Vol. 275, Issue 1, F46-F54, July 1998
Effect of dietary salt on neuronal nitric oxide synthase in
the inner medullary collecting duct
Agnes
Roczniak1,
Joseph
Zimpelmann2, and
Kevin D.
Burns1,2
Departments of 2 Medicine and
1 Cellular and Molecular Medicine,
University of Ottawa and Ottawa General Hospital, Ottawa, Ontario,
Canada K1H 8L6
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ABSTRACT |
Nitric oxide (NO)
derived from neuronal NO synthase (nNOS) in the kidney inner medulla
has been implicated in the regulation of arterial blood pressure. The
purpose of the present study was to determine the effect of high
dietary NaCl on the expression of nNOS in the rat inner medullary
collecting duct (IMCD). After 3 days or 3 wk of high (4.0%)-NaCl diet
in rats, urinary
NO
2/NO
3 excretion significantly increased. In freshly microdissected IMCD, nNOS
was readily detected by immunofluorescence with polyclonal antibody, an
effect that was completely blocked by neutralization of antibody with
immunizing antigen. In rats fed a 4.0% NaCl diet for 3 days, IMCD nNOS
mRNA, detected by RT-PCR, did not change from control values (0.3%
NaCl, 19.84 ± 1.57 × 103, vs. 4.0% NaCl, 20.44 ± 3.14 × 103 cpm;
P = not significant,
n = 3). By Western blotting however, nNOS protein expression significantly increased (0.3% NaCl, 0.51 ± 0.12, vs. 4.0% NaCl, 0.92 ± 0.14 arbitrary units;
P < 0.05, n = 5). After 3 wk of 4.0% dietary
NaCl, expression of nNOS mRNA and protein in IMCD did not differ
significantly from control values. In contrast to these data, renal
cortical expression of nNOS mRNA and protein was significantly
decreased after 4.0% NaCl diet for 3 days. High dietary NaCl had no
significant effect on expression of mRNA for inducible NO synthase
(iNOS) in IMCD after either 3 days or 3 wk. In summary, our data
indicate that nNOS mRNA and protein are expressed in IMCD and that high
dietary NaCl differentially regulates nNOS expression in IMCD and
cortex. The early increase in nNOS protein in IMCD may contribute to
enhanced local production of NO and thereby represent an adaptive
response to salt intake.
nitric oxide; messenger ribonucleic acid expression; nitrates; inducible nitric oxide synthase
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INTRODUCTION |
NITRIC OXIDE (NO) regulates glomerular hemodynamics and
renal blood flow and induces natriuresis (11). Within the kidney, the
capacity to synthesize NO has been demonstrated in both tubular epithelium and vascular endothelium. Recently, the renal medulla has
been shown to produce the highest levels of NO in the kidney (26). In
this regard, there are at least three isoforms of nitric oxide synthase
(NOS) that could contribute to medullary NO production: neuronal NOS
(nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS).
In the medulla, abundant nNOS mRNA has been demonstrated by reverse
transcription-polymerase chain reaction (RT-PCR) in inner medullary
collecting ducts (IMCD), with lesser expression in the vasa recta and
the inner medullary thin limb (22). Bachmann et al. (3), however,
demonstrated immunoreactivity for nNOS in the macula densa and afferent
arteriole but did not detect nNOS in IMCD. With regard to iNOS mRNA,
studies have demonstrated its presence in IMCD and inner medullary
interstitial cells (1). Histochemical analysis of renal tissue,
however, has not detected basal iNOS protein expression in the IMCD
(2). In contrast, eNOS mRNA and protein are distributed in endothelial
cells throughout the renal vasculature, although expression of this
isoform in renal tubules is not consistently detected (20, 25).
In rats, chronic infusion of an inhibitor of NOS directly into the
renal medullary interstitium results in a selective decrease in
medullary blood flow, associated with salt and water retention and
development of hypertension (14). Inhibition of nNOS expression by
intramedullary antisense oligonucleotide administration increases blood
pressure in rats maintained on a high-salt diet, suggesting that the
ability of the kidneys to excrete sodium may be impaired in the absence
of medullary nNOS activity (12). Furthermore, during high dietary salt
intake, the serum concentration and urinary excretion of the NO
decomposition products NO
2 and
NO
3 increase (18), suggesting that
enhanced NO production represents an adaptive mechanism that induces
vasodilatation and natriuresis, thereby contributing to the maintenance
of normal blood pressure.
The potential importance of nNOS-derived NO in the inner medulla led us
to examine whether this isoform is expressed in tubular cells of the
rat IMCD and to test the hypothesis that high dietary NaCl increases
nNOS expression in this segment. Our results demonstrate that nNOS
protein is readily detected in IMCD by immunofluorescence and that high
dietary NaCl differentially regulates nNOS mRNA and protein expression
in the IMCD and renal cortex. Whereas high dietary NaCl inhibits nNOS
mRNA and protein expression in the cortex, it increases nNOS protein
expression without affecting mRNA levels in the IMCD. In contrast, high
dietary NaCl has no effect on iNOS mRNA expression in the IMCD.
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MATERIALS AND METHODS |
Animals. Male Sprague-Dawley rats
(200-250 g) were fed a standard (0.3% NaCl, control) or high-salt
(4.0% NaCl) diet (Harlan Teklad, Madison, WI) for either 3 days or 3 wk, with unlimited access to water. The diets were equivalent in
contents of all other electrolytes. For 24 h prior to death, the rats
were in metabolic cages for urine collection. After death, the kidneys were immediately removed for dissection of cortices and for isolation of IMCD. Kidneys from two rats were pooled for each IMCD preparation.
IMCD preparation. Rat IMCDs were
isolated using a modification of the method of Grenier and Smith (8),
essentially as described (10). Briefly, renal papillary tissue was
rapidly minced and suspended in Krebs buffer containing (in mM) 118 NaCl, 14 glucose, 25 NaHCO3, 4.7 KCl, 2.5 CaCl2, 1.8 MgSO4, and 1.8 KH2PO4;
0.1% collagenase A (type I; Boehringer-Mannheim, Laval, Quebec,
Canada); and 0.01% deoxyribonuclease (Boehringer-Mannheim). The minced tissue was incubated for 120 min at 37°C in 5%
CO2-95% air with gentle
agitation. After collagenase digestion, the tubule suspension was
pelleted by centrifugation at 100 g
for 30 s at room temperature and washed three times with Krebs buffer.
The pellet was then reconstituted in a hypotonic solution consisting of
Krebs buffer and sterile water (1:2 respectively, vol/vol), gently
mixed for 4 min, and centrifuged at 100 g for 30 s. This procedure has been shown to disrupt all cells, except those of the IMCD (8). The tubules
were washed three times with Krebs buffer and then used immediately for
either RNA or protein isolation. Cell viability was assessed by trypan
blue exclusion, which demonstrated that at least 95% of IMCD cells
were viable.
Reverse transcription-polymerase chain reaction
(RT-PCR). Total RNA was isolated from cortex and
suspensions of IMCD using a commercially available kit (RNeasy; Qiagen,
Chatsworth, CA). Prior to RT-PCR, residual genomic DNA was digested by
incubating RNA with amplification grade DNase I (Life Technologies,
Burlington, ON, Canada) for 15 min at room temperature. DNase I was
inactivated by adding ethylenediaminetetraacetic acid (final
concentration, 1.6 mM) followed by incubation at 65°C for 15 min.
RNA quality was determined by running samples on 1%
agarose-formaldehyde gels stained with ethidium bromide, and RNA
concentration and purity were determined by optical density measurement
at 260 and 280 nm. RNA samples were uniformly of high quality by these
standards.
Serial dilutions of total RNA (6, 30, 150, and 750 ng) were prepared in
RNase-free water and reverse transcribed in a buffer containing 5 mM
MgCl2, 1× PCR buffer (50 mM
KCl, 10 mM Tris · HCl, pH 8.3), 1 mM each of
deoxynucleoside triphosphates, 1 µCi [
-32P]dCTP (sp act,
3,000 Ci/mmol; Amersham, Mississauga, ON), 20 U RNase inhibitor, 50 U
Moloney murine leukemia virus reverse transcriptase, and 2.5 µM
random hexamers (GeneAmp RNA PCR kit; Perkin-Elmer, Norwalk, CT) in a
total volume of 20 µl. After reverse transcription, reactions were
brought up to 100 µl with 1× PCR buffer supplemented with
(final concentrations) 2 mM MgCl2,
2.5 U AmpliTaq DNA polymerase
(Perkin-Elmer), and 1 µM of either nNOS- or iNOS-specific
oligonucleotide primers. After initial denaturation at 94°C for 3 min, 33 cycles of amplification [94°C for 30 s, 68°C
(nNOS) or 60°C (iNOS) for 30 s, and 72°C for 30 s] were
performed, followed by final extension at 72°C for 10 min, in a
Perkin-Elmer GeneAmp PCR system 2400 apparatus. The PCR products were
separated on ethidium bromide-stained 2% agarose gels, the gel bands
were excised, and the incorporated radioactivity was determined by liquid scintillation counting.
To determine relative differences in NOS mRNA abundance in rats on
0.3% and 4.0% NaCl diets, we used the titration analysis method as
described (17). This is a noncompetitive, semiquantitative method that
utilizes the linear relationship between the logarithm of the initial
amount of target mRNA and the logarithm of the amount of amplification
product generated, providing that the PCR reactions have not reached
plateau phase. To ensure that all data were collected before PCR
reactions reached this plateau, we measured
32P incorporation into PCR
products as a function of cycle number for both nNOS and iNOS mRNA
amplifications. In both cases, PCR product amplification was
exponential for at least 40 cycles. Accordingly, we used 33 cycles of
amplification for all further experiments. To minimize tube-to-tube
variation in PCR efficiency, we used a master mix of reaction
components in all experiments. Under these conditions, the logarithm of
incorporated radioactivity varied linearly with the logarithm of
initial RNA amount (not shown). To control for variations in RNA
isolation and for the efficiency of the reverse transcription reaction,
-actin mRNA was amplified at the time of nNOS and iNOS mRNA
amplifications. Total RNA (150 ng) was reverse transcribed and
amplified as described above, in the presence of specific primers for
-actin (20 nM). As a control for any possible genomic DNA
contamination, a reaction lacking the reverse transcriptase enzyme was
included in all experiments.
The primer sequences for nNOS, iNOS, and
-actin products are
reported in Table 1. The primers for nNOS
and iNOS were chosen from the rat brain (5) and murine macrophage (28)
cDNA sequences, respectively. The specificity of the primers was
verified through GenBank. The
-actin primers were selected according
to the human cDNA sequence (9).
The identity of the nNOS PCR product was confirmed by restriction
enzyme digestion with either Bgl II or
Sca I, which generated the expected
products (210 and 389 bp and 101 and 498 bp, respectively). To confirm
the fidelity of iNOS RT-PCR, the rat IMCD iNOS PCR product was
subcloned, and DNA derived from a single bacterial colony was partially
sequenced (92 bp, corresponding to bp 1194-1286; Ref. 28), using
the Taq Cyclist DNA sequencing kit
(Stratagene, La Jolla, CA). This product demonstrated 98% nucleotide
identity to the murine macrophage iNOS cDNA (28) and 100% nucleotide identity to rat vascular smooth muscle iNOS cDNA (7) in the sequenced
region.
Western blotting. IMCDs from 0.3% and
4.0% NaCl-fed rats were homogenized with a cell disrupter in boiling
lysis buffer [1% sodium dodecyl sulfate (SDS), 10 mM
Tris · HCl, pH 7.4]. The lysate was then boiled
for 10 min, followed by centrifugation at 12,000 g for 2 min to remove insoluble
debris. Protein concentrations in the supernatant were determined by
the Bradford method (Bio-Rad, Montreal, PQ), using bovine serum albumin
(BSA; Sigma, St.Louis, MO) as standard. Tubule lysates (20, 40, or 60 µg) were separated on 7.5% SDS-polyacrylamide gels and transferred
onto nitrocellulose membranes (Bio-Rad). The membranes were blocked
with 10% skimmed milk in Tris-buffered saline (pH 7.6) containing
0.1% Tween 20 (TBS-T) and 0.01% sodium azide for 3 h at room
temperature. The membranes were then incubated for 18 h at 4°C with
1:1,000 dilution of mouse monoclonal antibody to nNOS or 1:400 dilution
of mouse monoclonal antibody to iNOS (Transduction Laboratories,
Lexington, KY). The mouse monoclonal antibody to nNOS was raised
against a human peptide fragment of nNOS and reacts against both human and rat nNOS at 160 kDa. By Western blot analysis, we determined that
this antibody did not cross-react with endothelial cell (eNOS) protein
lysates (Transduction Laboratories) but did detect a 130-kDa band,
clearly discernible from nNOS, from cytokine-stimulated macrophages
(iNOS), as reported by the manufacturer. In some experiments, two
separate rabbit polyclonal antibodies to iNOS were used (Transduction Laboratories; Santa Cruz Biotechnology, Santa Cruz, CA). The membranes were then incubated with 1:2,000 dilution of either anti-mouse or
anti-rabbit secondary antibodies conjugated to horseradish peroxidase
(Amersham). Primary antibodies were diluted in TBS-T supplemented with
5% skimmed milk and 0.01% sodium azide, whereas the secondary
antibodies were diluted in TBS-T supplemented with 2% milk. Proteins
were detected by enhanced chemiluminescence (ECL, Amersham) on
Hyperfilm (Amersham), according to the manufacturer's instructions.
Prestained standards were used as molecular weight markers (Bio-Rad),
and rat pituitary and macrophage lysates (4 µg, Transduction
Laboratories) were used as positive controls for nNOS and iNOS,
respectively. To control for protein loading, all membranes were
stripped and probed with a monoclonal anti-
-actin antibody (mouse
ascites fluid, Sigma). Signals on Western blots were quantified by
densitometry and corrected for the
-actin signal, using an image
analysis software program (NIH Image 1.47).
Immunofluorescence studies. IMCD
segments were microdissected from rat kidney medulla, as described
(16), dried onto microscope slides for 1 h at room temperature, and
then fixed in a 1:1 (vol/vol) acetone-methanol mixture for 15 min at
room temperature. After fixation, the tubules were permeabilized with
0.3% Triton X-100 in phosphate-buffered saline (PBS, pH 7.4) for 30 min at room temperature. Slides were then incubated in blocking buffer
consisting of PBS supplemented with 1% BSA for 30 min. A 1:200
dilution of polyclonal nNOS antibody (Santa Cruz Biotechnology) was
added to the slides and incubated for 18 h at 4°C. In separate
experiments, this antibody did not cross-react with protein lysates
(Transduction Laboratories) from cytokine-stimulated macrophages (iNOS)
or endothelial cells (eNOS) on Western blots (not shown), confirming
the manufacturer's description regarding its specificity. In contrast,
the antibody detected the expected nNOS protein band of 160 kDa in the
pituitary and IMCD lysates. IMCDs incubated in the presence of primary
antibody that had been previously neutralized with the immunizing
antigen were used as negative controls (Santa Cruz Biotechnology). The slides were then washed three times for 5 min each in PBS and incubated
with donkey anti-rabbit secondary antibody (1:20 dilution, Amersham)
conjugated to fluorescein isothiocyanate (FITC) for 60 min at 37°C.
All antibodies were diluted in PBS supplemented with 0.3% Triton
X-100. After three washes with PBS, the slides were covered with an
anti-fade compound (PBS containing 0.1 mM phenylaminediamine and 10%
glycerol) and viewed with a Zeiss Axioplan fluorescent microscope.
Photographs were taken with Kodak Tri-X black and white film.
Urinary nitrate/nitrite content.
Urinary nitrate/nitrite content was determined by the nitrate
reductase/Griess reaction assay as described (18). First, nitrate
present in the urine samples was reduced to nitrite in the presence of
nicotinamide adenine dinucleotide phosphate (150 µg/ml), flavin
adenine dinucleotide (3.0 µg/ml), and 0.4 U nitrate reductase
(Boehringer-Mannheim) for 30 min at room temperature. Nitrite was then
measured by combining reduced urine aliquots with a 1:1 mixture
(vol/vol) of 1% sulfanilamide (in 30% acetic acid) and 0.1%
N-(1-naphthyl)ethylenediamine
dihydrochloride (in 60% acetic acid). Absorbances were read on an
ELISA reader at 550 nm. Nitrate and nitrite content was calculated
using nitrate and nitrite standard curves.
Urinary sodium excretion. Urinary
sodium concentration was determined by flame photometry (model IL-943;
Instrumentation Laboratory, Milan, Italy), kindly performed by the
laboratory of Dr. D. Z. Levine (University of Ottawa).
Statistics. Results are presented as
means ± SE. Data were analyzed by the unpaired Student
t-test. A value of
P < 0.05 was considered significant.
 |
RESULTS |
Effect of high dietary NaCl on nNOS mRNA
and protein expression in IMCD and kidney cortex.
Relatively high levels of nNOS mRNA are found in the IMCD (22), yet
histochemical methods have been unable to detect nNOS protein in this
segment (3). As shown in Fig.
1A,
using a polyclonal antibody to nNOS, cells within freshly
microdissected IMCDs stained strongly for nNOS, with a cytoplasmic
pattern. The specificity of the response was verified by staining IMCD
with nNOS antibody, which had been previously neutralized with
immunizing antigen, which resulted in complete loss of
immunofluorescence (Fig. 1B). A
monoclonal nNOS antibody (Transduction Laboratories) also strongly
stained microdissected IMCD (not shown).

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Fig. 1.
Immunolocalization of neuronal nitric oxide synthase (nNOS) in inner
medullary collecting duct (IMCD). Shown are fluorescence micrographs of
microdissected IMCD segments labeled with a polyclonal nNOS antibody
(A) or with nNOS polyclonal antibody
neutralized by preincubation with immunizing antigen
(B). Similar results were obtained
in 2 additional experiments.
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The effect of high dietary NaCl on the expression of nNOS mRNA and
protein in IMCD was determined, using RT-PCR and Western blotting,
respectively. In rats fed a 4.0% NaCl diet for 3 days or 3 wk, urinary
sodium excretion significantly increased (Table 2, P < 0.001 vs. 0.3% NaCl diet). This was associated with significant increases in urinary
NO
2/NO
3 excretion (Fig. 2: 3 days of 0.3% NaCl,
2.82 ± 0.31, vs. 4.0% NaCl, 3.79 ± 0.43 µmol · 100 g body
wt
1 · 24 h
1,
P < 0.05, n = 12; 3 wk of 0.3% NaCl, 1.21 ± 0.26, vs. 4.0% NaCl, 1.92 ± 0.08 µmol · 100 g body
wt
1 · 24 h
1,
P < 0.01, n = 7). Three days of 4.0%
NaCl diet had no effect on nNOS mRNA expression in IMCD (Fig.
3) [0.3% NaCl, 19.84 ± 1.57 × 103, vs. 4.0% NaCl, 20.44 ± 3.14 × 103 cpm,
P = not significant (NS),
n = 3] but significantly
increased nNOS protein expression in IMCD (Fig.
4) (0.3% NaCl, 0.51 ± 0.12, vs. 4.0%
NaCl, 0.92 ± 0.14 arbitrary units,
P < 0.05, n = 5).

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Fig. 2.
Effect of high-NaCl diet on urinary excretion of
NO 2 + NO 3.
A: 24-h urinary content of
NO 2 + NO 3 after 3 days of 4.0% NaCl intake.
B: 24-h urinary content of
NO 2 + NO 3 after 3 wk of 4.0% NaCl intake.
Results are means ± SE. A:
* P < 0.05, n = 12. B:
* P < 0.01, n = 7.
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Fig. 3.
Effect of 3 days of 4.0% NaCl diet on expression of nNOS mRNA in IMCD.
A: representative ethidium
bromide-stained agarose gel of IMCD nNOS RT-PCR products (599 bp) from
0.3% (lanes 1-4) and 4.0%
(lanes 5-8) NaCl-fed rats.
Dilutions of total RNA were (from
left) 750, 150, 30, and 6 ng.
-Actin PCR products (350 bp) from IMCD of 0.3% and 4.0% NaCl-fed
rats are shown in lanes 9 and
10, respectively.
B: radioactivity incorporated into
nNOS mRNA amplification products. Results are means ± SE for 30 ng
of initial total RNA (n = 3).
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Fig. 4.
High-NaCl diet increases nNOS protein expression in IMCD at 3 days.
A: representative Western blot of nNOS
protein expression in IMCD of rats fed a 4.0% NaCl diet for 3 days.
Lanes 1 and
3: proteins from 0.3% NaCl-loaded
rats, 40 and 20 µg of total protein, respectively.
Lanes 2 and
4: proteins from 4.0% NaCl-loaded
rats, 40 and 20 µg of total protein, respectively. Corresponding
bands for -actin are depicted, demonstrating equal loading.
B: densitometric quantification of
Western blot signals for nNOS protein in IMCD. Results are means ± SE (* P < 0.05, n = 5).
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After 3 wk of 4.0% NaCl intake, both nNOS mRNA (Fig.
5) (0.3% NaCl, 11.02 ± 1.05 × 103, vs. 4.0% NaCl, 13.71 ± 3.02 × 103 cpm,
P = NS,
n = 3) and nNOS protein (Fig.
6: 0.3% NaCl, 1.03 ± 0.16, vs. 4.0%
NaCl, 1.13 ± 0.12 arbitrary units;
P = NS,
n = 3) in the IMCD did not differ from
control values, indicating that the salt-induced nNOS protein
upregulation is transient.

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Fig. 5.
Effect of 3-wk 4.0% NaCl diet on expression of nNOS mRNA in IMCD.
A: representative ethidium
bromide-stained agarose gel of IMCD nNOS RT-PCR products (599 bp) from
0.3% (lanes 1-4) and 4.0%
(lanes 5-8) NaCl-fed rats.
Dilutions of total RNA were (from
left) 750, 150, 30, and 6 ng.
-Actin PCR products (350 bp) from 0.3% and 4.0% NaCl-fed rats are
shown in lanes 9 and
10, respectively.
B: radioactivity incorporated into
nNOS mRNA amplification products. Results are means ± SE for 30 ng
of initial total RNA (n = 3).
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Fig. 6.
Effect of 3-wk 4.0% NaCl diet on nNOS protein expression in IMCD.
A: representative Western blot of nNOS
protein expression in IMCD of rats fed 4.0% NaCl diet for 3 wk.
Lane 1: rat pituitary lysate (positive
control). Lanes 2 and
4: proteins from 0.3% NaCl-fed rats,
20 and 40 µg total protein, respectively. Lanes
3 and 5: proteins from
4.0% NaCl-fed rats, 20 and 40 µg total protein, respectively.
B: densitometric quantification of
Western blot signals for nNOS protein in IMCD. Results are means ± SE (n = 3).
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Previous studies have demonstrated downregulation of renal cortical
nNOS mRNA by high dietary salt (4, 20). In agreement with these studies
and in contrast to our results in the IMCD, kidney cortical nNOS mRNA
significantly decreased after 3 days of 4.0% NaCl intake (Fig.
7) (0.3% NaCl, 30.88 ± 4.36 × 103, vs. 4.0% NaCl, 16.4 ± 4.70 × 103 cpm;
P < 0.05, n = 5). This was associated with a
significant decrease in kidney cortical nNOS protein expression,
determined by Western blot analysis (Fig.
8) (0.3% NaCl diet, 1.88 ± 0.14, vs.
4.0% NaCl, 0.92 ± 0.20 arbitrary units;
P < 0.05, n = 5).

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Fig. 7.
High-NaCl diet decreases nNOS mRNA expression in kidney cortex at 3 days. A: representative ethidium
bromide-stained agarose gel of kidney cortex nNOS RT-PCR products (599 bp) from 0.3% (lanes 1 and
2) and 4.0% (lanes
3 and 4) NaCl-fed
rats. Dilutions of total RNA were (from
left) 150 and 30 ng. -Actin PCR
products (350 bp) from 0.3 and 4.0% NaCl-fed rats are shown in
lanes 5 and
6, respectively.
B: radioactivity incorporated into
nNOS mRNA amplification products. Results are means ± SE for 150 ng
initial total RNA. * P < 0.05, n = 5.
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Fig. 8.
High-NaCl diet decreases nNOS protein expression in kidney cortex at 3 days. A: representative Western blot
of nNOS protein expression in kidney cortex of rats fed 4.0% NaCl diet
for 3 days. Lane 1: 0.3% NaCl-fed
rats (40 µg total protein); lane 2:
4.0% NaCl-fed rats (40 µg total protein).
B: densitometric quantification of
Western blot signals for nNOS protein from kidney cortex. Results are
means ± SE. * P < 0.05, n = 5.
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In all experiments, to control for possible variations in RNA isolation
and reverse transcription reactions,
-actin mRNA was PCR amplified
along with the nNOS mRNA amplifications. There were no differences in
radioactivity incorporated into
-actin PCR products among samples
derived from 0.3 or 4.0% NaCl-fed rats (not shown). Similarly, there
were no differences in
-actin protein abundance among samples
derived from 0.3 or 4.0% NaCl-fed rats by Western blot analysis.
Effect of high dietary NaCl on iNOS mRNA expression in
IMCD. Basal expression of iNOS mRNA has been reported
in the IMCD (15). High dietary NaCl intake had no effect on IMCD iNOS
mRNA expression after either 3 days (Fig.
9) (0.3% NaCl, 3.43 ± 0.14 × 103, vs. 4.0% NaCl, 3.14 ± 0.52 × 103 cpm;
P = NS,
n = 5) or 3 wk (Fig.
10) (0.3% NaCl, 3.90 ± 0.32 × 103, vs. 4.0% NaCl, 4.44 ± 0.74 × 103 cpm;
P = NS,
n = 3).

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Fig. 9.
Effect of 3-day 4.0% NaCl diet on expression of iNOS mRNA in IMCD.
A: representative ethidium
bromide-stained agarose gel of IMCD iNOS RT-PCR products (705 bp)
from 0.3% (lanes 1-4) and
4.0% (lanes 5-8) NaCl-fed
rats. Dilutions of total RNA were (from
left) 750, 150, 30, and 6 ng.
-Actin PCR products (350 bp) from IMCD of 0.3% and 4.0% NaCl-fed
rats are shown in lanes 9 and
10, respectively.
B: radioactivity incorporated into
IMCD iNOS mRNA PCR products. Results are means ± SE for 6 ng
initial total RNA (n = 5).
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Fig. 10.
Effect of 3-wk 4.0% NaCl diet on expression of iNOS mRNA in IMCD.
A: representative ethidium
bromide-stained agarose gel of IMCD iNOS RT-PCR products (705 bp) from
0.3% (lanes 1-4) and 4.0%
(lanes 5-8) NaCl-fed rats.
Dilutions of total RNA were (from
left) 750, 150, 30, and 6 ng.
-Actin PCR products (350 bp) from IMCD of 0.3% and 4.0% NaCl-fed
rats are shown in lanes 9 and
10, respectively.
B: radioactivity incorporated into
IMCD iNOS PCR products. Results are means ± SE for 6 ng of initial
total RNA (n = 3).
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Western blots, using a mouse monoclonal antibody to iNOS (Transduction
Laboratories) and with loading of up to 100 µg protein per lane,
failed to detect the expected 130-kDa iNOS protein in lysates from IMCD
in either 0.3 or 4.0% NaCl-fed rats. Two other polyclonal antibodies
to iNOS also did not detect the iNOS protein on Western blots.
 |
DISCUSSION |
The present studies determined the effect of high dietary salt on nNOS
expression in the IMCD. Our data clearly demonstrate that nNOS mRNA and
protein are expressed in the IMCD. In addition, we show that high
dietary salt intake differentially regulates nNOS mRNA and protein
expression in the IMCD and renal cortex but has no effect on expression
of iNOS mRNA in the IMCD.
Localization of nNOS. Utilizing
RT-PCR, Terada et al. (22) detected the highest levels of intrarenal
nNOS mRNA in the IMCD. The results of the present study, in which we
readily detected nNOS mRNA in normal rat IMCD by RT-PCR, are consistent
with this study and with the data of Singh et al. (20). Previous
immunolocalization studies, however, have not detected nNOS protein
expression in the IMCD, although an abundance of nNOS is present in the
macula densa (3). In contrast, in the present studies,
immunofluorescence revealed intense cytoplasmic staining for nNOS in
cells of freshly microdissected IMCD, and this staining was specific
for nNOS, since preincubation of the polyclonal nNOS antibody with
immunizing antigen resulted in complete elimination of
immunofluorescence. Furthermore, Western blot analysis of proteins
extracted from IMCD revealed a single band of ~160 kDa that
comigrated with nNOS from a rat pituitary lysate, used as a positive
control. Taken together, therefore, our data clearly demonstrate
presence of nNOS mRNA and protein in IMCD. The explanation for the lack
of detection of nNOS protein in IMCD by Bachmann et al. (3) is unclear,
although we speculate that differences in either tissue processing
methods, antibodies, or immunodetection methods may account for the
varying results.
Regulation of nNOS by dietary salt in IMCD and
cortex. Inhibition of renal medullary nNOS activity
increases mean arterial pressure in rats maintained on high-salt diets,
suggesting that this NOS isoform may be involved in the long-term
regulation of blood pressure (12). In the present study, nNOS protein
expression in the IMCD significantly increased after 3 days of high
dietary NaCl, but nNOS mRNA levels remained unchanged. This suggests
either increased translation of nNOS mRNA into protein or enhanced
protein stability due to posttranslational modification. The regulation of nNOS gene transcription and translation, however, is incompletely understood. In this regard, nNOS mRNAs with distinct
5'-untranslated regions that are encoded by separate promoters
have been described in human cerebellum (27). These mRNAs likely encode
identical nNOS proteins but could differ in stability, processing, or
translation efficiency. Accordingly, it is possible that high dietary
salt could induce a conversion to formation of nNOS mRNA transcripts with increased efficiency of translation, via as yet undefined signaling mechanisms. Alternatively, the possibility exists that the
semi-quantitative RT-PCR method utilized in the present study was not
sensitive enough to detect small increases in mRNA expression that
could have accounted for the increased expression of nNOS at 3 days.
Our results, however, are in agreement with those of Singh et al. (20),
who did not detect an effect of high-salt diet for 7 days on nNOS mRNA
expression in the rat IMCD by competitive RT-PCR.
In rats on high-NaCl diet for 3 wk, IMCD nNOS mRNA and protein
expression did not significantly change from control values, suggesting
that the increase in nNOS protein observed at 3 days is transient.
Although information regarding factors regulating nNOS expression is
limited (reviewed in Ref. 11), our data suggest that the molecular
signals for stimulation of nNOS protein expression are either no longer
present or are counteracted by other factors after 3 wk of high-salt
diet. In rat total inner medullary tissue, Mattson and Higgins (13)
reported that protein expression of eNOS, nNOS, and iNOS increased
significantly after 3 wk of high dietary salt intake. We cannot readily
provide an explanation for these differences from our present data,
although it is conceivable that high dietary salt might increase nNOS
expression in other inner medullary structures, such as within renal
nerves, vasa recta, or inner medullary thin limbs (3, 22). It must be noted, furthermore, that the Mattson and Higgins (13) study utilized a
single monoclonal antibody to detect both nNOS and iNOS by Western blot
analysis, and, in contrast to our results, they did not detect nNOS in
renal cortical homogenates.
Within the renal cortex, the macula densa contains the highest levels
of nNOS mRNA and protein (3, 20). In rats, salt deprivation stimulates
nNOS mRNA expression in the macula densa, whereas salt loading
significantly decreases mRNA expression (4, 20). Our data on nNOS mRNA
expression in renal cortex, performed as a positive control for our
assay system, are in agreement with these observations. In addition, we
have extended these findings by demonstrating that renal cortical nNOS
protein expression is significantly downregulated by high dietary salt
intake. The cellular signaling mechanisms mediating the inverse
relationship between salt intake and macula densa nNOS expression are
unknown, but a recent study revealed that blockade of systemic
AT2 ANG II receptors significantly
inhibited sodium depletion-induced increases in renal interstitial cGMP
levels, suggesting that AT2
receptors may be linked to stimulation of renal NO production (21). In addition to the possibility that differential regulation of nNOS expression between IMCD and renal cortex is due to separate cell signaling pathways, variable expression patterns may also be secondary to transcription of mRNAs from separate promoters, as discussed above
(27). Furthermore, alternative splicing of nNOS mRNA yielding a novel
nNOSµ protein isoform has been reported in skeletal muscle, suggesting that tissue-specific regulation of nNOS mRNA may occur (19).
Our data demonstrate an increase in urinary excretion of NO
decomposition products after 3 days of high dietary salt intake, a time
at which IMCD nNOS protein expression is increased but at which renal
cortical nNOS is inhibited. However, urinary excretion of
NO
2/NO
3
remained significantly increased after 3 wk of high salt intake,
despite normalization of IMCD nNOS protein expression. In rats fed a
high-salt diet for 2 wk, an increase in both serum concentration and
urinary excretion of NO
2 and
NO
3 has also been reported (18). Thus
our data suggest a dissociation between IMCD nNOS protein expression
and excretion of urinary NO products. Furthermore, although absolute
levels of urinary NO
2/NO
3
were comparable in rats at 3 days and 3 wk, we observed decreased
excretion at 3 wk, when correction for body weight was made. This
suggests there may be age-dependent effects on urinary excretion of NO
products.
The urinary excretion of NO products is complex, representing the
contributions from glomerular filtration of serum
NO
2 and
NO
3, tubular handling, and de novo
intrarenal NO synthesis. Thus the effects of enhanced nNOS protein in
IMCD on intrarenal NO production cannot be estimated from our data. The
gene expression and activity of eNOS have been shown to be upregulated
by shear stress (24), suggesting that increased urinary levels of NO
decomposition products with high salt intake could also reflect
increased intrarenal activity of this isoform. In addition, the
activity of nNOS protein may be affected by levels of intracellular
calcium or endogenous inhibitors (reviewed in Ref. 11), independent of
protein expression. Whether these mechanisms are involved in the
regulation of nNOS activity in the IMCD with high salt is unknown.
Dietary NaCl and iNOS in IMCD. Our
data demonstrate tonic expression of iNOS mRNA in the IMCD of normal
rats, with levels not affected by high dietary salt. Abundant iNOS mRNA
has previously been detected in rat IMCD by in situ hybridization (1).
Restriction mapping of iNOS RT-PCR products in rat kidney revealed that
the murine macrophage homologue of iNOS was the principal isoform expressed in tubular cells, including the IMCD, with lesser amounts of
the rat vascular isoform of iNOS (15). The present studies were not
designed to determine the relative amounts of these two mRNAs in IMCD
cells. Rather, we chose PCR primers to examine total iNOS transcript
levels in this segment. In this regard, it is of interest that a recent
study identified four alternatively spliced iNOS mRNAs in human
tissues, including kidney, with upregulation of all splice variants by
cytokines (6). Accordingly, it is possible that multiple splice
variants of iNOS exist in IMCD, although our data suggest that dietary
salt intake does not affect total mRNA transcript levels.
In the present studies, Western blotting with three different
antibodies failed to detect iNOS protein in IMCD. Recently, immunohistochemistry using antibodies to the macrophage-type iNOS in
rat kidney demonstrated strong staining in outer medulla, cortical collecting ducts, and some proximal tubules, with no detection in IMCD
(2). Tojo et al. (23), using a vascular smooth muscle iNOS antibody in
rat kidney, did not report iNOS staining in IMCD, although there was
immunolabeling in terminal afferent arteriole, distal tubule, and thick
ascending limb. Our results are in agreement with these studies and
suggest that iNOS mRNA is not efficiently translated into protein in
IMCD under basal conditions or with high dietary salt.
In summary, the present studies demonstrate that cells of the normal
rat IMCD express nNOS mRNA and protein. Protein expression of nNOS but
not mRNA is upregulated by high dietary salt after 3 days. In contrast,
high-salt diet decreases renal cortical mRNA and protein expression of
nNOS. These data suggest that selective activation of inner medullary
nNOS could be involved in the early adaptive response to high dietary
salt intake.
 |
ACKNOWLEDGEMENTS |
We thank Drs. W. Staines and W. Ross (University of Ottawa) for
assistance with the immunofluorescence studies and for the use of the
ELISA reader, respectively.
 |
FOOTNOTES |
This work was supported by a grant from the Medical Research Council of
Canada (MT-11560) (to K. D. Burns).
A portion of this work was presented at the annual meeting of the
American Society of Nephrology, New Orleans, LA, November 1996, and has
been published in abstract form (J. Am. Soc.
Nephrol. 7: 1541, 1996).
Address for reprint requests: K. D. Burns, 501 Smyth Rd., Rm. N-8,
Ottawa, ON, Canada K1H 8L6.
Received 2 September 1997; accepted in final form 27 February
1998.
 |
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