Immunohistochemical localization of H-K-ATPase alpha 2c-subunit in rabbit kidney

Jill W. Verlander, Robin M. Moudy, W. Grady Campbell, Brian D. Cain, and Charles S. Wingo

Nephrology and Hypertension, Department of Veterans Affairs Medical Center, Gainesville, Florida 32608-1197


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The rabbit kidney possesses mRNA for the H-K-ATPase alpha 1-subunit (HKalpha 1) and two splice variants of the H-K-ATPase alpha 2-subunit (HKalpha 2). The purpose of this study was to determine the specific distribution of one of these, the H-K-ATPase alpha 2c-subunit isoform (HKalpha 2c), in rabbit kidney by immunohistochemistry. Chicken polyclonal antibodies against a peptide based on the NH2 terminus of HKalpha 2c were used to detect HKalpha 2c immunoreactivity in tissue sections. Immunohistochemical localization of HKalpha 2c revealed intense apical immunoreactivity in a subpopulation of cells in the connecting segment, cortical collecting duct, and outer medullary collecting duct in both the outer and inner stripe. An additional population of cells exhibited a thin apical band of immunolabel. Immunohistochemical colocalization of HKalpha 2c with carbonic anhydrase II, the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger AE1, and HKalpha 1 indicated that both type A and type B intercalated cells possessed intense apical HKalpha 2c immunoreactivity, whereas principal cells and connecting segment cells had only a thin apical band of HKalpha 2c. Labeled cells were evident through the middle third of the inner medullary collecting duct in the majority of animals. Immunolabel was also present in papillary surface epithelial cells, cells in the cortical thick ascending limb of Henle's loop (cTAL), and the macula densa. Thus in the rabbit kidney, apical HKalpha 2c is present and may contribute to acid secretion or potassium uptake throughout the connecting segment and collecting duct in both type A and type B intercalated cells, principal cells, and connecting segment cells, as well as in cells in papillary surface epithelium, cTAL, and macula densa.

hypokalemia; anatomy; renal function


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE KIDNEY IS UNIQUE IN THAT it expresses more than one H-K-ATPase alpha -subunit isoform. Whereas the stomach expresses only the H-K-ATPase alpha 1 (HKalpha 1)-isoform and the colon expresses only the H-K-ATPase alpha 2 (HKalpha 2)-isoform, both genes are transcribed in the kidney. Identification of the specific cell types that express HKalpha 1 or HKalpha 2 gene products has been the focus of a number of studies from separate laboratories, and in some cases the data are conflicting. Evidence for the presence of the HKalpha 1-subunit in the intercalated cells of both rat and rabbit collecting duct has been provided by immunohistochemical colocalization with the Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger AE1 (23). HKalpha 2 immunoreactivity has been detected in principal cells in the rat outer medullary collecting duct (OMCD) but not in intercalated cells (15). In experiments using in situ hybridization, mRNA for the HKalpha 1-subunit (1), the HKalpha 2-subunit (2), and the HKbeta -subunit (6) has been detected in intercalated cells. In these experiments, principal cells were only weakly labeled for HKalpha 1 and HKbeta mRNA (1, 6), whereas principal cells of the rat cortical collecting duct (CCD) were more strongly labeled for HKalpha 2 mRNA (2). Weak labeling in the thick ascending limb was also reported in all of the studies using in situ hybridization (1, 2, 6).

It is now apparent that the HKalpha 2 gene of both rat and rabbit gives rise to two distinct mRNA species and protein products as a result of alternative splicing (5, 10). In the rabbit, these two splice variants are referred to as HKalpha 2a and HKalpha 2c. The HKalpha 2c isoform is identical to HKalpha 2a throughout most of the coding sequence, but HKalpha 2c possesses a unique NH2-terminal sequence of 61 amino acids (5). The functional roles of both of these HKalpha 2 splice variants are presently unknown.

H-K-ATPase activity has been detected in various renal tubule segments by physiological and enzymatic methods. Although differences among the H-K-ATPase activities have been detected with respect to sensitivity to inhibitors and response to potassium depletion, the molecular identities and the cellular distribution of the proteins responsible for these specific activities have not been unequivocally established. Thus the purpose of the present study was to begin to identify the potential role of HKalpha 2c in renal transport processes by determining the cellular distribution of HKalpha 2c in the kidney. To do so, we performed immunohistochemistry using anti-peptide antibodies to a sequence in the NH2-terminal portion that is specific for HKalpha 2c and colocalized HKalpha 2c immunoreactivity with established markers for principal cells and intercalated cells of the collecting duct.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Female New Zealand White rabbits, weighing 1.5-2.0 kg, were used in this study (n = 6). Animals were maintained on tap water and standard rabbit chow ad libitum. The rabbits were anesthetized with ketamine-HCl (66.7 mg/kg im; Ketaset, Fort Dodge Laboratories) and xylazine-HCl (6.7 mg/kg im; Butler), followed by pentobarbital sodium (10-30 mg/kg iv) as needed to maintain an adequate plane of anesthesia. The kidneys were fixed by retrograde aortic perfusion with periodate-lysine-2% paraformaldehyde (12), cut transversely into several 2- to 4-mm-thick slices, and immersed overnight at 4°C in the same fixative.

Antibodies. Chicken polyclonal antibodies were used for immunolocalization of the HKalpha 2c-subunit (Lofstrand Laboratories, Bethesda, MD). These antibodies were raised against a synthetic peptide (Univ. of Florida ICBR Protein Core, Gainesville, FL) corresponding to amino acids 13-25 of the HKalpha 2c protein (CGEERKEGGGRWRA). Two chickens were inoculated and then received boost inoculations at 21-day intervals until the final bleed at day 73. Antisera from the two were designated LLC26 and LLC27. Eggs were collected during the 2-wk interval preceding the final bleed. Yolks were pooled, then purified using the EGGstract IgY Purification System (Promega, Madison, WI). Purified yolks from the two were designated LLC26egg and LLC27egg. The majority of the immunohistochemistry experiments were performed using the LLC26egg antibody.

Immunolocalization of kidney AE1 was accomplished using a mouse monoclonal antibody against human erythrocyte band 3 protein (IVF-12) that was kindly provided by Dr. Michael Jennings (Univ. of Arkansas, Little Rock, AR). We have used this antibody in several studies previously to localize the basolateral Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger in rabbit type A and medullary intercalated cells (16, 19).

Immunolocalization of carbonic anhydrase II (CA II) was performed using a mouse monoclonal antibody (7C6) raised against chicken CA II, which was kindly provided by Dr. Paul Linser (Whitney Marine Laboratory, Univ. of Florida, Gainesville, FL). We have used this antibody previously to identify intercalated cells in rabbit kidney (19).

Immunolocalization of Na-K-ATPase alpha 1 (NaKalpha 1)-subunit was performed using a commercially available mouse monoclonal antibody against rabbit NaKalpha 1 (Upstate Biotechnology, Lake Placid, NY).

Localization of HKalpha 2c immunoreactivity. Samples of kidney from each animal were embedded in polyester wax, and 5-µm sections were cut and mounted on glass slides coated with gelatin. Immunolocalization of HKalpha 2c was accomplished using immunoperoxidase procedures and a commercially available kit (Vectastain Elite, Vector Laboratories, Burlingame, CA). The slides were dewaxed in ethanol and rehydrated, then microwaved at medium heat in 0.1 M sodium citrate, 0.1 M citric acid, pH 6.0, for 10 min. The sections were rinsed in PBS, treated for 15 min with 5% goat (Vector Laboratories) or donkey serum (Jackson ImmunoResearch Laboratories, West Grove, PA) in PBS, then incubated at 4°C overnight with the anti-HKalpha 2c antibody, LLC26egg diluted 1:1,000 to 1:8,000 in PBS or LLC27 serum diluted 1:1,000 to 1:4,000. The sections were then washed in PBS, and endogenous peroxidase activity was blocked by incubating the sections in 0.3% H2O2 for 30 min. The sections were washed in PBS, incubated for 30 min with either a biotinylated goat anti-chicken IgG (IgY) secondary antibody (Vector Laboratories) or biotin-Sp-affinipure donkey anti-chicken IgG (Jackson ImmunoResearch Laboratories) diluted 1:200 in PBS, and then washed again with PBS. The sections were treated for 30 min with the avidin-biotin complex reagent, rinsed with PBS, then exposed to diaminobenzidine (0.5 mg/ml) in imidazole buffer (3.4g/l imidazole in 0.05 Tris buffer, pH 7.6) with 0.3% H2O2. The sections were washed in distilled water and counterstained with hematoxylin. The sections were then dehydrated with xylene, mounted using Permount (Fisher Scientific, Fair Lawn, NJ), and observed by light microscopy.

Colocalization of HKalpha 2c and kidney AE1 immunoreactivity. Colocalization was accomplished using sequential immunoperoxidase procedures and a commercially available kit (Vectastain Elite). Five-micrometer sections were dewaxed in ethanol and rehydrated, then microwaved at medium heat in 0.1 M sodium citrate, 0.1 M citric acid, pH 6.0, for 10 min. The sections were rinsed in PBS, and endogenous peroxidase activity was blocked by incubation of the sections in 0.3% H2O2 for 30 min. The sections were rinsed in PBS, treated for 20 min with 5% goat or donkey serum in PBS, and then incubated at 4°C overnight with the anti-HKalpha 2c antibody (LLC26egg) diluted 1:1,000 in PBS. The sections were washed in PBS for 1 min, then in 0.1% SDS in PBS for 10 min, and then again in PBS for 1 min. The sections were then incubated for 30 min with the biotinylated goat or donkey anti-chicken IgG secondary antibody diluted 1:200 in PBS, then washed with PBS. The sections were treated for 30 min with the avidin-biotin complex reagent, rinsed with PBS, then exposed to diaminobenzidine (0.5 mg/ml) in imidazole buffer (304 g/l imidazole in 0.05 Tris buffer, pH 7.6) with 0.3% H2O2. The sections were washed in glass-distilled water, then in PBS, and incubated in 0.3% H2O2 for 30 min. The sections were again washed in PBS and incubated for 20 min with 5% normal horse serum in PBS. The sections were treated for 60 min with the anti-band 3 protein (AE1) antibody (IVF-12) diluted 1:100 in PBS, washed in PBS, and incubated with the biotinylated horse anti-mouse secondary antibody. The sections were washed with PBS, incubated with the avidin-biotin complex reagent, and washed with PBS. For detection of kidney AE1 using the anti-band 3 protein antibody, Vector SG (Vector Laboratories) was used as the chromogen to produce a blue label. This label was easily distinguishable from the brown label produced by the diaminobenzidine used for detection of the anti-HKalpha 2c antibody. The sections were washed with glass-distilled water, dehydrated with xylene, mounted using Permount, and observed by light microscopy.

Colocalization of HKalpha 2c and CA II immunoreactivity. For colocalization of HKalpha 2c and CA II, the immunohistochemical procedure described previously for the localization of HKalpha 2c was repeated, then the anti-CA II antibody was used in the second immunolocalization procedure in place of the anti-band 3 antibody. Specifically, after the localization of HKalpha 2c, the sections were washed in PBS and 0.3% H2O2 and treated with 5% normal horse serum as described above, then incubated for 60 min with the anti-CA II antibody diluted 1:10 in PBS. The sections were rinsed, treated with the horse anti-mouse secondary antibody for 30 min, then rinsed with PBS. The sections were incubated with the avidin-biotin complex, rinsed with PBS, and exposed to the chromogen vector SG for 5 min. The sections were washed with glass-distilled water, dehydrated, mounted, and observed by light microscopy.

Colocalization of HKalpha 2c and NaKalpha 1 immunoreactivity. For colocalization of HKalpha 2c and NaKalpha 1, the immunohistochemical procedure described previously for the localization of HKalpha 2c was repeated, and then the anti-NaKalpha 1 antibody was used in the second immunolocalization procedure in place of the anti-band 3 antibody. Specifically, after the localization of HKalpha 2c, the sections were washed in PBS and 0.3% H2O2 and treated with 5% normal horse serum as described, then incubated for 60 min with the anti-NaKalpha 1 antibody diluted 1:400 in PBS. The sections were rinsed, treated with the horse anti-mouse secondary antibody for 30 min, then rinsed with PBS. The sections were incubated with the avidin-biotin complex, rinsed with PBS, and exposed to the chromogen Vector SG for 5 min. The sections were washed with glass-distilled water, dehydrated, mounted, and observed by light microscopy.

Controls. For controls in each of the immunolocalization procedures, preimmune purified yolk protein diluted at the same concentration as the primary antibody in PBS or PBS only was substituted for the primary antibody. In addition, to ensure that the immunolocalization was specific for the antigenic peptide, in some experiments the LLC26egg antibody was incubated with an excess of the peptide before it was applied to the tissue section.

Identification of nephron and collecting duct subsegments. The intercalated and principal cells were identified in the following segments of the collecting duct: the initial collecting tubule (ICT), the CCD, the OMCD outer stripe (OMCDo), the OMCD inner stripe (OMCDi), and the inner medullary collecting duct (IMCD). The CCD segments were contained within the medullary ray. The point of disappearance of the glomeruli and the presence of the arcuate arteries were used to define the corticomedullary junction. The ICT was located outside of the medullary ray. This segment was distinguished from the connecting segment (CNT) on the basis of the height of its epithelium, which is significantly lower than that of the CNT. Occasionally, the transition to the distal convoluted tubule (DCT) was observed and could also be used to identify the CNT. The OMCDo was located in the outer stripe of the medulla, using the point of disappearance of the proximal convoluted tubules as the border between OMCDo and OMCDi. The OMCDi was located in the inner stripe of the outer medulla, using the point of transition from thick ascending limbs to thin limbs as the inner border. The IMCD was located from this transition point to the tip of the papilla, not including the papillary surface epithelium.

The cortical thick ascending limb (cTAL) was identified by its location within the medullary ray and was distinguished from the CCD by the lower height of the epithelium. The macula densa was identified by its characteristic tall, columnar cells clustered at its distinctive location within the cTAL at its contact with the vascular pole of the glomerulus. The medullary thick ascending limb (mTAL) was distinguished from the medullary collecting ducts by the regularity of its profile.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Immunohistochemical localization of HKalpha 2c. Immunohistochemical localization demonstrated HKalpha 2c immunoreactivity in cells throughout the rabbit collecting duct system. In the majority of collecting duct segments, there was heterogeneity in the pattern and intensity of label; however, only apical label was observed. In all experiments, control sections incubated with PBS only or nonimmunized egg yolk protein in place of the primary antibody exhibited no labeling (Fig. 1e). Preincubation of the primary antibody with the antigenic peptide also eliminated all immunolabeling (Fig. 1, f and g). The pattern of immunohistochemical localization in the collecting duct was essentially the same with the LLC26egg and LLC27 serum antibodies. The LLC26egg antibody produced immunolocalization with more intense signal and less diffuse background staining and thus was used for the majority of experiments.


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Fig. 1.   a: Light micrograph of rabbit outer cortical collecting duct. A broad, intense band of apical HKalpha 2c immunoreactivity is present in a subpopulation of cells (arrows) in the collecting duct. The majority of cells (principal cells) exhibit a thin apical band of HKalpha 2c immunoreactivity (arrowheads). b: Higher magnification of a portion of Fig. 1a. c: Light micrograph of medullary ray that demonstrates heterogeneous labeling in the collecting duct (C), as described for Fig. 1a. In addition, the cortical thick ascending limb of Henle's loop (cTAL) exhibits patchy apical immunoreactivity, suggesting heterogeneity in the cellular distribution of HKalpha 2c within the cTAL. Proximal tubules (P) are negative. d: Higher magnification of bottom right corner of Fig. 1c. e: Negative control. Light micrograph of rabbit kidney cortex subjected to the immunohistochemical procedure with nonimmunized egg yolk protein substituted for the primary antibody is shown. No labeling of any structures was observed. f and g: Results of peptide-blocking experiment, with f (positive control) demonstrating immunolocalization in a heterogeneous pattern in connecting segments and cortical collecting ducts (*) as well as in the macula densa (arrow) and g showing results when primary antibody is incubated with excess antigenic peptide before treatment of the tissue section. No immunolabel is present in any structures, including the macula densa (arrow).

In the CCD segments, a clear distinction could be made among cells on the basis of the pattern of immunoreactive label. We consistently observed a subpopulation of cells with a broad, intense apical band of label, whereas the majority of cells exhibited a thin, apical band (Fig. 1, a and c). The fact that the cells with the broad apical immunoreactivity were less abundant and had a more prominent apical profile than the cells with the thin apical band of label suggested that the former cells represented intercalated cells and the latter represented principal cells. This pattern was observed in all segments of the collecting duct within the renal cortex as well as in the CNT. However, the greatest intensity of both patterns of labeling was most often observed in the CNT.

HKalpha 2c immunoreactivity was also observed in the cTAL (Figs. 1, c and d, and 2b). As in collecting duct cells, the immunoreactivity in the cTAL was apical. In general, the label was less prominent than that observed in the collecting duct, and the distribution within cTAL profiles was heterogeneous, suggesting that the abundance of the protein varied among cTAL cells. In addition, in experiments using the LLC26egg antibody, macula densa cells exhibited distinct apical immunolabel (Fig. 2).


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Fig. 2.   a: Light micrograph demonstrating distinct HKalpha 2c immunoreactivity along the apical edge of macula densa cells (arrow) and extending along a portion of the cTAL (arrowhead). b: Lower power light micrograph demonstrating HKalpha 2c immunoreactivity along the apical edge of macula densa cells (arrow) and occasional cTAL cells (arrowheads).

In the OMCDo, the pattern of immunolabeling was similar to that seen in the cortex, although typically the pattern was more discrete than in the CCD. A broad, intense apical band of HKalpha 2c immunoreactivity was observed in a subpopulation of cells that exhibited the characteristic profile of intercalated cells (Fig. 3). The remaining cells typically exhibited a thin, continuous apical band of immunoreactivity (Fig. 3). These cells had a lower, flatter profile than the cells with the broad apical band of immunolabel and constituted the majority of the cells in the OMCDo; thus they were considered to be principal cells.


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Fig. 3.   a: Light micrograph of rabbit outer stripe of outer medullary collecting duct. A broad, intense band of apical HKalpha 2c immunoreactivity is present in a subpopulation of cells (arrows), identified as intercalated cells in other experiments by colocalization with carbonic anhydrase II (CA II) and AE1 immunoreactivity. The majority of cells (principal cells) exhibit a thin apical band of HKalpha 2c immunoreactivity (arrowheads). b: Higher magnification of lower portion of Fig. 3a.

In the OMCDi, the pattern of labeling among tubules was heterogeneous, particularly in the deep inner stripe. In the OMCDi in the outer and middle regions of the inner stripe, apical label for HKalpha 2c was evident in a subpopulation of cells, whereas other cells were negative for HKalpha 2c immunoreactivity (Fig. 4a). The proportion of labeled and unlabeled cells varied widely among individual rabbits and collecting duct profiles. However, in OMCDi profiles located deep in the inner stripe, the majority of cells exhibited apical HKalpha 2c immunoreactivity. In some collecting ducts, there was a continuous band of label along the apical surface of homogeneous cells, whereas in others the collecting duct apical surface exhibited a cobblestone appearance and heterogeneous apical label for HKalpha 2c (Fig. 4b).


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Fig. 4.   a: Mid-inner stripe. In the collecting duct in the middle and outer regions of the inner stripe of the outer medulla, apical labeling for HKalpha 2c was present in a subpopulation of cells (arrows), whereas other cells were negative (arrowheads). The proportion of labeled and unlabeled cells varied among individual rabbits and collecting duct profiles. b: Deep inner stripe. In the deep region of the inner stripe of the outer medulla, the majority of cells in the collecting ducts exhibited apical HKalpha 2c immunoreactivity. In some collecting ducts, there was a continuous band of label along the apical surface of homogeneous cells (large arrowheads), whereas in others, the collecting duct apical surface exhibited a cobblestone appearance and heterogeneous apical label for HKalpha 2c. In these collecting ducts, some cells had intense apical label (small arrows), others were weakly positive (large arrows), and occasional cells were negative (small arrowheads).

In the inner medulla, the pattern of label in the collecting ducts showed regional heterogeneity. In the initial portion of the IMCD (IMCD1), similar to the OMCDi in the deep inner stripe, the pattern of HKalpha 2c immunoreactivity was predominantly a continuous band of apical label in cells with a flat apical profile (Fig. 5a). Occasional cells with a broad apical band of immunoreactivity were observed, similar to those seen in more proximal collecting duct segments and identified as intercalated cells (Fig. 5b). Immunolabeled cells were progressively less frequent in the more distal portions of the IMCD. In the IMCD at the papillary tip, labeled cells were rare or absent.


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Fig. 5.   a: Initial portion of the inner medullary collecting duct (IMCD1). As in the collecting duct in the deep inner stripe of the outer medulla, the pattern of HKalpha 2c immunoreactivity in the IMCD1 was predominantly a continuous band of apical label (arrows). Other cells that appeared to be intercalated cells exhibited a broad, intense apical band of immunolabel (arrowheads). b: Proximal segment of the IMCD (IMCD2). In the more distal segments of the IMCD, cells positive for HKalpha 2c immunoreactivity (arrow) were progressively less frequent and ultimately disappeared.

Immunolabeling was also observed in the cells of the papillary surface epithelium and the pelvic epithelium. The label was most intense and most frequent near the pelvic sulcus and diminished toward the papillary tip. Distinct label was observed in the apical region of individual cells, whereas in other cells the lateral borders appeared to be labeled (Fig. 6).


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Fig. 6.   Light micrograph of papillary surface epithelium in the pelvic sulcus. Cells with intense apical label for HKalpha 2c (arrows) or more diffuse staining with apparent accentuation along the lateral cell borders (arrowheads) were consistently observed in this region of the papillary surface epithelium and extended distally along the papillary surface to a variable extent.

Colocalization studies. Colocalization of immunoreactivity for HKalpha 2c and CA II, kidney AE1, and NaKalpha 1 was done to confirm the identification of HKalpha 2c-positive cells in the cortex and outer medulla. Cytoplasmic CA II immunoreactivity was used to identify intercalated cells. We observed apical HKalpha 2c immunoreactivity in the vast majority of CA II-positive cells, that is, in virtually all intercalated cells. Apical HKalpha 2c immunoreactivity was also present in CA II-negative cells, but the intensity and width of the apical HKalpha 2c label were less than in the CA II-positive cells (Fig. 7, a and b). These findings confirm that the cells exhibiting the broad apical band of HKalpha 2c immunoreactivity represent intercalated cells, and the cells with the thin apical band of HKalpha 2c immunoreactivity were principal cells or, in the CNT, connecting segment cells.


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Fig. 7.   a and b: Light micrographs of rabbit cortex (a) and outer medulla (b) labeled for both HKalpha 2c and CA II. Intercalated cells are identified by cytoplasmic CA II immunoreactivity (blue label). Apical HKalpha 2c immunoreactivity (brown label) is present in both CA II-positive (arrows) and CA II-negative cells (arrowheads), but the intensity and width of the apical HKalpha 2c label are greater in the intercalated cells. c: Light micrograph of rabbit connecting segment labeled for both HKalpha 2c and Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger (AE1) protein. Type A intercalated cells are identified by AE1-immunoreactivity (blue label). Apical HKalpha 2c immunoreactivity (brown label) is present in both AE1-positive (arrows) and AE1-negative cells. Although the majority of AE1-negative cells have only a thin apical band of HKalpha 2C immunolabel (small arrowheads), occasional AE1-negative cells exhibit broad, intense apical HKalpha 2C immunostaining (large arrowheads). d: Light micrograph of rabbit connecting segment labeled for both HKalpha 2c and NaKalpha 1. Connecting segment cells are identified by extensive basolateral NaKalpha 1 immunoreactivity (blue label). Apical HKalpha 2c immunoreactivity (brown label) is present in both NaKalpha 1-positive (arrowheads) and NaKalpha 1-negative cells (arrows). However, the intensity and width of the apical HKalpha 2c label are greater in the NaKalpha 1-negative cells, which represent intercalated cells.

In rabbit kidney labeled for both HKalpha 2c and kidney AE1, type A intercalated cells in the cortex and OMCD intercalated cells were identified by AE1 immunoreactivity. In the renal cortex, a broad apical band of apical HKalpha 2c immunoreactivity was observed in both AE1-positive and some AE1-negative cells (Fig. 7c). The majority of AE1-negative cells exhibited a thin apical band of HKalpha 2c immunoreactivity (Fig. 7c). Because HKalpha 2c and CA II colocalization demonstrated that the vast majority of intercalated cells exhibited broad apical HKalpha 2c immunoreactivity, the presence of some AE1-negative cells with broad apical label for HKalpha 2c is consistent with the interpretation that these represent type B intercalated cells. In the OMCD, where type B intercalated cells are rarely present, only AE1-positive cells exhibited a broad apical band of HKalpha 2c immunoreactivity, whereas AE1-negative cells, i.e., principal cells, had only a thin band of apical label. These findings support the conclusion that the intercalated cells in both the cortical and outer medullary collecting duct segments exhibit more intense HKalpha 2c immunoreactivity than do principal cells or connecting segment cells.

In additional experiments, HKalpha 2c and NaKalpha 1 were colocalized. In the CNT and ICT, connecting segment cells and principal cells were identified by basolateral NaKalpha 1 immunoreactivity. Apical HKalpha 2c immunoreactivity was observed in both NaKalpha 1-positive and NaKalpha 1-negative cells. However, the intensity and width of the apical HKalpha 2c label was typically greater in the NaKalpha 1-negative cells (Fig. 7d). Thus these separate observations support the conclusion that intercalated cells, principal cells, and connecting segment cells have apical HKalpha 2c immunoreactivity but that the protein is more abundant in intercalated cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The findings of this study demonstrate that a unique and novel splice variant of the HKalpha 2 gene, HKalpha 2c (5), is distributed throughout most of the collecting duct system, beginning in the CNT and extending to the IMCD. By morphological criteria and colocalization studies, the heterogeneous labeling pattern observed throughout the collecting duct was attributable to more intense and broader localization of HKalpha 2c in intercalated cells compared with only a narrow apical band of HKalpha 2c in the apical region of principal cells and connecting segment cells. Finally, the evidence for HKalpha 2c in the apical region of the cTAL, the macula densa, and papillary surface epithelium suggests that cell types in addition to intercalated cells may participate in potassium and proton transport via this H-K-ATPase isoform.

Our findings are consistent with physiological, enzymatic, and molecular data from other laboratories that have examined the renal distribution of H-K-ATPase. First, we observed the most intense immunoreactivity in the CNT and ICT, which correlates with data from studies of enzyme activity that found the highest K-ATPase activity in these segments (8, 9).

Second, our data suggest that both type A and type B intercalated cells exhibit apical, but not basolateral, HKalpha 2c immunoreactivity. These observations are supported by several physiological studies (13, 21, 24). It has been demonstrated that luminal inhibition of H-K-ATPase activity inhibits intracellular pH recovery by both type A and type B intercalated cells, suggesting the presence of apical H-K-ATPase in both cell types (13, 21). The same investigators found no evidence for a basolateral H-K-ATPase in the type B intercalated cell (21). The presence of an apical H-K-ATPase in type B intercalated cells is also consistent with physiological studies of the rabbit CCD that demonstrate inhibition of chloride absorption by luminal Sch-28080 (24). In contrast, H-ATPase transport activity (13, 21) and immunoreactivity (17, 18) have been localized to the apical plasma membrane in type A intercalated cells and to the basolateral plasma membrane in type B intercalated cells.

The finding that both the intercalated cells and the principal cells of the OMCDi exhibit immunoreactivity for HKalpha 2c is supported by physiological observations that all cells in this segment exhibit H-K-ATPase activity, defined as ethylisopropylamiloride-insensitive pH recovery that was inhibited by both luminal potassium removal and by Sch-28080 (22). In addition, the rate of pH recovery attributable to H-K-ATPase activity was significantly greater in intercalated cells than in principal cells (22), in keeping with the greater intensity of immunoreactivity that we observed in intercalated cells compared with principal cells.

In the IMCD1, the incidence of cells with the appearance of intercalated cells that exhibited strong apical HKalpha 2c immunoreactivity is consistent with the occurrence of intercalated cells in the the IMCD1 of the rabbit determined by immunocytochemical studies (11). In addition, many cells in the IMCD1 and the more proximal portion of the papillary IMCD (IMCD2) exhibited a thin band of HKalpha 2c immunoreactivity. No physiological studies of acid or potassium transport by the rabbit IMCD have been reported. However, studies of the isolated perfused IMCD of rats with chronic metabolic acidosis demonstrated a luminal proton secretory process that was inhibited ~50% by luminal addition of 10 µM Sch-28080 or removal of luminal K but was not inhibited by 5 nM bafilomycin A1 (20). These findings suggest the presence of apical H-K-ATPase activity in this segment (20).

Our observation of HKalpha 2c immunoreactivity in the cTAL may correspond to the physiological data that demonstrate a Sch-28080-sensitive K-ATPase activity in the TAL that is ouabain sensitive but distinct from Na-K-ATPase (4). Inhibition of this enzyme, referred to as type II K-ATPase, requires concentrations of Sch-28080 approximately sevenfold higher than does the collecting duct type I K-ATPase (IC50 of 1.7 µM for TAL type II K-ATPase vs. 0.25 µM for collecting duct type I K-ATPase) (4). These data are in agreement with studies in the rat kidney using in situ hybridization to determine the distribution of HKalpha 2 (2). These latter studies reported signal for HKalpha 2 mRNA in the rat cTAL, using a probe that would detect both HKalpha 2c as well as HKalpha 2a. Furthermore, the heterogeneous distribution of HKalpha 2c immunoreactivity that we observed may be due to differences in the abundance of HKalpha 2c in different cell types, which have been described in the cTAL (3).

In experiments using one of the anti-HKalpha 2c antibodies (LLC26egg), we also observed immunoreactivity in the apical region of the macula densa, the specialized group of cells in the cTAL that is involved in tubuloglomerular feedback and control of afferent arteriolar resistance. Recent findings from Dr. Darwin Bell's laboratory (Univ. of Alabama at Birmingham) indicate that these cells possess a luminal, but not basolateral, ouabain-sensitive sodium extrusion in macula densa cells attributable to an H-K-ATPase activity (14). An apical HKalpha 2c, as suggested by our observations, could be responsible for these physiological findings.

Our immunohistochemical evidence for HKalpha 2c in the papillary surface epithelium is also consistent with physiological studies (7). In studies of acid transport by this structure, the rabbit papillary surface epithelium acidified at ~60% of the rate of the OMCDi, and removal of apical potassium inhibited a component of proton secretion (7).

In conclusion, our findings indicate that HKalpha 2c is present in the apical region of the majority of cells throughout the collecting duct and in cells of the papillary surface epithelium, where it may mediate contributions to both potassium and acid-base balance by these structures. In addition, the presence of HKalpha 2c in the cTAL and macula densa suggests a novel role for this protein not only in electrolyte homeostasis but also in the regulation of glomerular filtration.


    ACKNOWLEDGEMENTS

We gratefully acknowledge the technical assistance of Melissa Lewis, Jeannette Lynch, Lance Parker, and Wendy Wilber.


    FOOTNOTES

These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49750 (C. S. Wingo) and funds from the Department of Veteran Affairs Merit Review Program (C. S. Wingo). They were presented in part at the 31st Annual Meeting of the American Society of Nephrology in Philadelphia, PA, 1998 and were published in abstract form (J Am Soc Nephrol 9: 13A, 1998).

Address for reprint requests and other correspondence: C. S. Wingo, Nephrology and Hypertension (111G), Dept. of Veterans Affairs Medical Center, Gainesville, FL 32608-1197 (E-mail: wingocs{at}ufl.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 18 October 1999; accepted in final form 20 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 281(2):F357-F365