Bile acid-induced necrosis in primary human hepatocytes and in patients with obstructive cholestasis

Abstract

Accumulation of bile acids is a major mediator of cholestatic liver injury. Recent studies indicate bile acid composition between humans and rodents is dramatically different, as humans have a higher percent of glycine conjugated bile acids and increased chenodeoxycholate content, which increases the hydrophobicity index of bile acids. This increase may lead to direct toxicity that kills hepatocytes, and promotes inflammation. To address this issue, this study assessed how pathophysiological concentrations of bile acids measured in cholestatic patients affected primary human hepatocytes. Individual bile acid levels were determined in serum and bile by UPLC/QTOFMS in patients with extrahepatic cholestasis with, or without, concurrent increases in serum transaminases. Bile acid levels increased in serum of patients with liver injury, while biliary levels decreased, implicating infarction of the biliary tracts. To assess bile acid-induced toxicity in man, primary human hepatocytes were treated with relevant concentrations, derived from patient data, of the model bile acid glycochenodeoxycholic acid (GCDC). Treatment with GCDC resulted in necrosis with no increase in apoptotic parameters. This was recapitulated by treatment with biliary bile acid concentrations, but not serum concentrations. Marked elevations in serum full-length cytokeratin-18, high mobility group box 1 protein (HMGB1), and acetylated HMGB1 confirmed inflammatory necrosis in injured patients; only modest elevations in caspase-cleaved cytokeratin-18 were observed. These data suggest human hepatocytes are more resistant to human-relevant bile acids than rodent hepatocytes, and die through necrosis when exposed to bile acids. These mechanisms of cholestasis in humans are fundamentally different to mechanisms observed in rodent models.

Keywords: Bile acids; Biomarkers; HMGB1; Inflammation; Obstructive cholestasis; Primary human hepatocytes.

Figure 1

Infarction of the biliary tracts mediates the initial injury in cholestatic human patients. Serum (A) and bile (B) samples obtained from patients thought to have biliary obstruction during routine ERCP. Total and individual bile acid levels were measured in these samples via UPLC/MS. Patient data are presented in Table 1. H&E staining was performed in liver sections from human patients with confirmed extrahepatic cholestasis (C,D) and mice 72 h after bile duct ligation (E,F). Frank necrosis is seen in the image (C) in an area of biliary infarction. Bile (marked by black arrow) leaks into adjacent hepatic parenchyma (D). Bile infarcts in mice subjected to 72 h bile duct ligation show similar histological features (E,F).

Figure 2

Bile acid exposure results in cell death in human hepatocytes. LDH was measured 3, 6 or 24 h after exposure to GCDC (A) or TCA (B). N=4 batches of isolated cells; *P< 0.05 (compared to controls). Propidium iodide and Hoechst stain were applied to hepatocytes 6 h after treatment with 1 mM GCDC (C). Images were overlaid using Image J software.

Figure 3

Bile acid exposure results in hepatic necrosis in vitro. Caspase-3 activity was measured after exposure to the indicated concentration of GCDC or TCA for 6 h or after 12 h galactosamine/TNF treatment (G/T) in human (A) and rat (B) hepatocytes pretreated with vehicle (DMSO) or z-VAD-fmk. Caspase-3 activation was assessed via western blot in human (C) or rat (D) hepatocytes after GCDC treatment. Galactosamine/TNF-treated human hepatocytes were used as controls. LDH release was measured in untreated cells or after pretreatment with 10μM z-VAD-fmk in human (E) or rat hepatocytes (F). N=5 batches of isolated cells for human hepatocytes, n=3 for rat hepatocytes; *P<0.05 (versus control). ^#P<0.05 (versus vehicle).

Figure 4

Biliary concentrations of bile acids and not serum concentrations cause toxicity in human hepatocytes. LDH release was measured in human hepatocytes after exposure to a complete serum BA milieu or a derivation of this concentration (A) or a complete biliary bile acid milieu or a derivation of this concentration for 6 or 24 h (B). N=3 batches of isolated cells;. *P<0.05 (versus control).

Figure 5

Necrosis predominates during cholestatic liver injury in man. Serum caspase-3 activities were measured in human patients and in galactosamine/endotoxin-treated mice (A). Full length cytokeratin 18 (FL-CK18) (M65 antigen values) and caspase-cleaved (cCK18) (M30 antigen values) were measured in serum of human patients (B). Patient data are presented in Table 1. *P<0.05 (versus control). ^#P<0.05 (versus UI patients).

Figure 6

Elevated serum HMGB1 levels in injured patients during cholestatic liver injury in man. HMGB1 (A) and acHMGB1 levels (B) were measured in serum of human patients. The ratio of acHMGB1 to HMGB1 was also calculated (C). Patient data are presented in Table 1. *P<0.05 (versus UI group).