Study On The Role And Mechanism Of Glycogen Synthase Kinase 3β In Regulation Of Renal Aging

Aging is a process of gradual functional deterioration at the cellular and organismal levels,which leaves an individual susceptible to environmental or internal stress followed by increasing rates of disease and death.Renal aging is also a complex interaction between genetics,environmental changes and cellular dysfunction,leading to structural and functional changes.Although aging per se does not cause kidney injury,physiologic changes associated with normal aging processes are likely to impair the reparative capacity of the kidney and thus predispose older people to acute kidney injury（AKI）,chronic kidney disease（CKD）,and other renal diseases.Moreover,renal aging affects the healthcare providers’choice of medications when treating diseases,and is a major determinant of the outcome of kidney transplant recipients and donors.Age-related renal decline is associated with the development of age-related glomerulosclerosis.An estimated 6000–6500 nephrons will be lost every year after30 years old,due to nephrosclerosis especially glomerulosclerosis.A crucial and still largely unresolved question is why nephrons perish during healthy aging?As terminally differentiated cells,podocytes play a central role in renal aging.Podocytes are highly differentiated neuron-like epithelial cell with limited capacity for cell division and replacement.There is already evidence that shows the glomerulosclerosis of aging is“a podocyte disease”.With the increase of global population aging,renal aging or age-related renal impairment has become an imminent challenge to clinical practice.Therefore,it is of great significance to find a new therapeutic target to delay the aging of kidney.Glycogen synthase kinase-3s（GSK3s）are ubiquitously expressed in mammalian eukaryotic cells,constitutively active,proline-directed serine/threonine kinases involved in diverse cellular processes and was discovered in the 1980s.More and more studies have found that GSK3 is an important regulator of cells and a variety of signaling pathways,regulating cell survival,proliferation,differentiation and apoptosis,and regulating a variety of signaling pathways,and thus participating in embryogenesis,tissue damage,repair and regeneration,inflammation and immune regulation.There are two subtypes of GSK3,αandβ,which have very similar primary molecular structures,but they have different molecular"tails,"which are slightly different at the N-and C-ends,resulting in completely different biological roles.GSK3αis a longevity regulator,while GSK3βis a regulator that promotes aging.There is growing evidence that GSK3 is involved in a range of diseases,including Type 2 diabetes,Alzheimer’s disease,Cancer,Inflammatory diseases,Mitochondrial diseases,Schizophrenia,and bipolar disorder.Micro-dose of lithium,a GSK3 inhibitor,have been shown to extend the expectancy of humans,nematodes and fruit flies.Low-dose lithium was found to promotes Drosophila longevity through GSK3 inhibition and subsequent Nrf2 activation,suggesting that GSK3 may be a potential drug target for aging.Converging evidence points to GSK3βas a key player in the pathogenesis of podocytopathy and proteinuria.In accordance with this,blockade of GSK3βhas been shown to attenuate proteinuria,podocyte injury,and glomerulosclerosis in a number of experimental glomerular diseases,including diabetic Nephropathy,adriamycin（ADR）nephropathy and nephrotoxic serum（NTS）nephritis.Very little,however,is known about the role of GSK3βin renal aging.Therefore,we observed the changes of GSK3βexpression and its effect on renal aging in human normal renal tissues,podocyte-specific GSK3βgene knockout and Li Cl treatment animal models,and in vitro cultured podocytes.Part one The relationship between GSK3βoverexpression and kidney aging in human normal kidney tissuesObjectiveTo observe the pathological changes of kidney in normal kidney specimens from different aged patients,examine the expression of GSK3βand the aging marker p16^INK4A,analyze the relationship between GSK3βand renal aging,and clarify the role of GSK3βin human kidney aging.Methods1.Nontumor kidney tissues were collected from 3 different aged patients who had renal cell carcinoma and underwent nephrectomy,and they were divided into young group,middle-aged group,and older subjects group according to age<30years old,30-59 years old and 60-79 years old.2.The renal pathological changes in the 3 groups were observed by Periodic acid-Schiff（PAS）,Masson staining and electron microscopy.3.According to the results of PAS,Masson staining and electron microscopy,the proportion of sclerotic glomeruli,interstitial fibrosis score and podocyte foot process width were quantitatively analyzed in the three groups.4.The co-localization of Podocin and WT-1 was detected by dual color immunofluorescent staining.5.According to the results of immunofluorescence staining,the average number of WT-1 positive cells per glomerular cross section per patient of the 3 groups were quantitatively counted.Linear regression was used to analyze the average number of WT-1 positive cells per glomerulus and the estimated glomerular filtration rate（estimated glomerular filtration rate,e GFR）.6.Gene Set Enrichment Analysis（GSEA）was used to detect the relationship between GSK3βgene expression and renal aging.7.Immunohistochemical staining was used to examine the expression of GSK3βand p16^INK4A.8.Linear regression analyses of the relative glomerular staining intensity of GSK3βand that of p16^INK4A or the average number of WT-1 positive podocytes per glomerular cross section per subject or the percentage of global glomerulosclerosis or estimated glomerular filtration rate（e GFR）.Results1.The percentage of glomeruli with global glomerulosclerosis in glomeruli was higher in older subjects group than the others（P<0.01）.2.Morphometric scoring of interstitial fibrosis by Masson staining of kidney tissue was increased in the three group with age（P<0.01）.3.The mean width of foot process width increased with age in the three different aged groups（P<0.001）.4.Estimated glomerular filtration rate（e GFR）and the average number of WT-1positive cells per glomerulus significantly decreased with age in the three groups（P<0.001）.5.Scatter plots with linear regression showed a positive correlation between the average number of WT-1 positive cells per glomerulus and e GFR in three different aged groups（R=0.65,P<0.01）.6.The results of gene enrichment analysis showed a significant enrichment of GSK3βgene sets associated with kidney aging progression（P<0.001）.7.The average immunohistochemical staining score of GSK3βand p16^INK4A in each glomerulus was calculated by Image-pro Plus Image analysis software,which was expressed by the ratio of cumulative optical density（IOD）)/observed area,representing the relative expression levels of GSK3βand p16^INK4A.The correlation between GSK3βexpression levels and p16^INK4A expression levels was analyzed by scatter plots with linear regressio in young,middle-aged and older subjects’groups,and the results showed they had a significantly positive correlation（R=0.87,P<0.0001）.8.Linear regression showed that GSK3βexpression level was significantly positively correlated with glomerulosclerosis in the three groups（R=0.89,P<0.0001）,and an inverse correlation between GSK3βexpression level and WT-1expression level（R=-0.67,P<0.0001）and e GFR（R=-0.72,P<0.001）.Conclusions1.The characteristic changes of kidney aging,such as glomerulosclerosis,interstitial fibrosis and foot processes effacement,podocyte detachment and cytoplasmatic absorption droplets increase with age,whereas,the e GFR gradually decreases in human.2.GSK3βexpression in glomeruli gradually increases with age and is positively correlated with aging marker p16^INK4A and glomerular sclerosis,whereas negatively correlated with the average number of WT-1 positive cells per glomerulus and e GFR,suggesting that GSK3βexpression is closely related to human kidney aging.Part two Effect of targeted inhibition of GSK3βon renal aging in C57BL/6 miceObjective1.To observe the pathological changes of kidney and verify the correlation between GSK3βand renal aging in 2 months old,12 months old,and 24 months old C57BL/6 mice.2.To examine whether podocyte specific knockout of GSK3βand systemic administration of Li Cl could regulate renal aging in C57BL/6 mice.Methods1.The C57BL/6 mice were divided into 3 groups by age:2 months,12 months,24 months.The mice urine was subjected to SDS-PAGE followed by Coomassie brilliant blue staining.Quantification of serum creatinine（Scr）level and urine albumin level adjusted by urine creatinine concentration.The changes of renal pathology in mice were observed by PAS staining and Electron microscopy.Senescence-associatedβ-galactosidase（SA-β-gal）staining of glomerular was detected in the 3 mice groups.2.The expression of WT-1,Podocin and Fibronectin were detected by immunofluorescent staining in glomeruli.The protein levels of Podocin and Fibronectin were detected by immunoblot analysis.Immunohistochemical staining was adopted to test the expression of GSK3β,and p16^INK4A in glomeruli.3.The protein levels of GSK3β,phosphorylated GSK3βat serine 9(p-GSK3β^S9),p16^INK4A,p53,p21,and p-Rb were tested by western blot analysis.4.Using Cre/lox P Technique to Construct of Conditional GSK3βgene Knockout Mice.Immunohistochemical staining was used to test whether podocyte specific deletion of GSK3βis successful.5.The mice were divided into 4 groups:12 months old wild type mice（12-Con）,12 months old knockout mice（12-KO）,24 months old wild type mice（24-Con）,and24 months old knockout mice（24-KO）.The mice urine was subjected to SDS-PAGE followed by Coomassie brilliant blue staining.Quantification of serum creatinine level and urine albumin level adjusted by urine creatinine concentration.PAS staining and Electron microscopy was used to observe the changes of renal pathology in the four groups.Senescence-associatedβ-galactosidase（SA-β-gal）staining of glomerular was detected in the four groups.6.The expression of WT-1,Podocin and Fibronectin were detected by immunofluorescent staining in glomeruli.The protein levels of Podocin and Fibronectin were examined by immunoblot analysis and the expression of p16^INK4Awas detected by immunohistochemical staining.7.The protein levels of p16^INK4A,p53,p21,Podocin,Fibronectin,and p-Rb were tested by immunoblot analysis.8.12 months old C57BL/6 mice were randomly divided into two groups,Li Cl or Na Cl were given weekly through subcutaneous injection for 3 or 6 months,that is,the end points of mice were 15 months and 18 months.9.The mice urine was subjected to SDS-PAGE followed by Coomassie brilliant blue staining.Quantification of urine albumin level adjusted by urine creatinine concentration and serum creatinine level.Immunofluorescent staining was used to examine the expression of WT-1,and SA-β-gal staining of glomerular was detected in mice.10.The protein levels of p16^INK4A,p53,p21,and p-Rb were analyzed by western blot analysis.Results1.The urine albumin/creatinine ratio and serum creatinine levels were gradual increased in 2 months,12 months,and 24 months groups（P<0.001）.2.The percentage of glomeruli with global glomerulosclerosis in glomeruli was higher in 24 months group than other groups（P<0.001）.The average width of foot process width and the number of SA-β-gal positive cells per glomerulus both increased with age（P<0.01）.3.The average number of WT-1 positive cells per glomerulus and the protein level of Podocin decreased,while Fibronectin increased in glomeruli with age（P<0.001）.4.The average expressions of GSK3βand p16^INK4A in each glomerulus increase with age in 2 months,12 months and 24 months groups and have a significantly positive correlation（R=0.88,P<0.0001）.Linear regression also showed that GSK3βexpression level was significantly positively correlated with glomerulosclerosis（R=0.84,P<0.0001）or serum creatinine（R=0.72,P<0.001）.5.The urine albumin/creatinine ratio and serum creatinine levels in 12 months old knockout mice（12-KO）group decreased significantly contrast to 12-Con group（P<0.05）,24-KO group also was significantly lower than that in 24-Con group（P<0.05）.6.The average width of podocyte foot process in 12-KO or 24-KO group was significantly narrower than that in 12-Con or 24-Con group（P<0.05）.7.The protein level of Fibronectin,p16^INK4A,p53,and p21,as well as the average number of SA-β-gal positive cells in each glomerulus of 12-KO or 24-KO group were significantly lower than that in 12-Con or 24-Con group（P<0.05）.However,the protein level of p-Rb was significantly higher than that in 12-Con or24-Con group（P<0.05）.8.The protein levels of Podocin in glomeruli and the average number of WT-1positive cells per glomerulus of 12-KO or 24-KO group were significantly higher than that in 12-Con or 24-Con group（P<0.05）.9.The average WT-1 positive cells in each glomerulus of 15M-Li Cl or 18M-Li Cl group were significantly higher than that in 15M-Na Cl or 18M-Na Cl group（P<0.05）.10.The urine albumin/creatinine ratio,serum creatinine level,the average SA-β-gal positive cells per glomerulus,and the protein level of p16^INK4A,p53,and p21 in the glomeruli of 15M-Li Cl or 18M-Li Cl group were significantly lower than that in 15M-Na Cl or 18M-Na Cl group（P<0.05）.However,the protein level of p-Rb was significantly higher than that in 15M-Na Cl or 18M-Na Cl group（P<0.05）.Conclusions1.The glomerular senescence of C57BL/6 mice gradually becomes worse with age.The expression and activity of GSK3βin glomeruli gradually increases,and is significantly positively correlated with aging marker p16^INK4A and glomerulosclerosis,and negatively correlated with the average number of WT-1 positive cells per glomerulus and e GFR,suggesting that GSK3βis closely related to mouse renal aging.2.Podocyte specific knockout of GSK3βattenuates renal aging in C57BL/6mice.3.Inhibition of GSK3βby Li Cl ameliorates renal aging in C57BL/6 mice.Part three In vitro,effect of targeted activation or inhibition of GSK3βon podocyte senescence and cellular senescence signal pathwaysObjective1.To observe the changes of podocyte senescence through primary culture of podocytes,conditionally immortalized murine podocytes were transiently lipotransfected with a control empty plasmid vector（EV）,or plasmids encoding the HA-conjugated dominant-negative kinase dead（KD）mutant of GSK3βor constitutively active（S9A）mutant of GSK3βin the presence or absence of lithium chloride（Li Cl,10m M）or an equal volume of vehicle.2.To further decipher the molecular mechanism underlying the GSK3βmodulated senescence signaling,the physical relationships between GSK3βand diverse senescence signaling molecules such as p16^INK4A or p53 were examined in isolated glomeruli and in cultured podocytes.Methods1.Primary podocytes were cultured from glomeruli isolated from 12-month-old control mice（Con）and mice with podocyte-specific GSK3βknockout（KO）.Primary podocytes were subjected to electroporation-based transfection with either an empty plasmid vector（EV）or a plasmid encoding the HA-conjugated WT GSK3βby using the Amaxa Nucleofection kit.The cells were divided into 3 groups:Con+EV group,KO+EV group,and KO+WT group.2.The expression of synaptopodin,andγH2AX counterstained with rhodamine phalloidin staining of F-actin were detected by immunofluorescence staining.SA-β-gal staining was used to detect SA-β-gal activity.Absolute count of the number ofγH2AX positive cells expressed as percentages of the total number of cells per microscopic field.Quantification of the SA-β-gal positive cells as percentages of the total number of cells per microscopic field.The protein levels of synaptopodin,γH2AX,GSK3β,p16^INK4A,p53,p21,and p-Rb were detected by Western blot.3.GPS 5.0（http://gps.biocuckoo.cn/）was used to predict GSK3βphosphorylation consensus motifs in p16^INK4Aand p53 proteins.4.To examine potential interactions between GSK3βand p16^INK4A or p53,homogenized isolated renal glomeruli and whole cell lysates were subjected to immunoprecipitation for GSK3β.The antibodies against p16^INK4A or p53 was used to probe the immunoprecipitates by immunoblot analysis.5.Immunofluorescence staining for GSK3βand p16^INK4A or p53 in mouse kidney tissues or in cultured podocytes was conducted.6.Cultured immortalized murine podocytes were transiently transfected with vectors encoding the empty vector（EV）,constitutively active GSK3βor（S9A）dominant negative kinase-dead（KD）.After transfection,cells were treated with 10mmol/L lithium chloride（Li Cl）or an equal volume of vehicle.The cells were divided into 4 groups:EV group,S9A group,KD group,EV+Li Cl group.7.Immunofluorescence was used to detect the expression of synaptopodin andγH2AX counterstained with rhodamine phalloidin staining of F-actin.SA-β-gal staining was used to detect SA-β-gal activity.Absolute count of the number ofγH2AX positive cells expressed as percentages of the total number of cells per microscopic field.Quantification of the SA-β-gal positive cells as percentages of the total number of cells per microscopic field.8.The protein levels of Synaptopodin,HA,γH2AX,GSK3β,p16^INK4A,p53,p21,and p-Rb in each group were analyzed by western blot analysis.9.Cell lysates were subjected to immunoprecipitation for p16^INK4A or p53.Immunoprecipitates were processed for immunoblot analysis for phosphorylated serine（p-Ser）.Results1.The protein level of Synaptopodin or p-Rb in KO+EV group was higher than in Con+EV group or KO+WT group（P<0.05）.The protein level ofγH2AX,p16^INK4A,p53 or p21 in KO+EV group was significantly lower than in Con+EV group or KO+WT（P<0.05）.The cytoskeleton integrity of KO+EV group was better than that of Con+EV group or KO+WT group.2.The number ofγH2AX or SA-β-gal positive podocytes expressed as percentages of the total number per microscopic field in KO+EV group was less than that in Con+EV group or KO+WT group（P<0.05）.3.In silico revealed that a number of amino acid residuals in p16^INK4A or p53reside in the consensus motifs for phosphorylation by GSK3βwith statistically significant prediction scores,inferring that p16^INK4A or p53 are likely putative cognate substrates for GSK3β.4.P16^INK4A and p53 were found to coprecipitate with GSK3βconsistently in both glomerular homogenates and podocyte lysates.5.Laser scanning confocal fluorescence microscopy of dual color fluorescent immunocytochemistry staining of mouse kidney specimens and cultured podocytes for GSK3βand p16^INK4A or p53 showed that p16^INK4A or p53 staining colocalized with a discrete pool of GSK3βstaining,which distributed mainly in cytoplasm and,to a much lesser extent,in nuclei of podocytes.6.The protein level of Synaptopodin or p-Rb in KD group and EV+Li Cl group was higher than EV group and S9A group（P<0.05）.The protein levels ofγH2AX,p16^INK4A,p53,and p21,as well as the number ofγH2AX or SA-β-gal positive podocytes expressed as percentages of the total number per microscopic field in KD group and EV+Li Cl group were significantly lower than EV group and S9A group（P<0.05）.7.The protein levels of p-Ser-p16^INK4Aand p-Ser-p53 in KD group and EV+Li Cl group were lower than EV group and S9A group（P<0.05）,and S9A group was significantly higher than EV group（P<0.05）.Conclusions1.Podocyte specific GSK3βgene knockout has a protective effect on podocyte senescence,possibly by inhibiting p16^INK4A/p-Rb and p53/p21 signaling pathways.2.Continuous activation of GSK3βenhances p16^INK4A/p-Rb and p53/p21signaling pathways,and accelerates podocyte senescence.3.GSK3βinhibition by transfection of plasmid KD inhibits p16^INK4A/p-Rb and p53/p21 signaling pathways,and alleviates podocyte senescence.4.GSK3βinhibition by Li Cl inhibits p16^INK4A/p-Rb and p53/p21 signaling pathways and alleviates podocyte senescence.