Interruption of bile acid uptake by hepatocytes after acetaminophen overdose ameliorates hepatotoxicity.

Background & aimsAcetaminophen (APAP) overdose remains a frequent cause of acute liver failure, which is generally accompanied by increased levels of serum bile acids (BAs). However, the pathophysiological role of BAs remains elusive. Herein, we investigated the role of BAs in APAP-induced hepatotoxicity.MethodsWe performed intravital imaging to investigate BA transport in mice, quantified endogenous BA concentrations in the serum of mice and patients with APAP overdose, analyzed liver tissue and bile by mass spectrometry and MALDI-mass spectrometry imaging, assessed the integrity of the blood-bile barrier and the role of oxidative stress by immunostaining of tight junction proteins and intravital imaging of fluorescent markers, identified the intracellular cytotoxic concentrations of BAs, and performed interventions to block BA uptake from blood into hepatocytes.ResultsPrior to the onset of cell death, APAP overdose causes massive oxidative stress in the pericentral lobular zone, which coincided with a breach of the blood-bile barrier. Consequently, BAs leak from the bile canaliculi into the sinusoidal blood, which is then followed by their uptake into hepatocytes via the basolateral membrane, their secretion into canaliculi and repeated cycling. This, what we termed 'futile cycling' of BAs, led to increased intracellular BA concentrations that were high enough to cause hepatocyte death. Importantly, however, the interruption of BA re-uptake by pharmacological NTCP blockage using Myrcludex B and Oatp knockout strongly reduced APAP-induced hepatotoxicity.ConclusionsAPAP overdose induces a breach of the blood-bile barrier which leads to futile BA cycling that causes hepatocyte death. Prevention of BA cycling may represent a therapeutic option after APAP intoxication.Lay summaryOnly one drug, N-acetylcysteine, is approved for the treatment of acetaminophen overdose and it is only effective when given within ∼8 hours after ingestion. We identified a mechanism by which acetaminophen overdose causes an increase in bile acid concentrations (to above toxic thresholds) in hepatocytes. Blocking this mechanism prevented acetaminophen-induced hepatotoxicity in mice and evidence from patients suggests that this therapy may be effective for longer periods after ingestion compared to N-acetylcysteine

Spatio-Temporal Multiscale Analysis of Western Diet-Fed Mice Reveals a Translationally Relevant Sequence of Events during NAFLD Progression

Mouse models of non-alcoholic fatty liver disease (NAFLD) are required to define therapeutic targets, but detailed time-resolved studies to establish a sequence of events are lacking. Here, we fed male C57Bl/6N mice a Western or standard diet over 48 weeks. Multiscale time-resolved characterization was performed using RNA-seq, histopathology, immunohistochemistry, intravital imaging, and blood chemistry; the results were compared to human disease. Acetaminophen toxicity and ammonia metabolism were additionally analyzed as functional readouts. We identified a sequence of eight key events: formation of lipid droplets; inflammatory foci; lipogranulomas; zonal reorganization; cell death and replacement proliferation; ductular reaction; fibrogenesis; and hepatocellular cancer. Functional changes included resistance to acetaminophen and altered nitrogen metabolism. The transcriptomic landscape was characterized by two large clusters of monotonously increasing or decreasing genes, and a smaller number of ‘rest-and-jump genes’ that initially remained unaltered but became differentially expressed only at week 12 or later. Approximately 30% of the genes altered in human NAFLD are also altered in the present mouse model and an increasing overlap with genes altered in human HCC occurred at weeks 30–48. In conclusion, the observed sequence of events recapitulates many features of human disease and offers a basis for the identification of therapeutic targets

Inhibition of the Renal Apical Sodium Dependent Bile Acid Transporter Prevents Cholemic Nephropathy in Mice with Obstructive Cholestasis.

BACKGROUND & AIMS Cholemic nephropathy (CN) is a severe complication of cholestasis-associated liver diseases, with no specific treatment. We revisited the pathophysiology to identify therapeutic strategies. METHODS Cholestasis was induced by bile duct ligation (BDL) in mice. Bile flux in kidneys and livers was visualized by intravital imaging, supported by MALDI-MSI and LC-MS/MS. The effect of AS0369, a systemically bioavailable apical sodium-dependent bile acid transporter (ASBT) inhibitor, was evaluated by intravital imaging, RNA-sequencing, histological, blood, and urine analyses. Translational relevance was assessed by ASBT immunostaining in kidney biopsies of CN patients, analysis of mice with humanized BA spectrum, and by analysis of serum bile acids (BA) and kidney injury molecule (KIM-1) in liver disease and hyperbilirubinemia patients. RESULTS Proximal tubular epithelial cells (TEC) reabsorbed and enriched BA, leading to oxidative stress and death of proximal TEC, casts in distal tubules and collecting ducts, peritubular capillaries leakiness, and glomerular cysts. Renal ASBT inhibition by AS0369 blocked BA uptake into TEC and prevented kidney injury up to 6 weeks after BDL. Similar results were obtained in mice with humanized BA composition. In advanced liver disease patients, serum BA were the main determinant of KIM-1 levels. ASBT expression in TEC was preserved in biopsies from CN patients, further highlighting the translational potential of targeting ASBT for treatment of CN. CONCLUSIONS BA enrichment in proximal TEC followed by oxidative stress and cell death is an early key event in CN. Inhibiting renal ASBT and consequently BA enrichment in TEC prevents CN and systemically decreases BA concentrations. IMPACT AND IMPLICATIONS Cholemic nephropathy (CN) is a severe complication of cholestasis with an unmet clinical need for therapy. We demonstrate that CN is triggered by the renal accumulation of bile acids (BA)- that are considerably increased in the systemic blood. Specifically, the proximal tubular epithelial cells (TEC) of the kidney take up BA via the apical sodium-dependent bile acid transporter (ASBT). We developed a therapeutic compound that blocks ASBT in the kidneys, prevents BA overload in TEC, and almost completely abolished all disease hallmarks in a CN mouse model. Renal ASBT inhibition represents a potential therapeutic strategy for CN patients