Complementary Role of Fibroblast Growth Factor 21 and Cytokeratin 18 in Monitoring the Different Stages of Nonalcoholic Fatty Liver Disease

www.nature.com/scientificreports OPEN Received: 18 January 2017 Accepted: 25 May 2017 Published: xx xx xxxx Complementary Role of Fibroblast Growth Factor 21 and Cytokeratin 18 in Monitoring the Different Stages of Nonalcoholic Fatty Liver Disease Guangyu Wu1,2, Huating Li1, Qichen Fang1, Jing Zhang1,2, Mingliang Zhang1, Lei Zhang1,2, Liang Wu1,2, Xuhong Hou1, Junxi Lu1, Yuqian Bao1 & Weiping Jia1 Fibroblast growth factor 21 (FGF21) and cytokeratin 18 (CK18) were previously reported to be elevated in nonalcoholic fatty liver disease (NAFLD). We aim to analyze the differential roles of FGF21, cell apoptosis marker CK18 fragment M30 and total cell death marker CK18 M65ED in monitoring the different stages of NAFLD spectrum in a population-based prospective cohort comprising 808 Chinese subjects. Predictive performances for monitoring the different stages of NAFLD were assessed by logistic regression and receiver-operating characteristic (ROC) curves. We found baseline FGF21 but not CK18 level was an independent predictor for the development of simple steatosis. NAFLD patients who had remission during follow-up had significantly lower baseline M30 levels than those who sustained NAFLD (84.74U/L [53.26–135.79] vs. 118.47U/L [87.16–188.89], P = 0.012). M65ED was independently predictive of progressing to suspected non-alcoholic steatohepatitis (NASH) in NAFLD patients. These results suggest that FGF21 can be used for early identification of hepatic steatosis. On the other hand, CK18 including M30 and M65ED, are predictive of the prognosis of NAFLD patients. FGF21 and CK18 might play differential roles and have complementary value in non-invasive identification and monitoring the outcome of NAFLD patients. Non-alcoholic fatty liver disease (NAFLD) is defined by the presence of liver fat accumulation exceeding 5% of hepatocytes, in the absence of significant alcohol intake, viral infection, or any other specific etiology of liver disease1. NAFLD is divided into simple steatosis and nonalcoholic steatohepatitis (NASH) which is distinguished from the former by additional presence of hepatocellular injury with or without fibrosis2. The majority of patients with simple steatosis are stable in term of liver histology over time, while NASH is associated with progressive liver disease3. The gold standard for diagnosing and differentiating simple steatosis and NASH is liver biopsy, but it is invasive and limited by sampling error4. In clinical practice, ultrasonography is the most commonly used imaging technique for diagnosing NAFLD, while it shows low accuracy in diagnosing NAFLD in obese patients. The sensitivity also decreases dramatically for mild steatosis5. Magnetic resonance spectrophy (MRS) is more sensitive and accurate but it is expensive and not broadly available6. Neither ultrasonography nor MRS can differentiate between patients with simple steatosis and NASH. In an attempt to overcome biopsy and to monitor NAFLD patients leading to better intervention decisions, seeking potential novel biomarkers based on current knowledge of the pathophysiology of NAFLD are yet far from being accomplished. Previous studies reported FGF21 and CK18 fragment are potential NAFLD biomarkers6, 7. Fibroblast growth factor 21 (FGF21) is predominantly released from hepatocytes and in a lesser extent from adipocytes and other tissues8. FGF21 binds to fibroblast growth factor receptor and co-receptor beta-klotho and exerts its hormone-like 1 Department of Endocrinology and Metabolism, Shanghai Jiao Tong University Affiliated Sixth People’s Hospital; Shanghai Key Laboratory of Diabetes Mellitus, Shanghai Clinical Center of Diabetes, Shanghai, 200233, China. 2 Department of Medicine, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. Guangyu Wu and Huating Li contributed equally to this work. Correspondence and requests for materials should be addressed to W.J. (email: ) Scientific Reports | 7: 5095 | DOI:10.1038/s41598-017-05257-5 1 www.nature.com/scientificreports/ metabolic effects8. FGF21 knockout mice are refractory to the beneficial insulin-sensitizing effects of the peroxisome proliferator-activated receptor (PPAR)γ agonist rosiglitazone9. FGF21 deficiency also exacerbated accumulation of triglycerides (TG), impaired activation of fatty acids and oxidation in the liver and increased inflammation and fibrosis10. Despite the beneficial effect of FGF21 found in animal studies, circulating FGF21 levels in human are elevated in obesity, metabolic syndrome, type 2 diabetes and coronary artery disease11. We previously found FGF21 level was increased in NAFLD patients and was an independent predictor of NAFLD7, 12. The elevation of circulating FGF21 levels in over-nutrition may indicate the presence of compensatory responses of FGF21 to the underlying metabolic stress11. Cytokeratin 18 (CK18) is a major intermediate filament protein of hepatocytes13. CK18 is cleaved by caspases and released to the circulation during hepatocyte apoptosis, which is a characteristic feature of liver injury and disease progression in NAFLD13, 14. The serum level of cleaved CK18 fragment is representative of the degree of hepatocyte apoptosis and can be measured by M30 level, which is an epitope generated during CK18 cleavage. In contrast, M65 antigen, existing on both cleaved and uncleaved CK18 protein, is used as a marker for total death of hepatocytes, including both apoptosis and necrosis15. M65 EpiDeath (M65ED) ELISA uses inverse capture antibody and detection antibody compared to M65 ELISA and further improves binding specificity15. Recent clinical studies demonstrated that M30 and M65ED are potentially useful to diagnose fatty liver, NASH and fibrosis13, 15. A two-step approach using M30 and FGF21 was demonstrated to further improve the accuracy in diagnosing NASH16. Based on the above mentioned research, it seems that FGF21 and CK18 are involved in the different stages in view of the pathophysiology of NAFLD. However, no longitudinal epidemiological study focused on the differential roles of FGF21 and CK18 in NAFLD spectrum. In this prospective study, we aimed at assessing FGF21, CK18 M30 and M65ED levels during different stages in the spectrum of NAFLD and evaluating their respective role in early identification and predicting the prognosis of NAFLD patients to investigate their clinical significance. Results Clinical Characteristics of subjects and serum FGF21, M30 and M65ED levels at baseline. As described in our previous study7, 660 subjects from baseline survey (257 men and 403 women; aged 49.15 ± 12.60 years) were included in this follow-up reassessment after a mean duration of 3.1 ± 0.1 years, 148 subjects were not followed up due to emigration, refusal or death. Subjects enrolled in the follow-up study were compared to those who were not included and no difference was observed between groups in demographic, anthropometric and biochemical indexes. Ninety-five subjects wer (...truncated)