In vitro Urate Uptake Assay in URAT1-Expressing 293A Cells

The 293A cells were seeded on a 12-well plate (Becton Dickenson & Co., Franklin Lakes, NJ, USA) at 2 × 10⁵ cells/well. Twenty-four hours after seeding, the cells were transiently transfected with the FLAG-URAT1 pcDNA3.1(+) vector or empty vector using PEI-MAX (0.4 μg plasmid/4 μL PEI-MAX/100 μL serum free DMEM/2 × 10⁵ cells) as described previously (Stiburkova et al., 2016). Forty-eight hours after the transfection, the cells were washed twice with Buffer T2 (125 mM Na-gluconate, 4.8 mM K-gluconate, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 1.3 mM Ca-gluconate, 25 mM HEPES, 5.6 mM D-glucose, and pH 7.4) and preincubated in Buffer T2 for 15 min at 37°C. Then, the buffer was exchanged to fresh Buffer T2 containing 2 μM [8-¹⁴C]-urate, and the cells were further incubated for the indicated periods. Subsequently, the cells were washed with ice-cold Buffer T2 twice and then lysed with 500 μL 0.2 M NaOH on ice with gently shaking for 1 h. The resulting lysates were transferred to 1.5-mL tubes, neutralized with 100 μL 1 M HCl, and then the radioactivity were measured using a liquid scintillator. The protein concentrations were determined using a bicinchoninic acid assay kit (Life Technologies) according to the manufacturer’s instruction. The urate transport activity was calculated as the incorporated clearance (μL/mg protein/min): (incorporated level of urate [DPM/mg protein/min]/urate level in the incubation mixture [DPM/μL]). URAT1-dependent urate transport activity was calculated by subtracting the urate transport activity of mock cells from that of the URAT1-expressing cells.