Gene Expression Profiles In Anti-Thy1 Rat Model

ObjectiveMesangial proliferative glomerulonephritis (MsPGN) is one of the kidney diseases with highest incidence in China, with main pathological changes including mesangial cells (MC) proliferation and mesangial matrix increase, which may eventually lead to the irreversible glomerulosclerosis. To inhibit the mesangial proliferation is an important treatment strategy of the treatment of glomerular diseases. Previous researches focused on searching for genes promoting mesangial cell proliferation for prevention and treatment of the disease. However, the occurrence and development of the disease process will involve not only a large number of genes upregulated, but also a large number of genes downregulated. We hypothesized that the progression of pathological mesangial proliferation may be a net result of down-regulation of the proliferation-inhibiting genes. Thus the main purpose of out study is to screen out proliferation-inhibiting genes in the anti-Thy1.1 animal model of mesangial proliferation, and to verify their mesangial proliferation-inhibiting effects.MethodsOX-7 cells were used to produce anti-Thy1.1 antibodies which, injected into SD rats through tail vein to establish anti-Thy1.1 MsPGN animal models. At different time points (day 0, 3, 5, 7, 10,and 14), the following was processed, including detection of renal function and urinary protein, renal glomerular tissue isolation, and extraction of total RNA. Rat U2302.0 chip scanning, and SAM andGominer softwares were used for analyzing the data, focusing on the proliferation period (day 5 to day 7) which was associated with expression of proliferation-relating genes. Fluorescence quantitative PCR (Taqman probe) verified the the proliferation-related differentially expressed genes (Ratio≥1.50 or≤0.67, and Score≥2 or≤-2, q-value <0.05, FDR<5%). To screen out candidate genes which may inhibit the proliferation of mesangial cells. At the same time, cultured rat mesangial cells (RMC) were also analyzed with siRNA of KLF15 (Kruppel-Like factor 15). Groups dividing: 1) serum-free group; 2) normal control group; 3) siCon group; 4) siKLF group. 24h or 48h later,MTT test and flow cytometry were used for analysis of cell proliferation and cell cycle changes, and Western blot and indirect immunofluorescence for detection of cell cycle protein p21,CDK2,cyclinD1 expression changes.ResultsAnti-Thy1 antibody was successfully purified with high specificity, and the anti-Thy1 nephritis rat model was established. Compared with the control group, the 24h urinary protein was significantly higher in the model group rats on Day 5 and Day 7,(p<0.05), which began to decrease at Day 10. The gene chip detected 9033 known genes among which 1001 genes displayed differential expression at least one time point compared with the control group. Among the genes displaying differential expression, 619 genes have known function including catalysis,molecular transduction, transport, transcriptional regulation, and regulation, etc.; Further analysis with GOminer software showed genes with differences during the proliferative phase and recovery phase, which were 19 genes at Day 5, 21 genes at Day 7, 21 genes at Day 10,21 genes at Day 14. KLF15 and MXI-1,which were upregulated at Day 5 and Day 7, may have potential proliferation-inhibiting functions. Genes that were upregulated at Day 5 and Day 7 with potential proliferation-inhibiting functions were TIMP-1, Ccl-2, and S100A4, etc.; Fluorescence quantitative PCR verified the above t trends and changes with the Pearson correlation coefficient> 0.90, p<0.05. The synthesized siRNA of KLF-15 gene inhibited the mRNA and protein expression levels (p<0.05). MTT test results indicate that cells of siKLF group increased, and flow cytometry analysis confirmed increase of cells in S phase (p<0.05). Western blot and indirect immunofluorescence test results showed increase expression of CDK2, cyclinD1 and of p21 in siKLF group (p<0.05).Conclusions1. In this study, anti- Thy1 antibody was successfull obtained from OX-7 hybridoma cell lines with high purity and specificity to establish the the Thy1 rat model of experimental mesangial proliferative glomerulonephritis.2. In the model, based on the use of DNA microarray technology and bioinformatics analysis, gene expression profile with differences during the disease progression was found, and selection of proliferation-related genes was made;3. Inhibition of KLF15 expression changed the expression of CDK2,cyclinD1 and p21,and increased mesangial cells in S phase indicating that KLF15 may play an important role in the process of mesangial cell proliferation, which may have provided a new way for controlling the proliferation of mesangial cells.