Warm ischemia-induced alterations in oxidative and inflammatory proteins in hepatic Kupffer cells in rats.
The aim of the study was to investigate the impact of ischemia/reperfusion injury on the proteome of Kupffer cells. Lean Zucker rats (n = 6 each group) were randomized to 75 min of warm ischemia or sham operation. After reperfusion for 8 h, Kupffer cells were isolated by enzymatic perfusion and density gradient centrifugation. Proteins were tryptically digested into peptides and differentially labeled with iTRAQ (isobaric tags for relative and absolute quantitation) reagent. After fractionation by cation exchange chromatography, peptides were identified by mass spectrometry (ESI-LC-MS/MS). Spectra were interrogated against the Swiss-Prot database and quantified using ProteinProspector. The results for heat shock protein 70 and myeloperoxidase were validated by ELISA. Quantitative information for more than 1559 proteins was obtained. In the ischemia group proteins involved in inflammation were significantly up-regulated. The ratio for calgranulin B in the ischemia/sham group was 1.81 +/- 0.97 (p < 0.0001), for complement C3 the ratio was 1.81 +/- 0.49 (p < 0.0001), and for myeloperoxidase the ratio was 1.30 +/- 0.32. Myeloperoxidase was only recently documented in Kupffer cells. The antioxidative proteins Cu,Zn-superoxide dismutase (1.34 +/- 0.19; p < 0.001) and catalase (1.23 +/- 0.43; p < 0.001) were also elevated. In conclusion, ischemia/reperfusion injury induces alterations in the Kupffer cell proteome. Isotope ratio mass spectrometry is a powerful tool to investigate these reactions. The ability to simultaneously monitor several pathways involved in reperfusion stress may result in important mechanistic insight and possibly new treatment options.
Hirsch
Jan
J
Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA. hirschj@anesthesia.ucsf.edu
Hansen
Kirk C
KC
Choi
Soojinna
S
Noh
Joonhwa
J
Hirose
Ryutaro
R
Roberts
John P
JP
Matthay
Michael A
MA
Burlingame
Alma L
AL
Maher
Jacquelyn J
JJ
Niemann
Claus U
CU
D000818:Animals; D002135:Calcium-Binding Proteins / Q000032:analysis; D040502:Calgranulin B / Q000032:analysis; D003176:Complement C3 / Q000032:analysis; D005982:Glutathione Transferase / Q000032:analysis; D018840:HSP70 Heat-Shock Proteins / Q000032:analysis; D007728:Kupffer Cells / Q000737:chemistry*; D008099:Liver / Q000098:blood supply / Q000166:cytology; D008840:Microfilament Proteins; D023421:Models, Animal; D009195:Peroxidase / Q000032:analysis; D010544:Peroxidases / Q000032:analysis; D054464:Peroxiredoxins; D040901:Proteomics; D051381:Rats; D011924:Rats, Zucker; D015427:Reperfusion Injury / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013482:Superoxide Dismutase / Q000032:analysis
C100745:Aif1 protein, rat; D002135:Calcium-Binding Proteins; D040502:Calgranulin B; D003176:Complement C3; D018840:HSP70 Heat-Shock Proteins; D008840:Microfilament Proteins; D010544:Peroxidases; D054464:Peroxiredoxins; D009195:Peroxidase; D013482:Superoxide Dismutase; D005982:Glutathione Transferase