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Proteomic investigation of urinary markers of carbon-tetrachloride-induced hepatic fibrosis in the Hanover Wistar rat

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Abstract

Proteomic techniques such as two-dimensional gel electrophoresis (2-DGE) and mass spectrometry have become important tools for the identification of novel biomarkers of toxicity and disease. Ideally, such biomarkers need to be sensitive and organ specific, but, recently, it has become apparent that it would be an additional benefit to be able to measure biomarkers in samples obtained using non-invasive methods. The present study is concerned with the identification of novel urinary markers of hepatic fibrosis. In a carbon-tetrachloride-induced liver fibrosis rat model, analysis of urine by 2-DGE revealed an increase in the concentration of a number of proteins in animals with hepatic fibrosis. Using in-gel trypsin digest and nano-scale liquid chromatography combined with electrospray ionisation tandem mass spectrometry, protein spots were identified as copper/zinc superoxide dismutase, d-dopachrome tautomerase, beta-2-microglobulin and neutrophil gelatinase associated lipocalin. These proteins are known to have important roles in the inflammatory response.

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Abbreviations

ALT:

alanine aminotransferase

AST:

aspartate aminotransferase

β-2m precursor:

beta-2-microglobulin precursor

CCl4 :

carbon tetrachloride

CHAPS:

3-(3-cholamidopropyl)dimethylammonio)-1-propanesulfonate

Cu/Zn SOD:

copper/zinc superoxide dismutase

DDA:

data dependent analysis

2-DGE:

two-dimensional gel electrophoresis

DTT:

dithiothreitol

ECM:

extracellular matrix

GLDH:

glutamate dehydrogenase

FA:

formic acid

HGF:

hepatic growth factor

HSCs:

hepatic stellate cells

H & E:

haematoxylin and eosin

IPG:

immobilised pH gradient

MALDI:

matrix assisted laser desorption ionisation

MHC:

major histocompatibility complex

MIF:

macrophage migration inhibitory factor

MMP:

metalloproteinase

nano-LC-ESI-MS/MS:

nano-liquid chromatography-electrospray tandem mass spectrometry

NGAL:

neutrophil gelatinase associated lipocalin

PDGF:

platelet-derived growth factor

SDS-PAGE:

sodium dodecyl sulphate polyacrylamide gel electrophoresis

α-SMA:

alpha smooth muscle actin

SOD:

superoxide dismutase

TCA:

trichloroacetic acid

TFA:

trifluoroacetic acid

TGF-β1:

transforming growth factor beta-1

TIMP:

tissue inhibitors of metalloproteinase

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Acknowledgements

We acknowledge the assistance of the technical staff at the School of Pharmacy for their care of the animals. GlaxoSmithKline and the School of Pharmacy funded this work.

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Author notes

  1. Christopher J. Clarke

    Present address: Novartis Pharma AG, Auhafenstrasse, Muttenz, CH-4132, Muttenz, Switzerland

  2. William Griffiths

    Present address: Institute of Mass Spectrometry, Swansea University, Swansea, SA2 8PP, UK

Authors and Affiliations

  1. Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, 29/39 Brunswick Square, London, WC1N 1AX, UK

    Rosemary Smyth, Catherine S. Lane, Rukhsana Ashiq, William Griffiths & Michael R. Munday

  2. Centre for Toxicology, Department of Pharmacology, The School of Pharmacy, University of London, 29/39 Brunswick Square, London, WC1N 1AX, UK

    John A. Turton

  3. GlaxoSmithKline Research and Development, Park Road, Ware, Hertfordshire, SG12 ODP, UK

    Christopher J. Clarke, Theo O. Dare & Malcolm J. York

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Smyth, R., Lane, C.S., Ashiq, R. et al. Proteomic investigation of urinary markers of carbon-tetrachloride-induced hepatic fibrosis in the Hanover Wistar rat. Cell Biol Toxicol 25, 499–512 (2009). https://doi.org/10.1007/s10565-008-9104-8

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