Tissue-specifc Expression Of SARS-CoV-2 Receptor, Angiotensin-converting Enzyme 2, in Mouse Models Of Chronic Kidney Disease
Mar 22, 2022
Txij li thaum Lub Ib Hlis 2020, tus kab mob coronavirus 2019 (COVID-19), tshwm sim los ntawm tus mob hnyav ua pa nyuaj rau tus mob coronavirus 2 (SARS-CoV-2), tau ua teeb meem kev noj qab haus huv thoob ntiaj teb. SARS-CoV-2 kab mob yog tsim los thaum tus kab mob S protein khi los tuav angiotensin-hloov enzyme 2 (ACE2) thiab nkag mus rau hauv cell1,2. Yog li ntawd, nce ACE2 kev qhia nyob rau hauv lub cev uas yog lub hom phiaj ntawm SARS-CoV{15}} tuaj yeem ua rau muaj kev pheej hmoo ntawm tus kab mob COVID{16}}. ACE2 yog ib qho enzyme uas ua lub luag haujlwm tseem ceeb hauv renin-angiotensin system (RAS), qhov uas nws hloov angiotensin (Ang) II rau Ang{19}} thiab tsim ib feem ntawm ACE2-Ang{21. }}MAS receptor axis3. Nws kuj tseem ua rau lub cev tiv thaiv kab mob los ntawm kev tawm tsam kev ua haujlwm ntawm ACE-Ang II-angiotensin type 1 (AT1) receptor axis4-7. Cov ntaub ntawv dhau los tau qhia tias ACE2 hloov pauv nws cov qauv qhia tshwj xeeb hauv cov ntaub so ntswg hauv ntau yam mob, suav nrog cov kab mob plawv, ntshav qab zib mellitus (DM), thiabkab mob raum 8–11. Pulmonary ACE2 qhia, uas yog qhov tseem ceeb tshwj xeeb rau SARS-CoV-2 kab mob, txo qis hauv lipopolysaccharide-induced acute respiratory distress syndrome12. Lwm daim ntawv tshaj tawm qhia tias ACE2 qhia tau siab dua hauv cov txiv neej laus dua 13. Hauv Department of Medical Science thiab Cardiorenal Medicine, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004, Nyiv. 2 Kev Pabcuam Mob plawv thiab Metabolic Disorders, Duke-NUS Medical School, Singapore, Singapore. 3 Department of Medicine, Mount Sinai Beth Israel, New York, NY, USA. 4 Department of Molecular Biology, Yokohama City University Graduate School of Medicine, Yokohama, Nyiv. 5 Department of Otorhinolaryngology, Head and Neck Surgery, School of Medicine, Yokohama City University, Yokohama, Nyiv. 6 Internal Medicine, Kanagawa Dental University.
Cov lus tseem ceeb;kab mob raum; ob lub raum; raum ACE2; cov ntaub so ntswg raum; raum tsis ua haujlwm
CISTANCHE yuav txhim kho rau lub raum/ raum mob
Ntxiv mus, qhov ntswg thiab ceg ACE2 qhia tau siab dua rau cov neeg laus dua li cov menyuam yaus 14. Txawm li cas los xij, rau qhov zoo tshaj plaws ntawm peb txoj kev paub, ob peb txoj kev tshawb fawb tau tshuaj xyuas seb qhov pulmonary ACE2 qhia tau hloov pauv li cas hauv lwm cov kab mob. Cov neeg mob uas mob ntevmob raum(CKD) muaj kev pheej hmoo ntawm tus kab mob COVID-19 hnyav, tab sis tsis muaj pov thawj ntawm qhov muaj ntau dua ntawm COVID-19 hauv CKD cov neeg mob15–21. Xav txog qhov tseeb tias ACE2 yuav tsum tau ua rau tus kab mob COVID-19, cov kev tshawb pom no yuav qhia tau tias cov kab mob pulmonary ACE2 tsis tau txhim kho rau cov neeg mob CKD. Tshwj xeeb tshaj yog, cellular ACE2 kev qhia yog suav tias muaj feem cuam tshuam nrog kev pheej hmoo ntawm kev kis kab mob los ntawm SARS-CoV, uas cuam tshuam nrog SARS-CoV-222. Qhov tseeb tias cov menyuam yaus uas tsis tshua muaj ACE2 qhia tau tsawg dua rau COVID-19 dua li cov neeg laus23 kuj txhawb cov kev xav no. Yog li, peb tau ua cov kev sim los tshuaj xyuas cov kev hloov pauv hauv pulmonary ACE2 qhia hauv ob hom CKD qauv nas: adenine-induced (ie, adenine nas) thiab aristolochic acid (AA)-induced (ie, AA nas) los hais txog qhov kev xav no. Kev sib raug zoo ntawm ACE2 qhia nrog RAS blockers kuj tau tshawb xyuas. Txog tam sim no, kev siv RAS blockers tau pom tias txhim kho lub cev ACE2 qhia hauv qee qhov xwm txheej24-27. Kev tshawb nrhiav kev kis kabmob tsis ntev los no tau qhia tias kev siv RAS blockers tsis ua rau muaj kev pheej hmoo ntawm COVID-1928–30. Tsis tas li ntawd, ACE2 qhia hauv lub ntsws tsis tau txhim kho thaum RAS blockers tau muab rau cov nas ib txwm muaj 31. Txawm li cas los xij, tsis tau muaj lus ceeb toom txog qhov cuam tshuam ntawm RAS blockers ntawm pulmonary ACE2 qhia hauv cov ntsiab lus ntawm CKD; cov ntsiab lus no yog qhov tseem ceeb rau kev tswj hwm yav tom ntej ntawm COVID-19 thiab nws cov sequelae. Ntawm no, peb tau tshawb xyuas cov kev hloov pauv hauv pulmonary ACE2 qhia vim RAS blocker kho hauv CKD qauv nas.
Cov txiaj ntsigAdenine-induced CKD qauv nas pom qhov poob ceeb thawj, lub raum ua haujlwm poob qis, thiab nce ntshav siab (BP), piv nrog kev tswj hwm. Baseline lub cev hnyav (BW) thiab systolic BP tau zoo ib yam ntawm cov tswj thiab adenine pawg. BW nce ntxiv rau lub sijhawm hauv pawg tswj hwm, thaum nws txo qis hauv pawg adenine; Qhov kev nce BW no sib txawv ntawm cov pab pawg (Fig. 1A). Systolic BP tau nce siab hauv pawg adenine, piv nrog rau pawg tswj hwm, dhau sijhawm (Daim duab 1B). Thaum 2 lub lis piam, urinary albumin excretion tau nce siab hauv pawg adenine, piv nrog rau pawg tswj hwm (adenine nas 41.0± 8.3 ug / hnub vs. tswj 13.6 ± 1.7 ug / hnub, P<0.001; fig. 1e).="" there="" were="" also="" signifcant="" enhancements="" of="" plasma="" creatinine="" and="" blood="" urea="" nitrogen="" (bun)="" levels="" in="" the="" adenine="" group,="" compared="" with="" the="" control="" group="" at="" 2="" or="" 4 weeks="" (creatinine:="" 2 weeks:="" adenine="" mice="" 0.22±0.01 mg/dl="" vs.="" control="" 0.10±0.01 mg/="" dl,=""><0.05; 4 weeks:="" adenine="" mice="" 0.31±0.06 mg/dl="" vs.="" control="" 0.11±0.01 mg/dl,=""><0.01; bun:="" 2 weeks:="" adenine="" mice="" 65.0±7.0 mg/dl="" vs.="" control="" 25.7±0.5 mg/dl,=""><0.01; 4 weeks:="" adenine="" mice="" 61.7±12.5 mg/dl="" vs.="" control="" 25.6±1.6 mg/dl,=""><0.01; fig. 1c,="" d).="" moreover,="" creatinine="" clearance="" also="" showed="" a="" signifcant="" reduction="" in="" the="" adenine="" group="" (2 weeks:="" adenine="" mice="" 170±15 μl/min="" vs.="" control="" 397±25 μl/min,=""><0.001; 4 weeks:="" adenine="" mice="" 136±29 μl/min="" vs.="" control="" 357±31 μl/min,=""><0.001;>
ACE2 qhia hauv lub raum tau txo qis hauv adenine-induced CKD qauv nas, piv nrog cov tswj, thaum ACE2 qhia hauv lub ntsws tsis txawv ntawm pawg..Lub raum histology pom heterogeneous o thiab atrophy ntawm tubules, nrog rau cov cellular infiltration rau hauv lub cev.lub rauminterstitium, nyob rau hauv adenine nas. ACE2 staining feem ntau tau pom nyob rau hauv cov tubules ze ze ntawm ob pawg, tab sis qhov kev staining tau txo qis hauv pawg adenine (Fig. 2A). ACE2 qib protein ntau hauv covlub raum(kwv yees los ntawm western blotting tsom xam) pom qhov txo qis hauv pawg adenine, piv nrog pawg tswj hwm, ntawm ob lub lis piam 2 thiab 4 (2 lub lis piam: adenine nas 0.55±0.{{ 9}}4 vs. tswj 1.00± 0.10, P<0.001; 4 weeks:="" adenine="" mice="" 0.32±0.07="" vs.="" control="" 0.90±0.07,=""><0.001; fig. 2c).="" in="">raum ACE2mRNA qhia tau pom qhov txo qis hauv pawg adenine ntawm 2 lub lis piam (adenine nas 0.61±0.04 vs. tswj 1.00±0.03, P<0.001; fig. 2e);="" a="" similar="" tendency="" for="" reduction="" was="" observed="" at="" 4 weeks,="" although="" the="" difference="" was="" not="" statistically="" signifcant="" (fig. 2e).="" lung="" histology="" findings="" did="" not="" differ="" between="" groups;="" ace2="" staining="" was="" observed="" in="" type="" 2="" alveolar="" epithelial="" cells="" (fig. 2b).="" there="" were="" no="" differences="" in="" ace2="" protein="" or="" mrna="" expression="" levels="" in="" the="" lungs="" at="" 2="" or="" 4 weeks="" (fig. 2d,="" f).="" the="" difference="" in="" plasma="" ace2="" activity="" was="" not="" statistically="" signifcant="">
CISTANCHE yuav txhim kho lub raum / raum tsis ua haujlwm
AA-induced CKD qauv nas pom qhov poob ceeb thawj thiab lub raum ua haujlwm tsis zoo, piv nrog rau pawg tsheb, thaum BP tsis txawv ntawm pawg.Baseline BW thiab systolic BP tau zoo ib yam ntawm cov tsheb thiab AA pawg. BW tau nce ntau lub sijhawm hauv pawg tsheb, thaum nws txo qis hauv pawg AA; Qhov kev nce BW no sib txawv ntawm cov pab pawg (Fig. 3A). Txawm li cas los xij, tsis muaj qhov sib txawv ntawm systolic BP ntawm pawg (Fig. 3B). Piv nrog rau pawg tsheb, muaj qhov tseem ceeb ntawm kev txhim kho cov ntshav creatinine thiab BUN qib, nrog rau cov zis albumin tso tawm, hauv pawg AA (creatinine: AA nas 0.40±{{6}). }.03 mg/dL vs. tsheb 0.14 ± 0.01 mg/dL, P<0.001; bun:="" aa="" mice="" 62.8±4.6 mg/="" dl="" vs.="" vehicle="" 29.0±1.4 ="" mg/dl,=""><0.001; urinary="" albumin="" excretion:="" aa="" mice="" 61.3±7.3 ="" μg/day="" vs.="" vehicle="" 9.5±0.9 μg/day,=""><0.001; fig. 3c–e).="" creatinine="" clearance="" also="" showed="" a="" signifcant="" reduction="" in="" the="" aa="" group="" (aa="" mice="" 72±12="" μl/min="" vs.="" vehicle="" 159±13="" μl/min,=""><0.001;>
ACE2 qhia hauv lub raum tau txo qis hauv AA-induced CKD qauv nas, piv nrog cov tswj, thaum pulmonary ACE2 protein ntau tsis txawv ntawm cov pab pawg.. Lub raum histology tsom xam pom tubular atrophy thiab cellular infiltration rau hauvlub rauminterstitium hauv pawg AA. ACE2 staining feem ntau tau pom nyob rau hauv cov tubules ze ze ntawm ob pawg, tab sis qhov kev staining tau txo qis hauv pawg AA (Fig. 4A). ACE2 qib protein ntau hauv covlub raumpom qhov txo qis hauv pawg AA, piv nrog pawg tsheb (AA nas {{0}}}.22±0.03 vs. tsheb 1.00±0.08, P<0.001; fig. 4c).="" in="" addition,="">raum ACE2mRNA qhia tau txo qis hauv pawg AA (AA nas {{0}}}.30±0.02 vs. tsheb 1.00±0.06, P<0.001; fig. 4e).="" lung="" histology="" findings="" did="" not="" differ="" between="">
ACE2 staining tau pom nyob rau hauv hom 2 alveolar epithelial hlwb (Fig. 4B) thiab tsis muaj qhov txawv ntawm pulmonary ACE2 protein theem ntawm pawg (Fig. 4D). ACE2 mRNA kev qhia hauv lub ntsws tau qis dua hauv pawg AA dua li hauv pawg tsheb (AA nas 0.76±0.04 vs. tsheb 1.{{11} ± 0.04, P<0.01; fig. 4f).="" moreover,="" there="" was="" a="" signifcant="" reduction="" in="" plasma="" ace2="" activity="" in="" the="" aa="" group="" (aa="" mice="" 84.8±2.6="" rfu/min="" vs.="" vehicle="" 98.3±3.0="" rfu/min,=""><0.01;>
Olmesartan attenuated enhancements ntawm BP thiab urinary albumin excretion thiab txo qhov hnyav poob nyob rau hauv adenine-induced CKD qauv nas.Kev kho Olmesartan txo qhov hnyav hauv cov nas adenine (Fig. 5A). Kev kho Olmesartan kuj txo qis systolic BP hauv ob pawg tswj thiab adenine pawg (adenine nas 131.5±0.4 mmHg vs. tswj 121.2±0.7 mmHg, P<0.001; olmesartan="" mice="" 111.7±1.1 ="" mmhg="" vs.="" control="" 121.2±0.7 ="" mmhg,=""><0.001; adenine+olmesartan="" mice="" 117.2±0.9 ="" mmhg="" vs.="" adenine="" mice="" 131.5±0.4 mmhg,=""><0.001; fig. 5b).="" in="" adenine="" mice,="" olmesartan="" treatment="" improved="" plasma="" creatinine="" level="" (adenine="" mice="" 0.19±0.02="" vs.="" control="" 0.13±0.01,="" p="" <="" 0.01;="" adenine+olmesartan="" mice="" 0.14±0.01="" vs.="" adenine="" mice="" 0.19±0.02,="" p="" <="" 0.01;="" fig. 5c)="" and="" suppressed="" urinary="" albumin="" excretion="" (adenine="" mice="" 67.9="" ±6.7 μg/day="" vs.="" control="" 18.0±3.6 μg/day,="" p=""><0.001; adenine+olmesartan="" mice="" 52.5±2.3="" vs.="" olmesartan="" mice="" 17.4±3.7 μg/day,=""><0.001; adenine-olmesartan="" mice="" 52.5±2.3 μg/day="" vs.="" adenine="" mice="" 67.9±6.7 μg/day,=""><0.05;>
Olmesartan tsis cuam tshuam rau ACE2 kev qhia.Hauv kev tswj hwm thiab adenine pawg, tsis yog mRNA lossis cov protein qhia qib ntawm ACE2 hauv lub raum tau hloov pauv los ntawm kev kho olmesartan (Fig. 6A, C). Tsis muaj kev hloov pauv hauv pulmonary ACE2 mRNA lossis protein qhia theem ntawm cov tswj thiab adenine pawg (Fig. 6B, D). Kuj tsis muaj qhov sib txawv ntawm cov ntshav ACE2 kev ua haujlwm (Fig. 6E). ACE2 mRNA qhia nyob rau hauv lub ntsws sab sauv (pharynx) kuj tau soj ntsuam, tab sis tsis muaj qhov sib txawv tseem ceeb ntawm cov pab pawg (Cov duab ntxiv S1).
Kev sib thamTe COVID-19 tus kab mob kis thoob ntiaj teb tau muaj kev cuam tshuam loj heev. Rau cov neeg laus thiab cov neeg uas muaj cov kab mob sib kis, cov ntaub ntawv hais txog kev pheej hmoo kis tus kab mob thiab qhov hnyav ntawm COVID-19 yog qhov tseem ceeb heev. Muaj ntau qhov kev tshawb fawb txog kev kis tus kab mob COVID-19 thoob ntiaj teb, uas tau pom tias cov neeg mob CKD feem ntau yuav kis tus kab mob COVID-1917 tab sis qhov mob ntawm COVID-19 yuav tsis nce siab hauv cov neeg mob. nrog CKD15–21. Qhov tseem ceeb, cov txiaj ntsig ntawm ntau qhov kev tshuaj ntsuam xyuas meta tau qhia tias qhov tshwm sim ntawm CKD yog 1.7–5.2 feem pua hauv cov neeg mob uas muaj COVID-1915–17,21. Ntxiv rau, kev tshuaj xyuas ntawm 5,700 tus neeg mob nrog COVID-19 hauv New
York-cheeb tsam tau pom tias 8.5 feem pua muaj cov hauv pausmob raum 19. Muab hais tias qhov kwv yees muaj ntau ntawm CKD yog 9.1 feem pua ntawm thoob ntiaj teb18 thiab kwv yees li 15 feem pua hauv Tebchaws Meskas20, qhov kev pheej hmoo ntawm tus kab mob SARS-CoV-2 yuav tsis nce ntxiv rau qhov muaj CKD. ACE2 ua lub luag haujlwm tseem ceeb hauv kev sib kis ntawm COVID-191,2. Tsis tas li ntawd, theem ntawm ACE2 qhia hauv cov hlab ntsws muaj feem cuam tshuam nrog kev pheej hmoo ntawm kev kis mob los ntawm SARS-CoV, zoo ib yam li tus kabmob coronavirus rau tus neeg sawv cev ntawm COVID-1922. Vim COVID-19 yog kab mob ua pa, ACE2 qhia hauv lub ntsws yog ib feem tseem ceeb ntawm kev kis kab mob. Hauv txoj kev tshawb fawb tam sim no, peb tau xav tias qhov tsis muaj qhov nce hauv pulmonary ACE2 qhia hauv CKD yog txuas nrog qhov tsis muaj qhov nce siab ntawm COVID{17}} morbidity. Qhov kev xav no tau txais kev txhawb nqa los ntawm qhov tseeb tias cov menyuam yaus uas tsis tshua muaj ACE2 kev qhia tsis tshua muaj kev cuam tshuam rau COVID{19}} ntau dua li cov laus. Peb siv ob hom CKD qauv nas hauv peb qhov kev sim los tshuaj xyuas cov kev hloov pauv hauv pulmonary ACE2 qhia txog kev sib txuas nrog CKD pathogenesis. Adenine-induced CKD yog tus qauv tsiaj sawv cev ntawm tus kab mob32. Kev tswj hwm adenine ntau dhau tuaj yeem ua rau CKD los ntawm qhov ua raulub raumtubular obstruction thiab degeneration, thiablub rauminterstitial fibrosis, vim qhov tso tawm ntawm 2-8 dihydroxyadenine32,33. Hauv qhov sib piv, AA-induced CKD yog tshwm sim los ntawm inducing fibrosis los ntawm AA-induced tubular puas tsuaj34. Ob tus qauv no tau raug xaiv vim tias BP tau pom tias nce hauv adenine-induced CKD model35, thaum nws tsis nce hauv AA-induced CKD model36. Qhov tseem ceeb, ntshav siab yog ib qho teeb meem tshwm sim hauv cov neeg mob CKD; peb xav tias nws yuav muaj txiaj ntsig los tshawb xyuas seb qhov muaj lossis tsis muaj kev kub ntxhov cuam tshuam rau pulmonary ACE2 qhia hauv CKD pathogenesis. Cov txiaj ntsig tau pom tias covraum ACE2Cov qib protein tau txo qis hauv ob qho tib si CKD qauv. Hauv cov nas adenine, tsis muaj qhov sib txawv ntawm mRNA qib ntawm 4 lub lis piam, tab sis muaj qhov txo qis hauv cov qib protein. Ib qho ntawm cov laj thawj rau qhov tsis sib xws no yuav yog kev koom tes ntawm kev tawm tsam kev txhais lus. Lambert DW, et al. qhia tau tias miR-421 txwv tsis pub txhais ntawm ACE2 protein tsis cuam tshuam rau ACE2 cov ntawv sau tseg qib 37. Lwm txoj kev tshawb fawb kuj tau pom tias cov ntshav miR-421 tau nce hauv CKD lossis cov neeg mob ntshav siab 38,39. Yog li, hauv pawg nas adenine nrog lub sijhawm ntev ntawm CKD thiab kub siab (4 lub lis piam), miR-421
Tej zaum yuav nce thiab txo qis kev txhais cov protein, ua rau muaj qhov sib txawv ntawm mRNA thiab cov protein qhia. Pulmonary ACE2 protein ntau tsis txawv hauv ob qho tib si CKD qauv piv rau cov tswj thiab tsheb. Txawm hais tias nws yog qhov xav tsis thoob tias qhov kev qhia pulmonary ACE2 mRNA raug txo qis hauv AA-induced CKD qauv, peb qhov kev tshawb pom tseem ceeb dua vim peb pom tsis muaj qhov sib txawv ntawm pulmonary ACE2 protein ntau, uas ncaj qha cuam tshuam rau SARS-CoV-2 kab mob. Tsis tas li ntawd, plasma ACE2 kev ua haujlwm tsis txawv ntawm ob qho tib si CKD qauv piv rau cov tswj thiab tsheb. Soluble ACE2 hauv plasma yog suav tias yog cleaved thiab tso tawm los ntawm cov ntaub so ntswg. Kev nce ntshav ntshav ACE2 kev ua haujlwm tau raug tshaj tawm tias muaj feem cuam tshuam nrog kev nce hauv kev tso tawm ACE2 protein los ntawm cov ntaub so ntswg, qhia txog kev txo qis ntawm ACE2 protein hauv cov ntaub so ntswg 40. Yog li, cov txiaj ntsig ntawm kev tshawb fawb tam sim no uas cov ntshav ACE2 tsis tau hloov pauv hauv CKD lub xeev qhia tau hais tias kev tso tawm ACE2 cov protein qhia hauv cov ntaub so ntswg tsis hloov pauv hauv CKD lub xeev, uas txhawb kev tshawb pom tias ACE2 protein qhia hauv cov ntaub so ntswg tsis hloov pauv. lub ntsiab lus ntawm CKD. Kev sib raug zoo ntawm COVID-19 thiab RAS blockers yog qhov tseem ceeb ntawm kev tshawb fawb kev sib tham. Cov kev tshawb fawb tsiaj yav dhau los tau qhia tias RAS blockers upregulate cov ntaub so ntswg ACE2 qhia, uas ua rau muaj kev ntshai tias kev siv RAS blockers tuaj yeem ua rau muaj kev pheej hmoo ntawm SARS-CoV{18}} kab mob. Txawm li cas los xij, kev siv RAS blockers hauv cov kev tshawb fawb ua ntej tsuas yog ua rau kev txhim kho ntawm cov ntaub so ntswg ACE2 raws li cov kab mob tshwj xeeb 24,27; qhov kev txhim kho no tsis tau tshaj qhov qub. Lwm txoj kev tshawb nrhiav pulmonary thiabraum ACE2kev qhia nyob rau hauv cov nas ib txwm kho nrog RAS blockers pom tsis muaj kev nce ntxiv, piv nrog pawg tswj hwm 31. Ntawm qhov tod tes, muaj tsab ntawv ceeb toom tias RAS blockers xws li candesartan thiab captopril nce pulmonary ACE2 qhia hauv cov nas ib txwm muaj 41. Hauv kev xyaum kho mob hauv ntiaj teb tiag tiag, qee qhov kev tshawb fawb txog kev kis mob ntsig txog kev siv RAS blockers hauv cov neeg mob nrog COVID-19 tau pom tias kev siv cov tshuaj no tsis ua rau muaj kev pheej hmoo ntawm COVID-1928-30. Txawm li cas los xij, cov nyhuv ntawm RAS blockers ntawm pulmonary ACE2 qhia tseem muaj teeb meem. Tsis muaj kev tshawb fawb yav dhau los tau tshuaj xyuas qhov cuam tshuam ntawm RAS blockers ntawm pulmonary ACE2 qhia hauv cov ntsiab lus ntawm CKD, txawm hais tias RAS blockers yog qhov tseem ceeb rau kev kho mob ntawm cov neeg mob CKD. Kev tshawb fawb tam sim no tau ua los daws qhov sib txawv ntawm cov ntaub ntawv. Txhawm rau ua qauv zoo tshaj plaws hauv ntiaj teb kev kho mob, peb tau siv tus qauv adenine-induced CKD, uas ua rau muaj kev kub ntxhov. Cov txiaj ntsig tau pom tias kev tswj hwm ntawm olmesartan, RAS blocker, tsis txhim kho pulmonary ACE2 qhia, piv nrog kev tswj hwm. Tsis tas li ntawd, ACE2 mRNA qhia nyob rau hauv lub ntsws sab sauv, lub hom phiaj thawj zaug ntawm SARS-CoV{14}} kev kis tus kab mob, tsis tau txhim kho hauv kev teb rau kev kho olmesartan.Lub raum ACE2cov protein ntau tau txo qis hauv CKD qauv; kev tswj hwm ntawm olmesartan tsis tau nce lub raum ACE2 protein ntau hauv kev tswj hwm thiab adenine nas. Qhov tsis muaj kev kho dua tshiab hauv lub raum ACE2 hauv olmesartan-kho adenine nas tuaj yeem yog vim kev siv cov qauv CKD sib txawv, piv nrog cov kev tshawb fawb yav dhau los27, nrog rau lub sijhawm luv luv ntawm angiotensin receptor blocker kho25,27. Txawm li cas los xij, kev kho olmesartan tau txhim kho ntau lub raum tsis ua haujlwm, suav nrog kev tso zis albumin. Cov txiaj ntsig no txhawb nqa txuas ntxiv siv RAS blockers hauv cov neeg mob nrog COVID-19 uas muaj CKD.
CISTANCHE yuav txhim kho lub raum / raum kab mob
Txoj kev tshawb no muaj qee qhov kev txwv. Ua ntej, nws tsuas yog tshuaj xyuas cov kev hloov pauv hauv pulmonary ACE2 qhia hauv CKD qauv nas, qhov uas nws tsis ncaj qha tshawb xyuas SARS-CoV-2 kab mob. Qhov thib ob, peb tau soj ntsuam cov nas adenine rau 4 lub lis piam thiab AA nas rau 8 lub lis piam hauv txoj kev tshawb no, tab sis cov txiaj ntsig yuav txawv nyob rau lub sijhawm ntev ntawm kev soj ntsuam. Qhov thib peb, ACE2 qhia hauv lub ntsws yuav txawv ntawm cov nas thiab tib neeg. Plaub, hauv cov neeg mob CKD, COVID-19 qhov hnyav yuav zoo dua, tab sis peb tsis tuaj yeem xa mus rau lub hauv paus txheej txheem ntawm qhov hnyav. Thib tsib, nws tsis yog tsuas yog qhia theem ntawm pulmonary ACE2 uas txiav txim siab qhov cuam tshuam rau COVID-19 tab sis nws kuj yuav cuam tshuam los ntawm ntau lwm yam. Hauv kev xaus, peb pom tsis muaj kev hloov pauv hauv pulmonary ACE2 protein qhia hauv CKD qauv nas, tsis hais txog kev kub ntxhov. Tsis tas li ntawd, kev siv cov tshuaj angiotensin receptor blocker kho hauv CKD qauv nas tsis tau nce pulmonary ACE2 qhia. Cov txiaj ntsig no qhia txog qhov kev xav tias qhov kev pheej hmoo ntawm COVID{11}} morbidity yuav tsis nce siab hauv cov neeg mob CKD vim tias lawv cov kab mob pulmonary ACE2 ruaj khov. Cov kev tshawb pom kuj tseem muab cov pov thawj tseem ceeb hauv kev tshawb fawb uas RAS blockers tuaj yeem siv tau nyab xeeb hauv kev kho cov neeg mob nrog COVID-19 uas muaj CKD.
Khoom siv thiab cov txheej txheem Tsiaj.Txoj kev tshawb no tau ua raws li National Institutes of Health cov lus qhia rau kev siv cov tsiaj sim. Txhua qhov kev sim tsiaj tau raug tshuaj xyuas thiab pom zoo los ntawm Pawg Saib Xyuas Tsiaj Studies ntawm Yokohama City University (Pom Zoo Naj Npawb: FA{{{{10}}}}), uas tau ua raws li ARRIVE cov lus qhia. Kev siv zog ua kom txo qis cov tsiaj siv thiab ua kom muaj kev txom nyem tsawg. Cov nas tau nyob hauv qhov chaw tswj hwm nrog 12-h lub voj voog tsaus ntuj ntawm qhov kub ntawm 25 degree . Cov nas tau tso cai nkag mus rau zaub mov thiab dej dawb. CKD qauv nas. Cov kev sim no tau ua tiav siv 8–9-cov txiv neej hnub nyoog ib lim tiam C57BL/6 J nas, tom qab 1-lub lim tiam acclimatization hauv txhua pab pawg. Hauv kev sim adenine, nas tau noj cov zaub mov sib xyaw nrog 0.2 feem pua adenine (0.2 feem pua adenine ntxiv rau 0.3 feem pua NaCl, 3.6 kcal/g, CE{{ 18}}; CLEA, Tokyo, Nyiv; pawg adenine) lossis cov khoom noj khoom haus (0.3 feem pua NaCl, 3.6 kcal/g, CE-2; CLEA; pawg tswj) rau 2 lossis 4 lub lis piam. Hauv kev sim AA, nas tau txhaj tshuaj intraperitoneally nrog AA (3 mg / kg) ob zaug hauv ib lub lis piam rau 4 lub lis piam, tom qab ntawd los ntawm 4-lub lim tiam rov qab los; pawg tsheb tau txhaj tshuaj nrog lub tsheb (75 feem pua dimethyl sulfoxide). Hauv kev sim tshuaj olmesartan, nas tau muab faib ua plaub pawg: (1) pawg tswj; (2) pab pawg adenine, noj zaub mov sib xyaw nrog 0.2 feem pua adenine; (3) pawg tswj-olmesartan, kho nrog AT1 receptor antagonist olmesartan hauv dej haus (4 mg / kg / hnub; Daiichi Sankyo Chemical Pharma Co., Ltd, Tokyo, Nyiv); thiab (4) adenine-olmesartan pawg, uas koob tshuaj zoo ib yam rau cov neeg hauv pawg 2 thiab 3.
Kev ntsuas BP.Systolic BP tau ntsuas los ntawm txoj kev tail-cuf (BP-Monitor MK-2000; Muromachi Kikai Co., Tokyo, Nyiv), raws li tau piav qhia yav dhau los 42,43. Txhua qhov kev ntsuas tau ua nyob nruab nrab ntawm 9:00 thiab 14:00 h. Tsawg kawg yog 10 qhov ntsuas tau ua nyob rau hauv txhua tus nas thiab tus nqi nruab nrab yog siv rau kev tshuaj xyuas.Real-time quantitative reverse transcription-polymerase chain reaction analysis.Tag nrho RNA tau muab rho tawm los ntawm lub ntsws,lub raum, thiab pharynx ntaub so ntswg siv ISOGEN (Nippon Gene, Tokyo, Nyiv); cDNA tau tsim los siv SuperScript III First-Strand System (Invitrogen, Carlsbad, CA, USA). Real-time quantitative reverse transcription-polymerase chain reaction analysis tau ua los ntawm ABI PRISM 7000 Sequence Detection System; rov qab cov khoom siv tau raug tsim nrog TaqMan PCR Master Mix thiab kev sojntsuam TaqMan (Applied Biosystems, Foster City, CA, USA), raws li tau piav qhia yav dhau los 44,45. TaqMan sojntsuam hauv qab no tau siv: ACE2 (Mn01159003_m1). mRNA qib tau normalized rau cov ntawm 18S rRNA.
Western blotting tsom xam.Protein qhia tau soj ntsuam los ntawm sab hnub poob blotting siv cov ntaub so ntswg homogenates, raws li tau piav ua ntej 45,46. Luv luv, tag nrho cov protein extract tau npaj los ntawm cov ntaub so ntswg nrog sodium dodecyl sulfate-muaj cov qauv tsis. Cov protein concentration ntawm txhua tus qauv raug ntsuas nrog NanoDrop Ib (Thermo Fisher Scientific), siv bovine serum albumin raws li tus qauv. Qhov sib npaug ntawm cov protein rho tawm los ntawm txhua cov ntaub so ntswg (cov ntaub so ntswg ntsws: 24 ug, cov ntaub so ntswg raum: 10 ug) tau muab faib rau ntawm 5-20 feem pua polyacrylamide gel (Atto, Tokyo, Nyiv). Te cais cov proteins tau raug xa mus rau polyvinylidene difluoride membrane siv iBlot Dry Blotting System (Invitrogen). Membranes raug thaiv rau 1 teev ntawm chav tsev kub nrog phosphate-buffered saline uas muaj 5 feem pua skim mis hmoov. Membranes tau incubated nrog thawj cov tshuaj tiv thaiv rau ACE2 (Ab108252 1: 500 [lub ntsws] lossis 1: 1000 [lub raum], Abcam, Cambridge, MA, USA) thiab -actin (A{{16}) }: 10,000, Sigma-Aldrich, St. Louis, MO, USA). Membranes raug ntxuav thiab tom qab ntawd incubated nrog lwm cov tshuaj tiv thaiv rau 60 min ntawm chav tsev kub. Te qhov chaw ntawm cov tshuaj tiv thaiv kab mob-antigen tau pom los ntawm kev txhim kho chemiluminescence substrate (Merck, Kenilworth, NJ, USA). Cov duab tau txheeb xyuas ntau npaum li cas siv ChemiDoc Touch (Bio-Rad, Hercules, CA, USA). Txhawm rau sib piv ACE2 protein qhia qib hauv lub ntsws thiablub raum cov ntaub so ntswgNyob rau hauv cov nas ib txwm, ntxiv western blotting tau ua rau tib daim nyias nyias (Ntxiv daim duab S2). Tus nqi ntawm cov protein tau piav qhia tau muab faib ua gels ((A) lub ntsws: 24/10/1 ug, raum: 24/10/1 ug, (B) ntsws: 24/12 ug , raum: 0.2/0.15/0.1 ug); Tib yam tshuaj tiv thaiv kab mob rau ACE2 (Ab108252 1:1000, Abcam, Cambridge, MA, USA) tau siv. Txhawm rau tshuaj xyuas qhov tshwj xeeb ntawm cov tshuaj tiv thaiv, ib qho ACE2- xaiv thaiv peptide (ab198988) tau siv. Western blotting pom ib pawg protein ntau, uas tau tshem tawm los ntawm ACE2- xaiv thaiv peptide (Cov duab ntxiv S3).
Immunohistochemical tsom xam.Lub ntsws thiablub raumCov ntaub so ntswg los ntawm nas tau kho nrog 4 feem pua ntawm paraformaldehyde thiab tom qab ntawd muab tso rau hauv paraffin. Plaub-micrometer-thick seem tau dewaxed thiab rehydrated; Antigen retrieval tau ua los ntawm microwave cua sov. Te ntu raug thaiv kom txo qis kev ua haujlwm biotin endogenous siv peroxidase thaiv cov tshuaj reagent (Dako, Carpinteria, CA, USA) thiab kho rau 60 min nrog 10 feem pua tshuaj tshis ib txwm nyob hauv phosphate-buffered saline. Tom qab ntawd cov seem tau muab tshuaj tua kab mob anti-ACE2 diluted rau 1:500 (Ab15348, Abcam). Txhawm rau tshuaj xyuas qhov tshwj xeeb ntawm cov tshuaj tiv thaiv kab mob, kev tiv thaiv kab mob kuj tau tshuaj xyuas los ntawm kev tshem tawm thawj Ab thiab siv ACE2- xaiv thaiv peptide (ab15352). ACE2 staining hauv ntsws thiablub raum cov ntaub so ntswgs tau pom, uas tsis tau pom thaum cov tshuaj tiv thaiv kab mob preabsorbed nrog ACE2- xaiv thaiv peptide (ab15352) lossis kev tso tseg ntawm ACE2 antibody. (Cov duab ntxiv S4).
CISTANCHE yuav txhim kho lub raum / raum mob
Biochemical tsom xam.Tom qab nqus tau 5 feem pua ntawm isoflurane anesthesia, cov ntshav tau sau los ntawm cov hlab plawv hauv lub xeev pub los ntawm lub plawv punctures. Cov tsiaj sim raug tua tib neeg tom qab tshuaj loog. Tag nrho cov ntshav kuaj tau centrifuged ntawm 3000 rpm (MR-150, Tomy Seiko Co., Ltd., Tokyo, Nyiv) ntawm 4 degree rau 10 min kom cais cov ntshav. Cov txiaj ntsig ntshav plasma tau khaws cia ntawm -80 degree kom txog thaum siv. Plasma creatinine, BUN, urinary creatinine, thiab urinary albumin theem tau ntsuas siv Hitachi 7180 autoanalyzer (Hitachi, Tokyo, Nyiv).
Plasma ACE2 kev ua haujlwm.ACE2 kev ua haujlwm tau ntsuas los ntawm TechnoPro R&D Tuam Txhab (Tokyo, Nyiv) siv ACE2 Cov Khoom Siv Kev Ua Haujlwm (SensoLyte 390) ntawm qhov kub ntawm 27 degree . Cov qauv tau diluted 1: 10 siv qhov ntsuas tsis tau muab nrog ACE2 Cov Khoom Siv Ntsuas Kev Ua Haujlwm. Te assay tau ua nyob rau hauv 384- zoo daim hlau. Cov qauv ntshav ntshav, kuaj tsis tsuas yog (tom qab), lossis 4-methyl-coumarin-7-amide (0.16–5 μM) raws li tus qauv siv tau ntxiv rau OptiPlate-384 F cov tshuaj tiv thaiv phaj ntawm 10 μL / zoo. Kaum, ACE2 substrate tov (0.05 mM 4-methylcoumaryl-7-amide/Dnp nyob rau hauv kev soj ntsuam buffer) tau ntxiv ntawm 10 μL/zoo. Tom qab cov tshuaj tiv thaiv, fluorescence intensity (Ex/ Em{22}} nm/390 nm) raug ntsuas ntawm 5-min intervals rau 3 h siv tus EnSpire phaj nyeem ntawv (PerkinElmer, Waltham, MA, USA). Qhov kev siv fluorescence ntawm cov qauv ntawm txhua lub sij hawm taw tes yog xam los ntawm fluorescence siv ntawm cov qauv. Qhov nqes hav ntawm txhua tus qauv (RFU / min) tau txiav txim siab los ntawm ib qho kev kwv yees ntawm cov phiaj xwm hauv lub sijhawm ntawm 30 txog 90 min tom qab pib ntsuas.
Kev txheeb cais.Cov ntaub ntawv raug nthuav tawm raws li qhov ntsuas ± tus qauv yuam kev ntawm qhov ntsuas. Qhov sib txawv tau txheeb xyuas raws li hauv qab no. Ob txoj kev tsom xam ntawm qhov sib txawv, ua raws li Bonferroni post hoc tsom xam, tau ua los txiav txim siab qhov sib txawv ntawm lub sijhawm ntawm adenine thiab tswj pawg (Daim duab 1, 2), AA thiab pawg tsheb (Fig. 3A, B), lossis adenine thiab olmesartan pab pawg (Fig. 5, 6 thiab S1). Unpaired t-tests tau siv los txiav txim qhov sib txawv ntawm AA thiab lub tsheb nas (Figs. 3C–F, 4). P tus nqi<0.05 were="" considered="" statistically="">
