Reciprocal feedback regulation of kidney angiotensinogen and renin mRNA expressions by angiotensin II HERIBERT SCHUNKERT, JULIE R. INGELFINGER, HOWARD JACOB, BRUCE JACKSON, BOUTROS BOUYOUNES, AND VICTOR J. DZAU Cardiovascular Research Center, Stanford University School of Medicine, Stanford, California 94305-5246; and Division of Pediatric Nephrology, Massachusetts General Hospital, Boston, Massachusetts 02114 Schunkert, Heribert, Julie R. Ingelfinger, Howard Jacob, Bruce Jackson, Boutros Bouyounes, and Victor J. Dzau. Reciprocal feedback regulation of kidney angiotensinogen and renin mRNA expressions by angiotensin II. Am. J. Physiol. 263 (Endocrinol. Metab. 26): E863-E869, 1992.-The present study asks whether angiotensin II (ANG II), a potent inhibitor of renal renin synthesis and release, regulates renal angiotensinogen synthesis. ANG II (or vehicle) was intravenously infused into male Sprague-Dawley rats for 3 days (vehicle or 100,300, and 1,000 ng kg-l min-l, n = g/group), significantly increasing mean plasma ANG II concentrations and raising mean arterial blood pressure (MAP). ANG II dose dependently suppressed plasma renin concentration, kidney renin concentration, and renal renin mRNA levels. In contrast, ANG II infusion increased renal angiotensinogen mRNA levels stepwise to 122,136 (P < 0.05), and 150% (P < 0.05) of control and also increased both liver mRNA levels (P < 0.05) and plasma angiotensinogen concentration (P < 0.05). Three days of angiotensin-converting enzyme inhibition (10 mg kg-l day-l quinapril in drinking water, n = 8) significantly decreased MAP (P < 0.05) and increased both mean plasma renin concentration (P c 0.05) and renal renin mRNA levels (P < 0.005). Plasma ANG II concentration tended to decrease (not significant), and neither renal nor hepatic angiotensinogen mRNA levels displayed significant difference. However, when data from ANG II-infused and quinapril-treated rats were analyzed together, correlation between plasma ANG II concentrations and renal angiotensinogen mRNA levels was highly significant (P < 0.005, r = 0.585). Thus plasma ANG II upregulates renal angiotensinogen gene expression and downregulates renal renin gene expression, a reciprocal feedback regulation that may have important physiological consequences. messenger ribonuclease l
l
l
l
EXTENSIVE WORK in recent years has demonstrated that the kidney is not just a target organ of the circulating renin-angiotensin system (RAS) but contains a functionally important (l4,20-22,3l, 38) and independently regulated tissue RAS (8, 15, 26). Furthermore, the concept of an intrarenal RAS is supported by the observations that angiotensin II (ANG II) concentrations in renal lymph (1) and proximal tubule fluid (55) far exceed the plasma concentration. All components of the RAS have been detected by immunohistochemical staining in the kidney (50, 59). Angiotensinogen has been detected in renal tissue using enzymatic assay (43). The coexpression of renin (9, 41), angiotensinogen (26), and angiotensin-converting-enzyme (ACE) mRNAs (58) has provided even stronger evidence of the existence of an intrarenal RAS. Our knowledge of the regulation of renal angiotensinogen synthesis and the interaction between the circulating and intrarenal components of the RAS is incomplete. It has been shown that a low-sodium diet (26) 0193-1849/92
$2.00 Copyright
increases renal angiotensinogen mRNA expression. Furthermore, various hormones have effects on the steadystate expression of angiotensinogen. For example, administration of a mixture of glucocorticoids plus thyroxine and estrogen (8) or testosterone (15) stimulates renal angiotensinogen mRNA expression. However, Kalinyak and Perlman (30) found no increase in renal angiotensinogen mRNA with glucocorticoid stimulation alone. In the past, the role of ANG II on angiotensinogen mRNA expression has been approached largely indirectly by means of ACE inhibition. Gomez et al. (17) reported that ACE inhibition had no significant effect on renal angiotensinogen expression in normal rats receiving regular Chow. However, it has been observed that ACE inhibition can reduce renal angiotensinogen mRNA levels in animals with experimental heart failure (53) or on a low-sodium diet (28). These observations suggest that angiotensin, under the appropriate circumstances (e.g., when the intrarenal RAS is activated), may play a role in the feedback regulation of the intrarenal renin and angiotensinogen gene expression. To examine directly the effect of ANG II, we measured renal angiotensinogen mRNA as well as renal renin mRNA and their respective protein concentrations after a 3-day intravenous ANG II infusion. In a parallel experiment the effects of the blockade of ANG II production by the ACE inhibitor quinapril were studied. Our data demonstrate that high concentrations of ANG II exert a positive feedback stimulation on renal angiotensinogen mRNA levels. On the other hand, renal renin mRNA level, enzyme activity, and plasma renin concentration were suppressed by ANG II in a dose-dependent manner. METHODS Male Sprague-Dawley rats (8 wk old), weighing 250-300 g, were studied. In the first protocol, the effects of ANG II infusion for 3 days (100, 300, or 1,000 nge kg-l amin-l, n = 8 for each group; Sigma Chemicals, St. Louis, MO) were compared with a control group (n = 8, saline infusion). In the second protocol, rats were treated for 3 days with either quinapril (10 mg kg-l day-l in drinking water, n = 8) or water alone (n = 7). Animals were allowed free access to a 0.4% sodium diet and water. The protocols were approved by the Institutional Standing Committee on Animals and were performed according to National Institutes of Health guidelines. ANG II infusion and blood pressure measurements. Under pentobarbital sodium anesthesia, PE-10 tubing was inserted in both femoral vein and artery. The venous line was filled with the respective infusate and attached to an Alzet osmotic minipump (Alza, Palo Alto, CA; flow rate 1.02 PI/h). The arterial line was cannulated subcutaneously to the neck for subsequent direct blood pressure measurements. Mean arterial blood pressure was l
l
0 1992 the American Physiological
Society
ES63
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E864
RECIPROCAL
FEEDBACK
measured on day 3 of the study with a Gould Statham pressure transducer connected to a polygraph (Grass Instruments, Quincy, MA). Urinary sodium and creatinine clearance. On the 2nd to 3rd day of the study, urine was collected for sodium and creatinine measurements. Sodium ion was measured using a flame photometer (Instrumentation Laboratory 443, Lexington, MA). Creatinine was determined in urine and serum using a modified Jaffe reaction (Sigma Chemicals). Creatinine clearance was calculated from the 24-h urinary excretion of creatinine divided by the serum creatinine concentration. Measurement of renin, ANG II, and angiotensinogen. Plasma renin concentration was measured in rat plasma obtained from trunk blood collected in ice-chilled tubes containing Na-EDTA (final concn of 4 mM). Plasma was incubated with excess exogenous angiotensinogen at 37”C, pH 7.4, in the presence of inhibitors [3.4 mM 8hydroxyquinolone sulfate, 0.25 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1.6 mM dimercaprol, and 5 mM sodium tetrathionate (NaTT)] for l-3 h. The source of angiotensinogen was renin-free plasma (diluted 1:3) from sheep that had been nephrectomized 48 h previously. The ANG I generated was measured by radioimmunoassay (19). Tissue renin concentration was similarly determined. The tissue was homogenized in 0.1 M tris(hydroxymethyl)aminomethane*HCl, pH 7.4, containing the above inhibitors plus 0.1% Triton X-100. After centrifugation to remove debris, the supernatant was assayed as above. Tissue protein concentration was determined by the method of Bradford (6). Tissue renin concentration was expressed as pmol ANG I h-l mg protein-l. Plasma angiotensinogen concentration was determined using an “exhaustion” assay as previously described (15). Briefly, plasma was incubated with excessexogenous renin at 37”C, pH 6, in the presence of protease inhibitors (in mM: 3.4 &hydroxyquinolone sulfate, 0.25 EDTA, 0.1 PMSF, 5 NaTT, and 1.6 dimercaprol) for l-3 h, which was found to be adequate for complete hydrolysis of ANG I from angiotensinogen. The source of renin was mouse submandibular gland renin, partially purified by precipitation with ammonium sulfate (80%), subsequent dialysis, and purification with pepstatin column (Pharmacia Biotechnology, Piscataway, NJ). ANG I generated was measured by radioimmunoassay. Plasma ANG II was measured in rat plasma obtained by centrifugation at 4°C of blood collected in ice-chilled tubes containing EDTA, PMSF, and NaTT, after which the plasma was snap-frozen in liquid nitrogen. Subsequently, plasma was partially purified on a SepPak column (Waters, Millis, MA) conditioned with 3 ml methanol and 10 ml 0.01% trifluoroacetic acid (TFA) before sample was loaded and washed with 10 ml 0.01% TFA. Peptides were eluted with 3 ml 80% acetonitrile in 0.01% TFA and lyophilized. Recovery of ANG II standard (Sigma Chemicals) over a range of lo-200 fmol/ml by this procedure was 95%. ANG II was then measured by radioimmunoassay using a commercial rabbit ANG II antibody (Arnel, New York, NY) and a donkey anti-rabbit second antibody (Amersham, Arlington Heights, IL) for magnetic separation of bound and unbound tracer. RNA studies. Rats were killed by decapitation. Organs were snap-frozen in liquid nitrogen within 3 min, and tissue was stored at -80°C until used (maximum storage = 2 days). Homogenization of various tissues and extraction of total RNA were carried out using the guanidium thiocyanate method (10). RNA was resuspended and precipitated in 2 vol ethanol. The precipitated RNA was dissolved in water and the amount quantified by absorbance at 260 nm in duplicate. The same amount of total RNA applied per sample permitted subsequent comparison of relative mRNA levels, as further detailed below. l
l
REGULATION
For quantitative studies, we performed slot-blot hybridization analysis. Samples were denatured with formaldehyde and diluted serially in 15~ standard saline citrate (SSC). Three concentrations from each sample (8,4, and 2 pg) were blotted in duplicate onto nylon filters using a slot-blot apparatus (Schleicher and Schuell, Peterborough, NH) after which they were cross-linked by ultraviolet light. After prehybridization for 4 h, the blots were hybridized overnight in a buffer to which [a-32P] cDNA probes were added. Blots were then washed in 0.2~ SSC with 0.1% sodium dodecyl sulfate at room temperature for 10 min, then three times at 65°C for 30 min. Blots were exposed for 5 days to X-ray film (Kodak XAR, Eastman Kodak, Rochester, NY). All studies of angiotensinogen mRNA were performed with a full-length rat liver angiotensinogen cDNA probe (pRang 1650). For renin mRNA determinations, we used a full-length rat renin cDNA probe. RNA from rat testes served as a negative control for angiotensinogen mRNA, and rat hepatic RNA served as a negative control for renin mRNA. To control for possible sample variability, identical slot blots were performed and hybridized with a @actin cDNA probe. P-actin mRNA levels did not change under the current experimental conditions. Quantification of mRNA and statistical analysis. Autoradiograms generated by slot blots were scanned with an LKB microdensitometer (Paramus, NJ) with background set to zero for each autoradiograph. Regression lines were calculated from the integral values obtained by scanning each sample at various serial concentrations of total RNA applied. The relative signals of the specific mRNA were estimated from the slope of the regression line, and only r values 20.90 were accepted. Exposures of the films were such that, in all cases,the values were in the linear range for the Beer-Lambert law, and a standard curve from 0.5 to 16 pg total RNA from rat kidney or liver (depending on tissue) was constructed for each blot. The derived slopes of specific mRNA for each condition were compared as relative ratios. Sufficient material was available to permit multiple sample applications from each specimen. RNA from four adult male rat livers or kidneys were run on each slot blot as interblot reference standards to normalize calculations. Our intra- and interblot coefficients of variation were 8 and 9%, respectively. Average slopes were directly compared by Student’s unpaired t tests or analysis of variance for multiple comparisons in case of comparisons between three or more groups. Relationships between plasma ANG II concentration, creatinine clearance, or sodium excretion and renal angiotensinogen mRNA levels were determined using rank correlation analysis (Spearman’s rho). Significance was accepted for P < 0.05. RESULTS Changes in blood pressure and plasma ANG II with administration of ANG II or ACE inhibitor. Infusion of
intravenous ANG II by osmotic minipumps increased plasma ANG II concentrations in a dose-related manner (Table 1). Mean arterial blood pressure increased in rats with ANG II infusion as well (Table 2) but was more variable with the highest dose of ANG II. ACE inhibition with quinapril was followed by a slight decrease in plasma ANG II concentrations (Table 1). Quinapril treatment decreased blood pressure by an average of 12 mmHg (Table 2). Effects of ANG II on circulating and tissue renin. ANG II infusion resulted in a significant reduction of renal renin mRNA level (Fig. 1). Kidney renin concentration
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RECIPROCAL
Table 1. Plasma concentrations
FEEDBACK
E865
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and kidney renin content ANG
II Infusion,
ng - kg-l . min-l
ACE Inhibition
Sham 100
300
1,000
Control
Quinapril
Plasma ANG II, fmol/ml 62&13 101~10 126&17* 168+12$ 66k14 49t8 Plasma angiotensinogen concentration, pmol/ml 1,218+173 1,773+119* 1,558+145 2,068+348* 1,231+110 835+90§ PRC, pmol ANG I h-l. ml-l 7.6&l 3.5+0.6-f 2.9+0.4$ 2.6kO.31 6.8& 1 14.Ok2.59 KRC, pmol ANG I h-l. mg protein-l 1,171+312 705t233 523t108* 347+467 1,108&123 1,039&181 Values are means t SE. In ANG II infusion studies, n = 8 rats/group; in quinapril study, n = 8 quinapril-treated rats; and n = 7 rats in controls. Plasma concentrations of ANG II, angiotensinogen, and renin (PRC) as well as kidney renin content (KRC) in animals with ANG II infusion or angiotensin-converting enzyme (ACE) inhibition for 3 days and respective controls. * P < 0.05, t P < 0.01, and $ P < 0.005 by analysis of variance (ANOVA) as compared with sham. $ P c 0.05 by Student’s unpaired t test as compared with sham. l
l
Table 2. Mean arterial blood pressure, sodium excretion, and creatinine clearance in animals with ANG II infusion or ACE inhibition ANG
II Infusion,
ng - kg-l - min-l
ACE Inhibition
Sham 100
300
1,000
Control
Quinapril
Mean arterial blood pressure, mmHg 10624.1 119t1.2” 134t9.6* 125t11.3 lllt3.6 99k4.25 Urinary Na+ excretion, meq/day 1.99t0.13 1.64&0.19* 1.46t0.20 1.22&0.31* 1.93t0.17 2.03t0.21 Creatinine clearance, ml min-l kg-l 6.37kO.41 5.31&0.54* 4.39kO.437 3.02+0.43$ 5.85kO.22 5.8t0.2 1 Values are means t SE. In ANG II infusion studies, n = 8 rats/group; in quinapril study, n = 8 quinapril-treated rats; and n = 7 rats in controls. Mean arterial blood pressure, sodium excretion, and creatinine clearance in animals with ANG II infusion or ACE inhibition for 3 days and respective controls. * P < 0.05, t P c 0.01, and $ P < 0.005 by ANOVA as compared with sham. § P < 0.05 by Student’s unpaired t test as compared with sham. l
l
increased plasma renin concentration twofold (Table 1). Kidney renin concentrations showed no difference between rats treated with quinapril for 3 days and control (Table 1). Effects of ANG II on angiotensinogen. The effects of ANG II on renal and hepatic angiotensinogen mRNA levels are shown in Fig. 2. ANG II infusion increased angiotensinogen mRNA levels significantly in both tissues, whereas the slight reduction of plasma ANG II concentrations by quinapril was not associated with significant changes in tissue angiotensinogen mRNA levels. Overall, renal angiotensinogen mRNA levels correlated significantly with plasma ANG II concentrations (r = 0.585;P < 0.005),providing further evidence for a positive feedback regulation of ANG II on its precursor (Fig. SHAM ANG II CONTROL WIN ANG II ANG II 100 300 1000 3). We also observed an increase in plasma angiotensinogen concentrations in response to ANG II infusion (Table ?cts of ANG II and angiotensin-converti] ng enzyme (ACE) inhibition on renal renin in RNA levels as calculated from slope of 1). There was no significant correlation between blood densitometric analysis of slot-blot hybridization. Infusion of ANG II for pressure and angiotensinogen mRNA levels in either kid3 days (100, 300, and 1,000 ngekg-l l rein-l, respectively) decreased ney or liver [r = 0.038, P = not significant (NS), and r = renal renin mRNA levels in an apparent dose-dependent manner. Thus 0.033, P = NS], suggesting that the changes in mRNA renal renin mRNA levels decreased to 60, 44 (P < 0.05), and 29% (P < 0.05) of control, respectively, with each dose of ANG II. On the other levels after ANG II infusion were not mediated by alterhand, ACE inhibition with quinapril (Quin; 10 mg. kg-l *day-l) for 3 ations in blood pressure. days increased renal renin mRNA levels (134% of control). *P < 0.05 by Renal functional effects of ANG II. Sodium excretion analysis of variance (ANOVA); t P < 0.005 by Student’s t test, as and creatinine clearance studied on the 3rd day of the compared with sham or control. In ANG II infusion studies, n = 8 rats/group; in quinapril study, n = 8 quinapril-treated rats/group; and IZ experiment decreased significantly and dose dependently = 7 rats/group in control study. in animals receiving ANG II infusion (Table 2). Interestingly, there was a significant inverse correlation between and plasma renin concentration decreased in parallel (Ta- renal angiotensinogen mRNA levels and Na+ excretion (r ble 1). There was a significant inverse relationship be- = 0.488, P = O.Ol),as well as creatinine clearance (r = tween plasma ANG II concentrations and plasma renin 0.663, P = 0.0006). On the other hand, the correlation concentration (r = 0.46;P = 0.005), thereby documenting between plasma ANG II concentrations and 24-h sodium a negative feedback effect of ANG II on renin synthesis excretion (r = 0.106, P = NS) or creatinine clearance (r = and release. Quinapril, on the other hand, stimulated re- 0.412, P = 0.05) displayed only a weak relationship. nal renin mRNA level to 134% of control (Fig. 1) and Quinapril treatment for 3 days had no significant effect +
J
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RECIPROCAL
FEEDBACK
REGULATION
p
l
l
l
EXTENSIVE WORK in recent years has demonstrated that the kidney is not just a target organ of the circulating renin-angiotensin system (RAS) but contains a functionally important (l4,20-22,3l, 38) and independently regulated tissue RAS (8, 15, 26). Furthermore, the concept of an intrarenal RAS is supported by the observations that angiotensin II (ANG II) concentrations in renal lymph (1) and proximal tubule fluid (55) far exceed the plasma concentration. All components of the RAS have been detected by immunohistochemical staining in the kidney (50, 59). Angiotensinogen has been detected in renal tissue using enzymatic assay (43). The coexpression of renin (9, 41), angiotensinogen (26), and angiotensin-converting-enzyme (ACE) mRNAs (58) has provided even stronger evidence of the existence of an intrarenal RAS. Our knowledge of the regulation of renal angiotensinogen synthesis and the interaction between the circulating and intrarenal components of the RAS is incomplete. It has been shown that a low-sodium diet (26) 0193-1849/92
$2.00 Copyright
increases renal angiotensinogen mRNA expression. Furthermore, various hormones have effects on the steadystate expression of angiotensinogen. For example, administration of a mixture of glucocorticoids plus thyroxine and estrogen (8) or testosterone (15) stimulates renal angiotensinogen mRNA expression. However, Kalinyak and Perlman (30) found no increase in renal angiotensinogen mRNA with glucocorticoid stimulation alone. In the past, the role of ANG II on angiotensinogen mRNA expression has been approached largely indirectly by means of ACE inhibition. Gomez et al. (17) reported that ACE inhibition had no significant effect on renal angiotensinogen expression in normal rats receiving regular Chow. However, it has been observed that ACE inhibition can reduce renal angiotensinogen mRNA levels in animals with experimental heart failure (53) or on a low-sodium diet (28). These observations suggest that angiotensin, under the appropriate circumstances (e.g., when the intrarenal RAS is activated), may play a role in the feedback regulation of the intrarenal renin and angiotensinogen gene expression. To examine directly the effect of ANG II, we measured renal angiotensinogen mRNA as well as renal renin mRNA and their respective protein concentrations after a 3-day intravenous ANG II infusion. In a parallel experiment the effects of the blockade of ANG II production by the ACE inhibitor quinapril were studied. Our data demonstrate that high concentrations of ANG II exert a positive feedback stimulation on renal angiotensinogen mRNA levels. On the other hand, renal renin mRNA level, enzyme activity, and plasma renin concentration were suppressed by ANG II in a dose-dependent manner. METHODS Male Sprague-Dawley rats (8 wk old), weighing 250-300 g, were studied. In the first protocol, the effects of ANG II infusion for 3 days (100, 300, or 1,000 nge kg-l amin-l, n = 8 for each group; Sigma Chemicals, St. Louis, MO) were compared with a control group (n = 8, saline infusion). In the second protocol, rats were treated for 3 days with either quinapril (10 mg kg-l day-l in drinking water, n = 8) or water alone (n = 7). Animals were allowed free access to a 0.4% sodium diet and water. The protocols were approved by the Institutional Standing Committee on Animals and were performed according to National Institutes of Health guidelines. ANG II infusion and blood pressure measurements. Under pentobarbital sodium anesthesia, PE-10 tubing was inserted in both femoral vein and artery. The venous line was filled with the respective infusate and attached to an Alzet osmotic minipump (Alza, Palo Alto, CA; flow rate 1.02 PI/h). The arterial line was cannulated subcutaneously to the neck for subsequent direct blood pressure measurements. Mean arterial blood pressure was l
l
0 1992 the American Physiological
Society
ES63
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (130.237.165.040) on December 4, 2018. Copyright © 1992 the American Physiological Society. All rights reserved.
E864
RECIPROCAL
FEEDBACK
measured on day 3 of the study with a Gould Statham pressure transducer connected to a polygraph (Grass Instruments, Quincy, MA). Urinary sodium and creatinine clearance. On the 2nd to 3rd day of the study, urine was collected for sodium and creatinine measurements. Sodium ion was measured using a flame photometer (Instrumentation Laboratory 443, Lexington, MA). Creatinine was determined in urine and serum using a modified Jaffe reaction (Sigma Chemicals). Creatinine clearance was calculated from the 24-h urinary excretion of creatinine divided by the serum creatinine concentration. Measurement of renin, ANG II, and angiotensinogen. Plasma renin concentration was measured in rat plasma obtained from trunk blood collected in ice-chilled tubes containing Na-EDTA (final concn of 4 mM). Plasma was incubated with excess exogenous angiotensinogen at 37”C, pH 7.4, in the presence of inhibitors [3.4 mM 8hydroxyquinolone sulfate, 0.25 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), 1.6 mM dimercaprol, and 5 mM sodium tetrathionate (NaTT)] for l-3 h. The source of angiotensinogen was renin-free plasma (diluted 1:3) from sheep that had been nephrectomized 48 h previously. The ANG I generated was measured by radioimmunoassay (19). Tissue renin concentration was similarly determined. The tissue was homogenized in 0.1 M tris(hydroxymethyl)aminomethane*HCl, pH 7.4, containing the above inhibitors plus 0.1% Triton X-100. After centrifugation to remove debris, the supernatant was assayed as above. Tissue protein concentration was determined by the method of Bradford (6). Tissue renin concentration was expressed as pmol ANG I h-l mg protein-l. Plasma angiotensinogen concentration was determined using an “exhaustion” assay as previously described (15). Briefly, plasma was incubated with excessexogenous renin at 37”C, pH 6, in the presence of protease inhibitors (in mM: 3.4 &hydroxyquinolone sulfate, 0.25 EDTA, 0.1 PMSF, 5 NaTT, and 1.6 dimercaprol) for l-3 h, which was found to be adequate for complete hydrolysis of ANG I from angiotensinogen. The source of renin was mouse submandibular gland renin, partially purified by precipitation with ammonium sulfate (80%), subsequent dialysis, and purification with pepstatin column (Pharmacia Biotechnology, Piscataway, NJ). ANG I generated was measured by radioimmunoassay. Plasma ANG II was measured in rat plasma obtained by centrifugation at 4°C of blood collected in ice-chilled tubes containing EDTA, PMSF, and NaTT, after which the plasma was snap-frozen in liquid nitrogen. Subsequently, plasma was partially purified on a SepPak column (Waters, Millis, MA) conditioned with 3 ml methanol and 10 ml 0.01% trifluoroacetic acid (TFA) before sample was loaded and washed with 10 ml 0.01% TFA. Peptides were eluted with 3 ml 80% acetonitrile in 0.01% TFA and lyophilized. Recovery of ANG II standard (Sigma Chemicals) over a range of lo-200 fmol/ml by this procedure was 95%. ANG II was then measured by radioimmunoassay using a commercial rabbit ANG II antibody (Arnel, New York, NY) and a donkey anti-rabbit second antibody (Amersham, Arlington Heights, IL) for magnetic separation of bound and unbound tracer. RNA studies. Rats were killed by decapitation. Organs were snap-frozen in liquid nitrogen within 3 min, and tissue was stored at -80°C until used (maximum storage = 2 days). Homogenization of various tissues and extraction of total RNA were carried out using the guanidium thiocyanate method (10). RNA was resuspended and precipitated in 2 vol ethanol. The precipitated RNA was dissolved in water and the amount quantified by absorbance at 260 nm in duplicate. The same amount of total RNA applied per sample permitted subsequent comparison of relative mRNA levels, as further detailed below. l
l
REGULATION
For quantitative studies, we performed slot-blot hybridization analysis. Samples were denatured with formaldehyde and diluted serially in 15~ standard saline citrate (SSC). Three concentrations from each sample (8,4, and 2 pg) were blotted in duplicate onto nylon filters using a slot-blot apparatus (Schleicher and Schuell, Peterborough, NH) after which they were cross-linked by ultraviolet light. After prehybridization for 4 h, the blots were hybridized overnight in a buffer to which [a-32P] cDNA probes were added. Blots were then washed in 0.2~ SSC with 0.1% sodium dodecyl sulfate at room temperature for 10 min, then three times at 65°C for 30 min. Blots were exposed for 5 days to X-ray film (Kodak XAR, Eastman Kodak, Rochester, NY). All studies of angiotensinogen mRNA were performed with a full-length rat liver angiotensinogen cDNA probe (pRang 1650). For renin mRNA determinations, we used a full-length rat renin cDNA probe. RNA from rat testes served as a negative control for angiotensinogen mRNA, and rat hepatic RNA served as a negative control for renin mRNA. To control for possible sample variability, identical slot blots were performed and hybridized with a @actin cDNA probe. P-actin mRNA levels did not change under the current experimental conditions. Quantification of mRNA and statistical analysis. Autoradiograms generated by slot blots were scanned with an LKB microdensitometer (Paramus, NJ) with background set to zero for each autoradiograph. Regression lines were calculated from the integral values obtained by scanning each sample at various serial concentrations of total RNA applied. The relative signals of the specific mRNA were estimated from the slope of the regression line, and only r values 20.90 were accepted. Exposures of the films were such that, in all cases,the values were in the linear range for the Beer-Lambert law, and a standard curve from 0.5 to 16 pg total RNA from rat kidney or liver (depending on tissue) was constructed for each blot. The derived slopes of specific mRNA for each condition were compared as relative ratios. Sufficient material was available to permit multiple sample applications from each specimen. RNA from four adult male rat livers or kidneys were run on each slot blot as interblot reference standards to normalize calculations. Our intra- and interblot coefficients of variation were 8 and 9%, respectively. Average slopes were directly compared by Student’s unpaired t tests or analysis of variance for multiple comparisons in case of comparisons between three or more groups. Relationships between plasma ANG II concentration, creatinine clearance, or sodium excretion and renal angiotensinogen mRNA levels were determined using rank correlation analysis (Spearman’s rho). Significance was accepted for P < 0.05. RESULTS Changes in blood pressure and plasma ANG II with administration of ANG II or ACE inhibitor. Infusion of
intravenous ANG II by osmotic minipumps increased plasma ANG II concentrations in a dose-related manner (Table 1). Mean arterial blood pressure increased in rats with ANG II infusion as well (Table 2) but was more variable with the highest dose of ANG II. ACE inhibition with quinapril was followed by a slight decrease in plasma ANG II concentrations (Table 1). Quinapril treatment decreased blood pressure by an average of 12 mmHg (Table 2). Effects of ANG II on circulating and tissue renin. ANG II infusion resulted in a significant reduction of renal renin mRNA level (Fig. 1). Kidney renin concentration
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RECIPROCAL
Table 1. Plasma concentrations
FEEDBACK
E865
REGULATION
and kidney renin content ANG
II Infusion,
ng - kg-l . min-l
ACE Inhibition
Sham 100
300
1,000
Control
Quinapril
Plasma ANG II, fmol/ml 62&13 101~10 126&17* 168+12$ 66k14 49t8 Plasma angiotensinogen concentration, pmol/ml 1,218+173 1,773+119* 1,558+145 2,068+348* 1,231+110 835+90§ PRC, pmol ANG I h-l. ml-l 7.6&l 3.5+0.6-f 2.9+0.4$ 2.6kO.31 6.8& 1 14.Ok2.59 KRC, pmol ANG I h-l. mg protein-l 1,171+312 705t233 523t108* 347+467 1,108&123 1,039&181 Values are means t SE. In ANG II infusion studies, n = 8 rats/group; in quinapril study, n = 8 quinapril-treated rats; and n = 7 rats in controls. Plasma concentrations of ANG II, angiotensinogen, and renin (PRC) as well as kidney renin content (KRC) in animals with ANG II infusion or angiotensin-converting enzyme (ACE) inhibition for 3 days and respective controls. * P < 0.05, t P < 0.01, and $ P < 0.005 by analysis of variance (ANOVA) as compared with sham. $ P c 0.05 by Student’s unpaired t test as compared with sham. l
l
Table 2. Mean arterial blood pressure, sodium excretion, and creatinine clearance in animals with ANG II infusion or ACE inhibition ANG
II Infusion,
ng - kg-l - min-l
ACE Inhibition
Sham 100
300
1,000
Control
Quinapril
Mean arterial blood pressure, mmHg 10624.1 119t1.2” 134t9.6* 125t11.3 lllt3.6 99k4.25 Urinary Na+ excretion, meq/day 1.99t0.13 1.64&0.19* 1.46t0.20 1.22&0.31* 1.93t0.17 2.03t0.21 Creatinine clearance, ml min-l kg-l 6.37kO.41 5.31&0.54* 4.39kO.437 3.02+0.43$ 5.85kO.22 5.8t0.2 1 Values are means t SE. In ANG II infusion studies, n = 8 rats/group; in quinapril study, n = 8 quinapril-treated rats; and n = 7 rats in controls. Mean arterial blood pressure, sodium excretion, and creatinine clearance in animals with ANG II infusion or ACE inhibition for 3 days and respective controls. * P < 0.05, t P c 0.01, and $ P < 0.005 by ANOVA as compared with sham. § P < 0.05 by Student’s unpaired t test as compared with sham. l
l
increased plasma renin concentration twofold (Table 1). Kidney renin concentrations showed no difference between rats treated with quinapril for 3 days and control (Table 1). Effects of ANG II on angiotensinogen. The effects of ANG II on renal and hepatic angiotensinogen mRNA levels are shown in Fig. 2. ANG II infusion increased angiotensinogen mRNA levels significantly in both tissues, whereas the slight reduction of plasma ANG II concentrations by quinapril was not associated with significant changes in tissue angiotensinogen mRNA levels. Overall, renal angiotensinogen mRNA levels correlated significantly with plasma ANG II concentrations (r = 0.585;P < 0.005),providing further evidence for a positive feedback regulation of ANG II on its precursor (Fig. SHAM ANG II CONTROL WIN ANG II ANG II 100 300 1000 3). We also observed an increase in plasma angiotensinogen concentrations in response to ANG II infusion (Table ?cts of ANG II and angiotensin-converti] ng enzyme (ACE) inhibition on renal renin in RNA levels as calculated from slope of 1). There was no significant correlation between blood densitometric analysis of slot-blot hybridization. Infusion of ANG II for pressure and angiotensinogen mRNA levels in either kid3 days (100, 300, and 1,000 ngekg-l l rein-l, respectively) decreased ney or liver [r = 0.038, P = not significant (NS), and r = renal renin mRNA levels in an apparent dose-dependent manner. Thus 0.033, P = NS], suggesting that the changes in mRNA renal renin mRNA levels decreased to 60, 44 (P < 0.05), and 29% (P < 0.05) of control, respectively, with each dose of ANG II. On the other levels after ANG II infusion were not mediated by alterhand, ACE inhibition with quinapril (Quin; 10 mg. kg-l *day-l) for 3 ations in blood pressure. days increased renal renin mRNA levels (134% of control). *P < 0.05 by Renal functional effects of ANG II. Sodium excretion analysis of variance (ANOVA); t P < 0.005 by Student’s t test, as and creatinine clearance studied on the 3rd day of the compared with sham or control. In ANG II infusion studies, n = 8 rats/group; in quinapril study, n = 8 quinapril-treated rats/group; and IZ experiment decreased significantly and dose dependently = 7 rats/group in control study. in animals receiving ANG II infusion (Table 2). Interestingly, there was a significant inverse correlation between and plasma renin concentration decreased in parallel (Ta- renal angiotensinogen mRNA levels and Na+ excretion (r ble 1). There was a significant inverse relationship be- = 0.488, P = O.Ol),as well as creatinine clearance (r = tween plasma ANG II concentrations and plasma renin 0.663, P = 0.0006). On the other hand, the correlation concentration (r = 0.46;P = 0.005), thereby documenting between plasma ANG II concentrations and 24-h sodium a negative feedback effect of ANG II on renin synthesis excretion (r = 0.106, P = NS) or creatinine clearance (r = and release. Quinapril, on the other hand, stimulated re- 0.412, P = 0.05) displayed only a weak relationship. nal renin mRNA level to 134% of control (Fig. 1) and Quinapril treatment for 3 days had no significant effect +
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