This pre-clinical study was performed according to the recommendations in the Guide for the Care and Use of Laboratory Animals of Fenyang College Shanxi Medical University (Fenyang, China). All experimental protocols on animals were in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Ethics Committee of Fenyang College Shanxi Medical University. All surgeries and euthanasia were performed in a manner of causing minimal suffering.

A total of 60 C57BL/KsJ db/+ (db/+) mice with type II diabetes (age, 6–8 weeks) were purchased from Charles River Laboratories (Sulzfeld, Germany). All mice were housed in a temperature-controlled room (25±1°C) with a 12-h light/dark cycle. The mice received insulin once a day for a 365-day observation. The concentration of creatinine in the serum was analyzed to identify those mice in which chronic renal failure was induced by type II diabetes. The mice with chronic renal failure induced by type II diabetes were divided into three groups (n=20 in each) and received resveratrol (10 mg/kg; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), angiotensin-converting enzyme inhibitor (ACEI) aldosterone (10 mg/kg, Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) or PBS by intravenous injection, and 20 healthy C57BL/KsJ+/+ (wild-type) mice (6–8 weeks old; Beijing University, Beijing, China) were used as controls. The treatments were continued for 8 weeks at one-day intervals. The renal cells were isolated from experimental mice for further analysis on day 60.

Mice with chronic renal failure induced by type II diabetes were fasted 6 h and injected intraperitoneally with glucose at a dose of 2 g/kg for the glucose tolerance test. Blood glucose concentrations were analyzed using an ACCU-CHEK Advantage glucometer (Roche Diagnostics, Basel, Switzerland). The results of the glucose tolerance tests were recorded at baseline and after glucose injection (120 min). For the insulin tolerance tests, mice with GDM insulin were injected with insulin intraperitoneally at 0.75 U/kg body weight. The blood glucose concentration was measured at baseline and 30 min after GDM mice received resveratrol, aldosterone or PBS.

Indirect calorimetric analysis was used to evaluate the physical activity of mice with chronic renal failure induced by type II diabetes by using the Comprehensive Laboratory Animal Monitoring System (Oxymax/CLAMS; Columbus Instruments Corp., Columbus, OH, USA). On day 60 after treatment with resveratrol, ACEI or PBS, physical activities of mice were monitored every 30 min for 24 h to analyze the therapeutic effects of the treatments on chronic renal failure induced by type II diabetes. The respiratory exchange ratio, volume of oxygen (VO₂), VCO₂, heat production and physical activity were measured according to the manufacturers' instructions.

Glucose-6-phosphatase activity of mice with chronic renal failure induced by type II diabetes was analyzed as previously described (28). Total protein in the blood of mice with chronic renal failure induced by type II diabetes was analyzed by the Bradford method. Glucose-6-phosphatase activity was normalized by subtracting nonspecific phosphatase activity determined by para-nitrophenylphosphate (p-NPPSt, Sigma-Aldrich; Merck KGaA).

Renal sections from experimental mice were prepared for apoptotic analysis. Renal cells were incubated with terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) stain (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) for 60 h according to the protocol of previous study (29). The apoptotic renal cells were analyzed by fluorescence microscopy as described in a previous study (30).

ELISA kits (Huiying, Shanghai, China) were used to determine interleukin (IL)-6 (HY1738), IL-10 (HY1798), IL-17 (HY2013), IL-1β (HY0028), vascular endothelial growth factor (VEGF, HY3014) and tumor necrosis factor (TNF)-α (HY62203) levels in the sera of the mice. The procedures were performed according to the manufacturer's instructions. The final results were recorded at 450 nm on an ELISA plate reader.

Renal cells from experimental mice (1.0×10³) were seeded in 96-well plates in MEM with 10% FBS in a final volume of 100 µl/well at 37°C. Following 24 h, cell viability was evaluated using the MTS assay (CellTiter 96^® AQueous One Solution Cell Proliferation Assay; Promega Corporation) according to the manufacturer's instructions. Cells morphology was observed using a confocal microscope (Carl Zeiss, Zen 2010) (magnification, ×40). The final absorbance was recorded using a microplate reader (BMG Labtech Ltd., Aylesbury, UK) at a wavelength of 450 nm. Cells viability was determined by the absorbance of cells at 450 nm.

Total mRNA was isolated from renal cells by using an RNA Easy Mini Extract kit (Sigma-Aldrich; Merck KGaA). RNA was reversed transcribed using a PrimeScript RT Master Mix kit (Takara Bio, Inc., Otsu, Japan). The expression of caspase-3, −8 and −9 as well as apoptotic protease activating factor 1 (Apaf-1) in renal cells was determined by using an RT-qPCR kit (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's instructions with β-actin expression as an endogenous control. All procedures were performed according to the manufacturer's instructions. All of the primers were from Invitrogen (Thermo Fisher Scientific, Inc.) and their sequences are listed in Table I. Relative mRNA expression levels were determined by using the 2^−ΔΔCq method (31). The final results were presented as the fold of β-actin.

Renal tissues were isolated from mice with chronic renal failure induced by type II diabetes and homogenized in 1X radioimmunoprecipitation assay buffer on day 60. Subsequently, western blotting was performed to analyze the expression of analyte proteins. Protein concentrations were examined using a BCA protein assay, and protein samples (40 µg) were loaded and separated using 15% SDS-PAGE. Protein were subsequently blotted on a nitrocellulose membrane and hybridized using primary antibodies against AMPK (1:2,000; 9839), phosphorylated AMPK (1:2,000; 4186), and β-actin (1:500; 3700; Cell Signaling Technologies, Inc., Danvers, MA, USA) were added for 12 h at 4°C after blocking with 5% skimmed milk for 1 h at 37°C, and membranes were then incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG mAb (1:5,000; PV-6001; ZSGB-BIO, Beijing, China) for 24 h at 4°C. The blots were visualized by using a chemiluminescence detection system (32209; Invitrogen; Thermo Fisher Scientific, Inc.). Densitometric quantification of the immunoblot data was performed by using the software of Quantity-One (version 3.2; Bio-Rad Laboratories, Inc., Hercules, CA, USA).

Immunohistochemical staining was performed by an avidin-biotin-peroxidase technique. Paraffin-embedded tissue sections were prepared and epitope retrieval was performed for further analysis. The paraffin sections were incubated with hydrogen peroxide (3%) for 15 min at 37°C and subsequently blocked with 5% skimmed milk for 15 min at 37°C. Finally, the sections were incubated in caspase-3 (1:2,000; 9662; Cell Signaling Technologies, Inc.) at 4°C for 12 h. All sections were washed 3 times and incubated with horseradish peroxidase-conjugated secondary antibodies (1:2,000; 12768; Cell Signaling Technologies, Inc.) for 1 h at 37°C. A total of 6 randomly selected fields of view were observed under the fluorescence microscope (ECLIPSE TE300; Nikon Corporation; Tokyo, Japan). Tissue sections were also stained with 4-hydroxy-2-nonenal (HNE; Cell Signaling Technologies, Inc.) for 2 h at 37°C and measured using ELISA with commercially available kits (MBS027502; MyBioSource; San Diego, CA, USA).

Renal tissues and cells were isolated from experimental mice and homogenized in buffer containing 1 mM EDTA, 0.5 mM phenylmethane sulfonyl fluoride, 100 mM Tris-HCl (pH 7.4) and 0.05% Triton X-100 with sonication. The homogenates were centrifuged at 8,000 × g for 10 min at 4°C and the supernatants were used to measure the total thiol, reduced glutathione (GSH), lipid peroxidation and superoxide dismutase (SOD) activity. Total thiol, GSH, lipid peroxidation and superoxide dismutase and reactive oxygen species (ROS) activity in tissue homogenates was evaluated according to a previous study (32). All of the spectrophotometric data were recorded by a Spectramax spectrophotometer (Molecular Devices, Inc., Sunnyvale, CA, USA).

Values are expressed as the mean ± standard deviation from three independent experiments. All data were analyzed using SPSS Statistics 19.0 (IBM Corp., Armonk, NY, USA). Statistical significance of differences between groups was analyzed using a two-tailed Student's t-test. Unpaired data were analyzed by analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

The inflammatory response is crucial for renal failure induced by type II diabetes. Therefore, the present study first analyzed the inflammatory factors in the renal cells from the experimental mice. It was observed that inflammatory factors IL-1β, IL-17, IL-10 and TNF-α in the serum in mice with chronic renal failure induced by type II diabetes were significantly increased compared with those in healthy mice, while IL-6 and VEGF were decreased (Fig. 1). However, in comparison with those in the PBS group, the plasma concentrations of IL-1β, IL-17, IL-10 and TNF-α were significantly decreased after resveratrol treatment. Furthermore, resveratrol treatment led to increased IL-6 and VEGF levels compared with those in the PBS group. ACEI treatment also significantly decreased IL-1β, IL-17, IL-10 and TNF-α and increased IL-6 and VEGF levels compared with those in the PBS group, but the effect was significantly lower than that of resveratrol. These results indicated that resveratrol regulates inflammatory factors, which may be beneficial for mice with chronic renal failure induced by type II diabetes.

To investigate the efficacy of resveratrol on chronic renal failure induced by type II diabetes, its effect on glucose metabolism and insulin tolerance in the experimental mice was assessed. First, changes in the body weight of mice with chronic renal failure induced by type II diabetes were analyzed. The results revealed that in the PBS group, the body weight was significantly decreased compared with that in the Control group, which was inhibited by resveratrol and, to a significantly lesser extent, by ACEI (Fig. 2A). In addition, resveratrol significantly improved the fasting blood glucose concentration in mice with chronic renal failure induced by type II diabetes (Fig. 2B). Resveratrol treatment also significantly alleviated glucose intolerance after the initial glucose injection compared with that in the PBS and ACEI groups (Fig. 2C). Furthermore, resveratrol treatment decreased food intake of mice with chronic renal failure induced by type II diabetes compared with that in the ACEI and PBS groups (Fig. 2D). Of note, insulin levels in mice treated with resveratrol were significantly higher than those in the ACEI and PBS-treated groups (Fig. 2E). Furthermore, the insulin tolerance test indicated that resveratrol treatment significantly improved insulin tolerance in mice with chronic renal failure induced by type II diabetes (Fig. 2F). These results suggested that resveratrol improves body weight, fasting blood glucose concentration and insulin tolerance in mice with chronic renal failure induced by type II diabetes.

Apoptosis has an important role in mammals with chronic renal failure induced by type II diabetes (33). In the present study, the inhibitory effects of resveratrol on renal cell apoptosis in experimental mice with chronic renal failure were assessed. A TUNEL staining assay revealed that apoptotic renal cells were markedly decreased in mice treated with resveratrol compared with those in the PBS- and ACEI-treated groups (Fig. 3A). Furthermore, resveratrol treatment markedly inhibited the expression of apoptosis-associated genes (Apaf-1, caspase-3, caspase-8 and caspase-9) in renal cells with chronic renal failure (Fig. 3B-E). Immunohistochemical analysis demonstrated that resveratrol treatment significantly decreased caspase-3 expression, which confirmed the protective effect of resveratrol on renal cells from experimental mice (Fig. 3F). Collectively, these results suggested that resveratrol treatment inhibited apoptosis of renal cells by inactivation of caspase-3 in mice with chronic renal failure induced by type II diabetes.

As AMPK is likely to be a target of resveratrol, via which it exerts its pharmacodynamic function, the AMPK signaling pathway in renal cells from experimental mice was examined. The expression and phosphorylation levels of AMPK were decreased after treatment with resveratrol (Fig. 4A and B). In addition, morphological observation revealed that resveratrol treatment improved the viability of renal cells determined by cells viability (Fig. 4C). Furthermore, immunostaining demonstrated that immunocyte numbers were decreased in renal cells of resveratrol-treated mice compared with those in the ACEI- and PBS-treated groups (Fig. 4D). AMPK activation was also decreased in renal cells of experimental mice after resveratrol treatment compared with that in the ACEI- and PBS-treated groups (Fig. 4E). In addition, glucose-6-phosphate activity was significantly increased in the serum of experimental mice after treatment with resveratrol (Fig. 4F). These findings suggested that decreased AMPK activation by resveratrol contributes to the enzymatic capacity for glucose production in mice with chronic renal failure induced by type II diabetes, which may proceed via the AMPK-mediated signaling pathway.

Oxidative stress contributes to type II diabetes-induced chronic renal failure, leading to increased apoptosis of renal cells in experimental mice. Hence, the present study investigated the oxidative stress and anti-oxidant status in renal cells after treatment with resveratrol. As presented in Fig. 5A, total thiol levels were decreased in renal cells after resveratrol treatment. Lipid peroxidation in renal cells of resveratrol-treated mice was increased compared with that in ACEI- and PBS-treated experimental mice (Fig. 5B). In addition, resveratrol treatment increased SOD activity and ROS levels in renal cells from experimental compared with that in ACEI- and PBS-treated experimental mice (Fig. 5C and D). In addition, increases of GSH in renal cells of experimental mice were found to be included in the benefits of resveratrol treatment (Fig. 5E). Furthermore, immunohistochemical detection of 4-hydroxy-2-nonenal (HNE) revealed that resveratrol treatment decreased the intense staining of HNE in renal cells compared with that in the controls, which may also indicate the enhancement of the anti-oxidant status (Fig. 5F). These results indicated that resveratrol treatment decreased oxidative stress and improved the anti-oxidant status in renal cells from mice with chronic renal failure induced by type II diabetes.