Urine Proteome in Patients With Metabolic Associated Fatty Liver Disease

Abstract

Background: In patients with Metabolic associated fatty liver disease (MAFLD), hepatic steatosis is an independent predictor for insulin resistance and cardiovascular risk and hence mortality. Urine biomarker is less complex and has a relatively lower dynamic range with less technical challenges compared to blood. However, little was known whether the urine sample could reflect the altered metabolic/lipid feature in liver and the severity of hepatic steatosis. Therefore, the aim of present study was to investigate the urine molecular pattern and to verify new urine biomarker for hepatic steatosis in MAFLD patients. Method: Hepatic steatosis was measured by Magnetic resonance imaging (MRI) that measure the proton density fat fraction (MRI-PDFF). Mild hepatic steatosis defined as MRI-PDFF 5-10% and the severe hepatic steatosis was defined as MRI-PDFF>10%. Urine samples of 7 healthy controls, 8 MAFLD patients with mild hepatic steatosis, and 12 MAFLD patient with severe steatosis were collected. Proteomic measurements of urine samples were done by mass spectrometry. Western blot and Elisa were performed for validation of potential urine biomarker. Results: There were 53 proteins identified with significantly different expressions among the healthy control, mild steatosis and severe steatosis in MAFLD patients by proteomic data. GO and KEGG analysis of these significantly changed urinary molecular features implied that the MAFLD could result in the dysregulation of carbohydrate derivative catabolic process, glycosaminoglycan process, aminoglycan metabolic process, catabolic process, inflammatory response, insulin-like growth factor receptor, GTPase complex, PI3K−Akt signaling pathway and cholesterol metabolism. Nextly, Alpha-1-acid glycoprotein 1 (ORM1) and ceruloplasmin were the most correlated with clinical parameters in MAFLD, including liver fat content, fibrosis, ALT, triglyceride, glucose, HOMA-IR, and C-reactive protein. Finally, the validation of urine sample (30 cases) by Elisa showed that both ceruloplasmin (healthy control 3.02 ± 1.43 vs. mild steatosis 4.27 ± 1.18 vs. severe steatosis 5.61 ± 1.31, p<0.001) and ORM1 (1.19 ± 1.085 vs. 3.41 ± 2.61 vs. 4.68 ± 3.154, p=0.011) showed an increased tendency from healthy control to mild steatosis and severe steatosis, those results were further validated by western blot. Conclusion: Proteomics profiling allowed us to reveal molecular patterns accompanying hepatic steatosis in MAFLD. Urine ORM1 and ceruloplasmin are potential biomarkers for population-based screening, diagnosing and follow-up for mild/severe hepatic steatosis. Funding Information: This study was supported by the Post-Doctor Research Project of West China Hospital of Sichuan University (grant numbers 2020HXBH079) and National Natural Science Foundation of China (grant numbers 81900512). Declaration of Interests: The authors declare that there are no competing interests associated with the manuscript. Ethics Approval Statement: The study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki. The Institutional Review Board Committee of West China Hospital of Sichuan university approved the study protocol. The study was performed by following the ethical guidelines expressed in the Declaration of Helsinki and the International Conference on Harmonization Guidelines for Good Clinical Practice. Informed consent was obtained from all subjects.