Urolithiasis is a serious, debilitating and costly problem in societies throughout the world. The role of urinary proteins in calcium oxalate (CaOx) kidney stone formation, particularly intracrystalline proteins, remains a mystery. The planned work will clarify the role of superficial and intracrystalline CaOx proteins, especially OPN and UPTF1, in the attachment of urinary crystals to renal epithelial cells, as well as their subsequent disintegration and dissolution. Using a Madin-Darby canine kidney (MDCK) cell line model of crystal-cell interaction, the studies will: (1) determine the effects of intracrystalline and surface-bound OPN and UPTF1 on the binding, phagocytosis, intracellular disintegration and dissolution of CaOx crystals. (2) (a) identify which lysosomal proteases digest intracrystalline OPN and UPTF1 and (b) determine the effects of crystal phagocytosis on their individual specific activities, as well as that of total protease specific activity. (3) (a) determine the effects of intracrystalline OPN and UPTF1 on the gene expression (mRNA) of individual proteases identified in Aim 2 and on the gene expression (mRNA) and further cellular production of UPTF1 and OPN. (b) Determine the effects of [Ca] and [Ox] on the gene expression (mRNA) of OPN and UPTFI. (4) (a) compare the ability of urinary CaOx crystals from male and female stone formers and controls to be bound, phagocytosed and destroyed intracellularly; (b) determine the effects of those crystals on the specific activity and cellular synthesis of the lysosomal enzymes identified in Aim 2, as well as the cellular synthesis of OPN and UPTF1; (c) quantify the urinary activities of those enzymes in stone formers and healthy subjects. The proposed studies will use a combination of cell culture, high-resolution microscopy, enzymatic analysis, protein chemistry and molecular biological techniques. The data generated will offer boundless promise in the design of drugs for stone prophylaxis, the construction of porous crystals for medical and industrial applications, and for understanding the cycle of biomineral formation, destruction and reclamation throughout the natural world.
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