Role Of SIRT6 In High Glucose-induced Mitochondrial Dysfunction In Podocyte

Objective:Diabetic nephropathy(DN)is one of the most serious microvascular complications of diabetes and one of the important causes of end-stage renal disease.Podocyte is an important part of the formation of glomerular filtration barrier.Numerous studies have shown that podocyte injury plays a key role in the occurrence and development of proteinuria in diabetic nephropathy.Podocyte injury is considered to be one of the important factors in the prognosis of diabetic nephropathy.Therefore,exploration of the molecular mechanisms of podocyte injury and the molecular target of podocyte protection is very important for delaying the development of diabetic nephropathy.In recent years,various studies have shown that mitochondria damage plays an important role in the development of podocyte injury and diabetic nephropathy.The molecular mechanisms and signaling pathway involved in mitochondrial damage in high glucose-stimulated podocyte remains to be further studied.Sirtuins(silent information regulator 2 proteins)are evolutionarily conserved nicotinamide Adenine Dinucleotide(NAD+)-dependent histone deacetylases,a family which includes SIRT 1-7,which functions in many different biology.Sirtuins 6(SIRT6)is an important member of the Sirtuins family,localized in the nucleus,which has diverse enzymatic activities: deacetylating histones H3K9 and H3K56,with mono-ADP-ribosyltransferase activity and long-chain acylation.Studies have shown that SIRT6 can alleviate high glucose-induced podocyte injury through multiple mechanisms,including inhibiting high glucose-stimulated inflammatory factor secretion and podocyte apoptosis,improving cytoskeletal rearrangement and enhancing podocyte autophagy.At present,studies have suggested that SIRT6 participates in the protection of mitochondrial function in cardiac and skeletal muscle cells.However,whether SIRT6 is involved in the regulation of high glucose-induced mitochondria damage in podocytes is still unclear.In this study,we observed the changes of podocyte mitochondrial morphology and function and evaluated SIRT6 expression in podocytes of DN patients and diabetic mice as well as cultured human podocytes.The effects of SIRT6 on mitochondrial morphology and function of cultured human podocytes stimulated by high glucose were observed by transfecting SIRT6 plasmid and SIRT6 si RNA.We explored the mechanisms by which SIRT6 mediates high-glucose induced mitochondrial damage by transfecting WT SIRT6 plasmid or SIRT6 H133 Y mutant plasmid and down-regulating AMPK expression.Methods:Part 1: 12 male C57BL/6 mice were randomly divided into diabetic nephropathy(DN)group and normal group.Mice in DN group were injected with streptozocin(STZ)buffer for the first time(75mg/kg/10ml)and injected with STZ buffer(150 mg/kg/10 ml)in the subsequent 5 days.Normal control mice were injected with normal saline.Body weight,24 h urine volume,blood glucose,urine microalbumin/urinary creatinine(ACR)were monitored every weekend.Animals were sacrificed at the end of the 12 th week.Kidney weight was recorded;blood samples,24 h urine specimen and kidney tissue specimen were collected for the subsequent biochemical and pathological analysis.Differentiated cultured human podocytes were divided into normal glucose(NG)group(5.0 mmol/L glucose for 24 h)and high glucose(HG)group(30 mmol/L glucose for 24h).The renal specimens from patients with DN diagnosed by renal biopsy were collected.The control samples were para-carcinoma tissues from individuals who underwent tumor nephrectomies.HE and PAS staining were used to observe the pathological changes of kidney in mice.Ultrastructural changes of mice glomerular podocytes and cultured human podocyte were observed by transmission electron microscope(TEM).Mito Tracker Green staining was conducted to observe the number of mitochondria in cultured human podocyte.Mito SOX Red staining was performed to observe the mitochondrial ROS content in human podocyte.The changes of mitochondrial membrane potential in podocyte from each group were detected by JC-1 staining.The apoptosis of podocyte in each group was evaluated by flow cytometry.Immunofluorescence was employed to detect the expression of SIRT6 in glomerular podocyte of patients with DN,glomerular podocyte of mouse and cultured human podocyte in vitro.Western blotting was conducted to assess the expression of SIRT6 in mouse glomeruli and cultured human podocytes.Part 2: Cultured human podocytes transfected with WT SIRT6 plasmid(for 24h)or SIRT6 si RNA(for 48h)were stimulated by high glucose for 24 h,then divided into normal group,high glucose group,high glucose + pc DNA3.1 SIRT6 group,high glucose + empty plasmid group,normal + SIRT6 si RNA group and normal + empty vector group.The ultrastructural changes of mouse glomerular podocyte and cultured human podocyte were observed by TEM.The mitochondrial superoxide content of human podocyte was detected by Mito SOX Red staining.The changes of podocyte mitochondrial membrane potential in each group were determined by JC-1 staining.The apoptosis of podocyte in each group was assessed by flow cytometry.Part 3: Western blotting was performed to assess the expression of p-AMPK and AMPK in mouse glomeruli and high glucose-treated human podocytes.Cultured human podocytes were transfected with WT SIRT6 plasmid or SIRT6 H133 Y mutant plasmid.The human podocytes transfected with WT SIRT6 plasmid were then treated by AMPK inhibitor Compound C.Subsequently podocytes were then cultured under high glucose conditions for 24 h after the above pretreatment,and were divided into normal group,high glucose group,high glucose + pc DNA3.1 SIRT6 group,high glucose + pc DNA3.1 group,high glucose + pc DNA3.1 SIRT6-H133 Y group,high glucose + pc DNA3.1 SIRT6 + Compound C group.The ultrastructural changes of mouse glomerular podocyte and cultured human podocyte were observed by TEM.The mitochondrial superoxide content of human podocyte was detected by Mito SOX Red staining.The changes of podocyte mitochondrial membrane potential in each group were determined by JC-1 staining.The podocyte apoptosis in each group was assessed by flow cytometry.Western blotting was conducted to evaluate the expression of SIRT6,p-AMPK,AMPK,H3K9 AC and H3K56 AC in cultured human podocytes from different group.ResultsPart 1:(1)DN mice showed obvious mesangial cell proliferation,basement membrane thickening and extensive fusion of glomerular foot processes.Compared with the normal group,the body weight of DN mice was significantly reduced(P<0.05),the blood glucose was increased(P<0.05),kidney/body weight ratio was increased(P<0.05),and ACR was increased(P<0.05);(2)high glucose stimulation resulted in abnormal mitochondrial morphology as well as mitochondria swelling,vacuolization,rupture and disappearance;(3)high glucose stimulation caused a decrease of mitochondria number(P<0.05),an increase of mitochondria superoxide level(P<0.05),a decrease of mitochondrial membrane potential(MMP)(P<0.05)and increased apoptosis(P<0.05)in cultured human podocytes;(4)SIRT6 was widely distributed in renal glomeruli,including podocytes.The expression level of SIRT6 in podocyte from DN patients was significantly decreased(P<0.05).SIRT6 expression was significantly reduced in podocytes of DN mice and high-glucose treated human podocytes.Part 2:(1)SIRT6 plasmid transfection can alleviate the mitochondrial morphological disorder in high-glucose induced human podocytes,mitochondrial swelling and vacuoles formation of podocytes were alleviated;(2)Compared with high glucose group,SIRT6 plasmid transfection reduced mitochondrial ROS level(P<0.05),increased mitochondrial membrane potential(MMP)(P<0.05)and decreased podocyte apoptosis(P<0.05);(3)Mitochondria became swelling in the SIRT6 si RNA transfected group compared with normal group,and the mitochondrial cristae became disorder;(4)Compared with normal group,SIRT6 si RNA transfection increased the mitochondrial ROS levels(P<0.05),decreased mitochondrial membrane potential(MMP)(P<0.05),and increased podocyte apoptosis(P<0.05).Part 3:(1)An increased AMPK dephosphorylation level was observed in glomeruli from DN mouse and high-glucose induced podocytes;(2)Transfection of podocytes with WT SIRT6 plasmid or SIRT6 H133 Y mutant plasmids can alleviate high glucose-induced mitochondria damage and podocyte apoptosis in human podocytes.Also,AMPK phosphorylation level was up-regulated;(3)Inhibition of AMPK had no effect on SIRT6 expression,which could attenuate the mitochondrial protective function mediated by WT SIRT6 plasmid transfection in podocytes under high glucose treatment;(4)WT SIRT6 plasmid transfection can inhibit high glucose-induced H3K9 and H3K56 acetylation in human podocytes.SIRT6 H133 Y mutant plasmids transfection had no effect on H3K9 and H3K56 acetylation levels.Conclusion: AMPK is the downstream signaling molecule of SIRT6.SIRT6 mediates high glucose-induced mitochondrial damage in podocytes by regulating AMPK signaling pathway.This effect is independent of H3K9 and H3K56 deacetylation by SIRT6.