Regulation of rENaC mRNA by dietary NaCl and steroids: organ, tissue, and steroid heterogeneity

John B. Stokes and Rita D. Sigmund

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

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

Rats on a low-NaCl diet have a high Na+ channel activity in colon and kidney. To address the mechanism of this increased activity, we measured mRNA levels of three Na+ channel subunits in epithelial tissue (rENaC) from rats having been fed either a low (0.13%)- or high (8%)-NaCl diet for 2-3 wk. The size of the mRNA for each of the rENaC subunits as determined by Northern blot was unaffected by diet. RNase protection assay showed heterogeneity of response by organs and subunit. In lung, there was no effect of diet on any of the three subunits. In descending colon, the low-NaCl diet increased beta - and gamma -rENaC mRNA, with no effect on alpha -rENaC mRNA. In the kidney, the response to dietary NaCl was dependent on the region. In cortex and outer medulla, diet had no effect on any of the subunits. Rats fed the low-NaCl diet had greater alpha -rENaC in inner medulla but not beta - or gamma -rENaC mRNA. We next asked whether acute administration of pure glucocorticoid (GC) or mineralocorticoid (MC) hormones to adrenalectomized rats reproduced the effects of a low-NaCl diet. Six hours after administration of GC or MC, a somewhat different heterogeneity occurred. In lung, alpha -rENaC mRNA was increased but only in response to GC. In colon, either GC or MC increased beta - or gamma -rENaC, and there was no effect on alpha -rENaC. In kidney, either GC or MC increased alpha -rENaC, without an effect on beta - or gamma -rENaC. In contrast to the response to a low-NaCl diet, all three regions were similarly affected by acute steroids. These results demonstrate a striking heterogeneity in response to physiological stimuli that regulate ENaC function. The mRNA levels of each of the rENaC subunits can be determined by the type of steroid and by factors unique to the organ and even to the specific region of the kidney.

kidney; kidney medulla; colon; lung; sodium channel; aldosterone; RU-28362; dietary salt

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

THE DISCOVERY AND MOLECULAR CLONING of the three homologous proteins that comprise the epithelial Na+ channel (ENaC) have provided investigators with a critically important tool to further explore the mechanisms involved in Na+ homeostasis (10, 11, 27). The ENaC complex is present in the descending colon, distal nephron, and airway epithelia, where it permits the entry of Na+ into the cell across the apical cell membrane (9, 20, 39). The physiological regulation of Na+ transport by cells of the colon and renal collecting duct is greatly influenced by the amount of dietary Na+ and circulating levels of aldosterone (6, 20, 39). There is general agreement that a low-Na+ diet increases Na+ absorption by the target epithelial cells, at least in part, by increasing circulating aldosterone levels. The molecular details of the effects of low dietary NaCl and the extent to which aldosterone can account for its effects on target epithelial cells are incompletely understood.

Most or all of the effect of aldosterone on the apical Na+ channel requires new protein synthesis (2, 25). However, the specific proteins involved in the regulation of electrogenic Na+ transport are not known. A leading possibility is the ENaC complex. The simplest hypothesis linking dietary NaCl to the magnitude of electrogenic Na+ transport would be as follows. Aldosterone levels increase in response to dietary NaCl restriction (3, 33). The occupied mineralocorticoid (MC) receptors translocate to the nucleus, where they interact with hormone response elements to increase ENaC mRNA transcription and subsequently ENaC subunit protein synthesis. The increased amount of one or more of the three subunit proteins increases the assembly of functional channels in the apical membrane. More Na+ channels in the apical membrane increase the rate of Na+ entry and thus the magnitude of transepithelial Na+ transport. This simple scenario does not exclude other important events, such as alteration in the function of the Na+ pump, as playing additional roles in this process.

Portions of this hypothesis have begun to be examined. Elevated steady-state mRNA levels for beta - and gamma -subunits of rat ENaC (rENaC) have been detected in colons from animals eating a low-NaCl diet (3, 34). Steroids have been implicated in ENaC mRNA regulation in lung, but there is some disagreement about whether the target is the alpha -subunit alone (38) or all three subunits (34). The effects of dietary NaCl and steroids on ENaC mRNA in the kidney are unclear (3, 34).

The purpose of these studies was to test the idea that acute steroid administration to adrenalectomized (ADX) rats would reproduce the effects of chronic dietary NaCl restriction on steady-state mRNA levels in lung, colon, and kidney. Because the collecting duct in the cortex, outer medulla, and inner medulla serves somewhat different functions (37), we also sought to determine the response to these maneuvers in these three regions of the kidney. Finally, we designed these studies to separate the acute effects of pure glucocorticoid (GC) hormone from pure MC hormone.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Preparation of rats. For the studies comparing low- and high-NaCl diets, normal Wistar rats of either sex were purchased from Harlan Sprague Dawley (Indianapolis, IN) at the age of ~5 wk. On arrival at the University of Iowa, the shipments were randomly divided into those to receive a low (0.13%)-NaCl diet (ICN, Nutritional Biochemicals) or an otherwise identical diet but high in NaCl (8%). After 2-3 wk, some rats were placed in a metabolic cage, and their 24-h Na+ and K+ excretion rates were measured. Rats eating the low-NaCl diet excreted 264 ± 18 µmol Na+ and 1,207 ± 288 µmol K+. Rats on the high-NaCl diet excreted 6,481 ± 862 µmol Na+ and 1,834 ± 242 µmol K+.

Rats to be given steroids were obtained from Harlan Sprague Dawley 1 wk after undergoing ADX. They were fed 0.9% NaCl solution to drink and a normal rat diet (1.3% NaCl). ADX rats were maintained for 2-3 days at the University of Iowa Animal Care unit before being killed. On the morning of the study, the rats weighed ~220 g and received either the pure GC RU-28362 (1.0 mg/kg) or a combination designed to activate MC receptors consisting of aldosterone (1.5 mg/kg) plus the GC antagonist RU-38486 (1.8 mg/kg). Control animals received ethanol vehicle only (400 µl/kg ip). These protocols were chosen because this dose of RU-28362 saturates ~50% of the GC receptors, and this dose of aldosterone plus RU-38486 saturates ~90% of the MC receptors without crossover binding to the GC receptor (30). Organs from these rats were harvested 6 h after steroid administration.

Preparation of RNA. Rats were anesthetized with methoxyflurane and decapitated, and the lung, descending colon, and kidneys were rapidly removed. Lungs were immersed in liquid nitrogen. The descending colon was rinsed with ice-cold PBS, and the most distal 1.5 cm were trimmed and frozen. The kidneys were chilled and dissected into cortex, outer medulla, and papilla (inner medulla). Small pieces (<0.25 cm3) of cortex and outer medulla and the entire inner medulla were rapidly frozen in liquid nitrogen.

Total RNA was isolated from tissues by using the method of Chomczynski and Sacchi (13). An amount of tissue estimated to yield 25-50 µg total RNA was homogenized using a 92/Polytron PT-DA 3012/2 TS probe in 1.0 ml of the guanidinium isothiocyanate solution for 3-5 s, and the RNA was extracted in phenol-chloroform, acidified to pH 4.0 by addition of 2 M sodium acetate. After precipitation with isopropanol, the RNA was rinsed with 75% ethanol and resuspended in diethyl pyrocarbonate-treated water. RNA from the inner medulla was subjected to an additional step by centrifugation through RNeasy (Qiagen, Chatsworth, CA) to reduce contaminating materials that seem to be unique to this tissue. This technique permits recovery of >90% of starting RNA and greatly facilitates electrophoresis.

RNA analysis. Northern blot analysis was conducted on lung, ascending and descending colon, and each kidney region as previously described (40). Approximately 25 µg total RNA was fractionated by electrophoresis through a 1.5% agarose-6.6% formaldehyde gel buffered with 10 mM NaH2PO4 (pH 6.5) using a Hoefer HE-99 horizontal submarine unit (Hoefer Instruments, San Francisco, CA). RNA was transferred onto nylon (Hybond N, Amersham, Arlington Heights, IL) by capillary transfer in 10× standard saline citrate and ultraviolet (UV) cross-linked (UV Stratalinker, Stratagene, La Jolla, CA). The blots were prehybridized and hybridized in a buffer containing 1% BSA, 1 mM EDTA, 500 mM NaH2PO4 (pH 7.2), and 7% SDS according to Church and Gilbert (14).

The probes used for the Northern blots were those previously described for alpha -, beta -, and gamma -rENaC and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (40). These constructs were modified for the RNase protection assay (RPA) as follows. The alpha -rENaC construct was a 422-nt segment subcloned into the pKS(-) vector (Stratagene) 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 Bst XI. The rat GAPDH construct was a 140-nt segment extending from the translation start site to the first Sty I restriction site. All of the probes were directed against 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, 750, and 220 nt for alpha -, beta -, gamma -rENaC, and GAPDH, respectively (Fig. 1).


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Fig. 1.   Northern blots of RNA from regions of kidney, colon, and lung from rats fed a high (H)- or a low (L)-NaCl diet for 2 wk. Each blot hybridized twice, first with rat epithelial Na+ channel (rENaC) probe and again with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe. KC, kidney cortex; KOM, outer medulla; KIM, inner medulla; Asc Colon, ascending colon; Des Colon, descending colon.

The hybridization of ~1 ng of each of these probes with 25 µg total RNA from each of the tissues was conducted using the RPAII RNase protection assay kit (Ambion). RNase A and RNase T1 were diluted to 250 and 10,000 U/l, respectively, and the reaction was incubated for 18 h. The products were subjected to electrophoresis through a 5% denaturing polyacrylamide-8 M urea gel buffered with Tris borate for 2.5 h at 250 V and transferred to a nylon membrane (BrightStar Plus, Ambion) using a semidry electroblotter (Fisher, Itasca, IL). The membrane was subsequently UV cross-linked, 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.

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 <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 all quantitation 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 each of the bands on a given gel fell into the linear range of the instrument. In general, gels were arranged so that organs from randomly paired rats were analyzed together. In this way, we could appreciate the quantitative differences in the amount of each subunit in each of the tissues as well as between treatment groups. An additional set of three pairs of inner medullae from rats fed high- and low-NaCl diets were analyzed without other tissues.

Statistical analysis was conducted by use of the Student's paired t-test comparing treatment groups analyzed in the same assay. All values were normalized to the GAPDH band (exposed for a shorter period to account for considerably larger amount of mRNA). In general, the GAPDH bands were exposed for 1.0 min and all other bands for 15 min. The experimental conditions were designed to test for increases in mRNA produced by a low-NaCl diet or steroids; therefore a one-tailed analysis was used. P < 0.05 was considered significant.

Materials. Unless otherwise specified, chemicals were purchased from Sigma (St. Louis, MO). Methoxyflurane was purchased from Mallinckrodt Veterinary (Mundelein, IL) and BSA from Boehringer-Mannheim (Indianapolis, IN). RU-28362 and RU-38486 were gifts from Roussel-UCLAF (Romainville, France), and the rat GAPDH construct was a gift from Dr. Christie Thomas (University of Iowa).

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effects of dietary NaCl on rENaC mRNA. We initially used Northern blot analysis to assess the effect of dietary NaCl on mRNA from lung, ascending and descending colon, kidney cortex, and outer and inner medullae. An example is shown in Fig. 1. We reached two preliminary conclusions. First, dietary NaCl intake had no detectable effect on the size of alpha -, beta -, or gamma -rENaC mRNA for any of the organs. Second, there appeared to be a greater abundance of rENaC mRNA in the descending colon and inner medulla of rats fed a low-NaCl diet. We therefore proceeded to quantitate the abundance of the alpha -, beta -, and gamma -rENaC subunits by RPA.

Figure 2 shows a representative RPA of alpha -, beta -, and gamma -rENaC mRNA from lung, descending colon, and three regions of the kidney taken from rats fed either a low- or high-NaCl diet. We did not perform extensive analysis on the ascending colon because the amount of each transcript seemed to be too small to quantitate reliably (and amount was variable). Table 1 displays the calculated ratios for all RPAs factored for the GAPDH signal. There was no effect of dietary NaCl on GAPDH mRNA (based on loading approximately same amount of RNA in each lane). There was no effect of diet on any rENaC mRNA abundance in lung. In descending colon, beta - and gamma -rENaC were much greater in rats fed the low-NaCl diet, but alpha -rENaC mRNA abundance was not different. The dietary NaCl effects on the kidney depended on region. There was no effect on any subunit in renal cortex or outer medulla. There was also no effect of diet on beta - or gamma -rENaC abundance in inner medulla. However, rats fed the low-NaCl diet had greater alpha -rENaC mRNA abundance in the inner medulla.


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Fig. 2.   Example of an RNase protection assay (RPA) for alpha -, beta -, and gamma -rENaC and GAPDH. Rats were fed either H- or L-NaCl diet for 2 wk. Labels on left show position of protected fragments. Labels on right show position and length of unprotected fragments. All such autoradiograms were quantitated by scanning densitometry. Intensity of GAPDH probe was measured from shorter exposures. DC, descending colon; other abbreviations as in Fig. 1.

                              
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Table 1.   Relative abundance of alpha -, beta -, and gamma -rENaC mRNA in colon, lung, and kidney region in rats fed either a low- or high-NaCl diet

Table 1 also shows the relative tissue abundance of alpha -, beta -, and gamma -rENaC mRNA factored for GAPDH expression. For rats fed a high-NaCl diet, the abundance of alpha -, beta -, and gamma -rENaC followed the general rank order lung > descending colon > cortex > outer medulla > inner medulla. For rats fed a low-NaCl diet, there was the same rank order for alpha -rENaC. However, in these rats, the descending colon had the lowest relative amount of beta - and gamma -rENaC mRNA.

The relative amounts of alpha -, beta -, and gamma -rENaC mRNA are graphically shown in Fig. 3 for the two tissues in which we detected an effect. The difference was most dramatic in beta - and gamma -rENaC mRNA from descending colon, where the low- to high-NaCl ratio was 6.1 ± 2.5 for beta -rENaC and 28 ± 12 for gamma -rENaC. Interestingly, the pattern for the inner medulla was quite different. Although there was no significant effect on beta - or gamma -rENaC, alpha -rENaC mRNA abundance was 4.3 ± 1.1-fold higher in the low-NaCl group.


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Fig. 3.   Mean ratios of alpha -, beta -, or gamma -rENaC mRNA from descending colon (A) and inner medulla (B) taken from rats fed either L- or H-NaCl diet. Values are from RPA. All ratios factored for GAPDH expression. Primary data in Table 1. In this analysis, L and H results were analyzed in pairs according to each assay. * P < 0.01 by paired analysis.

Acute effects of steroid hormones on rENaC mRNA. We performed at least one Northern blot analysis on each rENaC subunit for each tissue from untreated ADX rats and from ADX rats treated with GC or MC. A sample of the results is shown in Fig. 4; treatments did not alter the apparent length of the mRNA for any of the transcripts in any tissue. We therefore proceeded with the quantitative RPA.


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Fig. 4.   Examples of Northern blots of RNA from regions of kidney, colon, and lung from adrenalectomized (ADX) rats treated for 6 h with vehicle or glucocorticoid (GC) or mineralocorticoid hormone (MC) as described in METHODS.

Table 2 shows the ratio of the mRNA abundance for each of the subunits factored for GAPDH expression. In contrast to the effects of low-NaCl diet, every tissue responded to at least one steroid hormone. The mean ratios of GC to ADX and MC to ADX for all tissues and subunits are shown in Fig. 5. It is clear that the most dramatic effect is seen in the descending colon. GC and MC increased beta -rENaC mRNA 20- and 13-fold, respectively, and increased gamma -rENaC mRNA 12- and 5-fold, respectively. Neither GC nor MC had an effect on alpha -rENaC mRNA.

                              
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Table 2.   Relative abundance of alpha -, beta -, and gamma -rENaC mRNA in colon, lung, and kidney region in response to steroids


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Fig. 5.   Acute response to GC or MC exposure in ADX rats as assayed by RPA. Rats were injected with appropriate steroid, and organs were harvested 6 h later. A: descending colon; B: lung; C: kidney cortex; D: outer medulla; E: inner medulla. Values are mean ratios of GC or MC to ADX assayed in same group. Primary data in Table 2. * P < 0.02 by paired analysis.

In the lung, GC treatment but not MC treatment increased alpha -rENaC mRNA (by 2.7-fold); there was no effect of either GC or MC on beta - or gamma -rENaC mRNA. Each region of the kidney responded to GC and to MC, but only alpha -rENaC mRNA, and not beta - or gamma -rENaC, was increased. The magnitude of the increase in the three regions of the kidney was similar to the increase in the lung and smaller than the increase in beta - or gamma -rENaC mRNA in descending colon. In contrast to the lung, alpha -rENaC mRNA in kidney was increased by both GC and MC, although the magnitude of the stimulation by MC was consistently smaller than the stimulation by GC.

    DISCUSSION
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Abstract
Introduction
Methods
Results
Discussion
References

These results demonstrate that dietary NaCl and acute steroids produce a striking heterogeneous effect in the rENaC mRNA levels in lung, colon, and kidney. The distal colon responds to a low-NaCl diet and acute GC or MC exposure by greatly increasing the abundance of beta - and gamma -rENaC but not alpha -rENaC mRNA. The inner medulla responds in a completely different fashion. Dietary NaCl restriction or acute GC or MC increases alpha -rENaC but not beta - or gamma -rENaC mRNA. The responses in the lung, kidney cortex, and outer medulla show variations on the pattern of the inner medulla.

Colon. There is now excellent agreement that dietary NaCl restriction or MC agonists increase the abundance of beta - and gamma -rENaC mRNA in colon, with little or no effect on alpha -rENaC mRNA (3, 26, 34). Other investigators have demonstrated a good temporal correlation between the increase in Na+ current and the magnitude of gamma -rENaC mRNA levels (26). Taken together, these results lend strong support to the idea that a major mechanism of aldosterone's action to increase electrogenic Na+ transport is mediated through this increase in mRNA abundance.

The fact that GC administration had an effect on rENaC (in colon) similar to MC is somewhat surprising. It is well known that large doses of dexamethasone can increase electrogenic Na+ transport (32) and beta - and gamma -rENaC mRNA levels (34). However, this action of dexamethasone on Na+ transport is mediated via the MC receptor and thus is not strictly a GC effect (4). GC hormones, administered to ADX rats in doses that allow binding exclusively to GC (type II) receptors, stimulate electroneutral not electrogenic Na+ transport (5, 7). In this setting, pure GCs actually inhibit electrogenic Na+ transport (8). Taken together, these results suggest that an increase in beta - and gamma -rENaC mRNA in colonic epithelia will not inevitably cause an increase in electrogenic Na+ transport. Clearly, posttranscriptional events must play an important role in modulating the steroid effects leading to altered Na+ transport.

Lung. The importance of the rENaC complex, and specifically the alpha -rENaC subunit, to lung function is dramatically illustrated in the mouse with a homozygous deletion of the alpha -ENaC gene. These mice are born normally but die within 48 h because they fail to absorb fluid from their lungs (22). The magnitude of alpha -rENaC mRNA detected by our RPA is consistent with the amount detected by in situ hybridization in airway epithelial cells (19, 29). The lack of change in rENaC abundance by dietary NaCl restriction (Table 1) is consistent with that reported by others (34).

Why are the rENaC mRNA subunits not regulated by dietary NaCl? A teleological answer to this question would be that conservation of Na+ and fluid is not a major responsibility of the lung but, rather, of the kidney and colon. The molecular explanation must incorporate our observations that neither chronically elevated levels of aldosterone (from low-NaCl diet) nor acute MC administration increases lung rENaC mRNA but that acute GC administration does (Tables 1 and 2). The simplest explanation for the lack of response of rENaC mRNA to aldosterone is that lungs do not have MC receptors. However, MC receptors have been demonstrated in rat lung tissue (1, 24), and aldosterone can hyperpolarize the membrane voltage in cultured type II alveolar cells (12).

How can we reconcile the presence of MC receptors in the lung with the failure of aldosterone to produce an effect on rENaC mRNA abundance? There are three possible explanations. First, the MC receptors might be confined to cells that make little or no rENaC mRNA. In this regard, we note that alpha -rENaC mRNA, the lung subunit responsive to GC (Table 2), is present in nearly every epithelial cell (19, 29). However, there is no information to date on the localization of MC receptors in lung tissue. A second possibility is that the responses to MC receptor occupancy in lung cells do not include activation of the system responsible for increasing alpha -rENaC mRNA levels. This explanation would invoke an unprecedented degree of diversity between the actions of GC and MC receptors in the same cell. Finally, the amount of MC receptor protein in lung tissue is small (24). Thus there may be too few receptors in airway epithelial cells to mount a significant physiological response to NaCl restriction.

The observation that lung ENaC mRNA does not respond to the MC actions of aldosterone is not discrepant with the previous report of aldosterone's action in cultured type II alveolar cells. Champigny et al. (12) found that high concentrations of aldosterone increased alpha -rENaC mRNA and hyperpolarized membrane voltage. The effect of aldosterone, at least on the mRNA levels, was inhibited by RU-38486, an antagonist of the GC receptor (12). Thus, in these lung cells as in others (36), aldosterone can activate GC receptors when used in high enough concentrations. The failure of aldosterone to produce an effect in the presence of a GC antagonist is consistent with the interpretation that there are few MC receptors expressed in lung.

The acute response to GC infusion is consistent with an important role for GCs in regulating alpha -rENaC mRNA. Other investigators (12, 31, 38) have documented an upregulation of lung alpha -rENaC mRNA in response to GCs. Longer exposure (2 days) to GCs in ADX rats appears to increase levels of beta - and gamma -rENaC mRNA as well (34). The most important physiological role for these steroid effects is probably the induction of Na+ channels at the time of birth (31, 38). The correlation between the increase in production of fetal steroids (28) and the increase in alpha -rENaC mRNA and surfactant proteins (41) makes a compelling argument for a concerted effect of GCs on lung maturation.

Kidney. There is disagreement about the effect of aldosterone on rENaC mRNA subunits in kidney cortex. Asher et al. (3) found that aldosterone or dexamethasone infused into normal rats for 3 days increased alpha -rENaC but not beta - or gamma -rENaC mRNA. On the other hand, Renard et al. (34) found that neither a low-NaCl diet nor 2 days of dexamethasone infusion into normal rats produced a change in any rENaC mRNA abundance. The failure to detect an effect of dietary NaCl or aldosterone on rENaC mRNA in rat kidney cortex is surprising in view of the well-studied effect of these maneuvers on electrogenic Na+ transport by rat cortical collecting duct (CCD), one of the major targets for aldosterone action in the kidney (33, 35).

Our results indicate that there is no effect of chronic dietary NaCl restriction on alpha -, beta -, or gamma -rENaC mRNA abundance in renal cortex or outer medulla. In contrast, there is an increase in alpha -rENaC, but not beta - or gamma -rENaC mRNA, from inner medulla (Table 1, Fig. 2). These results uncover unexpected intrarenal heterogeneity in rENaC mRNA regulation.

The observation that dietary NaCl restriction increases alpha -rENaC and not beta - or gamma -rENaC mRNA in inner medulla is consistent with our previous reports demonstrating that aldosterone increases electrogenic Na+ transport and alpha -rENaC mRNA (but not beta - or gamma -rENaC mRNA) of inner medullary collecting duct (IMCD) cells in primary culture (23, 40). Thus the responses of the intact inner medulla and the target cells for aldosterone's action in this region of the kidney are similar.

Why is the response to dietary NaCl restriction different in the regions of the kidney? In the cortex, rENaC mRNA is localized to both the distal convoluted tubule (DCT) and the CCD (15, 18). It seems possible that a large constitutive expression of rENaC mRNA in the DCT could render a small but important effect of aldosterone in the CCD undetectable in the entire tissue. Given the magnitude of the increase in Na+ transport by the CCD in response to NaCl restriction or to aldosterone (33, 35), such a possibility seems unlikely. Furthermore, this heterogeneity does not apply to the outer medulla, where NaCl restriction also had no effect. It seems more likely that the differences in response to NaCl restriction relate to the underlying function of ENaC in the cortex and inner medulla.

In our view, a more plausible explanation for the different responses in the cortex and inner medulla relates to the regional differences in the role of rENaC in Na+ and K+ balance. The CCD and DCT are the major sites from which K+ is secreted. K+ secretion requires Na+ absorption through ENaC (37). The IMCD does not share with the CCD the capacity for K+ secretion (17, 23). Because dietary NaCl restriction does not alter the requirements for K+ secretion, it seems possible that rENaC mRNA subunits in the DCT and CCD might be more directly influenced by factors related to regulation of K+ balance rather than Na+ balance.

In contrast to the CCD, Na+ transport by the IMCD is related more to Na(Cl) balance than K+ balance. From this teleological perspective, one might suppose that dietary NaCl could have more of an effect on rENaC mRNA in IMCD than in CCD. However, this functional difference does not explain why CCD segments taken from animals with high circulating MC levels absorb more Na+ in vitro than do segments from normal rats. The conclusion we draw from the aggregate of these results is that an increase in rENaC mRNA abundance is not necessary for aldosterone to increase Na+ transport by CCD. Therefore aldosterone must be producing other effects to enhance Na+ transport by CCD.

Despite the difference in the regional response to chronic changes in circulating aldosterone levels, all regions of the kidney are able to respond to steroid hormones acutely, at least in ADX rats, in which the basal level of circulating steroids is low (Table 2). These results, taken together, suggest that basal levels of aldosterone and/or corticosterone are necessary to produce sufficient rENaC mRNA to respond to needs relating to K+ balance (in DCT and CCD) and that chronically elevated circulating levels of aldosterone produce different effects dependent on the specific target tissue.

General deductions. These results reveal several levels of heterogeneity in the tissue response to NaCl restriction and steroid hormones. The most striking heterogeneity is the opposite responses of the descending colon and inner medulla to NaCl restriction (Fig. 2). The intrarenal heterogeneity in the response to NaCl restriction but not to acute steroid administration provides evidence for another layer of complexity. The dissociation between rENaC mRNA levels and magnitude of electrogenic Na+ transport in response to steroids (in colon and kidney cortex) suggests that posttranscription events are important in modulating the steroid effects on Na+ transport.

The levels of heterogeneity might not be limited to tissues of the rat. There is evidence that responses might be species dependent. For example, cultured rabbit CCD cells respond to aldosterone by increasing gamma -rENaC mRNA (16), a response not detected in rat cortex (Fig. 3). In the chicken, the intestinal response to dietary NaCl restriction includes an increase in alpha -rENaC mRNA (21), a response quite different from that of the rat colon (Fig. 2).

Despite the complexities of steroid hormone action on ENaC mRNA abundance, the demonstration that a critically important mRNA can be regulated by physiological maneuvers provides a valuable opportunity to further explore the cellular mechanisms responsible for regulating electrogenic Na+ transport.

    ACKNOWLEDGEMENTS

We appreciate the assistance of Russ Husted, Chong Zhang, and Ken Volk in preparing the tissue samples.

    FOOTNOTES

This work was supported in part by National Institutes of Health Grants HL-55006 and DK-52617 and a Merit Award from the Department of Veterans Affairs.

Address for reprint requests: J. B. Stokes, Dept. of Internal Medicine, University of Iowa College of Medicine, Iowa City, IA 52242.

Received 28 February 1997; accepted in final form 23 February 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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