Resveratrol:Sigma,CAS#v900386;Adriamycin:Haizheng Pfizer Pharmaceutical Co.,Ltd.,CAS#131101;Lotensin:Solarbio,CAS#YZ100768; Reynoutria japonica:
Guangzhou Kangmei Pharmaceutical Co.,Ltd.; Cisplatin:Solarbio,CAS#15663271; MDA Reagent:Solarbio,CAS#BC0025100;SOD Reagent:Solarbio,CAS#BC0175100; TRI Reagent®:Sigma,CAS#93289;SYBR® Premix Ex Taq TM II:Takara, CAS#RR-820A;dNTP mix:Sigma,CAS#D7295;96 well PCR plate:Axygen,CAS#96M2HSC; MicroAmp™ Optical Adhesive Film:Thermo,CAS#4311971;DAB:Solarbio,CAS#S-W1010;light cycleR 96 RT-PCR:Roche Diagnostics;EDTA:Solarbio,CAS#C1034;PV-600:Beijing Zhongshan Jinqiao Biotechnology Co.,Ltd.;SIRT1 antibody:Abcam,CAS #ab110304;GAPDH antibody:Abcam,CAS#ab9485;Pathological slicer:Thermo HM-340E(American);Microscope and camera system:OLYMPUS(Japan);Digital patholo-gical image analysis system:MoticMed 6.0;Urinary albumin/creatinine Test: BECK-MAN COULTER iRICELL3000 Automatic urinalysis line;Urinary Creatinine/BUN Test:HITACHI 7600 automatic biochemical analyzer;Pentobarbital sodium:Sigm-a,CAS#P3761;4% Paraformaldehyde:Sigma,CAS#P6148.
2.2 The animals for the experiment
Fifty male SD rats (180–200 g) were purchased from SPF Biotechnology Co., Ltd. (Beijing, China) on September 13, 2019 with the production licence number SCXK (Beijing 2016-0002), Certificate No. 1103241911005725. The experimental protocol was approved by the Animal Care and Animal Ethics Committee of the Luohe Central Hospital, Certification No. 2019027. And this study was performed according to the National Institute of Health guidelines. All animals were housed in a standard feeding environment (room temperature 21-23℃, 12 hours of day/night cycle) and were free to eat standard feed and drinking water.
Fifty adult male SD rats were randomly divided into two groups: a blank group (10 rats) and a model group (40 rats). The model group rats were injected with cisplatin (10 mg/kg) via the tail vein. Three days later, the proteinuria test strip was green or orange, which proved that proteinuria was positive. The successfully modelled rats were randomly divided into a blank group, model group, Reynoutria japonica group, resveratrol group and lotensin group, with ten rats in each group.
2.4 Gastric perfusion method and collection of specimens
The Reynoutria japonica group was given 2.7g/kg/d, which converted from human dose, resveratrol group was given 40 mg/kg/d.respectively, while the lotensin group was given 0.90 mg/kg. The blank and model groups were given double distilled water at the same time and gavaged twice daily for four weeks. Doses of cisplatin and resveratrol  were based on previous literature. The rats were weighed on the first morning of each week to adjust the dosage. On the morning of the seventh day of each week, random urine was collected with a metabolic cage for urinary protein excretion measurement. All rats were anaesthetized with 3% sodium pentobarbital (0.1～0.2 ml/100g) on the fourth weekend to obtain blood taken from the heart to measure serum creatinine and urea nitrogen and their kidneys. The kidneys of both sides were divided into two halves along the sagittal line on ice and preserved in three parts: ① the kidneys were frozen in liquid nitrogen after being infiltrated with RNA protective solution and stored in -80℃ for real-time PCR experiment; ②for detection of oxidative stress index after tissue homogenization saved as above; ③ the kidneys were fixed in paraformaldehyde for immunohistochemistry and PAS staining.
2.5 Immunohistochemistry and pathological section observation
Part of the right kidney was paraffin-embedded, dewaxed, dehydrated, soaked and washed. The negative control used PBS instead of primary and secondary antibodies. For observation, DAB was used for coloration, as described previously.
Pathological section observation: The kidneys were observed under a light microscope as described previously.
2.6 Detection of urinary albumin/urinary creatinine
Urinary albumin/urinary creatinine (mg/g) was detected by an BECKMAN COULTER iRICELL3000 Automatic urinalysis line on November 12, 2019.
2.7 Detection of serum creatinine and urea nitrogen
Serum creatinine (µmol/L) and urea nitrogen (mmol/L) were detected by an HITACHI 7600 automatic biochemical analyzer on November 28, 2019.
2.8 Detection of oxidative stress index and plasma vasoactive substances
The levels of malonic dialdehyde (nmol/L) and superoxide dismutase (U.ml-1) in the kidney were measured by mature commercial kits on November 18, 2019.
2.9 Quantitative real-time PCR
Real-time polymerase chain reaction analysis was performed as described previously.Oligonucleotide PCR primers for rat genes were as Table 1.
3. Statistical analysis
The entire analyses used SPSS version 24.0. All values are expressed as the means ± SD. Single factor analysis of variance for statistical analysis of data. P<0.05 was considered statistically significant.