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.
Highlights
Cell death and amounts of secreted proteins reported in Fig. 1A–C, respectively, we selected 20 h as the optimal time-point for subsequent secretome study of polarized Mardin-Darby Canine kidney (MDCK) cells under the serum-free condition
We have proposed that changes in secretion of proteins from basolateral compartment of renal tubular epithelial cells induced by COM crystals are associated with kidney stone pathogenic mechanisms, crystal invasion into renal interstitium and subsequent inflammatory process
MDCK (Type II) cell line was used as a cell model in this study because of its properties, including i) stable epithelial phenotype, ii) lack of gap junctions, and iii) large and tall cell morphology, making this cell line suitable for secretome study[14]
Summary
Spot matching and quantitative intensity analysis revealed significant changes in levels of six protein spots in basolateral secretome of COM-treated cells. We confirmed the increase of enolase-1 secretion in basolateral compartment of MDCK polarized cells after exposure to COM crystals.
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