Adhesion at calcium oxalate crystal surfaces and the effect of urinary constituents. Kidney stone disease, which occurs in approximately 10% of the U.S. population, causes substantial suffering and occasional renal failure, yet the disease mechanism is poorly understood. Kidney stones are aggregates, most commonly containing microcrystals of calcium oxalate monohydrate (COM) as the primary inorganic constituent. Stones also contain small amounts of embedded proteins, which are thought to play an adhesive role in these aggregates, and they often are found attached to the tip of renal papilla, presumably through adhesive contacts. Although crystallogenesis is essential to stone formation, calcium oxalate crystal growth rates are sluggish, to the extent that during typical urine transit times it is unlikely that single crystals will grow large enough to become lodged in the terminal collecting duct of the kidney based on size alone. Therefore, crystal aggregation and attachment to interfaces at the papillary tip must play a significant role in crystal retention and stone formation. Identification of the most important functional group-crystal face adhesive combinations is crucial to understanding the stability of COM aggregates and the strength of their attachments to epithelial cell surfaces under flow in the renal tubules of the kidney.

Using shared facilities acquired through the UMN MRSEC, investigators Xiaoxia Sheng, Taesung Jung, and Michael Ward, in collaboration with Jeffrey Wesson of the Medical College of Wisconsin used atomic force microscopy (AFM) to measure directly the adhesion forces between various functional groups immobilized on the AFM tip, including biologically relevent ones, and select faces of COM crystals immersed in aqueous media. Adhesion at single crystal faces has not been examined extensively, and this study represents the first demonstration of adhesion force measurements for different crystal faces of a material. Tip-immobilized carboxylate and amidinium groups, chosen to mimic aspartate and arginine residues of urinary proteins, displayed the largest adhesion forces of various functional groups examined during these studies. Furthermore, the adhesive strength of the COM crystal faces decreased in the order (100) > (12-1) > (010). This order, which scales with the concentrations of calcium and oxalate ions on the respective crystal faces, demonstrates that adhesion is sensitive to the structure and composition of crystal faces. Notably, the (100) face is the most prominent one in vivo, which may explain the tendency of COM to aggregate readily into kidney stones and attach to renal epithelial cells. The influence of certain urinary proteins on adhesion was examined, and curiously, osteopontin, a suspected regulator of stone formation, increased the adhesion force between a carboxylate tip and the (100) crystal face. This behavior was unique among the various combinations of additives and COM crystal faces examined here, suggesting that osteopontin regulates aggregation and attachment in a distinct manner. Collectively, the force measurements demonstrate that adhesion of functional groups and binding of soluble additives, including urinary macromolecules, to COM crystal surfaces are highly specific in nature, suggesting a path toward a better understanding of kidney stone disease and the eventual design of therapeutic agents. [X. Sheng, T. Jung, J. A. Wesson, M. D. Ward, Proc. Nat. Acad. Sci., 2005, 102, 267.]

Figure 1. (upper panels) Scanning electron microscopy images of COM crystals viewed perpendicular to the (100), (12-1), and (010) faces. (bottom panels) AFM lattice images, displayed as raw (upper half) and Fourier-filtered (lower half) data. The AFM images were acquired in aqueous solutions (pH È 7) saturated with calcium oxalate (0.11 mM)

Figure 2. Schematic representation of the contact between an idealized hemispherical gold-coated AFM tip, modified with a monolayer of organosulfur molecules of the same kind, the COM crystal surface (not drawn to scale).

Figure 3. (top) The adhesion forces measured for Au:S(CH₂)₁₀COO^- tips and COM crystal surfaces in 0.15 mM calcium oxalate solution (pH È 7.0) after addition of OPN (5 mg/ml). (bottom) Comparison of the effect of several urinary proteins on the adhesion force. The inset in the top panel is a proposed schematic to explaing the enhanced adhesion of a tip modified with carboxylate groups to a COM surface decorated with adsorbed osteopontin. The binding is mediated by the divalent calcium ions in solution, which bridge the carboxylates on the tip and protein side chains.