100, group of animals treated with both naringin (100 mg/kg, orally) and gentamicin (120 mg/kg, i.p) daily for 7 consecutive days. # p b 0.001 vs. vehicle control group, ⁎⁎p b 0.01 and ⁎⁎⁎p b 0.001 vs. GM control group.

marked DNA smearing with fragmentation in the gentamicin alone treated rats. Addition of naringin (100 mg/kg) treatment along with gentamicin effectively reduced the gentamicin-induced renal expression of Bax, p53, cleaved caspase 3 and activities of caspases and normalized the Bcl-2 expression when compared to gentamicin alone treated rats. Furthermore, naringin treatment also prevented the gentamicin-induced DNA shearing (Fig. 6).

Effect of naringin on kidney tissue levels of gentamicin The gentamicin consists of four components at three different molecular weights (Fig. 7). The lower limit of quantification for gentamicin was found to be 2 ng/10 mg of tissue. The method was found to be linear over 2–4000 ng/ml. Fig. 8 shows the U-HPLC/ESI-MS-EIC of gentamicin components. The C1, C2 and C1a components of gentamicin at m/z 450.2945, 464.3094 and 478.3232, respectively, were eluted at 0.80 min. and Neomycin, internal standard, at m/z 615.3224 was eluted at 0.95 min. The concentration of gentamicin in gentamicin alone treated rats, naringin 50 mg/kg and 100 mg/kg along with gentamicin treated rats showed 205 ± 29.6, 193.5 ± 14.2 and 199.4 ± 25.5 ng/mg of kidney tissue respectively (Fig. 9). The UHPLC-MS data revealed that co-administration of naringin along with gentamicin did not alter the uptake and accumulation of gentamicin in kidney tissues of different experimental rats.

Discussion Gentamicin is an effective and widely used antibiotic against serious and life-threatening infections caused by Gram-negative aerobes in clinical practice (Bledsoe et al., 2006). However, the major dose limiting side effect of gentamicin is its nephrotoxicity, which limits its clinical utility (Juan et al., 2007; Sue et al., 2009). Thus, a therapeutic approach to protect or reverse gentamicin-induced kidney injury would have significant clinical value. Naringin, a flavanone type of polyphenolic bioflavonoid is abundantly present in citrus species. The content of naringin in various sources of citrus fruits is reported to be as follows; grapefruit (varies from 17 to 76 mg/100 ml juice) (Chanet et al., 2012), commercial sweet orange (2.31 mg/100 ml juice), commercial lemon (0.38 mg/100 ml of juice) and commercial bergamot juice (26.1 mg/100 ml of juice) (Gattuso et al., 2007). It has also been reported that the daily intake of flavanones in adults from different countries was estimated to range from 2.7 to 78 mg of aglycone equivalent (Chanet et al., 2012). In the present study, we aimed to investigate the effect and to elucidate the mechanism of action of naringin, against gentamicin-induced nephrotoxicity, in order to gain new insights into the treatment modality of gentamicin-induced nephrotoxicity. Gentamicin administration at the dose of 120 mg/kg (i.p.) for 7 consecutive days produced a significant elevation of blood urea nitrogen and creatinine levels in serum and renal tubular degeneration with

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Fig. 6. Effect of naringin on gentamicin-induced changes in apoptotic markers in kidney tissues of rats. Immunoblot analysis of (A) cleaved caspase 3, p53, Bax and Bcl-2 protein expression in the kidney tissue of rats treated with naringin and/or gentamicin. β-actin expression was used as a loading control. Representative bar diagram showing quantitative results for relative levels of (B) cleaved caspase 3, (C) p53, (D) Bax and (E) Bcl-2 proteins for vehicle, GM and GM and naringin treated rats. Values are the means ± SD (n = 3). (F) DNA fragmentation using agarose gel electrophoresis of DNA isolated from kidney tissues. NC lane, showing intact DNA; GM lane, showing both smearing and laddering pattern of DNA damage; GN50 lane, showing decreased amount of DNA laddering; and GN100 lane, showing without smearing and laddering pattern of DNA damage. Caspase 3 (G) and caspase 9 (H) activities of kidney tissue of rats treated with naringin and/or gentamicin. Values are the means ± SD (n = 8). Control, group of animals treated with gum acacia (2%) along with normal saline (i.p.) daily for 7 consecutive days; GM, group of animals treated with gentamicin (120 mg/kg, i.p.) daily for 7 consecutive days; GN50, group of animals treated with both naringin (50 mg/kg, orally) and gentamicin (120 mg/kg, i.p.) daily for 7 consecutive days; GN100, group of animals treated with both naringin (100 mg/kg, orally) and gentamicin (120 mg/kg, i.p) daily for 7 consecutive days. #p b 0.01 vs. vehicle control group, ⁎p b 0.05, ⁎⁎p b 0.01 and ⁎⁎p b 0.001 vs. GM control group.

pink color fluid accumulation along with massive necrosis and foci of inflammation containing infiltration of inflammatory cells in kidney tissues. A marked reduction in body weight without any mortality was observed in response to gentamicin administration for 7 consecutive days (data not shown). The decrease in body weight could be attributed to reduction of food intake and inhibition of protein synthesis due to gentamicin treatment. Since we have not observed any significant difference of serum levels of BUN and creatinine and kidney histomorphology between vehicle control and naringin control animals, we excluded naringin per se treated group for further biochemical and immunoblot analysis. In the present study, addition of naringin (100 mg/kg) treatment to gentamicin significantly reduced the BUN and creatinine levels, decreased kidney to body weight ratios and produced apparently normal histo-architecture with mild tubular degeneration and without any inflammatory infiltration when compared to gentamicin alone treated rats. It is well established that overproduction of reactive oxygen species (ROS) damage the protein molecules and degrade the membrane bound phospholipids through the process of lipid peroxidation (Sahu et al., 2013). In this study, gentamicin treated rats showed an increased lipid

peroxidation and protein oxidation compared to vehicle control group of rats. These findings favor the hypothesis that gentamicin induced excess free radical production stimulates lipid peroxidation and alters cellular membrane integrity. Naringin (100 mg/kg) treatment along with gentamicin significantly ameliorated this increase in lipid peroxidation and protein carbonylation compared to gentamicin alone treated rats. Reduced glutathione (GSH) is an important and most abundant cellular detoxifying antioxidant in body. Gentamicin increases the generation of reactive oxygen species like hydroxyl radicals, superoxide anions and hydrogen peroxides and reactive nitrogen species in the renal cortex and aggravate renal structural deterioration (Balakumar et al., 2010). In the present study, kidney tissue antioxidant enzyme (SOD, CAT, GPx, GR and GST) activities and the levels of non-enzymatic antioxidants (GSH and vitamin C) were significantly decreased in gentamicin alone treated rats. Here, we believe that decreased amount of GSH levels in gentamicin administered rats due to excessive production of gentamicin induced free radicals or its increased utilization in protecting \SH group containing proteins from free radicals. Similarly, decreased amounts of superoxide dismutase and catalase activities may be

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Fig. 7. Chemical structure and MS fragmentation pattern of gentamicin components.

attributed to excessive formation of superoxide anions and hydrogen peroxide and/or their inactivation by excessive gentamicin induced oxidants (Pedraza-Chaverri et al., 2000). Addition of naringin treatment at a dose of 100 mg/kg body weight along with gentamicin significantly restored the gentamicin-induced decrease in the antioxidant levels when compared to gentamicin alone treated rats. It has also been reported that naringin activates Nrf2 (nuclear factor-erythroid 2-related factor-2) and induces phase II detoxifying and antioxidant genes in an in vivo model (Gopinath and Sudhandiran, 2012). These observations support the hypothesis that the protective effect imposed by naringin might be attributed to its intrinsic antioxidant activity (directly neutralizing reactive species) and/or its ability to enhance antioxidant defense system in renal tissues. Our findings corroborate those of earlier studies in which enhanced endogenous oxidative stress has a major role in the severity of gentamicin-induced nephrotoxicity (Lee et al., 2012; Morales et al., 2010). Mitochondrial dysfunction is one of the earliest indices of gentamicin-induced nephrotoxicity, which produces excessive ROS, resulting in morphological and functional changes (Jia et al., 2013; Morales et al., 2010). Simmons et al. (1980) demonstrated that gentamicin inhibited oxidative phosphorylation and reduced ATP levels in renal tubular cells. In the present study, gentamicin administration caused a marked impairment in mitochondrial respiratory enzyme activities compared to vehicle control group. The decrease in the activities of NADH dehydrogenase enzymes might be due to depletion of reducing equivalents like NADH and NADPH which are utilized for the formation of reduced glutathione to counter oxidative damage of mitochondrial components (Karthikeyan et al., 2007). Administration of naringin at 100 mg/kg dose along with gentamicin significantly improved mitochondrial function as evident from the increase in activities of mitochondrial respiratory enzymes compared to gentamicin alone treated rats. It is known that the MTT assay is a metabolic activity assay that measures mitochondrial function and is used to detect alteration in mitochondrial redox activity due to drugs or toxicants (Negrette-Guzman et al., 2013). Therefore, it is speculated that the observed decrease in MTT reduction activity in gentamicin treated rats is due to gentamicin-induced impairment in mitochondrial function. The increase in mitochondrial redox activity (increased MTT reduction) with naringin treatment is due to restoration in mitochondrial functions and respiration rate. This finding supports our hypothesis that naringin prevents gentamicin-

induced nephrotoxicity partly by restoration of mitochondrial function. Earlier studies have reported that renal injury as a consequence of gentamicin-induced tubular necrosis stimulates inflammatory events at the site of injury and enhances the migration of monocytes and macrophages to the site of tissue damage (Balakumar et al., 2010). Activation and nuclear translocation of NF-κB, in response to oxidative stress, are thought to be the key factors in the renal inflammatory process by regulating the gene expression of cytokines, chemokines and adhesion molecules (Bae et al., 2013). In the present study, addition of naringin (100 mg/kg) treatment along with gentamicin significantly decreased the renal nuclear protein expression of NFκB and NF-κB-DNA binding activities and levels of pro-inflammatory cytokines (TNF-α and IL-6) compared to gentamicin alone treated rats. Our findings corroborate those of earlier studies demonstrating that an increase in NF-κB activation is also followed by an increase in the concentration of inflammatory cytokines like TNF-α and IL-6 (Bae et al., 2013; Kalayarasan et al., 2009). Myeloperoxidase (MPO) activity in the renal cortex is an index of neutrophil accumulation; therefore an increase in MPO activity reflects the inflammatory infiltration to the site of tissue injury (Guo et al., 2013). In the present study, gentamicin treatment increased the levels of renal MPO activity, indicating neutrophil infiltration. Naringin treatment decreased renal neutrophil infiltration as evidenced by suppression of renal MPO activity and the improvement of histological features. The above findings indicate the ameliorative effect of naringin against gentamicin-induced nephrotoxicity through its anti-inflammatory effect. Apoptosis plays an important role not only in the physiological processes of the kidney, but also in various human renal diseases and drug-induced nephrotoxicity (Chen et al., 2011; Servais et al., 2008). Gentamicin treatment causes acute renal failure with acute tubular necrosis in about 20% of patients (Juan et al., 2007). Caspasedependent apoptotic signaling plays a major role in gentamicininduced apoptotic injury. Caspase-3 is an executioner caspase that can be activated by caspase-9 in the mitochondrial pathway (Chen et al., 2011). It is well known that during apoptosis, Bax acts as a proapoptotic protein, whereas Bcl-2 acts as an anti-apoptotic protein. The Bcl-2 proteins bind to the outer membrane of mitochondria and block cytochrome c activation (Kalkan et al., 2012). In this contest, oxidative stress seems to play a major role in mitochondrial dysfunction which is an important early event in the intrinsic pathway of apoptosis

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Fig. 8. U-HPLC-ESI-MS extracted ion chromatogram (EIC) of different components of gentamicin. (A) C1a: m/z 450, (B) C2, C2a: m/z 464 and (C) C1: m/z 478 and (D) internal standard neomycin: m/z 615. Each chromatogram is the sum of three ions. Kidney tissue level of gentamicin was calculated based on the sum of total ion counts from a standard curve.

(Morales et al., 2010). Gentamicin administration increased the protein expression of cleaved caspase 3, Bax and p53 and down regulated Bcl-2 expression. The gel electrophoresis of DNA isolated from different

experimental groups confirmed both smearing and laddering pattern of DNA break. Our findings clearly revealed that gentamicin treatment triggers both apoptosis and necrosis in kidney tissues. Addition of

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Conflict of interest The authors declare that they have no conflicts of interest. Acknowledgment This work was partially supported by research grant from CSIR project ‘SMiLE’ (CSC 0111). The authors thank Director, IICT, Hyderabad for constant support and encouragement. B.D.S. thanks the Council of Scientific and Industrial Research (CSIR), New Delhi for financial assistance in the form of Senior Research Fellowship. Fig. 9. Effect of naringin on kidney tissue levels of gentamicin. Values are the means ± SD (n = 6). Control, group of animals treated with gum acacia (2%) along with normal saline (i.p.) daily for 7 consecutive days; GM, group of animals treated with gentamicin (120 mg/kg, i.p.) daily for 7 consecutive days; GN50, group of animals treated with both naringin (50 mg/kg, orally) and gentamicin (120 mg/kg, i.p.) daily for 7 consecutive days; GN100, group of animals treated with both naringin (100 mg/kg, orally) and gentamicin (120 mg/kg, i.p.) daily for 7 consecutive days. The concentration of gentamicin in kidney tissue was expressed as ng/mg of kidney tissue.

naringin treatment to gentamicin significantly prevented renal tubular apoptosis/necrosis when compared to gentamicin alone treated rats. Earlier studies have shown that NF-κB activation promotes gentamicin-induced apoptosis in rat renal tubular cells (Chen et al., 2011; Juan et al., 2007). Our findings also revealed NF-κB activation upon gentamicin administration in renal tissue of rats. Hence, it is speculated that the pro-apoptotic character of NF-κB might be due to its direct activation of apoptotic genes including p53, or down regulation of the activities of some anti-apoptotic proteins like Bcl-2 (Chen et al., 2011). Thus, the result of the present study revealed that naringin attenuated NF-κB activation and renal tubular apoptosis/necrosis associated with gentamicin-induced nephrotoxicity. Our findings are also in agreement with previously reported literature (Jia et al., 2013). In all these investigations related to oxidative stress, mitochondrial respiratory enzyme activity, inflammation and apoptosis, naringin was found to offer better protection at a dose of 100 mg/kg compared to a lower dose. The lower efficacy of naringin at a low dose of 50 mg/kg might be due to its low intestinal absorption (bioavailability of approximately 4%). It is well documented that accumulation of gentamicin in the proximal tubular cells of the kidney is responsible for its deleterious effects (Quiros et al., 2011; Sawada et al., 2012). Electrostatic interaction between gentamicin and negatively charged membrane phospholipids i.e. megalin/cubulin complex facilitates the gentamicin uptake to tubular cells through endocytosis (Quiros et al., 2011). Thus, if at all there is involvement of megalin/cubulin complex in the present study, we expect alterations in gentamicin levels in kidney tissue samples. Gentamicin concentrations in the kidney tissues of different experimental groups were estimated employing U-HPLC-MS. The data obtained from U-HPLC-MS revealed that coadministration of naringin at both doses (50 and 100 mg/kg) along with gentamicin did not alter the kidney tissue levels of gentamicin. As we have not observed any statistical significant difference in gentamicin concentrations among gentamicin per se, gentamicin with 50 mg/kg naringin and gentamicin with 100 mg/kg naringin treated rats, the role of megalin/cubulin complex in the present study is questionable. Therefore, we believe that naringin, by virtue of its antioxidant properties, attenuates the gentamicin-induced nephrotoxicity in rats. In conclusion, the present study suggests that the flavonoid naringin has a renal protective potential. The nephroprotective effect of naringin against gentamicin-induced renal toxicity may be ascribed to its anti-oxidant, anti-apoptosis and anti-inflammatory properties. Collectively, our results suggest that naringin may be a promising molecule for the treatment of gentamicin-induced nephrotoxicity.

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