Ryuzo Tsugawa

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.

Renal Cell Carcinoma of the Native Kidney after Renal Transplantation

We report a patient with small renal cell carcinoma of the native kidney long after renal transplantation, which was considered to have developed before transplantation, and review the characteristics

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.

Hypertension Due to Coexisting Pheochromocytoma and Aldosterone-Producing Adrenal Cortical Adenoma

A 49-year-old male was diagnosed as having primary aldosteronism at age 39, and he was treated with antihypertensive drugs. In 1995, a computed tomogram revealed a mass in the right adrenal gland. Radiological examinations and endocrinological data revealed the presence of a pheochromocytoma in the right and an adrenocortical tumor in the left adrenal gland. Right adrenalectomy and left partial adrenalectomy were performed. Histologically, the right adrenal mass was compatible with pheochromocytoma, and the left adrenal mass was an adrenocortical adenoma. Endocrinological data as well as blood pressure returned to normal after operation.

A Simple Method for Determining the Metastable Limit of Calcium Oxalate

The purpose of this paper is to evaluate the simple microplate method to determine the metastable limit of calcium oxalate in urine reported by Ryall et al. (1), to determine its limits under various conditions, and to determine the effect of various inhibitors on the whole urine system.

Inhibitory Potency of Crystal Matrix Protein on the Crystallization of Calcium Oxalate

The presence of macromolecular substances is among the multiple factors that may influence the complex process of urinary stone formation. The aim of this study was to evaluate the inhibitory potency of crystal matrix protein (CMP). Purification of CMP consisted of calcium oxalate crystal formation, dissolution of crystals, electrodialysis and high–performance liquid chromatography (HPLC). The inhibitory potency of crystal aggregation was examined by the seed crystal method, the undiluted urine method, and the use of scanning electron microscopy (SEM). CMP showed the protein band of 31 kDa in SDS–PAGE. Anti–CMP polyclonal antibody and antihuman prothrombin antibody cross–reacted well with human prothrombin and CMP in Western blotting. CMP and human prothrombin had high inhibitory potency by the seed crystal method and undiluted urine method. Using SEM, we were able to observe the high inhibitory potency of human prothrombin and undiluted CMP on the aggregation of calcium oxalate crystals.

Excretion of Hippuran into Acquired Renal Cysts in Chronic Hemodialysis Patient

Isao Ishikawa, MD, Division of Nephrology, Department of Internal Medicine, Kanazawa Medical University, Uchinada, Kahoku, Ishikawa 920-02 (Japan) Dear Sir, Acquired renal cystic disease (ARCD) is becoming important because of its complications such as bleeding and renal cell carcinoma [1–3]. These cysts are considered to originate mainly from proximal tubular epithelium, based on analysis of the chemical composition of cyst fluid [4,5] and the immuno histochemical examination [6,7]. We examined the excretion of Hippuran, a substance handled by glomerular filtration and tubular secretion, into the acquired cysts 20 and 100 min after intravenous injection in 2 patients. The 1st patient, a 64-year-old man on hemodialysis fo 13 years, underwent right-side nephrectomy because o ARCD complicated with renal cell carcinoma on June ∏ 1985. Twenty minutes before nephrectomy (renal arter clamping) 70 μCi of 131I Hippuran were administers intravenously, and the blood was drawn at the tim of artery clamping. Serum isotopic counts wer 4,730 cpm/ml and the counts in four cysts were 270, 332 420 and 427 cpm/ml in 1.3,1.7,1.0 and 1.5 ml of cyst Ωuic respectively (table 1). Therefore, the cyst fluid/serur ratio of Hippuran was 0.06–0.09 20 min after the adminis tration.

Asymmetric hypertrophy of renal graft after transplantation.

Isao Ishikawa, MD, Division of Nephrology, Department of Internal Medicine, Kanazawa Medical University, Uchinada, Kahoku, Ishikawa 920-02 (Japan) Since the cortical thickness of the graft is thinner at contact with the psoas muscle [1], it is suggested that the local mechanical effect of the recipient affects the graft hypertrophy, i.e. the upper part of the graft facing the upper abdomen hypertrophies more than the lower part facing the minor pelvis where there is no space to move. In order to prove this hypothesis, renal volume was measured using CT scan in its upper, hilar and lower parts. Six recipients (25.3 ± 7.9 years of age, mean ± SD) received grafts from their parents (53.2 ± 5.6 years of age). CT scans were performed from the upper pole to lower pole of the kidney sequentially with a constant slice width (8 mm), 77 ± 43 days before transplantation and 75 ± 28 days after transplantation for grafts and 42 ± 36 days after transplantation for donor’s remaining kidneys. The donor’s left kidney was transplanted in the right iliac fossa of the recipient inside out. The serum creatinine levels in 6 recipients were 1.0–1.5 mg/dl at the time of CT examination after transplantation. The upper or lower parts of the kidney were defined as kidney part above the uppermost scan level or below the lowest scan level where the hilus is included. The hilar part was defined as the renal part with scan slices including the renal hilus. The volumes of the upper, hilar and lower parts were measured in 35 recipients who received the donor’s left kidney in their right iliac fossa besides 6 donor and recipient pairs. The angle of the axis of the graft to the median line in 6 recipients (1.2 ± 5.7°) is significantly more upright than the axis of the left kidney in the donor (-12.4 ± 7.6°) (p < 0.05). Therefore, the volume of the upper part of the graft is underestimated and the volume of the lower part is overestimated because the axis of the graft is more upright.

[A study of the risk factors in calcium oxalate stone formation--simple method for measuring metastable limits by the microplate method].

The stone forming urinary environment may be conducive to spontaneous nucleation of calcium oxalate, since it is generally characterized by a reduced metastable limit. This study indicates that the microplate method offers simple and reliable measures for estimating calcium oxalates propensity for spontaneous nucleation. In measuring the metastable limit by the microplate method, 200 microliters aliquots of each urine sample were treated with sodium oxalate to give final concentrations of 0-1.5 mmol. The urine samples were then incubated at 37 degrees C for 20 min and the minimum amount of oxalate necessary to induce nucleation detectable by inverted microscopy was taken to be the measured metastable limit of each urine. The metastable limit by microplate method positively was correlated (p less than 0.001) with the metastable limit found by the Coulter counter method and inversely correlated (p less than 0.001) with the concentration product ratio. The metastable limits were significantly lower (p less than 0.001) in recurrent stone formers than in the control subjects. The metastable limit rose significantly (p less than 0.01) during treatment with thiazides. Moreover, the metastable limit was inversely correlated (p less than 0.01) with the stone episode rate. It is concluded that the metastable limits by the microplate method are useful in stone forming potential identification in urine as well as in the assessment of the response to the therapy.

The Inhibitory Effect of Sodium Pentosan Polysulfate on Calcium-Oxalate Crystal Formation in vitro and in vivo

Calcium-oxalate (CaOx) stone disease is the most-common human urinarystone disease. Spontaneous CaOx urolithiasis is very rare in rats. However, because the rat is one of the most-easily-available and commonly-used laboratory animals, a number of experimental models have been developed for the study of CaOx urolithiasis.