Kiyoshi Tamaki

Differential Inhibition of Smad6 and Smad7 on Bone Morphogenetic Protein- and Activin-mediated Growth Arrest and Apoptosis in B Cells

Smad6 and Smad7 prevent ligand-induced activation of signal-transducing Smad proteins in the transforming growth factor-β family. Here we demonstrate that both Smad6 and Smad7 are human bone morphogenetic protein-2 (hBMP-2)-inducible antagonists of hBMP-2-induced growth arrest and apoptosis in mouse B cell hybridoma HS-72 cells. Moreover, we confirmed that the ectopic expressions of Smad6 and Smad7 inhibited the hBMP-2-induced Smad1/Smad5 phosphorylation. We previously reported that Smad7 is an activin A-inducible antagonist of activin A-induced growth arrest and apoptosis in HS-72 cells. Interestingly, although mRNA expression of Smad6 was induced by activin A in HS-72 cells, Smad6 showed no antagonistic effect on activin A-induced growth arrest and apoptosis. Moreover, we found that the ectopic expression of Smad7, but not Smad6, inhibited the activin A-induced Smad2 phosphorylation in HS-72 cells. Thus, Smad6 and Smad7 exhibit differential inhibitory effects in bone morphogenetic protein-2- and activin A-mediated signaling in B lineage cells.

Early Induction of Transforming Growth Factor-β via Angiotensin II Type 1 Receptors Contributes to Cardiac Fibrosis Induced by Long-term Blockade of Nitric Oxide Synthesis in Rats

We previously reported that the chronic inhibition of nitric oxide (NO) synthesis increases cardiac tissue angiotensin-converting enzyme expression and causes cardiac fibrosis in rats. However, the mechanisms are not known. Transforming growth factor-beta (TGF-beta) is a key molecule that is responsible for tissue fibrosis. The present study investigated the role of TGF-beta in the pathogenesis of cardiac fibrosis. The development of cardiac fibrosis by oral administration of the NO synthesis inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME) to normal rats was preceded by increases in mRNA levels of cardiac TGF-beta1 and extracellular matrix (ECM) proteins. TGF-beta immunoreactivity was increased in the areas of fibrosis. Treatment with a specific angiotensin II type 1 receptor antagonist, but not with hydralazine, completely prevented the L-NAME-induced increases in the gene expression of TGF-beta1 and ECM proteins and also prevented cardiac fibrosis. Intraperitoneal injection of neutralizing antibody against TGF-beta did not affect the L-NAME-induced increase in TGF-beta1 mRNA levels but prevented an increase in the mRNA levels of ECM protein. These results suggest that the early induction of TGF-beta1 via the angiotensin II type 1 receptor plays a major role in the development of cardiac fibrosis in this model.

Carbonic-adsorbent AST-120 reduces overload of indoxyl sulfate and the plasma level of TGF-β1 in patients with chronic renal failure

BackgroundWe previously reported a significant increase in plasma TGF-β1 in patients with chronic renal failure (CRF). Progression of CRF may be caused by persistent renal production of TGF-β1. In CRF rat models, an oral carbonic absorbent (AST-120) reduces the expression of the TGF-β1 gene in the kidney, and delays the progression of CRF, in part by alleviating the overload of indoxyl sulfate. The aim of this study was to evaluate the effect of AST-120 on plasma levels of indoxyl sulfate and TGF-β1 in CRF patients.MethodsTen CRF patients (aged 59.3 ± 9.5 years, 5 men, serum creatinine 4.37 ± 1.72 mg/dl) were enrolled in this study. All patients maintained a regular dietary therapy and the same medication throughout the study. AST-120 was added at a dose of 6 g/day. Parameters including the slope of the reciprocal of the serum creatinine – time plot, plasma indoxyl sulfate level, and plasma and urinary levels of TGF-β1 were compared before and after the treatment with AST-120. The mean observation periods before and after the treatment were 9.7 ± 2.8 and 6.5 ± 2.9 months, respectively.ResultsAdministration of AST-120 significantly reduced the plasma levels of indoxyl sulfate (1.42 ± 1.50 vs. 1.26 ± 1.40 mg/dl, P < 0.05) and TGF-β1 (17.9 ± 7.2 vs. 10.6 ± 4.7 ng/ml, P < 0.05) and improved the slope of the reciprocal of serum creatinine (−0.061 ± 0.041 vs. −0.032 ± 0.055 dl/mg/year, P < 0.05).ConclusionsThese results support the notion that indoxyl sulfate and TGF-β1 may be involved in the progression of CRF, and that the oral adsorbent AST-120 may suppress the progression, at least in part, by reducing overproduction of TGF-β1.

Short- or long-term effects of a low-protein diet on fibronectin and transforming growth factor-β synthesis in adriamycin-induced nephropathy

Increased synthesis and gene expression of fibronectin or transforming growth factor-beta (TGF-beta) have been reported to be involved in the progressive process of doxorubicin hydrochloride (Adriamycin)-induced nephropathy. In the present study, the effects of dietary protein restriction on the synthesis and gene expression of fibronectin or TGF-beta were investigated by immunoprecipitation, Northern blotting, and TGF-beta bioassay in this model after subjects were given either short- or long-term low-protein diets. In the long-term diet experiment, either a normal protein diet (NPD, 20%) or low-protein diet (LPD, 5%) was fed to the Adriamycin rats for 8 weeks after the injection of Adriamycin. An 8-week LPD significantly ameliorated kidney destruction and remarkably reduced the fibronectin synthesis. Furthermore, the significant decreases of the latent TGF-beta secretion and the expression of TGF-beta 1 mRNA were observed in the Adriamycin rats fed an 8-week LPD. In the short-term diet experiment, an NPD or LPD was fed to the Adriamycin rats for 2 weeks at weeks 4, 8, or 16 after the injection of Adriamycin. A 2-week LPD did not ameliorate kidney damage. Although fibronectin synthesis by the renal cortex in the Adriamycin rats was remarkably reduced by a 2-week LPD, there was no significant decrease in the latent TGF-beta secretion in the Adriamycin rats. The mRNA expressions of fibronectin or TGF-beta 1 were not affected by a 2-week LPD in the Adriamycin rats at any stage. In conclusion, decreased fibronectin and TGF-beta synthesis may be one of the mechanisms by which the long-term dietary protein restriction ameliorates kidney damage. On the other hand, a 2-week LPD affected the only fibronectin synthesis, which thus suggested that an LPD might exert a quicker influence on the protein synthesis of fibronectin than on the transcriptional events of fibronectin.

Adult-onset acute tubulointerstitial nephritis and uveitis with Fanconi syndrome. Case report and review of the literature.

We report a case of tubulointerstitial nephritis and uveitis (TINU syndrome) with full type Fanconi syndrome. A 32-year-old woman presented with fatigue, anorexia and weight loss. Laboratory findings showed anemia, polyclonal hypergammaglobulinemia and moderate renal dysfunction. Tubular function abnormalities were normoglycemic glucosuria, panaminoaciduria, phosphaturia and kaliuresis leading to hypokalemia. Renal tubular acidosis and hypouricemia were also evident. Serum antistreptolysin O titer was high. Ocular symptoms (bilateral anterior uveitis) emerged soon after admission. Renal biopsy showed diffuse tubulointerstitial infiltration by lymphocytes and plasma cells without granuloma. Treatment with systemic steroids was given and renal function, and ocular symptom returned to normal with 3 months. Although tubular abnormalities involving TINU syndrome has already been reported, the disease associated with full type Fanconi syndrome has rarely been seen, and systemic steroid may be beneficial in reducing the development of tubulointerstitial injury.

Crescentic glomerulonephritis due to rifampin treatment in a patient with pulmonary atypical mycobacteriosis.

A 64-year-old male was treated continuously with rifampin, isoniazid and streptomycin for pulmonary atypical mycobacteriosis, Mycobacterium kansasii. Five weeks after beginning the treatment, the patient suddenly developed acute renal failure. A renal biopsy showed crescentic lesions characteristic of rapidly progressive glomerulonephritis with moderate interstitial changes. Serum antirifampin antibody was detected, and the cessation of rifampin treatment was followed by a rapid spontaneous recovery of the patient’s renal function. This is, to our knowledge, the first case of rapidly progressive crescentic glomerulonephritis associated with rifampin treatment where circulating antirifampin antibody is demonstrated and the renal function spontaneously improved after discontinuing rifampin treatment.

Ectopic expression of Smad7 inhibits transforming growth factor-β responses in vascular smooth muscle cells

Vascular injury stimulates the cytokine-growth factor network in the vascular wall, including transforming growth factor-beta (TGF-beta). Reportedly, the intracellular signaling of TGF-beta is mediated by Smad proteins. We tested the effects of the ectopic expression of inhibitory Smads in cultured rat smooth muscle cells (SMC) to identify the role of TGF-beta/Smad signaling on the phenotypic modulation of SMC. The cells exposed to human recombinant TGF-beta1 (10 ng/ml) were stimulated Smad2 phosphorylation. Infection with the replication-deficient adenovirus vector expressing Smad7, but not bacterial beta-galactosidase or Smad6, was found to inhibit TGF-beta-induced Smad2 phosphorylation in a dose-dependent manner. TGF-beta suppressed the serum-induced proliferation of SMC from 36.3% to 51.0% (p<0.01), as measured by hand-counting, and this inhibition was attenuated by the ectopic expression of Smad7 (from 30.7% to 74.8% of the reduction of TGF-beta-response, p<0.05), but not Smad6. A BrdU incorporation assay also showed that TGF-beta-mediated growth inhibition was attenuated by exogenous Smad7 and that this inhibition can be reversed by an additional expression of exogenous Smad2. TGF-beta increased the expression of alpha-smooth muscle actin and myosin heavy chain by 1.3-fold and 1.6-fold in comparison to the control, respectively, and these increases were attenuated by exogenous Smad7, but not Smad6. Our data indicate that Smads mediate TGF-beta responses on SMC phenotypes. Smad7, but not Smad6, may specifically act as an inhibitor of TGF-beta responses.

Effect of Endothelin 1 on Fibrinolysis and Plasminogen Activator Inhibitor 1 Synthesis in Rat Mesangial Cells

Endothelins (ETs) have been known to have a variety of biological functions such as mitogenic stimulation, natriuresis and the stimulation of the proteolytic activity in addition to vasoconstrictive action, which may participate in the process of glomerular diseases pathophysiologically. In this study, the effects of ET-1 on fibrinolysis, plasminogen activator inhibitor 1 (PAI-1) synthesis and PAI-1 mRNA expression were examined in cultured rat mesangial cells (MCs). The addition of ET-1 (10(-11) to 10(-7) M) into MC cultures reduced fibrinolytic activities assayed by fibrin autography in a dose-dependent manner. For the assay of PAI-1 synthesis, MC culture media metabolically labeled with 35S-methionine were analyzed by SDS-PAGE with fluorography and immuno-precipitation using rabbit antirat PAI-1 antibody. Exposure to ET-1 for 24 h produced a clear dose-dependent effect on the PAI-1 release from the MCs. PAI-1 mRNA expression was also enhanced in parallel with the concentration of ET-1 in the conditioned media. These findings indicate that ET-1 participates in fibrinolysis and the PAI-1 synthesis by MCs, which may thus regulate the degradation of the extracellular matrix in the glomerular microenvironment.

Influence of a High Salt Diet on Glomerular Injury and the Preventive Effects of Amiloride in Adriamycin Nephropathy

The effects of a high salt diet on glomerular hypertrophy and sclerosis were examined in a focal glomerular sclerosis rat model. Sprague-Dawley rats were injected twice with Adriamycin (ADR, 2.5 mg/kg body weight) and then divided into 3 groups: (1) ADR rats fed a 1% sodium chloride (NaCI) diet (control ADR rats); (2) ADR rats fed an 8% NaC1 diet (ADR-NaC1 group), and (3) ADR rats fed a 10% sodium bicarbonate (NaHCO3) diet (the ADR-NaHCO3 group), and were then observed for 8 weeks. There were no differences in the blood pressure levels between the control ADR and ADR-NaC1 groups. The urinary protein excretion was significantly less in the ADR-NaC1 and ADR-NaHCO3 groups than in the control ADR group. However, a progressive increase in the blood urea nitrogen and serum creatinine associated with extensive glomerular sclerosis and hypertrophy was only observed in the ADR-NaC1 group. An increase in the glomerular diameter preceded the development of glomerulosclerosis in this group. Furthermore, a daily administration of amiloride, a Na+/H+ exchanger inhibitor, to the ADR-NaC1 rats prevented the development of glomerular hypertrophy and sclerosis. These results therefore suggest that the aggravated effect of a high salt diet on glomerular sclerosis may be related to glomerular hypertrophy which is associated with the stimulation of the Na+/H+ exchanger.

Roles of TGF-β and Latent TGF-β-Binding Protein in Glomerulosclerosis Induced by Two Consecutive Injections of Monoclonal Antibody 1-22-3 in Rats

The present study demonstrated the elevated synthesis and gene expressions of transforming growth factor β (TGF-β) or latent TGF-β binding protein (LTBP) in an irreversible glomerulosclerosis rat mode