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Biomedical subjects

N Kashihara

Publications and source records attributed to N Kashihara.

At least 19 recordsLinked to original sources
PubMed2000

Synthesis and biological activities of hapalosin derivatives with modification at the C12 position.

Among the hapalosin derivatives synthesized, the compounds carrying methyl (5a), methylthioethyl (5d) and phenylmethyl (5e) groups at the C12 position possess only the cis-peptide structure, in contrast to the cases of 5b and 5c. In addition to their conformational stability, the biological activities of the compounds were determined in relation of the P-glycoprotein-mediated MDR-reversing activity and induction of apoptosis.

Antineoplastic Agents↗
PubMed2000

Expression characteristics and relevance of sodium glucose cotransporter-1 in mammalian renal tubulogenesis.

Expression and role of sodium glucose cotransporter (SGLT-1) in tubulogenesis were investigated during renal development. A mouse SGLT-1 cDNA was cloned, and it had substantial homology with human and rat forms. Four mRNA transcripts were detected, which differed in size from other species. SGLT-1 transcripts were detected at day 13 of gestation, and their expression increased during later stages extending into the postnatal period. A high mRNA and protein expression of SGLT-1 was seen in tubular segments of the inner cortex and outer medulla at day 16, and it was developmentally regulated. Treatment with SGLT-1 antisense selectively decreased the population of tubules in the metanephric explants. Expression of glomerular mRNA and WGA binding were unchanged. SGLT-1 activity, as measured by [(14)C]methyl-alpha-D-glucopyranoside uptake, increased during gestation in the tubular segments where it is expressed. Glucose uptake was inhibited by the treatment with SGLT-1 antisense and D-galactose. The data suggest that SGLT-1 exhibits a restricted spatiotemporal expression with functional activity confined to the corresponding tubular segments, and it selectively maintains renal tubulogenesis during development.

Amino Acid Sequence↗
PubMed2000

Apoptosis and extracellular matrix-cell interactions in kidney disease.

Apoptosis and extracellular matrix-cell interactions in kidney disease. Extracellular matrix (ECM)-cell interactions have major effects on phenotypic features such as cell growth, differentiation, and gene expression. Apoptosis is an active form of cell death that is crucial for maintaining an appropriate number of cells as well as tissue organization. Recent reports have implied that ECM can influence survival and apoptosis of several cell lineages including glomerular mesangial cells (MC). Numerous glomerular diseases are associated with the expansion of the mesangial ECM, which may eventually produce glomerular scarring. Glomerular cell apoptosis is associated with the deletion of glomerular cells and the accumulation of ECM in the progression of glomerulosclerosis in rat remnant kidney model induced by 5/6 nephrectomy. Our recent study indicated that basement membrane matrix (a model for normal ECM components) prevented cultured MC from undergoing apoptosis after serum deprivation, thus promoting their survival, compared with type I collagen matrix (a model for abnormal ECM components). Inhibition of matrix-derived signals by antisense oligonucleotides against beta1 integrin increased MC apoptosis. Data suggest that the survival and death of MC are regulated by the surrounding ECM through integrin molecules. The mechanism of regulation of MC apoptosis by ECM requires further in vivo study to gain new insight into the treatment of glomerular diseases as well as the pathophysiology of the mesangium. Diabetic nephropathy is characterized by the abnormal ECM accumulation and the phenotypic change of MC. Some speculations on the possible involvement of apoptosis in diabetic nephropathy are also discussed.

Animals↗
PubMed1999

Tubulointerstitial nephritis antigen: an extracellular matrix protein that selectively regulates tubulogenesis vs. glomerulogenesis during mammalian renal development.

Tubulointerstitial nephritis antigen (TIN-ag) is an extracellular matrix protein and is expressed in the renal tubular basement membranes. Its role in metanephric development was investigated. TIN-ag cDNA, isolated from the newborn mouse library, had an ORF of 1,425 nucleotides, a putative signal sequence, and an ATP/GTP-binding site. The translated sequence had approximately 80% identity with rabbit TIN-ag. Among various tissues, TIN-ag mRNA was primarily expressed in the newborn kidney. In the embryonic metanephros, TIN-ag expression was confined to the distal convolution or pole of the S-shaped body, the segment of the nascent nephron that is the progenitor of renal tubules. Treatment with TIN-ag antisense oligodeoxynucleotide induced dysmorphogenesis of the embryonic metanephroi, malformation of the S-shaped body, and a decrease in the tubular population, whereas the glomeruli were unaffected. Treatment also led to a decrease of TIN-Ag mRNA, de novo synthesis of TIN-ag protein, and its antibody reactivity. The mRNA expression of glomerular epithelial protein 1 (a marker for renal podocytes), anti-heparan-sulfate-proteoglycan antibody reactivity, and wheat germ agglutinin lectin staining of the metanephros were unaffected. The anti-TIN-ag antibody treatment also caused deformation of the S-shaped body and a reduction in the tubular population, whereas the glomeruli were unchanged. The data suggest that the TIN-ag, unlike other basement membrane proteins, selectively regulates tubulogenesis, whereas glomerulogenesis is largely unaffected.

Amino Acid Sequence↗
PubMed1999

Cloning of rat fibrillin-2 cDNA and its role in branching morphogenesis of embryonic lung.

Fibrillin-2 is an extracellular matrix protein. It is associated with elastic fibers in several tissues and is believed to serve as a ligand for alphavbeta3 integrin, the latter being a known morphogen. In this study, the role of fibrillin-2 in lung development was investigated. Also, rat fibrillin-2 cDNA was isolated and sequenced and its spatiotemporal expression determined. It had approximately 88% homology with human fibrillin-2 and had Ca(2+) binding epidermal growth factor-like domains, transforming growth factor-beta binding protein motifs, and two RGD binding sites. Northern blot analysis revealed an approximately 10-kb transcript, and fibrillin-2 expression was developmentally regulated, and it paralleled that of tropoelastin. At day 13 of gestation, fibrillin-2 was expressed in the mesenchyme and at the epithelial:mesenchymal interface. From day 13 to 19 of gestation, its expression intensified and was confined around the tracheobronchial airways, while it lessened during the postnatal period. Immunoprecipitation revealed an approximately 350-kDa band by SDS-PAGE. Treatment with fibrillin-2 antisense oligodeoxynucleotide induced dysmorphogenesis of the lung explants. They were smaller and had rudimentary lung bud branches, collapsed conducting airways, and loose expanded mesenchyme. Concomitantly, fibrillin-2 mRNA, antibody reactivity in the explants, and fibrillin-2-specific radioincorporation were reduced. Anti-alphav and -laminin antibody reactivity and their respective incorporated specific radioactivities were unaltered. These data indicate that fibrillin-2 modulates organogenesis of the lung in the context of epithelial:mesenchymal interactions. Conceivably, the collapse of the conducting airways may also be related to the perturbed biology of the fibrillin-2 interacting protein, i.e., elastin, the latter being critical for the normal biophysiology of the lungs.

Actins↗
PubMed1999

Expression of the fibroblast growth factor receptor 1-4 genes in glomeruli in anti-Thy1.1 mesangial proliferative glomerulonephritis.

Basic fibroblast growth factor (FGF2) is generally known to induce proliferation of cultured mesangial cells and is expressed in proliferative mesangial cells in anti-Thy1.1 mesangial proliferative glomerulonephritis (anti-Thy1.1 GN). The distribution of the FGF receptor (FGFR) has not been studied in anti-Thy1.1 GN, so we used in situ hybridization to determine whether cells expressing FGFR1-4 mRNAs could be detected. In normal rats, all glomeruli were negative for FGFR1-4 mRNA, but those of the mesangial proliferative phase expressed FGFR1-4 mRNA in proliferative mesangial cells. Proliferation of mesangial cells has not been observed in normal rats injected with FGF2( )but it has been noted in anti-Thy1.1 rats injected with FGF2. These data and our results demonstrate that mesangial cells produce and release FGF2( )after injury and that during the proliferative phase these cells upregulate FGFR in vivo. This study is the first to demonstrate expression of FGFR1-4 mRNAs in pathological glomeruli of anti-Thy1.1 GN. The FGF2 and FGFR1-4 genes were expressed in the proliferative mesangial cells. Upregulation of FGFR is necessary for mesangial proliferation by FGF2.

Animals↗
PubMed1999

Role of membrane-type matrix metalloproteinase 1 (MT-1-MMP), MMP-2, and its inhibitor in nephrogenesis.

Extracellular matrix (ECM) proteins, their integrin receptors, and matrix metalloproteinases (MMPs), the ECM-degrading enzymes, are believed to be involved in various biological processes, including embryogenesis. In the present study, we investigated the role of membrane type MMP, MT-1-MMP, an activator pro-MMP-2, in metanephric development. Also, its relationship with MMP-2 and its inhibitor, TIMP-2, was studied. Since mRNAs of MT-1-MMP and MMP-2 are respectively expressed in the ureteric bud epithelia and mesenchyme, they are ideally suited for juxtacrine/paracrine interactions during renal development. Northern blot analyses revealed a single approximately 4.5-kb mRNA transcript of MT-1-MMP, and its expression was developmentally regulated. Inclusion of MT-1-MMP antisense oligodeoxynucleotide (ODN) in the culture media induced dysmorphogenetic changes in the embryonic metanephros. MMP-2 antisense ODN also induced similar changes, but they were relatively less; on the other hand TIMP-2 antisense ODN induced a mild increase in the size of explants. Concomitant exposure of MT-1-MMP and MMP-2 antisense ODNs induced profound alterations in the metanephroi. Treatment of TIMP-2 antisense ODN to metanephroi exposed to MT-1-MMP/MMP-2 antisense notably restored the morphology of the explants. Specificity of the MT-1-MMP antisense ODN was reflected in the selective decrease in its mRNA and protein expression. The MT-1-MMP antisense ODN also resulted in a failure in the activation of pro-MMP-2 to MMP-2. These findings suggest that the trimacromolecular complex of MT-1-MMP:MMP-2:TIMP-2 modulates the organogenesis of the metanephros, conceivably by mediating paracrine/juxtacrine epithelial:mesenchymal interactions.

Aging↗
PubMed1999

Treatment with a Ca(2+) channel blocker, barnidipine, reduces platelet-derived growth factor B-chain mRNA in glomeruli of spontaneously hypertensive rats.

We investigated the effect of barnidipine hydrochloride, a Ca(2+) channel blocker, on the glomerular level of mRNA expression of platelet-derived growth factor (PDGF) B-chain and transforming growth factor (TGF)-beta(1) in spontaneously hypertensive rats (SHR) with reverse transcription and polymerase chain reaction. Thirteen-week-old SHR were provided with food containing barnidipine (0.6 mg/g of food, average dose during treatment: 53 mg/kg of body mass/day) for 3 weeks. A stable reduction in systolic blood pressure relative to that of age-matched control SHR was recorded after week 1 of therapy. Although no renal histological changes were observed after 3 weeks of treatment with barnidipine, the level of expression of PDGF B-chain mRNA in glomeruli was significantly reduced relative to that in control SHR. The glomerular level of TGF-beta(1) mRNA expression was not affected by the treatment. Treatment with barnidipine significantly reduced the excretion of urinary protein. Thus, the stable reduction in systemic blood pressure by barnidipine is associated with a reduction in PDGF B-chain mRNA expression in the glomerulus and reduction in urinary protein excretion in SHR.

Animals↗
PubMed1998

Inhibition of mesangial cell proliferation by E2F decoy oligodeoxynucleotide in vitro and in vivo.

The transcription factor E2F coordinately activates several cell cycle-regulatory genes. We attempted to inhibit the proliferation of mesangial cells in vitro and in vivo by inhibiting E2F activity using a 25-bp decoy oligodeoxynucleotide that contained consensus E2F binding site sequence (E2F-decoy) as a competitive inhibitor. The decoy's effect on human mesangial cell proliferation was evaluated by [3H]thymidine incorporation. The E2F decoy inhibited proliferation in a concentration-dependent manner, whereas a mismatch control oligodeoxynucleotide had little effect. Electrophoretic mobility shift assays demonstrated that the decoy's inhibitory effect was due to the binding of the decoy oligodeoxynucleotide to E2F. The effect of the E2F decoy was then tested in a rat anti-Thy 1.1 glomerulonephritis model. The E2F decoy oligodeoxynucleotide was introduced into the left kidney 36 h after the induction of glomerulonephritis. The administration of E2F decoy suppressed the proliferation of mesangial cells by 71%. Furthermore, treatment with the E2F decoy inhibited the glomerular expression of proliferating cell nuclear antigen at the protein level as well as the mRNA level. These findings indicate that decoy oligonucleotides can suppress the activity of the transcription factor E2F, and may thus have a potential in treating glomerulonephritis.

Animals↗
PubMed1998

Regulation of survival and death of mesangial cells by extracellular matrix.

BACKGROUND: Cell-matrix interactions exert major effects on such phenotypic features as cell growth and differentiation. Apoptosis is an active form of cell death that is crucial for maintaining the appropriate number of cells as well as the organization of tissue. Recently, it has been suggested that apoptosis of the mesangial cells (MC) is important in glomerular remodeling after injury. The MC are surrounded by an extracellular matrix (ECM) in vivo. Since in disease conditions the mesangial matrix is altered quantitatively and qualitatively, it is of interest to determine whether cell-matrix interactions may influence apoptosis of the MC. METHODS: We first investigated the differences in the susceptibility to apoptotic stimuli of the MC cultured on various ECM components (type I collagen, fibronectin, basement membrane matrix). We then determined whether the inhibition of MC-matrix interactions would affect apoptosis. Finally, interactions between MC and matrix were disrupted by the inhibition of beta1-integrin expression with antisense oligonucleotides (ODN). RESULTS: When MC were cultured on type I collagen or fibronectin and deprived of serum for eight hours, the extracted DNA from the MC demonstrated an internucleosomal ladder pattern on gel electrophoresis that constituted the biochemical characteristic of apoptosis. However, no ladder pattern was apparent when MC were cultured on basement membrane matrix. The attachment of cells was completely inhibited when the MC were cultured on agarose-coated dishes for 24 hours. Gel electrophoresis of DNA extracted from these cells showed a ladder pattern. However, the MC attached to the substratum did not show any apoptosis. MC showed an increase in apoptotic cell death after treatment with antisense ODN against beta1-integrin molecule. CONCLUSIONS: These results indicate that normal ECM may prevent the MC from undergoing apoptosis and serve as a survival factor for MC. Signals from ECM that prevent apoptosis may be mediated by beta1-integrin molecules.

Apoptosis↗
PubMed1998

Cytokine interactions promote synergistic fibronectin accumulation by mesangial cells.

BACKGROUND: The development of glomerulosclerosis has been associated with the presence of the cytokines transforming growth factor-beta 1 (TGF-beta 1), tumor necrosis factor-alpha (TNF-alpha) and/or interleukin-1 beta (IL-1 beta), at some stage in the glomerulus. To better understand the role of these cytokines in the scarring process their effect on rat mesangial cell fibronectin production was investigated. METHODS: Mesangial cells were exposed to 10 ng/ml of either TGF-beta 1, TNF-alpha, or IL-1 beta or to TGF-beta 1 in combination with TNF-alpha or IL-1 beta. Tissue culture supernatants and cell lysates were assayed for fibronectin. Supernatants were also assayed for TGF-beta 1. Northern blot analyses probing for fibronectin, transin, TIMP-1 and TGF-beta 1 were carried out on RNA extracted from mesangial cells exposed to individual and combinations of cytokines. RESULTS: Individually these cytokines were only able to induce modest increases in fibronectin protein levels. However, when mesangial cells were exposed to TGF-beta 1 in combination with either TNF-alpha or IL-1 beta then fibronectin levels were synergistically up-regulated approximately fivefold over unstimulated levels. Northern analysis demonstrated that fibronectin mRNA levels in the combination were also synergistically increased. In contrast, rat transin gene expression in the combinations was reduced to well below levels induced by TNF-alpha and IL-1 beta individually. In addition, synergistic up-regulation of both TGF-beta 1 protein and message by the cytokine combinations was also observed. TGF-beta 1: TNF-alpha and TGF-beta 1: IL-1 beta induced additive increases in TIMP-1 (tissue inhibitor of metalloproteinases-1) mRNA levels. CONCLUSIONS: These data illustrate that complex interactions can occur between cytokines within the glomerulus modulating both matrix synthetic and degradation pathways. These could initiate the scarring process and the development of glomerulosclerosis.

Animals↗
PubMed1998

Antisense oligonucleotides targeting c-fos mRNA inhibit rheumatoid synovial fibroblast proliferation.

OBJECTIVE: To determine whether antisense oligonucleotides targeting c-fos mRNA have the ability to inhibit the growth of interleukin 1 (IL1) stimulated fibroblast-like cells from the synovium in rheumatoid arthritis (RA). METHODS: Fibroblast-like cells established from RA synovium were stimulated by IL1 with antisense or sense oligonucleotides complementary to c-fos mRNA, and the proliferation of these cells was determined by 3H-thymidine incorporation. Effect of antisense oligonucleotides on expression of activator protein 1 (AP1) activity was evaluated using electrophoretic mobility shift assay. RESULTS: C-fos antisense oligonucleotides inhibited IL1 stimulated synovial fibroblast proliferation. The expression of AP1 activity induced by IL1 was suppressed by treatment with antisense oligonucleotides. CONCLUSION: These results suggest the feasibility of antisense strategies designed to suppress c-fos expression as therapeutic agents for RA.

Arthritis, Rheumatoid↗
PubMed1998

Isolation of rat fibrillin-1 cDNA and its relevance in metanephric development.

The role of fibrillin-1 in metanephrogenesis was investigated. Fibrillin-1 cDNA was isolated from the rat kidney cDNA library and sequenced, and its spatiotemporal expression was studied. It had approximately 88% homology with human fibrillin-1 and had Ca2+ binding epidermal growth factor-like domains, transforming growth factor-beta binding protein motifs, and an RGD binding site. Northern blot analysis revealed an approximately 10-kb transcript, and fibrillin-1 expression was developmentally regulated. In situ hybridization and immunofluorescence studies indicated that at day 15 of gestation, fibrillin-1 is expressed in the metanephric mesenchyme. At day 18, its expression was confined to nascent blood vessels and glomeruli, and it increased in the newborn and neonatal kidneys. Immunoprecipitation revealed an approximately 300-kDa band by SDS-PAGE. Treatment with fibrillin-1 antisense oligodeoxynucleotide induced marked dysmorphogenesis of the embryonic metanephroi. Concomitantly, the fibrillin-1 mRNA, antibody reactivity in the metanephroi, and fibrillin-1-specific radioincorporation were reduced. These data indicate that, like alphavbeta3 integrin, a known morphogen and a putative receptor of fibrillin-1, the fibrillin-1 modulates events related to early organogenesis and possibly also the vascularization of the rat kidney.

Amino Acid Sequence↗
PubMed1998

Expression of interleukin-8 in human glomerulonephritis.

Interleukin-8 (IL-8) is a cytokine, which possesses both chemotactic and activating properties for neutrophils, lymphocytes and basophils. Various evidence has indicated IL-8 to be implicated in the pathophysiology of immune-mediated renal diseases. We thus examined the expression of IL-8 in renal diseases. We detected the expression of IL-8 both in mRNA and the protein levels in renal biopsy specimens obtained from patients with IgA nephropathy and lupus nephritis. A significant correlation was found between the expression of IL-8 mRNA and the number of neutrophils in the glomerulus. We also found a negative correlation between the expression of IL-8 mRNA and creatinine clearance. Our study thus suggested IL-8 to be involved in the pathophysiology of proliferative glomerulonephritis.

Adolescent↗
PubMed1997

Inhibition of rheumatoid synovial fibroblast proliferation by antisense oligonucleotides targeting proliferating cell nuclear antigen messenger RNA.

OBJECTIVE: To evaluate the feasibility of antisense oligonucleotides as therapeutic agents to inhibit synovial cell growth in rheumatoid arthritis (RA). METHODS: Fibroblast-like cells established from RA synovium were stimulated with interleukin-1beta (IL-1beta) and treated with antisense or sense oligonucleotides targeting proliferating cell nuclear antigen (PCNA) messenger RNA (mRNA). Proliferation of these cells was determined by 3H-thymidine incorporation. Effects of antisense oligonucleotides on the expression of mRNA and protein were evaluated by reverse transcriptase-polymerase chain reaction and immunohistochemical staining, respectively. RESULTS: Antisense oligonucleotides targeting PCNA inhibited IL-1-stimulated fibroblast proliferation, whereas sense oligonucleotides had no effect. Both mRNA and protein levels of PCNA were suppressed in the cells treated with antisense oligonucleotides, indicating that the antiproliferative effect was occurring through an antisense mechanism. CONCLUSION: These results suggest that antisense strategies designed to suppress PCNA expression have potential use as therapeutic agents for RA.

Arthritis, Rheumatoid↗