Bile Microinfarcts in Cholestasis Are Initiated by Rupture of the Apical Hepatocyte Membrane and Cause Shunting of Bile to Sinusoidal Blood

Abstract

Bile duct ligation (BDL) is an experimental procedure that mimics obstructive cholestatic disease. One of the early consequences of BDL in rodents is the appearance of so-called bile infarcts that correspond to Charcot-Gombault necrosis in human cholestasis. The mechanisms causing bile infarcts and their pathophysiological relevance are unclear. Therefore, intravital two photon-based imaging of BDL mice was performed with fluorescent bile salts (BS) and non-BS organic anion analogues. Key findings were followed up by matrix-assisted laser desorption ionization imaging, clinical chemistry, immunostaining, and gene expression analyses. In the acute phase, 1-3 days after BDL, BS concentrations in bile increased and single-cell bile microinfarcts occurred in dispersed hepatocytes throughout the liver caused by the rupture of the apical hepatocyte membrane. This rupture occurred after loss of mitochondrial membrane potential, followed by entry of bile, cell death, and a "domino effect" of further death events of neighboring hepatocytes. Bile infarcts provided a trans-epithelial shunt between bile canaliculi and sinusoids by which bile constituents leaked into blood. In the chronic phase, ≥21 days after BDL, uptake of BS tracers at the sinusoidal hepatocyte membrane was reduced. This contributes to elevated concentrations of BS in blood and decreased concentrations in the biliary tract. Conclusion: Bile microinfarcts occur in the acute phase after BDL in a limited number of dispersed hepatocytes followed by larger infarcts involving neighboring hepatocytes, and they allow leakage of bile from the BS-overloaded biliary tract into blood, thereby protecting the liver from BS toxicity; in the chronic phase after BDL, reduced sinusoidal BS uptake is a dominant protective factor, and the kidney contributes to the elimination of BS until cholemic nephropathy sets in.

A novel murine model of reversible bile duct obstruction demonstrates rapid improvement of cholestatic liver injury

“…While prior work has described the inflammatory changes in BDL, we provide important insight into the immune response after restoration of bile flow that may have significant application to human disease. Recent studies in murine BDL have shown that the influx of CD45+ immune cells near the site of bile infarcts occurs early, along the same timeframe as liver enzyme elevation, hepatocyte death, and release of damage‐associated molecular patterns (Ghallab et al., 2019). While the present study does not evaluate the microscopic changes in immune cell infiltration, our global assessment by histology and gene expression provides insight into the importance of similarly tracking temporal changes after restoration of bile flow.…”

“…Murine bile duct ligation (BDL) is an established model of obstructive cholestasis in which there is complete cessation of bile flow. Rupture of the apical membrane of hepatocytes in murine BDL has been shown as a trigger for immune cell infiltration within the first 3 days after murine BDL (Ghallab et al., 2019) and chemokine (C‐C motif) ligand 2 (CCL2)‐mediated cell recruitment contributes to hepatic injury in this model (Cai et al., 2017). In addition, other studies demonstrate impaired chemokine expression in the setting of infection in murine BDL that may contribute to increase susceptibility to infections in cholestasis (Rattay et al., 2018).…”

Bile Infarcts: New Insights Into the Pathogenesis of Obstructive Cholestasis

“…In a study described in this issue of Hepatology, Ghallab et al used an elegant intravital two-photon-based imaging system and fluorescent labeled bile acid and other cellular markers to capture, in real time, the live events of bile infarct formation in the liver of a bile-duct-ligated mouse. (4) During the acute phase (1-3 days after BDL), hepatic bile acid levels increased, resulting in loss of the cells' mitochondrial membrane potential. At this point, the apical canalicular membrane in focal areas ruptured, and bile was seen to initially regurgitate into single cells and then into adjacent sinusoids, creating a canalicular bile-sinusoidal shunt (#4 in Fig.…”

“…In a study described in this issue of Hepatology , Ghallab et al used an elegant intravital two‐photon‐based imaging system and fluorescent labeled bile acid and other cellular markers to capture, in real time, the live events of bile infarct formation in the liver of a bile‐duct‐ligated mouse . During the acute phase (1‐3 days after BDL), hepatic bile acid levels increased, resulting in loss of the cells’ mitochondrial membrane potential.…”

Highlight report: caspase 8 as a therapeutic target in chronic liver disease

“…It has been shown that cholestatic liver disease usually evolves over time with an ascending course of the disease process with first lesions in bile ducts, followed by damage of liver parenchyma (Jansen et al 2017). In this situation, concentrations of bile acids in the biliary tract increase, lead to ruptures of the apical and later basolateral hepatocyte membranes and thereby cause shunting of bile acids from canaliculi into the blood (Ghallab et al 2019). At the level of interlobular bile ducts, cholestasis leads to adaptive remodeling by cholangiocyte proliferation, branching as well as looping of ducts, also known as ductular reaction (Vartak et al 2016).…”