E2a Expression And Immune-related Study In Anti-glomerular Basement Membrane Glomerulonephritis In Mice

Objective:Crescentic glomerulonephritis (CGN) has a rapid onset and leads to rapidly progressive renal injury and poor prognosis. Anti-glomerular basement membrane (GBM) nephritis (anti-GBM GN)is the most severe pathological type. It is a kind of autoimmune disease, which is mainly mediated by anti-GBM antibody to induce extensive glomerular capillary damage. It is an important part in the immune process of crescent glomerulonephritis progression and outcome for the coordination Thl7 and Treg cells. In the process of lymphocyte maturation, survival, proliferation and differentiation, a helix-loop-helix (HLH) protein E2a is more obvious. Through elucidating the E2a regulatory mechanism of the differentiation of Th17 and Treg in anti-GBM GN, we want to intervene in immune response, reduce the pathological damage, and provide a foundation for anti-GBM GN.Methods:1.Induction of experimental anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). In different time points, mice were sacrificed to collect and detect serum creatinine (Scr), blood urea nitrogen (BUN) content, albumin and albumin-to-creatinine ratio(ACR); renal tissues were collected for the pathological analysis.2. In the groups between control and anti GBM GN, we evaluated Th17 and Treg by FACS. The levels of serum IL-17A and TGF-?1were determined by ELISA. Western blot and RT-PCR were used to assess the expression of protein and mRNA of E2a, Foxp3, Roryt from renal tissues.3. In vitro, the naive CD4+T cells were isolated, then stimulated to Th17 and Tregs differentiation for 6 days. FACS, ELISA, Western blot and RT-PCR were used for related indicators. The naive CD4+T cells were knocked down by E2a siRNAand were then induced to Th17 and Tregs differentiation.Results:1. Anti-GBM GN model was established successfully. Compared with control group mice, the irregular thickening and breakage of GBM, glomerular mesangial cells, matrix proliferation, and crescent formation were observed in anti-GBM GN group. Immunofluorescence showed that IgG was linearly deposited along the GBM. Scr, BUN, and ACR were increased significantly.2. The anti-GBM GN model group of cells, genes and protein expression levels of relevant indicators showed as followed:FACS showed Th17 cells declined after increasing, while Tregs were increased significantly; serum IL-17 and TGF-?1 were shown an inconsistent change. The expression of E2a gene and protein was relevant to the transcription factors related to Th17 cells and Tregs.3.In vitro, experiments showed that E2a was decreased in Tregs inducted, while Th17 cells was increased; and after E2a was knocked down, more Tregs were detected.Conclusion:E2a aggravates anti-GBM glomerulonephritislesions by promoting proinflammatory Th17 differentiation.