The Effects Of Silencing Renal P300,Egr-1,ATF3 And Gadd45β/γ Gene On GMC Apoptosis And Proliferation In The Rats With Thy-1 Nephritis

Part Ⅰ The expression of acetyltransferase(p300),Transcription factor(Egr-1,ATF3)and apoptotic related genes(Gadd45α/β/γ)in the renal tissue of rats with Thy-1 nephritisObjective:To investigate the expression of E1 A binding protein p300(p300),early growth response protein 1(Egr-1),activating transcription factor 3(ATF3)and growth arrest and DNA-damage-inducible gene 45(Gadd45)a/p/y in the renal tissue of rats with Thy-1N.And to further examine the interaction between acetyltransferase p300 and Egr-1 or ATF3,and the level of acetylated Egr-1 or ATF3.Methods:Firstly,an animal model of rats Thy-1N was reproduced by tail intravenous injection of anti-thymocyte serum(ATS),and the renal tissue of Thy-1N rats at fixed time(0h,1h,2h,3h,6h and 12h)was collected.Then,the mRNA and protein levels of p300,Egr-1,ATF3 and Gadd45α/β/γ in the renal tissue of Thy-1N rats were measured using quantitative real-time PCR(qRCR)and western blot(WB).Subsequently,the interaction between p300 and Egr-1 or ATF3 including acetylated level of Egr-1 or ATF3 in the renal tissue of rats with Thy-1N at fixed time was detected by immunoprecipitation(IP)and WB assay.Results:The expression levels of p300,Egr-1,ATF3 and Gadd45α/β/γ in the renal tissue of Thy-1N rats were gradually elevated in a time-dependent manner,with the maximum mRNA levels peaked at 1h or 3h(p300 at 3h,others at 1h)after the Thy-1N induction,while the protein levels peaked at 2h or 3h(Egr-1 at 2h,others at 3h),respectively.Moreover,an interaction between p300 and ATF3 but not Egr-1 was readily detected,and reached the maximum at 3h in the renal tissue of rats with Thy-1N.Meanwhile,the level of acetylated ATF3 was also increased in a time-dependent manner,peaked at 3h,but acetylated Egr-1 was not measured.Conclusion:In the renal tissue of Thy-lN rats,the expression of p300,Egr-1,ATF3 and Gadd45α/β/γ was significantly increased,and p300 could interact with ATF3 and acetylate ATF3.Part Ⅱ The effects of silencing renal p300,Egr-1,ATF3 and Gadd45p/y gene on GMC apoptosis and proliferation in the rats with Thy-1 nephritisObjective:To investigate the effects of silencing renal p300,Egr-1,ATF3 and Gadd45β/γ gene on GMC apoptosis and secondary proliferation as well as urinary protein secretion in the rats with Thy-1N.Methods:Firstly,lentiviral vectors of p300,Egr-1,ATF3 and Gadd45β/γ shRNA were packaged respectively(namely,LV-shp300,LV-shEgr-1,LV-shATF3 and LV-shGadd45β/γ),and then the virus titers were verified and the ability to infect rat GMC was determined by cell-infecting experiment.Thereafter,LV-shp300,LV-shEgr-1,LV-shATF3 and LV-shGadd45β/γ were infused into rat kidney through renal artery perfusion.Then,an animal model of rats Thy-1N was reproduced by tail intravenous injection of anti-thymocyte serum(ATS),and the renal tissue of Thy-1N rats at 2h,3h and on day 7(7d)as well as 24h-urine on 7d were collected.The protein levels of p300,Egr-1,ATF3 and Gadd45β/γ in the renal tissue of Thy-IN rats were measured using western blot(WB)to identify the interference effects.Meantime,the expression of apoptosis-related protein Gadd45p/y after silencing renal p300,Egr-1 and ATF3 was detected using WB,and the level of ATF3 acetylation in the renal tissue of rats with Thy-1N after renal p300 knockdown was also measured through IP and WB.In addition,the relevant pathological changes of the renal tissue in Thy-1N rats at 3h and on 7d were examined using pathological technology,and the 24-hour urine protein of Thy-IN rats on 7d was measured as well.Results:The packaged LV-shp300,LV-shEgr-1,LV-shATF3 and LV-shGadd45β/γcould infect rat GMC.The expression of apoptosis-related protein Gadd45p/y in the renal tissue of Thy-1N rats was significantly decreased after silencing renal p300,Egr-1 and ATF3 by infusing the LV-shRNAs into rat kidney through renal artery perfusion.Similarly,the level of ATF3 acetylation in the renal tissue of Thy-1N rats after silencing renal p300 was also markedly down-regulated.Besides,the number of apoptosis cells in glomerulus in the rats with Thy-1N at 3h was obviously reduced.Furthermore,the secondary GMC proliferation and extracellular matrix(ECM)secretion in rat renal tissue as well as the 24-hour urine protein of the rats were also notably diminished on 7d after Thy-1N induction.Conclusion:Silencing renal p300,Egr-1,ATF3 and Gadd45β/γ could obviously inhibit GMC early apoptosis and secondary proliferative lesion as well as urine protein secretion in Thy-lN rats.