Regulation of Aldosterone Biosynthesis by Adrenal Renin Is Mediated Through AT1 Receptors in Renin Transgenic Rats
Abstract The transgenic (TG) rat (mREN2)27 is characterized by overexpression of the additional mouse Ren-2d gene in the adrenal cortex with marked suppression of renal renin. We have previously shown that in salt-depleted TG rats enhanced activation of mineralocorticoid biosynthesis is associated with selective stimulation of adrenal renin. To investigate whether the local renin-angiotensin system regulates aldosterone biosynthesis in the adrenal cortex of TG rats, we studied the effects of the AT1–angiotensin subtype receptor antagonist DuP 753 on aldosterone production in 5-week-old TG rats during salt restriction. All the rats (n=56) were shifted from regular chow to a diet containing only 0.04% NaCl for 1 week. The AT1-receptor antagonist DuP 753 (10 mg/kg per day in drinking water) was administered to 27 of these rats during low-salt diet. Subgroups of rats were killed at 0, 4, and 7 days. Low-salt diet increased both adrenal renin activity (from 31±3 to 77±4 and 85±2 ng angiotensin I · h−1 · mg protein−1 at 4 and 7 days, respectively; P<.001) and mRNA (by 68.4±10% and 80±17% from baseline, P<.05). In addition, salt restriction was associated with increases of AT1-receptor subtype mRNA, aldosterone-synthase cytochrome P450 mRNA (by 130±56% and 227±33% at 4 and 7 days, respectively; P<.05), and plasma aldosterone (from 184±33 to 402±79 and 338±59 pg/mL, P<.05), whereas renal renin activity did not change, renin mRNA levels remained almost undetectable, and plasma renin activity showed a transient increase. In contrast, in TG rats treated with DuP 753 salt restriction was associated with increases of renal renin mRNA and renal and plasma renin activity, whereas the stimulation of aldosterone-synthase mRNA was markedly attenuated, and plasma aldosterone did not change. Separate analysis of mouse transgene and endogenous rat renin performed by RNase protection assay in 18 additional TG rats and in 18 age- and sex-matched Sprague-Dawley rats showed that the mouse transgene was prominently expressed in the adrenal glands of TG rats in all experimental conditions. In conclusion, our data show that AT1-receptor antagonism abolishes the adrenal renin–related stimulation of the aldosterone biosynthesis produced by salt restriction in TG rats. This indicates that in this model the adrenal renin-angiotensin system regulates the mineralocorticoid production via the AT1-receptor subtype.
Although all components of the renin-angiotensin system are present in the adrenal cortex, it is still controversial whether the local renin-angiotensin system plays an active role within the adrenal glands.1 Previous evidence obtained by Doi et al2 in the normal rat shows that the adrenal renin-angiotensin system is correlated with aldosterone production and indirectly supports a role of the local renin-angiotensin system in the control of aldosterone production and secretion.
The hypertensive renin transgenic (TG) rat (mREN2)27 provides a suitable experimental tool for the investigation of the pathophysiological significance of tissue renins.3 In particular, an overexpression of the additional mouse Ren-2d gene takes place in the zona glomerulosa and zona fasciculata of the adrenal cortex of TG rats. In contrast, renin gene expression in the kidney is markedly suppressed.4 The enhanced adrenal renin concentration has been previously indicated as a potential pathogenic mechanism in TG rats and is related to the higher excretion rate of mineralocorticoids observed in this strain.5 In addition, adrenal renin is highly susceptible to aldosterone secretagogues such as potassium and adrenocorticotropic hormone.1 We have recently observed that in TG rats sodium restriction enhances the expression of aldosterone-synthase cytochrome P450 in the adrenal cortex and produces a marked increase of aldosterone levels in the blood. These responses are associated with a selective increase of adrenal renin, whereas renal renin is not stimulated.6 These observations indirectly suggest that in this experimental model the tissue adrenal renin-angiotensin system is functionally related to the mineralocorticoid biosynthetic pathway, consistent with observations obtained in different animal models.2 7 8
To determine whether the adrenal renin-angiotensin system regulates the mineralocorticoid biosynthesis and secretion in TG rats (mREN2)27, in the present study we investigated the effects of the selective antagonism of the AT1–angiotensin II receptor subtype on the responses to salt depletion.
Materials and Methods
The study was approved by the Animal Care and Treatment Committee of our institution. For the main study, 56 male 5-week-old TG (mREN2)27 rats heterozygous for the Ren-2d transgene obtained from the Moellegard Breeding Centre Deutschland GmbH (Schonwalde, Germany) were used. The rats were housed under controlled conditions of light, temperature, and humidity and fed with a regular rat diet during the week of acclimatization. Subsequently, the rats were shifted to a diet containing 0.04% NaCl and 0.68% potassium (Laboratori dottori Piccioni). Twenty-seven of these rats concomitantly received the angiotensin II receptor antagonist DuP 753 (losartan, 10 mg/kg per day) in the drinking water. DuP 753 is a selective and specific nonpeptide antagonist of the AT1-receptor subtype.9 The dose used in the present study has been shown to provide complete inhibition of AT1-receptor subtype–mediated effects of angiotensin II on blood pressure and aldosterone secretion in normotensive rats.10 The drug was kindly provided by the DuPont Merck Pharmaceutical Company. Animals were randomly killed by decapitation (without premedication) to obtain plasma and tissues at baseline (n=14), after a 2-day pretreatment with DuP 753 (n=8), and on days 4 and 7 of the low-salt diet without (n=8 and 7, respectively) and with pharmacological treatment (n=12 and 7, respectively). Additional 18 male 5-week-old TG rats and 18 age- and sex-matched Sprague-Dawley (SD) normotensive rats obtained from the same source were used for separate measurements of endogenous and exogenous renin in our experimental conditions. Of these animals, six were killed at baseline, six on day 7 of the low-salt diet without pharmacological treatment, and six on day 7 of the low-salt diet under DuP 753 administration in both TG and SD groups.
Systolic blood pressure was measured in the conscious restrained rats by tail-cuff sphygmomanometry (PE-300, Narco Biosystems Inc) and recorded on a multichannel polygraph (Universal oscillograph, Harvard Instruments) before death. Sodium intake and excretion were monitored on a daily basis while the rats were housed in metabolic cages (Nalgene, Harvard Bioscience).
Truncal blood for hormonal measurements was collected in prechilled EDTA Vacutainer tubes, and plasma was stored at −40°C until the time of the assay.
Plasma renin activity (PRA) was measured by enzymatic incubation of plasma at 37°C and pH 6.4 for the rat renin measurement11 and at the optimal pH of 8.5 for the mouse renin assay in a subgroup of 18 TG rats and 18 SD rats. In fact, mouse renin has been reported to be more active on rat angiotensinogen at this pH.12 The angiotensin I (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. Angiotensinogen was measured by incubating 10 μL plasma in duplicate with excess hog renin (Sigma Chemical Co) for 1 hour at 37°C in the presence of 3 mmol/L phenylmethylsulfonyl fluoride (PMSF). Aldosterone was measured by a direct radioimmunoassay kit (Diagnostic Product Corp). The sensitivity of this assay is 16 pg/mL. The interassay and intra-assay variabilities were 5.1% and 7.1%, respectively. Corticosterone was determined by a radioimmunoassay kit (Eurogenetics). The sensitivity of this assay is 25 ng/mL. The interassay and intra-assay variabilities were 6.9% and 7.3%, respectively.
Serum and urinary electrolyte concentrations were measured by autoanalyzer (Beckman).
For measurement of renin activity, tissues were placed on ice after collection and immediately frozen at −70°C until the time of their processing. Three pools of adrenal capsular portions or kidney cortex were obtained at each time point of the study and homogenized as previously reported.13 Each pool included at least three adrenal capsules or 1 g of kidney cortex. Briefly, the tissues were homogenized by 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 PMSF and 6 mmol/L diisopropyl fluorophosphate were added.
Active renin in the tissue homogenates was measured by incubating 100 μL sample with a 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 either as nanograms or micrograms Ang I per milligram protein per hour. Tissue protein content was determined by the method of Lowry et al.14
To assess the specificity of the renin assay in tissue homogenates, samples of both adrenal and renal extracts were incubated 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). The antibody was kindly provided by Dr J.E. Sealey (Cornell University Medical College, New York, NY).
RNA Preparation and Northern Blot Analysis
The adrenal and kidney cortex were immediately frozen in liquid nitrogen and kept at −70°C until RNA extraction. These tissues were pooled as described for tissue renin. Total RNA was isolated by the guanidinium phenol chloroform method. For Northern blotting, total RNA (20 μg per lane) was electrophoresed on 1.0% agarose gel containing 2.2 mol/L formaldehyde and transferred to Hybond-N filter (Amersham).
Prehybridization and hybridization with the cDNA probes (rat renin, rat AT1 receptor, and rat GAPDH cDNAs) were performed at 65°C in a solution containing 7% SDS and 0.5 mol/L Na2HPO4 (pH 7.2). Filters were 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. Rat renin and AT1-receptor cDNA probes were kindly provided by Dr K.R. Lynch, University of Virginia Medical School, Charlottesville. Hybridization with an oligonucleotide specific for the aldosterone-synthase cytochrome P450 activity, manufactured by GENSET, was performed by following the procedure described by Tremblay et al.15 The end-labeled oligonucleotide corresponding to positions 857 to 891 of rat aldosterone-synthase P450 was hybridized at 50°C in a buffer containing 5× standard saline citrate (SSC), 20 mmol NaH2PO4 (pH 7.0), 3.5% SDS, 10× Denhardt’s solution, 10% dextran sulfate, 0.2 mg/mL denatured salmon sperm DNA, and an excess of unlabeled oligonucleotide corresponding to positions 863 to 882 of the rat 11β-hydroxylase cytochrome P450. The washing was performed with 2× SSC and 1% SDS at room temperature, followed by 0.1× SSC and 0.1% SDS at 42°C. After each hybridization, filters were exposed to preflashed Kodak XAR-5 film at −70°C by use of a fluorescence intensifying screen.
Quantitative analysis of the autoradiograms was performed by densitometric scanning and normalized by GAPDH levels. Data are expressed as percentage of the control value.
Measurements of Mouse Renin Transgene and Endogenous Rat Renin by RNase Protection Assay
To discriminate the endogenous rat renin from the mouse transgene under the different experimental conditions of the present study, we performed a separate analysis of renal and adrenal mouse and rat renin expression by the RNase protection assay in 18 additional TG rats and 18 SD rats. For this purpose, [α-32P]UTP antisense RNA fragments were prepared by using a Maxiscript kit (Ambion). Transcription from Acc I–linearized plasmid pSLM containing a Pst I–Sac I fragment of Ren-2d cDNA, using SP6 polymerase, yielded a mouse Ren-2d–specific 224-bp fragment plus 20 bp of vector-encoded sequence. Plasmid pGEM4 containing a Pst I–Kpn I fragment of rat renin cDNA was linearized with HindIII and transcribed with SP6 polymerase to obtain a rat renin-specific 295-bp fragment with 31 bp of vector-encoded sequence. A rat β-actin–specific antisense RNA fragment of 150 bp was obtained by linearization with Xba I of plasmid pSKβac followed by transcription with T7 polymerase. For the RNase protection assay, total RNA (40 μg from each kidney cortex and 2 μg from each adrenal capsule) was vacuum-dried and redissolved in 20 μL 80% formamide containing 40 mmol/L PIPES, 400 mmol/L NaCl, 1 mmol/L EDTA, 200 000 cpm gel-purified mouse or rat renin transcript, and 20 000 cpm rat β-actin transcript. The samples were denatured for 1 minute and allowed to hybridize overnight at 45°C. RNase digestion was performed in 200 μL buffer containing 2 μL of an RNase A/T1 mixture (RPA kit, Ambion). The protected fragments were then precipitated by following the RPA kit instructions, redissolved in 5 μL loading buffer, denatured at 94°C for 1 minute, and fractionated on a 5% polyacrylamide gel containing 7 mol/L urea. The protective probes were detected by autoradiography and quantified by a Phosphor Imager (Molecular Dynamics).
Data are expressed as mean±SEM. One-way ANOVA followed by appropriate a posteriori comparisons was used to test the responses to low-salt diet within the same group. Two-way ANOVA, factoring by group and time, was used for comparisons between the groups.
Effects of Low-Salt Diet in Untreated TG Rats
One week of low-salt diet caused a significant reduction of urinary sodium excretion (from 1.4±0.1 to 0.1±0.01 mEq for 24 hours, P<.001). Body weight increased from 136±2 g at baseline to 208±3 g on day 7 (P<.001). There was a slight increase of systolic blood pressure on day 7 (baseline, 149±4 mm Hg; day 7, 161±1 mm Hg; P<.05). During the low-salt diet, PRA increased only transiently on day 4 and returned to baseline on day 7 (Fig 1⇓, left panel, bottom bar graph), whereas plasma angiotensinogen levels remained unchanged (baseline, 821±26 ng/mL; day 7, 785±56 ng/mL). Tissue adrenal renin activity showed a threefold increase at the end of the low-salt diet (F=63.2, P<.001), whereas renal renin activity did not change (Fig 1⇓, left panel, top and middle bar graphs). Consistently, adrenal renin mRNA levels (Fig 2⇓) showed increases of 68.4±10% and 80±10% from baseline (each n=3, both P<.05), whereas renal renin mRNA remained almost undetectable throughout the study (Fig 3⇓). During the low-salt diet, AT1-receptor subtype mRNA was slightly stimulated in the adrenal cortex (P<.05 versus baseline) but remained unchanged in the kidney (Fig 4⇓).
The aldosterone-synthase gene expression in adrenal tissue was stimulated on day 4 (+130±56%) and day 7 (+227±38%) of the diet (both P<.05 versus basal values) (Fig 5⇓). Consistent with the stimulation of aldosterone-synthase, plasma aldosterone levels increased significantly on days 4 and 7 of the low-salt diet (Fig 6⇓, left panel) (F=3.6, P<.05), whereas plasma corticosterone levels did not change (baseline, 187±45; day 7, 182±30 ng/mL).
Effects of the AT1-Receptor Subtype Inhibition With DuP 753 During Low-Salt Diet in TG Rats
In the TG rats treated with the angiotensin II receptor antagonist, there was a comparable decrease in urinary sodium excretion during the low-salt diet (from 1.3±0.1 to 0.1±0.01 mEq for 24 hours, P<.001). Body weight increased to 223±7 g on day 7 of the study (P<.001 versus baseline). Blood pressure was significantly reduced by the pharmacological treatment (116±2 mm Hg on days 4 and 7 [P<.001] versus 149±4 mm Hg, n=8 at baseline) and was significantly lower than in the untreated animals at the end of the low-salt diet.
PRA was markedly stimulated (Fig 1⇑, right panel, bottom bar graph) and achieved an eightfold increase at the end of the study (F=8.6, P<.001), this response being significantly greater than that observed in the untreated TG rats (P<.001). Concomitantly, plasma angiotensinogen levels were significantly decreased on day 4 (618±37 ng/mL) and day 7 (609±29 ng/mL) (P<.001 compared with baseline and with the untreated TG rats), suggesting enhanced consumption of the substrate.
In the TG rats treated with DuP 753, the tissue renal renin activity was markedly augmented (F=26.2, P<.001) (two-way ANOVA, F=11.4, P<.001 versus the untreated group), whereas there was no further increase of adrenal renin activity compared with the untreated group of salt-restricted TG rats (Fig 1⇑, right panel). On the other hand, renin expression was transiently stimulated in the adrenal glands (by 179±78% versus baseline, P<.05) and returned to the baseline levels by the end of the study (Fig 2⇑). In addition, renin mRNA became largely detectable in the kidney after DuP 753 (Fig 3⇑).
In the salt-depleted TG rats treated with DuP 753, the expression of the AT1-receptor subtype was enhanced in both the adrenal cortex (+100%) and the renal tissue (+80%). The treatment with DuP 753 was associated with a reduced stimulation of the aldosterone-synthase expression on days 4 and 7 of the low-salt diet (−80% and −50%, respectively, compared with the corresponding days of the untreated TG rats) (Fig 5⇑). Accordingly, plasma aldosterone levels did not increase significantly in response to salt restriction, as observed in the untreated rats (Fig 6⇑). Two-way ANOVA, in fact, showed a difference also in the aldosterone response (F=3.2, P<.05). Plasma corticosterone concentration did not change in this group of rats during low-salt diet (from 202±57 to 217±82 ng/mL on day 7).
Effects of Low-Salt Diet and DuP 753 on Mouse and Rat Renin in TG Rats and in SD Rats
In TG rats, the circulating rat renin was not stimulated by the low-salt diet (baseline, 3.2±0.7 ng Ang I · mL−1 ·h−1; day 7, 3.2±0.4 ng Ang I · mL−1 · h−1), whereas it was significantly increased by DuP 753 administration (22.0±3.0 ng Ang I · mL−1 · h−1). The circulating mouse renin was 5.4±0.9 ng Ang I · mL−1 · h−1 at baseline and 6.2±1.5 ng Ang I · mL−1 · h−1 at the end of the low-salt diet and did not increase after the low-salt diet plus DuP 753 administration (7.0±1.7 ng Ang I · mL−1 · h−1).
In SD rats, no evidence of PRA was obtained at pH 8.5, as expected. Rat renin showed an increase at the end of the low-salt diet (from 4.1±0.6 to 12.0±1.2 ng Ang I · mL−1 · h−1) and a marked stimulation after treatment with DuP 753 (35.0±5.0 ng Ang I · mL−1 · h−1).
RNase Protection Assay
In the adrenal capsular tissue of the TG rats, mouse renin was highly expressed at baseline, significantly enhanced after low-salt diet, and comparable to the baseline level after the administration of DuP 753 (Fig 7⇓). Rat adrenal renin was undetectable in our assay conditions even when 60 to 70 μg of total RNA was used. In the kidneys of TG rats, rat renin mRNA was barely detectable both at baseline and after the low-salt diet, whereas it was significantly induced by DuP 753. In the same tissue, the mouse transgene was expressed at baseline, significantly stimulated after sodium restriction, and further increased under DuP 753.
In the kidneys of SD rats, renin expression was induced by sodium restriction and further increased after DuP 753. In the adrenal capsular tissue of these animals, renin was undetectable.
The present study demonstrates that in the TG (mREN2)27 rat model, local adrenal renin, and not circulating renin of renal origin, plays a pivotal role in the regulation of mineralocorticoid biosynthesis and secretion in response to salt restriction. The major finding of the present study is that the adrenal renin-angiotensin system regulates mineralocorticoid production through the AT1–angiotensin II receptor subtype. Our experiments also show that the mouse transgene and not the endogenous renin is involved in the regulation of aldosterone biosynthesis in the adrenals of TG rats.
The hypertensive rat strain TG (mREN2)27 is transgenic for murine Ren-2d gene, providing an excellent tool for the investigation of the function of renin-angiotensin systems in specific tissues. The transgene, in fact, is overexpressed particularly in extrarenal tissues, such as in the zona glomerulosa and fasciculata of the adrenal cortex.4 Since plasma steroid levels and urinary steroid concentrations markedly increase during the development of hypertension in this strain, in spite of the low renal and circulating renin levels,5 it has been hypothesized that local renin exerts a regulatory role on the steroidogenesis in these animals. Previous studies by Sander et al16 have shown that local renin has an important influence on the basal levels of aldosterone-synthase cytochrome P450, the enzyme responsible for aldosterone biosynthesis. The results of the same study, however, also showed that the amount of adrenal renin had little or no effect on the corticotropin-regulated expression of this enzyme. On the other hand, the concentrations of renin in the adrenal glands are largely affected by changes in sodium balance.2 17 18 19 20
With this in mind, in a recent study we explored the effect of sodium restriction on adrenal renin in TG rats.6 The results of that study show that adrenal renin is significantly stimulated by low salt intake in TG rats, whereas the adaptive response of renal renin to low-salt diet is defective. In addition, the stimulation of aldosterone-synthase expression and aldosterone production during the low-salt diet was strictly associated with the response of adrenal renin.
In the present study, the stimulation of the adrenal renin-angiotensin system in TG rats exposed to a low-salt diet is further indicated by the selective increase of the expression of the AT1-receptor subtype in the adrenal glands. Moreover, the inhibition of these receptors markedly reduced the response of aldosterone-synthase and prevented the increase of circulating aldosterone levels in response to salt restriction. These data, obtained in a model characterized by overexpression of the renin transgene in the adrenal cortex, support an important functional role of the adrenal renin-angiotensin system in the control of aldosterone biosynthesis through the excessive local production of angiotensin II. Thus, the AT1 receptors, which are well represented in the adrenal cortex,21 22 mediate the effect of adrenal renin activation on steroidogenesis during low salt intake. Consistent findings have been described in recent reports in in vitro preparations of rat and bovine adrenal capsules.23 24 25 It is also likely that the AT1 receptors mediate the paradoxical stimulatory effect of angiotensin II on the renin gene, previously described only in the adrenal tissue of transgenic rats.26 In fact, the inhibition of the receptor during salt restriction was not associated with increased renin expression by the end of the low-salt diet. The different responses of adrenal renin activity and mouse renin expression after AT1-receptor blockade might be at least partially due to the contribution of the rat renin component that was detectable in our enzymatic assay at the optimal pH for rat renin.
Our results also show that the low rat renal renin levels of TG rats can be modified by inhibition of AT1 receptors, since rat renal renin mRNA and protein content as well as AT1-receptor expression were markedly enhanced by angiotensin II antagonism during the low-salt diet. It is likely that the stimulation of rat renal renin synthesis observed under DuP 753 treatment was due to both the blood pressure decrease produced by the drug and the interruption of the negative feedback exerted by angiotensin II generated by the mouse transgene on the endogenous renin production. Studies by other authors, in fact, suggest that the angiotensin II negative feedback on renal renin is mediated by AT1 receptors in different animal models, including the TG rats.27 28
The present study cannot provide new insights into the potential importance of the tissue renin-angiotensin system or of aldosterone in the pathogenesis of malignant hypertension in TG rats. To rule out the possible interference of renal damage on the hormonal adaptations to salt-intake manipulation, in the present study the rats were in fact studied at a very young age before the development of overt hypertension. On the other hand, recent studies14 do not support a pathogenic role of mineralocorticoids in the development of malignant hypertension in this model.
Another limitation of the present study is that we could not assess the behavior of the endogenous adrenal rat renin. In fact, the RNase protection assay did not permit us to identify rat renin in both TG rats and SD rats. In this regard, however, we have previously reported that a moderate increase of adrenal renin activity in SD rats under sodium restriction was associated with a lesser degree of aldosterone production compared with renin transgenic rats, in spite of more consistent increases of circulating and renal renin.6 Although the approach adopted in the present study only permits an assessment of the adrenal mouse renin transgene, it could be hypothesized that the response of mouse renin to a low-salt diet in the adrenals of TG rats and its related functional consequences represent an amplification of the potential role of the endogenous rat adrenal renin-angiotensin system.
In conclusion, the present study provides evidence of a functional causal link between tissue adrenal renin of exogenous origin and mineralocorticoid biosynthesis in the TG rat model. Although the mechanisms underlying the stimulatory effect of sodium restriction on adrenal renin are not clarified in the present study, the selective increase of adrenal renin appears to be constantly associated with parallel increases of aldosterone-synthase and circulating aldosterone in these experimental conditions. Furthermore, inhibition of the AT1–angiotensin II receptor subtype, as documented by the hemodynamic changes and by the stimulation of renal renin and of receptor expression, can modulate mineralocorticoid biosynthesis in salt-depleted TG rats.
- Received July 28, 1994.
- Accepted March 24, 1995.
- © 1995 American Heart Association, Inc.
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