Departments of 1 Physiology and of 2 Pediatrics, Tulane University School of Medicine, New Orleans, Louisianna 70112
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ABSTRACT |
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The
relative contributions of ANG II and bradykinin (BK) to the renal
regional blood flow responses during angiotensin-converting enzyme
(ACE) inhibition remain unclear. This study was performed to evaluate
renal cortical (CBF) and medullary blood flow (MBF) responses to
intrarterial administration of enalaprilat (33 µg · kg1 · min
1) after
blockade of the ANG II AT1 receptors with candesartan (100 µg) in 7 dogs fed a low-salt diet (0.01%) for 5 days. Laser-Doppler flowmetry was used to measure relative changes in CBF and MBF. Candesartan alone increased CBF (+20 ± 2%) and MBF (+22 ± 7%). Enalaprilat infusion after candesartan administration resulted in
further increases in both CBF (+21 ± 5%) and MBF (+41 ± 8%). However, the relative changes in MBF were significantly greater (P < 0.01) than those in CBF. Administration of the BK
B2 receptor blocker icatibant (300 µg) after enalaprilat
returned CBF and MBF to values seen with candesartan alone. These data
support a substantive role for BK potentiation during ACE
inhibitor-induced renal vasodilation in dogs maintained on a low-sodium
diet, with a relatively greater effect on MBF compared to CBF.
candesartan; icatibant; enalaprilat; laser-Doppler flowmetry; angiotensin-converting enzyme
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INTRODUCTION |
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THE RELATIVE CONTRIBUTIONS of reduced ANG II and increased bradykinin (BK) levels to the renal vasodilatory responses to angiotensin-converting enzyme (ACE) inhibition have remained uncertain (2, 3, 6). We have recently reported that administration of a selective BK B2 receptor antagonist (icatibant) had no significant effect on the renal blood flow (RBF) responses to administration of the ACE inhibitor, enalaprilat, in dogs fed a normal salt diet (12). In these dogs, administration of icatibant did not significantly alter the observed increases in RBF, cortical (CBF), and medullary blood flow (MBF) in response to enalaprilat. The observations indicated that BK potentiation did not play a major role in these vasodilatory responses to ACE inhibition, thus supporting a primary role for reduced ANG II formation, in dogs receiving a normal salt diet. In contrast, in dogs fed a low-salt diet (0.01%, 5 days), pretreatment with icatibant prevented the renal vasodilation following administration of enalaprilat, indicating that these responses were dependent to a much greater extent on activation of BK B2 receptors (12). In those studies, enalaprilat was administered intra-arterially into the kidney to block local generation of ANG II while minimizing the other systemic effects of ACE inhibition. However, it was possible that renal responses to B2 receptor blockade observed during local administration of the ACE inhibitor could be influenced by systemically formed circulating ANG II acting on renal ANG II receptors. Additionally, sodium restriction has been shown to increase the activity of the renin-angiotensin system, and this could have led to further activation or upregulation of AT1 receptors, which have been shown to be the primary mediators of ANG II effects in the canine kidney (7, 17). Thus the relative responses of CBF and MBF caused by reduced ANG II vs. enhanced bradykinin remained undetermined.
In the present study, we further examined renal responses to ACE inhibition in dogs fed a low-salt diet, under conditions of selective ANG II receptor blockade with candesartan, a high-affinity, nonsurmountable AT1 receptor blocker (1, 8, 9). To determine if the residual ACE inhibition-induced vasodilation was due to enhanced B2 receptor activation, the effects of the B2 receptor blocker, icatibant (10, 16), were examined. The results allow a more complete characterization of the relative contributions of reduced ANG II activity vs. enhanced BK actions to the vasodilatory effects of ACE inhibition under conditions of reduced salt intake.
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METHODS |
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Experiments were conducted on 7 mongrel dogs, weighing 18-20 kg. The dogs were fed a low-salt diet (0.01%) for 5 days before the experiment, as previously described (12). On the day of the experiments, the dogs were anesthetized with pentobarbital sodium with an initial dose of 30 mg/kg body wt, intravenous, and supplementation was given as needed during the course of the experiment. Positive pressure ventilation was provided via a cuffed endotracheal tube with an artificial respirator at a rate of 18 strokes per min, and a stroke volume of 15 ml/kg body wt. Body temperature was maintained within the range of 99° to 101° F using an electric heating pad placed under the dog. Mean arterial pressure was measured from a catheter placed in the abdominal aorta inserted via the right femoral artery and recorded on a polygraph (model 7D; Grass Instruments, Quincy, MA). The left femoral artery was cannulated for the collection of blood samples. The femoral and jugular veins were cannulated for the administration of drugs, infusion of saline and inulin solutions, and additional doses of anesthesia as needed.
The left kidney was exposed through a flank incision, and the renal artery separated from the surrounding tissue. Renal denervation was performed by cutting all visible nerves projecting to the kidney from the aortico-renal ganglion. The rationale for renal denervation was to minimize the effects of alterations in renal sympathetic activity due to possible changes in arterial pressure following administration of candesartan and/or enalaprilat. The ureter was cannulated for the collection of urine samples. RBF was measured with an electromagnetic flow probe placed around the renal artery and connected to a square wave flowmeter (Carolina Medical Electronics, Kings, NC). The zero flow was obtained at the beginning and the end of the experiment by momentarily occluding the renal artery. A curved 23-gauge needle was inserted in the renal artery and connected to a pressure transducer to measure renal arterial pressure (RAP). In the present study, RAP was not reduced with an occluding clamp; therefore, RAP and mean arterial pressure are essentially the same, and thus only RAP is reported. A catheter connected to this needle cannula was used for the direct renal infusion of drug solutions and heparinized saline at the rate of 0.4 ml/min.
A dual channel laser-Doppler flowmeter (LDF; Periflux 4001, Perimed, Stockholm, Sweden) with two needle probes (500 µm diameter) was used to measure relative changes in blood flows in the renal cortex
and medulla (4, 5). The cortical probe was
inserted to a depth of 5 mm into the kidney to position the tip in the mid-cortical region; the medullary probe was inserted to a depth of
~15 mm to position the tip at the junction of the inner and outer
medulla. At the end of each experiment, the positions of the tips of
the needle probes were confirmed by dissecting the kidney and locating
the needle tracts. The flow probes were calibrated with a motility
standard of a colloidal suspension of latex particles (10-µm
microspheres). Brownian motion of the latex particles provides a
standard value of 250 perfusion units (PU), with one PU corresponding to an analog output of 10 mV. The data are reported as percent of the
basal levels recorded during the control periods, although the absolute
PU values were also monitored and recorded. The zero flow recordings of
the LDF probes were determined by occluding the renal artery
momentarily at the beginning and the end of each experiment. To avoid
respiratory movement artifacts in the recording of LDF signals, the
kidney was maintained in a fixed position by placing it on a plastic
holder similar to that used for micropuncture studies. Care was taken
not to cause any changes in the basal RBF due to such
fixation. After the completion of surgery, a 2.5% solution of
inulin in normal saline was administered via the jugular vein for at
least 45 min before the beginning of the experimental protocol. An
initial dose of 1.6 ml/kg body wt was followed by a continuous infusion
of 0.03 ml · min1 · kg body
wt
1.
The experimental period was started with urine collections for two
consecutive 10-min control periods, with an arterial blood sample (2 ml) taken at the mid-point of each collection period. This was followed
by a 100-µg intra-arterial bolus of candesartan. This dose was
effective in blocking blood flow responses to 100 ng of ANG II
administered intra-arterially into the kidney of 8 dogs. The RBF
changes in response to ANG II prior to candesartan administration
averaged 63 ± 6%, compared to
0.03 ± 0% observed after candesartan administration, indicating complete blockade of
AT1 receptors. After a 10-min stabilization period
following candesartan administration, two 10-min collections of urine
were made. An intra-arterial infusion of enalaprilat was then started at a dose of 33 µg · kg
1 · min
1 and continued for the duration of the experiment.
After 10 min of stabilization, another two 10-min urine samples were
collected. The BK B2 receptor blocker icatibant was then
given as a 300-µg bolus systemic dose followed by a 10-min
stabilization and two 10-min clearance periods.
At the end of each experiment, the electromagnetic flow probe was calibrated in situ by timed collections of blood into a graduated cylinder from a catheter placed in the renal artery. The kidney was then removed, stripped of all surrounding tissue, blotted dry and weighed so that the calculated parameters could be expressed per gram of kidney weight. Flame photometry (Instrumentation Laboratory, Watertown, MA) was used to determine the sodium concentrations in plasma and urine. Inulin concentrations in the samples were determined by the anthrone colorimetric technique (Gilford Instruments, Oberlin, Ohio).
Values are reported as means ± SE. Statistical comparisons of
differences in the responses were conducted with the use of ANOVA,
followed by the Newman-Keuls test. Differences in the mean values were
deemed significant at P 0.05.
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RESULTS |
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Figures 1 and 2 summarize the RBF, renal vascular resistance
(RVR), CBF and MBF responses to candesartan, enalaprilat, and icatibant. As shown in Fig.
1A, ATI receptor
blockade with candesartan resulted in a significant increase in RBF by
21% (3.8 ± 0.4 to 4.6 ± 0.4 ml · min1 · g
1, P < 0.01). ACE inhibition after ATI receptor blockade further increased RBF to 5.7 ± 0.5 ml · min
1
· g
1 (24%, P < 0.001). B2
receptor blockade with icatibant returned RBF back to 5.0 ± 0.5 ml · min
1 · g
1. Figure
1B shows the effects of AT1 receptor blockade,
ACE inhibition, and B2 receptor blockade on RVR.
Candesartan significantly decreased RVR by 22% (34.6 ± 3.8 to
26.9 ± 3.3 mmHg · ml
1 · min
1 · g
1, P < 0.001), and enalaprilat resulted in a further decrease of 34% to
17.8 ± 2.4 mmHg · ml
1 · min
1 · g
1. RVR partially returned
with icatibant significantly below the RVR during enalaprilat to a
value that was not significantly different from that observed with
candesartan alone (21.9 ± 3.6 mmHg · ml
1 · min
1 · g
1, P < 0.001) although this value was
still significantly different from the control value.
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Figure 2 shows the responses in CBF and
MBF. As shown in Fig. 2A, AT1 receptor blockade
increased CBF (+20 ± 2%, P < 0.05), and
administration of enalaprilat resulted in a further increase of 21%
(to +41 ± 8%). Blockade of B2 receptors returned CBF
to +24 ± 8%, an average not significantly different from those
observed with candesartan administration alone. Figure 2B
summarizes MBF responses to candesartan, enalaprilat, and icatibant.
Candesartan increased MBF by 22 ± 7% (P < 0.05). Enalaprilat infusion following candesartan further increased MBF
by 40% (to +61 ± 10%, P < 0.01). Blockade of
B2 receptors returned MBF to levels not different from
those seen prior to ACE inhibition (+14 ± 8%). The relative responses of MBF to enalaprilat after candesartan administration were
noted to be significantly greater than those of CBF (+21% vs. +41%,
respectively, P < 0.01).
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Table 1 summarizes the responses of RAP,
glomerular filtration rate (GFR), urine flow (UV), and sodium excretion
(UNaV). AT1 receptor blockade decreased RAP
with a further decrease due to subsequent administration of
enalaprilat. B2 receptor blockade using icatibant returned
the responses to levels not different from those observed with
candesartan administration alone. GFR, UV, UNaV, and
fractional excretion of sodium (FENa%) were not significantly altered by any of the treatments (Table 1).
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DISCUSSION |
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In this study, we have evaluated the total and regional blood flow responses to ACE inhibition in the presence of AT1 receptor blockade and how these are altered by blockade of B2 receptors. The objective was to examine the relative contributions of reduced ANG II influences and enhanced BK effects in the observed vasodilatory responses to ACE inhibition. Intrarenal blockade of ANG II AT1 receptors with candesartan significantly increased RBF, CBF and MBF, with the magnitude of the changes in each parameter not significantly different from each other, indicating that at the administered dose that was sufficient to effectively block ANG II-mediated vasoconstriction, CBF and MBF were equally sensitive to ANG II receptor blockade. This observation also confirmed a significant tonic role for ANG II in regulating total and regional RBF during sodium restriction. Subsequent administration of enalaprilat in the presence of candesartan resulted in further vasodilation, increasing RBF, CBF, and MBF. This further vasodilation in the presence of ANG II receptor blockade indicates that these latter responses were due to effects other than ACE inhibitor-induced reductions in ANG II concentrations. Although it is possible that part of these effects could be partially due to inhibition of residual effects of ANG II that were not blocked by candesartan, this seems unlikely because the responses to enalaprilat were partially or completely reversed by the B2 receptor blocker icatibant, returning blood flows to levels not significantly different from those observed with candesartan alone. These results indicate that the vasodilation induced by enalaprilat in the presence of candesartan was due primarily to activation of BK B2 receptors and provide further support to the concept that BK has a significantly greater role in mediating the RBF, CBF, and MBF responses to ACE inhibition during sodium restriction. These results extend our previous findings that although icatibant had no effect on ACE inhibition-induced renal vasodilation in dogs fed a normal salt diet, the B2 receptor blocker significantly attenuated the vasodilation caused by enalaprilat in dogs maintained on a low-salt diet (3, 6, 12, 18). The increased contribution of BK B2 receptor activation to ACE inhibition-induced vasodilation in salt-restricted dogs compared with dogs on a normal salt intake support an increased role of kinins in regulating intrarenal blood flow during sodium restriction, a condition shown to increase intrarenal BK levels (11, 15). There is evidence that elevated ANG II levels tonically stimulate BK production and subsequent nitric oxide generation via activation of AT2 receptors (13-15). The increased BK-dependent influence after ACE inhibition may reflect a greater modulating action of this interaction during sodium restriction.
Although the magnitudes of CBF and MBF responses to candesartan were similar, a comparatively greater increase in MBF than in CBF was observed when enalaprilat was administered after candesartan (+41 vs. +21%, respectively). This indicates that during sodium restriction, the medullary circulation may have an enhancement of BK forming capability, an increased BK trapping ability, or an enhanced sensitivity to BK, compared with the cortical circulation. These possibilities, however, remain to be explored further.
Neither AT1 receptor blockade nor ACE inhibition significantly affected GFR, UV, and UNaV. The AT1 receptor blocker was administered as a single intrarenal dose, which was able to block the vasoconstrictor effects of 100 ng of ANG II. It may be that although the vascular receptors were completely blocked, insufficient candesartan was available to block tubular receptors, resulting in an incomplete antagonism of ANG II-mediated tubular transport.
In conclusion, the present results provide further support to the concept that during sodium restriction there is an increased kinin-dependent influence via B2 receptors, which contributes to the vasodilatory effects of ACE inhibition. Furthermore, the renal medullary circulation is more sensitive to kinin potentiation than the cortical circulation.
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ACKNOWLEDGEMENTS |
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We would like to thank Karim Said and Abu Taher for technical assistance. We also thank Merck Research laboratories for providing us with enalaprilat and Astra for providing the candesartan.
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FOOTNOTES |
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This study was supported by a grant from the Louisiana Education Quality Support Fund and by National Heart, Lung, and Blood Institute Grants HL-18426 and HL-51306. S. S. El Dahr is supported by National Kidney Foundation Clinical Scientist Award and National Institute of Diabetes and Digestive and Kidney Diseases Grant R21DK-53595.
Address for reprint requests and other correspondence: L. G. Navar, Dept. of Physiology, SL39, Tulane Univ. School of Medicine, 1430 Tulane Ave., New Orleans, LA 70112 (E-mail: navar{at}mailhost.tcs.tulane.edu).
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.
Received 19 November 1999; accepted in final form 22 March 2000.
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