Neutrophil gelatinase-associated lipocalin (NGAL) promotes calcium oxalate crystallization, growth, aggregation, adhesion to renal cells and invasion through extracellular matrix via its oxalate-binding capability

Abstract

Neutrophil gelatinase-associated lipocalin (NGAL) is commonly found in the urine of patients with kidney diseases including nephrolithiasis. Nevertheless, its role in stone formation was unknown. Herein, we systematically examined the effects of NGAL on multiple calcium oxalate (CaOx) stone-development processes. Recombinant human NGAL was produced, purified and subjected to multiple crystal assays. The analyses demonstrated that all NGAL concentrations (0.01–10 μg/ml) concentration-dependently increased the size of CaOx crystals after initial crystallization. At later processes, crystal size expansion, aggregation and adhesion to renal cells were also concentration-dependently induced by NGAL. Additionally, NGAL at all concentrations enhanced extracellular matrix (ECM) invasion by crystals. Mechanistic examinations revealed that NGAL had an affinity to bind oxalate ions without affinity with calcium ions, and the binding of NGAL to CaOx crystal surfaces was confirmed by an immunofluorescence method. Finally, neutralization of the crystal-NGAL complex using a specific anti-NGAL antibody prior to crystal-cell adhesion assay revealed that NGAL functioned as an adhesive medium to enhance crystal-cell adhesion. In conclusion, our findings indicate that NGAL promotes CaOx stone development by enhancing CaOx crystallization, growth, aggregation, adhesion to renal cells and ECM invasion via its oxalate-binding capability.