Relative deficiency of acidic isoforms of osteopontin from stone former urine

Abstract

We have tested the relative electrophoretic mobility of osteopontin (OPN) isolated from urine obtained from normal individuals (NU) against similar samples derived from the urine of stone formers (SFU) using high-resolution isoelectric focusing (isoelectric point, pI range 3.5–4.5) in 2D electrophoresis, with Western blot detection. We also report the results from competitive ELISA analyses of these samples. We demonstrated that human urinary OPN has a discrete four band separation pattern that conforms to four previously documented OPN isoforms. The lower two Mr isoforms migrate to a greater degree toward the acidic end of the gel than do the higher two Mr isoforms. Densitometry of the signal reveals significant difference in the migration pattern of OPN from SFU as compared to that from NU based on an analysis of the spot intensities grouped in 0.1 pI unit increments. A novel method for the calculation of a weight-averaged pI based on the relative signal strength in an OPN 2D Western blot was developed. The analysis revealed a significantly increased weight-averaged pI values for the higher Mr forms of OPN in the stone former compared to normal population. Additionally, alkaline phosphatase-treated NU samples resulted in a significant average pI shift of 0.05 units in the alkaline direction, suggesting that a decrease in the average degree of phosphorylation could be responsible for the difference between NU and SFU pI.

The Way from Renal Calcifications and Urinary Crystals to Kidney Stones: An Important Aspect in the Pathogenesis of Calcium Nephrolithiasis

“…Further experiments which were performed with latex beads in almost electrolyte and especially Ca-free albumin solutions showed that the AGN of UM-coated particles too can base on a hydrophobic effect [19]. Pathological UMs with reduced anionic groups are found to be responsible for crystal AGN in urine [33][34][35] and were observed in urine of some stone patients [36][37][38]. However, also dialysis in a hemofilter abolishes the inhibitory activity of UMs [16].…”

Biomimetic Randall’s plaque as an in vitro model system for studying the role of acidic biopolymers in idiopathic stone formation

“…OPN is usually believed to be inhibitory to nucleation [55, 72–74], but with high supersaturations (or other conditions) it can induce the PILP process, and has been shown to do so in vitro (Figure 4b) [66]. In addition, OPN undergoes significant post-translational modification; the degree of phosphorylation, glycosylation, or both could change it from an inhibitor to a promoter of crystallization [17, 34, 40, 45, 72, 75]. In fact, studies have even shown that there are differences in the properties of OPN in urine from stone formers compared to non-stone formers [40].…”

“…In addition, OPN undergoes significant post-translational modification; the degree of phosphorylation, glycosylation, or both could change it from an inhibitor to a promoter of crystallization [17, 34, 40, 45, 72, 75]. In fact, studies have even shown that there are differences in the properties of OPN in urine from stone formers compared to non-stone formers [40]. Therefore, OPN was a protein of keen interest to initiate our studies.…”

From crystalluria to kidney stones, some physicochemical aspects of calcium nephrolithiasis

“…The inhibition of crystallization processes by UM's is mainly attributed to anionic residues like carboxyglutamic acid [44,45] , phosphate [46,47] and sialic acid [48,49] which have a high affinity to the Ca of crystals. Some of these anionic groups being responsible for the electro-negative charge of coated crystals were found to be reduced in UM's of stone patients.…”

“…Physicochemical aspects of calcium nephrolithiasis acid groups [47,49] . A reduction of anionic groups reinforces as mentioned above hydrophobic activity of proteins and thus the tendency to self AGN and to the binding to hydrophobic domains of other proteins.…”