Tib neeg Alpha Defensin 5 Kev Tshaj Tawm hauv Tib Neeg Lub raum Thiab Cov Kab Mob urinary
Mar 16, 2022
Yog xav paub ntxiv: ali.ma@wecistanche.com
John David Spencer et al
Abstract
Keeb kwm:Cov txheej txheem tswj kev ua kom tsis muaj menyuam hauv cov zis yog nkag siab tsis tiav. Cov kev tshawb fawb tsis ntev los no tau hais txog qhov tseem ceeb ntawm cov tshuaj tiv thaiv kab mob peptides (AMP) hauv kev tiv thaiv kab mob urinary los ntawm kev kis kab mob. Ntawm no, peb qhia txog kev qhia thiab qhov tseeb ntawm AMP human alpha-defensin 5 (HD5) hauv tib neegraumthiab urinary ib ntsuj av nyob rau hauv ib txwm thiab muaj kab mob.
Methodology/Principal Findings:Siv RNA cais los ntawm tib neegraum, ureter, thiab zais zis cov ntaub so ntswg, peb tau ua kom muaj nuj nqis ntawm PCR lub sij hawm los qhia tias DEFA5, cov noob encoding HD5, yog constitutively qhia thoob plaws hauv cov zis. Nrog pyelonephritis, DEFA5 qhia tau nce ntxiv hauv covraum. Siv cov tshuaj immunoblot, HD5production kuj nce nrog pyelonephritis. Immunostaining localized HD5 rau urothelium ntawm lub zais zis thiab ureter. Nyob rau hauv lub raum, HD5 feem ntau yog tsim nyob rau hauv lub distal nephron thiab sau tubules. Siv cov tshuaj immunoblot thiab ELISA, HD5 tsis niaj hnub kuaj pom nyob rau hauv tib neeg cov zis tsis muaj kab mob thaum lub ntsiab lus tso zis HD5 ntau ntxiv nrog E.coli urinary tract infection.
Cov ntsiab lus / qhov tseem ceeb:DEFA5 tau qhia thoob plaws hauv cov zis hauv cov neeg uas tsis muaj kab mob. Tshwj xeeb, HD5 yog qhia thoob plaws hauv lub urothelium ntawm sab urinary ib ntsuj av thiab nyob rau hauv lub sau tubules.raum. Nrog kev kis tus kab mob, HD5 kev qhia nce ntxiv hauv covraum, thiab qib yuav kuaj tau hauv cov zis. Rau peb txoj kev paub, peb qhov kev tshawb pom sawv cev rau thawj zaug kom muaj nuj nqis HD5 kev qhia thiab kev tsim khoom hauv tib neeg lub raum. Ntxiv mus, qhov no yog thawj daim ntawv tshaj tawm los kuaj pom muaj HD5 nyob rau hauv cov zis kuaj kab mob. Peb cov txiaj ntsig tau qhia tias HD5 tuaj yeem muaj lub luag haujlwm tseem ceeb hauv kev tswj cov zis tsis muaj menyuam.
Cistanche phiv thiab Cistanche tubulosa rauraumkab mob, nyem qhov no kom tau txais cov qauv
Taw qhia
Cov kab mob urinary, sib nrug los ntawm cov urethral meatus, feem ntau yog tsis muaj menyuam, txawm tias nws qhov sib thooj nrog fecal flora. Cov txheej txheem meej meej uas cov kab mob urinary tswj nws txoj kev tsis muaj menyuam tsis nkag siab zoo. Cov txheej txheem uas txhawb nqa kev tiv thaiv ntawm cov zis muaj xws li tso zis, hloov pauv hauv cov zis pH thiab osmolarity, lub zais zis tsis tu ncua, tshuaj tiv thaiv cov khoom ntawm uroepithelium, thiab epithelial shedding / influx ntawm cov tshuaj tiv thaiv kab mob nrog cov kab mob stimulation [1]. Tsis tas li ntawd, antimicrobial peptides (AMPs) tsis ntev los no tau pom tias muaj lub luag haujlwm tseem ceeb hauv kev tswj cov kab mob urinary sterility [2] .AMPs, uas yog cov tshuaj tua kab mob ntuj tsim los ntawm ze li ntawm txhua yam kab mob, yog ib qho tseem ceeb ntawm cov kab mob hauv lub cev. AMPs yog cationic molecules qhia los ntawm phagocytic dawb hlwb thiab epithelial hlwb. Hauv tib neeg thiab lwm yam tsiaj tsiaj, defensins yog ib tsev neeg loj ntawm AMPs. Defensins feem ntau muaj dav-spectrum antimicrobial kev ua ub no tiv thaiv kab mob gram-positive thiab gram-negative kab mob, kab mob, fungi, thiab protozoa [2,3]. Defensins tau pib tsim los ua preproteins thiab ua tiav los ua kom paub tab, lom neeg peptides [4]. Hauv tib neeg, cov tshuaj tiv thaiv tau muab faib ua ib qho ntawm ob tsev neeg nyob ntawm lawv cov kab mob disulfide bridging - cov alpha-defensins lossis beta-defensins [5].
Hauv cov zis, beta-defensins tau nthuav dav thoob plaws hauv uroepithelium. Cov kab mob epithelial nyob rau hauv cov ntaub so ntswgraumLoop ntawm Henle, distal tubule, thiab sau duct constitutively qhia human beta-defensin 1 (hBD1). Txawm hais tias urinary theem ntawmhBD1 tsis txaus los tua cov kab mob invading, hBD1 muab cov tshuaj tua kab mob ceev ceev ntawm tubular lumens thiab tiv thaiv kab mob los ntawm inhibiting kab mob txuas rau urothelium [6]. Cov kev tshawb fawb tsis ntev los no qhia tau hais tias redox-xeev ntawm hBD1 cuam tshuam rau cov tshuaj tiv thaiv kab mob, xws li qhov txo qis peptide muaj zog ntau dua li daim ntawv disulfide-txuas oxidized [7]. Qhov tseem ceeb ntawm qhov no ntawm qhov chaw urothelial tsis tau txiav txim siab. Lwm tus defensin, tib neeg beta-defensin 2 (hBD2) tsis yog qhia nyob rau hauv cov ntaub so ntswg noj qab nyob zoo; Txawm li cas los xij, hBD2 kev qhia yog tshwm sim los ntawm kev kis kab mob [8].
Tsis zoo li cov beta-defensins, lub luag haujlwm ntawm epithelial-derived alpha-defensins tsis tau piav qhia zoo hauv cov zis. Kev qhia thiab kev ua haujlwm ntawm alpha-defensins HD5 thiab HD6 feem ntau tau tshaj tawm hauv cov hnyuv me uas lawv tau zais los ntawm Panethcells rau hauv cov hnyuv crypts thiab pab txhawb kev sib npaug ntawm plab hnyuv microbiota [9]. HD5 kuj tau nyob hauv thaj chaw ntawm cov txiv neej thiab poj niam qhov chaw mos, nrog cov pov thawj qhia tias nws yog qhov tsis muaj peev xwm thiab tseem ceeb hauv kev tshem tawm cov kab mob [10,11,12]. Urinary HD5 tau raug kuaj pom nyob rau hauv cov neeg mob uas tau ua tiav ileal neobladder reconstruction thiab ileal conduit urinary diversion uas yog lub hauv paus ntawm HD5 ntau lawm feem ntau credit rau ileal Paneth hlwb [13,14]. HD5 muaj cov tshuaj tua kab mob tiv thaiv kab mob uropathogenic gram-zoo kab mob thiab kab mob gram-negative [15]. HD5 kuj muaj cov tshuaj tua kab mob tiv thaiv kab mob uropathogenic xws li adenovirus thiab BK virus [16,17,18]. Hauv txoj kev tshawb no, peb muab cov lus piav qhia thawj zaug thiab qhov ntau ntawm HD5 qhia hauv tib neegraumthiab ntxiv txhais nws cov lus qhia nyob rau hauv qis urinary ib ntsuj av thaum tsis muaj menyuam thiab kis kab mob.
Cov txiaj ntsig
DEFA5 mRNA yog tsim nyob rau hauv tib neeg lub zais zis, ureter, thiab raum.
Tus nqi PCR ntawm lub sijhawm tiag tiag qhia tau tias txhua qhov kev sim zais zis, ureter, thiabraumCov qauv uas tsis muaj kev kis kab mob tau hais tawm DEFA5. DEFA5 qhia tau ntau dua nyob rau hauv lub urinary ib ntsuj av qis dua lub urinary tract (p= 0.014). Hauv lub zais zis (n=4), txhais tau tias DEFA5 kev qhia yog 4,656637 cov ntawv teev lus ib 10 ng RNA thiab hauv cov zis (n=4) txhais tau tias DEFA5 qhia yog 4,1126170 cov ntawv sau ib 10 ngRNA (Daim duab 1A) . Hauv lub raum (n=6), qhov txhais tau tias DEFA5 qhia yog 2,9376274 cov ntawv sau tseg rau 10 ng RNA. DEFA5 kev qhia tau raug txheeb xyuas nyias nyob rau hauv lub raum cortex, medulla, thiab pelvis.DEFA5 kev qhia tsis sib txawv ntawm lub raum (p= 0.45).
DEFA5 qhia thiab HD5 peptide qhia nce nrog pyelonephritis
Kev txheeb xyuas PCR ntawm lub sijhawm tiag tiag ua raucov ntaub so ntswg nrogpyelonephritis pom tau tias muaj kev nce ntxiv hauv DEFA5 qhia piv rau cov ntaub so ntswg raum tsis muaj kab mob. Withpyelonephritis (n= 6), txhais tau hais tias DEFA5 qhia tau nce mus rau 7,82961,052 cov ntawv teev lus ib 10 ng RNA (p= 0.019) (Daim duab 2A).
Txhawm rau ntsuas qhov nce ntxiv hauv kev qhia nrog pyelone-phritis, peb tau ua qhov kev tshuaj ntsuam immunoblot ntawm tib yam.raumcov ntaub so ntswg siv rau lub sij hawm PCR tsom xam los ntsuam xyuas rau concurrent nce nyob rau hauv HD5 peptide ntau lawm (Daim duab 2B, nruab nrab vaj huam sib luag).Immunoblot tsom xam, siv polyclonal HD5 antisera, pom tau ntau dua HD5 peptide ntau lawm nyob rau hauv lub raum cov ntaub so ntswg nrog pyelonephritis (n {{5}). }) piv rau cov tsis muaj kab mobraumcov ntaub so ntswg (n=6). Tsis muaj kab mobraumcov ntaub so ntswg nthuav tawm 300625 ng HD5 / gram ntub cov ntaub so ntswg hnyav thaumraumcov ntaub so ntswg nrog pyelonephritisexpressed 600621 ng HD5/gram ntub cov ntaub so ntswg hnyav (p,0.0001). Blots tau rov soj ntsuam nrog GAPDH los ua tus tswj kev thauj khoom (Daim duab 2B, nruab nrab vaj huam sib luag) thiab cov nyiaj stain kuj tau ua kom paub tseeb tias cov khoom muaj protein sib npaug (Daim duab 2B, sab saum toj vaj huam sib luag).
HD5 peptide yog tsim thoob plaws hauv tib neeg lub raum thiab cov zis
Kev txhaj tshuaj tiv thaiv tau ua rau thaj chaw HD5 ntau lawm hauv cov zis. Immunohistochemistry (IHC) qhia tias HD5immunoreactivity muaj nyob thoob plaws hauv lub urothelium ntawm lub ureter thiab zais zis ntawm tag nrho cov soj ntsuam kuaj (n=4, daim duab 3).IHC kuj qhia tau hais tias HD5 tau tsim nyob rau hauv tubules ntawm lub raum cortex thiab medulla ntawm tag nrho cov. cov qauv (n= 6, Daim duab 4). Immunofluorescence (IF) qhia tau hais tias HD5 qhia tau zoo tshaj plaws nyob rau hauv lub distal nephron thiab cov tubules sau (Daim duab 5). Lub interstitium thiab glomeruli tsis qhia HD5 immunoreactivity. Kev tiv thaiv kab mob hauv daim duab 3, 4, thiab 5 tau ua los ntawm nas monoclonal anti-HD5 antibody (8C8) uas paub txog HD5 propeptide (Abcam, Cambridge, MA). Cov txiaj ntsig tau zoo sib xws thaum siv luav polyclonal anti-HD5 antibody uas paub txog HD5 propeptide thiab peptide paub tab (cov ntaub ntawv tsis qhia) - qhia tias HD5 yog khaws cia ua propeptide.
Nrog pyelonephritis, HD5 immunostaining markedly nce.HD5 immunoreactivity nce thoob plaws lub proximal nephron, lub distal nephron, thiab cov tubules sau (Daim duab 4 thiab daim duab 5). Raws li nyob rau hauv cov qauv uas tsis muaj kab mob, cov glomeruli thiab interstitium tsis pom HD5 qhia nrog tus kab mob. Kev tswj tsis zoo pom tsis muaj HD5 immunoreactivity.
Cov neeg mob cov zis muaj qhov ntsuas tau ntawm HD5 peptide
Immunoblot tsom xam, siv luav polyclonal antiserum uas kuaj pom tus precursor proHD5 thiab cov ntaub ntawv ua tiav ntxiv tau txheeb xyuas qib ntsuas HD5 hauv 13 ntawm 15 kuaj cov zis kuaj kab mob uropathogenic E.coli (Daim duab 6A thiab B). Thaum tam sim no, HD5 qib, normalized rau cov zis creatinine, nyob ntawm 299.8–669.7630 ng HD5 / mg zis Cr (110.67 ng / mL–276.67 ng / mL), uas sib raug rau 11.10–27.67 nmol / L. Innon-infected cov zis kuaj (n=15), HD5 nyob ntawm lossis qis dua qhov kev kuaj pom ntawm (,50 ng). Enzyme-linked immunosorbent assay (ELISA) ntawm tib cov qauv tso zis, siv cov nas monoclonal antibody (8C8) uas kuaj pom tsuas yog cov precursor proHD5, kuaj ntsuas qib ntawm proHD5 hauv 13 ntawm 15 cov kab mob kuaj. Urinary proHD5 concentrations ranges ntawm 122.78–490.060.03 ng HD5/mg zis Cr (30.02 ng/mL–200.5 ng/mL) thaum tam sim no (Daim duab 7).
Kev sib tham
Nyob rau hauv txoj kev tshawb no, peb muab thawj tus cwj pwm ntawm HD5 hauv tib neegraum, ureter, thiab zais zis. HD5 tau pom tias yog ib qho tseem ceeb AMP uas tiv thaiv kev kis kab mob hauv txoj hnyuv thiab tau tswj hwm hauv qhov chaw mos thaum kis kab mob[11,18,19,20,21]. Epithelial-derived AMPs, zoo li HD5, tau pom tias yog qhov tseem ceeb hauv kev tswj kom tsis muaj menyuam hauv cov zis [8,22,23,24]. Kev tsis txaus ntawm cov kab mob hauv lub cev tuaj yeem ua rau mob hnyav thiab / lossis mob ntev [25,26].
Peb cov txiaj ntsig ntawm PCR lub sij hawm muaj nuj nqis qhia tau tias DEFA5 tau tsim tawm hauv tib neeglub raum, ureters, thiab zais zis. DEFA5 qhia tau nce los ntawm cov urinary sab sauv mus rau sab urinary ib ntsuj av - tom qab qhov ntws ntawm cov kwj deg thiab qhov nce nyob ze rau microbiota qhov taw tes ntawm kev ntxeem tau. Rau peb txoj kev paub, qhov no yog thawj txoj kev tshawb fawb los ntsuas DEFA5 qhia hauv tib neeg lub raum thiab zais zis. Tsis ntev los no, Townes thiab al tau pom tias cov ureter ib txwm muaj peev xwm nthuav qhiaDEFA5 [14]. Peb cov txiaj ntsig kuj tseem qhia tau tias DEFA5 tau tsim los ntawm cov ureter distal. Txawm hais tias DEFA5 kev qhia nyob rau hauv cov zis yog ze li ntawm 100-fold qis dua qhov uas pom nyob rau hauv cov ntaub so ntswg plab, basal uroepithelial qhia ntawm DEFA5 yog piv rau cov lus piav qhia yav dhau los tso zis khiav amps xws li cathelicidin, hBD-1, hBD -2, thiab ribonuclease 7[6,8,22,24,27].
Hauv kev sib piv rau cov kab mob plab hnyuv loj uas DEFA5 tau qhia nyob rau theem siab hauv ob lub xeev ntawm kev noj qab haus huv thiab kev kis kab mob / mob, peb cov ntaub ntawv PCR ntau npaum li lub sijhawm qhia tau tias DEFA5 qhia hauvraumNws muaj peev xwm tswj tau tus kab mob [27,28,29]. Cov qauv ntawm kev qhia tsis zoo no yog qhov zoo sib xws rau DEFA5 kev qhia hauv poj niam cev xeeb tub qhov twg DEFA5 qhia nce nrog salpingitis [30]. Hauv cov zis, Townes et al tau qhia txog qhov sib txawv rau ntau dua ureteral qhia ntawm DEFA5 hauv cov neeg mob nrog UTIs uas tau dhau los ntawm ileal conduit urinary diversion [14]
Peb qhov kev tshuaj ntsuam immunoblot ua tiav cov ntaub ntawv PCR ntawm lub sijhawm tiag tiag los ntawm kev ua kom pom tias HD5 peptide ntau lawm nce nrog pyelonephritis. Qhov no inducible qauv ntawm HD5 ntau lawm zoo ib yam li uas pom nyob rau hauv ib co tswv cuab ntawm beta-defensin tsev neeg.hBD2 qhia tau hais tias ib tug inducible qauv ntawm zus tau tej cov kab mob ntawm lub urinary ib ntsuj av thiab / los yog mob pyelonephritis [8,31,32]. HD5peptide ntau lawm tau pom tias yuav nce hauv cov poj niam qhov chaw mos thiab cov txiv neej urethra nrog o [11,30].
Siv cov tshuaj tiv thaiv kab mob, peb pom tau tias HD5 yog ib yam tsim tawm thoob plaws hauv urothelium ntawm ureter thiab zais zis. Vim tias feem ntau ntawm UTIs tshwm sim los ntawm fecal flora uas nce mus rau hauv lub zais zis, cov txiaj ntsig no qhia tias HD5 qhia tau muaj nyob rau hauv qhov chaw uas muaj kab mob microbial tshwm sim feem ntau [33]. Yog li ntawd, HD5 yog qhov zoo tshaj plaws rau kev tiv thaiv kev kis kab mob microbial. Hauvraum, HD5 feem ntau yog tsim nyob rau hauv lub distal nephron thiab sau tubule. Cov kev tshawb pom no qhia tias HD5 yog tsim nyob rau hauv qhov chaw uas nws yuav tsum tau positioned kom muaj kev pom zoo tshuaj tiv thaiv kab mob. Cov kev tshawb fawb yav dhau los tau qhia tias cov tshuaj tua kab mob ntawm cov tshuaj tiv thaiv kab mob yog nyob ntawm cov ntsev concentration - nrog cov ntsev ntau dua txo cov tshuaj tiv thaiv kab mob [23,34,35]. Peb xav tias cov ntsev tsawg ntawm cov nephron distal thiab khaws cov tubules muab ib qho txiaj ntsig zoo rau HD5 cov tshuaj tua kab mob.
Immunoblot thiab ELISA tsom xam kuj qhia tau hais tias HD5 yog secreted rau hauv cov zis nyob rau theem qis. Muab qhov loj me (8.1 kDa thiab 3.7 kDa) thiab tus nqi zoo ntawm proHD5 thiab ua tiav HD5, nws muaj peev xwm hais tias qee qhov tso zis HD5 peptide tuaj, tsawg kawg ntawm ib feem, los ntawm cov ntshav plasma filtrate. Txawm li cas los xij, tsis muaj pov thawj me me uas qhia tias HD5 tseem nyob hauv cov ntshav [27]. Tsis tas li ntawd, kom tshwm sim hauv cov zis, HD5 yuav tsum tau khiav tawm cov txheej txheem peptide nqus tau zoo hauv cov tubule ze ze [6,36]. Thaum kawg, cov qauv tso zis siv underwent centrifugation ua ntej qhov kev ntsuam xyuas tau ua, tshem tawm cov xov tooj ntawm tes ntawm HD5.
Thaum kuaj pom cov concentrations, cov zis HD5 tsis zoo li yuav ncaj qha rau cov tshuaj tua kab mob tiv thaiv kab mob uropathogenic microorganisms raws li kev ua tiav HD5 muaj qhov tsawg kawg nkaus inhibitory concentration (MIC) nruab nrab ntawm 6-10 mg / mL tiv thaiv cov kab mob gram-negative uropatho genic [15]. Txawm li cas los xij, qhov concentration ntawm mucosal saum npoo yuav muaj ntau dua. Tsis tas li ntawd, kev tshuaj ntsuam immunoblot qhia tau hais tias HD5 concentrations muaj ntau dua nyob rau hauvraum. Cov txiaj ntsig no qhia tias HD5 koom nrog hauv kev tiv thaiv mucosal ntawm cov zis. Ib qho piv txwv piv tau pom nrog hBD1, qhov twg cov zis ntau ntawm hBD1 tsis txaus los ua kom pom cov tshuaj tiv thaiv kab mob tab sis pom muaj peptide ntau dua nyob ze ntawm lub raum tubules [6,31].
Vim tias IHC pom tau tias proHD5 yog ib daim ntawv loj ntawm HD5 hauvraumthiab vim hais tias ob leeg paub tab thiab proHD5 raug kuaj pom nyob rau hauv cov zis, peb xav tias HD5 feem ntau yog secreted ib tug precursor molecule thiab tom qab ntawd undergoes proteolytic ua los tsim mature forms - zoo ib yam li lub plab thiab genitourinary tracts [11,30,37]. Hauv cov hnyuv me, Paneth hlwb tsim trypsin, uas cleaves HD5 propeptide kom cov peptide ua tiav tag nrho yog cov ntawv tseem ceeb hauv plab hnyuv lumen [37]. Hauv cov txiv neej urethra, cov txheej txheem tseem ceeb thiab ua kom cov enzymes rau HD5 yog cov neutrophil proteases pab los ntawm neutrophils recruited mus rau qhov chaw kis kab mob [11]. Kev koom tes ntawm epithelial trypsinogen, urinary trypsin, thiab / los yog neutrophil proteases ntawm HD5 ua nyob rau hauv cov zis thaum lub sij hawm kis kab mob tseem yuav txiav txim siab. Ntxiv mus, qhov cuam tshuam ntawm kev hloov pauv hauv qhov chaw tso zis ntawm HD5 muaj nuj nqi (kev hloov pauv hauv osmolarity, pH, thiab cationic concentrations) kuj tseem raug txiav txim siab.
Hauv kev xaus, qhov no yog thawj txoj kev tshawb fawb los txheeb xyuas thiab ntsuas qhov kev qhia thiab tsim tawm ntawm HD5 hauv cov zis. Peb cov txiaj ntsig tau qhia tias HD5 yog ib qho epithelial-derived AMP uas tuaj yeem ua lub luag haujlwm tseem ceeb hauv kev tiv thaiv kab mob hauv lub cev ntawm tib neeg uroepithe lium tiv thaiv kev hloov pauv ntawm cov kab mob nkag mus rau hauv kev ncig. Elucidation ntawm cov yam ntxwv uas tswj hwm HD5 ntau lawm tuaj yeem qiv qhov kev pom tshiab rau hauv cov kab mob ntawm UTIs hauv cov neeg mob uas muaj kev pheej hmoo rau UTIs thiab cov neeg mob uas muaj kab mob ntev.
Cov txheej txheem
Kev pom zoo kawm
Kev lees paub sau ntawv tau txais los ntawm txhua tus neeg mob koom nrog hauv txoj kev tshawb no. Rau cov menyuam hnub nyoog qis dua 18 xyoo, tau txais daim ntawv tso cai niam txiv/tus saib xyuas. Lub Tsev Kho Mob NationwideChildren's Hospital (NCH) Institutional Review Board tau pom zoo txoj kev tshawb no nrog rau cov txheej txheem tso cai thiab cov ntaub ntawv (IRB07-00383).
Tib neeg cov ntaub so ntswg thiab cov zis kuaj
Cov ureter uas tsis muaj kab mob thiab lub zais zis (n= 4) tau txais los ntawm cov menyuam yaus uas tau muab cov ureteral re-implantation rau cov laj thawj uas tsis yog tus kab mob rov tshwm sim dua. Tsis muaj kab mobraumcov qauv (n=6) tau txais los ntawm cov neeg mob uas raug nephrectomy rau cov qog nqaij hlav hauv lub raum. Cov ntaub so ntswg kuaj tsis muaj cov tsos mob ntawm tus kab mob lossis o.Lub raumcov ntaub so ntswg los ntawm cov neeg mob uas muaj mob pyelonephritis tau muab los ntawm Cooperative HumanTissue Network (n=6) [38]. Ob tus kws kho mob ywj pheej tau lees paub qhov kev kuaj mob histopathologic ntawm pyelonephritis. Cov qauv ntaub so ntswg tau snap-kua los yog khaws cia raws li nruab nrab formalin-taw paraffin-embedded seem. Cov kab mob ntawm lub raum tsis muaj kab mob tau muab pov tseg rau hauv cortex, medulla, lossis lub raum pelvis ua ntej khaws cia (n=4).
Cov kuaj zis tsis muaj kab mob thiab kis tau los ntawm cov menyuam yaus tuaj rau NCH lub chaw haujlwm xwm txheej ceev lossis chaw kho mob nephrology. Kev kuaj mob ntawm UTI tau tsim los ntawm cov kab mob tso zis zoo raws li American Academy of PediatricsGuidelines [39]. Txhua qhov kuaj zis muaj kab mob muaj .106 CFU/mL ntawm E.coli. Cov zis pH nyob ntawm 5.5 mus rau 8.5 (txhais zis 6.9). Lub urinary ionic muaj pes tsawg leeg tsis tau ntsuas. Cov zis kuaj tau centrifuged kom tshem tawm cov zis tawm, thiab ntxiv cov tshuaj protease inhibitor cocktail (Thermo Scientific, Rockford, IL, USA).
Ribonucleic Acid Isolation thiab Reverse Transcription
Tag Nrho RNA raug cais tawm ntawm cov ntaub so ntswg khov siv PromegaTotal RNA Isolation System (Promega, Madison, WI, USA). ForcDNA synthesis, 4–8 mg ntawm tag nrho RNA tau thim rov qab nrogSuperscript III reverse transcriptase siv oligo-(dT)12–18primer raws li tus neeg muag khoom txoj cai (Invitrogen, Carlsbad, CA, USA).
Cloning ntawm Gene Specific Plasmids rau Standard Curves
Lub cDNA encoding DEFA5 thiab GAPDH tau cloned rau hauv ib tug 4-Topo plasmid vector (Invitrogen) raws li cov lus qhia manufacturers. Plasmids tau ua ntu zus kom paub meej tias qhov tseeb
kev tsim kho tau txais. Serial dilutions ntawm noob caj noob ces plasmids tau quantitated (los ntawm spectrophotometric absorbance ntawm 260 thiab ethidium bromide-stained agarose gel electrophoresis) thiab siv nyob rau hauv real-time PCR los tsim qauv nkhaus rau txhua cov tshuaj tiv thaiv.
Quantitative Real-time PCR
Lub sijhawm tiag tiag PCR tau ua tiav siv ib leeg-stranded cDNA los ntawm tib neegraum, ureter, thiab zais zis cov ntaub so ntswg nrog tshwj xeeb oligonucleotide primer khub siv 7500 Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA).PCR exon junction spanning primers tau tsim thiab ua ntu zus tau lees paub siv DNAstarH Laser Gene SeqBuilder (DEFA5 forward primer : 59- TCC CTC CTG CAG GTG ACC CCA-39 and DEFA5 reverse primer 59-GTG GCT CTT GCC TGA GAA CCT GA-39).
Luv luv, cDNA sib raug rau 10 ng RNA tau ua atemplate hauv 25 ml cov tshuaj tiv thaiv uas muaj 2.0 mM ntawm txhua tus primer thiab 16 Lub Teeb-Cycler-Fast Start DNA Master SYBR Green mix. PCR tej yam kev mob yog: thawj denaturation ntawm 95uC rau 10 feeb, ua raws li 40 cycles nrog txhua lub voj voog uas muaj denaturation ntawm 94uC rau 30 vib nas this, annealing ntawm 64uC rau 30 vib nas this, thiab ncua ntawm 72uC rau 30 vib nas this. Lub voj voog-rau-lub voj voog fluorescence emission raug saib xyuas ntawm 530 nm thiab tshuaj xyuas siv 7500Software V2.0.3 (Applied Biosystems). Gene-specific plasmid cov qauv tau suav nrog txhua qhov kev cuam tshuam. Raws li kev tswj hwm zoo, lub davhlau ya nyob twg ileum RNA tau suav nrog txhua qhov kev tawm tsam, thiab cov txiaj ntsig tau muab piv rau cov qauv luam tawm yav dhau los [27].
Txhawm rau kom paub meej tias PCR nthuav dav ntawm cov khoom npaj, cov qauv piv txwv tau txheeb xyuas los ntawm electrophoresis ntawm 1.5 feem pua agarose gel. Cov khoom tau pom los ntawm ethidium bromidestaining thiab piv rau DNA loj cov qauv kom paub tseeb tias cov khoom loj (tsis pom). Tsis tas li ntawd, lub meltingtemperature profile nkhaus ntawm txhua qhov PCR cov tshuaj tiv thaiv tau txiav txim siab thaum kawg ntawm txhua qhov tshuaj tiv thaiv.
HD5 Antibodies
Cov HD5 propeptide (AA20-94) thiab cov ntawv ua tiav ib nrab (AA36-94 thiab 56–94) tau txheeb xyuas siv cov nas muaj muag monoclonal (8C8) anti-HD5 antibody (Abcam).The HD5 propeptide thiab cov peptide paub tab (AA{10}}) tau txheeb xyuas siv yav dhau los piav txog luav polyclonal antiserum[13,40].
Immunoblot
Cov seem ntawm tib yamraumCov qauv siv rau kev txheeb xyuas PCR ntau npaum li cas (3-6 mg ntub qhov hnyav) tau pulverized siv cov tshuaj khib thiab pestle hauv cov kua nitrogen thiab yaj hauv RIPA tsis nrog protease inhibitors. Cov zis tso zis tau muab rho tawm los ntawm cov qauv hauv zis siv ProteospinTM Urine Protein Concentration Micro Kit (Norgen Biotek Corporation, Thorold, ON, Canada). Lub raum thiab cov zis muaj protein ntau tau suav nrog kev hloov kho Bradford Assay thiab lees paub tias siv kev nyeem ntawv anOD280 nm. Qhov sib npaug ntawm cov ntaub so ntswg hauv lub raum lossis cov zis tso zis tau thauj mus rau 18 feem pua sodium dodecyl sulfate gradient gels thiab raug rau electrophoresis. Txhawm rau kom muaj qhov sib npaug ntawm cov khoom noj protein, ib qho nyiaj stain tau ua.
Tom qab electrophoretic sib cais, cov proteins raug xa mus rau ib daim nyias nyias ntawm nitrocellulose. Tom qab kho nrog 0.05 feem pua glutaraldehyde hauv TBS, cov membranes tau thaiv hauv 5 feem pua cov mis tsis muaj rog rau 30 mus rau 60 feeb thiab incubated hauv 1: 1,000 dilution ntawm luav polyclonal HD5 antiserum thaum hmo ntuj . Tom qab ntxuav, cov tshuaj tiv thaiv thib ob, tshuaj tiv thaiv luav horseradish peroxidase-conjugated anti-luav IgG diluted 1:10,{15}} (Cell Signaling Technology, Danvers, MA, USA), tau thov rau 1 teev ntawm chav tsev kub. Immunoblots los ntawmraumcov ntaub so ntswg kuj tau soj ntsuam nrog cov tshuaj tiv thaiv-GAPDH antibody (Sigma Aldrich) rau ob teev ntawm chav tsev kub thiab tom qab ntawd incubated nrog cov tshuaj tiv thaiv thib ob uas tau piav qhia saum toj no. Cov proteins tau pom los ntawm kev siv ECL kev kuaj xyuas qhov system thiab chemiluminescence zaj duab xis raws li cov chaw tsim khoom cov lus qhia (BioExpress, Kaysville, UT, USA).
HD5 tau muab xam los ntawm kev sib piv cov qhab nia uas tau siv nrog kev sib txuas ntawm cov txheej txheem proHD5 protein (Peptides International, Louisville, KY, USA).Lub raumHD5concentrations tau standardized rau cov ntaub so ntswg hnyav. UrinaryHD 5 concentrations tau muab faib los ntawm cov zis creatinine los tsim cov qauv zis HD5-to-creatinine ratios (mg/mg) los suav rau cov zis dilution. Cov zis creatinine concentrations tau txiav txim siab siv Oxford Biomedical Research creatinine microplate assay (Rochester Hills, Michigan, USA).
Immunohistochemistry
Tom qab deparaffinization, rehydration, thiab antigen retrieval, biotin block thiab serum-free protein block tau ua (Superblock, ScyTek Laboratories, Logan, UT, USA). Cov slides tau incubated ib hmos ntawm 4uC nrog monoclonal nas HD5(8C8) antibody (1:2{{10}}}0; Abcam) los yog luav polyclonal antiserum (1:500) tom qab los ntawm anti-polyvalent biotinylated antibody adulterated Streptavidin / HRP (ScyTek Laboratories). Cov ntu tau tsim los siv 0.1 feem pua diaminobenzidine tetrachloride nrog 0.02 feem pua hydrogen peroxide thiab counterstained nrog hematoxylin. Cov kev tswj tsis zoo tau raug tsim nrog cov tshuaj tsis muaj tshuaj tiv thaiv kab mob hauv qhov chaw ntawm HD5 antibody.
Immunofluorescence
Ob-labeled immunofluorescence tau ua los pab hauv zos HD5 qhia hauvraum. Cov ducts sau tau muab ob npaug rau daim ntawv lo rau lub hauv paus tseem ceeb nrog tshis polyclonal anti-human aquaporin -2 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) [41]. Lub voj ntawm Henle yog ob-labeled nrog mousepolyclonal los tiv thaiv tib neeg uromodulin antibody (Sigma-Aldrich) thiab cov tubule ze ze yog ob-labeled nrog tshis polyclonalantihuman aquaporin -1 (Santa Cruz) [41,42]. Rhodamine nees luav polyclonal anti-tshis (Jackson ImmunoResearch Laboratories, West Grove, PA, USA), rhodamine tshis anti-nas (JacksonImmunoResearch Laboratories), thiab FITC nees luav polyclonal anti-luv (Santa Cruz) tau txais kev pabcuam thib ob.
Txhua ntu tau npaj raws li tau teev tseg saum toj no. Lawv tau tsim nrog kev sib xyaw ntawm nas antisera tiv thaiv HD5 (1:200)(Abcam), AQP-2 (1:500), uromodulin (1:500), AQP-1 (1:400) atroom kub rau 90 feeb. Cov tshuaj tiv thaiv kab mob thib ob tau siv rau 90 feeb ntawm chav tsev kub thiab cov ntu tau txuas nrog mounting media nrog DAPI. Tsis muaj tshuaj tiv thaiv kab mob tau siv los ua kev tswj tsis zoo. Cov slides tau tshuaj xyuas nrog aLeica DM4000B microscope thiab yees duab digitally siv Spot RT lub koob yees duab / software (Diagnostic Instruments, SterlingHeights, MI, USA).
ELISA
Txuas Biotin Antibody Labeling Kit, Novus Biologicals) nas monoclonal antibody rau 2 teev ntawm chav tsev kub, streptavidin-nees radish peroxidase (Biolegend, San Diego CA, USA) tau ntxiv rau 30 feeb. Tom qab incubation nrog TMB substrate tov rau 15 feeb (Kem-En-Tec Diagnostics), cov tshuaj tiv thaiv raug txiav nrog STOP tov (Cell Signaling Technology, Danvers, MA, USA) thiab nyeem ntawm ib tug wavelength ntawm 450 nm. HD5concentrations los ntawm ELISA qhov kev ntsuam xyuas tau muab faib los ntawm cov zis urinecreatinine los tsim cov qauv zis HD5-to-creatinine ratios(mg/mg) kom suav cov zis dilution raws li tau piav qhia saum toj no.
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