Ua kit

After the cells were cultured as described above for 24 h, the cells were placed in the upper chamber of ttranswell plate. The upper and lower chamber represents the apical side and basal side of kidney cells. Therefore, two different treatment methods were set for each group of cells to verify the effects of different treatment methods on UA excretion and UA reabsorption capacity of NRK-52E cells. One was treated with 300 μmol/L of UA only in the upper chamber for 24 h, and the UA level in the lower chamber was measured to reflect the effect of different intervention methods on the UA reabsorption capacity of NRK-52E cells. The other only added 300 μmol/L of UA in the lower chamber, and the effect of different intervention methods on the UA excretion ability of NRK-52E cells was reflected by measuring the UA level in the upper chamber. The UA level of upper and lower chambers was measured by colorimetric method with a UA kit (Nanjing Jiancheng Bioengineering Institute, C-012-2), aiming to reflect the UA reabsorption or excretion capacity of cells.

Hypoxanthine (HX, 99%) and oxonic acid potassium salt (OAS, 97%) were obtained from Sigma-Aldrich Co. LLC (USA). Allopurinol was purchased from Shanghai Xinyi Wanxiang Pharmaceutical Group Ltd. (Shanghai, China). UA kit was purchased from Nanjing Jian Cheng Bioengineering Institute (Nanjing, China). SP Rabbit HRP (DAB) immunohistochemical kit was obtained from Jiangsu Kang Wei Shi Ji Biotechnology Co. Ltd. (Jiangsu, China). The primers were purchased from Genscript (Nanjing, China), Trizol was commercially obtained from Invitrogen (USA), and Revert Aid First Strand cDNA Synthesis Kit and PowerUp™ SYBR™ Green Master Mix were obtained from Thermo Fisher Scientific Inc. (USA). Anti-OAT1 was purchased from Abcam Inc. (Cambridge, UK), Anti-OAT3 from Changzhou Xiangtai Biotechnology Co. Ltd. (Changzhou, China), Anti-URAT1 from Abbiotec Inc. (San Diego, CA, USA). Anti-GLUT9 was purchased from Millipore Inc. (USA). Other biochemical reagents and chemicals were of analytical grade.

The blood samples were collected from the eyeballs of the mice, and the serum was separated via centrifugation at 4000× g for 30 min. The serum samples were deposited into clean 200 μL EP tubes, and the levels of the renal function indicators blood urea nitrogen (BUN), creatinine (Cr), and uric acid (UA) were determined using the BUN kit, Cr kit, and UA kit (Nanjing Jiancheng bioengineering Institution, Nanjing, China), respectively, according to the manufacturers’ instructions.