The effect of renal dysfunction and haemodialysis on circulating liver specific miR-122.

AIMS: microRNA-122 (miR-122) is a hepatotoxicity biomarker with utility in the management of paracetamol overdose and in drug development. Renal dysfunction and haemodialysis have been associated with a reduction in circulating microRNA. The objective of this study was to determine their effect on miR-122. METHODS: Blood samples were collected from 17 patients with end-stage renal disease (ESRD) on haemodialysis, 22 healthy controls, 30 patients with chronic kidney disease (CKD) and 15 patients post-kidney transplantation. All had normal standard liver function tests. Samples from ESRD patients were collected immediately pre- and post-haemodialysis. Serum alanine transaminase activity (ALT), miR-122 and miR-885 (liver enriched) were compared. RESULTS: Circulating miR-122 was substantially reduced in ESRD patients pre-haemodialysis compared with the other groups (19.0-fold lower than healthy controls; 21.7-fold lower than CKD). Haemodialysis increased miR-122 from a median value of 6.7 × 103 (2.3 × 103 -1.4 × 104 ) to 1.6 × 104 (5.4 × 103 -3.2 × 104 ) copies ml-1 . The increase in miR-122 did not correlate with dialysis adequacy. miR-122 was reduced in the argonaute 2 bound fraction pre-haemodialysis; this fraction was increased post-dialysis. There was no change in miR-122 associated with extracellular vesicles. miR-885 was also reduced in ESRD patients (4-fold compared to healthy subjects) and increased by haemodialysis. CONCLUSION: miR-122 is substantially lower in ESRD compared to healthy controls, patients with CKD and transplanted patients. Haemodialysis increases the concentration of miR-122. These data need to be considered when interpreting liver injury using miR-122 in patients with ESRD on dialysis, and specific reference ranges that define normal in this setting may need to be developed.

Characterization of Triptolide-Induced Hepatotoxicity by Imaging and Transcriptomics in a Novel Zebrafish Model.

Triptolide is a vine extract used in traditional Chinese medicines and associated with hepatotoxicity. In vitro data suggest that inhibition of RNA synthesis may be the mechanism of toxicity. For studying drug-induced liver injury the zebrafish has experimental, practical and financial advantages compared with rodents. The aim of this study was to explore the mechanism of triptolide toxicity using zebrafish as the model system. The effect of triptolide exposure on zebrafish larvae was determined with regard to mortality, histology, expression of liver specific microRNA-122 and liver volume. Fluorescent microscopy was used to track toxicity in the Tg(-2.8lfabp:GFP)as3 zebrafish line. Informed by microscopy, RNA-sequencing was used to explore the mechanism of toxicity. Triptolide exposure resulted in dose-dependent mortality, a reduction in the number of copies of microRNA-122 per larva, hepatocyte vacuolation, disarray and oncotic necrosis, and a reduction in liver volume. These findings were consistent across replicate experiments. Time-lapse imaging indicated the onset of injury was 6 h after the start of exposure, at which point, RNA-sequencing revealed that 88% of genes were down-regulated. Immune response associated genes were up-regulated in the triptolide-treated larvae including nitric oxide synthase. Inhibition of nitric oxide synthase increased mortality. Triptolide induces hepatotoxicity in zebrafish larvae. This represents a new model of drug-induced liver injury that complements rodents. RNA sequencing, guided by time-lapse microscopy, revealed early down-regulation of genes consistent with previous invitro studies, and facilitated the discovery of mechanistic inflammatory pathways.