JAK Inhibitor Blocks COVID-19-cytokine-induced JAK-STAT-APOL1 Qhia hauv Glomerular Cells Thiab Podocytopathy hauv tib neeg lub raum Organoids

Abstract

COVID-19 ua rau muaj kab mobkev puas tsuaj ntawm glomerular capillariesthiabpoob ntawm podocytes, xaus hauv amob raum mob hnyavhu uaCOVID-19-sociated nephropathy(COV). Lub hauv paus txheej txheem ntawm COVID-19 tsis paub. Peb xav tias cytokines induced los ntawm COVID-19 ua rau kev nthuav tawm ntawm cov kab mob APOL1 los ntawm JAK-STAT signaling, ua rau podocyte poob thiab COVAN phenotype. Ntawm no, raws li cuaj qhov kev kuaj pom tseeb ntawm COVAN, peb tau pom thawj zaug uas APOL1 protein ntau nthuav tawm hauv podocytes thiabglomerular endothelial kab mobcells (GECs) ntawm COVAN raum tab sis tsis nyob hauv kev tswj. Ntxiv mus, feem ntau (77.8%) ntawm COVAN cov neeg mob nqa obAPOL 1phom alleles. Peb tau pom tias cov cytokines recombinant induced los ntawm SARS-CoV-2 ua synergistically tsavAPOL 1qhia los ntawm JAK-STAT txoj hauv kev hauv thawj tib neeg podocytes, GECs, thiablub raum microorganoidstau txais los ntawm tus neeg nqa khoom ntawm obAPOL 1pheej hmoo alleles tab sis raug thaiv los ntawm JAK1/2-inhibitor, baricitinib. Peb tau ua qauv qhia thawj zaug uas cytokine-induced JAK-STAT-APOL1 signaling txo qhov kev muaj peev xwm ntawm lub raum organoid podocytes tab sis tau cawm los ntawm baricitinib. Ua ke, peb cov txiaj ntsig txhawb qhov kev txiav txim siab ntawdCOVID-19-induced cytokinestxaus los tsav COVAN-koom nrog podocytopathy ntawm JAK-STAT-APOL1 signaling thiab tias JAK-inhibitor tuaj yeem thaiv cov txheej txheem pathogenic no. Cov kev tshawb pom no qhia tias JAK-inhibitors tuaj yeem muaj txiaj ntsig kho mob rau kev tswj hwm cytokine-induced APOL1-kev kho mob podocytopathy.

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Lub raum tsis ua hauj lwm yog ib qho teeb meem loj heev ntawmTus kab mob COVID-19. Txog li 50% ntawm cov neeg mob hauv tsev kho mob thiab 70% ntawm chav saib xyuas mob hnyav COVID-19 kev nkag mus yog qhov nyuaj los ntawm kev raug mob raum (AKI), uas ua rau muaj kev tuag ntau ntxiv los ntawm 30-50% (1, 2). Cov kab mob raum biopsy tau tshaj tawm tias kev sib tsoo glomerulopathy yog qhov kev kuaj mob histopathologic feem ntau hauv COVID-19- koom nrog AKI (3). Ib qho tshwj xeeb ntawm COVID-19- cuam tshuam nrog kev sib tsoo glomerulopathy (cov ntawv luv COVAN) yog nws qhov ze-tshwj xeeb rau cov neeg Asmeskas Dub lossis Dub uas muaj ob qho kev pheej hmoo ntawm Apolipoprotein L1 (APOL1) (3, 4). Ob qhov kev pheej hmoo alleles (lub npe G1 thiab G2) tau tshwm sim los ntawm coding variants nyob rau hauv APOL1 noob thiab conferencing tiv thaiv African trypanosomiasis. Txawm li cas los xij, kev thauj khoom ntawm G1G1, G2G2, lossis G1G2 (lub npe hu ua genotypes uas muaj kev pheej hmoo siab) ua rau muaj kev pheej hmoo ntawm cov kab mob raum thiab piav qhia ntau qhov kev pheej hmoo ntau dhau ntawm cov kab mob raum tsis zoo ntawm cov neeg Asmeskas Asmeskas (5-8). Kwv yees li ntawm 13% ntawm cov neeg Asmeskas cov neeg Asmeskas muaj kev pheej hmoo siab APOL1 genotypes (7). Thaum muaj tus kab mob COVID{30}} muaj thoob qhov txhia chaw, cov kev tshawb fawb pom tau hais tias 92% ntawm cov neeg mob qog nqaij hlav-cov pov thawj COVAN yog cov neeg uas muaj APOL1 genotypes uas muaj kev pheej hmoo siab, 61% ntawm cov neeg yuav tsum tau lim ntshav ntawm kev nthuav qhia (3, 9). Cov kev tshawb pom no tsim cov APOL1 qhov sib txawv raws li qhov tseem ceeb rau kev sib cais ntawm haiv neeg hauv COVID-19 cov txiaj ntsig kev noj qab haus huv. Txawm hais tias qhov kev koom tes zoo siab no, cov txheej txheem ntawm tes uas txuas cov APOL1 cov genotypes uas muaj kev pheej hmoo siab rau SARS CoV-2 kab mob thiab cov kab mob ntawm kev sib tsoo glomerupathies ntawm COVAN tseem tsis tau paub.

Lub koom haum muaj kev sib kis muaj zog ntawm cov neeg muaj kev pheej hmoo siab APOL1 genotype thiab COVAN tau coj mus rau qhov kev xav tias COVID-19-induced qhia ntawm APOL1 G1 los yog G2 nyob rau hauv podocytes thiab glomerular endothelial hlwb-lub raum hlwb cuam tshuam nyob rau hauv collapsing glomerulopathy-tsav pathogenesis ntawm COV. Qhov kev xav no tau txais kev txhawb nqa los ntawm cov ntaub ntawv tsis ntev los no hais tias transgenic overexpression ntawm APOL1 muaj kev pheej hmoo alleles nyob rau hauv nas podocytes los yog glomerular endothelial hlwb ua rau podocytopathy, endotheliopathy, glomerulopathy, thiab kev kho mob tshwm sim ntawm lub raum tsis ua hauj lwm (10-14). Cov qauv kab mob murine no qhia tias cov txheej txheem uas cuam tshuam txog COVID-19- ntxias APOL1 kev qhia yuav yog lub hom phiaj kho mob rau COVID-19. Txawm li cas los xij, muaj ob qhov tseem ceeb tsis paub. Ib qho, nws tsis paub tias APOL1 cov lus qhia protein tau raug tswj hwm hauv glomeruli ntawm cov neeg mob COVAN. Ob, nws tsis paub tias SARS-CoV-2 induces APOL1 qhia ncaj qha los ntawm tus kab mob kis kab mob ntawm lub raum los yog indirectly los ntawm cov teebmeem ntawm SARS-CoV-2-induced cytokine cua daj cua dub.

The fail to detect SARS-CoV-2 inraum biopsies of COVANCov neeg mob muab kev txhawb nqa tsis ncaj rau qhov kev xav tias COVAN yuav tshwm sim los ntawm cov teebmeem ntawm cytokine cua daj cua dub es tsis yog los ntawm kev kis kab mob ncaj qha ntawm lub raum parenchyma. Kev tshawb pom qee lub sijhawm ntawm SARS-CoV-2 cov kab mob kis tau nyob hauv cov kab mob hauv lub raum hauv lub cev uas qhov cuam tshuam ntawm cov ntaub so ntswg autolysis tsis tuaj yeem raug cais tawm (3, 4, 15, 16). Ob peb inflammatory cytokines thiab chemokines tau raug sau tseg kom tau upregulated nyob rau hauv lubSera ntawm cov neeg mob nrog COVID-19and/or COVAN (4, 17, 18). Cov npe no suav nrog cytokines xws li interferons alpha, beta, gamma, thiab TNF uas yav dhau los paub txog kev txhawb nqa APOL1 kev qhia. Txawm li cas los xij, daim ntawv teev npe kuj suav nrog ntau cov kab mob cytokines uas tau tswj hwm los ntawm COVID-19 tus kab mob. Nws tsis paub meej tias cov cytokines no muaj cov tshuaj ntxiv, kev sib koom ua ke, lossis kev tawm tsam ntawm APOL1 qhia thiab cuam tshuam nrog podocytopathy.

Hauv txoj kev tshawb fawb tam sim no, peb tau hais txog cov kev paub dhau los no los ntawm kev siv lub raum biopsies tau los ntawm cuaj tus neeg mob nrog COVAN thiab ob lub raum tswj tib neeg los tshawb xyuas seb thiab qhov twg APOL1 protein tau qhia hauv COVAN thiab tswj lub raum. Peb tau tshaj tawm kaum yim COVID-19-induced cytokines los txheeb xyuas yim cytokines uas txaus, thaum tsis muaj SARS-CoV-2, los ua kom muaj zog APOL1 qhia hauv cov tib neeg glomerular hlwb thiab ua rau podocytopathy hauv tib neeg lub raum micro- organoids. Txoj kev tshawb no tsis tsuas yog muab thawj cov pov thawj hauv tib neeg los ntawm kev sim ua qauv uas COVID-19 cytokine cua daj cua dub induces APOL1 kev qhia thiab podocytopathy, nws kuj txheeb xyuas cov kev taw qhia kev sib haum xeeb uas cuam tshuam cov kab mob. Txoj kev tshawb no muaj qhov cuam tshuam uas tuaj yeem cuam tshuam cov tswv yim rau kev tshuaj xyuas thiab kho COVID-19 hauv cov neeg mob Dub thiab Hispanic. Nws tsa cov lus nug txog kev nyab xeeb ntawm cov tshuaj interferons ntxiv raws li COVID{10}} kev kho mob hauv cov neeg Dub thiab Neeg Mev uas muaj kev pheej hmoo siab APOL1 genotypes (19, 20).

Cov txiaj ntsig

APOL1 qhia yog upregulated nyob rau hauv podocytes thiab glomerular endothelial hlwb ntawm COVAN cov neeg mob. Txhawm rau tshawb xyuas seb cov neeg mob uas muaj kev kuaj mob biopsy-pov thawj ntawm COVAN tau nce qhov kev qhia ntawm APOL1 protein ntau hauv lawv cov podocytes thiab glomerular endothelial cells (GECs), peb tau ua cov tshuaj tiv thaiv kab mob ntawm APOL1, synaptopodin (ib qho actin-associated protein ntawm txawv podocytes), thiab CD31 (ib qho endothelial cell marker) ntawm lub raum biopsies ntawm ob tus neeg mob nrog KEVAN kuaj mob (Daim duab 1). APOL1 kev nthuav qhia muaj ntau nyob rau hauv glomeruli ntawm ob qhov xwm txheej 1, uas tau biopsied kaum lub hlis tom qab COVID-19 kuaj mob (Daim duab 1A-F), thiab rooj plaub 6, uas tau biopsied cuaj hnub tom qab COVID-19 kuaj mob (Daim duab 1 G-L). Hauv ob tus neeg mob, muaj zog APOL1 staining nyob rau hauv synaptopodin-positive podocytes ( xub; Daim duab 1B, E, H, thiab K) thiab nrog CD31-zoo glomerular endothelium ( xub xub taub; Daim duab 1C, F, I, thiab L. ). Lub xub ntiag ntawm APOL1 protein nyob rau hauv podocytes thiab GECs ntawm lub sij hawm 9 hnub thiab 10 lub hlis tom qab kuaj pom tus kab mob COVID-19 qhia tau hais tias APOL1 qhia tau ua rau ntxov ntxov thiab tej zaum yuav nyob rau hauv lub glomeruli rau ob peb lub hlis, ntev tom qab ua rau COVID{{ 27}} kab mob tau daws lawm.

APOL1 kev qhia yog upregulated nyob rau hauv biopsy cov ntaub so ntswg ntawm COVAN mob tab sis tsis nyob rau hauv kev tswj. Txhawm rau ntsuas qhov dav dav ntawm cov kev tshawb pom immunohistochemical no, peb tau txheeb xyuas tag nrho ntawm cuaj tus neeg mob COVAN uas muaj cov ntaub so ntswg biopsy rau genotyping thiab IHC (Daim duab 2) nrog rau ob tus neeg mob tswj, suav nrog ib qho kev tswj xyuas lub cev ntawm tus neeg mob uas muaj COVID{{2 }} kab mob tab sis tsis tsim AKI (Daim duab 2B thiab 2C). Cov yam ntxwv histopathologic classic ntawm COVAN suav nrog glomerular capillary tuft vau nrog cov nyob ib sab podocyte hypertrophy thiab proliferation, feem ntau nrog podocyte protein reabsorption droplets txuam nrog glomerular proteinuria (Supple Fig1 thiab Supple Fig2). APOL1 IHC staining tsis muaj nyob rau hauv tag nrho cov glomeruli ntawm kev tswj (Daim duab AC) tab sis tam sim no nyob rau hauv lub glomeruli ntawm tag nrho cov cuaj tus neeg mob COVAN (Daim duab 2D-X). APOL1 staining muaj ntau nyob rau hauv cytoplasm ntawm podocytes, GECs, thiab nyob rau hauv ib co parietal epithelial hlwb (Daim duab 2N, hashed vajvoog) (Daim duab 1, daim duab 2 thiab Suppl Fig3). APOL1 protein tuaj yeem pom hauv glomeruli nrog qhib capillaries nrog rau hauv thaj chaw ntawm glomerular vau. Qhov pom tseeb ntawm APOL1 protein nyob rau hauv qee qhov capillary lumen yuav sawv cev ncig APOL1, uas yog tsim los ntawm daim siab (21). Ntxiv mus, APOL1 kuj tau pom nyob rau hauv peritubular capillaries thiab nyob rau hauv raug mob tubular epithelial hlwb (asterisks). Qhov tshwj xeeb thiab qhov tseem ceeb ntawm qhov kev tshawb nrhiav tom kawg no tsis meej. Qhov tseem ceeb, xya ntawm cuaj tus neeg mob COVAN tau muaj kev pheej hmoo siab APOL1 genotype (Daim duab 2D-V). Lwm ob kis, rooj plaub 9 thiab rooj plaub 4, nqa G{28}}G0 genotypes (Daim duab 2W thiab 2X). Txawm hais tias muaj kev pheej hmoo tsawg, cov ntaub ntawv 9 APOL1 qhia tau piv rau cov ntawm xya tus neeg muaj kev pheej hmoo siab (Daim duab 2W). Tsuas muaj ob lub glomeruli nyob rau hauv rooj plaub 4 raum biopsy swb, thiab APOL1 qhia tau qis dua hauv cov glomeruli (Daim duab 2X). Ua ke, cov kev tshawb pom no qhia tau hais tias lub raum raum APOL1 qhia tau qis hauv glomeruli ntawm cov tib neeg uas tsis muaj glomerular raug mob, txawm tias tus neeg muaj tus kab mob COVID-19; whereas APOL1 kev nthuav qhia dhau los ua kev tswj hwm hauv podocytes thiab GECs hauv kev teeb tsa COVAN hauv 89% ntawm peb kis.

Raws li pom nyob rau hauv Table 1, xya ntawm cuaj tus neeg mob (77.8%) nrog biopsy-proven COVAN tus kheej-tshaj tawm tias yog neeg Asmeskas Asmeskas. Rau ntawm xya tus neeg mob no (85.7%) tau muaj kev pheej hmoo siab APOL1 genotypes (plaub G1G1, ib G1G2, ib G2G2). Los ntawm kev sib piv, 13% ntawm African Asmeskas muaj kev pheej hmoo siab APOL1 genotype. Ob tus neeg mob uas tau txheeb xyuas tus kheej tias yog Neeg Mev Dawb, tab sis qhov tseem ceeb, ib tus ntawm lawv kuj muaj qhov pheej hmoo siab APOL1 genotype. Nyob rau hauv tag nrho, xya ntawm cuaj tus neeg mob COVAN (77.8%) tau muaj kev pheej hmoo siab APOL1 genotypes. Lub hnub nyoog nruab nrab ntawm cov neeg mob yog 51 xyoo (nqi 37-60). Tag nrho cuaj tus neeg mob tau tsim mob raum mob thiab muaj qhov sib txawv ntawm cov proteinuria, xws li subnephrotic mus rau nephrotic ntau yam (1.4- 14 g/24hrs). Feem ntau ntawm kev kuaj kab mob tau ua yam tsawg kawg 1 lub hlis tom qab tus kab mob COVID-19, tshwj tsis yog cov ntaub ntawv 6 uas tau kuaj pom 9 hnub tom qab kuaj PCR zoo. Ib tus neeg mob lub cev nqaij daim tawv tsis tau mus txog 10 lub hlis tom qab kis tus kab mob thib ob kom rov ua tsis tiav. Tag nrho cuaj tus neeg mob biopsies tau nthuav tawm kev sib tsoo glomerupathies, kev raug mob ntawm tubular, thiab mob sib kis. Endothelial tubular reticular inclusions tsis tau pom nyob rau hauv ib qho ntawm cov xwm txheej. Tshwj xeeb tshaj yog, nyob rau hauv ob lub COVAN biopsies uas tau sim rau SARS-CoV-2 tus kab mob kis ncaj qha los ntawm IHC thiab nyob rau hauv situ hybridization, tsis tau kuaj pom tus kab mob no (cov ntaub ntawv tsis qhia).

Recombinant COVID-19-Induced cytokines synergistically upregulate APOL1 qhia nyob rau hauv thawj tib neeg glomerular endothelial hlwb thiab podocytes. Txhawm rau tshawb xyuas seb COVID-19-induced cytokine cua daj cua dub txaus los ua rau APOL1 qhia nyob rau hauv tib neeg glomerular hlwb, peb kab lis kev cai tib neeg podocytes cais los ntawm cov neeg tuag pub raum thiab thawj tib neeg glomerular endothelial hlwb (GECs) hauv 1 ntawm 18 cytokines thiab chemokines yav dhau los qhia tias tau nce siab hauv cov ntshav ntawm cov neeg mob SARS-CoV-2 (Figure 3A) (4, 17). Podocyte tus kheej tau lees paub nrog ntau cov cim podocyte suav nrog Wilms qog1, synaptopodin, nephrin, thiab podocalyxin (Daim duab 3B thiab Suppl Fig 4A-B). GEC tus kheej tau lees paub los ntawm kev qhia ntawm PECAM1 txheeb ze rautib neeg lub raum embryonic293 cells (HEK) (Figure 3C). Induced APOL1 expression was quantitated by qPCR and immunoblot after 48hr treatment in GECs (Figure 3D and 3F) and podocytes (Figure 3E and 3G). Consistent with prior report (22), we found that interferons (gamma > beta > alpha) robustly induced expression of APOL1 in both GECs and in podocytes. Similarly, we found that TNF also induced a modest APOL1 expression in GECs and podocytes. Unexpectedly, we found that three cytokines-IL-6, IL-1β, and IL-18, which were previously unrecognized as inducers of APOL1 expression, also individually induced modest APOL1 expression in GECs or podocytes. Notably, the combination of all 18 recombinant cytokines produced a synergistic upregulation of APOL1 that was an order of magnitude higher than that produced by any of the interferons alone (Figure 3D). These effects were not only observed with cytokine concentration of 50ng/mL (Figures 3D and 3E) but also at 20ng/mL and 10ng/mL (Supplemental Figure 5). Cytokine conditions inducing >1.5 quav APOL1 cov ntaub ntawv pov thawj piv rau kev tshaj tawm kev tswj hwm tau raug tshuaj xyuas ntxiv rau qhov tseem ceeb. Qhov tseem ceeb tau raug ntsuas los ntawm kev siv qhov tsis sib xws t-test nrog Holm-Sidak kho rau ntau qhov sib piv. Cov P-tus nqi tshaj tawm yog cov hloov kho p-tus nqi. Cov txiaj ntsig no nthuav dav cov npe ntawm lub cev cytokines uas muaj peev xwm ua rau APOL1 qhia tshaj qhov paub zoo txog interferons thiab TNF. Qhov tseem ceeb, qhov kev tshawb pom kuj qhia tias kev sib koom ua ke ntawm COVID-19-induced cytokines tej zaum yuav muaj feem cuam tshuam rau APOL1 txoj cai ntau dua li qhov cuam tshuam ntawm ib qho cytokine cais ib leeg.

JAK-STAT signaling mediatesCOVID-19-cytokine-induced APOL1 qhia. Peb txuas ntxiv mus tshawb xyuas seb COVID-19 ntxias cov cytokines upregulate APOL1 kev qhia los ntawm ib qho kev qhia hauv lub cev uas tuaj yeem siv los ua lub hom phiaj kho mob. Nws tau tshaj tawm yav dhau los tias interferon induction ntawm APOL1 yog kho los ntawm JAK-STAT1/2 (22, 23). Kev taw qhia los ntawm IL-6 receptor tau pom tias muaj kev sib kho los ntawm STAT3, thiab ob qho tib si IL-1 thiab TNF tau tshaj tawm tias tsis ncaj qha qhib STAT3 (24, 25). Ntawm kev txaus siab, Meliambro et al tsis ntev los no tau tshaj tawm txog kev tswj hwm ntawm phospho-STAT3 hauv cov ntaub so ntswg biopsy ntawm cov ntaub ntawv ntawm COVAN thiab HIV-koom nrog nephropathy (HIVAN) piv rau kev tswj hwm (26). Raws li cov ntaub ntawv keeb kwm yav dhau los no, peb tau xav tias JAK1/2-STAT1/2/3 txoj hauv kev yog thawj tus neeg nruab nrab ntawm cov teebmeem ntawm COVID-19-induced cytokines hauv kev tsav tsheb APOL1. Txhawm rau ntsuas qhov kev xav no, peb tau txiav txim siab txog lub xeev ntawm cov kev taw qhia no los ntawm kev ntsuas cov phosphorylated STAT1, 2, thiab 3 hauv lysates ntawm GECs tom qab culturing lawv nyob rau hauv ib tug neeg los yog ua ke cytokines rau 48 teev (Daim duab 3F). Hom I interferons (IFN thiab IFN ) nce phosphorylation ntawm STAT1-3 thaum IFN upregulated phosphorylation ntawm STAT 1 thiab 3. IL-1 , TNF, thiab IL-6 nce phosphorylation nkaus xwb ntawm STAT3. Ua ke cytokines nce phosphorylation ntawm STAT1-3. Paub tias JAK1 thiab JAK2 yog thawj cov protein kinases uas phosphorylate STAT1-3, peb xav tias inhibition ntawm JAK1/2 yuav thaiv APOL1 kev qhia tawm los ntawm "tag nrho cov cytokines". Raws li qhov kev kwv yees no, peb pom tias JAK1/2- tshwj xeeb inhibitor, baricitinib, txo qis APOL1 mRNA thiab APOL1 qhia los ntawm tag nrho-cytokine-kho GECs thiab thawj podocytes (Daim duab 3D G). Ua ke, cov txiaj ntsig no qhia tau tias JAK-STAT teeb liab yog thawj txoj hauv kev uas kho COVID-19-cytokine-induced APOL1 qhia.

COVID-19-induced cytokines txaus los tsav APOL1 kev qhia hauv tib neeg iPSC-los ntawm lub raum micro-organoids ntawm txoj kev JAK-STAT. Tib neeg lub raum micro-organoid yog ib qho pov thawj platform rau kev ua qauv rau tib neeg lub raum kab mob thiab pab txhawb kev kho mob txhais lus. Peb nug seb cov txiaj ntsig peb tau txais los ntawm thawj tib neeg podocytes thiab GECs puas tuaj yeem ua tau dav dav thiab siv tau los ntawm tib neeg lub raum micro-organoid qauv. Yog li ntawd, peb tsim lub raum micro-organoids los ntawm induced pluripotent qia hlwb (iPSCs) ntawm ib tug neeg Asmeskas neeg Asmeskas cov cab kuj ntawm G1G2 APOL1 genotype los tshawb xyuas APOL1 kev cai, kev qhia, thiab cov txiaj ntsig hauv cov qauv no (Daim duab 4A). Peb cov kab mob raum microorganoids hauv IFN 10ng / mL los yog sib xyaw ntawm yim cytokines (IFN , IFN , IFN , IL-18, IL-8, IL-6, TNF, IL{18 }} ) txhua ntawm 10ng / mL txawm tias tsis muaj lossis muaj baricitinib, 10µM rau 24 teev. Cov yim cytokines no tau raug xaiv vim lawv txoj cai tswj hwm ntawm APOL1 qhia hauv kev sim ua ntej. Podocytes thiab tubular epithelial hlwb nyob rau hauv lub raum micro-organoids tau marker-confirmed (Daim duab 4B). Raws li cov ntaub ntawv (27), endothelial hlwb tsis muaj npe nyob rau hauv lub raum micro-organoids (cov ntaub ntawv tsis qhia). Peb pom tias basal APOL1 protein qhia tau qis hauv micro-organoids. IFN kev kho mob induced ntau heev APOL1 qhia, nrog lub siab tshaj plaws siv co-localized rau thaj chaw ntawm podocyte marker, podocalyxin. Lub cocktail ntawm cytokines induced ib tug outsized thiab robust APOL1 qhia thoob plaws hauv lub micro-organoid qauv thaum piv rau lwm yam kev kho mob. Cov pab pawg kho nrog IFN ntxiv rau baricitinib thiab tag nrho cov cytokines ntxiv rau baricitinib tsis pom muaj APOL1 qhia, zoo ib yam nrog ua tiav inhibition ntawm cytokine nyhuv. APOL1 qhia nyob rau hauv lub raum micro-organoid podocytes reminiscent ntawm uas pom nyob rau hauv podocytes ntawm COVAN cov neeg mob. Txawm li cas los xij, tsis zoo li COVAN ob lub raum uas tsis muaj qhov tseem ceeb APOL1 qhia tau pom nyob rau hauv cov kab mob hauv lub cev tsis zoo, lub raum micro-organoid E-Cadherin-zoo tubular epithelial hlwb qhia APOL1. Qhov sib txawv no tuaj yeem yog vim qhov sib txawv ntawm daim nyias nyias cytokine receptors lossis epigenetic yam cuam tshuam uas cuam tshuam cov protein qhia hauv cov tubules tsis paub qab hau ntawm lub raum micro-organoids. Hauv cov ntsiab lus, tib neeg iPSC-los ntawm lub raum micro-organoids kab lis kev cai nrog COVID-19-induced cytokines qhia muaj zog upregulation ntawm pathogenic G1G2 APOL1 protein thiab cov lus qhia tau thaiv los ntawm inhibition ntawm JAK-STAT signaling.

Cytokine-induced JAK-STAT-APOL1 signaling txo qhov kev muaj peev xwm ntawm lub raum micro-organoid podocytes uas tau cawm los ntawm JAK-inhibitor. Thaum kawg, peb tau nug yog tias G1G2 APOL1 qhia hauv cov kab mob hauv lub raum puas cuam tshuam rau kev muaj peev xwm ntawm podocyte. Peb kwv yees tias cytokine-induced variant APOL1 protein yuav ua rau podocyte poob-ib qho cim phenotype ntawm COVAN. Txhawm rau ntsuas qhov kev xav no, peb cais cov podocytes los ntawm lub raum micro-organoids tsim los ntawm iPSCs ntawm cov cab kuj ntawm G1G2. Cov podocytes tau coj mus rau hauv IFN (10ng / mL), los yog sib xyaw ntawm yim cytokines (10ng / mL txhua), ob qho tib si nyob rau hauv lub xub ntiag thiab tsis muaj baricitinib (10µM) rau 96 teev (Daim duab 5A thiab B). Cytokine kev kho mob robustly induced APOL1 qhia thiab cov lus qhia no tau thaiv los ntawm baricitinib, raws li peb cov kev sim ua ntej (Daim duab 5C). Concordantly, cytokine kev kho mob ua rau poob podocyte tseem ceeb raws li qhia los ntawm kev soj ntsuam muaj peev xwm thiab tag nrho cellular ATP (Daim duab 5D thiab 5E). Remarkably, baricitinib kiag li cawm cytokine-induced podocyte poob. Ua ke, cov txiaj ntsig no txhawb qhov kev txiav txim siab tias COVID-19-induced cytokines ua rau JAK-STAT-APOL1 signaling uas nyob rau hauv lem ua rau podocyte raug mob thiab poob. Kev tiv thaiv ntawm JAK-inhibition ntawm podocyte viability txhawb nqa qhov kev xav no.

Kev sib tham

Cov lus xaus tseem ceeb ntawm txoj kev tshawb fawb tam sim no yog tias ntau qhov KEVID-19-induced cytokines tshaj interferons ua synergistically ntawm JAK-STAT signaling los tsav pathogenic APOL1 qhia, ua rau podocyte raug mob thiab poob uas yog thaiv los ntawm JAK inhibition. Raws li cov ntaub ntawv xov xwm, peb pom thawj zaug tias APOL1 cov protein ntau tau nthuav tawm hauv podocytes thiab GECs ntawm cov neeg mob kuaj pom tias muaj tus kab mob COVID-19 tab sis tsis yog nyob rau hauv glomeruli ntawm kev noj qab haus huv tswj tsis yog ntawm COVID-19- zoo tab sis COVAN-negative control. Hauv peb qhov kev sim ua qauv, peb pom tau tias cov cytokines recombinant upregulated nyob rau hauv COVID-19 tus kab mob yog txaus los tsav lub zog APOL1 qhia, thiab poob nthav, uas muaj zog synergism uas ua los ntawm ib tug ua ke ntawm cytokines tau kho feem ntau los ntawm ib tug ntau intracellular signaling txoj kev. Sib sau ua ke, peb cov pov thawj kev sim tau txhawb nqa kev sib raug zoo ntawm cytokine-induced JAK-STAT-APOL1 signaling thiab hauv vivo COVAN glomerular phenotype thiab txhawb kev tshawb nrhiav ntxiv rau lub hom phiaj kho mob no.

Qhov ntau zaus ntawm kev pheej hmoo siab APOL1 genotype (77.8%) ntawm cov neeg mob COVAN uas peb tau tshaj tawm no cuam tshuam nrog kev tshuaj xyuas thoob ntiaj teb ntau lub chaw kuaj mob uas tau tshaj tawm tias muaj kev pheej hmoo siab APOL1 genotype hauv 91.7% ntawm cov neeg mob COVAN (3). Muab hais tias qhov zaus ntawm APOL1 genotype uas muaj kev pheej hmoo siab nyob rau hauv cov pej xeem neeg Asmeskas cov neeg Asmeskas yog 13% (28), pom qhov zaus ntawm 77-90% hauv COVAN yog qhov muaj txiaj ntsig thiab piv rau 60-70% zaus tshaj tawm hauv HIV-associated nephropathy (HIVAN) (28-31). Qhov muaj nyob ntawm qhov seem 20-30% ntawm COVAN (thiab HIVAN) cov neeg mob uas tsis muaj kev pheej hmoo siab APOL1 genotypes qhia txog qhov muaj peev xwm ntawm APOL{{20}}}kev ywj pheej pathomechanism lossis muaj peev xwm muaj nyob hauv qee qhov mob COVID-19-induced supraphysiologic expression ntawm G0 APOL1 kuj yuav ua rau podocytopathy. Kev txheeb xyuas qhov muaj peev xwm no yuav xav tau kev tshawb fawb ntxiv. Txawm li cas los xij, peb yav dhau los ua qauv qhia hauvtib neeg lub raum embryonic(HEK) cov hlwb nrog tetracycline-inducible APOL1 qhia qhov system uas cytotoxicity ntawm APOL1 yog ob qho tib si variant- thiab koob tshuaj-dependent (32, 33). Dose-dependent APOL1 cytotoxicity kuj tau tshaj tawm los ntawm lwm tus neeg soj ntsuam hauv cov kab ke ntawm tes zoo sib xws (34). Ntxiv mus, APOL1 transgenic nas qauv tsis tau tsuas yog validated lub causal txuas ntawm APOL1 txaus ntshai alleles thiab podocyte raug mob, tab sis tau pom tias cov degree ntawm podocytopathy correlated nrog APOL1 qhia theem (10, 12-14, 35) . Peb qhov kev tshawb pom tias yim tawm ntawm cuaj tus neeg mob COVAN pom tau tias muaj zog glomerular APOL1 qhia txog kev tswj hwm thiab cov pov thawj uas qhia tias endogenous APOL1 txaus ntshai alleles ua rau podocytopathy hauv tib neeg lub raum micro-organoids txhawb kev sib txuas ntawm APOL1 thiab podocytopathy. Hloov pauv, qhov tsis muaj APOL1 hauv lub glomeruli ntawm COVID-19 zoo tab sis AKI-negative G0G0 autopsy tswj qhia tias COVID-19 kis tsis tau APOL1 induction tsis yog tus tsav tsheb txaus ntawmCOVAN disease.