The importance of a clean face: the effect of different washing procedures on the association of Tamm–Horsfall glycoprotein and other urinary proteins with calcium oxalate crystals

Abstract

This study was undertaken to determine whether the use of different washing procedures could explain dissident findings in published studies examining the role of urinary macromolecules in urolithiasis. Calcium oxalate monohydrate (COM) crystals were deposited from or added to the same sieved urine, washed with copious or limited amounts of distilled water, or with methanol, and examined by field emission scanning electron microscopy (FESEM). Demineralized extracts were analysed by SDS-PAGE and Western blotting for Tamm-Horsfall glycoprotein (THG), human serum albumin (HSA), osteopontin (OPN) and prothrombin fragment 1 (PTF1). Synchrotron X-ray diffraction (SXRD) with Rietveld whole-pattern peak fitting and profile analysis was used to determine non-uniform crystal strain and crystallite size in crystals generated from inorganic solutions in the presence of increasing concentrations of THG and prothrombin (PT). HSA and PTF1 were present in all demineralized crystal extracts, confirming their inclusion within COM. OPN was present in all extracts except those derived from pure inorganic COM crystals, because of its occlusion within small numbers of calcium oxalate dihydrate (COD) crystals contaminating the COM population. THG was absent from the demineralized extracts of all crystals washed copiously with water, but present in those washed with methanol or limited amounts of water. FESEM showed extraneous organic material associated only with crystals whose extracts contained THG, confirming that the protein does not bind permanently to the COM crystal surface and is not occluded within the mineral bulk. This was confirmed by SXRD, which showed that non-uniform strain and crystallite size remained unaltered in crystals grown in the presence of increasing THG concentrations. However, non-uniform strain increased and crystallite size decreased with increasing PT concentrations, demonstrating unambiguously that PT is included in COM crystals. It was concluded that scrupulous care must be taken to ensure the complete removal of extraneous THG adventitiously associated with CaOx crystals in order to avoid inaccurate analysis of crystal matrix protein content and possible misinterpretation of experimental data.

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

“…A number of studies (1, 46) used high-resolution SXRD to demonstrate the presence of intracrystalline proteins in biogenic calcite crystals, a method that we have also previously applied to confirm that COM crystals precipitated from human urine contain intracrystalline proteins (17). Using the same technique, we have shown that urinary COM crystals have raised lattice strains and decreased crystallite sizes proportional to the concentration of crystal matrix macromolecules in the urine from which they were generated, thus confirming that proteins are incarcerated within the CaOx mineral bulk and substantiating our previous data demonstrating similar dose-response relationships with increasing concentrations of prothrombin (49). Increased lattice strain and reduced crystallite size result from the presence of intracrystalline defects and discontinuities, which destabilize the crystalline structure and thereby render it more susceptible to proteolytic invasion and dissolution.…”

“…The ultrafiltered (10 kDa) sample was divided into five portions, to which CME was added to give final concentrations of 0, 0.5, 1.0, 2.0, and 5.0 mg/l. Radiolabeled and nonradiolabeled urinary COM crystals were then precipitated from each sample (7), filtered (0.22 m), washed (49), lyophilized, and stored at Ϫ70°C. Before use, crystals were sterilized by ethylene oxide and suspended (400 g/ml) in PBS saturated with CaOx, which we have shown in unpublished work does not alter their binding to or internalization by cells, or their vulnerability to proteolytic degradation.…”

“…Suspensions of the unlabeled COM crystals were examined using a Philips XL30 field-emission scanning electron microscope (FESEM) as described earlier (47,49). Ten fields were randomly selected for each stub, and the length and breadth of 10 single crystals were measured in each field.…”

“…The lyophilized samples were mixed with reducing sample buffer, electrophoresed, and the gel was stained with silver as described previously (18,47,49). For Western blotting, after electrophoresis, proteins were electrophoretically transferred to nitrocellulose membrane followed by immunoblotting using antibodies detailed previously (18).…”

“…Dissolution experiments were carried out as described above, but in culture dishes containing sterile 13-mm-diameter Thermanox Plastic coverslips (Nalge Nunc). After 24 h the coverslips were mounted on aluminium stubs, dried overnight at 37°C, coated with platinum, and examined using FESEM as described earlier (47,49).…”