| The appearance of microalbuminuria,the progression to overt proteinuria,and the resultant renal dysfunction over several years to decades is the typical progressive course of diabetic nephropathy.The association between the onset of albuminuria and progressive loss of renal function is well described,but the underlying pathophysiological mechanisms are still unclear.Albuminuria could be the result of impaired function of the glomerular filtration barrier or tubular reabsorption.The glomerular filtration barrier consists of a highly specialized structure with three layers: the endothelium,basement membrane,and podocytes.Damage or loss of podocytes is a primary cause of the decreased ability of the barrier to filter out albumin.The density of podocytes per glomerulus is markedly reduced in patients with diabetes.In animal models of diabetes,podocytes depletion represents one of the earliest cellular injuries affecting the kidney.Podocytes are highly differentiated cells with limited potential for proliferation.Thus,podocytes that are lost from the glomerulus are slowly replenished leading to podocytopenia.This has focused attention on podocytes as the primary target for developing therapies against diabetic nephropathy?There are many evidences that reactive oxygen species(ROS)plays a key role in the most pathogenic pathways of diabetic nephropathy.ROS might induceinflammation and apoptosis.There is substantial evidence supporting that ROS is increased in kidney and retina either exposed to hyperglycemia or in diabetic animals.Podocytes are known to be susceptible to oxidative damage.Recent evidence has suggested that,enhanced expression of antioxidant protection in podocytes reduced apoptosis,preserved podocytes numbers,and prevented podocytes effacement.This was associated with attenuated glomerular pathology and reductions of glomerular and mesangial volumes.Therefore,we can conclude that reducing oxidative stress in podocytes is sufficient to ameliorate diabetic kidney disease,providing further support for a central role of the podocytes in this disorder.The generation of ROS,by damaged or dysfunctional mitochondria,has been postulated as the primary initiating event in the development of diabetic nephropathy.Therefore,decreasing mitochondrial ROS generation in podocytes has increasingly been considered a relevant aim in ameliorating the burden of diabetic nephropathy.Mitochondrial autophagy(Mitophagy),a type of specific autophagy,plays a vital role in selectively removing damaged mitochondria.Therefore,it is important that regulating the level of mitophagy in podocytes under diabetic conditions may contribute to alleviating oxidative injury in podocytes.PTEN-induced putative kinase1(PINK1),a key initiator of mitophagy,which stabilization or overexpression is sufficient to trigger Parkin translocation to mitochondria and initiate mitophagy.PINK1 senses mitochondrial damage and activates Parkin by phosphorylating Parkin and ubiquitin.When the membrane potential of a mitochondrial segment depolarized,PINK1 accumulates on the outer mitochondrial membrane,then its kinase activity recruits parkin to the mitochondria.Activated Parkin then builds ubiquitin chains on damaged mitochondria to tag them for degradation though mitophagy.Despite the importance of mitophagy to the regulation of oxidative stress,the effects of mitophagy in podoctyes in Diabetic Nephropathy have never been reported.Thus,we set out to discover the effects and mechanisms of PINK1-induced mitophagy on podocyte injury through the perspective of molecules,cells and animals,which will shed light on novel strategy of protecting from diabetic nephropathy.Part I Defective mitophagy driven by dysregulation of PINK1 inglomerular podocytes exposed to high glucose and in the kidney ofdiabetic mice ObjectiveTo observe the activation of mitophagy driven by PINK1 and to investigate the relationship of mitophagy and ROS production,podocytes injury and apotosis in podoctyes cultured in high glucose and in the kidney of diabetic mice.Methods1.The podocytes were cultured in high glucose for 24,48 or 72 h,respectively.Podocytes in normal glucose were served as normal control group.The expression of PINK1,Parkin,LC3 was determined by TaqMan probe-based qRT-PCR assay.The PINK1,Parkin,LC3,Nephrin,Podocalyxin and Desmin was determined by Westernblot.The mitochondrial membrane potential of podocytes was monitored using JC-1.Intracellular ROS production was measured using MitoSOX.The apoptosis induced by HG in podocytes were determined with Annexin V-FITC kit.2.6-week-old SPF male Kun Ming(KM)mice were injected with a single dose of STZ to induce diabetes(DM).At the end of 4,8 and 12 weeks,the expression of PINK1,Parkin,LC3 was determined by TaqMan probe-based qRT-PCR assay.The PINK1,Parkin,LC3,Nephrin,Podocalyxin and Desmin was determined by Westernblot and Immunohistochemistry.Renal cortex MDA levels were quantified using the Lipid Peroxidation MDAAssay Kit.Results1.Compared with NG,PINK1 was significantly down-regulated in HG in a time-dependent manner(all P<0.05).2.Compared with NG,activation of mitophagy was significantly decreaseed in renal cortex of diabetes mice in a time-dependent manner(all P<0.05).3.Compared with NG,MTP was significantly down-regulated in HG in a time-dependent manner(all P<0.05).4.Compared with NG,mtROS was significantly up-regulated in HG in a time-dependent manner(all P<0.05).5.Compared with NG,apoptosis of podoctyes was significantly up-regulated in HG in a time-dependent manner(all P<0.05).6.Compared with NG,the expression of Nephrin,Podocalyxin was significantly down-regulated in HG with a time-dependent manner(all P<0.05).ConclusionsDefective mitophagy driven by dysregulation of PINK1 in glomerular podocytes exposed to high glucose and in the kidney cortices of diabetic micePart II The effect of PINK1-Mediated mitophagy on oxidative injuryin high glucose-cultured podocytes ObjectiveTo investigate the effect and molecular mechanisms of PINK1-Mediated mitophagy on oxidative injury in high glucose-cultured podocytes MethodsPodocytes were stimulated with normal glucose(NG)medium(5.6 mM),HG medium(25 mM),HG plus LV-PINK1,NG plus LV-PINK1-shRNA,or HG plus the empty LV vector.The cells were collected at 72 h post-stimulation.The expression of PINK1,Parkin,LC3 was determined by TaqMan probe-based qRT-PCR assay.The PINK1,Parkin,LC3,Nephrin,Podocalyxin and Desmin was determined by Westernblot,Immunofluorescence.The mitochondrial membrane potential of podocytes was monitored using JC-1.Intracellular ROS production was measured using MitoSOX.The apoptosis induced by HG in podocytes were determined with Annexin V-FITC kit.Results1.Compared with NG,PINK1 was significantly down-regulated in HG(all P<0.05).Compared with HG,the expression of PINK1 and the activation of Parkin and LC3 was up-regulated in HG+LV-PINK1 group(all P<0.05).On the contrary,compared with HG,the expression of PINK1 and the activation of Parkin and LC3 was down-regulated in HG+LV-sh-PINK1 group(all P<0.05).2.Compared with NG,mtROS was significantly up-regulated in HG(all P<0.05).Compared with HG,the mtROS was down-regulated in HG+LV-PINK1group(all P<0.05).On the contrary,compared with HG,the mtROS was up-regulated in HG+LV-sh-PINK1 group(all P<0.05).3.Compared with NG,MTP was significantly down-regulated in HG(all P<0.05).Compared with HG,the MTP was up-regulated in HG+LV-PINK1 group(all P<0.05).On the contrary,compared with HG,the MTP was down-regulated in HG+LV-sh-PINK1 group(all P<0.05).4.Compared with NG,Nephrin,Podocalyxin was significantly down-regulated in HG(all P<0.05).Compared with HG,the expression of Nephrin,Podocalyxin was up-regulated in HG+LV-PINK1 group(all P<0.05).On the contrary,compared with HG,the expression of Nephrin,Podocalyxin was down-regulated in HG+LV-sh-PINK1 group(all P<0.05).5.Compared with NG,apoptosis of podocytes was significantly up-regulated in HG(all P<0.05).Compared with HG,the apoptosis of podocytes was down-regulated in HG+LV-PINK1 group(all P<0.05).On the contrary,compared with HG,the apoptosis of podocytes was up-regulated in HG+LV-sh-PINK1 group(all P<0.05).ConclusionsActivation of mitophagy through over-expression of PINK1 Protects against HG-induced ROS production and podocytes injury.Part III The effect of PINK1-Mediated mitophagy on oxidativeinjury in vivoObjectiveTo investigate the effect of PINK1-Mediated mitophagy on oxidative injury in vivo MethodsMice were were injected once intraperitoneally with 130 mg/kg STZ to induce diabetes.Mice injected with citrate buffer served as a normal control group(NG).Blood glucose was measured to confirm diabetes,defined as glycemia higher than16.7 mmol/L.After blood glucose levels stabilized for 5 days,50 mL recombinant LV(LV-NC or LV-PINK1)or normal saline was injected at different sites of the dorsal region of the right renal cortex.At 4,8 and 12 weeks post-injection,the expression of PINK1,Parkin,LC3 was determined by TaqMan probe-based qRT-PCR assay.The PINK1,Parkin,LC3,Nephrin,Podocalyxin and Desmin was determined by Westernblot and Immunohistochemistry.Renal cortex MDA levels were quantified using the Lipid Peroxidation MDAAssay Kit.Results1.Compared with NG,PINK1 was significantly down-regulated in mice kidney of DM(P<0.05).Compared with DM,the expression of PINK1 and the activation of Parkin and LC3 was up-regulated in DM+LV-PINK1 group(all P<0.05).2.Compared with NG,MDA was significantly up-regulated in mice kidney of DM.Compared with DM,the MDA was down-regulated in DM+LV-PINK1 group(P<0.05).3.Compared with normal NG,Nephrin,Podocalyxin was significantly down-regulated in mice kidney of DM(all P<0.05).Compared with DM,the expression of Nephrin,Podocalyxin was up-regulated in DM+LV-PINK1 group(allP<0.05).5.Compared with NG,apoptosis of podocytes was significantly up-regulated in mice kidney of DM(all P<0.05).Compared with DM,the apoptosis of podocytes was down-regulated in DM+LV-PINK1 group(P<0.05).ConclusionsActivation of mitophagy through over-expression of PINK1 Protects against HG-induced ROS production and podocytes injury and apotosis in vivo... |
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