SLC37A2, Phosphorus-related Moleculeincreases hauv Smooth Muscle Cellsin Calcified Aorma

Vascular calcificationyog lub hauv paus loj ntawmkab mob plawvnyob rau hauv cov neeg mob uas mob raum mob.Hyperphosphatemiaua rau muaj zog intracellular phosphorus influx, uas ua rau muaj kev nce hauv osteoblast zoo li cov hlwb hauv vascular du leeg hlwb. PiT-1 hloov phosphate hauv vascular du leeg hlwb. Txawm li cas los xij, cov txheej txheem ntawm vascular calcification tsis to taub tag nrho. Txoj kev tshawb no tshawb xyuas cov neeg sib tw phosphorus-txog cov molecules uas tsis yog PiT-1. Peb xav tias phosphorus-txog cov molecules uas muaj nyob rau hauvkua-carrier(SLC) superfamily yuav koom nrog hauv vascular calcification. Raws li qhov tshwm sim ntawm DNA microarray tsom xam, peb tau tsom mus rau SLC37A2 thiab pom tias mRNA qhia ntawm cov hlwb no tau nce ntxiv ntawm calcified aortic smooth muscle cells (AoSMC). SLC37A2 tau tshaj tawm tias thauj cov piam thaj ob qho tib si -6-phosphate/phosphate thiab phosphate/phosphate exchanges. Nyob rau hauv vitro tsom xam pom tias SLC37A2expression tsis cuam tshuam los ntawm o ntawm AoSMC. Kev nthuav qhia ntawm SLC37A2 mRNA thiab protein ntau ntxiv hauv calcified AoSMC. Hauv vivo, kev tsom xam pom tias SLC37A2 mRNA qhia hauv aorta ntawmmob raum mobnas tau correlated nrog osteogenic marker noob. Tsis tas li ntawd, SLC37A2 tau qhia nyob rau hauv vascular calcification cheeb tsam hauvmob raum nas nas. Yog li ntawd, peb tau pom tias SLC37A2 yog ib qho ntawm cov molecules uas nce nrogvascular calcificationhauv vitro thiab vivo.

Cov lus tseem ceebQauv: SLC37A2,vascular calcification, aortic smooth nqaij hlwb,mob raum mob, hyperphosphatemia

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Raws li tus naj npawb ntawm cov neeg mob nrogmob raum mob(CKD) nce, tus naj npawb ntawm cov neeg mobyuav tsum tau dialysisyuav nce ntxiv thiab. Feem ntau cov neeg tuag nyob hauv cov neeg moblim ntshavyog vimkab mob plawvxws liplawv tsis ua hauj lwm, mob cerebrovascular, los yog myocardial infarction. (,2) Ib qhov loj ntawmkab mob plawvyog ectopic calcification, () ib qho txawv txav tshwm sim los ntawm kev tsim thiab deposition ntawm calcium phosphate crystals nyob rau hauv cov ntaub so ntswg mos. (4 6) Ntau yam xws li mob. oxidative kev nyuaj siab, thiab nce nyob rau hauv plasma calcium (Ca) thiab phosphorus (P) ntau ntau koom tes nyob rau hauv kev loj hlob ntawm vascular calcification. Ntawm cov xwm txheej no, hyperphosphatemia feem ntau cuam tshuam nrog vascular calcification. (7.8) Hyperphosphatemia yog tshwm sim los ntawm kev noj ntau dhau ntawm P nyob rau hauv lub raum tsis ua haujlwm. Yog li ntawd, cov neeg mob ntshav qab zib yuav tsum txwv tsis pub noj P. (79-12) Txawm li cas los xij, nws tau tshaj tawm tiasvascular calcificationtwb tau nce mus lawm thaum pib lim ntshav; Nws kuj tau pom nyob rau hauv cov neeg mob nrog CKD thaum ntxov thaum hyperphosphatemia tsis pom tseeb. (3) Yog li ntawd, nws yog ib qho tseem ceeb los tiv thaiv vascular calcification nyob rau hauv cov neeg mob nrog CKD thaum ntxov los txhim kho lub neej prognosis.

uas ua rau muaj kev nce ntxiv ntawm P mus rau hauv vascular du leeg hlwb (VSMC). P influx yog nqa tawm ntawm PiT-1, tus thauj khoom ntawm lub cell membrane ntawm VSMC. PiT- yog ib tug tswv cuab ntawm cov neeg nqa khoom solute (SLC) superfamily, uas suav nrog sodium phosphate transporter encoded los ntawm SLC20A1.04 PiT- thauj phosphate hla Na-dependent cell membranes thiab tom qab matrix mineralization vim nce p theem.s) Raws li intracellular P qib nce, VSMC pib nthuav qhia cov yam ntxwv ntawm cov tshuaj osteogenic, xws li runt-related transcription factor 2(Runx2)(16) Cov nyhuv no yog ua raws li kev nthuav qhia ntawm osteoblast-specific genes, xws li osteopontin thiab osteocalcin. Tsis tas li ntawd, txo qhov kev qhia ntawm cov leeg nqaij tshwj xeeb proteins. xws li cov nqaij leeg nqaij du 22-0 (SM22-0), tshwm sim. Raws li qhov tshwm sim, VSMC tau hloov mus rau hauv cov hlwb zoo li osteoblast. (1718) Cov kev tshawb fawb soj ntsuam tsis ntev los no tau pom tias cov ntshav muaj calcium. protein hais (CPP), uas sawv cev rau calcium phosphate colloidal hais, nce nrog rau lub raum ua haujlwm. (19-21) Tsis tas li ntawd, kev sib raug zoo nrog cov ntshav CPP thiab P qib tau raug tshaj tawm.2o2i) Nws muaj peev xwm hais tias CpP tuaj yeem ua rau muaj kev loj hlob ntawm vascular calcification.22-24)

Txawm li cas los xij, vascular calcification tau pom nyob rau hauv cov neeg mob thaum ntxov CKD nrog cov ntshav plasma P qib. (13) Ntxiv rau.PiT-1 mRNA tau nthuav tawm thoob plaws hauv txhua cov ntaub so ntswg. Yog li lwm cov molecule tuaj yeem ua feem ntau hauv thaj tsam ntawm vascular calcification. Muaj lus ceeb toom tiasmobtxhawb vascular calcification. Inflammatory yam xws li interleukin (IL)-1B, IL-6, thiab qog necrosis factor-alpha (TNF-a) induced sib txawv ntawm VSMC rau hauv osteoblast-likes cells (2s26) TNF-a induces vascular calcification los ntawm txoj kev cAMP, uas ua lub luag haujlwm hauv osteoblast sib txawv.2728) IL-6 tswj ntau txoj hauv kev ua rau vascular calcification ntawm VSMC2-31) Tsis tas li ntawd. IllB, IL-6, thiab TNF-a kuj tseem muaj feem cuam tshuam nrog nce vascular calcification induced los ntawm siab p theem.3233) Txawm li cas los. cov ntaub ntawv tam sim no tsis tau piav qhia tag nrho cov txheej txheem ntawm vascular

calcification induced los ntawm cov xwm txheej no. Peb xav tias tej zaum yuav muaj lwm yam P-txog molecules xws li cov nyob hauv SLC superfamily, koom nrog hauv vascular calcification. Peb tsom mus rau daim nyias nyias cov proteins hauv SLC superfamily thiab tshawb xyuas cov neeg sib tw P-txog cov molecules uas tsis yog PiT-1 rau kev koom tes hauv vascular calcification. Tsis tas li ntawd, peb tau tshuaj xyuas qhov kev sib raug zoo ntawm cov neeg sib tw molecules thiab vascular calcification hauv video thiab hauv vivo.

Cov ntaub ntawv thiab cov txheej txheem

Cell kab lis kev cai. Ib txwm humanaorticsmoothmuscle hlwb (AoSMC) tau txais los ntawm Lonza (Basel, Switzerland) AoSMC yog kab lis kev cai raws li cov chaw tsim khoom cov lus qhia. AoSMC tau khaws cia nyob rau hauv ib qho kev loj hlob nruab nrab (cov leeg nqaij du basal nruab nrab ntxiv nrog 5% fetal bovine serum (FBS), epidermal growth factor, insulin, fibroblast growth factor-B, thiab gentamicin/amphotericin-B). Media tau hloov txhua hnub. AoSMC tau loj hlob nyob rau hauv ib qho av humidified 5% CO incubator ntawm 37 degree thiab khaws cia kom txog rau thaum lub confluence tau mus txog thiab tom qab ntawd siv hauv kev sim.

Calcified AoSMC.Calcification ntawm AoSMC yog induced raws li tau piav yav dhau los.435) AOSMC tau cultured nyob rau hauv Dulbecco'smodified Eagle's nruab nrab supplemented nrog 15% FBS thiab penicillin-streptomycin thiab cultured nrog 2.6 mM P rau 3, 6, 9, 12 . P concentration tau kho los ntawm P bufferNaH, PO/Na, thiab HPO. pH 7.2). Lwm yam AoSMCs tsis yog kab lis kev cai nrog 2.6 mM P.

DNA microarray. Tag nrho RNA ntawm AoSMC cov qauv coj mus kuaj thiab tsis yog kab lis kev cai nrog 2.6 mM P tau siv. Tag Nrho RNA 5.0 ug tau soj ntsuam siv DNA microarray (Agilent Technologies, Tokyo. Nyiv). SurePrint G3 Tib Neeg GE 8 x 60k ver. 2 Microarray tau siv los tshuaj xyuas txog 26, 000 noob. Qhov piv tau suav los ntawm kev faib cov theem qhia ntawm AoSMC kab lis kev cai nrog 2.6 mM rau 12 hnub los ntawm theem qhia ntawm AoSMC tsis yog kab lis kev cai nrog 2.6 mM P

DNA microarray

Tag nrho RNA ntawm AoSMC cov qauv coj mus kuaj thiab tsis yog kab lis kev cai nrog 2.6 mM P tau siv. Tag Nrho RNA 5.0 ug tau soj ntsuam siv DNA microarray (Agilent Technologies, Tokyo. Nyiv). SurePrint G3 Tib Neeg GE 8 x 60k ver. 2 Microarray tau siv los tshuaj xyuas txog 26, 000 noob. Qhov piv tau suav los ntawm kev faib cov theem qhia ntawm AoSMC kab lis kev cai nrog 2.6 mM rau 12 hnub los ntawm theem qhia ntawm AoSMC tsis yog kab lis kev cai nrog 2.6 mM P.Expression ntawm SLC37A2 hauv vascular o. Txhawm rau ua kom muaj cov lus teb inflammatory hauv AoSMC, cov hlwb raug coj los ntawm kev loj hlob nruab nrab kom txog thaum lawv mus txog qhov sib txuam thiab tom qab ntawd kho nrog 1 ugmL lipopolysaccharides (LPS) (LPS los ntawm escherichia coli O111:B4; Sigma, St. Louis, MO) rau 24 thiab 72 yam tsis muaj 2.6 mM P. Lwm yam AoSMC tsis yog kab lis kev cai nrog lub sijhawm tiag tiag polymerase saw tshuaj tiv thaiv (PCR). Tag nrho RNA tau muab rho tawm los ntawm AoSMC kho nrog LPS rau 24 thiab 72 teev thiab cov hlwb tsis kho nrog LPS. SYBR ntsuab-raws li PCR ntawm lub sijhawm tiag tiag tau ua los ntawm kev siv cDNA synthesized thiab tsim nyog primer teem rau txhua lub hom phiaj gene nrog aStepOnePlus real-time PCR system (Applied Biosystems, FosterCity, CA). Cov txheej txheem primer tau raws li hauv qab no: tib neeg IL-6.5-AATTCGGTACATCCTCGACGG-3' thiab 5-GCCATCTTTGGAAGGTTCAGG-3'; human SLC37A2,5TCACTTTAGTGCCAAAGGAGGC-3' thiab 5CCATTGGTGTAGTCAGAGACG-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH5'-CTGCACCACCAACTGCTTAGC- thiab 5-CTTCTGGGIGGCAGTGATGGC-3.IL-6 thiab SLC37A2 mRNA qhia theem tau xam tom qab normalization ntawm GAPD. AoSMC tag nrho tau muab rho tawm los ntawm AoSMC uas tsis yog kab lis kev cai nrog 2.6 mM P thiab AoSMC hlwb kab lis kev cai nrog 2.6 mM P rau 3, 6, 9, 12, thiab 14 hnub. raws li hauv qab no: human Runx2, 5-TGAGAGCCGCTTCTCCAACCC-3', thiab 5-CTCTGGCTTTGGAAGAGCCG-3. The Runx2 thiab SLC37A2 mRNA qhia theem tau xam tom qab normalization nrog APPH.

Western blotting tsom xam.

Tag nrho cov protein tau sau los ntawm AoSMC cov qauv kab lis kev cai nyob rau hauv tib lub sijhawm siv rau kev txheeb xyuas PCR tiag tiag. Cov qauv no tau lysed siv Tris-buffered saline (pH 75) ntxiv nrog 5% NP-40 thiab 1% protease inhibitor cocktail. Cov qauv kuaj pom muaj protein ntau tau ntsuas siv BCA Protein Assay Kit (Thermo FisherScientific.Waltham, MA). thiab cov concentration tau kho los ntawm sab hnub poob blotting, 10 ugs ntawm cov protein tau sib cais hauv 10% polyacrylamide gel siv sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lub membrane tau kho nrog SLC37A2antibody (Novus Biologicals, Centennial, CO) lossis GAPDH antibody (GeneTex, Irvine, CA) ua cov tshuaj tiv thaiv thawj, incubated nrog Rabbit lgG Horseradish peroxidase-conjugated antibody (R&D Systems, Minneapolis, MN). thiab pom kev siv Amersham ECL Xaiv (GE Healthcare. Buckinghamshire, England) thiab luminescent duab analyzer (LAS-1000 ntxiv, FUNIFILM, Tokyo, Nyiv). Lub band tau suav nrog lmagel.

Kev sim

Tag nrho cov txheej txheem kev kawm tau pom zoo los ntawm Pawg Neeg Saib Xyuas Kev Ncaj Ncees ntawm University of Hyogo, Tsev Kawm Ntawv ntawm Tib Neeg Kev Tshawb Fawb thiab Ib puag ncig. Kaum ob lub lis piam txiv neej, Sprague-Dawley nas tau yuav los ntawm Nyiv SLC (Shizuoka, Nyiv). Cov nas tau khaws cia rau ntawm 12- teeb, 12-h lub voj voog tsaus ntuj (09:00-21:00) thiab pub dawb nkag mus rau zaub mov thiab dej ntshiab ntxiv.

Cov kev sim noj zaub mov suav nrog kev tswj P noj zaub mov (1.0% P, 0.6Ca, 20% protein), noj adenine (0.75% adenine , 1. .6% Ca, 10% protein), thiab kev noj zaub mov siab P (HP) (1.0% P, 0.6% Ca, 10% protein) raws li AIN93-G noj (Oriental Poov xab, Tokyo, Nyiv) nrog casein raws li cov protein.3637) Kev noj zaub mov tsis muaj protein ntau tau tshaj tawm los txhawb vascular calcification hauv cov nas uas muaj adenine-induced lub raum tsis ua haujlwm, yog li kev txwv tsis pub muaj protein ntau tau ua.3738) Ua ntej pab pawg, txhua tus nas (n=23) tau noj cov zaub mov MF (Oriental Poov xab) rau ib lub lis piam kom haum. Thaum muaj hnub nyoog 13 lub lis piam, nas tau muab faib ua 2 pawg: pawg tswj (n=5) thiab pab pawg adenine-induced CKD (n=18). . Cov pab pawg adenine-induced CKD tau txais cov khoom noj adenine li 3 lub lis piam thiab tau muab faib ua 2 pawg: pawg CKD-LP (n=8) lossis CKDHP pawg (n=10). Cov nas ntawm pawg CKD-LP thiab CKD-HP pawg tau txais LP noj zaub mov thiab HP noj zaub mov rau 3 lub lis piam, raws li lub sijhawm sim, kev noj zaub mov tau sau tseg txhua hnub. Thaum noj adenine, cov ntshav tau sau los ntawm cov hlab ntsha ntawm tus Tsov tus tw ntawm 10:00 AM thiab 11:00 AM ib zaug ib asthiv mus txog rau lub lim tiam thib ob, thiab tom qab ntawd ib zaug txhua 2 hnub los saib xyuas cov ntshav plasma. Thaum lub sij hawm LP noj zaub mov lossis HP noj zaub mov, kuaj ntshav tau sau ib zaug ib lub lim tiam. Cov qauv zis tau sau tseg thiab cov zis ntim tau sau tseg ib zaug txhua lub lim tiam. Cov nas tsuag tau txi nrog tshuaj loog siv isoflurane (Wako Pure Chemical Industries. Osaka Nyiv) thiab laparotomies. Cov ntshav coj los ntawm qhov tsis zoo vena cava thiab thoracoabdominal aorta tau sau rau kev tshuaj xyuas.

SLC37A2 qhia ntawm lub aorta.

Kwv yees li ib feem peb ntawm cov thoracoabdominal aorta tau siv los ua kom pom kev calcification thiab ua kev tiv thaiv kab mob. Lub aorta tau muab tso rau hauv paraffin thiab 4-um ntu raug txiav. Cov seem tau stained siv Von Kossa Method rau Calcium Kit (Polysciences. Warrington, PA) raws li cov chaw tsim khoom cov lus qhia. immunostaining tsom xam tau ua nrog cov ntu ntu. Cov ntu tau tsim nrog SLC37A2 antibody thaum hmo ntuj ntawm 4 degree thiab tom qab ntawd kho nrog Histofine Simple Stain Rat MAX-POMULTI) (Nichirei Biosciences, Tokyo, Nyiv) rau 30 min ntawm chav tsev kub. Tom qab ntawd, ntu tau kho nrog cov khoom siv DABsubstrate (Nichirei).

Tag nrho RNA tau muab rho tawm los ntawm tus so ntawm cov khov thoracoabdominal aorta. Lub sijhawm PCR tiag tiag tau ua los ntawm kev siv cDNA synthesized. Cov primer sequences yog raws li nram no: nas osteo-pontin,5ACCCATCTCAGAAGCAGAATC-3' thiab 5ATCCATGTGGTCATGGCTTTTC-3' thiab; rat SLC37A2,5-TAGTCCCAGCTTCGAGTACGG-3' thiab 5'-CTGATGGGCTTTGACATG-3.The osteopontin mRNA thiab SLC37A2 mRNA qhia theem tau xam tom qab normalization nrog GAPPH

Kev txheeb cais.

Cov ntaub ntawv tau nthuav tawm raws li qhov nruab nrab + SE. Qhov sib txawv ntawm cov pab pawg tau txheeb xyuas siv t-test. Hauv kev sim phosphate-induced calcified AoSMC, qhov sib txawv tau txheeb xyuas siv ib txoj kev tsom xam ntawm qhov sib txawv raws li Dunnett txoj kev. Hauv CKD qauv kev sim, qhov sib txawv tau txheeb xyuas siv K ruskal-Wallis test ua raws li txoj kev Steel-Dwass. Correlation coefficients (rs) tau txiav txim siab siv Spearman's correlation coefficient rank test. Rau txhua qhov kev xeem. ob-tailed p nqi<0.05 were considered statistically significant.