EVALUATION OF THE PROTECTIVE EFFECT OF NIGELLA SATIVA (BLACK CUMIN) OIL AGAINST VANCOMYCIN-INDUCED NEPHROTOXICITY IN RATS

Objective: The study was designed to investigate a possible protective effect of Nigella sativa (NS) against vancomycin (VAN)-induced nephrotoxicity in rats. Methods: Twenty-eight adult male Albino rats were randomly divided into four groups; seven rats in each. Group I (control): The animals were treated with normal saline (2 ml/kg/day) given orally and intraperitoneally (IP); Group II: VAN was given at a dose of 400 mg/kg/day for 7 days IP and normal saline orally; Group III: NS oil was given at a dose of 2 ml/kg/day for seven days orally and normal saline IP; and Group IV: VAN 400 mg/kg/day IP in combination with NS oil 2 ml/kg/day orally for 7 days. Twenty-four hours after the last dose, the animals were sacrificed, and serum was collected to estimate urea and creatinine. Then, both kidneys were excised, one for homogenate preparation to estimate renal tissue malondialdehyde (MDA), glutathione (GSH) and neutrophil gelatinase-associated lipocalin (NGAL) and the other for histopathological examination. Results: NS significantly decreased serum urea and creatinine compared to VAN treated group, p<0.001. NS significantly increased renal tissue GSH compared to VAN treated group p<0.001. NS lowered MDA and NGAL levels in the homogenate of renal tissues compared to their elevated levels in rats treated with VAN, but this did not achieve statistical significance. NS also ameliorated renal histopathological changes induced by VAN. Conclusion: NS has a protective effect against VAN-induced nephrotoxicity.


INTRODUCTION
Vancomycin (VAN) is a glycopeptide antibiotic. It is an exceptionally important antibiotic which should be deserved for the treatment of severe infections such as methicillin-resistant Staphylococcus aureus (MRSA) [1]. VAN, in that context, is effective, but there are numerous side effects encountered with its use. Nephrotoxicity is among these side effects; it is dose and duration of use related and is practically unavoidable [2]. The incidence of VAN-induced nephrotoxicity was reported as (5-35%) according to different nephrotoxicity definitions [3]. The exact mechanism of VAN-induced nephrotoxicity is not fully known, but previous experimental studies revealed pro-inflammatory oxidation, mitochondrial dysfunction, and cellular apoptosis as the main mechanisms of renal injury [4]. VAN may increase oxidative phosphorylation in renal tubular cells, leading to the formation of superoxide, especially in the mitochondria. Furthermore, activation of caspase 6 in the presence of superoxide inside the mitochondria may lead to peroxidation of mitochondrial membrane phospholipids and specially cardiolipin leading to changes in mitochondrial membrane potential (depolarization) and an increase in the permeability. This will lead to releasing of oxygen reactive species with cytochrome c to the cytoplasm and activation of caspase 9 which, in turn, activates caspase 3. These actions are considered as the first step in the apoptosis process leading to cell death plus reduction in ATP content of the cell. These are possible causes of proximal tubular cell death and renal injury [4,5]. In general, the idea of searching for a drug, medicinal plant or a chemical compound that help protects against VAN-induced nephrotoxicity is well appreciated.
Nigella sativa (NS) is a medicinal plant which is known to have a lot of pharmacological actions, which for most of these actions, antioxidant activity is responsible [6].
The aim of the present study, therefore, was to investigate a possible protective effect of NS on VAN-induced nephrotoxicity in rats.
For the best of our knowledge, the present work is the first study designed to unveil the nephroprotective activity of NS oil against VAN-induced nephrotoxicity.

Chemicals
VAN as hydrochloride (1 g vial) was purchased from Julphar Gulf pharmaceutical industries, Ras Al-Khaimah, United Arab Emirate. NS oil was obtained by cold press extraction by a special machine. The opinion of an expert herbalist was taken to ensure authentication of the seed; nonetheless, a voucher specimen of the seed was kept in the Department of Pharmacology as a reference.

Animals
The protocol of the present study was approved by the Institutional Ethical Committee. Twenty-eight adult male Albino rats weighing 175-350 g were obtained from the animal house of the College of Veterinary Medicine/Basrah University and kept in the animal house of the College of Medicine/Basrah University for 2 weeks for acclimatization in separated cages under controlled conditions with free access to water and standard food. All measures were taken to minimize the suffering of the animals in accordance with the guidelines (National Institutes for Health USA publication, 1985).
The animals were treated for 7 consecutive days. Twenty-four hours after the last dose, the animals were sacrificed after short anesthesia with diethyl ether. Blood samples were collected directly from the heart and transferred into a tube containing (thrombin). The tubes were centrifuged for 20 min at 1000 round/min. Serum was obtained and stored at −20°C until assayed. Both kidneys were dissected. One kidney for homogenate preparation and the other was kept in 10% formalin for histopathological examination.

Estimation of serum urea and creatinine
Serum urea and creatinine were measured using a spectrophotometer (INTEGRA machine, Roche (Germany).

Estimation of renal tissue glutathione and malondialdehyde
• Renal tissue glutathione (GSH) and malondialdehyde (MDA) were measured using MYBIOSOURCE commercial ELISA kit • (China) Specific for rats. This kit uses competitive -ELISA method.

Estimation of renal tissue neutrophil gelatinase-associated lipocalin
Renal tissue neutrophil gelatinase-associated lipocalin (NGAL) concentration was measured using MYBIOSOURCE rat ELISA kit (China). The method is based on sandwich ELISA technology.

Histopathological examination
The slides were prepared from kidney specimens, stained with Hematoxylin and Eosin (H&E), coded and blindly examined by an unbiased histopathologist. Histopathological findings were classified according to the severity of changes to score 0, 1, 2, and 3 for no change, 0.25 change, 0.50 change, and >0.50 change observed in the specimens of the kidneys, respectively.

Statistical analysis
Statistical Package for Social Sciences version 20 was used. The results were expressed as mean±standard deviation. Differences between the groups were estimated using independent samples t-test. Statistical significance was considered when value of p˂0.05.

Effect of Nigella sativa on renal function test
There was a statistically significant increase in the levels of serum urea and creatinine following treatment with VAN as compared to the control, (p<0.001). When VAN and NS were given in combination for 7 days, the levels of serum urea and creatinine significantly decreased compared to VAN treated group, (p<0.001) ( Table 1).
The level of NGAL in renal tissue was increased to 1.026.84±682.17 pg/ml in VAN treated group compared to a level of 770.69±340.88 pg/ml in the control group. Treatment with VAN and NS combination lowered NGAL levels toward the control value. None of these changes in NGAL levels obtained statistical significance ( Table 1).

Effect on renal tissue malondialdehyde
There was a small and insignificant increase in renal tissue MDA in the rats treated with VAN. Treatment with the combination of VAN and NS showed a small and insignificant reduction in renal tissue MDA level. The only significant change in renal tissue MDA levels compared to the control values was observed in NS treatment alone, (p<0.033) ( Table 2).

Effect on renal tissue glutathione
There was a statistically significant decrease in renal tissue GSH following treatment with VAN compared to the control (p<0.0001). While treatment with a combination of NS and VAN significantly raised renal tissue GSH toward the control level (p<0.0001) ( Table 2).

Effect of nigella sativa and vancomycin on kidney morphology
In VAN-treated group, the kidneys looked pale, gray in color, bigger in size and easily detached from the animal body, whereas, in the VAN and NS combination treated group, the kidneys maintained normal, healthy color and firmly attached to the animal body.
Following treatment with VAN at a dose of 400 mg/kg/day for seven days, the homogenate of kidney tissue and the supernatant obtained following centrifugation of the homogenate looked pale, grey. While treatment with the combination of VAN and NS for seven days has resulted in a light red colour of supernatant which is similar to that of the control. This means that NS corrected the changes in kidney tissues induced by VAN.

Effect of Nigella sativa on kidney weight and length
The mean weight of the rat kidney was doubled after treatment with VAN. This elevation achieved statistical significance (p<0.0001). Treatment with the VAN and NS combination significantly decreased kidney weight toward the control. This obtained statistical significance (p<0.0001) ( Table 3).
Kidney length, as well, was significantly increased by VAN treatment compared to the control, p<0.001. Treatment with the combination of VAN and NS significantly reduced kidney length toward the control, p<0.001 (Table 3).

Control group
No histopathological changes were observed in both the control and NS groups (zero score) ( Fig. 1 and Table 4).

Vancomycin group
All VAN-treated animals showed variable degrees of histopathological changes in kidney specimens (sample size=7). Score 2 was observed in 3 animals, 4 animals were identified to have severe changes (score 3) ( Fig. 1 and Table 4).

Vancomycin + nigella sativa group
Treatment with NS oil and VAN together resulted in ameliorating the severity of histopathological changes; in 4 animals, there were no histopathological changes, mild changes were observed in 2 animals and moderate changes were observed in only one animal ( Fig. 1 and Table 4).

DISCUSSION
VAN is regarded as an indispensable antibiotic for the treatment of MRSA, which is one of the most resistant bacteria [1]. This achievement which is accredited for VAN is unfortunately confounded by harmful side effects. One of these side effects is nephrotoxicity which, despite all precautions, it cannot be overcome [2]. NS, a medicinal plant which is frequently reported having a nephroprotective activity against nephrotoxicities induced by several drugs such as methotrexate [11], gentamicin [12], and cisplatin [13]. These facts laid the basis for performing the current study to explore a possible protective effect of NS oil against VAN-induced nephrotoxicity in an animal model. In the current study, VAN was found to have a clear toxicity to the kidney of rats. The kidneys were directly inspected before removal from the animals. The color of the kidneys in VAN-treated animals, appeared pale gray-to-pink in color with a double increase in kidney weight, in addition, the kidneys were easily detached from the animals. A possible explanation for these changes in kidney color could be attributed to renal tubular ischemia caused by VAN [14], and the increased kidney volume and weight could be due to edema. A similar observation of increased kidney weight and volume was also reported by Takigawa et al. [15]. On the contrary, Bayomy found that administration of VAN in rats caused a significant reduction in rats' body and kidney weight. The author attributed these changes to anorexia, low food intake and high levels of catabolism as a result of acute renal failure and acidosis [16].
These findings suggest that the kidneys were severely damaged by VAN treatment. A similar result was also observed by   Naghibi et al. [17]. These changes were supported by histopathological examination. Histopathological changes were labeled as mild, moderate, and severe which characterized by dilatation in renal tubules with desquamation of renal tubular epithelial cells and cyst formation with the cast and the proliferation of inflammatory cells of renal tissues. The fact that nephrotoxicity was seen in all VAN treated animals made an evaluation of the effect produced by NS feasible; which means any effect which can be observed with NS is taken as a true effect. NS markedly ameliorated toxicity produced by the VAN. Histopathological changes have decreased significantly and also the levels of serum urea and creatinine declined significantly. NGAL was chosen for the present study as a valuable biomarker, more specific than serum creatinine and can discover early changes in acute tubular injury [18]. In the current study, changes in renal tissue NGAL following treatment with VAN were small and the effect of NS was small too. Therefore, a conclusive result cannot be satisfied.
These results are highly suggestive of the protective effect of NS in VAN-induced nephrotoxicity. In addition, the group of animals treated with VAN and NS, the kidneys returned to its normal color and the weight of the kidneys returned to that of the control group.
The observed nephrotoxic effect of VAN could involve oxidative stress since VAN adversely affects the parameters of oxidative stress (MDA and GSH) in the homogenate of kidney tissue. Despite decades of interest in VAN-induced nephrotoxicity, the underlying mechanism is still not well defined. In one study, the expression of the gene was studied to explore the most probable mechanism of VAN-induced nephrotoxicity [19]. This study has substantiated the importance of gene markers of toxicity of the kidney, but at the same time, this study could not eliminate the involvement of inflammation, necrosis and oxidative stress of proximal tubular cells as mechanisms for VAN-induced renal injury [19].
NS was found to have a renoprotective effect because of its anti-inflammatory and antioxidant properties. This effect is attributed to the active ingredients TQ, carvacol, and others [6]. In the present study, parameters of oxidative stress, MDA and GSH were evaluated in the homogenate of kidney tissue. Blood samples were not sufficient to perform the analysis on these parameters in the serum. However, there is an advantage to evaluate these parameters in target tissues since this reflects direct changes. There is evidence suggesting that oxidative stress plays a major role in VAN-induced nephrotoxicity and tubular damage and the reversal of oxidative stress with a thiol derivative mucolytic agent (erdosteine) reduces VAN-induced nephrotoxicity [20].
In the current study, administration of VAN at a dose of 400 mg/kg/day led to an increase in MDA level in the homogenate of kidney tissue which indicates the role of oxidative stress in renal damage due to lipid peroxidation; this result is in agreement with other studies [16,20]. On the other hand, administration of NS oil has resulted in a reduction in MDA level in kidney tissues which indicates its oxidative vanquishing ability and this is consistent with the study of Uz et al. [21].
GSH is an important antioxidant system in the cell which is responsible for the protection of the cell against intracellular free radicals, peroxides and toxic compounds by conjugation with toxic substances [22]. Depletion of GSH in renal tissues is considered as one of the major factors that permit lipid peroxidation [23]. In the current study, treatment with VAN resulted in a significant decrease in GSH renal tissue's level. This indicates renal injury due to oxygen free radical formation which depletes GSH defense system. This result is compatible with the results of Ahmida [23]. On the other hand, concomitant administration of NS oil with VAN has resulted in a significant elevation of GSH concentration in renal tissue. This effect confirms the antioxidant potential of NS and in addition may explain the protective effect of NS in VAN-induced nephrotoxicity. In one study, it has been observed that TQ, which is the active constituent of NS, caused an elevation in renal tissue GSH level in rats treated concomitantly with sodium nitrite and protect the animal renal system from injury. TQ is the most powerful antioxidant, antiinflammatory, and anti-apoptotic ingredient present in NS [24].

CONCLUSION
The present study demonstrates that NS has a nephroprotective effect against VAN-induced nephrotoxicity. This effect is attributed to the antioxidant potential inherited in NS.