2.1 Animals

Male albino rats weighing (130±20 gm) were obtained from the animal house of Al-Nile Pharmaceutical Company. They were housed in stainless steel cages under a 12 hours light / dark cycle at room temperature. Animals were kept under the same condltion with regard food and water all over the period of this study. Each individual animal was weighed before start of therapy and was clearly marked by gentian violet to indicate its weight. The doses of drugs were accurately calculated according to the weight of each animal.

2.2 Apparatuses

1. Centrifuge.VEB MLW Zentrifugenbav engelsdorf type T52.1.
2. Spectrophotometer “Shimadzu, UV-visible recording spectrophotometer UV-160, Shimadzu Corporation Kyoto-Japan”.

2.3 Drugs and chemicals

In this study, the following drugs and chemicals were used:

1. Gentamicin sulphate 80 mg /ml (Epigent) was obtained from: E.I.P.I.Co.
2. Losartan (50 mg. tab. - Amriya comp).
3. Urea and creatinine kits.

2.4 Induction of Nephropathy in Rats

The rats received ordinary rat diet for 6 weeks and were given gentamicin injection in a dose of 80 mg/kg intraperitoneal (IP) once daily) [4] during the last 8 consecutive days.

2.5 Study design

Animals were randomized after estimation of basal blood urea and serum creatinine into four groups as follows:

2.6 Samples collection

The animals were anaesthetized using diethyl ether. Blood samples from the venousplexus deep to the medial canthus of the palpebral fissure. 5 ml blood were added to an anticoagulant ( EDTA) centrifuged at 2,000 rpm for 10 min at 4⁰C to separate plasma from erythrocytes. The biochemical parameters were determined as follows.

2.7 Biochemical analysis and technique

1. Serum urea (measured in mg/dl).
2. Serum creatinine (measured in mg/dl).

2.8 Determination of serum urea

Blood urea levels were determined following urease modifled berthelot reaction [8] using kit supplied by Biomerieux- France.

2.9 Reagents

1. Reagent 1 (standard): urea 50 mg/dl.
2. Reagent 2 (enzyme): urease
3. Reagent 3 (color reagent): phosphate buffer (pH = 0.8), sodium salicylate, sodium nitroprusside and ethylenediaminotetra-acetic acid (EDTA).
4. Reagent 4 (alkaline reagent): sodium carbonate and sodium hypochloride
5. Working reagent: one bottel of reagent 3 was reconstituted with one vial of reagent 2 and shacked gently.

2.10 Procedure

1. 1 ml of working solution was pipetted into 3 test tubes (test, blank and standard)
2. 10 μl of tested serum sample and 10 μl of standard solution were added to their corresponding tubes.
3. The tubes were mixed and incubated at 37⁰C for 3 minutes.
4. 200 μl of reagent 4 was added to each ofthe three tubes.
5. The tubes were mixed and incubated at 37⁰C for 5 minutes
6. The absorbance was read using a spectrophotometer at wave length of 580 nm and setting the blank at zero.

2.11 Calculation

Serum urea =  A bsorbance A sample A bsorbance A standard ×50. mg/dl.

2.12 Determination of serum creatinine

Serum creatinine was determined by kinetic measurements according to the methods of [9] using kit supplied by Human-Germany.

2.13 Principle

The complex formed by creatinine and picric acid in an alkaline medium is measured for two minute

2.14 Reagents

1. Reagent 1 (standard): creatmine 2 mg/dl.
2. Reagent 2 (colour reagent): picric acid.
3. Reagent 3 (alkaline reagent): sodium hydroxide diluted with distilled water in the ratio 1:4.
4. Working reagent: one volume of reagent 2 was mixed with one volume of reagent 3.

2.15 Procedure

1. 1 ml of working solution was pipetted into 2 cuvettes (test and standard).
2. 100 μl of tested serum sample and 100 ul of standard solution were added to their corresponding cuvettes.
3. The cuvettes were mixed and stopwatch was started immediately.
4. The absorbance was read using a spectrophotometer at wave length of 492 nm between t= 20 sec and t= 80 sec where the zero adjustment was the air.

2.16 Caculation

Serum creatinme (mg/dl) =  Δ A sample Δ A standard ×2
ΔA= A₂-A₁
A₂: absorbance 60 seconds after first measurement A₁.
A₁: absorbance at 20 seconds.

2.17 Collection of kidney for histopathological examination

At the end of 6th week after drug or vehicles administration the animals were sacrificed and the kidneys were removed for histological and ultra structure examination. One kidney was cut longitudinally; one half was fixed in 10% buffered formalin and embedded in paraffin. Sections of 5-mm thickness were cut and stained with Mayer's hematoxyline and eosin (for examination of cell structure) and examined by light microscope [10].

2.18 Statistical analysis of results

The variability of results was expressed as the mean ± standard deviation (X ± SD). Statistical analysis of the difference between groups was performed by using Statistical package for the social Sciences (SPSS) version 20, using one-way analysis of variance (ANOVA), and paired T test. Charts were done using Excel program, Microsoft Office XP 2007.

2.19 Degree of significance

P > 0.05 = insignificant difference.
P < 0.05 = significant difference.

3.1 Serum urea

The mean of serum urea levels of the control group was 28.2±1.5mg/ dl, and in gentamicin treated group was 92.3±1.2mg/dl. This level showed a significant increase compared to the control group (P<0.05) (Figure 1).

Figure 1: Shows the effects of different treatments of groups: (control gp, gentamicin treated gp., Losartan gp., Losartan with Gentamicin gp) on the mean serum levels of urea (mg/dl).

Our study found that in Losartan treated group the mean serum levels of urea was 25.2±0.9mg/dl. This level showed no significant change compared to control group (28.2±1.5mg/dl), This study found that gentamicin and Losartan treated group, thier mean serum urea levels was 60.4±1.2 mg/dl. The statistical analysis indicate that the serum levels of urea showed a significant decrease as compared to that levels of gentamicin treated group which was 92.3±1.2mg/dl by using paired T test the P value < 0.05.

3.2 Serum creatinine

Our results expressed as mean ± SD (Table 1, Figure 2) show that in the control group, the mean of serum levels of creatinine was 0.62±0.05 mg/dl, and in group treated with gentamicin ,the mean of serum levels of creatinine was 2.7±0.09 mg/dl. This level showed a significant increase compared to control group (P<0.05).

Table 1: Shows the Effects of oral administration of Losartan ( in absence and presence of gentamicin ) on serum urea(mg/dl), and, serum creatinine (mg/dl) in rats treated groups.

Figure 2: Shows the effects of different treatment of groups : (control gp, gentamicin treated gp., Losartan gp., Losartan with Gentamicin gp. ) on the mean serum levels of creatinine (mg/dl).

The results obtained for group treated with Losartan, the mean of serum levels of creatinine was 0.63±0.03 mg/dl. This value showed no significant change compared to control group. In gentamicin and Losartan treated group, the mean of serum levels of creatinine was 0.98±0.08 mg/dl, this value showed a statistically significant decrease compared to gentamicin treated group (P<0.05). The results proved that Losartan has a nephroprotective effects.

3.3 Histopathology

In the control group, section of rat kidney shows normal appearance of glomeruli and tubules, (figure 3). In gentamicin treated group, section of rat kidney shows dilatation of the tubules with drop out of some epithelial lining (picture of tubular necrosis), (figure 4).

Figure 3: Section from the rat kidney of control group showed normal appearance of glomeruli and tubules,[( H& E) X 400].

Figure 4: Section from the rat kidney of gentamicin treated group shows dilatation of the tubules with drop out of some epithelial lining, (picture of tubular necrosis), [( H& E) X 400].

In Losartan treated rats, animals show normal appearance of glomeruli and tubules. While, that treated with gentamicin and, Losartan show apparently minimal glomerular atrophy and minimal tubular heamorrhge.