1 Division of Nephrology, Department of Medicine, Mount Sinai School of Medicine, New York, New York 10029; and 2 Division of Nephrology, St. Luc Academic Hospital, University of Louvain Medical School, B-1200 Brussels, Belgium
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ABSTRACT |
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During kidney organogenesis, the
Na+-K+-ATPase
pump is not restricted to the basolateral plasma membrane of the renal
epithelial cell but is instead either localized to the apical and
lateral membrane sites of the early nephron or expressed in a
nonpolarized distribution in the newly formed collecting ducts. The
importance of
Na+-K+-ATPase
-subunit expression in the translocation of the
Na+-K+-ATPase
to the plasma membrane raises the question as to which
-subunit
isoform is expressed during kidney organogenesis. Immunocytochemical, Western analysis and RNase protection studies showed that both
2-subunit protein and
2 mRNA are expressed in the early gestation to midgestation human metanephric kidney. In contrast, although
1
mRNA abundance is equivalent to that of the
2-subunit in the metanephric kidney, the
1-subunit protein was not detected in early
to midgestation metanephric kidney samples. Immunocytochemical analysis
revealed that both
1- and
2-subunits were present in the apical
epithelial plasma membranes of distal nephron segments of early stage
nephrons, maturing loops of Henle, and collecting ducts during kidney
development. We also detected a significant increase in
1 and
1
mRNA after birth with a marked reduction in
2 mRNA abundance
associated with an increase in
1- and
1-subunit proteins and loss
of
2 protein expression. These studies support the conclusion that
the expression of the
2-subunit in the fetal kidney may be an
important mechanism controlling polarization of the
Na+-K+-ATPase
pump in the epithelia of the developing nephron during kidney organogenesis.
sodium-potassium adenosinetriphosphatase; renal epithelium; sodium transport; nephron
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INTRODUCTION |
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THE
Na+-K+-ATPase
pump is a heterodimeric membrane protein composed of - and
-subunits, which catalyzes ATP-dependent sodium/potassium exchange
essential for the maintenance of cellular volume and ionic homeostasis
(36). The activity of
Na+-K+-ATPase
depends on the assembly of the 100-kDa
-catalytic subunit with a 40- to 60-kDa
-subunit prior to transport of functional pumps to the
plasma membrane (17). Assembly of
-
heterodimers is required for
intracellular transport of the
Na+-K+-ATPase
pump to the plasma membrane (41), and
-subunit structure influences
the function of the
-subunit in the catalysis of
Na+/K+
exchange (15, 30, 31). There are three unlinked
-isoform genes
(
1,
2,
3) (37, 48, 55) and two
-subunit genes (
1 and
2) (21, 40, 56) in the mammalian genome that exhibit cell- and
tissue-specific patterns of expression (59). This diversity of isoforms potentially allows the expression of six different
Na+-K+-ATPase
heterodimers, which may result in functional differences in the catalysis of
Na+/K+
exchange (7, 8) and fulfill the physiological requirements for specific
Na+-K+-ATPase
ion pumps in particular cell types.
In polarized reabsorptive epithelial cells in the intestine and kidney,
the expressed 1-
1 heterodimeric
Na+-K+-ATPase
(18) is mostly localized to the basolateral membrane domain as required
for its function in the vectorial transport of sodium across the tubule
(33, 34, 44). The molecular mechanism for targeting and retention of
the
Na+-K+-ATPase
to the basolateral membrane of kidney epithelia has been extensively
studied in cell model systems (46) and found to depend on basolateral
membrane-specific vesicle transport pathways, apical-to-basolateral
membrane transcytosis, and retention of pumps by stable binding to
localized ankyrin sites in the cytoskeleton of the basolateral membrane
(47). In one renal epithelial cell culture model system, it appears
that the
-subunit may contribute the dominant sorting signal for
basolateral membrane localization (45). In the retinal pigment
epithelium, apical distribution of the ankyrin/fodrin cytoskeleton
results in apical membrane localization of the
Na+-K+-ATPase
1-
1 heterodimers (25, 52) consistent with the model that
epithelial membrane localization of
Na+-K+-ATPase
is dependent on ankyrin binding and retention in an apical multiprotein
membrane cytoskeletal complex rather than on the function of an apical
membrane vesicle sorting pathway.
The possibility that developmental specification of -subunit isoform
expression may play a role in the plasma membrane localization of
Na+-K+-ATPase
in polarized epithelial cells or in neurons has not yet been
extensively studied. In the choroid plexus, which has apical membrane
localization of the
Na+-K+-ATPase
(49) associated with apical membrane ankyrin (39), there is expression
of both the
1- and
2-isoforms (62, 65) suggesting that
2-isoform expression may also influence membrane localization of the
Na+-K+-ATPase
in epithelia. Although these studies suggest that expression of the
2-subunit in the choroid plexus may be a potential mechanism for
directing apical membrane localization of the
Na+-K+-ATPase
in this tissue, both
1- and
2-subunits can be detected by
immunolocalization studies in the apical plasma membranes of this
epithelium using isoform-specific antisera (22, 39). In contrast, the
exclusive expression of the
1-subunit without the
2-subunit (21)
in the adult kidney might be important for the restricted localization
of the
Na+-K+-ATPase
to the basolateral membranes of the tubular epithelial cells, but this
question has not been addressed with appropriate studies in a suitable
experimental model system.
The
Na+-K+-ATPase
2-subunit is expressed in the central nervous system (CNS) glial
cells, pinealocytes, and retinal photoreceptors (3, 54),
where it assembles with the
2-subunit (21) and can direct the
assembly of functional
1-
2,
2-
2, and
3-
2 Na+-K+-ATPase
pumps (7, 8, 53).
2
/
Homozygous knockout mice die
from CNS lesions by postnatal day 18 (38). These results appear to be indicative of lethal region-specific
failures of cellular ionic homeostasis and establish the essential
function of the
2-subunit in CNS
Na+-K+-ATPase
catalytic activity but do not address its potential role in plasma
membrane targeting.
During human kidney organogenesis (26), the
Na+-K+-ATPase
-subunit has been shown to be partly localized to the apical
membrane domains of renal epithelial cells in the developing nephron.
Apical localization of the
Na+-K+-ATPase
has also been detected in a subset of collecting duct epithelia during
kidney development in other mammals (4, 28, 43) in contrast to the
largely basolateral membrane localization in the normal adult nephron
(33, 34). In the adult kidney, the predominant
Na+-K+-ATPase
subunit genes expressed are
1 (18) and
1 (11). Low-level transcriptional expression of
2- and
3-subunit genes and a
truncated
1 splice variant (11, 42) have also been reported in the rat kidney confirming previous reports of potential
-subunit diversity in the kidney (5, 27, 58). However, two independent studies
have demonstrated only full-length
1-subunit
Na+-K+-ATPase
protein in the adult kidney by Western blot (21, 61), and
transcriptional expression of the
-subunit is highly selective for
the
1 gene with only trace amounts of
2 mRNA detectable (11). The
absence of
2-subunit protein in the adult mammalian kidney has also
been documented in earlier studies (21). Previous studies have
demonstrated stable
1 and
1 mRNA levels during kidney development
(48) but have not analyzed transcriptional regulation of the
2-subunit gene or analyzed which
-isoform is expressed in the
apical membrane of the maturing nephron during kidney development.
We reasoned that 2-to-
1 isoform switching in kidney development
might be an important control mechanism for relocalization of the
Na+-K+-ATPase
from the apical to the basolateral membrane. The precedent of
developmental alterations in the ratio of
1/
2 expression is well
established in the cerebellum (3, 38). In addition, in autosomal
dominant polycystic kidney disease, aberrant apical distribution of
Na+-K+-ATPase
is associated with persistent expression of the
Na+-K+-ATPase
2-subunit after birth (P. D. Wilson, unpublished observations).
In this study, we demonstrate expression of the
Na+-K+-ATPase
2-subunit in newly formed nephrons in human fetal kidneys associated with apical membrane localization of the
Na+-K+-ATPase
1- and
2-subunits. These results support the hypothesis that
2-subunit expression during kidney development, and in the adult
choroid plexus, may result in apical membrane localization of the
Na+-K+-ATPase
through altered handling of the
1/
2 complex by the cellular mechanisms that direct nearly exclusive basolateral
membrane localization of the
1/
1
Na+-K+-ATPase.
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MATERIALS AND METHODS |
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Tissues and cell culture. Human fetal
(Anatomic Gift Foundation, Woodbine, GA) and normal adult
human kidneys (National Disease Research Interchange, Philadelphia, PA)
were procured under sterile conditions, flushed with neutral salts
solution (Collins or UW), clamped, and stored in salts
solution over ice for a maximum of 24 h prior to use. Parallel samples
were frozen immediately in liquid nitrogen and stored at
80°C until use. All tissue samples were divided into three
portions and used for 1)
microdissection and tissue culture,
2) protein or RNA extraction, and
3) fixed for pathology,
immunostaining, or in situ hybridization using 4% paraformaldehyde in
diethyl pyrocarbonate-treated PBS at 4°C for 4 h. Microdissected
human fetal collecting ducts were prepared for culture using previously
described methods (63, 64) and immortalized with the retrovirus
pZipneoTA58U19 (19). We packaged this vector in the
amphotropic packaging cell line psi-CRIP (12), which we have used
previously to derive immortalized human renal epithelial cell lines
from the adult kidney (50). This vector transduces a
temperature-sensitive allele of the SV40 large T antigen; after
transfection and G418 selection, clonal cell lines were derived by
limit dilution and selected on the basis of showing expression of the
2-subunit of the
Na+-K+-ATPase.
This led to the identification of the clones HFCT.6D and HFCT.6E used
in the experiments shown in Fig. 6. The cell lines were cultured at
33°C in 25-cm2 flasks coated
with type I (rat tail) collagen (Collaborative Research, Lexington, MA)
until subconfluent and then shifted to 37°C for 7-10 days
until harvest. Following aspiration of the cell culture media, T25
confluent monolayers were washed for 5 min with PBS (pH 7.4) at room
temperature, scraped, and centrifuged for 8,000 g for 90 s. The cell pellet was flash
frozen in liquid nitrogen and then stored at
80°C until use.
Antibodies. Polyclonal
isoform-specific antibodies were prepared in rabbits using
synthetic peptides specific for the
Na+-K+-ATPase
1-,
1-, and
2-isoforms (Immunodynamics). The
1
immunizing peptide sequence was
(
residues are marked
with underscore), corresponding to amino acids 3-16
of human
1 predicted sequence (PIR: locus A24414, accession no.
A24414). The
1 sequence is 93 residues
NH2-terminal to the first
transmembrane domain and therefore cytoplasmic; this
1 sequence is
50% conserved in
2 but entirely different (0/14 matches) from the
3 sequence. The
1 immunizing peptide sequence was
, corresponding to amino
acids 14-27 of the human
1 predicted sequence (GenBank: locus
HSU16799, accession no. U16799). The
1 peptide sequence is identical
in sheep, rat, dog, pig, and mouse. This peptide is part of the
NH2-terminal cytoplasmic portion
of the protein. A BLAST search using this peptide sequence resulted in
significant matches only with other
1-subunits. The homologous
2
sequence to the
1 immunizing peptide is
PRT
and is 43% identical (6/14 residues, conserved residues are marked
with underscore). The
2 immunizing peptide sequence was
, homologous to amino acids
85-98 of the human predicted
2 sequence (GenBank: locus
HUMATPBII, accession no. M81181). In contrast to the
1 peptide, the
2 peptide starts 20 amino acid residues COOH-terminal to the
transmembrane domain and is extracellular. A BLAST search with the
2
sequence identifies only other
2 sequences; only 2 of 14 residues of
this sequence are identical with the homologous
1 sequence. The
2
and
1 antibodies were purified from 15 ml crude antisera using
specific peptide-derivatized 4-ml thiosepharose affinity chromatography
columns. The
V anti-
Na+-K+-ATPase
subunit used in the immunoprecipitation and immunolocalization experiments was a gift from D. M. Fambrough, which was raised against
immunopurified chicken
Na+-K+-ATPase
(29, 35).
This monoclonal antibody (which recognizes a cytoplasmic epitope of the
Na+-K+-ATPase
-subunit shared by all isoforms) and the
6F anti-
monoclonal antibody (60) used in immunolocalization studies are also available from the Developmental Studies Hybridoma Bank
(http://www.uiowa.edu/~dshbwww/info.html). The
isoform-specific SpETB2 is anti-human
2 rabbit polyclonal antiserum
(a gift from P. Martin-Vasallo) and was raised against a human
2-subunit fusion protein (amino acid residues 54-290) expressed
in Escherichia coli. The
characterization of the SpETB2 antiserum has been published (22).
Western immunoblot analysis and
immunoprecipitation. Membrane extracts were prepared
from human fetal (12-24 wk gestational age) and normal adult
kidneys according to the method described by Jørgensen (32). After
washing in ice-cold PBS, pH 7.4, the kidneys were finely minced in
ice-cold homogenization buffer (300 mM sucrose, 25 mM HEPES made to pH
7.0 with 1 M Tris) containing the protease inhibitors 1 mM
4-(2-aminoethyl)-benzenesulfonyl fluoride (Boehringer), 1 mM
benzamidine (Sigma), 10 µg/ml leupeptin (Boehringer), 1 µg/ml
pepstatin A (Boehringer), 1 µg/ml aprotinin (Boehringer),
and 1 µg/ml chymostatin (Boehringer), and then homogenization was
performed in the cold using a Potter apparatus. The homogenate was
centrifuged at 1,000 g for 20 min at
4°C to remove nuclei and cell debris. The supernatant was further
centrifuged at 80,000 g for 30 min at
4°C. The pellet (whole cell membranes) was suspended in the
ice-cold homogenization buffer, and protein concentrations were
determined with the BCA protein assay (Pierce), using BSA as standard.
For
Na+-K+-ATPase
1-subunit detection by Western blots
(n = 5 fetal;
n = 5 adult kidneys), detergent
extraction of membranes was required, as follows: the 80,000 g pellet was resuspended in ice-cold
detergent extraction buffer [20 mM Tris · HCl,
120 mM NaCl, 2 mM EDTA, 2 mM EGTA, and 0.1 mM dithiothreitol (DTT), pH
7.4] containing the protease inhibitors described above,
incubated for 15 min on ice with either 1% octylglucoside (Pierce) or
0.5%
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS, Pierce), and then centrifuged at 100,000 g for 1 h at 4°C. For
Na+-K+-ATPase
2-subunit detection by Western blots
(n = 9 fetal;
n = 9 adult kidneys), detergent
extractions were made with the same protocol using either 1% SDS or
1% NP-40 as the detergent. For Na+-K+-ATPase
1-subunit (n = 9 fetal;
n = 9 adult kidneys), 1% SDS or 0.5%
Triton X-100 (Boehringer) was used as the detergent. Protein concentrations were determined on the supernatant, which contained the
solubilized membrane proteins, and the extracts were used immediately.
All extracts were solubilized for SDS-PAGE by heating either at
95°C for 2 min (for
1 and
2
Na+-K+-ATPase
isoforms) or at 60°C for 12 min (
1
Na+-K+-ATPase)
in sample buffer [1.5% SDS, 10 mM Tris · HCl,
pH 6.8, 0.6% DTT, and 6% (vol/vol) glycerol]. Proteins (20 µg/lane) were separated by electrophoresis through 0.1 × 9 × 6-cm 12% acrylamide slabs and transferred to nitrocellulose.
Membranes were blocked for 30 min at room temperature in blotting
buffer (50 mM NaPO4, 150 mM NaCl,
and 0.05% Tween 20, pH 7.4) containing 5% nonfat dry milk, followed
by incubation with the primary antisera and affinity-purified
antibodies (anti-
1, anti-
1, or anti-
2) in the blotting buffer
containing 2% BSA at 4°C for 18 h. The membranes were then washed
in several changes of blotting buffer, incubated for 30-60 min
with peroxidase-labeled goat anti-rabbit IgG (Kirkegaard & Perry),
washed again, and visualized after 1-min incubation with enhanced
chemiluminescence (Amersham) at room temperature.
For immunoprecipitations, whole cell extracts were prepared from frozen
cell pellets of cell lines HFCT.6D and HFCT.6E after lysis in 500 µl
cold lysis buffer [10 mM Tris, pH 7.2, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 0.2 mM vanadate, 0.5% NP-40, and 1% Triton X-100 containing
the protease inhibitors 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride
(Boehringer, Indianapolis, IN), 1 mM benzamidine (Sigma, St. Louis,
MO), 10 µg/ml leupeptin (Boehringer), 10 µg/ml pepstatin A
(Boehringer), 1 µg/ml aprotinin (Boehringer), and 1 µg/ml
chymostatin (Boehringer)]. After 30-min incubation on ice, the
suspension was dispersed by aspiration using an 18-gauge needle and
then centrifuged at 14,000 rpm for 15 min at 4°C. Cell lysates (200 µg protein) were incubated for 1 h at room temperature with 25 µl
of Protein A/G Plus agarose (Santa Cruz Biotechnology), and 10 µl of
the anti-
Na+-K+-ATPase
subunit monoclonal antibody
V in a total volume of 400 µl. The
pellet was collected at 8,000 rpm for 2 min and washed three
times with RIPA buffer (Ca2+- and
Mg2+-free PBS, pH 7.4, 1% NP-40,
0.5% sodium deoxycholate, 0.1% SDS, 1% phenylmethylsulfonyl
fluoride, 1 µg/ml aprotonin, and 1 mM vanadate), and the pellet was
resuspended in 40 µl 1× electrophoresis sample buffer [10
mM Tris, pH 6.8, 6% glycerol, 4.0% (vol/vol)
-mercaptoethanol, 1.5% SDS], boiled for 3 min,
centrifuged at 8,000 rpm for 2 min and analyzed by Western blot
following SDS-PAGE using an 8% polyacrylamide gel.
Immunocytochemistry. For light
microscopy, paraffin-embedded tissue sections on glass slides were
first dewaxed and rehydrated through a graded series of ethanols.
Tissues were then incubated in 0.3%
H2O2
in methanol to block endogenous peroxidase activity followed by
incubation with 10% normal goat serum in PBS for 20 min at room
temperature in a humidified atmosphere. Tissue sections were incubated
for 45 min at room temperature in a humidified chamber with the
following primary antibodies: anti-chicken
Na+-K+-ATPase
-subunit monoclonals (
V, and 6F12) from Dr. D. M. Fambrough (Johns Hopkins University, Baltimore, MD)
(n = 8 independent fetal kidney
blocks); or polyclonal anti-
1 (n = 27 independent fetal kidney blocks), anti-
2
(n = 28 independent fetal kidney
blocks) antisera raised in rabbits against
isoform-specific peptides as described above. Affinity-purified
anti-
2 antibody was used to confirm
2 immunolocalization
(n = 10 independent fetal kidney blocks used). Primary antibodies were diluted in PBS containing 2% BSA
(1:100 to 1:500); washed three times in PBS-Tween 20 (0.02%); incubated for 45 min with biotinylated goat anti-rabbit IgG (Vector Laboratories), washed twice for 5 min each in PBS-Tween and once for 5 min in PBS, incubated for 45 min with avidin-biotin peroxidase (Vectastain Elite, Vector Laboratories), and washed for 5 min in PBS
followed by two washes of 5 min each in Tris-buffered saline. Color
development was carried out for 10-45 min using
aminoethylcarbazole as substrate. Sections were mounted in Aquamount
(Polysciences) and viewed under a Nikon FXA-Microphot equipped with
Nomarski optics.
Preparation of riboprobes. The 1,
1, and
2 PCR products were subcloned into the vector
pBSIIKS(
); sequence and insert orientation were verified by DNA
sequencing. Antisense probes were prepared from
Xho I-restricted plasmids transcribed
in vitro with T7 RNA polymerase in the presence of
[32P]UTP (Amersham) as
follows: RNA polymerase concentration 2.5 U/µl in 40 mM Tris-Cl (pH
8.0), 25 mM NaCl, 8 mM MgCl2, 2 mM
spermidine-HCl3, 10 mM DTT, 400 µM ATP, 400 µM CTP, 400 µM GTP, 12.5 µM
[
-32P]UTP (400 Ci/mmol), and 2 U/µl placental RNase inhibitor (Boehringer) in a
reaction volume of 20 µl for 30 min at 37°C followed by a 15-min
37°C incubation with 0.5 U/µl RNase-free DNase (Boehringer). The
riboprobes were purified with
phenol/CHCl3/isoamyl
alcohol extraction and precipitated three times in 75%
ethanol plus 0.5 M
NH4OAC prior to use.
RNase protection assays. For
hybridizations, 20 µg of total RNA (or control tRNA) was resuspended
in 40 mM PIPES (pH 6.4), 400 mM NaCl, 1 mM EDTA, and 80% formamide in
the presence of 2 × 105 cpm
antisense probe and 2 × 105
cpm internal control 18S riboprobe (Ambion) in 30 µl for 16 h in a
45°C bath (for 1, n = 6 fetal
kidney, n = 7 adult kidney RNA
samples; for
1, n = 6 fetal kidney,
n = 7 adult kidney RNA samples; for
2, n = 7 fetal kidney,
n = 7 adult kidney RNA samples). A
volume of 350 µl of 10 mM Tris-Cl (pH 7.5), 300 mM NaCl, 5 mM EDTA
containing 40 µg/ml RNase A (Boehringer), 0.2 µg/ml RNase T1
(Boehringer) was then added to the hybridization mixture, and RNA
digestion was performed at 30°C for 30 min followed by proteinase K
digestion in 0.5% SDS,
phenol/CHCl3/isoamyl alcohol
extraction and ethanol precipitation. The assay products
were fractionated using a 6% urea-PAGE system, and dried gels were
examined by autoradiography with an intensifying screen for 12-72
h. The
1 undigested probe length was 496 bp,
1 was 506 bp, and
2 was 612 bp.
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RESULTS |
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Characterization of anti-1 and
anti-
2 isoform-specific
Na+-K+-ATPase
antisera.
The rabbit polyclonal anti-
1 raised against the
NH2-terminal peptide
was used for Western
immunoblot analysis of solubilized membrane proteins isolated from the
normal adult human kidney and compared with the
V anti-
1
monoclonal antibody raised against the chicken
Na+-K+-ATPase
1-subunit (gift of D. M. Fambrough, Johns Hopkins University). As
shown in Fig.
1A, the
rabbit polyclonal anti-
1
Na+-K+-ATPase
antiserum detected the same 100-kDa band expressed in the adult human
kidney (left two lanes) as was
detected by the control anti-
1
Na+-K+-ATPase
monoclonal antibody (right two lanes).
This 100-kDa band was not detected by the rabbit preimmune control
Western immunoblot (data not shown). The rabbit polyclonal anti-
1
Na+-K+-ATPase
antiserum also detected basolateral expression of the
1-subunit in
the renal tubules of adult rat kidneys (see Fig. 5L) and human kidneys (data not
shown). The characterization of the rabbit polyclonal antiserum raised
against the
1 peptide
demonstrated specific recognition of a 42-kDa protein in the adult
kidney not recognized by the preimmune antiserum control which was
specifically competed by the addition of the immunizing peptide. This
42-kDa protein was also detected by the anti-
1 antiserum following
affinity purification over a
1-peptide-containing column (data not
shown, available on request). The rabbit polyclonal antiserum raised
against the
2 peptide
detected a 50-kDa protein (Fig. 1B,
lanes 1 and
2) by Western immunoblotting of
solubilized membrane proteins from the human fetal kidney not detected
by preimmune antiserum (Fig. 1B,
lane 4); confirmation that this
50-kDa protein is the
2-subunit was obtained using affinity-purified
anti-
2 antiserum (Fig. 1B,
lane 3) which also detected the
50-kDa
2-subunit and a minor band at 30-35 kDa. The observed
molecular mass of the
2-subunit was in the range of 46-51 kDa
reported for the
2-subunit in the choroid plexus (65) and brain (3).
The difference between the predicted molecular mass of the
2 gene
product of 33.2 kDa and the observed molecular mass of 45-50 kDa
is likely due to N-linked glycosylation (21); the 30- to
35-kDa band detected in Fig. 1B,
lane 3, with the affinity-purified
antibody may reflect the presence of some nonglycosylated
2-subunit
core protein in the fetal kidney solubilized membrane extracts. Further
characterization of the
2-subunit anti-peptide antiserum showed that
it detects apical plasma membrane
2-subunit protein in human
2-subunit-expressing transfected MDCK cells but not in control MDCK
cells (data not shown; D. M. Fambrough, personal communication); The
specificity of the
2 antiserum and lack of cross-reactivity with the
1-subunit is demonstrated by the lack of detection of the
1-subunit in the adult kidney by immunocytochemistry (not shown), or
Western immunoblotting (Fig. 2). We have
also shown that the immunizing
2 peptide will specifically compete
the immunodetection of the
2-subunit detected by Western blotting
(data not shown). Our
2 anti-peptide antiserum detects the same
protein on Western immunoblots of fetal collecting duct cell lines cell
extracts (shown in Fig. 6A),
as does the previously characterized
2-isoform-specific SpETB2 rabbit polyclonal antiserum raised against
a human
2 fusion protein expressed in E. coli (22).
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The
Na+-K+-ATPase
2-subunit substitutes for the
1-isoform
during human kidney organogenesis.
Immunoblot analysis of
1 expression in human fetal kidney at 18, 20, and 24 wk of gestation demonstrated a 100- to 110-kDa band
(lanes 1-3, Fig.
2A) with increased expression in the
adult kidney as detected in membrane extracts prepared from kidney
tissue obtained from 17-, 20-, and 34-yr-old donors
(lanes 4-6, respectively, Fig.
2A). In contrast, Western immunoblot
studies demonstrated no
1 protein in fetal kidney at 16, 19, and 23 wk gestation (lanes 1-3, Fig.
2B), whereas the 42-kDa
1-subunit
was detected in kidney tissue membrane extracts prepared from 2, 16, and 31-yr-old donor kidneys (lanes
4-6, Fig. 2B)
(the 90- to 95-kDa and 140-kDa bands detected are antigenically
unrelated to the
1-subunit). Expression of the 50-kDa
2-subunit
protein in fetal kidneys was demonstrated at 18, 20, and 24 wk of
gestation (lanes 1-3, Fig.
2C) with absent expression evident
in the adult kidney obtained from 17-, 20-, and 34-yr-old donors
(lanes 4-6, Fig.
2C). On longer exposure of this
Western blot, faint expression of the
2-subunit was seen in two of
three adult kidneys. During kidney development, the level of
2-subunit expression appeared to be constant from 15-24 wk by
Western blot (data not shown). We have not yet determined when
1-subunit expression replaces
2, but downregulation of
2 mRNA
to undetectable levels has been observed in a newborn kidney (see Fig.
3C,
lane 3).
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A reduction in 2 mRNA abundance in
association with
1 translational activation results in
2/
1-isoform switching during maturation
of the nephron.
Quantitative analysis of
Na+-K+-ATPase
isoform mRNA abundance by RNase protection analysis supported the
conclusion that
2 mRNA levels are significantly increased in the
fetal kidney compared with the adult kidney (lane
1 vs. 2, Fig.
3A). This pattern of predominantly
fetal kidney expression of the
2 mRNA is reversed for the
1-
(Fig. 3B,
left) and
1-subunits (Fig.
3B,
right), which show marked induction
during kidney maturation in the fetus and after birth. As shown in Fig.
3C, some of the increase in abundance
of the
1- and
1-subunits mRNA occurs postnatally
(lane 1, newborn kidney
1 mRNA, vs.
lane 4,
1 mRNA detected in sample prepared from 2-yr-old donor kidney; lane
2, newborn kidney
1 mRNA, vs. lane
5,
1 mRNA detected in sample prepared from 2-yr-old donor kidney). Although we found variable expression of
2 mRNA by
RNase protection and RT-PCR with
2-specific primers (data not shown)
in some adult kidney samples, the
2 mRNA abundance was lower than
that detected in fetal kidney and a small fraction of the
1 mRNA
abundance in the adult (data not shown). The finding of low-level
expression of
2 mRNA in the adult rat kidney has been previously
reported by others (11). We next examined the relative abundance of
1,
1, and
2 mRNA during kidney development from 13 to 24 wk
gestational age (lane 1, 13 wk;
lane 2, 16 wk; lane
3, 20 wk; lane 4, 24 wk in Fig. 4,
A-C).
In these experiments, we found that the level of
1 and
2 mRNA was
nearly equal during kidney development with the
1 mRNA being
slightly more abundant than the
-isoform mRNAs after
20 wk of gestation (compare lanes 3 and 4 in Fig. 4,
A-C).
The reduction in
2 mRNA abundance in the adult kidney suggests that
transcriptional repression of the
2 promoter or a developmental
reduction in
2 mRNA stability during nephron maturation is a
potential control mechanism for switching off
2 protein expression
in the adult kidney. In contrast, the
1-subunit mRNA is easily
detectable in all stages of kidney organogenesis (Figs. 3,
B and
C, and
4B) despite the absence of detectable
1-subunit protein by immunoblot studies in the fetal kidney (Fig. 2B). We have not
determined whether alterations in
1 mRNA polyadenylation during
kidney development may control
1 protein expression as has been
shown during early Xenopus laevis development (9). There is also a definite increase in
1 and
1
mRNA abundance between fetal, newborn, and adult stages (Fig. 3,
B and
C), supporting a potential role for
either transcriptional activation of the
1- and
1-subunit
promoters or an increase in
1 and
1 mRNA stability in the
developmental regulation of renal
Na+-K+-ATPase
expression.
|
The 1- and
2-subunits are expressed in apical plasma membranes of
epithelial cells in the maturing nephron during
nephrogenesis.
Following induction of the mesenchymal cells of the metanephric
blastema by the ureteric bud, a morphogenetic program is initiated that, in association with epithelial cell fate specification and differentiation, results in the formation of nephrons (16). In this
developmental pathway, the acquisition of epithelial membrane polarity
is required to generate a tubular epithelium with basolateral Na+-K+-ATPase
essential for reabsorption of water and solute from the tubular lumen.
In previous reports, it has been noted that some of the
-subunit
Na+-K+-ATPase
is located in the apical membrane of fetal collecting ducts (43).
During human kidney organogenesis, nephron formation occurs principally
during the second trimester, and we therefore sought to analyze
1-
and
2-subunit membrane localization in the primitive renal vesicle,
early nephron, and maturing nephron in 12-24 wk metanephric
kidneys to determine the first stage of nephron development associated
with activation of
Na+-K+-ATPase
protein expression.
|
The 1- and
2-subunits assemble into an
Na+-K+-ATPase
holoenzyme protein complex in the apical plasma membranes of human
fetal collecting duct cells.
Although it is well established that the
1- and
2-subunits can
assemble into a functional of
Na+-K+-ATPase
holoenzyme complex (7, 8, 53), the formal possibility exists that
during fetal kidney development, the
2-subunit might be targeted to
the plasma membrane independently from the
1-subunit. To confirm
assembly of the
1- and
2-subunits in the fetal kidney collecting
duct epithelia, we performed immunoprecipitation experiments using the
V
anti-
-Na+-K+-ATPase
monoclonal antibody followed by
2 detection with
immunoblotting with either our anti-
2 peptide antiserum or with
the SpETB2 anti-human
2-isoform-specific antibody [gift of Dr.
P. Martin-Vasallo (23)], which has identical specificity to our
anti-peptide
2 antiserum (Fig.
6A). For
these experiments, we established the immortalized human fetal kidney
collecting duct cell line HFCT.6D using the retroviral
vector pZipneoTA58U19 (see MATERIALS AND
METHODS), which we have previously employed for the
immortalization of human adult kidney renal epithelial cell lines (50).
HFCT.6D expresses the
Na+-K+-ATPase
1- and
2-subunits in a pattern consistent with apical membrane
(data not shown). In contrast, we have not detected the typical
"chicken-wire" pattern indicative of basolateral membrane localization of the
Na+-K+-ATPase
in this cell line. As shown in Fig.
6B, the identical
2 doublet band
was detected in the immunoprecipitate by immunoblotting with the SpETB2
antiserum (lane 1) as was detected
in the cell lysate (lane 2) of the
HFCT.6D cell line. With omission of the
V monoclonal antibody
(lane 3), no
2-subunit was
recovered after precipitation with protein A/G agarose beads alone
excluding nonspecific binding of the
2-subunit to the agarose matrix
of the beads. We have also confirmed that our anti-peptide
2
antiserum detects the identical
2-subunit following
immunoprecipitation with the
V
anti-
-Na+-K+-ATPase
monoclonal antibody in other epithelial cell lines that express both
the
1- and
2-subunits (data not shown). In
addition, it is evident that the
2-subunit is heavily
glycosylated in two independent HFCT cell lines (HFCT.6D and HFCT.6E,
see Fig. 6); since full glycosylation of
Na+-K+-ATPase
-subunits depends on assembly with the
-subunit in the endoplasmic reticulum (1, 20), this strengthens the conclusion that the
1/
2 complexes detected in these experiments are associated with
the plasma membrane.
|
![]() |
DISCUSSION |
---|
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---|
Although the -subunit of the
Na+-K+-ATPase
is the catalytic subunit for
Na+/K+
exchange at the cell membrane, elucidation of the function of the
-subunit has revealed its essential role in the translocation of the
-subunit to the plasma membrane (41) and provided insights into its
function in the regulation of the kinetics of
Na+/K+
exchange by the
-subunit (30). The
-subunit also plays a role in
localization of the
Na+-K+-ATPase
to the basolateral membrane of renal epithelial cells mediated by
stable association with ankyrin sites (13) associated with the fodrin
cytoskeleton (46, 47) in the kidney. It also appears that the
NH2-terminal half of the
-subunit is important in sorting for basolateral membrane
localization of newly synthesized pumps (45). In the choroid plexus (2,
39) and retinal pigment epithelium (25), an extensive series of studies
has demonstrated the potential importance of binding of the
Na+-K+-ATPase
-subunit to an apical membrane ankyrin-cytoskeletal complex as a
principal mechanism for polarization of the sodium pump to the apical
membrane domains in these epithelial cell types. By comparison,
relatively little is known about the potential role of the
-subunit
in the regulation of cell membrane polarity of the
Na+-K+-ATPase,
although
-subunit signal sequence(s) might be important for
basolateral membrane vesicle transport of the
Na+-K+-ATPase
in renal epithelial cells. Alternatively,
-isoform structure might
influence binding of the
-subunit to the ankyrin cytoskeletal complex and influence membrane localization of the
Na+-K+-ATPase
through this mechanism.
The acquisition of basolateral membrane polarization of
Na+-K+-ATPase
necessary for tubular reabsorption in the adult kidney occurs well
after nephron formation since apical, as well as basolateral, membrane
localization of the -subunit is identified throughout at least the
12-24 wk gestational age range in human kidney development (this
study) and has been consistently seen in other studies of mammalian
kidney development (4, 28, 43). It is also important to recognize that
even in the adult kidney, biochemical and immunohistochemical studies
have consistently demonstrated the presence of small amounts of apical
membrane
Na+-K+-ATPase
in the kidney (33, 34, 44). Therefore, the developmentally acquired
molecular mechanisms that control cell polarization do not result in
the complete loss of apical membrane
Na+-K+-ATPase.
In this study we have shown that
2-subunit is induced in the stage
III-IV early nephrons as the loop of Henle is first formed where both
the
1- and
2-isoforms are found in the apical membrane domains.
We also made a surprising observation that there is little, if any,
apparent expression of the
1-subunit protein during early kidney
development detected by immunoblotting using our
1-subunit
isoform-specific antiserum. This result was unexpected, because
throughout nephrogenesis, there is a similar abundance of
1 and
2
mRNA by RNase protection analysis of total fetal kidney RNA. Although
we cannot completely exclude that the absence of detectable
1
protein in the fetal kidney might somehow derive from antigen masking,
we consider this unlikely, as we have employed multiple alternate
detergent extractions (including octylglucoside, CHAPS, NP-40, or SDS
in a range of concentrations) and have consistently failed to detect fetal kidney
1 protein by immunoblotting. It is
also possible that a low level of
1-subunit expression is present
during kidney organogenesis which is below the limit of sensitivity of
our Western blot assay. However, the lack of detectable expression of
fetal kidney
1 protein expression in our studies supports the
conclusion that selective translational silencing of the
1 mRNA may
occur during kidney development. A similar lack of
1 protein
expression due to developmental translational silencing was recently
demonstrated in the early development of the X. laevis embryo (9). Postnatal increases in the levels of
the
1-subunit have also been observed previously, although the
regulatory basis for this had not been defined (57). Recently, the
potential importance of posttranscriptional upregulation of
1-subunit expression has also been identified as an important mechanism of postnatal increases in the basolateral membrane
Na+-K+-ATPase
in the newborn guinea pig renal cortex (24). The loss of
2
expression and the activation of
1 protein biosynthesis apparently
occurs late in gestation and may be required for the acquisition of
basolateral polarization of the
Na+-K+-ATPase
-subunit in the kidney tubule. These data therefore suggest that
basolateral membrane polarization of the
Na+-K+-ATPase
during maturation of the renal tubule may be regulated at least in part
at the level of
-subunit isoform expression and support the
conclusion that the molecular structure of the
-subunit may
influence the sorting of the
Na+-K+-ATPase
into either specific basolateral or apical vesicle transport pathways.
The hypothesis that the 2-subunit contains a signal for apical
membrane localization of the
Na+-K+-ATPase
has recently been tested in MDCK cells. These studies have shown that
transfection of the human
2-subunit into MDCK cells results in the
apical localization of
2-subunits (Ref. 51; and D. M. Fambrough,
personal communication). In contrast, MDCK cell
transfection of the
1-subunit results in the assembly of
1-
1
heterodimers with basolateral membrane localization (17). This supports
the conclusion that sequence differences between the
1- and
2-subunit influence molecular mechanisms involved in vesicle
transport pathways or binding to components of the cytoskeleton important for the establishment and maintenance of membrane protein polarization in epithelia. The existence of a specific
basolateral vesicle transport pathway in renal epithelial cells has
been established (10, 46) and may in part be directly or indirectly
(through an effect on the
-subunit) dependent on
1 amino acid
sequences not found in the
2-subunit. The low homology between the
1- and
2-subunits in the cytoplasmic
NH2 terminus (30% identity
residues 1-30 of
2 vs.
1), which would be on the outer
surface of a transport vesicle, suggests the potential importance of
this sequence in regulating apical versus basolateral delivery of the
Na+-K+-ATPase
in renal epithelia cells. The expression of the
2-subunit in the
choroid plexus, which also demonstrates apical membrane localization of
the apical
Na+-K+-ATPase,
suggests that developmental selection of
-subunit isoform expression
may be a general mechanism for the regulation of membrane polarization
of the
Na+-K+-ATPase
in epithelial cells. A difficulty with this model is that the apical
membranes of the choroid plexus epithelium contain both
1- and
2-subunits, as shown by immunolocalization studies using isoform-specific antisera (22). However, if higher
order Na+-K+-ATPase
multimers [i.e.,
(
1
1)2] exist in cells
(see Ref. 36), (
1)2(
1
2)
heteromultimers could also potentially be formed and be
preferentially retained in the apical membrane domains.
Our finding of apical localization of
Na+-K+-ATPase
during development is not likely related to a failure of formation of
intercellular tight junctions, as ultrastructural studies have
demonstrated that even the epithelia of the early renal vesicle have
already acquired apical tight junctions (14). Our results, therefore, suggest that the membrane localization of the
Na+-K+-ATPase
may in part be developmentally controlled at the level of -isoform
expression and not only dependent on the binding of the
-subunit to
the ankyrin-fodrin cytoskeletal components. As a partial validation of
this model, it has recently been established that
2-subunit
expression in MDCK cells results in its apical membrane localization
under experimental conditions associated with a normally polarized
basolateral ankyrin-fodrin network.
It is also important to emphasize that in the early nephron the
1-isoform is also found in the basolateral membranes of fetal tubule
segments including the proximal tubule and the loop of Henle, where we
have not been able to detect expression of the
2-subunit (see Fig.
5G) which is consistently apical in
these segments. Although this might suggest the possibility that the
1-subunit may reach the basolateral plasma membrane without
association with the
2-subunit, we think that immunolocalization
studies alone are insufficient evidence for reaching this conclusion
given the uniform requirement for
-subunit association with the
-subunit for its stabilization and insertion into the plasma
membrane. In contrast, we find that although
2-subunit is
preferentially localized to the apical membrane of the maturing
collecting ducts (Fig. 5J), there is
also some basolateral membrane
2-subunit localization. For the
1-subunit, there is clearly both apical membrane as well as
basolateral localization in the maturing collecting duct (Fig.
5K), as previously seen in the rat
(4) and rabbit postnatal kidney (43). In sum, there exist in the fetal
kidney collecting ducts, as well as in more proximal
segments of the tubule in the early nephron, apparent differences in
the relative staining intensity of the
1- and
2-subunits in the
basolateral membrane (
1 greater than
2), which might indicate
different subunit stoichiometries in the apical versus basolateral
membrane in tubulogenesis.
A conservative interpretation of our results is that during
tubulogenesis, 1/
2
Na+-K+-ATPase
heterodimers are not exclusively directed or retained in the apical
membrane in all nephron segments, but may also be localized to the
basolateral membranes as found in the developing collecting duct
system. These results suggest that the
2-subunit does not have a
functionally dominant and exclusive apical membrane targeting sequence
active in all nephron segments during organogenesis despite the fact
that the
2-subunit shows preferential localization to the apical
membrane of transfected MDCK cells. Our findings are consistent with a
model that
1/
2 heterodimeric
Na+-K+-ATPase
pumps may partially escape the in vivo cellular control mechanisms that
ensure basolateral membrane localization, with the result that in
certain tubule segments, their localization is largely apical, whereas
in others (such as the collecting duct) there is
essentially a nonpolarized distribution of pumps throughout the plasma
membrane. In sum, this model predicts that the
1/
1 Na+-K+-ATPase
pump is subject to a much more stringent regulatory mechanism, which
results in highly selective basolateral membrane
localization with a far lower level of apical membrane
Na+-K+-ATPase
than is possible with the more relaxed control mechanisms governing
polarization of the
1/
2
Na+-K+-ATPase.
The functional role of apical
Na+-K+-ATPase
during tubulogenesis remains to be established. Expression of apical
Na+-K+-ATPase
in the loop of Henle during its formation in the early nephron appears
to precede formation of a functional glomerular tuft and supports the
possible importance of basal-to-luminal sodium transport in the
establishment of a lumen in the distal nephron. This model proposes
that the requirement for apical membrane Na+-K+-ATPase
during tubulogenesis is similar to the function of the apical membrane
Na+-K+-ATPase
in trophectodermal cells in the developing blastocyst (6). The
developmental regulatory mechanisms directing the reprogramming of
renal tubular epithelial cells for postnatal life are aimed at assuring
basolateral membrane localization of the
Na+-K+-ATPase.
Our data support the potential importance of analysis of the molecular
basis of transcriptional repression of the 2 Na+-K+-ATPase
gene and translational activation of
1 mRNA expression in
establishing basolateral membrane localization of the
Na+-K+-ATPase
in the renal epithelial cells of the developing nephron during kidney
organogenesis and in early postnatal life.
![]() |
ACKNOWLEDGEMENTS |
---|
Katherine Thornton, Samantha Wilson, Rebecca Zausmer, Lillian Kang,
and Lida Zhen are gratefully acknowledged for technical assistance in
these studies. We thank Dr. Douglas Fambrough for the gift of the V
anti-chicken
-subunit
Na+-K+-ATPase
monoclonal antibody, Dr. Pablo Martin-Vasallo for the anti-
2
Na+-K+-ATPase
antibody SpETB2, and Dr. Alicia McDonough for many helpful suggestions.
![]() |
FOOTNOTES |
---|
This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-44833 (to P. D. Wilson).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: C. R. Burrow, Box 1243, Mount Sinai School of Medicine, 1 Gustave L. Levy Place, New York, NY, 10029-6574 (E-mail: chris_burrow{at}smtplink.mssm.edu).
Received 12 January 1998; accepted in final form 27 April 1999.
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