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Am J Physiol Heart Circ Physiol 275: H1247-H1253, 1998;
0363-6135/98 $5.00
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Vol. 275, Issue 4, H1247-H1253, October 1998

Increased expression of glomerular AT1 receptors in rats with myocardial infarction

Peter F. Mento, Mary E. Pica, Jim Hilepo, Jaime Chang, Larissa Hirsch, and Barry M. Wilkes

Division of Nephrology and Hypertension, Department of Medicine, North Shore University Hospital, and Department of Medicine, New York University School of Medicine, Manhasset, New York 11030

    ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References

Rats with congestive heart failure demonstrate striking intrarenal vasoconstriction that contributes to reduced renal excretory function. We previously demonstrated that inhibition of angiotensin action reverses intrarenal vasoconstriction in rats 4-6 wk after coronary artery ligation. In the present study we tested the hypothesis that abnormalities in the expression and regulation of glomerular angiotensin receptors contribute to the intrarenal vasoconstriction. Because glomerular angiotensin type 1 (AT1) receptors normally downregulate in response to high local ANG II concentrations, we anticipated that glomerular AT1-receptor expression would be reduced in rats after myocardial infarction (MI). To our surprise, the density of glomerular AT1 receptors was nearly double (97% increase, P < 0.002) that of controls, indicating an acquired abnormality in angiotensin receptor regulation. This was specific for renal glomeruli, because the density of angiotensin receptors on renal vasculature was decreased in rats after MI compared with normal controls. Glomerular AT1-receptor expression was downregulated by an acute pharmacological infusion of ANG II and upregulated by acute angiotensin-converting enzyme inhibition to a similar extent in MI and control rats. Renal cortical mRNA expression showed an increase in the renin mRNA-to-actin ratio and angiotensinogen-to-actin ratio, indicating stimulation of the intrarenal angiotensin system in rats after MI. The data indicate a specific dysregulation of AT1 receptors in glomeruli but not blood vessels after MI.

angiotensin II receptors; glomeruli; heart failure; coronary artery ligation; renal vasculature

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

REDUCED CARDIAC MASS after myocardial infarction (MI) commonly leads to congestive heart failure in humans and in experimental animal models. In patients with congestive heart failure, renal perfusion and function are often compromised. The coronary artery ligation model of MI in rats is a good model for study of renal hemodynamic changes, since similar reductions in renal perfusion occur. We recently showed that the intense intrarenal vasoconstriction after coronary artery ligation in rats can be reversed by inhibition of ANG II formation with an angiotensin-converting enzyme inhibitor or a renin inhibitor (14) and that subtypes 1 and 2 of the angiotensin (AT1 and AT2) receptor contribute to the renal vasoconstriction (15). It is not known whether the intrarenal vasoconstriction is due to increased tissue angiotensin levels alone or to abnormalities in the expression and function of angiotensin receptors. In addition, the pharmacological studies indicating that AT1 and AT2 receptors need to be inhibited to fully restore renal hemodynamics suggested that AT2 receptors, which are not normally expressed in renal glomeruli, may be induced in the kidneys after coronary artery ligation.

The current study was designed to test the hypothesis that there is increased expression of angiotensin receptors in the kidneys of rats with reduced cardiac mass after coronary artery ligation. Using the coronary artery ligation model in rats, we studied ANG II receptor expression in renal glomeruli and vasculature. Using specific antagonists to determine whether the balance between AT1 and AT2 receptors was altered in congestive heart failure, we further subtyped glomerular ANG II receptors into AT1 and AT2 receptors. To our surprise, we found significant overexpression of glomerular AT1 receptor, despite evidence for local activation of the renin-angiotensin system. Additional experiments were performed to study the regulation of AT1-receptor expression in glomeruli and renal blood vessels by changes in local angiotensin concentrations.

    METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Studies were performed on 169 male Sprague-Dawley rats (Charles River, Wilmington, MA) weighing 210-300 g. The rats were fed standard Purina Rat chow and tap water ad libitum. All protocols were approved by the Institutional Animal Care Committee. Separate groups of rats were used for measuring glomerular angiotensin receptors, renal vascular angiotensin receptors, glomerular angiotensin receptor subtypes, and molecular components of the renin-angiotensin system.

MI

Rats were anesthetized with methohexital sodium (50 mg/kg; Brevital, Eli Lilly, Indianapolis, IN), intubated, and placed on a positive-pressure respirator. The left coronary artery was ligated 3-4 mm from its origin with 5-0 silk suture (21). Age-matched control rats were not subjected to surgery. Rats were included in the MI group if there was a transmural left ventricular scar at autopsy. Rats with MI had a significantly increased heart weight-to-body weight ratio compared with normal controls: 3.16 ± 0.04 mg/g in controls (n = 57) and 3.57 ± 0.08 mg/g in MI animals (n = 59, P < 0.001). There were no differences in body weight between MI and control groups at the time of study, since rats with MI gained weight at a rate comparable to control rats. Of ~225 rats subjected to surgery, 50% survived the immediate postoperative period (72 h). Of the 112 surviving rats, 59 animals met the criteria above to be included in the MI group, whereas 53 animals did not show gross evidence of a transmural scar. Fifty-seven control rats were not subjected to surgery. This second group is referred to as the "sham group" and consisted of rats in which the ligature did not occlude the coronary artery or in which there was a tiny MI without the development of a transmural scar. The heart weight-to-body weight ratio of this group (3.24 ± 0.05 mg/g, n = 53) was not significantly different from that of the control group (P = NS compared with controls, P < 0.001 compared with MI).

Isolation of Glomeruli for Binding Studies

Rats were killed by CO2 inhalation, and the kidneys were removed and placed in Tris · NaCl buffer (50 mM Tris in 154 mM NaCl, 4°C, pH 7.5). The outer cortex was minced to a pastelike consistency, and glomeruli were isolated by graded sieving using 90-, 180-, 150-, and 75-µm stacked phosphor bronze sieves. Glomeruli on the 75-µm sieve were >95% pure by light microscopy (22). The amount of protein was adjusted to 15 µg/100 µl using the Bradford reagent (2).

Isolation of Renal Vasculature

The renal vasculature was isolated from both kidneys using a modification of the technique of De Leon and Garcia (3). The kidneys were removed and pressed through a 300-µm sieve, and the material from the top of the sieve was washed with ice-cold PBS and centrifuged at 1,000 g for 5 min. The pellet was resuspended in PBS and pushed through a 20-gauge adapter three times. The suspension was centrifuged at 1,000 g for 5 min, the pellet was resuspended in PBS, and nonvascular material was pushed through a 90-µm sieve. Vessels on top of the sieve were spun at 1,000 g for 5 min and homogenized in a Polytron for binding studies (4 times at setting 8 for 15 s on ice). The homogenate was centrifuged at 1,000 g for 5 min, and the supernatant was spun at 40,000 g for 35 min. The resulting pellet was resuspended in 250 mM sucrose, and the amount of protein was measured with the Bradford reagent (2) and adjusted to 5 µg/100 µl using assay buffer.

Receptor-Binding Experiments

The binding of ANG II to isolated glomeruli was measured as previously described (23). Glomeruli were incubated at 22°C for 60 min with continuous gentle shaking. The incubation medium consisted of the following: 50 mM Tris base, 1 µM aprotinin, 0.1% bacitracin, 5 mM MgCl2, 0.5 mM phenylmethylsulfonyl fluoride, 0.4 µM phosphoramidon, and 0.5% BSA (3). Also present in the medium were 15 µg of glomerular protein (or 5 µg of renal vascular protein) and various amounts of 125I-[Sar1,Ile8]ANG II (2,200 Ci/mmol; New England Nuclear, Boston, MA) and unlabeled [Sar1,Ile8]ANG II (0.14-17 nM). Bound and free radioactivity were separated by filtration through a glass filter (no. 30, Schleicher and Schuell, Keene, NH). 125I was counted using an LKB 1277 Gammamaster (Pharmacia LKB Nuclear, Gaithersburg, MD) with 75% efficiency. Specific binding was calculated by subtracting the binding in the presence of 1 µM unlabeled [Sar1,Ile8]ANG II from the total binding.

Glomerular angiotensin receptors were subtyped using the specific nonpeptidic antagonists losartan (AT1-receptor antagonist) and PD-123319 (AT2-receptor antagonist). AT1 receptors were measured as specific binding in the presence of PD-123319 (10-6 M); AT2 receptors were measured as specific binding in the presence of losartan (10-6 M).

RT-PCR

Total cellular RNA was extracted from whole kidney cortex and first-strand synthesized from 1 µg of total RNA with the use of Moloney murine leukemia virus RT and oligo(dT). The reaction was carried out in the presence of first-strand buffer, 1 mM dNTPs and 20 mM dithiothreitol, at 42°C for 1 h. The PCR mixture contained 10 µl of the cDNA reaction mixture 10 pM cold 3'-specific primers, PCR buffer, 0.2 mM dNTPs, and 0.5 U Taq polymerase, and the reaction was carried out with a Perkin-Elmer DNA thermal cycler. The thermal cycle was 60 s at 94°C, 60 s at 54°C, and 120 s at 72°C repeated 30 times. The PCR products were separated on a 1.8% agarose gel and hybridized with digoxigenin-labeled probes, and intensity of the bands was measured using chemiluminescence on a molecular imaging system (model 525, Bio-Rad). A beta -actin-specific band was used to normalize the data. The primers for PCR were as follows: sense 5'-GTGGGGCGCCCCAGGCACCA-3' and antisense 5'-CTCCTTAATGTCACGCACGATTTC (500-bp product) for beta -actin, sense 5'-TGCCACCTTGTTGTGTGAGG and antisense 5'-ACCCGATGCGATTGTTATGCCG (PCR product 374 bp) for renin, and sense 5'-CTGACCCAGTTCTTGCTGCC and antisense 5'-TGGGGGTTATCCACTCTGCC (PCR product 724 bp) for angiotensinogen. Preliminary experiments verified that the amounts of starting RNA were appropriate (increasing amounts give a larger signal) and that the amplification was in the exponential phase.

Protocols

ANG II infusion. Rats were anesthetized with pentobarbital sodium (45 mg/kg ip), and catheters (PE-50) were placed in a jugular vein for ANG II infusion and in a carotid artery for blood pressure monitoring. Rats were infused with normal saline (0.02 ml/min) for 30 min after the surgical procedure to make up for fluid losses and to allow stabilization. ANG II was then infused at a rate of 100 ng · kg-1 · min-1 for 15 min (0.02 ml/min) with continuous blood pressure monitoring. This infusion protocol is sufficient to cause maximal downregulation of glomerular angiotensin receptors in normal rats (1). At the end of the infusion period the kidneys were quickly removed and glomeruli were isolated for measurement of angiotensin receptors.

Angiotensin-converting enzyme inhibition. Rats were gavaged with enalapril (10 mg/kg in a homogenized 0.1% starch suspension), and after 3 h they were killed by CO2 overdose and the kidneys were removed for measurement of angiotensin receptors in glomeruli as described above. Three hours after this dose of enalapril, plasma angiotensin levels are suppressed to undetectable levels (16).

Data Analysis

Values are means ± SE and were analyzed on an IBM computer using SAS software by ANOVA, the Student-Newman-Keuls procedure for multiple comparisons, and Student's t-test for paired samples. Equilibrium binding studies were analyzed by Scatchard transformation of binding data using the LIGAND program of Munson and Rodbard (17). The null hypothesis was rejected when P < 0.05.

Materials

All chemicals were of the purest commercial grade available. 125I-[Sar1,Ile8]ANG II was purchased from NEN Life Sciences Products (Boston, MA). Enalapril maleate was provided by Dr. Charles Sweet (Merck Institute for Research, West Point, PA). Losartan was a gift from Merck Pharmaceutical. PD-123319 {(S)-1-[[dimethylamino-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo-[4,5-c]pyridine-6-carboxylic acid} was a gift from Warner-Lambert (Ann Arbor, MI).

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Glomerular ANG II Receptors in MI Rats

The binding of 125I-[Sar1,Ile8]ANG II was significantly increased in glomeruli from MI rats compared with normal control or sham rats (Fig. 1). Binding in sham-operated rats was similar to that in control rats. When the data were transformed by Scatchard analysis, abnormalities in ANG II receptors emerged (Fig. 2). Receptor density was increased by 97% in MI rats compared with sham rats: 2,755 ± 94 and 1,402 ± 118 fmol/mg, respectively (P < 0.002; Fig. 2, top). Receptor affinity was not statistically different between the MI and sham groups [equilibrium dissociation constant (Kd) = 2.6 ± 0.1 and 1.8 ± 0.2 nM in MI and sham rats, respectively]. There were no differences in receptor affinity or density between sham and normal control rats (Kd = 1.1 ± 0.1 nM, receptor density = 1,605 ± 38 fmol/mg; Fig. 2, bottom). When the MI rats were compared with normal controls, there was a significant decrease in receptor affinity (P < 0.002) and an increase in receptor density (P < 0.002).


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  		Fig. 1.   Equilibrium binding of 125I-[Sar1,Ile8]ANG II in glomeruli from control and sham rats and rats subjected to myocardial infarction (MI). Specific binding was studied at 60 min using 0.14-17 nM [Sar1,Ile8]ANG II. In glomeruli from MI rats (n = 11) there was a significant increase in specific binding at the 4 highest incubated concentrations compared with normal control rats (n = 12). Specific binding in rats in which there was no evidence of MI (sham rats, n = 4) was similar to that in control rats. * P < 0.005 compared with control or sham rats.


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  		Fig. 2.   Scatchard analysis of equilibrium binding of 125I-[Sar1,Ile8]ANG II in glomeruli from control, sham, and MI rats. Scatchard analysis of equilibrium binding studies indicated one class of high-affinity angiotensin receptors in glomeruli of control, sham, and MI rats. Equilibrium binding data were transformed by plotting amount of ligand bound (x-axis) vs. amount of ligand bound divided by free ligand concentration (B/F, y-axis). x-Intercept corresponds to receptor density (Ro), and slope of line corresponds to inverse negative of equilibrium dissociation constant (Kd). Number of glomerular ANG II receptor sites was increased in MI rats (2,755 ± 94 fmol/mg) compared with sham rats (1,402 ± 118 fmol/mg, P < 0.002; top). Kd was similar in MI and sham rats, as indicated by parallel lines. There were no significant differences in binding parameters between control and sham rats (bottom). However, when MI and control rats were compared, there was a significant increase in Kd and an increase in receptor number in MI rats.

To test the hypothesis that glomerular angiotensin receptor subtypes were altered after MI, experiments were performed to determine which subtype of glomerular angiotensin receptors was expressed in rats after MI. As shown in Fig. 3, total specific binding was greater in the MI than in the control group in the absence of competing ligands. All the specific binding of 125I-[Sar1,Ile8]ANG II was displaceable with losartan, indicating that the receptors are exclusively of the AT1 subtype. Curve fitting indicated that the concentration of losartan needed to displace 50% of the binding (ED50) was approximately fourfold higher in MI than in control rats: 105 vs. 26 nM. PD-123319 did not displace 125I-[Sar1,Ile8]ANG II binding in either group of rats until the concentrations exceeded 10-5 M. At these concentrations, inhibition of binding by PD-123319 is likely to represent nonspecific inhibition of AT1-receptor binding. The ED50 for PD-123319 displacement of binding was similarly higher (by 3.4-fold) in MI than in control rats: 148 vs. 43 µM. These data indicate that glomerular angiotensin receptors in normal and MI rats were of the AT1 subtype.


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  		Fig. 3.   Competitive inhibition of 125I-[Sar1,Ile8]ANG II binding in glomeruli from normal control and MI rats. Specific binding was studied at 60 min using 1 nM 125I-[Sar1,Ile8]ANG II and various concentrations of losartan (L, 10-10-3 × 10-5 M) or PD-123319 (PD, 10-7-10-2 M). In glomeruli from MI rats (n = 10), ED50 for displacement of 125I-[Sar1,Ile8]ANG II by competing ligand was higher for rats treated with losartan (105 and 26 nM in MI and control rats, respectively) or PD-123319 (148 and 43 µM in MI and control rats, respectively) than for normal control rats (n = 9).

Renal Vascular ANG II Receptors in MI Rats

To determine whether the increased expression of angiotensin receptors after MI was specific for glomeruli, we also measured angiotensin receptors by Scatchard analysis of equilibrium binding studies on vasculature isolated from kidneys. In contrast to glomeruli, where ANG II receptors were upregulated, ANG II receptors in isolated vasculature were downregulated in MI rats (Fig. 4). There was no significant difference between sham and MI rats in the density [606 ± 28 (n = 9) and 929 ± 52 fmol/mg (n = 10) for MI and sham rats, respectively, P = NS] or affinity (Kd = 0.62 ± 0.06 and 0.99 ± 0.10 nM for MI and sham rats, respectively, P = NS) of angiotensin receptors. However, sham and MI rats expressed fewer ANG II receptors than normal controls. Renal vascular angiotensin receptor density was reduced by 67% in MI rats compared with normal controls, and there was also a 49% decrease in sham rats compared with normal controls [1,825 ± 169 fmol/mg (n = 9), P < 0.01 for MI vs. control, P < 0.02 for control vs. sham]. Receptor affinity was increased in MI rats compared with normal control rats (P < 0.05) but was not significantly different from sham rats (Kd = 0.62 ± 0.06, 2.3 ± 0.2, and 0.99 ± 0.10 nM for MI, control, and sham, respectively). The finding that renal vascular ANG II receptors were downregulated after MI is compatible with the expected homologous downregulation of angiotensin receptors by increased ANG II in kidneys from rats with MI (20).


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  		Fig. 4.   Equilibrium binding of 125I-[Sar1,Ile8]ANG II in renal vessels from control, sham, and MI rats. Specific binding was studied at 60 min using 0.14-17 nM [Sar1,Ile8]ANG II. In renal vessels from MI rats (n = 9), there was a significant decrease in specific binding at the 3 highest incubated concentrations compared with normal control rats (n = 9). Specific binding levels in sham rats (n = 10) were intermediate between MI and control rats. * P < 0.02 compared with other two groups.

Homologous Regulation of Glomerular ANG II Receptors

Additional experiments were performed to determine whether the increase in angiotensin receptor expression in glomeruli after MI was due to an overall increase in receptor number or an abnormality in the homologous regulation of angiotensin receptors. The effects of inhibiting or stimulating ANG II levels were studied in MI, sham, and normal control rats. In one group of experiments the effects of inhibiting ANG II with an acute dose of enalapril were measured. In a second group of experiments the effects of acutely increasing ANG II levels were studied. Baseline mean arterial pressure (MAP) was similar in MI, sham, and control rats (111.7 ± 6.5, 116.5 ± 7.4, and 108.1 ± 8.6 mmHg in MI, sham, and control, respectively, P = NS), and ANG II infusion (100 ng · kg-1 · min-1) caused a similar increase in systemic blood pressure (MAP = 37.8 ± 2.5, 39.8 ± 4.7, and 32.7 ± 3.9 mmHg in MI, sham, and control, respectively, P = NS). Rats receiving enalapril by gavage did not have indwelling catheters, and blood pressures were not recorded. ANG II infusion caused a significant decrease in the density of glomerular ANG II receptors in all three groups to a similar level, indicating that the maximal homologous downregulation of ANG II receptors was not affected in the MI group (Fig. 5). Inhibition of ANG II formation with enalapril caused a significant increase in the density of ANG II receptors to levels that were similar in all three groups (Fig. 5). In all three groups, enalapril caused a significant increase in the density of ANG II receptors compared with untreated rats: P < 0.02 (MI) and P < 0.002 (sham and control). Decreases and increases in ANG II caused similar changes in ANG II receptor density in MI, sham, and control rats.


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  		Fig. 5.   Ro of glomerular ANG II receptors in untreated, ANG II-infused, and enalapril-treated rats. Ro of ANG II receptors was significantly higher in MI rats than in control and sham rats (filled bars, * P < 0.002). Homologous downregulation of ANG II receptors was studied by infusion of ANG II (100 ng · kg-1 · min-1 for 15 min). In control, MI, and sham rats there was a significant decrease in Ro to final levels that were similar in all 3 groups, indicating that maximal homologous downregulation of ANG II receptors was not affected in MI group (P < 0.005 vs. untreated; open bars). Homologous upregulation was studied by gavage of 10 mg/kg of converting enzyme inhibitor enalapril (rats were studied 3 h later). In control, MI, and sham rats, there was a significant increase in Ro to final levels that were similar in all three groups (P < 0.002 vs. untreated; crosshatched bars).

ANG II infusion did not affect the Kd for glomerular ANG II receptors in any of the groups: 1.6 ± 0.2, 1.5 ± 0.2, and 1.6 ± 0.2 nM in MI, sham, and control, respectively, (P = NS vs. uninfused). However, treatment with enalapril was associated with a significant (P < 0.002) but similar increase in Kd in all groups of rats: 4.9 ± 0.7, 5.0 ± 0.3, and 4.9 ± 0.6 nM in MI, sham, and control, respectively (P = NS for group effect).

Evidence for a Stimulated Intrarenal Renin-Angiotensin System After MI

As an index of activity of the intrarenal renin-angiotensin system, steady-state expression of renin and angiotensinogen was measured by PCR analysis of RNA isolated from the renal cortex. Amounts of mRNA were standardized by comparison with simultaneously amplified messages for beta -actin. The mRNA angiotensinogen-to-actin ratio in MI rats was significantly increased by 35% compared with normal control rats (P < 0.025). The sham rats were intermediate and were not statistically different from MI or normal control rats. The renin-to-actin ratio was increased in MI (55%) and sham rats (46%) compared with normal control rats (P < 0.05; Fig. 6).


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  		Fig. 6.   Molecular components of renin-angiotensin system in renal cortex. Renal components of renin-angiotensin system were measured by PCR analysis of renin and angiotensinogen. Amounts of mRNA expressed were standardized by comparison with simultaneously amplified messages for actin. Expression of mRNA for angiotensinogen (P < 0.05, top) and renin (P < 0.05, bottom) was significantly increased in MI rats compared with controls. Expression of mRNA for renin was also increased in sham group compared with controls (P < 0.05). Expression of mRNA for angiotensinogen in sham group was intermediate between control and MI groups and was not significantly different from either group. Numbers within bars indicate numbers of rats studied. * P < 0.05.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The potential role of the renin-angiotensin system in modulating renal hemodynamics in congestive heart failure has long been suspected. Studies of glomerular dynamics in rats with congestive heart failure demonstrated that efferent arteriolar constriction in congestive heart failure could be reversed by inhibition of the renin-angiotensin system (9, 19). Studies from our laboratory suggested abnormalities in the expression of ANG II receptors in the kidney or their regulation in congestive heart failure (15). In the current study we tested the hypothesis that the expression and regulation of glomerular ANG II receptors were altered in rats with congestive heart failure. Glomerular and renal vascular ANG II receptors were studied in rats 4-6 wk after MI. The data indicate a significant increase in the expression of glomerular ANG II receptors in MI rats compared with sham or normal control rats. Additional experiments were performed to study the specific subtype(s) of angiotensin receptor expressed in the glomeruli after MI. The data indicated that the predominant, if not exclusive, receptor expressed was the AT1 subtype, and there was no evidence of increased AT2-receptor expression after MI. The finding that the data for sham rats were in some cases intermediate between data for normal control and MI rats is not surprising, since the sham group consisted of rats with ligatures that missed the coronary artery or rats that had a tiny MI without the development of a transmural scar.

The circulating renin-angiotensin system is likely to be important in the early phases of cardiac compensation in congestive heart failure. However, during chronic cardiac impairment the most striking increases occur in the intrarenal renin-angiotensin system (6, 8). Although these changes are initially effective in compensating for the reduced cardiac output, in the absence of correction of low cardiac output, increased activity of the renin-angiotensin system further aggravates congestive heart failure and hastens the development of morbid events. Numerous studies suggest an important role for increases in local components of the renin-angiotensin system in rats with MI (10, 11, 13). Because kidney ANG II content and renal angiotensinogen mRNA increased twofold in rats with MI (20), the finding of increased expression of glomerular ANG II receptors in the current study suggested that there may be abnormalities in the normal homologous downregulation response of glomerular ANG II receptors (1, 22, 24) in congestive heart failure. This hypothesis was further tested by studying the effects of inhibiting ANG II levels with the converting enzyme inhibitor enalapril or raising ANG II levels by infusion. The data indicate that the homologous downregulation response was intact in renal glomeruli after MI but that the number of ANG II receptors expressed in MI rats was set at an increased level.

To confirm the reports that there is a stimulated renin-angiotensin system in the kidney in congestive heart failure (20), we examined steady-state mRNA levels for renin and angiotensinogen in the renal cortex. As expected, mRNA for angiotensinogen and renin were increased in rats after MI. These data are compatible with observations that the intrarenal renin-angiotensin system is stimulated in congestive heart failure. When interpreted in the context of previous studies that demonstrate that stimulation of the intrarenal renin-angiotensin system is associated with downregulation of glomerular ANG II receptor sites (1, 24), the current observations indicate that the number of glomerular ANG II receptors expressed is much higher than anticipated. This finding may help explain the intense intrarenal vasoconstriction and abnormalities in glomerular hemodynamics characteristic of congestive heart failure. To test the specificity of the abnormalities in ANG II receptor expression in glomeruli, we measured the expression of renal vascular ANG II receptors in rats after MI. Vascular ANG II receptor expression was downregulated in rats with MI, an observation compatible with the expected downregulation of vascular AT1 receptors in response to elevated local levels of the hormone. Therefore, there was a dissociation between the downregulation of ANG II receptors in blood vessels and the upregulation of ANG II receptors in glomeruli in congestive heart failure. The mechanism for regional differences in ANG II receptor expression in congestive heart failure remains to be elucidated. Many factors are known to regulate glomerular ANG II receptor density independent of the local concentration of angiotensin. These include changes in glomerular size (larger glomeruli express more surface receptors) (23), glucose intolerance (glomeruli from diabetic rats have fewer receptor sites) (22), corticosteroid levels (5), and protein kinase C activity (high levels of protein kinase C promote internalization of ANG II receptors in response to elevated glucose levels) (25). Renal denervation reduces sympathetic output to the kidneys (4) and lowers renin and ANG II levels and upregulates glomerular ANG II receptors (24). Conversely, increased sympathetic output to the kidneys in congestive heart failure would be expected to raise local renin levels and ANG II production. It is unlikely that the abnormality in glomerular ANG II receptor regulation in congestive heart failure is due to changes in sympathetic nerve output. The cause for dysregulation of glomerular angiotensin receptors is likely to be related to a localized abnormality, since renal vascular ANG II receptors downregulated normally in congestive heart failure. The concentration of ANG II at the glomerular receptor sites may be reduced, possibly as a consequence of increased local degradation of angiotensin in glomeruli and not renal vessels. Nishimura et al. (18) described upregulation of angiotensin receptor gene expression in renal arterioles of mice by extracellular fluid volume depletion in the absence of an interaction between ligand and receptor. In our studies the upregulation of angiotensin receptors in glomeruli of MI rats could reflect a similar mechanism, since glomerular preparations may contain arterioles, which are important in determining renal resistance. However, routine examination of glomeruli for optical purity shows that attachment of arterioles to the glomerular tuft occurs infrequently. This makes it more likely that the observed abnormalities reside within the glomerulus. There also may be an acquired abnormality in regulatory sequences that determines the expression of ANG II receptors in renal glomeruli or the relative number of receptors internalized and expressed on the cell surface. ANG II receptors are known to be regulated dynamically by the interactions of the receptor with G proteins and intracellular signaling systems (7). Abnormalities in these systems can provide an explanation for the abnormalities in angiotensin receptors in the kidneys in congestive heart failure and will be the focus of future investigations.

    ACKNOWLEDGEMENTS

This work was supported in part by National Heart, Lung, and Blood Institute Grants HL-44373 and HL-40914 (B. M. Wilkes), the American Heart Association, New York State Affiliate (P. F. Mento), and the Heart Council of Long Island (P. F. Mento). J. Hilepo was the recipient of a 2-yr fellowship award from the National Kidney Foundation of NY/NJ, Inc.

    FOOTNOTES

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: B. M. Wilkes, Div. of Nephrology and Hypertension, North Shore University Hospital, 100 Community Dr., Great Neck, NY 11021.

Received 25 February 1998; accepted in final form 17 June 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Bellucci, A., and B. M. Wilkes. Mechanism of sodium modulation of glomerular angiotensin receptors in the rat. J. Clin. Invest. 74: 1593-1600, 1984.

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Am J Physiol Heart Circ Physiol 275(4):H1247-H1253
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society


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