CAJ | 학술논문

目的探讨晚期糖基化终产物(AGE)对大鼠肾小球内皮细胞(rGEnC)紧密连接的影响及激活细胞内肾素血管紧张素系统( RAS)在其中的作用.方法采用原代培养的rGEnC,予不同浓度AGE( 20、40、80 mg/L)分别作用6h、12 h和24 h,采用跨内皮细胞电阻抗和异硫氰酸荧光素标记的牛血清白蛋白滤过率观察通透性的变化；Western印迹检测晚期糖基化终产物受体(RAGE)、紧密连接蛋白[occludin、claudin-5、连接黏附分子A(JAM-A)和闭合小环蛋白1(ZO-1)]和细胞RAS组分[血管紧张素原、肾素和血管紧张素Ⅱ(AngⅡ)1型受体(AT1)]蛋白表达量的变化；免疫荧光技术显示紧密连接的完整性；紫外光法和酶免疫分析技术测定细胞内外血管紧张素转化酶( ACE)活性及AngⅡ水平.结果 AGE可引起内皮细胞通透性、RAGE表达量、ACE活性、AngⅡ浓度和AT1表达量的升高,occludin、claudin-5和JAM-A表达量的下降.加入抗RAGE抗体(100 mg/L)预处理后,上述AGE作用被阻断.AGE可引起上述紧密连接蛋白在细胞连接处的中断.予卡托普利(1 mmol/L)或缬沙坦(10μmol/L)预处理可部分阻断AGE上述效应.结论 AGE可通过上调RAGE表达,激活细胞内肾素血管紧张素系统,破坏肾小球内皮细胞紧密连接,导致其通透性的升高.
목적 탐토만기당기화종산물(AGE)대대서신소구내피세포(rGEnC)긴밀련접적영향급격활세포내신소혈관긴장소계통( RAS)재기중적작용.방법 채용원대배양적rGEnC,여불동농도AGE( 20、40、80 mg/L)분별작용6h、12 h화24 h,채용과내피세포전조항화이류청산형광소표기적우혈청백단백려과솔관찰통투성적변화；Western인적검측만기당기화종산물수체(RAGE)、긴밀련접단백[occludin、claudin-5、련접점부분자A(JAM-A)화폐합소배단백1(ZO-1)]화세포RAS조분[혈관긴장소원、신소화혈관긴장소Ⅱ(AngⅡ)1형수체(AT1)]단백표체량적변화；면역형광기술현시긴밀련접적완정성；자외광법화매면역분석기술측정세포내외혈관긴장소전화매( ACE)활성급AngⅡ수평.결과 AGE가인기내피세포통투성、RAGE표체량、ACE활성、AngⅡ농도화AT1표체량적승고,occludin、claudin-5화JAM-A표체량적하강.가입항RAGE항체(100 mg/L)예처리후,상술AGE작용피조단.AGE가인기상술긴밀련접단백재세포련접처적중단.여잡탁보리(1 mmol/L)혹힐사탄(10μmol/L)예처리가부분조단AGE상술효응.결론 AGE가통과상조RAGE표체,격활세포내신소혈관긴장소계통,파배신소구내피세포긴밀련접,도치기통투성적승고.
Objective To investigate the effect of advanced glycation end products (AGEs) on the disruption of tight junctions in rat glomerular endothelial cells (rGEnCs) and the role of renin-angiotensin system (RAS) in this pathological procedure.Methods Primary cultured rGEnCs were incubated with AGEs at concentrations of 20 mg/L,40 mg/L and 80 mg/L,for 6 h,12 h and 24 h respectively.The cells were treated with captopril (1 mmol/L) or valsartan (10 μ mol/L)to block RAS.The endothelial permeability was investigated by transendothelial electrical resistance and the flux of fluorescein isothiocyanate-conjugated bovine serum albumin.The expression of AGEs receptor (RAGE),tight junction proteins [occludin,claudin-5,junctional adhesion molecules A (JAM-A) and zona occludens-1 (ZO-1)]and RAS components [angiotensinogen,renin and angiotensin Ⅱ type 1 receptor (AT1)]were detected by Western blotting.Immunofluorescence was used to demonstrate the disruptions of the tight junction proteins.The activity of angiotensin converting enzyme (ACE) was evaluated by UV spectrophotometry.Angiotensin Ⅱ (Ang Ⅱ ) was measured by enzyme immunoassay.Results The monolayer permeability,the expression of RAGE,the activity of ACE,the concentration of Ang Ⅱ and the expression of AT1 of rGEnCs were increased after induced by AGEs.Meanwhile,AGEs decreased the expression of occludin,claudin5 and JAM-A and induced disruption of tight junction proteins.Pretreatment with anti-RAGE antibody (100 mg/L),captopril or valsartan could attenuate the detrimental effect of AGEs.Conclusion The changes of permeability induced by AGEs in glomerular endothelial cells are partly mediated by RAS through RAGE.