title: The calcium oxalate crystal growth inhibitor protein produced by mouse kidney cortical cells in culture is osteopontin.
abstract: Urine contains proteins that inhibit the growth of calcium oxalate (CaOx) crystals and may prevent the formation of kidney stones. We have identified a potent crystal growth inhibitor in the conditioned media from primary cultures of mouse kidney cortical cells. Conditioned media, incubated with the kidney cells for 6-72 h, was assayed for crystal growth inhibition; inhibitory activity increased 15-fold by 24 h. Inhibitory activity was purified from serum-free media containing proteinase inhibitors using anion-exchange and gel-filtration chromatography. A single band of molecular weight 80,000 daltons was seen after SDS-polyacrylamide gel electrophoresis. The sequence of the N-terminal 21 amino acids of this protein matched that of osteopontin (OP), a phosphoprotein initially isolated from bone matrix. Antisera raised to fusion proteins produced by plasmids containing the N-terminal or C-terminal portions of OP cDNA also cross-reacted with the protein purified from cell culture media on western blots. The effect of the purified protein on the growth of CaOx crystals was measured using a constant composition assay. A 50% inhibition of growth occurred at a protein concentration of 0.85 micrograms/ml, and the dissociation constant of the protein with respect to CaOx crystal was 3.7 x 10(-8) M. The concentration of OP in mouse urine, measured using antibodies raised to the purified protein, was approximately 8 micrograms/ml. We conclude that OP is synthesized by kidney cortical tubule cells and functions as a crystal growth inhibitory protein in urine.
authors.affiliation: Nephrology Section, Zablocki VA Medical Center, Milwaukee.
authors.forename: E M
authors.initials: EM
authors.lastname: Worcester
authors.forename: S S
authors.initials: SS
authors.lastname: Blumenthal
authors.forename: A M
authors.initials: AM
authors.lastname: Beshensky
authors.forename: D L
authors.initials: DL
authors.lastname: Lewand
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002129:Calcium Oxalate / Q000737:chemistry*; D002478:Cells, Cultured; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003460:Crystallization; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D007684:Kidney Tubules / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D053495:Osteopontin; D012795:Sialoglycoproteins / Q000096:biosynthesis* / Q000737:chemistry / Q000494:pharmacology / Q000652:urine
chemical_list: D000596:Amino Acids; D003470:Culture Media; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; D053495:Osteopontin; D002129:Calcium Oxalate