Molecular modulation of calcium oxalate crystallization by osteopontin and citrate

Abstract

Calcium oxalate monohydrate (COM), which plays a functional role in plant physiology, is a source of chronic human disease, forming the major inorganic component of kidney stones. Understanding molecular mechanisms of biological control over COM crystallization is central to development of effective stone disease therapies and can help define general strategies for synthesizing biologically inspired materials. To date, research on COM modification by proteins and small molecules has not resolved the molecular-scale control mechanisms. Moreover, because proteins directing COM inhibition have been identified and sequenced, they provide a basis for general physiochemical investigations of biomineralization. Here, we report molecular-scale views of COM modulation by two urinary constituents, the protein osteopontin and citrate, a common therapeutic agent. Combining force microscopy with molecular modeling, we show that each controls growth habit and kinetics by pinning step motion on different faces through specific interactions in which both size and structure determine the effectiveness. Moreover, the results suggest potential for additive effects of simultaneous action by both modifiers to inhibit the overall growth of the crystal and demonstrate the utility of combining molecular imaging and modeling tools for understanding events underlying aberrant crystallization in disease. Kidney stone disease is a common chronic disorder in humans with the majority of stones being primarily composed of calcium oxalate monohydrate (COM) crystals. COM is the most thermodynamically stable form of calcium oxalate and is the predominant calcium crystallite in many plants (1). Because normal urine is frequently supersaturated with respect to calcium oxalate, urinary crystals are often formed. In most humans, progression from crystalluria to stone disease is prevented by biologic control mechanisms. Normal urine contains inhibitors that decrease the formation, growth, and aggregation of COM crystals (2-4). However, the ways that these normal urinary proteins and small molecules modify COM have not been previously defined at a molecular level. In this article, our investigations using in situ atomic force microscopy (AFM) and molecular modeling provide molecular-scale views of COM modulation by two urinary constituents, a small organic anion, citrate, and a protein, osteopontin (OPN).Citrate and OPN were chosen for study because urinary levels of both citrate and OPN have been shown to inhibit the growth and change the gross morphology of calcium oxalate crystals (5-7). Citrate is a short-chain nonplanar molecule with three carboxylic acid groups. It is normally secreted into urine and is also widely used in the therapy of renal stone disease (8). OPN is a single-chain protein with a peptide molecular mass of Ϸ33 kDa. Normal human urine contains levels of OPN (Ͼ100 nM) that markedly inhibit several aspects of COM crystallization (9). OPN has an abundance of sequence domains rich in dicarboxylic acids. Because the acidic residues o...

Adhesion at calcium oxalate crystal surfaces and the effect of urinary constituents

“…AFM imaging has proven to be a powerful tool for real-time visualization of crystal growth on specific faces of organic and inorganic crystals (21)(22)(23)(24), including COM (15,25). AFM imaging also has been used to obtain images of kidney stones (26).…”

Dynamics of crystallization and dissolution of calcium orthophosphates at the near-molecular level

“…Four mechanisms have been proposed for crystallization modulation through impurity (including organic and inorganic molecules) interactions: (1) step pinning [45], (2) incorporation [34], (3) kink blocking, and (4) step-edge adsorption. Each of these major mechanisms for growth inhibition exhibits a characteristic dependence of step speed on supersaturation and impurity concentration [35].…”

Acceleration of Calcite Kinetics by Abalone Nacre Proteins

“…[1,2] One prevailing view is that mineral-associated macromolecules are responsible for initiating and stabilizing non-equilibrium crystal polymorphs and morphologies through interactions between anionic moieties and cations in solution or at mineral surfaces. [3][4][5][6] Indeed, numerous studies have demonstrated that bio-organic additives can dramatically alter crystal shapes and growth-rates in vitro. However, previous molecular-scale studies revealing mechanisms of growth modification focused on small molecules such as amnio acids or peptides and always observed growth inhibition.…”