高糖通过SGK_1通路促进人肾小球系膜细胞合成结缔组织生长因子

High glucose stimulates synthesis of CTGF in HMC via SGK_1 signaling pathway

目的研究血清和糖皮质激素诱导的蛋白激酶1(SGK1)在高糖诱导人肾小球系膜细胞(HMC)产生结缔组织生长因子(CTGF)中的作用,探讨SGK1在糖尿病肾病(DN)肾小球硬化中的作用机制。方法HMC分为低糖组(5·5mmol·L-1D-葡萄糖)、高糖组(25mmol·L-1D-葡萄糖)和甘露醇对照组(19·5mmol·L-1甘露醇和5·5mmol·L-1D-葡萄糖),刺激24、72h后,采用RT-PCR方法和West-ernblot方法检测CTGFmRNA及蛋白的表达;将带有SGK1显性激活型突变体质粒(pIRES2-EGFP-S422DhSGK1,SD)和带有SGK1显性失活型突变体质粒(pIRES2-EGFP-K127NhSGK1,KN)分别瞬时转染HMC;同时,设空质粒(PIRES2-EGFP,FP)转染组和未转染组(NT)为对照。分别用低糖(LG,5·5mmol·L-1D-葡萄糖)和高糖(HG,25mmol·L-1D-葡萄糖)刺激24、72h后,采用RT-PCR方法和Western blot方法检测CT-GF的mRNA及蛋白的表达。结果与低糖组和甘露醇组相比较,在高糖刺激下,HMC中CTGF mRNA及蛋白的表达明显上调;低糖环境下,转染SD的HMC与转染FP、NT组比较,其CTGFmRNA和蛋白的表达明显增加。转染KN的HMC与转染FP、NT组比较,其CTGF mRNA和蛋白的表达差异无显著性;高糖环境下转染SD的HMC与转染KN、转染FP、NT组比较,其CTGF mRNA和蛋白的表达明显增加。转染KN的HMC与转染FP、NT组比较,其CTGF mRNA和蛋白的表达明显减少。结论在DN中,高糖能促进HMC的CTGF mRNA及蛋白的表达,并且可以通过SGK1介导的信号通路来诱导人肾小球系膜细胞增加合成CTGF,这种新发现的信号通路,表明SGK1可能通过促进CTGF的合成来参与糖尿病肾病肾小球纤维化的发生。

Aim To investigate the role of Serum and glucocorticoid induced kinase 1(SGK1) pathways in Connective Tissue Growth Factor(CTGF)synthesis in cultured human mesangial cell(HMC) with high glucose and the mechanism by which SGK1 contributes to glomerulosclerosis in diabetic nephropathy(DN).Methods HMC were divided into three groups:low glucose group(5.5 mmol·L-1 D-glucose),high glucose group(25 mmol·L-1 D-glucose),mannitol control group(19.5 mmol·L-1 mannitol and 5.5 mmol·L-1 D-glucose).After stimulating for 24 h or 72 h,the expression of CTGF mRNA were measured by semi-quantitative RT-PCR.The expression of CTGF protein was detected by Western blot.Overexpression of SGK1 by transfected with pIRES2-EGFP-S422D hSGK1(SD) or pIRES2-EGFP-K127N hSGK1(KN) in HMC,we stimulate the HMC with low glucose(LG,5.5 mmol·L-1 D-glucose) or high glucose(HG,25 mmol·L-1 D-glucose) by using the HMC by transfected with pIRES2-EGFP(FP) and the non-transfected(NT) as control groups.After stimulating for 24 h or 72 h,the expression of CTGF mRNA was assessed by semi-quantitative RT-PCR.The expression of CTGF protein was measured by Western blot.Results The level of CTGF mRNA and protein was significantly elevated 24 h or 72 h after high glucose stimulating.Mannitol didn't significantly elevate the expression of CTGF mRNA and protein;Under LG condition,Overexpression of SGK1 by transfected with SD in HMC resulted in the stimulation of CTGF mRNA and protein synthesis compared with transfected with FP and NT groups;In HG condition,Overexpression of SGK1 by transfected with SD in HMC increased CTGF mRNA and protein synthesis compared with transfected with KN,FP and NT groups.Overexpression of SGK1 by transfected with KN in HMC resulted in decrease of CTGF mRNA and protein synthesis compared with transfected with FP and NT groups.Conclusion These results suggest that high glucose can elevate the expression of CTGF in HMC,and SGK1 could be involved in the signal transduction leading to the increase of CTGF production in DN and therefore might play an active part in glomerulosclerosis in DN.