1. The American Journal of Pathology
Hepatic Tmem30a Deficiency Causes Intrahepatic Cholestasis by Impairing Expression and Localization of Bile Salt Transporters 
Leiming Liu, Lingling Zhang, Lin Zhang, Fan Yang, Xudong Zhu, Zhongjie Lu, Yeming Yang, Haiqi Lu, Lifeng Feng, Zhuo Wang, Hui Chen, Sheng Yan, Lin Wang, Zhenyu Ju, Hongchuan Jin, Xianjun Zhu 
The American Journal of Pathology 
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Copyright © 2017 American Society for Investigative Pathology Terms and Conditions

2. Figure 1
Hyperbilirubinemia and hypercholanemia in Tmem30a liver-specific knockout (LKO) mice. A: Tmem30a LKO mice showed strong yellow pigmentation in their sera. B: Serum total bile acid (TBA) levels were dramatically elevated in Tmem30a LKO mice compared with the littermate controls. C: Serum total bilirubin (TBIL), direct bilirubin (DBIL), and indirect bilirubin (IBIL) levels were dramatically increased in Tmem30a LKO mice compared with their littermate controls. D: Serum γ-glutamyl transferase (GGT) levels were comparable between the two groups. E–G: Serum TBA (217.2 ± 53.1 μmol/L) (E), DBIL (18.7 ± 5.5 μmol/L) (F), and TBIL (23.7 ± 6.3 μmol/L) (G) levels peaked at 4 months of age, and their increased levels decreased progressively with age in Tmem30a LKO mice. All mice were at 2 months of age except for special indicationData are expressed as means ± SEM. n = 10 Tmem30a LKO mice (B); n = 11 littermate control mice (B and C); n = 12 Tmem30a LKO mice (C); n = 6 to 8 mice in both groups (D); n = 14 TBA samples (E); n = 13 samples (F), n = 12 samples (G); n ≥ 6 Tmem30a LKO mice per month (E–G). ∗P < 0.05, ∗∗P < 0.01 (t-test). WT, wild-type.
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3. Figure 2
Identification and quantification of bile salt (BS) species in wild-type (WT) and Tmem30a liver-specific knockout (LKO) mice. A: The plasma levels of unconjugated and conjugated BS species were significantly increased in Tmem30a LKO mice. B: The levels of MCA, cholic acid (CA), and TCA in Tmem30a-deficient livers were slightly increased. C: The levels of muricholate (MCA) and TUDCA in bile were slightly changed in Tmem30a LKO mice. All samples were from mice aged 2 to 3 months. Plasma levels are expressed in μmol/L, biliary levels in nmol/mL, hepatic levels in nmol/g liver, respectively. Error bars represent SEM. n ≥ 6 mice per group. ∗P < 0.05, ∗∗P < 0.01 (t-test). CDCA, chenodeoxycholate; DCA, deoxycholate; G, glycol; LCA, lithocholic acid; T, tauro; UDCA, ursodeoxycholate.
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4. Figure 3
Analysis of mRNA and protein levels of ATP8B1, ATP11C, bile salt (BS) transporters and related nuclear receptors. A: Mean relative mRNA levels of BS transporters, Atp8b1, Atp11c, related nuclear receptors, and enzymes in livers (relative mRNA levels were normalized to β-actin). B: The protein levels of ATP8B1, ATP11C, and BS transporters in Tmem30a-deficient livers were significantly reduced compared with the littermate controls. C: The protein levels of related nuclear receptors were also significantly reduced in Tmem30a-deficient livers. D: Phosphorylation of protein kinase C (PKC)ζ was also dramatically reduced in Tmem30a liver-specific knockout (LKO) mice. All samples were from mice aged 2 to 3 months. Protein levels were quantified by densitometry and normalized to β-actin. Error bars represent SEM. n ≥ 4 samples (A); n ≥ 4 samples per group (D). ∗P < 0.05, ∗∗P < 0.01 (t-test). BSEP, bile salt export pump; FXR, farnesoid X receptor; HNF, hepatocyte nuclear factor; LRH, liver receptor homolog; MRP, multidrug resistance-associated protein; NTCP, Na+-taurocholate cotransporting polypeptide; OATP, organic anion transporting polypeptide; RXR, retinoid X receptor; SHP, small heterodimer partner; WT, wild-type.
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5. Figure 4
The localization of ATP8B1, ATP11C, and bile salt (BS) transporters was altered in the Tmem30a liver-specific knockout (LKO) mice. A: Western blot analysis of membrane proteins extracted from the livers of the Tmem30a-deficient mice and littermate controls. B: Immunofluorescence images showing the reduced expression and localization of ATP8B1 and ATP11C in Tmem30a-deficient livers. C–E: Confocal images demonstrated that Na+-taurocholate cotransport protein (NTCP) (C), multidrug resistance-associated protein (MRP)2 (D), and BS export pump (BSEP) (E) in Tmem30a LKO mice were mainly localized intracellularly but not on the membrane compared with the littermate controls. White arrows indicate the membrane localization of NTCP, MRP2, and BSEP. All samples were from mice aged 2 to 3 months. Green: ATP8B1 antibody; red: primary antibodies as indicated; blue: DAPI. n ≥ 3 samples per group (C–E). ∗P < 0.05, ∗∗P < 0.01 (t-test). Scale bars: 100 μm (B–E). OATP, organic anion-transporting polypeptide; WT, wild-type.
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6. Figure 5
Bile formation was impaired in Tmem30a liver-specific knockout (LKO) mice. The endogenous bile salt (BS) pools were depleted from Tmem30a LKO mice and littermate controls for 90 minutes. Taurocholate (TC) was infused through the jugular vein, and the infusion rates increased in a stepwise manner every 30 minutes at 400 nmol/min per 100 g for each step (400, 800, 1200, 1600 nmol/min per 100 g). A–C: No difference was found in the biliary outputs of BS (A), cholesterol (B), and phospholipids (C) between Tmem30a LKO mice and littermate controls during BS depletion. However, the biliary outputs of BS, cholesterol, and phospholipids were significantly decreased in Tmem30a LKO mice with the increased infusion rates. D and E: The biliary outputs of glutathione (D) and alkaline phosphatase (ALP) (E) were significantly reduced in Tmem30a LKO mice during both bile depletion and TC infusion. All male mice were tested at 2 to 3 months age. Error bars represent SEM. n ≥ 3 male mice per group (D and E). ∗P < 0.05 (one-way analysis of variance analysis). WT, wild-type.
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7. Figure 6
Protein levels of bile salt (BS) transporters and related nuclear receptors were partially restored in Tmem30a liver-specific knockout (LKO) mice after treatment with the proteasome inhibitor. All mice were sacrificed 6 and 12 hours after treatment with the proteasome inhibitor bortezomib (0.5 mg/kg). A and B: Compared with their dimethyl sulfoxide (DMSO) control groups, wild-type (WT) mice treated with bortezomib showed no difference in the protein levels of ATP8B1, ATP11C and BS transporters (A), whereas Tmem30a LKO mice treated with bortezomib showed dramatically increased levels of these proteins (B). C and D: Compared with their DMSO control groups, WT mice treated with bortezomib showed no difference in the protein levels of farnesoid X receptor (FXR)α, retinoid X receptor (RXR)α, hepatocyte nuclear factor (HNF4)α, liver receptor homolog (LRH)-1, and small heterodimer partner (SHP) (C), whereas Tmem30a LKO mice treated with bortezomib showed dramatically increased levels of these nuclear receptors (D). All mice () were tested at 2 to 3 months age. Protein levels were quantified by densitometry and normalized to β-actin. n ≥ 3 mice per group. ∗P < 0.05, ∗∗P < 0.01 (t-test). BSEP, bile salt export pump; MRP, multidrug resistance-associated protein; NTCP, Na+-taurocholate cotransport protein; OATP, organic anion transporting polypeptide.
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8. Figure 7
Exacerbation of the Tmem30a-deficient phenotypes on cholic acid (CA)-supplemented diet. Male Tmem30a-deficient mice and male littermate controls (2 to 3 months) were fed a 0.5% CA-supplemented diet. A–C: Serum total bile acid (TBA) levels (A); serum total bilirubin (TBIL), direct bilirubin (DBIL), and indirect bilirubin (IBIL) levels (B); and serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels (C) were dramatically increased in Tmem30a liver-specific knockout (LKO) mice compared with the littermate controls after 9 days on CA-supplemented diet. D: The body weight ratios of male Tmem30a LKO mice were dramatically decreased compared with male littermate controls after 3 weeks on CA-supplemented diet. The body weight ratios were normalized to the weight on day 0 (weight before receiving CA). The mice were weighed at 3-day intervals. E: Death of male Tmem30a LKO mice and male littermate controls after 3 weeks on CA-supplemented diet. Mice were monitored every day for the survival curve. F: Hematoxylin and eosin (H&E) staining showed the presence of more multinucleate hepatocytes and smaller hepatocyte sizes in morbid male Tmem30a LKO mice but not the littermate controls on CA-supplemented diet. Error bars represent SEM. n = 15 male Tmem30a-deficient mice; n = 10 male littermate control mice. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 (t-test). Scale bars = 100 μm. Original magnification, ×200.
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9. Supplemental Figure S2
Severe inflammatory infiltration and liver damage in Tmem30a-deficient livers. A: The liver-to-body weight ratios of 2-month-old Tmem30a liver-specific knockout (LKO) mice and littermate controls were similar. B: As shown in the F4/80 immunohistochemistry analysis, macrophage infiltration was increased (red arrow) in the livers of the 2-month-old Tmem30a LKO mice. C: Hematoxylin and eosin (H&E) staining show obvious lymphocyte infiltration at 9 months of age. D: The levels of several inflammatory factors, including IL-6, IL-18, IL-10, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α, were increased in Tmem30a LKO mice. E: Masson blue staining was more apparent (black arrow), and mRNA levels of collagen type 1 (Col1)a1, collagen type 3 (Col3)a1, and transforming growth factor (Tgf)-β were increased in Tmem30a-deficient livers, F: Immunohistochemistry and mRNA levels of cytokine-19 (CK-19) indicated significant bile-duct proliferation in Tmem30a-deficient livers (black arrow). All mice were tested at 2 to 3 months age. Data are expressed as means ± SEM. n ≥ 3 mice per group. ∗P < 0.05 (t-test). Scale bars: 200 μm (B, D, and E); 500 μm (F). Original magnification: ×200 (B and C); ×100 (C). GM-CSF, granulocyte macrophage colony-stimulating factor; GRO, growth-related oncogene; MCP, macrophage chemotactic protein; MIP, macrophage inflammatory protein; RANTES, regulated on activation normal T cell expressed and secreted; VEGF, vascular endothelial growth factor.
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10. Supplemental Figure S3
Biochemical analysis of liver function in Tmem30a liver-specific knockout (LKO) mice and their littermate controls at various ages. A–C: Plasma levels of alanine aminotransferase (ALT) (A), aspartate aminotransferase (AST) (B), and alkaline phosphatase (ALP) (C) in Tmem30a LKO mice were increased compared with littermate controls at various ages. D and E: Serum total protein (TP) (D) and albumin (ALB) (E) levels were similar in Tmem30a LKO mice and littermate controls. Data are expressed as means ± SEM. n ≥ 6 mice (A–C); n ≥ 5 mice (D and E). ∗P < 0.05 (t-test). WT, wild-type.
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11. Supplemental Figure S4
Localization of bile acid (BA) transporter proteins were reduced on the membrane. A: The expression and localization of organic anion transporting polypeptide (OATP)1A4, OATP1B2, and Na+-taurocholate cotransport protein (NTCP) were decreased in Tmem30a-deficient livers. White arrow indicates portal area; red arrow, central area. B: The expression and localization of bile salt export pump (BSEP) and multidrug resistance-associated protein (MRP)2 were also impaired in Tmem30a liver-specific knockout (LKO) mice. All mice were tested at 2 to 3 months age. Red: primary antibodies as indicated; blue: DAPI. n ≥ 4 mice per group. Scale bars: 100 μm (A and B). WT, wild-type.
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12. Supplemental Figure S5
No significant changes in expression levels of bile-salt (BS)-related proteins and markers of kidney function in the Tmem30a liver-specific knockout (LKO) mice. A and B: The mRNA (A) and protein (B) levels in the intestines of Tmem30a LKO mice were similar to that of their littermate controls. C–E: Serum levels of creatinine (CREA) (C), blood urea nitrogen (BUN) (D), and uric acid (UA) (E) in Tmem30a LKO mice were similar to that of their littermate controls at various age. All mice were tested at the indicated age. Data are expressed as means ± SEM (t-test). n ≥ 4 mice. ABST, apical sodium-dependent bile salt transporters; FGF, fibroblast growth factor; FXR, farnesoid X receptor; MRP, multidrug resistance-associated protein; SHP, short heterodimer partner; WT, wild-type.
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13. Supplemental Figure S6
Hematoxylin and eosin (H&E) staining of heart and kidney in littermate control and Tmem30a liver-specific knockout (LKO) mice at 12 months of age. A: The hearts in littermate controls and Tmem30a LKO mice showed similar morphologic characteristics both in global scan (left) or in cardiocytes (right). B: The global scan (left) of kidneys showed normal morphologic characteristics between the two groups. The medulla and renal cortex also showed normal morphologic characteristics. Scale bars: 100 μm (A); 200 μm (B). Original magnification, ×200. WT, wild-type.
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14. Supplemental Figure S8
Hematoxylin and eosin (H&E) staining of muscle, pancreas, and intestine in littermate control and Tmem30a liver-specific knockout (LKO) mice at 12 months of age. A: H&E staining revealed similar morphologic characteristics in the skeletal muscle cells of littermate controls and Tmem30a LKO mice. B: H&E staining revealed similar morphologic characteristics in the pancreas cells of littermate controls and Tmem30a LKO mice. C: No differences was found in the morphologic characteristics of intestinal villus and crypts of littermate controls and Tmem30a LKO mice. Scale bars: 200 μm (A and B); 500 μm (C and D). WT, wild-type.
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15. Supplemental Figure S9
ATP8B1, ATP11C, and bile salt (BS) transporters were co-localized with protein disulfide isomerase (PDI) in the endoplasmic reticulum (ER) on proteasome inhibition by bortezomib. All mice were sacrificed at 12 hours on the proteasome inhibition by bortezomib (0.5 mg/kg). Confocal microscopy images showing the co-localization of ATP8B1 (A), ATP11C (B), organic anion-transporting polypeptide (OATP)1B2 (C), bile salt export pump BSEP (D), and MRP2 (E) with PDI in ER in Tmem30a liver-specific knockout (LKO) mice. White arrows indicate the membrane-bound proteins. n ≥ 4 mice. Scale bars = 10 μm. NTCP, Na+-taurocholate cotransporting polypeptide; WT, wild-type.
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16. Supplemental Figure S10
Analysis of bile salt (BS) transporter mRNAs after treatment with the proteasome inhibitor bortezomib. The mRNA levels of BS transporters in Tmem30a liver-specific knockout (LKO) mice were partially restored compared with the dimethyl sulfoxide (DMSO) controls. Mice were sacrificed at 12 hours after proteasome inhibition by bortezomib (0.5 mg/kg). Data are expressed as means ± SEM. n ≥ 4 mice. ∗P < 0.05 (t-test). Bsep, bile salt export pump; Mdr, multidrug resistance; Mrp, multidrug resistance-associated protein; Ntcp, Na+-taurocholate cotransporting polypeptide; Oatp, organic anion-transporting polypeptide; WT, wild-type.
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17. Supplemental Figure S11
Deterioration of the Tmem30a-deficient phenotype after feeding a cholic acid (CA)-supplement diet. A: Jaundice was observed in Tmem30a liver-specific knockout (LKO) mice but not their littermate controls. B: Serum total protein (TP) and albumin (ALB) levels were significantly reduced in Tmem30a LKO mice compared with their littermate controls after a 9-day CA-supplemented diet. C: The serum creatinine (CREA), blood urea nitrogen (BUN), and uric acid (UA) levels in the morbid Tmem30a LKO mice were comparable with the littermate controls after a 9-day CA-supplemented diet. D: The liver-to-body weight ratios of morbid male Tmem30a LKO mice were dramatically decreased compared with the littermate controls. E: The mRNAs levels of collagen type 1 α 1 chain (Col1a1), Col3a1 and transforming growth factor (Tgf)-β mRNAs were obviously increased in Tmem30a-deficient livers. F: Masson blue staining indicated severe liver fibrosis in Tmem30a-deficient livers. All male mice were tested at 2 to 3 months age. Data are expressed as means ± SEM. n ≥ 9 male mice. ∗P < 0.05, ∗∗P < 0.01 (t-test). Scale bar = 200 μm. WT, wild-type.
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18. Supplemental Figure S12
Hematoxylin and eosin (H&E) staining of heart and kidney in littermate control and Tmem30a liver-specific knockout (LKO) mice on cholic acid (CA)-supplemented diet. Male Tmem30a-deficient mice and male littermate controls (2 to 3 months) were fed a 0.5% CA-supplemented diet. A: The global scan (left) and cardiocytes (right) of the hearts indicated that there was no morphologic difference between littermate control and Tmem30a LKO mice. B: H&E staining revealed that there was no morphologic difference in the medulla and renal cortex of kidneys of littermate control and Tmem30a LKO mice. Scale bars: 100 μm (A); 200 μm (B). WT, wild-type.
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19. Supplemental Figure S13
Hematoxylin and eosin (H&E) staining of brain, intestine, and spleen in littermate control and Tmem30a liver-specific knockout (LKO) mice on cholic acid (CA)-supplemented diet. Male Tmem30a-deficient mice and male littermate controls (2 to 3 months) were fed a 0.5% CA-supplemented diet. A: H&E staining of the brains revealed similar morphologic characteristics in both groups. B: H&E staining of intestinal villi revealed similar morphologic characteristics in both groups. C: H&E staining of the spleens also revealed similar morphologic characteristics in both groups. Scale bars: 5000 μm (A); 500 μm (B); 200 μm (C). WT, wild-type.
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20. Supplemental Figure S14
Biochemical analysis of surviving Tmem30a liver-specific knockout (LKO) mice. A and B: (A) Plasma total bile acid (TBA), total bilirubin (TBIL) and direct bilirubin (DBIL) levels (A) and plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) levels (B) were increased in surviving Tmem30a LKO mice, but the increases were not as dramatic as in morbid Tmem30a LKO mice. C: Hematoxylin and eosin (H&E) staining showing the presence of unclear cell boundaries in the surviving Tmem30a LKO mice. All mice were tested at 2 to 3 months age. Data are expressed as means ± SEM. n ≥ 9 mice. ∗P < 0.05, ∗∗P < 0.01 (t-test). Scale bars = 100 μm. WT, wild-type.
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