Fetal Renal DNA Methylation and Developmental Programming of Stress-Induced Hypertension in Growth-Restricted Male Mice

Abstract

Fetal growth restriction (FGR) is associated with developmental programming of adult onset hypertension, which may be related to differences in nephron development. Prior studies showed that maternal nutrient restriction is associated with reduced nephrogenesis in rodents, especially in male progeny. We hypothesized that maternal genetic risk for FGR may similarly affect fetal kidney development, leading to adult onset hypertension. We employed an angiotensinogen (AGT) gene titration transgenic (TG) construct with 3 copies of the mouse AGT gene that mimics a common human genotype (AGT A[-6]G) associated with FGR. We investigated whether FGR in 2-copy (wild type, [WT]) progeny from 3-copy TG dams leads to developmental programming differences in kidney development and adult blood pressure compared with age- and sex-matched controls. Progeny were tested in the late fetal period (e17.5), neonatal period (2 weeks of age), and as young adults (12 weeks). We measured weights, tested for renal oxidative stress, compared renal DNA methylation profiles, counted the number of glomeruli, and measured adult blood pressure ± stress. Progeny from TG dams were growth restricted with evidence of renal oxidative stress, males showed fetal renal DNA hypermethylation, they had fewer glomeruli, and they developed stress-induced hypertension as adults. Their female siblings did not share this pathology and instead resembled progeny from WT dams. Surprisingly, glomerular counts in the neonatal period were not different between sexes or maternal genotypes. In turn, we suspect that differences in fetal renal DNA methylation may affect the long-term viability of glomeruli, rather than reducing nephrogenesis.

Keywords: Angiotensinogen; DNA methylation; Developmental programming; Hypertension; Nephrogenesis.

Fig. 1

Birthweight and catch-up gowth of progeny from TG and WT dams. The sex-specific mean body weight for 10 litters was averaged and presented as means ± SEM from birth through 12 weeks for the progeny of TG dams (closed triangles) relative to the body weight of age-matched WT controls (open circles). Data are separated by fetal sex (a and b). *p < 0.05; **p < 0.01; ***p < 0.001

Fig. 2

Resting and stressed-induced blood pressure in progeny of TG and WT dams. Mean arterial blood pressure (MAP) for a males and b females shown as means ± SEM for progeny of TG dams (pTG) and pWT controls. Data are from adult 2-copy progeny from TG (n = 6 and 5, males and females, respectively from six litters) and WT dams (n = 5 and 6, males and females); cross-hatched bar is data from 3-copy male positive controls [17] (n = 6) from the same TG litters. *Maternal genotype effect, P < 0.05

Fig. 3

Glomerular number in neonatal and adult progeny of TG and WT dams. Adult (a) and neonatal (c) kidney weight in grams (g), and total estimated nephron number in adult cohort (b) and in neonatal cohort (d). Data are shown as means ± SEM for male and female progeny from TG dams (pTG) and WT controls (4–8 per group from six litters). Interactions between maternal genotype and fetal sex: §p < 0.05, §§p < 0.01; sex alone: *p < 0.05

Fig. 4

Fetal kidney oxidative stress, cell proliferation, and apoptosis. Immunohistochemical staining levels for hypoxyprobe (a) in fetal kidneys of WT and TG dams reveals positive signs of oxidative stress in the proximal tubules of both males and females from TG dams (b). Panel a shows representative dam’s partially ligated left kidney as positive control compared with her unligated right kidney as negative control, and representative fetal kidneys from progeny of WT and TG dams. There is no difference in the Ki-67 proliferation index (c) or renal tubular apoptosis by ApopTag (d) between groups. Data are shown as means ± SEM for male and female progeny from 5 litters per group (pWT and pTG) controlling fetal genotypes from TG dams (only 2-copy, WT, genotypes from TG dams were used for analysis). ***Maternal genotype effect, p < 0.001

Fig. 5

Global promoter methylation in fetal kidney in progeny of TG and WT dams. a Distribution of log2-fold changes in promoter methylation in males (dotted line) and females (solid line) from TG dams compared with age- and sex-matched WT controls. Volcano plots show no significant difference in fetal renal DNA methylation between female groups (b), but marked hypermethylation in males from TG dams vs WT (c). Unsupervised cluster analysis using global DNA methylation (blue is hypermethylated) data (d) or rank ordered data (e) show good separation between groups with accentuation of a hypermethylated H19 locus in male progeny from TG dams