1 Department of Pediatrics, The apical secretory K+
(SK) channel in the principal cell represents the rate-limiting step
for K+ secretion across the
cortical collecting duct (CCD). Patch clamp analysis of maturing rabbit
principal cells identifies an increase in number of conducting SK
channels after the 2nd week of life [L. M. Satlin and L. G. Palmer. Am. J. Physiol. 272 (Renal Physiol. 41): F397-F404,
1997], ~1 wk after an increase in activity of the
amiloride-sensitive epithelial Na+
channel (ENaC) is detected. To correlate the postnatal increase in
channel activity with developmental expression of ROMK, the molecular
correlate of the SK channel, we used gene-specific probes to show a
developmental increase in abundance of renal ROMK mRNA and a
ROMK-specific antibody to examine the nephron distribution, localization, and abundance of this protein in developing rat kidney.
Using antibodies directed against aquaporin-3 (AQP-3) and Tamm-Horsfall
protein (THP), we confirmed that ROMK was expressed along the apical
membranes of principal cells and thick ascending limbs of Henle (TALH)
in adult kidney. Within the midcortex of the neonatal kidney,
ROMK-positive segments revealed weak coincident staining with the
TALH-specific antibody but did not colabel with an antibody directed
against distal and connecting tubule (CNT)-specific kallikrein or the
lectin Dolichos biflorus agglutinin
(DBA), which labels proximal tubules and collecting ducts. In inner
cortex and outer medulla of kidneys from 1-wk-old animals, ROMK protein was identified in medullary TALH (MTALH) and DBA-positive
collecting ducts. By 3 wk of age, coincident ROMK and DBA expression
was detected in midcortical and outer cortical CNTs and CCDs.
Immunoblot analysis of plasma membrane-enriched fractions of maturing
rat kidney revealed a developmental increase in a ~40-kDa band, the expected size for ROMK. Immunolocalization of
potassium channel; amiloride-sensitive sodium channel; maturation; collecting duct; principal cell
GROWING ORGANISMS MAINTAIN a state of positive
K+ balance (33). The final renal
regulation of K+ homeostasis in
the adult occurs in the collecting duct (12). Cortical collecting ducts
(CCDs) isolated from adult rabbits and microperfused in vitro at
physiological flow rates secrete net K+ at high rates (13, 26, 32). In
contrast, net K+ transport is not
detected in microperfused CCDs from newborn rabbits until the 3rd week
of postnatal life (26). Net Na+
absorption, however, absent at birth, rapidly increases to levels approximating 50% of those observed in the adult after the 1st week of
life (26, 34).
K+ secretion in the fully
differentiated CCD is accomplished by a two-step process. First,
K+ is actively taken up into the
principal cell at the basolateral membrane in exchange for
Na+, a process mediated by the
ubiquitous Na-K-ATPase (12). Thereafter, K+ is secreted through apical
K+-selective channels into the
tubular lumen by passive diffusion down a favorable electrochemical
gradient (12). The electrochemical gradient is determined by the
lumen-negative voltage, generated by apical
Na+ entry through
Na+-selective channels and its
electrogenic basolateral extrusion, and the high cell
K+ concentration (12). The apical
K+ and
Na+ channels constitute the
rate-limiting steps for K+
secretion and Na+ absorption, respectively.
The apical secretory K+ (SK)
channel in the rat principal cell has been shown to be a
low-conductance (30-40 pS) inwardly rectifying ATP-sensitive
channel (11, 12, 24, 37). Electrophysiological analysis of the apical
K+ permeability of the maturing
rabbit principal cell revealed a paucity of functional SK channels
immediately after birth, with channel activity increasing progressively
after the 1st week of postnatal life (28). Yet, in these same cells,
the mean number of conducting apical amiloride-sensitive
Na+ channels per patch reached the
mature level by 2 wk of age (27).
Cumulative functional and biophysical evidence (reviewed in Refs. 12,
25, 38) now identifies ROMK, a cDNA encoding a family of
KATP channels (14, 39), as the
molecular correlate of the apical SK channel. ROMK was originally
cloned from rat outer medulla where the apical low-conductance
KATP channel in the thick
ascending limb of the loop of Henle (TALH) recycles K+ across the apical membrane to
ensure an abundant supply of substrate for the Na-K-2Cl cotransporter
(36). Three renal ROMK isoforms (ROMK1-3), derived from
alternative splicing of the 5' end of the gene and differing only
in the sequence and length of their amino termini, have been identified
(2, 14, 39). ROMK mRNA has been found in all nephron segments beyond
and including the medullary TALH (MTALH) in rat kidney (2).
Immunofluorescence studies of adult rat kidney performed using
anti-ROMK carboxy-terminal antibodies revealed that ROMK protein is
present along the apical membranes of cortical TALH
(CTALH) and MTALH, distal convoluted tubule, connecting
tubule (CNT), and principal cells in the CCD and outer medullary
collecting duct (OMCD) (18, 22, 38). ROMK is not detected beyond the
initial portion of the inner medullary collecting duct (IMCD) (38).
The amiloride-sensitive epithelial
Na+ channel, ENaC, cloned from rat
distal colon using a functional expression strategy (4, 5), is a
multimeric channel comprising The purpose of the present study was to correlate the functional
expression of SK channel activity with appearance of ROMK message and
protein in the maturing kidney. To this end, we used gene-specific
molecular probes to assess ROMK mRNA abundance and a rabbit polyclonal
antibody raised against ROMK to examine the nephron distribution,
localization, and abundance of this protein in developing rat kidneys
by immunocytochemistry and immunoblotting. Immunolocalization of the
Animals. Adult female Sprague-Dawley
rats with their litters were obtained from Taconic Farms (Germantown,
NY) and raised in the animal facilities at the Albert Einstein College
of Medicine or Mount Sinai School of Medicine. Adult animals were
maintained on standard rat chow (Purina 5001; Ralston-Purina, St.
Louis, MO) and allowed free access to tap water. Newborn animals were raised with and fed by their mothers. Rats were studied at each week
during the first 6 wk of postnatal life; adult rats were defined as
animals Preparation of ROMK probe for Northern blot
analysis. The ROMK probe was prepared by RT-PCR of rat
renal medulla RNA, as described below, using gene-specific primers
designed to amplify a highly conserved region (bp 821-1123) of the
published ROMK1 sequence (sense:
5'-CAACAGCCCTTTCTTCCACATG-3'; antisense
5'-TGTCATAGCCTCTCTTCATCCTGG-3') (14). The amplification
product was subcloned into the pCR2.1 vector (Invitrogen, San Diego,
CA). Sequence analysis (Applied Biosystems model 373 fluorescent
sequencer) of the insert revealed its identity to the published ROMK
sequence (14). The cloned insert was restriction enzyme digested and
labeled with
[ To generate the probe, RT-PCR was performed using 2 µg of total RNA
prepared by the single-step acid guanidinium
thiocyanate-phenol-chloroform method (6). The RNA was treated with
DNase I (amplification grade, GIBCO-BRL; Life Technologies, Grand
Island, NY), according to the manufacturer's protocol, to eliminate
residual genomic DNA. To 10 µl of the RNA sample were added 2 µl
10× PCR buffer (GIBCO), 1 µl 50 mM
MgCl2, 2 µl oligo(dT) (80 pmol;
Pharmacia LKB Biotechnology, Piscataway, NJ), 2 µl deoxynucleotide
mixture (1.25 mM stock of each nucleotide; Pharmacia), 2 µl 0.1 M
dithiothreitol, and 1 µl (200 U/µl) Superscript II reverse
transcriptase (GIBCO). Reverse transcription proceeded at 25°C for
10 min, 42°C for 55 min, 95°C for 5 min, and the contents were
then cooled to 4°C. The reverse-transcribed cDNA was amplified
after a 3-min denaturation at 94°C using 40 cycles of PCR (Perkin
Elmer Thermocycler, model 480), each consisting of denaturation for 1 min at 94°C, annealing of primers for 1 min at 55°C, elongation
for 1 min at 72°C, and a final extension for 8 min at 72°C. The
sample was size fractionated by electrophoresis on a 2% agarose gel to
verify that the PCR product was of expected size (303 bp).
Northern blot analysis. Total RNA was
extracted from whole kidney homogenates using the method of Chomczynski
and Sacchi (6), and the integrity of the RNA was verified
spectrophotometrically by measuring absorbance at 260 and 280 nm.
Thirty micrograms of total RNA from each age group were size
fractionated by electrophoresis on a 1% agarose-3% formaldehyde gel
and transferred overnight to a Hybond-N filter (Amersham). Each filter
was prehybridized for 2 h at 42°C with 50% formamide, 5×
Denhardt's reagent, 5× standard saline citrate (SSC), 40 mM
sodium phosphate, pH 6.8, 0.1% SDS, and 200 µg/ml denatured salmon
sperm DNA in diethyl pyrocarbonate-treated water. The RNA was
hybridized overnight in the same solution with the
32P-labeled rat ROMK probe. The
membranes were washed successively with 1× SSC-0.5% SDS twice at
room temperature for 30 min, then 0.1× SSC-0.1% SDS three times
at 50°C. The filter was exposed at The relative intensities of bands were measured by analysis of scanned
autoradiographs using an image densitometer (Bio-Rad model GS-670) and
Molecular Analyst software (Bio-Rad, v. 2.12). To compensate for
differences in quantity of total RNA on each lane of the membrane, each
densitometric value for ROMK expression was normalized to its
respective value of Tissue preparation for immunofluorescence
microscopy. Animals were anesthetized, and their
kidneys were perfused either via the aorta (adults) or the heart
(newborns) with PBS followed by 2% paraformaldehyde for 10-15 min
at a pressure of 150 cmH2O. Kidneys were immediately removed, sliced into 2-mm sagittal sections, postfixed in 2% paraformaldehyde for 4 h, and immersed overnight at
4°C in 30% sucrose for cryoprotection. Blocks of tissue were then
embedded in OCT compound (Sakura Finetek, Torrance, CA), frozen in
liquid nitrogen, and cut into 4- to 5-µm sections using a Leica
CM3050 cryostat. Sections were collected on
poly-L-lysine-coated (Sigma, St.
Louis, MO) slides.
Antibodies. Rabbit antiserum directed
against a 23-amino acid synthetic peptide corresponding to the carboxy
terminus of rat ROMK and thus common to ROMK isoforms 1-3 (22) was
generously provided by L. Palmer. Additional tubule-specific probes
included an affinity-purified rabbit polyclonal antibody to rat
aquaporin (AQP-3) that recognizes principal cells of collecting ducts,
kindly provided by G. Frindt (8); goat anti-human Tamm-Horsfall
glycoprotein (THP; uromucoid) antiserum (ICN Cappel Pharmaceuticals,
Aurora, OH) that recognizes TALH cells (30); and sheep anti-rat
kallikrein antiserum that recognizes tissue kallikrein in distal
tubules and CNTs (gift from J. Chao) (9). A protein-A-purified rabbit polyclonal anti- Immunofluorescence microscopy.
Sections were hydrated with PBS for 10 min, blocked with FCS for 30 min, and permeabilized using 0.05% saponin in FCS in a 1:1 dilution
for 30 min. FCS was replaced by normal goat serum for subsequent
labeling with the anti-kallikrein antiserum. Slides were then washed
three times in PBS, 5 min each wash, and incubated for 30 min at 37°C with one of the following primary antibodies: anti-ROMK
(1:200 dilution) or anti- Fluorescence microscopy was performed either with an Olympus or Nikon
Diaphot inverted microscope equipped with infinity-corrected optics.
Images were collected from the Olympus microscope with a Photometrics
(Tuscon, AZ) PXL cooled charge-coupled device camera driven by I.P. Lab Spectrum software (Signal Analytics, Vienna, VA)
running on a Power Macintosh (Apple Computer, Elk Grove, CA). The green
and red fluorescence of FITC and rhodamine, respectively, were
visualized with Nikon filters (B and G) or, most often, with a Nikon
double-filter cassette allowing for simultaneous visualization of
fluorescein and rhodamine/Texas Red fluorescence. Photographs were
obtained with a Nikon 35-mm camera using Kodak Elite 400 film.
Western blotting. Whole kidney was
minced finely and homogenized three times for 15 s each
time using a Brinkmann tissue homogenizer (model PT
10/35) in ice-cold isolation solution (250 mM sucrose, 10 mM
triethanolamine, pH 7.4), containing 1 mM EDTA, 1 mM EGTA, and the
following protease inhibitors: 1 mM Pefabloc, 1 mM
leupeptin, 1 mM benzamidine, 1 µg/ml chymostatin, 10 µg/ml
pepstatin, and 1 µg/ml aprotinin. Homogenates were spun
in a Sorvall model RC5C centrifuge at 1,000 g for 15 min at 4°C to remove
nuclei and incompletely homogenized cells. After an initial spin at
4,000 g for 20 min, the supernatant
was respun at 17,000 g for 20 min in a
Beckman model LE 80K ultracentrifuge to enrich the fraction in plasma membranes, as previously described (8). The membrane fractions were
solubilized at 60°C for 15 min in Laemmli sample buffer. The total
protein concentration in spun fractions was measured using a protein
assay reagent kit (BCA; Pierce, Rockford, IL).
Aliquots of membrane extracts (40 µg/lane) were separated by SDS-PAGE
on 12% polyacrylamide precasted minigels (Bio-Rad, Hercules, CA) and
electrophoretically transferred to polyvinylidene fluoride membranes
(Immobilon-P; Millipore, Bedford, MA). Membranes were blocked with blot
wash buffer (42 mM
Na2HPO4,
8 mM
NaH2PO4,
150 mM NaCl, and 0.05% Tween 20, pH 7.5) containing 5% nonfat dried milk for 30 min and then incubated with the ROMK antibody at a 1:2,000
dilution overnight. After six washes in blot wash buffer, the secondary
antibody, a horseradish peroxidase-conjugated donkey anti-rabbit IgG
(Pierce), was applied at a 1:10,000 concentration for 1 h. After
washing, antibody binding was visualized by enhanced chemiluminescence
(ECL, Amersham) before exposure to X-ray film. Controls were performed
by replacing the primary antibody with ROMK antiserum preadsorbed with
a 50-fold molar excess of immunizing peptide. The relative intensities
of bands were measured by analysis of scanned images using the NIH
Image 1.61 software for Macintosh.
Statistics. Developmental comparisons
were performed by t-test, ANOVA,
and/or linear regression analysis using SigmaStat software (SPSS,
Chicago, IL). Values are means ± SE. Significance was asserted for
P < 0.05.
Northern analysis. The developmental
expression of ROMK mRNA in rat kidney was examined by Northern
analysis. As shown in the representative blot depicted in Fig.
1, a single band of expected size (14) for
ROMK (~3.1 kb) was detected at all ages. Densitometric analysis
revealed a biphasic increase in steady-state ROMK mRNA expression after
birth (Fig. 2). This pattern of
developmental increase in ROMK mRNA expression was similar to that
reported in rabbit (1), the species used for functional (26) and
electrophysiological (28) characterization of the ontogeny of net
K+ transport and SK channel
activity in the CCD, respectively.
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
-ENaC showed apical staining of a majority of cells in distal nephron segments after the
1st week of postnatal life. The
- and
-ENaC subunit expression was routinely detected in a mostly cytoplasmic distribution immediately after birth, albeit in low abundance;
-ENaC showed some apical polarization. These results suggest that the postnatal increases in a
principal cell apical SK and Na+
channel activity are mediated, at least in part, by increases in
abundance of ROMK message and protein and ENaC subunit proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
-,
-, and
-subunits. Coexpression of the three subunits in oocytes reconstitutes a channel
with ion-selective permeability, gating properties, and pharmacological
profile similar to the native channel (5). In the kidney of
Na+-depleted rats, the three
subunit mRNAs are coexpressed along the apical membranes of the
aldosterone-responsive segments of the distal nephron, including the
distal convoluted tubule, CNT, principal cells of the CCD and OMCD, and
IMCD (7), a pattern of localization that correlates well
with the expression of amiloride-sensitive electrogenic
Na+ absorption. Vehaskari et al.
(35) has shown that steady-state levels of
-,
-, and
-ENaC
mRNA in neonatal rat are comparable to those measured in the adult.
-,
-, and
-subunits of ENaC in rat cortex was also performed
to determine the pattern of postnatal expression of
Na+ channel proteins.
METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
6 wk of age. At least four litters of rat pups were used for
each group of studies. Animals were anesthetized or killed by
intraperitoneal injection of pentobarbital sodium (30 or 100 mg/kg body
wt, respectively).
-32P]dCTP
(Megaprime DNA-labeling system; Amersham, Arlington
Heights, IL).
80°C for 3 days to
Hyperfilm film (Amersham). The equality of loading and integrity of RNA
were confirmed by reprobing the membranes with a PCR
-actin-generated probe (3). The filter was exposed
overnight at
80°C.
-actin.
-rENaC antibody was obtained from P. Smith (31); antisera raised in rabbits and directed against the carboxy-terminal ends of
- and
-ENaC (7) were provided by C. Canessa. All antibodies have been well characterized and their specificity previously established.
-, anti-
-, or anti-
-ENaC (1:200
dilution). After three successive washes in PBS, the
sections were incubated with FITC-conjugated F(ab')2 fragment donkey
anti-rabbit IgG (1:200; Jackson Immunochemicals, W. Grove, PA).
Colabeling of some sections was performed by the subsequent application
of anti-AQP-3 (1:1,250), anti-THP (1:1,250), or anti-kallikrein (1:100)
antibodies, visualized with rhodamine-conjugated F(ab')2 fragment donkey
anti-rabbit IgG, Lissamine rhodamine-conjugated rabbit anti-goat, or
Texas Red-conjugated anti-sheep IgG secondary antibodies, as
appropriate (1:200; Jackson Immunochemicals). Other sections were
colabeled with the proximal tubule- and collecting duct-specific
rhodamine-conjugated Dolichos biflorus
agglutinin (DBA; 5 µg/ml; Vector Laboratories, Burlingame, CA) (15,
16). Control experiments consisted of either omitting the primary or secondary antibodies, or substituting the primary antibody with anti-ROMK or anti-ENaC antibody preincubated overnight at 4°C with
a 50-fold molar excess of peptide against which the antibody was
raised. All sections were mounted on coverslips with the Prolong Antifade Kit (Molecular Probes, Eugene, OR).
RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (48K):
[in a new window]
Fig. 1.
Maturational increase in ROMK mRNA expression. Thirty micrograms of
total RNA from kidneys of 1- to 6-wk-old rats were fractionated on a
denaturing formaldehyde gel and transferred to a nylon filter.
Hybridization with the 32P-labeled
ROMK probe revealed a single transcript at 3.1 kb. Northern blot was
then rehybridized to a -actin probe as a loading and transfer
control. Whereas
-actin mRNA expression was relatively constant
during postnatal life, ROMK mRNA abundance increased after birth.
View larger version (19K):
[in a new window]
Fig. 2.
Densitometric analysis of ROMK mRNA expression as a function of age.
Laser densitometry was used to obtain ROMK/ -actin mRNA ratios. For
each of 4 blots, ratios were expressed as a percentage of the value
obtained at 6 wk, with the latter set to equal 100%. ROMK mRNA
abundance increased significantly during postnatal life.
* P < 0.01 compared with 6 wk;
r = 0.94 by linear regression
analysis. Values are means ± SE.
Immunofluorescence. Indirect
immunofluorescence microscopy of the adult rat kidney labeled with
anti-ROMK antibody revealed apical ROMK expression in tubules within
medullary rays. Labeling in the outer medulla, localized to MTALH and
OMCD (22, 38), was more prominent than that detected in cortex (Fig.
3A).
Control experiments were performed using antiserum immunoadsorbed with the ROMK-immunizing peptide or secondary antibody alone. No
immunofluorescence above background was detected (data not shown), as
has been reported by Mennitt et al. (22) using this same antibody.
|
To confirm the tubular distribution and membrane localization of this protein, double antibody labeling using anti-ROMK and TALH- and principal cell-specific antibodies was performed. As reported by others (22, 38), ROMK staining was heterogeneous in THP-positive CTALH (Fig. 3B) with most, but not all, cells expressing the epitope. ROMK labeling in neighboring tubules (Fig. 3C), devoid of THP antigen and thus not TALHs, was not only restricted to the apical membrane but was apparent in the subapical cytoplasm. The presence of ROMK in only a subset of cells in such segments suggested it likely they represent CNTs or CCDs. In the outer medulla, the majority of ROMK-positive tubules cut in cross section (Fig. 3A) costained with anti-THP antibody (not shown), as has been reported by others (22, 38).
To verify that ROMK is present along the apical membrane of principal cells, other sections were colabeled with antibodies against ROMK and AQP-3. Apical ROMK was detected only in AQP-3-positive cells (Fig. 3D), consistent with expression of the channel protein in principal cells. Intercalated cells, which do not express ROMK channels (22, 38), are devoid of ROMK and AQP-3. As controls for these double labeling experiments, anti-AQP-3 or anti-THP antibodies were omitted in the labeling of some cryosections; application of their appropriate secondary antibodies to sections stained with anti-ROMK antibody and its corresponding secondary antibody showed no cross-reactivity (data not shown).
Thus, within the fully differentiated kidney, ROMK is selectively expressed along the apical membranes of TALH and principal cells. This pattern of localization of ROMK expression is identical to that previously described by others using the same (22) or different antibodies raised against the COOH-terminal portion of the protein (18, 38).
Developmental expression of ROMK antigen in maturing
rat kidney. Although immunofluorescence studies
indicated that ROMK was indeed expressed in the neonatal kidney, our
ability to identify the neonatal cortical segments expressing ROMK was
hampered by weak colabeling with the anti-THP antibody and almost
undetectable staining with the anti-AQP-3 antibody, presumably
reflecting low levels of antigen
expression.1
Attempts to increase the concentration of either of the latter two
primary antibodies were complicated by generation of high levels of
background fluorescence (see Fig.
4D).
To circumvent this technical difficulty and identify distal tubular
structures, we colabeled sections with anti-ROMK antibody and DBA, a
lectin shown to preferentially bind to the brush border of proximal
tubules and apical membrane and/or cytoplasm of cells showing
basolateral immunoreactivity for Na-K-ATPase in the CCD of rat kidney,
i.e., principal cells (15, 16).
|
Low-power magnification of cryosections cut from 1-wk-old rat kidney colabeled with anti-ROMK antibody and DBA (Fig. 4A) revealed DBA staining of two populations of tubular profiles: one displaying an intense uniform labeling along the luminal membrane, presumably representing proximal tubules (curved arrows), and the other showing a heterogeneous labeling of only some cells, likely representing CNT or collecting ducts, both of which include principal cells (20). CNTs, whether those of juxtamedullary or midcortical nephrons, which form arcades, or those of subcapsular nephrons, which directly join the collecting duct, course laterally through the cortex at some point and thus, in longitudinal section, may appear to be cut in cross section (23), as shown in Fig. 4C. ROMK was absent from the nephrogenic zone and present most often in DBA-negative tubules in middle and inner cortex (CTX; short arrows in Fig. 4A) and outer medulla (OM). Rarely, inner cortical and outer medullary tubules exhibiting a heterogeneous pattern of DBA staining revealed coincident apical ROMK (short arrows in OM; Fig. 4A).
High-power magnification of the midcortex at 1 wk of age revealed apical DBA binding on a discrete population of cells, presumably principal cells within CNTs or CCDs; ROMK was not expressed in these tubules (Fig. 4B). Nor was ROMK immunostaining evident in distal tubules or CNTs expressing kallikrein at this age (Fig. 4C). However, by 1 wk of age, CTALH expressed modest apical staining for ROMK (Fig. 4D).
By 3 wk of age, ROMK was clearly evident in DBA-positive cells in midcortical tubules presumed to be CCDs based on their heterogeneity of staining and linear profiles (Fig. 4E). Occasional outer cortical tubules, likely representing CNTs cut in cross section, also showed coincident labeling with ROMK and DBA at this stage of renal development (Fig. 4F).
In the medulla of the 1-wk-old newborn, ROMK protein was evident along the apical membranes of THP-positive MTALH (Fig. 4G) and occasional collecting ducts exhibiting heterogeneous DBA staining (Fig. 4A).
Western analysis. A representative
Western blot of plasma membrane-enriched fractions from maturing rat
kidneys using the anti-ROMK antibody is shown in Fig.
5
(left). Four prominent bands were
detected at ~76, 50, 45, and 40 kDa. Whereas the 76-kDa band, accompanied by a less intense band at 85 kDa, was present at all ages,
a developmental increase in intensity of the 40- (Fig.
6; r = 0.99 by linear regression analysis) and 45-kDa bands, consistent with
the predicted and reported molecular weights of core and glycosylated
ROMK (22, 38), respectively, was detected (Fig. 6). In contrast, a
maturational reduction in intensity of the 50-kDa band was apparent
(Fig. 5); the identity of this band is uncertain. Preadsorption of the
immune serum with ROMK peptide ablated all bands (Fig. 5,
right).
|
|
ENaC protein expression.
Immunofluorescence studies performed with antibodies directed against
-subunit of ENaC showed the absence of staining throughout the
cortex in 1-wk-old animals (Fig. 7).
Thereafter, antibody against
-ENaC was detected on the apical
membranes of numerous tubular profiles; the observation that most, but
not all, cells in these tubules labeled with the antibody was
consistent with their identity as CNTs and CCDs (Fig. 7), sites
previously reported to coexpress all three ENaC subunits (7). Both
-
and
-subunits were routinely detected after birth in a predominantly
cytoplasmic distribution in distal nephron segments (Fig.
8 and 9). Whereas
-ENaC labeling was
consistently cytoplasmic (Fig. 8), the
-subunit showed some apical
polarization (Fig. 9). Cells devoid of ENaC
label in these segments are likely intercalated cells. Control
cryosections, incubated with
-ENaC-antiserum preadsorbed with its
peptide or secondary antibody alone, were devoid of specific labeling
(data not shown).
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DISCUSSION |
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The present study demonstrates a postnatal increase in expression of ROMK protein in the rat kidney after the 2nd week of postnatal life (Fig. 6), closely following the early increase in steady-state ROMK mRNA abundance (Fig. 2). Immunolocalization studies suggest that this increase in protein abundance is likely due to increases in ROMK expression in the TALH and more distal segments, including CNT and collecting duct (Fig. 4). In the cortex of newborn rats, ROMK was evident in TALH, albeit at low abundance, but was not detected in distal tubules, CNT, and CCDs. By 3 wk of age, labeling of mid-CCDs and occasional outer cortical structures, believed to be CNTs, was clearly evident. In the medulla, ROMK was already present in MTALH and collecting ducts in the 1st week of life.
Our studies confirm the observations, published by others (18, 22, 38), that ROMK antibodies label the apical membranes of TALH and principal cells in adult rat kidney, as expected on the basis of electrophysiological analyses of the apical K+ conductances in these two segments (11, 12, 28, 36, 37). Because the ROMK antibody used in the present study was raised against the carboxy-terminal region of the protein, common to all alternatively spliced forms, it could not differentiate among the unique isoforms.
The temporal and spatial appearance of ROMK expression after birth correlates well with our patch clamp analysis of the apical K+ conductance of the differentiating principal cell in the mid-CCD (28). In the latter study, we identified a developmental increase in apical SK channel activity in the maturing rabbit principal cell. Conducting apical SK channels were not detected in CCDs isolated from animals in the 1st week of postnatal life. The mean number of open SK channels per patch (NPo) of principal cell, negligible at 2 wk of age, increased progressively thereafter. The increase in NPo appeared to be due primarily to a developmental increase in number (N) of channels; Po remained constant at ~0.5 for all channels identified after the 2nd week of life. To the extent that the program of channel development in the rat is similar to that in rabbit, the results of the present study provide support for the likelihood that the postnatal increase in SK channel activity is due to an increase in transcription and translation of ROMK message and protein, respectively. As the SK channel constitutes the rate-limiting step for K+ secretion in fully differentiated CCD, we speculate that the limited capacity of the immature CCD for K+ secretion is due, at least partially, to a scarcity of conducting ROMK channels in CNT/CCD.
Apical ROMK was detected in the TALH at an earlier developmental stage than in the collecting duct (Fig. 4). Although functional analysis of the K+ transport pathways in the TALH in the maturing nephron has not been performed, results of micropuncture studies (19) are consistent with functional immaturity of the loop of Henle early in life. Postnatal increases in diluting capacity (17, 40) and Na-K-ATPase activity (29) in rat TALH have been reported, suggesting that K+ transport pathways in this segment may undergo developmental regulation. Characterization of these pathways and the role of apical ROMK in ion transport in the neonatal segment has yet to be accomplished.
Immunoblotting confirmed a maturational increase in abundance of ~40- and 45-kDa proteins in plasma membranes harvested from maturing rat kidneys; the 76- and 85-kDa bands did not appear to be developmentally regulated. Other investigators using anti-ROMK carboxy-terminal antibodies have reported similar size bands in kidney, all of which were abolished by immunoadsorption with ROMK immunizing peptide. Whereas the 40- and 45-kDa bands are consistent with core and glycosylated ROMK monomer, the high-molecular-mass bands have been suggested to represent ROMK complexed with itself or other proteins that were not dissociated under the conditions used for Western blotting (22, 38). Against this speculation was the observation by Xu et al. (38) that these high-molecular-mass bands were absent in HEK-293 cells transfected with ROMK1. Our detection of a prominent 76-kDa band in the neonatal kidney, which showed only a trivial 40-kDa band, lends support to the hypothesis that the high-molecular-weight band represents a protein unrelated to, but sharing carboxy-terminal sequence homology with ROMK and does not represent a complex formed by ROMK isoforms. The identity of the developmentally regulated ~50-kDa protein is unknown at this time.
Coexpression of the -,
-, and
-subunits is required for
maximal and efficient cell-surface expression of ENaC activity (5, 10).
Whereas expression of the
-subunit alone allows for conduction of a
small amiloride-sensitive current,
- and
-subunits alone or
together do not induce a Na+
current (5). Recent studies indicate that synthesis of the
-subunit
is the limiting factor in the assembly and targeting of the channel
protein to the apical surface (10). The observation of coincident
increases in rate of synthesis of the
-subunit and short-circuit
current in A6 kidney cells 60 min after addition of aldosterone to the
bathing medium suggests that de novo synthesis of this
channel subunit may mediate the early increase in sodium transport
(21). In the present study, we found that
- and
-subunits were
already present in midcortical distal nephron segments at birth, albeit
at low abundance. The temporal relationship between appearance of
-ENaC subunit (Fig. 7) and conducting amiloride-sensitive Na+ channels (27) in the apical
membrane of the principal cell in the 2nd week of life of the rodent
provides further evidence that the
-subunit may be essential for
assembly, targeting, and activation of apical amiloride-sensitive
Na+ channels.
In summary, the results of our investigation suggest that the postnatal increases in number of conducting apical SK and Na+ channels in the differentiating principal cell are mediated, at least in part, by increases in abundance of ROMK message and protein and ENaC subunit proteins. The signal(s) mediating these developmental events remains to be identified.
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ACKNOWLEDGEMENTS |
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We thank Beth Zavilowitz for expert technical assistance and Shahana Khan for help with the Western blots.
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FOOTNOTES |
---|
Funding for the study was provided by the National Institute of Diabetes and Digestive and Kidney Diseases Research Grant DK-38470 and an American Heart Association Grant-in-Aid.
Portions of this work were presented at the 1998 Annual Meetings of the Society of Pediatric Research in New Orleans and American Society of Nephrology in Philadelphia.
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.
1
Although colabeling of single sections with
anti-ENaC and anti-ROMK antibodies would have provided a definitive
identification of CNT/CCD and insight into the temporal pattern of
expression of the two channel proteins during postnatal
differentiation, we encountered a major difficulty in performing these
studies with the available channel antibodies, both raised in rabbit. In preliminary experiments in which single cryosections were
sequentially labeled with anti-ROMK antibody followed by its
appropriate 2° antibody and then anti--ENaC antibody visualized
by its 2° antibody, we found that the
-ENaC labeling was no
longer specific to the apical membrane of a subpopulation of cells in
the CNT/CCD (as shown in Fig. 7). Similarly, cytoplasmic
-ENaC
staining could no longer be detected in sections initially labeled with
anti-ROMK antibody and its fluorescent 2° antibody. Based on the
loss of selective binding of the second 1° antibody in these double
labeling experiments, in which the two 1° antibodies were raised in
the same donor species, we elected to identify collecting ducts by their characteristic heterogeneous apical DBA staining pattern (Fig.
4).
Address for reprint requests and other correspondence: L. M. Satlin, Box 1664, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029-6574 (E-mail: lisa_satlin{at}smtplink.mssm.edu).
Received 9 November 1998; accepted in final form 12 February 1999.
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