Discovery, validation and sequencing of urinary peptides for diagnosis of liver fibrosis—A multicentre study
Ayman S. Bannaga,
Jochen Metzger,
Ioannis Kyrou,
Torsten Voigtländer,
Thorsten Book,
Jesus Melgarejo,
Agnieszka Latosinska,
Martin Pejchinovski,
Jan A. Staessen,
Harald Mischak,
Michael P. Manns,
Ramesh P. Arasaradnam
Affiliations
Ayman S. Bannaga
Department of Gastroenterology and Hepatology, University Hospital Coventry and Warwickshire NHS Trust, Clifford Bridge Road, Coventry CV2 2DX, UK; Warwick Medical School, University of Warwick, Coventry CV4 7HL, UK; Corresponding author at: Department of Gastroenterology and Hepatology, University Hospital Coventry and Warwickshire NHS Trust, Clifford Bridge Road, Coventry CV2 2DX, UK.
Jochen Metzger
Mosaiques-Diagnostics GmbH, Hannover, Germany
Ioannis Kyrou
Warwick Medical School, University of Warwick, Coventry CV4 7HL, UK; Aston Medical Research Institute, Aston Medical School, Aston University, Birmingham B4 7ET, UK; Warwickshire Institute for the Study of Diabetes, Endocrinology and Metabolism (WISDEM), University Hospital Coventry and Warwickshire NHS Trust, Coventry CV2 2DX, UK
Torsten Voigtländer
Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
Thorsten Book
Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
Jesus Melgarejo
Department of Cardiovascular Sciences, Studies Coordinating Centre, Research Unit Hypertension and Cardiovascular Epidemiology, KU Leuven, University of Leuven, Leuven, Belgium; Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands
Agnieszka Latosinska
Mosaiques-Diagnostics GmbH, Hannover, Germany
Martin Pejchinovski
Mosaiques-Diagnostics GmbH, Hannover, Germany
Jan A. Staessen
Department of Cardiovascular Sciences, Studies Coordinating Centre, Research Unit Hypertension and Cardiovascular Epidemiology, KU Leuven, University of Leuven, Leuven, Belgium; Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, Maastricht, the Netherlands
Harald Mischak
Mosaiques-Diagnostics GmbH, Hannover, Germany
Michael P. Manns
Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany
Ramesh P. Arasaradnam
Department of Gastroenterology and Hepatology, University Hospital Coventry and Warwickshire NHS Trust, Clifford Bridge Road, Coventry CV2 2DX, UK; Warwick Medical School, University of Warwick, Coventry CV4 7HL, UK; Faculty of Health and Life Sciences, Coventry University, Priory St, Coventry CV1 5FB, UK; School of Biological Sciences, University of Leicester, University Road, Leicester LE1 7RH, UK
Background: Liver fibrosis is a consequence of chronic inflammation and is associated with protein changes within the hepatocytes structure. In this study, we aimed to investigate if this is reflected by the urinary proteome and can be explored to diagnose liver fibrosis in patients with chronic liver disease. Methods: In a multicentre combined cross-sectional and prospective diagnostic test validation 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 analysed by capillary electrophoresis coupled to mass spectrometry (CE-MS) and a support vector machine marker model was established by integration of peptide markers for liver fibrosis. Findings: CE-MS enabled identification of 50 urinary peptides associated with liver fibrosis. When combined into a classifier, LivFib-50, it separated patients with liver fibrosis (N = 31) from non-liver disease controls (N = 123) in cross-sectional diagnostic phase II evaluation with an area under the curve (AUC) of 0.94 (95% confidence intervals (CI): 0.89–0.97, p<0.0001). When adjusted for age, LivFib-50 demonstrated an AUC of 0.94 (95% CI: 0.89–0.97, p<0.0001) in chronic liver disease patients with (N = 19) or without (N = 17) liver fibrosis progression. In this prospective diagnostic phase III validation set, 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 are 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.
