© 1999 American Heart Association, Inc.
Original Contributions |
In Vivo Enzymatic Assay Reveals Catalytic Activity of the Human Renin Precursor in Tissues
From the Laboratory of Molecular Biochemistry of Hypertension and Medical Research Canada Multidisciplinary Research Group on Hypertension (D.M., T.L.R.), Clinical Research Institute of Montreal, Montreal, and Centre de recherche en reproduction animal (D.W.S.), Faculty of Veterinary Medicine, University of Montreal, St-Hyacinthe, Quebec, Canada.
Correspondence to Timothy L. Reudelhuber, Laboratory of Molecular Biochemistry of Hypertension, Clinical Research Institute of Montreal, 110 Pine Ave West, Montreal, Quebec, H2W 1R7, Canada. E-mail reudelt{at}ircm.qc.ca
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Abstract |
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Abstract—The aspartyl protease renin is secreted into the circulation of mammals in 2 forms: the proteolytically processed active form of the enzyme and the precursor form, prorenin. Prorenin has no detectable enzymatic activity in the circulation, but it is the exclusive form of the enzyme produced by several tissues that also produce the other components of the renin enzymatic cascade (renin-angiotensin system). To test whether prorenin might be enzymatically active in these tissues, transgenic mice expressing the human renin substrate (angiotensinogen) exclusively in the pituitary gland were mated to mice expressing either active human renin or prorenin in the same tissue. Measurement of in vivo product formation in pituitary glands of double-transgenic mice revealed that human prorenin was enzymatically active, and Western blot analysis demonstrated that this prorenin was in the precursor form with its prosegment attached. This in vivo enzymatic assay demonstrates for the first time that human prorenin can be activated within tissues by nonproteolytic means, where it could contribute to the activity of a localized renin-angiotensin system.
Key Words: renin • prorenin • angiotensin
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Introduction |
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Many proteases are first synthesized as precursors containing a prosegment that serves not only as a folding catalyst or scaffold for the nascent protein, but also as an endogenous inhibitor to restrain the proteolytic activity of the protein until it has reached its target destination.1 Likewise, the aspartyl protease renin is synthesized as a proenzyme precursor (prorenin), and the renin present in the circulation is derived by the proteolytic removal of a 43-amino acid N-terminal prosegment exclusively in the kidney. Renin plays a key role in the regulation of blood pressure and in numerous cardiovascular pathologies, such as hypertension, cardiac hypertrophy, and renal defects,2 3 through the cleavage of angiotensinogen into angiotensin I (Ang I), which is the rate-limiting step in the renin-angiotensin system (RAS; Figure 1A
).
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Prorenin is also present in the circulation of mammals at levels that often largely exceed the concentration of renin.4 5 This prorenin is secreted not only from the kidney but also from numerous other tissues, including adrenal and pituitary glands, brain, eyes, ovaries, testes, uterus, and placenta. Moreover, these tissues express the other components of the RAS,6 7 8 raising the possibility that local RASs may exist that display activity only within tissues. However, these nonrenal tissues do not secrete active renin, because removal of the kidneys causes active renin to disappear from the circulation, whereas prorenin remains detectable even years later.9 10 11 Activation of prorenin does not occur once it is secreted into the circulation, given that injection of monkeys with large amounts of recombinant human prorenin does not lead to any significant increase in circulating renin activity, blood pressure, or plasma angiotensin II (Ang II).12 In contrast, transgenic rats engineered to secrete prorenin from the liver show tissue pathologies (renal and cardiac) in the absence of increases in circulating active renin.13 Taken together, these results raise the possibility that prorenin is activated locally within tissues and contributes to tissue RAS activity and cardiovascular pathologies.
We have developed a double-transgenic mouse approach to test for the
enzymatic activity of human prorenin (Figure 1B
). In this
system, the renin substrate (human angiotensinogen) and its
potential protease (either human active renin, human native prorenin,
or human noncleavable prorenin) are expressed in a specific cell type
(pituitary somatotrophs) of individual lines of mice. Selected lines of
single-transgenic mice were mated, and pituitary glands were excised
from double-transgenic offspring expressing both human
angiotensinogen and 1 of the human prorenins. Tissue
content of Ang I (the product of the reaction) was determined as an
indicator of renin enzymatic activity.
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Materials and Methods |
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Plasmid Constructions and Generation of Transgenic Mice
Human prorenin14 and human angiotensinogen15 cDNAs were expressed under the transcriptional control of a 320-bp fragment of rat growth hormone promoter.16 Intron and polyadenylation signals for the transcribed RNA are provided by a portion of the simian virus 40 large T antigen gene (Figure 2
).
Engineering of human active renin was achieved by modification of the
native prorenin cleavage site (PMKRL) to RMKRS,
so that it is cut by furin, a ubiquitous Golgi-anchored convertase,
resulting in the constitutive secretion of active renin from the
cell.17 In the human noncleavable prorenin, the Lys in
position 42 of the prosegment (PMKRL) was mutated to Ala,
which prevents cleavage by known proteases.14 The 4
transgenes were independently coinjected with the tyrosinase minigene
into the pronuclei of single-cell embryos from the FVB/N albino mouse
line, as previously described.18 Double-transgenic mice
were generated by mating single-transgenic mice expressing 1 of the
human prorenins (active renin, native prorenin, or noncleavable
prorenin) with single-transgenic mice expressing human
angiotensinogen. All animal manipulations were carried out
in accordance with institutional guidelines and were approved by a
local ethics committee.
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Expression of Transgenes
RNA was isolated from whole tissues of male mice by the acid
guanidinium thiocyanate-phenol chloroform method.19 To
prepare labeled RNA probes, human prorenin from nucleotides
401 to 650 of the cDNA and human angiotensinogen from
nucleotides 178 to 377 of the cDNA were subcloned in the
Bluescript II KS+ plasmid (Stratagene). RNase protection assays were
carried out by using the Promega Riboprobe Gemini System
(Promega Corp) according to the manufacturer's protocol. To compare
prorenin expression levels in the different transgenic lines,
unquantified total RNA from 2 pooled pituitary glands was hybridized
with 5x103 cpm 32P-labeled
RNA probe for human prorenin and 5x103 cpm
32P-labeled RNA probe for human
angiotensinogen overnight at 45°C in hybridization buffer
containing 80% formamide. Yeast RNA (5 µg) and RNA from
nontransgenic mouse pituitary glands were treated similarly to negative
controls, and RNA products from in vitro transcription of human
prorenin and human angiotensinogen were hybridized as
positive controls. To test for tissue specificity of transgene
expression, 5 µg of total RNA from brain, liver, kidney, and heart
were also hybridized under the same conditions. Protected fragments
after RNase A and T1 digestion were fractionated on a 6%
polyacrylamide-7 mol/L urea denaturing gel and were then
exposed to x-ray film for 5 days at -80°C using intensifying
screens. The protected fragments of prorenin and
angiotensinogen were 250 and 200 nucleotides in
length, respectively. Relative expression of prorenin in the various
double-transgenic lines was estimated by comparing the expression of
human prorenin with that of human angiotensinogen (all
animals were bred against the same human
angiotensinogen-producing line). Animals were classified as
expressing more prorenin than angiotensinogen (+++),
equivalent prorenin and angiotensinogen (++), or less
prorenin than angiotensinogen (+/–).
Determination of Pituitary Ang I Content
Pituitary glands surgically excised from 5 male mice were pooled
and immediately sonicated in cold 1% trifluoroacetic acid (TFA)
solution (pH 1.5) and cleared by centrifugation, and
their peptide content was separated by HPLC. The HPLC separation was
performed on a C18 column (Waters Corp). Solvent
A consisted of 0.1% TFA in water; solvent B, 0.1% TFA in
acetonitrile. Flow rate was 1 mL per 47 minutes, and 40 fractions were
collected using a gradient of 20% to 40% solvent B over 40 minutes.
Pituitary Ang I content of the eluted fractions was measured by an Ang
I RIA (DuPont NEN) and was detected in the identical fraction as a
commercial Ang I standard. The acid extraction used was designed to
avoid in vitro activation of prorenin, activity of released proteases,
and degradation of angiotensin peptides. Ang I recovery
using this method was determined to be 
70%. Statistical
analysis was performed by 1-way ANOVA with Dunnett
posttest.
Western Blot Analysis of Human Prorenin
Pituitary glands from 2 male transgenic mice were surgically
excised and lysed in 450 µL of a buffer containing (in mmol/L)
Tris (pH 8.0) 10, NaCl 10, and EDTA 1 and 0.1% SDS. The lysates were
passed repeatedly through a 25-gauge needle and were immunoprecipitated
with a rabbit polyclonal antibody to prorenin and renin.
Immunoprecipitated proteins were fractionated by SDS–10% PAGE, and
human prorenin and renin were detected by Western blot with a mouse
monoclonal antibody specific for human prorenin and renin (a gift from
D. Lamarre, Bio-Mega, Quebec, Canada) using a chemiluminescence kit
(SuperSignal, Pierce).
Renin Activity in Transfected Cells
Rat pituitary GH4C1 cells were grown in
DMEM supplemented with 5% (vol/vol) FCS, 0.1% serXtend (Irvine
Scientific), and 10 µg/mL gentamicin (Life Technologies, Inc) in a
humidified incubator (5% CO2, 95% air) at
37°C. GH4C1 cells, plated at
1x106 cells per 35-mm dish, were transfected 20
hours after plating with lipofectin (Life Technologies, Inc) in
serum-free medium using 18 µg of human angiotensinogen
plasmid DNA alone or in combination with either 2 µg of human active
renin plasmid DNA or 2 µg of human native prorenin plasmid DNA per
dish. After 20 hours, cells were transferred to 12-well plates in
medium with serum. Twenty-four hours later the supernatants were
collected and boiled for 2 minutes, and the cells were lysed in a
solution of 0.5% Triton X-100 in Tris-buffered saline and
boiled. Ang I content was measured by an Ang I RIA (DuPont NEN).
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Results |
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Transgene expression was targeted specifically to the somatotrophs of the pituitary gland by putting the human renin and angiotensinogen cDNAs under the transcriptional control of the rat growth hormone gene promoter. Human prorenin and human angiotensinogen mRNA were monitored in various tissues (brain, heart, kidney, liver, and pituitary gland) and were detectable only within the pituitary gland (Figure 3
), confirming the anterior pituitary
gland–specific activity of the rat growth hormone
promoter.16 20 21 We have studied 8 independent lines of
animals expressing the 3 different types of human prorenin transgenes.
The relative levels of transgene expression in each line were compared
by quantifying mRNA transcripts in the pituitary glands of transgenic
mice (Figure 4). Because all of these
lines were crossed with the same human
angiotensinogen-expressing line, the
angiotensinogen mRNA signal was used as an internal
reference to compare the level of prorenin mRNA expression in the
various lines. Because of the species-specific reactivity between renin
and angiotensinogen,22 23 it is possible to
express the 2 human proteins in the mouse without producing
compounding effects from the endogenous mouse RAS
components. Measurement of pituitary Ang I in this system (Figure 1B) is a direct indicator of renin activity within the same
tissue, given that Ang I released into the circulation would be rapidly
diluted and converted to Ang II by the
angiotensin-converting enzyme present in vascular
endothelium (Figure 1A). Human prorenin and
human renin were undetectable in the plasma of transgenic lines (data
not shown).
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To test for the Ang I–generating activity of human active renin in
this system, we engineered a mutation in human prorenin (Figure 2) that renders the prosegment sensitive to removal by a
ubiquitous protease (furin) present in the secretory
pathway.24 25 26 The advantage of this approach for
generating active renin is the conservation of the prosegment during
the early stages of protein biosynthesis, when it is required for
efficient protein folding,14 while allowing the removal of
the prosegment before secretion in a broad variety of cell
types17 (also D. Methot et al, unpublished results,
1998). Mice expressing both human active renin and human
angiotensinogen have marked increases in pituitary Ang I
content (Figure 5, hatched bars) as
compared with single-transgenic mice expressing only human
angiotensinogen (Figure 5, open bar). Pituitary Ang
I was not detectable in any of the transgenic mice expressing only the
various human prorenins (not crossed with
angiotensinogen-expressing mice) or in nontransgenic
controls. Repeated assays within the same transgenic lines yielded
highly reproducible results and demonstrate that transgenic mice can be
used as a sensitive measure for tissue-specific activity of human
renin.
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We then tested whether prorenin itself could generate Ang I under these
conditions by crossing the same line of
angiotensinogen-expressing mice with lines of mice
expressing human native prorenin. Interestingly, these
double-transgenic mice also have clearly elevated pituitary Ang I
content (Figure 5, solid bars), which suggests that human
prorenin is enzymatically active against its substrate within the mouse
pituitary gland. The relative amounts of Ang I generated in the
pituitary glands of the various crosses correlated well with the levels
of expression of prorenin mRNA in those lines (as
represented by +/– signs above the bars in Figure 5). To test whether prorenin had been rendered active in the
pituitary gland by the proteolytic removal of its prosegment, mice
expressing human angiotensinogen were crossed with a line
expressing human prorenin in which the native prosegment cleavage site
(Lys42, Arg43) was mutated (Ala42, Arg43) so as to be noncleavable
(Figure 2). The finding that Ang I content is also elevated in
the pituitary gland of these mice suggests that prorenin exhibits
enzymatic activity with the prosegment still in place (Figure 5, stippled bar).
To confirm the form of the enzymatically active prorenin present in
these pituitary glands, we performed Western blot analysis of
pituitary homogenates (Figure 6). Analysis of pituitary glands
from a line expressing human active renin (line 33) reveals 2 bands
corresponding in size to prorenin and proteolytically activated
renin. This result is expected, as furin cleavage of the modified
prorenin is seldom complete (T. Reudelhuber, unpublished observation,
1998). Analysis of pituitary glands from mice expressing
human native prorenin (line 50) reveals a single band that migrates
with the expected size of full-length prorenin. No band corresponding
to proteolytically activated renin could be detected in the
pituitary glands of native prorenin-expressing mice even at much longer
exposures. Thus, the catalytic activity seen with human native prorenin
is not due to proteolytic cleavage of the prosegment within the
pituitary gland.
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One possible explanation for the ability of prorenin to cleave its
substrate in the pituitary glands of double-transgenic mice is that the
2 proteins meet in the secretory pathway of expressing cells before the
prorenin has completed its folding to the repressed state. To test
whether Ang I generation could occur intracellularly, cultured
GH4C1 (rat somatotroph) cells were cotransfected
with expression vectors for human angiotensinogen either
alone or with one of the human prorenins. The results
(Table) show that coexpression of human
angiotensinogen and human active renin leads to the
detection of Ang I in the supernatant of transfected cells, but not in
cell lysates. In contrast, cotransfection of human
angiotensinogen and human native prorenin does not lead to
generation of Ang I either in the cell culture supernatant or within
the transfected cells. These results suggest that
angiotensinogen cleavage does not occur within the cell and
that, unlike renin and prorenin expressed in the in vivo model,
prorenin is unable to carry out the cleavage of
angiotensinogen after secretion into the serum-containing
culture supernatant.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Using a sensitive and specific in vivo enzymatic assay, we have obtained 2 independent lines of evidence that human prorenin does not need to be proteolytically cleaved to display enzymatic activity within the pituitary gland. First, both native prorenin and noncleavable prorenin are capable of generating Ang I in the in vivo assay (Figure 5
). Second, Western blot analysis of enzymatically
active prorenin shows that it retains its prosegment (Figure 6).
These findings strongly suggest that prorenin can be activated
within tissues by means other than proteolytic cleavage. Although the
actual mechanism whereby prorenin is activated in tissues
remains to be elucidated, it is highly likely that the prosegment,
which is predicted to fold over the substrate-binding cleft of the
enzyme,27 is able to unfold and to transiently expose the
active site of the enzyme. The conditions favoring this activation must
be particular to the extracellular space in tissues, because prorenin
catalytic activity is not detectable in either the
circulation12 or in the lysates or supernatants of cells
cotransfected with prorenin and angiotensinogen
(Table).
Ang II has been proposed to play a role in the modulation of secretion
of several pituitary hormones, including adrenocorticotrophic hormone,
prolactin, and growth hormone.7 However, in our study,
mice with dramatically elevated levels of pituitary Ang I (the
immediate peptide precursor of Ang II) show no reproducible effects on
growth, reproduction, blood pressure, or pituitary hormone
content or distribution by immunostaining (data not
shown). These results might be explained by one of the following
factors. First, we have targeted expression of the transgenes to
somatotropes in this study, whereas in rodents the
endogenous renin gene is expressed in
gonadotropes,38 perhaps leading to a different tissue
distribution of produced peptides. Second, we have noted that pituitary
Ang II levels were not reproducibly elevated in mice expressing very
high amounts of Ang I, which suggests that the necessary Ang
II–generating enzyme (see Figure 1) might be limiting. Finally,
our results could point to physiological
differences in the role of angiotensin peptides in
pituitary hormone secretion between the mice used in this study and the
much more thoroughly characterized rat model.
Prorenin is the only human aspartyl protease that does not cleave its own prosegment. Autocatalysis in other aspartyl proteases is thought to be initiated by a transient unfolding of the prosegment on contact of the precursors with the acid pH of their environment.28 Thus, although it is not capable of autocatalysis, it is possible that prorenin can also undergo transient unfolding under certain conditions, allowing it to catalyze the conversion of its substrate. In vitro, purified prorenin can be induced to exhibit reversible enzymatic activity by prolonged exposure to acid pH or cold storage.29 30 Thus, 1 possible explanation for the prorenin activity seen in tissues could be the slightly more acidic pH found in tissue interstitial spaces as compared with the circulation.31 Alternatively, serum proteins might be responsible for maintaining the inactive conformation of prorenin in both the circulation and in tissue culture supernatants.
In certain body fluids, such as ovarian follicular fluid,32 chorionic fluid,33 amniotic fluid,34 and seminal fluid,35 as well as in the ovarian and adrenal veins,36 prorenin concentrations reach many times the levels found in plasma. Ang II is also found in higher concentrations than in circulation in tissues such as the adrenal gland,37 the pituitary gland,38 and others,6 and the current study suggests that it may be produced locally by tissue prorenin. A transient unfolding of the prosegment of prorenin to exhibit enzymatic activity within tissues might also allow modulation by local physiological and pathophysiological conditions.
Heart and vascular tissues have also been shown to take up renin and prorenin from the circulation,39 40 and a specific receptor for these has been reported.41 The ability of tissues to activate prorenin by a nonenzymatic mechanism, as well as the ability to capture circulating prorenin and renin, strengthen the possibility that tissue RASs function in a locally restricted manner.
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Acknowledgments |
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This research was supported by a Medical Research Council grant. D.M. is the recipient of a Heart and Stroke Foundation of Canada scholarship. We thank I. Daneau, D. Raiwet, and C. Mercure for expert technical assistance; Drs P. Corvol and D. Lamarre for reagents; and Drs C.F. Deschepper, G. Thibault, and M. Bouvier for helpful comments.
Received September 30, 1999; accepted February 10, 1999.
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 N. Lochard, D. W. Silversides, J. P. van Kats, C. Mercure, and T. L. Reudelhuber Brain-specific Restoration of Angiotensin II Corrects Renal Defects Seen in Angiotensinogen-deficient Mice J. Biol. Chem., January 17, 2003; 278(4): 2184 - 2189. [Abstract] [Full Text] [PDF]
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 K. M. I. Caron, L. R. James, H.-S. Kim, S. G. Morham, M. L. S. S. Lopez, R. A. Gomez, T. L. Reudelhuber, and O. Smithies A genetically clamped renin transgene for the induction of hypertension PNAS, June 11, 2002; 99(12): 8248 - 8252. [Abstract] [Full Text] [PDF]
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J. J. Saris, M. M.E.D. van den Eijnden, J. M.J. Lamers, P. R. Saxena, M. A.D.H. Schalekamp, and A.H. J. Danser Prorenin-Induced Myocyte Proliferation: No Role for Intracellular Angiotensin II Hypertension, February 1, 2002; 39(2): 573 - 577. [Abstract] [Full Text] [PDF]
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 M. M. E. D. van den Eijnden, J. J. Saris, R. J. A. de Bruin, E. de Wit, W. Sluiter, T. L. Reudelhuber, M. A. D. H. Schalekamp, F. H. M. Derkx, and A. H. J. Danser Prorenin Accumulation and Activation in Human Endothelial Cells : Importance of Mannose 6-Phosphate Receptors Arterioscler Thromb Vasc Biol, June 1, 2001; 21(6): 911 - 916. [Abstract] [Full Text] [PDF]
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 J. J. Saris, F. H. M. Derkx, R. J. A. De Bruin, D. H. W. Dekkers, J. M. J. Lamers, P. R. Saxena, M. A. D. H. Schalekamp, and A. H. Jan Danser High-affinity prorenin binding to cardiac man-6-P/IGF-II receptors precedes proteolytic activation to renin Am J Physiol Heart Circ Physiol, April 1, 2001; 280(4): H1706 - H1715. [Abstract] [Full Text] [PDF]
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 G. PRESCOTT, D. W. SILVERSIDES, S. M. L. CHIU, and T. L. REUDELHUBER Contribution of circulating renin to local synthesis of angiotensin peptides in the heart Physiol Genomics, November 9, 2000; 4(1): 67 - 73. [Abstract] [Full Text] [PDF]
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