Tohru Umekawa

Molecular cloning and sequencing of cDNA encoding urinary stone protein, which is identical to osteopontin

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.

Effect of Angiotensin II Receptor Blockage on Osteopontin Expression and Calcium Oxalate Crystal Deposition in Rat Kidneys

Hyperoxaluria leads to calcium oxalate (CaOx) crystallization and development of tubulointerstitial lesions in the kidneys. Treatment of hyperoxaluric rats with angiotensin II (Ang II) type I receptor blocker (ARB) reduces lesion formation. Because Ang II mediates osteopontin (OPN) synthesis, which is involved in both macrophage recruitment and CaOx crystallization, it was hypothesized that ARB acts via OPN. Hyperoxaluria was induced in 10-wk-old male Sprague-Dawley rats, and they were treated with ARB candesartan. At the end of 4 wk, kidneys were examined for crystal deposits, ED-1-positive cells, and expression of OPN mRNA. PCR was used to quantify OPN, renin, and angiotensin-converting enzyme (ACE) mRNA in kidneys. RIA was used to determine renal, plasma, and urinary OPN; plasma renin; Ang II and ACE; and renal Ang II. For evaluating oxidative stress, malondialdehyde was measured. Urinary calcium, oxalate, creatinine, and albumin were also determined. Despite similar urinary calcium and oxalate levels, kidneys of hyperoxaluric rats on candesartan had fewer CaOx crystals, fewer ED-1-positive cells, reduced OPN expression, and reduced malondialdehyde than hyperoxaluric rats. Urinary albumin excretion and serum creatinine levels improved significantly on candesartan treatment. mRNA for OPN, renin, and ACE were significantly elevated in hyperoxaluric rats. OPN synthesis and production increased with hyperoxaluria but to a lesser extent in candesartan-treated hyperoxaluric rats. These results show for the first time that oxalate can activate the renal renin-angiotensin system and that oxalate-induced upregulation of OPN is in part mediated via renal renin-angiotensin system.

The effect of osteopontin on the adhesion of calcium oxalate crystals to Madin-Darby canine kidney cells

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.

Superoxide from NADPH oxidase as second messenger for the expression of osteopontin and monocyte chemoattractant protein-1 in renal epithelial cells exposed to calcium oxalate crystals.

To test the hypothesis that exposure of a renal epithelial cell line, NRK52E, to calcium oxalate monohydrate crystals (COM) would up‐regulate NADPH oxidase subunit p47phox, enhance superoxide production and increase monocyte chemoattractant protein‐1 (MCP‐1) and osteopontin mRNA levels.

Interaction between Osteopontin on Madin Darby Canine Kidney Cell Membrane and Calcium Oxalate Crystal

We recently reported that the addition of the protein osteopontin (OPN) resulted in an increase in the deposition of calcium oxalate (CaOx) crystals on the surface of Madin Darby canine kidney (MDCK) cells. To determine the degree to which this increased deposition is caused by OPN, we investigated the extent to which the CaOx crystal deposition produced by the expression of OPN at the cell surface was suppressed by 4 different methods prior to the determination of the level of CaOx crystal binding. MDCK cells (2 × 106 cells/well) were cultured to a confluent state, and the binding of OPN to the cellular surface was then inhibited by adding one of the following 4 substances: human OPN polyclonal antibody, thrombin, cyclic Arg-Gly-Asp (RGD) peptides and tunicamycin. The cells were cultured for 24 h. We then used a fluorescent antibody technique with an OPN polyclonal antibody to determined whether the expression of OPN at the cell surface was inhibited, and we measured the degree of CaOx crystal deposition using the isotope 45Ca. The degree of CaOx crystal deposition was inhibited by 80% or more in the antibody-treated group, by 50–80% in the thrombin-treated group, by 60–80% in the cyclic RGD-treated group, and by 50–60% in the tunicamycin-treated group. These results suggest that OPN in the extracellular matrix is the main cause of CaOx crystal deposition on the surface of MDCK cells.

The Role of Osteopontin on Calcium Oxalate Crystal Formation

OBJECTIVE We evaluated whether osteopontin (OPN) and other proteins with the RGD sequence as in OPN (RGD family proteins) that are present in renal tubular cells (fibronectin [FN], Tamm-Horsfall glycoprotein [THP], vitronectin [VN], and laminin [LN]) inhibit the aggregation and growth of calcium oxalate (CaOx) crystals by a novel seed crystal method using collagen granules (CG) with and without OPN adhered on the surface. We also evaluated the effect of solid phase OPN, FN and THP in which the relationship between their proteins and CaOx crystallization was reported. Moreover, the state and time-course changes in CaOx crystals adhered to CG were observed under scanning electron microscopy (SEM). METHODS The inhibitory activity (IA) on the aggregation and growth of CaOx crystals was measured in vitro by the conventional seed crystal method using isotopes. In this study, the following nine samples were used: OPN alone; FN alone; THP alone; VN alone; LN alone; CG alone; and CG with OPN, FN, or THP adhered on the surface (OPN/FN/THP-immobilized CG). In addition, the state and time-course changes in CaOx crystals adhered to CG were evaluated by SEM. RESULTS Using the conventional seed crystal method, the following values of IA were obtained: 91.7% (37.5 micro g/ml) for OPN, 5.0% (100 micro g/ml) for FN, 2.0% (100 micro g/ml) for THP, 3.0% (100 micro g/ml) for VN, and 1.0% (100 micro g/ml) for LN. However, the value of IA obtained by our seed crystal method using CG was 92.1% (180cm(2)/5ml PBS) when CG alone was used. Although the value of IA was decreased by 33.6% when OPN-immobilized CG was used, it did not significantly change when FN/THP-immobilized CG was used. When CG alone was used, the evaluation of CaOx crystallization by SEM demonstrated mild adherence and aggregation of CaOx crystal suspension (seed crystals) on the CG surface, although newly formed crystals only slightly adhered to the CG surface. When OPN-immobilized CG was used, marked adherence and aggregation of seed crystals were observed, in addition to the relatively increased adherence of newly formed crystals. When FN/THP-immobilized CG was used, newly formed crystals only slightly adhered to the CG surface, although the degree of seed crystal adherence and aggregation did not significantly change. CONCLUSIONS These findings suggest that the immobilization of OPN to the CG surface enhances the adherence and aggregation of seed crystals, as well as enhancing the adherence of newly formed crystals, resulting in decreased IA of CG (overall promotion of crystal deposition). Therefore, the results of this study clarified that OPN enhances the formation and aggregation of CaOx crystals in this experimental system.

Oxalate ions and calcium oxalate crystal‐induced up‐regulation of osteopontin and monocyte chemoattractant protein‐1 in renal fibroblasts

To examine the responses of renal fibroblasts to high oxalate (Ox) and calcium Ox (CaOx) crystals, as the latter are found in the renal interstitium of patients with primary or enteric hyperoxaluria, and in animals with experimental CaOx nephrolithiasis, and are associated with tubulointerstitial inflammation (TI). TI might begin with the production of chemoattractants by the renal epithelial cells exposed to high Ox and/or CaOx crystals; as Ox levels are also high in the renal interstitium and crystal deposition in nephrolithiasis might start in the interstitium, we hypothesized that renal fibroblasts might also be involved in the development of TI.

Prevalence of Urolithiasis in Kaizuka City, Japan—An Epidemiologic Study of Urinary Stones

Background: According to the results of nationwide surveys, the prevalence of urolithiasis has been steadily increasing in Japan. However, these surveys relied on hospital statistics, and there has been no survey of the prevalence of urolithiasis in Japan utilizing postal questionnaires. We surveyed the prevalence of urolithiasis among the inhabitants of Kaizuka City.

Clinical utility of the prostate cancer gene 3 (PCA3) urine assay in Japanese men undergoing prostate biopsy

It is known that a prostate cancer gene 3 (PCA3) urine assay is superior to serum PSA level or PSA‐related indices for predicting a positive biopsy result in European and US men. This is the first report on PCA3 in a large cohort of Japanese men. The diagnostic value of the PCA3 score in Japanese men was similar to those reported in European and US men. The study concludes that a combination of PSA density and PCA3 score may be useful for selecting patients who could avoid an unnecessary biopsy.

Analysis of bone mineral density in urolithiasis patients

Abstract  Background:  The association between hypercalciuria and bone mineral density (BMD) has been already recognized. The aim of the present study is to relate BMD to age and sex and to evaluate the calcium metabolism and hypercalciuria‐defined dietary or non‐dietary category in patients with urolithiasis.