ASSOCIATION OF URINARY MACROMOLECULES WITH CALCIUM OXALATO CRYSTALS INDUCED IN-VITRO IN NORMAL HUMAN AND RAT URINE

This study was undertaken to identify proteins which are found associa ted with calcium oxalate crystals induced in vitro in normal human and rat urine. Crystallization was initiated by adding sodium oxalate ind ividually to each urine sample without centrifugation and filtration. Crystals were collected and analyzed by scanning electron microscopy a nd X-ray diffraction. Crystal matrix proteins (CMPs) were obtained by demineralization of the crystals with ethylenediaminetetra-acetic acid (EDTA) and analyzed by western blotting technique for immunological i dentification. Crystals produced in human urine were found to be a mix ture of calcium oxalate monohydrate (COM) and calcium oxalate dihydrat e (GOD) while those produced in rat urine were exclusively GOD. CMPs e xtracted from crystals in human urine comprised, in addition to prothr ombin-related proteins, osteopontin and albumin. However, CMPs extract ed from crystals in rat urine contained only osteopontin and albumin. Prothrombin-related proteins were found only in trace amounts. In a se parate experiment, rat urine samples were supplemented with COM before inducing crystallization. Similar results were observed showing that CMP contained osteopontin, albumin and trace amounts of prothrombin-re lated proteins. We conclude that several urinary macromolecules includ ing not only prothrombin-related proteins, but also osteopontin and al bumin, become associated with CaOx crystals. The incorporation of thes e proteins in growing stones is not only due to the presence of gamma- carboxyglutamic acid as it was suggested for prothrombin-related prote ins, but may be due to other factors such as urinary chemistry, presen ce of glutamic and aspartic acid residues, and calcium-binding sites.