| Podocytes are terminally differentiated glomerular epithelial cells and play a critical role in maintaining glomerular filtration barriers.Studies from diabetic patients have revealed that podocyte injury is an early event in the progression of diabetic nephropathy(DN).In diabetic condition,podocytes lose specific markers of differentiation,including foot process effacement and detachment,and then reduce the capacity to maintain the glomerular filtration barrier,thereby resulting in proteinuria.Thus,therapies aimed at preventing podocyte injury or promoting podocyte repair are potential strategies for treating patients with DN.Mitochondria are important organelle in cells,the primary function of which is to generate energy.Mitochondrial dysfunction leads to alterations in cellular function and the loss of renal function.An increasing number of studies have revealed that mitochondrial dysfunction plays a critical role in the pathogenesis of diabetic nephropathy.Podocytes are sensitive to mitochondrial dysfunction.Abnormalities of mitochondria in podocytes have been found in animal models of DN,and high fat diet-induced glomerulopathy.PGRN,an autocrine growth factor,has multiple roles in the progression of many kinds of diseases,such as tumorigenesis and immunoinflammatory response.Our previous study has demonstrated that the expression level of PGRN was significantly reduced in the kidney from DN mice,and PGRN deficiency aggravated glomerular injury in mice with DN.However,the regulatory mechanism of PGRN in podocyte injury,especially whether it is involved in maintaining mitochondrial homeostasis is still unknown.Therefore,elucidating the role and mechanism of PGRN in mitochondrial homeostasis is crucial for the identification of potential therapeutic targets of podocyte injury.Objective1.To identify the protective role of PGRN in mitochondrial dysfunction of podocytes in diabetic nephropathy.2.To elucidate the molecular mechanism of PGRN in maintaining podocyte mitochondrial function and homeostasis.MethodPart I To identify the protective role of PGRN in mitochondrial dysfunction of podocytes in DN1.1 Effect of PGRN deficiency on podocyte injury in diabetic mice:To construct mouse model of diabetes,8 week-old male PGRN deficient mice and littermate wild type mice were selected randomly,and one week after the recovery of uninephrectomy,50mg of streptozotocin(STZ)was injected intraperitoneally and continuously for four days.Urine was collected for 24h in a metabolic cage.Caspase3 staining and TUNEL staining were performed to determine the cell death in glomeruli.WT1 staining was used to analyze the number of podocytes.1.2 Effect of PGRN deficiency on mitochondrial damage in diabetic mice:Transmission electron microscopy(TEM)was performed to determine mitochondrial damage in mice with diabetic nephropathy,and the expression of mitochondrial respiratory chain protein COXIV was detected by Immunohistochemical(IHC)analysis.1.3 The protective role of recombinant PGRN in mice with diabetic nephropathy:Human recombinant PGRN(rPGRN)protein was purified from HEK-293 cells stably transfected with pHis/Myc-PGRN.Then diabetic mice were injected with rPGRN(10mg/kg).Glomerular morphological change was analyzed by Periodic acid-Schiff stain(PAS);TUNEL staining was performed to observe glomerular cell death;the podocyte foot process was observed by TEM,and then ImageJ software was used to analyze the thickness of the glomerular basement membrane(GBM),foot process width and the number of foot process per micron of GBM(number of foot process/μm GBM).1.4 rPGRN attenuated mitochondrial damage in podocytes induced by high glucose:Human podocytes(HPC)were treated with high glucose(HG,40mM)in the presence or absence of rPGRN.The morphology of mitochondrial was observed by TEM and then the count of mitochondrial and mitochondrial diameter was analyzed by ImageJ software Mitochondrion-Selective Probe Mitotracker Green was used to study mitochondrial morphological integrity and mitochondrial breakage.Mitochondrial membrane potential(MMP)was detected by the mitochondrial membrane potential assay kit with JC-1.1.5 The expression pattern of PGRN in glomerular podocytes:The level of PGRN in renal biopsies from patients with diabetic nephropathy,normal people and patients with diabetic non-nephropathy were measured by IHC.The expression of PGRN in the glomerular podocyte of diabetic mice was detected by Immunofluorescence(IF)staining.For in vitro study,human podocyte(HPC),mouse podocyte(MPC)and human renal tubular epithelial cell(HK-2)were cultured and stimulated with high glucose(HG),and the expression of PGRN was detected by Western blot(WB).1.6 HG-reduced PGRN expression in podocytes is regulated by C/EBPβ:WB was used to confirm the expression level of C/EBPβ.Inhibiting the expression of C/EBPβ by small interfering RNA(siRNA),and the expression level of C/EBPβ and PGRN were detected by WB.Part Ⅱ To elucidate the molecular mechanism of PGRN in maintaining podocyte mitochondrial function and homeostasis1 PGRN restored mitochondrial biogenesis in HG-treated podocyte.Human podocytes were treated with HG with or without rPGRN(500ng/ml).the levels of mitochondrial biogenesis-related proteins PGC1α and TFAM were detected by WB and Real-time RT-PCR.Total DNA from podocytes was extracted with a DNA extraction kit and the relative copy number of mitochondrial DNA(mtDNA)was detected by real-time quantitative PCR.2 PGRN restored HG-reduced mitophagy in podocytes2.1 PGRN rescued HG-reduced mitophagy in podocyte:The level of PARK2 in the kidney was detected by WB.The levels of PINK1 and PARK2 in human podocytes were detected by WB and Real-time RT-PCR.TEM was performed to determine the number of mitophagosome in human podocytes;IF was used to label mitochondrial protein TOMM20 and autophagy protein LC3 to observe mitophagy.2.2 PGRN protected against podocyte injury by enhancing mitophagy:Inhibiting the expression of PARK2 by siRNA in human podocytes.Flow cytometry was performed to detect the percent of apoptotic podocytes,and the level of cleaved-PARP1 was detected by WB.2.3 PGRN restored HG-reduced autophagy in podocytes:Double immunostaining for LC3 or p-AMPKa and WT1 was used to determine the levels of LC3 or p-AMPKa in glomerular podocyte of diabetic mice;Flow cytometry was performed to detect the effect of autophagy inhibitor(3-MA)on PGRN-restored cell death of podocytes under HG condition.The effect of PGRN and PGRN-downstream AMPKa on autophagic flux in HG-treated podocyte was detected by using HPC and MPC infected with tandem GFP-RFP-LC3 adenovirus construct.3 PGRN restored Sirt1/PGC1α FoxO1 pathway in HG-treated podocytesThe levels of histone deacetylase Sirt1-7 and acetylated FoxO1 in HG-treated podocytes with or without rPGRN and Sirtl in the renal cortex from WT and PGRN deficient diabetic mice were detected by WB.Immunoprecipitation(IP)and WB were performed to detect the acetylation level of PGC1α in podocytes.The levels of Sirtl,PGC1α and PARK2 in the renal cortex from diabetic mice treated with rPGRN were detected by WB.4 PGRN maintained mitochondrial homeostasis and protected against podocyte injury by Sirtl4.1 PGRN restored HG-reduced mitophagy in podocytes by Sirtl:Inhibiting the expression of Sirtl in human podocytes by RNA interference.After HG and rPGRN treatment,the acetylation levels of FoxO1 and downstream PINK1/PARK2 were measured by WB and mitochondrial autophagosome was detected by IF.4.2 PGRN upregulated mitochondrial biogenesis in HG-treated podocytes by Sirtl:IP was performed to detect the acetylation level of PGC1α.Mitochondrial membrane potential assay kit with JC-1,real-time quantitative PCR and flow cytometry assay were used to determine MMP,mtDNA and cell death,respectively.Results Part Ⅰ To identify the protective role of PGRN in mitochondrial dysfunction of podocytes in DN1.1 Effect of PGRN deficiency on podocyte injury in diabetic mice:Compared with WT diabetic mice,the UACR was increased in PGRN deficient diabetic mice.PGRN deficiency led to more serious glomerular cell death and decreased the number of glomerular podocytes in diabetic mice,indicating PGRN deficiency aggravated podocyte injury in diabetic mice.1.2 Effect of PGRN deficiency on mitochondrial damage in diabetic mice:TEM revealed that PGRN deficiency further aggravated mitochondrial damage in diabetic mice.Compared with wild type diabetic mice,the level of mitochondrial respiratory chain protein COXIV in glomeruli of PGRN deficient diabetic mice was further reduced.1.3 The protective role of recombinant PGRN in mice with diabetic nephropathy:rPGRN administration reduced proteinuria levels,mesangial expansion,and glomerular cell death in diabetic mice.Importantly,rPGRN ameliorated podocyte injury as evidenced by alleviated pathological changes of foot process and restored podocyte number1.4 rPGRN attenuated mitochondrial damage in podocytes induced by high glucose:rPGRN treatment significantly inhibited HG-induced mitochondrial fission,and preserved HG-induced mitochondrial abnormalities in podocytes,in vitro1.5 The expression pattern of PGRN in glomerular podocytes:The levels of PGRN were reduced in renal biopsies from DN patients.In particular,the expression of PGRN in WT1-positive podocytes was dramatically reduced in glomeruli from diabetic mice.In vitro.HG treatment reduced the expression of PGRN in both HPC and MPC.1.6 HG-reduced PGRN expression in podocytes is regulated by C/EBPβ:The expression of C/EBPβ was significantly reduced in HPC treated with HG,while the expression of PGRN was further reduced after C/EBPβ silencing under HG condition.Part Ⅱ To elucidate the molecular mechanism of PGRN in maintaining podocyte mitochondrial function and homeostasis1 PGRN restored mitochondrial biogenesis in HG-treated podocyte.The expression of PGC1α and TFAM was significantly reduced under HG conditions and were partially restored by rPGRN treatment.rPGRN also restored HG-reduced mtDNA levels2 PGRN restored HG-reduced mitophagy in podocytes2.1 PGRN rescued HG-reduced mitophagy in podocyte:TEM and double immunofluorescence staining for TOMM20 and LC3 showed that rPGRN restored the number of mitophagosome in podocytes under HG conditions.PGRN rescued HG-reduced PINK1 and PARK2 expression.In addition,PGRN deficiency further reduced the level of PARK2 in the renal cortex from diabetic mice.2.2 PGRN protected against podocyte injury by enhancing mitophagy:Inhibition of PARK2 significantly reduced the protective effect of PGRN on podocyte apoptosis under HG conditions,indicating that mitophagy contributes to the protective role of PGRN in podocyte injury.2.3 PGRN restored HG-reduced autophagy in podocytes:PGRN deficiency further reduced the level of LC3 in WT1-positive podocyte from diabetic mice.In vitro,rPGRN restored HG-reduced autophagic flux,and 3-MA disrupted the protective effect of PGRN in podocytes injury.Mechanistically,PGRN-promoted autophagy was involved in the activation of AMPK.The role of PGRN in autophagy was also confirmed in MPC.3 PGRN restored Sirt1/PGC1α/FoxO1 pathway in HG-treated podocytesSirtl is extensively implicated in both mitochondrial biogenesis and mitophagy.Our data showed that,in sirtuins family,the levels of Sirtl,Sirt3,Sirt4,and Sirt6 were reduced under HG conditions,and rPGRN specifically restored Sirtl protein levels.In vivo,PGRN deficiency further reduced the expression level of Sirt1 in the renal cortex of diabetic mice.In HG-treated podocytes,rPGRN decreased the acetylation levels of PGC1α and FoxO1.Furthermore,administration of rPGRN enhanced the expression of Sirtl,PARK2 and PGC1α in the kidney from diabetic mice.4 PGRN maintained mitochondrial homeostasis and protected against podocyte injury by Sirtl4.1 PGRN restored HG-reduced mitophagy in podocytes by Sirtl:Inhibition of Sirtl significantly restricted PGRN-reduced FoxO1 acetylation of and PGRN-enhanced PINK1 and PARK2 expression under HG conditions.Functionally,gene silencing of Sirtl in HG-treated podocytes also disrupted PGRN-induced mitochondrial autophagosomes.4.2 PGRN upregulated mitochondrial biogenesis in HG-treated podocytes by Sirtl:The inhibition of Sirtl significantly restricted PGRN-reduced acetylation of PGC1α in podocytes treated with HG.Functionally,silencing Sirtl blocked the PGRN-mediated restoration of HG-declined mitochondrial content and MMP.Furthermore,the protective role of PGRN in HG-induced cell death was also dependent on Sirtl pathway.Conclusion and innovation1 For the first time we demonstrate the role of PGRN in maintaining mitochondrial homeostasis,which provides new insights into the novel mechanism of PGRN in diseases,including DN.2 PGRN enhances mitochondrial biogenesis and mitophagy via SIRT1/PGC1α/FoxO1 pathway in podocytes under diabetic conditions.3 rPGRN protects against podocyte injury via maintaining mitochondrial homeostasis,suggesting that PGRN may be an potential therapeutic strategy for treating podocyte injury-related diseases... |
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