Urine protein test kit

The kidney weight (KW)/body weight (BW) was calculated after all rats were sacrificed. The rats' FBG levels were estimated at 4th, 8th, 12^th, and 16th weeks with rapid blood glucose meter (Johnson & Johnson Medical Ltd., USA). The levels of creatinine (SCr), urea nitrogen (BUN), cholesterol (CHO), and triglyceride (TG) were determined by a Hitachi automatic biochemical analyzer (7600–020, Hitachi, Tokyo, Japan). A urine protein test kit (Nanjing Jiancheng Bioengineering Institute, Jiangsu, China) was used to measure urinary protein quantity, and the urinary protein quantity was expressed as milligrams per 24 h.

Fasting blood glucose levels were monitored with Glucometerusing one drop of tail blood every week after injection with STZ 2 weeks. Blood urea nitrogen (BUN) was measured using the Urea Assay Kit (C013, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). HbA1c was measured using Glycosylated hemoglobin assay kit (A056, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Proteinuria was measured using a Urine protein test kit (C035, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Serum creatinine (Scr) was measured using a Creatinine (Cr) Assay kit (C011, Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The NO levels from serum and renal tissue were detected to evaluate the oxidative stress and the magnitude of vascular damage in diabetic rats. This NO was quantified by Nitric Oxide (NO) assay kit (A013, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) and BCA Protein Quantification Kit (PC0020, Solarbio, Beijing, China).

Amomum tsao-ko (Hebei yishengshan traditional Chinese Medicine Co., Ltd.); GM sulfate injection; verapamil (abbvie Deutschland GmbH & Co. KG, Düsseldorf, Germany); creatinine (Cr), urea nitrogen (BUN), a urine protein test kit, lysozyme (LZM), β-b-acetyl-glucosaminidase (NAG), nitric oxide (NO), nitric oxide synthase (NOS), superoxide dismutase (SOD), malondialdehyde (MDA), and total protein quantitative test kit (BCA) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China); rat IL-6, rat IL-1β, and a rat TNF-α ELISA kit were purchased from Lianke Biology Co. Ltd., Hangzhou, China; P-SAPK/JNK antibody, p38/P-p38, NF-κB p65 were purchased from Cell Signaling Technology; ERK1/2 antibody, P-ERK1 + ERK2, caspase-3, Bax, Bcl-2, Goat anti-rabbit antibody, and Goat anti-mouse antibody were purchased from Wuhan bode Biology Co., Ltd., Wuhan, China; and GADPH TA-08 and β-Actin TA-09 were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd., Shanghai, China.

Body weight was measured every week. In the meantime, fasting blood glucose (FBG) was measured at 4-week intervals and monitored with a Roche ACCU-CHEK Active Glucometer (Roche Diabetes Care GmbH, Mannheim, Germany). Rats were housed in individual metabolic cages every 4 weeks for 24 h urine collection. Urinary protein excretion was determined by using a urine protein test kit (Nanjing Jiancheng Bioengineering Institute, China).
At the end of the study, blood samples were collected from the abdominal aorta. Serum was separated (3,500 r/min, 10 min) for the detection of Scr, BUN, total cholesterol (TC), triglycerides (TG), total protein (TP) and serum albumin (ALB) by an automatic biochemistry analyser (Chemray 240, Rayto Life and Analytical Sciences Co., Ltd, China).
The right kidneys of rats in each group were removed and weighed after blood collection. The kidney hypertrophy index (KHI) was calculated according to the method described previously, that is, KHI = kidney weight (KW)/BW^{57 (link)}.

The Creatinine Assay kit (C011-2-1, JianCheng, Nanjing, Jiangsu, China) and Urine Protein Test kit (C035-2-1, JianCheng) were used to analyze serum and urine creatinine and protein levels in mice. The protein-to-creatinine ratio in mice urine was also measured to assess changes in mouse renal function.

Axitinib was synthesized by the Pharmaceutical Design and Synthesis Laboratory of Tianjin University of Science and Technology. Losartan was purchased from Anaiji Chemical Co., Ltd. (Shanghai, China). Trypsin 0.25% solution (Hyclone), RPMI (Roswell Park Memorial Institute) medium 1640 basic (Gibco) and fetal bovine serum were obtained from Solarbio Co., Ltd. (Beijing, China). PBS (Phosphate Buffer Saline, 10×) solution was prepared by NaCl (80.00 g), Na₂HPO₄•12H₂O (29 g), KCl (2 g), KH₂PO₄ (2 g), which was dissolved in 1 L of sterile double distilled water. The formalin solution was made up of 100 mL of 40% formaldehyde solution and 900 mL of 1 × PBS solution. The aspartate aminotransferase assay kit, alanine aminotransferase assay kit, creatinine (Cr) assay kit (sarcosine oxidase), urine protein test kit, endothelin-1 assay kit, and endothelial nitric oxide synthase assay kit were purchased from Nanjing Jiancheng Bioengineering Institute. The 786-O (Human renal clear cell adenocarcinoma) was furnished from Peking Union Medical College. The Wistar rats, C57BL/6N mice, and BALB/c nude mice were purchased from Charles River Laboratories, Beijing, all animal experiments carried out in accordance with the National Institutes of Health guide for the care and use of Laboratory animals and approved by the Academic Committee of Tianjin University of Science and Technology.