Effects of Ca2+ channel activity on renal
hemodynamics during acute attenuation of NO synthesis in the rat
R. A.
Kramp,
P.
Fourmanoir,
L.
Ladrière,
E.
Joly,
C.
Gerbaux,
A.
el Hajjam, and
N.
Caron
Service de Physiologie et Pharmacologie, Faculté de
Médecine et de Pharmacie, Université de Mons-Hainaut,
7000 Mons, Belgium
 |
ABSTRACT |
In
cultured vascular muscle cells, nitric oxide (NO) has been shown to
inhibit voltage-dependent Ca2+ channels, which are involved
in renal blood flow (RBF) autoregulation. Therefore, our purpose was to
specify in vivo the effects of this interaction on RBF autoregulation.
To do so, hemodynamics were investigated in anesthetized rats during
Ca2+ channel blockade before or after acute NO synthesis
inhibition. Rats were treated intravenously with vehicle (n = 10), 0.3 mg/kg body wt
NG-nitro-L-arginine-methyl ester
(L-NAME; n = 7), 4.5 µg · kg body wt
1 · min
1 nifedipine
(n = 8) alone, or with nifedipine infused before (n = 8), after (n = 8), or coadministered with L-NAME
(n = 10). Baseline renal vascular resistance (RVR) averaged
14.0 ± 1.2 resistance units and did not change after vehicle. RVR
increased or decreased significantly by 27 and 29% after
L-NAME or nifedipine, respectively. Nifedipine reversed,
but did not prevent, RVR increase after or coadministered with
L-NAME. RBF autoregulation was maintained after
L-NAME, but the autoregulatory pressure limit
(PA) was significantly lowered by 15 mmHg. Nifedipine
pretreatment or coadministration with L-NAME limited
PA resetting or suppressed autoregulation at higher doses.
Results were similar with verapamil. Intrarenal blockade of
Ca2+-activated K+ channels also prevented
autoregulatory resetting by L-NAME (n = 8). These
findings suggest NO inhibits voltage-dependent Ca2+
channels and thereby modulates RBF autoregulatory efficiency.
autoregulation; renal vascular resistance; nifedipine; iberiotoxin
 |
INTRODUCTION |
THE ENDOTHELIUM-DERIVED RELAXING factor or nitric oxide
(NO) induces a continuous relaxation of vascular smooth muscle cells. To do so, NO is released from endothelial cells and diffuses very rapidly through the interstitial space to activate the soluble guanylate cyclase of the adjacent smooth muscle cell, maintaining high
levels of cGMP (19). cGMP activates, in turn, a protein kinase G that
maintains intracellular Ca2+ low by reducing
Ca2+ transport into the cytosol (18). In this
regard, it has been shown in vitro that the NO-cGMP pathway markedly
attenuates the activity of voltage-dependent Ca2+ channels
and that Ca2+-activated K+ channels may be
involved (1, 8, 24). Several studies in cultured smooth muscle cells
that originated from different vascular beds have also provided
evidence for a direct inhibitory effect of NO on the activity of
voltage-dependent Ca2+ channels (7, 8).
In the kidney, voltage-dependent Ca2+ channels are
selectively located in the preglomerular microvasculature, in
particular in the afferent arteriole (10). The afferent arteriole is
the main site for regulation of renal vascular resistance, which is rapidly adjusted when renal perfusion pressure (RPP) changes (26). Renal hemodynamics are therefore characterized by a powerful intrinsic capacity of the kidney to autoregulate renal blood flow (RBF) and
glomerular filtration rate (GFR). Although NO does not seem to play a
major role in the autoregulatory phenomenon of RBF, its in vitro
inhibitory effect, either direct or indirect, on voltage-dependent
Ca2+ channels of vascular smooth muscle cells nevertheless
suggests some implication of NO in autoregulation of RBF. Therefore,
the purpose of our study was to specify in vivo the impact of the interaction between NO and Ca2+ channel activity on renal
hemodynamics, in particular on RBF autoregulation. To address this
point, experiments on renal hemodynamics were undertaken in
anesthetized euvolemic rats during the acute inhibition of NO synthesis
induced with a low or a high dose of NG-nitro-L-arginine-methyl ester
(L-NAME) injected intravenously. Moreover, to evaluate in vivo
the involvement of NO in the activity of voltage-dependent
Ca2+ channels in the kidney, the effects of
Ca2+ channel blockade on renal hemodynamics were
investigated before or after inhibition of NO synthase, as well as
during the simultaneous inactivation of Ca2+ channels and
inhibition of NO synthase. RBF autoregulation was also evaluated during
intrarenal blockade of Ca2+-activated K+
channels, which have been localized in the preglomerular arterioles in
the rat and may be implicated in NO-cGMP pathway and voltage-dependent Ca2+ channel interactions (1, 14), as well as during the
intravenous infusion of angiotensin II because voltage-dependent
Ca2+ channels are then activated (31).
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METHODS |
Animal preparation.
Male Wistar rats were maintained on a rat food diet (25 g daily of
Muracon G. Ster.) containing 1.8 g/kg sodium and 9.6 g/kg potassium as
stated by the manufacturer (Trouw, Gent, Belgium). Before
experimentation, the rats, weighing ~300 g, were deprived of food
overnight but had free access to tap water. They were anesthetized with
Inactin (10 mg/100 g body wt ip; Byk-Gulden). The animals were placed
on a heated table to maintain rectal temperature between 37 and
38°C. The left femoral artery was first catheterized to determine
the initial hematocrit and to measure blood pressure. Blood pressure
was measured by using a Statham P23 ID pressure transducer connected to
a pressure monitor (Mennen Medical) and a recorder. The right femoral
artery, for subsequent blood sampling, and vein were then rapidly
catheterized. To avoid fluid shifts during further surgery, a 0.85%
saline solution containing 2.5% albumin was infused immediately into
the right femoral vein at a rate of 150 µl/min for 5 min and of 83 µl/min for the next 30 min. This infusion was maintained throughout
experimentation at a rate of 8 µl/min. After tracheostomy, the right
jugular vein was catheterized for subsequent infusions. The left kidney
was exposed through a midline and subcostal abdominal incision as previously described (21). The segment of the aorta located between the
two renal arteries as well as the left renal artery were then carefully
dissected from surrounding tissues, avoiding as much as possible
interference with nerves bundles (21). Finally, each ureter was
cannulated for urine collection. In some rats, a tapered and curved
thin catheter was introduced in the left iliac artery and pushed upward
until its tip was positioned in the left renal artery. Thereafter,
isotonic saline was continuously infused intrarenally at a rate of 5 µl/min. After completion of surgery and after a suitable prime, a
0.85% NaCl solution containing 3% inulin (Laevosan, Innsbruck,
Austria), except if otherwise stated, was infused at a rate of 48 µl/min to measure GFR. The kidneys were removed, decapsulated,
blotted dry, and weighed at the end of the experiment.
Renal hemodynamics.
An adjustable constriction clamp was placed around the aorta above the
left and just beneath the right renal artery to reduce RPP in a
stepwise manner. A small-diameter, noncannulating, and factory-precalibrated electromagnetic flow transducer (0.60 or 0.62 mm
ID), connected to a square-wave electromagnetic flowmeter (MDL 1401 compact; Skalar Medical, Delft, The Netherlands) and a recorder, was
vertically fitted around the left renal artery to continuously measure
RBF. Calibration and use of these flow sensors as well as the final
tests to check the accuracy of RBF measurements at the end of the
experiment were previously described in detail (21). No marked changes
in RBF measurements before or after these final tests were found. They
averaged <3%.
RBF autoregulatory efficiency was investigated by stepwise aortic
constrictions inducing 5-mmHg decrements in RPP from spontaneous mean
arterial pressure (MAP) down to 60 mmHg and by measurement of RBF
during 30-s periods. No measurements during increased MAP were undertaken.
Experimental protocol.
After 60- to 90-min equilibration, baseline measurements of
hemodynamics and renal function were carried out during three control
periods of 20-min duration each. Control autoregulatory maneuvers
(A1) were undertaken during the second control period. After the control periods, vehicle, L-NAME (Sigma
Chemical), nifedipine (Bayer), or verapamil (Isoptin, Knoll) was
administered intravenously. Moreover, iberiotoxin (Sigma Chemical), a
specific inhibitor of Ca2+-activated K+
channels, was infused in the renal artery of some rats. After a brief
equilibration period, six experimental periods of 20-min duration each
were then undertaken. Autoregulatory maneuvers were carried out during
the second (A2) and fifth (A3) experimental periods. After the first three experimental periods, an intravenous infusion of nifedipine was started in some of the rats previously injected with L-NAME, whereas L-NAME was
injected intravenously in some of the rats infused with nifedipine
since 1 h. The two drugs were simultaneously coadministered in other
rats after the last control period. In each rat, blood was periodically
sampled from the femoral artery, and urine was separately collected
from each kidney at the end of each period.
The following experimental groups were considered.
1) Eight rats were treated with vehicle (Vehicle; 0.85% NaCl
in bidistilled water injected iv as 200 µl/kg body wt or infused iv,
at a rate of 5 µl/min).
2) Seven rats were injected intravenously with
L-NAME at a dose of 0.3 mg/kg body wt (L-NAME).
3) Seven rats were infused intravenously with nifedipine at a
dose of 4.5 µg · kg body
wt
1 · min
1 (Nifedipine).
4) Eight rats were first injected intravenously
with L-NAME at a dose of 0.3 mg/kg body wt, and, after 1 h,
were infused intravenously with nifedipine at a dose of 4.5 µg · kg body
wt
1 · min
1
(L-NAME/Nifedipine).
5) Eight rats were infused intravenously with nifedipine at a
dose of 4.5 µg · kg body
wt
1 · min
1,
and, after 1 h, were injected intravenously with L-NAME at
a dose of 0.3 mg/kg body wt (Nifedipine/L-NAME).
6) Five rats were treated concomitantly at the start of the
first experimental period with nifedipine infused intravenously at a
dose of 4.5 µg · kg body
wt
1 · min
1,
and with L-NAME injected intravenously at a dose of 0.3 mg/kg body wt. Five other rats, subjected to a similar protocol, were infused intravenously with verapamil at a dose of 10 or 15 µg · kg body
wt
1 · min
1
instead of nifedipine [Nifedipine (Verapamil)+ L-NAME
coadministered]. The results were combined because there were no
marked differences between hemodynamic effects of the two
Ca2+ channel blockers.
7) In addition, seven rats were infused intravenously with
nifedipine at a dose of 18 µg · kg body
wt
1 · min
1
(n = 4) or with verapamil at a dose of 40 µg
· kg body
wt
1 · min
1
(n = 3), and were injected intravenously with
L-NAME at a dose of 10 mg/kg body wt at the start of the
first experimental period. Results were again combined. Five rats were
injected intravenously only with L-NAME at a dose of 10 mg/kg body wt.
8) Eight other rats were infused intrarenally with iberiotoxin
at a dose of 3.4 µg/min at the start of the first experimental period
and, after 1 h, they were injected intravenously with
L-NAME at a dose of 0.3 mg/kg body wt.
9) Finally, five rats, also prepared as described above and
submitted to a similar experimental protocol, were infused
intravenously with angiotensin II (Sigma Chemical) at a dose of 150 ng · kg body
wt
1 · min
1
during the six experimental periods.
Only renal hemodynamics were studied in the animals of groups
7, 8, and 9.
Analytic methods.
Urine volume from each kidney was estimated by gravimetry assuming a
specific activity for water of 1.0. Inulin in plasma and urine samples
was determined by using the anthrone method.
Calculations and statistics.
Renal hemodynamic data were obtained from the left kidney, henceforth
referred to as the experimental kidney. Mean values of MAP and RBF were
obtained by averaging measurements carried out every 5 min on the
recordings, except during autoregulatory maneuvers. Renal plasma flow
(RPF) was estimated by using the following equation, RPF = RBF(1
Hct), where Hct represents the hematocrit. Renal vascular
resistance (RVR) was calculated as MAP/RBF, where MAP refers to the
pressure in the femoral artery. RPP was arbitrarily equated to the
femoral MAP. The relationship between RBF and MAP was assessed
independently by two investigators in each experiment by applying
progressively linear regressions by using the least squares method, as
modified from the method described by Persson et al. (29). The
magnitude of the correlation coefficient, the slope, and the intercept
of the best-fit regression lines, as well as the amplitude of the
change in RBF, were taken as criteria to attribute a data point with a
precision of 5 mmHg to one of the following components of the RBF-MAP
relationship: 1) the autoregulatory plateau, which corresponds
to the pressure range in which autoregulatory efficiency was maximal;
2) a subautoregulatory zone, which corresponds to the pressure
range in which some autoregulatory efficiency persisted; 3) the
absence of autoregulation, which corresponds to the pressure range in
which RBF changes were fully pressure dependent. The perfusion
pressure, corresponding to the lower limit of the autoregulatory
plateau, was defined as the pressure limit of efficient RBF
autoregulation (PA). The perfusion pressure, corresponding
to disappearance of RBF autoregulation, was defined as the pressure
limit of no RBF autoregulation (Po). GFR and filtration
fraction (FF) were calculated as usually described. GFR and FF were
determined during two control and four experimental periods, which
respectively corresponded to the pre- and postautoregulatory periods as
indicated in the experimental protocol. Data obtained during
autoregulatory periods (A1, A2, A3)
were not considered because autoregulatory maneuvers transiently
reduced GFR from the experimental kidney.
One-way ANOVA was applied for multiple intergroup comparisons, and
one-way ANOVA for repeated measurements was applied for multiple
intragroup comparisons, followed by a post hoc t-test. Two-way
ANOVA for repeated measurements on one factor was used to compare the
effects of different treatments on RVR. Significant differences were
identified with the Newman-Keuls test for multiple pairwise comparisons
(37). The paired t-test was used for a single comparison within
a group. All results are presented as means ± SE. P < 0.05 was considered to be statistically significant.
 |
RESULTS |
General observations.
Body weight and left and right kidney weights averaged 292 ± 1, 1.156 ± 0.015, and 1.141 ± 0.012 g, respectively, in 73 rats. Plasma
sodium, potassium, and osmolality were in the normal range for rats
(data not shown). They did not differ from baseline at the end of the
experiment. Table 1 presents the mean
baseline values for Hct, MAP, RBF, RPF, RVR, GFR, and FF in rats
treated with vehicle (n = 8), L-NAME (n = 7), Nifedipine (n = 7), L-NAME/Nifedipine (n = 8), Nifedipine/L-NAME (n = 8), or
Nifedipine (Verapamil)+L-NAME coadministered (n = 10). The baseline values did not differ statistically among these
experimental groups [not significant (NS), ANOVA]. At the
end of the experiment, Hct averaged 44 ± 0.7, 44 ± 0.7, 45 ± 0.7, 46 ± 0.6, 45 ± 0.8, and 45 ± 0.9% in the respective experimental groups (NS from baseline, paired t-test).
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Table 1.
Baseline values for hematocrit, mean arterial pressure, renal blood and
plasma flow, renal vascular resistance, glomerular filtration rate and
filtration fraction in rats treated with vehicle, L-NAME, or nifedipine
alone, with L-NAME and nifedipine or nifedipine and L-NAME combined
during the second experimental hour, or with nifedipine (or verapamil)
and L-NAME coadministered
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Temporal evolution of MAP and RBF.
Figure 1 illustrates the temporal
variations of MAP (A) and RBF (B) starting before each
treatment until the end of the experiment. As shown in Fig. 1A,
MAP did not change after injection of vehicle, nor with time. MAP
increased slightly, but transiently, by 4 mmHg in the rats injected
with L-NAME alone and returned to baseline after 1-h
treatment without further changes. In contrast, MAP decreased
progressively and significantly in the rats infused only with
nifedipine, and averaged 94 ± 4 mmHg at the end of the experiment. In
the L-NAME/Nifedipine group, MAP increased again slightly
and rapidly during the first hour after injection of L-NAME
but decreased progressively and significantly to 98 ± 6 mmHg after
superimposition of the infusion of nifedipine. In the Nifedipine/L-NAME group, MAP decreased significantly to 105 ± 4 mmHg during the first hour of infusion of nifedipine but
increased rapidly and significantly after the superimposed injection of L-NAME, averaging 111 ± 4 mmHg at the end of the
experiment. Finally, MAP did not change during coadministration of
nifedipine (or verapamil) and L-NAME. As shown in
Fig.1B, RBF did not vary after injection of vehicle, nor with
time. However, RBF decreased rapidly and significantly after the
injection of L-NAME alone, and, thereafter, remained
relatively stable, averaging 6.65 ± 0.43 ml/min at the end of the
experiment. Conversely, RBF increased rapidly, but not significantly,
in the rats infused only with nifedipine, averaging 8.47 ± 0.76 ml/min at the end of the experiment. In the
L-NAME/Nifedipine group, RBF decreased again rapidly and
significantly after the injection of L-NAME but increased
significantly to 7.00 ± 0.30 ml/min after superimposition of the
infusion of nifedipine. In the Nifedipine/L-NAME group, RBF
increased slightly during the infusion of nifedipine and then decreased
rapidly and significantly to 6.84 ± 0.68 ml/min after the
superimposed injection of L-NAME. After coadministration of
nifedipine (or verapamil) and L-NAME, RBF decreased more
slowly and stabilized at 6.28 ± 0.36 ml/min within 1 h.

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Fig. 1.
Temporal variations of mean arterial pressure (MAP; in mmHg; A)
and renal blood flow (RBF; in ml/min; B) in rats treated
intravenously with vehicle ( ; n = 8), 0.3 mg/kg body wt
NG-nitro-L-arginine-methyl ester
(L-NAME; ; n = 7), 4.5 µg · kg
body
wt 1 · min 1
nifedipine ( ; n = 7), 0.3 mg/kg body wt L-NAME
and, during second experimental hour, with 4.5 µg · kg body
wt 1 · min 1
nifedipine ( ; n = 8), 4.5 µg · kg body
wt 1 · min 1
nifedipine and, at the start of the second experimental h, with 0.3 mg/kg body wt L-NAME ( ; n = 8), or 4.5 µg · kg body
wt 1 · min 1
nifedipine (or 10-15 µg · kg body
wt 1 · min 1
verapamil) coadministered with 0.3 mg/kg body wt L-NAME at
the start of the experimental periods ( ; n = 10). Values are
means ± SE and are illustrated for last control and 6 experimental
periods. They were arbitrarily set in the middle of each 20-min period.
Some SE are too small to be shown. Differences in temporal variations
of MAP and RBF were evaluated by ANOVA for repeated measurements
followed by Newman-Keuls test between control and experimental values
(statistical significance: * P < 0.05) and between
experimental value at the end of the first hour treatment and last 3 experimental values (statistical significance: + P < 0.05).
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In each of the six experimental groups, RPF followed a similar temporal
evolution as RBF, whereas GFR did not differ significantly from
baseline shown in Table 1, except that GFR increased to 1.235 ± 0.194 ml/min in the rats infused with nifedipine alone (NS). On the other
hand, FF was significantly enhanced to 0.29 ± 0.02 (P < 0.025) and to 0.41 ± 0.03 (P = 0.01) in the rats treated with
L-NAME alone or with nifedipine (or verapamil) and
L-NAME coadministered, respectively.
Changes in RVR.
Table 2 presents RVR before and 1 and 2 h
after treatment was started. Control values of RVR did not differ
statistically among the six experimental groups. There were no changes
in RVR after treatment with vehicle. In contrast, RVR was significantly increased 1 and 2 h after injection of L-NAME and decreased
progressively and significantly during the infusion of nifedipine. RVR
was significantly enhanced during the first experimental hour in the
L-NAME/Nifedipine group but was decreased to baseline
levels after the superimposed infusion of nifedipine. RVR was reduced
below baseline during the first experimental hour in the
Nifedipine/L-NAME group and was increased significantly
above baseline levels after the superimposed injection of
L-NAME. Finally, RVR was significantly enhanced 1 and 2 h
after starting coadministration of nifedipine (or verapamil) and
L-NAME. Figure 2 summarizes the
effects of the different treatments on RVR, expressed in percentage of
baseline values. Changes in RVR differed significantly between each
group, except between Vehicle and L-NAME/Nifedipine, and
between L-NAME alone and Nifedipine/L-NAME or
Nifedipine (Verapamil)+L-NAME coadministered. These two
last groups also did not differ between each other. Similar statistical results were obtained when the changes in RVR were expressed in absolute values.

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Fig. 2.
Percent changes ( ) from baseline in renal vascular resistance (RVR)
occurring 2 h after intravenous treatment with vehicle (filled bar),
0.3 mg/kg body wt L-NAME (open bar), 4.5 µg · kg body
wt 1 · min 1
nifedipine (hatched bar), 0.3 mg/kg body wt L-NAME and 4.5 µg · kg body
wt 1 · min 1
nifedipine (vertically striped bar), 4.5 µg · kg
body
wt 1 · min 1
nifedipine and 0.3 mg/kg body wt L-NAME (crosshatched bar),
or 4.5 µg · kg body
wt 1 · min 1
nifedipine (or 10-15 µg · kg body
wt 1 · min 1
verapamil) coadministered with 0.3 mg/kg body wt L-NAME
(horizontally striped bar). Values are means ± SE. RVR
changes averaged 2 ± 2, 27 ± 4, 19 ± 4, 2 ± 6, 24 ± 9, and 22 ± 5%, respectively, and differed significantly among groups
(P < 0.001, ANOVA). All RVR changes differed significantly
between each other, except between Vehicle and
L-NAME/Nifepidine, between L-NAME alone and
Nifedipine/L-NAME as well as Nifedipine
(Verapamil)+L-NAME coadministered, and between
Nifedipine/L-NAME and Nifedipine
(Verapamil)+L-NAME coadministered (* P < 0.05, Newman-Keuls test).
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RBF autoregulation.
Figure 3 illustrates the RBF-MAP
relationship 30 min before (A1) and 30 (A2) and
90 min (A3) after treatment with vehicle (A),
L-NAME (B), or nifedipine (C) alone was
started. As shown in Fig. 3A, the three autoregulatory curves
were superimposed and were essentially similar in the vehicle-treated
rats. The lower pressure limit of the RBF autoregulatory plateau, as
well as the pressure limit of no RBF autoregulation, did not change after vehicle treatment, or with time (Table
3). After injection of L-NAME
(Fig. 3B), the RBF autoregulatory plateau was maintained but
was shifted downward from control (A2 and A3).
Moreover, the autoregulatory plateau extended into the
subautoregulatory zone so that the autoregulatory pressure limit was
significantly lowered by >10 mmHg (Table 3). As illustrated in Fig.
3C, changes in RBF autoregulation occurred progressively in the
rats continuously infused with nifedipine. After 30 min, the RBF-MAP
relationship was slightly shifted upward from control (A2).
RBF autoregulation was maintained in five of the seven animals.
Thereafter, the autoregulatory plateau was abolished and the RBF-MAP
relationship was shifted upward and leftward from control
(A3). Figure 4, A and
B, illustrates, respectively, the RBF-MAP relationship in the
rats pretreated with L-NAME or with nifedipine during the
first experimental hour (A2), and, thereafter, submitted to
a combined treatment (A3). The RBF-MAP relationship and the
autoregulatory pressure limits, 30 min before (A1) and
after (A2) pretreatment, were as described above (Table 3).
As shown in Fig. 4A, the autoregulatory plateau was maintained
during the superimposed infusion of nifedipine but was shifted toward
control (A3). The autoregulatory pressure limit remained
significantly lowered (Table 3). As shown in Fig. 4B, RBF
remained autoregulated after the superimposed injection of
L-NAME, but the autoregulatory plateau was shifted downward (A3). The autoregulatory pressure limit was now less
lowered (Table 3). In rats submitted to the simultaneous
coadministration of nifedipine (verapamil) and L-NAME, the
RBF autoregulatory plateau was again maintained but was only slightly
shifted downward from control (Fig. 4C). It did not extend into
the subautoregulatory zone (A2 and A3), so that
pressure limits of the autoregulatory and subautoregulatory zones did
not vary significantly from baseline (Table 3). Note that
coadministration of 9 µg · kg body
wt
1 · min
1
nifedipine with the low dose of L-NAME (n = 4)
markedly attenuated RBF autoregulation (data not shown). The
autoregulatory pattern (A2 and A3) was as
illustrated below in Fig. 5B.

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Fig. 3.
RBF-MAP relationships during stepwise decrements by 5 mmHg in MAP in
rats treated intravenously with vehicle (n = 8; A), 0.3 mg/kg body wt L-NAME (n = 7; B), or 4.5 µg · kg body
wt 1 · min 1
nifedipine (n = 7; C). Autoregulatory curves were
determined 30 min before (A1; ), and 30 (A2;
) and 90 min (A3; ) after treatment was started.
Values are means ± SE.
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Table 3.
Pressure limits of efficient and no RBF autoregulation in rats 30 min
before and 30 and 90 min after vehicle, L-NAME, or nifedipine treatment
alone or combined during the 2nd experimental hour, or after a
concomitant administration of nifedipine (or verapamil) and L-NAME,
was started
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Fig. 4.
RBF-MAP relationships during stepwise decrements by 5 mmHg in MAP in
rats treated intravenously at 1-h interval with 0.3 mg/kg body wt
L-NAME and 4.5 µg · kg body
wt 1 · min 1
nifedipine (n = 8; A) or with 4.5 µg · kg body wt
1 · min 1
nifedipine and 0.3 mg/kg body wt L-NAME (n = 8;
B), or treated concomitantly with 4.5 µg · kg body
wt 1 · min 1
nifedipine (or 10-15 µg · kg body
wt 1 · min 1
verapamil) and 0.3 mg/kg body wt L-NAME (n = 10;
C). Autoregulatory curves were determined 30 min before
(A1; ), and 30 (A2; ) and 90 min
(A3; ) after treatment was started. Values are means ± SE.
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Fig. 5.
RBF-MAP relationships during stepwise decrements by 5 mmHg in MAP in
rats injected intravenously with 10 mg/kg body wt L-NAME
(n = 5; A) or treated concomitantly with 18 µg · kg body
wt 1 · min 1
nifedipine (or 40 µg · kg body wt
1 · min 1
verapamil) and 10 mg/kg body wt L-NAME (n = 7;
B). Autoregulatory curves were determined 30 min before
(A1; ), and 30 (A2; ) and 90 min
(A3; ) after treatment was started. Values are means ± SE. MAP and RBF averaged, respectively, 113 ± 4 and 133 ± 3 mmHg
(P = 0.017), and 7.59 ± 0.21 and 3.70 ± 0.17 ml/min
(P < 0.001) before and after L-NAME alone, and
109 ± 3 and 103 ± 3 mmHg (not significant), and 7.00 ± 0.35 and
4.06 ± 0.48 ml/min (P < 0.001) before and after
simultaneous coadministration.
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For comparative purposes, autoregulatory experiments were also
undertaken in rats injected intravenously with a high dose of
L-NAME alone (n = 5), or coadministered with very
high doses of nifedipine or verapamil (n = 7). As shown in Fig.
5A, the RBF autoregulatory plateau was maintained after
injection of L-NAME alone but was now markedly shifted
downward from control and extended into the subautoregulatory zone
(A2 and A3). There were no changes with time.
Pressure limits of the autoregulatory plateau (PA) and
subautoregulatory zone (Po) averaged, respectively,
100 ± 4 (A1), 84 ± 4 (A2), and 81 ± 5 mmHg (A3) (P = 0.001, ANOVA and Bonferroni
test), and 85 ± 4 (A1), 64 ± 2 (A2), and 64 ± 3 mmHg (A3) (P = 0.002). As illustrated in Fig. 5B, the RBF autoregulatory plateau was disrupted in the rats treated with high doses of either Ca2+channel blocker and L-NAME. RBF tended to
become pressure dependent at each pressure step, and autoregulatory
pressure limits could not be determined.
RBF autoregulation was also investigated in eight rats during
intrarenal infusion of iberiotoxin followed by an intravenous injection
of a low dose of L-NAME. As shown in Table
4, autoregulatory pressure limits,
PA and Po, did not change from control under these conditions. The autoregulatory profile was indeed similar to
control during the intrarenal infusion of iberiotoxin and was only
shifted downward from control after L-NAME. Note that the increase in MAP and RVR was more marked when L-NAME was
injected after pretreatment with iberiotoxin than after
L-NAME alone (see Fig. 1A and Table 2).
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|
Table 4.
Preautoregulatory values for Hct, MAP, RBF, RVR, and PA and
Po in 8 rats before and during intrarenal infusion of
iberiotoxin, followed by an injection of a low dose of L-NAME
|
|
Figure 6 illustrates the RBF-MAP
relationship in five other rats infused intravenously with 150 ng · kg body
wt
1 · min
1
of angiotensin II to increase MAP and to lower RBF. Under these conditions, the RBF-MAP relationship was markedly shifted downward from
control at each pressure decrement, 30 (A2) and 90 min
(A3) after the infusion of angiotensin II was started. The
RBF autoregulatory plateau was maintained, but the subautoregulatory
zone was markedly extended to the right. The lower autoregulatory
pressure limit was now significantly reset from 99 ± 2 (A1) to 115 ± 5 (A2; P < 0.01), and to 111 ± 6 mmHg (A3; P < 0.025, ANOVA and Bonferroni t-test). In contrast, the lower pressure
limit of the subautoregulatory zone did not vary, averaging 82 ± 1 (A1), 80 ± 5 (A2), and 83 ± 4 mmHg
(A3), respectively (NS, ANOVA).

View larger version (17K):
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[in a new window]
|
Fig. 6.
RBF-MAP relationships during stepwise decrements by 5 mmHg in MAP in 5 rats infused intravenously with 150 ng · kg body
wt 1 · min 1
angiotensin II. Autoregulatory curves were determined 30 min before
(A1; ), and 30 (A2; ) and 90 min
(A3; ) after infusion of angiotensin II was started.
Values are means ± SE. Baseline and end MAP and RBF averaged,
respectively, 116 ± 3 and 130 ± 2 mmHg (P < 0.005), and
8.68 ± 0.85 and 4.63 ± 0.15 ml/min (P < 0.005).
|
|
 |
DISCUSSION |
Treatment with the low dose of L-NAME selected after
preliminary dose-response tests diminished renal blood circulation by 25% in our rats, as previously found by Walder et al. (36). The renal
vasoconstriction was essentially due to a reduction in RBF because
blood pressure was only mildly and transiently elevated. The decrease
in RBF was stabilized within 15 min after injection of
L-NAME and remained so for more than 2 h, underlining the
effectiveness of the inhibition of the synthesis and release of
intrarenal NO throughout the time period of the experiment. The
concomitant increase in FF indicated that the vasomotor tone of the
pre- as well as of the postglomerular vessels was affected. There were,
however, no marked changes in GFR, nor in salt and water excretion
(data not shown). These conditions were considered to reflect an acute
attenuation of NO synthesis and to be of interest because they evoked
some of the pathophysiological consequences encountered during the
early stages of vascular and renal endothelial dysfunction in humans
(12, 30). In comparison, the renal vasoconstriction was more pronounced
after the acute and systemic administration of a high dose of
L-NAME (4, 6). MAP was then elevated by 20-30 mmHg,
and RBF was reduced by 50%. These conditions were assimilated to an
acute blockade of NO synthesis (5). To test the influence of
voltage-dependent Ca2+ channels on the hemodynamic
consequences of the attenuated synthesis of NO,
dihydropyridine-sensitive Ca2+ channels were blocked with
nifedipine. The dose of nifedipine was sufficiently effective to
enhance slightly and rapidly RBF until the end of the experiment and to
reduce blood pressure near the autoregulatory pressure range, thus
producing a true renal vasodilation. It was slightly higher than the
dose range of nifedipine that normalized blood pressure and abolished
RBF autoregulation in anesthetized, spontaneously hypertensive rats
(22). Higher doses of nifedipine reduced blood pressure below
autoregulatory limits and diminished RBF (unpublished
observations). Verapamil, another type of Ca2+
channel blocker, was infused in some rats to test the reproducibility of the nifedipine findings. It should be pointed out that very high
doses of Ca2+ channel blockers may interfere with other
Ca2+ transport systems (15).
An important feature of our study concerned the action of nifedipine on
the preestablished renal vasoconstriction induced by the attenuated
synthesis of NO. The renal vasoconstriction was, indeed, completely
reversed by the Ca2+ channel antagonist. Interestingly, the
effect of nifedipine was not limited to the systemic circulation, as
displayed by the decrease in blood pressure, but involved also the
renal vasculature because the reduction in RBF was reversed, at least
in part, by the Ca2+ channel antagonist. These findings are
in agreement with recent observations in human volunteers infused
intravenously with
NG-monomethyl-L-arginine, in whom
nifedipine completely restored systemic hemodynamics and reversed
partially renal vasoconstriction (13). Pretreatment with nifedipine or
simultaneous coadministration of nifedipine and the low dose of
L-NAME, however, did not hinder the increase in RVR. In
conscious rats, Baylis et al. (5) found that a high dose of verapamil
rapidly administered after the injection of a high dose of
L-NAME did not affect renal vasoconstriction. In contrast,
these authors observed that blood pressure and RVR did not change after
concomitant administration of the two drugs. In our anesthetized rats,
blood pressure also did not increase after coadministration of high
doses of nifedipine, or verapamil, and of L-NAME. However,
RBF decreased by 42% so that RVR was significantly enhanced.
Differences between the two studies could, at least in part, relate to
different experimental conditions, such as the use of conscious or
anesthetized rats.
An interesting finding of our study concerned the RBF-MAP relationship
during inhibition of NO synthesis. The autoregulatory pattern was then
maintained, but the pressure limit of the autoregulatory plateau was
significantly lowered by ~15 mmHg, whether a low or a high dose of
L-NAME was injected. This observation contrasts with
previous studies undertaken in conscious and anesthetized dogs as well
as in anesthetized rats indicating that the autoregulatory pressure
limit after treatment with L-arginine competitors was either unchanged or slightly reset to a higher pressure (3, 6, 23, 34).
Nevertheless, some evidence for improved autoregulation has been
reported in vivo, for example, in juxtamedullary glomeruli of the
hydronephrotic kidney in anesthetized female rats and in the kidney of
normal and two-kidney, one-clip, hypertensive anesthetized rats (16,
35). Also, a pressure-dependent increase in active wall tension of
interlobular arteries of in vitro perfused juxtamedullary nephrons was
found to be amplified during inhibition of NO synthesis (20).
Furthermore, NO donors have been shown to modulate autoregulatory responses of arcuate arteries and afferent arterioles in the in vitro
blood-perfused juxtamedullary nephron preparation (9). However, it
could be argued that resetting of the autoregulatory pressure limit to
a lower pressure was artifactual in the presence of a reduced RBF. This
possibility is unlikely because resetting to a higher pressure limit of
the autoregulatory plateau, by 15 mmHg, could also be disclosed in our
rats infused with angiotensin II. RBF was then decreased by 50%, and
blood pressure was markedly increased. Thus our autoregulatory
methodology was appropriate to detect variations of autoregulatory
pressure limits in the presence of a reduced renal blood circulation.
Autoregulation of blood flow in the kidney depends on the interplay of
the myogenic and tubuloglomerular feedback mechanisms (27). The
myogenic mechanism allows rapid and appropriate adjustments of RVR,
occurring in the preglomerular vasculature and predominantly in the
afferent arteriole (2, 11, 26). These adjustments are regulated by the
level of intracellular Ca2+, which depends, in part, on
Ca2+ influx through voltage-dependent Ca2+
channels because their blockade markedly attenuates autoregulatory responses in vivo as well as in vitro, and as also shown by the present
results (11, 17, 22, 28). In our study, the basic pattern of the
RBF-MAP relationship was maintained whether voltage-dependent Ca2+ channel activity was inhibited before or after
administration of the low dose of L-NAME. However,
resetting of the pressure limit of the autoregulatory plateau was
significantly reduced by pretreatment with nifedipine and was abolished
by simultaneous coadministration of nifedipine (or verapamil) and the
low dose of L-NAME. RBF autoregulation was even markedly
attenuated by increasing the dose of nifedipine, and during
coadministration of very high doses of nifedipine or verapamil and the
high dose of L-NAME. These findings suggest thus that
extension of the autoregulatory plateau occurring during inhibition of
NO synthesis was dependent, at least in part, on influx of
Ca2+ through dihydropyridine-sensitive Ca2+
channels. Although it cannot be excluded that our observations might be
coincidental, it should be pointed out that intrarenal blockade of
Ca2+-activated K+ channels also prevented the
autoregulatory resetting induced by inhibition of NO synthesis. These
observations do support interactions between the NO-cGMP pathway and
voltage-dependent Ca2+ channels involving
Ca2+-activated K+ channels (1). The role of
angiotensin II must also be taken into consideration because
angiotensin II interferes with NO and because renal vascular reactivity
to angiotensin II depends, in part, on voltage-dependent
Ca2+ channels (27, 31). Moreover, angiotensin II is
implicated in the setting of the autoregulatory pressure limit as shown
in our study. It is thus possible that extension of the autoregulatory plateau was facilitated during inhibition of NO synthesis, because mechanisms activating the synthesis of renin in afferent arteriolar cells are then ineffective (32).
The tubuloglomerular feedback mechanism that is "essential for
maximum autoregulatory efficiency" could also be involved in the
extension of the autoregulatory plateau detected under our experimental
conditions. Indeed, sensitivity and reactivity of the tubuloglomerular
mechanism are increased during inhibition of intrarenal NO synthesis
(27, 33). However, it is not known at present to what
extent the autoregulatory resetting occurring in our rats was
influenced by enhancement of tubuloglomerular feedback activity.
Interestingly, Ca2+ channel blockade decreases the
sensitivity of this mechanism (25). Therefore, hindrance by
Ca2+ channel blockade of the autoregulatory pressure limit
resetting induced by L-NAME may implicate changes in
myogenic as well as in tubuloglomerular feedback mechanisms. Their
respective contribution remains to be further investigated.
In summary, attenuation of endothelial synthesis of NO with a low dose
of L-NAME induced a significant renal vasoconstriction and
an enhanced efficiency of RBF autoregulation. The renal
vasoconstriction was reversed by inactivation of voltage-dependent
Ca2+ channels with nifedipine, whereas autoregulatory
resetting was hindered by prior or concomitant administration of
nifedipine (or verapamil) and L-NAME. RBF autoregulation
was also more efficient after blockade of NO synthesis with a high dose
of L-NAME but was attenuated by coadministration of very
high doses of nifedipine or verapamil. Moreover, intrarenal blockade of
Ca2+-activated K+ channels prevented the
autoregulatory resetting induced by L-NAME. In conclusion,
these hemodynamic findings suggest that NO and/or the NO-cGMP pathway
exerts inhibitory effects on the activity of voltage-dependent
Ca2+ channels, which may be important to modulate RVR in
the rat. Our pharmacological manipulations also suggest that the
release of intrarenal NO may reduce the efficiency of RBF
autoregulation at renal perfusion pressures below 100 mmHg.
 |
ACKNOWLEDGEMENTS |
We appreciate the dedicated technical assistance of B. Blairon, F. Coulon, and V. Jenart and the secretarial assistance of M. Fontaine. We
thank Bayer AG Belgium for the generous gift of nifedipine. This study
is dedicated to the late Dr. C. W. Gottschalk.
 |
FOOTNOTES |
This study was presented in part at the 2nd European Kidney Research
Forum in Baveno, Italy, on May 24-26, 1996, and at the 29th Annual
Meeting of the American Society of Nephrology in New Orleans, LA, on
November 3-6, 1996. It appeared in abstract form in Kidney
Int 50: 1785, 1996, and in J Am Soc Nephrol 7: 1566, 1996. E. Joly is a Collaborateur scientifique of the Fonds National de la
Recherche Scientifique.
Present address of L. Ladrière: Laboratoire de Médecine
Expérimentale, Faculté de Médecine de
l'Université Libre de Bruxelles, route de Lennik, 808, 1070 Bruxelles, Belgium.
Present address of P. Fourmanoir: Baxter R&D Europe, 7 rue du
Progrès, 1400 Nivelles, Belgium.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: R. A. Kramp,
Service de Physiologie et Pharmacologie, Faculté de
Médecine et de Pharmacie, Université de Mons-Hainaut, 7000 Mons, Belgium (E-mail: Ronald.Kramp{at}umh.ac.be).
Received 19 February 1999; accepted in final form 24 November
1999.
 |
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