Role of nNOS in regulation of renal function in hypertensive Ren-2 transgenic rats.

Abstract

The present study was performed to evaluate the role of neuronal nitric oxide synthase (nNOS)-derived nitric oxide (NO) during the developmental phase of hypertension in transgenic rats harboring the mouse Ren-2 renin gene (TGR). The first aim of the present study was to examine nNOS mRNA expression in the renal cortex and to assess the renal functional responses to intrarenal nNOS inhibition by S-methyl-L-thiocitrulline (L-SMTC) in heterozygous TGR and in age-matched transgene-negative Hannover Sprague-Dawley rats (HanSD). The second aim was to evaluate the role of the renal sympathetic nerves in mediating the renal functional responses to intrarenal nNOS inhibition. Thus, we also evaluated the effects of intrarenal L-SMTC administration in acutely denervated TGR and HanSD. Expression of nNOS mRNA in the renal cortex was significantly increased in TGR compared with HanSD. Intrarenal administration of L-SMTC decreased the glomerular filtration rate (GFR), renal plasma flow (RPF) and sodium excretion and increased renal vascular resistance (RVR) in HanSD. In contrast, intrarenal inhibition of nNOS by L-SMTC did not alter GFR, RPF or RVR and elicited a marked increase in sodium excretion in TGR. This effect of intrarenal L-SMTC was not observed in acutely denervated TGR. These results suggest that during the developmental phase of hypertension TGR exhibit an impaired renal vascular responsiveness to nNOS derived NO or an impaired ability to release NO by nNOS despite enhanced expression of nNOS mRNA in the renal cortex. In addition, the data indicate that nNOS-derived NO increases tubular sodium reabsorption in TGR and that the renal nerves play an important modulatory role in this process.

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PHYSIOLOGICAL RESEARCH
 2002 Institute of Physiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic
E-mail: physres@biomed.cas.cz
ISSN 0862-8408
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http://www.biomed.cas.cz/physiolres
Physiol. Res. 52: 571-580, 2002
Role of nNOS in Regulation of Renal Function in
Hypertensive Ren-2 Transgenic Rats
L. ČERVENKA1,5, H.J. KRAMER2, J. MALÝ1, I. VANĚČKOVÁ1,5, A. BÄCKER2,
D. BOKEMEYER2, M. BADER3, D. GANTEN3, K.D. MITCHELL4
1Department of Experimental Medicine, Institute for Clinical and Experimental Medicine, Prague,
Czech Republic, 2Section of Nephrology, Medical Policlinic, Department of Medicine, University of
Bonn, Bonn, 3Max Delbrück Center for Molecular Medicine, Berlin-Buch, Germany, 4Department
of Physiology, Tulane University School of Medicine, New Orleans, Louisiana, USA and 5Center
for Experimental Cardiovascular Research, Prague, Czech Republic
Received April 8, 2002
Accepted July 30, 2002
Summary
The present study was performed to evaluate the role of neuronal nitric oxide synthase (nNOS)-derived nitric oxide
(NO) during the developmental phase of hypertension in transgenic rats harboring the mouse Ren-2 renin gene (TGR).
The first aim of the present study was to examine nNOS mRNA expression in the renal cortex and to assess the renal
functional responses to intrarenal nNOS inhibition by S-methyl-L-thiocitrulline (L-SMTC) in heterozygous TGR and in
age-matched transgene-negative Hannover Sprague-Dawley rats (HanSD). The second aim was to evaluate the role of
the renal sympathetic nerves in mediating the renal functional responses to intrarenal nNOS inhibition. Thus, we also
evaluated the effects of intrarenal L-SMTC administration in acutely denervated TGR and HanSD. Expression of nNOS
mRNA in the renal cortex was significantly increased in TGR compared with HanSD. Intrarenal administration of
L-SMTC decreased the glomerular filtration rate (GFR), renal plasma flow (RPF) and sodium excretion and increased
renal vascular resistance (RVR) in HanSD. In contrast, intrarenal inhibition of nNOS by L-SMTC did not alter GFR,
RPF or RVR and elicited a marked increase in sodium excretion in TGR. This effect of intrarenal L-SMTC was not
observed in acutely denervated TGR. These results suggest that during the developmental phase of hypertension TGR
exhibit an impaired renal vascular responsiveness to nNOS derived NO or an impaired ability to release NO by nNOS
despite enhanced expression of nNOS mRNA in the renal cortex. In addition, the data indicate that nNOS-derived NO
increases tubular sodium reabsorption in TGR and that the renal nerves play an important modulatory role in this
process.
Key words
Hypertension • Transgenic rat • Neuronal nitric oxide synthase • Renal nerves • Renal hemodynamics
Introduction
Although the hypertension that occurs in rats
transgenic for the mouse Ren-2 renin gene (TGR) is
clearly due to the expression of the Ren-2 renin gene, the
exact pathophysiological mechanisms responsible for the
development of hypertension in this model remain
unclear (Mullins et al. 1990). Previous studies have

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580 Červenka et al.
Vol. 51
shown that the hypertension in TGR is angiotensin II
(ANG II)-dependent and that activation of ANG II
receptor subtype 1 (AT1) is largely responsible for the
development of hypertension in this model (Hirth-
Dietrich et al. 1994, Böhm et al. 1995, Gross et al. 1995,
Mitchell and Mullins1995). However, it has been
demonstrated that plasma and kidney ANG II levels are
not elevated during prehypertensive and developmental
phases of hypertension in TGR (Mitchell et al. 1997).
Therefore, the development of hypertension in this model
cannot be explained purely on the basis of increased
production of ANG II. However, it has been reported that
TGR exhibit exaggerated peripheral and renal vascular
responsiveness to ANG II (Jacinto et al. 1999). The
enhanced renal vascular responsiveness to ANG II could
contribute to the inability of the kidney to maintain
normal rates of salt and water excretion at normotensive
pressures and, thereby, to the hypertension in this model.
Nevertheless, the mechanisms responsible for the
augmented vascular responsiveness to ANG II in this
model remain uncertain.
It is well known that tonically produced nitric
oxide (NO) plays an important role in the maintenance of
systemic and vascular tone and that acute NO synthase
(NOS) inhibition causes dose-dependent increases in
blood pressure and renal vasoconstriction (for review see
Wilcox 2001). In addition, it has been shown that
enhanced NO formation counteracts the vasoconstrictor
influences of ANG II in ANG II-dependent forms of
hypertension (for review see Navar et al. 2000). It is
therefore possible that NO deficiency could contribute to
the development of hypertension. However, it has been
reported that overall intrarenal NOS expression and NO
production are increased in various models of
hypertension (Dubey et al. 1998, Hayakawa and Raij
1998, Vaziri et al. 1998). Nevertheless, it has been
reported that despite an increased overall intrarenal NO
production, ANG II-infused hypertensive rats exhibit an
impaired ability to produce NO by neuronal NOS (nNOS)
(Ichihara et al. 1999, Červenka et al. 2001). Thus, it is
possible that a selective deficit in nNOS function
accounts for the diminished nNOS-derived NO
renoprotective effects on renal hemodynamic function in
TGR. This notion is supported by previous in vitro and
in vivo studies that demonstrated a diminished role for
nNOS-derived NO in counteracting ANG II-mediated
vasoconstriction in ANG II-infused hypertensive and
spontaneously hypertensive rats (SHR) (Ichihara et al.
1999, Welch et al. 1999, Červenka et al. 2001). The
importance of NO derived from nNOS in the regulation
of blood pressure is also supported by the findings that
nNOS inhibition in Dahl salt-resistant rats caused the
development of salt-sensitive hypertension (Tan et al.
1999). Taken together, these data are consistent with the
possibility that decreased renal vascular effects of nNOS-
derived NO contribute to the enhanced renal vascular
responsiveness to ANG II in TGR. Such effects might
contribute to a compromised ability of the kidney to
respond to ANG II-mediated increase in arterial blood
pressure with appropriate increase in sodium excretion
and thereby contribute to the development of
hypertension in TGR. However, the specific contribution
of nNOS-derived NO to the impairment of renal function
in TGR remains uncertain.
The present study was performed to evaluate the
renal cortical expression of nNOS and to determine the
renal hemodynamic and sodium excretory responses to
selective intrarenal nNOS inhibition in hypertensive TGR.
Since the renal sympathetic nerves are important in
modulating renal hemodynamic and excretory function in
both normotensive and hypertensive states (Lundin et al.
1984, Khraibi 1995), and given that nNOS is expressed in
renal sympathetic nerves fibers (Liu and Barajas 1998),
additional experiments were performed to determine
whether renal denervation influences the renal functional
responses to intrarenal nNOS inhibition.
Methods
Our studies were performed in accordance with
guidelines and practice established by the Institute for
Clinical and Experimental Medicine Animal Care and
Use Committee and are in accordance with laws in the
Czech Republic and Federal Republic of Germany. The
experiments were performed in male heterozygous TGR
rats of the hypertensive line TGR(mRen2)27 (37 to 38-
day-old) and age-matched transgene-negative Hannover
Sprague-Dawley rats (HanSD). All animals used in the
present study were bred at the Center for Experimental
Cardiovascular Research of Institute for Clinical and
Experimental Medicine from stock animals supplied from
the Max Delbrück Center for Molecular Medicine of
Berlin, Germany. Animals were fed standard rat chow
(SEMED, Prague, Czech Republic) with NaCl content
0.4% and tap water ad libitum and were kept on a 12-
hour/12-hour light/dark cycle.

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Role of nNOS in Regulation of Renal Function 579
Experiment 1: Studies on nNOS mRNA expression in the
renal cortex
Experiments were performed to compare the
nNOS mRNA expression in TGR and HanSD. In
previous studies, a close correlation between nNOS
mRNA transcript abundance in the renal cortex and
nNOS transcript abundance in macula densa (MD) has
been demonstrated (Schricker et al. 1996, Welch et al.
1999). Thus, this study was performed in renal cortical
tissue with the assumption that possible differences
reflect changes in the MD region. TGR (n=5) and HanSD
(n=5) were anesthetized with thiopental sodium
(60 mg/kg i.p.), the abdomen was opened and a segment
of renal cortex was removed and immediately frozen in
liquid nitrogen. The samples were stored at –80 oC until
isolation of total RNA, which was extracted from the
frozen renal cortex using a commercially available kit
(RNeasy; Qiagen, Germany). The semiquantitative RT-
PCR was performed as described in detail previously
(Schricker et al. 1996). The sense primer for nNOS was
5´-GAATACCAGCCTGATCCA.-3´, and the antisense
primer was 5´-TCCAGGAGGGTGTCCACCGCA-3´.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
was used as an internal standard. The sequences of the
GAPDH primers were
GATGCAGGGATGATGTTC-3´ and the antisense, 5´-
CGCTAACATCAAATGGGGTG-3´. Because GAPDH
mRNA levels did not differ between these groups, nNOS
mRNA expression was evaluated as the nNOS
mRNA/GAPDH mRNA ratio.
as follows: sense, 5´-
Experiment 2: Renal functional studies – effects of acute
intrarenal nNOS inhibition
For studies designed to evaluate the effects of
intrarenal blockade of nNOS on renal function, rats were
fasted overnight. On the day of the experiment they were
anesthetized with thiopental sodium (50 mg/kg i.p) and
were placed on a thermoregulated table to maintain body
temperature at 37-37.5
performed to maintain a patent airway, and the exterior
end of the tracheal cannula was placed inside a small
plastic chamber into which humidified 95 % O2/5 % CO2
mixture was continuously passed. This procedure
improves the stability of arterial pressure in anesthetized
rats (Mitchell and Mullins 1995). The right jugular vein
was catheterized with PE-50 tubing for infusion of
solutions and additional anesthetic as required. The right
femoral artery was cannulated to allow continuous
monitoring of arterial blood pressure and blood sampling.
Mean arterial pressure (MAP) was monitored with a
oC. A tracheostomy was
pressure transducer (model MLT 1050) and recorded on a
computer using a computerized data-acquisition system
(PowerLab/4SP, ADInstruments, UK).
The left kidney was exposed via a flank incision,
isolated from the surrounding tissue, and placed in a
Lucite cup. A tapered PE-10 catheter was inserted into
the left renal artery via the left femoral artery for
selective intrarenal administration. This catheter was kept
patent by a continuous infusion of heparinized isotonic
saline at a rate of 4 µl/min throughout the experiment. In
a previous study, it was demonstrated that this procedure
allows selective administration of drugs without spillover
into the systemic circulation (Červenka et al. 2001).
During surgery, an isotonic saline solution containing
bovine serum albumin (6 %) (Sigma Chemical Co.,
Prague, Czech Republic) was infused at a rate of
20 µl/min. After surgery, isotonic saline solution
containing p-aminohippurate sodium (PAH; Merck,
Sharp & Dohme West Point, PA) (1.5 %), and
polyfructosan (Inutest, Laevosan, Linz/Donau, Austria)
(7.5 %) was infused at the same infusion rate.
After completion of the surgical procedures, an
equilibrium period of 45 min was allowed for the animals
to establish steady state before initiating two 30-min
control urine collections. Then, continuous intrarenal
infusion of the nNOS inhibitor (S-methyl-L-thiocitrulline,
L-SMTC; Sigma) at a rate of 0.3 mg/h (infusion rate
4 µl/min) was started. After a 10-min delay, two 30-min
experimental urine collections
Simultaneously with the urine collections, arterial blood
samples were collected to allow the determination of
whole kidney hemodynamic function. The dose of
L-SMTC used in the present study was chosen because
we had previously demonstrated that it elicited substantial
selective blockade of nNOS activity when infused
directly into the renal artery of ANG II-infused
hypertensive rats (Červenka et al. 2001). The effects of
intrarenal L-SMTC administration on renal hemo-
dynamics and excretory function were evaluated in both
TGR and HanSD (n=10 in both cases). For control
purposes, the effects of intrarenal infusion of isotonic
saline were assessed in separate groups of TGR (n=7) and
HanSD (n=8).
were obtained.
Experiment 3: Renal functional studies – effects of acute
intrarenal nNOS inhibition after acute renal denervation
In order to evaluate the role of the renal
sympathetic nerves in mediating the renal functional
responses to intrarenal nNOS inhibition, additional
experiments in which the effects of intrarenal L-SMTC

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administration in TGR (n=7) and HanSD (n=8) subjected
to acute renal denervation were examined. The rats were
surgically prepared for renal clearance studies as
described above. Renal denervation was performed as
described and validated in previous studies (Bello-Reus et
al. 1975, Quan et al. 2001). Briefly, all visible nerves
were sectioned, the adventitia of the left renal artery was
stripped by coating it with a solution of 10 % phenol in
absolute alcohol. During the application of phenol, the
left kidney and adjacent tissues were carefully protected
from exposure to the chemical and damage of the major
lymphatic vessels in the area was avoided. Rats that
exhibited a spasm of the renal artery during or following
phenol application were not included in the study. The
experimental protocol was identical to that described in
Experiment 2. For control purposes, the effects of
intrarenal infusion of isotonic saline were assessed in
acutely denervated TGR (n=8) and HanSD (n=7).
Urine volume was measured gravimetrically,
inulin and PAH concentrations in urine and plasma were
determined colorimetrically. Glomerular filtration rate
(GFR) and renal plasma flow (RPF) were calculated from
the clearances of inulin and PAH, respectively. Sodium
and potassium concentrations in plasma and urine were
determined by flame photometry. Renal vascular
resistance (RVR) and fractional sodium excretion rate
were calculated using standard formulae.
Statistical comparisons within groups were
conducted by the ANOVA
measurements, followed by Newman-Keuls test. One-
way ANOVA test was used for comparisons between
groups. Values exceeding the 95 % probability limits
(P<0.05) were considered statistically significant. All
data are expressed as mean ± S.E.M.
test for repeated
Results
Experiment 1: studies on nNOS mRNA expression in
renal cortex
As shown in Figure 1, semiquantitative analysis
of RT-PCR products of nNOS mRNA demonstrated by
expression of nNOS mRNA in the renal cortex was
significantly increased in TGR compared with HanSD
(0.35±0.04 vs. 0.22±0.02 densitometric units, P<0.05).
Experiment 2: renal functional studies – effects of acute
intrarenal nNOS inhibition
Basal values for
hemodynamics, and sodium excretion rates from
Experiment 2 are summarized in Table 1. There were no
blood pressure, renal
significant differences in hemodynamic function and
sodium excretion between TGR and HanSD.
Because experimental manipulations did not
cause significantly different responses in experimental
periods 1 and 2 in all groups, we decided, in order to
highlight our results, to show only data from the
experimental period 2.
Fig. 1. Expression of nNOS mRNA corrected for GAPDH
expression (nNOS mRNA/GAPDH mRNA ratio) in renal
cortical tissue of hypertensive TGR and normotensive
HanSD. * P<0.05 compared with HanSD.
Intrarenal infusion of L-SMTC did not alter
MAP in either TGR or HanSD (131±5 vs.133±4 mm Hg
and 119±3 vs.121±4 mm Hg, respectively). As shown in
Figure 2, intrarenal administration of L-SMTC did not
alter either GFR or RPF in TGR (–2±2 and –8±4 %,
respectively). In contrast, L-SMTC administration
elicited significant decreases in GFR and RPF in HanSD
(–20±3 and –23±3 %, P<0.05 in both cases). Thus,
L-SMTC infusion did not influence RVR in TGR (from
27±3 to 30±4 mm Hg.ml-1.g-1) but significantly increased
RVR in HanSD (from 29±2 to 38±3 mm Hg.ml-1.g-1,
P<0.05). Intrarenal infusion of L-SMTC caused marked
increases in absolute and fractional sodium excretion in
TGR (+565±88 and +573±64 %, P<0.05 in both cases).
In HanSD, the L-SMTC-mediated reductions in GFR
were associated with decreases in absolute and fractional
sodium excretion (–23±7 and –24±5 %, P<0.05 in both

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Role of nNOS in Regulation of Renal Function 579
Table 1. Basal values for mean arterial blood pressure, renal hemodynamics and excretory function in TGR and HanSD
with intact renal nerves (not subjected to acute renal denervation).
Group n MAP
(mm Hg)
GFR
(ml/min.g)
RPF
(ml/min.g)
UVNa
(µEq/min.g)
FENa
(%)
UF
(µl/min.g)
HanSD + SAL
HanSD + L-SMTC
TGR + SAL
TGR + L-SMTC
8
10
7
10
117±2
119±3
135±3*
131±5*
0.55±0.03
0.54±0.02
0.65±0.05
0.57±0.03
2.36±0.13
2.21±0.14
2.87±0.33
2.60±0.18
0.37±0.06
0.38±0.09
0.58±0.14
0.48±0.25
0.38±0.06
0.57±0.09
0.56±0.13
0.55±0.25
6.31±0.48
7.98±0.88
7.61±0.39
6.75±0.44
Values are mean ± SEM. SAL indicates intrarenal saline infusion; L-SMTC indicates intrarenal S-methyl-L-
thiocitrulline. GFR, glomerular filtration rate; RPF, renal plasma flow; UVNa, absolute sodium excretion;
FENa, fractional sodium xcretion; UF, urine flow. *P<0.05 compared with other groups.
Fig. 2. Renal hemodynamics (top) and sodium excretory
responses (bottom) to intrarenal infusion of L-SMTC in
hypertensive TGR and normotensive HanSD. Results are
taken from the second experimental period of Experiment
2. GFR, glomerular filtration rate; RPF, renal plasma
flow; UVNa, absolute sodium excretion; FENa, fractional
sodium excretion. * P<0.05 compared with HanSD.
cases) (Fig. 2). Intrarenal infusion of the vehicle (isotonic
saline) did not significantly influence MAP in either TGR
(135±3 vs. 133±4 mm Hg) or in HanSD (117±2 vs.
115±2 mm Hg). In addition, intrarenal infusion of saline
did not alter GFR or RPF in either TGR (+5±3 and
+2±2 %, respectively) or in HanSD (–5±3 and –1±1 %,
respectively). Thus, RVR did not exhibit any significant
changes in response to intrarenal saline infusion in TGR
or HanSD (25±3 vs. 24±3 mm Hg. ml-1.g-1 and 27±4 vs.
26±3 mm Hg. ml-1.g-1, respectively).
Experiment 3: Renal functional studies – effects of acute
intrarenal nNOS inhibition after acute renal denervation
As summarized in Table 2, acute renal
denervation resulted in about a two- to three-fold increase
in absolute and fractional sodium excretion in both TGR
and HanSD compared with denervated animals from
Experiment 2 (Table 1). Intrarenal saline infusion did not
significantly influence MAP in either denervated TGR
(131±3 vs. 130±5 mm Hg) or in denervated HanSD
(115±3 vs. 117±2 mm Hg). Similarly, intrarenal saline
infusion did not alter GFR and RPF in either denervated
TGR (+9±3 and +8±4 %, respectively) or in denervated
HanSD (–6±2 and –4±2 %, respectively). However,
intrarenal infusion of saline was associated with slight
reductions in both absolute and fractional sodium
excretion in denervated TGR (–27±11 % and –37±12 %,
respectively, P<0.05 in both cases) and in denervated
HanSD (–26±9 % and –38±14 %, respectively, P<0.05 in
both cases). Intrarenal administration of L-SMTC did not
significantly change GFR (+8±4 %) or RPF (–2±2 %) in
acutely denervated TGR.
administration elicited decreases in both GFR and RPF
(–19±5 % and –26±7 %, respectively, P<0.05 in both
cases) in denervated HanSD. Intrarenal infusion of
However, L-SMTC

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580 Červenka et al.
Vol. 51
Table 2. Basal values for mean arterial blood pressure, renal hemodynamics and excretory function in TGR and HanSD
subjected to acute renal denervation.
Groupn MAP GFR
(mm Hg)
RPF
(ml/min.g)
UVNa
(µEq/min.g)
FENa
(%)
UF
(µl/min.g) (ml/min.g)
HanSD + SAL
HanSD +L-SMTC
TGR +SAL
TGR + L-SMTC
7
8
8
7
115±3
112±3
131±3*
133±4*
0.59± 0.05
0.61±0.04
0.60±0.08
0.61±0.04
2.66±0.22
2.83±0.28
2.84±0.19
3.04±0.21
1.27±0.25
1.65±0.16
1.64±0.27
1.34±0.11
1.69±0.41
1.24±0.14
1.53±0.20
1.36±0.21
12.65±1.11
15.55±1.24
14.25±0.91
13.3±0.82
Values are mean ± S.E.M. SAL indicates intrarenal saline infusion; L-SMTC indicates intrarenal S-methyl-L-
thiocitrulline. GFR, glomerular filtration rate; RPF, renal plasma flow; UVNa, absolute sodium excretion;
FENa, fractional sodium excretion; UF, urine flow. *P<0.05 compared with other groups.
Fig. 3. Absolute (top) and fractional (bottom) sodium
excretion rate responses to intrarenal saline and L-SMTC
infusion in acutely denervated TGR and HanSD. Results
are taken from the second experimental period of
Experiment 3. SALINE indicates intrarenal saline
infusion; L-SMTC indicates intrarenal S-methyl-L-
thiocitrulline. * P<0.05 compared with TGR.
L-SMTC did not increase either absolute or fractional
sodium excretion rates in denervated TGR or in
denervated HanSD. Rather, intrarenal L-SMTC infusion
elicited decreases in both absolute and fractional sodium
excretion rates in denervated TGR (–32±7 % and –34±8
%, respectively, P<0.05 in both cases) as well as in
denervated HanSD (–47±5 % and –58±8 %, respectively,
P<0.05 in both cases) (Fig. 3).
Discussion
The present study was performed in order to
evaluate the role of nNOS-derived NO in the regulation
of renal function in TGR rats and its potential
contribution to the development of hypertension in this
model. We found that the expression of nNOS is
increased in the kidney cortex of the TGR. Despite this
enhanced nNOS expression in TGR the present data
demonstrate that selective blockade of intrarenal nNOS
with L-SMTC does not elicit marked alterations of renal
hemodynamic function in hypertensive TGR. The reason
for this apparent unresponsiveness to nNOS inhibition
could be related to reduced enzyme activity of nNOS in
the macula densa cells of TGR. Alternatively, it is
possible that intrarenal production of superoxide anion
(O2
interacts with NO to yield peroxynitrate so that the
biological half-life of NO could therefore be substantially
reduced. This second explanation is supported by a recent
study which demonstrated that enhanced production of
O2
tubuloglomerular feedback (TGF) responsiveness in SHR
(Welch et al. 2000, for review see Schnackenberg 2002).
In addition, it has been shown that the removal of O2
superoxide dismutase mimetic tempol restored the
-) in TGR is increased. It is well known that O2
-
- in the renal cortex is responsible for exaggerated
- by

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Role of nNOS in Regulation of Renal Function 579
impaired afferent arteriole responses to nNOS inhibition
in SHR (Ichihara et al. 2001). Moreover, it has been
demonstrated that ANG II stimulates O2
an AT1 receptor-dependent mechanism (for review see
Berry et al. 2001) and that AT1 receptor blockade
specifically attenuates oxidative stress and restores renal
vascular responsiveness to NO in SHR (Welch and
Wilcox 2001). In view of the findings that the
hypertension in TGR is ANG II-dependent and activation
of AT1 receptors is largely responsible for the
hypertension in this model (Hirth-Dietrich et al. 1994,
Böhm et al. 1995, Gross et al. 1995, Mitchell and
Mullins1995), it seems reasonable to assume that
enhanced degradation of NO via interaction with O2
contributes to the diminished renal vascular responses to
nNOS inhibition in TGR. However, additional studies are
required to address this issue.
In the present study, selective intrarenal
blockade of nNOS by L-SMTC markedly increased in
absolute and fractional sodium excretion without
influencing renal hemodynamics in TGR. These results
imply that nNOS-derived NO increases tubular sodium
reabsorption in TGR. This finding is apparently
controversial to the results of previous studies showing
that NO exerts a direct inhibitory action on sodium
channels in the distal tubule and collecting duct
epithelium (Stoos et al. 1994, 1995) and that NOS
inhibition decreases sodium excretion when associated
increases in renal perfusion pressure are prevented
(Takenaka et al. 1993). However, it has been shown that
luminal application of L-NAME decreased whereas NO
donors increased fluid and HCO3
proximal tubule (Wang 1997).
In addition, it has been reported that mice with
targeted disruption of the nNOS gene exhibit lower
tubular fluid and HCO3
tubule accompanied by lower blood pressure and
metabolic acidosis compared with corresponding wild-
type control mice (Wang et al. 2000). These findings
imply that endogenously produced NO enhance tubular
fluid and HCO3
Altogether, these data suggest that NO elicits disparate
effects on the proximal and distal nephron reabsorptive
function with NO stimulating
reabsorption rate while inhibiting reabsorption by the
distal tubule and collecting duct segments. The
mechanism responsible for the inhibitory action of NO in
distal and collecting duct nephron segments is well
established and involves an inhibitory action of NO on
the amiloride-sensitive apical membrane sodium channel
-
production via
-
- reabsorption in the rat
- reabsorption by the proximal
- reabsorption by the proximal tubule.
proximal tubular
as well as inhibition of the apical membrane sodium
chloride cotransporter in the early distal tubule (Majid
and Navar 1994, Stoos et al. 1994, 1995). In contrast, the
mechanism underlying the stimulation of proximal
tubular reabsorption by NO remains unclear. Whatever
the mechanism, it is possible that nNOS-derived NO may
act to maintain an inappropriately high proximal
reabsorptive rate in TGR and, thereby, may contribute to
the hypertension in this model.
It is generally recognized that the renal
sympathetic nerves play an important role in the
regulation of tubular sodium reabsorption (Quan and
Baum 2001) and that increased renal sympathetic nerve
activity contributes markedly to the renal functional
derangements that appear to be necessary for the
development of various forms of hypertension (for review
see DiBona and Kopp 2001). In view of this and given
that nNOS in the kidney is expressed in nerve bundles,
especially in the region of the proximal tubules (Liu and
Barajas 1998), we hypothesized that the effect of nNOS-
derived NO to increase tubular reabsorption rate is
mediated, at least in part, via increased activity of the
renal sympathetic nerves. In order to address this
hypothesis, we evaluated whether acute renal denervation
alters the renal functional responses to intrarenal
blockade of nNOS in both TGR and HanSD. Intrarenal
administration of L-SMTC in acutely denervated HanSD
decreased GFR and RPF to a similar extent as was
observed in intact HanSD. These decreases of renal
hemodynamic function in HanSD were accompanied by
decreases in both absolute and fractional sodium
excretion rates. In contrast, inhibition of nNOS by
L-SMTC in acutely denervated TGR did not significantly
alter renal hemodynamics and did not significantly
increase sodium excretion.
hemodynamic and excretory responses to nNOS
inhibition in denervated TGR were similar to those
elicited by intrarenal administration of isotonic saline.
Thus, the present findings indicate that the increase in
sodium excretion following nNOS inhibition is dependent
on intact renal sympathetic nerves. This suggests that
nNOS-derived NO acts to stimulate tubular reabsorption
via an interaction with the renal sympathetic nerves.
Whether this involves an increase in the release of
norepinephrine from sympathetic nerve terminals or an
increased tubular responsiveness to norepinephrine
remains to be determined. Regardless of this , the effects
of nNOS-derived NO to augment tubular reabsorption
rate would likely contribute to an inappropriately high
Indeed, the renal

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580 Červenka et al.
Vol. 51
reabsorptive status, to the elevation of arterial pressure
and, thus to the hypertension in TGR.
On the basis of these results we conclude that
during the developmental phase of hypertension, TGR
exhibit an impaired renal vascular responsiveness to
nNOS derived NO or an impaired ability to release NO
by nNOS despite enhanced expression of nNOS mRNA
in the renal cortex. In addition, our data indicate that
nNOS-derived NO increases tubular sodium reabsorption
in TGR. An attenuated vasodilator influence of nNOS-
derived NO on renal hemodynamic function combined
with nNOS-derived stimulation of sodium reabsorption
may contribute to the impaired renal excretory function
and, thus to the development of hypertension in this
model.
Acknowledgements
This study was supported by grants no. NE/6358-3,
305/00/0334, 139/1999 C awarded to L. Červenka and
J. Malý by the Internal Grant Agency of the Ministry of
Health of the Czech Republic, Grant Agency of Czech
Republic, Grant Agency of Charles University and partly
by financial support from the Center for Experimental
Cardiovascular Research (LN 00A069). H.J. Kramer,
D. Bokemeyer and A. Bäcker are supported by the
German Research Foundation (DFG 436 TSF) and by the
BONFOR Research Committee of the Medical Faculty of
the University of Bonn.
Selected Abbreviations and Acronyms
ANG II – angiotensin II
AT1 – angiotensin II receptor subtype 1
GFR – glomerular filtration rate
GAPDH – glyceraldehyde-3-phosphate dehydrogenase
HanSD – transgene-negative Hannover Sprague-Dawley
rats
L-SMTC – S-methyl-L-thiocitrulline, neuronal nitric
oxide synthase inhibitor
MAP – mean arterial pressure
MD – macula densa
nNOS – neuronal nitric oxide synthase
NOS – nitric oxide synthase
O2
PAH – p-aminohippurate sodium
RPF – renal plasma flow
RVR – renal vascular resistance
SHR – spontaneously hypertensive rats
TGF – tubuloglomerular feedback
TGR – transgenic rats for the mouse Ren-2 renin gene
- – superoxide anion
References
BELLO-REUS E, COLINDRES RE, PASTORIZA-MUŇOZ E, MUELLER R, GOTTSCHALK CW: Effects of acute
unilateral renal denervation in the rat. J Clin Invest 56: 208-217, 1975.
BERRY C, BROSNAN MJ, FENNELL J, HAMILTON CA, DOMINICZAK AF: Oxidative stress and vascular damage
in hypertension. Curr Opin Nephrol Hypertens 10: 247-255, 2001.
BÖHM M, LEE MA, KREUTZ MA, KIM S, SCHNINKE M, DJAVIDANI B, WAGNER J, KALING M, WIENEN
W, BADER M, GANTEN D: Angiotensin II receptor blockade in TGR(mRen2)27: effects on renin-
angiotensin-system gene expression and cardiovascular functions. J Hypertens 13: 891-899, 1995.
ČERVENKA L, KRAMER HJ, MALY J, HELLER J: The role of nNOS in regulation of renal function in angiotensin
II-induced hypertension. Hypertension 38: 280-285, 2001.
DIBONA GF, KOPP UC: Neural control of renal function. In: The Kidney, Physiology and Pathophysiology.
DW SELDIN, G. GIEBISCH (eds), Philadelphia, Lippincott Williams and Wilkins, 2001, pp 981-1005.
DUBEY RK, BOEGEHOLD MA, GILLESPIE DG, ROSSELLI M: Increased nitric oxide activity in early renovascular
hypertension. Am J Physiol 270: R118-R124, 1998.
GROSS V, LIPPOLDT A, SCHNEIDER W, LUFT FC: Effect of captopril and angiotensin II receptor blockade on
pressure natriuresis in transgenic TGR(mRen2)27 rats. Hypertension 26: 471-479, 1995.
HAYAKAWA H. RAIJ L: Nitric oxide synthase activity and renal injury in genetic hypertension. Hypertension 31:
266-270, 1998.
HIRTH-DIETRICH C, STASCH JP, GANTEN D, LUFT FC: Renal effects of captopril and nitrendipine in transgenic
rats with an extra renin gene. Hypertension 23: 626-631, 1994.

Page 9

2002
Role of nNOS in Regulation of Renal Function 579
ICHIHARA A, IMIG JD, NAVAR LG: Neuronal nitric oxide synthase-dependent afferent arteriolar function in angiotensin
II-induced hypertension. Hypertension 33: 462-466, 1999.
ICHIHARA A, HAYASHI M, HIROTA N, SARUTA T: Superoxide inhibits neuronal nitric oxide synthase influences
on afferent arterioles in spontaneously hypertensive rats. Hypertension 37: 630-634, 2001.
JACINTO SM, MULLINS JJ, MITCHELL KD: Enhanced renal vascular responsiveness to angiotensin II in
hypertensive ren-2 transgenic rats. Am J Physiol 276: F315-F322, 1999.
KHRAIBI AA: Role of renal nerves in natriuresis of L-NNMA infusion in SHR and WKY rats. Am J Physiol 269: F17-
F21, 1995.
LIU L, BARAJAS L: Evidence for NOS-containing renal neuronal somata transiently expressing a catecholaminergic
phenotype during development in the rat. Neurosci Lett 251: 161-164, 1998.
LUNDIN S, RICHSTEN SE, THOREN P: Renal sympathetic activity in spontaneously hypertensive rats and
normotensive controls, as studied by three different methods. Acta Physiol Scand 120: 265-272, 1984.
MAJID DSA, NAVAR LG: Blockade of distal nephron sodium transport attenuates pressure natriuresis in dogs.
Hypertension 23: 1040-1045, 1994.
MITCHELL KD, JACINTO SM, MULLINS JJ: Proximal tubular fluid, kidney, and plasma levels of angiotensin II in
hypertensive ren-2 transgenic rats. Am J Physiol 273: F246-F253, 1997.
MITCHELL KD, MULLINS JJ: ANG II dependence of tubuloglomerular feedback responsiveness in hypertensive ren-
2 transgenic rats. Am J Physiol 268: F821-F828, 1995.
MULLINS JJ, PETERS J, GANTEN D: Fulminant hypertension in transgenic rats harbouring the mouse Ren-2 gene.
Nature 344: 541-544, 1990.
NAVAR LG, ICHIHARA A, CHIN SY, IMIG JD: Nitric oxide-angiotensin II interactions in angiotensin II-dependent
hypertension. Acta Physiol Scand 168: 139-147, 2000.
QUAN A, BAUM M: The renal nerve is required for regulation of proximal tubule transport by intraluminally produced
ANG II. Am J Physiol 280: F524-F529, 2001.
SCHNACKENBERG CG: Physiological and pathophysiological roles of oxygen radicals in the renal microvasculature.
Am J Physiol 282: R335-R342, 2002.
SCHRICKER K, POTZL B, HAMANN M, KURTZ A: Coordinate changes of renin, and brain-type nitric oxide-
synthase (b-NOS) mRNA levels in rat kidneys. Pflügers Arch 432: 394-400, 1996.
STOOS BA, CARRETERO OA, GARVIN JL: Endothelial-derived nitric oxide inhibits sodium transport by affecting
apical membrane channels in cultured collecting duct cells. J Am Soc Nephrol 4: 1855-1860, 1994.
STOOS BA, GARCIA NA, GARVIN JL: Nitric oxide inhibits sodium reabsorption in the isolated perfused cortical
collecting duct. J Am Soc Nephrol 6: 89-94, 1995.
TAKENAKA T, MITCHELL KD, NAVAR LG: Contribution of angiotensin II to renal hemodynamic and excretory
responses to nitric oxide synthesis inhibition in the rat. J Am Soc Nephrol 4: 1046-1053, 1993.
TAN DY, MENG S, MANNING JR. RD: Role of neuronal nitric oxide synthase in Dahl salt-sensitive hypertension.
Hypertension 33: 456-461, 1999.
VAZIRI ND, NI Z, OVEISI F: Upregulation of renal and vascular nitric oxide synthase in young spontaneously
hypertensive rats. Hypertension 31: 1248-1254, 1998.
WANG T: Nitric oxide regulates HCO3
tubule. Am J Physiol 272: F242-F248, 1997.
WANG T, INGLIS FM, KALB RG: Defective fluid and HCO3
synthase-knockout mice. Am J Physiol 279: F518-F524, 2000.
WELCH WJ, WILCOX CS: AT1 receptor antagonist combats oxidative stress and restores nitric oxide signaling in the
SHR. Kidney Int 59: 1257-1263, 2001.
WELCH WJ, TOJO A, LEE JU, KANG DG, SCHNACKENBERG CG, WILCOX CS: Nitric oxide synthase in the
JGA of the SHR: expression and role in tubuloglomerular feedback. Am J Physiol 277: F130-F138, 1999.
- and Na+ transport by a cGMP-mediated mechanism in the kidney proximal
- absorption in proximal tubule of neuronal nitric oxide

Page 10

580 Červenka et al.
Vol. 51
WELCH WJ, TOJO A, WILCOX CS: Roles of NO and oxygen radicals in tubuloglomerular feedback in SHR. Am J
Physiol 278: F769-F776, 2000.
WILCOX CS: L-arginine-nitric oxide pathway. In: The Kidney, Physiology and Pathophysiology DW SELDIN,
G GIEBISCH (eds), Philadelphia, Lippincott Williams and Wilkins, 2001, pp 849-872.
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L. Červenka, M.D., Ph.D., Department of Experimental Medicine, Institute for Clinical and Experimental Medicine,
Vídeňská 1958/9, CZ-140 00 Prague 4, Czech Republic. Fax: +4202 41721666. E-mail: luce@medicon.cz