(-)-Epigallocatechin-3-gallate Inhibits Human and Rat Renal Organic Anion Transporters

Abstract

Organic anion transporter 1 (OAT1, SLC22A6) and 3 (OAT3, SLC22A8) are multispecific drug transporters highly expressed on the basolateral membranes of the renal proximal tubules. OAT1 and OAT3 mediate the tubular secretion of clinically significant drugs; thus, they influence the pharmacokinetics of drugs and further determine their efficacy and toxicity. OAT1 and OAT3 are also the target of drug-drug interactions. In this study, we examined the effects of the tea catechin (-)-epigallocatechin-3-gallate (EGCG) on human (h) and rat (r) OAT1 and OAT3 using the fluorescent organic anion 6-carboxyfluorescein (6-CF) and hOAT1-, hOAT3-, rOat1-, or rOat3-expressing HEK293 cells and on renal elimination of 6-CF in rats. 6-CF is transported by hOAT1, hOAT3, rOat1, and rOat3. 6-CF is urinary excreted by Oats in rats. EGCG, a dominant catechin in green tea leaf, inhibits human and rat OAT1 and OAT3 and reduces the renal elimination of 6-CF in rats. Our findings are useful for the assessment of food-drug interactions mediated by renal OATs.

Figure 1

6-CF uptake by rOat1 and rOat3. The uptake of 6-CF into HEK293 cells was measured for 2 min at 37 °C and pH 7.4 unless otherwise indicated. (A) Time-dependent uptake of 6-CF by rOat1 and rOat3. HEK293 cells transfected with vector control (HEK293-VC), rOat1 (HEK293-rOat1), or rOat3 (HEK293-rOat3) were incubated with 6-CF (5 μM) for the indicated periods. (B) Concentration-dependent uptake of 6-CF by rOat1 and rOat3. HEK293 cells were incubated for 2 min with various concentrations of 6-CF (1, 3, 10, 30, and 100 μM). The values obtained at the indicated concentrations from the HEK293-VC cells were subtracted from the corresponding values obtained from HEK293-rOat1 or HEK293-rOat3. Data are presented as the mean ± standard error (n = 3).

Figure 2

Effect of organic ions on 6-CF uptake by human and rat OAT1 and OAT3. (A) Effects of various compounds on hOAT1-, hOAT3-, rOat1-, or rOat3-mediated 6-CF uptake. The uptake of 6-CF (1 μM) was measured for 2 min at 37 °C and pH 7.4 in the presence of the indicated compounds (100 μM). The values obtained at the indicated concentrations from the HEK293-VC cells were subtracted from the corresponding values obtained from HEK293-rOat1 or HEK293-rOat3. Data are presented as the mean ± standard error (n = 3). (B–E) 2D scatter plot of the indicated inhibitory effects on human and rat OAT1 and OAT3. (B) hOAT1 vs hOAT3, (C) hOAT1 vs ratT1, (D) rOat1 vs rOat3, and (E) hOAT3 vs ratT3. PAH, p-aminohippurate; PSP, phenolsulfonphthalein; PRB, probenecid; ADF, adefovir; MTX, methotrexate; PGE₂, prostaglandin E₂; and TEA, tetraethylammonium.

Figure 3

Effect of EGCG on 6-CF uptake by human and rat OAT1 and OAT3. (A–D) Effects of EGCG on hOAT1- (A), hOAT3- (B), rOat1- (C), or rOat3- (D) mediated 6-CF uptake. The uptake of 6-CF (1–100 μM) was measured for 2 min at 37 °C and pH 7.4 in the absence or presence of EGCG. The values obtained at the indicated concentrations from the HEK293-VC cells were subtracted from the corresponding values obtained from HEK293-rOat1 or HEK293-rOat3. Data are presented as the mean ± standard error (n = 3–4) and plotted as the Eadie–Hofstee plot. The EGCG concentration is 1 mM for hOAT1 (A) and rOat1 (C) or 0.1 mM for hOAT3 (B) and rOat3 (D).

Figure 4

Effect of probenecid, d-malate, and EGCG on the plasma concentration and urinary excretion of 6-CF in rats. 5% mannitol in the absence (control, probenecid, and EGCG group) or presence of 2% d-malate was bolus-injected at 2.5 mL/kg via the femoral vein and then infused at 0.03 mL/min. Twenty minutes after the mannitol injection, 6-CF (1 mg/kg) was bolus-injected intravenously in the absence or presence of probenecid (100 mg/kg) or EGCG (60 mg/kg). (A) Mean plasma concentration–time curve of 6-CF in rats after a single dose of 6-CF. (B) Urinary excretion of 6-CF. Urine samples were collected 0–10, 10–20, 20–40, and 40–60 min after 6-CF injection. 6-CF concentration was determined, and cumulative amounts excreted into urine were calculated. Each point represents the mean ± standard deviations of 6–8 rats.