title: Calcium oxalate crystals increased enolase-1 secretion from renal tubular cells that subsequently enhanced crystal and monocyte invasion through renal interstitium.
abstract: Calcium oxalate monohydrate (COM) crystals cause kidney stone disease by still unclear mechanisms. The present study aimed to characterize changes in secretion of proteins from basolateral compartment of renal tubular epithelial cells after exposure to COM crystals and then correlated them with the stone pathogenesis. Polarized MDCK cells were cultivated in serum-free medium with or without 100 μg/ml COM crystals for 20 h. Secreted proteins collected from the lower chamber (basolateral compartment) were then resolved in 2-D gels and visualized by Deep Purple stain (n = 5 gels/group). Spot matching and intensity analysis revealed six protein spots with significantly altered levels in COM-treated samples. These proteins were then identified by tandem mass spectrometry (Q-TOF MS/MS), including enolase-1, phosphoglycerate mutase-1, actinin, 14-3-3 protein epsilon, alpha-tubulin 2, and ubiquitin-activating enzyme E1. The increased enolase-1 level was confirmed by Western blot analysis. Functional analysis revealed that enolase-1 dramatically induced COM crystal invasion through ECM migrating chamber in a dose-dependent manner. Moreover, enolase-1 bound onto U937 monocytic cell surface markedly enhanced cell migration through the ECM migrating chamber. In summary, our data indicated that the increased secretory enolase-1 induced by COM crystals played an important role in crystal invasion and inflammatory process in renal interstitium.
authors.affiliation: Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.|Graduate Program in Molecular Medicine, Faculty of Science, Mahidol University, Bangkok, Thailand.|Center for Research in Complex Systems Science, Mahidol University, Bangkok, Thailand.
authors.forename: Wararat
authors.initials: W
authors.lastname: Chiangjong
authors.affiliation: Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.|Center for Research in Complex Systems Science, Mahidol University, Bangkok, Thailand.
authors.forename: Visith
authors.initials: V
authors.lastname: Thongboonkerd
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D000818:Animals; D002129:Calcium Oxalate / Q000737:chemistry*; D002465:Cell Movement; D003460:Crystallization; D016895:Culture Media, Serum-Free / Q000737:chemistry; D004285:Dogs; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005109:Extracellular Matrix / Q000378:metabolism; D006801:Humans; D007249:Inflammation; D007684:Kidney Tubules / Q000166:cytology*; D061985:Madin Darby Canine Kidney Cells; D008856:Microscopy, Fluorescence; D009000:Monocytes / Q000166:cytology; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism; D040901:Proteomics; D053719:Tandem Mass Spectrometry; D014404:Tubulin / Q000378:metabolism; D020298:U937 Cells
chemical_list: D048948:14-3-3 Proteins; D016895:Culture Media, Serum-Free; D020543:Proteome; D014404:Tubulin; D002129:Calcium Oxalate; D010751:Phosphopyruvate Hydratase