http://informahealthcare.com/rnf ISSN: 0886-022X (print), 1525-6049 (electronic) Ren Fail, Early Online: 1–8 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/0886022X.2015.1006100
LABORATORY STUDY
Rutin attenuates gentamicin-induced renal damage by reducing oxidative stress, inflammation, apoptosis, and autophagy in rats Fatih Mehmet Kandemir1, Mustafa Ozkaraca2, Betul Apaydin Yildirim1, Basak Hanedan3, Akin Kirbas3, Kubra Kilic1, Esra Aktas1, and Fulya Benzer4
Ren Fail Downloaded from informahealthcare.com by University of Waterloo on 02/12/15. For personal use only.
1
Department of Biochemistry, Faculty of Veterinary Medicine, Ataturk University, Yakutiye, Erzurum, Turkey, 2Department of Pathology, Faculty of Veterinary Medicine, Ataturk University, Yakutiye, Erzurum, Turkey, 3Department of Internal Medicine, Faculty of Veterinary Medicine, Ataturk University, Yakutiye, Erzurum, Turkey, and 4Health Highschool, Tunceli University, Tunceli, Turkey Abstract
Keywords
Gentamicin is commonly used against gram-negative microorganisms. Its therapeutic use is mainly limited by nephrotoxicity. This study was aimed at evaluating the effect of rutin on oxidative stress, inflammation, apoptosis, and autophagy in gentamicin-induced nephrotoxicity in rats. The rats were treated with saline intraperitoneally (group I), 150 mg/kg of rutin orally (group II), 80 mg/kg of gentamicin intraperitoneally for 8 d (group III), or 150 mg/kg of rutin plus 80 mg/kg of gentamicin (group IV). The serum urea, creatinine, kidney malondialdehyde (MDA), and reduced glutathione (GSH) levels and superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activity and protein concentration were measured, and renal histopathology analysis and immunohistochemical staining were performed. Rutin pretreatment attenuated nephrotoxicity induced by gentamicin by reducing the urea, creatinine, and MDA levels and increasing the SOD, CAT, and GPx activity, and the GSH levels. The rutin also inhibited inducible nitric oxide synthase (iNOS), cleaved caspase-3 and light chain 3B (LC3B), as evidenced by immunohistochemical staining. The present study demonstrates that rutin exhibits antioxidant, anti-inflammatory, anti-apoptotic, and anti-autophagic effects and that it attenuates gentamicin-induced nephrotoxicity in rats.
Gentamicin, rutin, nephrotoxicity, oxidative stress, apoptosis, autophagy
Introduction Gentamicin, an aminoglycoside antibiotic, is commonly used against gram-negative microorganisms. The compound’s therapeutic use is mainly limited by nephrotoxicity.1 Gentamicin-induced nephrotoxicity is indicated by increases in the serum blood urea nitrogen, creatinine, and non-protein nitrogen levels2 and histopathological changes such as glomerular atrophy, tubular necrosis, tubular fibrosis, and inflammation.3 Gentamicin has been demonstrated to increase the generation of reactive oxygen species (ROS) such as superoxide anions, hydroxyl radicals, and hydrogen peroxides4 and reactive nitrogen species in the renal cortex, eventually leading to renal structural and functional deterioration.3 The intrinsic pathway of apoptosis is induced via gentamicin’s
Address correspondence to Assoc. Prof. Dr. Fatih Mehmet Kandemir, Department of Biochemistry, Faculty of Veterinary Medicine, Ataturk University, Yakutiye 25240, Erzurum, Turkey. Tel.: +90 442 2315535; Fax: +90 442 2315563. E-mail: [email protected]
History Received 29 September 2014 Revised 7 December 2014 Accepted 12 December 2014 Published online 23 January 2015
effects on the mitochondria.5 At the molecular level, apoptosis is mediated by the activation of caspases and other factors released from mitochondria.1 In contrast to apoptosis, autophagy protects cells by eliminating damaged organelles and toxic protein aggregates. However, as has been indicated in earlier studies, extensive autophagy can kill severely damaged cells through a caspase-independent form of cell death.6,7 Rutin is a flavonol glycoside consisting of the flavonol quercetin and the disaccharide rutinose. Rutin exhibits a number of pharmacological activities such as antioxidant, anti-cancer, anti-inflammatory, and anti-diabetic activity. Rutin can be regarded as a non-toxic and non-oxidiziable molecule.8 Gentamicin-induced nephrotoxicity principally consists of renal inflammatory cascades, elevated renal oxidative stress, and increases in associated pathological signalling mechanisms.3 Previous studies have suggested that agents with strong antioxidant and cellular anti-inflammatory properties may be able to halt the nephrotoxicity induced by gentamicin.9–12 Research into methods of ameliorating the toxicity of aminoglycosides remains of clinical interest.13 Thus, this
2
F. M. Kandemir et al.
study was aimed at evaluating the effect of rutin on oxidative stress, inflammation, apoptosis, and autophagy in gentamicininduced nephrotoxicity in rats.
Renal function analysis
Materials and methods
Analysis of oxidants and antioxidants
Drugs and chemicals
The kidney tissues were homogenized in a Teflon-glass homogenizer using a buffer of 1.15% KCl to obtain a 1:10 (w/v) homogenate. The malondialdehyde (MDA) levels in the kidney homogenate were measured using the thiobarbituric acid reaction according to the method of Placer et al.16 The MDA levels are expressed as nmol/g of tissue. Kidney catalase (CAT) activity was measured based on the decomposition of hydrogen peroxide at 240 nm according to the method of Aebi17 and is expressed as katal/g of protein. The protein concentration was also measured in the supernatant according to the method of Lowry et al.18 The generation of superoxide radicals produced by xanthine and xanthine oxidase, following the reaction of nitro blue tetrazolium and the formation of formazan dye, was used to measure superoxide dismutase (SOD) activity.19 SOD activity was measured as the level of inhibition of absorbance at 560 nm and is expressed as U/g protein. The glutathione (GSH) content of the kidneys was measured at 412 nm according to the method of Sedlak and Lindsay20 and is expressed as nmol/g of kidney tissue. The glutathione peroxidase (GPx) activity was determined according to the method of Lawrence and Burk21 and is expressed as U/g of protein in the kidney tissue.
Gentamicin sulphate was purchased from Eczacibasi (Gentasol flacon; Istanbul, Turkey), and rutin hydrate was purchased from the Sigma Chemical Company, USA. All other chemicals used were of analytical grade and were purchased from the Sigma Chemical Co. (St. Louis, MO). Animals Ren Fail Downloaded from informahealthcare.com by University of Waterloo on 02/12/15. For personal use only.
Ren Fail, Early Online: 1–8
In total, 24 adult male Sprague-Dawley rats, weighing 180–200 g, were obtained from the Animal Laboratory at the Experimental Research Centre of Ataturk University, Erzurum, Turkey. The animals were maintained in standard housing facilities (temperature: 24 ± 1 C, humidity: 45 ± 5%, and a 12 h light/dark cycle). The animals were supplied with standard laboratory chow and water ad libitum and were left to acclimatize for 1 week before the experiments. The experimental protocol was approved by the Local Animal Care Committee of Ataturk University, Erzurum, Turkey, and the experimental procedures were performed in accordance with the International Guidelines for the Care and Use of Laboratory Animals.
The serum urea and creatinine levels were measured using a commercial kit (Diasis Diagnostic Systems, Istanbul, Turkey).
Histopathological examination Experimental procedure The rats were randomly divided into four groups as follows, with each group consisting of six rats: (1) Group I was treated with sterile saline intraperitoneally for 8 d (control group). (2) Group II was treated with rutin orally at a dose of 150 mg/kg/d for 14 d.14 (3) Group III was treated with gentamicin intraperitoneally at a dose of 80 mg/kg/d for 8 d.15 (4) Group IV was treated with rutin orally at a dose of 150 mg/kg/d for 6 d, followed by gentamicin intraperitoneally at a dose of 80 mg/kg/d plus rutin orally at a dose of 150 mg/kg/d for 8 d. The rats in each group were sacrificed by decapitation 24 h after the last administration under sevoflurane (Sevorane liquid 100%, Abbott Laboratory, Istanbul, Turkey) anesthesia. Sample collection Blood samples were transferred into tubes without anticoagulant and allowed to clot at room temperature for 15 min. The blood was then centrifuged at 200 g for 5 min at 4 C, and serum was collected for analysis of urea and creatinine concentrations. One of the kidneys was removed, washed with ice-cold physiological saline, and stored at 20 C for enzymatic analysis. The other kidney was fixed with 10% buffered formalin solution for histopathological and immunohistochemical examinations.
The kidney tissues of the rats were fixed in a 10% buffered formalin solution. The tissues were subjected to routine alcohol-xylol processes and embedded in paraffin. The samples were then cut into 5-mm thick sections and stained with hematoxylin-eosin (H&E). The kidney sections were examined by light microscopy and assessed at 20 magnification in 10 randomly selected areas to determine changes in degeneration and necrosis. The changes were graded as follows: none (0), mild (1), moderate (2), severe (3) and extremely severe (4). Immunohistochemical examinations To demonstrate inflammation, apoptosis, and autophagy in the kidney tissues of the rats, the presence of inducible nitric oxide synthase (iNOS), cleaved caspase-3, and light chain 3B (LC3B) antibodies in the kidney sections was examined. The iNOS (polyclonal rabbit iNOS antibody, Abcam Cat. No. ab48394), cleaved caspase-3 (polyclonal rabbit active/cleaved caspase-3, Novus Biological Cat. No. NB600-1235), and LC3B (polyclonal rabbit LC3B antibody, Abcam Cat. No. ab15323) primary antibodies were diluted at 1:400. Secondary antibody was used according to manufacturer’s protocol (expose mouse and rabbit specific HRP/DAB detection IHC Kit, Abcam Cat. No. ab80436). The number of stained cells in the kidney sections was evaluated. The sections were scored from 0 to 4, with 0 denoting a lack of staining, 1 denoting cell staining of 25%, 2 denoting staining of 25–50% and 3 denoting staining of 50–75%, 4 denoting staining of 75%.
Rutin attenuates gentamicin-induced nephrotoxicity in rats
DOI: 10.3109/0886022X.2015.1006100
Statistical analysis The data were analyzed with one-way ANOVA using the SPSS package program (version 12.0; SPSS, Chicago, IL). Post-hoc Tukey’s test was used to compare the studied parameters between the groups. The data are presented as the mean ± standard error of means (SEM). Differences were considered significant when the p value was less than 0.05.
Results
Ren Fail Downloaded from informahealthcare.com by University of Waterloo on 02/12/15. For personal use only.
Assessment of kidney function There were no significant differences in the serum urea and creatinine levels between Groups I and II. The levels of serum urea and creatinine in the gentamicin-treated rats (group III) were significantly increased in comparison to those of the control and the rutin-treated rats (p50.05), and the levels of serum urea and creatinine in the gentamicin-plus-rutin-treated rats (group IV) were significantly decreased in comparison to those of the gentamicin-treated rats (group III) (p50.05) but were still significantly higher than those of the control group (p50.05) (Table 1). MDA levels There were no significant differences between the kidney MDA levels of the control and rutin-treated groups. The kidney MDA level was significantly increased in the gentamicin-treated group compared to the control and rutin-treated groups (p50.05), and the kidney MDA level in the gentamicin-plus-rutin-treated group was significantly decreased in comparison to that of the gentamicin-treated group (p50.05) (Table 1). Antioxidants Superoxide dismutase The SOD activity was significantly increased in the rutintreated group compared to the control group (p50.05). In contrast, the enzyme’s activity was significantly decreased in the gentamicin-treated group compared to the control and rutin-treated groups (p50.05). In the
gentamicin-plus-rutin-treated group, the SOD activity was significantly increased compared to the gentamicin-treated group (p50.05). There was no significant difference in SOD activity between the control group and the gentamicinplus-rutin-treated group. Catalase The CAT activity was significantly increased in the rutintreated group compared to the control group (p50.05). The enzyme’s activity level was significantly decreased in the gentamicin-treated group in comparison to the control group (p50.05). In the gentamicin-plus-rutin-treated group, the CAT activity was significantly increased, as compared to the gentamicin-treated group (p50.05) (Table 1). Glutathione peroxidase The GPx activity was significantly increased in the rutintreated group compared to the control group (p50.05). In contrast, the enzyme’s activity was significantly decreased in the gentamicin-treated group compared to the control group (p50.05). In the gentamicin-plus-rutin-treated group, the GPx activity was significantly increased in comparison to the gentamicin-treated group (p50.05). There was no significant difference in GPx activity between the control group and the gentamicin-plus-rutin-treated group, as shown in Table 1. Reduced glutathione There was no significant difference in the GSH content of the control group and the rutin-treated group. The GSH content was significantly decreased in the gentamicin-treated group, as compared to the control group (p50.05). The GSH level of the gentamicin-plus-rutin-treated group was significantly higher than that of the gentamicin-treated group (p50.05), as shown in Table 1. Histopathological evaluation Figure 1(A–D) show histopathological images of the kidney tissues of the control group, rutin-treated group, gentamicintreated group, and gentamicin-plus-rutin-treated group.
Table 1. Effect of rutin and gentamicin on serum markers of renal function and on MDA, GSH and antioxidant enzymes in rat kidney tissues. Parameter Urea (mg/dL) Creatinine (mg/dL) MDA (nmol/g tissue) SOD (U/g protein) CAT (katal/g protein) GPx (U/g protein) GSH (nmol/g tissue)
Group I 7.70 ± 0.22 0.60 ± 0.02 73.73 ± 1.46 35.24 ± 1.12 81.73 ± 0.82 41.31 ± 0.53 5.66 ± 0.13
3
Group II 6.22 ± 0.21 0.74 ± 0.02 75.55 ± 1.35 42.33 ± 1.14d 87.32 ± 0.79c 46.11 ± 1.19d 5.94 ± 0.17
Group III
Group IV a
37.33 ± 0.90 4.75 ± 0.15a 150.03 ± 3.70a 19.46 ± 0.87a 61.22 ± 1.14a 34.14 ± 0.36a 3.58 ± 0.08a
11.37 ± 0.29b 1.28 ± 0.03b 86.76 ± 2.03b 32.24 ± 1.03 74.29 ± 1.45b 41.31 ± 1.42 4.69 ± 0.06b
Notes: MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GSH, glutathione. Group I: control group; Group II: rutin-treated group; Group III: gentamicin-treated group; Group IV: gentamicinplus-rutin-treated group. All the values are expressed as the mean ± SEM of six rats in each group. a p50.05 versus control, rutin-treated and gentamicin-plus-rutin-treated groups. b p50.05 versus control, rutin-treated and gentamicin-treated groups. c p50.05 versus control group. d p50.05 versus control and gentamicin-plus-rutin-treated groups.
Ren Fail Downloaded from informahealthcare.com by University of Waterloo on 02/12/15. For personal use only.
4
F. M. Kandemir et al.
Ren Fail, Early Online: 1–8
Figure 1. Histopathological appearance in Group I (A), Group II (B), Group III (C), and Group IV (D). H&E-stained rat kidney sections.
Table 2. Assessment of degenerative and necrotic changes graded as none (0), mild (1), moderate (2), severe (3) and extremely severe (4) by light microscopy of kidney sections at 20 magnification in 10 randomly selected areas. Histopathological finding Necrosis and degeneration
Group I 0.00 ± 0.00
Group II a
Group III a
0.00 ± 0.00
Group IV b
3.77 ± 0.14
3.00 ± 0.00c
Notes: Group I: control group; Group II: rutin-treated group; Group III: gentamicin-treated group; Group IV: gentamicin-plusrutin-treated group. All the values are expressed as the mean ± SEM of six rats in each group. a p50.05 versus gentamicin-treated and gentamicin-plus-rutin treated groups. b p50.05 versus control, rutin-treated and gentamicin-plus-rutin-treated groups. c p50.05 versus control, rutin-treated and gentamicin-treated groups.
Necrosis and degeneration were not observed in the control group and rutin-treated group. Severe necrosis and degenerative changes were observed in the gentamicin-treated group. These necrosis and degenerative changes were decreased in the gentamicin-plus-rutin-treated group compared to the gentamicin-treated group (Table 2, p50.05). The extensive infiltration of mononuclear cells and presence of hyaline casts were observed in the gentamicin-treated group and rutin decreased the infiltration of mononuclear cells and presence of hyaline casts induced by gentamicin. Immunohistochemical evaluation iNOS, cleaved caspase-3, and LC3B immunopositivity was mild in the control and rutin-treated groups, as shown in Figures 2(A) and (B), 3(A) and (B) and 4(A) and (B), respectively. Severe iNOS immunopositivity in the glomerular, intertubular, and tubular regions was decreased in the gentamicin-plus-rutin-treated group compared to the gentamicin-treated group (p50.05). The increase in cleaved
caspase-3 and LC3B immunopositivity was attenuated in the glomerular and tubular regions of the gentamicinplus-rutin-treated group compared to the gentamicin-treated group (p50.05). A significant difference in the expression of iNOS, cleaved caspase-3, and LC3B was found between the gentamicin-treated group and the gentamicinplus-rutin-treated group (Table 3, p50.05).
Discussion Gentamicin is commonly used against gram-negative bacterial infections.13,22 Gentamicin causes renal damage such as structural changes and functional impairments of the plasma membrane, mitochondria and lysosomes.22 In the present study, the intraperitoneal administration of gentamicin at a dose of 80 mg/kg/d body weight for 8 d induced nephrotoxicity. The impairment of renal function was indicated by the significantly increased serum urea and creatinine levels in the gentamicin-treated group compared to the control group. Various studies report renal function impairment
Ren Fail Downloaded from informahealthcare.com by University of Waterloo on 02/12/15. For personal use only.
DOI: 10.3109/0886022X.2015.1006100
Rutin attenuates gentamicin-induced nephrotoxicity in rats
5
Figure 2. Expression of iNOS in Group I (A), Group II (B), Group III (C), and Group IV (D).
Figure 3. Expression of cleaved caspase-3 in Group I (A), Group II (B), Group III (C), and Group IV (D).
characterized by increased blood urea nitrogen and creatinine levels.11,12,23 Pretreatment with rutin significantly attenuated increases in the urea and creatinine levels for the gentamicintreated animals, a finding which is consistent with the results obtained by the combined administration of quercetin and gentamicin to rats in another study.15
ROS play an important role in the pathophysiology of renal diseases.24 In vivo and in vitro, gentamicin increases the production of ROS by altering mitochondria respiration.9 Khan et al. reported that free radicals and ROS mediate the peroxidation of polyunsaturated fatty acids (PUFAs).25 An overabundance of PUFAs increases the vulnerability of
Ren Fail Downloaded from informahealthcare.com by University of Waterloo on 02/12/15. For personal use only.
6
F. M. Kandemir et al.
Ren Fail, Early Online: 1–8
Figure 4. Expression of LC3B in Group I (A), Group II (B), Group III (C), and Group IV (D).
Table 3. Immunopositivity of iNOS, cleaved caspase 3, and LC3B in the kidney tissues of rats. Immunostaining iNOS Cleaved caspase 3 LC3B
Group I
Group II a
Group III a
Group IV b
1.57 ± 0.20 1.62 ± 0.26 4.00 ± 0.00 3.14 ± 0.14c 1.44 ± 0.17a 1.60 ± 0.24a 3.85 ± 0.14b 3.14 ± 0.14c 1.57 ± 0.20a 1.42 ± 0.20a 3.87 ± 0.12b 3.00 ± 0.00c
Notes: Group I: control group; Group II: rutin-treated group; Group III: gentamicin-treated group; Group IV: gentamicin-plus-rutin-treated group. All the values are expressed as the mean ± SEM of six rats in each group. a p50.05 versus gentamicin-treated and gentamicin-plus-rutin-treated groups. b p50.05 versus control, rutin-treated and gentamicin-plus-rutin-treated groups. c p50.05 versus control, rutin-treated and gentamicin-treated groups.
the kidneys to ROS.24 The present study showed that the MDA level in the kidney was significantly increased in the gentamicin-treated group compared to the control group and that the kidney SOD, CAT, and GPx activities and GSH content were significantly decreased in the gentamicin-treated group, in comparison to the control group. The pretreatment with rutin provided protection against gentamicin-induced nephropathy by significantly reducing lipid peroxidation and significantly increasing the activities of antioxidant enzymes. In agreement with the findings presented here, various studies have reported that antioxidants attenuate gentamicin-induced lipid peroxidation.10–12,23 Gentamicin-induced nephrotoxicity has been previously reported to cause glomerular atrophy, tubular degeneration
and necrosis, intracytoplasmic vacuolation, and mononuclear leukocyte cell infiltration.15 Quercetin treatment reverses most of the aforementioned histopathological alterations induced by gentamicin.15 As shown in Figure 1(C) in the histopathological view of the kidney tissues, the gentamicintreated group exhibited tubular degeneration and necrosis, hyaline casts, and mononuclear cell infiltration. The observed renal histopathological changes due to gentamicin-induced nephrotoxicity are in accord with those reported in other studies.10,12 In addition, this study revealed that severe iNOS, caspase-3 and LC3B immunopositivity occurred in the glomerular, intertubular, and tubular regions of the gentamicin-treated group. The pretreatment with rutin attenuated the kidney damage in the gentamicin-treated group by decreasing the immunopositivity of iNOS, cleaved caspase-3, and LC3B. Other studies of hexachlorobutadiene-induced nephrotoxicity,26 potassium bromate-induced nephrotoxicity,25 renal ischemia/reperfusion injury,27 high cholesterol diet-induced renal injury,28 and experimental diabetic nephropathy29 have reported the ameliorative effects of rutin. Gentamicin has also been reported to reduce renal blood flow,30 induce iNOS expression in glomeruli and mesangial cells,31 and cause peroxynitrite production, through the reaction of excessive iNOS-derived nitric oxide (NO) with superoxide anions32 and inflammation3. Lee et al.23 demonstrated that iNOS is synthesized during inflammation and that iNOS supports NO production. Increased iNOS-mediated NO concentrations may lead to DNA damage and apoptosis. Thus, induction of iNOS leads to tubular cytotoxicity and provokes renal failure.33 Previous studies have reported that various agents such as N-imino-ethyl lysine,34 macrophage stimulating protein,23 and Tephrosia purpurea (L.) Pers. leaves11
Ren Fail Downloaded from informahealthcare.com by University of Waterloo on 02/12/15. For personal use only.
DOI: 10.3109/0886022X.2015.1006100
inhibit iNOS in gentamicin-induced nephrotoxicity. In the present study, increased iNOS expression was observed in the gentamicin-treated group compared to the control group, and rutin pretreatment attenuated this gentamicininduced increase in iNOS, as observed in Figure 2(C) and (D). These findings suggest that increased iNOS expression may worsen gentamicin-induced kidney damage and that pretreatment with rutin can reduce the iNOS expression by decreasing inflammation induced by gentamicin. As demonstrated in a previous study, cytosolic gentamicin activates the intrinsic pathway of apoptosis by attacking mitochondria, inhibiting respiration, and producing oxidative stress.5 Next, specific proteases termed executioner caspases (caspase-3 and -7) are induced, and characteristic morphological signs of apoptosis such as cell shrinkage and DNA separation occur.35 Studies have reported that various agents such as macrophage stimulating protein,23 Zhibai Dihuang Wan,36 leptin,37 and tetramethylpyrazine38 attenuate gentamicin-induced apoptosis. In this study, the expression of the apoptotic molecule, cleaved caspase-3, was significantly increased in the gentamicin-treated group, and pretreatment with rutin significantly reduced its expression, as shown in Figure 3(C) and (D). These results suggest that pretreatment with rutin can suppress gentamicin-induced apoptosis in rat kidney tissues by reducing the expression of cleaved caspase3 and oxidative stress. Autophagy induced in response to stress signals plays a key role in cellular survival rather than cell death. However, excessive autophagy or the unsuitable induction of autophagy causes cell death.39,40 Consistent with the above-mentioned statements, autophagy is stated to increase in nephropathy conditions due to cisplatin and cyclosporine agents.41,42 In this study, LC3B immunopositivity was mild in the control and rutin-treated groups, as shown in Figures 4(A) and (B), due to the occurrence of low-level constitutive autophagy in response to normal physiological processes.39,40 The intense LC3B immunopositivity in the glomerular and tubular regions of the gentamicin-treated group suggested that autophagy persisted during renal injury (Figure 4C). Pretreatment with rutin was able to attenuate renal injury by reducing LC3B expression as shown in Figure 4(D), and reducing oxidative stress. These data suggest that the expression of iNOS, cleaved caspase-3, and LC3B, in addition to oxidative stress and inflammation, plays a significant role in the pathogenesis of gentamicin-induced nephropathy in rats. In conclusion, the present study demonstrated that rutin exhibits antioxidant, anti-inflammatory, anti-apoptotic, and anti-autophagic effects. By inhibiting iNOS, cleaved caspase-3, and LC3B and by increasing antioxidant activity, pretreatment with rutin could attenuate gentamicin-induced nephrotoxicity in rats.
Acknowledgements The authors acknowledge Assoc. Prof. Dr. Omer COBAN for statistical analysis, and Assoc. Prof. Dr. Sinan AKTAS, Director of Experimental Research Centre, and Assistant Prof. Dr. Orhan AKMAN, Assistant Director of Experimental Research Centre, for all types of help.
Rutin attenuates gentamicin-induced nephrotoxicity in rats
7
Declaration of interest The authors declare that there are no conflicts of interest. This study was supported by BAP of Ataturk University (Project No: 2012/366).
References 1. Quiros Y, Vicente-Vicente L, Morales AI, Lopez-Novoa JM. An integrative overview on the mechanisms underlying the renal tubular cytotoxicity of gentamicin. Toxicol Sci. 2011;119:245–256. 2. Plumb DC. Plumb’s Veterinary Drug Handbook. 5th ed. Stockholm, WI: Blackwell Publishing; 2005: 359–363. 3. Balakumar P, Rohilla A, Thangathirupathi A. Gentamicin-induced nephrotoxicity: Do we have a promising therapeutic approach to blunt it? Pharmacol Res. 2010;62:170–186. 4. Baliga R, Ueda N, Walker PD, Shah SV. Oxidant mechanisms in toxic acute renal failure. Drug Metab Rev. 1999;31:971–997. 5. Morales AI, Detaille D, Prieto M, et al. Metformin prevents experimental gentamicin-induced nephropathy by a mitochondriadependent pathway. Kidney Int. 2010;77:861–869. 6. Xie Z, Klionsky DJ. Autopgasome formation: Core machinery and adaptations. Nat Cell Biol. 2007;9:1102–1109. 7. Scarlatti F, Granata R, Meijer AJ, Codogno P. Does autophagy have a licence to kill mammalian cells? Cell Death Differ. 2009;16: 12–20. 8. Sharma S, Asgar A, Javed A, Jasjeet KS, Sanjula B. Rutin: Therapeutic potential and recent advances in drug delivery. Expert Opin Investig Drugs. 2013;22:1063–1079. 9. Koyner JL, Sher AR, Murray PT. Antioxidants. Do they have a place in the prevention or therapy of acute kidney injury? Nephron Exp Nephrol. 2008;109:e109–e117. 10. Manikandan R, Beulaja M, Thiagarajan R, Priyadarsini A, Saravanan R, Arumugam M. 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. 2011;670:578–585. 11. Jain A, Nahata A, Singhai AK. Effect of Tephrosia purpurea (L.) Pers. Leaves on gentamicin induced nephrotoxicity in rats. Sci Pharm. 2013;81:1071–1087. 12. Kang C, Lee H, Hah DY, et al. Protective effects of Houttuynia cordata Thunb. On gentamicin-induced oxidative stress and nephrotoxicity in rats. Toxicol Res. 2013;29:61–67. 13. Ali BH, Al Za’abi M, Blunden G, Nemmar A. Experimental gentamicin nephrotoxicity and agents that modify it: A mini-review of recent research. Basic Clin Pharmacol Toxicol. 2011;109: 225–232. 14. Arjumand W, Seth A, Sultana S. Rutin attenuates cisplatin induced renal inflammation and apoptosis by reducing NFkB, TNF-a and caspase-3 expression in wistar rats. Food Chem Toxicol. 2011;49: 2013–2021. 15. Abdel-Raheem IT, Abdel-Ghany AA, Mohamed A. Protective effect of quercetin against gentamicin-induced nephrotoxicity. Biol Pharm Bull. 2009;32:61–67. 16. Placer ZA, Cushmanni LL, Johnson BC. Estimation of products of lipid peroxidation (as malondialdehyde) in biochemical systems. Anal Biochem. 1966;16:359–364. 17. Aebi H. Catalase. In: Bergmeyer HU, ed. Methods in Enzymatic Analysis. New York: Academic Press; 1983: 276–286. 18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951; 193:265–275. 19. Sun Y, Larry WO, Ying L. A simple method for clinical assay of superoxide dismutase. Clin Chem. 1988;34:497–500. 20. Sedlak J, Lindsay RHC. Estimation of total protein bound and nonprotein sulfhydryl groups in tissue with Ellmann’s reagent. Anal Biochem. 1968;25:192–205. 21. Lawrence RA, Burk RF. Glutathione peroxidase activity in selenium-deficient rat liver. Bioch Biophys Res Comm. 1976;71: 952–958. 22. Nagai J, Takano M. Molecular aspects of renal handling of aminoglycosides and strategies for preventing the nephrotoxicity. Drug Metab Pharmacokinet. 2004;19:159–170. 23. Lee KE, Kim EY, Kim CS, et al. Macrophage-stimulating protein attenuates gentamicin-induced inflammation and apoptosis in
8
24. 25. 26. 27. 28.
Ren Fail Downloaded from informahealthcare.com by University of Waterloo on 02/12/15. For personal use only.
29. 30. 31. 32.
F. M. Kandemir et al. human renal proximal tubular epithelial cells. Biochem Biophys Res Commun. 2013;434:527–533. Singh D, Kaur R, Chander V, Chopra K. Antioxidants in the prevention of renal disease. J Med Food. 2006;9:443–450. Khan RA, Khan MR, Sahreen S. Protective effects of rutin against potassium bromate induced nephrotoxicity in rats. BMC Complement Altern Med. 2012;12:204. Sadeghnia HR, Yousefsani BS, Rashidfar M, Boroushaki MT, Asadpour E, Ghorbani A. Protective effect of rutin on hexachlorobutadiene-induced nephrotoxicity. Ren Fail. 2013;35:1151–1155. Korkmaz A, Kolankaya D. Inhibiting inducible nitric oxide synthase with rutin reduces renal ischemia/reperfusion injury. Can J Surg. 2013;56:6–14. Al-Rejaie SS, Abuohashaish HM, Alkhamees OA, Aleisa AM, Alroujayee AS. Gender difference following high cholesterol diet induced renal injury and the protective role of rutin and ascorbic acid combination in Wistar albino rats. Lipids Health Dis. 2012;11: 1–10. Hao H-H, Shao Z-M, Tang D-Q, et al. Preventive effects of rutin on the development of experimental diabetic nephropathy in rats. Life Sci. 2012;91:959–967. Sec¸ilmi¸s MA, Karata¸s Y, Yorulmaz O, et al. Protective effect of L-arginine intake on the impaired renal vascular responses in the gentamicin-treated rats. Nephron Physiol. 2005;100:13–20. Kiang JG, Tsen KT. Biology of hypoxia. Chin J Physiol. 2006;49: 223–233. Pedraza-Chaverri J, Barrera D, Maldonado PD, et al. S-allylmercaptocysteine scavenges hydroxyl radical and singlet oxygen in vitro and attenuates gentamicin-induced oxidative and nitrosative stress and renal damage in vivo. BMC Clin Pharmacol. 2004;4:5.
Ren Fail, Early Online: 1–8
33. Goligorsky MS, Gross SS. Nitric Oxide and the Kidney. New York: Chapman & Hall; 1997:22–38. 34. Ghaznavi R, Kadkhodaee M. Comparative effects of selective and non-selective nitric oxide synthase inhibition in gentamicin-induced rat nephrotoxicity. Arch Toxicol. 2007;81:453–457. 35. Servais H, Ortiz A, Devuyst O, Denamur S, Tulkens PM, MingeotLeclercq MP. Renal cell apoptosis induced by nephrotoxic drugs: Cellular and molecular mechanisms and potential approaches to modulation. Apoptosis. 2008;13:11–32. 36. Hsu Y-H, Chen T-H, Wu M-Y, et al. Protective effects of Zhibai Dihuang Wan on renal tubular cells affected with gentamicininduced apoptosis. J Ethnopharmacol. 2014;151:635–642. 37. Chen Y-C, Chen C-H, Hsu Y-H, et al. Leptin reduces gentamicininduced apoptosis in rat renal tubular cells via the PI3K-Akt signaling pathway. Eur J Pharmacol. 2011;658:213–218. 38. Juan S-H, Chen C-H, Hsu Y-H, et al. Tetramethylpyrazine protects rat renal tubular cell apoptosis induced by gentamicin. Nephrol Dial Transplant. 2007;22:732–739. 39. Kundu M, Thombson CB. Autophagy: Basic principles and relevance to disease. Annu Rev Pathol. 2008;3:427–455. 40. Levine B, Yuan J. Autophagy in cell death: an innocent convict? J Clin Invest. 2005;10:2679–2788. 41. Domitrovic R, Cvijanovic O, Pernjak-Pugel E, Skoda M, Mikelic L, Crncevic-Orlic Z. Berberine exerts nephroprotective effect against cisplatin-induced kidney damage through inhibition of oxidative/ nitrosative stress, inflammation, autophagy and apoptosis. Food Chem Toxicol. 2013;62:397–406. 42. Xiang Y, Piao SG, Zou HB, et al. L-carnitine protects against cyclosporine-induced pancreatic and renal injury in rats. Transplant Proc. 2013;45:3127–3134.
LABORATORY STUDY
Rutin attenuates gentamicin-induced renal damage by reducing oxidative stress, inflammation, apoptosis, and autophagy in rats Fatih Mehmet Kandemir1, Mustafa Ozkaraca2, Betul Apaydin Yildirim1, Basak Hanedan3, Akin Kirbas3, Kubra Kilic1, Esra Aktas1, and Fulya Benzer4
Ren Fail Downloaded from informahealthcare.com by University of Waterloo on 02/12/15. For personal use only.
1
Department of Biochemistry, Faculty of Veterinary Medicine, Ataturk University, Yakutiye, Erzurum, Turkey, 2Department of Pathology, Faculty of Veterinary Medicine, Ataturk University, Yakutiye, Erzurum, Turkey, 3Department of Internal Medicine, Faculty of Veterinary Medicine, Ataturk University, Yakutiye, Erzurum, Turkey, and 4Health Highschool, Tunceli University, Tunceli, Turkey Abstract
Keywords
Gentamicin is commonly used against gram-negative microorganisms. Its therapeutic use is mainly limited by nephrotoxicity. This study was aimed at evaluating the effect of rutin on oxidative stress, inflammation, apoptosis, and autophagy in gentamicin-induced nephrotoxicity in rats. The rats were treated with saline intraperitoneally (group I), 150 mg/kg of rutin orally (group II), 80 mg/kg of gentamicin intraperitoneally for 8 d (group III), or 150 mg/kg of rutin plus 80 mg/kg of gentamicin (group IV). The serum urea, creatinine, kidney malondialdehyde (MDA), and reduced glutathione (GSH) levels and superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activity and protein concentration were measured, and renal histopathology analysis and immunohistochemical staining were performed. Rutin pretreatment attenuated nephrotoxicity induced by gentamicin by reducing the urea, creatinine, and MDA levels and increasing the SOD, CAT, and GPx activity, and the GSH levels. The rutin also inhibited inducible nitric oxide synthase (iNOS), cleaved caspase-3 and light chain 3B (LC3B), as evidenced by immunohistochemical staining. The present study demonstrates that rutin exhibits antioxidant, anti-inflammatory, anti-apoptotic, and anti-autophagic effects and that it attenuates gentamicin-induced nephrotoxicity in rats.
Gentamicin, rutin, nephrotoxicity, oxidative stress, apoptosis, autophagy
Introduction Gentamicin, an aminoglycoside antibiotic, is commonly used against gram-negative microorganisms. The compound’s therapeutic use is mainly limited by nephrotoxicity.1 Gentamicin-induced nephrotoxicity is indicated by increases in the serum blood urea nitrogen, creatinine, and non-protein nitrogen levels2 and histopathological changes such as glomerular atrophy, tubular necrosis, tubular fibrosis, and inflammation.3 Gentamicin has been demonstrated to increase the generation of reactive oxygen species (ROS) such as superoxide anions, hydroxyl radicals, and hydrogen peroxides4 and reactive nitrogen species in the renal cortex, eventually leading to renal structural and functional deterioration.3 The intrinsic pathway of apoptosis is induced via gentamicin’s
Address correspondence to Assoc. Prof. Dr. Fatih Mehmet Kandemir, Department of Biochemistry, Faculty of Veterinary Medicine, Ataturk University, Yakutiye 25240, Erzurum, Turkey. Tel.: +90 442 2315535; Fax: +90 442 2315563. E-mail: [email protected]
History Received 29 September 2014 Revised 7 December 2014 Accepted 12 December 2014 Published online 23 January 2015
effects on the mitochondria.5 At the molecular level, apoptosis is mediated by the activation of caspases and other factors released from mitochondria.1 In contrast to apoptosis, autophagy protects cells by eliminating damaged organelles and toxic protein aggregates. However, as has been indicated in earlier studies, extensive autophagy can kill severely damaged cells through a caspase-independent form of cell death.6,7 Rutin is a flavonol glycoside consisting of the flavonol quercetin and the disaccharide rutinose. Rutin exhibits a number of pharmacological activities such as antioxidant, anti-cancer, anti-inflammatory, and anti-diabetic activity. Rutin can be regarded as a non-toxic and non-oxidiziable molecule.8 Gentamicin-induced nephrotoxicity principally consists of renal inflammatory cascades, elevated renal oxidative stress, and increases in associated pathological signalling mechanisms.3 Previous studies have suggested that agents with strong antioxidant and cellular anti-inflammatory properties may be able to halt the nephrotoxicity induced by gentamicin.9–12 Research into methods of ameliorating the toxicity of aminoglycosides remains of clinical interest.13 Thus, this
2
F. M. Kandemir et al.
study was aimed at evaluating the effect of rutin on oxidative stress, inflammation, apoptosis, and autophagy in gentamicininduced nephrotoxicity in rats.
Renal function analysis
Materials and methods
Analysis of oxidants and antioxidants
Drugs and chemicals
The kidney tissues were homogenized in a Teflon-glass homogenizer using a buffer of 1.15% KCl to obtain a 1:10 (w/v) homogenate. The malondialdehyde (MDA) levels in the kidney homogenate were measured using the thiobarbituric acid reaction according to the method of Placer et al.16 The MDA levels are expressed as nmol/g of tissue. Kidney catalase (CAT) activity was measured based on the decomposition of hydrogen peroxide at 240 nm according to the method of Aebi17 and is expressed as katal/g of protein. The protein concentration was also measured in the supernatant according to the method of Lowry et al.18 The generation of superoxide radicals produced by xanthine and xanthine oxidase, following the reaction of nitro blue tetrazolium and the formation of formazan dye, was used to measure superoxide dismutase (SOD) activity.19 SOD activity was measured as the level of inhibition of absorbance at 560 nm and is expressed as U/g protein. The glutathione (GSH) content of the kidneys was measured at 412 nm according to the method of Sedlak and Lindsay20 and is expressed as nmol/g of kidney tissue. The glutathione peroxidase (GPx) activity was determined according to the method of Lawrence and Burk21 and is expressed as U/g of protein in the kidney tissue.
Gentamicin sulphate was purchased from Eczacibasi (Gentasol flacon; Istanbul, Turkey), and rutin hydrate was purchased from the Sigma Chemical Company, USA. All other chemicals used were of analytical grade and were purchased from the Sigma Chemical Co. (St. Louis, MO). Animals Ren Fail Downloaded from informahealthcare.com by University of Waterloo on 02/12/15. For personal use only.
Ren Fail, Early Online: 1–8
In total, 24 adult male Sprague-Dawley rats, weighing 180–200 g, were obtained from the Animal Laboratory at the Experimental Research Centre of Ataturk University, Erzurum, Turkey. The animals were maintained in standard housing facilities (temperature: 24 ± 1 C, humidity: 45 ± 5%, and a 12 h light/dark cycle). The animals were supplied with standard laboratory chow and water ad libitum and were left to acclimatize for 1 week before the experiments. The experimental protocol was approved by the Local Animal Care Committee of Ataturk University, Erzurum, Turkey, and the experimental procedures were performed in accordance with the International Guidelines for the Care and Use of Laboratory Animals.
The serum urea and creatinine levels were measured using a commercial kit (Diasis Diagnostic Systems, Istanbul, Turkey).
Histopathological examination Experimental procedure The rats were randomly divided into four groups as follows, with each group consisting of six rats: (1) Group I was treated with sterile saline intraperitoneally for 8 d (control group). (2) Group II was treated with rutin orally at a dose of 150 mg/kg/d for 14 d.14 (3) Group III was treated with gentamicin intraperitoneally at a dose of 80 mg/kg/d for 8 d.15 (4) Group IV was treated with rutin orally at a dose of 150 mg/kg/d for 6 d, followed by gentamicin intraperitoneally at a dose of 80 mg/kg/d plus rutin orally at a dose of 150 mg/kg/d for 8 d. The rats in each group were sacrificed by decapitation 24 h after the last administration under sevoflurane (Sevorane liquid 100%, Abbott Laboratory, Istanbul, Turkey) anesthesia. Sample collection Blood samples were transferred into tubes without anticoagulant and allowed to clot at room temperature for 15 min. The blood was then centrifuged at 200 g for 5 min at 4 C, and serum was collected for analysis of urea and creatinine concentrations. One of the kidneys was removed, washed with ice-cold physiological saline, and stored at 20 C for enzymatic analysis. The other kidney was fixed with 10% buffered formalin solution for histopathological and immunohistochemical examinations.
The kidney tissues of the rats were fixed in a 10% buffered formalin solution. The tissues were subjected to routine alcohol-xylol processes and embedded in paraffin. The samples were then cut into 5-mm thick sections and stained with hematoxylin-eosin (H&E). The kidney sections were examined by light microscopy and assessed at 20 magnification in 10 randomly selected areas to determine changes in degeneration and necrosis. The changes were graded as follows: none (0), mild (1), moderate (2), severe (3) and extremely severe (4). Immunohistochemical examinations To demonstrate inflammation, apoptosis, and autophagy in the kidney tissues of the rats, the presence of inducible nitric oxide synthase (iNOS), cleaved caspase-3, and light chain 3B (LC3B) antibodies in the kidney sections was examined. The iNOS (polyclonal rabbit iNOS antibody, Abcam Cat. No. ab48394), cleaved caspase-3 (polyclonal rabbit active/cleaved caspase-3, Novus Biological Cat. No. NB600-1235), and LC3B (polyclonal rabbit LC3B antibody, Abcam Cat. No. ab15323) primary antibodies were diluted at 1:400. Secondary antibody was used according to manufacturer’s protocol (expose mouse and rabbit specific HRP/DAB detection IHC Kit, Abcam Cat. No. ab80436). The number of stained cells in the kidney sections was evaluated. The sections were scored from 0 to 4, with 0 denoting a lack of staining, 1 denoting cell staining of 25%, 2 denoting staining of 25–50% and 3 denoting staining of 50–75%, 4 denoting staining of 75%.
Rutin attenuates gentamicin-induced nephrotoxicity in rats
DOI: 10.3109/0886022X.2015.1006100
Statistical analysis The data were analyzed with one-way ANOVA using the SPSS package program (version 12.0; SPSS, Chicago, IL). Post-hoc Tukey’s test was used to compare the studied parameters between the groups. The data are presented as the mean ± standard error of means (SEM). Differences were considered significant when the p value was less than 0.05.
Results
Ren Fail Downloaded from informahealthcare.com by University of Waterloo on 02/12/15. For personal use only.
Assessment of kidney function There were no significant differences in the serum urea and creatinine levels between Groups I and II. The levels of serum urea and creatinine in the gentamicin-treated rats (group III) were significantly increased in comparison to those of the control and the rutin-treated rats (p50.05), and the levels of serum urea and creatinine in the gentamicin-plus-rutin-treated rats (group IV) were significantly decreased in comparison to those of the gentamicin-treated rats (group III) (p50.05) but were still significantly higher than those of the control group (p50.05) (Table 1). MDA levels There were no significant differences between the kidney MDA levels of the control and rutin-treated groups. The kidney MDA level was significantly increased in the gentamicin-treated group compared to the control and rutin-treated groups (p50.05), and the kidney MDA level in the gentamicin-plus-rutin-treated group was significantly decreased in comparison to that of the gentamicin-treated group (p50.05) (Table 1). Antioxidants Superoxide dismutase The SOD activity was significantly increased in the rutintreated group compared to the control group (p50.05). In contrast, the enzyme’s activity was significantly decreased in the gentamicin-treated group compared to the control and rutin-treated groups (p50.05). In the
gentamicin-plus-rutin-treated group, the SOD activity was significantly increased compared to the gentamicin-treated group (p50.05). There was no significant difference in SOD activity between the control group and the gentamicinplus-rutin-treated group. Catalase The CAT activity was significantly increased in the rutintreated group compared to the control group (p50.05). The enzyme’s activity level was significantly decreased in the gentamicin-treated group in comparison to the control group (p50.05). In the gentamicin-plus-rutin-treated group, the CAT activity was significantly increased, as compared to the gentamicin-treated group (p50.05) (Table 1). Glutathione peroxidase The GPx activity was significantly increased in the rutintreated group compared to the control group (p50.05). In contrast, the enzyme’s activity was significantly decreased in the gentamicin-treated group compared to the control group (p50.05). In the gentamicin-plus-rutin-treated group, the GPx activity was significantly increased in comparison to the gentamicin-treated group (p50.05). There was no significant difference in GPx activity between the control group and the gentamicin-plus-rutin-treated group, as shown in Table 1. Reduced glutathione There was no significant difference in the GSH content of the control group and the rutin-treated group. The GSH content was significantly decreased in the gentamicin-treated group, as compared to the control group (p50.05). The GSH level of the gentamicin-plus-rutin-treated group was significantly higher than that of the gentamicin-treated group (p50.05), as shown in Table 1. Histopathological evaluation Figure 1(A–D) show histopathological images of the kidney tissues of the control group, rutin-treated group, gentamicintreated group, and gentamicin-plus-rutin-treated group.
Table 1. Effect of rutin and gentamicin on serum markers of renal function and on MDA, GSH and antioxidant enzymes in rat kidney tissues. Parameter Urea (mg/dL) Creatinine (mg/dL) MDA (nmol/g tissue) SOD (U/g protein) CAT (katal/g protein) GPx (U/g protein) GSH (nmol/g tissue)
Group I 7.70 ± 0.22 0.60 ± 0.02 73.73 ± 1.46 35.24 ± 1.12 81.73 ± 0.82 41.31 ± 0.53 5.66 ± 0.13
3
Group II 6.22 ± 0.21 0.74 ± 0.02 75.55 ± 1.35 42.33 ± 1.14d 87.32 ± 0.79c 46.11 ± 1.19d 5.94 ± 0.17
Group III
Group IV a
37.33 ± 0.90 4.75 ± 0.15a 150.03 ± 3.70a 19.46 ± 0.87a 61.22 ± 1.14a 34.14 ± 0.36a 3.58 ± 0.08a
11.37 ± 0.29b 1.28 ± 0.03b 86.76 ± 2.03b 32.24 ± 1.03 74.29 ± 1.45b 41.31 ± 1.42 4.69 ± 0.06b
Notes: MDA, malondialdehyde; SOD, superoxide dismutase; CAT, catalase; GPx, glutathione peroxidase; GSH, glutathione. Group I: control group; Group II: rutin-treated group; Group III: gentamicin-treated group; Group IV: gentamicinplus-rutin-treated group. All the values are expressed as the mean ± SEM of six rats in each group. a p50.05 versus control, rutin-treated and gentamicin-plus-rutin-treated groups. b p50.05 versus control, rutin-treated and gentamicin-treated groups. c p50.05 versus control group. d p50.05 versus control and gentamicin-plus-rutin-treated groups.
Ren Fail Downloaded from informahealthcare.com by University of Waterloo on 02/12/15. For personal use only.
4
F. M. Kandemir et al.
Ren Fail, Early Online: 1–8
Figure 1. Histopathological appearance in Group I (A), Group II (B), Group III (C), and Group IV (D). H&E-stained rat kidney sections.
Table 2. Assessment of degenerative and necrotic changes graded as none (0), mild (1), moderate (2), severe (3) and extremely severe (4) by light microscopy of kidney sections at 20 magnification in 10 randomly selected areas. Histopathological finding Necrosis and degeneration
Group I 0.00 ± 0.00
Group II a
Group III a
0.00 ± 0.00
Group IV b
3.77 ± 0.14
3.00 ± 0.00c
Notes: Group I: control group; Group II: rutin-treated group; Group III: gentamicin-treated group; Group IV: gentamicin-plusrutin-treated group. All the values are expressed as the mean ± SEM of six rats in each group. a p50.05 versus gentamicin-treated and gentamicin-plus-rutin treated groups. b p50.05 versus control, rutin-treated and gentamicin-plus-rutin-treated groups. c p50.05 versus control, rutin-treated and gentamicin-treated groups.
Necrosis and degeneration were not observed in the control group and rutin-treated group. Severe necrosis and degenerative changes were observed in the gentamicin-treated group. These necrosis and degenerative changes were decreased in the gentamicin-plus-rutin-treated group compared to the gentamicin-treated group (Table 2, p50.05). The extensive infiltration of mononuclear cells and presence of hyaline casts were observed in the gentamicin-treated group and rutin decreased the infiltration of mononuclear cells and presence of hyaline casts induced by gentamicin. Immunohistochemical evaluation iNOS, cleaved caspase-3, and LC3B immunopositivity was mild in the control and rutin-treated groups, as shown in Figures 2(A) and (B), 3(A) and (B) and 4(A) and (B), respectively. Severe iNOS immunopositivity in the glomerular, intertubular, and tubular regions was decreased in the gentamicin-plus-rutin-treated group compared to the gentamicin-treated group (p50.05). The increase in cleaved
caspase-3 and LC3B immunopositivity was attenuated in the glomerular and tubular regions of the gentamicinplus-rutin-treated group compared to the gentamicin-treated group (p50.05). A significant difference in the expression of iNOS, cleaved caspase-3, and LC3B was found between the gentamicin-treated group and the gentamicinplus-rutin-treated group (Table 3, p50.05).
Discussion Gentamicin is commonly used against gram-negative bacterial infections.13,22 Gentamicin causes renal damage such as structural changes and functional impairments of the plasma membrane, mitochondria and lysosomes.22 In the present study, the intraperitoneal administration of gentamicin at a dose of 80 mg/kg/d body weight for 8 d induced nephrotoxicity. The impairment of renal function was indicated by the significantly increased serum urea and creatinine levels in the gentamicin-treated group compared to the control group. Various studies report renal function impairment
Ren Fail Downloaded from informahealthcare.com by University of Waterloo on 02/12/15. For personal use only.
DOI: 10.3109/0886022X.2015.1006100
Rutin attenuates gentamicin-induced nephrotoxicity in rats
5
Figure 2. Expression of iNOS in Group I (A), Group II (B), Group III (C), and Group IV (D).
Figure 3. Expression of cleaved caspase-3 in Group I (A), Group II (B), Group III (C), and Group IV (D).
characterized by increased blood urea nitrogen and creatinine levels.11,12,23 Pretreatment with rutin significantly attenuated increases in the urea and creatinine levels for the gentamicintreated animals, a finding which is consistent with the results obtained by the combined administration of quercetin and gentamicin to rats in another study.15
ROS play an important role in the pathophysiology of renal diseases.24 In vivo and in vitro, gentamicin increases the production of ROS by altering mitochondria respiration.9 Khan et al. reported that free radicals and ROS mediate the peroxidation of polyunsaturated fatty acids (PUFAs).25 An overabundance of PUFAs increases the vulnerability of
Ren Fail Downloaded from informahealthcare.com by University of Waterloo on 02/12/15. For personal use only.
6
F. M. Kandemir et al.
Ren Fail, Early Online: 1–8
Figure 4. Expression of LC3B in Group I (A), Group II (B), Group III (C), and Group IV (D).
Table 3. Immunopositivity of iNOS, cleaved caspase 3, and LC3B in the kidney tissues of rats. Immunostaining iNOS Cleaved caspase 3 LC3B
Group I
Group II a
Group III a
Group IV b
1.57 ± 0.20 1.62 ± 0.26 4.00 ± 0.00 3.14 ± 0.14c 1.44 ± 0.17a 1.60 ± 0.24a 3.85 ± 0.14b 3.14 ± 0.14c 1.57 ± 0.20a 1.42 ± 0.20a 3.87 ± 0.12b 3.00 ± 0.00c
Notes: Group I: control group; Group II: rutin-treated group; Group III: gentamicin-treated group; Group IV: gentamicin-plus-rutin-treated group. All the values are expressed as the mean ± SEM of six rats in each group. a p50.05 versus gentamicin-treated and gentamicin-plus-rutin-treated groups. b p50.05 versus control, rutin-treated and gentamicin-plus-rutin-treated groups. c p50.05 versus control, rutin-treated and gentamicin-treated groups.
the kidneys to ROS.24 The present study showed that the MDA level in the kidney was significantly increased in the gentamicin-treated group compared to the control group and that the kidney SOD, CAT, and GPx activities and GSH content were significantly decreased in the gentamicin-treated group, in comparison to the control group. The pretreatment with rutin provided protection against gentamicin-induced nephropathy by significantly reducing lipid peroxidation and significantly increasing the activities of antioxidant enzymes. In agreement with the findings presented here, various studies have reported that antioxidants attenuate gentamicin-induced lipid peroxidation.10–12,23 Gentamicin-induced nephrotoxicity has been previously reported to cause glomerular atrophy, tubular degeneration
and necrosis, intracytoplasmic vacuolation, and mononuclear leukocyte cell infiltration.15 Quercetin treatment reverses most of the aforementioned histopathological alterations induced by gentamicin.15 As shown in Figure 1(C) in the histopathological view of the kidney tissues, the gentamicintreated group exhibited tubular degeneration and necrosis, hyaline casts, and mononuclear cell infiltration. The observed renal histopathological changes due to gentamicin-induced nephrotoxicity are in accord with those reported in other studies.10,12 In addition, this study revealed that severe iNOS, caspase-3 and LC3B immunopositivity occurred in the glomerular, intertubular, and tubular regions of the gentamicin-treated group. The pretreatment with rutin attenuated the kidney damage in the gentamicin-treated group by decreasing the immunopositivity of iNOS, cleaved caspase-3, and LC3B. Other studies of hexachlorobutadiene-induced nephrotoxicity,26 potassium bromate-induced nephrotoxicity,25 renal ischemia/reperfusion injury,27 high cholesterol diet-induced renal injury,28 and experimental diabetic nephropathy29 have reported the ameliorative effects of rutin. Gentamicin has also been reported to reduce renal blood flow,30 induce iNOS expression in glomeruli and mesangial cells,31 and cause peroxynitrite production, through the reaction of excessive iNOS-derived nitric oxide (NO) with superoxide anions32 and inflammation3. Lee et al.23 demonstrated that iNOS is synthesized during inflammation and that iNOS supports NO production. Increased iNOS-mediated NO concentrations may lead to DNA damage and apoptosis. Thus, induction of iNOS leads to tubular cytotoxicity and provokes renal failure.33 Previous studies have reported that various agents such as N-imino-ethyl lysine,34 macrophage stimulating protein,23 and Tephrosia purpurea (L.) Pers. leaves11
Ren Fail Downloaded from informahealthcare.com by University of Waterloo on 02/12/15. For personal use only.
DOI: 10.3109/0886022X.2015.1006100
inhibit iNOS in gentamicin-induced nephrotoxicity. In the present study, increased iNOS expression was observed in the gentamicin-treated group compared to the control group, and rutin pretreatment attenuated this gentamicininduced increase in iNOS, as observed in Figure 2(C) and (D). These findings suggest that increased iNOS expression may worsen gentamicin-induced kidney damage and that pretreatment with rutin can reduce the iNOS expression by decreasing inflammation induced by gentamicin. As demonstrated in a previous study, cytosolic gentamicin activates the intrinsic pathway of apoptosis by attacking mitochondria, inhibiting respiration, and producing oxidative stress.5 Next, specific proteases termed executioner caspases (caspase-3 and -7) are induced, and characteristic morphological signs of apoptosis such as cell shrinkage and DNA separation occur.35 Studies have reported that various agents such as macrophage stimulating protein,23 Zhibai Dihuang Wan,36 leptin,37 and tetramethylpyrazine38 attenuate gentamicin-induced apoptosis. In this study, the expression of the apoptotic molecule, cleaved caspase-3, was significantly increased in the gentamicin-treated group, and pretreatment with rutin significantly reduced its expression, as shown in Figure 3(C) and (D). These results suggest that pretreatment with rutin can suppress gentamicin-induced apoptosis in rat kidney tissues by reducing the expression of cleaved caspase3 and oxidative stress. Autophagy induced in response to stress signals plays a key role in cellular survival rather than cell death. However, excessive autophagy or the unsuitable induction of autophagy causes cell death.39,40 Consistent with the above-mentioned statements, autophagy is stated to increase in nephropathy conditions due to cisplatin and cyclosporine agents.41,42 In this study, LC3B immunopositivity was mild in the control and rutin-treated groups, as shown in Figures 4(A) and (B), due to the occurrence of low-level constitutive autophagy in response to normal physiological processes.39,40 The intense LC3B immunopositivity in the glomerular and tubular regions of the gentamicin-treated group suggested that autophagy persisted during renal injury (Figure 4C). Pretreatment with rutin was able to attenuate renal injury by reducing LC3B expression as shown in Figure 4(D), and reducing oxidative stress. These data suggest that the expression of iNOS, cleaved caspase-3, and LC3B, in addition to oxidative stress and inflammation, plays a significant role in the pathogenesis of gentamicin-induced nephropathy in rats. In conclusion, the present study demonstrated that rutin exhibits antioxidant, anti-inflammatory, anti-apoptotic, and anti-autophagic effects. By inhibiting iNOS, cleaved caspase-3, and LC3B and by increasing antioxidant activity, pretreatment with rutin could attenuate gentamicin-induced nephrotoxicity in rats.
Acknowledgements The authors acknowledge Assoc. Prof. Dr. Omer COBAN for statistical analysis, and Assoc. Prof. Dr. Sinan AKTAS, Director of Experimental Research Centre, and Assistant Prof. Dr. Orhan AKMAN, Assistant Director of Experimental Research Centre, for all types of help.
Rutin attenuates gentamicin-induced nephrotoxicity in rats
7
Declaration of interest The authors declare that there are no conflicts of interest. This study was supported by BAP of Ataturk University (Project No: 2012/366).
References 1. Quiros Y, Vicente-Vicente L, Morales AI, Lopez-Novoa JM. An integrative overview on the mechanisms underlying the renal tubular cytotoxicity of gentamicin. Toxicol Sci. 2011;119:245–256. 2. Plumb DC. Plumb’s Veterinary Drug Handbook. 5th ed. Stockholm, WI: Blackwell Publishing; 2005: 359–363. 3. Balakumar P, Rohilla A, Thangathirupathi A. Gentamicin-induced nephrotoxicity: Do we have a promising therapeutic approach to blunt it? Pharmacol Res. 2010;62:170–186. 4. Baliga R, Ueda N, Walker PD, Shah SV. Oxidant mechanisms in toxic acute renal failure. Drug Metab Rev. 1999;31:971–997. 5. Morales AI, Detaille D, Prieto M, et al. Metformin prevents experimental gentamicin-induced nephropathy by a mitochondriadependent pathway. Kidney Int. 2010;77:861–869. 6. Xie Z, Klionsky DJ. Autopgasome formation: Core machinery and adaptations. Nat Cell Biol. 2007;9:1102–1109. 7. Scarlatti F, Granata R, Meijer AJ, Codogno P. Does autophagy have a licence to kill mammalian cells? Cell Death Differ. 2009;16: 12–20. 8. Sharma S, Asgar A, Javed A, Jasjeet KS, Sanjula B. Rutin: Therapeutic potential and recent advances in drug delivery. Expert Opin Investig Drugs. 2013;22:1063–1079. 9. Koyner JL, Sher AR, Murray PT. Antioxidants. Do they have a place in the prevention or therapy of acute kidney injury? Nephron Exp Nephrol. 2008;109:e109–e117. 10. Manikandan R, Beulaja M, Thiagarajan R, Priyadarsini A, Saravanan R, Arumugam M. 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. 2011;670:578–585. 11. Jain A, Nahata A, Singhai AK. Effect of Tephrosia purpurea (L.) Pers. Leaves on gentamicin induced nephrotoxicity in rats. Sci Pharm. 2013;81:1071–1087. 12. Kang C, Lee H, Hah DY, et al. Protective effects of Houttuynia cordata Thunb. On gentamicin-induced oxidative stress and nephrotoxicity in rats. Toxicol Res. 2013;29:61–67. 13. Ali BH, Al Za’abi M, Blunden G, Nemmar A. Experimental gentamicin nephrotoxicity and agents that modify it: A mini-review of recent research. Basic Clin Pharmacol Toxicol. 2011;109: 225–232. 14. Arjumand W, Seth A, Sultana S. Rutin attenuates cisplatin induced renal inflammation and apoptosis by reducing NFkB, TNF-a and caspase-3 expression in wistar rats. Food Chem Toxicol. 2011;49: 2013–2021. 15. Abdel-Raheem IT, Abdel-Ghany AA, Mohamed A. Protective effect of quercetin against gentamicin-induced nephrotoxicity. Biol Pharm Bull. 2009;32:61–67. 16. Placer ZA, Cushmanni LL, Johnson BC. Estimation of products of lipid peroxidation (as malondialdehyde) in biochemical systems. Anal Biochem. 1966;16:359–364. 17. Aebi H. Catalase. In: Bergmeyer HU, ed. Methods in Enzymatic Analysis. New York: Academic Press; 1983: 276–286. 18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem. 1951; 193:265–275. 19. Sun Y, Larry WO, Ying L. A simple method for clinical assay of superoxide dismutase. Clin Chem. 1988;34:497–500. 20. Sedlak J, Lindsay RHC. Estimation of total protein bound and nonprotein sulfhydryl groups in tissue with Ellmann’s reagent. Anal Biochem. 1968;25:192–205. 21. Lawrence RA, Burk RF. Glutathione peroxidase activity in selenium-deficient rat liver. Bioch Biophys Res Comm. 1976;71: 952–958. 22. Nagai J, Takano M. Molecular aspects of renal handling of aminoglycosides and strategies for preventing the nephrotoxicity. Drug Metab Pharmacokinet. 2004;19:159–170. 23. Lee KE, Kim EY, Kim CS, et al. Macrophage-stimulating protein attenuates gentamicin-induced inflammation and apoptosis in
8
24. 25. 26. 27. 28.
Ren Fail Downloaded from informahealthcare.com by University of Waterloo on 02/12/15. For personal use only.
29. 30. 31. 32.
F. M. Kandemir et al. human renal proximal tubular epithelial cells. Biochem Biophys Res Commun. 2013;434:527–533. Singh D, Kaur R, Chander V, Chopra K. Antioxidants in the prevention of renal disease. J Med Food. 2006;9:443–450. Khan RA, Khan MR, Sahreen S. Protective effects of rutin against potassium bromate induced nephrotoxicity in rats. BMC Complement Altern Med. 2012;12:204. Sadeghnia HR, Yousefsani BS, Rashidfar M, Boroushaki MT, Asadpour E, Ghorbani A. Protective effect of rutin on hexachlorobutadiene-induced nephrotoxicity. Ren Fail. 2013;35:1151–1155. Korkmaz A, Kolankaya D. Inhibiting inducible nitric oxide synthase with rutin reduces renal ischemia/reperfusion injury. Can J Surg. 2013;56:6–14. Al-Rejaie SS, Abuohashaish HM, Alkhamees OA, Aleisa AM, Alroujayee AS. Gender difference following high cholesterol diet induced renal injury and the protective role of rutin and ascorbic acid combination in Wistar albino rats. Lipids Health Dis. 2012;11: 1–10. Hao H-H, Shao Z-M, Tang D-Q, et al. Preventive effects of rutin on the development of experimental diabetic nephropathy in rats. Life Sci. 2012;91:959–967. Sec¸ilmi¸s MA, Karata¸s Y, Yorulmaz O, et al. Protective effect of L-arginine intake on the impaired renal vascular responses in the gentamicin-treated rats. Nephron Physiol. 2005;100:13–20. Kiang JG, Tsen KT. Biology of hypoxia. Chin J Physiol. 2006;49: 223–233. Pedraza-Chaverri J, Barrera D, Maldonado PD, et al. S-allylmercaptocysteine scavenges hydroxyl radical and singlet oxygen in vitro and attenuates gentamicin-induced oxidative and nitrosative stress and renal damage in vivo. BMC Clin Pharmacol. 2004;4:5.
Ren Fail, Early Online: 1–8
33. Goligorsky MS, Gross SS. Nitric Oxide and the Kidney. New York: Chapman & Hall; 1997:22–38. 34. Ghaznavi R, Kadkhodaee M. Comparative effects of selective and non-selective nitric oxide synthase inhibition in gentamicin-induced rat nephrotoxicity. Arch Toxicol. 2007;81:453–457. 35. Servais H, Ortiz A, Devuyst O, Denamur S, Tulkens PM, MingeotLeclercq MP. Renal cell apoptosis induced by nephrotoxic drugs: Cellular and molecular mechanisms and potential approaches to modulation. Apoptosis. 2008;13:11–32. 36. Hsu Y-H, Chen T-H, Wu M-Y, et al. Protective effects of Zhibai Dihuang Wan on renal tubular cells affected with gentamicininduced apoptosis. J Ethnopharmacol. 2014;151:635–642. 37. Chen Y-C, Chen C-H, Hsu Y-H, et al. Leptin reduces gentamicininduced apoptosis in rat renal tubular cells via the PI3K-Akt signaling pathway. Eur J Pharmacol. 2011;658:213–218. 38. Juan S-H, Chen C-H, Hsu Y-H, et al. Tetramethylpyrazine protects rat renal tubular cell apoptosis induced by gentamicin. Nephrol Dial Transplant. 2007;22:732–739. 39. Kundu M, Thombson CB. Autophagy: Basic principles and relevance to disease. Annu Rev Pathol. 2008;3:427–455. 40. Levine B, Yuan J. Autophagy in cell death: an innocent convict? J Clin Invest. 2005;10:2679–2788. 41. Domitrovic R, Cvijanovic O, Pernjak-Pugel E, Skoda M, Mikelic L, Crncevic-Orlic Z. Berberine exerts nephroprotective effect against cisplatin-induced kidney damage through inhibition of oxidative/ nitrosative stress, inflammation, autophagy and apoptosis. Food Chem Toxicol. 2013;62:397–406. 42. Xiang Y, Piao SG, Zou HB, et al. L-carnitine protects against cyclosporine-induced pancreatic and renal injury in rats. Transplant Proc. 2013;45:3127–3134.
