Significance of alanine aminopeptidase N (APN) in bile in the diagnosis of acute cellular rejection after liver transplantation

Background: Allograft dysfunction after liver transplantation requires histopathologic examination for confirmation of the diagnosis, however, the procedure is invasive and its interpretation is not always accurate. The aim of this study was to find novel protein markers in bile for the diagnosis of acute cellular rejection (ACR) after liver transplantation. Materials and Methods: Quantitative proteomic analysis using the ¹⁸O labeling method was used to search for bile proteins of interest. Nine recipients were selected who had liver dysfunction, diagnosed by liver biopsy, either with ACR (ACR group, n = 5) or without (LD group, n = 4). Donor bile samples were obtained from nine independent live liver donors. Enzyme activity in bile samples was assayed and liver biopsy specimens were immunostained for candidate protein of ACR. Results: The analysis identified 78 proteins, among which alanine aminopeptidase N (APN/CD13) was considered a candidate marker of ACR. Comparative analysis of the ACR and LD groups showed high APN enzyme activity in three (60%) of five cases of the ACR group, while it was as low as donor level in all patients of the LD group. APN enzyme activity in bile samples of liver dysfunction liver transplantation (LDLT) recipients of the ACR group collected within 3 d before biopsy-confirmed ACR (n = 10) was significantly higher (584 ± 434 U/g protein) than in those of recipients free of ACR (n = 96, 301 ± 271 U/g protein) (P = 0.004). APN overexpression along bile canaliculi was observed during ACR in all five cases of the ACR group. Conclusion: APN in bile seems to be a useful and noninvasive biomarker of ACR after liver transplantation.