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

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.

Introduction

Organic anion transporter 1 (OAT1, <span class="Gene">SLC22A6) and 3 (OAT3, SLC22A8) are organic anion/dicarboxylate exchangers, which are highly expressed in the kidney.[1] Using dicarboxylates such as α-ketoglutarate concentration gradient as a driving force, they contribute to active tubular secretion via the uptake of various anionic compounds from the blood into the renal tubular cells. OAT1 and OAT3 transport various endogenous and exogenous compounds, including prescribed drugs such as nonsteroidal anti-inflammatory drugs (NSAIDs), antifolates, and antiviral nucleoside analogues.[2,3] OAT1 and OAT3 inhibition causes either harmful or beneficial drug–drug interactions. For example, severe cytotoxicity-induced cell death has occurred with the co-administration of high doses of the antifolate methotrexate and NSAIDs, where the latter inhibit the renal tubular secretion of methotrexate by OAT1 and OAT3.[4−7] Conversely, the co-administration of the antiviral nucleoside analogue cidofovir and the strong OAT1 and OAT3 inhibitor probenecid prevents cidofovir-induced nephrotoxicity via cidofovir uptake inhibition.[8−11] Therefore, OAT1 and OAT3 inhibition is of considerable clinical importance.

The metabolomic analysis of Oat1 or <span class="Gene">Oat3 knockout mice revealed that OAT1 and OAT3 are not only involved in the urinary excretion of drugs but also involved deeply in the pharmacokinetics of endogenous compounds, dietary components, and their metabolites.[12,13] These findings suggest the importance of OAT-mediated food–drug interactions. Moreover, the renal handling of the flavonoid and its effect on renal transporters is an important issue from the standpoint of nutritional research. For OAT1 and OAT3, interactions with various dietary components, such as flavonoids and their conjugates, phenolic acids, and phenylpropanoids, are demonstrated by the inhibition of OAT-mediated transport of model substrates.[14−19] We previously investigated the effects of natural compounds on OAT1 and OAT3 function using human OAT1- and OAT3-expressing cells and the fluorescent organic anion 6-carboxyfluorescein (6-CF) and found that several phenylpropanoids inhibit OAT1 and OAT3 functions.[20]

Green tea (<span class="Species">Camellia sinensis) is a popular beverage in the world. It is receiving increased attention because of its beneficial antioxidant, antiobesity, antimicrobial, antiviral, and antitumor activities.[21−24] Green tea is rich in phenolic compounds such as catechins, which account for 30–40% of dry weight of green tea.[22] One of them, (−)-epigallocatechin-3-gallate (EGCG), is the most abundant catechins, representing 50–80% of the total catechins and believed to be the most potent component of green tea.[25] For example, EGCG has the most potent antioxidants and anticancer activities of the green tea catechins.[21] Furthermore, EGCG supplements are commercially available with the general public having the opportunity to consume them. Although green tea catechins including EGCG are beneficial for health, they may alter drug pharmacokinetics. Some EGCG–drug interactions have been reported. For example, orally co-administered EGCG decreases the maximum plasma concentration (Cmax) and area under the concentration versus time curve (AUC) of nadolol.[26] The inhibition of the intestinal uptake transporter organic anion transporting polypeptide 2B1 (OATP2B1, SLCO2B1) may contribute to this interaction. Inhibitions of other drug transporters by EGCG have also been reported. For instance, EGCG inhibits the efflux of daunorubicin and rhodamine 123 by P-glycoprotein (MDR1, ABCB1) and the uptake of tetraethylammonium (TEA) by multidrug and toxin extrusion 1 (MATE1, SLC47A1).[27,28]

This study focused on EGCG, which is the most abundant and biologically active component in the green <span class="Chemical">tea catechins. First, we evaluated that 6-CF would be a fluorescent substrate of rat Oat1 and Oat3. Then, we investigated the effects of EGCG on 6-CF transport mediated by human (h) and rat (r) OAT1 and OAT3. Finally, we investigated the effects of OAT inhibitors and EGCG on the renal elimination of 6-CF in rats to show the in vitro and in vivo interactions of the tea catechin EGCG and renal OATs.

Results

Effects of EGCG on 6-CF Transport by hOAT1, hOAT3, rOat1, and rOat3

We previously reported that 6-CF was transported by <span class="Gene">hOAT1 and hOAT3 in a time- and concentration-dependent manner.[20] The Km and Vmax values were 6.94 ± 0.33 and 38.4 ± 4.2 μM and 125.1 ± 1.6 and 70.0 ± 3.2 pmol/mg/2 min for hOAT1 and hOAT3, respectively.

Here, we first investigated the transport characteristics of 6-CF mediated by <span class="Gene">rOat1 and rOat3. 6-CF was transported in a time-dependent manner for up to 30 min, and the uptake was linear until 2 min (Figures A and S1). 6-CF uptake was transported in a concentration-dependent manner, but the uptake was saturable (Figure B). The Km values were 6.68 ± 0.62 and 2.52 ± 0.48 μM for rOat1 and rOat3, respectively. The Vmax values were calculated to be 131 ± 3 and 35.3 ± 1.3 pmol/mg/2 min for rOat1 and rOat3, respectively. From these results, the subsequent studies were conducted using 1 μM 6-CF for 2 min.

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).

6-CF uptake by <span class="Gene">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).

We next examined the effects of known OAT substrate/inhibitors and <span class="Chemical">TEA, a typical organic cation transporter substrate, on 6-CF transport mediated by human and rat OAT1 and OAT3 (Figure A). TEA showed no effects on human and rat OAT functions. Probenecid and prostaglandin E2 (PGE2) strongly (more than 80%) inhibited human and rat OAT1 and OAT3. The OAT1 versus OAT3 inhibition correlation plot showed a positive correlation but was not linear (r, 0.657 and 0.731, and p, 0.109 and 0.0619, for hOAT1 vs hOAT3 and rOat1 vs rOat3, respectively) (Figure B,D). In particular, the inhibitory effects of adefovir and p-aminohippurate were higher for OAT1 than those for OAT3, which bent the regression curves (Figure B,D). On the other hand, the human versus rat inhibition plot showed linearity, suggesting that each OAT isoform has no species difference between human and rat (r, 0.987 and 0.967, and p, 3.47 × 10–5 and 3.85 × 10–4, for hOAT1 vs rOat1 and hOAT3 vs rOat3, respectively) (Figure C,E). From these results, 6-CF is a suitable in vitro fluorescent substrate for rat Oats and for human OATs.

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.

Effect of organic ions on 6-CF uptake by <span class="Species">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.

EGCG inhibited <span class="Species">human and rat OAT1 and OAT3 (Figure ). While hOAT1 showed a mixed-type inhibition, hOAT3, rOat1, and rOat3 showed a competitive inhibition. The Ki values were estimated to be 334 ± 58, 162 ± 29, 595 ± 89, and 56.5 ± 19.2 μM for hOAT1, hOAT3, rOat1, and rOat3, respectively.

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).

Effect of EGCG on <span class="Chemical">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).

Effects of EGCG on the Renal Elimination of 6-CF in Rats

We then investigated whether 6-CF could be transported by r<span class="Species">Oats in vivo. At a dose of 1 mg/kg, the initial concentration of the total 6-CF (C0) was estimated to be 14.6 μg/mL = 38.8 μM (Figure A). Since the binding of carboxyfluorescein to serum protein was ∼90%,[30] the initial concentrations of the unbound forms of 6-CF (C0,u) were estimated to be 4 μM at this dose. From these results, the subsequent studies were conducted at 1 mg/kg to achieve an unbound plasma concentration (Cp,u) of ∼1 μM, an equivalent concentration to that of in vitro studies.

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.

Effect of probenecid, <span class="Chemical">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.

Next, we examined the effect of probenecid, an OAT inhibitor, and <span class="Chemical">d-malate, a reducing agent of α-ketoglutarate (a driving force for OATs),[29] on the plasma–time and urinary excretion profile of 6-CF. Probenecid (100 mg/kg) and d-malate (2.5 mL/kg bolus and infusion at 1.8 mL/h of 2% solution) significantly decreased the renal clearance (CLr) of 6-CF (∼6- and 4-fold, respectively) (Figure and Table ).

Table 1

Pharmacokinetic and Renal Parameters of 6-CF after the Intravenous Administration of a Bolus Dose of 1 mg/kg to Ratsa

	control	probenecid	d-malate	EGCG
N	6	6	6	7–8
6-CF AUC0–60 (μg·min/mL)	34.4 ± 6.4	163.5 ± 10.4*	62.4 ± 39.6*	73.9 ± 16.0*
6-CF CLr (mL/min/kg)	18.3 ± 6.8	2.8 ± 1.1*	4.9 ± 2.5*	7.4 ± 2.7*
Ccr (mL/min/kg)	3.29 ± 0.69	2.51 ± 0.78	2.92 ± 0.66	2.58 ± 0.82
BUN (mg/dL)	12.5 ± 1.6	12.6 ± 0.7	11.4 ± 1.1	11.9 ± 1.3

Each parameter represents the standard deviations. *p < 0.05, versus control by Dunnett’s test.

6-CF was co-administered with <span class="Chemical">EGCG (60 mg/kg) intravenously in rats, and the concentrations of 6-CF in the blood and urine were measured. EGCG increased (∼8-fold) the plasma concentration of 6-CF at 60 min. AUC until 60 min (AUC0–60) was also significantly increased by EGCG co-administration (∼2.2-fold) (Figure and Table ). By contrast, EGCG significantly (∼2.6-fold) decreased the renal clearance of 6-CF (Figure and Table ).

Another series of experiments were performed to elucidate the interaction between 6-CF and <span class="Chemical">EGCG which was not caused by its nephrotoxicity. With the administration of probenecid, d-malate, or EGCG, no significant change was observed in creatinine clearance (Ccr) and blood urea nitrogen (BUN) (Table ).

Discussion

Inhibitor Profile of Human and Rat OAT1- and OAT3-Mediated 6-CF Transport

Human and <span class="Species">rat OATs have similar topologies consisting of 12 putative transmembrane domains (TM), extracellular long loop in TM1–TM2, and intracellular long loop in TM6–TM7 (Figure S2A). The amino acid sequences of rOat1, hOAT3, and rOat3 exhibited 85.97, 48.92, and 47.41% identities to that of hOAT1 and that of rOat3 exhibited 79.66% identity to that of hOAT3, suggesting that the difference between isoforms was larger than that between species (Figure S2B,C). Both human and rat OAT1 and OAT3 localized on the basolateral membranes of the renal proximal tubules and contributed to numerous small-molecule xenobiotics. Besides the kidneys, both OAT1 and OAT3 were expressed in the choroid plexus, and OAT3 was also expressed in the brain endothelium, retina, and testes.[1]

The ligand recognition of OAT1 and <span class="Gene">OAT3 was broad and highly overlapped. Judged from the Km values, although hOAT1 and rOat1 showed similar affinity to 6-CF, rOat3 showed higher affinity than that of hOAT3. OAT1 and OAT3 tended to prefer hydrophilic and hydrophobic compounds, respectively.[31] We previously reported that this tendency is also observed in prenyloxyphenylpropanoids.[20] Although the compounds studied in this study were few in number and used only at one concentration (100 μM), the OAT1 versus OAT3 plot for inhibition (Figure B,D) suggested the different inhibitor profile for OAT1 and OAT3. Adefovir and p-aminohippurate suggested to be OAT1-selective, which is consistent with the aforementioned hydrophilic and hydrophobic tendency of ligand selectivity (logP values: adefovir, −4.5; p-aminohippurate, −0.5; methotrexate, −0.5; probenecid, 2.4; PGE2, 3.2; and phenolsulfonphthalein, 4.1). Furthermore, the linearity of human versus rat inhibitory activity plots suggests that there were few species differences between human and rat in inhibitory characteristics (OAT1 and OAT3; Figure C,E).

Human and Rat OAT1 and OAT3 Transport 6-CF

Multiple research groups have used the 6-CF as a model subst<span class="Species">rate to study the functions of hOAT1 and hOAT3 in vitro.[18,20,32−36] Furthermore, 6-CF was used for ex vivo assays as OAT-selective substrates.[35,37] Consequently, there is one report using 6-CF for in vivo study. Woolfrey et al. showed the time–plasma concentration profile of 6-CF in rats, but they did not investigate the urinary excretion.[38] In this study, we investigated the time–plasma concentration and urinary excretion of 6-CF in rats. We demonstrated that 6-CF is eliminated in the urine. 6-CF CLr/Ccr was over 1, showing active tubular secretion (Figure and Table ). We previously reported that d-malate interferes with the active tubular secretion of phenolsulfonphthalein.[29] The underlying mechanism was presumed that d-malate inhibited sodium-dependent dicarboxylate transporter 1 (NaDC1, SLC13A2)-mediated reabsorption of α-ketoglutarate, a driving force of OATs, and then decreased the amount of α-ketoglutarate in the kidney cortex. Here, the active secretion of 6-CF was inhibited by probenecid and d-malate, suggesting that it was mediated by renal rOat1 and rOat3. Phenolsulfonphthalein is often used to investigate renal organic anion transport in vivo.[16,29,39,40] For example, we previously examined the effects of lithium on the renal organic anion transport using phenolsulfonphthalein.[40] Sakurai et al. used phenolsulfonphthalein for cellular study,[41] but the determination of phenolsulfonphthalein by an absorption spectrometer showed a lower limit of detection of several μM. By contrast, 6-CF could be easily determined using a fluorescence spectrometer for the nM range. Therefore, 6-CF could be useful for both in vitro and in vivo studies to reveal the role of renal OATs in drug elimination.

Recently, Nigam et al. reported that the ligand overlaps of OAT1 and <span class="Gene">OAT3 are smaller than previously assumed and suggested that OAT1 and OAT3 have potential distinct physiological effects by machine learning of in vivo metabolomics data.[42] 6-CF is the universal substrate of OAT1 and OAT3, of which structural transformations leading to the selectivity are desired. The OAT1- or OAT3-selective fluorescent substrate will accelerate the research to clarify these potential differences.

EGCG Inhibits Renal OATs

Using 6-CF as a fluorescent subst<span class="Species">rate, we demonstrated that EGCG inhibits human and rat OAT1 and OAT3. A previous report suggested that flavonoids, such as quercetin, daidzein, and their conjugates, are transported by hOAT1 and hOAT3 and interact with them at physiologically relevant concentrations.[14] Conversely, Wang and Sweet reported that flavonoids catechin and epicatechin did not interact with hOAT1.[43] Wu et al. reported that epicatechin and epicatechin gallate inhibit mouse Oat3.[13] However, the effect of the dominant green tea catechin EGCG on human and rat OAT1 and OAT3 was not examined. In this study, we demonstrated that EGCG also inhibited human and rat OAT1 and OAT3. Moreover, intravenously administered EGCG significantly decreased the renal clearance of 6-CF (Figure and Table ). Renal OAT inhibitors are useful for protecting the kidneys from toxins such as antiviral nucleoside analogues and the uremic toxin indoxyl sulfate. Although the inhibitory effect of EGCG is weak, with antioxidant activity, EGCG (and its analogues) may be useful for renal OAT inhibitors for kidney protection.

Unlike the noninhibitors of OAT1 <span class="Chemical">catechin and epicatechin, EGCG has gallate moieties. Other than EGCG, compounds containing gallate moieties are also found in herbal medicines and food additives. For example, 1,2,3,4,6-pentagalloyl-β-d-glucose is a gallotannin and a precursor of ellagitannins. It is a component of tannic acid and herbal medicines such as gallnut and peony root. Moreover, propyl gallate is an antioxidant used to preserve and stabilize edible oils, fat-containing foods, cosmetics, and medicinal preparations. Further kinetic analyses of the inhibition of OAT1 and OAT3 by gallate-type compounds are significant for the prevention and prediction of food–drug and drug–drug interactions. Since the substrate-dependent interactions of EGCG on OATP1B3 has been reported,[44] further studies using multiple substrates are necessary to evaluate the potential of EGCG–drug interactions targeted to OAT1 and OAT3.

There were no significant changes in renal parameters (Ccr and BUN) in rats treated with <span class="Chemical">EGCG, suggesting that the short-term exposure of EGCG does not affect the renal function and it inhibits the renal OATs. However, in long-term exposure, the renal function could be altered by the induction of drug transporters and/or cumulative toxicity. For example, we previously reported that the exposure of auraptene, a coumarin-related compound occurring widely in citrus fruit, inhibits the P-glycoprotein function in the short-term exposure, and auraptene induces P-glycoprotein expression in the long-term exposure.[45,46] Further investigations are needed for the effects of the long-term exposure of EGCG on the expression of drug transporters.

In usual tea drinking, the low bioavailability of <span class="Chemical">EGCG due to inadequate gastrointestinal absorption during oral administration and high Ki values for renal OAT1 and OAT3 unlikely cause the EGCG–drug interactions at the renal excretion step. However, the potential EGCG–drug interactions are worth evaluating when considering the following situations: (1) people taking high-dose EGCG supplements instead of green tea, (2) people taking a mixture containing multiple inhibitors (e.g., epicatechin and epicatechin gallate) as a green tea extract supplement, (3) patients who tend to compete in the renal excretion process such as during polypharmacy, and (4) patients at high risk of drug–drug interactions due to the decreased renal function such as elderly and chronic kidney disease. Furthermore, to develop EGCG and its analogue for novel kidney-protecting agents, administration methods other than oral administration may be considered due to its low bioavailability. In this situation, the pharmacokinetic profile obtained in this study may be a useful fundamental knowledge.

The limitations of this study should be addressed to precisely predict the risk of EGCG–drug interactions. Although significant changes in AU<span class="Chemical">C0–60 and CLr were observed from the blood and urinary 6-CF concentrations up to 1 h were investigated after a single intravenous injection, we could not obtain the pharmacokinetic data of EGCG. The pharmacokinetic–pharmacodynamic profiles and simulations from various administration design (e.g., long-term, multiple dosing, and oral administration), with both the substrate and inhibitor (EGCG), should be useful to understand the contributions of renal transporters to the EGCG excretion and its effect on renal excretion of xenobiotics. Previously, many of the endogenous substrates of OAT1 and OAT3 suggested by metabolomics of Oat knockout mice were supported by in vitro assay data.[12,13] Therefore, these knockout mice could be useful for further investigation of the effects of EGCG on renal OATs. Currently, the process of establishing the determination of EGCG concentrations in plasma and urine using liquid chromatography–tandem mass spectrometry is being done for further studies of EGCG pharmacokinetics.

In conclusion, we demonstrated that (1) <span class="Chemical">6-CF is an in vitro substrate of rOat1 and rOat3, (2) EGCG is the inhibitor of human and rat OAT1 and OAT3, and (3) the co-administration of EGCG with 6-CF significantly decreased the renal clearance of 6-CF in rats. These results suggest that 6-CF is a useful fluorescent substrate of renal OAT1 and OAT3 in vitro and in vivo and EGCG may interact with renal OATs depending on the dosage and administration method.

Materials and Methods

Materials

Plasmid vectors pTCN-empty and <span class="Chemical">pTCN-hOAT1, pTCN-hOAT3, pTCN-rOat1, and pTCN-rOat3 were from transOMIC (Huntsville, AL, USA). 6-CF was purchased from Sigma-Aldrich (St. Louis, MO, USA). EGCG was obtained from Nagara Science (Gifu, Japan). Adefovir, p-aminohippurate, methotrexate, phenolsulfonphthalein, probenecid, PGE2, and TEA were obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). All other chemicals used were of the highest purity available.

Uptake Assay

Uptake assay was performed as previously reported, with some modifications.[20,29] Briefly, HEK293 cells (8.0 × 104 cells/well) were seeded. For transient expression, expression plasmid (<span class="Chemical">pTCN-hOAT1, pTCN-hOAT3, pTCN-rOat1, pTCN-rOat3, or pTCN-empty) was transfected with polyethylenimine according to the manufacturer’s instructions. Forty-eight hours after transfection, cells were washed twice with 100 μL of Waymouth buffer (in mM: NaCl, 135; KCl, 5; MgCl2, 1.2; CaCl2, 2.5; MgSO4, 0.8; d-glucose, 28; and HEPES-NaOH, 13; pH 7.4) and preincubated in Waymouth buffer without test compounds for 10 min at 37 °C. Next, the cells were incubated for 2 min in uptake buffer (Waymouth buffer containing 1 μM 6-CF and test compounds) at 37 °C and washed twice with 100 μL of ice-cold phosphate-buffered saline. Cells were lysed with 100 μL of 1% SDS, and the fluorescence intensity was measured using a microplate reader (Spark 10M, Tecan Group Ltd., Männedorf, Switzerland). Transporter-mediated uptake was defined as the difference between the total uptake by the transporter of interest-transfected HEK293 cells and that measured in parallel using the pTCN-empty-transfected HEK293 cells.

Pharmacokinetic Experiments Using Rats

6-CF pharmacokinetic experiments were performed according to our previous report, with some modifications.[29] Animals were treated in accordance with the regulations of the Institutional Animal Care and Use Committee of the School of Pharmacy, Aichi Gakuin University. Seven-week-old male Wistar/ST <span class="Species">rats were obtained from Chubu Kagaku Shizai (Nagoya, Japan). The rats were anesthetized with ethyl carbamate and α-chloralose. Catheters were then inserted into the femoral artery and vein with polyethylene tubes (SP-31; Natsume Seisakusho, Tokyo, Japan) filled with heparin solution (50 IU/mL) for blood sampling and drug administration, respectively. Urine was collected from the bladder that was catheterized with SP-31 polyethylene tubes.

All injection solution was prepared with saline as the solvent and administered via femoral vein. 5% <span class="Chemical">mannitol in the absence (control, probenecid, and EGCG group) or presence (d-malate group) of 2% d-malate was bolus-injected intravenously at 2.5 mL/kg and then infused at 0.03 mL/min until the last blood and urine samples were taken to maintain sufficient and constant urine flow. Twenty minutes after the bolus injection of mannitol, 6-CF (1 mg/kg) was bolus-injected intravenously in the absence or presence of probenecid (100 mg/kg) or EGCG (60 mg/kg). Blood was collected 1, 2, 5, 10, 30, and 60 min after 6-CF was administered and centrifuged for plasma sampling. Urine samples were collected 0–10, 10–20, 20–40, and 40–60 min after 6-CF administration. The fluorescence intensity of 6-CF was measured using a microplate reader (Spark 10M, Tecan).

AUC0–60 was calculated using the trapezoidal rule method. CLr was obtained by dividing <span class="Chemical">6-CF excreted amounts into the urine over 60 min by AUC0–60.

The BUN was determined using QuantiChrom Urea Assay Kit II DUR2-100 (BioAssay Systems, CA, USA). The concentrations of creatinine in plasma and urine samples were measured using a LabAssay Creatinine kit (FUJIFILM Wako Pure Chemical Corporation, Osaka, JAPAN). Ccr was calculated by dividing the urinary excretion rate of creatinine by the plasma creatinine concentration.

Data Analysis

Statistical differences were compared with one-way analysis of variance, followed by Dunnett’s test using KaleidaGraph (Synergy Software, Reading, PA, USA), and were considered significant at p < 0.05. The Pearson correlation coefficient (r) and the significance of the correlation coefficient (p) were also calculated using KaleidaGraph.

In all in vitro experiments, transporter-independent uptake was determined in HEK293 cells transfected with vector control (<span class="CellLine">HEK293-VC) and is either shown or subtracted from the total uptake to quantify transporter-mediated uptake. The Km and Vmax values were obtained by fitting the Michaelis–Menten equationwhere [S] is the concentration of 6-CF, Km is the Michaelis–Menten constant, and Vmax is the maximum uptake rate. The inhibitory constant Ki value was obtained using the following equations for competitive inhibition and mixed-type inhibition, respectivelywhere α is the binding modality ratio. Akaike’s Information Criterion was used for fitting equation selection.