(Pro)renin receptor contributes to pregnancy-induced sodium-water retention in rats via activation of intrarenal RAAS and α-ENaC

Abstract

The (pro)renin receptor (PRR) is a new component of the renin-angiotensin-aldosterone system (RAAS) and regulates renin activity. The objective of the present study was to test potential roles of the renal PRR and intrarenal RAAS in the physiological status of late pregnancy. Late pregnant Sprague-Dawley rats were studied 19-21 days after sperm was observed in vaginal smears. Experiments were performed using age-matched virgin rats and late pregnant rats treated with the specific PRR inhibitor PRO20 (700 μg·kg^-1·day^-1 sc for 14 days, 3 times/day for every 8 h) or vehicle. The indices of RAAS, including PRR, renin, angiotensin II, and aldosterone levels, were examined by immunoblotting, qRT-PCR, or ELISA. Further analyses of renal epithelial sodium channel (ENaC) expression, sodium-water retention, and plasma volume were performed. We first present evidence for the activation of intrarenal RAAS in late pregnant rats, including increases in urinary renin activity, active and total renin content, and prorenin content, angiotensin II and aldosterone excretion, in parallel with increased renal PRR expression and urinary soluble PRR excretion. Functional evidence demonstrated that PRR antagonism with PRO20 effectively suppressed the indices of intrarenal RAAS in late pregnant rats. In addition, our results revealed that renal α-ENaC expression, sodium-water retention, and plasma volume were elevated during late pregnancy, which were all attenuated by PRO20. In summary, the present study examined the renal mechanism of sodium-water retention and plasma volume expansion in late pregnant rats and identified a novel role of PRR in regulation of intrarenal RAAS and α-ENaC and thus sodium and fluid retention associated with pregnancy.

Keywords: (pro)renin receptor; ENaC; plasma volume expansion; pregnancy; renin-angiotensin-aldosterone system.

Fig. 1.

Renal (pro)renin receptor (PRR) expression is increased during normal late pregnancy. A: representative immunoblotting and densitometric analysis of full-length (fPRR) and soluble (sPRR) PRR protein in the renal cortex and renal inner medulla. The densitometry values are shown underneath the corresponding blots. Expression was normalized by β-actin; n = 8–12/group. B: ELISA measurement of urinary sPRR excretion; n = 8–12/group. C: representative immunofluorescence images of cortical and inner medullary PRR in control (CTR) and Pregnancy group rats. D: statistical results of semiquantitative analysis for the relative fluorescence intensity of all immunofluorescence images; n = 6/group. Values are means ± SE. **P < 0.01 vs. CTR.

Fig. 2.

Renal PRR primarily increases intrarenal renin activity but not renin expression during normal late pregnancy. The urine samples were assayed for urinary renin activity (A), urinary active renin content (B), urinary prorenin content (C), and urinary total renin content (D). The urine samples (E) and plasma samples (F) were assayed for total renin/prorenin by ELISA. Of note, the ELISA kit was unable to differentiate between prorenin and renin. qRT-PCR was performed to analyze renin mRNA expression in the renal cortex (G) and renal inner medulla (H); n = 8–to 12/group. **P < 0.01 vs. CTR. #P < 0.05 and ##P < 0.01 vs. Pregnancy.

Fig. 3.

Analysis of other renin-angiotensin-aldosterone system (RAAS) components. A: ELISA measurement of urinary angiotensin II excretion and plasma angiotensin II concentration. B: ELISA measurement of urinary aldosterone excretion and plasma aldosterone concentration; n = 8– to 12/group. *P < 0.05 vs. CTR. #P < 0.05 vs. Pregnancy.

Fig. 4.

Effect of PRO20 on renal epithelial sodium channel (ENaC) expression in normal late pregnant rats. Representative immunoblot and densitometric analysis of α-ENaC, β-ENaC, and γ-ENaC protein in the renal cortex (A) and renal inner medulla (B) are shown. The densitometry values are shown underneath the corresponding blots. Expression was normalized by β-actin; n = 8–12/group. Data are means ± SE. **P < 0.01 vs. CTR. #P < 0.05 and ##P < 0.01 vs. Pregnancy.

Fig. 5.

Summary of physiological data from metabolic cage experiment of CTR, Pregnancy group, and Pregnancy+PRO20 group. A: water intake. B: Na⁺ intake. C: urine volume. D: urinary Na⁺ excretion. E: water retention. F: Na⁺ retention; n = 8–12/group. **P < 0.01 vs. CTR. #P < 0.05 and ##P < 0.01 vs. Pregnancy.

Fig. 6.

A: effect of PRO20 on body weight (BW) in normal late pregnant rats. B: effect of PRO20 on plasma volume in normal late pregnant rats. C: effect of PRO20 on hematocrit (Hct) in normal late pregnant rats; n = 8–12/group. **P < 0.01 vs. CTR. #P < 0.05 vs. Pregnancy.