Crystal-cell Adhesion Research Articles

Biomolecular mechanism of urinary stone formation involving osteopontin

Urinary stones consist of two phases-an inorganic (mineral) phase and an organic (matrix) phase. Studies on the organic components of kidney stones have been undertaken later than those on the inorganic components. After osteopontin was identified as one of the matrix components, the biomolecular mechanism of urinary stone formation became clearer. It also triggered the development of new preventive treatments. Osteopontin expression is sporadically observed in normal distal tubular cells and is markedly increased in stone-forming kidneys. Calcium oxalate crystals adhering to renal tubular cells are incorporated into cells by the involvement of osteopontin. Stimulation of crystal-cell adhesion impairs the opening of mitochondrial permeability transition pores (mPTP) in tubular cells and produces oxidative stress, apoptosis, and osteopontin expression. Macrophages phagocytose and digest a small amount of crystals, but many crystals aggregate into a mass containing osteopontin and epithelial cell debris and are excreted into the renal tubular lumen, becoming nuclei of urinary stones. This biomolecular mechanism is similar to atherosclerotic calcification. Based on these findings, new preventive treatments have been developed. Dietary control such as low-cholesterol intake and the ingestion of antioxidative foods and vegetables have successfully reduced the 5-year recurrence rate. Osteopontin antibodies and cyclosporine A, which blocks the opening of mPTP, have markedly inhibited the expression of osteopontin and urinary stone formation in animal models.

Ceftriaxone crystallization and its potential role in kidney stone formation

Drug-induced nephrolithiasis contributes to 1–2% of the incidence of renal calculi. We examined whether ceftriaxone at therapeutic doses could be crystallized in the urine and also explored its role in kidney stone formation. Crystallization was induced by mixing ceftriaxone sodium at therapeutic urinary excretion levels (0.5–4.0 mg/ml) to calcium chloride at physiologic urinary concentration (5 mM) in deionized (dI) water or artificial urine (AU). The results showed that ceftriaxone was crystallized with free calcium in dose- and time-dependent manner. These ceftriaxone/calcium crystals showed birefringence property under polarized microscope. Individual crystals had needle-shape (5–100 μm in length), whereas the aggregated form had star-burst and irregular-plate shape (40–200 μm in diameter) (note that the crystal sizes were much larger than renal tubular lumens). Calcium-depletion assay revealed that crystallization required free calcium as a substrate. In AU, crystallization remained although it was partially inhibited when compared to that in dI water. Finally, these crystals could tightly adhere onto renal tubular cell surface. Our data demonstrated that ceftriaxone at therapeutic levels could be crystallized with free calcium in the urine under physiologic condition. We hypothesize that tubular occlusion and crystal–cell adhesion may play important role in pathogenic mechanisms of ceftriaxone-induced nephrolithiasis.

Sialylation of urinary prothrombin fragment 1 is implicated as a contributory factor in the risk of calcium oxalate kidney stone formation

Urinary glycoproteins are important inhibitors of calcium oxalate crystallization and adhesion of crystals to renal cells, both of which are key mechanisms in kidney stone formation. This has been attributed to glycosylation of the proteins. In South Africa, the black population rarely form stones (incidence < 1%) compared with the white population (incidence 12-15%). A previous study involving urinary prothrombin fragment 1 from both populations demonstrated superior inhibitory activity associated with the protein from the black group. In the present study, we compared N-linked and O-linked oligosaccharides released from urinary prothrombin fragment 1 isolated from the urine of healthy and stone-forming subjects in both populations to elucidate the relationship between glycosylation and calcium oxalate stone pathogenesis. The O-glycans of both control groups and the N-glycans of the black control samples were significantly more sialylated than those of the white stone-formers. This demonstrates a possible association between low-percentage sialylation and kidney stone disease and provides a potential diagnostic method for a predisposition to kidney stones that could lead to the implementation of a preventative regimen. These results indicate that sialylated glycoforms of urinary prothrombin fragment 1 afford protection against calcium oxalate stone formation, possibly by coating the surface of calcium oxalate crystals. This provides a rationale for the established roles of urinary prothrombin fragment 1, namely reducing the potential for crystal aggregation and inhibiting crystal-cell adhesion by masking the interaction of the calcium ions on the crystal surface with the renal cell surface along the nephron.

Cell-surface matrix proteins and sialic acids in cell-crystal adhesion; the effect of crystal binding on the viability of human CAKI-1 renal epithelial cells.

To investigate the role of sialic acids and cellular matrix proteins as crystal-binding molecules in human calcium-oxalate nephrolithiasis. The well-defined human renal cancer cell line CAKI-1 was used a standard cell culture system. After enzymatic digestion of various cell surface molecules, the binding of alpha2,6 (Sambucus nigra, SN-) and alpha2,3 (Maackia amurensis, MA)-specific lectins to CAKI-1 cells was analysed. Simultaneously, the effect on adhesion and release of calcium oxalate monohydrate crystals was investigated (eight replicates). The effect of crystal adhesion on cell viability was assessed using Trypan blue exclusion (five replicates). Neuraminidase decreased MA-lectin binding of CAKI-1 cells by 39% (P < 0.05) but elevated SN-lectin binding by 812% (P < 0.05). Simultaneously, crystal binding to CAKI-1 cells was increased by 28% (P > 0.05). Pretreatment with collagenase type I, trypsin and dispase II reduced crystal-binding by 61-74% (P < 0.05) with no effect on sialic acid-specific lectin-binding. However, only collagenase type I and dispase (ratio 4 : 1) were also able to release crystals from their receptor-binding sites (P < 0.05). An increase in the number of cell surface-bound crystals correlated significantly with a decrease in cell viability (P < 0.05). alpha2,3-linked sialic acids protect cells from crystal-binding. Much greater SN-lectin binding associated with only moderately increased crystal binding argues against alpha2,6-linked sialic acids as a main target structure of crystals. In contrast, collagen type I, type IV and/or fibronectin seem to be potent crystal-binding molecules on human renal epithelial cells, with collagen type I involved in a potential second step of crystal-cell interaction.