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Departments of *Pharmacology and Toxicology and Cell Physiology, Nijmegen Center for Molecular Life Sciences, Nijmegen, The Netherlands.
Correspondence to Dr. Frans G. M. Russel, Department of Pharmacology and Toxicology 233, Nijmegen Center for Molecular Life Sciences, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Phone: +31-24-3613691/3616892; Fax: +31-24-3614214; E-mail: F.Russel{at}farm.kun.nl
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-naphthyl-ß-D-glucuronide, and p-nitrophenyl-ß-D-glucuronide are also demonstrated. In kidneys of rats deficient in the apical anionic conjugate efflux pump Mrp2, Mrp4 expression is maintained at the same level. It is concluded that MRP4 is a novel apical organic anion transporter and the putative efflux pump for cAMP and cGMP in human kidney proximal tubules. |
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Monolayers of LLC-PK1 cells release both cAMP and cGMP preferentially across the apical membrane, via a probenecid-sensitive, temperature-sensitive, energy-dependent transport mechanism (8,9). Such a profile matches a member of the multidrug resistance protein (MRP/ABCC) subgroup of the ATP-binding cassette (ABC) protein superfamily (10,11). Mrp2 (ABCC2) functions as an ATP-dependent anionic conjugate efflux pump in the apical membrane of renal proximal tubules, small intestinal villi, and hepatocytes (12). The substrate specificity of MRP2, including leukotriene C4 (LTC4), S-(2,4-dinitrophenyl)-glutathione (DNP-SG), and estradiol-17ß-D-glucuronide (E217ßG), exhibits considerable overlap with that of MRP1, which does not interact with cAMP (12,13). Furthermore, rats deficient in functional Mrp2 (Eisai hyperbilirubinemic Spague-Dawley [EHBR]/transport-deficient Wistar[TR-]rats) do not display a clear defect in the renal excretion of several organic anions, suggesting the presence of a compensating apical organic anion transporter in the kidney (14). MRP5 (ABCC5) has been identified as a probenecid-sensitive, ATP-dependent transporter for cGMP and cAMP but is localized to the basolateral membrane upon expression in MDCKII cells (15,16). MRP4 (ABCC4) exhibits the closest resemblance to MRP5, on the basis of structural characteristics, and both proteins mediate resistance to several xenobiotic nucleosides, such as the antiviral therapeutic agent 9-(2-phosphonylmethoxyethyl)adenine (adefovir) (16–18). Pharmacokinetic studies with human subjects have indicated that the kidney is the main excretory pathway for adefovir and cidofovir [(S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine] (19). Furthermore, the excretory pathway is probenecid-sensitive and rate-limiting, which explains the nephrotoxic side effects observed with these drugs (19).
In this report, we provide evidence that MRP4 is a novel renal proximal tubule apical organic anion efflux pump for cAMP and cGMP. We demonstrate the expression and immunolocalization of MRP4 in human kidney and the substrate specificity of recombinant human MRP4 by functional expression.
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Materials and Methods |
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Isolation of Full-Length Human MRP4 and Partial Rat MRP4 cDNA
Total RNA was isolated from human kidney cortex by acid guanidinium isothiocyanate-phenol-chloroform extraction and was reverse-transcribed in 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 10 mM dithiothreitol, 3 mM MgCl2, supplemented with 0.5 mM dNTP, 10 µM reverse primer (see below), and 200 U of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Breda, The Netherlands). For amplification of two overlapping MRP4 cDNA fragments, primers were designed on the basis of the published human sequence (nucleotides 1 to 3979, GenBank accession number AF071202). The forward primer M4KK [5'-GGTACCGCCACCATGCTGCCCGTG-3'; the KpnI site is underlined, a partial Kozak sequence (21), introduced for future studies, is in bold, and ATG is italic] and the reverse primer M4R2 (5'-GGCTCTCCAGAGCACCATCTTTCAAGG-3', nucleotides 2003 to 2029) were used to amplify the 5' cDNA fragment. For amplification of the 3' cDNA fragment, the forward primer M4F2 (5'-TGGTGCAGAAGGGGA-CTTACACTGAGTTC-3', nucleotides 1832 to 1860) and the reverse primer M4R3 (5'-CCTTCGGAACGGACTTGACATTTT-3', nucleotides 3987 to 4010) were used. PCR was performed with the Advantage-2 PCR enzyme system (Clontech, Leusden, The Netherlands), which includes a nuclease-deficient Taq DNA polymerase and a proofreading polymerase. Amplifications were performed for 30 cycles of 94°C for 45 s, 55°C for 30 s, and 72°C for 3 min. The PCR fragments obtained were purified and cloned into pGEM-T Easy (Promega, Leiden, The Netherlands). Multiple clones were analyzed by cycle sequencing using an ABI 377 automated sequence (Applied Biosystems, Foster City, CA). The coding sequence was identical to the published sequence (GenBank/EMBL accession number AF071202) except for two 1-bp alterations (guanine for adenine at nucleotide positions 2030 and 3800), which resulted in substitution of arginine for glutamine at position 677 and glycine for serine at position 1267. A full-length human MRP4 cDNA in the EcoRI and SpeI sites of pGEM-T-Easy (pGTE-MRP4) was obtained by assembling the 5' (nucleotides 1 to 1993) and 3' (nucleotides 1993 to 4010) cDNA fragments.
Total RNA was isolated from rat kidney cortex and reverse-transcribed with 10 µM oligo(dT) as described above. PCR with the human MRP4 primers described by Kool et al. (22) was performed for 35 cycles of 94°C for 30 s, 30°C for 1 min, and 72°C for 1 min. The resulting 239-bp fragment was cloned into the SmaI site of pBluescript KS (pBS-Mrp4) and sequenced as described above. The partial rat Mrp4 cDNA has been submitted to GenBank/EMBL (accession number AF376781).
Antibodies
The linker region (amino acids 611 to 676) of human MRP4 was chosen as the epitope for a polyclonal antibody (pAb). With the M4F2 and M4R2 primers, the encoding cDNA was amplified from pGTE-MRP4 and cloned in-frame into the expression vector pGEX-3x (Pharmacia, Woerden, The Netherlands). A specific rat Mrp4 cDNA, which encodes a protein with 93% identity to amino acids 1165 to 1231 of human MRP4, was amplified from pBS-Mrp4 with the forward primer 5'-TAAAATGGACACTGAACTAG-3' and the reverse primer 5'-AATGGTGAGAACAGTGCA-3'. The resulting 202-bp fragment was cloned in-frame into pGEX-2T. Glutathione S-transferase (GST) fusion proteins were expressed in DH5 Escherichia coli cells and isolated by glutathione-Sepharose 4B (Amersham, Uppsala, Sweden) affinity chromatography. Rabbits were immunized and boosted three times with fusion protein (400 and 200 µg, respectively), followed by collection of serum, which was affinity-purified with a column of immobilized GST and then a column of immobilized GST-fusion protein. The eluted, affinity-purified, anti-rat Mrp4 (pAb rM4-p1; 2 mg/ml) and anti-human Mrp4 (pAb hM4-p3; 3 mg/ml) antibodies were used in immunohistochemical and immunoblot analyses (see below). Monoclonal antibody (mAb) M2III-6 is directed against rat Mrp2 and cross-reacts with human MRP2 (23), and pAb G34 is directed against the rat Na+/K+-ATPase (24). A pAb directed against the rat sodium/phosphate type IIa cotransporter was kindly provided by Dr. J. Biber (Institute of Physiology, University of Zurich, Zurich, Switzerland).
Immunohistochemical Analyses
Normal tissue from a nephrectomized human kidney was provided by the Department of Pathology; its use for medical study was approved by the Nijmegen University institutional review board. Kidney slices were fixed in 1% (vol/vol) periodate-lysine-paraformaldehyde (PLP) fixative for 2 h, washed with 20% (vol/vol) sucrose in phosphate-buffered saline (PBS), and subsequently frozen in liquid N2 (25). Sections were air-dried, pretreated for 5 min at 100°C with citrate buffer (10 mM citric acid, pH 6.0), blocked with 10% (vol/vol) goat serum for 30 min at room temperature, and incubated with pAb rM4-p1 or hM4-p3 and M2III-6 for 16 h at 4°C. Pretreatment with citrate buffer enhanced the labeling with pAb rM4-p1 and hM4-p3 and was essential for the use of mAb M2III-6 in double-labeling experiments. After being washed with PBS, sections were incubated with Alexa 488-conjugated goat anti-rabbit IgG or Alexa 594-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, OR) for 1 h at room temperature, for observation of MRP4 and MRP2, respectively. Finally, sections were washed with PBS and methanol, mounted in Mowiol, and analyzed by immunofluorescence microscopy.
Expression of Human MRP4 in Sf9 Cells
From the construct pGTE-MRP4, an EcoRI fragment containing the full-length MRP4 cDNA was cloned into the EcoRI site of the pFASTBAC1 multiple cloning site, to create pFB1-MRP4. Recombinant baculovirus encoding human MRP4 was generated by using the Bac-To-Bac baculovirus expression system (Life Technologies), and Sf9 cells were infected as described (26). For control experiments, Sf9 cells were infected with recombinant baculovirus encoding the ß-subunit of rat H+/K+-ATPase (HKß).
Preparation of Membrane Vesicles from Kidney and Sf9 Cells
Brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) from rat and human kidney cortex were isolated as described (27). Membrane vesicles from Sf9-MRP4 and control cells were isolated as described (28).
Deglycosylation Study and Immunoblot Analyses
Membrane vesicles were treated with PNGase F according to the instructions provided by the manufacturer. Immunoblotting was performed as described previously (26). Blots were incubated with pAb rM4-p1 (2 µg/ml), pAb hM4-p3 (6 µg/ml), G34 antiserum (diluted 1:1000), or M2III-6 (diluted 1:10) according to the same protocol. Densitometric analyses were performed by using Image Pro Plus 3.0 (Media Cybernetics, Silver Spring, MD).
Transport Studies in Membrane Vesicles
Freshly prepared membrane vesicles (150 µg protein-equivalents) from Sf9 cells expressing MRP4 or HKß were prewarmed at 37°C for 1 min and added to prewarmed transport buffer [10 mM MgCl2, 40 mM 3-(N-morpholino)propanesulfonic acid-Tris, pH 7.0, 50 mM KCl] supplemented with an ATP-regenerating mixture (4 mM ATP, 10 mM creatine phosphate, and 100 µg/ml creatine kinase) and a 3H-labeled compound (28). At indicated times, samples were withdrawn from the reaction mixture, diluted in cold transport buffer, and filtered through NC45 filters. Samples from [3H]adefovir uptake studies were filtered through ME25 filters. After being washed with 5 ml of transport buffer, filters were dissolved in liquid scintillation fluid for determination of bound radioactivity. In control experiments, ATP was replaced by the nonhydrolyzable analog ß,
-methylene-ATP (AMP-PCP). Net ATP-dependent transport was calculated by subtracting values measured in the presence of AMP-PCP from those measured in the presence of ATP.
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MTX is eliminated from the body largely via the kidneys, and it is a substrate for MRP2 (28). We investigated whether it is also transported by MRP4. In the presence of ATP, uptake of 0.1 µM [3H]MTX into Sf9-MRP4 membrane vesicles was linear up to 15 min (Figure 4A). The net ATP-dependent [3H]MTX uptake rate was approximately 10-fold higher than that for Sf9-HKß membrane vesicles. MRP4-mediated, ATP-dependent [3H]MTX uptake increased with increasing MTX concentrations. Fitting of the recorded values to the Michaelis-Menten equation yielded a Vmax value of 430 ± 30 pmol/mg per min and a Km value of 1.3 ± 0.2 mM (Figure 4B). Concentration-dependent inhibition of ATP-dependent [3H]MTX transport by MRP4 was observed for DNP-SG and NAc-DNP-Cys, both with IC50 values of approximately 10 µM (Table 2). MRP4 was not inhibited by LTC4 (Table 2), and Sf9-MRP4 membrane vesicles did not exhibit ATP-dependent uptake of [3H]LTC4 with concentrations of either 5 nM or 1 µM. MRP4 was also inhibited by the glucuronide conjugates -naphthyl-ß-D-glucuronide (IC50 < 100 µM), p-nitrophenyl-ß-D-glucuronide (IC50 < 1000 µM), and E217ßG (Table 2). Sf9-MRP4 membrane vesicles exhibited time- and ATP-dependent uptake of [3H]E217ßG at a concentration of 500 µM (17.6 mCi/mmol), but uptake was slow (Figure 5). At a tracer concentration of 0.2 µM, we did not observe MRP4-mediated, ATP-dependent [3H]E217ßG transport, although, under identical conditions, uptake was observed in membrane vesicles from Sf9 cells expressing human MRP2 (data not shown).
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Discussion |
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The localization of MRP4 and its ability to transport cAMP are in agreement with the results of previous functional studies that indicated the release of cAMP from renal proximal tubule cells upon stimulation by parathyroid hormone, leading to intracellular cAMP levels of at least 100 µM (9). Urinary cAMP has been suggested to originate from plasma cAMP by uptake via basolateral organic anion transporter 1 (OAT1) (1,30). However, plasma cAMP concentrations are in the nanomolar range (31), which may be too low for transport by OAT1; furthermore, infused [3H]cAMP is almost completely filtered by the glomeruli (1). Excreted cAMP has been demonstrated to be directly involved in the inhibition of phosphate reabsorption, after degradation to adenosine and subsequent uptake into the proximal tubules (3). Dipyridamole inhibits adenosine uptake and can be used to protect patients from renal phosphate leakage (4). Our results indicate that dipyridamole may have dual effects, i.e., reduction of adenosine uptake and also inhibition of cAMP release via MRP4, reducing urinary adenosine availability. The source of urinary cGMP has been localized primarily to the glomeruli (6). However, modulation of sodium reabsorption by cGMP in proximal tubules has also been reported, as well as the role of extracellular cGMP as a modulator of sodium transport in LLC-PK1 cells (7,8). The presence of MRP4 in proximal tubules, combined with the observed substrate specificity, suggests that this part of the nephron also contributes to urinary cGMP. Whereas a high but physiologic concentration of [3H]cAMP (100 µM) was used to demonstrate transport by MRP4, a much lower concentration of [3H]cGMP (1 µM) was required. These results are in line with previous observations indicating that intracellular levels of cGMP are approximately 20-fold lower than cAMP levels and that efflux of cGMP from cells is significantly more efficient, compared with that of cAMP (31,32). High concentrations of cAMP were needed to inhibit ATP-dependent [3H]cGMP transport by MRP4 (this study), similar to findings for MRP5 (although with a lower IC50 value) (15). During the final preparation of this manuscript, a study by Chen et al. (33), in which these cyclic nucleotides were identified as MRP4 substrates, was reported. Further studies with polarized monolayers of proximal tubule cells will be required to confirm that MRP4 is the renal proximal tubule efflux pump for cAMP and cGMP.
MRP4 and MRP5 have recently been associated with cellular resistance against adefovir (17,18). Furthermore, adefovir and cidofovir accumulate in proximal tubule cells, causing nephrotoxicity (19). The proximal tubule basolateral uptake carrier OAT1 was recently demonstrated to transport both adefovir and cidofovir (34). Pharmacologic studies with human subjects have suggested that the apical efflux pump, rather than the uptake carrier, represents the probenecid-sensitive, rate-limiting step in the urinary excretion of these compounds (19). Those data prompted us to speculate that MRP4 is the renal efflux pump for adefovir and cidofovir. However, those compounds did not inhibit MRP4-mediated transport, and we did not observe any evidence of ATP-dependent uptake of [3H]adefovir in membrane vesicles from MRP4-expressing Sf9 cells. It therefore remains unclear how MRP4 extrudes xenobiotic monophosphate nucleosides from the cell. Adefovir and cidofovir are monophosphates and are metabolized into di- and triphosphate (the active form) derivatives (35). HPLC analysis has suggested that MRP4 transports only the unmetabolized derivative and that both adefovir and cidofovir are excreted unchanged into the urine (17,19). However, the possibility that the di- or triphosphate metabolite is the actual substrate, which may be difficult to detect because of rapid degradation by extracellular enzymes, cannot be excluded. In the case of cidofovir, a cyclic metabolite has been identified in urine (36). With MRP4 being an efflux transporter for endogenous cyclic nucleosides, the cyclic metabolite, rather than cidofovir itself, may be transported. However, MRP4 and MRP5 may require a cofactor that is as yet unknown. In a similar manner, both MRP1 and MRP2 require GSH for ATP-dependent transport of the anticancer agents vincristine, vinblastine, and daunorubicin (29,37). Our results at least do not support a role for GSH in stimulating drug transport by MRP4.
Rats deficient in functional Mrp2 do not display clear differences in the renal excretion of many organic anions, compared with wild-type rats (14). Furthermore, transport studies with killifish renal proximal tubules have provided evidence for organic anion transporters in addition to MRP2 (38,39). We demonstrate that the substrate specificity of MRP4 overlaps with that of MRP2. The MRP2 substrates E217ßG and MTX are both transported by MRP4, and the affinity of MRP4 for MTX (Km of approximately 1 mM) is in the same range as that documented for MRP2 (28). However, E217ßG, a high-affinity MRP2 substrate (Km of approximately 7 µM), seems to be a low-affinity substrate for MRP4, because a concentration of 500 µM was required for detection of [3H]E217ßG transport by MRP4 and 100 µM E217ßG inhibited MRP4-mediated [3H]MTX transport by only 25%. On the basis of the results of inhibitor studies, we propose that -naphthyl-ß-D-glucuronide, p-nitrophenyl-ß-D-glucuronide, DNP-SG, and NAc-DNP-Cys are substrates for MRP4. Of interest, the excretion of -naphthyl-ß-D-glucuronide from TR- rat kidneys was found not to be different from that from wild-type kidneys (40). In rat kidneys, three additional organic anion transporters, i.e., organic anion transporter polypeptide 1 (Oatp1), kidney-specific organic anion-transporter 1 (Oat-k1), and sodium/phosphate cotransporter type 2 (Npt1), are expressed at the proximal tubule brush border membrane, with substrate specificities overlapping those of MRP2 and MRP4 (14,41). A human ortholog has been identified for rat Npt1; however, human counterparts of rat Oatp1 and Oat-k1 do not seem to exist (41,42). Whether rat Mrp4 in TR- rats and human MRP4 in patients with Dubin-Johnson syndrome (both lacking functional MRP2) (12) can represent an alternative pathway for the renal excretion of organic anions remains to be established and may even involve differences between species.
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) or ß,
). Net ATP-dependent uptake of [3H]cAMP (C) or [3H]cGMP (D) into membrane vesicles from Sf9 cells expressing MRP4 (
) or HKß (
) was determined. Data represent the mean ± SEM of three determinations.
