Labetalol and soluble endoglin aggravate bile acid retention in mice with ethinylestradiol-induced cholestasis

Abstract

Labetalol is used for the therapy of hypertension in preeclampsia. Preeclampsia is characterized by high soluble endoglin (sEng) concentration in plasma and coincides with intrahepatic cholestasis during pregnancy (ICP), which threatens the fetus with the toxicity of cumulating bile acids (BA). Therefore, we hypothesized that both labetalol and increased sEng levels worsen BA cumulation in estrogen-induced cholestasis. C57BL/6J, transgenic mice overexpressing human sEng, and their wild-type littermates were administrated with ethinylestradiol (EE, 10 mg/kg s.c., the mice model of ICP) and labetalol (10 mg/kg s.c.) for 5 days with sample collection and analysis. Plasma was also taken from healthy pregnant women and patients with ICP. Administration of labetalol to mice with EE cholestasis aggravated the increase in BA plasma concentrations by induction of hepatic Mrp4 efflux transporter. Labetalol potentiated the increment of sEng plasma levels induced by estrogen. Increased plasma levels of sEng were also observed in patients with ICP. Moreover, increased plasma levels of human sEng in transgenic mice aggravated estrogen-induced cholestasis in labetalol-treated mice and increased BA concentration in plasma via enhanced reabsorption of BAs in the ileum due to the upregulation of the Asbt transporter. In conclusion, we demonstrated that labetalol increases plasma concentrations of BAs in estrogen-induced cholestasis, and sEng aggravates this retention. Importantly, increased sEng levels in experimental and clinical forms of ICPs might present a novel mechanism explaining the coincidence of ICP with preeclampsia. Our data encourage BA monitoring in the plasma of pregnant women with preeclampsia and labetalol therapy.

Keywords: bile acids; cholestasis; ethinylestradiol (EE2); labetalol; soluble endoglin.

FIGURE 1

Ethinylestradiol and labetalol effects on parameters of liver injury. The activity of ALP (A) and ALT (B) and bilirubin levels (C) in plasma. Ratio liver to body weight (D) and mouse body weight (E). Plasma concentration of BAs (F). Biliary elimination of BAs (G) and fecal excretion of BAs (H). The data are presented as median with SEM (n = 8). **p < 0.01, ***p < 0.001, by One-Way ANOVA test comparing vehicle-administered control group with cholestatic groups. ^† p < 0.05, ^§ p = 0.07 using the unpaired t-test for cholestatic animals (EE) vs. cholestatic animals treated with labetalol (EEL).

FIGURE 2

Labetalol affects BA levels in plasma by modulation of BA basolateral Mrp4 transporter. Protein expression of BA transporters Ntcp (A), Mrp4 (B), and Bsep (C) in the liver. Protein expression of BA synthesizing enzymes Cyp7a1 (D), Cyp8b1 (E), and Cyp27a1 (F). The data are presented as median with SEM (n = 8). **p < 0.01, ***p < 0.001, by One-Way ANOVA test comparing vehicle-administered control group with cholestatic groups. ^† p < 0.05, ^††† p < 0.001 using the unpaired t-test for cholestatic animals (EE) vs. cholestatic animals treated with labetalol (EEL). The images of loading control (Gapdh) are re-used for illustrative purposes.

FIGURE 3

Ethinylestradiol and labetalol effects on endoglin homeostasis. Protein and mRNA expression of Eng in the liver (A, B). Plasma levels of sEng (C) and protein levels of MMP14 (D). The mRNA expression of transcription factor Klf6 (E). The data are presented as median with SEM (n = 8). *p < 0.05, ***p < 0.001, by One-Way ANOVA test comparing vehicle-administered control group with cholestatic groups. ^†† p < 0.05, ^††† p < 0.01, ^§ p = 0.059 using the unpaired t-test for cholestatic animals (EE) vs. cholestatic animals treated with labetalol (EEL).

FIGURE 4

sEng levels are increased in patients with intrahepatic cholestasis of pregnancy. Total bile acid concentration in plasma (A). Plasma levels of sEng (B). The data are presented as mean with SEM. *p < 0.05, ***p < 0.001, by Mann-Whitney test.

FIGURE 5

High levels of human hsEng in cholestatic animals treated with labetalol resulted in increased plasma concentration of BA. Plasma levels of human sEng (A), protein expression of Eng in the liver (B), and plasma levels of mouse sEng (C). Plasma activities of liver enzymes ALP (D), ALT (E), and AST (F). Bilirubin levels in plasma (G). Mouse weight (H) and ratio liver to body weight (I). Biliary secretion of BAs (J), plasma concentration (K), and fecal excretion (L) of BAs. The data are presented as median with SEM (n = 8). *p < 0.05, **p < 0.01, ***p < 0.001, by unpaired t-test.

FIGURE 6

Increased plasma concentration of BAs is due to increased enterohepatic reabsorption of BA. Protein expression of basolateral hepatic transporters Ntcp (A) and Mrp4 (B). Protein expression of canalicular hepatic transporter, Bsep (C). Protein expression of BA synthesizing enzymes Cyp8b1 (D), Cyp27a1 (E), and Cyp7a1 (F). Protein expression of ileal transporters Asbt (G), Ostα (H), and Ostβ (I). The data are presented as median with SEM (n = 8). *p < 0.05, **p < 0.01, by unpaired t-test. The images of loading control (Gapdh) are re-used for illustrative purposes.

FIGURE 7

Intrahepatic cholestasis (ICP) is a serious complication of pregnancy, threatening the fetuses with the toxicity of cumulating bile acids (BAs). In this study, we showed for the first time that ICP is accompanied by increased plasma levels of soluble endoglin (sEng). It may explain the increased coincidence of ICP with preeclampsia, a condition characterized by high soluble endoglin (sEng) concentrations. Increased sEng may increase BA plasma concentrations by stimulating their reabsorption in the ileum. In addition, labetalol, when used for therapy of hypertension during preeclampsia in patients with simultaneous ICP, may further increase BA plasma concentrations by enhancing their export from hepatocytes to plasma, increasing the toxicity risks for the fetus.