Functions of
Tamm-Horsfall protein (THP), the most abundant human urinary
protein, have been studied for decades. However, its precise roles in
kidney stone formation remain controversial. In this study, we aimed to clarify the roles of native human urinary THP in
calcium oxalate monohydrate (COM)
kidney stone formation. THP was purified from the human urine by adsorption method using
diatomaceous earth (DE). Its effects on stone formation processes, including COM crystallization, crystal growth, aggregation, crystal-cell adhesion and invasion through extracellular matrix (ECM), were examined. SDS-PAGE and Western blotting confirmed that DE adsorption yielded 84.9% purity of the native THP isolated from the human urine. Systematic analyses revealed that THP (at 0.4-40 μg/ml) concentration-dependently reduced COM crystal size but did not affect the crystal mass during initial crystallization. At later steps, THP concentration-dependently inhibited COM crystal growth and aggregation, and prevented crystal-cell adhesion only at 40 μg/ml. However, THP did not affect crystal invasion through the ECM. Sequence analysis revealed two large
calcium-binding domains (residues 65-107 and 108-149) and three small
oxalate-binding domains (residues 199-207, 361-368 and 601-609) in human THP. Immunofluorescence study confirmed the binding of THP to COM crystals. Analyses for
calcium-affinity and/or
oxalate-affinity demonstrated that THP exerted a high affinity with only
calcium, not
oxalate. Functional validation revealed that saturation of THP with
calcium, not with
oxalate, could abolish the inhibitory effects of THP on COM crystal growth, aggregation and crystal-cell adhesion. These data highlight the inhibitory roles of the native human urinary THP in COM crystal growth, aggregation and crystal-cell adhesion, which are the important processes for
kidney stone formation. Such inhibitory effects of THP are most likely mediated via its high affinity with
calcium ions.