Renal epithelial cells rapidly bind and internalize calcium oxalate monohydrate crystals.

Abstract

Renal tubular fluid is supersaturated with calcium and oxalate ions, which can nucleate to form crystals of calcium oxalate monohydrate (COM), the most abundant constituent of kidney stones. However, the mechanisms by which nascent crystals are retained in the nephron and then grow into kidney stones are unclear. An interaction of COM crystals with the surface of renal epithelial cells could be a critical initiating event in nephrolithiasis. To investigate this possibility we used cultures of monkey kidney epithelial cells (BSC-1 line) as a model system and found that [14CJCOM crystals bound to the cell surface within seconds. Scanning electron microscopy revealed that crystals bind first to apical microvilli, which subsequently migrate over the crystalline surface. When visualized by transmission electron microscopy, intracellular crystals were located within vesicles. Cytoskeletal responses to crystal uptake were sought by immunofuorescence microscopy, which revealed concentration of (3). Before use, crystals were sterilized by heating to 180TC overnight and then suspended in distilled water to form a slurry from which they were added to the culture medium (3). X-ray crystallography, performed by S. Deganello (University of Chicago), demonstrated that heating did not alter the structure of COM crystals.[14C]COM crystals (10-300 Mig/ml; 2.4-70.8 pg/cm2 cell surface) were added to high-density, quiescent cultures in 60-mm dishes (Nunc) to define the kinetics of association between a cell and crystal. After a specified period, the medium was aspirated, and the monolayer was washed three times with phosphate-buffered saline (PBS; 5 ml). Each culture was inspected under a microscope and the number of cells with adherent crystals was counted in five separate fields. Subsequently, the cell monolayer with adherent crystals was scraped directly into a scintillation vial containing 6 M HC (0.5 ml), 4.5 ml of Ecoscint (National Diagnostics) was added, and the amount of radioactivity was measured. To investigate the effect of crystal exposure for up to 15 days on renal epithelial cells, COM crystals (50 pg/ml) were added to near-confluent cultures of BSC-1 cells (106 cells per 60-mm dish) containing 0.5% calf serum. Every 4 days thereafter, the medium was aspirated and replaced with fresh medium containing 0.5% calf serum with no additional crystals. At 1, 8, and 15 days after addition of crystals, the medium was aspirated, and a solution of crystalline trypsin was used to detach the cells, which were then inspected under a microscope as described (4). The total number of cells in each culture was counted with a hemocytometer, and 100 cells from each culture were scored for the presence of internalized crystals.Abbreviations: COM, calcium oxalate monohydrate; TEM, transmission electron microscopy; SEM, scanning electron microscopy. tTo whom reprint requests should be addressed. 6987The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "adv...

Cytoprotective role of the aqueous extract of Terminalia chebula on renal epithelial cells

“…It is used to treat urolithiasis and is actively used in various drug formulations of kidney stone treatments like neeri (product information by AIMIL PHARMACEUTICALS (INDIA) LTD.). It is extensively explored for antimicrobial (7), antioxidant (8), anticarcinogenic (9), hypocholesterolemic (10) and diuretic (11,12) activities by various research groups.…”

Modulation of Proliferating Renal Epithelial Cell Affinity for Calcium Oxalate Monohydrate Crystals

“…However, binding of microcrystals to the apical surface of tubular cells (5,11) or perhaps nucleation on the cell surface (36) followed by cellular processing of the crystals (11,37) could be important determinants of intranephronal calcification. Our study supports the hypothesis that injury of tubular cells favors crystal deposition (33) and possibly promotes kidney stone formation.…”

“…The COM crystals that precipitated had a range in specific activity from 300 to 450 cpm/ g. Therefore, baseline crystal adhesion values could vary as much as 50%. Crystal size and shape were assessed by light and scanning electron microscopy (11). COM crystals were cuboidal to spindle shaped and uniformly small at 1 to 2 m in largest diameter.…”

“…In more marked hyperoxaluric states (e.g., enteric or primary hyperoxaluria), direct adhesion of calcium oxalate crystals to renal epithelial cells may predominate (4). Therefore, we and others have hypothesized that attachment of newly formed crystals to the tubular cell surface (5-10) and the cellular responses that follow (11)(12)(13) could result in crystal retention and thereby set in motion a series of events that lead to pathologic renal calcification. Adhesion of calcium oxalate crystals to anionic, sialic acid-containing molecules on the surface of renal epithelial cells is crystal-face specific (14) and can be blocked by competing soluble anions in tubular fluid such as glycosaminoglycans, citrate, or glycoproteins (5,6).…”

Annexin II Is Present on Renal Epithelial Cells and Binds Calcium Oxalate Monohydrate Crystals

“…In addition, it appears mathematically unlikely that nucleation and growth of an individual crystal could produce a particle large enough to occlude a nephron lumen (4). Therefore, our laboratory (5)(6)(7)(8)(9)(10)(11) and others (12)(13)(14)(15)(16)(17) have investigated the mechanism(s) whereby urinary crystals can interact with renal epithelial cells; once adherent to a cell, smaller particles could be retained and serve as a nidus for renal stone formation.…”