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Protective effect of allicin against gentamicin-induced nephrotoxicity in rats
INTIMP-03884; No of Pages 8 International Immunopharmacology xxx (2015) xxx–xxx
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Protective effect of allicin against gentamicin-induced nephrotoxicity in rats Dalia H. El-Kashef, Asmaa E. El-Kenawi, Ghada M. Suddek ⁎, Hatem A. Salem Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
a r t i c l e
i n f o
Article history: Received 23 July 2015 Received in revised form 15 August 2015 Accepted 13 September 2015 Available online xxxx Keywords: Gentamicin Allicin Nephrotoxicity Urinary bladder Nitric oxide TNF-α
a b s t r a c t In this study, the modulator effect of allicin on the oxidative nephrotoxicity of gentamicin in the kidneys of rats was investigated by determining indices of lipid peroxidation and the activities of antioxidant enzymes, as well as by histological analyses. Furthermore, the effect of allicin on gentamicin induced hypersensitivity of urinary bladder rings to ACh was estimated. Twenty-four male Wistar albino rats were randomly divided into three groups, control, gentamicin (100 mg/kg, i.p.) and gentamicin + allicin (50 mg/kg, orally). At the end of the study, all rats were sacrificed and then urine, blood samples and kidneys were taken. Gentamicin administration caused a severe nephrotoxicity as evidenced by an elevated kidney/body weight ratio, serum creatinine, blood urea nitrogen (BUN), serum lactate dehydrogenase (LDH) and proteinuria with a reduction in serum albumin and creatinine clearance as compared with control group. In addition, a significant increase in renal contents of malondialdehyde (MDA), myeloperoxidase (MPO), nitric oxide (NOx) and tumor necrosis factor-alpha (TNFα) concomitantly with a significant decrease in renal reduced glutathione (GSH) and superoxide dismutase (SOD) activities was detected upon gentamicin injection. Exposure to gentamicin increased the sensitivity of isolated urinary bladder rings to ACh and induced acute renal tubular epithelial cells necrosis. Administration of allicin significantly decreased kidney/body weight ratio, serum creatinine, LDH, renal MDA, MPO, NOx and TNF-α while it significantly increased creatinine clearance, renal GSH content and renal SOD activity when compared to gentamicin-treated group. Additionally, allicin significantly reduced the responses of isolated bladder rings to ACh and ameliorated tissue morphology as evidenced by histological evaluation. Our study indicates that allicin exerted protection against structural and functional damage induced by gentamicin possibly due to its antioxidant, anti-inflammatory and immunomodulatory properties in addition to its ability to retaining nitric oxide level. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Gentamicin (GNT) is commonly applied in human clinical practices for treatment of life-threatening Gram-negative infections [1,2]. However, the usefulness of GNT is limited by the development of nephrotoxicity. In some cases, this side effect is so severe that the use of the drug must be discontinued. In spite of the introduction of newer and less toxic antibiotics, GNT is still used clinically because of its rapid bactericidal action, broad-spectrum activity, chemical stability, and low cost [3,4]. GNT-induced nephrotoxicity is characterized by direct tubular necrosis, without morphological changes in glomerular structures [1]. The mechanism of gentamicin-induced nephrotoxicity is not completely known. However, studies have implicated reactive oxygen species particularly superoxide anion radical in the pathophysiology of gentamicin nephropathy [5]. It has been demonstrated that gentamicin administration increases renal cortical lipid peroxidation, nitric oxide generation and mitochondria hydrogen peroxide production [1,6,7,8]. Abnormal ⁎ Corresponding author at: Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt. E-mail address: [email protected] (G.M. Suddek).
production of such molecules may damage macromolecules, induce cellular injury and necrosis via several mechanisms including peroxidation, protein denaturation and DNA damage [6,9]. The alteration in kidney functions induced by lipid peroxidation is a proximal event in the injury cascade of gentamicin mediated nephrotoxicity [1]. Accordingly, the administration of compounds with antioxidant activity has been successfully used to prevent or ameliorate gentamicin-induced nephrotoxicity [1,5]. In the past few years, much interest has been laid on the role of naturally occurring dietary substances for the control and management of various chronic diseases [10]. Allicin (diallyl thiosulfinate), the major pharmacological component of garlic [11], has attracted attention of the international medical field gradually due to its potential for disease prevention and treatment. It is formed by the action of the enzyme alliinase on alliin in crushed fresh garlic cloves. It possesses antioxidant activity and is shown to cause a variety of actions potentially useful for human health. Allicin exhibits hypolipidemic, antiplatelet, antibacterial, and antifungal effects. It has been reported that allicin inhibits various cancer cells demonstrating anticancer and chemopreventive activities [12,13]. The present study aimed at studying the renoprotective properties of allicin against gentamicin-induced nephrotoxicity.
http://dx.doi.org/10.1016/j.intimp.2015.09.010 1567-5769/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: D.H. El-Kashef, et al., Protective effect of allicin against gentamicin-induced nephrotoxicity in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.010
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2. Materials and methods
The amount of the complex formed is directly proportional to the creatinine concentration. A kit from Biodiagnostics Co., Egypt was used.
2.1. Chemicals 2.5. Determination of blood urea nitrogen (BUN) Allicin was obtained as pharmaceutical drug (Allimax capsule containing 100% allicin powder, Allisure ® AC-23) obtained from (Allimax Nutriceuticals, US). Gentamicin was purchased as a pharmaceutical preparation (Gentamicin ampoules 80 mg, Alexandria Chemical Co., Egypt). All other chemicals and reagents used were of the highest analytical grade commercially available.
Urea was measured enzymatically in rat serum according to Fawcett and Scott [17]. Urea in the sample was hydrolyzed by urease enzyme to yield ammonia and carbon dioxide. In a modified Berthelot reaction, the blue dye indophenol product reaction absorbs light proportional to initial urea concentration and was measured spectrophotometrically at 550 nm. A kit from Biodiagnostics Co. was used.
2.2. Animals Twenty-four adult male Wistar rats weighing 160–200 g were used for the experimental procedures. Rats were allowed 1 week to adapt to the surroundings before beginning any experimentation. Animals were housed in individual plastic cages with bedding. Standard rat food and tap water were available ad libitum for the duration of the experiments unless otherwise noted. Temperature was maintained at 25 °C with 12/ 12-h light/dark cycle. All animal experiments described in this study comply with the ethical principles and guidelines for the care and use of laboratory animals adopted by “Research Ethics Committee, Faculty of Pharmacy, Mansoura University”. 2.3. Experimental design Gentamicin-induced acute kidney injury was established in male Wistar rats. The animals were randomly divided into three groups containing eight rats in each group. Group (1): Control group, rats did not receive any solvent or drug during the experiment and were on a usual diet. Group (2): Gentamicin (GNT) group, rats were injected with GNT (100 mg/kg, i.p.) for 7 consecutive days [14]. Group (3): Gentamicin/Allicin (GNT/AL) group, rats were injected with GNT (100 mg/kg, i.p.) and received AL (50 mg/kg, orally) [15] starting seven days before GNT injection till the end of experimental study. Allicin treatment schedule and dose level were chosen after pilot experiments. After the last dose, all control and experimental animals were immediately kept in individual metabolic cages for collection of 24 h urine samples. These samples were centrifuged for 15 min at 3000 rpm, and kept frozen until analyzed. Blood samples were obtained from overnight fasted animals through retro-orbital sinus, under diethyl ether anesthesia, into non-heparinized tubes. The collected blood samples were allowed to clot for 30 min at 25 °C. After clotting, they were centrifuged at 1000 ×g, 4 °C for 15 min. using cooling centrifuge (Damon/IEC Division, Model: CRU-5000, Needham, Mass., USA). Sera were collected and stored frozen for the determination of levels of Cr, BUN, albumin and LDH. The animals were sacrificed after anesthesia by cervical dislocation, then the lower abdomen was opened and the contractile response of the isolated urinary bladder rings towards ACh was tested. Fatty adherents from the kidneys were removed and the kidneys were weighed using a digital balance to calculate the kidney body weight ratio. The left kidneys were excised immediately, rinsed in ice-cold normal saline (0.9% w/v), homogenized in 0.1 M phosphate buffer (pH 7.4) to yield 10% w/v tissue homogenates that were then used for biochemical assays. The right kidneys were harvested for histopathological examination. 2.4. Determination of serum creatinine Creatinine was measured in rat serum as described by Bartels et al. [16]. Creatinine in alkaline solution reacts with picric acid to form a colored complex that was measured spectrophotometrically at 550 nm.
2.6. Determination of creatinine clearance (Ccr) Glomerular filtration was assessed by creatinine clearance based on serum and urine creatinine levels, with values expressed in ml/min, computed with the formula: Ccr = urine creatinine (mg/dl) × urine flow (ml/min)/Serum creatinine (mg/dl). Urine flow was calculated dividing 24 h of urine volume by 1440, which corresponds to the number of minutes in 24 h (60 min × 24 h = 1440): urine flow (ml/min) = value of urine volume (24 h) / 1440. 2.7. Determination of protein in urine Protein in urine was determined by Folin–Lowry colorimetric method; two reactions are involved: (a) an initial interaction of protein and Cu+2 in alkali (related to biuret reaction); (b) a reduction of the phosphotungstic and phosphomolybdic acids to molybdenum blue and tungsten blue both by the Cu–protein complex and by the tyrosine and tryptophan of the protein. The latter two give color in the absence of Cu+2 but the rest of the protein gives no color without Cu+2. About 75% of the color is dependent on the Cu+2. The maximum absorption of the colored products is at 750 nm [18]. 2.8. Determination of serum albumin Albumin, in the presence of bromocresol green at a slightly acid pH, produces a color change in the indicator from yellow-green to greenblue. The intensity of the color formed is proportional to the albumin concentration in the sample [19]. 2.9. Determination of serum lactate dehydrogenase Lactate dehydrogenase (LDH) activity was assessed according to the method described by Henry [20]. The method depends on the conversion of pyruvate to lactate by LDH consuming NADH+, which absorbs at 340 nm. Its consumption is directly proportional to serum LDH concentration. LDH activity was calculated as units/l (U/l). 2.10. Determination of lipid peroxidation Lipid peroxidation was determined by the method of Ohkawa et al. [21]. The principle of this method being that malondialdehyde (MDA), an end product of lipid peroxidation, reacts with thiobarbituric acid (TBA) to form a pink chromogen. For this assay, 0.2 ml of 8.1% sodium dodecyl sulfate (SDS), 1.5 ml of 20% acetic acid (pH 3.5) and 1.5 ml of 0.8% thiobarbituric acid aqueous solution were added in succession in a reaction tube. To this reaction mixture, 0.2 ml of the kidney homogenate was added, and the mixture was then heated in boiling water for 60 min. After cooling to room temperature, 5 ml of butanol: pyridine (15:1, v/v) solution was added. The mixture was then centrifuged at 2236 × g for 15 min following which the upper layer was separated, and the intensity of the resulting pink color was read at 532 nm.
Please cite this article as: D.H. El-Kashef, et al., Protective effect of allicin against gentamicin-induced nephrotoxicity in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.010
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Tetramethoxypropane was used as an external standard and the level of lipid peroxides was expressed as nmol of MDA formed/g wet tissue. 2.11. Estimation of superoxide dismutase (SOD) activity Superoxide dismutase activity in kidney homogenates, as an index of endogenous antioxidant activity, was measured spectrophotometrically by monitoring the SOD-inhabitable autooxidation of pyrogallol, as described by Marklund [22]. In brief, the reaction mixture consisted of 24 mmol/l of pyrogallol in 10 mM HCl, Tris buffer (pH 7.8). The reaction was carried out at 25 °C. The change in absorbance at 420 nm was recorded for 3 min at 1 min interval. One unit of enzyme activity is defined as 50% inhibition of pyrogallol autooxidation under the assay conditions. The SOD activity was expressed as Unit/g wet tissue. 2.12. Estimation of reduced glutathione (GSH) activity Reduced glutathione activity in kidney homogenates was determined according to the method described by Ellman [23]. The method based on the reduction of Ellman's reagent by (− SH) groups of GSH to produce a yellow colored 2-nitro-5-mercaptobenzoic acid. The produced chromogen is directly proportional to GSH concentration. The absorbance was measured spectrophotometrically at 412 nm and the concentrations were expressed as μmol/g wet tissue. − 2.13. Measurement of NO− 2 /NO3 concentration − Nitrite/nitrate (NO− 2 /NO3 ) production, an indicator of NO synthesis, was measured in the supernatant of the kidney homogenate using a commercially available NO assay kit (R&D Systems, Minneapolis, USA) following the manufacturer's instruction.
2.14. Measurement of TNF-α level Tumor necrosis factor-alpha concentrations were measured in kidney tissue homogenates using an enzyme-linked immunosorbent assay kit (Bender Med Systems GmbH, Vienna, Austria). Kidney tissue homogenate was added to a microtitre plate pre-coated with a monoclonal antibody specific for rat tumor necrosis factor-alpha. Incubation, plate washing and quenching of reactions were carried out according to the kit manufacturer's instruction and tumor necrosis factor-alpha levels were expressed as pg/mg.
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prior to the experiment under a resting load of 1 g [26]. During this time, the bath solution was replaced every 5 min. Isometric tension generated by the smooth muscle was measured by means of an isometric transducer (serial no. 88576, Biegestab K30, Hugo Sachs Elektronik, Fedral Republic of Germany) recorded with a Powerlab unit/400 linked to a PC running Chart v 4.2 software (ADInstruments Pty Ltd., Australia). Concentration–response curves to acetylcholine (ACh) were constructed. The rings were exposed to different concentrations of ACh in a cumulative manner. Exposure to each concentration of ACh was maintained until the maximal response to that concentration was reached. The responses of the bladder rings were calculated as g tension/g tissue.
2.17. Histological examinations The kidney tissues of rats were fixed in buffered 10% formalin solution for 24 h and embedded in a paraffin wax. Tissues were then sectioned at 5-μm, stained with hematoxylin eosin (H&E). A semiquantitative evaluation of renal tissues was accomplished by scoring the degree of severity according to the formerly published criteria [27].
2.18. Statistical analysis The values are expressed as mean ± standard error of mean (S.E.M.), for 8 rats in each group. Bladder contraction was calculated as g tension/ g tissue. The highest response obtained was considered as the maximum response (Emax). pEC50 (negative log the concentration producing 50% of maximal response) was determined from non-linear regression analysis (4-parameter curve fit). Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by a Tukey– Kramer post-hoc test. Differences were considered significant at P b 0.05. Statistical analyses were carried out using Graph pad Prism software (GraphPad Software Inc. V4.03, San Diego, CA, USA).
2.15. Determination of myeloperoxidase (MPO) activity The neutrophil accumulation in the kidney was measured by assaying MPO activity as described by Schierwagen et al. [24]. Myeloperoxidase activity was assayed by measuring the H2O2-dependant oxidation of tetramethylbenzidine. Tetramethylbenzidine has a blue color in its oxidized form, which can be monitored spectrophotometrically at 450 nm. MPO activity was calculated as milliunits/mg wet tissue (mU/mg). 2.16. Isolation and preparation of urinary bladder rings Following the collection of blood samples, the rats were sacrificed by cervical dislocation; the lower abdomen was opened and the urinary bladder was exposed, the connective tissue and accompanying blood vessels were cut away, and the bladder was cut into rings and placed in a warm physiological salt solution (PSS). The composition of the PSS [25] in g/l was as follows: NaCl, 6.9; NaHCO3, 2.1; KCl, 0.35; MgSO4, 0.15; KH2PO4, 0.16; CaCl2, 0.28; and glucose, 2.0. The rings were mounted horizontally between a clamp and a force transducer for the measurement of the isometric tension in an organ bath that was filled with 10 ml of the PSS at a temperature of 37 °C and gassed with 95% O2–5% CO2. The rings were allowed to equilibrate for 30 min
Fig. 1. Effect of AL (50 mg/kg) on kidney/body weight in GNT-treated rats. Values are expressed as means ± standard error of mean (n = 8). ANOVA, analysis of variance; GNT, gentamicin; AL, allicin; SEM, standard error of mean. Comparisons performed using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. ⁎p b 0.05 vs. control; #p b 0.05 vs. GNT-treated group.
Please cite this article as: D.H. El-Kashef, et al., Protective effect of allicin against gentamicin-induced nephrotoxicity in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.010
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3. Results 3.1. Kidney/body weight ratio The results in Fig. 1 show that gentamicin (GNT) produced a significant increase in the kidney body weight ratio compared to control rats. In the GNT/allicin (AL)-treated group, the kidney body weight ratio was significantly lower than that of the GNT-treated rats while it did not significantly differ from the control group. 3.2. Serum biochemical parameters 3.2.1. Kidney functions Gentamicin caused a significant elevation in serum creatinine and BUN and protein in urine compared to the control group, with a significant decrease in serum albumin and creatinine clearance (CCr). Administration of allicin at doses of 10 and 20 mg/kg had no significant effect on GNT-induced elevation in serum creatinine and BUN (data not shown). Administration of 50 mg/kg allicin significantly attenuated GNT-induced changes in serum creatinine, and BUN and CCr while it did not significantly affect serum albumin and proteinuria compared to GNT-treated rats (Table 1). 3.2.2. Serum LDH activity Fig. 2 shows that GNT produced a significant increase in LDH activity compared to control rats. In the GNT/AL-treated group, LDH activity significantly decreased compared to GNT-treated rats.
Fig. 2. Effect of AL (50 mg/kg) on serum LDH activity in GNT-treated rats. Values are expressed as means ± standard error of mean (n = 8). ANOVA, analysis of variance; GNT, gentamicin; AL, allicin; SEM, standard error of mean. Comparisons performed using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. ⁎p b 0.05 vs. control; #p b 0.05 vs. GNT-treated group.
3.3. Bladder reactivity 3.2.3. Antioxidant status Table 2 shows that after 7 days of treatment, GNT significantly increased the MDA levels in rat kidney homogenate but decreased both GSH and SOD activities. Allicin significantly decreased the GNTinduced changes in MDA levels and significantly increased GSH and SOD activities. 3.2.4. Renal nitric oxide (NOx) content The results in Fig. 3 show that GNT produced a significant increase in the renal NOx content compared to control rats. In GNT/AL-treated group, the renal NOx content decreased but did not significantly differ from the GNT group, but it was significantly higher than that of the control one. 3.2.5. Tumor necrosis factor-α (TNF-α) level in kidney tissue homogenate Fig. 4 shows that GNT produced a significant increase in the renal TNF-α level compared to control rats. In the GNT/AL-treated group, the renal TNF-α level decreased significantly from the GNT group. 3.2.6. Myeloperoxidase (MPO) activity in kidney tissue homogenate The results in Fig. 5 show that GNT produced a significant increase in the renal MPO activity compared to control rats. In the GNT/AL-treated group, the renal MPO activity decreased significantly from the GNT group.
The effects of GNT and AL on the responses of bladder rings to ACh are shown in Fig. 6. The average increments in the control urinary bladder rings tension after ACh of 0.1, 0.3, 1, 3 and 10 μM were 2.9 ± 0.4, 14. 2 ± 1.6, 37.9 ± 2.3, 75 ± 2.5 and 124.2 ± 3.6 g tension/g tissue, respectively. Treatment with GNT significantly enhanced the responsiveness of the rings towards ACh, as compared with the control group. Thus, the average increments in tension in response to ACh of 0.1, 0.3, 1, 3 and 10 μM were 26.5 ± 1.7, 74.6 ± 3.1, 188.5 ± 6.2, 270.1 ± 5.5 and 360 ± 8 g tension/g tissue, respectively. Urinary bladder rings, isolated from GNT/AL-treated rats showed a significant reduction in their responsiveness to ACh when compared to the GNT-treated ones. The average increments in their tensions in response to ACh of 0.1, 0.3, 1, 3 and 10 μM were 6.68 ± 0.7, 22.1 ± 1.65, 53.3 ± 2.4, 105.9 ± 5.4 and 165.8 ± 1.7 g tension/g tissue, respectively.
3.4. Histopathological results The kidney of control rats showed normal architecture of glomerulus and tubules (Fig. 7A). Kidney of gentamicin-induced rats showed acute tubular necrosis with sloughed necrotic cells, moderate diffuse tubular atrophy with hyaline casts in group II (Fig. 7B) animals. Pretreatment of rats with 50 mg/kg/day allicin for 14 days (group III: Fig. 7C), showed mild focal acute tubular injury with attenuated tubular lining and focal
Table 1 Effect of AL (50 mg/kg) on kidney function parameters in GNT-treated rats.
Control GNT GNT/AL
Serum creatinine (mg/dl)
BUN (mg/dl)
CCr (ml/min)
Proteinuria (mg/day)
Serum albumin (g/dl)
0.97 ± 0.05 3.6 ± 0.22⁎ 2.18 ± 0.21⁎,#
35.8 ± 2.1 121.1 ± 5⁎ 95.8 ± 8.3⁎,#
34.0 ± 3.3 14.7 ± 1.5⁎ 26.4 ± 1.6#
98.8 ± 8.2 257.1 ± 19.4⁎ 255.2 ± 14.5⁎
4.1 ± 0.2 3 ± 0.11⁎ 3.4 ± 0.11⁎
ANOVA, analysis of variance; GNT, gentamicin; AL, allicin; SEM, standard error of mean. Data expressed as means ± SEM (n = 8). Analyses performed using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. ⁎ p b 0.05 vs. control. # p b 0.05 vs. GNT.
Please cite this article as: D.H. El-Kashef, et al., Protective effect of allicin against gentamicin-induced nephrotoxicity in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.010
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Table 2 Effect of AL (50 mg/kg) on kidney anti-oxidant status in GNT-treated rats.
Control GNT GNT/AL
MDA (nmol/g)
GSH (μmol/g)
SOD (U/g)
4.6 ± 0.45 12.1 ± 0.47⁎ 7.8 ± 0.5⁎,#
0.06 ± 0.008 0.02 ± 0.0004⁎ 0.058 ± 0.006#
47.9 ± 2.2 38.7 ± 2.2⁎ 48.7 ± 1.9#
ANOVA, analysis of variance; GNT, gentamicin; AL, allicin; SEM, standard error of mean. Data expressed as means ± SEM (n = 8). Analyses performed using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. ⁎ p b 0.05 vs. control. # p b 0.05 vs. GNT.
sloughed cells associated with interstitial inflammatory cells. The glomeruli and arteries were unremarkable. 4. Discussion Intraperitoneal injection of gentamicin resulted in a significant increase in the kidney weight index in rats compared to the untreated control group. The increase in the kidney weight index of gentamicintreated rats probably resulted from the edema that was caused by drug-induced acute tubular necrosis [28]. Allicin administration with gentamicin showed a significant decrease in kidney weight index compared to gentamicin-treated group; this can be explained by the study of Son et al. [29] that has proved that allicin downregulates gamma IR-induced intercellular adhesion molecule-1 expression (ICAM-1). Bae et al. [30] reported that ICAM-1 is increased in the gentamicintreated kidneys. Since ICAM-1 plays a pivotal role in inflammatory responses, perhaps the down-regulation of this adhesion molecule by allicin could mediate its reducing effect on inflammation and edema. Additionally, allicin exhibits anti-inflammatory effect by inhibiting cell-mediated T-helper 1 and inflammatory cytokines (TNF-α, IL-1α, IL-6, IL-8, T-cell, interferon-γ and IL-2) while upregulating IL-10 production [31]. The results of this study shows that, gentamicin administration to Wistar rats produced a typical pattern of nephrotoxicity which was
Fig. 3. Effect of AL (50 mg/kg) on renal NOx content in GNT-treated rats. Values are expressed as means ± standard error of mean (n = 8). ANOVA, analysis of variance; GNT, gentamicin; AL, allicin; SEM, standard error of mean. Comparisons performed using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. ⁎p b 0.05 vs. control; #p b 0.05 vs. GNT-treated group.
Fig. 4. Effect of AL (50 mg/kg) on renal TNF-α level in GNT-treated rats. Values are expressed as means ± standard error of mean (n = 8). ANOVA, analysis of variance; GNT, gentamicin; AL, allicin; SEM, standard error of mean. Comparisons performed using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. ⁎p b 0.05 vs. control; #p b 0.05 vs. GNT-treated group.
manifested by marked increase in serum creatinine and blood urea nitrogen levels. This result agreed with Jafarey et al. [14]. Allicin treatment decreased serum levels of creatinine and blood urea nitrogen levels when compared to gentamicin-treated group. This result came in line with Wang et al. [32] who showed the oral administered allicin with a concentration of 5, 10, and 20 mg/kg b.w./day could significantly
Fig. 5. Effect of AL (50 mg/kg) on renal MPO activity in GNT-treated rats. Values are expressed as means ± standard error of mean (n = 8(. ANOVA, analysis of variance; GNT, gentamicin; AL, allicin; SEM, standard error of mean. Comparisons performed using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. ⁎p b 0.05 vs. control; #p b 0.05 vs. GNT-treated group.
Please cite this article as: D.H. El-Kashef, et al., Protective effect of allicin against gentamicin-induced nephrotoxicity in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.010
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Fig. 6. Effect of GNT (100 mg/kg) and AL (50 mg/kg) on contractions to acetylcholine (ACh) in isolated rat urinary bladder rings. Values are expressed as means ± standard error of mean (n = 5). ANOVA, analysis of variance; GNT, gentamicin; AL, allicin; SEM, standard error of mean. Cumulative dose–response curve to ACh (10−7–10−5 M) was measured. p b 0.05 for Emax value compared with control group (*) and compared with GNT group (#) using one way analysis of variance (ANOVA) followed by Tukey–Kramer's multiple comparison post hoc test.
decrease the damage indexes of AST, ALT and BUN. This renoprotective effect may be related to its effect as an antioxidant. In the present study, gentamicin resulted in a significant increase in serum level of lactate dehydrogenase (LDH), but a decrease in level of albumin. This result came in line with Galaly et al. [33] who showed that administration of gentamicin for 3 weeks produced a significant
elevation of LDH activity and decrease in albumin concentration. Allicin administration significantly reduced LDH activity when compared to gentamicin-treated group. This result agreed with Wang et al. [32] who reported the oral administration of allicin significantly decreased the damage indexes of AST, ALT and LDH. On the other hand, our results showed allicin failed to significantly increase the reduced levels of serum albumin. Serum albumin might not be a significant risk marker for GNT-induced nephrotoxicity according to our findings. Further studies should be performed to verify these results due to the limitations of our study. In the current study, gentamicin caused a significant increase in the MDA levels, while GSH and SOD levels were reduced in the kidney tissue. Similar results were also observed in earlier studies [34,35]. Depletion of renal GSH is one of the primary factors which permit lipid peroxidation, suggested to be closely related to gentamicin-induced tissue damage [10]. This observation is in line with many reports that demonstrated apparent elevation in kidney thiobarbituric acid reactive substances (TBARS) following administration of gentamicin [36]. Gentamicin directly increases the production of mitochondrial ROS from the respiratory chain [37]. As known, xanthine–xanthine oxidase produces ROS in the mitochondria through the NADPH oxidase system. Interaction of the ROS produced in excessive amount with lipids, nucleic acids, various proteins, and biomolecules such as polysaccharides leads to cell and tissue damages. ROS cause changes in the fluidity and permeability of the cell membrane giving rise to the lipid peroxidation when occurred in the environment. MDA that can be easily identified in the biologic structures in the terms of the lipid peroxidation and is the indicator of the peroxidative damage is generally used for the evaluation of the lipid peroxidation [38]. On contrast, allicin administration with gentamicin showed a significant decrease in MDA content compared to rats treated with gentamicin alone, a result that agreed with other reported data that demonstrated apparent decrease in TBARS after administration of allicin [39,40]. This effect may be due to the ability of allicin to scavenge hydroxyl and peroxyl radicals and superoxide
Fig. 7. Light micrographs of sections from rat kidneys stained with hematoxylin and eosin. (A) Normal kidney section from the control group (×100); (B) acute tubular necrosis with sloughed necrotic cells, moderate diffuse tubular atrophy with hyaline casts (arrow) after treatment with GNT (100 mg/kg/day) for 7 days (×100); (C) pretreatment of rats with AL (50 mg/kg/day) for 14 days showed mild focal acute tubular injury (×100). GNT, gentamicin; AL, allicin.
Please cite this article as: D.H. El-Kashef, et al., Protective effect of allicin against gentamicin-induced nephrotoxicity in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.010
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anion [41] and inhibit lipid peroxidation [42]. Also, treatment of rats with allicin showed a significant increase in the activity of GSH compared to the gentamicin-treated group, because it has antioxidant activity [43]. Also, it exerts its effect by increasing the activities of nonenzymatic antioxidant (GSH) and the detoxifying enzyme (GST) [42]. Allicin may provide the sulfur source required for the synthesis of GSH [44] and so it restores glutathione level and increases the activities of GSH and GST. Allicin administration with gentamicin resulted in significant increase in the activity of SOD compared to the gentamicintreated group. This may be related to its effect as an antioxidant [43]. In this study, gentamicin treatment produced a significant increase in the NO level. This result came in line with previous study that demonstrated that NO and peroxynitrite plays an important role in the acute renal failure caused by gentamicin [45]. This effect was reversed by allicin administration that resulted in significant decrease in levels of NO when compared to gentamicin-treated group. In the present study, treatment of rats with gentamicin resulted in significant increase in level of TNF-α. This result is in line with Galaly et al. [33]. The activation and nuclear translocation of NF-κB, in response to oxidative stress/nitrosative stress, are the key factors in the renal inflammatory process by regulating the gene expression of cytokines, chemokines, and adhesion molecules [30]. Administration of allicin with gentamicin significantly decreased the level of TNF-α compared with gentamicin-treated group. This result is in agreement with the investigation reported by Bruck et al. [46], who reported that allicin administration (300 ml/mouse/day) had the ability to decrease the elevation in serum level of TNF-α, and the hepatic necroinflammation was much improved. In this study, gentamicin treatment increased the levels of renal MPO activity, this result agreed with El Gamal et al. [47] who showed that gentamicin treatment increased the levels of renal MPO activity, indicating neutrophil infiltration. Administration of allicin resulted in significant decrease in level of MPO when compared to gentamicin-treated group. This result is in consistent with Wang et al. [31] who showed the oral administered allicin with a concentration of 5, 10, and 20 mg/kg b.w./day could significantly decrease the damage indexes of, ROS and MPO. The current study showed that gentamicin treatment produced reduction in GFR, manifested by the progressive reduction in creatinine clearance. This result agreed with Hosaka et al. [48] who reported that the decrease in creatinine clearance corresponds to the reduction in the glomerular filtration rate occurred. Also gentamicin treatment resulted in significant increase in proteinuria level. Allicin administration caused a significant increase in creatinine clearance which reflects the enhanced glomerular function. On the other hand, our results showed allicin failed to significantly decrease the elevated levels of protein in urine. In this study, treatment with gentamicin significantly enhanced the responsiveness of the isolated urinary bladder rings towards ACh at all concentrations used (10−7–10−5). ROS may play an important role in gentamicin-induced tubular damage [5]. NO• could be harmful to tubular cells due to its direct cytotoxic or its reaction with superoxide anion generating the oxidant peroxynitrite ions, which are known to be highly damaging [49]. Therefore, generating ROS, particularly NO•, in the renal system could be suggested as the pathway mediated gentamicininduced kidney dysfunction that manifested by increasing the responsiveness of the urinary bladder rings towards ACh. On the other hand, allicin administration retained normal response of urinary bladder rings towards ACh. The histological studies of kidney from gentamicin treated rats showed tubular necrosis, thickening of vessels and tubular atrophy with hyaline casts. Similar changes were also reported [50,51]. However, allicin attenuated that change. In summary, we have confirmed that allicin has a protective role against nephrotoxicity induced by gentamicin exposure. According to our biochemical findings, which were supported by histopathological and sensitivity of urinary bladder rings to ACh, administration of allicin
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rescued the cells from the effects of gentamicin. These findings indicate that allicin administration may reduce gentamicin-induced acute renal injury. Therefore, we propose that allicin might be a potential candidate agent against gentamicin-induced nephrotoxicity via its antioxidant, anti-inflammatory and immunomodulatory properties.
Conflict of interest statement The authors declare no conflict of interest.
References [1] I. Karahan, A. Atessahin, S. Yilmaz, A.O. Ceribassi, F. Sakin, Protective effect of lycopene on gentamicin-induced oxidative stress and nephrotoxicity in rats, Toxicology 215 (2005) 198–204. [2] W. Mwengee, T. Butler, S. Mgema, et al., Treatment of plague with gentamicin or doxycycline in a randomized clinical trial in Tanzania, Clin. Infect. Dis. 42 (2006) 614–621. [3] W.E. Siegenthaler, A. Bonetti, R. Luthy, Aminoglycoside antibiotics in infectious diseases. An overview, Am J Med. 80 (1986) 2–14. [4] T.H. Mathew, Drug-induced renal disease, Med J Aust. 156 (1992) 724–728. [5] S. Cuzzocrea, E. Mazzon, L.I. Dugo Serraino, R. Di Paola, D. Britti, A. De Sarro, S. Pierpaoli, A. Caputi, E. Masini, D. Salvemini, A role for superoxide in gentamicin mediated nephropathy in rats, Eur. J. Pharmacol. 450 (2002) 67–76. [6] H. Parlakpinar, S. Tasdemir, A. Polat, A. Bay-Karabulut, N. Vardi, M. Ucar, A. Acet, Protective role of caffeic acid phenethyl ester (cape) on gentamicin induced acute renal toxicity in rats, Toxicology 207 (2005) 169–177. [7] C. Yanagida, K. Ito, I. Komiya, T. Horie, Protective effect of fosfomycin on gentamicininduced lipid peroxidation of rat renal tissue, Chem. Biol. Interact. 148 (2004) 139–147. [8] C.L. Yang, X.H. Du, Y.X. Han, Renal cortical mitochondria are source of oxygen free radicals enhanced by gentamicin, Ren. Fail. 17 (1995) 21–26. [9] Z. BaligaZhang, M. Baliga, N. Ueda, S.V. Shah, In vitro and in vivo evidence suggesting a role for iron in cisplatin induced nephrotoxicity, Kidney Int. 53 (1998) 394–401. [10] R. Manikandan, M. Beulaja, R. Thiagarajan, A. Priyadarsini, R. Saravanan, M. Arumugam, 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 (2011) 578–585. [11] R.S. Rivlin, Historical perspective on the use of garlic, J. Nutr. 131 (2001) 951–954S. [12] K. Hirsch, M. Danilenko, J. Giat, et al., Effect of purified allicin, the major ingredient of freshly crushed garlic, on cancer cell proliferation, Nutr. Cancer 38 (2000) 245–254. [13] J. Jakubikova, J. Sedlak, Garlic-derived organosulfides induce cytotoxicity, apoptosis, cell cycle arrest and oxidative stress in human colon carcinoma cell lines, Neoplasma 53 (2006) 191–199. [14] M. Jafarey, S. Changizi Ashtiyani, H. Najafi, Calcium dobesilate for prevention of gentamicin-induced nephrotoxicity in rats, Iran J. Kidney Dis. 8 (2014) 46–52. [15] N.A. Ashry, N.M. Gameil, G.M. Suddek, Modulation of cyclophosphamide-induced early lung injury by allicin, Pharm. Biol. 51 (2013) 806–811. [16] H. Bartels, M. Böhmer, C. Heierli, Serum creatinine determination without protein precipitation, Clin. Chim. Acta 37 (1972) 193–197 (Article in German). [17] J.K. Fawcett, J.E. Scott, A rapid and precise method for the determination of urea, J. Clin. Pathol. 13 (1960) 156–159. [18] W.H. Daughaday, O.H. Lowry, N.J. Rosebrough, W.S. Fields, Determination of cerebrospinal fluid protein with the Folin phenol reagent, J Lab Clin. Med 39 (1952) 663–665. [19] B.T. Doumas, W.A. Watson, H.G. Biggs, Albumin standards and the measurement of serum albumin with bromocresol green, Clin. Chim. Acta 31 (1971) 87–96. [20] J.B. Henry, Todd Sanford Davidsohn, Clinical Diagnosis and Management by Laboratory Methods, 16th edn WB Saunders and Co., Philadelphia, 1974. [21] H. Okhawa, N. Ohishi, K. Yagi, Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction, Anal. Biochem. 95 (1979) 351–358. [22] S.L. Marklund, Superoxide dismutase isoenzymes in tissues and plasma from New Zealand black mice, nude mice and normal BALB/c mice, Mutat. Res. 148 (1985) 129–134. [23] G.L. Ellman, Tissue sulfa hydryl groups, Arch. Biochem. Biophys. 74 (1959) 214–226. [24] C. Schierwagen, A.C. Bylund-Fellenius, C. Lundberg, Improved method for quantification of tissue PMN accumulation measured by myeloperoxidase activity, J. Pharmacol. Methods. 23 (1990) 179–186. [25] A.M. Mostafa, M.N. Nagi, O.A. Al-Shabanha, H.A. El-Kashef, Effect of aminoguanidine and melatonin on the response of isolated urinary bladder to acetylcholine in normal and diabetic rats, Med. Sci. Res. 28 (2000) 33–37. [26] I. Nakamura, C. Takahashi, I. Miyagawa, The alterations of norepinephrine and acetylcholine concentrations in immature rat urinary bladder caused by streptozotocin induced diabetes, J. Urol. 148 (1992) 423–426. [27] R.B. Teixeira, J. Kelly, H. Alpert, V. Pardo, C.A. Vaamonde, Complete protection from gentamicin-induced acute renal failure in the diabetes mellitus rat, Kidney Int. 21 (1982) 600–612. [28] A. Erdem, N.U. Gundogan, A. Usubutun, K. Kilic, S.R. Erdem, A. Kara, et al., The protective effect of taurine against gentamicin-induced acute tubular necrosis in rats, Nephrol. Dial. Transplant. 15 (2000) 1175–1182.
Please cite this article as: D.H. El-Kashef, et al., Protective effect of allicin against gentamicin-induced nephrotoxicity in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.010
8
D.H. El-Kashef et al. / International Immunopharmacology xxx (2015) xxx–xxx
[29] E.W. Son, S.J. Mo, D.K. Rhee, S. Pyo, Inhibition of ICAM-1 expression by garlic component, allicin, in gamma-irradiated human vascular endothelial cells via downregulation of the JNK signaling pathway, Int. Immunopharmacol. 6 (2006) 1788–1795. [30] E.H. Bae, I.J. Kim, S.Y. Joo, et al., Renoprotective effects of the direct renin inhibitor aliskiren on gentamicin-induced nephrotoxicity in rats, J. Renin-AngiotensinAldosterone Syst. 15 (2014) 348–361. [31] G. Hodge, S. Hodge, P. Han, Allium sativum (garlic) suppresses leukocyte inflammatory cytokine production in vitro: potential therapeutic use in the treatment of inflammatory bowel disease, Cytometry 48 (2002) 209–215. [32] E.T. Wang, D.Y. Chen, H.Y. Liu, H.Y. Yan, Y. Yuan, Protective effect of allicin against glycidamide-induced toxicity in male and female mice, Gen. Physiol. Biophys. 34 (2015) 177–187. [33] S.R. Galaly, O.M. Ahmed, A.M. Mahmoud, Thymoquinone and curcumin prevent gentamicin-induced liver injury by attenuating oxidative stress, inflammation and apoptosis, J. Physiol. Pharmacol. 65 (2014) 823–832. [34] E. Ozbek, Y. Turkoz, E. Sahna, F. Ozugurlu, B. Mizrak, M. Ozbek, Melatonin administration prevents the nephrotoxicity induced by gentamicin, BJU Int. 85 (2000) 742–746. [35] M. Tavafi, H. Ahmadvand, Effect of rosmarinic acid on inhibition of gentamicin induced nephrotoxicity in rats, Tissue Cell 43 (2011) 392–397. [36] F.O. Aygün, F.Z. Akçam, O. Kaya, B.M. Ceyhan, R. Sütçü, Caffeic acid phenethyl ester modulates gentamicin-induced oxidative nephrotoxicity in kidney of rats, Biol. Trace Elem. Res. 145 (2012) 211–216. [37] A.I. Morales, D. Detaille, M. Prieto, A. Puente, E. Briones, M. Arévalo, X. Leverve, J.M. López-Novoa, M.Y. El-Mir, Metformin prevents experimental gentamicin-induced nephropathy by a mitochondria dependent pathway, Kidney Int. 77 (2010) 861–869. [38] P. Kovacic, R. Somanathan, Unifying mechanism for eye toxicity: electron transfer, reactive oxygen species, antioxidant benefits, cell signaling and cell membranes, Cell Membr Free Radic Res 2 (2008) 56–69. [39] K. Prasad, V.A. Laxdal, M. Yu, B.L. Raney, Antioxidant activity of allicin, an active principle in garlic, Mol. Cell. Biochem. 148 (1995) 183–189. [40] X.H. Li, C.Y. Li, Z.G. Xiang, et al., Allicin can reduce neuronal death and ameliorate the spatial memory impairment in Alzheimer's disease models, Neurosciences (Riyadh) 15 (2010) 237–243.
[41] K.M. Kim, S.B. Chun, M.S. Koo, et al., Differential regulation of NO availability from macrophages and endothelial cells by the garlic components S-allyl cysteine, Free Radic. Biol. Med. 30 (2001) 747–756. [42] G. Saravanan, J. Prakash, Effect of garlic (Allium sativum) on lipid peroxidation in experimental myocardial infarction in rats, J. Ethnopharmacol. 94 (2004) 155–158. [43] S.K. Banerjee, M. Maulik, S.C. Mancahanda, et al., Dose dependent induction of endogenous antioxidants in rat heart by chronic administration of garlic, Life Sci. 70 (2002) 1509–1518. [44] C.C. Wu, L.Y. Sheen, H.W. Chen, et al., Effects of organosulfur compounds from garlic oil on the antioxidation system in rat liver and red blood cells, Food Chem. Toxicol. 39 (2001) 563–569. [45] J.S. Christo, A.M. Rodrigues, M.G. Mouro, M.A. Cenedeze, M.J. Simões, N. Schor, E.M.S. Higa, Nitric oxide (NO) is associated with gentamicin (GENTA) nephrotoxicity and the renal function recovery after suspension of GENTA treatment in rats, Nitric Oxide 24 (2011) 77–83. [46] R. Bruck, H. Aeed, E. Brazovsky, et al., Allicin, the active component of garlic, prevents immune-mediated, concanavalin A-induced hepatic injury in mice, Liver Int. 25 (2005) 613–621. [47] A.A. El Gamal, M.S. AlSaid, M. Raish, M. Al-Sohaibani, S.M. Al-Massarani, A. Ahmad, M. Hefnawy, M. Al-Yahya, O.A. Basoudan, S. Rafatullah, Beetroot (Beta vulgaris L.) extract ameliorates gentamicin-induced nephrotoxicity associated oxidative stress, inflammation, and apoptosis in rodent model, Mediat. Inflamm. 2014 (2014) 983952. [48] E.M. Hosaka, O.F.P. Santos, A.C. Seguro, F.F. Vattimo, Effect of cyclooxygenase inhibitors on gentamicin-induced nephrotoxicity in rats, Braz. J. Med. Biol. Res. 37 (2004) 979–985. [49] R. Ghaznavi, M. Faghihi, M. Kadkhodaee, S. Shams, H. Khastar, Effects of nitric oxide on gentamicin toxicity in isolated perfused rat kidneys, J. Nephrol. 18 (2005) 548–552. [50] A.A. Al-Majed, A.M. Mostafa, A.C. Al-Rikabi, O.A. Al-Shabanah, Protective effects of oral Arabic gum administration of gentamicin-induced nephrotoxicity in rats, Pharmacol. Res. 46 (2002) 445–451. [51] K.V. Kumar, A.A. Shifow, U.U. Naidu, K.S. Ratnakar, Carvedilol: a beta blocker with antioxidant property protect against gentamicin-induced nephrotoxicity, Life Sci. 66 (2000) 2603–2611.
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Protective effect of allicin against gentamicin-induced nephrotoxicity in rats Dalia H. El-Kashef, Asmaa E. El-Kenawi, Ghada M. Suddek ⁎, Hatem A. Salem Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
a r t i c l e
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Article history: Received 23 July 2015 Received in revised form 15 August 2015 Accepted 13 September 2015 Available online xxxx Keywords: Gentamicin Allicin Nephrotoxicity Urinary bladder Nitric oxide TNF-α
a b s t r a c t In this study, the modulator effect of allicin on the oxidative nephrotoxicity of gentamicin in the kidneys of rats was investigated by determining indices of lipid peroxidation and the activities of antioxidant enzymes, as well as by histological analyses. Furthermore, the effect of allicin on gentamicin induced hypersensitivity of urinary bladder rings to ACh was estimated. Twenty-four male Wistar albino rats were randomly divided into three groups, control, gentamicin (100 mg/kg, i.p.) and gentamicin + allicin (50 mg/kg, orally). At the end of the study, all rats were sacrificed and then urine, blood samples and kidneys were taken. Gentamicin administration caused a severe nephrotoxicity as evidenced by an elevated kidney/body weight ratio, serum creatinine, blood urea nitrogen (BUN), serum lactate dehydrogenase (LDH) and proteinuria with a reduction in serum albumin and creatinine clearance as compared with control group. In addition, a significant increase in renal contents of malondialdehyde (MDA), myeloperoxidase (MPO), nitric oxide (NOx) and tumor necrosis factor-alpha (TNFα) concomitantly with a significant decrease in renal reduced glutathione (GSH) and superoxide dismutase (SOD) activities was detected upon gentamicin injection. Exposure to gentamicin increased the sensitivity of isolated urinary bladder rings to ACh and induced acute renal tubular epithelial cells necrosis. Administration of allicin significantly decreased kidney/body weight ratio, serum creatinine, LDH, renal MDA, MPO, NOx and TNF-α while it significantly increased creatinine clearance, renal GSH content and renal SOD activity when compared to gentamicin-treated group. Additionally, allicin significantly reduced the responses of isolated bladder rings to ACh and ameliorated tissue morphology as evidenced by histological evaluation. Our study indicates that allicin exerted protection against structural and functional damage induced by gentamicin possibly due to its antioxidant, anti-inflammatory and immunomodulatory properties in addition to its ability to retaining nitric oxide level. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Gentamicin (GNT) is commonly applied in human clinical practices for treatment of life-threatening Gram-negative infections [1,2]. However, the usefulness of GNT is limited by the development of nephrotoxicity. In some cases, this side effect is so severe that the use of the drug must be discontinued. In spite of the introduction of newer and less toxic antibiotics, GNT is still used clinically because of its rapid bactericidal action, broad-spectrum activity, chemical stability, and low cost [3,4]. GNT-induced nephrotoxicity is characterized by direct tubular necrosis, without morphological changes in glomerular structures [1]. The mechanism of gentamicin-induced nephrotoxicity is not completely known. However, studies have implicated reactive oxygen species particularly superoxide anion radical in the pathophysiology of gentamicin nephropathy [5]. It has been demonstrated that gentamicin administration increases renal cortical lipid peroxidation, nitric oxide generation and mitochondria hydrogen peroxide production [1,6,7,8]. Abnormal ⁎ Corresponding author at: Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt. E-mail address: [email protected] (G.M. Suddek).
production of such molecules may damage macromolecules, induce cellular injury and necrosis via several mechanisms including peroxidation, protein denaturation and DNA damage [6,9]. The alteration in kidney functions induced by lipid peroxidation is a proximal event in the injury cascade of gentamicin mediated nephrotoxicity [1]. Accordingly, the administration of compounds with antioxidant activity has been successfully used to prevent or ameliorate gentamicin-induced nephrotoxicity [1,5]. In the past few years, much interest has been laid on the role of naturally occurring dietary substances for the control and management of various chronic diseases [10]. Allicin (diallyl thiosulfinate), the major pharmacological component of garlic [11], has attracted attention of the international medical field gradually due to its potential for disease prevention and treatment. It is formed by the action of the enzyme alliinase on alliin in crushed fresh garlic cloves. It possesses antioxidant activity and is shown to cause a variety of actions potentially useful for human health. Allicin exhibits hypolipidemic, antiplatelet, antibacterial, and antifungal effects. It has been reported that allicin inhibits various cancer cells demonstrating anticancer and chemopreventive activities [12,13]. The present study aimed at studying the renoprotective properties of allicin against gentamicin-induced nephrotoxicity.
http://dx.doi.org/10.1016/j.intimp.2015.09.010 1567-5769/© 2015 Elsevier B.V. All rights reserved.
Please cite this article as: D.H. El-Kashef, et al., Protective effect of allicin against gentamicin-induced nephrotoxicity in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.010
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2. Materials and methods
The amount of the complex formed is directly proportional to the creatinine concentration. A kit from Biodiagnostics Co., Egypt was used.
2.1. Chemicals 2.5. Determination of blood urea nitrogen (BUN) Allicin was obtained as pharmaceutical drug (Allimax capsule containing 100% allicin powder, Allisure ® AC-23) obtained from (Allimax Nutriceuticals, US). Gentamicin was purchased as a pharmaceutical preparation (Gentamicin ampoules 80 mg, Alexandria Chemical Co., Egypt). All other chemicals and reagents used were of the highest analytical grade commercially available.
Urea was measured enzymatically in rat serum according to Fawcett and Scott [17]. Urea in the sample was hydrolyzed by urease enzyme to yield ammonia and carbon dioxide. In a modified Berthelot reaction, the blue dye indophenol product reaction absorbs light proportional to initial urea concentration and was measured spectrophotometrically at 550 nm. A kit from Biodiagnostics Co. was used.
2.2. Animals Twenty-four adult male Wistar rats weighing 160–200 g were used for the experimental procedures. Rats were allowed 1 week to adapt to the surroundings before beginning any experimentation. Animals were housed in individual plastic cages with bedding. Standard rat food and tap water were available ad libitum for the duration of the experiments unless otherwise noted. Temperature was maintained at 25 °C with 12/ 12-h light/dark cycle. All animal experiments described in this study comply with the ethical principles and guidelines for the care and use of laboratory animals adopted by “Research Ethics Committee, Faculty of Pharmacy, Mansoura University”. 2.3. Experimental design Gentamicin-induced acute kidney injury was established in male Wistar rats. The animals were randomly divided into three groups containing eight rats in each group. Group (1): Control group, rats did not receive any solvent or drug during the experiment and were on a usual diet. Group (2): Gentamicin (GNT) group, rats were injected with GNT (100 mg/kg, i.p.) for 7 consecutive days [14]. Group (3): Gentamicin/Allicin (GNT/AL) group, rats were injected with GNT (100 mg/kg, i.p.) and received AL (50 mg/kg, orally) [15] starting seven days before GNT injection till the end of experimental study. Allicin treatment schedule and dose level were chosen after pilot experiments. After the last dose, all control and experimental animals were immediately kept in individual metabolic cages for collection of 24 h urine samples. These samples were centrifuged for 15 min at 3000 rpm, and kept frozen until analyzed. Blood samples were obtained from overnight fasted animals through retro-orbital sinus, under diethyl ether anesthesia, into non-heparinized tubes. The collected blood samples were allowed to clot for 30 min at 25 °C. After clotting, they were centrifuged at 1000 ×g, 4 °C for 15 min. using cooling centrifuge (Damon/IEC Division, Model: CRU-5000, Needham, Mass., USA). Sera were collected and stored frozen for the determination of levels of Cr, BUN, albumin and LDH. The animals were sacrificed after anesthesia by cervical dislocation, then the lower abdomen was opened and the contractile response of the isolated urinary bladder rings towards ACh was tested. Fatty adherents from the kidneys were removed and the kidneys were weighed using a digital balance to calculate the kidney body weight ratio. The left kidneys were excised immediately, rinsed in ice-cold normal saline (0.9% w/v), homogenized in 0.1 M phosphate buffer (pH 7.4) to yield 10% w/v tissue homogenates that were then used for biochemical assays. The right kidneys were harvested for histopathological examination. 2.4. Determination of serum creatinine Creatinine was measured in rat serum as described by Bartels et al. [16]. Creatinine in alkaline solution reacts with picric acid to form a colored complex that was measured spectrophotometrically at 550 nm.
2.6. Determination of creatinine clearance (Ccr) Glomerular filtration was assessed by creatinine clearance based on serum and urine creatinine levels, with values expressed in ml/min, computed with the formula: Ccr = urine creatinine (mg/dl) × urine flow (ml/min)/Serum creatinine (mg/dl). Urine flow was calculated dividing 24 h of urine volume by 1440, which corresponds to the number of minutes in 24 h (60 min × 24 h = 1440): urine flow (ml/min) = value of urine volume (24 h) / 1440. 2.7. Determination of protein in urine Protein in urine was determined by Folin–Lowry colorimetric method; two reactions are involved: (a) an initial interaction of protein and Cu+2 in alkali (related to biuret reaction); (b) a reduction of the phosphotungstic and phosphomolybdic acids to molybdenum blue and tungsten blue both by the Cu–protein complex and by the tyrosine and tryptophan of the protein. The latter two give color in the absence of Cu+2 but the rest of the protein gives no color without Cu+2. About 75% of the color is dependent on the Cu+2. The maximum absorption of the colored products is at 750 nm [18]. 2.8. Determination of serum albumin Albumin, in the presence of bromocresol green at a slightly acid pH, produces a color change in the indicator from yellow-green to greenblue. The intensity of the color formed is proportional to the albumin concentration in the sample [19]. 2.9. Determination of serum lactate dehydrogenase Lactate dehydrogenase (LDH) activity was assessed according to the method described by Henry [20]. The method depends on the conversion of pyruvate to lactate by LDH consuming NADH+, which absorbs at 340 nm. Its consumption is directly proportional to serum LDH concentration. LDH activity was calculated as units/l (U/l). 2.10. Determination of lipid peroxidation Lipid peroxidation was determined by the method of Ohkawa et al. [21]. The principle of this method being that malondialdehyde (MDA), an end product of lipid peroxidation, reacts with thiobarbituric acid (TBA) to form a pink chromogen. For this assay, 0.2 ml of 8.1% sodium dodecyl sulfate (SDS), 1.5 ml of 20% acetic acid (pH 3.5) and 1.5 ml of 0.8% thiobarbituric acid aqueous solution were added in succession in a reaction tube. To this reaction mixture, 0.2 ml of the kidney homogenate was added, and the mixture was then heated in boiling water for 60 min. After cooling to room temperature, 5 ml of butanol: pyridine (15:1, v/v) solution was added. The mixture was then centrifuged at 2236 × g for 15 min following which the upper layer was separated, and the intensity of the resulting pink color was read at 532 nm.
Please cite this article as: D.H. El-Kashef, et al., Protective effect of allicin against gentamicin-induced nephrotoxicity in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.010
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Tetramethoxypropane was used as an external standard and the level of lipid peroxides was expressed as nmol of MDA formed/g wet tissue. 2.11. Estimation of superoxide dismutase (SOD) activity Superoxide dismutase activity in kidney homogenates, as an index of endogenous antioxidant activity, was measured spectrophotometrically by monitoring the SOD-inhabitable autooxidation of pyrogallol, as described by Marklund [22]. In brief, the reaction mixture consisted of 24 mmol/l of pyrogallol in 10 mM HCl, Tris buffer (pH 7.8). The reaction was carried out at 25 °C. The change in absorbance at 420 nm was recorded for 3 min at 1 min interval. One unit of enzyme activity is defined as 50% inhibition of pyrogallol autooxidation under the assay conditions. The SOD activity was expressed as Unit/g wet tissue. 2.12. Estimation of reduced glutathione (GSH) activity Reduced glutathione activity in kidney homogenates was determined according to the method described by Ellman [23]. The method based on the reduction of Ellman's reagent by (− SH) groups of GSH to produce a yellow colored 2-nitro-5-mercaptobenzoic acid. The produced chromogen is directly proportional to GSH concentration. The absorbance was measured spectrophotometrically at 412 nm and the concentrations were expressed as μmol/g wet tissue. − 2.13. Measurement of NO− 2 /NO3 concentration − Nitrite/nitrate (NO− 2 /NO3 ) production, an indicator of NO synthesis, was measured in the supernatant of the kidney homogenate using a commercially available NO assay kit (R&D Systems, Minneapolis, USA) following the manufacturer's instruction.
2.14. Measurement of TNF-α level Tumor necrosis factor-alpha concentrations were measured in kidney tissue homogenates using an enzyme-linked immunosorbent assay kit (Bender Med Systems GmbH, Vienna, Austria). Kidney tissue homogenate was added to a microtitre plate pre-coated with a monoclonal antibody specific for rat tumor necrosis factor-alpha. Incubation, plate washing and quenching of reactions were carried out according to the kit manufacturer's instruction and tumor necrosis factor-alpha levels were expressed as pg/mg.
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prior to the experiment under a resting load of 1 g [26]. During this time, the bath solution was replaced every 5 min. Isometric tension generated by the smooth muscle was measured by means of an isometric transducer (serial no. 88576, Biegestab K30, Hugo Sachs Elektronik, Fedral Republic of Germany) recorded with a Powerlab unit/400 linked to a PC running Chart v 4.2 software (ADInstruments Pty Ltd., Australia). Concentration–response curves to acetylcholine (ACh) were constructed. The rings were exposed to different concentrations of ACh in a cumulative manner. Exposure to each concentration of ACh was maintained until the maximal response to that concentration was reached. The responses of the bladder rings were calculated as g tension/g tissue.
2.17. Histological examinations The kidney tissues of rats were fixed in buffered 10% formalin solution for 24 h and embedded in a paraffin wax. Tissues were then sectioned at 5-μm, stained with hematoxylin eosin (H&E). A semiquantitative evaluation of renal tissues was accomplished by scoring the degree of severity according to the formerly published criteria [27].
2.18. Statistical analysis The values are expressed as mean ± standard error of mean (S.E.M.), for 8 rats in each group. Bladder contraction was calculated as g tension/ g tissue. The highest response obtained was considered as the maximum response (Emax). pEC50 (negative log the concentration producing 50% of maximal response) was determined from non-linear regression analysis (4-parameter curve fit). Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by a Tukey– Kramer post-hoc test. Differences were considered significant at P b 0.05. Statistical analyses were carried out using Graph pad Prism software (GraphPad Software Inc. V4.03, San Diego, CA, USA).
2.15. Determination of myeloperoxidase (MPO) activity The neutrophil accumulation in the kidney was measured by assaying MPO activity as described by Schierwagen et al. [24]. Myeloperoxidase activity was assayed by measuring the H2O2-dependant oxidation of tetramethylbenzidine. Tetramethylbenzidine has a blue color in its oxidized form, which can be monitored spectrophotometrically at 450 nm. MPO activity was calculated as milliunits/mg wet tissue (mU/mg). 2.16. Isolation and preparation of urinary bladder rings Following the collection of blood samples, the rats were sacrificed by cervical dislocation; the lower abdomen was opened and the urinary bladder was exposed, the connective tissue and accompanying blood vessels were cut away, and the bladder was cut into rings and placed in a warm physiological salt solution (PSS). The composition of the PSS [25] in g/l was as follows: NaCl, 6.9; NaHCO3, 2.1; KCl, 0.35; MgSO4, 0.15; KH2PO4, 0.16; CaCl2, 0.28; and glucose, 2.0. The rings were mounted horizontally between a clamp and a force transducer for the measurement of the isometric tension in an organ bath that was filled with 10 ml of the PSS at a temperature of 37 °C and gassed with 95% O2–5% CO2. The rings were allowed to equilibrate for 30 min
Fig. 1. Effect of AL (50 mg/kg) on kidney/body weight in GNT-treated rats. Values are expressed as means ± standard error of mean (n = 8). ANOVA, analysis of variance; GNT, gentamicin; AL, allicin; SEM, standard error of mean. Comparisons performed using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. ⁎p b 0.05 vs. control; #p b 0.05 vs. GNT-treated group.
Please cite this article as: D.H. El-Kashef, et al., Protective effect of allicin against gentamicin-induced nephrotoxicity in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.010
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3. Results 3.1. Kidney/body weight ratio The results in Fig. 1 show that gentamicin (GNT) produced a significant increase in the kidney body weight ratio compared to control rats. In the GNT/allicin (AL)-treated group, the kidney body weight ratio was significantly lower than that of the GNT-treated rats while it did not significantly differ from the control group. 3.2. Serum biochemical parameters 3.2.1. Kidney functions Gentamicin caused a significant elevation in serum creatinine and BUN and protein in urine compared to the control group, with a significant decrease in serum albumin and creatinine clearance (CCr). Administration of allicin at doses of 10 and 20 mg/kg had no significant effect on GNT-induced elevation in serum creatinine and BUN (data not shown). Administration of 50 mg/kg allicin significantly attenuated GNT-induced changes in serum creatinine, and BUN and CCr while it did not significantly affect serum albumin and proteinuria compared to GNT-treated rats (Table 1). 3.2.2. Serum LDH activity Fig. 2 shows that GNT produced a significant increase in LDH activity compared to control rats. In the GNT/AL-treated group, LDH activity significantly decreased compared to GNT-treated rats.
Fig. 2. Effect of AL (50 mg/kg) on serum LDH activity in GNT-treated rats. Values are expressed as means ± standard error of mean (n = 8). ANOVA, analysis of variance; GNT, gentamicin; AL, allicin; SEM, standard error of mean. Comparisons performed using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. ⁎p b 0.05 vs. control; #p b 0.05 vs. GNT-treated group.
3.3. Bladder reactivity 3.2.3. Antioxidant status Table 2 shows that after 7 days of treatment, GNT significantly increased the MDA levels in rat kidney homogenate but decreased both GSH and SOD activities. Allicin significantly decreased the GNTinduced changes in MDA levels and significantly increased GSH and SOD activities. 3.2.4. Renal nitric oxide (NOx) content The results in Fig. 3 show that GNT produced a significant increase in the renal NOx content compared to control rats. In GNT/AL-treated group, the renal NOx content decreased but did not significantly differ from the GNT group, but it was significantly higher than that of the control one. 3.2.5. Tumor necrosis factor-α (TNF-α) level in kidney tissue homogenate Fig. 4 shows that GNT produced a significant increase in the renal TNF-α level compared to control rats. In the GNT/AL-treated group, the renal TNF-α level decreased significantly from the GNT group. 3.2.6. Myeloperoxidase (MPO) activity in kidney tissue homogenate The results in Fig. 5 show that GNT produced a significant increase in the renal MPO activity compared to control rats. In the GNT/AL-treated group, the renal MPO activity decreased significantly from the GNT group.
The effects of GNT and AL on the responses of bladder rings to ACh are shown in Fig. 6. The average increments in the control urinary bladder rings tension after ACh of 0.1, 0.3, 1, 3 and 10 μM were 2.9 ± 0.4, 14. 2 ± 1.6, 37.9 ± 2.3, 75 ± 2.5 and 124.2 ± 3.6 g tension/g tissue, respectively. Treatment with GNT significantly enhanced the responsiveness of the rings towards ACh, as compared with the control group. Thus, the average increments in tension in response to ACh of 0.1, 0.3, 1, 3 and 10 μM were 26.5 ± 1.7, 74.6 ± 3.1, 188.5 ± 6.2, 270.1 ± 5.5 and 360 ± 8 g tension/g tissue, respectively. Urinary bladder rings, isolated from GNT/AL-treated rats showed a significant reduction in their responsiveness to ACh when compared to the GNT-treated ones. The average increments in their tensions in response to ACh of 0.1, 0.3, 1, 3 and 10 μM were 6.68 ± 0.7, 22.1 ± 1.65, 53.3 ± 2.4, 105.9 ± 5.4 and 165.8 ± 1.7 g tension/g tissue, respectively.
3.4. Histopathological results The kidney of control rats showed normal architecture of glomerulus and tubules (Fig. 7A). Kidney of gentamicin-induced rats showed acute tubular necrosis with sloughed necrotic cells, moderate diffuse tubular atrophy with hyaline casts in group II (Fig. 7B) animals. Pretreatment of rats with 50 mg/kg/day allicin for 14 days (group III: Fig. 7C), showed mild focal acute tubular injury with attenuated tubular lining and focal
Table 1 Effect of AL (50 mg/kg) on kidney function parameters in GNT-treated rats.
Control GNT GNT/AL
Serum creatinine (mg/dl)
BUN (mg/dl)
CCr (ml/min)
Proteinuria (mg/day)
Serum albumin (g/dl)
0.97 ± 0.05 3.6 ± 0.22⁎ 2.18 ± 0.21⁎,#
35.8 ± 2.1 121.1 ± 5⁎ 95.8 ± 8.3⁎,#
34.0 ± 3.3 14.7 ± 1.5⁎ 26.4 ± 1.6#
98.8 ± 8.2 257.1 ± 19.4⁎ 255.2 ± 14.5⁎
4.1 ± 0.2 3 ± 0.11⁎ 3.4 ± 0.11⁎
ANOVA, analysis of variance; GNT, gentamicin; AL, allicin; SEM, standard error of mean. Data expressed as means ± SEM (n = 8). Analyses performed using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. ⁎ p b 0.05 vs. control. # p b 0.05 vs. GNT.
Please cite this article as: D.H. El-Kashef, et al., Protective effect of allicin against gentamicin-induced nephrotoxicity in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.010
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Table 2 Effect of AL (50 mg/kg) on kidney anti-oxidant status in GNT-treated rats.
Control GNT GNT/AL
MDA (nmol/g)
GSH (μmol/g)
SOD (U/g)
4.6 ± 0.45 12.1 ± 0.47⁎ 7.8 ± 0.5⁎,#
0.06 ± 0.008 0.02 ± 0.0004⁎ 0.058 ± 0.006#
47.9 ± 2.2 38.7 ± 2.2⁎ 48.7 ± 1.9#
ANOVA, analysis of variance; GNT, gentamicin; AL, allicin; SEM, standard error of mean. Data expressed as means ± SEM (n = 8). Analyses performed using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. ⁎ p b 0.05 vs. control. # p b 0.05 vs. GNT.
sloughed cells associated with interstitial inflammatory cells. The glomeruli and arteries were unremarkable. 4. Discussion Intraperitoneal injection of gentamicin resulted in a significant increase in the kidney weight index in rats compared to the untreated control group. The increase in the kidney weight index of gentamicintreated rats probably resulted from the edema that was caused by drug-induced acute tubular necrosis [28]. Allicin administration with gentamicin showed a significant decrease in kidney weight index compared to gentamicin-treated group; this can be explained by the study of Son et al. [29] that has proved that allicin downregulates gamma IR-induced intercellular adhesion molecule-1 expression (ICAM-1). Bae et al. [30] reported that ICAM-1 is increased in the gentamicintreated kidneys. Since ICAM-1 plays a pivotal role in inflammatory responses, perhaps the down-regulation of this adhesion molecule by allicin could mediate its reducing effect on inflammation and edema. Additionally, allicin exhibits anti-inflammatory effect by inhibiting cell-mediated T-helper 1 and inflammatory cytokines (TNF-α, IL-1α, IL-6, IL-8, T-cell, interferon-γ and IL-2) while upregulating IL-10 production [31]. The results of this study shows that, gentamicin administration to Wistar rats produced a typical pattern of nephrotoxicity which was
Fig. 3. Effect of AL (50 mg/kg) on renal NOx content in GNT-treated rats. Values are expressed as means ± standard error of mean (n = 8). ANOVA, analysis of variance; GNT, gentamicin; AL, allicin; SEM, standard error of mean. Comparisons performed using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. ⁎p b 0.05 vs. control; #p b 0.05 vs. GNT-treated group.
Fig. 4. Effect of AL (50 mg/kg) on renal TNF-α level in GNT-treated rats. Values are expressed as means ± standard error of mean (n = 8). ANOVA, analysis of variance; GNT, gentamicin; AL, allicin; SEM, standard error of mean. Comparisons performed using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. ⁎p b 0.05 vs. control; #p b 0.05 vs. GNT-treated group.
manifested by marked increase in serum creatinine and blood urea nitrogen levels. This result agreed with Jafarey et al. [14]. Allicin treatment decreased serum levels of creatinine and blood urea nitrogen levels when compared to gentamicin-treated group. This result came in line with Wang et al. [32] who showed the oral administered allicin with a concentration of 5, 10, and 20 mg/kg b.w./day could significantly
Fig. 5. Effect of AL (50 mg/kg) on renal MPO activity in GNT-treated rats. Values are expressed as means ± standard error of mean (n = 8(. ANOVA, analysis of variance; GNT, gentamicin; AL, allicin; SEM, standard error of mean. Comparisons performed using one-way ANOVA followed by Tukey–Kramer multiple comparisons post hoc test. ⁎p b 0.05 vs. control; #p b 0.05 vs. GNT-treated group.
Please cite this article as: D.H. El-Kashef, et al., Protective effect of allicin against gentamicin-induced nephrotoxicity in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.010
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Fig. 6. Effect of GNT (100 mg/kg) and AL (50 mg/kg) on contractions to acetylcholine (ACh) in isolated rat urinary bladder rings. Values are expressed as means ± standard error of mean (n = 5). ANOVA, analysis of variance; GNT, gentamicin; AL, allicin; SEM, standard error of mean. Cumulative dose–response curve to ACh (10−7–10−5 M) was measured. p b 0.05 for Emax value compared with control group (*) and compared with GNT group (#) using one way analysis of variance (ANOVA) followed by Tukey–Kramer's multiple comparison post hoc test.
decrease the damage indexes of AST, ALT and BUN. This renoprotective effect may be related to its effect as an antioxidant. In the present study, gentamicin resulted in a significant increase in serum level of lactate dehydrogenase (LDH), but a decrease in level of albumin. This result came in line with Galaly et al. [33] who showed that administration of gentamicin for 3 weeks produced a significant
elevation of LDH activity and decrease in albumin concentration. Allicin administration significantly reduced LDH activity when compared to gentamicin-treated group. This result agreed with Wang et al. [32] who reported the oral administration of allicin significantly decreased the damage indexes of AST, ALT and LDH. On the other hand, our results showed allicin failed to significantly increase the reduced levels of serum albumin. Serum albumin might not be a significant risk marker for GNT-induced nephrotoxicity according to our findings. Further studies should be performed to verify these results due to the limitations of our study. In the current study, gentamicin caused a significant increase in the MDA levels, while GSH and SOD levels were reduced in the kidney tissue. Similar results were also observed in earlier studies [34,35]. Depletion of renal GSH is one of the primary factors which permit lipid peroxidation, suggested to be closely related to gentamicin-induced tissue damage [10]. This observation is in line with many reports that demonstrated apparent elevation in kidney thiobarbituric acid reactive substances (TBARS) following administration of gentamicin [36]. Gentamicin directly increases the production of mitochondrial ROS from the respiratory chain [37]. As known, xanthine–xanthine oxidase produces ROS in the mitochondria through the NADPH oxidase system. Interaction of the ROS produced in excessive amount with lipids, nucleic acids, various proteins, and biomolecules such as polysaccharides leads to cell and tissue damages. ROS cause changes in the fluidity and permeability of the cell membrane giving rise to the lipid peroxidation when occurred in the environment. MDA that can be easily identified in the biologic structures in the terms of the lipid peroxidation and is the indicator of the peroxidative damage is generally used for the evaluation of the lipid peroxidation [38]. On contrast, allicin administration with gentamicin showed a significant decrease in MDA content compared to rats treated with gentamicin alone, a result that agreed with other reported data that demonstrated apparent decrease in TBARS after administration of allicin [39,40]. This effect may be due to the ability of allicin to scavenge hydroxyl and peroxyl radicals and superoxide
Fig. 7. Light micrographs of sections from rat kidneys stained with hematoxylin and eosin. (A) Normal kidney section from the control group (×100); (B) acute tubular necrosis with sloughed necrotic cells, moderate diffuse tubular atrophy with hyaline casts (arrow) after treatment with GNT (100 mg/kg/day) for 7 days (×100); (C) pretreatment of rats with AL (50 mg/kg/day) for 14 days showed mild focal acute tubular injury (×100). GNT, gentamicin; AL, allicin.
Please cite this article as: D.H. El-Kashef, et al., Protective effect of allicin against gentamicin-induced nephrotoxicity in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.010
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anion [41] and inhibit lipid peroxidation [42]. Also, treatment of rats with allicin showed a significant increase in the activity of GSH compared to the gentamicin-treated group, because it has antioxidant activity [43]. Also, it exerts its effect by increasing the activities of nonenzymatic antioxidant (GSH) and the detoxifying enzyme (GST) [42]. Allicin may provide the sulfur source required for the synthesis of GSH [44] and so it restores glutathione level and increases the activities of GSH and GST. Allicin administration with gentamicin resulted in significant increase in the activity of SOD compared to the gentamicintreated group. This may be related to its effect as an antioxidant [43]. In this study, gentamicin treatment produced a significant increase in the NO level. This result came in line with previous study that demonstrated that NO and peroxynitrite plays an important role in the acute renal failure caused by gentamicin [45]. This effect was reversed by allicin administration that resulted in significant decrease in levels of NO when compared to gentamicin-treated group. In the present study, treatment of rats with gentamicin resulted in significant increase in level of TNF-α. This result is in line with Galaly et al. [33]. The activation and nuclear translocation of NF-κB, in response to oxidative stress/nitrosative stress, are the key factors in the renal inflammatory process by regulating the gene expression of cytokines, chemokines, and adhesion molecules [30]. Administration of allicin with gentamicin significantly decreased the level of TNF-α compared with gentamicin-treated group. This result is in agreement with the investigation reported by Bruck et al. [46], who reported that allicin administration (300 ml/mouse/day) had the ability to decrease the elevation in serum level of TNF-α, and the hepatic necroinflammation was much improved. In this study, gentamicin treatment increased the levels of renal MPO activity, this result agreed with El Gamal et al. [47] who showed that gentamicin treatment increased the levels of renal MPO activity, indicating neutrophil infiltration. Administration of allicin resulted in significant decrease in level of MPO when compared to gentamicin-treated group. This result is in consistent with Wang et al. [31] who showed the oral administered allicin with a concentration of 5, 10, and 20 mg/kg b.w./day could significantly decrease the damage indexes of, ROS and MPO. The current study showed that gentamicin treatment produced reduction in GFR, manifested by the progressive reduction in creatinine clearance. This result agreed with Hosaka et al. [48] who reported that the decrease in creatinine clearance corresponds to the reduction in the glomerular filtration rate occurred. Also gentamicin treatment resulted in significant increase in proteinuria level. Allicin administration caused a significant increase in creatinine clearance which reflects the enhanced glomerular function. On the other hand, our results showed allicin failed to significantly decrease the elevated levels of protein in urine. In this study, treatment with gentamicin significantly enhanced the responsiveness of the isolated urinary bladder rings towards ACh at all concentrations used (10−7–10−5). ROS may play an important role in gentamicin-induced tubular damage [5]. NO• could be harmful to tubular cells due to its direct cytotoxic or its reaction with superoxide anion generating the oxidant peroxynitrite ions, which are known to be highly damaging [49]. Therefore, generating ROS, particularly NO•, in the renal system could be suggested as the pathway mediated gentamicininduced kidney dysfunction that manifested by increasing the responsiveness of the urinary bladder rings towards ACh. On the other hand, allicin administration retained normal response of urinary bladder rings towards ACh. The histological studies of kidney from gentamicin treated rats showed tubular necrosis, thickening of vessels and tubular atrophy with hyaline casts. Similar changes were also reported [50,51]. However, allicin attenuated that change. In summary, we have confirmed that allicin has a protective role against nephrotoxicity induced by gentamicin exposure. According to our biochemical findings, which were supported by histopathological and sensitivity of urinary bladder rings to ACh, administration of allicin
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rescued the cells from the effects of gentamicin. These findings indicate that allicin administration may reduce gentamicin-induced acute renal injury. Therefore, we propose that allicin might be a potential candidate agent against gentamicin-induced nephrotoxicity via its antioxidant, anti-inflammatory and immunomodulatory properties.
Conflict of interest statement The authors declare no conflict of interest.
References [1] I. Karahan, A. Atessahin, S. Yilmaz, A.O. Ceribassi, F. Sakin, Protective effect of lycopene on gentamicin-induced oxidative stress and nephrotoxicity in rats, Toxicology 215 (2005) 198–204. [2] W. Mwengee, T. Butler, S. Mgema, et al., Treatment of plague with gentamicin or doxycycline in a randomized clinical trial in Tanzania, Clin. Infect. Dis. 42 (2006) 614–621. [3] W.E. Siegenthaler, A. Bonetti, R. Luthy, Aminoglycoside antibiotics in infectious diseases. An overview, Am J Med. 80 (1986) 2–14. [4] T.H. Mathew, Drug-induced renal disease, Med J Aust. 156 (1992) 724–728. [5] S. Cuzzocrea, E. Mazzon, L.I. Dugo Serraino, R. Di Paola, D. Britti, A. De Sarro, S. Pierpaoli, A. Caputi, E. Masini, D. Salvemini, A role for superoxide in gentamicin mediated nephropathy in rats, Eur. J. Pharmacol. 450 (2002) 67–76. [6] H. Parlakpinar, S. Tasdemir, A. Polat, A. Bay-Karabulut, N. Vardi, M. Ucar, A. Acet, Protective role of caffeic acid phenethyl ester (cape) on gentamicin induced acute renal toxicity in rats, Toxicology 207 (2005) 169–177. [7] C. Yanagida, K. Ito, I. Komiya, T. Horie, Protective effect of fosfomycin on gentamicininduced lipid peroxidation of rat renal tissue, Chem. Biol. Interact. 148 (2004) 139–147. [8] C.L. Yang, X.H. Du, Y.X. Han, Renal cortical mitochondria are source of oxygen free radicals enhanced by gentamicin, Ren. Fail. 17 (1995) 21–26. [9] Z. BaligaZhang, M. Baliga, N. Ueda, S.V. Shah, In vitro and in vivo evidence suggesting a role for iron in cisplatin induced nephrotoxicity, Kidney Int. 53 (1998) 394–401. [10] R. Manikandan, M. Beulaja, R. Thiagarajan, A. Priyadarsini, R. Saravanan, M. Arumugam, 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 (2011) 578–585. [11] R.S. Rivlin, Historical perspective on the use of garlic, J. Nutr. 131 (2001) 951–954S. [12] K. Hirsch, M. Danilenko, J. Giat, et al., Effect of purified allicin, the major ingredient of freshly crushed garlic, on cancer cell proliferation, Nutr. Cancer 38 (2000) 245–254. [13] J. Jakubikova, J. Sedlak, Garlic-derived organosulfides induce cytotoxicity, apoptosis, cell cycle arrest and oxidative stress in human colon carcinoma cell lines, Neoplasma 53 (2006) 191–199. [14] M. Jafarey, S. Changizi Ashtiyani, H. Najafi, Calcium dobesilate for prevention of gentamicin-induced nephrotoxicity in rats, Iran J. Kidney Dis. 8 (2014) 46–52. [15] N.A. Ashry, N.M. Gameil, G.M. Suddek, Modulation of cyclophosphamide-induced early lung injury by allicin, Pharm. Biol. 51 (2013) 806–811. [16] H. Bartels, M. Böhmer, C. Heierli, Serum creatinine determination without protein precipitation, Clin. Chim. Acta 37 (1972) 193–197 (Article in German). [17] J.K. Fawcett, J.E. Scott, A rapid and precise method for the determination of urea, J. Clin. Pathol. 13 (1960) 156–159. [18] W.H. Daughaday, O.H. Lowry, N.J. Rosebrough, W.S. Fields, Determination of cerebrospinal fluid protein with the Folin phenol reagent, J Lab Clin. Med 39 (1952) 663–665. [19] B.T. Doumas, W.A. Watson, H.G. Biggs, Albumin standards and the measurement of serum albumin with bromocresol green, Clin. Chim. Acta 31 (1971) 87–96. [20] J.B. Henry, Todd Sanford Davidsohn, Clinical Diagnosis and Management by Laboratory Methods, 16th edn WB Saunders and Co., Philadelphia, 1974. [21] H. Okhawa, N. Ohishi, K. Yagi, Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction, Anal. Biochem. 95 (1979) 351–358. [22] S.L. Marklund, Superoxide dismutase isoenzymes in tissues and plasma from New Zealand black mice, nude mice and normal BALB/c mice, Mutat. Res. 148 (1985) 129–134. [23] G.L. Ellman, Tissue sulfa hydryl groups, Arch. Biochem. Biophys. 74 (1959) 214–226. [24] C. Schierwagen, A.C. Bylund-Fellenius, C. Lundberg, Improved method for quantification of tissue PMN accumulation measured by myeloperoxidase activity, J. Pharmacol. Methods. 23 (1990) 179–186. [25] A.M. Mostafa, M.N. Nagi, O.A. Al-Shabanha, H.A. El-Kashef, Effect of aminoguanidine and melatonin on the response of isolated urinary bladder to acetylcholine in normal and diabetic rats, Med. Sci. Res. 28 (2000) 33–37. [26] I. Nakamura, C. Takahashi, I. Miyagawa, The alterations of norepinephrine and acetylcholine concentrations in immature rat urinary bladder caused by streptozotocin induced diabetes, J. Urol. 148 (1992) 423–426. [27] R.B. Teixeira, J. Kelly, H. Alpert, V. Pardo, C.A. Vaamonde, Complete protection from gentamicin-induced acute renal failure in the diabetes mellitus rat, Kidney Int. 21 (1982) 600–612. [28] A. Erdem, N.U. Gundogan, A. Usubutun, K. Kilic, S.R. Erdem, A. Kara, et al., The protective effect of taurine against gentamicin-induced acute tubular necrosis in rats, Nephrol. Dial. Transplant. 15 (2000) 1175–1182.
Please cite this article as: D.H. El-Kashef, et al., Protective effect of allicin against gentamicin-induced nephrotoxicity in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.010
8
D.H. El-Kashef et al. / International Immunopharmacology xxx (2015) xxx–xxx
[29] E.W. Son, S.J. Mo, D.K. Rhee, S. Pyo, Inhibition of ICAM-1 expression by garlic component, allicin, in gamma-irradiated human vascular endothelial cells via downregulation of the JNK signaling pathway, Int. Immunopharmacol. 6 (2006) 1788–1795. [30] E.H. Bae, I.J. Kim, S.Y. Joo, et al., Renoprotective effects of the direct renin inhibitor aliskiren on gentamicin-induced nephrotoxicity in rats, J. Renin-AngiotensinAldosterone Syst. 15 (2014) 348–361. [31] G. Hodge, S. Hodge, P. Han, Allium sativum (garlic) suppresses leukocyte inflammatory cytokine production in vitro: potential therapeutic use in the treatment of inflammatory bowel disease, Cytometry 48 (2002) 209–215. [32] E.T. Wang, D.Y. Chen, H.Y. Liu, H.Y. Yan, Y. Yuan, Protective effect of allicin against glycidamide-induced toxicity in male and female mice, Gen. Physiol. Biophys. 34 (2015) 177–187. [33] S.R. Galaly, O.M. Ahmed, A.M. Mahmoud, Thymoquinone and curcumin prevent gentamicin-induced liver injury by attenuating oxidative stress, inflammation and apoptosis, J. Physiol. Pharmacol. 65 (2014) 823–832. [34] E. Ozbek, Y. Turkoz, E. Sahna, F. Ozugurlu, B. Mizrak, M. Ozbek, Melatonin administration prevents the nephrotoxicity induced by gentamicin, BJU Int. 85 (2000) 742–746. [35] M. Tavafi, H. Ahmadvand, Effect of rosmarinic acid on inhibition of gentamicin induced nephrotoxicity in rats, Tissue Cell 43 (2011) 392–397. [36] F.O. Aygün, F.Z. Akçam, O. Kaya, B.M. Ceyhan, R. Sütçü, Caffeic acid phenethyl ester modulates gentamicin-induced oxidative nephrotoxicity in kidney of rats, Biol. Trace Elem. Res. 145 (2012) 211–216. [37] A.I. Morales, D. Detaille, M. Prieto, A. Puente, E. Briones, M. Arévalo, X. Leverve, J.M. López-Novoa, M.Y. El-Mir, Metformin prevents experimental gentamicin-induced nephropathy by a mitochondria dependent pathway, Kidney Int. 77 (2010) 861–869. [38] P. Kovacic, R. Somanathan, Unifying mechanism for eye toxicity: electron transfer, reactive oxygen species, antioxidant benefits, cell signaling and cell membranes, Cell Membr Free Radic Res 2 (2008) 56–69. [39] K. Prasad, V.A. Laxdal, M. Yu, B.L. Raney, Antioxidant activity of allicin, an active principle in garlic, Mol. Cell. Biochem. 148 (1995) 183–189. [40] X.H. Li, C.Y. Li, Z.G. Xiang, et al., Allicin can reduce neuronal death and ameliorate the spatial memory impairment in Alzheimer's disease models, Neurosciences (Riyadh) 15 (2010) 237–243.
[41] K.M. Kim, S.B. Chun, M.S. Koo, et al., Differential regulation of NO availability from macrophages and endothelial cells by the garlic components S-allyl cysteine, Free Radic. Biol. Med. 30 (2001) 747–756. [42] G. Saravanan, J. Prakash, Effect of garlic (Allium sativum) on lipid peroxidation in experimental myocardial infarction in rats, J. Ethnopharmacol. 94 (2004) 155–158. [43] S.K. Banerjee, M. Maulik, S.C. Mancahanda, et al., Dose dependent induction of endogenous antioxidants in rat heart by chronic administration of garlic, Life Sci. 70 (2002) 1509–1518. [44] C.C. Wu, L.Y. Sheen, H.W. Chen, et al., Effects of organosulfur compounds from garlic oil on the antioxidation system in rat liver and red blood cells, Food Chem. Toxicol. 39 (2001) 563–569. [45] J.S. Christo, A.M. Rodrigues, M.G. Mouro, M.A. Cenedeze, M.J. Simões, N. Schor, E.M.S. Higa, Nitric oxide (NO) is associated with gentamicin (GENTA) nephrotoxicity and the renal function recovery after suspension of GENTA treatment in rats, Nitric Oxide 24 (2011) 77–83. [46] R. Bruck, H. Aeed, E. Brazovsky, et al., Allicin, the active component of garlic, prevents immune-mediated, concanavalin A-induced hepatic injury in mice, Liver Int. 25 (2005) 613–621. [47] A.A. El Gamal, M.S. AlSaid, M. Raish, M. Al-Sohaibani, S.M. Al-Massarani, A. Ahmad, M. Hefnawy, M. Al-Yahya, O.A. Basoudan, S. Rafatullah, Beetroot (Beta vulgaris L.) extract ameliorates gentamicin-induced nephrotoxicity associated oxidative stress, inflammation, and apoptosis in rodent model, Mediat. Inflamm. 2014 (2014) 983952. [48] E.M. Hosaka, O.F.P. Santos, A.C. Seguro, F.F. Vattimo, Effect of cyclooxygenase inhibitors on gentamicin-induced nephrotoxicity in rats, Braz. J. Med. Biol. Res. 37 (2004) 979–985. [49] R. Ghaznavi, M. Faghihi, M. Kadkhodaee, S. Shams, H. Khastar, Effects of nitric oxide on gentamicin toxicity in isolated perfused rat kidneys, J. Nephrol. 18 (2005) 548–552. [50] A.A. Al-Majed, A.M. Mostafa, A.C. Al-Rikabi, O.A. Al-Shabanah, Protective effects of oral Arabic gum administration of gentamicin-induced nephrotoxicity in rats, Pharmacol. Res. 46 (2002) 445–451. [51] K.V. Kumar, A.A. Shifow, U.U. Naidu, K.S. Ratnakar, Carvedilol: a beta blocker with antioxidant property protect against gentamicin-induced nephrotoxicity, Life Sci. 66 (2000) 2603–2611.
Please cite this article as: D.H. El-Kashef, et al., Protective effect of allicin against gentamicin-induced nephrotoxicity in rats, Int Immunopharmacol (2015), http://dx.doi.org/10.1016/j.intimp.2015.09.010