Search

We previously reported the importance of osteopontin (OPN) in the formation of urinary calculus. Since OPN protein is present in normal kidneys, we investigated the difference in OPN at the DNA level between normal subjects and urolithiasis patients. There has not been any genetic investigation of OPN in familial urolithiasis previously reported worldwide. To confirm hereditary predisposing factors for urolithiasis, changes in OPN DNA within a family were investigated in relation to the presence or absence of urinary calculus. Leukocyte OPN DNA from two normal subjects and five patients with urinary calculus was investigated by SSCP analysis: OPN DNA nucleotide sequence was determined, based on the result of SSCP analysis. As a result, a mutation of GCC to GCT, encoding amino acid position 250 (Ala-250) was found. To confirm the frequency of mutation at this site, OPN DNA was extracted from peripheral blood in 36 normal subjects (Con group), 25 patients with familial urolithiasis (FSF), and 40 patients with recurrent urinary calculus and who had had two or more previous episodes (RSF). The degree of mutation at Ala-250 was then examined by restriction fragment length polymorphism (RFLP) method. As described above, the nucleotide codon encoding the amino acid sequence position 250, Ala-250, was GCC in two normal subjects. This is the original codon. In five patients with urolithiasis it was GCT, showing a substitution of C with T. On examining the frequency of this mutation, the ratio of normal homozygous GCC was 11/36 in the Con group, 1/25 in FSF and 1/40 in RSF. The ratio of heterozygous GCC/GCT was 16/36 in the Con group, 15/25 in FSF and 26/40 in RSF, and the ratio of homozygous GCT was 9/36 in the Con group, 9/25 in FSF and 13/40 in RSF. Furthermore, the gene frequency of the normal codon GCC was 0.528 in the Con group, 0.3 in FSF and 0.35 in RSF, showing a significantly higher incidence in the Con group (P < 0.05). The gene frequency of mutated GCT was 0.472 in Con group, 0.7 in FSF and 0.65 in RSF, showing a significantly higher incidence in urolithiasis patients (P < 0.05). On investigating the inheritance of Ala-250 in five families in which both parent and offspring demonstrated urolithiasis, the nucleotide substitution in Ala-250 in parents with urolithiasis was inherited by their offspring. In all five families the offspring developed urinary calculus. This study showed that there is no difference in OPN structure between the Con group and urolithiasis patients. However, it was predicted that due to the frequency of normally coded GCC being high in the Con group a difference in the amount of OPN might be caused by a difference in transcription velocity between the two groups. Furthermore, it was suggested that examining the inheritance of Ala-250 within a family is a diagnostic method for identifying the predisposing hereditary factors for urolithiasis patients.

Objective: We have recently reported that osteopontin protein (OPN) is present in calcium-containing urinary calculi and in rat distal tubular cells and that it is an important substance of the matrix in urinary stone formation. To investigate how OPN participates in urinary stone formation, we assessed the role of OPN in the adhesion of the surfaces of Madin-Darby canine kidney (MDCK) cells, which are distal tubular cells of the dog, to calcium oxalate crystals., Methods: We cultured MDCK cells with OPN, thrombin, which is an OPN inhibitor, Arg-Gly-Asp peptide and Arg-Gly-Glu peptide as a control for Arg-Gly-Asp peptide, and examined changes in adhesion of calcium oxalate crystals to the cells by scanning electron microscopy and the calcium (45Ca) concentration around the cells., Results: The 45Ca concentration in calcium oxalate crystals adhering to MDCK cells was about 1.4 times higher in MDCK cells incubated with OPN than in an MDCK-cell-negative group, and was about one half that in MDCK cells incubated with thrombin than in the group without MDCK cells., Conclusion: OPN is considered to be an accelerator of urinary stone formation because of the increase in adhesion of calcium oxalate crystals to the renal distal tubular epithelium in the presence of OPN. These findings suggest that OPN plays an important role in the formation of calcium-containing urinary stones.

We had sequenced a cDNA of calcium oxalate urinary stone protein extracted with 0.1 M EDTA. cDNA sequences showed complete homology between urinary stone protein and human osteopontin (OPN, bone sialoprotein I). In the present study, we investigated the expression of OPN mRNA in the rat kidney in an experimental model of renal stone formation using glyoxylic acid and 1,25-dihydroxyvitamin D3 (D3). In the renal stone formation model with and without renal failure, OPN mRNA was shown to be localized by in situ hybridization using an OPN cRNA probe mainly in the distal convoluted tubule of the kidney, and was enhanced compared with the normal control which was sporadically positive. By Northern blot analysis, the expression of OPN mRNA was shown to be increased by about 5.2-fold in the renal stone formation model and 2.3-fold in D3-administered rats compared with controls. However, no change in the expression of OPN mRNA was observed in an acute renal failure model induced by gentamicin or in rats administered glycoxylic acid alone. Therefore, the promotion of OPN mRNA expression was intrinsic to this stone formation model, and not secondary to acute renal failure because of obstruction by microcrystals in the renal tubules or gentamicin.

We have sequenced a cDNA of urinary stone protein. cDNA sequences show complete homology between urinary stone protein and human osteopontin (bone sialoprotein) (nucleotides 265-886 and 1183-1424). Osteopontin is a recently discovered bone matrix protein which has been implicated in mediating mineral formation within bone extracellular matrix. This result shows that osteopontin is presumably involved in stone formation as stone matrix.