title: Large-scale identification of calcium oxalate monohydrate crystal-binding proteins on apical membrane of distal renal tubular epithelial cells.
abstract: Adhesion of calcium oxalate monohydrate (COM) crystals onto apical surface of renal tubular epithelial cells is a crucial mechanism for crystal retention, leading to kidney stone formation. Various proteins on apical membrane may bind to COM crystals; however, these crystal-binding proteins remained unidentified. The present study therefore aimed to identify COM crystal-binding proteins on apical membrane of distal renal tubular epithelial cells. Madin-Darby Canine Kidney (MDCK) cells were cultivated to be polarized epithelial cells and apical membrane was isolated from these cells using a peeling method established recently. Enrichment and purity of isolated apical membrane were confirmed by Western blot analysis for specific markers of apical (gp135) and basolateral (Na(+)/K(+)-ATPase) membranes. Proteins derived from the isolated apical membrane were then resuspended in artificial urine and incubated with COM crystals. The bound proteins were eluted, resolved by SDS-PAGE, and analyzed by Q-TOF MS and MS/MS, which identified 96 proteins. Among these, expression and localization of annexin II on apical surface of MDCK cells were confirmed by Western blot analysis, immunofluorescence staining, and laser-scanning confocal microscopic examination. Finally, the function of annexin II as the COM crystal-binding protein was successfully validated by COM crystal-binding assay. This large data set offers many opportunities for further investigations of kidney stone disease and may lead to the development of new therapeutic targets.
authors.affiliation: Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Kedsarin
authors.initials: K
authors.lastname: Fong-Ngern
authors.forename: Paleerath
authors.initials: P
authors.lastname: Peerapen
authors.forename: Supachok
authors.initials: S
authors.lastname: Sinchaikul
authors.forename: Shui-Tein
authors.initials: ST
authors.lastname: Chen
authors.forename: Visith
authors.initials: V
authors.lastname: Thongboonkerd
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002129:Calcium Oxalate / Q000737:chemistry*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003460:Crystallization; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D007684:Kidney Tubules / Q000166:cytology* / Q000378:metabolism*; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D011485:Protein Binding; D040901:Proteomics / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D014357:Trypsin / Q000737:chemistry
chemical_list: D002129:Calcium Oxalate; D014357:Trypsin