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Cat calcium oxalate monohydrate kidney stone matrix proteome showed great similarity to human calcium oxalate monohydrate stone matrix proteome, but inference of mechanistic similarity was limited by the absence of cat urine proteomic data. In this study, urine proteome distributions were measured by the same methods in 7 healthy cats for comparison to both the published human urine and cat calcium oxalate stone matrix proteomes to assess for similar enrichment patterns in both species. Furthermore, proteomic distributions were determined in cat struvite stone matrix to test for similarity to calcium oxalate monohydrate stone matrix and urine proteomes. Cat urine proteins demonstrated a similar distribution of abundance as a function of isoelectric points or net charge to human urine samples, and consequently the similarly altered patterns of protein distributions seen in calcium oxalate monohydrate stone matrix seen from both cat and human stones likely derives from the same preferential adsorption mechanism. Furthermore, the fact that protein abundance patterns seen in cat struvite stone matrix samples differ from both urine and calcium oxalate monohydrate stone matrix proteomes in systematic ways suggests that a combination of protein-protein and protein crystal interactions underly the formation of the crystal aggregates that comprise stones., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Despite its critical nature, the role of matrix in calcium oxalate stone formation is poorly understood. The wide diversity of proteins comprising matrix has contributed to the ambiguity. This study compares the protein distributions measured by mass spectrometry in human calcium oxalate stone matrix to that observed in cat stone matrix, because cats share many clinical characteristics of their stone disease with humans. The observed protein distributions were analyzed in the context of a recent model based on the aggregation of strongly anionic and strongly cationic proteins which includes selective adsorption of other proteins based on total charge. Matrix protein distributions shared many common features between species, including enrichment of both strongly anionic and strongly cationic proteins, increased total charge in matrix proteins compared to urine proteins, and a high degree of similarity of prominent strongly anionic proteins in the matrix of both species. However, there was weaker overlap of the specific dominant proteins in other regions of the net charge distribution. Collectively, these observations support the conceptual model where the strongly anionic proteins associate most strongly with the calcium oxalate crystal surfaces, while the other proteins associate with the strongly anionic proteins through non-specific, charge interactions with each other to create stones. Also, cats appear to be the best animal model of human stone disease identified to date based on these similarities., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Despite the apparent importance of matrix proteins in calcium oxalate kidney stone formation, the complexity of the protein mixture continues to elude explanation. Based on a series of experiments, we have proposed a model where protein aggregates formed from a mixture containing both strongly charged polyanions and strongly charged polycations could initiate calcium oxalate crystal formation and crystal aggregation to create a stone. These protein aggregates also preferentially adsorb many weakly charged proteins from the urine to create a complex protein mixture that mimics the protein distributions observed in patient samples. To verify essential details of this model and identify an explanation for phase selectivity observed in weakly charged proteins, we have examined primary structures of major proteins preferring either the matrix phase or the urine phase for their contents of aspartate, glutamate, lysine and arginine; amino acids that would represent fixed charges at normal urine pH of 6-7. We verified enrichment in stone matrix of proteins with a large number of charged residues exhibiting extreme isoelectric points, both low (pI<5) and high (pI>9). We found that the many proteins with intermediate isoelectric points exhibiting preference for stone matrix contained a smaller number of charge residues, though still more total charges than the intermediate isoelectric point proteins preferring the urine phase. While other sources of charge have yet to be considered, protein preference for stone matrix appears to correlate with high total charge content., Competing Interests: The authors have declared that no competing interests exist.

Calcium oxalate monohydrate (COM) crystals are the primary constituent of most kidney stones, but urine proteins in stone matrix are believed to be critical elements for stone formation from these crystals. Recent data have shown that hundreds of proteins appear in the stone matrix with no explanation for inclusion of so many proteins. We have proposed a stone formation model with protein stimulated COM aggregation based on polyanion-polycation aggregation, which is supported by finding that matrix is highly enriched in strongly anionic and strongly cationic proteins. Many other proteins may be drawn to such aggregates due to their limited solubility in water or charge effects. Finding similar protein enrichment in both polyarginine (pR) induced aggregates of urine proteins and COM stone matrix would support this hypothesis. Purified proteins (PP) were obtained from random urine samples of six healthy adults by ultradiafiltration. Protein aggregation was induced by adding pR to PP solutions at two concentrations; 0.25 and 0.5 µg pR/µg of PP. Samples of each fraction and the original PP mixture were lyophilized and analyzed by tandem mass spectrometry. Aggregates induced by pR addition to PP samples collected a protein mixture that mimicked the protein distribution observed in COM matrix, supporting our hypothesis. The apparently discordant behavior of certain abundant anionic proteins preferentially joining the pR aggregate, when they had demonstrated reduced abundance in COM stone matrix, suggests that this model was overdriven to aggregate. The reversal of aggregate preference of albumin at low pR addition supports this interpretation., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Urine proteins are thought to control calcium oxalate stone formation, but over 1000 proteins have been reported in stone matrix obscuring their relative importance. Proteins critical to stone formation should be present at increased relative abundance in stone matrix compared to urine, so quantitative protein distribution data were obtained for stone matrix compared to prior urine proteome data. Matrix proteins were isolated from eight stones (> 90% calcium oxalate content) by crystal dissolution and further purified by ultradiafiltration (> 10 kDa membrane). Proteomic analyses were performed using label-free spectral counting tandem mass spectrometry, followed by stringent filtering. The average matrix proteome was compared to the average urine proteome observed in random urine samples from 25 calcium oxalate stone formers reported previously. Five proteins were prominently enriched in matrix, accounting for a mass fraction of > 30% of matrix protein, but only 3% of urine protein. Many highly abundant urinary proteins, like albumin and uromodulin, were present in matrix at reduced relative abundance compared to urine, likely indicating non-selective inclusion in matrix. Furthermore, grouping proteins by isoelectric point demonstrated that the stone matrix proteome was highly enriched in both strongly anionic (i.e., osteopontin) and strongly cationic (i.e., histone) proteins, most of which are normally found in intracellular or nuclear compartments. The fact that highly anionic and highly cationic proteins aggregate at low concentrations and these aggregates can induce crystal aggregation suggests that protein aggregation may facilitate calcium oxalate stone formation, while cell injury processes are implicated by the presence of many intracellular proteins.

Many urine proteins are found in calcium oxalate stones, yet decades of research have failed to define the role of urine proteins in stone formation. This urine proteomic study compares the relative amounts of abundant urine proteins between idiopathic calcium oxalate stone forming and non-stone forming (normal) cohorts to identify differences that might correlate with disease. Random mid-morning urine samples were collected following informed consent from 25 stone formers and 14 normal individuals. Proteins were isolated from urine using ultrafiltration. Urine proteomes for each sample were characterized using label-free spectral counting mass spectrometry, so that urine protein relative abundances could be compared between the two populations. A total of 407 unique proteins were identified with the 38 predominant proteins accounting for >82% of all sample spectral counts. The most highly abundant proteins were equivalent in stone formers and normals, though significant differences were observed in a few moderate abundance proteins (immunoglobulins, transferrin, and epidermal growth factor), accounting for 13 and 10% of the spectral counts, respectively. These proteins contributed to a cationic shift in protein distribution in stone formers compared to normals (22% vs. 18%, p = 0.04). Our data showing only small differences in moderate abundance proteins suggest that no single protein controls stone formation. Observed increases in immunoglobulins and transferrin suggest increased inflammatory activity in stone formers, but cannot distinguish cause from effect in stone formation. The observed cationic shift in protein distribution would diminish protein charge stabilization, which could lead to protein aggregation and increased risk for crystal aggregation.

Crystal growth rates have been extensively studied in calcium oxalate monohydrate (COM) crystallization, because COM crystals are the principal component in most kidney stones. Constant composition methods are useful for studying growth rates, but fail to differentiate concurrent nucleation and aggregation events. A constant composition method coupled with particle size determinations that addresses this deficiency was previously published for a calcium phosphate system, and this method was extended to COM crystallization in this report. A seeded constant composition experiment was combined with particle size determination and a separate near-equilibrium aggregation experiment to separate effects of growth rate, nucleation, and aggregation in COM crystal formation and to test the effects of various inhibitors relevant to stone formation. With no inhibitors present, apparent COM growth rates were heavily influenced by secondary nucleation at low seed crystal additions, but growth-related aggregation increased at higher seed crystal densities. Among small molecule inhibitors, citrate demonstrated growth rate inhibition but enhanced growth-related aggregation, while magnesium did not affect COM crystallization. Polyanions (polyaspartate, polyglutamate, or osteopontin) showed strong growth rate inhibition, but large differences in nucleation and aggregation were observed. Polycations (polyarginine) did not affect COM crystal growth or aggregation. Mixtures of polyanions and polycations produced a complicated set of growth rate, nucleation, and aggregation behaviors. These experiments demonstrated the power of combining particle size determinations with constant composition experiments to fully characterize COM crystallization and to obtain detailed knowledge of inhibitor properties that will be critical to understanding kidney stone formation.

Tamm-Horsfall protein (THP) is thought to protect against calcium oxalate monohydrate (COM) stone formation by inhibiting COM aggregation. Several studies reported that stone formers produce THP with reduced levels of glycosylation, particularly sialic acid levels, which leads to reduced negative charge. In this study, normal THP was treated with neuraminidase to remove sialic acid residues, confirmed by an isoelectric point shift to higher pH. COM aggregation assays revealed that desialylated THP (ds-THP) promoted COM aggregation, while normal THP inhibited aggregation. The appearance of protein aggregates in solutions at ds-THP concentrations ≥1 μg/mL in 150 mM NaCl correlated with COM aggregation promotion, implying that ds-THP aggregation induced COM aggregation. The aggregation-promoting effect of the ds-THP was independent of pH above its isoelectric point, but was substantially reduced at low ionic strength, where protein aggregation was much reduced. COM aggregation promotion was maximized at a ds-THP to COM mass ratio of ~0.025, which can be explained by a model wherein partial COM surface coverage by ds-THP aggregates promotes crystal aggregation by bridging opposing COM surfaces, whereas higher surface coverage leads to repulsion between adsorbed ds-THP aggregates. Thus, desialylation of THP apparently abrogates a normal defensive action of THP by inducing protein aggregation, and subsequently COM aggregation, a condition that favors kidney stone formation.

The attachment of calcium oxalate monohydrate (COM) crystals to renal tubules is thought to be one of the critical steps of kidney stone formation. Patterns of phosphatidylserine (DPPS) bilayers and osteopontin (OPN) were fabricated on silica substrates through the combination of a microcontact printing technique and fusion of lipid vesicles to create spatially organized surfaces of lipids and proteins that may mimic renal tubule surfaces while allowing direct visualization of the competition for COM attachment to compositionally different regions. In the case of DPPS-OPN patterns, micrometer-sized COM crystals dispersed in saturated aqueous calcium oxalate solutions attached preferentially to the OPN regions, in agreement with other in vitro studies that have suggested a binding affinity of OPN to COM crystal surfaces. COM crystals attached with nearly equal coverage to OPN and DPPS surfaces alone, suggesting that the preferential segregation of COM crystals to the OPN regions on the patterned surfaces reflects reversible attachment of micrometer-sized COM crystals capable of Brownian motion. These attached microcrystals then grow larger over time during immersion in the supersaturated calcium oxalate solutions. Free OPN, a major constituent in urine, adsorbs on COM crystals and suppresses attachment to DPPS, suggesting a link between OPN and reduced attachment of COM crystals to renal epithelium. This patterning protocol can be expanded to other urinary molecules, providing a convenient approach for understanding the effects of biomolecules on COM crystal attachment and the pathogenesis of kidney stones.

Attachment of stone crystals to tubular epithelium may initiate kidney stone formation. We previously reported that apical nucleolin related protein (NRP) expression during mitosis enhance attachment of Ca oxalate monohydrate crystals (COM). Some forms of injury may also increase affinity for crystals. We examined changes in subcellular localization of NRP during the course of cisplatin-induced apoptosis in cultured inner medullary collecting duct cells. Caspase-3 activation and chromatin condensation followed by nuclear fragmentation occurred after 20 h exposure to cisplatin, indicating the development of apoptosis. Cells were fixed without permeabilization and stained for surface NRP. Cells with condensed chromatin showed little or no cytoplasmic or apical NRP. Those at an early stage of nuclear fragmentation had cytoplasmic but not apical NRP and cells with advanced nuclear fragmentation were positively stained for apical NRP. Membrane proteins isolated by apical biotinylation and precipitated with avidin were analyzed by Western blot. Apical NRP was markedly increased after cisplatin compared to control, while expression of the apical marker, GP-135, and other putative attachment protein were unchanged. Hyaluronic acid was decreased. Cultures with apoptotic cells demonstrated increased adherence of COM that was inhibited by the polyanion (poly)aspartic acid. We conclude that pre-existing apoptotic injury may promote calcium oxalate crystals attachment to renal tubular epithelium via apical NRP expression.

Acidic macromolecules inhibit calcium oxalate nucleation, growth, aggregation and attachment to cells in vitro. To test for such an effect in vivo we used osmotic minipumps to continuously infuse several doses of the 5.1 kDa poly(acrylic acid) (pAA(5.1)) into rats fed a diet which causes renal calcium oxalate crystal deposition. Although kidneys of rats receiving the saline control contained calcium oxalate crystals, measured by polarized light microscopy, those of animals given pAA(5.1) had significantly lower numbers of crystals in various zones of the kidney. Delivery of pAA(5.1) to urine was confirmed by measuring excretion of infused biotinylated pAA(5.1). Both the derivatized and unlabelled pAA(5.1) had the same effects on crystallization in vitro. Our study shows that acidic polymers hold promise as effective therapies for kidney stones likely through prevention of calcium oxalate crystal aggregate formation.

Purpose of Review: Atomic force microscopy has been used recently to characterize the adhesion force between selected calcium oxalate crystal surfaces and biologically relevant chemical groups attached to the atomic force microscopy probe tip. These measurements have permitted comparisons of the adhesion properties of different, well defined crystal faces, as well as determination of the influence of solution-phase macromolecules on adhesion. These studies have produced new insight into the specific chemical interactions that regulate kidney stone formation., Recent Findings: The adhesion force measurements have demonstrated that the large hexagonal (100) face of calcium oxalate monohydrate is the most adhesive. In contrast, the large (101) face of calcium oxalate dihydrate is the least adhesive. Carboxylate and amidinium groups on the atomic force microscopy tip exhibit equivalently large adhesion at a given crystal face, implicating specific binding to crystal surface lattice ions. Solution-phase macromolecules modulate adhesion in a face-selective manner, dependent on their chemical structures., Summary: The low adhesion force for calcium oxalate dihydrate predicts a decreased ability of these crystals to aggregate or attach to cells, and correlates with the relative absence of calcium oxalate dihydrate in kidney stones. These measurements provide new understanding of the macromolecular regulation of crystal aggregation and attachment to cells in stone formation.

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.

Based on the structure of kidney stones, it is likely that they form as aggregations of preformed crystals, mostly calcium oxalate monohydrate (COM). In this study, we examined the ability of a macromolecular mixture isolated from the urine of normal individuals and stone formers to inhibit aggregation of preformed COM seed crystals in a simple ionic solution using measurements of changes in the particle size distribution (PSD) of preformed COM crystal aggregates. We also examined the effect in this assay of a number of synthetic homopolymers, naturally occurring urine macromolecules, and binary mixtures thereof. The macromolecular mixtures from urine of normals and most stone formers reduced the degree of aggregation of the seed crystals, whereas 22% of stone former urine macromolecules either did not disaggregate or actually promoted further aggregation. Stone formers within one family shared this property, but a non-stone forming sibling did not. Polyanions, either synthetic or naturally occurring, induced disaggregation to an extent similar to that exhibited by normal urine macromolecules, while polycations had no effect on the PSD. However, mixing a polyanion, either poly-aspartate or osteopontin, with the polycation poly-arginine, changed their behavior from disaggregation to aggregation promotion. The disaggregating behavior of normal urinary macromolecules provides a defense against aggregation, but a minority of stone forming individuals lacks this defense, which may contribute to stone formation.

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.

Crystals that form in tubular fluid must be retained in the kidney to become stones. Nucleolin-related protein (NRP) is found on the surface of inner medullary collecting duct (IMCD) cells in culture (cIMCD) and selectively adsorbs to calcium oxalate (CaOx). We proposed that NRP mediates attachment to the renal tubular epithelium of Ca stone crystals through an electrostatic interaction with a highly acidic region (acidic fragment [AF]) similar to those of other proteins that have been reported to affect urinary crystal formation. The current studies demonstrate that nucleolin is expressed on both apical and basolateral cell surfaces of cIMCD, reaching a peak in the late stages of mitosis and gradually declining to undetectable levels with maturation of the polarized epithelium. Scraping areas of mature monolayers stimulated the cells surrounding the defects to migrate and proliferate so as to repair them, and these areas demonstrate surface NRP expression and enhanced attachment of CaOx monohydrate crystals. Surface expression of the NRP AF was produced by cloning the NRP AF into a display vector. Transfected cIMCD demonstrating copious surface expression of AF enhanced CaOx attachment 6.7-fold compared with control cIMCD, whereas cells transfected with a vector without the AF did not differ from control. AF was also cloned into a replication-deficient adenovirus and expressed in 293 cells, resulting in AF secretion into the nutrient medium. This medium inhibited CaOx attachment to cIMCD, compared with conditioned medium from cells infected with wild-type virus. These results demonstrate that surface-bound AF can mediate CaOx attachment and that secreted AF can inhibit attachment. These results support the notion that surface-associated NRP could mediate attachment of CaOx to the renal tubule epithelium, thereby causing retention of crystals that might eventually become kidney stones.

Osteopontin (OPN) is a phosphorylated protein of wide tissue distribution that is found in association with dystrophic calcification including in the organic matrix of kidney stones. It is a strong inhibitor of crystal formation and growth in vitro, but there is still debate regarding its effects upon crystal adhesion to tubular epithelial cells. In this brief review, we will outline the evidence implicating OPN in stone disease with the primary emphasis being on the interaction of OPN with calcium oxalate (CaOx), the major constituent of calcium containing stones. Finally, preliminary data is presented regarding the amounts and features of OPN present in the urine of stone formers and normal individuals., (Copyright (c) 2004 S. Karger AG, Basel.)

Kidney stones are crystal aggregates, most commonly containing calcium oxalate monohydrate (COM) crystals as the primary constituent. Notably, in vitro studies have suggested that anionic molecules or macromolecules with substantial anionic functionality (e.g., carboxylate) play an important role in crystal aggregation and crystal attachment to renal epithelial cells. Furthermore, kidney stones contain measurable amounts of carboxylate-rich proteins that may serve as adhesives and promote aggregation of COM crystals. Atomic force microscopy (AFM) measurements of adhesion forces between tip-immobilized molecules and the COM (100) surface in aqueous media, described herein, reveal the effect of functional groups on adhesion and support an important role for the carboxylate group in processes responsible for kidney stone formation, specifically macromolecule-mediated adhesion of COM crystals to cells and crystal aggregation. The presence of poly(aspartic acid) during force measurements results in a reduction in the adhesion force measured for carboxylate-modified tips, consistent with the blocking of binding sites on the COM (100) surface by the carboxylate-rich polymer. This competitive binding behavior mimics the known reduction in attachment of COM crystals to renal epithelial cells in the presence of carboxylate-rich urinary macromolecules. These results suggest a feasible methodology for identifying the most important crystal surface-macromolecule combinations related to stone formation.

Calcium nephrolithiasis is the most common form of renal stone disease, with calcium oxalate (CaOx) being the predominant constituent of renal stones. Current in vitro evidence implicates osteopontin (OPN) as one of several macromolecular inhibitors of urinary crystallization with potentially important actions at several stages of CaOx crystal formation and retention. To determine the importance of OPN in vivo, hyperoxaluria was induced in mice targeted for the deletion of the OPN gene together with wild-type control mice. Both groups were given 1% ethylene glycol, an oxalate precursor, in their drinking water for up to 4 wk. At 4 wk, OPN-deficient mice demonstrated significant intratubular deposits of CaOx crystals, whereas wild-type mice were completely unaffected. Retained crystals in tissue sections were positively identified as CaOx monohydrate by both polarized optical microscopy and x-ray powder diffraction analysis. Furthermore, hyperoxaluria in the OPN wild-type mice was associated with a significant 2- to 4-fold upregulation of renal OPN expression by immunocytochemistry, lending further support to a renoprotective role for OPN. These data indicate that OPN plays a critical renoprotective role in vivo as an inhibitor of CaOx crystal formation and retention in renal tubules.

Purpose: We tested the effect of molecular weight and amino acid composition (aspartate versus glutamate) in model peptides on calcium oxalate dihydrate (COD) formation to understand how known urinary inhibitor proteins might control spontaneous crystallization., Materials and Methods: Supersaturated solutions of CaCl2 and Na2C2O4 in HEPES buffered saline solution were prepared at various calcium (Ca) to oxalate (Ox) ratios, but constant supersaturation, in the presence of protein inhibitors (polyaspartic acid molecular weight series or polyglutamic acid). The resulting crystals were collected and evaluated with optical microscopy., Results: With no added inhibitors, the crystal size increased with Ca to Ox ratio, while the number of crystals decreased. With protein inhibitors at equivalent mass concentrations, intermediate molecular weight proteins produced a greater proportion of COD in Ca rich conditions than did either extreme. In Ox rich conditions, the proportion of COD was directly related to protein molecular weight. However, at equivalent molar concentrations, the proportion of COD produced was directly related to molecular weight under all conditions. Larger protein concentrations were required to produce COD at high Ox conditions, in proportion to the increased number of crystals produced. Polyglutamic acid had a much weaker effect on crystal structure, but it changed the COM morphology., Conclusions: The results suggest that a discrete number of protein molecules per crystal were required to direct crystallization toward COD, and that a characteristic size of polypeptide chain can be defined. The charge of the side group was not the sole determinant of this effect, as polyglutamic and polyaspartic acids behaved differently. Calcium oxalate crystal nucleation rates appeared to increase with Ox content.

Crystal polymorphism is exhibited by calcium oxalates in nephrolithiasis, and we have proposed that a shift in the preferred crystalline form of calcium oxalate (CaOx) from monohydrate (COM) to dihydrate (COD) induced by urinary macromolecules reduces crystal attachment to epithelial cell surfaces, thus potentially inhibiting a critical step in the genesis of kidney stones. We have tested the validity of this hypothesis by studying both the binding of monohydrate and dihydrate crystals to renal tubule cells and the effect of macromolecular urinary solutes on crystal structure. Renal tubule cells grown in culture bound 50% more CaOx monohydrate than dihydrate crystals of comparable size. The effects of macromolecules on the spontaneous nucleation of CaOx were examined in HEPES-buffered saline solutions containing Ca2+ and C2O4(2-) at physiologic concentrations and supersaturation. Many naturally occurring macromolecules known to be inhibitors of crystallization, specifically osteopontin, nephrocalcin and urinary prothrombin fragment 1, were found to favor the formation of calcium oxalate dihydrate in this in vitro system, while other polymers did not affect CaOx crystal structure. Thus, the natural defense against nephrolithiasis may include impeding crystal attachment by an effect of macromolecular inhibitors on the preferred CaOx crystal structure that forms in urine.

The effect of poly-L-aspartic acid (PA) on the crystal structure of calcium oxalate crystals grown after spontaneous nucleation was evaluated as a function of relative supersaturation and calcium:oxalate ratio in a buffered salt solution, with pH and ionic strength in the range of normal human urine. PA was used as a model for naturally occurring acidic urine proteins that have been shown to inhibit nucleation and growth of calcium oxalate crystals. The crystals grown were characterized by optical microscopy and X-ray powder diffraction. It was observed that calcium oxalate monohydrate was the preferred crystalline form in the absence of added PA, and it was the only crystalline form obtained at most conditions tested without PA. However, the presence of PA favored the formation of calcium oxalate dihydrate crystals, when present in adequate quantities. The quantity of PA required to affect this change in preferred crystal structure was increased at higher supersaturations and at lower calcium:oxalate ratios, exhibiting a non-linear dependence on both variables. PA was also shown to be a kinetic inhibitor of calcium oxalate dihydrate crystallization. Aspartic acid monomer was found to cause no change in the preferred structure of calcium oxalate monohydrate at mass concentrations well beyond those required with PA to obtain 100% calcium oxalate dihydrate, indicating the critical importance of the polymeric nature of PA for this effect on crystal structure.