Download ATPase isoforms in human kidney - AJP

Am J Physiol Renal Physiol
281: F763–F768, 2001.
Detection and localization of H⫹-K⫹-ATPase isoforms
in human kidney
JEFFREY A. KRAUT,1,2 KERSTIN G. HELANDER,3 HERBERT F. HELANDER,2,4
NGOZI D. IROEZI,1,2 ELIZABETH A. MARCUS,2 AND GEORGE SACHS2
1
Division of Nephrology and 2Membrane Biology Laboratory, Research Service and Department of
Medicine, Veterans Affairs Greater Los Angeles Health Care System, and University of California
School of Medicine, Los Angeles, California 90073; 4AstraZeneca, Mölndal, Sweden;
and 3Department of Biomedical Laboratory Science, University of Göteborg, Göteborg, Sweden
Received 12 January 2001; accepted in final form 29 May 2001
-K⫹-ATPASE RESPONSIBLE for acidification of the
gastric lumen (HK␣1) is a P2-type ATPase that consists
of an ␣-subunit that subserves the catalytic function of
the enzyme and a glycosylated ␤-subunit that plays a
role in stabilization, maturation, and targeting of the
enzyme to the plasma membrane and perhaps in K
countertransport (12, 14, 24, 25, 33). Several additional isoforms of the ␣-subunit of the H⫹-K⫹-ATPase
have been described: HK␣2a was cloned from rat colon
(8, 9) and truncated versions, HK␣2b and HK␣2c (4, 20),
were cloned from rat and rabbit kidney, respectively;
HK␣3 was cloned from toad bladder (18); and HK␣4
(17), also termed ATP1AL1, was cloned from a human
skin library. All isoforms share a high homology with
each other at the amino acid level (60–87%) (5).
Previous studies performed in the rat and rabbit using
RT-PCR, in situ hybridization, and immunostaining have
localized two of the isoforms, HK␣1 and HK␣2, to principal and intercalated cells of the collecting ducts (11–13,
22, 32). Furthermore, microperfusion of isolated cortical
and medullary collecting ducts of both species has shown
that hydrogen and potassium transport by these segments is attenuated by exposure to SCH-28080, a specific
inhibitor of HK␣1 (15, 25, 31) and ouabain, an inhibitor of
HK␣2a, HK␣2b, and HK␣2c, at high concentrations (1 M)
(23, 25). These results indicate a probable role for the
H⫹-K⫹-ATPase in the transport of hydrogen and potassium by the rat and rabbit kidney.
The potential role of an H⫹-K⫹-ATPase in the regulation of acid-base and potassium balance by the human kidney remains unexplored. The present studies
were designed to examine whether various isoforms of
the H-K⫹-ATPase are present in the human kidney,
and, if so, to determine their location using immunocytochemical techniques. The results of these studies
demonstrate that HK␣1 and HK␣4 are present in the
cortical and medullary collecting ducts of the human
kidney. Both isoforms are primarily present within
intercalated cells and to a lesser extent in principal
cells. The results of these studies are consistent with a
role of the H⫹-K⫹-ATPase in regulation of potassium
and hydrogen balance by the human kidney. The precise functional role of the renal H⫹-K⫹-ATPase in the
human remains to be determined.
Address for reprint requests and other correspondence: J. A.
Kraut, Division of Nephrology 691/111L, VA Greater Los Angeles
Health Care System, 11301 Wilshire Blvd., Los Angeles, CA 90073
(E-mail: [email protected]).
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.
renal hydrogen-potassium-adenosinetriphosphatase; hydrogen; potassium; immunohistochemistry
THE H
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Kraut, Jeffrey A., Kerstin G. Helander, Herbert F.
Helander, Ngozi D. Iroezi, Elizabeth A. Marcus, and
George Sachs. Detection and localization of H⫹-K⫹-ATPase
isoforms in human kidney. Am J Physiol Renal Physiol 281:
F763–F768, 2001.—An H⫹-K⫹-ATPase contributes to hydrogen secretion and potassium reabsorption by the rat and
rabbit collecting ducts. Transport of these ions appears to be
accomplished by one or both of two isoforms of the H⫹-K⫹ATPase, HK␣1 and HK␣2, because both isoforms are found in
the collecting ducts and transport of hydrogen and potassium
is attenuated by exposure to inhibitors of these transport
proteins. To evaluate whether an H⫹-K⫹-ATPase is present
in the human kidney, immunohistochemical studies were
performed using normal human renal tissue probed with
antibodies directed against epitopes of three of the known
isoforms of the H⫹-K⫹-ATPase, HK␣1, HK␣2, and HK␣4, and
the V-type H⫹-ATPase. Cortical and medullary tissue probed
with antibodies against HK␣1 showed cytoplasmic staining of
intercalated cells that was less intense than that observed in
the parietal cells of normal rat stomach stained with the
same antibody. Also, weak immunoreactivity was detected in
principal cells of the human collecting ducts. Cortical and
medullary tissue probed with antibodies directed against
HK␣4 revealed weak, diffuse staining of intercalated cells of
the collecting ducts and occasional light staining of principal
cells. Cortical and medullary tissue probed with antibodies
directed against the H⫹-ATPase revealed staining of intercalated cells of the collecting ducts and some cells of the
proximal convoluted tubules. By contrast, no discernible
staining was noted with the use of the antibody against
HK␣2. These data indicate that HK␣1 and HK␣4 are present
in the collecting ducts of the human kidney. In this location,
these isoforms might contribute to hydrogen and potassium
transport by the kidney.
RENAL H⫹-K⫹-ATPASE IN HUMAN KIDNEY
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MATERIALS AND METHODS
Fig. 1. Immunoreactivity in renal sections probed with antibody 1218 directed against H⫹-K⫹-ATPase isoform
(HK␣1). A: renal cortex. B: renal medulla. In both areas of the kidney, immunostaining was localized to the collecting ducts (C), where intercalated
cells (arrow) generally stained more
intensely than principal cells. P, proximal tubule. The concentration of antibody 1218 was 2 ␮g/ml. The streptavidin-biotin-tyramide method (CSA;
DAKO) was used. Bar ⫽ 100 ␮m. C:
confocal images of renal medulla
probed with fluorescent-tagged 1218
antibody. Fluorescence was primarily
observed in cytoplasmic vesicles. Bar ⫽
10 ␮m. D: confocal images of renal medulla stained with mouse serum. No
fluorescence was detected in the
sections.
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Samples. For immunohistochemical analysis of normal
human kidney, tissue samples were obtained from tumorfree regions of nephrectomy specimens removed for treatment of localized renal cell carcinoma. Sections from the
tissues were examined histologically before immunostaining
to confirm the absence of residual tumor tissue. The protocol
was approved by the Institutional Review Board of the
Greater Los Angeles Veterans Affairs Health Care System.
Archival specimens of normal rat stomach were utilized to
confirm the sensitivity of the antibody against HK␣1.
Antibodies. For detection of HK␣1, antibody 1218, a monoclonal antibody directed against the separated ␣-subunit (26)
(generously provided by Dr. A Smolka, Univ. of South Carolina, Charleston, SC), was used in dilutions ranging from 0.4
to 10 ␮g/ml. For detection of HK␣4, a rabbit polyclonal antibody generated against a fusion protein containing the sequence from serine-14 to leucine-39 of the human ATP1AL1
sequence was utilized (kindly provided by Dr. Michael
Caplan, Yale Univ., New Haven, CT). For detection of H⫹ATPase, a rabbit polyclonal antibody generated against a
synthetic peptide, Cys-Pro-Gln-Asp-Thr-Glu-Ala-Asp-ThrAla-Leu, from the NH2 terminus of the bovine kidney 56-kDa
subunit of the vacuolar H⫹-ATPase, was used (kindly provided by Dr. Ira Kurtz, UCLA School of Medicine, Los Angeles, CA). For detection of HK␣2, a polyclonal antibody generated against a synthetic peptide consisting of amino acids
686–698 of the putative rat HK␣2 was used (21) (kindly
provided by Dr. A. Smolka)
Immunohistochemistry. Tissue specimens were fixed in
buffered 4% formaldehyde dehydrated in ethanol and embedded in paraffin. Four-micrometer sections were cut in a
microtome and placed on glass slides (SuperFrostPlus, Menzel). Tissue sections were deparaffinized in xylene and rehydrated in decreasing concentrations of ethanol, followed by
distilled water. Preliminary studies demonstrated weak
staining or no staining with conventional immunohistochemical techniques. Therefore, studies were performed using
antigen-retrieval methods that serve to unmask fixed
epitopes by making them accessible to the primary antibody.
Briefly, sections were placed in Target Retrieval Solution
(DAKO, Carpinteria, CA), pH 6.1, in a water bath at 95°C for
20 min and then allowed to reach near-room temperature
over 20 min. This procedure resulted in a considerable increase in immunoreactivity of the sections. To further enhance the immunohistochemical signal, two highly sensitive
techniques were used: a streptavidin-biotin method (LSAB⫹;
DAKO) and a streptavidin-biotin-tyramide method (CSA;
DAKO). Briefly, for the biotin-streptavidin method, the sections were rinsed in Tris-buffered saline (TBS) and then
covered with the primary antibody diluted with 1% BSA in
TBS buffer, usually for 15 min. Biotinylated secondary antibodies were then added, followed by streptavidin conjugated
to alkaline phosphatase. Visualization depended on the precipitation of fuchsia-colored New Fuchsin. The sections were
thoroughly rinsed with TBS between each step. For the
streptavidin-biotin-tyramide method, the sections were first
exposed to 3% H2O2 to block endogenous peroxidase activity,
then covered with a protein block. They were subsequently
exposed for 30 min to the primary monoclonal antibody
diluted with TBS, followed by biotinylated rabbit anti-mouse
IgG. The streptavidin, biotin, and tyramide were then added
as recommended by the manufacturer (DAKO). Finally, in
the presence of H2O2, peroxidase coupled to streptavidin
catalyzed the precipitation of diaminobenzidine, resulting in
a brown color. Rinsing between the various steps, except for
the step between the protein block and the primary antibody
where no rinsing took place, was carried out using TBS
containing NaCl and Tween 20.
Controls. For negative controls, primary antibodies were
replaced by normal mouse serum or normal rabbit IgG,
respectively, diluted to the same protein concentrations as
the respective antisera. Additional negative controls were
performed with DAKO’s mouse negative control antibody and
with 50 mM Tris buffer, pH 7.4, replacing the primary antibodies.
All procedures, except the antigen retrieval, were carried
out at room temperature. For contrast, the sections were
RENAL H⫹-K⫹-ATPASE IN HUMAN KIDNEY
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RESULTS
Fig. 2. Immunoreactivity in rat gastric corpus probed with antibody
1218 (0.4 ␮g/ml). Immunoreactivity was prominent in the parietal
cells and more intense than that observed in the sections of the
human kidney. The CSA method was used. Bar ⫽ 100 ␮m.
briefly stained with Mayer’s hematoxylin. Coverslips were
mounted with Faramount aqueous medium (DAKO), and
slides were viewed with a Zeiss microscope.
Confocal microscopy. To further examine the location of
the H⫹-K⫹-ATPase in the cell, tissues were examined using
confocal microscopy. Tissues were processed in a similar
fashion to the immunohistochemistry studies described
above up to the step of placing them in the Target Retrieval
Solution. The slides were washed in 100 mM phosphate
buffer, pH 7.4, and then exposed to the primary antibody
1218 overnight at 4°C, followed by three 15-min washes with
phosphate buffer. The secondary antibody, goat anti-mouse
IgG coupled to tetramethyl rhodamine isothiocyanate (American Qualex, San Clemente, CA), was utilized at a dilution of
1:100. For negative controls, the primary antibody was replaced by normal mouse serum diluted to the same protein
concentration as the antiserum.
Immunofluorescence of the sections was assessed with a
Zeiss 510 confocal microscope (Carl Zeiss, New York, NY)
The fluorophore was excited using the 543-nm line of the
helium-neon laser, and emission was detected using a
560-nm long-pass filter. The detector gain and amplitude
offset were set so that the negative control sections were just
above the threshold of detection. All subsequent confocal
observations used the same detector settings. Optical slices
(1 ␮m) were recorded.
AJP-Renal Physiol • VOL
Fig. 3. Immunoreactivity of renal sections probed with antibody
directed against HK␣4. A: renal cortex. B: renal medulla. Staining
was primarily localized to the intercalated cells (arrow). G, glomerulus. The streptavidin-biotin method (LSAB⫹; DAKO) was used.
Bar ⫽ 100 ␮m.
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As shown in Fig. 1, A and B, sections probed with
antibody 1218 directed against HK␣1 revealed staining
of a majority of the cells of the cortical and medullary
collecting ducts. In the intercalated cells, the staining
was diffuse in the cytoplasm. With a similar immunohistochemical method, but utilizing a secondary antimouse fluorescent antibody in the final step to allow
confocal visualization, staining of renal medullary tubules was observed to be present primarily on relatively large cytoplasmic vacuoles (Fig. 1C). No staining
was observed when the primary antibody was replaced
by mouse serum (Fig. 1D). In the principal cells, the
staining was generally weaker. Staining with this antibody in the human renal tissue was considerably less
prominent that that observed in the parietal cells of
positive control sections from the rat gastric mucosa
stained with the same antibody (Fig. 2), but at a lower
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RENAL H⫹-K⫹-ATPASE IN HUMAN KIDNEY
DISCUSSION
Fig. 4. Immunoreactivity of renal sections probed with antibody
directed against H⫹-ATPase. A: renal cortex. B: renal medulla.
Staining was most intense in the intercalated cells of the collecting
ducts (C). The LSAB⫹ method was used. Bar ⫽ 100 ␮m.
concentration.
As shown in Fig. 3, A and B, staining with antibodies
directed against HK␣4 was present primarily in intercalated cells of the cortex and medulla but was significantly less intense than with antibody against HK␣1.
Light staining was also occasionally observed in principal cells. By contrast, sections probed with antibody
31.7 directed against rat HK␣2 (not shown) revealed no
detectable staining in any portion of the kidney.
Strong immunoreactivity against H⫹-ATPase (Fig. 4,
A and B) was observed in the cytoplasm of the intercalated cells of the cortex and medulla, and in addition,
in some cells of the proximal tubules, as reported
previously (19).
Whereas sections stained with the primary antibodies revealed immunoreactivity, those stained with
mouse or rabbit serum, rabbit IgG (Fig. 5, A and B), the
mouse negative control antibody, or the buffer (not
shown) revealed no detectable staining of the renal
cells.
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Fig. 5. Renal control sections probed with normal rabbit serum
followed by the LSAB⫹ method or normal rabbit IgG followed by the
CSA method. No staining was observed in either cortical (A) or
medullary tissue (B). Bar ⫽ 100 ␮m.
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The results of the present studies demonstrate that
two of the known ␣-subunit isoforms of the H⫹-K⫹ATPase, HK␣1 and HK␣4, are present in the collecting
ducts of the human kidney. This conclusion is based on
the demonstration that sections probed with the primary antibodies had prominent immunoreactivity of
collecting duct cells, whereas those probed with mouse
or rabbit serum, rabbit IgG, negative control antibodies, or buffer had no staining. In this location, the
H⫹-K⫹-ATPase could contribute to renal handling of
protons and potassium, as has been demonstrated in
the rat and rabbit (12, 14, 23, 25, 31). Confocal imaging
of the renal cells demonstrated that the H⫹-K⫹ATPase was primarily located in relatively large vesicles in the cytoplasm of the cell. For the enzyme to be
active in the renal handling of hydrogen and potassium, it should be present in the plasma membranes of
the collecting duct cells. In the resting parietal cell in
RENAL H⫹-K⫹-ATPASE IN HUMAN KIDNEY
AJP-Renal Physiol • VOL
concentrations of bicarbonate or potassium to those in
normal controls (27).
Renal tubular acidosis is classically associated not
only with reduced hydrogen secretion but also with
hypokalemia (10). This constellation of findings is what
might be expected with reduced renal H⫹-K⫹-ATPase
activity if the H⫹-K⫹-ATPase contributed importantly
to the regulation of acid-base balance. At present, only
kindreds with mutations in AE1 and the H⫹-ATPase
(10) or patients with immunologically mediated renal
disease with absent H⫹-ATPase immunoreactivity (10)
have been reported with full-blown renal tubular acidosis. However, recently a small group of individuals in
Thailand has been described with distal renal tubular
acidosis and hypokalemia attributable to exposure to
vanadium, an inhibitor of P-type ATPases (28). Given
the presence of HK␣1 in the human collecting duct, it is
intriguing to speculate that genetic defects and clinical
disorders associated with defective H⫹-K⫹-ATPase activity might be described in the future, similar to those
reported with genetic defects in AE1 and H⫹-ATPase.
HK␣4 (ATP1AL1) was first cloned from the skin
axilla (17). Comparison with the other HK isoforms
revealed that HK␣4 was 87% identical to rat or rabbit
HK␣2. The level of dissimilarity is greatest within the
NH2-terminal domain, where identity is only ⬃50% (5).
HK␣4 was demonstrated in human brain, kidney, and
skin by RT-PCR but not by Northern blot analysis,
indicating a low abundance of this protein in these
tissues (17).
Studies in oocytes by Grishin and Caplan (16) demonstrated that Na⫹- K⫹ exchange via HK␣4 was 10-fold
that of H⫹-K⫹-exchange, data consistent with this isoform acting primarily as a Na⫹-K⫹-ATPase. Although
we have not localized this transporter to the luminal
membrane, its presence in the collecting ducts of the
kidney might indicate that it primarily conserves potassium under conditions of potassium depletion, as
proposed for HK␣2 in the rat kidney.
In summary, HK␣1 and HK␣4 are present in the
collecting ducts of the human kidney, a site where they
probably participate in regulating renal acid excretion
and potassium reabsorption.
We acknowledge the assistance of Dr. Thomas Stanley in providing the tissue sections; the excellent technical assistance of Katherine Handelsman and Joanne Paulsen in processing the tissue sections; the excellent technical assistance of Dr. David R. Scott in
obtaining the confocal images of the kidney; and the excellent administrative assistance of Dr. Eva Lacayo-Barquero.
These studies were supported in part by funds from the Research
Service of the Department of Veterans Affairs and a Senior Medical
Investigatorship (G. Sachs). G. Sachs was a Senior Medical Investigator of the Department of Veterans Affairs during the performance
of a portion of these studies. During the performance of these studies,
K. G. Helander was supported by a travel grant from the Gothenburg
Medical Association.
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