Time course of renal Na-K-ATPase, NHE3, NKCC2, NCC, and ENaC abundance changes with dietary NaCl restriction

Shyama Masilamani1, Xiaoyan Wang1, Gheun-Ho Kim1, Heddwen Brooks1, Jakob Nielsen1,2, Soren Nielsen2, Kenzo Nakamura3, John B. Stokes3, and Mark A. Knepper1

1 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1603; 2 The Water and Salt Institute, University of Aarhus, DK-8000 Aarhus C, Denmark; and 3 Department of Internal Medicine, University of Iowa, and Veterans Affairs Medical Center, Iowa City, Iowa 52242


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

We have used peptide-directed antibodies to each major renal Na transporter and channel proteins to screen renal homogenates for changes in Na transporter protein expression after initiation of dietary NaCl restriction. After equilibration on a NaCl-replete diet (2.0 meq · 200 g body wt-1 · day-1), rats were switched to a NaCl-deficient diet (0.02 meq · 200 g body wt-1 · day-1). Na excretion fell to 25% of baseline levels on day 1, followed by a further decrease <4% of baseline levels on day 3, of NaCl restriction. The decreased Na excretion at day 1 occurred despite the absence of a significant increase in plasma aldosterone level or in the abundance of any of the major renal Na transporters. However, after a 1-day lag, plasma aldosterone levels increased in association with increases in abundances of three aldosterone-regulated Na transporter proteins: the thiazide-sensitive Na-Cl cotransporter (NCC), the alpha -subunit of the amiloride-sensitive epithelial Na channel (alpha -ENaC), and the 70-kDa form of gamma -ENaC. RNase protection assays of transporter mRNA levels revealed an increase in renal alpha -ENaC mRNA coincident with the increase in alpha -ENaC protein abundance. However, there was no change in NCC mRNA abundance, suggesting that the increase in NCC protein in response to dietary NaCl restriction was not a result of altered gene transcription. These results point to early regulatory processes that decrease renal Na excretion without an increase in the abundance of any Na transporter, followed by a late aldosterone-dependent response associated with upregulation of NCC and ENaC.

aldosterone; distal convoluted tubule; collecting duct; type 3 sodium/hydrogen exchanger; bumetanide-sensitive type 2 sodium-potassium-2 chloride cotransporter; thiazide-sensitive sodium-chloride cotransporter; epithelial sodium channel


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

DAY-TO-DAY REGULATION OF SYSTEMIC Na balance occurs predominantly through the control of renal tubule Na reabsorption. Adjustments in Na excretion occur in part through adaptive changes in the abundances of apical Na transporter proteins expressed in the various renal tubule segments (3, 7, 17-20, 23, 39). The major apical Na transporters that contribute signficantly to this regulation are the type 3 Na/H exchanger in the proximal tubule (NHE3), the bumetanide-sensitive type 2 Na-K-2Cl cotransporter in the thick ascending limb of Henle (NKCC2), the thiazide-sensitive Na-Cl cotransporter in the distal convoluted tubule (NCC), and the amiloride-sensitive epithelial Na channel in the connecting tubules and collecting ducts (ENaC) (30). ENaC consists of a complex of three different subunits (alpha -, beta -, and gamma -ENaC), each coded by a different gene (14). Although the adrenal corticosteroid aldosterone has an essential role in the regulation of renal NaCl excretion, other mediators, such as angiotensin II, vasopressin, nitric oxide, and catecholamines, also have important regulatory effects (6). The interplay between these mediators is complex and has not yet been investigated at the level of the individual Na transporters expressed along the nephron.

A prototypical regulatory event is the renal response to dietary NaCl restriction, a response that involves changes in the abundances of several renal tubule Na transporter proteins (23). Although the changes in renal Na transporter abundances have been determined for the steady state, the dynamic response to dietary NaCl restriction has not been ascertained. Here, we employ semiquantitative immunoblotting using rabbit polyclonal antibodies to each of the major renal Na transporters (4, 19, 23) to investigate changes in the protein abundance of each Na transporter as a function of time after the switch from a NaCl-replete to a NaCl-deficient diet. We also employ RNase protection assays to investigate the corresponding changes in NCC and ENaC subunit mRNA levels.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
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Animals and Experimental Protocols

Experiments were conducted in male Sprague-Dawley rats (initial weight 216-276 g, Taconic Farms, Germantown, NY) kept in metabolism cages (approved animal protocol 8-KE-2). To investigate the time course of changes after initiation of dietary NaCl restriction, five different experiments were carried out in which the period of NaCl restriction was 1 day (2 experiments), 3 days (2 experiments), or 10 days (1 experiment). Separate control rats were studied in each experiment. The control rats were maintained on a NaCl-replete diet for the same period of NaCl restriction as for experimental rats.

All rats were ration-fed using a gelled diet (20), which permitted uniform intake of nutrients and water (except for NaCl) in experimental and control rats. The base gelled diet was prepared by combining commercially available synthetic rat chow containing no added NaCl (formula code 53140000; Zeigler Bros., Gardners, PA) with deionized water (25 ml/15 g rat chow) and agar (0.5%) for gelation. For control rats, NaCl was added to the base diet, giving them a daily Na intake of 2.0 meq/200 g body wt (NaCl-replete diet). For NaCl-restricted rats, no NaCl was added to the base diet, giving them 0.02 meq · 200 g body wt-1 · day-1 of Na (NaCl-deficient diet). All rats were fed the quantity of the gelled diet to give them 15 g · 200 g body wt-1 · day-1 of the synthetic chow and 25 ml · 200 g body wt-1 · day-1 of water. Thus water intake and caloric intake were equally maintained in control vs. low-NaCl rats. The amount of the food/water ration given in this protocol is marginally lower than what the rats would eat spontaneously on an ad libitum protocol, ensuring that virtually all of the ration is eaten each day as confirmed by direct observation.

To observe the time course of changes after initiation of NaCl restriction, all rats were initially placed in the control Na diet over a 3-day equilibration period, and on day 0 the experimental rats were switched to the low-NaCl diet. Control rats followed the same time course but were not switched to the low-NaCl diet. On the final day of each experiment, a 24-h urine sample was collected for analysis (see Serum and Urine Analysis). Control and experimental rats were euthanized at the same time by decapitation. Serum was collected from the neck for analysis, and the left kidneys were harvested for semiquantitative immunoblotting as described in the subsection below. In some experiments, the left kidney was used for mRNA extraction (see RNA preparation), whereas the right kidney was processed for immunoblotting. In one other experiment (10-day NaCl restriction vs. control), distal colon and lung were harvested from rats and processed for semiquantitative immunoblotting (see the subsection below) to compare the pattern of ENaC subunit response to that seen in kidney.

Antibodies

The present study used rabbit polyclonal antibodies, characterized in previous papers from this laboratory, directed to the major apical Na transporters expressed along the renal tubule, namely, NHE3 (10), NKCC2 (also known as BSC1) (18), NCC (also termed TSC) (20), and the alpha -, beta -, and gamma -subunits of ENaC (23). The antisera were affinity purified against the immunizing peptides as previously described (18, 20). The specificity of the antibodies has been demonstrated by showing unique peptide-ablatable bands on immunoblots and a unique distribution of labeling by immunocytochemistry.

We also used a commercially available mouse monoclonal antibody against the alpha 1-subunit of the Na-K-ATPase (Upstate Biotechnology, Lake Placid, NY).

Semiquantitative Immunoblotting

Tissue (renal cortex, lung, colon) was homogenized intact and prepared for immunoblotting as described in detail previously (20, 35). Equal loading was confirmed by staining identically loaded gels with Coomassie blue as described previously (20). Incubation with peroxidase-conjugated secondary antibodies (Pierce no. 31458 or Pierce no. 31434) was followed by band visualization using enhanced chemiluminescence substrate (LumiGLO for Western blotting, Kirkegaard and Perry no. VC110) before exposure to X-ray film (Kodak 165-1579). The band densities were quantitated by laser densitometry (model PDS1-P90, Molecular Dynamics). To facilitate comparisons, the densitometry values were normalized to control, defining the mean for the control group as 100%.

mRNA Measurements by RNase Protection Assay

RNA preparation. Total RNA was extracted from whole cortices dissected from the left kidneys of control and NaCl-restricted rats as described (5) using a RNeasy Midi Kit (Qiagen, Valencia, CA). RNA purity and concentration were assessed spectrophotometrically. RNA integrity was confirmed by inspection of ribosomal RNA bands on ethidium bromide-stained agarose gels.

RNase protection assay. The RNase protection assay (RPA) was conducted as previously described for ENaC subunits (16, 34) or NCC (39). Biotin-labeled probes for rat alpha -, beta -, and gamma -ENaC and for GAPDH were constructed from plasmids to yield protected fragments of 420, 249, 190, and 140 nucleotides, respectively. Biotin-labeled probes for NCC and beta -actin were 389 and 127 nucleotides in length, respectively (39). The RPA was carried out using an RPA assay kit (Ambion, RPA III, no. 1414) according to the protocol recommended by the manufacturer. Approximately 25 µg of total RNA were hybridized to 1 ng of labeled probe for the ENaC assay, whereas 10 µg total RNA were used with 1 ng labeled probe for the NCC assay. After hybridization and digestion with RNase A and T1, the products were subjected to electrophoresis through a 5% denaturing polyacrylamide-8 M urea gel buffered with Tris borate for 2.5 h and transferred to a nylon membrane (BrightStar Plus, Ambion). Chemiluminescence intensity was quantitated on a densitometer using Kodak software. ENaC mRNA subunit abundance was normalized to GAPDH abundance. NCC mRNA subunit abundance was normalized to beta -actin.

Serum and Urine Analysis

The final serum sample was collected and analyzed for serum aldosterone concentration by radioimmunoassay (Coat-A-Count; Diagnostic Products, Los Angeles, CA) and creatinine. The final urine sample was collected and analyzed for excretion of Na and creatinine (Monarch 2000 autoanalyzer, Instrumentation Laboratories, Lexington, MA).

Presentation of Data and Statistical Analyses

Quantitative data are presented as means ± SE. Statistical comparisons were accomplished by unpaired t-test (when variances were the same) or by the Mann-Whitney rank-sum test (when variances were significantly different between groups). P values <0.05 were considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

After equilibration on the NaCl-replete control diet, separate groups of rats were switched to the NaCl-deficient diet for 1, 3, or 10 days or maintained on the control diet for equal periods of time. Figure 1 summarizes the time course of changes in Na excretion and plasma aldosterone concentration for these experiments. Na excretion fell to 25% of control levels on the first day (Fig. 1A), whereas aldosterone concentration in plasma did not change significantly on the first day (Fig. 1B). There was a further decrease in Na excretion to very low levels by day 3 (0.06 meq Na/day) and day 10 (0.04 meq Na/day), in association with significant increases in plasma aldosterone concentration after 3 days of dietary NaCl restriction (to 2.58 ± 0.43 nM) and 10 days of dietary NaCl restriction (to 3.53 ± 1.26 nM). In these studies, ration feeding (see METHODS) was used to ensure that rats in both groups received identical water and caloric intakes.


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Fig. 1.   Na excretion and plasma aldosterone concentration. Time course of urinary sodium excretion (A) and plasma aldosterone concentration (B) in NaCl-replete control rats (dashed lines) and NaCl-restricted rats (solid lines). NaCl-restricted rats were switched from 2.0 to 0.02 mmol of NaCl in food/day on day 0. *Significantly different from control, P < 0.05.

Figure 2 shows examples of immunoblots for each of the three major renal Na transporters in these experiments (10-day time point). The abundances of Na transporters expressed in pre-macula densa segments, NHE3 and NKCC2, were unchanged. Three Na transporter proteins were significantly increased in abundance, namely, NCC (normalized band densities: control, 100 ± 13; Na-restricted, 358 ± 31; P < 0.05); alpha -ENaC (control, 100 ± 4; Na-restricted, 184 ± 7; P < 0.05); and the 70-kDa form of gamma -ENaC (control, 100 ± 7; Na-restricted, 148 ± 8; P < 0.05). [The 70-kDa form of gamma -ENaC has been proposed to be produced from the 85-kDa form by a physiological proteolytic cleavage of the extracellular loop (23).] In contrast, there were significant decreases in the abundances of both beta -ENaC (normalized band densities: control, 100 ± 12; Na-restricted, 52 ± 6; P < 0.05) and gamma -ENaC (85-kDa plus 70-kDa bands: control, 100 ± 8; Na-restricted, 57 ± 4; P < 0.05), findings that have not been previously reported.


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Fig. 2.   Profile of Na transporter and channel abundance changes in renal cortex in response to dietary NaCl restriction for 10 days. Each panel is an immunoblot loaded with samples from 6 control rats on a NaCl-replete diet (Na intake, 2 meq · 200 g body wt-1 · day-1) and 6 rats on a NaCl-restricted diet (Na intake, 0.02 meq · 200 g body wt-1 · day-1). Blots were probed with antibodies to the type 3 Na-H exchanger (NHE3) of the proximal tubule, the bumetanide-sensitive type 2 Na-K-2Cl cotransporter (NKCC2) of the thick ascending limb of Henle's loop, the thiazide-sensitive Na-Cl cotransporter (NCC) of the distal convoluted tubule, each of the 3 subunits of the amiloride-sensitive epithelial sodium channel (ENaC), and the Na-K-ATPase alpha 1-subunit. Equality of loading was confirmed by densitometry of parallel Coomassie-stained gels. Densitometric and statistical analysis is reported in Table 1 in the APPENDIX. *Significantly different from control, P < 0.05.

Figure 3 shows the time courses of changes in renal cortical abundance of each of the major apical Na transporters, listed in order of their expression along the renal tubule. NHE3 abundance was not significantly changed throughout the time course. NKCC2, although not changed in the steady state (day 10), underwent a significant transient decrease on day 1 after the shift to the low-NaCl diet. NCC was unchanged on day 1 but underwent a marked increase in abundance at subsequent time points in parallel with measured increases in plasma aldosterone concentration (cf. Fig. 1) in accordance with the known role of aldosterone to regulate its abundance (20). The other transporter protein that is known to be regulated by aldosterone (23), alpha -ENaC, also was unchanged on the first day but increased subsequently in parallel with changes in plasma aldosterone concentration. Interestingly, the abundance of beta -ENaC fell on day 1 and remained decreased throughout the time course. Similarly, total gamma -ENaC abundance was decreased, but the decrease did not reach significance until the day 10 time point (Fig. 3, bottom, black line). In contrast, the abundance of the 70-kDa form of gamma -ENaC increased in parallel with observed changes in plasma aldosterone concentration (Fig. 3, bottom, gray line), in accordance with the proposed role for aldosterone in triggering this change (23). (A full listing of densitometric analysis of the time course data is shown in Table 1 in the APPENDIX.)


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Fig. 3.   Time course of changes in apical Na transporter protein abundances after the switch from a NaCl-replete control diet to a NaCl-restricted diet. Values are normalized band densities from semiquantitative immunoblots (see table in the APPENDIX). Each time point had a separate set of controls allowing statistical analysis by t-test. Bottom: total gamma -ENaC (black line) and the 70-kDa form of gamma -ENaC (gray line). *Significantly different from control value, P < 0.05.

While the apical Na transporters vary among the tubule segments, the basolateral Na transporter, the Na pump (Na-K-ATPase), is common to each segment (37). Figure 4 reports changes in the abundance of the alpha 1-subunit in response to dietary NaCl restriction. On days 3 and 10, there was no change in Na-K-ATPase alpha 1-subunit relative to simultaneous control rats on the NaCl-replete diet. However, a transient decrease in Na-K-ATPase abundance was seen on day 1.


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Fig. 4.   Time course of changes in protein abundance Na-pump (alpha 1-subunit of Na-K-ATPase) after switch from NaCl-replete control diet to NaCl-restricted diet. Values are normalized band densities from semiquantitative immunoblots (see Table 1 in the APPENDIX). Each time point had a separate set of controls allowing statistical analysis by t-test. *Significantly different from NaCl-replete control value, P < 0.05.

Thus although Na excretion decreased 75% on the first day after the switch to a low-NaCl diet, none of the renal tubule Na transporters exhibited an increase in abundance, which could account for the observed decrease in Na excretion. One possible explanation for the decrease in Na excretion would be a decrease in the glomerular filtration rate on day 1. However, creatinine clearance (an index of glomerular filtation rate) was unchanged on day 1 [NaCl-replete, 52.8 ± 4.6 ml/h; NaCl-restricted, 53.6 ± 6.0 ml/h; not significant (NS)].

To determine whether the increases in NCC and alpha -ENaC protein abundance are associated with increases in the abundances of the corresponding mRNAs, we ran RPAs in additional rats studied after 1 and 3 days of dietary NaCl restriction. Figure 5 shows the results of RPAs for NCC mRNA abundance. The normalized means of the NCC/beta -actin band density ratios were not different between NaCl-replete and NaCl-restricted rats at either day 1 (NaCl-restricted, 117 ± 13, vs. NaCl-replete, 100 ± 13; NS) and day 3 (NaCl-restricted 86 ± 6 vs. NaCl-replete 100 ± 29; NS). Immunoblotting for NCC was carried out in the opposite kidneys from the day 3 rats to confirm that dietary NaCl restriction upregulated NCC protein abundance in these rats. Densitometry of the immunoblot showed that NCC protein abundance was significantly increased in response to NaCl restriction (normalized band densities for NaCl-restricted, 205 ± 20, vs. NaCl-replete, 100 ± 19) (immunoblot not shown).


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Fig. 5.   Assessment of NCC mRNA abundance by RPA after 1 (A) and 3 days of NaCl restriction (B). The NCC probe is 389 bp, and the beta -actin probe is 127 bp. The mean band density ratio (NCC/beta -actin) showed no significant difference at either day 1 or day 3 (see text).

Data from RPAs for the three ENaC subunits are shown in Fig. 6. The abundance of the alpha -ENaC mRNA was increased after 3 days of dietary NaCl restriction but not after 1 day, in parallel with demonstrated changes in alpha -ENaC protein. There were no increases in the abundances of beta -ENaC or gamma -ENaC mRNA at either time point, although the abundance of beta -ENaC mRNA was significantly decreased on day 1. An immunoblot done with homogenates from the opposite kidneys of the day 3 rats confirmed the changes in alpha -, beta -, and gamma -ENaC protein previously documented in Fig. 1 (immunoblots not shown). Thus the results are compatible with the view that increases in alpha -ENaC protein abundance after 3 days of dietary NaCl restriction are due at least in part to increases in alpha -ENaC mRNA. Furthermore, they suggest that the decrease in beta -ENaC protein abundance may be due in part to a decrease in beta -ENaC mRNA levels.


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Fig. 6.   Assessment of mRNA abundance for each of the 3 ENaC subunits [alpha (A), beta  (B), and gamma  (C)] by RPA after 1 and 3 days of NaCl restriction. Values are normalized band densities (subunit/GAPDH) from RNase protection assays. *Significantly different between NaCl-replete and NaCl-restricted values, P < 0.05.

To test whether changes in ENaC subunit protein abundances occur in other aldosterone-responsive epithelia, we carried out immunoblotting of homogenates from rat lung (Fig. 7) and distal colon (Fig. 8). In the lung, there were no significant changes in the protein abundances of any of the subunits of ENaC in response to dietary NaCl restriction. In the distal colon, 10 days of low-NaCl intake produced significant increases in the band densities of beta -ENaC (NaCl-replete control, 100 ± 23%; NaCl-restricted, 312 ± 31%; P < 0.05) and both the 85-kDa (NaCl-replete control, 100 ± 17%; NaCl-restricted, 2,355 ± 421%; P < 0.005) and the 70-kDa (NaCl-replete control, 100 ± 5%; NaCl-restricted, 843 ± 288%; P < 0.05) forms of gamma -ENaC. (Note that although no bands are visible in the control lanes for beta - or gamma -ENaC in Fig. 8, faint bands become visible with very long exposures of the film, permitting a densitometric comparison.) In contrast to the kidney, dietary NaCl restriction resulted in no change in the band density of alpha -ENaC in the distal colon. Thus each tissue appears to have a unique pattern of responses at a protein level, as previously seen at an mRNA level (34).


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Fig. 7.   Immunoblots showing the effect of dietary NaCl restriction for 10 days on abundance of each of the 3 ENaC subunits [alpha (A), beta  (B), and gamma  (C)] in the lung.



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Fig. 8.   Immunoblots showing the effect of dietary NaCl restriction for 10 days on abundances of each of the 3 ENaC subunits [alpha (A), beta  (B), and gamma  (C)] in the distal colon.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

This study extends our present series of renal tubule profiling studies investigating factors that result in adaptive regulation of Na transporter and channel protein abundance in rodents (3, 7, 8, 18, 20, 22, 23, 38, 39). The major objective here is to describe the patterns of adaptive response to known regulators of renal Na excretion and to look for these patterns in animal models of dysregulation of extracellular fluid volume or blood pressure regulation. The observations in this paper are consistent with an important role for aldosterone-mediated adaptation in the long-term response to dietary NaCl restriction but suggest a lesser role for aldosterone in the short-term response. Specifically, after 3 days, but not 1 day, of NaCl restriction, there were marked increases in the abundances of NCC, the alpha -subunit of ENaC, and the 70-kDa form of the gamma -subunit of ENaC. These transporter proteins have been identified in previous studies to be major targets of aldosterone-mediated regulation in the kidney (20, 23), and the time course of their increases parallels the observed increases in circulating aldosterone levels. These results are consistent with the conventional view that aldosterone plays a key role in the ability of the kidney to achieve maximal Na conservation. We emphasize that some effects of aldosterone on the collecting duct are not detectable as changes in ENaC subunit abundances but nevertheless contribute importantly to changes in Na transport in the collecting duct (14, 33). Such effects include trafficking and posttranslational modification of ENaC subunits.

On the first day after initiation of dietary NaCl restriction, Na excretion fell by 75% without a substantial change in circulating aldosterone level (Fig. 1). This result points to the possibility that a major portion of the initial decrease in Na excretion was due to factors other than aldosterone. It seems possible, however, that a very small increase in plasma aldosterone concentration could have occurred even on day 1 of this study without being statistically detectable (Fig. 1). In fact, in our previous study (12) we were able to detect significant increases in plasma aldosterone levels within 18 h of initiation of dietary NaCl restriction, albeit using a completely different feeding protocol. In the present study, the decrease in Na excretion on day 1 occurred in the absence of a measurable change in creatinine clearance, consistent with the view that the decrease was due to an increase in Na reabsorption. However, none of the major renal tubule transporters examined in this study manifested an increase in protein abundance on day 1. Instead, several transporters were decreased in abundance on the first day after initiation of dietary NaCl restriction (see below). These results imply that on the first day of dietary NaCl restriction, increased Na reabsorption in one or more tubule segments occurred by mechanisms not involving regulation of transporter abundance.

These observations lead us to emphasize two points discussed in the next two paragraphs. First, regulation of Na transport by the renal tubule occurs by multiple mechanisms, including processes that are independent of the action of aldosterone. On the first day after the switch to the NaCl-deficient diet, the rats excreted 0.4 meq/200 g body wt of Na, while taking in only 0.02 meq/200 g body wt of Na.1 Thus the rats were in negative NaCl balance during the first 24 h. Na losses would predict an extracellular fluid volume contraction of at least 2.7 ml/200 g body wt (0.38 meq/200 g body wt Na loss divided by the ECF Na concentration, 140 meq/l), or slightly more than 1% of body weight. This degree of ECF volume contraction could be expected to increase renin secretion, circulating and tissue angiotensin II levels, and sympathetic nerve traffic to the kidney, resulting in increased tissue norepinephrine levels (6). Clearly, these factors or other factors could be involved in the increased Na reabsorption occurring on the first day after the switch to an NaCl-deficient diet, i.e., before plasma aldosterone concentration was substantially increased.

Second, it is clear that the changes in renal tubule Na transport on the first day after initiation of dietary NaCl restriction do not depend on changes in the abundance of Na transporter and channel proteins or on changes in glomerular filtration rate. It is well established that Na transport activity can change by mechanisms that would not be manifested as changes in transporter abundance, such as trafficking of transporters to the plasma membrane from intracellular membrane-bound stores or posttranslational modification, e.g., phophorylation. An illustration of this point was seen in an earlier study in which we examined ENaC subunit abundances after only 18 h of dietary NaCl restriction (12). In that study, amiloride-sensitive whole cell currents were markedly increased despite the absence of an increase in the total abundances of the three ENaC subunits, correlating only with an increase in the abundance of the 70-kDa form of gamma -ENaC. Thus these findings emphasize the general observation that ENaC activity can be strongly upregulated without adaptive changes in subunit abundances. Furthermore, net NaCl transport can change without changes in Na transporter activity, for example, as a result of altered driving forces for passive NaCl backleak in the proximal tubule as a result of altered hemodynamics (21).

On the first day after initiation of NaCl restriction, several changes in Na transporter abundance occurred that were opposite of what would have been expected to achieve homeostasis. These changes included transient decreases in the abundance of NKCC2 (Fig. 3) and the alpha 1-subunit of the Na-K-ATPase (Fig. 4). In addition, there was a sustained fall in the total abundance of the beta - and gamma -subunits of ENaC (Fig. 3).

The decrease in total abundance of the beta -subunit of ENaC seen on day 1 was sustained throughout the time course of observation (Fig. 3). Total gamma -subunit abundance changes appear to parallel those of beta -ENaC, although the decrease was not statistically significant until the day 10 time point (Fig. 3). The mechanism for reduction in beta -ENaC abundance is not clear. This change is not likely caused by aldosterone, because the fall in beta -ENaC protein and mRNA occurred before aldosterone levels increased substantially (Fig. 1). Finally, in separate studies, spironolactone administration did not affect beta -ENaC protein levels (27). Three factors have thus far been identified that regulate beta -ENaC protein abundance independently of alpha -ENaC. First, increased circulating vasopressin levels are associated with selective upregulation of beta - and gamma -ENaC (7). Second, altered acid-base balance also selectively changes beta - and gamma -ENaC abundance, with alkali loading increasing, and acid loading decreasing, beta - and gamma -ENaC abundance (19). Finally, studies in AT1a receptor knockout mice support the conclusion that angiotensin II may directly decrease beta - and gamma -ENaC abundance (3). It is possible that one or more of these factors is involved in the changes produced by NaCl restriction.

Functional ENaC is a complex of alpha -, beta -, and gamma -subunits. The finding that the alpha -subunit is regulated independently of the beta - and gamma -subunits suggests that the stoichiometry of ENaC complexes within the cell is variable. This observation, together with the observation that under normal NaCl intake the intracellular distribution of alpha -ENaC is different from the distribution of beta - and gamma -ENaC (15), raises the question of how and where the ENaC complex is assembled. The work of May et al. (24) suggests that production of the alpha -subunit is rate limiting for the formation of mature, functional complexes at least under some conditions. However, the observation that beta - and gamma -ENaC can be regulated independently of alpha -ENaC in both kidney and colon suggests that the idea that the subunits are assembled in the endoplasmic reticulum with a single unique stoichiometry may be overly simplistic.

The abundance of NKCC2, expressed in thick ascending limb of Henle, was decreased on day 1 of dietary NaCl restriction but not beyond that point. From previous studies, we know that the abundance of NKCC2 in the thick ascending limb is upregulated by vasopressin (18) and by saline loading (9), which increases flow and Na delivery to the thick ascending limb. Conversely, its abundance is downregulated by PGE2 (11). In addition, the transient natriuresis seen during the vasopressin escape phenomenon, which is thought to be due to suppression of proximal tubule NaCl absorption, is also associated with a transient increase in renal NKCC2 abundance (9). From these considerations, it seems possible that decreased Na delivery from enhanced proximal tubular Na absorption and/or effects of enhanced PGE2 production might contribute to the reduction in NKCC2 abundance.

A transient fall in the renal expression of the alpha 1-subunit of Na-K-ATPase was also seen on the first day after initiation of NaCl restriction (Fig. 4). Na-K- ATPase provides the pathway for Na exit from the renal tubule epithelial cells in each segment. The transport function is mediated by the alpha -subunit, whereas the beta - and gamma -subunits provide regulatory and chaperone functions (25, 36). Synthesis of the alpha -subunit is believed to be rate limiting for assembly of the mature complex (25), and consequently our studies focus on this subunit rather than the beta - or gamma -subunits. While it is possible that the fall in Na-K-ATPase on day 1 was primarily localized to the thick ascending limb, the alpha 1-subunit of the Na-K-ATPase is expressed in all renal tubule segments (37), and thus it is not possible to deduce from these experiments which nephron site or sites are responsible for the observed fall. After 3 and 10 days of dietary NaCl restriction, there were no detectable changes in Na-K-ATPase alpha 1-subunit abundance. Of course, there is evidence that mineralocorticoids do mediate long-term regulation of Na-K-ATPase activity in collecting duct and connecting tubule (13, 26). However, possible changes in Na-K-ATPase alpha 1-subunit protein abundance in these segments may be obscured, because in the majority of the tubular mass (proximal tubules and thick ascending limbs), this subunit is probably not regulated.

Relationship Between mRNA and Protein Levels

NCC. In the present study, we confirmed the finding that dietary NaCl restriction increases the renal abundance of NCC protein (20) and demonstrated that this change correlates temporally with an increase in plasma aldosterone concentration. On the basis of the finding that infusion of aldosterone produced a similar increase in NCC abundance, we concluded that NCC protein abundance is regulated by aldosterone (20). Supporting this conclusion, several recent studies showed that the mineralocorticoid antagonist spironolactone decreases the abundance of the NCC protein in kidney (1, 2, 28). We anticipated that dietary NaCl restriction would also be associated with an increase in the abundance of NCC mRNA, a hypothesis that was ruled out (Fig. 5). The lack of an increase in NCC mRNA suggests that whatever role the mineralocorticoid receptor plays in mediating the response to dietary NaCl restriction, it is not likely that the response depends on direct effects on NCC gene transcription. Presumably, the role of the mineralocorticoid receptor is indirect and owes to activation of other genes that regulate NCC protein abundance by other mechanisms, e.g., through regulation of NCC mRNA translation or of NCC protein half-life.

alpha -ENaC. alpha -ENaC subunit mRNA levels in the kidney increased after 3 days, but not after 1 day, of NaCl restriction. Thus the onset of the increase in alpha -ENaC mRNA abundance coincided with the change in alpha -ENaC protein abundance, suggesting that alpha -ENaC protein abundance regulation is dependent, at least in part, on changes in alpha -ENaC mRNA. Although not specifically investigated in the present study, this effect presumably occurs via regulation of alpha -ENaC gene transcription (32). In this regard, it is well established that administration of aldosterone or synthetic glucocorticoid hormones can increase alpha -ENaC mRNA in the lung and kidney (34). However, the effects of dietary NaCl restriction are more complicated. An increase in alpha -ENaC mRNA abundance in the cortex and outer medulla in response to dietary NaCl restriction was seen in a previous study (40) but not in two other studies (31, 34), indicating that changes in alpha -ENaC mRNA abundance is not an obligatory concomitant of dietary NaCl restriction and may depend on ancillary factors that may have differed among the studies reported. Therefore, we speculate that the increase in alpha -ENaC protein abundance in the cortex was mediated, at least in part, by posttranscriptional mechanisms, e.g., regulation of alpha -ENaC mRNA translation (24). Different conclusions have been drawn for the renal medulla, in which dietary NaCl restriction has been found to consistently increase alpha -ENaC mRNA abundance (29, 34).

In contrast to alpha -ENaC mRNA, beta - and gamma -ENaC mRNA abundance did not increase after initiation of dietary NaCl restriction (Fig. 6). In fact, the abundance of beta -ENaC mRNA was significantly decreased on day 1 after initiation of dietary NaCl restriction, corresponding to the measured decrease in beta -ENaC protein abundance.

ENaC Subunits in the Colon and Lung

Both the distal colon and lung exhibit amiloride-sensitive Na transport and contain mRNA for each ENaC subunit. The pattern of response to NaCl restriction in both tissues was different from that in the kidney. In the distal colon, the abundance of beta - and gamma -ENaC were increased by NaCl restriction, whereas alpha -ENaC abundance was unaltered. In the lung, none of the three subunits exhibited changes in abundance in response to dietary NaCl restriction. Despite the differences in the pattern of ENaC protein expression in these three tissues, the changes in protein levels paralleled changes in mRNA levels (34), suggesting that the observed changes in protein abundances occur either through regulation of transcription of the subunit genes or possibly through regulation of mRNA stability. These results taken together imply that the regulation of ENaC subunit adundance by corticosteroids and NaCl intake depends on the cellular context.


    APPENDIX
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES


                              
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Table 1.   Densitometric analysis of immunoblots for major Na transporters and channels in the kidney after the shift from normal-NaCl to low-NaCl intake


    ACKNOWLEDGEMENTS

The authors thank David Caden, Clinical Chemistry Section, Laboratory of Animal Medicine and Surgery, National Heart, Lung, and Blood Institute (NHLBI), National Institutes of Health (NIH), for carrying out a careful analysis of the serum and urine samples. The authors gratefully acknowledge the contribution of Dr. Heddwen L. Brooks, who designed and produced the hybridization probe for measurement of NCC mRNA and carried out total RNA extractions.


    FOOTNOTES

This study was funded by the Intramural Budget of the NHLBI (Z01-HL-01282-KE to M.A. Knepper), by NIH Grant DK-52617 (to J. B. Stokes), and by a grant from the Department of Veterans Affairs (to J. B. Stokes). Studies at Aarhus University were supported by the Danish Medical Research Council, the Karen Elise Jensen Foundation, and the Commission of the European Union (EU-TMR Program and K.A. 3.1.2 Program). The Water and Salt Research Center, Aarhus University, is supported by the Danish National Research Foundation (Danmarks Grundforskningsfond). S. Masilamani was supported by NHLBI Career Transition Award K22-HL-66994. J. Nielsen was supported by the American Heart Association while at the NIH and the Human Frontier Science Program while at Aarhus University. K. Nakamura was supported in part by a postdoctoral fellowship grant from the National Kidney Foundation of Iowa.

Present address of K. Nakamura: Dept. of Urology, Yamanashi Medical School, Yamanashi 409-38, Japan.

1 While we measured Na intake and urinary output, we have neglected Na losses from other sources in this balance calculation. Consequently, it is possible that the net Na imbalance is somewhat greater than what is inferred here.

Address for reprint requests and other correspondence: M. A. Knepper, 10 Center Dr., MSC-1603, Bldg. 10, Rm. 6N260, NIH, Bethesda, MD 20892-1603 (E-mail: knep{at}helix.nih.gov).

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. Section 1734 solely to indicate this fact.

April 23, 2002;10.1152/ajprenal.00016.2002

Received 11 January 2002; accepted in final form 17 April 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
APPENDIX
REFERENCES

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Am J Physiol Renal Fluid Electrolyte Physiol 283(4):F648-F657