2.1 Animals and CIHH pretreatment
Male Sprague-Dawley (SD) rats, SPF grade, 24 in total (weight 160±10 g, buy from Hebei Ivivo Biotechnology Co., Ltd.), were purchased and kept in the laboratory of Hebei Medical University, where the laboratory temperature was constant at 22±1℃ and received light/dark time for 12 h each, with free access to water and food. At the beginning of the experiment, 24 male SD rats were randomly divided into 4 groups of 6 rats each: normal-controlled group (CTL, rats were kept in the laboratory environment at all times), CI-AKI group (rats were kept in the laboratory environment at all times), CIHH group (SD rats were placed in a hypobaric hypoxic chamber simulating 4000m altitude for 5h per day for 35 days), CIHH prevented CI-AKI group ( SD rats were placed in a hypobaric hypoxic chamber simulating 4000m altitude for 5 hours per day for 35 days before CI-AKI modeling). The experiment was approved by the ethics committee of Hebei Provincial People's Hospital and completed by the Department of physiology of Hebei Medical University. The study was carried out in compliance with the ARRIVE guidelines.
2.2 CI-AKI rats model establishment
The modeling method was referred to Bae et al., as follows: rats in the CI-AKI group and CIHH to prevent CI-AKI group were sequentially injected with indomethacin (10mg/kg, Aladdin,China,I106885-5g, purity98%), Nω-nitro-L-arginine methyl ester (10mg/kg, 15 min later, Sigma, USA, N5751-5G, purity>98%), and ioversol injection (8.3 mL/kg, 30 min later，350mgI/ml, Jiangsu Hengrui Pharmaceutical Co., Ltd, China) through the tail vein, and rats in the CTL and CIHH groups were injected with saline through the tail vein 0.5mL/time,3 times in total. After injection, all rats were kept in the laboratory environment and resumed free access to water and food.
3. Detection methods of various indicators.
3.1 Heart rate and mean arterial pressure measurements
Heart rate and mean arterial pressure were measured by a tail cuff manometer (LE5001, Panlab) every morning (7:00-10:00 am) from one week before injecting drug until 24 hours after contrast agent injection, or before blood collection if blood specimens were to be collected from the tail vein.
3.2 Testing of biochemical indicators
Blood was collected from the tail vein of rats before and 24 hours after contrast agent injection, respectively. Then centrifuged them for 15 min at 4℃ and 3000 rpm using a tabletop high-speed refrigerated centrifuge (DaLong, D3024R), and the supernatant was taken for the determination of serum creatinine and serum urea nitrogen by an automatic biochemical analyzer (Shenzhen Redu Life Science, Chemray800).
3.3 Measurement of Biomarkers of Oxidative Stress
The rats were killed by neck leading at 24 h after contrast agent injection, and the isolated right kidney tissues were washed with saline (pre-cooled on ice in advance) to remove residual blood and homogenized, then using a benchtop high speed frozen centrifuge (DaLong, D3024R) to centrifuge for 15 min at 4℃, 3000 rpm /min,, and the supernatant was collected. SOD activity and MDA levels were determined by using two commercial experimental kits (Nanjing Jiancheng Institute of Biological Engineering) according to the assay instructions of the kits.
3.4 Histological examination
Rats were executed 24 hours after drug injection, and the left kidney was fixed in 4% paraformaldehyde solution for 48 hours and then dehydrated in gradient alcohol, transparent in xylene, and embedded in paraffin wax. The paraffin kidney tissue block was cut into thin slices of approximately 3μm thickness using a microtome (Leica, Germany), then stained with hematoxylin eosin staining (HE staining), periodic acid Schiff reaction (PAS)and Masson trichrome after dewaxing in xylene and rehydrating in gradient alcohol. Last observed and photographed in positive White light microscope (Nikon (Japan), Eclipse Ci-L), and each tissue section was carefully observed under the microscope and histological changes were recorded.
3.5 Immunoblot analysis
Rats were executed 24 h after drug injection, the right kidney was taken and cut with tissue scissors. The kidney tissues were homogenized in tissue/cell lysate. Protein samples were separated by SDS–PAGE, transferred to a PVDF membrane (Millipore Corporation, USA) that was blocked for 1h with 5%(w/v) non-fat milk in Tris-buffered saline, and incubated with antibodies against HIF-1α (1:500, Wanleibio, China, WL01607), BNIP3 (1:1500, Wanleibio, China, WL01139), caspase3 (1:1500, Affinity, USA, AF6311)), PARP (1:750, Wanleibio, China, WL01932), overnight at 4 ℃ on a constant temperature refrigerator shaker. The same membrane was stripped and re-blotted with an β-actin antibody (1:5000, Proteintech, USA, 20536-1-AP) for normalization. Blots were developed by the chemiluminescent detection method (Amer sham ECL). The protein blots were quantified by densitometry using Image J software and normalized to β-actin.
3.6 Data analysis
All data were performed with GraphPad Prism 9.0.0（121）software. All groups with n=6 each were analyzed by one-way analysis of variance (ANOVA). Results are presented as mean±SEM and P<0.05was considered significant.