Maintenance of functional hepatocellular polarity determines recovery in acute-on-chronic liver failure

Background & Aims:

Submassive hepatic necrosis (SMHN) is the defining histological feature of acute-on-chronic liver failure (ACLF). In the condition of SMHN, liver progenitor cells (LPC)-mediated regeneration is crucial for survival and recovery of ACLF patients. LPC-mediated liver regeneration comprises three consecutive steps: formation of reactive ducts by activated LPC, LPC differentiation towards hepatocytes, and functional bile canaliculi formation by newly generated hepatocytes. In this study, we examined which steps play crucial roles in ACLF recovery.

Methods:

We investigated liver tissue specimens of 55 ACLF patients with a known interval between the first symptoms of acute decompensation and the time of tissue sampling. Immunostaining for Cytokeratin 7 and apical molecules, including BSEP, MDR1, CD10 and CD26, were used to identify LPC and bile canaliculi. Electronic microscopy was performed to detect the existence of bile canaliculi. Expression of CYP7A1, the rate-limiting enzyme for bile acid synthesis, and nuclear receptors such as farnesoid X receptor (FXR) and small heterodimer partner (SHP) was measured by immunohistochemistry. In collagen sandwich cultured primary hepatocytes, we examined how nuclear receptors regulate expression of apical molecules.

Results:

Rapid ductular reaction occurs in all ACLF patients regardless of clinical duration and outcome. LPCs differentiate into hepatocytes over time. However, LPC-derived hepatocytes do not improve liver function in irreversible ACLF patients. Electronic microscopy analysis reveals that although these hepatocytes maintain a typical polar structure, bile canaliculi present without microvilli. Immunostaining analyses further show that in contrast to recovered patients, hepatocytes in irreversible ACLF patients have lost functional molecules in the bile canali, including CD26, CD10, and BSEP, whereas MDR1 expression is maintained. The loss of hepatocyte membrane transporters, e.g. BSEP, in irreversible ACLF is associated with a failed feedback regulatory mechanism of bile acid synthesis, which is characterized by high levels of CYP7A1, but lack of nuclear FXR and SHP in hepatocytes. In vitro, FXR agonist GW4064 up-regulates expression of apical molecules, including BSEP and MRP2, whereas depleting FXR inhibits expression of BSEP and MRP2, but not MDR1. Furthermore, the effects of FXR are SHP-dependent.

Conclusions:

Functional bile canaliculi are required to restore intact liver function following SMHN, thus determining recovery in ACLF. Maintenance of functional hepatocellular polarity is at least partially controlled by nuclear receptors, in particular the FXR-SHP axis.