Organic cation/carnitine transporter OCTN2 (Slc22a5) is responsible for carnitine transport across apical membranes of small intestinal epithelial cells in mouse

The organic cation/carnitine transporter OCTN2 is responsible for renal tubular reabsorption of its endogenous substrate, carnitine, although its physiological role in small intestine remains controversial. Here we present direct evidence for a predominant role of OCTN2 in small intestinal absorption of carnitine based on experiments with juvenile visceral steatosis (jvs) mice, which have a hereditary deficiency of the octn2 gene. Uptake of carnitine, assessed with an Ussing-type chamber system, from the apical surface of the small intestine was saturable and higher than that from the basal surface in wild-type mice, whereas carnitine uptake having these characteristics was almost absent in jvs mice. Saturable uptake of carnitine was also confirmed in isolated enterocytes obtained from wild-type mice, and the Km value obtained (approximately 20 microM) was close to that reported for carnitine uptake by human embryonic kidney 293 cells stably expressing mouse OCTN2 (Slc22a5). The carnitine uptake by enterocytes was decreased in the presence of various types of organic cations, and this inhibition profile was similar to that of mouse OCTN2, whereas uptake of carnitine was quite small and unsaturable in enterocytes obtained from jvs mice. Immunohistochemical and immunoprecipitation analyses suggested colocalization of OCTN2 with PDZK1, an adaptor protein that functionally regulates OCTN2. Immunoelectron microscopy visualized both OCTN2 and PDZK1 in microvilli of absorptive epithelial cells. These findings indicate that OCTN2 is predominantly responsible for the uptake of carnitine from the apical surface of mouse small intestinal epithelial cells, and it may therefore be a promising target for oral delivery of therapeutic agents that are OCTN2 substrates.     

Biopharmaceutical studies on molecular mechanisms of membrane transport

By incorporating the transporter-mediated or receptor-mediated transport process in physiologically based pharmacokinetic models, we succeeded in the quantitative prediction of plasma and tissue concentrations of beta-lactam antibiotics, insulin, pentazocine, quinolone antibacterial agents, and inaperizone and digoxin. The author's research on transporter-mediated pharmacokinetics focuses on the molecular and functional characteristics of drug transporters such as oligopeptide transporter, monocarboxylic acid transporter, anion antiporter, organic anion transporters, organic cation/carnitine transporters (OCTNs), and the ATP-binding cassette transporters P-glycoprotein and MRP2. We have successfully demonstrated that these transporters play important roles in the influxes and/or effluxes of drugs in intestinal and renal epithelial cells, hepatocytes, and brain capillary endothelial cells that form the blood-brain barrier. In the systemic carnitine deficiency (SCD) phenotype mouse model, juvenile visceral steatosis (jvs) mouse, a mutation in the OCTN2 gene was found. Furthermore, several types of mutation in human SCD patients were found, demonstrating that OCTN2 is a physiologically important carnitine transporter. Interestingly, OCTNs transport carnitine in a sodium-dependent manner and various cationic drugs transport it in a sodium-independent manner. OCTNs are thought to be multifunctional transporters for the uptake of carnitine into tissue cells and for the elimination of intracellular organic cationic drugs.     

Acetyl-L-carnitine permeability across the blood-brain barrier and involvement of carnitine transporter OCTN2

OCTN2 (SLC22A5), an organic cation/carnitine transporter, is widely distributed throughout the body, including the brain. In the present study, the involvement of OCTN2 in acetyl-L-carnitine (ALCAR) permeation across the blood-brain barrier (BBB) was examined using a microdialysis method in mouse. OCTN2 function was examined by comparison of wild-type mice with jvs mice, which express defective OCTN2 and are considered a model for primary systemic carnitine deficiency. Zero-net-flux method analysis indicated higher in vivo recovery of ALCAR and lower physiological ALCAR concentration in thalamus extracellular fluid (ECF) in jvs mice compared with wild-type mice. Externally added ALCAR showed significantly slower initial uptake across the BBB in jvs mouse. These results indicated that OCTN2 is functionally involved in ALCAR transfer across the BBB. Total radioactivity in ECF after i.v. administration of radiolabelled ALCAR remained constant for the rest of the experimental period. Accordingly, our results indicate that ALCAR is transported from blood to brain ECF by OCTN2 at least in part, and its concentration in brain ECF is regulated by other events such as protein binding and anabolic reactions in the brain, as well as by transport across the BBB.     

Involvement of carnitine/organic cation transporter OCTN2 (SLC22A5) in distribution of its substrate carnitine to the heart

This study was designed to clarify the pharmacological role of carnitine/organic cation transporter (Octn) family members in mouse heart. Immunohistochemical analysis revealed that Octn1 was exclusively expressed on endothelial cells in blood vessels. Octn2 was detected on the plasma membrane of cardiac muscle cells by immunoelectron microscopy. Octn3 was not detected in the heart. Integration plot analysis showed that coadministration of unlabeled L-carnitine reduced distribution of L-[3H]carnitine to the heart. L-[3H]Carnitine uptake in heart slices was reduced by carnitine analogs and various Octn2 substrates. L-[3H]Carnitine uptake by heart slices from juvenile visceral steatosis (jvs) mice, which have a hereditary octn2 gene deficiency, was negligible. Distribution of [3H]quinidine, another Octn2 substrate, to the heart was not reduced by L-carnitine, and [3H]quinidine uptake in heart slices was Na(-)-independent and inhibited by cationic drugs, but not carnitine analogs. [3H]Quinidine uptake by heart slices from jvs mice was similar to that of wild-type mice. These results demonstrate that OCTN2 is functionally expressed on the plasma membrane of muscle cells and is involved in distribution of carnitine to the heart. Some mechanism(s) other than OCTN2 is involved in the distribution of quinidine to the heart.     

Characteristics of L-carnitine transport in cultured human hepatoma HLF cells.

The recently cloned organic cation transporter, OCTN2, isolated as a homologue of OCTN1, has been shown to be of physiological importance in the renal tubular reabsorption of filtered L-carnitine as a high-affinity Na+ carnitine transporter in man. Although the mutation of the OCTN2 gene has been proved to be directly related to primary carnitine deficiency, there is little information about the L-carnitine transport system in the liver. In this study, the characteristics of L-carnitine transport into hepatocytes were studied by use of cultured human hepatoma HLF cells, which expressed OCTN2 mRNA to a greater extent than OCTN1 mRNA. The uptake of L-carnitine into HLF cells was saturable and the Eadie-Hofstee plot showed two distinct components. The apparent Michaelis constant and the maximum transport rate were 6.59+/-1.85 microM (mean+/-s.d.) and 78.5+/-21.4 pmol/5 min/10(6) cells, respectively, for high-affinity uptake, and 590+/-134 microM and 1507+/-142 pmol/5 min/10(6) cells, respectively, for low-affinity uptake. The high affinity L-carnitine transporter was significantly inhibited by metabolic inhibitors (sodium azide, dinitrophenol, iodoacetic acid) and at low temperature (4 degrees C). Uptake of [3H]L-carnitine also required the presence of Na+ ions in the external medium. The uptake activity was highest at pH 7.4, and was significantly lower at acidic or basic pH. L-Carnitine analogues (D-carnitine, L-acetylcarnitine and gamma-butyrobetaine) strongly inhibited uptake of [3H] L-carnitine, whereas beta-alanine, glycine, choline, acetylcholine and an organic anion and cation had little or no inhibitory effect. In conclusion, L-carnitine is absorbed by hepatocytes from man by an active carrier-mediated transport system which is Na+-, energy- and pH-dependent and has properties very similar to those of the carnitine transporter OCTN2.     

Activities of cytochrome p450 enzymes in liver and kidney microsomes from systemic carnitine deficiency mice with a gene mutation of carnitine/organic cation transporter

Juvenile visceral steatosis (jvs) mice, isolated from the C3H-H-2 degrees strain, exibit a systemic carnitine deficiency (SCD) phenotype and develop fatty liver, hyperammonemia and hypoglycemia. This phenotype is caused by a missense mutation (Leu352Arg) of a sodium-dependent carnitine/organic cation transporter, Octn2 (Slc22a5). The jvs mouse could be a useful model for pharmacokinetics and drug metabolism studies concerning Octn2 substrate drugs. In the present study, the effects of the SCD phenotype on the cytochrome P450 (P450 or CYP) dependent activities of four endobiotic and seven xenobiotic oxidations catalyzed by liver and kidney microsomes from jvs mice were investigated. The jvs-type mutation was genotyped by PCR-RFLP. The contents of total P450 and NADPH-P450 reductase were similar in the the liver microsomes from male or female mice of the wild-type and those heterozygous or homozygous for the jvs-type mutation. The 6beta-hydroxylation activities of testosterone and progesterone (marker for Cyp3a) based on the protein contents were 1.2- to 2.0-fold higher in liver microsomes from jvs/jvs-type mice compared to jvs/wt- or wt/wt-type mice. Coumarin 7-hydroxylation activities (marker for Cyp2a) were decreased to 0.7-fold in the male jvs/jvs-type mice. The activities of lauric acid 12-hydroxylation (a marker for Cyp4a) and aniline p-hydroxylation (a marker for Cyp2e1) in liver microsomes were increased 1.4- to 1.9-fold in female jvs/jvs-type mice. Genotoxic activation of 2-aminofluorene (a marker for Cyp4b1) by male and female mouse kidney microsomes were not affected by the SCD phenotype. These results demonstrated that the SCD phenotype affected the P450-dependent catalytic activities in liver microsomes. The jvs mouse could provide valuable information in drug interaction and drug metabolism studies of OCTN2 substrate drugs and new compounds in development.     

Transport of organic cations across the blood-testis barrier

Throughout the mammalian spermatogenic pathway, differentiating spermatogenic cells remain in close contact with somatic Sertoli cells, and this has been considered to be essential for the proliferation, differentiation, and survival of spermatogenic cells. It is thought that Sertoli cells form tight junctions to protect developing spermatogenic cells against harmful agents and provide several nutrients for spermatogenesis from the blood stream. Accordingly, Sertoli cells should regulate the movement of various nutritious and xenobiotic compounds by selective membrane transporters. However, the information on membrane transporters in Sertoli cells is limited. In the present study, we characterized the transport systems of organic cations in Sertoli cells. Uptake of [14C]tetraethylammonium (TEA) was measured by primary-cultured rat Sertoli cells. Initial uptake of TEA was concentration dependent, and an Eadie-Hofstee plot indicated the involvement of two saturable transport systems. The apparent Km values of high- and low-affinity components were comparable to those of previously known organic cation transporter (OCT) or organic cation/carnitine transporter (OCTN). In addition, OCT1, OCT3, OCTN1, and OCTN2 were expressed in Sertoli cells. In conclusion, multiple organic cation transporters, OCTs and OCTNs are expressed in Sertoli cells and the cells regulate transport of cationic compounds to protect and/or maintain the spermatogenesis in testis as the blood-testis barrier.     

Synthesis, transport and mechanism of a type I prodrug: L-carnitine ester of prednisolone

Aerosol glucocorticoid medications have become more and more important in treating BA (bronchial asthma). Although these agents are dosed to directly target airway inflammation, adrenocortical suppression and other systematic effects are still seen. To tackle this problem in a novel way, two L-carnitine ester derivatives of prednisolone (as the model drug), namely, PDC and PDSC, were synthesized to increase the absorption of prednisolone across the human bronchial epithelial BEAS-2B cells by the organic cation/carnitine transporter OCTN2 (SLC22A5) and then to slowly and intracellularly release prednisolone. The transport of prednisolone, PDC and PDSC into the human bronchial epithelial BEAS-2B cells was in the order PDSC > prednisolone > PDC at 37 °C. It was found that PDSC displayed 1.79-fold increase of uptake compared to prednisolone. Transport of PDSC by BEAS-2B was temperature-, time-, and Na(+)-dependent and saturable, with an apparent K(m) value of 329.74 μM, suggesting the involvement of carrier-mediated uptake. An RT-PCR study showed that organic cation/carnitine transporters OCTN1 and OCTN2 are expressed in BEAS-2B cells, but little in HEK293T cells. The order of uptake by HEK293T was prednisolone > PDC > PDSC. In addition, the inhibitory effects of organic cations such as L-carnitine, ergothioneine, TEA(+) and ipratropium on PDSC uptake in BEAS-2B cells were in the order L-carnitine > ipratropium > TEA(+) > ergothioneine, whereas their inhibitory effects on PDSC uptake in HEK293T cells were negligible. Finally, in vitro LPS-induced IL-6 production from BEAS-2B was more and longer suppressed by PDSC than prednisolone and PDC. All of these results suggested PDSC may be an attractive candidate for asthma treatment.     

Functional characterization of ergothioneine transport by rat organic cation/carnitine transporter Octn1 (slc22a4)

It has been reported that organic cation/carnitine transporter 1 (OCTN1) is associated with rheumatoid arthritis and Crohn's disease. Additionally, we reported that OCTN1 is expressed in hematopoietic cells, and is associated with proliferation and differentiation of erythroid cells. However, physiological role of OCTN1 is still unclear. Ergothioneine, an anti-oxidant, was recently reported to be a good substrate of human OCTN1. However, the transport characteristics of ergothioneine in rat remains to be clarified. The present study, is to further investigate the role of rat Octn1 on transport of ergothioneine in rat Octn1 transfected cells and natively expressing cell line PC12 derived from rat adrenal pheochromocytoma. [(3)H]Ergothioneine uptake by rat Octn1 stably transfected HEK293 cells was saturable, sodium dependent with 1 : 1 stoichiometry of ergothioneine, and pH dependent. Since ergothioneine was reported to presumably play a protective role against oxidative stress-induced apoptosis in PC12 cells, its transport in this cell line was investigated. The expression of rat Octn1 and a saturable and Na(+)-dependent transport of ergothioneine were observed in PC12 cells, suggesting that ergothioneine transport in this cell line may be mediated by rat Octn1. These findings suggested that rat Octn1 may act as a survival factor by taking up ergothioneine to suppress oxidative stress in this cell line. In conclusion, functional characteristics of ergothioneine transport by rat Octn1 is similar to that of human OCTN1 and it is suggested that rat Octn1 is important by transporting anti-oxidant ergothioneine in PC12 cells, though its role in vivo is to be investigated.     

Functional regions of organic cation/carnitine transporter OCTN2 (SLC22A5): roles in carnitine recognition

The organic cation/carnitine transporter OCTN2 transports carnitine in a sodium-dependent manner, whereas it transports organic cations sodium-independently. To elucidate the functional domain in OCTN2, we constructed chimeric proteins of human OCTN2 (hOCTN2) and mouse OCTN3 (mOCTN3) and introduced mutations at several amino acids conserved among human, rat and mouse OCTN2. We found that transmembrane domains (TMD) 1-7 are responsible for organic cation transport and for sodium dependence in carnitine transport. Within TMD1-7, Q180 and Q207 of hOCTN2 are the critical amino acids for the sodium dependence, and double mutation of Q180 and Q207 resulted in minimal change in transport activity when sodium was removed from the uptake medium. We propose that sodium-dependent affinity for carnitine is dependent on sodium recognition by these critical amino acids in hOCTN2, whereas carnitine transport by OCTN2 requires functional linkage between TMD1-7 and TMD11.     

PDZ adaptor protein PDZK2 stimulates transport activity of organic cation/carnitine transporter OCTN2 by modulating cell surface expression.

A part of the organic cation transporter families (OCT3, OCTN1, and OCTN2) has recently been identified to physically interact with PDZ (PSD95, Dlg, and ZO1) domain-containing proteins, although the physiological relevance of such interaction has not yet been fully examined. Here we have examined the stimulatory effect of PDZK2 [also named NaPi-Cap2 and intestinal and kidney-enriched PDZ protein (IKEPP)] on those cation transporters. In HEK293 cells, coexpression with PDZK2 increased the uptake of carnitine by OCTN2 with minimal effect on its substrate recognition specificity, but not for transport activity of OCT3 or OCTN1. The stimulatory effect of PDZK2 on OCTN2 was compatible with an approximately 2 times increase in transport capacity and can be accounted for by the increase in cell surface expression of OCTN2. Coexpression of PDZK2 did not affect carnitine transport activity of OCTN2 with deletion of the last four amino acids, which were found to be important for the interaction, suggesting involvement of physical interaction of the two proteins in the increase of cell surface expression of OCTN2. In mouse kidney, colocalization of PDZK2 and OCTN2 occurred predominantly in the region that was close to, but not the same as, the surface of apical membranes where OCTN2 alone was observed, suggesting the existence of OCTN2 in the subapical compartment that interacts with PDZK2. The present data have thus proposed an "intracellular pool" for OCTN2 that may be relevant to the stabilization of cell surface expression of OCTN2, thereby increasing transport activity for carnitine.     

Regulation of Octn2 transporter (SLC22A5) by peroxisome proliferator activated receptor alpha

The tissue distribution and disposition of carnitine, which plays an important role in the transport of long-chain fatty acids across the mitochondrial inner membrane for beta-oxidation, are well controlled by carnitine transporter organic cation/carnitine transporter 2 (OCTN2). Since little information is available on regulation of the expression of the OCTN2 gene, we examined the factors that affect the expression level of rat Octn2 (rOctn2), focusing on nuclear receptor peroxisome proliferator activated receptor alpha (PPARalpha), which regulates expression of genes associated with beta-oxidation of fatty acids. mRNA of rOctn2 was induced by the PPARalpha ligand fenofibrate in primary-cultured rat hepatocytes. Further, the PPARalpha ligand Wy14643 increased the expression of Octn2 in wild-type mice, but not in PPARalpha knockout mice. Analysis of the rOctn2 promoter region suggested the presence of putative cis elements of PPARalpha. Wistar rats treated with intraperitoneal fenofibrate administration showed increased expression of rOctn2 mRNA in liver, and uptake of [3H]carnitine by freshly isolated hepatocytes derived from those rats was also increased. In conclusion, our results indicate that the nuclear receptor PPARalpha directly up-regulates the expression of rOctn2 and increases the hepatic uptake of carnitine via rOctn2.     

The organic cation transporter OCT2 mediates the uptake of beta-adrenoceptor antagonists across the apical membrane of renal LLC-PK(1) cell monolayers

Previous studies have shown that beta-adrenoceptor antagonists may be substrates of organic cation transporters in kidney and lung. In this study we examined the transport of the beta-adrenoreceptor antagonists propranolol and metoprolol, in renal LLC-PK(1) cell monolayers. Experiments with BCECF (2', 7'-bis(2carboxyethyl)-5(6)-carboxyfluorescein) loaded LLC-PK(1) cell monolayers demonstrated that metoprolol and propranolol flux across the basolateral membrane was consistent with non-ionic diffusion. Flux across the apical membrane consisted of both non-ionic diffusion and the uptake of the cationic form of the beta-adrenoceptor antagonists. Uptake of the cationic form of metoprolol across the apical membrane was Na(+)-independent, electrogenic and sensitive to external pH. Furthermore, uptake was sensitive to inhibition by Decynium-22 and the organic cations TEA (tetraethylammonium) and MPP(+) (1-methyl 4-phenylpyridinium). These results, allied with the apical location of the uptake mechanism suggest that beta-adrenoceptor antagonists may be substrates for the organic cation transporter, OCT2. To confirm beta-adrenoceptor antagonists as substrates for OCT2, we demonstrate, in cells transiently transfected with an epitope tagged version of hOCT2 (hOCT2-V5):(1) Decynium-22 sensitive [(14)C]-propranolol uptake, (2) cis-inhibition of OCT2 by a range of beta-adrenoceptor antagonists and (3) metoprolol induced intracellular acidification.     

Carnitine and γ‐Butyrobetaine Stimulate Elimination of Meldonium due to Competition for OCTN2‐mediated Transport

Meldonium (3‐(2,2,2‐trimethylhydrazinium)propionate) is the most potent clinically used inhibitor of organic cation transporter 2 (OCTN2). Inhibition of OCTN2 leads to a decrease in carnitine and acylcarnitine contents in tissues and energy metabolism optimization‐related cardioprotective effects. The recent inclusion of meldonium in the World Anti‐Doping Agency List of Prohibited Substances and Methods has raised questions about the pharmacokinetics of meldonium and its unusually long elimination time. Therefore, in this study, the rate of meldonium washout after the end of the treatment was tested with and without administration of carnitine, γ‐butyrobetaine (GBB) and furosemide to evaluate the importance of competition for OCTN2 transport in mice. Here, we show that carnitine and GBB administration during the washout period effectively stimulated the elimination of meldonium. GBB induced a more pronounced effect on meldonium elimination than carnitine due to the higher affinity of GBB for OCTN2. The diuretic effect of furosemide did not significantly affect the elimination of meldonium, carnitine and GBB. In conclusion, the competition of meldonium, carnitine and GBB for OCTN2‐mediated transport determines the pharmacokinetic properties of meldonium. Thus, due to their affinity for OCTN2, GBB and carnitine but not furosemide stimulated meldonium elimination. During long‐term treatment, OCTN2‐mediated transport ensures a high muscle content of meldonium, while tissue clearance depends on relatively slow diffusion, thus resulting in the unusually long complete elimination period of meldonium.     

Functional genetic diversity in the high-affinity carnitine transporter OCTN2 (SLC22A5)

Systemic carnitine deficiency (SCD) is a rare autosomal recessive disease resulting from defects in the OCTN2 (SLC22A5) gene, which encodes the high-affinity plasma membrane carnitine transporter. Although OCTN2 is fairly well studied in its relationship with SCD, little is known about the carrier frequency of disease-causing alleles of OCTN2, or of more common functional polymorphisms in this gene. To address these issues, we screened for genetic variants in the OCTN2 coding region by direct sequencing of the exons and flanking intronic region of OCTN2 in a large sample (n = 276) of ethnically diverse subjects. In addition, we established lymphoblastoid cell lines from subjects homozygous for either allele of the previously identified promoter region variant, -207G>C. We found eight amino acid sequence variants of OCTN2, of which three (Phe17Leu, Leu144Phe, and Pro549Ser) were polymorphic in at least one ethnic group. When assayed for functional activity by expression in human embryonic kidney 293 cells, using as probes both the endogenous substrate (l-carnitine) and the organic cation tetraethylammonium, three variants showed functional differences from the reference OCTN2 (Phe17Leu, Tyr449Asp, Val481Phe; p < 0.05). Further studies of the Phe17Leu polymorphism showed a reduced V(max) for l-carnitine transport to approximately 50% of the reference OCTN2. Confocal microscopy studies using an OCTN2-GFP fusion protein showed that Phe17Leu had distinct subcellular localization from the reference OCTN2, with diffuse cytoplasmic retention of Phe17Leu, in contrast to reference OCTN2, which localized specifically to the plasma membrane. Lymphoblasts from subjects homozygous for the -207G allele showed increased l-carnitine transport compared with the -207C/C homozygotes (p < 0.05). This study suggests that although loss-of-function mutations in OCTN2 are likely to be rare, common variants of OCTN2 found in healthy populations may contribute to variation in the disposition of carnitine and some clinically used drugs.     

Functional characterization of human organic cation transporter OCTN1 single nucleotide polymorphisms in the Japanese population

The organic cation transporter OCTN1 (SLC22A4) is expressed ubiquitously, with strong expression in kidney, trachea, bone marrow, and fetal liver, and it mediates transport of organic cations in a pH-dependent manner. Recent studies have identified single nucleotide polymorphisms (SNPs) of OCTN1 in the Japanese population. Two SNPs present in the exon regions, c1063t and g1531a, cause amino acid mutation, Thr306Ile (T306I) and Gly462Glu (G462E), respectively. We examined the influence of these SNPs on the intracellular localization, protein expression, and transport activity of OCTN1. Immunocytochemical analysis showed similar localizations of OCTN1 in cellular membranes of HEK293 cells transiently transfected with an expression plasmid DNA for OCTN1 or its SNP allelic variants. The Km and Vmax values for tetraethylammonium (TEA) uptake by T306I were similar to those of the wild-type even when the Vmax value was normalized for the expression level of OCTN1 protein. In contrast, G462E had almost negligible transport activity, although the protein expression level of G462E was equivalent to that of the wild-type. We conclude that the SNP that causes the single amino acid mutation T306I does not affect TEA transport activity, whereas the mutation G462E abrogates the TEA transport activity, presumably affecting the physiological function of OCTN1 and/or the pharmacological characteristics of its substrates.     

Wudang cherry ameliorates urate underexcretion and renal dysfunction in hyperuricemic mice

OBJECTIVE To investigate effects of Wudang cherry on urate excretion and renal function and examined whether renal organic ion transporters were involved in potassium oxonateinduced hyperuricemic mice.METHODS The model of hyperuricemic mice was induced by intraperitoneal injection of potassium oxonate(250 mg·kg~(-1))for 7 d.Water extracts of Wudang cherry at 500 mg·kg~(-1)were orally administered to hyperuricemic mice for 7 d,benzbromarone(20 mg·kg~(-1))and allopurinol(20 mg·kg~(-1))were given as positive controls,vehicle control group was given equal normal saline.Serum and urine levels of uric acid were measured in hyperuricemic and normal mice.Simultaneously,the m RNA and protein levels of mouse urate transporter 1(m URAT1),glucose transporter 9(mGLUT9),organic anion transporters(mOAT1 and mOAT3),ATP-binding cassette,subfamily G,membrane 2(mABCG2)and organic cation/carnitine transporters(m OCT1,m OCT2,m OCTN1 and m OCTN2)in the kidney were analyzed by Western blot,RT-PCR,immunohistochemical and immunofluorescent assay,respectively.RESULTS Wudang cherry significantly reduced serum uric acid levels and increased urine uric acid levels in hyperuricemic mice.And it effectively reversed potassium oxonate-induced alterations in renal m URAT1,mGLUT9,mOAT1,mOAT3 and mABCG2 m RNA and protein levels,resulting in the enhancement of renal urate excretion in mice.Moreover,Wudang Cherry increased renal m OCT1,m OCT2,m OCTN1 and m OCTN2 m RNA and protein levels,and improved renal impairment in this model.CONCLUSION Wudang cherry processes uricosuric and nephroprotective actions by regulating renal organic ion transporters in hyperuricemic mice.     

Involvement of uric acid transporter in increased renal clearance of the xanthine oxidase inhibitor oxypurinol induced by a uricosuric agent, benzbromarone.

Benzbromarone has been reported to increase the renal clearance of oxypurinol, an active metabolite of allopurinol. We examined the renal transport of oxypurinol to determine whether such a change in renal clearance could be explained by altered transporter-mediated reabsorption. Since the first step of reabsorption takes place at the renal epithelial apical membrane, we focused on membrane transporters. Benzbromarone is an inhibitor of reabsorption of uric acid mediated by the uric acid transporter (URAT) URAT1 (SLC22A12), which is expressed at the apical membrane of proximal tubular cells in humans. Uptake of oxypurinol by Xenopus oocytes injected with complementary RNA of URAT1 was significantly higher than that by water-injected oocytes, and the uptake was saturable, with a K(m) of about 800 microM. Moreover, benzbromarone inhibited the oxypurinol uptake by URAT1 at concentrations as low as 0.01 microM. The uptake of oxypurinol by another organic anion transporter (OAT), OAT4 (SLC22A11), which is also expressed at the apical membrane of proximal tubular epithelial cells, was negligible, whereas the uptake of [3H]estrone-3-sulfate by OAT4 was significantly inhibited by oxypurinol. Furthermore, neither the transport activity of organic cation/carnitine transporter (OCTN) 1 nor OCTN2 was affected by oxypurinol or benzbromarone. These results indicate that URAT1 is involved in renal reabsorption of oxypurinol, and the increment of renal clearance of oxypurinol upon concomitant administration of benzbromarone could be due to drug interaction at URAT1.     

PDZK1 directly regulates the function of organic cation/carnitine transporter OCTN2

Urinary excretion of cationic xenobiotics is believed to be mediated by organic cation transporter (OCT and OCTN) families expressed on both basolateral and brush-border membranes of renal tubules, although the molecular mechanisms for targeting of these transporters to each membrane are poorly understood. Here, to examine the regulatory mechanisms for cell-surface expression and function of these transporters, we evaluated the interaction of these transporters with several PDZ proteins. A pull-down study using recombinant C-terminal proteins of OCTs and OCTNs identified a specific interaction of apical transporters OCTN1 and OCTN2, but not basolateral transporters OCT1 and OCT2, with PDZK1, intestinal and kidney-enriched PDZ protein, and Na+/H+ exchanger regulatory factor 2 (also called E3KARP, SIP-1, or TKA-1). Both yeast two-hybrid and pull-down studies suggested a requirement of the last four amino acids in OCTN1 and OCTN2 for the interaction. The interaction of PDZK1 with the C terminus of OCTN2 was also confirmed in a pull-down study using kidney brush-border membrane vesicles. Immunohistochemical analysis revealed that both PDZK1 and OCTN2 are colocalized in brush-border membranes of the kidney. Finally, double transfection of OCTN2 with PDZK1 stimulated the uptake by OCTN2 of its endogenous substrate carnitine, and this increase could be accounted for by the 6-fold increase in transport capacity. Such an increase was not observed for OCTN2 with deletion of the last four amino acids. Biotinylation study of surface proteins revealed minimal effect of PDZK1 on cell-surface expression of OCTN2. The present findings are the first to identify PDZK1 as a functional regulator of OCTN2 through direct interaction with the C terminus.