Laboratory of Epithelial Transport, Departments of Internal Medicine and Pediatrics, University of Iowa; and Veterans Affairs Medical Center, Iowa City, Iowa 52242
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ABSTRACT |
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The epithelial Na+ channel
(ENaC) plays an important role in regulating
Na+ balance in neonatal and adult
life. Using in situ hybridization, we localized -,
-, and
-rat
ENaC (rENaC) mRNA in developing rat kidney and
uroepithelia. rENaC mRNA was first detectable on fetal day 16, and by
fetal day 17, mRNA was abundant in the
terminal collecting duct and uroepithelia. After birth, the intensity
of the signals for all three subunits increased in the cortical
collecting ducts and by 9 days after birth had diminished in the inner
medullary collecting ducts. Expression in uroepithelial cells was
different. mRNA for
- and
-rENaC, but not
-rENaC, was detected
in pelvis, ureters, and bladder at all stages of development beyond
fetal day 16. By RNase protection
assay (RPA), the greatest increase in subunit abundance in the kidney
occurred before birth. Between postnatal days
9 and 30, the
abundance of
- and
-rENaC decreased relative to
-rENaC in
outer and inner medulla. The urinary bladder, in contrast, demonstrated
the greatest increase in
- and
-rENaC mRNA abundance
after birth. We were generally unable to detect
-rENaC by RPA in
urinary bladder. Feeding weaned rats a diet of high or low NaCl did not
change the abundance of any of the subunit mRNAs in bladder. These
results demonstrate additional heterogeneity of developmental
expression and regulation of ENaC. The differences between the
collecting duct and uroepithelial cell rENaC mRNA regulation raise the
possibility of significant differences in function.
RNase protection assay; collecting duct; urinary bladder; ureter; pelvis; in situ hybridization
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INTRODUCTION |
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THE EPITHELIAL Na+ channel (ENaC) plays an important role in regulating Na+ excretion by the kidney. ENaC is located on the apical membrane of the collecting duct, and its activity is regulated by a variety of hormones that thereby contribute to the regulation of Na+ balance. Of the agents that regulate ENaC activity, aldosterone is the best studied. It is now clear that a part of the upregulation of ENaC activity by adrenocortical steroids is an increase in steady-state mRNA levels for one or more of the three subunits (1, 19, 25, 33).
ENaC plays an important role in the transition from fetal to neonatal
life. One particularly dramatic demonstration of its importance
involves the elimination of the -subunit by genetic recombination;
mice so manipulated die within 48 h of birth (14). The major role for
ENaC in the neonatal period seems to be to absorb lung fluid. However,
rescue of expression, when largely confined to the lung by a
cytomegalovirus (CMV) promoter, does not restore these mice to a normal
existence. They develop severe hyperkalemia and metabolic
acidosis, findings similar to those seen in pseudohypoaldosteronism
(15). Genetic studies of patients with this syndrome have detected
variants in the primary sequence on one or more of the subunits (7).
The importance of understanding the normal development of Na+ transport in the kidney is underscored by the fact that premature infants can develop severe electrolyte disturbances as a result of their inability to regulate Na+ excretion normally (13, 28). A portion of this dysregulation appears to involve insensitivity to aldosterone (28, 32, 35).
Recently, rat ENaC (rENaC) mRNA has been discovered in the epithelial cells of the urinary bladder, ureters, and pelvic epithelium (17, 31). The functional significance of this mRNA is not entirely clear. We hypothesized that, if the function of rENaC in the urinary bladder was similar to that of the collecting duct, there should be similar patterns of developmental expression and regulation by dietary NaCl.
The purpose of these experiments was to define the location of the ENaC subunits within the kidney and urinary bladder during development, to quantitate their expression, and to determine whether dietary NaCl influenced their expression in urinary bladder. We reasoned that information from these experiments would provide valuable clues regarding the mechanisms involved in regulating Na+ excretion in the neonatal period and the transition into adult life.
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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). Pilot experiments were conducted on whole fetus from days 12, 14, and 16 of gestation. (Gestation for rat is 21-22 days.) Kidneys and bladders were obtained from rats on days 17, 19, 20, and 21 of gestation (17f, 19f, 20f, and 21f) and postnatal days 1, 3, 9, 15, and 30 (D1, D3, D9, D15, and D30). Maternal animals were anesthetized with methoxyflurane and exsanguinated, and the fetuses were removed and placed in ice-cold Hanks' solution. Neonatal rats were also anesthetized with methoxyflurane. Fetal tissues were pooled for RPA as follows: fetal days 17 and 19, four kidney and six bladder specimens; fetal day 20, four kidney and four bladder specimens; fetal day 21, two kidney and four bladder specimens; postnatal day 1, two kidney and three bladder specimens; all other time points, one specimen/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.
For studies on the effects of dietary NaCl intake, newly weaned rats were randomly divided into those to receive a low-NaCl (0.13%) diet (ICN; Nutritional Biochemicals) or a high-NaCl (8%) diet, which was otherwise identical, for 2 wk. This protocol is similar to one we have previously reported (33) and is well tolerated by these animals.Preparation of RNA. The kidneys and bladders were rapidly removed and immersed directly in liquid nitrogen. Kidneys from rats D9 or older were sliced and some were dissected by region before freezing. Total RNA was isolated from tissues using a modification of the method of Chomczynski and Sacchi (8). An amount of tissue estimated to yield 50-100 µg total RNA was homogenized using a 92/Polytron PT-DA 3012/2 TS probe in 2 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 (DEPC)-treated water. Total RNA was measured by absorption spectrophotometry at 260 nm. The 260/280 ratio averaged 1.6-1.8.
Probe preparation for the RNase protection
assay.
The plasmids used to make the antisense probes for the RNase protection
assay (RPA) were modified from those previously described for -,
-, and
-rENaC (36).
RNase protection assay. 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 (diluted to 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 semi-dry electroblotter (Fisher, Itasca, IL). The membrane was subsequently UV crosslinked (UV Stratalinker; Stratagene), and the protected RNA fragments were developed 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.
TheIn situ hybridization.
Kidney and bladder samples were prepared for both paraffin embedding
and frozen sections for use in in situ hybridization experiments, as
previously reported (22). We obtained kidney and bladder tissue from
3-4 animals at each time point for paraffin and for frozen
sections. Tissues were immersed in 2% paraformaldehyde in PBS for 2 h
at 4°C, embedded in paraffin, sectioned to 5-µm thickness, and
mounted onto slides (Superfrost Plus; Fisher Scientific, Fair Lawn, NJ)
that were air dried and stored with desiccant at room temperature in an
airtight box until they were used for hybridization. Tissues for frozen
section were rapidly removed and frozen in liquid isopentane. Frozen
tissues were sectioned on a cryostat to 7-µm 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 at 20°C in an airtight box.
Probe preparation for in situ
hybridization.
The -rENaC cDNA used for the in situ hybridization studies was the
same as that used for the RPA described above. The
-rENaC cDNA was a
modification of a previously described construct (36) cloned into pCRII
TA vector (Invitrogen). The probe was restricted with
Spe I to remove the 3' poly-A
tail and religated. The plasmids containing the
- and
-rENaC
probes were linearized with the appropriate restriction enzymes, and
sense (T3) or antisense (T7) labeled probes were prepared using the
Riboprobe Gemini II Core System transcription kit (Promega, Madison,
WI) substituting 11 µM 35S-UTP
(Amersham, Arlington Heights, IL) for cold nucleotide. The
-rENaC
cDNA consisted of the first 567-nt segment of the open-reading frame
(36) subcloned into pCRII TA cloning vector (Invitrogen, San Diego,
CA). Purified plasmids were linearized with the appropriate restriction
enzyme, and sense (T7) or antisense (SP6)
35S-UTP,
35S-CTP (Amersham) double-labeled
probes were prepared.
Statistical analysis. All values determined by RPA were normalized to the 18S or the GAPDH band (exposed for a shorter period to account for the generally larger signal). In general, the denominator bands were exposed for 1-3 min and all other bands were exposed for 15 min. Significance was determined by two-way analysis of variance comparing time (development) effects and subunit differences or organ differences as indicated. The time effects on the abundance of the subunits was always highly significant (P < 0.01).
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RESULTS |
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Localization of rENaC mRNA.
In situ hybridization of whole fetus before day
16 showed no evidence of specific signal. On
fetal day 16, there was some evidence
of specific hybridization in kidney, but on fetal day 17 the localization was much clearer. As shown in Fig.
1, each of the three rENaC subunit
antisense riboprobes hybridized to collecting ducts. The most intense
signal was detected in the developing papilla. rENaC mRNA was also
detectable by fetal day 16 in
uroepithelium, but was much clearer by day
17. In contrast to the collecting ducts, only - and
-rENaC hybridized with the epithelium lining the pelvis, ureter, and
bladder. We detected no
-rENaC in fetal uroepithelia at any stage.
Sense (control) hybridization showed no localized signal in any region
(data not shown).
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Quantitation of rENaC in kidney.
We used an RPA to estimate the amount of each mRNA in whole kidney.
Figure 5 shows an example of a single assay
showing the developmental pattern at each timepoint. Figure
6 shows the densitometric analysis of four
such assays, demonstrating that each of the subunits increased in
abundance before birth and had a variable pattern after birth. When the
values for kidney were normalized to -rENaC abundance on
day 1 after birth (Fig.
6A), the rank order of abundance was
>
>
(P < 0.01). When
each subunit was normalized to its own abundance on
day 1 (Fig.
6B),
-rENaC showed a different pattern of expression than did
- or
-rENaC. Whereas the latter two subunits decreased after birth,
-rENaC increased
(P < 0.001).
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Quantitation of rENaC in urinary
bladder.
Figure 8 shows an example of an RPA of
bladder tissue during development. Because we did not detect an
-rENaC signal in these assays, we have not assigned a value to its
abundance. Figure 9 shows a quantitative
estimate of the developmental expression of
- and
-rENaC in
urinary bladder. There is considerably more
- than
-rENaC,
especially after birth, at which point the abundance of both
increases and plateaus between days 9 and 30. Although the amount of
-rENaC is greater than that of
-rENaC, the majority of the
increase in the abundance of each subunit occurs after birth.
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DISCUSSION |
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The present results demonstrate that the transcripts for all three
rENaC subunits are expressed in the kidney, with increasing abundance
in the final 3-4 days of fetal life. After birth, their overall
abundance changes modestly, with -rENaC increasing and
- and
-rENaC decreasing. The expression of all three subunits within the
kidney is limited primarily to the collecting ducts. In the immature
kidney, the greatest intensity is detected in the terminal collecting
duct. As the kidney matures, the signal in the inner medullary
collecting duct (IMCD) decreases while the signal in the cortical
distal nephron increases. This redistribution is largely complete by
about postnatal day 9.
The pattern of expression in the uroepithelium is completely different.
-rENaC mRNA is generally not detectable at any stage of development
and cannot be induced by reduction in dietary NaCl intake. In addition,
the greatest increase in the abundance of
- and
-rENaC mRNA
occurs after birth. These results add to the increasing evidence of
considerable heterogeneity of ENaC expression.
Developmental expression of rENaC is best understood in the lung, where
the abundance of -rENaC is much greater than that of either
- or
-rENaC mRNA (34, 37). As in the rat kidney, the abundance of
subunits in the rat lung increases most dramatically during the final
3-4 days of fetal life, with only modest changes after birth. The
pattern of renal
-rENaC in the present studies is similar to the
developmental expression of the
-subunit in mouse (10). To our
knowledge, there is no published information on renal ENaC expression
during development in any other species.
The lung and the kidney share another feature of rENaC development: localization of rENaC mRNA changes after birth. Whereas readily detectable mRNA diminishes in the IMCD and increases in the CCD, the mRNA in the lung increases in the alveoli (37). The reason(s) for this change in pattern after birth is not clear, but may be related to continuing maturation of these tissues. In the adult kidney, in situ hybridization readily detects all three subunits of rENaC mRNA, but detection in the inner medulla by this technique has not been reported (12, 22).
There are emerging data regarding the developmental expression of rENaC in colon tissue. Our studies show that the increase in rENaC expression is not limited to fetal life; mRNA abundance of all three subunits increases significantly after birth (37). This pattern is different from that of lung and kidney, but shares a general similarity with that of the urinary bladder.
The differences in the pattern of expression of rENaC mRNA in urinary
bladder and collecting duct are somewhat surprising at first glance.
After all, these tissues share similar embryonic origins. Furthermore,
there is strong evidence that the rabbit urinary bladder expresses
amiloride-sensitive Na+ transport,
at least when stretched (18). Our recent data indicate that -rENaC
mRNA is detectable in uroepithelial tissue using a very sensitive
technique, reverse transcriptase and polymerase chain reaction
(17). In addition, Smith et al. (31) have detected
-rENaC mRNA and protein in adult rat urinary bladder.
We tried two maneuvers to determine whether our failure to detect
-rENaC mRNA was because of an inadequate physiological stimulus.
First, we looked carefully at the period around weaning. We did this
because we detected two bladders having evidence of
-rENaC in rats
28 days old. We postulated that the process of weaning might produce a
physiological stimulus (such as a change in dietary
Na+ intake) that might induce the
mRNA. However, we failed to detect any
-rENaC in these studies.
Second, we manipulated the dietary NaCl in weaned rats. A low-NaCl diet
increased
-rENaC mRNA abundance in the inner medulla but not in
cortex (1, 25, 33). Neither diet allowed us to detect the
-rENaC
transcript using our RPA. We must conclude that, although dietary NaCl
can influence
-rENaC expression in IMCD, it does not do so in
urinary bladder. If there is a stimulus that can induce
-rENaC mRNA
in urinary bladder, we have not found it.
In attempting to integrate the present observations with the known functional aspects of collecting duct development, we note that there may be important species differences. The rabbit CCD is the best studied. There is general agreement that, during the first 1-2 wk of life, the capacity to reabsorb Na+ is less than in CCDs from more mature rabbits (30, 35). A similar sequence of events may occur in the rat collecting duct as well. Although there are no studies of the developing rat CCD that we know of, data from urine Na+/K+ ratios show that rats younger than 2 wk old are resistant to aldosterone (32). This resistance does not seem to be due to a lack of receptors or a failure to translocate them to the nucleus. To this data, we can now add that aldosterone resistance is not the result of the absence of ENaC subunit mRNA. Taken together, these data suggest that low Na+ transport by CCD and/or relative aldosterone resistance in early life is probably caused by the lack of a factor(s) that helps to coordinate the assembly and/or functional expression of Na+ channels. This process may also play a role in the maturation process resulting in the centripetal pattern of ENaC mRNA expression in the first weeks of life. It is important to note that these studies do not address the expression of rENaC proteins with development. When sufficiently sensitive and specific antibodies become available, they will add considerably to our understanding of how ENaC is assembled and functionally regulated.
The stimulus for the increase in ENaC mRNA expression between fetal day 17 and birth is not known. However, the pattern suggests that adrenal steroids may play an important role. This hypothesis is supported by two lines of evidence. First, steroid hormones (i.e., glucocorticoid and mineralocorticoid hormones) increase the abundance of ENaC subunit mRNA (1, 6, 19, 33). Thus steroids are logical candidates. Second, the increase in the abundance of subunit mRNA, particularly in lung (37), parallels the fetal surge in corticosterone (2, 21).
The hypothesis that the prenatal increase in kidney rENaC mRNA
abundance is caused by corticosterone could be challenged on the
following basis. This steroid has no effect on
Na+ transport by collecting ducts
because the enzyme 11-hydroxysteroid dehydrogenase (type 2) resides
in the collecting duct and inactivates the endogenous glucocorticoid
(4, 16). If the enzyme is active in the collecting duct, there should
be no effect on ENaC mRNA levels. The reason that lung rENaC levels
rise before birth is that the expression of this inactivating enzyme is
greatly reduced in lung tissue immediately before birth (5). It is not
inactivated in whole kidney, but cell-specific measurements have not
been conducted. From measurements in newborn rat kidneys, we know that the metabolizing activity on postnatal day
1 is considerably lower than on day
8 or in adults (3). Thus it is possible that the corticosteroid surge that occurs a few days before birth could play an
important role in the increase in rENaC mRNA levels in the kidney. The
subsequent rise in abundance may be related to increasing capacity of
the neonatal adrenal gland to produce steroids. In rodents, this
process evolves over the first 2 wk of life (21, 29).
The mechanism responsible for the shift in the intensity of ENaC
transcript intensity from the IMCD to the CCD and the distal nephron is
not clear. One possible factor is the increase in circulating aldosterone levels that begins 1-2 wk after birth (26). Our recent
experiments show that -rENaC mRNA can be increased by steroids when
basal levels are low (as in adrenalectomy). Whether a similar stimulus
might increase
- and
-rENaC in CCD is not so clear. In the adult,
the acute steroid effect in the cortex is limited to the
-subunit
(33). However, it may be possible for steroids to increase
-rENaC
mRNA in CCD under some conditions (11). The mechanism certainly exists
in the adult colon for steroids to increase
- and
-rENaC mRNA
abundance (1, 27, 33). The possibility that the mechanisms existing in
adult colon are transiently operative in the developing CCD or that
there is a different set operating in the collecting duct at different times during development deserve to be examined in greater detail.
The factors responsible for the increase in - and
-rENaC mRNA in
the urinary bladder after birth are more difficult to postulate. The
absence of an effect of dietary NaCl on any of the subunits makes it
unlikely that adrenal steroids play an important role in their
regulation. The lack of regulation by dietary NaCl suggests that rENaC
subunits in the urinary bladder may be playing a different role than
that ascribed to them in lung, colon, and collecting duct. The
magnitude of Na+ absorption by
urinary bladder is small, and the possibility that stretching might
activate its activity (20) raises intriguing possibilities regarding
its function. In this regard, it is useful to remember
that the ENaC proteins belong to a larger family, some of which appear
to be involved in mechanosensation (9). Perhaps there is another as yet
unidentified subunit that participates in the functioning of this
complex in urinary bladder.
These results further demonstrate the considerable heterogeneity of ENaC transcript expression in epithelial cells. The differences in expression during development add to the evidence from physiological stimuli suggesting that ENaC may serve different functions in different organs.
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ACKNOWLEDGEMENTS |
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We appreciate the technical advice and assistance of Rita Sigmund.
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FOOTNOTES |
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This work was supported, in part, by National Institutes of Health Grants DK-52617 and HL-55006, and by grants from the Department of Veterans Affairs, the American Lung Association, and the Children's Miracle Network Telethon. S. Watanabe was supported by a fellowship from the American Heart Association, Iowa Affiliate.
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: J. B. Stokes, Dept. of Internal Medicine, Univ. of Iowa College of Medicine, Iowa City, IA 52242.
Received 4 May 1998; accepted in final form 24 November 1998.
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