Four-week inhibition of the renin-angiotensin system in spontaneously hypertensive rats results in persistently lower blood pressure with reduced kidney renin and changes in expression of relevant gene networks

Abstract

Aims: Prevention of human hypertension is an important challenge and has been achieved in experimental models. Brief treatment with renin-angiotensin system (RAS) inhibitors permanently reduces the genetic hypertension of the spontaneously hypertensive rat (SHR). The kidney is involved in this fascinating phenomenon, but relevant changes in gene expression are unknown.

Methods and results: In SHR, we studied the effect of treatment between 10 and 14 weeks of age with the angiotensin receptor blocker, losartan, or the angiotensin-converting enzyme inhibitor, perindopril [with controls for non-specific effects of lowering blood pressure (BP)], on differential RNA expression, DNA methylation, and renin immunolabelling in the kidney at 20 weeks of age. RNA sequencing revealed a six-fold increase in renin gene (Ren) expression during losartan treatment (P < 0.0001). Six weeks after losartan, arterial pressure remained lower (P = 0.006), yet kidney Ren showed reduced expression by 23% after losartan (P = 0.03) and by 43% after perindopril (P = 1.4 × 10-6) associated with increased DNA methylation (P = 0.04). Immunolabelling confirmed reduced cortical renin after earlier RAS blockade (P = 0.002). RNA sequencing identified differential expression of mRNAs, miRNAs, and lncRNAs with evidence of networking and co-regulation. These included 13 candidate genes (Grhl1, Ammecr1l, Hs6st1, Nfil3, Fam221a, Lmo4, Adamts1, Cish, Hif3a, Bcl6, Rad54l2, Adap1, Dok4), the miRNA miR-145-3p, and the lncRNA AC115371. Gene ontogeny analyses revealed that these networks were enriched with genes relevant to BP, RAS, and the kidneys.

Conclusion: Early RAS inhibition in SHR resets genetic pathways and networks resulting in a legacy of reduced Ren expression and BP persisting for a minimum of 6 weeks.

Keywords: Hypertension; Prevention; Renin; SHR.

Graphical Abstract

Figure 1

Outline of the basic experimental design showing the five experimental groups, their treatments, and experimental rationale. Treatment periods are shown by the wide grey vertical bars against age. The data obtained are shown in boxes at 14 and 20 weeks of age, spanning the groups analysed.

Figure 2

Direct MAP in male SHR from 10 to 20 weeks of age. Animals received saline (VEH, n = 2, blue), losartan (LOS, n = 2, green), or perindopril (PER, n = 3, orange) between 10 and 14 weeks of age or hydralazine (HYD, n = 2, purple) between 10 and 20 weeks of age. Weekly values are summarized as estimated marginal means from the repeated measures ANOVA, and error bars represent the SEM. The P values represent the overall effect of treatment during (10–14 weeks) and after treatment for LOS and PER (15–20 weeks) by repeated measures ANOVA. Individual treatment comparisons with VEH are given in the text.

Figure 3

Changes in expression of 13 candidate genes at 14 and 20 weeks in losartan and control SHR animals. Gene expression (counts) is displayed individually (black points) and summarized as boxplots (vehicle, grey; losartan, red). FDR q-values (***<0.001; **<0.01; *<0.05; ns > 0.05) are from differential gene expression analyses.

Figure 4

Leading differentially expressed genes and their associations. Including 35 mRNAs (black text) from Table 1, 45 miRNAs (blue text) from Table 2, and 15 lncRNAs (gold text). Hub genes identified from the WGCNA analysis are in bold text. For each of the 35 mRNAs, the leading 2 negative correlations < −0.8 (if present) with non-coding genes are shown as thicker lines representing larger negative associations (see Supplementary material online, Figure S2 and Table S10).

Figure 5

Patterns of methylation across Ren and LOC102550525. Detailed view of methylation % by vehicle and losartan animals (point shading reflects count number per CpG). Green vertical lines show position of variants (see variant description in Supplementary material online, Table S18) in and around Ren. General promoter region for Ren is indicated by dark blue dashed lines. Position of CpG clusters (2–6) across Ren is shown which corresponds to the cluster number in CpG cluster analysis (see Supplementary material online, Table S16).

Figure 6

Renin immunolabelling in kidneys from 14-week (A and B) and 20-week-old (C–F) SHR treated with vehicle (n = 12), losartan (n = 13), perindopril (n = 8), and hydralazine (n = 6). Three μm paraffin sections counterstained with haematoxylin. G, glomerulus. Arrows denote renin immunolabelling (brown). Scale bar, 100 μm.