(Hypertension. 1995;26:272-278.)
© 1995 American Heart Association, Inc.
Articles |
Effects of Human Renin in the Vasculature of Rats Transgenic for Human Angiotensinogen
From the Franz Volhard Clinic, Rudolph Virchow University Hospitals, and the Max Delbrück Center for Molecular Medicine, Humboldt University, Berlin, Germany; University of Erlangen-Nürnberg (Germany); and Hoffmann–La Roche, Basel, Switzerland.
Correspondence to Friedrich C. Luft, MD, Franz Volhard Clinic, Wiltberg Strasse 50, 13122 Berlin, FRG.
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Abstract Transgenic rats, which express the human angiotensinogen gene, provide a unique model for studying local vascular effects of human renin. We examined the cleavage of human angiotensinogen to angiotensin I (Ang I) by human renin and its inhibition by a human renin inhibitor in an isolated perfused hindlimb preparation from such rats. Perfusion resulted in the sustained release of human angiotensinogen, which decreased from 19.4 to 11.8 pmol/mL over 45 minutes. Active human renin at doses of 3, 10, and 30 ng/mL perfusate for 15 minutes increased Ang I release from undetectable levels (mean±SEM) to 31.9±3.3, 147.1±26.2, and 206.4±17.1 fmol/mL, respectively, by 9 minutes. In separate experiments aimed at the quantification of renin-induced vasoconstriction, captopril decreased the perfusion pressure and lowered Ang II concentrations to nondetectable levels, whereas Ang I values increased sharply. When renin (30 ng/mL) was infused for 15 minutes, renin values in the perfusate decreased to barely detectable levels within minutes after termination of the infusion. However, Ang I values remained high for at least 30 minutes thereafter. The addition of a human renin inhibitor during renin infusion caused Ang I values to promptly decrease within minutes to undetectable levels. Hindlimbs from nontransgenic control rats released no detectable amounts of Ang I, with or without human renin. Finally, by in situ hybridization we documented the presence of human angiotensinogen message in the vessels of the hindlimb. We conclude that renin acts on angiotensinogen at a site in the vascular wall. The cleavage depends on renin and not on other lysosomal proteases. Transgenic rats are a novel model that may be used to test the functional importance of the local human renin-angiotensin system in experimental animals.
Key Words: rats, transgenic • angiotensinogen • angiotensin • renin • hindlimb • renin-angiotensin system
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Angiotensin II (Ang II) plays a major role in the regulation of blood pressure and fluid and electrolyte homeostasis. The tissues are a major site of Ang I and II formation, and the release of Ang I and II after local generation contributes to the circulating levels of these peptides.1 2 3 4 5 6 Local formation may result from an interaction between renin and angiotensinogen produced within the vessel wall or from the uptake and retention of renin and angiotensinogen from the plasma, on the cell surface, or in the interstitial fluid.5 6 7 8 9 10 The mRNA for both renin and angiotensinogen has been detected in various tissues.11 12 13 14 15 16 Much evidence for the functional importance of vascular renin derives from studies describing angiotensin release from the isolated perfused hindlimb vasculature. This evidence suggests that plasma renin of renal origin is the major source of functional renin in the vessels and determines the regulation of vascular angiotensin release.2 3 6 Since human renin does not cleave rodent angiotensinogen and rodent renin has no effect on human angiotensinogen, rats and mice transgenic for various components of the human renin-angiotensin system (RAS) are novel animal models for the study of the human RAS.17 18 19 In the present study we demonstrate that skeletal muscle resistance vessels of rats transgenic for human angiotensinogen synthesize and contain human angiotensinogen. We used an isolated perfused resistance vessel preparation from these rats with high gene expression19 to test the hypothesis that human renin can be taken up by the peripheral tissue to induce local angiotensin formation. The transgenic rat hindquarter as a pharmacological model allowed us to examine the interaction of human components of the RAS and of human-specific renin inhibitors in the vascular tissue in a nonprimate.
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Animals
Male Sprague-Dawley rats heterozygous for the complete human genomic angiotensinogen gene [TGR(hAOGEN) 1623] and Sprague-Dawley control rats (Ivanovas, Kislegg, FRG) weighing 250 to 400 g were used for all experiments. The transgenic line was developed as outlined elsewhere.19 The rats were kept in rooms at 24±2°C and fed a standard rat diet (No. C-1000, Altromin) containing 0.2% sodium by weight and were allowed free access to tap water. All procedures were done according to guidelines from the American Physiological Society and were approved by local authorities (AZ IV A4/5-G 0399/92).
Hindquarter Perfusion
Hindquarter preparation and perfusion were performed with rats
under thiopental anesthesia (75 mg/kg IP) as previously
described.4 5 Briefly, rats underwent median laparotomy.
After evisceration the abdominal aorta and inferior vena
cava were cannulated, and the perfusion was started immediately. The
hindquarters were perfused in a nonrecirculating system with a modified
Tyrode's solution containing 2 g/L glucose and 20 g/L of the
artificial colloid Ficoll 70 (Pharmacia). A 4% Ficoll solution was
used for the experiments designed to measure the effects on perfusion
pressure. The perfusate was gassed with a mixture of
O2/CO2 (95%/5%), adjusted to pH 7.4,
and maintained at 37°C. The hindquarter perfusion was performed at a
constant flow rate of 10 mL/min with a peristaltic pump (Abimed
Gilson), and the perfusion pressure was monitored by a pressure
transducer connected to an on-line computer system (TSE). Protocols
were started after 30 minutes of equilibration perfusion. All
experimental substances, including renin, were infused into the
perfusion system at a rate of 100 µL/min by means of a syringe pump
(HT Infusors).
Experimental Protocols
After an initial 30-minute period of baseline perfusion,
perfusate for measurement of peptides was collected after 3, 6, 9, 12,
15, 20, 25, 30, 35, 40, and 45 minutes. All perfusate samples were
collected over 9 seconds in the presence of a human-specific renin
inhibitor (remikiren, Hoffmann–La Roche)
(10-5 mol/L) to prevent any angiotensin
formation outside the hindlimb. Preliminary experiments (data not
shown) demonstrated that this remikiren concentration completely blocks
renin activity during sample collection and handling. Renin
measurements were performed without remikiren. Purified human
recombinant renin (Dr S. Mathews, Hoffmann–La Roche) was infused for
15 minutes. The samples were immediately frozen on dry ice and stored
at -80°C until assayed. One sample was obtained after the washout
period of each experiment to exclude contamination of the perfusion
system with human renin and angiotensin peptides.
Angiotensinogen Release
We conducted this protocol to examine the release of human
angiotensinogen from the hindlimb preparation. The effluent
perfusate was collected according to the same protocol for 45 minutes
from transgenic rats (n=5) without infusion of renin or other
substances.
Ang I Release
We performed this protocol to determine the dose-response
relationship between renin infusions and Ang I generation from
angiotensinogen in the hindlimb preparation. Human
recombinant renin (1, 3, 10, and 30 ng/mL) was infused for 9 minutes in
separate experiments and the perfusate collected over 9 seconds every 3
minutes in three to four hindlimb preparations for each dose. At the 10
and 30 ng/mL doses, the renin infusions were stopped after 15 minutes;
collections were continued to 45 minutes and in a separate experiment
for up to 105 minutes. The purpose of these longer observations was to
observe the renin and Ang I values over time after the renin infusion
was discontinued. The perfusate contained 10-5 mol/L
captopril to inhibit any conversion of Ang I to Ang II.
We also observed the effect of renin inhibition during renin infusion. In four separate experiments human renin (10 ng/mL) was infused for 15 minutes. Remikiren (10-5 mol/L) was infused after 10 minutes as well, and collections for Ang I were obtained every minute for 5 minutes.
Ang II Release
The 
-adrenoreceptor agonist
methoxamine (2x10-5 mol/L) was added to the
perfusate to increase vascular smooth muscle tone and sensitivity to
the pressure effects of Ang II as described for our model
elsewhere.20 The perfusate contained no
angiotensin-converting enzyme inhibitor. The
preparations (n=5) were allowed to equilibrate for 35 minutes. Ang I
and II were measured after an infusion of human renin (10 ng/mL).
Captopril (10-5 mol/L) was infused additionally in the
30th minute, and the infusion was continued for 10 minutes.
Total Protein
Total protein was determined by measurement of the shift of the
adsorption with a Cobas Mira apparatus (Hoffmann–La Roche)
of a pyrogallol-red-molybdate–amino acid complex. We used a
modification of the method of Fujita et al21 using
Microprotein-PR reagent (Sigma Chemical Co).
Enzyme Kinetic Determinations and Radioimmunoassay
Immunoreactive Ang I and II concentrations in the perfusate were
determined by direct radioimmunoassay. The cross-reactivity of the
anti–Ang I antibody to Ang II was less than 0.01% and less than
0.01% to [Val5]Ang II, 100% to
[des-Asp1]Ang I, 0.03% to Ang I/II-(1-7), and 0.02% to
Ang III. Values for anti–Ang II antibody were 6% for Ang I and 70%
for [Val5]Ang II, 0.1% for [des-Asp1]Ang
I, less than 0.01% for Ang I/II-(1-7), and 70% for Ang III. Peptides
(Bachem) represent human species except for the artificial
peptide [Val5]Ang II. Both assays can detect 2 to 3 fmol
angiotensin in 100 µL perfusate. Mean intra-assay
variabilities were 9% for Ang I versus 11% for Ang II, and mean
interassay variabilities were 13% versus 15%, respectively. There was
no interference of remikiren or captopril in these immunoassays.
Human angiotensinogen concentrations in the perfusate were determined by an in vitro enzyme kinetic assay.22 Human angiotensinogen was completely exhausted by cleavage with an excess of human recombinant renin. Twenty-five microliters of perfusate was incubated together with 470 µL of 0.15 mol/L citrate phosphate buffer (pH 5.7) containing 0.02 mol/L Na2-EDTA and 1.0 pmol/mL human recombinant renin with 5 µL phenylmethylsulfonyl fluoride (50 g/L ethanol) for 1 hour at 37°C. Ang I was measured by direct radioimmunoassay; human angiotensinogen concentrations were expressed as picomoles per milliliter based on an equimolar production of Ang I from angiotensinogen.
Human renin concentration was determined by a similar enzyme kinetic assay.23 Plasma from 48 hours, bilaterally nephrectomized TGR(hAOGEN) 1623 was used as a source of excess renin substrate. This plasma contained 85 nmol/mL of human and 4.1 nmol/mL of rat angiotensinogen. Absence of any detectable rat plasma renin activity was checked. From each sample, 50 µL was incubated together with 100 µL of bilaterally nephrectomized transgenic rat plasma, 295 µL of 0.15 mol/L citrate phosphate buffer containing 0.02 mol/L Na2-EDTA, and 5 µL phenylmethylsulfonyl fluoride at pH 5.7 and 37°C for 1 hour. Rat angiotensinogen was not cleaved during incubation because of the absence of any detectable rat renin in the perfusate. Ang I generated during the enzyme kinetic assays was measured by direct radioimmunoassay; human renin concentration was expressed as picomoles Ang I per milliliter per hour.
Tissue Preparation
For in situ hybridization the skeletal muscle was dissected
after the perfusion procedure and immediately snap-frozen in
isopentane. Sections 12 µm in thickness were cut in a cryostat (Jung
Frigocut, Leitz) and mounted onto APES (Sigma)–coated slides.
In Situ Hybridization
A human angiotensinogen cDNA fragment subcloned into
pGEM5 was used and has been described in detail.19 The
cDNA is complementary to nucleotides 1219 through 1531 of
the human angiotensinogen gene24 and to
nucleotides 133 through 424 of the rat
angiotensinogen gene showing approximately 60% sequence
homology.25 In situ hybridization was described elsewhere
in detail.26 Briefly, the sections were brought to room
temperature, fixed with 4% buffered paraformaldehyde,
washed in phosphate-buffered saline, and deproteinized in 0.1 mol/L
HCl. After additional washing the sections were acetylated to
avoid unspecific binding. After dehydration in graded ethanols the
sections were prehybridized in a humidified chamber with 150 µL
prehybridization buffer (50% deionized formamide, 50 mmol/L Tris-HCl
[pH 7.6], 25 mmol/L EDTA [pH 8.0], 20 mmol/L NaCl, 0.25 mg/mL yeast
tRNA, and 2.5x Denhardt's solution [0.05% Ficoll, 0.05%
polyvinylpyrrolidone, 0.05% bovine serum albumin]) for 2 to 4
hours. After the prehybridization buffer was drained off the slides,
the sections were hybridized according to the standard procedure with
0.15 ng labeled sense and antisense RNA, respectively. The
hybridization was done at 37°C in a buffer containing 50% formamide,
1x Denhardt's solution, 10% dextran sulfate, 0.5 mg/mL yeast tRNA,
0.1 mg/mL polyadenylate, and 0.2 mol/L dithiothreitol for 16 to
18 hours. Thereafter, the slides were washed in 0.5x SSC/50%
formamide at 48°C for 2 hours and in 1x SSC at 48°C for 20 minutes
and subsequently treated with RNAse (10 µg/mL) for 30 minutes at
37°C and washed several times in 1x SSC, 0.5x SSC, and 0.2x SSC at
48°C; dehydrated in graded ethanols; dried; and coated with Kodak
NTB2 emulsion. The slides were exposed at 4°C for 6 weeks on
emulsion. Emulsion-coated slides were counterstained with
hematoxylin.
The specificity of the method was determined by hybridization with
35S--UTP–labeled sense RNA at the same specific
activity, length, and concentration as the antisense RNA.
Ribonuclease Protection Assay
Total RNA was isolated from a transgenic rat aorta and a
nontransgenic rat liver by the lithium chloride precipitation
technique.27 Identification of mRNA specific for human or
rat angiotensinogen was performed by a ribonuclease
protection assay with an Ambion RPA II kit (ITC Biotechnology GmbH)
according to the manufacturer's description. Radioactively labeled
species-specific antisense RNA probes were prepared by T7
RNA-polymerase transcription of human and rat
angiotensinogen cDNA fragments subcloned into pGEM5 and
pGEM4 vectors.19 28 Vectors were linearized for
transcription by BstEII and EcoRI restriction
enzymes, respectively. The human-specific transcript spanned 132
nucleotides of antisense RNA and 51 nucleotides
of vector-ended sequences, which were labeled with
[32P]UTP to a specific activity of 3.3x108
cpm/µg RNA. The rat-specific transcript comprised 290
nucleotides of antisense RNA and 60 nucleotides
of vector-encoded sequences and was labeled to a specific activity of
4.1x108 cpm/µg RNA. One microgram of total sample RNA
was hybridized together with 1.3x105 cpm of human and
2.1x105 cpm of rat antisense probe. Probe fragments
subsequently protected from combined RNAseA/T1 digestion were separated
by electrophoresis on a 5% denaturing polyacrylamide gel and
visualized with a FUJIX BAS 2000 Phospho-Imager system after 3 hours of
autoradiography.
Statistical Analysis
Data are expressed as mean±SEM. We used
nonparametric tests (Wilcoxon) to determine statistical
significance. A value of P<.05 was accepted as
significant.
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Results |
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The perfusion pressure of the isolated rat hindquarters ranged from 18 to 22 mm Hg. No Ang I or II could be measured in samples taken before the infusion of human renin. Addition of renin to the perfusate led to release of readily detectable amounts of Ang I from the perfused rat hindquarters (Fig 1). The effects of renin were clearly dose dependent (Fig 1). These experiments were performed in the presence of captopril; no change of perfusion pressure was detected. Renin caused no Ang I formation if infused in nontransgenic rat hindquarters or in perfusion channels without hindquarter preparation.
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The time course of renin and Ang I concentrations after cessation of renin infusion (Fig 2) provided evidence for uptake of renin to vascular tissue. Renin in venous perfusate decreased sharply to barely detectable levels within 5 to 10 minutes, but Ang I levels did not change for at least 30 minutes (Fig 2). This discrepancy between Ang I levels and perfusate renin concentrations was observed with two different doses of renin (Fig 2, top and bottom). The enzyme was clearly still active at the vascular wall despite its absence from the perfusate. The renin-induced Ang I formation was abolished within less than 5 minutes with the specific human renin inhibitor remikiren (Fig 3).
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We also tested whether human renin can induce local Ang II formation
and vasoconstriction. These experiments were performed in the absence
of captopril and the presence of an -adrenoceptor agonist.
Methoxamine increased perfusion pressure from 26.2±1.8 to
68.8±4.9 mm Hg over 25 minutes. Renin infusion further increased
perfusion pressure by 15 mm Hg, whereas the pressure did not change
significantly in saline controls (Fig 4). Release of Ang
I and of higher amounts of Ang II paralleled the pressure response.
Addition of captopril decreased Ang II and perfusion pressure, whereas
Ang I increased.
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indicates 10 ng/mL human
renin; 
, 0.9% NaCl solution. Bottom, Line graph shows the
effects of human renin for 15 minutes at 10 ng/mL on
angiotensin (ANG) I and II concentrations and the effect of
captopril from the experiment shown in the top panel. Renin increased
both angiotensin I and II. Captopril at 30 minutes
decreased angiotensin II to nondetectable levels, whereas
angiotensin I values increased markedly after captopril.
*P<.05.High amounts of human angiotensinogen were released from perfused rat hindquarters (Fig 5). The released angiotensinogen did not interfere with our measurements of renin activity or angiotensin release, because renin measurements were performed in the presence of excess exogenous angiotensinogen and because remikiren in sampling vials abolished any angiotensin formation in vitro. The release of angiotensinogen followed a time course similar to that of the washout of total protein (Fig 5).
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However, in situ hybridization demonstrated the expression of the transgene in skeletal muscle resistance vessels (Fig 6). Human angiotensinogen mRNA was detected in the smooth muscle cell layer. The limited optical resolution did not allow us to determine whether the mRNA was also present in the endothelium (Fig 6).
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These findings are consistent with data obtained by an RNAse protection assay that was performed in transgenic aortic tissue with human and rat angiotensinogen–specific antisense RNA probes (Fig 7). Species specificity of the probes was demonstrated by the absence of a human-specific protected fragment after hybridization with RNA from nontransgenic rat liver. In transgenic aortic tissue, however, high levels of transgene transcription were discovered in the absence of a detectable rat angiotensinogen mRNA.
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Discussion |
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Transgenic rats with components of the human RAS provide an opportunity for the study of the human RAS and human renin inhibitors in a rodent model readily suitable for physiological experimentation. We used transgenic rats with a high overexpression of human angiotensinogen19 to study the formation of angiotensin peptides at the level of the vascular wall. Our data show that human angiotensinogen is present at the vascular wall in these rats. Furthermore, human renin can be taken up from the circulation and remains active much longer than its presence in the circulation would explain. Even after its disappearance from the circulation, the enzyme continued to cleave human angiotensinogen locally in the tissues, resulting in the release of Ang I and II. The enzymatic activity of human renin was promptly inhibited by a specific human renin inhibitor.
The species specificity of the renin-angiotensinogen interaction does not allow the study of human angiotensinogen or renin in rats.19 Specific inhibitors of human renin are barely active against rat renin.29 Evidence for vascular angiotensin formation in humans has been reported, including gene expression of the RAS in the vascular tissue30 and indirect estimates of local angiotensin formation based on arteriovenous angiotensin gradients2 31 and on angiotensin release from fetal human tissue.32 However, many pertinent studies cannot be conducted in humans. Thus, the rat has been a favorite animal model4 6 7 9 11 12 13 33 34 35 36 37 (see Reference 3838 for review). Therefore, we used the isolated perfused hindquarter of rats transgenic for human angiotensinogen as a pharmacological model to study the uptake of human renin to the vascular tissue and its local inhibition by a specific inhibitor. Rats with a high level of angiotensinogen were used.19 Pharmacological concentrations of human renin were infused to facilitate the detection of local angiotensin formation from human angiotensinogen.
We cannot state for certain whether the released angiotensinogen was derived from local synthesis or by uptake from the bloodstream. Angiotensinogen is produced in the livers of these rats in large quantities and is released into the circulation.19 Previous studies have shown that uptake of circulating angiotensinogen contributes to subsequent release from isolated perfused tissue.39 The protein content of the perfusate in our rats decreased at a rate similar to that of the angiotensinogen concentration. Had substantial amounts of angiotensinogen been released locally, we would have speculated that the protein concentration of the perfusate would have decreased more rapidly than the angiotensinogen concentration. However, locally synthesized angiotensinogen nevertheless could play an important role as a substrate for renin within the vascular tissue even if it were not released.
We were able to detect human angiotensinogen mRNA in resistance vessels by in situ hybridization and in the aorta by RNAse protection assay. These data are consistent with previous reports of angiotensinogen expression in human30 and rat11 12 13 vascular tissue. The RNAse protection assay demonstrated that there was a higher expression of the transgene compared with endogenous rat angiotensinogen. The absence of rat angiotensinogen detection by our assay from aortic tissue does not indicate that rat angiotensinogen is not expressed.11 12 13 Instead, it merely indicates that rat angiotensinogen mRNA was not detected under conditions that readily allow detection of the transgene. There is some controversy about the precise cellular localization of vascular angiotensinogen expression in the rat. Campbell and Habener40 localized angiotensinogen expression to the perivascular adipocytes and fibroblast-like cells, whereas Naftilan et al13 found angiotensinogen expression in the smooth muscle cell layer in addition to perivascular tissue. We observe that the human angiotensinogen transgene was clearly expressed in the smooth muscle cell layer of arterioles. The resolution of our in situ hybridization was not sufficient to allow conclusions on angiotensinogen expression in endothelial cells or to allow precise cellular localization of the perivascular hybridization signals. However, our results are consistent with transgene expression in both perivascular tissue and the smooth muscle cell layer. Because of the limitations of our method and the controversy in previous reports,13 40 we cannot state whether the expression pattern of the transgene is different from that of the rat angiotensinogen gene.
The presence of human angiotensinogen in the vascular tissue of our transgenic rats makes these rats a highly suitable model for the study of renin uptake to the vascular wall, regardless of the origin of the renin substrate. Despite many years of investigation, the vascular formation of angiotensin peptides remains a debated issue.16 41 Other researchers have already addressed the question of whether renin is synthesized in the vascular tissue5 6 7 8 30 42 43 (see References 16 and 3816 38 for review). We did not design the present study to address the issue of local renin synthesis. Instead, we investigated whether human renin can be taken up from the circulation into the vascular wall to cleave Ang I locally from angiotensinogen. Evidence for the notion that plasma-derived renin may be retained in the vessel wall was first reported by Thurston and Swales (Loudon et al9 and Thurston et al10 ). Recent reports of renin binding to vascular tissue44 have greatly enhanced the interest in renin uptake as a potential mechanism of local angiotensin formation. Our present data clearly demonstrate uptake of human renin into vascular tissue and a local action, since the effects of renin were still present after cessation of renin infusion, when renin activity in the perfusate was almost undetectable.
Using a similar hindquarter preparation, we4 5 7 and others6 have previously provided indirect evidence for renin uptake in the vascular wall. However, heterologous (hog) renin, which was only partially purified, had to be used in these studies; the highly specific inhibitors designed for human renin could not be used. In contrast, the transgenic rat model allowed us to use highly purified human renin and a species-specific inhibitor, which provides more specific evidence for activity of human renin in the vascular tissue. The local formation of angiotensin peptides by the endogenous rat RAS4 6 did not interfere with our measurements, because the concentrations of angiotensin peptides released in the absence of human renin were lower than the detection limits of the assay.4 6 7 Infusion of human renin induced high concentrations of angiotensin in the perfusate. The released angiotensin peptides were formed in the tissue and not by an interaction of released angiotensinogen with infused renin in the perfusate, as evidenced by the continuing angiotensin release after the disappearance of renin from the perfusate.
Uptake of human renin into the vascular tissue induced Ang II formation and vasoconstriction, in agreement with previous results.4 Converting enzyme inhibition increased Ang I and decreased both Ang II release and vasoconstriction. However, the renin-induced pressure response was not completely abolished by captopril, despite the apparent cessation of Ang II release. Several factors may account for this apparent discrepancy. First, the angiotensin assay used here could have missed low concentrations of persistent Ang II formation.7 Second, Ang II formation may have continued at a site that is not accessible to captopril and does not equilibrate with the circulation. Finally, irreversibly increased resistance due to edema formation or other problems related to artificial perfusion medium4 may have interfered. However, both vasoconstriction and Ang II formation induced by renin were significantly reduced by captopril and thus were dependent on converting enzyme activity.
The highly specific inhibitor remikiren inhibited renin-induced vascular angiotensin formation. These data support the indirect evidence for an action of remikiren at tissue sites in primates45 46 and confirm our previous results with remikiren obtained from guinea pig hindquarter perfusion experiments.42 Campbell and Valentijn44 reported that renin inhibitors may interfere with the binding of the enzyme to vascular tissue. Although remikiren may inhibit not only the activity but also the uptake of renin, we have no data to support this notion. At present, we cannot determine whether renin is taken up by a specific mechanism, as suggested by two recent reports,44 47 or is merely "trapped" in the vascular tissue as a result of its physical and chemical properties.
Whatever mechanism may promote renin uptake into the vascular wall, the enzyme is retained in the tissue in a manner that allows it to cleave local angiotensinogen. The newly formed Ang I is effectively converted to Ang II by local angiotensin-converting enzyme, resulting in vasoconstriction. Thus, renin uptake may be an important mechanism for the enhancement of angiotensin-dependent vascular tone or growth, regardless of the mechanism of uptake. For example, even a nonregulated uptake of renin might maintain a normal level of vascular angiotensin generation in the face of decreased plasma renin. Consistent with this hypothesis, we observed unchanged vascular angiotensin generation in models of low-renin hypertension.48 The investigation of the mechanism of renin uptake and its potential regulation will require further research. Transgenic models similar to the one we used here may prove useful for addressing these issues.
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Acknowledgments |
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This study was supported by a grant-in-aid from the Bundesministerium für Forschung und Technologie (BMFT) and Hoffmann–La Roche and by a grant-in-aid of the Deutsche Forschungsgemeinschaft (DFG) to K.F. Hilgers (Hi 51/5-2). K.F. Hilgers is the recipient of a DFG research fellowship (Hi 510/5-1). This work completes in part the requirements for the PhD degree of Dominik Müller. We gratefully acknowledge the expert technical assistance of Irene Strauss. We thank Dr Joël Ménard, Paris, France, for reading the manuscript and for helpful suggestions.
Received February 15, 1995; first decision March 8, 1995; accepted May 2, 1995.
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