Activation of ENaC by AVP Contributes to Urinary Concentrating

Articles in PresS. Am J Physiol Renal Physiol (November 12, 2014). doi:10.1152/ajprenal.00246.2014
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Activation of ENaC by AVP Contributes to Urinary Concentrating
Mechanism and Dilution of Plasma
Elena Mironova1, Yu Chen2, Alan C. Pao2, Karl P. Roos3, Donald E. Kohan3,
Vladislav Bugaj1, and James D. Stockand1
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Department of Physiology, University of Texas Health Science Center,
San Antonio, Texas 78229
Department of Medicine, Stanford University School of Medicine, Stanford, California
94305 & VA Palo Alto Health Care System, Palo Alto, California 94304
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Division of Nephrology, University of Utah Health Science Center,
Salt Lake City, Utah 84132
Correspond with James David Stockand:
University of Texas Health Science Center at San Antonio
Department of Physiology
7703 Floyd Curl Drive
San Antonio TX 78229
stockand@uthscsa.edu
ph. 210-567-4332
fx. 210-567-4410
Running title: ENaC contributes to dilution of plasma
Word count for abstract: 249
Word count for text: 2885
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Copyright © 2014 by the American Physiological Society.
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ABSTRACT
Vasopressin (AVP) activates the epithelial Na+ channel (ENaC). The physiological
significance of this activation is unknown. The current studies test if activation of ENaC
contributes to AVP-sensitive urinary concentration. Consumption of a 3% NaCl solution
induced hypernatremia and plasma hypertonicity in mice. Plasma [AVP] and urine
osmolality increased in hypernatremic mice in an attempt to compensate for increases
in plasma tonicity. ENaC activity was elevated in mice consuming 3% NaCl solution
compared to mice consuming a diet enriched in Na+ with ad libitum tap water. The latter
diet does not cause hypernatremia. To determine whether the increase in ENaC activity
in mice consuming 3% NaCl solution served to compensate for hypernatremia, mice
were treated with the ENaC inhibitor benzamil. Co-administration of benzamil with 3%
NaCl solution decreased urinary osmolality and increased urine flow so that urinary Na+
excretion increased with no effect on urinary [Na+]. This decrease in urinary
concentration further increased plasma [Na+], osmolality, and [AVP] in these already
hypernatremic mice. Benzamil similarly compromised urinary concentration in water
deprived mice and in mice treated with desmopressin. These results demonstrate that
stimulation of ENaC by AVP plays a critical role in water homeostasis by facilitating
urinary concentration, which can compensate for hypernatremia or exacerbate
hyponatremia. The current findings are consistent with ENaC in addition to serving as a
final effector of the renin-angiotensin-aldosterone system and blood pressure
homeostasis, also playing a key role in water homeostasis by regulating urine
concentration and dilution of plasma.
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INTRODUCTION
Arginine vasopressin (AVP) decreases renal water excretion (2; 3; 26; 27). AVP
is secreted by the posterior pituitary in response to decreases in arterial blood pressure
and increases in plasma tonicity. The latter is the primary stimulus controlling AVP
secretion. AVP promotes concentration of urine and consequent dilution of plasma as a
feedback response to increases in plasma tonicity. AVP signals through the AVP
receptor 2 (AVPR2) in the distal nephron to concentrate urine by increasing urinary
osmolality and decreasing urine flow. AVP drives urinary concentration through two
effects.
First, AVP promotes the formation and maintenance of an axial
corticomedullary hyperosmotic gradient. This hyperosmotic gradient draws water from
the urine by maximizing the trans-epithelial osmotic gradient between medullary
interstitial fluid and urine within the collecting duct. Second, AVP increases the water
permeability of the collecting duct by increasing the expression of aquaporin 2 water
channels (AQP2) in the luminal membrane of principal cells.
AVP induces anti-diuresis by stimulating a decrease in both urine water excretion
(anti-aquaresis) and urine Na+ excretion (anti-natriuresis) (5; 17; 27). The physiological
significance of AVP controlling urine Na+ excretion is unclear raising the question: if
AVP promotes renal Na+ conservation, then how does this process affect plasma
tonicity?
AVP via AVPR2 decreases Na+ excretion by stimulating the epithelial Na+
channel (ENaC) (4; 5; 7; 15; 19). ENaC, which is expressed along with AQP2 in the
luminal membrane of principal cells, serves as the final effector for hormone pathways
that fine-tune urinary Na+ excretion (9; 12; 21). Consequently, ENaC is an important
effector of the renin-angiotensin-aldosterone system (RAAS): aldosterone stimulates
ENaC to promote Na+ reabsorption and protect plasma volume and arterial blood
pressure. The importance of feedback regulation of ENaC by RAAS is highlighted by
abnormalities in blood pressure caused by gain and loss of function of ENaC and its
upstream regulators (13; 14). Gain of function of ENaC causes improper renal Na+
retention and associated increases in arterial blood pressure. Loss of function of ENaC
causes inappropriate renal sodium wasting and concomitant decreases in blood
pressure.
However, regulation of ENaC by both RAAS and AVP signaling creates a conflict
between regulation of plasma volume and tonicity because RAAS responds to and
controls arterial blood pressure by regulating plasma volume, whereas AVP responds to
and controls plasma tonicity by regulating urine concentration. Classically, ENaC has
been considered to be primarily under the control of RAAS for regulation of blood
pressure. Findings from our laboratory and others suggest that ENaC is also a key
effector of AVP for regulation of plasma tonicity (7; 15; 18; 19). For instance, in
adrenalectomized mice, which lack aldosterone, increases in plasma [AVP] maintain
ENaC activity (15). Thus, in certain instances, ENaC may respond to AVP signaling
and contribute to regulation of Na+ and water balance.
To better understand how ENaC contributes to regulation of plasma volume and
tonicity, we tested the hypothesis that AVP-mediated activation of ENaC facilitates urine
concentration and dilution of plasma. The current results demonstrate that AVPdependent activation of ENaC is necessary for compensatory urinary concentration
during hypernatremia and contributes to the dilution of plasma during hyponatremia. As
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such, these findings demonstrate that in addition to serving as a final effector of the
RAAS and blood pressure homeostasis, ENaC also plays a key role in water
homeostasis by regulating urine concentration and dilution of plasma.
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METHODS
Animals. All mouse use and welfare adhered to the NIH Guide for the Care and Use of
Laboratory Animals (8) following protocols reviewed and approved by Institutional
Animal Care and Use Committees at the University of Texas Health Science Center at
San Antonio, and the Veterinary Medical Unit of the VA Palo Alto Health Care System,
Palo Alto CA. Adult, aged and weight matched male C57BL/6J mice (~25g, 6-8 wk old)
were used for all experiments. Mice were purchased from Jackson Laboratories.
Control mice were maintained with standard chow (0.32% Na+; TD.7912, Harlan Teklad)
and ad libitum tap water. For some experiments, mice were maintained with a high Na+
diet (2% Na+; TD.92034, Harlan Teklad) and ad libitum tap water. Mice were made
hypernatremic by consumption of a 3% NaCl drinking solution for 3 days. In some
experiments, dDAVP (1 ng/hr) was provided in 0.9% saline for 3-6 days via sterile alzet
micro-osmotic pumps (model 1007D; Durect Corp.) implanted s.c. between the
scapulae. In others, mice were water deprived for 24 hrs. Benzamil (1.4 mg/kg bw)
was provided once a day for three days via i.p. injection in 100 μl water.
Metabolic cage studies. In vivo analysis of urine and plasma electrolyte and water
content followed standard methods published previously (15; 20). In brief, mice (n = 4-5
per condition) were housed in metabolic cages (Techniplast) for the duration of the
study. Mice were provided three days of free access to normal chow and tap water to
acclimate prior to experimentation. The duration of the experimental period was three
days. During the acclimation and experimental periods, temperature and lighting (12
hrs light and dark cycles) were controlled. All measurements and procedures were
made at a routine time and mice were provided measured amounts of drinking solution
and chow during the experimental period. Spontaneously voided urine was collected
under light mineral oil every 24 hrs. At the end of the experiment, mice were sacrificed
and trunk blood collected immediately in heparinized tubes via cardiac puncture.
Analysis of hormones and electrolytes. Urinary and plasma [Na+] and osmolality
were determined by using standard procedures with a PFP7 flame photometer (Techne)
and vapor pressure osmometer (Wescor Inc). Plasma [AVP] was quantified with a
competitive enzyme-linked immunoassay (Arg8-Vasopressin EIA kit; Enzo Life
Sciences) following standard proceures (15).
Medullary Na+ content was also
quantified by using standard procedures (1; 24). In brief, kidneys were removed from
mice, and the inner medulla was rapidly excised and dissected. Inner medulla samples
were blotted on pre-soaked filter paper, placed in a separate tube, and weighed.
Samples were then dried over a desiccant at 60° C for 4-5 hrs. After reweighing,
samples were soaked in 100 µl of deionized, distilled water, heated to 90° C for 3 min,
centrifuged, and stored at 4° C for 24 hrs. The next day, samples were centrifuged for 1
min at 8,000 g, and the supernatant was collected for Na+ concentration measurements.
Patch-clamp electrophysiology in isolated, split-open aldosterone-sensitive distal
nephron. A previously published method for isolating aldosterone-sensitive distal
nephron containing connecting tubule and cortical collecting duct suitable for patchclamp electrophysiology was followed (15). The activity of ENaC in principal cells of
murine aldosterone-sensitive distal nephron was quantified in cell-attached patches of
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the apical membrane made under voltage-clamp conditions (-Vp = -60 mV) by using
standard procedures (15). For the current studies, bath and pipette solutions were (in
mM): 150 NaCl, 5 mM KCl, 1 CaCl2, 2 MgCl2, 5 glucose and 10 Hepes (pH 7.4); and
140 LiCl, 2 MgCl2 and 10 Hepes (pH 7.4), respectively. For each experimental
condition, aldosterone-sensitive distal nephron from at least five different mice were
assayed.
Statistical Analysis and Data Presentation. Data are reported as mean ± SEM.
Unpaired data were compared with a two-sided unpaired Student t test and One Way
Analysis of Variance using the Student-Newman-Kuels post hoc test as appropriate.
Proportions were compared using a z-test. The criterion for significance was P ≤ 0.05.
For presentation of current traces, slow baseline drifts were corrected and data
software-filtered at 50 Hz.
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RESULTS
AVP activates ENaC. We and others have reported previously that AVP activates
ENaC (18; 19; 27). Results presented in figure 1 are in agreement with these earlier
findings. Shown in figure 1A is a continuous current trace before and after addition of
AVP to the bath solution of a cell-attached patch that contains at least five ENaC. This
patch was formed on the apical membrane of a principal cell in an isolated, split-open
murine distal nephron. In this representative experiment and in the summary results in
1B and table 1, AVP increases ENaC activity by significantly increasing both channel
number and open probability.
AVP maintains ENaC activity high during hypernatremia. In a recent study of
adrenalectomized mice, we demonstrated that ENaC was active in the absence of
aldosterone due to a rise in plasma [AVP] (15). Adrenalectomy, however, introduces a
multitude of changes, including a loss of all adrenal hormones and a marked decline in
plasma volume. In turn, these changes may induce a complex set of adaptations, all of
which may obscure a clear understanding of the physiological consequences of AVP
regulation of ENaC. In order to define the physiological consequences of AVP
stimulation of ENaC, we fixed sodium and water intake together by maintaining mice
with a 3% NaCl drinking solution. This maneuver simultaneously suppresses RAAS
signaling while stimulating AVP release because mice become volume expanded and
hypernatremic (see below). As a consequence, this experimental perturbation isolates
AVP regulation of ENaC and limits confounding influences from RAAS and other
signaling pathways. As shown by the summary graphs in figure 2, when mice are
forced to consume 3% NaCl solution, plasma tonicity, plasma and urinary [Na+], and
plasma [AVP] increase. While RAAS is suppressed under these conditions, the rise in
[AVP] is consistent with preservation of some ENaC activity in this state of
hypernatremia and with a role for AVP-stimulated ENaC in counteracting plasma
hypertonicity by facilitating renal water reabsorption, urine concentration and dilution of
plasma.
Results in figure 3 and table 1 reveal that ENaC is indeed active in hypernatremic
mice maintained with a 3% NaCl solution drinking solution. Shown here is ENaC
activity in distal nephron principal cells from mice maintained with normal chow and ad
libitum tap water (control), ad libitum tap water and a high (2%) Na+ diet, and 3% NaCl
solution and normal chow. Consistent with previous findings, ENaC activity is
suppressed when mice are allowed to consume water as they increase Na+ intake (6;
16; 30). When mice consume a Na+ rich diet, plasma volume expands and RAAS
signaling is suppressed, which leads to an increase in urine Na+ excretion associated
with a decrease in ENaC activity. With ad libitum water consumption, mice are able to
regulate water balance independently: plasma [Na+] and plasma [AVP] do not change.
However, when mice are maintained with a 3% NaCl solution drinking solution, Na+ and
water intake are fixed so that both volume expansion and hypernatremia ensue. In this
condition, an increase in serum [AVP] is able to preserve some ENaC activity despite
suppression of the RAAS. Consumption of a Na+ rich diet leads to volume expansion
and suppression of RAAS signaling, whereas the concomitant hypernatremia increases
plasma [AVP], which stimulates ENaC activity.
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Activation of ENaC by AVP facilitates concentration of urine. As shown in figure 4,
active ENaC contributes to compensatory water conservation during hypernatremia.
Shown here is urine osmolality and flow in mice maintained with ad libitum water and
normal chow compared to those maintained with 3% NaCl drinking solution in the
absence and presence of benzamil treatment. Consumption of hypertonic saline
promotes urine concentration reflected by an increase in urine osmolality. Treatment
with benzamil significantly decreases urine osmolality and increases urine flow in mice
drinking 3% NaCl solution.
ENaC-dependent urinary concentration contributes to dilution of plasma. As
shown above, forced consumption of hypertonic saline causes hypernatremia; benzamil
impedes urinary concentration in this condition. From these observations, we predicted
that treating mice consuming 3% NaCl drinking solution with benzamil should increase
plasma [Na+] further. As demonstrated by the results shown in figure 5, this is the case.
Shown in 5A is plasma [Na+] from mice maintained with ad libitum water and normal
chow compared to those maintained with 3% NaCl solution and 3% NaCl solution plus
benzamil treatment. Consumption of 3% NaCl solution increases plasma [Na+];
treatment of these mice with benzamil significantly increases plasma [Na+] further.
Similarly, as shown in 5B, administration of benzamil to mice consuming 3% NaCl
solution significantly increases further the already elevated plasma osmolality.
Mechanism: Inhibition of ENaC diminishes the draw for reabsorption of urinary
water. Results shown in figure 6 reveal the mechanism by which AVP-stimulated ENaC
contributes to urinary concentration. Consumption of hypertonic saline increases
medullary Na+ content and urinary Na+ excretion. Benzamil further significantly
increases renal Na+ excretion in mice maintained with a 3% NaCl solution drinking
solution without affecting medullary Na+ content. Thus, the mechanism by which
benzamil impairs urine concentration does not involve impairment of the medullary Na+
concentration gradient; rather, inhibition of ENaC impairs tubular transport of Na+ so
that Na+ is trapped in the urine lessening the osmotic gradient between urine and
interstitial fluid. Because this Na+ in the urine is osmotically active, inhibition of ENaC
results in retaining water in the urine without changing urinary [Na+]. Such observations
demonstrate that by inhibiting ENaC, benzamil promotes an osmotic diuresis and
consequently decreases urine concentrating ability, making the Na+ concentration in the
plasma more like the fluid being consumed. When this fluid is hypertonic saline,
inhibition of ENaC further increases plasma [Na+] and worsens hypernatremia by
limiting concentrating ability.
Facilitation of urinary concentrating ability is a general manifestation of AVP
stimulation of ENaC. The findings above are consistent with a role for AVP-stimulated
ENaC in facilitating reabsorption of urinary water to compensate for hypernatremia. To
test whether facilitation of urinary concentrating ability is a general manifestation of AVP
stimulation of ENaC, and to test whether AVP-stimulated ENaC plays a role in the
pathogenesis of hyponatremia, we water deprived mice and injected mice with
desmopressin (dDAVP) in mice in the presence and absence of benzamil. As shown in
figure 7, urine osmolality increases and urine flow decreases in both water deprived and
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dDAVP treated mice, demonstrating the expected concentration of urine in response to
these stimuli. During water deprivation, urinary concentration is an appropriate
compensatory response, whereas in dDAVP treated mice, urinary concentration is
causative for hyponatremia (11). Treatment with benzamil significantly reduces urine
osmolality and increases urine flow in water deprived mice; treatment with benzamil
significantly reduces urine osmolality and promotes a trend towards increased urine flow
in dDAVP treated mice. Thus, in both conditions inhibition of ENaC compromises
urinary concentrating ability. These results are consistent with a role for AVP-mediated
activation of ENaC in not only promoting renal water retention under conditions of
hypernatremia but also for causing hyponatremia by obligating renal water reabsorption
and urinary concentration. Thus, AVP activation of ENaC is a general response for
promoting renal water conservation and dilution of plasma.
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DISCUSSION
The following questions inspired the current studies: 1) what is the function of
ENaC when AVP signaling is active; 2) does AVP-mediated activation of ENaC serve to
increase renal water reabsorption during hypernatremia and AVP-driven hyponatremia;
or 3) does inhibition of ENaC relieve hypernatremia and exacerbate hyponatremia?
The answers to these questions are not obvious because ENaC is primarily regarded as
an end effector of RAAS signaling.
Moreover, inhibiting ENaC could relieve
hypernatremia and exacerbate hyponatremia by decreasing sodium content in the
plasma. However, the current results demonstrate that this is not the case. Inhibition of
ENaC exacerbates hypernatremia and relieves hyponatremia because the primary
action of AVP-mediated ENaC activation is to facilitate urine concentration and dilution
of plasma.
The current findings are consistent with the recent observation that AVP
maintains ENaC activity in adrenalectomized mice (15). Such animals are hypovolemic
and hyponatremic because mineralocorticoids are absent and AVP assumes a greater
role in stimulating both renal Na+ and water retention. However, due to the complex
physiological adaptations that occur with adrenal insufficiency, it remains unclear from
this earlier study whether stimulation of ENaC by AVP exacerbated hyponatremia while
compensating for volume depletion, or if activation of ENaC mitigated both decreases in
plasma volume and tonicity. This uncertainty highlights the inherent limitation of how all
biological organisms, including terrestrial vertebrates, must use solutes such as sodium
to absorb water and regulate water homeostasis. When water can move only through
osmosis, the need to protect plasma volume may compete with the need to protect
plasma tonicity. ENaC sits at the nexus of these two homeostatic systems, one
controlling plasma tonicity and the other plasma volume. A central question that
emerges from this paradigm: What is the activity of ENaC and the consequences of this
activity when the need to maintain volume competes against the need to maintain
tonicity?
The current findings show that AVP-mediated activation of ENaC facilitates
urinary concentration and dilution of plasma.
In instances where AVP drives
hyponatremia, ENaC is positioned to maintain or even exacerbate hyponatremia by
promoting urine concentration. This may seem counterintuitive if one only considers the
role of ENaC in RAAS signaling and plasma volume regulation. However, if one
considers a broader role of ENaC in mediating AVP action on water homeostasis, as
explored in the current studies, it becomes apparent that inhibitors of ENaC provoke an
osmotic diuresis because inhibition of ENaC compromises urinary concentrating ability
to some degree. In this sense, inhibitors of ENaC are similar to all diuretics in that they
compromise the ability of the kidney to regulate the Na+ concentration in urine and
consequently force the Na+ concentration of plasma to be more similar to that in the
fluid being ingested.
The current findings may have important implications for treatment of
hyponatremia associated with high AVP levels such as in decompensated heart failure,
cirrhosis, and SIADH. In these diseases, where hyponatremia is the clinical hallmark of
the nonosmotic release of AVP, inhibition of ENaC may serve to counter hyponatremia
at the same time as decreasing plasma volume. This strategy may be particularly
useful in heart failure and cirrhosis where aldosterone levels are concomitantly elevated
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(25; 28; 29; 31). Mineralocorticoid receptor (MR) antagonists are frequently used to
inhibit aldosterone action in these diseases, but these treatments do not necessarily
block the action of AVP, which is also increased. The increase in AVP levels in these
diseases may act in a synergistic manner with aldosterone to increase pathologic renal
Na+ and water retention. A combined regimen of antagonists against ENaC and MR
could remove the risk of hyponatremia by increasing both urine NaCl and water
excretion.
We propose that ENaC contributes to dilution of plasma when the channel, along
with AQP2, is activated by AVP. This notion is consistent with what was reported many
years ago when both aldosterone and AVP were considered to be required for maximal
urine concentration (reviewed in 26). As we and others have shown, ENaC activity is
maximal in the presence of both aldosterone and AVP (7; 10; 19; 22; 23). We argue
that this regulation contributes to the underlying basis for the standing clinical maxim
that the kidney protects volume over tonicity. AVP-mediated activation of ENaC serves
to increase renal Na+ retention and protect plasma volume but this can occur at the
expense of increasing renal water retention. This mechanism may contribute to
hyponatremia observed in individuals with significant volume depletion. In addition, we
propose that AVP-mediated activation of ENaC also serves to dilute plasma by
facilitating maximal urinary concentration.
This mechanism may exacerbate
hyponatremia in diseases with high AVP levels such as in heart failure, cirrhosis, or
SIADH.
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FIGURE LEGENDS
Figure 1. AVP stimulates ENaC. A. Continuous current trace from a representative
cell attached patch containing at least five ENaC before and after addition (arrow) of 1
uM AVP to the bath solution. This patch was on the apical membrane of a principal cell
in a freshly isolated and split-open murine distal nephron. Inward current is downwards.
Shown below on an expanded time scale are areas of the trace under the gray lines
marked 1 and 2 before and after AVP, respectively. Open (o) and closed (c) states are
noted. B. Summary graphs of the number of active ENaC (N; top left) in patches that
contained at least one active channel, the probability that ENaC is open (Po; top right);
the activity of ENaC (NPo; lower left); and total cellular ENaC activity (fNPo; lower right),
which is activity (NPo) multiplied by the frequency (f) of forming a viable seal for that
condition that contained at least one active channel. For these experiments, ENaC
activity was measured in freshly isolated and split-open distal nephron treated for 30
minutes with vehicle (gray bars) or 1 μM AVP (black bars). All other conditions were
identical to 1A. *Significantly greater compared to vehicle.
Figure 2. Drinking 3% NaCl solution causes hypernatremia and increases plasma
[AVP]. Summary graphs of plasma osmolality (A), plasma [Na+] (B), urinary [Na+] (C),
and plasma [AVP] (D) for mice provided tap water (control; gray bars) or a drinking
solution containing 3% NaCl (black bars) for three days. *Significant increase versus
control.
Figure 3. ENaC activity is high in hypernatremic mice. Summary graph of total
cellular ENaC activity in mice maintained with normal chow and ad libitum tap water
(control; light gray bar), 2% dietary Na+ and ad libitum tap water (dark gray bar), and
normal chow and 3% NaCl solution drinking solution (black bar) for three days. Patch
clamp conditions identical to fig. 1. *Significantly less than control. **Significantly
greater than 2% dietary Na+.
Figure 4. Inhibition of ENaC blunts concentration of urine and increases urine
flow in hypernatremic mice. Summary graphs of urine osmolality (top) and urine flow
(bottom) in matched mice drinking tap water (control; light gray bar), 3% NaCl solution
(black bar) or 3% NaCl solution plus injection with benzamil (dark gray bar) for three
days. *Significantly greater compared to control. **Significant decrease compared to the
absence of benzamil.
Figure 5. Inhibition of ENaC further increases PNa and POsm in hypernatremic
mice. Summary graphs of plasma [Na+] (A) and osmolality (B) in matched mice
maintained with tap water (control; light gray bar), 3% NaCl solution drinking solution
(black bar) or 3% NaCl solution plus injection with benzamil (dark gray bar) for 3 days.
*
Significantly greater compared to control. **Significantly greater compared to the
absence of benzamil.
Figure 6. Inhibition of ENaC decreases the draw for water reabsorption in
hypernatremic mice. Summary graphs of medullary Na+ content (A), urinary Na+
excretion (B) and urinary [Na+] (C) in matched mice maintained with tap water (control;
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light gray bars), 3% NaCl solution drinking solution (black bars) or 3% NaCl solution
plus injection with benzamil (dark gray bars) for 3 days. *Significantly greater compared
to control. **Significantly greater compared to the absence of benzamil.
Figure 7. Inhibition of ENaC blunts AVP-dependent urinary concentration.
Summary graphs of urine osmolality (top) and urine flow (bottom) in untreated control
mice (light gray bars) and matched mice water deprived for 24 hours (A), or
instrumented with a minipump releasing dDAVP for 3 days (B) in the absence (black
bars) and presence of injection with benzamil (dark gray bars). *Significantly different
from control. **Significant different compared to the absence of benzamil.
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TABLES
Table I. ENaC activity
1
Condition1
control
AVP
2% Na+
diet
3% NaCl
water
N
2.4 ± 0.3
3.8 ± 0.3*
1.5 ± 0.1*
Po
0.27 ± 0.04
0.43 ± 0.02*
0.16 ± 0.02*
NPo
0.73 ± 0.17
1.78 ± 0.22*
0.31 ± 0.06*
n (total)2
65 (141)
43 (60)
55 (153)
f3
0.46
0.72*
0.36
fNPo4
0.34 ± 0.08
1.28 ± 0.16*
0.11 ± 0.02*
2.0 ± 0.3**
0.15 ± 0.02
0.45 ± 0.13
26 (49)
0.53**
0.24 ± 0.06**
Control = tubules harvested from control mice provided 0.32% Na+ diet and ad libitum water;
AVP = tubules from control mice treated ex vivo with 1 μM AVP for 30 minutes; 2% Na+ diet =
tubules harvested from mice provided a 2% Na+ diet and ad libitum tap water for 5 days; 3%
NaCl water = tubules harvested form mice provided 3% NaCl drinking solution and 0.32% Na+
diet for 3-4 days. 2n = number of patches with at least one active ENaC; total = total number of
viable seals for that condition. 3f = n/total. 4fNPo = to cellular activity which is activity (NPo)
multiplied by the frequency (f) of forming a viable seal for that condition that contained at least
*
one active channel.
Significantly different compared to control; **significantly different
+
compared to 2% Na diet.
16
Figure 1
before
+ AVP
1
AVP
B
2
0.5 pA
1 min
2 sec
*
4
N
2
c
o1
o2
c
o1
o2
o3
o4
o5
0
Po
0.2
vehicle
AVP
0.0
*
2.0
NPo
1.0
0.0
*
0.4
fNPo
A
vehicle
AVP
1.6
1.2
0.8
0.4
0.0
vehicle
AVP
*
vehicle
AVP
Figure 2
*
POsm, mOsm/kg
400
300
200
B
*
200
PNa
(mmol/L)
100
100
0
0
control
C
*
400
UNa
(mmol/L)
200
0
3% NaCl
water
control
3% NaCl
water
D
plasma [AVP]
(pg/ml)
A
control
3% NaCl
water
*
40
30
20
10
0
control
3% NaCl
water
Figure 3
0.5
fNPo
0.4
**
0.3
0.2
*
0.1
0.0
control
2% Na+ 3% NaCl
diet
water
Figure 4
*
UOsm, mOsm/kg
2000
**
1500
1000
500
0
control
3%NaCl
water
+ benzamil
0.0
o
V, ml/day
1.0
2.0
3.0
4.0
5.0
**
Figure 5
A
*
150
PNa
(mmol/L)
**
B
*
400
**
POsm 300
(mOsm/kg)
100
200
50
100
0
0
control
3% NaCl + benzamil
water
control
3% NaCl + benzamil
water
Figure 6
B
*
0.20
0.10
0.00
control 3% NaCl +benzamil
water
**
1.6
1.2
0.8
*
0.4
C
UNa, mmol/L
0.30
UNaV, mol/min
Medullary Na+ content
mmol/g WT
A
600
*
400
200
0
0.0
control 3% NaCl +benzamil
water
control 3% NaCl +benzamil
water
Figure 7
1000
control
*
3000
2000
1000
control
0.0
0.0
0.4
0.4
0.8
1.2
*
**
**
0
water
+ benzamil
deprivation
o
o
**
UOsm, mOsm/kg
2000
0
V, ml/day
*
3000
B
V, ml/day
UOsm (mOsm/kg)
A
dDAVP
0.8
1.2
1.6
1.6
2.0
2.0
2.4
*
+ benzamil