(231a) Mechanisms of Citrate Derivatives As Inhibitors of Brushite Crystallization

Brushite (dicalcium phosphate dihydrate) is a key mineral associated with kidney stone disease. While calcium oxalate (CaOx) is the predominant constituent of renal calculi, brushite plays a crucial role as a primary precursor in pathological mineralization. It has been identified in early-stage mineral deposits within the renal papillae, particularly in Randall’s plaques, where it putatively facilitates heterogeneous nucleation of hydroxyapatite and CaOx contributing to stone growth. Brushite stones exhibit a higher recurrence rate than other kidney stone types, posing a greater challenge for long-term treatment of urolithiasis by use of foreign substances (modifiers), which interact with crystal surfaces and inhibit growth via various pathways. Over the past three decades, there has been an increase in the prevalence of calcium phosphate kidney stones in humans, which has led to a rising interest in identifying inhibitors of brushite crystallization, including metal ions, small molecules, and protein-based inhibitors. Citrate (CA) is a naturally occurring component of human urine and is known to inhibit the growth of calcium oxalate crystals while its analogue, hydroxycitrate (HCA) with an additional hydroxyl (-OH) group, was found to be more effective.

In this presentation, we discuss our recent findings on the effects of CA, HCA, and combinations thereof on bulk brushite crystallization and surface dynamics over a wide range of supersaturations using a multifaceted approach incorporating bulk crystallization assays, nucleation studies, in situ microfluidics and atomic force microscopy (AFM). Bulk studies reveal that both CA and HCA modify crystal morphology, while time-resolved analyses demonstrate their ability to inhibit both nucleation and subsequent crystal growth, with HCA showing a much higher inhibition efficacy. Combination studies of CA and HCA reveal an antagonistic cooperativity that is highly dependent on the ratio of modifiers and their net concentrations. Microfluidic experiments show that CA acts as a weak, direction-dependent inhibitor of brushite crystal growth, whereas HCA exhibits strong inhibition across both crystallographic directions. Furthermore, in situ AFM imaging of the brushite (010) basal surface reveals that at high supersaturation, CA and HCA both exhibit similar inhibitory effects, whereas at low supersaturation, HCA suppresses step growth via a unique mechanism. Collectively, our findings provide mechanistic insights into the inhibition of brushite crystallization and highlight potential strategies for controlling pathological mineralization in kidney stone diseases.