Urinary calculus formation is believed to be preceded by crystal nucleation in renal tubular fluid, growth and aggregation of these crystals, and subsequent attachment of the crystals of renal epithelium. Urinary proteins probably influence these processes, but the nature of the interaction between urinary proteins and calcium oxalate and phosphate crystals is only partly understood. Osteopontin (OPN) is a glycosylated phosphoprotein that is secreted by renal cortical cells in culture. It is a potent inhibitor of calcium oxalate crystal growth in vitro. OPN has been found in human urine, as well as in renal stone matrix; its role in renal stone formation is unclear. Regulation of OPN production has not been studied in the kidney; however, in other tissues production is regulated by a variety of hormones and growth factors, including PTH and 1,25(OH)2D3. The effect that these hormones have on the protein are complex, however, as they may change the types of post-translational modification that the protein undergoes, in a tissue-specific manner. For example, 1,25(OH)2D3 stimulates production of a non-phosphorylated form of OPN in one target tissue, and phosphorylation may change OPN's function. The investigator has isolated OPN from renal cortical cells in primary culture, and has used antibodies raised to the protein to localize the sites of OPN production in the kidney. This grant request seeks to address several questions related to the function of OPN in the kidney. Thus: 1) using the cell culture system, and assays for crystal growth, the nature of the post-translational modifications introduced into OPN by renal cells will be studied, as well as their effect on crystal growth. The effect of phosphorylation, glycosylation and sulfation will be investigated, as well as the post-translational processing of OPN by cleavage of a peptide from the C-terminal; 2) regulation of OPN production by hormonal stimuli will be studied in vitro and in vivo, using methods to detect hormonal effects on OPN message, as well as protein secretion. Effects on post-translational modifications will also be sought; and 3) a new assay will be characterized, that the applicant hopes will provide a sensitive way to detect effects of urinary macromolecules on crystal nucleation. It is likely that abnormalities of urinary crystal growth inhibitors contribute to the pathogenesis of renal stones in a subset of stone formers. A better understanding of the ways that proteins influence crystallization in fluids that are supersaturated with respect to calcium salts may lead to more effective therapy for these patients, and may also contribute to a better understanding of pathologic calcification occurring in other sites.
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