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


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.


CONCLUSIONS
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.
Ryall
Rosemary L
RL
Department of Surgery, Flinders Medical Centre and Flinders University School of Medicine, Bedford Park, South Australia. rose.ryall@flinders.edu.au
Chauvet
Magali C
MC
Grover
Phulwinder K
PK
D015153:Blotting, Western / Q000379:methods; D002129:Calcium Oxalate / Q000737:chemistry / Q000652:urine*; D002402:Cathepsin D / Q000737:chemistry; D003460:Crystallization; D004591:Electrophoresis, Polyacrylamide Gel; D019286:Endopeptidase K / Q000737:chemistry; D005260:Female; D006801:Humans; D008297:Male; D008855:Microscopy, Electron, Scanning; D053495:Osteopontin; D010446:Peptide Fragments / Q000032:analysis*; D011487:Protein Conformation; D011498:Protein Precursors / Q000032:analysis*; D011516:Prothrombin / Q000032:analysis*; D012795:Sialoglycoproteins / Q000032:analysis*; D014462:Ultrafiltration; D014545:Urinary Calculi / Q000378:metabolism / Q000652:urine*
D010446:Peptide Fragments; D011498:Protein Precursors; C506895:SPP1 protein, human; D012795:Sialoglycoproteins; D053495:Osteopontin; D002129:Calcium Oxalate; C029733:prothrombin fragment 1; D011516:Prothrombin; D019286:Endopeptidase K; D002402:Cathepsin D