Cytokeratin-18 is a sensitive biomarker of alanine transaminase increase in a placebo-controlled, randomized, crossover trial of therapeutic paracetamol dosing (PATH-BP biomarker substudy)

Drug-induced liver injury (DILI) is a challenge in clinical medicine and drug development. Highly sensitive novel biomarkers have been identified for detecting DILI following a paracetamol overdose. The study objective was to evaluate biomarker performance in a 14-day trial of therapeutic dose paracetamol. The PATH-BP trial was a double-blind, placebo-controlled, crossover study. Individuals (n = 110) were randomized to receive 1 g paracetamol 4× daily or matched placebo for 2 weeks followed by a 2-week washout before crossing over to the alternate treatment. Blood was collected on days 0 (baseline), 4, 7, and 14 in both arms. Alanine transaminase (ALT) activity was measured in all patients. MicroRNA-122 (miR-122), cytokeratin-18 (K18), and glutamate dehydrogenase (GLDH) were measured in patients who had an elevated ALT on paracetamol treatment (≥50% from baseline). ALT increased in 49 individuals (45%). All 3 biomarkers were increased at the time of peak ALT (K18 paracetamol arm: 18.9 ± 9.7 ng/ml, placebo arm: 11.1 ± 5.4 ng/ml, ROC-AUC = 0.80, 95% CI 0.71–0.89; miR-122: 15.1 ± 12.9fM V 4.9 ± 4.7fM, ROC-AUC = 0.83, 0.75–0.91; and GLDH: 24.6 ± 31.1U/l V 12.0 ± 11.8U/l, ROC-AUC = 0.66, 0.49–0.83). All biomarkers were correlated with ALT (K18 r = 0.68, miR-122 r = 0.67, GLDH r = 0.60). To assess sensitivity, biomarker performance was analyzed on the visit preceding peak ALT (mean 3 days earlier). K18 identified the subsequent ALT increase (K18 ROC-AUC = 0.70, 0.59–0.80; miR-122 ROC-AUC = 0.60, 0.49–0.72, ALT ROC-AUC = 0.59, 0.48–0.70; GLDH ROC-AUC = 0.70, 0.50–0.90). Variability was lowest for ALT and K18. In conclusion, K18 was more sensitive than ALT, miR-122, or GLDH and has potential significant utility in the early identification of DILI in trials and clinical practice

Glucocorticoid receptor alters isovolumetric contraction and restrains cardiac fibrosis

Corticosteroids directly affect the heart and vasculature and are implicated in the pathogenesis of heart failure. Attention is focussed upon the role of the mineralocorticoid receptor (MR) in mediating pro-fibrotic and other adverse effects of corticosteroids upon the heart. In contrast, the role of the glucocorticoid receptor (GR) in the heart and vasculature is less well understood. We addressed this in mice with cardiomyocyte and vascular smooth muscle deletion of GR (SMGRKO mice). Survival of SMGRKO mice to weaning was reduced compared with that of littermate controls. Doppler measurements of blood flow across the mitral valve showed an elongated isovolumetric contraction time in surviving adult SMGRKO mice, indicating impairment of the initial left ventricular contractile phase. Although heart weight was elevated in both genders, only male SMGRKO mice showed evidence of pathological cardiomyocyte hypertrophy, associated with increased myosin heavy chain-β expression. Left ventricular fibrosis, evident in both genders, was associated with elevated levels of mRNA encoding MR as well as proteins involved in cardiac remodelling and fibrosis. However, MR antagonism with spironolactone from birth only modestly attenuated the increase in pro-fibrotic gene expression in SMGRKO mice, suggesting that elevated MR signalling is not the primary driver of cardiac fibrosis in SMGRKO mice, and cardiac fibrosis can be dissociated from MR activation. Thus, GR contributes to systolic function and restrains normal cardiac growth, the latter through gender-specific mechanisms. Our findings suggest the GR:MR balance is critical in corticosteroid signalling in specific cardiac cell types

Exosome signalling in the kidney

Urine contains exosomes originating from the circulation and all cells lining the urinary tract. Exosomes are a route of inter-cellular communication along the nephron potentially able to transfer of protein and/or RNA. It is not known whether this is a regulated process analogous to other cell-to-cell signalling systems. The aims of this study were to develop nanoparticle tracking analysis (NTA) as a technique to quantify exosomes in urine. Secondly, the hormonal regulation of exosome uptake in vitro and in vivo was investigated. Thirdly, exosome excretion in a central diabetes insipidus (DI) patient and a patient group after radiocontrast exposure was measured to investigate exosome excretion along the kidney in injury. Using the fluorescent capabilities of NTA, urinary exosomes were quantified in urine samples. NTA was able to detect changes in aquaporin 2 levels in vitro and in vivo. Storage conditions for human urinary exosomes were also optimised using NTA. A kidney cortical collecting duct cell line (CCDs) was used to model regulation of exosome uptake in vitro. CCDs were stimulated with desmopressin, a vasopressin analogue, and uptake of fluorescently-loaded or microRNA-loaded exosomes was measured. Desmopressin stimulated exosome uptake into collecting duct cells via V2 receptor stimulation. Intra-cellular uptake of exosomes was confirmed by microRNA specific mRNA down-regulation. Mechanistically, exosome uptake in response to desmopressin required cyclic AMP production, was mediated by clathrin-dependent endocytosis and was selective for exosomes from kidney tubular cells. In mice, fluorescently-loaded exosomes were systemically injected before and after administration of the V2 antagonist, tolvaptan, and urinary exosome excretion was measured. Basally, 2.5% of injected exosomes were recovered in urine; tolvaptan treatment resulted in a 5-fold increase. By combining antibodies to nephron segment-specific proteins with NTA we measured human urinary exosome excretion in central diabetes insipidus (DI) and after radiocontrast exposure (n=37). In DI, desmopressin reduced the excretion of exosomes derived from upstream glomerular and proximal tubule cells. In patients exposed to radiocontrast, urinary exosomes from the glomerulus were positively correlated with the tubular injury markers KIM- 1 and NGAL. These findings therefore show that tubular exosome uptake is a specific, hormonally regulated process that is reduced with injury. Physiologically, exosomes are a mechanism of inter-cellular communication; therapeutically, exosomes represent a novel vehicle by which RNA therapy could be targeted for the treatment of kidney disease