Mercapturic Acids (<i>N</i>-Acetylcysteine<i>S</i>-Conjugates) as Endogenous Substrates for the Renal Organic Anion Transporter-1

Pombrio, James M.; Giangreco, Adam; Li, Liqiong; Wempe, Michael F.; Anders, M. W.; Sweet, Douglas H.; Pritchard, John B.; Ballatori, Nazzareno

doi:10.1124/mol.60.5.1091

Cited by 48 publications

(34 citation statements)

References 33 publications

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“…11B). The fact that DMPS is a substrate for OAT1 is further underlined by the recent findings of Pombrio et al (Pombrio et al, 2001). They concluded that selected mercapturic acids serve as high affinity substrates for OAT1.…”

Section: Discussionmentioning

confidence: 87%

Interaction of the Metal Chelator 2,3-Dimercapto-1-propanesulfonate with the Rabbit Multispecific Organic Anion Transporter 1 (rbOAT1)

et al. 2002

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The metal chelator DMPS (2,3-dimercapto-1-propanesulfonate) is used to treat heavy metal intoxication because it increases renal excretion of these toxins, which are accumulated in proximal tubule cells. To evaluate the involvement of the organic anion transporter 1 (OAT1) in the renal flux of DMPS, we examined the effect of DMPS on transport mediated by the rabbit ortholog of OAT1 and compared these characteristics with those observed in intact isolated rabbit proximal tubules. The rabbit OAT1 (rbOAT1) cDNA consisted of 2124 base pairs encoding a protein of 551 amino acids. Heterologous expression in COS-7 cells revealed rbOAT1-mediated transport of p-aminohippurate (PAH; K t ϭ 16 M). A 1 mM concentration of unlabeled PAH, ␣-ketoglutarate, urate, or probenecid inhibited [ 3 H]PAH uptake by 70 to 90%. cis-Inhibition and trans-stimulation experiments using several Krebs cycle intermediates implicated ␣-ketoglutarate as the main intracellular exchange anion. Reduced DMPS inhibited rbOAT1-mediated fluorescein transport with an apparent K i of 102 M. These characteristics paralleled those observed in isolated rabbit proximal tubules. PAH was transported into nonperfused single proximal tubule S 2 segments with a K t of 76 M. DMPS inhibited FL uptake into single tubule segments with a K i-app of 71 M. Fluorescein efflux from preloaded tubules was trans-stimulated by 1 mM PAH and 1 mM DMPS, consistent with DMPS entry into tubule cells by rbOAT1. In summary, rbOAT1 mediates basolateral uptake of DMPS into proximal tubule cells, implicating this process in the detoxification process of heavy metals in the kidneys.

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Section: Discussionmentioning

confidence: 87%

Interaction of the Metal Chelator 2,3-Dimercapto-1-propanesulfonate with the Rabbit Multispecific Organic Anion Transporter 1 (rbOAT1)

et al. 2002

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“…In fact, mercapturic acids have been shown previously to be secreted by the renal proximal tubule (Stevens and Jones, 1989). In addition, it was recently shown that several mercapturic acids [i.e., S-(2,4-dinitrophenyl)-N-cysteine] are specific hOAT1 substrates (Pombrio et al, 2001).…”

Section: Discussionmentioning

confidence: 99%

Human Renal Organic Anion Transporter 1-Dependent Uptake and Toxicity of Mercuric-Thiol Conjugates in Madin-Darby Canine Kidney Cells

et al. 2003

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Mercuric ions are highly reactive and form a variety of organic complexes or conjugates in vivo. The renal proximal tubule is a primary target for mercury uptake and toxicity, and circumstantial evidence implicates organic anion transporters in these processes. To test this hypothesis directly, the transport and toxicity of mercuric-thiol conjugates were characterized in a Madin-Darby canine kidney cell line stably transfected with the human organic anion transporter 1 (hOAT1). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-terazolium bromide assays (for mitochondrial dehydrogenase) confirmed that mercuric conjugates of the thiols N-acetylcysteine (NAC), cysteine, or glutathione were more toxic in hOAT1-transfected cells than in the nontransfected cells. The NAC-Hg 2ϩ conjugate was most cytotoxic, inducing greater than 50% cellular death over 18 h at a concentration of 100 M. The cytotoxic effects were fully reversed by probenecid (an OAT1 inhibitor) and partially reversed by p-aminohippurate (an OAT1 substrate). Toxicity of this conjugate was reduced by the OAT1-exchangeable dicarboxylates ␣-ketoglutarate, glutarate, and adipate, but not by succinate, a nonexchangeable dicarboxylate.203 Hg-uptake studies showed probenecid-sensitive uptake of mercury-thiol conjugates in the hOAT1-transfected cells. The apparent K m for the NAC-Hg 2ϩ conjugate was 44 Ϯ 9 M. Uptake of the NAC-Hg 2ϩ conjugate was cis-inhibited by glutarate, but not by methylsuccinate, paralleling their effects on toxicity. Probenecid-sensitive transport of the NAC-Hg 2ϩ conjugate was also shown to occur in Xenopus laevis oocytes expressing the hOAT1 or the rOAT3 transporters, suggesting that OAT3 may also transport thiolHg 2ϩ conjugates. Thus, renal accumulation and toxicity of thiolHg 2ϩ conjugates may depend in part on the activity of the organic transport system.

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“…The animals were anesthetized with 0.3% tricaine (Sigma), decapitated, pithed, and the ovaries were removed. Stage V and VI oocytes were isolated by collagenase digestion as previously described (6,13,26). All animal procedures were carried out in accord with protocols approved by the NIEHS Animal Care and Use Committee.…”

Section: Animalsmentioning

confidence: 99%

Stoichiometry of organic anion/dicarboxylate exchange in membrane vesicles from rat renal cortex and hOAT1-expressing cells

Aslamkhan¹,

Han²,

Walden³

et al. 2003

American Journal of Physiology-Renal Physiology

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of organic anion/dicarboxylate exchange in membrane vesicles from rat renal cortex and hOAT1-expressing cells. Am J Physiol Renal Physiol 285: F775-F783, 2003. First published July 1, 2003 10.1152/ajprenal.00140.2003.-Although membrane vesicle studies have established the driving forces that mediate renal organic anion secretion and the organic anion transporter Oat1 has now been cloned in several species, its stoichiometry has remained uncertain. In this study, we used electrophysiology, kinetic measurements, and static head experiments to determine the coupling ratio for Oat1-mediated organic anion/dicarboxylate exchange. Initial experiments demonstrated that uptake of PAH by voltage-clamped Xenopus laevis oocytes expressing rOat1 led to net entry of positive charge, suggesting that coupling was one-to-one. This conclusion was confirmed by kinetic analysis of PAH and glutarate fluxes in native basolateral membrane vesicles from the rat renal cortex, which showed a Hill coefficient of 1. Similarly, static head experiments on the rat vesicles also showed a 1:1 coupling ratio. To confirm these conclusions in a system expressing a single cloned transporter, MadinDarby canine kidney cells were stably transfected with the human exchanger hOAT1. The hOAT1-expressing cell line showed extensive PAH transport, which was very similar in all respects to transport expressed by hOAT1 in Xenopus oocytes. Its K m for PAH was 8 M and glutarate effectively trans-stimulated PAH transport. When stoichiometry was assessed using plasma membranes isolated from the hOAT1-expressing cells, both kinetic and static head data indicated that hOAT1 also demonstrated a 1:1 coupling between organic anion and dicarboxylate.

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Mercapturic Acids (N-AcetylcysteineS-Conjugates) as Endogenous Substrates for the Renal Organic Anion Transporter-1

Cited by 48 publications

References 33 publications

Interaction of the Metal Chelator 2,3-Dimercapto-1-propanesulfonate with the Rabbit Multispecific Organic Anion Transporter 1 (rbOAT1)

Interaction of the Metal Chelator 2,3-Dimercapto-1-propanesulfonate with the Rabbit Multispecific Organic Anion Transporter 1 (rbOAT1)

Human Renal Organic Anion Transporter 1-Dependent Uptake and Toxicity of Mercuric-Thiol Conjugates in Madin-Darby Canine Kidney Cells

Stoichiometry of organic anion/dicarboxylate exchange in membrane vesicles from rat renal cortex and hOAT1-expressing cells

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