Transporters involved in renal excretion of N-carbamoylglutamate, an orphan drug to treat inborn n-acetylglutamate synthase deficiency

2014 ◽  
Vol 307 (12) ◽  
pp. F1373-F1379 ◽  
Author(s):  
Elisabeth Schwob ◽  
Yohannes Hagos ◽  
Gerhard Burckhardt ◽  
Birgitta C. Burckhardt
Keyword(s):  
Orphan Drug ◽  
Organic Anion ◽  
Xenopus Laevis Oocytes ◽  
Human Embryonic Kidney ◽  
Estrone Sulfate ◽  
Anion Transporters ◽  
293 Cells ◽  
Acetylglutamate Synthase ◽  
Inborn Defects ◽  
The Impact

Inborn defects in N-acetylglutamate (NAG) synthase (NAGS) cause a reduction of NAG, an essential cofactor for the initiation of the urea cycle. As a consequence, blood ammonium concentrations are elevated, leading to severe neurological disorders. The orphan drug N-carbamoylglutamate (NCG; Carbaglu), efficiently overcomes NAGS deficiency. However, not much is known about the transporters involved in the uptake, distribution, and elimination of the divalent organic anion NCG. Organic anion-transporting polypeptides (OATPs) as well as organic anion transporters (OATs) working in cooperation with sodium dicarboxylate cotransporter 3 (NaDC3) accept a wide variety of structurally unrelated drugs. To test for possible interactions with OATPs and OATs, the impact of NCG on these transporters in stably transfected human embryonic kidney-293 cells was measured. The two-electrode voltage-clamp technique was used to monitor NCG-mediated currents in Xenopus laevis oocytes that expressed NaDC3. Neither OATPs nor OAT2 and OAT3 interacted with NCG, but OAT1 transported NCG. In addition, NCG was identified as a high-affinity substrate of NaDC3. Preincubation of OAT4-transfected human embryonic kidney-293 cells with NCG showed an increased uptake of estrone sulfate, the reference substrate of OAT4, indicating efflux of NCG by OAT4. In summary, NaDC3 and, to a lesser extent, OAT1 are likely to be responsible for the uptake of NCG from the blood. Efflux of NCG across the luminal membrane into the tubular lumen probably occurs by OAT4 completing renal secretion of this drug.

scholarly journals Transport of Kynurenic Acid by Rat Organic Anion Transporters rOAT1 and rOAT3: Species Difference between Human and Rat in OAT1

2013 ◽  
Vol 6 ◽  
pp. IJTR.S11206 ◽  
Author(s):  
Yuichi Uwai ◽  
Hiroaki Hara ◽  
Kikuo Iwamoto
Keyword(s):  
Kynurenic Acid ◽  
Organic Anion ◽  
Species Difference ◽  
Xenopus Laevis Oocytes ◽  
Organic Anion Transporters ◽  
Comparative Experiment ◽  
Transport Activity ◽  
Estrone Sulfate ◽  
Anion Transporters ◽  
Uptake Experiment

A tryptophan catabolite, kynurenic acid, is involved in schizophrenia and uremia; there is little information on the mechanism of its disposition. Recently, our laboratory showed that kynurenic acid is a good substrate of human organic anion transporters hOAT1 and hOAT3. In this study, we performed uptake experiment using Xenopus laevis oocytes to characterize the transport of kynurenic acid by rat homologs of the transporters, rOAT1, and rOAT3. These transporters stimulated the uptake of kynurenic acid into oocytes, and transport by rOAT3 was marked. The Km values of the transport were estimated to be 8.46 μM for rOAT1 and 4.81 μM for rOAT3, and these values are comparable to their human homologs. The transport activity of kynurenic acid by rOAT1 was about one quarter of that of p-aminohippurate, although they were at the similar levels in hOAT1. A comparative experiment with hOAT1 was added in this study, showing that uptake amounts of kynurenic acid by hOAT1-expressing oocytes were 4 times greater than rOAT1-expressing oocytes. rOAT3 transported kynurenic acid as efficiently as estrone sulfate; this phenomenon was also observed in hOAT3. In conclusion, transport of kynurenic acid by rOAT1 and rOAT3 was shown. The characteristics of rOAT3 were similar to hOAT3, but low transport activity of kynurenic acid by rOAT1 was exhibited compared with hOAT1.

Renal human organic anion transporter 3 increases the susceptibility of lymphoma cells to bendamustine uptake

2015 ◽  
Vol 308 (4) ◽  
pp. F330-F338 ◽  
Author(s):  
Yohannes Hagos ◽  
Philip Hundertmark ◽  
Volodymyr Shnitsar ◽  
Venkata V. V. R. Marada ◽  
Gerald Wulf ◽  
...  
Keyword(s):  
Organic Anion ◽  
Inhibitory Effect ◽  
Organic Anion Transporter ◽  
Lymphoma Cells ◽  
Estrone Sulfate ◽  
Anion Transporters ◽  
Lymphatic Leukemia ◽  
Anion Transporter ◽  
Nephritic Syndrome ◽  
The Impact

Chronic lymphatic leukemia (CLL) is often associated with nephritic syndrome. Effective treatment of CLL by chlorambucil and bendamustine leads to the restoration of renal function. In this contribution, we sought to elucidate the impact of organic anion transporters (OATs) on the uptake of bendamustine and chlorambucil as a probable reason for the superior efficacy of bendamustine over chlorambucil in the treatment of CLL. We examined the effects of structural analogs of p-aminohippurate (PAH), melphalan, chlorambucil, and bendamustine, on OAT1-mediated [3H]PAH uptake and OAT3- and OAT4-mediated [3H]estrone sulfate (ES) uptake in stably transfected human embryonic kidney-293 cells. Melphalan had no significant inhibitory effect on any OAT, whereas chlorambucil reduced OAT1-, OAT3-, and OAT4-mediated uptake of PAH or ES down to 14.6%, 16.3%, and 66.0% of control, respectively. Bendamustine inhibited only OAT3-mediated ES uptake, which was reduced down to 14.3% of control cells, suggesting that it interacts exclusively with OAT3. The IC50 value for OAT3 was calculated to be 0.8 μM. Real-time PCR experiments demonstrated a high expression of OAT3 in lymphoma cell lines as well as primary CLL cells. OAT3-mediated accumulation of bendamustine was associated with reduced cell proliferation and an increased rate of apoptosis. We conclude that the high efficacy of bendamustine in treating CLL might be partly contributed to the expression of OAT3 in lymphoma cells and the high affinity of bendamustine for this transporter.

Human organic anion transporter 1B1 and 1B3 function as bidirectional carriers and do not mediate GSH-bile acid cotransport

2007 ◽  
Vol 293 (1) ◽  
pp. G271-G278 ◽  
Author(s):  
Chitrawina Mahagita ◽  
Steven M. Grassl ◽  
Pawinee Piyachaturawat ◽  
Nazzareno Ballatori
Keyword(s):  
Bile Acid ◽  
Transport Mechanism ◽  
Organic Anion ◽  
Large Family ◽  
Organic Anion Transporter ◽  
Xenopus Laevis Oocytes ◽  
Facilitated Diffusion ◽  
Transport Activity ◽  
Estrone Sulfate ◽  
Anion Transporter

Organic anion transporting polypeptides (OATP/ SLCO) are generally believed to function as electroneutral anion exchangers, but direct evidence for this contention has only been provided for one member of this large family of genes, rat Oatp1a1/Oatp1 ( Slco1a1). In contrast, a recent study has indicated that human OATP1B3/OATP-8 ( SLCO1B3) functions as a GSH-bile acid cotransporter. The present study examined the transport mechanism and possible GSH requirement of the two members of this protein family that are expressed in relatively high levels in the human liver, OATP1B3/OATP-8 and OATP1B1/OATP-C ( SLCO1B1). Uptake of taurocholate in Xenopus laevis oocytes expressing either OATP1B1/OATP-C, OATP1B3/OATP-8, or polymorphic forms of OATP1B3/OATP-8 (namely, S112A and/or M233I) was cis-inhibited by taurocholate and estrone sulfate but was unaffected by GSH. Likewise, taurocholate and estrone sulfate transport were trans-stimulated by estrone sulfate and taurocholate but were unaffected by GSH. OATP1B3/OATP-8 also did not mediate GSH efflux or GSH-taurocholate cotransport out of cells, indicating that GSH is not required for transport activity. In addition, estrone sulfate uptake in oocytes microinjected with OATP1B3/OATP-8 or OATP1B1/OATP-C cRNA was unaffected by depolarization of the membrane potential or by changes in pH, suggesting an electroneutral transport mechanism. Overall, these results indicate that OATP1B3/OATP-8 and OATP1B1/OATP-C most likely function as bidirectional facilitated diffusion transporters and that GSH is not a substrate or activator of their transport activity.

scholarly journals Human organic anion transporter 2 is distinct from organic anion transporters 1 and 3 with respect to transport function

2015 ◽  
Vol 309 (10) ◽  
pp. F843-F851 ◽  
Author(s):  
Maja Henjakovic ◽  
Yohannes Hagos ◽  
Wolfgang Krick ◽  
Gerhard Burckhardt ◽  
Birgitta C. Burckhardt
Keyword(s):  
Organic Anion ◽  
Organic Anion Transporter ◽  
Substrate Uptake ◽  
Organic Anion Transporters ◽  
Counter Anion ◽  
Anion Transporters ◽  
Anion Transporter ◽  
293 Cells ◽  
High K ◽  
Resistance Protein

Phylogentically, organic anion transporter (OAT)1 and OAT3 are closely related, whereas OAT2 is more distant. Experiments with human embryonic kidney-293 cells stably transfected with human OAT1, OAT2, or OAT3 were performed to compare selected transport properties. Common to OAT1, OAT2, and OAT3 is their ability to transport cGMP. OAT2 interacted with prostaglandins, and cGMP uptake was inhibited by PGE2 and PGF2α with IC50 values of 40.8 and 12.7 μM, respectively. OAT1 (IC50: 23.7 μM), OAT2 (IC50: 9.5 μM), and OAT3 (IC50: 1.6 μM) were potently inhibited by MK571, an established multidrug resistance protein inhibitor. OAT2-mediated cGMP uptake was not inhibited by short-chain monocarboxylates and, as opposed to OAT1 and OAT3, not by dicarboxylates. Consequently, OAT2 showed no cGMP/glutarate exchange. OAT1 and OAT3 exhibited a pH and a Cl− dependence with higher substrate uptake at acidic pH and lower substrate uptake in the absence of Cl−, respectively. Such pH and Cl− dependencies were not observed with OAT2. Depolarization of membrane potential by high K+ concentrations in the presence of the K+ ionophore valinomycin left cGMP uptake unaffected. In addition to cGMP, OAT2 transported urate and glutamate, but cGMP/glutamate exchange could not be demonstrated. These experiments suggest that OAT2-mediated cGMP uptake does not occur via exchange with monocarboxylates, dicarboxylates, and hydroxyl ions. The counter anion for electroneutral cGMP uptake remains to be identified.

scholarly journals A role for the organic anion transporter OAT3 in renal creatinine secretion in mice

2012 ◽  
Vol 302 (10) ◽  
pp. F1293-F1299 ◽  
Author(s):  
Volker Vallon ◽  
Satish A. Eraly ◽  
Satish Ramachandra Rao ◽  
Maria Gerasimova ◽  
Michael Rose ◽  
...  
Keyword(s):  
Organic Anion ◽  
Basolateral Membrane ◽  
Tubular Secretion ◽  
Organic Anion Transporter ◽  
Xenopus Laevis Oocytes ◽  
Mean Values ◽  
Anion Transporters ◽  
Anion Transporter ◽  
Neutral Compounds ◽  
Quantitative Contribution

Tubular secretion of the organic cation, creatinine, limits its value as a marker of glomerular filtration rate (GFR) but the molecular determinants of this pathway are unclear. The organic anion transporters, OAT1 and OAT3, are expressed on the basolateral membrane of the proximal tubule and transport organic anions but also neutral compounds and cations. Here, we demonstrate specific uptake of creatinine into mouse mOat1- and mOat3-microinjected Xenopus laevis oocytes at a concentration of 10 μM (i.e., similar to physiological plasma levels), which was inhibited by both probenecid and cimetidine, prototypical competitive inhibitors of organic anion and cation transporters, respectively. Renal creatinine clearance was consistently greater than inulin clearance (as a measure of GFR) in wild-type (WT) mice but not in mice lacking OAT1 ( Oat1−/−) and OAT3 ( Oat3−/−). WT mice presented renal creatinine net secretion (0.23 ± 0.03 μg/min) which represented 45 ± 6% of total renal creatinine excretion. Mean values for renal creatinine net secretion and renal creatinine secretion fraction were not different from zero in Oat1−/− (−0.03 ± 0.10 μg/min; −3 ± 18%) and Oat3−/− (0.01 ± 0.06 μg/min; −6 ± 19%), with greater variability in Oat1−/−. Expression of OAT3 protein in the renal membranes of Oat1−/− mice was reduced to ∼6% of WT levels, and that of OAT1 in Oat3−/− mice to ∼60%, possibly as a consequence of the genes for Oat1 and Oat3 having adjacent chromosomal locations. Plasma creatinine concentrations of Oat3−/− were elevated in clearance studies under anesthesia but not following brief isoflurane anesthesia, indicating that the former condition enhanced the quantitative contribution of OAT3 for renal creatinine secretion. The results are consistent with a contribution of OAT3 and possibly OAT1 to renal creatinine secretion in mice.

Transport of cimetidine by flounder and human renal organic anion transporter 1

2003 ◽  
Vol 284 (3) ◽  
pp. F503-F509 ◽  
Author(s):  
Birgitta C. Burckhardt ◽  
Stefan Brai ◽  
Sönke Wallis ◽  
Wolfgang Krick ◽  
Natascha A. Wolff ◽  
...  
Keyword(s):  
Organic Anion ◽  
Human Kidney ◽  
Organic Anion Transporter ◽  
In Vivo Studies ◽  
Xenopus Laevis Oocytes ◽  
Organic Anion Transporters ◽  
Anion Transporters ◽  
Anion Transporter ◽  
Inward Currents

The H2-receptor antagonist cimetidine is efficiently excreted by the kidneys. In vivo studies indicated an interaction of cimetidine not only with transporters for basolateral uptake of organic cations but also with those involved in excretion of organic anions. We therefore tested cimetidine as a possible substrate of the organic anion transporters cloned from winter flounder (fROAT) and from human kidney (hOAT1). Uptake of [3H]cimetidine into fROAT-expressing Xenopus laevis oocytes exceeded uptake into control oocytes. At −60-mV clamp potential, 1 mM cimetidine induced an inward current, which was smaller than that elicited by 0.1 mM PAH. Cimetidine concentrations exceeding 0.1 mM decreased PAH-induced inward currents, indicating interaction with the same transporter. At pH 6.6, no current was seen with 0.1 mM cimetidine, whereas at pH 8.6 a current was readily detectable, suggesting preferential translocation of uncharged cimetidine by fROAT. Oocytes expressing hOAT1 also showed [3H]cimetidine uptake. These data reveal cimetidine as a substrate for fROAT/hOAT1 and suggest that organic anion transporters contribute to cimetidine excretion in proximal tubules.

Identification and functional assessment of the novel murine organic anion transporter Oat5 (Slc22a19) expressed in kidney

2004 ◽  
Vol 287 (2) ◽  
pp. F236-F244 ◽  
Author(s):  
Geri L. Youngblood ◽  
Douglas H. Sweet
Keyword(s):  
Ochratoxin A ◽  
Cdna Clone ◽  
Organic Anion ◽  
Organic Anion Transporter ◽  
Xenopus Laevis Oocytes ◽  
Hepatic Expression ◽  
Estrone Sulfate ◽  
Anion Transporter ◽  
Transporter Family ◽  
2,4 Dichlorophenoxyacetic Acid

An uncharacterized murine cDNA clone was identified and, through sequence, phylogenetic, and functional analysis, determined to encode the newest member of the organic anion transporter family, organic anion transporter 5 (Oat5; Slc22a19). The Oat5 cDNA clone contained an insert 1,964 bp in length with a predicted open reading frame (from bp 84 to bp 1,739) coding for a peptide 551 amino acids long. Slc22a19 was localized to mouse chromosome 19 near the genes encoding Oat1 ( Slc22a6) and Oat3 ( Slc22a8). Northern blot analysis revealed Oat5 is highly expressed in the kidney of adult mice and rats. No sexual dimorphism in renal or hepatic expression of Oat5 was observed. Unlike Oat1–3, Oat5 expression was not detected in the choroid plexus of either mice or rats. Murine Oat5-expressing Xenopus laevis oocytes supported increased accumulation of the mycotoxin ochratoxin A, compared with water-injected control oocytes. This uptake was significantly inhibited by probenecid and the organic anions 2,4-dichlorophenoxyacetic acid, salicylate, and estrone sulfate but not by para-aminohippurate or urate. Transport of ochratoxin A by murine Oat5 was saturable, with an estimated Km of 2.0 ± 0.45 μM. Oat5-mediated transport was neither cis-inhibited nor trans-stimulated by the dicarboxylate glutarate. Uptake was also completely unaffected by short-circuiting of the membrane potential. Thus the motive forces behind Oat5 function, which provide insight into its membrane localization, need to be further resolved. These data demonstrate for the first time that this newly identified gene encodes a protein that functions as an organic anion transporter.

scholarly journals Characterization of an organic anion transport system in a placental cell line

2003 ◽  
Vol 285 (5) ◽  
pp. E1103-E1109 ◽  
Author(s):  
Fanfan Zhou ◽  
Kunihiko Tanaka ◽  
Michael J. Soares ◽  
Guofeng You
Keyword(s):  
Cell Line ◽  
Transport System ◽  
Apical Membrane ◽  
Organic Anion ◽  
Phosphodiesterase Inhibitor ◽  
Anion Transport ◽  
Organic Anion Transport ◽  
Estrone Sulfate ◽  
Anion Transporters ◽  
Anion Transport System

Transporters within the placenta play a crucial role in the distribution of nutrients and xenobiotics across the maternal-fetal interface. An organic anion transport system was identified on the apical membrane of the rat placenta cell line HRP-1, a model for the placenta barrier. The apical uptake of 3H-labeled organic anion estrone sulfate in HRP-1 cells was saturable ( Km = 4.67 μM), temperature and Na+ dependent, Li+ tolerant, and pH sensitive. The substrate specificity of the transport system includes various steroid sulfates, such as β-estradiol 3,17-disulfate, 17β-estradiol 3-sulfate, and dehydroepiandrosterone 3-sulfate (DHEAS) but does not include taurocholate, p-aminohippuric acid (PAH), and tetraethylammonium. Preincubation of HRP-1 cells with 8-bromo-cAMP (a cAMP analog) and forskolin (an adenylyl cyclase activator) acutely stimulated the apical transport activity. This stimulation was further enhanced in the presence of IBMX (a phosphodiesterase inhibitor). Together these data show that the apical membrane of HRP-1 cells expresses an organic anion transport system that is regulated by cellular cAMP levels. This transport system appears to be different from the known taurocholate-transporting organic anion-transporting polypeptides and PAH-transporting organic anion transporters, both of which also mediate the transport of estrone sulfate and DHEAS.

Cl−-dependent upregulation of human organic anion transporters: different effects on transport kinetics between hOAT1 and hOAT3

2007 ◽  
Vol 293 (1) ◽  
pp. F391-F397 ◽  
Author(s):  
Harumasa Ueo ◽  
Hideyuki Motohashi ◽  
Toshiya Katsura ◽  
Ken-ichi Inui
Keyword(s):  
Organic Anion ◽  
Substrate Recognition ◽  
Chloride Ion ◽  
Transport Kinetics ◽  
Hek293 Cells ◽  
Organic Anion Transporters ◽  
Basolateral Membranes ◽  
Estrone Sulfate ◽  
Anion Transporters ◽  
Threefold Increase

Chloride ion has a stimulatory effect on the transport of organic anions across renal basolateral membranes. However, the exact mechanisms at molecular levels have been unclear as of yet. Human organic anion transporters hOAT1 and hOAT3 play important roles in renal basolateral membranes. In this study, the effects of Cl− on the activities of these transporters were evaluated by using HEK293 cells stably expressing hOAT1 or hOAT3 (HEK-hOAT1 or HEK-hOAT3). The uptake of p-[14C]aminohippurate by HEK-hOAT1 and [3H]estrone sulfate by HEK-hOAT3 was greater in the presence of Cl− than in the presence of SO42− or gluconate. Additionally, the uptake of various compounds by HEK-hOAT1 and HEK-hOAT3 was significantly higher in the Cl−-containing medium than the gluconate-containing medium, suggesting that the influences of Cl− are not dependent on substrate and that Cl− directly stimulates the functions of hOAT1 and hOAT3. The substitution of gluconate with Cl− did not change the Km value for the uptake of p-[14C]aminohippurate by HEK-hOAT1 but caused an approximately threefold increase in the maximal uptake rate (Vmax) value. On the other hand, replacement of gluconate with Cl− decreased the Km value for the uptake of [3H]estrone sulfate and cefotiam by HEK-hOAT3 to about one-third, while it did not change the Vmax value. In summary, Cl− upregulates the activities of both hOAT1 and hOAT3, but its effects on transport kinetics differ between these transporters. It was suggested that Cl− participates in the trans-location process for hOAT1, and the substrate recognition process for hOAT3.

Organic anion transporter 3 (Slc22a8) is a dicarboxylate exchanger indirectly coupled to the Na+gradient

2003 ◽  
Vol 284 (4) ◽  
pp. F763-F769 ◽  
Author(s):  
Douglas H. Sweet ◽  
Lauretta M. S. Chan ◽  
Ramsey Walden ◽  
Xiao-Ping Yang ◽  
David S. Miller ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Organic Anion ◽  
Renal Proximal Tubule ◽  
Organic Anion Transporter ◽  
Xenopus Laevis Oocytes ◽  
Estrone Sulfate ◽  
Anion Transporter ◽  
Renal Proximal Tubule Cells ◽  
Rat Renal Cortical Slices ◽  
Cortical Slices

Basolateral uptake of organic anions in renal proximal tubule cells is indirectly coupled to the Na+ gradient through Na+-dicarboxylate cotransport and organic anion/dicarboxylate exchange. One member of the organic anion transporter (OAT) family, Oat1, is expressed in the proximal tubule and is an organic anion/dicarboxylate exchanger. However, a second organic anion carrier, Oat3, is also highly expressed in the renal proximal tubule, but its mechanism is unclear. Thus we have assessed Oat3 function in Xenopus laevis oocytes and rat renal cortical slices. Probenecid-sensitive uptake of p-aminohippurate (PAH, an Oat1 and Oat3 substrate) and estrone sulfate (ES, an Oat3 substrate) in rat Oat3-expressing oocytes was significantly trans-stimulated by preloading the oocytes with the dicarboxylate glutarate (GA). GA stimulation of ES transport by oocytes coexpressing rabbit Na+-dicarboxylate cotransporter 1 and rat Oat3 was significantly inhibited when the preloading medium contained Li+ or methylsuccinate (MS) or when Na+ was absent. All these treatments inhibit the Na+-dicarboxylate cotransporter, but not rat Oat3. Li+, MS, and Na+ removal had no effect when applied during the ES uptake step, rather than during the GA preloading step. Concentrative ES uptake in rat renal cortical slices was also demonstrated to be probenecid and Na+ sensitive. Accumulation of ES was stimulated by GA, and this stimulation was completely blocked by probenecid, Li+, MS, taurocholate, and removal of Na+. Thus Oat3 functions as an organic anion/dicarboxylate exchanger that couples organic anion uptake indirectly to the Na+ gradient.

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