Article Figures & Data
Figures
- Fig. 1.
Species differences in the inhibition potencies of ANPs (adefovir, cidofovir, and tenofovir) for OAT1-mediated 6CF uptake. HEK293 cells stably expressing hOAT1, cyOAT1, rat OAT1, mouse OAT1, and dog OAT1 were incubated with HBSS buffer containing 6CF (1 μM) for 1 minute with or without designed concentrations of adefovir, cidofovir, and tenofovir. Data points represent the mean ± S.D. of 6CF uptake from three replicate determinations in a single experiment. The experiments were repeated three times and similar results were obtained. Representative curves of the OAT1-mediated 6CF uptake inhibition by ANPs: (A) Adefovir; (B) Cidofovir; (C) Tenofovir. The IC50 values for each of the analogs with each OAT1 ortholog are listed in Table 1.
- Fig. 2.
Kinetics of uptake of tenofovir for species orthologs of OAT1. (A) The uptake kinetics of [3H]-tenofovir in HEK293 cells expressing hOAT1, cyOAT1, rat OAT1, mouse OAT1, and dog OAT1. The uptake rate was evaluated at 3 minutes. Each point represents the mean ± S.D. uptake in the OAT1-transfected cells minus that in empty vector cells. (B) Correlation of the Vmax value of OAT1-mediated tenofovir uptake in HEK293 cells stably expressing five OAT1 species orthologs with total cell membrane-bound OAT1 protein quantity (R2 = 0.892).
- Fig. 3.
Chimera proteins of OAT1 created to assess the critical domains and residues involved in the species differences of tenofovir kinetics in cells stably expressing hOAT1 and cyOAT1. (A) Predicted membrane-bound hOAT1 structure showing 550 amino acids. The white color indicates amino acid residues that are conserved between hOAT1 and cyOAT1, the turquoise color shows residues that vary between hOAT1 and cyOAT1, and the orange color indicates residues that were mutated and evaluated for tenofovir transport kinetics. (B) Wild-type hOAT1, cyOAT1, and chimeric proteins with different combinations of hOAT1 and cyOAT1 amino acids. (C) [3H]-tenofovir uptake by OAT1 chimera proteins. Transiently transfected HEK293 cells overexpressing chimera proteins were incubated with [3H]-tenofovir (50 nM) for 3 minutes. (D) [3H]-tenofovir uptake by cyOAT1 mutants (cyOAT1 S198A, A203S, I254V, and V256A) and hOAT1 mutant (hOAT1 S203A). Transiently transfected HEK293 cells overexpressing mutant proteins were incubated with [3H]-tenofovir (50 nM) for 3 minutes. (E) Eadie-Hofstee plot for the [3H]-tenofovir uptake by stable cell lines overexpressing hOAT1 and cyOAT1 and their mutants, hOAT1 S203A and cyOAT1 A203S. (F) Comparison of the averaged Km values for OAT1 orthologs with S203 (N = 7, including human, chimpanzee, gorilla, orangutan, gibbon, galago, and cynomolgus monkey OAT1 A203S) with that for OAT1 orthologs with alanine at the equivalent position (N = 6, including cynomolgus monkey, squirrel monkey, mouse, rat, dog, and human OAT1 S203A). Student’s t test was performed to determine significant differences between the two groups. (G) The uptake rate of [3H]-adefovir and [3H]-cidofovir in cells overexpressing hOAT1 and cyOAT1 and the mutants, hOAT1 S203A and cyOAT1 A203S. Each column represents the mean ± S.D. uptake in the OAT1-transfected cells minus that in empty vector cells. Two experiments were conducted and there were four replicates for each experiment. Statistical analyses were performed by one-way analysis of variance followed by Tukey’s multiple comparisons test to determine significant differences between controls and treatment groups. (H) The rate of uptake of [3H]-tenofovir in HEK293 cells overexpressing hOAT1 or cyOAT1 with or without 120-minute preincubation with 5 mM α-ketoglutarate or 5 mM glutaric acid. Each bar represents uptake (mean ± S.D.) in the OAT1-transfected cells minus that in empty vector cells (N ≥ 2). *P value <0.05; **P value < 0.01; ***P value <0.001; ****P value = 0.0001. n.s., not significant.
- Fig. 4.
Association of OAT1-mediated tenofovir transport efficiency (Vmax/Km) with SUA levels and effect of amino acid substitutions in hOAT1 on tenofovir uptake rate. (A) Mean ± S.D. of SUA levels (black bar) and OAT1-mediated tenofovir transport efficiencies (gray bar) in species with (cynomolgus monkey, squirrel monkey, mouse, rat, and dog) and without (human, chimpanzee, gorilla, orangutan, and gibbon) functional uricase (* represents comparison of SUA levels in species with and without uricase; # represents comparison of tenofovir transport efficiencies in species with and without uricase; **P < 0.01; ####P < 0.0001). (B) [3H]-tenofovir uptake by hOAT1, cyOAT1, and mutants (hOAT1 S203T and hOAT1 S203A). Transiently transfected HEK293 cells overexpressing wild-type or mutant proteins were incubated with [3H]-tenofovir (50 nM) for 3 minutes. Transporter-mediated tenofovir uptake was obtained after subtracting the respective rate of uptake in empty vector cells.
- Fig. 5.
Uric acid uptake in HEK293 cells expressing hOAT1 or cyOAT1 as a function of time and concentration. (A) Uptake of [14C]-uric acid (20 μM) as a function of time in cells expressing hOAT1 and in empty vector cells. (B) Kinetics of [14C]-uric acid uptake by hOAT1 and cyOAT1. HEK293 cells overexpressing hOAT1 and cyOAT1 were incubated with [14C]-uric acid (20 μM) with various concentrations of unlabeled uric acid for 5 minutes. Stock of uric acid was dissolved in 0.1 N NaOH and added to obtain designed concentrations in HBSS buffer plus 10 mM HEPES to maintain pH 7.4. Each point represents the uptake in OAT1-expressing cells minus that in empty vector cells.
Tables
- TABLE 1
Potencies of ANPs in inhibiting 6CF uptake by OAT1 species orthologs from human, cynomolgus monkey, mouse, rat, and dog
Statistical analyses were performed by one-way analysis of variance followed by Dunnett’s multiple comparisons test. N ≥ 2.
Inhibitor IC50 Human Cynomolgus Monkey Mouse Rat Dog μM μM μM μM μM Adefovir 71 ± 15 220 ± 67* 399 ± 147*** 263 ± 66* 181 ± 4 Cidofovir 88 ± 25 181 ± 67 237 ± 69** 245 ± 19** 185 ± 27 Tenofovir 61 ± 14 343 ± 40**** 165 ± 27* 153 ± 17 223 ± 64** ↵* P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
- TABLE 2
Kinetic parameters of the tenofovir uptake by OAT1 species orthologs from human, cynomolgus monkey, mouse, rat, and dog
Statistical analyses were performed by one-way analysis of variance followed by Dunnett’s multiple comparisons test. N ≥ 2; &, tenofovir transport efficiency for OAT1 from each species was normalized to the corresponding membrane-bound OAT1 quantity (Supplemental Table 3).
Parameter Human Cynomolgus Monkey Mouse Rat Dog Km (μM) 72.6 ± 20 254 ± 0.1**** 138 ± 18*** 139 ± 7.6*** 157 ± 8.8**** Vmax (nmol/mg/min) 4.6 ± 1.4 2.5 ± 0.2 2.4 ± 0.6* 2.1 ± 0.6* 3.9 ± 0.9 Vmax/Km (μl/mg/min) 63 10 17 15 25 Normalized Vmax/Km (μl/mg/min)& 1.62 0.47 1.17 2.59 0.88 ↵* P < 0.05; ***P < 0.001; ****P < 0.0001.
- TABLE 3
Kinetic parameters for OAT1-mediated uptake by wild-type and mutant transporters
Mutations were created by site-directed mutagenesis of both hOAT1 and cyOAT1 at position 203. Statistical analyses were performed by one-way analysis of variance followed by Tukey’s multiple comparisons test. N ≥ 2; * represents comparison with human; # represents comparison between cynomolgus monkey and cynomolgus monkey A203S.
Parameter Human Cynomolgus Monkey Cynomolgus Monkey A203S Human S203A Km (μM) 72.6 ± 20 254 ± 0.1**** 105 ± 27### 216 ± 19*** Vmax (nmol/mg/min) 4.6 ± 1.4 2.5 ± 0.2 2.8 ± 0.1 3.6 ± 0.7 Vmax/Km (μl/mg/min) 63 10 27 17 ↵*** P < 0.001; ****P < 0.0001; ###P < 0.001.
- TABLE 4
Kinetic parameters of tenofovir uptake rate by OAT1 wild-type transporters and mutants at position 203
Species Amino Acid at position 203 or Equivalent Position Km μM Human S 72.6 ± 20 Chimpanzee S 44.2 ± 1.5 Gorilla S 56.0 ± 7.5 Orangutan S 79.0 ± 16 Gibbon S 65.0 ± 2.4 Galago S 84.9 ± 24 cyOAT1 A203S S 105 ± 27 Cynomolgus monkey A 254 ± 0.1 Squirrel monkey A 181 ± 60 Mouse A 138 ± 18 Rat A 139 ± 7.6 Dog A 157 ± 8.8 hOAT1 S203A A 216 ± 19 S, Serine; A, Alanine.
- TABLE 5
OAT1-mediated tenofovir transport efficiencies and SUA levels in species with or without functional uricase
*The mean value of SUA was calculated based on reported levels. The range of SUA is included in parenthesis. —, not applicable.
Species Without Uricase Activity With Uricase Activity SUA* Tenofovir Transport Efficiency SUA Tenofovir Transport Efficiency μM μl/mg/min μM μl/mg/min Human 318 (210–420) (Tan et al., 2016) 63 — — Chimpanzee 244 (120–360) (Fanelli and Beyer, 1974) 61 — — Gorilla 146 (130–160) (Fanelli and Beyer, 1974) 49 — — Orangutan 140 (110–170) (Fanelli and Beyer, 1974) 69 — — Gibbon 178 (120–300) (Fanelli and Beyer, 1974) 52 — — Cynomolgus monkey — — 36 (30–42) (Fanelli and Beyer, 1974) 10 Squirrel monkey — — 30 (12–60) (Fanelli and Beyer, 1974) 15 Mouse — — 53 (30–78) (Wu et al., 1994; So and Thorens, 2010) 17 Rat — — 67 (64–70) (Mazzali et al., 2001; Lu et al., 2016) 15 Dog — — 18 (Miller et al., 1951) 25 Mean 205 ± 75 58.9 ± 8.3 41 ± 19 16.5 ± 5.3 - TABLE 6
Kinetic parameters of [14C]-uric acid uptake by hOAT1 and cyOAT1 in HEK293 cells stably expressing the transporters
Student’s t test was performed to determine significant differences between the two groups. N ≥ 2.
Parameter Human Cynomolgus Monkey Km (μM) 571 ± 97.7 1070 ± 90** Vmax (pmol/mg/min) 1510 ± 243 1040 ± 106 Vmax/Km (μl/mg/min) 2.6 0.97 ↵** P < 0.01.
Data Supplement
- Supplemental Data -
Supplemental Figure 1 - Molecular model of the interaction of tenofovir with serine at the position 203 of hOAT1
Supplemental Figure 2 - Kinetics of PAH uptake by hOAT1 and cyOAT1
Supplemental Figure 3 - Correlation of serum uric acid concentration with tenofovir transport efficiency for OAT1
Supplemental Figure 4 - Regional plot of the SLC22A6 locus
Supplemental Table 1 - Primers
Supplemental Table 2 - Amino acid sequence identity between hOAT1 and orthologs from cynomolgus monkey, mouse, rat and dog
Supplemental Table 3 - Quantification of membrane-bound OAT1 protein in stable cell lines and kidney cortex from human, cynomolgus monkey, mouse, rat and dog
Supplemental Table 4 - List of the amino acid at position 203 in hOAT1 and the equivalent position in OAT1 orthologs from primates and more distant species
Supplemental Table 5 - Kinetic parameters of PAH uptake by hOAT1 and cyOAT1
Supplemental Table 7 - Summary of statistical analysis results for Table 1, Table 2, Table 3 and Table 6
- Supplemental Table 6 -
Allele frequency of each populations in 1000 genomes
- Supplemental Data -
