Disrupted Murine Gut–to–Human Liver Signaling Alters Bile Acid Homeostasis in Humanized Mouse Liver Models

Abstract

The humanized liver mouse model is being exploited increasingly for human drug metabolism studies. However, its model stability, intercommunication between human hepatocytes and mouse nonparenchymal cells in liver and murine intestine, and changes in extrahepatic transporter and enzyme expressions have not been investigated. We examined these issues in FRGN [fumarylacetoacetate hydrolase (Fah2/2), recombination activating gene 2 (Rag22/2), and interleukin 2 receptor subunit gamma (IL-2rg 2/2) triple knockout] on nonobese diabetic (NOD) background] and chimeric mice: mFRGN and hFRGN (repopulated with mouse or human hepatocytes, respectively). hFRGN mice showed markedly higher levels of liver cholesterol, biliary bilirubin, and bile acids (liver, bile, and plasma; mainly human forms, but also murine bile acids) but lower transforming growth factor beta receptor 2 (TGFBR2) mRNA expression levels (10%) in human hepatocytes and other proliferative markers in mouse nonparenchymal cells (Tgf-b1) and cholangiocytes [plasma membrane-bound, G protein-coupled receptor for bile acids (Tgr5)], suggestive of irregular regeneration processes in hFRGN livers. Changes in gene expression in murine intestine, kidney, and brain of hFRGN mice, in particular, induction of intestinal farnesoid X receptor (Fxr) genes: fibroblast growth factor 15 (Fgf15), mouse ileal bile acid binding protein (Ibabp), small heterodimer partner (Shp), and the organic solute transporter alpha (Osta), were observed. Proteomics revealed persistence of remnant murine proteins (cyotchrome P450 7a-hydroxylase (Cyp7a1) and other enzymes and transporters) in hFRGN livers and suggest the likelihood of mouse activity. When compared with normal human liver tissue, hFRGN livers showed lower SHP mRNA and higher CYP7A1 (300%) protein expression, consequences of tb-and ta-muricholic acid-mediated inhibition of the FXR-SHP cascade and miscommunication between intestinal Fgf15 and human liver fibroblast growth factor receptor 4 (FGFR4), as confirmed by the unchanged hepatic pERK/total ERK ratio. Dysregulation of hepatocyte proliferation and bile acid homeostasis in hFRGN livers led to hepatotoxicity, gallbladder distension, liver deformity, and other extrahepatic changes, making questionable the use of the preparation for drug metabolism studies.

Organic Anion–Transporting Polypeptide (OATP)–Mediated Drug-Drug Interaction Study between Rosuvastatin and Cyclosporine A in Chimeric Mice with Humanized Liver

“…Okumura et al (2007) investigated the gene expression of human transporters in human hepatocytes of PXB mice and reported that the expression of OATP1B1/1B3 was strong, whereas that of oatp1b2 was weak. They also reported that the mRNA expression levels of hepatic transporters in the PXB mice and donor hepatocytes, and the levels of OATP1B1/1B3 in PXB mice were almost same as, 0.58-1.58 higher than, those in donor hepatocytes, which would indicate that the intestinal-liver axis does not affect expressions of OATPs in the mouse model compared with the other (Chow et al, 2017). Ohtsuki et al (2014) also conducted the protein qualification of transporters using an LC-MS/MS technique and showed similar expression levels for human transporters between human hepatocytes of PXB mice and 17 human liver biopsy samples, with no significant deference of the protein expression of OATP1B1/1B3 among three different donors (2, 5, and 10 years old) of hepatocytes of PXB mice.…”

Alterations in gene expression in vitamin D‐deficiency: Down‐regulation of liver Cyp7a1 and renal Oat3 in mice

“…The mRNA expression levels of nuclear receptors, transporters and enzymes, particularly those responsive to Vdr (Chow et al, , ; Chow, Durk, Maeng, & Pang, ; Chow, Quach, et al, ; Chow, Sondervan, Jin, Groothuis, & Pang, ) and highly expressed in mouse tissues (Alnouti & Klaassen, ; Alnouti, Petrick, & Klaassen, ; Buckley & Klaassen, ; Buist & Klaassen, ; Cheng, Maher, Chen, & Klaassen, ; Lu & Klaassen, ) were examined. Total RNA extraction and cDNA synthesis procedures were performed as described (Chow et al, ; Chow, Durk, et al, ); specific primer sequences (Supplemental Table 2) were obtained from the literature (Chow, Durk, et al, ; Chow, Quach, et al, ; Chow et al, ; Chow et al, ; Chow et al, ; Durk et al, ; Gibson, Hossain, Richardson, & Aleksunes, ; Gong et al, ; Lee et al, ) or designed using Primer‐BLAST (Chow et al, ). The critical threshold cycle (C T ) values of target genes for mouse liver and kidney were normalized to that for cyclophilin while those of intestine (ileum) were normalized to that of villin, then expressed as the relative mRNA expression of the 0‐week of the normal vitamin D‐sufficient diet controls.…”

“…Human livers (n = 11) were provided by In Vitro ADMET Laboratories from the International Institute for the Advancement of Medicine (Edison, NJ) and the National Disease Research Interchange (Philadelphia, PA). These donor tissues were the same as those examined previously in another report, wherein information on the donors was described (Chow et al, 2017). 2.7 | Analysis 2.7.1 | Analysis of plasma 25(OH)D 3 , 1,25(OH) 2 D 3, calcium and PTH levels…”

In vitro and in vivo methods to assess pharmacokinetic drug– drug interactions in drug discovery and development

“…These models usually have high variability due to the different degrees of humanization of the liver (partially human and partially mouse) whereas the other organs (e.g., intestine, kidney) are still murine in nature, and have different enzyme and transporter specificities from humans. It has been suggested that species incompatibility or miscommunication between other liver cells or tissues versus human hepatocytes may pose the main reason for deficiency, and the remaining mouse enzymes/transporters could be upregulated or have higher affinities to certain drugs (Chow et al, ). Humanized mice are prepared with a genetically immunocompromised background that may alter their metabolic and transport capabilities.…”