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.

Acidic polyanion poly(acrylic acid) prevents calcium oxalate crystal deposition

“…4 Acidic urinary macromolecules that have been studied individually interact strongly with crystals and generally inhibit crystal formation, especially those of calcium oxalate (CaOx), in vitro. [5][6][7][8][9][10][11][12] Synthetic polyanions have similar effects on CaOx formation; however, polycations and neutral polymers are, in general, ineffective. [13][14][15][16][17] Among the synthetic polyanions, those with carboxylate side chains are more potent at inhibiting crystal formation than phosphoryl or sulfonyl, in that order.…”

OPN gene polymorphism (Ala250) and lower serum OPN levels are associated with urolithiasis

“…OPN is able to inhibit very early phases of CaOx crystallization at concentrations that may be present in the renal tubule, and at levels of supersaturation found in urine. 18 The urinary concentration of OPN is in the range within which its contribution to the inhibition of growth and aggregation is likely to be considerable. 4 Since OPN is an important molecule for kidney stone formation, SNPs in the gene that conduct an expression of OPN may possibly be a genetic predisposing factor to urolithiasis.…”

Deletion of the 8 Integrin Gene Does Not Protect Mice From Myocardial Fibrosis in DOCA Hypertension

“…A rabbit polyclonal antibody to collagen I (Biogenesis, Poole, England) was used at a dilution of 1:1,000. A rabbit polyclonal OPN antibody which was kindly provided by Dr. Kleinman (Milwaukee, WI) 26,27 was used at a dilution of 1:1,000.…”