Nephrocalcin (NC) is a calcium-binding glycoprotein that has been isolated from human urine and human kidney tissue culture, and that inhibits growth of the calcium oxalate crystals in vitro. Molecular abnormalities have been found in NC isolated from the pooled urine of calcium stone formers, which decrease its affinity for crystal surface, and thus its ability to slow crystal growth. This abnormality includes a deficiency of gamma-carboxglutamic acid (GLA). It is unknown whether abnormalities in the structure and function of NC contribute to kidney stone formation in certain individuals. The goal of this proposal is to begin to study the role of NC in stone-formation in individual calcium stone-formers, and to study the factors that regulate production of the protein. In previous studies by the PI, antibodies to the protein have been used to estimate excretion rates of NC in normal men and women, and to correlate this with the ability of the urine to inhibit calcium oxalate crystal growth in a seeded crystal growth system. Our studies have also shown that Vitamin D may play a role in regulation of NC excretion. Finally a similar protein has been isolated from kidney cortical tissue culture supernatants, providing a model for in vitro studies of regulation of protein production. To investigate the possibility that some stone-formers make an abnormal NC molecule, decreased either in amount or in ability to inhibit crystal growth, and to learn more about the abnormalities themselves, three types of studies are proposed; I) Calcium stone formers with known metabolic defects and stone composition will be studied using ELISA assay to measure NC excretion, and assays of crystal growth to detect abnormalities in inhibitory activity. Isolation and study of abnormal proteins will be done II) Patients taking coumadin will also be studied. If lack of GLA is the crucial defect, these patients may display abnormalities similar to those of stone-formers. III)Studies of regulation of protein production will be carried out in cell culture. Effect of vitamin D, PTH, medium calcium, vitamin K deficiency, etc. can be assessed. It is likely that inhibitor abnormalities contribute to the pathogenesis of stones in a subset of stone-formers. Knowledge about the specific abnormalities that lead to impaired ability to inhibit crystal growth and of the factors that regulate production of this protein may lead to effective therapeutic interventions.
