Keiji Fujita

Osteopontin regulates adhesion of calcium oxalate crystals to renal epithelial cells.

Abstract Background : The association of calcium crystals with renal tubular cells is an important factor during the formation of urinary stones. We previously reported the strong expression of osteopontin (OPN) on renal tubular cells in the stone‐forming kidney, suggesting that OPN plays a role in the crystal–cell interaction. In the present study, we examined the biological consequences of inhibiting OPN expression at the translational level on the formation and adhesion of crystals.

Expression of bone matrix proteins in urolithiasis model rats

Abstract Urinary calcium stones are a pathological substance, and they show similarities to physiological mineralization and other pathological mineralizations. The expression of messenger (m) RNAs of osteopontin (OPN), matrix Gla protein (MGP), osteonectin (ON) and osteocalcin (OC) in bones and teeth has been described. We previously identified OPN as an important stone matrix protein. In addition, the spontaneous calcification of arteries and cartilage in mice lacking MGP was recently reported, a finding which indicates that MGP has a function as an inhibitor of mineralization. Here, we examined the mRNA expressions of OPN, MGP, ON, and OC in the kidneys of stone-forming model rats administered an oxalate precursor, ethylene glycol (EG) for up to 28 days. The Northern blotting showed that the mRNA expressions of OPN and MGP were markedly increased with the administration of EG, but their expression patterns differed. The OPN mRNA expression reached the maximal level at day 7 after the initiation of the EG treatment and showed no significant difference after 14 and 28 days, whereas the MGP mRNA expression rose gradually to day 28. The in situ hybridization demonstrated that the cell type expressing OPN mRNA was different from that expressing MGP. We suggest that OPN acts on calcification and MGP acts on suppression.

Quantification of osteopontin in the urine of healthy and stone-forming men

Abstract Osteopontin (OPN) is one of the most important components in calcium stone matrix, but its role in stone formation is not clear. Since quantitative data regarding the excretion of OPN are necessary to assess its role, we have developed a quantitative enzyme-linked immunosorbent assay (ELISA) for OPN, and measured the urinary OPN concentrations in urolithiasis patients. Forty-seven men with urinary stones composed chiefly of calcium oxalate participated in the study. The controls were 13 normal healthy male volunteers. Urine samples were collected early in the morning and analyzed by a quantitative ELISA employing purified polyclonal antibodies to synthesized OPN aminopolypeptides. The urinary ratio of the concentrations of OPN and creatinine (OPN/Cre) in the urolithiasis patients (0.039 ± 0.029) was significantly lower than that in the control subjects (0.062 ± 0.030) (P<0.05). Single stone formers (n = 26; 0.050 ± 0.020) had significantly higher OPN/Cre ratios compared with recurrent stone formers (n = 21; 0.031 ± 0.021) (P<0.05). The results show that OPN excretion in urolithiasis patients was lowered, presumably because of the incorporation of OPN by kidney stones.

The effect of takusha, a kampo medicine, on renal stone formation and osteopontin expression in a rat urolithiasis model.

Abstract Kampo medicine is a traditional Japanese therapeutic system which originated in China and was used to treat various diseases for hundreds of years. Kampo medicine had been also used for the cure and the prevention of urinary calculi for many years, but the effect and the mechanism of this use of kampo medicine are unclear. We examined the inhibitory effect of the kampo medicine takusha on the formation of calcium oxalate renal stones induced by ethylene glycol (EG) and vitamin D3 in rats. We also investigated the effect of takusha on osteopontin (OPN) expression, which we previously identified as an important stone matrix protein. The control group rats were non-treated; the stone group rats were administered EG and vitamin D3, and the takusha group was administered takusha in addition to EG and vitamin D3. The rate of renal stone formation was lower in the takusha group than in the stone group; thus, the OPN expression in the takusha group was smaller than in the stone group. Takusha was effective in preventing oxalate calculi formation and OPN expression in rats. These findings suggest that takusha prevents stone formation including not only calcium oxalate aggregation but also proliferation.

Distribution of osteopontin and calprotectin as matrix protein in calcium-containing stone.

Abstract We recently reported that osteopontin (OPN) and calprotectin (CPT) are present in the matrix of urinary calcium stones, and that OPN mRNA is expressed in the renal distal tubular cells. In the present study, we examined the immunohistochemical distributions of OPN and CPT in urinary stones. The stones used in this study were passed spontaneously from the upper urinary tract. One half of each of the stones was analyzed with an infrared spectrophotometer, and were shown to be comprised of calcium oxalate, calcium phosphate, uric acid and cystine. The other half of each stone was immersed in tetrasodium ethylenediamine-tetraacetate (EDTA) solution. The half-stones were embedded in paraffin and cut into 5-μm sections. The avidin-biotin-peroxidase complex technique was employed. A monoclonal antibody to human milk-derived OPN and a monoclonal antibody to human granulocyte-derived CPT were used as primary antibodies. The immunochemical study using the OPN and CPT antibodies showed positive staining of the matrix of the urinary calcium stones. The stones showed staining in two distinct zones: a core area was stained with randomly aggregated OPN and CPT, and peripheral layers were stained in concentric circles. On the basis of our observations, it is reasonable to presume that OPN and CPT play roles as the matrix in the structure of urinary calcium stones.

Effects of citrate on renal stone formation and osteopontin expression in a rat urolithiasis model.

Abstract Previous studies have described the inhibitory effects of citrate on calcium oxalate crystallization in place of crystal growth, but the effects of citrate on matrix proteins of stones has not been studied in vivo. To examine the effect of citrate on the matrix, we investigated the effect of citrate on osteopontin (OPN) expression, which we had previously identified as an important stone matrix protein. Control rats were treated with saline while rats of the stone group were treated with ethylene glycol (EG) and vitamin D3, and the citrate groups (low-dose and high-dose groups) were treated with a citrate reagent compound of sodium citrate and potassium citrate, in addition to EG and vitamin D3. The rate of renal stone formation was lower in the citrate groups than in the stone group. This was associated with a low expression of OPN mRNA in citrate-treated rats relative to that in the stone group. Citrate was effective in preventing calcium oxalate stone formation and reduced OPN expression in rats. Our results suggest that citrate prevents renal stone formation by acting against not only the crystal aggregation and growth of calcium oxalate but also OPN expression.

Effects of Eicosapentaenoic Acid on Urinary Calcium Excretion in Calcium Stone Formers

Objectives: The low incidence of atherosclerosis and other degenerative disease, including urolithiasis, in the Greenland Eskimo has been attributed to their high consumption of oily fish with its high concentration of eicosapentaenoic acid (EPA). With a westernized diet, the oxygenated products of renal prostaglandin synthesis are metabolites of the n–6 series and these are known to play important roles in several pathophysiological processes involved in calcium stone formation. Buck’s group presented a hypothesis that the initiating factor for lithiasis triggers prostaglandin synthesis, and showed that this influenced by EPA treatment. Method: In order to ascertain the effects of EPA on plasma lipids and urinary parameters, we undertook a clinical study whereby a highly purified preparation was administrated (1,800 mg/day) to 88 patients with urinary stones for 3 months (short term) and 18 months (long term). Results: Hyperlipemia improved the affected individuals and urinary calcium was significantly reduced in the hypercalciuric but not in the normocalciuric group. Conclusion: The results suggest that EPA by reducing urinary calcium might favorably affect urine composition in a way that possibly reduces the risk of calcium stone formation.

Alendronate inhibits osteopontin expression enhanced by parathyroid hormone-related peptide (PTHrP) in the rat kidney

Abstract It has been reported that osteopontin (OPN) plays an important role during urolithiasis as well as bone formation. Generation of stones in the urinary tract may be associated with osteoporosis and bisphosphonates are potent inhibitors of bone resorption, being used with effect in the management of bone disease. We therefore investigated the relationship between alendronate, a bisphosphonate derivative, and OPN expression in the kidney. Alendronate was administered to rats made hypercalcemic by treatment with parathyroid hormone-related peptide (PTHrP). The renal expression of OPN was then evaluated at both protein and mRNA levels. OPN expression was enhanced in the distal tubular cells of hypercalcemic rats and was decreased by alendronate. The observed inhibition of OPN expression suggests an ability of alendronate and other bisphosphonates to act as inhibitors of stone formation in the urinary tract.

Effects of Allopurinol on Renal Stone Formation and Osteopontin Expression in a Rat Urolithiasis Model

Background: The inhibitory effect of allopurinol on calcium oxalate urolithiasis has been reported, but its effect on stone matrix proteins has not been studied in vivo. To clarify the effect of allopurinol on the matrix, we investigated its effect on the expression of osteopontin (OPN), which we previously identified as an important stone matrix protein. Methods: Control rats were not treated. Rats of the stone group were given ethylene glycol (EG) and vitamin D3, while the allopurinol groups (low-dose group and high-dose group) were treated with allopurinol in addition to receiving EG and vitamin D3. Results: The rate of renal stone formation was lower in the allopurinol groups than in the stone group. This was associated with a low expression of OPN mRNA in allopurinol-treated rats relative to that in the stone group. Conclusion: Allopurinol was effective in preventing calcium oxalate stone formation and reduced OPN expression in rats. Our results suggest that allopurinol prevents renal stone formation by acting against not only the control of oxalate but also OPN expression.

Calcium phosphate stones produced by Madin-Darby canine kidney (MDCK) cells inoculated in nude mice.

Abstract The canine renal distal tubular cell line Madin-Darby canine kidney (MDCK) forms calcium phosphate microliths during a long-term culture in vitro. We identified osteopontin (OPN) and calprotectin (CPT) from a urinary stone matrix. We recently also detected the expression of OPN and CPT in MDCK cells. The relationship between the mechanism of the stone formation and these stone matrix proteins is not yet known. Here, MDCK cells were cultured and inoculated in the subcutis of nude mice. After 4, 8 and 12 weeks, the inoculated tissues were resected, fixed and immunostained with polyclonal anti-human OPN and polyclonal anti-human CPT antibodies. Some serial specimens were stained with von Kossas procedure. MDCK cells formed some follicular formations in the subcutis of nude mice at least at 12 weeks after transplantation. At 8 weeks after the inoculation, we detected small calcium phosphate stones with MDCK cells trapped in the follicles. The cells forming the stones also expressed both OPN and CPT. The CPT expression sites coincided with the stone formation sites. We confirmed that MDCK cells inoculated in nude mice had stone-forming potential, and we speculate that OPN and CPT play important roles in stone formation by MDCK cells.