Prophylaxis With Diosmin Mitigates Kidney Damage Induced by Gentamicin in Rats


 Background Gentamicin is a crucial aminoglycoside antibiotic but it is used only to treat severe bacterial infections, because of its high nephrotoxicity among patients. We evaluated the preventive effects of diosmin (as a natural ingredient) on gentamicin-related renal damage in rats. MethodsIn this research, 28 male Wistar rats were assigned to 4 groups: control, gentamicin (100 mg/kg, (i.p.), daily for 1 week), and gentamicin plus diosmin (50mg/kg, p.o., daily for two weeks), diosmin (50mg/kg/day, p.o. for two weeks). After, the final gavage, blood specimens were gathered for determining serum blood urea nitrogen (BUN) and creatinine. kidneys used for biochemical, inflammation and histological test.Results Creatinine, BUN, nitric oxide, malondialdehyde, TNF-α and IL-1β concentrations significantly increased and glutathione, catalase, glutathione peroxidase, and superoxide dismutase activities decreased after gentamicin treatment. Creatinine, BUN, nitric oxide, malondialdehyde, tumour necrosis factor α (TNF-α), interleukin 1 beta (IL-1β) concentrations significantly reduced and glutathione level, catalase and glutathione peroxidase activities significantly increased via co-administration with diosmin. ConclusionDiosmin had ameliorative impacts against gentamicin-related kidney injury that can be owing to its antioxidant, and anti-inflammatory activities.

and homogenized (1/10 w/v) on cool Tris buffer (pH 7.4) and served at -20 °C regarding in ammation and biochemical tests.

Determination of serum Cr and BUN levels
Serum Cr concentration was measured by creatinine colorimetric kit (BioMérieux, France) [17].
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.

Bradford assay
The total protein content was calculated considering the Bradford's approach [18].

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 [21].

Renal GSH content assay
The GSH concentration of renal tissue was assessed based on Ellman's method [22]. 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 2nitrobenzoic 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-in ammatory cytokines
Pro-in ammatory 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.).

Histopathological evaluations
Following blood sampling, the kidneys were isolated immediately followed by xation in formalin (10%) and dehydrating in graded alcohol concentrations and, embedding in para n. 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, in ammatory cell in ltration, 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 eld by the overall count of tubules in the similar eld and the obtained value multiplied by 100. Accumulating in ammatory 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 eld was determined. We read slides in a "blind" manner and examining under light microscope with 400× magni cation.

Statistical analysis
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 ndings between groups. P values of p<0.05 were regarded as signi cant.

Impact of GEN and DIO on serum Cr and BUN Levels
Gentamicin administrated for seven days led to a significant increase in serum Cr and BUN concentrations (P <0.001) than normal saline group ( Figure 1A, B). Nonetheless, an elevation in the Cr and BUN levels were significantly (P<0.05 and P<0.01 respectively) reduced by DIO pretreatments compared to GEN group. Diosmin alone caused no change in the renal function examinations than the normal saline group ( Figure 1A, B).

Impact of GEN and DIO on renal oxidative stress biomarkers
Tissue MDA and NO concentrations significantly increaseed in the GEN-administrated renal injury group than the normal saline group (P< 0.001). An increase due to GEN were significantly reduced after  (Figure 2A, B).

Effect of GEN and DIO on renal antioxidants factors
In GEN group, the renal GSH content, SOD, GPx, and CAT activities indicated a signi cantly (P< 0.001) reduced than those in the normal saline rats. In the DIO + GEN group, the renal amount of GSH, GPx and CAT activities showed a signi cant increase than the GEN-intoxicated rats (P<0.05, P<0.01, P<0.05, respectively). Diosmin treatment alone caused no significant effect on GSH level, GPx, SOD and CAT activities of the normal saline group ( Figure 3A-D).

Effect of GEN and DIO on renal in ammatory cytokines
Regarding the normal saline group, GEN treatment signi cantly elevated the tumor necrosis factor -α and interleukin-1β concentrations in rats (P<0.001). However, pretreatment of rats with DIO caused a signi cant decrease in TNF-α and IL-1β concentrations than GEN-intoxicated group (P<0.01 and P<0.001 respectively). DIO alone caused no a significant impact on TNF-α and IL-1β levels ( Figure 4A, B).
Histopathological Analysis Figure 5 and Table 1

Discussion
Our ndings indicated that DIO protected kidneys to GEN-related nephrotoxicity in rats. Creatinine and BUN levels were considered as indices of damage to kidney. Reduced glomerular ltration causes an elevation in serum Cr and BUN concentrations [23,24]. We found that rats receiving GEN for 7 continuous days showed nephrotoxicity, because of an increase in serum Cr and BUN concentrations that is consistent with other reports [7,25]. In our study, there are a correlation between such changes in biochemical parameters and the renal histological characteristics. On the other hand, the elevated BUN and creatinine concentrations in rats receiving GEN were restored by DIO administration, which can be linked to antioxidant effect of DIO [26,27]. Oxidative stress and ROS due to GEN causes the structural and functional deterioration of the kidney. In an organism, GEN is metabolized and activates free radicals generation that affect proteins, membrane phospholipids, and nucleic acids, leading to alterations in the function and structure of these molecules resulting in tissue injury [28][29][30][31]. Malondialdehyde can disrupt enzyme activities, change the intracellular ion balance via in uencing ion exchange in the membranes, increase the cell membrane permeability and cause base modi cations in DNA structure [28]. Similar to other reports, we found that GEN could increase MDA concentration [32,33], and it reduced signi cantly after DIO administration.
Nitric oxide can be produced with nitric oxide synthase (NOS) and affects many patho-physiological conditions, like in ammation and oxidative injury[28, 34,35]. Nitric oxide plays a role to regulate the renal hemodynamics and renal tubular function [35]. Reduced glomerular damage (GD) and glomerulosclerosis (GS) were observed by decreasing nitric oxide production [36]. GEN-related oxidative damage causes NO generation and its reaction with superoxide radicals produces very toxic ROS, like per-oxynitrite, leading to renal damge [5,37]. The result of this study showed that GEN treatment could increase the NO concentrations in renal tissue of rats that may be due to oxidative stress. Pretreatment with DIO attenuated the NO concentrations and ameliorated the renal dysfunction because of the GEN-related oxidative damage.
Antioxidants show a direct and indirect protective effect against the damages of oxidative stress [38]. Nonetheless, GSH as a non-enzymatic anti-oxidant is a ROS scavenger protecting against the harmful effect of free radicals. Superoxide dismutase can catalyze the dismutation of superoxide radical to hydrogen peroxide (H 2 O 2 ), CAT can break down and inhibit H 2 O 2 change to free radical, whereas GPx can utilize GSH for preventing hydroperoxide generation [38][39][40].
Our ndings showed that GEN induces GSH concentration depletion because of extra production of ROS or elevated use in the formation of proteins with -SH group and reduced SOD, CAT, and GPx antioxidant enzymes activity linked to overproduction of ROS[28, 41,42]. Diosmin treatment restores the GSH concentrations, GPx and CAT activities in kidney tissue of GEN-related toxicity groups by its ROS scavenging and/or enhancing anti-oxidant effect. Diosmin showed no signi cant difference in SOD activity. Previous studies have shown that DIO could exert its anti-oxidant activity directly via ROS scavenging as well as indirectly via upregulation of anti-oxidant enzymes [10,14,15].
Gentamicin-related renal tubular necrosis may induces/stimulates in ammatory responses and promote migration of monocytes and macrophages tissue injury area [43]. The nuclear factor kappa B (NF-κB) activation against GEN-related oxidative stress and other disorders is an important transcription marker in the renal in ammatory actions via regulation of many gene expressions of cytokines, like IL-1β and TNFα [44][45][46]. The outcomes of this report showed that GEN administration could upregulate the in ammatory reaction evidenced by increased IL-1β and TNF-α concentration in kidney. Flavonoids inhibit the NF-κB pathway effective to regulate in ammatory markers, including IL-6, IL-1β and TNF-α [47,48]. Our ndings indicated that DIO prevented ranal damage by inhibition of IL-1β and TNF-α. So, IL-1β and TNF-α inhibition is a cornerstone to understand anti-in ammatory mechanism of DIO. Previous reports have revealed that the antiin ammatory impacts of DIO could mediate its protection to hepatotoxicity [49], gastric injury [10], ulcerative colitis [50], cognitive impairment [51] and brain oxidative damage [27].

Conclusion
Diosmin is effective against development of GEN-induced nephrotoxicity. The DIO effectiveness to treat GEN-related nephrotoxicity is associated with its antioxidant and antiin ammatory effects. Thus, DIO may be an appropriate strategy to prevent progression of nephrotoxicity and may be a drug employed for kidney damage.