The Effects Of GCN5L1-mediated MnSOD Acetylation On Oxidative Stress In Diabetic Kidney Disease

Increasing evidences have demonstrated that diabetic kidney disease(DKD)mainly caused by diabetes is one of the main causes for chronic kidney disease and end-stage renal disease.The main therapeutic strategies for the treatment of DKD are limited in regulating blood glucose,antihypertension and decreasing proteinuria via applying inhibitors of renin-angiotensin system(RAS).However,those ways had limited effect in delaying DKD progression and cannot reverse the injury caused by DKD.At the stage of end-stage renal disease,those patients had to be dependent on renal replacement therapy.Therefore,it is urgent to define the mechanism of DKD and find the promising therapeutic target.Hyperglycemia leads to the excessive production of generating nicotinamide adenine dinucleotide(NADH)and flavin adenine dinucleotide(FADH2),which contributes to generation of mitochondrial electron transport chain and reactive oxygen species(ROS).Evidences have demonstrated that overproduction of ROS is a significant pathway for the pathogenesis of DKD.The imbalance between the generation of ROS and the antioxidant system including glutathione redox system,superoxide dismutase system and peroxidase contributes to the accumulation of ROS.Manganese superoxide dismutase(SOD2,Mn-SOD)is a main antioxidant enzyme located in the mitochondrial matrix and catalyzes the conversion of O2·-to H2O2.Some papers have demonstrated that the elimination of ROS is dependent on the amount of Mn-SOD,However,increasing evidences have reported that enzymatic activity of Mn-SOD has a more significant role in regulating the antioxidant function.According to the researches,reversible lysine acetylation can orchestrate the enzymatic activity of Mn-SOD.However,the level of Mn-SOD acetylation and how it is regulated in DKD is unclear.General control of amino acid synthesis 5 like-1(GCN5L1)is an important protein sharing the sequence homology with the nuclear acetyltransferase GCN5.GCN5L1 is primarily found to bind several proteins in the cytoplasm and function in endo-lysosomal trafficking and biogenesis.Recently,researches have found that GCN5L1 is located at mitochondria and regulates the mitochondrial function by acetylating mitochondrial proteins.GCN5L1 participates in several mitochondria functions including mitosis,mitochondrial biogenesis and fatty acid oxidation(FAO).In addition,GCN5L1 functions to be an energy sensor and regulate several cellular functions including mitochondrial metabolism.For exampling,human embryonic kidney 293T cells cultured in the medium of lacking energy could regulate mitochondrial protein synthesis,mitochondrial respiration and energy metabolism by GCN5L1-induced acetylation of mitochondrial proteins in vitro.Several evidences have clarified that GCN5L1 is the key protein involved in the development and progression of several metabolic diseases including fatty liver disease,and myocardial damage in diabetes and ischemia reperfusion.Essentially,DKD is a disease with oversupplied energy.Therefore,we hypothesize that GCN5L1 may be involved in the progression of DKD.In this study,we tried to explore whether GCN5L1 can promote the progression of DKD by acetylating Mn-SOD and hope to find a promising therapeutic target for DKD.Objectives:In this study,DKD was applied to explore the role and specific molecular mechanism of GCN5L1 in the progression of diabetic nephropathy and tried to find a promising therapeutic target for DKD.This research objectives are as follows:(1)Find the expression of GCN5L1 in DKD.(2)Try to explore the mechanism of GCN5L1 regulating the acetylation of Mn-SOD.(3)Find the roles of GCN5L1-MnSOD-ROS pathway in the progression of DKD.(4)Exploring the upstream mechanism of GCN5L1 upregulation in DKD.Methods and Results:1.The expression of GCN5L1 is largely increased in DKD.(1)GCN5L1 is overexpressed in the tissues of DKD patients.We collected kidney biopsies and normal kidney tissues from DKD patients and renal tumor patients.Immunohistochemistry(IHC)was used to detect the expression of GCN5L1 in DKD tissues.The results showed that the expression of GCN5L1 was increased in those tissues.(2)GCN5L1 is upregulated in DKD mice models.In order to detect the expression of GCN5L1 in DKD mice models,we first established a type I diabetic nephropathy mice models by treating STZ.50mg/kg STZ was intraperitoneally injected after fasting for 5 consecutive days,and the same volume of sterile citrate buffer was intraperitoneally injected in control group.IHC and western blotting were used to detect the expression of GCN5L1 in the renal tissues of diabetic mice.The results showed that GCN5L1 was highly expressed in the renal tissues of diabetic nephropathy mice,which was coincident with the results of clinical pathological staining.(3)The glomerulus and renal tubule are damaged severely in diabetic mice.H/E staining,Masson staining and PAS staining were used to detect the degree of renal tissue damage in diabetic nephropathy mice.H/E staining showed glomerular hypertrophy and hyperplasia,thickening of the mesangial basement membrane,vacuolar degeneration of renal tubules,and empty cytoplasm in STZ group.Masson staining showed that a large amount of collagen fibers was deposited in the mesenchyme of glomeruli and renal tubules in STZ group,and the mesenchyme was widened with extensive fibrosis.PAS staining demonstrated the thickened glomerular basement membrane,mesangial hyperplasia,mesangial matrix precipitation,and increased glomerular volume in STZ group.During the time,urine was collected,and the urine protein and creatinine were determined by enzyme-linked immunosorbent assay(ELISA).The results showed increased urine protein to creatinine ratio(UACR)in STZ group,indicating that the kidney injury was obvious in diabetic mice.(4)GCN5L1 expression is increased in renal tubular epithelial cells(TECs).After the treatment of TECS with high glucose(30mM)for 48 hours,the expression of GCN5L1 was detected by Western blotting and immunofluorescence(IF)staining.The results showed that the expression of GCN5L1 was significantly increased in TECs treated with high glucose.2.Downregulation of GCN5L1 improves the kidney injury and albuminuria of DKD mice.(1)Knockdown of GCN5L1 improves the kidney injury and albuminuria of DKD mice.To further elucidate the importance of GCN5L1 in regulating renal injury,kidney-specific GCN5L1 knockdown(KD)in mice was performed by locally injecting the GCN5L1 shRNA-expressing adenovirus associated virus into the renal cortex of C57BL/6 mice,along with STZ treatment for about sixteen weeks.H/E staining,Masson staining and PAS staining were used to detect the degree of renal tissue injury.H/E staining showed that GCN5L1 knockdown can improve the glomerular hy pertrophy and hyperplasia,glomerular mesangial basement membrane thickening,renal tubular vacuolar degeneration,and cytoplasm vacuity caused by STZ treatment.Masson staining demonstrated that GCN5L1 knockdown decreased the large amount of collagen fiber deposition in the glomerulus and renal tubule interstitium,and the widened interstitium with extensive fibrosis caused by STZ treatment changes.PAS staining showed that the glomerular basement membrane was significantly thickened,mesangial hyperplasia,mesangial matrix precipitation and glomerular volume increased significantly in the kidney tissue of mice in the STZ group,which was significantly improved by knocked down GCN5L1 expression.At the same time,urine was collected and the UACR was determined by ELISA.The ELISA results showed that the UACR was increased in the STZ group and GCN5L1 knockdown can significantly decrease the UACR.These results suggested that downregulation of GCN5L1 can improve renal injury and proteinuria in DKD mice.3.The role and mechanism of GCN5L1 in regulating Mn-SOD acetylation under high glucose environment.(1)GCN5L1 interacts with Mn-SOD.In order to detect the relationship between GCN5L1 and Mn-SOD,co-precipitation test(CO-IP)and Proximity Ligation Assay(PLA)were used to detect whether GCN5L1 can interact with Mn-SOD.The results proved that there was mutual binding between GCN5L1 and Mn-SOD.(2)Downregulation of GCN5L1 inverses the high glucose-induced acetylation of Mn-SOD at K68,decreased enzymatic activity and increased ROS.In order to detect whether the acetylation level and enzyme activity of Mn-SOD were changed under high glucose conditions,we used western blotting and ELISA to detect the acetylation level and enzyme activity of Mn-SOD under high glucose conditions.The results showed that the acetylation level of Mn-SOD was increased and the enzyme activity of Mn-SOD was decreased under high glucose condition.ROS was detected by IF under high glucose condition,and it was found that the production of ROS in mitochondria was increased.However,the decreased acetylation level of Mn-SOD induced,increased enzyme activity of Mn-SOD,decreased mitochondrial ROS was observed when GCN5L1 was knocked down under high glucose condition.(3)Downregulation of GCN5L1 inverses increased acetylation of Mn-SOD at K68 and ROS level in DKD mice tissues.We further explored the effect and mechanism of GCN5L1 on diabetic renal injury in vivo.First,we measured the acetylation of Mn-SOD at K68 in mouse kidney tissues.IHC staining showed that GCN5L1 knockdown significantly reduced the acetylation level of Mn-SOD at K68 site in STZ-induced diabetic mice model.Then,we used DHE,a reactive dye sensitive to ROS,to detect the ROS.The results showed that GCN5L1 knockdown reduced ROS production in STZ-induced diabetic mice kidney tissues.4.Knockdown of GCN5L1 improves kidney injury in diabetic mice by inhibiting Mn-SOD/ROS pathway.(1)GCN5L1 promotes activation of NLRP3 inflammasome.The production of excessive mitochondrial ROS induced by diabetes was crucial for the activation of NLRP3 inflammasome.Our above works found that GCN5L1 was significantly overexpressed under high glucose conditions,and downregulation of GCN5L1 could decrease ROS level induced by high glucose stimulation.This hinted that GCN5L1 overexpression may promote the inflammatory response through ROS-induced NLRP3 inflammasome activation.To test this hypothesis,we upregulated GCN5L1 expression and then a ROS inhibitor N-acetyl-L-cysteine(NAC)was used in TECS.Western blotting showed that the expression levels of NLRP3,active-caspase-1,active-IL18 and active-IL1β were significantly upregulated after upregulation of GCN5L1.However,the treatment of ROS inhibitor NAC effectively blocked the up-regulation of NLRP3,active-caspase-1,active-IL18 and active-IL1βmediated by GCN5L1 overexpression.Those results suggested that GCN5L1 promoted the activation of NLRP3 inflammasome.(2)GCN5L1 Knockdown improves kidney injury in diabetic mice by disrupting Mn-SOD/ROS pathway.We then explored the molecular mechanism of GCN5L1-induced NLRP3 activation.Western blotting showed enhanced expression of NLRP3,caspase-1,IL18 and IL1β in TECs treated with high glucose.In contrast,GCN5L1 knockdown inhibited the expression of these proteins under high glucose conditions.This suggested that knockdown of GCN5L1 may inhibit the inflammatory response by eliminating cellular ROS production.We then transfected the Mn-SOD K68-R mutant plasmid(the lysine residue was replaced by arginine)into TECs with GCN5L1 knockdown.Western blotting showed that the expressions of NLRP3,caspase-1,IL18,and IL1β were significantly reduced,which suggested that GCN5L1-induced acetylation of Mn-SOD K68 promoted inflammatory response through ROS-induced oxidative stress.(3)GCN5L1 promotes Endothelial mesenchymal transformation(EMT).EMT triggered by ROS promoted renal fibrosis associated with diabetic nephropathy.Under this context,whether GCN5L1 can affect EMT through the Mn-SOD/ROS pathway under high glucose conditions was unclear.First,we examined the expression of epithelial marker E-cadherin and stromal marker a-SMA in GCN5L1 overexpressed TECs by Western blotting.The results showed the decreased E-cadherin and enhanced a-SMA expression in renal tubular epithelial cells with GCN5L1 overexpression.However,NAC treatment reversed GCN5L1-medicated E-cadherin and a-SMA expression.These results suggested that GCN5L1 can promote EMT of renal tubular epithelial cells.(4)GCN5L1 Knockdown reverses high glucose-induced EMT by disrupting Mn-SOD/ROS pathway.We used western blotting to detect the expression of E-cadherin and a-SMA in TECs.The results showed that the expression of E-cadherin was significantly decreased and a-SMA expression was largely enhanced in TECs under high glucose conditions.However,GCN5L1 knockout effectively reversed the expression of E-cadherin and a-SMA.Then,we detected whether GCN5L1-induced EMT was mediated by Mn-SOD acetylation.We co-transfected GCN5L1 siRNA and Mn-SOD K68-R mutant plasmid in TECs.Western blotting showed that GCN5L1 downregulation increased E-cadherin expression and decreased a-SMA expression,which was significantly reversed by the Mn-SOD K68-R mutant.These results indicated that GCN5L1 can acetylate Mn-SOD K68 under high glucose condition,and then promote EMT through mitochondrial ROS.(5)GCN5L1 Knockdown reverses high glucose-induced inflammatory and EMT by disrupting Mn-SOD/ROS pathway in kidney tissues of diabetic mice.We further verified the mechanism of GCN5L1-promoted diabetic nephropathy in vivo.IHC showed that NLRP3 was significantly upregulated in diabetic mice compared with control mice.However,NLRP3 expression was decreased in kidney tissues of diabetic mice with renal GCN5L1 knockdown.Western blotting exhibited similar results in kidney tissues.Furthermore,downregulation of E-cadherin expression and upregulation of a-SMA expression was observed by IHC and Western blotting.GCN5L1 knockdown improved the kidney fibrosis in diabetic mice.In summary,these results suggested that downregulation of GCN5L1 protected the kidney from hyperglycemia-induced EMT and inflammasome activation through the Mn-SOD/ROS pathway.5.STZ treatment increases GCN5L1 expression by reducing its ubiquitination.(1)GCN5L1 mRNA expression had no significant changes between control and STZ group.In order to study the mechanism of increased GCN5L1 expression with STZ treatment,we first detected the mRNA expression of GCN5L1 under STZ treatment.According to the results of RT-qPCR,we found that this mRNA expression had no significant changes between control and STZ group.Above works have demonstrated that GCN5L1 expression was upregulated in vitro and in vivo under hyperglycemia.Therefore,we hypothesized that STZ treatment may increase GCN5L1 expression through a post-translational mechanism.(2)Decreased ubiquitination level of GCN5L1 contributes to GCN5L1 upregulation.Ubiquitination is crucial for physiological processes which are controlled by Ubiquitin and deubiquitinating enzymes.One of the important cellular functions of the ubiquitin-proteasome system(UPS)is to selectively degrade damaged or abnormal proteins.Under these backgrounds,we used immunoprecipitation assay to detect the ubiquitination level of GCN5L1.The results showed that the ubiquitination level of GCN5L1 was decreased under the STZ treatment,which induced the GCN5L1 overexpression in protein level.Conclusions:1.GCN5L1 was overexpressed in diabetic mice.2.Knockdown of GCN5L1 improved the kidney injury triggered by STZ treatment in diabetic mice.3.Downregulation of GCN5L1 induced inhibition of Mn-SOD acetylation,increase of enzymatic activity of Mn-SOD,and promotion of ROS in vitro and in vivo.4.Downregulation of GCN5L1 improved the inflammatory and reversed EMT in vitro and in vivo.5.Decreased ubiquitination level of GCN5L1 contributes to GCN5L1 upregulation.Highlights in this studyIn this work,we first found that GCN5L1 was overexpressed in DKD patients and diabetic mice.Knockdown of GCN5L1 improved the kidney injury and albuminuria in diabetic mice.GCN5L1 downregulation impaired the inflammatory response and reversed EMT by inhibiting Mn-SOD acetylation and decreased ROS.Decreased ubiquitination level of GCN5L1 contributes to GCN5L1 upregulation.GCN5L1 may be a promising therapeutic target in DKD.