核受体相互作用蛋白140及其相关核受体辅助因子在足细胞高糖损伤中的表达分析

Role of high glucose in expression of RIP140and its related nuclear receptor cofactors in podocyte

目的探讨核受体相互作用蛋白140(RIP140)及其相关核受体辅助因子在足细胞高糖损伤中的表达及可能作用。方法分离培养小鼠原代肾脏足细胞,分为对照组(Con)、浓度为25mmol/L甘露醇高渗组(MT)及浓度为25mmol/L葡萄糖高糖组(HG),流式细胞仪检测细胞凋亡率。分别提取细胞mRNA、总蛋白、核蛋白及胞浆蛋白,RT-PCR检测辅助活化因子PGC-1α、PGC-1β、SRC-1、SRC-2、SRC-3、PCAF及辅助抑制因子N-COR1、RIP140表达,Western blot分析细胞总蛋白、核内蛋白及胞浆蛋白内RIP140表达。结果 HG组足细胞凋亡率明显增加(t=6.72,P<0.01),HG组足细胞内核辅助活化因子PGC-1α(t=6.22,P<0.01)、PGC-1β(t=11.41,P<0.01)、SRC-1(t=4.92,P<0.01)、SRC-2(t=5.40,P<0.01)及PCAF(t=6.57,P<0.01)mRNA表达下降,RIP140 mRNA表达(t=6.36,P<0.01)升高,辅助活化因子SRC-3及辅助抑制因子N-COR1表达差异无统计学意义(P>0.05)。HG组足细胞总蛋白及胞浆蛋白中RIP140表达增加,而胞核内RIP140表达下降。结论 RIP140及其相关核受体辅助因子表达变化可能参与调控高糖对足细胞的损伤。

Objective To investigate the expression of receptor interaction protein 140（RIP140）and its related nuclear receptor cofactors in podocyte under the condition of high glucose. MethodsPrimary mouse podocytes were isolated and cultured.Control group,hypertonic group and high glucose group were included in the experiment.Apoptosis rate was detected using flow cytometer in three groups.The mRNA level of nuclear receptor cofactors PGC-1α,PGC-1β,SRC-1,SRC-2,SRC-3,PCAF,N-COR1 and RIP140 were assessed using real-time PCR.The distribution of RIP140 protein in cytoplasm and nucleus of podocyte was analyzed by western-blot. Results Apoptosis rate increased while podocyte was treated with 25 mmol/L glucose（t=6.72,P〈0.01）.The mRNA levels of nuclear receptor coactivators PGC-1α（t=6.22,P〈0.01）,PGC-1β（t=11.41,P〈0.01）,SRC-1（t=4.92,P〈0.01）,SRC-2（t=5.40,P〈0.01）and PCAF（t=6.57,P〈0.01）decreased in high glucose group than those in control group or in hypertonic group.The expression level of corepressor RIP140 mRNA（t=6.36,P〈0.01）increasedin high glucose group.SRC-3 and N-COR1 mRNA expression had no difference between high glucose group and control group.With the decrease of RIP140 expression in the nucleus of podocyte,the expression of RIP140 increased in the cytoplasm following treatment with 25 mmol/L glucose. Conclusion Nuclear receptor coactivators and corepressors are changed in podocyte under the condition of high glucose,which may be involved in glucose toxicity induced podocyte injury.