P-aminohippurate Research Articles

RT-PCR-based evidence for the in vivo stimulation of renal tubular p-aminohippurate (PAH) transport by triiodothyronine (T3) or dexamethasone (DEXA) in kidney tissue of immature and adult rats

Our previous studies have shown that a pre-treatment of rats with triiodothyronine (T3) or dexamethasone (DEXA) increases renal PAH excretion significantly. This stimulation was accompanied by an enhanced protein synthesis within the renal cortex. To explore the molecular basis for this sub-chronic induction process, we investigated the stimulation of PAH accumulation in renal cortical slices as well as the expression level of organic anion transporter 1 (OAT1), the recently cloned renal basolateral PAH-transporter, using RT-PCR techniques under the applied conditions. 10- and 55-day-old Han:WIST rats were treated in vivo with T3 (20 microg/100 g b.wt.) or DEXA (60 microg/100 g b.wt.), both for 3 days, once daily. Renal cortical slices were incubated for 2 hours in Cross-Taggart medium and PAH uptake into kidney tissue was measured time dependently (slice to medium ratio, QS/M). The accumulation capacity is comparable between immature and mature rats (control-QS/M: 6.7 +/- 0.1 vs. 6.9 +/- 0.2, respectively). Both age groups showed a significant increase of PAH accumulation capacity after T3 treatment (10-day-old rats: 15.0 +/- 0.2; 55-day-old rats: 11.7 +/- 1.3). After DEXA pre-treatment, PAH accumulation was only slightly changed (10-day-old rats: 5.9 +/- 0.2; 55-day-old rats: 8.2 +/- 1.3). Semi-quantitative measurements of OAT1 mRNA expression level showed a significant increase of OAT1 mRNA after pre-treatment with both T3 and DEXA in the two age groups. Thus, this is the first evidence that T3 and DEXA pre-treatment induces the expression of OAT1.

Urate transport via human PAH transporter hOAT1 and its gene structure

We recently cloned the human organic anion transporter 1 (hOAT1) as a p-aminohippurate (PAH) transporter. Whether urate is transported by the PAH transporter in humans remains unclear. Familial juvenile gouty nephropathy (FJGN) is thought to develop as a result of an abnormality in the urate transporter. To determine if hOAT1 transported urate, the cellular uptakes of PAH and urate were determined, as were the inhibition profiles of inorganic anions, and uricosuric and antiuricosuric agents using a mouse S2 cell line expressing hOAT1. The hOAT1 gene was cloned from a genomic library using full-length hOAT1-1 cDNA as a probe. The coding regions of the hOAT1 genes of two sisters with FJGN were sequenced. Also, immunohistochemical fluorescence analysis of hOAT1 in the kidney of the younger sister with FJGN was performed. The Km and Vmax values of urate transport via hOAT1 were 943 +/- 84 micromol/L and 1286 +/- 162 pmol/mg protein/min, respectively. The order of the IC50 of urate transport via hOAT1 was benzbromarone < probenecid < salicylate or pyrazine carboxylic acid. The 10.9 kb hOAT1 gene was found to be interrupted by nine introns. Mutations in the coding region of the hOAT1 gene from the two sisters with FJGN were undetectable. Immunohistochemical fluorescent staining showed that hOAT1 in the kidney of the younger sister was similar to that of control individuals. Our data show that hOAT1 transports urate, and the inhibition profiles of uricosuric and antiuricosuric agents are defined. hOAT1 is not responsible for FJGN in the two sisters examined in this study.

Characterization and identification of steroid sulfate transporters of human placenta.

Human trophoblasts depend on the supply of external precursors, such as dehydroepiandrosterone-3-sulfate (DHEA-S) and 16 alpha-OH-DHEA-S, for synthesis of estrogens. The aim of the present study was to characterize the uptake of DHEA-S by isolated mononucleated trophoblasts (MT) and to identify the involved transporter polypeptides. The kinetic analysis of DHEA-(35)S uptake by MT revealed a saturable uptake mechanism (K(m) = 26 microM, V(max) = 428 pmol x mg protein(-1) x min(-1)), which was superimposed by a nonsaturable uptake mechanism (diffusion constant = 1.2 microl x mg protein(-1) x min(-1)). Uptake of [(3)H]DHEA-S by MT was Na(+) dependent and inhibited by sulfobromophthalein (BSP), steroid sulfates, and probenecid, but not by steroid glucuronides, unconjugated steroids, conjugated bile acids, ouabain, p-aminohippurate (PAH), and bumetanide. MT took up [(35)S]BSP, [(3)H]estrone-sulfate, but not (3)H-labeled ouabain, estradiol-17beta-glucuronide, taurocholate, and PAH. RT-PCR revealed that the organic anion-transporting polypeptides OATP-B, -D, -E, and the organic anion transporter OAT-4 are highly expressed, and that OATP-A, -C, -8, OAT-3, and Na(+)-taurocholate cotransporting polypeptide (NTCP) are not or are only lowly expressed in term placental tissue and freshly isolated and cultured trophoblasts. Immunohistochemistry of first- and third-trimester placenta detected OAT-4 on cytotrophoblast membranes and at the basal surface of the syncytiotrophoblast. Our results indicate that uptake of steroid sulfates by isolated MT is mediated by OATP-B and OAT-4 and suggest a physiological role of both carrier proteins in placental uptake of fetal-derived steroid sulfates.

Urate/alpha-ketoglutarate exchange in avian basolateral membrane vesicles.

Membrane transport pathways for transcellular secretion of urate across the proximal tubule were investigated in avian kidney. The presence of coupled urate/alpha-ketoglutarate exchange was investigated in basolateral membrane vesicles (BLMV) by [(14)C]urate and [alpha-(3)H]ketoglutarate flux measurements. An inward Na gradient induced accumulation of alpha-ketoglutarate of sufficient magnitude to suggest a Na-dicarboxylate cotransporter. An inward Na gradient also induced concentrative accumulation of urate in the presence of alpha-ketoglutarate but not in its absence, suggesting urate/alpha-ketoglutarate exchange. alpha-Ketoglutarate-dependent stimulation of urate uptake was not observed in brush-border membrane vesicles. An outward urate gradient induced concentrative accumulation of alpha-ketoglutarate. alpha-Ketoglutarate-coupled urate uptake was specific for alpha-ketoglutarate, Cl dependent, and insensitive to membrane potential. alpha-Ketoglutarate-coupled urate uptake was inhibited by increasing p-aminohippurate (PAH) concentrations, and alpha-ketoglutarate-coupled PAH uptake was observed. alpha-Ketoglutarate-coupled PAH uptake was inhibited by increasing urate concentrations, and an outward urate gradient induced concentrative accumulation of PAH. These results suggest a Cl-dependent, alpha-ketoglutarate-coupled anion exchange mechanism as a pathway for active urate uptake across the basolateral membrane of urate-secreting proximal tubule cells.

Identification of a mechanism by which the methylmercury antidotes N-acetylcysteine and dimercaptopropanesulfonate enhance urinary metal excretion: transport by the renal organic anion transporter-1.

N-Acetylcysteine (NAC) and dimercaptopropanesulfonate (DMPS) are sulfhydryl-containing compounds that produce a dramatic acceleration of urinary methylmercury (MeHg) excretion in poisoned animals, but the molecular mechanism for this effect is unknown. NAC and DMPS are themselves excreted in urine in high concentrations. The present study tested the hypothesis that the complexes formed between MeHg and these anionic chelating agents are transported from blood into proximal tubule cells by the basolateral membrane organic anion transporters (Oat) 1 and Oat3. Xenopus laevis oocytes expressing rat Oat1 showed increased uptake of [(14)C]MeHg when complexed with either NAC or DMPS but not when complexed with L-cysteine, glutathione, dimercaptosuccinate, penicillamine, or gamma-glutamylcysteine. In contrast, none of these MeHg complexes were transported by Oat3-expressing oocytes. The apparent K(m) values for Oat1-mediated transport were 31 +/- 2 microM for MeHg-NAC and 9 +/- 2 microM for MeHg-DMPS, indicating that these are relatively high-affinity substrates. Oat1-mediated uptake of [(14)C]MeHg-NAC and [(14)C]MeHg-DMPS was inhibited by prototypical substrates for Oat1, including p-aminohippurate (PAH), and was trans-stimulated when oocytes were preloaded with 2 mM glutarate but not glutamate. Conversely, efflux of [(3)H]PAH from Oat1-expressing oocytes was trans-stimulated by glutarate, PAH, NAC, DMPS, MeHg-NAC, MeHg-DMPS, and a mercapturic acid, indicating that these are transported solutes. [(3)H]PAH uptake was competitively inhibited by NAC (K(i) of 2.0 +/- 0.3 mM) and DMPS (K(i) of 0.10 +/- 0.02 mM), providing further evidence that these chelating agents are substrates for Oat1. These results indicate that the MeHg antidotes NAC and DMPS and their mercaptide complexes are transported by Oat1 but are comparatively poor substrates for Oat3. This is the first molecular identification of a transport mechanism by which these antidotes may enhance urinary excretion of toxic metals.

Facilitated diffusion of urate in avian brush-border membrane vesicles.

Membrane transport pathways mediating transcellular secretion of urate across the proximal tubule were investigated in brush-border membrane vesicles (BBMV) isolated from avian kidney. An inside-positive K diffusion potential induced a conductive uptake of urate to levels exceeding equilibrium. Protonophore-induced dissipation of membrane potential significantly reduced voltage-driven urate uptake. Conductive uptake of urate was inhibitor sensitive, substrate specific, and a saturable function of urate concentration. Urate uptake was trans-stimulated by urate and cis-inhibited by p-aminohippurate (PAH). Conductive uptake of PAH was cis-inhibited by urate. Urate uptake was unaffected by an outward alpha-ketoglutarate gradient. In the absence of a membrane potential, urate uptake was similar in the presence and absence of an imposed inside-alkaline pH gradient or an outward Cl gradient. These observations suggest a uniporter-mediated facilitated diffusion of urate as a pathway for passive efflux across the brush border membrane of urate-secreting proximal tubule cells.

Specificity of Anion Exchange Mediated by Mouse Slc26a6

Recently, CFEX, the mouse orthologue of human SLC26A6, was localized to the brush border membrane of proximal tubule cells and was demonstrated to mediate Cl(-)-formate exchange when expressed in Xenopus oocytes. The purpose of the present study was to examine whether mouse Slc26a6 can mediate one or more of the additional anion exchange processes observed to take place across the apical membrane of proximal tubule cells. Influx of [(14)C]formate into Slc26a6-expressing oocytes was inhibited by sulfate, oxalate, and p-aminohippurate (PAH), indicating affinity for these anions. Measurements of uptake of [(14)C]oxalate, [(14)C]PAH, and [(35)S]sulfate indicated that Slc26a6 can mediate transport of oxalate and sulfate but not PAH. Studies of the effect of external anions on [(14)C]oxalate efflux demonstrated Slc26a6-mediated Cl(-)-oxalate, oxalate-formate, oxalate-oxalate, and oxalate-sulfate exchange. Two-electrode voltage clamp measurements indicated that Slc26a6-mediated Cl(-)-oxalate exchange is electrogenic. Intracellular pH recordings demonstrated that Slc26a6 can mediate Cl(-)-HCO(3)(-) exchange, but Cl(-)-OH(-) exchange was not detected. The presence of 100 microm oxalate inhibited the rate of Cl(-)-HCO(3)(-) exchange by 60%. We conclude that mouse Slc26a6 has affinity for oxalate, sulfate, and HCO(3)(-) in addition to Cl(-) and formate and can function in multiple exchange modes involving pairs of these anions. In the presence of high oxalate concentrations as found in renal tubular fluid and urine, Slc26a6 may largely function as an electrogenic Cl(-)-oxalate exchanger.

Interaction of the organic base cimetidine with the renal basolateral p-aminohippurate (PAH) transport system

Interaction of the organic base cimetidine with the renal basolateral p-aminohippurate (PAH) transport system

Role of aromatic transmembrane residues of the organic anion transporter, rOAT3, in substrate recognition.

Organic anion transporters (OATs, SLC21) are important in the excretion of endogenous and exogenous compounds in the kidney. The rat organic anion transporter, rOAT3, mediates the transport of organic anions such as p-aminohippurate (PAH) and estrone sulfate as well as the basic compound, cimetidine. In the present study, we examined the role of conserved transmembrane aromatic amino acid residues of rOAT3 in substrate recognition and transport. Alanine scanning followed by amino acid replacements was used to construct mutants of rOAT3. The uptake of model compounds was studied in Xenopus laevis oocytes expressing the mutant transporters. We observed that four mutants in transmembrane domain 7 (TMD 7), W334A, F335A, Y341A, and Y342Q, and one mutant in transmembrane domain 8 (TMD 8), F362S, exhibited a less than 2-fold enhanced uptake of PAH and cimetidine in comparison to wild-type rOAT3, which exhibited a 16-fold enhanced uptake of PAH and an 8-fold enhanced uptake of cimetidine. Estrone sulfate uptake in oocytes expressing any one of these five mutants remained at least 8-fold enhanced. The data suggest that the five residues, W334, F335, Y341, Y342, and F362, contribute differently to the transport of the small hydrophilic organic substrates PAH and cimetidine in comparison to the large hydrophobic organic substrate estrone sulfate. The effects of side chains of these five residues on transporter functions were also evaluated by constructing conservative mutations. We observed that the residues contribute to PAH and cimetidine transport in different ways: the -OH group of Y342, the indole ring of W334, and the aromatic rings of F335, Y341, and F362 are important for PAH and cimetidine transport by rOAT3. These data suggest that there is an aromatic pocket composed mainly of residues in TMD 7 in the translocation pathway of rOAT3, which is important for the transport of PAH and cimetidine. Aromatic residues in this pocket may interact directly with substrates of rOAT3 through hydrogen bonds and pi-pi interactions.

Expression and functional characterization of rat organic anion transporter 3 (rOat3) in the choroid plexus.

We reported previously that an efficient efflux system for benzylpenicillin (PCG) is located on the choroid plexus (CP). In this study, we investigated the involvement of rat organic anion transporter 1 (rOat1; Slc22a6) and rOat3 (Slc22a8) in the uptake of PCG and p-aminohippurate (PAH) by the CP. Western blot analysis indicates the expression of rOat3, but not rOat1, on the CP, and immunohistochemical staining shows that rOat3 is localized on the brush border membrane of the choroid epithelial cells. PCG and PAH were found to be taken up by isolated rat CP, with K(m) values of 111 and 354 microM, respectively. A mutual inhibition study suggests that the same transporter is responsible for the uptake of PCG and PAH by isolated rat CP. This was confirmed by examining the effect of organic anions and cimetidine on their uptake. Estradiol-17beta-glucuronide and cimetidine were found to be selective inhibitors of rOat3. The inhibition constants of the inhibitors including estradiol-17beta-glucuronide and cimetidine were comparable for the uptake of PAH and PCG by isolated rat CP. In addition, these values were also comparable with those for rOat3, but not with those for rOat1. These results suggest that rOat3 is mainly responsible for the uptake of PCG and PAH by isolated rat CP, and it functions as one of the detoxification systems on the CP by removing its substrates from the cerebrospinal fluid.

Functional involvement of rat organic anion transporter 3 (rOat3; Slc22a8) in the renal uptake of organic anions.

Our previous kinetic analyses have shown that the transporter responsible for the renal uptake of pravastatin, an HMG-CoA reductase inhibitor, differs from that involved in its hepatic uptake. Although organic anion transporting polypeptides are now known to be responsible for the hepatic uptake of pravastatin, the renal uptake mechanism has not been clarified yet. In the present study, the involvement of rat organic anion transporter 3 (rOat3; Slc22a8) in the renal uptake of pravastatin was investigated. Immunohistochemical staining indicates the basolateral localization of rOat3 in the kidney. rOat1- and rOat3-expressed LLC-PK1 cells exhibited specific uptake of p-aminohippurate (PAH) and pravastatin, respectively, with the Michaelis-Menten constants (Km values) of 60 microM for rOat1-mediated PAH uptake and 13 microM for rOat3-mediated pravastatin uptake. Saturable uptake of PAH and pravastatin was observed in kidney slices with Km values of 69 and 11 microM, respectively. The difference in the potency of PAH and pravastatin in inhibiting uptake by kidney slices suggests that different transporters are responsible for their renal uptake. This was also supported by the difference in the degree of inhibition by benzylpenicillin, a relatively selective inhibitor of rOat3, for the uptake of PAH and pravastatin by kidney slices. These results suggest that rOat1 and rOat3 are mainly responsible for the renal uptake of PAH and pravastatin, respectively.

P-Aminohippurate transport at the apical membrane in the OK kidney epithelial cell line.

We investigated the characteristics of transport of an organic anion, p-aminohippurate (PAH), at the apical membrane in a kidney epithelial cell line OK. Efflux and uptake of [14C]PAH across the apical membrane were measured using OK cell monolayers grown on microporous membrane filters. PAH efflux to the apical side was greater than that to the basolateral side and significantly inhibited by probenecid. Diethyl pyrocarbonate (DEPC), an inhibitor of potential-sensitive organic anion transport, significantly decreased PAH efflux to the apical side. Moreover, PAH efflux to the apical side was significantly decreased on incubation with high potassium buffer, as compared with control condition. Extracellular pH and Cl- had no effect on PAH efflux across the apical membrane. PAH uptake from the apical side was inhibited by various organic anions, and the inhibition patterns of PAH uptake from the apical and basolateral sides by various dicarboxylates were similar. These results suggested that PAH efflux to the apical side in OK cells was mediated by a potential-sensitive transport system, but not by an anion exchanger. Moreover, PAH uptake from the apical side was mediated by a specific transport system, which interacts with various organic anions and dicarboxylates.

Rat Renal Organic Anion Transporter rOAT1 Mediates Transport of Urinary-Excreted Cephalosporins, but not of Biliary-Excreted Cefoperazone

Most cephalosporin antibiotics are excreted into urine via glomerular filtration and active tubular secretion by renal organic anion transporters. In this study, we investigated the interaction of cephalosporins with rat organic anion transporter rOAT1, mainly expressed at the basolateral membrane of the renal proximal tubules, using Xenopus laevis oocytes, to assess the roles of rOAT1 in renal excretion of cephalosporin antibiotics. The expression of rOAT1 significantly stimulated the uptake of cefazolin, cefotiam and cephalexin into oocytes, but not of cefoperazone. The inhibition constants of these cephalosporins to rOAT1-mediated p-aminohippurate (PAH) uptake were 72 microM for cefazolin, 298 microM for cefoperazone, 718 microM for cefotiam and 6 mM for cephalexin. Eadie-Hofstee plot analysis revealed that cefoperazone as well as cefotiam inhibited rOAT1-mediated PAH uptake competitively. These results suggest that rOAT1 mediates basolateral uptake of cephalosporin antibiotics in the renal tubules. Furthermore, it is suggested that a minor contribution of the kidney to cefoperazone excretion could be related to the finding that cefoperazone is a poor substrate of rOAT1.

Cationic amino acids involved in dicarboxylate binding of the flounder renal organic anion transporter.

Three conserved cationic amino acids in predicted transmembrane domains 1, 8, and 11, respectively, of the flounder renal organic anion transporter, fROAT, were changed by site-directed mutagenesis and the resulting mutants functionally characterized in Xenopus laevis oocytes. Uptake of p-aminohippurate (PAH) in oocytes that expressed mutant H34I, K394A, or R478D was markedly reduced compared with oocytes that expressed wild-type fROAT, but was still several-fold higher than that in water-injected control oocytes. Immunocytochemically, no decrease in cell surface expression of the mutants could be detected. Only mutant R478D appeared to have a lower PAH affinity than the wild type. Similar to wild-type-dependent PAH transport, uptake induced by mutant H34I was sensitive to glutarate (GA) cis-inhibition. In contrast, mutants K394A and R478D could not be significantly affected by up to 10 mM GA, although the cRNA-dependent PAH uptake could still be almost completely suppressed by probenecid. Moreover, again in contrast to the wild type, neither PAH influx nor PAH efflux mediated by these two mutants could be trans-stimulated by GA, nor did they induce GA transport. These data suggest that amino acids K394 and R478 in fROAT are required for dicarboxylate binding and PAH/dicarboxylate exchange.

Adenine nucleotide and calpain inhibitor I protect against atractyloside-induced toxicity in rat renal cortical slices in vitro.

Atractyloside is a compound with a documented nephrotoxicity. It induces renal tubular necrosis at high doses and apoptosis at lower doses. This study investigates the potential protective effect of some chemical agents against atractyloside-induced nephrotoxicity in vitro using the precision-cut rat renal cortical slices obtained from kidneys of Wistar rats. For co-incubation experiments, slices were incubated for 3 h at 37 degrees C on a rocker platform with various chemical agents: ADP (5 mM), calpain inhibitor I (CPI, 1 mM), stevioside (STV, 2.5 mM) or probenecid (PRB, 2.5 mM) in the presence or absence of atractyloside (2 mM). For pre-incubation experiments, slices were incubated with the same chemical agents for 1 h before exposure to atractyloside. The nephrotoxic effects of atractyloside (2 mM) alone were manifested in several ways: by a marked increase in lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) leakage, significant inhibition of p-aminohippurate (PAH) accumulation, marked depletion of intracellular ATP and reduced glutathione (GSH), and a significant reduction in pyruvate-stimulated gluconeogenesis. Co-incubation of slices with ADP or CPI and atractyloside completely blocked atractyloside-induced increase in LDH leakage, but not ALP leakage. Atractyloside-induced depletion of ATP and reduced gluconeogenesis was prevented by co-incubation with ADP or CPI. Furthermore, co-incubation of slices with STV and atractyloside, but not PRB, completely abolished atractyloside-induced depletion of ATP and decreased gluconeogenesis in the slices. Pre-incubation of slices with either ADP or CPI protected against atractyloside-induced increase in LDH leakage, reduced ATP and decreased gluconeogenesis. PAH uptake in the slices was inhibited by atractyloside and PRB in a time-dependent manner. While ADP and CPI were found to exert complete protection against atractyloside-induced toxicity irrespective of treatment schedule, STV is effective only under certain conditions, and PRB offer no protection at all. The results of this study demonstrate the usefulness of renal cortical slices as toxicology tool for evaluating and screening compounds for their potential protective effects, and are supportive of a role of adeninine nucleotide (ADP) and protease inhibitor (CPI) in protecting against atractyloside-induced cell injury.

Transient renal effects of sildenafil in male kidney transplant recipients.

Sildenafil (Viagra) improves erection by sustaining Guanosine 3', 5'-cyclic monophosphate (cGMP)-mediated smooth muscle relaxation in the corpus cavernosum. It also induces systemic vasodilation, resulting in a minor decrease in blood pressure. We evaluated the effect of one dose of sildenafil on graft function and hemodynamics in impotent male transplant recipients. Two sets of combined lithium, inulin, and p-amino hippurate clearance studies were conducted, with and without sildenafil (100 mg orally) in 11 male kidney transplant recipients (KTRs). Sildenafil increased glomerular filtration rate by 14+/-4 from the baseline value of 55+/-7 ml x min(-1) x 1.73 m2(-1) (P<0.01), whereas calculated renal vascular resistances decreased by 40+/-18 from the baseline value of 247+/-29 mmHg/L x min(-1) x 1.73 m2-1 (P<0.05). The oral administration of sildenafil in KTRs did not impair the function of the graft. In terms of renal physiology, the observed modifications did not warrant any specific precautions when offering sildenafil to KTRs suffering from erectile dysfunction.

Characterization of the mechanisms involved in the gender differences inp-aminohippurate renal elimination in rats

Gender differences in the renal handling on drugs and toxins have received too little attention. In the present study, a variety of preparations were used to examine the basis for the greater effectiveness of the male kidneys in the elimination of p-aminohippurate (PAH) in rats. Renal clearance of PAH was significantly lower in female rats as consequence of its smaller filtered and secreted load. The gender difference in the filtered load may be accounted for the lower value of glomerular filtration rate (GFR) displayed by female rats as compared with males. The lower value of the renal blood flow observed in females might explain, at least in part, the decrease in the GFR and in the secreted load of PAH. In females, maximal uptake for PAH transport into renal basolateral membrane vesicles decreased to 52+/-9% (P < 0.05) and Michaelis-Menten constant for PAH uptake into renal brush border membrane vesicles was increased to 163+/-8% (P < 0.05). These changes might also explain the lower secreted load of PAH. The sex difference in the renal clearance of PAH was also evidenced by the reduced systemic clearance observed in female rats.

Molecular physiology of renal p-aminohippurate secretion.

Renal proximal tubules secrete various organic anions, including drugs and p-aminohippurate (PAH). Uptake of PAH from blood into tubule cells occurs by exchange with intracellular alpha-ketoglutarate and is mediated by the organic anion transporter 1. PAH exit into tubule lumen is species specific and may involve ATP-independent and -dependent transporters.

Technetium‐99m‐MAG3 as a transport substrate of rat renal organic anion transporter‐1

AbstractThe first step of technetium‐99m‐mercaptoacetylglycylglycylglycine (99mTc‐MAG3) secretion by the proximal tubule cells is the extraction of 99mTc‐MAG3 from the peritubular plasma by proximal tubule cells through the basolateral membrane. The evidence for 99mTc‐MAG3 being excreted from renal proximal tubules via rat organic anion transporter 1 (OAT1) by molecular biological experiments with expressed OAT1 in Xenopus laevis oocytes was discussed. Inhibition studies of 99mTc‐MAG3 revealed that OAT1 mediated Na+‐independent 99mTc‐MAG3 uptake, and that this transport was energy‐independent. Accumulated dicarboxylate, such as glutarate, stimulated 99mTc‐MAG3 uptake. Tc‐99m‐MAG3, as well as paminohippurate (PAH), and o‐iodohippurate (OIH), was confirmed to be a transport substrate of OAT1 by the uptake of expressed oocytes.

Arginine 454 and lysine 370 are essential for the anion specificity of the organic anion transporter, rOAT3.

Organic anion transporters (OATs) and organic cation transporters (OCTs) mediate the flux of xenobiotics across the plasma membranes of epithelia. Substrates of OATs generally carry negative charge(s) whereas substrates of OCTs are cations. The goal of this study was to determine the domains and amino acid residues essential for recognition and transport of organic anions by the rat organic anion transporter, rOAT3. An rOAT3/rOCT1 chimera containing transmembrane domains 1-5 of rOAT3 and 6-12 of rOCT1 retained the specificity of rOCT1, suggesting that residues involved in substrate recognition reside within the carboxyl-terminal half of these transporters. Mutagenesis of a conserved basic amino acid residue, arginine 454 to aspartic acid (R454D), revealed that this amino acid is required for organic anion transport. The uptakes of p-aminohippurate (PAH), estrone sulfate, and ochratoxin A were approximately 10-, approximately 48-, and approximately 32-fold enhanced in oocytes expressing rOAT3 and were only approximately 2-, approximately 6-, and approximately 5-fold enhanced for R454D. Similarly, mutagenesis of the conserved lysine 370 to alanine (K370A) suggested that K370 is important for organic anion transport. Interestingly, the charge specificity of the double mutant, R454DK370A, was reversed in comparison to rOAT3-R454DK370A preferentially transported the organic cation, MPP(+), in comparison to PAH (MPP(+) uptake/PAH uptake = 3.21 for the double mutant vs 0.037 for rOAT3). These data indicate that arginine 454 and lysine 370 are essential for the anion specificity of rOAT3. The studies provide the first insights into the molecular determinants that are critical for recognition and translocation of organic anions by a member of the organic anion transporter family.