| Objective: Progressive renal tubulointerstitial fibrosis is a main pathological basis and final result of various chronic kidney diseases to end-stage renal failure.It has an adverse effect on the quality of life and prognosis of patients with nephropathy.Renal tubular epithelial cells transform into interstitial fibroblasts under the activation of persistent injury factors and participate the pathological process of renal fibrosis.Protein acetylation is a dynamic process controlled by histone acetyltransferase and deacetylase,which play an important role in the progression of many diseases.In our previous studies,we found that the overall acetylation level of renal tissues in the mouse model of renal interstitial fibrosis with unilateral ureteral obstruction(UUO)was significantly increased.Pre-experimental results showed that the expression and activity of histone acetyltransferase P300 was significantly increased in UUO mice and TGF-β1 cultured HK-2 cells.Therefore,we speculate that P300/CBP-targeted inhibition of acetylation modification may improve renal interstitial fibrosis.A-485 is a newly discovered small molecule inhibitor with high selectivity and efficiency.It inhibits the catalytic activity of acetyltransferase P300 by competing with acetyl-Co A,thereby regulating the acetylation modification.At present,A-485 has been proved to inhibit tumor growth,lipogenesis and inflammatory reaction,but whether A-485 plays a regulatory role in renal interstitial fibrosis not clear.In this study,we will first evaluate the effect of A-485 on epithelial-mesenchymal transition of renal tubular cells and renal interstitial fibrosis by using human renal tubular epithelial cell line HK-2 cells and unilateral ureteral obstruction mice.At the same time,point mutation and gene transfection was adopted to further explore the possible mechanisms of A-485 on renal fibrosis in chronic kidney disease.This study will provide experimental basis for the prevention and treatment of CKD.Methods: 1.Animal experiments: Male wild-type mice(C57BL/6)aged 6~8 weeks were randomly divided into four groups: Sham operation group(Sham),unilateral ureteral obstruction group(UUO),sham operation +A-485 group(Sham+A-485)and unilateral ureteral obstruction +A-485 group(UUO+A-485),with eight mice in each group.The UUO model was used to establish the chronic renal interstitial fibrosis model by free and ligating the unilateral ureters,while the Sham group did the same without ligating the ureters.After the successful establishment of the model,A-485 small molecule inhibitor(10 mg/kg/day)was intraperitoneally injected once every other day for in vivo intervention,and mice in the control group were intraperitoneally injected with the same amount of normal saline.The renal tissues were collected on the 3rd,7th and 14 th days after modeling,respectively.The morphological changes of kidney were observed by Masson staining and PAS staining.The expressions and localization of FN,Col-Ⅰ,α-SMA,E-cad,ac-STAT3(Lys685)and p-STAT3(Tyr705)were detected by western blot and immunohistochemistry.P300,CBP,STAT3 and p-STAT3(Ser727)protein expressions were detected by western blot quantitatively.P300 activity were detected by commercial kits.2.Cell experiment: The human renal tubular cell lines cells(HK-2)were used as object to detect the effects of A-485 on epithelial-mesenchymal transition,P300 enzyme activity,and STAT3 phosphorylation and acetylation modification under TGF-β1 stimulation.(1)Detection the condition of epithelial-mesenchymal transition in TGF-β1 stimulated HK-2 cells: HK-2 cells were randomly divided into control group(control)and TGF-β1 stimulated groups with different concentrations(TGF-β1).The cells were stimulated at 0 ng/ml,1 ng/ml,5 ng/ml,10 ng/ml,and 20 ng/ml respectively.Then the cells were harvested at 24,48 and 72 h after stimulation or not.Cell viability were detected by commercial CCK-8 kit,the morphological changes were observed and recorded by a light microscope.(2)To test the impact of A-485 on epithelial-mesenchymal transition in TGF-β1 stimulated HK-2 cells: Cells were divided into five groups: control group(control),10 ng/ml TGF-β1 group(TGF-β1),10 ng/ml TGF-β1+2.2 μM A-485 group(TGF-β1+2.2 μM A-485),10 ng/ml TGF-β1+6.6 μM A-485 group(TGF-β1+6.6 μM A-485)、10 ng/ml TGF-β1+13.2 μM A-485 group(TGF-β1+13.2 μM A-485)、 10 ng/ml TGF-β1+20 μM A-485 group(TGF-β1+20 μM A-485).The cells were collected after 48 h of stimulation in groups respectively.Cell viability were detected by CCK-8 kit.And cells morphological changes were observed and recorded by light microscope.P300 enzyme activity were detected by commercial kits.The expression levels of Col-Ⅰ,FN,α-SMA,E-cad,P300,CBP,STAT3,ac-STAT3(Lys685),p-STAT3(Tyr705)and p-STAT3(Ser727)proteins in HK-2 cells were observed by western blot.Expression and localization of α-SMA,E-cad,ac-STAT3(Lys685),and p-STAT3(Tyr705)were detected by cellular immunofluorescence.(3)In order to explore the interaction between ac-STAT3(Lys685)and p-STAT3(Tyr705),HK-2 cells was stimulated by 10 ng/ml TGF-β1 for 0~48 h and the protein expressions of p-STAT3(Tyr705),ac-STAT3(Lys685)and total STAT3 was detected.Furthermore,STAT3-sh RNA was then used to construct a STAT3 stable knockout cell line.After screening with G418,the plasmids of STAT3-WT,STAT3-K685 R,STAT3-K685 Q,STAT3-Y705 F and STAT3-Y705 D were transfected respectively,and the cells were collected after 48 h.The expression changes of total STAT3,ac-STAT3(Lys685)and p-STAT3(Tyr705)proteins were detected by western blot.The expression of ac-STAT3(Lys685),p-STAT3(Tyr705)and α-SMA was detected by cellular immunofluorescence staining.Results: 1.Effects of A-485 intervention on renal interstitial fibrosis in UUO mice;(1)Compared with Sham group,the volume of the kidney on the obstructive side(left side)in UUO group gradually increased with the prolongation of obstruction days,with obvious cystic sensation,enlarged cross-sectional anterior-posterior diameter of renal pelvis,renal papilla becomes even and disappears,and the renal cortex and medulla become thinner.The renal pelvis expands in the 3rd day and is most obvious in 14 th days.The kidney weight ratios of UUO mice(left kidney weight compared to right kidney weight of the same mouse in the UUO group)increased in a time-dependent manner(P<0.05 or P<0.01).(2)The results of PAS and Masson staining showed that,compared with Sham group,the renal tubular lumen of the UUO group was dilated,the epithelial cells get shorter,and a small part of collagen was secreted and deposited under mesenchyme the third day.With the prolongation of obstruction days,the renal tubules gradually atrophy and epithelial cells gradually necrotize and fall off,and interstitial fibrosis of different degrees occurs at the same time.A-485 intervention significantly improved the morphological changes of renal tissue in UUO mice(P<0.05 or P<0.01).(3)Western blot results indicated that expression levels of FN,Col-Ⅰ and α-SMA in the kidney tissue of UUO mice increased in a time-dependent manner,the expression of E-cad declined in a time-dependent manner when compared Sham group(P<0.01).A-485 intervention significantly inhibited the expressions of FN,Col-Ⅰ and α-SMA,and up-regulated the expression of E-cad.Immunohistochemical staining results were consistent with western blot results(P<0.05 or P<0.01).2.Effects of A-485 intervention on epithelial-mesenchymal transition of HK-2 cells induced by TGF-β1(1)The results of light microscope and CCK-8 showed that the optimal stimulation concentration and time of TGF-β1 was 10 ng/ml for 48 h.(2)A-485 intervention with concentrations of 2.2,6.6 and 13.2 μM reversed the undesired morphological changes in HK-2 cells stimulated with TGF-β1 at 10 ng/ml,and significantly inhibited the high expressions of FN,Col-Ⅰ and α-SMA as well as the low expression of E-Cad(P<0.05 or P<0.01).3.Inhibition of P300 enzyme activity by A-485 intervention;(1)Western blot indicated that the protein expressions of P300 and CBP in the renal of UUO mice increased in a time-dependent manner as compared with that in the Sham group(P<0.01),while A-485 intervention had no effect on the high expressions of P300 and CBP.(2)TGF-β1 at 10 ng/ml significantly up-regulated the protein expressions of P300 and CBP in HK-2 cells(P<0.01);However,A-485 intervention had no effect on the high expression of P300 and CBP proteins.(3)P300 enzyme activity in renal tissue of UUO 14 d mice was increased,while A-485 intervention significantly inhibited the P300 enzyme activity in UUO group.HK-2 cells stimulated by TGF-β1 in vitro produced the same results(P<0.05 or P<0.01).4.A-485 intervention inhibits the expression of p-STAT3(Tyr705)and ac-STAT3(Lys685);(1)Compared with Sham group,the protein expressions of p-STAT3(Tyr705),ac-STAT3(Lys685),p-STAT3(Ser727)and total STAT3 in the tissue of UUO mice increased in a time-dependent manner,and the increase was most significant on the 14 th day,accompanied by significant nuclear translocation(P<0.01).A-485 intervention significantly inhibited high expression of ac-STAT3(Lys685)and p-STAT3(Tyr705)and STAT3 nuclear translocation without significant inhibition of total STAT3 and p-STAT3(Ser727)expression.(2)Immunofluorescence and western blot showed that TGF-β1 up-regulated the protein expressions of STAT3,ac-STAT3(Lys685),p-STAT3(Tyr705)and p-STAT3(Ser727)in HK-2 cells along with the nuclear translocation of STAT3.A-485 intervention significantly decreased the expression of ac-STAT3(Lys685)and p-STAT3(Tyr705)and inhibited nuclear translocation of STAT3(P<0.01),while no obvious decrease on the expression of total STAT3 and p-STAT3(Ser727).5.Regulatory relationship of p-STAT3(Tyr705)and ac-STAT3(Lys685)in HK-2 cells under TGF-β1 stimulation.(1)Western blot results indicated that TGF-β1 at 10 ng/ml stimulated HK-2 cells,and the protein expression of p-STAT3(Tyr705)and ac-STAT3(Lys685)increased in a time-dependent manner.Ac-STAT3(Lys685)began to increase 1 h after TGF-β1 stimulation,while p-STAT3(Tyr705)began to increase 6 h after TGF-β1 stimulation,suggesting that TGF-β1-induced acetylation modification of STAT3 in HK-2 cells occurred earlier than phosphorylation modification.(2)Western blot results indicated that the STAT3 K685 R mutation down-regulated the expression levels of p-STAT3(Tyr705)and ac-STAT3(Lys685)proteins and inhibited the nuclear translocation of STAT3.P-STAT3(Tyr705)was promoted by the STAT3 K685 Q mutation.At the same time,immunofluorescence results showed that either STAT3 knockout or STAT3 K685 R mutation inhibited the expression of α-SMA in HK-2 cells induced by TGF-β1,with statistical significance(P<0.01).(3)Western blot results indicated that p-STAT3(Tyr705)protein expression was declined after STAT3 Y705 F site mutation(P<0.01),however,the expression of ac-STAT3(Lys685)was not significantly inhibited.Similarly,the STAT3 Y705 D mutation did not affect protein expression of ac-STAT3(Lys685).Conclusions: P300/CBP inhibitor,A-485,can relieve renal interstitial fibrosis by inhibiting P300-mediated STAT3 acetylation(Lys685),leading to expression and nuclear translocation of p-STAT3(Tyr705)was suppressed in chronic kidney disease.The result suggests that the specific inhibition of CBP/P300 HAT will provide a novel therapeutic approach for chronic fibrosis diseases. |
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