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RESEARCH ARTICLE

Saffron extract prevents vancomycin-induced nephrotoxicity

Background: Vancomycin, a glycopeptide antibiotic, is commonly used against methicillin-resistant Gram-positive cocci. However, the nephrotoxic side-effects of Vancomycin results in dose restriction and reduces the duration of administration. Objectives: The present study was designed to evaluate the protective effects of aqueous ( sativus) extract on vancomycin-induced nephrotoxicity in rats. Materials and methods: In this study 32 adult male Wistar rats were randomly divided into 4 groups of 8 rats; (i) control (ii) saffron (80 mg/kg, I.P.), (iii) vancomycin (200 mg/kg/BD, I.P.) and (iv) vancomycin plus saffron. The saffron treatment began 24 hours before the administration of the vancomycin therapy and lasted 8 days, while the duration of the vancomycin therapy was 7 days. Serum creatinine and blood urea nitrogen (BUN), renal malondialdehyde (MDA) levels, superoxide dismutase (SOD) activity and histopathological changes of renal tissue were analyzed. Results: Administration of vancomycin caused a significant increase in serum creatinine, BUN and renal MDA levels, whereas, SOD activity was decreased, when compared to the control group. Aqueous saffron extract, on the other hand, decreased serum creatinine, BUN concentration and renal MDA levels but increased the level of renal SOD activity. Substantial histopathological alterations including destruction of kidney tubules, interstitial edema, epithelia vacuolization and epithelial desquamation, were observed with the vancomycin-only treatment group. However, the administration of saffron extract resulted in a significant reduction of damage to the kidneys. Conclusion: The present study demonstrated that oxidative stress plays an important role in vancomycin induced nephrotoxicity and saffron extract has a potent renoprotective effect against vancomycin induced lipid peroxidation and nephrotoxicity.

KEYWORDS: vancomycin ■ nephrotoxicity ■ saffron extract Maryam Jenabi1*, Ali Asghar Hemmati2, Katayoon Hafezi3 & 4 Background antioxidants can be a potential therapeutic Esrafil Mansouri strategy to protect against renal damage caused

Vancomycin is a glycopeptide antibiotic by like vancomycin [8,9]. 1School of Pharmacy, Ahvaz which is commonly used against methicillin- Jundishapur University of Medical Medicinal with anti-inflammatory resistant gram-positive cocci. In recent years, it Sciences, constituents have been used to manage a 2Department of Pharmacology, has been used more frequently due to the School of Pharmacy, Medicinal number of inflammatory diseases. Grape seed increasing incidences of infections caused by Plant Research Center, Ahvaz extract and are two examples of Jundishapur University of Medical methicillin-resistant Staphylococcus aureus and the many herbs that have been documented as Sciences, Ahvaz, Iran Staphylococcus epidermidis [1,2]. 3Diabetes Research Center, Ahvaz effective for inflammatory diseases [10-12]. Jundishapur University of Medical One major concern with vancomycin is its L., commonly known as saffron, Sciences, Ahvaz, Iran considerable nephrotoxicity. The incidence of has traditionally been used as an 4Department of Anatomical Sciences, Faculty of Medicine, vancomycin-induced nephrotoxicity has been antidepressant, anti-inflammatory, antioxidant, Cellular and Molecular Research reported to be between 5% and 35%, thus, hypnotic agent, antispasmodic, expectorant, Center, Ahvaz Jundishapur restricting dose and duration of administration hepatoprotective agent and inducer of labor in University of Medical Sciences, Iran [3]. folk medicine [13,14]. *Author for correspondence [email protected] Although the exact mechanism of Recent studies have demonstrated that vancomycin is not fully understood, recent saffron has many pharmacological effects, such studies suggest that vancomycin has oxidative as, antioxidant, anti-tumor, antiplatelet, anti- effects on the proximal renal tubule and atherosclerosis, radical scavenger, production of reactive oxygen species (ROS) anticonvulsant, genoprotective, and which causes changes in the energy dependent chemoprotective properties. In addition, renal reabsorption and mitochondrial functions saffron has been linked to improved learning [2-8]. It has also been demonstrated that behavior and elevating the oxygen diffusivity in oxidative stress dysfunction is responsible for the various tissues. The majority of research the tubular necrosis associated with published on saffron has reported an acceptable vancomycin treatments. Therefore the use of safety profile. However, reports showed adverse

ISSN 1755-5191 Imaging Med. (2019) 11(1) 23 RESEARCH ARTICLE Jenabi M, Hemmati AA, Hafezi K, et al.

reactions such as dry mouth, headaches, nausea and then was dried at 35-40˚C. The extract [15-22]. Chemical studies have shown that was kept at 2–8°C. saffron extract has , and To prepare the solution for injection, the constituents that are responsible for its active ingredients in saffron were quantitatively pharmacological activity [23]. determined using a high-performance liquid Several investigations have reported that chromatography (HPLC). The powder was saffron extract protects renal tissue from first weighed and dissolved in distilled water, destruction caused by ischemia/reperfusion then filtered through a 0.2 mm filter, and then and nephrotoxicity induced by gentamicin and immediately administered to the rats. The cisplatin [13,24-28]. This protective effect is HPLC analyzed the saffron extract using a UV mainly due to the anti-oxidative properties of detector with wavelengths of 308 nm and 443 saffron. However, this present study is the first nm showed the presence of safranal, crocin and one regarding the effect of saffron extract on with a concentration of 0.520%, vancomycin nephrotoxicity. 94.98% and 3.01% respectively.

■Experimental design Objectives Adult male Wistar rats were randomly This study was designed to evaluate the divided into 4 groups of 8 rats: (i) control (ii) protective effects of aqueous saffron extract on saffron (iii) vancomycin (iv) vancomycin plus vancomycin-induced nephrotoxicity in rats. saffron. The results of this study were then used to suggest a potential new strategy for preventing Aqueous saffron extract was prepared and the nephrotoxicity of vancomycin. given to both group 2 and 4 at a dose of 80 mg/kg per day, i.p in 24 h intervals for 8 days. This dosage scheme was reported to protect Materials and Methods from marked nephrotoxicity caused by The use of animals in the study and the gentamicin and oxidative stress following renal experimental protocol was approved by the ischemia-reperfusion injury in rats [13,28]. Ethics Review Committee for Animal Vancomycin (500 mg, Exir Pharmaceutical Experimentation of Ahvaz Jundishapur Co.) was dissolved in water for injection and University of Medical Sciences. All efforts were administrated to Group 3 and 4 at a dose of made to minimize the number of animals 200 mg/kg, i.p., twice daily for 7 days. used. Particular attention was given to animal Vancomycin treatment was started 1 day after cruelty and all measures were taken to ensure the first administration of saffron. First, the humane treatment of all the rats used in this saffron treatment was given at 8:00 am then an study. Adult male Wistar rats (220-280 g) were hour later, at 9:00 am, the first dose of purchased from the animal house and research vancomycin was administrated. The second center of the Jundishapur University of dose of vancomycin was then administrated 12 Medical Sciences, Ahvaz, Iran. They were all h later at 21:00. An Isotonic saline solution, housed in the same room at a controlled with an equal volume to vancomycin, was temperature of 23 ± 2˚C and were exposed to a administrated to group 1 and an injection of 12 h light/dark cycle. All the rats were allowed sterile water, with an equal volume to saffron, access to food and water, ad libitum, for at was given i.p. to both groups 1 and 3. least one week before experiments. After 24 hours from the last injection, the animals were anaesthetized with xylazine (10 ■ Preparation of aqueous extract of saffron mg/kg/d; Chanelle Pharmaceutical Co., The saffron that was used in this experiment Galway, Ireland) and HCL (50 was supplied by Saharkhiz Saffron Co. The mg/kg/day; Parke Davis Pharmaceutical Co., component of the saffron that was used Cambridge, UK) and exterminated in a rapid for the formation of the medicine was and painless method. Blood samples were the . The stigmas were air-dried in the collected from the inferior vena cava. Serum shade and then powdered for 10 min with the samples were separated by centrifugation at use of a mortar and pestle. After grinding, 15 g 1600 g for 5 min and frozen (-80˚C) for of ground stigma was extracted with 400 ml of measurements of serum creatinine (Cr) and distilled water for 18 h in a Soxhlet extractor. blood urea nitrogen (BUN) levels. The aqueous extract was concentrated to 100 Concentration of BUN and Cr were ml with a rotary evaporator in low pressure, determined using a commercial kit (Pars Azmoon, Tehran, Iran) and Hitachi

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multianalyser (Hitachinaka, Japan) according minute. The product transformed the color of to the manufacturer’s instructions. the chromogen and SOD activity was determined colorimetrically to be 420 nm by The abdomen was opened and both kidneys using an ELISA plate reader [30]. Again, were removed, weighed and de-capsulated. The superoxide dismutase activity was evaluated left kidneys were placed in formaldehyde (10% using diagnostic Zell Bio kits in accordance to solution) and then embedded in paraffin wax, the manufacturer’s instructions (Zell Bio, sectioned (4 µm) and stained with Germany). Hematoxylin and Eosin (H&E) for the standard histopathological examination by ■Ethical issues light microscopy [28]. Coded slides from each group were examined by a pathologist, The research was both in compliance with whereby, the corresponding treatment the tenets of the Declaration of Helsinki and regimens remained undisclosed. was approved by the Ethics Committee of Ahvaz Jundishapur University of Medical The right kidneys were washed with Sciences, Ahvaz, Iran. Prior to the experiment, physiological saline and maintained at -80˚C the protocols were confirmed to be in and used for analysis of malondialdehyde accordance with the Guidelines of Animal (MDA) and superoxide dismutase (SOD). On Ethics Committee of Ahvaz Jundishapur the day of analysis, the kidney tissues were University of Medical Sciences. homogenized in a motor driven tissue homogenizer with a cold phosphate buffer ■Statistical analysis solution to give a 10% homogenate suspension. Unbroken cells, cells debris and Biochemical data were analyzed using the nuclei were sedimented by centrifugation at SPPS 16.0 software program (SPSS, Chicago, 4000rmp for approximately 20 min. The IL, USA) and presented as means ± SEM. supernatants were collect and used for Means of groups were compared by one-way biochemical assays. analysis of variance (ANOVA) then Post-Hoc analysis (Tukey test). The level of statistical ■Malondialdehyde measurement significance was accepted as p<0.05. Malondialdehyde (MDA) levels, an index of lipid peroxidation, were determined in renal Results tissues. MDA, as a thiobarbituric acid reactive substance (TBARS), reacts with thiobarbituric ■ Baseline characteristics acid (TBA) to generate a color complex that All the rats survived the experimental phase has a peak absorbance of 532 nm, that can be despite the considerable variance in kidney measured in the visible range using an ELISA weights amongst the different test groups. plate reader [29]. The MDA levels were Kidney weight was significantly higher in the evaluated, in accordance to the manufacturers vancomycin and the vancomycin plus saffron instructions (Zell bio, Germany), using study group in contrast to the control group diagnostic Zell Bio kits. (p<0.05). However, the ratio of kidney to body weight in the vancomycin plus saffron group ■ Superoxide dismutase activity measurement was significantly lower than the vancomycin- One unit of SOD activity was defined as the only group (p<0.05), and showed no amount of sample necessary to catalyze 1 significant change in comparison with control micromole of superoxide radicals to group (p>0.05) (TABLES 1 and 2). peroxide and oxygen in the span of one

Table 1. Mean of body weight, kidney weight and the ratio of kidney weight to body weight in each groups.

Control Saffron Vancomycin Vancomycin + saffron

Body weight (g) 264.5 ± 4.89 249.38 ± 7.82 248.75 ± 7.05 238.75 ± 4.70

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Kidney weight (g) 0.75 ± 0.05 0.87 ± 0.05# 1.82 ± 0.24* 1.35 ± 0.12*

Kidney weight to 0.28 ± 0.02 0.35 ± 0.02# 0.74 ± 0.09* 0.57 ± 0.06# body weight ratio in %

Data are the mean ± SEM (n=8). *P< 0.05 compared with control (group 1); #P< 0.05 compared with vancomycin treated rats (group 3)

Table 2. Blood urea nitrogen (BUN) and creatinine levels in serum and malondialdehyde (MDA) and superoxide dismutase activity (SOD) in kidney homogenates of rats.

Control Saffron Vancomycin Vancomycin + saffron

BUN (mg/dL) 18.81 ± 1.31 15.36 ± 1.23### 112.29 ± 17.68*** 49.18 ± 5.92###

Creatinine (mg/dL) 0.47 ± 0.03 0.48±0.02### 2.06 ± 0.49*** 0.77 ± 0.05##

MDA (nmol/g ) 62.38 ± 2.73 70.57±2.15## 89.43 ± 3.99** 60.23 ± 5.13###

SOD (U/g protein) 44.37 ± 3.60 49.49±2.86## 30.38 ± 2.12* 44.94 ± 3.64#

Data are the mean±SEM (n=8). BUN: Blood urea nitrogen, MDA: malondialdehyde, SOD superoxide dismutase. *p< 0.05 compared with control (group 1); **p< 0.01 compared with control (group 1); ***p< 0.001 compared with control (group 1); # p< 0.05 compared with vancomycin treated rats (group 3); ## p< 0.01 compared with vancomycin treated rats (group 3); ### p< 0.001 compared with vancomycin treated rats (group 3).

■ Effects of vancomycin and saffron extract compared with the control group (p<0.01). treatment on levels of renal MDA and activity of Conversely, the saffron treatment lowered the renal SOD levels of MDA in the vancomycin plus saffron As shown in FIGURE 1, the MDA levels in group compared to the vancomycin-only the vancomycin-only group were elevated group (p<0.001).

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Figure 1. Tissue malondialdehyde (MDA) levels per gram kidney weight; Data represent the mean ± SEM (n=8), ** p<0.01 as compared to the control group, ## P<0.01 and ### p<0.001 as compared to the vancomycin treated group.

Figure 2. Renal tissue superoxide dismutase (SOD) activity in the study groups; Data represent the mean ± SEM (n=8), * p< 0.05 as compared to the control group, # p< 0.05 and ## p< 0.01 as compared to the vancomycin treated group.

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In FIGURE 2, a significant reduction in (p<0.001) higher in the vancomycin-treated SOD activity was observed in the vancomycin group than in the control. In the vancomycin group when compared to the control group plus saffron group, saffron produced a marked (p<0.05). The saffron treatment caused a reduction in the serum BUN and creatinine significant elevation in SOD activities in the concentration, in contrast to the vancomycin- vancomycin plus saffron group when only group (p<0.001 BUN, p<0.01 compared with the vancomycin only group creatinine). (p<0.05).

■Effects of vancomycin and saffron extract treatment on blood urea nitrogen (BUN) and serum creatinine According to FIGURES 3 and 4, BUN and serum creatinine levels were substantially

Figure 3. Blood urea nitrogen (BUN) levels in the study groups; Data represent the mean ± SEM (n=8), *** p< 0.001 as compared to the control group, ### p< 0.001 as compared to the vancomycin treated group.

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Figure 4. Serum creatinine levels in the study groups; Data represent the mean ± SEM (n=8), *** p< 0.001 as compared to the control group, ## p< 0.01 and ### p< 0.001 as compared to the vancomycin treated group.

■Effects of vancomycin and saffron extract treatment on kidney histology Histological evaluations indicated that the control group had normal structure in renal corpuscles and tubules (FIGURE 5).

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Figure 5. Histological evaluations indicated that normal group had normal structure in renal corpuscles and tubules (H&E × 400).

The micrographs in the saffron-treated group also demonstrated no pathological changes (FIGURE 6).

Figure 6. Micrographs in Saffron group showed that kidney tissue had normal structure and was not observed pathological changes (H&E × 400).

Treatment with vancomycin, on the other destruction of kidney tubules, with the hand, caused tissue damage and partial accumulation of hyaline droplets, and

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diminishing tubule epithelial cytoplasm (FIGURE 7).

Figure 7. In vancomycin group, kidney tissue damage can clearly be observed that includes: the destruction of some kidney tubules, accumulation of hyaline droplets within tubules, clearing of tubule epithelial cytoplasm (H&E × 400).

Treatment with saffron dramatically tubules and renal corpuscles. Only negligible improved vancomycin nephropathy, and less amounts of hyaline droplets inside the tubules histological damage was observed in renal were detected (FIGURE 8).

Figure 8. Treatment with saffron extract dramatically improved the vancomycin nephropathy, and less histological damage was observed in renal tubules and renal corpuscles. Only a very small amount of hyaline droplets inside the tubules can be seen (H&E × 400).

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Discussion considered as an indicator of lipid peroxidation. There was a significant increase Vancomycin has considerable nephrotoxic of renal MDA concentration in the side effects causing serious complications when vancomycin-treated group when compared to administered. Therefore amelioration of the control group. Nishino et al., Oktem et al., nephrotoxicity would enhance its clinical use Celik et al. and Basarslan et al., reported [3]. Despite the fact that the mechanism of significant increases in the levels of renal MDA vancomycin nephrotoxicity is not fully known, in vancomycin-induced nephrotoxicity models several studies have suggested that the main in rats, and also suggested that oxidative mechanism of vancomycin nephrotoxicity damage and lipid peroxidation play important could be oxidative stress. Some studies have roles in the pathogenesis of nephrotoxicity also reported that vancomycin-induced induced by vancomycin [4,31,32,38]. In our nephrotoxicity was prevented with the use of study, saffron extract significantly attenuated different antioxidants [2,8,9,31,32]. In the the increase of MDA concentration in renal present study, we have explored the possible tissue. The nephroprotective effect of saffron protective effects of saffron extract, which has a may be due to its capacity to eliminate free powerful antioxidant effect, on nephrotoxic radicals. In addition, it’s a recognized powerful injury induced by vancomycin. After reviewing antioxidant, therefore, it reduces lipid the literature, we realized that our study was peroxidation of renal tubular cells in the the first experiment that considered the kidneys of rats [13]. Thus, our study protective effects of saffron extract on demonstrated that saffron extract could inhibit vancomycin-induced nephrotoxicity. renal toxicity induced by vancomycin and supports the theory that free radicals are In this study, with the systemic responsible for its pathogenesis. administration of high doses of vancomycin (400 mg/kg BW/day, i.p.), we created marked Studies on the mechanism of vancomycin- nephrotoxicity in the vancomycin group. This induced nephrotoxicity have mentioned the was corroborated by the significant histological role of renal tubules in the excretion of changes that were observed. Then we evaluated vancomycin [39]. Once administered, the protective effect of saffron using oxidative vancomycin moves out of the blood and and biochemical markers such as MDA, SOD, accumulates in the proximal tubular BUN and serum creatinine that are generally epithelium which is associated with used to survey the progress of renal injury. The nephrotoxicity [8]. Increased serum BUN and biological and histological results suggested creatinine levels indicate cell injury in the renal that saffron effectively protects against proximal tubules [3]. Several studies have vancomycin-induced nephrotoxicity. reported that vancomycin, as a treatment, significantly increases the levels of serum BUN Reactive oxygen species (ROS) have been and creatinine [2,9,32,40-42]. Levels of serum considered to be significant mediators of BUN and creatinine are indicators of renal aminoglycoside and cisplatin induced function and marked for the assessment of late nephrotoxicity [3,33,34]. Nishino et al., and early renal failure respectively [31,43]. considered that ROS play a very important Our study found that the levels of serum BUN role in the pathophysiology of nephrotoxicity and creatinine in the vancomycin group were induced by vancomycin treatment [4]. King significantly higher than that of the control and Smith, demonstrated that vancomycin group. This reveals that the administration of stimulates production of both free radicals and vancomycin causes serious nephrotoxicity. oxidative phosphorylation and increases Moreover, a comparison to the control group cellular adenosine triphosphate concentration showed a significant increase in the kidney and oxygen consumption [35]. Cellular and weight ratio and the histopathological DNA damage, protein denaturation and examination of the kidney demonstrated peroxidation of membrane lipids are caused by significant renal damage. Treatment with free radicals [36]. Lipid peroxidation can cause aqueous saffron extract lead to lower levels of damage in both membrane structure and serum BUN and creatinine than the function which results in the creation of vancomycin-treated group, demonstrating different end-products, such as, improvement in renal function. Antioxidant malondialdehyde (MDA) [31]. Therefore there molecules and enzymes are present in cells to is a direct relation between MDA and lipid protect against the destructive effects of free peroxidation [37]. In the present study we radicals. The treatment of vancomycin induced measured MDA levels, which is a result of free oxidative stress which suppressed and rendered radical damage to membrane structure and is

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most of the antioxidant enzymes inactive, curcumin and results supported the beneficial allowing the function of free radical scavenging effects of antioxidants in the prevention of [31]. Vancomycin either directly binds to vancomycin-induced nephrotoxicity enzyme active sites containing ̶ SH groups, or [2,9,38,40,47]. it displaces metal cofactors from active sites Yarijani et al., suggested that crocin has [44,45] SOD is the essential protective enzyme protective effects against gentamycin-induced against oxidative stress in renal tubules, and its nephrotoxicity and that might be due to the highest concentration is found in the proximal vasodilatory effect of the kidneys which tubules [46]. In our study, the vancomycin corrects the glomerular filtration rate and also group displayed a significant reduction in has antioxidant and anti-inflammatory SOD activity when compared to the control properties [25]. Recent studies have also group. Several of the latest studies have also reported the renoprotective effects of saffron reported a significant decrease in SOD activity extract on nephrotoxicities induced by in vancomycin nephrotoxicity rat models cisplatin, gentamycin and ceftazidime in rats [2,3,8,31,32,40,45]. In this study, treatment [17,26-28,34]. with saffron extract completely inhibited the decrease of SOD activity in the vancomycin Hosseinzadeh et al., evaluated the protective plus saffron group when compared to the effect of aqueous saffron extract and its active vancomycin-only group. constituent, crocin, on renal ischemia- reperfusion induced oxidative damage in rats. Although the biochemical mechanism of the He reported that although both components preventive effect of saffron extract on the might be useful agents for the prevention of inactivation of SOD activity has not yet been renal oxidative injury, saffron extract consist of proven, we suggested that its effectiveness may various constituents which have roles in be due to its free radical scavenging and protection [13]. antioxidant activity. In the present study, we demonstrated that Oklem et al., found erdosteine treatment vancomycin treatment causes kidney damage. caused an increase in the activity of SOD and a It raised the levels of serum creatinine, BUN decrease in MDA levels in vancomycin- and renal MDA and decreased SOD activity. induced nephrotoxicity in rats and rationalized In addition, the histological analysis clearly that it was due to the free radical scavenging showed partial destruction of kidney tubules, activity of erdostein [8]. Basarslan et al., have interstitial edema, epithelial vacuolization and shown that the administration of epithelial desquamation. Therefore, we thymoquinone protects the kidney from concluded that oxidative stress is the main damage that is induced by vancomycin. This is factor in vancomycin-induced nephrotoxicity. observed by the diminishing levels of MDA, Treatment with aqueous saffron extract BUN and creatinine and the increase in SOD significantly normalized biochemical and levels. Therefore, the efficacy of thymoquinone histological changes seen in vancomycin is a result of its interaction with ROS, which is treated rat. Saffron extract has been proved to a general radical scavenger, during vancomycin be a highly efficient scavenger of oxygen toxicity [31]. Sadeeshkumar et al., reported radicals and other excited species and it that pre-treatment with Gallic acid increased protects against the oxidation of lipid, protein the activity of SOD in vancomycin treated and DNA [17,22]. It can also decrease rats. This may be due to Gallic acid’s ability to oxidative stress by increasing the expression of act as a chelator and eliminate vancomycin antioxidant enzymes, such as, superoxide from renal tissue, as well as, reducing the dismutase and catalase [48]. Accordingly, it accumulation of free radical generation during seems that more than one mechanism of lipid peroxidation [45]. Cetin et al., found that protection involved. the treatment of erythropoietin on vancomycin treated rats caused a decrease in MDA, serum creatinine and BUN levels and an increase in Conclusion SOD activity. This indicated that the In conclusion, the present study renoprotective effect of erythropoietin was due demonstrated that saffron extract has a potent to the inhibition of apoptosis, as well as, acted renoprotective effect against vancomycin- as an antioxidant agent against vancomycin- induced nephrotoxicity and it is suggested as a induced renal damage [32]. Recent animal potential new therapeutic strategy. studies used various antioxidants, such as, E, , N-, caffeic acid phenethyl , atorvastatin and

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Conflicts of Interest The authors have declared that no conflict of interest exists.

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