Low-molecular-weight variants of osteopontin generated by serine proteinases in urine of patients with kidney stones

Abstract

Osteopontin (OPN) is a multifunctional glycosylated phosphoprotein found 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 quantify OPN levels in the urine, we developed an ELISA using a combination of a mouse monoclonal and rabbit polyclonal antibodies raised against a recombinant glutathione-S-transferase-human OPN fusion protein. In a group of 34 patients diagnosed with kidney stones compared with a control group of 23 normal individuals, we found that OPN levels in urine of the patient and control groups ranged from 0.01 to 2.7 micrograms/ml, with no significant difference in their medians (P > 0.8, Mann-Whitney test). OPN in urine was qualitatively assessed by Western blotting using a biotinylated monoclonal antibody to detect various molecular forms. 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 (< or = 40 kDa) compared to 2 of 23 for the control group (P < 0.03, chi 2 test). Mixing experiments indicated that urine samples with aberrant OPN contain proteases inhibitable with phenylmethylsulfonyl fluoride. Such proteases could break down normal urine OPN in vitro. Therefore, urine from a high frequency of kidney stone patients contains serine proteases that contribute to proteolytic cleavage of OPN.

Categorical meta-analysis of Osteopontin as a clinical cancer marker

“…It is therefore more cancer-specific than total Osteopontin. Fragments of the Osteopontin protein excreted into the urine have been used to diagnose ovarian cancer (31) and renal conditions (32). …”

Urine protein markers distinguish stone-forming from non-stone-forming relatives of calcium stone formers

“…Bautista et al (5) found aberrant LMM OPN variants more frequently in SF than normal controls due to increased serine proteases in their urine. We have not observed that urine PF1, ITI, CD59, or calgranulin is susceptible to rapid degradation in the absence of protease inhibitors (data not shown).…”

Localization and expression of osteopontin in the rotator cuff tendons in patients with calcifying tendinitis

“…OPN has been reported to be synthesized by the cells in mineralizing developmental tissues [13,25,26], and several biochemical studies suggest that OPN regulates cell attachment, migration [22,23], and tissue mineralization [4]. In addition, OPN has been reported to play an important role in the calcification that occurs in several pathological conditions in humans, including arteriosclerosis [8,10,12,18,27,28,32] and urinary stones [2,15,20,21,33]. However, no reports exist regarding the involvement of OPN in ectopic calcification in tendon tissues.…”