The protein -actin was used as a control to confirm equal loading of protein in the gel lanes and to correct western blot signals. experimental group (n=50), used for the generation of diabetic rat model. The experimental group fasted for 12 h and then a single dose of STZ (dissolved in citrate buffer, pH MLN1117 (Serabelisib) 4.5, 60 mg/kg body weight) (13) was injected into MLN1117 (Serabelisib) the abdominal cavity of rats to generate an STZ-induced diabetic rat model. The control group were injected with citrate buffer (pH 4.5). After 72 h, the tail blood was collected to test the levels of serum glucose and those with serum glucose concentrations of >16.7 mmol/l were deemed diabetic rats. After 1 week of observation, the diabetic rats were used in subsequent experiments, apart from 6 rats which MLN1117 (Serabelisib) died due to side effect of STZ injection and four rats were not successful for the diabetic rat model. All the experiments complied with the guidance by the animal use and care of The First Affiliated Hospital of Zhengzhou University and the agents were approved by the ethical committee of animal care and use. Experimental groups A total of 40 STZ-induced diabetic rats were housed for 16 weeks and randomly assigned into four groups: i) Sham-operated group (sham group), where rats were only treated by separating the bilateral renal arteries and veins and then treated with 10% dimethyl sulfoxide (DMSO, 1 ml/kg bw, i.v.) (14C16); ii) RI/RI group (vehicle group), where NEDD9 the rats were treated with ischemia through clamping the bilateral renal arteries and veins for 45 min followed by 24 h reperfusion with DMSO (1 ml/kg bw, i.v.); iii) I/R+ DAPT group (DAPT group), where DAPT (dissolved in DMSO, 15 mg/kg) was administered as a pretreatment for rats via a single-dose injection into the abdominal cavity at 30 min prior to the I/R procedure; and iv) I/R+ DAPT + cisplatin group (Cisplatin group), where cisplatin (15 mg/kg) was intraperitoneally administered to rats at 24 h prior the I/R procedure and DAPT was administered to the animals in the same way as the DAPT group. Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals of Zhengzhou University. The protocol was approved by the Committee on the Ethics of Animal Experiments of Zhengzhou University. Tissue collections Following reperfusion for 24 h, the MLN1117 (Serabelisib) animals were euthanized using CO2 in a flow rate lower than 30% chamber vol/min and then decapitated to collect blood samples from the abdominal aorta. The collected tissues were centrifuged at 4,000 g at 4C for 20 min to isolate the sera. The entire kidneys were removed and weighed and immediately placed on dry MLN1117 (Serabelisib) ice or kept at ?80C until further analysis. The kidneys from each group were homogenized in cold normal saline and centrifuged at 4,000 g at 4C for 20 min to obtain the supernatant, which was used for the determination of various parameters. Renal damage examination Renal function was evaluated based on the analysis of blood urea nitrogen (BUN) and serum creatinine (SCr). The concentration of BUN and SCr were analyzed using an automatic biochemistry analyzer (Hitachi 76000; Hitachi High-Technologies Corporation) according to the manufacturer’s protocols. Analysis of anti-oxidation in renal tissues Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in kidney tissues were used as two anti-oxidative markers. SOD activity and MDA were respectively examined at wavelengths of 550 nm and 532 nm according to the xanthine oxidase and thiobarbituric acid method (Roche Diagnostics GmbH). The level of lipid peroxides was expressed as U of SOD/mg protein and nmol of MDA/mg protein. ELISA Tumor necrosis factor (TNF) , interleukin (IL) 10 and hypoxia-inducible factor (HIF) 1a levels in kidney tissues were assessed. TNF- (cat. no. ab4607; Abcam) and IL-10 (cat. no. ab108872; Abcam) levels were measured using ELISA kits according to the manufacturer’s protocols. For the HIF1a evaluation, the quantitative sandwich ELISA method (cat. no. MAN0014317; Thermo Fisher Scientific, Inc.) following the manufacturer’s protocols was performed to test the level of HIF1a in diabetic rat model. Briefly, HIF1A standards and samples are captured by a polyclonal HIF1A antibody on the pre-coated plate and detected using a biotinylated monoclonal HIF1A antibody reactive to epitopes other.