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Mar 27, 2008 - Abstract. Background Renal ischemia/reperfusion (I/R) injury is a major cause of acute renal failure. Silymarin is extracted from Silybum ...
Int Urol Nephrol (2008) 40:453–460 DOI 10.1007/s11255-008-9365-4

ORIGINAL ARTICLE

Antioxidant and protective effects of silymarin on ischemia and reperfusion injury in the kidney tissues of rats Faruk Turgut Æ Omer Bayrak Æ Ferhat Catal Æ Reyhan Bayrak Æ Ali Fuat Atmaca Æ Akif Koc Æ Ali Akbas Æ Ali Akcay Æ Dogan Unal

Received: 21 June 2007 / Accepted: 4 March 2008 / Published online: 27 March 2008 Ó Springer Science+Business Media B.V. 2008

Abstract Background Renal ischemia/reperfusion (I/R) injury is a major cause of acute renal failure. Silymarin is extracted from Silybum marianum and Cynara cardunculus seeds and fruits. The aim of this study is to investigate whether silymarin administration prevents the damage induced by I/R in rat kidneys. Materials and methods Thirty male Wistar rats were randomly divided into five experimental groups

F. Turgut (&)  A. Akcay Department of Nephrology, Fatih University, School of Medicine, Hosdere cad no: 145, Y. Ayranci, 06540 Ankara, Turkey e-mail: [email protected] O. Bayrak  A. Koc  D. Unal Department of Urology, Fatih University, School of Medicine, Ankara, Turkey F. Catal Department of Pediatrics, Fatih University, School of Medicine, Ankara, Turkey R. Bayrak Department of Pathology, Fatih University, School of Medicine, Ankara, Turkey A. F. Atmaca First Urology Clinic, Ataturk Training and Research Hospital, Ankara, Turkey A. Akbas Department of Biochemistry, Gaziosmanpasa University, Medical Faculty, Tokat, Turkey

(n = 6, each) as follows; control group, sham-operated group, I/R group, silymarin group, and I/R + silymarin group. In the I/R and I/R + silymarin groups, both renal arteries were occluded using nontraumatic microvascular clamps for 45 min. Then, at the end of 24 h of reperfusion, the animals were killed. Kidney function tests, the serum and tissue antioxidant enzymes and oxidant products were determined. Results Animals that were subjected to I/R exhibited significant increase in serum urea, creatinine, and cystatin C levels compared with the rats treated with silymarin prior to the I/R process (P \ 0.001). The serum enzymatic activities of superoxide dismutase and glutathione peroxidase significantly decreased in the I/R group; however, this reduction was significantly improved by the treatment with silymarin (P \ 0.001 and P \ 0.05, respectively). Renal I/R produced a significant increase in serum and tissue malondialdehyde, nitric oxide, and protein carbonyl as compared with controls. Treatment with silymarin resulted in significant reduction in these markers (P \ 0.001). Conclusion Based on our findings, silymarin protects the kidneys against I/R injury. This finding may provide a basis for the development of novel therapeutic strategies for protection against the damages caused by I/R. Keywords Free radicals  Ischemia  Kidney  Oxidants  Reperfusion  Silymarin

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Introduction Acute renal failure (ARF) is among the most important complications observed in hospitalized patients. Despite decades of laboratory and clinical investigations, the overall mortality rate associated with ARF has changed little [1]. Renal ischemia/reperfusion (I/ R) injury, a major cause of acute renal failure, is frequently associated with shock or surgery, and is unavoidable in renal transplantation [2]. Ischemia and reperfusion may injure tubular cells by activating inflammatory pathways and generating free radicals. Inflammatory cells may augment renal injury in part by the release of oxidants and other vasoactive mediators [3]. Under normal conditions, the continuous production of free radicals is compensated by the powerful action of protective enzymes such as superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px), which are believed to be major antioxidant enzymes present in the human body. Antioxidants provide necessary defense against the oxidative-stress-induced damages. Given the clinical importance of renal I/R injury, scientists have sought to develop renoprotective therapy for patients with high risk of ARF. Silymarin is a poliphenolic compound extract from Silybum marianum and Cynara cardunculus seeds and fruits. It has been demonstrated that silymarin acts as an antioxidant, reducing free-radical-mediated damage in tissues and inhibiting lipid peroxidation [4]. It has been reported as having multiple pharmacological activities including antioxidant, hepatoprotectant and anti-inflammatory agent, antibacterial, antiallergic, antiviral, and antineoplastic effects [5–9]. The aim of our study is to investigate whether silymarin administration prevents the functional and structural damage induced by I/R in rat kidneys in vivo.

Materials and methods Animals Thirty male Wistar rats (150–200 g) were maintained under conditions with controlled temperature (22 ± 2°C) and relative humidity (65–70%) in a 12 h light–dark cycle and fed ad libitum with commercially available rat chow and water. The

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animals were housed at six animals per cage (50 9 35 9 20 cm) and had free access to water and diet. All animals were adapted to handling and the metabolic cages repeatedly during a 5-day period prior to the experiments. This study was approved by the Animal Ethics Committee of Fatih University, School of Medicine (date; 07.06.2007, meeting number; 12) and performed in accordance with the guidelines of the Research Committee of Fatih University. Experimental design Rats were randomly divided into five experimental groups (n = 6, each) as follows: control group (nonoperated rats), sham group (sham-operated; animals underwent exposure of both the renal pedicles but were not subjected to any I/R), I/R group (animals were subjected to 45 min of bilateral ischemia followed by 24 h of reperfusion), silymarin group (animals received only silymarin and were not exposed to any I/R), and I/R + silymarin group (animals received silymarin and subjected to 45 min of bilateral ischemia followed by 24 h of reperfusion). Silymarin administration Silymarin (Mikrogen Pharmaceuticals, Istanbul, Turkey), used in this study, is a dried extract of Silybum marianum. Silymarin, suspended in 0.5% methyl cellulose, was administered orally by feeding tube to six rats each of the silymarin and I/R + silymarin groups, at a dose of 100 mg/kg for 7 days before I/R. Silymarin was not given after the I/R procedure. Surgical procedure Following a 12 h fasting period, rats underwent surgery, using ketamine HCl anesthesia (50 mg/kg); anesthesia was maintained by supplementary injections of thiopental sodium as required. The abdominal region was sterilized with povidine iodine solution and the abdomen was entered through a midline minimal incision and both kidneys were isolated. In the sham group, the abdomen was closed without any further procedure. In the I/R and I/R + silymarin administered groups, both renal arteries were occluded using nontraumatic microvascular clamps for 45 min and

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occlusion of blood flow was confirmed by visual inspection of the kidneys. After declamping, we confirmed that renal blood flow had been restored prior to closing the incision. Body temperature was maintained close to 37.5°C with a heating lamp throughout the experiment. Fluid loss was replaced by the administration of 5 ml warm (37°C) saline intraperitoneally before abdominal closure in all rats. At the end of 24 h of reperfusion, the abdomen was reopened, 3 ml blood was drown from the heart and in the process the animal was killed. Blood samples were collected in heparinized microcentrifuge tubes and serum was isolated and stored at -70°C for subsequent analyses. Then, bilateral nephrectomies were performed. The right kidney was immediately stored at 70°C to use for further enzymatic analysis. The left kidney was stored in 10% formalin for histological examination. The same part of each kidney was used consistently for the same determinations in all groups. All specimens were coded in the research laboratory and were evaluated by the same individuals, who were blinded to group assignments. Biochemical analyses Serum urea, creatinine, and cystatin C concentrations were determined with an Abbott-Aeroset autoanalyzer (Chicago, IL, USA) using the original kits. Assays for antioxidant enzymes and oxidant products Tissue samples were homogenized in five volumes of ice-cold Tris–HCl buffer (50 mM, pH 7.4) containing 0.50 ml 1-1 Triton x-100; homogenization (homogenizer: IKA Ultra-Turrax t 25 Basic, Germany) was carried out for 2 min at 13,000 rpm. All procedures were performed at 4°C. Homogenate, supernatant, and extracted samples were prepared and the following determinations were made on the samples using commercial chemicals supplied by Sigma (St Louis, USA). In the kidney tissue samples, total (Cu–Zn and Mn) SOD activity was determined according to the method of Sun et al. [10]. The principle of the method is based on inhibition of nitroblue tetrazolium (NBT) reduction by the xanthine–xanthine oxidase system as a superoxide generator. Activity was

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assessed in the ethanol phase of the supernatant after 1.0 ml of ethanol–chloroform mixture (5:3, v/v) was added to the same volume of sample and centrifuged. One unit of SOD was defined as the amount causing 50% inhibition in the NBT reduction rate. The SOD activity is expressed as U mg-1 protein. The GSH-Px activity was measured by the method of Pagia and Valentine [11]. The enzymatic reaction in the tube—containing nicotineamide adenine dinucleotide phosphate (NADPH), reduced glutathione, sodium azide, and glutathione reductase—was initiated by addition of H2O2 and the change in absorbance at 340 nm was monitored by a spectrophotometer. Activity is expressed as U g-1 protein. The catalase (CAT) activity was determined according to Aebi’s method [12]. The principle of the method was based on determination of the rate constant k (s-1) of the H2O2 decomposition at 240 nm. Results are expressed as k g-1 protein. All samples were assayed in duplicate. NO measurement is very difficult in biological specimens; therefore tissue nitrite (NO2-) and nitrate (NO3-) were estimated as an index of NO production. Samples were initially deproteinized with Somogyi reagent. Total nitrite (nitrite + nitrate) was measured after conversion of nitrate to nitrite by copperized cadmium granules by a spectrophotometer at 545 nm [13]. Results were expressed as nmol/g wet tissue. The protein carbonyl contents were determined spectrophotometrically (Cintra 10 E, Austria) based on reaction of carbonyl group with 2,4-dinitrophenylhydrazine to form 2,4-dinitrophenylhydrazone [14]. The results were given as nanomoles of carbonyl per milligram of protein. The tissue thiobarbituric acid-reactive substance level was determined based on reaction with thiobarbituric acid (TBA) at 90–100°C [15]. In the TBA test reaction, malondialdehyde (MDA) or MDA-like substances and TBA react to produce a pink pigment with an absorption maximum at 532 nm. The reaction was performed at pH 2–3 and 90°C for 15 min. The sample was mixed with two volumes of cold 10% (w/v) trichloroacetic acid to precipitate the protein. The precipitate was pelleted by centrifugation and an aliquot of the supernatant was reacted with an equal volume of 0.67% (w/v) TBA in a boiling water bath for 10 min. After cooling, the absorbance was read at 532 nm. Results were expressed as nmol/g wet tissue,

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according to the standard graphic prepared from measurements with a standard solution. Estimation of serum total antioxidant capacity (TAC) and total oxidant status (TOS) TAC of serum was determined using a novel automated measurement method, developed by Erel [16]. Using this method, the antioxidative effect of the sample against the potent free radical reactions, which is initiated by the produced hydroxyl radical, is measured. The assay has excellent precision values lower than 3%. The results are expressed as mmol Trolox equiv./l. TOS of serum was determined using a novel automated measurement method, developed by Erel [17]. In this method, oxidants, present in the sample, oxidize the ferrous ion–o-dianisidine complex to ferric ion. The oxidation reaction is enhanced by glycerol molecules, which are abundantly present in the reaction medium. The ferric ion makes a colored complex with xylenol orange in an acidic medium. The color intensity, which can be measured spectrophotometrically, is related to the total amount of oxidant molecules present in the sample. The assay is calibrated with hydrogen peroxide and the results are expressed in terms of micromolar hydrogen peroxide equivalent per liter (lmol H2O2 equiv./l). Histopathological analysis Kidneys, decapsulated and fixed in a 10% neutral buffered formalin solution, were embedded in paraffin. Then, they were used for histopathological analysis. Five-micron-thick sections were cut by microtome, stained with H&E. An experienced pathologist who was unaware of the treatment conditions made histological evaluation under a light microscope. Histological changes were evaluated by quantitative measurements of tubular cell necrosis in ten separate fields in the outer medulla (4009). A numerical score was used to define the degree of tubular cell damage: 0, no damage; 1, unicellular, patchy isolated necrosis; 2, tubular necrosis less than 25%; 3, tubular necrosis between 25 and 50%; 4, more than 50% tubular necrosis and presence of infracted tissue.

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Statistical analyses All statistical analyses were performed using SPSS for Windows, version 11.5 (SPSS, Chicago, IL, USA). Data were analyzed using analysis of variance (ANOVA) followed by Bonferroni’s post test. The Kruskal Wallis one-way analysis of variance by ranks was used for a simultaneous statistical test of the pathologic score for I/R and I/R + silymarin groups. When the null hypothesis could be rejected, comparisons between the two groups were made with the Mann–Whitney U nonparametric test for independent samples.

Results Kidney function tests Table 1 shows the serum urea, creatinine, and cystatin C levels of all groups. Animals subjected to renal ischemia exhibited significant increase in serum urea, creatinine, and cystatin C levels compared with the control, sham, and silymarin groups, suggesting a significant decrease of glomerular function caused by renal I/R injury. However, the rats treated with silymarin had significantly lower levels of the serum urea, creatinine, and cystatin C compared with the I/R group (P \ 0.01). After renal I/R process, the serum enzymatic activities of SOD and GSH-Px significantly decreased in the I/R group. This reduction was significantly improved by the treatment with silymarin in the rats treated with silymarin (P \ 0.001 and P \ 0.05, respectively) (Table 2). The I/R process Table 1 Renal function parameters Groups

n

Urea (mg/dl)

Creatinine (mg/dl)

Cystatin C (mg/dl)

C

6

16.6 ± 1.3

0.6 ± 0.18

0.13 ± 0.02

S

6

17.1 ± 1.4

0.5 ± 0.06

0.18 ± 0.01

I/R

6

86.8 ± 8.5*

2.0 ± 0.21*

0.31 ± 0.07*

Silymarin

6

13.0 ± 0.6

0.4 ± 0.13

0.12 ± 0.01

I/R + silymarin

6

41.1 ± 13.1

1.4 ± 0.83

0.24 ± 0.05

C; control, S; sham operated, I/R; ischemia/reperfusion * P \ 0.001 compared R + silymarin

with

C,

S,

silymarin

and

I/

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Table 2 Serum malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), nitric oxide (NO), and protein carbonyl (PC) levels Groups (n = 6)

C

MDA

S

0.24 ± 0.02

SOD

I/R

0.30 ± 0.12

Silymarin

0.49 ± 0.08*

0.33 ± 0.01

I/R + silymarin 0.31 ± 0.04

4.16 ± 0.2

2.84 ± 0.5

2.03 ± 0.2*

3.01 ± 0.3

3.1 ± 0.1

2,108 ± 100

2,059 ± 135

1,630 ± 124**

1,929 ± 231

1,834 ± 72

NO

47.1 ± 5.1

56.3 ± 5.9

84.1 ± 7.5*

55.5 ± 5.3

59.5 ± 8.6

PC

669 ± 51

837 ± 63

981 ± 98*

667 ± 88

741 ± 78

GSH-Px

C; control, S; sham operated, I/R; ischemia/reperfusion * P \ 0.001 compared with C, S, silymarin, and I/R + silymarin; ** P \ 0.05 compared with C, S, silymarin, and I/R + silymarin

also caused significant decreases in tissue enzymatic activity of CAT when compared with the rats treated by silymarin prior to the process (P \ 0.05). Additionally, tissue SOD and GSH-Px activities decreased after I/R process; however, the difference was not significant statistically between the I/R and I/R + silymarin groups (Table 3). In addition, renal ischemia and reperfusion produced a significant increase in serum and tissue MDA, NO, and PC as compared with control and sham-operated animals. Treatment with silymarin resulted in significant reduction in these markers (P \ 0.001 for tissue and serum levels) (Tables 2, 3). As illustrated in Table 4, TAC was lower, while TOS was higher in I/R group than in the control, sham, and

silymarin groups (P \ 0.001). On the other hand, pretreatment with silymarin before I/R injury resulted in higher TAC and lower TOS levels than in the I/R group (P \ 0.001). In the control group, renal tissue sections had a normal morphology (Fig. 1). As expected, no morphological damage was observed in the shamoperated and silymarin groups. However, histological examination of the kidneys exposed to I/R showed the distinctive pattern of ischemic renal injury, which included widespread degeneration of tubular architecture, loss of brush border, sloughing tubular epithelial cells from the basement membrane, tubular cell necrosis, and intratubular cast formation especially in the outer medulla (Fig. 2).

Table 3 Enzyme activities in renal tissue Groups

Catalase

SOD

GSH-Px

NO

PC

MDA

C

0.59 ± 0.07

0.13 ± 0.32

0.14 ± 0.04

0.12 ± 0.03

0.70 ± 0.1

2.90 ± 0.2

S

0.46 ± 0.07

0.12 ± 0.18

0.14 ± 0.01

0.19 ± 0.02

1.11 ± 0.1

2.45 ± 0.2

I/R

0.23 ± 0.03**

0.03 ± 0.00

0.09 ± 0.01

0.28 ± 0.03*

2.10 ± 0.2*

4.77 ± 0.4*

Silymarin

0.47 ± 0.17

0.05 ± 0.00

0.10 ± 0.02

0.08 ± 0.02

0.79 ± 0.1

2.92 ± 0.7

I/R + silymarin

0.30 ± 0.05

0.04 ± 0.00

0.10 ± 0.02

0.11 ± 0.02

0.87 ± 0.3

3.37 ± 0.7

C; control; S; sham; I/R; ischemia/reperfusion; SOD; superoxide dismutase, GSH-Px; glutathione peroxidase, NO; nitric oxide, PC; protein carbonyl, MDA; malondialdehyde * P \ 0.001 compared with C, S, Silymarin, I/R + silymarin; ** P \ 0.001 compared with C, S, Silymarin Table 4 The serum total antioxidant capacity (TAC) and total oxidant status (TOS) of the groups Control TAC (mmol Trolox equiv./l) TOS (lmol H2O2 equiv./l)

3.9 ± 0.09 19.12 ± 1.6

Sham 2.7 ± 0.07 22.08 ± 1.0

I/R 1.6 ± 0.05* 39.86 ± 1.3*

Silymarin 2.8 ± 0.10 15.1 ± 2.2

I/R + silymarin 2.3 ± 0.07** 21.3 ± 1.9**

I/R; ischemia/reperfusion * P \ 0.001 as compared with the control, sham, and silymarin groups; ** P \ 0.001 as compared with the I/R group

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Renal sections obtained from the rats pretreated with silymarin demonstrated marked reduction of the histological features of renal injury, consisting of more focal and mild tubular necrosis (Fig. 3). Semiquantitative assessment of the histological changes were graded and I/R group had significantly higher score than I/R + silymarin group (2.01 ± 1.61 versus 3.61 ± 0.72) (Fig. 4).

Discussion

Fig. 1 Renal tissue section with a normal morphology from the control group

Fig. 2 Renal tissue section with the distinctive pattern of ischemic renal injury from the rat only subjected to I/R process

Fig. 3 Renal tissue section consisting of more focal and mild tubular necrosis from the rat treated with silymarin prior to the I/R process

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In the present study, we observed that silymarin treatment protects rat kidney against I/R injury. Salient findings of this study are that: (1) kidney function test results are significantly better in the rats treated with silymarin prior to I/R process than the rats only subjected to the I/R process, (2) the serum and tissue antioxidant enzymes levels are significantly high in the I/R + silymarin group compared with the I/R group, and (3) the tissue oxidant product levels were significantly lower in I/R + silymarin group than I/R group. ARF with an increasing prevalence is still associated with high mortality in humans [18]. Current therapy is limited to supportive measures and preventive strategies, none of which have been definitively shown to alter mortality. Ischemic ARF frequently occurs in hospitalized patients. Oxidative stress has been implicated in the pathogenesis of ischemic ARF [19, 20]. In the present study, we have shown that kidney function tests including urea, creatinine, and cystatin C significantly increased after

Fig. 4 Semiquantitative assessment of the histological changes in the ischemia/reperfusion (IR) and I/R + silymarin groups

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I/R process. Silymarin partly ameliorated I/R-induced alterations in parameters associated with glomerular function. Silymarin possesses anti-oxidant properties that have already been elucidated in hepatocytes challenged with a variety of radical-generating drugs [8, 9, 21]. GSH-Px, CAT, and SOD are well-known enzymatic antioxidants. In the present study, the decrease in GSH-Px, SOD, and CAT and increase in MDA and PC after I/R procedure compared with controls suggests an oxidative type of injury after I/R process. In a previous study, Soto et al. reported that, the protective effect of silymarin on pancreatic damage induced by alloxan may be due to an increase in the activity of antioxidant enzymes (SOD, GSH-Px) [22]. Sanhueza et al. demonstrated that silybin has protective effect against I/R injury by measuring the xanthine dehydrogenase/xanthine oxidase ratio in the rat kidney [23]. Oliveira et al. reported that silymarin protects liver against I/R injury [24]. In agreement with these previous studies, we observed that silymarin has antioxidant effect and protect kidney against I/R injury. In previous studies, lipid peroxidation is also implicated in the pathogenesis of postischemic tissue injuries [25, 26]. MDA is known a marker for lipid peroxidation [27]. Consistent with previous studies [25–27], we have found significantly increased both the serum and tissue activities of MDA in the rats subjected to I/R process. On the other hand, the rats given orally silymarin had significantly lower MDA levels. Increased NO level has been implicated in increased cellular effects of reactive oxygen species due to peroxynitrite [28]. Treatment with silymarin has also significantly decreased NO level compared with the rats only subjected to I/R in our study. Administration of silymarin increased antioxidants and decreased oxidants, so it has antioxidant activity in the present study. Oxidants and antioxidant capacity may be measured simultaneously to assess oxidative stress more exactly. In the present study, we observed that serum TOS levels were significantly higher and TAC levels were significantly lower in the rats subjected to I/R as compared with controls. However, pretreatment with silymarin resulted in lower TOS and higher TAC levels. A functional study such as the one presented here can only give limited evidence as to the mechanisms of damage or protection. Morphological examinations

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at the light microscopical level were performed to supplement the data derived from functional studies in the present study. Pretreatment with silymarin partly or totally protected the kidneys from I/R injury, and renal sections obtained from the rats pretreated with silymarin demonstrated marked reduction of the histological features of renal injury compared with the rats only subjected to I/R process. Furthermore, silymarin alone had no effect on renal morphology. In summary, our results allow us to draw the conclusion that silymarin would protect the kidneys against I/R injury. The protective effect of silymarin is associated with its antioxidant properties, as it possibly acts as a free-radical scavenger, lipid peroxidation inhibitor. These findings provide a basis for the development of novel therapeutic strategies, such as antioxidant supplementation with a flavonoid silymarin, for protection against the damages caused by ischemia and reperfusion.

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