Biomedical subjects

Iwao Waga

Publications and source records attributed to Iwao Waga.

7 recordsLinked to original sources

PubMed2004

Protein disulfide isomerase suppresses the transcriptional activity of NF-kappaB.

We report here that the transcriptional activity of NF-kappaB is negatively regulated by protein disulfide isomerase (PDI). Over-expression of PDI in RAW 264.7 cells strongly suppressed the LPS-induced production of inflammatory cytokines as well as NF-kappaB-dependent luciferase activity. This negative regulation of NF-kappaB was reversed by bacitracin, a PDI inhibitor. Interestingly, NF-kappaB/DNA complex formation and phosphorylation of NF-kappaB subunits was intact in PDI-expressing cells following stimulation with LPS. In addition, PDI and another redox regulator, thioredoxin (TRX), had opposite effects on NF-kappaB-dependent gene expression: activation of the NF-kappaB pathway by TRX was suppressed by expression of PDI in a dose-dependent manner. Finally, PDI expression was induced by the anti-inflammatory cytokine IL-10, and IL-10-mediated inhibition of LPS-induced IL-6 expression was reduced by bacitracin. These findings clearly demonstrate that PDI is a negative regulator of NF-kappaB, and may act downstream of IL-10 in this signaling pathway.

Animals↗

PubMed2004

Gene expression profiles of circulating leukocytes correlate with renal disease activity in IgA nephropathy.

BACKGROUND: The goal of these studies was to explore the possibility of using gene expression profiles of circulating leukocytes as a functional fingerprint of nephritic disease activity. METHODS: This feasibility study utilized IgA nephropathy (IgAN) as a model system. Genes differentially expressed in IgAN patients were identified by Affymetrix GeneChip microarrays, and compared with gene expression of focal segmental glomerulosclerosis (FSGS), minimal change disease, antineutrophil cytoplasmic antibody (ANCA) glomerulonephritis, and with healthy volunteers. Of the genes identified, 15 transcriptionally up-regulated were validated in a larger cohort of patients using TaqMan polymerase chain reaction (PCR). To test whether increased expression of these genes correlated with disease activity, cluster analyses were performed utilizing the TaqMan PCR values. Taking a mathematical approach, we tested whether gene expression values were correlative with kidney function, as reflected by serum creatinine and creatinine clearance values. RESULTS: We identified 15 genes significantly correlative with disease activity in IgAN. This gene signature of IgAN patients' leukocytes reflected kidney function. This was demonstrated in that mathematically generated theoretical values of serum creatinine and creatinine clearance correlated significantly with actual IgAN patient values of serum creatinine and creatinine clearance. There was no apparent correlation with hematuria and proteinuria. The expression levels of this same gene set in ANCA glomerulonephritis or Lupus nephritis patients were not correlative with serum creatinine or creatinine clearance values. CONCLUSION: These data indicate that leukocytes carry informative disease-specific markers of pathogenic changes in renal tissue.

Adolescent↗

PubMed2003

Molecular cloning, genomic structure, and expression analysis of MUC20, a novel mucin protein, up-regulated in injured kidney.

Immunoglobulin A nephropathy (IgAN) is the most common primary glomerulonephritis in the world. Here, we identify a cDNA encoding a novel mucin protein, shown previously to be up-regulated in IgAN patients, from a human kidney cDNA library. This protein contains a mucin tandem repeat of 19 amino acids consisting of many threonine, serine, and proline residues and likely to be extensively O-glycosylated; thus, this gene was classified in the mucin family and named MUC20. The human MUC20 gene contains at least four exons and is localized close to MUC4 on chromosome 3q29. We found variations in repeat numbers in the mucin tandem domain, suggesting polymorphism of this region. Northern blot and reverse transcription-PCR analyses revealed that human MUC20 mRNA was expressed most highly in kidney and moderately in placenta, colon, lung, prostate, and liver. Immunohistochemical analysis of human kidney revealed that MUC20 protein was localized in the proximal tubules. Immunoblotting analysis of MUC20 proteins produced in Madin-Darby canine kidney and HEK293 cells indicated the localization of MUC20 protein in a membrane fraction and extensive posttranslational modification. Immunoelectron microscopy of MUC20-producing Madin-Darby canine kidney cells demonstrated that MUC20 protein was localized on the plasma membrane. Expression of MUC20 mRNA in a human kidney cell line was up-regulated by tumor necrosis factor-alpha, phorbol 12-myristate 13-acetate, or lipopolysaccharide. Two species of MUC20 mRNA (hMUC20-L and hMUC20-S), resulting from alternative transcription, were identified in human tissue, whereas only one variant was observed in mouse tissues. Mouse MUC20 mRNA was expressed in the epithelial cells of proximal tubules, and the expression increased dramatically with the progression of lupus nephritis in the kidney of MRL/MpJ-lpr/lpr mice. Moreover, the expression of mouse MUC20 was augmented in renal tissues acutely injured by cisplatin or unilateral ureteral obstruction. These characteristics suggest that the production of MUC20 is correlated with development and progression of IgAN and other renal injuries.

Amino Acid Sequence↗

PubMed2003

Altered mRNA expression in renal biopsy tissue from patients with IgA nephropathy.

BACKGROUND: Immunoglobulin A (IgA) nephropathy (IgAN) is a renal disease characterized by glomerular deposition of IgA-dominant immune deposits that cause glomerular inflammation and sclerosis. Gene expression changes induced in renal tissues/cells as a result of the disease are largely uncharacterized. METHODS: A sensitive differential mRNA display technique, restriction endonucleolytic analysis of differentially expressed sequences (READS) compared similarly processed normal renal tissue to renal biopsy RNA from patients with IgAN, minimal change disease, and necrotizing crescentic glomerulonephritis. A subset of genes with altered expression in IgAN as identified by the READS technology was further characterized and expression levels confirmed using real-time quantitative polymerase chain reaction (RT-PCR) analysis (TaqMan) in all RNA. RESULTS: Initial READS analysis showed IgAN samples have lower mRNA levels relative to normal renal tissue mRNA samples based upon total RNA as measured by ribosomal RNA. One hundred seventy-five differentially expressed non-redundant fragments were found from 860 initial candidate fragments. Twenty genes were selected for additional TaqMan analysis, and 13 of 20 genes showed statistically different expression when comparing biopsies from normal individuals and IgAN patients. Expression differences were seen in these genes in biopsies of IgAN of differing clinical activities. Gene expression cluster analysis using the Ward method detailed disease- and gene-related clusters. Detailed examination of the promoter regions of the genes within two gene clusters revealed common gene transcriptional regulatory protein-binding sites. CONCLUSION: IgAN leads to significant changes in overall mRNA transcription levels within the renal tissue, in addition to gene-specific mRNA level changes. Disease-related patterns of expression were identified and gene-specific clusters suggest common mechanisms of transcriptional alteration.

Biopsy↗

PubMed2002

Symptomatic and asymptomatic benign prostatic hyperplasia: molecular differentiation by using microarrays.

Benign prostatic hyperplasia (BPH) is a disease of unknown etiology that significantly affects the quality of life in aging men. Histologic BPH may present itself either as symptomatic or asymptomatic in nature. To elucidate the molecular differences underlying BPH, gene expression profiles from the prostate transition zone tissue have been analyzed by using microarrays. A set of 511 differentially expressed genes distinguished symptomatic and asymptomatic BPH. This genetic signature separates BPH from normal tissue but does not seem to change with age. These data could provide novel approaches for alleviating symptoms and hyperplasia in BPH.

Adolescent↗

PubMed2002

Expression profile of leukocyte genes activated by anti-neutrophil cytoplasmic autoantibodies (ANCA).

BACKGROUND: Anti-neutrophil cytoplasmic autoantibodies (ANCA) induce neutrophil activation in vitro with release of injurious products that can mediate necrotizing vasculitis in vivo. The importance of ANCA IgG F(ab')2-antigen binding versus Fcgamma receptor engagement in this process is controversial. We propose that ANCA-antigen binding affects cell signaling pathways that can result in changes of gene expression. METHODS: Microarray GeneChip analysis and real-time, quantitative PCR (TaqMan(R)) was used to probe for transcripts in leukocytes from patients (in vivo gene expression study) and in leukocytes treated with ANCA IgG or ANCA-F(ab')2 (in vitro gene expression study). RESULTS: Microarray gene chip analysis showed that ANCA IgG and ANCA-F(ab')2 stimulate transcription of a distinct subset of genes, some unique to whole IgG, some unique to F(ab')2 fragments, and some common to both. DIF-2, COX-2, and IL-8 were identified as genes responsive to ANCA signaling and were selected for in depth evaluation. In vitro DIF-2 and IL-8 were increased by both ANCA IgG and F(ab')2, but COX-2 only by MPO-ANCA F(ab')2. In vivo DIF-2 levels were increased in leukocytes of ANCA patients, which correlated strongly with disease activity and ANCA titer. DIF-2 was not increased in patients in remission or in disease control patients (systemic lupus erythematosus and IgA nephropathy). COX-2 gene expression was significantly increased in patients with active disease, while IL-8 was increased in remission. CONCLUSIONS: The data indicate that leukocyte genes are activated in vitro by both ANCA Fc and ANCA F(ab')2 pathways and that in vitro activation mimics changes in circulating leukocytes of patients with ANCA disease. Increased levels of DIF-2 in patient leukocytes strongly correlate with severity of disease in kidney tissue. The observations indicate a previously unrecognized role for DIF-2 in ANCA-mediated inflammation, which raises the possibility that DIF-2 has an important role in other types of inflammation.

Antibodies, Antineutrophil Cytoplasmic↗

PubMed2002

Microarray studies of gene expression in circulating leukocytes in kidney diseases.

The molecular characterization of changes in mRNA expression in renal tissue during disease is hampered by the acquisition of sufficient mRNA to do genomewide expression profiling. In many renal diseases, such as systemic lupus erythematosus, IgA nephropathy, antineutrophil cytoplasmic autoantibody (ANCA) associated glomerulonephritis, and small-vessel vasculitis (ANCA disease), circulating leukocytes play a role in onset, progression, and severity of the condition. Circulating leukocytes are readily isolated and supply sufficient mRNA for analysis, allowing molecular investigation into their involvement in the disease process. Our laboratory has undertaken a systematic study of the genomewide expression profiles of the circulating leukocytes from patients with a variety of renal diseases (ANCA disease, IgA nephropathy, lupus nephritis, focal segmental glomerulosclerosis), using the Affymetrix high-density gene chip array technology. Analysis of the data showed clustering of expressed genes unique for each individual disease group. These results imply that significant gene expression changes occur in leukocytes that are circulating in patients with renal diseases. In addition, gene expression has been studied in leukocytes activated in vitro by mechanisms that mimic pathogenic events in vivo. The expression levels of genes identified in in vitro studies were compared with the patient leukocyte gene expression to determine whether similar pathological events were occurring in vivo.

Humans↗