CAJ | 학술논문

目的探讨体外近曲小管肝型脂肪酸结合蛋白(L-FABP)在IgA肾病(IgAN)中的上调机制及其对肾脏的保护作用.方法原代培养的小鼠系膜细胞(MC)与不同浓度的多聚IgA(AIgA)(10～250 mg/L)共孵育48 h,取上清作为AIgA-MC介质.分别应用不同浓度的AIgA、AIgA-MC介质、中和性抗肿瘤坏死因子α(TNF-α)抗体及重组鼠TNF-α刺激小鼠近曲小管上皮细胞系mProx和通过转染稳定表达人L-FABP(hL-FABP)基因的mProx (mProx-L)细胞.实时荧光定量PCR方法检测细胞中的hL-FABP和单核细胞趋化蛋白1(MCP-1)的mRNA表达.Western印迹方法检测细胞中的hL-FABP蛋白和4-羟壬烯醛(4-HNE)修饰蛋白的表达.结果 (1)AIgA-MC介质显著上调mProx-L细胞的hL-FABP mRNA和蛋白的表达(P＜0.01),而AIgA刺激不能上调hL-FABP的表达.(2)中和性抗TNF-α抗体(终质量浓度为1和5 mg/L)的预孵育可以显著抑制AIgA-MC介质对hL-FABP蛋白表达的上调效应(P＜0.05和P＜0.01).(3)重组鼠TNF-α(终质量浓度为50和250 ng/L)呈剂量依赖性显著上调hL-FABP蛋白的表达(P＜0.01).(4)AIgA-MC介质刺激后,mPmx-L细胞4-HNE修饰蛋白和MCP-1 mRNA的表达水平显著低于mProx细胞(P＜0.05和P＜0.01).结论 IgAN中系膜细胞源性TNF-α可以诱导肾小管L-FABP表达的上调.肾小管高表达的L-FABP抑制了氧化应激和炎性反应,发挥了肾脏保护作用.
목적 탐토체외근곡소관간형지방산결합단백(L-FABP)재IgA신병(IgAN)중적상조궤제급기대신장적보호작용.방법 원대배양적소서계막세포(MC)여불동농도적다취IgA(AIgA)(10～250 mg/L)공부육48 h,취상청작위AIgA-MC개질.분별응용불동농도적AIgA、AIgA-MC개질、중화성항종류배사인자α(TNF-α)항체급중조서TNF-α자격소서근곡소관상피세포계mProx화통과전염은정표체인L-FABP(hL-FABP)기인적mProx (mProx-L)세포.실시형광정량PCR방법검측세포중적hL-FABP화단핵세포추화단백1(MCP-1)적mRNA표체.Western인적방법검측세포중적hL-FABP단백화4-간임희철(4-HNE)수식단백적표체.결과 (1)AIgA-MC개질현저상조mProx-L세포적hL-FABP mRNA화단백적표체(P＜0.01),이AIgA자격불능상조hL-FABP적표체.(2)중화성항TNF-α항체(종질량농도위1화5 mg/L)적예부육가이현저억제AIgA-MC개질대hL-FABP단백표체적상조효응(P＜0.05화P＜0.01).(3)중조서TNF-α(종질량농도위50화250 ng/L)정제량의뢰성현저상조hL-FABP단백적표체(P＜0.01).(4)AIgA-MC개질자격후,mPmx-L세포4-HNE수식단백화MCP-1 mRNA적표체수평현저저우mProx세포(P＜0.05화P＜0.01).결론 IgAN중계막세포원성TNF-α가이유도신소관L-FABP표체적상조.신소관고표체적L-FABP억제료양화응격화염성반응,발휘료신장보호작용.
Objective To explore the mechanism of up-regulation of tubular liver-type fatty acid binding-protein (L-FABP) in IgA nephropathy (IgAN) and its renoprotective role.Methods Murine mesangial cells (MCs) from primary cell culture were cultured with aggregated IgA (AIgA) (10 to 250 mg/L) for 48 hours. The supernatant after culture was collected as AIgA-MC medium. Murine proximal tubular cell line (mProx) stably expressing human L-FABP (hL-FABP) by transfection (mProx-L) were cultured with AIgA, AIgA-MC medium and /or neutralizing anti-TNF-α antibody and recombinant murine TNF-α, respectively. AIgA-MC medium (AIgA final concentration was 25 mg/L) was cultured with mProx and mProx-L cells. The mRNA expressions of hL-FABP and MCP-1 of the cells were detected by real-time PCR. The protein expressions of hL-FABP and 4-HNE of the cells were detected by Western blotting. Results (1) The hL-FABP mRNA and protein expression stimulated by AIgA-MC medium was significantly higher as compared to AIgA (P＜0.01). (2) Pre-incubation of neutralizing anti-TNF-α antibody (final concentration was 1 and 5 mg/L) with mProx-L cells could significantly suppress the up-regulation of hL-FABP protein expression induced by AlgA-MC medium (P＜0.05 and P＜0.01).(3) Recombinant murine TNF-α (final concentration was 50 and 250 ng/L) also induced a significant up-regulation of hL-FABP expression (P＜0.01). (4) After the stimulation of AIgA-MC medium, both 4-HNE protein expression and MCP-1 mRNA expression were significantly suppressed in mProx-L cells compared to those of mProx cells (P ＜0.05 and P＜0.01). Conclusion Mesangial cell-derived TNF-α can induce up-regulation of tubular L-FABP expression. Overexpression of tubular L-FABP may lessen the progression of IgAN by reducing oxidative stress and inflammatory mediators.