Organic anion transporter 3 (OAT3) and renal transport of the metal chelator 2,3-dimercapto-1-propanesulfonic acid (DMPS)

Abstract

Recent investigations involving intact rabbit renal proximal tubules indicated that organic anion transporter 3 (OAT3) may be involved in the transport of 2,3-dimercapto-1-propanesulfonic acid (DMPS). Therefore, we evaluated the interaction of OAT3 with DMPS to determine the effect of OAT3 on basolateral DMPS uptake. We used stably transfected HEK293 cells expressing human and rabbit orthologs of the exchanger OAT1 and OAT3. Using 6-carboxyfluorescein (6-CF) as a substrate, the IC50 determinations for reduced DMPS (DMPSH) revealed a stronger interaction with OAT1 than with OAT3 (rbOAT1, 123.3 +/- 13.7; hOAT1, 85.1 +/- 8.8; rbOAT3, 171.7 +/- 22.3; and hOAT3, 172.2 +/- 36.4 micromol/L). However, inhibition of 6-CF uptake by the oxidized form of DMPS (DMPSS), the main form of DMPS in the blood, showed a greater affinity for OAT3 (rbOAT1, 237.4 +/- 23; hOAT1, 104.6 +/- 13.1; rbOAT3, 52.4 +/- 7.6; and hOAT3, 31.6 +/- 6.6 micromol/L). To determine whether DMPSH and DMPSS are substrates for OAT3, we performed efflux studies with [14C]glutarate and inwardly directed gradients of glutarate. The inhibitors trans-stimulated the efflux of [14C]glutarate, suggesting that OAT3 may be able to transport both forms of DMPS. On the basis of the substantial interaction of OAT3 with DMPSS, we conclude that OAT3 represents the dominant basolateral player in renal detoxification processes resulting from use of DMPS.

Fig. 1

Concentration dependence of rbOAT3 (A) and hOAT3 (B) mediated uptake of 5 µM 6-CF into HEK293-OAT cells using various concentrations of DMPSH for 10 min at RT. Each point represents the mean of triplicate measurements from 4 separate experiments.

Fig. 1

Concentration dependence of rbOAT3 (A) and hOAT3 (B) mediated uptake of 5 µM 6-CF into HEK293-OAT cells using various concentrations of DMPSH for 10 min at RT. Each point represents the mean of triplicate measurements from 4 separate experiments.

Fig. 2

Concentration dependence of rbOAT3 (A) and hOAT3 (B) mediated uptake of 5 µM 6-CF into HEK293-OAT cells using various concentrations of DMPSS for 10 min at RT. Each point represents the mean of triplicate measurements from 3 separate experiments.

Fig. 2

Concentration dependence of rbOAT3 (A) and hOAT3 (B) mediated uptake of 5 µM 6-CF into HEK293-OAT cells using various concentrations of DMPSS for 10 min at RT. Each point represents the mean of triplicate measurements from 3 separate experiments.

Fig. 3

Effect of inwardly directed gradients of glutarate, DMPSH, or DMPSS (each in a concentration of 500 µM) on ¹⁴C-glutarate efflux in stably transfected HEK293 cells (rbOAT3, hOAT3). HEK293-OAT cells were preloaded with ¹⁴C-glutarate for 1h at 37°C. The efflux was calculated as percentage of ¹⁴C content recovered in the supernatant compared with the sum of total radioactivity recovered in the medium and that remaining in the cells at the conclusion of a 10 min efflux period at 37°C, where OAT-expressing cells in Ringer’s solution were set to 100%. Data are expressed as means SE of triplicate measurements from 3 separate experiments. If not otherwise noticed all tested substances showed a highly significant (P < 0.001) efflux of [¹⁴C]glutarate. *P < 0.05; **P < 0.01;

Fig. 4

Model for basolateral entry of DMPSH and DMPSS via OATs and NaDC-3.