Ds. Bautista et al., LOW-MOLECULAR-WEIGHT VARIANTS OF OSTEOPONTIN GENERATED BY SERINE PROTEINASES IN URINE OF PATIENTS WITH KIDNEY-STONES, Journal of cellular biochemistry, 61(3), 1996, pp. 402-409
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.