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Abstract
Most of the biological effects of the renin-angiotensin system are mediated by the binding of angiotensin II (Ang II) to the type 1 Ang II (AT1) receptor, the predominant receptor subtype present after fetal life. To study tissue-specific regulation of the expression of the AT1 receptor in the rat, we altered activity of the renin-angiotensin system by feeding rats a low (0.07% NaCl), normal (0.3% NaCl), or high (7.5% NaCl) salt chow for 14 days; infusing Ang II (200 ng/kg per minute IP) or vehicle for 7 days; and administering an angiotensin-converting enzyme inhibitor (captopril, 100 mg/dL in the drinking water) or vehicle for 7 days. Renin, angiotensinogen, and total AT1 receptor mRNA levels were measured by slot-blot hybridization with cRNA probes, and AT1 receptor subtypes (A and B) were measured by reverse transcription–polymerase chain reaction in the presence of a cRNA internal standard. Plasma renin concentration and renal renin, renal and hepatic angiotensinogen, and hepatic AT1 receptor mRNA levels were all inversely related to salt intake; in contrast, renal AT1 receptor mRNA levels were significantly lower in rats fed low salt, a difference that was exclusively due to a decrease in the AT1A subtype. This difference did not appear to be mediated by a change in the circulating levels of Ang II, because Ang II infusion reduced plasma renin concentration and renal renin mRNA with no effect on either angiotensinogen or AT1 receptor mRNA levels in kidney or liver; renal Ang II receptor density (determined by in situ autoradiography) decreased, presumably via a posttranscriptional mechanism. Similarly, inhibition of Ang II generation with captopril increased plasma renin concentration and renal renin mRNA levels without altering renal or hepatic angiotensinogen mRNA or renal AT1 receptor mRNA levels. Thus, AT1 receptor gene expression is regulated in a tissue-specific manner that is distinct from other components of systemic and local renin-angiotensin systems and that appears to be mediated by a mechanism other than through changes in the circulating levels of Ang II.
Elegant experiments carried out over more than half a century have resulted in a detailed understanding of the biochemistry and physiology of the renin-angiotensin system. When viewed as an endocrine paradigm, successive cleavage of angiotensinogen by renin and ACE results in the generation of Ang II in the peripheral circulation. Ang II induces aldosterone secretion in adrenal glomerulosa, vasoconstriction in vascular smooth muscle, and salt reabsorption in the proximal tubule of the kidney, thereby contributing to blood pressure regulation and fluid and electrolyte homeostasis.1 The discovery that many of the genes encoding the components of the renin-angiotensin system are expressed in diverse tissues of the body, including the kidney,2 blood vessels,3 lung,4 heart,5 and brain,6 has led to the hypothesis that Ang II, in addition to its effects in the systemic circulation, may also be generated locally and control organ-specific functions through autocrine or paracrine mechanisms.7
The effects of Ang II are initiated by binding to high-affinity receptors located on the plasma membrane. Ang II receptors have been distinguished pharmacologically into two major types (AT1 and AT2) on the basis of different affinities for the benzylimidazole derivatives losartan and PD 123177.8 9 Losartan inhibits binding to the AT1 receptor, which is the predominant type found in kidney,10 adrenal cortex,8 11 vascular smooth muscle,11 liver,12 and intestine,13 and PD 123177 inhibits binding to the AT2 receptor, present primarily in adrenal medulla,8 uterus,11 ovary,14 brain,15 and the developing fetus.16 In the heart, AT1 and AT2 receptors are present in roughly equivalent amounts.17 cDNAs for both the AT1 and AT2 receptors have been cloned from tissue libraries obtained from several mammalian species, including humans.4 18 19 20 21 Both receptors are members of the seven-transmembrane domain G protein–coupled receptor superfamily but differ substantially, particularly with respect to signal transduction mechanisms.4 20 Recently, two AT1 receptor isoforms, designated AT1A and AT1B, have been further distinguished in rat and mouse tissues.22 23 These receptor isoforms exhibit no difference in binding to Ang II analogues, have a high degree of nucleotide sequence homology (91%) within the coding region, and have a lower sequence homology (58% and 62%, respectively) within the 5′- and 3′-untranslated regions. Differences between the tissue distribution and regulation24 25 26 27 of these AT1 receptor isoforms, as well as the presence of two additional sites for protein kinase C phosphorylation in the AT1B receptor molecule, suggest that they may mediate different physiological functions.
Our purpose in the present study was to investigate tissue-specific regulation of AT1 receptor mRNA levels in rats studied under experimental conditions known to alter the activity of the renin-angiotensin system. In addition, we designed the studies to determine whether any differences in AT1 mRNA levels observed could be accounted for by differences in the circulating levels of Ang II.
Methods
Animals
All experiments were performed with male Sprague-Dawley rats (Bantin Kingman, Fremont, Calif) weighing 300 to 350 g.
Experiment 1
Rats were pair-fed low (0.07% NaCl, Purina Mills, n=6), normal (0.3% NaCl, n=6), or high (7.5% NaCl, n=6) salt diets for 14 days. During this period, rats were housed in metabolic cages in climate-controlled conditions with a 12-hour light/dark cycle and provided with tap water ad libitum. Body weight, water consumption, urine volume, urinary sodium excretion, and blood pressure (Pulse Amplifier, ITTC Life Sciences) were measured. At the end of the experiment, rats were killed by decapitation, and the liver and kidneys were removed, snap-frozen in liquid nitrogen, and stored at −80°C for total RNA isolation. Trunk blood was collected in EDTA for measurement of plasma renin concentration.
Experiment 2
Rats were anesthetized with sodium pentobarbital (50 mg/kg IP, Anthony Products Co), and an osmotic minipump (model 2001, Alza Corp) was implanted intraperitoneally. [Ile5]Ang II (Peninsula Laboratories) was dissolved in saline containing 48 mg/mL bovine serum albumin (Sigma Chemical Co) and was infused at a rate of 200 ng/kg per minute for 7 days, a rate that has been reported to increase the sensitivity of Ang II receptors in the rat adrenal without altering systemic blood pressure.28 Control rats were infused with saline and bovine serum albumin. Rats were killed by decapitation, and the tissues were handled as described above. In addition to RNA isolation, Ang II receptor binding was assayed in kidney tissue obtained from these rats.10 Plasma renin concentration was measured on trunk blood obtained at the time of death.
Experiment 3
Rats received either captopril (Squibb & Sons) dissolved in the drinking water at a concentration of 100 mg/dL or vehicle. After 7 days, rats were decapitated, and the liver and kidneys were handled as described above.
RNA Isolation
Total cellular RNA was isolated from frozen tissue by a modification of the guanidine thiocyanate method of Chirgwin et al29 as described previously.30 The resultant RNA pellet was dissolved in sterile water and measured by UV absorbance at 260/280 nm. RNA integrity was verified by agarose gel electrophoresis.
Probe Synthesis
Rat angiotensinogen and renin antisense 32P-cRNA probes and a 32P-radiolabeled AT1 cDNA probe were synthesized as described previously.5 30 31 The AT1 probe (a gift of Kenneth Bernstein, Emory University, Atlanta, Ga)19 was synthesized by nick translation (N. 5500, Amersham) with the HindIII–Not I cDNA fragment of Cal8b. A 32P-labeled oligonucleotide complementary to bases 4011-4036 of human 28S rRNA was prepared as described previously.5 30
mRNA Analysis
Angiotensinogen, renin, and angiotensin receptor mRNAs were measured by slot-blot hybridization as described previously.5 30 Autoradiographs were obtained by exposure to Cronex x-ray film (DuPont) with intensifying screens at −80°C for 4 to 8 days and analyzed by scanning densitometry (model GS670 Imaging Densitometer, Bio-Rad). Duplicate blots were prepared and hybridized with the 28S rRNA probe for detection of RNA degradation and loading artifacts. 28S rRNA levels were comparable in all the rat groups in all three experiments.
Analysis of AT1A and AT1B mRNAs by RT-PCR
Renal AT1A and AT1B receptor mRNA levels were measured by quantitative RT-PCR as described previously.27 Oligonucleotide primers were designed corresponding to homologous coding regions of the rat AT1A and AT1B receptor genes (positions 739-719 for the reverse primer and positions 295-314 for the forward primer). A synthetic cRNA, harboring a 63-bp deletion removing a unique EcoRI site encoding the rat AT1A receptor cDNA, was used in both the RT and subsequent PCR as an internal standard. First-strand sscDNA synthesis was performed in a 20-μL reaction mixture containing 500 ng of total kidney RNA and 8×105 molecules of cRNA standard or 120 ng of total liver RNA and 4×105 molecules of cRNA standard, reverse primer at a final concentration of 0.4 μmol/L, 1× First Strand Synthesis Buffer (GIBCO-BRL), 2.5 mmol/L dNTP, 10 mmol/L dithiothreitol, 40 U RNAsin (Promega), and 200 U Moloney murine leukemia virus reverse transcriptase (GIBCO-BRL). After incubation for 90 minutes at 37°C, the reaction was terminated by further incubation for 10 minutes at 72°C. PCR was then performed in a 50-μL reaction mixture containing 5 μL of the RT mixture, 1× PCR buffer (Perkin-Elmer), forward and reverse primers at a final concentration of 80 nmol/L each, 1.75 mmol/L MgCl2, 1.25 mmol/L dNTPs, 0.148 mBq (4 μL) [1′,2′,5-3H]dCTP (Amersham), and 2.5 U Taq DNA polymerase (Perkin-Elmer). The reaction mixture was overlaid with mineral oil before thermal cycling. After an initial denaturation step of 5 minutes at 94°C, thermal cycling was performed for 29 cycles (94°C for 1 minute, 55°C for 1 minute, 72°C for 1.5 minutes) followed by one cycle in which the 72°C incubation was extended to 10 minutes. Thermal cycling was performed with a Thermal Cycler (model 4800, Perkin-Elmer).
After PCR amplification, 20 μL of each PCR reaction was restricted with EcoRI for 90 minutes at 37°C to distinguish the AT1A and AT1B cDNAs. Upon restriction, the AT1A cDNA yields two cDNAs of 269 and 175 bp, whereas the AT1B and AT1 internal standard cDNAs, which lack EcoRI sites, remain as single cDNA species of 444 and 384 bp, respectively.
Quantitative Analysis of PCR Products
The EcoRI PCR products were analyzed visually by electrophoresis of the restriction products through a 1.5% agarose gel in the presence of ethidium bromide. The restricted PCR products were quantified by fractionation of the restricted PCR products through an 8% polyacrylamide gel, excision of the appropriate gel bands, solubilization of the excised bands by the addition of 0.05 mol/L periodic acid, and incubation at 50°C followed by liquid scintillation counting. Results are presented as the ratio of the total counts per minute for the two AT1A EcoRI fragments and the counts per minute for the internal standard. The results for the single AT1B amplification product are presented similarly. We excluded the presence of potential genomic DNA contamination of the total RNA samples by performing PCR reactions using total RNA samples without prior RT and subsequent analysis by agarose gel electrophoresis in the presence of ethidium bromide.
In Situ Ang II Receptor Assay
The density of renal Ang II receptors was assessed by an in situ autoradiographic binding assay with 125I-labeled [Sar1,Ile8]Ang II (DuPont) as radioligand, as described previously.10 Nonspecific binding was determined by competition with 10 μmol/L unlabeled [Sar1]Ang II (Peninsula Laboratories). Saturation studies were performed in which adjacent sections were incubated with increasing concentrations of radioligand (from 10 to 1500 pmol/L), and the counts on the slide were determined in a gamma counter. Kd and Bmax values were calculated by Scatchard analysis with the ligand program.32
Plasma Renin Concentration
Plasma renin concentration was measured as the rate of Ang I generation (nanograms per milliliter per hour) in plasma incubated at pH 6.5 for 2 hours in the presence of excess rat angiotensinogen with the use of the method of Menard and Catt.33
Statistical Analysis
Data are presented as mean±SE. Comparisons between groups were done by one-way ANOVA or Student's t test for unpaired data with Bonferroni adjustment when appropriate (StatView, BrainPower). Differences were considered to be statistically significant at a value of P<.05.
Results
Experiment 1
After 2 weeks of the experimental diets, urinary sodium excretion reflected the differences in dietary NaCl intake. Systolic pressure did not differ significantly among the groups (Table 1⇓). Plasma renin concentration was significantly and inversely related with dietary salt intake, as were the levels of renal renin, renal and hepatic angiotensinogen, and hepatic AT1 receptor mRNAs (Table 1⇓). In contrast, renal AT1 mRNA levels increased with greater dietary salt content (Table 1⇓). The relationship between renal AT1 mRNA and dietary salt appeared to be exclusively due to a decrease in the AT1A receptor subtype, as shown by RT-PCR (Fig 1⇓).
Effect of variation of dietary sodium intake on renal AT1A and AT1B mRNA levels. AT1 receptor mRNA levels were determined by RT-PCR with an internal cRNA standard. Results are presented as the ratio of total counts per minute for the two AT1A EcoRI fragments and counts per minute for the internal standard. Results for the single AT1B amplification product are presented similarly. LS indicates low salt diet; HS, high salt diet; n=6 for both groups.
Effects of Dietary Salt on Urinary Sodium Excretion; Blood Pressure; Plasma Renin Concentration; and Renin, Angiotensinogen, and AT1 Receptor mRNA Levels in Normal Rats
Experiment 2
Plasma renin concentration and renal renin mRNA levels were significantly lower in the rats infused with Ang II than in those infused with vehicle (Table 2⇓). In contrast, renal and hepatic angiotensinogen and hepatic AT1 mRNA levels were not significantly different between groups (Table 2⇓). Renal AT1 mRNA levels were comparable, whereas the Bmax of renal Ang II receptors was significantly less in the rats infused with Ang II (Table 2⇓, Fig 2⇓). Kd values were comparable in rats chronically infused with Ang II or saline (Table 2⇓, Fig 2⇓).
Scatchard plots of Ang II receptor binding obtained from saturation studies performed on kidney sections of rats infused with Ang II (200 ng/kg per minute) or saline; n=5 for each group. Plots were linear, indicating the presence of a single class of receptors. Bmax was significantly lower (P<.05) in rats infused with Ang II. No significant difference was observed in Kd.
Effects of Ang II Infusion on Plasma Renin Concentration, Renin, Angiotensinogen, and AT1 mRNA Levels and Ang II Binding in Normal Rat Kidney
Experiment 3
At the end of the experimental period, captopril-treated rats had comparable rates of urinary sodium excretion (control, 1.6±0.3 mmol/d; captopril, 1.5±0.3). Captopril treatment resulted in a fourfold to fivefold increase in renin mRNA levels and no change in either renal or hepatic angiotensinogen mRNA levels (Table 3⇓). AT1 receptor Bmax, Kd, and mRNA levels were comparable in captopril-treated and control rats (Table 3⇓).
Effects of Captopril Treatment on Renin, Angiotensinogen, and AT1 mRNA Levels in Normal Rats
Discussion
The biochemistry and physiology of the proteins and peptides that comprise the renin-angiotensin system have been the subject of extensive investigation for decades; however, knowledge of the structure, function, and regulation of the receptors that mediate the biological effects of Ang II at the tissue level is relatively recent and has culminated with the expression cloning of the AT1 and AT2 receptors.4 18 19 20 21 Inactivation (ie, knockout) of the AT1 receptor gene in mice using the technique of homologous recombination resulted in a significant reduction in blood pressure, providing evidence for a tonic effect of the receptor in circulatory homeostasis.34 In contrast to the increasing body of knowledge concerning the AT1 receptor, the precise function of the AT2 receptor remains uncertain. The demonstration of AT2 receptors in great abundance in mesenchymal tissues of the developing fetus16 35 has suggested a role in development.
AT1 receptor expression is relatively minor during prenatal life but increases rapidly and substantially after birth.35 36 37 38 By day 10 of postnatal life, the AT1 receptor predominates and, with the exception of adrenal medulla, brain, and reproductive tissues, is virtually the only subtype present thereafter.16 38 In the adult animal, the AT1A receptor is the exclusive subtype in liver and the predominant subtype in kidney,10 27 39 indicating that this receptor subtype is involved in the majority of the hepatic and renal effects of Ang II. These changes in receptor density, as demonstrated by in situ autoradiography, occur in association with parallel changes in tissue mRNA levels, suggesting that the quantity of receptors is regulated at the level of gene expression.35 36 38
Tissue levels of AT1 receptor mRNA have been demonstrated to be altered during fetal development,35 36 37 38 cardiac hypertrophy,40 and renovascular hypertension27 ; after bilateral nephrectomy24 and dietary protein restriction41 ; and in response to administration of Ang II,42 losartan,26 cyclosporine,43 and steroids.44 To study tissue-specific regulation of AT1 receptor expression under more physiological conditions, we fed rats diets of differing salt content, an experimental maneuver that has been demonstrated to affect both circulating and tissue levels of several components of the renin-angiotensin system. Our results indicate that dietary sodium intake modulates AT1 mRNA levels in a tissue-specific manner (Table 1⇑). During dietary sodium restriction, AT1 mRNA levels decreased in the kidney while increasing in the liver in parallel with a concomitant increase in hepatic and renal angiotensinogen and renal renin mRNA levels. The decrease in renal AT1 mRNA levels was exclusively due to a decrease in the AT1A subtype because renal AT1B mRNA levels were unchanged (Fig 1⇑), indicating that the AT1 receptor subtypes are differentially regulated.
Differential regulation of AT1 receptor subtypes has been demonstrated also in some recent studies.23 24 25 26 27 44 45 46 Sandberg et al45 found that AT1A receptor mRNA is decreased and AT1B receptor mRNA is increased in the brain of rats fed a low salt diet. In adrenal glands, sodium depletion increased the mRNA levels of both AT1A and AT1B receptors,27 whereas in the heart, a low sodium diet increased AT1A gene expression.44 In contrast to our present findings, Kitami et al26 saw no differences in hepatic or renal AT1A or AT1B mRNA levels in salt-depleted rats (low sodium diet for 1 week plus furosemide for 4 days) compared with rats ingesting normal lab chow. In another study, Du et al,46 using Northern blotting and in situ hybridization, found increased AT1A and decreased AT1B mRNA levels in the kidney of rats fed a low salt diet (0.02% NaCl for 2 weeks) compared with controls. In that study, rats fed a low sodium diet had lower average plasma renin activity than controls. Although tissue differences in the regulation of AT1 receptor subtypes may exist, it is likely that at least some of the discrepancies between these studies are the result of differences in the weight of the animals (400 to 450 g in Kitami et al and 150 to 200 g in Du et al); in the degree of sodium depletion due, in turn, to differences in the duration and magnitude of dietary interventions; and in the methodology used for mRNA measurement. Finally, we would like to point out that in our study, inclusion of a high salt group permitted identification in both kidney and liver of a dose-dependent relationship between dietary salt intake and AT1 receptor gene expression, which was not found in the other two studies.
Since dietary sodium intake modulates plasma Ang II levels and Ang II in turn modulates Ang II receptor density, we performed additional experiments designed to chronically increase or decrease the plasma concentration of Ang II. Neither Ang II infusion nor administration of ACE inhibitors altered AT1 mRNA levels (Table⇑s 2 and 3), suggesting that circulating levels of Ang II do not directly affect AT1 receptor gene expression and that a different mechanism must account for the effects of dietary salt. Our results obtained during a 1-week infusion of Ang II are consistent with the results of previous studies in which AT1 mRNA levels were unaffected in kidney, aorta, and brain of rats infused with Ang II for up to 14 days42 and in hearts of rats with unilateral renal artery clipping.44 The results reported herein, in which administration of an ACE inhibitor failed to affect renal AT1 mRNA levels, complement the findings during long-term Ang II infusion.
In contrast to the absence of an effect on AT1 mRNA levels, however, Ang II binding in the kidney was decreased in rats chronically infused with Ang II (Fig 2⇑). This observation is in agreement with findings of previous studies47 and indicates that exogenous Ang II decreases renal Ang II receptor density through a posttranscriptional mechanism. Increased receptor internalization, sequestration, and degradation after Ang II binding is a likely explanation.48
The results of the present study confirm those of previous reports of the effect of manipulation of dietary salt intake, treatment with ACE inhibitors, and infusion of Ang II on the regulation of renin gene expression. In these previous studies, sodium depletion; high protein feeding; renal ischemia; unilateral ureteral obstruction; and administration of β-adrenergic receptor agonists, ACE inhibitors, and furosemide have been shown to increase renal renin mRNA levels, whereas sodium loading, Ang II infusion, and deoxycorticosterone and testosterone administration have an inhibitory effect.49
Like renin, angiotensinogen mRNA levels increase during development, after bilateral nephrectomy, and during dietary salt restriction.50 In the isolated perfused liver, Ang II infusion increases the rate of angiotensinogen formation,51 whereas in the present study, long-term infusion of Ang II did not affect angiotensinogen mRNA levels in either liver or kidney. The different durations of Ang II infusion, in addition to the differences in the experimental model, might explain these discrepancies. Also, different levels of plasma Ang II, which was not measured in these studies, might have been attained in the experiments. On the other hand, the absence of an effect of long-term changes in circulating levels of Ang II on angiotensinogen gene expression was further evidenced by the unaltered levels of angiotensinogen mRNA levels during administration of an ACE inhibitor.
In summary, this study shows that AT1 mRNA is regulated in a tissue-specific manner that is directionally distinct from other components of local renin-angiotensin systems. Dietary salt intake modulates the levels of AT1 mRNA through changes in AT1A receptor subtype that are independent of the circulating levels of Ang II. Ang II infusion and ACE inhibition do not affect AT1 receptor mRNA levels. Renin gene expression is increased by a low salt diet and administration of an ACE inhibitor, whereas it is decreased by long-term Ang II infusion. Angiotensinogen gene expression is increased in both kidney and liver by a low salt diet, whereas it is not affected by captopril treatment or Ang II infusion. Taken together, these results indicate that regulation of the activity of the renin-angiotensin system occurs at many levels, ranging from synthesis of the component proteins to expression of the receptors in target tissues, thereby providing a wide array of potential mechanisms for modulation of its physiological effects.
Selected Abbreviations and Acronyms
| ACE | = | angiotensin-converting enzyme |
| Ang I, II | = | angiotensin I, II |
| AT1, AT2 | = | type 1, type 2 angiotensin |
| PCR | = | polymerase chain reaction |
| RT | = | reverse transcription |
Acknowledgments
This research was supported by National Heart, Lung, and Blood Institute grant HL-11046. L.A. Sechi was the recipient of awards from the Italian Society of Hypertension, the International Juvenile Diabetes Foundation, and the Ferrero Foundation. G. Giacchetti was the recipient of an award from the Italian Society of Hypertension. J.-P. Valentin was a recipient of fellowship awards from ICI-Pharma and the American Heart Association, California Affiliate.
Footnotes
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Reprint requests to Leonardo A. Sechi, MD, Hypertension Unit, Department of Internal Medicine, University of Udine, School of Medicine, Ospedale Civile, Padiglione Medicine, 33100 Udine, Italy.
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Part of this work was presented at the 13th International Congress of Nephrology, Madrid, Spain, July 2-6, 1995, and has been published in abstract form (p 99).
- Received February 8, 1996.
- Revision received March 11, 1996.
- Accepted April 18, 1996.
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