Donate Help Contact The AHA Sign In Home
American Heart Association
Circulation Research
Search: search_blue_button Advanced Search
Circulation Research. 1999;84:1067-1072

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Methot, D.
Right arrow Articles by Reudelhuber, T. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Methot, D.
Right arrow Articles by Reudelhuber, T. L.
Related Collections
Right arrow Biochemistry and metabolism
Right arrow ACE/Angiotension receptors
Right arrow Genetically altered mice
(Circulation Research. 1999;84:1067-1072.)
© 1999 American Heart Association, Inc.


Original Contributions

In Vivo Enzymatic Assay Reveals Catalytic Activity of the Human Renin Precursor in Tissues

Danielle Methot, David W. Silversides, Timothy L. Reudelhuber

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


*    Abstract
up arrowTop
*Abstract
down arrowIntroduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
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


*    Introduction
up arrowTop
up arrowAbstract
*Introduction
down arrowMaterials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
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 1ADown).



View larger version (16K):
[in this window]
[in a new window]
 
Figure 1. A, Schematic representation of the RAS. AOGEN indicates angiotensinogen; ANG I, angiotensin; and ACE, angiotensin-converting enzyme. B, Schematic representation of the approach used to test for enzymatic activity of prorenin in vivo. Double-transgenic mice expressing human angiotensinogen (the renin substrate) and human active renin, human native prorenin, or human noncleavable prorenin in the pituitary gland were generated, and the mice were assayed for increased pituitary content of Ang I.

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 1BUp). 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.


*    Materials and Methods
up arrowTop
up arrowAbstract
up arrowIntroduction
*Materials and Methods
down arrowResults
down arrowDiscussion
down arrowReferences
 
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 2Down). 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.



View larger version (26K):
[in this window]
[in a new window]
 
Figure 2. Diagram of transgenes used in this study. Mutations relative to the native prorenin cleavage site are underlined. rGH indicates rat growth hormone promoter; PRO, human prorenin prosegment; AOGEN, angiotensinogen; and SV40, simian virus 40 T antigen splice and polyadenylation signal. Arrow indicates expected and putative cleavage sites for the various prorenins; question mark indicates questionable cleavage; and crossed-out arrow indicates no cleavage. Drawings are not to scale. See text for details.

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 {approx}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).


*    Results
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrowMaterials and Methods
*Results
down arrowDiscussion
down arrowReferences
 
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 3Down), 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 4Down). 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 1BUp) 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 1AUp). Human prorenin and human renin were undetectable in the plasma of transgenic lines (data not shown).



View larger version (57K):
[in this window]
[in a new window]
 
Figure 3. Pituitary gland–specific expression of the human renin transgene. Total RNA from the designated tissues of transgenic line 34 (expressing native human prorenin) was hybridized with a human prorenin-specific antisense RNA probe. *Nonspecific bands arising from digestion of the probe. PRO indicates the expected position for the human prorenin-protected fragment after RNase digestion.



View larger version (60K):
[in this window]
[in a new window]
 
Figure 4. Relative expression of the human transgenes in various transgenic lines. Comparison of the mRNA levels of 8 independent double-transgenic lines of animals expressing human angiotensinogen (AOGEN) and 1 of the 3 different human prorenins (active renin, native prorenin, or noncleavable prorenin) by RNase protection assay. Each of the lines generated with the different human prorenin transgenes was crossed with the same angiotensinogen-expressing line, and the angiotensinogen mRNA signal was used as an internal reference to compare the different prorenin mRNA levels. M indicates size marker; NT, nontransgenic control; and ST, standards. Expected positions for the human prorenin (PRO)–protected and human angiotensinogen-protected fragments are designated at the right. *Nonspecific bands.

To test for the Ang I–generating activity of human active renin in this system, we engineered a mutation in human prorenin (Figure 2Up) 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 5Down, hatched bars) as compared with single-transgenic mice expressing only human angiotensinogen (Figure 5Down, 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.



View larger version (25K):
[in this window]
[in a new window]
 
Figure 5. Pituitary content of Ang I in double-transgenic mice. Pituitary Ang I content was determined in pituitary glands of transgenic mice expressing either human angiotensinogen alone (open bar) or in combination with human active renin (hatched bar), human native prorenin (solid bar), or human noncleavable prorenin (stippled bar). Ang I content was not detectable from either nontransgenic mice or individual single-transgenic mice expressing only the various prorenins. Relative levels of prorenin transgene expression in the various lines (derived from Figure 4Up) are indicated by + and – signs shown above the bars (see Materials and Methods for classifications). Each data point is derived from a pool of 5 pituitary glands, and results are expressed as the quantity in picograms of Ang I/pituitary gland. Data are mean±SE. **P<0.01, *P<0.05 vs mice expressing only human angiotensinogen by 1-way ANOVA with Dunnett post-test. AOGEN indicates angiotensinogen.

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 5Up, 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 5Up). 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 2Up). 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 5Up, 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 6Down). 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.



View larger version (41K):
[in this window]
[in a new window]
 
Figure 6. Western blot analysis of human prorenin and human renin in pituitary glands of transgenic mice. Human renin/prorenin expressed in transgenic mouse pituitary gland was analyzed by coupled immunoprecipitation and Western blot analysis. Nontransgenic control (NT), single-transgenic line 50 expressing human native prorenin, and single-transgenic line 33 expressing human active renin are shown. Arrows indicate predicted sizes of prorenin (upper band) and renin (lower band) based on coelectrophoresis of molecular weight markers. Results are from a single experiment, the results of which were confirmed in a second, independent experiment.

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 (TableDown) 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.


View this table:
[in this window]
[in a new window]
 
Table 1. Ang I Values From Transfected GH4C1 Cells1


*    Discussion
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrowMaterials and Methods
up arrowResults
*Discussion
down arrowReferences
 
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 5Up). Second, Western blot analysis of enzymatically active prorenin shows that it retains its prosegment (Figure 6Up). 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 (TableUp).

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 1Up) 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.


*    Acknowledgments
 
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.


*    References
up arrowTop
up arrowAbstract
up arrowIntroduction
up arrowMaterials and Methods
up arrowResults
up arrowDiscussion
*References
 
1. Baker D, Shiau AK, Agard DA. The role of pro regions in protein folding. Curr Opin Cell Biol. 1993;5:966–970.[Medline] [Order article via Infotrieve]

2. Griendling KK, Murphy TJ, Alexander RW. Molecular biology of the renin-angiotensin system. Circulation. 1993;87:1816–1828.[Free Full Text]

3. Ingelfinger JR, Dzau VJ. Molecular biology of renal injury: emphasis on the role of the renin-angiotensin system. J Am Soc Nephrol. 1991;2:S9–S20.

4. Derkx FH, Schalekamp MA. Human prorenin: pathophysiology and clinical implications. Clin Exp Hypertens Theory Pract. 1988;10:1213–1225.

5. Kim S, Hosoi M, Nakajima K, Yamamoto K. Immunological evidence that kidney is primary source of circulating inactive prorenin in rats. Am J Physiol. 1991;260:E526–E536.[Abstract/Free Full Text]

6. Phillips MI, Speakman EA, Kimura B. Levels of angiotensin and molecular biology of the tissue renin angiotensin systems. Regul Pep. 1993;43:1–20.[Medline] [Order article via Infotrieve]

7. Ganong WF. Reproduction and the renin-angiotensin system. Neurosci Biobehav Rev. 1995;19:241–250.[Medline] [Order article via Infotrieve]

8. Wagner J, Jan Danser AH, Derkx FH, de Jong TV, Paul M, Mullins JJ, Schalekamp MA, Ganten D. Demonstration of renin mRNA, angiotensinogen mRNA, and angiotensin converting enzyme mRNA expression in the human eye: evidence for an intraocular renin-angiotensin system. Br J Ophthalmol. 1996;80:159–163.[Abstract/Free Full Text]

9. Campbell DJ, Kladis A, Skinner SL, Whitworth JA. Characterization of angiotensin peptides in plasma of anephric man. J Hypertens. 1991;9:265–274.[Medline] [Order article via Infotrieve]

10. Hosoi M, Kim S, Tabata T, Nishitani H, Nishizawa Y, Morii H, Murakami K, Yamamoto K. Evidence for the presence of differently glycosylated forms of prorenin in the plasma of anephric man. J Clin Endocrinol Metab. 1992;74:680–684.[Abstract]

11. Sealey JE, Moon C, Laragh JH, Atlas SA. Plasma prorenin in normal, hypertensive, and anephric subjects and its effect on renin measurements. Circ Res. 1977;40 (suppl I):I-41–I-45.

12. Lenz T, Sealey JE, Maack T, James GD, Heinrikson RL, Marion D, Laragh JH. Half-life, hemodynamic, renal, and hormonal effects of prorenin in cynomolgus monkeys. Am J Physiol. 1991;260:R804–R810.[Abstract/Free Full Text]

13. Véniant M, Ménard J, Bruneval P, Morley S, Gonzales M, Mullins J. Vascular damage without hypertension in transgenic rats expressing prorenin exclusively in the liver. J Clin Invest. 1997;98:1966–1970.[Medline] [Order article via Infotrieve]

14. Mercure C, Thibault G, Lussier-Cacan S, Davignon J, Schiffrin EL, Reudelhuber TL. Molecular analysis of human prorenin prosegment variants in vitro and in vivo. J Biol Chem. 1995;270:16355–16359.[Abstract/Free Full Text]

15. Gaillard I, Clauser E, Corvol P. Structure of human angiotensinogen gene. DNA. 1989;8:87–99.[Medline] [Order article via Infotrieve]

16. Lira SA, Crenshaw EB 3d, Glass CK, Swanson LW, Rosenfeld MG. Identification of rat growth hormone genomic sequences targeting pituitary expression in transgenic mice. Proc Natl Acad Sci U S A. 1988;85:4755–4759.[Abstract/Free Full Text]

17. Brechler V, Chu WN, Baxter JD, Thibault G, Reudelhuber TL. A protease processing site is essential for prorenin sorting to the regulated secretory pathway. J Biol Chem. 1996;271:20636–20640.[Abstract/Free Full Text]

18. Methot D, Reudelhuber TL, Silversides DW. Evaluation of tyrosinase minigene co-injection as a marker for genetic manipulations in transgenic mice. Nucleic Acids Res. 1995;23:4551–4556.[Abstract/Free Full Text]

19. Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987;162:156–159.[Medline] [Order article via Infotrieve]

20. Behringer RR, Mathews LS, Palmiter RD, Brinster RL. Dwarf mice produced by genetic ablation of growth hormone-expressing cells. Genes Dev. 1988;2:453–461.[Abstract/Free Full Text]

21. Borrelli E, Heyman RA, Arias C, Sawchenko PE, Evans RM. Transgenic mice with inducible dwarfism. Nature. 1989;339:538–541.[Medline] [Order article via Infotrieve]

22. Hatae T, Takimoto E, Murakami K, Fukamizu A. Comparative studies on species-specific reactivity between renin and angiotensinogen. Mol Cell Biochem. 1994;131:43–47.[Medline] [Order article via Infotrieve]

23. Oliver WJ, Gross F. Unique specificity of mouse angiotensinogen to homologous renin. Proc Soc Exp Biol Med. 1966;122:923–926.[Medline] [Order article via Infotrieve]

24. Schalken JA, Roebroek AJ, Oomen PP, Wagenaar SS, Debruyne FM, Bloemers HP, Van de Ven WJ. fur gene expression as a discriminating marker for small cell and nonsmall cell lung carcinomas. J Clin Invest. 1987;80:1545–1549.

25. Bresnahan PA, Leduc R, Thomas L, Thorner J, Gibson HL, Brake AJ, Barr PJ, Thomas G. Human fur gene encodes a yeast KEX2-like endoprotease that cleaves pro-beta-NGF in vivo. J Cell Biol. 1990;111:2851–2859.[Abstract/Free Full Text]

26. Hatsuzawa K, Hosaka M, Nakagawa T, Nagase M, Shoda A, Murakami K, Nakayama K. Structure and expression of mouse furin, a yeast Kex2-related protease: lack of processing of coexpressed prorenin in GH4C1 cells. J Biol Chem. 1990;265:22075–22078.[Abstract/Free Full Text]

27. Hsueh WA, Baxter JD. Human prorenin. Hypertension. 1991;17:469–479.[Abstract/Free Full Text]

28. Dunn B. Splitting image. Nat Struct Biol. 1997;4:969–972.[Medline] [Order article via Infotrieve]

29. Derkx FH, Schalekamp MP, Schalekamp MA. Two-step prorenin-renin conversion: isolation of an intermediary form of activated prorenin. J Biol Chem. 1987;262:2472–2477.[Abstract/Free Full Text]

30. Pitarresi TM, Rubattu S, Heinrikson R, Sealey JE. Reversible cryoactivation of recombinant human prorenin. J Biol Chem. 1992;267:11753–11759.[Abstract/Free Full Text]

31. Martin GR, Jain RK. Noninvasive measurement of interstitial pH profiles in normal and neoplastic tissue using fluorescence ratio imaging microscopy. Cancer Res. 1994;54:5670–5674.[Abstract/Free Full Text]

32. Glorioso N, Atlas SA, Laragh JH, Jewelewicz R, Sealey JE. Prorenin in high concentrations in human ovarian follicular fluid. Science. 1986;233:1422–1424.[Abstract/Free Full Text]

33. Itskovitz J, Rubattu S, Levron J, Sealey JE. Highest concentrations of prorenin and human chorionic gonadotropin in gestational sacs during early human pregnancy. J Clin Endocrinol Metab. 1992;75:906–910.[Abstract]

34. Skinner SL, Cran EJ, Gibson R, Taylor R, Walters WA, Catt KJ. Angiotensins I and II, active and inactive renin, renin substrate, renin activity, and angiotensinase in human liquor amnii and plasma. Am J Obstet Gynecol. 1975;121:626–630.[Medline] [Order article via Infotrieve]

35. Mukhopadhyay AK, Cobilanschi J, Schulze W, Brunswig-Spickenheier B, Leidenberger FA. Human seminal fluid contains significant quantities of prorenin: its correlation with the sperm density. Mol Cell Endocrinol. 1995;109:219–224.[Medline] [Order article via Infotrieve]

36. Sealey JE, Rubattu S. Prorenin and renin as separate mediators of tissue and circulating systems. Am J Hypertens. 1989;2:358–366.[Medline] [Order article via Infotrieve]

37. Husain A, DeSilva P, Speth RC, Bumpus FM. Regulation of angiotensin II in rat adrenal gland. Circ Res. 1987;60:640–648.[Abstract/Free Full Text]

38. Deschepper CF, Crumrine DA, Ganong WF. Evidence that the gonadotrophs are the likely site of production of angiotensin II in the anterior pituitary of the rat. Endocrinology. 1986;119:36–43.[Abstract/Free Full Text]

39. Danser AH, Schalekamp MA. Is there an internal cardiac renin-angiotensin system? Heart. 1996;76:28–32.

40. Muller DN, Hilgers KF, Bohlender J, Lippoldt A, Wagner J, Fischli W, Ganten D, Mann JF, Luft FC. Effects of human renin in the vasculature of rats transgenic for human angiotensinogen. Hypertension. 1995;26:272–278.[Abstract/Free Full Text]

41. Sealey JE, Catanzaro DF, Lavin TN, Gahnem F, Pitarresi T, Hu LF, Laragh JH. Specific prorenin/renin binding (ProBP): identification and characterization of a novel membrane site. Am J Hypertens. 1996;9:491–502.[Medline] [Order article via Infotrieve]




This article has been cited by other articles:


Home page
HypertensionHome page
A.H. J. Danser
Does Prorenin Exert Angiotensin-Independent Effects In Vivo?
Hypertension, December 1, 2009; 54(6): 1218 - 1220.
[Full Text] [PDF]


Home page
HypertensionHome page
C. Mercure, G. Prescott, M.-J. Lacombe, D. W. Silversides, and T. L. Reudelhuber
Chronic Increases in Circulating Prorenin Are not Associated With Renal or Cardiac Pathologies
Hypertension, June 1, 2009; 53(6): 1062 - 1069.
[Abstract] [Full Text] [PDF]


Home page
Am. J. Physiol. Endocrinol. Metab.Home page
J. Zhang, N. A. Noble, W. A. Border, R. T. Owens, and Y. Huang
Receptor-dependent prorenin activation and induction of PAI-1 expression in vascular smooth muscle cells
Am J Physiol Endocrinol Metab, October 1, 2008; 295(4): E810 - E819.
[Abstract] [Full Text] [PDF]


Home page
Exp PhysiolHome page
G. Nguyen and A. H. J. Danser
Prorenin and (pro)renin receptor: a review of available data from in vitro studies and experimental models in rodents
Exp Physiol, May 1, 2008; 93(5): 557 - 563.
[Abstract] [Full Text] [PDF]


Home page
J. Biol. Chem.Home page
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]


Home page
Proc. Natl. Acad. Sci. USAHome page
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]


Home page
HypertensionHome page
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]


Home page
Arterioscler. Thromb. Vasc. Bio.Home page
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]


Home page
Am. J. Physiol. Heart Circ. Physiol.Home page
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]


Home page
Physiol. GenomicsHome page
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]


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Methot, D.
Right arrow Articles by Reudelhuber, T. L.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Methot, D.
Right arrow Articles by Reudelhuber, T. L.
Related Collections
Right arrow Biochemistry and metabolism
Right arrow ACE/Angiotension receptors
Right arrow Genetically altered mice