Ren Hao, Hou Fanfan, Bi Ye, Zhang Xun
Division of Nephrology, Nanfang Hospital Affiliated to First Military Medical University Guangzhou 510515, China.
Zhonghua Yi Xue Za Zhi. 2002 Sep 10;82(17):1168-72.
To elucidate the relationship between the expression of receptors for AGE (RAGE) at the surface of monocyte and the plasma levels of tumor necrosis factor alpha (TNFalpha) and interleukin-1beta (IL-1beta), proinflammatory cytokines in patients with chronic renal failure (CRF) undergoing hemodialysis (HD).
Human serum albumin modified with AGE (AGE-HSA) was prepared in vitro and was radiolabeled with (125)I. Human peripheral blood monocytes were isolated from 59 CRF patients (37 of which underwent hemodialysis) and 30 normal volunteers by Ficoll-hypaque centrifugation technique. Specific binding of AGE-HSA to monocytes was analyzed by radioactive ligand-receptor binding assay. The degradation to AGE-HSA by monocytes was measured by trichloroacetic acid precipitation method. The expression of REGE in monocytes was determined by flow cytometry. The levels of TNFalpha and IL-1beta were detected by a sandwich enzyme immunoassay.
The specific binding of (125)I-AGE-HSA to monocytes was 41.4 fmol/10(5) cells +/- 2.2 fmol/10(5) cells, significantly higher than that in normal controls (31.6 fmol/10(5) cells +/- 4.1 fmol/10(5) cells, P < 0.05). The number of AGE conjugated protein at the monocytes' surface of CRF patients undergoing HD was 2.42 +/- 0.21 x 10(5)/cells, significantly higher than that in normal controls (1.74 +/- 0.29 x 10(5)/cells, P < 0.001), and the affinity constant (Kd) of the monocytes of CRF patients undergoing HD was 228 nmol/L +/- 100 nmol/L, not significantly different from that in normal controls (262 nmol/L +/- 108 nmol/L, P > 0.05). The degration rate of AGE-HAS by monocytes in CRF patients undergoing HD was 25.24% +/- 4.35%, significantly higher than that in normal controls (18% +/- 0.6%, P < 0.05). The fluorescent intensity of RAGE at the surface of monocytes in CRF patients undergoing HD was 3.68 +/- 0.42, significantly higher than that in normal controls (1.09 +/- 0.37, P < 0.05), When the monocytes were incubated in vitro with 50 microgram/ml of AGE-HSA, the levels of TNFalpha and IL-1beta in the supernatants of CRF patients undergoing HD were 197 +/- 98 ng/L and 357.0 +/- 140.1 ng/L respectively, both significantly higher than those in the normal controls (111 +/- 77 ng/L and 184 +/- 118 ng/L respectively, both P < 0.05). The secretion of proinflammatory cytokines was inhibited when the monocytes were preincubated with anti-RAGE. Increased RAGE level was accompanied by higher plasma levels of TNFalpha and IL-1beta.
The expression of AGE receptors in monocytes is up-regulated in patients with CRF. This may contribute to the enhanced plasma level of proinflammatory cytokines seen in HD patients.
阐明接受血液透析（HD）的慢性肾衰竭（CRF）患者单核细胞表面晚期糖基化终末产物受体（RAGE）的表达与肿瘤坏死因子α（TNFα）和白细胞介素-1β（IL-1β）这两种促炎细胞因子血浆水平之间的关系。
体外制备经晚期糖基化终末产物修饰的人血清白蛋白（AGE-HSA）并用（125）I进行放射性标记。采用Ficoll-泛影葡胺离心技术从59例CRF患者（其中37例接受血液透析）和30名正常志愿者中分离人外周血单核细胞。通过放射性配体-受体结合试验分析AGE-HSA与单核细胞的特异性结合。采用三氯乙酸沉淀法测定单核细胞对AGE-HSA的降解。通过流式细胞术测定单核细胞中RAGE的表达。采用夹心酶免疫测定法检测TNFα和IL-1β的水平。
（125）I-AGE-HSA与单核细胞的特异性结合为41.4 fmol/10（5）个细胞±2.2 fmol/10（5）个细胞，显著高于正常对照组（31.6 fmol/10（5）个细胞±4.1 fmol/10（5）个细胞，P<0.05）。接受HD的CRF患者单核细胞表面AGE结合蛋白数量为2.42±0.21×10（5）/细胞，显著高于正常对照组（1.74±0.29×10（5）/细胞，P<0.001），接受HD的CRF患者单核细胞的亲和常数（Kd）为228 nmol/L±100 nmol/L，与正常对照组（262 nmol/L±108 nmol/L，P>0.05）无显著差异。接受HD的CRF患者单核细胞对AGE-HAS的降解率为25.24%±4.35%，显著高于正常对照组（18%±0.6%，P<0.05）。接受HD的CRF患者单核细胞表面RAGE的荧光强度为3.68±0.42，显著高于正常对照组（1.09±0.37，P<0.05）。当单核细胞在体外与50μg/ml的AGE-HSA孵育时，接受HD 的CRF患者上清液中TNFα和IL-1β的水平分别为197±98 ng/L和357.0±140.1 ng/L，均显著高于正常对照组（分别为111±77 ng/L和184±118 ng/L，均P<0.05）。当单核细胞与抗RAGE预孵育时，促炎细胞因子的分泌受到抑制。RAGE水平升高伴随着血浆中TNFα和IL-1β水平升高。
CRF患者单核细胞中AGE受体表达上调。这可能导致HD患者促炎细胞因子血浆水平升高。
