Qhov ncauj Hypoxia-inducible Factor Prolyl Hydroxylase Inhibitor Enarodusta Counteracts Alterations nyob rau hauv lub raum zog Metabolism nyob rau hauv lub Early Stage ntawm Diabetic raum Kab Mob

Hu rau: emily.li@wecistanche.com

Taw qhia

Division of Nephrology thiab Endocrinology, Lub Tsev Kawm Ntawv Qib Siab Tokyo Kawm tiav Tsev Kawm Ntawv Tshuaj, Tokyo, Nyiv; Kev Tshawb Fawb Txog Kev Tshawb Fawb rau Cov Tub Ntxhais Hluas, Nyiv Lub Koom Haum rau Kev Txhawb Kev Tshawb Fawb, Tokyo, Nyiv; Biological thiab Pharmacological Research Laboratories,

Central Pharmaceutical Research Institute, Nyiv Tobacco Inc., Takatsuki, Nyiv; Department of Systems Pharmacology, University of Tokyo Graduate School of Medicine, Tokyo, Nyiv; Laboratory rau Synthetic Biology, RIKEN Center rau Biosystems Dynamics Research, Suita, Nyiv; thiab WPI International Research Center rau Neurointelligence, University of Tokyo Institutes for Advanced Study (UTIAS), University of Tokyo, Tokyo, Nyiv.

Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors, tseem hu ua HIF stabilizers, nce endogenous erythropoietin ntau lawm thiab ua haujlwm ua cov tshuaj kho tshiab tiv thaiv ntshav qab zib hauvmob ntevraumkab mob. HIFinduces kev qhia ntawm ntau yam noob muaj feem xyuam rau lub zog metabolism raws li kev hloov kho rau hypoxia. Txawm li cas los xij, nws tseem tsis paub meej li cas cov metabolic reprogramming sab hauv cov ntaub so ntswg los ntawm HIF stabilization cuam tshuam rau pathophysiology ntawmraum kab mob. Cov kev tshawb fawb yav dhau los qhia tias cov kab mob metabolic xws li hyperglycemia thiab dyslipidemia ua rau muaj kev hloov pauv ntawm lub raum metabolism, ua rau lub raum tsis ua haujlwm xws li mob ntshav qab zib raum. Ntawm no, peb txheeb xyuas qhov cuam tshuam ntawm enarodustat (JTZ-951), qhov ncauj HIF stabilizer, ntawm lub raum lub zog metabolism hauv cov theem pib mob ntshav qab zib mellitus, siv streptozotocin-induced diabetic nas thiab alloxan-induced diabetic nas. Kev txheeb xyuas cov ntaub ntawv tau qhia tias enarodustat cuam tshuam cov kev hloov pauv hauv cov ntshav qab zib lub raum metabolism. Kev txheeb xyuas cov ntaub ntawv qhia tau hais tias fatty acid thiab amino acid metabolism hauv cov ntshav qab zib lub raum cov ntaub so ntswg thiab downregulated los ntawm enarodustat, thaum cov piam thaj metabolism tau upregulated. Cov kev hloov pauv symmetric no tau lees paub los ntawm kev txheeb xyuas metabolome. Whereasglycolysis thiab tricarboxylic acid voj voog metabolites tau sau thiab cov amino acids txo qis hauv lub raum cov ntaub so ntswg ntawm cov tsiaj mob ntshav qab zib, cov kev cuam tshuam metabolic no tau txo los ntawm enarodustat. Tsis tas li ntawd, enarodustatin tau nce glutathione rau glutathione disulfide piv thiab txo cov kev ntxhov siab oxidative hauv lub raum cov ntaub so ntswg ntawm cov tsiaj mob ntshav qab zib. Yog li, HIF stabilization counteracts alterations nyob rau hauv lub raum zog metabolism tshwm sim nyob rau hauv incipient.mob ntshav qab zibraumkab mob.

Cistanche zoo heev rau lub raum ua haujlwm

Lus Txhais Lus

Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors (tseem hu ua HIF stabilizers) nce endogenous erythropoietin ntau lawm thiab ua haujlwm ua cov tshuaj kho tshiab tiv thaiv ntshav qab zib hauvmob ntevraumkab mob. Peb cov ntaub ntawv sau tseg thiab cov tshuaj metabolome ntawm lub raum cov ntaub so ntswg hauv nas thiab nas mob ntshav qab zib qauv tau qhia tias enarodustat (JTZ-951), qhov ncauj HIF stabilizer, tiv thaiv lub raum lub zog metabolism hauv cov theem pib ntawm cov kab mob raum. Cov txiaj ntsig tau muab cov ntaub ntawv tseem ceeb rau kev nthuav tawm qhov cuam tshuam ntawm HIF stabilizers ntawm lub raum lub zog metabolism hauv chaw kho mob, txawm hais tias xav tau kev tshawb fawb ntxiv los qhia meej tias qhov no lub raum metabolism reprogramming los ntawm HIF stabilizers cuam tshuam rau kev loj hlob ntawm kev kho mob.mob ntshav qab zibraumkab mob.

Kev taw qhia

Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors nce endogenous erythropoietin ntau lawm thiab ua haujlwm ua cov tshuaj kho tshiab tiv thaiv kab mob ntshav qab zib.raumkab mob. Cells tau txais txiaj ntsig nrog kev tiv thaiv kev tiv thaiv tiv thaiv hypoxia, thiab HIF yog tus tswj hwm kev tiv thaiv no. Lub raum yog physiologically raug rau hypoxia, thiab mob hypoxia tau lees paub tias yog txoj hauv kev kawg uas ua rau mob raum kawg. Xav tias HIF induces cov kev qhia ntawm ntau yam noob muaj feem xyuam rau cov lus teb hypoxia, HIF stabilizers tej zaum yuav muaj pleiotropic cuam tshuam rau kev loj hlob ntawm tus kab mob.raumkab mobnrog rau kev txhim kho ntawm anemia nyob rau hauv ntevraumkab mob. Interestingly, HIF induces qhov kev qhia ntawm glycolytic noob thiab pyruvate dehydrogenase kinase, uas inhibits pyruvate dehydrogenase los ntawm kev siv pyruvate rau roj mitochondrial tricarboxylic acid (TCA) cycle.6,7 Qhov no metabolic reprogramming los ntawm TCA voj voog rau glycolysis yog represses oxygen noj. rau adaptation ntawm hlwb raug rau hypoxic ib puag ncig. Txawm li cas los xij, nws tseem tsis paub meej li cas cov metabolic reprogramming ntawm lub raum cov ntaub so ntswg los ntawm HIF stabilization cuam tshuam rau pathophysiology ntawmraum kab mob. Mob raum mob ntshav qab zib mellitus (DKD) yog qhov ua rau mob raum raum kawg. Cov kab mob metabolic xws li hyperglycemia thiab dyslipidemia ua rau lub raum hloov pauv metabolism, ua rau lub raum tsis ua haujlwm nrog rau DKD. Cov kev tshawb fawb yav dhau los tau pom tias muaj zog metabolic flux thiab tsub zuj zuj ntawm cov piam thaj thiab TCA lub voj voog metabolites hauv cov ntshav qab zib lub raum cortical cov ntaub so ntswg, uas tej zaum yuav cuam tshuam rau mitochondrial dysfunction thiab DKD kev loj hlob. Peb xav tias HIF stabilizers tuaj yeem thim rov qab cov metabolism hauv kev hloov pauv hauv cov ntshav qab zib lub raum cortical cov ntaub so ntswg, xav tias HIF, raws li kev hloov kho rau hypoxia, txo cov metabolism hauv cov hlwb kom txo qis cov pa oxygen. Yog li, siv transcriptome thiab metabolome analyses, peb tau ua pov thawj-ntawm-lub tswv yim kawm kom nkag siab txog yuav ua li cas enarodustat (JTZ-951), qhov ncauj HIF stabilizer, cuam tshuam rau lub raum metabolism hauv kev hloov pauv tshwm sim nyob rau theem pib ntawm DKD.

Cov txiaj ntsig: Enarodusat induces metabolic reprogramming los ntawm TCA lub voj voog mus rau glycolysis hauv lub raum proximal tubule cells

Raws li lub raum cortex feem ntau yog tsim los ntawm cov tubules ze, peb thawj zaug tshuaj xyuas cov teebmeem ntawm enarodustat ntawm cov metabolic flux ntawm lub raum proximal tubule hlwb hauv vitro. Ua ntej, Mito Stress Test (Agilent Technologies, Inc., Santa Clara, CA) thiab Glycolytic Rate Assay (Agilent) tau ua nyob rau hauv kab lis kev cai HK-2 hlwb, ib tug tib neeg proximal tubule epithelial cell kab (Daim duab 1a). Enarodustat txo qis mitochondrial respiration (TCA lub voj voog) thiab nce basal glycolysis, qhia tias metabolic reprogramming los ntawm TCA mus rau glycolysis (Daim duab 1; Ntxiv daim duab S1). Peb kuj tau ua ib qho kev sim siv me me cuam tshuam RNA (siRNA) rau HIF-1 (Daim duab 2; Daim duab ntxiv S2). HIF-1 knockdown los ntawm siRNA thim rov qab cov kev hloov pauv metabolic (basal ua pa, ua pa siab tshaj plaws, muaj peev xwm ua pa, thiab adenosine triphosphate [ATP] ntau lawm) induced los ntawm enarodustat, uas pom tau tias cov metabolic reprogramming feem ntau yog los ntawm HIF-1 stabilization (Daim duab 2; Ntxiv daim duab S2). Pyruvate dehydrogenase kev ua si, ib qho tseem ceeb rau cov hlwb siv pyruvate rau roj TCA lub voj voog, kuj raug txo los ntawm enarodustat (Daim duab 2f), uas tau sib xws nrog cov kev soj ntsuam yav dhau los.

Cov ntaub ntawv keeb kwm yav dhau ntawm streptozotocin-induced ntshav qab zib nas

Los ntawm cov txiaj ntsig ntawm kev sim hauv vitro, peb xav tias HIF stabilizers tuaj yeem txo cov metabolism hauv cov ntshav qab zib lub raum cortical cov ntaub so ntswg los ntawm metabolic reprogramming los ntawm TCA mus rau glycolysis. Peb thawj zaug xaiv streptozotocin (STZ)-vim cov nas mob ntshav qab zib ua tus qauv rau kev ua pov thawj-ntawm-lub tswv yim sim sim cov lus hais saum toj no. Hauv cov qauv no, ntshav qab zib tau nrawm nrawm, tso cai rau peb saib xyuas cov txiaj ntsig ntawm ntshav qab zib thiab HIF stabilizers ntawm lub zog metabolism hauv lub raum cov ntaub so ntswg hauv lub sijhawm luv luv. Txoj kev kawm raws tu qauv thiab cov ntaub ntawv yooj yim los ntawm kev sim hauv STZ-induced ntshav qab zib nas yog qhia hauv daim duab 3a. Peb faib cov nas ua 3 pawg: pawg A (sham), pawg B (DKD), thiab pawg C (DKD þ enarodustat). Ntshav ntshav qabzib, glycosylated hemoglobin HbA1c, triglyceride, thiab tag nrho cov roj cholesterol nyob rau hnub 14 tau nce ntau hauv cov neeg mob ntshav qab zib xws li piv nrog pawg A, thaum tsis muaj qhov sib txawv ntawm pawg B thiab C (Daim duab 3d-g). Txawm hais tias plasma creatinine qib tsis txawv ntawm cov pab pawg, urinary albumin excretion tau nce zuj zus thiab glomerulomegaly tau pom nyob rau hauv pawg B raws li piv nrog pawg A, thiab enarodustat nyiam thim rov qab cov kev hloov no (Daim duab 4). Ntshav urea nitrogen ntau dua nyob rau hauv pab pawg neeg mob ntshav qab zib, qhia txog lub cev qhuav dej vim ntshav qab zib. Hauv kev xaus, lub raum ntawm STZ-kho nas hauv peb txoj kev tshawb fawb sawv cev rau theem pib ntawm DKD. Kev txheeb xyuas cov ntaub ntawv thiab cov tshuaj metabolome tau ua los ntawm kev siv lub raum cortical cov ntaub so ntswg ntawm cov nas.

Transcriptome tsom xam ntawm lub raum cortical ntaub so ntswg

Cov txiaj ntsig ntawm transcriptome tsom xam ntawm lub raum cortical cov ntaub so ntswg yog qhia nyob rau hauv daim duab 5 thiab 6. Thawj feem kev tsom xam thiab hierarchical pawg qhia tau hais tias pawg A, B, thiab C tau muab cais ua pawg sib txawv, raws li (Daim duab 5a thiab b). Cov caj ces txawv txawv tau xaiv los ntawm │log2 fold-hloov (FC) │ Ntau dua lossis sib npaug rau 0.5 thiab Q tus nqi < 0.05.="" gene="" ontology="" thiab="" canonical="" txoj="" kev="" txheeb="" xyuas="" tau="" qhia="" tias="" cov="" noob="" muaj="" feem="" xyuam="" rau="" cov="" roj="" ntsha="" fatty="" acid="" metabolism="" tau="" raug="" tswj="" hwm="" hauv="" pawg="" b="" piv="" nrog="" pawg="" a.="" hauv="" qhov="" sib="" piv,="" cov="" noob="" muaj="" feem="" xyuam="" rau="" cov="" piam="" thaj="" metabolism="" thiab="" cov="" lus="" teb="" hypoxia="" suav="" nrog="" hif-1="" network="" tau="" tswj="" hwm="" hauv="" pawg="" c="" piv.="" nrog="" pab="" pawg="" b="" (daim="" duab="" 5c="" thiab="" d).="" peb="" kuj="" tau="" ua="" qhov="" kev="" tshuaj="" ntsuam="" gene="" set="" enrichment="" (gsea)="" siv="" cov="" ntaub="" ntawv="" transcriptomic="" (daim="" duab="" 6,="" table="" 1="" thiab="" 2).="" cov="" noob="" caj="" noob="" ces="" ntawm="" fatty-acid="" thiab="" amino-acid="" metabolism="" tau="" raug="" tswj="" hwm="" hauv="" pawg="" b="" piv="" nrog="" pawg="" a="" (daim="" duab="" 6).="" nyob="" rau="" hauv="" sib="" piv,="" cov="" gene="" sets="" tau="" downregulated,="" thiab="" gene="" sets="" ntawm="" qabzib="" metabolism="" tau="" upregulated="" nyob="" rau="" hauv="" pawg="" c="" raws="" li="" piv="" nrog="" rau="" pawg="" b,="" uas="" qhia="" tau="" hais="" tias="" enarodustat="" reversed="" metabolism="" alterations="" tshwm="" sim="" los="" ntawm="" ntshav="" qab="" zib="" (daim="" duab="" 6).="" tsis="" tas="" li="" ntawd,="" cov="" noob="" caj="" noob="" ces="" ntawm="" tca="" lub="" voj="" voog="" raug="" txo="" qis="" hauv="" pawg="" c="" piv="" nrog="" pawg="" b="" (table="" 2),="" uas="" yog="" sib="" xws="" nrog="" cov="" kev="" xav="" ntawm="" enarodustat-induced="" metabolic="" reprogramming="" nyob="" rau="" hauv="" proximal="" tubules="" pom="" nyob="" rau="" hauv="" peb="" in="" vitro="" txoj="" kev="" tshawb="" no.="" hauv="" cov="" ntsiab="" lus,="" enarodustat="" tiv="" thaiv="" cov="" mob="" ntshav="" qab="" zib="" lub="" raum="" cov="" metabolism="" hauv="" kev="" hloov="" pauv="" los="" ntawm="" cov="" kev="" xav="">

Metabolome tsom xam ntawm lub raum cortical ntaub so ntswg

Peb ntsuas qhov tseeb ntawm 116 lub zog-txog metabolites nyob rau hauv lub raum cortical ntaub so ntswg ntawm nas (n =4, rau txhua pab pawg) random xaiv los ntawm txhua pab pawg neeg (Sab Ntxiv Table S1; Ntxiv daim duab S3). Ib feem tsawg squares discriminant tsom xam (PLS-DA) tau ua los ntsuas qhov tseem ceeb ntawm kev ntxub ntxaug hauv chav kawm (Daim duab 7). Peb tau ua metabolite teeb enrichment tsom xam (MSEA) siv cov metabolites nrog PLS-DA qhov tseem ceeb sib txawv hauv qhov projection (VIP) qhab nia $ 1. Qhov sib txawv ntawm cov metabolism ntawm cov amino acids, xws li glycine, serine, methionine, aspartate, glutamate, arginine, proline, thiab b-alanine ntawm pawg A thiab B tau sau tseg. Qhov sib txawv ntawm cov amino-acid metabolism kuj tau pom ntawm pawg B thiab C. Ntxiv mus, cov txheej txheem ntawm cov piam thaj hauv cov metabolism, xws li glycolysis thiab gluconeogenesis, pom qhov sib txawv ntawm pawg B thiab C (Daim duab 7).

Kev pom cov ntaub ntawv transcriptome thiab metabolome ntawm lub zog metabolic txoj hauv kev daim ntawv qhia

Peb pom cov ntaub ntawv transcriptome thiab metabolome rau kev nkag siab zoo ntawm kev hloov pauv ntawm lub zog metabolism (Daim duab 8). Glycolysis thiab TCA lub voj voog metabolites tau pom tias muaj nyob rau hauv pawg B piv nrog pawg A, uas tej zaum yuav yog vim muaj cov piam thaj ntau dhau thiab txhim kho cov rog-acid metabolism hauv DKD. Cov amino acid concentration tau txo qis hauv pawg B piv nrog rau pawg A, qhia txog kev txhim kho ntawm cov amino acid metabolism (Daim duab 8a). Nyob rau hauv sib piv, cov tsub zuj zuj ntawm glycolysis metabolites yog relieved los ntawm enarodustat, vim lub yooj yim khiav ntawm glycolysis. Enarodustat kuj thim rov qab cov ntshav qab zib vim kev hloov pauv hauv TCA cycle metabolites thiab amino acids (Daim duab 8b). Ntxiv mus, enarodustat alleviated lub tsub zuj zuj ntawm glutathione disulfide (GSSG) nyob rau hauv cov ntaub so ntswg mob raum thiab yog li pom ib tug ntau dua glutathione / GSSG piv, uas qhia tias enarodustat relieved oxidative kev nyuaj siab nyob rau hauv DKD (Daim duab 8a thiab b). Qhov txo qis hauv oxidative kev nyuaj siab tau lees paub los ntawm qib ntawm lipid peroxidation marker malondialdehyde hauv lub raum cortical cov ntaub so ntswg: enarodustat thim rov qab cov tsub zuj zuj ntawm malondialdehyde hauv cov ntshav qab zib raum cortical cov ntaub so ntswg (Ntxiv daim duab S4). Hauv cov ntsiab lus, kev koom ua ke ntawm cov ntaub ntawv transcriptome thiab metabolome tau pom tias enarodustat tiv thaiv lub raum lub zog metabolism hauv kev hloov pauv tshwm sim nyob rau theem pib ntawm DKD.

Symmetric metabolism alterations (diabetes vs. enarodustat) tau lees paub nyob rau hauv ib qho piv txwv

Peb tau soj ntsuam cov ntaub ntawv pov thawj hauv alloxan-induced ntshav qab zib nas, lwm hom tsiaj ntawm ntshav qab zib, kom paub meej tias peb qhov kev tshawb pom hauv tus qauv nas. Kev kawm raws tu qauv thiab cov ntaub ntawv keeb kwm yav dhau rau tus qauv nas yog qhia hauv daim duab 9. Cov zis albumin tso zis tau nce ntau hauv pawg B piv nrog pawg A, uas tau thim rov qab los ntawm enarodustat (Daim duab 9d). Tsis tas li ntawd, peb tau siv dav 3-kev tsom xam qhov ntev (Clear, Unobstructed Brain / Cocktails Imaging Cocktails, thiab Computational analysis [CUBIC] – raum) 16 kom pom glomeruli hauv lub raum. Glomerulomegaly tau pom nyob hauv pawg B piv nrog pawg A, uas tau thim rov qab los ntawm enarodustat (Daim duab 9e). Kev txheeb xyuas ntawm lub raum cov ntaub so ntswg nyob rau hauv alloxan-induced ntshav qab zib nas pom symmetric metabolism alterations (diabetes vs. enarodustat) ib yam li nyob rau hauv STZ-induced mob ntshav qab zib nas qauv (Daim duab 10): fatty-acid metabolism yog upregulated los ntawm ntshav qab zib, hos qabzib. Cov metabolism tau raug tswj los ntawm enarodustat. Tsis tas li ntawd, cov amino-acid metabolism tau nce los ntawm ntshav qab zib thiab txo qis los ntawm enarodustat. Yog li, enarodustat tiv thaiv lub raum lub zog metabolism hauv kev hloov pauv tshwm sim nyob rau theem pib ntawm DKD hauv cov qauv alloxan-induced ntshav qab zib nas nrog rau hauv STZ-induced ntshav qab zib nas qauv.

Kev sib tham

Hauv txoj kev tshawb no, peb tau pom tias enarodustat (JTZ-951), qhov ncauj HIF stabilizer, tiv thaiv lub raum lub zog metabolism hauv kev hloov pauv tshwm sim nyob rau theem pib ntawm DKD hauv nas thiab nas qauv ntawm ntshav qab zib. Transcriptome thiab metabolome analyses tau pom symmetric metabolism alterations nyob rau hauv lub raum cov ntaub so ntswg (diabetes vs. enarodustat): fatty-acid thiab amino-acid metabolisms tau upregulated nyob rau hauv DKD, whereas enarodustat downregulated cov kev thiab ntxiv upregulated qabzib metabolism (Daim duab 5-10).

Nws tsis tau paub meej meej tias HIF stabilization muaj kev tiv thaiv los ntawm pathophysiology ntawm DKD lossis tsis. Cov kev tshawb fawb yav dhau los tau pom tias cov ntaub so ntswg mob ntshav qab zib tau raug hypoxia17 thiab HIF qhia hauv cov ntaub so ntswg raum tsis txaus los teb rau lawv cov kev mob hypoxic, uas yog ib feem los ntawm oxidative stress.18 HIF stabilization los ntawm cobalt chloride txhim kho oxidative kev nyuaj siab thiab txo proteinuria thiab tubulointerstitial puas nyob rau hauv STZ-induced DKD.19 Tsiaj txoj kev tshawb fawb tau qhia tias HIF stabilization tiv thaiv kev loj hlob ntawm kev rog, 20 txhim kho insulin rhiab heev, 20 thiab txo qis cov roj (cholesterol) hauv cov ntshav.21 Cov txiaj ntsig no qhia txog kev tiv thaiv kev tiv thaiv ntawm HIF stabilization hauv kev txhim kho. DKD. Hauv qhov sib piv, supraphysiologic HIF stabilization los ntawm von Hippel-Lindau tshem tawm tau tshaj tawm los ua rau lub raum fibrosis.22 Lub luag haujlwm ntawm HIF stabilization hauv DKD kev loj hlob tuaj yeem yog cov ntsiab lus-raws li muab cov teebmeem pleiotropic ntawm HIF thiab muaj ntau yam DKD phenotypes. Lub hom phiaj ntawm txoj kev tshawb no yog los qhia meej qhov cuam tshuam ntawm HIF stabilization ntawm lub zog metabolism hauv cov ntshav qab zib raum. Cov ntaub ntawv dhau los tau qhia tias kev hloov pauv ntawm lub zog metabolism tshwm sim hauv DKD. Sas et al.10 pom tias muaj zog metabolic flux thiab tsub zuj zuj ntawm cov piam thaj metabolites nyob rau hauv cov ntshav qab zib raum cortical cov ntaub so ntswg, uas tej zaum yuav muaj feem xyuam rau mitochondrial dysfunction. Koj li al.23 tau tshaj tawm tias fumarate, TCA lub voj voog metabolite, tau sau rau hauv cov ntaub so ntswg mob ntshav qab zib thiab nws ncaj qha rau kev loj hlob ntawm DKD. Peb tau pom yav dhau los tias TCA lub voj voog metabolites khaws cia hauv cov ntshav qab zib raum cov ntaub so ntswg, uas tau thim rov qab los ntawm sodium-glucose cotransporter 2 inhibition lossis calorie txwv.11 Ntxiv mus, Qi thiab al.24 tau pom tias cov enzymes hauv glycolytic txoj hauv kev suav nrog pyruvate kinase M2 (PKM2), tau tswj hwm hauv cov neeg mob ntshav qab zib hom 1 yam tsis muaj DKD piv nrog cov neeg mob DKD. Kev ua kom PKM2 thim rov qab cov tsub zuj zuj ntawm glycolysis metabolites thiab rov qab ua haujlwm mitochondrial, ib feem los ntawm kev nce glycolytic flux.24,25 Cov ntaub ntawv dhau los no qhia tias kev sib xyaw ntawm glycolysis thiab TCA lub voj voog metabolites tuaj yeem cuam tshuam rau kev nce qib ntawm DKD, uas tuaj yeem txo qis los ntawm PKM2. ua kom. Hauv peb txoj kev tshawb fawb, HIF stabilization los ntawm enarodustat thim rov qab lub zog metabolism hauv kev hloov pauv thiab txo qis qhov sib xyaw ntawm glycolysis thiab TCA cycle metabolites hauv cov ntshav qab zib raum cortical (Daim duab 8). Kev nthuav qhia ntawm glycolytic enzymes suav nrog PKM2 kuj tau tswj hwm los ntawm enarodustat (Cov Duab Ntxiv S4B). Ntxiv mus, enarodustat alleviated GSSG tsub zuj zuj thiab nce glutathione / GSSG piv, uas qhia tias enarodustat relieved oxidative kev nyuaj siab nyob rau hauv DKD (Daim duab 8). Qhov no enarodustat-vim txo qis hauv oxidative kev nyuaj siab kuj tau lees paub los ntawm kev hloov pauv hauv malondialdehyde qib hauv lub raum cortical cov ntaub so ntswg (Cov duab ntxiv S4A). Cov txiaj ntsig no qhia tias HIF stabilization yuav tsum muaj lub luag haujlwm tiv thaiv ntawm pathophysiology ntawm DKD yam tsawg kawg los ntawm kev xav txog metabolic. Nws yog qhov tseeb tias peb tsis tuaj yeem kuaj xyuas seb qhov metabolic reprogramming los ntawm HIF stabilization muaj qhov cuam tshuam ncaj qha rau DKD kev loj hlob, vim tias nws yuav tsis tuaj yeem pom qhov cuam tshuam ntawm cov metabolism hauv kev hloov pauv ntawm lub raum tshwm sim vim yog lub luag haujlwm pleiotropic ntawm HIF. Txawm li cas los xij, hauv peb txoj kev tshawb fawb, cov zis me me albumin excretion thiab lub raum pathological txawv txav (glomerulomegaly thiab glomerular hauv qab daus daim nyias nyias nyias) vim yog ntshav qab zib tau txo qis los ntawm enarodustat, nrog rau kev ua haujlwm ntawm lub raum lub zog metabolism. Peb rho tawm cov noob uas nws cov kev hloov pauv cuam tshuam nrog cov qib albumin urinary (257 probes; 232 genes) los ntawm cov ntaub ntawv microarray hauv alloxan-induced ntshav qab zib nas qauv thiab ua txoj hauv kev tsom xam ntxiv (Cov duab ntxiv S5). Raws li qhov tshwm sim, txoj hauv kev cuam tshuam nrog mitochondrial daim nyias nyias thiab cov pa hluav taws xob thauj mus los tau raug kho nrog cov qib urinary albumin, qhia tias lub nra ntawm mitochondrial yog ze ze rau kev tso zis albumin. Yog li, muaj peev xwm ua kom cov metabolic normalization ntawm cov ntshav qab zib raum cov ntaub so ntswg los ntawm HIF stabilizers yuav txo tau lub nra ntawm mitochondrial thiab txo qhov kev loj hlob ntawm DKD. Peb cov ntaub ntawv qhia txog pathophysiology ntawm DKD los ntawm metabolic foundations raws li hauv qab no: Hyperglycemia tsim lub zog thov rau qabzib reabsorption nyob rau hauv lub raum proximal tubules; Yog li, TCA lub voj voog hauv mitochondria raug yuam kom ua kom tau raws li qhov xav tau ntawm lub zog nyob rau theem pib ntawm DKD (upregulation ntawm fatty-acid thiab amino-acid metabolism). Thiab HIF stabilizers yuav txo tau lub nra ntawm lub mitochondrial los ntawm cov metabolic reprogramming los ntawm TCA mus rau glycolysis (downregulation ntawm fatty-acid thiab amino-acid metabolism thiab upregulation ntawm glycolysis), uas tej zaum yuav muaj lub luag hauj lwm tiv thaiv kev loj hlob ntawm DKD. Cov kev tshawb fawb ntxiv nrog rau ib leeg-cell omics yog xav tau los qhia meej tias lub nra mitochondrial hauv lub raum puas cuam tshuam ncaj qha rau pathophysiology ntawm DKD. Lwm cov lus nug nthuav yog seb HIF stabilizers ncaj qha cuam tshuam rau kev nqus cov piam thaj hauv cov tubules ze. Peb cov ntaub ntawv hauv vitro tau pom tias ATP ntau lawm tau txo qis los ntawm enarodustat los ntawm metabolic reprogramming los ntawm TCA mus rau glycolysis (Figures 1 thiab 2). Xav txog tias ATP xav tau rau cov piam thaj reabsorption los ntawm sodium-glucose cotransporter 2, peb thawj zaug pom tias cov piam thaj reabsorption yuav tsis ua haujlwm los ntawm enarodustat. Txawm li cas los xij, cov piam thaj hauv zis tsis sib txawv ntawm pawg B thiab pawg C hauv ob qho tib si tsiaj qauv (Cov duab ntxiv S6). Tej zaum, qhov txo qis hauv ATP ntau lawm los ntawm enarodustat yog qhov mob me dua hauv qhov xwm txheej physiological piv nrog nws cov nyhuv hauv vitro; Yog li, HIF stabilization tsis cuam tshuam rau kev nqus cov piam thaj los ntawm sodium-glucose cotransporter 2, tsawg kawg hauv peb cov tsiaj sim. Peb txoj kev tshawb fawb muaj 2 qhov kev txwv. Ua ntej, peb tau siv STZinduced nas ntshav qab zib thiab alloxan-induced ntshav qab zib nas raws li cov qauv DKD thaum ntxov thiab pom cov teebmeem luv luv ntawm cov mob ntshav qab zib. Txawm li cas los xij, hauv qhov chaw kho mob, HIF stabilizers yuav raug muab rau cov neeg mob ntshav qab zib hauv theem kawg DKD. Cov kev tshawb fawb ntxiv yuav tsum tau nkag siab txog qhov cuam tshuam ntawm HIF stabilization ntawm lub raum metabolism hauv kev hloov pauv hauv DKD lig. Qhov thib ob, txoj kev tshawb no tsis muaj cov ntaub ntawv pov thawj txaus nrog rau kev cuam tshuam ntawm cov metabolites cov ntsiab lus hauv cov ntaub ntawv metabolome, thaum cov ntaub ntawv transcriptome muaj cov ntaub ntawv teev tseg loj txaus los qhia tag nrho cov duab ntawm lub zog metabolism. Vim tias tsis muaj ntau cov metabolites pom qhov sib txawv ntawm pawg, peb tau ua PLS-DA thiab xaiv cov metabolites nrog PLS-DA VIP qhab nia Ntau dua lossis sib npaug rau 1 rau MSEA. Txawm hais tias nws nyuaj rau kev txheeb xyuas qhov kev hloov pauv hauv metabolic siv cov ntaub ntawv metabolomics ib leeg, cov txiaj ntsig ntawm kev tshuaj ntsuam metabolome tau sib xws nrog cov ntaub ntawv transcriptomic (Daim duab 7 thiab 8), muab kev txhawb nqa ntxiv rau kev soj ntsuam cov txiaj ntsig ntawm kev hloov pauv ntawm lub raum lub zog metabolism. Hauv kev xaus, enarodustat (JTZ-951), qhov ncauj HIF stabilizer, tiv thaiv lub raum lub zog metabolism hauv kev hloov pauv hauv cov theem pib ntawm DKD. Peb txoj kev tshawb fawb qhia tias HIF stabilization tuaj yeem ua rau muaj peev xwm cuam tshuam rau lub hom phiaj ntawm lub raum lub zog metabolism hauv DKD.

Txoj kev

Mito Stress Test thiab Glycolytic Rate Assay

HK-2 hlwb tau muab noob rau ntawm 96-zoo microplate ntawm qhov ceev ntawm 1 * 10⁴cell/zoo. Hnub tom qab, metabolic flux tau ntsuas hauv lub sijhawm tiag tiag los ntawm Seahorse XFe96 Analyzer (Agilent) siv Mito Stress Test Kit (103015-100; Agilent) lossis Glycolytic Rate Assay Kit (103344- 100; Agilent). Cov ntsiab lus ntawm cov piam thaj, pyruvate, thiab glutamine hauv kab lis kev cai yog 10 mmol / l, 1 mmol / l, thiab 2 mmol / l, raws li.

siRNA kev hloov pauv

HIF-1 knockdown tau ua los ntawm Stealth RNAi rau tib neeg HIF1A (HSS104774 [#1] thiab HSS104775 [#2]; Thermo Fisher Scientific, Waltham, MA). Stealth RNAi siRNA Negative Control Med GC Duplex #2 (12935112; Thermo Fisher Scientific) tau siv los ua kev tswj tsis zoo.

Western blotting

Cov tshuaj tiv thaiv tseem ceeb uas siv rau staining yog cov tshuaj tiv thaiv tib neeg HIF-1 antibody (luv polyclonal, 1:500; Novus Biologicals, Littleton, CO) thiab anti-human actin antibody (luv polyclonal, 1: 1000; Sigma-Aldrich, St. . Louis, MO). Ib qho tshuaj tiv thaiv kab mob thib ob yog horseradish peroxidase-conjugated tshis los tiv thaiv luav IgG antibody (170-6515, 1:5000; Bio-Rad Laboratories, Hercules, CA).

Tsiaj sim

Crl: CD (Sprague Dawley) nas thiab Crl: CD1 (Institute of Cancer Research) nas tau txais los ntawm Charles River Laboratories Japan Inc. (Yokohama, Nyiv). Txhua qhov kev sim tau pom zoo los ntawm University of Tokyo Institutional Review Board (kev pom zoo tus naj npawb P17-110). Tag nrho cov txheej txheem tsiaj txhu tau ua raws li National Institutes of Health cov lus qhia (Cov Lus Qhia rau Kev Saib Xyuas thiab Kev Siv Cov Tsiaj Txhaum Cai). Cov txheej txheem kawm tau qhia nyob rau hauv daim duab 3 thiab 9.

Transcriptome tsom xam

Tag Nrho RNA los ntawm lub raum cortical cov ntaub so ntswg raug cais siv GenElute Mammalian Total RNA Miniprep Kit (RTN7{{10}}}; Sigma-Aldrich). Tag Nrho RNA (100 ng) tau sau npe siv Low Input Quick Amp Labeling Kit (Agilent) thiab tom qab ntawd hybridized rau SurePrint G3 Rat GE v2 8x60K Microarray (rau nas; Agilent) thiab SurePrint G3 Mouse GE v{{7 }}x60K Microarray (rau nas), feem. Txhua qhov kev sim microarray tau ua los ntawm DNA Chip Research Inc. (Tokyo, Nyiv). Cov ntaub ntawv nyoos tau ua tiav nrog R pob limma hauv Bioconductor (http://www.bioconductor.org/) los kho keeb kwm yav dhau thiab cov ntaub ntawv normalization siv txoj kev quantile normalization. Batch teebmeem raug tshem tawm los ntawm ComBat (Bioconductor) hauv kev sim nas. Cov noob sib txawv tau txiav txim siab los ntawm│log2 FC│ Ntau dua lossis sib npaug rau 0.5 thiab Q tus nqi <0.05. gene="" ontology="" thiab="" canonical="" txoj="" kev="" txheeb="" xyuas="" tau="" ua="" los="" ntawm="" basespace="" correlation="" engine="" (illumina,="" san="" diego,="" ca).="" microarray="" cov="" ntaub="" ntawv="" tau="" muab="" tso="" rau="" hauv="" national="" center="" for="" biotechnology="" information="" gene="" expression="" omnibus="" li="" series="" gse131221="" (rat)="" thiab="" gse139317="">

Gene Set Enrichment Analysis

GSEA yog ib txoj hauv kev suav uas txiav txim siab seb puas yog ib qho tseem ceeb ntawm cov noob caj noob ces qhia pom qhov tseem ceeb, sib txawv ntawm 2 lub xeev lom neeg. GSEA tau ua tiav siv GSEA v.3.0 (Broad Institute, Cambridge, MA). Txoj hauv kev nrog qhov kev tshawb pom tsis tseeb < 0.25="" tau="" suav="" tias="" yog="" qhov="" tseem="">

Kev tsom xam Metabolome

Kev ntsuas Metabolome tau ua los ntawm Human Metabolome Technologies Inc. (Tsuruoka, Nyiv). Metabolite concentrations raug xam los ntawm normalizing lub ncov cheeb tsam ntawm txhua metabolite nrog rau cov cheeb tsam ntawm tus txheej txheem sab hauv thiab los ntawm kev siv tus qauv nkhaus, uas tau los ntawm 3-point calibrations. Saib Cov Txheej Txheem Ntxiv.

Metabolite teem enrichment tsom xam

Kev soj ntsuam ntawm cov ntaub ntawv metabolomics tau ua los ntawm kev siv lub vev xaib sib koom ua ke MetaboAnalyst 4.0. PLS-DA tau ua tiav thiab suav nrog cov qhab nia VIP. Cov metabolites nrog PLS-DA VIP qhab nia Ntau dua lossis sib npaug rau 1 tau siv rau MSEA.

Quantification ntawm glomerular cheeb tsam nyob rau hauv lub raum pathologies

Peb random coj 3 daim duab pathological los ntawm txhua tus qauv ntawm cov kua qaub-Schiff staining duab thiab ntsuas txhua qhov glomerular cheeb tsam raws li kev siv Fiji (ib qho kev faib tawm ntawm ImageJ; National Institutes of Health, Bethesda, MD).

CUBIC-lub raum

Kev nthuav dav 3-Kev ntsuas qhov ntev ntawm glomeruli hauv cov ntaub so ntswg raum tau ua tiav siv CUBIC raws li peb cov ntaub ntawv dhau los. Nyob rau hauv luv luv, tsau nas lub raum tau muab tso rau hauv CUBIC-L rau delipidation thiab ces raug rau immunofluorescent staining. Thaum kawg, qhov refractive index tau sib phim los ntawm qhov chaw ntawm cov qauv hauv CUBIC-R ntxiv. Cov duab 3D-dimensional los ntawm lub raum pob tshab tau txais nrog kev cai tsim lub teeb-daim ntawv fluorescence microscopy (MVX10-LS; Olympus, Tokyo, Nyiv). Thawj cov tshuaj tiv thaiv uas siv rau staining yog cov tshuaj tiv thaiv-podocin antibody (Lavbit polyclonal, 1:100, P0372; Sigma-Aldrich). Cov tshuaj tiv thaiv kab mob thib ob yog Alexa Hmoov 555- conjugated nees luav los tiv thaiv luav IgG (1:100, A-31572; Invitrogen, Thermo Fisher Scientific).

Kev txheeb cais

Rau ntau qhov kev sib piv, {{0}}}txoj kev tsom xam ntawm qhov sib txawv ua raws li kev tshaj tawm hoc Tukey ntau qhov kev sib piv, yog tias tsim nyog, raug siv. Txhua qhov kev txheeb cais tau ua nrog GraphPad Prism 7 software (GraphPad Software, San Diego, CA). P <0.05 tau="" suav="" tias="" yog="" qhov="" tseem="" ceeb.="" cov="" ntaub="" ntawv="" raug="" nthuav="" tawm="" raws="" li="" qhov="" nruab="" nrab="" ±="">

Qhia tawm

MN tau txais kev qhuas, tus nqi pab tswv yim, lossis kev tshawb fawb nyiaj txiag los ntawm KyowaHakko-Kirin, Akebia, Astellas, Chugai, GlaxoSmithKline, Nyiv Tobacco Inc. (JT), Torii, Tanabe-Mitsubishi, Daiichi-Sankyo, Takeda, Ono, Bayer, Boehringer Inhegel , thiab Alexion. TT tau txais nyiaj pab tshawb fawb los ntawm JT. Enarodustat (JTZ-951) yog muab los ntawm JT, Tokyo, Nyiv. EAS thiab HRU yog coinventors ntawm patents muaj los ntawm RIKEN. Ib feem ntawm txoj kev tshawb no tau ua tiav hauv kev koom tes nrog Olympus Corporation thiab zoo software txhawb nqa los ntawm Bitplane. Lwm tus sau tau tshaj tawm tias tsis muaj kev sib tw nyiam.

TXOJ CAI

Peb ua tsaug rau Dr. Shuhei Yao thiab Dr. Shinji Tanaka rau kev sib tham tseem ceeb. Peb kuj ua tsaug rau Dr. Tsuyoshi Inoue, Dr. Satoru Fukuda, thiab Ms. Kahoru Amitani rau lawv txoj kev txhawb nqa. Txoj haujlwm no tau ua ib feem raws li kev txhawb nqa ntawm Isotope Science Center, University of Tokyo. Txoj haujlwm no tau txais kev txhawb nqa los ntawm Grant-in-Aid rau Nyiv Lub Koom Haum rau Kev Txhawb Kev Tshawb Fawb (JSPS) Kev Tshawb Fawb Kev Tshawb Fawb (JSPS KAKENHI muab 19J11928 rau SH), Grant-in-Aid for Scientific Research (B) (JSPS KAKENHI grant 18H02824 rau MN ), Grant-in-Aid for Scientific Research (C) (JSPS KAKENHI grant 17K09688 to TT), thiab Grant-in-Aid for Scientific Research on Innovative Areas (KAKENHI grant 26111003 to MN).