Biomedical subjects
Publications and source records attributed to Y Isaka.
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BACKGROUND: The evidence that transforming growth factor-beta (TGF-beta) is a key mediator in the pathogenesis of fibrotic diseases is now supported by several lines of investigation. This evidence provides a certain base for targeting TGF-beta as an antifibrotic agent. METHODS: We generated a chimeric cDNA, termed TGF beta RII/Fc, encoding an extracellular domain of the TGF-beta type II receptor fused to the IgG-Fc domain, and tested whether TGF beta RII/Fc could be a novel strategy for treating glomerular diseases. RESULTS: In cultured BNul-7 cells, recombinant TGF beta RII/Fc reversed the antiproliferative response induced by TGF-beta 1. In addition, TGF beta RII/Fc diminished the TGF-beta 1-induced production of EIIIA-positive fibronectin in cultured normal rat kidney cells. We then introduced the chimeric cDNA into the muscle of the nephritic rats by the hemagglutinating virus of Japan liposome-mediated gene transfer method in order to block the TGF-beta activity in nephritic glomeruli through systemic delivery of chimeric molecules. Treatment with TGF beta RII/Fc gene transfection could suppress the glomerular TGF-beta mRNA in nephritic rats with a comparable effect in the reduction of extracellular matrix accumulation. CONCLUSION: TGF beta RII/Fc successfully inhibited the action of TGF-beta in vitro and in vivo, and gene therapy by chimeric TGF beta RII/Fc might be feasible for the therapy of glomerulosclerosis.
BACKGROUND: With progressive renal disease, structural derangement increasingly encompasses the tubulointerstitial compartment. Tubulointerstitial injury is a critical determinant of renal functional reserve and prognosis in renal disease. Interstitial cells acquiring characteristic of myofibroblasts are an important contributor to interstitial fibrosis. Caldesmon, a calmodulin or actin binding protein, is a molecular marker of differentiation in smooth muscle cells and has recently been shown by us to be a good marker of mesangial cell activation in IgA nephropathy patients. METHODS. We studied whether the expression of caldesmon in interstitium of the kidney was enhanced in the process of glomerular disease and whether it would be a marker of interstitial activation in specific disease states. We performed immunohistochemical staining with anti-caldesmon antibodies in 38 biopsy specimens from IgA nephropathy patients and analysed them quantitatively with a computer-aided manipulator. Interstitial caldesmon expression were compared with histological changes and clinical parameters. RESULTS: Caldesmon expression was enhanced where interstitial cell infiltration and fibrosis were found. Immunoelectron microscopy revealed that caldesmon staining in the renal interstitium was cytoplasmic, and in the processes of myofibroblast-like cells. Caldesmon expression was more prominent in the intense CD68 infiltrated group than in the low positive cells infiltrated group. Patients showing high intensity of interstitial caldesmon expression had significantly higher urinary protein excretion than those showing low intensity of caldesmon expression. Next, 15 patients were treated with glucocorticoid and heparin for 4-8 weeks and re-biopsies were performed. Caldesmon expression was reduced in concomitant with decreased interstitial cell infiltration. Follow-up of these patients (average 24 months) revealed a significant suppression of urinary protein excretion and significant improvement of creatinine clearance. CONCLUSION: These results suggest that the interstitial caldesmon expression is associated with the progression of IgA nephropathy, and glucocorticoid--heparin therapy may reverse the phenotypic change of interstitial cells during the disease process of glomerulonephritis.
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Kidney targeted gene transfer has been a realistic goal for many researchers since 1991, but unfortunately, to date there is no reliable gene transfer technique for gene therapy of renal diseases. However, at the experimental level, several in vivo gene transfer methods have attempted to target certain renal structures, for example, the HVJ-liposome method and renal perfusion of adenovirus for glomerular cells, intravenous injection of oligonucleotides (ODNs) for proximal tubule, intra-arterial injection of adenovirus followed by cold incubation with a vasodilator for interstitial vasculature of the outer medulla, and adenoviral injection into the renal pelvis for the inner medullary collecting duct. As an ex vivo gene transfer method targeting the glomerulus, the transfusion of genetically-modified mesangial cells has been attempted. Implantation of genetically-modified tubular epithelial cells into the subcapsular region has been employed for ex vivo transfection to the interstitium. Gene therapy has focused particularly on the transplanted kidney, where an exogenous gene can transferred in advance. In the future, an inducible system and individual cell targeting strategy should be developed. The improvement of gene transfer techniques, especially vectors for delivering genes, is crucial. The potential application of gene transfer technologies is enormous while the therapeutic approaches have just begun to be explored. Therapeutic interventions of the process of progression of glomerulonephritis in the rat have been directed towards inhibiting the actions of growth factors. Obviously, molecular biological intervention is coming of age and there is a tremendous excitement over its potential. We believe that gene transfer techniques will become common tools for the dissection of molecular aspects of diseases and possibly for gene therapy in the field of nephrology.
BACKGROUND: Activation and consequent phenotypic modulation of mesangial cells is considered to play a crucial role in the process of glomerular disease progression. Caldesmon, a calmodulin and actin-binding protein, is a molecular marker of the phenotypic change in smooth-muscle cells. SUBJECTS AND METHODS: We studied whether the expression of caldesmon in mesangial cells was enhanced in the process of IgA nephropathy and whether it would be a marker of mesangial activation indicating prognostic significance in specific disease states. We performed immunohistochemical staining with anticaldesmon and alpha-smooth-muscle actin (alpha-SMA) antibodies in 32 biopsy specimens from IgA nephropathy patients and analysed them quantitatively with a computer-aided manipulator. RESULTS: The glomerular expression of caldesmon was enhanced in IgA nephropathy patients. We compared caldesmon expression with composite histological scores (cell score and matrix score), clinical parameters and expressions of alpha-SMA. There was a statistically significant correlation between the caldesmon score and the histological scores (cell score and matrix score, P<0.0001, P<0.01 respectively). Patients showing a high intensity of caldesmon expression (defined as caldesmon score > or = 35; H-group) had significantly higher urinary protein excretion than those showing a low intensity of caldesmon expression (defined as caldesmon score < 35; L-group) (1.2 +/- 1.2 g/24 h vs 0.41 +/- 0.53 g/24 h, P<0.05). Caldesmon and alpha-SMA expression had a statistically significant correlation (P<0.000). Next, 13 patients were treated with glucocorticoid-heparin for 4-8 weeks and re-biopsies were performed. After the therapy, the caldesmon and alpha-SMA scores were significantly lower than those before the therapy (P<0.01). DISCUSSION: These results suggest that the expression of caldesmon in glomeruli is associated with the progression of IgA nephropathy, and that glucocorticoid heparin therapy may reverse the phenotype of mesangial cells during the disease process of glomerulonephritis.
A number of structurally diverse compounds have been shown to be potent inhibitors of the DNA polymerase activity of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT). The compounds can be grouped into two broad classes; nucleoside analogs and nonnucleoside RT inhibitors. The nonnucleoside RT inhibitors are quite specific for HIV-1 RT but not human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus (SIV) RT. We have investigated the property of SIV/HIV-1 chimeric viruses in which portions of SIV(MAC) RT were exchanged with the corresponding domain of HIV-1 RT; amino acids 176-190, 176-383 and 176-495 of HIV-1 RT. The chimeric virus, which was substituted amino acids 176-190 of RT, had detectable RT activity, and this chimeric RT was sensitive to three nonnucleoside RT inhibitors [nevirapine, HEPT derivative (E-EBU-dM) and TIBO derivative (R82913)]. To further study this chimeric virus, we purified the chimeric RT enzyme expressed in Escherichia coli and determined its kinetic properties; the Km, and Vmax values, and the Ki value of HEPT derivative calculated for the DNA polymerase activity. This study reveals that amino acids 176-190 of SIV(MAC) RT were important for the enzymatic activity and the SIV/HIV-1 chimeric RT, which had amino acids 176-190 of HIV-1, was sensitive to the nonnucleoside RT inhibitor.
S-1153 is a new imidazole compound that inhibits human immunodeficiency virus (HIV) type 1 (HIV-1) replication by acting as a nonnucleoside reverse transcriptase inhibitor (NNRTI). This compound inhibits replication of HIV-1 strains that are resistant to nucleoside and nonnucleoside reverse transcriptase inhibitors. S-1153 has a 50% effective concentration in the range of 0.3 to 7 ng/ml for strains with single amino acid substitutions that cause NNRTI resistance, including the Y181C mutant, and also has potent activity against clinical isolates. The emergence of S-1153-resistant variants is slower than that for nevirapine, and S-1153-resistant variants contained at least two amino acid substitutions, including F227L or L234I. S-1153-resistant variants are still sensitive to the nucleoside reverse transcriptase inhibitors zidovudine (AZT) and lamivudine. In a mouse and MT-4 (human T-cell line) in vivo HIV replication model, S-1153 and AZT administered orally showed a marked synergy for the inhibition of HIV-1 replication. S-1153 shows a significant accumulation in lymph nodes, where most HIV-1 infection is thought to occur. S-1153 may be an appropriate candidate for two-to three-drug combination therapy for HIV infection.
Certain types of chemokine receptors have been identified as coreceptors for HIV-1 infection. The process of viral entry is initiated by the interaction between an envelope protein gp120 of HIV-1, CD4, and one of the relevant coreceptors. To understand the precise mechanism of the Env-mediated fusion and entry of HIV-1, we examined whether the V3 region of gp120 of T-cell line tropic (T-tropic) virus directly interacts with the coreceptor, CXCR-4, by using five synthetic V3 peptides: two cyclized V3 peptides (V3-BH10 and V3-ELI) which correspond to the V3 regions of the T-tropic HIV-1 IIIB and HIV-1 ELI strains, respectively, a linear V3 peptide (CTR36) corresponding to that of HIV-1 IIIB strain; and cyclized V3 peptides corresponding to that of the macrophage-tropic (M-tropic) HIV-1 ADA strain (V3-ADA) or the dualtropic HIV-1 89.6 strain (V3-89. 6). FACScan analysis with a CXCR-4(+) human B-cell line, JY, showed that V3-BH10, V3-ELI, and V3-89.6 but not CTR36 or V3-ADA blocked the binding of IVR7, an anti-CXCR-4 monoclonal antibody (MAb), to CXCR-4 with different magnitudes in a dose-dependent manner, while none of the V3 peptides influenced binding of an anti-CD19 MAb at all. Next, the effects of the V3 peptides on SDF-1beta-induced transient increases in intracellular Ca2+ were investigated. Three V3 peptides (V3-BH10, V3-ELI, and V3-89.6) prevented Ca2+ mobilization. Furthermore, the three peptides inhibited infection by T-tropic HIV-1 in a dose-dependent manner as revealed by an MTT assay and a reverse transcriptase assay, while the other peptides had no effects. These results present direct evidence that the V3 loop of gp120 of T-tropic HIV-1 can interact with its coreceptor CXCR-4 independently of the V1/V2 regions of gp120 or cellular CD4.
The uric acid concentration in blood has been widely accepted as a diagnostic indicator of hyperuricemia and gout, and its assay method is well established. In the present study, we developed a simple and rapid method for the determination of uric acid in hair, which can be obtained non-invasively. The concentration (nmol/mg hair) of uric acid extracted from 10-20 mg hair with 0.1 M potassium hydroxide was determined by an enzymatic method using uricase. The concentration of uric acid (nmol/mg hair, mean+/-S.D.: 0.49+/-0.157, n=16) in hair from hyperuricemic patients was significantly higher than that (0.26+/-0.107, n=8) in healthy volunteers (p<0.01). The within-run and between-day precision (CVs) of the assay was 9.6-10.3% (n=10 each) and 11.6-16.3% (n=7 each), respectively. The concentration (nmol/mg hair, y) of uric acid in hair correlated well with that in serum (mg/l, x): y=0.09x-0.12 (r=0.75, Syx=0.122, n=23). Changes in the concentration of uric acid in the hair of antihyperuricemic drug-treated patient paralleled that in serum, suggesting that the concentration of uric acid in hair is a reliable indicator of the metabolic control in hyperuricemia.
The rationale of the somatic gene therapy is the correction of diseases at the most fundamental level. Ideal gene therapy should be achieved by the replacement of the wrong gene sequence of genome with correct one. However, the gene technology to date is yet immature so as to correct the wrong gene sequence in vivo. Potentially, the present technology of gene transfer may provide: 1) correction of cellular dysfunction by expressing the deficient gene; 2) addition of new function for a cell by transferring an exogenous gene; 3) inhibition of unfavorable action of a cell by introducing a counteracting gene. In nephrology, the gene transfer targeted kidney has been challenged at the experimental level. HVJ-liposome method and recombinant adenovirus allow gene transfer to the particular cells in kidney in vivo. Ex vivo gene transfer using mesangial cells and macrophages are another option. Transplant kidney is also a good material for genetic engineering. The potential application of gene transfer is enormous while the therapeutic application have just begun to explored. We have been devoted to HVJ-liposome mediated gene transfer to the kidney and successfully demonstrated the suppression of the extracellular matrix accumulation of the glomeruli in the experimental glomerulonephritis through inhibition of the TGF-beta action by antisense oligonucleotides or soluble type receptor chimera for TGF-beta. We also applied this technology to the inhibition of interstitial fibrosis in unilateral ureter obstruction model. The new HVJ-liposome method improved in lipid composition allows gene transfer to tubulointerstitial fibroblast by retrograde approach from ureter. In consequence, introduced TGF-beta antisense suppressed the TGF-beta mRNA in concomitant with ameliorating interstitial fibrosis. We believe that the gene transfer technique will become common strategy to study the molecular aspect of the renal diseases and will be possibly applicable to molecular intervention in nephrology.
The aim of this study was to seek a promoter, transactivated selectively in renal cells in vivo by using transgenic (tg) mouse technology. We generated two kinds of tg mouse lines carrying a green fluorescence protein (GFP) cDNA driven either by cytomegalovirus enhancer and beta-actin/beta-globin promoter (CX-GFP) or by elongation factor 1alpha promoter (EF-GFP), and investigated the expression of GFP in the kidney. Microscopic examination of the renal tissues in CX-GFP-tg mice revealed that GFP was expressed only in glomeruli, mainly epithelial cells, but not in tubules, arteries and interstitium. Moreover, in situ hybridization demonstrated that GFP mRNA expression was localized in the glomerular cells. In contrast, GFP was not detectable in the kidney in any of the lines of EF-GFP-tg mouse. To exclude the possible involvement of the GFP cDNA as an enhancer, we constructed tg mice carrying the CX promoter driving a human CD4 cDNA. It was confirmed that the expression patterns of human CD4 in the kidney were quite similar to those of GFP in the kidney of CX-GFP-tg mice. These results strongly suggest that CX promoter could be transactivated in glomerular epithelial cells in vivo.
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BACKGROUND AND PURPOSE: The pathophysiological mechanisms that cause cerebral MR T2 high intensities in end-stage renal disease (ESRD) are unclear. We evaluated the incidence and the risk factors of T2-weighted MR brain high intensities in patients with ESRD. METHODS: We examined the degree of T2-weighted MR brain high intensities (high intensity score) and determined the variables that had an independent association with the occurrence of high intensities in 38 patients with ESRD before chronic dialysis treatment, 173 patients with essential hypertension, and 72 normotensive control subjects. RESULTS: The whole brain high intensity score was significantly higher in patients with ESRD than in the control subjects, but there was no significant difference in high intensity score between the ESRD and the hypertensive groups. Age, hypertension, and smoking were significant independent predictors of high intensities in a multiple logistic regression model. The distribution pattern of high intensities in ESRD patients was very similar to that obtained from hypertensive patients; the high intensity score was highest in the corona radiata and was lowest in the cerebellum. CONCLUSIONS: T2 high intensities on MR images of ESRD may reflect subcortical small-vessel alterations induced by hypertension.
BACKGROUND AND PURPOSE: The importance of MR imaging in carotid artery disease is unclear. We evaluated the sensitivity and specificity of the high signal intensity changes on MR images for diagnosis of hemodynamically compromised unilateral internal carotid artery disease. METHODS: We evaluated the association of high signal intensities on T2-weighted MR images with changes in cerebral perfusion reserve measured using 99mTc-hexamethylpropyleneamine oxime single-photon emission CT and acetazolamide in 23 patients. RESULTS: Eleven patients had a type I response (normal flow and normal perfusion reserve), 8 patients had a type II response (normal flow and decreased perfusion reserve), and 4 patients had a type III response (decreased flow and decreased perfusion reserve). High signal intensities in the centrum semiovale (11/12) and/or posterior periventricular white matter (6/12) were frequently seen in the hemodynamically compromised groups. Extensive high signal intensities were associated with severely impaired cerebral circulation. MR imaging had high sensitivity (0.92) and specificity (1.0) in predicting hemodynamically compromised patients when we used the presence of T2 high intensity in the centrum semiovale as a criterion. CONCLUSIONS: The centrum semiovale T2 hyperintensities lateralized to the side of carotid occlusion are specific and sensitive for the presence and severity of hemodynamic compromise from carotid occlusive disease.
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Uric acid in blood has been widely accepted as a reliable indicator of hyperuricemia and gout, and its assay method has been established. In the present study, we developed a simple and non-invasive rapid method for the determination of uric acid in hair. Concentration(nmol/mg hair) of uric acid extracted from 10-20 mg of hair(95% < extractability; 0.1N KOH, 37 degrees C, 2 hr) was determined by an enzymatic method using uricase. The concentration of uric acid(nmol/mg hair, mean +/- SD: 0.489 +/- 0.157, n = 16) in hair from hyperuricemic patients was significantly higher than that(0.258 +/- 0.107, n = 8) from healthy volunteers(p < 0.01). Within-run and between-day precisions(reproducibilities, CVs) for the assay were 9.6-10.3%(n = 10 each) and 11.6-16.3%(n = 7 each), respectively. The concentration(y, nmol/mg hair) of uric acid in hair correlated well with that in blood(x, g/l): y = 8.770x-0.123(r = 0.746, Syx = 0.122, n = 23). Changes in the concentration of uric acid in hair of hyperuricemic patient treated with allopurinol paralleled to those in blood. In conclusion, it was confirmed that the concentration of uric acid in hair reflected that in blood, suggesting that measuring uric acid in hair can be available for the metabolic control in hyperuricemia.