Characterization of gut microbiota in patients with stage 3-4 chronic kidney disease: a retrospective cohort study.

PURPOSE: Multiple factors, such as dietary patterns, pharmaceutical interventions, and exposure to harmful substances, possess the capacity to influence gut microbiota composition. Gut microbiota dysbiosis has emerged as a significant contributor to the progression of chronic kidney disease (CKD) and its associated complications. By comprehending the intricacies of the intestinal microbiota, this research endeavor holds the potential to offer novel perspectives on potential strategies for mitigating CKD progression. METHODS: In this retrospective analysis, we assessed gut microbiota composition in CKD patients. Fecal samples were collected from a cohort of 44 patients with stage 3-4 CKD, alongside a control group consisting of 132 healthy volunteers. Subsequently, 16 s rDNA sequencing was conducted to examine the composition of the gut microbiota. RESULTS: Our findings revealed significant alterations in the diversity of intestinal microbiota in fecal samples between patients with stage 3-4 CKD and healthy subjects. Among the 475 bacterial genera, 164 were shared, while 242 dominant genera were exclusive to healthy subjects and 69 to CKD stages 3-4 samples. Notably, healthy volunteers exhibited a prevalence of intestinal Firmicutes and Bacteroidetes, whereas stage 3-4 CKD patients displayed higher abundance of Proteobacteria and Actinobacteria. The presence of uncultured Coprobacillus sp. notably contributed to distinguishing between the two groups. ROC curve analysis identified distinct microbiota with superior diagnostic efficacy for discriminating stage 3-4 CKD patients from healthy individuals. Metabolic pathway analysis revealed differing dominant pathways between the two groups-the NADH dehydrogenase pathway in healthy individuals and the phosphate acetyltransferase pathway in stage 3-4 CKD patients. Moreover, the CKD cohort displayed a higher proportion of Gram-negative bacteria and facultative anaerobes. CONCLUSIONS: In conclusion, our study underscores the profound influence of gut microbiota dysbiosis on CKD progression. The distinct microbial profiles observed in CKD patients highlight the potential efficacy of microbiota-based interventions in mitigating CKD advancement.

Organic Anion Transporting Polypeptide (OATP) 1B3 is a Significant Transporter for Hepatic Uptake of Conjugated Bile Acids in Humans.

BACKGROUND & AIMS: OATP1B3/SLCO1B3 is a human liver-specific transporter for the clearance of endogenous compounds (eg, bile acid [BA]) and xenobiotics. The functional role of OATP1B3 in humans has not been characterized, as SLCO1B3 is poorly conserved among species without mouse orthologs. METHODS: Slc10a1-knockout (Slc10a1-/-), Slc10a1hSLCO1B3 (endogenous mouse Slc10a1 promoter-driven human-SLCO1B3 expression in Slc10a1-/- mice), and human SLCO1B3 liver-specific transgenic (hSLCO1B3-LTG) mice were generated and challenged with 0.1% ursodeoxycholic-acid (UDCA), 1% cholic-acid (CA) diet, or bile duct ligation (BDL) for functional studies. Primary hepatocytes and hepatoma-PLC/RPF/5 cells were used for mechanistic studies. RESULTS: Serum BA levels in Slc10a1-/- mice were substantially increased with or without 0.1% UDCA feeding compared with wild-type (WT) mice. This increase was attenuated in Slc10a1hSLCO1B3-mice, indicating that OATP1B3 functions as a significant hepatic BA uptake transporter. In vitro assay using primary hepatocytes from WT, Slc10a1-/-, and Slc10a1hSLCO1B3-mice indicated that OATP1B3 has a similar capacity in taking up taurocholate/TCA as Ntcp. Furthermore, TCA-induced bile flow was significantly impaired in Slc10a1-/- mice but partially recovered in Slc10a1hSLC01B3-mice, indicating that OATP1B3 can partially compensate the NTCP function in vivo. Liver-specific overexpression of OATP1B3 markedly increased the level of hepatic conjugated BA and cholestatic liver injury in 1% CA-fed and BDL mice. Mechanistic studies revealed that conjugated BAs stimulated Ccl2 and Cxcl2 in hepatocytes to increase hepatic neutrophil infiltration and proinflammatory cytokine production (eg, IL-6), which activated STAT3 to repress OATP1B3 expression by binding to its promoter. CONCLUSIONS: Human OATP1B3 is a significant BA uptake transporter and can partially compensate Ntcp for conjugated BA uptake in mice. Its downregulation in cholestasis is an adaptive protective response.

Unique DUOX2+ACE2+ small cholangiocytes are pathogenic targets for primary biliary cholangitis.

Cholangiocytes play a crucial role in bile formation. Cholangiocyte injury causes cholestasis, including primary biliary cholangitis (PBC). However, the etiology of PBC remains unclear despite being characterized as an autoimmune disease. Using single-cell RNA sequencing (scRNA-seq), fluorescence-activated-cell-sorting, multiplex immunofluorescence (IF) and RNAscope analyses, we identified unique DUOX2+ACE2+ small cholangiocytes in human and mouse livers. Their selective decrease in PBC patients was associated with the severity of disease. Moreover, proteomics, scRNA-seq, and qPCR analyses indicated that polymeric immunoglobulin receptor (pIgR) was highly expressed in DUOX2+ACE2+ cholangiocytes. Serum anti-pIgR autoantibody levels were significantly increased in PBC patients, regardless of positive and negative AMA-M2. Spatial transcriptomics and multiplex IF revealed that CD27+ memory B and plasma cells accumulated in the hepatic portal tracts of PBC patients. Collectively, DUOX2+ACE2+ small cholangiocytes are pathogenic targets in PBC, and preservation of DUOX2+ACE2+ cholangiocytes and targeting anti-pIgR autoantibodies may be valuable strategies for therapeutic interventions in PBC.

Gut Microbiota Deficiency Exacerbates Liver Injury in Bile Duct Ligated Mice via Inflammation and Lipid Metabolism.

Bile components play a critical role in maintaining gut microbiota homeostasis. In cholestasis, bile secretion is impaired, leading to liver injury. However, it remains to be elucidated whether gut microbiota plays a role in cholestatic liver injury. Here, we performed a sham operation and bile duct ligation (BDL) in antibiotic-induced microbiome depleted (AIMD) mice and assessed liver injury and fecal microbiota composition in these mice. Significant reductions in gut microbiota richness and diversity were found in AIMD-sham mice when compared to sham controls. Three-day BDL leads to great elevation of plasma ALT, ALP, total bile acids, and bilirubin where reduced diversity of the gut microbiota was also found. AIMD further aggravated cholestatic liver injury evidenced by significantly higher levels of plasma ALT and ALP, associated with further reduced diversity and increased Gram-negative bacteria in gut microbiota. Further analyses revealed increased levels of LPS in the plasma of AIMD-BDL mice where elevated expression of inflammatory genes and decreased expression of hepatic detoxification enzymes were also found in liver when compared to the BDL group. These findings indicate that gut microbiota plays a critical role in cholestatic liver injury. Maintaining its homeostasis may alleviate liver injury in patients with cholestasis.

Runt-related transcription factor-1 ameliorates bile acid-induced hepatic inflammation in cholestasis through JAK/STAT3 signaling.

BACKGROUND AND AIMS: Bile acids trigger a hepatic inflammatory response, causing cholestatic liver injury. Runt-related transcription factor-1 (RUNX1), primarily known as a master modulator in hematopoiesis, plays a pivotal role in mediating inflammatory responses. However, RUNX1 in hepatocytes is poorly characterized, and its role in cholestasis is unclear. Herein, we aimed to investigate the role of hepatic RUNX1 and its underlying mechanisms in cholestasis. APPROACH AND RESULTS: Hepatic expression of RUNX1 was examined in cholestatic patients and mouse models. Mice with liver-specific ablation of Runx1 were generated. Bile duct ligation and 1% cholic acid diet were used to induce cholestasis in mice. Primary mouse hepatocytes and the human hepatoma PLC/RPF/5- ASBT cell line were used for mechanistic studies. Hepatic RUNX1 mRNA and protein levels were markedly increased in cholestatic patients and mice. Liver-specific deletion of Runx1 aggravated inflammation and liver injury in cholestatic mice induced by bile duct ligation or 1% cholic acid feeding. Mechanistic studies indicated that elevated bile acids stimulated RUNX1 expression by activating the RUNX1 -P2 promoter through JAK/STAT3 signaling. Increased RUNX1 is directly bound to the promotor region of inflammatory chemokines, including CCL2 and CXCL2 , and transcriptionally repressed their expression in hepatocytes, leading to attenuation of liver inflammatory response. Blocking the JAK signaling or STAT3 phosphorylation completely abolished RUNX1 repression of bile acid-induced CCL2 and CXCL2 in hepatocytes. CONCLUSIONS: This study has gained initial evidence establishing the functional role of hepatocyte RUNX1 in alleviating liver inflammation during cholestasis through JAK/STAT3 signaling. Modulating hepatic RUNX1 activity could be a new therapeutic target for cholestasis.

Hepatic TNFRSF12A promotes bile acid-induced hepatocyte pyroptosis through NFκB/Caspase-1/GSDMD signaling in cholestasis.

Tumor necrosis factor receptor superfamily member-12A (TNFRSF12A) plays a critical role in inflammation and cell death. It is expressed in multiple tissues yet extremely low in normal liver. To date, little is known about its role in cholestasis. Therefore, we sought to delineate the role of TNFRSF12A in cholestasis and its underlying mechanisms. Human liver tissues were collected from patients with obstructive cholestasis (OC) or primary biliary cholangitis (PBC). Tnfrsf12a knockout (KO) mice were generated. Cholestasis was induced by bile-duct ligation (BDL) or 0.1% 5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-feeding. Human hepatoma PLC/PRF/5-ASBT and THP1 cell lines or primary mouse hepatocytes were used for mechanistic studies. Hepatic TNFRSF12A expression was markedly increased in OC or PBC patients. Genetic ablation of Tnfrsf12a in BDL- and 0.1%DDC-induced cholestatic mice significantly attenuated cholestatic liver injury with remarkable reduction of hepatocyte pyroptosis but without changing scores of necroptosis and apoptosis. Morphological features of hepatocyte pyroptosis and increased levels of pyroptosis-related proteins, NLRP3, cleaved-Caspase-1, and cleaved-GSDMD in OC patients and BDL-mice confirmed this observation. Further mechanistic studies revealed that bile acids (BAs) induced TNFRSF12A expression by enhancing the transcription factor c-JUN binding to the TNFRSF12A promoter and subsequently initiated hepatocyte pyroptosis by the NFκB/Caspase-1/GSDMD signaling. Interestingly, TWEAK, a typical ligand of TNFRSF12A, secreted by infiltrated macrophages in cholestatic livers, enhanced TNFRSF12A-induced hepatocyte pyroptosis. Taken together, we report, for the first time, that hepatic TNFRSF12A is dramatically increased in human cholestasis. Deletion of TNFRSF12A inhibits BAs-induced hepatocyte pyroptosis through the NFκB/Caspase-1/GSDMD signaling and thereby ameliorates cholestatic liver injury. As such, targeting TNFRSF12A could be a promising approach to treating cholestasis.

SEMA7AR148W mutation promotes lipid accumulation and NAFLD progression via increased localization on the hepatocyte surface.

Genetic polymorphisms are associated with the development of nonalcoholic fatty liver disease (NAFLD). Semaphorin7a (Sema7a) deficiency in mouse peritoneal macrophages reduces fatty acid (FA) oxidation. Here, we identified 17 individuals with SEMA7A heterozygous mutations in 470 patients with biopsy-proven NAFLD. SEMA7A heterozygous mutations increased susceptibility to NAFLD, steatosis severity, and NAFLD activity scores in humans and mice. The Sema7aR145W mutation (equivalent to human SEMA7AR148W) significantly induced small lipid droplet accumulation in mouse livers compared with WT mouse livers. Mechanistically, the Sema7aR145W mutation increased N-glycosylated Sema7a and its receptor integrin ß1 proteins in the cell membranes of hepatocytes. Furthermore, Sema7aR145W mutation enhanced its protein interaction with integrin ß1 and PKC-α and increased PKC-α phosphorylation, which were both abrogated by integrin ß1 silencing. Induction of PKCα_WT, but not PKCα_dominant negative, overexpression induced transcriptional factors Srebp1, Chrebp, and Lxr expression and their downstream Acc1, Fasn, and Cd36 expression in primary mouse hepatocytes. Collectively, our findings demonstrate that the SEMA7AR148W mutation is a potentially new strong genetic determinant of NAFLD and promotes intrahepatic lipid accumulation and NAFLD in mice by enhancing PKC-α-stimulated FA and triglyceride synthesis and FA uptake. The inhibition of hepatic PKC-α signaling may lead to novel NAFLD therapies.

Urinary exosomal vitronectin predicts vesicoureteral reflux in patients with neurogenic bladders and spinal cord injuries.

Neurogenic bladder (NGB) is an important complication of urinary tract dysfunction after spinal cord injury (SCI). However, using urodynamics and urography to guide therapy remains invasive and complicated. Therefore, the present study aimed to identify potential noninvasive biomarkers from urinary exosomes that can facilitate diagnosis and guide prognosis of patients with NGB subsequent to SCI. Urinary exosomes were isolated, and their proteome profile was analyzed by mass spectrometry. Transmission electron microscopy and Nanoparticle Tracking Analysis confirmed the size and morphological characteristics of urinary exosomes. In addition, bioinformatics analysis and parallel reaction monitoring (PRM) were used to screen candidate biomarkers. The selected biomarkers were validated using western blotting and ELISA. Mass spectrometry identified 134 upregulated proteins and 99 downregulated proteins between the vesicoureteral reflux (VUR) and non-VUR groups. A total of 18 candidate proteins were selected for PRM validation, but only vitronectin (VTN) and α-1 type I collagen (COL1A1) demonstrated significant differences. In the validation experiments using western blotting and ELISA, VTN was exclusively highly expressed in VUR patients compared with non-VUR patients. However, the ELISA results of COL1A1 revealed no significant difference when a larger sample size was used. Furthermore, a receiver operating characteristic curve of ELISA-based VTN demonstrated an area under the curve of 0.795 and 80% sensitivity at a threshold set to give 82.9% specificity. Collectively, these results suggested that VTN in urinary exosomes may be used as a biomarker to predict the progression and guide the prognosis of NGB.

A homozygous R148W mutation in Semaphorin 7A causes progressive familial intrahepatic cholestasis.

Semaphorin 7A (SEMA7A) is a membrane-bound protein that involves axon growth and other biological processes. SEMA7A mutations are associated with vertebral fracture and Kallmann syndrome. Here, we report a case with a mutation in SEMA7A that displays familial cholestasis. WGS reveals a SEMA7AR148W homozygous mutation in a female child with elevated levels of serum ALT, AST, and total bile acid (TBA) of unknown etiology. This patient also carried a SLC10A1S267F allele, but Slc10a1S267F homozygous mice exhibited normal liver function. Similar to the child, Sema7aR145W homozygous mice displayed elevated levels of serum ALT, AST, and TBA. Remarkably, liver histology and LC-MS/MS analyses exhibited hepatocyte hydropic degeneration and increased liver bile acid (BA) levels in Sema7aR145W homozygous mice. Further mechanistic studies demonstrated that Sema7aR145W mutation reduced the expression of canalicular membrane BA transporters, bile salt export pump (Bsep), and multidrug resistance-associated protein-2 (Mrp2), causing intrahepatic cholestasis in mice. Administration with ursodeoxycholic acid and a dietary supplement glutathione improved liver function in the child. Therefore, Sema7aR145W homozygous mutation causes intrahepatic cholestasis by reducing hepatic Bsep and Mrp2 expression.

The role of bile acids in cholestatic liver injury.

Clinical disorders that impair bile flow result in retention of bile acids and cholestatic liver injury, characterized by parenchymal cell death, bile duct proliferation, liver inflammation and fibrosis. However, the pathogenic role of bile acids in the development of cholestatic liver injury remains incompletely understood. In this review, we summarize the current understanding of this process focusing on the experimental and clinical evidence for direct effects of bile acids on each major cellular component of the liver: hepatocytes, cholangiocytes, stellate cells and immune cells. During cholestasis bile acids accumulated in the liver, causing oxidative stress and mitochondrial injury in hepatocytes. The stressed hepatocytes respond by releasing inflammatory cytokines through activation of specific signaling pathways and transcription factors. The recruited neutrophils and other immune cells then cause parenchymal cell death. In addition, bile acids also stimulate the proliferation of cholangiocytes and stellate cells that are responsible for bile duct proliferation and liver fibrosis. This review explores the evidence for bile acid involvement in these phenomena. The role of bile acid receptors, TGR5, FXR and the sphingosine-1-phosphate receptor 2 and the inflammasome are also examined. We hope that better understanding of these pathologic effects will facilitate new strategies for treating cholestatic liver injury.

The role of the retinoid receptor, RAR/RXR heterodimer, in liver physiology.

Activated by retinoids, metabolites of vitamin A, the retinoic acid receptors (RARs) and the retinoid X receptors (RXRs) play important roles in a wide variety of biological processes, including embryo development, homeostasis, cell proliferation, differentiation and death. In this review, we summarized the functional roles of nuclear receptor RAR/RXR heterodimers in liver physiology. Specifically, RAR/RXR modulate the synthesis and metabolism of lipids and bile acids in hepatocytes, regulate cholesterol transport in macrophages, and repress fibrogenesis in hepatic stellate cells. We have also listed the specific genes that carry these functions and how RAR/RXR regulate their expression in liver cells, providing a mechanistic view of their roles in liver physiology. Meanwhile, we pointed out many questions regarding the detailed signaling of RAR/RXR in regulating the expression of liver genes, and hope future studies will address these issues.

Hepatic NFAT signaling regulates the expression of inflammatory cytokines in cholestasis.

BACKGROUND & AIMS: The nuclear factor of activated T-cells (NFAT) plays an important role in immune responses by regulating the expression of inflammatory genes. However, it is not known whether NFAT plays any role in the bile acid (BA)-induced hepatic inflammatory response. Thus, we aimed to examine the functional role of NFATc3 in cholestatic liver injury in mice and humans. METHODS: Gene and protein expression and cellular localization were assessed in primary hepatocyte cultures (mouse and human) and cholestatic liver tissues (murine models and patients with primary biliary cholangitis [PBC] or primary sclerosing cholangitis [PSC]) by quantitative PCR, western blot and immunohistochemistry. Specific NFAT inhibitors were used in vivo and in vitro. Gene reporter assays and ChIP-PCR were used to determine promoter activity. RESULTS: NFAT isoforms c1 and c3 were expressed in human and mouse hepatocytes. When treated with cholestatic levels of BAs, nuclear translocation of NFATc3 was increased in both human and mouse hepatocytes and was associated with elevated mRNA levels of IL-8, CXCL2, and CXCL10 in these cells. Blocking NFAT activation with pathway-specific inhibitors or knocking down Nfatc3 expression significantly decreased BA-driven induction of these cytokines in mouse hepatocytes. Nuclear expression of NFATc3/Nfatc3 protein was increased in cholestatic livers, both in mouse models (bile duct ligation or Abcb4-/- mice) and in patients with PBC and PSC in association with elevated tissue levels of Cxcl2 (mice) or IL-8 (humans). Gene reporter assays and ChIP-PCR demonstrated that the NFAT response element in the IL-8 promoter played a key role in BA-induced human IL-8 expression. Finally, blocking NFAT activation in vivo in Abcb4-/- mice reduced cholestatic liver injury. CONCLUSIONS: NFAT plays an important role in BA-stimulated hepatic cytokine expression in cholestasis. Blocking hepatic NFAT activation may reduce cholestatic liver injury in humans. LAY SUMMARY: Bile acid induces liver injury by stimulating the expression of inflammatory genes in hepatocytes through activation of the transcription factor NFAT. Blocking this activation in vitro (in hepatocyte cultures) and in vivo (in cholestatic mice) decreased the expression of inflammatory genes and reduced liver injury.

A Positive Feedback Loop of TET3 and TGF-ß1 Promotes Liver Fibrosis.

Pathological activation of TGF-ß signaling is universal in fibrosis. Aberrant TGF-ß signaling in conjunction with transdifferentiation of hepatic stellate cells (HSCs) into fibrogenic myofibroblasts plays a central role in liver fibrosis. Here we report that the DNA demethylase TET3 is anomalously upregulated in fibrotic livers in both humans and mice. We demonstrate that in human HSCs, TET3 promotes profibrotic gene expression by upregulation of multiple key TGF-ß pathway genes, including TGFB1. TET3 binds to target gene promoters, inducing demethylation, which in turn facilitates chromatin remodeling and transcription. We also reveal a positive feedback loop between TGF-ß1 and TET3 in both HSCs and hepatocytes. Furthermore, TET3 knockdown ameliorates liver fibrosis in mice. Our results uncover a TET3/TGF-ß1 positive feedback loop as a crucial determinant of liver fibrosis and suggest that inhibiting TET3 may represent a therapeutic strategy for liver fibrosis and perhaps other fibrotic diseases.

Inflammasome Is Activated in the Liver of Cholestatic Patients and Aggravates Hepatic Injury in Bile Duct-Ligated Mouse.

BACKGROUND & AIMS: Inflammation plays an important role in the pathogenesis of cholestatic liver injury, but it is unclear whether the inflammasome is involved and is the objective of this study. METHODS: Gene expression was analyzed in the livers of patients with primary biliary cholangitis (n = 15) and primary sclerosing cholangitis (n = 15). Bile duct ligation (BDL) or sham operation was performed in wild-type (WT) and Caspase-1-/- (Casp1-/-) mice for 7 days. Mouse hepatocytes and macrophages were treated with bile acids. RESULTS: Caspase-1, NLRP1, NLRP3 and IL-1ß were significantly increased in the livers of cholestatic patients when compared to healthy control subjects (n = 9). Significantly higher levels of plasma IL-1ß (826 vs 345 pg/ml), ALT (674 vs 482 U/L) and ALP (900 vs 622 U/L) were seen in WT BDL mice compared to Casp1-/- BDL mice. Caspase-1 cleavage was found only in WT BDL livers. Assessment of liver histology indicated more fibrosis in Casp1-/- BDL mice than in WT BDL mice, confirmed by analyses of liver hydroxyproline content and the expression of fibrotic genes. Profiling of immune cells revealed that there were more macrophages in Casp1-/- BDL livers than in WT BDL livers. Further macrophage phenotype characterization indicated that Casp1-/- BDL livers had more M2 anti-inflammatory macrophages evidenced by more CD206 positive cells and higher expression of IL-4, CD163, Fizz1 and IL-33. When mouse hepatocytes and peritoneal macrophages were exposed to cholestatic levels of major endogenous bile acids (300µM TCA), neither IL-1ß induction nor procaspase-1 cleavage were detected. CONCLUSIONS: The inflammasome exacerbates cholestatic liver injury, but bile acids do not directly activate the inflammasome.

Cytoprotective Effect of Antioxidant Pentapeptides from the Protein Hydrolysate of Swim Bladders of Miiuy Croaker (Miichthys miiuy) against H2O2-Mediated Human Umbilical Vein Endothelial Cell (HUVEC) Injury.

In our previous research, ten antioxidant pentapeptides including FYKWP, FTGMD, GFEPY, YLPYA, FPPYERRQ, GFYAA, FSGLR, FPYLRH, VPDDD, and GIEWA were identified from the hydrolysate of miiuy croaker (Miichthys miiuy) swim bladder. In this work, their protective function on H2O2-induced oxidative damage to human umbilical vein endothelial cells (HUVECs) was studied. Results indicated that there was no significant difference in the HUVEC viability between the normal group and the treated groups with the 10 pentapeptides at the concentration of 100 µM for 24 h (p < 0.05). Furthermore, FPYLRH of 100 µg/mL extremely significantly (p < 0.001) increased the viability (80.58% ± 5.01%) of HUVECs with H2O2-induced oxidative damage compared with that of the model group. The protective mechanism indicated that FPYLRH could extremely significantly (p < 0.001) increase the levels of superoxide dismutase (SOD) (211.36 ± 8.29 U/mg prot) and GSH-Px (53.06 ± 2.34 U/mg prot) and decrease the contents of reactive oxygen species (ROS) (139.1 ± 11.8% of control), malondialdehyde (MDA) (13.66 ± 0.71 nM/mg), and nitric oxide (NO) (4.36 ± 0.32 µM/L) at the concentration of 100 µM in HUVECs with H2O2-induced oxidative damage compared with those of the model group. In addition, FPYLRH dose-dependently protected DNA in oxidative damage HUVECs model. These results suggested that FPYLRH could significantly attenuate the H2O2-induced stress injury in HUVECs and might be used as a potential natural antioxidant in the functional food industries.

A novel ASBT inhibitor, IMB17-15, repressed nonalcoholic fatty liver disease development in high-fat diet-fed Syrian golden hamsters.

The manipulation of bile acid (BA) homeostasis by blocking the ileal apical Na+-dependent bile salt transporter (ASBT/SLC10A2) may have therapeutic effects in nonalcoholic fatty liver disease. We developed a novel ASBT inhibitor, an N-(3,4-o-dichlorophenyl)-2-(3-trifluoromethoxy) benzamide derivative referred to as IMB17-15, and investigated its therapeutic effects and the molecular mechanisms underlying the effects. Syrian golden hamsters were challenged with high-fat diet (HFD) to induce NAFLD and were subsequently administered 400 mg/kg IMB17-15 by gavage daily for 21 days. Serum, liver, and fecal samples were collected for further analysis. Plasma concentration-time profiles of IMB17-15 were also constructed. The human hepatocyte cell line HL-7702 was treated with Oleic acid (OA) with or without IMB17-15. Western blotting and real-time PCR were used to study the molecular mechanisms of IMB17-15. We found that IMB17-15 inhibited ASBT and subsequently suppressed ileal farnesoid X receptor (FXR) and FXR-activated fibroblast growth factor15/19 (FGF15/19) expression, which reduced the hepatic phosphorylated extracellular regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) levels and upregulated the cholesterol 7α-hydroxylase (CYP7A1) activity. Additionally, IMB17-15 stimulated adenosine monophosphate (AMP)-activated protein kinase (AMPKα) phosphorylation and enhanced peroxisome proliferator activated receptor α (PPARα) expression and thus promoted triglyceride (TG) oxidation and high-density lipoprotein cholesterol (HDL-c) metabolism through an ASBT-independent mechanism. In conclusion, a novel ASBT inhibitor known as IMB17-15 protected hamsters against HFD-induced NFALD by manipulating BA and lipid homeostasis. IMB17-15 also reduced lipid deposition in human hepatic cell lines, indicating that it may be useful as a therapy for NAFLD patients.

H19 Is Expressed in Hybrid Hepatocyte Nuclear Factor 4α+ Periportal Hepatocytes but Not Cytokeratin 19+ Cholangiocytes in Cholestatic Livers.

Long noncoding RNA (lncRNA) H19 is abundantly expressed in fetal liver. Its expression is significantly diminished in adult healthy liver but is re-induced in chronic liver diseases, including cholestasis. In this study, we developed a new method with combined in situ hybridization (ISH) and immunofluorescence (IF) colabeling to establish an H19 expression profile with both parenchymal and nonparenchymal cell-specific markers in the livers of cholestatic mouse models and patients with cholestasis. H19RNA+ cells showed no colocalization with biliary epithelial cell marker cytokeratin 19 (CK19)+ cholangiocytes but were immediately adjacent to biliary structures in bile duct ligation (BDL), 3,5-diethoxycarbony1-1,4-dihydrocollidine (DDC), and multidrug-resistant gene 2 knockout ( Mdr2 -/- ) mouse models and in human primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC) liver specimens. In contrast, double-positive H19RNA+/sex-determining region Y (SRY)-box 9 (SOX9)+ ductal progenitor cells, H19RNA+/hepatocyte nuclear factor 4α (HNF4α)+ hepatocytes, H19RNA+/F4/80+ Kupffer cells, HNF4α+/SOX9+ hybrid hepatocytes, as well as triple-positive H19 RNA+/HNF4α+/SOX9+ periportal hepatocytes were identified. In addition, H19 RNA could not be detected in mesenchymal cell marker desmin+ cells. Furthermore, H19 RNA was predominately detected in cytoplasm with a small amount at the interspace with neighboring cells. Conclusion: H19RNA is localized in HNF4α+ periportal hepatocytes, SOX9+ ductal progenitor cells, and F4/80+ Kupffer cells but not in CK19+ cholangiocytes and desmin+ stellate cells in cholestatic livers.

Deacetylation of NAT10 by Sirt1 promotes the transition from rRNA biogenesis to autophagy upon energy stress.

Anabolism and catabolism are tightly regulated according to the cellular energy supply. Upon energy stress, ribosomal RNA (rRNA) biogenesis is inhibited, and autophagy is induced. However, the mechanism linking rRNA biogenesis and autophagy is unclear. Here, we demonstrate that the nucleolar protein NAT10 plays a role in the transition between rRNA biogenesis and autophagy. Under normal conditions, NAT10 is acetylated to activate rRNA biogenesis and inhibit autophagy induction. Mechanistic studies demonstrate that NAT10 binds to and acetylates the autophagy regulator Che-1 at K228 to suppress the Che-1-mediated transcriptional activation of downstream genes Redd1 and Deptor under adequate energy supply conditions. Upon energy stress, NAT10 is deacetylated by Sirt1, leading to suppression of NAT10-activated rRNA biogenesis. In addition, deacetylation of NAT10 abolishes the NAT10-mediated transcriptional repression of Che-1, leading to the release of autophagy inhibition. Collectively, we demonstrate that the acetylation status of NAT10 is important for the anabolism-catabolism transition in response to energy stress, providing a novel mechanism by which nucleolar proteins control rRNA synthesis and autophagy in response to the cellular energy supply.

Solute Carrier Organic Anion Transporter Family Member 3A1 Is a Bile Acid Efflux Transporter in Cholestasis.

BACKGROUND & AIMS: Bile acid transporters maintain bile acid homeostasis. Little is known about the functions of some transporters in cholestasis or their regulatory mechanism. We investigated the hepatic expression of solute carrier organic anion transporter family member 3A1 (SLCO3A1, also called OATP3A1) and assessed its functions during development of cholestasis. METHODS: We measured levels of OATP3A1 protein and messenger RNA and localized the protein in liver tissues from 22 patients with cholestasis and 21 patients without cholestasis, using real-time quantitative polymerase chain reaction, immunoblot, and immunofluorescence analyses. We performed experiments with Slco3a1-knockout and C57BL/6J (control) mice. Mice and Sprague-Dawley rats underwent bile duct ligation (BDL) or a sham operation. Some mice were placed on a 1% cholic acid (CA) diet to induce cholestasis or on a control diet. Serum and liver tissues were collected and analyzed; hepatic levels of bile acids and 7-α-C4 were measured using liquid chromatography/mass spectrometry. Human primary hepatocytes and hepatoma (PLC/PRF/5) cell lines were used to study mechanisms that regulate OATP3A1 expression and transport. RESULTS: Hepatic levels of OATP3A1 messenger RNA and protein were significantly increased in liver tissues from patients with cholestasis and from rodents with BDL or 1% CA diet-induced cholestasis. Levels of fibroblast growth factor 19 (FGF19, FGF15 in rodents) were also increased in liver tissues from patients and rodents with cholestasis. FGF19 signaling activated the Sp1 transcription factor and nuclear factor κB to increase expression of OATP3A1 in hepatocytes; we found binding sites for these factors in the SLCO3A1 promoter. Slco3a1-knockout mice had shorter survival times and increased hepatic levels of bile acid, and they developed more liver injury after the 1% CA diet or BDL than control mice. In hepatoma cell lines, we found OATP3A1 to take prostaglandin E2 and thyroxine into cells and efflux bile acids. CONCLUSIONS: We found levels of OATP3A1 to be increased in cholestatic liver tissues from patients and rodents compared with healthy liver tissues. We show that OATP3A1 functions as a bile acid efflux transporter that is up-regulated as an adaptive response to cholestasis.