(Background) I investigated which structural segment of osteopontin (OPN), a matrix component of urinary stones, is significantly related to the formation of urinary stones.
(Methods) I prepared several kinds of OPNs under various conditions and compared the effects of these OPNs on calcium oxalate (CaOx) crystal using RI counts obtained by the seed crystal method and diluted urine method. Furthermore, I performed scanning electron microscopic (SEM) observation of CaOx crystals used in these experiments and evaluated the effects of OPN based on morphological changes in CaOx crystals. The following OPNs were used in this study: human recombinant OPN (rOPN), human native OPN (nOPN) purified from human milk, denatured OPN (dOPN) obtained by adding organic solvent during the course of nOPN purification, and asiaro OPN (aOPN) obtained by removing sialic acid after enzymatic digestion of nOPN.
(Results) When the effects of OPNs (15μg/ml) were evaluated by the seed crystal method, the following inhibitory activities were observed: nOPN (82%), aOPN (56%), dOPN (49%) and rOPN (15%). When the effects of OPNs (150μg/ml) were evaluated by the undiluted urine method, the following inhibitory activities were observed: nOPN (38%), aOPN (27%), dOPN (21%) and rOPN (0%). Furthermore, using nOPN, I performed SEM observation of CaOx crystals and found that nOPN mainly inhibited CaOx crystal aggregation.
(Conclusion) Since the inhibitory activity of nOPN was observed not only in the seed crystal method, but also in the undiluted urine method, it was suggested that nOPN may play an important role in the living body during the course of urinary stone formation. Moreover, the inhibitory activity of OPN was not due to its primary structure, but it was closely related to its higher-order structure and side chains including sialic acid. Furthermore, it was clarified that the inhibitory activity of OPN mainly resulted from inhibition of CaOx crystal aggregation rather than growth inhibition in these crystals.