INTRODUCTION

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The majority of the filtered load of HCO₃ is reabsorbed in the kidney proximal tubule via the luminal Na⁺/H⁺ exchanger NHE-3 (1-4). The exit of HCO₃ across the basolateral membrane of proximal tubule is via the Na⁺:HCO₃ cotransporter (5-8). The Na⁺:HCO₃ cotransporter (NBC)¹ mediates an electrogenic process with an stoichiometry of 3 eq of HCO₃ per Na⁺ (9, 10). Recent studies have indicated that the actual ionic mechanism involves the cotransport of Na⁺, HCO₃, and CO₃² in a 1:1:1 ratio (11). In addition to reabsorption of HCO₃ in proximal tubule, NBC also plays an important role in cell pH regulation in several tissues, including brain, liver, heart, and lung (12-17).

Functional studies support the presence of more than one NBC isoform as judged by direction and stoichiometry of the cotransporter. In kidney, NBC activity leads to cell acidification, whereas in other tissues (such as liver and heart) its function leads to cell alkalinization (5-8, 13, 14). Furthermore, NBC has a stoichiometry of 3 eq of HCO₃ per Na⁺ ion in the kidney (9, 10) but shows a stoichiometry of 2 eq of HCO₃ per Na⁺ in other tissues (12).

We recently cloned and functionally expressed a human kidney Na⁺:HCO₃ cotransporter (18). In addition to the kidney, the Na⁺:HCO₃ cotransporter (called here NBC-1) is highly expressed in pancreas, with detectable levels in brain (18). The human NBC-1 (18) shows 80% homology to the amphibian Na⁺:HCO₃ cotransporter (19). Both the human NBC-1 (18) and the amphibian NBC (19) mediate Na⁺-dependent HCO₃ cotransport in a DIDS-sensitive manner. The purpose of the current study was to examine the functional properties of the human kidney NBC. Accordingly, cultured HEK-293 cells were transiently transfected with the NBC-1 cDNA and studied.

	EXPERIMENTAL PROCEDURES

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Cell Culture Procedures-- HEK-293 cells were cultured in Dulbecco's modified Eagle's medium containing 100 units/ml penicillin-G and supplemented with 10% fetal bovine serum. Cultured cells were incubated at 37 °C in a humidified atmosphere of 5% CO₂ in air. The medium was replaced every other day.

Transient Transfection-- Cultured HEK-293 cells were plated on coverslips and transfected at 60% of confluence with 8 µg of the full-length human NBC-1 cDNA construct (in the cloning/expression vector pCMV.SPORT1) by calcium phosphate-DNA coprecipitation (20). Cells were assayed 44-52 h after transfection.

Intracellular pH Measurement-- Changes in intracellular pH (pH_i) were monitored using the acetoxymethyl ester of the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) as described (21-23). HEK-293 cells were grown to confluence on coverslips and incubated in the presence of 5 µM BCECF in a Na⁺-free solution consisting of 115 mM tetramethylammonium-Cl and 25 mM KHCO₃, pH 7.4 (solution A, Table I). pH_i was measured in a thermostatically controlled holding chamber (37 °C) in a Delta Scan dual excitation spectrofluorometer (Photon Technology International, South Brunswick, NJ). The monolayer was then perfused with the appropriate solutions (Table I). The fluorescence ratio at excitation wavelengths of 500 and 450 nm was utilized to determine intracellular pH values in the experimental groups by comparison with the calibration curve that was generated by KCl/nigericin technique. The fluorescence emission was recorded at 525 nm. The Na⁺:HCO₃ cotransporter activity was determined as the initial rate of the DIDS-sensitive, Na⁺-dependent pH_i recovery (dpH_i/dt, pH/min) in a HCO₃-containing solution following an acid load induced by NH₃/NH₄⁺ withdrawal. The experiments were performed in the presence of 1 mM amiloride to block the Na⁺/H⁺ exchanger activity. The experiments were repeated in the absence of HCO₃ and CO₂ and bubbled with O₂ to determine whether NBC can mediate Na⁺:OH (hydroxyl) cotransport. The dpH_i/dt was calculated by fitting to a linear equation the first 2 min of the time course of intracellular pH recovery. Correlation coefficients for these linear fits averaged 0.984 ± 0.004

Materials-- Dulbecco's modified Eagle's medium was purchased from Life Technologies, Inc. BCECF-AM was from Molecular Probes Inc. Nigericin, DIDS, amiloride, and other chemicals were purchased from Sigma.

Statistics-- Results are expressed as means ± S.E. Statistical significance between experimental groups was determined by Student's t test or by one-way analysis of variance.

	RESULTS

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Na⁺ and HCO₃ Dependence of the Cloned Human Kidney NBC (Influx Mode)-- In the first series of experiments, cells were incubated and loaded with BCECF in Na⁺-free solution (solution A, Table I), exposed to NH₄⁺ for 10 min (solution B, Table I), and acid-loaded by switching to an NH₄⁺-free solution (solution A, Table I). The base-line pH_i in sodium-free solution was 7.11 ± 0.02. In the presence of 1 mM amiloride (to block Na⁺/H⁺ exchange), switching to a Na⁺ (115 mM) and HCO₃-containing solution (solution C, Table I) resulted in rapid pH_i recovery from acidosis in transfected cells (Fig. 1A), which was completely inhibited by 300 µM DIDS. The rate of pH_i recovery was 0.190 ± 0.013 pH/min in transfected cells (n = 5). This recovery was HCO₃-dependent, as shown by lack of significant pH_i recovery in the absence of HCO₃ (solution D, Table I) (Fig. 1B). In HCO₃-free solution, pH_i recovery was 0.015 ± 0.003 pH/min in transfected cells (p < 0.001 versus HCO₃-containing solution, n = 5). Nontransfected cells showed little Na⁺-dependent HCO₃ cotransport (Fig. 1C) with pH_i recovery of 0.017 ± 0.002 pH/min (p < 0.001 versus transfected cells, n = 5).

View this table: [in this window] [in a new window]   Table I Composition of experimental solutions Concentrations are in mM. Solutions (A, B, C, E, F) were bubbled with 5% CO2, 95% O2; solution D was gassed with 100% O2. The pH was adjusted to 7.40 with tris(hydroxymethyl)aminomethane. For Cl-free solution, NH4Cl was replaced with NH4SCN and other Cl-containing chemicals were replaced with gluconate salts.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Na+ and HCO3 dependence of the cloned human kidney NBC (influx mode). Transfected cells were acid loaded by NH4+ withdrawal and monitored for pHi recovery. A, in Na+ and HCO3-containing solution, in the presence of 300 µM DIDS (n = 4) or its vehicle (n = 5); B, in HCO3-free Na+-containing solution (n = 4); C, nontransfected cells were exposed to Na+ and HCO3-containing solution (n = 5). Amiloride, 1 mM, was present to block Na+/H+ exchange.

Na⁺ and HCO₃ Dependence of NBC (Efflux Mode)-- NBC activity in transfected cells was also measured in the efflux mode. As shown in Fig. 2, switching the cells from a Na⁺ and HCO₃-containing medium (solution C, Table I) to a Na⁺-free solution (solution A, Table I) resulted in rapid cell acidification,² with pH of 0.27 pH unit (n = 3). This cell acidification was reversible, with pH_i returning to base line upon switching back to the Na⁺-containing solution (solution A, Table I) (Fig. 2A), with pH of 0.31 pH unit (n = 3). The pH_i recovery back to base line was completely inhibited in the presence of 300 µM DIDS (Fig. 2A) (n = 3). Neither cell acidification nor pH_i recovery were observed in the absence of HCO₃ (Fig. 2B), indicating the dependence of the transporter on Na⁺ and HCO₃. Taken together with Fig. 1, these studies indicate that the cloned cDNA encodes a Na⁺:HCO₃ cotransporter.

View larger version (21K): [in this window] [in a new window]   Fig. 2.   Na+ and HCO3 dependence of the cloned NBC (efflux mode). A, transfected cells incubated in Na+ and HCO3-containing solution were exposed to a Na+-free solution (resulting in rapid cell acidification). Thereafter, cells were switched back to the Na+-containing solution (resulting in pHi recovery back to normal) (n = 3), in the presence of 300 µM DIDS or its vehicle (n = 3). Amiloride, 1 mM, was present to block Na+/H+ exchange. B, transfected cells were subjected to the same maneuver as above in the absence of HCO3 in the environment.

Interaction of Lithium (Li⁺) with the Cloned NBC-- The ability of NBC to mediate Li⁺:HCO₃ cotransport was next tested. As shown in Fig. 3A, exposing the cells to a Li⁺-containing solution (solution E, Table I) caused significant recovery from intracellular acidosis. Switching from the Li⁺-containing solution to the Na⁺-containing solution (solution A, Table I) further increased the rate of pH_i recovery (Fig. 3A). At comparable acidic pH_i (nadir pH of 6.282 ± 0.039 and 6.32 ± 0.027 for Li⁺ and Na⁺ experiments, respectively, p > 0.05, n = 4) the rate of pH_i recovery caused by Na⁺ was 4-fold higher then Li⁺ (Fig. 3, A and B) (pH_i recovery was 0.185 ± 0.003 pH/min in the presence of Na⁺ and 0.045 ± 0.003 in the presence of Li⁺, p < 0.001, n = 4 for each group). The Li⁺-dependent HCO₃ cotransport was completely inhibited in the presence of 300 µM DIDS (Fig. 3C) and was not detected in nontransfected cells (Fig. 3D). These results indicate that Li⁺ can substitute for Na⁺ on NBC, with Li⁺ showing lower rates in mediating HCO₃ transport. This is in contrast to the proximal tubule luminal NHE, where Li⁺ has much stronger affinity than Na⁺ (1).

View larger version (35K): [in this window] [in a new window]   Fig. 3.   Interaction of lithium (Li+) with the cloned NBC. A, transfected cells were acidified and then exposed to a Li+ or Na+-containing solution (n = 4). B, the rate of pHi recovery caused by Li+ or Na+ at the same acidic pHi (n = 4 for each). C, cells were exposed to a Li+-containing solution in the absence or presence of 300 µM DIDS (n = 4). D, nontransfected cells were acidified and then exposed to a Li+-containing solution (n = 4). HCO3 was present during the duration of the experiment. Amiloride, 1 mM, was present to block Li+/H+ exchange.

Inhibition of the Cloned NBC by Harmaline-- The purpose of the next series of experiments was to examine the interaction of harmaline with NBC. As indicated in Fig. 4, the presence of 0.2 mM harmaline completely blocked the Na⁺-dependent pH_i recovery in HCO₃-containing media (the experiments were performed in the presence of 1 mM amiloride to block Na⁺/H⁺ exchange). The inhibition by harmaline was reversible as shown by recovery from cell acidosis upon switching to a harmaline-free solution (Fig. 4). pH_i recovery from acidosis was 0.184 ± 0.005 pH/min in the absence of harmaline and 0.007 ± 0.002 in the presence of harmaline (p < 0.001, n = 4 for each group). pH_i recovery upon removal of harmaline was 0.178 ± 0.006 pH/min (n = 4).

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Inhibition of the cloned NBC by harmaline. Transfected cells were acidified and then exposed to a Na+-containing solution in presence of 200 µM harmaline or its vehicle (n = 4). HCO3 was present during the duration of the experiment. Amiloride, 1 mM, was present to block Na+/H+ exchange.

Effect of External pH on the Cloned NBC-- NBC in rabbit kidney cortex is absolutely dependent on HCO₃ (24) and does not demonstrate any affinity for OH whereas in colon it can also accept OH (25). To examine the interaction of human kidney NBC with OH, cells were transfected and assayed for pH_i recovery from an acid load in the presence of varying pH_o and the absence of HCO₃. As shown in Fig. 5A, in the absence of HCO₃ and at pH_o 7.4, transfected cells showed little recovery from an acid load, indicating that at normal pH, NBC-1 has very low affinity for OH (pH_i recovery from acidosis was 0.020 ± 0.004 pH/min in the absence of HCO₃ (p < 0.001 versus HCO₃-containing solution, n = 5 for each group). Increasing the inward OH gradient by increasing the external pH to 7.8 caused significant Na⁺-dependent pH_i recovery from an acid load (0.085 ± 0.005, p < 0.001 versus pH_o 7.4, n = 5) that was abolished in the presence of DIDS (Fig. 5B).

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Effect of external pH on the cloned NBC. Cells were acidified and pHi recovery caused by Na+ addition was monitored at varying pHo and in the absence of HCO3. A, Na+-dependent pHi recovery at pHo 7.40 (n = 5). B, Na+-dependent pHi recovery at pHo 7.80 in the presence of 300 µM DIDS (n = 4) or its vehicle (n = 5). Nontransfected cells were exposed to Na+ at pHo 7.80 in the absence (C) (n = 4) or presence of 300 µM DIDS (D) (n = 3). Fig. 5E shows the Na+-dependent and Na+-independent pHi recovery at pHo 7.8 in transfected cells.

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Interaction of K+ with the cloned NBC. A, transfected cells were acidified and then exposed to a K+ (solution F, Table I) or Na+-containing solution (solution C, Table I) (n = 4). B, the rate of pHi recovery caused by Na+ or K+ addition in the same monolayer (n = 4). HCO3 was present during the duration of the experiment. Amiloride, 1 mM, was present to block possible K+/H+ exchange.

	DISCUSSION

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The results of current experiments indicate that the cloned human kidney Na⁺:HCO₃ cotransporter (NBC-1) accepts Na⁺ and HCO₃ and is inhibited by DIDS (Figs. 1 and 2). The results further indicate that Li⁺ can substitute for Na⁺ on NBC-1, with Li⁺ showing decreased capacity to mediate HCO₃-dependent pH_i recovery compared with Na⁺ (Fig. 3). NBC-1 is inhibited by DIDS and harmaline (Fig. 4) and can accept OH in place of HCO₃, with HCO₃ showing much higher affinity than OH (Fig. 5). NBC does not function in K⁺:HCO₃ cotransport mode (Fig. 6).

We recently cloned the human kidney NBC-1 (18). Based on the deduced amino acid sequence, the cDNA encodes a protein with a molecular mass of ~116 kDa. Northern blot analysis reveals that NBC-1 encodes a 7.6-kilobase mRNA that is highly expressed in kidney and pancreas, with lower levels of expression in the brain (18). NBC-1 mRNA was not detected in the liver, lung, and heart (18) despite functional studies indicating the presence of Na⁺:HCO₃ cotransport in these tissues. These results strongly suggest that the Na⁺:HCO₃ cotransport in these latter tissues might be another isoform from this family. The human kidney NBC cDNA is highly homologous with the amphibian kidney NBC and shares striking similarities at both structural and functional levels with that cotransporter (19).

We have cloned rat NBC-1 and examined its mRNA distribution in different tissues as well as various nephron segments (28). Rat NBC-1 is highly expressed in kidney and brain but shows low levels of expression in stomach and colon (28). Nephron segment distribution studies revealed that NBC-1 is predominantly expressed in proximal tubules (28).

The results in Fig. 1 demonstrate that the Na⁺:HCO₃ cotransport does not mediate complete pH_i recovery from acidosis to baseline values in the presence of amiloride. Whereas in the absence of amiloride, intracellular pH_i returns to base-line values (data not shown), further studies are needed to determine whether NHE activity is indeed essential for complete pH_i recovery from acidosis.

The results of Fig. 3 demonstrate the interaction of Li⁺ with NBC and indicate that the human kidney NBC can accept Li⁺ in place of Na⁺, with Li⁺:HCO₃ cotransport demonstrating lower transport rate than Na⁺:HCO₃ cotransport (Fig. 3). This is very similar to the NBC in basolateral membranes of rabbit kidney proximal tubule (11, 29) and is opposite to the luminal NHE, which has much higher affinity for Li⁺ than Na⁺ (1).

The human kidney NBC was completely inhibited in the presence of harmaline (Fig. 4) and DIDS (Fig. 1), indicating that it has an inhibitory profile similar to the mammalian NBC in basolateral membranes of rat or rabbit kidney proximal tubule (11, 30). Harmaline is known to inhibit the rabbit kidney proximal tubule NBC by binding to the Na⁺ site (11), whereas DIDS binds to the HCO₃ site (5-8). This latter conclusion is based on the fact that DIDS inhibits both Na⁺-dependent as well as Na⁺-independent HCO₃ transporters.

The anion exchanger AE1 (band-3) in red blood cells can mediate the transport of Na⁺ and HCO₃ via ion pair (Na⁺:CO₃²) (31-33). According to this mechanism, the positive charge of the Na⁺ ion is being shielded by a CO₃² (31), allowing the cotransport to be mediated as an anion via AE1. In support of the ion pair functioning as an anion, we find that cation inhibitors do not inhibit the transport of Na⁺ via band-3 (31). NBC was cloned based on homology to anion exchanger isoforms (18), raising the possibility that the Na⁺:CO₃² ion pair may be the actual substrate for transport on this protein. The results of the experiments in Figs. 3 and 4, however, argue against Na⁺:CO₃² ion pair as the substrate. The first argument against ion pair formation concerns the relative affinities of Na⁺ and Li⁺. The association constant of Li⁺ for ion pair formation with CO₃² is 4-fold greater than of Na⁺ (32), consistent with Li⁺ having higher affinity than Na⁺ for transport via red cell band-3 (33). The results of Fig. 3 indicate that the ability of Li⁺ to mediate HCO₃ transport via NBC is significantly lower than Na⁺. This difference is opposite to that for ion pair formation with CO₃². The other argument against ion pair formation is inhibition of NBC by alkaloid harmaline (Fig. 4). Harmaline is an organic cation known to compete at the Na⁺ site of several Na⁺-coupled transport systems in kidney proximal tubules such as the Na⁺/H⁺ exchanger and the Na⁺/glucose cotransporter. Although we have not studied the kinetics of NBC inhibition by harmaline, we suggest that based on similarities with the renal basolateral Na⁺:HCO₃ cotransporter in rabbit and rat kidney (11, 30), harmaline inhibits the cloned human NBC by interaction with the Na⁺ site. These results, in conjunction with the results of Li⁺ studies, strongly argue that Na⁺ directly interacts at a distinct site on the NBC rather than via ion pair formation with CO₃².

The human kidney NBC accepts OH in place of HCO₃, with OH demonstrating much lower Na⁺-dependent transport rates than HCO₃ (Fig. 5). These results are in agreement with functional studies in rabbit colon, indicating lower affinity of NBC for OH (25). The rabbit kidney NBC on the other hand shows absolute dependence on HCO₃ and does not demonstrate any affinity for OH (24). It is worth mentioning that functional studies in rabbit kidney cortex were performed at physiologic pH (pH_o 7.4) (24). As such, the affinity of rabbit kidney cotransporter for OH at external pH values similar to current experiments (i.e. pH_o 7.8) remains unknown.

Kidney NBC functions in an outwardly directed mode under physiologic conditions (5-8), resulting in trans-vectorial transport of HCO₃ from lumen to blood. As such, the physiologic role of the Na⁺:OH cotransport mode remains speculative, as carbonic anhydrase activity in the kidney proximal tubule cells couples the CO₂ with OH to generate HCO₃, thereby diminishing the concentration of intracellular OH. We propose that the physiologic significance of Na⁺:OH cotransport mode may be in the colon, where secretion of acid into the lumen via NHE-3 may not lead to the reabsorption of HCO₃, as the luminal [HCO₃] is negligible (34), but rather results in generation of intracellular OH. The Na⁺:OH cotransporter will then exit the cell across the basolateral membrane, thereby regulating cell pH.

In conclusion, based on functional properties (Figs. 1-6) and nephron segment distribution studies (28), we propose that NBC-1 is the kidney proximal tubule Na⁺:HCO₃ cotransporter.