The selective mineralocorticoid receptor antagonist eplerenone prevents decompensation of the liver in cirrhosis

Abstract

Background and purpose: The mineralocorticoid receptor (MR) contributes to fibrosis in various tissues, and MR antagonists, like eplerenone, are used to prevent fibrosis. The role of MR antagonists in hepatic fibrosis and cirrhosis is unknown. Here, we investigated the role of MRs and eplerenone in cirrhosis development.

Experimental approach: Liver fibrosis (5 weeks) and cirrhosis, without (8 weeks) and with ascites (12 weeks), were induced by CCl₄ in rats and comprehensively analysed. The effect of eplerenone on the development of cirrhosis with ascites was assessed. MR expression, cellular and subcellular distribution and impact of hypoxia were investigated in vivo and ex vivo. Primary rat hepatocytes and cell lines were used to investigate MR trafficking and transcriptional activity mechanistically.

Key results: In cirrhosis with ascites, MR mRNA and protein expressions were reduced in hepatocytes of hypoxic areas. While in normoxic areas MRs were mainly cytosolic, the remaining MRs in hypoxic areas were mainly localized in the nuclei, indicating activation followed by translocation and degradation. Accordingly, eplerenone treatment prevented nuclear MR translocation and the worsening of cirrhosis. Exposing hepatocytes ex vivo to hypoxia induced nuclear MR translocation and enhanced transcriptional MR activity at response elements of the NF-κB pathway.

Conclusions and implications: We showed for the first time that hypoxia leads to a pathogenetic ligand-independent activation of hepatic MRs during cirrhosis resulting in their nuclear translocation and transcriptional activation of the NF-κB pathway. Treatment with eplerenone prevented the worsening of cirrhosis by blocking this ligand-independent activation of the MR.

Figure 1

Expression of MRs in primary hepatocytes (Hep), primary HSC and primary sinusoidal endothelial cells and MR expression in cirrhosis. (A) In normal livers (n = 5) and liver cells of normal rats (n = 6 animals per group), the copy number of MR mRNA μg⁻¹ RNA was determined by ddPCR. (B) Western blot analysis of the MR in normal livers and isolated liver cells of normal rats. (C) Immunohistochemistry of a normal liver showing the distribution of the MR (green). (D) Western blot analysis of whole liver lysates and isolated hepatocytes from control (liver/hepatocytes: n = 8), fibrotic (5 weeks; liver: n = 6; hepatocytes: n = 8), cirrhotic without ascites (8 weeks; liver/hepatocytes: n = 8) and cirrhotic with ascites (12 weeks; liver/hepatocytes: n = 8) rats. The amount of MR protein in livers and hepatocytes related to the housekeeping protein vinculin is shown. (E) Relative mRNA quantification of the MR in liver and primary hepatocytes (n = 8 animals per group) at different stages of liver cirrhosis. (F) A representative Western blot against MR of primary hepatocytes. ^* P < 0.05 versus control.

Figure 2

Immunohistochemistry against MR and markers for hypoxia either CA9 or HIF1α. Sirius Red staining in cirrhotic animals without and with eplerenone treatment. (A) Immunohistochemistry of livers of control animals showing increased amount of CA9‐positive cells (brown) around the central vein showing areas with less oxygen. Expression of MR (green) was higher, and MR was located in the cytoplasm in CA9‐negative cells. In CA9‐positive cells, the MR was located mainly in the nucleus. (B) Immunohistochemistry of cirrhotic livers with ascites (12 weeks of CCl₄ administration) showing a reduced amount of MR (green) compared with control. MR (green) was located in the nucleus, or no MR was detected in CA9‐positive cells. In CA9‐negative cells, MR was located in the cytoplasm or the nucleus. (C) Immunofluorescence of primary control hepatocytes in vitro. MR (green) is mainly located in the cytoplasm. Note that there is almost no expression of HIF1α (red stain), a marker of hypoxia. (D) Immunofluorescence of primary cirrhotic hepatocytes in vitro. MR (green) is located in the cytoplasm and nucleus in cells with a low amount of red stain (HIF1α) (right side). In cells with a higher amount of HIF1α, MR is located in the nucleus (left side). Nuclei are stained in blue (DAPI). (E and F) Animals with cirrhosis (8 weeks of CCl₄ inhalation) were treated with and without eplerenone, and inhalation with CCl₄ was continued until week 12. (E) Sirius Red staining of a cirrhotic liver without eplerenone treatment. (F) Sirius Red staining of a cirrhotic liver with eplerenone treatment.

Figure 3

Distribution of the MR in non‐hypoxic and hypoxic areas of the liver in normal, untreated cirrhotic and eplerenone‐treated animals. MR expression in primary hepatocytes under hypoxic conditions. Animals with cirrhosis (8 weeks of CCl₄ inhalation) were treated with and without eplerenone, and inhalation with CCl₄ was continued until week 12. (A and B) Immunohistochemistry of cirrhotic livers with treatment of eplerenone illustrating the distribution of the MR (green) in non‐hypoxic (CA9‐negative staining; A) and hypoxic (CA9 staining, brown; B) areas. (C) Relative MR localization in the nucleus in CA9‐negative (non‐hypoxic) and CA9‐positive (hypoxic) areas of normal livers (control; n = 9; P < 0.05), cirrhotic livers without treatment with eplerenone (cirrhosis; n = 5; P < 0.05) and cirrhotic livers with treatment of eplerenone (n = 5; P < 0.05; chi‐squared test). (D–F) Primary hepatocytes were exposed to hypoxia (1% O₂). (D) Relative mRNA quantification of serpine‐1 in normoxia and hypoxia. (E) Relative mRNA quantification of MR in normoxia and hypoxia. (F) Amount of MR protein in hepatocytes related to the housekeeping protein in normoxia and hypoxia. ^* P < 0.05 versus control normoxia; n = 8 per group.

Figure 4

Hypoxia affects transcriptional MR activity. SEAP activity of HEK cells that were contransfected with positive GRE‐SEAP, positive NFAT‐SEAP, positive AP‐1‐SEAP or positive NF‐κB‐SEAP and MR construct, or empty vector. Twenty‐four hours after transfection, cells were submitted to normoxia (20% O₂) or hypoxia (1% O₂) (A) and normoxia (20% O₂, dark) or hypoxia (1% O₂) with or without supplementation with aldosterone (aldo) (B). AP‐1 (n = 13), NF‐κB (n = 12), NFAT (n = 8) and GRE (n = 6), ^* P < 0.05.

Figure 5

Proposed mechanism of the MR and effect of eplerenone on the development of cirrhosis from cirrhosis without ascites (cirrhosis) to cirrhosis with ascites. Hypoxia induces ligand‐independent MR activation with a shift in response element activation from mainly GRE to increased NF‐κB activation. Eplerenone blocks this hypoxia‐dependent activation and attenuates fibrosis and worsening to cirrhosis with ascites.