Peb-dimensional Architecture Ntawm Nephrons nyob rau hauv Qhov Ib txwm Thiab Cystic raum

Hu rau: emily.li@wecistanche.com

Thomas Blanc1,2,7, Nicolas Goudin3,7, Mohamad Zaidan1,4,7, Meriem Garfa Traore3, Frank Bienaime1,5, Lisa Turinsky1, Serge Garbay1, Cle´ment Nguyen1, Martine Burtin1, Ge´rard Friedlander1,6, Fabi Terzi1,8 thiab Marco Pontoglio1,8

Cistanche yog qhov zoo rau lub raum

KEYWORDS: cystic raum kab mob; raum; nephron; nephronophthisis;cov ntaub so ntswg clearing

Lub raum ua haujlwm tseem ceeb yog nyob ntawm cov qauv peb-dimensional ntawm nephrons. Txhua yam distortion ntawm lawv cov duab yuav ua rauraum tsis ua haujlwm. Cov txheej txheem histological ib txwm muaj kev txwv loj rau kev tsim kho cov ntaub so ntswg peb sab. Ntawm no, peb ua ke cov ntaub so ntswg clearing, multi-photon microscopy thiab digital tracing rau lub reconstruction ntawm ib leeg nephrons nyob rau hauv physiological thiab pathological mob. Teeb ntawmnephronstxawv ntawm qhov chaw, cov duab thiab qhov loj me raws li lawv txoj haujlwm tau txheeb xyuas. Interestingly, nephrons nyiam pw hauv dav hlau. Thaum cov txheej txheem no tau siv rau ib qho qauv ntawmkab mob cystic raum, cysts tau pom los tsim tsuas yog hauv cov ntu tshwj xeeb nephron. Nyob rau tib ntu, cov hlwv yog txuas mus rau hauv ib txwm tsis-dilated tubules. Ntxiv mus, cov duab ntawm cov cysts txawv raws li nephron ntu. Yog li, peb cov fifindings muab lub tswv yim tseem ceeb rau kev pom cov qauv ntawm ob lub raum ntawm ib theem nephron thiab, qhov tseem ceeb tshaj, muab lub hauv paus rau kev nkag siab txog cov txheej txheem pathological xws li cystogenesis.

Lus Txhais Lus

Cov txheej txheem tshem tawm muab cov cuab yeej tshwj xeeb rau kev nkag siab tias kev hloov pauv morphological ua rau muaj qhov tshwm sim ntawm pathological. Lub raum yog ib qho tseem ceeb hauv nruab nrog cev uas tswj lub cev homeostasis los ntawm dej, metabolite, thiab electrolyte tuav, uas yog ib qho tseem ceeb nyob rau hauv lub complex 3-dimensional qauv ntawm nephrons. Thaum cov txheej txheem no hloov pauv, lub raum pathophysiology tshwm sim. Nyob rau hauv txoj kev tshawb no, peb tau tsim ib txoj kev muaj zog raws li kev kho qhov muag,

multiphoton microscopy, thiab digital tracing los kawm lub raum ntawm ib leeg-nephron qib nyob rau hauv physiological thiab pathological mob. Tshwj xeeb tshaj yog, peb muab thawj 3-dimensional reconstruction ntawm ib lub raum polycystic ntawm qhov ntsuas ntawm ib leeg nephrons. Txoj kev no tuaj yeem siv rau ntau lub ntsiab lus pathological, ua rau peb nkag siab zoo dua cov txheej txheem ntawm lub raum deterioration thiab, yog li ntawd, txhawm rau txhim kho cov tswv yim kho mob ntau dua.

Lub raum tswj lub cev homeostasis ntawm nws cov complex 3-dimensional (3D) nephron qauv. Thaum cov txheej txheem no hloov pauv, lub raum pathophysiology tshwm sim.Mob raum mobyog yus muaj los ntawm kev loj hlob ntawm lub raum lesions. Intriguingly, cov kws kho mob tau tshaj tawm txog qhov dav dav ntawm kev faib cov kab mob thaum lub raum mob ntev.1,2 Txawm hais tias qhov no cuam tshuam qhov tseeb tias cov nephron tshwj xeeb tuaj yeem sib txawv, lossis lwm yam, tias qee qhov nephrons muaj qhov sib txawv ntawm qhov raug mob tag nrho. , tsis paub. Txhawm rau daws qhov teeb meem no, nws yog qhov yuav tsum tau rov tsim kho 3D duab ntawm nephrons hauv cov ntsiab lus pathological.

Rau peb txoj kev paub,nephronstsuas yog tau rov tsim kho tag nrho siv serial 2D seem.3–5 Txawm hais tias tus qauv histological sectioning muab kev daws teeb meem siab, 3D reconstructions yog ib qho nyuaj thiab nyuaj kom tau txais. Qhov no feem ntau yog vim muaj kev cuam tshuam ntawm kev siv tshuab, uas yog qhov cuam tshuam tsis tau ntawm cov txheej txheem slicing. Tsis tas li ntawd, 3D reconstruction los ntawm 2D dluab tsis tso cai rau cov duab ncaj qha ntawm 3D qauv nyob rau hauv tag nrho-mounted ntaub so ntswg.

Multiphoton microscopy tau txhim kho peb lub peev xwm los txheeb xyuas cov kev hloov pauv hauv cov ntu tuab. Txawm li cas los xij, qhov kev txwv tseem ceeb yog qhov tob nkag tau yooj yim vim lub teeb tawg. Los ntawm kev txo qis qhov sib txawv ntawm qhov ntsuas qhov sib txawv, cov tshuaj tshem tawm tau txhim kho lub peev xwm ntawm cov duab qhov tob.6,7 Txawm hais tias qhov tseeb tias thawj tus neeg tshem tawm thawj zaug tau qhia txog ib puas xyoo dhau los, 8 nws tsuas yog tsis ntev los no uas muaj ntau qhov kev tshem tawm tau tsim, feem ntau rau lub hlwb. .9–17 Cov kev tshawb fawb tsis ntev los no tau pom tias lawv muaj peev xwm ua rau pom lwm yam kabmob, xws li daim siab, txiav, lossis raum.18–26

cistanche rau mob raum

Polycystic raum kab mob, ib yam kab mob sib kis uas muaj feem cuam tshuam nrog kev hloov pauv hauv ntau cov noob ciliary, yog cov kab mob ntau tshaj plaws ntawm lub raum.27,28 Polycystic raum kab mob yog tus cwj pwm los ntawm kev loj hlob ntawm cov hlwv uas ua rau lub raum puas tag.28 Cov kev tshawb fawb Microdissection qhia tias cov hlwv tuaj yeem. txhim kho xws li "tawm thawb" ntawm ntu nephron, xws li hauv autosomal dominant polycysticmob raum; raws li ectatic expansion ntawm sau ducts (CDs), xws li nyob rau hauv autosomal recessive polycysticmob raum; los yog tshwj xeeb tshaj yog nyob rau hauv medullary tubules, xws li nephronophthisis (NPHP).

Ntawm no, peb ua ke optical clearing nrog multiphoton microscopy los muab kev nkag siab tshiab rau yuav ua li casnephronsyog cov zoo li tus thiab muaj nyob rau hauv physiological tej yam kev mob thiab yuav ua li cas lawv hloov thaum lub sij hawm tus kab mob, xws li cystogenesis. Peb cov txiaj ntsig tau pom tias muaj 3 hom nephrons thiab cov hlab ntsha tuaj yeem cuam tshuam rau nephron spatial lub koom haum. Interestingly, peb pom tias nephrons zoo li dag hauv cov dav hlau tshwj xeeb. Kev npaj txhij txog, thaum peb siv cov txheej txheem no rau jck nas, peb pom tias cov hlwv tsim tsuas yog nyob rau hauv tshwj xeeb nephron ntu nrog fusiform cysts intermingled nrog ib txwm tubules.

TSEEM CEEB

Kev sim teeb tsa

Kawm cov duab ntawm tag nrhonephronsNyob rau hauv kev sib raug zoo rau lawv ib puag ncig, peb tau txais ib tug txheej txheem raws li kev kho qhov muag clearing thiab multiphoton microscopy (Supplementary Figure S1A). Los ntawm kev sib piv cov txheej txheem kho qhov muag sib txawv, peb txiav txim siab tias rau lub raum, benzyl cawv / benzyl benzoate (BABB) yog qhov tsim nyog tshaj plaws ntawm kev ua tau zoo (Cov duab ntxiv S1B thiab C thiab Cov Lus Ntxiv S1).

3D reconstruction thiab digital tracing xav tau cov duab zoo nrog cov kev daws teeb meem siab thiab siab teeb liab-rau-tom qab piv. Peb pom tias cov teeb liab kho qhov muag muab los ntawm haiv neeg fluorescence tsis zoo ib yam ntawm lub raum. Tshwj xeeb, cov duab los ntawm medulla tsis zoo sib xws nrog cov teeb liab qis-rau-tom qab piv (Cov Duab Ntxiv S2). Txhawm rau txhim kho qhov sib piv ntawm cov teeb liab-rau-tom qab, peb stained ntu nrog ntu ntu ntawm cov kua qaub-Schiff (Cov Duab Ntxiv S1D), vim peb tau sau tseg tias qhov staining no tau ua rau muaj qhov sib txawv hauv cov ntu nyias thaum lub sijhawm 2-photon fluorescence. Txawm li cas los xij, lub teeb liab tsis zoo ib yam thoob plaws hauv cov kua qaub-Schiff-stained raum seem (Cov duab ntxiv S1E). Yog li ntawd, peb stained raum seem nrog flfluorochrome-coupled lectins: txiv laum huab xeeb agglutinin thiab hom qoob mog agglutinin, uas stain glomeruli thiab tubules, thiab Griffonia simplicifolia agglutinin, uas cim cov hlab ntsha.30 Zoo kawg li, nrog rau kev sib xyaw ntawm cov lecttoins-, cov teeb liab. keeb kwm piv txwv tau nce ntau hauv tag nrhokab mob raum, nrog kev txhim kho tseem ceeb hauv ob lub dav hlau xy thiab z axis (Cov duab ntxiv S1E thiab Supplementary Movie S1).

3D nephron ntu visualization

Txhawm rau pom cov duab ntawm tag nrho cov nephrons, peb taug qab lawv txoj hauv kev, pib ntawm qhov tso zis ntawm lub glomerulus thiab xaus rau ntawm CD junction (Supplementary Movie S2). Txawm hais tias qhov tseeb tias lectins siv thoob ntiaj teb khi tag nrho cov nephron ntu, peb pom tias thaum ua tib zoo tshuaj xyuas, cov lectins no tau tshwm sim los ntawm cov qauv tshwj xeeb hauv txoj kev uas lawv stained basal lossis luminal membranes nyob rau hauv qhov sib txawv nephron ntu. Hauv lwm lo lus, peb tau coj kom zoo dua ntawm cov qauv stereotypical ntawm lectin staining los txheeb xyuas qhov sib txawv nephron ntu. Nyob rau hauv txoj kev no, peb tuaj yeem txheeb xyuas tau yooj yim 6 qhov chaw: proximal tubule (PT), nyias limb (TL) ntawm Henle voj (HL), tuab ascending limb (TAL) ntawm HL, distal convoluted tubule (DCT), txuas tubule (CNT). ), thiab CD. Kev hloov pauv ntawm PT thiab TL tau tshwm sim los ntawm kev poob tam sim ntawm PT txhuam ciam teb teeb liab thiab los ntawm kev txo qis hauv lumen dav, uas yuav luag tsis tuaj hauv TL (Daim duab 1a). Hloov pauv, kev hloov pauv ntawm TL thiab TAL tau pom los ntawm qhov tseem ceeb nce ntxiv hauv txoj kab uas hla sab nraud thiab cov lumen dav tsis tu ncua (Daim duab 1b). Nyob rau hauv tag nrhonephrons, kev hloov ntawm TAL rau DCT tau pom muaj nyob rau ntawm lub vascular ncej ntawm glomerulus. Qhov kev hloov pauv no tau tshwm sim los ntawm kev loj hlob sai ntawm sab nraud tubular txoj kab uas hla uas feem ntau yog vim muaj qhov loj ntawm lumen txoj kab uas hla (Daim duab 1c). Kev hloov pauv ntawm DCT thiab CNT tau pom los ntawm kev txo qis ntawm ob lub raj raj thiab lumen (Daim duab 1d). Thaum kawg, kev sib txuas ntawm CNT thiab cortical CD yog qhov tshwm sim los ntawm kev nce sai ntawm ob lub raj raj thiab lumen (Daim duab 1e). Qhov ntau ntawm cov kev hloov pauv ntawm cov morphological no yog qhov tseem ceeb (Daim duab 1f), thiab nws qhov kev cuam tshuam tau raug txheeb xyuas nrog kev staining nrog cov cim tshwj xeeb tubular (Cov Duab Ntxiv S3-S5 thiab Ntxiv Movie S3-S5).

Cov duab thiab qhov loj ntawm PTs txawv raws li lawv qhov tob

Lub staining ntawm tuabkab mob raumtso cai rau peb taug qab cov kev sib koom ua ke ntawm spatial evolution ntawm tag nrho PT ntu. Interestingly, peb pom tias lawv cov duab yog tsis tshua muaj variation thiab txiav txim los ntawm txoj hauj lwm ntawm lawv glomeruli nyob rau hauv lub cortex. Thoob plaws ntiaj teb, peb tau txheeb xyuas 3 tus qauv. Thawj cortex tau teb rau feem ntau cov nephrons (SNs) (Daim duab 2a; Supplementary Movie S6), uas nyob rau sab nraud feem ntau ntawm lub cortex (feem ntau sab nraud 30 feem pua ​​​​nyob ze rau lub raum capsule). Lawv qhov chaw convoluted tsim compact qauv nrog tsuas yog 6 mus rau 7 convolutions nyob ib ncig ntawm lawv tus kheej glomerulus, occupying heev me me thiab nruj packed qhov chaw. Lub convolution ntawm cov SNs no xaus nyob rau hauv ntev thiab ncaj pars recta uas nqis mus rau hauv lub medulla. Nyob rau ntawm qhov kawg ntawm qhov spectrum no, peb pom tus qauv thib ob ntawmnephronsnyob rau hauv qhov tob tshaj plaws ntawm lub cortex (40 feem pua ​​​​ntawm qhov tob hauv), nyob ib sab ntawm lub medulla (juxtamedullary nephrons [JNs]) (Daim duab 2a; Daim duab ntxiv S6 thiab Supplementary Movie S6). Lawv cov PTs tau tshwm sim los ntawm qhov pib luv luv uas tau nqis los rau ntawm papilla thiab tom qab ntawd U tig mus nce mus rau lub raum tshuaj ntsiav. Nyob rau hauv sib piv rau SNs, JN proximal convoluted tubules tau tus cwj pwm los ntawm cov coils loj uas hloov zuj zus nyob ib ncig ntawm lawv tus kheej glomerulus thiab nyob rau hauv loj, xoob packed domains nyob rau hauv lub juxtamedullary cortex. Lub convoluted ib feem ntawm PT ntawm JNs muaj 10 mus rau 15 convolutions, tsim loj domains. Thaum kawg, tus qauv thib peb suav nrog nephrons nyob hauv nruab nrab ntawm lub cortex (nruab nrab nephrons [MNs]) (Daim duab 2a; Supplementary Movie S6). Interestingly, cov tubules no muaj cov duab thiab spatial orientation uas sawv cev rau kev hloov ntawm SNs thiab JNs. Qhov tseeb, lawv muaj 8 txog 9 convo lutions nrog coils uas loj dua SNs, tab sis me dua JNs. Tsis tas li ntawd, MNs tau pars recta ntev dua li JNs. Qhov zoo siab, kev sib piv thoob ntiaj teb ntawm cov duab ntawm PTs tau pom tias SNs thiab MNs muaj cov qauv zoo sib xws thaum JNs tsis tshua muaj siab (Cov duab ntxiv S6). Tsis tas li ntawd, qhov kev tsim kho qhov chaw ntawm ntau lub nephrons tau qhia tias PTs yeej tsis cuam tshuam (Supplementary Movie S6), qhia tias txhua tus nephron nyob ntawm nws tus kheej qhov chaw hauv lub cortex.

Kev soj ntsuam Morphometric tau lees paub qhov sib txawv loj ntawm qhov loj thiab cov duab ntawm SNs thiab JNs, nrog rau qhov nruab nrab ntawm MNs. PT ntawm JNs yog ntau dua 2-fold ntev dua PT ntawm SNs (Daim duab 2b); Txawm li cas los xij, qhov ncaj yog ntau dua hauv SNs dua li hauv JNs (Daim duab 2c), zoo ib yam nrog kev soj ntsuam tias JN tubules tau ntau dua tortuous (Daim duab 2a; Ntxiv Figure S6).

Daim duab 1|Cov txheej txheem morphological siv los txheeb xyuas ntu nephron. (a–e) Morphology ntawm qhov sib txawv ntawm cov nephron hauv cov nas tswj. Nephrons tau taug qab los ntawm cov urinary ncej ntawm lub glomerulus mus rau lub duct (proximal tubule [PT]: turquoise; nyias limb [TL] ntawm lub voj Henle: lub teeb grey; tuab ascending limb [TAL] ntawm lub voj Henle: tsaus grey; distal convoluted tubule [DCT]: liab dawb; txuas tubule [CNT]: daj; cortical sau duct [CCD]: txiv kab ntxwv). Cov xub qhia txog txoj kab uas hla ntawm qhov sib txawv nephron ntu. (Txuas ntxiv)

Daim duab 1|(Txuas ntxiv) (f) Quantifification ntawm sab tubular txoj kab uas hla ntawm qhov sib txawv nephron ntu. Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias SEM. Kev tshuaj xyuas qhov sib txawv ua raws li kev xeem Tukey-Kramer: **P < 0.01,="" ***p=""><>

Daim duab 3|Peb-dimensional (3D) nephron reconstruction qhia 3 ntau hom nephrons. (a) Cov neeg sawv cev 3D duab ntawm tag nrho nephron (sab laug panels) los ntawm glomeruli mus khaws cov duct nyob rau hauv lub cortex superficial (xiav), nruab nrab cortex (ntsuab), thiab cheeb tsam juxtamedullary (liab) hauv cov nas tswj. Nephron segmentation (txoj cai panels) rau 3 ntau hom nephrons. Bar ¼ 150 mm. (b) Quantifification ntawm interdistance ntawm 2 libs ntawm lub voj Henle rau 3 hom nephrons. (c) Quantification ntawm nephron ntev rau qhov (ntxiv)

Daim duab 3|(txuas ntxiv) 3 hom nephrons. (d) Kev ntsuas qhov ntev ntawm qhov sib txawv ntawm nephron raws li hom nephron. Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias -SEM. Kev soj ntsuam ntawm qhov sib txawv ua raws li kev ntsuas Tukey-Kramer: juxtamedullary nephron (JN) piv rau superfificial nephron (SN): ###P < 0.001;="" jn="" piv="" rau="" nruab="" nrab="" nephron="" (mn):="" *="" p="">< 0.05,="" **p="">< 0.01,="" ***p="">< 0.001.="" cnt,="" txuas="" tubule;="" dct,="" distal="" convoluted="" tubule;="" pt,="" lub="" cev="" sib="" npaug;="" tal,="" tuab="" ascending="" limb="" ntawm="" lub="" voj="" henle;="" tl,="" nyias="" caj="" npab="" ntawm="" lub="" voj="">

Glomerular loj sib txawv nrog qhov tob

Tom qab ntawd peb txheeb xyuas qhov tob thiab txoj kab uas hla ntawm glomeruli xaiv los ntawm txhua qhov ntawm 3 thaj chaw. Kev soj ntsuam Morphometric qhia tau hais tias glomeruli nyob ntawm qhov nruab nrab 783 thiab 355 hli los ntawm lub raum tshuaj ntsiav hauv JNs thiab SNs, feem. Raws li yav dhau los tau tshaj tawm, peb pom tias qhov loj ntawm glomeruli sib txawv raws li lawv qhov tob. Tshwj xeeb, qhov ntim glomerular ntawm JNs yog 3 npaug ntau dua li ntawm SNs thiab MNs (Daim duab 2d).

3D nephron reconstruction

Peb mam li taug qab qhov spatial evolution ntawm tag nrho cov nephrons los ntawm PT mus rau CNT. Kev pom thoob ntiaj teb ntawm lawv qhov kev tsim kho 3D tau lees paub tias cov duab nephron txawv ntawm SNs, MNs, thiab JNs (Daim duab 3a; Ntxiv Movie S7). Qhov sib txawv no feem ntau yog los ntawm cov qauv ntawm PT. Tsis tas li ntawd, peb pom tias HL muaj lub koom haum 3D spatial sib txawv. Tshwj xeeb, TL thiab TAL tau sib nrug deb ntawm JNs ntau dua li hauv SNs thiab MNs (Daim duab 3b). Morphometric tsom xam tau qhia tias JNs tau thoob ntiaj teb 2-fold ntev dua SNs (Daim duab 3c). Qhov ntev ntev tau muab faib ua feem faib rau txhua ntu, tshwj tsis yog TAL thiab DCT, uas zoo li muaj qhov ntev zoo sib xws hauv txhua hom nephron (Daim duab 3d). Qhov no tau qhia tias nephron elongation yog ib qho kev xav tsis thoob uas yog homogenously txheej txheem.

Cov hlab ntsha arcuate cuam tshuam juxtamedullary PT convolution

Txhawm rau kom tau txais kev pom dav ntxiv ntawm covlub raum qauv, peb coj kom zoo dua ntawm Griffonia simplicifolia agglutinin staining, uas tso cai rau peb taug qab cov hlab ntsha arcuate thiab lawv cov ceg mus rau hauv cov hlab ntsha cortical radiate (Daim duab 4a; Ntxiv Movie S8). Interestingly, lub concomitant 3D reconstruction ntawm nephrons thiab cov hlab ntsha qhia tau hais tias cov hlab ntsha arcuate zoo li nyob rau hauv ib tug surprise parallel spatial lub koom haum nrog cov tubules nyob ze. Tshwj xeeb, peb tau pom tias txoj hauv kev ua raws li JNs tshwj xeeb ( txwv tsis pub JNs) nyiam ua raws li txoj hauv kev ntawm lub nkoj nyob ze (Daim duab 4b; Ntxiv Movie S9). Remarkably, qhov kev tsis ncaj ncees no (tsuas yog pom nyob rau hauv ib pawg ntawm JN nephrons) suav txog qhov loj heterogeneity nyob rau hauv cov duab ntawm PT (Cov duab ntxiv S6). Kev soj ntsuam Morphometric qhia tau hais tias "tsis muaj kev txwv" PTs tau ntev dua thiab muaj kev cuam tshuam ntau dua (txo qhov ncaj) dua li "tsis muaj kev txwv" (Daim duab 4c thiab d). Nws yog ib nqi sau cia hais tias cov convoluted qhov chaw ntawm ob qho tib si SNs thiab MNs tau positioned saum toj no arcuate hlab ntsha thiab tsis constrained.

Tubules nyiam pw hauv dav hlau

Interestingly, kev tshuaj xyuas ntawm 3D duab ntawm nephrons tau qhia tias lawv nyiam pw hauv cov dav hlau (Daim duab 5a; Ntxiv Movie S10). Txhawm rau ntsuas qhov ntsuas qhov ntsuas no thiab ntsuas qhov ua tau raws li kev txheeb cais ntawm qhov kev sib haum xeeb, peb ntsuas qhov dav dav ntawm tubules khoov tawm ntawm lub dav hlau txhais los ntawm lawv cov loops. Peb cov kev ntsuas tau pom tias piv rau cov qauv simulated ntawm kev taug kev tsis sib xws uas tau tsim nrog tib txheej ntsuas sib law liag thiab qhov ntev ntawm cov ntsiab lus sib law liag (Daim duab 5b), cov tubules tau hloov zuj zus nrog qhov sib txawv me me ntawm lub dav hlau (Daim duab 5c). Qhov no qhia tau hais tias tubules nyiam txwv lawv txoj kev raws lub dav hlau. Cov kev sib txawv tau pom muaj qhov tseem ceeb heev (P <>

cistanche tuaj yeem txhim kho lub raum ua haujlwm

3D nephron reconstruction qhia txog tus qauv tshwj xeeb rau kev loj hlob cyst

Txhawm rau txiav txim siab seb peb cov txheej txheem tso cai rau kev taug qab nephrons hauv cov ntaub so ntswg tsis sib haum xeeb, peb pom cov duab thiab qhov chaw faib tawm ntawm cov hlwv hauv cov nas jck, tus qauv siv dav ntawm NPHP thiab kab mob cystic.31–33 3D cov duab pom tias txawm tias ntawm cov tsos mob ntawm cov hlwv tseem ceeb, cov chav kawm 3D dav dav ntawm nephrons tsis txawv qhov tseem ceeb ntawm cov nas tswj hwm (Supplementary Movie S11). Ib yam li ntawd, kev tshuaj ntsuam morphometric tau lees paub tias lub ntiaj teb ntev ntawm nephrons, glomerular ntim, thiab ntev ntawm txhua.nephron uantu tau muab piv nrog cov nas tswj hwm (Cov duab ntxiv S7). Nco ntsoov, vim tias kev txhim kho cyst, peb tsis tuaj yeem sib txawv TL ntawm TAL hauv HL. Tag nrho cov nephrons tsim fusiform cysts (Cov tsos mob ntxiv S11 thiab S12). Ib qho tshwj xeeb tshaj plaws yog qhov kev soj ntsuam uas fusiform cystic dilations tshwm sim nyob rau hauv ntau qhov chaw nyob rau tib yam.nephron uantu (Sab ntxiv tsos S12 thiab S13). Intriguingly, peb pom tias cysts yeej tsis tsim nyob rau hauv PTs thiab HL descending libs (Daim duab 6a; Supplementary Movie S12). Nyob rau hauv sib piv, lawv tau pom feem ntau nyob rau hauv HL ascending libs, DCTs, CNTs, thiab nyob rau hauv sab sauv ntawm CDs, nyob rau hauv conti nuity nrog CNT (Daim duab 6a; Supplementary Movie S12). Tshwj xeeb, peb tau pom tias DCT, nrog rau cov ntu CNT, muaj qhov tshwj xeeb nce qhov tshwm sim los tsim cov hlwv hauv lawv qhov chaw seem ntau dua (Daim duab 6b). Quantitative morphometric tsom xam ntawm 3D reconstruction dluab qhia tias tag nrho cov cyst ntim ibnephron uatshwj xeeb tshaj yog nyob rau hauv CNT (Daim duab 7a). Tsis tas li ntawd, peb tau pom tias cyst en loj hauv HL thiab DCT muaj ntau dua hauv JNs dua li hauv SNs thiab MNs (Daim duab 7a; Ntxiv Movie S12). Ntxiv mus, qhov tshwm sim ntawm cyst yog qhov tseem ceeb hauv JNs dua li lwm hom nephrons (Daim duab 7b). Peb kuj tau pom tias qhov nruab nrab glomerular interdistance (xws li ntawm qhov ze tshaj 5 glomeruli) nyiam tshwj xeeb tshaj yog nyob rau hauv cov nas cystic, tshwj xeeb tshaj yog nyob rau hauv lub cortex ntau superficial (Cov duab ntxiv S8).

Kev tsim kho 3D ntawm ntau lub hlwv tau qhia tias lawv muaj qhov sib txawv ntawm cov duab thiab ntim (Daim duab 6c; Ntxiv Movies S11 thiab S13). Nyob rau hauv HL ascending limb, cysts muaj ib tug fusiform zoo nrog ib tug me me txoj kab uas hla. Hauv DCT, cov hlwv muaj lub ntsej muag zoo nkauj thiab loj dua thiab tau sib cuam tshuam ntawm cov tubules uas tsis muaj tshuaj lom. Interestingly, qhov kev hloov ntawm DCT thiab CNT yog qhov systematically tus yam ntxwv los ntawm ib tug ib txwm nondilated qauv. Nyob rau hauv sib piv, nyob rau hauv CNT, peb systematically pom ib tug contiguous dilated cystic qauv ncaj qha nyob rau hauv kev sib cuag nrog CD. Interestingly, nyob rau hauv CD, cov qauv no nce zuj zus mus rau ib tug ib txwm tubule loj. Ntau qhov distally, tsis muaj cov hlwv ntxiv tuaj yeem kuaj pom hauv qhov sib txuas ntawm CD. Lwm qhov zoo sib xws yog lub koom haum peculiar spatial ntawm cysts hauv HL ascending limb. Qhov tseeb, txawm hais tias cysts tau tob dua thiab ze rau lub voj voog hauv SNs, lawv tau pom ntau dua thiab ze rau DCT hauv JNs (Daim duab 6c). Ib zaug ntxiv, cov hlwv nyob hauv ib qho chaw nruab nrab hauv MNs (Daim duab 6c).

Daim duab 5|Nephron tubules zoo li hloov zuj zus raws li cov qauv sib txawv. (a) Ib txoj hauv kev ntawm nephron proximal tubule. Txoj kev no qhia txog nws txoj kev nyiam pw hauv lub dav hlau. Lub hom phiaj ntawm evolution ntawm txoj kev tuaj yeem pom zoo dua thaum tig thiab ua raws li cov neeg soj ntsuam qhov pom (saib sab xis-sab sawv cev ntawm tib lub tubular ntu, uas, ntawm sab laug-tes, sawv cev ntawm qhov sib txawv. lub kaum sab xis). (b) Schematic sawv cev ntawm txoj kev tubular muaj li ntawm 4 ntu (nruab nrab ntawm 5 lub ntsiab lus txhais ib txoj hauv kev muab tubular qhia ua piv txwv). Cov qib uas txoj kev yuav mus "tawm ntawm lawv lub dav hlau" tuaj yeem sawv cev los ntawm lub kaum sab xis qhia ntawm no li "beta." (Txuas ntxiv)

Daim duab 5|(Txuas ntxiv) Lub kaum sab xis no yog ntsuas nruab nrab ntawm ntu p3-p4 nrog rau lub dav hlau (txhais los ntawm cov ntsiab lus ua ntej tam sim p3, p2, thiab p1) thiab sawv cev los ntawm cov duab plaub grey tsaus hauv lub tswv yim. Kev suav ntawm beta cov ces kaum tau recursively xam tag nrho raws li cov ntsiab lus nyob rau hauv ib txoj kev tubular. Txhawm rau ntsuas qhov kev tsis ncaj ncees ntawm cov beta cov ces kaum, peb simulated taug kev random (Monte Carlo simulation [MCs]) los ntawm kev siv tib txheej sib law liag alpha kaum ntawm txhua txoj hauv tubular ( ntsuas ntawm txhua ntu sib law liag). Qhov kev taug kev random no tsim txoj hauv kev nrog cov txheej txheem zoo ib yam ntawm alpha cov ces kaum thiab randomized beta cov ces kaum. (c) Violin plots ntawm lub ntiaj teb no tis ntawm beta ces kaum thiab tshwm sim ntawm MCs nyob rau hauv lub proximal tubule (PT), nyias limb (TL) ntawm lub Henle voj, tuab ascending limb (TAL) ntawm lub Henle voj, distal convoluted tubule ( DCT), thiab txuas tubule (CNT). MCs, P <>

Kev sib tham

Cov txheej txheem histological yog txwv nyob rau hauv lawv lub peev xwm los kuaj xyuas cov kev hloov pauv uas cuam tshuam rau lub ntiaj teb cov duab ntawm nephrons. Ntawm no, peb nthuav qhia txoj hauv kev muaj zog raws li kev tshem tawm cov ntaub so ntswg uas muaj peev xwm los daws cov lus nug tseem ceeb ntawm lub raum architecture nrog cov ntsiab lus tsis tau pom dua nyob rau hauv ib txwm muaj thiab cov kab mob pathological. Peb cov txiaj ntsig tau lees paub qhov muaj nyob ntawm 3 hom nephrons, uas txawv ntawm lawv qhov chaw, cov duab, thiab qhov loj me, raws li lawv qhov tshwj xeeb ua haujlwm. Ntxiv mus, peb pom tau hais tias nephrons zoo li dag nyob rau hauv dav hlau thiab hloov mus rau lub nkoj spatial lub koom haum. Intriguingly, thaum peb siv cov txheej txheem no rau ib tug qauv ntawmkab mob cystic raum, peb pom tias cysts tsim nyob rau hauv tag nrho cov

nephrons, tab sis tsuas yog hauv ntu tshwj xeeb. Interestingly, peb tau pom tias cov cyst zoo sib txawv raws li nephron ntu, thiab nyob rau tib lub nephron, cov hlwv intercalate nrog cov tubules tsis zoo. Ua ke, cov txiaj ntsig no muab thawj 3D tus yam ntxwv ntawm qhov chaw ntawm nephrons thiab cov hlab ntsha thiab tseem ceeb muab lub hauv paus rau kev nkag siab txog cov txheej txheem pathological, xws li cystogenesis.

Lub raum kho qhov muag tshem tawm yog qhov nyuaj vim tias cov qib siab ntawm autoflfluorescence thiab cell ceev. Los ntawm kev sib piv cov txheej txheem tshem tawm sib txawv, peb tau txheeb xyuas hauv BABB cov txheej txheem muaj zog rau kev siv qhov kev pom thiab kev tsim kho ntawm cov qauv nyob rau hauv qhov tob tshaj plaws ntawm cov txheej txheem.raum, uas yog medulla. Ntxiv mus, BABB yog ceev thiab scalable thiab, ib zaug tshem tawm, cov qauv tuaj yeem khaws cia rau lub hlis ua ntej tau txais cov duab. Lwm qhov zoo dua ntawm cov txheej txheem no yog nws tus nqi qis. Cov tshuaj tshem tawm tej zaum yuav ua rau lub ntsws loj zuj zus tuaj.9–17 Txawm li cas los xij, vim tias cov kev hloov kho ntawm lub raum loj yog isotropic, cov kev ntsuas txheeb ze tsis xav tias yuav cuam tshuam lossis tsis ncaj ncees rau lawv txoj kev txhais. Ib qho ntawm cov kev txwv tseem ceeb tshaj plaws yog cov lus piav qhia ntawm cov qauv uas siv sijhawm ntev heev. Txawm li cas los xij, muaj ntau txoj hauv kev los ntawm kev kawm tob uas yuav tsum tau kov yeej qhov kev txwv no sai sai.

Raws li cov txiaj ntsig tau los ntawm cov txheej txheem classical, peb txoj kev tshawb fawb pom tias nephrons txawv ntawm lawv cov duab thiab ntev raws li lawv txoj haujlwm. Tshwj xeeb, peb tau pom tias JNs tau tsim kho ntau dua PTs thiab cov glomeruli loj dua thiab ntev dua 2 npaug ntawm SNs. Qhov ntev ntev yog qhov tshwm sim ntawm cov txheej txheem sib haum xeeb vim tias lengthening cuam tshuam rau txhua qhov nephron ntu. Peb kuj pom tias JNs muaj HL loj dua. Ua ke, cov ntaub ntawv no qhia meej meej tias microanatomy ntawm SNs thiab JNs yog qhov sib txawv thiab tias MNs qhia cov yam ntxwv nruab nrab. Interestingly, physiological kev tshawb fawb pom tau hais tias nephrons kuj ua hauj lwm txawv.2 Cov xwm txheej morphogenetic uas underlies qhov txawv no tseem tsis tau paub. Nws yog li kev ntxias kom xav tias cov qauv tshwj xeeb ntawm JNs tuaj yeem suav rau qhov tshwj xeeb ntawm lawv cov haujlwm.

Interestingly, los ntawm kev muab thawj zaug ntawm 3D kev tsim kho ntawm nephrons thiab lawv cov hlab ntsha nyob ib puag ncig, peb tau pom ib qho kev txwv ntawm cov hlab ntsha arcuate thiab ib pawg ntawm nephrons. Yog li, nws yog qhov xav tias cov hlab ntsha tuaj yeem hais txoj hauv kevnephronslengthen thaum lub sij hawm kev loj hlob.34,35 Kev tshuaj xyuas ntawm 3D cov duab ntawm nephrons ua ke nrog kev sim simulations kuj qhia tau hais tias nephrons yeej tsis intermingle thiab hais tias txhua nephron nyhav nyob rau hauv ib lub dav hlau. Lub luag haujlwm tseem ceeb ntawm qhov kev soj ntsuam no tseem yuav tau piav qhia.

Cyst kev loj hlob thaum lub sij hawmpolycystic raum kab mobtseem yog txheej txheem intriguing. NPHP, kab mob pathological tshwm sim los ntawm kev loj hlob cyst, yog cov kab mob ntau tshaj plaws - ua rau mob raum raum kawg hauv cov menyuam yaus thiab cov tub ntxhais hluas. Nees nkaum NPHP genes tau txheeb xyuas txog tam sim no.36 Ntawm lawv, NEK8 encodes ib tug tswv cuab ntawm cov tsis tau muaj nyob rau hauv mitosis A-txog kinase tsev neeg, uas plays lub luag hauj lwm nyob rau hauv cilia muaj nuj nqi thiab cell voj voog txoj kev loj hlob.37 Nek8 yog Ameslikas cim raws li cov noob mutated nyob rau hauv. jck nas.32 Tshwj xeeb tshaj yog, kev hloov pauv hauv tib lub npe protein tau pom tias ua rau NPHP9 hauv tib neeg. , hauv NPHP, cysts zoo li tau muab los ntawm CD thiab DCT.31 Txawm hais tias cov ntaub ntawv qhia, cov kev tshawb fawb txog kev tiv thaiv kab mob tsis tuaj yeem sib cav txog qhov ua tau tias qhov poob ntawm cov cim tubular tshwj xeeb 39 artificially suav rau qhov kev soj ntsuam no. Yog li, peb txoj kev tshawb fawb 3D muab cov pov thawj tseeb thawj zaug tias kev txhim kho cyst yog cov txheej txheem uas yuav koom nrog tsuas yog ntu nephron. Interestingly, txawm hais tias NIMA (Tsis Muaj Hauv mitosis gene A)-Related Kinase 8 (NEK8) tau qhia nyob rau hauv cytoplasm ntawm txhua ntu nephron, nws cov lus qhia hauv cilia yog txwv rau DCT thiab CD. Vim tias tsuas yog cov ntu no tuaj yeem tsim cov hlwv, 31 peb tuaj yeem tshaj tawm tias kev cuam tshuam ntawm NEK8 ciliary muaj nuj nqi yog qhov tseem ceeb rau kev tsim cov cyst. Intriguingly, peb kuj tau pom tias fusiform cysts nyob rau hauv txuas ntxiv nrog cov tubules ib txwm tsis muaj. Qhov tseeb tias qhov kev hloov pauv ntawm cov kab mob tsis zoo ua rau muaj kab mob phenotype hauv ib pawg ntawm cov hlwb tsuas yog qhia tias qhov tshwm sim thib ob tuaj yeem ua rau muaj kev loj hlob cyst hauv cov hlwb, raws li tau hais tseg rau autosomal dominant polycystic.mob raum.40,41

Peb kuj tau pom tias qhov cyst loj dua yog qhov tseem ceeb hauv JNs dua li hauv SNs, tsawg kawg yog xav txog cov hlwv uas nyob nruab nrab ntawm PT thiab CNT. Raws li txoj cai, nws tau raug tshaj tawm tias nyob rau hauv cov ntsiab lus ntawm glomerulosclerosis, glomerular lesions pom ntau zaus hauv JNs dua li hauv SNs.1,21,42 Txawm hais tias kev ua haujlwm dhau los vim qhov nce ib leeg.nephron uaglomerular fifiltration rates thiab thauj / enzymatic kev ua ub no tso nyiaj rau qhov nce siab ntawm JNs kom deteriorate yog ib qho kev xav zoo uas tsim nyog tshawb xyuas ntxiv.

Hauv cov ntsiab lus, peb piav qhia txog cov txheej txheem tshiab rau cov duab rau lub raum hauv 3D nrog cov qauv molecular txaus thiab kev daws teeb meem los sau ncaj qha rau spatial thiab quantitative faib ntawm cov cim tshwj xeeb hauv cov qauv (nephrons, hlab ntsha, thiab cysts). Muab qhov yooj yim, ceev, thiab muaj peev xwm ua kom muaj nuj nqis ncaj qha, peb cia siab tias cov txheej txheem no yuav dhau los ua cov cuab yeej muaj zog rau kev nkag siab txog lub raum pathophysiology.

Daim duab 6|Peb-dimensional (3D) nephron reconstruction qhia tau hais tias cov hlwv loj hlob tsuas yog nyob rau hauv tshwj xeeb nephron ntu. (a) Cov duab sawv cev 3D nrog cyst ntim rendering ntawm tag nrho nephron (sab laug vaj huam sib luag) los ntawm glomeruli mus rau sau duct nyob rau hauv lub cortex superficial (xiav), nruab nrab cortex (ntsuab), thiab juxtamedullary cheeb tsam (liab) nyob rau hauv jck nas. Nephron segmentation (txoj cai panels) rau 3 ntau hom nephrons. Bar ¼ 150 mm. (b) Muaj peev xwm tsim cov hlwv hauv qhov sib txawv nephron ntu hauv jck nas. (Txuas ntxiv)

Daim duab 6|(Txuas ntxiv) Kab rov tav axis yog qhov ntev ntawm nephrons sib haum rau 50 bins (proximal tubule [PT], turquoise; Henle voj [HL], dawb; distal convoluted tubule [DCT], liab dawb; txuas tubule [CNT], daj) . (c) Cov neeg sawv cev 3D hlwv rov tsim kho cov duab nrog cov cyst ntim rendering ntawm lub nce toj ceg ntawm HL (sab laug panels), DCTs (middle panels), thiab CNTs thiab cortical collecting ducts (CCDs) (txoj cai panels) nyob rau hauv superficial (sab laug panels), nruab nrab (middle panels), thiab juxtamedullary (cov vaj huam sib luag qis) nephrons hauv jck nas. Bar ¼ 50 mm.

Txoj kev

Tsiaj txhu

Tag nrho cov txheej txheem tsiaj tau pom zoo los ntawm "Services Vétérinaires de la Préfecture de Police de Paris" thiab los ntawm pawg neeg ncaj ncees ntawm Université Paris Descartes. Ob lub hlis FVB/N nas (n¼ 20) tau siv los teeb tsa qhov kev sim rau lub raum tshem tawm. Txoj kev tshawb no tau ua nyob rau hauv 2-lub lim tiam jck txiv neej nas (n ¼ 4) thiab tswj cov phooj ywg (n ¼ 3).

Kev npaj ntawm lub raum cov ntaub so ntswg

Ua ntej tua, nas tau perfused, ntawm intracardiac catheterization, nrog 25 ml ntawm heparinized saline (1000 IU / l) ua raws li 75 ml ntawm 4 feem pua ​​​​ntawm paraformaldehyde hauv phosphate-buffered saline (PBS). Kev sim tau ua nyob rau hauv xylazine (Rompun 2 feem pua, Bayer, Leverkusen, Lub teb chaws Yelemees, 6 mg / g ntawm lub cev hnyav) thiab ketamine (Clorketam 1000, Vetoquinol SA, Lure, Fabkis, 120 mg / g ntawm lub cev hnyav) tshuaj loog.

Rau kev tshem tawm cov kev tshawb fawb, ob lub raum tau kho nyob rau hauv 4 feem pua ​​​​paraformaldehyde rau 4 teev thiab embedded nyob rau hauv 4 feem pua ​​agarose thiab 1.5-mm-thick seem nyob ib ncig ntawm lub raum hilum raug txiav thiab khaws cia nyob rau hauv PBS ntawm 4 C.Lub raum seemyog thawj zaug stained thiab ces cleared.

Rau kev tshawb fawb immunohistochemical ntawm cov seem nyias,lub raumtau kho nyob rau hauv 4 feem pua ​​paraformaldehyde thaum hmo ntuj thiab paraffin-embedded thiab 4-mm seem raug txiav.

Kev pleev xim

Periodic acid-Schiff staining. Lub 1.5- hli lub raum seem tau incubated nyob rau hauv ntshiab los yog 1:100 PBS-diluted periodic acid-Schiff rau 5 feeb ntawm chav tsev kub ua ntej clearing.

Immunohistochemistry ntawm nyias paraffin-embedded seem. Plaub-micrometer seem ntawm paraffifin-embedded ob lub raum tau ua kom sov rau antigen retrieval thiab incubated thaum hmo ntuj ntawm 4 C nrog flfluorochrome-coupled lectins mus taug qab cov tubules (rhodamine txiv laum huab xeeb agglutinin [RL-1072-5] thiab rhodamine{wheat germ agglutinin [RL{201}] 6}}], Vector Laboratories, Burlingame, CA, diluted ntawm 1:200) thiab ntu tshwj xeeb thawj cov tshuaj tiv thaiv los piav qhia txog cov kab mob sib txawv (biotinylated Lotus tetragonolobus lectin [B-1325], Vector Laboratories, diluted ntawm 1: 100; nas anti-Calbindin D28K [D-4], Santa Cruz, Heidelberg, Lub teb chaws Yelemees, diluted ntawm 1:200; tshis anti AQP2 [C-17], Santa Cruz, diluted ntawm 1:200) . Hnub tom qab, cov seem tau muab tso nrog cov tshuaj tiv thaiv thib ob rau 1 teev ntawm chav tsev kub (Alexa Fluor 488 conjugate [S32354], Invitrogen; antigoat Alexa Fluor 488 [A-11055], Invitrogen; anti-nas Alexa Fluor 488 [A-21202], Invitrogen, Carlsbad, CA; tag nrho cov diluted ntawm 1:500) ua ntej yuav xim nrog 40,6-diamidino-2-phenylindole. Tag nrho cov duab tau txais nrog Nikon Eclipse E800 microscope (Champigny sur Marne, Fabkis) thiab npaj siv FiJi software (version 1.50).

Lectin staining ntawm cov ntu tuab. Lub 1.5- hli-thick raum seem tau incubated ntawm 4 C rau 1 lub hlis nyob rau hauv Texas Liab los yog flfluorescein isothiocyanate coupled lectins: txiv laum huab xeeb agglutinin (RL-1072-5) thiab hom qoob mog agglutinin (RL{{6}) }), diluted ntawm 1: 100 hauv 0.1 feem pua ​​PBS azide thiab 0.1 feem pua ​​Triton-X. Cov seem raug ntxuav txhua hnub rau 2 lub lis piam nrog PBS ua ntej tshem tawm.

Optical clearing

Rau Scale clearing, 1. (8 M urea, 0.1 feem pua ​​Triton X-100) rau 2 lub lis piam, mus txog 1 xyoos, ntawm 4 C.

Rau BABB tshem tawm, 1. Cov qauv no tau muab tso rau hauv BABB (Sigma-Aldrich, Saint Louis, MO) hauv 1: 2 piv rau tsawg kawg 2 hnub ntawm 4 C.

Ob-photon microscopy duab tau

Cov ntaub so ntswg tau duab nrog aqueous gel ntawm ib qho inverted multiphoton microscope (LaVision BioTec) nruab nrog lub Mai Tai HP Titanium-Sapphire Laser (Spectra-Physics, Santa Clara, CA) (Cov duab ntxiv S1A). Lub excitation wavelength yog 815 nm ntawm 8 feem pua ​​​​ntawm lub zog. Peb siv lub hom phiaj 20 dej raus dej (XLUMPLFL20XW, Olympus [Tokyo, Nyiv]; tus lej aperture, 0.95; ua haujlwm nrug, 2.0 hli). Rau kev nrhiav tau ntawm fluorescence, peb siv lub tshuab ntsuas tsis-descanned ntawm 80 feem pua ​​​​ntawm cov bandpass filter 593/40 nm.

Thawj kauj ruam yog los ntawm 2D mosaic duab nrhiav tau ntawm nruab nrab z pawg los ntawm kev siv cov khoom tsis zoo (400 mm 400 mm thiab 1011 1011 pixels, nrog 10 feem pua ​​​​ntawm kev sib tshooj thiab kab nruab nrab ntawm 2) los coj kev xaiv ntawm qhov tseem ceeb tshaj plaws hauv nruab nrab fifields (Cov duab ntxiv S9A). Thaum xy daim phiaj thiab z fifields ntawm kev txaus siab tau raug txheeb xyuas, qhov ntsuas tau raug kho kom tau txais cov duab zoo. Xws li ib txoj hauv kev txo thaj tsam ntawm kev txaus siab rau 5 12 fifields ib z pawg. Tom qab ntawd, peb tau txais 850 z stacks / optical slices (1- hli kauj ruam loj) nyob ib puag ncig lub raum hilum nyob rau hauv nruab nrab ntawm qhovlub raum ntu.

Duab stitching, ua, thiab tracing

Txhua pawg tau tiled raws li txoj kev tsim los ntawm Preibisch li al., 43 uas tso cai rau lub stitching ntawm ib tug loj sau ntawm dluab los ntawm kev siv lub "Grid/Collection stitching" plugin ntawm Fiji software (http:// fifiji.sc/Fiji) . Vim tias peb tau pom qhov kev hloov pauv tseem ceeb ntawm 2 cov duab uas nyob ib sab tom qab stitching, peb tau sau ib qho kev cai sib xws hauv Python. Qhov kev pab cuam no them nyiaj rau cov duab hloov, tso cai rau peb kom haum cov duab (S9B). Stitched-kho cov duab raug tshuaj xyuas siv Imaris version 8.4.2 (Bitplane, Zurich, Switzerland). Tubules thiab cov hlab ntsha tau taug qab siv cov fifilament tracer pob phau ntawv hom ntawm Imaris thiab koom nrog Imaris Xtension uas peb tsim nrog MATLAB (https://www.dropbox.com/s/sm9u5em3orjrpmc/Standalone_Koom{{12} }} Filament_Tool.zip?dl¼0) (Cov duab ntxiv S9C). Glomeruli thiab hlwv tau 3D reconstructed siv phau ntawv hom ntawm pob nto ntawm Imaris. Lub koom haum 3D glomeruli spatial tau tshwm sim siv hom kev qhia ntawm pob pob ntawm Imaris.

Morphometry

Tubular ntu ntev, nephron ntev, thiab ncaj ncees tau txiav txim siab siv lub pob filament tracer ntawm Imaris. Raws li Imaris software, kev ncaj ncees tau suav tias yog qhov sib piv ntawm qhov kev ncua deb ntawm 2 cov ntsiab lus thiab qhov ntev ntawm txoj kev nephron ntu. Peb caug-ib PTs thiab 12 tag nrho cov nephrons tau tsim kho thiab ntsuas hauv 3 cov nas qus, whereas 37 PTs thiab 17 tag nrho cov nephrons tau rov tsim dua thiab ntsuas hauv 4 jck nas. Cyst thiab

glomerular ntim tau txiav txim siab siv "nto pob" ntawm Imaris. Peb caug-ib thiab 34 glomeruli tau ntsuas hauv cov tsiaj qus thiab jck nas, raws li. Xya caum-plaub cysts tau ntsuas tag nrho.

Qhov "tawm ntawm lub dav hlau" cov ces kaum ntawm tubular lem (qhia raws li "beta") tau ntsuas ntawm lub dav hlau (txhais tau los ntawm 3 lub ntsiab lus sib law liag) thiab cov kev taw qhia nrog rau qhov sib law liag thib plaub hauv qhov chaw (Daim duab 5b). Txhawm rau ntsuas qhov tseem ceeb ntawm qhov kev tsis pom zoo, peb simulated qhov kev faib tawm ntawm cov kaum sab xis nrog kev sib hloov ntawm cov kaum sab xis ntawm cov beta (Monte Carlo simulation) hauv cov qauv uas tau tsim los ntawm tib ntu nrog tib lub kaum sab xis ntawm alpha.

Rau kev xam qhov tshwm sim los tsim ib qho cystic lesion nrognephron uantu, peb sawv cev txhua nephron los ntawm cov ntsiab lus hauv qhov chaw. Txhua lub ntsiab lus tau piav qhia txog qhov muaj cyst piv rau cov qauv qub thiab hom ntu. Qhov tshwm sim ntawm txhua tus qauv ntev (teeb tsa ntawm 50 bins) tau xam raws li qhov sib piv ntawm cov lej ntawm cov ntsiab lus nrog cystic annotation thiab tag nrho cov ntsiab lus hauv lub thawv.

Rau kev suav ntawm qhov nruab nrab glomerular interdistance, peb txiav txim siab rau txhua lub glomerulus qhov nruab nrab glomerular interdistance xam ntawm qhov ze tshaj 5 glomeruli.

Cov ntaub ntawv txheeb xyuas thiab txheeb cais

Cov ntaub ntawv raug nthuav tawm raws li txhais tau tias SEM. Qhov sib txawv ntawm cov pab pawg sim tau raug ntsuas los ntawm kev txheeb xyuas qhov sib txawv ua raws, thaum qhov tseem ceeb (P < {{0}}.05),="" los="" ntawm="" tukey-kramer="" test.="" thaum="" tsuas="" yog="" 2="" pawg="" tau="" muab="" piv,="" qhov="" kev="" sim="" mann-whitney="" tau="" siv.="" kev="" txheeb="" xyuas="" txheeb="" cais="" tau="" ua="" tiav="" siv="" graph="" prism="" software="" (san="" diego,="" ca,="" version="">

Qhia tawm

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

TXOJ CAI

Peb ua tsaug rau Laboratoire Experimentation Animale thiab Transgenese (LEAT), Histology thiab Imaging Platforms of Structure Federative de Recherche Necker rau kev pab. Peb ua tsaug rau Pierre Isnard, Marie-Claire Gubler, thiab Nicolas Kuperwasser rau kev nyeem ntawv tseem ceeb. Txoj haujlwm no tau txais kev txhawb nqa los ntawm Institut National de la Santé et de la Recherche Médicale, Université Paris Descartes, Assistance Publique – Hôpitaux de Paris, Agence Nationale de la Recherche, Whoami Laboratoire d'Excellence Whoam I, Roche Pharma Research thiab Early Development (Basel , Switzerland), thiab Institut Roche de Recherche et de Médecine Translationnelle (Paris, Fabkis).

AUTHOR CONTRIBUTIONS

TB, NG, thiab MZ tau tsim thiab ua qhov kev sim thiab tshuaj xyuas cov ntaub ntawv. FB, LT, MGT, thiab SG kuj tau ua qee qhov kev sim thiab tshuaj xyuas cov ntaub ntawv. MB thiab CN tau ua qhov kev sim nas. TB thiab MZ kuj tau pab sau cov ntawv sau. GF tau hloov kho cov ntawv sau. FT thiab MP tau muab lub tswv yim tsim thiab tsim kev kawm, saib xyuas qhov project, thiab sau ntawv.

cistanche tuaj yeem tonify raum

SUPPLEMENTARY KHOOM

Cov ntaub ntawv ntxiv (PDF)

Daim duab S1. Kev sim teeb tsa, kev yuav khoom, thiab kev ua duab.

Daim duab S2. Kev txwv ntawm autoflfluorescence teeb liab hauv xy thiab z daws teeb meem.

Daim duab S3. Daim duab ob sab ntawm kev hloov pauv ntawm PT thiab TL ntawm 4-mm paraffin-embedded raum seem.

Daim duab S4. Daim duab ob sab ntawm kev hloov pauv ntawm TAL thiab DCT ntawm 4-mm paraffin-embedded raum seem.

Daim duab S5. Daim duab ob sab ntawm kev hloov pauv ntawm DCT thiab CNT ntawm 4-mm paraffififin-embedded raum seem.

Daim duab S6. Peb-dimensional reconstruction nthuav tawm 3 qhov sib txawv ntawm cov tubules ze ze.

Daim duab S7. Peb-dimensional reconstruction qhia tau hais tias qhov ntev thiab segmentation ntawm nephrons tsis cuam tshuam los ntawm cysts.

Daim duab S8. Qhov ntom ntawm glomeruli hauv kev tswj thiab cystic ob lub raum.

Daim duab S9. Cov txheej txheem tau txais thiab tom qab kho cov duab ua tiav.

Tab S1. Kev sib piv ntawm txoj kev tshem tawm. Cov ntaub ntawv ntxiv (Movies)

Movie S1. Lectin staining markedly txhim kho qhov kev daws teeb meem thiab teeb liab-rau-tom qab piv.

Movie S2. Tracing ntawm txoj kev ntawm ib tug tubule.

Movie S3. Peb-dimensional reconstruction ntawm lub raum cleared qhia qhov hlws ris ntawm lub proximal tubule thiab nyias ceg ntawm lub Henle voj.

Movie S4. Peb-dimensional reconstruction ntawm lub raum cleared qhia qhov sib txuas ntawm cov tuab ascending limb ntawm Henle voj thiab lub distal convoluted tubule.

Movie S5. Peb-dimensional reconstruction ntawm lub raum cleared qhia qhov kev sib tshuam ntawm lub distal convoluted tubule thiab lub connecting tubule.

Movie S6. Cov duab thiab qhov loj ntawm proximal tubules txawv raws li lawv qhov tob.

Movie S7. Peb-dimensional nephron reconstruction nyob rau hauv tswj nas.

Movie S8. Tracing txoj kev ntawm lub nkoj los ntawm lub nkoj arcuate mus rau lub nkoj cortical radiate.

Movie S9. Cov hlab ntsha arcuate qauv cov duab ntawm cov juxtamedullary proximal tubules.

Movie S10. Nephrons nyiam pw hauv lub dav hlau.

Movie S11. Peb-dimensional nephron reconstruction qhia tau hais tias tus qauv tshwj xeeb rau kev loj hlob cyst.

Movie S12. Peb-dimensional nephron segmentation qhia tias cov hlwv loj hlob hauv cov nephron tshwj xeeb.

Movie S13. Kev sib koom ua ke thiab kev sib cuam tshuam ntawm nephrons thiab cov hlab ntsha hauv lub raum polycystic

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