Nephrology and Hypertension, Department of Veterans Affairs Medical Center, Gainesville, Florida 32608-1197
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ABSTRACT |
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The rabbit kidney
possesses mRNA for the H-K-ATPase 1-subunit
(HK
1) and two splice variants of the H-K-ATPase
2-subunit (HK
2). The purpose of this
study was to determine the specific distribution of one of these, the
H-K-ATPase
2c-subunit isoform (HK
2c), in
rabbit kidney by immunohistochemistry. Chicken polyclonal antibodies
against a peptide based on the NH2 terminus of
HK
2c were used to detect HK
2c
immunoreactivity in tissue sections. Immunohistochemical localization
of HK
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
HK
2c with carbonic anhydrase II, the
Cl
/HCO
1 indicated that both type A and type B intercalated
cells possessed intense apical HK
2c immunoreactivity,
whereas principal cells and connecting segment cells had only a thin
apical band of HK
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 HK
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
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INTRODUCTION |
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THE KIDNEY IS UNIQUE IN
THAT it expresses more than one H-K-ATPase -subunit isoform.
Whereas the stomach expresses only the H-K-ATPase
1
(HK
1)-isoform and the colon expresses only the H-K-ATPase
2 (HK
2)-isoform, both genes
are transcribed in the kidney. Identification of the specific cell
types that express HK
1 or HK
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 HK
1-subunit in the intercalated cells of both rat and rabbit collecting duct has been provided by
immunohistochemical colocalization with the
Cl
/HCO
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 HK
1-subunit (1), the HK
2-subunit (2), and
the HK
-subunit (6) has been detected in intercalated
cells. In these experiments, principal cells were only weakly labeled
for HK
1 and HK
mRNA (1, 6), whereas
principal cells of the rat cortical collecting duct (CCD) were more
strongly labeled for HK
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 HK2 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 HK
2a and
HK
2c. The HK
2c isoform is identical to
HK
2a throughout most of the coding sequence, but
HK
2c possesses a unique NH2-terminal
sequence of 61 amino acids (5). The functional roles of
both of these HK
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 HK2c in renal transport
processes by determining the cellular distribution of HK
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
HK
2c and colocalized HK
2c
immunoreactivity with established markers for principal cells and
intercalated cells of the collecting duct.
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MATERIALS AND METHODS |
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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
HK2c-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 HK
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.
Localization of HK2c 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 HK
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-HK
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 HK2c 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-HK
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-HK
2c antibody. The
sections were washed with glass-distilled water, dehydrated with
xylene, mounted using Permount, and observed by light microscopy.
Colocalization of HK2c and CA II immunoreactivity.
For colocalization of HK
2c and CA II, the
immunohistochemical procedure described previously for the localization
of HK
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
HK
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 HK2c and NaK
1
immunoreactivity.
For colocalization of HK
2c and NaK
1, the
immunohistochemical procedure described previously for the localization
of HK
2c was repeated, and then the
anti-NaK
1 antibody was used in the second
immunolocalization procedure in place of the anti-band 3 antibody.
Specifically, after the localization of HK
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-NaK
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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Immunohistochemical localization of HK2c.
Immunohistochemical localization demonstrated HK
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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Colocalization studies.
Colocalization of immunoreactivity for HK2c and CA II,
kidney AE1, and NaK
1 was done to confirm the
identification of HK
2c-positive cells in the cortex and
outer medulla. Cytoplasmic CA II immunoreactivity was used to identify
intercalated cells. We observed apical HK
2c immunoreactivity in the vast majority of CA II-positive cells, that is,
in virtually all intercalated cells. Apical HK
2c
immunoreactivity was also present in CA II-negative cells, but the
intensity and width of the apical HK
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 HK
2c
immunoreactivity represent intercalated cells, and the cells with the
thin apical band of HK
2c immunoreactivity were principal
cells or, in the CNT, connecting segment cells.
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DISCUSSION |
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The findings of this study demonstrate that a unique and novel
splice variant of the HK2 gene, HK
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
HK
2c in intercalated cells compared with only a narrow
apical band of HK
2c in the apical region of principal cells and connecting segment cells. Finally, the evidence for HK
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, HK2c
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 HK2c
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 HK2c
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 HK
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 HK2c 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
HK
2 (2). These latter studies reported signal for HK
2 mRNA in the rat cTAL, using a probe that
would detect both HK
2c as well as HK
2a.
Furthermore, the heterogeneous distribution of HK
2c
immunoreactivity that we observed may be due to differences in the
abundance of HK
2c in different cell types, which have
been described in the cTAL (3).
In experiments using one of the anti-HK2c 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 HK
2c, as suggested by our
observations, could be responsible for these physiological findings.
Our immunohistochemical evidence for HK2c 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 HK2c 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 HK
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.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the technical assistance of Melissa Lewis, Jeannette Lynch, Lance Parker, and Wendy Wilber.
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FOOTNOTES |
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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.
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