Intracrystalline proteins and urolithiasis: a comparison of the protein content and ultrastructure of urinary calcium oxalate monohydrate and dihydrate crystals

Abstract

OBJECTIVE To compare the ultrastructure and protein content, particularly prothrombin fragment 1 and osteopontin, of calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD) crystals precipitated from human urine, and their susceptibility to proteolysis, to try to clarify the role of intracrystalline proteins in urolithiasis, as differences between these types of crystal may determine whether calcium oxalate crystals nucleated in urine progress to stone formation. MATERIALS AND METHODS Sodium dodecyl sulphate gel electrophoresis and Western blotting were used to analyse demineralized extracts of COM and/or COD crystals deposited from the same centrifuged and filtered urine (which contains abundant urinary proteins) by adjusting the calcium concentration to 2 and 7 mmol/L, respectively. Similar analyses were performed on COM and COD crystals deposited from ultrafiltered urine (which contains only proteins of < 10 kDa) and then incubated in centrifuged and filtered urine, as well as crystals generated in the presence of increasing concentrations of proteins derived from the organic matrix of urinary calcium oxalate crystals. Field‐emission scanning electron microscopy was used to assess effects of proteinase K and cathepsin D on internal and superficial crystal structure. RESULTS Osteopontin was undetectable in COM extracts, but clearly visible in COD. Prothrombin fragment 1 was abundant in COM, but present in COD in lesser amounts than osteopontin. The selectivity was also the same with crystals from ultrafiltered urine that were incubated in centrifuged and filtered urine: prothrombin fragment 1 binding was favoured by low calcium concentration, while osteopontin bound at higher levels. Scanning electron microscopy of COM and COD digested with proteinase K and cathepsin D revealed superficial and internal texture, as wells as surface erosion, in crystals from centrifuged and filtered urine, thus confirming the presence of intracrystalline proteins. Such features were absent from crystals precipitated from ultrafiltered urine. CONCLUSION Binding of osteopontin and prothrombin fragment 1 to calcium oxalate is dictated primarily by ambient calcium concentration. Each protein may inhibit urolithiasis by inhibiting crystallization of its preferred crystal habit, and by facilitating the intracellular disintegration and dissolution of crystals attached to and internalized by renal epithelial cells.

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

“…Twenty-four-hour urine samples collected from six healthy laboratory colleagues were pooled and processed as described earlier (18,47). This study was reviewed and approved by the Flinders Clinical Research Ethics Committee.…”

“…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.…”

“…CME was prepared from COM crystals precipitated from pooled centrifuged and filtered (0.22 m) urine (47). An aliquot was retained for protein determination (47) and SDS-PAGE and Western blot analysis, and the remainder was stored at Ϫ70°C for later use.…”

“…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).…”