Proteomic analysis of calcium oxalate monohydrate crystal-induced cytotoxicity in distal renal tubular cells.
Calcium oxalate monohydrate (COM) is the major crystalline component found in kidney stones and its adhesion to renal tubular cells provokes tubular injury, which in turn enhances COM crystal adhesion. However, COM-induced toxic effects in these tubular cells remain largely unknown. We performed a proteomics study to characterize changes in the cellular proteome in MDCK distal renal tubular cells after an exposure to high-dose (1000 microg/mL) COM crystals for 48 h, at which percentage of cell death was significantly increased. Proteins were extracted from MDCK cells cultured with COM-containing or COM-free medium ( n = 5 individual flasks per group), resolved in individual 2-D gels, and stained with SYPRO Ruby fluorescence dye. Quantitative and statistical analyses revealed 53 proteins whose abundance levels were altered (25 were increased, whereas other 28 were decreased) by COM-induced toxicity. Among these, 50 were successfully identified by quadrupole time-of-flight (Q-TOF) mass spectrometry (MS) and/or tandem MS (MS/MS) analyses. The proteomic data were clearly confirmed by 2-D Western blot analysis. While three chaperones (GRP78, Orp150 and Hsp60) were increased, other proteins involved in protein biosynthesis, ATP synthesis, cell cycle regulator, cellular structure, and signal transduction were decreased. These data provide some novel mechanistic insights into the molecular mechanisms of COM crystal-induced tubular toxicity.
Thongboonkerd
Visith
V
Medical Proteomics Unit & Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. thongboonkerd@dr.com
Semangoen
Theptida
T
Sinchaikul
Supachok
S
Chen
Shui-Tein
ST
D000818:Animals; D002129:Calcium Oxalate / Q000633:toxicity*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003470:Culture Media / Q000737:chemistry; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D007686:Kidney Tubules, Distal / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D010449:Peptide Mapping; D020543:Proteome / Q000032:analysis* / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D013997:Time Factors
D003470:Culture Media; D020543:Proteome; D002129:Calcium Oxalate