Characterization of uronic-acid-rich inhibitor of calcium oxalate crystallization isolated from rat urine

Human urine contains several macromolecules which inhibit calcium oxalate crystallization. Uronic-acid-rich protein (UAP), a glycoprotein with a molecular weight of approximately 35 kDa, is one such inhibitor. Here we report the characterization of UAP extracted from rat urine using three chromatographic steps including diethylaminoethanol (DEAE)-Sephacel, Sephacryl S-300 and Mono Q column and compare it with human UAP. The molecular weight of rat UAP (UAP_r) is similar to that of human UAP (UAP_h), being approximately 35 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Their amino acid compositions are identical, they contain a high percentage of aspartic and glutamic acids and they react positively in the carbazole reaction, suggesting that they contain uronic acid. The inhibitory activities of UAP_h and UAP_r were assayed on a calcium oxalate crystallization system in vitro using [⁴⁵Ca]calcium chloride. Both exert a strong inhibition, suggesting that UAP_r, like UAP_h, plays an important role in preventing and reducing calcium oxalate crystallization in the urine. On Western blot analysis, both UAP_h and UAP_r immunoreact with inter--trypsin inhibitor (ITI) antibody. Nevertheless, using the Ouchterlony immunodiffusion technique, there was no precipitation 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 superfamily of macromolecules which contribute to the regulation of the calcium oxalate crystallization process.