Hu rau:joanna.jia@wecistanche.com/ WhatsApp: 008618081934791

Imane Bensaada, Blaise Robin3, Joeille Perez', Yann Salemkour, Anna Chipont, Marine Camus, Mathilde Lemoine', Lea Guyonnet', Helene Lazareth', Emmanuel Letavernier, Carole Henique', Pierre-Louis Tharaux' thiab Olivia Lenoir'

'Université de Paris, PARCC, Inserm, Paris, Fabkis; thiab Université Paris Descartes, Sorbonne Paris Cite, Paris, Fabkis

Lub zog kwv yees tus nqi ntawm cov proteinuria nyob rau hauv cov mob glomerulopathies yog ruaj khov, nrog rau lub luag haujlwm pathogenic ntawm angiotensin ll txhawb kev loj hlob ntawm cov kab mob glomerular nrog kev hloov pauv glomerular pom teeb meem, podocyte raug mob thiab caws pliav ntawm glomeruli. Ntawm no peb pom tias mob angiotensin Il-induced hypertension inhibited autophagy flux hauv nas glomeruli. Kev rho tawm ntawm Atg5 (ib cov noob encoding ib cov protein koom nrog autophagy) tshwj xeeb hauv cov podocyte ua rau muaj kev ceev angiotensin I-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-Il-induced calpain kev ua hauv podocytes inhibits autophagy flux. Podocytes los ntawm cov nas nrog kev qhia txog kev hloov pauv ntawm endogenous calpain inhibitor calpastatin pom ntau dua podocyte autophagy ntawm lub hauv paus uas tiv taus angiotensin Il-dependent inhibition. Tsis tas li ntawd, txhawb nqa autophagy nrog calpastatin txwv podocyte kev puas tsuaj 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 Il-mediated hypertension inhibits autophagy ntawm calcium-induced recruitment ntawm calpain nrog rau cov kab mob tshwm sim nyob rau hauv rooj plaub ntawm qhov tsis txaus ntseeg los ntawm kev ua haujlwm calpastatin. Yog li, tiv thaiv calpain-mediated txo qis hauv autophagy tej zaum yuav yog ib qho kev cog lus tshiab kho mob rau nephropathies cuam tshuam nrog kev ua haujlwm siab renin-angiotensin system.

Cistanchedeserticola tiv thaivraumkab mob

Lus Txhais Lus

Muab lub luag haujlwm tseem ceeb ntawm autophagy hauv kev txhim kho ntawmraumkab mob, pharmacological modulation ntawm autophagy tej zaum yuav yog ib lub tswv yim zoo rau kev tiv thaiv thiab kev kho mob ntawm ob peb lub raum kab 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 Il rau kev cuam tshuam tsis zoo ntawm autophagy hauv podocytes thiab qhia tias calpain inhibition tuaj yeem yog lub hom phiaj kho mob rau cov kab mob podocyte ib nrab los ntawm kev saib xyuas ntawm podocyte autophagy.

Ntshav siab yog qhov thib ob nkaus xwb rau ntshav qab zib yog qhov ua rau muaj kev vam meejmob ntevraumkab mob-thiab txawm tias qhov nce siab hauv cov ntshav siab yog ib qho kev pheej hmoo ntawm kev mob raum kawg. Ib qho kev nce ntxiv ntawm cov kev tshawb fawb sim tau qhia txog qhov tseem ceeb ntawm podocytes hauv kev loj hlob ntawmraumraug mob. Progressive poob ntawm podocytes thiab microvascular alterations tshwm sim thaum ntxov nrog rau lub raum ua hauj lwm poob nyob rau hauv kev sim hypertensive nephropathy. Hauv cov neeg mob, cov zis tso zis ntawm cov podocytes siv tau tau pom tias yog ib qho cim tshwj xeeb rau preeclampsia, thiab cov neeg mob nephrosclerosis muaj qhov ntom ntom ntawm glomerular podocytes ntau dua li cov neeg mob raum. Tsis tas li ntawd, lub luag haujlwm pathogenic ntawm angiotensin II (Angel) 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.

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 signaling thiab hloov kho lawv cov duab raws li AnglI hom 1 receptor (AT1)-28Cov kev hloov pauv no nyob ntawm kev txhawb nqa.24-28 ua rau tsis zoo 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 cov cellular homeostasis, tshwj xeeb tshaj yog nyob rau hauv postmitotic thiab tshwj xeeb tshaj yog nyob rau hauv podocytes.1- Autophagy yog ib tug cll9olysosomal-kawg degradation system rau lub neej ntev cytoplasmic proteins thiab dysfunctional organelles55 thiab koom nrog thiab sequestration ntawm proteins. hauv autophagosomes. Kev tsim ntawm autophagosomes yog nyob ntawm qhov induction ntawm ntau cov noob suav nrog Mapllc3a/b, Berlin 1, thiab Atgs. Muaj ntau cov pov thawj tias dysregulation ntawm txoj kev autophagic cuam tshuam rau hauv lub pathogenesis ntawmraumlaus thiab ob pebraumCov kab mob xws li mob raum raug mob, kab mob polycystic raum, kev laus, thiab mob ntshav qab zib nephropathy.

Kev tswj hwm ntawm autophagy hauv podocytes, physiological thiab, qhov tseem ceeb tshaj plaws, hauv cov ntsiab lus pathological, 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) hauv cov kab mob physiological, ua rau hom cell no tshwj xeeb. 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 txoj kev tshawb no, peb tsom mus rau lub luag haujlwm ntawm AnglI signaling hauv podocyte autophagy kev tswj hwm. Peb tau txheeb xyuas cov calcium-activated proteases calpains mediating ib qho kev cuam tshuam ntev ntawm AnglI ntawm podocyte autophagy. Tsis tas li ntawd, peb pom tias endogenous calpain inhibitor calpastatin tuaj yeem tiv thaiv AngI-dependent autophagy inhibition thiab podocyte raug mob thaum kub siab.

Cov kev tshawb pom no nthuav tawm cov txheej txheem qub uas yog AnglI-mediated hypertension inhibits autophagy ntawm calcium-induced recruitment ntawm calpain nrog rau cov kab mob tshwm sim nyob rau hauv rooj plaub ntawm kev tsis txaus siab los ntawm kev ua calpastatin.

Txoj kev

Tsiaj txhu

Calpastatin transgenic (CST18) nas tau ua siab zoo los ntawm Dr. E. Letavernier. Cov nas uas muaj qhov cuam tshuam tshwj xeeb ntawm podocyte ntawm Atg5) tau tsim tawm raws li tau piav qhia yav dhau los gene (Nphs2.cre Atg5'o los ntawm hla Nphs2.cre nas nrog Ag5lxlomice5 ntawm keeb kwm C57BL6/J. Nphs2.cre Atg5'oxlox nas thiab tswj cov txiv neej littermates. , hnub nyoog 10 mus rau 12 lub lis piam, tau siv nyob rau hauv txoj kev tshawb no. Tus qauv mob ntshav siab tau tshwm sim los ntawm sc infusion ntawm AnglI (Sigma-Aldrich, A9525) ntawm koob tshuaj 1 ug / 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. Tag nrho cov kev tshawb fawb. Rau deoxycorticosterone acetate (DOCA) ntsev nrog cov nephron txo tus qauv, cov txiv neej laus nas tau txais unilateral sab laug nephrectomy. Ob lub lis piam tom qab nephrectomy, lawv tau txais DOCA pellets nrog rau 21- hnub tso tawm (Innovative Research of America) cog sc Qhov thib ob pellet tau cog rau 3 lub lis piam tom qab cog thawj zaug. Txhua tus nas rov tau txais 0.9 feem pua ​​NaCl hauv cov dej haus ad libitum thiab raug tua tom qab 6 lub lis piam ntawm kev tswj hwm DOC.6 Kev sim tau ua raws li Fabkis cov kws kho tsiaj cov lus qhia thiab cov tsim los ntawm European Community rau kev siv tsiaj sim (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 hauv nroog (APAFIS-7646 thiab -22373).

Thawj qhov kev sim podocyte kab lis kev cai

Qhov sib txawv thawj podocytes tau coj los ua kab lis kev cai raws li tau piav qhia yav dhau los./Nyob rau hauv luv luv, lub raum cortex tshiab tau muab sib xyaw thiab zom los ntawm collagenase I(Gibco, {0}}) hauv Roswell Park Memorial Institute 1640 (Lub Neej Technologies, {{2 }}). Cov ntaub so ntswg tau dhau los ntawm 70 μm thiab 40 μm cell strainers (BD Falcon, 352340 thiab 352350). Glomeruli, uas ua raws li 40 um cell strainer, raug tshem tawm nrog phosphate-buffered saline (PBS; Life Technologies, 10010023) ntxiv rau 0.5 feem pua ​​​​bovine serum albumin (Eurobio, HALALB07-65) txhaj rau hauv siab thiab tom qab ntawd ntxuav ob zaug. hauv PBS. Freshly cais glomeruli tau plated nyob rau 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 (Cov Duab Ntxiv S1). Podocytes tau coj mus kuaj thaum tsis muaj lossis muaj bafilomycin Al (100 nmol /, 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 dhau los. Tag nrho ntawm 100,000 hlwb raug coj mus rau hauv 24-cov ntaub so ntswg kab lis kev cai tais 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) kev daws teeb meem (pH 7.4) uas muaj 4 mM CaCl2, nrog lossis tsis muaj 10 uM 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-methycoumarin (Sigma-Aldrich, S6510). Tom qab 90-feeb incubation lub sij hawm, calpain kev ua ub no tau txiav txim raws li qhov sib txawv ntawm 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 ul ntawm xov tooj cua-immunoprecipitation assay tsis muaj phosphatase thiab protease inhibitor. Protein concentration tau ntsuas nrog BCA Protein Assay Kit (Merck Biochemistry, 71285). Nees nkaum micrograms ntawm cov proteins 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, ab39170 ), 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 tshawb pom ntawm cov teeb liab 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). Urinary albumin excretion raug ntsuas los ntawm kev ntsuas tshwj xeeb enzyme-linked immunosorbent assay rau kev txiav txim siab ntau ntawm albumin hauv cov zis nas (Crystal Chem, 80630).

Histology

Lub raum tau sau thiab kho hauv 4 feem pua ​​​​PBS-buffered formalin. Paraffin-embedded sections (3-} um tuab) tau stained los ntawm Masson's trichrome 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 tau soj ntsuam los ntawm kev kuaj qhov muag tsis pom qhov tsawg kawg yog 50 glomeruli ib ntu ntawm lub raum.

Immunofluorescence staining ntawm lub raum seem thiab thawj podocytes

Tsau thawj podocytes raug thaiv hauv TBS-T 3 feem pua ​​​​bovine serum albumin thiab incubated thaum hmo ntuj ntawm 4 degree nrog thawj cov tshuaj tiv thaiv kab mob guinea npua anti-SQSTM1/P62 (1: 1000, PROGEN, GP-62C) thiab luav anti-ntsuab fluorescent protein (GFP; 1:500, Abcam, ab290). Tom qab TBS-T yaug, fluorophore-conjugated secondary antibodies nees luav anti-guinea npua IgG AF594-conjugated antibody(Jackson ImmunoResearch,706-585-148) thiab luav anti-luv IgG AF{19}}conjugated antibody( Invitrogen, A21206) tau thov. Cov duab tau muab coj los siv Zeiss 2 fluorescent microscope, AxioCam HRc lub koob yees duab, thiab Axiovision 4.3 software.

Rau formalin-taw paraffin-embedded (FFPE) ob lub raum, ntu (3 um) 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 nyob rau hauv TBS-T3 feem pua ​​​​bovine serum albumin ua ntej hmo ntuj tshuaj tiv thaiv incubation ntawm 4 degree . Peb siv tshis anti-nephrin (1:100, PROGEN, GP-N2), guinea npua 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 Alexa 488- thiab Alexa 568-conjugated antibodies los ntawm Invitrogen. Nuclei tau stained xiav siv Hoechst. Slides tau teeb tsa siv qhov nruab nrab fluorescent mounting (Dako, S3023). Photomicrographs tau coj nrog Zeiss Axiophot photomicroscope thiab Axiovision software. Semiautomatic quantifications ntawm Fiji tau siv rau qhov ntau ntawm nephrin-positive thiab PODXL ntxiv rau thaj chaw ntawm glomerular seem ntawm tsawg kawg 30 glomeruli ib tus nas. Tus lej Podocyte raug suav tias yog tus lej ntawm WT1 ntxiv rau 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 hli) tau kho hauv 3 feem pua ​​​​glutaraldehyde EM 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 te-troxide 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 nyob rau hauv qhov sib piv ntawm 1: 1 rau 30 feeb tom qab sib tov Epikote 812 thiab propylene oxide nyob rau hauv ib tug ratio 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 lub qhov cub rhuab mus rau 60 degree C. Ultrathin seem raug txiav nrog ib tug UFC7 ultramicrotome (Leica Microsystems GmbH) 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 Digi-talMicrograph software (Gatan) kom tau.

Quantitative polymerase chain reaction array

Freshly cais glomeruli tau khov hauv QIAzol Lysis reagent (Qiagen) ntawm -80 degree . Tag nrho RNA rho tawm siv cov phenol-raws li txoj kev tau ua tiav raws li cov chaw tsim khoom cov lus pom zoo.cDNAs tau tsim los siv RT²First Strand Kit (Qiagen, 330401), thiab lub sijhawm ntawm cov tshuaj tiv thaiv polymerase saw (PCR) tau ua los ntawm kev siv RT²Profiler PCR Array ( Qiagen, CLAM36771C) nrog RT'SYBR Green qPCR Mastermix(Qiagen, 330502). Qhov ntau PCR daim hlau tau khiav ntawm Applied Bio-systems 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-△△CT txoj kev nrog kev pab los ntawm GeneGlobe Data Analysis Center (www. Qiagen. com/shop/genes-and-pathways/data-analysis-center-overview-page) thiab nthuav tawm raws li Log2 quav hloov pauv hauv cov noob qhia.

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 parametric Student 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 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 Sidak kho. Tus nqi ntawm P<0.05 were="" considered="" significant.=""><><><>

Daim duab 1|Angiotensin I (Angel) ntxiv rau kev noj zaub mov muaj ntsev ntau (HSD) vim muaj ntshav siab inhibits glomerular autophagy. (ac)

Immunofluorescence ntawm Podocalyxin (PODXL; liab) thiab P62 (ntsuab) hauv glomeruli (a, a') los ntawm cov nas qus (WT), (b, B) los ntawm WT nas tom qab 6 lub lis piam ntawm Angel ntxiv rau HSD, thiab (c) los ntawm Nphs2.cre Atgsl/ox nas uas qhia txog kev sib sau ntawm P62 hauv podocytes thaum kub siab thiab hauv podocyte-specific ATG5- cov nas tsis muaj zog. Cov xub xub qhia P62 ntxiv rau cov teev hauv WT hauv (a'). Nuclei tau counterstained nrog Hoechst (xiav). Daim duab subparts nrog prime qhia ntau dua magnification. Bars =50 μm. (d) Associated quantification of the P62 plus is expressed as the percentage of the glomerular area.n =4 WT nas thiab n=5 WT nrog Angel plus HSD thiab Nphs2.cre Atgsoxlo 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.

Angel ntxiv rau kev noj zaub mov uas muaj ntsev ntau vim muaj ntshav siab inhibited podocyte autophagy

Podocytes nthuav tawm qib siab ntawm autophagy hauv vivo, raws li qhia los ntawm GFP kev qhia muaj zog hauv cov nas transgenic nrog GFP fusion rau LC3 (GFP-LC3 nas), tus cim tseem ceeb ntawm autophagy (Cov duab ntxiv S2A). Autophagy yog cov txheej txheem dynamic nrog kev tsim tas li ntawm autophagosomes thiab degradation ntawm autophagolysosomes. Thaiv autophagosomal degradation nrog chloroquine ua rau cov khoom ntawm GFP ntxiv rau cov dots, qhia tias muaj cov kab mob autophagic hauv podocytes (Cov duab ntxiv S2A thiab B). Kev lees paub tias GFP ntxiv cov dots yog autophagosomes tau pom los ntawm ob npaug ntawm kev tiv thaiv kab mob-fluorescence rau GFP thiab SQSTM1 / P62, chaperone protein degraded los ntawm autophagy (Cov duab ntxiv S2C thiab D). Ib zaug ntxiv, kev kho mob chloroquine ua rau muaj kev sib txuam ntawm GFP ntxiv rau P62 ntxiv rau cov dots, ua kom pom qhov tseem ceeb autophagic flux hauv podocytes. Thaum kawg, siab autophagic flux 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 GFP ntxiv thiab P62 ntxiv cov dots nyob rau hauv bafilomycin Al kev kho mob, lwm blocker ntawm autophagosomal degradation (Ntxiv daim duab S2E- H).

Lub peev xwm ntawm kev kub siab los hloov kho cov lus teb podocyte autophagic tau soj ntsuam hauv cov nas infused nrog AngII nrog kev noj zaub mov muaj ntsev (HSD) rau 6 lub lis piam thiab hauv kev tswj tsis muaj ntshav siab. Raws li pom nyob rau hauv daim duab l, AnglI plus HSD induced P62 tsub zuj zuj nyob rau hauv glomeruli nrog muaj zog tsub zuj zuj nyob rau hauv podocytes, yog li qhia tias Angl plus HSD yog lub luag hauj lwm rau podocyte autophagy blockade (Daim duab av). Interestingly, P62 tsub zuj zuj nyob rau hauv podocytes zoo ib yam li ib tug pom nyob rau hauv nas tsis muaj rau podocyte autophagy (Nphs2.cre Atg5'olox nas). Nyob rau hauv lwm cov qauv ntawm kub siab, tus qauv DOCA- ntsev, peb kuj soj ntsuam zuj zus P62 tsub zuj zuj nyob rau hauv podocytes raws lub lub sij hawm kawm ntawm tus kab mob (Ntxiv daim duab S3).

Kev rho tawm ntawm Atg5 tshwj xeeb hauv podocytes ua rau muaj ntau ntxiv albuminuria, podocyte poob, thiab glomerular raug mob hauv Angel ntxiv rau HSD qauv.

Peb mam li tshuaj xyuas seb puas yog autophagy blockade tsuas yog hauv podocytes (Nphs2.cre Atg5laxlox nas) cuam tshuam rau glomerular raug mob hauv AnglI ntxiv rau HSD qauv. Peb xub lees paub tias Nphs2. cre Atg5mice muaj ntshav siab, lub raum ua haujlwm tsis zoo, thiab tsis muaj cov kab mob glomerular histological mus txog 10 lub hlis ntawm lub hnub nyoog, raws li tau tshaj tawm yav dhau los (Supplementary lox (WT) thiab Nphs2.cre Atgslox Figure S4).2 Tom qab ntawd, Atg5'cl 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 AnglI infusion thaum lub sijhawm kawm (Daim duab 2a), txawm hais tias tus Tsov tus tw-cuff txoj kev siv los ntsuas ntshav siab yuav tsis muaj peev xwm los daws qhov sib txawv me me ntawm cov ntshav siab. Angl infusion nrog HSD tau nce urinary albumin-to-creatinine piv hauv WT nas, thiab cov nyhuv no tau nce ntxiv hauv cov nas (Daim duab 2b). Hypertensive Nphs2.cre Atg5lox/lox Nphs2.cre Atgsoxlox nas kuj pom muaj qhov nce glomerular sclerosis thaum piv nrog WT littermates (Daim duab 2c-e). Raws li kev pom zoo nrog kev ntsuas proteinuria, proteinaceous casts thiab tubular dilatation tau muaj ntau dua nyob rau hauv cov nas uas muaj qhov tsis txaus podocyte hauv ATG5 (Daim duab 2f-h).

Podocyte tus naj npawb ib glomerulus tau txo qis hauv Nphs2.cre Atg5loxlox (Daim duab 2i-k).Podocyte raug mob hauv Nphs2.cre Atgsoxlo nas nrog AngII infusion ntxiv rau HSD txawm nce mus rau focal thiab segmental glomerulosclerosis raws li qhia los ntawm parietalPEC epithelial cell. ) 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 tsis txaus thaum mob AngII infusion nrog HSD, suav nrog cov txheej txheem ko taw hauv… nas. Los ntawm qhov sib piv, ob peb tus neeg mob ntshav siab Nphs2. cre Atg5'oxlx structural defects tau pom nyob rau 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 tiv thaiv Angel ntxiv rau HSD-vim kev puas tsuaj. Ua ke, peb cov txiaj ntsig tau qhia tias hauv AnglIl plus HSD qauv, autophagy inhibition aggravates podocyte raug mob thiab poob thiab induces tom qab focal thiab segmental glomerulosclerosis.

Daim duab 2|Kev tshem tawm ntawm Atg5 tshwj xeeb hauv podocytes ua rau muaj kev nce ntxiv hauv albuminuria, mob raum, thiab podocyte poob tom qab 6 lub lis piam ntawm angiotensin Il (Angel) infusion ntxiv rau cov khoom noj ntsev ntau (HSD). (a) Systolic ntshav siab nyob rau hauv Atgslo/b thiab Nphs2.cre Ataslo nas thaum 36 hnub ntawm Angel ntxiv rau HSD.n =9 nas ib genotype. Cov nqi tau nthuav tawm raws li txhais tau tias ± SEM. Ob txoj kev tsom xam ntawm qhov sib txawv (ANOVA): ns. Hauv (bm), 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) Angel plus HSD (txuas ntxiv

Daim duab 3|Calpain qhia thiab kev ua si 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 ll (Angel; 100 nM) rau 24 teev nrog lossis tsis muaj calpeptin (10 uM), 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 Angll piv rau lub hauv paus, P=0.0056 rau Angll plus calpeptin piv rau Angll.(c) Calpain kev ua haujlwm tau ntsuas ntawm thawj podocytes los ntawm cov tsiaj qus (WT) lossis calpastatin transgenic (CST') nas kho lossis tsis kho nrog Angel (100 nM) rau 24 teev. n {{18} } 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 Angll versus baseline,*P=0.0420 for CST'9 Angll versus baseline, *P=0.0424 for WT versus CST9 Angll.

Pls nyem qhov no mus rau Ntu 2