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(Hypertension. 1995;26:279-284.)
© 1995 American Heart Association, Inc.

Articles

Altered Angiotensinogen Amino Acid Sequence and Plasma Angiotensin II Levels in Genetically Hypertensive Rats

A Study on Cause and Effect

Norbert Hübner; Reinhold Kreutz; Saori Takahashi; Detlev Ganten; Klaus Lindpaintner

From the Cardiovascular Division, Department of Medicine, Brigham and Women's Hospital, and the Department of Cardiology, Children's Hospital, Harvard Medical School, Boston, Mass (N.H., R.K., K.L.); the Department of Applied Biology, Kyoto (Japan) Institute of Technology (S.T.); and the Max Delbrück Center for Molecular Medicine, Berlin, Germany (D.G.).

Correspondence to Klaus Lindpaintner, MD, Cardiovascular Division, Brigham and Women's Hospital, 75 Francis St, Boston, MA 02115.

	Abstract

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Abstract The components of the renin-angiotensin system have been implicated in the development of primary hypertension in humans and genetically hypertensive rats. In humans a mutation in the angiotensinogen gene and elevated plasma angiotensinogen levels have been linked to primary hypertension. Although we had previously excluded a linkage of blood pressure to the angiotensinogen gene in the stroke-prone spontaneously hypertensive rat (SHRSP), elevated angiotensin II (Ang II) levels in this strain compared with the normotensive reference, the Wistar-Kyoto rat (WKY), prompted us to investigate further into the origins and effects of altered Ang II regulation using a range of physiological, biochemical, molecular, and genetic approaches. Ang II plasma levels determined by radioimmunoassay were confirmed to be significantly elevated in SHRSP compared with WKY. Sequence comparison among the two rat strains revealed a mutation in the coding region of the angiotensinogen gene that results in an isoleucine-to-valine substitution in SHRSP at amino acid position 154 (I154V). We performed a cosegregation analysis in an F₂ intercross cohort bred from SHRSP and WKY from the University of Heidelberg (SHRSP_HD and WKY_HD) to address the following questions: (1) whether this or another mutation of the angiotensinogen gene may be causally related to the observed differential Ang II plasma levels, (2) whether Ang II plasma levels may be correlated with blood pressure or organ hypertrophy, and (3) whether genetic linkage to the renin or angiotensin-converting enzyme (ACE) gene loci (the two classic regulatory enzymes of the renin-angiotensin system) may provide an explanation for elevated Ang II plasma levels. We measured Ang II plasma concentrations and determined systolic and diastolic pressures as well as heart rate by indwelling arterial catheters in conscious rats before and after dietary sodium loading. We found (1) no linkage of the angiotensinogen gene mutation to Ang II plasma levels, (2) no cosegregation of Ang II plasma levels with blood pressure or morphometric parameters, and (3) no linkage of Ang II plasma levels to either the renin gene or the ACE gene locus, excluding a genetically determined regulatory effect on Ang II plasma levels of either enzyme. Of all other parameters measured in this cross, only plasma renin activity showed a significant correlation with Ang II plasma levels. Thus, the demonstrated angiotensinogen mutation has no effect on plasma Ang II levels; Ang II plasma levels do not account for, do not contribute to, and are not caused by elevated blood pressure in SHRSP_HD; and altered Ang II plasma levels in SHRSP_HD are not genetically determined by the renin or ACE gene. We conclude that elevated Ang II levels in SHRSP_HD are likely to represent an independent, unrelated phenomenon that is of no direct relevance to the pathogenesis of hypertension.

Key Words: angiotensin II • hypertension, primary • genetics • linkage (genetics) • renin-angiotensin system

	Introduction

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The spontaneously hypertensive rat¹ and its close relative the SHRSP² have been used extensively as models for human primary hypertension. Numerous differences among a wide range of morphometric, physiological, and biochemical characteristics have been reported between these rats and normotensive reference strains, commonly with the implication that such differences are relevant to the pathogenesis of hypertension. It has become clear, however, that an abundance of coincidental and disease-unrelated differences exists between hypertensive and normotensive strains^{3 4} and that only approaches based on the principles of genetic linkage will be capable of recognizing biologically relevant differences. Given two inbred strains that differ in a trait (phenotype) of interest, a genetic, biochemical, morphological, etc, difference can be viewed as contributing to this trait only if it can be shown, in an intercross or backcross population bred from the two contrasting progenitor strains, to cosegregate with the trait of interest.

The RAS plays a central role in electrolyte and volume regulation and the maintenance of vascular tone. Stimulation or inhibition of this system has profound effects on circulatory parameters, in particular blood pressure. Thus, the genes encoding the components of the RAS have been viewed and investigated as prime candidate genes for hereditary hypertension in humans and experimental animals. Positive^{5 6 7 8 9 10} and negative^{11 12 13 14 15 16} linkage results have been reported for individual genes. We have previously reported that neither the renin nor the angiotensinogen gene shows cosegregation with blood pressure in the SHRSP_HD/WKY_HD cross^{11 15} (the subscript indicates origin at the University of Heidelberg) but found that a region on chromosome 10 carrying the ACE gene (among other candidates) was significantly linked to blood pressure .^{7 8} Here, we report that Ang II plasma levels in SHRSP_HD and WKY_HD are markedly different and that a point mutation in the coding region of the angiotensinogen gene results in the expression of an altered angiotensinogen protein in SHRSP_HD compared with WKY_HD. We investigated whether altered regulation of Ang II plasma levels may be explained by this difference in the amino acid sequence and whether they are related to blood pressure levels. Also, as Ang II has been postulated to have important growth-promoting effects on the cardiovascular system,^{17 18 19} we tested for cosegregation of Ang II plasma levels with a number of morphometric parameters. Last, to test whether Ang II plasma levels are genetically determined by either renin or ACE, the two enzymes necessary for the generation of Ang II, we performed linkage analysis for these gene loci and Ang II plasma levels.

	Methods

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Experimental Animals
Rats were housed three to a cage with free access to food and water during the entire study. Regular 12-hour diurnal cycles were kept. During the maintenance of an indwelling arterial catheter, rats were housed one per cage. The WKY_HD and SHRSP_HD used were obtained from the colony at the University of Heidelberg (Germany), which is derived from original Aoki-Okamoto¹ and Okamoto-Yamori² stock and has been maintained under strict inbreeding conditions for more than 40 generations.

Breeding Experiment
One male WKY_HD and one male SHRSP_HD were mated with two female SHRSP_HD (cross I) and two female WKY_HD (cross II), respectively (a reciprocal crossbreeding strategy that allows investigation of sex chromosomal effects). The F₁ progeny (n=41) were mated brother to sister, again, one male with two females each. The resulting F₂ offspring (n=115) were divided into groups according to their ancestry from a WKY_HD (n=56) or an SHRSP_HD (n=59) grandfather (reciprocal cross I and cross II). Starting with the founders, all rats were ear tagged with a running number. At the end of the experiment the rats were killed by decapitation under ether anesthesia. Several other studies conducted in this cohort have previously been communicated.^{7 8 11 15 20} In addition, 12 male and 12 female SHRSP_HD and WKY_HD each at the same age were subjected to the identical investigational protocol.

Hemodynamic Determinations
All hemodynamic measurements were carried out as previously described.¹⁵ Briefly, at 16 weeks of age all F₂ rats underwent catheterization of the right femoral artery under light ether anesthesia. After the operation the rats were allowed to recover for 24 hours. On 2 consecutive days, three sets of blood pressure and heart rate measurements were taken: twice between midnight and 4 AM and once between 9 AM and 1 PM with a Statham model P23PD pressure transducer (Gould-Statham) connected to a model 7B polygraph (Grass Instruments). During the recordings rats were shielded from the observer, and measurements were taken only while the rats were resting quietly. All measurements were taken by the same observer, who was unaware of each rat's genotype. The rats then had 1% NaCl added to their drinking water for 12 days. After cannulation of the left femoral artery, a second set of three hemodynamic measurements was obtained. On completion of the second set of measurements, NaCl was discontinued and the rats were killed 6 weeks later at the age of 24 weeks.

Plasma Ang II Concentrations and Plasma Renin Activity
For Ang II measurements, 0.5 mL blood was withdrawn from the arterial cannula at the end of the first recording session (before NaCl loading) and at the end of the last recording session (after NaCl loading). Volume was replaced by slow infusion of an identical volume of heparinized 0.9% saline. Blood samples were immediately chilled on ice. The plasma was separated by centrifugation at 10 000 rpm for 10 minutes at 4°C and stored at -80°C until analysis. Plasma Ang II concentrations were measured by radioimmunoassay as described previously.²¹ Plasma renin activity was determined by an in vitro activation assay as described in detail elsewhere.²²

Preparation of Genomic DNA
DNA was prepared from liver according to a previously published method.^{23 24} Integrity, purity, and quantity of the extracted DNA were assessed by spectrophotometric analysis and agarose gel electrophoresis.

Detection of Angiotensinogen Molecular Variant
The following primers were used to amplify, by polymerase chain reaction, a region encompassing bases 385 through 770 of exon 2 of the rat angiotensinogen gene (Genbank Accession No. L00091); sense and antisense primer sequences were 5'-TGCTGAGTGAGGCAAGAGGTGTAG-3' (nucleotides 385 through 408) and 5'-GGCAGCAAGAACTGGGTCAGTG-3' (nucleotides 749 through 770), respectively. Automated fluorescent dye dideoxyterminator sequencing was carried out subsequently with a model 373A DNA sequencing apparatus (Applied Biosystems, Inc).

Polymorphic Markers
To test for cosegregation of the angiotensinogen molecular variant I154V with Ang II plasma concentrations, we used a previously described polymerase chain reaction–typeable restriction fragment length polymorphism¹¹ that is coinherited in complete linkage disequilibrium with the mutation. Likewise, previously described polymorphisms in the renin gene (typed by restriction fragment length polymorphism analysis on Southern blots)¹⁵ and of the ACE gene (typed using a mouse-homologous CA-repeat marker)²⁵ were used to carry out linkage analysis for these genes with Ang II plasma concentrations.

Statistical Analysis
Ang II plasma concentrations in WKY_HD and SHRSP_HD rats were examined by two-way ANOVA to account for the effect of cross and sex. Pearson correlations of Ang II plasma concentrations with blood pressure values and other parameters were calculated as well as correlations with plasma renin activity in each case with values obtained both before and after sodium loading. For comparison of systolic and diastolic blood pressures, the mean value of all observations made either at baseline or after sodium loading was used for each rat. Of 115 F₂ rats phenotyped, 112 were available for genotype analysis. After the code was broken the rats were divided into different subgroups according to angiotensinogen, renin, or ACE allele pattern (WKY homozygotes, SHRSP homozygotes, or heterozygotes); sex (male or female); and type of progenitor cross (cross I or II). Three-way ANOVA was carried out (accounting for the effects of the reciprocal cross, sex, and candidate gene allele status) to detect cosegregation between each gene locus and Ang II plasma levels, again at baseline and after sodium loading. A statistical software package (Crunch Software Co) was used to carry out the calculations, and the LINKAGE package²⁶ was used to calculate Lod scores.

	Results

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Ang II Plasma Concentrations in SHRSP and WKY
Ang II plasma concentrations were significantly elevated in SHRSP_HD compared with WKY_HD both at baseline (P=.0036) and after NaCl loading (P=.025, Fig 1).

View larger version (36K): [in this window] [in a new window]   Figure 1. Bar graphs show Ang II plasma concentrations in parental WKYHD and SHRSPHD (mean±SEM; n=24 in each group).

Mutation in Exon 2 of the Angiotensinogen Gene
On resequencing the region of the angiotensinogen gene previously screened in the search for genotyping markers, we detected a single base substitution at position 513 in exon 2 replacing adenine (WKY_HD) with guanine (SHRSP_HD). This point mutation results in a predicted amino acid substitution at position 154 from isoleucine (WKY_HD) to valine (SHRSP_HD), I154V (Fig 2). The presence of this mutation was confirmed by repeated sequence analysis of additional amplicons prepared in separate reactions and of polymerase chain reaction products generated from DNA from several rats from each strain.

View larger version (18K): [in this window] [in a new window]   Figure 2. Schematic shows comparison of nucleotide and amino acid sequence between WKYHD and SHRSPHD. The bar represents the coding sequence of the angiotensinogen gene and indicates exonic structure as well as the site where Ang I is cleaved from the protein and its relation toward the Pvu II restriction fragment length polymorphism (RFLP) (C-to-T substitution at nucleotide position 495)11 and the molecular variant I154V (reflecting the A-to-G substitution at nucleotide position 513).

Cosegregation Analysis of Plasma Ang II Concentrations and Phenotype
In F₂ intercross hybrids, blood pressure values and Ang II plasma concentrations were normally distributed around the arithmetic mean of values observed in the progenitor strains, SHRSP_HD and WKY_HD. No cosegregation was found between systolic or diastolic blood pressure and Ang II plasma concentrations either at baseline or after sodium loading (Table 1). Likewise, no cosegregation was observed between Ang II plasma concentrations and any of the morphometric parameters obtained (Table 1). In addition, subgroup analysis after partitioning for sex or cross failed to show cosegregation in any particular subgroup (data not shown). In all analyses male rats had significantly higher systolic and diastolic blood pressures and lower heart rates than female rats (P<.0001), as reported previously.¹⁵ No such sexual dimorphism was seen with regard to Ang II plasma concentrations. A significant correlation was present between plasma renin activity and Ang II plasma concentrations at baseline (P<.01) and after dietary sodium loading (P<.001, Fig 3).

View this table: [in this window] [in a new window]   Table 1. Regression Coefficients and Probability Values of Plasma Ang II Levels on Hemodynamic and Morphometric Parameters

View larger version (14K): [in this window] [in a new window]   Figure 3. Scatterplots show Ang II plasma concentrations against plasma renin activity before and after dietary sodium loading.

Linkage Analysis of Plasma Ang II Concentrations and RAS Gene Allele Status
The genotype distribution for each of the RAS genes examined was not statistically different from the expected ratio for mendelian inheritance of 1:2:1 in the 112 F₂ rats investigated (see Table 2 for distribution). ANOVA and linkage analysis adjusted for the effects of sex and reciprocal cross excluded an effect of the angiotensinogen gene locus on Ang II plasma levels of the magnitude of the difference seen among the progenitor strains with a power of 88% (=.05, Table 2). Similarly, no linkage of Ang II plasma levels to the renin and ACE genes was observed. Adjustment of the analysis for two of the genes simultaneously to test for a possible interaction did not change these negative results (data not shown).

View this table: [in this window] [in a new window]   Table 2. Ang II Plasma Concentrations According to Genotype in F2 Progeny

	Discussion

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The renin-angiotensin system has been implicated in the pathogenesis of primary hypertension in humans and hereditary hypertensive rat models. Here, we report marked differences in Ang II plasma levels between SHRSP and WKY and a mutation of the coding sequence of the angiotensinogen gene that results in differential gene products in these two strains. To test the hypothesis that the mutated angiotensinogen protein may be causally related to differential Ang II levels or that Ang II levels may be associated with hypertension or organ hypertrophy, we conducted a cosegregation and linkage study. Our results lead us to reject these hypotheses. Thus, the I154V angiotensinogen mutation does not affect Ang II plasma levels, and elevated Ang II levels neither contribute to nor are the result of hypertension or organ hypertrophy in SHRSP_HD. Failure to detect linkage of Ang II plasma levels to the renin or ACE gene loci excludes the possibility that Ang II levels are genetically determined by either of these two genes and indicates that the observed correlation with plasma renin activity is due to other mechanisms.

The I154V molecular variant of the angiotensinogen gene was found in the process of defining a polymorphism for genotyping. Although the mutation is at considerable distance from the NH₂-terminal location of the cleavage site for Ang I release, its possible effects on protein folding could theoretically affect the kinetics of renin-mediated catalysis. Based on our findings that the angiotensinogen gene locus is not linked to plasma Ang II levels, we can rule out the possibility that the marked difference in Ang II levels among SHRSP_HD and WKY_HD is caused by this (or any other) sequence difference among the two strains in the coding or flanking regions of the angiotensinogen gene. Thus, the mutation is most likely biologically inert. The theoretical possibility that a specific interaction between the renin gene, which had previously been implicated in the development of hypertension in other crosses,^{6 27 28} and the angiotensinogen variant was operative was excluded by ANOVA that took both renin and angiotensinogen genotypes into account.

Studies in human hypertension have recently provided evidence that the angiotensinogen gene is linked to primary hypertension and that particular molecular variants of angiotensinogen may play a causative role in the development of hypertension.⁵ The associated finding of higher angiotensinogen plasma levels in affected subjects led to the speculation that the hypertensive angiotensinogen genotype results in higher Ang II plasma levels, providing a pathogenetic mechanism for the hypertensive effect. We have previously shown that no such genetic linkage of the angiotensinogen gene locus to blood pressure is present in the SHRSP_HD/WKY_HD cross.¹¹ Our failure to demonstrate a cosegregation of Ang II plasma levels and blood pressure indicates that there is no effect of circulating Ang II levels on blood pressure in the rat and that conversely, plasma Ang II levels are not a consequence of altered blood pressure. A number of investigations in experimental animals and humans have indicated that differential regulation of RAS components, in particular angiotensinogen^{17 18} and ACE,¹⁹ contributes to the pathogenesis of vascular and cardiac hypertrophy. In addition, there is experimental evidence from in vitro studies for growth-promoting actions of Ang II.²⁹ Therefore, the possibility that Ang II plasma levels may affect cardiac hypertrophy independent of hypertension was considered. The results of these analyses also fail to show an effect attributable to Ang II plasma levels. The negative findings concerning a role of plasma Ang II concentrations on hemodynamic and morphometric parameters do not necessarily rule out a role of the peptide on the tissue level: it has been argued that tissue RASs rather than the circulating RAS may play the premier role in long-term, tonic effects on blood pressure and organ hypertrophy.³⁰

Last, we examined whether the elevated Ang II plasma levels seen in SHRSP_HD may result from a primary, genetically determined difference in processing by either of the two enzymes involved in the activation of the catalytic cascade, renin and ACE. Linkage analysis carried out for both loci against Ang II plasma levels before and after NaCl loading did not confirm such an effect. Thus, the observed differences of plasma Ang II levels among SHRSP_HD and WKY_HD must be attributed to the direct or indirect effects of another as yet uncharacterized gene.

The correlation observed between Ang II plasma levels and plasma renin activity may point to a regulatory effect of renin on plasma Ang II levels, which would be commensurate with the classic view of this enzyme as the rate-limiting step in the cascade. The previous finding that the renin gene is not linked to either blood pressure or plasma renin activity in this cross¹⁵ is compatible with the present finding of a correlation of Ang II and renin plasma values and a lack of cosegregation of either with blood pressure. Plasma renin activation has previously been postulated to represent a secondary phenomenon, reflecting the degree of renal impairment encountered in SHRSP during the late phase of severe hypertension.³¹ If such a mechanism were to be operative, then plasma renin activity as well as Ang II levels should ultimately be found to cosegregate with the gene/genetic defect that is linked to renal impairment in these rats. This may, but need not necessarily, correspond or overlap with genes found to be responsible for blood pressure elevation. Although possible, such a mechanism does not necessarily apply to the relatively young F₂ rats studied here.

Certain caveats are in order in interpreting our results, as with any study showing a negative outcome. Although our findings rule out an effect of angiotensinogen on plasma Ang II levels of the magnitude observed in the progenitor strains with a ß error of only .12, a lesser effect of this locus on phenotype cannot be excluded because of the limited number of rats and the error inherent in measurements of the sort used in this study. Also, our results apply, strictly speaking, only to a particular developmental stage (16 weeks of age), when the major rise in blood pressure has occurred. We cannot exclude the possibility that Ang II plasma levels may be correlated to blood pressure or cardiac and vascular growth during other developmental stages or that the genes investigated may transiently influence Ang II plasma levels during developmental phases other than the one addressed here. Also, because of the differences in genetic makeup responsible for elevated blood pressure in different rat models of hypertension, the results of this investigation are not necessarily applicable to other rat strains or even to SHRSP originating from different colonies.^{4 32}

In summary, we report the characterization of an amino acid substitution in the angiotensinogen gene that distinguishes SHRSP_HD and WKY and the finding of differential levels of plasma Ang II levels in these two strains. A cosegregation analysis indicated that the mutation has no effect on altered Ang II plasma levels, that Ang II plasma levels are neither a cause nor a consequence of hypertension or cardiac hypertrophy, and that altered Ang II levels are not linked to the renin or ACE gene loci. Therefore, plasma Ang II levels appear to be influenced directly or indirectly by an as yet unknown genetic factor that is not part of the classic catalytic cascade of the RAS, and elevated Ang II plasma levels among SHRSP and WKY are a blood pressure–unrelated phenomenon. These results emphasize the importance of applying appropriate genetic testing paradigms to phenomenological differences among animal models of hereditary diseases and their respective nondiseased reference strains, before concluding that observed differences are of pathogenetic relevance.

	Selected Abbreviations and Acronyms

 

ACE = angiotensin-converting enzyme Ang = angiotensin RAS = renin-angiotensin system SHRSP = stroke-prone spontaneously hypertensive rat(s) WKY = Wistar-Kyoto rat(s)

	Acknowledgments

 
R.K. is the recipient of a Howard Hughes Postdoctoral Research Fellowship for Physicians. K.L. was supported by Research Career Development Award HL-03138 from the National Heart, Lung, and Blood Institute and by a Harcourt Charitable Foundation Young Investigator's Award.

Received February 21, 1995; first decision April 17, 1995; accepted April 26, 1995.

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