Gene expression analysis of high-glucose stimulated human mesangial cells and tubular epithelial cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE52734 | GEO | 2013/12/16
SECONDARY ACCESSION(S): PRJNA229946
REPOSITORIES: GEO
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Project description:we performed genome-wide transcriptome profiling in mesangial cells and tubular epithelial cells (TECs), which were stimulated by high glucose (HG) and detected the expression of inflammation associated genes. HG increased the mRNA expression of oxidative stress, inflammasome and mammalian target of rapamycin (mTOR) related genes in mesangial cells. Pro-inflammatory/Th1 gene expression was upregulated, but Th2 related gene expression was downregulated in mesangial cells. In TECs, HG stimulation increased pro-inflammatory/Th1/Th2 gene expression. mRNA profiles of human mesangial cells and tubular epithelial cells, which were stimuated HG for 24 hours or mannitol contorol.
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Project description:IgA nephropathy (IgAN) is the most common glomerulonephritis in the world. The disease is characterized by galactose deficient IgA (gd-IgA) in the circulation forming immune complexes. The complexes are deposited in the glomerular mesangium leading to inflammation and loss of renal function, but the pathophysiology of the disease is still not fully understood. Using an integrated global transcriptomic and proteomic profiling approach we investigated the role of the mesangium in the onset and progression of IgAN. Global gene expression was investigated by microarray analysis of the glomerular compartment of renal biopsies from patients with IgAN. The influence of galactose deficient IgA (gd-IgA) on mesangial cells was investigated by proteomic profiling. By utilizing the previous published literature curated glomerular cell type-specific genes, we found that mesangial cells and their positive standard genes play a more dominant role in IgAN comparing to the podocyte standard genes. Additionally, the patient clinical parameters (serum creatinine values and estimated glomerular filtration rate - eGFR) significantly correlate with z-scores derived from expression profile of mesangial cell positive standard genes. 22 common pathways were identified both from in vivo microarray data and in vitro mesangial cell mass spectrometry data and the main part was inflammatory pathways. The correlation between clinical data and mesangial standard genes allows for a better understanding of the onset of IgAN. The genes, proteins and their corresponding pathways identified in this paper give us novel insights into the pathophysiological mechanisms leading to progression of IgAN. RNA from glomerular compartment was extracted and processed for hybridization on Affymetrix microarrays. This dataset is part of the TransQST collection.
2017-07-03 | E-GEOD-93798 | biostudies-arrayexpress
Project description:The renal mesangial cells play an important role in the development of diabetic glomerulosclerosis and renal failure. We have previously demonstrated some of the effects of high glucose are mediated via the hexosamine biosynthesis pathway (HBP) in which fructose-6-phosphate is converted to glucosamine-6-phosphate by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT). Using Affymetrix murine expression U430 2.0 chips, we examined the global effects of high glucose (HG) and glucosamine (GlcN) on the transcriptomes of a mouse mesangial cell line (MES-13). Of the 34,000 genes on the chip, ~55-60% genes are detected in MES-13 cells. HG induces the expression of ~369 genes at >2-fold where 263 genes are up and 106 genes down regulated. Similarly, GlcN increases the expression of 120 genes and decreases 94 genes. Seventy-two genes are commonly regulated by HG and GlcN, in which 33 genes are up and 39 genes are down. The differential expressions of several genes found in the microarray are also confirmed by quantitative PCR. Significant pathways co-modulated by HG and GlcN are the thioredoxin system (a 20-fold increase in thioredoxin interacting protein expression), endoplasmic reticulum (ER) stress, extracellular matrix and interferon-inducible genes. Furthermore, HG and GlcN target various intracellular pathways including the mitogen-activated protein kinase, TOLL-like receptor, fructose and mannose metabolism, and the biosynthesis of steroids and N-glycans. We conclude from this microarray data and other experimental results that the HBP mediates several effects of high glucose on mesangial cell metabolism, which promotes ER stress, oxidative stress and the interferon-inducible gene expression to cause cell cycle arrest, ECM gene expression and apoptosis. Cell culture: Stable murine mesangial (MES-13) cells transformed with non-capsid-forming SV-40 virus were obtained from the ATCC, Manassas, VA. These cells display a differentiated mesangial cell phenotype including the typical spindle-like appearance, positive staining for vimentin and desmin, and contraction in response to ANG II and expression of AT1 receptor. The cells were maintained in DMEM and F-12 Nutrient Mixture (Ham's) (4:1 ratio) (GIBCO BRL, Gaithersburg, MD) containing a normal D-glucose concentration of 5.5 mmol/L, 2% FCS, 100 µg/ml streptomycin, 100 U/ml penicillin, and 2 mmol/L glutamine (26). The cells were incubated in a humidified incubator of 5% CO2 at 37 °C and routinely passaged at confluence every 3 days by trypsinization using 10-cm culture dishes. Approximately 50% confluent monolayers were starved in the above medium without FCS for 1 day and then incubated in the starvation medium with the desired concentrations of glucose and GlcN for 48h (LG, 5.5 mM; HG, 25 mM and GlcN, 1.5 mM + LG). Total RNA isolation, cDNA and cRNA synthesis and genechip hybridization: Total RNA was isolated using Trizol reagent (Life Technologies, Inc., Massachusetts). cDNA and cRNA synthesis, genechip hybridization, and scanning of the Affymetrix murine expression U430 2.0 chips (the Affymetrix U430 2.0 chip contains 39,000 transcripts targeted at 34,000 well characterized genes) were performed according to the manufacturer's protocol (Affymetrix, Santa Clara, CA). Briefly, 5 mg of RNA (from LG, HG and GlcN treated MES-13 cells) was converted into double-stranded cDNA by reverse transcription using a cDNA synthesis kit (SuperScript Choice, Life Technologies, Inc., MA) with an oligo(dT) 24 primer containing a T7 RNA polymerase promoter site added 3' of the poly(T) (Genset, La Jolla, CA). After second-strand synthesis, labeled cRNA was generated from the cDNA sample by an in vitro transcription reaction supplemented with biotin-11-CTP and biotin-16-UTP (Enzo, Farmingdale, NY). The labeled-cRNAs were purified in RNeasy spin columns (Qiagen, Valencia, CA). Fifteen mg of each cRNA was fragmented at 94 °C for 35 min in a fragmentation buffer (40 mM Tris acetate, pH 8.1, 100 mM potassium acetate, 30 mM magnesium acetate). These cRNA fragments were then used to prepare 300 ml of hybridization mixture (100 mM MES, 0.1 mg/ml herring sperm DNA (Promega), 1M sodium chloride, 10 mM Tris, pH 7.6, 0.005% Triton X-100). The solution was equilibrated at 45 °C for 5 min and clarified by centrifugation (14,000 x g) at room temperature for 5 min. Aliquots of each sample (10 mg of cRNA in 200 ml of the mater mix) were hybridized to GeneChip Mouse Genome U430 2.0 Array at 45 °C for 16 h in a rotisserie oven set at 60 rpm. After the overnight hybridization, the chips were washed with a non-stringent wash buffer (6x saline/sodium phosphate/EDTA) at 25 °C, followed by a stringent wash buffer (100 mM MES (pH 6.7), 0.1 M NaCl, 0.01% Tween-20) at 50 °C. The GeneChips were then stained with streptavidin-phycoerythrin (Molecular Probe), washed with 6x saline/sodium phosphate/EDTA, incubated with biotinylated anti-streptavidin lgG, followed by a second staining with streptavidin-phycoerythrin. Finally, a third wash with 6x saline/sodium phosphate/EDTA was performed using the GeneChip Fluidics Station 450. The arrays were then scanned in a GeneChip Scanner 3000 (Affymetrix). Each experiment was repeated twice. Microarray Data analysis: The chips were read with Affymetrix GCOS v1.2, and the probe intensity files were modeled with DChip 1.3 (Harvard School of Public Health) in both PM-only and PM-MM modes. Consensus differentially regulated genes were initially derived from repeat experiments on the bases of a 90% CI of greater than 2-fold change in expression and with p<0.05 of error in paired t-test across repeats. This was further validated through modeling of variance between sample groups (one-way ANOVA) conducted in GeneSpring 6 (Silicon Genetix). The consensus gene-set was clustered in DChip and GeneSpring 6.0, with OntoExpress (Wayne State University) to explore ontological associations. Further ontological and pathway analyses were conducted with the GeneGo software and NIH's DAVID bioinformatics programs (http://appls.niaid.nih.gov/david). Keywords = glucose Keywords = glucosamine Keywords = mesangial cells Keywords = hexosamine Keywords: repeat sample
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Project description:Sanchez2017 - Inflammatory responses during acute hyperinsulinemia
This model is described in the article:
The CD4+ T cell regulatory
network mediates inflammatory responses during acute
hyperinsulinemia: a simulation study
Mariana E. Martinez-Sanchez, Marcia
Hiriart, Elena R. Alvarez-Buylla
BMC Systems Biology
Abstract:
Obesity is frequently linked to insulin resistance, high
insulin levels, chronic inflammation, and alterations in the
behaviour of CD4+ T cells. Despite the biomedical importance of
this condition, the system-level mechanisms that alter CD4+ T
cell differentiation and plasticity are not well understood. We
model how hyperinsulinemia alters the dynamics of the CD4+ T
regulatory network, and this, in turn, modulates cell
differentiation and plasticity. Different polarizing
microenvironments are simulated under basal and high levels of
insulin to assess impacts on cell-fate attainment and
robustness in response to transient perturbations. In the
presence of high levels of insulin Th1 and Th17 become more
stable to transient perturbations, and their basin sizes are
augmented, Tr1 cells become less stable or disappear, while
TGF? producing cells remain unaltered. Hence, the model
provides a dynamic system-level framework and explanation to
further understand the documented and apparently paradoxical
role of TGF? in both inflammation and regulation of immune
responses, as well as the emergence of the adipose Treg
phenotype. Furthermore, our simulations provide new predictions
on the impact of the microenvironment in the coexistence of the
different cell types, suggesting that in pro-Th1, pro-Th2 and
pro-Th17 environments effector and regulatory cells can
coexist, but that high levels of insulin severely diminish
regulatory cells, especially in a pro-Th17 environment. This
work provides a first step towards a system-level formal and
dynamic framework to integrate further experimental data in the
study of complex inflammatory diseases.
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MODEL1606020000.
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2017-06-27 | MODEL1606020000 | BioModels
Project description:IgA nephropathy (IgAN) is the most common glomerulonephritis in the world. The disease is characterized by galactose deficient IgA (gd-IgA) in the circulation forming immune complexes. The complexes are deposited in the glomerular mesangium leading to inflammation and loss of renal function, but the pathophysiology of the disease is still not fully understood. Using an integrated global transcriptomic and proteomic profiling approach we investigated the role of the mesangium in the onset and progression of IgAN. Global gene expression was investigated by microarray analysis of the glomerular compartment of renal biopsies from patients with IgAN. The influence of galactose deficient IgA (gd-IgA) on mesangial cells was investigated by proteomic profiling. By utilizing the previous published literature curated glomerular cell type-specific genes, we found that mesangial cells and their positive standard genes play a more dominant role in IgAN comparing to the podocyte standard genes. Additionally, the patient clinical parameters (serum creatinine values and estimated glomerular filtration rate - eGFR) significantly correlate with z-scores derived from expression profile of mesangial cell positive standard genes. 22 common pathways were identified both from in vivo microarray data and in vitro mesangial cell mass spectrometry data and the main part was inflammatory pathways. The correlation between clinical data and mesangial standard genes allows for a better understanding of the onset of IgAN. The genes, proteins and their corresponding pathways identified in this paper give us novel insights into the pathophysiological mechanisms leading to progression of IgAN.
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