The Mechanism Of Oxidative Stress-Induced MtDNA Damage In Ischemic AKI

Background and Objective:Acute Kidney Injury（AKI）is a most commonly seen in critically ill patients,and defined as a rapid deterioration of renal function in a short period of time（a few hours to a few days）caused by numerous factors.AKI is broadly defined as an abrupt decline in glomerular filtration rate withing a rise in serum creatinine and urea nitrogen,disorder of water,electrolyte and acid-base balance.AKI is associated with high morbidity and mortality rates.According to incomplete statistics,about 2million patients die from AKI every year in the world.Survival of patients after an episode of AKI contributed to future development of progressive Chronic Kidney Disease（CKD）and end-stage renal disease（ESRD）,which may lead to a serious socio-economic burden.Clinically,an ischemic insult may arise in a number of ways,including trauma,surgery,sepsis,nephrotoxic drugs.All of the above factors can cause ischemia and hypoxia injury of kidneys.Thus,tissue damage also occurs after ischemia and reperfusion,which can lead to further aggravation of tissue damage and cause ischemia-reperfusion injury（IRI）.IRI breaks the balance of cell reduction reactions,especially the accumulation of metabolites during reperfusion leads to a large amount of reactive oxygen species（ROS）,which triggers a series of cell events such as mitochondrial damage,energy depletion,apoptosis and necrosis.At present,there is no effective treatment to reduce the damage of AKI kidney tissue and promote the repair of damaged tissue except renal replacement therapy（RRT）and nutritional supplement support.Therefore,it is urgent to investigate the pathogenesis of AKI and search for new therapeutic targets.The pathogenesis of AKI is complex,involving oxidative stress,initial immune response,inflammation,and autophagy.Oxidative stress is common feature of ischemic AKI.The burst of ROS after IRI is involved in kidney mitochondrial failure,energy depletion,calcium Ion overload,inflammation,apoptosis and necrosis which can aggravate kidney damage.Mitochondria are the main source of ROS.About 90%of ROS are produced in mitochondria,especially mitochondrial respiratory chain complexes I and III.The outbreak of mitochondrial reactive oxygen species（mtROS）not only drives acute kidney injury,but also cause pathological changes in the kidney from several days to several months after reperfusion.A variety of pathological mechanisms have been reported to be involved AKI,such as mitochondrial lipid and protein peroxidation;release of cytochrome C via mitochondrial membrane depolarization related apoptosis signaling pathway;pro-inflammatory signaling pathways such as Toll-like receptors（TLRs）and NLRP3,but the mechanism by which oxidative stress drives kidney mitochondrial damage and inflammation is not fully understood.Mitochondria contain their own DNA（mitochondrial DNA,mtDNA）,that encodes 13 respiratory chain proteins,22 transfer RNA（t RNA）and 2 ribosomal RNA（r RNA）,therefore mitochondrial function is partly dependent on mtDNA.mtDNA is a double-stranded circular DNA,which is organized in DNA-protein complexes called nucleoids.Mitochondrial transcription factor A（TFAM）is an important nucleoid-related protein,which is encoded and synthesized by the nucleus,and then imported to the mitochondria through the HSP60/70 complex,however,mtDNA-free TFAM is degraded by mitochondrial matrix protease Lon.TFAM is essential for maintenance and transcription of mtDNA.Under pathology（such as aging,stress）,loss of TFAM can lead to mtDNA depletion and impaired mitochondrial biogenesis.mtDNA damage triggers inflammatory response,for example,instability and cytoplasmic release of mtDNA activates TLR9 and c GAS-STING pathways resulting in the production of pro-inflammatory factors.Therefore,we propose that the burst of mtROS after AKI may lead to mitochondrial dysfunction and inflammation by disturbing the structural homeostasis of mtDNA,but its specific mechanism remains unclear.In this study,we aimed to investigate the specific role of mtROS in mtDNA damage and inflammation during AKI.The changes of renal function,mitochondrial function,mtDNA maintenance,and TFAM expression were analyzed in the cells and AKI mouse models with or without mtROS inhibition.In addition,the effect of mtROS on signaling pathways related to mtDNA maintenance and TFAM turnover were also analyzed.TFAM might be served as a target to promote renal recovery after AKI.Materials and Methods:1 In vitro1.1 Effect of oxidative stress on the damage of renal tubular epithelial cellsOxidative stress model was established by Hypoxia/Reoxygenation（H/R）or tert-butyl hydroperoxide（t-BHP）in human renal tubular epithelial cells（HK-2）,respectively,and the excessive mtROS was removed by mitochondrial antioxidants（Mito-Tempo,MT）:The cell viability,mtROS level,mitochondrial morphology,the expression of mitochondrial related genes（PGC-1ɑ/ATP5a-1/NDUFS8/UQCRC1）Level and mitochondrial respiratory function were measured in HK-2 cells.In addition,RAW264.7 macrophages chemotaxis experiment was performed to explore the role of mtROS on oxidative stress-induced damage of HK-2 cells.1.2 Effect of mtROS on the transcription and degradation of TFAM in HK-2cellsFirstly,the expression level of TFAM in HK-2 cells were determined by q-PCR and Western Blotting.Secondly,the co-localization of TFAM with mitochondria were observed by immunofluorescence staining.Furthermore,to confirms that sort of ROS lead to the reduction of expression of TFAM protein,mitochondrial antioxidants（MT）,catalase（CAT）,NADPH oxidase 1/4（Nox1/4）Inhibitors（GKT）,NADPH oxidase inhibitors（APO）,and nitric oxide synthase inhibitors（1400w）were used in the study.Meanwhile,to confirm that TFAM protein degradation is depended on Lon protease,interfering RNA（si-Lon）or protease inhibitor bortezomib were used in HK-2 cells,and the protein expression levels of Lon and TFAM were detected by Western Blotting.Finally,the mtDNA copy number was assayed by q-PCR.1.3 Effect of lowered TFAM on mitochondrial function of renal tubular epithelial cellsAfter treated with tfam si RNA,the co-location of TFAM and mitochondria,the level of mtROS and mitochondrial respiratory function were analyzed in HK-2 cells.In addition,the obstruction of mtDNA nucleoids size and cytoplasmic mtDNA were detected by immunofluorescence staining in HK-2 cells.1.4 Effect of mtROS on TFAM-mediated mtDNA homeostasisHK-2 cells were transfected with tfam si RNA under oxidative stress,and the ROS level were detected by DCFH-DA fluorescent probe in HK-2 cells.The protein expression of TFAM were measured by Western Blotting.To explore the TFAM pathway in oxidative stress-induced damage in HK-2 cells,co-localization of TFAM and mitochondria were analyzed by immunofluorescence,and then,mitochondrial respiratory function（OCR）were detected by seahorse.Besides,the average mtDNA nucleoid size and cytoplasmic mtDNA were determined by immunofluorescence staining.the mtDNA copy number were measured by q-PCR.What’s more,Bax/mito/ds DNA fluorescence staining was used for mtDNA escape analysis.Finally,the m RNA expression of inflammatory factor（TNF-ɑ/IL-1β）and RAW264.7Macrophage chemotaxis numbers were evaluated to explore the mechanism of damage caused by mtROS disrupting mtDNA stability in HK-2 cells.2 In vivo2.1 Establishment of ischemia-reperfusion-induced AKI（IRI-AKI）modelThe mouse model of IRI-AKI was established by bilateral clamping of the renal pedicle for 30 min,while mice in the control group received laparotomy only.At day5 after surgery,mice in each group were sacrificed by overdose of anesthesia,and the serum and renal samples were respectively collected for further analysis.Kidney histopathology were confirmed by stained with hematoxylin and eosin（H&E）and the kidney tissue m RNA expressions of pro-inflammatory,pro-apoptosis,mitochondria biogenesis and mtDNA copy number were measured by q-PCR.2.2 The role and mechanism of mtROS involved in kidney injury in IRI-AKI miceMice were randomly divided into three groups:normal control group（CON）,model group（IRI-AKI）,intervention group（IRI-AKI+MT）.After successful reperfusion,25μL of MT was injected into kidney by insulin syringe,and then mice were given intraperitoneal injection of MT（5 mg/kg）once per day,while mice in AKI group received equal volume of vehicle（DMSO/PBS）.At day 5 after surgery,the blood and kidney tissue were collected for further analysis.In addition,the renal pathology and apoptosis were observed by H&E and TUNEL staining,respectively.The renal inflammatory factors,mitochondrial-related genes,and mtDNA copy number were detected by q-PCR.To confirm the renal injury,immunofluorescence and immunohistochemistry（IHC）were performed.Finally,the mitochondrial morphology,the average size of mtDNA nucleoid or cytoplasmic mtDNA and the expressions of mitochondrial protein（Lon/TFAM/TOM20）were analyzed to explore the role of mtROS in IRI-AKI.3 TFAM expression and mtDNA nucleoid size in kidney tissues of patients with AKIThe kidney tissue was obtained by surgery,then the kidney tissue damage,mitochondrial mass,the protein expression of TFAM,and mtDNA nucleoid size were evaluated by immunofluorescence staining.4 Effect of mesenchymal stem cells-derived extracellular vesicles（MSC-EV）on reducing mtDNA damage caused by oxidative stress in renal tubular epithelial cells4.1 Extraction and identification of MSC-EVHuman umbilical mesenchymal stem cells（h MSCs）were isolated and identified by the Sichuan Stem Cell Bank and cultured in DMEM medium containing 10%EV-depletion FBS,and then the cells divided into three groups:normal control group（NC）,knockdown of tfam group（TFAM-KD）,H₂O₂group（nonspecific downregulation of TFAM by H₂O₂,Aged）.The culture supernatant was collected and separated by centrifugation（300 g for 10 min;2,000 g for 15 min;10,000 g for30 min）and ultracentrifugation（100,000 g for 1.5 h）to obtain MSC-EV.The morphology,size distribution and particle number of MSC-EV were observed by TEM and NTA analysis,respectively.Furthermore,the surface markers of MSC-EV were detected by Western Blotting.PKH26 and RNASelect were used for labeling MSC-EV to observe its uptake by HK-2 cells.4.2 Detection of TFAM and mitochondrial components in MSC-EVThe tfam mRNA expression and mtDNA copy number of MSC and MSC-EV were measured by q-PCR,and the mitochondrial components of MSC-EV were determined by Western Blotting.The mtDNA fragments were confirmed by RT-PCR.4.3 Effect of MSC-EV on TFAM expression and mtDNA copy number in renal tubular epithelial cellsHK-2 cells were incubated with H₂O₂to establish an oxidative stress model,and with or without different MSC-EV treatment.Following,the tfam m RNA and mtDNA copy number were detected by q-PCR.HK-2 cells were pretreated by protein synthesis inhibitors CHX or tfam si RNA followed by MSC-EV treatment.The protein expressions of TFAM and TOM20 were determined to explore the effect of MSC-EV on the TFAM pathway in HK-2 cells.4.4 Effect of MSC-EV on mtDNA homeostasis and mitochondrial function in renal tubular epithelial cellsThe co-localization of TFAM and mitochondria,mtDNA nucleoid morphology and mitochondrial respiratory function were evaluated to explore the effect of MSC-EV on mtDNA damage and mitochondrial function.Results:1 In vitro1.1 Oxidative stress induced mitochondrial impairment and pro-inflammatory cytokines release in renal tubular epithelial cellsHK-2 cells were exposed to H/R or t-BHP（ROS inducer）stimulation.The elevated mtROS in HK-2 cells,induced by H/R or t-BHP,could be effectively reduced by MT treatment.Oxidative stress lowered the cell viability in HK-2 cells,while MT could recover the cell viability in a dose-dependent manner.Moreover,oxidative stress caused an elevation of mitochondrial fragmentation,and a reduction of mitochondrial biogenesis-related genes（ATP5a1/PGC-1α/NDUFS8/UQCRC1）expression and overall mitochondrial respiratory capacity in HK-2 cells.By contrast,MT reduced these mitochondrial lesions caused by mtROS in HK-2 cells.In addition,the increased level of pro-inflammatory cytokines（IL-1β/TNF-α）m RNA expression and macrophage chemotaxis in HK-2 cells induced by oxidative stress could be suppressed by MT treatment.These results indicate that mtROS is involved in the damage and inflammation in HK-2 cells induced by oxidative stress.1.2 mtROS suppressed tfam transcription and promoted Lon-mediated TFAM degradation in renal tubular epithelial cells under oxidative stressWe next determined whether the TFAM expression could be affected by oxidative stress in HK-2 cells.Indeed,the protein levels of TFAM and ATP5α-1were down-regulated by mtROS in HK-2 cells,whereas their protein levels were partly recovered after MT treatment.The m RNA level of tfam was suppressed by mtROS and further restored by MT.The majority of TFAM was evenly co-located with mitochondrial marker TOM20,but mtROS reduced the abundance of TFAM in mitochondria,while it was further recovered by MT.To reveal the impact of different ROS sources or types on TFAM protein,HK-2 cells were treated with different ROS inhibitors under oxidative stress.The declined TFAM protein under oxidative stress was only restored by MT and APO in HK-2 cells under t-BHP.The impact of mtROS on TFAM degradation was also analyzed.Interestingly,the protein level of Lon in HK-2 cells was unchanged in response to ROS,MT or tfam si RNA but its protease activity was up-regulated by mtROS,as indicated by the elevated TFAM protein in t-BHP plus bortezomib（Lon protease inhibitor）compared to t-BHP alone.In contrast,the reduction of TFAM protein under t-BHP was restored by MT or bortezomib,and there was no difference in TFAM protein expression between the two groups.As a result,the reduction of mtDNA copy number in HK2 cells under t-BHP could be partly recovered by MT or bortezomib.1.3 Knockdown of tfam led to mitochondrial dysfunction in renal tubular epithelial cellsSince mtROS-induced TFAM loss has been revealed in HK-2 cells,we sought to explore the role of TFAM protein deficiency in mtDNA and mitochondrial function.tfam knockdown cells were generated,and the reduction of TFAM was confirmed by q PCR and Western Blotting.Knockdown of tfam led to a reduction of TFAM abundance in mitochondria and elevated level of mtROS in HK-2 cells.Loss of TFAM led to a declined mitochondrial respiratory capacity in HK-2 cells.1.4 mtROS disrupted TFAM-mediated mtDNA homeostasis and induced cytokines release in renal tubular epithelial cells under oxidative stressThe impact of mtROS on mtDNA stability was evaluated,and mtDNA nucleoid was visualized using TFAM and ds DNA co-staining.Under oxidative stress,HK-2cells displayed aberrant mtDNA package and reduced mtDNA copy number.Conversely,inhibition of mtROS by MT attenuated the aberrant mtDNA package and restored mtDNA copy number in HK-2 cells under oxidative stress,but the beneficial effects of MT was eliminated when knockdown of tfam.In addition,we observed that BAX proteins were co-localized with Mito-Tracker and they formed macropore-like structures,while mtDNA was appeared within the pores.The upregulation of BAX under oxidative stress in HK-2 cells was suppressed by MT,while the inhibitory effect of MT on Bax was abolished when knockdown of TFAM.Meanwhile,oxidative stress induced cytokines（IL-1β/TNF-α/ICAM-1）expression and macrophage chemotaxis in HK-2 cells,while these adverse effects were reduced by MT.However,knockdown of TFAM enhanced the cytokines expression and macrophage chemotaxis in HK-2 cells even in the presence of MT.2 In Vivo2.1 Establishment of kidney ischemia-reperfusion-induced AKI（IRI-AKI）modelCompared with the CON groups,the IRI-AKI groups and the IRI-AKI+DMSO groups showed significantly increased serum CREA and BUN levels.Pathological examination revealed that renal tubular damage and pro-inflammatory factors（ICAM-1/MCP-1）were increased.In addition,the pro-apoptosis（Bax）m RNA expression was also increased,however,the m RNA expressions of mitochondrial related genes（tfam/PGC-1α/ATP5a1）and mtDNA copy number were decreased.2.2 mtROS involved in renal dysfunction of IRI-AKI mice2.2.1 mtROS promoted kidney injury and inflammation in IRI-AKI miceCompared with the CON groups,AKI mice showed higher serum CREA/BUN,tubular necrosis,kidney injury molecule-1（Kim-1）,and TUNEL⁺apoptotic cells,as well as elevated levels of TNF-α/IL-6/MCP-1 m RNA and CD68⁺macrophages.By contrast,inhibition of mtROS with MT significantly ameliorated the renal dysfunction and inflammatory response in AKI mice.These results indicate that mtROS is an important pathological factor involved in IRI-AKI.2.2.2 mtROS promoted mitochondrial damage in IRI-AKI mice.Compared with the CON groups,AKI mice exhibited severe mitochondrial injury in kidneys,as evidence by increased mtROS and decline of ATP,PGC-1α/ATP5α1/TOM20 expression,mtDNA copy number,mitochondrial area,and mitochondrial length/width ratio.Conversely,MT treatment significantly reduced mtROS and mitochondrial fragmentation,and restored the ATP production and expression of mitochondrial proteins in kidneys of AKI mice.Occurrence,especially mitochondrial morphology and mtDNA damage to accelerate pathological changes in kidney tissue.2.2.3 mtROS induced TFAM reduction and mtDNA damage in IRI-AKI miceIn line with the results in vitro,compared with the CON groups,TFAM mainly expressed in renal tubules,and the m RNA and protein level of TFAM was significantly reduced in kidneys of IRI-AKI mice.Meanwhile,the percentage of mtDNA nucleoids with aberrant package was increased in renal tubules of AKI mice.In addition,Bax expression was found in kidneys of AKI mice compared to CON groups,Meanwhile,the up-regulated Bax and declined TFAM protein in kidneys of AKI mice were partly reversed by MT.However,there was no difference in Lon protein level among these groups.3 TFAM deletion and mtDNA damage in the kidneys of AKI patientsTo validate our findings from cell and mouse AKI models,the changes of TFAM and mtDNA nucleoids were analyzed in kidneys of AKI patients.Compared with the Controls,the kidneys from AKI patients exhibited higher level of NGAL in tubules,while the levels of renal TFAM and TOM20 protein was lower than that of controls.Moreover,we observed the presence of enlarged mtDNA nucleoids in kidneys of AKI patients.4 MSC-EV improves oxidative stress-induced cell damage via mtDNA maintenance in renal tubular epithelial cells4.1 Extraction and identification of MSC-EVThe isolated MSC-EV from the culture medium of h MSCs were typical double-layered membrane vesicles by TEM,and their average diameter was¹60 nm.The positive markers of EV（ALIX,TSG101,and HSP70）were observed in MSC-EV,while GM130（a negative marker）was not detectable in these EV.In addition,MSC-EV labeled with PKH26 and RNASelect can be taken up by HK-2cells.4.2 MSC-EV contained tfam m RNA and mitochondrial componentswe extracted the different types of contents（RNA and DNA）from MSC-EVs and detected the expression of tfam,mtDNA,and mitochondrial proteins.Using q-PCR with specific primers,we detected the tfam m RNA in EVs from both mouse MSC（m MSC）and h MSC.However,the TFAM protein was not detected in the MSC-EVs and supernatant,while some other mitochondrial proteins,such as ATP5a1,COX IV,TOM20 were observed in the MSC-EV.Further reverse transcription-PCR（RT-PCR）analysis detected a large fragment of mtDNA in EV.In addition,some PKH26-labeled MSC-EVs were observed within the mitochondria of recipient cells.4.3 MSC-EV restored TFAM expression and mtDNA copy number in renal tubular epithelial cellsMSC-EV reversed the decrease in levels of the TFAM/TOM20 protein and mtDNA copy number in HK-2 cells exposed to H₂O₂.Higher concentrations of MSC-EVs exerted better rescue effects.MSC-EVs showed a reduced capacity to restore TFAM and TOM20 protein expression in the presence of protein synthesis inhibitor Cycloheximide（CHX）and tfam si RNA.But MSC-EVs partially restored the mtDNA copy number in injured HK-2 cells with tfam si RNA.Taken together,these results indicated that the TFAM signaling pathway might be an important target of MSC-EV.4.4 MSC-EV attenuated oxidative stress-induced mitochondrial dysfunction and the expression of inflammatory factors in renal tubular epithelial cellsMSC-EV improved mitochondrial fragmentation,respiratory capacity,and abnormal mtDNA morphology in HK-2 cells exposed to H₂O₂.In addition,MSC-EV reduced lipopolysaccharide（LPS）-induced inflammatory factors（HMGB1/IL-6）protein expression in primary renal tubular cells.Instead,MSC-EV from TFAM-KD and Aged h MSC showed a limited capacity to reserve the levels.Conclusion:our results demonstrate that mtROS are one of the drivers of mitochondrial dysfunction and inflammation of the renal tubules in IRI-AKI.Mechanistically,mtROS reduced the abundance of TFAM in renal tubular cells by inhibiting its transcription and promoting its Lon-mediated degradation.TFAM deficiency further reduced mtDNA synthesis and mitochondrial biogenesis and thus induced mtDNA depletion and mitochondrial respiratory defects in renal tubular cells during IRI-AKI.At the same time,the loss of TFAM also impaired the stability of mtDNA nucleoids and promoted the release of mtDNA fragments and cytokine production in renal tubular cells during IRI-AKI.Therefore,mtROS-induced TFAM depletion plays an essential role in the pathology of IRI-AKI,and defective TFAM may serve as a therapeutic target for the promotion of renal recovery after IRI-AKI.Furthermore,the application of MSC-EVs restored TFAM protein and TFAM-mtDNA complex（nucleoid）stability,thereby reversing mtDNA deletion and mitochondrial oxidative phosphorylation（OXPHOS）defects in injured renal tubular cells.