Expression of unfolded protein response genes in post-transplantation liver biopsies

Abstract

Background: Cholestatic liver diseases are a major source of morbidity and mortality that can progress to end-stage liver disease and hyperbilirubinemia is a hallmark of cholestasis. There are few effective medical therapies for primary biliary cholangitis, primary sclerosing cholangitis and other cholestatic liver diseases, in part, due to our incomplete understanding of the pathogenesis of cholestatic liver injury. The hepatic unfolded protein response (UPR) is an adaptive cellular response to endoplasmic reticulum stress that is important in the pathogenesis of many liver diseases and recent animal studies have demonstrated the importance of the UPR in the pathogenesis of cholestatic liver injury. However, the role of the UPR in human cholestatic liver diseases is largely unknown.

Methods: RNA was extracted from liver biopsies from patients after liver transplantation. RNA-seq was performed to determine the transcriptional profile and hepatic UPR gene expression that is associated with liver injury and cholestasis.

Results: Transcriptome analysis revealed that patients with hyperbilirubinemia had enhanced expression of hepatic UPR pathways. Alternatively, liver biopsy samples from patients with acute rejection had enhanced gene expression of LAG3 and CDK1. Pearson correlation analysis of serum alanine aminotransferase, aspartate aminotransferase and total bilirubin levels demonstrated significant correlations with the hepatic expression of several UPR genes, as well as genes involved in hepatic bile acid metabolism and inflammation. In contrast, serum alkaline phosphatase levels were correlated with the level of hepatic bile acid metabolism gene expression but not liver UPR gene expression.

Conclusions: Overall, these data indicate that hepatic UPR pathways are increased in cholestatic human liver biopsy samples and supports an important role of the UPR in the mechanism of human cholestatic liver injury.

Keywords: Cholestasis; ER stress; Hyperbilirubinemia; Transcriptome.

Fig. 1

Principal component analysis of RNA-seq from liver biopsies from post-transplantation patients. AR acute rejection, NR:HBR non-rejection with hyperbilirubinemia (serum total bilirubin > 2.5 mg/dL), NR:Mild non-rejection with total bilirubin ≤ 2.5 mg/dL and serum ALT, AST and ALP ≤ 1.67 × ULN, NR:Others non-rejection with total bilirubin ≤ 2.5 mg/dL and serum ALT, AST and ALP > 1.67 × ULN

Fig. 2

Volcano plot and pathway analysis examining hepatic gene expression in patients with hyperbilirubinemia. A Volcano plot comparing the hepatic gene expression of the NR:Mild group to the NR:HBR group. Expression of CYP7A1 was significantly higher, while LOXL4, CFTR and ADGRG2 expression was lower in the NR:Mild group. B Differentially expressed hepatic pathways in patients with hyperbilirubinemia. GSEA study using the Hallmark pathway database comparing the NR:HBR group to the NR:Mild group. C The Unfolded_Protein_Response pathway had a normalized enrichment score of 1.431951 comparing the NR:HBR group to NR:Mild group, although the FDR q-value was 0.051

Fig. 3

RNA-seq comparing hepatic gene expression in patients with and without acute rejection. A Volcano plot demonstrated that the acute rejection (AR) group had increased hepatic gene expression of lymphocyte activating 3 (LAG3) and cyclin dependent kinase 1 (CDK1) compared to non-rejection (NR) groups. B Hepatic gene expression of LAG3 and CDK1 in patients with AR and NR. *P-adj < 0.05