Ken-ichi Inui

Identification and Functional Characterization of a New Human Kidney–Specific H+/Organic Cation Antiporter, Kidney-Specific Multidrug and Toxin Extrusion 2

A cDNA coding a new H+/organic cation antiporter, human kidney-specific multidrug and toxin extrusion 2 (hMATE2-K), has been isolated from the human kidney. The hMATE2-K cDNA had an open reading frame that encodes a 566-amino acid protein, which shows 94, 82, 52, and 52% identity with the hMATE2, hMATE2-B, hMATE1, and rat MATE1, respectively. Reverse transcriptase-PCR revealed that hMATE2-K mRNA but not hMATE2 was expressed predominantly in the kidney, and hMATE2-B was ubiquitously found in all tissues examined except the kidney. The immunohistochemical analyses revealed that the hMATE2-K as well as the hMATE1 was localized at the brush border membranes of the proximal tubules. HEK293 cells that were transiently transfected with the hMATE2-K cDNA but not hMATE2-B exhibited the H+ gradient-dependent antiport of tetraethylammonium (TEA). Transfection of hMATE2-B had no affect on the hMATE2-K-mediated transport of TEA. hMATE2-K also transported cimetidine, 1-methyl-4-phenylpyridinium (MPP), procainamide, metformin, and N1-methylnicotinamide. Kinetic analyses demonstrated that the Michaelis-Menten constants for the hMATE2-K-mediated transport of TEA, MPP, cimetidine, metformin, and procainamide were 0.83 mM, 93.5 microM, 0.37 mM, 1.05 mM, and 4.10 mM, respectively. Ammonium chloride-induced intracellular acidification significantly stimulated the hMATE2-K-dependent transport of organic cations such as TEA, MPP, procainamide, metformin, N1-methylnicotinamide, creatinine, guanidine, quinidine, quinine, thiamine, and verapamil. These results indicate that hMATE2-K is a new human kidney-specific H+/organic cation antiporter that is responsible for the tubular secretion of cationic drugs across the brush border membranes.

Cisplatin and oxaliplatin, but not carboplatin and nedaplatin, are substrates for human organic cation transporters (SLC22A1-3 and multidrug and toxin extrusion family).

We have examined the role of the human organic cation transporters [hOCTs and human novel organic cation transporter (hOCTN); SLC22A1–5] and apical multidrug and toxin extrusion (hMATE) in the cellular accumulation and cytotoxicity of platinum agents using the human embryonic kidney (HEK) 293 cells transiently transfected with the transporter cDNAs. Both the cytotoxicity and accumulation of cisplatin were enhanced by the expression of hOCT2 and weakly by hOCT1, and those of oxaliplatin were also enhanced by the expression of hOCT2 and weakly by hOCT3. The hOCT-mediated uptake of tetraethylammonium (TEA) was markedly decreased in the presence of cisplatin in a concentration-dependent manner. However, oxaliplatin showed almost no influence on the TEA uptakes in the HEK293 cells expressing hOCT1, hOCT2, and hOCT3. The hMATE1 and hMATE2-K, but not hOCTN1 and OCTN2, mediated the cellular accumulation of cisplatin and oxaliplatin without a marked release of lactate dehydrogenase. Oxaliplatin, but not cisplatin, markedly decreased the hMATE2-K-mediated TEA uptake. However, the inhibitory effect of cisplatin and oxaliplatin against the hMATE1-mediated TEA uptake was similar. The release of lactate dehydrogenase and the cellular accumulation of carboplatin and nedaplatin were not found in the HEK293 cells transiently expressing these seven organic cation transporters. These results indicate that cisplatin is a relatively good substrate of hOCT1, hOCT2, and hMATE1, and oxaliplatin is of hOCT2, hOCT3, hMATE1, and hMATE2-K. These transporters could play predominant roles in the tissue distribution and anticancer effects and/or adverse effects of platinum agent-based chemotherapy.

Cloning and Functional Characterization of a Novel Rat Organic Anion Transporter Mediating Basolateral Uptake of Methotrexate in the Kidney

We have cloned a cDNA coding for a novel member of organic anion transporter, designated OAT-K1, expressed specifically in the kidney of rats. The rat OAT-K1 cDNA (2788 base pairs) had an open reading frame encoding for a 669-amino acid protein (calculated molecular mass of 74 kDa) which shows 72% identity with the cloned rat liver organic anion transporter, oatp. Northern hybridization and reverse transcription-coupled polymerase chain reaction revealed that the rat OAT-K1 messenger RNA transcript is expressed predominantly in the kidney. By use of stable LLC-PK1 cell monolayers transfected with the rat OAT-K1 cDNA, the transporter was suggested to mediate basolateral uptake of methotrexate, an anionic anticancer drug, but not taurocholate, p-aminohippurate, prostaglandin E2, and leukotriene C4. The methotrexate transport by rat OAT-K1 was unaffected by the presence of Na+ or Cl− gradient. The methotrexate accumulation by the OAT-K1-expressing cells showed saturability with the apparent Km value of 1.0 μM. Folate, sulfobromophthalein, and 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) inhibited the methotrexate accumulation markedly. These findings suggest that the rat OAT-K1 is localized in the basolateral membranes of renal tubules, where it mediates renal clearance of methotrexate from the blood.

Fibroblast Growth Factor (FGF)-23 Inhibits Renal Phosphate Reabsorption by Activation of the Mitogen-activated Protein Kinase Pathway

The homeostasis of the plasma phosphate level is essential for many biological processes including skeletal mineralization. The reabsorption of phosphate in the kidney is a major determinant of the plasma levels of phosphate. Phosphatonin is a hormone-like factor that specifically inhibits phosphate uptake in renal proximal epithelial cells. Recent studies on tumor-induced osteomalacia suggested that phosphatonin was potentially identical to fibroblast growth factor (FGF)-23. However, as purified recombinant FGF-23 could not inhibit phosphate uptake in renal proximal epithelial cells, the mechanism of action of FGF-23 remains to be elucidated. Therefore, we examined the mechanism of action of FGF-23 in cultured renal proximal epithelial cells, opossum kidney cells. FGF-23 was found to require heparin-like molecules for its inhibitory activity on phosphate uptake. FGF-23 binds to the FGF receptor 3c, which is mainly expressed in opossum kidney cells, with high affinity. An inhibitor for tyrosine kinases of the FGF receptor, SU 5402, blocked the activity of FGF-23. FGF-23 activated the mitogen-activated protein kinase (MAPK) pathway, which is the major intracellular signaling pathway of FGF. Inhibitors of the MAPK pathway, PD98059 and SB203580, also blocked the activity of FGF-23. The present findings have revealed a novel MAPK-dependent mechanism of the regulation of phosphate uptake by FGF signaling.

C3435T polymorphism in the MDR1 gene affects the enterocyte expression level of CYP3A4 rather than Pgp in recipients of living-donor liver transplantation.

The bioavailability of structurally unrelated drugs is limited by active secretion via the multidrug resistance gene (MDR1) product P-glycoprotein (Pgp) from enterocyte into lumen as well as intestinal metabolism by cytochrome P450 IIIA4 (CYP3A4). In the present study, we analyzed whether genetic polymorphism of the MDR1 had some influence on the intestinal expression levels of Pgp and CYP3A4 and the tacrolimus concentration/dose ratio over the first postoperative days in recipients of living-donor liver transplantation (LDLT). Genotyping assays were performed for the major 10 polymorphisms in the MDR1 gene by the polymerase chain reaction-restriction enzyme length polymorphism method. The allele frequencies of variations at five positions were almost comparable with those in the former studies in Caucasians and Japanese, but there was no variation at the other five positions. Although no polymorphism correlated with the intestinal expression of MDR1 mRNA or the tacrolimus concentration/dose ratio in the LDLT recipients, the C3435T polymorphism significantly affected the intestinal expression level of CYP3A4 mRNA as follows; 3435C/C>3435C/T (P < 0.05 vs. 3435C/C)>3435T/T (P < 0.01 vs. 3435C/C). Therefore, the identified polymorphisms including C3435T in the MDR1 gene were indicated to have no influence on the intestinal expression level of Pgp or the tacrolimus concentration/dose ratio in the recipients of LDLT. On the other hand, the C3435T polymorphism of MDR1 was suggested to correlate with the enterocyte expression of CYP3A4 rather than Pgp linking unknown genetic variation in CYP3A4 gene.

Effect of clarithromycin on renal excretion of digoxin: Interaction with P‐glycoprotein

We present a digoxin‐clarithromycin interaction in two patients in whom digoxin concentrations were unexpectedly increased. The ratio of renal digoxin clearance to creatinine clearance in one patient was lower during the concomitant administration of clarithromycin (0.64 and 0.73) than that after cessation of clarithromycin administration (1.30 ± 0.20; mean ± SD). Because P‐glycoprotein could play an important role in the renal secretion of digoxin, we hypothesized that clarithromycin decreases renal digoxin excretion by inhibiting P‐glycoprotein‐mediated transport. Digoxin transport was evaluated with use of a kidney epithelial cell line, which expresses the human P‐glycoprotein on the apical membrane by transfection with MDR1 complementary deoxyribonucleic acid. Clarithromycin inhibited the transcellular transport of digoxin from the basolateral to the apical side in a concentration‐dependent manner and concomitantly increased the cellular accumulation of digoxin. These results suggest that clarithromycin may inhibit the P‐glycoprotein‐mediated tubular secretion of digoxin, and this interaction mechanism may contribute to an increase in the serum digoxin concentration.

MOLECULAR CLONING AND TISSUE DISTRIBUTION OF RAT PEPTIDE TRANSPORTER PEPT2

A cDNA encoding rat H(+)- coupled peptide transporter PEPT2 was isolated. The cDNA encoded a protein of 729 amino acids with 48% amino acid identity to the rat PEPT1. The mRNA expression of rat PEPT2 was predominant in the kidney. When expressed in Xenopus oocytes, rat PEPT2 stimulated the uptake of bestatin, a dipeptide-like drug.

Creatinine Transport by Basolateral Organic Cation Transporter hOCT2 in the Human Kidney

AbstractPurpose. Creatinine is excreted into urine by tubular secretion in addition to glomerular filtration. The purpose of this study was to clarify molecular mechanisms underlying the tubular secretion of creatinine in the human kidney. Methods. Transport of [14C]creatinine by human organic ion transporters (SLC22A) was assessed by HEK293 cells expressing hOCT1, hOCT2, hOCT2-A, hOAT1, and hOAT3. Results. Among the organic ion transporters examined, only hOCT2 stimulated creatinine uptake when expressed in HEK293 cells. Creatinine uptake by hOCT2 was dependent on the membrane potential. The Michaelis constant (Km) for creatinine transport by hOCT2 was 4.0 mM, suggesting low affinity. Various cationic drugs including cimetidine and trimethoprim, but not anionic drugs, markedly inhibited creatinine uptake by hOCT2. Conclusion. These results suggest that hOCT2, but not hOCT1, is responsible for the basolateral membrane transport of creatinine in the human kidney.

(Section A: Molecular, Structural, and Cellular Biology of Drug Transporters) Peptide Transporters: Structure, Function, Regulation and Application for Drug Delivery

Proton-coupled peptide transporters, localized at brush-border membranes of intestinal and renal epithelial cells, play important roles in protein absorption and the conservation of peptide-bound amino nitrogen. These transporters also have significant pharmacological and pharmacokinetic relevance to the transport of various peptide-like drugs such as beta-lactam antibiotics. The identification and molecular characterization of H(+)/peptide cotransporters (PEPT1 and PEPT2) have facilitated the clarification of many aspects of these transporters such as the structure/function relationship and regulation. Recent findings that intestinal PEPT1 can transport l-valine ester prodrugs such as valacyclovir provided a major step forward toward the development of novel drug delivery systems. It has been demonstrated that peptide transporters, which have a similar substrate specificity to PEPT1 and PEPT2, but possess other distinct functional properties, are localized at basolateral membranes of intestinal and renal epithelial cells. This review highlights the recent advances in our knowledge of the cellular and molecular nature of PEPT1, PEPT2 and the basolateral peptide transporters.

Carrier-mediated transport systems of tetraethylammonium in rat renal brush-border and basolateral membrane vesicles.

Transport of [3H]tetraethylammonium, an organic cation, has been studied in brush-border and basolateral membrane vesicles isolated from rat kidney cortex. Some characteristics of carrier-mediated transport for tetraethylammonium were demonstrated in brush-border and basolateral membrane vesicles; the uptake was saturable, was stimulated by the countertransport effect, and showed discontinuity in an Arrhenius plot. In brush-border membrane vesicles, the presence of an H+ gradient ( [H+]i greater than [H+]o) induced a marked stimulation of tetraethylammonium uptake against its concentration gradient (overshoot phenomenon), and this concentrative uptake was completely inhibited by HgCl2. In contrast, the uptake of tetraethylammonium by basolateral membrane vesicles was unaffected by an H+ gradient. Tetraethylammonium uptake by basolateral membrane vesicles was significantly stimulated by a valinomycin-induced inside-negative membrane potential, while no effect of membrane potential was observed in brush-border membrane vesicles. These results suggest that tetraethylammonium transport across brush-border membranes is driven by an H+ gradient via an electroneutral H+-tetraethylammonium antiport system, and that tetraethylammonium is transported across basolateral membranes via a carrier-mediated system and this process is stimulated by an inside-negative membrane potential.