Clinical Science (1994) 86, 405-409 (Printed in Great Britain)
405
Effects of nitric oxide inhibition on the renal papillary
blood flow response to salineinduced volume expansion
in the rat
Noemi M. ATUCHA, Ana RAMiREZ, Tomis QUESADA and Joaquin GARCIA-ESTAN
Departamento de Fisiologio, Facultad de Medicina, Universidod de Murcia, Murcia, Spain
(Received 2 July/27 October 1993; accepted
I I November 1993)
1. Evidence indicates that nitric oxide (NO) exerts a
paracrine influence in the renal medulla. Increases in
papillary blood flow are thought to be an important
determinant of the renal response to extracellular
volume expansion. Therefore, in the present study, we
have evaluated the role of NO in mediating papillary
blood flow (laser-Doppler flowmetry) and excretory
responses to volume expansion with isotonic saline
(3% body weight, 15min).
2. Infusion of the NO synthesis inhibitor
Nu-nitro-L-arginine methyl ester (10 pg min- kg-'),
significantly attentuated the renal diuretic and
natriuretic responses to volume expansion as well as
the renal hydrostatic interstitial pressure increase
induced by this manoeuvre. The percentages of the
water and sodium excreted in 1h by the
N"-nitro-L-arginine methyl ester-pretreated animals
were 36% and 40% of the load, whereas those of the
control animals were 44% and 65%, respectively.
3. In similar experiments performed in the exposed
papilla of Munich Wistar rats, the same dose of
N"-nitro-L-arginine methyl ester reduced basal papillary blood flow and blunted the elevation in papillary
blood flow induced by volume expansion (6% versus
16% in the control animals).
4. These results indicate that the inhibition of NO
synthesis blunts the renal excretory and papillary
responses to volume expansion, suggesting that NO
modulates these responses through changes in papillary blood flow and renal interstitial hydrostatic
pressure.
~
~~
INTRODUCTION
Extracellular volume expansion (VE) with saline
induces an integrated response, involving physical,
hormonal and neural factors [1, 21, which allows
the kidney to eliminate the additional load. Since
the renal response to VE can be observed in the
absence of changes in the main renally active hormones [1, 31, numerous studies have evaluated the
role of intrarenal mechanisms as mediators of the
response to VE [4-91. Am ng these, special ir erest
has been devoted to change in intrarenal blood flow
distribution as a factor contributing to the renal
response to VE [4-7, 91. Thus, it has been shown
that VE with saline increases papillary blood flow
(PBF) [4, 7, 91 and this mechanism may be
mediated by the release of renal autacoids, such as
prostaglandins and kinins [10, 111. Of special interest to the renal medulla is the recently discovered
endothelium-derived nitric oxide (NO), which is an
important controller of vascular tone. Several
studies have shown that pharmacological blockade
of NO synthesis in experimental animals decreases
renal blood flow (RBF) and influences sodium and
water excretion [12-141, indicating that NO is an
important contributor to normal haemodynamic
and tubular function. Moreover, studies in laboratory animals have revealed that inhibition of NO at
non-pressor doses or by intrarenal infusion [15-1 81
influences renal haemodynamic and excretory responses, suggesting that NO can affect renal function in a paracrine fashion.
The aim of the present study was to evaluate the
role of NO as a mediator of the renal excretory and
PBF responses to VE in the rat. Previous data
suggest that NO is involved in the renal response to
VE [19]. However, the role of NO in the PBF
changes induced by VE is unknown. The hypothesis
is supported by results showing that VE increases
blood flow in the renal medulla [7, 9, 111 and it has
been suggested that the endothelial cells of the renal
medulla produce NO [20]. Moreover, intrarenal
infusion of a NO synthesis inhibitor selectively
reduces PBF in rats [17, 211. These results suggest
that NO could participate in the control of renal
vascular tone with a preferential effect on the
medulla.
METH0DS
Experiments were performed on rats born and
raised in the animal house of the Universidad de
Murcia, according to the guidelines for the ethical
Key words: extracellular volume expansion, laser-Doppler flowmetry, renal interstitial pressure, renal papilla, sodium excretion.
Abbreviations: GFR, glomerular filtration rate; MAP, mean arterial pressure; t-NAME, NW-nitreL-arginlne methyl ester; PBF, papillary blood flow; REF, renal blood flow; RIHP,
renal interstitial hydrostatic pressure; VE, volume expansion,
Correspondence: Dr Joaquin Garcia-Estai, Departamento de Fisiologia, Facultad de Medicina, 30100 Murcia. Spain.
406
N. M. Atucha
treatment of research animals of the European
Community and the Ministerio of Agricultura,
Pesca y Alimentacion of Spain.
Protocol I: effect of NO synthesis inhibition on the renal
excretory response to extracellular VE
All experiments were performed on SpragueDawley rats (40W25g) fasted the night before. The
rats were anaesthetized with Inactin (100mg/kg,
intraperitoneally) and placed on a heated surgical
table to maintain body temperature at 37°C. The
trachea was cannulated to facilitate respiration.
Cannulae were placed on the right jugular vein for
intravenous infusions and in the right femoral artery
for blood collection and for measurement of blood
pressure (transducer, Hewlett-Packard 1280; amplifier, Hewlett-Packard 8805D). All the animals
received a maintenance intravenous infusion of 0.9%
NaCl at a rate of 1.0mlh-' 1OOg-I throughout the
experiment. [ 3H]In~lin(1 pCi/ml) was added to the
infusate to allow measurement of glomerular filtration rate (GFR). After a midline abdominal incision, the left ureter was cannulated to obtain urine
samples. A 2.5mm flow probe was placed around
the left renal artery to measure RBF by means of an
electromagnetic flowmeter (model 501D; Carolina
Instruments, King, NC, U.S.A.). Renal interstitial
hydrostatic pressure (RIHP) was measured by the
acutely implanted capsule method as described previously [9, 221 with similar equipment to that described above for measurement of blood pressure.
The abdominal opening was covered with a piece of
Parafilm to prevent evaporation, and at least 60 min
were allowed before starting the experiment. Two
groups of rats were studied.
(1) Renal response to VE in control rats (n=7).
After the stabilization period, two basal 15min
clearance periods were taken and then a 0.9% NaCl
infusion (3% body weight) was administered in
15 min. Four more 15 min clearance periods were
obtained from the start of VE. Mean arterial pressure (MAP), RBF and RIHP were continuously
recorded throughout the experiment on a HewlettPackard polygraph (model 7754A). Urine and blood
samples were collected in every period. At the end
of the experiment, the animals were killed by a
pentobarbital overdose.
(2) Renal response to VE in animals pretreated
with the NO synthesis inhibitor N"-nitro-L-arginine
methyl ester (L-NAME) ( n = 7). After the stabilization period, two basal clearance periods were
obtained and then L-NAME was intravenously
infused (10 pg min- kg- ') for the duration of the
experiment. Thirty minutes later, two more clearance periods were taken and then a 0.9% NaCl
infusion (3% body weight) was administered in
15min. Four more 15min clearance periods were
obtained from the start of VE. The rest of the
protocol was similar to that described above.
et al.
Protocol 2 effect of NO synthesis inhibition on the PBF
response to extracellular VE
The experiments were performed in Munich
Wistar rats (175-200g), a strain of animals that
have a renal papilla protruding into the ureter. The
animals, surgically prepared in a similar way to that
described in protocol 1, were prepared for measurement of PBF as described by Roman et al. [23]
with some modifications. After tracheostomy and
cannulating the left femoral artery and vein, the
animals were placed in a lateral position and the left
kidney was exposed through a flank incision and
gently placed in a holder, specially built to isolate
the kidney from respiratory motion. Then, the renal
papilla was exposed by excising the ureter and was
surrounded by moistened cotton. PBF was measured using a laser-Doppler flowmeter (Periflux
PF3; Perimed, Sweden). The laser probe was fixed
to a micromanipulator and was placed on the
papillary surface at an angle of about 30". The
abdominal incision and the kidney were covered
with a piece of Parafilm to minimize evaporation.
MAP and PBF were continuously recorded and
averaged every minute throughout the experiment.
The animals received a maintenance saline infusion
throughout the experiment at the rate described in
protocol 1, and they were allowed to stabilize for at
least 60 min. The experimental protocol consisted of
15 min periods, two basal, one during an isotonic
NaCl infusion (3% body weight for 15min) and the
last during the recovery of VE. Two groups of
animals were studied:
(1) Control group (n=6). The experiment was
performed as described above.
(2) L-NAME-treated group (n= 6). After two
basal periods, L-NAME (10 pg min- kg- ') was
infused and 60 min later, without stopping the infusion, the saline load was administered.
'
Analytical techniques
The [3H]insulin concentration in urine and
plasma samples was measured using a liquid scintillation counter (Betamatic Basic, Kontron, Madrid).
GFR was calculated from the urine to plasma inulin
concentration ratio and urine flow, and was
expressed per g kidney weight. Urine flow was
determined gravimetrically. Sodium and potassium
concentrations were measured by flame photometry
(Corning 435, Izasa, Spain). Fractional excretions
were calculated using standard formulae.
Statistical analysis
Data are expressed as means_+SEM. Statistical
differences within a group were evaluated by a
repeated measures analysis of variance and posterior
Duncan test. Statistical differences between groups
were evaluated by a two-way analysis of variance
and a posterior Duncan test. A P level lower than
Effects of
NO
inhibition on papillary blood flow
407
Table 1. Systemic and renal parameters in control and t-NAME-treated animals subjected t o VE induced by 0.9% NaCl (3%
body weight). Abbreviations: UF, urine flow; UN,V, sodium excretion; UV
, , potassium excretion. B, basal period previous t o VE; VE,
volume expansion period; RI, R2 and R3, recovery periods after VE. Statistical significance: *P <0.05 versus basal period; t P 40.05
versus the same period in the control group. Values are means+SEM.
MAP (mmHg)
Control
t-NAME
RBF (mlmin-lg-l kidney wt.)
Control
L-NAME
B
VE
RI
R2
R3
I 14.3 k2.0
119.9 k2.0
I 14.3 k2.4
119.8 k2.9
119.0 k2.1
I17.5 f1.9
I 14.9 k2.9
116.5 k2.2
I12.6 k2.9
114.5 k2.3
6.3 k0.4
7.3 k0.3
6.6 k 0 . 4
7.2 +0.3
6.6 k 0 . 4
7.2 k0.3
6.4 +0.4
7.0 +0.3
6.3 k0.4
6.9 k0.3
1.og.1
I.4 g.2*
I.5 k0.I*
1.2g.1
1.2 kO.1
I. I +o. I
I.o +o. I
1.1 kO.1
0.9 k0.I
15.8 _+0.9*
10.8 +0.9*t
7.4 _+0.7*
5.0 +0.6t
6.2k0.5
4. I +0.7t
5.8 k0.7
4.6 k0.7
23.4k4.3
18.2 +I .3
126.0 kI4.7*
103.2 +9.3*
171.4k 17.9*
121.5+5.8*t
71.8+9.1*
57.8+4.5*t
56.5 +8.4*
39. I +4.3*
4.5 f0.8
3.5 k0.2
24.3 +I .8*
16.5k1.5*t
37.9 +4. I*
18.1 +1.3*t
14.8+1.4*
10.4 k0.6*
13.6 +2.6*
7.6 +0.6*
I.9,0.2*
1.2 kO.1t
I.4 kO.2
I.o +o. I
I.3 k 0 . 2
0.9 +O. I
GFR (mlmin-'g-' kidney wt.)
Control
t-NAME
RIHP (mmHg)
Control
t-NAME
0.9 +O. I
5.3 50.3
4.0 k 0 . 5 t
UF (plmin-'g-I)
Control
t-NAME
UN,V (mmol min-' g-)
Control
t-NAME
UV, (mmolmin-lg-l)
Control
L-NAME
I .o *o. I
0.9 +O. I
2.1 +_0.2*
1.5 kO.l*t
0.05 was considered to indicate a significant difference [24].
T
RESULTS
Protocol I
Mean body weight (control, 450.6 f6.9 g; LNAME, 413.4+ 6.8 g) and kidney weight (control,
1.38k0.04 g; L-NAME, 1.45 & 0.06 g) were not statistically different between groups. There were no
differences in mean arterial pressure (MAP), GFR,
RBF, packed cell volume, urine flow or sodium
excretion between the two groups of animals
(Table 1). Infusion of L-NAME did not change
GFR or MAP, but decreased RBF from 8.74k0.33
to 7.34+0.37mlmin-'g-'.
RIHP was also decreased by L-NAME pretreatment from 5.02 f0.3 to
3.79 f0.43 mmHg. Saline expansion decreased
packed cell volume similarly in both groups
(control, from 45.9 fO.4% to 41.2+0.4%, L-NAME,
from 46.2 & 0.5% to 41.4 f0.5%) and induced significant increases in water, sodium and potassium
excretion in both groups of animals, but those of
the L-NAME-treated rats were significantly blunted
(Table 1 and Fig. 1). In both groups of animals,
GFR increased only during the VE period and RBF
was unchanged during the experiment. VE significantly elevated RIHP in both groups, but the
increase was also significantly lower in L-NAMEpretreated rats than in control animals. The percent-
81
82
VE
Periods
RI
R2
R3
Fig. 1. Effects of extracellular VE with isotonic saline on the
percentages of the filtered water and sodium excreted in control
(0-0)
and 1-NAME-pretreated rats (.---a) (protocol I).
Abbreviations: BI, B2, basal periods; VE, volume expansion period; RI, RZ
and R3, recovery periods; FE, fractional excretion. Statistical significance:
*P<0.05 versus basal; t P <0.05 versus control group. Values are means
+SEM (n=7 in each group).
N. M. Atucha et al.
408
I40
-
- I30
-
E
-
M
I
E 120
2
x
-
110
-
100
-
130
-
120 -
a3
M
5 110
-
aP
loo
-
90
-
r
0
80
I-
I
I
Saline
I
Saline
or L-NAME
I
VE
I
I
Recovery
Fig. 2. MAP and PBF responses to extracellular VE with isotonic
saline in control (0)
and 1-NAME-pretreated (@--- 0) rats.
Statistical significance: *P <0.05 versus basal; t P <0.05 versus control
group. Values are means k S E M (n =6 in both groups).
ages of the saline load excreted in 1 h by the control
group were 44.2+4.9% of the water load and
64.9*4.8% of the sodium load. In contrast, LNAME-pretreated animals excreted 36.6 f 1.4% of
the water load (not significantly different compared
with the controls) and 40.9k 1.5% of the sodium
administered ( P < 0.05 versus controls).
Protocol 2
The results of these experiments are shown in
Fig. 2. There were no differences in blood pressure
between the control and the L-NAME-pretreated
groups at any time during the experiment. VE
elevated PBF by 16.3&3.4% (from 2.73k0.06 to
3.02k0.11 units) in control animals. In L-NAMEpretreated animals, PBF decreased during L-NAME
infusion from a basal value of 2.54k0.09 to
2.15 k0.09 units and the VE-induced increase in
PBF was blunted, since it changed by only
6.3_+3.5% to 2.30k0.14 units.
DISCUSSION
Recently there have been many reports emphasizing the importance of intrarenal blood flow redistri-
bution in different physiological and pathological
conditions [2, 9, 11, 17, 23, 25-27]. Extracellular VE
with saline has been shown, by different techniques,
to increase blood flow in the renal medulla
[7, 9, 25, 271 and this may be an important mechanism contributing to the subsequent increase in
urine flow and sodium excretion. Moreover, the
magnitude of the increase in PBF and the renal
response to VE are affected by the intrarenal level
of hormones, such as angiotensin 11, kinins and
prostaglandins [8, 10, 111, suggesting that the
haemodynamic changes may be secondary to
changes in the intrarenal release of renal autacoids.
In the present study, we have tested the hypothesis
that endothelium-derived N O is involved in the
renal response to VE through modulation of intrarenal haemodynamics. Thus, our results show that
pretreatment of the animals with the NO synthesis
inhibitor L-NAME abolishes the VE-induced
increase in PBF and RIHP and significantly reduces
the amount of the saline load excreted.
The dose of the inhibitor of NO synthesis used in
the present study is probably only partially inhibiting NO synthesis. However, larger doses would
have increased blood pressure and produced a
pressure-dependent elevation of sodium and water
excretion [12, 18, 221 that would have affected the
renal response to VE. Other studies using lower
doses of L-NAME have shown significant effects on
sodium and water excretion [15-18]. In the present
study, the dose of 10 pg min- kg- significantly
reduced RBF and RIHP, although it did not affect
sodium or water excretion. These results agree with
those of previous studies using the same dose and
type of NO synthesis inhibitor [16]. Our results
also indicate that this dose of L-NAME was not
selectively inhibiting the medullary circulation since
there was a reduction in total RBF. However, it is
unlikely that this effect can contribute to the
reduced excretory response to VE since different
studies have shown that the normal renal response
to saline-induced VE is not accompanied by
changes in RBF [l, 5, 7, 191.
The present data suggest that N O is released into
the papillary circulation during VE and this may be
a mechanism contributing to the subsequent excretion of the saline load. This idea is supported by the
RIHP measurements. As assessed by the change in
packed cell volume, the degree of saline-induced VE
was similar in both experimental groups and it is
likely that the dilution of plasma protein concentration was also similar. Thus, the VE-induced
decrease in plasma oncotic pressure should have
contributed similarly in both groups to the elevation
in RIHP. However, the increases in both RIHP and
PBF induced by VE were lower in L-NAMEpretreated animals, thereby suggesting that the
lower elevation of PBF in these animals was responsible for the lower increase in RIHP and secondary
to the decrease in NO in the renal medulla. This
suggestion agrees with the view that changes in the
'
Effects of NO inhibition on papillary blood flow
papillary circulation, in the absence of changes in
the cortical circulation, modulate the RIHP level
[l, 2, 22, 231. As suggested by some investigators,
RIHP may be the link between the medullary
circulation and sodium excretion. This effect can be
mediated through changes in the medullary solute
gradient, by increasing the backleak of solutes in
juxtamedullary nephrons or by the release of intrarenal autacoids [l, 2, 7, 281. An alternative possibility is that the increase in filtration fraction after
L-NAME administration caused the lower increase
in RIHP and sodium excretion. In support of this
possibility are recent results indicating that an
increase in proximal tubule reabsorption may be
responsible for the lower response to VE in LNAME-pretreated animals [19]. Finally, it is also
possible that these effects may be due to the direct
tubular effects of N O [29].
Recent results indicate that the rise in PBF after
saline infusion can be blocked by a B,-kinin receptor antagonist [l 11, suggesting that changes in
medullary kinins are involved in papillary haemodynamic changes. Our results do not contradict
these data since it has been shown that the haemodynamic effects of intrarenal bradykinin are
mediated by N O [30]. Thus, it is possible that the
effect reported in the present study might be secondary to a kinin-mediated release of NO.
In summary, the present results indicate that the
VE-induced increase in sodium excretion is partly
due to N O and that the medullary release of NO is
necessary for the elevation in PBF, RIHP and
sodium excretion that accompanies saline-induced
VE.
ACKN OWLEDGMENTS
This study was supported by grants from the
Direccibn General de Investigacibn Cientifica y
TCcnica (88/0229) and Fondo de Investigaciones
Sanitarias de la Seguridad Social (93/1227).
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