Creatinine (Cr) assay kit (R&D Systems, Minneapolis, MN, USA); blood urea nitrogen (BUN) assay kit (R&D Systems); xanthine oxidase assay kit used to determine the activity of superoxide dismutase (SOD; R&D Systems); TUNEL kit (R&D Systems); dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis, MO, USA), TRIzol kit (Invitrogen, Carlsbad, CA, USA), reverse transcription kit (Invitrogen); rabbit anti-Bax-beta, rabbit anti-Bax and rabbit anti-GAPDH primary antibodies, and horseradish peroxidase labeled anti-rabbit secondary antibody (Cell Signaling Technology, Danvers, MA, USA); ECL luminescent substrate (Invitrogen); color developing powder (Invitrogen); pipettes (Eppendorf, Hamburg, Germany); PCR instrument (Applied Biosystems, Inc., Foster City, CA, USA); UV imaging system (Biometra GmbH, Göttingen, Germany); electronic balance (Sartorious BP121S; Sartorius AG, Goettingen, Germany); −80°C refrigerator (Thermo Fisher Scientific, Schwerte, Germany); low temperature centrifuge (Thermo Fisher Scientific) and frozen slicers (Leica Microsystems, Wetzlar, Germany). The sources of other related instruments and reagents are described in the relevant section.

Male Sprague-Dawley (SD) rats (220–250 g) were purchased from the Shanghai Medical Laboratory Animal Center. Laboratory animal certificate number: SCXK (Shanghai) 2013–0015. Rats were raise in quiet environment with free access to food and water following the circadian rhythm for 1 week to adapt to the environment. Thirty-six SD rats were randomly divided into three groups (n=12) including sham operation (S) group, ischemia-reperfusion group (I/R) group and ischemic preconditioning (IP) group.

Rats were fasted for 12 h before surgery. After anesthesia with intraperitoneal injection of 4% chloral hydrate at a dose of 10 ml/kg, abdominal hair was removed and an incision was made on abdomen along the mediventral line to open abdominal cavity. Renal capsule was bluntly separated to separate the kidneys. Arterial clip was then used to clip bilateral kidney pedicles. The dark red color of kidneys indicated the successfully established ischemia model. In I/R group, bilateral renal pedicles were clipped for 45 min, followed by perfusion for 6 h to establish I/R model. Both kidneys in rats of S group were separated and exposed for 45 min, but renal pedicles were not clipped. In IP group, bilateral renal pedicles were clipped for 5 min, followed by perfusion for 5 min, this procedure was repeated 3 times, after that rats were subjected to the treatment described in the I/R group.

Peripheral blood was collected after reperfusion for 6 h. Blood samples were centrifuged 2,650 × g for 15 min to collect supernatant. Supernatant was transferred to tubes and stored at −80°C. After blood sample collection, rats in each group were sacrificed, and both kidneys were separated and fixed in 10% formalin for subsequent pathologic examination and other studies.

After H&E staining, pathological changes were observed under optical microscope (BX-42; Olympus, Tokyo, Japan) and scores of renal pathological damage were evaluated. Scoring was performed according to the standard of renal injury: normal renal tissue, 0 point; area of renal tissue damage in renal tubular area <25%, 1 point; area of renal tissue damage in renal tubular between 25 and 50%, 2 points; area of renal tissue damage in renal tubular between 50 and 75%, 3 points; area of renal tissue damage in renal tubular between >75%, 4 points.

Contents of Cr and BUN in serum of rats in each group were determined according to the instructions of the Cr assay kit and BUN assay kit. Cr content was expressed as number + µmg/ml and BUN content expressed as number + mmol/ml. SOD activity in serum of rats in each group was determined according to the instructions of SOD activity assay kit and expressed as number + U/mg.

TUNEL assay was performed according to the instructions of kit and apoptotic cells were observed under an optical microscope. Cells with brown nucleus were positive cells. Five visual fields were randomly selected (magnification, ×400) to count the number of apoptotic cells. The proportion of the number apoptotic cells to the number of total cells was used as apoptotic index.

Total RNA was extracted from renal tissue using the TRIzol kit. The quality of total RNA was checked by agarose gel electrophoresis. Only RNA samples with satisfactory qualities were used in subsequent experiments. Reverse transcription was performed using a kit to synthesize cDNA and expression levels of Bax and Bcl-2 were detected by semi-quantitative PCR with GAPDH as endogenous control. Reaction conditions were: 95°C for 4 min, followed by 35 cycles of 95°C for 30 sec, 64°C for 25 sec and 72°C for 30 sec. Primers were synthesized by Tiangen Biotech Co., Ltd. (Beijing, China). All primers are listed in Table I. PCR products were subjected to agarose gel electrophoresis and results were observed using a UV imaging system. The relative expression levels of Bax and Bcl-2 were represented by the ratio of Bax/GAPDH and Bcl-2/GAPDH.

Renal tissue was mixed with RIPA lysate with a ratio of 100 mg:1 ml. The mixture was centrifuged (10,500 × g) at 4°C for 10 min to collect the supernatant. Protein concentration was measured using the DAB Protein Quantitative kit (Invitrogen). After that, protein samples were subjected to SDS-PAGE gel electrophoresis, followed by transmembrane to PVDF membrane. After blocking with 5% skim milk, membranes were incubated with primary rabbit polyclonal Bax antibody (dilution, 1:500; cat. no. ab53154) and rabbit monoclonal Bcl-2 antibody (dilution, 1:500; cat. no. ab32124) overnight at 4°C. After washing with TBST 3 times, 5 min for each time, membranes were incubated with secondary goat anti-rabbit (HRP) IgG antibody (dilution, 1:2000; cat. no. ab6721) for 2 h. All antibodies were all purchased from Abcam (Cambridge, MA, USA). After washing with TBST 3 times, proper amount of ECL luminescent substrate (ratio of A and B was 1:1) was added and incubated with the membranes in the dark. The results were scanned and processed by ImageJ to calculate the expression level of each protein.

Data were expressed as mean ± standard deviation, and processed using the SPSS 19.0 software (SPSS, Inc., Chicago, IL, USA). Comparisons between 2 groups were performed using t-test, and comparisons among multiple groups were performed using variance analysis. If the variance was homogeneous, the comparisons between two groups were performed using Bonferronic method. If the variance was not homogeneous, Welch method was used. Multiple comparisons were performed using Dunnett's T3 method. P<0.05 was considered to be statistically significant.

Levels of Cr and BUN in peripheral blood of rats in each group were detected by Cr and BUN detection kit. As shown in Figs. 1 and 2, compared with group S, levels of Cr and BUN were significantly increased in I/R group and IP group (P<0.01). Compared with IP group, levels of Cr and BUN were significantly increased in the I/R group (P<0.01).

Activity of SOD was detected by SOD activity test kit. As shown in Fig. 3, activity of SOD in the I/R and IP groups was significantly lower than in the S group (P<0.01), and activity of SOD in the I/R group was significantly lower than in the IP group (P<0.05).

Pathological changes of renal tissue in each group after H&E staining were observed under an optical microscope. Structure of renal tissue in group S was clear, renal tubular and glomerular were normal, degeneration and atrophy were not observed in renal tubular epithelial cells, and the cavity was not expanded. In the I/R and IP groups, renal tissue glomerular telangiectasia and congestion, renal tubular epithelial cell edema, and granular degeneration or vacuolar degeneration were observed and the cavity was narrowed. After scoring (Table II), renal injury was found to be more serious in the I/R and IP groups than in the S group (P<0.01), and renal injury was more serious in the I/R group than in the IP group (P<0.05).

Apoptotic cells were detected using TUNEL kit. Number of positive cells was counted and the apoptotic index was calculated. As shown in Fig. 4, compared with S group, number of apoptotic cells in I/R group and IP group was significantly increased (P<0.01). Compared with IP group, the number of apoptotic cells in I/R group was significantly increased (P<0.01).

Expression of Bcl-2 and Bax mRNA was detected by semi-quantitative PCR. As shown in Fig. 5, compared with S group, expression level of Bcl-2 mRNA was significantly decreased, expression level of Bax mRNA was significantly increased, and the ratio of Bcl-2/Bax was significantly decreased in IP group and I/R group (P<0.01). Compared with I/R group, expression level of Bcl-2 mRNA was significantly increased, expression level of Bax mRNA was significantly deceased, and the ratio of Bcl-2/Bax was significantly increased in IP group (P<0.05).

Expression of Bcl-2 and Bax protein was detected by western blot analysis. As shown in Fig. 6, compared with S group, expression level of Bcl-2 protein was significantly decreased, expression level of Bax protein was significantly increased, and the ratio of Bcl-2/Bax was significantly decreased in IP group and I/R group (P<0.01). Compared with I/R group, expression level of Bcl-2 protein was significantly increased, expression level of Bax protein was significantly deceased, and the ratio of Bcl-2/Bax was significantly increased in IP group (P<0.01).