In Vivo Antioxidant And Kidney Protective Potential Of Atorvastatin In Rat Cadmium-Induced Toxicity

Purpose: Environmental and occupational exposure to cadmium chloride is known to cause nephrotoxicity linked with oxidative stress in humans and animals. This study used Atorvastatin to examine its effect on cadmium chloride-induced nephrotoxicity in rat model using biochemical and histological methodologies. Methods: Experiments were performed on 56 adult male Wistar rats (200 ±20 g), randomly assigned to eight groups. Atorvastatin was administered by oral for 15 days at 20 mg/kg/day, started 7 days before cadmium chloride intraperitoneal administration (1, 2, and 3 mg/kg) for eight days. On day 16, blood samples were collected, and kidneys were excised to evaluate the biochemical and histopathological changes.Cadmium chloride signicantly increased malondialdehyde (MDA), serum creatinine (Cr), blood urea nitrogen (BUN), and decreased superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GPx) levels. Results: Administration of Atorvastatin (20 mg/kg) signicantly improved lipid peroxidation, glutathione and activities of antioxidant enzymes and signicantly decreased BUN and Creatinine. Atorvastatin clearly improved the histological changes, demonstrating its protective role against Cadmium chloride-induced kidney injury. Conclusion: Treatment with Atorvastatin signicantly improves all biochemical parameters and suggests a protecting role against cadmium chloride-induced oxidative stress and histological changes in rat kidney.


Introduction
Cadmium (Cd) is frequently used in various industrial processes and considered as one of toxic metals to environment. Occupational and environmental exposures to Cd are linked to industrial activities. (Mezynska and Brzóska, 2018) The kidney is known as the main target organ for chronic Cd exposure. (Rana et al., 2018) The biological half-life of Cd in humans reported 10 to 30 years in the kidney cortex which may account for a higher occurrence of nephrotoxicity. (Moitra et al., 2014) In the kidney, the accumulation of 50 µg Cd/g, wet tissue weight resulted in renal dysfunction (Satarug et al., 2000).
Evidence suggests that Cd absorption may result in oxidative stress. Cd-induced oxidative injury includes a difference between production and removal of reactive oxygen species (ROS) in kidney. Furthermore, the production and accumulation of ROS are associated with hydrogen peroxide (H2O2), hydroxyl radical  (Şişman et al., 2003) Cd is ltered by the glomeruli and is then reabsorbed by the epithelial cells of the proximal tubule, possessing a potent toxic effect on the kidney. (Rana et al., 2018;Yang and Shu, 2015) Cd is known to cause physiological and biochemical effects in animals and humans. ( Overall, the widespread emission of Cd has increased exposure in working and general population and demanded further research to prevent health-related hazards. However, few experimental studies have investigated and reported the protective role of atorvastatin on toxic metals in animal model with renal failure. As per our knowledge, the protective role of AT on Cadmium-induced nephrotoxicity in rats has not been demonstrated. Therefore, the main goal of the current experiment was to investigate in vivo biochemical and histological changes induced by CdCl2 on the kidney of adult rat model. We evaluated the possible role of Atorvastatin in protecting CdCl2-induced renal toxicity in rats and in ameliorating the renal oxidative stress biomarkers and histological changes in rats.

Experimental Animals
Adult Wistar male rats (n = 56) weighing between 200 and 220 g were used. The animals were housed in metal cages (7 rats in each cage) under hygienic conditions and maintained at 22 ± 2°C and 12-hour lightdark cycles with free access to food and water. Rats were acclimatized for one week before treatment commenced.

Ethics
Prior to the experiment, we reviewed and ensured that the protocols were in accordance with the guidelines of animal acts proposed by the institutional Ethical Review Board of Semnan University of Medical Sciences (IR.SEMUMS.REC.1395.177).

Drug and Chemicals
AT was purchased from Tehran Chemie pharmaceutical Co. Cadmium chloride (CdCl2) was obtained from Merck (Darmstadt, Germany). Biochemical markers were assessed using Rat SOD, GPx, MDA, GSH ELISA Kits from ZellBio, GmbH, (Gerrmany) as instructed by manufacturer. BUN and Creatinine in the kidney tissues were detected as instructed by kits obtained from Pars Azmun Pharmaceutical (Iran). Purchased Chemicals were of standard grade and purity for performing experimental tests.

Experimental groups
The wistar rats were randomly assigned into eight groups of seven rats in each group. The rst group of rats received physiologic saline. The second group treated with oral gavages a dose of 20 mg/ kg /day AT for 15 days. The third, fth, and seventh groups received intraperitoneal (i.p.) CdCl2 with doses of 1, 2 and 3 mg/kg, while the fourth, sixth, and eighth groups pretreated with oral gavages containing 20 mg AT/kg body weight, 30 minutes prior to the intraperitoneal (i.p.) administration of CdCl2 at 1, 2 and 3 mg/kg. All animals received Cdcl2 from day 8 to day 15. After 24 hours of the last administration blood samples were taken from the heart under anesthesia with sodium pentobarbital (50 mg/kg). Excised Kidneys were washed thoroughly with saline solution. Obtained parts of kidney tissues were xed and examined by light microscopy, using hematoxylin and eosin (H&E) staining technique and a 0.5⋅0.5-cm 2 slices of additional half was cut for the measurement of GSH, SOD, MDA and GPx.

Histological analyses
Renal tissues were xed in buffered formalin (10%) for 48 hours and embedded in para n wax. Slices of 10 µm were cut and placed on glass slides. After making and drying kidney tissue slides, they were stained with hematoxylin and eosin (H&E) method. Kidney cell injury was examined based on dilated nuclei, loss of staining capacity and swelling of kidney tubular cells. Five elds of each slide were randomly selected and photographed under a magni cation of x400 and evaluated by a pathologist and a histologist.

Biochemical analyses
The samples of kidney tissue were washed and homogenized with phosphate buffered saline. The supernatants were prepared by centrifuging the homogenates at 800× g for 10 min at 4°C, and preserved the samples in -80°C. The activities of GSH, MDA, GPx, and SOD were measured in the obtained supernatants,(Yang and Shu, 2015) Serum was separated from blood samples by centrifugation at 3000 rpm for 20 minutes and stored at − 20°C until the measurement of parameters. Serum levels of BUN, CR and the renal tissues levels of GSH, MDA, GPx, and SOD were measured as instructed by relevant kits.
Serum level of BUN was assayed using colorimetric kit according to the method described. Serum level of Cr was measured by Jaffe's method. (Brouwers et al., 2013) The Concentrations of GSH, MDA, GPx, and SOD in kidney tissues were measured as per the instructions of kits from ZellBio GmbH (Ulm, Germany).
Thiobarbituric acid reaction with MDA Assay kit estimated the level of lipid peroxidation. Adding thiobarbituric acid to MDA results in the formation of red complex product that was measured at 532 nm by a NanoDrope Spectrophotometer.. The detection limit of MDA was 0.1 µM.(Pirmoradi et al., 2019) An assay kit (Zellbio Co) was used to measure the activity of SOD in kidney tissue. Measurement of SOD was based on an enzyme reacting with superoxide anion to produce oxygen and hydrogen peroxide. GSH and GPx were quanti ed by colorimetric method at 412 nm, using chemical assay kits ZellBio GmbH, (Ulm, Germany) with a 0.1 mM detection limit. (Sheikh, 2016) The content of renal Gpx was assessed by measuring NADH catalyzed one micromole GSH per minute to Oxidized glutathione (GSSG). The protein content of supernatant was assayed according to method described by Bradford method using standard bovine serum albumin at 560 nm. (Verdi et al., 2005) 2.8. Statistical analyses Statistical analyses were performed using the Graphpad Prism 8 software. Experimental data were processed to present the Standard Error Means. Data were analyzed by one-way analysis of variance (ANOVA), and Tukey's multiple comparisons tests were performed to compare the means of all groups to the mean of every other group. Differences were statistically considered signi cant at P < 0.05.

− 1. Effects of Cd and Atorvastatin on kidney MDA level
MDA level in the rat kidney homogenates increased signi cantly in the group treated with CdCl2 compared to control group (Cd 3 mg/kg/day, **P = .0011 and Cd 2 mg/kg/day *P = 0.0428). Atorvastatin at dose of 20mg/kg/day signi cantly decreased MDA level in CdCl2 treated rats (Cd 3 mg/kg/day + AT 20mg/kg/day, *P = 0.0268) as illustrated in Fig. 2.
The activities of non-enzymatic antioxidants (GSH) and, enzymatic (SOD and GPx) in the kidney of rats are shown in Figures 3-a, 3-b, and 3-c.

Glutathione GSH
The effect of Cdcl2 on GSH levels and treatment with a combination of CdCl2 plus AT on GSH concentration of rat renal tissues is depicted in Fig. 3-a. The administration of Cdcl2 signi cantly decreased GSH concentration compared to control values in kidney (Cd 3 mg/kg/day, **P = .0078). The combination of Cdcl2 with AT signi cantly altered kidney GSH content compared to CdCl2 treated rats (Cd 2 mg/kg/day + AT 20mg/kg/day, *P .0401 and Cd 3 mg/kg/day + AT 20mg/kg/day, *P = .0390).

Superoxide dismutase (SOD)
In rats given Cdcl2 at a dose of 3 mg/kg, SOD activity signi cantly lowered compared to control group (**P = 0.0056). Equally, pretreatment of AT (20 mg/kg/day) signi cantly increased SOD enzymatic activity (*P = 0.0323) compared to Cdcl2-treated group (1 mg/kg/day) as depicted in Fig. 3 The effect of Cdcl2 on rat kidney tissues GPx activity and treatment with a combination of Cdcl2 plus AT on the rat tissue GPx activity is depicted in Fig. 3-c. Administration of Cdcl2 signi cantly reduced GPx concentration in kidney compared to control values (Cd 2 mg/kg/day, *P = 0.0138 and Cd 3 mg/kg/day, **P < 0.0078). The combination of Cdcl2 plus AT increased kidney GPx content compared to CdCl2 treated rats, but the difference was not signi cant.

3-3. Effects of treatments on the serum level of BUN
A dose of 3-mg/kg Cdcl2 signi cantly increased BUN compared to the rats treated with saline (**P < 0.01) but not signi cantly at dose of 2 mg as shown in Fig. 4. Pretreatment with AT (20 mg/kg/day) signi cantly decreased BUN level in Cd-induced changes of 1 and 3 mg/kg (*P < 0.05) and control group (***P < 0.01) but not signi cantly at Cd dose of 2 mg/kg. Figure 5 shows the status of creatinine serum level of control group and experimental group. CdCl2 administration (3 mg/kg/day) signi cantly increased creatinine in the serum as compared to control rats (**P = 0.0029). Increased levels of creatinine due to CdCl2 challenge were signi cantly decreased at doses of 1, 2, and 3 mg/kg/day upon the pre-treatment with Atorvastatin 20 mg/kg/day (Cd1 + AT **P = 0.0072, Cd2 + AT **P = 0. 0048, Cd3 + AT *P = .0155).

3-5. Histological changes in the rat kidney:
The Histological examination on the renal tissues of the control rats demonstrated normal architecture and regularly arranged kidney tissue cells in both lateral cortex and medullary segments, respectively. In the central part of the tissue, normal epithelial cells were arranged in the collecting duct. The appearance of epithelial cells and glomerular size were observed normal in the proximal convoluted tubule and the distal convoluted tubule (Fig. 6-a).
The kidney tissue of CdCl2-treated rats group demonstrated severe histological damages, where renal glomerular size was reduced compared to normal tissue and hemorrhage was observed inside the Bowman's capsule. Lymphocytic cells increased in the renal tissues and the death of epithelial cells in the wall of Bowman's capsule was observed in glomeruli. Disrupted epithelial cells were deposited in the collector duct. In the central part of the tissue, the number of renal cells in the proximal and distal convoluted tubules was indistinguishable, indicating histopathological alterations in the cellular structure. Further, in rats treated with Cdcl2, the aggregation of interstitial lymphocyte in ltration was increased in renal tubular cells, and vacuolated cytoplasm was observed, which ultimately led to cellular death ( Fig. 6-b). In the central part of the tissue, abnormal epithelial cells were placed in the collection duct.
In contrast, these histopathological changes were reduced in the kidney of rats following administration of CdCl2 and AT (20mg/kg/day). The results of this study in the rat groups treated with CdCl2 and AT 20 mg/kg/day showed that Bowman's capsule wall epithelial cells in the kidney glomeruli had a normal appearance. Bowman space is slightly larger in the distance between the epithelial cells and the vessel wall thicker than in the control groups. The percentage of lymphocyte cells in tissue was normal and interstitial hemorrhage was not observed. In the cortical tissue, the entire complex tubes were seen around and near the epithelial wall and the cells were aligned. The epithelial cells near the convoluted tubules and the surrounding tubules were normal ( Fig. 6-c).

Discussion
This in-vivo experimental study examined the potential effects of AT on Cdcl2-induced kidney toxicity in rat. Our results showed that administration of Cdcl2 signi cantly increased MDA levels, and decreased the enzymatic and non-enzymatic antioxidants in the kidney of rats. Similarly, Renugadevi et al. Our results showed the administration of CdCl2 signi cantly decreased GPx (doses of 2 and3 mg/kg), SOD, and GSH (dose of 3 mg/kg ) activities in the kidney compared to a control group and resulted in oxidative stress in the rat kidney that was re ected by the renal histopathological and biochemical changes. Our results were in agreement with a rat experimental study by Adi et al. (Adi et al., 2016) concluded that Cd exposure (20 mg/kg bodyweight for 30 days) meaningfully reduced CAT, GR, SOD, and GPx activities and improved LPx and GST activities. Also, Hormozi et al. (Hormozi et al., 2018) demonstrated that concurrent workplace exposure to lead and cadmium in tile industry might result in a remarkable increase in lipid peroxidation, and altered antioxidant enzymes (CAT, SOD, and GPx) and oxidative stress.
In our study, the functional nephrotoxicity was indexed through BUN and creatinine levels, which were increased in CdCl2-treated rats as matched to control rats. Our results con rm the study by Wallin  A study reported opposite results showed that administration of AT (30 mg/kg/day for 8 weeks) induced adverse effect in renal tissues and a post-treatment of Arjunolic acid (20 mg/kg for 4 days) and vitamin C might protect kidney from AT-induced severe tissue toxicity. (Pal et al., 2015) Three major differences between our ndings and the results of Pal et al. (Pal et al., 2015) could be due to the discrepancy between dose, exposure time, and animals. In the current study, rats were exposed to 20 mg/kg AT for 15 days, while in Pal et al. (Pal et al., 2015) study mice were given 30 mg/kg/day AT for 8 weeks.
Our results highlighted that the administration of CdCl2 in rat model may lead to nephrotoxicity. Histological examination showed many alterations in renal tissue structure following exposure to CdCl2 (Fig. 6b). These results consistent with those of Mohammad et al. (Mohammed and Hashem, 2019) which described CdCl2 (5 mg/kg b.w, orally for 4 weeks) induced glomerular injury, acute dilatation of Bauman's capsule, congestion of the renal blood vessels, and injury to glomerular epithelial in rat.
A previous report concluded that CdCl2 administration (5 mg/kg for 30 days) indicated adverse effect on cortical blood ow and renal parenchyma replacement with numerous lymphocytes in ltrates, and dilation of glomeruli in rat. (

Conclusion
This study indicated that varying doses of Cdcl2 caused oxidative stress and accounted for decreasing enzymatic activity in neutralizing free radicals and histological changes. Our results highlighted AT might protect rats against CdCl2-induced oxidative stress. Workers must be informed about the potential health effects related to Cd exposure. We proposed that AT might be clinically relevant to CdCl2-induced renal disorders due to its potential therapeutic use in industrial workers. AG, ARB, SY conducted experiments, AD, EK, ZG, AG, ARB performed data analyses and interpretations. AD, EK, ZG contributed to writing and revising manuscript. AD supervised the study and nal approval of the version to be published. The authors declare that all data were generated in-house and that no paper mill was used.  Effects of AT on MDA levels in kidney tissues of rats treated with Cdcl2 at doses of 1, 2, and 3 mg/kg. Administration of Cdcl2 (2 and 3 mg/kg) signi cantly increased levels of MDA in serum compared to the rats received saline. Pretreatment with AT decreased the effect of Cdcl2 (3 mg/kg). Presented gures are mean ± S.E.M (n=7) 0.05999. *P <0.05, **P <0.01. pretreatment improved SOD at the level of control group and signi cantly suppressed the effect of Cdcl2 (1 mg/kg). Results are mean ± S.E.M (n=7) 0.4352. *P <0.05, **P <0.01. c. Effects of AT on GPx activity in renal tissues of rats received Cdcl2 with doses of 1, 2, and 3 mg/kg. Data showed no signi cant difference of GPx levels between rats pretreated with AT and the rats received the various doses of Cdcl2.

Figure 4
Effects of AT on BUN concentrations in the rats exposed to Cdcl2 with doses 1,2, and 3mg/kg.  Effects of AT on creatinine in rat kidney tissues exposed to Cdcl2. Administration of Cdcl2 (3 mg/kg), induced signi cant increase in creatinine level compared to the rats treated with saline and AT pretreatment signi cantly decreased creatinine and the effect of Cdcl2 (1, 2 and 3 mg/kg) . Results are presented as mean ± S.E.M (n=7) 0.1018. *P <0.02, **P <0.01. Figure 6 a. Light microscopy of rat renal tissue of the control group under normal saline treatment illustrating the healthy architecture of Bowman's capsule (black arrow), glomeruli, distal tubules, and collector duct. b.
Light microscopy of rat kidney received CdCl2 displaying renal damages: degeneration of glomeruli (G), hemorrhage (H), deposited epithelial cells in collector duct (C). c. Light microscopy of rat kidney structure following treatment of AT (20 mg/kg) and 30 min before administration of CdCl2. AT protective effect on injury in the kidney tissue, exhibiting normal kidney tissue structure with glomeruli, renal distal convoluted, and collecting duct.

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