Immunohistochemical localization of colonic H-K-ATPase to the apical membrane of connecting tubule cells

Géza Fejes-Tóth and Anikó Náray-Fejes-Tóth

Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies indicate that the colonic H-K-ATPase mRNA is expressed as the distal nephron. However, the exact intrarenal localization of the colonic H-K-ATPase protein is still unclear. The goal of the present study was to determine the cellular and subcellular localization of the colonic H-K-ATPase protein in the rabbit kidney. We used three monoclonal antibodies (MAbs) directed against different epitopes of the rabbit colonic H-K-ATPase alpha -subunit (HKalpha 2) to localize HKalpha 2 protein by immunofluorescence labeling of kidney sections and laser-scanning confocal microscopy. The specificity of the MAbs was confirmed by reaction with a single ~100-kDa band on Western blots of distal colon. Specific immunohistochemical reaction with the apical membrane of surface epithelial cells was observed with all three MAbs on distal colon sections. In rabbit kidney, immunofluorescence was detected only on the apical membrane of connecting tubule cells. Immunofluorescence was not detected in the cortical-, outer-, and inner-medullary collecting ducts. Furthermore, costaining with principal- and intercalated cell-specific MAbs and a MAb against the thick ascending limb suggests that these cell types express HKalpha 2 protein at levels that are below the detection limit with this method. We conclude that in the rabbit kidney, under normal dietary conditions, the HKalpha 2 protein is expressed in the apical membrane of connecting tubule cells.

potassium transport; confocal microscopy; rabbit kidney


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE KIDNEY PLAYS A CRITICAL role in maintaining potassium homeostasis and acid-base balance. The fine regulation of both K+ and H+ excretion takes place in the collecting duct system. A number of functional studies indicate that a family of H-K-ATPases participates in both K+ and H+ transport in the distal nephron (for review see Refs. 31 and 33). At least two genes encoding H-K-ATPase alpha -subunits seem to be expressed in rat kidney: the gastric isoform (or HKalpha 1) (27) and the colonic (or HKalpha 2) H-K-ATPases (8, 17). Additional diversity in the H-K-ATPase family is generated by alternative splicing. Recently, two NH2-terminal splice variants of the HKalpha 2 mRNA were cloned from both the rat (17) and the rabbit (6) kidney, designated HKalpha 2a and HKalpha 2b (rat) and HKalpha 2a and HKalpha 2c (rabbit).

HKalpha 1 mRNA has been localized to the connecting segment and the entire collecting duct of rats (2). Results from several laboratories, including ours, demonstrated that HKalpha 2 mRNA is expressed in cells of the distal nephron. In situ hybridization studies with rats on normal diets showed strong labeling in the connecting tubule (CNT) and cortical collecting duct (CCD) but only very low levels of expression in the outer medullary collecting duct (OMCD) (1). Our previous studies demonstrated that the HKalpha 2 mRNA is present in the CNT, CCD, and OMCD of rabbits (13, 14) and that the expression in CNT plus CCD cells is increased after in vivo base loading of the animals (14). Several studies have demonstrated that the expression of HKalpha 2 mRNA is increased in the rat medulla by chronic hypokalemia (1, 16, 25).

Information on the intrarenal localization of the HKalpha 2 protein is scanty. Only one immunohistochemical study has been published thus far by Sangan and co-workers (25). These authors reported that a polyclonal antibody against the rat colonic H-K-ATPase alpha -subunit labeled the apical membrane of principal cells in the rat OMCD, whereas no specific labeling was observed in the CNT. Localization of HKalpha 2 to OMCD principal cells was an unexpected finding because previous functional studies indicated that H-K-ATPase activity resides primarily in intercalated cells (28-30, 33). This finding also conflicts with previous in situ hybridization data showing intense signals in the CNT and CCD (1) and a recent report that shows that the HKalpha 2 protein is present in the renal cortex in the rabbit (6). The above immunohistochemical study was done in rat kidney, and the intrarenal and subcellular localization of the HKalpha 2 protein in other species remains unknown.

Despite clear evidence that HKalpha 2 is expressed in the kidney, its exact function is still unclear, in part because no isoform-specific inhibitors against the various H-K-ATPases are available. A recent study with HKalpha 2-deficient mice indicates that during K+ deprivation this transporter plays a critical role in the colon to maintain K+ homeostasis (20). On the basis of its presumed function (i.e., K+ reabsorption and H+ secretion), one would expect the colonic H-K-ATPase to be present in the apical membrane of OMCD cells, because this nephron segment exhibits net K+ reabsorption and acidification and this is the nephron segment in which dietary K+ seems to regulate HKalpha 2 mRNA expression (1). Indeed, Codina and co-workers (7) showed that HKalpha 2 protein, which was undetectable in the kidney in control rats, becomes detectable on immunoblots from the medulla (but not the cortex) of rats subjected to chronic hypokalemia.

In this study, we examined the cellular and subcellular localization of the HKalpha 2 protein in rabbit kidney, under conditions of normal K+ homeostasis. We used three new monoclonal antibodies (MAbs) against different epitopes on the rabbit HKalpha 2 protein, in combination with laser-scanning confocal microscopy. Our results demonstrate that the HKalpha 2 resides in the apical membrane of connecting tubule cells in the rabbit kidney.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of antibodies against the rabbit HKalpha 2. MAbs were generated against a fusion protein containing the NH2-terminal region of the rabbit HKalpha 2a (amino acids 1-99). The rabbit HKalpha 2a cDNA (14) was used as a template to amplify the corresponding region using a sense primer (5'-TCC GAA TTC ACA TGC GCC AGA GAA AGC TGG-3') and an antisense primer (5'-CAA ACA GAC CCC AGA GAT CAT CTA GGT ACC CAA-3'), which add KpnI and EcoRI restriction sites. After digestion with these enzymes, the PCR product was ligated into the pPROEx-1 vector (GIBCO-BRL), which contains an NH2-terminal polyhistidine tag and a tobacco etch virus (TEV) protease cleavage site. Direct sequencing was performed to verify that the insert is in the correct reading frame and is identical to the appropriate region of the rabbit HKalpha 2 cDNA (13, 14). Production of the fusion protein was induced with isopropyl-beta -D-thiogalactopyranoside in Escherichia coli DH5alpha . The fusion protein was purified on Ni-NTA resin according to the manufacturer's instructions (GIBCO-BRL). The purified fusion protein was then cleaved with recombinant TEV protease (GIBCO-BRL) and passed over a Ni-NTA column again to remove the polyhistidine tag. Balb/c female mice were immunized with 100 µg of the cleaved fusion protein in complete Freund's adjuvant (ip) once and then twice with 50 µg of fusion protein in incomplete Freund's adjuvant at 2-wk intervals. Blood was obtained from the tail for the determination of antibody titer by enzyme-linked immunosorbent assay (ELISA) using the purified fusion protein as antigen. MAbs were generated using the spleen of the mouse with the highest serum titer, as described previously (9, 11, 12). Hybridomas, which were positive in ELISA, were tested for Western blotting and immunohistochemical staining (see below).

Immunoblotting. Tissue homogenates were prepared from rabbit distal colon, kidney cortex, medulla, stomach, and heart by snap freezing the tissues after dissection and homogenization in 1% SDS-containing solubilization buffer (12). Rabbit CCD cells were isolated by immunodissection (9, 11) and lysed in solubilization buffer. Homogenates of E. coli expressing the fusion protein were obtained by sonication and centrifuged at 10,000 g. Protein concentrations were determined by the bicinchoninic acid method (Pierce), and then dithiothreitol was added to a final concentration of 1 mM. Ten micrograms of protein per lane were electrophoresed on 12.5% SDS-polyacrylamide gel with a 4% stacking gel and transferred to polyvinylidene difluoride Immobilon membranes. The membranes were blocked in 5% nonfat milk in 10 mM Tris · HCl, pH 7.4, 150 mM NaCl, and 0.02% Tween-20 (TBST) for 1 h and probed with the mouse MAbs (undiluted culture supernatant) at room temperature for 1 h. Membranes were washed with TBST four times and then incubated with a 1:20,000-fold dilution of alkaline phosphatase-conjugated rabbit anti-mouse IgG at room temperature for 1 h. After membranes were washed again with TBST, antibody binding was localized by the enhanced chemiluminescence method (Amersham).

Immunoprecipitation was performed by covalently linking MAb 0121 to an affinity support using a Seize X protein A immunoprecipitation kit (Pierce) after the manufacturer's instructions.

Immunohistochemistry and confocal microscopy. For these experiments, five male New Zealand rabbits were used. The rabbits were anesthetized and the kidneys were perfused with periodate-lysine-paraformaldehyde (PLP) fixative (19), kept in PLP for an additional 2 h at room temperature, and finally embedded in paraffin. Distal colon was fixed similarly. Four-micrometer-thick sections were cut and deparaffinized, followed by quenching of endogenous peroxidase activity by incubation with 3% H2O2. Immunohistochemistry was performed using culture supernatants of hybridomas followed by 1:100 dilution of a horseradish peroxidase-conjugated anti-mouse IgG (Zymed). Colonic H-K-ATPase immunoreactivity was visualized using the tyramide signal amplification system (TSA-Indirect, NEN; Ref. 3) and Cy-5-conjugated streptavidin (1:500). The following antibodies were used as segment and cell-specific markers: MAb DT17, which reacts with principal cells in the CCD (11); MAb F13/483, which reacts with CNT cells and principal cells in the CCD; ST.12, which reacts with rabbit CNT and CCD (9, 11); and peanut lectin agglutinin (PNA), a marker of beta -intercalated cells in the rabbit (18). alpha -Intercalated cells were labeled with either a MAb against H-ATPase (34) or a MAb against the basolateral HCO3/Cl exchanger, band 3 (26). As a CNT marker, we used a MAb against the Ca/Na exchanger (23). In dual-label immunohistochemistry, the second antibody was either directly labeled with FITC or Texas red (MAbs DT.17, F13/483, ST.12) or visualized using Alexa-568 or Texas red-conjugated species-specific antibodies in 1:2,000-1:4,000 dilution (for antibodies against H-ATPase and the basolateral HCO3/Cl exchanger, band 3). In the latter experiments, either isotype-specific fluorochrome-labeled secondary antibody was used, or in the case of directly labeled MAbs, binding to the secondary antibody was prevented by blocking the sections with 10% mouse serum before the second mouse antibody was applied. Appropriate controls with the second antibody alone were performed in each case. Sections were mounted in Vectashield (Vector Laboratories). Fluorescence images were captured on a PXL-cooled charge-coupled device camera (Photometrics) attached to an Olympus IMT2 microscope equipped with an epifluorescence attachment and standard FITC, Cy-5, and Texas red filter sets. Fluorescence laser scanning confocal microscopy was performed on a Bio-Rad MRC-1024 confocal system as described (22).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation and characterization of MAbs. MAbs were generated against the rabbit HKalpha 2 by immunization of mice with a fusion protein encompassing amino acids 1-99 in the rabbit HKalpha 2a (13). By using ELISA, we identified several hybridoma clones that produced antibodies reacting with the fusion protein. MAbs were tested on Western blots of lysates of bacteria expressing the fusion protein and lysates of rabbit distal colon. Three of the antibodies, designated 121, 132, and 233, recognized a major protein band with the appropriate size (14,700 kDa) in lysates of E. coli expressing the recombinant rabbit HKalpha 2 (Fig. 1A, lanes 2-4). Omission of the primary antibody or substitution with an irrelevant antibody that did not react with the HKalpha 2 antigen in ELISA (MAb 11) yielded no detectable signal, as shown in Fig. 1A, lane 1. The faint smaller band recognized by MAb 121 (Fig. 1A, lane 2) most likely corresponds to a degradation product or an early termination product. This band was not observed with the other two MAbs. MAbs 121, 132, and 233 reacted with a single band with an apparent molecular mass of ~100 kDa on immunoblots originating from the distal colon (Fig. 1B, lanes 2-4). We made numerous, unsuccessful attempts to detect HKalpha 2 on blots prepared with total kidney, cortex, or medulla. It is important to note that these blots did not reveal any nonspecific reaction of the MAbs either (not shown). To compensate for the apparent low abundance of the HKalpha 2 protein in the kidney, we thus enriched for HKalpha 2 protein by immunoprecipitation. Subsequent immunoblotting of the protein precipitated with MAb 121 yielded a strong band around 100 kDa in the distal colon (Fig. 1C, lane 1), and a weaker but still readily detectable band in the renal cortex (lane 2). However, even with prior immunoprecipitation, we failed to detect immunoreactivity in the medulla (lane 3).


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Fig. 1.   Immunoreactivity of anti-H-K-ATPase alpha -subunit (HKalpha 2) monoclonal antibodies (MAbs) on Western blots. Ten micrograms of protein per lane were electrophoresed, blotted, and probed with the MAbs as indicated. A: bacterial lysate of Escherichia coli expressing the pProEx-rabbit HKalpha 2 (amino acids 1-99) fusion protein. B: membranes from rabbit distal colon. C: rabbit distal colon (lane 1), kidney cortex (lane 2), or medulla (lane 3) was immunoprecipitated using MAb 121 and electrophoresed, blotted, and probed with MAb 121. D: rabbit heart. E: rabbit stomach. Proteins in D and E were blotted and probed with the MAbs as in A and B.

One of the three antibodies (MAb 121) reacted very strongly with an ~100-kDa band in the heart (Fig. 1D, lane 2), which might correspond to the heart isoform of Na-K-ATPase. The other two antibodies that reacted with the distal colon (MAbs 132 and 233) as well as the irrelevant MAb 11 were nonreactive in the heart. These data indicate that the three antibodies (MAb 121, 132, and 233) recognize at least two different epitopes on HKalpha 2. This conclusion is also supported by the fact that the antibodies have different isotypes (MAb 132 and 233 are IgG1 and MAb 121 is IgG2b).

Importantly, no signal could be detected with either antibody in the stomach (Fig. 1E). Immunoreactivity was very weak with membranes of isolated CCD plus CNT cells and showed only degradation products (data not shown). This finding confirms our previous observation that HKalpha 2 expression levels are much lower in the CCD and CNT than in the distal colon (14). The fact that only degradation products could be detected in these isolated cells is probably due to the use of proteases during the immunodissection procedure.

Immunohistochemistry. All three MAbs reacted strongly with the apical membrane of surface epithelial cells in the distal colon (Fig. 2). This localization is very similar to that observed in the rat colon (25).


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Fig. 2.   Immunoreactivity of the 3 anti-HKalpha 2 MAbs in distal colon. Periodate-lysine-paraformaldehyde (PLP)-fixed rabbit colon sections were stained with the anti-HKalpha 2 MAbs using the tyramide signal amplification-Indirect system, as described in MATERIALS AND METHODS. A, C, and E: phase-contrast images. B, D, and F: corresponding fluorescence images of sections stained with MAbs 121,132, and 233, respectively. Specific labeling of the apical membranes of surface epithelial cells was observed with all 3 MAbs. F: the immunoreactivity (top right) that is not localized to the apical membrane corresponds to red blood cells (due to their endogenous peroxidase activity).

In the kidney, immunoreactivity of MAbs 121, 132, and 233 were very similar, as shown on the images in Fig. 3, B, D, and E. Figure 3, A and C, shows phase- contrast images corresponding to Fig. 3, B and D, respectively. With all three MAbs, immunoreactivity (red fluorescence in Fig. 3, B, D, and E) was restricted to a small number of tubules in the cortical labyrinth, whereas tubules in the medullary rays were negative. The pattern of immunostaining on samples obtained from different animals was remarkably similar, and in each case immunoreactivity was abolished by preincubating the antibodies with the immunizing antigen (data not shown).


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Fig. 3.   Immunoreactivity of the 3 anti-HKalpha 2 MAbs in rabbit kidney. A and C: phase-contrast images corresponding to B and D, respectively. B: a small magnification image of a rabbit renal cortex labeled with HKalpha 2 MAb 233 (red fluorescence) and FITC-peanut lectin agglutinin (PNA; green fluorescence). The medullary ray and cortical collecting ducts (CCDs) are negative, whereas the PNA-positive tubules in the cortical labyrinth are positive for MAb 233. D: red fluorescence corresponds to HKalpha 2 MAb 121, green fluorescence to MAb DT.17 that reacts only with principal cells in the CCD. E: confocal image of a rabbit renal cortex labeled with HKalpha 2 MAb 132 (red fluorescence) and FITC-PNA (green fluorescence).

Glomeruli and other cortical tubules, as well as outer and inner medullary collecting tubules, were also negative. Figure 4B illustrates the absence of labeling with the anti-HKalpha 2 MAb in the medulla, whereas Fig. 4C demonstrates that the same tubules stained strongly with MAb ST.12, which labels the entire collecting duct (9, 11).


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Fig. 4.   Lack of immunoreactivity with an anti-HKalpha 2 MAb in the medulla. A: phase-contrast image of the outer medulla. B: corresponding immunofluorescence image of the same section stained with the anti-HKalpha 2 MAb 121 and a Cy-5-labeled 2nd antibody. C: immunofluorescence image of the same section stained with MAb ST.12, a collecting duct marker.

As shown in Fig. 3, B and E, the tubules that reacted with HKalpha 2 antibodies contained cells positive for PNA, a beta -intercalated cell marker in the rabbit (18). beta -Intercalated cells are present in the CNT and CCD. The position (cortical labyrinth) and morphology (arcades) of the HKalpha 2-positive tubules suggest that they are CNTs. This conclusion was confirmed by costaining rabbit kidney sections with MAb DT.17, which reacts exclusively with principal cells in the CCD (10). As shown in Figs. 3D and 5A, cells positive for HKalpha 2 antibodies (red fluorescence) were negative for MAb DT.17 (green fluorescence), indicating that HKalpha 2 in the rabbit is expressed in the CNT but not in the CCD. Immunoreactivity with all three anti-HKalpha 2 MAbs was restricted to the apical membrane. The fact that we never observed basolateral staining even with MAb 121 that recognized a protein in Western blots with heart tissue indicates that these MAbs do not cross-react with renal Na-K-ATPase isoforms.


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Fig. 5.   HKalpha 2 immunofluorescence in rabbit renal cortex. A: red fluorescence, anti-HKalpha 2 MAb 233; green fluorescence, CCD principal cell marker MAb DT.17. B: cross section of a connecting tubule (CNT) labeled with HKalpha 2 MAb 121 (red fluorescence) and a MAb against Ca/Na exchanger, a marker for the CNT (green fluorescence). HKalpha 2 immunoreactivity predominantly localizes to the apical membranes of cells that expressed Ca/Na exchanger in the basolateral membrane. C: confocal image of a region of the renal cortex labeled with HKalpha 2 MAb 121 (red fluorescence) and a MAb specific for the thick ascending limb (green fluorescence). The staining (C) for Tamm-Horsfall protein within the glomerular cells corresponds to red blood cells and is nonspecific.

The identity of the immunoreactive cells as CNT cells was further verified by staining sections with a MAb against a Ca/Na exchanger, a marker for the CNT (23). As shown in Fig. 5B, HKalpha 2 immunoreactivity (red fluorescence) was restricted to the apical membranes of those cells that expressed a Ca/Na exchanger in the basolateral membrane (green fluorescence). HKalpha 2 immunoreactivity was observed in most connecting tubules. Figure 5C illustrates that cells of the thick ascending limb that react with an antibody against Tamm-Horsfall protein, a specific marker for the thick ascending limb of the loop of Henle (green fluorescence), are negative for HKalpha 2 (red fluorescence).

The reactivity within the positive tubules was not homogenous. Surprisingly, HKalpha 2 immunoreactivity was undetectable in either alpha - or beta -intercalated cells. A mutually exclusive staining pattern was observed between the anti-HKalpha 2 MAb 121 and the beta -intercalated cell marker, PNA (Fig. 3, B and E; green fluorescence). Similarly, cells with diffuse cytoplasmic labeling for H-ATPase, which correspond to beta -intercalated cells, were negative for HKalpha 2 (Fig. 6A, arrowhead). No HKalpha 2 immunoreactivity could be detected in alpha -intercalated cells, which were labeled apically with an antibody against H-ATPase (Fig. 6A, green fluorescence, arrows; red fluorescence corresponds to the colonic HKalpha 2 MAb 121). Figure 6B shows a cross section of a CNT, which was costained with three antibodies: MAb 0121 (HKalpha 2; blue fluorescence); F13/483, a MAb that reacts with both CNT and CCD (red fluorescence); and a MAb against the basolateral HCO3/Cl exchanger band 3 (green fluorescence). The purple fluorescence is due to the overlap of blue and red fluorescence, again indicating that the HKalpha 2 antigen is expressed in CNT cells. On the other hand, alpha -intercalated cells (green fluorescence) were negative for HKalpha 2. Cells that are negative for all three markers (arrowheads) most likely correspond to beta -intercalated cells.


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Fig. 6.   Confocal images of CNTs labeled with the anti-HKalpha 2 MAb 121. A: alpha -intercalated cells (strong apical staining with a MAb against the 31-kDa subunit of H-ATPase, green fluorescence; arrows) are negative for HKalpha 2 (red fluorescence). Similarly, cells with diffuse cytoplasmic labeling for H-ATPase, which correspond to beta -intercalated cells, were negative for HKalpha 2 (arrowhead). B: cross section of a CNT costained with three MAbs: MAb 121 (HKalpha 2; blue fluorescence); F13/483, a MAb that reacts with both CNT and CCD (red fluorescence); and a MAb against the basolateral HCO3/Cl exchanger, band 3 (green fluorescence). The purple fluorescence is due to the overlap of blue and red fluorescence, indicating that the HKalpha 2 antigen is expressed in connecting tubule cells. alpha -Intercalated cells (green fluorescence) were negative for HKalpha 2. Cells that are negative for all 3 markers (arrowheads) most likely correspond to beta -intercalated cells.

In the kidney, just like in the colon, no immunoreactivity was observed with MAb 11 (data not shown), which was also negative on Western blots (Fig. 1).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although expression of H-K-ATPases in the kidney is well established, the exact cellular localization and function mediated by the individual H-K-ATPase isoforms are still unclear. The focus of the present study is the colonic H-K-ATPase, which, according to a recent study with knockout mice, plays a role in K conservation in the colon during K deprivation (20). Results of mRNA localization studies indicate that HKalpha 2 is expressed in the CNT (1, 13), CCD (2, 13, 14), OMCD, and inner medullary collecting duct (IMCD) (2).

The major finding of this study is that the HKalpha 2 protein is present in the apical membrane of connecting tubule cells in rabbit kidney. This conclusion is based on results obtained with MAbs that seem to be directed against different epitopes on the rabbit HKalpha 2 subunit. The specificity of these antibodies was confirmed by immunoblotting (Fig. 1) and specific immunohistochemical reaction on distal colon sections (Fig. 2).

The observation that in the rabbit kidney HKalpha 2 is expressed in the apical membrane of CNT cells was unexpected for several reasons. First, functional studies indicated that H-K-ATPase activity is present in intercalated cells (28, 30, 33), where it was assumed to participate in luminal acidification and K+ reabsorption. However, in the rabbit kidney, we did not observe immunoreactivity in intercalated cells that were clearly labeled with various cell-specific markers (Figs. 3-6). On the other hand, our data that connecting tubular cells express HKalpha 2 protein are in agreement with earlier in situ hybridization studies in rats showing strong reactivity in CNT (1). However, in the same report (1), mRNA signals were observed in the medulla, whereas we failed to detect HKalpha 2 protein anywhere else than in a subpopulation of cells within the CNT. Our data also differ from those obtained with a polyclonal antibody generated against the NH2-terminal region of the rat colonic H-K-ATPase (25). In that study, no staining was observed in the CNT or any other cortical tubule of rats on a normal K+ diet, whereas principal cells in the OMCD were labeled (25). Similarly, a recent report showed that immunoblots from rats subjected to chronic hypokalemia reacted with an anti-HKalpha 2 antibody, whereas the cortex remained negative (7).

There are several possible explanations for the apparent discrepancies between our data and those cited above. First, the diverging results from the studies by Ahn and coworkers (1) might be related to the different methods applied (detection of mRNA in their study vs. immunohistochemistry in ours). The discrepancies between earlier reports (1, 7, 25) and our findings might also be related to species differences. The rat and the rabbit have very different natural diets and metabolism, with respect to both K+ intake and acid excretion. Because the relative contribution of H-K-ATPases to K+ and acid-base homeostasis might vary, depending on the localization, it would seem logical that the intrarenal or cellular localization of this enzyme varies in species with different needs, to conserve K+ or secrete acid.

A further possibility is that the antibodies used in this study vs. those used by Sangan et al. (25) recognize different splice variants of HKalpha 2 (17). The splicing pattern is different between rat (17) and rabbit (6). Although the antibodies used in this study were directed against a region that is common to the two splice variants of HKalpha 2 in the rabbit, HKalpha 2a and HKalpha 2c (6), it is not clear that the anti-rat antibodies are also directed against shared epitopes.

It is also possible that the HKalpha 2 antigens are not equally accessible to our MAbs in all nephron segments. If this were the case, one would expect that antigen-unmasking techniques would reveal immunoreactivity at additional sites. However, using an antigen-retrieval method (5) we failed to reveal additional immunoreactivity (data not shown). Finally, although with the above described immunohistochemical techniques we could not detect any labeling in the CCD, OMCD, and IMCD of rabbit kidneys, these data do not exclude the possibility that HKalpha 2 is expressed in these segments at lower levels.

What might be the function of an H-K-ATPase located in the apical membrane of connecting tubule cells? A recent study with knockout mice indicates that, under normal dietary conditions, the elimination of this isoform does not result in major consequences in either K or acid-base balance. However, if these mice are K deprived, they lose excessive amounts of K, suggesting that the main function of the colonic H-K-ATPase is K conservation (20). The CNT and CCD exhibit net K secretion, whereas an apical H-K-ATPase is expected to reabsorb K. One possibility is that an H-K-ATPase in the apical membrane of CNT cells might participate in K+ recycling, thereby decreasing the obligatory K+ loss associated with Na+ reabsorption by the kidney. Such an idea is compatible with the observations of Wang and coworkers (32) that HKalpha 2 mRNA expression is upregulated by Na depletion in the renal cortex. H-K-ATPase expressed in the apical membrane of K+-secreting cells might also exert a local "paracrine" effect by acidifying the luminal microenvironment. Because an acidic luminal pH inhibits K+ secretion (4), an H-K-ATPase, besides recycling secreted K, may also limit K secretion itself. Such a mechanism might be particularly beneficial under conditions of K deprivation.

Another possible role of the colonic H-K-ATPase is to mediate enhanced secretion of NH<UP><SUB>4</SUB><SUP>+</SUP></UP> (and H+) into the lumen, similarly as in IMCD as suggested by a recent paper by Nakamura and co-workers (21).

In summary, our immunohistochemical data obtained with MAbs directed against different epitopes on the rabbit colonic H-K-ATPase indicate that, in the rabbit kidney under normal dietary conditions, the HKalpha 2 protein is expressed in the apical membrane of connecting tubule cells. The function of the colonic H-K-ATPase in this nephron segment remains to be identified.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-39523, DK-55845, and DK-41841.


    FOOTNOTES

Address for reprint requests and other correspondence: G. Fejes-Tóth, Dept. of Physiology, Dartmouth Medical School, Lebanon, NH 03756 (E-mail: geza.fejes-toth{at}dartmouth.edu).

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.

Received 2 August 2000; accepted in final form 17 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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