The effects of intracrystalline and surface‐bound proteins on the attachment of calcium oxalate monohydrate crystals to renal cells in undiluted human urine

Abstract

and 5.0 mg/L. The amount of protein associated with the crystals was qualitatively assessed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis and Western blotting, using prothrombin fragment 1 (PTF1) as a marker. Protein concentration was determined in sieved, centrifuged and filtered, and ultrafiltered fractions of 10 additional urine samples. RESULTSThe median crystal attachment in the six urine types decreased in the order iCOM > ufCOM > cfCOM = sCOM, in inverse proportion to the concentration of protein in the solution or urine from which they were precipitated. sCOM and cfCOM crystals bound ≈ 23% less than iCOM crystals. The attachment of COM crystals generated in the presence of increasing concentrations of CME proteins was unaffected up to a concentration of 5 mg/L, but binding of crystals containing the same concentrations of intracrystalline + surface-bound proteins decreased proportionally at protein concentrations from 0 to 5.0 mg/L. CONCLUSIONInorganic COM crystals bind significantly more strongly to MDCK-II cells than urinary crystals precipitated from sieved, centrifuged and filtered, and ultrafiltered urine, and binding affinity is inversely related to the concentration of protein in the urine in which they are formed. While both intracrystalline and superficial CME proteins reduce the attachment of COM crystals to MDCK-II cells, those located on the crystal surface have a greater influence than those incarcerated within the mineral bulk. Future cell-crystal interaction studies should use urinary crystals and be performed in human urine. KEYWORDS MATERIALS AND METHODSUrinary COM crystals were generated in sieved (sCOM), centrifuged and filtered (cfCOM), and ultrafiltered (ufCOM) portions of a pooled human urine and their adhesion to MDCK-II cells was compared using six different ultrafiltered urine samples as the binding medium. Crystal matrix extract (CME) was prepared by demineralizing calcium oxalate crystals precipitated from human urine and used to prepare COM crystals with intracrystalline, and intracrystalline + surface-bound CME at protein concentrations of 0, 0.05, 0.1, 0.5

Alpha-enolase on apical surface of renal tubular epithelial cells serves as a calcium oxalate crystal receptor

“…6). Our findings were consistent with those reported in previous studies, which demonstrated that macromolecules present in human urine could block the adhesion of COM crystals to renal tubular cells by coating crystal surface to prevent crystal retention inside the kidney293233.…”

“…Approximately 2.5% of total dry weight of kidney stone composes of organic matrices, of which >50% are enriched with urinary proteins that can affect various steps of kidney stone formation293031. The effects of urinary proteins on binding ability of COM crystals to α-enolase on renal tubular cells were examined (Fig.…”

In vitro Dissolution of Calcium Oxalate Stones with Ethylenediaminetetraacetic Acid and Snake Venom Thrombin-Like Enzyme

“…The matrix itself is comprised of both net and membranous structures that serve to both induce calcium crystal formation and to protect the crystal structure. If the protective effects could be eliminated, the efficacy of stone dissolution therapy would be greatly enhanced [16]. Many proteins have been identified in the organic matrix of calcium calculi, although relatively few of these have been studied in detail.…”

Physicochemical mechanisms of stone formation

“…Of importance is that UMMs have an ability to modulate the adhesion process [26,28,40,41] by forming a protective coating over crystals, thereby retarding the latter's capacity to participate in attractive interactive forces with renal cells [40,41]. As with the SB and SG mechanisms described earlier, different UMMs have different properties.…”