Modulatory Effect of the 23-kD Calcium Oxalate Monohydrate Binding Protein on Calcium Oxalate Stone Formation during Oxalate Stress

Abstract

Aims: To isolate, characterize, and quantify the 23-kD calcium oxalate monohydrate (COM) binding protein in the urine of controls and calcium oxalate stone formers and to study its role in kidney stone formation. Methods: Calcium oxalate crystals were prepared and allowed to interact with human control kidney homogenate as well as urine of controls and calcium oxalate stone formers. EDTA extract was used for the separation of the 23-kD COM-binding protein (partially purified). This partially purified 23-kD COM-binding protein was further separated by DEAE-cellulose column chromatography. SDS-PAGE confirmed the molecular weight. An antibody was raised against the renal 23-kD COM-binding protein in rabbits. The 23-kD COM-binding protein was quantified in the urine from controls and stone formers by ELISA. Thiol group quantification, oxalate-binding assay, and calcium oxalate crystal nucleation and aggregation were performed. Morphological changes of the calcium oxalate crystals induced by the urinary 23-kDa protein were determined using scanning electron microscopy. The expression of this protein using different concentrations of oxalate was also determined in an in vitro model. Results: The urinary excretion of the 23-kD COM-binding protein varies between 0.5 and 1.5 mg/24 h in controls, while in stone former its excretion was found to range from 5 to 7 mg/24 h. The protein isolated from urine was found to inhibit crystal nucleation and aggregation in controls, while the protein isolated from stone formers exhibited less inhibitory activity with reduced thiol groups. The 23-kD COM-binding protein derived from control urine formed COM crystals and intertwined calcium oxalate dihydrate crystals in a crystal growth system, while protein isolated from stone formers’ urine induced aggregation of COM crystals. This protein expression was found to be increased with increasing concentration of oxalate in renal epithelial cells of the African green monkey kidney (VERO) cell line. Conclusions: Increased expression and excretion of the 23-kD protein was observed in oxalate stress conditions, and in stone formers this protein exhibited a promoting activity. The increased excretion of this protein with promoting activity favors the lithogenic process in stone formers.