Tsib Amino Acids Deletion hauv NKCC2 Ntawm C57BL / 6 Cov nas cuam tshuam rau kev soj ntsuam ntawm NKCC2 Phosphorylation Tab sis Tsis cuam tshuam rau lub raum ua haujlwm
Feb 19, 2022
Taw qhia
Qhov tuab ascending limb (TAL) ntawm cov tsiaj nyegraumNws yog ib qho tseem ceeb rau kev tswj hwm cov kua dej hauv lub cev, ur-nary concentration, calcium (Ca 2 ntxiv) thiab magnesium (Mg 2 ntxiv) homeostasis, nrog rau pH sib npaug. 1 Txoj kev loj ntawm Na ntxiv rau kev thauj mus los hauv TAL yog Na ntxiv - K ntxiv rau - 2Cl − cotransporter (NKCC2), uas tshwj xeeb yog inhibited los ntawm loop- diuretics. 1 Lub cotransport ntawm Na plus, K ntxiv, thiab 2Cl - ions ntawm NKCC2 yog electroneutral tab sis nyob ntawm electrogenic K ntxiv secretion ntawm apical lub raumsab nrauv medulla K ntxiv channel ROMK. 2 Kev sib cuam tshuam ntawm NKCC2 nrog ROMK tsim lub lumen-zoo electrochemical gradient, uas txhim kho Na ntxiv rau reabsorption thiab tsav tsheb thauj mus los ntawm Ca 2 ntxiv thiab Mg 2 ntxiv. 3 Qhov tseem ceeb ntawm NKCC2 rau TAL thiablub raum ua haujlwmyog ua piv txwv los ntawm Bartter type I syndrome, ib qho kev mob caj ces tshwm sim los ntawm kev hloov pauv ntawm kev ua haujlwm hauv NKCC2, tshwm sim nrog mob hnyav.lub raumntsev thiab kua dej nkim, hypokalemia metabolic alkalosis thiab hypercalciuria. 3
NKCC2 yog tus tswv cuab ntawm cov neeg nqa khoom solute tsev neeg 12 (Slc12) ntawm daim nyias nyias thauj cov proteins uas tseem suav nrog cov NKCC1 uas tau hais tawm thoob plaws lub cev thiab qhov sib txawv ntawm cov tubules (DCT) tshwj xeeb Na plus - Cl − cotransporter (NCC). 2 Cov haujlwm ntawm NKCC2 zoo sib raug zoo nrog phosphorylation ntawm cotransporter ntawm ntau cov serine thiab threonine residues ntawm cytoplasmic N-terminal tail. 4 Cov amino acids ua ntu zus nyob ib puag ncig cov chaw phosphorylation no tau txais txiaj ntsig zoo thiab qhia tau tias muaj kev sib luag ntawm NKCC2, NKCC1 thiab NCC. 2 Lub phosphorylation ntawm NKCC2 yog txhawb los ntawm ntau txoj hauv kev hormonal thiab non-hormonal. Cov tshuaj hormones xws li arginine vasopressin (AVP) thiab angiotensin II (ANGII) txhawb nqa NKCC2 los ntawm kev nce cAMP ntau lawm thiab ua kom cov protein kinase A (PKA). 5 PKA tau xav tias ncaj qha phosphorylate NKCC2 ntawm Ser126, uas txhim kho cov exocytosis ntawm NKCC2 uas muaj cov vesicles thiab yog li qhov kev nplua nuj ntawm cov khoom thauj hauv apical plasma membrane. 6 Ib yam li ntawd, kev hloov pauv hauv intracellular chloride con-centrations tswj NKCC2 phosphorylation. Kev txo qis hauv tracellular Cl − concentration stimulates lub with- no- lysine (K) kinases WNK1 thiab WNK4 rau phosphorylate thiab qhib cov dej ntws qis Ste20- ntsig txog proline alanine nplua nuj kinase (SPAK) lossis oxidative stress responsive kinase 1 (OSSR1. ). 7- 9 Tom qab khi rau "RFxV" motif hauv NKCC2, cov kinases no ces phosphorylate NKCC2 ntawm ob threonine residues (T96 thiab T101). 2 Dephosphorylation ntawm cov residues no yog kho los ntawm phosphatase calcineurin, qhia tias NKCC2 yog lub hom phiaj rau ntau txoj hauv kev qhia. 10
Txhawm rau kawm NKCC2 muaj nuj nqi, ntau pab pawg suav nrog peb cov tsim phosphoform- cov tshuaj tiv thaiv tshwj xeeb tiv thaiv Thr96 thiab / lossis Thr101. 11- 17 Cov tshuaj tiv thaiv tau siv los ntsuas NKCC2 phosphorylation hauv cov kab ke heterologous qhia 7,11,17,18,19,20 thiab cov qauv tsiaj xws li nas 10,21,22,23,24,25 thiab nas. 14,15,16,17,19,26,27 Txawm li cas los xij, thaum siv peb cov tshuaj tiv thaiv pT96/101 NKCC2, peb tau txais cov txiaj ntsig tsis zoo hauv peb cov qauv nas sib txawv. Thaum peb pom muaj zog pNKCC2 cov cim qhia hauv lub raum los ntawm cov nas nyob rau hauv 129Sv caj ces keeb kwm yav dhau los, peb tsis tau txais kev ntseeg siab pNKCC2 cov cim qhia hauv lub raum los ntawm C57BL / 6 nas tab sis pom qhov sib txawv ntawm pNKCC2 cov tshuaj tiv thaiv nrog pNCC. Lwm lub chaw soj nstuam, suav nrog cov los ntawm JA McCormick thiab AA McDonough (kev sib txuas lus ntawm tus kheej) tau pom zoo sib xws. 18,28,29 Tsis tas li ntawd, qhov sib txawv ntawm qhov sib txawv tau piav qhia hauv nas raulub raummuaj nuj nqi 30 suav nrog txo qis zis Ca 2 ntxiv rau kev tso tawm hauv C57BL / 6 piv rau 129Sv nas. 31 Yog li ntawd, peb xav tias C57BL/6 nas qhia txog cov caj ces ntawm NKCC2 uas cuam tshuam rau NKCC2 phosphorylation thiab tej zaumlub raum ua haujlwmuas piav qhia txog qhov sib txawv ntawm qhov sib txawv. Cov kev tshawb fawb hauv qab no tau tsim los ntsuas qhov kev xav no.
Ntsiab lus:ion homeostasis, raum, NKCC2, phosphorylation, strain sib txawv
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2.1|C57BL / 6 nas muaj tsib amino acids tshem tawm hauv NKCC2 uas cuam tshuam nrog kev tshawb pom ntawm phosphorylated NKCC2Ua ntej, peb tau ua raws li ntau hom kev luam tawm N-terminal amino acids sequences (transcription IDs nyob rau hauv Table S1) ntawm NKCC2 thiab NCC (Daim duab 1A, B, feem) uas sib xws rau cov epitopes lees paub los ntawm anti- pNCC thiab anti- pNKCC cov tshuaj tiv thaiv. (Daim duab 1C). Kev sib raug zoo tau lees paub qhov siab ntawm kev txuag ntawm qhov chaw phosphorylation ntawm NKCC2 thiab NCC 7 uas yuav piav qhia txog kev sib cuam tshuam ntawm pNKCC2 thiab pNCC cov tshuaj tiv thaiv uas peb thiab lwm tus. 18,28,29 Txaus siab thiab ua tau raws li peb qhov kev xav, qhov kev sib raug zoo kuj tau rov ua dua ib qho kom deb li deb tsis txaus siab rau tsib amino acids deletion nyob rau hauv cov kab ke ntawm NKCC2 los ntawm C57BL/6 nas (ΔF97- T101) (Daim duab 1A) . Kev sib luag ntawm DNA los ntawm ob qho sib txawv C57BL / 6 nas cov cheeb tsam khaws cia ntawm Jackson Laboratory hauv Tebchaws Meskas lossis Janvier Labs hauv Fabkis tau lees paub qhov kev tshem tawm nucleotide hauv exon 2 ntawm C57BL / 6 NKCC2 uas sib haum rau cov lus hais saum toj no hais txog kev tshem tawm cov amino acids (Daim duab S1 , Tab S2).
Txhawm rau txheeb xyuas qhov cuam tshuam ntawm tsib cov amino acids de-letion ntawm kev tiv thaiv kab mob ntawm tag nrho thiab phosphorylated NKCC2, peb tau kawm txog lub raum ntawm C57BL / 6 thiab 129Sv nas los ntawm immunohistochemistry thiab immunoblotting. Immunofluorescence nrog cov tshuaj tiv thaiv kab mob tiv thaiv tag nrho NKCC2 tau pom muaj zog fluorescent teeb liab nyob rau hauv apical plasma membrane ntawm TALs ntawm 129Sv thiab C57BL / 6 nas. Txawm li cas los xij, peb yav dhau los tsim pT96 / T101 NKCC2 cov tshuaj tiv thaiv kab mob pom tias muaj zog apical daim ntawv lo ntawm TALs tsuas yog hauv 129Sv nas, thaum TALs ntawm C57BL / 6 nas muaj qhov tsis muaj zog tiv thaiv kab mob (Daim duab 1D). Raws li qhov no, Western blot tsom xam tau qhia ib pab pawg muaj zog rau tag nrho NKCC2 hauv ob hom nas, thaum pT96 / T101 NKCC2 cov tshuaj tiv thaiv tau kuaj pom ib pawg muaj zog ntawm qhov xav tau loj (170 kDa) tsuas yog hauv 129Sv nas (Daim duab 1E). Hloov chaw, lub raum ntawm C57BL / 6 nas tau nthuav tawm ib pawg ntawm 130 kDa uas sib haum rau qhov xav tau loj ntawm NCC (Daim duab 1E). Cov txiaj ntsig zoo sib xws tau txais nrog lwm cov tshuaj tiv thaiv yav dhau los siv los kawm NKCC2 phosphorylation (Daim duab S2). Lub pT212/T217 NKCC1 R5 antibody 11 tau qhia ntxiv rau pNCC teeb liab teeb liab ntawm qhov xav tau loj rau pNKCC2 (Daim duab S2). Cov kab mob tiv thaiv kab mob rau pNKCC2 tau mob siab rau thaum tshiabraumlysates tau siv. Kev siv cov lysis tsis nrog cov tshuaj detergents (RIPA buffer) tsis txhim kho tab sis txo qis cov teeb liab siv (Daim duab S3).
FIGURE 1 Lub pT96/T101 NKCC2 antibody tsis ntes pom pNKCC2 teeb liab hauv C57BL/6 nas. A, NKCC2 amino acids ua raws li kev sib txawv ntawm ntau hom thiab ob hom nas 129Sv thiab C57BL/6. NKCC2 kab zauv ID rau ntau hom muaj npe nyob rau hauv Table S1. Lub cheeb tsam kev txuag tau zoo sib xws rau murine Thr96 rau murine Thr101 yog cim rau hauv grey. B, Amino acids ib ntus kev sib raug zoo ntawm NCC hauv ntau hom. NCC kab zauv IDs rau ntau hom muaj npe nyob rau hauv Table S1. C, Cov npe ntawm cov epitopes uas nquag siv thiab luam tawm cov tshuaj tiv thaiv pom pT53 lossis pT58NCC, nrog rau pNKCC2 ntawm qhov chaw khaws cia ib ncig ntawm Thr96 txog Thr101. D, Immunostaining ntawm tag nrho thiab pT96/T101 NKCC2 nyob rau hauv sib law liag cryo- ntu ntawm nas nrog 129Sv thiab C57BL/6 keeb kwm yav dhau. Scale bar=50 µm. E, Immunoblot ntawm tag nrho thiab pT96 / T101 NKCC2 ntawm nas nrog 129Sv thiab C57BL / 6 keeb kwm yav dhau. Qhov tseem ceeb yog txhais tau tias ± SEM, n=5 nas rau ib pab pawg, cov xub ntsuab taw qhia rau cov kab tshwj xeeb thaum tus xub liab- taub hau taw tes rau cov kab tsis tshwj xeeb. Cov cim ntsuas qhov hnyav molecular suav nrog. Tag nrho thiab pT96 / T101 NKCC2 bands yuav tsum nyob ntawm 170 kDa. F, Immunoblots rau tag nrho thiab pT96/T101 NKCC2, tag nrho thiab pT53 thiab pT58 NCC hauv NCC WT thiab NCC knockout (KO) nas ntawm ntau haiv neeg. Cov xub ntsuab taw qhia rau cov kab tshwj xeeb thaum cov xub liab- taub hau taw tes rau cov tsis tshwj xeeb. Cov cim ntsuas qhov hnyav molecular suav nrog. Tag nrho thiab pT58 thiab pT53 NCC bands yuav tsum nyob ntawm 130 kDa, tag nrho thiab pT96 / T101 NKCC2 bands yuav tsum nyob ntawm 170 kDa
Txhawm rau kuaj seb peb cov tshuaj tiv thaiv pT96 / T101 NKCC2 tiag tiag cuam tshuam nrog phosphorylated NCC hauv C57BL / 6 nas, peb tau soj ntsuamraumlysates los ntawm 129Sv thiab C57BL / 6 cov nas qus thiab C57BL / 6 NCC knockout nas nrog cov tshuaj tiv thaiv tag nrho NKCC2, pT96 / T101 NKCC2, tag nrho NCC, thiab phosphorylated NCC (pT53 thiab pT58 NCC) (Daim duab 1F). Lub pT96/T101 NKCC2 cov tshuaj tiv thaiv kab mob thiab cov tshuaj tiv thaiv tag nrho NCC thiab pNCC tsis pom cov kab ntawm 130- 170 kDa hauv C57BL/6 NCC knockout nas (Daim duab 1F). Interestingly, lub immunoblot nrog rau ntxiv immunohistochemistry thwmsim (Daim duab S4) qhia tias tsis tsuas yog ua cov pNKCC2 tshuaj tiv thaiv cross-react nrog NCC nyob rau hauv C57BL/6 nas tab sis peb pNCC tshuaj tiv thaiv (pT53 NCC thiab pT58 NCC) cross-reacts nrog NKSCC2 nyob rau hauv 129. nas ntawm cov concentrations siv.
Calcium thiab magnesium excretion sib txawv ntawm 129Sv thiab C57BL / 6 nas
Txhawm rau txiav txim siab seb qhov kev tshem tawm (F97- T101) hauv NKCC2 tuaj yeem koom nrog qhov sib txawv ntawm lub cev ntawm 129Sv thiab C57BL/6 nas, peb piv cov dej kom tsawg, qib ntshav ions, thiab cov kua dej tso zis thiab ion excretion ntawm ob hom nas. Raws li tau piav qhia hauv Table 1, feem ntau ntawm cov kev ntsuas ntsuas tsis txawv ntawm cov kab mob. Txawm li cas los xij, C57BL / 6 nas pom qhov tso zis qis dua Ca 2 ntxiv thiab cov zis ntau dua Mg 2 ntxiv rau kev tso tawm ntau dua 129Sv nas. Cov qib plasma Ca 2 ntxiv thiab Mg 2 ntxiv tau zoo sib xws rau ob pawg, qhia tias cov nas nyob hauv lub xeev tsis tu ncua ntawm qhov homeostatic tshuav nyiaj li cas ntawm txoj hnyuv reabsorption, cov ntaub so ntswg mob (xws li los ntawm pob txha), thiablub raumKev tshem tawm ntawm Ca 2 ntxiv thiab Mg 2 ntxiv ions tau mus txog. Txhawm rau kuaj seb qhov sib txawv ntawm cov zis Ca 2 ntxiv thiab Mg 2 ntxiv rau qhov tso tawm los ntawm qhov sib txawv ntawm txoj hnyuv tso tawm ntawm cov ions, peb piv cov khoom noj txhua hnub, cov zis tawm, thiab faecal Ca 2 ntxiv thiab Mg 2 ntxiv rau kev tso tawm rau ob. cov nas. Raws li pom nyob rau hauv daim duab S5, kev noj zaub mov thiab faecal Ca 2 ntxiv tso tawm tau zoo sib xws, tab sis txhua hnub faecal Mg 2 ntxiv rau kev tso tawm qis dua hauv C57BL / 6 nas dua li hauv 129Sv nas qhia tias C57BL / 6 nas muaj cov hnyuv siab dua Mg 2 ntxiv rau reabsorption. tshaj 129 Sv nas. Raws li qhov kev xav no, C57BL / 6 nas tau muaj zog colonic qhia ntawm magnesium channel TRPM6 (Daim duab S6), nrog rau kev sib piv rau cov ntshav plasma Mg 2 ntxiv rau qib piv rau 129Sv nas, txawm hais tias qhov sib txawv tsis ncav cuag qhov tseem ceeb. Txhawm rau ntsuas qhov ua tau ntawm cov pob txha, peb txheeb xyuas los ntawm micro-CT cov pob txha pob txha pob txha hauv ob hom. Zoo ib yam li cov ntawv tshaj tawm dhau los, 32 C57BL / 6 nas muaj cov pob txha pob txha tsawg tsawg (Daim duab S7), uas yuav piav qhia vim li cas cov nas no nyiam khaws Ca 2 ntxiv rau ions ntawm kev txo qis.lub raumCa 2 plus excretion. Raws li ib qho kev txhim kho tubular Ca 2 ntxiv rau reabsorption tus nqi, lub raum ntawm C57BL / 6 nas.
qhia ntau dua TRPV5 Ca 2 ntxiv rau channel ntawm cov protein ntau dua raum ntawm 129Sv nas (Daim duab 2). Cov nas C57BL/6 kuj tseem muaj ntau dua NCC mRNA thiab cov protein qhia ntau dua 129Sv nas (Daim duab 2A). Ib yam li ntawd, cov protein ntau ntawm tag nrho NKCC2, tag nrho thiab phosphorylated NCC thiab ntawm tag nrho - EnaC tau siab dua hauv C57BL / 6 dua li ntawm 129Sv nas (Daim duab 2B, C). Cov kev nplua nuj ntawm cov proteolytically cleaved thiab yog li cov ntaub ntawv nquag ntawm - thiab - ENaC tsis txawv qhov qhia tiaslub raumKev ua ENaC zoo ib yam rau ob hom kab mob (Daim duab 2B, C).
Tsis muaj pov thawj rau qhov hloov pauv tuab nce siab ua haujlwm hauv C57BL / 6 nas piv nrog 129Sv nas
Qhov ntau dua ntawm tag nrho NKCC2, tag nrho NCC thiab phosphorylated NCC nyob rau hauv ob lub raum ntawm C57BL / 6 nas tej zaum yuav yog ib tug compensatory teb rau txo NKCC2 phosphorylation. Txhawm rau kuaj seb qhov kev ua ntawm NKCC2 thiab NCC txawv ntawm cov kab nas, peb tau ua ib qho bumetanide- thiab thiazide- kuaj raws li. Ib qho kev txhaj tshuaj ntawm bumetanide lossis hydrochlorothiazide ua rau muaj zog natriuresis uas tau tshaj tawm rau bumetanide dua li hydrochlorothiazide. Txawm li cas los xij, cov lus teb diuretic tau zoo ib yam hauv ob hom kev qhia tias kev ua haujlwm ntawm NKCC2 thiab NCC tsis txawv ntawm C57BL/6 thiab 129Sv nas (Daim duab 3A-D). Pom tau tias, cov protein ntau thiab phosphorylation tsis tas yuav tsum sib haum nrog cov haujlwm ua haujlwm ntawmlub raumion thauj cov proteins raws li tsis ntev los no kuj tau taw qhia los ntawm Hunter thiab cov neeg ua haujlwm. 33 Txhawm rau ntsuas TAL ntxiv, peb tau ua qhov kev ntsuas dej txwv uas cov nas muaj 24 teev nkag mus rau cov khoom noj uas ntub dej, tab sis tsis txhob siv dej. Nyob rau hauv cov xwm txheej no, ob leeg nas hom nce zis osmolarity rau qhov zoo sib xws (Table 1). Tsis tas li ntawd, C57BL / 6 nas thiab 129Sv nas tau pom tib lub tso zis ntawm uromodulin (Tamm- Horsfall protein) (Table 1), uas yog tsim thiab tawm mus rau hauv cov zis nyob ntawm qhov ua haujlwm ntawm TAL hlwb. 3
Kev sib tw ntawm 129Sv thiab C57BL / 6 nas qhia tias tsib amino acids tshem tawm hauv NKCC2 tsis cais cov phenotype tshwj xeeb
Cov ntaub ntawv nthuav tawm qhia tias qhov sib txawv ntawm cov zis ion excretions thiablub raumprotein ntau tsis muaj feem xyuam rau tsib amino acids deletion nyob rau hauv C57BL/6 NKCC2, tab sis muaj feem xyuam rau lwm yam genetic sib txawv ntawm ob nas hom. Txhawm rau kuaj qhov no ntxiv, peb tau hla 129Sv thiab C57BL / 6 nas mus rau F2 tiam uas muaj kev sib txawv ntawm cov noob caj noob ces (xws li cov hauv NKCC2) yuav tsum random.
faib rau nas. Peb mam li muab piv rau F2 nas uas yog homozygous rau tag nrho-ntev NKCC2 (f/f), homozy-gous rau NKCC2 deletion (Δ/Δ), lossis heterozygous rau NKCC2 deletion (f / Δ). Nrog rau qhov tshwj xeeb ntawm qhov kev cia siab sib txawv ntawm cov qauv sib txawv ntawm cov phosphoform- cov tshuaj tiv thaiv tshwj xeeb tiv thaiv NKCC2 thiab NCC (thiab qhov tsis meej qhov txawv ntawm ROMK qhia ntawm mRNA, tab sis tsis yog qib protein), tsis muaj qhov sib txawv ntawm ob hom kab mob yav dhau los tau tshwm sim hauv lub f/f, Δ/Δ, los yog f/Δ nas ntawm F2 tiam (Daim duab 4 thiab Table 2). Tshwj xeeb, tsis muaj qhov sib txawv ntawm cov zis Ca 2 ntxiv lossis Mg 2 ntxiv rau kev tso tawm, nrog rau tsis muaj qhov sib txawv hauv cov ntshav Ca 2 ntxiv lossis Mg 2 ntxiv, qhia tias qhov sib txawv ntawm 129Sv thiab C57BL / 6 nas yog ywj siab ntawm tsib amino acids tshem tawm hauv NKCC2.
Ib qho tshiab pT96 NKCC2 antibody pom pNKCC2 hauv ob hom kab mob
Txij li thaum tsib cov amino acids tshem tawm hauv NKCC2 cuam tshuam qhov kev tshawb nrhiav txhim khu kev qha ntawm NKCC2 phosphorylation hauv C57BL / 6 nas thaum siv cov tshuaj tiv thaiv muaj, peb tsim cov tshuaj tiv thaiv tshiab tiv thaiv T96 phospho-site hauv C57BL / 6 nas. Cov luav tau txhaj tshuaj nrog phospho-peptide uas sib raug rau cov amino acid sib lawv liag ntawm C57BL/6 NKCC2 nyob ib puag ncig T96 (YYLQ(p) TMDA). Cov tshuaj antisera tau affinity-purified thiab tus yam ntxwv ntawm immunoblotting thiab immunohistochemistry (Daim duab 5). Cov tshuaj tiv thaiv tau kuaj pom ib pawg ntawm qhov xav tau loj ntawm 170 kDa hauv lub raum ntawm 129Sv, C57BL / 6 hom tsiaj qus thiab C57BL / 6 NCC knock-out
nas (Daim duab 5A). Cov qhab tau meej meej sib txawv ntawm qhov kuaj pom rau NCC ntawm 130 kDa. Dephosphorylation ntawm 129 Svraumcov qauv nrog calf intestine alkaline phosphatase (CIAP) tau tshem tawm tag nrho cov teeb liab tau txais nrog pT96 NKCC2 antibody tshiab, thaum lub teeb liab nrog cov non-phosphoform- tshwj xeeb NKCC2 antibody tau pom nyob rau hauv kev tswj thiab dephosphorylated kuaj (Daim duab 5B). Interestingly, NKCC2 phosphorylation kuj tseem raug tshem tawm ntau thaumraumlysates tau incubated ntawm 37 degree rau 1 teev yam tsis muaj CIAP (tej zaum vim yog kev ua ntawm endogenous alkaline phosphatase los ntawm cov tubules ze). Kev sim Dephosphorylation siv lambda phosphatase ntawm qhov sib txuas paraffin- raum ntu ntawm C57BL / 6 nas ntxiv tau lees paub tias cov tshuaj tiv thaiv tshiab pom tsuas yog phosphorylated NKCC2 (Daim duab 5C). Ntxiv mus, immuno fluorescence ntawm kev sib law liag sib law liag tau pom tias tshiab cov tshuaj tiv thaiv kab mob tsuas yog apical plasma membrane ntawm NKCC2- zoo TALs tab sis tsis yog NCC- zoo DCTs hauv 129Sv thiab C57BL/6 nas (Daim duab 5D), qhia tias tus tshiab pNKCC2 antibody tsis cuam tshuam nrog pNCC. Qhov kev txiav txim siab no kuj tau lees paub tias siv cell lysates los ntawm MDCKI hlwb qhia tib neeg NKCC2 (hNKCC2) thiab tib neeg NCC (hNCC). Immunoblots ntawm immunoprecipitations (IP) thiab tag nrho cov cell lysates tau pom tias cov tshiab pT96 NKCC2 antibody lees paub tib neeg NKCC2 tab sis tsis yog tib neeg NCC (Daim duab S8A). Interestingly, txawm hais tias cov tshuaj tiv thaiv tshiab tau raug tsa tawm tsam pNKCC2 ntawm C57BL / 6 nas, cov tshuaj tiv thaiv tshiab tau kuaj pom NKCC2 phosphorylation sib npaug hauv ob lub raum ntawm ob hom nas los ntawm ob qho tib si immunohistochemistry (Daim duab 6A) thiab immunoblotting (Daim duab 6B).
Tus tshiab pT96 NKCC2 antibody tso cai tsom xam ntawm tswj NKCC2 phosphorylation
Lub phosphorylation thiab li no cov kev ua ntawm NKCC2 thiab NCC yog tswj los ntawm extracellular ion concentrations. Ib qho qis [Cl −] ex nce phosphorylation ntawm NKCC2 ntawm Thr96 thiab Thr101 thiab ntawm NCC ntawm Thr53 thiab Thr58 los ntawm kev ua kom lub WNK / SPAK / OSR1 txoj kev. 7,8,18,29,35 Ib yam li ntawd, siab [K ntxiv] txo qis cov phosphorylation ntawm NCC, tab sis xav tias yuav muaj tsawg lossis tsis muaj txiaj ntsig rau NKCC2 phosphorylation. 12,36,37,38 Txhawm rau kuaj seb tus tshiab pT96 NKCC2 cov tshuaj tiv thaiv kab mob puas tuaj yeem kuaj pom qhov sib txawv ntawm NKCC2 phosphorylation,raumcov nplais ntawm C57BL/6 nas tau ex-posed ex vivo rau tog twg los 110 los yog 5 mM [Cl −] ex thiab mus rau 3 los yog 10 mM [K ntxiv] ex feem. Raws li kev cia siab, qhov qis [Cl −] ex nce ntxiv NCC thiab NKCC2 phosphorylation (Daim duab 7A, B), thaum lub siab [K ntxiv] ex tsuas yog txo NCC phosphorylation tab sis tsis hloov NKCC2 phosphorylation (Daim duab 7C, D). Ib yam li ntawd, cov tshuaj tiv thaiv kab mob tshiab tau pom zoo sib npaug
Daim duab 4 Tsis muaj kev hloov pauv tseem ceeb hauv mRNA thiab cov protein qhia ntawm qhov lojlub raumion transporters thiab channels nyob rau hauv crossbred F2 tiam. A, Bar zaj duab xis ntawm cov txheeb ze mRNA qhia theem hauv peb pawg nas. Tag nrho cov txiaj ntsig tau normalized rau f/f nas. Tus nqi yog txhais tau tias ± SEM. B, Protein abundance nyob rau hauv nasraumlysates los ntawm cov tsiaj ntawm F2 tiam. Peb pawg sib raug mus rau tag nrho-ntev NKCC2 (f / f), NKCC2 homozygous rau kev tshem tawm (Δ / Δ) lossis NKCC2 heterozygous rau kev tshem tawm (f / Δ /). Cov cim ntsuas qhov hnyav molecular suav nrog. NKCC2 thiab pNKCC2 yuav tsum ntawm 170 kDa, NCC thiab pNCC ntawm 130 kDa, TRPM6 ntawm 260 kDa, - ENaC ntawm 95 kDa thiab 34 kDa (cleaved), - ENaC ntawm 100 kDa, - ENaC ntawm 1080 kDa (ENaC ntawm 108 kDa) , ROMK ntawm 55 kDa (mature glycosylated), 43 kDa (core glycosylated) thiab 34 kDa (unglycosylated). Cov xub ntsuab taw qhia rau cov kab tshwj xeeb thaum cov xub liab- taub hau taw tes rau cov kab tsis tshwj xeeb. C, Densitometric tsom xam ntawm cov protein ntau sib piv f / f thiab Δ / Δ nas. Cov qhab siv los ntawm Δ / Δ nas yog normalized rau cov los ntawm f / f. (Nqis txhais tau tias ± SEM; n=5 nas/pawg; *P< .05,="">< .01,=""><>
NKCC2 phosphorylation uas tau provoked nyob rau hauv MDCKI hlwb qhia tib neeg NKCC2 (hNKCC2) los yog nas NKCC2 (mNKCC2(C57BL/6)) los ntawm hypotonic tsawg extracellular chloride tej yam kev mob (Daim duab S8B). Hauvraumcov hlais incubated ntawm 5 mM [Cl −] ex , kuj tau piav qhia yav dhau los los tiv thaiv nas pT96 / T101 NKCC2 39 thiab tiv thaiv tib neeg pT212 / T217 NKCC1 R5 11 cov tshuaj tiv thaiv pom faint signals rau NKCC2 thiab NCC, uas tau pom tseeb dua thaum muaj cov protein ntau ntxiv thiab cov tshuaj tiv thaiv kab mob tau pom ntawm qhov chaw kho kom zoo (Daim duab S9).
Kev sib tham
C57BL/6 thiab 129Sv nas yog ob hom nas uas nquag siv rau kev tshawb fawb biomedical. Cov genome ntawm C57BL / 6 nas yog thawj zaug uas tau ua tiav rau nas 40 thiab tau ua haujlwm txij li thaum siv genome rau kev tshuaj ntsuam genetic thiab phenotypic variations ntawm nas hom. 41- 44 Rauraumkev tshawb fawb, ib qho tseem ceeb sib txawv ntawm C57BL / 6 thiab 129Sv nas txhawj xeeb txog kev qhia ntawm renin noob. Zoo li tib neeg, C57BL / 6 nas tsuas yog ib qho renin gene xwb, thaum 129Sv nas muaj ob daim ntawv luam, uas yuav piav qhia txog qhov kev pheej hmoo siab dua ntawm tus nas no rau ntshav siab 45,46 thiab raug mob hnyav. 47 Nrog rau txoj kev tshawb fawb tam sim no, peb ntxiv lwm qhov tseem ceeb ntawm cov noob caj noob ces uas yuav tsum tau xav txog. Peb piav qhia txog tam sim no tsis txaus siab rau tsib cov amino acids tshem tawm hauv NKCC2 (ΔF97- T101), tam sim no hauv C57BL/6 tab sis tsis nyob hauv 129Sv nas, uas cuam tshuam nrog kev tshawb pom ntawm NKCC2 phosphorylation nrog feem ntau cov tshuaj tiv thaiv yav dhau los, tab sis tsis zoo. cuam tshuamlub raum ua haujlwm.
Cov nas NKCC2 N- davhlau ya nyob twg phosphorylation qhov chaw T96 thiab T101 tau txais kev saib xyuas zoo thoob plaws cov tsiaj thiab nruab nrab ntawm NKCC2, NKCC1 thiab NCC (Daim duab 1). 48 Yog li ntawd, nws tsis yog qhov xav tsis thoob tias cov tshuaj tiv thaiv kab mob tiv thaiv cov chaw phosphorylation no cuam tshuam nrog lwm cov cotransporters. Cov kev tshawb fawb yav dhau los tau tshaj tawm tias cov tshuaj tiv thaiv kab mob tiv thaiv phosphorylated NCC kuj tshawb pom phosphorylated NKCC2 7,18,28,29 thiab tias pNKCC1 cov tshuaj tiv thaiv tso cai rau kev soj ntsuam ntawm qib phosphorylation ntawm NKCC1 thiab NKCC2. Raws li qhov no, peb tau pom qhov sib txawv ntawm peb cov pNCC (pT53 thiab pT58) cov tshuaj tiv thaiv nrog phosphorylated NKCC2 thiab ntawm peb pT96 / pT101 NKCC2 antibody nrog phosphorylated NCC. Txawm li cas los xij, qhov cross-reactivity ntawm pNCC cov tshuaj tiv thaiv nrog NKCC2 tsuas yog pom nyob rau hauv ob lub raum ntawm 129Sv nas, tab sis tsis nyob rau hauv ob lub raum ntawm C57BL / 6 nas. Hloov pauv, peb yav dhau los tsim cov tshuaj tiv thaiv rau NKCC2 pT96 / pT101 pom tias tsis muaj lossis qhov zoo tshaj plaws lub teeb liab tsis muaj zog rau pNKCC2 hauv ob lub raum ntawm C57BL / 6 nas thiab kuaj pom cov phosphorylated NCC hauv cov kab mob no. Tam sim no, peb txuas cov kev soj ntsuam tsis meej pem rau tsib cov amino acids tshem tawm hauv NKCC2 ntawm C57BL6 nas (ΔF97- T101). Cov kev cai ntawm NKCC2 phosphorylation tau raug soj ntsuam-
ied nyob rau hauv ntau yam kev sim thiab rau ntau yam stimuli xws li vasopressin, 14,21,24,25,49 uromodulin 19,20,50 thiab calcineurin inhibitors. 10,26 Thaum feem ntau ntawm cov kev tshawb fawb tau ua nyob rau hauv heterologous qhia systems 10,19,20,25,50 los yog nyob rau hauv nas 10,24 thiab nas 19,26 qhia tag nrho ntev NKCC2, ib co kev tshawb fawb kuj tau ua nyob rau hauv C57BL/6 nas. 7,18,27,28,29,51 Raws li peb tau tshaj tawm ntawm no, C57BL / 6 nas qhia txog NKCC2 variant nrog tsib amino acids tshem tawm uas cuam tshuam nrog kev tshawb pom zoo ntawm NKCC2 phosphorylation thiab uas muaj kev pheej hmoo tias tus qauv pT96 / T101 NKCC2 antibodies thiab R5 anti phospho- NKCC1 antibody cross-react nrog phosphorylated NCC tsawg kawg nyob rau hauv cov xwm txheej siv hauv peb txoj haujlwm. Nws tsis tuaj yeem txiav txim siab qhov cuam tshuam ntawm qhov kev cuam tshuam no ntawm cov kev sim yav dhau los, tab sis raws li peb cov kev soj ntsuam, cov ntaub ntawv los ntawm C57BL/6 nas yuav tsum tau rov mus xyuas thiab txhais nrog ceev faj. Txawm hais tias qhov tseeb, tias tsib amino acids tshem tawm tau muaj kev cuam tshuam zoo rau kev tshawb nrhiav NKCC2 phosphorylation, peb tsis muaj pov thawj tias kev tshem tawm cuam tshuam rau kev ua haujlwm ntawm cotransporter. C57BL / 6 nas muaj nyob rau hauv kev ua haujlwm siab dua urinary Mg 2 ntxiv rau kev tso tawm thiab qis tso zis Ca 2 ntxiv rau kev tso tawm 31 dua li 129Sv nas, tab sis peb pom tias qhov sib txawv no tej zaum tsis cuam tshuam txog kev hloov pauv paracellular thauj ntawm cov cations raws TAL, tab sis yog qhov tshwm sim ntawm kev txhim kho TRPM6- nyob ntawm Mg 2 ntxiv rau kev nce hauv cov hnyuv thiab nce TRPV5- nyob ntawm Ca 2 ntxiv rau reabsorption hauv DCT2 thiab CNT. Qhov kawg ua rau muaj qhov zoo Ca 2 ntxiv rau qhov sib npaug uas tuaj yeem pab ua kom cov pob txha pob txha tsawg ntawm C57BL / 6 nas. Qhov kev tshawb pom tias C57BL/6 thiab 129Sv nas qhia tib yamlub raumCov lus teb rau lub voj- thiab thiazide diuretics thiab kev txwv dej ntxiv qhia tias tsib amino acids tshem tawm hauv NKCC2 tsis cuam tshuam TAL ua haujlwm. Qhov tseem ceeb tshaj plaws, cov nas ntawm F2 tiam ntawm cross-bred C57BL / 6 thiab 129Sv nas muaj tib yam.lub raumphenotype ywj siab ntawm seb cov nas qhia tag nrho ntev (f/f) NKCC2 lossis NKCC2
homozygous (Δ / Δ) rau kev tshem tawm. Cov kev soj ntsuam hauv cov nas no zoo ib yam nrog cov ntaub ntawv ua haujlwm yav dhau los los ntawm cov kab lus heterologous. Thaum kev hloov pauv ntawm ib qho residues hauv NKCC1 (T217 hauv tib neeg, T211 hauv nas) lossis NCC (T60 hauv tib neeg, T58 hauv nas) tshem tawm cov haujlwm ntawm cov cotrans porters, kev hloov pauv ntawm cov khoom seem hauv NKCC2 (T105 hauv tib neeg, T101 hauv nas. ) muaj qhov me me lossis txawm tias tsis cuam tshuam rau hauv paus thiab txhawb nqa NKCC2 kev ua haujlwm. 35,52,53 Txawm tias thaum ob qho tib si threonine residues hauv NKCC2 tau hloov mus rau alanine, kev ua haujlwm ntawm NKCC2 tseem nce mus txog ~ 70 feem pua ntawm cov dej num siab tshaj plaws, 35 qhia tias phosphorylation ntawm cov chaw no yog qhov qhia tab sis tsis tseem ceeb rau NKCC2 kev ua.
Phosphorylation sites feem ntau yog homologous ntawm cov proteins. Yog li ntawd, qhov teeb meem ntawm cross-reactivity ntawm phosphoform- cov tshuaj tiv thaiv tshwj xeeb tsis yog tshwj xeeb rau cov tshuaj tiv thaiv kab mob tiv thaiv phosphorylated NCC, NKCC2 thiab NKCC1, tab sis kuj tau tshaj tawm rau lwm cov proteins xws li cyclin dependent kinases 1 thiab 2 54 thiab extracellular. teeb liab- tswj kinases 1 thiab 2. 55 Tsis tu ncua, thaum twg phosphorylation xeev- cov tshuaj tiv thaiv tshwj xeeb raug siv, yuav tsum muaj kev sim nruj tswj xyuas kom paub meej qhov tshwj xeeb ntawm cov teeb liab pom. Qhov no yuav suav nrog (a) kev lees paub tias cov neeg ua haujlwm tshuaj tiv thaiv kab mob kuaj pom cov protein ntawm qhov hnyav molecular (immunoblotting) thiab / lossis ntawm qhov xav tau ntawm cellular thiab subcellular localization (immunofluorescence), (b) pov thawj tias cov tshuaj tiv thaiv kab mob cuam tshuam tshwj xeeb nrog phosphorylated epitope los ntawm siv cov kev ntsuam xyuas kev sib tw nrog cov phosphorylated thiab non-phosphorylated peptides, thiab (c) ua qauv qhia tias
cov tshuaj tiv thaiv yog phosphoform tshwj xeeb los ntawm kev sib piv cov qauv phosphorylated thiab dephosphorylated cov ntaub so ntswg. Cov kev tswj no tuaj yeem ua tiav los ntawm kev tswj hwm caj ces uas suav nrog kev sim cov ntaub so ntswg los ntawm cov tsiaj txhu thiab / lossis cov hlwb heterologously qhia cov protein ntawm kev txaus siab. Hauv txoj kev tshawb fawb tam sim no, peb tau siv cov kev qhia saum toj no-hais kom meej meej tus yam ntxwv tshiab pT96 NKCC2 antibody uas kuaj pom phosphorylated NKCC2 hauvraumkuaj los ntawm C57BL/6 thiab 129Sv nas sib npaug zoo. Cov tshuaj tiv thaiv kab mob yog phosphoform- tshwj xeeb thiab tsis pom muaj kev sib txuam nrog phosphorylated NCC nyob rau hauv ib qho ntawm cov kev sim. Siv cov tshuaj tiv thaiv kab mob no peb tau lees paub tias cov tshuaj chloride qis qis ua rau cov phosphorylation ntawm NKCC2 ntawm qhov chaw no, 7,8 thaum hloov pauv cov tshuaj potassium ntxiv tsis muaj lub luag haujlwm tseem ceeb. 24 Hauv kev xaus, peb txoj haujlwm qhia txog qhov sib txawv tseem ceeb ntawm cov amino acids ntu ntawm nas NKCC2 uas cuam tshuam rau kev kuaj pom thiab yog li kev tshuaj xyuas ntawm NKCC2 phosphorylation thiab kev tswj hwm. Ib tug tshiab tsim pT96 NKCC2 antibody hla qhov kev caveat no thiab tso cai rau ib tug txhim khu kev qha tshawb pom ntawm hloov NKCC2 phosphorylation ywj siab los ntawm cov caj ces keeb kwm ntawm nas qauv. Ntxiv mus, peb txoj kev tshawb fawb tau hais ntxiv tias qhov sib txawv ntawm keeb kwm ntawm caj ces yuav tsum tau xav txog thaum txheeb xyuas cov qauv nas.
Cov khoom siv thiab cov txheej txheem
Tsiaj txhu,Txiv neej hnub nyoog sib tw (6- 8 lub lis piam) nas yog ib hom kab mob inbred 129S6/SvEvTac, C57BL/6J, ntawm no tom qab hu ua 129Sv thiab C57BL/6, los yog sib xyaw hybrid hom 129S/B6 hauv F2 tiam. Txhua qhov kev sim tsiaj tau ua raws li Swiss Animal Welfare txoj cai thiab pom zoo los ntawm cov kws kho tsiaj hauv Canton of Zurich, Switzerland (Daim ntawv tso cai: 141/2014, 185/2017 thiab 135/2018). Cov nas tau genotyped rau kev tshem tawm ntawm 15 lub hauv paus siv tus qauv PCR nrog primers muab hauv Table S3. Tag Nrho-ntev (f/f) NKCC2 muaj qhov xav tau PCR cov khoom lag luam ntawm 87 bp thaum tshem tawm NKCC2 ib ntus (Δ / Δ) xav tau ntawm 70 bp.
Kev ua cov ntaub so ntswgCov nas tau muab tshuaj loog nrog kev sib xyaw ntawm isoflurane (2 feem pua - 4 feem pua, 0.5 l/min; Provet; CH) thiab Temgesic (Buprenorphine; 0.05- 0. 4 mg / kg lub cev hnyav; Indivor; CH). Rau kev soj ntsuam biochemical, nas tau tshuaj loog thiab perfused ntawm sab laug ventricle ntawm lub plawv nrog phosphate buffered saline (PBS). Lub raum thiab cov plab hnyuv siab raum tau sau tseg, snap khov hauv cov kua nitrogen thiab khaws cia hauv −80 degree kom txog thaum ua tiav. Rau immunohistochemistry, ob lub raum raug kho siv paraformaldehyde (PFA) 3 feem pua hauv 0.1 M phosphate tsis (pH 7.4, 300} mOsm) los ntawm retrograde perfusion ntawm cov nas anaesthetized tob ntawm lub plab aorta ua raws li kev yaug luv nrog 0.1 M phos phate buffer (0.2 M NaH 2 PO 4 x H 2 O, 0.2 M NaH 2 PO 4 x 2H 2 O, 0.1 M CaCl 2 ). Tom qab ntawd, lub raum raug tshem tawm, txiav rau hauv daim thiab khov rau hauv cov kua propane thiab khaws cia ntawm -80 degree.
Kev tshawb nrhiav cov kab mob metabolicTom qab hloov mus rau cov kab mob metabolic (Techniplast), cov nas tau khaws cia rau hauv cov kab mob metabolic rau plaub hnub sib law liag nrog kev nkag tau dawb rau cov dej kais dej thiab cov qauv kuaj chow (Ssniff, Spezialdiäten GmbH). Lub cev hnyav, zaub mov thiab dej tau sau tseg txhua hnub thiab 24 teev tso zis thiab quav.
Cov txheej txheem txwv dejCov nas tau khaws cia rau 24 teev nyob rau hauv cov kab mob metabolic (Techniplast) yam tsis muaj kev nkag mus rau cov kais dej. Standard lab chow hmoov (Ssniff, Spezialdiäten GmbH) tau tov nrog dej 1: 1, 24 teev cov zis thiab quav tau sau thiab tshuaj xyuas. Tom qab 24 teev, cov nas tau muab tso rau hauv lub tawb qub (T 2L (IVC), LASC, UZH).
Kev ntsuas biochemicalCov zis ntau ntawm Na plus , K ntxiv , Ca 2 ntxiv tau txheeb xyuas los ntawm nplaim taws photometry (EFOX 5053, Eppendorf). Cov zis thiab ntshav ntshav ntau ntawm Mg 2 ntxiv tau ntsuas ntawm Zurich Center for Integrative Rodent Physiology nrog SYNCHRON LX ® System(s), UniCel ® DxC 800 Synchron ® Clinical System thiab Synchron ® System Multi Calibrator. Cov zis creatinine tau ntsuas raws li txoj kev Jaffe. Ntshav roj thiab ion concentrations tau ntsuas hauv heparinized tag nrho cov ntshav siv ABL825Flex Blood Gas Analyzer (Radiometer) tam sim ntawd tom qab sau ntshav los ntawm vena cava. Plasma aldosterone qib thiab tso zis concentration ntawm uromodulin (UMOD) tau ntsuas nrog cov khoom siv ELISA muag (Aldosterone ELISA cov khoom siv los ntawm Cayman; # 501090 thiab Mouse Uromodulin ELISA cov khoom siv los ntawm Abcam; ab245726 raws li).
Lub sijhawm tiag tiag kom muaj nuj nqis polymerase saw cov tshuaj tiv thaiv (RT-qPCR)
RNA raug cais tawm ntawm ob lub raum thiab cov plab hnyuv siab raum siv SV tag nrho RNA cais cov khoom siv (PROMEGA). Qhov sib npaug ntawm qhov sib txawv ntawm RNA (250 ng hauv 129Sv vs C57BL / 6 lossis 500 ng hauv f / f, Δ / Δ thiab f / Δ) tau thim rov qab rau hauv cDNAs nrog cov khoom siv GoScript™ Reverse Transcriptase (PROMEGA). Cov khoom tsim tawm cDNA tau ntxiv nrog rau DNAse / RNAse-dawb dej (1: 5). Primers tau tsim los kom muaj nuj nqis ntawm tag nrho Slc12a1, Slc12a3, Trpm6, Trpv5, Scnn1a, Scnn1b, Scnn1g thiab Kcnj1 (Table S3). Kev tsom xam PCR ntau tau ua tiav siv LightCycler ® 480 (Roche) los ntsuas cov qib qhia ntawm cov noob paj. Kev txheeb ze cov noob qhia tau suav nrog siv delta Ct txoj kev siv -actin raws li cov noob siv.
Western blot tsom xamLub raum tau homogenized nyob rau hauv ib tug detergent-free lysis buffer (DFLB) uas muaj mannitol 200 mM, HEPES [4- (2- hydroxylethyl)- 1piperazineethanesulfonic acids] 80 mM, potas sium hydroxide 41 mM, protease inhibitors (Ua tiav Ultra, Roche) thiab phosphatase inhibitors (PhosSTOP, Roche) siv 32 Magna Lyser Green Beads (Roche) hauv Precellys 24 cov ntaub so ntswg homogenizer (Bertin Instruments, Montigny-le- Bretonneux). Cov hlaws tau sedimented los ntawm centrifugation rau 10 feeb (1987 rcf) thiab cov protein uas muaj supernatant tau khaws cia rau hauv ib leeg-siv- aliquots ntawm -80 degree. Rau Western blot, 50 µg ntawm cov protein tau denatured hauv Laemmli Buffer, sib cais los ntawm SDS- PAGE siv 8 feem pua - 12 feem pua gels thiab tom qab ntawd hloov mus rau nitrocellulose membranes. Cov chaw tsis tshwj xeeb khi tau raug thaiv siv 1x Odyssey Blocking Solution (Li-COR Biosciences) ua ntej cov membranes tau incubated nrog thawj cov tshuaj tiv thaiv (Table S4) diluted hauv 0.2x Odyssey Blocking Solution ntawm 4 degree rau 12 teev. Tom qab ntxuav hauv PBS- 0.1 feem pua Tween, cov membranes tau incubated nrog lwm cov tshuaj tiv thaiv kab mob (tshis- anti- luav IRDye 800 los yog tshis- anti- nas IR dye 680, 1:10'000, LI - COR Biosciences) diluted hauv 0.1x Casein Blocking buffer (Sigma). Kev kuaj pom bands tau pom nrog Odyssey infrared imaging system (Li-COR Biosciences). Txhawm rau kom muaj qhov sib npaug ntawm cov khoom noj protein, daim nyias nyias tau ua ke nrog cov tshuaj tiv thaiv kab mob tiv thaiv - actin lossis tag nrho cov protein raug stained siv REVERT Total Protein Stain (Li-COR Biosciences) ua ntej kuaj cov tshuaj tiv thaiv. Immunoreactive bands tau txheeb xyuas nyob rau hauv Fiji (ImageJ) thiab normalized tiv thaiv - actin teeb liab los yog REVERT Tag nrho cov protein stain feem.
ImmunohistochemistryCov ntu Cryostat (thickness 4 µm) raug thaiv rau nonspecific antibody khi nrog 10 feem pua cov tshis ib txwm muaj ntshav qab zib thiab tom qab ntawd incubated nrog thawj cov tshuaj tiv thaiv (Table S4)
hmo ntuj ntawm 4 degree. Tom qab ntxuav nrog 1X PBS, slides tau incubated nrog lwm cov tshuaj tiv thaiv kab mob (Cy3 – conjugated tshis- anti- luav IgG, khoom naj npawb 111- 165- 144, 1:1000 thiab FITC – conjugated tshis- anti-nas IgG , khoom naj npawb 115- 095- 068, 1:100 raws, ob qho tib si los ntawm Jackson Immuno Research Laboratories) rau 2 teev ntawm chav tsev kub. Tag nrho cov tshuaj tiv thaiv tau diluted hauv 1 feem pua BSA / 1xPBS. Cell nuclei raug stained nrog DAPI (4′, 6- Diamidino- 2- phentyl- indole, Sigma- Aldrich, Co.) nyob rau hauv ib tug concentration ntawm 0.1 µg/mL. Kev yees duab tau ua tiav siv Leica DM6000 B fluorescence microscope (Leica Microsystems, 35578) nrog Leica DFC350 FX fluorescence monochrome digital koob yees duab (Leica Microsystems, 35578). Cov duab tau ua tiav hauv Fiji (ImageJ).
Generation ntawm affinity purified luav- tiv thaiv nas pT96 NKCC2 antibodyKev tsim tshuaj tiv thaiv kab mob tau ua los ntawm Pineda antibody kev pabcuam (Berlin, Lub Tebchaws Yelemees). Cov luav tau txhaj tshuaj nrog phospho-peptide suav nrog cov amino acids 105- 114 ntawm C57BL/6 nas NKCC2 ntu (NH2- YYLQ(p) TMDAV- COOH). Monospecific IgG feem yog affinity purified tiv thaiv phospho-peptide thiab tom qab preabsorbed nrog dephospho-peptide uas ua rau ib tug phosphoform- tshwj xeeb antibody feem. Cov tshuaj tiv thaiv tshwj xeeb ntawm cov feem no tau kuaj los ntawm immunohistochemistry thiab Western blot tsom (Daim duab 5 thiab daim duab S4).
Kev kho phosphataseProtein lysates tau incubated rau 1 teev ntawm 37 degree nrog calf plab hnyuv alkaline phosphatase (CIAP) (1 U/1 µg protein) kom induce lub hydrolysis ntawm 5′- phosphate pawg ntawm phos-phorylated proteins, uas inhibited qhov tshwj xeeb khi ntawm pT96. NKCC2 antibody rau cov proteins ntawm nitrocellulose Western blot membrane (Daim duab 5B). Archived paraffinembedded raum los ntawm C57BL6 nas tau siv. Kev kho mob ntawm cov ntu uas siv calf plab hnyuv alkaline phosphatase (Sigma Aldrich) tau ua raws li tau piav qhia ua ntej nrog kev hloov kho me me. 56 Tom qab thaiv cov pab pawg dawb aldehyde, ntu tau muab yaug tsib zaug hauv alkaline phosphatase tsis (100} mm Tris, 50 µm CaCl 2, 0.1 mm MgCl 2 , 8.4 µm leupeptin, 4 mm Pefablock pH 9.0). Tom qab ntawd, cov seem tau incubated nyob rau hauv alkaline phosphatase tsis nrog los yog tsis calf plab hnyuv alkaline phosphatase (~ 130 units / mL) nyob rau hauv lub incubator ntawm 35 degree rau 2 teev. Immunohistochemistry thiab lub teeb microscopy tau ua tom qab ua tiav raws li ntxaws. 57
Amino acids sequence alignmentCov amino acids sequences rau ntau hom thiab nas hom tau txais los ntawm cov ntaub ntawv qhib "Ensembl Genome Browser" (www.ensem bl.org, Sanger Institute). 42 Cov peev txheej ntawm daim npav ID tau teev tseg hauv Table S1. Cov kab ke tau ua raws li Qiagen © CLC Main Workbench 20.
Lub raum hlaisLub raumcov nplais ntawm C57BL/6 nas tau siv rau ex vivo thwmsim raws li tau piav qhia dhau los. 12 luv luv, nas tau yoo mov thaum hmo ntuj nrog kev nkag mus rau dej dawb kom tsis txhob muaj kev cuam tshuam ntawm kev noj haus ion kom tsawg ntawm phosphorylation ntawm NKCC2 thiab NCC. Lub raum raug tshem tawm los ntawm cov tsiaj anaesthetized, txiav rau hauv 280 µm tuab slices siv lub vibrating microslicer (Vibratome, Microm; Thermo Scientific), thiab muab tso rau hauv Ringer's tov (hauv mM): 98.5 NaCl, 25 NaHCO 3, 3 KCl, 1 Na 2. HPO 4, 2.5 CaCl 2, 1.8 MgCl 2 thiab 25 qabzib rau 30 feeb ntawm 30.5 degree nrog bubbling tas li nrog 95% O 2 thiab 5% CO 2. Tom qab ntawd, cov tshuaj Ringer tau hloov los ntawm cov kev daws teeb meem zoo sib xws tab sis nrog qhov sib txawv Cl − (5 mM rau qis Cl − , 110 mM rau ib txwm Cl − ) thiab K ntxiv concentrations (3 mM rau ib txwm K ntxiv, 10 mM rau siab K ntxiv) rau ntxiv 30 feeb. Thaum kawg, cov hlais tau khov rau hauv cov kua nitrogen.
Kev txheeb caisUnpaired Student's t test thiab Welch's test in case of uneven variances are used to comparison two group (GraphPad Prism, Version 8.0.2). Rau ntau qhov kev sib piv, ib-txoj kev lossis ob-txoj kev ANOVA ua raws li Tukey qhov kev sib piv ntau qhov kev xeem tom qab tau ua tiav (GraphPad Prism, Version 8.0.2). Cov ntaub ntawv raug tso tawm raws li txhais tau tias ± SEM (hauv Table thiab Figures). Qhov sib txawv tau suav tias yog qhov tseem ceeb thaum P <>
TXOJ CAI Cov kws sau ntawv ua tsaug rau Dr Gary Shull (University of Cincinnati, Cincinnait, OH) rau kev muab NCC-KO nas, Dr Biff Forbush (Yale School of Medicine, New Haven, CT) rau nws "R5" pNKCC1 antibody, thiab Dr Henrik Dimke ( University of Southern Denmark, Odense, Denmark) rau nws nas monoclonal tag nrho NCC antibody. Cov kws sau ntawv kuj lees paub Monique Carrell thiab Inger Merete Paulsen rau kev pab cuam thiab Dr Eszter Banki rau kev muabraumcov qauv. Kev ntsuas cov pob txha pob txha pob txha thiab ib feem ntawm cov zis thiab ntshav tsom xam tau ua los ntawm Dr Petra Seebeck thiab Nadine Nägele (Zurich Integrative Rodent Physiology, University of Zurich) raws li. Txaus siab heev yog cov tswv yim pab tau zoo ntawm Dr Alicia McDonough (University of Southern California) thiab Dr Olivier Devuyst (University of Zurich).
Kev tsis sib haum xeeb ntawm kev txaus siab Cov pab pawg ntawm JL tau txais cov nuj nqis rau cov ntawv tso cai tshuaj tiv thaiv kab mob tiv thaiv NCC thiab phosphorylated Nedd 4- 2 los ntawm Milipore thiab Abcam feem. Ob qho tshuaj tiv thaiv tsis tau siv hauv txoj kev tshawb fawb tam sim no. Ntxiv mus, JL tau txais 2019 ib qho kev qhuas los ntawm VIFOR rau kev nthuav qhia qhov ncauj ntawm lub rooj sib tham los ntawm VIFOR.
