Eight-week-old SPF C57BL / 6J mice weighing 20-23 g, were purchased from Sandscobes Biotechnology Co., Ltd. All the mice were housed in 12h light/dark alternating regular illumination, ambient temperature (20 ± 1) ℃, the humidity was (48 ± 10) % and free access to food and water. During the experiment period, animals were treated strictly according to the rules in the Ethics Committee of the first affiliated hospital of the University of Science and Technology of China (Anhui Provincial Hospital). Every effort was made to minimize the number of animals used and their suffering.
2. experimental Grouping
After 7 days of acclimatization, mice were randomly divided into normal control group (n=8, fed with an ordinary diet until the end of the experiment) and high-fat diet (HFD) group (n=30, fed with HFD (containing 20 % protein, 20 % carbohydrate, 60 % fat until the end of the experiment)). After 16 weeks of feeding on a regular and high-fat diet, HFD mice had received a small dose of STZ (50 mg/kg was dissolved in 0.1mol/L citrate buffer, pH=4.2 and 3 days), and the NC group was injected with an equal amount of citrate buffer, and after a week, random blood glucose ≥16.7mmol/L for T2DM. Then the T2DM model mice (n=22) were randomly divided into 3 groups as follows：(1) T2DM group (n=7), in which the mice were received by gavage of the equivalent volume of normal saline for 8 weeks; (2) MET group (n = 8), in which the mice were received by gavage of Metformin (Shanghai shimeigun Pharmaceutical Company) at a dose of 250 mg/kg/day for 8 weeks. (3) GLIB group (n = 7), in which the mice were received by gavage of Glibenclamide (Tianjin Pacific Pharmaceutical Co., Ltd) at a dose of 2.5 mg/kg/day for 8 weeks. The mice in the normal control group were received a subcutaneous injection of the equivalent volume of normal saline for 8 weeks. After 8 weeks, the mice were weighed, fasting blood glucose was measured. Furthermore, blood, urine, and kidney tissue specimens were collected for further examination. The tissue specimens were stored at ‑80℃ before further use.
3. Detection of biochemical indicators
Before the mice were sacrificed, they were placed in a metabolic cage to collect urine. Samples were used to assay for Alb, RBP, PCX, 8-OHdG, and 8-iso-PG by ELISA method and were tested for urine creatinine (UCR) by the picric acid method. Urinary Alb/UCR (UACR), urinary RBP /UCR (URCR), urinary PCX /UCR (UPCR), urinary 8-OHdG/UCR (UOHCR), and urinary 8-iso-PG/UCR (UISOCR) were calculated.
4. Electric microscope observation
The renal cortex was harvested and fixed with 2.5% glutaraldehyde to make ultrathin sections. Five visual fields of sections were examined in a JEM-1230 transmission electron microscope. GBMT (glomerular basement membrane thickness) was measured at five sites randomly. The total length of fused foot processes / total length of the basement membrane was measured and recorded as FPFR (foot process fusion rate). All parameters were measured by the Image J image analysis system.
5. Statistical analysis
Statistical analysis was performed using the Graphpad and spss 26.0 software. Data were expressed as mean ± standard deviation (means ± SD) and comparisons between two groups were performed using Student's t-test. Multi-group comparisons were analyzed using a one-way ANOVA test. If data were shown normally distributed, pairwise comparisons were conducted by LSD test. If not, pairwise comparisons were conducted by the Dunnett T3 test. All P values were two-sided, and a value of P less than 0.05 was considered statistically significant.