Unknown,Transcriptomics,Genomics,Proteomics

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Gene expression profiling of peripheral blood mononuclear cells from patients with minimal change nephrotic syndrome

ABSTRACT: Background: Minimal change nephrotic syndrome (MCNS) is considered to be associated with T cell dysfunction, via unknown mechanisms. Experimental observations suggest that some humoral factors alter the permeability of glomerular filtration barrier. However, the nature of such factors remains still uncertain. Methods: Using cDNA microarrays, we performed gene expression profiling of peripheral blood mononuclear cells (PBMC) from three patients with MCNS during nephrosis and remission phases. To confirm the cDNA microarray results, we performed quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses in nephrosis and remission samples from 20 MCNS patients and six patients with nephrotic syndrome due to membranous nephropathy. Results: Out of 24,446 genes screened, 33 genes were up-regulated (at least 1.5-fold) in PBMC from these MCNS patients during the nephrosis phase. Up-regulated genes mainly encoded proteins involved in signal transduction and cytokine response. For further examination, we selected two genes encoding provable secretary proteins, chemokine (C-C) ligand 13 (also known as monocyte chemotactic protein-4) (CCL13) and a novel galectin-related protein (HSPC159). The results of RT-PCR showed that expressions of CCL13 and HSPC159 mRNA in nephrosis PBMC samples are higher than those in remission PBMC samples from all 20 MCNS patients examined. On the other hand, these mRNA expression patterns were variable among six patients with membranous nephropathy. Conclusions: We conclude that CCL13 and HSPC159 mRNA expressions in PBMC is up-regulated in MCNS patients during the nephrosis phase. These expression changes in PBMC might be involved in the pathophysiologic processes of MCNS. Using cDNA microarrays, we performed gene expression profiling of peripheral blood mononuclear cells (PBMC) from three patients with MCNS during nephrosis and remission phases. To confirm the cDNA microarray results, we performed quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) analyses in nephrosis and remission samples from 20 MCNS patients and six patients with nephrotic syndrome due to membranous nephropathy.

ORGANISM(S): Homo sapiens

SUBMITTER: Atsushi Komatsuda

PROVIDER: E-GEOD-7349 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Background: The early stages of D. melanogaster embryogenesis involve cell migration and pattern formation, and lead to the formation of three germ layers (the ectoderm, mesoderm and endoderm). These developmental events are controlled by differential gene activity. In the current study we used a suppressive subtractive hybridization (SSH) procedure to identify a group of genes potentially involved in D. melanogaster early embryogenesis and to study the temporal activity of developmentally regulated genes at five different intervals covering 12 stages of embryogenesis. Results: Macroarrays were constructed to confirm induction of expression and determine the temporal profile of isolated subtracted cDNAs during embryo development. We identified a set of 118 genes that significantly increased their expression levels at least at one developmental interval compared with the reference interval. 53.4% of them have a phenotype and/or molecular function reported in the literature, whereas 46.6% are essentially uncharacterized. Clustering analysis revealed demarcated transcript groups with maximum gene activity at distinct developmental intervals. In situ hybridization assays were carried out on 30 of the transcripts and of these, 19 (63%) proved to have restricted expression patterns. 11 of the uncharacterized genes that exhibited temporal and spatially restricted patterns of expression in developing embryos encode putative secreted and transmembrane proteins. For three of them we validated our protein sequence predictions by expressing their cDNAs in S2R+ cells and analyze the subcellular distribution of recombinant proteins. Conclusions: Our data provides a list of developmentally regulated D. melanogaster genes and their expression profiles during embryogenesis, including novel information on the temporal and spatial expression patterns of several previously uncharacterized genes. In particular, we recovered a significant number of novel genes encoding putative secreted and transmembrane proteins, suggesting new components of signalling pathways that might be incorporated within the existing regulatory networks controlling Drosophila embryogenesis; they are also good candidates for more functional targeted analyses. Keywords: Drosophila melanogaster, gastrulation, macroarray, developmental time course We generated a subtracted stage specific cDNA library representing genes differentially expressed between Drosophila melanogaster synciyial blastoderm and gastrula stages. Clones were spotted onto nylon membranes and hybridized against cDNA probes representing mRNA from embryos at five different developmental intervals, in order to obtain time course expression data covering the first 12 stages of Drosophila development. We designed macroarrays containing either 302 non-redundant cDNAs, including protein-coding sequences, ncRNA, transposons, introns and intergenic regions (membranes A, B and C) and the complete subtracted library (579 cDNAs, membrane D). Each clone was spotted in duplicate. The following controls were spotted onto membranes: (1) a fragment of the vector pBluescript II obtained by amplification with the T7 and SP6 universal primers; (2) several spots of DMSO 50% (solvent used); (3) PCR amplified fragment of genes serendipity Î±, twist, tinman, fog and snail as positive controls, and (4) actin, tubulin, and RP49 as housekeeping, and (5) four dilutions of a PCR amplified fragment from a B. subtilis dap cDNA (ATCC; number 87486), which hybridizes to an in vitro synthesized poly(A)-RNA that was added to the embryo RNA samples (dilution 1/200) prior to the labelling process and used as spike mRNA (Kane et al., 2000).

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Gene expression profiling of peripheral blood mononuclear cells from patients with minimal change nephrotic syndrome

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