TNF-α ELISA kit was purchased from Diaclone (Besançon, France). SOD and GPx kits were from Randox Lab (Crumlin, UK). Antibody against IL-1β and horseradish peroxidase conjugated secondary antibody were from Zellbio GmbH (Germany). p-Coumaric acid (purity >98%) and other reagents were obtained from Sigma–Aldrich (St. Louis, Mo, USA).

Thirty Sprague-Dawley rats weighing from 190 to 210g were purchased from the animal house of Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran. The rats were kept in controlled temperature (22±2°C) with humidity 50–55% and under 12h light/12h dark cycle. During the period of experiment the animals were permitted free access to water and a conventional diet ad libitum.

The rats were randomly divided into 5 groups of 6 rats each.

Group I (control), animals received distilled water.

Group II (CA), animals received p-cumaric acid (100mg/kg/d).

Group III (I/R), animals were exposed to I/R.

Group IV (Vehicle+I/R), animals in this group were given propylene glycol 10% and were exposed to I/R.

Group V (CA+I/R), animals in this group were treated with p-cumaric acid (100mg/kg/d) followed by I/R.

CA was dissolved in 10% propylene glycol and also, all treatments were oral. Animals were pre-treated for 2 weeks and then were subjected to I/R.

All groups except those in groups I and II were anesthetized intraperitoneally with ketamine (75mg/kg) and xylazine (10mg/kg), a midline incision was made, and both left and right renal pedicles were identified and occluded with two smooth atraumatic vascular clamps for 45min. After 45min, the vessel clamps were removed and the kidneys were checked for blood flow restoration. After unclamping of the renal pedicles, the abdominal wall was sutured and the animals were placed in individual metabolic cages. Animals in groups I and II endured a similar surgical procedure without renal pedicle occluding. After reperfusion for 24h, rats were anesthetized, blood and kidney samples were taken for more studies.

Blood samples were collected from right ventricular and then centrifuged with 6000×g for 3min. Concentrations of blood urea nitrogen (BUN) and serum creatinine (Cr) were measured spectrophotometrically using a commercially available kit (parsazmon kit, Iran).

Right kidneys were cut into two halves. One half of kidneys weighed and homogenized 1:20 in 0.1M potassium phosphate buffer (pH 7.4) then, homogenates were centrifuged at 16,000×g for 20min at 4°C. The supernatants were used for measurement of biomarkers of oxidative stress include: MDA, SOD, CAT and GPx.

MDA level in renal tissue was determined by thiobarbituric acid reactivity as previously described.17 An aliquot (500μl) of the supernatant to formation of protein precipitate was added to 1500μl trichloroacetic acid (10%) and centrifuged at 4000×g for 10min and 1500μl of supernatant was mixed with 2ml thiobarbituric acid (0.67%). Then the mixture was boiled for 30min at 100°C. After cooling, centrifuged at 4000×g for 15min and the supernatant absorbency was read at 535nm by spectrophotometry. Values were expressed as μmol/mg protein, based on the standard curve prepared from standard solution of MDA.

SOD activity was measured using Ransod kit (Randox Labs, Crumlin, UK). This method uses xanthine oxidase and xanthine to produce superoxide radicals that react with 2-(4-iodophenyl)-3-(4-nitrophenol)-5-phenyltetrazolium chloride (INT) to create a red formazan. One unit of SOD was described as the amount causing 50% inhibition in the INT reduction rate. SOD activity is expressed as units/mg protein.

GPx activity was measured using Ransel kit (Randox Labs, Crumlin, UK). GPx accelerates the oxidation of glutathione by cumene hydroperoxide. The oxidized glutathione in the presence of NADPH and glutathione reductase, changed to the diminished form associated with oxidation of NADPH to NADP+. The reduce in absorbance was determined at 340nm with a spectrophotometer. Activity is expressed as U/mg protein.

CAT activity was determined according to the method that described previously.18 Briefly, 2.9ml of H2O2 (19mM) in phosphate buffer (50mM, pH 7.0) was placed in a cuvette and the reaction started by adding supernatant (100μml). The changes of absorbance per min was recorded at 240nm. CAT activity was determined by using the extinction coefficient of 0.0436cm2μmol−1 H2O2. The CAT activity was calculated as U/mg protein.

Measurement of protein concentration carried out by Bradford's method using bovine serum albumin as protein standard.19

Left kidneys from rats of all the groups were fixed in 10% formaldehyde, dehydrated in graded ethanol and embedded in paraffin and fine (5μm) sections were prepared and mounted on glass slides. The sections were stained with PAS (periodic acid schiff) for light microscopy (Olympus, Tokyo, Japan) evaluation at 300× magnification. Histological injury was analyzed using a tubular injury score.20 Briefly, injury was scored in a blinded method based on the injury percentage included tubular dilatation, destroy of brush border, interstitial tissue hemorrhage and intratubular cast: 0, no injury; 1, <25%; 2, 25–50%; 3, 50–75%; 4, >75%. Histological damage was examined in 10 microscopic fields.

TNF-α concentrations were measured in the kidney tissue21 using enzyme-linked immunosorbent assay (ELISA) kit specific for rats according to the manufacturer's instructions. 100mg of renal tissue was homogenized in 1ml of PBS contain anti-protease cocktail for preventing of protein destruction. The samples were then centrifuged at 6000×g for approximately 10min and the supernatant used for ELISA assay.

Detection of IL-1β in kidney sections was determined by immunohistochemistry as previously described.22 Briefly, sections were incubated overnight with primary antibody (rabbit anti-IL-1β at the dilution 1:400) at 4°C. Negative controls were incubated with normal serum instead of primary antibody. After washing with PBS sections were incubated with secondary conjugated antibody (goat anti-rabbit IgG/HRP conjugate) for 60min and then were washed with PBS. Finally, location of antigen-antibody complex was revealed by chromogen diaminobenzidine. The results of the immunohistochemical staining were categorized into 4 categories namely light (+), moderate (++), severe (+++) and very severe (++++). For each slide, 15 different microscopic fields were evaluated.

Data were expressed as mean±SEM. Study groups data were compared with one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. P values <0.05 was considered to be significant.

Animals in groups of I/R and Vehicle+I/R exhibited significant increase in the serum concentrations of Cr (p<0.005) and BUN (p<0.001) levels as compared to control animals. Pretreatment with CA (Group V) significantly improved the Cr and BUN levels (p<0.005) and (p<0.001). The Cr and BUN levels of CA group (Group II) were similar to control group (p>0.05) (Table 1).

As illustrated in Table 2, there were statistically significant increase in renal tissue MDA level of I/R and Vehicle+I/R groups compared to control (p<0.01). Pretreatment with CA significantly improved MDA concentration (p<0.05). Also, the activities of antioxidant enzymes (SOD, GPx, CAT) decreased in groups of I/R and Vehicle+I/R compared to control group (p<0.001). Pretreatment with CA significantly increased antioxidant enzymes activity in Group V (p<0.01, p<0.01 and p<0.05). But, MDA concentration and antioxidant enzymes activity in CA group were similar to control group.

In the control group, renal tissue had a normal structure. No significant pathological changes were observed in the CA group. Tissue from the I/R and Vehicle+I/R groups revealed, tubular lumen dilation, vacuolization of tubule cells, degeneration of some of tubules and the accumulation of castes in the tubular lumen. Tissue sections from rats pretreated with CA showed marked decline of the structural features of renal damage (Fig. 1A). The histopathological changes were graded (Fig. 1B) and the kidneys of I/R and Vehicle+I/R rats had a score of 3.2±0.2 and 3.8±0.2 while treatment with CA nearly preserved normal structure of the kidney with a score of 1.2±0.37 (p<0.001).

Fig. 1.

(A) Light micrographs of the rats kidney sections (PAS). Group I (control), received distilled water; Group II (CA), received p-cumaric acid (100mg/kg/d); in Group III (I/R), rats were exposed to I/R; in Group IV (Vehicle+I/R), rats received propylene glycol 10% and were exposed to I/R for 45min and in Group V (CA+I/R), rats were treated with p-cumaric acid (100mg/kg/d for 2 weeks) followed by I/R. Kidney sections from control and p-coumaric acid (CA) groups with normal renal structure (I, II), I/R and Vehicle+I/R groups show severe renal injury such as; tubular lumen dilation, vacuolization of tubule cells, degeneration of some of tubules and The accumulation of castes in the tubular lumen (III, IV), the rats treated with CA prior to I/R process show relatively well-preserved structure (V). (B) Renal injury was scored. Groups III and IV compared to control and Group V compared to Group III. Values are shown as mean±SEM; *p<0.001 (n=6).

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TNF-α level was significantly higher in groups of I/R and Vehicle+I/R compared with the control group (p<0.001). The TNF-α level were significantly lower in CA+I/R group than group I/R (p<0.001), however there was no significant difference between groups of control and CA. (Fig. 2).

Fig. 2.

Effect of CA pretreatment on renal TNF-α in different groups. Groups III and IV compared to control and Group V compared to Group III. Values are shown as mean±SEM; *p<0.001 (n=6).

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Staining for IL-1β by immunohistochemistry demonstrated that the expression of IL-1β was increased after IR in groups of III and IV when compared to control group (p<0.001). A marked reduction in the staining for IL-1β was observed in kidneys obtained from CA+I/R group when compared with kidneys from I/R group (p<0.005). There was no significant difference in IL-1β expression between groups of control and CA (Fig. 3A). Immunoreactivity degrees of IL-1β is summarized in Fig. 3B.

Fig. 3.

Effect of CA pretreatment on IL-1β expression in kidney. (A) Immunohistochemistry for IL-1β demonstrated that the expression of IL-1β was increased after IR in groups of ІІІ and IV when compared to control group (p<0.001). A marked reduction in the IL-1β expression was observed in kidney tissues of CA+I/R group when compared with kidneys from I/R group. (B) Expression degree of IL-1β. Groups III and IV compared to control and Group V compared to Group III. Values are shown as mean±SEM; *p<0.001, **p<0.0005 (n=6).

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