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Lub raum Pericyte Hypoxia-inducible Factor Regulates Erythropoiesis Tab sis Tsis Raum Fibrosis

Mar 13, 2022

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

Szu-Yu Pan1,2,3 ua al

Prolyl hydroxylase domain enzyme (PHD) inhibitors muaj txiaj ntsig zoo hauv kev kho mobmob raum mob(CKD) - txuam nrog ntshav qab zib los ntawm stabilizing hypoxia-inducible factor (HIF), yog li ua rau erythropoietin thiab thiaj li erythropoiesis. Txawm li cas los xij, kev txhawj xeeb txog kev mob CKD yuav tsum tau hais hauv kev sim tshuaj. Txawm hais tias cov kev tshawb fawb preclinical tau pom tias muaj cov nyhuv anti-inflammatory hauvraumkab mob qauv, cov nyhuv ntawm PHD inhibitors rauraumfibrosisyog qhov tsis sib xws tej zaum vim tias qhov cuam tshuam ntawm HIF yog hom cell thiab cov ntsiab lus-dependent. Qhov lojraumerythropoietin-tsim hlwb yog pericytes uas tsim erythropoietin los ntawm HIF-2ib-dependent gene transcription. Kev txhawj xeeb rau qhov cuam tshuam ntawm HIF hauv pericytes rauraumfibrosistshwm sim los ntawm qhov tseeb tias pericytes yog cov hlwb ua ntej ntawm myofibroblasts hauv CKD. Txij li thaum cov hlwb qhia Gli1 ua tiav cov txheej txheem morphologic thiab anatomic rau pericytes, peb ntxias Gli1 ntxiv rau cov xov tooj ntawm tes tshwj xeeb HIF stabilization lossis knockout los kawm txog qhov cuam tshuam ntawm HIF hauv pericytes ntawmraumpathology ntawm nas nrog los yog tsis muaj fibrotic raug mob los ntawm unilateral ureteral obstruction. Piv nrog rau cov littermate tswj, nas nrog pericyte-specific HIF stabilization vim von Hippel-Lindau protein los yog PHD2 knockout pom muaj zog erythropoietin thiab polycythemia ntau dua li qhov sib txawv hauv kev pom.raumfibrosis. Piv nrog Gli1ntxivpericytes txheeb los ntawm littermate tswj, Gli1ntxivpericytes txheeb los ntawm PHD2 knockout nas tau pom muaj zog erythropoietin noob qhia ntau dua li pom kev hloov pauv hauv Col1a1 lossis Acta2 qhia. Tsis tas li ntawd, pericyte-specific knockout ntawm HIF-1a lossis HIF-2a tsis cuam tshuamraumfibrosis. Yog li, peb txoj kev tshawb fawb txhawb nqa qhov tsis muaj qhov tsis zoo ntawm PHD inhibitors rauraumfibrosisntawm cov nas txawm tias HIF stabilization hauv pericytes.


KEYWORDSerythropoietin;fibrosis; hypoxia-inducible yam; pericyte


Cistanche for treating kidney fibrosis

Cistancheyog qhov zoo raulub raummuaj nuj nqi

Lus Txhais Lus

Prolyl hydroxylase domain enzyme (PHD) inhibitors muaj txiaj ntsig zoo hauv kev kho mob ntshav qab zibmob ntevraumkab mob(CKD) los ntawm stabilizing hypoxia-inducible factor (HIF), yog li ua rau erythropoietin (EPO) thiab erythropoiesis. Lub raum pericytes tsim EPO los ntawm HIF-2ib qho hauv lub cev ib txwm muaj, tab sis lawv sib txawv rau cov caws pliav tsim myofibroblasts hauv CKD. Nws tsis paub meej tias HIF qhia hauv lub raum pericytes cuam tshuamlub raumfibrosis. Los ntawm preclinical murine qauv ntawm HIF manipulation thiab raum fibrosis, peb qhia tau hais tias Gli1 ntxiv rau pericyte tshwj xeeb HIF manipulation cuam tshuam EPO ntau lawm thiab erythropoiesis tab sis tsis.raum fibrosis. Peb txoj kev tshawb fawb pom zoo qhov tsis muaj qhov tsis zoo ntawm PHD inhibitors ntawm lub raum fibrosis.

Lub ntsiab tseem ceeb ntawm kev kho mob ntshav qab zib hauv cov neeg mob nrogmob ntevraumkab mob(CKD) yog erythropoiesis-stimulating cov neeg ua haujlwm thiab cov tshuaj hlau. 1,2 Tsis ntev los no, prolyl hydroxylase domain enzyme (PHD) inhibitor tshwm sim los ua tus neeg sawv cev kho mob zoo rau CKD-koom nrog ntshav qab zib feem ntau los ntawm stabilizing lub hypoxia-inducible factor (HIF) thiab yog li nce erythropoietin (EPO) ntau lawm.3-5 Lub chaw kho mob. Daim ntawv thov ntawm PHD inhibitors lossis HIF stabilizers hauv cov neeg mob nrog CKD yog qhov yuav tshwm sim yav tom ntej yog tias muaj kev txhawj xeeb txog qhov ua tau ntawm angiogenesis, qog loj hlob, cov piam thaj tsis zoo hauv cov metabolism, thiab txo qis ntawm cov ntshav qab zib.raumkev ua haujlwm yuav tsum tau hais hauv kev sim tshuaj.

HIF yog qhov tseem ceeb tshaj plaws ntawm lub cellular oxygen-sensing machinery.6,7 Lub HIF ua haujlwm muaj xws li a thiab b subunits. HIF-a yog cov pa oxygen labile subunit uas yog degraded sai heev ntawm ib tug mob normoxic nyob rau hauv muaj cov cofactors. HIF-b tsis yog tswj los ntawm oxygen nro. Lub degradation ntawm HIF-a yog ua ntej los ntawm proline hydroxylation thiab polyubiquitination. Lub proline hydroxylation yog catalyzed los ntawm PHD nyob rau hauv muaj oxygen, hlau, thiab 2-oxoglutarate ua cofactors. Lub polyubiquitination yog tom qab ntawd catalyzed los ntawm E3 ubiquitin ligase tsim los ntawm von Hippel Lindau protein (pVHL)-Elongin BC-CUL2 complex. Qhov yuav tsum tau muaj hlau, PHD, thiab pVHL rau HIF degradation ua rau nws muaj peev xwm stabilize HIF nyob rau hauv tus mob normoxic nrog rau inhibition los yog txo ntawm ib yam ntawm cov tseem ceeb Cheebtsam.

Lub raum pericytes yog embedded nyob rau hauv lub microvascular hauv qab daim nyias nyias thiab nyob rau hauv kev sib cuag nrog endothelial hlwb los txhawb microvasculature thiab tswj cov ntshav khiav.8–13 Peb tau tshaj tawm tias lub raum pericytes yog lub raum EPO-producing cells (REPCs) uas tsim EPO los ntawm HIF{{3 }}a-dependent gene transcription induced los ntawm anemia lossis hypoxia.14 Pericytes tau pom tias yog qhov loj ntawm caws pliav tsim myofibroblasts hauv CKD, 8, 10, 11, 15, 16 qhov kev tshawb pom txhawb los ntawm lwm cov ntaub ntawv ywj pheej.17, 18 Txawm hais tias kev hloov pauv mus rau myofibroblasts ua rau muaj kev cuam tshuam ntawm Epo transcription, overexpression ntawm HIF-2 ib feem rov qab los ntawm EPO ntau lawm hauv myofibroblasts.14,17

Peb cov kev tshawb fawb tau qhia tiasraum fibrosistuaj yeem txo qis los ntawm kev cuam tshuam ntawm kev hloov pauv ntawm pericyte-myofibroblast los ntawm kev kho tshuaj thaiv kev hloov pauv kev loj hlob-b, 19 platelet-derived txoj kev loj hlob, 20 vascular endothelial kev loj hlob yam, 21 lossis WNT / b-catenin pathways.22

Hais txog qhov kev txhawj xeeb rau qhov nrawm nrawm ntawmraumkev ua haujlwm hauv cov neeg mob nyob rau hauv kev kho mob nrog PHD inhibitors, me ntsis paub txog cov teebmeem ntawm HIF kev ua haujlwm hauv pericytes lossis myofibroblasts ntawmraum fibrosis.23 Hauv qhov sib piv, qhov cuam tshuam ntawm HIF overexpression lossis knockout rauraum fibrosistau kawm ntau, tsis yog xaiv 24–27 los yog xaiv nyob rau hauv tubular hlwb, 28,29 endothelial hlwb, 30 thiab podocytes.31,32 Txawm li cas los xij, cov txiaj ntsig tsis sib xws tej zaum vim tias qhov cuam tshuam ntawm HIF yog hom cell thiab cov ntsiab lus-dependent.

Cov kev tshawb fawb hauv vitro siv cov tib neeg lub raum fifibroblasts33 thiab nas lub raum medullary interstitial cells34 qhia txog lub luag haujlwm profibrotic ntawm HIF-1a. Txawm li cas los xij, Souma et al.17 tau tshaj tawm tias kev sib koom ua ke ntawm PHD1, PHD2, thiab PHD3 hauv REPCs ntawm Tg (Epo-Cre) nas uas qhia Cre recombinase nyob rau hauv kev tswj hwm ntawm Epo txhawb nqa tsis cuam tshuam.raum fibrosisnyob rau hauv tus qauv ntawm unilateral ureteral obstruction (UUO). Txawm li cas los xij, lub raum Epo qhia qis qis thiab lub pas dej loj ntawm REPCs me me nyob rau hauv lub xeev khov kho yam tsis muaj hypoxia lossis ntshav ntshav, 35 qhia tias Epo-Cre transgene tsuas yog txhais ib feem me me ntawm tag nrho REPCs. Tsis ntev los no, lub raum Gli1- nthuav tawm cov hlwb tau pom tias yog pericytes thiab sib txawv rau myofibroblasts ntawmraumraug mob.11,36 Ablation ntawm Gli1 ntxiv rau cov hlwb ameliorates UUO-inducedlub raumfibrosis.11 Ntawm no peb tau siv nas nrog Cre recombinase nyob rau hauv kev tswj ntawm Gli1 txhawb nqa / txhim kho los kawm txog cov teebmeem ntawm pericyte-specific HIF stabilization lossis knockout ntawmrmob fibrosis.

cistanche for treating renal fibrosis

cistanche yog qhov zoo rauraum

Txoj kev

Cov nas

Gli 1CreERT2 / ntxiv, Tg(UBC-CreERT2), VhlF/F,ROSA 26fstdTomato/fstdTxiv lws suav, Hwv 1aF/F, thiab Hif2aF/F Cov nas tau yuav los ntawm Jackson Laboratory (Bar Harbor, ME). Egln 1F/F Cov nas tau ua siab zoo los ntawm xibfwb Guo-Hua Fong (University of Connecticut Health Center, Farmington, CT). Txhua qhov kev tshawb fawb tau ua tiav raws li txoj cai pom zoo los ntawm Pawg Saib Xyuas Tsiaj Tsiaj thiab Siv Tshuaj, National Taiwan University College of Medicine (IACUC 20160499).

Unilateral ureteral obstruction

Cov txheej txheem tau piav qhia hauv qhov kev tshawb fawb yav dhau los.14 Hauv luv luv, hauv cov nas laus hnub nyoog 7 txog 9 lub lis piam hauv qab tshuaj loog nrog ketamine/xylazine (ketamine 100 mg/kg, xylazine 10 mg/kg), sab laug ureters ntawm cov nas raug nthuav tawm thiab ligated nrog nylon suture los ntawm kev txiav ntawm sab laug flank. Hnub tim ntawm UUO kev phais raug txheeb xyuas yog hnub 0 ntawm qhov kev sim.

Kev txheeb cais

Cov ntaub ntawv tau nthuav tawm raws li lub ntsiab lusntxivSD. Kev txheeb xyuas txheeb cais tau ua tiav siv GraphPad Prism (GraphPad Software, San Diego, CA). Qhov tseem ceeb ntawm tus lej raug txiav txim los ntawm Tub Ntxhais Kawm t-test lossis Mann-Whitney U xeem raws li qhia hauv cov ntawv sau.

Cov txheej txheem ntxiv

Tag nrho cov txheej txheem suav nrog nas, UUO, tamoxifen tswj hwm, ntau cov tshuaj tiv thaiv polymerase saw, immunoblot, immunohistochemistry, RNA hauv situ hybridization, microscopy, flow cytometry, thiab kev rho tawm ntawm lub raum Gli1 ntxiv rau cov hlwb nyob hauv Cov Txheej Txheem Ntxiv.

TSEEM CEEB

Nce EPO ntau lawm thiab erythropoiesis hauv nas nrog Gli1 ntxiv rau pericyte-specific PHD2 knockout

Txhawm rau ua kom pom tias Gli1 ntxiv rau pericytes tsim EPO ua ntej peb mus kawm txog cov txiaj ntsig ntawm pericyte-specific HIF manipulation ntawmraum fibrosis, peb tsim Gli1CreERT2 / ntxiv; Egln 1F/Fnas los ntxias PHD2 knockout hauv Gli1 ntxiv rau pericytes conditionally (Daim duab 1a). Piv nrog littermate Egln1F/Fmus, Gli1CreERT2 / ntxiv; Egln 1F/FCov nas pom cov ntshav EPO ntau dua (Daim duab 1b), hnyav splenic hnyav (Daim duab 1c), ntau dua TER-119ntxiverythroid kab mob nyob rau hauv splenocytes los ntawm hnub 14 tom qab tamoxifen (Daim duab 1d), tab sis zoo li lub cev hnyav hloov tom qab tamoxifen tswj hwm (Cov duab ntxiv S1A). Hematocrit (Hct) siab dua hauv Gli1CreERT2 / ntxiv; Egln 1F/Fnas nyob rau hnub 21 (Daim duab 1e). Peb soj ntsuam lub raum Epo mRNA thiab cov ntshav peripheral suav 14 hnub tom qab tamoxifen. Piv nrog littermate Egln1F/Fmus, Gli1CreERT2 / ntxiv; Egln 1F/FCov nas tsuag tau nce lub raum Epo qhia (Daim duab 1f) thiab peripheral reticulocytes (Daim duab 1g), thaum peripheral cov ntshav dawb suav thiab platelet suav tsis txawv (Cov duab ntxiv S1B thiab C). Txhawm rau txheeb xyuas lub raum lub luag haujlwm rau Epo kev qhia, peb tau ua hauv situ hybridization ntawm Epo rauraumntu ntawm Egln1F/F thiab Gli1CreERT2/ntxiv; Egln 1F/Fnas (Cov duab ntxiv S2A–F). Ua kom Epo-expressing interstitial hlwb tuaj yeem txheeb xyuas feem ntau nyob rau hauv corticomedullary hlws ris, thiab kom tsawg dua nyob rau hauv lub puab medulla thiab cortex hauv Gli1.CreERT2/ntxiv; Egln 1F/Fnas. Cov txiaj ntsig no tau qhia tias Gli1 ntxiv rau pericyte-specific PHD2 knockout induced lub raum EPO ntau lawm thiab erythropoiesis.

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Txhawm rau txiav txim siab seb PHD2 tswj hwm EPO ntau lawm hauv hom cell thiab cov kabmob uas tsis yog pericytes hauv covraum, peb tsim Tg(UBC-CreERT2); Egln 1F/Fnas los ntxias thoob ntiaj teb PHD2 knockout (Ntxiv daim duab S3A). Tom qab kev tswj hwm tamoxifen, knockout ntawm Egln1 noob nyob rau hauvraum, daim siab, tus po, lub plawv, thiab lub ntsws tuaj yeem kuaj pom (Cov duab ntxiv S4). Hauvraum, qhov kev qhia ntawm Egln1 mRNA txo mus rau 4 feem pua, qhia tias kev ua haujlwm siab ntawm PHD2 knockout (Cov duab ntxiv S3B). Raws li hauv Gli1CreERT2 / ntxiv; Egln 1F/Fnas, Tg(UBC-CreERT2); Egln 1F/Fnas pom qhov nce ntxiv ntawm Hct piv nrog littermate Egln1F/Fnas (Ntxiv daim duab S3C). Qhov tseem ceeb, Tg(UBC-CreERT2); Egln 1F/FCov nas pom qhov poob ntawm lub cev hnyav (Cov duab ntxiv S3D). Remarkably siab ntawm cov ntshav EPO, nyob ib ncig ntawm 130,000 pg/ml, tau pom nyob rau hauv Tg(UBC-CreERT2); Egln 1F/F nas, uas siab dua qhov ua tiav los ntawm phlebotomy (tsawg dua 5,000 pg/ml) hauv cov nas qus (Cov duab ntxiv S3E). Txhawm rau txheeb xyuas lub hauv paus ntawm cov ntshav EPO, peb tau kuaj Epo mRNA qib hauv cov kabmob sib txawv los ntawm Tg (UBC-CreERT2); Egln 1F/F,thiab littermate tswj nas nyob rau hnub 15. Nyob rau hauv littermate tswj nas, Epo mRNA tsis tau kuaj pom nyob rau hauv ib lub cev. Hauv 5 lub cev loj (raum, siab, ntsws, plawv, spleen) ntawm Tg(UBC-CreERT2); Egln 1F/F nas, tsuas yograumpom qhov nce zoo kawg li ntawm Epo mRNA, implying lubraumraws li ib tug loj qhov chaw ntawm cov ntshav EPO (Ntxiv daim duab S3F thiab G). Hauv qhov chaw hybridization ntawm Epo mRNA ntawmraumcov seem qhia tias lub raum hlwb nrog Epo kev qhia yog cov hlwb interstitial nrog pericyte morphology hauv Tg (UBC-CreERT2); Egln 1F/Fnas (Ntxiv daim duab S5). Yog li, cov ntaub ntawv no tau lees paub tias lub raum Gli1 ntxiv rau pericytes tsim EPO ntawm PHD2 knockout, yog li nce erythropoiesis thiab Hct.

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Gli1 ntxiv rau pericyte-specific pVHL knockout ua rau polycythemia tab sis tsis cuam tshuam rau lub raum fibrosis

Raws li cov ntaub ntawv dhau los, 11,36 nce Gli1ntxivpericytes tau pom nyob rau hauv corticomedullary hlws ris thiab medulla tom qab UUO raug mob hauv Gli1CreERT2/ntxiv; ROSA 26fstdTomato/ fstdTomatoCov nas sau xov xwm (Cov duab ntxiv S6), qhia tias Gli1 ntxiv rau pericytes proliferate thiab pab txhawb rau myofibroblasts thaum lub sijhawmraum fibrosis.Yog li peb tau kawm txog cov txiaj ntsig ntawm HIF stabilization lossis knockout hauv Gli1ntxivpericytes rauraum fibrosis.

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Peb siv nas nrog Gli1ntxivpericyte-specific knockout ntawm pVHL, tsis yog PHD2 vim muaj peev xwm redundancy hauv 3 txawv PHDs. Txhawm rau tshawb xyuas qhov cuam tshuam ntev ntawm pericyte-specific HIF stabilization raulub raumHauv nas tsis muaj CKD, Gli1CreERT2/ntxiv; VhlF/Fnas, vhlF/FCov neeg littermates tau muab tamoxifen thaum muaj hnub nyoog 6 lub lis piam thiab tom qab ntawd saib mus txog 30 lub lis piam ntawm hnub nyoog (Cov duab ntxiv S7A). Ib qho kev tuag npaj txhij txog hauv Gli1CreERT2 /ntxiv; VhlF/Fnas tau tshwm sim thaum muaj hnub nyoog 25 lub lis piam (14.3 feem pua ​​​​ntawm cov neeg tuag), tab sis tsis muaj neeg tuag hauv VhlF/Fcov phooj ywg. Tsis muaj zog thiab txo lub cev hnyav nce tau pom hauv Gli1CreERT2 / ntxiv; VhlF/Fnas thaum muaj hnub nyoog 16 lub lis piam (Ntxiv daim duab S7B). Vim yog emaciation thiab tsis xav txog kev tuag, qee cov nas tau euthanized ua ntej 30 lub lis piam. Pathology qhia tawm qhov mob hnyav erythropoiesis nyob rau hauv tus po thiab congestion ntawm cov qe ntshav liab hauv daim siab, ntsws, thiabraumthaum muaj hnub nyoog 18 lub lis piam (Ntxiv daim duab S8). Tsuas yog cov ntshav liab congestion, tsis paub meejfibrosistau pom nyob rau hauv lub cev loj xws lilub raum. Cov theem ntawm Hct thiab cov ntshav EPO tau nce mus rau 87 ntxiv rau -2.6 feem pua ​​thiab 1604 ntxiv rau -141 pg/ml thaum muaj hnub nyoog 30 lub lis piam (piv txwv li, 24 lub lis piam tom qab tamoxifen) (Cov duab ntxiv S9A thiab B)

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Vim tsis pom kevraum fibrosisHauv cov nas nrog Gli1 ntxiv rau pericyte-specific pVHL knockout, peb ntxias UUO hauv cov nas thiab ntsuas qhov mob raum fibrosis 7 lossis 14 hnub tom qab (Daim duab 2a). Deleted Vhl alleles hauv Gli1CreERT2/þ; VhlF/Fnas tau kuaj pom hauvraumgenomic DNA los ntawm polymerase saw cov tshuaj tiv thaiv, qhia tias knockout ntawm pVHL hauv pericytes (Cov duab ntxiv S10). Hct tau nce hauv Gli1CreERT2 / ntxiv; VhlF/Fnas txawm tias tom qab UUO phais (Daim duab 2b). Qhov tseem ceeb, kev qhia ntawm EPO mRNA tau nce hauv ob qho tib si contralateral thiab UUOlub raumntawm Gli1CreERT2 / ntxiv; VhlF/Fnas (Daim duab 2c), qhia tias HIF stabilization tuaj yeem qhib Epo transcription hauv ob qho tib si pericytes thiab myofibroblasts. Txawm li cas los xij, tsis muaj qhov sib txawv hauvraum fibrosisnruab nrab ntawm Gli1CreERT2 / ntxiv; VhlF/Fthiab littermate VhlF/F Cov nas tuaj yeem raug kuaj pom, raws li ntsuas los ntawm picrosirius liab-stained cheeb tsam (Daim duab 2d thiab 3a thiab b) thiab kev qhia ntawm Col1a1 mRNA (Daim duab 2e) hauvraum. Tsis tas li ntawd, mRNA thiab cov protein ntau ntawm cov leeg nqaij leeg tsis sib txawvlub raum(Daim duab 2f thiab g thiab ntxiv daim duab S11). Txhawm rau kom paub meej tias myofibroblasts khaws Epo kev qhia muaj peev xwm tom qab pVHL knockout, peb tau ua hauv situ hybridization ntawm Epo thiab Acta2 ntawmraumseem (Cov duab ntxiv S12 thiab S13). Renal Acta2 qhia tau nce siab heev thiab sib piv tom qab UUO raug mob hauv ob qho tib si Gli1CreERT2 / ntxiv; VhlF/Fthiab VhlF/Fnas. Ntau interstitial Epo-expressing hlwb tuaj yeem pom hauv Gli1CreERT2 / ntxiv; VhlF/Fnas tshaj VhlF/F nas, tsis hais seb puas muaj UUO raug mob. Tsis tas li ntawd, cov hlwb nrog kev qhia ua ke ntawm Acta2 thiab Epo tuaj yeem raug txheeb xyuas hauv UUOraumlos ntawm Gli1CreERT2 / ntxiv; VhlF/Fnas tab sis tsis VhlF/Fnas. Qhov tseeb, peb tsis tuaj yeem txheeb xyuas cov Epo-expressing hlwb hauv UUOraumlos ntawm VhlF/Fnas. Cov txiaj ntsig no tau qhia tias Gli1 ntxiv rau pericyte-txo pVHL knockout txhawb Epo kev qhia tab sis tsis yog qhov sib txawv ntawm myofibroblast lossis collagen ntau lawm txawm tias tom qab UUO raug mob.

Vim tias UUO raug mob ua rau muaj kev puas tsuaj ntawm cov tubular cov hlwb thiab kev nrhiav neeg ua haujlwm ntawm macrophages, 37 mRNA qib ntawm Havcr1 (encodingraumkev raug mob molecule-1) (Daim duab 4a) thiab Adgre1 (encoding endothelial kev loj hlob yam zoo li module-muaj mucin-zoo li hormone receptor-zoo li 1, kuj hu ua F4/80) (Daim duab 4b) nce hauv UUOlub raum, tab sis tsis muaj qhov sib txawv tuaj yeem kuaj pom ntawm Gli1CreERT2 / ntxiv; VhlF/Fthiab littermate VhlF/F nas. Cov qib mRNA ntawm Vegfa, Hmox1, Egln1, thiab Egln3 (encoding vascular endothelial cell growth factor-A, heme oxygenase-1, PHD2, thiab PHD3, feem) hauvlub raumtsis tau hloov pauv (Daim duab 4c–f), tej zaum yuav xav txog tias cov ntawv sau tseg no tsis yog tsim los ntawm Gli1 ntxiv rau pericytes lossis tsis hloov tom qab Gli1 ntxiv rau pericyte-txo VHL knockout. Peb soj ntsuam kev raug mob tubulointerstitial nyob rau hauv periodic acid-Schiff-stainedraumntu, thiab tsis muaj qhov sib txawv ntawm Gli1-CreERT2 / ntxiv; VhlF/F,thiab littermate VhlF/Fnas (Daim duab 4g thiab 5a thiab b). Vim tias tamoxifen tau tshaj tawm kom txo qisfibrosis, 38,39 peb ncua lub sijhawm ntxuav tawm ntawm qhov kawg tamoxifen kev tswj hwm thiab UUO phais los ntawm 2 lub lis piam mus rau 4 lub lis piam (Cov duab ntxiv S14A). Ib zaug ntxiv, tsis muaj qhov sib txawv ntawm Gli1CreERT2 / ntxiv; VhlF/F thiab littermate VhlF/F nas (Cov duab ntxiv S14B–E). Cov kev tshawb pom no tau qhia tias Gli1 ntxiv rau pericyte-specific pVHL knockout ua rau EPO ntau lawm thiab polycythemia tab sis tsis muaj kev cuam tshuam raulub raumfibrosis.


cistanche is good for kidney

cistanche yog qhov zoo rauraum

Gli1 ntxiv rau pericyte-specific PHD2 knockout nce EPO ntau lawm tab sis tsis yog collagen ntau lawm lossis myofibroblast sib txawv

Txhawm rau kom paub meej qhov cuam tshuam ntawm HIF stabilization hauv Gli1 ntxiv rau pericytes ntawm qib cellular, peb txheeb tdTomato ntxiv cov hlwb los ntawmlub raumntawm Gli1CreERT2 / ntxiv; ROSA 26fstdTomato/fstdTomatothiab Gli1CreERT2 / ntxiv; Egln 1F/F; ROSA 26fstdTomato/fstdTomatonas tom qab UUO raug mob (Cov duab ntxiv S15 thiab S16). Nyob rau hauv kev tshuaj xyuas microscopic, cov sorted hlwb emission txiv kab ntxwv-liab fluorescence ntawm excitation (Ntxiv daim duab S17A-C). Piv nrog rau cov los ntawm Gli1CreERT2 / ntxiv; ROSA 26fstdTomato/fstdTomatonas, Egln1 mRNA theem hauv tdTomato ntxiv cov hlwb txheeb los ntawm contralateral thiab UUOlub raumntawm Gli1CreERT2 / ntxiv; Egln 1F/F; ROSA 26fstdTomato/fstdTomatonas 7 hnub tom qab UUO raug mob txo mus rau 46 feem pua ​​​​thiab 33 feem pua ​​​​, raws li (Cov duab ntxiv S17D thiab E).

Peb txheeb xyuas cov qib mRNA ntawm Epo, Col1a1, Col3a1, thiab Acta2 hauv kev txheeb tdTomato ntxiv rau Gli1 ntxiv rau pericytes los ntawm Gli1CreERT2 / ntxivROSA 26fstdTomato/fstdTomatothiab Gli1CreERT2 / ntxiv; Egln 1F/F; ROSA 26fstdTomato/fstdTomatonas. Raws li cov txiaj ntsig tau los ntawm nas nrog Gli1 ntxiv rau pericyte-specific pVHL knockout, PHD2 knockout hauv Gli1 ntxiv rau pericytes ua rau muaj Epo nthuav tawm, tab sis tsis muaj kev cuam tshuam rau kev qhia ntawm Col1a1, Col3a1, thiab Acta2 ntawm qib cellular (Daim duab 6) , lees paub qhov nruab nrab ntawm HIF stabilization ntawm cov khoom profibrotic ntawm pericytes / myofibroblasts.

Gli1þ pericyte-specific HIF knockout tsis cuam tshuam rau lub raum fibrosis

Vim tias cov kev sim no tau lees paub qhov nruab nrab ntawm HIF stabilization ntawm profibrotic thaj chaw ntawm pericytes / myofibroblasts, peb tsim Gli1CreERT2 / ntxiv ; Hif 1aF/F; Hif 2 aF/F nas los kawm txog cov txiaj ntsig ntawm HIF knockout hauv pericytes / myofibroblasts rauraum fibrosis(Daim duab 7a). Deletion ntawm Hif1a thiab Hif2a hauv lubraumtuaj yeem tshawb pom hauvraumgenomic DNA tom qab kev tswj hwm tamoxifen (Ntxiv daim duab S18). Piv nrog rau kev tswj littermate, Gli1CreERT2 / ntxiv; Hif 1aF/F; Hif 2 aF/F Cov nas muaj qhov sib txawv ntawm lub cev qhov hnyav thiab Hct theem 7 hnub tom qab UUO (Daim duab 7b thiab c). Nyob rau hauv cov nqe lus ntawmraum fibrosis, tsis muaj qhov sib txawv tuaj yeem kuaj pom hauv thaj chaw picrosirius liab-stained, mRNA qib ntawm Col1a1 thiab Acta2 hauvraum(Daim duab 7d–f). Cov qib mRNA ntawm Havcr1 thiab Adgre1 kuj tsis txawv (Daim duab 7g thiab h). Peb hloov cov koob tshuaj thiab lub sij hawm ntxuav tawm ntawm tamoxifen (Cov Duab Ntxiv S19A thiab S20A), thiab cov txiaj ntsig rau collagen tso tawm thiab cov noob qhia tau zoo sib xws (Cov duab ntxiv S19B-E, S20B-E). Peb rov ua qhov kev sim hauv Gli1CreERT2 / ntxiv; Hif 1aF/F(Daim duab 8a–c) thiab Gli1- CreERT2/ ntxiv ; Hif 2 aF/F(Daim duab 8d–f) nas thiab tsis pom qhov txawv ntawm UUO-inducedraum fibrosisthaum piv nrog kev tswj littermate.

figure 8-1

figure 8-2

Kev sib tham

Cov kev tshawb pom tseem ceeb ntawm txoj kev tshawb fawb no suav nrog cov hauv qab no: (i) lub raum Gli1 ntxiv rau pericyte-specific HIF stabilization ua rau EPO ntau lawm thiab erythropoiesis tab sis tsis muaj kev cuam tshuam rau pathology ntawm ob lub raum nrog lossis tsis muaj UUO raug mob; thiab (ii) raum Gli1 ntxiv rau pericyte-specific HIF-1a thiab HIF-2 tsis muaj kev cuam tshuamraum fibrosislos yog.

Peb yav dhau los tau tshaj tawm tias lub raum Foxd1 ntxiv rau cov progenitor-derived pericytes yog REPCs thiab tswj hwm los ntawm HIF-2a. 14 Hauv txoj kev tshawb no, peb tau pom tias lub raum Gli1 ntxiv rau cov hlwb kuj yog REPCs. Txawm hais tias tsis muaj pov thawj ncaj qha piav qhia txog kev sib raug zoo ntawm FOXD1 thiab GLI1 hauv pericytes, cov kev tshawb fawb ua ntej tau pom tias ob lub raum Foxd1 ntxiv rau progenitor-derived cells thiab Gli1 ntxiv cov hlwb yog pericytes thiab muaj peev xwm sib txawv rau myofibroblasts ntawm qhov raug mob.10,11,36 Peb. cov ntaub ntawv qhia tau hais tias knockout ntawm PHD2 los yog pVHL tshwj xeeb hauv Gli1 ntxiv rau pericytes ua rau muaj zog rau lub raum EPO ntau lawm, erythropoiesis, thiab polycythemia, uas yog raws li tsab ntawv ceeb toom tsis ntev los no los ntawm Greenwald li al.40 Ntxiv rau, piv nrog rau qhov txo qis ntawm Epo. qhia nyob rau hauv UUO raum ntawm cov tsiaj qus-hom littermates, Epo qhia nyob rau hauv UUO raum ntawm nas nrog Gli1- tshwj xeeb pVHL knockout yog piv rau theem qhia nyob rau hauv kev tswj contralateralraum, pom zoo HIF stabilization tuaj yeem kov yeej cov txheej txheem ntawm Epo kev tawm tsam hauv fibrotic raum myofibroblasts.14,17 Raws li peb qhov kev tshawb pom, Souma li al.17 tau tshaj tawm tias EPO synthesis rov qab los hauv UUO-raummyofibroblasts hauv nas nrog PHD2 knockout. Hauv kev sau ntawv nrog peb cov kev tshawb pom, kev tshawb fawb tsis ntev los no 41 tau tshaj tawm tias lub raum Gli1 ntxiv rau cov hlwb yog cov neeg subpopulation ntawm platelet-derived kev loj hlob ntawm receptor-b ntxiv rau REPCs thiab khaws lub peev xwm ntawm EPO ntau lawm hauv UUO.lub raum.

Cov kev sim tshuaj ntsuam xyuas tau ua pov thawj qhov ua tau zoo ntawm PHD inhibitors hauv kev kho mob ntawm CKD-associated anemia.3–5,42–45 Tseem ceeb, kev txheeb xyuas ntawm theem III kev sim ntawm roxadustat pom tau tias tsis yog qhov qis dua lossis zoo dua ntawm cov hlab plawv.46 Txawm li cas los xij, tsis muaj kev cai lij choj. tsab ntawv ceeb toom hais txog cov txiaj ntsig ntawm PHD inhibitors ntawm CKD kev nce qib tau tshaj tawm txog tam sim no. Kev sim, lub luag haujlwm ntawm hypoxia thiab HIF hauvraum fibrosisyog controversial.47,48 Hauv cov nas raug mob raum raug mob (AKI) tshwm sim los ntawm ischemia-reperfusion raug mob, Kapitsinou li al.24 qhia tias PHD inhibition ua ntej AKI amelioratesfibrosis, thaum inhibition nyob rau hauv lub sij hawm rov qab los ntawm AKI tsis. Ntxiv mus, Uchida et al.26 tau qhia tias PHD inhibitor tuaj yeem ua kom zoo duaraum fibrosisHauv kev koom tes nrog kev txo qis cov cytokines thiab kev kho dua tshiab ntawm capillary ntom nyob rau hauv lub raum ntawm tus nas CKD qauv induced los ntawm 5/6 subtotal nephrectomy, tab sis tsis muaj kev cuam tshuam rau proteinuria thiab ntshav creatinine qib. Hauv qhov sib piv, ib daim ntawv tshaj tawm tsis ntev los no tau qhia tias PHD inhibitor pab txhawb rau polycythemia thiab txo qis ntawm tubulointerstitial nephritis, tab sis nws tsis muaj kev cuam tshuam rau lub raum fibrosis hauv tus qauv ntawm adenine-induced chronic tubulointerstitial nephritis.25 Cov tshuaj tiv thaiv kab mob ntawm PHD inhibitors kuj tau pom. nyob rau hauv lub raum ntawm rog rog hom 2 mob ntshav qab zib murine qauv.27 Seb PHD inhibitors ameliorate lub raum fibrosis los ntawm attenuation ntawm interstitial o warrants ntxiv kawm. Txawm hais tias cov kev tshawb fawb no tsis tau tshaj tawm txog qhov cuam tshuam ntawm PHD inhibitors ntawm lub raum myofibroblast activation, qhov tsis muaj qhov tsis zoo ntawm lub raum fibrosis tuaj yeem txhawb nqa qhov tsis pom zoo ntawm HIF qhia ntawm collagen ntau lawm hauv lub raum pericytes. Kev soj ntsuam cov txiaj ntsig ntawm HIF rau lub raum yog sophisticated.49 Ua ntej, cov xov tooj sib txawv yuav qhia tau txawv HIF isoforms.50 Thib ob, txawm tias tib lub HIF isoform tuaj yeem tswj cov hom phiaj sib txawv hauv ntau hom cell.51 Thib peb, kev qhia thiab kev tswj hwm ntawm HIF Nyob rau hauv physiologic mob tej zaum yuav txawv ntawm lub raum kab mob.50 Tsis tas li ntawd, qhov degradation ceev ntawm HIF ntawm normoxic tej yam kev mob tsub kom qhov nyuaj ntawm kev soj ntsuam tsis raug ntawm HIF protein ntau ntau nyob rau hauv cov ntaub so ntswg.52 Yog li ntawd, yav tas los inconsistent tshwm sim hais txog cov nyhuv ntawm lub raum fibrosis yog tej zaum. vim tias cov teebmeem ntawm HIF yog hom cell thiab cov ntsiab lus-dependant. Piv txwv li, pov thawj tau pom tias PHD inhibitor ua ntej ischemia-reperfusion raug mob-vim AKI ua kom rov qab-AKI fibrosis los ntawm endothelial HIF-2a-dependent mechanism, 24,30 thaum HIF-1a-dependent o , fibrosis, thiab lub raum cell dysplasia tau pom nyob rau hauv Tg(Hoxb7-Cre); VhlF/Fnas uas muaj pVHL knockout nyob rau hauv cov ducts sau thiab ib tug subset ntawm distal tubules.29 Nyob rau hauv peb txoj kev tshawb no tsom rau seb HIF kev ua si nyob rau hauv pericytes modulatedraum fibrosis,Peb tau pom tias EPO ntau lawm tau nce ntxiv hauv lub raum pericytes hauv nas nrog Gli1 ntxiv rau pericyte-specific HIF stabilization los ntawm cell-specific pVHL lossis PHD2 knockout, thiab qhov tseem ceeb, Epo qhia tau khaws cia hauv UUO-mob raum myofibroblasts. Txawm li cas los xij, peb tsis tau pom tias pericyte-specific HIF stabilization cuam tshuam rau kev hloov pauv ntawm pericyte-myofibroblast, collagen ntau lawm hauv myofibroblasts, lossis UUO-induced.raumfibrosis. Ntxiv mus, cov lus qhia ntawm Havcr1 thiab Adgre1 tsis cuam tshuam rau hauv UUO raum, txhais tau hais tias qhov hnyav ntawm lub raum tubular raug mob thiab lub raum mob tsis cuam tshuam rau cov nas nrog pericyte-specific HIF stabilization. Vim hais tias cov myofibroblast yog cov caws pliav tsim cov cell thaum lub sijhawmraum fibrosis, peb cov txiaj ntsig yuav muab cov pov thawj sim rau qhov tsis muaj qhov tsis zoo ntawm HIF stabilization los ntawm PHD inhibitors ntawm lub raum fibrosis. Raws li peb txoj kev tshawb fawb, Souma et al.17 kuj tau tshaj tawm tiasraum fibrosistsis cuam tshuam los ntawm concomitant knockout ntawm PHD1, PHD2, thiab PHD3 hauv REPCs ntawm Tg (Epo-Cre) nas.

cistanche for treating renal fibrosis

cistanche yog qhov zoo raulub raum

Hauv peb txoj kev tshawb fawb, Gli1CreERT2/ ntxiv ; VhlF/F Cov nas tau pom los ntawm ob qho tib si nce endogenous EPO ntau lawm thiab polycythemia. Serum nce EPO qib ntawm polycythemia cuam tshuamraum fibrosismuaj teeb meem. Muaj ntau cov kev tshawb fawb txog cov teebmeem ntawm exogenous recombinant EPO ntawm AKI los yog CKD.53–57 Hauv cov kev tshawb fawb ua ntej, cov koob tshuaj recombinant EPO tau tshaj tawm tias ua rau polycythemia, kub siab, thiab kev ua haujlwm ntawm lub raum tsis zoo.56,58 Txaus siab, qee qhov kev tshawb fawb qhia. uas tsis tshua muaj tshuaj exogenous EPO analog, tsawg li tsis txhob ua rau erythropoiesis, ua rau cov teebmeem renoprotective hauv cov seem.raumthiab db/db nas cov qauv.59,60 Tsis tas li ntawd, phlebotomy kom normalize polycythemia tau pom tias ameliorate lub nce albuminuria hauv db/db nas raug rau high-dose exogenous EPO analog.60 Nyob rau hauv ib qho chaw kho mob cohort ntawm cov neeg mob polycythemia vera, a kab mob uas muaj polycythemia yam tsis tau nce endogenous EPO, 61 zaus ntawm CKD (txhais tau tias kwv yees glomerular pom tus nqi<60 ml/min="" per="" 1.73="" m2="" )="" was="" 27%,="" which="" was="" higher="" than="" in="" the="" general="" population.62,63="" however,="" the="" annual="" decline="" rate="" of="" estimated="" glomerular="" filtration="" rate="" was="" slower="" in="" patients="" with="" polycythemia="" vera="" than="" with="" other="" myeloproliferative="" diseases.="" in="" the="" study="" of="">CreERT2 / ntxiv; VhlF/F nas raug UUO raug mob, peb cov ntaub ntawv qhia tau hais tias muaj zog rau lub raum Epo qhia thiab polycythemia, tab sis tsis muaj kev hloov hauvraum fibrosis, mob, thiab raug mob tubular. Li no, tag nrho cov teebmeem ntawm pericytespecific HIF stabilization ntawm lub raum raug mob, o, thiab fibrosis yog nruab nrab, txhawb kev nyab xeeb ntawm PHD inhibitors hauv CKD kev loj hlob. Lub raum pericytes tej zaum yuav txhais tau tias yog ib tug heterogeneous pejxeem ntawm fibroblasts nyob rau hauv kev sib cuag nrog endothelial hlwb nyob rau hauv lubraum.8–13 Vim hais tias ntawm txoj kev loj hlob complexity, 64,65 heterogeneous lom muaj nuj nqi, 8,11–13,23 thiab qhov sib txawv PHD thiab HIF kev cai, 17,41,66 qhov zoo tshaj plaws genetic marker rau lub raum pericytes tseem tsis tau txhais. Gli1 ntxiv rau cov hlwb ua tiav cov txheej txheem morphologic thiab anatomic rau pericytes. Tom qab UUO raug mob, lub raum Gli1 ntxiv rau cov hlwb proliferate thiab sib txawv rau myofibroblasts. Lub raum Gli1 ntxiv rau cov hlwb suav txog kwv yees li 45 feem pua ​​​​ntawm tag nrho lub raum myofibroblast pas dej thiab ablation ntawm lub raum Gli1 ntxiv rau cov hlwb txo.raum fibrosis.11,36 Peb tau paub tias lub raum Gli1 ntxiv rau cov hlwb yog cov subpopulation ntawm platelet-derived growth factor receptor-b plus cells11,36,41 thiab hais tias cov teebmeem ntawm Gli1 ntxiv rau pericyte-txo cov noob caj noob ces manipulation tej zaum yuav npog los ntawm lwm cov fibroblasts. Txawm li cas los xij, peb qhov kev sim ntawm kev txheeb xyuas lub raum Gli1 ntxiv rau cov hlwb ua pov thawj tias HIF stabilization tuaj yeem nce Epo tab sis tsis yog collagen lossis Acta2 qhia ntawm qib cellular. Peb tsis tuaj yeem tshem tawm qhov ua tau tias HIF stabilization uas tau tsav los ntawm lwm cov cim pericyte yuav muaj qhov sib txawv. Cov nas nrog Cre recombinase yog tsav los ntawm Col1a1, Foxd1, lossis Pdgfrb tus txhawb nqa tsis tau siv los txhais cov pericytes hauv txoj kev tshawb no vim tias lawv Cre recombinase kuj tseem ua haujlwm hauv glomerular podocytes, 8 glomerular mesangial hlwb, 67 vascular du leeg hlwb, 68 thiab tej zaum qee cov tubular. epithelial cells.69 pVHL knockout hauv podocytes thiab tubular cells tau tshaj tawm tias ua rau lub raum pathologies xws li crescentic glomerulonephritis31 thiab lub raum cysts.70 Tsis tas li ntawd, peb siv inducible Cre rau knockout PHD2 lossis pVHL tom qab 5 lub lis piam ntawm hnub nyoog vim tias Foxd1 tau qhia nyob rau hauv lub embryonic. theem thiab knockout ntawm pVHL lossis PHD2 hauv Foxd1- qhia txog pericytes tau pom tias ua rau tsis zoo rau nephrogenesis.71,72

Hauv kev txheeb xyuas lub raum Gli1 ntxiv rau cov hlwb nrog PHD2 knockout siv Gli1CreERT2 / ntxiv; Egln 1F/F; ROSA 26fstdTomato/fstdTomatonas, peb tau pom qhov nce ntxiv ntawm cellular Epo mRNA qhia tab sis tsis yog Col1a1 lossis Acta2. Qhov 50-fold nce ntawm Epo mRNA tau lees paub tias HIF overexpression nyob rau hauv sorted raum Gli1 ntxiv rau pericytes nrog PHD2 knockout. Tsis tas li ntawd, hauv vivo pVHL lossis PHD2 knockout hauv Gli1 ntxiv rau pericytes ua rau muaj zog rau lub raum Epo qhia thiab polycythemia tab sis tsis yog.raum fibrosis. Peb ntseeg tias yog HIF downstream molecules hauv pericytes cuam tshuamraum fibrosis, nws yuav raug soj ntsuam hauv peb txoj kev tshawb fawb. Cov teebmeem ntawm PHD2 inactivation ntawm HIF stabilization tej zaum yuav npog los ntawm kev lom zem ntawm PHD1 thiab PHD3. Txawm li cas los xij, peb tau pom qhov cuam tshuam zoo ib yam ntawm EPO thiab collagen ntau lawm hauv Gli1 ntxiv rau pericytes nrog pVHL lossis PHD2 knockout, ntxiv txhawb HIF stabilization hauv pericytes txhawb erythropoiesis tab sis tsis yog.fibrosis. Peb tsis tuaj yeem tsim Gli1CreERT2 / ntxivvhl ;uaF/F; ROSA 26fstdTomato/fstdTomatonas los ntawm tus ntoo khaub lig ntawm Gli1CreERT2/þ; VhlF/Fthiab ROSA26fstdTomato/fstdTomatonas vim Vhl thiab ROSA26 nyob ntawm chromosome 6.

Peb siv UUO ua ib qhoraumkev raug mob qauv los soj ntsuam cov teebmeem ntawm pericyte-specific manipulation ntawm HIF rauraum fibrosisvim tias nyob rau hauv UUO qauv feem ntau Gli1 ntxiv rau pericytes sib txawv rau myofibroblasts thiab ablation ntawm Gli1 ntxiv cov hlwb ameliorated raum fibrosis.11,36 UUO qauv kuj tau ua tiav siv los ua kom pom kev hloov pauv ntawm collagen thiab EPO ntau lawm thaum lub sijhawm hloov pauv pericyte-myofibroblast los ntawm peb pawg thiab others.14,17,41 Txawm li cas los xij, UUO muaj nws tus kheej cov kev txwv raws li tus qauv CKD thiab peb qhov kev tshawb pom hauv qhov kev tshawb fawb no yuav tsis raug nthuav dav rau lwm cov qauv CKD. Cov kev tshawb fawb yav tom ntej tau lees paub los ntsuas qhov cuam tshuam ntawm pericyte-specific HIF manipulation hauv cov qauv CKD sib txawv.

Hauv kev xaus, peb tau pom tias Gli1 ntxiv rau pericytespecific HIF stabilization nce EPO, erythropoiesis, tab sis tsis cuam tshuam.fibrosishauv cov qauv nrog lossis tsis muaj UUO raug mob. Ntawm qib cellular, Gli1 ntxiv rau pericyte-specific HIF stabilization nce Epo qhia, tab sis tsis yog ib qho kev qhia ntawm Col1a1, Col3a1, thiab Acta2. Pericyte-specific HIF knockout tsis cuam tshuamraum fibrosis, ib. Cov txiaj ntsig no muab pov thawj kev sim rau qhov tsis muaj qhov tsis zoo ntawm PHD inhibitors ntawm lub raum fibrosis thiab CKD kev loj hlob.

Cistanche for improve kidney function

Cistanche rau kev txhim khoraummuaj nuj nqi

Qhia tawm

Txhua tus kws sau ntawv tshaj tawm tsis muaj kev sib tw txaus siab.

TXOJ CAI

SYP tau txais kev txhawb nqa los ntawm Ministry of Science thiab Technology (cov nyiaj pab 106-2314-B-418-006, 107-2314-B-418-001, thiab 108-2314-B-418-004) thiab Far Eastern Memorial Tsev Kho Mob (cov nyiaj pab 2017-C-037 thiab 2020-C- 037). SLL tau txais kev txhawb los ntawm Ministry of Science thiab Technology (cov nyiaj pab 108-2314-B-002-078-MY3 thiab 109-2314-B-002-260), National Health Research Institutes (grant EX108-10633 SI), National Taiwan University Tsev Kho Mob (NTUH; nyiaj pab 108-T16 thiab 109-T16), NTUH thiab NTU College of Medicine (grant NSCCMOH-131-43), Mrs. Hsiu-Chin LeeLub raumTshawb nrhiav Foundation, thiab Taiwan Health Foundation.

Peb ua tsaug rau xibfwb Guo-Hua Fong (University of Connecticut Health Center) rau Egln1F/Fnas; Dr. Yi-Ting Tsai (NTU College of Medicine) rau kev kuaj kab mob ntawm Gli1CreERT2 / ntxiv; VhlF/Fthiab VhlF/Fnas; thiab Department of Medical Research ntawm NTUH, Cell Sorting and Imaging Core Facility of the First Core Laboratory, Transgenic Mouse Model Core Facility thiab Gene Knockout Mouse Core Laboratory ntawm NTU College of Medicine rau kev txhawb nqa khoom thiab kev pab cuam.

AUTHOR CONTRIBUTIONS

SYP, PZT, YHC, YTC, FCC, YLC, thiab WCC tau ua cov kev sim thiab tshuaj xyuas cov ntaub ntawv. SYP, YLC, TH, YMC, thiab TSC tau koom nrog kev sim tsim thiab tshuaj xyuas cov ntaub ntawv. SLL tsim thiab coj qhov project. SYP thiab SLL tau sau cov ntawv sau.

SUPPLEMENTARY KHOOM

Cov ntaub ntawv ntxiv (PDF)

Cov txheej txheem ntxiv.

Daim duab S1. Lub cev hnyav, peripheral cov ntshav dawb suav, thiab cov platelet suav tsis tau hloov pauv hauv cov nas nrog pericyte-specific PHD2 knockout.

Daim duab S2. Cov duab sawv cev ntawm Epo mRNA kuaj pom los ntawm RNA hauv situ hybridization hauv lubraumntawm Egln1F/F thiab Gli1CreERT2/þ; Egln 1F/F nas. Daim duab S3. Ntiaj teb no PHD2 knockout txhawb EPO ntau lawm thiab

erythropoiesis.

Daim duab S4. PCR tsom xam ntawm genomic DNA muab rho tawm los ntawm lubraum, daim siab, tus po, lub plawv, thiab ntsws hauv Tg(UBC-CreERT2); Egln1F/F thiab littermate tswj nas.

Daim duab S5. Cov duab sawv cev ntawm Epo mRNA kuaj pom los ntawm RNA hauv situ hybridization hauv lubraumntawm Egln1F/F nas.

Daim duab S6. Cov duab sawv cev ntawm Gli1þ pericytes hauv CLK thiab UUOlub raumtom qab UUO raug mob.

Daim duab S7. Lub sij hawm ntev los ntawm pericyte-specific HIF stabilization hauv

Gli1CreERT2/þ;VhlF/F nas.

Daim duab S8. Pathology qhia ntawm Gli1CreERT2/ ntxiv; VhlF/F nas.

Daim duab S9. nce hematocrit thiab ntshav EPO qib hauv Gli1CreERT2 / ntxiv rau; VhlF/Fnas.

Daim duab S10. PCR tsom xam ntawmraumgenomic DNA tom qab tamoxifen induction.

Daim duab S11. Tag nrho cov duab gel rau immunoblotting ntawm a-SMA thiab a[1]tubulin.

Daim duab S12. Renal Epo thiab Acta2 qhia hauv VhlF/F thiab Gli1CreERT2 / ntxiv ; VhlF/Fnas 7 hnub tom qab UUO raug mob.

Daim duab S13. Renal Epo thiab Acta2 qhia hauv VhlF/F thiab Gli1CreERT2 / ntxiv ; VhlF/Fnas 14 hnub tom qab UUO raug mob.

Daim duab S14.Lub raum fibrosisnyob rau hauv lub ncua tamoxifen washout qauv.

Daim duab S15. Kev sim schema rau sau lub raum Gli1þ hlwb nrog tdTomato.

Daim duab S16. Gating zoo rau kev txheeb xyuas lub raum tdTomatoþ hlwb.

Daim duab S17. Validation of tdTomato expression thiab Egln1 deletion in the sorted renal tdTomatoþ cells.

Daim duab S18. PCR tsom xam ntawmraumgenomic DNA los ntawm Gli1CreERT2 / ntxiv; Hif 1aF/F; Hif 2 aF/Fthiab littermate Hif1aF/F; Hif 2 aF/Ftswj cov nas tom qab kev tswj hwm tamoxifen.

Daim duab S19. UA inducedraum fibrosishauv Gli1CreERT2 / ntxiv; Hif 1aF/F; Hif 2 aF/Fnas tom qab noj tshuaj ntau thiab lub sijhawm luv luv ntawm tamoxifen.

Daim duab S20. UA inducedraum fibrosishauv Gli1CreERT2 / ntxiv; Hif 1aF/F; Hif 2 aF/F nas tom qab ib nrab koob tshuaj thiab ncua sij hawm ntxuav tawm tamoxifen.

Tab S1. Primers siv rau genotyping.

Tab S2. Primers siv rau ntau PCR.

Ntxiv Macro. Macro rau tsis siv neeg quantification ntawm picrosirius liab-stainedraumcheeb tsam los ntawm ImageJ.

Cistanche for treating renal fibrosis

Cistanche khoom yog zoo raulub raum


REFERENCES

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