Developmental expression of the epithelial Na+ channel in kidney and uroepithelia

Shigeru Watanabe, Kazumichi Matsushita, Paul B. McCray Jr., and John B. Stokes

Laboratory of Epithelial Transport, Departments of Internal Medicine and Pediatrics, University of Iowa; and Veterans Affairs Medical Center, Iowa City, Iowa 52242


    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The epithelial Na+ channel (ENaC) plays an important role in regulating Na+ balance in neonatal and adult life. Using in situ hybridization, we localized alpha -, beta -, and gamma -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 beta - and gamma -rENaC, but not alpha -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 beta - and gamma -rENaC decreased relative to alpha -rENaC in outer and inner medulla. The urinary bladder, in contrast, demonstrated the greatest increase in beta - and gamma -rENaC mRNA abundance after birth. We were generally unable to detect alpha -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


    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 alpha -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.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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 alpha -, beta -, and gamma -rENaC (36).

The alpha -rENaC construct was a 422-nt segment corresponding to nucleotides 223-645 of the coding region (23) subcloned into the pKS(-) vector (Stratagene, La Jolla, CA) using restriction sites EcoR I and Pst I. The beta -rENaC construct was a 249-nt segment subcloned into the vector pCR-Script SK(+) (Stratagene) using restriction sites Pst I and Sac I. The gamma -rENaC construct was a 675-nt segment subcloned into the pCRII-TA vector (Invitrogen, Carlsbad, CA) using restriction sites Xba I and BstX I. All of the probes used for rENaC subunits were directed against unique segments within the open-reading frame.

Antisense 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 alpha -, beta -, and gamma -rENaC, respectively.

For the development studies, 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 GAPDH because these latter probes might be developmentally regulated in an organ-specific fashion, making comparison 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 matched well with experimentally determined ratios. For the effect of dietary NaCl intake on bladder rENaC mRNA, we used GAPDH to normalize for RNA abundance. We have previously shown that dietary NaCl has no effect on GAPDH abundance (33).

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.

The gamma - and alpha -rENaC probes occasionally showed degradation products when the amount of the respective mRNA was large. The magnitude of these products was less than 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 products was conducted on the major protected fragments. The 18S probe protected a major fragment of ~82 nt. Because it was the most dominant and consistent one, we used it for normalizing all densitometric quantitation.

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. We point out that RPA may not provide precise quantitative information regarding the relative abundance of each subunit compared with the other subunits. However, the relative abundance of alpha -, beta -, and gamma -rENaC mRNA by this method is similar to that obtained by Northern analysis (24, 34, 37).

In 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.

Paraffin and frozen sections were prepared for in situ hybridization as previously described (22). Hybridization 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 (39) as modified by this laboratory (22, 38). After hybridization, sections were washed at high stringency (50% formamide, 2× 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 µg/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 for in situ hybridization. The alpha -rENaC cDNA used for the in situ hybridization studies was the same as that used for the RPA described above. The beta -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 alpha - and beta -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 gamma -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.

All in situ hybridization reactions were carried out using serial sections for sense and antisense probes. 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. We detected no consistent difference between the results from the two types of fixation except that paraffin sections had better morphology and frozen sections had lower background.

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).


    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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 beta - and gamma -rENaC hybridized with the epithelium lining the pelvis, ureter, and bladder. We detected no alpha -rENaC in fetal uroepithelia at any stage. Sense (control) hybridization showed no localized signal in any region (data not shown).


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Fig. 1.   In situ hybridization of rat kidney at fetal day 17. Brightfield (left) and darkfield photos (right) of same fields are shown for hybridization with alpha - (top), beta - (middle), and gamma -rat EnaC (rENaC) probes (bottom). Arrows indicate ureter and arrowheads indicate pelvic epithelium. Magnification, ×16.

By 3 days after birth, the more fully developed kidney continued to show specific signals for all three subunits in all regions of the kidney (Fig. 2). There was more intense hybridization in the cortex, probably reflecting the greater degree of maturation of the cortical collecting ducts (CCD). At this early time, there was the suggestion that the relative intensity of the signal in the papilla was beginning to decline. This shift from papilla to cortex appeared more pronounced for alpha -rENaC than for beta - or gamma -rENaC. The pelvis, ureter, and bladder continued to demonstrate specific signals for beta - and gamma -rENaC but not alpha -rENaC.


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Fig. 2.   In situ hybridization of rat kidney 3 days after birth. Brightfield (left) and darkfield photos (right) of same fields are shown for hybridization with alpha - (top), beta - (middle), and gamma -rENaC probes (bottom). Papilla (p) is more fully developed, and kidney is larger. Arrowheads indicate pelvic epithelium. Magnification, ×16.

Nine days after birth, the intensity of the signal for all three subunit mRNAs was markedly diminished in the inner medulla, but strongly evident in the CCD and possibly the distal and connecting tubules (Fig. 3). We did not specifically identify (using special markers) these structures beyond the light microscopic appearance. The pattern of distribution of all three subunits did not change substantially after postnatal day 9. The cortical distal nephron continued to show specific localization that disappeared in the outer medulla and was undetectable in the papilla (data not shown). This pattern is the same as has been reported in adult kidney (12, 22). The pelvic epithelium continued to be negative for alpha -rENaC and positive for beta - and gamma -rENaC throughout the period of study.


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Fig. 3.   In situ hybridization of rat kidney 9 days after birth. Brightfield (left) and darkfield photos (right) of same fields are shown for hybridization with alpha - (top), beta - (middle), and gamma -rENaC probes (bottom). Signal for all 3 subunits is diminished in papilla (p). beta - and gamma -rENaC, but not alpha -rENaC, continue to demonstrate a signal in pelvic epithelium. Magnification, ×16.

Figure 4 shows an example of beta - and gamma -rENaC signals in the uroepithelial cells of the urinary bladder. We never detected a signal for alpha -rENaC in any bladder at any time point. The beta - and gamma -rENaC mRNA were diffusely expressed in the uroepithelial cells and did not appear to be present in the underlying smooth muscle cells. The pattern for alpha -, beta -, and gamma -rENaC expression did not change throughout the period of development.


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Fig. 4.   In situ hybridization of rat urinary bladder at fetal day 19. The specific signal was confined to uroepithelial cells. No specific signal for alpha -rENaC was detected in any bladder at any point in development. Orientation of Fig. 4 is as in Figs. 1-3. Arrows denote uroepithelium.

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 alpha -rENaC abundance on day 1 after birth (Fig. 6A), the rank order of abundance was beta  > alpha  > gamma  (P < 0.01). When each subunit was normalized to its own abundance on day 1 (Fig. 6B), alpha -rENaC showed a different pattern of expression than did beta - or gamma -rENaC. Whereas the latter two subunits decreased after birth, alpha -rENaC increased (P < 0.001).


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Fig. 5.   Example of RNase protection assay (RPA) of alpha -, beta -, and gamma -rENaC from whole kidney at various days of fetal life (17f-21f) and postnatal days (D1-D30). Protected bands run as indicated on left markers; unprotected bands are shown in right lane. The 18S ribosome was used as a denominator to factor for amount of total RNA in reaction.


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Fig. 6.   Quantitation of RPA using RNA from whole kidney. Timepoints as defined in Fig. 5. A: relative expression. Values normalized to intensity of 18S band and also to intensity of alpha -rENaC band on D1. Each subunit is different from each other by 2-way ANOVA and subsequent Newman-Keuls (P < 0.01). B: relative pattern of expression. Each value is normalized to 18S and to value of its own level of expression at D1. alpha -rENaC pattern is significantly different from beta - and gamma -rENaC primarily because of divergence of expression after D3 (+ P < 0.001, *P < 0.01); n = 4 assays.

To quantitate the intrarenal distribution of each subunit, we dissected the kidneys into cortex, outer medulla, and inner medulla. The earliest time point where the kidney was large enough to permit this dissection was postnatal day 9. As shown in Fig. 7, there were modest but significant quantitative differences in the expression of each of the subunits over time. The pattern of alpha -rENaC expression was different from that of beta - and gamma -rENaC in the outer and inner medulla, but not statistically significant in the cortex.


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Fig. 7.   Quantitation of RPA using RNA from regions of kidney on days 9, 15, and 30. Values on left were normalized to 18S and value of alpha -rENaC on day 9. Values on right were normalized to 18S and to their own individual value on day 9. Values with different symbols (*, +, #) are significantly different from each other (P < 0.05) by 2-way ANOVA and subsequent Newman-Keuls; n = 3 assays. bullet , alpha -rENaC; , beta -rENaC; black-triangle, gamma -rENaC.

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 alpha -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 beta - and gamma -rENaC in urinary bladder. There is considerably more beta - than gamma -rENaC, especially after birth, at which point the abundance of both increases and plateaus between days 9 and 30. Although the amount of beta -rENaC is greater than that of gamma -rENaC, the majority of the increase in the abundance of each subunit occurs after birth.


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Fig. 8.   Example of RPA of alpha -, beta -, and gamma -rENaC from urinary bladder at various days of fetal life (17f-21f) and postnatal days (D1-D30). Location of protected bands of beta - and gamma -rENaC are indicated on left; unprotected bands are shown on right. The 18S ribosome was used as a denominator to factor for amount of total RNA in reaction.


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Fig. 9.   Quantitation of RPA using RNA from urinary bladder. Timepoints are defined in Fig. 8. A: relative expression. Values normalized to intensity of respective 18S band and to beta -rENaC valueon D1. * beta -rENaC is more abundant than gamma -rENaC (P < 0.0005 by ANOVA). B: relative pattern of expression. Each value normalized to 18S and to value of its own level of expression at D1. Relative patterns are different (P < 0.0005 by ANOVA; n = 4).

We searched extensively for evidence of alpha -rENaC mRNA in bladder. Of the 36 bladders examined, we detected a band where alpha -rENaC should migrate in only 2 tissues. Both of these tissues were taken from rats that were ~30 days old. We therefore undertook a detailed examination of urinary bladders around the weaning period. Bladders were removed from rats on postnatal days 17, 19, 21, 23, 24, 25, 26, 27, 28, 30, 32, and 34. Normal weaning occurs gradually from days 22 to 28. We detected no major changes in the abundance of beta - or gamma -rENaC over this time period. In addition, we did not detect any alpha -rENaC mRNA in two sets of these experiments (data not shown).

In a final effort to detect alpha -rENaC in the urinary bladder, we examined the effects of dietary NaCl. Rats fed a low-NaCl diet had an increased abundance of beta - and gamma -rENaC mRNA in colon (1) and an increased alpha -rENaC mRNA abundance in inner medulla (33). The effects of the high- and low-NaCl diets on beta - and gamma -rENaC is shown in Fig. 10. There was no significant effect on either mRNA. Furthermore, we were unable to detect any alpha -rENaC.


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Fig. 10.   Effect of dietary NaCl on beta - and gamma -rENaC abundance in urinary bladder of weaned rats. Rats were randomized to either an 8% NaCl diet (HS) or a 0.13% NaCl diet (LS) for 2 wk. Quantitation of subunits was normalized to GAPDH mRNA. Diet had no significant effect on abundance of either subunit (n = 6).


    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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 alpha -rENaC increasing and beta - and gamma -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. alpha -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 beta - and gamma -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 alpha -rENaC is much greater than that of either beta - or gamma -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 alpha -rENaC in the present studies is similar to the developmental expression of the alpha -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 alpha -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 alpha -rENaC mRNA and protein in adult rat urinary bladder.

We tried two maneuvers to determine whether our failure to detect alpha -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 alpha -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 alpha -rENaC in these studies. Second, we manipulated the dietary NaCl in weaned rats. A low-NaCl diet increased alpha -rENaC mRNA abundance in the inner medulla but not in cortex (1, 25, 33). Neither diet allowed us to detect the alpha -rENaC transcript using our RPA. We must conclude that, although dietary NaCl can influence alpha -rENaC expression in IMCD, it does not do so in urinary bladder. If there is a stimulus that can induce alpha -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 11beta -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 alpha -rENaC mRNA can be increased by steroids when basal levels are low (as in adrenalectomy). Whether a similar stimulus might increase beta - and gamma -rENaC in CCD is not so clear. In the adult, the acute steroid effect in the cortex is limited to the alpha -subunit (33). However, it may be possible for steroids to increase gamma -rENaC mRNA in CCD under some conditions (11). The mechanism certainly exists in the adult colon for steroids to increase beta - and gamma -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 beta - and gamma -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.


    ACKNOWLEDGEMENTS

We appreciate the technical advice and assistance of Rita Sigmund.


    FOOTNOTES

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.


    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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