Katsuhito Miyazawa

Isolation and partial characterization of crystal matrix protein as a potent inhibitor of calcium oxalate crystal aggregation : Evidence of activation peptide of human prothrombin

In order to clarify the characteristics of crystal matrix protein (CMP), which exhibits a remarkable affinity for calcium oxalate crystals and may be important in stone pathogenesis, we have isolated CMP from macromolecular matrix substances of newly-formed calcium oxalate crystals. Purification of CMP consisted of calcium oxalate crystal formation, dissolution of crystals, electrodialysis, anion exchange chromatography and high-performance liquid chromatography. CMP showed the protein band of 31 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of CMP was identical to that of human prothrombin. Both anti-CMP polyclonal antibody and antihuman prothrombin antibody cross-reacted well with human prothrombin and CMP in Western blotting. Its amino acid composition and its molecular weight of 31 kDa strongly suggest that CMP is the activation peptide of human prothrombin.

Inhibitions of urinary oxidative stress and renal calcium level by an extract of Quercus salicina Blume/Quercus stenophylla Makino in a rat calcium oxalate urolithiasis model.

Objectives:  To clarify the mechanism of Urocalun, an extract of Quercus salicina Blume/Quercus stenophylla Makino (QS), in the treatment of urolithiasis.

Malignant epithelioid angiomyolipoma of the kidney in a patient with tuberous sclerosis: An autopsy case report with p53 gene mutation analysis

We report an autopsy case of malignant epithelioid angiomyolipoma in a 36-year-old male tuberous sclerosis patient. He had been diagnosed to have a bilateral renal tumor 20 years previously. The left kidney had been surgically resected at the age of 34, and the left renal tumor was pathologically diagnosed as classic angiomyolipoma and epithelioid angiomyolipoma. He suddenly died of cardiac arrest, and at autopsy the right kidney weighed 7120 g. The tumor presented with massive necrosis invading the inferior vena cava, but was not hemorrhagic. Microscopic examination revealed tumor cells varying in size with a predominantly solid proliferation pattern and marked atypical large cells with vesicular nuclei and abundant eosinophilic cytoplasm. Mitotic figures were often encountered, and atypical forms were also present. Metastatic lesions were identified in the right lung, liver, diaphragm, and mesentery. Immunohistochemical examination showed epithelioid angiomyolipoma cells that were focally reactive for HMB-45 and showed diffuse positive staining for Melan-A. No mutation was detected in the p53 gene by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis despite diffuse immunoreactivity for p53. This case was proven to be malignant because of the occurrence of distant metastases, and showed that p53 mutations are not always associated with malignant transformation in epithelioid angiomyolipoma.

Epidemiology of stone disease across the world

Nephrolithiasis is a highly prevalent disease worldwide with rates ranging from 7 to 13% in North America, 5–9% in Europe, and 1–5% in Asia. Due to high rates of new and recurrent stones, management of stones is expensive and the disease has a high level of acute and chronic morbidity. The goal of this study is to review the epidemiology of stone disease in order to improve patient care. A review of the literature was conducted through a search on Pubmed®, Medline®, and Google Scholar®. This review was presented and peer-reviewed at the 3rd International Consultation on Stone Disease during the 2014 Société Internationale d’Urologie Congress in Glasgow. It represents an update of the 2008 consensus document based on expert opinion of the most relevant studies. There has been a rising incidence in stone disease throughout the world with a narrowing of the gender gap. Increased stone prevalence has been attributed to population growth and increases in obesity and diabetes. General dietary recommendations of increased fluid, decreased salt, and moderate intake of protein have not changed. However, specific recommended values have either changed or are more frequently reported. Geography and environment influenced the likelihood of stone disease and more information is needed regarding stone disease in a large portion of the world including Asia and Africa. Randomized controlled studies are lacking but are necessary to improve recommendations regarding diet and fluid intake. Understanding the impact of associated conditions that are rapidly increasing will improve the prevention of stone disease.

Immunohistochemical expression of four different stem cell markers in prostate cancer: High expression of NANOG in conjunction with hypoxia‑inducible factor‑1α expression is involved in prostate epithelial malignancy

Cancer stem cells (CSCs) have been identified in a variety of cancer types, including prostate cancer. The aim of the present study was to evaluate the immunohistochemical expression of NANOG, octamer 4 (OCT4), cluster of differentiation 133 (CD133) and NESTIN, which are all CSC markers, and assess their function in prostate carcinogenesis. A total of 114 patients were referred to the Kanazawa Medical University Hospital (Uchinada, Japan) having presented with elevated serum prostate-specific antigen levels and/or abnormal digital rectal examinations, and underwent transrectal ultrasound sonography guided eight core biopsies. The prostate pathological specimens were re-evaluated for selection in this study. When specimens were diagnosed as prostate cancer, immunohistochemical analysis of the four different stem cell markers (NANOG, OCT4, CD133 and NESTIN) and hypoxia-inducible factor (HIF)-1α was performed. Prostate cancer was found in 38 cases (33.3%), while the other patients had benign prostate hyperplasia with prostatitis. All prostate cancers were histopathologically identified as adenocarcinomas of various grades, and cancer cells and intraepithelial neoplasia (high grade) were immunohistochemically shown to express NANOG and OCT4, but not CD133 and NESTIN. The intensity of NANOG expression was much greater than that of OCT4, and the positivity and intensity of the four stem cell markers, including NANOG, were elevated with high Gleason scores. A significant correlation was observed between the NANOG- and HIF-1α-positive regions. The CSC markers, in particular OCT4 and NANOG, were immunohistochemically expressed in prostate cancers. Furthermore, HIF-1α expression may affect NANOG and/or OCT4 expression. The findings of the current study suggested that NANOG expression may be a biomarker for the diagnosis of prostate cancer, and the coexpression of NANOG and HIF-1α may be involved in prostate carcinogenesis.

Pathobiology and Chemoprevention of Bladder Cancer

Our understanding of the pathogenesis of bladder cancer has improved considerably over the past decade. Translating these novel pathobiological discoveries into therapies, prevention, or strategies to manage patients who are suspected to have or who have been diagnosed with bladder cancer is the ultimate goal. In particular, the chemoprevention of bladder cancer development is important, since urothelial cancer frequently recurs, even if the primary cancer is completely removed. The numerous alterations of both oncogenes and tumor suppressor genes that have been implicated in bladder carcinogenesis represent novel targets for therapy and prevention. In addition, knowledge about these genetic alterations will help provide a better understanding of the biological significance of preneoplastic lesions of bladder cancer. Animal models for investigating bladder cancer development and prevention can also be developed based on these alterations. This paper summarizes the results of recent preclinical and clinical chemoprevention studies and discusses screening for bladder cancer.

Renal protective effects of erythropoietin on ischemic reperfusion injury.

While the problem of organ shortage has not yet been solved, the number of patients who need to be treated with dialysis due to end-stage renal disease (ESRD) is increasing each year. With the aim of eliminating dialytic therapy as much as possible, the opportunities for organ donation from expansive criteria donor (ECD) or marginal donors due to cardiac death have been increasing. With the purpose of extracting organs in a state in which the function is preserved as much as possible, we reexamined the conditions of tissue disorders resulting from temporary ischemia of the organs as well as changes in tissue function and the effects on the preservation of renal function over time by using rat models in order to clinically utilize erythropoietin, which has inhibitory effects on ischemia-reperfusion disorder, as has been conventionally reported. With 8- to 9-week-old Wister male rats, after the right kidney had been resected under general anesthesia, the left renal artery was clamped to inhibit the blood flow for 45 min. At 30 min before inhibiting the blood flow and after releasing the inhibited blood flow, 100 U/kg of recombinant human erythropoietin (rhEPO) was administered via the inferior vena cava and the abdominal cavity, and then the tissues and blood samples were extracted at 6 and 24 h after the release. The renal tissue specimens were evaluated using H&E staining and TUNEL staining in order to observe differences in the expression of apoptosis as well as the renal function and changes in the emergence of active oxygen were investigated by using samples that had been obtained from drawn blood. Moreover, we examined the degree of renal dysfunction by means of neutrophil gelatinase-associated lipocalin (NGAL) in the spot urine samples. The changes in renal function, which were observed according to the serum creatinine level, showed that the renal function was preserved with a significant difference in the rhEPO administration group. The liver deviation enzymes, which had also shown increases in the serum as well as the occurrence of renal dysfunction, showed clear decreases in the serum, even though changes with a significant difference were not observed in the rhEPO administration group. The active oxygen did not show changes before and after ischemia-reperfusion nor changes due to the rhEPO administration. When examining the status of apoptosis in the tissues, apoptosis was shown to be inhibited due to the rhEPO administration. It is believed that the main preservation effects of rhEPO are the elimination of cytopathy/cell death, as derived from the resulting ischemic condition that extends to the target organ before ischemia occurs. In this examination, no direct effects of rhEPO administration on the emergence of active oxygen were observed. It is therefore suggested that there is a possibility of preserving the renal function in marginal donors with a longer agonal stage by effectively using rhEPO.

Effects of oxalate exposure on Madin-Darby canine kidney cells in culture: renal prothrombin fragment-1 mRNA expression

It has been suggested that renal tubular cell damage induced by oxalic acid, one of the components of urinary calculi, may be involved in a variety of ways in the development of urolithiasis. During our study on a calculus related protein, renal prothrombin fragment-1 (RPTF-1), we noted that this is an inflammation related substance that mediates an acute inflammatory reaction, one of the original roles of prothrombin. RPTF-1 is a part of prothrombin that is a coagulation factor known to be expressed in the renal tubule. We examined whether oxalic acid may cause cytotoxic effects on tubular epithelial cells and whether such chemical stimulation may promote the translation of RPTF-1 mRNA into RPTF-1 proteins. We used Madin-Darby canine kidney (MDCK) cells derived from the distal tubule of a dog kidney. In this study, the effects of oxalic acid in culture solution at different concentrations on cytotoxicity were assessed using a MTT assay. The location of active oxygen species was identified using dichlorofluorescein diacetate. After the prothrombin sequence of RPTF-1 was confirmed in MDCK cells, RPTF-1 mRNA expression was determined by RT-PCR. The gene sequence of the same promoter area was ligated, and a luciferase sequence was inserted downstream of the vector. The target sequence was transfected into MDCK cells and the relation between oxalic acid and prothrombin promoter was examined. In addition, the variable expression of RPTF-1 mRNA was quantitatively compared depending on oxalic acid concentrations using real-time PCR. When cytotoxicity was investigated, cells were not damaged but, by contrast, were stimulated and activated under oxalic acid below a certain concentration. The relation between cytotoxicity on the cultured MDCK cell membrane and active oxygen species was confirmed. Luminescence in MDCK cells containing the luciferase gene was detected by the addition of oxalic acid, which activated the prothrombin promoter. A part of the prothrombin gene sequence in the MDCK cells was detected and an increase in the expression of RPTF-1 mRNA in MDCK cells by the addition of oxalic acid was confirmed using real-time PCR. Increased expression of prothrombin by adding oxalic acid has already been demonstrated in previous studies. In this study, however, RPTF-1 mRNA was promoted by oxalic acid and a direct association between oxalic acid and RPTF-1 is indicated. This finding shows that increased oxalic acid in urine induces the expression of RPTF-1 in tubular epithelial cells and thereby causes the generation of active oxygen species.

BACKSCATTERED ELECTRON IMAGING OF CRYSTAL MATRIX PROTEIN ON THE SURFACE OF CALCIUM OXALATE CRYSTALS USING COLLOIDAL GOLD

In order to clarify the presence and localization of crystal matrix protein (CMP) upon calcium oxalate crystals, scanning electron microscopy (SEM) and backscattered electron imaging (BEI) techniques were used. This protein exhibits a remarkable affinity with calcium oxalate crystals and may be important in stone pathogenesis. In this paper, rabbit anti‐human CMP polyclonal antibody was used as first antibody, and for the second antibody, goat anti‐rabbit IgG conjugated with 20 nm immunogold was used. Freshly prepared crystals from male urine were fixed in SEM fixative, then blocked and washed with phosphate‐buffered saline and bovine serum albumin (PBS/BSA). First and second antibodies were reacted in PBS/BSA. Crystals were then dehydrated and finally coated for SEM study. The SEM technique showed bipyramidal shaped dihydrate calcium oxalate crystals in every sample and even at high magnification, colloidal gold could barely be seen. BE 1 clearly demonstrated the presence and localization of the gold on the surface of the crystals as well as on the macromol‐ecules eluted from the crystals by dissolving them in ethylenediamminetetraacetic acid solution.

Factors influencing patient satisfaction with antimuscarinic treatment of overactive bladder syndrome: results of a real-life clinical study.

To investigate patient satisfaction with antimuscarinic treatment of overactive bladder syndrome, and to identify factors having a significant influence on satisfaction.