The majority of human urinary stones are primarily composed of calcium salts. Although normal urine is frequently supersaturated with respect to calcium oxalate, most humans do not form stones. Inhibitors are among the multiple factors that may influence the complex process of urinary stone formation. We have isolated an inhibitor of calcium oxalate crystal growth from human urine by monoclonal antibody immunoaffinity chromatography. The N-terminal amino acid sequence and acidic amino acid content of this aspartic acid-rich protein, uropontin, are similar to those of other pontin proteins from bone, plasma, breast milk, and cells. The inhibitory effect of uropontin on calcium oxalate crystal growth in vitro supports the concept that pontins may have a regulatory role. This function would be analogous to that of other members of the aspartic acid-rich protein superfamily, which stereospecifically regulate the mineralization fronts of calcium-containing crystals.Urinary tract stone disease is a common human malady, and the vast majority of stones formed in the urinary space are mineralized with calcium salts (1, 2). The elements contained in urine also provide a potential model system for evaluating the biologic control of mineralization in other body fluids. Although normal urine is frequently supersaturated with respect to calcium oxalate, most humans do not form stones. Urinary stone formation is a complex process involving multiple factors, and the precise role of the inhibitors that are present within urine is uncertain. The majority of the inhibition of crystal growth observed in normal urine is due to the presence of protein macromolecules rather than to the presence of lower molecular weight molecules (3). We approached the problem of identifying other crystal inhibitor proteins by preparing monoclonal antibodies from rats immunized with the main inhibitory peak of human urine protein (3). One of these monoclonal antibodies was used to purify an inhibitor of calcium oxalate crystal growth from human urine by immunoaffinity chromatography. METHODS Protein Purification. Human urine samples were carried through all procedures in the presence of 0.02% sodium azide and two protease inhibitors, 0.5 mM phenylmethanesulfonyl fluoride and 1.0 mM N-ethylmaleimide, and were partially depleted of the most abundant protein in normal urine, Tamm-Horsfall protein (TH), by salt precipitation followed by centrifugation at 5000 x g for 30 min (4). TH-depleted urine was adsorbed to DEAE-cellulose, batch eluted, and fractionated by DEAE-cellulose column chromatography, using a 0.1-0.4 M NaCl linear gradient in Tris buffer (3). The TH depletion step was performed since we found that TH is present within the main inhibitory peak from DEAE-cellulose and does not inhibit crystal growth in the assay used in the present study (5). Inhibitory activity of fractions was assayed by measuring the inhibition of incorporation of [14C]oxalate (Amersham) into calcium oxalate monohydrate seed crystals from a metastable calcium oxalat...

Therapeutic Level I. See Instructions for Authors for a complete description of levels of evidence.

The authors provide an ethical framework for considering researchers' obligations to human research participants in low-income countries.

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