INTRODUCTION

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IN SEVERAL ORGANS, Na(HCO₃)_n cotransporters participate in intracellular pH (pH_i) regulation and bicarbonate transport (5, 9, 10, 14, 23, 25). In the kidney, Na(HCO₃)_n cotransport has been localized by functional studies to the basolateral membrane of the proximal tubule, where it plays an important role in mediating electrogenic basolateral bicarbonate efflux (1, 5, 37). Studies by Soleimani et al. (35) have demonstrated that the renal transporter in the rabbit has a stoichiometry of 1 Na⁺:1 H:1 , whereas Seki et al. (32) have suggested that the coupling ratio is of 1 Na⁺:2 H. Although Na⁺-dependent and -independent Cl/base exchangers also contribute to basolateral bicarbonate transport in the proximal tubule (2, 13, 19, 24, 30), current evidence suggests that electrogenic Na(HCO₃)_n cotransport mediates the majority of bicarbonate efflux in this nephron segment (1, 6, 8, 21, 29).

The localization of Na(HCO₃)_n cotransport in the rabbit proximal tubule has remained controversial. It is currently accepted that this transporter mediates bicarbonate efflux in the superficial S₁ and S₂ segments (1, 29, 30). In neonatal and adult juxtamedullary S₁ proximal tubules, Baum (3, 4) demonstrated basolateral Na(HCO₃)_n cotransport, whereas Geibel et al. (12) reported that juxtamedullary S₁ and S₂ tubules lacked basolateral Na(HCO₃)_n cotransport. In the S₃ segment, Kondo et al. (17) have reported that basolateral bicarbonate efflux is mediated by Na⁺-independent Cl/base exchange. These results have led to the view that the S₃ segment may be unique, in that all basolateral bicarbonate transport in this segment is Cl dependent (11, 33, 34). However, Kurtz (19), Geibel et al. (12), and Nakhoul et al. (24) were able to demonstrate basolateral Na(HCO₃)_n cotransport in the S₃ portion of the rabbit proximal tubule.

Romero et al. (26) have recently cloned a renal electrogenic sodium-bicarbonate cotransporter (NBC) from rat (26) and salamander kidney (27). Burnham et al. (7) have recently cloned a sodium-bicarbonate cotransporter from human kidney. To date, NBC localization in the proximal tubule has not been reported. To further increase our understanding of the role of NBC in proximal tubule bicarbonate transport, we determined the expression of NBC in the rabbit proximal tubule by in situ hybridization combined with functional studies of basolateral Na(HCO₃)_n cotransport.

	METHODS

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Tubule perfusion. Male New Zealand rabbits (mean body wt, 2 kg) were used. They were allowed free access to standard laboratory diet and tap water. Superficial and juxtamedullary S₁, S₂, and S₃ tubules were dissected within 15 min and then perfused in vitro as previously described (18). pH_i was monitored using the fluorescent probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and a microfluorometer coupled to the tubule perfusion apparatus (18). Calibration of intracellular BCECF was performed at the end of each experiment by monitoring the 500/440-nm fluorescence excitation ratio at various values of pH_i in the presence of high K⁺-nigericin standards.

Equivalent base flux¹ was calculated as J_base = dpH_i/dt ×  × V, where dpH_i/dt represents the initial rate of change pH_i following basolateral Na⁺ removal (measured in the initial 10 s),  is the total cell buffer capacity, and V is the cell volume per tubule length. V was obtained by measuring external and internal tubule diameters, as previously described (20). The values for each tubule segment are depicted in Table 2. The intrinsic cell buffer capacity was measured between pH_i 6.8 and 7.0, using the NH₄Cl prepulse technique in HEPES-buffered solutions (28). Cellular buffering due to CO₂/H was calculated as 2.3 × intracellular H (28). Intracellular H was calculated using the initial pH_i measured immediately prior to basolateral Na⁺ removal. Total cell buffer capacity was calculated by adding the intrinsic cell buffer capacity to the CO₂/H buffer capacity. The total cell buffer capacity for each tubule segment is shown in Table 2.

Solutions. The composition of the perfusate and bathing solutions used in this study are listed in Table 1. All experiments were done in Cl-free solutions. The lumen was perfused with solution A, and the tubules were bathed in solution B containing 10 µM ethylisopropylamiloride (EIPA) to inhibit basolateral Na⁺/H⁺ exchange. The tubules were exposed to Cl-free conditions for ~1 h. After a steady state was achieved, the bath solution was changed to a Na⁺-free solution (solution A). dpH_i/dt was measured in the initial 10 s following the bath solution change. Basolateral Na⁺-dependent equivalent base flux under these conditions was completely DIDS inhibitable (250 µM, bath) in all proximal tubule segments.

Cloning and preparation of riboprobes. A 159-bp PCR (2,695-2,854 bp in human kidney NBC) was generated using the human pancreas EST clone W39298 (I.M.A.G.E clone) as a template, with random primer labeled with ³²P, and used to screen a human pancreas gt10 cDNA library (Clontech, Palo Alto, CA). Standard hybridization conditions were employed (42°C; 50% formamide, 5× standard saline phosphate EDTA, 5× Denhardt's solution, 0.5% SDS, and 0.2 mg/ml prehybridized herring sperm DNA). The filters were washed three times with 1× standard sodium citrate (SSC)/0.1% SDS (42°C) and once with with 0.1× SSC/0.1% SDS (25°C). Positive clones were verified by sequencing. The longest cDNA clone (9-2.1) was found to contain a 2,780-bp insert (1,854-4,633 in human kidney NBC). The nucleotide sequence of this region of human NBC was found to be 95% homologous to the same region of rabbit NBC and identical to human kidney NBC (7). To prepare the riboprobe, the insert was subcloned into pGem13zf+ (Promega, Madison, WI). Riboprobes were synthesized by in vitro transcription and labeled with [³⁵S]CTP. For generation of the antisense riboprobe, the plasmid was linearized with Sst I and transcribed by T7 RNA polymerase. For generation of the sense riboprobe, the plasmid was linearized with Kpn I and transcribed with T3 RNA polymerase. The RNA transcripts were purified by phenol-chloroform extractions and Sephadex G-50 spin columns (Sigma, St Louis, MO). The final products were suspended in Tris-EDTA buffer with 0.1 M dithiothreitol. The RNA transcripts were then sheared by alkaline hydrolysis at 68°C for 5 min. After shearing, the reaction was neutralized by adding 3 M sodium acetate, pH 5, to make a final acetate concentration of 0.3 M. Slices of rabbit kidney (1 mm) were fixed in 4% formalin, and 5-µm sections were attached to glass slides (Fisher Scientific, Pittsburgh, PA). The slides were prewashed and digested for 15 min at 37°C with proteinase K. To reduce nonspecific background staining, the slides were succinylated with succinic anhydride and acetylated with acetic anhydride. The ribopropes were hybridized for 18 h at 45°C. The slides were then washed for 15 min in 2× SSC at room temperature, followed by a wash (15 min) in 1× SSC/50% formamide at 45°C, then three washes in 2× SSC/0.1% Triton X-100 at 60°C for 15 min each, followed by two washes in 0.1 SSC at 60°C for 15 min each. The slides were then digested by RNase A (25 µg/ml, Sigma) and RNase T1 (25 U/ml, Sigma) for 40 min at 37°C. The slides were washed twice in 2× SSC at 60°C for 15 min each and then dehydrated in 0.3 M ammonium acetate/70% ethanol for 5 min, followed by a further 5 min of dehydration in 0.3 M ammonium acetate/95% ethanol. The slides were dipped into NTB2 emulsion solution (Kodak, Rochester, NY) and exposed for three days at 4°C, followed by hematoxylin/eosin staining. The sections were imaged using a Zeiss Axiophot microscope (Max Erb, Los Angeles, CA) and digitized using a Sony 3 charge-coupled device color video camera (model DXC-960MD, Compix Imaging Systems, Tuscon, AZ) with C Imaging software (Compix Imaging Systems).

Statistics. Results are reported as means ± SE. Dunnett's t-test was used to compare group means. P < 0.05 was considered significant.

	RESULTS AND DISCUSSION

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Figure 1 demonstrates that, in the kidney, NBC is expressed predominantly in the cortex, with less expression in the outer medulla. NBC expression was not detected in the inner medulla. The level of expression in the cortex is extremely high in comparison with the remaining regions of the kidney. Figure 2 is a microautoradiograph of the superficial cortex (A-D) and outer medulla (E and F) showing expression of NBC in greater detail. Specific NBC hybridization is shown in all proximal tubule segments. The greatest level of expression in superficial cortex was detected in the superficial S₁ proximal tubule. Superficial S₂ tubules also expressed NBC at a high level; however, the intensity was less than the S₁ segment. S₃ proximal tubules in the outer medulla all expressed NBC but at much lower levels. Figure 3 is a microautoradiograph of the deep cortex (A-D). The expression of NBC in the juxtamedullary S₁ segment was greater than all other proximal tubule segments. Juxtamedullary S₂ tubules also had a high level of NBC expression; however, the level was lower than the juxtamedullary S₁ segment.

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Localization of sodium-bicarbonate cotransporter (NBC) mRNA in rabbit kidney detected by in situ hybridization (dark-field photogragh). A: antisense probe; B: sense probe. NBC mRNA expression is highest in the cortex (C) with less labeling in the outer medulla (OM). No labeling was detected in inner medulla (IM). Magnification, ×3.

View larger version (K): [in this window] [in a new window]   Fig. 2.   NBC mRNA expression in rabbit proximal tubule. A-D: superficial cortex; antisense probe (A and C), sense probe (B and D). E and F: outer medulla; antisense probe (E) and sense probe (F). Note that both superficial and juxtamedullary S3 tubules are present in outer stripe of outer medulla. Bright-field microphotograph; magnification, ×370.

View larger version (K): [in this window] [in a new window]   Fig. 3.   NBC mRNA expression in rabbit proximal tubule. A-D: deep cortex; antisense probe (A and C) and sense probe (B and D). Bright-field microphotograph; magnifications, ×370 (A and B) and ×185 (C and D).

Further experiments were done to determine the axial distribution of basolateral Na(HCO₃)_n cotransport in superficial and juxtamedullary S₁, S₂, and S₃ proximal tubules. All experiments were done in Cl-free solutions in the presence of basolateral EIPA (10 µM) to inhibit the basolateral Na⁺/H⁺ antiporter. Basolateral DIDS (250 µM) completely blocked the basolateral Na⁺-dependent equivalent base flux in all proximal tubule segments (not shown). The DIDS-inhibitable rate of basolateral Na⁺-dependent equivalent base flux is shown in Figs. 4 and 5 and in Table 2. The rate of base efflux mediated by basolateral Na(HCO₃)_n cotransport followed a similar pattern in both superficial and juxtamedullary proximal tubules, i.e., S₁ > S₂ > S₃. The juxtamedullary S₁ segment had the highest rate of basolateral Na(HCO₃)_n cotransport of all proximal tubule segments. The flux of base equivalents via the Na(HCO₃)_n cotransporter in the juxtamedullary S₁ segment was approximately two times the value obtained in the superficial S₁ segment (P < 0.05). Superficial and juxtamedullary S₂ tubules had similar rates of basolateral Na(HCO₃)_n cotransport, as did S₃ tubules. Of interest, Geibel et al. (12) have previously reported that the juxtamedullary S₁ and S₂ proximal tubules lack basolateral Na(HCO₃)_n cotransport in tubules perfused in HEPES-containing, nominally H-free solutions. In contrast, Baum (3, 4), in agreement with the present study, has previously measured a high rate of basolateral Na(HCO₃)_n cotransport in rabbit juxtamedullary S₁ tubules perfused in H-containing solutions. Whether the affinity of NBC for H or methodological differences can account for these different results is unknown.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Axial distribution of basolateral Na(HCO3)n cotransport in isolated perfused superficial proximal tubule segments. Data represent the 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS)-inhibitable, basolateral Na+-dependent equivalent base flux in superficial S1, S2, and S3 tubule segments. Values are mean of 4-5 experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Axial distribution of basolateral Na(HCO3)n cotransport in isolated perfused juxtamedullary proximal tubule segments. Data represent the DIDS-inhibitable, basolateral Na+-dependent equivalent base flux in juxtamedullary S1, S2, and S3 tubule segments. Values are mean of 4-6 experiments.

The present results indicate that NBC mRNA is highly expressed in rabbit superficial and juxtamedullary S₁ and S₂ proximal tubules, with less expression in the S₃ segment. NBC was not evident in cortical thick ascending limbs, cortical collecting ducts, or the glomerulus. In the outer medulla, NBC expression was only detectable in the S₃ segment. NBC expression was undetectable in the inner medulla. The level of NBC mRNA expression in the proximal tubule correlated with the magnitude of basolateral Na(HCO₃)_n cotransport function. The juxtamedullary S₁ segment had the greatest rate of basolateral Na(HCO₃)_n cotransport and the highest level of NBC expression in the proximal tubule. Superficial and juxtamedullary S₁ tubules were found to have a significantly greater rate of basolateral Na(HCO₃)_n cotransport than the remaining proximal tubule segments.

These functional differences in basolateral Na(HCO₃)_n cotransport in the various proximal tubule segments are in qualitative agreement with previous studies from which the axial heterogeneity of rabbit proximal tubule transepithelial bicarbonate transport can be determined (6, 15, 21, 22, 31, 36). In general, transepithelial bicarbonate absorption is greatest in the S₁ proximal tubule. The high level of expression of NBC in superficial and juxtamedullary S₁ proximal tubules is in keeping with the high rate of transepithelial bicarbonate transport in these segments. The juxtamedullary S₁ segment has the greatest rate of basolateral Na(HCO₃)_n cotransport and the highest level of NBC expression in the proximal tubule. The rate of transepithelial bicarbonate absorption in the juxtamedullary S₁ tubule is approximately twice the value in the superficial S₁ segment (15, 31). This finding is of interest, given our results, which indicate that the flux of base equivalents via the Na(HCO₃)_n cotransporter in the juxtamedullary S₁ segment was approximately two times the value obtained in the superficial S₁ segment. Several additional factors, including differences in luminal Na⁺/H⁺ antiport activity and basolateral Na⁺-K⁺-ATPase activity, likely also play a role in determining the axial heterogeneity of proximal tubule transepithelial bicarbonate transport (3, 16).

The results of the present study confirm our previous finding (19) and studies by Geibel et al. (12) that the S₃ proximal tubule possesses a basolateral Na(HCO₃)_n cotransporter. The low level of NBC expression in the S₃ tubule likely explains the failure of some groups to detect Na(HCO₃)_n cotransport in this nephron segment. Indeed, it has been suggested that all basolateral bicarbonate transport in the S₃ segment is mediated by a Na⁺-independent anion exchange process (11, 17, 33, 34). Our data indicate that bicarbonate efflux in this portion of the proximal tubule is, in part, Na⁺ dependent. The greater role of Na(HCO₃)_n cotransport in mediating bicarbonate efflux in the S₁ segment is underscored by the high level of NBC expression in this portion of the proximal tubule.