Megalin, Proton Pump Inhibitors and the Renin-Angiotensin System in Healthy and Pre-Eclamptic Placentas

Abstract

Soluble Fms-like tyrosine kinase-1 (sFlt-1) is increased in pre-eclampsia. The proton pump inhibitor (PPI) lowers sFlt-1, while angiotensin increases it. To investigate whether PPIs lower sFlt-1 by suppressing placental renin-angiotensin system (RAS) activity, we studied gene expression and protein abundance of RAS components, including megalin, a novel endocytic receptor for prorenin and renin, in placental tissue obtained from healthy pregnant women and women with early-onset pre-eclampsia. Renin, ACE, ACE2, and the angiotensin receptors were expressed at identical levels in healthy and pre-eclamptic placentas, while both the (pro)renin receptor and megalin were increased in the latter. Placental prorenin levels were upregulated in pre-eclamptic pregnancies. Angiotensinogen protein, but not mRNA, was detectable in placental tissue, implying that it originates from maternal blood. Ex vivo placental perfusion revealed a complete washout of angiotensinogen, while prorenin release remained constant. The PPI esomeprazole dose-dependently reduced megalin/(pro)renin receptor-mediated renin uptake in Brown Norway yolk sac epithelial cells and decreased sFlt-1 secretion from placental villous explants. Megalin inhibition blocked angiotensinogen uptake in epithelial cells. In conclusion, our data suggest that placental RAS activity depends on angiotensinogen taken up from the maternal systemic circulation. PPIs might interfere with placental (pro)renin-AGT uptake/transport, thereby reducing angiotensin formation as well as angiotensin-induced sFlt-1 synthesis.

Keywords: megalin; pre-eclampsia; proton pump inhibitors; renin–angiotensin system.

Figure 1

Gene expression levels of RAS components (A–G) and megalin (H) in healthy and early-onset pre-eclamptic (EoPE) placental tissue. (I,J): AGT expression was confirmed with different pairs of primers. Data are mean ± SEM of n = 12. * p < 0.05. (P)RR, (pro)renin receptor; AGT, angiotensinogen; ACE, angiotensin-converting enzyme; AT1R, angiotensin II type 1 receptor; AT2R, angiotensin II type 2 receptor; A.U., arbitrary unit.

Figure 2

Total renin (A), renin (B) and prorenin (C) levels (n = 16) measured by immunoradiometric assay and angiotensinogen (AGT; D,E) and megalin (F,G) protein abundance (n = 12) measured by Western blot in healthy and early-onset pre-eclamptic (EoPE) placental tissue. Panels (D,F) are representative blots of AGT and megalin protein. Data were normalized versus GAPDH (E) or β-actin (G). Data are mean ± SEM. * p < 0.05, ** p < 0.005, *** p < 0.0005 vs. Healthy. AGT, angiotensinogen.

Figure 3

Prorenin (A), renin (B) and angiotensinogen (C) release from isolated perfused healthy (n = 5) and early-onset pre-eclamptic (EoPE, n = 6) cotyledons. AGT: angiotensinogen. Perfusate collection from two of the pre-eclamptic placentas could not be continued over the full 180 min due to fetal-to-maternal leakage. In such cases, only the samples prior to the occurrence of leakage were used. Data are mean ± SEM. * p < 0.05.

Figure 4

Panels (A–C): representative blots of His-AGT protein abundance in BN16 cells treated with negative control (siNC) or siRNA against megalin (siMegalin). Panels, (D–G): cell-associated renin levels after incubating BN16 cells with 100 U/L renin at 37 °C (D,F) or 4 °C (E,G) for 2 h in the presence of increasing concentrations of esomeprazole (D,E) or bafilomycin A1 (F,G). Data are mean ± SEM of n = 5–9. ** p < 0.005, *** p < 0.0005 vs. control.

Figure 5

sFlt-1 levels in medium (A) and mRNA levels in explants (B) after incubating healthy or pre-eclamptic (EoPE) placental villous explants with 100 μM of esomeprazole (Eso) for 48 h. sFlt-1 level: healthy n = 18, EoPE n = 8; Flt-1 mRNA level: healthy n = 7, EoPE n = 4. * p < 0.05, ** p < 0.005, *** p < 0.0005 vs. 0. A.U., arbitrary unit.

Figure 6

Schematic overview. AGT, angiotensinogen; , inhibition; PPI, proton pump inhibitor; (P)RR, (pro)renin receptor; Ang I, angiotensin I; ACE, angiotensin-converting enzyme; Ang II, angiotensin II; AT1R, angiotensin II type 1 receptor; PE, pre-eclampsia; sFlt-1, soluble Fms-like tyrosine kinase-1; +, normal sensitivity to Ang II; +++, increased sensitivity to Ang II.