Grape seed proanthocyanidin (purity exceeds 96%, Lot no. G050412) was purchased from Tianjin Jianfeng Natural Product R&D Co., Ltd. (Tianjin, China). STZ was purchased from Sigma. TUNEL staining kit (in situ Cell Death Detection kit) was obtained from Roche Diagnostics, (Indianapolis, IN, USA). Primary antibodies used in this study include: Rabbit anti-Caspase-12, rabbit anti-GRP78 (Abcam Ltd., Hong Kong, China), rabbit anti-p-ERK, rabbit anti-ERK antibodies (Cell Signaling Technology, Inc., Danvers, MA, USA), mouse anti-β-actin (Santa Cruz Biotechnology, Inc., Dallas, TX, USA). Anti-mouse and anti-rabbit secondary antibodies were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA).

Fifty-five adult male SD rats (8–12 weeks of age), weighing 120–160 g were provided by the Beijing Vital River Laboratory Animal Technology Co., Ltd (Beijing, China). The current study was approved by the Animal Ethics Committee of Shandong University (no. DWLL-2013-053; Jinan, China).

Forty-five randomly selected rats were given a high-fat diet (45% fat, 20% protein and 35% carbohydrate, as a percentage of total kcal; product no. D12451, provided by the Beijing Vital River Laboratory Animal Technology Co., Ltd.) for one month, and then intraperitoneally injected with 40 mg/kg STZ dissolved in pH 4.5 citrate buffer, while the ten remaining rats were given the same dosage of citrate buffer at pH 4.5 and given a normal diet (N group). Hyperglycemia was confirmed by measuring the venous circulating plasma concentration of glucose. Seven days post-STZ injection, blood samples were obtained from the rat tail vein after 12 h of fasting, and the glucose concentration was determined with an automatic analyzer (a Comfore automatic analyzer purchased from Shanghai). Forty rats showed the diabetic rat standard of a fasting blood glucose level higher than 300 mg/dl, while the value in the control group of rats injected with citrate buffer ranged from 90 to 130 mg/dl. Then, twenty randomly selected rats were continued on the high-fat diet (DM group) until 16 weeks, and the other twenty rats received intragastric administration of 250 mg/kg/day GSPE as well as the high-fat diet (GSPE group) until 16 weeks. At the end of the experiment, 16 rats survived in the DM group and 15 in the GSPE group. Three rats in the GSPE group died of asphyxiation due to gavage errors. The animals were placed in individual metabolic cages for 24 h to collect urine samples before the experimental rats were sacrificed. No difference of total water intake among groups was observed. Total urinary protein (g/l) was measured by the sulfosalicylic acid method (722N; China) (13). At the end of the experiments, the animals were fasted overnight for 18 h and then anesthetized with intraperitoneal injection of sodium pentobarbitone (60 mg/kg; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) and sacrificed. All animals were weighed before sacrifice. Blood was collected from the heart before sacrifice, and the serum was separated by centrifugation (912 × g at 4°C for 20 min) and stored at −80°C until it was assayed. Each kidney was cut in half by coronal plane after excision and weighed. Two pieces of kidney were stored at −80°C for a Western blotting analysis. The other pieces were fixed in 4% buffered paraformaldehyde at 4°C and embedded in paraffin for immunohistochemical histopathologic observation, and a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay.

Blood urea nitrogen (BUN) and serum creatinine (Scr) were analyzed on a Cobas 8000 (Roche Diagnostics) in Qilu Hospital, Shandong University. The renal index (RI) was calculated as follows: both kindey weights (g)/animal's weight (g) ×100.

The renal pathological changes were determined by periodic acid-Schiff (PAS) staining. The kidneys were sliced and fixed in 4% paraformaldehyde for no more than 24 h and then taken to 0.5% paraformaldehyde for long-term preservation. They were embedded in paraffin and cut into 4-µm-thick sections for staining with PAS or immunohistochemical staining.

The PAS-positive area present in the mesangial region excluding cellular elements indicated mesangial matrix expansion. The percentage of the PAS-positive area in the glomerulus was analyzed by using Leica QWin version 3 image analysis software (Leica Microsystems GmbH, Wetzlar, Germany).

For immunohistochemical analysis, the tissue slices were microwaved for 10 to 15 min in 0.01% sodium citrate buffer (pH 6.0) for antigen retrieval. The tissue slices were cooled at room temperature or in ice water and then washed with PBS three times. The tissue slices were immersed in 0.1% Triton X-100 for 15 min. To block endogenous peroxidase, all tissue slices were incubated with 3% hydrogen peroxide for 10 min in the dark. The tissue slices were incubated with 10% goat serum for 60 min at 37°C, then with primary antibody at 4°C overnight (anti-GRP78 1:200, anti-p-ERK 1:50, anti-Caspase-12 1:100). The negative control sections were incubated with PBS instead of the primary antibody. All sections were incubated with secondary antibodies for 60 min at 37°C and then stained with DAB and hematoxylin. The stained slides were analyzed by light microscopy. Brown areas were designated as positive. A semi-quantitative analysis was performed on the colored sections using an Image-Pro Plus 5.0.

TUNEL staining was performed according to the manufacturer's instructions. The sections were incubated with proteinase K at 37°C for 15 min, then washed in PBS for 5 min; the enzyme solution and label solution were then mixed (1:9) at 37°C for 60 min in the dark. The sections immersed in label solution served as negative controls. The sections were then stained with DAPI for 10 min. TUNEL-positive nuclei were expressed as a percentage of the total nuclei per field. Ten fields per section and two sections per kidney were assayed in each experiment.

The tissue samples were homogenized in TRIzol (50–100 mg tissue per 1 ml TRIzol). After chloroform was added (0.2 ml chloroform per 1 ml TRIzol), the homogenates were centrifuged at 12,000 × g for 15 min at 4°C. The supernatants were discarded, isopropanol was added (1.5 ml isopropanol per 1 ml TRIzol) to the lower phase, and then the samples were centrifuged at 12,000 × g for 15 min at 4°C. The pellets were washed with 0.3 M guanidine hydrochloride (2 ml guanidine hydrochloride per 1 ml TRIzol) three times, then dissolved in 1% SDS (100 µl SDS per 1 ml TRIzol) at 50°C for 30 min. The concentration of protein was determined using the Pierce BCA assay kit.

Protein (50 µg) was subjected to 10% or 12% SDS-polyacrylamide gel electrophoresis and transferred to cellulose acetate membranes. The membranes were blocked at room temperature for 1 h or more with 5% skim milk, then incubated with the primary antibodies (anti-GRP78 1:250, anti-p-ERK 1:1,000, anti-ERK 1:1,000, anti-caspase 12 1:500, anti-β-actin 1:2,500) at 4°C overnight. After incubation with the secondary antibodies at room temperature for 1 h, the membranes were soaked with enhanced chemiluminescence (ECL) reagent and exposed to X-ray film. Quantification of the luminosity of each identified protein band was performed using Adobe Photoshop software (Adobe Photoshop 7.0; Adobe, San Jose, CA, 2002). The Western blotting analysis used a ratio of GRP78, p-ERK, and Caspase-12 in the N group to the other groups.

Data were displayed as means ± standard deviation. Differences between groups were analyzed using the post hoc test used for multiple comparisions following one-way ANOVA. P<0.05 was considered to indicate a statistically significant difference.

As shown in Table I, the blood glucose in the DM group and the GSPE group was higher during the entire experiment, and it was statistically significant compared with the control group. As shown in Table II, 10 rats survived in the N group, 16 rats survived in the DM group, and 15 rats survived in the GSPE group at the end of the study. The BUN level and Scr had no significant changes among the three groups, but the 24-h urinary albumin level and the RI increased in the DM group compared with the N group. Compared with the DM group, the level of 24-h urine albumin and RI significantly decreased in the GSPE group.

The histopathology results are shown in Fig. 1. PAS staining indicated the presence of glomerular hypertrophy and thickening of the glomerular basement membrane in the DM group, which showed that the diabetic rats suffered from diabetic nephrology. In the GSPE group, the glomerular changes were significantly reduced, which indicated that the GSPE had a protective effect against diabetic nephrology. Quantitative analysis of the percentage of the PAS-positive area in the glomeruli is summarized in the histogram. The extracellular matrix (ECM) accumulation was significantly higher in the glomeruli of the D group than that of the N group (P<0.05), and the G group was significantly shorter than that of the D group (P<0.05).

To assess whether GSPE inhibited cell apoptosis in DN, the tissue sections were labeled with an in situ TUNEL assay. As shown in Fig. 2, apoptotic cells were visible with a red color. The DM group had high expression of TUNEL-positive cells compared with the N group (P<0.05). In the GSPE group the TUNEL-positive cells were significantly reduced compared with the DM group (P<0.05). In the N group and the GSPE group, little apoptosis was evident.

Apoptosis occurred in the cells with DN. To investigate whether GSPE protects against DN by attenuating ERS-induced apoptosis, we determined the expression of p-ERK, GRP78, and Caspase-12, important participants in ERS-induced apoptosis, by Western blotting and immunohistochemical methods. The two approaches showed that GRP78, p-ERK and Caspase-12 were highly expressed in the DM group, while their levels were significantly reduced in the GSPE group (P<0.05; Figs. 3 and 4). These proteins showed limited expression in the N group.