Homozygous Dab1-// Yog Ib Qhov Tseem Ceeb Novel Ua Rau Autosomal Recessive Congenital Anomalies Ntawm Cov nas rau raum thiab zis

edmund.chen@wecistanche.com

Taw qhia

Yotari mutant nas, tau los ntawm kev nrhiav tau ntawm kev hloov pauv hauv Dab1 gene, nthuav tawm histological txawv txav hauv nruab nrab paj hlwb [1,2], zoo ib yam li cov reeler (reelin //) nas, tawm tswv yim tias reelin thiab DAB1 koom nrog. tib txoj kev taw qhia [1–4]. Cov aberrations hauv txoj kev reelin/DAB1 tau raug tshaj tawm tias muaj feem xyuam nrog ntau yam kev puas siab puas ntsws [5–7]. Interestingly, dhau li ntawm lub hauv nruab nrab lub paj hlwb, lub xub ntiag ntawm DAB1 thiab reelin proteins kuj tau lees paub nyob rau hauv lub peripheral paj hlwb thiab ib co extraneural ntaub so ntswg. Lub xub ntiag ntawm DAB1 tau pom nyob rau hauv nas podocytes [8] thiab tib neeg fetallub raum[9] thaum reelin qhia tau raug tshaj tawm thaum lub sij hawm nas thiab tib neeg fetal txoj kev loj hlob, nrog rau qee lub hlwb endothelial nrog cov hlab ntsha hauv cov neeg laus nas [9-11]. Txoj hauv kev canonical reelin / DAB1 tuaj yeem ua rau muaj qhov sib txawv hauv qab cov proteins, suav nrog NOTCH2 receptors, uas ua lub luag haujlwm tseem ceeb hauv kev txiav txim siab txoj hmoo ntawm tes thiab kev sib txawv thaum lub sijhawm.raumkev loj hlob [12–14]. NOTCH2 receptors yuav tsum tau tsim cov tubules thiab podocytes [13], tab sis ib zaug nephron maturation tiav, txoj kev no feem ntau silenced [15]. Hauv cov xwm txheej mob hnyavkab mob raum,Kev nthuav qhia ntau ntxiv ntawm NOTCH2 tuaj yeem ua rau muaj feem cuam tshuam rau kev tsim dua tshiab, thaum cov lus qhia txuas ntxiv yog ua rau cuam tshuam nrog interstitial fifibrosis thiab glomerulosclerosis [15]. Downregulation ntawm NOTCH signaling tuaj yeem txo qis autophagy thiab ua rau podocyte sib txawv tsis zoo thaum lub sij hawm kev loj hlob [16].

Yog li ntawd, podocytes ua haujlwm tsis zoo ua lub luag haujlwm tseem ceeb hauv cov kab mob glomerular, tshwj xeeb tshaj yog nyob rau hauv tib neeg idiopathic nephrotic syndrome [17,18] thiab hauv kev hloov pauv nephrotic syndrome (MCNS) [19]. Li no, autophagy tswj cellular homeostasis nyob rau hauv ib txwm physiological tej yam kev mob, whereas nyob rau hauv pathological mob, nws muaj peev xwm mus rau hauv autophagic tuag [20]. Ib tug dav siv autophagy biomarker yog LC3B, cov qauv protein ntawm autophagosomal daim nyias nyias [21]. Complex crosstalk ntawm autophagy thiab apoptosis kuj tseem txhawb nqa kev saib xyuas ntawm homeostatic tshuav nyiaj li cas raws li cov lus teb rau lub cell microenvironment. Nws yog qhov paub zoo tias cov xwm txheej apoptotic nce hauv tus lej thaum lub sijhawmraumkev loj hlob [22] thiab poob qis heev thaum maturation dhau lawm, tshwj tsis yog nyob rau hauv tej yam kev mob ntawmmob raum[23]. Undeniably, apoptosis yog ib txoj hauv kev tswj hwm los ntawm kev sib cuam tshuam ntawm ntau txoj hauv kev tab sis feem ntau ua tiav nrog kev ua kom muaj peev xwm ntawm caspase3 (CASP3), uas nws qhov kev ua haujlwm yog ib qho ntawm feem ntau siv cov cim rau kev kuaj pom apoptosis [23].

Hauv tsab ntawv tshaj tawm no, peb tsom los tshuaj xyuas seb Dab1 gene functional silence influences li casraummorphology thiab kev qhia thiab hauv zos ntawm reelin, NOTCH2, LC3B, thiab cleaved CASP3 proteins hauv cov nas tom qab yug me nyuamlub raum. Peb xav tias cov proteins no tau nthuav tawm hauv cov nas tom qab yug menyuamraum,thiab lawv cov kev sib txuam ua haujlwm pab txhawb rau kev saib xyuas ntawm lawv cov qauv thiab kev ua haujlwm. Tsis tas li ntawd, raws li lub xub ntiag ntawm DAB1 tau raug lees paub thaum lub sij hawm cev xeeb tub tib neegraumtxoj kev loj hlob [9], nws tuaj yeem xav tias nws qhov kev tsis ua haujlwm tuaj yeem ua rau muaj kev tsis sib haum xeeb nyob rau hauv ib qho kev nthuav dav ntawm congenital anomalies.raumthiab urinary tract (CAKUT).

Ntsiab lus:yotari; lub raum ua haujlwm; postnatal raum kev loj hlob; immunoflfluorescence staining; kis tau tus mob electron microscopy

CISTANCHE yuav txhim kho lub raum / raum ua haujlwm

Cov ntaub ntawv thiab cov txheej txheem

Qauv SauRaws li Dab1 null pa mutants, peb siv yotari (Dab1 / ) nas yav dhau los piav qhia los ntawm Howell li al. [24]. PCR primers siv rau genotyping ntawm nas yog yotari: GCC CTTCAGCATTCACCATGCT thiab CAGTGAGTACATATTGTGTGAGTTCC, tsiaj qus-hom Dab1 locus: GCCCTTCAGCATTCACCATGCT thiab CCTTGTTTCTTTTTTTTTAA-GGCTGT [5]. C57BL / 6 N nas tau tsa thiab pab pawg nyob hauv cov txheej txheem polycarbonate tawb (suav nrog tsawg kawg yog ib qho ntawm txhua tus genotype) nrog ad libitum nkag mus rau zaub mov thiab dej hauv qhov ntsuas kub (23 ± 2 ◦C) chav nrog lub teeb 12 h / lub voj voog tsaus. Hnub tom qab yug menyuam 4, 11 thiab 14 (P4, P11, thiab P14), nas tau muab tshuaj loog nrog pentobarbital thiab transcardially perfused rau 10 min nrog phosphate buffer saline (PBS, pH 7.2) thiab 4 feem pua ​​paraformaldehyde (PFA. ) hauv 0.1 M PBS.Lub raumraug tshem tawm thiab kho nrog 4 feem pua ​​​​PFA hauv 0.1 M PBS thaum hmo ntuj rau cov kev tshuaj ntsuam xyuas histological (hematoxylin–eosin (H&E), immunohistochemical thiab immunoflfluorescence staining) thiab hauv 2 feem pua ​​​​PFA ntxiv rau 2.5 feem pua ​​glutaraldehyde (GA) sib tov rau kev ntsuam xyuas electron microscope. Tom qab fifixation, cov ntaub so ntswg tau npaj rau kev kuaj histological ntxiv, raws li peb tau piav qhia yav dhau los [25,26].

Immunoflfluorescence thiab Immunoperoxidase StainingTom qab deparaffinization thiab rehydration, ntu tau rhaub rau 20 min nyob rau hauv lub 0.01 M citrate buffer (pH 6.0) nyob rau hauv ib tug dej steamer thiab, tom qab ntawd, txias mus rau chav tsev kub. Kev txwv tsis pub (ab 64226, Abcam, Cambridge, UK) tau thov rau 30 feeb kom tsis suav cov xim tsis muaj tseeb. Tom qab ntawd cov seem tau muab tso rau hauv cov av noo chamber thaum hmo ntuj nrog cov tshuaj tiv thaiv thawj (Table 1). Tom qab ntxuav hauv PBS, cov tshuaj tiv thaiv kab mob thib ob (Table 1) tau thov rau ib teev thiab ntxuav hauv PBS dua. Tom qab ntawd, cov nuclei tau stained nrog 4060 -diamidino-2-phenylindole (DAPI) rau 2 min, ntxuav hauv PBS, thiab npog. Peb tau ua qhov kev sim preadsorption kom txhua tus thawj cov tshuaj tiv thaiv tau siv nrog cov peptide sib raug thiab siv lawv cov kev sib xyaw ua ke rau cov ntu. Cov txiaj ntsig tau pom tias tsis muaj tshuaj tiv thaiv kab mob. Tsis tas li ntawd, kev tswj hwm nrog kev tshem tawm ntawm thawj cov tshuaj tiv thaiv kab mob los txiav txim siab qib ntawm kev sib khi tsis tshwj xeeb ntawm cov tshuaj tiv thaiv theem nrab tau ua.

Rau immunoperoxidase staining, luav polyclonal anti-CASP3 (1: 100 dilutions, ab13847, Abcam, Cambridge, UK) tau siv los ua thawj cov tshuaj tiv thaiv rau ib teev nyob rau hauv cov av noo chamber tom qab kho cov seem nrog 0.1 feem pua ​​H2O2. Tom qab ntxuav hauv PBS, kev tshawb nrhiav theem nrab tau ua tiav siv qhov txuas thiab streptavidin peroxidase, txhua lub sijhawm kaum tsib feeb (DakoCytomation, CA 93013 USA, LOT 03477) thiab diaminobenzidine (Dako, CA 93013 USA, LOT 10051369), ua tib zoo tswj xyuas keeb kwm yav dhau los. Nuclei tau stained nrog hematoxylin, yaug hauv dej kais dej rau 10 feeb, luv luv lub cev qhuav dej nyob rau hauv ascending series ntawm ethanol daws, thiab npog.

Kev npaj cov ntaub so ntswg rau Transmission Electron Microscope (TEM)Tom qab fifixation nrog sib tov ntawm 2 feem pua ​​PFA thiab 2.5 feem pua ​​GA hauv 0.1 M PBS rau 2 h, cov qauv raug ntxuav nrog PBS thiab postfixed nyob rau hauv ib tug aqueous tov ntawm 2 feem pua ​​osmium tetroxide rau 2 h. Tag nrho cov qauv raug ntxuav ob zaug hauv PBS, lub cev qhuav dej hauv qib nce qib ntawm ethanol thiab kos rau hauv Epon 812 (TAAB Laboratories Equipment, Reading, UK). Cov ntu ntu (70 nm hauv thickness) raug txiav ntawm Ultracut UCT ultramicrotome (Leica Microsystems, Wetzlar, Lub teb chaws Yelemees) thiab stained nrog 1 feem pua ​​​​uranyl acetate thiab lead citrate.

Kev Tshawb Fawb thiab Kev Tshawb FawbKev tsom xam tau ua nrog lub tshuab ntsuas epiflfluorescence (Olympus BX51, Tokyo, Nyiv) nruab nrog lub koob yees duab DP71 (Olympus), JEM 1400 kis tau tus mob electron microscope (JEOL, Tokyo, Nyiv) ua haujlwm ntawm 80 kV thiab thaij duab nrog JEOL them- coupled device (CCD) lub koob yees duab system (Advanced Microscopy Techniques, Danvers, MA, USA) thiab kawg, lub teeb ci-fifield microscope (BX40, Olympus, Tokyo, Nyiv). Tag nrho cov duab txheeb xyuas tau ua tiav nrog ImageJ software thiab Adobe Photoshop (Adobe, San Jose, CA, USA). Ib pab pawg neeg soj ntsuam, peb siv peb mus rau plaub tus tsiaj. Tag nrho cov saulub raumraug txiav ntawm 5 µm tuab seem, thiab siab tshaj plawsraum length determined by using these samples was reported. Mean proximal convoluted tubules (PCT), distal convoluted tubules (DCT) and glomeruli diameters were determined by averaging 100 structure diameters per analyzed sample. As nephron segments have irregular shapes, we took the largest diameter of every examined segment as representative. The staining intensity was semiquantitatively evaluated at four degrees: the absence of any reactivity (( ), mild reactivity (+), moderate reactivity (++), and strong reactivity (+++) (Table 2). The number of DAB1, reelin, NOTCH2, LC3B, and cleaved CASP3 immunoreactive cells was counted and expressed as a percentage of total cells. For each sample, we analyzed twenty PCT, DCT, and G at ×40 objective magnifification. We averaged the number of positive cells per group. Any level of nuclear, cytoplasmic, or membrane staining was regarded as positive. Three investigators analyzed the images independently. Interrater agreement was tested with interclass correlation analysis, which yielded a coeffificient >0.75, qhia pom zoo heev [27].

Kev txheeb caisKev kuaj ob-tailed t-test tau ua los tshuaj xyuas qhov sib txawv ntawm qhov nruab nrab txoj kab uas hla ntawm PCT, DCT thiab G ntawm cov tsiaj qus thiab yotari. Txoj kab uas hla tau nthuav tawm raws li txhais tau tias ± tus qauv sib txawv (SD). Qib ntawm qhov tseem ceeb tau teeb tsa ntawm p < 0.05.="" ib="" qho="" kev="" xeem="" anova="" ob="" txoj="" kev="" ua="" raws="" li="" tukey="" qhov="" kev="" sib="" piv="" ntau="" yam="" tau="" siv="" los="" tshuaj="" xyuas="" qhov="" sib="" txawv="" ntawm="" feem="" pua="" ​​​​ntawm="" cov="" hlwb="" zoo="" ntawm="" pct,="" dct,="" thiab="" glomeruli="" ntawm="" txhua="" lub="" sijhawm.="" qhov="" feem="" pua="" ​​​​ntawm="" cov="" hlwb="" zoo="" tau="" qhia="" raws="" li="" qhov="" nruab="" nrab="" ±="" tus="" qauv="" yuam="" kev="" ntawm="" qhov="" nruab="" nrab="" (sem).="" qib="" ntawm="" qhov="" tseem="" ceeb="" tau="" teeb="" tsa="" ntawm="" p=""><0.05. kev="" tsom="" xam="" tau="" ua="" tiav="" hauv="" graphpad="" software="" (graphpad="" software,="" la="" jolla,="" ca,="">

Cov txiaj ntsig

Hematoxylin-Eosin Staining (H&E) thiab ntsuas lub raum txoj kab uas hlaH&E staining ntawm midsagittal seem ntawm lublub raumnyob rau txhua lub sij hawm soj ntsuam qhia qhov me meraumphenotype ntawm yotari nas piv rau cov tsiaj qus (Daim duab 1a). Hauv 4P txhais tau tiasraumtxoj kab uas hla ntawm Yotari tsiaj yog nyob ib ncig ntawm 55 µm tshaj li ntawm 75 µm. Tsis tas li ntawd, qhov sib txawv no tau pom nyob rau tom qab lub sijhawm cov ntsiab lus vim qhov kev loj hlob qeeb qeeblub raumnyob rau hauv yotari piv rau cov kev loj hlob ntawm covlub raumnyob rau hauv cov tsiaj qus. Txhawm rau txiav txim siab seb qhov txo qis hauv tag nrhoraumqhov loj me yog tshwm sim los ntawm kev txo qis hauv nephron ntu loj, peb nruab nrab ntawm txoj kab uas hla ntawm PCT, DCT thiab G ib pab pawg. Qhov tseeb, muaj qhov txo qis ntawm txoj kab uas hla G, PCT thiab DCT hauv pawg yotari piv rau hom tsiaj qus (Daim duab 1b, p < 0.05).="" tsis="" tas="" li="" ntawd,="" h&e="" staining="" qhia="" thinner="" cortex="">lub raumpelvis extension hauv yotari tsiaj thiab me ntsis diffusely dilated DCT ntawm 14P. Cov qauv yooj yim thiab cov qauv ntawm glomerular maturation yog tib yam nyob rau hauv ob qho tib si kuaj tsiaj pawg.

Kev piav qhia Histological Analysis Raws li TEM MicrophotographsNtawm microphotographs tau los ntawm TEM, glomeruli ntawm cov nas qus tau pom cov tsos mob ntawm txhua qhov chaw ntawm fifiltration barrier, uas ib txwm tsim thiab pom tau (Daim duab 2a). Podocytes, cov txheej txheem podocyte ko taw thiab pedicles, fifiltration slits, glomerular hauv qab daim nyias nyias, thiab cov capillary lumen nrog fenestrated endothelium tau yooj yim txawv (Daim duab 2a). Ntawm qhov tod tes, nyob rau hauv glomeruli ntawm yotari nas, kev loj hlob ntawm podocyte kev puas tsuaj nrog ko taw txheej txheem effacement thiab tsis muaj fifiltration slits yuav raug soj ntsuam (Daim duab 2b-d). Cov kev hloov ultrastructural no tau pom nyob rau hauv tag nrholub raumkuaj thiab koom nrog feem ntau ntawm cov tshuaj glomeruli. Tsis muaj qhov txawv txav tau pom hauv PCT lossis DCT ntawm cov nas yotari dua li cov tsiaj qus (Daim duab 2e–h). Hauv PCT, cell interfaces tau txhais tsis zoo vim yog qhov tsis sib xws ntawm cell membranes thiab cell interdigitation nrog lawv cov neeg nyob ze. Lub cytoplasm muaj ntau, muaj cov nuclei zoo sib xws, elongated mitochondria, basal infoldings, thiab ntau lub qhov hauv tubular nruab nrab ntawm microvilli, uas tsim ib qho txhuam ciam teb ntawm apical membrane. Kev nthuav dav ntau dua tau nthuav tawm qhov apical nto ntawm PCT epithelial hlwb uas muaj cov microvilli ntev los tsim cov txhuam ciam teb thiab nruj junctions ntawm luminal cell ciam teb ntawm cov nyob sib ze tubular epithelial hlwb (Daim duab 2e, f). Ntawm qhov tod tes, qhov chaw apical ntawm DCT epithelial hlwb muaj ob peb lub microvilli luv thiab ntau lub vesicles (Daim duab 2g, h).

Daim duab 2. Transmission electron microscope (TEM) microphotographs of wild-type and yotarilub raum.Glomeruli ntawm cov tsiaj qus (a) pom ib txwm tsim fifiltration barrier nrog fenestrated endothelium (E) ntawm capillary (C), hauv qab daus membrane (BM), podocytes (P) nrog pedicles (PE), thiab fifiltration slits (FS ). Hauvlub raumntawm yotari tsiaj, effacement ntawm pedicles thiab tsis muaj fifiltration slits yuav pom (asterisks, b-d). Tsis muaj qhov txawv txav tau pom nyob rau hauv cov kab mob sib kis (PCT) lossis cov kab mob sib kis (DCT) ntawm cov nas yotari piv rau cov tsiaj qus (e–h). Hauv PCT, cov cytoplasm muaj ntau, muaj cov nuclei zoo sib xws (N), elongated mitochondria (M), basal infoldings (BI), thiab ntau cov tubular pits (TP) ntawm microvilli, uas tsim ib tug txhuam ciam (BB) ntawm lub apical membrane (e, f). Kev nthuav dav ntau dua tau nthuav tawm qhov chaw apical ntawm PCT epithelial hlwb uas muaj cov microvilli ntev los ua tus txhuam ciam teb thiab nruj junctions (TJ) ntawm cov luminal cell ciam teb ntawm cov nyob sib ze tubular epithelial hlwb (e,f). Ntawm qhov tod tes, lub apical nto ntawm DCT epithelial hlwb uas muaj ob peb luv microvilli thiab ntau vesicles (g, h). Qhov ntsuas ntsuas hauv cov duab (a–d) thiab insets (e–h) yog 1 µm; qhov ntsuas bar hauv cov duab (e–h) yog 2 µm.

Localization and Colocalization of Reelin and DAB1Reelin tau hais tawm tsis muaj zog nrog me me mus rau nruab nrab reactivity (Table 2) nyob rau hauv lub glomeruli thiab DCT ntawm tag nrho cov tsiaj kuaj nyob rau hauv tag nrho cov soj ntsuam lub sij hawm cov ntsiab lus (p < 0.05,="" daim="" duab="" 3a).="" tsuas="" yog="" nyob="" rau="" hauv="" pct="" ntawm="" yotari="" nas="" yog="" feem="" pua="" ​​​​ntawm="" cov="" hlwb="" zoo="" dua="" li="" cov="" tsiaj="" qus="" (p=""><0.05, daim="" duab="" 3a).="" hauv="" cov="" hlwb="" glomerular,="" qhov="" chaw="" ntawm="" staining="" yog="" perinuclear,="" thaum="" nyob="" hauv="" pct="" thiab="" dct,="" nws="" tau="" tawg="" thoob="" plaws="" hauv="" cytoplasm="" (daim="" duab="">

igure 4. Immunofluorescence staining ntawm postnatal yotarilub raumnrog reelin marker (a) thiab ob chav immunofluorescence staining ntawm postnatal qus-homlub raumnrog DAB1 thiab reelin markers (b). (a,b) Nuclear DNA DAPI staining merged nrog DAB1, thiab reelin immunofluorescence yog qhia nyob rau hauv parallel (merge). Cov ntsiab lus pom lub sijhawm yog P4, P11, P14. Kev nthuav qhia ntawm cov cim kuaj hauv lub glomeruli (G), cov tubules sib thooj (PCT) thiab cov tubules distal convoluted (DCT) yog cim nrog cov xub, thaum lub hnub qub qhia txog kev qhia ntawm reelin hauv extracellular matrix (a). Inserts qhia cov lus qhia tseem ceeb tshaj plaws hauv daim duab. DAPI nuclear staining qhia tsis zoo colocalization ntawm DAB1 thiab reelin, feem ntau nyob rau hauv DCT (lub taub hau). Scale bar yog 20 µm thiab hais txog tag nrho cov duab.

Yuav luag tsis muaj lub cev tiv thaiv kab mob ntawm DAB1 hauv glomeruli thiab PCT ntawm cov tsiaj qus ntawm P4, thaum feem pua ​​​​ntawm cov hlwb zoo nrog me me reactivity (Table 2) tau nce ntau ntawm P11 thiab P14 (p < 0.05,="" daim="" duab="" 3b).="" hauv="" dct,="" dab1="" feem="" ntau="" tau="" nthuav="" tawm="" ntawm="" apical="" thiab="" lateral="" seem="" ntawm="" cell="" membranes="" (daim="" duab="" 4b)="" nrog="" muaj="" zog="" reactivity="" (table="" 2).="" qhov="" feem="" pua="" ​​​​ntawm="" cov="" hlwb="" zoo="" tau="" nce="" siab="" dua="" hauv="" cov="" qauv="" dhau="" los,="" nyob="" ib="" ncig="" ntawm="" 60="" feem="" pua="" ​​(daim="" duab="" 3b),="" tshwj="" xeeb="" tshaj="" yog="" nyob="" rau="" hauv="" macula="" densa="" (daim="" duab="" 4b).="" yuav="" luag="" tsis="" muaj="" colocalization="" ntawm="" dab1="" thiab="" reelin="" tshwj="" tsis="" yog="" hauv="" dct="" ntawm="" cov="" tsiaj="" qus="" ntawm="" p14="" (lub="" taub="" hau,="" daim="" duab="" 4b).="" biomolecules="" 2021,="" 11,="" 609="" 8="" ntawm="" 14="" daim="" duab="" 4.="" immunoflfluorescence="" staining="" ntawm="" postnatal="">lub raumnrog reelin marker (a) thiab ob chav immunoflfluorescence staining ntawm postnatal qus-homlub raumnrog DAB1 thiab reelin markers (b). (a,b) Nuclear DNA DAPI staining merged nrog DAB1, thiab reelin immunoflfluorescence yog qhia nyob rau hauv parallel (merge). Cov ntsiab lus pom lub sijhawm yog P4, P11, P14. Kev nthuav qhia ntawm cov cim kuaj hauv lub glomeruli (G), cov tubules sib thooj (PCT) thiab cov tubules distal convoluted (DCT) yog cim nrog cov xub, thaum lub hnub qub qhia txog kev qhia ntawm reelin hauv extracellular matrix (a). Inserts qhia cov lus qhia tseem ceeb tshaj plaws hauv daim duab. DAPI nuclear staining qhia tsis zoo colocalization ntawm DAB1 thiab reelin, feem ntau nyob rau hauv DCT (lub taub hau). Scale bar yog 20 µm thiab hais txog tag nrho cov duab. Yuav luag tsis muaj lub cev tiv thaiv kab mob ntawm DAB1 hauv glomeruli thiab PCT ntawm cov tsiaj qus ntawm P4, thaum feem pua ​​​​ntawm cov hlwb zoo nrog me me reactivity (Table 2) tau nce ntau ntawm P11 thiab P14 (p <0.05, daim="" duab="" 3b).="" hauv="" dct,="" dab1="" feem="" ntau="" tau="" nthuav="" tawm="" ntawm="" apical="" thiab="" lateral="" seem="" ntawm="" cell="" membranes="" (daim="" duab="" 4b)="" nrog="" muaj="" zog="" reactivity="" (table="" 2).="" qhov="" feem="" pua="" ​​​​ntawm="" cov="" hlwb="" zoo="" yog="" qhov="" tseem="" ceeb="" tshaj="" li="" cov="" qauv="" yav="" dhau="" los,="" nyob="" ib="" ncig="" ntawm="" 60="" feem="" pua="" ​​(daim="" duab="" 3b),="" tshwj="" xeeb="" tshaj="" yog="" nyob="" rau="" hauv="" macula="" densa="" (daim="" duab="" 4b).="" yuav="" luag="" tsis="" muaj="" colocalization="" ntawm="" dab1="" thiab="" reelin="" tshwj="" tsis="" yog="" nyob="" rau="" hauv="" dct="" ntawm="" cov="" tsiaj="" qus="" ntawm="" p14="" (lub="" taub="" hau,="" daim="" duab="">

Spatial thiab Temporal Expression Patterns ntawm NOTCH2 thiab LC3BQhov feem pua ​​​​ntawm NOTCH{{0}} cov hlwb zoo yog tsawg dua 20 feem pua ​​​​hauv glomeruli ntawm ob hom tsiaj ntawm txhua lub sijhawm pom. Tsuas yog ntawm P14 feem pua ​​​​ntawm cov hlwb zoo tau nce siab dua hauv glomeruli ntawm cov tsiaj yotari dua li cov tsiaj qus (p <0.05, daim="" duab="" 3c).="" qhov="" kev="" siv="" staining="" yog="" me="" me="" mus="" rau="" nruab="" nrab="" (table="" 2),="" feem="" ntau="" nyob="" hauv="" perinuclearly="" (daim="" duab="" 5a–f).="" hauv="" pct="" thiab="" dct="" ntawm="" ob="" qho="" tib="" si="" tsiaj="" genotypes="" feem="" pua="" ​​​​ntawm="" cov="" hlwb="" zoo="" tau="" nce="" mus="" txog="" lub="" sijhawm.="" ntawm="" p14,="" qhov="" feem="" pua="" ​​​​ntawm="" cov="" hlwb="" zoo="" tau="" nce="" siab="" dua="" hauv="" pct="" thiab="" dct="" ntawm="" cov="" tsiaj="" yotari="" dua="" li="" pct="" thiab="" dct="" ntawm="" cov="" tsiaj="" qus="" (p=""><0.05, daim="" duab="" 3c).="" lub="" teeb="" liab="" siv="" ntawm="" txhua="" lub="" sijhawm="" pom="" cov="" ntsiab="" lus="" yog="" me="" me="" mus="" rau="" nruab="" nrab="" (table="" 2)="" thiab="" tawg="" mus="" thoob="" plaws="" hauv="" cytoplasm="" (daim="" duab="" 5a–f).="" daim="" duab="" 5.="" immunofluorescence="" staining="" ntawm="" postnatal="" qus-hom="" thiab="">lub raumnrog NOTCH2 thiab LC3B cov cim thiab immunoperoxidase staining nrog cleaved caspase3 (CASP3) marker. (a–f) Nuclear DNA DAPI staining yog merged nrog NOTCH2 thiab LC3B. Cov ntsiab lus pom lub sijhawm yog P4, P11, P14. Kev nthuav qhia ntawm NOTCH2 thiab LC3B cov cim hauv glomeruli (G), proximal convoluted tubules (PCT) thiab distal convoluted tubules (DCT) yog cim nrog cov xub. Tshwj xeeb tshaj yog muaj zog LC3B reactivity tuaj yeem pom hauv Bowman capsule ntawm glomeruli decaying (asterisk, e). Inserts qhia cov lus qhia tseem ceeb tshaj plaws hauv daim duab. CASP3 tau qhia tsis zoo nyob rau hauv txhua lub sijhawm pom zoo. Qhov tseem ceeb tsuas yog qhia nyob rau hauv lub glomeruli ntawm yotari nas ntawm P14 ( xub). Scale bar yog 20 µm thiab hais txog tag nrho cov duab. Biomolecules 2021, 11, 609 9 ntawm 14 3.4. Spatial thiab Temporal Expression Patterns ntawm NOTCH2 thiab LC3B Qhov feem pua ​​​​ntawm NOTCH2- cov hlwb zoo yog tsawg dua 20 feem pua ​​​​hauv glomeruli ntawm ob qho tib si tsiaj genotypes ntawm txhua lub sijhawm pom. Tsuas yog ntawm P14 feem pua ​​​​ntawm cov hlwb zoo yog qhov tseem ceeb hauv cov glomeruli ntawm cov tsiaj yotari dua li cov tsiaj qus (p <0.05, daim="" duab="" 3c).="" qhov="" kev="" siv="" staining="" yog="" me="" me="" mus="" rau="" nruab="" nrab="" (table="" 2),="" feem="" ntau="" nyob="" hauv="" perinuclearly="" (daim="" duab="" 5a–f).="" hauv="" pct="" thiab="" dct="" ntawm="" ob="" qho="" tib="" si="" tsiaj="" genotypes="" feem="" pua="" ​​​​ntawm="" cov="" hlwb="" zoo="" tau="" nce="" mus="" txog="" lub="" sijhawm.="" ntawm="" p14,="" qhov="" feem="" pua="" ​​​​ntawm="" cov="" hlwb="" zoo="" yog="" qhov="" tseem="" ceeb="" hauv="" pct="" thiab="" dct="" ntawm="" cov="" tsiaj="" yotari="" dua="" li="" pct="" thiab="" dct="" ntawm="" cov="" tsiaj="" qus="" (p=""><0.05, daim="" duab="" 3c).="" lub="" teeb="" liab="" siv="" ntawm="" txhua="" lub="" sijhawm="" pom="" cov="" ntsiab="" lus="" yog="" me="" me="" mus="" rau="" nruab="" nrab="" (table="" 2)="" thiab="" tawg="" mus="" thoob="" plaws="" hauv="" cytoplasm="" (daim="" duab="">

Daim duab 5. Immunoflfluorescence staining ntawm postnatal qus-hom thiab yotarilub raumnrog NOTCH2 thiab LC3B cov cim thiab immunoperoxidase staining nrog cleaved caspase3 (CASP3) marker. (a–f) Nuclear DNA DAPI staining yog merged nrog NOTCH2 thiab LC3B. Cov ntsiab lus pom lub sijhawm yog P4, P11, P14. Kev nthuav qhia ntawm NOTCH2 thiab LC3B cov cim hauv glomeruli (G), proximal convoluted tubules (PCT) thiab distal convoluted tubules (DCT) yog cim nrog cov xub. Tshwj xeeb tshaj yog muaj zog LC3B reactivity tuaj yeem pom hauv Bowman capsule ntawm glomeruli decaying (asterisk, e). Inserts qhia cov lus qhia tseem ceeb tshaj plaws hauv daim duab. CASP3 tau qhia tsis zoo nyob rau hauv txhua lub sijhawm pom zoo. Tsuas yog qhov tseem ceeb tshaj plaws yog nyob rau hauv glomeruli ntawm yotari nas ntawm P14 ( xub). Scale bar yog 20 µm thiab hais txog tag nrho cov duab.

Hauv glomeruli thiab DCT ntawm ob hom tsiaj genotypes, feem pua ​​​​ntawm LC3B-zoo hlwb tau nce los ntawm lub sijhawm (p < 0.05,="" daim="" duab="" 3d).="" ntawm="" p11="" thiab="" p14,="" qhov="" feem="" pua="" ​​​​ntawm="" cov="" hlwb="" zoo="" yog="" qhov="" tseem="" ceeb="" hauv="" cov="" glomeruli="" ntawm="" cov="" tsiaj="" yotari="" dua="" li="" cov="" glomeruli="" ntawm="" cov="" tsiaj="" qus="" (p="">< 0.05,="" daim="" duab="" 3d).="" nyob="" rau="" hauv="" lub="" glomeruli="" ntawm="" yotari="" tsiaj="" staining="" siv="" feem="" ntau="" yog="" nruab="" nrab="" mus="" rau="" muaj="" zog="" (table="" 2)="" nyob="" rau="" hauv="" lub="" perinuclear="" thiab="" nuclear="" chaw="" (daim="" duab="" 5a-f),="" nyob="" rau="" hauv="" tag="" nrho="" cov="" pom="" lub="" sij="" hawm="" cov="" ntsiab="" lus,="" thaum="" nyob="" rau="" hauv="" lub="" glomeruli="" ntawm="" cov="" tsiaj="" qus-hom="" nas,="" qhov="" kev="" siv="" yog.="" feem="" ntau="" nruab="" nrab="" (table="" 2).="" pct="" ntawm="" yotari="" thiab="" cov="" tsiaj="" qus="" muaj="" nyob="" ib="" ncig="" ntawm="" 20="" feem="" pua="" ​​​​ntawm="" cov="" hlwb="" zoo="" ntawm="" txhua="" lub="" sijhawm="" pom="" (p=""><0.05, daim="" duab="">

Cleaved CASP-3 Cov lus qhiaYuav luag tsis muaj kev qhia txog CASP-3 nyob rau hauvlub raumntawm cov tsiaj qus ntawm txhua lub sijhawm pom. Hauv PCT thiab DCT ntawm cov nas yotari, ntau lub hlwb yog CASP-3-zoo (Daim duab 3e). Tsuas yog nyob rau hauv glomeruli ntawm lub raum yotari ntawm P14 (Daim duab 5e), qhov feem pua ​​​​ntawm cov hlwb zoo tau nce ntxiv piv raulub raumntawm cov nas qus (p < 0.05, Daim duab 3e).

Kev sib tham

Lub raummorphogenesis thiab kev loj hlob yog cov txheej txheem nyuaj precisely sib koom tes los ntawm kev sib cuam tshuam ntawm ntau cov noob. Congenital anomalies ntawm tus kab mobraumthiab urinary tract (CAKUT) yog qhov kev yug me nyuam ntau tshaj plaws uas muaj 23 feem pua ​​​​ntawm tag nrho cov teeb meem no thiab sawv cev rau qhov ua rau muaj qhov kawg.mob raumhauv me nyuam [28,29]. Cov kab mob ib leeg tuaj yeem yog qhov tseem ceeb ntawm CAKUT, thiab txog tam sim no, kev hloov pauv hauv ntau dua 20 cov noob tau raug txheeb xyuas tias yog qhov ua rau CAKUT [30]. Raws li peb cov hauj lwm yav dhau los tau qhia zoo heev ntawm DAB1 thaum lub sij hawm cev xeeb tub tib neegraumkev loj hlob [9], peb xav tias DAB1 tuaj yeem ua lub luag haujlwm tseem ceeb thaum lub sij hawm mammalianraumkev loj hlob. Txhawm rau ua pov thawj peb qhov kev xav, peb tau tshawb xyuas qhovraummorphology thiab qhia cov qauv ntawm reelin, NOTCH2, LC3B, thiab qhib CASP3 proteins hauv Dab1 knockout nas.

CISTANCHE yuav txhim kho lub raum / raum mob

Sectioning los ntawm cov nas yotarilub raumnthuav tawm lub cortex thinner thiab ib qho tseem ceeb me meraumthiab PCT, DCT thiab G diameters piv rau cov tsiaj qus. Qhov txo qis hauvraumqhov loj los ntawm qhov tsis txaus nephron endowment yog hu ualub raumhypoplasia, ib qho ntawm feem ntau CAKUT mob, uas predisposes neeg laus-pib kub siab thiab mob raum kab mob [31]. Tsis tas li ntawd, cov microphotographs ntes nrog TEM tau qhia tias cov nas yotari tau nthuav tawm cov kev puas tsuaj podocyte tseem ceeb nrog cov txheej txheem ko taw effacement thiab tsis muaj fifiltration slits. Tom qab qhov raug mob, podocytes tau txais cov txheej txheem ntawm effacement uas lawv poob lawv cov qauv, ua rau txo qis hauv lawv cov fifiltration barrier muaj nuj nqi [32]. Txhua hom mob nephrotic syndrome, nrog rau focal segmental glomerulosclerosis (FSGS), yog tus cwj pwm los ntawm qhov tsis xws luag hauv cov qauv podocyte lossis kev ua haujlwm [33].

Peb txoj kev tshawb fawb tam sim no tau pom qhov kev qhia siab tshaj plaws ntawm DAB1 ntawm apical thiab lateral seem ntawm cell membranes ntawm DCT, thaum REELIN feem ntau tau tawg mus thoob plaws hauv cytoplasm ntawm PCT. Ntawm lub sijhawm tshawb xyuas cov ntsiab lus, tsis muaj kev hloov pauv morphological tau pom hauv tubuli ntawm cov nas yotari, tab sis kev tshawb nrhiav ntxiv yog tsim nyog los piav qhia lub luag haujlwm ntawm DAB1 hauv tubulointerstitial compartments. Muaj qee zaus colocalization ntawm DAB1 thiab REELIN proteins, feem ntau hauv DCT. Cov fifindings no yog ua raws li peb cov hauj lwm yav dhau los hais txog kev qhia ntawm ob cov proteins thaum lub sij hawm cev xeeb tub tib neegraumkev loj hlob [9]. Qhov no yuav qhia tau tias DAB1 thiab reelin muaj lub luag haujlwm zoo sib xws hauv tib neeg thiab naslub raumlos ntawm kev ua kom muaj qee yam ntawm txoj kev qis qis, xws li Crk, MAPK thiab PI3K/Akt/mTOR signaling cascades [34–36]. Peb cov ntaub ntawv kuj tau qhia txog kev nthuav dav reelin hauv cov glomeruli ntawm cov nas yotari. Interestingly, ib qho kev tshawb fawb yav dhau los tau qhia tias DAB1 phosphorylation thiab nce reelin qhia nyob rau hauv glomeruli yog nrog los ntawm kub siab, proteinuria, thiab podocyte raug mob nyob rau hauv Ang II-infused nas [10].

Tsis tas li ntawd, immunofluorescence staining qhia ib qho kev nthuav qhia ntawm NOTCH2 receptors nyob rau hauv lub glomeruli ntawm P14 yotari nas. Nws yog qhov paub zoo tias NOTCH2 kev qhia yog downregulated ib zaug nephron maturation tiav, tshwj tsis yog nyob rau hauv cov xwm txheej ntawmmob raum,xws li ntshav qab zib nephropathy thiab FSGS [15,37]. Cov nas uas muaj Adriamycin-induced nephrotic syndrome kuj pom tau tias muaj kev nthuav qhia ntau ntxiv ntawm activated NOTCH2, uas ameliorates fifibrosis [38]. Ntawm lub sijhawm tshawb xyuas cov ntsiab lus, peb tsis tau pom tias muaj fifibrosis ntau ntxiv, tab sis nws tseem yuav tsum tau qhia meej lub luag haujlwm ntawm NOTCH2 hauv postnatal yotari.lub raum.Kev soj ntsuam ntxiv rau seb puas muaj kev hloov pauv fifibrotic tshwm sim nyob rau theem tom ntej yuav tsim nyog. Txawm li cas los xij, twb muaj qee qhov kev tshawb nrhiav uas qhia tias qhov cuam tshuam luv luv ntawm kev ua kom NOTCH2 nce ntxiv yog cuam tshuam nrog kev muaj sia nyob muaj txiaj ntsig zoo rau cov neeg raug mob podocytes, uas tau ploj mus ntev, xws li mob ntshav qab zib nephropathy [39].

Siab dua LC3B qhia nyob rau hauv lub glomeruli ntawm P11 thiab P14 yotari nas tej zaum yuav re-flflated ib tsub zuj zuj ntawm autophagosomes nyob rau hauv lub podocytes. Kom meej meej qhia tias autophagy tau nrawm. Nws yog ib qho tsim nyog los txheeb xyuas qhov piv ntawm LC3-II thiab LC3-I cov kab lus protein. Raws li ib txwm muaj, autophagy yog qhov tsim nyog rau qhov qublub raum ua haujlwm[40], tshwj xeeb tshaj yog nyob rau hauv podocytes. Vim tias nws muaj peev xwm tsawg rau kev faib tawm ntawm tes thiab hloov pauv, lawv pom muaj qib siab ntawm autophagy [41]. Txawm hais tias qhov kev tshawb nrhiav no, qhov nce hauv LC3B qhia tau raug tshaj tawm hauv ntau cov kab mob glomerular [42-44], feem ntau yog koom nrog cov txheej txheem ko taw effacement thiab nephrotic syndrome txoj kev loj hlob, raws li pom hauv prorenin receptor conditional knockout nas [45,46]. Tag nrho cov kev tshawb fawb no qhia txog kev tiv thaiv lub luag haujlwm ntawm kev txhim kho autophagy hauv cov kab mob pathological, txhawb ntxiv los ntawm kev tshawb nrhiav ntawm Adriamycin-induced nephropathy, qhov twg autophagy tau qhib los tiv thaiv kev raug mob podocyte [43], nrog rau cov hnub nyoog-nyob ntawm cov kab mob glomerular uas nws qeeb. qhov kev nce qib ua haujlwm poob ntawm lublub raum ua haujlwm[40]. Kev tsom xam ntawm tib neegraumbiopsies kuj tau pom cov pov thawj ntawm kev nce autophagy hauv kev koom tes nrog cov txheej txheem ko taw effacement hauv ntau cov kab mob glomerular, suav nrog kev hloov pauv nephrotic syndrome (MCNS), uas yog ib qho ntawm feem ntau ua rau idiopathic nephrotic syndrome [19]. Hauv cov mob ntshav qab zib nephropathy thiab lipopolysaccharide-induced mob hnyavmob raum,Txhim kho autophagy nrog qee qhov kev kho mob los ntawm inhibition ntawm PI3K / AKT / mTOR txoj hauv kev ua kom lub raum ua haujlwm [47,48]. Tag nrho cov fifindings hais tias autophagy muaj ib qho tseem ceeb cytoprotective lub luag hauj lwm rau kev nyuaj siab hloov nyob rau hauvmob raum, thiab nws qhov kev hloov pauv yuav yog qhov kev cog lus kho mob zoo, txawm hais tias, nyob rau qee qhov xwm txheej, autophagy tuaj yeem ua rau muaj kev puas tsuaj thiab nce mus rau hauv kev tuag ntawm tes.

CISTANCHE yuav txhim kho lub raum / raum tsis ua haujlwm

Tsuas yog qhov nce hauv autophagy, nce qib ntawm apoptosis kuj tuaj yeem pom nyob rau hauv glomeruli ntawm P14 yotari nas. Autophagy feem ntau tiv thaiv apoptosis, tab sis nyob rau hauv cov xwm txheej tshwj xeeb, nws tuaj yeem muaj qhov cuam tshuam rau kev ciaj sia ntawm tes los ntawm kev txhim kho thiab txhawb kev apoptosis [42]. Tom qab yug me nyuam, apoptosis nyob rau hauv lublub raumyog heev diminished, tsuas yog nyob rau hauv tej yam kev mob ntawmmob raum,xws li idiopathic nephrotic syndrome uas pab txhawb kev tsim tus kab mob [23]. muaj peev xwm tshiab ua rau autosomal recessive congenital anomalies ntawm lubraumthiab tso zis. Txawm li cas los xij, lub luag haujlwm tseem ceeb ntawm DAB1 thaum lub sijhawmraumtxoj kev loj hlob tseem tsis meej. Tsis tas li ntawd, cov nas no tau pom cov txheej txheem ko taw txawv txav thiab nce qib ntawm reelin, NOTCH2, LC3B thiab cleaved CASP3 proteins nyob rau hauv glomeruli uas yuav ua rau glomerular raug mob, xws li nephrotic syndrome, uas nyob rau hauv correlation nrog hypoplasia, tej zaum yuav ua rau tuag taus. ntawm cov tsiaj no thaum lub sij hawm weaning. Txhawm rau txheeb xyuas qhov kev xav no, nws yuav tsum tau muab cov ntaub ntawv ntxiv txog ntshav siab thiab lwm yam kev kuaj mob hauv tsev kho mob, xws li qib ntshav creatinine, albumin, thiab cov proteins.