Structure and expression of the mRNA encoding urinary stone protein (osteopontin)

Abstract

The chemical nature of urinary stone protein is poorly understood. We have sequenced a cDNA of urinary calcium oxalate stone protein extracted with EDTA. cDNA sequences showed complete identity between urinary stone protein and human osteopontin. Osteopontin protein was detected by staining with Stains-All, which specifically stains phosphoproteins, and by digestion with the highly specific protease thrombin, demonstrating that urinary calcium oxalate stones consist of osteopontin protein. We used a technique of in situ hybridization to detect osteopontin mRNA in the kidney. In control rats, distal tubular cells were sporadically positive, and proximal tubular cells and glomeruli were negative for osteopontin mRNA. A rat model of stone formation was induced with glyoxylic acid. In stone-forming rats, staining of distal tubular cells was remarkably increased, but proximal tubular cells and glomeruli were still negative. Immunostaining for the osteopontin protein also revealed that epithelial cells of distal tubules were weakly positive in control rats and significantly increased in stone-forming rats, although proximal tubular cells and glomeruli were negative. Northern blot analysis showed a significant increase of osteopontin mRNA in stone-forming rats in proportion to the dosage and the duration of the stone-inducing drugs. These results show that osteopontin in the kidney is presumably involved in urinary stone formation as the stone matrix.