Identification and Functional Characterization of a New Human Kidney–Specific H+/Organic Cation Antiporter, Kidney-Specific Multidrug and Toxin Extrusion 2
* Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine; and Department of Urology, Graduate School of Medicine, Kyoto University, Kyoto, Japan
Address correspondence to: Prof. Ken-ichi Inui, Department of Pharmacy, Kyoto University Hospital, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. Phone: +81-75-751-3577; Fax: +81-75-751-4207; inui{at}kuhp.kyoto-u.ac.jp
Received for publication March 7, 2006.
Accepted for publication May 15, 2006.
A cDNA coding a new H+/organic cation antiporter, human kidney-specificmultidrug and toxin extrusion 2 (hMATE2-K), has been isolatedfrom the human kidney. The hMATE2-K cDNA had an open readingframe that encodes a 566–amino acid protein, which shows94, 82, 52, and 52% identity with the hMATE2, hMATE2-B, hMATE1,and rat MATE1, respectively. Reverse transcriptase–PCRrevealed that hMATE2-K mRNA but not hMATE2 was expressed predominantlyin the kidney, and hMATE2-B was ubiquitously found in all tissuesexamined except the kidney. The immunohistochemical analysesrevealed that the hMATE2-K as well as the hMATE1 was localizedat the brush border membranes of the proximal tubules. HEK293cells that were transiently transfected with the hMATE2-K cDNAbut not hMATE2-B exhibited the H+ gradient–dependent antiportof tetraethylammonium (TEA). Transfection of hMATE2-B had noaffect on the hMATE2-K–mediated transport of TEA. hMATE2-Kalso transported cimetidine, 1-methyl-4-phenylpyridinium (MPP),procainamide, metformin, and N1-methylnicotinamide. Kineticanalyses demonstrated that the Michaelis-Menten constants forthe hMATE2-K–mediated transport of TEA, MPP, cimetidine,metformin, and procainamide were 0.83 mM, 93.5 µM, 0.37mM, 1.05 mM, and 4.10 mM, respectively. Ammonium chloride–inducedintracellular acidification significantly stimulated the hMATE2-K–dependenttransport of organic cations such as TEA, MPP, procainamide,metformin, N1-methylnicotinamide, creatinine, guanidine, quinidine,quinine, thiamine, and verapamil. These results indicate thathMATE2-K is a new human kidney–specific H+/organic cationantiporter that is responsible for the tubular secretion ofcationic drugs across the brush border membranes.
Vectorial secretion of cationic compounds across the tubularepithelial cells is an important function of the kidney. Usingthe stopped flow tubular microperfusion method, cultured renalepithelial cells, and isolated membrane vesicles, it was suggestedthat two functionally distinct organic cation transporters wereexpressed in the basolateral and brush border membranes, respectively(1,2). Organic cations are suggested to accumulate in the renaltubular cells via a basolateral organic cation transport systemthat is sensitive to membrane potential differences. The intracellularcationic compounds were secreted by the apical H+/organic cationantiport system, which was driven by an oppositely directedH+ gradient. A prototype substrate, tetraethylammonium (TEA),has been used consistently for the functional characterizationof these organic cation transport systems in the kidney (3–7).
Since cloning of the organic cation transporter OCT1 from therat kidney (8), many related transporter proteins have beencloned and characterized. It is widely known that the basolateralentry of cationic compounds is mediated mainly by hepatic hOCT1(SLC22A1) and renal hOCT2 (SLC22A2) in humans, depending onthe membrane potential (9–11). However, attempts at molecularidentification of the apical H+/organic cation antiporter havefailed for more than a decade. Recently, in silico homologyscreening of the human cDNA database identified a H+/organiccation antiporter, human multidrug and toxin extrusion 1 (hMATE1),from the human brain using the NorM Na+/multidrug antiporterin Vibrio parahaemolyticus as a reference (12). hMATE1 was expressedin the liver, kidney, and skeletal muscle and weakly in thebrain and the heart. Although an isoform of hMATE1, hMATE2,was reported simultaneously to be expressed primarily in thekidney, the functional characteristics of hMATE2 were not examined.
In this study, we cloned hMATE2 cDNA from the human kidney forfunctional characterization. Sequence analysis revealed thatthe newly cloned cDNA was an alternative splice variant of hMATE2.It is interesting that real-time PCR and reverse transcriptase–PCR(RT-PCR) analyses clearly indicated that the newly cloned hMATE2but not the original hMATE2 was expressed only in the kidney,showing a function of oppositely directed H+ gradient–dependentantiport of organic cations; therefore, we designated the newclone human kidney-specific multidrug and toxin extrusion (hMATE2-K).
[14C]TEA (2.035 GBq/mmol), [14C]creatinine (2.035 GBq/mmol),[14C]procainamide (2.035 GBq/mmol), [methyl-14C]choline (2.035GBq/mmol), [9-3H]quinidine (740 GBq/mmol), [3H]quinine (740GBq/mmol), [3H (G)]thiamine (370 GBq/mmol), l-[N-methyl-3H]carnitine(3.145 TBq/mmol), [N-methyl-14C]nicotine (2.035 TBq/mmol), [N-methyl-3H]verapamil(2.96 TBq/mmol), and [7-3H (N)]tetracycline (185 GBq/mmol) wereobtained from American Radiolabeled Chemicals Inc. (St. Louis,MO). [14C]Metformin (962 MBq/mmol) and [14C]guanidine hydrochloride(1.961 GBq/mmol) were purchased from Moravek Biochemicals Inc.(Brea, CA). [3H]1-Methyl-4-phenylpyridinium acetate (MPP; 2.7TBq/mmol), [14C]p-aminohippuric acid (1.9 GBq/mmol), and [3H]glycylsarcosine(148 GBq/mmol) were from PerkinElmer Life Analytical Sciences(Boston, MA). [N-Methyl-3H]Cimetidine (451 GBq/mmol) was fromAmersham Biosciences (Uppsala, Sweden). [14C]Levofloxacin (1.07GBq/mmol) was from Daiichi Pharmaceutical Co., Ltd. (Tokyo,Japan). N1-Methylnicotinamide (NMN) was obtained from Sigma(St. Louis, MO). [14C]Captopril (0.115 GBq/mmol; Sankyo Co.,Tokyo, Japan), cephalexin (Shionogi, Osaka, Japan), cefazolin(Astellas Pharma Inc., Tokyo, Japan), and cephradine (SankyoCo.) were gifts from the respective suppliers. All other chemicalsused were of the highest purity available.
Isolation of hMATE2-K cDNA
The hMATE2 cDNA was cloned by RT-PCR from Marathon-Ready humankidney cDNA (Clontech, Palo Alto, CA). Primers that are specificfor hMATE2 were designed on the basis of the sequence informationof FLJ31196 (NM_152908). The forward and reverse primers, withmutations creating restriction enzyme sites (italics) for cloningthe hMATE2 cDNA, were 5'-AGGGTACCCAGTGCCCCGGCCAGGAATGGA-3' and5'-CTGTCTAGACCCCTCTGAGTGTCACCACAA-3', respectively. Simultaneously,hMATE2 cDNA was cloned using the Marathon-Ready human braincDNA (Clontech) as above. The PCR product was subcloned intothe expression vector pcDNA3.1 (+) (Invitrogen, Carlsbad, CA)and sequenced using a multicapillary DNA sequencer RISA384 system(Shimadzu, Kyoto, Japan). Because both hMATE2 from the humankidney and brain were suggested to be splice variants of hMATE2(FLJ31196) (12), we redesignated newly isolated clones fromthe human kidney and the human brain as hMATE2-K and hMATE2-B,respectively (Figure 1). The nucleotide sequences of hMATE2-Kand hMATE2-B have been submitted to the DDBJ/EMBL/GenBank DataBank as accession nos. AB250364 and AB250701, respectively.
Figure 1. Comparison of the nucleotide sequences of the exon 7 region among human kidney-specific multidrug and toxin extrusion 2 (hMATE2-K), human brain-specific multidrug and toxin extrusion 2 (hMATE2-B), and human multidrug and toxin extrusion 2 (hMATE2). Conserved nucleotides among three transporters are indicated by dots. The nucleotides are numbered starting at the first residue of the ATG putative initiation codon.
Real-Time PCR and RT-PCR
Total RNA from various human tissues was purchased from BioChain(Hayward, CA). RT of the total RNA (500 ng/20 µl reaction)and real-time PCR were carried out as described previously (13).The primer-probe sets used for hMATE1 and hMATE2 are summarizedin Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)mRNA was checked for the quality of the total RNA of human tissuesused with GAPDH Control Reagent (Applied Biosystems). To detectindependently both hMATE2-K and hMATE2 mRNA, we designed qualitativePCR primers as summarized in Table 1.
Table 1. Primer sets and probes for real-time PCR and RT-PCR a
Polyclonal Antibodies and Immunohistochemical Analyses
Polyclonal antibodies were raised against the synthetic peptidethat corresponded to the intracellular domains of hMATE1 (CQQAQVHANLKVN,no. 466-478) or hMATE2-K (YSRSECHVDFFRTPEE, no. 543-558) asdescribed, respectively (14,15). For immunofluorescence histochemistry,the human renal specimens were fixed with 4% paraformaldehydein PBS at 4°C for 30 min (13). Fixed tissues were embeddedin OCT compound (Sakura Finetechnical, Tokyo, Japan) and frozenrapidly in liquid nitrogen. Sections (6 µm thick) werecut and covered with 2% FBS and 1 mg/ml RNase A (Nacalai Tesque,Kyoto, Japan) for 1 h. The covered sections were incubated for1 h with antiserum (1:100 dilution) specific for hMATE1, hMATE2-K,hOCT2, a control of basolateral transporter, or hOCTN2, a controlof luminal transporter, and then incubated with Cy3-labeleddonkey anti-rabbit IgG (CALTAG Laboratory, San Francisco, CA),5 U/ml Alexa 488-phalloidin (Molecular Probe, Eugene, OR), and4',6-diamidino-2-phenylindole for 1 h. These sections were examinedwith a BX-50-FLA fluorescence microscope (Olympus, Tokyo, Japan)at x100 magnification. Images were captured with a DP-50 CCDcamera (Olympus) using Studio Lite software (Olympus). As controls,specific rabbit antibodies were replaced with preimmune rabbitserum.
Cell Culture, Transfection, and Uptake Experiments
HEK293 cells (American Type Culture Collection CRL-1573, Manassas,VA) were cultured as described previously (16,17). pcDNA3.1(+) plasmid vector DNA that contained hMATE2-K cDNA and hMATE2-BcDNA were transfected into HEK293 cells using LipofectAMINE2000 Reagent (Invitrogen), as described (16,17). At 48 h afterthe transfection, the cells were used for uptake experiments.
Cellular uptake of cationic compounds was measured with monolayercultures of HEK293 cells that were grown on poly-d-lysine–coated24-well plates (16,17). Typically, the cells were preincubatedwith 0.2 ml of incubation medium (pH 7.4) for 10 min at 37°C.The medium then was removed, and 0.2 ml of incubation mediumthat contained radiolabeled substrates was added. The mediumwas aspirated off at the end of the incubation, and the monolayerswere rinsed rapidly twice with 1 ml of ice-cold incubation medium.The cells were solubilized in 0.5 ml of 0.5 N NaOH, and thenthe radioactivity in aliquots was determined by liquid scintillationcounting. The cellular uptake of cephalexin, cefazolin, andcephradine was described previously (18). For the cellular uptakeof NMN, the HEK293 cells that were transfected with the hMATE2-KcDNA were incubated with NMN for 10 min, washed twice, and scrapedwith 0.5 ml of incubation medium (pH 7.4). The cellular accumulationof NMN was determined by HPLC according to the method of Musfeldet al. (19). For manipulation of the intracellular pH, intracellularacidification was performed by pretreatment with ammonium chloride(30 mM, 20 min at 37°C, pH 7.4) (20,21). The protein contentof the solubilized cells was determined using a Bio-Rad ProteinAssay Kit (Bio-Rad Laboratories, Hercules, CA) with bovine -globulinas a standard.
Statistical Analyses
Data are expressed as the mean ± SEM. Data were analyzedstatistically using an unpaired t test. Significance was setat P < 0.05. In all figures, when error bars are not shown,they are smaller than the symbols.
After the sequencing of hMATE2 cDNA that was isolated from thehuman kidney (hMATE2-K) and human brain (hMATE2-B), it was revealedwith the BLAST program that both hMATE2-K and hMATE2-B transcriptsconsist of 17 exons. However, a deletion of 108 bp in exon 7of hMATE2-K and an insertion of 46 bp in exon 7 of hMATE2-Bwere found compared with hMATE2 (Figure 1). The open readingframe of the cloned hMATE2-K cDNA was 1698 bp, coding for a566–amino acid protein with a calculated molecular massof 61,012, and that of hMATE2-B was 660 bp and a 220–aminoacid protein with a calculated molecular mass of 23,357. Figure 2shows the deduced amino acid sequences of hMATE2-K and its alignmentwith its homologues hMATE2-B, hMATE2, hMATE1, or rat (r) MATE1,which was cloned recently in our laboratory from rat kidneywith an accession no. AB248823 (22). hMATE2-K showed 82% aminoacid identity with hMATE2-B, 94% with hMATE2 (12), 52% withhMATE1 (12), and 52% with rMATE1.
Figure 2. Comparison of the deduced amino acid sequences among hMATE2-K, hMATE2-B, hMATE2, hMATE1, and rat multidrug and toxin extrusion 1 (rMATE1). The conserved residues in hMATE2-K are indicated by dots. The accession numbers for hMATE2-K, hMATE2-B, hMATE2, hMATE1, and rMATE1 are AB250364, AB250701, NM_152908, AK001709, and AB248823, respectively.
The hMATE1 mRNA was expressed strongly in the adrenal glandas well as in the kidney; weakly in the fetal liver, testis,skeletal muscle, liver, and uterus; and faintly in various othertissues (Figure 3A). In contrast, the obtained data showed thatthe transcript of the hMATE2 gene was expressed in the kidney,because the real-time PCR condition could cross-react hMATE2-K,hMATE2-B, and hMATE2 (Figure 3A, Table 1). Although RT-PCR showedthat the amplification of both products derived from hMATE2-K(505 bp) and hMATE2-B (659 bp) among 18 tissues examined, onlythe 505-bp band that corresponded to hMATE2-K was found in thekidney (Figure 3B). The 613-bp band that corresponded to hMATE2was not amplified. After sequencing, the PCR product of 659bp was confirmed to be identical to hMATE2-B.
Figure 3. Real-time PCR for hMATE1 and hMATE2 (A), and the splice variant-specific reverse transcriptase–PCR (RT-PCR) analysis for hMATE2-K and hMATE2-B (B). (A) Total RNA isolated from various human tissues were reverse transcribed, and mRNA levels of hMATE1 and hMATE2 with their spliced variants were determined by real-time PCR with the oligonucleotides summarized in Table 1 using an ABI PRISM 7700 sequence detector. The mRNA expression level of hMATE1 and hMATE2 was calculated by the absolute standard method as described (13). As described in the legend for Table 1, the primer and probe set for hMATE2 cross-reacts with both hMATE2-K and hMATE2-B; therefore, the data are the mRNA levels of hMATE2, hMATE2-K, and hMATE2-B. Each column represents the mean of two separate experiments. (B) The RT-PCR amplification of hMATE2-K and hMATE2-B mRNA with the primer sets covering exon 7 of each gene shown in Table 1. The PCR products of 505 bp that corresponded to hMATE2-K (arrow) and 659 bp that corresponded to hMATE2-B (arrowhead) were separated by 2% agarose gel electrophoresis and visualized by ethidium bromide staining. No signal was observed of 613 bp that corresponded to hMATE2. The quality of the sample RNA also was checked by RT-PCR for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an internal control (dotted line). The mixture of plasmid DNA coding hMATE2-K and hMATE2-B was used as a positive control for hMATE2-K and hMATE2-B and as a negative control for GAPDH.
To visualize the intrarenal distribution of hMATE2-K, we examinedthe immunohistochemical analysis (Figure 4). Staining with theantibodies specific for hMATE1 and hMATE2-K revealed that bothtransporters were localized in the brush border membranes ofproximal tubules (Figure 4, A through F). Although the greensignals for F-actin with phalloidin were shown in all sections,red signal was not observed in the section stained with thepreimmune serum (Figure 4G). The red signals for hOCT2, whichis a basolateral organic cation transporter, and the hOCTN2,which is a luminal Na+/carnitine co-transporter, were confirmedin the basolateral and brush border membranes, respectively(Figure 4, H and I).
Figure 4. Immunofluorescence localization of hMATE1 and hMATE2-K in the human kidney. The hMATE1 (red; A), F-actin (green; B), and merged picture including the purple signals of 4',6-diamidino-2-phenylindole (DAPI; C) were observed in the same section. The hMATE2-K (red; D), F-actin (green; E), and merged picture including DAPI (F) were in the same section. The yellow signals consist of hMATE1 or hMATE2-K and F-actin and were concentrated in the brush border membranes of proximal tubules (C and F). No positive staining for hMATE2-K (red) was observed using the preimmune serum (G). The basolateral localization of hOCT2 (H) and the apical localization of hOCTN2 (I) were confirmed with each antibody, respectively. *Glomeruli. Magnification, x100.
The oppositely directed H+ gradient–dependent uptake of[14C]TEA was examined in HEK293 cells that were transfectedwith the pcDNA3.1 (+) empty vector, hMATE2-K cDNA, and hMATE2-BcDNA. The pH gradient–dependent uptake of [14C]TEA wasstimulated in cells that were transfected with hMATE2-K cDNAbut not with hMATE2-B cDNA (Figure 5A). In addition, intracellularacidification by ammonium chloride markedly stimulated the hMATE2-K–mediateduptake of [14C]TEA, but no response was observed in hMATE2-B–transfectedcells (Figure 5B). The hMATE2-K–mediated [14C]TEA uptakewas not affected by co-transfection of hMATE2-B cDNA (Figure 6).Therefore, the protein that was coded by hMATE2-B seemed tohave no influence on the activity of hMATE2-K or its own activityas an organic cation transporter. We subsequently focused onthe functional characterization of hMATE2-K. The hMATE2-K–mediateduptake of [14C]TEA was increased in accordance with the extracellularpH between 6.0 and 9.0 (Figure 7A). Because of the apparentlinearity of the time course of the hMATE2-K–mediateduptake of [14C]TEA until 5 min (Figure 7B), the transport characteristicsof hMATE2-K at 2 min were selected to examine the kinetics.The linearity of the uptakes of [14C]metformin, [3H]MPP, and[3H]cimetidine also were confirmed until 5 min (data not shown).The uptake of [14C]TEA, [14C]metformin, [3H]MPP, [3H]cimetidine,and [14C]procainamide by hMATE2-K exhibited saturable kinetics,following the Michaelis-Menten equation. The apparent Km valuesof TEA, metformin, MPP, cimetidine, and procainamide were estimatedat 0.83 ± 0.15 mM, 1.05 ± 0.29 mM, 93.5 ±4.9 µM, 0.37 ± 0.14 mM, and 4.10 ± 0.30mM, respectively (Figure 8).
Figure 5. Oppositely directed H+ gradient–dependent uptake of [14C]tetraethylammonium (TEA) by hMATE2-K but not hMATE2-B in the transiently transfected HEK293 cells. (A) The pcDNA3.1(+) empty vector (), hMATE2-K cDNA (), and hMATE2-B cDNA () were transfected into the HEK293 cells. Two days after transfection, uptake of [14C]TEA (5 µM, 7.4 kBq/ml) was examined at the extracellular pH 7.4 or 8.4. (B) HEK293 cells that were transfected with the empty vector, hMATE2-K cDNA, and hMATE2-B cDNA were preincubated with incubation medium (pH 7.4) in the absence () or presence () of 30 mM ammonium chloride for 20 min. Then, the preincubation medium was removed, and the cells were incubated with 5 µM [14C]TEA (7.4 kBq/ml, pH 7.4) in the absence () or presence () of 30 mM ammonium chloride for 2 min at 37°C. Each column represents the mean ± SE of three monolayers.
Figure 6. Transfection of hMATE2-B cDNA in HEK293 cells did not stimulate [14C]TEA uptake and did not affect hMATE2-K–mediated [14C]TEA uptake. HEK293 cells that were transfected with the empty vector (800 ng/well), hMATE2-K cDNA (400 ng/well) with the empty vector (400 ng/well), hMATE2-B cDNA (400 ng/well) with the empty vector (400 ng/well), and the combination of both hMATE2-K (400 ng/well) and hMATE2-B (400 ng/well) cDNA were incubated with 5 µM [14C]TEA (7.4 kBq/ml) at pH 7.4 () or pH 8.4 (). Each point represents the mean ± SE of three monolayers.
Figure 7. Oppositely directed H+ gradient dependence (A) and time course (B) of [14C]TEA uptake by hMATE2-K in the transiently transfected HEK293 cells. (A) HEK293 cells that were transfected with the empty vector () or hMATE2-K (•) were incubated for 15 min at 37°C with incubation medium of various pH that contained 5 µM of [14C]TEA. Each point represents the mean ± SE of six monolayers by two independent experiments. (B) Time course of [14C]TEA uptake by hMATE2-K. HEK293 cells that were transiently transfected with the empty vector () or hMATE2-K cDNA (•) were incubated with [14C]TEA (5µM, 7.4kBq/ml, pH 8.4) for 15 min at 37°C. Each point represents the mean ± SE of three monolayers.
Figure 8. Concentration dependence of hMATE2-K–mediated uptake of [14C]TEA (A), [14C]metformin (B), [3H]1-methyl-4-phenylpyridinium (MPP; C), [3H]cimetidine (D), and [14C]procainamide (E). HEK293 cells that were transfected with hMATE2-K cDNA were incubated with various concentrations of [14C]TEA (pH 8.4; A) or [14C]metformin (pH 8.4; B). For the kinetic analyses of [3H]MPP (C), [3H]cimetidine (D), and [14C]procainamide (E) at pH 7.4, ammonium chloride (30 mM, pH 7.4, 20 min) was pretreated to generate the condition of intracellular acidification. Uptake experiments were performed in the absence () or presence of each unlabeled substrate at 10 mM (•) for 2 min at 37°C. Each point represents the mean ± SE of four independent experiments.
Next, we examined the substrate specificity of hMATE2-K. ThehMATE2-K mediated the oppositely directed H+ gradient–dependenttransport of structurally diverse organic cations such as [14C]TEA,[3H]MPP, [3H]cimetidine, [14C]metformin, and NMN at extracellularpH 8.4 (Table 2) or at pH 7.4 after pretreatment of ammoniumchloride (Table 3). The uptake of [14C]procainamide was stimulatedin hMATE2-K–expressing cells only after pretreatment ofammonium chloride (Tables 2 and 3). In addition, the uptakeof [14C]creatinine, [3H]quinidine, [3H]thiamine, and [3H]verapamilwas increased in hMATE2-K–expressing cells with treatmentof ammonium chloride. However, the anionic compounds p-aminohippuricacid, dipeptide glycylsarcosine, and -lactam antibiotics werenot transported by hMATE2-K.
In this study, we cloned an alternatively spliced variant ofhMATE2 from the human kidney, hMATE2-K (Figures 1 and 2). ThehMATE2 that originated from the human brain was not expressedin the kidney (Figure 3). In addition, another variant was clonedfrom the human brain, hMATE2-B, but original hMATE2 was notcloned from either kidney or brain. hMATE2-B cDNA contains a154-bp insertion in exon 7 of hMATE2-K and a 46-bp insertionin exon 7 of hMATE2. The hMATE2-B has only 220 amino acids bythe 46-bp insertion in exon 7 of MATE2; therefore, the truncatedproduct of the gene, hMATE2-B, seems to lack the transport activity(Figures 5 and 6). Although an RT-PCR band that correspondedto hMATE2-B but not hMATE2 was ubiquitously found, the expressionlevel of the gene was markedly low in consideration of the resultof real-time PCR (Figure 3). Considering the 108- and 154-bpdeletions in hMATE2-K compared with hMATE2 and hMATE2-B, respectively,the splicing site differed between the kidney and other tissues.
Examination of the tissue distribution clearly indicated thathMATE2-K was a kidney-specific type H+/organic cation antiporter,whereas hMATE1 mRNA was found in several tissues (Figure 3A).Although hMATE1 mRNA was strongly expressed in the kidney, italso was preferentially expressed in the adrenal gland, liver,skeletal muscle, and testis, corresponding to the report byOtsuka et al. (12). Comparing promoter sequences between hMATE1and hMATE2-K should help to reveal the molecular mechanismsbehind the kidney-specific expression of hMATE2-K as well asother renal organic ion transporters hOCT2 (SLC22A2), hOAT1(SLC22A6), and hOAT3 (SLC22A8) (10). Immunohistochemical examinationsclearly demonstrated the apical localization of hMATE2-K proteinas well as hMATE1 protein in the proximal tubules (Figure 4,A through F) (12). Considering the functional characteristicsand the membrane localization, hMATE2-K was indicated to mediatetubular secretion of cationic compounds at the brush bordermembranes. These results suggest that some cationic drugs thatare preferentially recognized by hMATE2-K are eliminated predominantlythrough urine via tubular secretion, whereas those that arerecognized by hMATE1 are excreted into both bile and urine.Therefore, the substrate specificities between these two transportersshould be clarified to understand kidney/liver selectivity inthe elimination route of the organic cations.
Although the uptake of procainamide was not observed at pH 8.4,it was stimulated by the pretreatment with ammonium chloride(Tables 2 and 3). These results suggest that hMATE2-K requiresa strong driving force, a counteracting H+ gradient across theplasma membrane, for the transport of procainamide. Stoichiometricdetermination would clarify the coupling ratio between the substrateand H+ ion or the mass charge of the substrate in combinationwith the H+ ion. In addition, further transport studies shouldbe carried out to clarify the precise requirement(s) of thechemical structure(s) to understand the substrate specificityof hMATE2-K.
In this study, an antihyperglycemic agent, metformin, was demonstratedfor the first time to be a good substrate for hMATE2-K (Figure 8B,Tables 2 and 3). There is no information available about theH+ gradient–dependent transport of metformin in renalbrush border membrane vesicles. Actually, approximately 70%of metformin in the circulation was eliminated in urine mainlyvia tubular secretion, and when co-administered, cimetidinedecreased the renal clearance of metformin (23,24). Recently,we demonstrated that metformin is a superior substrate for renalOCT2 rather than hepatic OCT1 (17,25). Renal distribution ofmetformin immediately after intravenous administration was almost23-fold higher than the plasma concentration in the rats (1.29µg/ml in plasma versus 29.8 µg/g in kidney at 3min after intravenous administration) (17). This backgroundand our results strongly suggest that renal hMATE2-K plays akey role in the tubular secretion of metformin after basolateralaccumulation by renal hOCT2.
An active variant, hMATE2-K, and a longer variant, hMATE2-B,were cloned and characterized. It is indicated that hMATE2-Kis the first kidney-specific H+/organic cation antiporter tomediate the tubular secretion of a wide range of cationic compoundsacross the brush border membranes in the proximal tubules. Thephysiologic roles of hMATE2-B and hMATE2, including the expressionalcharacteristics of these genes, still are unclear. Further studiesshould be performed to elucidate the physiologic and pharmacologicsignificance of hMATE2-K as well as hMATE1 in the renal handlingof ionic drugs.
Acknowledgments
This work was supported in part by a grant-in-aid for Researchon Advanced Medical Technology from the Ministry of Health,Labor and Welfare of Japan; by the Japan Health Science Foundation"Research on Health Sciences Focusing on Drug Innovation"; bya grant-in-aid for Scientific Research from the Ministry ofEducation, Science, Culture and Sports of Japan; and by the21st Century COE program "Knowledge Information Infrastructurefor Genome Science." A.Y. was supported as a Research Assistantby the 21st Century COE program "Knowledge Information Infrastructurefor Genome Science."
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Top
Abstract
Introduction
-globulin as a standard. 
-lactam antibiotics were not transported by hMATE2-K. 
), hMATE2-K cDNA (
) were transfected into the HEK293 cells. Two days after transfection, uptake of [14C]TEA (5 µM, 7.4 kBq/ml) was examined at the extracellular pH 7.4 or 8.4. (B) HEK293 cells that were transfected with the empty vector, hMATE2-K cDNA, and hMATE2-B cDNA were preincubated with incubation medium (pH 7.4) in the absence (
) of 30 mM ammonium chloride for 2 min at 37°C. Each column represents the mean ± SE of three monolayers.
) or hMATE2-K (•) were incubated for 15 min at 37°C with incubation medium of various pH that contained 5 µM of [14C]TEA. Each point represents the mean ± SE of six monolayers by two independent experiments. (B) Time course of [14C]TEA uptake by hMATE2-K. HEK293 cells that were transiently transfected with the empty vector (