Upregulation of hepatic angiotensin-converting enzyme 2 (ACE2) and angiotensin-(1-7) levels in experimental biliary fibrosis

Abstract

Background/aims: Angiotensin-converting enzyme 2 (ACE2), its product, angiotensin-(1-7) and its receptor, Mas, may moderate the adverse effects of angiotensin II in liver disease. We examined the expression of these novel components of the renin angiotensin system (RAS) and the production and vasoactive effects of angiotensin-(1-7) in the bile duct ligated (BDL) rat.

Methods: BDL or sham-operated rats were sacrificed at 1, 2, 3 and 4 weeks. Tissue and blood were collected for gene expression, enzyme activity and peptide measurements. In situ perfused livers were used to assess angiotensin peptide production and their effects on portal resistance.

Results: Hepatic ACE2 gene and activity (P<0.0005), plasma angiotensin-(1-7) (P<0.0005) and Mas receptor expression (P<0.01) were increased following BDL compared to shams. Perfusion experiments confirmed that BDL livers produced increased angiotensin-(1-7) (P<0.05) from angiotensin II and this was augmented (P<0.01) by ACE inhibition. Whilst angiotensin II increased vasoconstriction in cirrhotic livers, angiotensin-(1-7) had no effect on portal resistance.

Conclusions: RAS activation in chronic liver injury is associated with upregulation of ACE2, Mas and hepatic conversion of angiotensin II to angiotensin-(1-7) leading to increased circulating angiotensin-(1-7). These results support the presence of an ACE2-angiotensin-(1-7)-Mas axis in liver injury which may counteract the effects of angiotensin II.

Fig. 1

Schematic representation of the role of ACE2 in the renin angiotensin system (RAS) showing the predominant pathway in solid lines for the generation of hypotensive and anti-fibrotic peptide angiotensin-(1–7) (Ang-(1–7)) from the substrate Ang I via Ang II, catalysed by angiotensin converting enzyme (ACE) and ACE2, and its degradation by ACE into inactive peptide Ang-(1–5). AT1 receptor, Ang II type 1 receptor.

Fig. 2

Hepatic angiotensin converting enzyme 2 (ACE2) gene expression (a) and ACE2 activity (b) in sham-operated control (open bars) and bile duct ligated (BDL) (filled bars) rats. QPCR derived ACE2 gene expression values were normalized to ribosomal 18S and the shams were given a value of 1 at each time point. ACE2 activity was determined by measuring nmol of ACE2 substrate (Mca-APK-(Dnp)-OH) cleaved by solubilized membrane fractions. Each bar represents the mean ± SEM expression and activity from 8 to 10 rats (a) and from 7 to 8 rats (b), respectively, per treatment group. ^∗∗∗∗P < 0.0005, BDL vs. sham.

Fig. 3

Hepatic angiotensin converting enzyme (ACE) gene expression (a), ACE-specific radioligand ¹²⁵I-MK351A binding (b) and ACE activity (c) in sham-operated control (open bars) and bile duct ligated (BDL) (filled bars) rats. QPCR derived ACE gene expression values were normalized to ribosomal 18S and the shams were given a value of 1 at each time point. ACE binding density was determined by computerised densitometry. ACE activity was determined by measuring nmol of ACE substrate (Hippuryl-His-Leu) cleaved by solubilized membrane fractions. Each bar represents the mean ± SEM expression, staining and activity from 8 to 10 rats (a), from 4 to 5 rats (b) and from 7 to 8 rats (c), respectively, per treatment group. ^∗∗∗∗P < 0.0005, ^∗∗∗P < 0.001, ^aP < 0.06, BDL vs. sham.

Fig. 4

Hepatic angiotensin-(1–7) receptor Mas (a) and angiotensin II type I receptor, AT1 (b) gene expressions in sham-operated control (open bars) and bile duct ligated (BDL) (filled bars) rats. QPCR derived gene expression values were normalized to ribosomal 18S and the shams were given a value of 1 at each time point. Each bar represents the mean ± SEM expression from 9 to 10 rats (a) or from 8 to 10 rats (b) per treatment group. ^∗∗∗∗P < 0.0005, ^∗∗P < 0.01, ^∗P < 0.05, BDL vs. sham.

Fig. 5

Plasma angiotensin converting enzyme (ACE) activity (a) and ACE2 activity (b) in sham-operated control (open bars) and bile duct ligated (BDL) (filled bars) rats. ACE and ACE2 activity were determined by measuring nmol of ACE substrate (Hippuryl-His-Leu) and pmol of ACE2 substrate (Mca-APK-(Dnp)-OH), respectively, cleaved by solubilized membrane fractions. Each bar represents the mean ± SEM activity from 9 to 10 rats per treatment group. ^∗∗∗∗P < 0.0005, ^∗P < 0.05, BDL vs. sham.

Fig. 6

Plasma concentrations of angiotensin-(1–7) (a) and angiotensin II (Ang II) (b) in sham-operated control (open bars) and bile duct ligated (BDL) (filled bars) rats. Angiotensin peptide levels were determined by radioimmunoassay. Each bar represents the mean ± SEM concentration from 9 to 10 rats per treatment group. ^∗∗∗∗P < 0.0005, ^∗∗∗P < 0.001, ^∗∗P < 0.01, BDL vs. sham.

Fig. 7

In situ perfused rat liver and angiotensin-(1–7) production in response to angiotensin II (Ang II) bolus injection by bile duct ligated (BDL) rat liver (closed circles) and control rat liver (open circles). Livers were perfused with oxygenated Krebs Henseleit solution (1% BSA) for 25 min, two outflow effluent samples were collected and Ang II bolus injection (60 pmol) was given, followed by sample collection for 75 s (a). The same livers were perfused again with Krebs for 5 min, and then with angiotensin-converting enzyme (ACE) inhibitor lisinopril (10⁻⁶ mol/L) throughout sample collection (b). Baseline corrected total area under the angiotensin-(1–7) curve for BDL livers (filled bars) and control livers (open bars) with or without ACE inhibitor is shown in panel (c). Each circle/bar represents the mean ± SEM concentration from 4 to 5 rats per treatment group. ^∗∗∗P < 0.001, ^∗∗P < 0.01, ^∗P < 0.05, ^aP = 0.05, baseline-corrected BDL vs. control.

Fig. 8

Vasoactive responses of angiotensin peptides in normal and cirrhotic rat livers. The livers were perfused as described in Section 2.2.2. Panel (a) depicts the change in portal resistance in in situ perfused livers from rats 4-weeks after bile duct ligation (BDL) (filled bars, n = 4) or age-matched controls (open bars, n = 6) in response to varying bolus doses of angiotensin-(1–7). The responsiveness of the liver to angiotensin-(1–7) was confirmed at the end of each experiment by the administration of a bolus dose of phenylephrine (PE). Panel (b) depicts the percentage of relaxation of pre-constricted control livers (n = 5) in response to a vehicle administration (closed circle) followed by varying bolus doses of angiotensin-(1–7) (open circles). Panel (c) depicts the vascular response of sham (open bar, n = 5) and BDL (filled bar, n = 6) livers in response to a bolus dose (10⁻⁸ M/L) of angiotensin II. Each circle/bar represents the mean ± SEM concentration. ^∗∗∗P < 0.005, ^∗P < 0.05, BDL vs. sham.