Jeffrey A. Wesson

Osteopontin Is a Critical Inhibitor of Calcium Oxalate Crystal Formation and Retention in Renal Tubules

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.

In situ polymerization of tetraethoxysilane in polymers: chemical nature of the interactions

Abstract The formation of molecular composites by the in situ polymerization of tetraethoxysilane in various organic polymers has been studied. The results show that highly homogeneous, transparent composites can be formed for polymers such as poly (methyl methacrylate), poly (vinyl acetate), poly (vinyl pyrrolidone), and poly (N,N-dimethylacrylamide). These composites have improved mechanical properties and increased solvent resistance. Strong interactions between the SiO2 networks and these polymers were observed, and the data suggest hydrogen bonding between residual hydroxyls on the SiO2 and carbonyl groups on the polymer chains as the major source of these interactions.

Crystal Growth Inhibitors for the Prevention of l-Cystine Kidney Stones Through Molecular Design

Taking the Cystine Kidney stones that form from l-cystine are much less common than those forming from calcium oxalate monohydrate, but are more likely to cause chronic kidney disease. Rimer et al. (p. 337; see the cover; see the Perspective by Coe and Asplin) designed two structural mimics for l-cystine. Atomic force microscopy showed that at low concentrations, the mimics could change the l-cystine crystal habit and inhibit overall crystal growth. These structural mimics may thus offer hope for treating cystinuria. Structural mimics for l-cystine may provide drug treatments for certain types of kidney stones. 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.

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.

Pathological Biomineralization of Kidney Stones

Kidney stones are aggregates of microcrystals, most commonly containing calcium oxalate monohydrate (COM) as the primary constituent. The formation of these aggregates in the renal tubules of the kidney and their attachment to epithelial cells lining the renal tubules are thought to involve adhesion events between COM crystal surfaces and urinary species that bind to these surfaces. The pathological behavior of COM is in stark contrast to calcium oxalate dihydrate (COD), a different mineral phase commonly found in voided urine but much less frequently in stones, and whose presence is thought to protect against stone formation. This observation suggests that the structure and composition of calcium oxalate crystal surfaces and the fundamental interactions of these surfaces with urinary species are crucial to unraveling the complex pathology of this debilitating disease.

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.

ROLE OF ANIONIC PROTEINS IN KIDNEY STONE FORMATION: INTERACTION BETWEEN MODEL ANIONIC POLYPEPTIDES AND CALCIUM OXALATE CRYSTALS

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.

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.

Regulation by macromolecules of calcium oxalate crystal aggregation in stone formers

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.

Role of crystal surface adhesion in kidney stone disease.

Purpose of reviewAtomic 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 findingsThe 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. SummaryThe 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.