Receptor interacting protein kinase 1 (RIPK1) in hepatocytes does not mediate murine acetaminophen toxicity
Authors: Anne T. Schneider, Jérémie Gautheron, Frank Tacke, Mihael Vucur, Tom Luedde
Journal: Hepatology (2015)

Potential conflict of interest: Nothing to report. To the Editor: Dara et al. reported in Hepatology that receptor interacting protein kinase 1 (RIPK1)—a kinase harboring pleiotropic functions in programmed cell death1—mediates acetaminophen‐induced necrosis and liver injury.2 To show this, the investigators used an experimental approach featuring repetitive injections of an antisense oligonucleotide to facilitate a knockdown of RIPK1 in liver cells, leading to protection of mice. To stimulate the discussion on this important and timely topic, we would like to add our own findings on RIPK1 in this context. Using cre‐loxP technology, we have generated a new mouse line with conditional ablation of Ripk1 specifically in liver parenchymal cells (LPCs; RIPK1LPC‐KO). With this gold‐standard method, we could reach a complete loss of RIPK1 expression in hepatocytes, as demonstrated by western blotting analysis from whole liver extracts and extracts from isolated and cultured primary hepatocytes (Fig. 1A,B). Importantly, acetaminophen treatment with a similar dose as used by Dara et al. (250 mg/kg intravenously [IV]) did not reveal any differences in liver enzymes and degree of necrosis between RIPK1LPC‐KO and wild‐type (WT) controls (cre‐negative, sex‐matched litter mates; Fig. 1C‐F), arguing against a hepatocyte‐specific function of RIPK1 in the mediation of acetaminophen toxicity.Figure 1: RIPK1 in hepatocytes does not mediate acetaminophen toxicity. Mice with conditional ablation of Ripk1 in liver parenchymal cells (RIPK1LPC‐KO mice) were generated by intercrossing RIPK1‐floxed mice (generation will be described elsewhere) with alfp‐cre transgenics. (A) Efficient deletion of Ripk1 in parenchymal liver cells was demonstrated by western blotting analysis in whole liver protein extracts from RIPK1LPC‐KO and WT controls (cre‐negative litter mates), showing a slight remnant band from nondeleted nonparenchymal liver cells. GAPDH serves as a loading control. (B) Complete ablation of Ripk1 in primary hepatocytes from RIPK1LPC‐KO mice was further demonstrated by western blotting analysis from isolated and cultured primary hepatocytes. β‐Actin serves as a loading control. (C‐E) Overnight fasted 7‐week‐old male RIPK1LPC‐KO mice and WT control littermates were IV injected with 250 mg/kg of acetaminophen. Liver injury was measured at the indicated time points after injection by assessment of glutamate dehydrogenase (GLDH), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) levels. Results are shown as mean ± standard error of the mean; n = 8. (F) Histological analysis and quantification of liver cell necrosis at 48 hours after acetaminophen injection. Results are shown as mean; n = 8. Abbreviations: APAP, acetaminophen; GAPDH, glyceraldehyde 3‐phosphate dehydrogenase; n.s., not significant.Although this finding provides further evidence for a lack of function for necroptosis in acetaminophen toxicity,2 it does not support the hypothesis that necroptosis‐independent functions of RIPK1 in hepatocytes are important in this context. These obvious differences are most likely owing to the different technical approaches used to modify hepatic RIPK1 expression. It cannot be excluded that other intra‐ and extrahepatic cell compartments than hepatocytes—such as monocytes or T cells—are targeted by repetitive injections of antisense oligonucleotides.3 Of note, macrophages lacking Ripk1 show reduced cytokine production, impaired nuclear factor kappa B activation, and greatly compromised caspase 1 processing,4 which might contribute to the beneficial effects upon oligomer injections.2 This would also explain the similarity between the findings from Dara et al. and previous results using the chemical inhibitor Nec‐1 in the acetaminophen model.5 Taken together, hepatocytic RIPK1 does neither directly mediate nor indirectly modulate acute toxicity of acetaminophen in mice.