Establishment of a novel colorimetric assay for high-throughput analysis of calcium oxalate crystal growth modulation

Abstract

Crystal growth is one of the most important mechanisms of calcium oxalate (CaOx) kidney stone formation. However, identification and characterization of CaOx crystal growth modulators were under-investigated in the past, probably due to the lack of an assay that could measure modulatory activities of a large number of candidates simultaneously. We therefore developed such a high-throughput assay based on colorimetric measurements of free calcium ions using Arsenazo III in a 96-well microplate, and a microplate reader (λ620 nm). CaOx monohydrate (COM) was crystallized in the absence (blank control) or presence of 5 mM sodium citrate (Na₃Citrate) (positive control) or 5 mM sodium chloride (NaCl) (negative control). The results showed that Na₃Citrate, but not NaCl, could significantly inhibit COM crystal growth. The reliability of this novel assay was validated by quantitative analysis of COM crystal size. Degree of the inhibitory activity of Na₃Citrate obtained from our novel assay was comparable to that obtained from the reference method (34.5 ± 4.3 vs. 40.0 ± 1.6%Inhibition, respectively; p was not significant). The high-throughput capability of the novel assay was demonstrated by simultaneous measurement of 33 samples (a triplicated set of 11 different concentrations of Na₃Citrate) with linear correlation between Na₃Citrate concentrations and COM crystal growth inhibitory activities (r = 0.893; p < 0.0001). In summary, we have successfully established a novel assay for high-throughput analysis of CaOx crystal growth modulatory activities of compounds that will be very useful for a large-scale analysis of CaOx crystal growth modulators.