| Diabetic Nephropathy(DN)is one of the most common and severe microvascular complications of diabetes,and is the main cause of renal failure in patients with endstage renal disease(ESRD).Diabetes Mellitus(DM)patients are ten times more likely to develop to ESRD than normal controls.Progressive proteinuria is one of the earliest clinical features of diabetic nephropathy.Glomerular podocyte injury is an imperative step in the development of DN;this process is significantly related to proteinuria and the progression of glomerulosclerosis.Numerous studies have shown that multiple pathways,including the infiltration of inflammatory mediators,accumulation of advanced glycation end products(AGEs),autophagy and apoptosis are involved in the podocyte injury process of DN patients.However,the pathogenesis of DN has not been fully understood.In clinical practice,the strict glycemic control is the key measures for the prevention and treatment of DN.However,one-third of diabetic patients still progress to develop DKD despite intensive glycemic control,evidenced by a number of longterm epidemiological follow-up studies.The reason for this phenomenon involves a series of pathogenic factors related to diabetes complications,including AGEs,oxidative stress,inflammation and epigenetic modification,etc.Epigenetics refers to the changes of gene expression and function and the generation of heritable phenotype without alteration of DNA sequences,including methylation,histone modification,non-coding RNA and chromatin remodeling.Nutritional status and other environmental factors can influence the progression of diabetes and diabetic nephropathy,which in turn changes the epigenetic status,thus epigenetic modifications may play vital role in the pathogenesis of DN.As one of the important patterns of epigenetic modification,long noncoding RNA(lncRNA)plays crucial role in the pathogenesis of DM and DN.In our previous study,we collected the blood,urine and kidney samples of DN patients confirmed by renal biopsy from the biobank of kidney disease to perform Array star human LncRNA CHIP V3.0.When compared with the normal control group,we found that LncRNA Distalless homeobox 6 antisense 1(DLX6-AS1,highly homologous to Dlx6os1 in the mouse)was significantly higher in DN patients.Our previous study also found that DLX6-AS1 was correlated with increased proteinuria excretion in DN patients.In Streptozotocin(STZ)-induced and db/db diabetic mouse models,podocyte specific Dlx6os1 deletion plays a crucial role in alleviating podocyte inflammatory response and reversing renal injury.However,the regulatory factors of elevated DLX6-AS1 expression in DN remain unclear.Therefore,in this study,we cultured immortalized human and murine podocytes in vitro,and then JASPAR database and DNA pull down combined subsequent liquid chromatography-tandem mass spectrometry were used to identify potential regulatory factors for DLX6-AS1.The transcription factor cAMP-response element binding protein(CREB)were found highly expressed in DN and were verified to bind with the gene sequence of DLX6-AS1/Dlx6os1.CREB,a 43 kDa stimulus-induced transcription factor,has been proved to participate the pathogenesis of various tumors and liver gluconeogenesis by targeting the expression of lncRNAs.However,whether the correlation between CREB and DLX6-AS1 involved in the podocyte injury of DN has not been reported.In this study,dual-luciferase reporter assay and chromatin immunoprecipitation were performed to confirm whether exist binding sites in the promoter region of DLX6AS1 or not.We then conducted in vitro and in vivo experiment to explore the role and underlying mechanism of CREB in podocyte injury.In addition,gene intervention and 666-15 targeted inhibition were used to regulate the expression of CREB in diabetic db/db mice and cultured human podocyte and to explore the mechanism of participating in DN inflammation and podocyte injury through targeting DLX6-AS1/Dlx6os1.Part Ⅰ:Effect of transcription factor CREB on the expression of DLX6-AS1/Dlx6os1 and glomerular podocyte injuryObjectiveTo identity the potential transcriptional regulatory factors of elevated DLX6AS1/Dlx6os1 in the context of DN;To observe the role of podocytes specific CREB overexpression in the elevated Dlx6os1 level and mouse podocytes injury;To explore the mechanism of CREB in podocyte injury in cultured human podocyte in vitro.Methods1.Human and murine immortalized podocytes were cultured with high glucose in vitro.DNA-pull down combined liquid chromatography-tandem mass spectrometry was used to filtrate the binding proteins of DLX6-AS1/Dlx6os1 gene sequence.The potential transcription factors binding with DLX6-AS1 were further screened by JASPAR database.2.The potential transcription factor binding with DLX6-AS1/Dlx6os1 was further confirmed in renal tissue samples of DN patient and diabetic db/db mice as well as podocytes cultured in high glucose.3.RNA in situ hybridization,luciferase reporter gene assay and chromatin immunoprecipitation were used to investigate whether CREB could bind with DLX6AS1/Dlx6os1 promoter and to verify the sequence of binding sites.4.Ten-week-old db/m mice were randomly divided into control group(db/m group),injected control vectors group(db/m+Vehicle group),and transfected podocyte-specific CREB overexpression adeno-associated virus group(db/m+CREBOE group).According to experimental requirements,mice were sacrificed at baseline level or at 4 and 12 weeks after injection,and kidney tissue was collected for use.Blood glucose,body weight and urinary albumin creatinine ratio(uACR)were tested regularly.PAS staining and electron microscopy were used to observe renal pathological changes in different group mice.5.Immunofluorescence staining was used to detect the expression level and colocation of WT-1 and Flag.Immunofluorescence staining and Western blot were used to detect the expression of podocyte markers(nephrin、podocin、synaptopodin),podocyte injury indicator(desmin)and inflammatory ptotein(B7-1).Quantitative Realtime PCR(qPCR)and RNA fluorescence in situ hybridization combined with immunofluorescence staining were used to detect the expression of Dlx6osl in podocytes of different group mice.6.Human podocytes were cultured in vitro and divided into NG group,NG+control vector group(NG+Vehicle group),NG+CREB overexpressed adenovirus group(NG+CREB OE group)and NG+CREB overexpressed adenovirus+DLX6-AS1 interfering lentivirus group(NG+CREB OE+DLX6-AS1 KD)and the expression of DLX6-AS1、CREB in each group was detected by qPCR and RNA fluorescence in situ hybridization combined with immunofluorescence staining.7.Western blot and immunofluorescence staining were used to detect CREB、pCREB、podocyte marker protein,podocyte injury indicator and cytoskeleton protein expression in each group.Results1.In high glucose cultured human and mouse podocytes,2756 and 2664 protein were found to binding with the gene sequence of DLX6-AS1/Dlx6os1 by DNA-pull down and mass spectrometry,respectively.131 and 73 transcription factors were predicted to binding with the promoter sequence of DLX6-AS1/Dlx6os1 by the JASPAR database,respectively.Among these,the expression level and activity of transcription factor CREB increased compared with the control group in the context of DN(P<0.05),and it can play a transcriptional regulatory role by binding to the promoter sequence of DLX6-AS1/Dlx6os1.2.The transfection efficiency of CREB overexpressed adeno-associated virus in mouse podocytes was about 60%,the transfection effect was fine and the effect was still stable at the point of 12 weeks after injection.Twelve weeks after podocytespecific CREB overexpression in db/m mice,uACR level was significantly higher than that in control group(db/m and db/m+Vehicle)mice(P<0.05),but there was no significant difference in blood glucose and kidney weight/body weight ratio(P>0.05).PAS staining and electron microscopy showed that:compared with the control group mice,the glomerular basement membrane was significantly thickened,and the podocyte processes were extensively fused and even disappeared in db/m+CREB-OE group(P<0.05).3.Compared with the control group mice,the expression of podocyte marker proteins was reduced and the expression of podocyte injury protein desmin and inflammation-related protein B7-1 were increased in db/m+CREB-OE group.The degree of podocyte injury was aggravated in db/m+CREB-OE group.In addition,the expression level of Dlx6os1 in db/m+CREB-OE groups was significantly higher than that in control group(P<0.05).4.In cultured podocytes,the expression of DLX6-AS1,the expression and activity of CREB in the NG+CREB OE group was higher than that in the NG+Vehicle group(P<0.05),while the expression of DLX6-AS1 in the NG+CREB OE+DLX6-AS1 KD group was lower than that in the NG+CREB OE group(P<0.05);and there’s no significant difference of CREB between the NG+CREB OE+DLX6-AS1 KD group and the NG+CREB OE group.5.Compared with the NG+Vehicle group,the expression of podocyte marker proteins was decreased and the expression of the podocyte injury protein was increased in the NG+CREB OE group;however,the effect was reversed in the NG+CREB OE+DLX6-AS1 KD group.Conclusions1.In the context of DN,the transcription factor CREB can upregulate the expression of DLX6-AS1/Dlx6os1 by binding to its promoter.2.The overexpression of CREB can significantly increase the excretion of urinary albumin and lead to podocyte inflammation and injury by upregulating DLX6AS1/Dlx6os1 expression.Part Ⅱ:Effect of podocyte-specific CREB knockdown on the expression of DLX6-AS1/Dlx6os1 and podocyte injury in diabetic nephropathyObjectiveTo observe the expression of DLX6-AS1 and the renoprotective role in podocytes cultured with high glucose when CREB was downregulated;To investigate whether podocyte-specific knockdown of CREB can regulate Dlx6os1 levels and alleviate renal injury and albuminuria excretion in diabetic db/db mice.Methods1.The qPCR and Western blot were used to detect the interference effect of three CREB shRNA adenoviruses in high glucose induced podocytes.The qPCR and RNA fluorescence in situ hybridization were used to detect the expression level of DLX6AS1 after transfecting CREB shRNA adenovirus.Western blot and immunofluorescence staining were used to detect the expression of podocyte markers,injury associated proteins and cytoskeleton protein and to observe the effect on inflammatory response and damage in high glucose induced podocytes after transfecting CREB shRNA adenovirus.2.Ten-week-old db/db mice were randomly divided into control group(db/db group),injected control vectors group(db/db+Vehicle group),and transfected podocyte-specific CREB knockdown adeno-associated virus group(db/db+CREB-KD group).The general conditions,body weight,fasten glucose,uACR were detected at regular intervals.According to experimental requirements,mice were sacrificed at baseline level or at 4 and 12 weeks after injection,and kidney tissue was collected for further examination.PAS staining and electron microscopy were used to observe renal pathological changes in different group mice.3.Immunofluorescence staining,immunohistochemistry staining and Western blot assay were used to detect the transfection efficiency of podocyte-specific CREB knockdown adeno-associated virus in db/db mice.4.qPCR and RNA fluorescence in situ hybridization combined with immunofluorescence staining were used to detect the expression of Dlx6osl in podocytes of different group mice.5.Immunofluorescence staining and Western blot were used to detect the expression of podocyte markersb(nephrin、podocin、synaptopodin),podocyte injury indicatorb(desmin)and inflammatory ptotein(B7-1).Results1.The results of qPCR and Western blot assay showed that all three CREB knockdown adenoviruses had interference effect when transfected into high glucose induced podocytes;and the knockdown effect of sh3 was most significant.2.Compared with the HG+Vehicle group,the expression level of DLX6-AS1 in the HG+CREB-KD group was significantly decreased(P<0.05).In addition,the expression of podocyte marker proteins and cytoskeletal protein F-actin were increased and the expression of damaged proteins was decreased in the HG+CREB-KD group3.The uACR level of db/db+CREB-KD 4w and 12w group was significantly lower than that in control group(db/db and db/db+Vehicle group)(P<0.05),but there was no significant difference in blood glucose and kidney weight/body weight ratio(P>0.05).The expression levels of CREB and pCREB in the db/db+CREB-KD 4w and 12w groups were also significantly decreased(P<0.05).4.The results of PAS staining and electron microscopy showed that:compared with the control group mice,the proliferation of mesangial matrix was reduced,the thickened glomerular basement membrane was significantly relieved,and the fusion of podocyte processes was disappeared in db/db+CREB-KD group(P<0.05).5.The expression level of Dlx6osl in db/db+CREB-KD group was significantly lower than that in control group(P<0.05).The expression of podocyte marker proteins was increased and the expression of podocyte injury protein desmin and inflammationrelated protein B7-1 were reduced in db/db+CREB-KD group.Conclusions1.In high glucose induced podocytes,the knockdown of CREB can down-regulate DLX6-AS1 levels and alleviate podocyte inflammation and injury.2.In db/db mouse model,podocyte specific knockdown of CREB can downregulate the expression of Dlx6os1,reduce the excretion of albuminuria and alleviate podocyte injury and inflammation.Part Ⅲ:Effect of targeted inhibition of CREB by 666-15 on the expression of DLX6-AS1/Dlx6osl and podocyte injury in diabetic nephropathyObjectiveTo explore whether the expression of DLX6-AS1/Dlx6os1 in podocytes decreased and whether the podocyte injury and inflammatory response were alleviated when treated with the selective CREB inhibitor 666-15 in diabetic db/db mice and high glucose induced podocytes.To investigate whether the renoprotected role of 666-15 can be abolished by Dlx6osl overexpression in podocytes or not.Methods1.High glucose induced human podocytes were treated with 666-15 solution at concentrations of 0.25,0.5 and 1μM,respectively.Western blot was used to detect the expression levels of CREB and pCREB,podocyte marker proteins,injury associated protein desmin and inflammatory indicator B7-1;and qPCR was used to detect the expression of DLX6-AS1.2.Ten-week-old db/db mice were randomly divided into control group(db/db group),solvent group(db/db+Saline group),and mice treated with 666-15 intraperitoneally(db/db+666-15 group).The general conditions,body weight,fasten glucose,uACR were detected at regular intervals.According to experimental requirements,mice were sacrificed at baseline level or at 3 and 7 weeks after injection,and kidney tissue was collected for further examination.PAS staining and electron microscopy were used to observe renal pathological changes in different group mice.3.qPCR and RNA fluorescence in situ hybridization combined with immunofluorescence staining were used to detect the expression of Dlx6osl in podocytes of different group mice.4.Immunofluorescence staining and Western blot were used to detect the expression of podocyte markers(nephrin、podocin、synaptopodin),podocyte injury indicator(desmin)and inflammatory protein(B7-1).Results1.The expression level of CREB、pCREB and DLX6-AS1 decreased following the increased concentration of 666-15 in high glucose induced podocytes(P<0.05).2.Compared with the high glucose group,the expression levels of podocyte marker proteins nephrin and podocin were increased,and the expression levels of B71 and desmin were reduced in HG+666-15 group(P<0.05).3.The uACR level of db/db+666-15 group was significantly lower than that in control group(db/db and db/db+Saline)mice(P<0.05),but there was no significant difference in blood glucose and kidney weight/body weight ratio(P>0.05);however,the uACR level of db/db+666-15+Dlx6os1 OE group was significantly higher than that in db/db+666-15 group(P<0.05).4.The results of PAS staining and electron microscopy showed that:compared with the control group mice,the proliferation of mesangial matrix was reduced,the thickened glomerular basement membrane was significantly relieved,and the fusion of podocyte processes was disappeared in db/db+666-15 group(P<0.05);however,the beneficial effect of 666-15 on DN was significantly abolished in the db/db+66615+Dlx6os1 OE group.5.The expression level of Dlx6osl in db/db+666-15 groups were significantly lower than that in control group mice(P<0.05).Additionally,the expression level of Dlx6osl in db/db+666-15+Dlx6os1 OE group was significantly increased when compared with db/db+666-15 group(P<0.05).6.Compared with the control group mice,the expression of podocyte marker proteins was increased and the expression of podocyte injury protein desmin and inflammation-related protein B7-1 were reduced in db/db+666-15 group,as demonstrated by immunofluorescence staining and Western blot assay.However,the expression of nephrin was lower and the expression of B7-1 was higher in the db/db+666-15+Dlx6os1 OE group than that in db/db+666-15 group;but there was no significant difference in the expression and activity of CREB.Conclusions1.In high glucose induced podocytes,the use of selective CREB inhibitor 666-15 can reduce the expression of DLX6-AS1 in a dose-dependent manner,and then alleviate podocyte injury caused by high glucose2.In db/db mouse model,the intraperitoneal injection of666-15 can downregulate the Dlx6os1 level in glomerular podocytes,alleviate renal injury and reduce podocyte inflammation;however,podocyte-specific Dlx6osl overexpression can partially abolish the beneficial effect of 666-15 on renal injury and podocyte inflammation.Full-text Conclusions:1.In the context of DN,the transcription factor CREB can upregulate the expression of DLX6-AS1/Dlx6os1 by binding to its promoter.2.The overexpression of CREB can significantly increase the excretion of urinary albumin and lead to podocyte inflammation and injury by upregulating DLX6AS1/Dlx6os1 expression.3.Podocyte specific knockdown or 666-15 targeted inhibition of CREB can downregulate the DLX6-AS1/Dlx6os1 levels and thus alleviate podocyte injury in diabetic nephropathy.4.Podocyte-specific Dlx6osl overexpression can partially abolish the beneficial effect of 666-15 on renal injury and podocyte inflammation. |
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