FIGURE 2.

Inhibitory effects of compound 4 on type-2 11β-hydroxysteroid dehydrogenase (11β-HSD2) using rat kidney microsome (A), and the uptake of 4 into rat kidney slices (B) or into HEK293 cells transfected with organic anion transporter (OAT) 1 (C) or OAT3 (D). (A) [³H] cortisone and glycyrrhetinic acid (GA) or 4 were mixed with rat kidney microsome fractions, and incubated at 37°C for 30 min. Then, the amount of [³H] cortisol was measured. Data are means ± standard error (S.E: n = 4) of percentage [³H] cortisol in mixtures without samples. *p < 0.05, **p < 0.01 and ***p < 0.001 compared with groups without samples using Dunnett’s multiple t-test (B) Female Eisai hyperbilirubinuria rats (EHBRs) were orally treated with GA (200 mg/kg), and their plasma samples were collected 24 h after the treatment. Concentration of 4 in plasma sample was 115 µM. Kidneys were collected from normal Sprague-Dawley (SD) rats and slices were incubated with plasma samples of female EHBRs at 37°C or 4°C for 2 h, and then homogenized. Samples were those described in our previous study (Ishiuchi et al., 2019). Concentrations of 4 in kidney slice homogenates were measured using liquid chromatography-tandem mass spectrometry (LC-MS/MS). **p < 0.01 vs 4°C group using Student’s t-test (C) Male EHBRs were orally treated with GA (200 mg/kg) and their plasma samples were collected 24 h after treatment. Concentrations of 4 in plasma sample was 189 μM. HEK293 cells transfected with OAT1 or OAT3 were incubated with 1:2 diluted plasma samples of male EHBRs at 37°C for 15 min. Then, concentrations of 4 in cells were measured using LC-MS/MS. Data are means ± S.E. (n = 6). *p < 0.05 vs mock cells using Dunnett’s multiple t-test.