The Brain Renin-Angiotensin System in Transgenic Mice Carrying a Highly Regulated Human Renin Transgene
We previously reported the generation of 2 novel transgenic mouse models containing the human renin (hREN) gene encoded on P1 artificial chromosomes (PAC) containing large amounts of 5′-flanking DNA. These mice exhibit a very narrow tissue-specific expression profile and exhibit tightly regulated expression in kidney in response to physiological cues. In brain, transcription of hREN occurs from an alternative upstream promoter, causing translation to initiate within exon-II and potentially generating an intracellular form of active renin. Double transgenic mice containing a PAC transgene and the human angiotensinogen (hAGT) gene (P+/A+) are moderately hypertensive. We tested whether increased RAS activity in the brain contributes to the mechanism of hypertension in P+/A+ double transgenic mice. Expression of hREN mRNA in brain was confirmed in 4 independent PAC transgenic lines and utilization of the alternative transcription start site in brain was confirmed in each line. Human REN immunostaining was observed in the dorsal cochlear nucleus, hypothalamus, and cortex. P+/A+ mice exhibited a greater fall in mean arterial pressure after intracerebroventricular injection of losartan than controls. P+/A+ mice exhibited a greater drop in arterial pressure after intravenous injection of a vasopressin V1 receptor antagonist, and an equivalent drop in arterial pressure after intravenous injection of a ganglion blocker compared with controls. These results support the hypothesis that renin is endogenously expressed in the brain and suggest that increased brain RAS activity may contribute to the maintenance of moderate hypertension in P+/A+ transgenic mice at least in part by a vasopressin-dependent mechanism.
Several lines of evidence suggest that the brain renin-angiotensin system (RAS) contributes to the hypertensive state in spontaneously hypertensive rats, DOCA-salt hypertensive rats, Dahl-salt sensitive rats, and renal hypertensive rats.1–4⇓⇓⇓ We have reported that hAGT and hREN expression is observed in the brains of mice overexpressing a hAGT transgene spanning a region from approximately 1.2 kb 5′ to 1.4 kb 3′ of the gene (A+) and in mice containing a hREN genomic sequence spanning a region from approximately 900 bp 5′ to 400 bp 3′ of the gene (R+), respectively.5,6⇓ Transgenic mice overexpressing both transgenes (R+/A+) are chronically hypertensive.7 Intracerebroventricular (ICV) injection of a selective angiotensin II (Ang II) type 1 (AT1) receptor antagonist, losartan, in R+/A+ mice causes a greater fall in blood pressure (BP) than in control nontransgenic mice, suggesting that increased activation of central AT1 receptors by locally overproduced Ang II may contribute to the maintenance of hypertension in these mice.6
We and others have reported that R+ transgenic mice exhibit high-level expression of the transgene in a number of ectopic sites, and further, the transgene is poorly regulated in the kidney.8–11⇓⇓⇓ These data suggest that 900 bp of hREN 5′-flanking sequence does not contain all the regulatory elements required for appropriate tissue-specific expression and regulation of gene expression in response to physiological cues. Therefore, it is unclear if the hREN mRNA detected in the brain of these mice is expressed appropriately or ectopically. Because of this, we generated 2 new hREN transgenic mice each containing a different large P1 artificial chromosome (PAC) encoding the entire hREN (P+) gene.12 PAC140 contains a 140-kb insert consisting of 35 kb of 5′- and 90 kb of 3′-flanking DNA, and PAC160 contains a 160-kb insert consisting of 75 kb of 5′- and 70 kb of 3′-flanking DNA. Expression of both PAC constructs was restricted exclusively to kidney and only a very low-level expression was detected in ectopic sites. Importantly, expression of hREN mRNA among 6 different lines of PAC140 and PAC160 mice was found to be proportional to copy number. This usually indicates the presence of sequences able to manipulate chromatin at the local level and suggests that all sequences required for appropriate tissue-specific expression are present in each construct.13 Moreover, hREN expression in the kidney of PAC140 and PAC160 mice was appropriately upregulated by low salt diet and angiotensin converting enzyme (ACE) inhibition and downregulated by high salt diet and Ang II. Moreover, expression of renal hREN in double transgenic P+/A+ mice was also downregulated, thus accounting for lower plasma Ang II and lower arterial pressure than R+/A+ mice, which contain the poorly regulated hREN transgene.
More recently, we detected hREN mRNA in brain in one line of PAC160 mice containing a high number of transgene copies.14 Interestingly, transcription of hREN mRNA in brain (but not kidney) occurred from an alternative transcription start site located about 1.3 kb upstream of the classic promoter and encoding a new exon 1 (termed exon 1b) that splices directly to exon 2. An identical form of renin mRNA was found in RNA isolated from human fetal brain suggesting this is not an artifact of transgenesis.14 This form of the mRNA lacks the normal translational initiation codon in exon-I thus forcing the use of an in-frame ATG present in exon-II. The predicted translation product lacks the signal peptide and the first 15 amino acids of the prosegment, suggesting the formation of an intracellularly active renin. In support of our finding, alternative transcripts have also been identified for renin in rat adrenal gland and brain, and biochemical studies suggest that this form of renin is active enzymatically.15,16⇓
The present study was undertaken to accomplish several specific goals: (1) to determine whether hREN mRNA is detectable in the brain of different lines of PAC140 and PAC160 mice, (2) to assess which transcriptional isoform of hREN is used, (3) to take advantage of the overexpression of hREN in a PAC160 line with a high number of transgene copies to determine which cells in the brain express renin endogenously, and (4) to examine whether increased activation of central AT1 receptors due to local Ang II overproduction in brain contributes to the mechanism of moderate hypertension in the P+/A+ transgenic mice.
Materials and Methods
Experimental Mice and Husbandry
PAC140, PAC160, and hAGT transgenic mice genes were generated and genotyped as previously described.12,17⇓ All mice (University of Iowa Transgenic Animal Facility) used for the experiments were 15 to 20 weeks of age and were fed standard mouse chow (LM-485; Teklad Premier Laboratory Diets) and water ad libitum. Care of the mice used in the experiments met the standards set forth by the National Institutes of Health in their guidelines for the care and use of experimental animals. All procedures were approved by the University Animal Care and Use Committee at the University of Iowa.
RNase Protection Assay and Reverse Transcriptase–Polymerase Chain Reaction
Tissues were harvested after CO2 asphyxiation, snap-frozen in liquid nitrogen, and RNA was purified with TriReagent (Molecular Research Center Inc). Total RNA (50 μg) was hybridized to antisense probes for hREN and mouse β-actin labeled with [α-32P]UTP by in vitro transcription as described.18 RNase protection assays were performed using the RPA III kit (Ambion Inc). Protected fragments were 300 (hREN) and 245 (β-actin) bp, respectively. For reverse transcriptase–polymerase chain reaction (RT-PCR), 20 μg of RNA was treated with DNase and subjected to RT-PCR as described.19 Ten percent of the RT reaction was used for PCR amplification. Upstream primers for the PCR analysis of exon 1a and exon 1b were ATGGATGGATGGAGAAGG and ACCACACAACAGCAAG, respectively. The gene-specific primer (GSP, 5′-GGTCTGGG-GTGGGGTGCCG-3′) hybridizes to a region of hREN exon-III and was used as the downstream primer in both experiments. The expected size of the amplification products for exon 1a and exon 1b were 307 bp and 241 bp, respectively.
Mice were killed and perfused transcardially with 20 mL phosphate-buffered saline (PBS) followed by 50 mL 4% formaldehyde in PBS. The brain was removed, postfixed at 4°C overnight, and placed in 30% sucrose solution at 4°C. The following day, the brain was frozen and cut coronally (30 μm) using a Reichert-Jung Cryostat. Brain sections were permeabilized by rinsing with 0.1% Triton X-100 in PBS at 25°C and incubated at 4°C 16 to 18 hours with a rabbit polyclonal antibody against hREN (1:2000 dilution, kindly provided by Professor Pierre Corvol, INSERM U36, College de France) that had been preadsorbed with nontransgenic brain sections at 4°C for 48 hours. Sections were also incubated with mouse monoclonal antisera (Chemicon International Inc, Temecula, Calif) against either GFAP (glial marker, 1:1000 dilution) or neuronal nuclei (NeuN, neuronal marker, 1:500 dilution). Samples were incubated with biotinylated anti-rabbit IgG (1:250 dilution), avidin D (1:50), biotinylated anti-avidin (1:125), fluorescein avidin D (1:250, all from Vector Laboratories Inc), and rhodamine-conjugated anti-mouse IgG (1:100, Chemicon International Inc); each incubation was performed at 25°C for 1 hour followed by rinsing with 0.1% Triton X-100 in PBS except the last rinse using PBS.
Mice were implanted with catheters in the carotid artery and jugular vein for direct measurement of arterial pressure (AP) and heart rate (HR) and for administration of drugs, respectively, and with ICV cannulae for microinjection of drugs into the lateral ventricles as described in detail previously.6 All experiments were performed in conscious, unrestrained mice in their home cages after at least 48 hours recovery time. After connection of the arterial line to a Transpac pressure transducer (Abbott Laboratory), mice were allowed at least a 30-minute adaptation period before testing. Baseline AP and HR were measured continuously for 1 hour/day for 3 consecutive days. ICV or intravenous (IV) infusion studies were performed following at least a 30-minute stabilization period after insertion of the microinjector into the ventricular guide cannula or connection of the venous line to a syringe. AP and HR were monitored for 1 hour after infusion of drugs, and each infusion study was performed on different days. Cardiovascular responses to ICV Ang II were recorded on the 1st day of measurement to confirm correct placement of the ICV injector. Four mice (2 P+/A+ and 2 controls) did not respond to ICV Ang II and were not used for further studies as it suggested incorrect cannula placement.
The effect of ICV injection of losartan (10 μg, generous gift of Merck Research Laboratories, Rahway, NJ) and artificial cerebrospinal fluid (ACSF, in mmol/L: NaCl 136, KCl 5.6, NaHCO3 16.2, NaH2PO3 1.2, MgCl2 1.2, and CaCl2 2.2, pH 7.4) on basal AP and HR was examined. IV injection of losartan (10 μg), hexamethonium (HEX, 5 mg/kg), and of an antagonist of peripheral vasopressin V1 receptors (AVPX, Manning Compound [d(CH2)5Tyr2(ME)Arg8]-vasopressin, 10 μg/kg) on baseline AP and HR were determined. Isotonic saline was injected as control. ICV and IV infusions were done over a 30-second period with compounds dissolved in ACSF and saline and delivered in a volume of 0.5 and 10 μL, respectively. All hemodynamic data were collected and analyzed on a computer using Chart version 4.0.1.
Plasma AVP Assay
Mice were anesthetized with pentobarbital (50 mg/kg, IP), decapitated immediately, and trunk blood was collected in chilled tubes containing EDTA. Plasma was separated and stored at −80°C until processed. Plasma proteins were extracted with acetone and petroleum ether as described previously.6 Levels of AVP in plasma extracts were determined by using 125I-AVP RIA kit according to the manufacturer’s instructions (Peninsula Laboratories Inc).
Data were expressed as mean±SEM. Group comparisons were made with unpaired t tests and ANOVA with repeated measures. A value of P<0.05 was considered statistically significant.
We previously reported that PAC140 and PAC160 mice exhibit strong but tightly regulated hREN expression in the kidney.12 Low-level hREN expression in brain was observed in two lines (6919/1 and 7217/2) that had the highest number of transgene copies. In the present study, using 50 μg of total brain RNA, hREN mRNA was reproducibly detected in the brain of all 4 lines examined for expression including 2 lines that contain a low number of transgene copies where hREN mRNA was previously undetected (Figure 1). Equivalent hREN expression was observed in brains of the PAC160 line 6407/1 as either a single transgenic or when made double transgenic (P+/A+). The detection of hREN mRNA in the brain of PAC140 and PAC160 mice that otherwise exhibit tightly restricted and regulated expression strongly suggests this is not an artifact of transgenesis but likely represents appropriate expression in brain. This is further supported by the observation that, like kidney, expression of hREN mRNA in brain is proportional to copy number (Figure 1).
We next determined the isoform of hREN mRNA expressed in the brain in these 4 lines of PAC140 and PAC160 transgenic mice. RT-PCR was performed by using primer sets designed to amplify exon 1a and 1b, respectively. As anticipated, an exon 1a–specific band, but no exon 1b–specific band, was amplified from kidney RNA of a PAC160 transgenic mouse (Figures 2A and 2B). Brain RNAs from all 4 PAC lines exhibited both the exon 1a– and 1b–specific products. When combined into a competitive PCR reaction, it is clear that hREN mRNA in kidney primarily contains exon 1a, whereas hREN mRNA in brain prefers exon 1b over exon 1a (Figure 2C). hREN mRNA in the brain of R+ mice does not contain exon 1b because the location of the transcription start site for this exon is further upstream than that contained in that transgene.14
The copy number proportional and high-level expression of hREN in the brain of one line (7217/2) of PAC160 mice provided a novel opportunity to examine the cell-specific expression of renin in the brain. Incubation of sections from nontransgenic animals with anti-hREN antisera resulted in little or no staining in the brain. Some very light immunostaining of glial-like elements that persisted even after preincubation of the antibody with purified hREN was consistently seen (Figures 3E, 3J, and 3O). However, this immunostaining was generally distributed throughout the brain, and except for the cerebellum where this staining appeared restricted to glial-like elements in the Purkinje cell layer, showed no regional specificity. Similar light background staining was seen in sections from transgenic animals but was virtually eliminated by preincubating the anti-hREN antisera with brain sections from nontransgenic animals.
In PAC160 mice, anti-hREN immunoreactivity showed a fairly restricted distribution and was associated with both glia and neurons (Table). In the brain stem, anti-hREN immunoreactivity was found only in neurons in the dorsal cochlear nucleus, mostly in large diameter cells in its deeper layers but also in some cells in the granular layer (Figure 3A). Double-immunostaining with either NeuN or GFAP showed colocalization of hREN staining with NeuN staining (Figures 3A through 3D). Very little staining was seen elsewhere in the brain stem except the hypothalamus, where many hREN-immunostained processes, also immunoreactive for GFAP, were observed in the medial nuclear group (Figures 3F through 3I). In the forebrain, immunostained glial-like elements were seen in the cortex, particularly the piriform cortex and possibly in the hippocampus. In the hippocampus, however, small numbers of hREN immunoreactive neurons were consistently seen (Figures 3K through 3N). These were large diameter neurons located mostly in the strata oriens and radiatum and only rarely in the stratum pyramidale. All these cells were also NeuN-immunoreactive.
AGT mRNA is expressed in astrocytes and some neurons in the brain.5,20⇓ Therefore, detection of hREN mRNA expression in the brain of PAC140 and PAC160 mice prompted us to hypothesize that increased production of Ang II and its interaction with AT1 receptors in the brain may be important in the maintenance of elevated arterial pressure in P+/A+ double transgenic mice. To exclude artificial effects due to gross overexpression of renin in the brain, we chose to examine the contribution of the brain RAS in double transgenic mice using the PAC160 6407/1 line, which contains a low transgene copy number and low expression of hREN in brain (Figure 1). Baseline mean arterial pressure (MAP) was significantly higher in P+/A+ (138±4 mm Hg, n=5, P<0.01) compared with nontransgenic mice (116±3 mm Hg, n=6). Baseline HR was not significantly different between groups (604±17 versus 624±17 bpm).
AT1 is the predominant Ang II receptor subtype in cardiovascular control regions in the brain.21,22⇓ To determine whether stimulation of brain AT1 receptors contributes to the BP elevation in P+/A+ mice, effects of ICV injection of selective AT1 receptor antagonist, losartan, was examined. ICV injection of losartan (10 μg) decreased MAP by approximately 20 mm Hg in P+/A+ double transgenic mice, reducing MAP in these animals nearly to the baseline of nontransgenic mice (Figures 4A and 4B). ICV losartan caused a slight, insignificant increase in HR in P+/A+ double transgenic mice. In contrast, IV administration of this dose of losartan had no effect on MAP or HR in P+/A+ mice (data not shown). In control mice, neither ICV nor IV injection of losartan (10 μg) significantly altered MAP or HR. As a result, the reduction in MAP and percent change in MAP was significantly greater in P+/A+ than in control mice (Figures 4B and 4C). These results suggest that there is an exaggerated contribution of central AT1 receptor activity to the maintenance of the elevated MAP in the P+/A+ double transgenic mice.
Because of the possible contribution of increased brain RAS to the elevated BP in these double transgenic mice, we wanted to investigate the mechanism of chronic BP elevation downstream of the central nervous system. Because the major mechanisms of pressor response to ICV Ang II are via activation of sympathetic nervous system and/or vasopressin secretion,23 we examined the cardiovascular effects of IV treatment with a ganglionic blocking agent, hexamethonium, and a vasopressin V1 receptor antagonist, AVPX. IV injection of hexamethonium significantly reduced MAP in both the P+/A+ and control groups (Figure 5). The actual decrease and percent change in MAP was approximately 60 mm Hg and −50%, respectively, and these prominent depressor responses were equivalent in the P+/A+ and control groups. Although the reduction was modest when compared with that of hexamethonium, IV infusion of AVPX caused a significantly greater decrease in MAP in P+/A+ mice than in control mice (Figure 6). There was a trend toward increased circulating AVP in P+/A+ mice (65.8±21.9 pg/mL, n=7) over control mice (45.7±5.0 pg/mL, n=9), although this difference did not reach statistical significance (P=0.331).
In the present study, hREN mRNA expression was detected in the brain of 4 independent lines of transgenic mice encoding the hREN gene on a large PAC clone. These clones consist of the entire coding region of the gene and extend far upstream and downstream and are therefore very likely to contain all sequence requirements for appropriate expression and regulation of the hREN gene. Indeed, we previously reported that expression of hREN from these large transgenes is highly restricted to kidney, expressed exclusively in renal juxtaglomerular cells, tightly regulated in response to physiological cues that upregulate and downregulate the gene, and exhibit copy number proportional expression.12 These data all strongly suggest that the PAC mice are excellent models from which to examine the regulation and localization of hREN expression in brain.
Immunoreactive REN has been previously reported in the hypothalamus and cerebellar cortex in the mouse and rat brain and in neurons in all areas of the human brain.24,25⇓ Despite these observations and the clear documentation of the expression of the other RAS genes in the brain, the notion that REN is endogenously expressed in the brain remains very controversial. Most likely, this is because the level of expression of REN in the brain is well below the threshold detectable by standard assays. The copy number proportional expression of hREN detected in the kidney and brain of our PAC mice provided a unique opportunity to examine renin expression in a model where (1) the correct sites of expression are likely retained, and (2) the level of expression is amplified. hREN immunoreactivity was detected in neurons only in the dorsal cochlear nucleus and hippocampus. In the thalamus and hypothalamus, as well as in the forebrain and possibly in the cerebellum, hREN immunoreactivity appeared confined to glia. It is worth pointing out that even under the conditions of elevated expression, rigorous controls such as preadsorbing the hREN antisera with brain sections from nontransgenic mice were necessary to ensure the accuracy and fidelity of the results.
The physiological relevance of REN expression in neurons of the dorsal cochlear nucleus and hippocampus remains unclear as we are not aware of any specific role played by these regions in cardiovascular homeostasis. In previous studies examining brain-specific expression of hAGT in transgenic mice, hAGT was found in glia in some hippocampal layers as the hREN positive neurons seen here were.5 The hREN positive neurons in the dorsal cochlear nucleus were located in a region containing Purkinje cell-like neurons.26 Cerebellar Purkinje cells have been shown to contain Ang II, probably as a result of uptake from cerebellar astrocytes, which are well documented to contain endogenous AGT.27 Though these observations superficially suggest a potential association between the observed hREN expression and cells containing compounds of the brain RAS system, they do not lead to an association with cardiovascular homeostasis.
On the other hand, the expression of REN in the hypothalamus is of significant interest as there are several nuclei in this region controlling blood pressure, water, and electrolyte homeostasis. Similarly, AGT mRNA has been reported in glia and neurons in some regions of the brain containing AT1 receptors, suggesting the potential for local synthesis and action of Ang II.5,28,29⇓⇓ Indeed, AT1 receptors are widely distributed in hypothalamic nuclei, such as the paraventricular nucleus, and brain stem nuclei, such as the ventrolateral medulla and the nucleus of the solitary tract, in addition to cardiovascular control centers located at the blood-brain interface.21,22,30,31⇓⇓⇓
We and others have reported that in brain, REN mRNA can exist in two forms: the classical form encoding preprorenin and a second form using an alternative promoter resulting in a mRNA encoding a form of REN lacking the signal peptide and a third of the prosegment.14,16⇓ In vitro experiments suggest this form of the protein has REN activity.15 Both forms of hREN are detected in the brain in 4 independent lines of PAC transgenic mice. However, as we currently have no assays capable of distinguishing between these species of hREN mRNA or protein at the cellular level, it is difficult to determine which cells in brain express each form. Nevertheless, we are currently examining the relevance of this observation in a new model selectively expressing the nonsecreted form of hREN in the brain. Indeed, it is attractive to speculate on the existence of an intracellular pathway for the generation of Ang I. Intracellular generation of Ang II has been reported in juxtaglomerular and endothelial cells, but has been disputed in other cell types.32–34⇓⇓ We reported that AGT containing neurons with extensions from the parabrachial nucleus to the amygdala also contain immunoreactive Ang II.5 Of course, we recognize that this result can be explained by the extracellular generation of Ang II and resultant internalization of the Ang II/AT1 receptor complex.35
Central administration of losartan lowers blood pressure to baseline in P+/A+ demonstrating the importance of Ang II and central AT1 receptors in the maintenance of high blood pressure in the P+/A+ model. Pharmacological experiments suggest that this AT1-dependent increase in blood pressure may occur via the actions of arginine vasopressin, and the trend toward increased plasma AVP supports this notion. Indeed, it is well established that vasopressin secretion from the posterior pituitary is increased by the actions of Ang II, via an AT1-dependent mechanism, in both circumventricular organs and intrinsic brain sites.36–39⇓⇓⇓ Both the R+/A+ and P+/A+ models exhibit elevated plasma Ang II, although its level is significantly lower in the P+/A+ model. Therefore, it remains unclear if the actions of Ang II on central AT1 receptors is due to the local or systemic production of Ang II. Although the localization of RAS components both systemically and within the brain would support both possibilities, we cannot presently distinguish between them. Microinjection and lesion studies are currently planned to address this important issue. Nevertheless, transgenic experiments by us and others involving brain-specific overproduction of either AGT or AGT antisense clearly demonstrate that AGT produced in the brain can be cleaved locally to Ang II, and that the resultant brain Ang II has an important contribution to cardiovascular control.18,40⇓
This work was funded by grants from the National Institutes of Health (HL58048, HL61446, and HL55006). C.D. Sigmund was an Established Investigator of the American Heart Association. S. Morimoto is funded by a Postdoctoral Fellowship from Uehara Memorial Foundation in Japan. Transgenic mice were generated and maintained at the University of Iowa Transgenic Animal Facility, supported in part by the College of Medicine and the Diabetes and Endocrinology Research Center. We would like to thank Norma Sinclair, Lucy Robbins, Patricia Lovell, Brandon Campbell, Debbie Davis, and Xiaoji Zhang for their excellent technical assistance.
Original received July 31, 2001; revision received October 23, 2001; accepted November 6, 2001.
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