单核细胞RAGE表达上调:慢性肾功能衰竭微炎症的机制

Activation of receptor for advanced glycation end products:a mechanism for monocyte-mediated inflammation in chronic renal failure

目的 探讨慢性肾功能衰竭 (CRF)时细胞表面晚期糖基化终产物 (AGE)受体 (RAGE)的表达及其在单核细胞介导的炎症反应中的作用。方法 96例非糖尿病CRF患者的断面队列研究。用密度梯度离心加免疫磁珠法分离外周血单核细胞 (PMC) ,用特异性抗RAGE抗体染色、流式细胞仪检测PMC表面RAGE表达 ,Scatchard印迹法分析PMC结合AGE的位点数和亲和力 ,用ELISA法测定循环新蝶呤、TNF α、C反应蛋白 (CRP)和AGE水平。结果 CRF患者PMC表面RAGE表达明显上调并随肾功能恶化而增加 (r2 =0 73) ,RAGE上调程度与血浆AGE水平呈正相关 (r =0 71) ;CRF患者PMC与AGE的结合位点数和亲和力明显高于正常人 ,在相同剂量AGE刺激下 ,CRF患者PMC生成的TNF α明显多于正常PMC ,且AGE诱导的TNF α分泌可被抗RAGE抗体所抑制 ;患者PMC表面RAGE表达水平与循环TNF α水平呈正相关 (r=0 6 1) ,与单核细胞活化标志物———新蝶呤 (r=0 6 5 )和急性时相反应蛋白———CRP(r =0 4 4 )呈正相关。结论 CRF患者RAGE表达增加 ,RAGE上调可能通过促发炎症正反馈环导致单核细胞持续活化 ,从而参与CRF由单核细胞介导的全身微炎症反应。

Objective To investigate the expression of receptor for advanced glycation end products (RAGE) in chronic renal failure (CRF) and its role in monocyte mediated inflammation associated with chronic renal failure (CRF). Methods Peripheral monocytes (PMC) were isolated from 96 non diabetic patients with varying severity of CRF. RAGE expression on monocytes was quantitated by flow cytometry. The binding capacity of monocytes with advanced glycation end products (AGE) was determined by 125 I AGEs HSA binding assay. The plasma level of pentosidine, a marker of AGE, was determined by competitive ELISA. Commercially available kits were used for measuring the plasma levels of neopterin, TNF α and C reactive protein (CRP), a systemic acute phase reactant. Results Flow cytometry showed that the RAGE expression at the PMC surface of CRF patients was 8.02±0.43, significantly higher than that of the normal controls ( P <0.001). The number of functional sites to bind 125 I AGEs HSA at the surface of PMC of CRF patients was increased in comparison with the normal control group. The biding capacity (Ka) at the surface of PMC of CRF patients was 2 times that of normal control group. Stimulated by AGEs HAS, the TNF α level in the supernatant of PMC increased dose dependently in both the normal control and CRF patients, especially in the latter ( P <0.01). After pretreatment of anti RAGE or non immune rabbit IgG and then by AGEs HAS the levels of TNF α in the PMC supernatants of CRF patients and normal controls decreased form 90.52 pg/(10 5 cell)±2.82 pg/(10 5 cell)to 17.86 pg/(10 5 cell)±1.05 pg/(10 5 cell)and from 26.38 pg/(10 5 cell)±1.54 pg/(10 5 cell) to 6.76 pg/(10 5 cell)±0.20 pg/(10 5 cell). HAS not modified by AGEs and non immune rabbit IgG showed no influence on the secretion of TNF α. The plasma levels of TNF α, neopterin, and CRP increased along with the worsening of renal function. The RAGE expression and pentosidine level at the surface of PMC in CPR patients without hemodialysis were positively correlated with plasma neopterin, TNF α, and 和CRP levels, even after correction of creatine clearance rate ( r =0.53, P <0.001; r =0.58, P <0.001; r =0.40, P =0.001). The expression of RAGE in CRF patients with hemodialysis was positively correlated with the plasma TNF α level ( r =0.33, P =0.029, n =36),however, not correlated with neopterin or CRP. Conclusion Enhanced RAGE may trigger a positive feed back loop of AGEs induced monocyte perturbation, and may contribute to the monocyte mediated systemic inflammation in CRF.