LAUSR

Hyperuricosuria is well documented to get involved in the pathogenesis of kidney stone disease, especially uric acid (UA) and calcium oxalate (CaOx) types. Nevertheless, hyperuricosuria-induced alterations in renal tubular cells and cascade mechanisms that subsequently trigger kidney stone formation remained largely unknown. This study aimed to examine changes in cellular proteome and function in renal tubular cells after treatment with high-dose UA. MDCK cells were incubated with 3.5 mM uric acid for 48-h. Cellular proteins were extracted and subjected to quantitative proteomics analysis using 2-D PAGE followed by nanoLC-ESI-ETD MS/MS. The proteome data were confirmed by Western blotting, whereas functional analyses were performed using various assays. Quantitative proteomics analysis revealed significant changes in levels of 22 proteins in the UA-treated cells that were related to ATPase activity, mitochondrial transport, cell adhesion and molecule binding, cell projection organization and maintenance of location network, metabolic process, response to unfold proteins, and vesicle-mediated transport. Western blotting confirmed changes in levels of cellular proteins identified by quantitative proteomics. Functional assays revealed an increase in intracellular ATP level and enhancement of tissue repair capability in the UA-treated cells. Interestingly, isolation/purification of apical membranes followed by Western blotting showed the increased levels of HSP70 and HSP90 (the known receptors for CaOx crystals) on apical membranes of the UA-treated cells. CaOx crystal-cell adhesion assay revealed significant increase in CaOx-binding capability of the UA-treated cells, whereas neutralization of the surface HSP70 and HSP90 using their specific monoclonal antibodies caused significant reduction in such binding capability (even below the basal level). These findings highlighted changes in renal tubular cells in response to high-dose UA that might be related to the pathogenic mechanisms of kidney stone disease associated with hyperuricosuria.