Protective Effect of Resveratrol Against Renal Oxidative Stress in Cholestasis

Abstract

Background/aims. This experimental study was designed to evaluate histological changes of the kidney and renal tissue levels of malondialdehyde (MDA), reduced glutathione (GSH), and nitric oxide (NO) and the effect of resveratrol on these metabolites after bile duct ligation in rats. Methods. Secondary biliary cirrhosis was induced by bile duct ligation for 28 days. Swiss albino rats were divided into three groups. Group 1: Sham (n = 7), Group 2: Bile duct ligation (n = 7), Group 3: Bile duct ligation plus resveratrol (n = 7). Bile duct ligation (BDL) plus resveratrol group received 10 mgr/kg dose of resveratrol intraperitoneally daily throughout 28 days. Kidney tissues were harvested to determine the tissue levels of MDA, GSH, and NO activity. Liver and kidney tissues were removed for light microscopic evaluation. Results. Cholestasis was determined by biochemical and pathologic examination. In the resveratrol-treated rats, levels of MDA were significantly lower than those of the BDL group (p < 0.04). The levels of GSH in the resveratrol-treated rats were significantly higher than those in the BDL group (p < 0.01). The levels of NO in the resveratrol group were significantly lower than those in the BDL group (p < 0.01). Conclusion. The present study demonstrates that intraperitoneal administration of resveratrol in bile duct ligated rats maintains antioxidant defenses and reduces kidney oxidative damage. This effect of resveratrol may be useful in the preservation of renal oxidative stress in cholestasis.

• bile duct ligation
• renal oxidative stress
• resveratrol

Introduction

Oxidative stress and increased lipid peroxidation are also present in the kidney of animal models of cholestatic liver disease.[1] The underlying basis of oxidative stress in the kidney is complex, involving intraorgan generation of reactive oxygen species (ROS), possibly mediated by endotoxin,[2] bile acids,[1] hyperbilirubinemia,[3] and accumulation of degradative products of lipid peroxidation, such as lipid peroxides and malondialdehyde.[4]

The cholestasis induced renal failure in kidney,[5] tubular handling of electrolytes was significantly changed, and an increased fractional excretion of sodium and natriuresis, following bile duct ligation, was accompanied by vasodilation of inner medullary peritubular capillaries.[6]

Resveratrol (3, 5, 4′-trans-trihydroxy stilbene) is a natural phytoalexins that is present in grapes and red wine, which possess a variety of biological activities, including antiinflammatory, anticarcinogenic, and antioxidative activities.[7-9] In addition, resveratrol suppresses the proteinuria, hypoalbuminemia, and hyperlipidemia induced by anti-rat kidney antiserum.[10] The effect of resveratrol on renal injury in biliary obstruction has not been investigated yet.

The aim of this study was to evaluate whether resveratrol administration could protect kidney tissue against oxidative stress in rats after experimental bile duct ligation (BDL). To asses the ability of resveratrol to function as an antioxidant in rats with biliary obstruction, we measured lipid peroxidation, levels of reduced GSH and NO. Furthermore, kidney injury in bile duct ligated rats and the effects of resveratrol administration were evaluated by histological examination of the kidney.

Materials and Methods

A total of 21, 3-month-old Swiss albino rats weighing 300–350 g were included in the study. Rats were obtained from Firat University, Animal Laboratory, Elazig Turkey. The study was approved by the Inonu University Ethics Committee. Rats were kept in stainless steel cages, allowed free access food and water ad libitum, and quarantined 14 days before surgery. Food was withheld 8 h prior to surgery, but free access to water was allowed. They were subjected to controlled conditions of temperature and humidity and controlled animal quarters with a 12 h light–dark cycle. All surgical procedures were performed while the rats were under intraperitoneal ketamine (50 mg/kg) and xylazine HCl (10 mg/kg) anesthesia.

A total of 21 rats were divided into three groups:

• Group 1: Sham (n = 7)

• Group 2: BDL (n = 7)

• Group 3: BDL plus resveratrol (n = 7)

Only laparotomy was performed to the sham group. Secondary biliary cirrhosis was induced in animals by double ligation and division of the common bile duct.[11] Abdominal layers were closed with appropriate suture materials. All animals were maintained under the same conditions after surgery. We administered resveratrol at the dose of 10 mg/kg per day i.p., which is reported to cause marked antioxidative activity.[12] Animals in the resveratrol group were treated with a 10 mg/kg dose of resveratrol (Sigma Chemicals USA) intraperitoneally once a day for 28 days. To eliminate complications arising from the diurnal effects, all rats were sacrificed under anesthesia at the same time of day. The blood samples were taken by vena cava inferior puncture. Parts of the liver and kidney were preserved in formalin for histopathological examination. The kidney was preserved in liquid nitrogen and stored at − 85°C for the analysis of malondialdehyde (MDA), GSH, and NO levels.

The plasma was used to measure total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT) as parameters indicative of hepatic function (Olympus Diagnostica Gmbt, Ireland). The kidney tissues were homogenized, and the MDA contents of homogenates were determined spectrophotometrically.[13] The amounts of lipid peroxides were calculated as thiobarbituric acid reactive substances of lipid peroxidation and are given as nmol/g tissue. As tissue nitrite (NO₂⁻) and nitrate (NO₃⁻) levels can be used to estimate NO production, we measured the concentration of these stable NO oxidative metabolites. Quantitation of NO₂⁻ and NO₃⁻ was based on the Griess reaction, in which a chromophore with a strong absorbance at 545 nm is formed by reaction of NO₂⁻ with a mixture of naphthlethylenediamine and sulfanilamide.[14] Results were expressed as umol/g tissue.

Glutathione was determined by the spectrophotometric method, which was based on the use of Elman's reagent.[15] Results were expressed as nmol/g tissue.

Histopathologic Evaluation

Liver and kidney tissue were stained with hematoxylin and eosin and Masson trichrome dye. Histologic chances were assessed under the light microscope by a pathologist without prior knowledge of the groups. For assessment of inflammation and necrosis, the histologic activity index of Ishak et al.[16] was applied.

Statistical Analysis

The statistical package for social sciences (SPSS) version 10.0 was used for statistical analysis. Individual group parameters were assessed using the Mann–Whitney U test. The results are given in the text as means ± standard deviation (STD) for all comparisons; statistical significance was defined as p < 0.05.

Results

Total bilirubin, AST, and ALT levels in bile duct ligated rats (53.6 ± 18.1, 140.5 ± 12.3, 2.7 ± 0.7, respectively) (Table 1) were significantly higher than those of the sham group (p < 0.01). The results of MDA, GSH, and NO are shown in Table 2. In the resveratrol-treated rats, the MDA and NO levels were significantly lower than those in the BDL group (p < 0.04, p < 0.01, respectively). The levels of GSH in the resveratrol-treated rats were significantly higher than those of the BDL group (p < 0.01).

Table 1. Plasma total bilirubin, AST, and ALT levels in the groups.

Groups	Total bilirubin (mg/dL)	AST (U/L)	ALT (U/L)
1. Sham	1.8 ± 0.2	39 ± 4	96 ± 4
2. Bile duct ligation	2.7 ± 0.7	53 ± 18	140 ± 12
3. Resveratrol	2.3 ± 0.3	45 ± 13	110 ± 9
p values
1 vs. 2	0.01	0.01	0.01
2 vs. 3	0.01	0.4	0.01

AST: aspartate aminotransferase; ALT: alanine aminotransferase.

^*p < 0.05 was considered to be statistically significant.

Table 2. Tissue levels of GSH, NO, and MDA activities in the groups.

Groups	MDA (nmol/g tissue)	GSH (nmol/g tissue)	NO (umol/g tissue)
I. Sham (n = 7)	244.8 ± 13.8	2.05 ± 0.1	70 ± 7.3
II. Bile duct ligation (n = 7)	313.02 ± 34.7	1.57 ± 0.18	130 ± 11.1
III. Resveratrol (n = 7)	258.08 ± 23.48	2.78 ± 0.35	97.50 ± 6.0949
p values
I vs. II	0.01	0.01	0.01
II vs. III	0.04	0.001	0.001

GSH: Reduced glutathione, NO: nitric oxide, MDA: malondialdehyde.

^*p < 0.05 was considered to be statistically significant.

With the sham group, the mild lymphoid portal inflammation was the only histopathologic change. There was no ductular proliferation and fibrosis. In the bile duct ligated rats, there was marked bile duct proliferation with incomplete/complete nodule formation (scores 3) and mild to moderate portal lymphocytic inflammation (Figure 1). Pathologic examination of the kidneys in resveratrol-treated rats did not reveal any significant differences compared with those of the BDL group.

Figure 1 Marked ductuler proliferation with incomplete nodule formation (score 3) and moderate lymphocytic infiltration in the portal area in BDL group Masson Trickrom × 100.

Discussion

Several reports showed that oxidative stress associated with lipid peroxidation is involved in the development of kidney injury with extrahepatic cholestasis after bile duct ligation.[4], [17&18]

The present study indicates that intraperitoneal administration of resveratrol at a dose of 10 mg/kg per day markedly causes a decrease in MDA and NO values and an increase in GSH values in the renal tissue after bile duct ligation in rats. However, histopathologic improvement was not detected in kidney.

Lipid peroxidation was quantitated by measuring the production of MDA. In most tissues, MDA is formed as a result of the peroxidative decomposition of polyunsaturated fatty acids. Therefore, measurements of the amount of MDA provide an index of oxidative stress and lipid peroxidation.[19] In the present study, the levels of MDA in the BDL group were significantly higher than in the resveratrol-treated rats. Our results are in agreement with previous works reporting high levels of MDA.[4], [18], [20&21] In cholestasis, different mechanisms may contribute to oxidative stress in renal tissue. For instance, bile acids accumulate in the diseased liver and plasma of patients with cholestatic liver diseases[22] and are pro-oxidants, causing direct tissue damage mediated directly by ROS[23&24] or indirectly through activation of tissue-resident macrophages.[25] Bile acids can also accumulate intracellularly by virtue of their ability to bind to plasma lipoproteins, specifically low-density lipoproteins[26] and thus, low-density lipoproteins may play a major role in the transport of potentially toxic bile acids from plasma into the intracellular milieu of extrahepatic tissues to generate ROS when the integrity of the enterohepatic circulation is disrupted due to portosystemic shunts or bile acid spillover into the systemic circulation.[21] In the resveratrol-treated rats, levels of MDA were significantly lower than in the BDL group.

This effect of resveratrol is not clear, because antioxidant properties of resveratrol are well documented. The decreased MDA levels in the resveratrol-treated rats may be related to its antioxidant and free-radical scavenging effect.[27]

By protecting cell membranes, resveratrol probably reduces the deleterious effects of oxidative stress in living cells. Sun et al.[28] reported that resveratrol protected the cells from peroxidative stress and lipid peroxidation-induced cell death. Furthermore, Chanvitayapongs et al.[29] showed that resveratrol not only possessed antioxidant properties but could also reduce the cell death induced by oxidized lipoproteins.

GSH plays a pivotal role in the defense against oxidative stress, as a cofactor of glutathione peroxidases (selenium dependent and independent) participates in the elimination of hydrogen peroxide and lipid hydroperoxides.[30&31]

In the current study, levels of GSH in the BDL group were significantly lower than in the resveratrol-treated group. These results are concordant with previous studies.[4], [17] In contrast to our study, high levels of GSH were previously reported in bile duct ligated rats.[18], [20]

The decreased GSH levels in the BDL group might suggest that the kidney is unable to recycle reduced glutathione from oxidized glutathione by the glutathione reductase enzyme.[4] Additionally, GSH is inhibited by superoxide anion,[32] and lipid hydroperoxides may inactivate gluthione peroxidase (GPx), presumably by binding to the active site of the enzyme.[33]

The increased GSH levels in the resveratrol-treated group may be related to its antioxidant and free-radical scavenging effect. Another explanation of this significant increase in GSH levels seen in the resveratrol-treated rats is the effect of resveratrol upon the enzymes involved in glutathione synthesis, whereby resveratrol may maintain the levels of GSH during oxidative stress.[34] This increased GSH would be expected to reflect the fact that kidney tissue is better protected by resveratrol against oxidative damage, and that depletion of tissue GSH content enhances cellular injury caused by oxidative stress.

NO is a free radical synthesized from l-arginine by nitric oxide synthase (NOS) in biological systems.[35] Nitric oxide (NO) plays an important role as a mediator of the systemic and renal alterations of liver cirrhosis.[36] NO inhibition results in a reduction of the hyperdynamic circulation[37&38] and produces beneficial effects on renal excretory function.[39] In our study, increased production of NO in bile duct ligated rats is concordant with previous studies.[40&41] On the other hand, the levels of NO in resveratrol-treated rats, were significantly lower than those of the BDL group. The low levels of NO in resveratrol-treated rats may be due to inhibition of NO by resveratrol.[42] Resveratrol has peroxynitrite (ONOO⁻) scavenger effects.[43] Possible explanations for our findings include reduction of excessive nitric oxide formation or neutralization of NO, once formed; this would lower oxidative damage and, therefore, allow resveratrol to reduce renal oxidative stress after BDL in rats.

In conclusion, renal oxidative stress is observed in bile duct ligated rats. The present study demonstrates that intraperitoneal administration of resveratrol maintains antioxidant defenses and reduces kidney oxidative damage. This effect of resveratrol may be useful in lowering renal oxidative stress in patients with biliary obstruction. Nevertheless, more investigations are required to evaluate the antioxidant renal protective effect of resveratrol in clinical and experimental models.