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Aim 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 6.7x103 (2.3x103–1.4x104) to 1.6x104 (5.4x103–3.2x104) copies/mL. 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 extra-cellular vesicles. miR-885 was also reduced in ESRD patients (4-fold compared to health) and increased by haemodialysis. Conclusion miR-122 is substantially lower in ESRD compared to health, 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. This article is protected by copyright. All rights reserved.

Summary Vascular and kidney dysfunction commonly co-exist. There is a need for biomarkers of vascular health. Circulating microRNAs are biomarkers; miR-126 is endothelial cell-enriched. We measured circulating miR-126 in rats with nephrotoxic nephritis (NTN) and humans with acute endothelial and renal injury (vasculitis associated with autoantibodies to neutrophil cytoplasm antigens (ANCAs)). We compared these findings to those from patients with chronic kidney disease (CKD) and end-stage renal disease (ESRD) and explored the relationship between miR-126 and vascular dysfunction. In NTN, miR-126 was reduced. In ANCA vasculitis (N = 70), pre-treatment miR-126 was reduced compared to health (N = 60) (88-fold). miR-126 increased 3.4-fold post-treatment but remained lower than in health (∼26-fold). Argonaute 2-bound miR-126 increased with ANCA vasculitis treatment. miR-126 did not differ between CKD (N = 30) and health but its concentration correlated with endothelial dysfunction. miR-126 was reduced in ESRD (N = 15) (∼350 fold). miR-126 may be a marker of vascular inflammation and could aid decision-making., Graphical abstract, Highlights • New biomarkers are needed that report vascular function in patients with kidney disease • The endothelial microRNA, miR-126, was reduced with vascular inflammation (vasculitis) • miR-126 correlated with vascular health in chronic kidney disease • Circulating miR-126 may be a marker of vascular dysfunction, Molecular Physiology; Molecular Genetics; Molecular Biology; Immunology

D.C. is funded by a Diabetes Research & Wellness Foundation Sutherland-Earl Clinical Fellowship (RG68554). R.W.H. is supported by the Wellcome Trust–University of Edinburgh Institutional Strategic Support Fund. N.D. is supported by a British Heart Foundation Intermediate Clinical Research Fellowship (FS/13/30/29994). R.S. is funded by the Wellcome Trust (grant WT098498).

SummaryBackgroundParacetamol poisoning is common worldwide. It is treated with intravenous acetylcysteine, but the standard regimen is complex and associated with frequent adverse effects related to concentration, which can cause treatment interruption. We aimed to ascertain whether adverse effects could be reduced with either a shorter modified acetylcysteine schedule, antiemetic pretreatment, or both.MethodsWe undertook a double-blind, randomised factorial study at three UK hospitals, between Sept 6, 2010, and Dec 31, 2012. We randomly allocated patients with acute paracetamol overdose to either the standard intravenous acetylcysteine regimen (duration 20·25 h) or a shorter (12 h) modified protocol, with or without intravenous ondansetron pretreatment (4 mg). Masking was achieved by infusion of 5% dextrose (during acetylcysteine delivery) or saline (for antiemetic pretreatment). Randomisation was done via the internet and included a minimisation procedure by prognostic factors. The primary outcome was absence of vomiting, retching, or need for rescue antiemetic treatment at 2 h. Prespecified secondary outcomes included a greater than 50% increase in alanine aminotransferase activity over the admission value. Analysis was by intention to treat. This trial is registered with ClinicalTrials.gov (identifier NCT01050270).FindingsOf 222 patients who underwent randomisation, 217 were assessable 2 h after the start of acetylcysteine treatment. Vomiting, retching, or need for rescue antiemetic treatment at 2 h was reported in 39 of 108 patients assigned to the shorter modified protocol compared with 71 of 109 allocated to the standard acetylcysteine regimen (adjusted odds ratio 0·26, 97·5% CI 0·13–0·52; p

INTRODUCTION: MicroRNA 122 (miR-122) is a new circulating biomarker for liver injury, which increases earlier than conventional markers in patients with acetaminophen hepatotoxicity. However, as co-ingestion of ethanol is common with drug overdose, a confounding effect of acute ethanol consumption on serum miR-122 must be examined.METHODS: Blood was collected from healthy volunteers before and after recreational consumption of ethanol. Routine biochemistry and haematology measurements were performed, and serum miR-122 was measured by qPCR. The primary outcome was the difference in serum miR-122 with ethanol consumption.RESULTS: We recruited 18 participants (72% male). Their mean serum ethanol concentration was 113 mg/dl (95% confidence interval [CI] 91-135 mg/dl) after consuming ethanol. Serum miR-122 increased from a mean of 71.3 million (95% CI 29.3-113.2 million) to 139.1 million (95% CI 62.6-215.7 million) copies/ml (2.2-fold increase). There was no significant difference in serum alanine aminotransferase activity before and after ethanol consumption.CONCLUSION: miR-122 increased with moderate ethanol consumption, but the fold change was modest. As increases with acetaminophen toxicity are 100- to 10 000-fold, moderate ethanol intoxication is unlikely to confound the use of this biomarker of hepatotoxicity.

We have developed a single probe method for detecting microRNA from human serum using single molecule arrays, with sequence specificity down to a single base, and without the use of amplification by polymerases. An abasic peptide nucleic acid (PNA) probe—containing a reactive amine instead of a nucleotide at a specific position in the sequence—for detecting a microRNA was conjugated to superparamagnetic beads. These beads were incubated with a sample containing microRNA, a biotinylated reactive nucleobase—containing an aldehyde group—that was complementary to the missing base in the probe sequence, and a reducing agent. When a target molecule with an exact match in sequence hybridized to the capture probe, the reactive nucleobase was covalently attached to the backbone of the probe by a dynamic covalent chemical reaction. Single molecules of the biotin-labeled probe were then labeled with streptavidin-β-galactosidase (SβG), the beads were resuspended in a fluorogenic enzyme substrate, loaded into an array of femtoliter wells, and sealed with oil. The array was imaged fluorescently to determine which beads were associated with single enzymes, and the average number of enzymes per bead was determined. The assay had a limit of detection of 500 fM, approximately 500 times more sensitive than a corresponding analog bead-based assay, with target specificity down to a single base mis-match. This assay was used to measure microRNA-122 (miR-122)—an established biomarker of liver toxicity—extracted from the serum of patients who had acute liver injury due to acetaminophen, and control healthy patients. All patients with liver injury had higher levels of miR-122 in their serum compared to controls, and the concentrations measured correlated well with those determined using RT-qPCR. This approach allows rapid quantification of circulating microRNA with single-based specificity and a limit of quantification suitable for clinical use., Quanterix Corporation provided support in the form of salaries and research materials for authors [D.M.R. and D.C.D.]., DestiNA Genomics Ltd. provided support in the form of salaries and research materials for authors [B.L.-L., S.P., and H.I.].

Drug-induced liver injury (DILI) is a major challenge in clinical medicine and drug development. New models are needed for predicting which potential therapeutic compounds will cause DILI in humans, and new markers and mediators of DILI still need to be identified. This review highlights the strengths and weaknesses of using zebrafish as a high-throughput in vivo model for studying DILI. Although the zebrafish liver architecture is different from that of the mammalian liver, the main physiological processes remain similar. Zebrafish metabolize drugs using similar pathways to those in humans; they possess a wide range of cytochrome P450 enzymes that enable metabolic reactions including hydroxylation, conjugation, oxidation, demethylation and de-ethylation. Following exposure to a range of hepatotoxic drugs, the zebrafish liver develops histological patterns of injury comparable to those of mammalian liver, and biomarkers for liver injury can be quantified in the zebrafish circulation. The zebrafish immune system is similar to that of mammals, but the zebrafish inflammatory response to DILI is not yet defined. In order to quantify DILI in zebrafish, a wide variety of methods can be used, including visual assessment, quantification of serum enzymes and experimental serum biomarkers and scoring of histopathology. With further development, the zebrafish may be a model that complements rodents and may have value for the discovery of new disease pathways and translational biomarkers.