Kidney stones, aggregates of microcrystals, most commonly contain calcium oxalate monohydrate (COM) as the primary constituent. The aggregation of COM microcrystals and their attachment to epithelial cells are thought to involve adhesion at COM crystal surfaces, mediated by anionic molecules or urinary macromolecules. 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. Here, we describe direct measurements of adhesion forces, by atomic force microscopy, between various functional groups and select faces of COM crystals immersed in aqueous media. Tip-immobilized carboxylate and amidinium groups displayed the largest adhesion forces, and the adhesive strength of the COM crystal faces decreased in the order (100) > (121) > (010), demonstrating that adhesion is sensitive to the structure and composition of crystal faces. The influence of citrate and certain urinary proteins on adhesion was examined, and it was curious that 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. 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.

https://www.pnas.org/content/pnas/102/2/267.full.pdf

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

Thermal treatment of whewellite—a thermal analysis and Raman spectroscopic study

Kidney stones are crystal aggregates, most commonly containing calcium oxalate monohydrate (COM) microcrystals as the primary constituent. Macromolecules, specifically proteins rich with anionic side chains, are thought to play an important role in the regulation of COM growth, aggregation, and attachment to cells, all key processes in kidney stone formation. The microscopic events associated with crystal growth on the {010}, {121}, and {100} faces have been examined with in situ atomic force microscopy (AFM). Lattice images of each face reveal two-dimensional unit cells consistent with the COM crystal structure. Each face exhibits hillocks with step sites that can be assigned to specific crystal planes, enabling direct determination of growth rates along specific crystallographic directions. The rates of growth are found to depend on the degree of supersaturation of calcium oxalate in the growth medium, and the growth rates are very sensitive to the manner in which the growth solutions are prepared and ...

Probing crystallization of calcium oxalate monohydrate and the role of macromolecule additives with in situ atomic force microscopy.

Renal tubular fluid in the distal nephron of the kidney is supersaturated with calcium oxalate (CaOx), which crystallizes in the tubules as either calcium oxalate monohydrate (COM) or calcium oxalate dihydrate (COD). Kidney stones are aggregates, most commonly containing microcrystals of 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. Voided urine, however, often contains COD in the form of single micron-sized crystals. This suggests that COD formation protects against stone disease because of its reduced capacity to form stable aggregates and strong adhesion contacts to renal epithelial cells. Using atomic force microscopy configured with tips modified with biologically relevant functional groups, we have compared the adhesion strengths of the morphologically important faces of COM and COD. These measurements provide direct experimental evidence, at the near molecular level, for poorer adhesion at COD crystal faces, which explains the benign character of COD and has implications for resolving one of the mysteries of kidney stone formation.

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Crystal Surface Adhesion Explains the Pathological Activity of Calcium Oxalate Hydrates in Kidney Stone Formation

The molecular recognition and interactions governing site-specific adsorption of growth inhibitors on crystal surfaces can be tailored in order to control the anisotropic growth rates and physical properties of crystalline materials. Here we examine this phenomenon in calcium oxalate monohydrate (COM) crystallization, a model system of calcification with specific relevance for pathological mineralization. We analyzed the effect of three putative growth inhibitors—chondroitin sulfate, serum albumin, and transferrin—using analytical techniques capable of resolving inhibitor–crystal interactions from interfacial to bulk scales. We observed that each inhibitor alters surface growth by adsorbing on to distinct steps emanating from screw dislocations on COM surfaces. Binding of inhibitors to different crystallographic faces produced morphological modifications that are consistent with classical mechanisms of layer-by-layer crystal growth inhibition. The site-specific adsorption of inhibitors on COM surfaces was...

Specificity of Growth Inhibitors and their Cooperative Effects in Calcium Oxalate Monohydrate Crystallization

Whewellite, CaCrOo'HrO, and weddellite, CaCzO+' (2 + x) HrO where x = 0'5, occur in sediments, in plants, and in urinary stones' Their crystal structures have been refined to R : 0.033 and R : 0.032 respectively, using new sets of X-ray diffraction data, collected on a single-crystal di-ffractometer. Refined cell parameterc are'. Y2,/c, a:6.29O(l), D: l4'583(l), c: 10'116(l)A' B: 109.46(2)\", Z : 8 for whewellite; I4/m, a : 12.371(3), c :7'35't(2)A, Z : 8 for weddellite' During refinement of whewellite, three out of four H atoms could be located, and split positions, p\".tiutty occupied, for the two independent water molecules were found. In weddellite refinement, it was possible to locate all the H atoms, and a split position for the \"zeolitic\" water was found: a maximum water content of 2'5 HrO was confirmed' The comparison of the structures explains the relationships existing between some repeats of the two minerals and shows the differences between the Ca coordination polyhedra. A possible correlation between the structural features and the mechanism of formation of the two mineral species is suggested. The symmetry and planarity of the oxalate groups are discussed' Introduction Whewellite and weddellite are hydrated oxalates of calcium, respect ively CaCrOo' H'O and CaCrOo'(2+x)HrO (with x = 0.5), found naturally in plant tissues, in sediments as a mineral of organic origin, and in urinary stones. About 70 percent of human urinary stones contain whewellite and/or weddellite, either singly or mixed with other components, mostly phosphates, uric acids, or urates. The frequency with which the two minerals are found in association in natural sources suggested a re-examination and a comparison of their crystal structure as the first stage of an investigation on their genetic relationshiPs. Previous structure analyses were carried out by Cocco (1961) and Cocco and Sabelli (1962) for whewellite (two-dimensional photographic data, R : 0.14) and by Sterling (1965) for weddellite (three-dimensional photographic data, R : 0.13). Experimental Single crystals of whewellite and weddellite were obtained from urinary calculi. A Philips PW ll00 single-crystal di.ffractometer was used to collect the X-ray diffraction data. Table I presents the experimenial details. On the crystals of both species three standard reflections monitored at three-hour intervals showed less than 3.5 percent intensity variation of figures by Germain et al. (1971), S'u5ing ef a/' (1g62),and Johnson (1965) were employed' The scatiering curves for neutral atoms given by the International Tablesfor X-ray Crystallography (1974) were used. High-precision methods using an co scan of four intense reflections from each of several lattice rows provided accurate data for unit-cell parameter computations. Results of the structure refinements VVhewellite The orientation and the dimensions of the cell axes (Table l) are consistent with one of the two alternaiive orientations proposed by Arnott et al' (1965) 0np3-ffi4x / 80 / 0304-0327$o2.oo 32'.1 328 TAZZOLI AND DOMENEGHETTI: Table l Crystal and diffraction data WHEWELLITE AND WEDDELLITE from them). The occupancy of these positions W(10) and W(20) and, in turn, that of the main positions W(l) and W(2) were refined with other cycles of least squares, together with the coordinates and the anisotropic thermal factors of the non-hydrogen atoms. Fixed isotropic temperature factors were used for the atoms H(ll), H(21), H(22), W(10), and W(20). The final atomic parameters are shown in Tables 2 and 3; bond lengths and angles in Table 4. The observed and computed structure factors are compared in Table 5.' The final value of the discrepancy index was R : 0.033 for the 2864 observed reflections. lleddellite The values of the cell parameters (Table l) agree well with those of Sterling (1965). The atomic coordinates of this author, with the exclusion of those relative to oxygen of the \"zeolitic\" water, were used to start the refinement. During the first cycles, carried out using isotropic thermal factors, the examination of the difference Fourier map confirmed the presence of zeolitic water, with incomplete occupancy, in the channel running along the four-fold axis, with coordinates similar to those given by Sterling. In the last stages of refinement, difference Fourier maps allowed us to locate the hydrogen atoms of the two molecules of non-zeolitic water and to find a split po-, sition W(30) for the oxygen of the zeolitic water, the occupilncy of which was refined alternately with that of the main position W(3) (he distance between the two positions was 0.574). Fixed isotropic temperature factors were used for the atoms H(5), H(6), and W(30). The final atomic parameters are given in Tables 6 and 7, lengths and bond angles in Table 8. The observed and computed structure factors are compared in Table 9.'The final value of the discrepancy index was R : 0.032 for the 889 observed reflections. Discussion and comparison of the structures lMhewellite In whewellite (Fig. l) the coordination polyhedra of the pseudo-equivalent atoms Ca(l) and Ca(2) are distorted square antiprisms: in each of them seven of the oxygens belong to five oxalic groups and one to a water molecule. Each Ca polyhedron shares three edges with three adjacent Ca polyhedra. In this way I To receive a copy of Tables 5 and 9, order Document AM-?9116 from the Business Office, Mineralogical Society of America, 2000 Florida Avenue, NW, Washington, DC 20009. please remit $1.00 in advance for the microfiche. P n o p e r t y W h e w e l l i t e W e d d e l l i t e

C. Domeneghetti

Papers

The crystal structures of whewellite and weddellite; re-examination and comparison