Protective Effect of Calcium Dobesilate Administration Against Hepatorenal Damage Induced By CCL4 In Male Mice

doi:10.21203/rs.3.rs-840854/v1

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Protective Effect of Calcium Dobesilate Administration Against Hepatorenal Damage Induced By CCL4 In Male Mice

https://doi.org/10.21203/rs.3.rs-840854/v1

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Purpose: Calcium dobesilate (CaD) has antioxidant and anti-inflammatory properties. In this study, the protective effects of CaD against hepatorenal damage induced by CCL4 in male mice were evaluated.

Methods: Thirty male mice randomly were divided into five groups: Control, CaD 100 mg/kg, CCL4, CCL4+CaD 50 mg/kg, and CCL4+CaD 100 mg/kg. Drugs were administered orally once a day for 4-weeks. The liver and kidney indices (serum creatinine, blood urine nitrogen, alanine aminotransferase and aspartate aminotransferase levels) were determined. Also, hepatic and renal tissue oxidant/antioxidant markers (glutathione peroxidase, malondialdehyde, total antioxidant capacity, and superoxide dismutase) were measured. Cleaved caspase-3, Bax, and Bcl-2 protein levels were measured by immunoblotting method. The liver and kidney histopathological changes were evaluated by H&E staining.

Results: CCL4 induces significant oxidative stress in the kidney and liver that was concomitant with functional and histopathological abnormalities in these organs in the CCL4 group versus the control (P<0.05). CaD could significantly improve the histopathological change in the liver and kidney tissues of CCL4+CaD 100 mg/kg mice versus the CCL4 group (P<0.05). In addition, CaD attenuated apoptosis in the liver and kidney tissues (P<0.05).

Conclusion: The protective effect of CaD may be related to its anti-inflammatory and antioxidant properties.

Clinical Pharmacology

CCL4

oxidative stress

apoptosis

mice

calcium dobesilate

Carbon tetrachloride (CCl4), an industrial solvent, has been used in animal models to explore renal injury in rats (Khan et al., 2009, Khan et al., 2010). It was indicated that the kidney is not the only objective organ of CCl4 toxicity, it causes damage in other organs, such as the liver (Lin et al., 2008), brain (Anand et al., 2011, Guzik-Kornacka et al., 2011), testis (Fadhel and Amran, 2002, Manjrekar et al., 2008), lungs (Mizuguchi et al., 2006), and blood (Soliman and Fahmy, 2011) by generation of free radicals. During the metabolism of CCL4, trichloromethyl (CCl₃) free radicals cause lipid peroxidation (Janbaz et al., 2002, Abdel-Kader et al., 2018) and trichloromethyl peroxyl radical (CCl₃O₂) causes renal injuries (Ruprah et al., 1985). Damage by CCl₄ includes altering the endogenous antioxidants in tissues (Alshammari et al., 2017), which is manifested by histopathological lesions. Furthermore, by CCl₄ administration rat liver and kidney triglycerides, cholesterol, and free fatty acids were increased (Marimuthu et al., 2013). Antioxidants are essential substances that are capable of guarding the body from free radical-induced oxidative stress damage (Kaeidi et al., 2020a, Kaeidi et al., 2020c). CaD (calcium 2, 5-dihydroxybenzenesulfonate) is a vascular protective drug that is used for treatment of diabetic retinopathy and chronic venous insufficiency (Tejerina and Ruiz, 1998, Berthet et al., 1999). As an angioprotective drug, CaD can inhibit platelet activity and reduce blood viscosity and also capillary permeability (Liu et al., 2019). In addition, recent studies have demonstrated that CaD exerts protective effects against diabetic nephropathy (Janbaz et al., 2002). Additionally, CaD has multiple mechanisms of action that include antioxidant properties which reduce lipid pre-oxidation caused by free oxygen radicals and anti-inflammatory properties that decrease release of inflammatory cytokines, such as platelet-activating factor (Bozkurt et al., 2002, Cihan Ozbek et al., 2009, Leal et al., 2010, Seker et al., 2016). Moreover, it has been shown in many studies that CaD corrects capillary dysfunctions, decreases free oxygen radicals, increases nitric oxide synthase, and prevents desquamation in endothelium cells (Ruiz et al., 1997, Suschek et al., 1997). Considering the anti-inflammatory and antioxidant properties of CaD, we evaluated the protective effect of calcium dobesilate administration against hepatorenal damage induced by CCL4 in male mice.

Animal

In this experimental study, 30 male BALB/c mice (30 ± 5 g) were acquired from Rafsanjan University of Medical Sciences Animal House, Rafsanjan, Iran. The male BALB/c mice were kept in a room with 12 hr light/dark cycle, humidity of 45%, and a temperature of 20–23°C with access to food and water ad libitum. The BALB/c mice were maintained in the colony room for one week before starting the experiment. The ethics committee of Rafsanjan University of Medical Sciences approved the study (IR.RUMS.REC.1399.055). Also, this study was done in agreement with the guidelines for animal care and use (National Institutes of Health Publication No. 85 − 23) revised in 2010.

Drugs

CCL4 (10 mmol/kg) (Sigma Chemical Co., St. Louis, MO, USA) was freshly dissolved in 50% olive oil (1:1) for intraperitoneal (i.p.) administration (Hermenean et al., 2013). CaD was procured from Sigma-Aldrich Company, Germany. CaD (50 and 100 mg/kg) was freshly prepared each day during the experiment, and it was dissolved in saline solution for oral administration (Unal et al., 2019a).

Experimental groups

Animals were randomly divided into 5 groups (six mice/group) as follows:

Group 1 (Control): This group received olive oil (as a vehicle of CCL4) by i.p. injection on the 1st day, then an hour later, animals were treated with saline (oral gavage) daily for 4 weeks (Fu et al., 2008).

Group 2 (CaD): This group received olive oil by i.p. injection on the 1st day, then an hour later, animals were treated with CaD (100 mg/kg) by oral gavage daily for 4 weeks (Unal et al., 2019a).

Group 3 (CCL4): This group received CCL4 (10 mmol/kg) in 50% olive oil (1:1) by i.p. injection on the 1st day, then an hour later, animals were treated with saline (oral gavage) daily for 4 weeks (Fu et al., 2008, Hermenean et al., 2013).

Group 4 (CCL4 + CaD 50 mg/kg): This group received CCL4 (10 mmol/kg) by i.p. injection on the 1st day, then an hour later, animals were treated with CaD (50 mg/kg) (oral gavage) daily for 4 weeks.

Group 4 (CCL4 + CaD 100 mg/kg): This group received CCL4 (10 mmol/kg) by i.p. injection on the 1st day, then an hour later, animals were treated with CaD (100 mg/kg) (oral gavage) daily for 4 weeks (Unal et al., 2019a).

All groups were given same volumes of saline or CaD during the experiment. 24 hr after the last administration, mice were anesthetized using ether, and blood samples were taken by heart puncture. Serum samples were prepared via blood centrifugation (6000 g for 15 min). Then mice were killed after deep anesthesia, and livers and kidneys were harvested and divided into two parts. One part was kept in formalin (10%) for histological evaluations by Hematoxylin and Eosin (H&E) staining. The other part was homogenized in ice-cold buffer solution and centrifuged for 20 min at 6000 g; then the supernatant was prepared and kept in -80°C for oxidative parameter evaluation (by the relevant kit) and some biochemical parameters of apoptosis (via western blotting method).

Biochemical parameters

Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels were measured using the relevant kits (Pars Azmoon, Co., Tehran, Iran). Serum creatinine (Cr) and blood urine nitrogen (BUN) levels were determined via quantitative commercial kits (Pars Azmoon, Co., Tehran, Iran).

Oxidative stress parameters

To evaluate the oxidative stress status in the liver and kidney tissues, the glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity levels, malondialdehyde (MDA) concentration, and total antioxidant capacity (TAC) status were measured via available commercial assay kits (ZellBio, Germany) according to the manufacturer’s instructions.

Apoptosis parameters

The immunoblotting method was done to evaluate Bax and Bcl-2 protein expression levels as two apoptosis parameters in the liver and kidney tissues. In brief, each protein sample was isolated via 12.5% polyacrylamide gel electrophoresis, and subsequently transferred to polyvinylidene difluoride membrane by electric current. The membranes were incubated overnight (at the temperature of 4°C and pH 7.4) in Tris-buffered saline and Tween-20 (20 mM Tris–HCl, 150 mM NaCl, 0.1% Tween 20) with 5% nonfat milk. Then each polyvinylidene difluoride membrane was incubated with rabbit polyclonal anti-Bax and anti-Bcl-2 (1:1000) antibodies for 3 hr at the temperature of 20 to 22°C. Subsequently, each blot was washed with Tween-20 thrice and incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody (Abcam, 1:5000) at room temperature for another hour. Then every blot was detected via an enhanced chemiluminescence method. Band densitometry analysis was done using the ImageJ software. Beta (β)-actin immunoblotting (1:5000) was applied as a loading control.

Histopathological analysis

The liver and kidney sections were stained using the routine H&E method (magnification 100×). Each stained slide was observed via a light microscope (Nikon Labophot, Japan) in a blind manner to score kidney and liver tissue damage (KTDS and LTDS) for histopathological analysis. Every kidney section was measured for inflammatory cell infiltration, glomerular atrophy, and tubular necrosis. The renal tubulointerstitial injury was evaluated semi-quantitatively according to previous studies (Kaeidi et al., 2020b, Kaeidi et al., 2020c). Every liver section was measured for congestion and pyknosis. KTDS and LTDS were graded from 0 to 3 by a pathologist blinded to our study (0-0.5 = normal, 1 = mild, 2 = moderate, and 3 = severe).

Data analysis

Data were analyzed using the GraphPad Prism software package (ver. 6.01, GraphPad Software, USA). The normality of the results was analyzed using the Kolmogorov-Smirnov test. The results are expressed as mean ± SD. For parametric data, one-way ANOVA followed by Tukey’s test was done to evaluate the significance level between the groups. For non-parametric data, the Kruskal-Wallis test was performed. P < 0.05 is considered a significant level.

Effect of calcium dobesilate on biochemical parameters

The results showed that serum Cr, BUN, AST, and ALT levels were increased in the CCL4-treated mice compared with the control group (P < 0.01) (Table 1).

CaD (50 and 100 mg/kg) did significantly affect BUN as compared with the CCL4 administrated group. In addition, CaD reduced the Cr level in the CCL4 + CaD 100 mg/kg group compared with CCL4-treated mice (P < 0.05). Furthermore, CaD (100 mg/kg) could significantly reduce both AST (P < 0.05) and ALT (P < 0.01) levels in the CCL4 administrated mice compared with the CaD 100 mg/kg treated group (Table 1).

Effect of calcium dobesilate on oxidative stress parameters

Free radical injury after CCL4 administration was evaluated using lipid peroxidation in the kidney and liver tissues, which was assessed by MDA concentration. According to Table 2, CCL4 significantly increased the MDA concentration in kidney and liver tissues in mice in comparison with the control group (P < 0.05). Administration of CaD (100 mg/kg) significantly decreased the MDA concentration in the kidney (P < 0.05) and liver (P < 0.01) tissues in the CCL4 + CaD 100 mg/kg group versus CCL4 mice (Table 2). Moreover, CaD (50 mg/kg) could decrease the MDA concentration significantly in the liver tissue in CCL4 + CaD 50 mg/kg group when compared with the CCL4-group (P < 0.05, Table 2).

According to results, CCL4 significantly decreased SOD and GPx activity (as two important antioxidant enzymes) in the kidney and liver tissues in the CCL4-group compared with the control mice (P < 0.001, Table 2). As shown in Table 2, administration of CaD (50 and 100 mg/kg) could significantly increase the SOD activity level in the kidney and liver tissues in CCL4 + CaD 50 mg/kg and CCL4 + CaD 100 mg/kg mice versus CCL4 mice (P < 0.05). Furthermore, CaD (50 and 100 mg/kg) significantly elevated the GPx activity in the liver tissue in CCL4 + CaD 50 mg/kg and CCL4 + CaD 100 mg/kg mice when compared with CCL4 mice (P < 0.05, Table 2). Also, administration of CaD (100 mg/kg) significantly increased the GPx activity (P < 0.05) in CaD treated mice compared with the CCL4-group. However, no significant difference was seen in the kidney GPx activity level in CCL4 + CaD 50 mice versus the CCL4 group (Table 2).

According to Table 2, CCL4 significantly reduced the TAC level in kidney (P < 0.01) and liver (P < 0.05) tissues in mice rather than in controls. Also, CaD (100 mg/kg) could significantly increase the TAC level in kidney and liver tissues in the CCL4 + CaD 100 mg/kg group compared with the CCL4-group (P < 0.05, Table 2).

Effect of calcium dobesilate on kidney and liver apoptosis

Our result also showed that CCL4 significantly increased the Bax protein expression level in the kidney and liver tissues in the CCL4 administered mice compared with the control group (P < 0.01, Figs. 1 and 2). However, CaD (100 mg/kg) could decrease the Bax protein expression level in the kidney and liver tissues in the CCL4 + CaD 100 mg/kg treated group versus the CCL4 group (P < 0.05, Figs. 1A and 2A).

Moreover, as shown in Figs. 1B and 2B, CCL4 significantly decreased the Bcl-2 protein expression level in the kidney and liver tissues in the CCL4 administered group rather than the control (P < 0.05). Also, CaD at doses of 100 mg/kg increased the Bcl-2 protein expression level in the kidney and liver tissues in CCL4 + CaD 100 mg/kg treated mice compared with CCL4 administered mice (P < 0.05, Figs. 1B and 2B).

As shown in Figs. 1C and 2C, CaD at dose of 100 mg/kg significantly decreased the Bax:Bcl-2 ratio in the kidney tissue in CCL4 + CaD 100 mg/kg group compared with CCL4 administered mice. (P < 0.001). Also CaD at doses of 50 and 100 mg/kg significantly decreased the Bax:Bcl-2 ratio in the liver tissue (P < 0.01 and P < 0.001, respectively) in CaD-treated groups (CCL4 + CaD 50 mg/kg and CCL4 + CaD 100 mg/kg) compared with CCL4 administered mice.

Effect of calcium dobesilate on kidney and liver tissue damage

The H&E staining results showed that there was no pathologic condition in the kidney and liver tissues of the control group (Figs. 3A and 4A). Furthermore, the histopathologic results showed that CCL4 increases kidney and liver damage, so that there were mild and severe kidney and liver damages in CCL4 administered mice when compared with the control group (P < 0.001, Figs. 3B and 4B), and CaD (100 mg/kg) could attenuate the damage caused by CCL4 (Figs. 3A and 4A). Therefore, KTDS and LTDS were significantly decreased in the CCL4 + CaD 100 mg/kg treated mice when compared with the CCL4 administered group (P < 0.05 and P < 0.01, respectively, Figs. 3B and 4B).

In the present study, we examined the protective effects of CaD treatment against hepatorenal damages caused by CCL4 in mice. In line with former studies, it has been shown that CCL4 treatment causes significant hepatorenal injuries as indicated by the heightened levels of liver and kidney indices, namely BUN, ALT, AST, and Cr (Venkatanarayana et al., 2012, Mohseni et al., 2019). Our results also showed that CCL4 increases the MDA levels as well as GPx, SOD, and TAC activities in the liver and kidney. Moreover, the liver and kidney histological results are in accord with the abovementioned changes in the biochemical and oxidative factors. The results of our study confirm that administration of CaD at the dose of 100 mg/kg reduced the hepatorenal injuries induced by CCL4 via reducing the levels of BUN, Cr, AST, ALP, and MDA as well as increasing the activities of GPx, SOD, and TAC. Still, all biochemical and oxidative results were corroborated by histological findings of liver and kidney tissues. Hepatorenal damages can be verified via changes in serum biochemical parameters and tissue histopathology.

In accordance with our results, it has been reported that CCL4 induces pathological lesions that are related to biochemical alterations such as increased serum ALT, AST, ALP, Cr, and BUN quantities in the liver and kidney (Venkatanarayana et al., 2012, Mohseni et al., 2019). In the present study, renal tubular necrosis, glomerular atrophy, renal leukocyte infiltration, hepatic congestion, sinusoid dilatation, and pyknosis of hepatocytes, in addition to increased abovementioned biochemical parameters in mice getting CCL4, can be regarded as proof of hepatorenal damage.

In our study, CaD treatment could reverse abnormal situation of serum Cr, BUN, ALT, and AST to normal levels, as well as histopathological lesions of liver and kidney tissues in CCL4 administered mice treated with CaD (100 mg/kg). Previous reports revealed that CaD has antioxidant properties (Garay et al., 2005). Seker et al. found that CaD has protective effects against intestinal ischemia-reperfusion injury via increasing the TAC indicator (Seker et al., 2016). CaD is a potent free radical scavenger and reduces the level of MDA (as a marker of lipid peroxidation) (Unal et al., 2019b). Besides, over-generation of MDA could result in functional and pathological changes in the kidneys and liver (Mehrzadi et al., 2018, Kaeidi et al., 2020b). The process of renal damage caused by CCl4 entails peroxidation of the cell membrane fatty acids, which initiates destruction of the cell and their intracellular organelles (Boll et al., 2001). It is widely recognized that oxidative stress triggers apoptosis (Hassanshahi et al., 2020), which results in liver and kidney degeneration (Hagen, 2003). Our results showed that CCL4 could induce apoptosis (via increasing Bax protein expression and lowering Bcl-2 protein expression level) in the kidney and liver organs (Figs. 1 and 2). However, CaD (100 mg/kg) could attenuate apoptosis in these organs (Figs. 1 and 2). In agreement with our study, Zhang et al. reported that CaD has an important role in inhibiting apoptosis factors in these organs (Zhang et al., 2015). Sola-Adell et al. found that CaD prevents neurodegeneration in diabetic retinopathy (Solà-Adell et al., 2017). In another study, it was revealed that treatment of diabetic rats with CaD reduced the retinal injury induced by ischemia/reperfusion by increasing the content of reduced glutathione and oxidized glutathione (Szabo et al., 2001). In another study, CaD increased kidney ferric reducing antioxidant power in nephrotoxicity induced by gentamicin (Jafarey et al., 2014). Besides, latest studies have verified that CAD exerts protective effects against diabetic nephropathy (Zhang, 2013) and gentamicin-induced acute kidney injury (Jafarey et al., 2014).

Conclusion: The present study measured the protective effects of CaD with specific reference to its impacts against oxidative stress (using levels of MDA as well as activities of CAT and GPx) after CCL4-induced damage in mice liver and kidneys. CaD treatment gave sufficient protection through serum and tissue biochemical and histopathological alterations. But, additional studies must be performed to improve understanding of the mechanisms and degree of this protective effect.

Supplementary Information

The online version will contain supplementary material available.

Acknowledgment

The authors thank Rafsanjan University of Medical Sciences for its financial support of the project.

Author contributions

JH conceived and designed the experiments; EH, AK, and JH performed the experiments; AK, MR, MA, and JH analyzed the data; MA, MR and JH contributed reagents/materials/analysis tools; and AK, EH and JH wrote the article.; All authors agree to be accountable for all aspects of work ensuring integrity and accuracy. The authors declare that all data were generated in-house and that no paper mill was used.

Funding

This study was supported by Rafsanjan University of Medical Sciences, Rafsanjan, Iran (Grant # 98274).

Data availability

Data is included in the form of a GraphPad file.

Code availability

Not applicable.

Declarations Ethical standards

The ethics committee of Rafsanjan University of Medical Sciences approved the study (IR.RUMS.REC.1399.055).

Consent to participate

Not applicable.

Conflicts of interest

The authors declare no conflicts of interest.

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Table 1. Effect of calcium dobesilate on functional indicators of kidney and liver in mice with CCL4 administration. Values are expressed as mean ± SD. n= 6 per group. ٭٭P˂0.01 versus the control group. #P˂0.05 and ##P˂0.01 versus CCL4 group. CCL4: Carbon tetrachloride, CaD: Calcium dobesilate, BUN: Blood urea nitrogen, Cr: Creatinine, AST: Aspartate aminotransferase, ALT: Alanine transaminase.

Table 2. Effect of calcium dobesilate on oxidative stress parameters of kidney and liver tissues in mice with CCL4 administration. Values are expressed as mean ± SD. n= 6 per group. ٭P˂0.05 and ٭٭P˂0.01 versus the control group. #P˂0.05 and ##P˂0.01 versus the CCL4 group. CCL4: Carbon tetrachloride, CaD: Calcium dobesilate, MDA: Malondialdehyde, GPx: Glutathione peroxidase, SOD: Superoxide dismutase, TAC: Total antioxidant capacity.

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Protective Effect of Calcium Dobesilate Administration Against Hepatorenal Damage Induced By CCL4 In Male Mice

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Abstract

Figures

Introduction

Materials And Methods

Animal

Drugs

Experimental groups

Biochemical parameters

Oxidative stress parameters

Apoptosis parameters

Histopathological analysis

Data analysis

Results

Effect of calcium dobesilate on biochemical parameters

Effect of calcium dobesilate on oxidative stress parameters

Effect of calcium dobesilate on kidney and liver apoptosis

Effect of calcium dobesilate on kidney and liver tissue damage

Discussion

Declarations

References

Tables

Supplementary Files

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