Epigenetic Regulatory Mechanism Of Upregulated HDAC3 Inhibits Klotho Transcription To Promote Renal Fibrosis

Background:Renal fibrosis is an indispensable pathohistological feature of renal aging and chronic kidney disease(CKD).It is an over-repairing result of kidney stimulated by continuous harmful substances and characterized by myofibroblast trans-differentiation(MTD)of renal parenchymal cells.Renal cells,including tubular epithelial cells,vascular endothelial cells,fibroblasts and mononuclear phagocytes,lose their own phenotypes and transdifferentiate to myofibroblasts,resulting in excessive production and secretion of extracellular matrix(ECM)proteins which deposited in the renal interstitium,progressively deteriorate renal structure and function.At the molecular level,renal fibrosis is regulated by several profibrotic signaling pathways(TGF?/Smad,Wnt/?-catenin)and antifibrotic factors(Klotho,RASAL1,BMP-7).Recent studies have showed that these fibrosis-related genes are influenced by epigenetic modifications,thereby affecting the process of renal fibrosis.Among these epigenetic modifications,various pan-and class-selective inhibitors of histone deacetylase(HDAC)all exhibit anti-renal fibrosis properties in various animal models,indicating that increased HDAC expressions control the development and progression of renal fibrogenesis.In mammals,HDAC is divided into four major families(Class)according to its structure,subcellular distribution and enzymatic characteristics: Class I(HDAC1,HDAC2,HDAC3 and HDAC8),Class IIa(HDAC4,HDAC5,HDAC7 and HDAC9),Class IIb(HDAC6 and HDAC10),Class III(Sirt1–7)and Class IV(HDAC11).HDACs inhibit gene transcription mainly by deacetylating lysine residues and restoring the positive charges of chromatin histones,leading to a condensed chromatin.Notably,Class I HDACs often form a transcription repressor complex consisting of transcription factors and actively silence gene transcription.In addition,Class I-selective inhibitors can improve the expressions of renal fibrosis-related proteins and alleviate renal fibrosis injury,suggesting that Class I HDACs is the main cause of renal fibrogenesis among all four classes,but the essential HDAC isoforms and the key targets that involved in renal fibrosis are still unclear.Renal fibrosis-related proteins such as profibrotic growth factors,ECM proteins and renal antifibrotic factors can be regulated by HDAC,among them,for example ECM proteins,are upregulated in fibrotic kidney,seemly opposing the inhibitory effects of HDAC.Studies have showed that the level of anti-fibrosis protein Klotho is suppressed in almost all patients and animal models of renal fibrosis-related diseases.Overexpression of endogenous Klotho and exogenous supplementation of Klotho can alleviate renal fibrosis in animal models and improve renal function.What's more,the down-regulation of Klotho in kidney disease can be reversed by HDAC inhibitors,indicating that Klotho is the key effector protein modified by HDAC aberration,so we speculate that the epigenetic loss of Klotho caused by aberrant upregulation of HDAC may be involved in the development and progression of renal fibrosis.Purpose:This study takes unilateral ureteral obstruction(UUO)and aristolochic acid nephropathy(AAN)induced renal fibrosis models as the research objects,to determine the main HDAC subtype that cause renal fibrosis and the effect of its epigenetic regulation of Klotho on renal fibrosis,also to explore whether HDAC inhibitors can alleviate renal fibrosis by reversing the loss of Klotho from the following aspects.Methods:1)By constructing a UUO-induced renal fibrosis model to determine the m RNA and protein changes of Class I HDAC(HDAC1,HDAC2,HDAC3 and HDAC8)and confirm the distribution of highly-expressed HDAC3 through immunohistochemistry and immunofluorescent staining of kidney sections.2)The HDAC3 promoter contains a Smad binding site.The up-regulation mechanism of HDAC3 in renal fibrosis was explored by in vivo administration of TGF? receptor inhibitors to UUO mice,in vitro administration of Smad3 phosphorylation inhibitors to TGF?-stimulated cells and construction of HDAC3 promoter reporter plasmids.3)By constructing HDAC3 gene knockout mice,UUO and AAN-induced renal fibrosis models,and administration of HDAC3 specific inhibitor RGFP966 to observe the changes of kidney damage and Klotho expression in different groups.4)To explore the transcriptional regulation of HDAC3 on Klotho by overexpression of HDAC3 and inhibition of HDAC3 with RGFP966,and also the construction of Klotho promoter reporter plasmid.5)Through protein immunoprecipitation and chromatin immunoprecipitation experiments to determine the regulatory method and modification sites by which HDAC3 inhibiting Klotho in fibrotic kidneys.6)Through the method of knocking down Klotho in vivo by small interfering RNA(si RNA),to compare the difference of RGFP966 in improving renal fibrosis of si RNA-Control mice and si RNA-Klotho mice,and explore whether Klotho is the key mediator protein of HDAC3 promoting renal fibrosis and RGFP966 reversing renal fibrosis.Results:1)HDAC3 knockout mice resist UUO-induced renal fibrosis,indicating that HDAC3 is an important HDAC subtype in promoting renal fibrosis.2)In fibrotic kidney and renal tubular fibrosis models,the aberrant upregulation of HDAC3 can inhibited by TGF? receptor inhibitor and Smad3 phosphorylation inhibitor respectively,therefore,the TGF?/Smad3 signaling pathway is the direct upstream inducer of the abnormally high-expressed HDAC3.3)The down-regulation of Klotho,abnormal expressions of renal fibrosis-related proteins and the renal fibrosis injury in the kidneys of UUO and AAN models can be alleviated by RGFP966,which is the HDAC3 selective inhibitor,indicating that HDAC3 inhibition might exert the anti-renal fibrosis effects via derepressing Klotho.4)HDAC3 is an upstream regulatory protein of Klotho.HDAC3 can form a transcription repressor complex by combining with transcription factors NF-?B and NCo R to act on the NF-?B binding site of Klotho promoter to exert a transcriptional inhibitory effect.5)HDAC3 selective inhibitor RGFP966 has significant anti-renal fibrosis effect on control UUO mice,but after Klotho knockdown,its anti-kidney fibrosis effect disappeared,indicating that Klotho is the key target of both the pro-renal fibrosis of abnormally upregulated HDAC3 and the anti-renal fibrosis of RGFP966.Conclusion:At present,a lot of studies have revealed that the abnormal activation of HDAC is closely related to the occurrence and development of renal fibrosis,but the key pro-fibrotic HDAC subtypes,targeted protein and regulatory mechanisms are not clear.Our study shows that TGF?-incurred HDAC3 aberration and its transcriptional inhibition of Klotho contributes significantly to renal fibrogenesis,HDAC3 inhibitor can reverse Klotho repression and alleviate fibrotic kidney damage in a Klotho-dependent manner.Our findings not only reveal important epigenetic regulatory pathways in renal fibrosis,but also provide more strategies for clinical treatment of renal fibrosis-related diseases.