(Hypertension. 1997;30:337.)
© 1997 American Heart Association, Inc.
Articles |
Renal Angiotensin II Receptor Regulation in Two-Kidney, One Clip Hypertensive Rats
Effect of ACE Inhibition
From the Laboratory of Experimental Hypertension and Vasoactive Peptides, Clinical Research Institute of Montreal; also affiliated with Université de Montréal (Canada).
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Abstract Local renal and plasma renin-angiotensin systems (RAS) both play an important role in blood pressure regulation during the development of two-kidney, one clip Goldblatt hypertension (2K1C) through their vasoactive component, angiotensin II (Ang II). Our goal was to characterize glomerular and preglomerular vascular Ang II receptors during the different stages of development of hypertension in 2K1C rats (2-, 4-, 8-, and 16-weeks postoperative) using Ang II antagonists [Sar1,Ile8]-Ang II, losartan, and PD 123319 and their regulation after angiotensin-converting enzyme (ACE) inhibition by captopril. Competitive binding studies showed that the only Ang II receptor detected on both glomeruli and preglomerular vessels of all groups (2-, 4-, 8-, and 16-week 2K1C rats, control rats, and captopril-treated rats) was the Ang II type 1 receptor (AT1). Vascular AT1 receptor density (Bmax) was significantly lower in only the 16-week 2K1C group, whereas glomerular Bmax was significantly lower in 2K1C rats at 2-, 4-, and 8-weeks. Vascular and glomerular receptor densities were both significantly higher in captopril-treated rats than in nontreated rats. We therefore conclude that in 2K1C rats, Ang II receptors on preglomerular vessels and glomeruli are regulated differentially during the development of hypertension and after ACE inhibition. Our results suggest that glomerular Ang II receptors are regulated by systemic plasma Ang II levels, whereas vascular Ang II receptors are not. However, when renal and systemic RASs are both blocked, these receptors are upregulated but are no longer differentially regulated.
Key Words: angiotensin II • hypertension, renovascular • angiotensin-converting enzyme inhibition • renin-angiotensin system • kidney • rats
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Introduction |
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The RAS plays an important role in BP regulation and fluid homeostasis. It mediates these functions through the production of Ang II,1 the active element of the systemic and renal RAS for which all components, namely angiotensinogen, Ang I, ACE, and Ang II, have been localized in the kidney.2 Thus, both systemic and intrarenally formed Ang II have the ability to increase renal vascular resistance by constricting the afferent and efferent arterioles and interlobular arteries.3 4 5 The effects of Ang II are mediated through the binding of high affinity membrane-bound receptors, namely AT1 and AT2, which have been recently classified with the aid of specific nonpeptide antagonists.6 7 All the known effects of Ang II have been attributed to AT1, which has a high affinity for the selective nonpeptide antagonist losartan. On the other hand, no functional correlate has been found for AT2, which has a high affinity for the selective nonpeptide antagonist PD 123319. In addition to these two receptor types, it has been reported that in rodents, AT1 has two subtypes, namely AT1a and AT1b,6 8 both of which are present in the rat kidney.9 However, these isoforms cannot be distinguished pharmacologically.10
Both Ang II receptor types have been localized in humans and rats, but their distribution is not uniform in all somatic tissues. Some tissues, such as the liver, lung, and kidneys, have a nearly homogeneous population of AT1 receptors, whereas others, such as the pancreas and human uterus, contain the AT2 subtype almost exclusively.11 12 Certain tissues such as the adrenals and heart are characterized by a mixture of both receptor subtypes.11 12 In addition to these localization studies, our laboratory has recently characterized Ang II receptors on glomeruli13 and preglomerular vessels14 and found them to be predominantly, if not exclusively, of the AT1 subtype.
Ang II, like many other peptides, has the ability to modulate the density of its receptors in several organs, including those involved in cardiovascular regulation, such as the vascular wall, heart, adrenals, and kidneys.15 Sollott et al16 have provided evidence that intrarenally generated Ang II is a more important modulator of glomerular Ang II receptors than plasma Ang II. We thus became interested in studying the regulation of renal Ang II receptors in a model of renovascular hypertension in which the influence of systemic RAS could be differentiated from that of intrarenal RAS. With this goal in mind, we chose the 2K1C hypertensive model,17 which is the experimental counterpart of unilateral renal artery stenosis that affects humans.18
It is well accepted that the development of hypertension after reducing flow to one kidney, as in the 2K1C model, is mediated by the increased activities of both systemic and intrarenal RAS,19 20 although their quantitative contributions vary depending on the time elapsed after constriction of the renal artery.18 This model of renovascular hypertension has been separated into three theoretical temporal phases: Phase I or the acute phase (which occurs 2 to 4 weeks after placement of the renal artery clip) and phase II or the moderate phase (which occurs 5 to 9 weeks after placement of the clip) are both renin-dependent phases, indicating the prominent role of systemic RAS in raising BP. These two phases are characterized by an elevation of PRA, which is an indirect indicator of plasma Ang II levels.18 21 Phase III or the chronic phase (which usually occurs 9 weeks or more after placement of clip) is known as the volume-dependent phase. In this phase, PRA returns to normal, and hypertension is maintained by a rise in plasma volume, the action of local RAS, or both.18 Also, unlike phases I and II, phase III is the only phase that demonstrates renal retention of both salt and water, factors involved in the increase of plasma volume.22 In addition to these differences, 2K1C hypertension is characterized by augmented renal renin activity in the clipped kidney with a decrease in that of its contralateral nonclipped counterpart.23
The BP elevation can be blocked or reversed by several methods, such as removal of the renal artery clip,24 the administration of Ang II receptor antagonists,25 or the administration of ACE inhibitors.25 26 The purpose of this study was to characterize glomerular and preglomerular vascular receptors in 2K1C rats during the different stages of hypertension development and subsequently to investigate their regulation after administration of the ACE inhibitor captopril. We chose captopril because it has been shown to markedly reduce the formation of both systemic and intrarenally formed Ang II.27
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Methods |
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Animals
All studies performed met the standards of the Canadian Council on Animal Care for the use of laboratory animals.
Ang II Receptor Characterization Studies
2K1C hypertension was produced in male Sprague-Dawley rats (125
to 150 g; Charles River Laboratories, St-Constant, Quebec, Canada)
(n=10 in each group per experiment, three experiments in total) under
sodium pentobarbital anesthesia (60 mg/kg body wt
IP) by partial constriction of the left renal artery with a rigid
silver clamp (0.2 mm internal gap). The right kidney was left
untouched. Sham-operated rats, which underwent the same surgical
procedure except for placement of the renal artery clip, served as
age-matched controls. All animals were fed standard Purina rat chow
(Ralston Purina Co) and tap water ad libitum and kept on a 12-hour
light/dark cycle.
Systolic BP was measured by the tail-cuff method under light ether anesthesia. Rats that developed hypertension (BP >140 mm Hg) were killed by decapitation either 2, 4, 8, or 16 weeks after surgery.
ACE Inhibition Studies
Similar to rats in the characterization studies, the 2K1C
animals used in the treatment experiments were prepared by constricting
the left renal artery with a silver clamp and leaving the right renal
artery untouched (n=15 rats in each group per experiment, three
experiments in total). Rats that developed hypertension (BP >140
mm Hg) after 2 weeks were randomly divided into two groups, one group
received captopril (75 to 85 mg/kg body wt) in their drinking
water and the other group was given plain water ad libitum. After 2
weeks of treatment, both groups were killed by decapitation.
Biochemical Methods
Trunk blood was collected in ice-chilled tubes containing
10-5 mol/L EDTA for the measurement of
PRA and ANF. The blood samples were immediately centrifuged at
1000g for 10 minutes at 4°C. C-terminal ANF [ANF
(99-126)] was extracted from plasma with Vycor glass beads (Corning
Glass Works) and was measured by radioimmunoassay. Similarly, PRA was
assessed by radioimmunoassay of Ang I generation.28
Histological Preparation
Decapsulated kidney halves were fixed for 24 hours in Bouin’s
solution and embedded in paraffin. Sections (5 µm) were cut and
stained with hematoxylin, lithium carbonate, and eosin. Readings of
each kidney section were done by a renal pathologist (Dr Pierre Russo,
St Justine Hospital, Montreal, Canada) in a single blind manner. Each
kidney section was classified as normal or presenting vascular
lesions either associated with or not associated with nephrotic
ischemia.
Isolation of Preglomerular Vessels
Once the animals were killed, their kidneys were decapsulated,
excised, and placed in ice-cold 0.9% NaCl solution. The kidneys were
then dissected longitudinally, and the medulla and papilla were
discarded. Kidney halves were pressed against a 0.3-mm stainless steel
grid. Interlobar arteries and their subsequent attached branches,
arcuate and interlobular arteries and afferent arterioles, were
retained on the grid surface, whereas glomeruli and tubules passed
through and were kept at 4°C.14
Preparation of Vascular Membranes
Preglomerular vessels were recovered immediately
after isolation and minced into small pieces to facilitate the
detachment of tubular and connective tissues. The isolated vessels were
then placed on a 75-µm nylon mesh and washed with ice-cold 0.9% NaCl
solution to eliminate adhering tubules and connective tissues. The
purity of the preparation was assessed by light microscopy and was
estimated to be approximately 95%. The microvessels were then
homogenized in fresh ice-cold 0.25 mol/L sucrose
solution with a Polytron (setting 7.2x30 seconds) and
centrifuged at 1000g for 10 minutes at 4°C. The
supernatant was kept on ice and the process was repeated. Both
supernatants were combined, filtered through a 20-µm nylon mesh, and
centrifuged at 100 000g for 30 minutes at 4°C.
The pellet was resuspended in 50 mmol/L Tris-HCl buffer, pH
7.4. Aliquots were taken for binding assays. Protein concentration was
assessed by a modification of Bradford’s method as described by
Spector.29 Vascular membranes were used immediately
thereafter for radioligand studies.
Vascular Membrane Binding Assay
Optimal conditions for binding dependency on incubation time,
temperature, and the protein concentration of vascular membrane
preparations were ascertained as described previously.14
In competition experiments, 30 to 35 pmol/L
[Sar1,Ile8] Ang II was incubated with
increasing concentrations of unlabeled displacing compounds, from
10-12 to 10-6
mol/L for both [Sar1,Ile8]-Ang II and
PD 123319 and from 10-11 to
10-5 mol/L for losartan. The
assay buffer contained 50 mmol/L Tris-HCl (pH 7.4), 1
µmol/L aprotinin, 0.1% bacitracin, 5 mmol/L
MgCl2, 0.5 mmol/L PMSF, 0.4
µmol/L phosphoramidon, and 0.5% bovine serum
albumin. Incubations were undertaken with 40 µg of membrane
protein at 22°C for 90 minutes in a final volume of 250 µL. The
reaction was stopped by dilution with 3.5 mL ice-cold Tris-HCl and
rapid filtration through Whatman GF/C filters using a Cell Harvester
(Brandel). The filters were then rinsed three times with 3 mL Tris-HCl,
allowed to dry, and counted in a LKB gamma counter with 65%
efficiency. Nonspecific binding was determined by the amount of tracer
bound in the presence of 1 µmol/L of unlabeled
[Sar1,Ile8]-Ang II, and specific binding was
defined as total binding less nonspecific binding. All
radioligand-receptor binding assays were conducted in
duplicate, and at least three separate binding experiments were
performed for each group.
Preparation of Glomerular Membranes
Glomeruli were isolated as described previously,13
by filtration with ice-cold 0.9% NaCl solution through a 200-, 150-,
120-, and 50-µm nylon mesh. Those retained on the sieve were
collected, washed by centrifugation (4°C,
2000g), suspended in 50 mmol/L Tris-HCl (pH
7.4), and snap-frozen with liquid nitrogen for assay the following day.
We found no significant differences in either Bmax or
Kd values when freshly prepared
glomerular membranes were compared with snap-frozen
preparations (data not shown). The purity of the glomerular
suspension was assessed by light microscopy and estimated to be about
95% at the end of each preparation. The next day the
glomerular suspensions were defrosted at room temperature,
homogenized for 1 minute in a Polytron (setting 7),
centrifuged at 40 000g for 20 minutes, and
resuspended in 50 mmol/L Tris-HCl (pH 7.4). Protein
concentration was assessed by a modification of Bradford’s
method.29
Glomerular Membrane Binding Assay
Optimal conditions for binding dependency on incubation time,
temperature, and the protein concentration of glomerular
membrane preparations were determined previously.13 Thus,
the radioligand-receptor binding assay of
glomerular membranes was performed similarly to that of
vascular membranes, except that 35 µg of glomerular
protein was assayed in a final volume of 1 mL binding buffer. As with
vascular membrane radioligand-receptor binding assays, all
these assays were conducted in duplicate and at least three separate
experiments were done for each group.
Chemicals
All materials were of the highest reagent grade available.
Bacitracin, PMSF, phosphoramidon, captopril, and bovine
serum albumin were purchased from Sigma Chemical Co; aprotinin
was obtained from Miles Laboratories; and sucrose came from JT Baker.
[Sar1,Ile8]-Ang II was procured from Bachem
California.
2-N-butyl-4-chloro-5-hydroxymethyl-1-(2-(H-tetrazole-5-yl)biphenyl-4-yl-methyl)
imidazole, potassium salt, losartan potassium (DuP 753), and PD
123319 were synthesized at EI DuPont Nemours & Co. Losartan
potassium and PD 123319 were generous gifts from the DuPont Merck
Pharmaceutical Co (Wilmington, Del) and Parke-Davis (Ann Arbor, Mich),
respectively. Eosin, hematoxylin, and lithium carbonate were all
purchased from Fisher Scientific.
Statistical Analysis
Binding data were analyzed by processing the raw data
with the EBDA program (Elsevier-Biosoft). The density
(Bmax) and affinity (Kd) of binding
sites were then determined with the LIGAND program
(Elsevier-Biosoft).30 Statistical analysis was
carried out with the SigmaStat program (Jandel Scientific) with one-way
ANOVA followed by the Student-Newman-Keuls t test to
determine significance, whereas for comparisons between 2K1C and sham
groups at different time periods an unpaired Student’s t
test was used. In addition, two-way ANOVA followed by
multivariate analysis was performed to
determine significance among the different tissues and the different
phases (normal or activated systemic RAS) or treatments (with
or without ACE inhibition). The values presented are mean±SEM.
Values of P<.05 were considered to be significant.
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Results |
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Ang II Receptor Characterization Studies
Table 1 enumerates several physiological characteristics of 2-, 4-, 8-, and 16-week hypertensive rats and their age-matched sham-operated controls. BP was significantly elevated (P<.0001) in 2K1C rats when compared with their age-matched sham-operated counterparts in all time periods. Similarly, heart weight, expressed in milligrams per 100 grams of body weight, was significantly increased in all 2K1C rats, whereas body weight was significantly reduced. PRA was significantly augmented (P<.0001) in 2-, 4-, and 8-week 2K1C rats but was not significantly different (P>.05) from values seen in control animals at 16 weeks. Although there was no significant difference in PRA in the latter group, it was the only group that showed a significant rise (P<.05) in plasma ANF (99-126) concentration. As in all other groups (2-, 4-, and 8-week), the 16-week 2K1C group presented no significant difference (P>.05) in hematocrit values when compared with sham-operated control animals.
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Table 2 gives the kidney weights of all age groups. Clipped kidneys weighed less (P<.05) than those from sham-operated rats in all groups except the 16-week group. Contralateral nonclipped kidneys weighed significantly more than kidneys of control rats (P<.05) in all but the 4-week group. There were also significant differences between the weights of the clipped and nonclipped kidneys within the same age group at all time periods.
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Fig 1 depicts representative competitive binding curves of preglomerular vascular membranes with the nonspecific Ang II antagonist [Sar1,Ile8]-Ang II and the specific Ang II receptor antagonists losartan and PD 123319. They revealed that on preglomerular vessels of clipped kidneys in 16-week 2K1C rats, only the AT1 receptor was present.
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), and the AT2 receptor antagonist PD
123319 (
) were used. B and Bo represent binding in the
respective presence and absence of the competitor.Fig 2a and 2b demonstrates the Bmax values of AT1 receptors on preglomerular vessels and glomeruli, respectively. The density of AT1 receptors in preglomerular vessels of 2K1C was significantly reduced (P<.05) in only the 16-week group when compared with the control animals. There was also a significant difference (P<.05) between the Bmax of the nonclipped and clipped kidneys in these animals, with that of the nonclipped kidney being lower (Fig 2a). Even though the Bmax values of preglomerular vascular receptors in 16-week hypertensive 2K1C rats showed significant differences, no significant difference in Kd was observed in this or any other group, with values ranging from 0.8±0.1 to 2.6±0.9 nmol/L for preglomerular vessels and from 1.4±0.1 to 2.6±0.5 nmol/L for glomeruli (data not shown). When we compared the effects of the different activation states of the systemic RAS (phases I and II versus phase III) on the glomerular and preglomerular vascular Bmaxs using two-way ANOVA, we found that Ang II receptors on preglomerular arterioles are significantly downregulated (P<.05) when the systemic RAS has returned to its normal activity (phase III), whereas glomerular Ang II receptors are significantly (P<.05) downregulated when the systemic RAS is activated (phases I and II).
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On the other hand, when glomerular values were considered, the Bmax of both clipped and nonclipped kidneys was significantly lower (P<.05) in 2K1C rats than in their sham-operated counterparts in the 2-, 4-, and 8-week groups (Fig 2b). No significant difference was noted in the Bmax of 16-week 2K1C animals when compared with their sham-operated controls (Fig 2b). Although the density of glomerular AT1 receptors of both kidneys was significantly lower in 2K1C rats at 2-, 4-, and 8-weeks, no significant difference (P>.05) in glomerular Bmax values was observed when clipped and nonclipped kidneys within the same group were compared (Fig 2b).
ACE Inhibition Studies
Table 3 illustrates some
physiological parameters of
captopril-treated and nontreated 2K1C rats. There were no significant
differences (P>.05) in body weight before or after
treatment between the two groups. However, both BP and relative heart
weight in nontreated rats were significantly higher (P<.01)
than in captopril-treated animals after the 2-week treatment period,
whereas PRA was significantly elevated (P<.01) in the
treated group.
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Table 4 shows the weight of clipped and nonclipped kidneys of treated and nontreated 2K1C rats. Nonclipped kidneys weighed significantly more (P<.05) than clipped organs in both nontreated and treated animals. Treatment with captopril further reduced (P<.05) the weight of the clipped kidney and increased (P<.05) that of the nonclipped organ when compared with the respective kidneys of nontreated animals.
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Vascular and glomerular Bmax values are presented in Table 5. No significant differences (P>.05) were seen in vascular AT1 receptor density when the clipped and nonclipped kidneys of the same treatment group were compared. However, clipped and nonclipped kidneys from treated animals both had significantly higher (P<.05) vascular receptor densities than their nontreated counterparts. On the other hand, glomerular Bmax was slightly but not significantly lower (P>.05) in nonclipped kidneys when compared with clipped kidneys in nontreated rats. Even though significant differences were evident in Bmax values, no significant differences (P>.05) were observed in Kd values of either vascular or glomerular receptors between kidneys of the same treatment groups or between different treatment groups, with values for nontreated animals ranging from 1.9±0.1 to 3.0±0.3 nmol/L and for treated animals from 1.6±0.2 to 3.1±0.1 nmol/L (data not shown). Hence, captopril upregulated vascular AT1 receptors in both clipped and nonclipped kidneys. Furthermore, treatment upregulated glomerular AT1 receptors in the nonclipped kidney of 2K1C rats. This upregulation was not differential since no significant differences (P>.05) were observed after we performed two-way ANOVA and treated both the tissue type and the activation state of the systemic and renal RAS.
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Histological Evaluations
The most important change was the presence of tubular dilatation,
interstitial inflammation, and focal
glomerulosclerosis in the captopril-treated
clipped kidneys. The presence of interstitial focal
fibrosis was also detected in clipped kidneys of nontreated rats. All
other groups, sham-operated rats and nonclipped kidneys of treated and
nontreated rats, presented no vascular lesions or
glomerulosclerosis. Also, no vascular lesions
were detected in either clipped or nonclipped kidneys.
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Discussion |
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In the present study, we have shown that Ang II receptors on preglomerular vessels and glomeruli are differentially regulated during different developmental stages of the 2K1C hypertensive rat and after blockade of the RAS by an ACE inhibitor.
As described previously,17 31 placement of a constricting clip on the left renal artery induces significant BP elevation in rats. As a physiological indicator of this BP increase, significant heart hypertrophy was observed in all 2K1C animals when compared with their age-matched sham-operated controls. In agreement with previous studies,18 21 PRA (an indirect indicator of both systemic RAS activity and plasma Ang II concentration) was significantly augmented in phases I (2- and 4-week groups) and II (8-week group). Again in agreement with other studies,18 32 PRA in phase III (16-week group) returned to basal values, whereas plasma ANF [99-126] concentration was significantly increased, suggesting an expanded blood volume. These results suggest that phases I and II are both renin-dependent, whereas phase III is probably volume-dependent. Our data are in total agreement with previous investigations33 34 that also demonstrated that in the chronic phase (phase III) of the 2K1C model, hypertension is maintained by the slow pressor component of RAS, eliciting an increase in plasma and blood volume mediated by the renal actions of Ang II. With respect to the physical effect of renal artery constriction and in accordance with previous studies,35 36 clipped kidneys weighed significantly less than those from control animals in all but the 16-week group, whereas nonclipped kidneys weighed significantly more than clipped kidneys in all groups.
Competitive binding studies with the specific AT1 antagonist losartan and the specific AT2 antagonist PD 123319 revealed that AT1 was the only Ang II receptor detected on preglomerular vessels and glomeruli in the kidneys of all groups, whereas the AT2 receptor was not detected. These results are in total agreement with previous studies,37 38 which have clearly demonstrated the absence of renal AT2 receptors and mRNA, respectively, but are in contradiction to the findings of Chatziantoniou and Arendshorst,39 who have shown the presence of two functional Ang II receptors on preglomerular vessels in young spontaneously hypertensive rats. Furthermore, no significant differences in either Bmax or Kd values were observed when freshly prepared glomerular membranes were compared with snap-frozen preparations (data not shown). These binding studies also revealed no significant differences in Kd values among the various age groups or kidneys within groups.
When the density of vascular AT1 receptors from control and 2K1C rats was considered, significant downregulation was observed in the 16-week 2K1C group. Even though there were significant differences, the wide variability seen in the control group at this time period did not allow us to draw any conclusions. Henceforth, the results from the statistical test were only suggestive and not conclusive. Moreover, vascular AT1 receptor Bmax of the nonclipped kidney was significantly lower than that of the contralateral organ in this time period. Hence, it appears that during the early phases of 2K1C hypertension, elevated plasma Ang II levels did not induce the expected downregulation of renal vascular AT1 receptors. However, when systemic RAS returns to normal in phase III but renal RAS remains stimulated the results seem to suggest a downregulation of vascular Ang II receptors. In view of the large SEM seen in the 16-week sham-operated group, we can only suggest a decrease in the preglomerular vascular Ang II receptor Bmax in this phase. Since vascular Ang II receptors seem to be decreased in both clipped and nonclipped kidneys and it has been shown that renal Ang II concentrations are increased in the latter,40 we can propose that vascular AT1 receptors could possibly be regulated by the intrarenal RAS when the systemic RAS is no longer activated. In support of this proposition, Mai et al41 have recently shown that AT1 mRNA expression of the nonclipped kidney is significantly lower than that of the clipped kidney.
On the other hand, the density of glomerular Ang II receptors was downregulated in 2-, 4-, and 8-week 2K1C rats, but no significant differences were seen when clipped and nonclipped kidneys were compared within the same age group. Therefore, it is proposed that plasma Ang II concentration is the major regulating factor of glomerular Ang II receptor density in 2K1C rats with RAS activation (phases I and II), but when this system is not overexpressed, as is the case in phase III, no such regulation occurs. Our results are in agreement with previously published results of Della Bruna et al,42 who have shown that the systemic RAS has no regulatory influence on renal AT1 receptor gene expression located in juxtaglomerular cells.
To dissect the role of systemic and intrarenal RAS, 2-week hypertensive 2K1C rats were treated with the ACE inhibitor captopril. Unlike other studies,25 26 hypertension was allowed to develop before captopril was administered, because as shown previously35 placement of the clip around the renal artery does not necessarily increase BP. Hence, the BP decrease that was observed in 2K1C rats after captopril treatment was due to inhibition of RAS and not to an unsuccessful surgical procedure. Also, as expected, treated animals had significantly higher PRA than nontreated control animals. These results are both in accordance with previous studies showing that ACE inhibitors decrease BP in 2K1C rats25 while at the same time increasing PRA due to removal of the negative feedback effect of Ang II.43 Furthermore, the physiological effect of a decrease in BP was manifested by a significant fall in the heart-to–body weight ratio in captopril-treated rats, although it could also have been a direct consequence of ACE inhibition in cardiac hypertrophy. In addition to BP-lowering effects, captopril significantly reduced the weight of the clipped kidney while at same time compensatory hypertrophy was found in the contralateral nonclipped organ. These results are concordant with previous studies, which have also shown that ACE inhibitors induce renal atrophy in the clipped kidney of 2K1C rats.36
As in the characterization studies, competitive binding assays with AT1 and AT2 antagonists revealed that only AT1 was present in preglomerular vessels and glomeruli of clipped and nonclipped kidneys from captopril-treated animals. No significant differences were observed in vascular and glomerular Ang II receptor affinities between clipped and nonclipped kidneys from treated or nontreated rats.
When vascular Bmax values of clipped and nonclipped kidneys from treated animals were compared with their respective nontreated counterparts, receptor density was significantly higher in both clipped and nonclipped kidneys of captopril-treated rats. This observed upregulation of vascular Ang II receptors after ACE inhibition was in total agreement with previous studies that showed that low plasma Ang II concentration upregulated AT1 receptors in vascular smooth muscle.15 43 However, since the renal RAS was blocked by captopril, no differences were seen in vascular Bmax values between the clipped and nonclipped kidneys of treated 2K1C rats. On the other hand, we saw that nonclipped kidneys from treated animals had a significantly higher glomerular AT1 receptor Bmax when compared with their nontreated counterparts. It therefore appears that when systemic RAS is suppressed by an ACE inhibitor, there is the usual decrease in Ang II formation, causing receptor upregulation, which has been documented previously15 44 in both kidneys. Also of interest was the absence of any type of regulation with respect to receptor density in the clipped kidney of treated rats when compared with their nontreated counterparts. This absence of effect could be secondary to glomerular atrophy present in the kidney.
To ascertain the existence of differential regulation for preglomerular vascular and glomerular Ang II receptors in the different phases of 2K1C hypertension and after ACE inhibition, we performed two-way ANOVA followed by multivariate analysis. These statistical analyses demonstrated that Ang II receptors on these structures are differentially regulated in the sense that preglomerular vascular Ang II receptors seem to be regulated by the renal RAS when the systemic RAS is no longer activated. Even though these statistical results are significant, we cannot draw any significant conclusions from them because the only difference in 2K1C vascular Bmax that was observed with respect to control animals was seen at the phase at which the variability in the control animals was very large. On the other hand, glomerular Ang II receptors are regulated by the systemic RAS. However, when the renal and systemic RAS are both blocked, there is no differential regulation of either glomerular or preglomerular vascular Ang II receptors.
In conclusion, we report that in 2K1C rats, renal Ang II receptors on preglomerular vessels and glomeruli are regulated differentially during the various developmental stages of hypertension and after the administration of an ACE inhibitor. We found that glomerular Ang II receptors are regulated by systemic RAS, whereas it seems that vascular Ang II receptors are not. However, when the systemic and tissue RAS are both blocked with an ACE inhibitor, Ang II receptors on both glomeruli and preglomerular vessels are upregulated compared with their respective nontreated kidneys but are no longer differentially regulated.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by a grant from the Medical Research Council of Canada (MT-11558). The authors thank Suzanne Diebold for her excellent technical assistance, Dr Pierre Russo for his pathological analysis, Angie Poliseno and Micheline Caron for their invaluable secretarial help, and Ovid Da Silva for his editorial input.
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Footnotes |
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Reprint requests to Raul Garcia, MD, Clinical Research Institute of Montreal, 110 Pine Ave W, Montreal, Quebec H2W 1R7, Canada.
Received October 18, 1996; first decision November 19, 1996; accepted February 13, 1997.
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References |
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