Isolation from human calcium oxalate renal stones of nephrocalcin, a glycoprotein inhibitor of calcium oxalate crystal growth. Evidence that nephrocalcin from patients with calcium oxalate nephrolithiasis is deficient in gamma-carboxyglutamic acid.

We have determined that the organic matrix of calcium oxalate kidney stones contains a glycoprotein inhibitor of calcium oxalate crystal growth (nephrocalcin) that resembles nephrocalcin present in the urine of patients with calcium oxalate stones and differs from nephrocalcin from the urine of normal people. Pulverized calcium oxalate renal stones were extracted with 0.05 M EDTA, pH 8.0; nephrocalcin eluted in five peaks using DEAE-cellulose column chromatography, and each peak was further resolved by Sephacryl S-200 column chromatography. Four of the five DEAE peaks corresponded to those usually found in nephrocalcin from urine; the fifth eluted at a lower ionic strength than any found in urine. Amino acid compositions and surface properties of nephrocalcins isolated from kidney stones closely resembled those of nephrocalcins isolated from urine of stone-forming patients: they differed from normal in lacking gamma-carboxyglutamic acid residues, and in forming air-water interfacial films that were less stable than those formed by nephrocalcin from normal urine.

Interaction between nephrocalcin and calcium oxalate monohydrate: a structural study.

Study of crystals of calcium oxalate monohydrate grown from gels exposed to 0, 5.6x, 12.5x, 26.2x, 52.5x, 100x, 200 x 10(-7) M nephrocalcin indicate that this protein profoundly affects their habit, size, and crystal structure. By the time nephrocalcin concentration is 26.2 x 10(-7) M calcium oxalate monohydrate undergoes a phase change in its basic structure and both crystal size as well the resolution of its diffraction pattern are severely curtailed. These effects are magnified when the protein is 52.5 x 10(-7) M, since long-range disorder becomes extreme and, out of the entire diffraction pattern, only the 0k0's, h00's and a few other nonaxial reflections remain from the ordered part of the crystal structure. Finally, once the concentration of nephrocalcin is raised to 100 and 200 x 10(-7) M, growth is so inhibited that calcium oxalate monohydrate no longer grows as distinct individuals but rather as aggregates of very small crystallites. All of this is caused by the ability on the part of nephrocalcin to disturb the juxtaposition of the (101) layers along c by disrupting the organization of both the C(3)-C(4) oxalate groups and the water molecules. Such interaction is modulated by the efficiency with which nephrocalcin adsorbs upon the (101) planes; this process is stereospecific.

The interaction between nephrocalcin and Tamm-Horsfall proteins with calcium oxalate dihydrate.

Studies of crystals of calcium oxalate dihydrate (COD) grown by vapor diffusion from solutions containing 5.1 x 10(-7), 1.5 x 10(-6), and 1.0 x 10(-5) M nephrocalcin (NC), indicate that NC profoundly affects COD's habit, size and structure. The decrease in COD size is such that at 1.0 x 10(-5) M NC, the dimensions of the crystals are reduced about five-fold with respect to those of a NC-free control. In addition, the planes of the (101) form disappear, the original habit is lost, and the diffraction pattern deteriorates to such an extent that only the 200 reflections are recorded. The results are quite different when NC is adsorbed upon rigid substrates. Under such conditions, NC acts as a promoter and not as an inhibitor of growth and thus nucleates COD from its (100) planes. Consequently, COD grows systematically juxtaposed on NC. This effect is highly reproducible and stereospecific. COD crystals grown by vapor diffusion from solutions exposed to increasing concentrations of Tamm-Horsfall protein (THP) exhibit a drastic decrease in COD's self-association. In sharp contrast with the results obtained for NC, precession photographs taken of COD samples exposed to 1.2 x 10(-5) M THP do not show evidence of deterioration of COD diffraction patterns with respect to a protein-free control. Furthermore, THP neither nucleates COD, nor does it appear to influence its growth or habit even when THP is immobilized upon a rigid substrate.

Phase transitions of calcium oxalate trihydrate and epitaxy in the weddellite-whewellite system.

The phase changes calcium oxalate trihydrate-weddellite, weddellite-calcium oxalate monohydrate and calcium oxalate trihydrate-whewellite are individually examined at the atomic level from a theoretical point of view; concomitantly the topological requirements necessary for phase stability are clarified for each structure type. In solution a sequential series of phase transitions according to the steps calcium oxalate trihydrate-weddellite-whewellite is not likely to be energetically favoured; direct conversion of calcium oxalate trihydrate to whewellite should be, instead, ordinarily expected. It is formally demonstrated that along two axial directions a set of atoms is in essentially identical positions in both weddellite and whewellite. This notwithstanding, it is concluded that epitactic catalysis cannot and should not be considered a common mechanism for the formation of whewellite from weddellite (and vice versa) or of kidney stones in general.

The uric acid-whewellite association in human kidney stones.

Human kidney stones, composed almost exclusively of uric acid and whewellite, were studied using x-ray (powder and single-crystal) as well as scanning electron-diffraction techniques. Whewellite--showing as a concentric aggregate characteristically marked by radial striations--is enclosed within a mass of uric acid, the crystallites of which grow with their b axis parallel to the radial direction of the striations. That axis corresponds to b (2 X 7.294 A) and tends to systematically superimpose over its uric acid counterpart (b = 7.40 A). Nonetheless no other such dimensional match was found for the other set of periodicities that characterize the uric acid whewellite interfaces, raising questions that a systematic epitaxial interaction could there take place. Selected uric acid-whewellite contacts and the crucial role of the "matrix" were also investigated.

The uric acid-whewellite association in human kidney stones.

A total of twelve human kidney stones, composed almost exclusively of uric acid, whewellite and organic matrix were initially studied by x-ray (powder; single crystal), scanning and transmission electron diffraction techniques prior to as well as after exposure to 0.25 M EDTA solutions (96 hours; pH 7.1). Subsequent high-resolution scanning ion probe analyses eventually detected a phosphate phase not resolved by any of those techniques. Templates of organic matter lay above and in between the radial striations of whewellite. Often juxtaposing sets of striations are not in correct register with respect to one another. A sharp transition exists in the cores of the stones and separates an area characterized by the random deposition of whewellite from one in which the latter commences to grow in the form of radial striations. This transition is expected to be mediated by a physico-chemical and/or structural control. In laminae of organic matrix sectioned from EDTA-untreated cores well developed spherules are detected. Those superimpose on a substrate characterized by a considerable degree of crosslinking. Maximum spherule width is of the order of 0.8 micron. There is evidence of deposition in between the spherules. That deposition pattern appears to be controlled by the morphology and location of the spherules, thus suggesting that it is secondary to spherule formation.

Crystal growth of calcium oxalate monohydrate and calcium carbonate from dilute solutions.

By controlling evaporation, calcium carbonate (calcite) is precipitated together with calcium oxalate monohydrate from equimolar (1 x 10(-5) M) solutions of calcium chloride and sodium oxalate under decreasing (37.5 degrees - 32.7 degrees C) and increasing temperature (22 degrees-42 degrees, 22 degrees-45 degrees, 22 degrees-50 degrees C), and initial pH's of 6.6 and 6, respectively. If, however, the pH of the solutions is, respectively, 8 and 8.5 and the temperature is 32 degrees C, oxalate breaks down to carbonate and calcium precipitates solely as calcite. This process materializes as the pH of both solutions initially adjusts to about 7.5. Both the quality and the size of the crystals of calcite and calcium oxalate monohydrate varied markedly. Euhedral crystals of calcite, measuring in excess of 200 micrometers in cross-section, and grown at 37.5 degrees C were remarkably ordered as shown by the lack of twinning, streaks and/or diffuseness around the diffracta, which is evidenced by single-crystal x-ray diffraction analysis. In contrast, calcium oxalate monohydrate was almost always disordered due to twinning along the (101) plane and never exceeded 100-150 micrometers along a coordinate axis.

Nucleation, adhesion, and internalization of calcium-containing urinary crystals by renal cells.

Renal tubular fluid in the distal nephron is supersaturated favoring nucleation of the most common crystals in renal stones, which are composed of calcium oxalate and calcium phosphate. The mechanisms whereby these newly formed crystals can be retained in the nephron and develop into calculi are not known. Calcium oxalate monohydrate and hydroxyapatite (calcium phosphate) crystals rapidly adhere to anionic sites on the surface of cultured renal epithelial cells, but this process is inhibited by specific urinary anions such as citrate, glycosaminoglycans, uropontin, or nephrocalcin, each of which can coat the crystals. Therefore, competition for the crystal surface between soluble anions in tubular fluid and anions anchored on the apical cell surface could determine whether a crystal binds to a tubular cell. Crystals of calcium oxalate dihydrate can also nucleate directly on the surface of cultured BSC-1 cells in a face-specific manner, suggesting another potential pathway for crystal deposition in the nephron. Once present on the cell surface, calcium oxalate monohydrate, calcium oxalate dihydrate, and hydroxyapatite crystals are quickly internalized by renal cells; alterations in gene expression and initiation of proliferation may then ensue. Calcium oxalate crystals can also dissolve after renal cells internalize them, but this process may require up to several weeks. Increased knowledge about cell-crystal interactions, including identification of molecules in tubular fluid and on the cell surface that modulate the process, appear critical for understanding the pathogenesis of nephrolithiasis.

Sialic acid-containing glycoproteins on renal cells determine nucleation of calcium oxalate dihydrate crystals.

BACKGROUND: The interaction between the surfaces of renal epithelial cells and calcium oxalate dihydrate (COD), the most common crystal in human urine, was studied to identify critical determinants of kidney stone formation. METHODS: A novel technique utilizing vapor diffusion of oxalic acid was employed to nucleate COD crystals onto the apical surface of living cells. Confluent monolayers were grown in the inner 4 wells of 24-well culture plates. To identify cell surface molecules that regulate crystal nucleation, cells were pretreated with a protease (trypsin or proteinase K) to alter cell surface proteins, neuraminidase to alter cell surface sialoglycoconjugates, or buffer alone. COD crystals were nucleated on the surface of cells by diffusion of oxalic acid vapor into a calcium-containing buffer overlying the cells. Crystal face-specific nucleation was evaluated by scanning electron microscopy. RESULTS: Nucleation and growth of a COD crystal onto an untreated control cell occurred almost exclusively via its (001) face, an event rarely observed during COD crystallization. In contrast, when COD crystals were nucleated onto protease- or neuraminidase-treated cells, they did so via the (100) face of the crystal. CONCLUSIONS: Specific sialic acid-containing glycoproteins, and possibly glycolipids (sialoglycoconjugates), appear to be critical determinants of face-specific nucleation of COD crystals on the apical renal cell surface. We hypothesize that crystal retention within the nephron, and the subsequent development of a kidney stone, may result when the number or composition of these cell surface molecules is modified by genetic alterations, cell injury, or drugs in tubular fluid.

Direct nucleation of calcium oxalate dihydrate crystals onto the surface of living renal epithelial cells in culture.

BACKGROUND: The interaction of the most common crystal in human urine, calcium oxalate dihydrate (COD), with the surface of monkey renal epithelial cells (BSC-1 line) was studied to identify initiating events in kidney stone formation. METHODS: To determine if COD crystals could nucleate directly onto the apical cell surface, a novel technique utilizing vapor diffusion of oxalic acid was employed. Cells were grown to confluence in the inner four wells of 24-well plates. At the start of each experiment, diethyloxalate in water was placed into eight adjacent wells, and the plates were sealed tightly with tape so that oxalic acid vapor diffused into a calcium-containing buffer overlying the cells. RESULTS: Small crystals were visualized on the cell surface after two hours, and by six hours the unambiguous habitus of COD was confirmed. Nucleation onto cells occurred almost exclusively via the (001) face, one that is only rarely observed when COD crystals nucleate onto inanimate surfaces. Similar results were obtained when canine renal epithelial cells (MDCK line) were used as a substrate for nucleation. Initially, COD crystals were internalized almost as quickly as they formed on the apical cell surface. CONCLUSIONS: Face-specific COD crystal nucleation onto the apical surface of living renal epithelial cells followed by internalization is a heretofore unrecognized physiological event, suggesting a new mechanism to explain crystal retention within the nephron, and perhaps kidney stone formation when this process is dysregulated or overwhelmed.

Face-selective adhesion of calcium oxalate dihydrate crystals to renal epithelial cells.

The interaction between the most common urinary crystal, calcium oxalate dihydrate (COD) and the surface of monkey renal epithelial cells of the BSC-1 line was investigated. The [100] face of exogenous COD crystals bound selectively and rapidly to the kidney cell surface. Cellular processes extended preferentially over the [100] face initially, and then progressively covered the crystal so that by 24 hours some crystals were observed beneath the plasma membrane. When nucleated from solution onto the surface of the cell monolayer, COD crystals oriented preferentially so that their [100] faces were in direct contact with the cell surface. In contrast, when siliconized glass was used as a substrate, nucleated COD crystals oriented randomly. Therefore, structures on the apical surface of renal tubular cells that mediate a stereospecific interaction with the molecular array presented by the [100] face of a COD crystal may be important determinants of crystal adhesion that could contribute to crystal retention and formation of kidney stones.

Cell-crystal interactions and kidney stone formation.

BACKGROUND: Renal tubular fluid in the distal nephron is supersaturated with calcium and oxalate ions that nucleate to form crystals of calcium oxalate monohydrate (COM), the most common crystal in renal stones. How these nascent crystals are retained in the nephron to form calculi in certain individuals is not known. METHODS: The results of experiments conducted in this and other laboratories that employ cell culture model systems to explore renal epithelial cell-urinary crystal interactions are described. RESULTS: COM crystals rapidly adhere to anionic sites on the surface of cultured renal epithelial cells, but this process can be inhibited, if specific urinary anions such as glycosaminoglycans, uropontin, nephrocalcin, or citrate are available to coat the crystalline surface. Therefore, competition for the crystal surface between soluble anions in tubular fluid and anions on the apical cell surface could determine whether or not a crystal binds to the cell. A similar paradigm describes adhesion of calcium phosphate (hydroxyapatite) crystals, also a common constituent of human stones. Once bound, COM and hydroxyapatite crystals are quickly internalized by renal cells; reorganization of the cytoskeleton, alterations in gene expression, and initiation of proliferation may then ensue. Each of these cellular events appears to be regulated by a different set of extracellular factors. Over several weeks in culture, renal cells (BSC-1 line) dissolve internalized crystals, although once a cell binds a crystal, additional crystals are more likely to bind, possibly forming a positive feedback loop that results in kidney stone formation. CONCLUSIONS: Increased knowledge about the cell-crystal interaction, including identification of molecules in tubular fluid and on the cell surface that modulate the process, and understanding its mechanism of action appear critical for explaining the pathogenesis of nephrolithiasis.

Evidence that nephrocalcin and urine inhibit nucleation of calcium oxalate monohydrate crystals.

Human urine, and nephrocalcin (NC), a glycoprotein of probable kidney cell origin, greatly reduce consumption of calcium and oxalate from metastably supersaturated solutions seeded with calcium oxalate crystals, a phenomenon usually referred to as inhibition of crystal growth. We seeded metastably supersaturated calcium oxalate solutions with calcium oxalate monohydrate crystals under conditions of ion clamping to maintain constant composition and measured ion consumption from pump delivery rates. Consumption rates increased continuously with time as if the solutions were autocatalytic. After incubation, the seeds were covered with innumerable crystallites, which were also free and numerous in the solution, reflecting self-nucleation. The addition of 20% whole, dialyzed urine, or purified NC reduced ion consumption rates markedly, and the only crystals observed at the end of incubation were the large original seeds. Crystals precoated with concentrated dialyzed urine or NC also showed reduced ion consumption. Urine and NC from patients with nephrolithiasis inhibited nucleation less than normal controls. Self-nucleation seems to be the preferred response in sparsely seeded, ion-clamped, supersaturated solutions, such as exist in the nephron. Urine and NC suppress self-nucleation in vitro by adsorbing to the surface of calcium oxalate crystals.

The 2.5 A crystal structure of a dimeric phospholipase A2 from the venom of Crotalus atrox.

The crystal structure of the dimeric (alpha 2) phospholipase A2 from Crotalus atrox has been determined by multiple isomorphous replacement to 2.5 A resolution. A skeletal model was fit to the electron density, and the stereochemistry of the backbone was idealized. The dimeric molecule is a well defined oblate ellipsoid composed of two covalently identical subunits related by a local dyad axis which is essentially "exact" except for deviations at the periphery induced by ionic lattice contacts with neighboring dimers. As expected, the basic architecture of the individual protomers is similar to the structure of the homologous monomeric bovine enzyme (Dijkstra, B. W., Drenth, J., Kalk, K., and Vandermaalen, P. J. (1978) J. Mol. Biol. 124, 53-60). The intramolecular contact surface between the protomers is extensive and involves the catalytic and calcium-binding sites. Access to an internal cavity formed by the enclosed and abutting active center regions is quite restricted. The putative interfacial recognition surfaces of each protomer are exposed to the solvent but are on opposing surfaces of the ellipsoid, suggesting that both of these regions cannot interact with the same membrane surface simultaneously unless the membrane is distorted from planarity and/or the dimer is significantly modified.