Hepatic Zonation Research Articles

Fast formation and maturation enhancement of human liver organoids using a liver-organoid-on-a-chip.

Background: Spatial and functional hepatic zonation, established by the heterogeneous tissue along the portal-central axis of the liver, is important for ensuring optimal liver function. Researchers have attempted to develop reliable hepatic models to mimic the liver microenvironment and analyze liver function using hepatocytes cultured in the developed systems. However, mimicking the liver microenvironment in vitro remains a great challenge owing to the lack of perfusable vascular networks in the model systems and the limitation in maintaining hepatocyte function over time. Methods: In this study, we established a microphysiological system that operated under continuous flush medium flow, thereby allowing the supply of nutrients and oxygen to liver organoids and the removal of waste and release of cytokines therefrom, similar to the function of blood vessels. Results: The application of microphysiological system to organoid culture was advantageous for reducing the differentiation time and enhancing the functional maturity of human liver organoid. Conclusion: Hence, our microphysiological culture system might open the possibility of the miniaturized liver model system into a single device to enable more rational in vitro assays of liver response.

Unconventional strategies for liver tissue engineering: plant, paper, silk and nanomaterial-based scaffolds.

The paper highlights how significant characteristics of liver can be modeled in tissue-engineered constructs using unconventional scaffolds. Hepatic lobular organization and metabolic zonation can be mimicked with decellularized plant structures with vasculature resembling a native-hepatic lobule vascular arrangement or silk blend scaffolds meticulously designed for guided cellular arrangement as hepatic patches or metabolic activities. The functionality of hepatocytes can be enhanced and maintained for long periods in naturally fibrous structures paving way for bioartificial liver development. The phase I enzymatic activity in hepatic models can be raised exploiting the microfibrillar structure of paper to allow cellular stacking creating hypoxic conditions to induce in vivo-like xenobiotic metabolism. Lastly, the paper introduces amalgamation of carbon-based nanomaterials into existing scaffolds in liver tissue engineering.

Single-Cell RNA Sequencing Reveals the Spatial Heterogeneity and Functional Alteration of Endothelial Cells in Chronic Hepatitis B Infection.

Chronic Hepatitis B virus (CHB) infection is a global health challenge, causing damage ranging from hepatitis to cirrhosis and hepatocellular carcinoma. In our study, single-cell RNA sequencing (scRNA-seq) analysis was performed in livers from mice models with chronic inflammation induced by CHB infection and we found that endothelial cells (ECs) exhibited the largest number of differentially expressed genes (DEGs) among all ten cell types. NF-κB signaling was activated in ECs to induce cell dysfunction and subsequent hepatic inflammation, which might be mediated by the interaction of macrophage-derived and cholangiocyte-derived VISFATIN/Nampt signaling. Moreover, we divided ECs into three subclusters, including periportal ECs (EC_Z1), midzonal ECs (EC_Z2), and pericentral ECs (EC_Z3) according to hepatic zonation. Functional analysis suggested that pericentral ECs and midzonal ECs, instead of periportal ECs, were more vulnerable to HBV infection, as the VISFATIN/Nampt- NF-κB axis was mainly altered in these two subpopulations. Interestingly, pericentral ECs showed increasing communication with macrophages and cholangiocytes via the Nampt-Insr and Nampt-Itga5/Itgb1 axis upon CHB infection, which contribute to angiogenesis and vascular capillarization. Additionally, ECs, especially pericentral ECs, showed a close connection with nature killer (NK) cells and T cells via the Cxcl6-Cxcr6 axis, which is involved in shaping the microenvironment in CHB mice livers. Thus, our study described the heterogeneity and functional alterations of three subclusters in ECs. We revealed the potential role of VISFATIN/Nampt signaling in modulating ECs characteristics and related hepatic inflammation, and EC-derived chemokine Cxcl16 in shaping NK and T cell recruitment, providing key insights into the multifunctionality of ECs in CHB-associated pathologies.

Cross-species variability in lobular geometry and cytochrome P450 hepatic zonation: insights into CYP1A2, CYP2D6, CYP2E1 and CYP3A4.

There is a lack of systematic research exploring cross-species variation in liver lobular geometry and zonation patterns of critical drug-metabolizing enzymes, a knowledge gap essential for translational studies. This study investigated the critical interplay between lobular geometry and key cytochrome P450 (CYP) zonation in four species: mouse, rat, pig, and human. We developed an automated pipeline based on whole slide images (WSI) of hematoxylin-eosin-stained liver sections and immunohistochemistry. This pipeline allows accurate quantification of both lobular geometry and zonation patterns of essential CYP proteins. Our analysis of CYP zonal expression shows that all CYP enzymes (besides CYP2D6 with panlobular expression) were observed in the pericentral region in all species, but with distinct differences. Comparison of normalized gradient intensity shows a high similarity between mice and humans, followed by rats. Specifically, CYP1A2 was expressed throughout the pericentral region in mice and humans, whereas it was restricted to a narrow pericentral rim in rats and showed a panlobular pattern in pigs. Similarly, CYP3A4 is present in the pericentral region, but its extent varies considerably in rats and appears panlobular in pigs. CYP2D6 zonal expression consistently shows a panlobular pattern in all species, although the intensity varies. CYP2E1 zonal expression covered the entire pericentral region with extension into the midzone in all four species, suggesting its potential for further cross-species analysis. Analysis of lobular geometry revealed an increase in lobular size with increasing species size, whereas lobular compactness was similar. Based on our results, zonated CYP expression in mice is most similar to humans. Therefore, mice appear to be the most appropriate species for drug metabolism studies unless larger species are required for other purposes, e.g., surgical reasons. CYP selection should be based on species, with CYP2E1 and CYP2D6 being the most preferable to compare four species. CYP1A2 could be considered as an additional CYP for rodent versus human comparisons, and CYP3A4 for mouse/human comparisons. In conclusion, our image analysis pipeline together with suggestions for species and CYP selection can serve to improve future cross-species and translational drug metabolism studies.

Deletion of Mitogen-inducible gene-6 (Mig6) in the Liver Alters Glucose Homeostasis and Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) Progression

Metabolic dysfunction-associated steatotic liver disease (MASLD) is a spectrum of chronic liver disease that can progress from benign steatosis to metabolic dysfunction-associated steatohepatitis (MASH) - both of which, if left untreated, can progress to cirrhosis and hepatocellular carcinoma. The strongest risk factor for progression of MASLD to MASH is insulin resistance. Previous work in our lab identified that the adaptor protein mitogen-inducible gene-6 (Mig6) is increased in obese mice and that deletion of Mig6 in the liver (LKO) improves glucose tolerance and insulin action during hyperinsulinemic-euglycemic clamps in diet-induced obese mice. Mig6 is an endogenous feedback inhibitor of epidermal growth factor receptor activation (EGFR) and regulates cellular repair and survival. Thus, we sought to elucidate the extent to which loss of Mig6 modulates the progression of MASLD to MASH. We hypothesized that liver-specific loss of Mig6 during early MASLD improves whole-body glucose homeostasis and protects against MASLD-mediated liver damage. LKO mice and matched, control littermates (CON) were fed a MASH diet (40% fat, 40% carbohydrate, 2% cholesterol; CON n=13-19, LKO n=16-22) or a low-fat-matched diet (LFD; 10% fat, 70% carbohydrate; CON n=8-16, LKO n=16-21) for up to 40 weeks. Body weights were recorded weekly, and body composition assessment, glucose and insulin tolerance tests, and hepatic histological analysis were performed after 20-, 30- and 40-weeks on each diet. Compared to the MASH-fed CON mice, MASH-fed LKO mice had lower body weights starting at 10-weeks and persisting throughout the study. Body composition analysis established these differences in body weight were due to decreases in fat, but not lean, mass. Interestingly, whereas intraperitoneal glucose tolerance tests did not identify significant differences in glucose handling between the LFD and MASH fed mice, oral glucose tolerance was significantly improved in 10- and 20-week MASH-fed LKO mice, suggesting alterations in the incretin response in this MASH model. Lastly, histological analysis revealed MASH-fed LKO mice had a preservation of hepatic lipid zonation and blunted levels of circulating alanine aminotransferase (ALT) compared to MASH-fed CON, indicating less hepatic damage in LKO mice. These results suggest that liver-specific loss of Mig6 improves whole-body glucose handling, which in turn ameliorates hepatic damage during MASLD progression. In conclusion, Mig6 prevents, or at least delays, the progression of MASLD, which may reveal a new therapeutic area for the prevention and treatment of MASH. MS was supported by a National Cancer Institute Cancer Metabolism Training Program Postdoctoral Fellowship (T32CA221709). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Spatial transcriptomics unravels palmitoylation and zonation-dependent gene regulation by AEG-1 in mouse liver

Obesity-induced metabolic dysfunction-associated steatohepatitis (MASH) leads to hepatocellular carcinoma (HCC). Astrocyte-elevated gene-1/Metadherin (AEG-1/MTDH) plays a key role in promoting MASH and HCC. AEG-1 is palmitoylated at residue cysteine 75 (Cys75) and a knock-in mouse representing mutated Cys75 to serine (AEG-1-C75S) showed activation of MASH- and HCC-promoting gene signature when compared to wild-type littermates (AEG-1-WT). The liver consists of 3 zones, periportal, midlobular and pericentral, and zone-specific dysregulated gene expression impairs metabolic homeostasis in the liver, contributing to MASH and HCC. Here, to elucidate how palmitoylation influences AEG-1-mediated gene regulation in regards to hepatic zonation, we performed spatial transcriptomics (ST) in the livers of AEG-1-WT and AEG-1-C75S littermates. ST identified six different clusters in livers and using zone- and cell type-specific markers we attributed specific zones and cell types to specific clusters. Ingenuity Pathway Analysis (IPA) of differentially expressed genes in each cluster unraveled activation of pro-inflammatory and MASH- and HCC-promoting pathways, mainly in periportal and pericentral hepatocytes, in AEG-1-C75S liver compared to AEG-1-WT. Interestingly, in AEG-1-C75S liver, the midlobular zone exhibited widespread inhibition of xenobiotic metabolism pathways and inhibition of PXR/RXR and LXR/RXR activation, vs AEG-1-WT. In conclusion, AEG-1-C75S mutant exhibited zone-specific differential gene expression, which might contribute to metabolic dysfunction and dysregulated drug metabolism leading to MASH and HCC.

A spatial map of hepatic mitochondria uncovers functional heterogeneity shaped by nutrient-sensing signaling

In the liver, mitochondria are exposed to different concentrations of nutrients due to their spatial positioning across the periportal and pericentral axis. How the mitochondria sense and integrate these signals to respond and maintain homeostasis is not known. Here, we combine intravital microscopy, spatial proteomics, and functional assessment to investigate mitochondrial heterogeneity in the context of liver zonation. We find that periportal and pericentral mitochondria are morphologically and functionally distinct; beta-oxidation is elevated in periportal regions, while lipid synthesis is predominant in the pericentral mitochondria. In addition, comparative phosphoproteomics reveals spatially distinct patterns of mitochondrial composition and potential regulation via phosphorylation. Acute pharmacological modulation of nutrient sensing through AMPK and mTOR shifts mitochondrial phenotypes in the periportal and pericentral regions, linking nutrient gradients across the lobule and mitochondrial heterogeneity. This study highlights the role of protein phosphorylation in mitochondrial structure, function, and overall homeostasis in hepatic metabolic zonation. These findings have important implications for liver physiology and disease.

Endothelial Slc35a1 Deficiency Causes Loss of LSEC Identity and Exacerbates Neonatal Lipid Deposition in the Liver in Mice

BACKGROUND & AIMSThe functional maturation of the liver largely occurs after birth. In the early stages of life, the liver of a newborn encounters enormous high-fat metabolic stress caused by the consumption of breast milk. It is unclear how the maturing liver adapts to high lipid metabolism. Liver sinusoidal endothelial cells (LSECs) play a fundamental role in establishing liver vasculature and are decorated with many glycoproteins on their surface. The Slc35a1 gene encodes a CMP-sialic acid transporter responsible for transporting CMP-sialic acids between the cytoplasm and the Golgi apparatus for protein sialylation. This study aimed to determine whether endothelial sialylation plays a role in hepatic vasculogenesis and functional maturation. METHODSEndothelial-specific Slc35a1 knockout mice were generated. Liver tissues were collected for histological analysis, lipidomic profiling, RNA sequencing, confocal immunofluorescence, and immunoblot analyses. RESULTSEndothelial Slc35a1-deficient mice exhibited excessive neonatal hepatic lipid deposition, severe liver damage, and high mortality. Endothelial deletion of Slc35a1 led to sinusoidal capillarization and disrupted hepatic zonation. Mechanistically, VEGFR2 in LSECs was desialylated and VEGFR2 signaling was enhanced in Slc35a1-deficient mice. Inhibition of VEGFR2 signaling by SU5416 alleviated lipid deposition and restored hepatic vasculature in Slc35a1-deficient mice. CONCLUSIONSOur findings suggest that sialylation of LSECs is critical for maintaining hepatic vascular development and lipid homeostasis. Targeting VEGFR2 signaling may be a new strategy to prevent liver disorders associated with abnormal vasculature and lipid deposition.

Strategic Replication of the Hepatic Zonation In Vitro Employing a Biomimetic Approach.

The varied functions of the liver are dependent on the metabolic heterogeneity exhibited by the hepatocytes within the liver lobule spanning the porto-central axis. This complex phenomenon plays an important role in maintaining the physiological homeostasis of the liver. Standard in vitro culture models fail to mimic this spatial heterogeneity of hepatocytes, assuming a homogeneous population of cells, which leads to inaccurate translation of results. Here, we demonstrate the development of an in vitro model of hepatic zonation by mimicking the microarchitecture of the liver using a 3D printed mini bioreactor and decellularized liver matrix to provide the native microenvironmental cues. There was a differential expression of hypoxic and metabolic markers across the developed mini bioreactor, showing the establishment of gradients of oxygen, Wnt/β-catenin pathway, and other metabolic pathways. The model also showed the establishment of zone-dependent toxicity on treatment with acetaminophen. The developed model would thus be a promising avenue in the field of tissue engineering for understanding the liver physiology and pathophysiology and for drug screening to evaluate the potential of new pharmaceutical interventions.

From complement to complosome in non-alcoholic fatty liver disease: When location matters.

Non-alcoholic fatty liver disease (NAFLD) is a growing public health threat and becoming the leading cause of liver transplantation. Nevertheless, no approved specific treatment is currently available for NAFLD. The pathogenesis of NAFLD is multifaceted and not yet fully understood. Accumulating evidence suggests a significant role of the complement system in the development and progression of NAFLD. Here, we provide an overview of the complement system, incorporating the novel concept of complosome, and summarise the up-to-date evidence elucidating the association between complement dysregulation and the pathogenesis of NAFLD. In this process, the extracellular complement system is activated through various pathways, thereby directly contributing to, or working together with other immune cells in the disease development and progression. We also introduce the complosome and assess the evidence that implicates its potential influence in NAFLD through its direct impact on hepatocytes or non-parenchymal liver cells. Additionally, we expound upon how complement system and the complosome may exert their effects in relation with hepatic zonation in NAFLD. Furthermore, we discuss the potential therapeutic implications of targeting the complement system, extracellularly and intracellularly, for NAFLD treatment. Finally, we present future perspectives towards a better understanding of the complement system's contribution to NAFLD.

Extracellular matrix modulates the spatial hepatic features in hepatocyte-like cells derived from human embryonic stem cells

BackgroundHuman pluripotent stem cell (hPSC)-derived hepatocyte-like cells (HLCs) can provide a valuable in vitro model for disease modelling and drug development. However, generating HLCs with characteristics comparable to hepatocytes in vivo is challenging. Extracellular matrix (ECM) plays an important role in supporting liver development and hepatocyte functions, but their impact on hepatocyte differentiation and maturation during hPSC differentiation remains unclear. Here, we investigate the effects of two ECM components—Matrigel and type I collagen on hepatic differentiation of human embryonic stem cells (hESCs).MethodshESC-derived HLCs were generated through multistage differentiation in two-dimensional (2D) and three-dimensional (3D) cultures, incorporating either type I collagen or Matrigel during hepatic specification and maturation. The resulting HLCs was characterized for their gene expression and functionality using various molecular and cellular techniques.ResultsOur results showed that HLCs cultured with collagen exhibited a significant increase in albumin and alpha-1 anti-trypsin expression with reduced AFP compared to HLCs cultured with Matrigel. They also secreted more urea than Matrigel cultures. However, these HLCs exhibited lower CYP3A4 activity and glycogen storage than those cultured with Matrigel. These functional differences in HLCs between collagen and Matrigel cultures closely resembled the hepatocytes of periportal and pericentral zones, respectively.ConclusionOur study demonstrates that Matrigel and collagen have differential effects on the differentiation and functionality of HLCs, which resemble, to an extent, hepatic zonation in the liver lobules. Our finding has an important impact on the generation of hPSC-HLCs for biomedical and medical applications.

Huntingtin loss in hepatocytes is associated with altered metabolism, adhesion, and liver zonation.

Huntington's disease arises from a toxic gain of function in the huntingtin (HTT) gene. As a result, many HTT-lowering therapies are being pursued in clinical studies, including those that reduce HTT RNA and protein expression in the liver. To investigate potential impacts, we characterized molecular, cellular, and metabolic impacts of chronic HTT lowering in mouse hepatocytes. Lifelong hepatocyte HTT loss is associated with multiple physiological changes, including increased circulating bile acids, cholesterol and urea, hypoglycemia, and impaired adhesion. HTT loss causes a clear shift in the normal zonal patterns of liver gene expression, such that pericentral gene expression is reduced. These alterations in liver zonation in livers lacking HTT are observed at the transcriptional, histological, and plasma metabolite levels. We have extended these phenotypes physiologically with a metabolic challenge of acetaminophen, for which the HTT loss results in toxicity resistance. Our data reveal an unexpected role for HTT in regulating hepatic zonation, and we find that loss of HTT in hepatocytes mimics the phenotypes caused by impaired hepatic β-catenin function.

Determination of key events in mouse hepatocyte maturation at the single-cell level

Hepatocytes, the liver's predominant cells, perform numerous essential biological functions. However, crucial events and regulators during hepatocyte maturation require in-depth investigation. In this study, we performed single-cell RNA sequencing (scRNA-seq) and single-nucleus RNA sequencing (snRNA-seq) to explore the precise hepatocyte development process in mice. We defined three maturation stages of postnatal hepatocytes, each of which establishes specific metabolic functions and exhibits distinct proliferation rates. Hepatic zonation is gradually formed during hepatocyte maturation. Hepatocytes or their nuclei with distinct ploidies exhibit zonation preferences in distribution and asynchrony in maturation. Moreover, by combining gene regulatory network analysis with invivo genetic manipulation, we identified critical maturation- and zonation-related transcription factors. This study not only delineates the comprehensive transcriptomic profiles of hepatocyte maturation but also presents a paradigm to identify genes that function in the development of hepatocyte maturation and zonation by combining genetic manipulation and measurement of coordinates in a single-cell developmental trajectory.

Utility of Minimal Physiologically Based Pharmacokinetic Models for Assessing Fractional Distribution, Oral Absorption, and Series-Compartment Models of Hepatic Clearance.

Minimal physiologically based pharmacokinetic (mPBPK) models are physiologically relevant, require less information than full PBPK models, and offer flexibility in pharmacokinetics (PK). The well-stirred hepatic model (WSM) is commonly used in PBPK, whereas the more plausible dispersion model (DM) poses computational complexities. The series-compartment model (SCM) mimics the DM but is easier to operate. This work implements the SCM and mPBPK models for assessing fractional tissue distribution, oral absorption, and hepatic clearance using literature-reported blood and liver concentration-time data in rats for compounds mainly cleared by the liver. Further handled were various complexities, including nonlinear hepatic binding and metabolism, differing absorption kinetics, and sites of administration. The SCM containing one to five (n) liver subcompartments yields similar fittings and provides comparable estimates for hepatic extraction ratio (ER), prehepatic availability (Fg ), and first-order absorption rate constants (ka ). However, they produce decreased intrinsic clearances (CLint ) and liver-to-plasma partition coefficients (Kph ) with increasing n as expected. Model simulations demonstrated changes in intravenous and oral PK profiles with alterations in Kph and ka and with hepatic metabolic zonation. The permeability (PAMPA P) of the various compounds well explained the fitted fractional distribution (fd ) parameters. The SCM and mPBPK models offer advantages in distinguishing systemic, extrahepatic, and hepatic clearances. The SCM allows for incorporation of liver zonation and is useful in assessing changes in internal concentration gradients potentially masked by similar blood PK profiles. Improved assessment of intraorgan drug concentrations may offer insights into active moieties driving metabolism, biliary excretion, pharmacodynamics, and hepatic toxicity. SIGNIFICANCE STATEMENT: The minimal physiologically based pharmacokinetic model and the series-compartment model are useful in assessing oral absorption and hepatic clearance. They add flexibility in accounting for various drug- or system-specific complexities, including fractional distribution, nonlinear binding and saturable hepatic metabolism, and hepatic zonation. These models can offer improved insights into the intraorgan concentrations that reflect physiologically active moieties often driving disposition, pharmacodynamics, and toxicity.

Transcriptomic and proteomic studies suggest the establishment of advanced zonation-like profiles in human-induced pluripotent stem cell-derived liver sinusoidal endothelial cells and carboxypeptidase M-positive liver progenitor cells cocultured in a fluidic microenvironment.

Hepatic zonation is a physiological feature of the liver, known to be key in the regulation of the metabolism of nutrients and xenobiotics and the biotransformation of numerous substances. However, the reproduction of this phenomenon remains challenging in vitro as only part of the processes involved in the orchestration and maintenance of zonation are fully understood. The recent advances in organ-on-chip technologies, which allow for the integration of multicellular 3D tissues in a dynamic microenvironment, could offer solutions for the reproduction of zonation within a single culture vessel. An in-depth analysis of zonation-related mechanisms observed during the coculture of human-induced pluripotent stem cell (hiPSC)-derived carboxypeptidase M-positive liver progenitor cells and hiPSC-derived liver sinusoidal endothelial cells within a microfluidic biochip was carried out. Hepatic phenotypes were confirmed in terms of albumin secretion, glycogen storage, CYP450 activity, and expression of specific endothelial markers such as PECAM1, RAB5A, and CD109. Further characterization of the patterns observed in the comparison of the transcription factor motif activities, the transcriptomic signature, and the proteomic profile expressed at the inlet and the outlet of the microfluidic biochip confirmed the presence of zonation-like phenomena within the biochips. In particular, differences related to Wnt/β-catenin, transforming growth factor-β, mammalian target of rapamycin, hypoxia-inducible factor-1, and AMP-activated protein kinase signaling, to the metabolism of lipids, and cellular remolding were observed. The present study shows the interest in combining cocultures of hiPSC-derived cellular models and microfluidic technologies for reproducing in vitro complex mechanisms such as liver zonation and further incites the use of those solutions for accurate reproduction of in vivo situations.

A Spatial Atlas of Wnt Receptors in Adult Mouse Liver

Hepatic zonation is critical for most metabolic functions in liver. Wnt signaling plays an important role in establishing and maintaining liver zonation. Yet, the anatomic expression of Wnt signaling components, especially all 10 Frizzled (Fzd) receptors, has not been characterized in adult liver. To address this, the spatial expression of Fzd receptors was quantitatively mapped in adult mouse liver via multiplex fluorescent in situ hybridization. Although all 10 Fzd receptors were expressed within a metabolic unit, Fzd receptors 1, 4, and 6 were the highest expressed. Although most Wnt signaling occurs in zone 3, expression of most Fzd receptors was not zonated. In contrast, Fzd receptor 6 was preferentially expressed in zone 1. Wnt2 and Wnt9b expression was highly zonated and primarily found in zone 3. Therefore, the current results suggest that zonated Wnt/β-catenin signaling at baseline occurs primarily due to Wnt2 and Wnt9b rather than zonation of Fzd mRNA expression. Finally, the study showed that Fzd receptors and Wnts are not uniformly expressed by all hepatic cell types. Instead, there is broad distribution among both hepatocytes and nonparenchymal cells, including endothelial cells. Overall, this establishment of a definitive mRNA expression atlas, especially of Fzd receptors, opens the door to future functional characterization in healthy and diseased liver states.

Exploring the Pharmacokinetic Mysteries of the Liver: Application of Series Compartment Models of Hepatic Elimination.

Among the basic hepatic clearance models, the dispersion model (DM) is the most physiologically sound compared with the well-stirred model and the parallel tube model. However, its application in physiologically-based pharmacokinetic (PBPK) modeling has been limited due to computational complexities. The series compartment models (SCM) of hepatic elimination that treats the liver as a cascade of well-stirred compartments connected by hepatic blood flow exhibits some mathematical similarities to the DM but is easier to operate. This work assesses the quantitative correlation between the SCM and DM and demonstrates the operation of the SCM in PBPK with the published single-dose blood and liver concentration-time data of six flow-limited compounds. The predicted liver concentrations and the estimated intrinsic clearance (CLint ) and PBPK-operative tissue-to-plasma partition coefficient (Kp ) values were shown to depend on the number of liver sub-compartments (n) and hepatic enzyme zonation in the SCM. The CLint and Kp decreased with increasing n, with more remarkable differences for drugs with higher hepatic extraction ratios. Given the same total CLint , the SCM yields a higher Kp when the liver perivenous region exhibits a lower CLint as compared with a high CLint at this region. Overall, the SCM nicely approximates the DM in characterizing hepatic elimination and offers an alternative flexible approach as well as providing some insights regarding sequential drug concentrations in the liver. SIGNIFICANCE STATEMENT: The SCM nicely approximates the DM when applied in PBPK for characterizing hepatic elimination. The number of liver sub-compartments and hepatic enzyme zonation are influencing factors for the SCM resulting in model-dependent predictions of total/internal liver concentrations and estimates of CLint and the PBPK-operative Kp . Such model-dependency may have an impact when the SCM is used for in vitro-to-in vivo extrapolation (IVIVE) and may also be relevant for PK/PD/toxicological effects when it is the driving force for such responses.

Single-cell transcriptomics shows dose-dependent disruption of hepatic zonation by TCDD in mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) dose-dependently induces the development of hepatic fat accumulation and inflammation with fibrosis in mice initially in the portal region. Conversely, differential gene and protein expression is first detected in the central region. To further investigate cell-specific and spatially resolved dose-dependent changes in gene expression elicited by TCDD, single-nuclei RNA sequencing and spatial transcriptomics were used for livers of male mice gavaged with TCDD every 4 days for 28 days. The proportion of 11 cell (sub)types across 131 613 nuclei dose-dependently changed with 68% of all portal and central hepatocyte nuclei in control mice being overtaken by macrophages following TCDD treatment. We identified 368 (portal fibroblasts) to 1339 (macrophages) differentially expressed genes. Spatial analyses revealed initial loss of portal identity that eventually spanned the entire liver lobule with increasing dose. Induction of R-spondin 3 (Rspo3) and pericentral Apc, suggested dysregulation of the Wnt/β-catenin signaling cascade in zonally resolved steatosis. Collectively, the integrated results suggest disruption of zonation contributes to the pattern of TCDD-elicited NAFLD pathologies.

Liver Acinus Dynamic Chip for Assessment of Drug-Induced Zonal Hepatotoxicity.

Zonation along the liver acinus is considered a key feature of liver physiology. Here, we developed a liver acinus dynamic (LADY) chip that recapitulates a key functional structure of the liver acinus and hepatic zonation. Corresponding to the blood flow from portal triads to the central vein in vivo, gradual flow of oxygen and glucose–carrying culture medium into the HepG2 cell chamber of the LADY chip generated zonal protein expression patterns in periportal (PP) zone 1 and perivenous (PV) zone 3. Higher levels of albumin secretion and urea production were obtained in a HepG2/HUVECs co-culture LADY chip than in HepG2 mono-culture one. Zonal expression of PEPCK as a PP marker and CYP2E1 as a PV marker was successfully generated. Cell death rate of the PV cells was higher than that of the PP cells since zonal factors responsible for metabolic activation of acetaminophen (APAP) were highly expressed in the PV region. We also found the co-culture enhanced metabolic capacity to process APAP, thus improving resistance to APAP toxicity, in comparison with HepG2 mono-culture. These results indicate that our LADY chip successfully represents liver zonation and could be useful in drug development studies as a drug-induced zonal hepatotoxicity testing platform.

Interplay Between GH-regulated, Sex-biased Liver Transcriptome and Hepatic Zonation Revealed by Single-Nucleus RNA Sequencing.

The zonation of liver metabolic processes is well-characterized; however, little is known about the cell type-specificity and zonation of sexually dimorphic gene expression or its growth hormone (GH)-dependent transcriptional regulators. We address these issues using single-nucleus RNA-sequencing of 32 000 nuclei representing 9 major liver cell types. Nuclei were extracted from livers from adult male and female mice; from males infused with GH continuously, mimicking the female plasma GH pattern; and from mice exposed to TCPOBOP, a xenobiotic agonist ligand of the nuclear receptor CAR that perturbs sex-biased gene expression. Analysis of these rich transcriptomic datasets revealed the following: 1) expression of sex-biased genes and their GH-dependent transcriptional regulators is primarily restricted to hepatocytes and is not a feature of liver nonparenchymal cells; 2) many sex-biased transcripts show sex-dependent zonation within the liver lobule; 3) gene expression is substantially feminized both in periportal and pericentral hepatocytes when male mice are infused with GH continuously; 4) sequencing nuclei increases the sensitivity for detecting thousands of nuclear-enriched long-noncoding RNAs (lncRNAs) and enables determination of their liver cell type-specificity, sex-bias and hepatocyte zonation profiles; 5) the periportal to pericentral hepatocyte cell ratio is significantly higher in male than female liver; and 6) TCPOBOP exposure disrupts both sex-specific gene expression and hepatocyte zonation within the liver lobule. These findings highlight the complex interconnections between hepatic sexual dimorphism and zonation at the single-cell level and reveal how endogenous hormones and foreign chemical exposure can alter these interactions across the liver lobule with large effects both on protein-coding genes and lncRNAs.