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

OBJECTIVE To compare the binding to Madin-Darby canine kidney (MDCK)-II cells of: (i) inorganic calcium oxalate monohydrate (iCOM) crystals and COM crystals precipitated from urine containing different concentrations of protein; and (ii) urinary COM crystals containing intracrystalline and intracrystalline + surface-bound protein. MATERIALS AND METHODS Urinary 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 and 5.0 mg/L. The amount of protein associated with the crystals was qualitatively assessed by sodium dodecyl sulphate-polyacrylamide 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. RESULTS The 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. CONCLUSION Inorganic 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.Phulwinder K. Grover, Lauren A. Thurgood, Tingting Wang and Rosemary L. Ryal