SP094
ROLES OF ALPHA-TUBULIN IN RENAL TUBULAR EPITHELIAL CELL FOR CELL VIABILITY, PROLIFERATION, TISSUE REPAIR AND CRYSTAL ADHESION IN CALCIUM OXALATE KIDNEY STONE DISEASE

Introduction and Aims: Adhesion of calcium oxalate crystal on renal tubular epithelial cells is a crucial process that triggers many cascades of cellular response and alterations in protein expression. From our previous study, calcium oxalate caused changes in levels of a number of cellular proteins, including a decrease level of tubulin. However, their functional significance remained largely unknown. In the present study, we aimed to address functional significance of tubulin in kidney stone disease.

Methods: MDCK renal tubular cells were transfected with pcDNA-TUBA1A, whereas the cells transfected with empty vector served as a control for the transfection system. The transfected and non-transfected cells were then treated with or without 1,000 µg/mL calcium oxalate crystals for up to 15 h. The cells were then examined by Western blot analyses, cell death/viability assay, total cell count, scratch assay, and crystal adhesion assay.

Results: Overexpression of alpha-tubulin caused a significant reduction of crystal-induced cell death to a basal level, comparable to the non-transfected controlled (untreated) cells. In addition, alpha-tubulin overexpression enhanced cell proliferation and tissue repair in both crystal-treated and untreated cells (with a greater degree in the untreated cells). Moreover, alpha-tubulin overexpression caused a reduction in crystal-cell adhesion. Western blot analyses of apical membrane fraction (purified by a recently established peeling method) showed decreased levels of some crystal-binding proteins, including alpha-enolase, HSP70 and HSP90, in the alpha-tubulin-overexpressed cells.

Conclusions: In summary, alpha-tubulin overexpression could prevent calcium oxalate crystal-induced cytotoxicity, promote cell viability, proliferation and tissue repair, and attenuate crystal-cell adhesion by decreasing levels of potential crystal receptors. These findings implicate that alpha-tubulin may have protective roles in calcium oxalate kidney stone disease, particularly at the crystal-cell adhesion step.