Proc. Nadl. Acad. Sci. USA Vol. 89, pp. 7806-7810, August 1992

Medical Sciences

Species specificity of renin kinetics in transgenic rats harboring the human renin and angiotensinogen genes (renin anglotensin system/renin inhibitors/blood pressure)

DETLEV GANTEN*, JUERGEN WAGNER, KARIN ZEH, MICHAEL BADER, JEAN BAPTISTE MICHEL, MARTIN PAUL, FRANK ZIMMERMANN, PAUL RUF, ULRICH HILGENFELDT, URSULA GANTEN, MICHAEL KALING, SEBASTIAN BACHMANN, AKIYOSHI FUKAMIZU, JOHN J. MULLINS, AND KAZUO MURAKAMI German Institute for High Blood Pressure Research and Department of Pharmacology, University of Heidelberg, Im Neuenheimer Feld 366, W-6900

Heidelberg, Germany Communicated by Alexander G. Bearn, February 28, 1992

ABSTRACT The renin-anglotensin system (RAS) is the most important regulatory system of electrolyte homeostasis and blood pressure. We report here the development of transgenic rats carrying the human angiotensinogen TGR(hAOGEN) and human renin TGR(hREN) genes. The plasma levels and tissue distribution of the transcription and translation products from both genes are described. A unique species specificity of the enzyme kinetics was observed. The human RAS components in the transgenic rats did not interact with the endogenous rat RAS in vivo. Insead, infusions of exogenous human RAS components specifically interacted with human transgene translation products. Thus, iion of human renin in TGR(hAOGEN) led to an increase of angioensin H and an elevation of blood pressure, which could not be antagonized by the human-specific renin enzyme inhibitor Ro 42-5892. Rat renin also elevated blood pressure and angiotensin H in TGR(hAOGEN); however, this effect was not antagonized by the human renin inhibitor. Compared to mice, rats offer the advantage of chronic instrumentation and repetitive, sophisticated, hemodynamic, and endocrinological investigations. Thus, transgenic rat models with human-specific enzyme kinetics permit primate-specific analyses in non-primate in vivo and in vitro experimental systems.

EXPERIMENTAL PROCEDURES Transgenic Techniques. Linear DNA fiagments consisting of either the entire human renin gene (1, 7) or the entire human angiotensinogen gene (8), as described elsewhere (1, 3), were injected into the male pronuclei of fertilized, outbred Sprague-Dawley (SD) rat eggs. Immnnalyses of Renin and en. Human renin was immunologically measured in transgenic rat plasma. The direct and specific measurement of human renin used two pairs of monoclonal antibodies in an immunoradiometric assay. One pair of antibodies was directed at the active human renin: 3E8 and 4G1 (9), whereas the other pair served to directly measure total renin: 3E8 and 4E1, as described (10). Determination of human plasma angiotensinogen was performed with an enzyme-linked immunosorbent assay (ELISA) using a human-specific angiotensinogen antibody (U.H., unpublished data). The concentration of rat renin and angiotensin II (ANG II) concentrations were determined as described (11, 12). Nucleic Acid Analyses. RNase protection and in situ hybridization assay for human renin. 32P-labeled RNA transcripts were prepared by transcription of a 291-nucleotide antisense RNA from a Sac I/EcoRV fragment of the human renin cDNA subcloned into pGEM4 vector using T7 RNA polymerase. This transcript comprised 225 nucleotides of human renin antisense RNA and 66 nucleotides of vectorencoded sequence. RNase protection assay and in situ hybridizationfor human angiotensinogen. To demonstrate transgene-specific gene expression, a Stu I/Ava II fragment of human angiotensinogen cDNA subcloned into pGem5 was used. Antisense RNA was transcribed by means of T7 RNA polymerase. This transcript spanned a 361-nucleotide antisense RNA and 51 nucleotides of vector-encoded sequence. Total RNA was isolated by lithium chloride precipitation (13). RNase protection assays were performed according to Goedert et al. (14). In Situ Hybrid on and inmu sohstry. Animals were perfusion-fixed by aortic cannulation using 3% paraformaldehyde in phosphate-buffered saline (PBS) for 5 min. For immunohistochemistry, kidneys were postfixed for 12 hr and paraffin-embedded. For in situ hybridization, organs were rinsed with sucrose/PBS (800 mosmol) and shockfrozen. For generation of the mouse renin antisense probe, a 330-nucleotide Sac I/Pst I fragment of mouse renin cDNA was subcloned into the pSP65 vector, linearized by Acc I and transcribed by the use of T7 RNA polymerase. For the rat

The successful incorporation of renin-angiotensin (RAS) genes into transgenic mice has been reported (1, 2). Rats, on the other hand, present specific technical problems with respect to the generation of transgenic animals. The transgenic rats TGR(mREN2)27, which harbor a mouse renin gene and exhibit fulminant hypertension, provided evidence for a monogenetic form of hypertension (3). Transgenic mice harboring the genes of mouse renin and angiotensinogen (2), rat renin and angiotensinogen (4), as well as human renin (1) have been described. Thus far, most of the RAS gene constructs used for the generation of transgenic animals interacted directly with the host RAS (2, 4). Since the genetic background for the transgenes appears to be of primary importance for the development of hypertension (5), we developed transgenic rats harboring the complete human renin TGR(hREN) and human angiotensinogen TGR(hAOGEN) genes. These rats allow studies into the regulation of human genes in non-primate animal models (6). In vitro, the species specificity of human renin and angiotensinogen is well known. Transgenic animals permit the study of specific interactions with the human transgene products in vivo, without interference from the host RAS.

Abbreviations: RAS, renin-angiotensin system; ANG I, angiotensin I; ANG II, angiotensin II; SD, Sprague-Dawley. *To whom reprint requests should be addressed at: Max-Delbrfick Centrum fur Moleculare Medizin, Robert-Rossle-Strasse 10, 0-1115 Germany.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 89 (1992)

Table 1. Human renin in plasma and tissue-specific gene expression in TGR(hREN) rats TGR(hREN) Parameter Control 1936 1988 Plasma RAS Active human renin (pg/ml) 0.0 4.9 ± 1.1 226.0 ± 0.7 Total human renin (pg/ml) 6820 ± 290 0.0 129.9 ± 17.8 Rat renin (ANG I, ng/ml 45.5 ± 4.3 51.4 ± 11.9 58.9 ± 16.9 per hr) Tissue mRNA ++ +++ Kidney + Adrenals ++ +++ Lung Heart ++ +++ Intestines ++ + Spleen Liver + ++ Thyroid + ++ Thymus + Brain + Medulla + Hypothalamus Human renin gene expression was determined by RNase protection assay as described in the legend to Fig. 2. The strength of the signal is approximated by the following symbols: -, negative; +, weak; + +, medium; +++, strong. Human renin was directly measured immunologically in transgenic rat plasma (see text). ANG I, angiotensin I.

angiotensinogen probe, a Pvu II/BamHI fragment of rat angiotensinogen cDNA (290 nucleotides) was subcloned into pGEM4, linearized by EcoRI, and transcribed by the use of SP6 polymerase in the presence of UTP[a-35S] (DuPont). In situ hybridization on cryostat sections was carried out as described (15). For immunohistochemistry, rabbit polyclonal antibodies (dilutions 1:1000-1:10,000) were applied on deparaffinized sections. Bound antibodies were detected by a peroxidase-antiperoxidase system. Blood Pressure Measurements. Direct blood pressure measurements were performed 48 hr after implantation of chronic femoral artery and vein catheters in unrestrained, conscious animals. Continuing recordings were monitored by a Senso Nor 840 transducer connected to a Hellige polygraph Recomed 330P. 100-

-E 0.

75

5 50C

E V 25-

I

I

7807

Values are given as mean + SEM with n = 6 for any group. Statistical analysis was performed by ANOVA; *** indicates P < 0.001.

RESULTS Transgenic Rats Harboring the Human Genomic Renin Gene TGR(hREN). The entire human renin gene containing 3 kilobases (kb) of 5' flanking sequences, 10 exons, 9 introns, and 1.2 kb of 3' flanking sequence (overall size, 17.6 kb) and cloned in a modified pUC19 vector (1, 7) was separated from vector sequences and microinjected into the male pronuclei of fertilized oocytes from outbred SD rats. From 87 rat eggs implanted, 15 progeny were obtained, of which 5 were found to carry the renin transgene. Two of these founders, with the designations TGR(hREN) 1936 and TGR(hREN) 1988, transmitted the transgene to their progeny, and line TGR(hREN) 1936 has been bred to homozygosity in the F3 generation. The data reported here are those from heterozygous animals. Human plasma renin and plasma prorenin, measured with specific antibodies directed against the human enzyme, were absent in control transgene-negative SD rats but could be demonstrated in both lines of TGR(hREN) (see Table 1). The enzyme activity of the human renin in rat plasma was 18 ng of ANG I per ml per hr in TGR(hREN) 1988; this value compares to about 1.5 ng of ANG I per ml per hr in human plasma (9). Rat prorenin, rat renin, ANG I, ANG II, and angiotensinogen levels were unchanged in TGR(hREN) animals as compared to SD rats, indicating that the human renin did not react with rat angiotensinogen to generate angiotensins. To demonstrate the response of human renin to physiologic stimuli, TGR(hREN) 1936 animals were sodium depleted, raising their human active renin concentration from 4.8 ± 1.1 pg/ml to 58.3 ± 9 pg/ml. Rat renin was also stimulated following sodium depletion, but there was no difference between animals with or without the transgene (Fig. 1). These data clearly indicate that the human renin transgene was expressed and translated and the active protein was secreted into the plasma in both transgenic lines. The specificity of human renin prevented reaction with the endogenous rat angiotensinogen, leaving the plasma ANG I and ANG II levels unchanged. Transgene expression was studied by a human reninspecific RNase protection assay. High-level expression of human renin was found in the kidney and positive signals were present in the lung, thymus, and gastrointestinal tract in both transgenic lines (Fig. 2A, Table 1). In situ hybridization

A

NS /.fUU

CX

C)' 500-

or.0 O SD rats * TGRthREN) 1936

_

0 v!

r

Control

Sodium depletion

Control

Sodium depletion

FIG. 1. Human renin concentration (A) and rat renin concentration (B) before and after sodium depletion of TGR(hREN) rats. The values represent the means and standard errors of eight male animals for each determination. Statistical analysis was done by ANOVA and showed the following significance: human renin concentration, P < 0.001, and rat renin concentration, P < 0.001, between the transgenic animals and the corresponding controls. NS, not significant. Human renin concentration is increased -11-fold by sodium depletion in TGR(hREN) rats. Animals were sodium depleted by intraperitoneal injection of furosemide at 20 mg/kg of body weight and placement on a sodium-restricted diet (0.8 meq/day) for 7 days. Blood samples were obtained at days 0 (control) and 7 (sodium depletion) retroorbitally under slight ether anesthesia.

7808

Proc. Nad. Acad. Sci. USA 89 (1992)

Medical Sciences: Ganten et al. A

TGR(hREN)

291-

X

'1'(iR(hAO(GEN'

B 361-

*.6

225 -

A.

310 -

ILTs

t' so

P

K! KI I.1 LU HE SP (1 PA SM BR TG; -

1

T1

1 A TG( -

1.

IT Hi SP .11-.

PA\

%N1 BH

FIG. 2. RNase protection assay analysis of gene expression of human renin (hREN) and human angiotensinogen (hAOGEN) in transgenic rats (TGR). (A) TGR(hREN): Expression of human renin mRNA in various tissues. Lanes: P., undigested human renin cRNA probe; T, tRNA; KI TG-, rat kidney RNA of transgenic negative littermates as negative control; KI, kidney; LI, liver; LU, lung; HE, heart; SP, spleen; GI, gastrointestinal tract; PA, pancreas; SM, submandibular gland; BR, brain. Each lane carries 50 Hg of total RNA. (B) TGR(hAOGEN): Tissue-specific expression of human angiotensinogen mRNA. For abbreviations see A. In each lane 50 Pg of total RNA was used except for liver (5 pg). Specificity was checked by 50 $Lg of liver transgenic negative RNA as control. P here represents undigested human angiotensinogen cRNA probe. Sizes are indicated in nucleotides.

of renin in the kidney showed transgene expression in the juxtaglomerular apparatus similar to the endogenous rat renin (Fig. 3). Systolic blood pressure in both TGR(hREN) lines was normal (133 ± 3 mmHg). Transgenic Rats Harboring the Human Genomnc Angiotensinogen Gene TGR(hAOGEN). The entire human angiotensinogen gene (8) containing 1.1 kb of 5' flanking sequences, five exons, four introns, and 2.4 kb of 3' flanking sequences (overall size, 16.3 kb) and cloned in pUC19 was freed from vector sequences and used to generate transgenic rats in SD outbred background. From 167 rat eggs implanted, 24 progeny were obtained, of which 7 were found to carry the human angiotensinogen transgene. Four founders with the designations TGR(hAOGEN) 1623, 1663, 1670, and 1671 transmitted the transgene to their progeny, and the line 1623 has been bred to homozygosity in the F3 generation. Data reported here are those obtained in the heterozygous animals TGR(hAOGEN). Plasma levels of human angiotensinogen in the four founders and their offspring were extremely elevated in TGR(hAOGEN) lines 1623, 1663, 1670, and 1671, ranging from 5185, 465, 397, to 120 jig of human angiotensinogen per ml of plasma, respectively, which compares to undetectable levels in transgene-negative rats and 60 ,ug of human angiotensinogen per ml (17) in normal human plasma (Table 2). The parameters ofthe rat RAS, prorenin, renin, angiotensinogen, ANG II, and converting enzyme remained unchanged in all lines, indicating that there is no reaction between human angiotensinogen and rat renin, even at very high plasma levels. Transgene expression was studied by a RNase protection assay specific for human angiotensinogen (Table 2, Fig. 2B). High-level expression in all founder animals and their progeny was found in the liver, kidney, brain, lung, heart, and gastrointestinal tract. Variable expression was found in the salivary glands, spleen, and muscle. In situ hybridization showed that the localization of gene expression in liver and kidney corresponds to the one known for endogenous rat angiotensinogen (Fig. 3). Transgenic Rats Harboring the Human Genomic Renin and Angiotensinogen Genes TGR(hRENxhAOGEN). Heterozygous human renin transgenic rats of line 1936 were crossbred with rats transgenic for the human angiotensinogen gene of line 1671 or 1670, respectively. From their offspring, rats harboring the human renin gene and human angiotensinogen gene could be obtained, which were subsequently named TGR(hREN xhAOGEN). Preliminary results indicate that these rats do not spontaneously develop hypertension.

Specificity of in Vitro and in Vivo Enzyme Kintis. In vitro, incubation of the plasma from sodium-depleted TGR(hREN) 1936 with the human-specific renin enzyme inhibitor Ro 42-5892 (1 AM; Hoffmann-La Roche) resulted in complete inhibition of human renin but did not influence rat plasma renin. In vivo infusion of Ro 42-5892 at a dosage of 1.5 mg/kg of body weight had no effect on blood pressure in sodiumdepleted TGR(hREN) 1936, even if human renin had been stimulated by sodium depletion (Fig. 1A). In contrast, the angiotensin receptor antagonist DUP 753 lowered blood pressure by 20 + 5 mmHg. These results indicate that even under stimulated conditions human renin did not cross-react with rat angiotensinogen nor did Ro 42-5892 inhibit rat renin activity at the used dosage. Rat renin infusions in TGR(hAOGEN) elicited a hypertensive response that was not affected by Ro 42-5892. This finding is in contrast to that observed with infusions of human renin (Fig. 4), which, given intravenously at a dosage of 5 jug Table 2. Human angiotensinogen in plasma and tissue-specific gene expression in TGR(hAOGEN) rats

TGR(hAOGEN) Parameter Plasma RAS Human AOGEN

(,tg/ml)

Control

1663

1670

0.0

397.4 ± 33.4

465.3 ± 44.9

46.3 ± 3.8

41.0 ± 3.4

43.6 ± 3.7

Rat AOGEN

(tAg/ml)

ANG II

30.5 ± 11.1 48.2 ± 6.5 42.6 ± 0.8 (fmol/ml) Tissue mRNA +++ +++ Liver ++ ++ Kidney + + Lung Pancreas ++ ++ Jejunum ++ ++ Heart Spleen + ++ Brain + SMG Human angiotensinogen (AOGEN) mRNA expression was studied using an RNase protection assay as described in the legend to Fig. 2. The strength of the signal is approximated by the following symbols: -, negative; +, weak; ++, medium; +++, strong. Determination of human plasma angiotensinogen was performed by an ELISA using a monoclonal antibody that is specific for human angiotensinogen.

Proc. Natl. Acad. Sci. USA 89 (1992)

Medical Sciences: Ganten et al.

7809

A

I

HR, 1 per min

40° 200-

BP, mmHg 100-

I

Iz

Human renin

Ro 42-5892

B HR, 1

per

min 400

006

200-

L

BP, mmHg 100lRat renin

S

s

753

FIG. 4. In vivo specificity of the human renin-angiotensinogen reaction and of the human renin inhibitor Ro 42-5892 in TGR(hAOGEN) rats. Blood pressure (BP) and heart rate (HR) recordings in an individual, conscious, unrestrained TGR(hAOGEN) rat are shown. (A) Blood pressure increases to plateau levels of 200 mmHg after infusion of purified human renin at a dosage of 5 ,ug of ANG I per ml per hr as an intravenous bolus over 5 min. Intravenously given Ro 42-5892 at 1000 ,ug/kg of body weight rapidly normalizes blood pressure to pretreatment values. (B) Infusion of renin from rat kidneys into TGR(hAOGEN) animal induces a rapid hypertensive response to about 200 mmHg. However, injection of Ro 42-5892 under steady-state conditions in the same dose remains without effect on the blood pressure elevation. The blood pressure increment is abolished by the angiotensin receptor antagonist DUP 753 at 10 mg/kg of body weight as an intravenous bolus over 5 min. Infusion of vehicle did not raise blood pressure.

I,

, .,~

DUP

Ro 42-5892

fr.

per ml per hr, raised blood pressure in TGR(hAOGEN) from 142 4 mmHg to 192 8 mmHg. This increase was lowered to pretreatment values by the intravenous administration of Ro 42-5892 (Figs. 4 and 5A). The angiotensin receptor antagonist DUP 753 significantly lowered blood pressure in a concentration of 10 mg/kg of body weight in both instances following rat and human renin infusions (Fig. 4). The rise in blood pressure induced by human renin infusion was paralleled by an increase in ANG II formation in transgene-positive rats. However, no change in blood pressure and ANG II generation occurred when human renin was infused into transgene-negative rats (Fig. 5).

of ANG I

±

±

DISCUSSION

FIG. 3. In situ hybridization and immunohistochemistry of renin and angiotensinogen gene products in transgenic rat tissues. (a and b) Kidney renin mRNA expression in a TGR(hREN) rat using an 35S-radiolabeled human (a) and mouse (b) renin probe on consecutive sections of the same tissue block. The mouse renin probe crosshybridizes with rat renin mRNA to nearly 100%o. The human renin probe cross-hybridizes with rat renin mRNA to about 60%o. Labeling with either probe shows renin transcripts exclusively over the glomerular afferent arteriole (arrowheads). (c and d) Immunohistochemistry of renin protein in the kidney of a TGR(hREN) rat using specific antibodies against human renin (c) (16) as well as rat renin (d) on consecutive sections of the same tissue. Under either condition, only the juxtaglomerular granular cells are positive (arrowheads; interference-contrast microscopy). The data show that the

The most important finding in these experiments was the fact that the human renin and angiotensinogen genes were expressed in the newly established transgenic rats and that the translation products renin and angiotensinogen did not crossreact enzymatically with the rat RAS and vice versa. This finding illustrates the unique species specificity of the RAS and allows for human-specific testing of in vivo and in vitro enzyme kinetics in these transgenic rats. The transgenic rat strain TGR(mREN2)27 harboring the mouse renin gene exhibited fulminant hypertension (3), whereas TGR(hREN) 1936 and 1988 did not, despite qualihuman and rat translation products are exclusively localized in the juxtaglomerular apparatus. (e) Liver angiotensinogen mRNA expression in a TGR(hAOGEN) rat using a radiolabeled human angiotensinogen probe (0.05 ng of antisense RNA per section; exposition time, 8 days). High density of silver grains is found over the parenchyma cells only; L indicates lumen of the central vein. (f) Labeling of rat angiotensinogen mRNA is considerably weaker in a control SD rat liver using a rat angiotensinogen probe compared to e under the same conditions. In control experiments both probes produce similar signal intensities when hybridized to angiotensinogen transcripts from either species. (a-d, x285; e and f, x430.)

Medical Sciences: Ganten et al.

7810

'A

Proc. Natl. Acad Sci. USA 89 (1992)

-

F

7

( ten11tl

I

Iliflml renill

IZ () 4 -" -;N I 1) -'

6000

*-i. 411AlFl --

ll

holz~~~~~~~~~~~~'7 1.

,_

FIG. 5.

_~~~~~~~~02111

In vivo

specificity of the human renin-angiotensinogen

reamn

reaction and of the human

(hAOGEN)

min

rats.

inhibitor Ro 42-5892 in TGR-

(A) Steady-state blood pressure levels before and 10

injection of purified human renin and renin inhibitor positive rats (hatched columns) and littermates (open columns). Blood pressure values are

after bolus

Ro 42-5892 in TGR(hAOGEN)

negative

markedly elevated in

response to human renin in

TGR(hAOGEN) but

not in control rats. Pretreatment with Ro 42-5892 3 hr

before human

reamn infusion inhibits blood pressure response completely but has no effect in control animals. (B) ANG II plasma levels in the

same

experiments as in A: ANG generation parallels blood pressure increase in TGR(hAOGEN) rats. Infusion of human renin does not alter ANG II levels in transgene-negative rats. ANG I formation was

essentially

tatively

identical to the ANG II levels.

similar

renin

vation demonstrates system, also does

transgenic

a

expression patterns.

human tissue renin,

This obser-

as a

paracrine

not react with the local rat substrate. The

animals

1671 exhibited

gene

that

TGR(hAOGEN)

1623, 1663, 1670, and

large variation of plasma levels from 120 to The transgene expression in tissue

icg/ml, respectively.

5000

corresponded qualitatively to endogenous rat angiotensinogen distribution, with the major localization in liver, brain, and

kidney.

confirms

These animals

once

were

also normotensive, which

again the specificity

of the

human angioten-

sinogen and its inability to react with rat reanI. The expression pattern of the human genes in experiments

using

the natural promoters was similar in mice and rats. This finding is in agreement with previous data indicating a

constant

gene

expression in different specificity

animal

species (6).

The unique species of the renin substrate reaction is advantageous for developing and testing humanspecific renin inhibitors. Both types of transgenic animals can be used to administer exogenous human ream or angioten-

sinogen, respectively, to then test the efficacy of the drugs in vivo.

Blood pressure

responses

may

be

observed instantly.

By means of crossbreeding experiments, transgenic rats were obtained that harbored the human renin gene and the angiotensinogen gene. In preliminary studies these TGR(hRENXhAOGEN) animals did not spontaneously develop hypertension compared to transgenic animals carrying the mouse Ren-2 gene (3). The rat renin and human renin transgenes, corresponding to the Ren-) gene, may be expressed and regulated in a similar fashion by angiotensin H feedback mechanisms, thereby resulting in a normal net ANG II formation. However, it cannot be excluded that the comparatively low amounts of human plasma renin concentrations expressed in TGR(hREN) 1936 prevent the development of arterial hypertension in the TGR(hRENxhAOGEN) animals. Investigations that manipulate the RAS, such as alterations in sodium homeostasis, are necessary to elucidate the interaction of human transgenes and its effect on blood pressure development in this model. In summary, transgenic rats harboring the genes for human renin and human angiotensinogen have been developed. The species specificity of the RAS prevented the interaction of human renin and angiotensinogen with the host rat RAS components. This system offers a unique opportunity for the specific testing of both human genes in rats. Further, the model will also be helpful to overcome the problems and limitations inherent in primate or human research directed at the development of human-specific drugs to interfere with the RAS. These transgenic rats will also offer an opportunity to study the regulation of human RAS genes at the tissue level in a rat model. Finally, the nature of the model provides an unprecedented opportunity for functional observations. This work was supported by Deutsche Forschunpemeinschaft (DFG), the National Heart, Lung, and Blood Institute (NHLBI), and European Community (Trans-Gen-Eur). 1. Fukamizu, A., Seo, M. S., Hatae, T., Yokoyama, M., Nomura, T., Katsuki, M. & Murakami, K. (1989) Biochem. Biophys. Res. Commun. 165, 826-832. 2. Miller, C. C., Samani, N. J., Carter, A. T., Brooks, J. I. & Brammar, W. J. (1989) J. Hypertens. 7, 861-863. 3. Mullins, J. J., Peters, J. & Ganten, D. (1990) Nature (London) 344, 541-544. 4. Ohkubo, H., Kawakami, H., Kakehi, Y., Takumi, T., Arai, H., Yokota, Y., Iwai, M., Tanabe, Y., Masu, M., Hata, J., Iwao, H., Okamoto, H., Yokoyama, M., Nomura, T., Katsuki, M. & Nakanishi, S. (1990) Proc. Nati. Acad. Sci. USA 87, 5153-5157. 5. Ganten, D., Lindpaintner, K., Ganten, U., Peters, J., Zimmermann, F., Bader, M. & Mullins, J. (1991) Hypertension 17, 843-855. 6. Mullins, J. J., Sigmund, C. D., Kane-Haas, C., McGowan, R. A. & Gross, K. W. (1989) EMBO J. 8, 4065-4072. 7. Miyazaki, H., Fukamizu, A., Hirose, S., Hayashi, T., Hon, H., Ohkubo, H., Nakanishi, S. & Murakami, K. (1984) Proc. Nati. Acad. Sci. USA 81, 5999-6003. 8. Fukamizu, A., Takahashi, S., Seo, M. S., Tada, M., Tanimoto, K., Uehara, S. & Murakami, K. (1990) J. Biol. Chem. 265, 7576-7582. 9. Menard, J., Guyenne, T.-T., Corvol, P., Pau, B., Simon, D. & Roncucci, R. (1985) J. Hypertens. 3, Suppl. 3, S275-S278. 10. Toffelmire, E. B., Slater, K., Corvol, P., M~nard, J. & Schambclan, M. (1989) J. Clin. Invest. 83, 679-687. 11. Schelling, P., Ganten, U., Sponer, G., Unger, T. & Ganten, D. (1980) Neuroendocrinology 31, 297-308. 12. Hermann, K., Ganten, D., Unger, T., Bayer, C. & Lang, R. E. (1988) Clin. Chem. 34, 1046-1051. 13. Auffray, C. & Rougeon, F. (1980) Eur. J. Biochem. 107, 303-314. 14. Goedert, M., Spillantini, M. G., Potier, M. C., Ulrich, J. & Crowther, R. A. (1989) EMBO J. 8, 393-399. 15. Bachmann, S., Metzger, R. & Bunnemann, B. (1990) Histochemistry 94, 517-523. 16. Galen, F. X., Devaux, C., Guyene, T. T., Menard, J. & Corvol, P. (1979) J. Biol. Chem. 254, 4848. 17. Gardes, J., Bouhnik, J., Clauser, E., Corvol, P. & M6nard, J. (1982) Hypertension 4, 185-189.