2.2. Cell Viability Assay

HK-2 cells were seeded in 96-well plates (Corning Incorporated) at a concentration of 4 × 10³ cells per well. Cells were then exposed to ioversol (25 to 150 mg iodine/mL) for 30 minutes. After exposure to ioversol, the cells were washed twice using Krebs-Ringer solution and then incubated in DMEM/F12 medium with 10% fetal bovine serum for 24 hours, 48 hours, and 72 hours at 37°C in 5% CO₂ air. Cell viability was detected using a cell counting Kit-8 kit (CCK-8, Dojindo, Japan) in accordance with the manufacturer's protocol.

After the above results were analyzed, the intervention concentration of ioversol (100 mg iodine/mL, Hengrui Medicine Co., Ltd., China) was selected. To test the effects of recombinant human H2 relaxin (Protech, USA) and LY294002 on contrast-induced cell viability, HK-2 cells were exposed to RLX (10 ng/mL) and LY294002 (50 μM, Selleck Chemicals) for 1.5 hours and then treated with ioversol. The absorbance was measured at 450 nm using a microplate reader (Tecan, Durham, NC).