Sirt6 Ameliorates Podocyte Injury In Mice With Adriamycin-induced Focal Segmental Glomerulosclerosis

Focal segmental glomerulosclerosis(FSGS)is a common glomerular disease in children and adult nephrotic syndrome(NS)and it is one of the main causes of end stage renal disease(ESRD).Podocyte injury is the main pathological manifestation of FSGS.As a component of glomerular filtration barrier,podocytes maintain the renal filtration function.Therefore,finding a target with podocyte protection is beneficial for diagnosing and treating focal segmental glomerulosclerosis.Recent studies have shown that epigenetic modifications such as DNA methylation,histone modifications and micRNA play important roles in focal segmental glomerulosclerosis.And histone modifications have received more and more attention.The histone acetylation modification is regulated by histone acetyltransferase(HAT)and histone deacetylases(HDACs).Histone deacetylases are divided into four subfamilies.Class Ⅰ,Ⅱ,and IV are Zn1+-dependent histone deacetylases,and the class Ⅲ subfamily,Sirtuin(SIRTs)family is dependent on NAD+ levels.SIRTs family includes the members of Sirtl-Sirt7.Studies have shown that Sirtl can play a protective role in the kidney through anti-apoptosis,anti-inflammatory and so on.However,the function of SIRTs in focal segmental glomerulosclerosis is not very clear.Objective1.Detect the expression patterns of SIRTs in FSGS and elucidate the role of Sirt6 in podocyte injury under FSGS.2.Investigate the mechanism by which Sirt6 protects against podocyte injury.Methods and ResultsPart1:The expression pattern and the role of Sirt6 in the pathogenesis of focal segmental glomerulosclerosis1.Detected the expression pattern of Sirt6 In the kidney from ADR miceThe mouse model of adriamycin(ADR)-induced FSGS was successfully constructed by tail intravenous injection.The levels of SIRTs were detected by Western blot(WB).Our results showed that levels of Sirtl,Sirt5 and Sirt6 were reduced in the kidney from ADR mice.However,the levels of Sirtl and Sirt6 were lower than that of other SIRTs.The expression of Sirt6 in podocytes was detected by double labelling immunofluorescence(IF).And the result showed that the expression of Sirt6 was decreased in podocytes from ADR mice.2.The expression of Sirt6 was detected in different renal parenchymal cells with ADR treatmentHuman podocyte cell(HPC),glomerular endothelial cells(GENC),human renal proximal tubular cells(HK-2)and mesangial cells(MC)were treated by ADR.WB analysis showed that the levels of Sirt6 were reduced in GENC,HK-2 and HPC.3.Podocyte-specific deletion of Sirt6 in micePodocyte-specific deletion of Sirt6 mice were generated by Cre-Loxp recombination system.Podocin-cre Sirt6fl/fl(Cre+/Sirt&/flfl)mice were identified by genotyping of mouse tail and the mRNA levels of Sirt6 in isolated glomeruli.Our results showed that Cre+/Sirt6fl/fl mice were successful generated.4.The podocyte-specific deletion of Sirt6 exacerbated podocyte injury in ADR miceEight weeks old wild male C57BL/6J mice and Cre+/Sirt6fl/fl mice were divided into four groups randomly and the model of FSGS was constructed.Urine albumin/creatinine ratio was analyzed after sample collection.Then glomerular morphology was observed by PAS staining and podocyte injury was detected by Transmission Electron Microscope(TEM).Immunofluorescence staining was used to observe the levels of Nephrin and Podocin.Our results showed that urine albumin/creatinine ratio was significantly increased and podocyte injury was exacerbated in ADR Cre+/Sirt6fl/fl mice.5.Sirt6 protected against podocyte injury in ADR miceSirt6 overexpression mice were constructed by Sirt6-lentivirus transfection.Real-time RT-RCR was used to detect the level of Sirt6 mRNA in glomeruli,confirming the successful construction of overexpression mice.Urine albumin/creatinine ratio was analyzed after sample collection.Then glomerular morphology was observed by PAS staining and podocyte morphology was detected by TEM.Immunofluorescence staining was used to observe the levels of Nephrin and Podocin.Our results showed that overexpression of Sirt6 decreased the urine albumin-to-creatinine ratio in ADR Cre+/Sirt6fl/fl mice and attenuated messangial matrix proliferation.The results of TEM and IF showed that overexpression of Sirt6 attenuated foot process fusion and detachment and increased the levels of Podocin and Nephrin.In vitro,the result of WB showed that overexpression of Sirt6 increased the levels of Podocin and Nephrin in podocytes with ADR treatment.The apoptosis ratio was detected by flow cytometry.The results showed that overexpression of Sirt6 decreaced ADR-induced podocyte apoptosis.Part 2:The mechanism by which Sirt6 ameliorates podocyte injuryThe levels of Notchl and Notch4 in podocytes were detected by double labelling immunofluorescence.The results showed that podocyte-specific deletion of Sirt6 increased the levels of Notch1 and Notch4 in podocytes from ADR mice and overexpression of Sirt6 inhibited the expression of Notch1 and Notch4 in podocytes frome ADR Cre+/Sirt6fl/fl mice.In vitro,the results of WB showed that the levels of Notch 1,Notch4 and the downstream taget genes,HES1 and Snail1 was up-regulated in podocytes with ADR treatment and overexpression of Sirt6 alleviated this.Conclusion and innovation1.Our study has identified for the first time that Sirt6 was significantly decreased in the kidney from ADR-induced FSGS mice.Further research found that podocyte-specific deletion of Sirt6 exacerbated podocyte injury and overexpression of Sirt6 ameliorated podocyte injury.2.Sirt6 protects against podocyte injury by negatively regulating Notch signaling pathway.This study helps to further elucidate the pathophysiological mechanisms of kidney injury in FSGS and provides animal experimental basis and theoretical basis for the design and development of Sirt6 related drugs.