Sirt6 Protects Against Podocyte Injury In Diabetic Nephropathy

Diabetic nephropathy(DN)is one of the major microvascular complications of diabetes mellitus and is the most common cause of end--stage renal diseases.Podocyte injury is an early event in the development of DN.Podocytes are highly specialized,terminally differentiated epithelial cells that are integral components of the renal glomerular filtration barrier,which are vulnerable to a variety of injuries and as a result,they undergo a series of changes ranging from hypertrophy,detachment,autophagy to apoptosis 1.The genetic or acquired impairment of podocytes leads to proteinuria and contributes to the development and progression of proteinuric kidney disease.As podocytes have limited ability to repair and regenerate,the extent of podocyte injury is considered as a major prognostic determinant in end-stage renal disease2.There is mounting evidence of podocyte injury in a variety of other renal diseases such as focal segmental glomerulosclerosis(FSGS),membranous glomerulonephritis(MGN),diabetic nephropathy(DN)and IgA nephropathy.In these conditions,podocytes lose specific markers of differentiation,undergo foot process effacement and eventual detachment,and reduce the capacity to maintain the glomerular filtration barrier,thereby resulting in proteinuria.Therefore,identifying the key and universal molecules involved in the different forms of podocytopathies may provide clues to develop new therapeutic strategies for patients with proteinuric kidney disease.Recently,studies have indicated that epigenetic modification such as histone modification,DNA methylation and microRNA may play important roles in the regulation of metabolic memory.Among them,studies have highlighted the importance of histone deacetylase(HDAC)-mediated histone and non-histone protein acetylation in the development of renal injury.HDACs can be divided into 4 groups.Our lab has clarified the expression patterns of zinc-dependent HD AC in DN,we found HDAC4 contributes to podcyte injury in diabetic nephropathy.SIRTs,the class III HD AC including at least 7 members,are NAD+ depended histone deacetylase.Although studies have suggest that Sirtl has protective renal effects by anti-inflammation or anti-fibrosis,the function of other SIRTs in the kidney is not very clear.Therefore,exploring the function of other SIRTs in the podocyte may provide clues to develop new therapeutic strategies for patients with proteinuric kidney disease.Objective1.Detect the expression patterns of SIRTs in the kidney from DN.2.Explore the role of Sirt6 in podocyte injury under DN.3.Clarify the mechanisms by which Sirt6 protects against podocyte injury.MethodsPart 1:The expression patterns of SIRTs in the kidney from DN1.1 Detected the expression patterns of SIRTs in the kidney from STZ-induced diabetic miceWe used a STZ-treated uninephrectomized mice model on a HFD to hasten the development of DN.The levels of SIRTs were detected by western blot in the kidney from STZ-induced diabetic mice.1.2 Determined the levels of Sirt6 in renal biopsies from DN,FSGS,MGN and IgA nephropathy,as well as the correlations between Sirt6 and estimaaed GFR or proteinuriaWe firstly confirmed the levels of Sirt6 in renal biopsies from DN,FSGS,MGN and IgA nephropathy by immunohistochemistry(IHC)staining and real time RT-PCR analysis.Then,the correlations between Sirt6 and estimated GFR or proteinuria in all subjects were also analyzed.1.3 In vitro,the protein level of Sirt6 were detected in podocyte with different stimulus treatment.Human podocyte were cultured in vitro.The protein level of Sirt6 in podocytes with different stimulus treatment,such as HG and AGE,were tested by western blot.Part 2:The role of Sirt6 in podocyte injury under diabetic nephropathy condition2.1 Generated conditional knockout mice in which Sirt6 is specifically ablated in podocytes by using Cre-LoxP recombination system.Podocin-Cre mice were crossed with Sirt6fl/fl to generate Podocin-Cre Sirt6fl/fl mice(Cre+/Sirt6fl/fl mice),which was identified by tail genotyping,immunofluorescent analysis in podocytes and the protein levels of Sirt6 in isolated glomeruli from Cre+/Sirt6fl/fl mice were carried out by western blot.We analyzed Cre+/Sirt6fl/fl mice and control mice in age-matched groups.Firstly,urinary albumin excretion were analyzed.Secondly,glomerular mesangium expansion were detected by PAS staining.Thirdly,the GBM thickness,foot process width and the number of foot processes per μm of GBM were determined by TEM.2.2 The role of Sirt6 in podocyte injury under diabetic nephropathy condition Firstly,urinary albumin excretion were analyzed after sample selection.Then,glomerular mesangium expansion and the GBM thickness,foot process width and the number of foot processes per μm of GBM were detected by PAS staining or TEM,respectively.In addition,the levels of key podocyte differentiation markers including nephrin and podocin in diabetic Cre+/Sirt6fl/fl mice were assessed by immunofluorescen analysis.The uPAR expression in podocytes in different groups of mice were further detectedand by immunofluorescent analysis.2.3 Pleiotropic protective actions of Sirt6 were studied in podocytes in vitro In vitro,overexpression of Sirt6 by a Sirt6-adenovirus transfection were used to study protective actions of Sirt6 in podocytes.The levels of pro-inflammatory mediators in podocytes with different treatments were detected by real time RT-PCR.Podocytes with different treatments were stained with fluorescein isothiocyanate(FITC)-conjugated Annexin V and propidium iodide(PI),and analyzed by flow cytometry to evaluate the role of Sirt6 in the prevention of apoptosis.Actin cytoskeleton derangement as evidenced by the loss of actin filaments in immunofluorescen analysis.The relative protein levels of autophagy-associated proteins in podocytes with different treatments were assessed by western blot.In addition,we utilized the tandem RFP-GFP-LC3 adenovirus construct to confirm autophagy induction by form punctate that represent autophagosome formation.Lastly,the number of typical autophagosomes with double membranes were counted in podocytes by TEM.Part 3:Mechanisms by which Sirt6 ameliorates podocytes injury3.1 Sirt6 is a highly specific deacetylase that targets H3K9 in podocyteThe levels of H3K9 acetylation(H3K9ac)in renal biopsies from patients with DN and FSGS were measured by IHC staining.In animal studies,we detected the levels of H3K9 acetylation in podocytes from diabetic Cre+/Sirt6fl/fl mice immunofluorescen analysis.In vitro,the levels of H3K9ac were analyzed by western blot after overexpression of Sirt6 in podocytes.3.2 The changes of Notch signaling in HG-treated podocytes with or without Sirt6 overexpression in podocyteWe observed the changes of Notch signaling in HG-treated podocytes with or without Sirt6 overexpression by Agilent Whole Human Genome Oligo Microarray for global gene expression analysis.Real time RT-PCR analysis were further to confirm the changes of mRNA levels of Notch isoforms in HG-treated podocytes.The levels of Notchl and Notch4 in diabetic Cre+/Sirt6fl/fl mice were assessed by immunofluorescen analysis.In vitro,the levels of Notch 1 and Notch4 were analyzed by western blot after silence of Sirt6 in podocytes.Then,Notch signaling downstream target genes such as HES1 and Snail 1 in diabetic Cre+/Sirt6fl/fl mice were detected by immunofluorescen analysis.And mRNA analysis were further to confirm the changes of HES1 and Snail 1 in podocytes with HG treatments.3.3 Sirt6 inhibits Notchl and Notch4 transcription by deacetylating H3K9Firstly,we examined whether Sirt6 deficiency results in the increased H3K9ac in the promoters of Notch genes by chromatin immunoprecipitation(ChIP).Then,to confirm whether Sirt6 can recruit and deacetylate H3K9 on these promoters,we performed ChIP assay in podocytes with overexpression of Sirt6.3.4 Notch inhibition rescues Sirt6-deficient podocytes injuryThe levels of pro-inflammatory mediators in podocytes with different treatments were detected by real time RT-PCR.Podocytes with different treatments were stained with fluorescein isothiocyanate(FITC)-conjugated Annexin V and propidium iodide(PI),and analyzed by flow cytometry to evaluate the role of inhibition of Notch signaling in Sirt6-deficient podocytes injury.Actin cytoskeleton derangement as evidenced by the loss of actin filaments in immunofluorescen analysis.The relative protein levels of autophagy-associated proteins,such as atg5,in podocytes with different treatments were assessed by western blot.Furthermore,we utilized the tandem RFP-GFP-LC3 adenovirus construct to confirm autophagy induction by form punctate that represent autophagosome formation.ResultsPart 1:The expression pattern of SIRTs in the kidney from DN1.1 Sirt6 is reduced in the kidney from DN miceOur results showed that the levels of Sirtl,Sirt3,Sirt4 and Sirt6 were reduced in the kidney from STZ-induced diabetic mice.However,the levels of Sirtl and Sirt6 were lower than that of other SIRTs in the kidney from diabetic nephropathy mice.1.2 Reduction of Sirt6 in various podocytopathies renal biopsies We firstly confirmed the reduction of Sirt6 in renal biopsies from DN and FSGS subjects compared with normal subjects or diabetic patients without nephropathy by immunohistochemistry(IHC)staining and real time RT-PCR analysis.Importantly,the Sirt6 level was also markedly reduced in renal biopsies from patients with other different forms of podocytopathies such as MGN and IgA nephropathy.Notably,the mRNA levels of Sirt6 were positively correlated with estimated GFR and negatively correlated with proteinuria in all subjects.1.3 In vitro,the protein levels of Sirt6 were decreased under different pathological conditions.In vitro,HG or AGE significantly reduced podocyte Sirt6 expression in a concentration dependent manner.Part 2;The role of Sirt6 in podocyte injury under diabetic nephropathy condition2.1 Generated conditional knockout mice in which Sirt6 is specifically ablated in podocytes by using Cre-LoxP recombination system.Podocin-Cre mice were crossed with Sirt6fl/fl to generate Podocin-Cre Sirt6fl/fl mice(Cre+/Sirt6fl/fl mice),which was identified by tail genotyping,immunofluorescent analysis in podocytes and a significant reduction in the protein levels of Sirt6 in isolated glomeruli from Cre+/Sirt6tfl/fl mice.Firstly,we analyzed Cre+/Sirt6fl/fl mice and control mice in age-matched groups.Up to 9-month after birth,Cre+/Sirt6fl/fl mice were indistinguishable from control littermates as analyzed by renal histology,albuminuria,and glomerular ultrastructure.In a 12-month follow-up,the ageing Cre+/Sirt6fl/fl mice developed a slight albuminuria,mesangial matrix expansion and podocyte injury as evidenced by glomerular basement membrane(GBM)thickening,and podocyte foot process broadening and effacement but significantly higher than in control mice.2.2 Sirt6 deletion exacerbates podocyte injury in diabetic mice STZ-induced diabetic mice had hyperglycemia and lower body weight compared with their non-diabetic counterparts,no difference in blood pressure among these groups.Diabetic Cre+lSirt6fl/fl mice exhibited a significant increase in urinary albumin excretion as compared with diabetic Cre+/Sirt6+/+ mice.Morphological examinations showed the glomerular mesangium expansion and podocyte injury from diabetic Cre+/Sirt6+/+ mice,all of which were exacerbated in diabetic Cre+/Sirt6fl/fl mice.Podocyte injury was further confirmed by a significant loss of key podocyte differentiation markers including nephrin and podocin in diabetic Cre+/Sirt6fl/fl mice.In addition,diabetic Cre+/Sirt6+/+ mice had a significant increase in podocyte uPAR expression,an even stronger induction of uPAR expression was fournd in podocytes from diabetic Cre+/Sirt6fl/fl mice.2.3 Sirt6 has pleiotropic protective actions in podocytesIn vitro,overexpression of Sirt6 reduced the levels of proinflammatory mediators,attenuated podocyte apoptosis,and ameliorated actin cytoskeleton derangement in podocytes with HG treatment.Moreover,we observed that overexpression of Sirt6 restored autophagy in podocytes as evidenced by the increased levels of autophagy-related proteins.In addition to accumulation of LC3,more red puncta were present in podocytes with Sirt6 overexpression than in controls under HG condition.In addition,the number of typical autophagosomes with double membranes was increased in podocytes with Sirt6 overexpression.Part 3:Mechanisms by which Sirt6 ameliorates podocytes injury3.1 Sirt6 is a highly specific deacetylase that targets H3K9 in podocyte We found that the H3K9ac levels were significantly increased in renal biopsies from patients with DN and FSGS by IHC staining.In animal studies,a more significant increase in H3K9ac in podocytes from diabetic Cre+/Sirt6fl/fl mice was also observed as compared with diabetic Cre+/Sirt6+/+ mice.In vitro,HG-induced H3K9ac was attenuated by overexpression of Sirt6.3.2 Sirt6 inhibits Notch signaling in podocyteWe observed the changes of Notch signaling in HG-treated podocytes with or without Sirt6 overexpression by Agilent Whole Human Genome Oligo Microarray for global gene expression analysis.Real time RT-PCR analysis found that among Notch isoforms,only Notch 1 and Notch4 were upregulated in HG-treated podocytes,which could be attenuated by overexpression of Sirt6.The same tendency was also found in podocytes from diabetic Cre+/Sirt6tfl/fl mice as compared with diabetic Cre+/Sirt6+/+ mice.Moreover,we found that gene silencing of Sirt6 can regulate the basal levels of Notch 1 and Notch4 in podocytes.In addition,overexpression of Sirt6 reduced HG-induced Notch signaling downstream target genes such as HES1 and Snaill by mRNA analysis as well as the results from podocytes treated with DAPT that is a γ-secretase inhibitor and indirectly inhibit Notch signaling pathways,which was consistent with in vivo studies showing more significant increases in the levels of HES1 and Snaill in podocytes from diabetic Cre+/Sirt6fl/fl mice.3.3 Sirt6 inhibits Notchl and Notch4 transcription by deacetylating H3K9 Our results revealed the increased levels of H3K9ac in the promoters of Notchl and Notch4 in podocytes with HG treatment.We also found that Sirt6 significantly reduced the level of H3K9ac in the promoter region of Notchl and Notch4.Moreover,Sirt6 was dissociated from the promoters of these genes in podocytes and recruited to them after Sirt6-adenovirus transfection by ChIP analysis.3.4 Notch inhibition rescues Sirt6-deficient podocytes injury Inhibition of Notch signaling reduced inflammatory responses and apoptosis,ameliorated actin cytoskeleton derangement,and recovered the expression levels of nephrin and podocin in Sirt6-deficient podocytes.As well as gene silencing of Notchl restored the levels of autophagy-associated proteins in podocytes with HG treatment,gene silencing of Notchl can also rescued the autophagy defect in Sirt6-deficient podocytes as evidenced by the increased the autophagy-associated protein levels and autophagy flux.Conclusion and innovation1.Sirt6 is downregulated in renal biopsies from patients with podocytopathies.Notably,the mRNA levels of Sirt6 were positively correlated with estimated glomerular filtration rate and negatively correlated with proteinuria in all subjects.2.Podocyte-specific deletion of Sirt6 exacerbates podocyte injury and proteinuria in diabetic nephropathy mice.Sirt6 has pleiotropic protective actions in podocytes,including anti-inflammatory and anti-apoptotic effects,is involved in actin cytoskeleton maintenance and promotes autophagy.3.Mechanistically,Sirt6 negatively regulated Notch signaling,which was associated with the suppression of the transcription of Notchl and Notch4 genes by deacetylating H3K9.4.Our findings provide new insights into the pivotal role of Sirt6 in maintaining podocyte function.In addition,we found that Sirt6 inhibits Notchl and Notch4 transcription by deacetylating histone H3K9 in improving podocyte injury.We propose Sirt6 as a potential therapeutic target for the treatment of proteinuric kidney disease.