IDENTIFICATION OF PROTEINS EXTRACTED FROM CALCIUM-OXALATE AND CALCIUM-PHOSPHATE CRYSTALS INDUCED IN THE URINE OF HEALTHY AND STONE FORMING SUBJECTS

The purpose of our study was to identify the proteins and investigate the differences, if any, between protein components of the matrices of calcium oxalate (CaOx) and calcium phosphate (CaP) crystals induced i n vitro in whole human urine of healthy individuals and kidney stone p atients. In addition, preliminary studies were performed to understand the effect of centrifugation and filtration of urine on its protein c ontents. Crystallization in urine was induced by addition of an oxalat e or phosphate load. Crystals were collected, washed, and analyzed by scanning electron microscopy, X-ray diffraction, and energy dispersive X-ray microanalysis. Matrix proteins were obtained by demineralizatio n with ethylene diamine tetraacetic acid (EDTA), analyzed by polyacryl amide gel electrophoresis, and identified by western blotting techniqu e. No significant differences were detected between protein components of the matrices of CaOx and CaP crystals and between the crystal matr ices obtained from the urine of normal and stone forming subjects. Alb umin (AB), inter-alpha-inhibitor (I alpha I) related proteins, alpha-1 microglobulin (alpha-1 m), osteopontin (OPN), prothrombin (PT)-relate d proteins and Tamm-Horsfall protein (THP) were identified in matrices of both CaOx and CaP crystals induced in urine from both the normal s ubjects and stone formers. AB, PT-related proteins and OPN were the ma in constituents. The other proteins were present in smaller but detect able amounts. However, CaP crystal matrix, contained a large amount of THP. In addition Cap crystals contained significantly more proteins t han CaOx crystals. Centrifugation and/or filtration of the urine resul ted in reduction of many high molecular weight proteins including THP, AB and OPN in the urine.