A total of 40 male Sprague Dawley rats (weight, 250–300 g; age, 9–12 weeks) were purchased from Wuhan University, Animal Biosafety Level 3 Laboratory (Wuhan, China). All rats were housed under standard laboratory conditions at 22–24°C, a relative humidity of 50±15%, and a 12 h light/dark cycle. All animals had free access to standard rat chow and water. The experimental protocol of the present study was reviewed and approved by the Animal Care and Use Committee of Wuhan University (Wuhan, China), and was in accordance with the Guide for the Care and Use of Laboratory Animals by the National Institutes of Health. Streptozotocin (STZ) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Antibodies against DJ-1 were purchased from Cell Signaling Technology, Inc. (cat no. 5933; Beverly, MA, USA). Antibodies to Nrf2 were purchased from Santa Cruz Biotechnology, Inc. (cat no. sc722; Dallas, TX, USA). GAPDH and Lamin B were purchased from Cell Signaling Technology, Inc. (cat nos. 5174 and 13435), and secondary antibodies were purchased from Santa Cruz Biotechnology, Inc. (cat no. sc2357). All other chemicals were obtained from commercial sources and were of the highest grade available.

Experimental diabetes was induced in the animals by a single intraperitoneal administration of STZ dissolved in 0.1 mol/l citrate buffer (pH 4.5) at a dose of 65 mg/kg. Normal rats received an equal volume of citrate buffer. Three days post-STZ injection, tail vein blood samples were collected and glucose levels were measured with a Onetouch glucometer (Johnson & Johnson, New Brunswick, NJ, USA). The rats were considered diabetic and used for the study only if their glucose levels were >15 mmol/l (15). Rats were maintained for 8 weeks after vehicle or STZ injection.

Animals were intraperitoneally anesthetized with pentobarbital sodium (50 mg/kg) (16) followed by a tracheotomy and artificial ventilation. Blood pressure was recorded from the left femoral artery using a pressure transducer with the heart rate monitored by an electrocardiogram throughout the procedure. The left femoral vein was cannulated for administration of drugs. A fourth-intercostal space thoracotomy was performed and the pericardium was excised to expose the heart. The left anterior descending coronary artery (LAD) was ligated 2 mm above the left auricle by a 6–0 silk suture. A small polypropylene tube was placed between the ligature and the LAD. The artery was occluded for 30 min by tightening the ligature. After ischemia for 30 min, the ligature was loosened to allow reperfusion for 2 h. The sham group underwent the same surgical procedures, apart from tying the 6–0 silk suture. After 2 h of reperfusion, the rats were sacrificed, and renal tissues and blood samples were obtained for further analysis.

Rats were randomly assigned into one of four experimental groups (n=8 per group) as follows: i) Sham-operated group (sham group), non-diabetic rats that underwent isolation of the LAD without occlusion as a control; ii) MIR group, non-diabetic rats subjected to 30 min of myocardial ischemia and 2 h of reperfusion after the LAD had been isolated and occluded (sham + MIR group); iii) diabetic rats that underwent isolation of the LAD without occlusion (diabetic group); iv) diabetic rats subjected to 30 min of myocardial ischemia and 2 h of reperfusion after the LAD had been isolated and occluded (diabetic + MIR group).

Blood samples were collected at the end of reperfusion and centrifuged at 1,000 × g for 10 min at 4°C. Serum was separated and stored at −20°C. HbA1c was measured using an Olympus automatic analyzer (cat no. AU5400; Olympus Corporation, Tokyo, Japan). CK-MB and LDH levels were measured using commercial kits (cat nos. 01010605082 and 01010113477; Beijing Kemeidongya Biotechnology Ltd., Beijing, China) according to the manufacturer's instructions.

The left kidney was cut into sections and fixed in 4% formaldehyde. After embedding in paraffin, 4-µm sections were stained with hematoxylin at 37°C for 3 min and eosin at 37°C for 10 sec for light microscopy (original magnification, ×200; Olympus BX50; Olympus Corporation).

Histological assessment of tubular necrosis was semi-quantitatively determined using a method modified from McWhinnie et al (17) using the following scoring system: 0, normal histology; 1, tubular cell swelling, brush border loss and nuclear condensation, with up to one-third of the tubular profile exhibiting nuclear loss; 2, same as for score 1, but more than one-third and less than two-thirds of the tubular profile displaying nuclear loss; and 3, same as for score 1, but more than two-thirds of the tubular profile exhibiting nuclear loss.

Blood samples were collected at the end of reperfusion and centrifuged at 3,000 rpm, for 10 min at 4°C. Serum was separated and stored at −20°C. Cys C and β2-MG levels were measured using ELISA assay kits (cat nos. E-EL-R783 and E-EL-R1085c; Elabscience Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer's instructions.

The renal tissues were harvested and immediately homogenized on ice in 5 volumes of normal saline. The homogenates were centrifuged at 1,200 × g for 10 min. The T-AOC and MDA levels in the supernatant were measured using T-AOC and MDA assay kits (cat nos. A015-2 and a003-2; Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions.

Paraffin-embedded renal sections were stained using the streptavidin-biotin complex (streptavidin peroxidase, SP) immunohistochemistry technique for DJ-1 and Nrf2 detection using SP immunohistochemistry kits (cat no. I003-2; Nanjing Jiancheng Bioengineering Institute) according to the manufacturer's protocol. Brown staining in the cytoplasm and/or nucleus was considered an indicator of positive expression. The results were semi-quantitatively evaluated with Image-Pro^® Plus version 6.0 software (Media Cybernetics, Rockville, MD, USA) according to optical density values of positive staining.

Cytoplasmic and nuclear proteins were extracted from frozen renal tissues with a nuclear extraction kit (cat no. P0027; Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's instructions. GAPDH was used as internal control of cytoplasmic protein and Lamin B was used as an internal control of nuclear protein. A BCA protein assay kit was used to determine the protein concentration (cat no. P0012S; Beyotime Institute of Biotechnology). Equal amounts of protein (30 µg) were separated by 12% SDS-PAGE at 100 V for 3 h. After electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes at 200 mA for 2 h. The transferred membranes were incubated overnight at 4°C with rabbit anti-mouse polyclonal antibodies for DJ-1 and Nrf2 (each at 1:800 dilution) in Tris-buffered saline containing Tween-20 (TBS-T) containing 5% skimmed milk. After washing three times in TBS-T, membranes were incubated with anti-rabbit immunoglobulin G conjugated to horseradish peroxidase at a dilution of 1:2,000 in TBS-T containing 5% skimmed milk for 2 h at room temperature. The immunoreactive bands were visualized by enhanced chemiluminescence (PerkinElmer, Inc., Waltham, MA, USA) and captured on X-ray film. The optical density of the bands was measured with a BandScan V4.0 imaging analysis system (http://bandscan.software.informer.com/).

Mean ± standard error of the mean values were calculated to summarize all outcome measurements. Statistical analysis was performed using GraphPad Prism software version 5.0 (GraphPad Software, Inc., La Jolla, CA, USA). The statistical significance of the differences between groups was evaluated using unpaired Student's t-tests for pair-wise comparisons, or a two-way analysis of variance followed by the Tukey post hoc test for multiple comparisons. The level of significance was set at P<0.05 for all statistical tests.

At the end of the study, the body weight of diabetic rats was significantly decreased as compared with that of normal rats. The change of kidney weight was not obvious in diabetic rats during the experimental period. The ratio of kidney weight to body weight in diabetic rats was significantly increased as compared to that in the normal rats. A significant increase in fasting blood glucose in diabetic rats was observed when compared with that in the control group. HbA1c was markedly higher in diabetic rats than that in normal rats (Table I).

To examine damage of cardiomyocytes induced by MIR, the activities of the cardiac enzyme CK-MB and LDH as indices of myocardial cellular injury were measured at the end of reperfusion in the different groups. Compared with the sham group, MIR caused a significant increase in LDH and CK-MB in the sham + MIR group, as did the diabetic condition, but to a significantly lesser extent (P<0.05). The damage evoked by MIR was further enhanced in the diabetes + MIR group (P<0.05 vs. all other groups; Fig. 1).

As presented in Fig. 2, the renal tubules exhibited pathological changes, including edema, necrosis and vacuolization in the sham + MIR and diabetic groups. A significant aggravation of histological edema, necrosis and vacuolization was seen in the diabetes + MIR group.

When compared with the renal histological evaluation score of kidneys obtained from normal rats, the sham + MIR and the diabetic groups exhibited in a significant increase in renal histologic evaluation score individually (P<0.05). This increase was significantly enhanced in the diabetes + MIR group (P<0.05 vs. all other groups).

Serum Cys C and β2-MG levels were measured to evaluate the renal injury induced by MIR in diabetic rats. MIR and diabetes individually significantly increased serum Cys C and β2-MG levels in comparison with those in the sham group, which was further aggravated in the diabetes + MIR group (P<0.05; Fig. 3).

As presented in Fig. 4, MIR and the diabetic condition significantly decreased the T-AOC and significantly increased MDA levels in rats, compared with those in the sham group, and these changes were significantly aggravated in the diabetes + MIR group (P<0.05).

Immunohistochemical analysis of the expression of DJ-1 in the sham group revealed small brown granules in the cytoplasm, while significant expression of DJ-1, as indicated by brown staining in the cytoplasm, was observed in the diabetes + MIR group (P<0.05 vs. sham group; Fig. 5). No significant difference was observed between Sham + MIR and Diabetic groups (Fig. 5).

The expression of Nrf2 in the sham group displayed as light brown immunostaining in the cytoplasm and no staining in the nuclei. Significant expression of Nrf2, as indicated by strong brown staining in the cytoplasm and nuclei, was observed in the diabetes + MIR group (P<0.05; Fig. 6). No significant difference was observed between Sham + MIR and Diabetic groups (Fig. 6).

In line with these results, western blot analysis revealed weak expression of DJ-1 and Nrf2 in the kidneys of animals of the sham group. By contrast, significant increases in the protein expression of Nrf2 and DJ-1 were found in the diabetes + MIR group (P<0.05; Fig. 7). No significant difference was observed in Nrf2 expression between Sham + MIR and Diabetic groups (Fig. 7); however, the expression of DJ-1 was significantly higher in the diabetic group compared with the Sham + MIR group (P<0.05; Fig. 7).