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We have previously shown that correction of hypomagnesemia by magnesium (Mg) supplementation ameliorates chronic cyclosporine A (CsA) nephropathy via inhibiting gene expression of fibrogenic molecules. Experiments were conducted to further elucidate upstream mechanism of the beneficial effects upon CsA nephrotoxicity.

Background/Aim:Investigators have reported that the nonmuscle myosin heavy chain B (SMemb) expression is enhanced in various types of glomerular diseases which develop into nephrosclerosis. In renal transplantation, transplant glomerulitis is often recognized during acute rejection. Therefore, we hypothesized that SMemb plays important roles in acute kidney rejection. To evaluate the role of SMemb in the development of kidney rejection, we examined its expression in rat kidney transplantation models. Methods:We used Lewis rats as recipients and Wistar rats as donors. Group I: controls; group II: isograft model; group III: allograft model; group IV: as group III +10 mg/kg/day of ciclosporin A (CsA), and group V: as group III + CsA administration for 5 days postoperatively. Histopathological and SMemb immunohistochemical studies were completed. Results:Clear enhancement of SMemb expression was found on day 3 in group III. In groups I, II, IV, and V, SMemb was faintly expressed in the glomerular cells. However, after termination of CsA treatment, the SMemb expression increased. The expression of SMemb was higher in the allograft model than in either isograft or CsA-treated models. Conclusions:Immunohistological investigations show that the SMemb expression was significant from an early stage at which histopathological reactions were hardly identifiable. This, therefore, could be useful for an earlier diagnosis of acute rejection.

We recently identified a novel gene, termed klotho (kl) that is involved in the development of a syndrome in mice resembling human aging. A defect of the kl gene expression in mice leads to multiple disorders including arteriosclerosis, osteoporosis, ectopic calcification, and skin atrophy together with short life-span and infertility. Patients with chronic renal failure (CRF), develop multiple complications that are reminiscent of phenotypes observed in kl mutant mice. Furthermore, the kl gene is mainly expressed in kidney and brain. These evidences above suggest the possible involvement of Klotho function in the complications arising in CRF patients. To investigate the above possibility, we examined the kidneys of 10 clinically or histologically diagnosed CRF cases. The level of kl gene expression was measured by utilizing RNase protection assay. The expression of Klotho protein was assayed by utilizing Western blot analysis and by immunohistochemistry. The levels of kl mRNA expression were greatly reduced in all CRF kidneys. Moreover, the production of Klotho protein was also severely reduced in all CRF kidneys. These results suggest that the decrease in kl gene expression in CRF patients may underlie the deteriorating process of multiple complications in the CRF patients.

Abstract: Background. Growth factors such as insulin-like growth factor, epidermal growth factor, and hepatocyte growth factor have been reported to be effective in the treatment of acute renal failure, but their effectiveness in chronic renal failure has not been reported. Thymosin, a glycoprotein extract obtained from calf thymus, is also known to have growth factor-like activity. In this study, we investigated the protective effect of thymosin fraction 5 (TF5), isolated from thymosin, in mice and rats with experimentally induced uremia. Methods. Uremia was induced in mice by administering cis-Diammine-dichloroplatinum CDDP; 5 mg/kg, intraperitoneally once a week for 5 weeks. From the start of CDDP administration, mice in groups 1, 2 and 3, respectively, were given subcutaneous injections of saline or 1 mg/kg or 3 mg/kg of TF5 three times a week for 5 weeks. Uremia was induced in rats using the 5/6 renal ablation model. Rats in groups 1 and 2, respectively, were given subcutaneous injections of saline or 3 mg/kg of TF5 three times a week from the fifth to the twelfth week after removal of the right kidney, for a total of 8 weeks. Results. In uremic mice, 6 weeks after the start of CDDP administration, serum urea nitrogen (SUN) levels were 40.1 ± 16.4, 20.6 ± 6.6, and 20.2 ± 5.3 mg/dl in groups 1, 2, and 3, respectively, and the level was significantly lower in groups 2 and 3 than in group 1 (P < 0.05). In the uremic rats, 13 weeks after the removal of the right kidney, SUN levels were 105.2 ± 20.1 and 82.9 ± 11.6 mg/dl in groups 1 and 2, respectively, and serum creatinine levels were 2.22 ± 0.89 and 1.56 ± 1.06 mg/dl in groups 1 and 2, respectively, with a significant difference in both SUN and serum creatinine between groups 1 and 2 (P < 0.05). Conclusion. These results suggest the possibility that TF5 has a protective effect against progression of uremia.

The gene of the human fatty acid ω-hydroxylase, CYP4A11, has been isolated from a human BAC library, and its complete genomic sequence has been determined. The CYP4A11 gene spanned 12,568 bp and contained 12 exons. The known PPAR recognition elements (PPRE), which were reported to be involved in the induction of CYP4A6 by clofibric acid, were not observed within the 5′-flanking region of the CYP4A11 gene. The recombinant CYP4A11 protein expressed in Escherichia coli using the pCWOri expression vector was purified to an almost electrophoretically homogeneous state with a specific content of 6.4 nmol of P450/mg of protein. This P450 exhibited ω-hydroxylation activity toward laurate, with a turnover number of 14.7 nmol/min/nmol of P450. The apparent Km and Vmax values were 56.7 μM and 15.2 nmol/min/nmol of P450, respectively. It also showed ω-hydroxylation activity toward palmitate, with a turnover number of 0.78 nmol/min/nmol of P450. Although several reports from other groups described that CYP4A11 preparations catalyzed ω-hydroxylation of arachidonic acid, our purified recombinant protein exhibited no activity toward arachidonic acid nor prostaglandin A1.

Chemopreventive effects of bovine lactoferrin (bLF), which is found at high concentrations in colostrum, on rat bladder carcinogenesis were investigated using a rat bladder medium‐term bioassay. In experiment 1, a total of 80 F344 male rats, 6 weeks old, were divided into 5 groups. Groups 1 and 2 were treated with 0.05%N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN) in the drinking water for 8 weeks and after a 1‐week interval, received dietary supplementation with 2% and 0.2% bLF, respectively. Group 3 received 0.05% BBN for 8 weeks and then no treatment. Group 4 was administered 2% bLF alone from week 9, without prior carcinogen exposure. Group 5 was maintained without any treatment throughout the experiment. All rats were killed at the end of week 36. Group 1 demonstrated a significantly decreased multiplicity of the bladder tumors (carcinomas and papillomas) as compared with group 3, Maximum cut surface areas of bladder tumors were also significantly decreased in groups 1 and 2 compared with group 3. No bladder tumors were observed in groups 4 or 5. In experiment 2, a total of 60 rats were divided into two groups (30 rats each); both were treated with 0.05% BBN for 4 weeks and after a 1‐week interval, one received 2% bLF (group 1) and the other, basal diet (group 2) for 4 weeks. Group 1 demonstrated a tendency for decrease of the 5‐bromo‐2′‐deoxyuridine (BrdU) labeling index. bLF was detected in the urine of rats fed bLF by ELISA as well as western blot analysis. The findings indicate that 2% bLF can inhibit BBN‐induced rat bladder carcinogenesis, and that this may be due to bLF in the urine.

Multifocal synchronous or metachronous tumor development is a common observation in human urothelial cancer cases. However, the underlying mechanism has remained obscure. We have employed a new tool to investigate the p53gene status, the yeast p53 functional assay, in combination with immunohistochemistry in a total of 50 tumor samples from 32 cases with urothelial cancers, including 8 with multiple synchronous tumor development and 2 demonstrating metachronous tumors. p53mutations were found in 13 cases (9 with missense mutations, 3 with deletion, 1 with splicing mutation) by the yeast p53 functional assay. p53 protein overexpression was seen in all 9 cases with missense mutations, but in only one of the 4 cases with nonsense mutations. Two tumors without p53mutation also showed positive p53 immunoreactivity. Overall, p53 abnormalities including mutations and/or protein overexpression were found in 15 (47%) cases. p53 abnormalities were significantly more frequent in non‐papillary and in high grade tumors. Loss of the wild type allele in addition to a p53mutation was suggested in 8 of the 15 (53%) cases. All 4 cases with mutations in multiple synchronous tumors had identical p53mutations in the separate urothelial cancers, strongly suggestive of monoclonality. The one case with multiple metachronous tumors, in contrast, was characterized by variation in the p53status, indicative of different clonal origins. In conclusion, combined assessment for p53status as used here (yeast p53 functional assay plus immunohistochemistry) may provide insights into the molecular mechanisms of urothelial carcinogenesis.

It is well known that cyclosporin, rapamycin and FK‐506 (tacrolimus) are metabolized by the liver microsomal cytochrome P450 enzyme system. Although there have been reports of interaction between these drugs and the renal P450 enzyme system, differences among these immunosuppressants has not been comprehensively demonstrated. We have studied the individual capacities of these immunosuppressants to induce renal microsomal P450 enzymes similar to CYP2B4 and CYP4A2 by examining renal function in treated rats, and have correlated the results by means of biochemical, immunological and immunohistochemical assays of renal P450 enzymes.

We have isolated a cDNA clone coding for CYP3A5from a human prostate cDNA library. The human prostate CYP3A5cDNA had a unique 5′-untranslated sequence, suggesting that the prostate specific regulation of CYP3A5is different from liver. Hybridization screening using a human genomic BAC library yielded four positive clones, two of which were shown to contain the unique 5′-untranslated sequence by Southern blot analysis. The CYP3A5recombinant protein expressed in Escherichia coliusing the pCWOri expression vector was purified to an almost electrophoretically homogeneous state with a specific content of 4.4 nmol of P450/mg of protein. This P450 exhibited 6β-hydroxylation activity toward both testosterone and progesterone. No polar metabolite of 5α-dihydrotestosterone (DHT) was detected. The apparent Kmvalues for testosterone and progesterone 6β-hydroxylation were 143 and 114 μM, respectively, with Vmaxvalues of 0.48 and 0.21 nmol/min/nmol of P450, respectively. This is the first report that a particular form of P450, CYP3A5,has been isolated from human prostate and that the purified recombinant protein of CYP3A5has been shown to be active in the metabolism of sex hormones.

5′-Deoxy-5-fluorouridine (5′-dFUrd) is a prodrug of 5-fluorouracil (5-FUra) activated by pyrimidine nucleoside phosphorylase (PyN Pase), mainly by uridine phosphorylase (Urd Pase) in rodents and by thymidine phosphorylase (TdR Pase) in humans, which is preferentially located in tumor tissues compared to normal tissues. It has been reported that PyN Pase is induced by cytokines such as tumor necrosis factor (TNF), interleukin-1α (IL-1α) and interferon (IFN). Thymosin is a glycoprotein extract obtained from the calf thymus and is a potent immunopotentiating preparation. In this study, the antiproliferative activity of 5′-dFUrd used in combination with thymosin fraction 5 (TF5) was investigated in mouse bladder cancer cell line MBT-2 in vitro and in vivo. In vitro TF5 enhanced the activity of 5′-dFUrd by up to 4.11-fold, whereas the activity of other cytostatics such as 5-FUra, mitomycin C, adriamycin, cis-platinum, etoposide, vinblastine and methrotrexate was not changed. In vivo when the effects of combination therapy with 5′-dFUrd and TF5 in C3H/HeN mice implanted with MBT2 were studied, tumor growth was not suppressed by TF5 alone while tumor growth was suppressed to some degree by 5′-dFUrd alone. However, tumor growth suppression was enhanced when 5′-dFUrd was used in combination with TF5. In order to investigate this mechanism, Urd Pase in MB2 was measured, and it was found that TF5 increased enzyme activity by up to 1.8-fold in MBT2. This increased susceptibility might be a result of the induction of Urd Pase, which is the essential enzyme for the conversion of 5′-dFUrd to 5-FUra. These results suggested that the therapeutic benefit of 5′-dFUrd would be improved by its use in combination with TF5 and the modulation of converting enzymes for antitumor prodrugs could be a novel therapeutic strategy for treating human cancers.

Background:

Summary The authors examined interleukin-2 (IL-2) production and interferon ? (IFN?) production of peripheral blood mononuclear cells in 28 patients with renal cell carcinoma and 17 control subjects. The peripheral blood was obtained prior to the initiation of therapeutic procedures. The patients were divided into two groups according to tumor size, ?5 cm and >5 cm. The production of IL-2 and IFN? was measured by immunoradiometric assay. As a result, in the patients with tumors >5 cm, IL-2 and IFN? production was impaired. However, in the patients with tumors ?5 cm, IFN? production was enhanced, though IL-2 production was not significantly different from that of the control subjects. There was no significant correlation between IL-2 production and IFN? production.

The platelet adhesion on adhesive protein-coated surfaces was significantly reduced by the addition of the synthetic tetrapeptide, RGDS(Arg-Gly-Asp-Ser), which was identified as the common amino acid sequence of adhesive site of adhesive proteins. The inhibitory effect was also observed for leukocyte(WBC) in complement-inactivated serum. No significant effect was observed for WBC in complement-activated serum. These indicate that the RGDS ligand-receptor mechanism operates on adhesive protein-adsorbed surfaces for both cellular systems and that activated complement factor(C3b)-membrane receptor(CR3) interaction operates for WBC as the complement is activated.

A case featuring a well differentiated adenocarcinoma mixed with a transitional cell carcinoma (TCC) arising in the renal pelvis of a 63-year-old woman is presented. Daughter tumors, located in the ureter and the uretero-vesical junction, were entirely TCC in character. Immunohistochemical assessment of cell cycle-related proteins revealed overexpression of cyclin D1 but reduced p21WAF1/Cip1or PCNA expression in the adenocarcinomatous regions. Conversely, expression of p21WAF1/Cip1and PCNA was high in the TCC components. Immunohistochemical staining for p53 was negative and PCR—SSCP analyses confirmed the absence of any mutation. Therefore, assessments on the altered expression of cell cycle-related elements may contribute to our understanding of tumor biology in adenocarcinomas and TCCs of the renal pelvis and to identifying the similarities and differences between the two different cell types.

Hepatocyte growth factor (HGF) is a mesenchymal-derived growth factor which predominantly targets various epithelial and endothelial cells. We here investigated the biological function of HGF in rat dorsolateral prostatic tissue. HGF stimulated proliferation and DNA synthesis of prostatic epithelial cells in primary culture. c-Met/HGF receptor mRNA was specifically expressed in isolated epithelial cells. An immunohistochemical study revealed that HGF is located in prostatic stromal cells. When stromal cells were isolated and cultured, they produced significant amounts of HGF, which was further stimulated by prostaglandin E2, 12-O-tetradecanoylphorbol-13-acetate, and dibutyryl cyclic AMP, but not by testosterone. These results indicate that HGF is a stromal-derived potent mitogen for dorsolateral prostatic epithelial cells which may function as a paracrine regulator for growth of the prostatic glands.

Environmental contamination by endocrine-disrupting chemicals (EDC) has been a major focus of recent research and policy discussions. EDC-suspected man-made chemicals used as raw materials or plasticizers have been shown to elute from plastic products. To examine whether the dialysate for continuous ambulatory peritoneal dialysis (CAPD) is contaminated with EDC, we determined bisphenol A (BPA), nonylphenol (NP), di-(2-ethylhexyl)phthalate (DEHP) and di-n-butyl phthalate (DBP) in the pre-used dialysate and in the peritoneal effluent from renal failure patients by gas chromatography/mass spectrometry. Concentrations of BPA, NP, DEHP and DBP were 0.02–0.23 ppb (μg/l), 0.09–0.22, 1.1–3.7, and <0.1–2.1 ppb, respectively, in the pre-used dialysate, and <0.01–0.07, <0.1–0.45, 0.35–1.23, and 0.42– 1.76 ppb, respectively, in the effluent, from which the maximal daily contamination of BPA and NP by CAPD was estimated at the microgram level and that of phthalate esters at the 10-μg level. These concentrations are far less than the toxic dosage reported so far, so that CAPD is unlikely to contaminate patients seriously.