The calcium oxalate crystal growth inhibitor protein produced by mouse kidney cortical cells in culture is osteopontin.
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.
Worcester
E M
EM
Nephrology Section, Zablocki VA Medical Center, Milwaukee.
Blumenthal
S S
SS
Beshensky
A M
AM
Lewand
D L
DL
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
D000596:Amino Acids; D003470:Culture Media; D012795:Sialoglycoproteins; C506896:Spp1 protein, mouse; D053495:Osteopontin; D002129:Calcium Oxalate