Departments of Medicine and Physiology and Biophysics, University of Texas Medical Branch, Galveston, Texas 77555
Submitted 7 November 2003 ; accepted in final form 8 March 2004
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ABSTRACT |
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K+-HCO3 cotransport; Na+/H+ exchange; Cl/HCO3 exchange; acid-base transport; KCC
As in other nephron segments, transepithelial HCO3 absorption in the thick ascending limb is accomplished as the combined result of secretion of protons from cell to tubule lumen across the apical membrane and transport of base equivalents (HCO3) from cell to interstitial fluid across the basolateral membrane (2, 18). In the MTAL of the rat, virtually all of the H+ secretion necessary for HCO3 absorption is mediated by the apical membrane Na+/H+ exchanger NHE3 (3, 5, 21, 27, 41). Neither an H+-ATPase nor the Na+/H+ exchanger isoform NHE2 appears to contribute significantly to H+ secretion and HCO3 absorption in this segment (18, 21, 41). However, whether other HCO3/OH/H+ transport pathways are present in the apical membrane of the MTAL and influence HCO3 absorption is unclear. Studies using membrane vesicles prepared from digested tubule fragments suggested that the apical membrane of the rat MTAL contains a Cl/HCO3 exchanger (13) and a K+/H+ exchanger (4). Apical Cl/HCO3 exchange was also postulated in the mouse cortical thick ascending limb based on the observations that transepithelial Cl absorption was enhanced by HCO3/CO2 and inhibited by luminal addition of the disulfonic stilbene SITS (14). However, there have been no direct studies of these transport pathways in the apical membrane of intact thick ascending limbs. Also, interpretation of the vesicle studies is complicated due to significant contamination of the apical membrane preparation with basolateral membrane vesicles (4, 13, 29).
The aims of the present study were 1) to determine whether HCO3/OH/H+ transport pathways in addition to Na+/H+ exchange are present in the apical membrane of the rat MTAL and 2) to define the role of these pathways in transepithelial HCO3 absorption. The results show that the apical membrane contains a K+-dependent HCO3 transport pathway that opposes HCO3 absorption. No evidence for apical Cl/HCO3 exchange or apical K+/H+ exchange was found.
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METHODS |
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MTALs from male Sprague-Dawley rats (6080 g; Taconic, Germantown, NY) were perfused in vitro as previously described (16, 21, 38, 39). The tubules were dissected from the inner stripe of the outer medulla, transferred to a bath chamber on the stage of an inverted microscope, and mounted on micropipettes for perfusion at 37°C. The composition of the perfusion and bath solutions for specific protocols is given below.
Measurement of Net HCO3 Absorption
To measure transepithelial HCO3 absorption rates, tubules were perfused and bathed in control solution that contained (in mM) 146 Na+, 4 K+, 122 Cl, 25 HCO3, 2.0 Ca2+, 1.5 Mg2+, 2.0 phosphate, 1.2 SO42, 1.0 citrate, 2.0 lactate, and 5.5 glucose (equilibrated with 95% O2-5% CO2; pH 7.45 at 37°C). Bath solutions also contained 0.2% fatty acid-free bovine albumin. Experimental agents were added to the luminal solution as described in results. DIDS and its H2 analog (H2DIDS) were obtained from Molecular Probes (Eugene, OR) and dissolved directly into perfusion solutions.
The protocol for study of transepithelial HCO3 absorption was as described (16, 21). The tubules were equilibrated for 2030 min at 37°C in the initial perfusion and bath solutions, and the luminal flow rate (normalized per unit tubule length) was adjusted to 1.61.9 nl·min1·mm1. One to three 10-min tubule fluid samples were then collected for each period (initial, experimental, and recovery). The tubules were allowed to reequilibrate for 1015 min after an experimental agent was added to or removed from the luminal solution. The absolute rate of HCO3 absorption (JHCO3; pmol·min1·mm1) was calculated from the luminal flow rate and the difference between total CO2 concentrations measured in perfused and collected fluids (16). Single tubule values are presented in the figures. Control measurements made at the beginning and end of an experiment (initial and recovery periods) were combined to obtain a mean control transport rate for each tubule. The individual tubule values were averaged to obtain the group means ± SE presented in the text (n = number of tubules).
Measurement of Intracellular pH and Calculation of Equivalent Net Base Flux
Intracellular pH (pHi) was measured in isolated, perfused MTALs by use of the pH-sensitive dye BCECF and a computer-controlled spectrofluorometer (CM-X, SPEX Industries) coupled to the perfusion apparatus, as described elsewhere (39, 40). The tubules were perfused in the same manner used for HCO3 transport experiments except that the lumen and bath solutions were delivered via rapid flow systems that permit complete exchange of the solutions in less than 2 s (39). Intracellular dye was excited alternately at 500- and 440-nm wavelengths, and emission was monitored at 530 nm using a photon counter. Intracellular dye was calibrated using high K+-nigericin standards at the end of each experiment to convert fluorescence excitation ratios (F500/F440) to pHi values, as previously described (39, 40). Initial rates of net base flux (pmol·min1·mm1) were calculated as dpHi/dt x x V, where dpHi/dt is the initial slope of the pHi record vs. time (pH U/min) measured over the first 4 s following an experimental maneuver,
is the intracellular buffering power (mmol·l1·pH U1, see below), and V is cell volume per millimeter of tubule length (nl/mm). For experiments using HCO3/CO2-free solutions, intrinsic buffering power (
i) was measured as a function of pHi as described (40). For experiments with HCO3/CO2-containing solutions, total buffering power (
T) was the sum of
i and the HCO3/CO2-buffering power (computed as 2.3 [HCO3]i) (38). V was determined from inner and outer tubule diameters as described (38, 40).
Two main solutions were used for pHi experiments. To examine K+-dependent transport pathways, tubules were perfused and bathed in K+ solution that contained (in mM) 120 K+, 25 N-methyl-D-glucammonium (NMDG+), 137 gluconate, 25 HCO3, 7 Ca2+, and 1.5 Mg2+. To examine Na+- and Cl-dependent pathways, tubules were perfused and bathed in Na+- and Cl-free solution that contained (in mM) 72 K+, 75 NMDG+, 139 gluconate, 25 HCO3, 7 Ca2+, and 1.5 Mg2+. All HCO3-containing solutions were gassed with 95% O2-5% CO2. In some experiments, variants of these solutions were prepared that were buffered with 25 mM HEPES and were nominally HCO3/CO2 free (Figs. 1B and 2D). The HCO3-free solutions were gassed with 100% O2 and titrated to pH 7.4. Tubules were equilibrated in HEPES-buffered solutions for 40 min before measurements were taken. In individual protocols, NMDG+ replaced K+ or Na+, and gluconate replaced Cl (see RESULTS). The total calcium concentration of gluconate solutions was adjusted so that Ca2+ activity matched that of Cl-containing solutions (40). All bath solutions also contained 5 mM glucose. Furosemide or EIPA was present in the luminal solution in some experiments (see RESULTS). H2DIDS was used in pHi experiments because of its insensitivity to light. Quinidine and glybenclamide were prepared as stock solutions in ethanol and diluted into luminal solutions to final concentrations given in RESULTS.
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Results are presented as means ± SE. Differences between means were evaluated using the paired Student's t-test or analysis of variance with the Newman-Keuls multiple range test, as appropriate. P < 0.05 was considered statistically significant.
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RESULTS |
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K+-dependent equivalent net base flux. To determine whether the MTAL expresses an apical K+-dependent acid-base transport pathway, pHi was monitored in response to luminal K+ removal and readdition. Tubules were perfused and bathed in 120 mM K+ solution that contained 25 mM HCO3, 5% CO2, and no Na+ or Cl (see METHODS). The luminal solutions also contained 104 M furosemide. Initial pHi was 7.46 ± 0.02 (n = 4). Removal of luminal K+ (K+ replaced with NMDG+) induced a rapid decrease in pHi (Fig. 1A). The initial rate of net base efflux was 23.2 ± 2.7 pmol·min1·mm1 (n = 4; Fig. 1E). The cell effect was reversed when K+ was readded to the tubule lumen (Fig. 1A). The cell acidification induced by lumen K+ removal was unaffected by the presence of 50 µM lumen EIPA (initial rate of net base efflux = 21.5 ± 1.4 pmol·min1·mm1, n = 3; data not shown).
Dependence on HCO3/CO2. To test whether the K+-induced equivalent net base flux was dependent on the presence of HCO3/CO2, the K+ removal protocol in Fig. 1A was repeated using HEPES-buffered solutions that were nominally HCO3/CO2 free. Initial pHi in the HCO3-free solution (7.42 ± 0.02; n = 5) was similar to that in HCO3-containing solution (Fig. 1, A and B). In the absence of HCO3/CO2, the pHi decrease in response to lumen K+ removal was nearly eliminated and the initial rate of K+-dependent net base efflux was reduced by 85% (Fig. 1, B and E). These results argue against the presence of an apical K+/H+ exchanger (or K+-OH cotransporter) and are consistent with the coupled transport of K+ and HCO3 across the apical membrane.
Effect of H2DIDS. In the presence of 25 mM HCO3 and 5% CO2, 1 mM luminal H2DIDS inhibited the pHi decrease induced by luminal K+ removal, reducing the initial rate of net base efflux by 60% (Fig. 1, C and E). These results are consistent with the previous finding that DIDS inhibited a HCO3-dependent K+ flux in rat MTAL suspensions (28) (see DISCUSSION).
Effects of K+ channel inhibitors. To test for the possible involvement of electrically coupled K+- and HCO3-conductive pathways, we examined the effects of the K+ channel blockers quinidine and glybenclamide. These inhibitors block the activity of both the intermediate- and low-conductance K+ channels in the apical membrane of the MTAL (6, 37) and prevent changes in apical membrane voltage induced by changing the luminal K+ concentration (6, 22, 23). In MTAL studied in the 120 mM K+ solution containing HCO3/CO2, the pHi decrease and the initial rate of net base efflux induced by lumen K+ removal were unaffected by the combination of 0.1 mM quinidine and 0.25 mM glybenclamide in the luminal fluid (Fig. 1, D and E). These results argue against the presence of electrically coupled K+ and HCO3 fluxes and identify an apical K+-dependent HCO3 transport pathway that operates independently of apical K+ channels.
Evidence Against Na+- and Cl-Dependent Apical Transport Pathways
To test for the presence of Na+- or Cl-dependent acid-base transport pathways, MTALs were perfused and bathed in Na+- and Cl-free solution (see METHODS), and pHi was monitored in response to luminal Na+ or Cl addition. In Na+ addition experiments or when Cl was added in the presence of Na+, 50 µM EIPA was present in the lumen to block apical Na+/H+ exchange (40, 41). In the presence of 25 mM HCO3 and 5% CO2, addition of either 50 mM Na+ (Na+ replaced NMDG+; Fig. 2A) or 120 mM Cl (Cl replaced gluconate; Fig. 2B) to the lumen had no effect on pHi. Luminal Cl addition also did not affect pHi in the presence of HCO3/CO2 and 145 mM Na+ (Fig. 2C) or in HCO3/CO2-free solution buffered with 25 mM HEPES (Fig. 2D). In Cl addition protocols, results were similar in the absence or presence of 104 M luminal furosemide. The lack of pHi response to lumen Na+ or Cl addition is not the result of a detrimental effect of the experimental conditions on the tubules because 1) removing 72 mM K+ from the lumen using the Na+- and Cl-free solution induces a rapid decrease in pHi with an initial rate of net base efflux (19.5 ± 1.9 pmol·min1·mm1; n = 3) similar to that observed (Fig. 1) using the K+ solution; 2) if the order of experimental conditions in Fig. 2A is reversed so that tubules are begun in the 50 mM Na+ solution, removing lumen Na+ has no effect on pHi (data not shown); and 3) lumen Na+ addition induces a rapid pHi increase in the absence of luminal EIPA using similar solutions (method 2 in Ref. 40). These experiments provide no evidence that Na+-HCO3(OH) cotransport, Cl/HCO3(OH) exchange, or Na+-dependent Cl/HCO3 exchange contributes significantly to the acid-base flux across the apical membrane.
Role in Transepithelial HCO3 Absorption
Effect of luminal DIDS on HCO3 absorption. The results in Fig. 1 are consistent with the presence of a DIDS-sensitive K+-dependent HCO3 transporter in the apical membrane of the MTAL. Under physiological conditions, the cell-to-lumen K+ concentration difference would be expected to drive the coupled transport of K+ and HCO3 from cell to tubule lumen, resulting in luminal HCO3 addition that would detract from net HCO3 absorption [the apical [HCO3] gradient is small compared with the [K+] gradient, based on measurements of pHi (21, 22)]. To test this, we examined the effect on HCO3 absorption of inhibiting apical K+-dependent HCO3 transport with DIDS. In MTAL studied in control solution (see METHODS), adding 1 mM DIDS or 1 mM H2DIDS to the lumen increased HCO3 absorption by 39%, from 10.7 ± 0.7 to 14.9 ± 0.7 pmol·min1·mm1 (P < 0.001; Fig. 3A). A similar increase in HCO3 absorption is observed in the presence of luminal furosemide (Fig. 3B). The stimulation was complete within 1520 min after the addition of DIDS, was stable for up to 60 min, and was reversible. Luminal DIDS also increased HCO3 absorption in tubules in which phosphate, sulfate, citrate, and lactate were omitted from the luminal solution (replaced with Cl) (results not shown). These results are consistent with the presence of a DIDS-inhibitable K+-dependent HCO3 transporter that mediates transfer of HCO3 into the tubule lumen and opposes net HCO3 absorption.
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DISCUSSION |
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Transepithelial absorption of HCO3 by the MTAL depends on proton secretion mediated by apical membrane Na+/H+ exchange (NHE3) (3, 5, 18, 21, 41). The identity and function of other apical acid-base transporters in this segment are poorly defined. In the present study, we found that the MTAL contains an apical K+-dependent base transport pathway that is dependent on HCO3/CO2, inhibited by DIDS, and independent of apical K+ channels. Adding DIDS to the tubule lumen to inhibit this pathway increases net HCO3 absorption and steady-state pHi. These results provide functional evidence for an apical membrane K+-dependent HCO3 transporter that mediates the coupled transfer of K+ and HCO3 from cell to tubule lumen. This transporter functions in parallel with apical membrane Na+/H+ exchange and opposes transepithelial HCO3 absorption.
Evidence for coupled transport of K+ and HCO3 has been presented previously in two systems: suspensions of rat MTAL and squid giant axon. In alkali-loaded MTALs in suspension, a HCO3 efflux pathway was described that was dependent on cell K+, independent of Na+ and Cl, and inhibited by DIDS (28). Also, K+ efflux from the cells was enhanced by a HCO3 gradient (28). In a detailed series of studies examining pHi regulation in the squid giant axon, a pathway for coupled transport of K+ and HCO3 was identified that was best explained by a K+-HCO3 cotransporter (24, 25, 42). In both the MTAL suspensions and squid giant axon, the K+-coupled HCO3 flux was unrelated to membrane voltage and could not be explained by K+, H+, or HCO3 conductances (24, 25, 28). Consistent with these findings, our results show that the K+-induced HCO3 flux in the apical membrane of the MTAL did not differ when the luminal K+ concentration was changed in the absence or presence of K+ channel blockers, conditions associated with marked differences in the apical membrane voltage (6, 22, 23, 37). Thus these studies suggest that the putative K+-HCO3 cotransporter(s) may be electroneutral. K+-HCO3 cotransport in the squid axon was not inhibited by DIDS, suggesting a transport pathway different from that in the MTAL (25). Studies using MTAL suspensions cannot localize transporters to the apical or basolateral membrane. However, it was presumed that K+-coupled HCO3 transport was basolateral where it would mediate HCO3 efflux for HCO3 absorption (28). Our results show, however, that K+-dependent HCO3 transport is apically located, where it detracts from net HCO3 absorption (see below). Whether the basolateral membrane may also contain a K+-dependent HCO3 transport mechanism is unclear (8, 28, 29).
A transport protein that mediates coupled transport of K+ and HCO3 has not been molecularly identified, but some possibilities can be considered. First, K+ could replace Na+ on a Na+-HCO3 cotransporter. This possibility is unlikely because 1) we found no evidence for Na+-dependent HCO3 transport in the apical membrane of the MTAL and 2) members of the Na+-HCO3 cotransporter gene family (NBC) do not have significant affinity for K+ (32, 33). Second, HCO3 could replace Cl on a K+-Cl cotransporter. Consistent with this possibility, three of the four known members of the K+-Cl cotransporter family are present in the kidney (KCC1, 3, and 4) (11, 31, 35), and KCC1 and KCC4 are inhibited by DIDS (30). KCC4 has been localized to the basolateral membrane of the MTAL, where it may contribute to transcellular Cl absorption (22, 31, 35). However, evidence for KCC expression in the apical membrane of kidney cells has not been presented. Also, to our knowledge, it has not been established whether KCCs can transport HCO3. Thus the possible role of K+-Cl cotransporters in mediating K+-dependent HCO3 flux requires further study. Highly selective inhibitors of K+-Cl cotransporters have not yet been identified. A third possibility is that K+-HCO3 cotransport may be mediated by a novel transporter distinct from the cation chloride cotransporter (11) or Na+-dependent bicarbonate cotransporter (10, 32) gene families. Our data suggest that the MTAL is a viable model to investigate this hypothesis.
Absence of Other Apical HCO3/OH/H+ Transport Pathways
We found no evidence that transport pathways other than Na+/H+ exchange (21, 40, 41) and K+-dependent HCO3 transport contribute significantly to the acid-base flux across the apical membrane of the MTAL. Using three different protocols involving luminal Cl addition, we found no evidence for a measurable component of apical Cl/HCO3 (OH) exchange in the rat MTAL. Based on the finding that 50% of net Cl absorption was dependent on HCO3/CO2, it was proposed that apical Cl/HCO3 exchange functioned in parallel with apical Na+/H+ exchange to mediate a portion of NaCl absorption in the mouse cortical thick ascending limb (14). However, in a separate study of the same segment, no dependence of NaCl absorption on HCO3/CO2 was found (12). Although the explanation for the differing results in the mouse cortical TAL is unclear, it is conceivable that HCO3/CO2 could influence NaCl absorption under certain conditions in this segment through cellular mechanisms other than apical Cl/HCO3 exchange. In the MTAL, the Cl/HCO3 exchanger AE2 has been localized to the basolateral membrane, where it likely mediates HCO3 efflux for HCO3 absorption (1, 8, 34). The Na+-HCO3 cotransporter NBCnl, which is electroneutral and insensitive to amiloride or EIPA (8, 10, 26), also has been localized to the basolateral membrane of the MTAL (8, 26, 36). We found no evidence for Na+-HCO3 cotransport activity in the apical membrane, consistent with the absence of apical NBC staining in the immunolocalization studies. Last, our findings that the apical K+-dependent net base flux in the MTAL is eliminated in the absence of HCO3/CO2 and is inhibited by DIDS argue against the involvement of an apical K+/H+ exchanger (or an H+-K+-ATPase). It is possible that the K+/H+ exchange activity reported previously in apical membrane vesicles from MTAL suspensions reflects transport activity present in basolateral membranes or contaminating membranes from other cell types or intracellular organelles (4, 13, 29). Alternatively, K+/H+ exchange observed in membrane vesicles may not be functionally active in intact MTALs. Our finding that the lumen K+-induced pHi change is not affected by luminal EIPA indicates that transport of K+ on the apical Na+/H+ exchanger does not contribute significantly to the apical acid-base flux.
Role of Apical K+-Dependent HCO3 Transport in HCO3 Absorption
Under physiological conditions, a K+-HCO3 cotransporter in the apical membrane should mediate the net transfer of K+ and HCO3 into the tubule lumen, driven by the large cell-to- lumen K+ concentration difference (22). This HCO3 transport would be expected to diminish net HCO3 absorption. Consistent with this prediction, luminal DIDS significantly increased HCO3 absorption in the MTAL. Although DIDS is a nonselective inhibitor of anion transport pathways, several lines of evidence support the view that DIDS increased HCO3 absorption through inhibition of apical K+-dependent HCO3 transport: 1) DIDS caused a sizable inhibition of the K+-induced apical net base flux; 2) no evidence for Na+- or Cl-dependent HCO3/OH transport pathways was found in the apical membrane; 3) DIDS increased HCO3 absorption in the presence of luminal furosemide, ruling out an indirect effect on Na+-K+-2Cl cotransport; 4) DIDS increased HCO3 absorption when Cl and HCO3 were the only luminal anions, ruling out potential effects on organic anion or divalent anion transporters; and 5) DIDS has no direct effect on the activity of the apical Na+/H+ exchanger NHE3 (9). In the presence of DIDS, HCO3 absorption was eliminated by luminal EIPA, consistent with mediation of the HCO3 absorption by apical Na+/H+ exchange. It appears, therefore, that the HCO3 absorption rate increases with DIDS because transport of HCO3 into the lumen via apical K+-HCO3 cotransport is inhibited and no longer opposes HCO3 absorption mediated by apical Na+/H+ exchange. We cannot be certain from our data whether the apical Na+/H+ exchange rate is increased, decreased, or unchanged when K+-HCO3 cotransport is inhibited. However, if it is decreased, then this decrease must be quantitatively less than the decrease in K+-HCO3 cotransport to result in an increase in net HCO3 absorption. An important area for future work will be to examine coupling between K+-dependent HCO3 transport and Na+/H+ exchange in the apical membrane. Based on the small effect of luminal DIDS on pHi, and the relative insensitivity of apical Na+/H+ exchange to physiological changes in pHi (21, 40), it is unlikely that K+-HCO3 cotransport influences apical Na+/H+ exchange activity through effects on pHi.
Our studies provide the first evidence that K+-dependent HCO3 transport plays a role in transepithelial acid-base transport and suggest that the control of HCO3 absorption in the MTAL can be achieved through regulation of apical Na+/H+ exchange, apical K+-dependent HCO3 transport, or both processes. Factors that regulate K+-HCO3 cotransport currently are unknown. Acute changes in extracellular K+ concentration do not appear to be a major influence on MTAL HCO3 absorption, because increasing K+ concentration in both luminal and bath solutions did not affect HCO3 absorption in vitro (15). This likely is because changing extracellular K+ concentration has multiple effects that are not observed with luminal DIDS, such as changes in intracellular K+ or Cl activity or membrane voltage that may influence basolateral HCO3 efflux. A broad range of physiological factors and signal transduction pathways has been identified that regulates MTAL HCO3 absorption through effects on apical Na+/H+ exchange activity (7, 18, 19, 21, 27, 38, 40, 41). Future studies aimed at identifying hormones and other factors that modulate K+-dependent HCO3 transport will be necessary to understand the role of this pathway in acid-base regulation. One interesting possibility is that downregulation of apical K+-HCO3 cotransport activity could result in an increase in MTAL HCO3 absorption that contributes to metabolic alkalosis in chronic K+ depletion.
In summary, we obtained functional evidence for a K+-dependent HCO3 transport pathway, possibly a K+-HCO3 cotransporter, in the apical membrane of the rat MTAL. This pathway functions in parallel with apical Na+/H+ exchange and decreases the rate of transepithelial HCO3 absorption, presumably by transporting K+ and HCO3 from cell to tubule lumen. The molecular identity of the K+-dependent HCO3 transporter and physiological factors that regulate this pathway remain to be identified.
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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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REFERENCES |
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