Gestational protein restriction increases angiotensin II production in rat lung

Abstract

Gestational protein restriction (PR) alters the renin-angiotensin system in uterine arteries and placentas and elevates plasma levels of angiotensin II in pregnant rats. To date, how PR increases maternal plasma levels of angiotensin II remains unknown. In this study, we hypothesize that the expression and/or the activity of angiotensin I converting enzyme (peptidyl-dipeptidase A) 1 (ACE) in lungs, but not kidneys and blood, largely contribute to elevated plasma angiotensin II levels in pregnant rats subject to gestational PR. Time-scheduled pregnant Sprague-Dawley rats were fed a normal or low-protein diet from Day 3 of pregnancy until euthanized at Day 19 or 22. Expressions of Ace and Ace2 (angiotens in I converting enzyme [peptidyl-dipeptidase A] 2) in lungs and kidneys from pregnant rats by quantitative real-time PCR and Western blotting, and the activities of these proteins in lungs, kidneys, and plasma, were measured. The mRNA levels of Ace and Ace2 in lungs were elevated by PR at both Days 19 and 22 of pregnancy. The abundance of ACE protein in lungs was increased, but ACE2 protein was decreased, by PR. The activities of ACE, but not ACE2, in lungs were increased by PR. PR did not change expressions of Ace and Ace2, the activities of both ACE and ACE2 in kidneys, and the abundance and activity of plasma ACE. These findings suggest that maternal lungs contribute to the elevated plasma levels of angiotensin II by increasing both the expression and the activity of ACE in response to gestational PR.

FIG. 1

Quantitative real-time PCR analysis of Ace (A) and Ace2 (B) in lungs from pregnant rats with PR at Days 19 and 22 of pregnancy. CT, control; PR, protein restriction. The error bar represents the mean ± SEM expressed as relative units of mRNA standardized against R18s (n = 4–5). *P < 0.05; **P < 0.01.

FIG. 2

Western blotting analysis of ACE and ACE2 proteins in lungs from pregnant rats with PR at Days 19 and 22 of pregnancy. A) ACE proteins shown as 150-kDa bands. B) Relative abundance of ACE protein. C) ACE2 proteins shown as 90-kDa bands. D) Relative abundance of ACE2 protein. ACTB, beta-actin; CT, control; PR, protein restriction. The error bar represents the mean ± SEM expressed as the ratio of density of the ACE or ACE2 band to that of ACTB (n = 4–5). *P < 0.05.

FIG. 3

In vitro analysis of ACE (A) and ACE2 (B) activities in lungs from pregnant rats with PR. CT, control; PR, protein restriction. The error bar represents the mean ± SEM (n = 4–5). *P < 0.05; **P < 0.01.

FIG. 4

Quantitative real-time PCR analysis of Ace (A), Ace2 (B), and Ren (C) in kidneys from pregnant rats with PR at Days 19 and 22 of pregnancy. CT, control; PR, protein restriction. The error bar represents the mean ± SEM expressed as relative units of mRNA standardized against R18s (n = 4–5).

FIG. 5

Western blotting analysis of ACE and ACE2 proteins in lungs from pregnant rats with PR at Days 19 and 22 of pregnancy. A) ACE proteins shown as 150-kDa bands. B) Relative abundance of ACE protein. C) ACE2 proteins shown as 90-kDa bands. D) Relative abundance of ACE2 protein. ACTB, beta-actin; CT, control; PR, protein restriction. The error bar represents the mean ± SEM expressed as the ratio of density of the ACE or ACE2 band to that of ACTB (n = 4–5). *P < 0.05; **P < 0.01.

FIG. 6

In vitro analysis of ACE (A) and ACE2 (B) activities in kidneys from pregnant rats with PR. CT, control; PR, protein restriction. The error bar represents the mean ± SEM (n = 4–5).

FIG. 7

Western blotting analysis of ACE proteins in plasma from pregnant rats with PR at Days 19 and 22 of pregnancy. A) ACE proteins shown as 150-kDa bands. B) Relative abundance of ACE protein. CT, control; PR, protein restriction. The error bar represents the mean ± SEM of the density of ACE bands (n = 4–5). ***P < 0.001.

FIG. 8

In vitro analysis of ACE activity in plasma from pregnant rats with PR. CT, control; PR, protein restriction. The error bar represents the mean ± SEM (n = 4–5). *P < 0.05; ***P < 0.001.