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

Abstract

The angiotensinogen M235T polymorphism in humans is linked to differential expression of the human angiotensinogen gene (AGT) gene and hypertension, but the homeostatic responses resulting from this polymorphism are not known. We therefore investigated how mice respond to five genetically determined levels of mouse angiotensinogen gene (Agt) expression covering the range associated with the M235T variants. By using high-throughput molecular phenotyping, tissue RNAs were assayed for expression of 10 genes important in hypertension. Significant positive and negative responses occurred in both sexes as Agt expression increased twofold, including a three-fold increase in aldosterone synthase expression in adrenal gland, and a two-fold decrease in renin expression in kidney. In males, cardiac expression of the precursor of atrial natriuretic peptide B and of adrenomedullin also increased approximately twofold. The relative expression of all genes studied except Agt differed significantly in the two sexes, and several unexpected relationships were encountered. A highly significant correlation between renal expression of the angiotensin type 1a receptor and kallikrein, independent of Agt genotype, is present in females (P < 0.0001) but not males (P = 0.4). The correlation between blood pressure (BP) and liver Agt expression within the five Agt genotypes is significant in females (P = 0.0005) but not in males (P = 0.2), whereas correlation of BP with differences between the genotypes is less in females (P = 0.06) than in males (P = 0.001). The marked gender differences in gene expression in wild-type mice and the changes induced by moderate alterations in Agt expression and BP emphasize the need to look for similar differences in humans.

Figure 1

AGT mRNA levels in mice with different Agt genotypes. The levels of mRNAs in the livers are presented as percentages of wild type (1/1). Five males and five females were used for each genotype. The bars show means ± SEM.

Figure 2

Comparisons of gene expression and BP in individual female mice. The left lower half of the figure presents the data in the form of a bivariate scatterplot matrix of the individual measurements and the corresponding 95% confidence ellipses. In those pairwise comparisons that have a significant correlation, the dots in the squares represent individual values; the right upper half of the figure tabulates the corresponding probabilities. In the pairwise comparisons without a significant correlation, only the 95% confidence ellipses are shown. More detailed versions of Figs. 2 and 3 are provided as supporting information.

Figure 3

Comparisons of gene expression and BP in individual male mice.

Figure 4

Comparison of correlation between BP and Agt expression. The data in A (males) and B (females) are from an analysis by ANOVA of BPs and Agt genotypes. C (males) and D (females) present leverage plots of BP against hepatic Agt mRNA expression nested within the Agt genotypes. The Agt genotypes in C and D are 1/1 (○), 1/2 (●), and 2/2 (■).