Discovery, Validation and Sequencing of Urinary Peptides for Diagnosis of Liver Fibrosis —A Multicentre Study

Abstract

Background: Liver fibrosis is a consequence of chronic inflammation and is associated with protein changes within the hepatocytes structure. We therefore investigated urinary peptides in patients with liver fibrosis. Methods: Multicentre case-control prospective study. 129 patients with varying degrees of liver fibrosis and 223 controls without liver fibrosis were recruited. Additionally, 41 patients with no liver, but kidney fibrosis were included to evaluate interference with expressions of kidney fibrosis. Urinary low molecular weight proteome was then analysed by capillary electrophoresis coupled to mass spectrometry (CE-MS). Findings: CE-MS enabled identification of 50 urinary peptides associated with liver fibrosis. When combined into a classifier, LivFib-50, it separated liver fibrosis from controls with an area under the curve (AUC) of 0.95 (95% confidence intervals (CI): 0.90-0.98, p<0.0001) with 83.5% sensitivity and 95.1% specificity. In the first validation cohort, LivFib-50 demonstrated an AUC of 0.94 (95% CI: 0.89-0.97, p<0.0001). In a second validation cohort, LivFib-50 was adjusted for age and demonstrated an AUC of 0.91 (95% CI: 0.76 -0.98, p<0.0001). Age-adjusted LivFib-50 showed 84.2% sensitivity (95% CI: 60.4 - 96.6) and 82.4% specificity (95% CI: 56.6 - 96.2) for detection of liver fibrosis. The sequence-identified peptides were mainly fragments of collagen chains, uromodulin and Na/K-transporting ATPase subunit γ. We also identified ten putative proteolytic cleavage sites, eight were specific for matrix metallopeptidases and two for cathepsins. Interpretation: In liver fibrosis, urinary peptides profiling offers potential diagnostic markers and leads to discovery of proteolytic sites that could be targets for developing anti-fibrotic therapy. Funding Statement: This manuscript was supported by research grant from the medical and life sciences research fund (MLSRF) which was awarded to AB. It is charity that supports research and education to enhance human health and had no role in study design or methodology. Declaration of Interests: Harald Mischak is founder and co-owner of Mosaiques Diagnostics GmbH, which developed the CE-MS technology. Jochen Metzger and Martin Pejchinovski are employees of Mosaiques Diagnostics GmbH. All other authors declare no conflict of interest. Ethics Approval Statement: The study was approved by the relevant Ethics and R&D Committees (Ref 18717 and Ref 260179). Study was conducted according to the World Medical Association Declaration of Helsinki, with all study participants providing written informed consent.