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Molecular genetics links renin to hypertension
Molecular and Cellular Endocrinology, 75 (1991) C13-C18 © 1991 Elsevier Scientific Publishers Ireland, Ltd. 0303-7207/91/$03.50
C13
MOLCEL 02464
At the Cutting Edge Molecular genetics links renin to hypertension Brian J. Morris Molecular Biology & Hypertension Laboratory, Department of Physiology, The University of Sydney, N.S. IV. 2006, A ustralia
(Accepted 13 December 1990)
Key words: Restriction fragment length polymorphism; Dahl rat; Spontaneously hypertensive rat; Renin transgene; Gene control
Essential hypertension Renin is pivotal to blood pressure regulation, being rate limiting in a dual enzyme cascade in which renin generates angiotensin I from which converting enzyme then produces angiotensin II, which in turn exerts the ultimate physiological actions. Whether renin also has a role in the pathogenesis of the genetic predisposition to essential hypertension has been a vexed question. Restriction fragment length polymorphisms (RFLPs) of the renin gene correlate with differences in plasma renin activity between AfroCarribean and European subjects (Webb et al., 1990), though segregation with essential hypertension could not be shown (Morris and Griffiths, 1988; Naftilan et al., 1989; Webb et al., 1990). Given the extent of human genetic heterogeneity and the expectation that more than one gene will be involved in essential hypertension, large multigenerational pedigrees with multiple affected individuals will be required for appropriate analyses. That the renin gene might have a role is nevertheless supported by recent findings with genetically pure strains of rats commonly used as genetic models of hypertension, the spontaneously hypertensive rat (SHR) and the Dahl salt-sensitive hypertensive (DS) rat. The DS rat resembles 'low renin' essential hypertension, whereas in the SHR,
Address for correspondence: Dr B.J. Morris, Molecular Biology&Hypertension Laboratory, Department of Physiology (Building F13), The University of Sydney, N.S.W. 2006, Australia.
renal function and renin content resemble those in 'nonmodulating', highly hereditable essential hypertension (Dluhy et al., 1988). In DS and S H R 4 independently segregating loci have been estimated to contribute to the elevated blood pressure (Knudsen et al., 1970; Harrap, 1986). Dahl salt-sensitive rats In the Sprague-Dawley strain of Rattus norvegicus there are 46 tandem 37 bp repeats in the first intron of the renin gene (Fukumizu et al., 1988), which thus represents a hot spot for recombination. This 1.7 kb region is flanked by B g l I I sites 2.3 kb apart (Fig. 1). In DS rats BglII gives a 2.7 kb band, indicating an insertion, and in D R (a control strain resistant to most hypertensive stimuli) the band is 1.5 kb, indicating a deletion (Wang and Rapp, 1989). Studies of F 2 and F 1 x DS populations have shown that one dose of the DS allele was associated with a 10 m m Hg rise in blood pressure, and two with a 20 m m Hg rise, i.e., 20% of the blood pressure elevation ( R a p p et al., 1989, 1990; R a p p and Wang, 1990). Blood pressure did not increase in F t x DR, which are 75% influenced by D R genes, suggesting that genes at other unidentified loci modify the blood pressure effect of the DS renin allele (Rapp et al., 1990). Renin is low and relatively unresponsive in DS rats, but high and hyper-responsive in DR. Although high blood pressure causes physiological suppression of renin, in DS rats renin is low before the rats become hypertensive ( R a p p et al., 1989), consistent with inherently low transcrip-
C14
DAHL RATS Salt-senslUve (Hypertensive)
t
(sT)
t
Salt-resistant (Normotenslve) (25)
NORMAL LABORATORY RATS l]n
Be. II
Exon I
SPONTANEOUSLY HYPERTENSIVE RAT WISTAR-KYOTO NORMOTENSIVE RAT
repeated sequence)
I
~ ~1,
(17)
Exon 2
1 kb
Exon 3
I
~1'
(34)
Fig. 1. Repetitive DNA sequences in the first intron of the rat renin gene (which has 9 exons). The estimated number of repeats for each strain is indicated in parentheses. Strain differences were detected as a result of a Bglll RFLP, where Bglll sites are indicated by arrows. The fragment lengths shown for SHR differ from those reported by Samani et al. (1989a) and Kurtz et al. (1990), who indicated a band of 1.7 kb for SHR and 2.8 kb for Sprague-Dawley, whereas restriction map and nucleotide sequence data predict a 2.3 kb band (Fukamizu et al., 1988). Samani and coworkers also found a 2.8 kb band for Wistar and Lewis rats; for WKY it was 2.3 kb. tional activity of the renin gene. Since repetitive D N A can s u p p r e s s t r a n s c r i p t i o n in a d o s e - d e p e n d e n t m a n n e r ( H e r r a n d Clarke, 1986) it is t e m p t i n g to s p e c u l a t e that the i n t r o n 1 r e p e a t s m a y have a silencer function. A l t e r n a t i v e l y , this R F L P m a y be m e r e l y serving as a m a r k e r for o t h e r difference(s) in the gene ( W a n g a n d R a p p , 1989) that d o affect t r a n s c r i p t i o n . W h i c h e v e r is the case, h o w low renin might reflect a causative role is n o t clear.
Spontaneously hypertensive rat T h e r e are o n l y 17 copies o f the t a n d e m r e p e a t in S H R , c o m p a r e d with 34 in the n o r m o t e n s i v e W i s t a r - K y o t o ( W K Y ) c o n t r o l ( S a m a n i et al., 1989a) (Fig. 1). S H R have increased renin, consistent with the d e l e t i o n of repetitive, p u t a t i v e ' s i l e n c e r ' D N A ; K u r t z et al. (1990) thus specu-
l a t e d that h y p e r t e n s i o n in the S H R a n d n o n m o d ulating essential h y p e r t e n s i o n m a y be r e l a t e d to the renin gene. In the s t r o k e - p r o n e S H R , however, L i n d p a i n t e r et al. (1990) c o u l d n o t d e m o n s t r a t e cosegregation of b l o o d p r e s s u r e with r e n i n genotype. In the studies b y K u r t z et al. (1990) a n F 2 p o p u l a t i o n was d e r i v e d from i n b r e d S H R a n d i n b r e d n o r m o t e n s i v e Lewis rats, a n d s h o w e d that b l o o d pressure in rats i n h e r i t i n g a single S H R r e n i n allele was significantly g r e a t e r ( b y - 20 m m Hg) t h a n t h a t in rats inheriting o n l y the Lewis allele ( K u r t z et al., 1990). Similarly, r e c o m b i n a n t i n b r e d strains d e r i v e d f r o m S H R × B r o w n - N o r w a y rats also s h o w e d c o s e g r e g a t i o n of the S H R allele with increase in b l o o d p r e s s u r e ( P r a v e n e c et al., 1990). Studies with o t h e r genetically h y p e r t e n s i v e strains, however, suggest that the repetitive D N A is n o t functional: the L y o n rat, for e x a m p l e , has
C15 28 copies of the repeat in hypertensive and hypotensive, but not in normotensive strains, and Milan hypertensive and normotensive rats are similar with respect to the BgllI R F L P (Samani et al., 1990). The R F L P may thus have no functional significance in the SHR, but serve as a linkage marker of a site elsewhere in the renin gene, its regulatory DNA, or of a neighbouring gene that contributes to the hypertension. In this context, it is of interest that at the 13th International Society of Hypertension Meeting (Montreal, June 1990), R a p p reported that when S H R are crossed with DS rats, blood pressure increases only when salt is given, thus demonstrating predomination of the 'salt-hypertension gene(s)' over those responsible for 'spontaneous' hypertension; presumably, renin may be one of these genes. Gene control mechanisms There have been no direct studies of cis-acting influences of intron 1 D N A in the renin gene; only the 5'-flanking D N A has been studied, and then only for human and mouse in cell lines that do not synthesize renin. In primary cultures of chorion, which produce renin from contaminating decidual cells (Shaw et al., 1989), the first 0.6 kb of human renin (REN) upstream D N A was sufficient to direct the transient expression of a reporter gene (Duncan et al., 1987). That this D N A may also be sufficient for tissue-specificity of expression is supported by the lack of expression of constructs in amnion cells. This active proximal 0.6 kb segment also displays high inter-species homology, whereas D N A further upstream has low homology and does not affect promoter activity (Smith and Morris, 1990). Within the active region an enhancer resides in the - 0 . 5 to - 0 . 1 kb D N A , and a negative element is located within 0.1 kb of the promoter (Burt et al., 1989; Smith and Morris, 1990). Similar studies for mouse have led to a different conclusion, however - - that inactivity of the renin gene in ceils that do not normally produce renin is not due to repression, but rather, cells that synthesize renin do so because they possess specific trans-acting factor(s) that target enhancer(s) close to the renin gene (Ekker et al., 1989a). Ekker et al. made this suggestion because when
they introduced a SV40 enhancer (which is utilized by all cells) into their constructs the activity of the renin promoter changed from zero to a level that was similar over a range of cells, none of which normally synthesize renin. Activity was similarly conferred on the human promoter in amnion cells by insertion of a SV40 enhancer (Duncan et al., 1987). N o r m a l mouse upstream D N A differs from human (and rat) by the presence of a 0.5 kb transposon-like insertion (Tronik et al., 1988), which appears to be a target for tissue-specific trans-acting-factor(s), and the existence of which has been used to explain why renin expression is high in mouse submandibular gland but completely absent from the same tissue of rats (Morris et al., 1980; Tronik et al., 1988). If enhancer(s) in this D N A have a dominating influence, then the mechanism Ekker et al. propose might apply only to mouse. The site of the insertion, which is at - 7 5 and - 9 2 relative to rat and human, respectively, is in the vicinity of the negative regulatory element found in the h u m a n studies and if disrupted would further account for species differences. The implications for hypertension are that putative variants of the renin gene that involve disrupted enhancer or silencer elements would give altered renin production; eventually one or other of the R F L P s may turn out to reflect such alterations in the renin D N A . Conserved synteny group Even if renin R F L P s serve only as markers for some other nearby gene that is causal in the rat models, this is still relevant to hypertension in humans since the section of genome containing the renin gene is part of a conserved synteny group - - located in humans at chromosome lq21.3-32.3, in mice as a 30 cM segment of distal chromosome 1 (Seldin et al., 1988), and in rats as linkage group X on chromosome 13 (Pravenec et al., 1990). Other members of the synteny group include genes for C4 binding protein, fumarate hydratase, peptidase 3 (PEPC), a-spectrin and antithrombin III, and for the latter we have found odds of 6 : 1 in favour of linkage in one essential hypertensive pedigree (R.Y.L. Zee, L.-H. Ying, B.J. Morris and L.R. Griffiths, unpublished). Genes so far shown to be linked to R E N include
C16
those for the B subunit of coagulation factor XIII (Griffiths et al., 1989) and C4 binding protein (Searle et al., 1989). Obviously a myriad of other genes will eventually be identified in this region of the genome; the existence of the synteny group may well help in applying rat data to humans should the hypertension gene not be that for renin itself.
Renin expression in hypertension The manifestation of a hypertensive renin genotype is most likely to involve abnormal gene regulation and therefore renin production in the kidney, or some other renin-synthesizing tissue, or both (Morris, 1986; Ekker et al., 1989b; Lenz and Sealey, 1990). Extrarenal tissues seem to synthesize exclusively prorenin (Morris and Lumbers, 1972; Catanzaro et al., 1983; Morris, 1986), which might nevertheless generate angiotensin by unfolding transiently at local tissue sites (Sealey and Rubattu, 1989; Lenz and Sealey, 1990). An elevatioin in renin gene expression, as reflected in increased renin mRNA, occurs in the kidneys of SHR before they develop hypertension, although once hypertension becomes established renin m R N A reverts to control (WKY) levels (Samani et al., 1989b). Brain, adrenal, liver, heart and aorta also showed this early increase; however, measurements in these tissues need to be confirmed, especially as the exquisitively sensitive PCR technique could not detect transcripts in heart and aorta (Ekker et al., 1989b) - - which fits with criticisms of previous data levelled by Lenz and Sealey (1990). By PCR, adrenal, hypothalamus and pituitary, contained, respectively, 1%, 0.1% and 0.01% of the concentration in kidney, where production is by cells occupying less than 0.01% of kidney weight, so that it is hardly surprising that measurements in extrarenal tissues by older techniques have been called into question.
Transgenic experiments The amount of D N A needed for expression of the renin gene in vivo, as well as strain- and tissue-specificity, has been studied by transgenic experiments in which renin genes from strains of mice with one (Ren-1 c) or two (Ren-1 a and Ren-2)
renin genes have been introduced into the germline. For example, the 'duplicated' Ren-2 gene, including either 2.5 kb (Tronik et al., 1987) or 5.3 kb (Mullins et al., 1989) of upstream DNA, has been introduced into the Ren-1 c mouse embryo, and since the resulting transgenic mice display tissue-specific expression of the transgene, DNA extending no more than 2.5 kb upstream is sufficient to achieve this. Although R e n - U mice do not normally express renin in their adrenal, the Ren-2 transgene is expressed; and since this was restricted to the X zone in females, whereas cyclical expression in the X-zone and zona fasciculata occurs during oestrus in wild-type Ren-2 mice, Mullins et al. (1989) suggested that cell-specific expression in the adrenal is mediated in trans by at least one additional locus. In the kidney, dietary NaCI loading can suppress expression of a Ren-1 d transgene, indicating that responsiveness to this physiological stimulus is mediated somewhere between < 5 kb upstream and < 4 kb downstream (Miller et al., 1989). Human renin transgenes (with 3 kb of 5'- and 1.2 kb of 3'-flanking DNA) also display tissue-specific expression in mice (Fukamizu et al., 1989), suggesting that transcriptional activation is not species-specific in the tissues examined. As discussed earlier, tlae amount of upstream DNA actually involved in directing the promoter in a tissue-specific manner may in fact be as little as 0.6 kb. The most exciting transgenic work to date, however, was reported recently by Mullins et al. (1990) who produced fulminating hypertension in rats by making them transgenic for the duplicated mouse gene Ren-2, which is normally expressed at a very high level in the mouse submandibular gland (Morris et al., 1980; Catanzaro et al., 1983, 1985). The stably integrated transgene was expressed only at very low levels in the kidney; though plasma renin, angiotensin I and angiotensin II were depressed the rats developed chronic sustained hypertension. In the adrenal cortex, however, renin m R N A was elevated 1000-fold; since adrenal renin content was increased only 4-fold, it is possible that rapid secretion was occurring. The high adrenal renin may have accounted for an elevation seen in urinary aldosterone and corticosterone, although such steroids were regarded as contributing to only a
C17 p o r t i o n of the rise in b l o o d pressure. T h e transgene was also expressed in brain, pituitary, aorta, a nd m a n y o t h er tissues, but, n o t a b l y (in relation to the discussions above), n o t in the s u b m a n d i b u lar gland. C o n s i s t e n t with high e x t r a r e n a l expression, circulating p r o r e n i n was m a r k e d l y e l e v a t e d and, if it i n d e e d has transient activity, w o u l d c o n t r i b u t e to the h y p e r t e n s io n . W h a t e v e r the m e c h a n i s m , it is nevertheless clear that the raised b l o o d pressure reflects the presence of the renin t r a n s g e n e and, in particular, its extrarenal expression. Finally, a l t h o u g h this is clearly a f a s c i n a ti n g ne w m o d e l of h y p er t e n s io n , there is n o t h i n g to suggest that it will tell us what causes essential h y p e r t e n s i o n in h u m a n s .
Conclusion T h e d e m o n s t r a t i o n of linkage of allelic variants of the renin gene to h y p e r t e n s i o n in D a h l - s a l t sensitive an d s p o n t a n e o u s l y h y p e r t e n s i v e rats, a n d the c a p a c i t y o f a renin t r a n s g e n e to cause h y p e r tension in rats, has o p e n e d up n e w p r o s p e c t s for a role of renin in h u m a n essential h y p e r t e n s i o n . Studies p r o c e e d i n g in parallel to d e t e r m i n e the m e c h a n i s m of c o n t r o l o f the renin gene at the m o l e c u l a r level co u ld in time c o n v e r g e with the genetic studies in describing the specific D N A v a r i a n t responsible. S h o u l d this o c c u r then o n e piece in the genetic puzzle of essential h y p e r t e n sion will h a v e b e e n solved.
References Burt, D.W., Nakamura, N., Kelley, P. and Dzau, V.J. (1989) J. Biol. Chem. 264, 7357-7362. Catanzaro, D.F., Mullins, J.J. and Morris, B.J. (1983) J. Biol. Chem. 258, 7364-7368. Catanzaro, D.F., Mesterovic, N. and Morris, B.J. (1985) Endocrinology 117, 872-878. Dluhy, R.G., Hopkins, P., Hollenberg, N.K., Williams, G.H. and Williams, R.R. (1988) J. Cardiovasc. Pharmacol. 12 (Suppl. 3), S149-S154. Duncan, K.G., Haidar, M.A., Baxter, J.D. and Reudelhuber, T.L. (1987) Trans. Am. Assoc. Physicians 100, 1-9. Ekker, M., Sola, C. and Rougeon, F. (1989a) FEBS Lett. 255, 241-247. Ekker, M., Tronik, D. and Rougeon, F. (1989b) Proc. Natl. Acad. Sci. U.S.A. 86, 5155-5158. Fukamizu, A., Nishi, K., Cho, T., Saitoh, M., Nakayama, K., Ohkubo, H., Nakanishi, S. and Murakami, K. (1988) J. Mol. Biol. 201,443-450.
Fukamizu, A., Seo, M.S., Hatae, T., Yokoyama, M., Nomura, T., Katsuki, M. and Murakami, K. (1989) Biochem. Biophys. Res. Commun. 165, 826-832. Gilffiths, L.R., Board, P.G., Zwi, M.B., Morris, B.J., McLeod, J.G. and Nicholson, G.A. (1989) Hum. Hered. 39, 107-109. Hardman, J.A., Hort, Y.J., Catanzaro, D.F., Tellam, J.T., Baxter, J.D., Morris, B.J. and Shine, J. (1984) DNA 3, 457-468. Harrap, S.B. (1986) Hypertension 8, 572-582. Herr, W. and Clarke, J. (1986) Cell 45, 461-470. Knudsen, K.D., Dahl, L.K., Thompson, K., Iwai, J. and Leitl, G. (1970) J. Exp. Med. 132, 976-980. Kurtz, T.W., Simonet, L., Kabra, P.M., Wolfe, S., Chan, L. and Hjelle, B.L. (1990) J. Clin. Invest. 85, 1328-1332. Lenz, T. and Sealey, J.E. (1990) in Hypertension, Pathophysiology, Diagnosis, and Management (Laragh, J.H. and Brenner, B.M., eds.), pp. 131-328, Raven Press, New York. Lindpainter, K., Takahashi, S. and Ganten, D. (1990) J. Hypertens. 8, 763-773. Miller, C.J., Samani, N.J., Carter, A.T., Brooks, J.l. and Brammar, W.J. (1989) J. Hypertens. 7, 861-863. Morris, B.J. (1986) Clin. Sci. 71,345-355. Morris, B.J. and Griffiths, L.R. (1988) Biochem. Biophys. Res. Commun. 150, 219-224. Morris, B.J. and Lumbers, E.R. (1972) Biochim. Biophys. Acta 289, 385-391. Moils, B.J., de Zwart, R.T. and Young, J.A. (1980) Experientia 36, 1333-1334. Mullins, J.J., Sigmund, C.D., Kane-Haas, C., McGowan, R.A. and Gross, K.W. (1989) EMBO J. 8, 4065-4072. Mullins, J.J., Peters, J. and Ganten, D. (1990) Nature 344, 541-544. Naftilan, A.J., Williams, R., Burt, D., Paul, M., Pratt, R.E., Hobart, P., Chirgwin, J. and Dzau, V.J. (1989) Hypertension 14, 614-618. Pravenec, M., Simonet, L., Kren, V., Kunes, J., Levan, G., Szpirer, J., Szpirer, C. and Kurtz, T. (in press) Genomics. Rapp, J.P. and Wang, S.-M. (1990) in Hypertension, Pathophysiology, Diagnosis, and Management (Laragh, J.H. and Brenner, B.M., eds.), pp. 955-964, Raven Press, New York. Rapp, J.P., Wang, S.-M. and Dene, H. (1989) Science 243, 542-544. Rapp, J.P., Wang, S.-M. and Dene, H. (1990) Am. J. Hypertens. 3, 391-396. Samani, N.J., Brammar, W.J. and Swales, J.D. (1989a) J. Hypertens. 7, 249-254. Samani, N.J., Swales, J.D. and Brammar, W.J. (1989b) Clin. Sci. 77, 629-636. Samani, N.J., Vincent, M., Sassard, J., Henderson, I.W., Kaiser, M. and Swales, J.D. (1990) J. Hypertens. 8 (Suppl. 3), $62 (abstract). Sealey, J.E. and Rubattu, S. (1989) Am. J. Hypertens. 2, 358-366. Searle, A.G., Peters, J. and Lyon, M.F. (1989) Ann. Hum. G~net. 53, 89-140. Seldin, M.F., Morse, H.C., Reeves, J.P., Scilbner, C.L., LeBoeuf, R.C. and Steinberger, A.D. (1988) J. Exp. Med. 167, 688-693.
C18 Shaw, K.J., Do, Y.S., Kjos, S., Anderson, P.W., Shinagawa, T., Dubeau, L. and Hseuh, W.A. (1989) J. Clin. Invest. 83, 2085-2092. Smith, D.L. and Morris, B.J. (1990) Submitted for publication. Tronik, D.A., Dreyfus, M., Babinet, C. and Rougeon, F. (1987) EMBO J. 6, 983-987.
Tronik, D., Ekker, M. and Rougeon, F. (1988) Gene 69, 71-80. Wang, S.-M. and Rapp, J.P. (1989) Mol. Endocrinol. 3, 288294. Webb, D.J., Cruikshank, J.K., Barley, J. and Carter, N.D. (1990) J. Hypertens. 8 (Suppl. 3), $71 (abstract).
C13
MOLCEL 02464
At the Cutting Edge Molecular genetics links renin to hypertension Brian J. Morris Molecular Biology & Hypertension Laboratory, Department of Physiology, The University of Sydney, N.S. IV. 2006, A ustralia
(Accepted 13 December 1990)
Key words: Restriction fragment length polymorphism; Dahl rat; Spontaneously hypertensive rat; Renin transgene; Gene control
Essential hypertension Renin is pivotal to blood pressure regulation, being rate limiting in a dual enzyme cascade in which renin generates angiotensin I from which converting enzyme then produces angiotensin II, which in turn exerts the ultimate physiological actions. Whether renin also has a role in the pathogenesis of the genetic predisposition to essential hypertension has been a vexed question. Restriction fragment length polymorphisms (RFLPs) of the renin gene correlate with differences in plasma renin activity between AfroCarribean and European subjects (Webb et al., 1990), though segregation with essential hypertension could not be shown (Morris and Griffiths, 1988; Naftilan et al., 1989; Webb et al., 1990). Given the extent of human genetic heterogeneity and the expectation that more than one gene will be involved in essential hypertension, large multigenerational pedigrees with multiple affected individuals will be required for appropriate analyses. That the renin gene might have a role is nevertheless supported by recent findings with genetically pure strains of rats commonly used as genetic models of hypertension, the spontaneously hypertensive rat (SHR) and the Dahl salt-sensitive hypertensive (DS) rat. The DS rat resembles 'low renin' essential hypertension, whereas in the SHR,
Address for correspondence: Dr B.J. Morris, Molecular Biology&Hypertension Laboratory, Department of Physiology (Building F13), The University of Sydney, N.S.W. 2006, Australia.
renal function and renin content resemble those in 'nonmodulating', highly hereditable essential hypertension (Dluhy et al., 1988). In DS and S H R 4 independently segregating loci have been estimated to contribute to the elevated blood pressure (Knudsen et al., 1970; Harrap, 1986). Dahl salt-sensitive rats In the Sprague-Dawley strain of Rattus norvegicus there are 46 tandem 37 bp repeats in the first intron of the renin gene (Fukumizu et al., 1988), which thus represents a hot spot for recombination. This 1.7 kb region is flanked by B g l I I sites 2.3 kb apart (Fig. 1). In DS rats BglII gives a 2.7 kb band, indicating an insertion, and in D R (a control strain resistant to most hypertensive stimuli) the band is 1.5 kb, indicating a deletion (Wang and Rapp, 1989). Studies of F 2 and F 1 x DS populations have shown that one dose of the DS allele was associated with a 10 m m Hg rise in blood pressure, and two with a 20 m m Hg rise, i.e., 20% of the blood pressure elevation ( R a p p et al., 1989, 1990; R a p p and Wang, 1990). Blood pressure did not increase in F t x DR, which are 75% influenced by D R genes, suggesting that genes at other unidentified loci modify the blood pressure effect of the DS renin allele (Rapp et al., 1990). Renin is low and relatively unresponsive in DS rats, but high and hyper-responsive in DR. Although high blood pressure causes physiological suppression of renin, in DS rats renin is low before the rats become hypertensive ( R a p p et al., 1989), consistent with inherently low transcrip-
C14
DAHL RATS Salt-senslUve (Hypertensive)
t
(sT)
t
Salt-resistant (Normotenslve) (25)
NORMAL LABORATORY RATS l]n
Be. II
Exon I
SPONTANEOUSLY HYPERTENSIVE RAT WISTAR-KYOTO NORMOTENSIVE RAT
repeated sequence)
I
~ ~1,
(17)
Exon 2
1 kb
Exon 3
I
~1'
(34)
Fig. 1. Repetitive DNA sequences in the first intron of the rat renin gene (which has 9 exons). The estimated number of repeats for each strain is indicated in parentheses. Strain differences were detected as a result of a Bglll RFLP, where Bglll sites are indicated by arrows. The fragment lengths shown for SHR differ from those reported by Samani et al. (1989a) and Kurtz et al. (1990), who indicated a band of 1.7 kb for SHR and 2.8 kb for Sprague-Dawley, whereas restriction map and nucleotide sequence data predict a 2.3 kb band (Fukamizu et al., 1988). Samani and coworkers also found a 2.8 kb band for Wistar and Lewis rats; for WKY it was 2.3 kb. tional activity of the renin gene. Since repetitive D N A can s u p p r e s s t r a n s c r i p t i o n in a d o s e - d e p e n d e n t m a n n e r ( H e r r a n d Clarke, 1986) it is t e m p t i n g to s p e c u l a t e that the i n t r o n 1 r e p e a t s m a y have a silencer function. A l t e r n a t i v e l y , this R F L P m a y be m e r e l y serving as a m a r k e r for o t h e r difference(s) in the gene ( W a n g a n d R a p p , 1989) that d o affect t r a n s c r i p t i o n . W h i c h e v e r is the case, h o w low renin might reflect a causative role is n o t clear.
Spontaneously hypertensive rat T h e r e are o n l y 17 copies o f the t a n d e m r e p e a t in S H R , c o m p a r e d with 34 in the n o r m o t e n s i v e W i s t a r - K y o t o ( W K Y ) c o n t r o l ( S a m a n i et al., 1989a) (Fig. 1). S H R have increased renin, consistent with the d e l e t i o n of repetitive, p u t a t i v e ' s i l e n c e r ' D N A ; K u r t z et al. (1990) thus specu-
l a t e d that h y p e r t e n s i o n in the S H R a n d n o n m o d ulating essential h y p e r t e n s i o n m a y be r e l a t e d to the renin gene. In the s t r o k e - p r o n e S H R , however, L i n d p a i n t e r et al. (1990) c o u l d n o t d e m o n s t r a t e cosegregation of b l o o d p r e s s u r e with r e n i n genotype. In the studies b y K u r t z et al. (1990) a n F 2 p o p u l a t i o n was d e r i v e d from i n b r e d S H R a n d i n b r e d n o r m o t e n s i v e Lewis rats, a n d s h o w e d that b l o o d pressure in rats i n h e r i t i n g a single S H R r e n i n allele was significantly g r e a t e r ( b y - 20 m m Hg) t h a n t h a t in rats inheriting o n l y the Lewis allele ( K u r t z et al., 1990). Similarly, r e c o m b i n a n t i n b r e d strains d e r i v e d f r o m S H R × B r o w n - N o r w a y rats also s h o w e d c o s e g r e g a t i o n of the S H R allele with increase in b l o o d p r e s s u r e ( P r a v e n e c et al., 1990). Studies with o t h e r genetically h y p e r t e n s i v e strains, however, suggest that the repetitive D N A is n o t functional: the L y o n rat, for e x a m p l e , has
C15 28 copies of the repeat in hypertensive and hypotensive, but not in normotensive strains, and Milan hypertensive and normotensive rats are similar with respect to the BgllI R F L P (Samani et al., 1990). The R F L P may thus have no functional significance in the SHR, but serve as a linkage marker of a site elsewhere in the renin gene, its regulatory DNA, or of a neighbouring gene that contributes to the hypertension. In this context, it is of interest that at the 13th International Society of Hypertension Meeting (Montreal, June 1990), R a p p reported that when S H R are crossed with DS rats, blood pressure increases only when salt is given, thus demonstrating predomination of the 'salt-hypertension gene(s)' over those responsible for 'spontaneous' hypertension; presumably, renin may be one of these genes. Gene control mechanisms There have been no direct studies of cis-acting influences of intron 1 D N A in the renin gene; only the 5'-flanking D N A has been studied, and then only for human and mouse in cell lines that do not synthesize renin. In primary cultures of chorion, which produce renin from contaminating decidual cells (Shaw et al., 1989), the first 0.6 kb of human renin (REN) upstream D N A was sufficient to direct the transient expression of a reporter gene (Duncan et al., 1987). That this D N A may also be sufficient for tissue-specificity of expression is supported by the lack of expression of constructs in amnion cells. This active proximal 0.6 kb segment also displays high inter-species homology, whereas D N A further upstream has low homology and does not affect promoter activity (Smith and Morris, 1990). Within the active region an enhancer resides in the - 0 . 5 to - 0 . 1 kb D N A , and a negative element is located within 0.1 kb of the promoter (Burt et al., 1989; Smith and Morris, 1990). Similar studies for mouse have led to a different conclusion, however - - that inactivity of the renin gene in ceils that do not normally produce renin is not due to repression, but rather, cells that synthesize renin do so because they possess specific trans-acting factor(s) that target enhancer(s) close to the renin gene (Ekker et al., 1989a). Ekker et al. made this suggestion because when
they introduced a SV40 enhancer (which is utilized by all cells) into their constructs the activity of the renin promoter changed from zero to a level that was similar over a range of cells, none of which normally synthesize renin. Activity was similarly conferred on the human promoter in amnion cells by insertion of a SV40 enhancer (Duncan et al., 1987). N o r m a l mouse upstream D N A differs from human (and rat) by the presence of a 0.5 kb transposon-like insertion (Tronik et al., 1988), which appears to be a target for tissue-specific trans-acting-factor(s), and the existence of which has been used to explain why renin expression is high in mouse submandibular gland but completely absent from the same tissue of rats (Morris et al., 1980; Tronik et al., 1988). If enhancer(s) in this D N A have a dominating influence, then the mechanism Ekker et al. propose might apply only to mouse. The site of the insertion, which is at - 7 5 and - 9 2 relative to rat and human, respectively, is in the vicinity of the negative regulatory element found in the h u m a n studies and if disrupted would further account for species differences. The implications for hypertension are that putative variants of the renin gene that involve disrupted enhancer or silencer elements would give altered renin production; eventually one or other of the R F L P s may turn out to reflect such alterations in the renin D N A . Conserved synteny group Even if renin R F L P s serve only as markers for some other nearby gene that is causal in the rat models, this is still relevant to hypertension in humans since the section of genome containing the renin gene is part of a conserved synteny group - - located in humans at chromosome lq21.3-32.3, in mice as a 30 cM segment of distal chromosome 1 (Seldin et al., 1988), and in rats as linkage group X on chromosome 13 (Pravenec et al., 1990). Other members of the synteny group include genes for C4 binding protein, fumarate hydratase, peptidase 3 (PEPC), a-spectrin and antithrombin III, and for the latter we have found odds of 6 : 1 in favour of linkage in one essential hypertensive pedigree (R.Y.L. Zee, L.-H. Ying, B.J. Morris and L.R. Griffiths, unpublished). Genes so far shown to be linked to R E N include
C16
those for the B subunit of coagulation factor XIII (Griffiths et al., 1989) and C4 binding protein (Searle et al., 1989). Obviously a myriad of other genes will eventually be identified in this region of the genome; the existence of the synteny group may well help in applying rat data to humans should the hypertension gene not be that for renin itself.
Renin expression in hypertension The manifestation of a hypertensive renin genotype is most likely to involve abnormal gene regulation and therefore renin production in the kidney, or some other renin-synthesizing tissue, or both (Morris, 1986; Ekker et al., 1989b; Lenz and Sealey, 1990). Extrarenal tissues seem to synthesize exclusively prorenin (Morris and Lumbers, 1972; Catanzaro et al., 1983; Morris, 1986), which might nevertheless generate angiotensin by unfolding transiently at local tissue sites (Sealey and Rubattu, 1989; Lenz and Sealey, 1990). An elevatioin in renin gene expression, as reflected in increased renin mRNA, occurs in the kidneys of SHR before they develop hypertension, although once hypertension becomes established renin m R N A reverts to control (WKY) levels (Samani et al., 1989b). Brain, adrenal, liver, heart and aorta also showed this early increase; however, measurements in these tissues need to be confirmed, especially as the exquisitively sensitive PCR technique could not detect transcripts in heart and aorta (Ekker et al., 1989b) - - which fits with criticisms of previous data levelled by Lenz and Sealey (1990). By PCR, adrenal, hypothalamus and pituitary, contained, respectively, 1%, 0.1% and 0.01% of the concentration in kidney, where production is by cells occupying less than 0.01% of kidney weight, so that it is hardly surprising that measurements in extrarenal tissues by older techniques have been called into question.
Transgenic experiments The amount of D N A needed for expression of the renin gene in vivo, as well as strain- and tissue-specificity, has been studied by transgenic experiments in which renin genes from strains of mice with one (Ren-1 c) or two (Ren-1 a and Ren-2)
renin genes have been introduced into the germline. For example, the 'duplicated' Ren-2 gene, including either 2.5 kb (Tronik et al., 1987) or 5.3 kb (Mullins et al., 1989) of upstream DNA, has been introduced into the Ren-1 c mouse embryo, and since the resulting transgenic mice display tissue-specific expression of the transgene, DNA extending no more than 2.5 kb upstream is sufficient to achieve this. Although R e n - U mice do not normally express renin in their adrenal, the Ren-2 transgene is expressed; and since this was restricted to the X zone in females, whereas cyclical expression in the X-zone and zona fasciculata occurs during oestrus in wild-type Ren-2 mice, Mullins et al. (1989) suggested that cell-specific expression in the adrenal is mediated in trans by at least one additional locus. In the kidney, dietary NaCI loading can suppress expression of a Ren-1 d transgene, indicating that responsiveness to this physiological stimulus is mediated somewhere between < 5 kb upstream and < 4 kb downstream (Miller et al., 1989). Human renin transgenes (with 3 kb of 5'- and 1.2 kb of 3'-flanking DNA) also display tissue-specific expression in mice (Fukamizu et al., 1989), suggesting that transcriptional activation is not species-specific in the tissues examined. As discussed earlier, tlae amount of upstream DNA actually involved in directing the promoter in a tissue-specific manner may in fact be as little as 0.6 kb. The most exciting transgenic work to date, however, was reported recently by Mullins et al. (1990) who produced fulminating hypertension in rats by making them transgenic for the duplicated mouse gene Ren-2, which is normally expressed at a very high level in the mouse submandibular gland (Morris et al., 1980; Catanzaro et al., 1983, 1985). The stably integrated transgene was expressed only at very low levels in the kidney; though plasma renin, angiotensin I and angiotensin II were depressed the rats developed chronic sustained hypertension. In the adrenal cortex, however, renin m R N A was elevated 1000-fold; since adrenal renin content was increased only 4-fold, it is possible that rapid secretion was occurring. The high adrenal renin may have accounted for an elevation seen in urinary aldosterone and corticosterone, although such steroids were regarded as contributing to only a
C17 p o r t i o n of the rise in b l o o d pressure. T h e transgene was also expressed in brain, pituitary, aorta, a nd m a n y o t h er tissues, but, n o t a b l y (in relation to the discussions above), n o t in the s u b m a n d i b u lar gland. C o n s i s t e n t with high e x t r a r e n a l expression, circulating p r o r e n i n was m a r k e d l y e l e v a t e d and, if it i n d e e d has transient activity, w o u l d c o n t r i b u t e to the h y p e r t e n s io n . W h a t e v e r the m e c h a n i s m , it is nevertheless clear that the raised b l o o d pressure reflects the presence of the renin t r a n s g e n e and, in particular, its extrarenal expression. Finally, a l t h o u g h this is clearly a f a s c i n a ti n g ne w m o d e l of h y p er t e n s io n , there is n o t h i n g to suggest that it will tell us what causes essential h y p e r t e n s i o n in h u m a n s .
Conclusion T h e d e m o n s t r a t i o n of linkage of allelic variants of the renin gene to h y p e r t e n s i o n in D a h l - s a l t sensitive an d s p o n t a n e o u s l y h y p e r t e n s i v e rats, a n d the c a p a c i t y o f a renin t r a n s g e n e to cause h y p e r tension in rats, has o p e n e d up n e w p r o s p e c t s for a role of renin in h u m a n essential h y p e r t e n s i o n . Studies p r o c e e d i n g in parallel to d e t e r m i n e the m e c h a n i s m of c o n t r o l o f the renin gene at the m o l e c u l a r level co u ld in time c o n v e r g e with the genetic studies in describing the specific D N A v a r i a n t responsible. S h o u l d this o c c u r then o n e piece in the genetic puzzle of essential h y p e r t e n sion will h a v e b e e n solved.
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