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Clinical Reviews in Allergy & Immunology

, Volume 53, Issue 1, pp 105–116 | Cite as

A Critical Evaluation of Liver Pathology in Humans with Danon Disease and Experimental Correlates in a Rat Model of LAMP-2 Deficiency

  • Lu Wang
  • Jingbo Wang
  • Weile Cai
  • Yongquan Shi
  • Xinmin Zhou
  • Guanya Guo
  • Changcun Guo
  • Xiaofeng Huang
  • Zheyi Han
  • Shuai Zhang
  • Shuoyi Ma
  • Xia Zhou
  • Daiming Fan
  • M. Eric GershwinEmail author
  • Ying HanEmail author
Article

Abstract

Danon disease is a genetic deficiency in lysosome-associated membrane protein 2 (LAMP-2), a highly glycosylated constituent of the lysosomal membrane and characterized by a cardiomyopathy, skeletal muscle myopathy, and cognitive impairment. Patients, however, often manifest hepatic abnormalities, but liver function has not been well evaluated and the syndrome is relatively uncommon. Hence, we have taken advantage of a rat that has been deleted of LAMP-2 to study the relative role of LAMP-2 on liver function. Interestingly, rats deficient in LAMP-2 develop a striking increase in serum alkaline phosphatase (ALP) and a decrease in bile flow compared with wild-type littermates. Importantly and by ultrastructural analysis, deficient rats manifest dilated canaliculi that lack microvilli with evidence of bile-containing bodies. Moreover, following bile duct ligation, LAMP-2-deficient rats develop rapid and severe evidence of advanced cholestasis, with an increase in serum bilirubin, as early as 6 h later. In wild-type control rats, multidrug resistance-associated protein 2 (Mrp2) normally concentrates at the bile canalicular membranes to secrete conjugated bilirubin into bile. However, in LAMP-2y/− rats, Mrp2 was detected in hepatocytes compared with other canalicular proteins including P-glycoproteins, dipeptidyl peptidase IV (CD26), and aminopeptidase (CD13). Our data further suggest that LAMP-2 interacts with the membrane cytoskeletal proteins radixin and F-actin in determining the localization of integral membrane proteins.

Keywords

Lysosome-associated membrane protein 2 Intrahepatic cholestasis Multidrug resistance-associated protein 2 

Abbreviations

ALP

Alkaline phosphatase

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

BDL

Bile duct ligation

Bsep

Bile salt export pump

CMA

Chaperone-mediated autophagy

DAPI

4′,6-Diamidino-2-phenylindole

DBIL

Unconjugated bilirubin

GAPDH

Glyceraldehyde 3-phosphate dehydrogenase

LAMP-2

Lysosome-associated membrane protein 2

Mrp2

Multidrug resistance-associated protein 2

RT-PCR

Real-time polymerase chain reaction

TALEN

Transcription activator-like effector nuclease

TBIL

Total bilirubin

TGN

Trans-Golgi network

ZO-1

Zonula occludens-1

Notes

Acknowledgements

We thank Wanjun Chen (Mucosal Immunology Section, National Institutes of Health) for the technical guidance during the experiment and reviewing the manuscript. We thank Professor Stieger (Department of Clinical Pharmacology and Toxicology, University Hospital, Zürich, Switzerland) for the gift of anti-BSEP antibodies. The study was funded by the National Natural Science Foundation of China (Nos. 81600451, 81470835, and 81370519), National Science and Technology Major Projects for New Drugs Innovation and Development (Nos. 2014ZX09508002-001 and 2014ZX09104002-003), and Natural Science Foundation of Shaanxi Province (Nos. 2013HM-01 and 2011KTCL03-09).

Author Contributions

YH and EG conceived and designed the experiments and wrote the manuscript; LW performed the experiments and wrote the manuscript; JBW and WLC assisted in some experiments and revised the manuscript; YQS, XMZ, GYG, and CCG assisted in some experiments; LW, XFH, and ZYH collected samples and interpreted the data; and SZ, SYM, XZ, and DMF collected samples.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Ethical Approval

This article contains studies with animals. All animal experimental protocols were approved by our local animal care and use committee according to criteria outlined in Guide for the Care and Use of Laboratory Animals from the National Academy of Sciences (National Institutes of Health publication 86-23, 1985 revision).

Supplementary material

12016_2017_8598_Fig7_ESM.gif (313 kb)
Supplementary Figure 1

LAMP-2 deficiency causes increased mortality and loss of weight. a, The representative LAMP-2 heterozygote female, with one pink eye at two weeks. b, Loss of weight in LAMP-2y/− rats. Hemizygote mutant male (LAMP-2y/−, left); heterozygote female (LAMP-2+/−, middle); wild-type male (LAMP-2y/+, right). c, Loss of weight in early dying (n = 6 males) and surviving LAMP-2-deficient rats (n = 12 males) in comparison with control rats (n = 15 males) monitored for 50 days after birth. d, Increased mortality in LAMP-2 deficient rats. Fifty-four control (full line) and LAMP-2-deficient rats (males, dotted line) were monitored over 90 days. (GIF 313 kb)

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High Resolution Image (TIFF 3.13 mb)
12016_2017_8598_Fig8_ESM.jpg (2.1 mb)
Supplementary Figure 2

Light micrographs of sections of the liver, kidney, heart, and small and large intestine of wild-type and LAMP-2y/− rats stained with hematoxylin and eosin, which showed no significant difference under light microscopy. Scale bars, 50 μm. (JPG 2.11 MB)

12016_2017_8598_MOESM2_ESM.tif (4.2 mb)
High Resolution Image (TIFF 2.11 mb)
12016_2017_8598_Fig9_ESM.gif (346 kb)
Supplementary Figure 3

Accumulation of autophagic vacuoles in LAMP-2 deficient tissues. Ultrastructure of autophagic vacuoles (red arrow) in pancreas, kidney, skeletal muscle and cardiac muscle. Mitochondria had reduced density, were swollen and had deformed cristae (green arrow head) in the heart of LAMP-2y/− rats. Thin line arrow, the widening of perinuclear space in the myocardial cells of LAMP-2y/− rats. Scale bars, 1 μm. (GIF 346 kb)

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High Resolution Image (TIFF 3.33 mb)
12016_2017_8598_Fig10_ESM.gif (58 kb)
Supplementary Figure 4

Dynamically assessed markers of AST and ALT in LAMP-2-deficient rats after bile duct ligation. (GIF 57.6 kb)

12016_2017_8598_MOESM4_ESM.tif (447 kb)
High Resolution Image (TIFF 447 kb)
12016_2017_8598_Fig11_ESM.gif (508 kb)
Supplementary Figure 5

Immunohistochemical detection of Mdr1, Bsep, CD26 and CD13 in the liver of wild-type (LAMP-2y/+) and LAMP-2y/− rats. The concentrations of P-glycoproteins including Bsep, Mdr1, as well as CD26 (dipeptidyl peptidase IV) and CD13 (aminopeptidase), had not changed significantly around bile canaliculi at 4 wk. Scale bars, 20 μm. (GIF 507 kb)

12016_2017_8598_MOESM5_ESM.tif (5.7 mb)
High Resolution Image (TIFF 5.69 mb)

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Lu Wang
    • 1
  • Jingbo Wang
    • 1
  • Weile Cai
    • 1
  • Yongquan Shi
    • 1
  • Xinmin Zhou
    • 1
  • Guanya Guo
    • 1
  • Changcun Guo
    • 1
  • Xiaofeng Huang
    • 2
  • Zheyi Han
    • 1
  • Shuai Zhang
    • 1
  • Shuoyi Ma
    • 1
  • Xia Zhou
    • 1
  • Daiming Fan
    • 1
  • M. Eric Gershwin
    • 3
    Email author
  • Ying Han
    • 1
    Email author
  1. 1.Xijing Hospital of Digestive DiseasesThe Fourth Military Medical UniversityXi’anChina
  2. 2.Center of Electron MicroscopeThe Fourth Military Medical UniversityXi’anChina
  3. 3.Division of Rheumatology, Allergy and Clinical ImmunologyUniversity of California at Davis School of MedicineDavisUSA

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