Tamm-Horsfall protein (THP) is thought to protect against calcium oxalate monohydrate (COM) stone formation by inhibiting COM aggregation. Several studies reported that stone formers produce THP with reduced levels of glycosylation, particularly sialic acid levels, which leads to reduced negative charge. In this study, normal THP was treated with neuraminidase to remove sialic acid residues, confirmed by an isoelectric point shift to higher pH. COM aggregation assays revealed that desialylated THP (ds-THP) promoted COM aggregation, while normal THP inhibited aggregation. The appearance of protein aggregates in solutions at ds-THP concentrations ≥1 µg/mL in 150 mM NaCl correlated with COM aggregation promotion, implying that ds-THP aggregation induced COM aggregation. The aggregation-promoting effect of the ds-THP was independent of pH above its isoelectric point, but was substantially reduced at low ionic strength, where protein aggregation was much reduced. COM aggregation promotion was maximized at a ds-THP to COM mass ratio of ~0.025, which can be explained by a model wherein partial COM surface coverage by ds-THP aggregates promotes crystal aggregation by bridging opposing COM surfaces, whereas higher surface coverage leads to repulsion between adsorbed ds-THP aggregates. Thus, desialylation of THP apparently abrogates a normal defensive action of THP by inducing protein aggregation, and subsequently COM aggregation, a condition that favors kidney stone formation.

No 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.