P-aminohippurate Transport Research Articles

Abstract 4364: Oncometabolite L-2-hydroxyglutarate blocks differentiation of renal proximal tubule cells in matrigel

Abstract Previously, the Sudarshan laboratory has identified elevations in the level of the oncometabolite L-2-hydroxyglutarate (L-2HG) in human renal cell carcinomas (RCCs). Of particular interest in these regards, both L-2HG inhibits 2-oxoglutarate-dependent dioxygenases (2-OGDs), including histone demethylases, which regulate the epigenetic landscape of cells, and cellular differentiation. Thus, it was of interest to determine whether the accumulation of L-2HG alters the differentiation of the normal renal proximal tubule (RPT), ultimately affecting the phenotype of the RCC. Towards these ends, L-2HG dehydrogenase (L2HGDH) was knocked down in primary cultures of normal rabbit RPT cells, and their capacity for cellular differentiation was examined. Initially, we examined the ability of primary RPT cells to form tubules in matrigel following an L2HGDH knockdown (KD). While tubulogenesis was stimulated by Epidermal Growth Factor (EGF) in primary RPT cells transduced with a control lentiviral vector, tubulogenesis in matrigel was dramatically impaired in primary RPT cells when L2HGDH was knocked down by lentiviral L2HGDH shRNA. In order to determine whether the expression of differentiated transport functions was affected, RealTime PCR (RTPCR) was conducted. The results indicated that an L2HGDH knockdown (80%) resulted in a reduction in the expression of the Na+/Pi cotransporter NaPi2a (81%), the Na+/glucose cotransporter SGLT2 (88%), the water transporter Aquaporin 1 (AQP1) (95%), and the Na,K-ATPase ß1 subunit (atp1b1) (43%), whereas the expression of the p-Aminohippurate transporter OAT1 was not significantly affected. Similar results were obtained when using L2HGDH siRNA and lentiviral shRNA. Not only is tissue architecture critical for the maintenance of functional differentiation, but alterations in tissue architecture are a necessary component of tumor formation. Thus, we are examining the underlying causes for the reduced tubulogenesis in matrigel cultures with an L2HGDH KD. In our initial studies, we examined the expression of differentiated transport functions in matrigel vs. monolayer cultures. Notably, while the expression such transporters as AQP2 increased dramatically in matrigel (as opposed to monolayer cultures), the expression of other transporters such as NaPi2a, SGLT2 and atp1b1 was affected to a much smaller extent. Of particular interest in these regards, is the known role of AQP1 in the migration of renal proximal tubule cells, tumor spread, and wound healing. We are currently studying the effects of an L2HGDH KD on the expression of such genes as AQP1 during tubulogenesis in matrigel.This work has been funded by Grant # 1RO1CA200653-01A1 to Dr. Sunil Sudarshan (PI) with a subaward to Dr. Mary Taub. Citation Format: Mary L. Taub, Sunil Sudarshan. Oncometabolite L-2-hydroxyglutarate blocks differentiation of renal proximal tubule cells in matrigel [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4364.

Effect of Sulfhydryl Modification on Rat Kidney Basolateral Plasma Membrane Transport Function

Transport processes are the hallmark of functioning kidney. Various nephrotoxicants disrupt the transport processes to manifest nephrotoxicity. Of several nephrotoxicants, mercuric chloride (HgCl(2)) depletes the reduced glutathione (GSH) in kidney and has been observed to affect the in vitro p-aminohippurate (PAH) transport by basolateral (BL) membrane vesicles. The role of renal nonprotein sulfhydryls such as, reduced GSH has been demonstrated to affect the PAH transport by BL membrane vesicles. The role of protein sulfhydryls in transport process of PAH by BL membrane is not known. Due to mercury mediated effects on sulfhydryls, the effects of protein-sulfhydryls (-SH) modifying reagents in the current study were investigated on PAH transport by BL membrane. It was observed that modification of -SH by p-chloromercuribenzoate sulphate (pCMBS), and mercuric chloride (HgCl(2)) decreased while recovering the protein -SH with dithiothreitol treatment provided protection against the effects of pCMBS, and HgCl(2) on PAH transport by BL membrane vesicles.

Transport of anionic drugs across the basolateral membrane of proximal S2 segments of the rabbit kidney. Inverse relationship between the affinity to the p-aminohippurate transport system and the transport rate.

The renal p-aminohippurate (PAH) transport system, which resides in the S2 segments of proximal tubules, is a main tubular secretory system for drugs. Previous studies have shown that the hydrophobicity of drugs is positively correlated with their affinity for the basolateral PAH transporter. Affinity was deduced from inhibition of tubular 3H-PAH uptake. However, up to now, no studies are available which tested the relationship between hydrophobicity and transport rate of drugs. Therefore, in the present study, the basolateral transport rates of several drugs (probenecid, CAS 57-66-9, furosemide, CAS 54-31-9, bumetanide, CAS 28395-03-1, mefruside, CAS 7195-27-9) which are substrates of the PAH transporter and which differ markedly regarding their hydrophobic properties (log P values ranging from 1-3) were determined on non-perfused proximal S2 segments microdissected from rabbit kidneys without the use of enzymatic solutions. To measure the transport rate advantage of the finding was taken that the PAH transporter in the basolateral membrane of proximal S2 segments acts as a countertransporter which exchanges PAH with dicarboxylates (glutarate or alpha-keto-glutarate). Hence, the stimulatory effect of a drug on the 14C-glutarate efflux rate of 14C-glutarate preloaded S2 segments is a measure of its translocation across the basolateral cell membrane. The results revealed that with increasing hydrophobicity (mefruside < furosemide < bumetanide < probenecid) the stimulating effect of the drugs on 14C-glutarate efflux decreased indicating that the compounds are more slowly translocated although their potency to inhibit 3H-PAH uptake increased. Thus, an inverse relationship between hydrophobicity and hence affinity of the drugs for the PAH transporter and transport rate could be established.

Moment Analysis of Drug Disposition in Rat Kidney: Role of Basolateral Membrane Transport in Renal Transepithelial Transport of p-Aminohippurate

The determining step in transepithelial transport of p-aminohippurate (PAH) in renal tubular secretion has been elucidated in the rat isolated perfused kidney. The method was based upon a multiple indicator dilution experiment and non-compartmental moment analysis. Single-pass dilution curves were obtained from venous and urinary effluents after simultaneous intra-arterial injections of Evans blue with albumin, [3H]inulin and [14C]PAH. Probenecid was used as a transport inhibitor and dissolved in both perfusate and injection solution. The urinary excretion of PAH decreased depending on the probenecid concentration, while that of inulin was not affected. No correlation was observed between the amount of secretion and the mean residence time of secreted PAH in renal epithelial cells (Tcell). Since Tcell ought to be affected by the rate of secretion from cells to lumen, it was suggested that the secretion rate was independent of the amount secreted. In contrast, the amount of PAH excreted via tubular secretion showed a linear correlation with the volume of distribution in the kidney and the apparent rate constant for tubular uptake of PAH. Since these kinetic parameters reflect the transport from blood into cells across the basolateral membranes of renal epithelial cells, the present results demonstrated that basolateral membrane transport is a determining step in the transcellular transport of PAH and that the major effect of probenecid is the inhibition of transport from blood into cells.

Inhibitory Effect of Caffeic Acid on Human Organic Anion Transporters hOAT1 and hOAT3: A Novel Candidate for Food–Drug Interaction

Several kinds of food have been shown to influence the absorption and metabolism of drugs, although there is little information about their effect on the renal excretion of drugs. In this study, we performed uptake experiments using Xenopus laevis oocytes to assess the inhibitory effects of chlorogenic acid, caffeic acid and quinic acid, which are contained in coffee, fruits and vegetables, on human organic anion transporters hOAT1 and hOAT3; these transporters mediate renal tubular uptake of anionic drugs from blood. Injection of hOAT1 and hOAT3 cRNA into oocytes stimulated uptake of typical substrates of hOAT1 and hOAT3 (p-aminohippurate and estrone sulfate, respectively); among the three compounds tested, caffeic acid most strongly inhibited these transporters. The apparent 50% inhibitory concentrations of caffeic acid were estimated to be 16.6 µM for hOAT1 and 5.4 µM for hOAT3. Eadie-Hofstee plot analysis showed that caffeic acid inhibited both transporters in a competitive manner. In addition to the transport of p-aminohippurate and estrone sulfate, that of antifolates and antivirals was inhibited by caffeic acid. These findings show that caffeic acid has inhibitory potential against hOAT1 and hOAT3, suggesting that renal excretion of their substrates could be affected in patients consuming a diet including caffeic acid.

新規ヒト電位駆動性有機酸トランスポーターＮＰＴ４（ＳＬＣ１７Ａ３）による薬物輸送

In kidney, the secretion of organic anions (OAs) takes place in the renal proximal tubular cells via at least two steps. The first is the transport of OAs from the blood across the basolateral membrane into the proximal tubules. The transport of p-aminohippurate (PAH), a prototypical substrate for the renal organic anion transport systems, across the basolateral membrane of proximal tubular cells against the electrochemical gradient occurs in exchange for intracellular dicarboxylates. Two organic anion transporters, OAT1 (SLC22A6) and OAT3 (SLC22A8), have been proposed to be responsible for this step. The second step is the exit of OAs across the apical membrane at the tubular epithelial cells into the urine. Voltage-driven organic anion transport plays an important role for this step. However, the molecular nature and precise functional properties of these efflux systems were largely unknown. Recently, the characteristics of human type I sodium-phosphate transporter hNPT4 (SLC17A3), an orphan transporter, has been analyzed using Xenopus oocyte expression system. hNPT4 acts as a voltage-driven transporter for several OAs such as PAH, estrone sulfate, diuretic drugs and urate. This finding will complete a model of the secretion of OAs in the renal tubular cell, where OAs in the blood are taken up via OAT1 and/or OAT3 and intracellular OAs exit from the cell into the tubular lumen via hNPT4.

Impairment of the renal p-aminohippurate transport induced by 6-hydroxydopamine.

In this study, the effects of 6-hydroxydopamine (6-OHDA) on renal p-aminohippurate transport were investigated in-vitro using rat renal cortical slices. Cisplatin, a known nephrotoxin, was used as positive control. Renal cortical slices were incubated for 60 min in a cisplatin-containing medium (0.83-5.0 microM) at 37 degrees C under a 100% O(2) atmosphere. In another series of experiments, renal cortical slices were incubated in a 3.33 microM cisplatin-containing medium for 15-120 min or in a cisplatin-free medium. Subsequently, for each series of experiments, kidney slices were incubated at 25 degrees C for 90 min in a media containing p-aminohippurate. In a further series of experiments, renal cortical slices were incubated for 60 min in a 6-OHDA containing medium (3.125-100 microM) at 37 degrees C under a 100% O(2) atmosphere. In another series of experiments, renal cortical slices were incubated in a 50 microM 6-OHDA-containing medium for 15-120 min or in 6-OHDA-free medium. Subsequently, for each series of experiments, kidney slices were incubated at 25 degrees C for 90 min in a media containing p-aminohippurate. The results of this study where slices were incubated in 6-OHDA- or cisplatin-containing media indicate that both 6-OHDA and cisplatin induced a time- and concentration-dependent decrease in p-aminohippurate accumulation by renal cortical slices. Therefore, similarly to cisplatin, 6-OHDA causes functional injury of renal proximal tubule cells, leading to impairment of transport processes across the cell membrane.

Roles of Rat Renal Organic Anion Transporters in Transporting Perfluorinated Carboxylates with Different Chain Lengths

Perfluorinated carboxylates (PFCAs) are generally stable to metabolic and environmental degradation and have been found at low concentrations in environmental and biological samples. Renal clearance of PFCAs depends on chain length, species, and, in some cases, gender within species. While perfluoroheptanoate (C7) is almost completely eliminated renally in both male and female rats, renal clearance of perfluorooctanoate (C8) and perfluorononanoate (C9) is much higher in female rats. Perfluorodecanoate (C10) mainly accumulates in the liver for both genders. Therefore, we tested whether PFCAs with different chain lengths are substrates of rat renal transporters with gender-specific expression patterns. Inhibition of uptake of model substrates was measured for the basolateral organic anion transporter (Oat)1 and Oat3 and the apical Oat2, organic anion transporting polypeptide (Oatp)1a1, and Urat1 with 10microM PFCAs with chain lengths from 2 to 18 (C2-C18) carbons. Perfluorohexanoate (C6), C7, and C8 inhibited Oat1-mediated p-aminohippurate transport, with C7 being the strongest inhibitor. C8 and C9 were the strongest inhibitors for Oat3-mediated estrone-3-sulfate transport, while Oatp1a1-mediated estradiol-17beta-glucuronide uptake was inhibited by C9, C10, and perflouroundecanoate (C11), with C10 giving the strongest inhibition. No strong inhibitors were found for Oat2 or Urat1. Kinetic analysis was performed for the strongest inhibitors. Oat1 transported C7 and C8 with K(m) values of 50.5 and 43.2microM, respectively. Oat3 transported C8 and C9 with K(m) values of 65.7 and 174.5microM, respectively. Oatp1a1-mediated transport yielded K(m) values of 126.4 (C8), 20.5 (C9), and 28.5microM (C10). These results suggest that Oat1 and Oat3 are involved in renal secretion of C7-C9, while Oatp1a1 can contribute to the reabsorption of C8 through C10, with highest affinities for C9 and C10.

Interaction of Organic Cations with Organic Anion Transporters

Studies of the organic anion transporters (Oats) have focused mainly on their interactions with organic anionic substrates. However, as suggested when Oat1 was originally identified as NKT (Lopez-Nieto, C. E., You, G., Bush, K. T., Barros, E. J., Beier, D. R., and Nigam, S. K. (1997) J. Biol. Chem. 272, 6471-6478), since the Oats share close homology with organic cation transporters (Octs), it is possible that Oats interact with cations as well. We now show that mouse Oat1 (mOat1) and mOat3 and, to a lesser degree, mOat6 bind a number of "prototypical" Oct substrates, including 1-methyl-4-phenylpyridinium. In addition to oocyte expression assays, we have tested binding of organic cations to Oat1 and Oat3 in ex vivo assays by analyzing interactions in kidney organ cultures deficient in Oat1 and Oat3. We also demonstrate that mOat3 transports organic cations such as 1-methyl-4-phenylpyridinium and cimetidine. A pharmacophore based on the binding affinities of the tested organic cations for Oat3 was generated. Using this pharmacophore, we screened a chemical library and were able to identify novel cationic compounds that bound to Oat1 and Oat3. These compounds bound Oat3 with an affinity higher than the highest affinity compounds in the original set of prototypical Oct substrates. Thus, whereas Oat1, Oat3, and Oat6 appear to function largely in organic anion transport, they also bind and transport some organic cations. These findings could be of clinical significance, since drugs and metabolites that under normal physiological conditions do not bind to the Oats may undergo changes in charge and become Oat substrates during pathologic conditions wherein significant variations in body fluid pH occur.

Characterization of Human Orgnic Anion Transporter 4 (hOAT4) as a Low Affinity p-aminohippurate (PAH) Transporter

Organic anion transporters play an essential role in the elimination of numerous endogenous and exogenous organic anions from the body. In t he present study, we investigated the transport property of human organic anion transporter 4 （hOAT4） as a p-aminohippurate （PAH） transporter. For this purpose, we established and utilized cells derived from the second segment of mice proximal tubule stably expressing hOAT4 （S 2 hOAT4） . RT-PCR result shows the expression of hOAT4 in S2 hOAT4. ［ 3 H］ estrone sulfate （ES） and ［ 14 C］PAH uptake via h OAT4 were increased with time and concentration dependent manner. Km of ES and PAH were 9.9μM and 2.2 mM, respectively. ES uptake via hOAT4 was inhibited by PAH with competetive manner （Ki = 4.3 mM） . hOAT4-mediated PAH uptake was strongly inhibited by sulfate conjugates, non-steroidal anti-inflammatory drugs, bile acids, penicillin G, BSP, probenecid, ouabain and quinidine and moderately inhibited b y cAMP, cGMP and cimetidine. No inhibitory effect by tetraethylammonium and β-estradiol was observed. The present study may provide the molecular basis of drug efflux pathway in human kidneys. 杏林医会誌 38 巻 2・3 号 61 〜 68 2007 年 9 月 原 著

Identification of a New Urate and High Affinity Nicotinate Transporter, hOAT10 (SLC22A13)

The orphan transporter hORCTL3 (human organic cation transporter like 3; SLC22A13) is highly expressed in kidneys and to a weaker extent in brain, heart, and intestine. hORCTL3-expressing Xenopus laevis oocytes showed uptake of [(3)H]nicotinate, [(3)H]p-aminohippurate, and [(14)C]urate. Hence, hORCTL3 is an organic anion transporter, and we renamed it hOAT10. [(3)H]Nicotinate transport by hOAT10 into X. laevis oocytes and into Caco-2 cells was saturable with Michaelis constants (K(m)) of 22 and 44 microm, respectively, suggesting that hOAT10 may be the molecular equivalent of the postulated high affinity nicotinate transporter in kidneys and intestine. The pH dependence of hOAT10 suggests p-aminohippurate(-)/OH(-), urate(-)/OH(-), and nicotinate(-)/OH(-) exchange as possible transport modes. Urate inhibited [(3)H]nicotinate transport by hOAT10 with an IC(50) value of 759 microm, assuming that hOAT10 represents a low affinity urate transporter. hOAT10-mediated [(14)C]urate uptake was elevated by an exchange with l -lactate, pyrazinoate, and nicotinate. Surprisingly, we have detected urate(-)/glutathione exchange by hOAT10, consistent with an involvement of hOAT10 in the renal glutathione cycle. Uricosurics, diuretics, and cyclosporine A showed substantial interactions with hOAT10, of which cyclosporine A enhanced [(14)C]urate uptake, providing the first molecular evidence for cyclosporine A-induced hyperuricemia.

Overlapping in vitro and in vivo specificities of the organic anion transporters OAT1 and OAT3 for loop and thiazide diuretics

Organic anion transporter (OAT) genes have been implicated in renal secretion of organic anions, but the individual in vivo contributions of OAT1 (first identified as NKT) and OAT3 remain unclear. Potential substrates include loop diuretics (e.g., furosemide) and thiazide diuretics (e.g., bendroflumethiazide), which reach their tubular sites of action mainly by proximal tubular secretion. Previous experiments in Oat1 knockout (-/-) mice revealed an almost complete loss of renal secretion of the prototypic organic anion p-aminohippurate (PAH) and a role of OAT1 in tubular secretion of furosemide (Eraly SA, Vallon V, Vaughn D, Gangoiti JA, Richter K, Nagle M, Monte JC, Rieg T, Truong DM, Long JM, Barshop BA, Kaler G, Nigam SK. J Biol Chem 281: 5072-5083, 2006). In this study we found that both furosemide and bendroflumethiazide inhibited mOat1- and mOat3-mediated uptake of a labeled tracer in Xenopus oocytes injected with cRNA, consistent with their being substrates for mouse OAT1 and OAT3. Experiments in Oat3(-/-) mice revealed intact renal secretion of PAH, but the dose-natriuresis curves for furosemide and bendroflumethiazide were shifted to the right and urinary furosemide excretion was impaired similar to the defect in Oat1(-/-) mice. Thus, whereas OAT1 (in contrast to OAT3) is the classic basolateral PAH transporter of the proximal tubule, both OAT1 and OAT3 contribute similarly to normal renal secretion of furosemide and bendroflumethiazide, and a lack of either one is not fully compensated by the other. Although microarray expression analysis in the kidneys of Oat1(-/-) and Oat3(-/-) mice revealed somewhat altered expression of a small number of transport-related genes, none were common to both knockout models. When searching for polymorphisms involved in human diuretic responsiveness, it may be necessary to consider both OAT1 and OAT3, among other genes.

Rat organic solute carrier protein 1 (rOscp1) mediated the transport of organic solutes in Xenopus laevis oocytes: Isolation and pharmacological characterization of rOscp1

Rat organic solute carrier protein 1 (rOscp1) was isolated from a rat testis cDNA library. Isolated rOscp1 cDNA consisted of 1089 base pairs that encoded a 363-amino acid protein, and the amino acid sequence was 88% and 93% identical to that of human OSCP1 (hOSCP1) and mouse Oscp1 (mOscp1), respectively. The message for rOscp1 is highly detected in rat testis. When expressed in X. oocytes, rOscp1 mediated the high affinity transport of p-aminohippurate (PAH) with a K m value of 15.7 ± 1.9 μM, and rOscp1-mediated organic solutes were exhibited in time- and Na +-independent manners. rOscp1 also transported various structurally heterogenous compounds such as testosterone, dehydroepiandrosterone sulfate (DHEA-S), and taurocholate with some differences in substrate specificity compared with hOSCP1. Immunohistochemical analysis revealed that the rOscp1 protein is localized in the basal membrane side of Sertoli cells as observed in mouse testis [Kobayashi et al., 2007; Kobayashi, Y., Tsuchiya, A., Hayashi, T., Kohyama, N., Ohbayashi, M., Yamamoto, T., 2007. Isolation and characterization of polyspecific mouse organic solute carrier protein 1 (mOscp1). Drug Metabolism and Disposition 35 (7), 1239–1245]. Thus, the present results indicate that a newly isolated cDNA clone, rOscp1, is a polyspecific organic solute carrier protein with some differences in substrate specificity compared with human and mouse OSCP1.

The Chloride Dependence of the Human Organic Anion Transporter 1 (hOAT1) Is Blunted by Mutation of a Single Amino Acid

Organic anion transporter 1 (OAT1) is key for the secretion of organic anions in renal proximal tubules. These organic anions comprise endogenous as well as exogenous compounds including frequently used drugs of various chemical structures. The molecular basis for the polyspecificity of OAT1 is not known. Here we mutated a conserved positively charged arginine residue (Arg(466)) in the 11(th) transmembrane helix of human OAT1. The replacement by the positively charged lysine (R466K) did not impair expression of hOAT1 at the plasma membrane of Xenopus laevis oocytes but decreased the transport of p-aminohippurate (PAH) considerably. Extracellular glutarate inhibited and intracellular glutarate trans-stimulated wild type and mutated OAT1, suggesting for the mutant R466K an unimpaired interaction with dicarboxylates. However, when Arg(466) was replaced by the negatively charged aspartate (R466D), glutarate no longer interacted with the mutant. PAH uptake by wild type hOAT1 was stimulated in the presence of chloride, whereas the R466K mutant was chloride-insensitive. Likewise, the uptake of labeled glutarate or ochratoxin A was chloride-dependent in the wild type but not in R466K. Kinetic experiments revealed that chloride did not alter the apparent K(m) for PAH but influenced V(max) in wild type OAT1-expressing oocytes. In R466K mutants the apparent K(m) for PAH was similar to that of the wild type, but V(max) was not changed by chloride removal. We conclude that Arg(466) influences the binding of glutarate, but not interaction with PAH, and interacts with chloride, which is a major determinant in substrate translocation.

Isolation and Characterization of Polyspecific Mouse Organic Solute Carrier Protein 1 (mOscp1)

We succeeded in isolating the cDNA-encoding mouse organic solute carrier protein 1 (mOscp1) from a mouse testis cDNA library. mOscp1 consisted of 1137 base pairs that encoded a 379-amino acid protein, and the amino acid sequence was 85% identical to that of human OSCP1 (hOSCP1). Northern blot analysis revealed that the gene coding for mOscp1 is highly expressed in the testis, but not in other tissues. When expressed in Xenopus laevis oocytes, mOscp1 mediated the high-affinity transport of p-aminohippurate (PAH) (K(m) = 18.8 +/- 4.1 microM) with Na(+) independence. mOscp1 transported various kinds of structurally dissimilar drugs and chemicals such as probenecid, dehydroepiandrosterone sulfate, and glutarate with some differences in substrate specificity compared with hOSCP1. Cyclophosphamide inhibited the mOscp1-mediated PAH uptake. Immunohistochemical analysis revealed that the mOscp1 protein is localized in the plasma membrane side of Sertoli cells in the testis. Our results indicate that isolated mOscp1 is a polyspecific organic solute carrier protein and may be a key molecule for the testicular handling of organic solutes.

Hydrocortisone upregulates organic anion (OA) transport in the opossum kidney (OK) cell line

In rats and humans corticosteroids upregulate renal OA transport, but the mechanism of this upregulation has not been characterized. An increase in uptake may involve increasing capacity (Jmax) for, or affinity (Km) of substrate for a transporter. Here we characterized the effect of long-term (5 day) exposure to hydrocortisone on the basolateral membrane transport kinetics of p-aminohippurate (PAH) in OK cell cultures. Five-day treatment of OK cell cultures with 10 μM hydrocortisone produced a two-fold increase in PAH transport compared to ethanol-treated controls. The regulation of OA transport by treatment with 10 μM hydrocortisone was further characterized by measuring the kinetics of basolateral PAH uptake in OK cell cultures. The capacity for PAH uptake in OK cell cultures increased more than five-fold (ethanol-treated vs. hydrocortisone-treated, Jmax = 0.5 mol·cm−2·min−1 vs. 2.8 mol·cm−2min−1), whereas no significant change in the affinity for basolateral PAH uptake was observed (ethanol-treated vs. hydrocortisone-treated, Km = 71 vs. 123 μM). The marked increase in the Jmax for basolateral PAH uptake with minimal change in Km is consistent with an upregulation of OA transport protein expression. Thus, our data suggest long-term exposure to hydrocortisone may upregulate PAH transport by an increase in OA transporter protein expression. Supported by DK062097.

Isophosphoramide Mustard-Lysine (ZIO-201): Active Moiety of Ifosfamide (IFOS) without Kidney Toxicity.

Ifosfamide (IFOS) and cyclophosphamide (CPA) are widely used anti-cancer drugs. Both are pro-drugs which must be metabolized before their metabolites cross-link DNA. Use of these drugs, especially high-dose IFOS, is complicated by substantial adverse effects of metabolites not directly involved in DNA-cross-linking including kidney, bladder and CNS toxicities. Recently, a lysine-stabilized form of isophosphoramide mustard (IPM-lysine; ZIO-201), an IFOS-metabolite, was developed. ZIO-201 directly cross-links DNA and is active against human cancer cell lines and in mice with human cancer xenografts. To evaluate nephrotoxicity of ZIO-201 we used an in vitro primary rabbit kidney proximal tubule (RPT) cell culture system, which retains many of the characteristics of renal proximal tubule cells including a polarized morphology, a Na+/glucose cotransport system and a p-aminohippurate transport system, glutathione status, and hormone responses (including a parathyroid hormone sensitive adenylate cyclase). We studied effects of ZIO-201 and other IFOS metabolites on primary RPT cells. IFOS is activated by the cytochrome P450 system producing a mixture of 2- and 3-dechloroethylifosfamide and resulting in formation of chloroacetaldehyde (CAA). Also, metabolism of IFOS by the cytochrome P450 system produces 4-hydroxyifosfamide, ultimately resulting in the production of acrolein (ACR). CAA and ACR are IFOS metabolites implicated in kidney toxicity in humans. Effects of ZIO-201 and CAA on the viability of confluent monolayers of primary rabbit kidney proximal tubule (RPT) cells were tested at 15–100 μM. Viability was determined by neutral red dye uptake by the primary cultures after 8 h incubation. ZIO-201 did not significantly reduce viability of primary RPT cells; the LD50 for CAA was about 40 μM with complete inhibition at 75 μM. Effects of 4-hydroperoxyifosfamide (HIFA), a synthetic prodrug of 4-hydroxyifosfamide and ACR were also studied. Although ACR had an LD50 of 80 μM HIFA did not significantly affect viability. The IFOS metabolite 4-hydroxyifosfamide, was prepared from 4-hydroperoxyifosfamide (1). At 200 μM, 4-hydroxyifosfamide resulted in extensive cell death after 4 h incubation. These data show that ZIO-201 avoids the kidney toxicity caused by metabolites of pro-drugs like IFOS. Whereas 2 IFOS metabolites, CAA and ACR were cytotoxic to kidney cells, ZIO-201 was not. An explanation for the toxic effects of 200 μM 4-hydroxyifosfamide t might be metabolism to ACR in vitro.

Sex Differences in the mRNA, Protein, and Functional Expression of Organic Anion Transporter (Oat) 1, Oat3, and Organic Cation Transporter (Oct) 2 in Rabbit Renal Proximal Tubules

Sex differences in transport of the organic anion (OA) substrate p-aminohippurate (PAH) and the organic cation (OC) substrate tetraethylammonium (TEA) have been recognized for some time. In the rat kidney, androgens up-regulate and estrogens down-regulate PAH and TEA transport, which correlate with similar changes in mRNA and protein expression for the renal basolateral membrane transporters organic anion transporter (Oat) 1 and organic cation transporter (Oct) 2. However, these sex differences are not readily demonstrated in other species. The present study characterizes the kinetics of basolateral membrane PAH, estrone sulfate (ES), and TEA uptake in renal proximal tubule (RPT) suspensions isolated from female and male rabbits to compare functional expression of transport with mRNA and protein expression for rbOat1, rbOat3, and rbOct2. Although rbOat1-rbOat3 mRNA expression exhibited developmental differences, no sex differences in mRNA levels were observed. Oat1 and Oat3 protein expression in RPT suspensions also was similar between adult female and male rabbits. In contrast, rbOct1 and rbOct2 mRNA levels did not show developmental differences, but rbOct2 mRNA expression was greater in adult male than female rabbits. However, the sex difference in rbOct2 mRNA level did not translate to rbOct2 protein expression. Importantly, functional expression of Oat1, Oat3, and Oct2 transport as measured by kinetics (J(max) and K(t)) of PAH, ES, and TEA uptake was similar between adult male and female rabbits, and correlated with rbOat1, rbOat3, and rbOct2 protein expression. Thus, unlike rodents, rabbit renal OA and OC transport does not exhibit sex differences, pointing to the need for caution in extrapolating transport-related sex differences between species.

Isolation and Functional Characterization of a Novel Organic Solute Carrier Protein, hOSCP1

We succeeded in isolating a novel organic solute carrier from a human placenta cDNA library. The isolated cDNA consisted of 1137 base pairs that encoded a 379-amino acid protein, hOSCP1. Northern blot and reverse transcription PCR analyses revealed that the hOSCP1 mRNA is expressed in the placenta and testis and weakly expressed in the thymus and small intestine. When expressed in Xenopus laevis oocytes, hOSCP1 mediated the high affinity transport of p-aminohippurate (PAH) (K(m) = 35.0 +/- 7.5 microm) and tetraethylammonium (K(m) = 62.3 +/- 12.2 microm) in a sodium-independent manner. However, the hOSCP1-expressing oocyte did not mediate the transport of L-carnitine. The transport of PAH by hOSCP1 was sensitive to pH, but the tetraethylammonium was not transported at the high pH examined. hOSCP1 transported prostaglandin E(2), prostaglandin F(2alpha), estrone sulfate, glutarate, L-leucine, L-ascorbic acid, and tetracycline. Thus, hOSCP1 also showed broad substrate specificity. A wide range of structurally unrelated organic compounds inhibited the hOSCP1-mediated PAH uptake. Immunohistochemical analysis revealed that the hOSCP1 protein is localized in the basal membrane of the syncytiotrophoblast in the human placenta. Our results suggest that hOSCP1 is a novel polyspecific organic solute carrier protein responsible for drug clearance from the human placenta.

Molecular Cloning and Functional Analyses of OAT1 and OAT3 from Cynomolgus Monkey Kidney

The functional characterization of monkey OAT1 (SLC22A6) and OAT3 (SLC22A8) was carried out to elucidate species differences in the OAT1- and OAT3-mediated transport between monkey and human. The cDNAs of monkey OAT1 and OAT3 were isolated from monkey kidney, and their stable transfectants were established in HEK293 cells (mkOAT1- and mkOAT3-HEK). Transport studies were performed using cDNA transfectants, and kinetic parameters were compared among rat, monkey and human. The amino acid sequences of mkOAT1 and mkOAT3 exhibit 97% and 96% identity to their corresponding human orthologues. For OAT1, there was no obvious species difference in the K(m) values and the relative transport activities of 11 substrates with regard to p-aminohippurate transport. For OAT3, there was no species difference in the K(m) values and in the relative transport activities of nine substrates with regard to benzylpenicillin transport between monkey and human. However, the relative transport activities of indoxyl sulfate, 3-carboxy-4-methyl-5-propyl-2-furanpropionate, and estrone-3-sulfate showed a difference between primates and rat and gave a poor correlation. These results suggest that monkey is a good predictor of the renal uptake of organic anions in the human.