Takahiro Yasui

Transcriptional regulation of osteopontin gene in vivo by PEBP2αA/CBFA1 and ETS1 in the skeletal tissues

Osteopontin (Opn) and polyoma enhancer-binding protein (PEBP) 2αA/core binding factor (CBFA) 1 have been suggested to play important roles in ossification. The overlapping localization of opn and PEBP2αA/CBFA1 mRNA, and the marked decrease of opn mRNA expression in PEBP2αA knockout mice, indicated that the transcription of opn gene was controlled by PEBP2αA. In the present study, we determined the direct regulation of PEBP2αA on the opn promoter activity. Opn promoter activity was markedly enhanced by PEBP2αA and ETS1 in a synergistic manner. The synergistic effect was diminished when either the PEBP2αA or ETS1 binding site was mutated, or the spatial arrangement of these sites was mutated by a 4-nt insertion. The distance between these sites was important for transactivation but not protein-DNA binding. The direct interaction between PEBP2αA and ETS1 was depended on protein-DNA binding. These results suggested that the specific spatial arrangement of both sites and direct interaction between PEBP2αA and ETS1, were essential for promoter function. Furthermore, endogenous opn mRNA was decreased with the introduction of dominant negative PEBP2αA to MC3T3/E1 cells expressing endogenous PEBP2αA, ETS1 and opn. These findings suggest that PEBP2αA and ETS1 cooperate in vivo to regulate expression of the opn gene in the skeletal tissue. Cell type-specific regulation of Opn gene expression will also be discussed.

Renal macrophage migration and crystal phagocytosis via inflammatory-related gene expression during kidney stone formation and elimination in mice: Detection by association analysis of stone-related gene expression and microstructural observation†

Mice have a strong ability to eliminate renal calcium oxalate crystals, and our previous examination indicated a susceptibility in which monocyte‐macrophage interaction could participate in the phenomenon. To clarify the macrophage‐related factors playing roles in the prevention of crystal formation in mouse kidneys, morphologic and expression studies based on microarray pathway analysis were performed. Eight‐week‐old male C57BL/6N mice were administered 80 mg/kg of glyoxylate by daily intraabdominal injection for 15 days, and the kidneys were extracted every 3 days for DNA microarray analysis. Based on the raw data of microarray analysis, pathway analyses of inflammatory response demonstrated macrophage activation through the increased expression of chemokine (C‐X‐C) ligand 1, fibronectin 1, and major histocompatability (MHC) class II. Association analysis of related gene expression values by quantitative reverse transcription polymerase chain reaction (RT‐PCR) indicated the high association of chemokine (C‐C) ligand 2, CD44, colony‐stimulating factor 1, fibronectin 1, matrix gla protein, secreted phosphoprotein 1, and transforming growth factor β1 (TGF‐β1) with the amount of both renal crystals and F4/80, a macrophage marker. Immunohistochemically, interstitial macrophages increased during the experimental course, and CD44 and MHC class II were upregulated around crystal‐formation sites. Ultrastructural observation of renal macrophages by transmission electron microscopy indicated interstitial macrophage migration with the phagocytosis of crystals. In conclusion, increased expression of inflammation‐related genes of renal tubular cells induced by crystal formation and deposition could induce monocyte‐macrophage migration and phagocytosis via the interaction of CD44 with osteopontin and fibronectin. Such crystal‐removing ability of macrophages through phagocytosis and digestion might become a new target for the prevention of stone formation.

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.

Efficacy of selective α1A adrenoceptor antagonist silodosin in the medical expulsive therapy for ureteral stones

Recently, we reported that α1A adrenoceptor (AR) is the main participant in phenylephrine‐induced human ureteral contraction. We therefore decided to carry out a prospective randomized study to evaluate the effects of silodosin, a selective α1A AR antagonist, as a medical expulsive therapy for ureteral stones. A total of 187 male patients, who were referred to our department for the management of symptomatic unilateral ureteral calculi of less than 10 mm, were randomly divided into two groups: group A (92 patients), who were instructed to drink 2 L of water daily, and group B (95 patients), who received the same instruction and were also given silodosin (8 mg/daily) for a maximum of 8 weeks. Expulsion rate, mean expulsion time and need for analgesics were examined. Overall, the mean expulsion time was 15.19 ± 7.14 days for group A and 10.27 ± 8.35 days for group B (P = 0.0058). In cases involving distal ureteral stones, the mean expulsion time was 13.40 ± 5.90 and 9.29 ± 5.91 days, respectively (P = 0.012). For stones of 1–5 mm in diameter, the mean expulsion time was 14.28 ± 6.35 and 9.56 ± 8.45 days, respectively (P = 0.017). For stones of 6–9 mm in diameter, the stone expulsion rate was 30.4% and 52.2% (P = 0.036), and the mean expulsion time was 21.00 ± 9.9 and 11.33 ± 8.31 days, respectively (P = 0.038). Herein, we report the first on silodosin in the management of ureteral lithiasis. Our findings suggest that silodosin might have potential as a medical expulsive therapy for ureteral 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.

Biomolecular mechanism of urinary stone formation involving osteopontin

Urinary stones consist of two phases—an inorganic (mineral) phase and an organic (matrix) phase. Studies on the organic components of kidney stones have been undertaken later than those on the inorganic components. After osteopontin was identified as one of the matrix components, the biomolecular mechanism of urinary stone formation became clearer. It also triggered the development of new preventive treatments. Osteopontin expression is sporadically observed in normal distal tubular cells and is markedly increased in stone-forming kidneys. Calcium oxalate crystals adhering to renal tubular cells are incorporated into cells by the involvement of osteopontin. Stimulation of crystal–cell adhesion impairs the opening of mitochondrial permeability transition pores (mPTP) in tubular cells and produces oxidative stress, apoptosis, and osteopontin expression. Macrophages phagocytose and digest a small amount of crystals, but many crystals aggregate into a mass containing osteopontin and epithelial cell debris and are excreted into the renal tubular lumen, becoming nuclei of urinary stones. This biomolecular mechanism is similar to atherosclerotic calcification. Based on these findings, new preventive treatments have been developed. Dietary control such as low-cholesterol intake and the ingestion of antioxidative foods and vegetables have successfully reduced the 5-year recurrence rate. Osteopontin antibodies and cyclosporine A, which blocks the opening of mPTP, have markedly inhibited the expression of osteopontin and urinary stone formation in animal models.

Renal tubular epithelial cell injury and oxidative stress induce calcium oxalate crystal formation in mouse kidney.

Objectives:  To clarify the role of renal tubular cell (RTC) injury and oxidative stress in the early stage of renal calcium oxalate crystal formation in a mouse model.

Efficacy of Endoscopic Combined Intrarenal Surgery in the Prone Split-Leg Position for Staghorn Calculi

UNLABELLED Abstract Purpose: To evaluate the efficacy of endoscopic combined intrarenal surgery (ECIRS) using retrograde flexible ureteroscopy and miniature percutaneous nephrolithotomy (PNL) for the treatment of patients with staghorn calculi in the prone split-leg position. PATIENTS AND METHODS We retrospectively reviewed the records of 42 patients with staghorn calculi (45.8±3.2 mm) who underwent ECIRS using retrograde flexible ureteroscopy and miniature PNL in the prone split-leg position for the treatment of staghorn calculi in our center between December 2010 and August 2013. A flexible ureteroscope with a laser fiber was inserted through a ureteral access sheath, and lithoclast lithotripsy was performed through a mini-percutaneous tract. Both procedures were performed simultaneously by two urologists. Surgical parameters, including surgical time, stone-free (SF) rates, modified Clavien complication grades, and risk factors for residual stones, were analyzed. RESULTS Fifteen patients (35.7%) had complete staghorn calculi. Among the 42 staghorn calculi treated, 23 had 0 to 5 stone branches, 14 had 6 to 10 stone branches, and 5 had ≥11 stone branches. All procedures were performed successfully using a single lithotripsy tract with the patient in the prone split-leg position. The mean surgical time was 143.2±9.2 minutes. The initial SF rate was 71.4%, and the final SF rate was 83.3% after further treatment. One patient required a blood transfusion (2.4%), but no patient experienced a ≥3 Clavien grade complication. Risk factors for residual stones were stone size, stone surface area, complete staghorn calculi, and the number of stone branches. CONCLUSIONS ECIRS for staghorn calculi in the prone split-leg position is a safe, efficient, and versatile method for the effective management of staghorn calculi without the creation of multiple percutaneous tracts.