| Hyperhomocysteinemia is a key pathogenic factor in inducing the occurrence and development of end-stage renal disease,which can cause glomerular podocyte damage and thus impaired renal function.When the podocytes are damaged,the foot processes will fuse and eliminate,which will further lead to the apoptosis and shedding of the podocytes,resulting in proteinuria.However,the mechanism of hHcys-induced podocyte injury has not yet been fully clarified,so in-depth exploration is still needed.In recent years,people have paid more and more attention to the role of epigenetics in a variety of diseases,and its mechanisms mainly involve DNA methylation,histone modification,and chromatin remodeling.There are four types of histone deacetylases,of which,type Ⅰ,Ⅱ and Ⅳ are Zn2+-dependent enzyme activities,and type III is nicotinamide adenine nucleotide(NAD+)-dependent enzyme activity.The previous study of our research group found that Zn2+-dependent histone deacetylase HDAC4 can specifically promote the damage of podocytes in diabetic nephropathy.However,the role of HD AC family in renal podocyte injury caused by hyperhomocysteinemia has not been reported,so it is of important research significance to clarify its role and related mechanisms.The previous experimental results of the research group showed that the expression level of HDAC9 was specifically increased in hHcys model mice,suggesting that HDAC9 may be involved in the occurrence and development of hyperhomocysteinemia-induced kidney injury.Therefore,this topic will focus on exploring the role and potential mechanism of HDAC9 in hyperhomocysteinemia-induced kidney injury.ObjectiveThis topic will establish a model of hyperhomocysteinemia in vivo and in vitro,to study the role and mechanism of HDAC9 in hyperhomocysteinemia-induced podocyte injury.1.Clarify the role of HDAC9 in hyperhomocysteinemia-induced podocyte injury.2.Explore the mechanism of HDAC9 in hyperhomocysteinemia-induced podocyte injury.MethodsPart I:The role of HDAC9 in hyperhomocysteinemia-induced podocyte injury1.1 The effect of HDAC9 on proteinuria in mice with hyperhomocysteinemia Urine of mice was taken for analysis,and the effect of deletion or inhibition of HDAC9 gene in the formation of hyperhomocysteinemia induced by folate-free diet on proteinuria in mice was determined by ELISA.1.2 The effect of HDAC9 on podocyte injury in mice with hyperhomocysteinemia Paraffin sections of mouse kidneys were stained by periodic acid Schiff’s staining to observe the glomerular differences between HDAC9 gene deletion or suppression and wild-type mice.Next,transmission electron microscopy was used to observe the glomerular basement membrane thickness,the number of foot processes,and the fusion of the foot processes between HDAC9 gene deletion or suppression and wild-type mice.At the same time,the immunofluorescence staining experiment was used to study the expression of Nephrin in podocyte split membrane proteins between HDAC9 gene deletion or suppression and wild-type mice.1.3 In vitro,the effect of HDAC9 gene silencing on podocyte injury stimulated by L-Hcys was detected.Western blot was used to detect the the level of Nephrin,Podocin,Desmin,Vimentin,Chop and Cleaved caspase-12 in HPC after silencing HDAC9.At the same time,the effect of silencing HDAC9 on podocyte apoptosis was detected by flow cytometry.Part II:The mechanism of HDAC9 in hyperhomocysteinemia-induced podocyte injury2.1 Detected the regulation of HDAC9 on the acetylation level of H3K9 in podocytesImmunofluorescence double staining experiment was performed to detect the the acetylation level of H3K9 site in podocytes after HDAC9 knockout in hyperhomocysteinemia mice.In vitro,Western blot was used to detect the effect of HDAC9 gene deletion on the acetylation level of H3K9 locus stimulated by L-hHcys.2.2 Explored the regulation of HDAC9 on the expression level of Klotho in podocytesWe used Immunofluorescence double staining to detect the level of Klotho in the podocytes of hyperhomocysteinemia mice and used western blot to detect the effect of the deletion of HDAC9 gene in L-hHcys-treated podocytes on Klotho protein levels.Real-time PCR detected the mRNA level of Klotho by the deletion of HDAC9 gene in L-hHcys-treated podocytes.2.3 Explored whether HDAC9 can regulate the acetylation level of H3K9 and affect Klotho transcriptionThe chromatin immunoprecipitation method detected whether the Klotho promoter region binds to H3K9ac,and observed the regulation of the acetylation level of the H3K9 site in the Klotho promoter region under the stimulation of L-hHcys after HDAC9 silencing in HPC.2.4 Detected the effect of Klotho on L-Hcys-induced podocyte injuryWe used western blot to detect the effect of Klotho on L-Hcys-induced podocyte injury,and we used flow cytometry to explore the effect of Klotho on podocyte apoptosis under L-Hcys stimulation.2.5 Explored whether the klotho signaling pathway is involved in HDAC9-mediated podocyte injuryWestern blot detected the changes in the expression levels of Nephrin,Podocin,Desmin,Vimentin,Chop and Cleaved caspase-12 induced by L-hHcys in HPC after HDAC9 silencing,and then observed the changes in the expression of the above proteins after silencing Klotho.At the same time,we used flow cytometry to detect the effect of silencing Klotho on L-hHcys-induced podocyte apoptosis.Research resultsPart 1:The role of HDAC9 in hyperhomocysteinemia-induced podocyte injury1.1 Knockout of HDAC9 can reduce proteinuria levels in mice with hyperhomocysteinemiaELISA showed that the ratio of urinary albumin to creatinine in mice was increased after feeding the mice on folate-free diet.Sodium valproate(SVA),a non-specific inhibitor of HDAC9,was administered to mice in vivo,and it was found that the proteinuria level of mice was also significantly reduced.1.2 Knockout of HDAC9 ameliorated podocyte injury in mice with hyperhomocysteinemiaPAS showed that the glomerular mesangial hyperplasia in hyperhomocysteinemia mice was increased.TEM showed that the thickening of glomerular basement membrane,the fusion of foot processes,and the decrease of the number of foot processes in hyperhomocysteinemia mice,and the above characteristics were restored after HDAC9 knockout or inhibition.IF found that in the model of hyperhomocysteinemia,the expression level of the podocyte slit membrane protein Nephrin was decreased,and its expression level was recovered after the HDAC9 gene was knocked out or suppressed.1.3 HDAC9 ameliorated podocyte injury under the stimulation of L-HcysWestern blot showed that silencing HDAC9 in podocytes can inhibit the protein levels of Nephrin,Podocin,Desmin,Vimentin,Chop and Cleaved caspase-12 in podocytes induced by L-hHcys.In addition,flow cytometry showed that silencing HDAC9 can reduce podocyte apoptosis induced by L-hHcys.Part Ⅱ:The mechanism of HDAC9 in hyperhomocysteinemia-induced podocyte injury2.1 Knockout of HDAC9 in podocytes up-regulated the acetylation level of H3K9Immunofluorescence double staining showed that the knockout of HDAC9 can restore the expression level of H3K9ac in the podocytes of hyperhomocysteinemia.At the same time,after silencing HDAC9 in HPC,the decrease in the expression level of H3K9ac induced by L-Hcys was also reversed.2.2 HDAC9 in podocytes reversely regulated the expression level of KlothoImmunofluorescence double staining showed that the knockout of HDAC9 can restore the expression level of Klotho in hyperhomocysteinemia podocytes.RT-PCR showed that the silencing of HDAC9 can restore the decrease of Klotho expression level under L-hHcys stimulation conditions.Western blot experiments showed that silencing HDAC9 in podocytes can reverse the decrease of Klotho level under L-hHcys stimulation conditions,which showed that HDAC9 regulated the expression level of Klotho.2.3 HDAC9 in podocytes inhibitd Klotho transcription by regulating the acetylation level of H3K9 siteCHIP showed that the Klotho promoter region was bound to H3K9ac,and the acetylation level of H3K9 in the Klotho promoter region was significantly reduced under L-hHcys stimulation,and the acetylation level of H3K9 was increased after silencing HDAC9.2.4 Klotho ameliorated podocyte injury induced by L-HcysWestern blot showed that Klotho can reverse the down-regulation of Nephrin and Podocin protein levels induced by hHcys,and it can also reverse the increase of Desmin,Chop,Cleaved caspase 12 induced by hHcys.Flow cytometry showed that hHcys can induce podocyte apoptosis,and Klotho inhibited L-Hcys-induced podocyte apoptosis,indicating that Klotho can ameliorate the podocyte injury induced by L-Hcys.2.5 Gene silencing of HDAC9 ameliorated L-Hcys-induced podocyte injury through KlothoWestern blot found that silencing HDAC9 can reduce the down-regulation of Nephrin and Podocin in k:idney tissue induced by L-hHcys and can significantly reduce the protein expression level of Desmin.After silencing Klotho,the expression of Nephrin,Podocin and Desmin recovered.After flow cytometry,it was found that L-hHcys induced podocyte apoptosis.Silencing HDAC9 could reduce L-hHcys-induced podocyte apoptosis.After silencing Klotho,it was found that podocyte apoptosis increased again,indicating that HDAC9 can regulate Klotho signaling pathway.Conclusion and innovation1.In the study of the role of HDAC9 in hyperhomocysteinemia-induced podocyte injury,we found that HDAC9 deficiency ameliorated L-Hcys-induced podocyte injury.2.In the study of the mechanism of HDAC9 in hyperhomocysteinemia-induced podocyte injury,we found that HDAC9 inhibited Klotho transcription by deacetylating H3K9,and then aggravated L-Hcys-induced podocyte injury through inhibition of Klotho.3.The study proved for the first time that HDAC9 regulated the Klotho signaling pathway and played an important role in podocyte injury.This provided new ideas for the study of the downstream effects and mechanisms of HDAC9,and also served as a therapeutic target for HDAC9 in kidney-related diseases. |
