1Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75390; 2Veterans Administration Medical Center, Dallas 75216; and 3Department of Physiology, University of Texas Medical Branch, Galveston, Texas 77555
Submitted 12 February 2003 ; accepted in final form 11 February 2004
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ABSTRACT |
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citrate; acid base; nephrocalcinosis; nephrolithiasis; opossum kidney cells
Substantial evidence supports the idea that this apical membrane 3Na+-citrate2 cotransporter is encoded by the Na-dicarboxylate cotransporter-1 (NaDC-1) (14). NaDC-1 expressed in Xenopus oocytes exhibits many characteristics of the apical membrane 3Na+-citrate2 transporter, including Na-coupled electrogenic transport, specificity for di- and tricarboxylates, pH-dependent citrate transport, and pH-independent succinate transport (14). NaDC-1 protein has been localized to the apical membrane of the proximal tubule (2). Chronic metabolic acidosis, which is known to increase proximal tubule citrate absorption as well as the activity of the apical membrane 3Na+-citrate2 cotransporter, also increases renal cortical NaDC-1 mRNA and apical membrane protein abundance (2, 5, 7).
OKP cells are an opossum kidney cell line that possesses many characteristics of the renal proximal tubule. These cells have proved extremely useful in studying the mechanisms of regulation of proximal tubule H+, Na+, and phosphate transport. Recently, OK cells, another opossum kidney cell line, were demonstrated to exhibit Na-dependent citrate transport with many of the characteristics of NaDC-1 (6). The purpose of our study was to clone the OKP NaDC-1 (oNaDC-1) cDNA and examine its characteristics. Results demonstrate that OKP cells express a 2.4-kb NaDC-1 mRNA. Injection of oNaDC-1 cRNA into Xenopus oocytes leads to expression of a Na-dicarboxylate cotransporter with functional characteristics similar to those of NaDC-1. When OKP cells are exposed to acidic media, there is no change in oNaDC-1 mRNA abundance at 12 and 24 h. When the cells are transfected with a green fluorescent protein (GFP)-oNaDC-1 construct, media acidification increases oNaDC-1 activity, demonstrating a nontranscriptionally regulated, chronic adaptation in the cotransporter.
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METHODS |
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OKP cells were passaged in high-glucose DMEM with 10% fetal bovine serum (FBS). For experimentation, wild-type (untransfected) cells were grown to confluence, rendered quiescent by serum removal for 48 h, and harvested in guanidinium thiocyanate, and then total cellular RNA was extracted with phenol-chloroform-ethanol precipitate as previously described (2). Poly(A)+ RNA was isolated by oligo(dT) cellulose chromatography. Poly(A)+ RNA (5 µg) was size-fractionated by agarose formaldehyde gel electrophoresis and transferred to nylon membranes. Radiolabeled probes were synthesized from the appropriate cDNA using the random hexamer method. Filters were prehybridized in 5x SSC (0.75 M NaCl and 0.075 M Na-citrate, pH 7.0), 5x Denhardt's solution, 0.1 mg/ml salmon sperm DNA, and 50% formamide for 2 h at 42°C; hybridized in the same solution containing radiolabeled probe at 42°C overnight; and washed three times in 2x SSC containing 0.1% sodium dodecyl sulfate (SDS) at room temperature for 20 min at 55°C. Filters were then exposed to film overnight at 70°C, and labeling was quantitated by densitometry.
Reverse Transcription and Degenerate Polymerase Chain Reaction
First-strand cDNA was synthesized from 5 µg of poly(A)+ RNA using Moloney murine leukemia virus reverse transcriptase (SuperScript II; GIBCO BRL, Grand Island, NY) with oligo(dT) primers. DNA was then amplified by PCR using 0.2 mM dNTP, 2 mM Mg, and 2 mM each of degenerate primers: forward primer, GGCATTGCCACGCTGACTGGNACN(A/G)CNCCNAA; reverse primer, GGNTA(A/G/A)(G/A)ANCCNAGGTACCGGGTCCGGTAGAC.
Primers were designed on the basis of the conserved amino acid sequences in NaDC-1 using the consensus-degenerate hybrid oligonucleotide primers (CODEHOP) method (19). PCR was initiated by incubation at 94°C for 2 min, followed by 28 cycles of 94°C for 1 min, 62°C for 1 min, and 72°C for 1 min, with annealing temperature decreasing by 1°C every four cycles. Because no band was seen after agarose gel electrophoresis, a secondary PCR was performed as described above using the product of the first PCR as the template. In this PCR, the annealing temperature was 57°C, which again was lowered by 1°C every four cycles. Agarose gel electrophoresis of this second PCR product showed a band of the expected size. This PCR product was subcloned into a TA cloning vector (Invitrogen, Carlsbad, CA) and sequenced.
5' Rapid Amplification of cDNA Ends
First-strand cDNA was synthesized from 1 µg of poly(A)+ RNA using SuperScript II with a gene-specific primer, GSP5-1 (AGGATCTGGAGCCATATCCA), designed from the sequence obtained by degenerate PCR. Template RNA was digested with 1 µl of RNase Mix (GIBCO BRL); cDNA was purified using a GlassMax DNA isolation spin cartridge (GIBCO BRL); and poly(dC) was added to the 5' end of the cDNA using dCTP and terminal deoxynucleotidyl transferase (TdT). dC-tailed cDNA was PCR-amplified using an abridged anchor primer (GIBCO BRL), 5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3, and a nested gene-specific primer, GSP5-2 (CCAAACCAGGAAGCAAAGTT), designed from the previous PCR product, with 30 cycles of 94°C for 1 min, 54°C for 1 min, and 72°C for 1 min. Because no bands were seen on agarose gel electrophoresis, a second PCR was performed with the PCR product using the same primers, resulting in five bands. To determine which was the band of interest, Southern blot analysis was performed using a full-length rabbit NaDC-1 cDNA as the probe. One band was positive, which was gel-extracted, cloned into a TA cloning vector, and sequenced.
3' Rapid Amplification of cDNA Ends
First-strand cDNA was synthesized from 1 µg of poly(A)+ RNA using SuperScript II with an adapter primer, 5'-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-3'. RNA was digested with RNase Mix, and PCR was performed using Taq polymerase with an abridged universal amplification primer (AUAP), 5'-GGCCACGCGTCGACTAGTAC-3', and a gene-specific primer, GSP3-3 (CAGCCTACCAGGTTATCCAGACTG), designed from the sequence of the initial PCR product. Nested PCR was performed with AUAP and GSP3-4 (CTTTTCTAATGAGGATGGGGAAA). The PCR product was size-fractionated by gel electrophoresis and transferred to a nylon membrane. Southern blot analysis was performed using full-length rabbit NaDC-1 cDNA as the probe. The positive band was gel-extracted, cloned into a TA cloning vector, and sequenced.
Full-Length cDNA
The products from degenerate PCR, 5' rapid amplification of cDNA ends (RACE), and 3' RACE were assembled to yield the 2.4-kb product of oNaDC-1.
Xenopus Oocytes
Stages V and VI oocytes from Xenopus laevis were dissected, treated with collagenase, and cultured as described previously (13). The oNaDC-1 cDNA in pSP64LA plasmid, containing the Xenopus -globin untranslated regions, was used as a template for cRNA synthesis (13). Plasmids were linearized with XbaI, and in vitro cRNA transcription was performed using the SP6 mMessage mMachine kit (Ambion, Austin, TX). cRNA was resuspended in water to a final concentration of 2040 ng/µl, and each oocyte was injected with 50 nl (total injection of 12 ng/oocyte).
Transport Experiments
Oocytes. Transport of [3H]succinate (DuPont NEN, Boston, MA) and [14C]citrate (Moravek Biochemicals, Brea, CA) was measured 23 days after oocyte injection as described (13). Na and choline buffers were as follows (in mM): 100 NaCl or choline-Cl, 2 KCl, 1 MgCl2, 1 CaCl2, and 10 HEPES-Tris, pH 7.5. The oocytes were rinsed briefly with choline buffer to remove Na and serum. Transport was initiated by replacement of the choline rinse with 0.4 ml of the appropriate transport buffer, as described in the figure legends. Transport was stopped by addition of 4 ml of ice-cold choline buffer, followed by removal of extracellular radioactivity with three additional washes in cold choline buffer. Individual oocytes were transferred to scintillation vials and dissolved in 0.5 ml of 10% SDS. Scintillation cocktail was added, and radioactivity was counted. Counts in uninjected control oocytes were subtracted from counts in cRNA-injected oocytes. Data are presented as means ± SE, except for kinetic data, in which the error represents the standard error of the curve fit. Statistical analysis was performed with the SigmaStat software program (Jandel Scientific, Chicago, IL).
Control studies were performed to compare Na-dependent uptake in uninjected vs. cRNA-injected oocytes. Uptake in uninjected oocytes ranged from 10 to 16 pmol·oocyte1·h1, and in cRNA-injected oocytes, it ranged from 437 to 1,084 pmol·oocyte1·h1. There were no differences between water and uninjected oocytes.
OKP cells. OKP cells were passaged in high-glucose DMEM with 10% FBS. For experimentation, cells were plated on six-well culture plates, grown to 5070% confluence, and then transfected for 5 h with 1 µg of DNA [pEGFP-C3 vector (Clontech Laboratories, Palo Alto, CA) with or without full-length oNaDC-1 inserted into the multiple cloning site at the EcoR I restriction site] using the Lipofectamine Plus kit (Invitrogen). Serum (10% FBS) was then added to the wells for 19 h, after which the cells were 95100% confluent. The cells were rendered quiescent within 24 h by the removal of serum and then were exposed to control (pH 7.4) or acidic (pH 6.8) media for 6 h. [14C]citrate (DuPont NEN) uptake was measured at pH 7.4 for 5 min in the presence (136 mM) or absence of NaCl (NaCl replaced with 136 mM choline-Cl) using a solution containing, in addition to either NaCl or choline-Cl, 1.2 mM CaCl2, 1 mM MgSO4, 3 mM KCl, 25 mM HEPES, 1.39 mM citric acid, and 0.5 µCi/ml [14C]citrate. The uptake reaction was stopped, and the remaining extracellular [14C]citrate was removed by washing the cells three times with an ice-cold 0.1 M MgCl2 solution. After the last wash, the cells were lysed in 300 µl of 0.1 N NaOH and scraped with a rubber policeman. A 10-µl aliquot was used to measure protein content by Bradford assay (Bio-Rad Laboratories, Hercules, CA), the remaining 290 µl were put into 5 ml of scintillation fluid, and [14C]citrate was counted with a Beckman scintillation counter (model LS 3801; Fullerton, CA). Uptake is reported as picomoles of citrate per 5 min per milligram of protein.
Statistics
Statistical significance was determined by performing the appropriate t-test. Differences between means were considered significant at P 0.05.
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RESULTS |
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To determine whether OKP cells express NaDC-1 mRNA, Northern blot analysis using OKP poly(A)+ RNA was performed using rabbit NaDC-1 cDNA as the probe. A 2.4-kb band the size of NaDC-1 mRNA in other species was visualized (data not shown).
We next attempted to clone the oNaDC-1 using degenerate PCR. Degenerate PCR primers were designed on the basis of amino acid sequences conserved across rat, rabbit, and human NaDC-1 proteins. A specific PCR product was not obtained, probably because of marked codon degeneracy and low mRNA abundance. We next tried the CODEHOP method, which uses hybrid primers consisting of a relatively short 3' degenerate core and a 5' nondegenerate consensus sequence. Reducing the length of the 3' core to a minimum decreases the total number of individual primers in the degenerate primer pool. Hybridization of the 3' degenerate core with the target template is stabilized by the 5' nondegenerate consensus sequence, allowing higher annealing temperatures without increasing the degeneracy of the pool. By this method, PCR primers were designed on the basis of amino acid sequences conserved in rat, rabbit, and human NaDC-1, corresponding to amino acids 233246 (GIATLTGTAPN) and 485495 (PILASMAQAIC) of the human sequence. The PCR product obtained using 5 µg of OKP poly(A)+ RNA was 784 bp, consistent with the predicted size. Sequencing of the 784-bp product revealed 80% sequence identity to human NaDC-1. The PCR clone hybridized to a 2.4-kb transcript in OKP poly(A)+ RNA and was able to detect a 2.8-kb transcript in rat kidney cortex total RNA at high stringency.
5' and 3' sequences were then determined by 5' and 3' RACE, respectively, with gene-specific primers designed from the above PCR product. The sequences obtained by 5' RACE and 3' RACE also revealed 80% homology to human NaDC-1. oNaDC-1 mRNA obtained by combining the sequences of the three PCR yielded an oNaDC-1 cDNA of 2,404 bp in length with an open reading frame of 1,818 bp that encoded a protein of 605 amino acid residues (Fig. 1A). oNaDC-1 shows 71, 67, 68, and 72% amino acid identity to human, rabbit, rat, and mouse NaDC-1, respectively (Fig. 1B). The hydropathy plot predicts that oNaDC-1 has 13 transmembrane domains (TMDs; Fig. 1C).
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Anion specificity. The OKP cell Na+-dicarboxylate cotransporter oNaDC-1 transports both succinate and citrate with kinetic values that are similar to those of the rabbit NaDC-1 (16). In a typical experiment (averaging 5 oocytes from a single frog for each data point) (Fig. 2A), the Km for succinate was 150 µM. In three separate experiments, the mean Km was 180 ± 28 µM. The Km for citrate in the single experiment shown in Fig. 2B was 1.7 mM, and the mean Km for three experiments was 1.5 ± 0.3 mM.
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In untransfected cells, we were unable to reliably demonstrate Na-dependent citrate uptake at pH 7.4 or 6.8, probably because of the low level of native NaDC-1 expression (data not shown). Thus, to determine whether media acidification regulates oNaDC-1 activity, cells were transfected with oNaDC-1 as a GFP-NaDC-1 construct, grown to confluence, and rendered quiescent as described in METHODS. Cells were then exposed to either control (pH 7.4) or to acidic (pH 6.8) media for 6 h, and Na-dependent [14C]citrate uptake was measured as described in METHODS. As shown in Fig. 7, media acidification led to a 30% increase in NaDC-1 activity.
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DISCUSSION |
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Chronic metabolic acidosis is associated with nephrolithiasis and nephrocalcinosis, in part because of hypocitraturia (2, 11, 22). In rats, metabolic acidosis has been shown to increase proximal tubule citrate absorption, apical membrane Na+-citrate2 cotransporter activity, and cortical NaDC-1 mRNA and proximal tubule apical membrane protein abundances (2, 5, 7).
To further study the mechanisms responsible for NaDC-1 regulation, it would be extremely useful to possess a tissue culture model. OKP is an opossum kidney cell line that has proved extremely valuable in examining the regulation of proximal tubular hydrogen and phosphate transport. Hering-Smith et al. (6) recently demonstrated that incubation in acidic media causes an increase in Na-coupled citrate uptake in OK cells. To determine whether OKP cells express a Na-dicarboxylate transporter, we performed Northern blot analysis using the rabbit NaDC-1 as a probe. Although a band was visualized, it required 5 µg of poly(A)+ mRNA.
We therefore decided to clone the OKP NaDC-1 cDNA by performing PCR. The OKP NaDC-1 cDNA is a 2,404-bp sequence, has 80% sequence identity to the human NaDC-1 cDNA, and labels a 2.4-kb transcript in OKP mRNA. The open reading frame is 1,818 bp and encodes a protein of 605 amino acids that has 71, 67, 68, and 72% amino acid identity to human, rabbit, rat, and mouse NaDC-1, respectively. The major difference between oNaDC-1 and NaDC-1 of the other four species is that oNaDC-1 has nine additional amino acids (residues 204212), which are predicted to be in the fourth hydrophilic loop (see below).
Depending on the program used, the predicted hydropathy plot for oNaDC-1 contains 13 (3 programs), 14 (1 program), or 15 (1 program) TMDs. All five programs predicted the first TMD and nine COOH-terminal TMDs. The variation in the predicted topologies was in the size and/or number of domains between amino acids 40 and 156. Figure 1C is based on the programs that predicted 13 TMDs.
For comparison, human, rabbit, and mouse NaDC-1 are all predicted to have at least 11 TMDs (13, 14, 18). All four species vary in the sizes of the individual hydrophilic domains. The major difference between the oNaDC-1 and human, rabbit, or mouse predicted hydropathy plots is that there is essentially no hydrophilic loop between TMD 10 and 11 and TMD 11 and 12 in oNaDC-1, whereas the hydrophilic domains in the other three species are larger.
Hydropathy plots are only predictions. Pajor and colleagues (15, 24, 25) used cysteine mutagenesis, chemical modifications, antibodies, and epitope tagging to verify the locations of specific amino acids. They confirmed that the NH2 terminus is intracellular, which is predicted by all hydropathy plots, and that amino acids 164233 of rabbit NaDC-1 are part of hydrophilic loop 4 (cytoplasmic). These amino acids correspond to amino acids 164243 in oNaDC-1, with amino acids 164233 predicted to be part of hydrophilic loop 4 (cytoplasmic) and amino acids 234243 on the cytoplasmic side of TMD 5. This is in the region of the additional amino acids in oNaDC-1 (see above; Ref. 3). Amino acids R349 and D373 of rabbit NaDC-1, both involved in substrate affinity and cation binding, are near the extracellular side of TMD 7 and 8, respectively. These amino acids correspond to R360 and D384 in oNaDC-1, which are also predicted to be near the extracellular side of TMD 7 and 8, respectively. These arginine and aspartic acid residues are conserved in all five species. Amino acid S372 of rabbit NaDC-1, also conserved in all five species, is near the extracellular side of TMD 8. It corresponds to amino acid 383 in oNaDC-1, also predicted to be near the extracellular edge of TMD 8. Rabbit NaDC-1 amino acids F473, T474, E475, S478, N479, A480, A481, and T482 are predicted to be on the extracellular side of TMD 9. All except A481 are conserved in all five species. Rabbit A481 is a valine residue in the other four species. These eight amino acids correspond to amino acids F483, T484, E485, S488, N489, V490, A491, and T492 in oNaDC-1. In oNaDC-1, however, these amino acids are predicted to be on the cytoplasmic side of TMD 10 (F483, T484, and E485) and 11 (S488, N489, V490, A491, and T492). As noted above, the predicted hydropathy plot for oNaDC-1 in this region (Fig. 1C) does not predict an intracellular loop between TMD 10 and 11 or an extracellular loop between TMD 11 and 12. Because this whole region of the protein is highly conserved among the five species, it is possible that the oNaDC-1 predicted hydropathy plot is in error.
To confirm that this transcript encodes an NaDC-1 analog, protein was expressed and studied functionally in Xenopus oocytes. The transporter has a high affinity for succinate and a lower affinity for citrate and is Na selective. A number of dicarboxylic acids inhibited succinate transport, and Li could substitute for Na, but poorly. Na affinity is 22 mM with a stoichiometry of 3 Na:1 citrate. Most important, transport of citrate is pH dependent, whereas succinate is not. This pH dependence is characteristic of NaDC-1 and the proximal tubule apical membrane Na-dicarboxylate transporter and likely is due to the fact that the transporter carries only dicarboxylates. All of the above functional characteristics confirm that this transcript encodes oNaDC-1.
The oNaDC-1 Km for citrate is similar to that of other NaDC-1 orthologs, which provides additional support for its being the oNaDC-1 ortholog. This Km is higher than the luminal citrate concentration. However, Km is calculated on the basis of rates analyzed as a function of total luminal citrate concentration, whereas the transported substrate is citrate2. Along the proximal tubule, pH drops from 7.4 to 6.5, with most of the decrease occurring in the early proximal tubule. This pH drop would raise the concentration of citrate2 and thus lower the apparent Km 10-fold, bringing it into the range of plasma citrate concentration (0.10.2 mM).
Ingestion of acid in vivo leads to enhanced NaDC-1 activity, mRNA abundance, and protein abundance (2, 7). OKP cells provide a model for studying the mechanisms that mediate acid-induced NaDC-1 regulation. We (1) previously showed that exposing OKP cells to acidic media for 24 h increased NHE-3 mRNA abundance. However, incubation at pH 6.8 for 12 or 24 h had no effect on endogenously expressed oNaDC-1 mRNA. There are a number of possible explanations for the apparent discrepancy between this observation and our results in vivo. First, regulation of NaDC-1 mRNA abundance in OKP cells may require more than 24 h, although in rats, NaDC-1 mRNA was upregulated 16 h after gavage and 24 h after NH4Cl was added to the drinking water. Second, it is possible that regulation requires participation of a second cell type present in vivo that secretes a hormone or a paracrine factor. Third, this form of regulation may be species specific. Given the similarities between rat, rabbit, mouse, and opossum cells in other forms of pH regulation, however, the latter explanation seems unlikely.
We also examined the effect of pH on Na-coupled citrate transport in OKP cells. In the past, we have been unable to demonstrate Na-dependent citrate uptake in wild-type cells in a manner that we thought was convincing; this remained so in the present study. One possible reason for this is the low level of NaDC-1 expression in our cells. Even in the kidney proximal tubule, rates of citrate transport are significantly lower than those of other Na-dependent processes, such as H+, glucose, or phosphate transport. In addition, cultured cells tend to have poorly developed brush border membranes and thus would be expected to have even lower expression levels of brush border membrane proteins. We therefore decided to overexpress oNaDC-1 in OKP cells and measure transport. In cells transiently transfected with a GFP-oNaDC-1 construct, Na-dependent citrate uptake was clearly evident and upregulated by acid incubation. These results demonstrate that acid stimulates oNaDC-1 by a posttranscriptional regulatory mechanism, but an additional transcriptional regulatory component cannot be ruled out. Possible mechanisms of posttranscriptional regulation include regulation of mRNA stability, protein synthesis or degradation, trafficking to the apical membrane, and posttranslational modification. The OKP cell is a good cell line in which to study such regulation because it has the same signaling mechanisms as the intact proximal tubule. It is unfortunate that NaDC-1 expression is low, making it necessary to transfect the cells with NaDC-1 to study regulation.
Our finding of acid regulation is slightly different from what Hamm and colleagues (6, 8) found. In their studies using OK cells, acute decreases in pH had no effect on citrate transport, whereas acute increases raised transporter activity. As these authors (8) noted, this response to changes in pH is not typical of the proximal tubule apical membrane citrate transporter. In further studies (6), they did find that both acute and chronic (48 h) decreases in media pH increased transporter activity. However, these studies were conducted with total extracellular calcium concentrations of <200 µM. At higher extracellular calcium concentrations, Hamm and colleagues found that transporter activity was significantly lower and did not demonstrate competition between citrate and succinate, another characteristic of the apical membrane transporter. Our functional as well as acid regulation studies, showing competition between citrate and succinate and chronic regulation by pH, were performed in the presence of 1.0 or 1.2 mM Ca, respectively. The reason for this difference in the effect of Ca on transporter function and regulation is not apparent. Our results suggest that the transporter cloned represents the opossum NaDC-1. The transporter studied by Hamm and coworkers may represent a different NaDC isoform.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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