Effect of Vitamin E on Serum Urea Level on Gentamicin Induced Nephrotoxicity in Long Evans Rats

Background: The kidneys have an important role in eliminating the final products of metabolic activities, excreting the drugs and chemicals. A variety of frequently used drugs have been demonstrated to produce nephrotoxic effects. Objective: This study was carried out to observe the effect of vitamin E on gentamicin-induced nephrotoxicity by assessing serum urea level in Long Evans rats. Materials and method: The experimental study was carried out on 40 healthy Long Evans rats of both sex with the weight ranges from 172-255 gm and the age ranges from 7 to 10 weeks. The rats were divided into four groups Group A (normal control) received normal saline, group B, C and D received gentamicin for 6 days, rats of group C received vitamin E capsule for total 9 days with gentamicin whereas group D received vitamin E capsule for total 10 days with gentamicin. Serum urea level was measured at the end of the experiment. Results: The (mean±SD) serum urea levels in group A, B, C and D were 4.79±0.32, 12.41±1.22, 7.56±1.11 and 7.15±1.09 mmol/L respectively. The differences between groups were highly significant (p<0.001) for group A & B, A & C, A & D, B & C, B & D whereas the difference between C & D (p>0.01) was not significant. Serum urea level of the normal saline control group (group A) was within the normal limit (4.79 mmol/L). Serum urea level in gentamicin treated rats (group B) was more in comparison to gentamicin and vitamin E treated rats (group C & D) and pretreatment with longer duration group (group D) showed lower serum urea value than shorter one (group C) though the groups showed no significant difference. Conclusion: Vitamin E treatment showed some protective effect against gentamicin-induced nephrotoxicity. The results also indicated that effectiveness of vitamin E depends on duration of pretreatment that means the pretreatment duration must be increased to a suitable period for better protection against gentamicin-induced nephrotoxicity.


Introduction
The kidneys are one of the vital organs of our body.Kidney is an important organ because of its role in eliminating the final products of metabolic activities, excreting the drugs and chemicals. 1,2A variety of drugs have been demonstrated to produce nephrotoxic side effects.With regard to intrarenal structural processes, several factors make the kidney especially susceptible to toxic injury.The high rates of delivery of compounds to the kidney, concentration of drugs in tubule lumens and interstitial, and transcellular transport of toxins by the kidney make the renal tubular cells especially vulnerable to toxic injury and causes toxic nephropathy. 3Acute renal failure complicates approximately 5% of hospital admissions and up to 30% of admissions to intensive care units.Gentamicin causes acute renal failure in 10% to 15% of all cases.The incidence of renal involvement due to gentamicin is estimated about 4 to 10%.[6][7] Gentamicin is the aminoglycoside of first choice because of its low cost and its reliable activity against all but the most resistant gram negative aerobes.This drug is excreted mainly in urine. 6he renal clearance of gentamicin averages 85% of total clearance, indicating that the drugs are primarily eliminated by kidney. 8The range between effective and toxic blood level is narrow.0] It is apparent that gentamicin nephrotoxicity is reversible but that renal function may take up to several months to return to pre-treatment values. 11tamin E is an antioxidant and probably acts as a free radical scavenger in cell membranes to protect membrane polyunsaturated fatty acids from peroxidation. 12Vitamin E is used in this study because dietary vitamin E supplementation ameliorates renal injury in chronic puromycin aminonucleoside nephropathy, and suppresses peroxidation of membrane phospholipids. 13,14The important cause of cell damage is injury induced by accumulation of such free radicals in the body. 15Very recently it was found that vitamin E protects against gentamicin-induced nephrotoxicity in rats. 16,17

Materials and method
This experimental study was carried out on 40 healthy Long Evans rats of both sex with the weight ranges from 172-255 gm and the age ranges from 7 to 10 weeks.They were allowed to live at normal room temperature under the condition of normal natural light and dark schedule and were fed on pellets of standard rat food.Drugs used were gentamicin, vitamin E and normal saline (0.9%).Distilled water was used as vehicle for both the drugs.
The rats were divided into four groups and randomly selected.Grouping of the rats was done according to treatment pattern.

Group A (Normal control)
The rats of this group received normal diet and injection normal saline intramuscularly (2 mL/kg/day) for 6 days and no drug treatment were given.
All the rats were sacrificed on the 7th day.

Group B (Experimental control)
The rats of this group received normal diet and injection gentamicin intramuscularly (80 mg/kg/day) for 6 days.
All the rats were sacrificed on the 7th day.

Group C (Experimental)
The rats of this group received normal diet and vitamin E capsule orally (1mg/g bw/day) for total 9 days (pretreatment for 3 days + along with gentamicin for 6 days) and gentamicin were injected intramuscularly (80 mg/kg/day) during the last 6 days of the 9-day period.
All the rats were sacrificed on the 10th day that is 24 hours after gentamicin administration.

Group D (Experimental)
The rats of this group received normal diet and vitamin E capsule orally (1mg/g bw/day) for total 10 days (pretreatment for 4 days + along with gentamicin for 6 days) and gentamicin were injected intramuscularly during the last 6 days of the 10-day period.
All the rats were sacrificed on the 11th day that is 24 hours after gentamicin administration.
The animals were sacrificed on the fixed day of each group under chloroform anaesthesia by cervical dislocation.Kidneys were collected after opening the abdomen.Blood was obtained by cardiac puncture by 5 cc syringe from each rat in separate test tubes and allowed to clot for 1 hour, at room temperature.The serum was separated and preserved.Estimation of serum urea was done by chemical method that is Diacetylmonoxime method.Data were analyzed by SPSS version 12.0 for Windows.This study was conducted in the department of Anatomy in Dhaka Medical College, Dhaka, Bangladesh.

Results
The (mean±SD) serum urea levels in group A, B, C and D were 4.79±0.32,12.41±1.22,7.56±1.11and 7.15±1.09mmol/L respectively.The differences between groups were highly significant (p<0.The result indicates that serum urea level was more in gentamicin treated rats (group B) in comparison to simultaneous vitamin E (group C & D) treated rats but the urea level is high in relation to normal saline control group (group A).In the present study the results indicate that serum urea level of normal saline control group (group A) of rats was within normal limit (4.79 mmol/L).But serum urea level was abnormally high (12.41mmol/L) in the gentamicin treated rats (group B).On the other hand simultaneous vitamin E treated rats showed that the values are relatively lower in comparison to only gentamicin treated rats.But the values of serum urea level in group C and D were significantly higher (p<0.001) in comparison to normal saline control rats (group A).

Discussion
In spite of being one of the most nephrotoxic aminoglycoside, gentamicin is still frequently used as a first and second choice drug in a vast variety of clinical situations.Moreover, this aminoglycoside has been widely used as a model to study the nephrotoxicity of this family of drugs, both in experimental animals and human being.However, due to its toxicities, prolonged use is restricted to the therapy of life threatening infections. 19e present study demonstrated the severity of nephrotoxicity by assessing serum urea level in different group of rats.The mean serum urea level was significantly raised in all the groups treated with gentamicin in comparison to normal control group.Alarifi et al. 20 reported that serum urea was significantly increased in the gentamicin treated groups as compared to normal saline control group.This was the biochemical proof of nephrotoxicity produced by gentamicin and conforms to the findings of Cohen et al. 21, Erdem et al. 22 , Sepehri et al. 23 , Babu et al. 24 , Kore et al. 25 and many other researchers.
Abdel-Naim et al. 16 found that pretreatment with intramuscular vitamin E at a dose of 250 mg/kg/day for 3 consecutive days prior to gentamicin administration significantly lowered the elevated serum urea and creatinine levels.In the current study also vitamin E treatment showed some protective effect against gentamicin-induced nephrotoxicity induced by gentamicin administered during the last 6 days of treatment with vitamin E by returning the urea concentration to near normal.The results also indicate that vitamin E was less effective in less duration of pretreatment that means the pretreatment duration must be increased to a suitable period for better protection against gentamicin induced nephrotoxicity.
001) for group A & B, A & C, A & D, B & C, B & D, whereas the difference between C & D (p>0.01) was not significant. 18