Mandal AK et al. (2017), Uricosuric targets of tranilast.

XB-ART-53172

Pharmacol Res Perspect 2017 Feb 06;52:e00291. doi: 10.1002/prp2.291.

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Uricosuric targets of tranilast.

Mandal AK , Mercado A , Foster A , Zandi-Nejad K , Mount DB .

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Uric acid, generated from the metabolism of purines, has both proven and emerging roles in human disease. Serum uric acid in humans is determined by production and by the net balance of reabsorption and secretion in kidney and intestine. In the human kidney, epithelial reabsorption dominates over secretion, such that in normal subjects there is at least 90% net reabsorption of filtered urate resulting in a fractional excretion of <10%. Tranilast, an anti-inflammatory drug with pleiotropic effects, has a marked hypouricemic, uricosuric effect in humans. We report here that tranilast is a potent inhibitor of [14C]-urate transport mediated by the major reabsorptive urate transporters (URAT1, GLUT9, OAT4, and OAT10) in Xenopus oocytes; this provides an unequivocal molecular mechanism for the drug's uricosuric effect. Tranilast was found to inhibit urate transport mediated by URAT1 and GLUT9 in a fully reversible and noncompetitive (mixed) manner. In addition, tranilast inhibits the secretory urate transporters NPT1, OAT1, and OAT3 without affecting the secretory efflux pump ABCG2. Notably, while benzbromarone and probenecid inhibited urate as well as nicotinate transport, tranilast inhibited the urate transport function of URAT1, GLUT9, OAT4, OAT10, and NPT1, without significantly affecting nicotinate transport mediated by SMCT1 (IC 50 ~1.1 mmol/L), SMCT2 (IC 50 ~1.0 mmol/L), and URAT1 (IC 50 ~178 μmol/L). In summary, tranilast causes uricosuria by inhibiting all the major reabsorptive urate transporters, selectively affecting urate over nicotinate transport. These data have implications for the treatment of hyperuricemia and gout, the pharmacology of tranilast, and the structure-function analysis of urate transport.

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Species referenced: Xenopus
Genes referenced: abcg2 gnl3 kcnk3 slc22a13 slc2a9 slc5a8 tirap

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	Figure 1. Tranilast inhibits urate transport mediated by the human urate transporter URAT1 expressed in Xenopus laevis oocytes. (A) Time course of [14C]‐urate uptake by control and URAT1‐expressing oocytes. [14C]‐urate uptake by URAT1‐expressing oocytes was trans‐stimulated by preinjecting with 50 nL of 100 mmol/L PZA or nicotinate (Nico) 2 h before [14C]‐urate uptake experiment. Urate uptakes in the control oocytes were identical, resulting in superimposed data. Inset, western blot analysis of URAT1 protein expressed in oocytes. (B) [14C]‐urate transport properties of URAT1: voltage sensitivity and Na+‐dependence was examined by replacing extracellular NaCl by KCl or LiCl. Chloride effects were also examined; for the 0 Cl− bath, NaCl was replaced by Na‐gluconate (Na‐Gluc), KCl by K‐gluconate (K‐Gluc), MgCl2 by Mg‐gluconate, and CaCl2 by Ca‐gluconate. P < 0.001 compared with NaCl (ND96). (C) Trans‐stimulatory effects of preloaded organic anions on [14C]‐urate uptake by URAT1‐expressing oocytes: [14C]‐urate uptake rate via URAT1 was measured in URAT1‐expressing oocytes preloaded with 50 nL of 100 mmol/L Lac, Nico, PZA, PAH, BHB or salicylate (Sal). P < 0.001 compared with water‐injected control; NS, not significant. (D). Inhibition of [14C]urate by URAT1 in the presence of extracellular organic anions or uricosuric drugs, including tranilast: the uptake of [14C]‐urate (40 μmol/L) by URAT1‐expressing oocytes in exchange of preloaded intracellular nicotinate was determined after 1 h in the absence or presence of inhibitors (uricosuric drugs or organic anions) that were added to the extracellular medium (pH 7.4) at the indicated concentrations. *P < 0.001 compared with DMSO. Tran, Benz, Prob, DMSO. Data are mean ± S.E. with n = 12–15. (E) The 50% inhibitory concentration (IC 50) curve of tranilast for [14C]‐urate uptake‐mediated by URAT1 in oocytes preloaded with nicotinate. PZA, pyrazine carboxylate; PAH, para‐aminohippurate; Tran, tranilast; Benz, benzbromarone; Prob, probenecid; DMSO, dimethylsulfoxide.
	Figure 2. Tranilast inhibits urate transport mediated by the human urate transporter GLUT9a. (A) Time course of [14C]‐urate uptake by control and GLUT9a‐expressing oocytes. Inset, western blot analysis of GLUT9a protein expressed in oocytes. (B) [14C]urate transport properties of GLUT9a: voltage sensitivity and Na+‐dependence was examined by replacing extracellular NaCl by KCl or LiCl. We also assessed [14C]‐urate uptake by GLUT9a in the absence of extracellular Cl− ion (0 Cl−); in 0 Cl− bath, NaCl was replaced by Na‐gluconate (Na‐Gluc), KCl by k‐gluconate (K‐Gluc), MgCl2 by Mg‐gluconate, and CaCl2 by Ca‐gluconate. Asterisk, P < 0.001 compared with NaCl (ND96); NS, not significant. (C) Inhibition of [14C]‐urate uptake by GLUT9a in the presence of antiuricosuric or uricosuric drugs: The uptake of [14C]‐urate (40 μmol/L) by GLUT9a‐expressing oocytes was determined in complete absence of Na+ (NaCl of ND96 medium was replaced by KCl) after 1 h in the absence or presence of inhibitors (uricosuric drugs) which were added to the extracellular medium (pH 7.4) at the indicated concentrations. P < 0.001 compared with DMSO. (D) Inhibition of [14C]‐urate uptake by GLUT9a in depolarized cells. The uptake of [14C]‐urate (40 μmol/L) by GLUT9a‐expressing oocytes was determined in the complete absence of Na+ (NaCl of ND96 medium was replaced by KCl) after 1 h in the absence or presence of inhibitors (uricosuric drugs) at the indicated concentrations. P < 0.001 compared with DMSO. Tran, Benz Prob, Sal, DMSO. Data are mean ± S.E. with n = 12–15. Data are mean ± S.E. with n = 12–15. (D) The 50% inhibitory concentration (IC 50) curve of tranilast for [14C]‐urate uptake via GLUT9a. Tran, tranilast; Benz, benzbromarone; Prob, probenecid; Sal, salicylate; DMSO, dimethylsulfoxide.
	Figure 3. Tranilast inhibits urate transport mediated by the human urate/dicarboxylate exchanger OAT4. (A) The uptake of [14C]‐urate by OAT4‐expressing oocytes was performed in ND96 medium (pH 7.4) at ~25°C for 1 h. Na+‐dependence was examined by replacing extracellular NaCl by KCl. The [14C]‐urate uptake by OAT4‐expressing oocytes was found to be significantly trans‐stimulated when OAT4‐expressing oocytes were preinjected with 50 nL of 100 mmol/L maleate (Mal); preinjection with 50 nL of 100 mmol/L succinate (Succi), α‐ketogluterate (α‐KG), nicotinate (Nico), or PZA had no significant effects. Asterisk, P < 0.001 compared with water‐injected control. (B) The inhibition of [14C]‐urate uptake by OAT4 was examined in the presence of extracellular organic anions (for cis‐inhibition) or uricosuric drugs at the indicated concentrations. Asterisk, P < 0.001 compared with DMSO/NaCl(ND96). Tran, Benz, Prob, DMSO. Data are mean ± S.E. with n = 12–15. PZA, pyrazine carboxylate; Tran, Tranilast; Benz, Benzbromarone; Prob, probenecid; DMSO, dimethylsulfoxide; Lac, lactate; Nico, nicotinate.
	Figure 4. Tranilast inhibits urate transport mediated by the human urate transporter OAT10. (A) Time course of [14C]‐urate uptake by control and URAT1‐expressing oocytes. [14C]‐urate uptake by OAT10‐expressing oocytes was trans‐stimulated by preinjecting with 50 nL of 100 mmol/L PZA 2 h. Inset, western blot analysis of OAT10 protein expressed in oocytes. (B) Trans‐stimulatory effects of preloaded organic anions on [14C]‐urate uptake by OAT10‐expressing oocytes: [14C]‐urate uptake rate mediated by OAT10 was measured in OAT10‐expressing oocytes preloaded with 50 nL of 100 mmol/L Lac, Nico, PZA, PAH, BHB, α‐ketoglutarate (α‐KG) or succinate (Succi). P < 0.001 compared with water‐injected control. (C) [14C]‐urate transport properties of OAT10: voltage sensitivity and Na+‐dependence was examined by replacing extracellular NaCl by KCl or LiCl. Existence of urate/Cl− exchange mechanism or Cl− dependence or cis‐inhibitory effects of extracellular Cl− on was verified by [14C]‐urate uptake via OAT10 in the absence of extracellular Cl− ion (0 Cl−). In 0 Cl− bath, NaCl was replaced by Na‐gluconate (Na‐Gluc), KCl by k‐gluconate (K‐Gluc), MgCl2 by Mg‐gluconate, and CaCl2 by Ca‐gluconate. Asterisk, P < 0.001 compared with NaCl (ND96); NS, not significant. (D) Inhibition of [14C]‐urate by OAT10 in the presence of extracellular organic anions or uricosuric drugs: The uptake of [14C]‐urate (40 μmol/L) by OAT10‐expressing oocytes in exchange of preloaded intracellular PZA was determined after 1 h in the absence or presence of inhibitors (uricosuric drugs) that were added to the extracellular medium at the indicated concentrations. P < 0.001 compared with NaCl (ND96). Tran, Benz, Prob, DMSO. Data are mean ± S.E. with n = 12–15. (E) The 50% inhibitory concentration (IC 50) curve of tranilast for [14C]‐urate uptake via OAT10 in oocytes preloaded with PZA. PZA, pyrazine carboxylate; PAH, para‐aminohippurate; Lac, lactate; Nico, nicotinate; PZA, pyrazine carboxylate; Tran, tranilast; Benz, benzbromarone; Prob, probenecid; DMSO, dimethylsulfoxide.
	Figure 5. Tranilast inhibits urate transport activity of URAT1 and GLUT9a in a noncompetitive (mixed) manner. (A) The kinetic curve of URAT1‐mediated [14C]‐urate uptake in the absence or presence of inhibitor (cold urate or tranilast). (B) The kinetic curve of GLUT9a‐mediated [14C]‐urate uptake in the absence or presence of inhibitor (cold urate or tranilast). (C) Eadie–Hofstee plot of URAT1‐mediated [14C]‐urate uptake in the absence or presence of inhibitor. (D) Eadie–Hofstee plot of GLUT9a‐mediated [14C]‐urate uptake in the absence or presence of inhibitor. V, the [14C]‐urate uptake rate in pmol/oocyte/h; V/S, [14C]‐urate uptake rate per concentration (μmol/L) of [14C]‐urate; open circle, in the absence of inhibitor; open circle, in the absence of inhibitor; open triangle, in the presence 50 μmol/L cold urate; closed circle 15 or 20 μmol/L tranilast; closed triangle 30 or 40 μmol/L tranilast. Tran, Benz, Prob, SalDMSO, W, oocytes were washed out of drugs with uptake medium. Data are mean ± S.E. with n = 12–15. Tran, Tranilast; Benz, Benzbromarone; Prob, probenecid; Sal, salicylate; DMSO, dimethylsulfoxide; W, oocytes were washed out of drugs with uptake medium.
	Figure 6. Functional characterization of human URAT1 as a nicotinate transporter; effects of uricosuric drugs. (A) Trans‐stimulatory effects of preloaded organic anions on [14C]‐nicotinate uptake by URAT1‐expressing oocytes: [14C]‐nicotinate transport rate via URAT1 was measured in URAT1‐expressing oocytes preloaded with 50 nL of 100 mmol/L Lac, PZA, PAH, BHB, or urate. P < 0.001 compared with water‐injected control; NS, not significant. (B) Inhibition of [14C]‐nicotinate by URAT1 in the presence of extracellular urate or uricosuric drugs: The uptake of [14C]‐nicotinate (40 μmol/L) by URAT1‐expressing oocytes in exchange of preloaded intracellular PZA was determined after 1 h in the absence or presence of inhibitors (uricosuric drugs) that were added to the extracellular medium (pH 7.4) at the indicated concentrations. P < 0.001 compared with DMSO/NaCl(ND96). Tran, Benz, Prob, DMSO. (C) The 50% inhibitory concentration (IC 50) curve of tranilast for [14C]‐nicotinate uptake by URAT1‐expressing oocytes preinjected with 50 nL of 100 mmol/L PZA 2 h before [14C]‐nicotinate uptake experiment. Data are mean ± S.E. with n = 12–15. BHB, β‐hydroxy butyrate; PAH, para‐aminohippurate; Lac, lactate, PZA, pyrazine carboxylate; PAHe , para‐aminohippurate; Tran, Tranilast; Benz, Benzbromarone.
	Figure 7. Functional and pharmacological characterization of human nicotinate transporters, hSMCT1, SMCT2, and OAT10. The uptake of [14C]‐nicotinate (40 μmol/L) by SMCT1‐, SMCT2‐ or OAT10‐expressing oocytes was performed in ND96 medium (pH 7.4) at ~25°C. [14C]‐nicotinate transport properties of nicotinate transporters: voltage sensitivity and Na+‐dependence was examined by replacing extracellular NaCl by KCl or LiCl. Chloride ion‐dependence was also assessed; in 0 Cl− bath, NaCl was replaced by Na‐gluconate (Na‐Gluc), KCl by K‐gluconate (K‐Gluc), MgCl2 by Mg‐gluconate, and CaCl2 by Ca‐gluconate. (A) [14C]‐nicotinate transport properties of human SMCT1 (B) [14C]nicotinate transport properties of human SMCT2. P < 0.001 compared with NaCl (ND96). (C) Inhibition of [14C]‐nicotinate uptake via SMCT1, and SMCT2 in the presence of uricosuric drugs added into the extracellular medium (pH 7.4) at the indicated concentrations. P < 0.01 compared with NaCl (ND96); NS, not significant. (D) [14C]‐nicotinate transport properties of human OAT10 in oocytes preloaded with PZA. P < 0.001 compared with uptake in the absence of inhibitor; NS, not significant. (E) Inhibition of [14C]‐nicotinate uptake via OAT10 (preloaded with PZA) in the presence of uricosuric drugs added into the extracellular medium (pH 7.4) at the indicated concentrations. P < 0.01 compared with NaCl (ND96); NS, not significant. Tran, Benz, Prob, DMSO. Data are mean ± S.E. with n = 12–15. PZA, pyrazine carboxylate; Tran, Tranilast; Benz, Benzbromarone; Prob, probenecid; DMSO, dimethylsulfoxide.
	Figure 8. Tranilast inhibits urate transport mediated by the human urate transporter NPT1. The uptake of [14C]‐urate by NPT1‐expressing oocytes was performed in ND96 medium (pH 7.4) at ~25°C for 1 h. The [14C]‐urate uptake by NPT1‐expressing oocytes was found little trans‐stimulated when NPT1‐expressing oocytes were preinjected with 50 nL of 100 mmol/L nicotinate (Nico) 2 h before [14C]‐urate uptake experiment. The [14C]‐urate uptake by NPT1 was found substantially inhibited in the presence of extracellular organic anions or uricosuric drugs. Organic anions or uricosuric drugs were added to the extracellular medium (pH 7.4) at the indicated concentrations. *P < 0.001 compared with uptake in the presence of DMSO or in the absence of inhibitor. Tran, Benz, Prob, DMSO, PZA, Sal. Data are mean ± S.E. with n = 12–15. PZA, pyrazine carboxylate; Tran, Tranilast; Benz, Benzbromarone; Prob, probenecid; DMSO, dimethylsulfoxide.
	Figure 9. Urate efflux mediated by the human urate transporter ABCG2 is unaffected by tranilast. Control or ABCG2‐expressing oocytes were preinjected with 50 nL of 500 μmol/L 14C]‐urate [dissolved in efflux medium (ND96, pH 7.4)], incubated in ND96 medium for 1 h at 16°C for recovery. Oocytes were washed in cold efflux medium four times and then subjected to efflux for 1 h at room temperature (~25°C) in ND96 medium (pH 7.4) in the absence or presence of drug. The amount of [14C]‐urate retained in each oocyte after 60 min of efflux is shown here. *P < 0.001 compared with urate efflux in the absence of any inhibitor. Tran, Benz, Prob, DMSO. All data are mean ± S.E.M. with n = 12–15. PZA, pyrazine carboxylate; Tran, Tranilast; Benz, Benzbromarone; Prob, probenecid; DMSO, dimethylsulfoxide. Prob, probenecid; DMSO, dimethylsulfoxide.
	Figure 10. Tranilast inhibits urate transport mediated by urate transporter OAT3 and OAT1. The uptake of [14C]‐urate (40 μmol/L) was performed in ND96 medium (pH 7.4) at ~25°C for 1 h. (A) The [14C]‐urate uptake by mouse OAT3 in oocytes remained unaffected if oocytes were preinjected with 50 nL of 100 mmol/L PZA but cis‐inhibited by 10 mmol/L PZA, nicotinate (Nico), or salicylate (Sal). The [14C]‐urate uptake by OAT3‐expressing oocytes was effectively inhibited by 50 μmol/L tranilast (Tran), 100 μmol/L benzbromarone (Benz) or 1.0 mmol/L probenecid (Prob). (B) The [14C]‐urate uptake by human OAT1 in oocytes was effectively inhibited by 100 μmol/L tranilast (Tran), 100 μmol/L benzbromarone (Benz) or 1.0 mmol/L probenecid (Prob). Data are mean ± S.E. with n = 12–15. *P < 0.001 compared with uptake in the presence of DMSO or in the absence of inhibitor. PZA, pyrazine carboxylate; Tran, Tranilast; Benz, Benzbromarone; Prob, probenecid; DMSO, dimethylsulfoxide.

References [+] :

Anzai, Plasma urate level is directly regulated by a voltage-driven urate efflux transporter URATv1 (SLC2A9) in humans. 2008, Pubmed, Xenbase