CHARACTERIZATION OF URONIC ACID-RICH INHIBITOR OF CALCIUM-OXALATE CRYSTALLIZATION ISOLATED FROM RAT URINE

Human urine contains several macromolecules which inhibit calcium oxal ate crystallization. Uronic-acid-rich protein (UAP), a glycoprotein wi th a molecular weight of approximately 35 kDa, is one such inhibitor. Here we report the characterization of UAP extracted from rat urine us ing three chromatographic steps including diethylaminoethanol (DEAE)-S ephacel, Sephacryl S-300 and Mono Q column and compare it with human U AP. The molecular weight of rat UAP (UAP(r)) is similar to that of hum an UAP (UAP(h)), being approximately 35 kDa as estimated by sodium dod ecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Their amin o acid compositions are identical, they contain a high percentage of a spartic and glutamic acids and they react positively in the carbazole reaction, suggesting that they contain uronic acid. The inhibitory act ivities of UAP(h) and UAP(r) were assayed on a calcium oxalate crystal lization system in vitro using [Ca-45] calcium chloride. Both exert a strong inhibition, suggesting that UAP(r), like UAP(h), plays an impor tant role in preventing and reducing calcium oxalate crystallization i n the urine. On Western blot analysis, both UAP(h) and UAP(r) immunore act with inter-alpha-trypsin inhibitor (ITI) antibody. Nevertheless, u sing the Ouchterlony immunodiffusion technique, there was no precipita tion line between ITI antibody and UAP. Therefore, we hypothesize that UAP is related to ITI and that they may have the same epitope but are not completely identical. We conclude that UAP belongs to the ITI sup erfamily of macromolecules which contribute to the regulation of the c alcium oxalate crystallization process.