Maternal protein restriction reduces expression of angiotensin I-converting enzyme 2 in rat placental labyrinth zone in late pregnancy

Abstract

Both the systemic and the uteroplacental renin-angiotensin system (RAS) display dramatic changes during pregnancy. However, whether gestational protein insufficiency affects the expressions of RAS in the placenta remains unknown. In this study, we hypothesized that the expression of Ace2 in the placental labyrinth was reduced by maternal protein restriction. Pregnant Sprague-Dawley rats were fed a normal diet or a low-protein diet (LP) from Day 1 of pregnancy until they were killed at Day 14 or Day 18. The labyrinth zone (LZ) of the placenta was then dissected and snap frozen for expression analysis by quantitative real-time PCR of Ace, Ace2, Agtr1a, Agtr1b, and Agtr2. Formalin-fixed placentas were used for immunohistochemical analysis on ACE and ACE2 proteins. The findings include 1) the expression of Ace2 in rat LZ was reduced by maternal protein restriction in late pregnancy; 2) ACE protein was mainly present in syncytiotrophoblasts, whereas ACE2 protein was found predominantly in fetal mesenchymal tissue and fetal capillaries; 3) Agtr1a was predominant in the rat LZ, and its mRNA levels, but not protein levels, were reduced by LP; 4) expressions of Ace, Ace2, and Agtr1a in the rat LZ and their response to LP occurred in a gender-dependent manner. These results may indicate that a reduced expression of Ace2 and perhaps an associated reduction in angiotensin (1-7) production in the placenta by maternal protein restriction may be responsible for fetal growth restriction and associated programming of adulthood hypertension.

FIG. 1.

Fetal weights in rats fed with the normal diet (CT) and LP at Days 14 and 18 of pregnancy. The error bar represents the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

FIG. 2.

Quantitative real-time PCR analysis of Ace (A), Ace2 (B), and Agtr1a (C) in the rat LZ. The error bar represents the mean ± SEM expressed as relative units of mRNA standardized against Gapdh (n = 6). *P < 0.05, **P < 0.01.

FIG. 3.

Western blotting analysis of ACE protein in the rat LZ. A) ACE proteins shown in 150-kDa bands. F, female; M, male. B) Relative abundance of ACE protein. The error bar represents the mean ± SEM expressed as the ratio of density of the ACE band to that of ACTB (n = 6). *P < 0.05, ***P < 0.001.

FIG. 4.

Western blotting analysis of ACE2 protein in the rat LZ. A) ACE2 proteins shown in 90-kDa bands. F, female; M, male. B) Relative abundance of ACE2 protein. The error bar represents the mean ± SEM expressed as the ratio of density of ACE bands to those of ACTB. *P < 0.05, **P < 0.01; ***P < 0.001.

FIG. 5.

Western blotting analysis of AGTR1 protein in the rat LZ. A) AGTR1 proteins shown in 27-kDa bands. F, female; M, male. B) Relative abundance of AGTR1 protein. The error bar represents the mean ± SEM expressed as the ratio of density of ACE bands to those of ACTB. *P < 0.05, **P < 0.01.

FIG. 6.

Immunohistochemical analysis of ACE protein in the rat LZs. A representative negative control for immunostaining (IgG from nonrelevant rabbit) and three representative images for immunostaining in trophoblast cell and fetal capillary in a female LZ at Day 18 of pregnancy (shown in high magnification) were present in the bottom row in the micrograph. F, female; M, male; En, endothelial cell; FC, fetal capillary; MBS, maternal blood space; RBC, red blood cell; ST, syncytiotrophoblasts; TGC, trophoblast giant cell. Bar = 10 μm.

FIG. 7.

Immunohistochemical analysis of ACE2 protein in the rat LZs. A representative negative control for immunostaining (IgG from nonrelevant rabbit) and three representative images for immunostaining in trophoblast cells and fetal capillary in a female LZ at Day 18 of pregnancy (shown in high magnification) were present in the bottom row in the micrograph. F, female; M, male; En, endothelial cell; FC, fetal capillary; MBS, maternal blood space; RBC, red blood cell; ST, syncytiotrophoblast; TGC, trophoblast giant cell. Bar = 10 μm.