Cell and Transport Physiology

The Renal-Specific Transporter Mediates Facilitative Transport of Organic Anions at the Brush Border Membrane of Mouse Renal Tubules

Tomoki Imaoka, Hiroyuki Kusuhara, Satomi Adachi-Akahane, Maki Hasegawa, Naomi Morita, Hitoshi Endou and Yuichi Sugiyama
JASN August 2004, 15 (8) 2012-2022; DOI: https://doi.org/10.1097/01.ASN.0000135049.20420.E5

Abstract

ABSTRACT. The renal secretion of organic anions across the proximal tubules is achieved by a coordination of uptake and efflux transporters. This study reports the expression, localization, and functional properties of mouse renal-specific transporter (RST). Mouse RST mRNA is predominantly expressed in the kidney and localized on the brush border membrane of mouse kidney proximal tubules. Mouse RST-expressing HEK293 cells exhibited saturable uptake of p-aminohippurate (Km ∼234 μM), which was increased by an increase in K+ concentration or in the presence of Ba2+ and ouabain and decreased by diethylpyrocarbonate, a histidine modifier. An increase in K+ concentration enhanced the uptake of benzylpenicillin, 2,4-dichlorophenoxyacetate, and dehydroepiandrosterone sulfate, suggesting polyspecific substrate specificity of mouse RST. Vectorial transport of 2,4-dichlorophenoxyacetate was observed in the basal-to-apical direction in rat organic anion transporter 3-expressing LLC-PK1 cells (rOat3-LLC); however, coexpression of mouse RST in rOat3-LLC caused a 1.3-fold increase in the basal-to-apical transport. In addition, the basal-to-apical transport of benzylpenicillin and urate was 3- and 2.5-fold greater than that in the opposite direction in the double-transfected cells, respectively, whereas their transepithelial transport in vector- or rOat3-LLC was symmetrical. Furthermore, the basal-to-apical transport of benzylpenicillin was saturable and reduced by increasing extracellular K+ concentration and ouabain. These results suggest that mouse RST mediates the efflux of organic anions including urate and works as exit for organic anions in the proximal tubules. In addition to the kidney, mouse RST was detected in the brain capillaries and the choroid plexus, and it may also play a role in efflux transport of organic anions across the barriers of the central nervous system.

The kidney plays important roles in the detoxification of xenobiotics and endogenous wastes and maintains the correct balance of ions and nutrients in the body (1–6⇓⇓⇓⇓⇓). Urinary excretion is the major detoxification mechanism in the kidney and consists of glomerular filtration in the glomeruli, tubular secretion across the proximal tubules, and reabsorption. Cumulative studies have revealed the importance of transporters in the tubular secretion of a large number of organic compounds. Vectorial transport across the renal tubules is achieved by an interplay between the uptake and efflux transport at the basolateral membrane (BLM) and brush border membrane (BBM), respectively, especially for hydrophilic compounds (1–6⇓⇓⇓⇓⇓). Tubular secretion has been characterized by organic anion and cation transport systems, and recent progress in the molecular cloning of transporters has revealed the important role played by members of solute carrier family (SLC) 22A in the tubular secretion of organic compounds (1–6⇓⇓⇓⇓⇓). Organic cation transporters (OCT) and OCTN exhibit broad substrate specificity for hydrophilic organic cations such as tetraethylammonium (TEA). OCT are facilitative transporters and responsible for the renal uptake of organic cations at the BLM (1,3,5⇓⇓). OCTN have been suggested to be candidate transporters for apical secretion of organic cations since the reduced renal clearance of TEA was decreased in the juvenile visceral steatosis mouse, an animal model of primary carnitine deficiency caused by functional loss of Octn2 (7), and the transport properties of OCTN1 are similar to that in the BBM, i.e., an organic cation and proton exchanger (8). Although there is no general consensus whether OCTN1 and OCTN2 represent the predominant apical pathway for renal organic cations, it is likely that OCT and OCTN play important roles in the vectorial transport system for organic cations in the kidney.

As far as organic anions are concerned, two organic anion transporters have been identified on the BLM. Sekine et al. (9) and Sweet et al. (10) cloned organic anion transporter 1 (Oat1/Slc22a6) from rat kidney as the classical renal organic anion transporter, and, subsequently, Oat3 (Slc22a8) (11) has been cloned from rat brain by homology screening. Along the proximal tubules, Oat1 is abundantly expressed in the S2 segment (12), whereas Oat3 is expressed in all of the segments (S1 to S3) (13,14⇓). Both transporters have broad substrate specificity, and kinetic analysis using kidney slices has suggested that rat Oat1 is mainly responsible for the renal uptake of hydrophilic and small organic anions, whereas rat Oat3 is responsible for more bulky organic anions (14,15⇓). A transporter that accounts for subsequent excretion of organic anions across the BBM of proximal tubules remains to be identified, although a series of studies using BBM vesicles suggests the presence of two transport mechanisms (16–19⇓⇓⇓): (1) facilitated transporter(s) driven by a luminal positive membrane voltage or (2) an exchanger which exports organic anions in exchange for luminal Cl−.

Renal-specific transporter (RST) was isolated from mouse kidney using the signal sequence trap method without any functional report (20). Its amino acid sequence exhibits relatively high homology to recently cloned human urate transporter (URAT1; 74% identity in amino acid level (21)). Both human URAT1 and mouse RST are localized on the BBM of the proximal tubules (21,22⇓) and have been hypothesized to be involved in the reabsorption of urate in the kidney, because human URAT1 from the patients who had hypouricemia lacked the transport function (21). Hosoyamada et al. (22) showed that mouse RST accepts urate as substrate and has transport property similar to human URAT1. In this study, we established a stable transfectant of mouse RST in HEK293 cells (mRST-HEK) and examined the uptake of organic anions. In a preliminary experiment, we found that increasing extracellular K+ concentration increased the uptake of p-aminohippurate (PAH) by mRST-HEK. A facilitative transporter for organic anions has been suggested in the BBM of the proximal tubules (16–19⇓⇓⇓), and it is possible that mouse RST is a candidate transporter. When expressed in LLC-PK1 cells, rat Oat3, one of the basolateral organic anion transporters, is mainly localized on the basolateral membrane. For examining whether mouse RST can mediate the efflux of organic anions, mouse RST cDNA was introduced into the LLC-PK1 cells that stably express rat Oat3 (rOat3-LLC), and transcellular transport of common substrates of rat Oat3 and mouse RST was determined in control, rOat3-LLC, and the double-transfectant rOat3/mRST-LLC.

Materials and Methods

Materials

[3H]PAH (4.08 Ci/mmol), [3H]dehydroepiandrosterone sulfate (DHEAS; 74.0 Ci/mmol), [3H]benzylpenicillin (19.0 Ci/mmol), and [14C]TEA (2.40 mCi/mmol) were purchased from Perkin Elmer Life Science Products (Boston, MA). [3H]2,4-dichlorophenoxyacetate (2,4-D; 20.0 Ci/mmol) was purchased from Amersham Biosciences (Little Chalfont, Buckinghamshire, UK). [3H]ochratoxin A (18.0 Ci/mmol) and [14C]urate (50.0 mCi/mmol) were from Moravek (Brea, CA). All other chemicals and reagents were of analytical grade and were readily available from commercial sources.

Northern Blot Analysis

A commercially available hybridization blot containing poly(A)+ RNA from various mouse tissues (Clontech) was used for Northern blot analysis. The filter was hybridized in hybridization solution at 42°C with a full-length cDNA of mouse RST randomly labeled with [32P]dCTP as a probe. The filter was then washed with 0.1% SSC/0.1% SDS at 55°C.

Reverse Transcription–PCR Analysis

On the basis of the nucleotide sequence (NM_009203), the following primers were designed: forward primer, 5′-atggcctttcctgaactcctg-3′; reverse primer, 5′-ggtcacgattgtggacctgaa-3′. PCR was performed using cDNA from mouse choroid plexus, brain capillaries, and kidney as template according to the following protocol: 94°C for 30 s, 57°C for 30 s, and 72°C for 60 s; 40 cycles. The nucleotide sequence of the reverse transcription–PCR (RT-PCR) products was confirmed by direct sequencing.

Preparation of Anti-Mouse RST Serum

Anti-mouse RST serum was raised in rabbits against a synthetic peptide consisting of the 15 carboxyl-terminal amino acids of mouse RST coupled to keyhole limpet hemocyanine at its carboxyl-terminal via an additional tyrosine.

Western Blot Analysis

The BBM fraction from mouse kidney was prepared according to the methods reported by Lahjouji et al. (23). The purity was checked by measurement of alkaline phosphatase activities. The enrichment factor of alkaline phosphatase in the BBM fraction compared with that in the homogenate was 9.2 ± 0.6 (mean ± SE of three preparations), which was comparable with previous reports (23). The brain capillary–enriched fraction was prepared from mouse brain according to the previously described method (24,25⇓). The enrichment factor of γ-GTP in the brain capillary–enriched fraction compared with that in the homogenate was 6.7 ± 0.8 (mean ± SE of three preparations). Crude membrane was prepared from the control or cDNA transfected cells according to the previously described method (14).

These specimens were subjected to electrophoresis by 10% SDS-PAGE. Separated proteins were electroblotted onto a polyvinylidene difluoride membrane (Pall, NY). The membrane was blocked with Tris-buffered saline that contained 0.05% Tween 20 (TBS-T) and 5% skim milk for 1 h at room temperature, and, after washing with TBS-T, it was incubated with anti-mouse RST serum (dilution 1:1000). The membrane was allowed to bind a horseradish peroxidase–labeled anti-rabbit IgG antibody (Amersham Pharmacia Biotech, Buckinghamshire, UK) diluted 1:2000 in TBS-T for 1 h at room temperature.

Immunofluorescence Study

Frozen sections from mouse kidney (strain ddY, male, 7 wk old) for the immunofluorescence study were prepared after being fixed in acetone (−20°C). Nonspecific protein binding was blocked by incubation with Nonspecific Staining Blocking Reagent (DAKO, Carpinteria, CA). Sections were incubated with anti-mouse RST serum (1:200) for 1 h at room temperature, washed three times with TBS-T, and incubated with the secondary antibodies labeled with FITC for 1 h at room temperature. They were then mounted in VECTASHIELD Mounting Medium with propidium iodide (Vector Laboratories, Burlingame, CA). The specificity of the antibody reaction was verified by incubating negative controls with antiserum that had been blocked with antigenic peptide.

For immunocytochemistry, cells were grown on a coverglass coated with poly-lysine (Matsunami, Osaka, Japan). After fixation with 4% paraformaldehyde for 10 min and permeabilization in 1% Triton X-100 in PBS for 10 min, cells were incubated with the antiserum against rat Oat3 or mouse RST (diluted 50-fold in PBS) for 30 min at room temperature. Cells were then washed three times with PBS and incubated with Goat anti-rabbit IgG (Alexa 488; Molecular Probes, Eugene, OR; diluted 250-fold in PBS) for 30 min at room temperature. Nuclei were stained with propidium iodide.

Functional Expression of Mouse RST cDNA and Cell Culture

The cDNA of mouse RST was cloned from mouse kidney cDNA library according to the protocols described previously (9). The cDNA ligated to the pcDNA3.1 was introduced into HEK293 cells by lipofection with FuGENE6 reagent, and stably transfected cells (mRST-HEK) were selected by G418 (Invitrogen). HEK293 cells were grown in DMEM supplemented with 10% FBS (Sigma) and G418 sulfate (400 μg/ml) at 37°C with 5% CO2 and 95% humidity on the bottom of a dish. Cells were cultured for 48 h with culture medium without sodium butyrate on the bottom of a dish and for an additional 24 h with culture medium supplemented with sodium butyrate (5 mM) before starting the transport studies.

Uptake Studies

Uptake was initiated by adding the radiolabeled ligands to the medium in the presence and absence of inhibitors after cells had been washed three times and preincubated with Krebs-Henseleit buffer at 37°C for 15 min. The Krebs-Henseleit buffer contains (in mM) 118 NaCl, 23.8 NaHCO3, 4.83 KCl, 0.96 KH2PO4, 1.20 MgSO4, 12.5 HEPES, 5 glucose, and 1.53 CaCl2 adjusted to pH 7.4. The uptake was terminated at designated times by adding ice-cold Krebs-Henseleit buffer. Ligand uptake is given as the amount of ligand associated with the cells divided by the medium concentration.

The effect of changing the concentration of K+ in the uptake buffer (high K+) on the uptake by mRST-HEK was studied. The ion composition was the same with Krebs-Henseleit buffer except NaCl (4.83 mM) and KCl (118 mM). For investigating the effect of Na+, low Na+ buffer was prepared, which contains (in mM) 238 mannitol, 23.8 NaHCO3, 4.83 KCl, 0.96 KH2PO4, 1.20 MgSO4, 12.5 HEPES, 5 glucose, and 1.53 CaCl2 adjusted to pH 7.4. For examining the effect of Ba2+, MgCl2 was substituted for equimolar MgSO4 to avoid sedimentation of BaSO4.

Intracellular Electrical Potential Recordings

The complete cell patch-clamp recording was obtained to study the effects of substitution of K+ for Na+ on the resting intracellular electrical potential in HEK293 cells. Intracellular potential was measured using a patch/whole-cell clamp amplifier (Axopatch 200B; Axon Instruments, Union City, CA) by means of an analog-to-digital converter (Digidata 1200; Axon Instruments). Voltage-clamp protocols and data acquisition were carried out using pCLAMP6 software (Axon Instruments). The patch electrodes were filled with the pipette solution containing (in mM) 130 KCl, 5 Na2-phosphocreatine, 5 Mg-ATP, 0.1 ethylenedioxybis(o-phenylenenitrilo)tetraacetic acid, and 10 HEPES (pH 7.3).

Construction of the Double Transfectant and Transcellular Transport Study

Mouse RST cDNA ligated to the pcDNA3.1(zeo) was introduced into the LLC-PK1 cells expressing rat Oat3 (rOat3-LLC), which had been established previously (26). Stably transfected cells (rOat3/mRST-LLC) were selected by zeocin. The cells were seeded in a cell-culture insert at a density of 1.4 × 105 cells/insert and cultured for 4 d. The culture medium was replaced with that containing 10 mM sodium butyrate 24 h before starting the experiments. After cells were washed three times and preincubated with Krebs-Henseleit buffer, the experiments were initiated by addition of [3H]2,4-D, [3H]benzylpenicillin or [14C]urate to either the apical or the basal side of the cell layer. The cells were incubated at 37°C, and radioactivities accumulated in the opposite side were determined.

Statistical Analyses

Statistical differences were determined using one-way ANOVA followed by Fisher least significant difference method.

Results

Cloning of Mouse RST

The nucleotide sequence of the cloned mouse RST was identical to that of mouse RST (NM 009203) except for three nucleotide changes (T429C, A1166G, and C1653T). A1166G resulted in the amino acid change (Asp389Gly).

Tissue Distribution of Mouse RST

Northern blot analysis showed that a band was detected at 2.4 kb predominantly in the kidney (Figure 1A). The expression of mouse RST protein was confirmed by Western blot analysis. As shown in Figure 1B, the mouse RST protein was detected at ∼55 kDa in mouse BBM and in the mRST-HEK cells but not in vector-transfected cells. The bands were abolished when the preabsorbed antiserum for mouse RST was used, suggesting that the positive signals were specific for the antigen peptide. The localization of mouse RST in the kidney was investigated by immunofluorescence analysis (Figure 1C). Mouse RST specific staining was observed in the BBM of the proximal tubular cells, and no positive signal was detected when preabsorbed antibody against mouse RST was used (data not shown).

Figure

Figure 1. Northern, Western blot, and immunohistochemical analyses of mouse renal-specific transporter (RST). (A) Northern blot analysis. Mouse multiple tissue blot that contains 1 μg of poly(A) + RNAs from eight mouse tissues in each of the lanes was probed with a [32P]-labeled mouse RST cDNA fragment. Lane 1, heart; lane 2, brain; lane 3, spleen; lane 4, lung; lane 5, liver; lane 6, skeletal muscle; lane 7, kidney; lane 8, testis. (B) Western blot analysis. Crude membrane prepared from mouse RST-expressed (lanes 1 and 4; 50 μg), vector-transfected HEK cells (lanes 2 and 5; 50 μg) and mouse brush border membrane (BBM; lanes 3 and 6; 20 μg) were used in the Western blot analysis. The membrane was incubated with anti-mouse RST serum (lanes 1 to 3) or with preabsorbed anti-mouse RST serum (lanes 4 to 6). (C) Immunofluorescence localization of mouse RST in the kidney. Cryosections of the mouse kidney were incubated with anti-mouse RST serum. Nuclei were stained with propidium iodide (red). The BBM of the kidney was stained with anti-mouse RST serum (green fluorescence). The signal was abolished when preabsorbed antibody against mouse RST was used.

Characterization of Mouse RST-Mediated Uptake

Figure 2 shows the time profiles of the uptake of PAH by vector- and mRST-HEK. The uptake of PAH by mRST-HEK was slightly greater than that by vector-transfected cells. However, substitution of K+ for Na+ in the incubating buffer caused a significant increase in the uptake of PAH by mRST-HEK, but this effect was not observed in vector-HEK (Figure 2A). The uptake of PAH by mouse RST in a high K+ buffer was saturable and followed the Michaelis-Menten equation with Km and Vmax values of 234 ± 19 μM and 730 ± 43 pmol/min per mg protein, respectively (Figure 2B). Direct measurement confirmed that increasing the extracellular K+ concentration significantly depolarized the intracellular voltage (−53 to −6 mV). For investigating the effect of perturbation of K+ gradient on the uptake via mouse RST, [3H]PAH uptake was determined in the presence of ouabain (a Na+-K+ ATPase inhibitor) and Ba2+ (a nonspecific K+ channel blocker). Treatment with ouabain (1 mM) or Ba2+ (2 mM) increased PAH uptake two- and threefold, respectively (Figure 2C).

Figure

Figure 2. Uptake of p-aminohippurate (PAH) by mRST-HEK and effect of K+ on the PAH uptake. (A) The time course of the uptake of PAH (500 nM) in mRST-HEK (filled symbols; High K+, •; Na+, ▪) and vector-HEK (open symbols; High K+, ○; Na+, □) was determined. Each point represents the mean ± SE (n = 3). (B) The uptake of PAH by mRST-HEK and by vector-HEK in the high K+ buffer were measured for 2 min at different concentrations. Specific uptake was obtained by subtracting the uptake by vector-HEK from that by mRST-HEK. Following the Michaelis-Menten equation, the Km and Vmax values were determined by least-squares regression analysis with weighting as the reciprocal of the observed values, and the Damping Gauss Newton Method algorithm was used for fitting. The solid line represents the fitted line. Each point represents the mean ± SE (n = 3). (C) Effect of external K+, Na+, ouabain, and Ba2+ was examined for the uptake of PAH by mouse RST- (▪) and vector-HEK (□). Cells were preincubated in the Krebs-Henseleit in which MgCl2 was substituted for MgSO4 with various concentrations of NaCl and KCl: low Na+ (23.8 mM), high K+ (124 mM). The composition of the buffer is described in Materials and Methods. The effect of ouabain (1 mM) and BaCl2 (2 mM) was tested in the presence of control concentrations of KCl and NaCl. Cells were preincubated with either agent for 30 min before the uptake experiment. The uptake was determined for 15 min. Each bar represents the mean ± SE (n = 3). **P < 0.01 significant difference. (D) Effect of diethylpyrocarbonate (DEPC) on the uptake of [3H]PAH via mouse RST. mRST-HEK were treated with 1 mM DEPC in the presence or absence of 100 μM unlabeled PAH for 15 min, and, subsequently, PAH transport activity was determined for 15 min. □ and ▪ represent uptake by vector-HEK (control) and by mRST-HEK cells, respectively. Each bar represents the mean ± SE (n = 3). *P < 0.05, **P < 0.01 significant difference.

A previous study using BBM vesicles from rat kidney revealed that the membrane voltage–sensitive transport of PAH was selectively inhibited by a histidyl modifier, diethylpyrocarbonate (DEPC) (27). Therefore, the effect of DEPC on the transport of PAH via mouse RST was examined (Figure 2D). DEPC pretreatment abolished the uptake of PAH by mRST-HEK in a high K+ buffer, and this effect was protected by addition of 100 μM unlabeled PAH during the preincubation with DEPC.

In addition to PAH, a similar effect by high K+ was observed in the uptake of other organic anions such as ochratoxin A (a mycotoxin; Figure 3A), DHEAS (a steroid conjugate; Figure 3B), benzylpenicillin (a β-lactam antibiotic; Figure 3C), and 2,4-D (an organic herbicide; Figure 3D). There was no significant difference in the uptake of TEA (Figure 3E), estrone sulfate, estradiol 17β glucuronide, folate, and methotrexate (data not shown).

Figure

Figure 3. Mouse RST-mediated uptake of organic anions. The uptake of radiolabeled compounds ([3H]ochratoxin A [500 nM; A], [3H]dehydroepiandrosterone sulfate [DHEAS; 500 nM; b], [3H]benzylpenicillin [500 nM; C], [3H]2,4-dichlorphenoxyacetate [2,4-D; 500 nM; D], and [14C]tetraethyl ammonium [TEA; 1 μM; E]) by mRST-HEK and vector-HEK were measured for 15 min in high K+ buffer (indicated by K+) or normal buffer (indicated by Na+). □ and ▪ represent uptake by vector-HEK cells (control) and by mRST-HEK, respectively. Each bar represents the mean ± SE (n = 3). *P < 0.05, **P < 0.01 significant difference.

Inhibition of Mouse RST-Mediated [3H]PAH Uptake by Various Compounds

To investigate the substrate selectivity of mouse RST, we conducted inhibition studies. Probenecid was found to be a potent inhibitor of mouse RST, whereas 2,4-D, benzylpenicillin and DHEAS were moderate or weak inhibitors (Figure 4A). Estrone sulfate, TEA, or urate did not exhibit any inhibitory activity (Figure 4A). Some uremic toxins exhibited a weak inhibitory effect (Figure 4B), and acidic metabolites of neurotransmitters slightly inhibited the uptake of PAH by mRST-HEK (Figure 4C).

Figure

Figure 4. Inhibitory effect of various compounds on mouse RST-mediated [3H]PAH uptake. The uptake of [3H]PAH (500 nM) by mRST-HEK and vector-HEK was determined in the absence or presence of compounds at the concentrations indicated in the figures. Specific uptake was obtained by subtracting the uptake by vector-HEK from that by mRST-HEK. Each bar represents the mean ± SE (n = 3). Statistical comparisons: *P < 0.05, **P < 0.01, significant difference from control.

Transcellular Transport of [3H]2,4-D, [3H]Benzylpenicillin, and [14C]Urate across an LLC-PK1 Monolayer

For investigating whether mouse RST is involved in the secretion of organic anions, mouse RST was stably expressed in the rat Oat3-LLC. The expression of rat Oat3 and mouse RST in the double-transfected cells was determined using confocal immunofluorescence laser scanning microscopy. As shown in Figure 5, mouse RST was localized on the apical membrane of the double-transfected cells, whereas intense signals associated with rat Oat3 were mainly localized to the basal membrane, but weak signals were also detected at the apical membrane. To support membrane localization of rat Oat3, we determined the uptake of benzylpenicillin from the apical and basal sides of rat Oat3-LLC cultured on a porous membrane. The uptake from the basal side was much greater than that from the apical side (32 ± 3.8 versus 7.0 ± 0.4 μl/mg protein per 5 min). The uptake from the apical side in rat Oat3-LLC was slightly but significantly greater than that in vector control (7.0 ± 0.4 versus 4.4 ± 0.2 μl/mg protein per 5 min).

Figure

Figure 5. Immunolocalization of recombinant rat organic anion transporter 3 (Oat3) and mouse RST in stably transfected cells. LLC-PK1 cells transfected with empty-vector (A and C) and both rat Oat3 and mouse RST (double transfectant; B and D) cDNA were stained with the polyclonal antibody against the carboxyl terminus of rat Oat3 (A and B) or mouse RST (C and D; green fluorescence). Nuclei were stained with propidium iodide (red fluorescence). (Top) Optical sections (0.8 μm) in the x-y plane. (Bottom) Vertical sections in the x-z plane indicated by the red lines in top panel. AP, apical membrane; BL, basolateral membrane.

Transcellular transport of 2,4-D, benzylpenicillin, and urate across an LLC-PK1 monolayer was determined in vector, rat Oat3-, and rat Oat3/mouse RST-LLC. Basal-to-apical transport of 2,4-D was slightly greater than that in the opposite direction in vector-LLCC (Figure 6A), but expression of rat Oat3 increased the basal-to apical transport (Figure 6B). However, expression of mouse RST cDNA to rOat3-LLC caused a slight increase in the basal-to-apical and apical-to-basal transport of 2,4-D compared with those in rOat3-LLC (1.3- and 1.5-fold, respectively; Figure 6, B and C). Although there was no vectorial transport in the vector- and rOat3-LLC (Figure 6, D, E, G, and H), the basal-to-apical transport of benzylpenicillin and urate was 3- and 2.5-fold greater than that in the opposite direction in the double-transfected cells, respectively (Figure 6, F and I). To characterize mouse RST transport in efflux mode, we examined the effect of high K+ and ouabain for the basal-to-apical transport of benzylpenicillin (Figure 7). The basal-to-apical transport of benzylpenicillin was saturable and was decreased by high K+ as well as ouabain treatment (Figure 7).

Figure

Figure 6. Time profiles for the transcellular transport of [3H]2,4-D, [3H]benzylpenicillin, and [14C]urate across LLC-PK1 monolayers. Transcellular transport of [3H]2,4-D (0.5 μM; A through C), [3H]benzylpenicillin (0.5 μM; D through F), and [14C]urate (1 μM; G through I) across LLC-PK1 monolayers expressing rat Oat3 (B, E, and H) and both rat Oat3 and mouse RST (rat Oat3/mouse RST: double transfectant; C, F, and I) was compared with that across the control LLC-PK1 monolayer (A, D, and G). ○ and • represent the transcellular transport in the apical-to-basal and basal-to-apical directions, respectively. Each point represents the mean ± SE (n = 3).

Figure

Figure 7. Effect of external K+ and ouabain on the transcellular transport of [3H]benzylpenicillin across the monolayer of rOat3/mRST-LLC. Transcellular transport of [3H]benzylpenicillin was measured for 180 min in control buffer (Na+), high K+ buffer (K+), and control buffer that contained 1 mM ouabain (+ouabain). Excess amount of nonradiolabeled benzylpenicillin was added as a control (+1 mM benzylpenicillin). □ and ▪ represent the transcellular transport in the basal-to-apical and apical-to-basal directions, respectively. Each bar represents the mean ± SE (n = 3). **P < 0.01 significant difference.

Expression of Mouse RST in the Brain

RT-PCR was carried out to examine the expression of mouse RST in the choroid plexus and brain capillaries (Figure 8A). The size of the PCR product in these organs was ∼400 bp as expected from the nucleotide sequences of mouse RST. To identify the product, we confirmed the nucleotide sequence of the RT-PCR products by sequencing analysis. The expression was also confirmed by Western blot analysis (Figure 8B). Signals were also detected by anti-mouse RST serum in the mouse choroid plexus and brain capillary–enriched fraction as well as in the BBM from mouse kidney, which was abolished when the preabsorbed antiserum was used.

Figure

Figure 8. Reverse transcription–PCR (RT-PCR) and Western blot analysis of expression of mouse RST in choroid plexus and brain capillary endothelial cells. (A) Total RNA from mouse brain capillary endothelial cells, choroid plexus and kidney, or distilled water was subjected to RT-PCR analysis using primers specific for the mouse RST gene. (B) Mouse choroid plexus (lanes 1 and 3) and mouse brain capillary (lanes 2 and 4; 25 μg) were used in the Western blot analysis. The membrane was incubated with anti-mouse RST serum (lanes 1 and 2) or with preabsorbed anti-mouse RST serum (lanes 3 and 4). CP, choroid plexus; BBB, brain capillary.

Discussion

In the present study, we demonstrated that mouse RST is an organic anion transporter with polyspecific substrate specificity and suggested that it is a candidate for the classical facilitative transporter at the BBM of the renal proximal tubules, which has been hypothesized to be involved in the export of organic anions. The substrate specificity of mouse RST was investigated in mRST-HEK. The uptake of PAH by mRST-HEK was slightly greater than that by vector-HEK, but substitution of K+ for Na+ caused an increase only in the saturable uptake of PAH by mRST-HEK (Figure 2, A and B). Preincubation of DEPC inactivated the mouse RST-mediated transport of PAH. Addition of an excess of nonradiolabeled PAH blocked DEPC-inactivation (Figure 2D), suggesting that amino acid residue, possibly histidine, plays a key role in the substrate recognition of PAH by mouse RST. In addition to PAH, substitution of K+ for Na+ also induced the uptake of other organic anions (Figure 3). Inhibition studies suggested that these substrate compounds exhibit moderate or large Km values for mouse RST as in the case of PAH (Figure 4A). In addition, acidic metabolites and uremic toxins are potential substrates of mouse RST (Figure 4, B and C).

The effect of ion substitution was investigated in mRST-HEK (Figure 2C). Incubating the cells with low Na+ buffer had no effect (Figure 2C), and, thus, the effect is likely to be ascribed to direct perturbation of the K+ gradient across the plasma membrane. Treatment with ouabain or Ba2+, which has been reported to cause a perturbation of the K+ gradient by indirect mechanisms (28–30⇓⇓), also induced the mouse RST-mediated uptake of PAH (Figure 2C). It has been reported that perturbation of the K+ gradient causes depolarization of the membrane voltage (29,30⇓), and, indeed, direct measurement revealed that the membrane voltage was depolarized in the high K+ buffer. It is likely that mouse RST is a facilitative transporter, and the transport direction depends on the concentration gradient of the substrate compounds and the membrane voltage. Considering the negative membrane voltage, it is possible that mouse RST mediates the efflux of organic anions under physiologic conditions.

Because organic anions generally show poor membrane permeability, both uptake and efflux transporters are involved in the vectorial transport across the epithelial cells. The substrate specificity of mouse RST overlaps that of basolateral organic anion transporters (Oat1/OAT1 and Oat3/OAT3), which could be involved in the tubular secretion of organic anions together with mouse RST. To investigate this possibility, we established LLC-PK1 cells expressing both rat Oat3 and mouse RST on the basal and apical membrane, respectively (Figure 5), and the transcellular transport of their common substrates was determined. The basal-to-apical transport of 2,4-D was markedly greater than that in the opposite direction even in rOat3-LLC (Figure 6B). Expression of mouse RST in rOat3-LLC caused a slight increase in the basal-to-apical transport of 2,4-D (Figure 6C). The small increase in the basal-to-apical transport of 2,4-D could be accounted for by the following kinetic consideration. In the case in which the efflux at the apical membrane is large enough, the transcellular transport is governed by the uptake process at the basal side, and the alteration in the efflux transport activity at the apical side hardly affects the transcellular transport. In LLC-PK1 cells, expression of endogenous efflux transporter at the apical side may satisfy this condition. Vectorial transport of benzylpenicillin was clearly observed in the double transfectant (Figure 6F), whereas there was no directional transport in vector- and rOat3-LLC (Figure 6, D and E). The basal-to-apical transport of benzylpenicillin was saturable and significantly reduced in high K+ buffer or after ouabain treatment (Figure 7). These results were in good agreement with the results obtained using mRST-HEK and support the hypothesis that mouse RST is involved in the tubular secretion of organic anions in conjunction with the uptake transporters.

In addition to basolateral Oat/OAT isoforms, there are similarities and differences in the substrate specificity of mouse RST and human OAT4, the other OAT isoform identified on the BBM of the proximal tubules (31). Human OAT4 accepts sulfo-conjugates such as estrone sulfate and DHEAS as substrates and is inhibited by other sulfo-conjugates (32,33⇓). Mouse RST transports DHEAS with a Ki value greater than its Km value for human OAT4 (0.63 μM (33)), but estrone sulfate is not transported by mouse RST and does not inhibit mouse RST. Indoxyl sulfate has an inhibitory effect on mouse RST, which suggests that this sulfate conjugate is a potential substrate of both mouse RST and OAT4 (33). Weak or minimal inhibition by p-nitrophenyl sulfate and minoxidil sulfate is seen in the case of both mouse RST and human OAT4. Furthermore, mouse RST shows saturable uptake of PAH, whereas it has no inhibitory effect on OAT4-mediated [3H]estrone sulfate uptake. These differences and similarities in substrate specificity may help us to understand the kinetics of the renal elimination of organic anions.

Recently, urate has been shown to be an endogenous substrate of mouse RST (22). However, urate does not have any inhibitory effect on PAH uptake by mouse RST (Figure 4). Taking into consideration the Km value of urate for mouse RST (1.2 mM (22)), urate should show, at least, weak inhibition of mouse RST-mediated PAH uptake at the concentration examined. However, PAH has no effect on urate uptake by mouse RST/human URAT1 even at concentrations sufficient to saturate its own uptake based on its Km value determined in this study (21,22⇓). The assumption that mouse RST has multiple substrate recognition sites for PAH and urate may account for the lack of mutual inhibition between PAH and urate as reported for another organic anion transporter, rat Oatp2 (34). The difference in the response to ion substitution in the incubating buffer may support this possibility; the mouse RST-mediated uptake of PAH was increased in high K+ buffer, which did not affect the human URAT1-mediated uptake of urate (21).

The coordinated effects of rat Oat3 and mouse RST facilitate the vectorial transport of urate from the basal to apical side (Figure 6I), suggesting that mouse RST can mediate the efflux of urate as well as organic anions. The apical-to-basal transport of urate in the double transfectant was comparable with that in rOat3-LLC, although human URAT1 and mouse RST have been suggested to be involved in the reabsorption of urate in the kidney (21,22⇓), and mouse RST and Oat3 can also mediate the efflux of urate, respectively (22,36⇓). Another basolateral transporter may increase the efficacy of the apical-to-basal transport, or, because mouse RST/human URAT1-mediated urate uptake is stimulated by intracellular lactate (21,22⇓), a difference in the outward concentration gradient of endogenous ligands may account for the low degree of apical-to-basal transport. Further studies are necessary to elucidate the role of mouse RST in the renal transport of urate.

Recently, primary active transporters, such as multidrug resistance associated proteins, e.g., MRP2 (ABCC2) and MRP4 (ABCC4), and rodent breast cancer resistance protein (BCRP/ABCG2), have been identified on the BBM of the renal tubules (36–38⇓⇓). The substrates of MRP2 include glutathione and glucuronide conjugates and certain types of unconjugated amphipathic organic anions as well as PAH (36,39⇓), whereas MRP4 has been shown to accept cyclic nucleotides (37), estradiol 17β glucuronide, and methotrexate. BCRP was shown recently to accept sulfate conjugates of drugs and steroids (40). Masereeuw et al. (41) showed that Mrp2 is involved in the renal secretion of fluorescence Mrp2 substrates by comparing the renal clearances of normal and Mrp2-deficient mutant rats (TR−). In addition to facilitative transporters and exchangers, these primary active transporters are candidate transporters for the renal secretion of organic anions. Further studies are necessary to assess the importance of mouse RST in the renal secretion of organic anions in the kidney.

RT-PCR and Western blotting detected the expression of mouse RST mRNA and protein in the brain capillaries and choroid plexus. It is well established that the brain capillaries and choroid plexus epithelial cells constitute a tight monolayer acting as a barrier to protect the central nervous system from invasion by xenobiotics from the circulating blood (42–45⇓⇓⇓). The presence of efflux transport systems for hydrophilic organic anions, such as PAH and benzylpenicillin, in these barriers has been demonstrated (46,47⇓). Rat Oat3 has been identified on the brain side of the plasma membrane of the brain capillaries and choroid plexus and is involved in the removal of hydrophilic organic anions from the brain and cerebrospinal fluid, respectively (26,46⇓). Breen et al. (48) suggested the involvement of a membrane voltage-sensitive excretion mechanism for fluorescein in the choroid plexus, and mouse RST may play at least some role in the excretion of organic anions, including the acidic metabolites of neurotransmitters, from the central nervous system.

In conclusion, the present study demonstrates that mouse RST is an organic anion transporter that is driven by an intracellular negative membrane voltage and is involved in the tubular secretion of organic anions together with basolateral organic anion transporters (rat Oat1 and rat Oat3). Mouse RST is localized on the apical membrane of the renal tubules, and its protein is also expressed in brain capillary endothelial cells and the choroid plexus. Identification of mouse RST will provide new insights into the detoxification system present in the kidney and the barriers in the central nervous system.

Acknowledgments

This work was supported by a Grant-in-Aid for Young Scientist (A) (KAKENHI 00302612) and performed in the context of the Advanced and Innovational Research Program in Life Sciences from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Footnotes

  • T.I. and H.K contributed equally to this work.

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The Renal-Specific Transporter Mediates Facilitative Transport of Organic Anions at the Brush Border Membrane of Mouse Renal Tubules
Tomoki Imaoka, Hiroyuki Kusuhara, Satomi Adachi-Akahane, Maki Hasegawa, Naomi Morita, Hitoshi Endou, Yuichi Sugiyama
JASN Aug 2004, 15 (8) 2012-2022; DOI: 10.1097/01.ASN.0000135049.20420.E5
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