| Chronic kidney disease(CKD)is becoming a global public health problem with its increasing morbidity,limited treatment,poor prognosis and expensive medical expenses.A large cross-sectional study of adults in 2012 showed that the incidence of CKD was 10.8%,involving about 119 million people in China.Epidemiological studies have shown that acute renal injury(AKI)is an independent risk factor for CKD,the degree,frequency and duration of acute renal injury are related to the progression of CKD,and the transition from AKI to CKD is an important cause of progressive renal deterioration.AKI to CKD transition is a complex pathophysiological process,and current pathophysiological mechanisms involved incomplete regeneration of tubular cells(EMT and G2/M cell cycle arrest,etc.),persistent chronic inflammatory(M1 macrophage phenotype to M2),mitochondrial dysfunction,genetic modifications(DNA methylation and microRNA expression),RAS activation,endothelial cell dysfunction and microvascular rarefaction.Renal tubular epithelial cells are metabolically active,especially sensitive to the imbalance of redox reaction,a large number of studies have shown that the oxidative damage of renal tubular epithelial cells plays an important role in the development of AKI,AKI-CKD and CKD.The vital regulatory factor for maintaining redox balance is Nuclear Factor-erythroid 2-related Factor 2(Nrf2),participates in anti-oxidant,anti-inflammatory,detoxification and other cytoprotective mechanisms.Nrf2 signaling pathway is a complex,highly conservative pathophysiological response,the classical regulatory response is Keap1-dependent signaling pathway,under physiological state,Keap1 and Nrf2 combined,the complex is anchored on the actin cytoskeleton,so that Nrf2 expressed in a low concentration and located in the cytoplasm.Under oxidative stress state,Nrf2 was dissociated from Keap1,then activated and translocated into nucleus,binded ARE DNA sequence and induced antioxidant genes.However,Nrf2 non-classical regulatory response,that is,Keap1-independent signaling pathway,glycogen synthase kinase 3?(GSK3?)was considered to be the "Common Effector" of multiple Nrf2 inducer,the pivotal mechanism of Nrf2 regulation in delayed stage.GSK3? is a serine/threonine kinase,widely expressed in diverse organs,can regulate Nrf2 in aging,type 2 diabetes mellitus,neurological lesions,liver diseases and other diseases.But is GSK3? also involved in regulating Nrf2-mediated antioxidant response in AKI to CKD transition? There are no reports yet.Therefore,by using folic acid-induced kidney injury mouse model,hydrogen peroxide-induced cell oxidative damage model and human renal samples,conditional GSK3? knockout and GSK3? inhibitor lithium chloride treatment,this study explored the mechanism of GSK3?-mediated Nrf2 signaling in AKI to CKD transition in a murine model of folic acid induced kidney injury and in cultured renal tubular epithelial cells in vitro.Part one: Nrf2 antioxidant response is impaired in folic acid-injured mice and patients with AKI to CKD transition and associated with sustained GSK3? hyperactivity in renal tubules ObjectiveTo observe the expression of GSK3? and Nrf2 mediated antioxidant response in renal tubular epithelial cells by using folic acid-induced renal injury model and patient's kidney samples,and to clarify its role in AKI to CKD transition.Methods1.A retrospective analysis model of folic acid-injured mice,based on the median value of serum creatinine at the 28 th day,the mouse was classified for the high creatinine group and low creatinine group,the serum creatinine at different time points were analyzed retrospectively,kidney morphology and kidney/body weight ratio were observed,the pathological changes of kidney were observed by Masson staining,the protein expressions of fibronectin,8-OHDG,p-histone H3 and others were detected by using western blot and immunohistochemistry staining.2.A prospective analysis model of folic acid-injured mice,the blood creatinine was screened on the 3rd day of folic acid injury.According to the median value of blood creatinine in the 7th,14 th and 28 th days respectively,mouse were divided into high creatinine group and low creatinine group,renal pathological changes were observed by Masson staining,the expressions of 8-OHdG?GSK3??Nrf2?HO-1 in kidneys was examined by immunohistochemistry staining,the protein expressions of GSK3??p-GSK3?(S9)?Nrf2?HO-1?Keap1?Fibronectin??-SMA?Collagen I?Lipocalin-2?Nitrotyrosine by western blot analysis,arbitrary value of 1-p-GSK3?(S9)/GSK3? in the kidney for each subgroup by quantitative analysis.3.Examined the protein expressions of 8-OHdG?GSK3??Nrf2?HO-1 in kidney biopsy specimens from normal control,normal allograft and patients with progressive CKD by immunohistochemistry staining.Results1.In the retrospective model of folic acid-injured mice,there was a statistically significant difference(P<0.05)between high creatinine group and low creatinine group at the 28 th day.Compared with the low creatinine group,the kidney from high creatinine group demonstrated a shrunk kidney shape with prominent granular surface and decreased kidney/body weight ratio(P<0.01),increased extracellular matrix accumulation in tubulointerstitium,as revealed by immunblot analysis of kidney homogenates for fibronectin and collagen I and by Masson trichrome staining and fluorescent immunohistochemistry staining for fibronectin.Moreover,shown by immunoblot analysis and immunohistochemistry staining for phosphorylated histone H3(p-Histone H3),a hallmark of cell cycle arrest at the G2/M phase,compared with the low creatinine group,high creatinine group demonstrated more renal tubular cell growth arrest.Immunoblot analysis of kidney homogenates for nitrotyrosine,a marker of protein oxidation,and immunohistochemistry staining for 8-hydroxydeoxyguanosine(8-OHdG),an oxidized nucleoside of DNA,revealed that the high creatinine group had significantly more oxidative kidney injury than the low creatinine group.2.In the prospective analysis model of folic acid-injured mice,beginning day 14,the low and high serum creatinine subgroups started to exhibit significantly different serum creatinine levels.Semi-quantitative morphometric analysis indicated that the difference of renal histologic changes between the two subgroups did not reach statistical significance until day 14.Moreover,oxidative insults in renal parenchyma were revealed by immunohistochemistry staining for 8-OHdG,during the acute phase on day 3,oxidative injury was abundant,but it progressively receded in the low serum creatinine subgroup.In contrast,the high serum creatinine subgroup demonstrated a persistent oxidative damage.Morphometric analysis revealed that the difference in oxidative damage between the two subgroups reached statistical significance at day 7th(P<0.05).This finding was further corroborated by immunoblot analysis of kidney homogenates for nitrotyrosine.The persistent oxidative stress in the high serum creatinine subgroup was consistent with a sustained renal tubular injury,marked by prolonged expression of lipocalin-2 as detected by immunoblot analysis of kidney homogenates.3.In the prospective analysis model of folic acid-injured mice,during the acute phase of folic acid injury(day 3),there was a drastic Nrf2 induction in injured renal tubules that was predominantly located to the nuclei,denoting an activated Nrf2 antioxidant response.Accordingly,expression of HO-1 in injured tubules was elevated.Nrf2 nuclear accumulation and HO-1 induction persisted in renal tubules in the low serum creatinine subgroup.These morphologic findings were further validated by immunoblot analysis of whole kidney homogenates and nuclear fractions for HO-1 and Nrf2 respectively.The impaired Nrf2 nuclear accumulation was unlikely attributable to Nrf2 because of the expression of Keap1 was consistently comparable between the low and the high serum creatinine subgroups.The immunohistochemistry staining for GSK3? shown GSK3? was expressed by renal tubules to a low extent in normal and acutely injured kidney.This pattern and intensity of renal GSK3? expression were negligibly altered in the low serum creatinine subgroup.In contrast,in the high serum creatinine subgroup,there was a progressively augmented expression of GSK3? in renal tubules.This finding was further corroborated by immunoblot analysis of kidney homogenates followed by densitometric analysis.As shown by immunoblot analysis of kidney homogenates,GSK3? phosphorylation at serine 9 was suppressed during the AKI phase,but variably reinstated at the chronic phase of folic acid nephropathy.To estimate the relative activity of GSK3? in the kidney,the value of 1-p-GSK3?/GSK3? was estimated based on densitometric analysis of immunoblots and revealed a progressive correction of GSK3? hyperactivity in the low serum creatinine subgroup but a sustained GSK3? overexpression and hyperactivity in the high serum creatinine subgroup.4.In kidney biopsy samples,marked by 8-OHdG staining was noted in protocol biopsy tissues derived from normal allograft kidneys,and markedly increased in CKD patients.This coincided with a functional Nrf2 response,marked by nuclear accumulation of Nrf2 and HO-1 induction that was associated with normal GSK3? expression in the same renal tubules in the consecutive section.In CKD patients,HO-1 induction was blunted in renal tubules and Nrf2 nuclear accumulation was diminished,concomitant with GSK3? overexpression in the same renal tubules,as shown by immunohistochemistry staining of consecutive sections.Conclusions1.The long-term renal outcome after folic acid-elicited AKI in mice is contingent on the extent of oxidative damage in renal tubules.2.AKI to CKD transition in folic acid-injured mice is associated with persistent oxidative injury.3.Nrf2 antioxidant response is impaired during AKI to CKD transition in folic acid-injured mice and associated with sustained GSK3? hyperactivity in renal tubules.4.GSK3? hyperactivity in renal tubules coincides with impaired Nrf2 antioxidant response in patients with AKI to CKD transition.Part two: Effects of targeted inhibition of GSK3? on Nrf2 activityand cell function in hydrogen peroxide-induced oxidative stressmodel of renal tubular epithelial cells ObjectiveTo explore if a possible cause effect relation exists between GSK3? hyperactivity and impaired Nrf2 response in renal tubules,and effects of GSK3? on cell apoptosis,differentiation and G2/M cell cycle arrest.MethodsIn cultured renal tubular epithelial cells,empty vector(EV,pcDNA3.1),a hemagglutinin(HA)-conjugated dominant-negative kinase-dead(KD)mutant of GSK3?,or an HA-conjugated constitutively active mutant(S9A)of GSK3? was transfected by Lipofectamine 2000,followed by oxidative insults elicited by hydrogen peroxide(200?mol/L)exposure 48 hours.1.The changes of nuclear expression in Nrf2 were observed by Nrf2 and DAPI dual immunofluorescence.2.Understand cell apoptosis by cell TUNEL staining,double immunofluorescence staining of E-cadherin and vimentin represented cell differentiation changes,p-histone H3 fluorescence staining showed G2/M cell cycle arrest.3.Total protein and nuclear protein were extracted from each group,western blot analyzed Nrf2 expression in nuclear protein,the expressions of HO-1,Keap1,HA,GSK3?,Collagen I,E-Cadherin,Vimentin,Cleaved Caspase 3 and p-Histone H3 were detected in total protein.Results1.Immunofluorescence staining and immunoblot analysis for HA revealed a satisfactory transfection efficiency.2.In empty vector(EV)-transfected cells,Nrf2 mainly located in the cytoplasm without hydrogen peroxide injury,hydrogen peroxide induced some Nrf2 nuclear accumulation.Immunoblot analysis demonstrated an increased nuclear fraction of Nrf2 and an amplified expression of HO-1.These effects were largely abrogated in cells expressing S9 A but enhanced in KD expressing cells.S9A-and KD-expressing cells expressed Keap1 has no obvious changes either under basal condition or after injury.3.Hydrogen peroxide induced changes in renal tubular cells,including apoptosis and cell cycle arrest,as detected by TUNEL staining and immunofluorescent staining for phosphorylated histone H3.The findings were further confirmed by immunoblot analysis of cell lysates for activated caspase 3 and phosphorylated histone H3.Immunofluorescent staining and immunoblot analysis showed loss of E-cadherin,which are typical signs of tubular cell dedifferentiation and increased vimentin after hydrogen peroxide injury.Furthermore,production of extracellular matrix collagen I was up-regulated.The hydrogen peroxide elicited tubular cell apoptosis,growth arrest,dedifferentiation and extracellular matrix synthesis were reinforced in S9 A expressing cells but attenuated in KD expressing cells.ConclusionsIn hydrogen peroxide induced renal tubular epithelial cells,GSK3? mediated Keap1-independent Nrf2 nuclear expression and antioxidant respose,and affected cell apoptosis,differentiation,cell cycle and other functions.Part three: Conditional ablation of GSK3? and LiCl treatment regulate Nrf2 activity and mitigates AKI to CKD transition in folic acid-injured mice ObjectiveTo observe whether the function of Nrf2 is affected and whether the AKI prognosis has a positive effect in folic acid-induced kidney injury model by conditional knockout and LiCl administration,and further verifies whether GSK3? mediated Nrf2 antioxidant response can be used as an effective therapeutic target for preventing AKI to CKD transition.Methods1.Using Cre/Loxp technology to construct conditional GSK3? gene knockout mice.PCR and immunohistochemistry methods examined whether GSK3? was successful knockout in the kidney tubules.2.Use of conditional GSK3? gene knockout mice to establish folic acid induced kidney injury model by a higher dose(300mg/kg)folic acid intraperitoneal injection,then mouse was divided into two groups: KO group and Con group.Blood creatinine was detected at the 3,7,14 and 28 days after folic acid injury.The pathological changes of kidney in mice were observed by PAS staining.3.In folic acid induced kidney injury model by using gene knockout mice,the expressions of Nrf2 ? HO-1 ? 8-OHdG in both groups were observed by immunohistochemical staining,the expressions of E-cadherin and Vimentin were observed by immunofluorescence staining,and the Nrf2 expression was detected in nuclear protein.The expressions of Keap1,HO-1,Lipocalin-2,Nitrotyrosine,Fibronectin,E-Cadherin,Vimentin were detected in total protein by using western blot.4.On day 7 after folic acid injury,mice randomly received a subcutaneous injection of microdose lithium chloride(LiCl,40mg/kg).GSK3? and p-GSK3?(S9)were detected by western blot at LiCl treatment every other day.5.In the folic acid induced kidney injury model,mice were randomly divided into NaCl group,LiCl group,LiCl+Trig Group at day 7th after injury.Observe the morphological changes of the kidney at 28 th day.The levels of serum creatinine in the different days were detected.Masson staining was observed in various groups,immunohistochemical staining was detected for Fibronectin,p-histone H3,GSK3?,Nrf2,HO-1,Nitrotyrosine,immunofluorescence stained for E-cadherin and vimentin.The changes of Nrf2 nucleus expression were detected in nuclear protein,total protein was used to detect Fibronectin,Collagen I,p-histone H3,E-cadherin,Vimentin,GSK3?,p-GSK3?,HO-1 and Nitrotyrosine.Results1.PCR gel results showed that the conditioned knockout mice expressed the Cre gene and the floxed-GSK3? base simultaneously.Immunohistochemical staining showed that GSK3? was ablated selectively in renal tubules with without affecting glomerular expression of GSK3?.2.At 28 th day of folic acid injury,compared with the Con group,the KO group creatinine decreased significantly(P<0.05);PAS staining and semi-quantitative renal injury score analysis showed that compared with Con group,the lesions of kidney fibrosis and renal tubular atrophy in KO group were alleviated and has the statistically difference between two group(P<0.05).3.Nrf2 antioxidant response in the injured kidney,marked by Nrf2 nuclear accumulation and HO-1 induction in renal tubules,was prominently enhanced in KO mice.This was concomitant with diminished oxidative damages of renal tubules,marked by 8-OHdG staining.Morphologic findings were further validated by immunoblot analysis of kidney homogenates.KO kidneys displayed much more Nrf2 nuclear accumulation than control kidneys since day 7 after folic acid injury.The expression of nitrotyrosine and lipocalin-2 were consistently attenuated in KO kidneys,associated with lessened deposition of extracellular matrix like fibronectin.4.On day 7 after folic acid injury,mice received a subcutaneous injection of microdose lithium chloride(LiCl,40mg/kg)treatment,lithium treatment resulted in a drastic induction of phosphorylation of GSK3? at serine 9.Accordingly,a therapeutic regimen of once a week injection of microdose lithium was set up.5.Delayed lithium versus sodium treatment significantly improved the gross kidney morphology and serum creatinine levels.Tubular atrophy,interstitial fibrosis and inflammation,were abrogated after lithium treatment,as demonstrated by immunoblot analysis of kidney homogenates for fibronectin and collagen I and verified by Masson trichrome staining and by immunohistochemistry staining for fibronection.G2/M cell cycle arrest in renal tubular cells,as detected by immunoblotting or immunohistochemistry staining for phosphorylated histone H3,and tubular cell dedifferentiation,featured by loss of E-cadherin and increased expression of vimentin,were evident in NaCl-treated mice but strikingly diminished after lithium therapy.An increase in Nrf2 nuclear accumulation and a reinforced HO-1 induction,as shown by immunoblot analysis of kidney homogenates and by immunohistochemistry staining.LiCl treatment induced an augmented inhibitory phosphorylation of GSK3? at serine 9 and a blunted GSK3? induction in renal tubules.Simultaneously treatment of mice with trigonelline(Trig),a small molecule inhibitor of Nrf2,largely abolished the lithium's effect on improving kidney function,histologic injury and oxidative stress.Conlusions1.Conditional ablation of GSK3? in renal tubules reinforces Nrf2 antioxidant response in a Keap1 independent mode and mitigates AKI to CKD transition in folic acid-injured mice.2.Delayed targeting of GSK3? by microdose lithium once a week inhibited the activity of GSK3? in folic acid-injured mice.3.Delayed targeting of GSK3? by microdose lithium reinstates Nrf2 antioxidant responses and improves the long-term renal outcome of folic acid-induced AKI in mice,lithium is an effective drug to prevent AKI to CKD transition. |
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