Arginine vasopressin modulates expression of neuronal NOS in
rat renal medulla
Pierre-Yves
Martin,
Mathieu
Bianchi,
Frank
Roger,
Laurent
Niksic, and
Eric
Féraille
Division of Nephrology, Hôpital Cantonal
Universitaire, CH-1211 Geneva 14, Switzerland
 |
ABSTRACT |
Arginine vasopressin (AVP) plays a
central role in water balance. In principal cells of the collecting
duct system, AVP controls the expression of several genes, including
aquaporin-2. Because nitric oxide (NO) participates in the regulation
of water reabsorption by the collecting duct system, we analyzed the
effect of AVP on the expression of NO synthase (NOS) isoforms in the
kidney. Rats were either water restricted or water loaded to modify the
circulating AVP levels, and expressions of NOS isoforms were assessed
by Western blot analysis. In water-restricted rats, endothelial NOS
(eNOS) expression increased in the outer medulla, and neuronal NOS
(nNOS) expression rose in both the outer medulla and the papilla.
Conversely, water loading induced a decrease in expression of nNOS in
the outer medulla and papilla but did not alter eNOS expression. Oral administration of the specific V2-receptor antagonist
SR-121463B decreased nNOS expression in the outer medulla and papilla
but did not alter eNOS expression levels. Finally, the very low nNOS expression levels observed in AVP-deficient Brattleboro rats was restored by AVP infusion for 1 wk. Thus AVP specifically increases nNOS
expression levels in the renal outer medulla and papilla. Because nNOS
is specifically expressed in principal cells of the collecting duct
system, the stimulation of nNOS expression by AVP may participate in
the control of water reabsorption.
principal cells; aquaporin-2; collecting duct; nitric oxide
synthase
 |
INTRODUCTION |
THE MAINTENANCE OF
CONSTANT body fluid osmolality is achieved by complex homeostatic
mechanisms in which the kidney plays a central role through the control
of water balance. In higher vertebrates, water balance can be achieved
mainly because the kidney has the ability to excrete either diluted or
concentrated urine. Imbalance between water intake and water excretion
due to impaired kidney function leads to life-threatening disorders such as hyponatremia or hypernatremia.
Once filtered by the glomerulus, water is reabsorbed by the kidney
tubule. Quantitatively, the most important segments are the proximal
tubule and the thin descending limb of Henle. In these segments,
transcellular water reabsorption is achieved through aquaporin-1 (AQP1)
water channels constitutively expressed in both apical and basolateral
membrane domains (4, 21, 30). In addition, water
reabsorption occurs along the paracellular route in proximal tubules
(30). Regulated water reabsorption takes place in the
collecting duct system, i.e., the connecting tubule and the collecting
duct (14, 16). The principal cells of the collecting duct
system express different water channels in their apical and basolateral
membrane domains. AQP3 and AQP4 are constitutively located in the
basolateral membrane domain (8, 16, 33), whereas AQP2
shuttles between intracellular stores and the apical membrane domain
(16, 19).
Arginine vasopressin (AVP) is the major hormone that controls
water reabsorption by the collecting duct system. Indeed, the collecting duct system is virtually impermeable to water in the absence
of AVP and becomes highly permeable to water in the presence of AVP
(10, 14, 16). This effect has been demonstrated in vivo
(17) and by in vitro microperfusion of isolated collecting ducts (10). In principal cells, AVP acts through
V2 receptors coupled to the stimulation of adenylyl cyclase
(11, 16, 25). The subsequent increase in cellular cAMP
concentration activates protein kinase A and induces AQP2 translocation
from intracellular stores to the apical plasma membrane
(15). Besides short-term stimulatory effects on water
permeability (10), sodium reabsorption (9),
and potassium secretion (3), AVP also controls the expression of a specific subset of genes in principal cells of the
collecting duct (28). For instance, increasing and
decreasing circulating AVP levels by water restriction or water loading
in rats alters AQP2 expression levels (34). In addition,
infusion of AVP into Brattleboro rats increases AQP2 expression
(5). On the other hand, in vitro AVP treatment of a rat
cortical collecting duct cell line increased the expression of the
Na-K-ATPase
-subunit and the epithelial sodium channel
- and
-subunits (6, 7).
The three nitric oxide synthase (NOS) isoforms, inducible NOS (iNOS),
endothelial NOS (eNOS), and neuronal NOS (nNOS), have been found to be
expressed in the kidney (1, 18). It appears that the
constitutive NOS isoforms nNOS and eNOS are responsible for modulating
the physiological function of renal cells, whereas iNOS is involved in
pathophysiological events (20, 26). Recent studies
indicate that NO mimics the effect of AVP on AQP2 translocation by
activating protein kinase G through increased intracellular cGMP levels
(2). Together with the expression of nNOS and the absence
of eNOS in collecting duct principal cells (36), these results suggest that endogenous NO from nNOS may participate in the
regulation of water reabsorption by the collecting duct. In addition,
AVP has been shown to induce transcription of several proteins that may
play a role in physiological responses (28).
The purpose of this study was to investigate the effect of AVP on renal
NOS activity and protein expression in rodents.
 |
MATERIAL AND METHODS |
Animals and study design.
Male Wistar (Service de Zootechnie, Centre Medical Universitaire,
Geneva, Switzerland), Brattleboro, and Long-Evans rats (Harlan, Indianapolis, IN) weighing between 150 and 300 g were used for experiments. Wistar rats were randomly assigned to either control or
experimental groups, were pair-fed, and had unrestricted access to
water. Two days before the start of the experimental protocol, rats
were housed in metabolic cages with powdered chow. Experimental group 1 consisted of water-restricted rats that were allowed
to drink 10 ml of water for 3 days and 5 ml for 2 days and were
deprived of water for the remaining 24 h. Experimental group
2 consisted of water-loaded rats fed for 6 days a mixture of
hydrated agar and powdered standard laboratory rat chow containing
3.33 g water/g of dry food; each animal was provided with 18 g dry food/day. Experimental group 3 consisted of rats
receiving a daily intragastric administration of 1 mg/kg body weight of
the specific V2-receptor antagonist SR-121463B (a kind gift
from Dr. Serradeil-Le Gal, Sanofi Research, Toulouse, France) for 7 days (31); rats were killed 12 h after the last
administration of SR-121463B. Measurements of water and food intake,
body weight, and urine volume were undertaken at days 0 and
7. Urine specimens were collected under oil, and samples
taken at days 0 and 7 were frozen at
80°C for
later analysis.
Brattleboro and Long-Evans (control) rats were kept under the same
conditions as the other groups. One group of Brattleboro rats was
infused for 1 wk with AVP (5 µg/day) delivered by an intraperitoneal
osmotic minipump (Alzet). An intraperitoneal osmotic minipump
delivering solvent alone (1 µl/h of NaCl 0.9%) was implanted in both
control Long-Evans and nontreated Brattleboro rats. Control Long-Evans
and Brattleboro rats were then placed in individual metabolic cages for
7 days with free access to water and powdered chow, and physiological
parameters were recorded as described above.
Each urine sample was centrifuged to remove any solid matter before
analysis. Urine osmolality was determined by freezing point depression,
and urine sodium levels were measured by flame photometry. Sodium
balance was calculated as the ratio of sodium intake to urinary sodium.
The protocols were approved by the Ethical Commission of the Faculty of
Medicine of the University of Geneva.
Isolation of tissue.
After anesthesia with pentobarbital sodium (50 mg/kg body wt), the two
kidneys were infused with incubation solution containing (mM) 120 NaCl,
5 RbCl, 4 NaHCO3, 1 CaCl2, 1 MgSO4,
0.2 NaH2PO4, 0.15 Na2HPO4, 5 glucose, 10 lactate, 1 pyruvate, 4 essential and nonessential amino acids, and 20 HEPES, as well as 0.1%
(wt/vol) bovine serum albumin and vitamins (1× MEM; Life
Technologies), pH 7.45. Afterward, the cortex, inner stripe of the
outer medulla, and papilla were separated, frozen into liquid nitrogen,
and stored at
80°C until used for Western blotting or NOS activity
measurement (see below).
Western blotting.
Frozen tissues were pulverized and glass homogenized in 1 ml of
homogenization buffer containing (in mM) 20 Tris · HCl (pH 7.4), 2 EDTA, 2 EGTA, 1 phenylmethylsulfonyl fluoride, 1 of
4-(2-aminoethyl)-benzenesulfonyl fluoride, 30 NaF, and 30 Na
pyrophosphate, as well as 25 µg/ml leupeptin, 25 µg/ml aprotinin,
0.1% (wt/vol) SDS, and 1% (vol/vol) Triton X-100. After protein
concentration determination (BCA assay, Pierce), equal amounts of
protein (50-100 µg) were mixed with an equal volume of Laemmli
2× buffer and analyzed by SDS-PAGE. After electrophoresis on 7%
polyacrylamide gels, proteins were electrotransferred on polyvinylidine
difluoride membranes (Immobilon-P, Millipore, Bedford, MA) and
incubated overnight at 4°C with primary antibody diluted in
Tris-buffered saline (TBS) Nonidet P-40 (NP-40) medium (TBS-NP-40:
150 mM NaCl, 50 mM Tris, 0.2% NP-40, pH 7.4) with 5% (wt/vol) nonfat
dry milk. After washing in TBS-NP-40, membranes were incubated with a
secondary antibody coupled to horseradish peroxidase (HRP) diluted in
TBS-NP-40 with 5% (wt/vol) nonfat dry milk. The antigen-antibody
complexes were detected by chemiluminescence with the Super Signal
Substrate method (Pierce, Rockford, IL) according to the
manufacturer's instructions. For detection of nNOS, we used rabbit
polyclonal antibodies (Biomol) diluted 1:1,000. For detection of eNOS,
we used a mouse monoclonal antibody (Transduction Laboratories,
Lexington, KY) diluted 1:1,000. For detection of iNOS, we used a mouse
monoclonal antibody (Transduction Laboratories) diluted 1:1,000 or a
rabbit polyclonal antibody (Biomol). With both antibodies, iNOS protein
expression was not detectable in the cortex, outer medulla, or inner
medulla (data not shown). For detection of AQP2, we used previously
characterized rabbit polyclonal antibodies (38). Rabbit
polyclonal antibodies were detected by HRP-conjugated anti-rabbit Ig
diluted 1:20,000 (Transduction Laboratories), and mouse monoclonal
antibodies were detected by HRP-conjugated anti-mouse Ig diluted
1:20,000 (Transduction Laboratories). Results were quantified by laser
densitometry (Molecular Dynamics, Sunnyvale, CA) using Image Quant
software. Data were expressed as percentage of the paired control value.
NOS activity.
The NOS activity measurement assay was performed on the basis of the
biochemical conversion of L-arginine to
L-citrulline and NO (37). Renal cortex, outer
medulla, and papilla from control and experimental rats were
homogenized in a buffer containing 25 mM Tris · HCl, pH 7.4, 1 mM EDTA, and 0.1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride.
Thereafter, 50-100 µg protein contained in 10 µl of homogenization buffer were mixed with 40 µl of reaction buffer containing 50 mM Tris · HCl, pH 7.4, 6 µM BH4, 2 µM FAD, 1 mM reduced NADP, 0.1 µM calmodulin, 0.6 mM
CaCl2, 100 µM L-arginine, and trace amounts
(0.1 µCi) of L-[3H]arginine (Amersham,
Little Chaffont, UK). Samples were incubated for 2 h at 37°C,
and the reaction was stopped by the addition of 1 ml of 50 mM HEPES, pH
5.5, and 5 mM EDTA. Samples were then incubated for 10 min at 4°C
with equilibrated AG 50W-X8 (Na+ form) to bind the
unreacted L-arginine. Citrulline was eluted by
centrifugation, and radioactivity was measured by liquid scintillation counting. In each experiment, a blank value, consisting of the radioactivity measured in samples incubated in the absence of tissue
homogenate, has been determined and subtracted from each experimental
value. For each experimental series, all samples were freshly
homogenized, and measurement of NOS activity was performed the same day
with the same batches of buffers. The NOS activity was measured in
duplicate and expressed as femtomoles L-citrulline per
milligram protein per minute. Time course experiments have shown that
NOS activity was linear for at least 2 h, using 100 µg protein
from either renal cortex or medulla (data not shown). Dose-dependence
experiments have shown that NOS activity measured after 2 h
incubation was linear from 25 to 200 µg protein (data not shown).
Statistics.
Statistical analyses of NOS isoforms, Na-K-ATPase
-subunit, and AQP2
immunoreactivity were done using the Mann-Whitney U-test. Statistical analysis for NOS activity was by unpaired Student's t-test; P < 0.05 was considered
significant. Results are expressed as means ± SE from several
independent animals.
 |
RESULTS |
Effects of water restriction on NOS activity and expression levels
of NOS isoforms.
To increase endogenous AVP circulating levels, rats were water
restricted. As shown in Table 1, urinary
volume was dramatically decreased and urinary osmolality rose close to
5,000 mosmol/kgH2O in water-restricted rats. In addition to
these alterations in water handling, it is noteworthy to mention that
water-restricted rats also retained significant amounts of sodium, as
reflected by the 60% excretion of the amounts of ingested sodium as
shown in Table 2. This sodium retention
is most likely mediated by an adaptive response to hypovolemia
consecutive to severe water restriction (32). Accordingly,
water-restricted rats weighed less than control rats despite similar
amounts of food intake, reflecting the negative water balance. The
renal effect of AVP was indirectly assessed by the increase in
expression of AQP2 in the inner stripe of the outer medulla
from water-restricted rats (Fig.
1A).
View this table:
[in this window]
[in a new window]
|
Table 1.
Physiological parameters of water-restricted, water-loaded,
SR-121463B-treated, and Brattleboro rats compared with their
respective controls
|
|
View this table:
[in this window]
[in a new window]
|
Table 2.
Sodium balance parameters of water-restricted, water-loaded,
SR-121463B-treated, and Brattleboro rats compared with their
respective controls
|
|

View larger version (104K):
[in this window]
[in a new window]
|
Fig. 1.
Aquaporin-2 (AQP2) expression in control and water-restricted
(A), control and water-loaded (B), control and
V2-receptor antagonist SR-121463B-treated (C),
and Long-Evans (Control) and arginine vasopressin (AVP)-infused
Brattleboro rats (D), as well as their respective controls.
After completion of experimental protocols, tissues were lysed, and
equal amounts of protein from the inner stripe of the outer medulla of
control and experimental rats were separated by 10% SDS-PAGE. After
electrotransfer, AQP2 was detected by Western blotting. Representative
Western blots showing results from 3 control and 3 experimental rats
are shown.
|
|
The effect of increased AVP circulating levels on renal NOS activity
and NOS expression was assessed in the cortex, inner stripe of the
outer medulla, and papilla from control and water-restricted rats. As
indicated in Table 3, NOS activity
increased to 219 ± 8% of the control value in the outer medulla
of water-restricted rats but remained unchanged in the cortex and
papilla. Figure 2 shows that eNOS
expression levels increased to 245 ± 14% of the control value
(P < 0.05) in the inner stripe of the outer medulla
but remained unchanged in the cortex and papilla from water-restricted
rats (Fig. 2). On the other hand, nNOS expression rose to 710 ± 34 (P < 0.05) and 159 ± 9% (P < 0.05) of the control value in the outer medulla and papilla of
water-restricted rats, respectively (Fig.
3). It is worth mentioning that nNOS
expression levels in the cortex of both control and water-restricted
rats were below the minimal detection levels of our Western blot assay.
View this table:
[in this window]
[in a new window]
|
Table 3.
Nitric oxide synthase activity measured in cortex, outer medulla, and
papilla of water-restricted, water-loaded, anti-V2-treated,
and Brattleboro rats compared with their respective controls
|
|

View larger version (36K):
[in this window]
[in a new window]
|
Fig. 2.
Effect of water restriction on endothelial nitric oxide
synthase (eNOS) expression levels. Equal amounts of protein from the
cortex, inner stripe of the outer medulla, and papilla of control (C)
and water-restricted rats (WR) were separated by 7% SDS-PAGE. After
electrotransfer, eNOS was detected by Western blotting. A:
Western blot showing results from 5 control and 5 water-restricted
rats. B: densitometric quantification of theWestern blot
shown in A. Values are means ± SE expressed as the
percentage of the optical density value of control animals in each
experiment. * P < 0.05 vs. control.
|
|

View larger version (33K):
[in this window]
[in a new window]
|
Fig. 3.
Effect of water restriction on neuronal NOS (nNOS)
expression levels. Equal amounts of protein from the inner stripe of
the outer medulla and papilla of control and water-restricted rats were
separated by 7% SDS-PAGE. After electrotransfer, nNOS was detected by
Western blotting. A: Western blot showing results from 5 control and 5 water-restricted rats. B: densitometric
quantification of the Western blot shown in A. Values are
means ± SE expressed as the percentage of the optical density
value of control animals in each experiment. * P < 0.05 vs. control.
|
|
Effects of water loading on NOS activity and expression of NOS
isoforms.
A water-loading protocol by ingestion of hydrated agar was applied to
decrease the endogenous circulating levels of AVP. Table 1 shows that
increasing the water intake of rats increased urinary volume fivefold
and decreased urinary osmolality to <400 mosm/kgH2O. Sodium balance and growth were not altered by the water-loading protocol (Tables 1 and 2). Inhibition of AVP secretion was
confirmed by the decrease in AQP2 expression levels in the inner stripe of the outer medulla from water-loaded rats (Fig. 1B).
Water loading did not alter NOS activity in the cortex, inner stripe of
the outer medulla, and papilla (Table 3). Similarly, renal expression
of eNOS was not altered in water-loaded rats (Fig.
4). Figure
5 shows that water loading decreased
nNOS expression to 54 ± 23% of the control value (not
significant) in papilla but did not alter nNOS expression in the outer
medulla (91 ± 37% of the control value).

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 4.
Effect of water loading on eNOS expression levels. Equal
amounts of protein from the cortex, inner stripe of the outer medulla,
and papilla of control and water-restricted rats were separated by 7%
SDS-PAGE. After electrotransfer, eNOS was detected by Western blotting.
A: Western blot showing results from 5 control and 5 water-loaded rats (WL). B: densitometric quantification of
the Western blot shown in A. Values are means ± SE
expressed as the percentage of the optical density value of control
animals in each experiment.
|
|

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of water loading on the expression levels of nNOS.
Equal amounts of protein from the inner stripe of the outer medulla and
papilla of control and water-restricted rats were separated by 7%
SDS-PAGE. After electrotransfer, nNOS was detected by Western blotting.
A: Western blot showing results from 5 control and 5 water-loaded rats. B: densitometric quantification of the
Western blot shown in A. Values are means ± SE
expressed as the percentage of the optical density value of control
animals in each experiment.
|
|
Effects of V2-receptor blockade on NOS activity and
expression of NOS isoforms.
To determine whether the effect of endogenous AVP was mediated through
the V2 receptor, we assessed the effect of
SR-121463B, a specific V2-receptor antagonist
(31). Table 1 shows that SR-121463B increased
urinary volume ninefold and decreased urinary osmolality to 145 mosm/kgH2O. As already demonstrated (31), administration of SR-121463B did not change the sodium balance and growth of the animals (Tables 1 and 2). The inhibition of V2-receptor-mediated effects of AVP was illustrated by the
decrease of AQP2 expression levels in the inner stripe of the outer
medulla from SR-121463B-treated rats (Fig. 1C).
SR-121463B did not alter NOS activity (Table 3) or eNOS expression
(Fig. 6) in the cortex, inner stripe of
the outer medulla, and papilla. In contrast, Fig.
7 shows that SR-121463B decreased nNOS
expression to 19 ± 24 (not significant) and 42 ± 24%
(P < 0.05) of the control value in the outer medulla
and papilla, respectively.

View larger version (41K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of the V2-receptor antagonist
SR-121463B on eNOS expression levels. Equal amounts of protein from the
cortex, inner stripe of the outer medulla, and papilla of control and
water-restricted rats were separated by 7% SDS-PAGE. After
electrotransfer, eNOS was detected by Western blotting. A:
Western blot showing results from 5 control and 5 SR-121463B-treated
rats (SR) is shown. B: densitometric quantification of the
Western blot shown in A. Values are means ± SE
expressed as the percentage of the optical density value of control
animals in each experiment.
|
|

View larger version (34K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of SR-121463B on nNOS expression levels. Equal
amounts of protein from the inner stripe of the outer medulla and
papilla of control and water-restricted rats were separated by 7%
SDS-PAGE. After electrotransfer, nNOS was detected by Western blotting.
A: Western blot showing results from 5 control and 5 SR-121463B-treated rats. B: densitometric quantification of
the Western blot shown in A. Values are means ± SE
expressed as the percentage of the optical density value of control
animals in each experiment. * P < 0.05 vs.
control.
|
|
Comparison of NOS activity and expression of NOS isoforms in
Long-Evans and Brattleboro rats.
To further assess the effect of AVP on renal NOS isoforms, we compared
NOS activity and expression in Brattleboro rats and in control
Long-Evans rats, their normal strain counterpart, and we assessed the
effect of AVP infusion in Brattleboro rats. Table 1 shows that
Brattleboro rats had a higher urinary output (14-fold) and lower
urinary osmolality compared with Long-Evans rats but no difference in
sodium balance and growth (Tables 1 and 2). The absence of endogenous
AVP in Brattleboro rats was illustrated by very low AQP2 expression
levels in the inner stripe of the outer medulla (Fig. 1D).
AVP infusion into Brattleboro rats markedly decreased urinary output
and increased urinary osmolality but did not alter sodium balance and
growth (Tables 1 and 2). The effect of AVP infusion on water excretion
was associated with an increase in expression levels of AQP2 in the
inner stripe of the outer medulla (Fig. 1D).
Compared with control Long-Evans rats, Brattleboro rats exhibit a
significantly lower NOS activity in the outer medulla (46 ± 15%
of the control value, P < 0.05) and papilla (42 ± 15% of the control value, P < 0.05). Outer
medullary and papillary NOS activity of Brattleboro rats returned to
the levels of control rats after 1 wk of AVP infusion. In contrast,
cortical NOS activity was similar in control and Brattleboro rats
(Table 3).
As shown in Fig. 8, eNOS expression was
similar in the cortex, inner stripe of the outer medulla, and papilla
of Brattleboro and control Long-Evans rats. In contrast (Fig.
9), compared with Long-Evans rats,
Brattleboro rats displayed low levels of nNOS expression in the outer
medulla (17 ± 31% of the control value, P < 0.05) and papilla (34 ± 27% of the control value,
P < 0.05). AVP infusion significantly increased
Brattleboro rat NOS expression to 54 ± 24 (P < 0.05) and 99 ± 20% (P < 0.05) of the control
value in the outer medulla and papilla, respectively.

View larger version (56K):
[in this window]
[in a new window]
|
Fig. 8.
Expression levels of eNOS in Long-Evans (Control; C), untreated
(B), and AVP-infused Brattleboro rats (B+A). Equal amounts of protein
from the cortex, inner stripe of the outer medulla, and papilla of
Long-Evans and untreated or AVP-infused Brattleboro rats were separated
by 7% SDS-PAGE. After electrotransfer, eNOS was detected by Western
blotting. A: Western blot showing results from 5 control
Long-Evans rats, 5 untreated, and 5 AVP-infused Brattleboro rats.
B: densitometric quantification of the Western blot shown in
A. Values are means ± SE expressed as the percentage
of the optical density value of control animals in each experiment.
|
|

View larger version (42K):
[in this window]
[in a new window]
|
Fig. 9.
Expression levels of nNOS in Long-Evans and Brattleboro rats. Equal
amounts of protein from the inner stripe of the outer medulla and
papilla of Long-Evans and untreated or AVP-infused Brattleboro rats
were separated by 7% SDS-PAGE. After electrotransfer, nNOS was
detected by Western blotting. A: Western blot showing
results from 5 control Long-Evans rats, 5 untreated Brattleboro, and 5 AVP-infused Brattleboro rats is shown. B: densitometric
quantification of the Western blot shown in A. Values are
means ± SE expressed as the percentage of the optical density
value of control animals in each experiment. * P < 0.05 vs. control; &P < 0.05 vs.
Brattleboro.
|
|
 |
DISCUSSION |
Our study has investigated the relationship between renal
expression of NOS isoforms and AVP. The results demonstrate that AVP
specifically modulates the expression of renal nNOS through V2 receptors but does not influence the expression of renal
eNOS. In addition, our results confirm the renal expression pattern of
eNOS and nNOS with a predominant expression of eNOS throughout the
kidney, whereas nNOS is mainly restricted to the renal medulla and papilla.
The present study shows that nNOS, specifically expressed in the renal
outer medulla and papilla, follows the same pattern of expression as
AQP2, the AVP-regulated water channel, in response to water deprivation
or water loading. Our observations are consistent with previous results
obtained by Shin et al. (32), who showed an increase in
nNOS mRNA levels in the outer medulla and papilla of water-deprived
rats. We are adding more information by demonstrating that protein
expression of nNOS is significantly altered by water deprivation or
loading. Moreover, administration of the specific V2-receptor antagonist SR-121463B (31)
decreased expression of nNOS, strengthening the link with AVP through
its action on the basolateral V2 receptor of principal
cells. Finally, Brattleboro rats, which are genetically AVP deficient,
had very low renal nNOS expression levels that significantly increased
after chronic AVP infusion. Altogether, these observations demonstrate
that in vivo AVP stimulates nNOS protein expression through
V2 receptors. However, our results cannot discriminate
between the direct or indirect effects of AVP. This is the limitation
of in vivo studies, as already mentioned with the results suggesting a
long-term control of AQP2 (4a, 5, 34) and epithelial sodium channel
subunits (7) expression by AVP. Nevertheless, one could
conceive that AVP increases expression of nNOS through paracrine and/or
autocrine factors or by decreasing the action of an endogenous protein
inhibitor of nNOS (12). Importantly, the variations of
nNOS protein expression are independent of alterations in food intake,
growth, or sodium balance because, with the exception of
water-restricted rats, these parameters were similar in both control
and experimental groups of rats. In contrast, eNOS expression does not
appear to be under AVP control. Indeed, neither decreasing endogenous
circulating levels of AVP by water loading nor treatment with a
V2-receptor antagonist altered renal eNOS expression.
Furthermore, Brattleboro rats exhibited similar eNOS expression levels
to control rats despite the complete absence of circulating AVP, and
AVP infusion did not significantly alter eNOS expression levels. Thus
the increase in eNOS expression restricted to the outer medulla of
water-restricted rats, confirming previous observations
(32), was most likely secondary to dehydration and
subsequent hypovolemia, which was illustrated by oliguria, weight loss,
and sodium retention in these rats. This interpretation is consistent
with increased plasma and kidney angiotensin II levels in
water-deprived rats (32).
In addition to hormones and neuromediators (9), renal
sodium and water handling is also controlled by local factors such as
NO, which plays an increasingly recognized role (18).
Active sodium transport has been shown to be regulated through an
increase in NO and cGMP (22, 35). Our study suggests that
nNOS may be the major NOS isoform involved in the control of sodium and water handling by NO. Quantitatively, however, eNOS seems to be responsible for most renal NO production (13), but
principal cells of the collecting duct system and inner medullary
collecting duct cells express nNOS (18, 36). A strong body
of evidence suggests that nNOS plays an important role in this renal
segment in which fine regulation of sodium and water balance occurs.
Indeed, high-salt intake increases renal nNOS expression
(29). In addition, NO exerts AVP-like effects in principal
cells of the collecting duct system through indirect stimulation of the
epithelial sodium channel by apical membrane hyperpolarization
(22) and direct stimulation of phosphorylation and apical
membrane insertion of AQP2 through activation of protein kinase G by
cGMP (2). Therefore, the increase in expression of nNOS,
leading to increased NO production, may participate in the effects of
AVP on water (16) and sodium transport (27)
in the collecting duct system. Besides direct effects on transporters,
NO can also modulate renal water and sodium handling through its
hemodynamic effects. Indeed, NO regulates medullary blood flow and
administration of
NG-nitro-L-arginine methyl ester
into the medullary interstitial space, decreasing sodium and water
excretion without modifying glomerular filtration rate or blood
pressure (23).
Our results confirm the distribution pattern of renal eNOS and nNOS
protein expression (1, 18). In agreement with most authors, eNOS protein expression is detected throughout the kidney, and
nNOS protein expression is predominantly located in the medulla and
papilla. In the renal cortex, nNOS expression is restricted to macula
densa cells (1) and cortical collecting duct principal cells (36) and is below the detection threshold of Western
blot analysis in crude cortex homogenates. With the exception of
Brattleboro rats, NOS activity measured at Vmax
was parallel to eNOS expression levels. The absence of AVP-dependent
modulation of NOS activity as well as the parallel increase in NOS
activity and eNOS protein expression in the outer medulla of
water-restricted rats suggests that eNOS generated the majority of the
renal NO, thus occulting changes in nNOS-generated NO production that
are quantitatively less important in Wistar rats. However, compared
with Long-Evans rats, NOS activity is significantly lower in
Brattleboro rats and varies in parallel with nNOS expression levels.
This finding may suggest that the contribution of nNOS to overall NOS
activity is larger in this specific rat strain. Finally, we did not
find any protein expression of iNOS isoform, which does not exclude that a low level of iNOS protein was present, because iNOS mRNA has
been demonstrated in the cortex and inner and outer medulla (18,
20). However, such a low level may not have a role in this setting.
In conclusion, our results indicate that AVP specifically increases the
expression of nNOS in the renal medulla and papilla. This effect may
participate in long-term control of water and sodium reabsorption by
AVP in the collecting duct system.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Serradeil-Le Gal for the gift of SR-121463B.
 |
FOOTNOTES |
This work was supported in part by Swiss National Foundation for
Science Grant 31-56504 and the Sir Jules Thorn Charitable Overseas
Trust Shaan (P.-Y. Martin).
Address for reprint requests and other correspondence:
P.-Y. Martin, Div. of Nephrology, Hôpital Cantonal
Universitaire, 24 rue Micheli du Crest, 1211 Geneva 14, Switzerland
(E-mail: Pierre-Yves.Martin{at}hcuge.ch).
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.
March 19, 2002;10.1152/ajprenal.00309.2001
Received 3 October 2001; accepted in final form 11 March 2002.
 |
REFERENCES |
1.
Bachmann, S,
Bosse HM,
and
Mundel P.
Topography of nitric oxyde synthesis by localizing constitutive NO synthases in mammalian kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
268:
F885-F898,
1995[Abstract/Free Full Text].
2.
Bouley, R,
Breton S,
Sun TX,
McLaughlin M,
Nsumu NN,
Lin HY,
Ausiello DA,
and
Brown D.
Nitric oxide and atrial natriuretic factor stimulate cGMP-dependent membrane insertion of aquaporin 2 in renal epithelial cells.
J Clin Invest
106:
1115-1126,
2000[Abstract/Free Full Text].
3.
Cassola, AC,
Giebisch G,
and
Wang W.
Vasopressin increases density of apical low-conductance K channels in rat CCD.
Am J Physiol Renal Fluid Electrolyte Physiol
264:
F502-F509,
1993[Abstract/Free Full Text].
4.
Chou, CL,
Knepper MA,
van Hoek AN,
Brown D,
Yang B,
Ma T,
and
Verkman AS.
Reduced water permeability and altered ultrastructure in thin descending limb of Henle in aquaporin-1 null mice.
J Clin Invest
103:
491-496,
1999[Abstract/Free Full Text].
4a.
Christensen, BM,
Marples D,
Jensen UB,
Frøkiaer J,
Sheikh-Hamad D,
Knepper MA,
and
Nielsen S.
Acute effects of vasopressin V2-receptor antagonist on kidney AQP2 expression and subcellular distribution.
Am J Physiol Renal Physiol
275:
F285-F297,
1998[Abstract/Free Full Text].
5.
DiGiovanni, SR,
Nielsen S,
Christensen EI,
and
Knepper MA.
Regulation of collecting duct water channel expression by vasopressin in Brattleboro rat.
Proc Natl Acad Sci USA
91:
8984-8988,
1994[Abstract].
6.
Djelidi, S,
Fay M,
Cluzeaud F,
Escoubet B,
Eugene E,
Capurro C,
Bonvalet JP,
Farman N,
and
Blot-Chabaud M.
Transcriptional regulation of sodium transport by vasopressin in renal cells.
J Biol Chem
272:
32919-32924,
1997[Abstract/Free Full Text].
7.
Ecelbarger, CA,
Kim GH,
Terris J,
Masilamani S,
Mitchell C,
Reyes I,
Verbalis JG,
and
Knepper MA.
Vasopressin-mediated regulation of epithelial sodium channel abundance in rat kidney.
Am J Physiol Renal Physiol
279:
F46-F53,
2000[Abstract/Free Full Text].
8.
Ecelbarger, CA,
Terris J,
Frindt G,
Echevarria M,
Marples D,
Nielsen S,
and
Knepper MA.
Aquaporin-3 water channel localization and regulation in rat kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
269:
F663-F672,
1995[Abstract/Free Full Text].
9.
Féraille, E,
and
Doucet A.
Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the kidney: hormonal control.
Physiol Rev
81:
345-418,
2001[Abstract/Free Full Text].
10.
Grantham, JJ,
and
Burg MB.
Effect of vasopressin and cyclic AMP on permeability of isolated collecting tubules.
Am J Physiol
211:
255-259,
1966[Free Full Text].
11.
Imbert-Teboul, M,
Chabardes D,
Montegut M,
Clique A,
and
Morel F.
Vasopressin-dependent adenylate cyclase activities in the rat kidney medulla: evidence for two separate sites of action.
Endocrinology
102:
1254-1261,
1978[Abstract].
12.
Jaffrey, SR,
and
Snyder SH.
PIN: an associated protein inhibitor of neuronal nitric oxide synthase.
Science
274:
774-777,
1996[Abstract/Free Full Text].
13.
Kakoki, M,
Zou AP,
and
Mattson DL.
The influence of nitric oxide synthase 1 on blood flow and interstitial nitric oxide in the kidney.
Am J Physiol Regul Integr Comp Physiol
281:
R91-R97,
2001[Abstract/Free Full Text].
14.
Kishore, BK,
Mandon B,
Oza NB,
DiGiovanni SR,
Coleman RA,
Ostrowski NL,
Wade JB,
and
Knepper MA.
Rat renal arcades segment expresses vasopressin-regulated water channel and vasopressin V2 receptor.
J Clin Invest
97:
2763-2771,
1996[Abstract/Free Full Text].
15.
Klussman, E,
Maric K,
Wiesner B,
Beyermann M,
and
Rosenthal W.
Protein kinase A anchoring proteins are required for vasopressin-mediated translocation of aquaporin-2 into cell membrane of renal principal cells.
J Biol Chem
274:
4934-4938,
1999[Abstract/Free Full Text].
16.
Knepper, MA.
Molecular physiology of urinary concentrating mechanism: regulation of aquaporin water channels by vasopressin.
Am J Physiol Renal Physiol
272:
F1-F12,
1997[Free Full Text].
17.
Knepper, MA,
and
Burg M.
Organization of nephron function.
Am J Physiol Renal Fluid Electrolyte Physiol
244:
F579-F589,
1983[Abstract/Free Full Text].
18.
Kone, BC,
and
Baylis C.
Biosynthesis and homeostatic roles of nitric oxide in the normal kidney.
Am J Physiol Renal Physiol
272:
F561-F578,
1997[Abstract/Free Full Text].
19.
Marples, D,
Knepper MA,
Christensen EI,
and
Nielsen S.
Redistribution of aquaporin-2 water channels induced by vasopressin in rat kidney medullary collecting duct.
Am J Physiol Cell Physiol
269:
C655-C664,
1995[Abstract].
20.
Martin, PY,
and
Féraille E.
Nitric oxide in renal disease.
Adv Nephrol Necker Hosp
29:
93-113,
1999[Medline].
21.
Maunsbach, AB,
Marples D,
Chin E,
Ning G,
Bondy C,
Agree P,
and
Nielsen S.
Aquaporin-1 water channel expression in human kidney.
J Am Soc Nephrol
8:
1-14,
1997[Abstract].
22.
Ming, L,
Giebisch G,
and
Wang W.
Nitric oxide-induced hyperpolarization stimulates low-conductance Na+ channel of rat CCD.
Am J Physiol Renal Physiol
272:
F498-F504,
1997[Abstract/Free Full Text].
23.
Miyata, N,
Zou AP,
Mattson DL,
and
Cowley AW, Jr.
Renal medullary interstitial infusion of L-arginine prevents hypertension in Dahl salt-sensitive rats.
Am J Physiol Regul Integr Comp Physiol
275:
R1667-R1673,
1998[Abstract/Free Full Text].
25.
Nonoguchi, H,
Owada A,
Kobayashi N,
Takayama M,
Terada Y,
Koike J,
Ujiie K,
Marumo F,
Sakai T,
and
Tomita K.
Immunohistochemical localization of V2 receptor along the nephron and functional role of luminal V2 receptor in terminal inner medullary collecting ducts.
J Clin Invest
96:
1768-1778,
1995[ISI][Medline].
26.
Raij, L,
and
Baylis C.
Glomerular actions of nitric oxide.
Kidney Int
48:
20-32,
1995[ISI][Medline].
27.
Reif, MC,
Troutman SL,
and
Schafer JA.
Sodium transport by rat cortical collecting tubule.
J Clin Invest
77:
1291-1298,
1986[ISI][Medline].
28.
Robert-Nicoud, M,
Flahaut M,
Elalouf JM,
Nicod M,
Salinas M,
Bens M,
Doucet A,
Wincker P,
Artiguenave F,
Horisberger JD,
Vandewalle A,
Rossier BC,
and
Firsov D.
Transcriptome of a mouse kidney cortical collecting duct cell line: effects of aldosterone and vasopressin.
Proc Natl Acad Sci USA
98:
2712-2716,
2001[Abstract/Free Full Text].
29.
Roczniak, A,
Zimplemann J,
and
Burns KD.
Effect of dietary salt on neuronal nitric oxide synthase in the inner medullary collecting duct.
Am J Physiol Renal Physiol
275:
F46-F54,
1998[Abstract/Free Full Text].
30.
Schnermann, J,
Chou CL,
Ma T,
Traynor T,
Knepper MA,
and
Verkman AS.
Defective proximal tubular fluid reabsorption in transgenic aquaporin-1 null mice.
Proc Natl Acad Sci USA
95:
9660-9664,
1998[Abstract/Free Full Text].
31.
Serradeil-Le Gal, C,
Lacour C,
Valette G,
Garcia G,
Foulon L,
Galindo G,
Bankir L,
Pouzet B,
Guillon G,
Barberis C,
Chicot D,
Jard S,
Vilain P,
Garcia C,
Marty E,
Raufaste D,
Brossard G,
Nisato D,
Maffrand J-P,
and
Le Fur G.
Characterization of SR 121463A, a highly potent and selective orally active vasopressin V2 receptor antagonist.
J Clin Invest
98:
2729-2738,
1996[Abstract/Free Full Text].
32.
Shin, SJ,
Lai FJ,
Wen JD,
Lin SR,
Hsieh MC,
Hsiao PJ,
and
Tsai JH.
Increased nitric oxide synthase mRNA expression in the renal medulla of water-deprived rats.
Kidney Int
56:
2191-2202,
1999[ISI][Medline].
33.
Terris, J,
Ecelbarger CA,
Marples D,
Knepper MA,
and
Nielsen S.
Distribution of aquaporin-4 water channel expression within rat kidney.
Am J Physiol Renal Fluid Electrolyte Physiol
269:
F775-F785,
1995[Abstract/Free Full Text].
34.
Terris, J,
Ecelbarger CA,
Nielsen S,
and
Knepper MA.
Long-term regulation of four renal aquaporins.
Am J Physiol Renal Fluid Electrolyte Physiol
271:
F414-F422,
1996[Abstract/Free Full Text].
35.
Wang, T.
Nitric oxide regulates HCO
and Na+ transport by a cGMP-dependent mechanism in the kidney proximal tubule.
Am J Physiol Renal Physiol
272:
F242-F248,
1997[Abstract/Free Full Text].
36.
Wang, X,
Lu M,
Gao Y,
Papapetropoulos A,
Sessa WC,
and
Wang W.
Neuronal nitric oxide synthase is expressed in principal cell of the collecting duct.
Am J Physiol Renal Physiol
275:
F395-F399,
1998[Abstract/Free Full Text].
37.
Wu, F,
Park F,
Cowley AW, Jr,
and
Mattson DL.
Quantification of nitric oxide synthase activity in microdissected segments of rat kidney.
Am J Physiol Renal Physiol
276:
F874-F881,
1999[Abstract/Free Full Text].
38.
Xu, DL,
Martin PY,
Ohara M,
St. John J,
Pattison T,
Meng X,
Morris K,
Kim JK,
and
Schrier RW.
Upregulation of aquaporin-2 water channel expression in chronic heart failure rat.
J Clin Invest
99:
1500-1505,
1997[Abstract/Free Full Text].
Am J Physiol Renal Fluid Electrolyte Physiol 283(3):F559-F568
0363-6127/02 $5.00
Copyright © 2002 the American Physiological Society