高脂诱导大鼠肾组织整合素连接激酶表达及足细胞损害

目的 研究高脂诱导的大鼠肾小球足细胞损害及肾组织整合素连接激酶(ILK)的表达。 方法 36只Wistar大鼠分3组：高脂饮食组给予高脂饲料喂养;辛伐他汀组在高脂饲料喂养同时,予辛伐他汀10 mg&#8226;kg-1&#8226;d-1干预;正常对照组予正常饮食。于实验第4、10周各处死6只鼠,留取血清及肾组织,采用酶法测定血清三酰甘油（TG）及总胆固醇(TC)水平;电镜观察足细胞结构变化;Western 印迹法检测肾组织ILK及结蛋白（desmin）的蛋白表达; RT-PCR检测ILK mRNA表达;免疫组化染色观察肾组织ILK的阳性分布。 结果 (1)与正常对照组比较,高脂饮食组及辛伐他汀组大鼠血脂水平升高,足细胞足突明显融合,结蛋白表达显著上调;辛伐他汀组上述指标均显著轻于高脂饮食组,组间两两比较,差异有统计学意义（P < 0.01）。(2)与正常对照组比较,高脂饮食组和辛伐他汀组大鼠肾组织ILK mRNA及蛋白表达显著增加;辛伐他汀组肾组织ILK mRNA及蛋白表达显著低于高脂饮食组,组间两两比较,差异有统计学意义（P < 0.01）。免疫组化染色显示ILK主要表达在足细胞和肾小管上皮细胞,各组ILK阳性染色与Western 印迹结果一致。(3)直线回归结果显示,肾组织结蛋白表达与ILK蛋白表达呈正相关（r = 0.93107,R2=0.8669,P < 0.01)。 结论 高脂饮食导致大鼠肾小球足细胞损害,该损害与ILK表达增高有关。辛伐他汀可抑制ILK表达,减轻高脂饮食大鼠足细胞的损害。     

人类关节滑膜细胞表达晚期糖基化终产物受体

目的 进一步探讨晚期糖基化终产物修饰的β２－微球蛋白（ＡＧＥ－β２ｍ）对关节固有细胞的生物学作用,确定人类关节滑膜细胞是否表达对ＡＧＥ特异的受体。方法 分离、培养人关节Ａ型和Ｂ型滑膜细胞,用免疫组织化学法及流式细胞仪法分别观察滑膜细胞表面ＡＧＥ受体１（ＡＧＥ－Ｒ１）,ＡＧＥ受体２（ＡＧＥ－Ｒ２）、ＡＧＥ受体３（ＡＧＥ－Ｒ３）及ＡＧＥ受体（ＲＡＧＥ）的表达,用逆转录－聚合酶链反应（ＲＴ－ＰＣＲ）技术     

内质网应激对树突状细胞表面晚期糖基化终产物受体表达的影响

目的:晚期糖基化终产物受体(RAGE)与其配体结合在炎症及免疫反应中具有重要作用.本研究探讨内质网应激(ERS)在高迁移率族蛋白B1(HMGB1)诱导树突状细胞(DC)表面RAGE上调中的作用及意义.方法:分离正常BALB/c小鼠脾脏DC进行体外培养,给予HMGB1刺激后检测DC表面RAGE表达水平及细胞ERS相关分子...     

晚期糖基化终末产物与其受体相互作用对足细胞凋亡的影响

目的 探讨可溶性与复合型晚期糖基化终末产物(AGE)与晚期糖基化终末产物受体（RAGE）的相互作用对足细胞凋亡的影响。 方法 以可溶性（CML-BSA、AGE-BSA）和复合型（AGE修正胶原Ⅳ）AGE刺激小鼠足细胞，并用浓度分别为10、50、100 mg/L的AGE刺激细胞，应用TUNEL染色和荧光激活细胞分类（FACS）法来计数凋亡和坏死的足细胞。用RAGE iRNA转染足细胞后，以同样剂量的可溶性和复合型AGE刺激足细胞，观察凋亡情况的改变。 结果 可溶性和复合型AGE均可诱导小鼠足细胞凋亡，复合型AGE引起的足细胞凋亡是可溶性AGE的2~3倍（均P < 0.01）。AGE呈剂量依赖性引起足细胞凋亡。用RAGE iRNA转染足细胞，降低60%~70%RAGE基因活性后，可溶性AGE引起的凋亡率明显下降，复合型AGE诱导的凋亡有下降趋势，但不明显。只有在AGE 100 mg/L刺激后才发生细胞坏死。结论 可溶性AGE主要通过与RAGE相互作用引起足细胞凋亡，复合型AGE部分通过与RAGE相互作用诱导足细胞凋亡。减少AGE生成和RAGE表达可能是预防肾脏病进展的重要途径。     

循环晚期糖基化终产物的检测方法和评价

晚期糖基化终产物（AGE）是蛋白质的氨基组、脂质和脂蛋白与糖的醛基组之间的非酶性糖基化／氧化反应的终产物。反应早期生成可逆的Schiff碱，该早期产物经过结构重建，转化成较为稳定的amadori产物，而后再经过一系列化学重排和脱水反应，产生不可逆的晚期产物即ACE。正常人体内该反应进行得非常缓慢，通常只发生于某些转换率很低的蛋白质如基质蛋白。蛋白质一旦被AGE修饰，即丧失其生理功能，将通过巨噬细胞等细胞表面AGE受体而被摄取、降解、清除。因此，正常情况下AGE修饰蛋白作为一种信号参与了机体清除衰老组织以及结构重建的…     

晚期糖基化终产物修饰的β2微球蛋白对人滑膜细胞黏附、伸展和增殖的影响

目的探讨晚期糖基化终产物(AGE)修饰的β2微球蛋白(AGE-β2m)对人滑膜细胞黏附、伸展、增殖功能的影响.方法分离、培养正常人关节B型滑膜细胞,使其与AGE-β2m或正常基质成份包被的固相表而接触.用同位素标记细胞计数法比较滑膜细胞的黏附功能,染色法比较细胞伸展功能,3H-TdR掺入法和直接细胞计数比较细胞增殖功能.结果滑膜细胞贴附于AGE-β2m、β2m和AGE-胶原蛋白时其黏附能力较之与纤维连接蛋白、Ⅰ型胶原等正常基质成份接触时明显降低.伸展现象延迟,细胞增殖功能受抑.结论与AGE-β2m等非正常基质成份的接触将影响人类B型关节滑膜细胞的功能,这可能是透析相关性淀粉样变(DRA)时关节病变的原因之一.     

氟伐他汀对晚期糖基化终产物诱导的人近端肾小管上皮细胞转分化的影响

目的：研究氟伐他汀对晚期糖基化终产物（AGE）诱导的人近端肾小管上皮细胞（HK-2）转分化的影响及核因子（NF）-κB在其中所起的作用。方法：将培养细胞分为5组：（1）BAS组;（2）AGE-BAS组;（3）AGE-BAS＋NF-κB特异性阻断剂（PDTC）组;（4）AGE-BAS＋氟伐他汀组;（5）AGE-BAS＋氟伐他汀＋PDTC组。应用EMSA法测定NF-κB活性变化。Westernblot法检测α-平滑肌肌动蛋白（α-SMA）、E-钙黏着糖蛋白（E-cadherin）表达。结果：氟伐他汀在一定浓度范围内,呈剂量依赖性抑制AGE-BSA诱导的HK-2细胞NF-κB活化。AGE-BSA刺激后随浓度增加,时间延长,α-SMA蛋白表达明显升高、E-adherin蛋白表达明显降低。NF-κB特异性阻断剂PDTC及氟伐他汀均能部分阻断AGE-BSA诱导的α-SMA蛋白表达升高及E-adherin蛋白表达降低。氟伐他汀＋PDTC进一步抑制AGE-BSA诱导的α-SMA蛋白表达升高及E-adherin蛋白表达降低。结论：NF-κB参与了AGE-BSA诱导的HK-2细胞转分化。氟伐他汀抑制HK-2细胞转分化除通过抑制NF-κB活化,可能还有其他机制。     

整合素连接激酶和肾小管-间质纤维化

整合素连接激酶(Integrin-linkedkinase,ILK)是一种近期发现的Ser/Thr蛋白激酶。ILK能够通过与整合素β1亚单位的结合介导细胞与胞外基质的连接,以依赖于PI3K的方式激活,并通过磷酸化下游底物PKB/AKT、GSK3等使胞外信号得以向下游传递,参与多种信号传导通路,与肾间质纤维化的发生相关。     

整合素连接激酶和肾小管-间质纤维化

整合素连接激酶(Integrin-linked kinase,ILK)是一种近期发现的Ser/Thr蛋白激酶.ILK能够通过与整合素β1亚单位的结合介导细胞与胞外基质的连接,以依赖于PI3K的方式激活,并通过磷酸化下游底物PKB/AKT、GSK3等使胞外信号得以向下游传递,参与多种信号传导通路,与肾间质纤维化的发生相关.     

整合素连接激酶在前列腺癌组织中的表达及意义

目的: 探讨整合素连接激酶(ILK)在前列腺癌组织中的表达及其临床意义。　方法: 应用免疫组化SP法测定 50例前列腺癌及 16例良性前列腺增生组织中ILK的表达。　结果: 前列腺癌组织中ILK阳性表达率46. 0%(23 /50),肿瘤病理分级:高分化组阳性表达率为9. 0% ( 1 /11 ),中分化组为35. 7% ( 5 /14 ),低分化组68. 0% ( 17 /25),随肿瘤病理分级高分化组到低分化组,阳性表达率有趋势性增高。临床分期A+B期为22. 5% (7 /31)和C+D期为84. 2% (16 /19),临床分期程度的增高,癌细胞ILK阳性表达率明显增加。良性前列腺增生组织ILK阳性表达率仅为6. 2% (1 /16),明显低于前列腺癌组织(χ2 =8. 27,P<0. 01)。　结论: ILK异常表达在前列腺癌的恶性进展中起重要作用,检测ILK的表达有帮助于判断病期及预后。     

晚期糖基化终末产物对人肾小管上皮细胞表达结缔组织生长因子和纤维连接蛋白的影响

目的：观察AGE-BSA认对体外培养的人近端肾小管上皮细胞株HK-2表达结缔组织生长因子（CTGF）和纤维连接蛋白（FN）的影响。方法：将HK-2细胞在体外与不同浓度的AGE—BSA和BSA共同培养。以酶联免疫法（ELISA）检测转化生长因子-β1（TGF-β1）的分泌，以逆转录-聚合酶联反应法（RT—PCR）测定CTGF mRNA和FN mRNA的表达。结果：100μg／ml的AGE—BSA刺激HK-2细胞24h，细胞上清中TGF-β1的浓度是对照组的2.37倍。AGE—BSA刺激HK-2细胞48h后，CTGF mRNA的表达开始出现显著升高（P〈0.01）。AGE—BSA能够剂量依赖性的上调HK-2细胞CTGF mRNA和FN mRNA的表达。BSA刺激组没有以上作用。结论：AGE—BSA可上调HK-2细胞CTGF和FN的表达，这可能在糖尿病肾病肾小管间质纤维化形成中起到一定作用。     

A Receptor‐Based Bioadsorbent to Target Advanced Glycation End Products in Chronic Kidney Disease

The accumulation of advanced glycation end products (AGEs) has been reported to be a major contributor to chronic systemic inflammation. AGEs are not efficiently removed by hemodialysis or the kidney of a chronic kidney disease (CKD) patient. The goal of this study was to develop a receptor for AGEs (RAGE)‐based bioadsorbent device that was capable of removing endogenous AGEs from human blood. The extracellular domain of RAGE was immobilized onto agarose beads to generate the bioadsorbent. The efficacy of AGE removal from saline, serum, and whole blood; biological effects of AGE reduction; and hemocompatibility and stability of the bioadsorbent were investigated. The bioadsorbent bound AGE‐modified bovine serum albumin (AGE‐BSA) with a binding capacity of 0.73 ± 0.07 mg AGE‐BSA/mL bioadsorbent. The bioadsorbent significantly reduced the concentration of total AGEs in serum isolated from end‐stage kidney disease patients by 57%. AGE removal resulted in a significant reduction of vascular cell adhesion molecule‐1 expression in human endothelial cells and abolishment of osteoclast formation in osteoclast progenitor cells. A hollow fiber device loaded with bioadsorbent‐reduced endogenous AGEs from recirculated blood to 36% of baseline levels with no significant changes in total protein or albumin concentration. The bioadsorbent maintained AGE‐specific binding capacity after freeze‐drying and storage for 1 year. This approach provides the foundation for further development of soluble RAGE‐based extracorporeal therapies to selectively deplete serum AGEs from human blood and decrease inflammation in patients with diabetes and/or CKD.     

Skin Autofluorescence,a Measure of Cumulative Metabolic Stress and Advanced Glycation End Products,Decreases During the Summer in Dialysis Patients

Tissue advanced glycation end products (AGEs) are a measure of cumulative metabolic and oxidative stress and cytokine‐driven inflammatory reactions. AGEs are thought to contribute to the cardiovascular complications of hemodialysis (HD) patients. Skin autofluorescence (SAF) is related to the tissue accumulation of AGEs and rises with age. SAF is one of the strongest prognostic markers of mortality in these patients. The content of AGEs is high in barbecue food. Due to the location in northern Sweden, there is a short intense barbecue season between June and August. The aim of this study was to investigate if seasonal variations in SAF exist in HD patients, especially during the barbecue season. SAF was measured noninvasively with an AGE Reader in 34 HD‐patients (15 of those with diabetes mellitus, DM). Each time the median of three measures were used. Skin‐AF was measured before and after each one HD at the end of February and May in 31 patients (22 men/9 women); the end of May and August in 28 (20 m/8 w); the end of August and March in 25 (19 m/6 w). Paired statistical analyses were performed during all four periods (n = 23, 17 m/6 w); as was HbA1c of those with DM. There was at a median 5.6% increase in skin‐AF during the winter period (February–May, P = 0.004) and a 10.6% decrease in the skin‐AF during the summer (May–August, P < 0.001). HbA1c in the DM rose during the summer (P = 0.013). In conclusion, skin‐AF decreased significantly during the summer. Future studies should look for favorable factors that prevent skin‐AF and subsequently cardiovascular diseases.     

Advanced Glycation End Products in Hemodialyzed Patients with Diabetes Mellitus Correlate with Leptin and Leptin/Body Fat Ratio

Advanced glycation end products (AGEs) and other carbonyl and oxidative stress compounds are supposed to play a critical role in the pathogenesis of several diseases and their complications, i.e., diabetes mellitus, diabetic retinopathy, atherosclerosis, and chronic renal failure. In the present investigation, we were interested in the relationship of AGEs in plasma to other prominent factors in the patients on chronic hemodialysis treatment—27 patients with diabetes mellitus, 35 patients without diabetes mellitus. AGE-group reactivity was estimated using a spectrofluorometric method (excitation 350 nm, emission 430 nm) and is expressed in arbitrary units (AU). We found significantly higher AGEs levels in diabetics than in non-diabetics on regular hemodialysis treatment both before (2.7 ± 0.7 × 10⁴ AU vs. 2.2 ± 0.6 × 10⁴ AU, p<0.001) and after the dialysis session (2.3 ± 0.5 × 10⁴ AU vs. 1.8 ± 0.7 × 10⁴ AU, p<0.005). AGEs were significantly reduced during hemodialysis in both groups of patients—by 15.4 % in the diabetic go (p<0.001) and by 17.3% in non-diabetics (p<0.005). In the patients with diabetes mellitus, AGEs did not correlate with parameters of the glucose metabolism correction (blood glucose, HbA1c). We observed a significant correlation between AGEs and leptin (r = 0.48, p < 0.05) as well as the leptin/body fat ratio (r = 0.56, p < 0.05) only in hemodialyzed patients with diabetes mellitus. These findings suggest more detailed studies to identify the molecular links between carbonyl stress, i.e., advanced glycation end products, and leptin metabolism, sign of microinflammation and hypertension.     

Advanced glycation end-product expression is upregulated in the gastrointestinal tract of type 2 diabetic rats

AIM:To investigate changes in advanced glycation end products(AGEs) and their receptor(RAGE) expression in the gastrointestinal(GI) tract in type 2 diabetic rats.METHODS:Eight inherited type 2 diabetic rats GotoKakizak(GK) and ten age-matched normal rats were used in the study.From 18 wk of age,the body weight and blood glucose were measured every week and 2 wk respectively.When the rats reached 32 wk,twocentimeter segments of esophagus,duodenum,jejunum,ileum,and colon were excised and the wet weight was measured.The segments were fixed in 10% formalin,embedded in paraffin and five micron sections were cut.The layer thickness was measured in Hematoxylin and Eosin-stained slides.AGE [N epsilon-(carboxymethyl) lysine and N epsilon-(carboxyethyl)lysine] and RAGE were detected by immunohistochemistry staining and image analysis was done using Sigmascan Pro 4.0 image analysis software.RESULTS:The blood glucose concentration(mmol/L) at 18 wk age was highest in the GK group(8.88 ± 1.87 vs 6.90 ± 0.43,P 0.001),a difference that continued to exist until the end of the experiment.The wet weight per unit length(mg/cm) increased in esophagus,jejunum and colon from the normal to the GK group(60.64 ± 9.96 vs 68.56 ± 11.69,P 0.05 for esophagus; 87.01 ± 9.35 vs 105.29 ± 15.45,P 0.01 for jejunum; 91.37 ± 7.25 vs 97.28 ± 10.90,P 0.05 for colon).Histologically,the layer thickness of the GItract was higher for esophagus,jejunum and colon in the GK group [full thickness(μm):575.37 ± 69.22 vs 753.20 ± 150.41,P 0.01 for esophagus; 813.51 ± 44.44 vs 884.81 ± 45.31,P 0.05 for jejunum; 467.12 ± 65.92 vs 572.26 ± 93.60,P 0.05 for colon].In esophagus,the AGE and RAGE mainly distributed in striated muscle cells and squamous epithelial cells.The AGE distribution was much stronger in the GK group compared to the normal group both in the striated muscle layer and mucosa layer(immuno-positive area/ total measuring area %:4.52 ± 0.89 vs 10.96 ± 1.34,P 0.01 for muscle; 8.90 ± 2.62 vs 22.45 ± 1.26,P 0.01 for mucosa).No visible difference was found for RAGE distribution between the two groups.In the intestine AGE and RAGE distributed in epithelial cells of villi and crypt.RAGE was also found in neurons in the myenteric and submucosal plexus.The intensity of AGE staining in mucosa of all segments and RAGE staining in neurons in all segments were strongest in the diabetes group.Significant difference for AGE was found in the epithelial cells of villi and crypt in duodenum(immunopositive area/total measuring area %:13.37 ± 3.51 vs 37.48 ± 8.43,P 0.05 for villi; 0.38 ± 0.12 vs 1.87 ± 0.53,P 0.05 for crypt) and for RAGE in neurons of all segments(e.g.,for jejunum:no staining neurons% 0 vs 0,mild 36.0 ± 5.2 vs 28.7 ± 3.5,moderate 53.2 ± 4.8 vs 55.8 ± 5.4,strong 10.7 ± 1.1 vs 15.4 ± 2.0,P 0.05).In the colon,RAGE was primarily found in neurons in the myenteric and submucosal plexus.It was stronger in the diabetes group than in the normal group(no staining neurons% 6.2 ± 0.2 vs 0.3 ± 0.04,mild 14.9 ± 2.1 vs 17.6 ± 1.5,moderate 53.1 ± 4.6 vs 44.7 ± 4.4,strong 25.6 ± 18 vs 43.6 ± 4.0,P 0.05).In the rectum,RAGE was primarily found in the mucosa epithelial cells.CONCLUSION:The AGE and RAGE expression was upregulated in the GI tract of GK diabetic rats and may contribute to GI dysfunction in type 2 diabetic patients.