Adult male Sprague-Dawley rats (n=120), weighing 200±20 g, were obtained from Xuzhou Medical College Laboratory Animal Center (Xuzhou, China). Rats were housed in standard stainless steel hanging cages with controlled temperature (22–25°C), humidity (35–50%), and photocycle (12-h light/dark) conditions. Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals published in P.R. China and with the approval of Xuzhou Medical Ethics Committee.

Rats were randomly divided into four equal groups: Control (CON; n=30), CIN-treated (CIN; n=30), normal rats treated with NAC (NAC; n=30), and CIN rats treated with NAC (NAC+CIN; n=30). A total of 10 mg/kg indomethacin (INDO), 10 mg/kg Nv-nitro-L-arginine methyl ester (both Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) and 2.9 g I/kg iohexol (Jiangsu Yangtze River Pharmaceutical Group Co., Ltd., Taizhou, China) was administered intravenously into the tail vein of rats in the CIN and NAC+CIN groups every 15 min, as described by Yokomaku et al (13). Rats in the NAC+CIN group received intraperitoneal injection of 10 mg/kg NAC (Sigma-Aldrich; Merck Millipore) 12 h prior to INDO treatment. Rats in the CON group received the same amount of saline injections into their abdomen and tail vein. Rats in the NAC group were pretreated with 10 mg/kg NAC by intraperitoneal injection, and the same amount of saline as delivered to the CIN group was administered 12 h later.

Rats were sacrificed at 2, 12, 24, 48 and 72 h by anesthetic overdose (10% chloral hydrate, 0.5 ml/100 g; Sangon Biotech Co., Ltd., Shanghai, China) after the procedure. Blood samples were harvested and centrifuged at 1,500 × g for 15 min at 4°C. Scr values was measured by an automatic biochemical analyzer (Olympus Corp., Tokyo, Japan), and the concentration of serum NGAL was evaluated via an enzyme linked immunosorbent assay (ELISA) with an ELISA kit (Westang Biotech, Shanghai, China).

Kidneys were harvested and separated into two pieces, one of which was fixed in 10% formalin for 24 h, and the other was kept at −80°C for subsequent western blot analysis and to study oxidative status. The fixed piece of kidney was processed and embedded in paraffin, cut into 4-µm-thick section, embedded in paraffin sections and stained with HE. Biopsies were examined by light microscopy in a blinded manner, and scored with a semi-quantitative scale designed to evaluate the degree of tubular injury, according to the tubular necrosis, tubular dilatation and/or atrophy, inflammatory cell infiltration, or cellular edema. Injury was graded on a scale: 0, normal kidney; 1, minimal damage (unicellular, patchy isolated damage); 2, mild damage (<25%); 3, moderate damage (25–50%); and 4, severe damage (>50%) (14). A minimum of 10 fields at ×400 magnification were assessed and graded in each biopsy.

Following deparaffinization, antigen retrieval was performed in citrate buffer. Non-specific binding was blocked with 0.3% H₂O₂ for 10 min and, after blocking for 30 min with 10% goat serum at room temperature, anti-NGAL antibody (1:150 in PBS; PB0641; Boster Biological Technology, Ltd., Wuhan, China) was applied overnight at 4°C. Samples were washed in buffer and incubated with the goat anti-rabbit IgG-Biotin secondary antibody (1:200 in PBS; BA1003; Boster Biological Technology, Ltd.) for 30 min, followed by the diaminobenzidine-peroxidase reaction and counterstaining with hematoxylin. Negative control sections were stained under identical conditions by omitting the primary antibody instead of PBS. Stained sections were subsequently visualized using light microscopy at ×200 magnification and the integrated optical density (IOD) was measured using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Total protein extraction was performed by homogenization and centrifugation at 1,500 × g for 20 min at 4°C, and the concentration of the protein was determined by the bicinchoninic acid assay (BCA) kit (Biyuntian Biotechnological Co., Shanghai, China). Protein samples were separated by 12% SDS-PAGE and transferred onto nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and blocked with 5% skimmed milk for 2 h. Primary antibodies against rabbit polyclonal anti-NGAL (1:1,000; ab63929; Abcam, Cambridge, UK) and sheep anti-mouse anti-GAPDH (1:500; sc-25778; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) were respectively added, and incubated at 4°C overnight. Following washing in TBST for 5 min three times, the membranes were then incubated with secondary antibody (1:5,000; P/N926-32231; ODYSSEY; LI-COR Biosciences, Inc., Lincoln, NE, USA) for 2 h in the dark. Protein bands were analyzed via an ODYSSEY Laser Imaging system (LI-COR Biosciences, Inc.).

Renal cortex was homogenized with ice-cold saline homogenization buffer, and centrifuged at 1,500 × g for 20 min at 4°C. The supernatant was analyzed by commercial malondialdehyde (MDA) and superoxide dismutase (SOD) kits (cat. nos. A003-1 and A001-3, respectively; Jiancheng Bioengineering Institute, China). MDA levels were measured via the thiobarbituric acid reactive substances method (15). SOD activity was measured by a method based on the nitro blue tetrazolium (NBT) reduction rate. One unit for SOD activity was expressed as the enzyme protein amount that was able to cause a 50% inhibition in NBT reduction rate (15). Sample absorbances were measured at 532 nm and 550 nm respectively. Protein levels of the supernatants were measured using a BCA assay kit.

Statistical analysis was performed using SPSS 17.0 software (SPSS Inc., Chicago, IL, USA). All data were expressed as mean ± standard deviation. Statistical comparisons were executed by one-way analysis of variance followed by Student-Newman-Keuls analysis. P<0.05 was considered to indicate a statistically significant difference.

At 2, 12 and 24 h following administration, there were no significant differences in the Scr values among the four groups (P>0.05). Scr levels significantly increased in the CIN group at 48 and 72 h (P<0.05), which was significantly higher than the NAC+CIN group at the same time point (P<0.05; Fig. 1). However, compared with the CON group, the value of serum NGAL in the CIN group had markedly increased, with a significant increase at 2 h after administration (P<0.05; Fig. 2). NGAL values in the NAC+CIN group were significantly decreased, as compared with the CIN group (P<0.05; Fig. 2).

In contrast-injected rats, vacuolar degeneration of tubular epithelial cells, tubular dilation, protein cast (protein deposition in the tubular lumen), tubular brush border loss, increased epithelial cell shedding, and necrosis of partial tubular epithelial cells were observed (Fig. 3). Semi-quantitative analysis demonstrated no difference between the renal injury scores of the NAC and CON groups. Renal injury scores in the NAC+CIN group were higher than that of the CON group, but significantly lower than that of the CIN group (P<0.05; Table I).

Immunohistochemistry findings demonstrated that NGAL was predominantly expressed in the cytoplasm of proximal tubular cells (Fig. 4). Compared with the CON group, the positive staining IOD value of NGAL significantly increased 2 h after administration in the CIN group (P<0.05; Table II), and was significantly higher than that of the NAC+CIN group at same time point (P<0.05; Table II).

According to the western blotting results, the expression levels of NGAL in the kidney tissue of the CIN group began to significantly increase 2 h after the procedure (P<0.05), and were significantly decreased in the NAC+CIN group at the same time point (P<0.05). No significant differences were detected between the NAC and CIN groups (P>0.05; Fig. 5).

As shown in Tables III and IV, a significant decline of kidney SOD was observed in CIN rats compared with the CON group (P<0.05), and there was also a significant decline between the CIN and NAC+CIN groups (P<0.05). This was accompanied by an increase of MDA in the CIN group, which was significantly higher than the other three groups (P<0.05).