Calpastatin Tiv Thaiv Angiotensin II-mediated Podocyte Injury Los Ntawm Kev Kho Ntawm Autophagy
Mar 17, 2022
Yog xav paub ntxiv:ali.ma@wecistanche.com
Imane Bensaada1,3, Blaise Robin1,3, Joe¨lle Perez2, Yann Salemkour1, Anna Chiv1, Marine Camus1, Mathilde Lemoine1, Lea Guyonnet1, He'le`ne Lazareth1, Emmanuel Letavernier2, Carole Henique1, Pierre-Louis Tharaux1thiab Olivia Lenoir1
1Université de Paris, PARCC, Inserm, Paris, Fabkis; thiab2Université Paris Descartes, Sorbonne Paris Cité, Paris, Fabkis
Lub zog kwv yees tus nqi ntawm cov proteinuria nyob rau hauv cov kab mob glomerular ntev yog ruaj khov, nrog rau lub luag haujlwm pathogenic ntawm angiotensin II txhawb kev loj hlob ntawm cov kab mob glomerular nrog kev hloov pauv glomerular fi-filtration barrier, podocyte raug mob thiab caws pliav ntawm glomeruli. Ntawm no peb pom tias mob angiotensin II-induced hypertension inhibitedautophagyefflux nyob rau hauv nas glomeruli. Deletion ofAtg5 (ib tug noob encoding ib cov protein koom nrog autophagy) tshwj xeeb hauv cov podocyte ua rau kom ceev dangio tensin II-induced podocytopathy, accentuated albuminuria, thiab glomerulosclerosis. Qhov no qhia tau hais tias autophagy yog ib qho tseem ceeb tiv thaiv mechanism nyob rau hauv lub podocyte nyob rau hauv tus mob no. Angiotensin-II induced calpain kev ua si hauv podocytes inhibitsautophagyefflux. Podocytes los ntawm nas nrog transgenic qhia ntawm endogenous calpain inhibitortshuaj calpastatintso tawm ntau dua podocyteautophagyNyob rau hauv lub hauv paus kab uas tiv taus angiotensin II-dependent inhibition. Tsis tas li ntawd, sustained autophagy nrogtshuaj calpastatintxwv podocyte puas thiab albuminuria. Cov kev tshawb pom no qhia tias kev kub siab muaj cov kab mob tshwm sim ntawm cov qauv glomerular thiab kev ua haujlwm, ib feem los ntawm kev ua kom cov calpains ua rau blockade ntawm podocyte autophagy. Cov kev tshawb pom no nthuav tawm cov txheej txheem qub uas yog angiotensin II-mediated hypertension inhibits autophagy ntawm calcium-induced recruitment ntawm calpain nrog rau cov teeb meem tshwm sim nyob rau hauv rooj plaub ntawm kev tsis txaus siab los ntawmtshuaj calpastatinkev ua si. Yog li, tiv thaiv calpain-mediated txo nyob rau hauvautophagyTej zaum yuav yog qhov kev cog lus tshiab kho tshiab rau nephropathies cuam tshuam nrog kev ua haujlwm siab renin-angiotensin system.
KEYWORDS: angiotensin II;autophagy; tshuaj calpastatin; ntshav siab; podocyte
Lus Txhais Lus
Muab lub luag haujlwm tseem ceeb ntawm autophagy hauv kev txhim kho ntawmraumkab mob, kev kho tshuaj pharmacological ntawmautophagytej zaum yuav yog ib lub tswv yim zoo rau kev tiv thaiv thiab kev kho mob ntawm ob pebraumkab mob. Nyob rau tib lub sijhawm, overactivation ntawm calpain kev ua si hauv podocytes tau pom tias ua rau muaj kev cuam tshuam tsis zoo ntawm podocyte muaj nuj nqi thaum nws cov txheej txheem tsis zoo ntawm kev ua tsis tau txheeb xyuas. Ntawm no, peb muab pov thawj tias calpain txuas qhov kev txiav txim siab ntawm angiotensin II mus rau kev cuam tshuam ntawm kev cuam tshuam.autophagyhauv podocytes thiab qhia tias calpain inhibition tuaj yeem yog lub hom phiaj kho mob zoo rau cov kab mob podocyte ib nrab los ntawm kev saib xyuas ntawm podocyte autophagy.
Nyem rauCistanche deserticola ma rau mob raum mob
Ntshav siab yog qhov thib ob nkaus xwb rau ntshav qab zib yog qhov ua rau muaj mob hnyavraumkab mob 1-3 thiab txawm tias qhov nce siab hauv cov ntshav siab yog ib qho kev pheej hmoo ntawm cov kab mob raum kawg.4 Kev tshawb fawb ntau ntxiv tau qhia txog qhov tseem ceeb ntawm podocytes hauv kev loj hlob ntawm cov kab mob hauv lub raum.raumraug mob. Kev poob ntawm podocytes thiab microvascular alterations tshwm sim ntxov nrog rau lub raum tsis ua hauj lwm poob rau hauv kev sim hypertensive nephropathy.5 Hauv cov neeg mob, tso zis tawm ntawm cov podocytes uas siv tau tau pom tias yog ib qho cim tshwj xeeb rau preeclampsia, 6,7 thiab cov neeg mob nephrosclerosis muaj qhov cuam tshuam loj heev. txo qis ntawm glomerular podocytes dua li uaraumCov neeg pub.8,9 Tsis tas li ntawd, lub luag haujlwm pathogenic ntawm angiotensin II (AngII) txhawb txoj kev loj hlob ntawm cov kab mob glomerular yog tsim tau zoo, tsis yog nyob rau hauv cov mob hnyav xwb tab sis kuj muaj ntau yam kab mob glomerular.10-21.
Glomerular hypertension ua rau glomerular capillary stretching, endothelial puas, thiab siab glomerular protein filtration ua rau glomerular collapse thiab glomerulosclerosis. Nws kuj tseem ua haujlwm ncaj qha rau ntawm cov qauv glomerular, ua rau muaj teeb meem kev tswj xyuas cov lus teb txhawm rau them nyiaj. Ib qho activated systemic thiab hauv zos renin-angiotensin-aldosterone system (RAAS) txhawb nqa mesangial hyperplasia thiab synthesis ntawm vascular permeability yam. Concomitantly, podocytes qhia calcium signaling22,23 thiab hloov lawv cov duab raws li AngII hom 1 receptor (AT1)- nyob ntawm stimulation.24-28 Cov kev hloov kho no ua rau lub sijhawm ntev, thaum kawg ua rau glomerulosclerosis. AT1 mediates tseem ceeb RAAS kev koom tes rau ntshav siab thiab ntsev thiab dej homeostasis. Angiotensin-hloov enzyme inhibitors thiab AT1 blockers tau siv kho mob rau kev kho mob ntshav siab thiab plawv tsis ua haujlwm hauv cov neeg mob. Interestingly, ob qho tib si blockers kuj qhia txog kev tiv thaiv ntawmraummuaj nuj nqi.
Autophagy tau pom tias yog qhov tseem ceeb rau kev saib xyuas ntawm cellular homeostasis, tshwj xeeb tshaj yog nyob rau hauv postmitotic cells29,30 thiab tshwj xeeb hauv podocytes.31–34.Autophagyyog ib qho lysosomal-koom nrog degradation system rau lub neej ntev cytoplasmic proteins thiab dysfunctional organelles35,36 thiab koom nrog sequestration ntawm cov proteins thiab organelles hauv autophagosomes. Kev tsim ntawm autophagosomes yog nyob ntawm qhov induction ntawm ntau cov noob suav nrog Map1lc3a/b, Beclin 1, thiab Cim npe. 37 Muaj cov pov thawj loj zuj zus tuaj tias kev tsis sib haum xeeb ntawm txoj kev autophagic cuam tshuam rau hauv lub raum kev laus thiab ntau yam.raumkab mob xws li mob raum raug mob, kab mob polycystic raum, kev laus, thiab mob ntshav qab zib nephropathy.31,32,38–41
Kev cai ntawmautophagynyob rau hauv podocytes, physiological thiab, saum toj no tag nrho, nyob rau hauv ib tug pathological ntsiab lus, tsis paub zoo. Peb tsis ntev los no tau pom tias podocyte autophagy yog ywj siab ntawm lub hom phiaj ntawm kev tswj hwm ntawm rapamycin (mTOR) nyob rau hauv physiological tej yam kev mob, ua rau hom cell no muaj kev zam.42 AT1 activation stimulates protein synthesis thiab protein turnover hauv hlwb. Yog li, peb tau xav tias kev ua kom RAAS kuj tseem cuam tshuam rau kev hloov pauv ntawm cov protein thiab proteostasis.
Hauv kev tshawb fawb tam sim no, peb tau tsom mus rau lub luag haujlwm ntawm AngIIsignaling hauv podocyteautophagykev cai. Peb tau txheeb xyuas cov calcium-activated proteases calpains mediating ib qho kev cuam tshuam ntev ntawm AngII ntawm podocyte.autophagy. Tsis tas li ntawd, peb pom tias endogenous calpain inhibitortshuaj calpastatinmuaj peev xwm tiv thaiv AngII-dependent autophagy inhibition thiab podocyte raug mob thaum kub siab.
Cov kev tshawb pom no nthuav tawm cov txheej txheem qub uas yog AngII-mediated hypertension inhibitsautophagyLos ntawm calcium-vim kev nrhiav neeg ua haujlwm ntawm calpain nrog rau qhov tshwm sim ntawm pathogenic tshwm sim nyob rau hauv rooj plaub ntawm kev tsis txaus siab los ntawm kev ua haujlwm calpastatin.
Txoj kev
Tsiaj
CalpastatinCov nas transgenic (CSTTg) tau ua siab zoo los ntawm Dr. E. Letavernier.43 Cov nas uas muaj qhov cuam tshuam ntawm podocyte tshwj xeeb ntawm Atg5 noob (Nphs2.cre Atg5lox/lox) tau tsim raws li tau piav qhia dhau los31 los ntawm kev hla Nphs2.cre nas44 nrog Atg5lox/lox nas 45 ntawm C57BL6 / J keeb kwm yav dhau. Nphs2.cre Atg5lox/lox nas thiab tswj littermate txiv neej, hnub nyoog 10 txog 12 lub lis piam, tau siv rau hauv txoj kev tshawb no. Tus qauv ntshav siab tau raug ntxias los ntawm sc infusion ntawm AngII (Sigma-Aldrich, A9525) ntawm koob tshuaj 1 mg / kg / min rau 4 mus rau 6 lub lis piam ntawm osmotic minipumps (Alzet Corp, qauv 2006). Cov twj tso kua mis tau cog rau hauv qab ntawm lub xub pwg hniav thiab lub duav. Cov nas tau txais ntsev ntxiv (3 feem pua NaCl) hauv cov zaub mov. Atg5lox/lox (wild-type [WT]) nas tau siv los tswj hauv txhua qhov kev tshawb fawb. Rau deoxycorticosterone acetate (DOCA) ntsev nrog nephron txo tus qauv, cov neeg laus txiv neej nas underwent unilateral sab laug nephrectomy. Ob lub lis piam tom qab nephrectomy, lawv tau txais DOC pellets nrog rau 21- hnub tso tawm (Innovative Research of America) implanted sc Ib tug thib ob pellet tau cog rau 3 lub lis piam tom qab thawj implant. Txhua tus nas tau txais 0.9 feem pua NaCl hauv cov dej haus ad libitum thiab raug tua tom qab 6 lub lis piam ntawm DOC tswj hwm.46 Kev sim tau ua raws li Fabkis cov kws kho tsiaj cov lus qhia thiab cov tsim los ntawm European Community rau kev sim siv tsiaj (L358-86/ 609EEC) thiab tau txais kev pom zoo los ntawm Fab Kis Lub Tsev Haujlwm Saib Xyuas Kev Tshawb Fawb thiab pawg neeg tshawb fawb hauv tsev kawm qib siab (APAFIS-7646 thiab -22373).
Thawj qhov kev sim podocyte kab lis kev cai
Kev sib txawv thawj podocytes tau coj los ua kab lis kev cai raws li tau piav qhia yav dhau los.47,48 Luv luv, lub raum cortex tshiab tau muab sib xyaw thiab zom los ntawm collagenase I (Gibco, 17100-017) hauv Roswell Park Memorial Institute 1640 (Lub Neej Technologies, 61870-044). Cov ntaub so ntswg tau dhau los ntawm 70 mm thiab 40 mm cell strainers (BD Falcon, 352340 thiab 352350). Glomeruli, uas ua raws li 40 mm cell strainer, raug tshem tawm nrog phosphate-buffered saline (PBS; Life Technologies, 10010023) þ 0.5 feem pua bovine serum albumin (Eurobio, HALB07-65) txhaj ob zaug hauv siab thiab tom qab ntawd ntxuav ob zaug. hauv PBS. Freshly cais glomeruli tau plated hauv 6-cov tais diav hauv Roswell Park Memorial Institute 1640 (Gibco, 61870036) ntxiv nrog 10 feem pua fetal calf serum thiab 1 feem pua penicillin/streptomycin (Life Technologies, 15140122) thiab loj hlob. Podocyte enrichment tau txheeb xyuas los ntawm Western blot tsom xam raws li yav dhau los tau piav qhia 31,48,49 (Cov Duab Ntxiv S1). Podocytes tau coj mus kuaj thaum tsis muaj lossis muaj bafilomycin A1 (100 nmol / l, Sigma-Aldrich, B1793) rau 4 teev. Rau kev sim tshuaj immunofluorescence, thawj podocytes tau plated ntawm 4 daim ntawv tais diav (Dutcher, 055071). Podocytes tau kho nyob rau hauv paraformaldehyde 4 feem pua rau 10 feeb thiab ua tiav rau immunofluorescence.
Calpain kev ntsuam xyuas
Intracellular calpain kev ua haujlwm tau txiav txim siab hauv thawj podocytes, raws li tau piav qhia yav dhau los.50–52 Tag nrho ntawm 100,000 cov hlwb raug coj los rau hauv 24-cov ntaub so ntswg kab lis kev cai hauv Roswell Park Memorial Institute 1640 ntxiv nrog 10 feem pua fetal calf serum thiab 1 feem pua penicillin/streptomycin. Tom qab lub sijhawm kab lis kev cai, qhov nruab nrab tau hloov nrog Krebs-Ringer HEPES bicarbonate (KRH) cov tshuaj (pH 7.4) uas muaj 4 mM CaCl2, nrog lossis tsis muaj 10 mM calpain inhibitor-1, thiab incubated rau 10 feeb ua ntej ntxiv ntawm 50 mM calpain substrate N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methyl coumarin (Sigma-Aldrich, S6510). Tom qab ib tug 90-feeb incubation lub sij hawm, calpain kev ua ub no tau txiav txim raws li qhov sib txawv ntawm FL fluorescence ( ntsuas ntawm 360 nm excitation thiab 430 nm emission) nrog thiab tsis muaj calpain inhibitor -1.
Western blot
Thawj podocytes raug khawb nrog 80 ml ntawm radioimmunoprecipitation assay tsis muaj phosphatase thiab protease inhibitor. Protein concentration tau ntsuas nrog BCA Protein Assay Kit (Merck Biochemistry, 71285). Nees nkaum micrograms ntawm cov protein tau electrophoresed ntawm Criterion XT precast gel (12 feem pua Bis-Tris, Bio-Rad, 3450124). Proteins raug xa mus rau polyvinylidene difluoride membrane (Thermo Fischer Scientific, 88518). Tom qab thaiv hauv 5 feem pua ntawm cov mis nyuj hauv Tris Buffer Saline 0.1 feem pua Tween (TBS-T), cov ntaub so ntswg tau incubated nrog luav polyclonal anti-LC3 (1: 1000, Cell Signaling Technology, 2575), luav polyclonal anti-ATG5 (1:2000, Cell Signaling Technology, 2630), guinea pig polyclonal anti-Sequestosome 1 (SQSTM1)/P62 (1:10,000, PROGEN, GP62), luav polyclonal anti-calpain 1 domain IV (1:1000, Abcam, ab39) ), luav polyclonal anti-calpain 2 amino-terminal kawg ntawm sau kuv (1: 1000, Abcam, ab39165), nas monoclonal IgG1 anti-calpain 4 (1: 1000, Santa Cruz Biotechnology, sc-32325), luav anti podocin (1: 1000, Abcam, ab50339), guinea pig anti-nephrin (1: 500, PROGEN, GP-N2), thiab nas monoclonal anti-tubulin (1: 5000, Abcam, ab6160) antibody. Tom qab ntxuav, daim nyias nyias tau incubated nrog horseradish peroxidase-txuas antibody (1: 2000, Cell Signaling Technology, 7074, 7076, 7077). Kev kuaj pom cov cim tshwj xeeb tau ua tiav siv ECL Chemiluminescent Kit (Bio-Rad, 170-5070) ntawm LAS 4000 ntaus ntawv (Fuji). Kev tsom xam Densitometry nrog ImageJ software (National Institutes of Health) tau siv los ntsuas qhov ntau.
Kev ntsuas ntshav siab thiab ntsuas physiological
Cov systolic ntshav siab ntawm nas tau sau tseg siv txoj kev tail-cuff (Visitech Systems Inc., BP-2000). Kaum qhov kev ntsuas ntawm txhua tus nas raug coj mus, thiab tom qab ntawd tus nqi nruab nrab tau txiav txim siab. Systolic ntshav siab tau ntsuas ntawm lub hauv paus (12 lub lis piam ntawm hnub nyoog) thiab tom qab ntawd txhua lub lim tiam mus txog rau thaum xaus ntawm lub sijhawm kho. Tag nrho cov nas tau muab tso rau hauv cov kab mob metabolic nrog kev nkag mus rau dej dawb rau 6- teev tso zis. Cov zis creatinine thiab plasma urea concentrations tau soj ntsuam spectrophotometrically los ntawm kev siv txoj kev colorimetric (Olympus, AU400). Cov zis albumin tso zis tau ntsuas los ntawm kev siv cov tshuaj tiv thaiv kab mob sib txuas tshwj xeeb rau kev txiav txim siab ntau ntawm albumin hauv cov zis nas (Crystal Chem, 80630).
Histology
Lub raumtau sau thiab kho hauv 4 feem pua PBS-buffered formalin. Paraffin-embedded seem (3-mm tuab) tau stained los ntawm Masson'strichrome los ntsuas lub raum morphology. Qhov txawv txav hauv lub raum tau muab qhab nia raws li qhov muaj thiab qhov hnyav ntawm cov khoom txawv txav, suav nrog glomerulosclerosis, mesangial expansion, tubular atrophy lossis casts, thiab fibrosis. Qhov kev faib ua feem ntawm sclerotic glomeruli raug soj ntsuam los ntawm kev kuaj qhov muag tsis pom qhov tsawg kawg yog 50glomeruli ib.raumntu.
Immunofluorescence staining ntawm lub raum seem thiab thawj podocytes
Tsau thawj podocytes raug thaiv nyob rau hauv TBS-T 3 feem pua bovine serum albumin thiab incubated thaum hmo ntuj ntawm 4 degree nrog thawj anti-bodies guinea npua anti-SQSTM1/P62 (1:1000, PROGEN, GP-62C) thiab luav anti -green FL fluorescent protein (GFP; 1:500, Abcam, ab290). Tom qab TBS-T yaug, FL fluorophore-conjugated thib ob antibodies nees luav anti-guinea npua IgG AF594-conjugated antibody (JacksonImmunoResearch, 706-585-148) thiab luav anti-luv IgG AF488-conjugated antibody ( Invitrogen, A21206) tau thov. Cov duab tau muab coj los siv Zeiss 2 ft fluorescent microscope, AxioCam HRccamera, thiab Axiovision 4.3 software.
Rau formalin-taw paraffin-embedded (FFPE)lub raum, ntu (3 hli) tau deparaffinized thiab hydrated thiab antigen retrieval tau ua nyob rau hauv rhuab citrate tsis (pH 6). Cov seem tau permeabilized nrog Triton 0.1 feem pua (Euromedex) thiab thaiv hauv TBS-T 3 feem pua bovine serum albumin ua ntej hmo ntuj tshuaj tiv thaiv incubation ntawm 4 C.Peb siv tshis anti-nephrin (1:100, PROGEN, GP. -N2), guinea pig anti-SQSTM1/P62 (1:1000, PROGEN, GP-62C), luav anti-GFP(1:1000, Abcam, ab290), tshis anti-Podocalyxin (PODXL; 1: 1000, Bio-Techne, AF-1556), thiab luav anti-Wilm's Tumor 1 (WT1; 1:100, Abcam, ab192) antibody. Cov tshuaj tiv thaiv thib ob yog Alexa488- thiab Alexa 568-conjugated antibodies los ntawm Invitrogen. Nuclei tau stained xiav siv Hoechst. Cov zawv zawg tau teeb tsa siv qhov nruab nrab fluorescent mounting (Dako, S3023). Photomicrographs tau raug coj los nrog Zeiss Axiophot photomicroscope thiab Axiovisionsoftware. Semiautomatic quantifications ntawm Fiji tau siv rau kom muaj nuj nqis ntawm nephrin-positive thiab PODXLþ thaj chaw ntawm glomerular seem ntawm tsawg kawg 30 glomeruli ib tus nas. Podocytenumber raug suav tias yog tus naj npawb ntawm WT1þ nuclei ib ntu glomerular ntawm tsawg kawg 30 glomeruli ib tus nas.
Transmission electron microscopy txheej txheem
Cov me me ntawm lub raum cortex (1 mm3) tau kho hauv 3 feem pua glutaraldehyde qib (Electron Microscopy Sciences) rau 1 txog 30 hnub thiab ntxuav peb zaug hauv PBS. Cov qauv raug kho tom qab 1 feem pua osmium tetroxide 0.1 M (Electron Microscopy Science) hauv 0.1 M PBS (pH 7.4) thiab ntxuav hauv dej. Cov qauv tau qhuav dej hauv qib cawv thiab 100 feem pua propylene oxide (Electron Microscopy Science). Resin infiltration tau ua raws li hauv qab no: sib tov Epikote 812 thiab propylene oxide hauv qhov sib piv ntawm 1: 1 rau 30 feeb tom qab sib xyaw Epikote 812 thiab propylene oxide hauv qhov sib piv ntawm 1: 2 rau hmo ntuj chav tsev kub. Cov qauv tau muab tso rau hauv 4 hli gelatin capsules hauv 100 feem pua Epikote 812 thiab polymerized nyob rau hauv qhov cub rhuab mus rau 60 C. Ultrathin seem raug txiav nrog ib tug UFC7 ultramicrotome (Leica MicrosystemsGmbH) thiab muab tso rau ntawm Gilder Grids 200 mesh (Electron Microscopy Science). Lawv tau counterstained nrog uranyl acetate 7 feem pua (LFG Distribution) thiab Reynold's lead citrate (LFG). Cov qauv raug kuaj xyuas hauv JEM1011 kis tau tus mob electron microscope (JEOL) nrog Orius SC1000 CCD lub koob yees duab (Gatan), ua haujlwm nrog Digital Micrograph software (Gatan) kom tau.
Cistanche-mob kab mob
Quantitative polymerase chain reaction array
Freshly cais glomeruli tau khov hauv QIAzol Lysis reagent (Qiagen) ntawm -80 degree . Tag nrho RNA rho tawm siv phenol-raws li txoj kev tau ua tiav raws li cov chaw tsim khoom cov lus pom zoo. cDNAs tau tsim los siv RT2 Thawj Strand Cov Khoom Siv (Qiagen, 330401), thiab lub sijhawm ntawm cov tshuaj tiv thaiv polymerase saw (PCR) tau ua los ntawm kev siv RT2 Profiler PCR Array (Qiagen, CLAM36771C) nrog RT2 SYBR Green qPCR Mastermix (Qiagen, 330) . Cov PCR daim hlau ntau tau khiav ntawm Applied Biosystems StepOnePlus cycler. Txhua qhov array muaj kev tswj xyuas zoo rau kev rov qab hloov pauv kev ua haujlwm thiab kev sib kis DNA genomic. Kev txheeb xyuas PCR ntau tau ua tiav siv 2-DDCT txoj kev nrog kev pab los ntawm GeneGlobe Data Analysis Center (www. Qiagen. com/shop/genes-and-pathways/data-analysis-center-overview-page) thiab qhia raws li Log2 quav hloov pauv hauv cov kev qhia gene.
Hauv kev soj ntsuam silico proteomic
Hauv silico twv ua ntej ntawm calpain cleavage site tau ua tiav nrog DeepCalpain (http://deepcalpain.cancerbio.info/help.php), GPS-CCD (http://ccd.biocuckoo.org), thiab CaMPDB (http:// calpain.org/) cov cuab yeej online. Mouse protein amino acid sequence tau los ntawm Uniprot (https://www.uniprot.org). Cov txiaj ntsig tau rov pib dua hauv Cov Lus Ntxiv S1 thiab S2.
Kev txheeb cais
Txhua daim duab sawv cev rau tus kheej tus nqi thiab txhais tau tias ± SEM. Kev txheeb xyuas txheeb cais tau ua tiav siv GraphPad Prism software, version 9. Kev sib piv ntawm 2 pawg tau ua los ntawm parametricStudent t-test thaum cov qauv dhau qhov kev xeem Anderson-Darling thiab D'Agostino normality tests thiab F test rau qhov sib npaug ntawm qhov sib txawv. Tsis tas li ntawd, ib qho kev xeem Mann-Whitney nonparametric tau siv. Kev sib piv ntawm ntau pab pawg tau ua los ntawm kev siv 1-txoj kev lossis 2- txoj kev tsom xam ntawm qhov sib txawv ua raws li kev sib piv ntau yam nrog Simak txoj kev kho. Tus nqi ntawm P < 0.05="" tau="" suav="" tias="" yog="" qhov="" tseem="" ceeb.*p="">< 0.05,="" **p="">< 0.01,="" ***p=""><>
Daim duab 1|Angiotensin II (AngII) D high-salt noj zaub mov (HSD)-vim ntshav siab inhibits glomerularautophagy. (a–c) Immunofluorescence ntawm Podocalyxin (PODXL; liab) thiab P62 (ntsuab) hauv glomeruli (a, a0 ) los ntawm cov nas qus (WT), (b, b0 ) los ntawm WT nas tom qab 6 lub lis piam ntawm AngII þ HSD, thiab (c,c0 ) los ntawm Nphs2.cre Atg5lox/lox nas uas qhia txog kev sib sau ntawm P62 hauv podocytes thaum kub siab thiab hauv podocyte-specific ATG{{1{{12} }}}}cov nas tsis txaus. Cov xub xub qhia P62þ dots hauv WT hauv (a0 ). Nuclei tau counterstained nrog Hoechst (xiav). Daim duab subparts nrog prime qhia ntau dua magnification. Bars =50 hli. (d) Associated quantification ntawm P62þ cheeb tsam qhia raws li feem pua ntawm glomerular cheeb tsam. n=4 WT nas thiab n=5 WT nrog AngII þ HSD, thiab Nphs2.cre Atg5lox/lox nas. Cov txiaj ntsig tau nthuav tawm raws li tus kheej cov phiaj xwm thiab txhais tau tias ± SEM. Mann-Whitney test: *P=0.0159. Txhawm rau ua kom zoo saib cov duab no, thov mus saib hauv online version ntawm tsab xov xwm no ntawm www.raum-international.org.
TSEEM CEEB
AngII D kev noj zaub mov muaj ntsev-ua rau ntshav siab inhibited podocyte autophagy
Podocytes nthuav tawm qib siab ntawmautophagynyob rau hauv vivo, raws li qhia los ntawm muaj zog GFP qhia nyob rau hauv cov nas transgenic nrog GFP fusion rau LC3 (GFP-LC3 nas), ib tug tseem ceeb cim ntawm autophagy (Ntxiv daim duab S2A). Autophagy yog cov txheej txheem dynamic nrog kev tsim tas li ntawm autophagosomes thiab degradation ntawm autophagolysosomes. Blocking autophagosomal degradation nrog chloroquine ua rau cov tsub zuj zuj ntawm GFPþ dots, qhia tias muaj autophagic efflux hauv podocytes (Cov duab ntxiv S2A thiab B). Kev lees paub tias GFPþ dots yog autophagosomes tau pom los ntawm ob chav immuno- FL fluorescence rau GFP thiab SQSTM1 / P62, chaperone protein degraded los ntawmautophagy(Cov duab ntxiv S2C thiab D). Ib zaug ntxiv, kev kho mob chloroquine ua rau muaj kev sib txuam ntawm GFPþ P62þ dots, ua kom pom qhov tseem ceeb ntawm cov autophagic efflux hauv podocytes. Thaum kawg, siab autophagic efflux tau khaws cia hauv vitro raws li qhia los ntawm GFP thiab P62 qhia hauv thawj podocytes cais los ntawm GFP-LC3 nas thiab muaj zog tsub zuj zuj ntawm GFPþ thiab P62þ dots nyob rau hauv kev kho mob bafilomycin A1, lwm blocker ntawm autophagosomal degradation (Supplementary Figure) .
Lub peev xwm ntawm kev kub siab los hloov cov lus teb podocyte autophagic tau soj ntsuam hauv cov nas uas muaj AngII nrog kev noj zaub mov muaj ntsev ntau (HSD) rau 6 lub lis piam thiab hauv kev tswj tsis muaj ntshav siab. Raws li pom hauv daim duab 1, AngII þ HSD induced P62 tsub zuj zuj hauv glomeruli nrog muaj zog accu muulation hauv podocytes, yog li qhia tias AngII þ HSD yog lub luag haujlwm rau podocyte.autophagyblockade (Daim duab 1a–d). Interestingly, P62 tsub zuj zuj hauv podocytes zoo ib yam li cov nas uas tsis muaj rau podocyte autophagy (Nphs2.cre Atg5lox/lox nas). Hauv lwm tus qauv ntawm kev kub siab, tus qauv DOC-ntsev, peb kuj tau pom kev nce qib P62 nyob rau hauv podocytes raws li lub sijhawm ntawm tus kab mob (Cov duab ntxiv S3).
Kev rho tawm ntawm Atg5 tshwj xeeb hauv podocytes ua rau muaj albuminuria, podocyte poob, thiab glomerular raug mob hauv AngII D HSD qauv.
Peb mam li tshuaj xyuas sebautophagyblockade nkaus xwb hauv podocytes (Nphs2.cre Atg5lox/lox nas) cuam tshuam glomerular raug mob hauv AngII þ HSD qauv. Peb xub lees paub tias Nphs2. cre Atg5lox/lox nas muaj ntshav siab, ib txwmraummuaj nuj nqi, thiab tsis muaj glomerular histological lesions mus txog rau 10 lub hlis ntawm lub hnub nyoog, raws li yav tas los qhia (Ntxiv daim duab S4).32 Ces, Atg5lox/lox (WT) thiab Nphs2. cre Atg5lox/lox nas tau infused nrog AngII nrog HSD rau 6 lub lis piam. Qhov tseem ceeb, systolic ntshav siab tau zoo ib yam hauv 2 pawg tom qab AngII infusion thaum lub sijhawm kawm (Daim duab 2a), txawm hais tias tus Tsov tus tw-cuff siv los ntsuas ntshav siab yuav tsis muaj peev xwm los daws qhov sib txawv ntawm cov ntshav siab me me. AngII infusion nrog HSD tau nce urinary albumin-to-creatinine piv hauv WT nas, thiab cov nyhuv no tau nce ntxiv hauv Nphs2.cre Atg5lox/lox nas (Daim duab 2b). Hypertensive Nphs2.cre Atg5lox/lox nas kuj pom muaj qhov nce glomerular sclerosis thaum piv nrog WT littermates (Daim duab 2c–e). Raws li kev pom zoo nrog cov ntsuas proteinuria, cov proteinaceous casts thiab tubular dilatation tau tshwm sim ntau dua hauv cov nas uas muaj qhov tsis txaus podocyte hauv ATG5 (Daim duab 2f-h).
Daim duab 2|Kev tshem tawm ntawm Atg5 tshwj xeeb hauv podocytes ua rau muaj kev nce ntxiv hauv albuminuria,raumraug mob, thiab podocyte poob tom qab 6 lub lis piam ntawm angiotensin II (AngII) infusion D high-ntsev noj zaub mov (HSD). (a) Systolic ntshav siab hauv Atg5lox/lox thiab Nphs2.cre Atg5lox/lox nas thaum 36 hnub ntawm AngII þ HSD. n=9 nas ib genotype. Cov txiaj ntsig tau nthuav tawm raws li txhais tau tias ± SEM. Ob txoj kev tsom xam ntawm qhov sib txawv (ANOVA): ns. Hauv (b–m), n=10 nas ib genotype. Hauv (b, g, h, k), qhov tseem ceeb tau nthuav tawm raws li tus kheej cov phiaj xwm thiab txhais tau tias ± SEM. (b) AngII þ HSD (txuas ntxiv)
Daim duab 2|(txuas ntxiv) ua rau muaj qhov nce ntawm albuminuria hauv Nphs2. cre Atg5lox/lox nas piv nrog Atg5lox/lox tswj nas. Ob txoj kev ANOVA: genotype, P=0.013; sijhawm, P=0.0018. (c–f) Cov duab sawv cev ntawm Masson's trichrome-stained seem ntawm glomeruli los ntawm Atg5lox/lox tswj thiab Nphs2.cre Atg5lox/lox nas tom qab 6 lub lis piam ntawm AngII þ HSD. Bars =50 hli hauv (c,d). Bars =100 hli hauv (e,f). (g, h) Kev sib piv (g) ntawm qhov kev faib ua feem ntawm sclerotic glomeruli thiab (h) ntawm tus naj npawb ntawm cov tubular casts ib thaj chaw microscopic. Mann-Whitney test: **P=0.0026 in (h), ***P=0.0003 in (g). (i,j) Tus neeg sawv cev immunofluorescence cov duab ntawm kev qhia ntawm Wilm's Tumor 1 (WT1; liab) thiab Podocalyxin (PODXL; ntsuab) hauv glomeruli los ntawm Atg5lox/lox tswj thiab Nphs2.cre Atg5lox/lox nas tom qab AngII þ HSD rau 6 lub lis piam. Nuclei tau stained nrog Hoechst (xiav). Bars =50 hli. (k) Kev ntsuas tus naj npawb ntawm WT1þ hlwb ib ntu glomerular. Mann-Whitney test: **P=0.0029 in (l,m). Cov neeg sawv cev immunofluorescence cov duab ntawm kev qhia ntawm CD44 (ntsuab) hauv glomeruli los ntawm Atg5lox/lox tswj thiab Nphs2.cre Atg5lox/lox nas tom qab AngII þ HSD rau 6 lub lis piam. Nuclei tau stained nrog Hoechst (xiav). Bars =50 hli. (n,o) Tus neeg sawv cev xa xov hluav taws xob electron microscopy photomicrographs ntawm ntu ntawm glomeruli los ntawm Atg5lox/lox tswj thiab Nphs2.cre Atg5lox/lox nas tom qab AngII þ HSD rau 6 lub lis piam, qhia podocyte ko taw txheej txheem effacement (arrowheads) hauv hypertensive Nphs2. cre Atg5lox/lox nas. Bars =1 hli. n=3 nas ib genotype. Txhawm rau ua kom pom cov duab no zoo dua, thov mus saib hauv online version ntawm tsab xov xwm no ntawm www.kidney-international.org.
Daim duab 3|Calpain qhia thiab kev ua haujlwm hauv podocytes. (a) Western blot tsom xam ntawm kev qhia ntawm calpain-1, calpain-2, thiab calpain-4 hauv thawj podocytes. Tubulin qhia ua haujlwm li normalization. (b) Calpain kev ua haujlwm tau ntsuas ntawm thawj podocytes kho lossis tsis kho nrog angiotensin II (AngII; 100 nM) rau 24 teev nrog lossis tsis muaj calpeptin (10 mM). n=5 kev sim ywj pheej. Cov txiaj ntsig tau nthuav tawm raws li tus kheej cov phiaj xwm thiab txhais tau tias ± SEM. Ib txoj kev tsom xam ntawm qhov sib txawv (ANOVA): kev kho mob, P=0.0035. Sidak ntau qhov kev sib piv: *P=0.0128 rau AngII piv rau lub hauv paus, ##P=0.0056 rau AngII þ calpeptin piv AngII. (c) Calpain kev ua haujlwm tau ntsuas ntawm thawj podocytes los ntawm hom tsiaj qus (WT) lossistshuaj calpastatintransgenic (CSTTg) nas kho lossis tsis kho nrog AngII (100 nM) rau 24 teev. n=7 kev sim ywj pheej. Cov txiaj ntsig tau nthuav tawm raws li tus kheej cov phiaj xwm thiab txhais tau tias ± SEM. Ob txoj kev ANOVA ua khub rau kev kho mob: genotype, P=0.0483. Sidak ntau qhov kev sib piv: ***P=0.0009 rau WT AngII piv rau lub hauv paus, *P=0.0420 rau CSTTg AngII piv rau lub hauv paus, #P=0.0424 rau WT piv rau CSTTg Ang II. Txhawm rau ua kom zoo saib cov duab no, thov mus saib hauv online version ntawm tsab xov xwm no ntawm www.raum-international.org.
Tus lej Podocyte ib glomerulus tau txo qis hauv Nphs2. cre Atg5lox/lox nas kho nrog AngII þ HSD (Daim duab 2i–k). Podocyte raug mob nyob rau hauv Nphs2.cre Atg5lox/lox nas nrog AngII infusion þ HSD txawm nce mus rau focal thiab segmental glomerulosclerosis raws li qhia los ntawm kev qhia ntawm parietal epithelial cell (PEC) activation marker CD44 hauv glomeruli (Daim duab 2l thiab m). Electron microscopy tsom xam pom cov kev hloov pauv tseem ceeb cuam tshuam nrog ATG5 deficiency thaum mob AngII infusion nrog HSD, suav nrog cov txheej txheem ko taw effacement hauv hypertensive Nphs2. cre Atg5lox/lox nas. Los ntawm qhov sib txawv, ob peb qhov tsis xws luag ntawm cov txheej txheem ultrastructural tau pom hauv podocytes los ntawm WT nas txawm tias tom qab 10 lub lis piam ntawm AngII infusion nrog HSD (Daim duab 2n thiab o), qhia tias kev ua haujlwm ntawm autophagic ntawm podocytes yuav tsum tau ua rau lawv tsis kam rau AngII þ HSD-vim kev puas tsuaj. Ua ke, peb cov txiaj ntsig tau qhia tias hauv AngII þ HSD qauv,autophagyinhibition aggravates podocyte raug mob thiab poob thiab induces tom qab focal thiab segmental glomerulosclerosis.
AngII D HSD activates calpain kev ua si hauv podocytes uas ua rau autophagy blockade
Raws li kev ua haujlwm calpain tau pom (i) kom qhib los ntawm AngII hauv ntau hom cell thiab (ii) kom tshem tawm ntau yamautophagy-related proteins, 53 peb xav paub yog tias AngII þ HSD- mediated autophagy blockade tuaj yeem ua rau muaj zog AngII-induced calpain kev ua. Peb thawj zaug pom tias thawj podocytes qhia txog 3 ubiquitous daim ntawv ntawm calpains (Daim duab 3a). Tom qab ntawd peb tau pom tias AngII txhawb kev ua haujlwm calpain hauv thawj podocytes. Qhov nce hauv calpain kev ua ub no tau thaiv los ntawm kev xaiv calpain inhibitor (Daim duab 3b). Peb siv tus nas khob nrog ntxivtshuaj calpastatintransgene qhia (ua rau txo qis kev ua haujlwm calpain) 43 los ntsuas lub luag haujlwm ntawm calpains hauv AngII þ HSD-mediatedraumkev raug mob thiab autophagy blockade. Thawj cov podocytes los ntawm CSTTg nas pom qhov txo qis ntawm calpain kev ua haujlwm hauv cov lus teb rau AngII thaum piv nrog cov podocytes los ntawm cov nas tswj (Daim duab 3c). Yog li,tshuaj calpastatinoverexpression nyob rau hauv podocytes txo AngII-mediated calpain activation.
Peb soj ntsuam tom ntejautophagytheem hauv podocytes los ntawm CSTTg nas. Peb tsim CSTTg nas nrog GFP-LC3 transgene. LC3-GFP tus neeg sau xov xwm tso cai suav cov autophagosomes li GFPþ/P62þ dots. P62 yuav sib sau ua ke hauv cov hlwb thaum autophagic efflux raug thaiv. Ntawm lub xeev basal, peb suav tsawg dua GFPþ P62þ dots (Daim duab 4a, b, thiab e) thiab tsawg dua P62 tsub zuj zuj (Daim duab 4c, d, thiab f) hauv podocytes ntawm CSTTg GFP-LC3 nas dua li hauv podocytes ntawm cov nas GFP-LC3 ib txwm , qhia tias nce autophagic efflux nyob rau hauv podocytes ntawm nas nrog siab calpastatin abundance.
Daim duab 4|Kev ntsuam xyuas ntawm autophagic efflux hauv podocytes ntawmtshuaj calpastatintransgenic (CSTTg) nas. (a,b) Tus neeg sawv cev immunofluorescence dluab ntawm kev qhia ntawm ntsuab FL fluorescent protein (GFP) (ntsuab) thiab P62 (liab) nyob rau hauv glomeruli los ntawm 12-lub lim tiam-laus GFP-LC3 thiab CSTTg GFP-LC3 nas. Cov xub xub qhia GFPþ P62þ autophagosomes. (c,d) Tus neeg sawv cev immunofluorescence dluab ntawm kev qhia ntawm P62 (ntsuab) thiab Podocalyxin (PODXL; liab) nyob rau hauv glomeruli los ntawm 12-lub lim tiam-laus GFP-LC3 thiab CSTTg GFP-LC3 nas. Daim duab subparts nrog prime qhia ntau dua magnification. Nuclei tau stained nrog Hoechst (xiav). Bars =50 hli. (e) Quantification ntawm tus naj npawb ntawm LC3þ P62þ dots ib podocyte. Mann-Whitney test: **P= 0.0065. (f) Quantification ntawm P62þ cheeb tsam ib glomerular seem. Mann-Whitney test: *P=0.0420. Hauv (e,f), n=5 GFP-LC3 nas thiab n=8 CSTTg GFP-LC3 nas. Cov txiaj ntsig tau nthuav tawm raws li tus kheej cov phiaj xwm thiab txhais tau tias ± SEM. Txhawm rau ua kom zoo saib cov duab no, thov mus saib hauv online version ntawm tsab xov xwm no ntawm www.raum-international.org.
Autophagic efflux tuaj yeem saib xyuas los ntawm kev ntsuas ntawm kev hloov pauv ntawm cytoplasmic daim ntawv ntawm LC3, LC3-I, mus rau autophagosomal cleaved thiab phosphatidylethanolamine-ua ke daim ntawv ntawm LC3, LC3-II, ntawm Western blot . Podocytes los ntawm CSTTg nas pom tau nce LC3-I rau LC3-II hloov dua siab tshiab thiab txo P62 qhia, ob qho tib si nyob rau hauv lub xub ntiag thiab tsis muaj bafilomycin A1 (Daim duab 5a thiab b), qhia tias muaj zog autophagic efflux hauv podocytes nrog calpastatin overexpression. Thaum kawg, kev sib sau ntawm GFPþ P62þ dots hauv podocytes tom qab kev tswj hwm chloroquine tseem ceeb dua hauv CSTTg GFP-LC3 nas dua li cov nas GFP-LC3, yog li lees paub tiastshuaj calpastatinoverexpression induces autophagic efflux hauv podocytes hauv vivo (Daim duab 5c–e). Ua ke, peb cov ntaub ntawv qhia tias AngII txhawb kev ua haujlwm calpain hauv podocytes, uasautophagyyog inhibited los ntawm calpain hauv podocytes, thiab qhov inhibition ntawm endogenous calpain kev ua los ntawm calpastatin overexpression yog txaus los txhawb autophagic efflux hauv podocytes.
CSTTg nas raug tiv thaiv los ntawm AngII D HSD-induced podocyte raug mob
CSTTg nas tsis tau qhiaraumKev hloov pauv kom txog thaum muaj hnub nyoog tsawg kawg yog 12 lub hlis (Ntxiv daim duab S5). Peb soj ntsuam kev raug mob podocyte hauv CSTTg nas thaum kho AngII þ HSD. Txawm hais tias WT nas tsim mob me me glomerulosclerosis thiab podocyte raug mob tom qab 4 lub lis piam ntawm kev kub siab, raws li qhia los ntawm qhov txawv txav ntawm podocalyxin thiab nephrin, CSTTg nas tau nthuav tawm tsawg dua cov kab mob glomerular (Daim duab 6a thiab b) thiab khaws cia podocalyxin thiab nephrin qhia (Daim duab) 6. . Xws li qhov sib txawv ntawm podocyte phenotype tau pom nyob rau theem uas qhov ntom ntawm podocyte nuclei tsis txawv ntawm WT thiab hypertensive CSTTg nas (Daim duab 6i–k).
Daim duab 5|Thaiv autophagosomal degradation paub tseeb tias nce podocyte autophagic efflux nyob rau hauvtshuaj calpastatintransgenic (CSTTg) nas. (a) Western blot tsom xam ntawm kev qhia ntawm LC3, Sequestosome 1 (SQSTM1)/P62, thiab ATG5 hauv thawj podocytes los ntawm cov tsiaj qus (WT) lossis CSTTg nas. Tubulin qhia ua haujlwm li normalization. Podocytes tau kho lossis tsis kho nrog bafilomycin A1 (BafA1; 100 nM) rau 4 teev ua ntej kab lis kev cai raug ntes. (b) Quantification of the LC{{10}}II/tubulin and P62/tubulin ratios. n=10 WT nas thiab n=8 CSTTg nas. Cov txiaj ntsig tau nthuav tawm raws li tus kheej cov phiaj xwm thiab txhais tau tias ± SEM. Kev tsom xam ob txoj kev sib txawv ua ke rau kev kho mob: rau LC3-II/tubulin: genotype, P=0.{{30}}008; kev kho mob, P <0.0001; rau="" p62/tubulin:="" genotype,="" p="0.0884;" kev="" kho="" mob,="" p=""><0.0001. sidak="" ntau="" qhov="" kev="" sib="" piv:="" rau="" lc3-ii/tubulin:="" ***p=""><0.0001 rau="" wt="" ntxiv="" rau="" bafa1="" piv="" rau="" wt="" þ="" bafa1,="" ***p=""><0.0001 rau="" csttg="" ntxiv="" rau="" bafa1="" piv="" rau="" csttg="" þ="" bafa1,="" ##p{="" {34}}.0028="" rau="" wt="" þ="" bafa1="" versus="" csttg="" þ="" bafa1;="" rau="" p62/tubulin:="" ***p=""><0.0001 rau="" wt="" ntxiv="" rau="" bafa1="" piv="" rau="" wt="" þ="" bafa1,="" ***p=""><0.0001 rau="" csttg="" ntxiv="" rau="" bafa1="" piv="" rau="" csttg="" þ="" bafa1.="" (c,d)="" tus="" neeg="" sawv="" cev="" immunofluorescence="" cov="" duab="" ntawm="" kev="" qhia="" ntawm="" ntsuab="" fl="" fluorescent="" protein="" (gfp;="" ntsuab)="" thiab="" p62="" (liab)="" hauv="" glomeruli="" los="" ntawm="" 12-="" lub="" lim="" tiam-laus="" gfp-lc3="" thiab="" csttg="" gfp-lc3="" nas.="" daim="" duab="" subparts="" nrog="" prime="" qhia="" ntau="" dua="" magnification.="" nuclei="" tau="" stained="" nrog="" hoechst="" (xiav).="" bars="50" hli.="" cov="" nas="" raug="" kho="" nrog="" chloroquine="" (cq;="" 80="" mg="" kg)="" 4="" teev="" ua="" ntej="" tua.="" cov="" xub="" xub="" qhia="" gfpþ="" p62þ="" autophagosomes.="" (e)="" quantification="" ntawm="" tus="" naj="" npawb="" ntawm="" lc3þ="" p62þ="" dots="" ib="" podocyte.="" n="5" nas="" ib="" genotype.="" cov="" txiaj="" ntsig="" tau="" nthuav="" tawm="" raws="" li="" tus="" kheej="" cov="" phiaj="" xwm="" thiab="" txhais="" tau="">±SEM. Unpaired t-test nrog sib npaug SD: *P=0.0302. Txhawm rau ua kom zoo saib cov duab no, thov mus saib hauv online version ntawm tsab xov xwm no ntawm www.raum-international.org.
Daim duab 6 |Calpastatinoverexpression tiv thaiv angiotensin II (AngII) D high-ntsev noj zaub mov (HSD)-mediated podocyte raug mob. (a,b) Cov duab sawv cev ntawm Masson's trichrome-stained seem ntawm glomeruli los ntawm cov tsiaj qus (WT) thiab calpastatin transgenic (CSTTg) nas tom qab 6 lub lis piam ntawm AngII þ HSD. Bars =50 hli. (c,d,f,g) Cov neeg sawv cev immunofluorescence cov duab ntawm kev qhia ntawm (c,d) Podocalyxin (PODXL) thiab (f,g) nephrin (NPHS1) hauv WT thiab CSTTg nas tom qab 6 lub lis piam ntawm AngII þ HSD thiab (e , h) cuam tshuam cov lej. Bars =50 hli. (i,j) Tus neeg sawv cev immunofluorescence dluab ntawm kev nthuav qhia ntawm Wilm's Tumor 1 (WT1; ntsuab) thiab PODXL (liab) hauv glomeruli los ntawm WT thiab CSTTg nas tom qab 6 lub lis piam ntawm AngII þ HSD thiab (k) cuam tshuam qhov muaj pes tsawg leeg ntawm WT1þ cell ntawm glomerular seem. Nuclei tau stained nrog Hoechst (xiav). Bars =50 hli. Hauv (e, h, k), qhov tseem ceeb tau nthuav tawm raws li tus kheej cov phiaj xwm thiab txhais tau tias ± SEM. n=9 WT nas thiab n=7 CSTTg nas. Unpaired t-test nrog sib npaug SD: **P=0.0095 hauv (e), *P=0.0249 hauv (h), P=0.7891 hauv (k). Txhawm rau ua kom zoo saib cov duab no, thov mus saib hauv online version ntawm tsab xov xwm no ntawm www.raum-international.org.
Ib yam li ntawd, tom qab 6 lub lis piam ntawm kev kub siab, GFP-LC3 thiab CSTTg GFP-LC3 nas tau nthuav tawm cov kab mob podocyte tab sis cov kab mob tseem ceeb dua hauv GFP-LC3 nas (Daim duab 7). Cov zis albumin-to-creatinine piv tau siab dua hauv GFP-LC3 nas tom qab 4 lub lis piam ntawm AngII þ HSD (Daim duab 7a). Podocyte tus lej tsis txawv ntawm 2 genotypes, tab sis nephrin qhia tau txo qis dua hauv GFP-LC3 (tswj) nas (Daim duab 7b–e). Correlating nrogtshuaj calpastatin-mediated tiv thaiv, kev tsom xam ultrastructural pom me me thiab focal podocyte ko taw txheej txheem effacement nyob rau hauv GFP-LC3 nas kho nrog AngII þ HSD nrog zoo preservation ntawm ko taw txheej txheem effacement nyob rau hauv CSTTg nas, txawm hais tias lub ntiaj teb no quantification ntawm tus naj npawb ntawm ko taw txheej txheem ib glomerular qab daus. daim nyias nyias (GBM) ntev tsis sib txawv, feem ntau yuav yog vim qhov raug mob podocyte yog focal thiab segmental hauv peb cov qauv (Daim duab 7f–h). Nco ntsoov, macrophages thiab T-lymphocyte infiltration hauvlub raumtsis txawv ntawm cov pab pawg (Cov duab ntxiv S6). Ua ke, cov txiaj ntsig no tau pom tiastshuaj calpastatintiv thaiv AngII þ HSD-induced podocyte raug mob.
Calpastatin overexpression restores autophagic efflux hauv podocytes los ntawm nas tom qab AngII D HSD kho
Thaum kawg, peb xav paub sebtshuaj calpastatin-mediated glomerular tiv thaiv nyob rau hauv AngII þ HSD qauv cuam tshuam autophagy tu hauv podocytes. Interestingly, AngII þ HSD-induced P62 tsub zuj zuj hauv podocytes tau tiv thaiv hauv CSTTg thiab GFP-LC3 CSTTg nas (Daim duab 8a–d), qhia tias calpastatin tiv thaiv kev thaiv ntawm podocyte.autophagy. Ntawm qhov ceeb thawj, tus naj npawb ntawm GFPþ P62þ dots labeling ntawm autophagosomes zoo ib yam hauv podocytes los ntawm hypertensive GFP-LC3 thiab GFP-LC3 CSTTg nas, yog li qhia tias AngII þ HSD tau ua rau qeeb ntawm podocyte autophagic efflux es tsis yog ua tiav (Daim duab 8e- g).
Hauv silico twv ua ntej ntawm calpain cleavage site hauv podocyte proteins
Peb siv ntau yam hauv silico cov cuab yeej los kwv yees lub hom phiaj calpain hauv podocytes (Cov Lus Ntxiv S1 thiab S2). Peb cov ntaub ntawv hauv online tau muab piv rau: GPS-CCD, 54 CaMPDB, 55, thiab DeepCalpain.56 Hauv kev tshuaj xyuas silico tau txheeb xyuas ntau yam podocyte proteins, nephrin thiab podocin ntawm lawv, uas tuaj yeem cleaved los ntawm calpains. Yog li,tshuaj calpastatin-mediated glomerular tiv thaiv tuaj yeem txuas nrog txo qis ntawm calpain enzymatic kev ua haujlwm, ua rau txo qis ntawm qee cov podocyte proteins.
Tsis tas li ntawd, tsawg kawg yog 3autophagy-related proteins yog lub hom phiaj ncaj qha ntawm calpains. ATG5 yog cleaved los ntawm calpains, ua rau muaj kev cuam tshuam hauv ATG12-ATG5 complex formation.57,58 Kev tswj hwm ntawm calpain inhibitors hauv vivo kuj tiv thaiv cleavage ntawm autophagy protein Beclin-1.59 Hauv kev tshuaj xyuas silico ntawm lub putative cleavage site ntawm autophagy-related proteins txhawb kev xav tias calpain tuaj yeem tswj hwm autophagy los ntawm cov enzymatic cleavage ntawm autophagy proteins.
Cistanche-lub raum ua haujlwm
mRNA qhia ntawm endoplasmic reticulum (ER) thiab oxidative stress markers hauv glomeruli los ntawm nas kho nrog AngII ntxiv rau HSD
Peb soj ntsuam endoplasmic reticulum (ER) kev nyuaj siab thiab oxidative kev nyuaj siab los ntawm quantitative PCR hauv glomeruli thaum AngII þ HSD kev kho mob (Table 1). Nyob rau hauv lub hauv paus, peb tsis tau pom ib qho kev hloov pauv hauv mRNA qhia ntawm cov tshuaj ntsuam genes hauv glomeruli los ntawm WT thiab Nphs2.cre Atg5lox/lox nas (Ntxiv daim duab S7). Tom qab 6 lub lis piam kub siab, glomeruli los ntawm Nphs2. cre Atg5lox/lox nas pom txawv mRNA profile ntawm cov noob ntawm ER kev nyuaj siab thiab oxidative kev nyuaj siab txoj kev nrog nce kev qhia ntawm Sod1, Prdx1, Atf4, Gpx1, thiab Hsp90b1 piv nrog WT glomeruli, yog li qhia tiasautophagydepletion hauv podocytes nyiam AngII þ HSD-induced ER kev nyuaj siab thiab oxidative kev nyuaj siab. Conversely, glomeruli los ntawm CSTTg nas qhia downregulation ntawm ob peb noob ntawm ER kev nyuaj siab thiab oxidative kev nyuaj siab txoj kev raws li tau zoo raws li kev txo qis ntawm ib co pro-apoptotic genes (Daim duab 9).
Ua ke, cov txiaj ntsig no qhia tau tiastshuaj calpastatinoverexpression tuaj yeem tiv thaiv glomerular raug mob los ntawm kev txo qis AngII þ HSD-induced ER thiab oxidative stress.
Kev sib tham
Hauv kev tshawb fawb tam sim no, peb tau pom tias hauv AngII þ HSD-induced hypertension, podocyte autophagy yog qhov tsis txaus ntseeg. Tsis tas li ntawd, nas nrog podocyte-tshwj xeeb tshem tawm ntawm Atg5 tau ua rau AngII þ HSD-induced glomerulosclerosis thiab podocyte poob, yog li qhia tiasautophagyhauv podocytes tiv thaiv kev txhim kho ntawm hypertensive nephropathy, qhia txog lub luag haujlwm tseem ceeb ntawm autophagy hauv AngII þ HSD-induced podocyte raug mob. Tsis paub me ntsis txog cov stimuli extracellular uas tswj cov cellular autophagy, thiab cov txiaj ntsig tau ua rau lub teeb pom kev ntawm cov txheej txheem pathophysiological ntawm podocyte autophagy los ntawm AngII.
Nyob rau hauv feem ntau tib neeg glomerulopathies, podocyte ko taw txheej txheem effacement yog ib qho cim ntawm glomerular raug mob ua rau proteinuria. Autophagy zoo li yuav ua lub luag haujlwm tseem ceeb hauv kev tswj hwm kev ua haujlwm ntawm podocyte vim tias cov hlwb sib txawv ntawm qhov sib txawv no pom muaj tus nqi siab.autophagytxawm tias tsis muaj kev ntxhov siab. Ib txoj kev tshawb fawb yav dhau los tau qhia tias AngII txhawb nqa autophagy los ntawm kev tsim cov pa oxygen reactive nyob rau hauv cov kab mob uas tsis txawj tuag murine podocyte cell line.60 Reactive oxygen hom kev tsim tawm yog qhov tseeb ntawm inducer ntawm autophagy hauv ntau hom cell thiab cov laj thawj rau qhov tsis sib xws nrog peb qhov kev tshawb pom yog tsis meej. Tsis zoo li qhov kev tshawb fawb yav dhau los no, peb siv murine thawj kab lis kev cai podocytes khaws podocin thiab nephrin qhia thiab hauv vivo txoj hauv kev thiab tsis yog murine cell kab. Peb lees paub cov ntawv tshaj tawm yav dhau los uas postmitotic podocytes pom qhov txawv txav siab ntawm kev tsimautophagy. Kev ntsuas qhov nce ntawm LC3-II tom qab AngII stimulation nyob rau hauv lub xub ntiag los yog tsis muaj lysosomal inhibitors yog tsim nyog los txiav txim seb puas autophagic efflux yog nce los yog thaiv. Cov kev tshawb fawb yav dhau los tau tsis quav ntsej qhov tshwm sim.60
Daim duab 7 |Calpastatinoverexpression tiv thaiv angiotensin II (AngII) D high-ntsev noj zaub mov (HSD)-kev kho mob podocyte raug mob hauv ntsuab FL fluorescent protein (GFP)–LC3 nas. (a) AngII þ HSD ua rau muaj kev nce siab ntawm albuminuria hauv GFP-LC3 nas piv nrog CSTTg (calpastatin transgenic) GFP-LC3 nas. n=8 mus rau 13 nas ib genotype. Cov txiaj ntsig tau nthuav tawm raws li tus kheej cov phiaj xwm thiab txhais tau tias ± SEM. Kev tsom xam ob txoj kev sib txawv: genotype, P=0.0429; sijhawm, P=0.0165. Sidak ntau qhov kev sim sib piv: *P=0.0453 rau GFP-LC3 piv rau CSTTg GFP-LC3 nas nyob rau hnub 28. (b,c) Tus neeg sawv cev immunofluorescence dluab ntawm kev qhia ntawm Wilm's Tumor 1 (WT1; ntsuab) thiab nephrin (NPHS1) (liab) hauv glomeruli los ntawm 18-lub lim tiam-laus GFP-LC3 thiab CSTTg GFP-LC3 nas tom qab 6 lub lis piam ntawm AngII infusion þ HSD. Nuclei tau stained nrog Hoechst (xiav). Bars =50 hli. Quantification ntawm (d) NPHS1þ cheeb tsam ib glomerular seem thiab (e) tus naj npawb ntawm WT1þ hlwb ib glomerular seem hauv 18-lub lim tiam-laus GFP-LC3 thiab CSTTg GFP-LC3 nas tom qab 6 lub lis piam ntawm AngII infusion þ HSD. n=19 GFP-LC3 nas thiab n=25 CSTTg GFP-LC3 nas. Cov txiaj ntsig tau nthuav tawm raws li tus kheej cov phiaj xwm thiab txhais tau tias ± SEM. Unpaired t-test nrog sib npaug SD: **P=0.0010 hauv (d), P= 0.1158 hauv (e). (f,g) Kev xa tawm cov duab hluav taws xob microscopy ntawm glomeruli los ntawm GFP-LC3 thiab CSTTg GFP-LC3 nas tom qab 6 lub lis piam ntawm AngII þ HSD. Cov xub xub taw qhia cov txheej txheem ko taw effacement. Bars =1 hli. (h) Kev ntsuas tus naj npawb ntawm cov txheej txheem ko taw ib micrometer ntawm glomerular hauv qab daus daim nyias nyias (GBM). n=3 nas ib genotype. Cov txiaj ntsig tau nthuav tawm raws li tus kheej cov phiaj xwm thiab txhais tau tias SEM. Txhua daim phiaj sawv cev rau tus lej nruab nrab ntawm podocytes ib micrometer ntawm GBM ntawm 1 txuas ntxiv ntev ntawm GBM. Unpaired t-test nrog sib npaug SD: P=0.7512. Txhawm rau ua kom zoo saib cov duab no, thov mus saib hauv online version ntawm tsab xov xwm no ntawm www.raum-international.org.
Ntawm no, peb siv tus qauv hypertensive raws li AngII perfusion thiab HSD. Podocytes nthuav tawm AT1 receptors thiab raug rau cov lim dej dawb peptides xws li AngII.13,16,26,61–65 Nws tau xav tias qhov pom kev cuam tshuam ntawm RAAS inhibition tuaj yeem yog-tsawg kawg ib nrab-vim muaj kev cuam tshuam ntawm cov podocyte- tshwj xeeb RAAS. Ib yam li ntawd, hauv vivo cov kev tshawb fawb tau lees paub qhov cuam tshuam ntawm AngII ntawm kev raug mob ntawm podocyte thiab podocyte-tshwj xeeb noob hom phiaj ntawm AT1 pom tau tias ua kom AT1 receptors hauv glomerulus hauv kev sim lupus nephritis yog txaus los ua kom nrawm.raumraug mob thaum tsis muaj ntshav siab.66,67 Calpain-mediatedautophagydysregulation hauv peb cov qauv tuaj yeem txuas rau ncaj qha AngII-AT1 signaling ntawm podocytes los yog ua rau muaj kev kub ntxhov. Peb tuaj yeem muab cov lus teb ntxov rau lo lus nug no. Tseeb, nyob rau hauv DOC-ntsev qauv, peb kuj pom P62 tsub zuj zuj nyob rau hauv podocytes los ntawm hypertensive nas (Ntxiv daim duab S3). Qhov no qhia tias autophagy blockade tshwm sim hauv podocytes hauv cov qauv no, uas yuav tsum muaj kev ywj pheej ntawm AngII.68 Cov kev tshawb fawb ntxiv los soj ntsuam cov kev raug mob podocyte cuam tshuam txog kev ua haujlwm ntawm calpain thiab autophagic efflux hauv cov nas tsis txaus rau AT1 hauv podocytes xaiv yuav tsum tau txiav txim siab yog tias cov kev cai no ntawm podocyte autophagy nyob ntawm ncaj qha lossis tsis ncaj qha cuam tshuam ntawm AngII.
Daim duab 8 |Calpastatinoverexpression tiv thaiv angiotensin II (AngII) D high-salt noj zaub mov (HSD)-inducedautophagydownregulation nyob rau hauv podocytes. (a, b) Tus neeg sawv cev immunofluorescence duab ntawm kev qhia ntawm P62 (ntsuab) thiab Podocalyxin (PODXL; liab) hauv glomeruli los ntawm ntsuab FL fluorescent protein (GFP)–LC3 thiab CSTTg (tshuaj calpastatintransgenic) GFP-LC3 nas tom qab 6 lub lis piam ntawm AngII þ HSD. Daim duab subparts nrog prime qhia ntau dua magnification. Nuclei tau stained nrog Hoechst (xiav). Bars =50 hli. (c,d) Quantification ntawm P62þ cheeb tsam ib glomerular seem. n=9 cov nas qus (WT) thiab n=7 CSTTg nas hauv (c), thiab n=16 GFP-LC3 nas thiab n=16 CSTTg GFP-LC3 nas hauv (d). Cov txiaj ntsig tau nthuav tawm raws li tus kheej cov phiaj xwm thiab txhais tau tias ± SEM. Mann-Whitney test: **P=0.0033 in (c), **P=0.0011 in (d). (e,f) Tus neeg sawv cev immunofluorescence cov duab ntawm kev qhia ntawm GFP (ntsuab) thiab P62 (liab) hauv glomeruli los ntawm GFP-LC3 thiab CSTTg GFP-LC3 nas tom qab 6 lub lis piam ntawm AngII þ HSD. Daim duab subparts nrog prime qhia ntau dua magnification. Cov xub xub qhia GFPþ P62þ autophagosomes. (g) Kev ntsuas tus naj npawb ntawm LC3þ P62þ dots ib podocyte. n=11 GFP-LC3 nas thiab n=16 CSTTg GFP-LC3 nas. Cov txiaj ntsig tau nthuav tawm raws li tus kheej cov phiaj xwm thiab txhais tau tias ± SEM. Unpaired t-test nrog sib npaug SD: P=0.1014. Txhawm rau ua kom zoo saib cov duab no, thov mus saib hauv online version ntawm tsab xov xwm no ntawm www.raum-international.org.
Table 1|Kev cai RT2 Profiler PCR Array
Calpain-1 thiab calpain-2 yog cov pro-inflammatory proteases nyob rau txhua qhov chaw, uas nws cov dej num yog tswj los ntawmtshuaj calpastatin, lawv tshwj xeeb inhibitor. Tseeb, calpastatin xaiv inhibits calpains thiab tsis muaj lwm yam proteases rau hnub tim. Kev ua kom Calpain tau txuas tsis ntev los no rauraumraug mob nyob rau hauv ob peb pathological contexts.69,70 Cov calcium channel transporter transient receptor tej zaum channel C6 tau pom los qhib calpain-1 hauv podocytes ntawm Ca2þ/calcineurin ua kom. Lub raum ntawm cov neeg mob uas muaj focal thiab segmental glomerulosclerosis tau nce kev hloov pauv hloov pauv muaj peev xwm channel C6 qhia, nce calpain thiab calcineurin kev ua haujlwm, thiab txo qis kev qhia ntawm calpain phiaj talin -1, uas yog ib qho tseem ceeb rau podocyte cytoskeletal stability.71 Transient receptor muaj peev xwm channel. C6 kuj txuas ncaj qha rau calpain-1 thiab calpain{10}}. Qhov kev cuam tshuam no yog qhov tseem ceeb rau kev tswj hwm ntawm talin{11}} cleavage thiab tswj motility ntawm podocytes.72
Transgenic nas overexpressingtshuaj calpastatinmuaj kev tiv thaiv tawm tsam vascular remodeling thiab AngII-dependent inflammation73; tiv thaiv kev mob hauv cov qauv ntawm glomerulonephritis, 43 sepsis, 74 lossis allograft rejection75; thiab tiv thaiv kev mob uas muaj hnub nyoog.76 Podocyte raug mob hauv cov nas no tsis tau tshawb pom. Peltier et al. qhia tias overexpression ntawmtshuaj calpastatintiv thaiv AngII-dependent perivascular o nyob rau hauv ob lub raum.43 Yog li, kev tiv thaiv raum hauv CSTTg nas tuaj yeem kho tau, tsawg kawg yog ib feem, los ntawm kev siv tshuaj tiv thaiv kab mob. Peb soj ntsuam macrophages thiab lymphocytes infiltration hauv peb tus qauv thiab pom tsis muaj qhov sib txawv loj hauvraumo ntawm CSTTg thiab tswj cov nas thaum txheeb xyuas thoob ntiaj tebraumleukocyte infiltration (Ntxiv daim duab S6).
Calpain tau koom nrog tsis ntev los no hauv kev tswj hwm autophagy (saib xyuas tsis ntev los no hauv Weber li al.53) nrog kev nthuav dav thiab cov khoom siv bioprotective ntawm calpain inhibition hauv cov ntshav qab zib cov ntsiab lus los ntawm kev kho dua tshiab.autophagy.77 Ntawm no, peb qhia tias calpain inhibition los ntawmtshuaj calpastatinoverexpression (i) tiv thaiv podocyte raug mob thaum kub siab thiab (ii) rov qab autophagy hauv podocytes, yog li qhia txog lub luag haujlwm tshiab ntawm calpain activation thaum kub siab los ntawm inhibition ntawmautophagy.
Yuav luag tag nrho cov ATG proteins tau pom tias tau cleaved los ntawm calpains hauv vitro. 78 Ntawm no peb postulated qhov ntawdautophagykev saib xyuas hauv cov nas hypertensive CSTTg tau kho los ntawm calpain inhibition. Txhawm rau txhawb peb qhov kev xav, peb tau pom tias podocytes los ntawm CSTTg muaj qhov txo qis ntawm calpain kev ua haujlwm thaum sib tw nrog AngII hauv vitro (Daim duab 3c). Peb pom ATG5 protein ntau ntxiv hauv podocytes los ntawm CSTTg nas, uas qhia tias calpastatin overexpression tiv thaiv calpain-mediated ATG5 cleavage nyob rau hauv cov ntsiab lus no (Daim duab 5a). Piv txwv li, nws tau pom tiastshuaj calpastatin-mediated calpain inhibition tuaj yeem ua rau ywj pheej ntawm inhibition ntawm lawv cov haujlwm protease79; Yog li, peb tsis tuaj yeem suav nrog kev tswj hwm ntawm autophagy los ntawm calpastatin ywj siab rau calpain enzymatic kev ua haujlwm.
Hauv cov ntsiab lus, cov kev tshawb pom no tau nthuav tawm lub luag haujlwm yav dhau los tsis paub txogtshuaj calpastatinnyob rau hauv kev cai ntawm podocyteautophagythiab muab tus thawj coj rau kev tshawb nrhiav cov tswv yim kho tshiab los txhim kho podocyte ciaj sia thaum lub sij hawm hypertensive nephropathies.
Qhia tawm
Txhua tus kws sau ntawv tshaj tawm tsis muaj kev sib tw txaus siab.
TXOJ CAI
Txoj haujlwm no tau txhawb nqa los ntawm Lub Tsev Haujlwm National de la Santé Et de la recherche Médicale (Inserm) thiab Université de Paris. IB tau txais kev txhawb nqa los ntawm kev kawm tiav kev sib raug zoo los ntawm Ministère de l'EducationNationale, de la Recherche thiab de la Technologie. OL tau txais nyiaj los ntawm European Foundation for the Study of Diabetes (EFSD) qhov khoom plig txhawb nqa los ntawm EFSD / Novo Nordisk Program rau Kev Tshawb Fawb Kev Mob Ntshav Qab Zib hauv Tebchaws Europe thiab nyiaj pab los ntawm Société Francophone duDiabète (SFD). BR thiab CH tau txais nyiaj los ntawm Pib Grant 107037 los ntawm European Research Council thiab European Union (P-LT). YS tau txais kev txhawb nqa los ntawm kev kawm tiav kev sib raug zoo los ntawm Fondation de France.
Peb ua tsaug rau Elizabeth Huc, Nicolas Perez, Corina Suldac, thiab pab pawg ERIU970 (Université de Paris, PARCC, Inserm, Paris, Fabkis) kev pabcuam tsiaj thiab tuav, Nicolas Sorhaindo forbiochemical ntsuas (ICB-IFR2, Laboratoire de Biochimie, Hôpital Bichat , Paris, Fabkis), thiab Alain Schmitt thiab Jean-Marc Massefor kis tau tus mob electron microscopy (Institut Cochin, Paris, Fabkis).Peb ua tsaug Morgane Le Gall (Cochin proteomic facility 3P5, Paris, Fabkis) rau kev pab nyob rau hauv silico tsom xam. Peb lees paub kev txhawb nqa kev tswj hwm los ntawm Véronique Oberweis, Bruno Pillard, thiab Cyrille Mahieux (Université de Paris, PARCC, Inserm, Paris, Fabkis).
SUPPLEMENTARY KHOOM
Cov ntaub ntawv ntxiv (PDF)
Daim duab S1. Thawj kab lis kev cai podocyte qhia cov cim podocyte. Western blot tsom xam ntawm kev qhia ntawm podocyte cov cim NPHS1 thiab NPHS2 hauv thawj kab lis kev cai podocyte los ntawm WT thiab CSTTg nas. Tubulin (TUBA) ua haujlwm tswj kev thauj khoom. Tus neeg sawv cev ntawm n=4 nas ib genotype.
Daim duab S2. Qhov siab basal theem ntawm podocyteautophagy. (A, B) Tus neeg sawv cev immunofluorescence cov duab ntawm kev qhia ntawm GFP (ntsuab) thiab NPHS1 (liab) hauv glomeruli los ntawm GFP-LC3 nas kho lossis tsis nrog CQ (80mg / kg) 4 teev ua ntej tua. Arrowheads qhia GFPþ autophagosomes. (C,D) Cov neeg sawv cev immunofluorescence cov duab qhia txog GFP (ntsuab) thiab P62 (liab) hauv glomeruli los ntawm GFP-LC3 nas kho lossis tsis nrog CQ (80mg / kg) 4 teev ua ntej tua. Arrowheads qhia GFPþ P62þ autophagosomes. (A–D) (0) sawv cev ntau dua magnification. Nuclei tau stained nrog Hoechst (xiav). Bar =50 hli. N=4 nas ib yam mob (E–H) Cov neeg sawv cev immunofluorescence cov duab ntawm kev qhia ntawm GFP (ntsuab) thiab P62 (liab) hauv thawj podocytes los ntawm GFP-LC3 nas kho (F, H) lossis tsis (E, G ) nrog Bafifilomycin A1 (100 nM) rau 4 teev. N=5 nas rau ib yam mob.
Daim duab S3. P62 accumulates nyob rau hauv podocytes hauv DOC-ntsev qauv ntawm kub siab. (A–C) Tus neeg sawv cev immunofluorescence dluab ntawm kev qhia ntawm P62 (ntsuab) thiab PODXL (liab) hauv glomeruli los ntawm WT nas tom qab 2 mus rau 6 lub lis piam ntawm DOC-ntsev qauv. (0 ) sawv cev ntau dua magnification. Nuclei tau stained nrog Hoechst (xiav). Bar ¼ 50 mm. (D) Kev Ntsuas ntawm P62 cheeb tsam ib cheeb tsam podocyte (%). N=5–6 nas rau ib qho mob. Ib txoj kev tsom xam ntawm qhov sib txawv: lub sij hawm P=0.0059, Sidak ntau qhov kev sib piv sim: D42 piv rau D14 **P=0.0055, D28 piv D14 P=0.8590.
Daim duab S4. Podocyteautophagyyog dispensable rau kev loj hlob podocyte. (A) Systolic ntshav siab, (B) zis albumin-to-creatinine piv, thiab (C) ntshav urea nitrogen theem hauv Atg5lox/lox thiab Nphs2.cre Atg5lox/lox nas. N=5–6 nas ib genotype. Cov txiaj ntsig tau nthuav tawm raws li tus kheej cov phiaj xwm thiab txhais tau tias±SEM. Mann-Whitney test: P=0.7273 (A), P=0.4286 (B), thiab P=0.6623 (C). (D–E) Cov duab sawv cev ntawm Masson's trichrome-stained seem ntawm glomeruli los ntawm Atg5lox/lox thiab Nphs2.cre Atg5lox/lox nas. (F–G) Cov neeg sawv cev immunofluorescence cov duab qhia ntawm PODXL (ntsuab) thiab WT1 (liab) hauv Atg5lox/lox thiab Nphs2.cre Atg5lox/lox nas. Nuclei tau stained nrog Hoechst (xiav). (D–G) Bar =50 hli. N ¼ 6 nas ib genotype. (H–I) Cov neeg sawv cev photomicrographs ntawm kis tau tus mob electron microscopy seem ntawm glomeruli los ntawm Atg5lox/lox thiab Nphs2.cre Atg5lox/lox nas. Bar =1 hli. N=3 nas ib genotype.
Daim duab S5.Calpastatinoverexpression tsis cuam tshuamraumua haujlwm ntawm lub hauv paus. (A) ntshav urea nitrogen thiab (B) plasma albumin qib hauv 12-lub lim tiam-laus GFP-LC3 thiab CSTTg GFP-LC3 nas. N=5 GFP-LC3 thiab N=6 CSTTg GFP-LC3. Cov txiaj ntsig tau nthuav tawm raws li tus kheej cov phiaj xwm thiab txhais tau tias±SEM. Mann-Whitney test: P=0.6623 (A) thiab P=0.9307 (B). (C,D) Cov duab sawv cev ntawm Masson's trichrome-stained seem ntawm glomeruli los ntawm GFP-LC3 thiab CSTTg GFP-LC3 nas. (E–H) Tus neeg sawv cev immunofluorescence duab ntawm kev qhia ntawm PODXL (E, F) thiab NPHS1 (G, H) hauv GFP-LC3 thiab CSTTg GFP-LC3 nas. (C–H) Bar =50 hli. (I, J) Associated quantification of PODXL and NPHS1 area per glomerular section. N=5 GFP-LC3 thiab N=6 CSTTg GFP-LC3. Cov txiaj ntsig tau nthuav tawm raws li tus kheej cov phiaj xwm thiab txhais tau tias±SEM. Mann-Whitney test: P=0.1898 (A) thiab P=0.8413 (B).
Cistanche-lub raum ua haujlwm
Daim duab S6.Calpastatinoverexpression tsis cuam tshuam rau lub raum mob thoob ntiaj teb. Tus neeg sawv cev immunohistochemistry ntawm kev qhia ntawm F4/80 (A, B) thiab CD3 (D-E) hauv GFP-LC3 thiab CSTTg GFP-LC3 nas tom qab 6 lub lis piam ntawm Angiotensin II þHSD. Bar =200 hli. (C, F) Associated quantification of F4/80 and CD3 area perraumntu. N=7 CSTTg GFP-LC3 thiab N=8 GFP-LC3 nas. Cov txiaj ntsig tau nthuav tawm raws li tus kheej cov phiaj xwm thiab txhais tau tias SEM. Mann-Whitney test: P=0.3969 (C) P= 0.3357 (F).
Daim duab S7. Podocyteautophagydeficiency does not induce ER stress or oxidative stress in young adults at baseline. qPCR analysis of the mRNA expression of genes of the ER stress, oxidative stress, and apoptosis pathway by Qiagen qPCR array in glomeruli from WT and Nphs2.cre Atg5lox/lox mice (A) and WT and CSTTg mice (B). N=4 mice per genotype. For Nox3, Ct >33.
Tab S1. Hauv silico prevision ntawm calpain cleavage sites. Calpain cleavage sites tau kwv yees hauv podocyte-related thiab autophagy-related proteins nrog GPS-CCD (http://ccd.biocuckoo.org), CaMPDB (HTTP:// calpain.org) thiab DeepCalpain Predict (http://deepcalpain. mob cancerbio.info/help.php). Cov ntaub ntawv ntxiv (Excel)
Ntxiv Table S2. Cov ntaub ntawv tag nrho ntawm hauv silico prevision ntawm calpain cleavage sites. Calpain cleavage sites tau kwv yees hauv podocyte ntsig txog thiabautophagy-related proteins nrog GPS-CCD (http://ccd. biocuckoo.org), CaMPDB (http://calpain.org), thiab DeepCalpain Predict (http://deepcalpain.cancerbio.info/help.php). Kev twv ua ntej cleavage sites tau rov pib dua rau txhua qhov protein rau GPS-CCD thiab DeepCalpain Predict.
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