Chronic liver injury in rats and humans upregulates the novel enzyme angiotensin converting enzyme 2

Abstract

Background: Angiotensin converting enzyme (ACE) 2 is a recently identified homologue of ACE that may counterregulate the actions of angiotensin (Ang) II by facilitating its breakdown to Ang 1-7. The renin-angiotensin system (RAS) has been implicated in the pathogenesis of cirrhosis but the role of ACE2 in liver disease is not known.

Aims: This study examined the effects of liver injury on ACE2 expression and activity in experimental hepatic fibrosis and human cirrhosis, and the effects of Ang 1-7 on vascular tone in cirrhotic rat aorta.

Methods: In sham operated and bile duct ligated (BDL) rats, quantitative reverse transcriptase-polymerase chain reaction was used to assess hepatic ACE2 mRNA, and western blotting and immunohistochemistry to quantify and localise ACE2 protein. ACE2 activity was quantified by quenched fluorescent substrate assay. Similar studies were performed in normal human liver and in hepatitis C cirrhosis.

Results: ACE2 mRNA was detectable at low levels in rat liver and increased following BDL (363-fold; p < 0.01). ACE2 protein increased after BDL (23.5-fold; p < 0.05) as did ACE2 activity (fourfold; p < 0.05). In human cirrhotic liver, gene (>30-fold), protein expression (97-fold), and activity of ACE2 (2.4 fold) were increased compared with controls (all p < 0.01). In healthy livers, ACE2 was confined to endothelial cells, occasional bile ducts, and perivenular hepatocytes but in both BDL and human cirrhosis there was widespread parenchymal expression of ACE2 protein. Exposure of cultured human hepatocytes to hypoxia led to increased ACE2 expression. In preconstricted rat aorta, Ang 1-7 alone did not affect vascular tone but it significantly enhanced acetylcholine mediated vasodilatation in cirrhotic vessels.

Conclusions: ACE2 expression is significantly increased in liver injury in both humans and rat, possibly in response to increasing hepatocellular hypoxia, and may modulate RAS activity in cirrhosis.

Figure 1

Schematic diagram of the renin angiotensin system (RAS) showing the main pathway for angiotensin (Ang) II generation from Ang I via angiotensin converting enzyme (ACE), and the role of ACE2 to degrade Ang I to Ang 1–9, and Ang II to the vasodilator Ang 1–7.

Figure 2

Angiotensin converting enzyme 2 (ACE2) expression and activity in sham operated control and bile duct ligated (BDL) rat liver. (A) Quantification of ACE2 mRNA by quantitative reverse transcriptase-polymerase chain reaction, with control values arbitrarily standardised to 1. Values are mean (SEM), n = 6 in each group; *p<0.01, BDL versus control. (B) Western blot of ACE2 protein in control and BDL livers. (C) Quantification of western blots. Values are mean (SEM); *p<0.05, BDL versus control. (D) ACE2 activity in sham and BDL. Values are mean (SEM), n = 9 in each group; *p<0.05, BDL versus control.

Figure 3

Angiotensin converting enzyme 2 (ACE2) expression and activity in normal and cirrhotic (HepC) human liver. (A) Quantification of ACE2 mRNA by quantitative reverse transcriptase-polymerase chain reaction of normal human (n = 3) and cirrhotic livers (n = 20) with control values arbitrarily standardised to 1. Values are mean (SEM); *p<0.01, cirrhosis versus control. (B) Western blotting of ACE2 protein in normal and cirrhotic liver. (C) Quantitation of western blotting in normal and cirrhotic liver. Values are mean (SEM), n = 3 and 4 per group, respectively; *p<0.01, cirrhotics versus normal. (D) ACE2 activity in normal and cirrhotic liver. Values are mean (SEM), n = 3 and n = 9, respectively; *p<0.01, cirrhotics versus normal.

Figure 4

Immunohistochemical staining of angiotensin converting enzyme 2 (ACE2) protein in rat and human liver. In rat liver, pimonidazole immunostaining was used as a marker of cellular hypoxia. Normal rat liver is shown in the top row (A–C). There was no hepatic ACE2 staining detectable following ACE2 peptide preabsorption (A), confirming the specificity of the antibody used to detect ACE2. In normal livers. ACE2 immunolabelling (B) and staining of pimonidazole adducts (C, arrow) were restricted to perivenular hepatocytes. Bile duct ligated (BDL) rat livers are shown in the second row (D–F). In rat livers at seven days post BDL (D), ACE2 expression was more heterogenous and widespread than in normal livers. At 21 days post BDL, immunostaining of both ACE2 (E) and pimonidazole adducts (F) was detected in more than 80% of hepatocytes, with ACE2 also detected in bile duct cells and endothelial cells. (G–I) Human liver. In normal liver, ACE2 was detected in hepatocytes surrounding occasional larger central veins (G, arrow). In cirrhotic liver, there was widespread ACE2 expression throughout cirrhotic nodules (H) and in bile duct cells (BD) and endothelial cells lining small blood vessels (BV) (I). Original magnifications ×850, except (4G) and (4H) × 450, and (4I) ×1700.

Figure 5

Effects of angiotensin (Ang) 1–7 on responses to acetylcholine (Ach) in aortic rings from normal (Con) and bile duct ligated (BDL) rats. Addition of Ang 1–7 to the organ bath enhanced Ach mediated vasodilatation in vessels from BDL rats. **p<0.01, ***p<0.001 versus control; †p = 0.043 versus BDL.