Briefly, the cells were collected by trypsinization, fixed and permeabilized with ice-cold 70% ethanol, and incubated about snow for 30?min. function. Until now, kidney stone disease is still a general public health problem in almost all areas around the world. The disease causes substantial suffering and ultimately end-stage renal disease (ESRD). Regrettably, the disease mechanisms remain poorly recognized. Calcium oxalate (CaOx) is the major chemical component found in clinical stones1. This type of the stones can be originated from supersaturation of calcium and oxalate ions, leading to crystallization inside renal tubular fluid or urine2. CaOx crystals can then nucleate to form stone nidus and adhere directly onto apical surface of renal tubular epithelial cells3,4,5. Adhesion of crystals onto the cells is definitely a critical event, which causes many cascades of cellular response, e.g. cytotoxicity, injury, proliferation and apoptosis, that ultimately lead to kidney stone formation6,7. CaOx crystals also evoke inflammatory processes that can lead to fibrosis, loss of nephron and eventually ESRD8,9. Even with the aforementioned knowledge, molecular mechanisms of the downstream cellular response remain mainly unfamiliar. From our earlier expression proteomics study7, we have identified a number of proteins with modified levels in MDCK renal tubular cells in response to CaOx crystals. Those modified proteins were involved in various biological processes, i.e. ubiquitination pathway, transmission transduction, cellular structure, purine biosynthesis, metabolic enzyme, retinol biosynthesis, cellular transportation, protein degradation, RNA rate of metabolism, RNA binding protein, cell surface antigen, nucleic Rabbit Polyclonal to STEA3 acid metabolism, antioxidant enzyme, chaperone, carrier protein, Parimifasor and protein biosynthesis. However, functional significance of those altered proteins had not been investigated. In the present study, we thus performed global protein network analysis of those altered proteins. Subsequently, overexpression of a protein, which was one of the central nodes of such protein-protein interactions network, was performed. Moreover, functional investigations were performed to address functional significance of the central-node protein and its associated partners in kidney stone disease. Results Parimifasor Global protein network analysis From our previous expression proteomics study7, a number of differentially expressed proteins were recognized in CaOx-treated MDCK cells. However, their functional functions in kidney stone disease had not been investigated. Our present study thus aimed to address functional significance of such altered proteins. First, they were submitted to global protein network analysis using STRING software (version 10) (http://string.embl.de/)10. The protein-protein interactions network exhibited that -tubulin was one of the central nodes of such protein-protein interactions (Fig. 1). We thus focused our attention on functional significance of -tubulin in association with kidney stone formation. Open in a separate window Physique 1 Global protein network analysis of altered proteins in MDCK renal tubular cells induced by CaOx crystals.All the altered proteins identified in our previous study7 were subjected to global protein network analysis using STRING tool (version 10) (http://string.embl.de/)10. Upward and downward arrows indicate up-regulation and down-regulation induced by the crystals, respectively. The connecting lines between protein nodes indicate protein-protein interactions. -tubulin overexpression (pcDNA6.2-TUBA1A) in MDCK cells and confirmation of -tubulin level To address functional significance of -tubulin, of which level was decreased in CaOx-treated MDCK cells, overexpression of -tubulin was performed Parimifasor using Gateway Technology (Invitrogen). Physique 2A summarizes schematic approach of -tubulin overexpression by using this technology, which is based on pcDNA6.2-TUBA1A. Western blot analysis revealed that -tubulin level was increased (approximately 1.5-fold) in pcDNA6.2-TUBA1A cells as compared to the unmodified (WT) cells, confirming that this overexpression of -tubulin using this technique was successful (Fig. 2B). Open in a separate window Physique 2 Overexpression of -tubulin in MDCK cells.(A) Schematic diagram of -tubulin overexpression (pcDNA6.2-TUBA1A) by Gateway Technology. (B) Efficacy of -tubulin overexpression was confirmed by Western blot analysis. GAPDH served as the loading control. The data are reported as mean??SEM (n?=?3 independent experiments). *gene, the cDNA was prepared from MDCK cells. Briefly, MDCK cells were grown.