Intracrystalline urinary proteins facilitate degradation and dissolution of calcium oxalate crystals in cultured renal cells

Abstract

134: 5-14, 2001. The aim of this investigation was to determine the effect of increasing concentrations of intracrystalline protein on the rate of CaOx crystal dissolution in Madin-Darby canine kidney (MDCKII) cells. Crystal matrix extract (CME) was isolated from urinary CaOx monohydrate (COM) crystals. Cold and [ 14 C]oxalate-labeled COM crystals were precipitated from ultrafiltered urine containing 0 -5 mg/l CME. Crystal surface area was estimated from scanning electron micrographs, and synchrotron X-ray diffraction was used to determine nonuniform strain and crystallite size. Radiolabeled crystals were added to MDCKII cells and crystal dissolution, expressed as radioactive label released into the medium, was measured. Increasing CME content did not significantly alter crystal surface area. However, nonuniform strain increased and crystallite size decreased in a dose-response manner, both reaching saturation at a CME concentration of 3 mg/ and demonstrating unequivocally the inclusion of increasing quantities of proteins in the crystals. This was confirmed by Western blotting. Crystal dissolution also followed saturation kinetics, increasing proportionally with final CME concentration and reaching a plateau at a concentration of ϳ2 mg/l. These findings were complemented by field emission scanning electron microscopy, which showed that crystal degradation also increased relative to CME concentration. Intracrystalline proteins enhance degradation and dissolution of CaOx crystals and thus may constitute a natural defense against urolithiasis. The findings have significant ramifications in biomineral metabolism and pathogenesis of renal stones. nephrocalcinosis; urolithiasis THE PATHOGENESIS OF RENAL calculi requires the sequential combination of two processes, namely, the nucleation of crystals and their retention within the kidney. While crystal nucleation requires supersaturation of urine with calcium oxalate (CaOx), renal crystal trapping has been explained by either the "free particle" or "fixed particle" theory. Although both these theories appear equally plausible (26), current consensus favors a fixed particle mechanism. This view is supported by the results of studies performed in the early 1990s, which demonstrated for the first time that CaOx crystals irreversibly adhere to and are phagocytosed by cultured renal epithelial cells (35,39). These findings introduced a new paradigm to urolithiasis research because they validated the credibility of using this experimental approach to examine and manipulate factors affecting the regulation of crystal retention, which had not previously been possible. Consequently, there followed a series of reports investigating factors affecting interactions between CaOx monohydrate (COM) crystals and renal cells (reviewed in Refs. 30 and 38). Those studies showed that COM crystals, which are the predominant form of CaOx occurring in human kidney stones (41), are highly membranolytic (55), and also that their adherence to renal epithelial cells is very rapid, concentration ...

Modulation of Calcium Oxalate Dihydrate Growth by Selective Crystal-face Binding of Phosphorylated Osteopontin and Polyaspartate Peptide Showing Occlusion by Sectoral (Compositional) Zoning

“…Taken together, these results demonstrate that poly-Asp 86 -93 (and very likely other acidic peptides of OPN generated by proteolytic cleavage) binds to COD, inhibits growth normal to the {110} face, and exclusively occludes into its {110} growth sectors. These results are consistent with a variety of other in vitro studies suggesting intracrystalline OPN in urinary COD crystals (1,23,24,36).…”

“…Some macromolecules, including urinary osteopontin (OPN), contain polyanionic regions and net negative charges that have been shown to inhibit calcium oxalate crystallization (14, 18 -21) and influence calcium oxalate growth in favor of COD (3,10,22,23). Although there appears to be preferential inhibition of COM, several studies present evidence for higher affinity of OPN binding to COD, and here we investigate this further to show the effects of OPN (and a peptide of OPN) on COD crystal growth and provide information on peptide/protein occlusion that might facilitate crystal dissolution as originally proposed by Ryall et al (see below) (23,24).…”

“…In addition, emerging data show that OPN may potentially direct the formation of COD and in the process incorporate (occlude) into the crystal structure (1,23,36). Interesting proposals for this occlusion include facilitating dissolution of the crystals from the inside-out by providing intracrystalline channels for fluid movement and facilitating phagocytosis of crystals by renal tubular cells for clearance of crystals from the urine (24,36,60,61).…”

“…In a rat model of urolithiasis, although increased OPN mRNA expression was associated with increased renal calculi formation, the urinary excretion level of OPN was less than in controls, discussed as being attributable to incorporation of OPN into stones (37,38). In general, the inclusion of OPN plus other urinary macromolecules into renal calculi has been suggested to be part of a cellular defense mechanism designed to inhibit crystal growth and limit the growth of kidney stones, to interrupt inorganic crystal structure of the calcium oxalate minerals, and to provide an organic volume whose degradation by permeating proteases creates channels facilitating dissolution of the mineral phase (23,24). Given these possibilities, our aim was to determine whether fulllength phosphorylated OPN and a poly-Asp peptide of OPN affect COD crystal growth and morphology, and if so, we further aimed to identify the contribution of face-specific binding and intracrystalline incorporation (occlusion) of OPN peptide into COD.…”

Oxalate: From the Environment to Kidney Stones

“…However, the Tamm-Horsfall protein obtained from recurrent stone formers did not modulate the rate of nucleation, while the aggregation of the crystals was even promoted and not inhibited (68). In addition to these specifi c proteins, urine proteins in general can be incorporated in the stone matrix dose-dependently, thus enhancing the degradation or dissolution of stones (69). Furthermore, some polypeptides can display similar effects on CaOx crystallisation.…”