Comparison of cat and human calcium oxalate monohydrate kidney stone matrix proteomes.

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.

Protein primary structure correlates with calcium oxalate stone matrix preference.

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.

Exploring mechanisms of protein influence on calcium oxalate kidney stone formation.

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.

Selective protein enrichment in calcium oxalate stone matrix: a window to pathogenesis?

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.

Stone former urine proteome demonstrates a cationic shift in protein distribution compared to normal.

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.

The role of macromolecules in the formation of kidney stones.

The formation of crystal aggregates, one of the critical processes in kidney stone pathogenesis, involves interactions between crystals (predominantly calcium oxalate monohydrate, COM) and urinary constituents (e.g., proteins), which serve as an adhesive "glue" between crystals in stones. To develop a better understanding of the protein-crystal interactions that lead to crystal aggregation, we have measured the effect of model proteins on bulk COM crystal properties as well as their adsorption on crystal surfaces using three synthetic polyanions: poly(aspartic acid) (polyD), poly(glutamic acid) (polyE), and poly(acrylic acid) (polyAA). These anionic macromolecules reduced the amount of COM crystal aggregation in bulk solution to an extent similar to that observed for mixture of proteins from normal urine, with little difference between the polymers. In contrast, the polymers exhibited differences in measures of COM crystal growth. Polycations such as poly(arginine) (polyR) and poly(lysine) (polyK) reduced aggregation weakly and exerted negligible effects on crystal growth. All polyions were found to associate with COM crystal surfaces, as evidenced by changes in the zeta potential of COM crystals in electrophoretic mobility measurements. On the other hand, COM aggregation and possibly growth can be promoted by many binary mixtures of polycations and polyanions, which appeared to be mediated by polymer aggregate formation rather than loss of crystal charge stabilization. Similarly, crystal aggregation promotion behavior can be driven by forming aggregates of weakly charged polyanions, like Tamm-Horsfall protein, suggesting that polymer (protein) aggregation may play a critical role in stone formation. Sensitivity of polyanion-COM crystal surface interactions to the chemical composition of polymer side groups were demonstrated by large differences in crystal aggregation behavior between polyD and polyE, which correlated with atomic force microscopy (AFM) measurements of growth inhibition on various COM surfaces and chemical force microscopy (CFM) measurements of unbinding forces between COM crystal surfaces and AFM tips decorated with either carboxylate or amidinium moieties (mimicking polyanion and polyR side chains, respectively). The lack of strong interaction for polyE at the COM (100) surface compared to polyD appeared to be the critical difference. Finally, the simultaneous presence of polyanions and polycations appeared to alter the ability of polycations to mediate unbinding forces in CFM and promote crystal growth. In summary, polyanions strongly associated with COM surfaces and influenced crystallization, while polycations did not, though important differences were observed based on the physicochemical properties of polyanions. Observations suggest that COM aggregation with both polyanion-polycation mixtures and weakly charged polyanions is promoted by polymer aggregate formation, which plays a critical role in bridging crystal surfaces.

Exploring calcium oxalate crystallization: a constant composition approach.

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.

Polyisobutylene Urolithiasis Due to Ileal Conduit Urostomy Appliance: An Index Case.

Polyisobutylene (PIB) is a synthetic elastomer that is a component of sealants, adhesives, and chewing gum base. We report a case of bilateral PIB urolithiasis in a patient with an ileal conduit urinary diversion due to neurogenic bladder from spinal cord injury. Infrared spectroscopy confirmed the composition of bilateral stones and adhesive from the patient's urostomy appliance to be PIB. No previous cases of PIB urolithiasis are reported in the literature.

Calcium oxalate monohydrate aggregation induced by aggregation of desialylated Tamm-Horsfall protein.

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.

Crystal growth inhibitors for the prevention of L-cystine kidney stones through molecular design.

Crystallization of L-cystine is a critical step in the pathogenesis of cystine kidney stones. Treatments for this disease are somewhat effective but often lead to adverse side effects. Real-time in situ atomic force microscopy (AFM) reveals that L-cystine dimethylester (L-CDME) and L-cystine methylester (L-CME) dramatically reduce the growth velocity of the six symmetry-equivalent {100} steps because of specific binding at the crystal surface, which frustrates the attachment of L-cystine molecules. L-CDME and L-CME produce l-cystine crystals with different habits that reveal distinct binding modes at the crystal surfaces. The AFM observations are mirrored by reduced crystal yield and crystal size in the presence of L-CDME and L-CME, collectively suggesting a new pathway to the prevention of L-cystine stones by rational design of crystal growth inhibitors.

Attachment of calcium oxalate monohydrate crystals on patterned surfaces of proteins and lipid bilayers.

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.

Induction of apoptosis with cisplatin enhances calcium oxalate crystal adherence to inner medullary collecting duct cells.

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 polyanion poly(acrylic acid) prevents calcium oxalate crystal deposition.

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.

Role of crystal surface adhesion in kidney stone disease.

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.

Crystal surface adhesion explains the pathological activity of calcium oxalate hydrates in kidney 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.

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

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.

Osteopontin and calcium stone formation.

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.

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

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 introduced to the AFM cell. The addition of macromolecules with anionic side chains, specifically poly(acrylic acid), poly(aspartic acid), and poly(glutamic acid), results in inhibition of growth on the hillock step planes. The magnitude of this effect depends on the macromolecule structure, macromolecule concentration, and the identity of the step site. Poly(acrylic acid) was the most effective inhibitor of growth. Whereas poly(aspartic acid) inhibited growth on the (021) step planes of the (100) hillocks more than poly(glutamic acid), the opposite was found for the same step planes on the (010) hillocks. This suggests that growth inhibition is due to macromolecule binding to both planes of the step site or pinning of the steps due to binding to the (100) and (010) faces alone. The different profiles observed for these three macromolecules argue that local binding of anionic side chains to crystal surface sites governs growth inhibition rather than any secondary polymer structure. Growth inhibition by cationic macromolecules is negligible, further supporting an important role for proteins rich in anionic side chains in the regulation of kidney stone formation.

An acidic peptide sequence of nucleolin-related protein can mediate the attachment of calcium oxalate to renal tubule cells.

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.

Adhesion between molecules and calcium oxalate crystals: critical interactions in kidney stone formation.

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.