| Background and ObjectivesThe major function of the kidney is to limit the passage of albumin and plasma proteins into the urinary space.During this process,a glomerular visceral epithelial cell,also termed as podocyte,plays an essential role as the ultimate filtration layer by the formation and maintenance of podocyte foot processes(FPs)and the interposed slit diaphragm.The podocyte is unable to proliferate and divide since it is a highly specialized and polarized as well as terminally differentiated cell type.It has been widely accepted that loss of podocytes due to apoptosis and/or detachment from the glomerular basement membrane is the key mechanism of proteinuria initiation and kidney disease progression to end stage renal failure.However,there is very limited insight into the nature of events that induce or promote podocyte apoptosis.Mitochondria(mt)are important organelles that ensure the energy supply by producing and delivering adenosine triphosphate(ATP).Moreover,mitochondria exert a key role in some cellular events such as ion homeostasis,reactive oxygen species(ROS)generation and cell death/apoptosis.Accumulating studies showed the involvement of mitochondrial dysfunction and excessive ROS generation in many types of kidney diseases.It was re-ported that a considerable number of patients with mitochondrial cytopathy,especially those with mitochondrial gene mutations,develop focal segmental glomerulosclerosis(FSGS).Puromycin aminonucleoside(PA)-induced podocyte injury in mice exhibited a decrease of mRNA expres-sion of mitochondrial transcription factor-A(mtTfa)and nuclear respiratory factor-1(Nrf-1),and reduction of mtDNA copy number as well as downregulation of the cytochrome c oxidase(Cox I)level.Recently,PGC-1 suppression,mitochondrial damage and cellular apoptosis were detected in aldosterone-induced podocyte injury,which was ameliorated by a SIRT1 activator via increasing PGC-1 expression.Moreover,TGF-?1 could induce mitochondrial Nox4 activation through the TGF-? receptor–Smad2/3 signaling pathway necessary for ROS generation,mitochondrial dysfunction,and cellular apoptosis in cultured mouse podocytes.These findings from human as well as experimental cell and animal models indicated that mitochondrial damage is closely related to podocyte injury and glomerular diseases.Nevertheless,the underlying molecular mechanisms are still unclear and should be further elucidated.Therefore,the current study was designed to address the potential proximal signaling related to in vitro and in vivo mitochondrial dysfunction in a PA-induced podocyte injury model.Our findings demonstrated that Smad3/Nox4 axis activation leads to podocyte injury by inducing mitochondrial damage,ROS generation and cellular apoptosis through activating the cytochrome c–caspase9–caspase3 signaling pathway.Methods and materialsIn vitro: An immobilized mouse podocyte cell line(a kind gift from Prof.Peter Mundel)was maintained at 33 °C for proliferation in RPMI 1640 media containing 10% fetal bovine serum(Gibco)and 10 U/ml of recombinant mouse ?-interferon(Invitrogen).podocytes were stimulated with puromycin aminonucleoside(PA)(Sigma)for the indicated time periods.To evaluate podocyte cell apoptosis by using Annexin V-FIT and Propidium Iodide(PI)double staining apoptosis detection kit(BD Biosciences).To down regulate the expression of mouse Smad3,Nox4,and Dab2,lentiviral short hairpin RNAs(shRNAs)were used.Images were taken by using a laser scanning confocal microscope equipped with a 60× oil immersion objective lens(Zeiss).The intensity of internalized FITC-albumin was quantified with Image J 1.47.The absorbance of FITC-albumin was deter-mined at 490 nm with a Fluorescence Multi-well plate reader(PerSeptive Biosystems)to indicate the filtration/influx function of monolayer podocytes.In vivo: The PAN rat model was produced by a single intraperitoneal injection of 10 mg/100 g body weight of PA(Sigma).Control rats were injected with an equal volume of 0.9% saline.From the second day after PA injections,prednisone was administered(0.6 mg/100 g body weight)by gastric gavage once a day until sacrifice.The urinary albumin and creatinine were determined by using the enzyme-linked immunosorbent assay(Exocell).The oxygen consumption level in whole cells was analyzed using Oxygen Consumption Rate Assay Kit(Cayman Chemical Company).The mitochondrial membrane potential(MMP)level was evaluated with JC-1 dye by comparing the ratio of J-aggregates/monomer.Reactive oxygen species(ROS)generation was determined by DCFDA Cellular ROS Detection Kit(Abcam).Mitochondrial complex I and complex IV activity were determined with Microplate Assay Kit(Abcam).Western blot detected caspase3,Smad3,Nox4,CoxI,Cox II,Cox III,Cox IV,mtTfa,cyt-c,Dab2 signaling pathway changing.Using the above-described method to investigated the potential proximal signaling related to in vitro and in vivo mitochondrial dysfunction in PA induced podocyte injury model.Statistical analysis-Data are shown as mean ± SD.Statistical evaluation was performed using One-Way or Two-Way ANOVA.Test by SPSS 15.0 software(SPSS Inc.,Chicago,IL,USA).A p value of less than 0.05 was considered as a significant difference.Results1.PA induces cellular injury in cultured podocytesDifferentiated podocytes were stimulated with different concentration PA for 24 h.Compared to control,the percentage of apoptotic cells was increased significantly,displaying time-and dose-dependent increase in podocyte apoptosis.Compared to control,not only the abundance of active caspase3 was increased significantly at 24 h and 48 h following PA application,but also the average intensity of intracellular FITC-albumin was increased remarkably in podocytes stimulated with PA.2.PA causes mitochondrial DNA damage in cultured podocytesQ-PCR data revealed that mtDNA copy number was decreased obviously at 12 h,and continued to decrease at 24 h and 48 h following PA application.PA led to a dramatic reduction of complex I and complex IV activity at both 12 h and 24 h.The Cox III protein showed no alteration at any time points,while Cox I and Cox II levels were decreased significantly within 6 h to 24 h,and CoxIV was decreased at 12 h and 24 h after PA application.As the major transcription factor in mitochondria,a robust reduction of mitochondrial transcription factor-A(mtTfa)was detected at 12 h and 24 h following PA application.Nuclear respiratory factor-1(Nrf-1)was decreased at 6 h,and continued to decrease at 12 h and 24 h following PA application.The abundance of SDHA and cyt-c in mitochondrial fractions was decreased significantly at 6 h,and continued to decrease at 12 h and 24 h in PA-stimulated podocytes.3.PA results in mitochondrial dysfunction in cultured podocyCompared to 0 h,PA induced a significant time-dependent reduction of the oxygen consumption level at 6h,12 h,and 24 h.Compared to 0 h,relative MMP levels were decreased obviously at 6 h,12 h and 24 h after PA application,displaying a time dependent fashion.Additionally,the generation of reactive oxygen species(ROS)was investigated by a cell permeant fluorogenic dye DCFDA.Relative ROS levels were increased remarkably by PA application at 12 h and 24 h.The current study showed by immunoblotting that the amounts of MnSOD and catalase were decreased significantly at 6h and continued to decrease at12 h and 24 h following PA application.4.PA induces nuclear translocation of p-Smad3 and upregulation of mitochondrial Nox4 in cultured podocytesour data showed that the Nox4 protein level in mitochondrial fractions was increased significantly at 12 h and 24 h after PA application.Immunoblotting assay revealed that PA led to a remarkable decrease of p-Smad3 in cytosolic fractions,but a significant increase in nuclear fractions at 12 h and 24 h,implying that PA induced a translocation of p-Smad3 from the cytosol to nuclei.Here,the amounts of the cyt-c protein in cytosolic fractions were increased remarkably at 12 h and 24 h following PA application,which is consistent with the above result that PA obviously reduced the level of cyt-c in mitochondrial fractions.These data demonstrated a release of cyt-c from mitochondria to the cytoplasm by PA application.In PA-stimulated podocytes,a noticeable increase of activated caspase9 and cleaved caspase8 was observed at both 12 h and 24 h.5.Dab2 is involved in PA-induced internalization of albumin in cultured podocytesImmunoblotting assay revealed that PA time-dependently increased Dab2 expression.In nuclear fractions,the amount of p-Smad3 was increased notably in podocytes stimulated with PA,which was not affected by Dab2 knockdown,implying that Dab2 may be not involved in PA-induced nuclear translocation of p-Smad3.Similarly,Dab2 knockdown showed no effect on PA-induced podocyte apoptosis.FITC-albumin uptake assay showed that the intensity of intracellular FITC-albumin was increased significantly in PA-stimulated podocytes,which was dramatically prohibited by Dab2 knockdown,suggesting that Dab2 is likely related to PA-induced albumin endocytosis in podocytes.Moreover,analysis showed that colocalization of FITC-albumin and early endosomal marker EEA1 was increased obviously in PA-stimulated podocytes,which was effectively reduced by Dab-shRNA not control-shRNA.These data demonstrate that Dab2 may be involved in albumin endocytosis,not Smad3 signaling-mediated podocyte apoptosis following PA treatment.6.Knockdown of Smad3 and Nox4 attenuates PA-induced podocyte damagePodocytes were infected with Smad3-shRNA or Nox4-shRNA for 24 h,induction of Nox4 by PA administration was significantly blocked by Smad3 knockdown.Nevertheless,PA-induced upregulation of p-Smad3 still occurred in Nox4-shRNA expressing podocytes.These findings imply that Nox4 may be the downstream signaling of Smad3 activation in PA-induced podocyte damage.The reduction of the PA-induced mtDNA and oxygen consumption level were partially prohibited in either Smad3-shRNA or Nox4-shRNA expressing cells.In addition,JC-1 assay displayed that the decrease of the relative MMP level in PA-stimulated podocytes was partially prevented by Smad3-shRNA,while completely blocked by Nox4-shRNA.Consistently,PA-induced ROS generation was dramatically reduced in either Smad3-shRNA or Nox4-shRNA expressing cells.The percentage of cells undergoing apoptosis was obviously increased in PA-stimulated podocytes,which was remarkably prohibited by Smad3-shRNA and Nox4-shRNA.Additionally,induction of the activated caspase3 and cyt-c in the cytosol by PA application was completely blocked in podocytes expressing Smad3-shRNA or Nox4-shRNA.Our results strongly indicate that PA-induced podocyte damage may be mediated by the activation of the Smad3–Nox4 signaling pathway.7.Mitochondrial damage occurs in the PA-induced nephropathy ratQuantification analysis showed that glomerular mtDNA levels were decreased remarkably at day 3 and continued to decrease at day 10 following PA injections.However,the mtDNA level partially recovered at day 15.This result indicates that PA also led to in vivo mitochondrial DNA damage.In PAN rats,relative MMP levels were decreased significantly at day 10 and then partially recovered at day 15.Additionally,mitochondrial complex I enzymatic activity and complex IV enzymatic activity were obviously reduced at day 10,and partially recovered at day 15 after PA injections.In mitochondrial fractions,immunoblotting analysis showed that CoxI and mtTfa levels were decreased at day 3 and continued to decrease at day 10,then partially recovered at day 15 in PAN rats,further indicating that mitochondrial DNA damage occurred in PA-induced nephropathy rats.In consistent with the in vitro findings,Nox4 protein levels were increased significantly at day 3,and peaked at day 10,then partially recovered at day 15.Notably,the abundance of nuclear p-Smad3 was increased hugely at day 10,and then partially recovered at day 15 in PAN rats.In cytosolic fractions,cyt-c and activated caspase3 levels were increased significantly at day 3,and peaked at day 10,then partially recovered at day 15 in PAN rats,suggesting that PA also resulted in podocyte apoptosis via activating caspase3 by cyt-c released from mitochondria.8.Prednisone improves mitochondrial function in PA-induced nephropathy ratPA injection-induced proteinuria was significantly reduced by prednisone treatment.Moreover,JC-1 assay showed that the reduction of the MMP level by PA injections was obviously prevented by prednisone.Similarly,prednisone remarkably prohibited the decrease of the mt DNA level in PAN rats.Additionally,the induction of mitochondrial Nox4 and the nuclear translocation of p-Smad3 were significantly reduced in PAN rats that received prednisone treatment.Conclusions? PA-induced cellular injury in cultured podocytes display time-and dose-dependent? Mitochondrial DNA damage and dysfunctionI are found both in vitro and in vivo PA-induced podocyte injury model? Smad3–Nox4 signaling-mediated mitochondrial dysfunction is involved in both in vitro and in vivo PA-induced podocyte damage via increasing ROS generation and activating the cyt-c–caspase9–caspase3 apoptosis pathway? The increase of albumin permeability in the PA-induced podocyte injury model may be related to the Dab2-mediated endocytosis pathway... |
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