O2152: Single nuclear RNA sequencing reveals a unique transcriptional profile of hepatocytes associated with small bowel resection induced liver injury

Background: Short gut syndrome (SGS) results from massive small bowel resection (SBR) that is required for treatment of many gastrointestinal conditions. Intestinal failure associated liver disease (IFALD) is the leading indication for intestinal and multivisceral transplantation in children with SGS, with an incidence of 50% in pediatric patients on parenteral nutrition. IFALD is unique from non-alcoholic fatty liver disease due to relative enteral starvation and lack of insulin resistance. In our TPN-independent SBR murine model, we have found evidence of progressive liver injury leading to steatosis and fibrosis which mimics IFALD. The purpose of this study is to identify differential gene expression profiles of key regulatory pathways involved in the pathogenesis of IFALD.

Methods: 8 week old C57BL/6J mice underwent sham (n=3) or 75% proximal SBR (n=3). Livers were harvested at 2 weeks following surgery, single nuclei were isolated, and single nuclear RNA sequencing (snRNAseq) was performed using the 10x chromium platform. Bioinformatics analysis was performed on RStudio version 2023.12.1+402 with packages Seurat v5, Harmony, presto, tidyverse, ggplot2, AnnotationHub, Sctype, SingleR, WGCNA, DESeq2, and Enrichr. Animal studies were approved by the institution’s Animal Studies Committee (Protocol #20-0197).

Results: SBR mice demonstrated liver injury with elevated serum transaminase levels and increased gene expression of oxidative and ER stress genes NAD(P)H quinone dehydrogenase and DNA damage inducible transcript 3 by q-RT-PCR (Nqo1 and Ddit3; Figure 1A). SnRNAseq revealed significant differences in many cell types in the SBR mice when compared to shams, among which we identified a cluster of hepatocytes unique to SBR (Cluster 6; Figure 1B). Using cell trajectory inferencing we targeted these alterations to pericentral hepatocytes and confirmed zonal specificity by immunohistochemistry of Annexin2A (Anxa-2; Figure 1C) which is a specific marker for hepatocytes in cluster 6 and has been associated with disease associated hepatocytes in preclinical models of NAFLD/NASH. Gene enrichment analysis of cluster 6 hepatocytes revealed a reduction in normal physiologic pathways and increase in cancer-associated pathways (Figure 1C).

Conclusion: Massive SBR is associated with progressive liver injury, with significant transaminitis and inflammatory changes as early as two weeks after SBR. A novel hepatocyte population emerges with a cluster of resection associated hepatocytes characterized by genes associated with fibrosis, cancer progression, fat metabolism, and autophagy. These findings highlight several potential novel mechanisms underlying the pathogenesis of IFALD.