James W. Dear

Circulating microRNAs as potential markers of human drug‐induced liver injury

New biomarkers of liver injury are required in the clinic and in preclinical pharmaceutical evaluation. Previous studies demonstrate that two liver‐enriched microRNAs (miR‐122 and miR‐192) are promising biomarkers of acetaminophen‐induced acute liver injury (APAP‐ALI) in mice. We have examined these molecules, for the first time, in humans with APAP poisoning. Serum miR‐122 and miR‐192 were substantially higher in APAP‐ALI patients, compared to healthy controls (median ΔΔCt [25th, 75th percentile]) (miR‐122: 1,265 [491, 4,270] versus 12.1 [7.0, 26.9], P < 0.0001; miR‐192: 6.9 [2.0, 29.2] versus 0.44 [0.30, 0.69], P < 0.0001). A heart‐enriched miR‐1 showed no difference between APAP‐ALI patients and controls, whereas miR‐218 (brain‐enriched) was slightly higher in the APAP‐ALI cohort (0.17 [0.07, 0.50] versus 0.07 [0.04, 0.12]; P = 0.01). In chronic kidney disease (CKD) patients, miR‐122 and ‐192 were modestly higher, compared to controls (miR‐122: 32.0 [21.1, 40.9] versus 12.1 [7.0, 26.9], P = 0.006; miR‐192: 1.2 [0.74, 1.9] versus 0.44 [0.30, 0.69], P = 0.005), but miR‐122 and ‐192 were substantially higher in APAP‐ALI patients than CKD patients (miR‐122: P < 0.0001; miR‐192: P < 0.0004). miR‐122 correlated with peak ALT levels in the APAP‐ALI cohort (Pearson R = 0.46, P = 0.0005), but not with prothrombin time. miR‐122 was also raised alongside peak ALT levels in a group of patients with non‐APAP ALI. Day 1 serum miR‐122 levels were almost 2‐fold higher in APAP‐ALI patients who satisfied Kings College Criteria (KCC), compared to those who did not satisfy KCC, although this did not reach statistical significance (P = 0.15). Conclusion: This work provides the first evidence for the potential use of miRNAs as biomarkers of human drug‐induced liver injury. (HEPATOLOGY 2011;)

Discovery of Protein Biomarkers for Renal Diseases

Animal models and human studies have been useful in dissecting the molecular mechanisms of renal disease and finding new disease targets; however, translation of these findings to new clinical therapeutics remains challenging. Difficulties with detecting early disease, measuring drug effectiveness, and the daunting cost of clinical trials hampers the development of new therapeutics for renal diseases. Many existing laboratory tests were discovered because of inspired recognition that a particular protein might prove useful in clinical practice. New unbiased genomic and proteomic techniques identify many constituents present in biologic samples and thus may greatly accelerate biomarker research. This review focuses on the steps needed to develop new biomarkers that are useful in laboratory and clinical investigations, with particular focus on new proteomic screening technologies. New biomarkers will speed the laboratory and clinical development of new treatments for renal diseases through mechanistic insights, diagnoses that are more refined, early detection, and enhanced proof of concept testing.

Mechanistic biomarkers provide early and sensitive detection of acetaminophen‐induced acute liver injury at first presentation to hospital

Acetaminophen overdose is a common reason for hospital admission and the most frequent cause of hepatotoxicity in the Western world. Early identification would facilitate patient‐individualized treatment strategies. We investigated the potential of a panel of novel biomarkers (with enhanced liver expression or linked to the mechanisms of toxicity) to identify patients with acetaminophen‐induced acute liver injury (ALI) at first presentation to the hospital when currently used markers are within the normal range. In the first hospital presentation plasma sample from patients (n = 129), we measured microRNA‐122 (miR‐122; high liver specificity), high mobility group box‐1 (HMGB1; marker of necrosis), full‐length and caspase‐cleaved keratin‐18 (K18; markers of necrosis and apoptosis), and glutamate dehydrogenase (GLDH; marker of mitochondrial dysfunction). Receiver operator characteristic curve analysis and positive/negative predictive values were used to compare sensitivity to report liver injury versus alanine transaminase (ALT) and International Normalized Ratio (INR). In all patients, biomarkers at first presentation significantly correlated with peak ALT or INR. In patients presenting with normal ALT or INR, miR‐122, HMGB1, and necrosis K18 identified the development of liver injury (n = 15) or not (n = 84) with a high degree of accuracy and significantly outperformed ALT, INR, and plasma acetaminophen concentration for the prediction of subsequent ALI (n = 11) compared with no ALI (n = 52) in patients presenting within 8 hours of overdose. Conclusion: Elevations in plasma miR‐122, HMGB1, and necrosis K18 identified subsequent ALI development in patients on admission to the hospital, soon after acetaminophen overdose, and in patients with ALTs in the normal range. The application of such a biomarker panel could improve the speed of clinical decision‐making, both in the treatment of ALI and the design/execution of patient‐individualized treatment strategies. (Hepatology 2013;58:777–787)

Molecular forms of HMGB1 and keratin-18 as mechanistic biomarkers for mode of cell death and prognosis during clinical acetaminophen hepatotoxicity

BACKGROUND & AIMS Full length keratin-18 (FL-K18) and High Mobility Group Box-1 (HMGB1) represent circulating indicators of necrosis during acetaminophen (APAP) hepatotoxicity in vivo. In addition, the caspase-cleaved fragment of K18 (cK18) and hyper-acetylated HMGB1 represent serum indicators of apoptosis and immune cell activation, respectively. The study aim was to assess their mechanistic utility to establish the balance between apoptosis, necrosis, and immune cell activation throughout the time course of clinical APAP hepatotoxicity. METHODS HMGB1 (total, acetylated) and K18 (apoptotic, necrotic) were identified and quantified by novel LC-MS/MS assays in APAP overdose patients (n=78). RESULTS HMGB1 (total; 15.4±1.9ng/ml, p<0.01, acetylated; 5.4±2.6ng/ml, p<0.001), cK18 (5649.8±721.0U/L, p<0.01), and FL-K18 (54770.2±6717.0U/L, p<0.005) were elevated in the sera of APAP overdose patients with liver injury compared to overdose patients without liver injury and healthy volunteers. HMGB1 and FL-K18 correlated with alanine aminotransferase (ALT) activity (R(2)=0.60 and 0.58, respectively, p<0.0001) and prothrombin time (R(2)=0.62 and 0.71, respectively, p<0.0001). Increased total and acetylated HMGB1 and FL-K18 were associated with worse prognosis (Kings College Criteria) or patients that died/required liver transplant compared to spontaneous survivors (all p<0.05-0.001), a finding not reflected by ALT and supported by ROC analysis. Acetylated HMGB1 was a better predictor of outcome than the other markers of cell death. CONCLUSIONS K18 and HMGB1 represent blood-based tools to investigate the cell death balance clinical APAP hepatotoxicity. Activation of the immune response was seen later in the time course as shown by the distinct profile of acetylated HMGB1 and was associated with worse outcome.

Chloroquine and inhibition of Toll-like receptor 9 protect from sepsis-induced acute kidney injury

Mortality from sepsis has remained high despite recent advances in supportive and targeted therapies. Toll-like receptors (TLRs) sense bacterial products and stimulate pathogenic innate immune responses. Mice deficient in the common adapter protein MyD88, downstream from most TLRs, have reduced mortality and acute kidney injury (AKI) from polymicrobial sepsis. However, the identity of the TLR(s) responsible for the host response to polymicrobial sepsis is unknown. Here, we show that chloroquine, an inhibitor of endocytic TLRs (TLR3, 7, 8, 9), improves sepsis-induced mortality and AKI in a clinically relevant polymicrobial sepsis mouse model, even when administered 6 h after the septic insult. Chloroquine administration attenuated the decline in renal function, splenic apoptosis, serum markers of damage to other organs, and prototypical serum pro- and anti-inflammatory cytokines TNF-alpha and IL-10. An oligodeoxynucleotide inhibitor (H154) of TLR9 and TLR9-deficient mice mirror the actions of chloroquine in all functional parameters that we tested. In addition, chloroquine decreased TLR9 protein abundance in spleen, further suggesting that TLR9 signaling may be a major target for the protective actions of chloroquine. Our findings indicate that chloroquine improves survival by inhibiting multiple pathways leading to polymicrobial sepsis and that chloroquine and TLR9 inhibitors represent viable broad-spectrum and targeted therapeutic strategies, respectively, that are promising candidates for further clinical development.

Blood Kidney Injury Molecule-1 Is a Biomarker of Acute and Chronic Kidney Injury and Predicts Progression to ESRD in Type I Diabetes

Currently, no blood biomarker that specifically indicates injury to the proximal tubule of the kidney has been identified. Kidney injury molecule-1 (KIM-1) is highly upregulated in proximal tubular cells following kidney injury. The ectodomain of KIM-1 is shed into the lumen, and serves as a urinary biomarker of kidney injury. We report that shed KIM-1 also serves as a blood biomarker of kidney injury. Sensitive assays to measure plasma and serum KIM-1 in mice, rats, and humans were developed and validated in the current study. Plasma KIM-1 levels increased with increasing periods of ischemia (10, 20, or 30 minutes) in mice, as early as 3 hours after reperfusion; after unilateral ureteral obstruction (day 7) in mice; and after gentamicin treatment (50 or 200 mg/kg for 10 days) in rats. In humans, plasma KIM-1 levels were higher in patients with AKI than in healthy controls or post-cardiac surgery patients without AKI (area under the curve, 0.96). In patients undergoing cardiopulmonary bypass, plasma KIM-1 levels increased within 2 days after surgery only in patients who developed AKI (P<0.01). Blood KIM-1 levels were also elevated in patients with CKD of varous etiologies. In a cohort of patients with type 1 diabetes and proteinuria, serum KIM-1 level at baseline strongly predicted rate of eGFR loss and risk of ESRD during 5-15 years of follow-up, after adjustment for baseline urinary albumin-to-creatinine ratio, eGFR, and Hb1Ac. These results identify KIM-1 as a blood biomarker that specifically reflects acute and chronic kidney injury.

Management of paracetamol poisoning

#### Summary points Paracetamol (acetaminophen) is an effective oral analgesic, with few adverse effects when used at the recommended dose. Nevertheless, paracetamol poisoning is common and potentially fatal.1 It is a leading cause of acute liver failure in the United Kingdom2 and the United States.3 Potential liver damage, predicted from blood paracetamol concentration and time from ingestion, can be prevented by prompt treatment with antidote. However, young and otherwise healthy patients still risk serious liver injury, especially if they present more than a few hours after overdose or take staggered overdoses over hours or days.4 #### Sources and selection criteria We based our review on a PubMed search for articles on paracetamol (or acetaminophen) and acetylcysteine or ( N- acetylcysteine) published between 1 January 1990 and 31 December 2010, without language limits. The search was limited to human clinical trials, meta-analyses, randomised controlled trials, reviews, and case reports. We also searched a newspaper database for reports published after 1988 of coroners’ inquests and procurators’ fiscal inquiries into fatal cases of paracetamol poisoning. In addition, we used a bibliography and our own collections of relevant references.5 The …

Urinary exosomes: A reservoir for biomarker discovery and potential mediators of intrarenal signalling

Over the last decade, there has been increasing research interest in urinary exosomes and their relationship with kidney physiology and disease. Protocols for isolating urinary exosomes have been refined and the exosomal proteome has been extensively catalogued and reported to contain proteins from the kidneys glomerulus and all sections of the nephron. In animal and human biomarker discovery studies, this proteome changes to reflect the underlying pathophysiology of certain kidney diseases. In addition to proteins, exosomes from urine have been demonstrated to contain RNA species, another new reservoir for biomarker discovery. Exosomes have the capacity to shuttle their cargo between kidney cells and change the recipient cells proteome and function, and may represent a mechanism for cell‐to‐cell signalling along the nephron. Significant challenges remain; methods for urinary exosome collection need optimisation if “real‐life” clinical utility is to be achieved, consensus is needed regarding normalisation of changes in exosomal protein and RNA, larger scale exosome biomarker validation studies remain to be performed, and whether exosomes signal between cells in vivo remains an intriguing, but untested, hypothesis.

Reduction of adverse effects from intravenous acetylcysteine treatment for paracetamol poisoning: a randomised controlled trial

BACKGROUND Paracetamol 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. METHODS We 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). FINDINGS Of 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<0·0001), and in 45 of 109 patients who received ondansetron compared with 65 of 108 allocated placebo (0·41, 0·20-0·80; p=0·003). Severe anaphylactoid reactions were recorded in five patients assigned to the shorter modified acetylcysteine regimen versus 31 who were allocated to the standard protocol (adjusted common odds ratio 0·23, 97·5% CI 0·12-0·43; p<0·0001). The proportion of patients with a 50% increase in alanine aminotransferase activity did not differ between the standard (9/110) and shorter modified (13/112) regimens (adjusted odds ratio 0·60, 97·5% CI 0·20-1·83); however, the proportion was higher with ondansetron (16/111) than with placebo (6/111; 3·30, 1·01-10·72; p=0·024). INTERPRETATION In patients with paracetamol poisoning, a 12 h modified acetylcysteine regimen resulted in less vomiting, fewer anaphylactoid reactions, and reduced need for treatment interruption. This study was not powered to detect non-inferiority of the shorter protocol versus the standard approach; therefore, further research is needed to confirm the efficacy of the 12 h modified acetylcysteine regimen. FUNDING Chief Scientist Office of the Scottish Government.

Quantification of human urinary exosomes by nanoparticle tracking analysis

•  Exosomes are vesicles that are released from the kidney into the urine. They contain RNA and protein from the cell of origin and can track changes in renal physiology non‐invasively. •  Current methods for the identification and quantification of urinary exosomes are time consuming and only semi‐quantitative. •  In this study, we applied nanoparticle tracking analysis to human urine and identified particles with a range of sizes, including a subpopulation of characteristic exosomal size that labelled positively with antibodies to exosome proteins. •  Nanoparticle tracking analysis was able to track an increase in exosomal aquaporin 2 concentration following desmopressin treatment of a kidney cell line, a rodent model and a patient with central diabetes insipidus. •  With appropriate sample storage, nanoparticle tracking analysis has potential as a tool for the rapid characterization and quantification of exosomes in human urine. This new method can be used to develop urinary extracellular vesicles further as a non‐invasive tool for investigating human renal physiology.