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(Circulation Research. 1997;81:857-864.)
© 1997 American Heart Association, Inc.

Articles

Role of Tissue Renin in the Regulation of Aldosterone Biosynthesis in the Adrenal Cortex of Nephrectomized Rats

Massimo Volpe, Bruna Gigante, Iolanda Enea, Antonio Porcellini, Rosaria Russo, Min Ae Lee, Paola Magri, Gerolama Condorelli, Carmine Savoia, Klaus Lindpaintner, , Speranza Rubattu

From the Istituto di Medicina Interna e Cardiologia (M.V., B.G., S.R., R.R., P.M., C.S.) and Patologia Cellulare e Molecolare (A.P., G.C.), Universita' di Napoli (Italy) "Federico II"; the Istituto Neurologico Mediterraneo "Neuromed" (M.V., I.E., S.R.), Pozzilli (IS), Italy; the Cardiovascular Division (K.L., M.A.L.), Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston Mass); and the Dipartimento di Medicina Sperimentale e Patologia (M.V.), Universita' di Roma (Italy) "La Sapienza."

Correspondence to Massimo Volpe, MD, 1a Clinica Medica, Via S. Pansini 5, 80131 Naples, Italy. E-mail volpema{at}cds.unina.it

	Abstract

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Abstract The aim of the study was to investigate whether the adrenal renin-angiotensin system plays an independent role in the regulation of mineralocorticoid biosynthesis in the adrenal gland and to explore the mechanisms of this action. Twelve-week-old male Sprague-Dawley rats were studied: 22 rats were maintained on a regular diet; 27 and 22 rats received a low salt diet with and without treatment, respectively, with the angiotensin II (Ang II) AT₁-subtype receptor antagonist losartan (10 mg/kg per day). A fraction of each group of rats underwent bilateral nephrectomy (n=12, 15, and 10, respectively) and was killed 48 hours later. In an additional group of 24 (12 intact and 12 nephrectomized) rats, the effects of the Ang II AT₂-subtype receptor antagonist PD123319 were investigated. In intact rats, plasma renin activity (PRA) and adrenal renin activity and expression were progressively raised by salt restriction and losartan, whereas aldosterone synthase mRNA and plasma aldosterone (PA) levels were increased by salt restriction and reduced by losartan. Forty-eight hours after nephrectomy, PRA fell to undetectable levels; in contrast, adrenal renin expression, assessed by semiquantitative reverse-transcriptase polymerase chain reaction (using GAPDH as a standard for gene expression), showed an 18-fold increase and was further increased after salt restriction and losartan (all P<.05). Also, adrenal renin activity was raised after nephrectomy and further increased after salt restriction (P<.05) and losartan. Cytochrome P450 aldosterone synthase expression in the adrenal cortex was stimulated by nephrectomy alone and by nephrectomy combined with low salt intake (P<.05), with consequent increases in PA concentrations. In losartan-treated salt-restricted nephrectomized rats, cytochrome P450 aldosterone synthase expression (P<.05 versus nephrectomy alone and nephrectomy plus salt restriction) and PA concentrations were diminished (P<.05) in spite of the observed increases of adrenal renin expression. The AT₂-receptor antagonism did not significantly affect PRA, adrenal renin, and aldosterone biosynthesis and production in either intact or nephrectomized salt-restricted rats. These results demonstrate that the adrenal renin-angiotensin system plays an independent role in the regulation of mineralocorticoid biosynthesis in vivo. This action is mediated primarily via the Ang II AT₁-subtype receptors.

Key Words: aldosterone • angiotensin receptor • hypertension • kidney • mineralocorticoid

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The existence and the functional relevance of a local renin-angiotensin system in the adrenal gland have been a topic of active investigation for >10 years. The presence of all the components of the renin-angiotensin system has been demonstrated in the adrenal tissue^{1 2 3 4 5 6 7} and has been shown to be localized in the zona glomerulosa cells.⁸ In addition, a number of studies have demonstrated that the adrenal renin-angiotensin system is modulated by factors such as salt balance,⁹ potassium intake,^{10 11 12} ACTH,^{11 12 13 14} Ang II,¹⁵ and nephrectomy.^{16 17}

The presence of a regulated renin-angiotensin system in the adrenals indicates the possibility that it may have a local function. Indirect evidence supporting a role of the adrenal renin-angiotensin system in the control of mineralocorticoid production and secretion is provided by earlier experimental findings¹⁰ showing a positive correlation between the stimulation of adrenal renin activity and aldosterone production in nephrectomized rats. In vitro studies¹⁸ have also supported the hypothesis that locally generated Ang II regulates ACTH and potassium-stimulated aldosterone secretion in rat and bovine zona glomerulosa cells. Finally, studies performed in our laboratory have recently shown that in the renin transgenic rat¹⁹ aldosterone biosynthesis and secretion are selectively regulated by exogenous mouse adrenal renin and that this modulation is mediated through Ang II AT₁-subtype receptors. Taken together, these studies suggest a regulatory function of adrenal renin in the control of aldosterone production. A direct demonstration of such an effect of the adrenal renin-angiotensin system in vivo, however, has hitherto not been available.

Thus, the aim of the present study was to investigate the role of the adrenal renin-angiotensin system in the regulation of mineralocorticoid biosynthesis in the rat. For this purpose, we studied the effects of Ang II antagonism on adrenal renin and cytochrome P450 aldosterone synthase expression in sodium-restricted Sprague-Dawley rats before and 48 hours after bilateral nephrectomy.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Experimental Design
The present study was performed according to the guidelines for animal care and treatment of the European Community and was approved by the local committee of our institution. Ninety-five male Sprague-Dawley rats (age, 12 weeks) weighing between 250 and 300 g were purchased (Morini, Polo D'Enza, Reggio Emilia, Italy). All rats were housed under controlled conditions of light, temperature, and humidity and were fed regular rat chow during 1 week of acclimatization. Subsequently, a first cohort of 71 animals undergoing the experiment with losartan was divided into six subgroups. The first three subgroups were as follows: 10 rats were used as a sham group (control) and were maintained on regular chow (NaCl, 0.5%; K⁺, 0.68%) (Laboratori Piccioni, Gessate); 12 rats were shifted to a salt-restricted regimen (SR group) (NaCl, 0.04%; K⁺, 0.68%) (Laboratori Piccioni); and 12 rats received the low salt diet along with the Ang II AT₁-subtype receptor antagonist losartan potassium (DuP753, 10 mg/kg per day) in the drinking water (SR+Los group). All dietary and treatment regimens were maintained for 1 week. In the remaining three subgroups of rats, bilateral nephrectomy was performed after the week of diet and treatment: 12 rats were nephrectomized while on regular rat chow (Nx group); 10 rats, during the low salt diet (Nx+SR group); and 15 rats, during the low salt diet plus losartan (Nx+SR+Los group) at the same dosage indicated above. The dosage of losartan used in the present study had been shown to provide a complete inhibition of AT₁ subtype–mediated effects of Ang II on blood pressure and aldosterone secretion in normotensive rats.²⁰ The drug was kindly provided by the DuPont Merck Pharmaceutical Co (Wilmington, Del). Dietary regimens and treatment with losartan were continued for 48 hours in the preceding three subgroups until death. A second cohort of 24 rats was included in the study to investigate the potential influence of the inhibition by PD123319 (PD subgroups) of the Ang II AT₂-subtype receptors, which account for 20% of Ang II receptors in the adrenal cortex.²¹ Twelve intact rats (control=4, SR=4, and SR+PD=4) and 12 nephrectomized rats (Nx=4, Nx+SR=4, and Nx+SR+PD=4), previously instrumented with a jugular polyethylene cannula and studied while they were conscious, received a constant infusion of the AT₂-subtype receptor antagonist PD123319 at a dosage of 50 µg/kg per minute. This dosage has been shown to provide a selective inhibition of the Ang II AT₂-subtype receptors.^{22 23} The drug was kindly provided by Parke-Davis (Ann Arbor, Mich).

Rats were kept for 24 hours in metabolic cages in order to estimate food and water consumption and to measure diuresis and natriuresis before decapitation. Serum and urinary electrolytes were measured by flame photometry. Bilateral nephrectomy was performed under a brief anesthesia of the animals induced by intramuscular injection of a mixture of ketamine (Ketalar, 160 mg/kg body wt) and xylazine (Rompun, 10 mg/kg body wt). Care was taken to preserve the adrenals upon removal of the kidneys. Rats from all experimental groups were killed by decapitation (without premedication) to obtain plasma and tissues. Systolic blood pressure was measured at least 12 hours before decapitation in the conscious restrained rats by tail-cuff sphygmomanometry (PE-300, Narco Biosystem Inc) and recorded on a multichannel polygraph (Universal Oscillograph, Harvard Instruments).

Hormonal Measurements
Blood Samples
Truncal blood for hormonal measurements was collected in prechilled EDTA Vacutainer tubes (Becton-Dickinson), and plasma was stored at -40°C until the time of the assay.

PRA was measured by enzymatic incubation of plasma at 37°C and pH 6.4. The Ang I generated during the incubation step was quantified by radioimmunoassay and expressed as nanograms Ang I per milliliter per hour. The radioimmunoassay sensitivity is 10 pg per tube. The intra-assay and interassay variabilities of this method were 3.7% and 7.6%, respectively. Aldosterone was measured by a direct radioimmunoassay kit (Diagnostic Product Corp). The sensitivity of this assay is 16 pg/mL. The intra-assay and interassay variabilities were 7.1% and 5.1%, respectively.

Tissues
From each animal, one adrenal was frozen and stored at -70°C for RNA extraction, whereas the contralateral adrenal was used for renin activity measurements performed in pool tissues derived from at least 3 animals each. For the latter purpose, adrenal capsules were homogenized as previously reported.²⁴ Thus, three pools were obtained from the 10 control and Nx+SR groups, four pools from the SR, SR+Los, and Nx groups (all n=12), and five pools for the Nx+SR+Los group (n=15). Briefly, the tissues were homogenized by the use of a polytron (Tekmar Co) with a 50 mmol/L Tris-HCl buffer (pH 8.0) containing 100 mmol/L NaCl and 3 mmol/L EDTA. During the homogenization, 6 mmol/L phenylmethylsulfonyl fluoride and 6 mmol/L diisopropyl fluorophosphate were added.

Active renin in the tissue homogenates was measured by incubating 100-µL samples with 50 mmol sodium maleate buffer (pH 6.4) and nephrectomized rat plasma as a source of substrate (final concentration, 1000 ng/mL). Samples were incubated for 0 and 1 hour. Results are expressed as nanograms Ang I per milligram protein per hour. Tissue protein content was determined by a commercially available kit (Bio-Rad).

Specificity of the renin assay in tissue homogenates was assessed by incubation for 1 hour at 37°C with a polyclonal antibody raised against human renin, which also recognizes both rat and mouse renins (50% precipitation at 1:100 dilution, almost 100% precipitation at 1:10 dilution), kindly provided by Dr J.E. Sealey (Cornell University Medical College, New York, NY).

RNA Preparation and Northern Blot Analysis
Adrenal capsules were immediately frozen in liquid nitrogen and kept at -70°C until RNA extraction. Total RNA was extracted by each individual adrenal capsule by the guanidinium thiocyanate–phenol–chloroform method.²⁵ The RNA was quantified by scanning spectrophotometry, and the Å₂₆₀/Å₂₈₀ ratio of all preparations included in the present experiments was >1.8. Thus, the following numbers of adrenal samples were obtained for each experimental group: control group, n=6; SR, n=8; SR+Los, n=4; Nx, n=5; Nx+SR, n=6; and Nx+SR+Los, n=9. In the PD123319 experiment, four capsules were included in each group.

For Northern blotting, total RNA (15 µg per lane) was electrophoresed on 1% agarose gel containing 2.2 mol/L formaldehyde and transferred to Hybond-N filters (Amersham North America).

Hybridizations for the two isoenzymes, cytochrome P450 aldosterone synthase and 11ß-hydroxylase cytochrome P450, were performed as previously described,²⁶ with the specific oligonucleotides corresponding to positions 857 to 891 of rat aldosterone synthase and to positions 863 to 882 of rat 11ß-hydroxylase. The two oligonucleotides were manufactured by GENSET and labeled with [-³²P]dATP using the polynucleotide kinase (New England Biolabs). Prehybridizations and hybridizations were carried out at 50°C in a buffer containing 5x SSC, 20 mmol/L NaH₂PO₄ (pH 7.3), 3.5% SDS, 10x Denhardt's solution, 10% dextran sulfate, and 0.2 mg/mL denatured salmon sperm DNA. To avoid cross hybridization between the two isoenzymes, an excess of the corresponding unlabeled oligonucleotide was added to each hybridization mixture. Washing was performed with 2x SSC and 1% SDS at room temperature, followed by 0.1x SSC and 0.1% SDS at 42°C.

Hybridization with the rat GAPDH cDNA was performed at 65°C in a solution containing 7% SDS and 0.5 mol/L Na₂HPO₄ (pH 7.3). The filter was then washed at room temperature in 1% SDS and 40 mmol/L Na₂HPO₄, followed by a stringent washing at 65°C with the same solution. GAPDH cDNA was labeled by random primers (Megaprime, Amersham) using [-³²P]dATP.

After each hybridization, filters were exposed for 24 to 48 hours to preflashed Kodak X-AR 5 film at -70°C using intensifying screens. The autoradiographic bands were analyzed by densitometric scanning and normalized for GAPDH levels. Data are expressed as mean±SEM of values obtained in three independent experiments.

cDNA Synthesis and rt-PCR
To avoid the contamination of RNA with genomic DNA, the samples were treated with RNase-free DNase for 15 minutes at 37°C, followed by an incubation at 94°C for 5 minutes to inactivate the enzyme. To verify the absence of genomic DNA contamination of RNA, an aliquot (100 ng) of each sample was subjected to PCR amplification without the reverse-transcriptase step.

Single-strand cDNA synthesis was performed on 1 µg of total RNA. The following samples were obtained for each group: control, n=6; SR, n=7; SR+Los, n=4; Nx, n=6; Nx+SR, n=6; and Nx+SR+Los, n=7. In the PD123319 experiment, four samples were included in each group. The reaction, performed three times for each sample, was carried out in 20 µL of reaction buffer (50 mmol/L Tris-HCl [pH 8.3], 75 mmol/L KCl, 15 mmol/L MgCl₂, 10 mmol/L dithiothreitol, and 500 µmol/L dNTPs) containing 20 pmol of random primers (pDN₆, Boehringer-Mannheim) and 200 U of reverse transcriptase (Superscript, GIBCO BRL). The reaction was stopped by adding 5 µL of 0.5 mol/L EDTA, and a final volume of 50 µL was achieved with sterile water.

cDNA (5 µL) covered with 20 µL of mineral oil was amplified in a 70-µL reaction buffer containing 10 mmol/L Tris-HCl (pH 8.3), 50 mmol/L KCl, 1.5 mmol/L MgCl₂, 200 µmol/L dNTPs, and 20 pmol of each oligonucleotide primer, and 1 U of TaqDNA polymerase (Boehringer-Mannheim) was added. The thermal profile used on a Perkin-Elmer/Cetus thermal cycler consisted of 5 minutes at 97°C followed by 29 cycles, the first five of which consisted of 1-minute denaturation at 94°C, 1-minute annealing at 60°C, and 1-minute extension at 72°C; the remaining 24 cycles consisted of 50 seconds at 94°C, 50 seconds at 55°C, and 45 seconds at 72°C. The final extension was carried out for 10 minutes.

In preliminary studies, we found that the amplification reaction reached a plateau phase after 22 cycles for GAPDH and 29 cycles for renin; therefore, the GAPDH primers were added, in the same reaction tube, after the first seven cycles. In these conditions, the reaction is linearly related to the initial cDNA concentration, and the renin/GAPDH ratio is not dependent on the amount of cDNA used for the coamplification reaction, as shown in Table 1. Semiquantitative rt-PCR was performed by coamplifying renin and GAPDH. All the rt-PCRs (three independent reactions for each sample) were coamplified in duplicate, at the same time in the same reaction, for renin and GAPDH.

View this table: [in this window] [in a new window]   Table 1. Representative Data Showing the Renin/GAPDH Ratio in Two Samples of Two Different Experimetal Groups (Control and Nephrectomy)

Aliquots (10 µL) of the rt-PCR products were taken up at 26 and 29 cycles and run on 1% agarose gel with TBE buffer. The samples were loaded twice on the same gel at a distance of 7 cm and blotted on N-Hybond filters (Amersham). After Southern blot analysis, the filter was cut in two parts, each containing all the rt-PCR products. The two filters were then hybridized: one with renin cDNA (kindly provided by Dr K.R. Lynch, University of Virginia Medical School, Charlottesville) and the other with GAPDH cDNA. Hybridizations were performed by following the same procedure described above for the GAPDH cDNA. The radioactive signals were detected and quantified by a PhosphorImager (ImageQuaNT, Molecular Dynamics). To test the linearity of the amplification reaction, the renin/GAPDH ratio was measured throughout the actual experiment. The samples in which the renin/GAPDH ratio was not constant between cycles 26 and 29 were not considered in the analysis. All the rt-PCR procedures were performed three times in duplicate, from three independent reverse-transcription reactions, as shown in Table 1. For each rt-PCR cycle, all the samples from the different experimental groups were processed at the same time. The samples in which the renin/GAPDH ratio was not comparable (ie, the variability among the three rt-PCR was >20%) were not included in the study.

The interassay variability was constantly <10%.

Choice of Primers
For the renin gene, two oligonucleotide primers were chosen on the cDNA sequence. The first is located at nucleotide 819 (5'-GATGGAGTCATCCCTGTCTTCG-3), and the second is located at nucleotide 1262 (5'-GTCATCGTTCCTGAAGGGATTC-3'), thus amplifying a cDNA fragment of 464 bp.

The GAPDH oligonucleotide primers are located at nucleotide 369 (5'-TTCACCACCACCATGGAGAAGGCT-3') and at nucleotide 715 (5'-ACAGCCTTGGCAGCACCAGT-3'), thus obtaining an amplification product of 346 bp of GAPDH cDNA.

Statistical Analysis
Data are expressed as mean±SEM. Multiple-comparison analysis was performed by two-way ANOVA factoring by group and treatment; a nonparametric post hoc test (Kruskal-Wallis) was used to detect significance among different experimental conditions.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Table 2 summarizes the behavior of blood pressure, serum, and urinary electrolytes in the losartan experiment. Systolic blood pressure was not significantly altered by salt restriction, but it was reduced significantly by treatment with losartan. Forty-eight hours after bilateral nephrectomy, blood pressure significantly fell, and no further changes were observed in the animals receiving the low salt diet. Treatment with losartan was also associated with a further reduction in blood pressure in nephrectomized rats.

View this table: [in this window] [in a new window]   Table 2. Effects of Salt Restriction and Losartan Treatment on Systolic Blood Pressure and Serum and Urinary Electrolytes in Intact and Nephrectomized Rats

Serum potassium levels did not change significantly in response to low salt diet but increased during the concomitant administration of losartan. As expected, serum potassium levels rose significantly in nephrectomized rats and did not further change in the nephrectomized rats receiving a low salt diet and losartan. Urinary sodium excretion rate decreased in salt-restricted rats and in losartan-treated salt-restricted rats. Finally, urinary potassium excretion rate did not change significantly in the salt restricted and losartan-treated rats.

Fig 1 shows the behavior of PRA and adrenal renin activity in the experimental groups. In intact animals, PRA increased during low salt diet (P<.05) and rose further after the administration of the Ang II AT₁-subtype receptor antagonist losartan (P<.05 versus control and salt-restricted rats) (Fig 1, top). In nephrectomized animals, PRA fell to almost undetectable levels and remained barely detectable in the nephrectomized rats fed with low salt diet (Nx+SR group) (both P<0.001 versus the corresponding values in intact rats). In contrast, in salt-restricted nephrectomized rats receiving losartan, PRA increased to 3.2±0.63 ng Ang I/mL per hour (P<0.05 versus nephrectomy and salt-restricted-nephrectomized rats and P<.001 versus the corresponding value in losartan-treated and salt-restricted intact rats) (Fig 1, top). Adrenal renin activity (Fig 1, bottom) and adrenal renin mRNA (Fig 2) showed parallel increases in response to low salt diet (P<.05) and during concomitant administration of losartan in the intact animals. After nephrectomy, adrenal renin activity (Fig 1, bottom) and mean adrenal renin mRNA (Fig 2) were markedly increased compared with control (both P<.05) and were further raised by low salt diet. In fact, renin mRNA showed progressive increases in nephrectomized rats, in salt-restricted nephrectomized rats (P<.05 versus Nx group), and in losartan-treated salt-restricted nephrectomized rats, respectively (P<.05 versus Nx and Nx+SR groups), whereas adrenal renin activity was increased by salt restriction (P<.05) and only tended to further increase after losartan treatment, although this latter change did not achieve a statistical significance.

View larger version (13K): [in this window] [in a new window]   Figure 1. Effects of salt restriction (SR) and salt restriction combined with losartan (SR+Los) on plasma (top) and adrenal (bottom) renin activity in intact rats (open bars) and in rats 48 hours after nephrectomy (solid bars). Bars represent mean±SEM for 10 rats (intact control group), 12 rats (intact SR, intact SR+Los, and nephrectomized control groups), 10 rats (nephrectomized SR group), and 15 rats (nephrectomized SR+Los group). *P<.05 vs intact control group; P<.05 vs intact SR group; P<.05 vs nephrectomized control group; P<.05 vs nephrectomized control and nephrectomized SR groups; ++P<.001 vs the corresponding intact group; and +P<.05 vs the corresponding intact group.

View larger version (30K): [in this window] [in a new window]   Figure 2. Representative Southern blot (top) of adrenal renin and GAPDH expression in salt-restricted (SR) and in losartan-treated salt-restricted (SR+Los) intact (open bars, bottom) and nephrectomized (Nx) (solid bars, bottom) rats. Each lane is identified by the corresponding bar of the densitometric scanning (bottom). Data are normalized by GAPDH levels and expressed as mean±SEM of values obtained in three independent experiments performed in duplicate from 6 rats (control [C], Nx, and Nx+SR groups), 7 rats (SR and Nx+SR+Los groups), and 4 rats (SR+Los group). *P<.05 vs intact C group; P<.05 vs intact SR group; P<.05 vs the corresponding intact group; P<.05 vs Nx group; and P<.05 vs Nx and Nx+SR groups.

Figs 3 and 4 show the effects of salt restriction and losartan on cytochrome P450 aldosterone synthase expression in the adrenal cortex and plasma aldosterone concentrations, respectively, in the intact and nephrectomized rats. When the intact rats were exposed to low salt diet, aldosterone synthase expression was enhanced (Fig 3) and plasma aldosterone levels rose from 252±35 to 914±98 pg/mL (P<0.001) (Fig 4). The concomitant administration of losartan was associated with a slight reduction of cytochrome P450 aldosterone synthase mRNA, whereas plasma aldosterone fell significantly to 642±96 (P<0.05 versus control and salt-restricted rats). After nephrectomy and nephrectomy combined with salt restriction, cytochrome P450 aldosterone synthase expression was markedly stimulated (both P<.05 versus the corresponding control conditions). Similarly, plasma aldosterone levels increased to 1272±524 pg/mL in the nephrectomized rats and to 2716±208 pg/mL in the salt-restricted nephrectomized rats (P<0.001 versus the corresponding control condition). Treatment with losartan resulted in reductions of cytochrome P450 aldosterone synthase mRNA (P<.05) (Fig 3) and of plasma aldosterone levels (P<.05), which were reduced to levels not significantly different from those measured in the untreated nephrectomized rats (Fig 4). No correlation was found between PRA and aldosterone synthase mRNA (r=.32, n=9, P=NS) or plasma aldosterone concentrations (r=.49, n=9, P=NS) in the losartan-treated salt-restricted nephrectomized rats. In the same animals, no correlation was found between PRA levels and adrenal renin mRNA (r=.19, n=7, P=NS).

View larger version (34K): [in this window] [in a new window]   Figure 3. Representative Northern blot (top) of cytochrome P450 aldosterone synthase and GAPDH mRNA in salt restricted (SR) and losartan-treated salt-restricted (SR+Los) intact (open bars, bottom) and nephrectomized (Nx) (solid bars, bottom) rats. Each lane is identified by the corresponding bar of the densitometric analysis. O.D. indicates optical density. Data are normalized by GAPDH levels and expressed as mean±SEM of values obtained in three independent experiments from 6 rats (control [C] and Nx+SR groups), 8 rats (SR group), 4 rats (SR+Los group), 5 rats (Nx group), and 9 rats (Nx+SR+Los group). *P<.05 vs intact C group; P<.05 vs the corresponding intact group; and P<.05 vs Nx and Nx+SR groups.

View larger version (12K): [in this window] [in a new window]   Figure 4. Effects of salt restriction (SR) and losartan treatment (Los) on plasma aldosterone concentrations in intact (open bars) and nephrectomized (Nx) (solid bars) rats. Each bar represents mean±SEM (number of rats as in Fig 1). **P<.001 vs intact control group; *P<.05 vs intact control group; P<.05 vs intact SR group; ++P<.001 vs the corresponding intact group; and P<.05 vs Nx+SR group.

Cytochrome P450 11ß-hydroxylase mRNA in the adrenal capsules was not modified by low salt intake or losartan administration in intact and nephrectomized rats.

In order to detect the potential influence of Ang II AT₂-subtype receptors in the regulation of mineralocorticoid biosynthesis by the adrenal renin-angiotensin system, we explored the effects of the AT₂-receptor antagonist PD123319 on aldosterone synthase expression and plasma aldosterone levels.

As shown in Fig 5, PD123319 treatment had no influence on aldosterone synthase expression in both intact and nephrectomized rats. Consistently, plasma aldosterone levels were not modified by the treatment with the AT₂-receptor antagonist (for intact rats: control, 160±40 pg/mL; SR, 635±122 pg/mL [P<.05]; and SR+PD, 588±137 pg/mL [P<.05 versus control, P=NS versus SR]; for nephrectomized rats: Nx, 2050±580 pg/mL; Nx+SR, 2060±299 pg/mL [P=NS versus Nx]; and Nx+SR+PD, 2263±169 pg/mL [P=NS versus Nx and Nx+SR]).

View larger version (32K): [in this window] [in a new window]   Figure 5. Representative Northern blot (top) of cytochrome P450 aldosterone synthase and GAPDH mRNA in salt-restricted (SR) and PD123319-treated salt-restricted (SR+PD) intact (open bars, bottom) and nephrectomized (Nx) (solid bars, bottom) rats. Each lane is identified by the corresponding bar of the densitometric analysis. O.D. indicates optical density. Data are normalized by GAPDH levels and expressed as mean±SEM of values obtained in three independent experiments. Four capsules derived from individual animals were used in each experimental group. *P<.05 vs intact control [C] group; P<.05 vs the corresponding intact group.

Finally, PD123319 had no significant influence on PRA in intact salt-restricted rats (from 6.7±1.0 to 6.6±1.0 ng Ang I/mL per hour after PD123319) and nephrectomized salt-restricted rats (from 0.18±0.01 to 0.18±0.01 ng Ang I/mL per hour after PD123319) and on adrenal renin mRNA.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The aim of the present study was to define whether the adrenal renin-angiotensin system directly regulates mineralocorticoid biosynthesis in the rat. To elucidate this action of the adrenal renin-angiotensin system, we studied the effects of salt restriction and angiotensin receptor antagonism in intact and nephrectomized rats. Consistent with previous findings after bilateral nephrectomy, we observed suppression of circulating renin and significant stimulation of locally generated renin in the adrenals. Increases in adrenal renin expression and activity were enhanced by salt restriction and were accompanied by progressive increases of cytochrome P450 aldosterone synthase expression in the adrenal capsules and of circulating aldosterone levels. Replication of these maneuvers during Ang II AT₁-subtype receptor blockade by losartan resulted in significant reductions of aldosterone synthase mRNA and plasma aldosterone levels in both intact and nephrectomized rats. In contrast, administration of the Ang II AT₂-subtype receptor PD123319 did not modify aldosterone synthase mRNA and plasma aldosterone concentrations and circulating and tissue renin.

These findings are the first demonstration that adrenal renin plays an independent regulatory role in the control of aldosterone biosynthesis and production in normal rats and that this action is primarily mediated through the Ang II AT₁-subtype receptors.

Previous studies have identified a regulated adrenal renin-angiotensin system in the rat. In particular, Brecher et al⁹ described a 2-fold increase of renin expression in the adrenals of nephrectomized rats during low salt diet, and Wang et al¹² showed that renin mRNA increases in zona glomerulosa cells after treatment with potassium and ACTH. In addition, Doi et al¹⁰ observed a marked increase in adrenal renin activity and aldosterone concentrations 20 hours after bilateral nephrectomy and described a significant correlation between adrenal renin and aldosterone in this model.

On the basis of these and other observations,^{13 16} it has been hypothesized that the local renin-angiotensin system may play a role in the regulation of aldosterone biosynthesis in the adrenals.^{1 2} However, so far, the correlative nature of these findings did not permit the conclusion that the adrenal renin-angiotensin system directly regulates mineralocorticoid biosynthesis.

More recently, we demonstrated that in a transgenic rat model, TGR(mREN2)27, which carries copies of the mouse renin gene,²⁷ low sodium intake is associated with a stimulation of renin and aldosterone synthase mRNA in the adrenals and with an increase of plasma aldosterone levels in the absence of significant changes of renin of renal origin.²⁸ In the same hypertensive animal model, we have shown that the exogenous renin expressed by the transgene controls aldosterone biosynthesis largely through the Ang II AT₁-subtype receptor.¹⁹ This latter experimental approach, however, did not provide information on the potential regulatory function of native adrenal renin in the control of adrenal aldosterone biosynthesis. In addition, even though in TGR(mREN2)27 rats renin is prominently expressed in extrarenal tissues, including the adrenals, the function of adrenal renin could not be completely dissected from that of renal renin, which is markedly suppressed but yet present in this model.

Tissue renin-angiotensin systems have represented an interesting and attractive field of research over the last few years. Although components of the system and its regulation have been shown in various tissues, including the adrenal, a clear identification and demonstration of the putative function in the different tissues has represented a difficult task. The experimental model used in our present experiment provides a selective approach to dissect the role of tissue renin-angiotensin system.

To define whether the adrenal renin-angiotensin system plays a direct and independent regulatory function in the control of mineralocorticoid biosynthesis, in fact, it is necessary to investigate the influence of the local system in the absence of the circulating renin-angiotensin system.

Bilateral nephrectomy, as used in our present study, permits a clear-cut dissection of the function of tissue renin from the influence of circulating renal system. Although this model has been criticized for its potential to introduce additional covariates such as uremia, the observation that the adrenal renin-angiotensin system in nephrectomized animals was still modulated by low salt intake and Ang II AT₁-subtype receptor antagonism demonstrates that the functional state of the system was sufficiently preserved under these experimental conditions. In fact, the stimulation of adrenal renin-angiotensin system observed 48 hours after nephrectomy, which is consistent with previous observations,^{10 16 17} was further enhanced by exposure to low salt intake and to losartan. These observations suggest that the adrenal renin-angiotensin system may be downregulated under the influence of the renal renin-angiotensin system, although we cannot exclude the possibility that the increases of ACTH and potassium serum levels associated with nephrectomy may have partially contributed to stimulate renin in the adrenals, as previously suggested.^{10 16}

The mechanisms leading to the stimulation of renin expression and activity in the adrenal cortex during salt restriction cannot be clarified by our present studies and may include several factors, such as activation of the sympathetic nervous system, which is known to accompany sodium restriction,²⁹ and changes in potassium levels and ACTH. These latter two factors do not seem to play a role in the response of adrenal renin to salt restriction observed in our experiments. In fact, potassium serum levels did not further increase in response to low salt intake in the intact and nephrectomized rats. On the other hand, the observation that 48 hours after nephrectomy cytochrome P450 aldosterone synthase increased and 11ß-hydroxylase remained unchanged does not support a significant intervention of ACTH. Such a conclusion is indirectly suggested by observations reported by Baba et al,¹⁶ who showed that ACTH infusion does not modify adrenal renin in intact rats, and by Sander et al,¹⁴ who described a reduction in aldosterone synthase expression in response to ACTH in intact rats.

The further stimulation of adrenal renin mRNA observed during losartan (and not during PD123319) administration indicates that Ang II exerts its negative feedback on adrenal renin synthesis through the AT₁-subtype receptors. Although we cannot completely exclude the possibility that in the losartan-treated salt-restricted nephrectomized rats Ang II is partially formed in other extrarenal tissues and taken up from plasma, the observation that plasma renin levels were not correlated with adrenal renin mRNA suggests that locally generated Ang II plays a prominent role in the modulation of local renin synthesis.

Our present studies in nephrectomized rats showed that the stimulation of adrenal renin mRNA and the activity produced by salt restriction were associated with increases of aldosterone synthase mRNA and of plasma aldosterone levels in the absence of any measurable circulating renin. This observation indicates that adrenal renin plays a direct and independent role in the modulation of aldosterone production. The selective modulatory action of adrenal renin on mineralocorticoid biosynthesis is secondary to the stimulation of Ang II AT₁-subtype receptors, since it was selectively reduced by losartan and not by PD123319. In the losartan-treated nephrectomized rats, circulating renin levels were not correlated with aldosterone synthase mRNA or with plasma aldosterone levels, thus supporting the postulated modulatory role of Ang II formed within the adrenals. Consistent with this hypothesis are in vitro observations^{18 30} obtained in the zona glomerulosa cells. These latter in vitro studies^{18 30} demonstrated that losartan but not PD123319 reduced potassium-stimulated aldosterone production, thus suggesting that Ang II generated in the adrenal tissue regulates aldosterone production through the stimulation of Ang II AT₁-subtype receptors.

With regard to the suppression of plasma aldosterone concentrations by losartan, it should be noted that although the absolute falls were comparable in intact and nephrectomized rats, in these latter animals aldosterone was reduced to levels not significantly different from those measured in the untreated nephrectomized rats, although this reduction was only 13%, whereas it was 30% in the intact rats with high PRA levels. One obvious interpretation of these data is that nephrectomy markedly attenuated the effect of losartan on plasma aldosterone in salt-restricted rats because circulating renin of renal origin is a major modulatory factor of aldosterone formation. It is interesting to note, however, that aldosterone synthase mRNA was even more suppressed by losartan in nephrectomized than in intact rats. This raises the possibility that other factors may have also contributed to determine the different plasma aldosterone reductions in intact and nephrectomized rats. In this regard, it is likely that Ang II AT₁-subtype receptor blockade could not interfere with the stimulation of aldosterone caused by the doubled levels of serum potassium observed in the nephrectomized rats. In addition, the lack of renal metabolism and clearance of aldosterone^{31 32} may have attenuated the fall in plasma aldosterone as witnessed by the elevated levels measured in all nephrectomized groups. Finally, it is important to mention that a reduction of aldosterone of a magnitude consistent with that observed in vivo in our present study was reported by exposing superfused zona glomerulosa cells to losartan (10 µmol/L).³⁰ In spite of these factors that may have limited the reduction of plasma aldosterone, it is evident that enhancement of the adrenal renin system by salt restriction led to a definite stimulation of aldosterone biosynthesis and that selective blockade of Ang II AT₁-subtype receptors significantly reduced both aldosterone synthase mRNA and plasma aldosterone levels in the nephrectomized rats.

In conclusion, the present study is the first in vivo demonstration that the adrenal renin-angiotensin system plays a selective and independent role in the regulation of aldosterone biosynthesis and production through the specific stimulation of Ang II AT₁-subtype receptors. Our findings also confirm and extend the observation that adrenal renin biosynthesis is regulated by systemic stimuli, including circulating renin and sodium balance, as well as by a local negative feedback exerted by Ang II generated within the adrenal tissue, which is also mediated through the AT₁-subtype receptors.

	Selected Abbreviations and Acronyms

 

ACTH = adrenocorticotropic hormone Ang I, Ang II = angiotensin I and II PCR = polymerase chain reaction PRA = plasma renin activity rt-PCR = reverse-transcriptase PCR

Received November 12, 1996; accepted August 1, 1997.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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