Berrou J, Tostivint I, Verrecchia F, Berthier C, Boulanger E, Mauviel A, Marti H P, Wautier M P, Wautier J L, Rondeau E, Hertig A
INSERM U702, Hôpital Tenon AP-HP, Paris, France.
Int J Mol Med. 2009 Apr;23(4):513-20. doi: 10.3892/ijmm_00000159.
Advanced glycation end products (AGEs) may play a role in the pathogenesis of diabetic nephropathy, by modulating extracellular matrix turnover. AGEs are known to activate specific membrane receptors, including the receptor for AGE (RAGE). In the present study, we analyzed the various receptors for AGEs expressed by human mesangial cells and we studied the effects of glycated albumin and of carboxymethyl lysine on matrix protein and remodelling enzyme synthesis. Membrane RAGE expression was confirmed by FACS analysis. Microarray methods, RT-PCR, and Northern blot analysis were used to detect and confirm specific gene induction. Zymographic analysis and ELISA were used to measure the induction of tPA and PAI-1. We show herein that cultured human mesangial cells express AGE receptor type 1, type 2 and type 3 and RAGE. AGEs (200 microg/ml) induced at least a 2-fold increase in mRNA for 10 genes involved in ECM remodelling, including tPA, PAI-1 and TIMP-3. The increase in tPA synthesis was confirmed by fibrin zymography. The stimulation of PAI-1 synthesis was confirmed by ELISA. AGEs increased PAI-1 mRNA through a signalling pathway involving reactive oxygen species, the MAP kinases ERK-1/ERK-2 and the nuclear transcription factor NF-kappaB, but not AP-1. Carboxymethyl lysine (CML, 5 microM), which is a RAGE ligand, also stimulated PAI-1 synthesis by mesangial cells. In addition, a blocking anti-RAGE antibody partially inhibited the AGE-stimulated gene expression and decreased the PAI-1 accumulation induced by AGEs and by CML. Inhibition of AGE receptors or neutralization of the protease inhibitors TIMP-3 and PAI-1 could represent an important new therapeutic strategy for diabetic nephropathy.
晚期糖基化终末产物（AGEs）可能通过调节细胞外基质更新在糖尿病肾病的发病机制中发挥作用。已知AGEs可激活特定的膜受体，包括AGE受体（RAGE）。在本研究中，我们分析了人系膜细胞表达的各种AGE受体，并研究了糖化白蛋白和羧甲基赖氨酸对基质蛋白和重塑酶合成的影响。通过流式细胞术分析证实了膜RAGE的表达。使用微阵列方法、逆转录聚合酶链反应（RT-PCR）和Northern印迹分析来检测和确认特定基因的诱导。使用酶谱分析和酶联免疫吸附测定（ELISA）来测量组织型纤溶酶原激活物（tPA）和纤溶酶原激活物抑制剂-1（PAI-1）的诱导情况。我们在此表明，培养的人系膜细胞表达1型、2型和3型AGE受体以及RAGE。AGEs（200微克/毫升）使参与细胞外基质重塑的10个基因的mRNA至少增加了2倍，包括tPA、PAI-1和金属蛋白酶组织抑制因子-3（TIMP-3）。通过纤维蛋白酶谱分析证实了tPA合成的增加。通过ELISA证实了PAI-1合成的刺激。AGEs通过涉及活性氧、丝裂原活化蛋白激酶细胞外信号调节激酶-1/细胞外信号调节激酶-2（ERK-1/ERK-2）和核转录因子核因子κB（NF-κB）而非激活蛋白-1（AP-1）的信号通路增加PAI-1 mRNA。羧甲基赖氨酸（CML，5微摩尔）作为一种RAGE配体，也刺激系膜细胞合成PAI-1。此外，一种阻断性抗RAGE抗体部分抑制了AGE刺激的基因表达，并减少了AGEs和CML诱导的PAI-1积累。抑制AGE受体或中和蛋白酶抑制剂TIMP-3和PAI-1可能代表糖尿病肾病一种重要的新治疗策略。
