UPLC-MS-Based Serum Metabolomics Reveals Potential Biomarkers of Ang II-Induced Hypertension in Mice

Abstract

Hypertension is caused by polygenic inheritance and the interaction of various environmental factors. Abnormal function of the renin-angiotensin-aldosterone system (RAAS) is closely associated with changes in blood pressure. As an essential factor in the RAAS, angiotensin II (Ang II) contributes to vasoconstriction and inflammatory responses. However, the effects of overproduction of Ang II on the whole body-metabolism have been unclear. In this study, we established a hypertensive mouse model by micro-osmotic pump perfusion of Ang II, and the maximum systolic blood pressure reached 140 mmHg after 2 weeks. By ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry, the metabolites in the serum of hypertensive model and control mice were analyzed. Partial least squares discriminant analysis (PLS-DA) in both positive and negative ionization modes showed clear separation of the two groups. Perfusion of Ang II induced perturbations of multiple metabolic pathways in mice, such as steroid hormone biosynthesis and galactose metabolism. Tandem mass spectrometry revealed 40 metabolite markers with potential diagnostic value for hypertension. Our data indicate that non-targeted metabolomics can reveal biochemical pathways associated with Ang II-induced hypertension. Although researches about the clinical use of these metabolites as potential biomarkers in hypertension is still needed, the current study improves the understanding of systemic metabolic response to sustained release of Ang II in hypertensive mice, providing a new panel of biomarkers that may be used to predict blood pressure fluctuations in the early stages of hypertension.

Keywords: LC-MS; angiotensin II; biomarkers; hypertension; metabolomics; mice; serum metabolites.

Figure 1

Sustained rise of systolic blood pressure (A) and diastolic blood pressure (B) in mice induced by the continuous release of Ang II (data are the mean ± SEM, n = 10, ***P < 0.001 vs. Control, two-way ANOVA).

Figure 2

(A,B) Total ion chromatograms of QC samples in Control (A) and Ang II groups (B) in ESI+ and ESI– mode. (C,D) Plots of PCA scores for serum samples from test mice and QC samples showing metabolites obtained in ESI+ mode (C) and ESI– mode (D). (E,F) Scatter plots of PCA scores of metabolites from the LC-MS/MS fingerprints in ESI+ mode (E) and ESI– mode (F).

Figure 3

(A,B) 3-D plots of scores from partial least-squares discriminant analysis based on the metabolic profiling data from the plasma of Ang II-induced hypertensive mice and healthy (control) mice in ESI+ (A) and ESI– mode (B) (black triangles, Ang II-induced hypertensive mice; blue circles, control mice). (C,D) Volcano plots based on P-values and fold-changes of single-dimensional tests in ESI+ mode (C) and ESI– mode (D). (E,F) Heatmaps of the differential metabolites for Ang II vs. control in ESI+ mode (E) and ESI– mode (F).

Figure 4

Metabolic pathway enrichment analysis in ESI+ mode (A) and ESI– mode (B). The size of each circle represents the number of metabolites enriched in the pathway.