Proteomic analysis of altered proteins in distal renal tubular cells in response to calcium oxalate monohydrate crystal adhesion: Implications for kidney stone disease.
Adhesion of calcium oxalate (CaOx) crystals to renal tubular cells is a critical event that triggers a cascade of responses, leading to the development of kidney stones. However, the molecular mechanisms of these cellular responses remain largely unknown. We performed gel-based, differential proteomics study to examine cellular responses (as determined by altered protein expression) in Madin-Darby Canine Kidney (MDCK) cells during CaOx monohydrate (COM) crystal adhesion. Approximately 3-million MDCK cells were inoculated in each culture flask and maintained for 24 h. A total of 10 semiconfluent flasks were then divided into two groups (n = 5 per group) and the culture medium was replaced by either COM-containing (with 100 μg/mL COM crystals) or COM-free medium. The cells were grown further for 48 h. Crystal adhesion on the cell surface was clearly demonstrated using phase-contrast and scanning electron microscopy. Cell death assay using annexin V/propidium iodide double staining showed that all these samples had comparable % cell death. Cellular proteins were then extracted, resolved with 2-DE, and visualized by SYPRO Ruby staining (n = 5 gels per group). Quantitative intensity analysis revealed significantly increased abundance of 15 protein spots, whereas the other 5 were decreased. These altered proteins were then identified by quadrupole TOF (Q-TOF) MS and/or MS/MS analyses, including transcription/translation regulators, signal transduction proteins, metabolic enzymes, nuclear membrane proteins, carrier protein, cellular structural protein, chaperones, and proteins involved in biosynthesis, enzyme activation, and growth regulation. These data may lead to better understanding of the cellular responses in distal renal tubular cells during COM crystal adhesion.
Medical Proteomics Unit and Medical Molecular Biology Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; Department of Immunology and Immunology Graduate Program, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, Thailand.
Theptida
T
Semangoen
Supachok
S
Sinchaikul
Shui-Tein
ST
Chen
Visith
V
Thongboonkerd