LIM-domain protein AJUBA suppresses malignant mesothelioma cell proliferation via Hippo signaling cascade.

Malignant mesothelioma (MM) is one of the most aggressive neoplasms usually associated with asbestos exposure and is highly refractory to current therapeutic modalities. MMs show frequent activation of a transcriptional coactivator Yes-associated protein (YAP), which is attributed to the neurofibromatosis type 2 (NF2)-Hippo pathway dysfunction, leading to deregulated cell proliferation and acquisition of a malignant phenotype. However, the whole mechanism of disordered YAP activation in MMs has not yet been well clarified. In the present study, we investigated various components of the NF2-Hippo pathway, and eventually found that MM cells frequently showed downregulation of LIM-domain protein AJUBA, a binding partner of large tumor suppressor type 2 (LATS2), which is one of the last-step kinases of the NF2-Hippo pathway. Although loss of AJUBA expression was independent of the alteration status of other Hippo pathway components, MM cell lines with AJUBA inactivation showed a more dephosphorylated (activated) level of YAP. Immunohistochemical analysis showed frequent downregulation of AJUBA in primary MMs, which was associated with YAP constitutive activation. We found that AJUBA transduction into MM cells significantly suppressed promoter activities of YAP-target genes, and the suppression of YAP activity by AJUBA was remarkably canceled by knockdown of LATS2. In connection with these results, transduction of AJUBA-expressing lentivirus significantly inhibited the proliferation and anchorage-independent growth of the MM cells that harbored ordinary LATS family expression. Taken together, our findings indicate that AJUBA negatively regulates YAP activity through the LATS family, and inactivation of AJUBA is a novel key mechanism in MM cell proliferation.

Induction of continuous ambulatory peritoneal dialysis after retroperitoneoscopic nephrectomy.

Induction of continuous ambulatory peritoneal dialysis (CAPD) as treatment of end-stage renal disease is difficult for patients requiring nephrectomy with traditional surgery, and usually hemodialysis is selected for these patients. In a 61-year-old woman with end-stage renal failure a left renal tumor was diagnosed by abdominal ultrasonography, enhanced computed tomography and magnetic resonance imaging. Following an urology consultation, we decided to perform left kidney nephrectomy. We estimated that she had to undergo dialysis permanently after nephrectomy. She desired to be treated by CAPD, however, we decided after allowing for a postoperative period for complete healing of the peritoneum to avoid complications. This is why during the interim period between surgery and induction of CAPD she underwent hemodialysis (HD) in a local outpatient HD center and in our hospital. We selected a retroperitoneoscopic approach for nephrectomy. Pathology evaluation revealed a renal cell carcinoma. 4 months after nephrectomy, CAPD catheter implantation was performed by using laparoscopy and CAPD was started. At the present time, the patient is doing well on CAPD. To our knowledge, there are no clear indications regarding initiation of peritoneal dialysis after nephrectomy. The retroperitoneoscopic approach for nephrectomy allows for initiation of peritoneal dialysis after nephrectomy within a relative short postoperative period.

Expression of BMP-7 and USAG-1 (a BMP antagonist) in kidney development and injury.

Once developed, end-stage renal disease cannot be reversed by any current therapy. Bone morphogenetic protein-7 (BMP-7), however, is a possible treatment for reversing end-stage renal disease. Previously, we showed that the BMP antagonist uterine sensitization-associated gene-1 (USAG-1, also known as ectodin and sclerostin domain-containing 1) negatively regulates the renoprotective action of BMP-7. Here, we show that the ratio between USAG-1 and BMP-7 expression increased dramatically in the later stage of kidney development, with USAG-1 expression overlapping BMP-7 only in differentiated distal tubules. Examination of USAG-1 expression in developing kidney indicated that a mosaic of proximal and distal tubule marker-positive cells reside side by side in the immature nephron. This suggests that each cell controls its own fate for becoming a proximal or distal tubule cell. In kidney injury models, the ratio of USAG-1 to BMP-7 expression decreased with kidney damage but increased after subsequent kidney regeneration. Our study suggests that USAG-1 expression in a kidney biopsy could be useful in predicting outcome.

A comparative study of the diagnostic accuracy of ELISA systems for the detection of anti-neutrophil cytoplasm antibodies available in Japan and Europe.

OBJECTIVES: Primary systemic vasculitis associated with anti-neutrophil cytoplasm antibodies (ANCA) differs in its frequency and clinical expression between Japan and Europe. We sought to ascertain whether such differences arise from the performance of enzyme-linked immunosorbent assays (ELISAs) for ANCA. METHODS: Plasma samples from 64 consecutive Japanese patients with a clinical and histological diagnosis of primary systemic vasculitis including microscopic polyangiitis (MPA; n=52), Churg-Strauss syndrome (CSS; n=1), and Wegener's granulomatosis (WG; n=11), or those from disease controls with non-vasculitic glomerulonephritis (n=54) and healthy controls (n=55) were tested for the presence of myeloperoxidase (MPO) by ELISAs available in Japan (Nipro and MBL) and compared with those in Europe (Wieslab). The sensitivity and specificity were calculated for each ELISA, and its diagnostic performance was assessed by receiver operating characteristic curve analysis. RESULTS: The sensitivity and specificity of either MPO-ANCA assays for a diagnosis of MPA were 90.4% and 98.2% (Nipro), 88.2% and 96.3% (MBL), and 86.5% and 99.1% (Wieslab). The overall diagnostic performance, assessed as the area under curve of the MPO-ANCA ELISAs for MPA were 0.946+/-0.022 (Nipro), 0.970+/-0.017 (MBL), and 0.971+/-0.017 (Wieslab), while that of PR3-ANCA ELISAs for WG were 0.986+/-0.025 (Nipro), 0.993+/-0.017 (MBL), and 0.916+/-0.059 (Wieslab). CONCLUSIONS: The MPO-ANCA ELISAs commercially available in Japan exhibited high sensitivity and specificity for the diagnosis of ANCA-associated vasculitides and provided similar diagnostic value to those in Europe. These results facilitate further international comparison of ANCA-associated vasculitides between Japanese and European populations.

NADPH oxidase inhibitor, apocynin, restores the impaired endothelial-dependent and -independent responses and scavenges superoxide anion in rats with type 2 diabetes complicated by NO dysfunction.

OBJECTIVE: We investigated the effect of apocynin, an NADPH oxidase inhibitor, in the impairment of vascular responses in Otsuka Long-Evans Tokushima Fatty (OLETF) rats (type 2 diabetic rat model) with or without (w/wo) N-nitro-l-arginine methyl ester treatment. METHODS: Male OLETF and littermate Long-Evans Tokushima Otsuka (LETO) (28 weeks old) rats were separated as follows: LETO w/wo apocynin (Gp C, Gp C-apo), OLETF w/wo apocynin (Gp DM, Gp DM-apo) and OLETF plus l-nitro arginine acetate ester w/wo apocynin (Gp DMLN, Gp DMLN-apo). Five days after, peritoneal macrophages were stimulated with thioglycolate. Two days after, they were evaluated. RESULTS: Plasma glucose and lipid levels remained unchanged. Acetylcholine-induced nitric oxide-dependent (NO-dependent) relaxation and nitroglycerin-induced NO-independent relaxation were improved in the Gp DMLN-apo, compared with that in Gp DMLN. Tone-related basal NO release and plasma NO(2) (-) and NO(3) (-) tended to be lower in Gp DM and Gp DMLN groups. The increased amount of superoxide anion released from macrophages in Gp DM and Gp DMLN was restored by apocynin. Intimal thickening was observed in aortae of Gp DM and Gp DMLN animals; however, there was little in aortae of Gp DM-apo and Gp DMLN(-) apo rats. Increased tumour necrosis factor-alpha (TNF-alpha) in the Gp DM and Gp DMLN was also restored by apocynin treatment. CONCLUSION: Apocynin restores the impairment of endothelial and non-endothelial function in diabetic angiopathy in OLETF without changing plasma glucose and lipid levels. NO and O(2) (-) may play a role in this process by decreasing TNF-alpha levels.

[Laparoscopic extraperitoneal bladder diverticulectomy: an initial case report].

We report a case with a large bladder diverticulum caused by the bladder outlet obstruction, which was successfully treated with laparoscopic extraperitoneal diverticulectomy. The patient was a 71-year-old man, who had had dysuria, micturition pain and cloudy urine. We found the patient to have benign prostatic hyperplasia and a large diverticulum of the bladder. We removed the diverticulum by laparoscopic procedure and then the prostate by transurethral resection on September 24th, 1998. Before the diverticulectomy, we placed a ureteral stent in the left ureter and 8Fr Foley catheters in the diverticulum and bladder. A working space was made by dilation using a balloon dilator. After removing the diverticulum, we made 5 stitches to close the bladder wall. After the surgery, a 22Fr Foley catheter was placed and removed on the 9th postoperative day. Patient had a satisfactory micturition and was discharged on the postoperative 15th day. Laparoscopic diverticulectomy might be a modality for symptomatic bladder diverticulum, because it is minimally invasive and can completely remove bladder diverticulum.

Gas6 induces mesangial cell proliferation via latent transcription factor STAT3.

Mesangial cell proliferation is essential for the pathogenesis and progression of glomerular disease. Previously, we showed that Gas6 plays a pivotal role in mesangial cell proliferation in vitro and in vivo. In the present study, we identified downstream targets of Gas6 signaling to examine the role in mesangial cell proliferation in vitro and in vivo. We found that Gas6 tyrosine phosphorylates STAT3 (signal transducers and activators of transcription) with concomitant translocation to the nucleus and induces STAT3-dependent transcriptional activation in cultured mesangial cells. Expressing dominant negative STAT3 inhibited Gas6-mediated transcriptional activation of STAT3 and abolished Gas6-induced mesangial cell proliferation. In a model of mesangial proliferative glomerulonephritis, STAT3 is phosphorylated in mesangial cells, and its phosphorylation peaks at day 8 after the injection of anti-Thy1.1 antibody. Inhibition of Gas6 by warfarin and the extracellular domain of its receptor, Axl, abolished phosphorylation of STAT3 in vivo. Thus, our in vitro and in vivo findings indicate that autocrine growth factor Gas6 induces mesangial cell proliferation via latent transcription factor STAT3. Therefore, STAT3 might be a new therapeutic target for kidney disease induced by mesangial proliferation.

A vitamin D analog ameliorates glomerular injury on rat glomerulonephritis.

OCT (22-oxa-calcitriol), a vitamin D analog, has been reported to show strong inhibitory effects on mesangial cell proliferation in vitro. In the present study, we report a study of the effect of OCT on anti-thy-1 glomerulonephritis. Both OCT and 1,25(OH)(2)D(3) significantly inhibited mesangial cell proliferation, the degree of glomerulosclerosis, and albuminuria at day 8 compared to the disease control group. The OCT-treated group showed normal calcium levels but the 1,25(OH)(2)D(3)-treated group showed higher levels. The disease control group showed a marked increase of type I and type IV collagens, and alpha-smooth muscle actin (alpha-SMA) compared to the normal group. The treatment of OCT or 1,25(OH)(2)D(3) significantly reduced the expression of these proteins. The mRNA of the glomeruli of anti-thy-1 model expressed significantly higher levels of type I and type IV collagens, and alpha-SMA at day 8 compared to normal rats. Treatment with OCT or 1,25(OH)(2)D(3) inhibited the mRNA expressions of type I and type IV collagens, as well as that of alpha-SMA. These data demonstrate that OCT inhibits mesangial cell proliferation and extracellular matrix expansion with a low calcemic activity. Disease control rats showed significantly increased levels of transforming growth factor-beta1 protein in the glomeruli, but treatment with OCT or 1,25(OH)(2)D(3) markedly reduced this expression. The levels of mRNA in glomeruli were also consistent with these protein levels. Therefore, the suppressive effect of OCT may be mediated by inhibition of transforming growth factor-beta1. The present results suggest that OCT has potential for use in therapeutic strategy for the treatment of glomerulonephritis without inducing hypercalcemia.

Gas6 regulates mesangial cell proliferation through Axl in experimental glomerulonephritis.

Proliferation of mesangial cells is a hallmark of glomerular disease, and understanding its regulatory mechanism is clinically important. Previously, we demonstrated that the product of growth arrest-specific gene 6 (Gas6) stimulates mesangial cell proliferation through binding to its cell-surface receptor Axl in vitro. We also showed that warfarin and the extracellular domain of Axl conjugated with Fc portion of human IgG1 (Axl-Fc) inhibit mesangial cell proliferation by interfering the Gas6/Axl pathway in vitro. In the present study, therefore, we examined in vivo roles of Gas6 and Axl in an experimental model of mesangial proliferative glomerulonephritis induced by the injection of anti-Thy1.1 antibody (Thy1 GN). In Thy1 GN, expression of Gas6 and Axl was markedly increased in glomeruli, and paralleled the progression of mesangial cell proliferation. Administration of warfarin or daily injection of Axl-Fc inhibited mesangial cell proliferation, and abolished the induction of platelet-derived growth factor-B mRNA and protein in Thy1 GN. Moreover, the anti-proliferative effect of warfarin was achieved at lower concentrations than those in routine clinical use. These findings indicate that the Gas6/Axl pathway plays a key role in mesangial cell proliferation in vivo, and could be a potentially important therapeutic target for the treatment of renal disease.

Expression of basic helix-loop-helix proteins in the glomeruli.

BACKGROUND: Basic helix loop helix (bHLH) proteins play a critical role in the differentiation of not only striated muscle cells but also adipocytes, neuron cells and smooth muscle cells. Previous studies have established in vitro mouse mesangial cells (MCs) to maintain the differentiated smooth muscle phenotype. MATERIALS AND METHODS: The purpose of the present study was to clone bHLH proteins from these MCs using the primers designed from a homologous sequence specific to bHLH, and to analyze the presence of bHLH proteins in normal kidney in vivo. From the cloning of MCs in vitro, we identified myf5 and herculin mRNA but not myoD. The expression of bHLH proteins in vivo was examined by immunohistochemistry with each specific antibody. RESULTS: The MCs in newborn mice possessed Id but did not express either protein herculin or myoD. On the other hand, mature MCs expressed both myf5 and herculin. The Id protein disappeared in mature glomeruli. CONCLUSION: These results suggest that bHLH proteins are an important factor for mature MCs in vivo.

Clinical benefits and tolerability of increased fill volumes in Japanese peritoneal dialysis patients.

BACKGROUND: Increasing fill volume is an effective means of improving clearances in patients on peritoneal dialysis (PD). Since Japanese PD patients are physically smaller than their Western counterparts, there is some concern that PD patients in Japan may be unable to tolerate larger fill volumes. OBJECTIVE: To determine patient tolerance and changes in solute clearance and net ultrafiltration resulting from increased fill volumes in Japanese patients on PD. DESIGN: Prospective double-blind study, randomizing patients to three different fill volumes (2.5% dextrose solution: 1.5 L, 2.0 L, or 2.5 L) administered in random order on three different occasions separated by 1 week. RESULTS: Twenty-one patients with a mean age of 55.4 +/- 2.1 years and a mean body surface area of 1.66 +/- 0.03 m2 were studied. On a scale of 0 to 10, patients' mean discomfort scores were 2.14 +/- 0.59, 3.48 +/- 0.54, and 3.81 +/- 0.63 (p = 0.047) at the end of the 1.5-L, 2.0-L, and 2.5-L dwells, respectively. There were no reports of cramps or shortness of breath with any fill volume. Patients were able to correctly guess the actual fill volume for only 34 of the 63 total exchanges (54.0%). Increasing fill volume resulted in an incremental improvement in peritoneal creatinine clearance, from 3.74 +/- 0.16 to 4.49 +/- 0.21 (p < 0.001, 2.0 L vs 1.5 L) to 5.12 +/- 0.20 mL/minute (p< 0.001, 2.5 L vs 2.0 L) for 1.5-L, 2.0-L, and 2.5-L dwells, respectively. Peritoneal urea clearance also increased significantly, from 5.65 +/- 0.13 to 7.04 +/- 0.17 (p < 0.001, 2.0 L vs 1.5 L) and 8.16 +/- 0.29 mL/minute (p < 0.001, 2.5 L vs 2.0 L), with incremental increases in fill volume. Similarly, net ultrafiltration in a 4-hour dwell increased significantly with fill volume, from 255.24 +/- 24 mL with 1.5 L, to 356 +/- 24 (p < 0.004, 2.0 L vs 1.5 L) and 392 +/- 29 mL (p < 0.086, 2.5 L vs 2.0 L) in patients receiving 2.0 L and 2.5 L, respectively. CONCLUSION: Increasing the fill volume results in improvement in solute clearance and net ultrafiltration in Japanese PD patients, with minimal increase in patient discomfort. A large percentage of patients were unable to identify the actual fill volume.

Effect of the lipoprotein lipase activator NO-1886 on adriamycin-induced nephrotic syndrome in rats.

Hyperlipidemia associated with nephrotic syndrome may play a role in the deterioration of renal function. Tsutsumi et al have previously reported that the novel compound NO-1886 increases lipoprotein lipase (LPL) activity, resulting in a reduction of plasma triglycerides and an elevation of high-density lipoprotein (HDL) cholesterol in normal rats. The aim of this study was to ascertain whether NO-1886 suppresses the renal injury by treatment of the hyperlipidemia in an Adriamycin (Kyowa Hakko Kogyo, Tokyo, Japan) induced nephrosis rat model fed a high-protein diet that induced renal dysfunction and tubulointerstitial injury. Administration of Adriamycin caused hyperlipidemia, proteinuria, and edema with ascites in rats in 4 weeks. Furthermore, a combination of Adriamycin and a high-protein diet increased plasma creatinine and blood urea nitrogen (BUN) and decreased plasma albumin. Histologically, in Adriamycin-treated rats, marked interstitial cellular infiltration, tubular lumen dilation, and tubular cast formation in the kidney were observed. NO-1886 decreased plasma triglyceride and increased HDL cholesterol in Adriamycin-induced nephrotic rats. NO-1886 treatment reduced plasma creatinine and BUN levels and increased plasma albumin in Adriamycin-treated rats; it also ameliorated the ascites and proteinuria. Histologically, NO-1886-treated rats showed a quantitatively significant preservation of tubulointerstitial lesions. These data suggest that NO-1886 may have a protective effect against Adriamycin-induced nephrosis with tubulointerstitial nephritis in rats by a modification of the plasma lipid disorder.

Protective role of extracellular superoxide dismutase in hemodialysis patients.

BACKGROUND: The superoxide anion and other oxygen radicals have been implicated in the progression of chronic renal failure, and are removed by extracellular superoxide dismutase (EC-SOD) in the extracellular space on the surface of the endothelium. A single-base substitution of the EC-SOD gene which reduces the binding capability to endothelial cells resulting in an increased serum concentration, has been identified in healthy persons and hemodialysis patients. RESULTS: The proportion of patients with this mutation among hemodialysis patients in each 20 months' duration after the initiation of hemodialysis was retrospectively studied. The percentage of substitution-positive patients declined 80 months after the start of hemodialysis in non-DM patients. In contrast, in DM patients, the rapid decrease was obvious as early as 40 months after the initiation of hemodialysis. By prospective study for 5 years, there were significant differences in the survival rate between patients with and without R213G in DM, but not in non-DM patients. Among those who died, the incidence of ischemic heart disease and cerebrovascular disease in cases with R213G was significantly higher than in cases without R213G. CONCLUSION: These results suggest that the presence of a substitution in the EC-SOD gene at the heparin-binding domain could be a prognostic marker of dialysis patients.

A lipoprotein lipase activator, NO-1886, improves endothelium-dependent relaxation of rat aorta associated with aging.

Endothelial function is closely related to development of atherosclerosis and is impaired with aging. The novel compound NO-1886, 4-diethoxyphosphorylmethyl-N-(4-bromo-2-cyanophenyl)benzamid e, is a lipoprotein lipase activator and its long term administration protects against the development of experimental atherosclerosis in animals. The aim of this study was to ascertain whether NO-1886 ameliorates the impaired endothelium-dependent relaxation of rat aorta associated with aging. NO-1886 (50 mg/kg p.o.) was administered to 7-month old rats for 3 months. Plasma lipid, glucose and insulin levels in old control rats (10 months of age) were significantly higher than those of young rats (2 months of age). NO- 1886 decreased plasma triglyceride levels (old rats, 233+/-10 mg/dl; old rats + NO-1886, 172+/-16 mg/dl, P < 0.01) and increased plasma high density lipoprotein (HDL) cholesterol level (old rats, 72+/-6 mg/dl; old rats + NO-1886, 142+/-6 mg/dl, P < 0.001) in old rats, but had no effects on plasma glucose or insulin. The endothelium-dependent relaxation of the thoracic aorta caused by histamine was significantly impaired in old rats (% relaxation at 10(-5.5) M histamine: young rats 25.4+/-3.1%; old rats 14.1+/-1.9%, P < 0.01), an effect completely prevented by NO-1886 (old rats + NO-1886; 22.8+/-2.8%, P < 0.05 vs. old rats). In contrast, NO-1886 showed no effect on the endothelium-independent relaxation by sodium nitroprusside. These results indicate that NO-1886 improves impaired endothelium-dependent relaxation of rat aorta associated with aging, possibly by correcting lipid metabolism.

Polymorphism of extracellular superoxide dismutase (EC-SOD) gene: relation to the mutation responsible for high EC-SOD level in serum.

Extracellular superoxide dismutase (EC-SOD) with amino acid substitution R213G generated by the nucleotide substitution 760C-->G in the heparin binding domain is responsible for the high EC-SOD level in serum. We identified the two DNA polymorphic sites in the coding region of EC-SOD gene related to the 760C-->G and determined the allele frequencies. The polymorphism were A and G at nucleotide position (nt.) 241 and C and T at nt. 280 near the N-terminal. The haplotype frequencies in Japanese were 241A280C: 0.45, 241G280T: 0.37, and 241G280C: 0.18. The haplotype of 241A280T did not exist. The mutation 760C-->G must occur on the allele having the haplotype of 241G280T.