Protective role of cyclosporine A and minocycline on

12.Arystarkhova E,Wetzel RK,Sweadner KJ.Distribution and oligomeric as-sociation of splice forms of Na(+)-K(+)-ATPase regulatory gamma-subunit in rat kidney.Am J Physiol Renal Physiol2002;282:F393–F40713.Adalat S,Woolf AS,Johnstone KA et al.HNF1B mutations associate withhypomagnesemia and renal magnesium wasting.J Am Soc Nephrol2009;20:1123–113114.Ferre S,Veenstra GJ,Bouwmeester R et al.HNF-1B specifically regulates thetranscription of the gamma-subunit of the Na+/K+-ATPase.Biochem Bio-phys Res Commun2011;404:284–29015.Ferre S,de Baaij JH,Ferreira P et al.Mutations in PCBD1cause hypomag-nesemia and renal magnesium wasting.J Am Soc Nephrol2014;25: 574–58616.Yang L,Frindt G,Palmer LG.Magnesium modulates ROMK channel-mediated potassium secretion.J Am Soc Nephrol2010;21:2109–211617.Huang CL,Kuo E.Mechanism of hypokalemia in magnesium deficiency.J Am Soc Nephrol2007;18:2649–265218.Devuyst O,Konrad M,Jeunemaitre X.Tubular disorders of electrolyteregulation.In:Avner ED,Harmon WE,Niaudet P,Yoshikawa N(eds), Pediatric Nephrology,6th edn.New York:Springer,2009,pp.929–978.19.Riveira-Munoz E,Chang Q,Godefroid N et al.Transcriptional and func-tional analyses of SLC12A3mutations:new clues for the pathogenesis of Gitelman’s syndrome.J Am Soc Nephrol2007;18:1271–128320.Marples D,Frøkiaer J,Dørup J et al.Hypokalemia-induced downregulationof aquaporin-2water channel expression in rat kidney medulla and cortex.J Clin Invest1996;97:1960–1968Received for publication:22.9.2014;Accepted in revised form:15.12.2014Nephrol Dial Transplant(2015)30:957–969doi:10.1093/ndt/gfv015Advance Access publication1February2015Protective role of cyclosporine A and minocycline on mitochondrial disequilibrium-related podocyte injuryand proteinuria occurrence induced by adriamycinNa Guan1,*,Ya-Li Ren2,*,Xiao-Ya Liu1,Ying Zhang3,Pei Pei3,Sai-Nan Zhu4and Qingfeng Fan1,5,*1Department of Pediatrics,Peking University First Hospital,Beijing100034,China,2Department of Electron Microscopy,Peking University First Hospital,Beijing100034,China,3Department of Central Laboratory,Peking University First Hospital,Beijing100034,China,4Department of Biostatistics,Peking University First Hospital,Beijing100034,China and5Present address:Renal-Electrolyte and Hypertension Division,Perelman School of Medicine,University of Pennsylvania,Philadelphia,PA19104,USACorrespondence and offprint requests to:Na Guan;E-mail:guanna@*These authors contributed equally to this work.A B S T R AC TBackground.Dysfunction of mitochondria is involved in po-docyte injury in some kidney diseases,but the relationship be-tween abnormal mitochondrial morphology and podocyte injury as well as the underlying mechanism is still unclear. This study aims to investigate dynamic changes of mitochon-drial morphology and the potential molecular events in an adriamycin(ADR)-induced podocyte injury model. Methods.Podocyte apoptosis was evaluated by annexin V assay.Podocyte mitochondrial membrane potential(MMP) was measured with MitoCapture kit.Double staining was used to show the distribution changes of mitochondria and actinfilament as well as mitofusin proteins and podocin.Mito-chondrial shape descriptors were obtained using analySIS Image system.Effects of cyclosporine A(CsA)or minocycline (Mcy)on mitochondrial morphology were explored in ADR-induced nephropathy rats.Results.ADR caused podocyte damage displaying as inductionof cellular apoptosis and increase of activated caspase3and cytochrome c.The MMP level was decreased remarkably inADR-treated podocytes.Mitochondrial morphological changes induced by ADR occurred rapidly from large and ellipsoidshape to the small,long and irregular.ADR significantly decreased surface area,perimeter and circularity,while increas-ing aspect ratio of mitochondria.In addition,mitochondria number transiently increased at6h following ADR application. Mitochondria intensity was increased along with punctate mito-chondria formation,which co-localized with polymerized actin cytoskeleton in ADR podocytes.In ADR-induced nephropathyrats,24-h proteinuria was decreased significantly by CsA orORIGINALARTICLE©The Author2015.Published by Oxford University Press on behalf of ERA-EDTA.All rights reserved.957 at Shanghai Jiao Tong University on December 19, 2015 / Downloaded fromMcy.ADR-induced abnormal changes of mitochondrial morphology were restored by CsA or Mcy.The induction of mi-tofusin proteins and the reduction of podocin in ADR rat glom-eruli were rescued by CsA or Mcy.Conclusions.Mitochondrial dysfunction may be an early event in ADR-induced podocyte damage,and the protective role of CsA or Mcy may be mediated partially by improving mitochon-drial function through inhibiting the induction of mitofusin proteins.Keywords:cyclosporine A,minocycline,mitochondria,mito-fusin,podocyte I N T RO D U C T I O N The podocyte plays an important role in maintaining the integ-rity of the glomerular filtration barrier.Injury of the podocyte leads to proteinuria in a variety of glomerular diseases [1].It has been demonstrated that the loss of glomerular podocytes due to cell death/apoptosis or detachment is a hallmark for the pro-gression of kidney diseases to end stage renal failure [2],while the underlying mechanisms remain elusive.The apoptotic events of cells can be initiated and/or propa-gated by diverse intracellular stress conditions,including oxida-tive stress,DNA damage,cytoplasmic Ca 2+release,endoplasmic reticulum stress,mitochondrial dysregulation and many other stimulators [3].The mitochondria are essential to multicellular life as a crucial organelle for ensuring the en-ergy supply and cell homeostasis,as well as the production and clearance of reactive oxygen species (ROS)[4].It has been revealed that the glomerular podocyte has abundant and complex mitochondrial networks [5].Just recently,several studies indicated that mitochondria exert a key role for main-taining podocyte homeostasis,and their failure could lead to podocyte damage [5].Morphological changes of podocyte mitochondria are de finitively observed in patients with mito-chondrial cytopathy or even focal segmental glomerular scler-osis (FSGS)[6].In cultured podocytes,aldosterone application caused remarked dysfunction of mitochondria,including re-duction of mitochondrial membrane potential (MMP)and ATP levels as well as mitochondrial DNA copy number,which may be responsible for decrease of the key podocyte slit diaphragm protein nephrin [7].In addition,TGF β1couldinduce podocyte damage by stimulating mitochondrial oxida-tive phosphorylation and generation of ROS via the mTOR sig-naling pathway [8].It was also reported that up-regulation of mitochondrial Nox4may mediate TGF β1-induced apoptosis in cultured podocytes.These findings emphasize again mito-chondrial functions in podocytes.Nevertheless,quantitative morphological changes of mitochondria in damaged podocytes have not yet been reported.Furthermore,it is also worth inves-tigating whether anti-proteinuric drugs commonly used in the clinic decrease proteinuria and enhance kidney functions by im-proving mitochondrial functions.In this study,stereology analyses of mitochondria morpho-logical changes based on electron microscopy were performed for the first time in an in vitro podocyte injury model induced by adriamycin (ADR).Effects of ADR on mitochondrial func-tion were also investigated by evaluating MMP level in cultured podocytes.Distribution change of mitochondria and actin cytoskeleton was revealed by confocal microscopy.Mitochon-drial remodeling is regulated by continuous fission and fusion that determine mitochondrial morphologies/shapes:long and filamentous (fused),or short and punctated (fragmented)[9].Moreover,the morphologic state of mitochondria could affect their response ability to injury.Several studies suggest that fila-mentous mitochondria are resistant to apoptotic events,while fragmented mitochondria are susceptible to apoptotic stimula-tions [10,11].Two isoforms of mitofusin proteins,namely mi-tofusins 1and 2,both located on the outer mitochondrial membrane,share 60%aminoacid identity with common func-tional domains and appear to function coordinately to regulate mitochondrial fusion [9].Nevertheless,mitofusins 1and 2may also implicate distinct functions in modulating numerous mito-chondrial biological processes with mitofusin-1-dominant mitochondria exhibiting greater tethering ef ficiency than mito-fusin-2-enriched mitochondria [12].Here,expression pattern and distribution change of mitofusins 1and 2were analyzed by using immuno fluorescence staining in ADR nephropathy rat glomeruli.As an immunosuppressant drug,cyclosporine A (CsA)is widely used in kidney diseases to decrease proteinuria or prevent rejection of kidney transplant [13].It was also reported that CsA could affect mitochondrial function by preventing the mitochon-drial permeability transition pore (MPTP)from opening,thus in-hibiting cytochrome c release,a potent apoptotic stimulation factor [14].However,it is unclear whether the anti-proteinuric role of CsA is mediated by improving mitochondrial function in kidney diseases.In addition,some studies suggested that mino-cycline (Mcy)has some favorable effects related to its ability to in fluence mitochondrial functioning [15].There is no report about whether Mcy could be used as an anti proteinuric drugin glomerular diseases.Therefore,the role of anti proteinuric medication CsA and mitochondria targeted drug Mcy was inves-tigated in ADR-induced rat nephropathy.Currently,we provide evidence that mitochondrial disequi-librium may be an early event in ADR-induced podocyte dam-age,and the protective role of CsA and Mcy may be mediated at least partially by improving mitochondrial function via inhibit-ing the induction of mitofusins 1and 2.M AT E R I A L S A N D M E T H O D SPodocyte culture and ADR inductionMouse podocyte cell line MPC5was a kind gift from Peter Mundel.Podocytes were cultured in RMPI 1640medium sup-plemented with 10%fetal bovine serum (Gibco),10U/mL IFN-γ(Sigma)and 1%penicillin/streptomycin at 33°C in a 5%CO 2/95%air atmosphere.After 1week,cells were transferred to the non-permissive conditions of 37°C and differentiated by re-moval of IFN-γfor 14days [16].The podocytes were treated with doxorubicin hydrochloride (ADR;Sigma)at 0.25,0.5and 1.0µg/mL for 24h,respectively.Or,the podocytes were treated with 0.5µg/mL of ADR for 6,12and 24h,respectively.O RIGINALARTICLE958N.Guan et al. at Shanghai Jiao Tong University on December 19, 2015/Downloaded fromApoptosis detectionPodocyte apoptosis was measured with annexin V-FITC Apoptosis Detection Kit(BD Pharmingen).Briefly,1×106 cells were washed twice with cold phosphate-buffered saline (PBS),and re-suspended in1mL of1×binding buffer.Then, 100µL was transferred to a5mL culture tube,and5µL of FITC-annexin V and5µL of propidium iodide were added. The tube was incubated for15min at room temperature.Final-ly,400µL of1×binding buffer was added and analyzed byflow cytometry(BD FACSAria).MMP assayPodocyte MMP was measured with MitoCapture according to the manufacturer’s instructions(BioVision).MitoCapture,a cationic dye,can accumulate and aggregate in mitochondria thus giving off a bright redfluorescence in healthy cells.In apop-totic cells,MitoCapture cannot aggregate in mitochondria due to the altered MMP,and thus it remains in the cytoplasm in its monomer form,fluorescing green color.Podocytes were col-lected,and incubated with MitoCapture dye at37°C for 15min.Greenfluorescence signal was detected immediately by flow cytometry(BD FACSAria)[17].Immunofluorescence stainingPodocytes were grown on collagen I-coated coverslips,and treated with0.5µg/mL of ADR for3,6,12and24h,respective-ly.Cells werefixed with4%paraformaldehyde(PFA),and per-meabilized with0.25%TritonX-100.Mitochondria and actin cytoskeleton were revealed with MitoTracker(Cell Signaling) and Alexa488-conjugated phalloidin(Invitrogen),respectively.Tissue immunofluorescence staining was performed on fro-zen kidney sections from ADR-induced nephropathy rats.Tis-sue sections werefixed with4%PFA,and permeabilized with 0.25%TritonX-100.To remove or decrease non-specific bind-ing,sections were incubated with10%goat serum.Then,mouse anti-mitofusin1or2antibody(Abcam)and rabbit anti-podocin antibody(Abcam)were applied for2h at room temperature. After three washes with PBS,Alexa488-conjugated goat anti-mouse IgG and Alexa594-conjugated goat anti-rabbit IgG(Invi-trogen)were added and incubated for1h.After three washes, sections were mounted with ProLong gold antifade reagent(In-vitrogen).Images were taken by using laser scanning confocal microscope(Olympus Fluoview FV1000)equipped with a 100×oil immersion objective lens(NA1.40).Immunoblotting assayTotal protein was isolated with RIPA buffer(150mM NaCl, 1%NP-40,0.1%sodium dodecyl sulfate,0.5%sodium deoxy-cholate,50mM Tris–HCl pH7.4,15%glycerol)supplemented with protease cocktail inhibitor(Roche).Protein concentration was measured using a BCA assay kit(Bio-Rad).Totally,50µg of protein was loaded on12.5%SDS-PAGE,and then transferred to nitrocellulose membrane.Membranes were rinsed once with Tris-buffered saline containing0.05%Tween-20(TTBS),and then blocked for30min in5%BSA/TTBS.The membranes were incubated overnight with the indicated primary antibodies at4°C.Afterfive washes with TTBS,membranes were incubated with HRP-conjugated secondary antibody for1h.The blots were developed with an ECL chemiluminescence de-tection kit(Pierce),and the specific band was scanned andquantified with Image J1.49d(/ij).Establishment of ADR rat nephropathyAll animal protocols were approved by the Animal Experi-mentation Committee of Peking University First Hospital.Therats were fed with standard diet,and water was given at ad libi-tum.Alternating12-h cycles of light and dark were maintainedfor rats.Male Sprague-Dawley rats weighing120–160g receivedone tail vein injection of ADR(Sigma)at0.7mg/100g body wt diluted in0.9%saline[18].An equal volume of saline was in-jected by tail vein in rats as control(Ctl).Four weeks after in-jections,the ADR-injected rats were further divided into three groups:(i)ADR alone,(ii)ADR plus CsA and(iii)ADR plusMcy.CsA(1mg/100g body wt)was intraperitoneally injected daily,and Mcy(5mg/100g body wt)was fed daily for a total of2weeks.After initial injections,the rats were killed at2weeks(Ctl group,n=3;ADR group,n=3),4weeks(Ctl group,n=3;ADR group,n=6)and6weeks(Ctl group,n=8;ADR group,n=7;ADR plus CsA group,n=8;ADR plus Mcy group,n=7).At each time point,24-h urine was collected from each groupand proteinuria was assessed by using a Pyrogallol red-molyb-date dye-binding method.Kidneys were harvested immediatelyandfixed with ice-cold1%gluteraldehyde in0.1M PBS for transmission electron microscopy(JEOL-1230,Japan).Electron microscopy in cultured podocytesA total of1×106cells werefixed with ice-cold1%gluteral-dehyde in0.1M PBS for24h at4°C followed by washing threetimes with0.2M sucrose.Cells were then post-fixed in1%os-mium tetroxide at room temperature for1h,dehydrated in graded ethanol,embedded with epon812,sectioned using an ultramicrotome(Leica,Germany),and stained with2%uranyl acetate and lead citrate.The sections were photographed with a transmission electron microscopy(JEOL-1230,Japan).Stereology analysis of mitochondrial morphologyForty photographs in each case were taken according to therule of equidistant zigzag movement under the magnification of15000in cell specimen and25000in the rat glomerular area.The grid size with2000×2000nm2was superimposed on eachcell photograph.Because of the lower number of mitochondriain rat glomerular podocytes,equidistant grid lines of1000nm spa-cing on each picture were set in order to get satisfied number of∼50.One grid was a test frame.The intersections of horizontal and vertical lines were testpoints that were used in the following.Mitochondria that inter-sect with the grids,whether horizontal or vertical lines,were de-fined as effective measure targets.The target number of mitochondria is about200in each case.Mitochondrial shape descriptors and size measurements were obtained using ana-lySIS Image Processing software,including(i)surface area (mitochondrial size)that is reported in squared nanometers;(ii)circumference(mitochondrial perimeter)in nanometers,(iii)aspect ratio that is computed as(major axis/minor axis)re-flecting the length-to-width ratio of mitochondria,(iv)ORIGINALARTICLEM i t o c h o n d r i a l d y s f u n c t i o n i n a d r i a m y c i n-d a m a g e d p o d o c y t e s959 at Shanghai Jiao Tong University on December 19, 2015 / Downloaded fromcircularity that is calculated as [4π·(surface area/perimeter 2)],a two-dimensional index of sphericity with values of 1indicating perfect spheroids and (v)mitochondrial density calculated as the number of mitochondria cross-sections per square nano-meter in the whole cell area or only in the plasma area excluding nuclei.The density value is obtained by dividing the area of all test points in the case by the number of effective mitochondria in the same case.According to the rule of stereology,the asso-ciated area of one test point is equivalent to the area of one test frame [19,20].Computed values were imported into Microsoft Excel and Prism 6(GraphPad software)for data analysis.Statistical analyses All data are expressed as means ±SEM.ANOV A was used to compare differences of multiple groups.A P-value of <0.05was considered as signi ficant differences.R E S U LT S Apoptosis induction and mitochondrial dysfunction in ADR-treated podocytes Podocyte damage was firstly analyzed by assessing the cellu-lar apoptosis with FITC-annexin V assay in cultured podocytes treated with different concentrations of ADR for -pared with non-treated cell (5.5±0.9),the percentage of apoptotic cells was signi ficantly increased (P <0.01)in podocytes treated with 0.25(21.9±2.5),0.5(37.7±6.4)and 1.0µg/mL (49.7±8.7)of ADR,respectively.In addition,0.5and 1.0µg/mL of ADR in-duced much more (P <0.01)podocyte apoptosis than 0.25µg/mL.Furthermore,the percentage of apoptotic cells was dramatically in-creased (P <0.05)in podocytes treated with 1.0µg/mL of ADR in comparison with 0.5µg/mL,displaying a dose-dependent effect of ADR on podocyte apoptosis (Figure 1A).Caspase 3is activated in the apoptotic cell,where cyto-chrome c that is released from mitochondria plays an essential role in processing procaspase 3[21].Here,total cellular protein was isolated from podocytes treated with 0.5µg/mL of ADR,and the protein level of cleaved caspase 3and cytochrome c was detected using immunoblot assay.In comparison with Ctl,the cleaved caspase 3and cytochrome c level were increased signi ficantly (P <0.01)at 12and 24h following ADR applica-tion (Figure 1B and C).Disruption of MMP is one of the earliest intracellular events that occur following induction of apoptosis [3,7].Here,MMP level was assessed with a cationic dye MitoCapture.As com-pared with non-treated cell (189.0±11.27),data from flow cy-tometry showed a signi ficant increase (P <0.05)of green fluorescent signal in podocytes treated with 0.5(257.3±27.2)or 1.0µg/mL (241.3±14.15)of ADR (Figure 1D),indicating re-duction of MMP level.Dynamic alteration of mitochondrial morphology in ADR-treated podocytes Timing effects of 0.5µg/mL of ADR on mitochondrial morphology were investigated in cultured podocytes.Electron microscopy images were used to evaluate morphological changes of mitochondria following ADR rge and round mitochondria were present in normal or non-treated cells,whereas mitochondrial fragmentation was observed in ADR-treated podocytes as evidenced by a signi ficant increase of small,long and irregular mitochondria (Figure 2A).Quanti-fication of mitochondrial shape is required to determine if changes in mitochondrial morphology are linked to the speci fic pathophysiological functions of podocytes [5].The stereology parameters of mitochondria were obtained using analySIS Image Processing software.Quantitative analyses showed that ADR-trea-ted podocytes exhibited 42.4,32.1and 30.2%reduction (P <0.01)of mitochondrial surface area at 6,12and 24h,respectively.In comparison with Ctl (2191±60.80nm),mitochondrial perimeter decreased signi ficantly (P <0.01)at 6(1735±68.14nm),12(1866±55.72nm)and 24h (1840±75.56nm)following ADR applica-tion.These data indicate that ADR-induced small mitochon-dria formation in podocytes.In addition,circularity,a two-dimensional index of sphericity with values of 1indicating per-fect spheroids,obviously decreased (P <0.01)at 6(0.76±0.01),12(0.78±0.01)and 24h (0.79±0.01)after ADR administra-tion against Ctl cell (0.82±0.01),providing the evidence that ir-regular and fragmented mitochondria were present.Consistently,compared with Ctl (1.56±0.04),aspect ratio that re flects the length-to-width ratio of mitochondria,remarkably increased (P <0.01)at 6(1.91±0.05),12(1.81±0.05)and 24h (1.72±0.06)in ADR-treated podocytes,supporting the formation of long mitochondria.Moreover,mitochondrial density both in whole cell area and only in plasma area was transiently increased (P <0.05)only at 6h following ADR application (Figure 2B).Dose-dependent effects of ADR on mitochondrial morpho-logical changes were also studied at 24h in cultured podocytes.Electron microscopy images disclosed that in comparison with normal cells,ADR induced dynamic changes of mitochondrial shapes from large and round morphology to the small,thin and irregular (Supplementary Figure S1A).Stereological quantita-tive analyses showed that compared with non-treated cell,po-docyte treated with 1.0µg/mL of ADR exhibited 25%reduction (P <0.05)of mitochondrial circumference,and mitochondrial surface area was respectively decreased (P <0.01)∼27.2,35.8and 44.2%in 0.25,0.5and 1.0µg/mL of ADR-treated podo-cytes.In addition,circularity was signi ficantly decreased (P <0.05)in 0.25(0.73±0.01),0.5(0.77±0.01)and 1.0µg/mL (0.79±0.01)of ADR-treated podocytes against Ctl cell (0.84±0.01).Consistently,compared with Ctl (1.39±0.04),aspect ratio was remarkably increased (P <0.01)in ADR-treated podocytes (0.25µg/mL:1.93±0.07;0.5µg/mL:1.80±0.06;1.0µg/mL 1.71±0.06)(Supplementary Figure S1B).Dynamic changes of mitochondria and actin cytoskeleton in ADR-treated podocytesDistribution of mitochondria and actin cytoskeleton was analyzed in podocytes treated with 0.5µg/mL of ADR at differ-ent time periods.Normal podocytes displayed large and com-plex mitochondrial networks as well as filamentous actincytoskeleton.In ADR-treated cells,mitochondria fluorescence in-tensity increased obviously along with remarked morphological alterations.Against Ctl,mitochondria became short and frag-mented at 3h after ADR application.Since 6h,linear mitochon-drial network disappeared,and aggregated mitochondria were O RIGINALARTICLE960N.Guan et al. at Shanghai Jiao Tong University on December 19, 2015/Downloaded fromobserved persisting to 24h.Rearrangement of actin cytoskeleton also occurred in ADR-treated cells.At 3and 6h following ADR application,actin polymerization was revealed displaying as the increased and aggregated staining of actin cytoskeleton at cell per-iphery.In comparison with the untreated cells,intracellular actin filaments disappeared in ADR-treated podocytes.Moreover,co-localization of punctate mitochondria and polymerized actin cytoskeleton was observed in ADR-treated cells,especially at 3and 6h (Figure 3).Protective role of CsA and Mcy in ADR-induced rat nephropathy Rat nephropathy was induced by a single tail vein injection of ADR.Twenty-four hour proteinuria was assessed at 2,4and 6weeks.As compared with the corresponding Ctl,induction ofproteinuria was signi ficantly detected (P <0.01)at 4weeks(167.92±23.56mg versus 12.50±1.82mg)and 6weeks (226.86±40.42mg versus 12.0±1.57mg)in ADR rats (Fig-ure 4A).Ultrastructural changes of podocyte foot processes (FPs)were revealed using transmission electron microscopy.In Ctl kidneys,thin and long FPs were well conserved,whereas FPs diffusion occurred at 2weeks,and effacement was widely observed at 4and 6weeks after ADR injections (Figure 4B).CsA is an anti proteinuric medication commonly used in kidney diseases [13].Several studies reported that Mcy could improve mitochondrial function [15].Therefore,the effects of CsA and Mcy were explored in ADR-injected rats.At 4weeks following ADR injections,CsA and Mcy were administrated once a day totally for 2weeks.Quantitative results of 24-h pro-teinuria showed that compared with rats injected with ADRalone (226.86±40.42mg),proteinuria was decreased sig-ni ficantly (P <0.05)in CsA-treated (113.10±35.18mg)or Mcy-treated (153.6±26.71mg)rats (Figure 4C).Electronmicroscope image showed that ADR led to widely diffusion and effacement of podocyte FPs,which was restored or recov-ered effectively by CsA or Mcy treatment (Figure 4D).Effects of CsA and Mcy on podocyte mitochondrial morphology in ADR-induced rat nephropathyIn saline-injected Ctl rat,round or ellipsoid mitochondria were orderly arranged in podocytes with intact mitochondrial membrane and regularly arranged cristae in the formof F I G U R E 1:Mitochondrial dysfunction in ADR-induced podocyte damage.Mouse podocytes were differentiated for 10days at 37°C.(A )Podocytes were treated for 24h with different concentrations of ADR.The percentage of apoptotic cells was analyzed using FITC-annexin V pared with non-treated cell (0µg/mL),ADR induced a dose-dependent increase of cellular apoptosis.n =3independent experiments.*P <0.01versus 0µg/mL;#P <0.01versus 0.25µg/mL;†P <0.05versus 0.5µg/mL.(B and C )Podocytes were treated with ADR for the indicated time periods.Total cellular protein was extracted with RIPA buffer,and immunoblotting was performed for expression of activated caspase 3(casp3)and cytochrome c (cyt c ).n =3independent experiments.*P <0.01versus 0h.(D)Podocytes were treated for 24h with different concentrations of ADR.MMP was measured with a cationic dye MitoCapture.In apoptotic cell,MitoCapture remains in the cytoplasm in its monomer form and emits green fluorescence,indicating reduction of MMP level.MitoCapture level was detected by using flow cytometry,displaying a signi ficant increase of green fluorescent signal in 0.5and 1.0µg/mL of ADR-treated podocytes.n =4independent experiments.*P <0.05versus 0µg/mL.O R I G I N A L A R T I C L E M i t o c h o n d r i a l d y s f u n c t i o n i n a d r i a m y c i n -d a m a g e d p o d o c y t e s 961 at Shanghai Jiao Tong University on December 19, 2015/Downloaded fromconcentrical ring or vertical line (Figure 5).At 2weeks after ADR injections,mitochondrial membrane in podocyte cell body seemed disappear and intra-mitochondrial structure became unclear.At 4weeks,podocyte mitochondria became swollen with vague or partly ruptured mitochondrial mem-brane,and disorganized cristae.At 6weeks,podocyte F I G U R E 2:Time-dependent alteration of mitochondrial morphology in ADR-treated podocytes.Differentiated mouse podocytes were treated with 0.5µg/mL of ADR for the indicated time periods.Electron microscopy images were used to evaluate mitochondria morphological changes.(A )Representative images are shown,and the typical mitochondrion was enlarged.Bar =2µm.(B )Mitochondrial shape descriptors and size measurements were obtained using analySIS Image Processing,including (i)surface area,(ii)circumference,(iii)aspect ratio (major axis/minor axis)re flecting the length-to-width ratio of mitochondria,(iv)circularity [4π·(surface area/perimeter 2)],a two-dimensional index of sphericity with values of 1indicating perfect spheroids and (v)mitochondria density calculated as the number of mitochondria in whole cell area or only in the plasma area.Totally,200mitochondria each group were puted values were imported into Prism 6for data analysis.ADR resulted in a reduction of mitochondrial area,circumference and circularity,while led to an increase of aspect ratio of mitochondria.In addition,mito-chondria number both in whole cell area and in only plasma area increased remarkably only at 6h following ADR application.*P <0.01versus 0µg/mL;#P <0.05versus 0µg/mL.ORIGINALARTICLE962N.Guan et al. at Shanghai Jiao Tong University on December 19, 2015/Downloaded from。