The Role Of Histone Deacetylase 9 In Hyperhomocysteinemia-induced Renal Injury

ObjectiveThough hyperhomocysteinemia(hHcys)is an independent and critical pathogenic risk factor in the progression of end stage renal disease(ESRD),the mechanisms by which hHcys induces renal injury are still not very clear.In recent years,epigenetics research in kidney disease gets more and more attention,for The control of acetylation or deacetylation of lysine residues of histone by histone acetyltransferase(HAT)and histone deacetylase(HDAC)is one of the important content.The HDAC is divided into four classes of 18 subtypes:the Class ?,which is Rpd3 like,includes HDAC1,HDAC2 and HDAC8.Class ? is Hdal like.Class Ila includes HDAC4,HDAC5,HDAC7,and HDAC9.Class ?b includes HDAC6 and HDAC 10.Class ? is Ser2 like,including sirtl-7.Class IV includes HDAC11.Class ?,?,and IV are Zn2+ dependent.And Class III is NAD+ dependent.An increasing amount of evidence also suggests that HDAC can regulate deacetylation of non-histones.Although,broad-spectrum HDAC inhibitors have shown good application prospects in tumor therapy.But the shortcoming is also very obvious,such as lack of specificity of HDAC subtypes,which leads to serious side effects.Also,studies have found that HDAC involved in the process of renal fibrosis.Although mechanism by which HDAC mediates renal fibrosis is not very clear.And it may be related to regulating the expression of inflammatory cytokines,fibrosis factors and activating signaling pathways mediated renal fibrosis.Therefore,in the case of hHcys,the role and mechanism of subtypes of HDAC have to be clarified and discussed.Hippo signaling pathway plays an important role in renal injury including renal fibrosis.The present study was desigened to determine the role of individual HDACs in hHcys-induced renal injury and our results showed that HDAC9 contributes to hHcys-induced renal fibrosis and podocyte injury,which is associated with Hippo signaling pathway.Methods and Results1 The expression of HDAC9 in the kidney of animal with hHcysWild-type C57BL/6J male mice(10 w)were operated for uninephrectomy.After a 1-week recovery,they were divided into two groups randomly.One group of mice were fed on a normal diet,and another group of mice were fed on folate-free(FF)diet.Both are maintained for 8 weeks.By high performance liquid chromatography(HPLC)analysis we measured plasma total Hcys(tHcys)and found that plasma tHcys levels of FF diet group markedly increased compared with normal diet group.Significantly increase of urine albumin-to-creatinine ratio was observed in FF diet group mice which was tested by inspection centre.Glomerular morphology changes were observed By periodic acid-schiff stain(PAS)staining and observation by electron microscope,we found that in hHcys mice,varying degrees of enhanced mesangial expansion,glomerular basement membrane thickening and podocyte foot processes fusion appeared,indicating glomerular injury.By real-time RT PCR analysis,it was found that the expression of HDAC9 was prominently induced among Zn2+-dependent HDACs in the kidney from mice and rats with hHcys,which was further confirmed by Western blot(WB),and immunohistochemistry(IHC)analyses.Furthermore,immunofluorescence staining confirmed HDAC9 was expressed and indueced in in podocytes and renal epithelial cells under hHcys condition as well as in vitro studies showing that L-Hcys significantly induced the HDAC9 expression in human podocytes(HPC)and human renal tubular epithelial cells(HK-2).2 The role of HDAC9 in hyperhomocysteinemia-induced renal injuryInjection of the shRNA-HDAC9 lentivirus by kidney local injections was used to gene silencing of HDAC9.We found that gene silencing of HDAC9 ameliorated the hHcys-induced renal fibrosis by Masson staining.In addition,HDAC9-silencing ameliorated the hyperhomocysteinemia-induced renal fibrosis.PAS staining showed that the thickening of GBM and exceeded mesangial proliferation could be ameliorated by gene silencing of HDAC9.At molecular levels,the down-regulation and integrity impairment of nephrin could be reversed by knockdown of HDAC9,and the hHcys-induced fibronectin expression,a profibrotic factor,was inhibited by gene silencing of HDAC9.3 HDAC9 mediated Hippo signaling pathwayIHC analysis showed that FF diet induced up-regulation of YAP in the kidney.In vitro,treatment of HK-2 with L-Hcys induced the increase of YAP.Moreover silence of HDAC9 inhibited the expression of YAP responded to L-Hcys to some extent and its downstream effector Smad2/3.ConclusionsOur results for the first time found that HDAC9 was prominently up-regulated among Zn2+-dependent HDAC members in the kidney from mice or rats with hHcys and gene silencing of HDAC9 ameliorated renal fibrosis and glomerular injury.We further demonstrated that Hippo signaling pathway is one of potential signaling pathways that links HDAC9 to hHcys-induced renal injury.Targeting HDAC9 may have potency for the treatment of patient with hHcys and the design of HDAC inhibitors.