Tshooj 2: Cov Khoom Siv Lub Hom Phiaj OSBPL7 nce ABCA1- Nyob ntawm Cov Roj Cholesterol Efflux Preserving raum Function hauv Ob Hom Kab Mob Raum

Yog xav paub ntxiv. tiv taujtina.xiang@wecistanche.com

Kev sib tham

Txoj kev tshawb no piav qhia txog kev txheeb xyuas cov chav kawm ntawm cov tshuaj sib txuas los ntawm phenotypic tshuaj nrhiav pom (PDD) uas txhawb nqa ABCA1-nyob ntawm cov roj cholesterol efflux. Lub hom phiaj deconvolution qhia OSBPL7 los ua lub hom phiaj molecular. Hauv cov tsiaj qauv ntawm cov kab mob raum, cov tshuaj no txo ​​cov raum lipid tsub zuj zuj, tiv thaiv podocyte poob, normalized proteinuria, thiab txo lub raum ua haujlwm poob.

Cov kev kho mob tam sim no rau cov mob ntevmob raumtxwv rau ACEi, ARBs, thiab, tsis ntev los no, sodium-glucose cotransporter-type 2 inhibitors (SLGT2i). Txawm hais tias qhov ua tau zoo ntawm cov neeg ua haujlwm no hauv kev ncua sijhawm ntawm kev mob raum mob ntev, feem ntau hauv cov neeg mob uas muaj cov kab mob hauv lub raum proteinuric, qhov kev xav tau kev kho mob tsis zoo hauv CKD tseem muaj ntau 3,3940.Nyob rau hauv kev sim randomized ntawm 4300 tus neeg mob, SGLT2i tau npaj txhij txog pom tias yuav ua tau zoo txawm tias tsis muaj teeb meem. ntawm qhov muaj ntshav qab zib 4, tawm tswv yim tias cov txheej txheem ntawm kev tiv thaiv kev tiv thaiv lwm yam tsis yog kev txo qis hauv qabzib tau koom nrog. Qhov tseeb, ib txoj kev tshawb fawb tsis ntev los no tau pom tias SGLT2i tuaj yeem ua rau cov ntshav HDL nce ntxiv thiab tuaj yeem txo cov rog rog 42. Qhov kev tshwm sim tsis ntev los no ntawm SGLT2i qhia txog qhov txiaj ntsig ntawm kev tsom mus rau ntau yam kev tsav tsheb CKD los tsim cov kev kho mob zoo dua.

Peb, thiab lwm tus, tau piav qhia txog glomerular cholesterol nyob rau hauvkab mob raumtshwm sim los ntawm ob qho tib si metabolic thiab nonmetabolic keeb kwm. Glomerular lipid tsub zuj zuj, nrog rau kev txo qis ABCAl-mediated cholesterol efflux, tshwm sim hauv DKD7-11, FSGS12-14, thiab AS13. Cov ntaub ntawv no qhia tias kev ua kom rov qab ua haujlwm ABCAl tuaj yeem ua tau zoo hauv kev kho cov kab mob raum. Peb tsis ntev los no tau pom tias lub raum tsis txaus cov roj cholesterol nrog cyclodextrin, lossis caj ces ABCAl overexpression, tiv thaiv los ntawmlub raum tsis ua haujlwmhauv cov qauv ntawm DKD9, FSGS13,14, thiab ASl3. Peb kuj tau pom tias qhov tsis muaj peev xwm ntawm podocyte-specific ABCAl deficiency ua rau lub raum phenotype hauv DKD7. Kev tshawb fawb nrog LXR agonists, xws li T1317, kuj tau muab kev txhawb zog rau lub luag haujlwm tseem ceeb ntawm ABC Al, uas tseem yog lub hom phiaj kho kom zoo nkauj txawm tias muaj kev txwv ntawm cov neeg ua haujlwm no.

Peb tau tshawb nrhiav cov neeg sawv cev los ntawm kev tshawb nrhiav tshuaj phenotypic thiab kev ua kom zoo ntawm cov neeg sib tw me me molecule drugstore. Peb tau txheeb xyuas ntau yam ntawm 5-arylnicotinamides, ntau tus neeg sawv cev uas nce qib ABCAl protein ntau thiab cov roj cholesterol los ntawm cov roj cholesterol. Qhov tsis muaj efflux kev ua haujlwm hauv fibroblasts los ntawm tus neeg mob uas muaj tus kab mob Tangier, thiab qhov tsis muaj qhov nce hauv ABCAl mRNA, tau nthuav tawm cov txheej txheem tshiab ntawm kev ua.

Lub hom phiaj deconvolution kev siv zog txheeb xyuas OSBPL7 yog lub hom phiaj kev txaus siab siab. Kev sojntsuam molecular (³H-Cpd K) ua haujlwm tau zoo ntawm OSBPL7 hauv cov hlwb thiab tuaj yeem sib tw zoo nrog cov tshuaj tsis muaj npe. Qhov tseem ceeb tshaj, peb tau ua kom pom kev sib raug zoo "kev ua haujlwm-kev ua haujlwm" kev sib raug zoo rau cov txheej txheem loj dua ntawm lawv lub peev xwm los ua kom ABCAl cholesterol efflux thiab lawv lub peev xwm los sib tw rau kev khi ntawm kev sojntsuam rau OSBPL7. Kev txheeb xyuas kev hloov pauv, ua ke nrog kev ua qauv molecular thiab kev tshawb fawb docking, qhia ncaj qha kev cuam tshuam ntawm cov tebchaw nrog kev kwv yees OSBPL7 sterol binding hnab ris. Thaum kawg, cov kev tshawb fawb siRNA tau lees paub qhov kev koom tes ntawm OSBPL7 hauv ABC Al ua haujlwm.

Nyem kom paub cistanche qhov twg yuav thiab dab tsi cistanche siv rau  

Peb kuj tau txheeb xyuas CBIR raws li lub hom phiaj muaj peev xwm. Ntau cov ntawv tshaj tawm qhia txog lub luag haujlwm rau endocannabinoid signaling hauv cellular cholesterol tswj. Rimonabant tau pom tias txo qis kev tswj hwm cov khoom siv scavenger B1 (SR-B1) thiab ABCGl, tab sis tsis yog ABC Al, raws li peb cov ntaub ntawv45. Cov hluavtaws cannabinoid WIN55, 212-2 tau pom los txo qhov kev qhia ntawm ABC Al thaum nce scavenger receptor CD36 hauv oxLDL-loaded RAW264.7 macrophages46. Ua ke cov ntaub ntawv no tsis txhawb nqa lub luag haujlwm muaj zog rau CBIR hauv kev tswj hwm ntawm ABCA1.Hauv peb cov kev tshawb fawb, rimonabant thiab lwm yam CBIR agonists / antagonists suav nrog AM251, WIN55-212-2, thiab arachidonic choloroethylamine (ACEA), tsis muaj zog los ntxias ABCA1. cholesterol efflux (Fig. 3a, 4a).

Mammalian oxysterol binding proteins (OSBPs) yog Ameslikas cuam tshuam rau hauv biosynthesis ntawm cov roj cholesterol thiab sphingomyelin47-49. Ntau qhov kev tshawb fawb tau txuas cov OSBPs tshwj xeeb nrog kev tswj hwm ntawm ABC Al protein stability thiab ABCAl-mediated cholesterol efflux50,51. Tsis muaj ntaub ntawv ua ntej daim ntawv tshaj tawm tam sim no tau cuam tshuam OSBPL7 ua tus tswj hwm ntawm ABCA1. Peb cov ntaub ntawv qhia tias OSBPL7 tuaj yeem muab cov kev hloov pauv hloov pauv hloov pauv hloov pauv rau ABC Al. Kev sim ua ntej co-immunoprecipitation hauv cov hlwb tsis tau txheeb xyuas qhov sib cuam tshuam ncaj qha ntawm ob cov proteins. Txawm li cas los xij, ib qho kev soj ntsuam ntawm cov kev tshawb fawb genome-wide koom haum (GWAS) tau txheeb xyuas OSBPL7 ntawm 60 genetic loci cuam tshuam rau cov qib roj cholesterol hauv tib neeg.

Peb qhov kev txaus siab mus sij hawm ntev hauv lub luag haujlwm ntawm ABC Al hauv cov kab mob raum ua rau peb ntsuas cov 5-arylnicotinimides hauv vitro thiab hauv vivo qauv ntawm cov kab mob raum. Peb pom tias OSBPL7 tau qhia hauv ob lub raum, suav nrog hauv glomeruli thiab podocytes, thiab tias Cpd A thiab Cpd G upregulate apo Al-mediated cholesterol efflux los ntawm cov roj cholesterol-loaded podocytes. Cpd A thiab Cpd G txhawb kev nce ntawm ABCAl ntawm cov plasma membrane yam tsis muaj kev cuam tshuam rau mRNA kev qhia. Cov txheej txheem no sib piv nrog LXR agonists, uas txhawb nqa ABCAl mRNA ua rau muaj kev nce hauv ABC Al hauv cov ntshav thiab cov ntaub so ntswg. Peb tau soj ntsuam cov kev kho mob ntawm Cpd A thiab Cpd G hauv vivo hauv tus qauv nas ntawm FSGS (ADR-induced nephropathy). Ob tus neeg ua haujlwm tau pom tias txo qis cov roj cholesterol hauv lub raum, cuam tshuam nrog txo cov proteinuria, poob phaus, thiab lub raum pathological hloov. Cpd G tau txais txiaj ntsig zoo los tiv thaiv ADR-induced podocyte poob thiab glomerular kev hloov pauv. Qhov tseem ceeb txo qis ntawm ABCAl hauv ob lub raum, nrog rau kev nce hauvmobcov proteins, tau tiv thaiv los ntawm Cpd G, txhawb nqa lub luag haujlwm tseem ceeb rau ABCA1. Cpd G txo qis albuminuria, BUN, thiab ntshav creatinine qib hauv AS nas. Qhov tseem ceeb tshaj, Cpd G ua rau lub neej ntev nyob rau hauv cov laus Col4a3 cov tsiaj uas muaj lub raum tsis ua haujlwm.

Cov ntaub ntawv no txhawb nqa cov peev txheej kho mob ntawm cov tebchaw tsom rau ABCAl thiab xav qhia tias hom kev ua haujlwm tshiab no yuav muab ntxiv, lossis ua kom muaj txiaj ntsig zoo rau cov tshuaj tsom RAAS, SGLT2, NRF2, thiab cov roj cholesterol synthesis lossis nqus. Tshwj xeeb tshaj yog, nyob rau hauv sib piv rau cov nyhuv ntawm Cpd G, RAAS blockade tsis ntev lifespan nyob rau hauv cov laus Col4a3 nas, txawm yog ib tug zoo tshaj plaws kev kho mob rau kev cuam tshuam thaum ntxov nyob rau hauv cov qauv no53,54. Cov tshuaj tiv thaiv kev ua haujlwm ntawm Cpd G tau zoo dua rau cov teebmeem ntawm ramipril uas peb tau tshaj tawm yav dhau los hauv cov qauv sib xyaw ntawm AS38. Cpd G tej zaum yuav zoo dua lub hom phiaj rau cov pej xeem ntawm cov neeg mob AS thiab proteinuria thaum piv rau bardoxolone, tus NRF-2 agonist uas tau pom tias yuav nce GFR tab sis tsis muaj kev cuam tshuam rau proteinuria, qhov kev kwv yees muaj zog tshaj plaws ntawm 10- xyoo ESRD txaus ntshai nyob rau hauv cov pej xeem 55,56. Statins tau pom tias muaj kev tiv thaiv hauv kev sim AS57, thiab tau pom zoo rau txhua tus neeg mob CKD kom txo qis kev pheej hmoo ntawm cov hlab plawv, tab sis tau ua pov thawj tias tsis muaj txiaj ntsig ua rau qeeb kev loj hlob ntawm cov kab mob raum 8. Thaum kawg, peb tsis ntev los no tau tshaj tawm cov txiaj ntsig zoo ntawm ezetimibe hauv AS los ntawm kev txo qis. fatty acid thiab triglyceride absorption. Txawm li cas los xij, ezetimibe tsis cuam tshuam rau lub raum cov roj cholesterol cov ntsiab lus, thiab nws qhov ua tau zoo los txhim kho GFR thiab txo cov proteinuria tsis zoo rau cov teebmeem uas peb tau tshaj tawm ntawm no rau Cpd G.

Hauv cov ntsiab lus, daim ntawv tshaj tawm tam sim no piav qhia txog kev txheeb xyuas los ntawm kev tshawb pom cov tshuaj phenotypic ntawm thawj-hauv-chav tshuaj zoo li cov tshuaj me me uas ua rau ABCAl-mediated cholesterol efflux. Cov tshuaj lom neeg biology tau txheeb xyuas OSBPL7 raws li lub hom phiaj molecular thiab pom tau tias cov molecules sib cuam tshuam nyob rau hauv nws qhov kev kwv yees oxysterol binding domain. Rau peb txoj kev paub, qhov no tsuas yog ib qho ntawm ob peb yam piv txwv ntawm cov tshuaj zoo li cov tshuaj pib pom los ntawm phenotypic mus kom ze, uas nws lub hom phiaj tom qab tau txheeb xyuas tau zoo 44. Cov tshuaj G thiab A tau txais txiaj ntsig zoo los txhim kho lub raum ua haujlwm hauv cov qauv nas qhia txog ob hom kev sib txawv ntawm cov kab mob hauv lub raum. Lawv zoo li muaj kev nyab xeeb thiab ua tau zoo, raws li kev xav tau los ntawm lawv lub peev xwm los txo cov qib AST hauv ADR cov nas uas nyuaj thiab, ua kom muaj zog dua, ua kom lub neej ntev ntawm Col4A3 KO nas. Cov tebchaw no tuaj yeem muab cov kev kho tshiab rau lub raum kab mob los ntawm kev tsom mus rau cellular cholesterol metabolism los ntawm kev ua haujlwm tshiab. Cov neeg sawv cev no, nrog rau cov tshuaj tam sim no tsom rau ntshav siab lossis tswj ntshav qabzib, tuaj yeem hais txog qhov xav tau tseem ceeb hauv CKD.

Cov txheej txheem

Cov tshuaj. Cov tebchaw siv nyob rau hauv txoj kev tshawb no yog sau tseg nyob rau hauv Ntxiv Table 5 thiab tau synthesized ntawm F.Hoffmann-La Roche Ltd, Basel, Switzerland tshwj tsis yog hais tias lwm yam. Rau kev sim hauv vitro, lyophilized compounds tau rov tsim dua hauv DMSO (Sigma) thiab aliquots tau khaws cia ntawm -20 degree . Rau hauv vivo thwmsim, lyophilized compounds tau resuspended nyob rau hauv lub tsheb uas muaj 1.25 feem pua ​​hydroxypropyl methylcellulose, 0.10 feem pua ​​docusate sodium ntsev,0.18 feem pua ​​propylparaben sodium, thiab 0.02 feem pua ​​citric acid monohydrate, ntawm pH 6. Ib tug zoo particle ncua kev kawm ntawv yog generated los ntawm peb luv luv mem tes sonications ua rau dej khov.

Synthetic chemistry cov txheej txheem thiab cov ntaub ntawv txheeb xyuas. Thov xa mus rau Cov Ntaub Ntawv Ntxiv 1 cov ntaub ntawv.

Chemical biology/candidate target screening Luv luv, plasmid qhia tus neeg sib tw lub hom phiaj ntawm kev txaus siab fused nrog FLAG tag tau kis mus rau HEK293 hlwb. Tom qab 24 teev, tritiated compound K ntawm qhov concentration muaj peev xwm ua rau ABCA1-mediated cholesterol efflux (1.0 μM) tau ntxiv rau 3h, ces cov hlwb nyob tau raug UV-teeb (K{{ 7}} nm) txhawm rau nug cov peev txheej sib txuas / lub hom phiaj sib txuas hauv qhov chaw. Cell lysates tau npaj, cov protein uas qhia tau raug xaiv los ntawm immunoprecipitated siv anti-FLAG antibody ua ke rau sepharose hlaws, khi cov proteins tau sib cais los ntawm SDS-PAGE, thiab ob lub blots zoo tib yam tau npaj thiab soj ntsuam los ntawm sab hnub poob blot thiab autoradiography kom pom cov protein qhia. thiab muaj los yog tsis muaj kev sib txuas ntawm crosslinking, feem. Daim ntawv qhia khiav ntawm lub vijtsam no yog qhia hauv daim duab 2. Daim duab 2d qhia txog lub peev xwm ntawm Cpd A thiab Cpd G kom tshem tawm tritiated Cpd K los ntawm overexpressed thiab immuno-precipitated OSBPL7. Daim duab 3b qhia txog kev siv qhov kev ntsuam xyuas no txhawm rau txheeb xyuas qhov kev sib khi ntawm tritiated Cpd K rau overexpressed thiab immunoprecipitated variants ntawm OSBPL7 uas muaj cov kev hloov pauv hauv qhov kwv yees oxysterol binding hnab tshos ntawm OSBPL7. Qhov zoo ntawm txoj kev no yog i. kev siv cov plasmid transfection ua rau muaj kev qhia siab ntawm txhua tus neeg sib tw uas txo qhov kev pheej hmoo kom nco tau lub hom phiaj tseeb rau cov uas yuav raug qhia nyob rau theem qis endogenous, iii. Kev sib cuam tshuam ntawm cov tshuaj soj ntsuam nrog nws lub hom phiaj tshwm sim nyob rau hauv cov ntsiab lus tseem ceeb ntawm cov roj ntsha (cov hlwb nyob) ntawm cov tshuaj pharmacologically active concentration, thiab iii. immunoprecipitation tso cai rau cov protein qhia ntawm kev txaus siab kom huv los ntawm cov proteins tom qab, txhim kho cov teeb liab / suab nrov piv.

Cell kab lis kev cai. Cov kab hluav taws xob tau yuav los ntawm ATCC thiab subcultured tsis pub dhau nqe 15, tshwj tsis yog rau tib neeg podocytes, uas yog keeb kwm los ntawm Dr. Jochen Reiser. Cov tib neeg uas tsis txawj tuag podocytes tau raug coj los ua raws li kev tso cai ntawm 33 degree, hauv RPMI nruab nrab (Corning) uas muaj 10 feem pua ​​FBS (GIBCO), 1 feem pua ​​penicillin/streptomycin,0.01 mg/ml recombinant human insulin, 0.0055 mg/ml human transferrin (tsis muaj hlau ntau), thiab 0.005 ug/ml sodium selenite (ITS, Corning). Txhawm rau ua kom muaj kev sib txawv, podocytes tau coj los ntawm 37 degree rau 14 hnub hauv RPMI nruab nrab uas muaj 10 feem pua ​​FBS (GIBCO) thiab 1 feem pua ​​penicillin / streptomycin. Cov podocytes sib txawv tau tshaib plab hauv RPMI-0.2 feem pua ​​​​FBS rau 18h thiab tom qab ntawd kho nrog 1 uM, 5 uM, thiab 10 μM ntawm cov tebchaw tshiab npaj hauv DMSO rau 18 teev ntawm 37 degree raws li qhia.

Cov roj (cholesterol) nyob rau hauv macrophages thiab fibroblasts. Lub peev xwm ntawm cov tebchaw los ntxias ABCAl-mediated cholesterol efflux tau txiav txim siab hauv cov kab lis kev cai ntawm THP1 hlwb lossis fibroblasts hauv 96-zoo microplates. Cells raug plated ntawm qhov ntom ntawm 150,000 cell/well. THP1 monocytes tau sib txawv rau macrophages los ntawm kev sib ntxiv ntawm PMA (100 nM) rau 72 h hauv RPMI nruab nrab uas muaj 10 feem pua ​​​​fetal bovine serum thiab 3 pl ntawm 2-mercaptoethanol. Cells raug ntxuav ib zaug nrog RPMI-1640 thiab tom qab ntawd ntim nrog 50 ug/ml acetylated LDL, thiab 10μCi/ml 【'H】-cholesterol hauv RPMI-1640 nruab nrab uas muaj 2 feem pua ​​FCS, rau 48h ntawm 37 degree . Tom qab thauj khoom, cov hlwb raug ntxuav ib zaug nrog RPMI-1640 thiab incubated nrog cov tshuaj ntsuam xyuas ntxiv rau 24h hauv RPMI-1640 nruab nrab uas muaj l mg/ml fatty acid dawb-bovine serum albumin (BSA). Cells raug ntxuav ib zaug, thiab cov roj cholesterol efflux raug ntxias los ntawm kev sib ntxiv ntawm 10 ug / ml ntawm tib neeg apoAI lossis 30 ug / ml ntawm HDL2, HDL3, lossis LDL hauv RPMI -1640 uas muaj I mg / ml BSA rau ib qho ntxiv 6h ib. Radioactivity nyob rau hauv kab lis kev cai supernatants tau txiav txim los ntawm scintillation suav. Cov roj cholesterol efflux tau qhia raws li tus nqi txheeb ze piv rau kev tswj hwm incubated thaum tsis muaj cov cholesterol txais. Dose-response curves raug xam siv XLfit3 (ID Business Solutions Ltd. UK).

Cov roj (cholesterol) nyob rau hauv podocytes. Tib neeg podocytes sib txawv rau 13 hnub tau sau npe rau 24h ntawm 37 degree hauv RPMI nruab nrab uas muaj 2 feem pua ​​FBS thiab [PH]-cholesterol (1 uCi / ml, American Radiolabeled Chemicals). Cells raug ntxuav 3 zaug nrog PBS thiab incubated nrog RPMI ntxiv nrog 0.2 feem pua ​​fat-free-BSA (Sigma) nrog los yog tsis muaj tebchaw rau 18-h ntawm 37 degree . Cov tebchaw tau siv los ntawm cov ntsiab lus hauv qab no∶ Cpd C (1 μM), Cpd A (1 μM, 5 uM), thiab Cpd G (l uM, 5 uM, 10 μM). Tom qab 18-h incubation, tib neeg apoAI (Calbiochem) tau ntxiv rau hauv xov xwm, (20 ug/mL zaum kawg concentration) thiab cov hlwb raug incubated rau lwm 18 teev ntawm 37 degree . Aliquots ntawm nruab nrab sau ua ntej (T =0h) thiab tom qab (T=18h) ntxiv ntawm apoAI tau centrifuged ntawm 12,00 × g rau 5 min thiab cov xov tooj cua hauv cov supernatant tau suav los ntawm cov kua scintillation. Cells raug ntxuav nrog PBS, lysed 0.1 feem pua ​​SDS, 0.1M NaOH thiab cov xov tooj cua hauv cov lysates tau ntsuas los ntawm cov kua scintillation. apo Al-mediated cholesterol efflux, qhia raws li feem pua ​​, raug xam raws li tus nqi ntawm daim ntawv tso tawm rau nruab nrab tom qab ntxiv apo Al (qhov sib txawv ntawm cov xov tooj cua hauv nruab nrab ua ntej thiab tom qab ntxiv ApoA1) muab faib los ntawm tus nqi ntawm tag nrho daim ntawv lo hauv txhua qhov dej. (Radioactivity tso tawm rau hauv xov xwm ntxiv rau cov xov tooj cua hauv cov hlwb lysed).

siRNA kev tshawb fawb. siRNA pas dej ua ke tshwj xeeb rau tib neeg OSBPL7, tib neeg CBIR, thiab nontarget siRNA kev tswj tsis zoo tau sau tseg hauv Cov Lus Ntxiv 6. THPI monocytes tau noob ntawm qhov ntom ntawm 80,000 hlwb/zoo hauv {{4} }zoo daim hlau thiab sib txawv nrog PMA raws li tau piav saum toj no. Tom qab 72 teev, qhov nruab nrab tau hloov los ntawm 50 ul ntawm qhov nruab nrab ntawm ib qhov dej. Cells tau hloov pauv siv Viromer Blue reagent (Origene) ua raws li cov chaw tsim khoom cov lus qhia. Luv luv, 10 ul ntawm siRNA (5 μM) tau tov nrog 90 ul ntawm Viromer Blue (diluted 1 feem pua ​​​​v / v hauv cov khoom tsis muaj). Tom qab 15 min incubation, siRNA-transfection reagent complexes tau diluted ntawm 1:6.5 hauv nruab nrab tshiab thiab ntxiv rau ib ntim ntawm 50 ul ib qhov dej. Tom qab 24 teev, cov hlwb tau ntim nrog cov roj cholesterol los ntawm kev tsim cov acetylated LDL (25 ug / ml) hauv RPMI nruab nrab, 1 feem pua ​​FBS, 100 nM PMA, 2 μCi H-cholesterol rau 24 teev. Ib txheej ntawm lub qhov dej uas muaj cov kab mob sib kis tau muab tso tseg los txheeb xyuas qhov ua tau zoo ntawm RT-PCR. Cov cholesterol-loaded macrophages ces incubated nyob rau hauv lub xub ntiag ntawm Cpd G 5 uM, los yog lub tsheb (0.1 feem pua ​​DMSO) nyob rau hauv RPMI nruab nrab uas Phenol liab, 0.2 feem pua ​​BSA) rau ntxiv 24 teev. Tom qab ntawd cov roj (cholesterol) efflux tau ntsuas raws li tau piav qhia saum toj no.

RT-PCR. RNA raug rho tawm siv cov khoom siv RNEasy Plus Mini (Qiagen). Kev hloov pauv rov qab tau ua tiav siv qScript cDNA SuperMix (Quantabio) raws li cov chaw tsim khoom cov lus qhia. Ntau PCR lub sijhawm tiag tiag tau ua tiav siv TaqMan Fast Universal PCR Master Mix thiab Taqman gene qhia kev soj ntsuam (Table Ntxiv 7).

Western blot. Lysates los yog cell fractions tau incubated nyob rau hauv Laemmli tsis nyob ntawm 55 degree rau 10 min nyob rau hauv txo tej yam kev mob. Tag nrho ntawm 30 ugs ntawm cov protein los ntawm cov cell lysates los yog ib feem peb ntawm cov ntim uas tau sau nyob rau hauv cell fractions npaj tau muab cais los ntawm SDS-PAGE nyob rau hauv 4-20 feem pua ​​​​gradient gels (Biorad) thiab cov proteins ces pauv mus rau PVDF daim nyias nyias. Tom qab thaiv nrog 5 feem pua ​​​​TBS-mis nyuj, daim nyias nyias tau soj ntsuam nrog thawj cov tshuaj tiv thaiv ib hmos ntawm 4 degree. Cov tshuaj tiv thaiv hauv qab no tau siv: nas anti-ABCA1(Abcam; 1:1000), luav anti-GAPDH antibody (Milli-pore; 1:10,000), nas anti-actin( CP01, Millipore 10, 000), anti-MEK, anti-ERp72, anti-V5 thiab anti-Na ntxiv / K ntxiv rau ATPase (Cell Signaling; tag nrho 1:100), thiab luav anti-OSBPL7 (Sigma 1: 1000). Tom qab ntxuav, daim nyias nyias tau incubated nrog anti-luav los yog anti-nas IgG-HRP cov tshuaj tiv thaiv (Promega, 1: 10,00). Cov teeb liab raug kuaj pom tom qab incubation nrog Westernbright ECL HRP substrate (Advansta) thiab luminescence signals raug ntes nrog Azure C600 Gel Imaging workstation (Azure Biosystems Inc, USA) lossis X-ray films.

Cell fractionation. Sib txawv podocytes tau kho nrog cov tebchaw Cpd C (l μM), Cpd A (5 μM), lossis Cpd G (10 μM) rau 18 h, ntxuav, thiab tom qab ntawd sau rau hauv dej khov-txias PBS. Cells raug centrifuged ntawm 1000×g rau 5 min, lub supernatant tau ua tib zoo tshem tawm, thiab cov pellet resuspended nyob rau hauv hypotonic buffer (15 mM KCl, 1.5 mM MgClz, 10mM HEPES, thiab I mM DTT) ntxiv nrog ib tug protease inhibitor cocktail (Roche). Cov hlwb raug cuam tshuam nrog lub khob douncer, thiab sucrose tau ntxiv kom ua tiav qhov kawg ntawm 227mM. Cell lysates tau centrifuged ntawm 1000×g rau 30 min ntawm 4 degree . Tom qab ntawd tus supernatant tau pauv mus rau lub raj tshiab thiab centrifuged ntawm 10,000 × g rau 15 min ntawm 4 degree. Cov pellet, uas muaj cov microsomal feem raug sau thiab cov supernatant raug xa mus rau lub raj tshiab thiab centrifuged ntawm 100,000 × g rau 1 h ntawm 4 degree. Cov pelleted plasma membrane feem tau sau thiab cov supernatant, uas muaj cov organelle-dawb cytosolic feem, tau concentrated hauv Vivaspin 500 lim kab. Western blot tau ua tiav siv cov tshuaj tiv thaiv kab mob rau ABCAl thiab cov cim tshwj xeeb rau txhua daim nyias nyias raws li tau piav qhia saum toj no. Nat/K ntxiv rau ATPase tau siv los txheeb xyuas cov plasma membrane feem, ERp72 ua tus cim rau cov kab mob microsomal-enriched feem, thiab MEK raws li kev tswj rau cov tsis-membranous cytosolic feem.

ABC AT co-immunoprecipitation. ABCA{{0}}Flag tsim tau ua siab zoo los ntawm Dr.Michael Fitzgerald, Harvard University. OSBPL7-V5 construct (pcDNA3.1-V5-ORP7) tau muas los ntawm Addgene. Mammalian kev qhia vectors PCMV6-AN-GFP thiab pCMV6-AC-3DDK, los ntawm Origene, tau siv los ua kev tswj tsis zoo. HEK293 hlwb tau hloov pauv hloov pauv mus ib ntus nrog ib qho ntawm cov plasmid ua ke hauv qab no: (1) ABCA1-Flag plus OSBPL7-V5;(2) ABCA1-Flag plus PCMV{19 }}AN-GFP; los yog (3) OSBPL7-V5＋pCMV6-AC-3DDK siv Fugene 6 transfection reagent (Promega) ua raws li cov chaw tsim khoom cov lus qhia. 48h tom qab hloov pauv, cov hlwb tau lysed nrog 1 ml immunoprecipitation tsis (1 feem pua ​​​​Triton X-100,50 mM Tris-Cl pH 7.0,150mM NaCI) ntxiv nrog cov tshuaj tiv thaiv protease inhibitor cocktail (Roche). Cov lysates raug tshem tawm los ntawm centrifugation thiab sib npaug ntawm tag nrho cov proteins los ntawm txhua tus supernatant tau incubated hmo ntuj ntawm 4 degree nrog 30 ul ntawm anti-Flag M2 Affinity gel (Sigma Aldrich). Cov hlaws tau pelleted los ntawm centrifugation thiab ntxuav 5 zaug nrog 1 ml ntawm immunoprecipitation tsis. Immunoprecipitated proteins tau eluted los ntawm incubating cov hlaws dai nrog 50 ul ntawm 2x qauv tsis rau 15 min ntawm 56 degree. Lub xub ntiag ntawm ABC A1 thiab OSBPL7-V5 hauv lysates thiab eluates tau txheeb xyuas los ntawm western blot raws li tau piav qhia saum toj no.

Kev tshawb fawb tsiaj. Tsiaj txhu tau nyob hauv 12 h / 12h lub teeb / tsaus voj voog thiab nyob rau hauv kev tswj kub (18-23 degree ) thiab av noo (40-60 feem pua ​​) nyob rau hauv ib qho chaw tsis muaj kab mob tsiaj ntawm Division of Veterinary Resources ntawm University ntawm Miami, Miller Tsev Kawm Ntawv Kho Mob. Cov nas tau muab tus qauv 18 feem pua ​​​​cov protein nas chow noj zaub mov thiab dej ad libitum. Txoj kev tshawb fawb raws tu qauv tau pom zoo los ntawm Pawg Saib Xyuas Tsiaj thiab Siv Tshuaj (IAACUC) ntawm University of Miami, uas tau lees paub los ntawm Lub Koom Haum rau Kev Ntsuas thiab Kev Pom Zoo ntawm Laboratory Animal Care International (AAALAC). Tag nrho cov txheej txheem tau ua raws li tsoomfwv thiab lub xeev cov cai tswj hwm kev coj noj coj ua, suav nrog Txoj Cai Tsiaj txhu Kev Noj Qab Haus Huv (AWA) kev tswj hwm los ntawm Tebchaws Meskas Lub Tsev Haujlwm Saib Xyuas Kev Ua Liaj Ua Teb (USDA) thiab Txoj Cai Pabcuam Kev Noj Qab Haus Huv rau Tib Neeg thiab Kev Siv Cov Tsiaj Looj (PHS) Txoj Cai) tswj hwm los ntawm National Institutes of Health (NIH), Office of Laboratory Animal Welfare (OLAW). Poj niam Balb/c nas tau yuav los ntawm Jackson Laboratories rau Adriamycin-induced nephropathy kev tshawb fawb. Thaum muaj hnub nyoog 8 lub lis piam, nas tau txais ib koob tshuaj Adriamycin (Sigma-Aldrich, 12mg/Kg) lossis tsheb (0.9 feem pua ​​NaCI) los ntawm kev txhaj tshuaj rau hauv cov leeg. Cov nas tau muab cais ua rau 6 pawg kev sim thiab, pib ntawm 24 teev tom qab ADR txhaj tshuaj, kho nrog lub tsheb lossis cov tshuaj sib txawv ntawm cov tshuaj txhua hnub ntawm qhov ncauj gavage rau 28 hnub. Cov koob tshuaj hauv qab no tau siv: Cpd C (LXR agonist) 10mg / Kg; Cpd A 30mg/Kg; Cpd G 100 mg/Kg. Txhua tus tsiaj tau txi 28 hnub tom qab txhaj tshuaj ADR. Col4a3 KO nas (Col4a3tmiDec strain 129X1/SvJ) tau yuav los ntawm Jackson Labs. Cov nas tau muab cais ua 2 pawg. Pib thaum muaj hnub nyoog 4 lub lis piam, cov nas tau muab tshuaj los ntawm qhov ncauj ib zaug ib hnub nrog lub tsheb lossis Cpd G (100 mg / Kg) rau 4 lub lis piam tom qab kev txi ntawm 8 lub lis piam. Ib txoj kev tshawb fawb thib ob tau ua hauv Col4a3 KO nas nrog kev kho mob pib thaum muaj hnub nyoog 6 lub lis piam. Txoj kev tshawb no tau ua tiav txhawm rau txheeb xyuas seb Cpd G kev kho cov nas uas muaj lub raum tsis ua haujlwm tuaj yeem ncua sijhawm kawg ntawm lub raum kab mob thiab yog li ua kom muaj sia nyob ntev. Rau kev kho mob ramipril, Col4a3 KO nas pib tau txais ramipril thaum muaj hnub nyoog 4 lub lis piam. Ramipril tau ntxiv rau cov dej haus ntawm qhov concentration uas yuav ua rau ib koob ntawm 10 mg / Kg / hnub 8.

Phenotypic tsom xam ntawm nas. Txhua lub lim tiam cov zis kuaj tau raug sau. Urinary albumin tau ntsuas los ntawm ELISA (Bethy! Laboratories) thiab creatinine los ntawm kev tshuaj ntsuam colorimetric (Stanbio). Albuminuria tau qhia raws li qhov piv txwv ntawm albumin / creatinine. Cov nas tsuag tau muab tshuaj loog nrog Ketamine (90 mg/Kg)/Xylazine (10mg/Kg), cov ntshav tau sau rau hauv cov hlab ntsha heparinized, thiab cov tsiaj tau muab tso rau ntawm sab laug ventricle nrog 0.9 feem pua ​​NaCl tov. Lub raum cortex tau ua tib zoo tshem tawm thiab muab cais rau hauv cov qauv uas yog snap khov, kos rau hauv OCT, lossis kho hauv 10 feem pua ​​formalin rau kev soj ntsuam tom ntej.

Kev txiav txim siab ntawm lipid deposition nyob rau hauv lub raum cortex seem. OCT embedded raum cortex kuaj tau cryosectioned ntawm 4 μm thickness thiab stained nrog roj. Liab O-isopropanol tov (Electron Microscopy Science, PA) diluted 6: 4 hauv dej thiab ces counterstained nrog Hematoxylin Harris Hg Dawb (VWR, PA). Tom qab ntawd cov khoom raug soj ntsuam los ntawm kev siv lub tshuab ntsuas lub teeb (Olympus BX 41, Tokyo, Nyiv).

Kev txiav txim siab ntawm cov roj cholesterol thiab cov ntsiab lus triglyceride. Snap khov raum cortex kuaj tau homogenized nyob rau hauv 2 mM poov tshuaj phosphate tsis, pH 7,(1{{10}}} pul buffer/mg cov ntaub so ntswg) siv lub khob douncer rau dej khov. 100 ul Kev sib xyaw ua ke ntawm cov homogenate tau sib xyaw kom zoo nrog I ml ntawm hexane: isopropanol (3: 2; vv) rau ib lub ntim ntawm cov ntaub so ntswg homogenate. Cov theem aqueous (pellet) thiab organic (supernet) tau sib cais los ntawm centrifugation ntawm 12, 000 × g rau 5 min. Cov pellets (aqueous phase) tau qhuav thiab rov tsim dua hauv 8 M urea, 0.1 feem pua ​​SDS, 0.1 M NaOH, thiab tag nrho cov proteins tau ntsuas los ntawm txoj kev BCA (Thermofisher). Lipids tau rov tsim dua hauv 100 ul isopropanol∶NP-40 (9: 1; vv). Triglyceride cov ntsiab lus tau soj ntsuam siv cov khoom siv xim xim raws li cov chaw tsim khoom cov lus qhia (Cayman Chemical USA). Tag nrho cov roj cholesterol thiab CE cov ntsiab lus tau soj ntsuam siv. ib qho enzymatic fluorometric method59. Rau tag nrho cov cholesterol ntsuas, cov extracts tau diluted 1:100 hauv assay buffer (100 mM potassium phosphat e, 50mM NAC, 5mM cholic acid, 0.1 feem pua ​​Triton X-10, pH 7.4) muaj cov roj cholesterol oxidase (1 U / ml), cholesterol esterase (1 U / ml), horseradish peroxidase (1 U / ml), thiab Amplex Liab (75 μM). Cov tshuaj tiv thaiv tau tsim nyob rau ntawm 37 degree rau 30 min nyob rau hauv ib tug dub opaque 96-zoo phaj (Greiner) thiab fluorescence tau ntsuas nyob rau hauv ib tug microplate nyeem ntawv (Spectramax i3X, Molecular Devices) ntawm 530 nm excitation thiab 580 emissions.

Rau kev txiav txim siab CE, 150 ul ntawm qhov ntsuas tsis tau piav qhia saum toj no, ntxiv nrog bovine daim siab catalase (45 U / ml) thiab cov roj cholesterol oxidase (1 U / ml), tau ntxiv rau 25 ul ntawm cov qauv thiab incubated hmo ntuj ntawm 37 degree kom deplete dawb Cov roj (cholesterol) tsuas tshuav cov roj cholesterol xwb. Tom qab ntawd, 75 ul ntawm cov roj cholesterol ester nrhiav reagent (1 U / ml cholesterol oxidase, 4 U / ml cholesterol esterase, 24 U / ml horseradish peroxidase, 300 μM Amplex Red) tau ntxiv. Cov txheej txheem fluorescence tau ntsuas raws li tau piav qhia saum toj no. Cov qauv sab hauv suav nrog cov qauv uas muaj 1 mM ntawm cov roj cholesterol thiab 5 uM ntawm cov roj cholesterol oleate txhawm rau txheeb xyuas qhov ua tiav ntawm cov enzymatic decomposition ntawm cov roj cholesterol dawb nrog rau kev soj ntsuam rhiab heev thiab tshwj xeeb.

Kev ntsuam xyuas histological. Formalin-taw paraffin-embedded raum cortex seem 4 um tuab tau stained nrog Periodic acid-Schiff (PAS). Cov swb tau txheeb xyuas los ntawm tus kws kho mob lub raum hauv kev tsim ob qhov muag tsis pom kev. Glomerular sclerosis, (ntiaj teb thiab segmental), podocyte hypertrophy, thiab podocyte hyperplasia tau qhia raws li feem pua ​​​​ntawm glomeruli pom nrog cov kev txawv txav no. Tubular microcysts thiab interstitial o tau qhab nia ntawm qhov ntsuas 0-4 qhov twg 0=0 feem pua ​​,05=1-10 feem pua ​​,1=11-25 feem pua ​​,2:26-50 feem pua ​​,3:{{ 12}} feem pua ​​, thiab 4= ntau dua 75 feem pua ​​​​kev puas tsuaj. Kev nthuav dav mesangial tau qhab nia raws li kev tsom xam ib nrab (teev 0-5) lossis feem pua ​​​​ntawm glomeruli nthuav tawm mesangial expansion (feem pua).

Immunofluorescence microscopy. Rau kev tshawb pom ntawm ABC Al kev qhia, 4 um paraffin raum cortex seem tau deparaffinized thiab rehydrated. Antigens tau muab rov qab los ntawm boiling rau 30 min hauv citrate tsis pH 6.0(Target Retrieval Solution, Citrate pH 6, Sigma, USA). Cov seem tau permeabilized rau 30 feeb nrog 0.1 feem pua ​​Triton X-100 hauv TBS. Cov ntaub so ntswg tau tsim nrog 10 feem pua ​​​​tws cov ntshav (miv #G9023, Millipore Sigma) thiab 1 feem pua ​​​​BSA (cat # SP-5050-500, Vector, USA) hauv TBS-T rau 1hr ntawm chav tsev kub los thaiv qhov tsis yog tshwj xeeb. kev khi. Tom qab ntawd cov qauv raug muab tso rau ib hmos ntawm 4 degree nrog Rat anti-ABCAl (Novus, 1: 200) thiab Luav anti-WI1 (Abcam, 1: 200) antibodies nyob rau hauv ib lub chamber humidified. Tom qab ntxuav ntau, cov qauv tau muab tso rau hauv I h ntawm RT nrog fluorochrome-conjugated Secondary antibodies (Invitrogen, 1: 500). Antifade mounting media uas muaj DAPI (Vectashield Plus) tau ntxiv rau cov ntu ua ntej yuav tshuaj xyuas

Cov duab tau txais los ntawm kev siv FluoView FV1000 Confocal Microscope (Olympus) nrog lub hom phiaj roj x63 hauv cov dav hlau sib txawv siv tus qauv Z-series nrog cov kauj ruam loj ntawm 0.18 um. Thaum lub sij hawm kev soj ntsuam, ib tug neeg lub dav hlau tau deconvoluted thiab stacked los tsim ib tug siab tshaj plaws projected duab siv tshuaj tiv thaiv khaub thuas saib software version 4.3b (Olympus). Yam tsawg kawg ntawm 5 cov duab raug ntes los ntawm 5 qhov sib txawv ntawm ib qho qauv. Kev ntsuas ntawm ABCAl glomerular txhais tau tias fluorescence siv (MFI) tau ua tiav siv Fiji-ImageJ software.

Rau kev tshawb pom ntawm OSBPL7,4 um khov raum cortex seem embedded hauv OCT, tau kho nrog PFA-sucrose (2 feem pua ​​,4 feem pua) hauv PBS rau 20min ntawm RT thiab permeabilized nrog 0.3 feem pua ​​Triton-PBS rau 15 min thiab thaiv nrog thaiv tsis pub (5 feem pua ​​​​FBS hauv PBS) rau 1h. Tom qab ntawd cov ntaub so ntswg tau incubated nrog luav anti-OSBPL7 (Sigma, 1:50) thiab tshis anti-Synaptopodin (Santa Cruz, 1:800) antibodies nyob rau hauv ib tug humidified chamber thaum hmo ntuj ntawm 4 degree. Tom qab ntxuav ntau, cov qauv tau muab tso rau hauv cov tshuaj fluorochrome-conjugated thib ob cov tshuaj tiv thaiv kab mob, thiab npog nrog cov xov xwm mounting raws li tau piav qhia saum toj no. Cov duab raug ntes siv Leica DMI 6000B fluorescent microscope.

Kev txheeb cais. GraphPad Prism ver 6 lossis 7 tau siv los ua txhua qhov kev ntsuas ntsuas. Cov pab pawg tau muab piv nrog kev siv ob-tailed t-test lossis One-way ANOVA ua raws li Dunnett's lossis Tukey's test. F, Brown-Forsythe, lossis Bartlett qhov kev sim tau siv los txiav txim siab qhov sib txawv ntawm cov pab pawg, yog tias P<0.05, then="" groups="" were="" compared="" using="" two-tailed="" mann-whitney,="" or="" kruskal-wallis="" followed="" by="" dunn's="" tests.="" the="" correlation="" between="" albuminuria="" and="" accumulation="" of="" cholesterol="" ester="" in="" kidney="" cortexes="" was="" assessed="" using="" the="" spearman="" coefficient="" test.="" p=""><0.05 were="" considered="" statistically="" significant.="" only="" data="" from="" independent="" experiments="" were="">

Txhua tus tsiaj tau muab tso rau hauv pawg kho mob.