Molecular phenotyping for analyzing subtle genetic effects in mice: Application to an angiotensinogen gene titration

Abstract

Essential hypertension is a deleterious condition with a multifactorial etiology that includes both genetic and environmental determinants. However, despite decades of intense research into the physiological mechanisms that regulate blood pressure (BP), the primary genetic determinants of essential hypertension remain elusive. A substantial source of the difficulties associated with attempts to understand the regulation of BP is the existence of sophisticated homeostatic systems that permit desirable physiological changes in biological variables in vivo but prevent the changes from extending into pathological ranges. These homeostatic changes, combined with environmental effects and the difficulties of obtaining measurements of BP sufficiently precise to detect small differences, make dissection of the hypertensive phenotype difficult.

Because humans are genetically very heterogenous, polymorphic forms of genes are common. Consequently, among the polymorphic forms, it is reasonable to expect that different alleles will exist that dictate expression at different intrinsic levels. Such genetic variations may cause BP changes if the recruiting homeostatic adjustments are incomplete. The M235 and T235 alleles of the human angiotensinogen gene (AGT), described by Jeunemaitre et al. (1), affect BP. Thus, it is important to understand in detail the homeostatic adjustments that occur in response to this type of genetic alteration and to identify genes that are called into action during homeostasis. The responding genes themselves then become candidates for having effects on BP. Obviously, however, a precise analysis of this type is difficult in humans because of their heterogeneous genetic background.

For these reasons, we have recently been carrying out experiments in mice with the aim of identifying genes whose quantitative expression affects BP or induces homeostatic compensations. To do this, we first use targeted gene disruption and gene duplication to generate mice having different numbers of the target gene (2) and then use this “gene titration” series of mice to uncover changes that directly influence BP and those that represent homeostatic compensations. Our first application of this strategy has centered on the renin-angiotensin system (RAS), which plays a central role in salt and water homeostasis and the maintenance of vascular tone. Angiotensinogen (AGT) is the sole source of angiotensin II (Ang II), the most active peptide of the RAS, which is produced from AGT by the serial actions of renin and the angiotensin-converting enzyme. Of all of the components in the RAS, AGT is the one most definitively identified as genetically important in the causation of essential hypertension in humans (1, 3) and in animals (4–6).

The mice we have studied here carry one, two, three, or four functional copies of the murine wild-type mouse angiotensinogen gene (Agt) at its normal chromosomal location (2, 6). Plasma AGT levels increase progressively from 35% in the one-copy mice to 145% of normal in the four-copy animals, and their BPs show significant and almost linear increases of ≈8 mmHg (1 mmHg = 133 Pa) per gene copy, even though their normal homeostatic systems are intact. Thus, the ranges of AGT alteration and BP in the mice include those observed in humans with variant AGT alleles. We have reported on the nature of the homeostatic compensations that occur in the one-copy mice (7). In the present article we extend this work to include increases as well as decreases in the expression of AGT, and we move from studying expression at the level of proteins and peptides to the study of expression at the level of mRNA. We do so for several reasons. First, the quantitation of the proteins and peptides requires a unique method for each component. Second, distinguishing between related components is often difficult. Third, sensitivities and precision are not always adequate. Fourth, high-throughput determinations are only available for some components. These difficulties can all be circumvented in experimental animals when expression is studied at the mRNA level and quantitative reverse transcription (RT)-PCR is used in conjunction with automated RNA preparation. Our application of this molecular phenotyping system to animals in the Agt gene titration series illustrates its power for recognizing subtle phenotypic changes in animals with minimal genetic differences and reveals several unexpected findings.

Values are pg/μg of total RNA ± SEM; n = 5 for each genotype and gender.

Acknowledgments

Abbreviations