Intracrystalline urinary proteins facilitate degradation and dissolution of calcium oxalate crystals in cultured renal cells.
We have previously proposed that intracrystalline proteins would increase intracellular proteolytic disruption and dissolution of calcium oxalate (CaOx) crystals. Chauvet MC, Ryall RL. J Struct Biol 151: 12-17, 2005; Fleming DE, van Riessen A, Chauvet MC, Grover PK, Hunter B, van Bronswijk W, Ryall RL. J Bone Miner Res 18: 1282-1291, 2003; Ryall RL, Fleming DE, Doyle IR, Evans NA, Dean CJ, Marshall VR. J Struct Biol 134: 5-14, 2001. The aim of this investigation was to determine the effect of increasing concentrations of intracrystalline protein on the rate of CaOx crystal dissolution in Madin-Darby canine kidney (MDCKII) cells. Crystal matrix extract (CME) was isolated from urinary CaOx monohydrate (COM) crystals. Cold and [14C]oxalate-labeled COM crystals were precipitated from ultrafiltered urine containing 0-5 mg/l CME. Crystal surface area was estimated from scanning electron micrographs, and synchrotron X-ray diffraction was used to determine nonuniform strain and crystallite size. Radiolabeled crystals were added to MDCKII cells and crystal dissolution, expressed as radioactive label released into the medium, was measured. Increasing CME content did not significantly alter crystal surface area. However, nonuniform strain increased and crystallite size decreased in a dose-response manner, both reaching saturation at a CME concentration of 3 mg/ and demonstrating unequivocally the inclusion of increasing quantities of proteins in the crystals. This was confirmed by Western blotting. Crystal dissolution also followed saturation kinetics, increasing proportionally with final CME concentration and reaching a plateau at a concentration of approximately 2 mg/l. These findings were complemented by field emission scanning electron microscopy, which showed that crystal degradation also increased relative to CME concentration. Intracrystalline proteins enhance degradation and dissolution of CaOx crystals and thus may constitute a natural defense against urolithiasis. The findings have significant ramifications in biomineral metabolism and pathogenesis of renal stones.
Grover
Phulwinder K
PK
Urology Unit, Department of Surgery, School of Medicine, Flinders University, Bedford Park, South Australia, Australia. pk.Grover@flinders.edu.au
Thurgood
Lauren A
LA
Fleming
David E
DE
van Bronswijk
Wilhelm
W
Wang
Tingting
T
Ryall
Rosemary L
RL
D000818:Animals; D002129:Calcium Oxalate / Q000737:chemistry / Q000378:metabolism* / Q000652:urine; D002478:Cells, Cultured; D003460:Crystallization; D004285:Dogs; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D053495:Osteopontin / Q000032:analysis; D010316:Particle Size; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D011498:Protein Precursors / Q000032:analysis / Q000737:chemistry; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D011516:Prothrombin / Q000032:analysis / Q000737:chemistry; D012709:Serum Albumin / Q000032:analysis; D013499:Surface Properties; D014545:Urinary Calculi / Q000737:chemistry / Q000378:metabolism*; D014961:X-Ray Diffraction
D010446:Peptide Fragments; D011498:Protein Precursors; D011506:Proteins; D012709:Serum Albumin; D053495:Osteopontin; D002129:Calcium Oxalate; C029733:prothrombin fragment 1; D011516:Prothrombin