Ohashi A , Mamada K , Harada T , Naito M , Takahashi T , Aizawa S , Hasegawa H .

1 Sato Fund Nihon University School of Dentistry, 1 Dental Research Center Nihon University School of Dentistry, 1 Uemura Fund in Nihon University School of Dentistry, JP16K20429 Japan Society for the Promotion of Science

	Fig. 1. Uptake of 6RBH4 by kidney slices in the presence or absence of typical ligands of organic anion transporters. The kidney slices were prepared as described in “Materials and methods” section. The slices took up 6RBH4 and the uptake was inhibited by OAT ligands. The reagents used were 6RBH4 (10 µM and 3 mM), penicillin G (PCG, 1 mM), probenecid (PBC, 1 mM) and p-aminohippuric acid (PAH, 1 mM). The uptake of BH4 for 15 min was expressed as a portion of the clearance. *P < 0.05, **P < 0.01 (Holm’s test); each point represents the mean ± S.D. (n = 3–7)
	Fig. 2. Biopterin uptake by rOat1- and rOat3-expressing LLC-PK1 cells and naïve LLC-PK1 cells. Tetrahydrobiopterin uptake by OAT-expressing or naïve LLC-PK1 cells was examined under a monolayer culture (4 × 104 cells/well, 96-well analytical culture plate). a LLC-PK1 cells transfected with rOat1 (left) or with rOat3 (right) were used. The cells were exposed for 1 h to 50 µM 6RBH4 in the absence (gray bars, control labeled “none”) or in the presence (open bars) of OAT ligands (1 mM each except for methotrexate (MTX) at 80 µM). The rOat1- and rOat3-expressing LLC-PK1 cells took up 6RBH4 and the uptake was inhibited by the ligands of OAT1 and OAT3. The OAT ligands used were probenecid (PBC), estronesulfate (ES), p-aminohippuric acid (PAH), penicillin G (PCG), methotrexate (MTX) and cimetidine (CIM). b rOat1-LLC-PK1 cells (left) or rOat3- LLC-PK1 cells (right) were given 50 µM each of 6RBH4, dihydrobiopterin (BH2) or sepiapterin (SP) in the absence (gray bars) or presence (open bars) of 1 mM PBC for 1 h. The resultant biopterin accumulations of BH2 + BH4 were then compared between those in the absence of PBC vs the presence of PBC, and levels of BH4 vs. BH2 and of BH2 vs SP. c Uptakes of 6RBH4 and BH2 (50 µM each) by naïve LLC-PK1 cells were also compared (left panel). The uptake of 6RBH4 (50 µM) for 1 h (right panel) was analyzed in the absence (gray bar, labeled “none”) or presence (open bars) of 200 µM nitrobenzylthioinosine (NBMPR) or 1 mM PBC. The uptake of the pterins was expressed as a portion of the clearance. *P < 0.05, **P < 0.01 (Holm’s test); †† P < 0.01 (Student’s t-test); each point represents the mean ± S.D. (n = 5–6)
	Fig. 3. Uptake of 6RBH4, BH2, and sepiapterin by hOAT1- and hOAT3-expressing Xenopus oocytes. Xenopus oocytes were individually injected (gray bars) with 50 nL of hOAT1 (a) or hOAT3 (b) cRNA (1 ng/nL). As the control (hatched bars), oocytes were injected with 50 nL of distilled water. The oocytes were then allowed to express the respective transporters at 19 °C for 2 days. In the uptake experiment, all pterins were used at 50 µM. All oocytes injected with either cRNA took up significantly more pterins than the control and the uptake was inhibited by OAT ligands (1 mM each). The OAT ligands used were probenecid (PBC), p-aminohippuric acid (PAH), estronesulfate (ES) and penicillin G (PCG). a 6RBH4, BH2, and sepiapterin (SP) were taken up by hOAT1-expressing oocytes (main panel) for 1 h, and the BH2 uptake was analyzed in the absence (gray bar, labeled “none”) or presence (open bars) of OAT1 ligands (upper panel). (b) Uptake of 6RBH4, BH2, and SP by hOAT3-expressing oocytes (main panel) and inhibition of BH4 uptake by OAT3 ligands in the absence (gray bar, labeled “none”) or presence (open bars) of OAT3 ligands (upper panel). The uptake of the pterins was expressed as a portion of the clearance. *P < 0.05, **P < 0.01 (Holm’s test); each point represents the mean ± S.D. (n = 4–9)
	Fig. 4. Elevation of blood BP accompanied by a decrease in the urinary loss of endogenous BP with a single dose of probenecid. Rats were given probenecid (PBC, 200 mg/kg, i.p.), and the blood (a) and urine (b) were collected sequentially from individual rats at the indicated times under sustained anesthesia for 6 h, then the kidney and liver (c) were dissected from the same rats at 6 h after the PBC dosing. The 0-time samples were taken from the rats without PBC treatment. The 0-time amounts of BH2 + BH4 (BP, open symbols) were compared with those of PBC-treated rat samples (grey symbols). In a and b, *P < 0.05, **P < 0.01 (“0-time” vs. “PBC-treated”, paired Student’s test), and in c, **P < 0.01, or n.s., no significant difference (“before” vs. “PBC-treated”, Williams’ test). Data are mean ± S.E. (n = 4)

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