Diosmin (CAS Number: 520-27-4), Gentamicin (CAS Number: 1405-41-0) and other chemicals and reagents prepared from Sigma-Aldrich (Germany).
Twenty-eight male Wistar albino rats (250–300g) were obtained from the animal house of Ahvaz Jundishapur University of Medical Sciences (AJUMS). Then, for seven days the rats were placed in a 12:12 h sleed-wake cycle in ideal states (normal moisture, 65±5%; temperature, 23±3°C).
The animals were allocated to 4 groups of 7 rats at random.
Group I received normal saline (10ml/kg of DIO vehicle) for fourteen days, and 0.9% normal saline (GEN vehicle, i.p.) from the eighth day to the fourteenth day.
Group II was used as GEN group and received 100mg/kg gentamicin for 7 continuous days, from day 8 to day 14.
Group III was used as GEN + DIO group and received DIO (50mg/kg, p.o.)  for 14 continuous days, from 7 consecutive days before GEN (100mg/kg, i.p.) administration as well as 7 days together with GEN administration.
Group IV was used as DIO group, rats received DIO (50mg/kg, p.o.) alone for 14 continuous days.
After 24h from final dose administration, all the rats were anesthetized by xylazine-ketamine (8/80 mg/kg). Blood sampling was taken from the heart and the serum samples was isolated by centrifuging (15 min / 1500 rpm / +4 °C) and kept at −20 °C to determine serum kidney markers, Cr and BUN. The rats were sacrificed with cervical dislocation and their kidneys were removed. For histopathological evaluation, the right kidney removed and fixed in formalin (10%), whereas the left kidney was promptly and homogenized (1/10 w/v) on cool Tris buffer (pH 7.4) and served at –20 °C regarding inflammation and biochemical tests.
Determination of serum Cr and BUN levels
Serum Cr concentration was measured by creatinine colorimetric kit (BioMérieux, France). Accordingly, Cr in alkaline solution is reacted with picrate for forming a colored complex, whose color was assessed at 492 nm. Blood urea nitrogen concentration was determined by urea enzymatic colorimetric kit (Linear Chemicals, S.L., Spain). Accordingly urea is hydrolyzed with water and urease for producing ammonia and carbon dioxide. The ammonia ions are reacted by hypochlorite and salicylate for forming green dye (2, 2 dicarboxyl-indophenol), whose color was assessed at 580 nm.
The total protein content was calculated considering the Bradford’s approach.
Renal malondialdehyde (MDA) assay
The tissue MDA concentration was measured based on previous reports[19, 20]. In brief, tissue homogenate (0.5 ml) was added to trichloroacetic acid (TCA, 10%, w/v; 1.5 ml), followed by centrifugation (5000 rpm / 12 min), and transferring 1.5 ml of each specimen supernatant into a test tube including thiobarbituric acid (TBA) solution (0.67% w/v; 2 ml). We then centrifuged (4000 rpm / 15 min) and its absorbance was read by the microplate-reader (at 532 nm). The standard curve was built in the concentration of 1 to 10 μM of tetraethoxypropane.
Renal nitric oxide (NO) assay
Griess assay was used to identify NO level, so that 100 µl of the sample was mixed with 100 µl acidic Griess reagent and the absorbance was determined by an ELISA reader (RayBiotech, Canada) at 540 nm.
Renal GSH content assay
The GSH concentration of renal tissue was assessed based on Ellman’s method. In summary, supernatant 1 mL was blended with 1 mL of 4% sulfosalicylic acid, then it was centrifuged at 1200 rpm for a quarter-hour at 4 °C. Then, 2.7 mL of 0.1 M phosphate buffer (pH 7.4) and 0.2 mL of 5,5-dithiobis 2-nitrobenzoic acid (DTNB) (40 mg/10 mL of 0.1 M phosphate buffer, pH 7.4) were mixed. Afterward, the yellow color was investigated instantly at 412 nm by an ELISA reader (RayBiotech, Canada).
Renal CAT, SOD and GPx assay
CAT, SOD and GPx activities were assessed using a commercial kit designed for rat, based on the producer’s guideline (ZellBio GmbH, Germany).
Assessment of pro-inflammatory cytokines
Pro-inflammatory cytokines of the kidney tissues were assessed through ELISA and commercial kits. Tumour necrosis factor α (TNF-α) and interleukin 1 beta (IL-1β) concentrations were measured by ELISA kits (IBL International Co.).
Following blood sampling, the kidneys were isolated immediately followed by fixation in formalin (10%) and dehydrating in graded alcohol concentrations and, embedding in paraffin. The obtained sections (4 to 6µm) were stained by hematoxylin and eosin (H&E) and 6 microscopy slides per rat were assessed for histological alterations, like red blood cells (RBCs) congestion, inflammatory cell infiltration, glomeruli and proximal tubule cells injury (degeneration, cell swelling). To assess proximal tubule injuries, the mean rate of injured tubules were calculated via dividing the count of tubules in a random microscopic field by the overall count of tubules in the similar field and the obtained value multiplied by 100. Accumulating inflammatory cells and RBCs was categorized into four classes: normal (0), mild (1), medium (2) or severe (3) and the mean values were regarded. Regarding each slide, the average of 6 field was determined. We read slides in a “blind” manner and examining under light microscope with 400× magnification.
Values are provided as mean ± S.D. The one way ANOVA was applied for comparing the results of the groups, Tukey post-hock test was employed to compare the findings between groups. P values of p<0.05 were regarded as significant.