Receptor interacting protein kinase 3 is a critical early mediator of acetaminophen-induced hepatocyte necrosis in mice

Abstract

Acetaminophen (APAP) overdose is a major cause of hepatotoxicity and acute liver failure in the US, but the pathophysiology is incompletely understood. Despite evidence for apoptotic signaling, hepatic cell death after APAP is generally considered necrotic in mice and in humans. Recent findings suggest that the receptor interacting protein kinase 3 (RIP3) acts as a switch from apoptosis to necrosis (programmed necrosis). Thus, the aim of the current investigation was to determine if RIP3 is involved in APAP-induced liver cell death. APAP (200–300 mg/kg) caused glutathione depletion and protein adduct formation, oxidant stress, mitochondrial release of apoptosis inducing factor, and nuclear DNA fragmentation resulting in centrilobular necrosis in C57Bl/6J mice. Inhibiting RIP3 protein induction with anti-sense morpholinos in wild-type animals or using RIP3-deficient mice had no effect on protein adduct formation but attenuated all other parameters, including necrotic cell death, at 6h after APAP. In addition, cultured hepatocytes from RIP3-deficient mice showed reduced injury compared to wild-type cells after 24h. Interestingly, APAP-induced mitochondrial translocation of dynamin-related protein 1 (Drp1), the initiator of mitochondrial fission, was inhibited by reduced RIP3 protein expression and the Drp1 inhibitor MDIVI reduced APAP-induced cell death at 24h. All of these protective effects were lost after 24h in vivo or 48h in vitro. Conclusion: RIP3 is an early mediator of APAP hepatotoxicity, involving modulation of mitochondrial dysfunction and oxidant stress. Controlling RIP3 expression could be a promising new approach to reduce APAP-induced liver injury, but requires complementary strategies to control mitochondrial dysfunction for long-term protection.

The pseudokinase MLKL mediates programmed hepatocellular necrosis independently of RIPK3 during hepatitis

“…However, the role of programmed hepatocellular death in acetaminophen-induced (APAP-induced) murine liver damage remains controversial. Although inhibition of necroptosis by deficiency of RIPK3 (17) or pharmacological blockage of RIPK1 kinase activity (18) reduced cell death at early time points during APAP-induced hepatic injury, RIPK3 and MLKL deficiency was unable to prevent liver injury in this model (19). Other studies further demonstrated that ethanol-induced hepatic injury is independent of RIPK1 kinase activity but dependent on RIPK3, suggesting that necroptosis does not always require RIPK1 function (20).…”

Caspase Inhibition Prevents Tumor Necrosis Factor-α–Induced Apoptosis and Promotes Necrotic Cell Death in Mouse Hepatocytes in Vivo and in Vitro

“…RIP3 À/À mice were generously provided by Dr. Vishva Dixit (Genentech, San Francisco, CA) as we described previously. 18 All mice were housed in cages (five mice per cage) receiving a 12-hour light/dark cycle. Mice were injected intraperitoneally with 100 mg/kg LPS and 700 mg/kg GalN for 6 hours.…”

“…Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed as described previously. 18 …”

Highlight report: acetaminophen hepatotoxicity

“…Much effort has been invested to gain a better understanding of the molecular mechanisms of APAP hepatotoxicity (Saito et al 2010;Kon et al 2004;Hwang et al 2015;Sjogren et al 2014;Schyschka et al 2013;Singh et al 2013) and to identify biomarkers of APAP overdose (McGill et al 2014;Beger et al 2015). APAP is known to be metabolically activated to the reactive N-acetyl-p-benzoquinone imine (NAPQI), which forms protein adducts including mitochondrial proteins leading to mitochondrial oxidative stress (Ramachandran et al 2013;Cohen et al 1997). As a consequence c-jun N-terminal kinase is translocated to the mitochondria, which enhances generation of reactive oxygen species (Ramachandran et al 2013;Hanawa et al 2008;Saito et al 2010).…”

“…APAP is known to be metabolically activated to the reactive N-acetyl-p-benzoquinone imine (NAPQI), which forms protein adducts including mitochondrial proteins leading to mitochondrial oxidative stress (Ramachandran et al 2013;Cohen et al 1997). As a consequence c-jun N-terminal kinase is translocated to the mitochondria, which enhances generation of reactive oxygen species (Ramachandran et al 2013;Hanawa et al 2008;Saito et al 2010). This may lead to opening of the mitochondrial membrane transition pore and cause a form of cell death which differs from classical apoptosis and has been named 'necroptosis' (Kon et al 2004;Gujral et al 2002;Ni et al 2012).…”