Science China Life Sciences

, Volume 61, Issue 11, pp 1369–1381 | Cite as

Early urinary candidate biomarker discovery in a rat thioacetamide-induced liver fibrosis model

  • Fanshuang Zhang
  • Yanying Ni
  • Yuan Yuan
  • Wei Yin
  • Youhe GaoEmail author
Research Paper


Biomarker is the change associated with the disease. Blood is relatively stable because of the homeostatic mechanisms of the body. However, urine accumulates changes of the body, which makes it a better early biomarker source. Liver fibrosis is a reversible pathological condition, whereas cirrhosis, the end-stage of liver fibrosis, is irreversible. Consequently, noninvasive early biomarkers for fibrosis are desperately needed. In this study, differential urinary proteins were identified in the thioacetamide liver fibrosis rat model using tandem mass tagging and two-dimensional liquid chromatography tandem mass spectrometry. A total of 766 urinary proteins were identified, 143 and 118 of which were significantly changed in the TAA 1-week and 3-week groups, respectively. Multiple reaction monitoring (MRM)-targeted proteomics was used to further validate the abundant differentially expressed proteins. A total of 40 urinary proteins were statistically significant, 15 of which had been previously reported as biomarkers of liver fibrosis, cirrhosis or other related diseases and 10 of which had been reported to be associated with the pathology and mechanism of liver fibrosis. These differential proteins were detected in urine before the alanine aminotransferase and aspartate transaminase changes in the serum and before fibrosis was observed upon hematoxylin and eosin (HE) and Masson’s staining.


urine proteomics biomarker animal model liver fibrosis 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.



This work was supported by the National Key Research and Development Program of China (2016YFC1306300), the National Basic Research Program of China (2013CB530850), Beijing Natural Science Foundation (7173264, 7172076) and Funds from Beijing Normal University (11100704, 10300-310421102).

Supplementary material

11427_2017_9268_MOESM1_ESM.docx (73 kb)
Table S1. Differential urinary proteins in TAA 1-week group.
11427_2017_9268_MOESM2_ESM.docx (43 kb)
Table S2. Differential urinary proteins in TAA 3-week group.


  1. An, M., and Gao, Y. (2015). Urinary biomarkers of brain diseases. Genomics Proteomics Bioinformatics 13, 345–354.CrossRefPubMedGoogle Scholar
  2. Ariza, X., Solà, E., Elia, C., Barreto, R., Moreira, R., Morales-Ruiz, M., Graupera, I., Rodríguez, E., Huelin, P., Solé, C., et al. (2015). Analysis of a urinary biomarker panel for clinical outcomes assessment in cirrhosis. PLoS ONE 10, e0128145.CrossRefPubMedPubMedCentralGoogle Scholar
  3. Ariza, X., Graupera, I., Coll, M., Solà, E., Barreto, R., García, E., Moreira, R., Elia, C., Morales-Ruiz, M., Llopis, M., et al. (2016). Neutrophil gelatinase-associated lipocalin is a biomarker of acute-on-chronic liver failure and prognosis in cirrhosis. J Hepatol 65, 57–65.CrossRefPubMedGoogle Scholar
  4. Aydin, A.F., Küskü-Kiraz, Z., Doğru-Abbasoğlu, S., Güllüoğlu, M., Uysal, M., and Koçak-Toker, N. (2010). Effect of carnosine against thioacetamide-induced liver cirrhosis in rat. Peptides 31, 67–71.CrossRefPubMedGoogle Scholar
  5. Balkan, J., Dogğru-Abbasoğlul, S., Kanbaglil, Ö, Çevikbas, U., Aykaç-Toker, G., and Uysal, M. (2001). Taurine has a protective effect against thioacetamide-induced liver cirrhosis by decreasing oxidative stress. Hum Exp Toxicol 20, 251–254.CrossRefPubMedGoogle Scholar
  6. Beretov, J., Wasinger, V.C., Millar, E.K.A., Schwartz, P., Graham, P.H., and Li, Y. (2015). Proteomic analysis of urine to identify breast cancer biomarker candidates using a label-free LC-MS/MS approach. PLoS ONE 10, e0141876.CrossRefPubMedPubMedCentralGoogle Scholar
  7. Bostick, B., Yue, Y., Long, C., and Duan, D. (2008). Prevention of dystrophin-deficient cardiomyopathy in twenty-one-month-old carrier mice by mosaic dystrophin expression or complementary dystrophin/utrophin expression. Circul Res 102, 121–130.CrossRefGoogle Scholar
  8. Bracht, T., Schweinsberg, V., Trippler, M., Kohl, M., Ahrens, M., Padden, J., Naboulsi, W., Barkovits, K., Megger, D.A., Eisenacher, M., et al. (2015). Analysis of disease-associated protein expression using quantitative proteomics—fibulin-5 is expressed in association with hepatic fibrosis. J Proteome Res 14, 2278–2286.CrossRefPubMedGoogle Scholar
  9. Carter, W.G., Vigneswara, V., Newlaczyl, A., Wayne, D., Ahmed, B., Saddington, S., Brewer, C., Raut, N., Gerdes, H.K., Erdozain, A.M., et al. (2015). Isoaspartate, carbamoyl phosphate synthase-1, and carbonic anhydrase-III as biomarkers of liver injury. Biochem Biophys Res Commun 458, 626–631.CrossRefPubMedPubMedCentralGoogle Scholar
  10. Chai, Y.C., Jung, C.H., Lii, C.K., Ashraf, S.S., Hendrich, S., Wolf, B., Sies, H., and Thomas, J.A. (1991). Identification of an abundant S-thiolated rat liver protein as carbonic anhydrase III characterization of S-thiolation and dethiolation reactions. Arch Biochem Biophys 284, 270–278.CrossRefPubMedGoogle Scholar
  11. Cheng, Z.X., Huang, X.H., Wang, Q., Chen, J.S., Zhang, L.J., and Chen, X. L. (2012). Clinical significance of decreased nidogen-2 expression in the tumor tissue and serum of patients with hepatocellular carcinoma. J Surg Oncol 105, 71–80.CrossRefPubMedGoogle Scholar
  12. Chilakapati, J., Shankar, K., Korrapati, M.C., Hill, R.A., and Mehendale, H. M. (2005). Saturation toxicokinetics of thioacetamide: role in initiation of liver injury. Drug Metab Dispos 33, 1877–1885.PubMedGoogle Scholar
  13. Cho, H.J., Kim, S.S., Ahn, S.J., Park, J.H., Kim, D.J., Kim, Y.B., Cho, S. W., and Cheong, J.Y. (2014). Serum transferrin as a liver fibrosis biomarker in patients with chronic hepatitis B. Clin Mol Hepatol 20, 347–354.CrossRefPubMedPubMedCentralGoogle Scholar
  14. Chu, S.C., Wang, C.P., Chang, Y.H., Hsieh, Y.S., Yang, S.F., Su, J.M., Yang, C.C., and Chiou, H.L. (2004). Increased cystatin C serum concentrations in patients with hepatic diseases of various severities. Clin Chim Acta 341, 133–138.CrossRefPubMedGoogle Scholar
  15. Dai, H.Y., Hong, C.C., Liang, S.C., Yan, M.D., Lai, G.M., Cheng, A.L., and Chuang, S.E. (2008). Carbonic anhydrase III promotes transformation and invasion capability in hepatoma cells through FAK signaling pathway. Mol Carcinog 47, 956–963.CrossRefPubMedGoogle Scholar
  16. El Saadany, S.A., Ziada, D.H., Farrag, W., and Hazaa, S. (2011). Fibrosis severity and mannan-binding lectin (MBL)/MBL-associated serine protease 1 (MASP-1) complex in HCV-infected patients. Arab J Gastroenterol 12, 68–73.CrossRefPubMedGoogle Scholar
  17. Fitzhugh, O.G., and Nelson, A.A. (1948). Liver tumors in rats fed thiourea or thioacetamide. Science 108, 626–628.CrossRefPubMedGoogle Scholar
  18. Friedman, S.L. (2008). Hepatic fibrosis—overview. Toxicology 254, 120–129.CrossRefPubMedGoogle Scholar
  19. Gajbhiye, A., Dabhi, R., Taunk, K., Vannuruswamy, G., RoyChoudhury, S., Adhav, R., Seal, S., Mane, A., Bayatigeri, S., Santra, M.K., et al. (2016). Urinary proteome alterations in HER2 enriched breast cancer revealed by multipronged quantitative proteomics. Proteomics 16, 2403–2418.CrossRefPubMedGoogle Scholar
  20. Gao, Y.H. (2013). Urine—an untapped goldmine for biomarker discovery? Sci China Life Sci 56, 1145–1146.CrossRefPubMedGoogle Scholar
  21. Gao Y. (2014). Roadmap to the urine biomarker era. MOJ Proteom Bioinform 1, p.00005.Google Scholar
  22. Glückmann, M., Fella, K., Waidelich, D., Merkel, D., Kruft, V., Kramer, P. J., Walter, Y., Hellmann, J., Karas, M., Kröger, M. (2007). Prevalidation of potential protein biomarkers in toxicology using iTRAQ reagent technology. Proteomics 7, 1564–1574.CrossRefPubMedGoogle Scholar
  23. Guo, J., and Friedman, S.L. (2007). Hepatic fibrogenesis. Semin Liver Dis 27, 413–426.CrossRefPubMedGoogle Scholar
  24. Henkel, C., Schwamborn, K., Zimmermann, H.W., Tacke, F., Kühnen, E., Odenthal, M., Groseclose, M.R., Caprioli, R.M., and Weiskirchen, R. (2011). From proteomic multimarker profiling to interesting proteins: thymosin-β4 and kininogen-1 as new potential biomarkers for inflammatory hepatic lesions. J Cell Mol Med 15, 2176–2188.CrossRefPubMedPubMedCentralGoogle Scholar
  25. Huang, J.T.J., Chaudhuri, R., Albarbarawi, O., Barton, A., Grierson, C., Rauchhaus, P., Weir, C.J., Messow, M., Stevens, N., McSharry, C., et al. (2012). Clinical validity of plasma and urinary desmosine as biomarkers for chronic obstructive pulmonary disease. Thorax 67, 502–508.CrossRefPubMedPubMedCentralGoogle Scholar
  26. Hwang, S., Hong, H.N., Kim, H.S., Park, S.R., Won, Y.J., Choi, S.T., Choi, D., and Lee, S.G. (2012). Hepatogenic differentiation of mesenchymal stem cells in a rat model of thioacetamide-induced liver cirrhosis. Cell Biol Int 36, 279–288.CrossRefPubMedGoogle Scholar
  27. Kountouras, J., Billing, B.H., and Scheuer, P.J. (1984). Prolonged bile duct obstruction: a new experimental model for cirrhosis in the rat. Br J Exp Pathol 65, 305–311.PubMedPubMedCentralGoogle Scholar
  28. Krishnan, A., Li, X., Kao, W.W.Y., Viker, K., Butters, K., Masuoka, H., Knudsen, B., Gores, G., and Charlton, M. (2012). Lumican, an extracellular matrix proteoglycan, is a novel requisite for hepatic fibrosis. Lab Invest 92, 1712–1725.CrossRefPubMedGoogle Scholar
  29. Ladero, J.M., Cárdenas, M.C., Ortega, L., González-Pino, A., Cuenca, F., Morales, C., and Lee-Brunner, A. (2012). Serum cystatin C: a noninvasive marker of liver fibrosis or of current liver fibrogenesis in chronic hepatitis C. Ann Hepatol 11, 648–651.PubMedGoogle Scholar
  30. Laleman, W., Vander Elst, I., Zeegers, M., Servaes, R., Libbrecht, L., Roskams, T., Fevery, J., and Nevens, F. (2006). A stable model of cirrhotic portal hypertension in the rat: thioacetamide revisited. Eur J Clin Invest 36, 242–249.CrossRefPubMedGoogle Scholar
  31. Lee, N.P., Poon, R.T., Shek, F.H., Ng, I.O., and Luk, J.M. (2010). Role of cadherin-17 in oncogenesis and potential therapeutic implications in hepatocellular carcinoma. Biochim Biophys Acta 1806, 138–145.PubMedGoogle Scholar
  32. Li, M.L., Zhao, M.D., and Gao, Y.H. (2014). Changes of proteins induced by anticoagulants can be more sensitively detected in urine than in plasma. Sci China Life Sci 57, 649–656.CrossRefPubMedGoogle Scholar
  33. Li, X., Benjamin, I.S., and Alexander, B. (2002). Reproducible production of thioacetamide-induced macronodular cirrhosis in the rat with no mortality. J Hepatol 36, 488–493.CrossRefPubMedGoogle Scholar
  34. Li, X.N., Huang, C.T., Wang, X.H., Leng, X.S., Du, R.Y., Chen, Y.F., and Hou, X. (1990). Changes of blood humoral substances in experimental cirrhosis and their effects on portal hemodynamics. Chin Med J (Engl) 103, 970–977.Google Scholar
  35. Liu, E., Nisenblat, V., Farquhar, C., Fraser, I., Bossuyt, P.M., Johnson, N., and Hull, M.L. (2015). Urinary biomarkers for the non-invasive diagnosis of endometriosis. Cochrane Database Syst Rev (12), CD012019.Google Scholar
  36. Liu, S., Yang, Z., Wei, H., Shen, W., Liu, J., Yin, Q., Li, X., and Yi, J. (2010). Increased DJ-1 and its prognostic significance in hepatocellular carcinoma. Hepatogastroenterology 57, 1247–1256.PubMedGoogle Scholar
  37. Low, T.Y., Leow, C.K., Salto-Tellez, M., and Chung, M.C.M. (2004). A proteomic analysis of thioacetamide-induced hepatotoxicity and cirrhosis in rat livers. Proteomics 4, 3960–3974.CrossRefPubMedGoogle Scholar
  38. Ma, L., Lin, J., Qiao, Y., Weng, W., Liu, W., Wang, J., and Sun, F. (2015). Serum CD166: a novel hepatocellular carcinoma tumor marker. Clin Chim Acta 441, 156–162.CrossRefPubMedGoogle Scholar
  39. MacLean, B., Tomazela, D.M., Shulman, N., Chambers, M., Finney, G.L., Frewen, B., Kern, R., Tabb, D.L., Liebler, D.C., and MacCoss, M.J. (2010). Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26, 966–968.CrossRefPubMedPubMedCentralGoogle Scholar
  40. Magalhães, P., Mischak, H., and Zürbig, P. (2016). Urinary proteomics using capillary electrophoresis coupled to mass spectrometry for diagnosis and prognosis in kidney diseases. Curr Opin Nephrol Hypertens 25, 494–501.CrossRefPubMedGoogle Scholar
  41. Mani, S., Cao, W., Wu, L., and Wang, R. (2014). Hydrogen sulfide and the liver. Nitric Oxide 41, 62–71.CrossRefPubMedGoogle Scholar
  42. Mehendale, H.M. (2005). Tissue repair: an important determinant of final outcome of toxicant-induced injury. Toxicol Pathol 33, 41–51.CrossRefPubMedGoogle Scholar
  43. Mondal, G., Saroha, A., Bose, P.P., and Chatterjee, B.P. (2016). Altered glycosylation, expression of serum haptoglobin and alpha-1-antitrypsin in chronic hepatitis C, hepatitis C induced liver cirrhosis and hepatocellular carcinoma patients. Glycoconj J 33, 209–218.CrossRefPubMedGoogle Scholar
  44. Natarajan, S.K., Thomas, S., Ramamoorthy, P., Basivireddy, J., Pulimood, A.B., Ramachandran, A., and Balasubramanian, K.A. (2006). Oxidative stress in the development of liver cirrhosis: a comparison of two different experimental models. J Gastroenterol Hepatol 21, 947–957.CrossRefPubMedGoogle Scholar
  45. Nesvizhskii, A.I., Keller, A., Kolker, E., and Aebersold, R. (2003). A statistical model for identifying proteins by tandem mass spectrometry. Anal Chem 75, 4646–4658.CrossRefPubMedGoogle Scholar
  46. Noda, S., Masumi, S., Moriyama, M., Kannan, Y., Ohta, M., Sugano, T., and Yamate, J. (1996). Population of hepatic macrophages and response of perfused liver to platelet-activating factor during production of thioacetamide-induced cirrhosis in rats. Hepatology 24, 412–418.CrossRefPubMedGoogle Scholar
  47. Okuyama, H., Nakamura, H., Shimahara, Y., Uyama, N., Kwon, Y.W., Kawada, N., Yamaoka, Y., and Yodoi, J. (2005). Overexpression of thioredoxin prevents thioacetamide-induced hepatic fibrosis in mice. J Hepatol 42, 117–123.CrossRefPubMedGoogle Scholar
  48. Okuyama, H., Son, A., Ahsan, M.K., Masutani, H., Nakamura, H., and Yodoi, J. (2008). Thioredoxin and thioredoxin binding protein 2 in the liver. IUBMB Life 60, 656–660.CrossRefPubMedGoogle Scholar
  49. Popper, H., and Kent, G. (1975). Fibrosis in chronic liver disease. Clin Gastroenterol 4, 315–332.PubMedGoogle Scholar
  50. Price, C.P., Newall, R.G., and Boyd, J.C. (2005). Use of protein:creatinine ratio measurements on random urine samples for prediction of significant proteinuria: a systematic review. Clin Chem 51, 1577–1586.CrossRefPubMedGoogle Scholar
  51. Russ, K.B., Stevens, T.M., and Singal, A.K. (2015). Acute kidney injury in patients with cirrhosis. J Clin Transl Hepatol 3, 195–204.CrossRefPubMedPubMedCentralGoogle Scholar
  52. Sawai, Y., Tamura, S., Fukui, K., Ito, N., Imanaka, K., Saeki, A., Sakuda, S., Kiso, S., and Matsuzawa, Y. (2003). Expression of ephrin-B1 in hepatocellular carcinoma: possible involvement in neovascularization. J Hepatol 39, 991–996.CrossRefPubMedGoogle Scholar
  53. Seow, T.K., Liang, R.C.M.Y., Leow, C.K., and Chung, M.C.M. (2001). Hepatocellular carcinoma: from bedside to proteomics. Proteomics 1, 1249–1263.CrossRefPubMedGoogle Scholar
  54. Shao, C., Li, M., Li, X., Wei, L., Zhu, L., Yang, F., Jia, L., Mu, Y., Wang, J., Guo, Z., et al. (2011). A tool for biomarker discovery in the urinary proteome: a manually curated human and animal urine protein biomarker database. Mol Cell Proteomics 10, M111.010975.Google Scholar
  55. Sherwood, C.A., Eastham, A., Lee, L.W., Risler, J., Mirzaei, H., Falkner, J. A., and Martin, D.B. (2009). Rapid optimization of MRM-MS instrument parameters by subtle alteration of precursor and product m/z targets. J Proteome Res 8, 3746–3751.CrossRefPubMedPubMedCentralGoogle Scholar
  56. Sirnes, T.B. (1953). Voluntary consumption of alcohol in rats with cirrhosis of the liver a preliminary report. Q J Stud Alcohol 14, 3–18.PubMedGoogle Scholar
  57. Smith, E.R., Zurakowski, D., Saad, A., Scott, R.M., and Moses, M.A. (2008). Urinary biomarkers predict brain tumor presence and response to therapy. Clin Cancer Res 14, 2378–2386.CrossRefPubMedGoogle Scholar
  58. Stankovic, Z. (2016). Four-dimensional flow magnetic resonance imaging in cirrhosis. World J Gastroenterol 22, 89–102.CrossRefPubMedPubMedCentralGoogle Scholar
  59. Su, M.C., Yuan, R.H., Lin, C.Y., and Jeng, Y.M. (2008). Cadherin-17 is a useful diagnostic marker for adenocarcinomas of the digestive system. Mod Pathol 21, 1379–1386.CrossRefPubMedGoogle Scholar
  60. Sun, W., Li, F., Wu, S., Wang, X., Zheng, D., Wang, J., and Gao, Y. (2005). Human urine proteome analysis by three separation approaches. Proteomics 5, 4994–5001.CrossRefPubMedGoogle Scholar
  61. Tan, X., Chen, F., Wu, S., Shi, Y., Liu, D., and Chen, Z. (2010). Proteomic analysis of differentially expressed proteins in mice with concanavalin A-induced hepatitis. J Zhejiang Univ Sci B 11, 221–226.CrossRefPubMedPubMedCentralGoogle Scholar
  62. Tangkijvanich, P., Yee, H.F. (2002). Cirrhosis—can we reverse hepatic fibrosis. Eur J Surg Suppl (587), 100–112.Google Scholar
  63. Tennakoon, A.H., Izawa, T., Wijesundera, K.K., Murakami, H., Katou-Ichikawa, C., Tanaka, M., Golbar, H.M., Kuwamura, M., and Yamate, J. (2015). Immunohistochemical characterization of glial fibrillary acidic protein (GFAP)-expressing cells in a rat liver cirrhosis model induced by repeated injections of thioacetamide (TAA). Exp Toxicol Pathol 67, 53–63.CrossRefPubMedGoogle Scholar
  64. Topic, A., Ljujic, M., and Radojkovic, D. (2012). Alpha-1-antitrypsin in pathogenesis of hepatocellular carcinoma. Hepat Mon 12, e7042.CrossRefPubMedPubMedCentralGoogle Scholar
  65. Twigt, J.M., Bezstarosti, K., Demmers, J., Lindemans, J., Laven, J.S.E., and Steegers-Theunissen, R.P. (2015). Preconception folic acid use influences the follicle fluid proteome. Eur J Clin Invest 45, 833–841.CrossRefPubMedGoogle Scholar
  66. Wang, J., Chen, L., Li, Y., and Guan, X.Y. (2011). Overexpression of cathepsin Z contributes to tumor metastasis by inducing epithelial-mesenchymal transition in hepatocellular carcinoma. PLoS ONE 6, e24–967.CrossRefGoogle Scholar
  67. Wang, X., Gong, G., Yang, W., Li, Y., Jiang, M., and Li, L. (2013). Antifibrotic activity of galangin, a novel function evaluated in animal liver fibrosis model. Environ Toxicol Pharmacol 36, 288–295.CrossRefPubMedGoogle Scholar
  68. Weber, L.W.D., Boll, M., and Stampfl, A. (2003). Hepatotoxicity and mechanism of action of haloalkanes: carbon tetrachloride as a toxicological model. Crit Rev Toxicol 33, 105–136.CrossRefPubMedGoogle Scholar
  69. Wiśniewski, J.R., Zougman, A., Nagaraj, N., and Mann, M. (2009). Universal sample preparation method for proteome analysis. Nat Methods 6, 359–362.CrossRefPubMedGoogle Scholar
  70. Wu, J., and Gao, Y. (2015). Physiological conditions can be reflected in human urine proteome and metabolome. Expert Rev Proteomics 12, 623–636.CrossRefPubMedGoogle Scholar
  71. Wu, T., Du, Y., Han, J., Singh, S., Xie, C., Guo, Y., Zhou, X.J., Ahn, C., Saxena, R., and Mohan, C. (2013). Urinary angiostatin—a novel putative marker of renal pathology chronicity in lupus nephritis. Mol Cell Proteomics 12, 1170–1179.CrossRefPubMedPubMedCentralGoogle Scholar
  72. Wynn, T.A. (2008). Cellular and molecular mechanisms of fibrosis. J Pathol 214, 199–210.CrossRefPubMedPubMedCentralGoogle Scholar
  73. Zhang, X., Xu, L., Yin, L., Qi, Y., Xu, Y., Han, X., and Peng, J. (2015). Quantitative chemical proteomics for investigating the biomarkers of dioscin against liver fibrosis caused by CCl4 in rats. Chem Commun 51, 11064–11067.CrossRefGoogle Scholar

Copyright information

© Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Fanshuang Zhang
    • 1
    • 2
  • Yanying Ni
    • 2
  • Yuan Yuan
    • 2
    • 3
  • Wei Yin
    • 2
  • Youhe Gao
    • 4
    Email author
  1. 1.Department of Pathology, National Cancer Center/Cancer HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijingChina
  2. 2.Department of Pathophysiology, Institute of Basic Medical SciencesChinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical CollegeBeijingChina
  3. 3.Department of Pathology, Beijing Tiantan HospitalCapital Medical UniversityBeijingChina
  4. 4.Department of Biochemistry and Molecular Biology, Beijing Normal UniversityGene Engineering Drug and Biotechnology Beijing Key LaboratoryBeijingChina

Personalised recommendations