A comparison of the binding of urinary calcium oxalate monohydrate and dihydrate crystals to human kidney cells in urine

Abstract

To compare the binding kinetics of urinary calcium oxalate monohydrate (COM) and dihydrate (COD) crystals to human kidney (HK-2) cells in ultra-filtered (UF), and centrifuged and filtered (CF) human urine; and to quantify the binding of COM and COD crystals to cultured HK-2 cells in UF and CF urine samples collected from different individuals. Urine was collected from healthy subjects, pooled, centrifuged and filtered. (14) C-oxalate-labelled COM and COD crystals were precipitated from the urine by adding oxalate after adjustment of two aliquots of the urine to 2 and 8 mm Ca(2+), respectively. For the kinetic study, the crystals were incubated with HK-2 cells for up to 120 min in pooled CF urine adjusted to 2 and 8 mm Ca(2+). Identical experiments were also carried out in UF urine samples collected from the same individuals. For the quantitative study, the same radioactively labelled COM and COD crystals were incubated with HK-2 cells for 50 min in separate CF and UF urines collected from eight healthy individuals at the native Ca(2+) concentrations of the urines. Field emission electron microscopy and Fourier transform-infrared spectroscopy were used to confirm crystal morphology. COM and COD crystals generally bound more strongly at 8 mm than at 2 mm Ca(2+). The kinetic binding curves of COM were smooth, while those of COD were consistently biphasic, suggesting that the two crystal types induce different cellular metabolic responses: HK-2 cells crystals appear to possess a transitory mechanism for detaching COD, but not COM crystals. In UF urine, COM binding was significantly greater than that of COD at 2 mm Ca(2+), but at 8 mm Ca(2+) the binding of COD was greater at early and late time points. COD also bound significantly more strongly at early time points in CF urine at both 2 and 8 mm Ca(2+). In both CF and UF urine, there was no difference between the binding affinity of urinary COM and COD crystals. Binding of both COM and COD crystals to cultured human renal epithelial cells is influenced by urinary macromolecules and ambient Ca(2+) concentration. HK-2 cells appear to possess a mechanism for the rapid detachment of bound COD crystals, making it difficult to show any unambiguous overall difference between the binding affinity of COM and COD crystals.