Cell and Transport Physiology

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

Satohiro Masuda, Tomohiro Terada, Atsushi Yonezawa, Yuko Tanihara, Koshiro Kishimoto, Toshiya Katsura, Osamu Ogawa and Ken-ichi Inui
JASN August 2006, 17 (8) 2127-2135; DOI: https://doi.org/10.1681/ASN.2006030205

Abstract

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 μM, 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.

Vectorial secretion of cationic compounds across the tubular epithelial cells is an important function of the kidney. Using the stopped flow tubular microperfusion method, cultured renal epithelial cells, and isolated membrane vesicles, it was suggested that two functionally distinct organic cation transporters were expressed in the basolateral and brush border membranes, respectively (1,2). Organic cations are suggested to accumulate in the renal tubular cells via a basolateral organic cation transport system that is sensitive to membrane potential differences. The intracellular cationic compounds were secreted by the apical H+/organic cation antiport system, which was driven by an oppositely directed H+ gradient. A prototype substrate, tetraethylammonium (TEA), has been used consistently for the functional characterization of these organic cation transport systems in the kidney (3–7).

Since cloning of the organic cation transporter OCT1 from the rat kidney (8), many related transporter proteins have been cloned and characterized. It is widely known that the basolateral entry of cationic compounds is mediated mainly by hepatic hOCT1 (SLC22A1) and renal hOCT2 (SLC22A2) in humans, depending on the membrane potential (9–11). However, attempts at molecular identification of the apical H+/organic cation antiporter have failed for more than a decade. Recently, in silico homology screening of the human cDNA database identified a H+/organic cation antiporter, human multidrug and toxin extrusion 1 (hMATE1), from the human brain using the NorM Na+/multidrug antiporter in Vibrio parahaemolyticus as a reference (12). hMATE1 was expressed in the liver, kidney, and skeletal muscle and weakly in the brain and the heart. Although an isoform of hMATE1, hMATE2, was reported simultaneously to be expressed primarily in the kidney, the functional characteristics of hMATE2 were not examined.

In this study, we cloned hMATE2 cDNA from the human kidney for functional characterization. Sequence analysis revealed that the 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 hMATE2 but not the original hMATE2 was expressed only in the kidney, showing a function of oppositely directed H+ gradient–dependent antiport of organic cations; therefore, we designated the new clone human kidney-specific multidrug and toxin extrusion (hMATE2-K).

Materials and Methods

[14C]TEA (2.035 GBq/mmol), [14C]creatinine (2.035 GBq/mmol), [14C]procainamide (2.035 GBq/mmol), [methyl-14C]choline (2.035 GBq/mmol), [9-3H]quinidine (740 GBq/mmol), [3H]quinine (740 GBq/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) were obtained 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; 2.7 TBq/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 from Amersham Biosciences (Uppsala, Sweden). [14C]Levofloxacin (1.07 GBq/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 (Sankyo Co.) were gifts from the respective suppliers. All other chemicals used were of the highest purity available.

Isolation of hMATE2-K cDNA

The hMATE2 cDNA was cloned by RT-PCR from Marathon-Ready human kidney cDNA (Clontech, Palo Alto, CA). Primers that are specific for hMATE2 were designed on the basis of the sequence information of FLJ31196 (NM_152908). The forward and reverse primers, with mutations creating restriction enzyme sites (italics) for cloning the hMATE2 cDNA, were 5′-AGGGTACCCAGTGCCCCGGCCAGGAATGGA-3′ and 5′-CTGTCTAGACCCCTCTGAGTGTCACCACAA-3′, respectively. Simultaneously, hMATE2 cDNA was cloned using the Marathon-Ready human brain cDNA (Clontech) as above. The PCR product was subcloned into the expression vector pcDNA3.1 (+) (Invitrogen, Carlsbad, CA) and sequenced using a multicapillary DNA sequencer RISA384 system (Shimadzu, Kyoto, Japan). Because both hMATE2 from the human kidney and brain were suggested to be splice variants of hMATE2 (FLJ31196) (12), we redesignated newly isolated clones from the human kidney and the human brain as hMATE2-K and hMATE2-B, respectively (Figure 1). The nucleotide sequences of hMATE2-K and hMATE2-B have been submitted to the DDBJ/EMBL/GenBank Data Bank as accession nos. AB250364 and AB250701, respectively.

Figure 1.
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 summarized in Table 1. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was checked for the quality of the total RNA of human tissues used with GAPDH Control Reagent (Applied Biosystems). To detect independently both hMATE2-K and hMATE2 mRNA, we designed qualitative PCR primers as summarized in Table 1.

View this table:
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 peptide that corresponded to the intracellular domains of hMATE1 (CQQAQVHANLKVN, no. 466-478) or hMATE2-K (YSRSECHVDFFRTPEE, no. 543-558) as described, respectively (14,15). For immunofluorescence histochemistry, the human renal specimens were fixed with 4% paraformaldehyde in PBS at 4°C for 30 min (13). Fixed tissues were embedded in OCT compound (Sakura Finetechnical, Tokyo, Japan) and frozen rapidly in liquid nitrogen. Sections (6 μm thick) were cut and covered with 2% FBS and 1 mg/ml RNase A (Nacalai Tesque, Kyoto, Japan) for 1 h. The covered sections were incubated for 1 h with antiserum (1:100 dilution) specific for hMATE1, hMATE2-K, hOCT2, a control of basolateral transporter, or hOCTN2, a control of luminal transporter, and then incubated with Cy3-labeled donkey anti-rabbit IgG (CALTAG Laboratory, San Francisco, CA), 5 U/ml Alexa 488-phalloidin (Molecular Probe, Eugene, OR), and 4′,6-diamidino-2-phenylindole for 1 h. These sections were examined with a BX-50-FLA fluorescence microscope (Olympus, Tokyo, Japan) at ×100 magnification. Images were captured with a DP-50 CCD camera (Olympus) using Studio Lite software (Olympus). As controls, specific rabbit antibodies were replaced with preimmune rabbit serum.

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-B cDNA were transfected into HEK293 cells using LipofectAMINE 2000 Reagent (Invitrogen), as described (16,17). At 48 h after the transfection, the cells were used for uptake experiments.

Cellular uptake of cationic compounds was measured with monolayer cultures of HEK293 cells that were grown on poly-d-lysine–coated 24-well plates (16,17). Typically, the cells were preincubated with 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 medium that contained radiolabeled substrates was added. The medium was aspirated off at the end of the incubation, and the monolayers were 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 then the radioactivity in aliquots was determined by liquid scintillation counting. The cellular uptake of cephalexin, cefazolin, and cephradine was described previously (18). For the cellular uptake of NMN, the HEK293 cells that were transfected with the hMATE2-K cDNA were incubated with NMN for 10 min, washed twice, and scraped with 0.5 ml of incubation medium (pH 7.4). The cellular accumulation of NMN was determined by HPLC according to the method of Musfeld et al. (19). For manipulation of the intracellular pH, intracellular acidification was performed by pretreatment with ammonium chloride (30 mM, 20 min at 37°C, pH 7.4) (20,21). The protein content of the solubilized cells was determined using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories, Hercules, CA) with bovine γ-globulin as a standard.

Statistical Analyses

Data are expressed as the mean ± SEM. Data were analyzed statistically using an unpaired t test. Significance was set at P < 0.05. In all figures, when error bars are not shown, they are smaller than the symbols.

Results

After the sequencing of hMATE2 cDNA that was isolated from the human kidney (hMATE2-K) and human brain (hMATE2-B), it was revealed with the BLAST program that both hMATE2-K and hMATE2-B transcripts consist of 17 exons. However, a deletion of 108 bp in exon 7 of hMATE2-K and an insertion of 46 bp in exon 7 of hMATE2-B were found compared with hMATE2 (Figure 1). The open reading frame of the cloned hMATE2-K cDNA was 1698 bp, coding for a 566–amino acid protein with a calculated molecular mass of 61,012, and that of hMATE2-B was 660 bp and a 220–amino acid protein with a calculated molecular mass of 23,357. Figure 2 shows the deduced amino acid sequences of hMATE2-K and its alignment with its homologues hMATE2-B, hMATE2, hMATE1, or rat (r) MATE1, which was cloned recently in our laboratory from rat kidney with an accession no. AB248823 (22). hMATE2-K showed 82% amino acid identity with hMATE2-B, 94% with hMATE2 (12), 52% with hMATE1 (12), and 52% with rMATE1.

Figure 2.
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 gland as well as in the kidney; weakly in the fetal liver, testis, skeletal muscle, liver, and uterus; and faintly in various other tissues (Figure 3A). In contrast, the obtained data showed that the 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 showed that the amplification of both products derived from hMATE2-K (505 bp) and hMATE2-B (659 bp) among 18 tissues examined, only the 505-bp band that corresponded to hMATE2-K was found in the kidney (Figure 3B). The 613-bp band that corresponded to hMATE2 was not amplified. After sequencing, the PCR product of 659 bp was confirmed to be identical to hMATE2-B.

Figure 3.
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 examined the immunohistochemical analysis (Figure 4). Staining with the antibodies specific for hMATE1 and hMATE2-K revealed that both transporters were localized in the brush border membranes of proximal tubules (Figure 4, A through F). Although the green signals for F-actin with phalloidin were shown in all sections, red signal was not observed in the section stained with the preimmune serum (Figure 4G). The red signals for hOCT2, which is a basolateral organic cation transporter, and the hOCTN2, which is a luminal Na+/carnitine co-transporter, were confirmed in the basolateral and brush border membranes, respectively (Figure 4, H and I).

Figure 4.
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, ×100.

The oppositely directed H+ gradient–dependent uptake of [14C]TEA was examined in HEK293 cells that were transfected with the pcDNA3.1 (+) empty vector, hMATE2-K cDNA, and hMATE2-B cDNA. The pH gradient–dependent uptake of [14C]TEA was stimulated in cells that were transfected with hMATE2-K cDNA but not with hMATE2-B cDNA (Figure 5A). In addition, intracellular acidification by ammonium chloride markedly stimulated the hMATE2-K–mediated uptake of [14C]TEA, but no response was observed in hMATE2-B–transfected cells (Figure 5B). The hMATE2-K–mediated [14C]TEA uptake was not affected by co-transfection of hMATE2-B cDNA (Figure 6). Therefore, the protein that was coded by hMATE2-B seemed to have no influence on the activity of hMATE2-K or its own activity as an organic cation transporter. We subsequently focused on the functional characterization of hMATE2-K. The hMATE2-K–mediated uptake of [14C]TEA was increased in accordance with the extracellular pH between 6.0 and 9.0 (Figure 7A). Because of the apparent linearity of the time course of the hMATE2-K–mediated uptake of [14C]TEA until 5 min (Figure 7B), the transport characteristics of 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 values of TEA, metformin, MPP, cimetidine, and procainamide were estimated at 0.83 ± 0.15 mM, 1.05 ± 0.29 mM, 93.5 ± 4.9 μM, 0.37 ± 0.14 mM, and 4.10 ± 0.30 mM, respectively (Figure 8).

Figure 5.
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.
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.
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.
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. The hMATE2-K mediated the oppositely directed H+ gradient–dependent transport of structurally diverse organic cations such as [14C]TEA, [3H]MPP, [3H]cimetidine, [14C]metformin, and NMN at extracellular pH 8.4 (Table 2) or at pH 7.4 after pretreatment of ammonium chloride (Table 3). The uptake of [14C]procainamide was stimulated in hMATE2-K–expressing cells only after pretreatment of ammonium chloride (Tables 2 and 3). In addition, the uptake of [14C]creatinine, [3H]quinidine, [3H]thiamine, and [3H]verapamil was increased in hMATE2-K–expressing cells with treatment of ammonium chloride. However, the anionic compounds p-aminohippuric acid, dipeptide glycylsarcosine, and β-lactam antibiotics were not transported by hMATE2-K.

View this table:
Table 2.

Uptake of various organic ions in the HEK293 cells that expressed hMATE2-K at extracellular pH 8.4a

View this table:
Table 3.

Uptake of various organic ions in HEK293 cells that expressed hMATE2-K at extracellular pH 7.4 after pretreatment of ammonium chloridea

Discussion

In this study, we cloned an alternatively spliced variant of hMATE2 from the human kidney, hMATE2-K (Figures 1 and 2). The hMATE2 that originated from the human brain was not expressed in the kidney (Figure 3). In addition, another variant was cloned from the human brain, hMATE2-B, but original hMATE2 was not cloned from either kidney or brain. hMATE2-B cDNA contains a 154-bp insertion in exon 7 of hMATE2-K and a 46-bp insertion in exon 7 of hMATE2. The hMATE2-B has only 220 amino acids by the 46-bp insertion in exon 7 of MATE2; therefore, the truncated product of the gene, hMATE2-B, seems to lack the transport activity (Figures 5 and 6). Although an RT-PCR band that corresponded to hMATE2-B but not hMATE2 was ubiquitously found, the expression level of the gene was markedly low in consideration of the result of real-time PCR (Figure 3). Considering the 108- and 154-bp deletions 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 that hMATE2-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, it also was preferentially expressed in the adrenal gland, liver, skeletal muscle, and testis, corresponding to the report by Otsuka et al. (12). Comparing promoter sequences between hMATE1 and hMATE2-K should help to reveal the molecular mechanisms behind the kidney-specific expression of hMATE2-K as well as other renal organic ion transporters hOCT2 (SLC22A2), hOAT1 (SLC22A6), and hOAT3 (SLC22A8) (10). Immunohistochemical examinations clearly demonstrated the apical localization of hMATE2-K protein as well as hMATE1 protein in the proximal tubules (Figure 4, A through F) (12). Considering the functional characteristics and the membrane localization, hMATE2-K was indicated to mediate tubular secretion of cationic compounds at the brush border membranes. These results suggest that some cationic drugs that are preferentially recognized by hMATE2-K are eliminated predominantly through urine via tubular secretion, whereas those that are recognized by hMATE1 are excreted into both bile and urine. Therefore, the substrate specificities between these two transporters should be clarified to understand kidney/liver selectivity in the 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 requires a strong driving force, a counteracting H+ gradient across the plasma membrane, for the transport of procainamide. Stoichiometric determination would clarify the coupling ratio between the substrate and H+ ion or the mass charge of the substrate in combination with the H+ ion. In addition, further transport studies should be carried out to clarify the precise requirement(s) of the chemical structure(s) to understand the substrate specificity of hMATE2-K.

In this study, an antihyperglycemic agent, metformin, was demonstrated for the first time to be a good substrate for hMATE2-K (Figure 8B, Tables 2 and 3). There is no information available about the H+ gradient–dependent transport of metformin in renal brush border membrane vesicles. Actually, approximately 70% of metformin in the circulation was eliminated in urine mainly via tubular secretion, and when co-administered, cimetidine decreased the renal clearance of metformin (23,24). Recently, we demonstrated that metformin is a superior substrate for renal OCT2 rather than hepatic OCT1 (17,25). Renal distribution of metformin immediately after intravenous administration was almost 23-fold higher than the plasma concentration in the rats (1.29 μg/ml in plasma versus 29.8 μg/g in kidney at 3 min after intravenous administration) (17). This background and our results strongly suggest that renal hMATE2-K plays a key role in the tubular secretion of metformin after basolateral accumulation by renal hOCT2.

Conclusion

An active variant, hMATE2-K, and a longer variant, hMATE2-B, were cloned and characterized. It is indicated that hMATE2-K is the first kidney-specific H+/organic cation antiporter to mediate the tubular secretion of a wide range of cationic compounds across the brush border membranes in the proximal tubules. The physiologic roles of hMATE2-B and hMATE2, including the expressional characteristics of these genes, still are unclear. Further studies should be performed to elucidate the physiologic and pharmacologic significance of hMATE2-K as well as hMATE1 in the renal handling of ionic drugs.

Acknowledgments

This work was supported in part by a grant-in-aid for Research on 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”; by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Culture and Sports of Japan; and by the 21st Century COE program “Knowledge Information Infrastructure for Genome Science.” A.Y. was supported as a Research Assistant by the 21st Century COE program “Knowledge Information Infrastructure for Genome Science.”

Footnotes

  • Published online ahead of print. Publication date available at www.jasn.org.

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Identification and Functional Characterization of a New Human Kidney–Specific H+/Organic Cation Antiporter, Kidney-Specific Multidrug and Toxin Extrusion 2
Satohiro Masuda, Tomohiro Terada, Atsushi Yonezawa, Yuko Tanihara, Koshiro Kishimoto, Toshiya Katsura, Osamu Ogawa, Ken-ichi Inui
JASN Aug 2006, 17 (8) 2127-2135; DOI: 10.1681/ASN.2006030205
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