Advanced glycation end products cause epithelial-myofibroblast transdifferentiation via the receptor for advanced glycation end products (RAGE)

Tubulointerstitial disease, a prominent phenomenon in diabetic nephropathy, correlates with decline in renal function. The underlying pathogenic link between chronic hyperglycemia and the development of tubulointerstitial injury has not been fully elucidated, but myofibroblast formation represents a key step in the development of tubulointerstitial fibrosis. RAGE, the receptor for advanced glycation end products (AGEs), induces the expression of TGF-beta and other cytokines that are proposed to mediate the transdifferentiation of epithelial cells to form myofibroblasts. Here we report specific binding of (125)I-AGE-BSA to cell membranes prepared from a rat proximal tubule cell line and show that the binding site was RAGE. AGE exposure induced dose-dependent epithelial-myofibroblast transdifferentiation determined by morphological changes, de novo alpha smooth-muscle actin expression, and loss of epithelial E-cadherin staining. These effects could be blocked with neutralizing Ab's to RAGE or to TGF-beta. Transdifferentiation was also apparent in the proximal tubules of diabetic rats and in a renal biopsy from a patient with type 1 diabetes. The AGE cross-link breaker, phenyl-4,5-dimethylthiazolium bromide (ALT 711) reduced transdifferentiation in diabetic rats in association with reduced tubular AGE and TGF-beta expression. This study provides a novel mechanism to explain the development of tubulointerstitial disease in diabetic nephropathy and provides a new treatment target.     

Soluble receptor for advanced glycation end products (sRAGE) in plasma and synovial fluid is inversely associated with disease severity of knee osteoarthritis

Objectives:The aim of this study was to measure soluble receptor for advanced glycation end products (sRAGE) in plasma and synovial fluid of knee osteoarthritis (OA) patients and to determine the correlation between sRAGE levels and disease severity.Design and methods:Thirty-six OA patients and 15 healthy controls were enrolled in this study. OA grading was performed using the Kellgren–Lawrence classification. sRAGE levels in plasma and synovial fluid were analyzed by enzyme-linked immunosorbent assay.Results:Plasma sRAGE levels were significantly lower in OA patients than in healthy controls (P = 0.01). sRAGE levels in plasma were remarkably higher with regard to paired synovial fluid (P = 0.001). Additionally, sRAGE concentrations in plasma and synovial fluid showed significant inverse correlation with disease severity (r = ?0.65, P < 0.001 and r = ?0.55, P = 0.001, respectively). Further analysis showed that there was a strong positive correlation between plasma and synovial sRAGE concentration (r = 0.81, P < 0.001).Conclusions:sRAGE levels were significantly lower in OA patients compared with controls, and sRAGE levels in plasma and synovial fluid also decreased significantly as the disease severity increased. Accordingly, sRAGE levels could be used as a biochemical marker for assessing the severity and progression of knee OA.     

Decreased plasma levels of soluble receptor for advanced glycation endproducts (sRAGE) in patients with nonalcoholic fatty liver disease

Objectives

Levels of soluble receptor for advanced glycation endproducts (sRAGE) have been linked to several components of the metabolic syndrome. We tested the hypothesis that plasma levels of sRAGE may be associated with non-alcoholic fatty liver disease.

Design and methods

We enrolled subjects with definite nonalcoholic steatohepatitis (NASH, n = 40), borderline NASH (n = 8), simple fatty liver (n = 9) and healthy controls (n = 14). Plasma levels of sRAGE were measured by ELISA.

Results

Concentrations of sRAGE were significantly lower in patients with definite NASH (1080 ± 392 pg/mL, P < 0.01) and borderline NASH (1050 ± 278 pg/mL, P < 0.05) compared to controls (1480 ± 387 pg/mL). Levels of sRAGE were significantly and inversely correlated with ALT (r = − 0.30, P < 0.05) and AST (r = − 0.23, P < 0.05).

Conclusion

Plasma levels of sRAGE are significantly reduced in definite and borderline NASH.     

Targeting the receptor for advanced glycation end products (RAGE) in type 1 diabetes

Type 1 diabetes (T1D) is one of the most common chronic diseases manifesting in early life, with the prevalence increasing worldwide at a rate of approximately 3% per annum. The prolonged hyperglycaemia characteristic of T1D upregulates the receptor for advanced glycation end products (RAGE) and accelerates the formation of RAGE ligands, including advanced glycation end products, high-mobility group protein B1, S100 calcium-binding proteins, and amyloid-beta. Interestingly, changes in the expression of RAGE and these ligands are evident in patients before the onset of T1D. RAGE signals via various proinflammatory cascades, resulting in the production of reactive oxygen species and cytokines. A large number of proinflammatory ligands that can signal via RAGE have been implicated in several chronic diseases, including T1D. Therefore, it is unsurprising that RAGE has become a potential therapeutic target for the treatment and prevention of disease. In this review, we will explore how RAGE might be targeted to prevent the development of T1D.     

脂质体槲皮素对糖尿病大鼠视网膜糖基化终产物生成及受体表达的影响

目的观察脂质体槲皮素（LQ）对糖尿病（DM）大鼠视网膜糖基化终产物（AGEs）生成及受体（RAGEmRNA）表达的影响。方法采用蒸发-高压均质法制备LQ悬浊液,链脲佐菌素（STZ）法诱导DM大鼠模型,并随机分为DM模型组、LQ低、中、高剂量及氨基胍灌胃组;连续给药12周后,免疫荧光法、RT—PCR方法分别检测DM大鼠视网膜中AGEs沉积情况及RAGEmRNA的表达。结果与DM模型组结果（AGEs：0．922±0．005;RAGEmRNA：1．192±0．098）比较,LQ各组及氨基胍组视网膜中AGEs沉积减少,RAGEmRNA表达也降低（P〈0．05）,以LQ中剂量组（AGEs：0．734±0．007;RAGEmRNA：0．724±0．028）降低最为明显（P〈0．01）。结论LQ可通过有效减少DM大鼠视网膜中AGEs的沉积,下调RAGEmRNA的表达。     

抗晚期糖基化终产物受体抗体对晚期糖基化终产物引起心肌细胞胞内游离钙瞬变的影响

目的:观察抗晚期糖基化终产物受体(RAGE)抗体对晚期糖基化终产物刺激引起的体外培养乳鼠心肌细胞胞游离钙瞬间升高的影响。方法:实验于2004-07/12在南方医科大学基础部中心实验室及南方医科大学心血管内科实验室进行。①取新生1-3dSD大鼠心脏在Petridish培养皿中行原代心肌细胞培养。②培养3-5d的原代心肌细胞分为2大组,第1大组:人血清白蛋白组、AGEs-HSA组、抗RAGE抗体10,50mg/L组;第2大组:人血清白蛋白组、AGEs-HSA组、丙种球蛋白10,50mg/L组。处理组试验前用相应浓度抗体提前预孵2h。③将处理好的细胞用荧光钙离子指示剂Fluo-3/AM标记后,于激光共聚焦显微镜下观察刺激后细胞内游离钙浓度(以荧光值表示)变化。人血清白蛋白组给予人血清白蛋白200mg/L刺激,其他各组给予晚期糖基化终产物修饰的人血清白蛋白200mg/L刺激。结果:①第1大组组间比较:AGEs-HSA组细胞荧光峰值和荧光值升高幅度均高于人血清白蛋白组[147.47±21.72,98.66±28.51;(78.47±22.94)%,(26.93±6.72)%;P均<0.05],抗RAGE抗体10,50mg/L组细胞荧光峰值和荧光值升高幅度均小于AGEs-HSA组[68.48±20.94,59.78±5.95;(-33.21±21.57)%,(1.80±7.00)%;P均<0.05]。②第2大组组间比较:AGEs-HSA组和丙种球蛋白10,50mg/L组细胞荧光峰值均高于人血清白蛋白组(61.72±28.62,59.60±17.20,39.04±5.62,P<0.05),后3组间比较无差异。结论:抗RAGE抗体可以阻断晚期糖基化终产物引起的心肌细胞胞内游离钙离子浓度升高,提示晚期糖基化终产物引起心肌细胞胞内游离钙离子浓度升高是由RAGE介导的。     

Soluble receptor for advanced glycation end products in physiological and pathological pregnancy

ObjectiveThe receptor for advanced glycation end products, RAGE, has been implicated in pathogenesis of many diseases. Soluble RAGE, sRAGE, extracellular domain of RAGE, is new biomarker. The aim of the study was to determine sRAGE levels in physiological pregnancy and their changes in pregnancies complicated by preterm labor or preeclampsia.Design and methodsSerum levels of sRAGE were determined in 79 healthy pregnant women, 42 pregnant women in preterm labor or with preeclampsia and 24 non-pregnant controls.ResultssRAGE serum levels are decreased in physiological pregnancy compared to healthy non-pregnant controls (p < 0.001). Serum sRAGE concentrations are higher in the 2nd trimester of physiological pregnancy, compared to the 1st and 3rd trimesters of pregnancy (p < 0.001). sRAGE levels in women with preterm labor are decreased (p < 0.05) and correlate negatively with the leukocyte count (r = -0.47, p < 0.05). In women with preeclampsia, sRAGE is elevated (p < 0.05) and correlates with serum creatinine concentration (r = 0.54, p < 0.05) and with uric acid concentration (r = 0.51, p < 0.05).ConclusionOur results clearly demonstrate significant differences in serum sRAGE levels in physiological pregnancy and in pathological states in pregnancy, however, further studies are required demonstrate the usefulness and significance of sRAGE.     

膝骨关节炎滑液中可溶性晚期糖基化终末产物受体水平与其病变程度的相关性

背景：研究表明骨关节炎患者关节滑液内可溶性晚期糖基化终末产物受体水平可能与关节炎病变的严重程度存在负相关,但在中国报道较少。目的：观察膝关节骨关节炎患者关节滑液内可溶性晚期糖基化终末产物受体水平与其病变严重程度的关系。方法：共纳入46名膝关节骨关节炎患者及14名健康对照者,纳入的骨关节炎患者符合美国风湿病学会骨关节炎的临床诊断标准。采用Kellgren-Lawrence的标准对膝关节骨关节炎病变严重程度进行分级,使用人可溶性晚期糖基化终末产物受体水平酶联免疫吸附试剂盒在酶标仪下检测测关节滑液的可溶性晚期糖基化终末产物受体水平。结果与结论：膝关节骨关节炎患者关节滑液可溶性晚期糖基化终末产物受体水平较健康对照组显著降低（P〈0.01）,且与膝关节骨关节炎病变严重程度呈显著独立负相关（r=-0.587,P〈0.01）。结果表明关节滑液可溶性晚期糖基化终末产物受体水平可能与膝关节骨关节炎病变的严重性和进展程度相关。     

膝骨关节炎滑液中可溶性晚期糖基化终末产物受体水平与其病变程度的相关性

背景:研究表明骨关节炎患者关节滑液内可溶性晚期糖基化终末产物受体水平可能与关节炎病变的严重程度存在负相关,但在中国报道较少。目的:观察膝关节骨关节炎患者关节滑液内可溶性晚期糖基化终末产物受体水平与其病变严重程度的关系。方法:共纳入46名膝关节骨关节炎患者及14名健康对照者,纳入的骨关节炎患者符合美国风湿病学会骨关节炎的临床诊断标准。采用Kellgren-Lawrence的标准对膝关节骨关节炎病变严重程度进行分级,使用人可溶性晚期糖基化终末产物受体水平酶联免疫吸附试剂盒在酶标仪下检测测关节滑液的可溶性晚期糖基化终末产物受体水平。结果与结论:膝关节骨关节炎患者关节滑液可溶性晚期糖基化终末产物受体水平较健康对照组显著降低(P<0.01),且与膝关节骨关节炎病变严重程度呈显著独立负相关(r=-0.587,P<0.01)。结果表明关节滑液可溶性晚期糖基化终末产物受体水平可能与膝关节骨关节炎病变的严重性和进展程度相关。     

急性肺损伤时晚期糖基化终末产物受体变化的实验研究

目的 观察晚期糖基化终末产物受体(RAGE)在不同程度急性肺损伤(ALI)大鼠血浆和肺泡灌洗液(BALF)中的变化,探讨RAGE在判断ALI严重程度方面的价值.方法 30只SD大鼠随机分为3组:低剂量组、高剂量组和对照组.Western blot法检测BALF和血浆中RAGE相对含量,同时测定PaO2、肺组织湿干质量比值(W/D)、BALF蛋白含量、血浆和BALF中肿瘤坏死因子-α(TNF-α)水平,结合病理改变评价各组肺损伤程度.结果致伤组均出现肺损伤,高剂量组损伤更明显;与对照组比较,两致伤组血浆和BALF中RAGE含量均有所上升(P﹤0.05),且高剂量组BALF中RAGE含量又高于低剂量组(P<0.05),而血浆中RAGE含量在低剂量组与高剂量组间差异无统计学意义.结论 ALI时,BALF和血浆中RAGE水平升高,BALF中RAGE水平升高的程度可反应肺损伤的程度,RAGE可作为ALI/ARDS的预警指标.     

Advanced glycation end products on stored red blood cells increase endothelial reactive oxygen species generation through interaction with receptor for advanced glycation end products

BACKGROUND: Recent evidence suggests that storage‐induced alterations of the red blood cell (RBC) are associated with adverse consequences in susceptible hosts. As RBCs have been shown to form advanced glycation end products (AGEs) after increased oxidative stress and under pathologic conditions, we examined whether stored RBCs undergo modification with the specific AGE N‐(carboxymethyl)lysine (N^ε‐CML) during standard blood banking conditions. STUDY DESIGN AND METHODS: Purified, fresh RBCs from volunteers were compared to stored RBCs (35‐42 days old) obtained from the blood bank. N^ε‐CML formation was quantified using a competitive enzyme‐linked immunosorbent assay. The receptor for advanced glycation end products (RAGE) was detected in human pulmonary microvascular endothelial cells (HMVEC‐L) by real‐time polymerase chain reaction, Western blotting, and flow cytometry. Intracellular reactive oxygen species (ROS) generation was measured by the use of 5‐(and 6‐)chloromethyl‐2′,7′‐dichlorodihydrofluorescein diacetate, acetyl ester–based assays. RESULTS: Stored RBCs showed increased surface N^ε‐CML formation when compared with fresh RBCs. HMVEC‐L showed detectable surface RAGE expression constitutively. When compared to fresh RBCs, stored RBCs triggered increased intracellular ROS generation in both human umbilical vein endothelial cells and HMVEC‐L. RBC‐induced endothelial ROS generation was attenuated in the presence of soluble RAGE or RAGE blocking antibody. CONCLUSIONS: The formation of the AGE N^ε‐CML on the surface of stored RBCs is one functional consequence of the storage lesion. AGE‐RAGE interactions may be one mechanism by which transfused RBCs cause endothelial cell damage.     

晚期糖基化终产物在糖尿病诱发动脉粥样硬化中的作用

糖尿病的发病率正日益提高。高血糖作为糖尿病的重要指征,通过非酶促的糖基化反应促进晚期糖基化终产物（AGEs）的生成。而AGEs在血液和组织中沉积,通过直接修饰蛋白质使基质功能和脂质代谢紊乱,通过受体介导的信号传导机制与参与动脉粥样硬化的细胞作用,促进动脉粥样硬化病变。     

糖尿病前期患者晚期糖基化终末产物受体与氧化应激的关系

目的 通过测定糖耐量正常者、糖尿病前期及糖尿病患者血浆可溶性晚期糖基化终末产物受体(sRAGE)、内源性分泌型晚期糖基化终末产物受体(esRAGE)及氧化应激的水平,探讨糖尿病前期血浆sRAGE、esRAGE水平的变化与氧化应激的相关性,为深入了解糖尿病前期血管并发症的发病机制提供依据.方法 研究对象均行75 g标准口服葡萄糖耐量实验(OGTT)和胰岛素释放试验,按血糖水平分为糖耐量正常(NGT)组、糖尿病前期(Pre-DM)组、糖尿病(DM)组,采用酶联免疫吸附检测(ELISA)法检测血浆sRAGE、esRAGE、8-异前列腺素F2α(8-isoPGF2α);硫代巴比妥酸(TBA)法测定丙二醛(MDA),羟胺法测定超氧物岐化酶(SOD),比色法测定血浆总抗氧化能力(TAOC).结果 Pre-DM组和DM组的sRAGE、esRAGE、SOD水平明显低于NGT组,Pre-DM组和DM组的8-iso-PGF2α、MDA水平明显高于NGT组,差异均有统计学意义(P＜0.05);Pre-DM组和NGT组的TAOC水平明显高于DM组(P＜0.05);血浆esRAGE水平与sRAGE、TAOC呈正相关(r=0.39、0.64,P＜0.05),与MDA呈负相关(r =-0.45,P＜0.05),与年龄、体质量指数(BMI)、收缩压、舒张压、空腹血糖、空腹胰岛素、糖化血红蛋白、甘油三酯、低密度脂蛋白、高密度脂蛋白、8-iso PGF2α无明显相关性(P＜0.05).结论 糖尿病前期患者已发生sRAGE、esRAGE及氧化应激水平的改变,sRAGE、esRAGE在机体氧化应激与抗氧化防御平衡间可能发挥重要作用.     

吡哆胺对体外晚期糖基化及其终产物形成的抑制作用

目的体外观察吡哆胺对晚期糖基化和晚期糖基化终产物(AGEs)形成的抑制作用。方法应用牛血清白蛋白-葡萄糖试验和N-乙酰-甘氨酰-赖氨酸甲基酯-核糖两种体外试验,观察吡哆胺对糖基化反应及AGEs形成的抑制效果。结果牛血清白蛋白-葡萄糖试验显示,吡哆胺对糖基化反应具有明显抑制效果。在50 mmol/L和200 mmol/L葡萄糖浓度下,当吡哆胺浓度为50 mmol/L时,相对抑制率均在50%以上。200 mmol/L吡哆胺对糖基化的抑制作用最强,其相对抑制率分别达到92.12%和86.73%。N-乙酰-甘氨酰-赖氨酸甲基酯-核糖试验证明,吡哆胺能明显抑制晚期糖基化产物AGEs的形成。随着吡哆胺剂量的增大,吡哆胺对AGEs形成的抑制作用明显增强。结论吡哆胺对体外晚期糖基化反应和AGEs形成有明显抑制作用。