Departments of 1 Internal Medicine and 2 Pediatrics, University of Iowa College of Medicine and Veterans Affairs Medical Center, Iowa City, Iowa 52242
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ABSTRACT |
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Na+
absorption via amiloride-sensitive
Na+ channels is of critical
importance in the transition between fetal and neonatal life in several
tissues, including the colon, lung, and kidney. To characterize and
contrast the mRNA expression of each of the three epithelial
Na+ channel complex (ENaC)
subunits, we conducted RNase protection assays (RPA) and in situ
hybridization in colon and lung in fetal (17, 19, 20, and 21 days) and
postnatal (1, 3, 9, 15, and 30 days) rats (r). In the colon the -,
-, and
-rENaC subunits showed quantitatively different but
qualitatively similar expression. All three subunits gradually
increased in abundance from fetal day
19 through day 30 of
life. The amount of each subunit on day 30 was approximately three times the amount at
day 1. In situ hybridization showed
that each subunit was localized to the surface epithelial cells with
minimal expression in the crypts. The lung showed a completely
different pattern. In contrast to the colon, the total amount of
-rENaC mRNA (by RPA) in the lung increased dramatically from
fetal day 19 to
21, whereas
- and
-rENaC showed modest prenatal increases. The amounts of all three mRNAs fell after
birth through day 9 (to about 75% of
the day 1 value). On days 15 and
30 the amount of mRNA rose to approach
the values on day 1.
-rENaC mRNA
abundance always exceeded
- and
-rENaC, and the quantitative
expression was different for
- than for
- and
-rENaC. In situ
hybridization studies showed that all three subunits were expressed in
epithelial cells of the bronchi, bronchioles, and alveoli and not in
blood vessels. These studies show striking developmental heterogeneity
in rENaC mRNA expression between lung and colon, probably reflecting
different developmental regulatory mechanisms in these organs.
glucocorticoid; mineralocorticoid; epithelial sodium channel complex
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INTRODUCTION |
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ELECTROGENIC TRANSPORT of
Na+ occurs across many epithelial
tissues, including the colon, lung, and kidney. This
process, which involves the translocation of
Na+ across cell membranes through
specific ion channels, plays an important role in the absorption of
fluid. The ability to absorb Na+
through epithelial channels is of critical importance in the transition
between fetal and neonatal life. In the colon and kidney, Na+ absorption is critically
important for the regulation of fluid and electrolyte homeostasis and
nutritional status during the early postnatal period. In the fetal
lung, net secretion of (Na)Cl and fluid is replaced by net absorption
of Na(Cl) and fluid at the time of birth, a process essential for
perinatal adaptation (44). This absorptive process is inhibited by
amiloride, suggesting that it takes place through amiloride-sensitive
Na+ channels (34, 44). The
molecular pathway believed to be responsible for this
Na+ transport is the epithelial
Na+ channel complex (ENaC). A
striking example of the role of ENaC is seen in genetically engineered
mice lacking the -subunit; these animals die within 48 h of birth
from failure to clear fetal lung fluid (20).
Electrogenic Na+ transport by the
colon occurs via ENaC, and it is regulated in the postnatal period
(38). Although the genes encoding the -,
-, and
-subunits of
rat (r) ENaC were originally cloned from colon (9, 10), less is known
about the process of developmental regulation of ENaC in the colon than
in the lung. Because the epithelial
Na+ channel encoded by rENaC plays
a significant role in fluid transport in the colon and lung, we
hypothesized that the mRNA expression pattern during the transition
from fetal to postnatal life would reflect the roles they play during
this period. To understand the ontogeny of the rENaC subunit expression
and factors that may regulate expression we determined steady-state
mRNA expression in rat colon and lung and localized the mRNAs using in
situ hybridization.
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METHODS |
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Tissue Preparation
Timed pregnant (sperm positive day 0) Sprague-Dawley rats were obtained from Sasco (Omaha, NE). Maternal animals were anesthetized with methoxyfluorane, exsanguinated, and the fetuses were removed and placed in ice-cold Hank's solution. Neonatal rats were also anesthetized with methoxyfluorane. Fetal lung and descending colon tissues were obtained from days 17, 19, 20, and 21 of gestation and postnatal days 1, 3, 9, 15, and 30. Fetal tissues were pooled for analysis as follows: fetal day 17 and 19, four lung specimens, six colon specimens; fetal day 20 and 21, two lung specimens, four colon specimens; postnatal day 1, one lung specimen, two colon specimens; all other time points, one specimen per analysis. Pooled samples were used only for a single measurement. The protocol was approved by the Animal Care and Use Committee of the University of Iowa.Preparation of RNA
The descending colon was rinsed with ice-cold PBS, and the most distal tissue was trimmed and frozen. The lungs were rapidly removed and immersed directly in liquid nitrogen. Total RNA was isolated from tissues using a modification of the method of Chomczynski and Sacchi (12). An amount of tissue estimated to yield ~100 µg total RNA was homogenized using a 92/Polytron PT-DA 3012/2 TS probe in 2.0 ml of TRI reagent (Molecular Research Center, Cincinnati, OH) for 3-5 s, and the RNA was extracted in 0.2 ml chloroform. After precipitation with isopropanol, the RNA was rinsed with 75% ethanol, centrifuged for 5 min at 7,500 g, air dried for 5-10 min, and resuspended in diethyl pyrocarbonate-treated water. Total RNA was measured by absorption spectrophotometry at 260 nm. The 260/280 ratio averaged 1.6-1.8.RNase Protection Assay
The plasmids used to make the antisense probes for the RNase protection assays (RPA) were constructed as previously described forAntisense probes for the RPA were synthesized from the appropriate
constructs using the BrightStar BIOTINscript nonisotopic in vitro
transcription kit (Ambion, Austin, TX). The amount of biotin-labeled
CTP was adjusted to give the highest possible specific activity. The
lengths of the biotin-labeled, unprotected fragments were 480, 280, and
750 nt for -,
-, and
-rENaC, respectively.
We used an 18S antisense probe to normalize for the amount of RNA assayed. This denominator probe was selected over the more traditional actin or glyceraldehyde-3-phosphate dehydrogenase because these latter probes might be developmentally regulated in an organ-specific fashion, thus making comparisons difficult. The human 18S cDNA (obtained from Dr. Jean Robillard) consisted of an unprotected fragment of 110 nt. Because the abundance of the 18S mRNA is several orders of magnitude greater than that of the rENaC subunits, we constructed separate biotin-labeled and unlabeled antisense RNA probes. Based on extensive preliminary testing, we determined that a mixture of 1:1,000 (labeled:unlabeled, total of 1 µg antisense RNA) saturated the 18S mRNA in the reaction and provided a signal that was easily detectable under our experimental conditions. The calculated molar ratio of 18S probe to 18S mRNA was 5:1. This value agreed well with experimentally determined ratios.
The hybridization of ~1 ng of each of the rENaC probes and 1 µg of 18S probe with 25 µg total RNA from each of the tissues was conducted using the RPAII ribonuclease protection assay kit (Ambion). Hybridization was conducted overnight at 45°C. Digestion with RNase A and RNase T1 (at a final concentration of 250 and 10,000 U/l, respectively) was conducted at 37°C for 30 min. The products were subjected to electrophoresis through a 5% denaturing polyacrylamide-8 M urea gel buffered with Tris borate for 2 h at 250 V and transferred to a nylon membrane (BrightStar Plus, Ambion) using a semidry electroblotter (Fisher, Itasca, IL). The membrane was subsequently ultraviolet (UV) cross-linked (UV Stratalinker, Stratagene), and development of the protected RNA fragments was conducted using the BrightStar Biodetect nonisotopic detection kit (Ambion) with minor modifications. To reduce background noise the wash times after incubation with the streptavidin-alkaline phosphatase conjugate solution were increased threefold. The developed blots were exposed to Kodak XAR-5 film (Eastman Kodak, Rochester, NY) for 1-45 min, depending on the intensity of the product.
The - and
-rENaC probes and the 18S probe occasionally showed
degradation products when the amount of the respective mRNA was large.
The magnitude of these products was <10% of the completely protected
fragment, and the shorter fragments did not interfere with the ability
to quantitate any of the other bands of interest. Therefore
quantitation of these transcripts was conducted on the major protected
fragments. The autoradiograms were quantitated with a PDI scanning
densitometer using Quantity One software (Huntington Station,
NY). The exposure time of the autoradiograms was adjusted so that the density of the majority of the bands on a given gel fell
into the linear range of the instrument.
In Situ Hybridization
Colon and lung samples were prepared from four animals from each time point (2 for paraffin, 2 for frozen section) for use in in situ hybridization as previously reported (27). We used both frozen and paraffin sections for all probes, recognizing that there might be some advantages to one form of fixation over the other. Tissues were immersed in 2% paraformaldehyde in PBS for 2 h at 4°C, embedded in paraffin, sectioned to 5-mm thickness, and mounted onto slides (Superfrost Plus, Fisher Scientific, Fair Lawn, NJ), which were air dried and stored with desiccant at room temperature in an airtight box until they were used for hybridization. Postnatal lung tissues for frozen section were rapidly removed and inflated via trachea with 5 ml of OCT compound (Miles, Elkhart, IN) diluted 1:1 with PBS and frozen in liquid isopentane. Frozen tissues were sectioned on a cryostat to 7-mm thickness and thaw-mounted onto slides. After air drying for a few minutes, slides containing sections were fixed in freshly prepared 4% paraformaldehyde in PBS, pH 7.4, at 4°C for 10 min, dehydrated in graded ethanols, and stored with desiccant atHybridization was performed on triplicate sections from the same tissue for each of three exposures (4, 8, and 13 wk). Tissues from different animals were used for paraffin-embedded and frozen sections. The prehybridization and hybridization steps were conducted according to standard methods (50) as modified by this laboratory (27). After hybridization, sections were washed at high stringency [50% formamide, 2× saline-sodium citrate (SSC), and 25 mM dithiothreitol] at 60°C for 30 min, rinsed with 4 M NaCl, 10 mM Tris, 5 mM EDTA, and 25 mM dithiothreitol (pH 8), and treated with 20 mg/ml RNase A and 1 U/ml RNase T1 for 30 min at 37°C. After a second series of high-stringency washes and dehydration, the sections were then coated with NTB-2 autoradiography emulsion (Eastman Kodak, Rochester, NY), diluted 1:1 in distilled H2O, air dried, and exposed at 4°C before being developed. Sections were counterstained with toluidine blue.
Probe Preparation
TheStatistical Analysis
All values determined by RPA were normalized to the 18S band (exposed for a shorter period to account for the generally larger signal). In general, the 18S bands were exposed for 1-3 min and all other bands for 15 min. Significance was determined by two-way analysis of variance comparing time (development) effects and subunit differences or organ differences as indicated. P < 0.05 was considered significant. ![]() |
RESULTS |
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rENaC mRNA Expression Is Regulated in Tissue-Specific Manner
Colon.
To characterize and quantitate the mRNA expression of each of the three
rENaC subunits, we conducted RPA in colon and lung in fetal and
postnatal rats. The developmental time course of rENaC mRNA expression
in the colon and lung were completely different. The features that
characterize colon rENaC expression during development are shown in
Figs. 1 and 2. Figure
1A shows
an example of an RPA for all three rENaC subunits in colon tissue
during development. All three subunits showed a gradual increase in
abundance throughout the period examined. The quantitative assessment
of the amount of each of the subunits relative to the abundance of
-rENaC on day 1 is shown in Fig.
2A.
-rENaC appeared to have the greatest abundance throughout
development. The amount of mRNA for each subunit was greater on
day 30 than on day
1.
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Lung.
The lung showed a completely different developmental time course of
rENaC mRNA expression from the colon. Figure
1B shows an example of an RPA for all
three rENaC subunits in lung tissue during development. Figure
3A shows
the analysis of the amount of each of the subunits relative to the
abundance of -rENaC on day 1. This
analysis indicates that the amount of
-rENaC is several-fold greater
than
- or
-rENaC at all time points after fetal
day 19. It also shows the biphasic nature of
-rENaC
mRNA expression with peaks around the time of birth and again 2-4
wk later.
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rENaC mRNA Expression Is Epithelial Cell Specific in Developing Colon and Lung
We used in situ hybridization to localize rENaC subunit mRNA expression. Localization was detectable in the early postnatal period in both the colon and lung. The in situ hybridization pattern in the colon was quantitatively similar to what we might have expected from the RPA analysis. We were unable to detect any signal before postnatal day 9. By day 15, expression of each subunit was localized to the surface epithelial cells with little signal in the crypts (Fig. 5, A-F).
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Representative photographs of the in situ hybridization results are
shown in Fig. 6. All three subunits were
expressed in epithelial cells of the bronchi and bronchioles during the
perinatal period. No expression was detected in blood vessels.
-rENaC was evident in alveolar epithelia at
postnatal day 1, whereas
- and
-rENaC were undetectable in alveoli (Fig. 6,
B, E,
and H). The pattern on
postnatal day 15 was similar; however,
at this point both
- and
-rENaC mRNAs were detected in alveolar
epithelia (Fig. 7,
B, E,
and H). The expression pattern in
the alveoli was similar to that seen in the adult rat lung and suggests
expression in type II pneumocytes (16, 27).
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DISCUSSION |
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The present studies show marked developmental heterogeneity in rENaC mRNA expression between colon and lung. In the colon prenatal increases in rENaC mRNA expression in the descending colon are modest, but a significant postnatal rise occurs between days 3 and 30. In contrast, lung rENaC mRNA upregulation begins after fetal day 19 with peak expression by the first postnatal day. The cellular localization patterns for rENaC mRNA in colon and lung are similar to that reported in adult rat tissues (15, 27). These results show that rENaC developmental expression is regulated in a temporal and tissue-specific manner, reflecting different regulatory mechanisms in these organs.
There are few data on rENaC expression in the colon during development.
Expression of -rENaC mRNA increases during development (14, 49), but
the ontogeny of the
- and
-rENaC subunits in the colon has not
been described. A recent report identified a developmental aspect that
we did not detect (14). A truncated (1.2 kb)
-ENaC transcript
missing the first ~800 nt of the coding region was noted in mouse
colon transiently before birth (14). Because our riboprobe hybridizes
to this region, we would not have detected this shorter transcript.
However, it is not likely that this shorter transcript would have a
similar function as the full length because it would be predicted to be
missing the first membrane spanning domain.
Aspects of the ontogeny of ENaC subunit mRNA expression in the lung
have been reported in the rat (14, 33, 45) and human (29, 30, 47). Our
results are similar to those of O'Brodovich and co-workers (33, 45)
who showed a biphasic expression of rat -ENaC with peaks around the
time of birth and again in adult life. The timing of the second
increase was not reported in their work. Dagenais and colleagues (14)
also noted a perinatal increase in
-ENaC mRNA expression in
developing mouse lung but had limited postnatal time points.
However, our results differ from those previously reported for the
pattern of - and
-rENaC expression in the lung. Whereas the
previous report (45) did not note a biphasic response of these
subunits, we can clearly detect this response (Fig.
3B). The reason may be due to the
differences in rat strain or to the nature of the assay. We suspect
that the latter explanation is correct. Ribonuclease protection assays
are 10- to 100-fold more sensitive in detecting low abundance mRNAs
than Northern hybridization. Thus our study might have detected more
subtle changes in the expression of the lower abundance
- and
-rENaC mRNAs in the lung.
Functional Importance of ENaC in Development
The epithelial-specific expression patterns of the rENaC subunits in the colon and lung are consistent with the ENaC complex being important in mediating developmentally regulated electrogenic Na+ absorption in these organs. Amiloride-sensitive Na+ absorption across the rat descending colon begins at the end of the first week of life and increases through the weaning period (38). The gradual increase in ENaC subunit mRNA expression reported here (Fig. 2) correlates well with the presumed functional consequences (Na+ transport). Studies in humans suggest that colonic salt absorption is present by late in the third trimester and that this route of Na+ conservation may be more important than the kidney in the neonatal period, especially in the preterm infant (1, 21). Amiloride-sensitive current is also present in the surface epithelium of the descending colon where rENaC mRNA is expressed in the neonate (this study) and adult (15, 40). Thus colonic Na+ absorption via ENaC may be an important nutritional source of Na+ to support the rapid growth that takes place in the neonatal period (17).Although there are few studies of ion transport across rat airways in vivo or in vitro (7, 23), amiloride-sensitive short-circuit current has been reported in the rat trachea (23). In other species amiloride-sensitive Na+ transport is well documented throughout the pulmonary epithelium. For example, studies of the ion transport pathways across airway epithelia of human (5, 6, 22) and mouse airways document the presence of significant amiloride-sensitive short-circuit current. Amiloride-sensitive Na+ transport also accounts for the majority of the short-circuit current present in monolayer cultures of alveolar type II cells from adult rats (13, 26). Measurements of the bioelectric properties of distal lung epithelia from fetal rats (35, 39) or humans (3, 28) also show evidence of significant amiloride-sensitive apical membrane Na+ current. Thus these in situ studies support the notion that rENaC is responsible for electrogenic Na+ transport in colonic and pulmonary epithelia.
Factors Potentially Responsible for ENaC Expression
The contrast in the onset of rENaC mRNA expression between the colon and lung implies that different regulatory factors are involved. There are several developmental aspects that might explain these differences. For purposes of simplicity, we will discuss the early (prenatal) response separately from the later (postnatal) response.We consider the prenatal increase in -rENaC mRNA to be caused by the
prenatal surge of corticosterone. Although this hypothesis has yet to
be proven conclusively, there are two lines of evidence that strongly
support it. First, the increase in fetal corticosterone occurs
immediately before the increase in the abundance of
-,
-, and
-rENaC (4, 25, and Fig. 3), indicating a strong temporal correlation. Second, synthetic glucocorticoid given before the normal
glucocorticoid surge causes an increase in
-rENaC mRNA in fetal rat
lung (33, 45). Furthermore, cultures prepared from rat fetal lung
epithelia (11, 45) or tissue explants (29, 46) show a similar response
to synthetic glucocorticoids. Thyroid hormone might also influence ENaC
expression in fetal lung, although the evidence for this is conflicting
(11, 46).
If the fetal corticosteroid surge increases rENaC mRNA in the lung, why
does it not do so in the colon? There are two possible explanations.
First, there may be an inadequately developed response mechanism in the
colon. For example, exogenous dexamethasone must be given after
fetal day 17 for it to produce its
effect on -rENaC mRNA in the lung. If given earlier (i.e., before
the canalicular stage), dexamethasone apparently has no effect (45).
Thus there seems to be a combination of events necessary to effect the
increase in
-rENaC mRNA in lung. A similar scenario might also occur
in the colon, but the events enabling colonic glucocorticoid
responsiveness might not be activated by fetal day
19. A second possibility derives from our understanding
of organ responsiveness to corticosterone and aldosterone. Both steroid
hormones bind to and activate the mineralocorticoid receptor. However,
in classic aldosterone-responsive target tissues, such as kidney
collecting duct and colon, corticosterone (and cortisol) is inactivated
by type II 11-
-hydroxysteroid dehydrogenase (8, 31). It is possible
that the differential effect of corticosterone on lung might be owing
to the presence of this metabolizing enzyme in colon as early as
fetal day 18. One study presents
evidence of type II 11-
-hydroxysteroid dehydrogenase activity in
midgestation human fetal lung and colon (42), whereas another
demonstrates conversion of cortisol to cortisone by first and second
trimester human fetal lung (32), raising the possibility that the
activity of this enzyme influences glucocorticoid responsiveness in
fetal tissues.
After the peak expression of rENaC mRNA in the lung around the time of
birth, the levels for all three subunits decline. After postnatal day 9 they begin to rise
again. These later changes parallel the increases in circulating
corticosterone in neonatal life (19). The fall in circulating
corticosterone levels in newborn rats probably reflects the switch from
predominantly maternal corticosterone production to that of the
newborn. During the first 1-2 wk of life rodents are relatively
unresponsive to stimuli that increase corticosterone in adults, a
period some call the "stress hyporesponsive period" (25, 41).
This second parallel increase in lung rENaC mRNA and circulating
corticosterone adds to the evidence that this adrenal steroid is a
major regulator of lung rENaC. In support of this notion, studies in
adrenalectomized adult rats also show that the mRNAs for -,
- and
-ENaC in the lung increase in response to dexamethasone (40).
The mechanism responsible for the increase in rENaC mRNA in
the colon after birth is not entirely clear. One possibility is that an
increase in circulating aldosterone produces this effect. It is well
established that aldosterone levels increase after the first week of
life in the rat (37). In addition, there is a strong correlation
between the aldosterone levels and the magnitude of amiloride-sensitive
short-circuit current across the colon, a measure of ENaC-mediated
Na+ transport (37, 38). The
temporal relationships between aldosterone levels, the effector mRNA,
and the functional effects provide good circumstantial evidence for
this explanation. In addition, exogenous aldosterone increases
amiloride-sensitive short-circuit current in colons from adult rats
(18) and aldosterone administered to 10-day-old rats produces a modest
increase in colonic -rENaC mRNA levels (49). From this analysis, one
might presume that whereas corticosterone is a major regulator of rENaC
expression in lung, aldosterone is the major regulator of rENaC in the colon.
Despite the attractiveness of this scenario, there are two lines of
evidence that raise important questions regarding its adequacy. First,
administering synthetic glucocorticoid to 10-day-old rats did not
increase -rENaC mRNA levels in colon (49). One might
explain this lack of a glucocorticoid effect as the result of
differential effects of glucocorticoid and mineralocorticoid hormones;
they do interact with different receptors. However, it would be the
first example of a tissue that responded to mineralocorticoid that did
not respond similarly to synthetic (i.e., nonmetabolizable) glucocorticoids. Furthermore, in adult rats dexamethasone increases colonic rENaC mRNA in a pattern similar to that of aldosterone (2, 40).
Second, it is now clear that either aldosterone or dexamethasone
produces an increase in
- and
-rENaC in adult rat colon without
any change in
-rENaC mRNA (2, 24, 40). If such steroid effects play
a role in the increase of
- and
-rENaC mRNA during development,
what causes the increase in
-rENaC mRNA in the postnatal period
(Fig. 2)? If aldosterone is producing these postnatal effects, it must
be doing it via a mechanism that is not currently recognized or that is
lost in adults. Clearly, further study is necessary to determine the
specific mechanisms responsible for the regulation of these mRNAs and
the relationship between the expression of ENaC subunit mRNAs and proteins.
That the ontogeny of rENaC mRNA expression is discordant in colon and lung is consistent with the needs of the organism at specific developmental time points. The increase in postnatal Na+ absorption by the colon is thought to be an important source of nutritional Na+ during a time of rapid growth (1, 21, 38). Upregulation of rENaC in the lung before birth allows the absorption of Na+, which drives the absorption of lung fluid at the time of birth. The absorption of Na+ in the lung via ENaC is essential for survival (20, 34-36).
Because both the ability to absorb Na+ from the colon and the clearance of fetal lung fluid are important for perinatal survival and growth, it is likely that abnormalities in the regulation of ENaC expression might be relevant to disease in premature infants. Colonic and pulmonary Na+ transport may be functionally immature in preterm infants born before the signals regulating ENaC expression are normally active. Thus immaturity of Na+ transport via ENaC may contribute to the pulmonary, electrolyte balance, and nutritional problems of the preterm infant.
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ACKNOWLEDGEMENTS |
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We thank Monica Driscoll for the -rENaC and
-rENaC cDNA
clones. We thank Rita Sigmund for technical assistance.
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FOOTNOTES |
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This work was supported in part by the National Institutes of Health Grant HL-02767 and the Children's Miracle Network Telethon (P. B. McCray) and the National Institutes of Health Grant HL-55006 (J. B. Stokes), the O'Brien Kidney Disease Center National Institutes of Health Grant DK-52617 (J. B. Stokes and P. B. McCray) and a merit award from the Department of Veterans Affairs (J. B. Stokes). S. Watanabe was supported in part by a fellowship from the Iowa Affiliate of the American Heart Association.
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: P. B. McCray, Jr., Dept. of Pediatrics, Univ. of Iowa College of Medicine, Iowa City, IA 52242.
Received 23 March 1998; accepted in final form 12 August 1998.
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