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. 2017 Apr 26;161(1):34–47. doi: 10.1093/toxsci/kfx078

Sortilin 1 Loss-of-Function Protects Against Cholestatic Liver Injury by Attenuating Hepatic Bile Acid Accumulation in Bile Duct Ligated Mice

Jibiao Li 1,#, Benjamin L Woolbright 1,#, Wen Zhao 1, Yifeng Wang 1, David Matye 1, Bruno Hagenbuch 1, Hartmut Jaeschke 1, Tiangang Li 1,✉
PMCID: PMC5837720  PMID: 28453831

Abstract

Sortilin 1 (Sort1) is an intracellular trafficking receptor that mediates protein sorting in the endocytic or secretory pathways. Recent studies revealed a role of Sort1 in the regulation of cholesterol and bile acid (BA) metabolism. This study further investigated the role of Sort1 in modulating BA detoxification and cholestatic liver injury in bile duct ligated mice. We found that Sort1 knockout (KO) mice had attenuated liver injury 24 h after bile duct ligation (BDL), which was mainly attributed to less bile infarct formation. Sham-operated Sort1 KO mice had about 20% larger BA pool size than sham-operated wildtype (WT) mice, but 24 h after BDL Sort1 KO mice had significantly attenuated hepatic BA accumulation and smaller BA pool size. After 14 days BDL, Sort1 KO mice showed significantly lower hepatic BA concentration and reduced expression of inflammatory and fibrotic marker genes, but similar degree of liver fibrosis compared with WT mice. Unbiased quantitative proteomics revealed that Sort1 KO mice had increased hepatic BA sulfotransferase 2A1, but unaltered phase-I BA metabolizing cytochrome P450s or phase-III BA efflux transporters. Consistently, Sort1 KO mice showed elevated plasma sulfated taurocholate after BDL. Finally, we found that liver Sort1 was repressed after BDL, which may be due to BA activation of farnesoid x receptor. In conclusion, we report a role of Sort1 in the regulation of hepatic BA detoxification and cholestatic liver injury in mice. The mechanisms underlying increased hepatic BA elimination in Sort1 KO mice after BDL require further investigation.

Keywords: cholestasis, bile duct ligation, CYP7A1, FXR, sulfotransferase, fibrosis

Disrupted bile flow out of the liver causes cholestasis, a pathological condition where accumulation of bile acids (BAs) and elevated biliary pressure cause bile duct rupture, leading to liver inflammation and injury (Fickert and Stieger, 2014; Kim et al., 2000; Woolbright and Jaeschke, 2016). Chronic cholestasis may be associated with the development of liver fibrosis and cirrhosis (Hirschfield et al., 2010; Kisseleva and Brenner, 2011). The liver responds to cholestasis by down-regulating BA synthesis genes and upregulating genes involved in phase-I BA metabolism, phase-II BA conjugation and phase-III BA excretion. However, these adaptive responses are usually not sufficient to fully maintain BA transport function, and chronic cholestasis was associated with reduced expression and function of some BA uptake and efflux transporters (Trauner and Boyer, 2003). The BA-activated nuclear receptor farnesoid x receptor (FXR) plays important roles in these adaptive responses in the liver (Li and Chiang, 2014). Studies have shown that BA activation of FXR inhibits the cholesterol 7α-hydroxylase (CYP7A1) to decrease BA synthesis (Goodwin et al., 2000; Inagaki et al., 2005; Li et al., 2006) and induces the canalicular BA efflux transporter bile salt export pump (BSEP) (Ananthanarayanan et al., 2001; Zollner et al., 2003) in both human liver cells and in mice. Bile acids have also been shown to inhibit the basolateral BA uptake transporter sodium/taurocholate cotransporting polypeptide (NTCP) in rat hepatocytes (Denson et al., 2001). The FXR agonist obeticholic acid was recently approved in the United States for the treatment of primary biliary cholangitis (Hirschfield et al., 2015).

Sortilin 1 (Sort1) is a single trans-membrane sorting receptor that mediates the vesicle trafficking in various cell types (Hermey, 2009). The luminal domain of Sort1 interacts with “cargo” proteins while the cytoplasmic domain of Sort1 recruits adaptor proteins to the trafficking vesicles. The major function of Sort1 is to transport various proteins to the endocytic compartments. Sort1 interacts with numerous functionally unrelated proteins and has been implicated in the regulation of various cellular pathways in distinct cell types (Lin et al., 1997; Mazella et al., 1998; Morris et al., 1998; Ni and Morales, 2006; Nielsen et al., 1999). Recently, Sort1 has been functionally implicated in the development of dyslipidemia, atherosclerosis and fatty liver disease, and studies have shown that Sort1 interacts with and regulates the trafficking of factors involved in hepatic and plasma cholesterol and lipid metabolism (Bi et al., 2013; Kjolby et al., 2010; Li et al., 2014, 2017; Musunuru et al., 2010). Bile acid metabolism is tightly linked to cholesterol homeostasis (Li and Chiang, 2014). Interestingly, Sort1 knockout (KO) mice also showed altered hepatic BA synthesis (Li et al., 2017). Furthermore, Sort1 loss-of-function in macrophages was shown to prevent macrophage activation and cytokine production, suggesting a role of Sort1 in modulating inflammatory response (Mortensen et al., 2014; Patel et al., 2015). These recent findings promoted us to investigate the possible role of Sort1 in modulating hepatic BA metabolism and inflammation in a bile duct ligated mouse model of obstructive cholestasis. Our results showed that Sort1 KO mice were protected against cholestatic liver injury after bile duct ligation (BDL), which may be attributed to decreased hepatic BA accumulation and bile infarct formation.

MATERIALS AND METHODS

Reagents

AntiSORT1 (ab16640), antiSULT2A1 (ab38416), and antiACTIN (ab8226) were purchased from Abcam (Cambridge, Massachusetts). AntiSULT1A1 (AF5546) was purchased from RnD Systems (Minneapolis, Minnesota). AntiHISTONE3 (No. 9715) and antiGAPDH (No. 2118) were purchased from Cell Signaling Technology (Danvers, Massachusetts). Cholic acid and GW4064 were purchased from Sigma (St Louis, Missouri). The alanine aminotransferase (ALT) assay kit was purchased from Pointe Scientific (Canton, Michigan). Bilirubin assay kit and BA assay kit were purchased from Diazyme Laboratories (Poway, California). Phosphatidylcholine assay kit and hydroxyproline assay kit were purchased from Biovision (Milpitas, Caliornia). Taurocholic acid 3-sulfate and taurolithocholic acid 3-sulfate (T-LCA 3-sulfate) were purchased from Cayman Chemical (Ann Arbor, Michigan).

Mice and experimental procedures

Global Sort1 knockout mice (Sort1 KO) on 129/SvEv/C57BL/6 mixed genetic background were obtained from Taconic Biosciences Inc (Hudson, New York). The Sort1 gene (accession: NM_019972) was silenced by inserting a stop codon into the second intron of the Sort1 gene through gene trapping technology. Mice used in this study have been backcrossed to C57BL/6J (The Jackson Lab, Bar Harbor, Maine) for 10 generations. Male age-matched (10–14 weeks of age) Sort1 KO mice and littermate wildtype (WT) mice were used in this study. Mice were housed under normal light-dark cycle. BDL and sham operation were performed as previously described in Woolbright et al. (2013). C57BL/6J WT male mice (10 weeks of age) were fed a 0.5% cholate containing chow diet for 7 days. C57BL/6J WT male mice (10 weeks of age) were administered via oral gavage vehicle (10% 2-hydroxypropyl-β-cyclodextrin) or GW4064 (30 mg/kg, twice a day) for 7 days. All animal protocols were approved by the Institutional Animal Care and Use Committee.

Hepatic neutrophil immunohistochemistry and bile infarct quantification

Liver tissue sections were used for immunohistochemistry for the neutrophil specific marker Ly6B2 as previously described (Woolbright et al., 2013). Bile infarcts were quantified by assessing the number of infarcts in 10 high powered fields. Infarcts were defined as areas of frank necrosis with an obvious inflammatory infiltrate. ImageJ software was used to calculate the relative area of each individual infarct. Five random infarcts were selected on each slide. These analyses were performed by BLW in a blinded fashion.

Measurement of tissue and plasma BA concentration and gallbladder BA composition

Tissue BAs were extracted in 90% ethanol at 55 °C overnight. Gallbladder bile was diluted in 90% ethanol. Serum was used directly. Total BAs were measured with a BA assay kit. Bile acid composition was measured with mass spectrometry as described previously in Li et al. (2012).

Western blotting

Liver tissues were homogenized in ice cold 1× RIPA buffer containing 1% SDS and protease inhibitors, and incubated for 1 h on ice followed by brief sonication. Supernatant after centrifugation was used for SDS-PAGE and immunoblotting. Densitometry was performed with ImageJ software.

Quantitative proteomics

This was performed by MS Bioworks (Ann Arbor, Michigan). Liver lysates containing the same amount of total protein from 16 h fasted male WT and Sort1 KO mice were pooled (n = 6/group). The samples were digested with trypsin and then analyzed by nano LC-MS/MS with a Waters NanoAcquity HPLC system interfaced to a ThermoFisher Q Exactive mass spectrometer. Samples were loaded on a trapping column and eluted over a 75 μm analytical column at 350 nl/min; both columns were packed with Jupiter Proteoresin (Phenomenex, Torrance, California). The mass spectrometer was operated in data-dependent mode with MS and MS/MS performed at 70 000 and 17 500 FWHM, respectively. For data processing, a local copy of Mascot was used with the following parameters: Enzyme: Trypsin/P; Database: SwissProt Mouse (Appended with common contaminants, reversed, and concatenated); Fixed modification: Carbamidomethyl; Variable modifications: Oxidation, Acetyl (N-term), Deamidation (N/Q), Pyro-Glu (N-term Q); Mass values: Monoisotopic; Peptide Mass Tolerance: 10 ppm; Fragment Mass Tolerance: 0.02 Da; Max Missed Cleavages: 2. Mascot DAT files were passed into the Scaffold software for validation, filtering and to create a non-redundant list per sample. Data were filtered using at 1% protein and peptide FDR and requiring at least 2 unique peptides per protein. Results were expressed as total spectral count (SpC) for each protein.

Real-time PCR

Total RNA was isolated with tri-reagent (Thermo Fisher Scientific, Waltham, Massachusetts). SYBR master mix (Bio-Rad Laboratory, Hercules, California) was used in real-time PCR. Amplification of 18S was used for normalization. Relative mRNA expression was calculated using the comparative CT (Ct) method and expressed as 2−ΔΔCt.

Coimmunoprecipitation

Ad-Null were purchased from Vector Biolabs (Philadelphia, Pennsylvania). Ad-hSort1-FLAG was generated as described previously in Li et al. (2015). C57BL/6J mice were injected with 1 × 109 pfu/mouse Ad-Null or Ad-hSort1-FLAG via tail vein (3 mice/group). Livers were collected 5 days postinjection. Pooled liver lysates were used for coimmunoprecipitation with antiFLAG (M2) antibody conjugated to magnetic beads (Sigma). Precipitated proteins were used for SDS-PAGE and Western blotting.

Statistical analysis

Results were expressed as mean ± SE. Statistical analysis was performed by either student’s t test or ANOVA and Tukey post hoc tests with the GraphPad Prism 7 software. A P < .05 was considered statistically significant.

RESULTS

Sort1 KO Mice Showed Attenuated Liver Injury After BDL

We first performed sham operation and short-term BDL in mice for 6 and 24 h. After 6-h BDL, plasma levels of ALT and bilirubin already were increased significantly compared with sham controls (Figs. 1A and B). Sort1 KO mice showed similar levels of plasma ALT and lower plasma bilirubin compared with WT mice 6 h after BDL (Figs. 1A and B). After 24-h BDL, plasma ALT and bilirubin levels continued to increase in WT mice, but were significantly lower in Sort1 KO mice (Figs. 1A and B). Consistently, BDL for 6 h and 24 h significantly increased hepatic cytokine mRNA levels in WT mice (Figs. 1C–F). Hepatic cytokine mRNA expressions were significantly lower in Sort1 KO mice than WT mice 24 h after BDL (Figs. 1C–F). These results suggest reduced liver injury in Sort1 KO mice after 24-h BDL.

Figure 1.

Figure 1

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Sort1 KO mice showed attenuated liver injury after 24-h BDL. WT and Sort1 KO mice were subjected to 24 h sham operation or 6- and 24-h BDL. A, Plasma ALT levels. B, Plasma direct bilirubin. C–F, Real-time PCR analysis of liver cytokine mRNA expression. TNFα, tumor necrosis factorα; IL1β, interleukin-1β; IL-6, interleukin-6; MCP-1, monocyte chemoattractant protein-1. All results were expressed as mean ± SE (n = 4–6). “#”P < .05, versus sham of the same genotype. “*”P < .05 versus WT of the same experimental condition.

Sort1 KO Mice Showed Reduced Hepatic Bile Acid Accumulation After BDL

Histological analysis of liver sections found that the number of bile infarcts was similar in WT and Sort1 KO mice at 6 h after BDL, but was significantly less in Sort1 KO mice at 24 h after BDL (Figure 2A). The average infarct size was similar between WT mice and Sort1 KO mice after 24-h BDL (not shown). Because fewer bile infarcts indicated reduced hepatic BA accumulation and attenuated bile duct injury, we therefore analyzed BA levels in WT and Sort1 KO mice. In sham-operated groups, WT and Sort1 KO mice had similar hepatic BA levels (Figure 2B), but Sort1 KO mice tended to have slightly more BAs in the gallbladder (P = .07) and in the intestine (P = .14) (Figs. 2C and D), which resulted in an approximately 20% larger BA pool size (P < .05) (Figure 2E). Upon 6-h BDL, the total BA pool was reduced to the same size in WT and Sort1 KO mice (Figure 2E), but Sort1 KO mice had approximately 1.0 μmol more BAs in the gallbladder and approximately 0.8 μmol less BAs in the intestine compared with WT mice (Figs. 2C and D), suggesting that Sort1 KO mice secreted more BAs into the bile during the 6 h period after BDL. After 24-h BDL, hepatic BA levels continued to increase in WT mice, but did not further increase in Sort1 KO mice, resulting in significantly lower hepatic BAs in Sort1 KO mice (Figure 2B). In addition, BA levels in the gallbladder, small intestine, plasma and therefore the total BA pool size were all significantly lower in Sort1 KO mice compared with WT mice after 24-h BDL (Figs. 2C–F). The level of gallbladder phospholipids (PLs), which play a critical role in attenuating BA toxicity in the bile, was modestly but significantly higher in sham-operated and 6 h bile duct ligated Sort1 KO mice, but not in 24-h BDL Sort1 KO mice (Figure 2G). The biliary BA compositions were similar in chow-fed WT and Sort1 KO mice (Figure 2H). However, biliary BA compositions in Sort1 KO mice after 6-h BDL showed a higher percentage of taurocholic acid (T-CA) and taurodeoxycholic acid and a lower percentage of tauromuricholic acid (T-MCA) than WT mice (Figure 2I). The mRNA expression of hepatic sterol 12α-hydroxylase (CYP8B1), which can affect the ratio of cholic acid (CA) and muricholic acid (MCA), was not different between WT and Sort1 KO mice after 6-h BDL (Figure 3B). The underlying causes of altered BA composition in 6 h bile duct ligated Sort1 KO mice remain to be determined by future studies.

Figure 2.

Figure 2

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Sort1 KO mice showed decreased hepatic BA accumulation after 24-h BDL. A, Left panel: Representative H&E staining of liver sections. Bile infarcts are indicated by arrows. Scale bar: 100 µm. Right panel, quantification of bile infarct numbers in mice (10 HPF/mouse, 4–6 mice/group). HPF, high power field. “*”P < .05, versus WT at the same time point. B–F, Tissue and plasma BA. BA pool was calculated as the sum of total BA amount in the liver, gallbladder and intestine. G, Gallbladder PLs. H and I, Bile acid composition in the bile of chow-fed mice and mice after 6-h BDL. T, taurine-conjugated; MCA, muricholic acid; CDCA, chenodeoxycholic acid; CA, cholic acid; DCA, deoxycholic acid; LCA, lithocholic acid. T-MCA represents the sum of T-αMCA, T-βMCA, and T-ωMCA. All results were expressed as mean ± SE. (n = 4–6). “#”P < .05, versus sham of the same genotype. “*”P < .05, versus WT of the same experimental condition.

Figure 3.

Figure 3

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Hepatic mRNA expression in mice after sham operation and BDL. WT and Sort1 KO mice were subjected to 24 h sham operation or 6 h and 24-h BDL. A–I, Real-time PCR analysis of liver mRNA expression. All results were expressed as mean ± SE (n = 4–6). “#” P < .05, versus sham of the same genotype. “*”P < .05, versus WT of the same experimental condition. CYP7A1, cholesterol 7α-hydroxylase; CYP8B1, sterol 12α-hydroxylase; BSEP, bile salt export pump; MRP, multi-drug resistance-associated protein; NTCP, sodium-taurocholate cotransporting polypeptide; OST, organic solute transporter; SHP, small heterodimer partner.

Analysis of hepatic gene expression showed that the mRNA of BA repressed genes CYP7A1, CYP8B1 and NTCP gradually decreased after BDL, and such repression was significantly less in the Sort1 KO mice after 24-h BDL (Figs. 3A–C). In contrast, the BA/FXR induced genes small heterodimer partner (SHP) and organic solute transporter β (Frankenberg et al., 2006) were gradually induced after BDL, but significantly less in Sort1 KO mice after 24-h BDL (Figs. 3D and E). This mRNA expression pattern reflected less hepatic BA accumulation and reduced FXR activation in Sort1 KO mice after 24-h BDL. The mRNA levels of BSEP, the bilirubin and BA transporter multidrug resistance-associated protein 2 (MRP2) and the basolateral BA efflux transporters MRP3 and MRP4 were all similar in sham-operated and bile duct ligated WT and Sort1 KO mice, and were all down-regulated after BDL (Figs. 3F–I). Taken together, these results suggest that in response to obstructed bile flow, Sort1 KO mice may have enhanced BA elimination from the liver, which led to rapidly decreased BA pool, attenuated hepatic BA build-up, fewer infarcts and reduced cholestatic liver injury.

Sort1 KO Mice Showed Reduced Neutrophil Infiltration After BDL

Neutrophil infiltration plays an important role in inflammatory-mediated cell death and liver injury in cholestasis after initial BA insults (Woolbright and Jaeschke, 2012). We found that neutrophil counts were similar between WT and Sort1 KO mice 6 h after BDL (Figure 4A). Neutrophil counts were further increased in WT mice after 24-h BDL compared with WT mice after 6-h BDL, but neutrophil counts were significantly lower in Sort1 KO mice than WT mice after 24-h BDL (Figure 4A). These results correlated with lower mRNA levels of neutrophil chemokines mouse keratinocyte-derived chemokine, macrophage inflammatory protein 2 (MIP-2) and intracellular adhesion molecule-1 (ICAM-1) in these mice (Figs. 4B–D). The number of neutrophils recruited to individual bile infarct appeared to be similar between WT and Sort1 KO mice (Figure 4A). These results are consistent with reduced hepatic BA accumulation and less bile infarcts in these mice.

Figure 4.

Figure 4

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Sort1 KO mice showed decreased hepatic neutrophil infiltration after 24-h BDL. A, Representative images of hepatic neutrophils after 6 or 24-h BDL. Lower left panel, quantification of neutrophils in mice (10 HPF/mouse, 4 mice/group). Lower right panel, average neutrophils per infarct (5 infarcts/mouse; 4 mice/group). PMN, polymorphonuclear cell. B–D, Real-time PCR analysis of liver mRNA expression. mKC, mouse keratinocyte-derived chemokine; MIP-2, macrophage inflammatory protein 2; ICAM-1, intracellular adhesion molecule-1 (n = 4–6). All results were expressed as mean ± SE. “#”P < .05, versus sham of the same genotype. “*”P < .05, versus WT of the same experimental condition.

Sort1 KO Mice Had Elevated Hepatic Bile Salt Sulfotransferase SULT2A1 and Plasma T-CA 3-Sulfate

Due to limited existing knowledge on the role of Sort1 in the regulation of BA metabolism, we took an unbiased approach and performed quantitative proteomics using pooled liver lysates of chow-fed WT and Sort1 KO mice. Naive mice were used for this analysis to avoid secondary changes caused by differential accumulation of hepatic BAs in WT and Sort1 KO mice after BDL. Among approximately 2800 proteins detected, we found similar protein abundance of BA transporters BSEP, NTCP, PL transporter multidrug resistance protein 3 (MDR3) and MRP2 in the livers of WT and Sort1 KO mice (<1.5-fold change) (Table 1). The organic anion transporting polypeptide (OATP) isoforms that participate in hepatic uptake of BAs and bilirubin were also similar in WT and Sort1 KO mice (Lu et al., 2008; van de Steeg et al., 2010). In contrast, the major phase-II bile salt sulfotransferase SULT2A1 (Radominska et al., 1990) as well as 2 SULT1 isoforms SULT1A1 and SULT1D1 were higher in Sort1 KO mice (Table 1). In comparison, phase-II UDP-glucuronosyltransferases and phase-I cytochrome p450s, especially the BA metabolizing enzymes CYP3A11 and CYP2B10, were not higher in Sort1 KO mice (Table 1). Western blotting further confirmed increased hepatic SULT2A1 and SULT1A1 protein levels in Sort1 KO mice (Figure 5A). In addition, SULT2A1 protein in Sort1 KO mice was higher after 6-h BDL, but not after 24-h BDL (Figure 5B). Hepatic mRNA of SULT2A1 was increased 6 and 24 h after BDL, but was not significantly higher in Sort1 KO mice (Figure 5C), suggesting that higher SULT2A1 protein levels in Sort1 KO mice were independent of transcriptional changes. SULT1A1 mRNA was also not different between chow-fed WT and Sort1 KO mice (not shown). To determine if higher SULT2A1 resulted in increased BA sulfation in Sort1 KO mice, we measured plasma concentrations of T-CA 3-sulfate and T-LCA 3-sulfate as markers. Plasma T-LCA 3-sulfate could not be detected in sham or BDL groups by our assay method. Plasma T-CA 3-sulfate was only detected in plasma of bile duct ligated mice but not sham-operated mice. After 6-h BDL, plasma T-CA 3-sulfate were similar between WT and Sort1 KO mice (Fig 5D). However, plasma T-CA 3-sulfate was approximately 5-fold higher in Sort1 KO mice after 24-h BDL (Fig 5D). After 6- or 24-h BDL, the T-CA 3-sulfate accounted for approximately 1% or less of total plasma BAs in WT mice (Figure 5E). The percentage of T-CA 3-sulfate was significantly higher in Sort1 KO mice than WT mice 24 h after BDL (Figure 5E). Sort1 has been shown to mediate cellular protein degradation by receptor-ligand interaction (Hermey, 2009). However, coimmunoprecipitation assays failed to detect Sort1 interaction with SULT2A1 in mouse livers (Figure 5F), suggesting that loss of Sort1 affected sulfotransferases via indirect mechanisms yet to be identified.

Table 1.

Hepatic SpCs in WT and Sort1 KO Mice

Protein ID Accession Number WT (SpC) Sort1 KO (SpC) Fold Change
Transporters
 BSEP sp|Q9QY30|ABCBB_MOUSE 193 176 0.91
 ABCB4 (MDR3) sp|P21440|MDR3_MOUSE 108 99 0.92
 ABCC2 (MRP2) sp|Q8VI47|MRP2_MOUSE 168 149 0.89
 NTCP sp|O08705|NTCP_MOUSE 24 29 1.21
 OATP1A1 sp|Q9QXZ6|SO1A1_MOUSE 101 123 1.22
 OATP1B2 sp|Q9JJL3|SO1B2_MOUSE 134 116 0.87
Sulfotransferase
 SULT2A1 sp|P52843|ST2A1_MOUSE 7 13 1.86
 SULT1A1 sp|P52840|ST1A1_MOUSE 43 62 1.44
 SULT1D1 sp|Q3UZZ6|ST1D1_MOUSE 18 33 1.83
UDP-glucuronosyltransferase
 UGT1A1 sp|Q63886|UD11_MOUSE 154 134 0.87
 UGT1A9 sp|Q62452|UD19_MOUSE 53 52 0.98
 UGT2A3 sp|Q8BWQ1|UD2A3_MOUSE 75 70 0.93
 UGT2B17 sp|P17717|UDB17_MOUSE 141 132 0.94
Cytochrome p450
 CYP3A11 sp|Q64459|CP3AB_MOUSE 174 133 0.76
 CYP2B10 sp|P12791|CP2BA_MOUSE 3 3 1.0
 CYP2A5 sp|P20852|CP2A5_MOUSE 63 57 0.90
 CYP2A12 sp|P56593|CP2AC_MOUSE 68 67 0.99
 CYP2C29 sp|Q64458|CP2CT_MOUSE 138 142 1.03
 CYP2C37 sp|P56654|CP237_MOUSE 61 52 0.85
 CYP2C50 sp|Q91X77|CY250_MOUSE 142 121 0.85
 CYP2C54 sp|Q6XVG2|CP254_MOUSE 86 88 1.02
 CYP2D10 sp|P24456|CP2DA_MOUSE 82 75 0.91
House keeping
 ACTIN sp|P63260|ACTG_MOUSE 391 412 1.05
 HISTONE H4 sp|P62806|H4_MOUSE 180 193 1.07
Total number of spectra count 98201 96027
Total number of identified proteins 2886 2801
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Pooled liver lysate of 6 mice per group was used

Figure 5.

Figure 5

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Sort1 KO mice showed higher hepatic SULT2A1 protein abundance and BA sulfation. A and B, Western blot of liver protein expression in chow-fed mice and in mice after 6 or 24-h BDL. The numbers indicate mean band intensity that was normalized to ACTIN. Results were expressed as mean ± SE. A “*” indicates statistical significance. C, Real-time PCR measurement of hepatic SULT2A1 mRNA expression. Results were expressed as mean ± SE (n = 4–6). “#”P < .05, versus sham of the same genotype. D, Plasma T-CA 3-sulfate concentrations. All results were expressed as mean ± SE (n = 4–6). “#”P < .05, versus 6-h BDL of the same genotype. “*”P < .05, versus WT of the same experimental condition. ND, Not detected. E, Plasma T-CA 3-sulfate as percentage of total plasma BAs. All results were expressed as mean ± SE (n = 4–6). “#”P < .05, versus 6-h BDL of the same genotype. “*”P < .05, versus WT of the same experimental condition. F, Coimmunoprecipitation in mouse liver lysates was described in “Materials and Methods”. Western blot was performed with antibody against FLAG, SULT2A1, and SULT1A1. Total liver lysates was used as “INPUT” controls. NS, nonspecific band.

Sort1 KO Mice Showed Lower Hepatic Bile Acid Concentration and Inflammatory and Fibrosis Gene Expression, but Not Fibrosis, 14 Days After BDL

We next investigated if Sort1 KO mice could still be protected against hepatic BA accumulation and possibly the development of liver fibrosis after long-term BDL. After 14-day BDL, the plasma ALT levels in WT mice were significantly lower than the plasma ALT levels in WT mice at 24 h after BDL (Figs. 1A and 6A). Plasma ALT levels were similar between the WT and Sort1 KO mice 14 days after BDL (Figure 6A). Hepatic BA levels, but not plasma BA levels, were still approximately 50% lower in Sort1 KO mice than WT mice 14 days after BDL (Figs. 6B and C). Consistently, hepatic cytokine expressions were significantly lower in Sort1 KO mice than WT mice 14 days after BDL (Figure 6D). Sort1 KO mice showed significantly lower mRNA levels of fibrosis markers tissue inhibitor of metalloproteinases-1 (TIMP1), collagen type-1 α1 (COL1A1), α smooth muscle actin (αSMA), transforming growth factor β1 (TGFβ1), and matrix metallopeptidase 2 (MMP2), MMP9, and MMP13 (Figure 6G). These genes are known to be induced during liver inflammation and fibrosis, and lower expression of these genes in Sort1 KO mice was consistent with lower hepatic BA accumulation and inflammatory cytokines that are known drivers of their expression (Allen et al., 2011; Trautwein et al., 2015). However, liver fibrosis, as evaluated by Sirius red staining and hydroxyproline measurement, was similar in WT and Sort1 KO mice 14 days after BDL (Figs. 6E and F), suggesting that lower hepatic BA accumulation and inflammation in Sort1 KO mice was not sufficient to attenuate the development of liver fibrosis after 14-day BDL. After 14-day BDL, hepatic SULT2A1 was strongly down-regulated to undetectable levels by Western blotting (Figure 6G). SULT2A1 mRNA was markedly induced after 14-day BDL, suggesting SULT2A1 was subjected to a posttranscriptional down-regulation after chronic BDL (Figure 6H).

Figure 6.

Figure 6

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Sort1 KO mice showed attenuated liver BA accumulation and lower inflammatory and fibrosis gene expression 14 days after BDL. A, Plasma ALT in WT and Sort1 KO mice after 14-day BDL. B and C, Liver and plasma BA. D, Real-time PCR measurement of liver mRNA expression. TNFα, tumor necrosis factorα; IL1β, interleukin-1β; IL-6, interleukin-6. TIMP, tissue inhibitor of metalloproteinases-1; COL1A1, collagen type-1 α1; αSMA, α smooth muscle actin; TGFβ1, transforming growth factor β1; MMP, matrix metallopeptidase. E, Representative images of hepatic Sirius red staining. Right panel, quantification of Sirius red positive area (average of 15–20 fields/mouse, 7–9 mice/group). Image analysis was performed with ImageJ software. Scale bar: 100 µm. F, Liver hydroxyproline measurement. G, Western blotting of liver protein. H, Real-time PCR measurement of liver SULT2A1 mRNA. Sham, n = 4; BDL, n = 7–9. All results were expressed as mean ± SE. “#”P < .05, versus sham of the same genotype. “*”, P < .05, versus WT of the same experimental condition.

Hepatic Sort1 Is a Novel Target of FXR and Is Down-Regulated After BDL in Mice

Interestingly, Western blotting revealed that hepatic Sort1 protein was about 50% lower in WT mice after 14-BDL (Figure 6G). Additional analysis showed that both hepatic Sort1 protein and mRNA were decreased after 24-h BDL (Figs. 7A and B). Furthermore, feeding mice a 0.5% CA—containing diet for 7 days reduced both Sort1 mRNA and protein by approximately 50% in the liver (Figs. 7C and D). To determine if this transcriptional repression by BAs was mediated by FXR, we administered a specific FXR agonist GW4064 to mice, which repressed liver Sort1 mRNA and protein by approximately 50% (Figs. 7E and F). As positive controls, CA feeding and GW4064 treatment decreased CYP7A1 mRNA and increased SHP mRNA (Figs. 7D and F). These results suggest that BA-activated FXR plays a role in mediating transcriptional repression of liver Sort1 after BDL. We have conducted further experiments to investigate the molecular mechanism of FXR regulation of Sort1 gene transcription. We cloned a luciferase reporter construct containing approximately 1.5 kb mouse Sort1 gene promoter. However, the Sort1 promoter/luciferase activity was not repressed by 24 h treatment of GW4064 or chenodeoxycholic acid in HepG2 cells cotransfected with FXR and RXR expression plasmids (Supplementary Figure 1). The mechanisms by which BAs and FXR repressed liver Sort1 still require further investigation.

Figure 7.

Figure 7

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Liver Sort1 was down-regulated by BAs in cholestasis. A, Western blotting of liver proteins in 24 h sham and 6- and 24-h BDL WT mice. B, Liver Sort1 mRNA. C and D, Liver protein and mRNA in WT mice fed chow and chow containing 0.5% CA for 7 days. Mice were fasted for 16 h before tissue collection. E and F, Liver protein and mRNA in WT mice administered vehicle (10% 2-hydroxypropyl-β-cyclodextrin) or GW4064 (30 mg/kg, oral gavage, twice a day) for 7 days. All results were expressed as mean ± SE (n = 4–6). “*”P < .05, versus control group (sham, Chow or Vehicle). Western blot band intensity was quantified with ImageJ software and normalized values (to ACTIN) were expressed as mean ± SE.

DISCUSSION

This study reports a new role of Sort1 in the regulation of BA detoxification mechanism after BDL in mice, a model of obstructive cholestasis. Our previous studies have documented that the early necrotic liver injury (bile infarcts) after BDL in mice is not caused by direct BA toxicity because the major murine BAs are not cytotoxic to mouse hepatocytes even at mM concentrations (Allen et al, 2011; Zhang et al., 2012). In contrast, BDL-induced liver injury is caused by an inflammatory response involving mainly neutrophils (Gujral et al., 2003, 2004). The mechanism of neutrophil recruitment after BDL includes an initial build-up of pressure within the biliary system, which leads to localized rupture and leakage of bile into the parenchyma (Fickert et al., 2002). The local exposure of hepatocytes to biliary concentrations of BAs triggers inflammatory mediator production in hepatocytes including the chemokine MIP-2, the adhesion molecule ICAM-1 (Allen et al., 2011) and cleaved osteopontin (Yang et al., 2014). Consequently, this local gradient of neutrophil chemotactic factors recruits neutrophils to cause injury (Gujral et al., 2003, 2004). In our experiments, reduced liver injury in Sort1 KO mice after BDL was mainly manifested as a reduction in the number of bile infarcts. Although the overall number of neutrophils recruited into the liver was reduced, the number of neutrophils per infarct was the same between the Sort1 KO and WT animals, which suggests that the overall inflammatory response was not impaired. Therefore, a reduction in the number of bile infarcts puts the cause of the protection at the level of bile leakage upstream of the inflammatory response. Consistently, the BA levels in the liver, gallbladder, intestine and plasma were rapidly reduced in Sort1 KO mice 24 h after BDL. At this point, much less BAs would be transported into the liver and excreted into the bile, which reduced the biliary pressure and rupture points of the biliary system in Sort1 KO mice. Consequently, this would cause less chemoattractant formation and reduced neutrophil-mediated bile infarcts.

After hepatic bile outflow was blocked by BDL for 6 h, Sort1 KO mice had approximately 70% more BAs in the gallbladder (Figure 2C), which suggests that Sort1 KO mice may have enhanced biliary BA elimination in response to BDL. In addition, biliary PL levels were significantly higher in sham operated and 6-h BDL Sort1 KO mice, which could decrease BA toxicity in the bile. As an adaptive response to cholestasis, BAs can be secreted into the blood circulation via direct hepatocyte basolateral efflux and via cholehepatic shunt after biliary BA uptake by the cholangiocytes (Xia et al., 2006). Bile acids in the blood circulation can be eliminated via the renal excretion route (Li and Chiang, 2014). Consistently, plasma BAs increased by approximately 200-fold while the BA pool size (not including plasma BA) rapidly shrank by approximately 40% in both WT and Sort1 KO mice after 6-h BDL. After 24-h BDL, Sort1 KO mice had even smaller BA pool size and significantly lower plasma BA concentration than WT mice. These results suggest that rapid reduction of BA pool may play a role in preventing bile infarct formation in Sort1 KO mice immediately after BDL. This initial rapid reduction of BA pool, together with down-regulation of hepatic de novo BA synthesis and basolateral and canalicular BA transporters, may provide adaptive protection against BA build-up in the biliary system at later time points after BDL.

So far, there has been no previous report of a role of Sort1 in the regulation of BA metabolism and detoxification. The unbiased quantitative proteomics approach revealed that several phase-II sulfotransferase isoforms, including the major hepatic BA sulfotransferase SULT2A1, were higher in Sort1 KO mice even at the baseline level before BDL. In cholestasis, BA sulfation decreases BA toxicity and promotes BA secretion into the bile via MRP2 and BSEP or into the systemic circulation via MRP3 and MRP4 (Alnouti, 2009). However, it should be noted that sulfated BAs generally accounted for relatively minor portions of total plasma BA despite elevated concentrations after cholestasis (Alnouti, 2009). A previous study showed that sulfated BAs increased initially in response to BDL, but decreased after chronic BDL in mice (Zhang et al., 2012). This is consistent with our results showing that liver SULT2A1 protein was significantly reduced 14 days after BDL (Figure 6G). Furthermore, Sort1 KO mice continued to show lower hepatic BA levels even after liver SULT2A1 became undetectable after 14-day BDL (Figure 7B). Additional mechanisms underlying the lower hepatic BAs need to be investigated by future studies. Our study does not necessarily rule out the possible involvement of phase-III BA transporters in reduced hepatic BA accumulation in Sort1 KO mice because Sort1 deletion may potentially alter their cellular localization without altering their protein abundance. Such studies will require the establishment of suitable in vivo and in vitro cell models and thus are outside the scope of our current study. The BA-activated FXR regulates a network of genes involved in liver BA synthesis and detoxification in cholestasis (Li and Chiang, 2014). Further mechanistic insights into the role of Sort1 in the regulation of BA detoxification in cholestasis may also help understand the significance of the FXR repression of hepatic Sort1, which could be an adaptive response to cholestasis.

Although our article was in preparation, a study reported that Sort1 deficient mice had attenuated ductular reaction and fibrosis after BDL (Hubel et al., 2017). Although the major focuses of the 2 studies were somewhat different, both studies consistently pointed to novel anticholestasis effects of Sort1 loss-of-function in mice. However, some mechanistic aspects in the 2 studies need to be discussed here. As mentioned earlier, the initial trigger of liver injury and inflammatory infiltration after BDL is BA build-up in the biliary system (Calmus and Poupon, 2014; Katz et al., 2011; Kennedy et al., 2014; Woolbright and Jaeschke, 2012). Our study suggested that a reduced BA pool was a potential mechanism underlying the anticholestasis effect of Sort1 KO. However, Sort1 KO and WT mice developed comparable fibrosis 14 days after BDL despite lower hepatic inflammatory and fibrosis gene expression. These data suggest that, at least in our hands, lower hepatic BA accumulation and inflammation in Sort1 KO mice were not sufficient to attenuate 14-day BDL-induced fibrosis, which can also be driven by factors in addition to liver inflammation and thus can sometimes dissociate from liver injury and inflammation (Moon et al., 2009; Seki and Schwabe, 2015). In contrast, Hubel et al. reported reduced hepatic injury and fibrosis in Sort1 KO mice after chronic BDL (Hubel et al., 2017). This study did not focus on changes in BA metabolism, but partially attributed the antifibrotic effect of Sort1 deficiency to reduced acid sphingomyelinase (aSMase) activity. This is based on a previous in vitro finding that Sort1 acted as an alternative receptor in addition to mannose 6-phosphate receptor to deliver aSMase to the lysosome for ceramide production (Ni and Morales, 2006). It is well known that aSMase activity and sphingolipid synthesis increase in response to various cellular stressors and inflammatory cytokines, and increased ceramide signaling in turn promotes inflammation (Bikman and Summers, 2011). This could provide an intriguing mechanism to explain reduced cholestatic liver injury in Sort1 KO mice. Because hepatic BA accumulation and inflammation are expected to induce aSMase activity after BDL (Bikman and Summers, 2011), future studies may need to further define the causal relationship between Sort1 loss-of-function and hepatic aSMase metabolism in cholestasis.

In summary, this study reports new findings that genetic Sort1 deletion resulted in protection against cholestatic liver injury in a BDL mouse model of obstructive cholestasis, which may be attributed to reduced hepatic BA accumulation and infarct formation. Our study found increased hepatic SULT2A1 in Sort1 KO mice, which showed increased plasma levels of sulfated BAs after 24-h BDL. Additional studies are necessary to further delineate the novel role and mechanisms of the Sort1 regulation of BA metabolism and transport in normal physiology and in cholestatic liver diseases.

SUPPLEMENTARY DATA

Supplementary data are available at Toxicological Sciences online.

FUNDING

This work was supported in part by an American Diabetes Association Junior Faculty Award (7-12-JF-35 to T.L.) and the National Institutes of Health (T32 ES007079-26A2 to B.L.W.; 1R01DK102487-01 to T.L.; 1R01DK102142 to H.J.; R01 GM077336 to B.H.; and P20GM103549 and P30GM118247).

Supplementary Material

Supplementary Data
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