The Gene And Protein Expression Profiles In Anti-Thy-1 Nephritis

Background and purpose:Mesangial proliferation is a common pathological phenomenon in chronic glomerular nephritis, which lacks effective therapy due to unclear pathogenesis. Anti-Thy-1 nephritis, as a traditional model of mesangial proliferation and matrix expansion, is wildly used in mesangial proliferation research. In this study we screen the genes and proteins related with mesangial proliferation by analyzing the gene and protein expression profiles to reveal the molecular pathogenesis of mesangial cells proliferation.Materials and methods:1.200g male Wistar rats were randomly allocated to either control or model group. Model group were induced with intravenous injection of anti-Thy-1 antibody at a dose of 2.5mg/kg. Control group were injected with same volume of isotonic saline. Anti-Thy-1 treated animals were sacrificed at 3d,5d, 7d, 10d and 14d. Control group were sacrificed at Od.2. Cortical regions of the kidney from each group were collected for RNA isolation. Affymetrix U230 2.0 chips (31000 probe sets,28000 well-substantiated rat genes.) were applied for microarray analysis. SAM (Significant Analysis of Microarray) software was performed to analyze significantly changed genes in every timepoint during anti-Thy-1 nephrites (vs. 0d); Gominer and MAS system (KEGG database) were performed to analyze GO (gene ontology) and pathway (KEGG) separately; Hierarchical clustering analysis (Spearman rank correlation) was performed by cluster 3.0; Quantitative PCR was performed for detecting the mRNA expression of TIMP-1, CCL2, S100A4, KLF15, MXI.3. Proteins labeled by Cy2, Cy3, or Cy5 were separated in the pH3-11 range 24cm Non-linear strips in the first dimension and 12.5% SDS-polyacrylamide gel in the second dimension. Decyder software was performed for data processing and statistic analysis; The protein spots from preparation gel were identified by MALDI-TOF MS (Matrix-assisted laser desorption inoization-time of flight mass spectrometry) or LTQ (Linear trap quadrupole). Hierarchical clustering analysis (Spearman rank correlation) was performed by cluster 3.0. Western blot was performed to detect the expression of FHL2 and NIT2 protein.Results:1. (1)The histological injury in anti-thyl nephritis began with an initial complement-dependent mesangiolysis at 3d, followed by mesangial cell proliferation at 5d. Mesangial cell proliferation peaked at 7d with mesangial matrix expansion and began to recover at 10d. Mesangial cell proliferation was decreased significantly at 14d. (2)The level of 24hr urine protein was increased significantly at 3d,5d,7d, 10d and peaked at 3d during anti-Thy-1 nephritis. Scr and BUN was increased significantly at 5d,7d and peaked at 7d during anti-Thy-1 nephritis.2. (1) 30,952,494,154,861 differential genes were identified at 3d,5d,7d, 10d, 14d respectively. (2)Differential genes in anti-Thy-1 nephritis were divided into five clusters (cluster 1:gene expression was upregulated at 5d and/or 7d, recovered at 10d and/or 14d; cluster 2:gene expression was downregulated at 5d and/or 7d, and recovered at 10d and/or 14d; cluster 3:gene expression was downregulated at 5d and/or 7d, upregulated at 10d, then downregulated at 14d; cluster 4:gene expression began to decrease at 5d and/or 7d, and reached the lowest value at 10d and/or 14d; cluster 5 gene expression was upregulated at 5d and/or 7d and peaked at 10d and/or 14d). There exited genes related with cell proliferation or apoptosis in every cluster. (3) Differential genes in anti-Thy-1 model were mainly involved in biological process such as metabolic process, biological regulation, signaling, response to stimulus and molecular function such as catalytic activity, binding. (4) Transcription genes were mainly assigned to cluster 1 and cluster 3. Cluster 1, in which gene expression was upregulated at 5d and/or 7d, and recovered at 10d and/or 14d, included genes inducing apoptosis and genes inhibiting cell proliferation. Clulster3, in which gene expression began to decrease at 5d and/or 7d, and reached the lowest value at 10d and/or 14d, included genes inducing apoptosis. (5) 328,24,9,22 pathways were affected at 3d,5d,7d, 10d,14d, respectively. Cell proliferation related pathways including focal adhesion, tight junction, gap junction, regulation of actin cytoskeleton, p53 signaling pathway, cell cycle, MAPK signaling pathway et al were activated in the phase of mesangial proliferation. Focal adhesion, gap junction and cell cycle were inhibited in the recovery phase of mesangial proliferation. (6) The mRNA expression trends of TIMP-1, CCL2, S100A4, MXI1, KLF15 detected by QPCR were consistent with the data in gene chip.3. (1) 40 differential proteins identified by Maldi-Tof-MS or LTQ were involved in biological process such as metabolic process, biological regulation, response to stimulus, signaling and molecular function such as catalytic activity, binding. (2) Differential proteins were assigned to two clusters:one cluster, in which genes expression was downregulated at lOd and/or 14d, included both genes promoting cell proliferation and genes inhibiting proliferation; The another one cluster, in which genes expression was upregulated at 10d and/or 14d, included genes inhibiting cell proliferation. (3) Protein expression trends of FHL2, NIT2 detected by western blot were consistent with the data in DIGE.Conclusion:Genes related with cell proliferation and apoptosis (including interrelated transcription genes) are regulated during the whole process of anti-Thy-1 model. Differential genes and proteins are mainly involved in metabolic process, biological regulation, signaling, response to stimulus, catalytic activity and binding in anti-Thy-1 nephritis. Pathway networks played a crucial role in regulating mesangial cell proliferation. Inhibiting the activation of pathways that can promote cell proliferation may suppress the mesangial cell proliferation, which may be the new therapy strategy for mesangial proliferation nephritis.