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
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ABSTRACT |
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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
wt1 · 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
-subunit of the amiloride-sensitive epithelial Na channel
(
-ENaC), and the 70-kDa form of
-ENaC. RNase protection assays of
transporter mRNA levels revealed an increase in renal
-ENaC mRNA
coincident with the increase in
-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
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INTRODUCTION |
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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 (-,
-, and
-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.
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METHODS |
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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
wt1 · 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 theWe also used a commercially available mouse monoclonal antibody against
the 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 -,
-, and
-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
-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
-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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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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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); -ENaC (control, 100 ± 4; Na-restricted, 184 ± 7; P < 0.05); and the
70-kDa form of
-ENaC (control, 100 ± 7; Na-restricted,
148 ± 8; P < 0.05). [The 70-kDa form of
-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
-ENaC (normalized band densities: control,
100 ± 12; Na-restricted, 52 ± 6; P < 0.05)
and
-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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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), -ENaC, also was unchanged on the first day but
increased subsequently in parallel with changes in plasma aldosterone
concentration. Interestingly, the abundance of
-ENaC fell on
day 1 and remained decreased throughout the time course.
Similarly, total
-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
-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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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 1-subunit in
response to dietary NaCl restriction. On days 3 and
10, there was no change in Na-K-ATPase
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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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 -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/
-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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Data from RPAs for the three ENaC subunits are shown in Fig.
6. The abundance of the -ENaC mRNA was
increased after 3 days of dietary NaCl restriction but not after 1 day,
in parallel with demonstrated changes in
-ENaC protein. There were
no increases in the abundances of
-ENaC or
-ENaC mRNA at either
time point, although the abundance of
-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
-,
-, and
-ENaC protein previously documented in Fig. 1
(immunoblots not shown). Thus the results are compatible with the view
that increases in
-ENaC protein abundance after 3 days of dietary
NaCl restriction are due at least in part to increases in
-ENaC
mRNA. Furthermore, they suggest that the decrease in
-ENaC protein
abundance may be due in part to a decrease in
-ENaC mRNA levels.
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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 -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
-ENaC. (Note
that although no bands are visible in the control lanes for
- or
-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
-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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DISCUSSION |
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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 -subunit of ENaC, and the 70-kDa form of the
-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 -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
1-subunit of the Na-K-ATPase (Fig. 4). In addition, there was a sustained fall in the total abundance of the
- and
-subunits of ENaC (Fig. 3).
The decrease in total abundance of the -subunit of ENaC seen on
day 1 was sustained throughout the time course of
observation (Fig. 3). Total
-subunit abundance changes appear to
parallel those of
-ENaC, although the decrease was not statistically
significant until the day 10 time point (Fig. 3). The
mechanism for reduction in
-ENaC abundance is not clear. This change
is not likely caused by aldosterone, because the fall in
-ENaC
protein and mRNA occurred before aldosterone levels increased
substantially (Fig. 1). Finally, in separate studies, spironolactone
administration did not affect
-ENaC protein levels
(27). Three factors have thus far been identified that
regulate
-ENaC protein abundance independently of
-ENaC. First,
increased circulating vasopressin levels are associated with selective
upregulation of
- and
-ENaC (7). Second, altered
acid-base balance also selectively changes
- and
-ENaC abundance,
with alkali loading increasing, and acid loading decreasing,
- and
-ENaC abundance (19). Finally, studies in
AT1a receptor knockout mice support the conclusion that
angiotensin II may directly decrease
- and
-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 -,
-, and
-subunits. The
finding that the
-subunit is regulated independently of the
- and
-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
-ENaC is different from the distribution of
- and
-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
-subunit is rate limiting for the formation
of mature, functional complexes at least under some conditions.
However, the observation that
- and
-ENaC can be regulated
independently of
-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
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
-subunit, whereas
the
- and
-subunits provide regulatory and chaperone functions
(25, 36). Synthesis of the
-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
- or
-subunits. While it is possible that the fall in Na-K-ATPase on
day 1 was primarily localized to the thick ascending limb,
the
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
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
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.
-ENaC.
-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
-ENaC mRNA abundance coincided with the change in
-ENaC protein
abundance, suggesting that
-ENaC protein abundance regulation is
dependent, at least in part, on changes in
-ENaC mRNA. Although not
specifically investigated in the present study, this effect presumably
occurs via regulation of
-ENaC gene transcription (32).
In this regard, it is well established that administration of
aldosterone or synthetic glucocorticoid hormones can increase
-ENaC
mRNA in the lung and kidney (34). However, the effects of
dietary NaCl restriction are more complicated. An increase in
-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
-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
-ENaC protein abundance in the cortex was mediated, at
least in part, by posttranscriptional mechanisms, e.g., regulation of
-ENaC mRNA translation (24). Different conclusions have
been drawn for the renal medulla, in which dietary NaCl restriction has
been found to consistently increase
-ENaC mRNA abundance (29,
34).
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 ![]() |
APPENDIX |
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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