The Epigenetic Legacy of Maternal Protein Restriction: Renal Ptger1 DNA Methylation Changes in Hypertensive Rat Offspring

Abstract

Nutrient imbalances during gestation are a risk factor for hypertension in offspring. Although the effects of prenatal nutritional deficiency on the development of hypertension and cardiovascular diseases in adulthood have been extensively documented, its underlying mechanisms remain poorly understood. In this study, we aimed to elucidate the precise role and functional significance of epigenetic modifications in the pathogenesis of hypertension. To this end, we integrated methylome and transcriptome data to identify potential salt-sensitive hypertension genes using the kidneys of stroke-prone spontaneously hypertensive rat (SHRSP) pups exposed to a low-protein diet throughout their fetal life. Maternal protein restriction during gestation led to a positive correlation between DNA hypermethylation of the renal prostaglandin E receptor 1 (Ptger1) CpG island and high mRNA expression of Ptger1 in offspring, which is consistently conserved. Furthermore, post-weaning low-protein or high-protein diets modified the Ptger1 DNA hypermethylation caused by fetal malnutrition. Here, we show that this epigenetic variation in Ptger1 is linked to disease susceptibility established during fetal stages and could be reprogrammed by manipulating the postnatal diet. Thus, our findings clarify the developmental origins connecting the maternal nutritional environment and potential epigenetic biomarkers for offspring hypertension. These findings shed light on hypertension prevention and prospective therapeutic strategies.

Keywords: DNA methylation; Ptger1; epigenetics; hypertension; kidney; low protein diet; maternal nutrition; nutrigenomics; offspring; postnatal nutrition.

Figure 1

Identification of potential candidate genes and Ptger1 CpG island DNA methylation levels in D28 pup kidneys with fetal exposure to a maternal low-protein diet. (A) Experimental scheme. SHRSP, stroke-prone spontaneously hypertensive pups. (B) Integrated methylome and transcriptome analyses were used to identify genes with coupled differential expression. Ninety-five genes were identified near the CpG sites in the methylome analysis, whereas 581 genes were identified in the transcriptome analysis. Six genes were found to show fluctuating expression in both the omics analyses. (C) mRNA expression of Gnas, Ptger1, Runx1, and Tbx3. The mRNA levels were normalized to those of Actb and expressed as fold-change values. (D) Ptger1 CpG island regions and location of three primer sets (CpG①, CpG②, and CpG③). (E,F) Total DNA methylation levels of the Ptger1 CpG island measured using (E) methylome and (F) bisulfite sequencing. (G) Bisulfite sequencing analysis of the Ptger1 CpG island ①. The horizontal axis shows each evaluated CpG site. ○, unmethylated cytosine; ●, methylated cytosine. (H) Percentages of DNA methylation levels of CpG site in the total Ptger1 CpG island ① were calculated using the methylome data shown in (G). Data in (C,F,H) are expressed as mean ± standard error (SE). ** p < 0.01, * p < 0.05 vs. the D28-CN group according to Student’s t-test. CN, control sample sizes in (C,E) were n = 5, and those in (F,H) were n = 3 rats per group.

Figure 2

Ptger1 mRNA expression and Ptger1 CpG island DNA methylation levels from D5 to D28 in pup kidneys exposed to a fetal low-protein diet. (A) Experimental scheme. SHRSP: stroke-prone spontaneously hypertensive. (B) Mean normalized mRNA expression of Ptger1 over time in pup kidneys from D5 to D28 according to maternal diet and offspring age. * p < 0.05 vs. corresponding CN group; # p < 0.05 vs. D5-LP group; ### p < 0.001 vs. D5-LP group. (C) Total DNA methylation levels of Ptger1 CpG island ① in pup kidneys from D5 to D28 according to maternal diet and offspring age. ** p < 0.01, * p < 0.05 vs. each CN group; ## p < 0.01 vs. corresponding D5 group; ++ p < 0.01, + p < 0.05 vs. corresponding D10 group. (D,F) Bisulfite sequencing analysis of Ptger1 CpG island ① in D5 and D10. The horizontal axis shows each evaluated CpG site. ○: unmethylated cytosine; ●: methylated cytosine. (E,G) Percentages of DNA methylation levels of CpG site in the total Ptger1 CpG island ① were calculated from the bisulfite sequencing data shown in (D,F). ** p < 0.01, * p < 0.05 vs. the D5-CN group. Data are expressed as mean ± standard error (n = 5). Statistical analysis was performed using Student’s t-test (E,G) and Tukey’s test (B,C).

Figure 3

Effects of fetal exposure to a maternal low-protein diet and postnatal protein intake on Ptger1 mRNA expression and Ptger1 CpG island DNA methylation status in offspring kidneys (D42). (A) Experimental scheme. mCN/mLP: maternal control diet/maternal low protein diet; CN/LP/HP: offspring CN/LP/HP diet. (B) Mean normalized mRNA expression of Ptger1 in offspring kidneys at D42 according to maternal diet and offspring age. ** p < 0.01, * p < 0.05 vs. corresponding mCN group. (C) Total DNA methylation levels of Ptger1 CpG island in offspring kidneys at D42 according to maternal diet and offspring age. ** p < 0.01, * p < 0.05 vs. corresponding mCN group; ++ p < 0.01, + p < 0.05 vs. mLP-CN group. (D) Bisulfite sequencing analysis of the Ptger1 CpG island at D42. The horizontal axis shows each evaluated CpG site. ○: unmethylated cytosine; ●: methylated cytosine. (E) Percentages of DNA methylation levels of CpG site in the total Ptger1 CpG island were calculated from the bisulfite sequencing data shown in (D). ** p < 0.01, * p < 0.05 vs. each mCN group; ## p < 0.01, # p < 0.05 vs. corresponding CN group. Data in (B,C,E) are expressed as mean ± standard error. Statistical analysis was performed using a two-way analysis of variance followed by Tukey’s test. Sample sizes in (B) were n = 5, and those in (C,E) were n = 3 rats per group.

Figure 4

Schematic of the proposed mechanism of Ptger1 DNA methylation and mRNA expression after fetal exposure to a low-protein maternal diet. Under fetal low-protein diet exposure, renal Ptger1 DNA is hypermethylated and stably conserved. This epigenetic variation in Ptger1 is associated with disease risk imprinted during fetal life and can be reprogrammed by the postnatal dietary environment.