Differential Gene Expression of Renin and Angiotensinogen in the TGR(mREN-2)27 Transgenic Rat
Abstract Transgenic rats carrying the murine Ren-2 gene represent a monogenetic model of hypertension characterized by low plasma renin and high extrarenal expression of the transgene. The hypothesis has been raised that stimulated local renin-angiotensin systems may be responsible for the development of hypertension in this model. This study analyzes the effects of the converting enzyme inhibitor lisinopril, which specifically interferes with the renin-angiotensin system, and the direct vasodilator dihydralazine on the renal and extrarenal expression of renin and angiotensinogen. A comparison of gene expression between heterozygous and homozygous transgenic and normal Sprague-Dawley rats was also performed. We demonstrate high sensitivity of blood pressure toward converting enzyme inhibition in transgenic TGR(mREN-2)27 rats. In the kidney, expression of the transgene and the endogenous renin gene increased, suggesting that both are modulated by lisinopril in a similar manner. On the other hand, blood pressure reduction by dihydralazine did not abolish renal renin suppression in transgenic rats, indicating that mechanisms different from direct effects of blood pressure account for renin suppression. Homozygosity for the transgene led to increased Ren-2 expression and higher blood pressure and had opposite effects on angiotensinogen expression compared with heterozygous rats. Cardiac hypertrophy was reduced by lisinopril but not dihydralazine and was positively correlated with cardiac angiotensinogen expression. Increased angiotensin II in the adrenal gland of TGR(mREN-2)27 rats, which overexpresses the transgene, provides evidence that this leads to enhanced generation of tissue angiotensin II. We conclude that expression of the mouse transgene, the endogenous rat renin gene, and the angiotensinogen gene is subject to differential tissue-specific regulation. Reversal of cardiovascular damage with the converting enzyme inhibitor but not dihydralazine suggests that angiotensin II generated locally may be involved in the pathogenesis of hypertension and structural changes in TGR(mREN-2)27 rats.
The renin-angiotensin system (RAS) plays a major role in the regulation of blood pressure and electrolyte homeostasis. In its classic definition, the RAS functions as an endocrine system. However, accumulating evidence suggests that angiotensin II (Ang II) can be generated locally, serving paracrine and autocrine functions that are regulated independently of the circulating RAS. Tissue-specific expression of RAS components–renin,1 angiotensinogen,2 converting enzyme (CE),3 and angiotensin receptors4 –as well as local generation of Ang II have been demonstrated in organs involved in the control of cardiovascular homeostasis, including brain, heart, adrenal gland, and kidney.5 6 7 Furthermore, tissue-specific function and modulation have been shown for several organs such as the brain8 and adrenal gland.9
The transgenic rat line TGR(mREN-2)27 has been established by introducing the murine Ren-2 gene into the genome of the rat with the use of microinjection techniques.10 These rats are characterized by fulminant hypertension, with values of up to 230 mm Hg at 5 to 6 weeks of age despite low circulating renin. Whereas renin expression is suppressed in the kidney, the transgene is highly expressed in extrarenal tissues such as adrenal gland, brain, and thymus and the gastrointestinal and urogenital tracts.11 Thus, overexpression of renin in the kidney does not account for the phenotype. Since all transgenic founder animals were hypertensive irrespective of the site of insertion into the genome10 and since the presence of the transgene segregates with the hypertensive phenotype, an insertional mutagenesis seems unlikely. Therefore, it is hypothesized that hypertension results from enhanced local Ang II generation in extrarenal tissues. Although the additional renin gene is the only genetic difference compared with nontransgenic controls, the mechanisms underlying the development of hypertension are still elusive. In the adrenal gland, high Ren-2 expression is associated with increased urinary steroid excretion in young animals,12 and high circulating prorenin levels are, to a large extent, of adrenal origin.13 In addition, transgenic rats exhibit early morphological signs of pathological alterations within the cardiovascular system, including cardiac hypertrophy and glomerulosclerosis.14 15 Thus, this monogenetic model of hypertension offers the possibility of studying the mechanisms by which a single gene contributes to the pathogenesis of hypertension and cardiovascular disease.
The aim of this study was to investigate the regulation of renin and angiotensinogen gene expression in TGR(mREN-2)27 rats. Since expression of the renin gene in the kidney is subject to feedback inhibition by increased circulating Ang II and elevated blood pressure, we examined the modulation of gene expression in renal and extrarenal tissues of heterozygous transgenic rats after intervention with the CE inhibitor lisinopril compared with the direct vasodilator dihydralazine, focusing on the mechanisms involved in renin suppression in the kidney. In addition, we examined the influence of gene dose on renin and angiotensinogen gene expression and on tissue Ang II.
Transgenic rats were generated and bred in our laboratory.10 Heterozygous transgenic rats were obtained by crossbreeding male homozygous transgenic rats with female Sprague-Dawley (SD) rats. All pharmacological experiments were performed in male heterozygous animals. Normotensive SD rats were purchased from the Zentralinstitut für Versuchstierkunde (Hannover, Germany). Two to three animals were kept in one cage under alternating 12-hour light/dark cycles at constant temperatures of 20° to 22°C. Animals were fed a standard laboratory chow.
Four groups of heterozygous male TGR(mREN-2)27 rats (9 weeks of age) were treated either with lisinopril (Zeneca) in daily doses of 10, 2, and 0.5 mg/kg body wt (n=11 in each group) for 7 weeks or with dihydralazine in a daily dose of 30 mg/kg body wt (n=8) for 6 weeks. Age-matched heterozygous transgenic rats (n=11) as well as homozygous (n=11) and SD (n=11) rats were kept under the same conditions without treatment. The average nighttime water intake was determined over 4 days, and drugs were administered accordingly. Daytime water intake was ad libitum. Systolic pressure was determined weekly in the morning with the use of tail-cuff plethysmography. Blood samples were obtained every fortnight by retro-orbital puncture during light short-term anesthesia and transferred into chilled tubes containing 5% phenanthroline-EDTA for measurement of renin, angiotensinogen, and Ang I or 1% heparin sodium for measurement of CE activity. At the end of treatment, rats were killed by decapitation. Tissues were removed, immediately frozen in liquid nitrogen, and stored at −80°C. Whole hearts were washed in 0.9% NaCl, blotted dry, and weighed. The ratio of cardiac mass and body weight was determined as a parameter for cardiac hypertrophy.
Plasma concentrations of inactive and active renin were determined according to Glorioso et al.16 Briefly, for activation of inactive renin, 20 μL plasma was incubated with 40 μL trypsin (400 U/mL, dissolved in TES buffer: 0.1 mol/L TES, 10 mmol/L EDTA, 1 mg/mL bovine serum albumin, 0.01% Neomycin, pH 7.2) on ice for 10 minutes. The reaction was stopped with 40 μL soybean trypsin inhibitor (600 U/mL TES buffer). For determination of active renin, 20 μL plasma was dissolved in 80 μL TES buffer. Samples were then incubated with renin substrate isolated from nephrectomized rat plasma (final concentration, 80 mg/mL; 0.11% 2,3-dimercapto-1-propanol, 1.15 mg/mL 8-OH-chinolin in TES buffer). The reaction was stopped with 0.1 mol/L Tris-acetate, pH 7.4, immediately before and 1 hour after incubation at 37°C, and generated Ang I was measured by radioimmunoassay.17 Prorenin was calculated by subtraction of active renin concentration from total renin concentration. CE activity was measured with a fluorometric assay using carboxy-phenyl-alanyl-histidyl-leucine as substrate.18
Measurement of renal and adrenal Ang II contents was carried out as previously described.19 Briefly, pieces of kidneys and whole adrenals were boiled in distilled water for 5 minutes, homogenized in 0.05N HCl, and centrifuged. The supernatant was applied to a Sep-Pak C18 cartridge column, and the retained peptide was eluted with 80% methanol/0.1% trifluoroacetic acid and subjected to high-performance liquid chromatography followed by radioimmunoassay.
Creatinine and blood urea nitrogen were determined by a colorimetric assay (Hitachi autoanalyzer).
RNase Protection Assays
Total RNA was isolated according to Auffray and Rougeon.20 For RNase protection assays, [32P]UTP-labeled cRNA probes were made from the following plasmids using the riboprobe Gemini II Kit (Promega)10 21 : pSLM, yielding a Ren-2–specific 224-bp fragment plus 20 bp of vector-encoded sequence; pRen412, yielding a rat renin–specific 295-bp fragment plus 31 bp; pRag0.3, yielding a rat angiotensinogen–specific 300-bp fragment plus 40 bp; and pSKrbac, yielding a rat β-actin–specific 150-bp fragment plus 28 bp of vector-encoded sequence. Transcription was carried out according to the manufacturer’s protocol using 50 μCi [32P]UTP (3000 Ci/mmol, Amersham) for labeling of renins and angiotensinogen and 24 μCi [32P]UTP plus 13 μmol/L unlabeled UTP for β-actin to reduce signal intensity. Total RNA samples were mixed in 30 μL hybridization buffer (final concentration, 80% mL formamide, 40 mmol/L piperazine-N,N′-bis-2-ethanesulfonic acid, 400 mmol/L NaCl, and 1 mmol/L EDTA, pH 8.0) containing 200 000 cpm Ren-2, rat renin or angiotensinogen cRNA, respectively, and 20 000 cpm β-actin cRNA. The samples were denatured at 100°C and allowed to hybridize overnight at 45°C. Digestion was carried out with 300 μL RNase buffer (300 mmol/L NaCl, 5 mmol/L EDTA, 10 mmol/L Tris-HCl, pH 7.4) containing 12 μg RNase A (Sigma Chemical Co) and 0.6 μg RNase T1 (Calbiochem) per sample at 37°C for 1 hour. After incubation with 10 μL proteinase K (10 mg/mL) and 10 μL 10% sodium dodecyl sulfate at 37°C for 30 minutes, protected fragments were extracted with phenol/chloroform/isoamylalcohol and ethanol-precipitated with 18 μg tRNA as carrier. The pellet was dissolved in 8 μL of 100% formamide-dye mix, denatured, and run on a 5% polyacrylamide gel containing 7 mol/L urea. Protected fragments were detected by autoradiography and quantified by scanning densitometry. For quantitative analysis of mRNA content, the ratio of the density of specific probe and β-actin was used.
Morphology and Histochemistry
Three rats of each group treated with lisinopril and dihydralazine as well as three heterozygous control rats were anesthetized by an injection of pentobarbital sodium (40 mg/kg body wt IP) and perfusion-fixed with 2% paraformaldehyde/phosphate-buffered saline after cannulation of the abdominal aorta.14 Kidney slices were either frozen in liquid nitrogen–cooled isopentane or postfixed for subsequent paraffin embedding. For immunohistochemistry, deparaffinized kidney sections were incubated with a rabbit polyclonal antibody against purified rat kidney renin (gift of Dr E. Hackenthal, Heidelberg, Germany) at a 1:10 000 dilution or with 1% nonimmune rabbit serum and were visualized with a peroxidase-antiperoxidase staining kit (Dako). For in situ hybridization, cryostat sections were incubated with digoxigenin-UTP–labeled riboprobes derived from the same plasmids that were used for RNase protection assays. Detection was carried out with an alkaline phosphatase–coupled anti-digoxigenin antibody and nitroblue tetrazolium/X-phosphate (Boehringer). For double labeling of renin protein and mRNA, a mixture of anti-renin and anti-digoxigenin antibody (dilution 1:200 and 1:500, respectively) was used. Protein signal was detected by Texas red fluorescence followed by phosphatase detection. Morphological analysis was performed on periodic acid–Schiff–stained paraffin sections. Renal damage was assessed by determining the percentage of damaged glomeruli characterized by collapse as well as glomerulosclerosis with three grades of intensity identified by hyaline deposits and mesangial proliferation.22 For this purpose, approximately 100 glomeruli on each of two coronal sections, one from the right and one from the left kidney, were evaluated with a Polyvar microscope with interference contrast optics (Reichert).
Statistical analysis was performed with the crunch statistical software program. Results are expressed as mean±SEM. Between-group differences were analyzed by the nonparametric Mann-Whitney U test. Values of P<.05 were considered significant.
When treatment was started, heterozygous TGR(mREN-2)27 rats were 9 weeks old and hypertension was already established. Lisinopril reduced blood pressure significantly compared with untreated controls even with the lowest dose of 0.5 mg/kg body wt (185±3 versus 230±5 mm Hg, P<.0005). Higher doses of 2 and 10 mg/kg led to normotensive blood pressure values (148±3 and 138±4 mm Hg, respectively, P<.0005) within the first week of treatment. Dihydralazine was also effective, and normotensive levels (134±10 mm Hg, P<.0005, Fig 1⇓) were reached. Body weight development was similar in all groups throughout the experiment, with values of 448±11 g in untreated controls; 416±7, 445±5, and 412±13 g in rats treated with 0.5, 2, and 10 mg/kg lisinopril, respectively; and 419±14 g in rats treated with dihydralazine at the end of treatment.
In transgenic rats treated with lisinopril, plasma active renin increased after the first week of treatment with all three doses and was 9-, 25-, and 23-fold elevated at 0.5, 2, and 10 mg/kg, respectively, after 6 weeks, whereas prorenin levels were not affected (Fig 2A⇓ and 2B⇓). This was accompanied by a parallel increase of Ang I in plasma, reaching 2.9-, 3.5-, and 6.3-fold higher levels with 0.5, 2, and 10 mg/kg lisinopril, respectively, at the end of treatment compared with untreated controls (Fig 2C⇓). Lisinopril led to an increase of plasma CE activity after 1 week of treatment in all groups (control, 321.3±47.5; 0.5, 2, and 10 mg/kg lisinopril, 677.5±31.9, 569.7±26.9, and 535.0±38.9 nmol His-Leu/mL per minute, respectively; P<.0005 for all groups; Fig 2D⇓). Plasma angiotensinogen levels gradually decreased in rats treated with 2 and 10 mg/kg lisinopril compared with control animals by 59% and 46%, respectively, whereas no changes were observed in rats treated with the lowest lisinopril dose (Fig 2E⇓). In rats treated with dihydralazine, plasma active renin and prorenin as well as CE activity were unchanged compared with control rats (data not shown).
Homozygosity for the transgene was associated with higher blood pressure. Plasma active renin was suppressed to an extent similar to that seen in heterozygous animals, whereas prorenin elevation was significantly higher (Fig 3A⇓ through 3C). Plasma Ang I was not affected by doubling of the transgene dose and was equally decreased in heterozygous and homozygous rats (Fig 3D⇓). Plasma CE activity was significantly reduced in homozygous rats compared with normotensive SD rats or heterozygous transgenic rats (Fig 3E⇓).
Effects on the Kidney
Renin activity in the kidney increased significantly in a dose-dependent manner, from 51.5±12.6 pmol Ang I/mL per hour in untreated transgenic controls to 267.8±19.8, 440.56±49.28, and 496.3±44.4 pmol Ang I/mL per hour after treatment with 0.5, 2, and 10 mg/kg lisinopril, respectively (P<.005 for all groups). To assess the relative contribution of renin mRNA from either the endogenous gene or the transgene, we used RNase protection assays that distinguish the two mRNA species based on the protected length of 295 bp for rat renin and 224 bp for mouse Ren-2, respectively. Treatment of heterozygous rats with 0.5, 2, and 10 mg/kg lisinopril was associated with a 2.4-, 3.3-, and 9-fold induction of Ren-2 mRNA, respectively, and a 9-, 28-, and 35-fold induction of the endogenous rat renin mRNA, whereas no changes in renin gene expression were observed with dihydralazine (Fig 4A⇓ and 4B⇓). Both lisinopril and dihydralazine treatments had no influence on renal angiotensinogen expression (Fig 4C⇓). In contrast to the induction of plasma CE activity after lisinopril administration, renal CE activity decreased from 1.75±0.14 nmol His-Leu/mg per minute in untreated transgenic controls to 1.23±0.01, 0.81±0.1, and 0.56±0.03 nmol His-Leu/mg per minute after treatment with 0.5, 2, and 10 mg/kg lisinopril, respectively (P<.005 for 0.5 and 2 mg/kg lisinopril, and P<.0005 for 10 mg/kg lisinopril).
The comparison of heterozygous and homozygous rats revealed that doubling of gene dose was accompanied by a 13-fold increase of the mouse transgene mRNA in the kidney, whereas endogenous rat renin mRNA was suppressed in heterozygous and homozygous rats to a similar extent (Fig 4A⇑ and 4B⇑). Interestingly, the presence of the transgene in heterozygous TGR(mREN-2)27 rats was associated with a significant decrease of renal angiotensinogen mRNA, which was more pronounced in homozygous rats compared with nontransgenic controls (Fig 4C⇑). Renal CE activity was similar in SD and heterozygous rats (1.82±0.6 and 1.75±0.1 nmol His-Leu/mg per minute) but decreased in homozygous transgenic rats (1.29±0.12 nmol His-Leu/mg per minute, P<.05 versus SD rats).
Measurement of renal tissue Ang II content revealed markedly reduced levels in heterozygous transgenic rats compared with SD rats (422.7±29.7 versus 734.8±46.6 pg Ang II/g, P<.0005, Fig 5⇓). Surprisingly, in homozygous rats, renal Ang II content did not differ from that of nontransgenic SD rats despite suppression of renin angiotensinogen and CE in the kidney. After CE inhibition, Ang II levels were reduced to 63% of the levels of untreated heterozygous controls, whereas in dihydralazine-treated rats, no changes in renal Ang II content occurred.
Immunohistochemical analysis of renal renin distribution after CE inhibition revealed a prominent increase of immunoreactive renin stored in the afferent arteriolar wall over considerable distances toward the branching points from interlobular arteries (Fig 6A⇓). Antihypertensive treatment with dihydralazine did not significantly increase renin immunostaining compared with untreated control rats, in which only few juxtaglomerular apparatuses were labeled (Fig 6B⇓ and 6C⇓). The immunohistochemical findings were confirmed by in situ hybridization that demonstrated a paucity of juxtaglomerular renin mRNA in untreated control rats, whereas increased staining in lisinopril-treated animals was accompanied by a renin mRNA signal that colocalized to the immunoreactive sites shown by double labeling of the same section (Fig 6D⇓ and 6E⇓). For in situ hybridization, the probes were not able to discriminate between the two species, and the same results were obtained with either mouse or rat renin probes. Although renin mRNA expression was occasionally found in extra-arteriolar sites, the highest accumulations of signal were present within glomeruli of rats treated with a high dose of lisinopril. The enhancement of renin expression under CE inhibition was dose dependent and less pronounced with the two lower doses of 2 and 0.5 mg/kg lisinopril.
Effects on the Adrenal Gland
Mouse renin mRNA was increased in the adrenal gland only after treatment with the highest lisinopril dose of 10 mg/kg (P<.05), whereas it was not affected by dihydralazine (Fig 7A⇓). Doubling of gene dose in homozygous animals was associated with a fourfold increase of transgene mRNA compared with untreated heterozygous rats. Transcripts of the endogenous rat renin gene were not detectable with 50 μg total RNA.
Ang II measurements in the adrenal gland demonstrated a significant increase in transgenic rats. Although homozygosity was associated with an unproportionally higher transgene expression in the adrenal gland when compared with heterozygous animals, tissue Ang II levels were similar in homozygous and heterozygous rats compared with SD rats. Lisinopril treatment resulted in a reduction of Ang II in the adrenal gland.
Effects on the Heart
Ren-2 mRNA is also found in cardiac tissue, and homozygosity is associated with doubling of its mRNA. Administration of lisinopril or dihydralazine showed no effect on the expression of Ren-2 or rat renin (Fig 8A⇓ and data not shown). Interestingly, presence of the transgene showed opposing effects on angiotensinogen mRNA in the heart. Whereas in heterozygous rats angiotensinogen mRNA was significantly suppressed, it was markedly increased in homozygous rats. Furthermore, angiotensinogen gene expression was negatively influenced by CE inhibition and decreased by 44%, 38%, and 48% with 0.5, 2, and 10 mg/kg lisinopril, respectively, whereas treatment with dihydralazine caused a 5.8-fold increase of angiotensinogen mRNA (Fig 8B⇓). This was positively correlated with cardiac hypertrophy. Treatment with all lisinopril doses significantly reduced ratios of heart weight to body weight, whereas dihydralazine increased cardiac hypertrophy (Fig 8C⇓). In addition, an increased cardiac angiotensinogen gene expression in homozygous rats was also accompanied by a higher cardiac mass index.
Renin and Angiotensinogen Gene Expression in Other Extrarenal Tissues
The thymus belongs to the tissues with the highest Ren-2 expression in TGR(mREN-2)27 rats, and doubling of the gene dose was associated with a threefold higher transgene expression. Long-term treatment with lisinopril or dihydralazine had no influence on Ren-2 expression (Fig 9A⇓). As opposed to mouse renin, endogenous rat renin mRNA was not detectable in SD rats, untreated heterozygous or homozygous transgenic rats, or dihydralazine-treated rats but was dose dependently induced after CE inhibition (Fig 9B⇓), suggesting a negative regulatory influence of Ang II on the endogenous renin gene expression in the rat. Angiotensinogen gene expression was not affected by either treatment, although in untreated heterozygous animals, angiotensinogen mRNA was significantly suppressed compared with SD or homozygous rats (Fig 9C⇓).
Ren-2 transcripts were also present in testes of male transgenic rats and were strongly enhanced in homozygous rats carrying two copies of the transgene. An induction of transgene expression was observed in heterozygous rats after treatment with 10 mg/kg lisinopril but not with dihydralazine (Fig 10⇓), whereas no changes in endogenous rat renin or angiotensinogen gene expression occurred (data not shown).
High expression of the transgene in the gastrointestinal tract was not affected by lisinopril or dihydralazine (Fig 11A⇓). In contrast to this, endogenous rat renin gene expression was dose dependently induced in stomach by lisinopril but not by dihydralazine (Fig 11B⇓). Angiotensinogen gene expression in stomach was not affected by gene dose or antihypertensive treatment (data not shown).
Morphological Analysis of Kidneys
Morphological studies revealed that CE inhibition led to a reduction of structural damages in the kidney. Quantitative evaluation of periodic acid–Schiff–stained paraffin sections of kidneys from untreated transgenic rats demonstrated focal glomerulosis, mesangial proliferation, widening of Bowman’s space, and occasionally collapsed glomeruli. After lisinopril treatment for 7 weeks, the histological aspect was almost normalized. As shown in Table 1⇓, only 60.9% of the total number of glomeruli were intact in the untreated group, whereas with high-dose lisinopril, 82.6% of the glomeruli were intact. The lower doses of lisinopril produced only minor morphological changes compared with controls. Conversely, dihydralazine treatment did not cause obvious changes in renal morphology when compared with untreated controls. The improvement of renal damage observed with CE inhibition was accompanied by a lack of progression of renal dysfunction, as evidenced by constant levels of blood urea nitrogen and plasma creatinine in lisinopril-treated rats, whereas in untreated controls both parameters and in dihydralazine-treated rats blood urea nitrogen were significantly increased (Table 2⇓).
The transgenic TGR(mREN-2)27 rat represents a monogenetic model of hypertension with low plasma and renal renin and high expression of renin in extrarenal tissues, thus offering a model for the study of the functional significance of local RAS in vivo. Although an increased steroid excretion parallels hypertension development, the role of a mineralocorticoid excess as a major pathogenic factor is questionable, because spironolactone did not reduce or prevent hypertension.12 This suggests that other factors contribute to the pathogenesis of hypertension in these animals.
The present study demonstrates that expression of renin and angiotensinogen in the kidney and extrarenal tissues is differentially regulated under the influence of the transgene and in response to the administration of a CE inhibitor or a direct vasodilator. Since it is known that CE inhibition interferes with the negative feedback mechanism, leading to increased synthesis and release of renin via decreased levels of circulating Ang II,6 it was of particular interest to investigate the influence of CE inhibition on the renal RAS in TGR(mREN-2)27 rats. We therefore examined the effects on blood pressure and plasma and tissue RAS and demonstrated a high sensitivity of TGR(mREN-2)27 rats to CE inhibition. Although kinin-potentiating effects of CE inhibitors cannot be excluded, they seem not to be of major importance because angiotensin receptor blockade has also been shown to be effective in reducing blood pressure.23
Increased circulating active renin results from the induction of renal expression of both the endogenous renin gene and the transgene in response to CE inhibition and implies similar regulating mechanisms that are independent of blood pressure because dihydralazine did not affect renin expression despite its antihypertensive effect. The physiological role of prorenin is unknown, and whether activation in vivo occurs is controversial. In spontaneously hypertensive rats and monkeys, exogenously administered prorenin is not converted within the circulation but taken up by the kidney and converted to active renin without affecting blood pressure.24 25 As elevated prorenin in TGR(mREN-2)27 rats is to a large extent of mouse transgene origin,13 the lack of any effect of CE inhibition on plasma prorenin suggests that it is not subject to a negative feedback control by Ang II. Higher prorenin levels accompanied by a suppressed plasma RAS and high expression of extrarenal renin in homozygous TGR(mREN-2)27 rats correlate with higher blood pressure values, thus rendering prorenin a marker for tissue renin.
The presence of the transgene is associated with reduced renal angiotensinogen gene expression in TGR(mREN-2)27 rats that is more pronounced in homozygous rats. Renal CE activity is likewise suppressed, although only in homozygous rats. Consequently, tissue Ang II is significantly decreased in transgenic compared with SD rats. Interestingly, homozygous rats show renal Ang II levels similar to those in heterozygous rats despite greater suppression of angiotensinogen and CE. A pressure-independent mechanism by which Ang II inhibits renin release by a direct intrarenal action, also referred to as short feedback loop, has been demonstrated.26 Therefore, overexpression of the transgene in the kidney may be responsible for relatively high levels of Ang II in homozygous animals, which in turn mediate suppression of renin independently of the elevated blood pressure. In this context, decreased renal Ang II in heterozygous rats that do express the transgene in the kidney may still be inappropriately high given the suppression of the endogenous renin gene that is equally strong irrespective of gene dose. The notion that suppression of the endogenous renin gene in the kidney is not mediated by the increased blood pressure alone is supported by the finding that normotension induced by dihydralazine was not sufficient to abolish renal renin suppression.
As an arteriolar vasodilator, dihydralazine tends to cause reflex tachycardia, renin release,27 28 and fluid retention, counteracting its antihypertensive effect, yet it is still unclear whether this is due to increased sympathetic discharge evoked by arterial vasodilation or a direct renal mechanism.29 Dihydralazine treatment of transgenic rats did not affect renin release or body weight, suggesting an absence of significant volume retention.
Transgenic rats exhibit severe glomerulosclerosis with accompanying proteinuria.15 In the present study the decrease of renal Ang II induced by CE inhibition was accompanied by an improvement of renal function, as demonstrated by the maintenance of stable renal function parameters (Table 2⇑). Since with dihydralazine renal function deteriorated, one may speculate that the effects of the CE inhibitor reflect the preservation of adequate renal function rather than control of hypertension by interfering with an abnormal regulation of the intrarenal RAS.7
As a prerequisite for an activated intra-adrenal RAS, we demonstrated increased local formation of Ang II in the adrenal gland of transgenic rats. The decline in adrenal Ang II content paralleled blood pressure reduction, suggesting effective tissue CE inhibition.
Overexpression of renin in the heart of TGR(mREN-2)27 rats may lead to increased Ang II formation despite decreased angiotensinogen mRNA found in heterozygous animals. Regression of cardiac hypertrophy after CE inhibition may thus result in part from the prevention of Ang II–mediated muscular hypertrophy.30 Opposite effects of transgene dose on cardiac angiotensinogen gene expression may occur as a result of mechanisms counteracting or interfering with an activated RAS. Circulating Ang II has been implicated in the upregulation of renal angiotensinogen mRNA,31 and because plasma Ang II levels of transgenic rats are unchanged compared with those in nontransgenic controls,10 it seems likely that differential effects on angiotensinogen expression are under tissue-specific regulation. Because normotension was achieved with both substances, the effect on cardiac hypertrophy and the improvement of renal damage seen with specific inhibitors of the RAS but not with dihydralazine suggest that inhibition of tissue CE may have an important long-term effect on the improvement of cardiovascular damage in this model. Chronic lisinopril treatment leads to an induction of plasma CE, whereas tissue CE is effectively inhibited, further suggesting that circulating CE is not the only determinant for the long-term effects of chronic CE inhibition.
Interestingly, Ren-2 and rat renin in the gastrointestinal tract are not regulated in parallel as in the kidney. After CE inhibition, endogenous rat renin mRNA, although undetectable in untreated TGR(mREN-2)27 or SD rats, was massively induced, whereas the highly expressed mouse transgene was not affected at all. A similar pattern was observed in thymus, a major site of high Ren-2 expression, where only the endogenous rat renin expression is positively regulated by CE inhibitors. In contrast to this, only mouse renin mRNA was induced in testes. The absence of inducibility of the transgene in stomach and thymus suggests either the presence of rat species–specific trans-acting factors or the absence of cis-acting factors within the transgene. Possible functions of an intestinal RAS, besides water and electrolyte absorption, include regulation of mesenteric conductance32 and microvascular permeability.33 Zhao et al34 recently showed that the Ren-2 gene is regulated in the intestine of mice by dietary changes and in transgenic rats during ontogeny.11 Whether overexpression of renin leads to increased resorption of water via enhanced Ang II generation and whether this effect contributes to the pathogenesis of hypertension in the transgenic rat remains to be investigated.
In conclusion, we demonstrated that the transgene not only is expressed in an organ-specific manner but also is subject to tissue-specific regulation by CE inhibition. Differential expression of renin and angiotensinogen suggests regulatory mechanisms on the transcriptional level dependent on cell-specific properties. We have no proof for every tissue overexpressing Ren-2, but the facts that this overexpression is accompanied by increased local generation of Ang II, high intra-adrenal Ang II as described here, and continuous release of Ang II from perfused hindquarter preparations of transgenic rats after bilateral nephrectomy35 strongly suggest that enhanced Ang II formation is also present in other tissues. The physiological significance of localized RAS in tissues, which seem not to be directly involved in blood pressure control, is still unclear, and this study cannot address causal relations of differential gene expression with functional responses. However, we speculate that they function as paracrine systems mediating local tissue perfusion, hormone release, resorptive processes, or inflammatory responses, thereby regulating individual tissue function independent of the circulating counterpart.
This work was supported by the Deutsche Forschungsgemeinschaft, SFB 317, and by Zeneca Co, Plankstadt, Germany. We wish to thank Drs Ursula Ganten, Klaus Lindpaintner, and Maria Minuth for their support and helpful discussions.
Reprint requests to Min Ae Lee, MD, Department of Medicine, Cardiovascular Division, Brigham and Women’s Hospital, 75 Francis St, Boston, MA 02115.
- Received July 11, 1994.
- Revision received August 10, 1994.
- Accepted November 29, 1994.
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