| Part I The renal lesions of the rats with Thy-1 nephritis and effects of sublytic C5b-9 on GMCs proliferation, ECM secretion and synthesis of TSP-1 and TGF-?1Objective: To investigate the lesions of glomerular mesangial cells (GMCs) in Thy-1 nephritis (Thy-1N) rats and the effects of sublytic C5b-9 complexes in vitro on the proliferation of GMCs, secretion of extracelluar matrix (ECM) and production of thrombospondin-1 (TSP-1) and transforming growth factor-?1 (TGF-?1). Methods: Firstly, an animal model of rat Thy-1N was induced, then the expression of cyclin D2, proliferative cell nuclear antigen (PCNA), fibronectin (FN), collagen IV and the two cytokines (TSP-1 and TGF-?1) in the renal tissue were observed at fixed time. Meanwhile, the proliferation of glomerular cells in rats was assessed by BrdU incorporation, light microscopy (LM) and electron microscopy (EM). Furthermore, the rat GMCs were cultured and divided into different groups. The mRNA and protein levels of cyclin D2, PCNA, FN, collagen IV, TSP-1 and TGF-?1 in GMCs with sublytic C5b-9 stimulation were measured by real-time PCR and Western blot. In addition, the contents of total and active TGF-?1 in the cultured supernatant were also detected by ELISA. Then the GMCs proliferation was evaluated by 3H-thymidine incorporation (3H-TdR) analysis. Results: The expression of cyclin D2 and PCNA in the renal tissue and the number of glomerular BrdU positive cells were increased at 18 h in Thy-1N rats. But the expression of FN, collagen IV and production of TSP-1 and TGF-?1 in the renal tissue were increased on days 3 (3d), and more increased on days 7 (7d). Moreover, the number of GMCs and the secretion of ECM were increased on 3d and became more obvious on 7d under LM and EM. The mRNA and protein levels of cyclin D2, PCNA, FN and collagen IV as well as TSP-1 and TGF-?1 including the activation of TGF-?1 and proliferative response of GMCs induced by sublytic C5b-9 were significantly increased at 18h. Conclusion: Glomerular lesions in rats with Thy-1N include early and secondary proliferation, while sublytic C5b-9 complexes in vitro can promote GMCs proliferation, ECM secretion, as well as TSP-1 and TGF-?1 production. Part?The effects of silencing TSP-1 gene and inhibiting TGF-?1 activation on GMCs proliferation and ECM secretion induced by sublytic C5b-9Objective: To explore the effects of silencing TSP-1 gene on sublytic C5b-9-induced GMCs proliferation and ECM secretion as well as the relationship between TSP-1 and TGF-?1. Methods: Four pairs of small interference RNAs (siRNA) were designed against different regions of TSP-1 gene, then the most optimized TSP-1 siRNA was chosen to silence TSP-1 gene in the experiment. The cultured rat GMCs were transfected with TSP-1 siRNA, and then stimulated with sublytic C5b-9 complexes. The mRNA and protein levels of TSP-1, TGF-?1, cyclin D2, PCNA, FN and collagen IV in GMCs and the contents of total and active TGF-?1 in cultured supernatant were assessed by real-time PCR and Western blot or ELISA respectively. At the same time, GMCs proliferation was also evaluated by 3H-TdR analysis. Moreover, the synthetic blocking peptide of TSP-1 (GGWSHW) and TGF-?1 neutralizing antibody were utilized to explore the role of TSP-1 and TGF-?1 in GMCs proliferation and ECM secretion induced by sublytic C5b-9, as well as the relationship between TSP-1 and TGF-?1. Results: Transfection of TSP-1 siRNA into rat GMCs could markedly decrease not only the expression of TSP-1 gene but also the proliferation of GMCs and the production of FN and collagen IV. The blocking peptides of TSP-1 (GGWSHW) could significantly reduce TGF-?1 activation and inhibit the proliferation of GMCs as well as the production of FN and collagen IV. In addition, TGF-?1 neutralizing antibody could also obviously inhibit the proliferation of GMCs and the expression of FN and collagen IV mediated by sublytic C5b-9.Conclusion: Silencing TSP-1 gene in the GMCs with TSP-1 siRNA can significantly inhibit sublytic C5b-9-induced GMCs proliferation and ECM secretion as well as TSP-1 and TGF-?1 production. The GMCs proliferation and ECM secretion stimulated by sublytic C5b-9 are possibly mediated in part by TSP-1 and TGF-?1. Part III The inhibitory effects of silencing TSP-1 gene on lesions and proteinuria of the rat with Thy-1 nephritisObjective: To explore the effects of silencing renal TSP-1 gene using siRNA on suppression of renal tissue lesions and urinary protein secretion in rats with Thy-1 nephritis (Thy-1N). Methods: TSP-1 siRNA expressing plasmids were transferred into rat kidneys by renal artery perfusion followed by electroporation, and then Thy-1N was reproduced by injection of anti-thymocyte serum (ATS). The mRNA and protein levels of TSP-1, TGF-?1, cyclin D2, PCNA, FN and collagen IV in the renal tissue were assessed by real-time PCR, Western blot and immunohistochemistry at 18h, 3d and 7d after ATS administration respectively. The contents of total and active TGF-?1 in the urine of rats were examined by ELISA. Meanwhile, the proliferation of glomerular cells was assessed by BrdU incorporation. In addition, the renal histopathologic changes were observed by light microscopy (LM) and electron microscopy (EM). The total content of 24h urinary protein was also detected. Results: The expression of TSP-1 in the renal tissue transferred with TSP-1 siRNA followed by induction of Thy-1N was significantly reduced compared with Thy-1N model rats. The treatment of TSP-1 siRNA could also reduce the content of active TGF-?1 in the urine of rats with Thy-1N. In addition, TSP-1 siRNA could markedly reduce the expression of cyclin D2, PCNA, FN and collagen IV in the renal tissue of Thy-1N rats. BrdU incorporation analysis and morphological observation under LM and EM showed that the GMCs proliferation and ECM accumulation in TSP-1 siRNA + Thy-1N group were markedly reduced compared with Thy-1N model group. The secretion of 24h urinary protein in TSP-1 siRNA + Thy-1N group was also less than that in Thy-1N model group. Conclusion: Silencing renal TSP-1 gene can not only reduce the activation of TGF-?1 but also diminish the proliferation of GMCs, the production of ECM (FN and collagen IV) and the secretion of proteinuria in rats with Thy-1N. |
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