Urolithiasis is a serious, debilitating and costly problem in societies throughout the world. The role of urinary proteins in calcium oxalate (CaOx) kidney stone formation, particularly intracrystalline proteins, remains a mystery. The planned work will clarify the role of superficial and intracrystalline CaOx proteins, especially OPN and UPTF1, in the attachment of urinary crystals to renal epithelial cells, as well as their subsequent disintegration and dissolution. Using a Madin-Darby canine kidney (MDCK) cell line model of crystal-cell interaction, the studies will: (1) determine the effects of intracrystalline and surface-bound OPN and UPTF1 on the binding, phagocytosis, intracellular disintegration and dissolution of CaOx crystals. (2) (a) identify which lysosomal proteases digest intracrystalline OPN and UPTF1 and (b) determine the effects of crystal phagocytosis on their individual specific activities, as well as that of total protease specific activity. (3) (a) determine the effects of intracrystalline OPN and UPTF1 on the gene expression (mRNA) of individual proteases identified in Aim 2 and on the gene expression (mRNA) and further cellular production of UPTF1 and OPN. (b) Determine the effects of [Ca] and [Ox] on the gene expression (mRNA) of OPN and UPTFI. (4) (a) compare the ability of urinary CaOx crystals from male and female stone formers and controls to be bound, phagocytosed and destroyed intracellularly; (b) determine the effects of those crystals on the specific activity and cellular synthesis of the lysosomal enzymes identified in Aim 2, as well as the cellular synthesis of OPN and UPTF1; (c) quantify the urinary activities of those enzymes in stone formers and healthy subjects. The proposed studies will use a combination of cell culture, high-resolution microscopy, enzymatic analysis, protein chemistry and molecular biological techniques. The data generated will offer boundless promise in the design of drugs for stone prophylaxis, the construction of porous crystals for medical and industrial applications, and for understanding the cycle of biomineral formation, destruction and reclamation throughout the natural world.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK064050-03
Application #
7109203
Study Section
Special Emphasis Panel (ZRG1-UROL (01))
Program Officer
Rasooly, Rebekah S
Project Start
2004-04-01
Project End
2009-03-31
Budget Start
2006-04-01
Budget End
2007-03-31
Support Year
3
Fiscal Year
2006
Total Cost
$181,516
Indirect Cost
Name
Flinders University of South Australia
Department
Type
DUNS #
756172482
City
Adelaide
State
Country
Australia
Zip Code
5001
Thurgood, Lauren A; Sørensen, Esben S; Ryall, Rosemary L (2012) The effect of intracrystalline and surface-bound osteopontin on the degradation and dissolution of calcium oxalate dihydrate crystals in MDCKII cells. Urol Res 40:1-15
Thurgood, Lauren A; Sorensen, Esben S; Ryall, Rosemary L (2012) The effect of intracrystalline and surface-bound osteopontin on the attachment of calcium oxalate dihydrate crystals to Madin-Darby canine kidney (MDCK) cells in ultrafiltered human urine. BJU Int 109:1100-9
Thurgood, Lauren A; Ryall, Rosemary L (2010) Proteomic analysis of proteins selectively associated with hydroxyapatite, brushite, and uric acid crystals precipitated from human urine. J Proteome Res 9:5402-12
Thurgood, Lauren A; Cook, Alison F; Sørensen, Esben S et al. (2010) Face-specific incorporation of osteopontin into urinary and inorganic calcium oxalate monohydrate and dihydrate crystals. Urol Res 38:357-76
Grover, Phulwinder K; Thurgood, Lauren A; Wang, Tingting et al. (2010) The effects of intracrystalline and surface-bound proteins on the attachment of calcium oxalate monohydrate crystals to renal cells in undiluted human urine. BJU Int 105:708-15
Wang, Tingting; Thurgood, Lauren A; Grover, Phulwinder K et al. (2010) A comparison of the binding of urinary calcium oxalate monohydrate and dihydrate crystals to human kidney cells in urine. BJU Int 106:1768-74
Thurgood, Lauren A; Wang, Tingting; Chataway, Timothy K et al. (2010) Comparison of the specific incorporation of intracrystalline proteins into urinary calcium oxalate monohydrate and dihydrate crystals. J Proteome Res 9:4745-57
Cook, Alison F; Grover, Phulwinder K; Ryall, Rosemary L (2009) Face-specific binding of prothrombin fragment 1 and human serum albumin to inorganic and urinary calcium oxalate monohydrate crystals. BJU Int 103:826-35
Thurgood, Lauren A; Grover, Phulwinder K; Ryall, Rosemary Lyons (2008) High calcium concentration and calcium oxalate crystals cause significant inaccuracies in the measurement of urinary osteopontin by enzyme linked immunosorbent assay. Urol Res 36:103-10
Grover, Phulwinder K; Thurgood, Lauren A; Fleming, David E et al. (2008) Intracrystalline urinary proteins facilitate degradation and dissolution of calcium oxalate crystals in cultured renal cells. Am J Physiol Renal Physiol 294:F355-61

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