LOW-MOLECULAR-WEIGHT VARIANTS OF OSTEOPONTIN GENERATED BY SERINE PROTEINASES IN URINE OF PATIENTS WITH KIDNEY-STONES

Osteopontin (OPN) is a multifunctional glycosylated phosphoprotein fou nd in body fluids, including urine, and has been implicated in urinary stone formation. We tested the hypothesis that OPN levels in urine of patients with kidney stones differed from normal individuals. To quan tify OPN levels in the urine, we developed an ELISA using a combinatio n of a mouse monoclonal and rabbit polyclonal antibodies raised agains t a recombinant glutathione-S-transferase-human OPN fusion protein. In a group of 34 patients diagnosed with kidney stones compared with a c ontrol group of 23 normal individuals, we found that OPN levels in uri ne of the patient and control groups ranged from 0.01 to 2.7 mu g/ml, with no significant difference in their medians (P > 0.8, Mann-Whitney test). OPN in urine was qualitatively assessed by Western blotting us ing a biotinylated monoclonal antibody to detect various molecular for ms. The urine of most individuals contained OPN species within in the 55- to 66-kDa electrophoretic mobility range. However, a significantly higher proportion of individuals in the patient group (13 of 34) was found to have aberrant urine OPN species (less than or equal to 40 kDa ) compared to 2 of 23 for the control group (P < 0.03, chi(2) test). M ixing experiments indicated that urine samples with aberrant OPN conta in proteases inhibitable with phenylmethylsulfonyl fluoride. Such prot eases could break down normal urine OPN in vitro. Therefore, urine fro m a high frequency of kidney stone patients contains serine proteases that contribute to proteolytic cleavage of OPN. (C) 1996 Wiley-Liss, I nc.