Sublyticc5b-induced Gadd45¦Ã Express Thy-1 Nephritis The Gmcs Apoptotic Lesions

Objective: To construct growth arrest and DNA damage-inducible gene 45γ(Gadd45γ) or its short hairpin RNA (shRNA) expression vectors and to assess their function on rat glomerular mesangial cells (GMCs). Methods: The cds area of Gadd45γor four 19~21bp reverse repeated motifs targeting of Gadd45γgene were synthesized and cloned into eukaryotic expression plasmid pcDNA3.1/HA or pGCsi.U6.neo.GFP. After being screened and confirmed, the recombinant plasmids were transfected into GMCs, then the levels of Gadd45γprotein in rat GMCs were measured using Western blot or immunocytochemistry to prove its expression and find out the optimal shRNA. Results: It was verified by partial nucleotide sequencing and restriction endonuclease digestion that the constructed eukaryotic vectors were correct. The results by Western blot or immunocytochemistry showed that the constructed pcDNA3.1/Gadd45γplasmid could express correctly and the optimal shRNA, which could effectively silence the target gene, was Gadd45γshRNA-3.Conclusion: The pcDNA3.1/Gadd45γplasmid and its shRNA were constructed successfully. These data provide the foundation for studying biological functions of Gadd45γgene in the future. Objective: To explore the role of Gadd45γin sublytic C5b-9-induced apoptosis in glomerular mesangial cells (GMCs). Methods: Firstly, the rat GMCs were cultured and divided into different groups. The GMCs apoptosis was evaluated by flow cytometry analysis.The mRNA and protein levels of Gadd45γin GMCs with sublytic C5b-9 stimulation were measured by Real-time PCR and Western blot. Then the effects of Gadd45γoverexpression by pGadd45γplasmid or knockdown by Gadd45γshRNA on the GMCs apoptosis induced by sublytic C5b-9 were determined using the corresponding methods. Results: (1) The mRNA transcripts of Gadd45γfrom GMCs with sublytic C5b-9 attack were found to be significantly up-regulated at 3h compared to control by Real-time PCR, which was similar to changes in previous microarray experiments. And meanwhile, the Gadd45γprotein from GMCs with sublytic C5b-9 treatment was markedly increased at 3h. The relative apoptosis number of GMCs following sublytic C5b-9 attack was markedly increased compared with that of control. (2) In sublytic C5b-9-treated GMCs and pGadd45γ-transfected GMCs, the ratio of apoptotic cells was increased. This was more obvious in pGadd45γ-transfected GMCs plus sublytic C5b-9 attack. In constract, the numbers of apoptotic GMCs, in response to sublytic C5b-9 attack were subdued mostly by Gadd45γshRNA through knock-down of Gadd45γgene. Conclusion: GMCs apoptosis mediated by sublytic C5b-9 in vitro is partially dependent on the up-regulation of Gadd45γexpression. Objective: To explore the effects of silencing renal Gadd45γgene using shRNA on suppression of renal tissue lesions in rats with Thy-1 nephritis (Thy-1N). Methods: Firstly, an animal model of rat Thy-1N was induced, then the expression of Gadd45γin the renal tissue were observed at fixed time. Furthermore, Gadd45γshRNA 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 (anti-Thy-1 Ab). The mRNA and protein levels of Gadd45γin the renal tissue were assessed by Real-time PCR, Western blot and immunofluorescence at 3h and 5d after anti-Thy-1 Ab administration respectively. Meanwhile, the renal histopathologic changes were observed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) and electron microscopy (EM). The total content of 24h urinary protein was also detected at 5d. Results: The expression of Gadd45γin the renal tissue of rats with Thy-1N was increased at 40min and more increased at 3h. And the expression of Gadd45γin the renal tissue transferred with Gadd45γshRNA followed by induction of Thy-1N was significantly reduced compared with Thy-1N model rats. TUNEL analysis and morphological observation under EM showed that the GMCs apoptosis in Gadd45γshRNA + Thy-1N group were markedly reduced compared with Thy-1N model group. The secretion of 24h urinary protein in Gadd45γshRNA + Thy-1N group was also less than that in Thy-1N model group at 5d. Conclusion: Silencing renal Gadd45γgene can diminish the apoptosis of GMCs in rats with Thy-1N.