Abstract: Background and Objectives: The nephrotoxicity due to gentamicin is well established in man and experimental animals. Silibinin a bioactive flavanone in milk thistle possess anti-inflammatory and antioxidant activity. Therefore, the present study investigated the renoprotective effect of silibinin against gentamicin induced nephrotoxicity in rats. Method: Thirty rats were randomly divided into five equal groups (n = 6). Group I served as a control and treated orally with vehicle and Group II as a gentamicin control and administered vehicle two days before and then treated with gentamicin intraperitonially (100 mg/kg/day) for eight days. Group III-V were received silibinin orally at three dose levels (20, 40 and 80 mg/kg/day) two days before and eight days concomitantly with gentamicin intraperitonially (100 mg/kg/day). The silibinin was suspended in CMC (1% w/v). At the end of the treatment urine and blood was collected to assess the kidney functions as well as renal tissue processed for antioxidant and histopathological study. Results: Eight days of gentamicin treatment significantly increased levels of BUN, serum creatinine and decreased urinary creatinine and creatinine clearance. Further it increased MDA levels and decreased SOD and CAT activity as well as GSH levels. Silibinin treatment (40 and 80 mg kg-1) reversed the gentamicin induced alterations dose dependently. Further necrosis and degenerative changes in glomeruli and tubules observed in gentamicin treated rats were significantly restored with silibinin treatment at a dose of 40 and 80 mg kg-1. Conclusion: Silibinin dose dependently protected the kidney functions and normalize biochemical parameters and histopathological changes.
INTRODUCTION
Gentamicin is a powerful antibiotic intravenously administered to the patients in hospitals, nursing homes and home healthcare settings to combat severe infections. Acute tubular necrosis is a common complication of gentamicin therapy, occurring in 10 to 20% of patients (Moore et al., 1984). The nephrotoxicity of gentamicin is well established in man and experimental animals. Aminoglycosides are freely filtered across the glomerulus and then partially taken up and concentrated in proximal tubular cells (Laurent et al., 1990). Renal tubular cell injury produced by gentamicin evolves sub-acutely over several days and clinical manifestation of gentamicin toxicity is manifested by an increase in creatinine, urea and electrolyte alterations (Werner et al., 1995; Mingeot-Leclercq and Tulkens, 1999; Rougier et al., 2003). The generation of free radical species and alteration of mitochondrial function in the renal proximal convoluted tubules are associated with gentamicin therapy. In addition, induction of acute tubular necrosis, glomerular damage and renal inflammation are the major events implicated in gentamicin nephrotoxicity (Yanagida et al., 2004). Several reports indicate antioxidants significantly protect the rats against this toxicity (Morales et al., 2002).
Flavonoids are phenolic compounds widely distributed in fruits, vegetables; plant extracts as well as plant derived beverages. These have generated interest because of their broad pharmacological effects. Many of these effects are related to their antioxidant properties which may be due to their ability to scavenge free radicals. Silibinin, a flavanone is the major and most active component, constitutes about 60-70% in silymarin (Saller et al., 2001). Various preclinical reports suggest the myriad pharmacological activities of silibinin. It has been reported for antioxidant and hepatoprotective in nonalcoholic steatohepatitis (Haddad et al., 2011). Silibinin markedly improves endothelial dysfunction in db/db mice by reducing circulating and vascular ADMA levels (Li et al., 2011). Recently, Marrazzo et al. (2011) reported its neuroprotective effect due to DNA protection and antioxidant activity in diabetic mice (Marrazzo et al., 2011). In addition, several recent studies have shown the potential cancer preventive and therapeutic efficacy of silibinin in different animal models and cell culture systems (Raina et al., 2007; Singh et al., 2008).
Therefore, the present investigation was carried out to study the possible protective effect and to elucidate the mechanism of action of silibinin on gentamicin induced renal dysfunction, in order to gain new insights into the prophylaxis of gentamicin nephrotoxicity which is a common problem in its use.
MATERIALS AND METHODS
Drugs and chemicals: Gentamicin was purchased from local market of Pune (Genticyn, Piramal Healthcare, India), silibinin, malondialdehyde (MDA), tetrabutyl ammonium and superoxide dismutase (Sigma-Aldrich, St. Louis), Catalase (Hi Media Laboratories Pvt. Ltd., Mumbai) and all other reagents and chemical were of analytical grade and purchased from local suppliers of Pune.
Animals: Sprague Dawley (SD) rats (150-200 g) were procured from National Institute of Biosciences, Pune. Rats were placed separately in polypropylene cages with paddy husk as bedding. The animals were maintained under standard laboratory conditions at temperature 23±2°C with relative humidity 55±10% in a 12 h light and 12 h dark cycle throughout the experiment. Animals had free access to water and standard laboratory feed ad libitum (Nutrivet Lab, India). All the experimental procedures and protocols used in this study were reviewed and approved (IAEC/2011-12/33) by the Institutional Animal Ethics Committee (IAEC). Ethical guidelines were strictly followed during all the experimental procedures.
Experimental design: Thirty rats were randomly divided into five equal groups (n = 6). Group I served as a control and treated orally with vehicle (CMC, 1% w/v in water) and Group II as a gentamicin control and administered vehicle two days before and then treated intraperitonially with gentamicin (100 mg/kg/day) for eight days. Group III-V received silibinin orally at three different dose levels (20, 40 and 80 mg/kg/day) two days before and eight days concomitantly with gentamicin intraperitonially (100 mg/kg/day). The silibinin was suspended in CMC (1% w/v) (Parlakpinar et al., 2006; Harlalka et al., 2007).
One day before sacrifice each rat was individually placed in metabolic cage for 24 h urine collection. Urine was centrifuged at 1000 rpm for 10 min to remove cells and debris. Blood was collected from retro orbital plexus under light anesthesia and the serum was separated by centrifugation at 3000 rpm for 15 min. At the end of the experiment rats were killed by cervical dislocation under ether anesthesia. The abdominal cavity was immediately opened and both kidneys were removed and processed for antioxidant activity as well as histological examinations.
Body and kidney weight change: The body weight of all animals after the experiment was taken and their difference was expressed as body weight change. After sacrificing the animal one of the kidneys was rinsed in chilled saline, decapsulated blotted on filter paper and quickly weighed. For standardization, total kidney weight was normalized as kidney/body-weight ratio:
Biochemical estimations in serum and urine: Blood Urea Nitrogen (BUN) (Fawcett and Scott, 1960), serum and urinary levels of creatinine (Bartels et al., 1972) were estimated as per the instruction of commercial diagnostic kits. Whereas creatinine clearance was calculated as per the following equation:
Ccr (mL/min/kg) = (urinary Cr (mg dL-1)× urinary volume (mL)/serum Cr (mg dL-1)) (1000/body weight (g))×(1/1440 (min))
Determination of oxidative stress biomarkers in renal tissues: Left kidneys was rinsed, decapsulated, blotted on filter paper and quickly weighed. Then, it was homogenized in chilled 50 mM phosphate buffer saline (pH 7.4) in volume of nine times of its weight to yield 10% (w/v) tissue homogenate. The homogenates were centrifuged at 10500 rpm for 15 min at 4°C. The homogenate was then used for determination of the levels of Malondialdehyde (MDA) (Ohkawa et al., 1979), reduced glutathione (GSH) (Beutler et al., 1963) and activities of SOD (Sun et al., 1988) and catalase (CAT) (Luck, 1971). Protein concentrations of homogenates were determined according to Lowry et al. (1951).
Histopathological studies: Right kidney of individual rat stored in 10% formalin solution were embedded with paraffin and stained with Haematoxylin-Eosin (HE). HE stained sample was observed under light microscope (100x).
Statistical analysis: All the data were expressed as the Mean±SEM (n = 6). Data were subjected to one-way analysis of variance (ANOVA) followed by the Tukey’s multiple comparison test. Level of significance was set at P<0.05 and the analysis were made using computerized Graph Pad Prism version 5.0 (Graph pad software, USA).
RESULTS
Body and kidney weight change: Gentamicin treatment produced significant loss in body weight and increase in kidney weight compared to control (p<0.01). Treatment with silibinin (40 and 80 mg kg-1 body weight) resulted in significant increase in body weight (p<0.05 and p<0.01, respectively) and decrease in kidney weight (p<0.01 and p<0.001, respectively) compared to gentamicin control rats. However, silibinin at dose of 20 mg kg-1 body weight could not produce significant changes in body weight and kidney weight compared to gentamicin control rats (Table 1).
Biochemical estimations in serum and urine: As shown in Table 2, gentamicin produced significant elevation in blood urea nitrogen and serum creatinine levels as well as decrease urinary creatinine levels compared to control rats (p<0001). Treatment with silibinin at dose of 40 and 80 mg kg-1 significantly reduced elevated levels of BUN (p<0.001) and serum creatinine (p<0.01 and p<0.001, respectively) and increased levels of urinary creatinine (p<0.01 and p<0.001, respectively) compared to gentamicin control rats. Further, gentamicin induced decrease in creatinine clearance was significantly increased by silibinin treatment at a dose of 80 mg kg-1 (p<0.001) (Fig. 1).
Renal antioxidant biomarkers: Gentamicin control rats exhibited increased renal MDA levels (p<0.001). Treatment with silibinin (20, 40 and 80 mg kg-1) attenuated MDA levels associated with gentamicin treatment (p<0.01, p<0.01 and p<0.001, respectively). Moreover, gentamicin treatment decreased the activity of renal SOD (p<0.001) and CAT (p<0.01) as well as levels of GSH (p<0.001) compared to control rats. Treatment with silibinin increased the activity of SOD (p<0.05 and p<0.001, respectively) and CAT (p<0.05 and p<0.01, respectively) as well as GSH levels (p<0.01 and p<0.001, respectively) compared to gentamicin control rats (Table 3).
Histopathological studies: Gentamicin treated animals showed more extensive and marked tubular necrosis, inflammation, blood vessel congestion and disintegrated nucleus. However, no abnormalities were observed in control rats. Treatment with silibinin (40 and 80 mg kg-1) dose dependently attenuated these progressions. Accordingly, there were no marked microscopical differences among the control and silibinin treated group (80 mg/kg/day) (Fig. 2).
DISCUSSION
Therapeutic use of aminoglycosides, gentamicin for more than seven days being used in clinical practice to combat severe infections is the commonest cause of nephrotoxicity in 20-30% of patient (Pedraza-Chaverri et al., 2003). Clinically, renal failure with a slow rise in serum creatinine, urea nitrogen along with reduction in Glomerular Filtration Rate (GFR) is the characteristic manifestation of gentamicin induced nephrotoxicity. The generation of free radical species and alteration of mitochondrial function in the renal proximal convoluted tubules are associated with gentamicin therapy. Therefore, antioxidants associated with renoprotective activity have been extensively studied against gentamicin induced nephrotoxicity.
In the present investigation intraperitoneal administration of gentamicin (100 mg kg-1) produced significant reduction in body weight which is in agreement of previous reports (Lakshmi and Sudhakar, 2010). In acute renal failure increased catabolism results in acidosis which is accompanied by anorexia responsible for decreased food intake and causes body weight loss (Ali et al., 1992). Further, renal tubular injury leads to subsequent loss of the tubular cells that take part in renal water reabsorption leading to dehydration and loss of body weight (Ali et al., 2005). The increase in the kidney weight of gentamicin treated rats probably resulted from the edema caused by drug induced acute tubular necrosis (Erdem et al., 2000). Treatment with silibinin at a dose of 40 and 80 mg kg-1 significantly restored the change in body weight and kidney weight associated with gentamicin.
Several reports suggest serum creatinine concentration is a more potent indicator than urea in the first phases of kidney disease (Gilbert et al., 1989). It was reported that gentamicin produced prominent kidney damage as evidenced by significantly higher levels of serum creatinine, blood urea nitrogen and decreased urine creatinine as well as marked reduction in creatinine clearance (Silan et al., 2007; Soliman et al., 2007). In the present study, we observed the significantly higher levels of serum creatinine, blood urea nitrogen and decreased urine creatinine as well as marked reduction in creatinine clearance following gentamicin treatment which is in agreement of the previous reports. On the other hand administration of silibinin at dose levels of 40 and 80 mg kg-1 two days prior and eight days concomitant with gentamicin provided marked improvement in renal functions. Silibinin dose dependently restored the elevated levels of serum creatinine, blood urea nitrogen and decreased urinary creatinine levels.
Gentamicin has been found to increase the generation of Reactive Oxygen Species (ROS) like superoxide anions, hydroxyl radicals and hydrogen peroxides and Reactive Nitrogen Species (RNS) in the renal cortex that eventually lead to renal damage and necrosis via several complex mechanisms including peroxidation of membrane lipids, protein denaturation and DNA damage (Nagai and Takano, 2004; Nagai, 2006). In the present study we observed that gentamicin induced oxidative stress as a result of marked elevation in MDA levels and decreased SOD and CAT activity as well as GSH levels. Free radical scavengers or agents interfere with the production of ROS have been used successfully to ameliorate gentamicin nephropathy. Antioxidant enzymes such as superoxide dismutase (SOD) and catalase protect the cells against oxidative stress mediated cellular injury by converting the toxic radicals to non-toxic end products. Treatment with silibinin at dose levels of 40 and 80 mg kg-1 significantly restored the oxidative stress by decreased MDA formation and increased GSH concentration as well as SOD and CAT activity. Moreover, histopathological examination of gentamicin treated rats supported the biochemical results indicating the structural abnormalities revealed by the presence of tubular necrosis, inflammation, blood vessel congestion and disintegrated nucleus in the gentamicin treated rats. Treatment with silibinin (40 and 80 mg kg-1) was found to reduce such changes induced by gentamicin.
Thus, treatment with silibinin at dose levels 40 and 80 mg kg-1 showed dose dependant renoprotective effect against gentamicin induced nephrotoxicity and the effect may be related to the antioxidant properties, since it has been found that reactive oxygen species may be involved in the impairment kidney function.
CONCLUSION
In conclusion, co-administration of silibinin (40 and 80 mg kg-1) along with gentamicin protect both functional and histological changes through inhibiting free-radical formation and restoration of the antioxidant systems.
ACKNOWLEDGMENTS
The authors would like acknowledge Prof. M.N. Navale, Founder President, Sinhgad Technical Education Society, Vadgaon (BK), Pune, India and Dr. K.N. Gujar, Principal, Sinhgad College of Pharmacy, Vadgaon (BK), Pune, India for providing necessary facilities to carry out the present study.