摘要
目的越来越多的国内外学者认为糖尿病肾病是一种炎症性疾病,其疾病的各个阶段在肾组织都伴有白细胞的浸润。趋化因子可参与招募炎症细胞亚群到局部肾组织,其中的单核细胞趋化蛋白-1(MCP-1)具有招募单核/巨噬细胞(M/M)的重要作用,这一观点已经在糖尿病肾病动物模型以及1型和2型糖尿病患者肾活检中多次被证实。浸润的M/M释放各种物质包括溶酶体酶、一氧化氮、反应氧中介物或转化生长因子-β1等介导肾脏局部的炎症反应,导致肾脏固有细胞增生,细胞外基质聚集,伴或不伴有肾小管纤维化,最终导致肾小球硬化。噻唑烷二酮类(TZDs)是一类通过能刺激过氧化物质体増殖活化受体γ增强胰岛素敏感性,减少胰岛素抵抗达到控制血糖目的的药物,它可以通过多种机制保护肾脏,包括降糖、降压、改善血管内皮功能、抗增殖及改变肾血流动力学等多个方面。近年来,各国学者开始关注TZDs的抗炎作用在糖尿病肾脏中的保护作用。据文献报道,TZDs可减少肾脏系膜细胞中白介素-1(IL-1)介导的IL-6和肿瘤坏死因子-α表达增加;还可通过降低核因子κB的活性和MCP-1的生成从而抑制单核细胞的趋化;另外,TZDs还具有减少肾组织内氧化应激的作用。本实验旨在观察马来酸罗格列酮(TZDs类抗糖尿病药物)对链脲霉素(STZ)诱导的糖尿病大鼠肾脏保护作用及其对尿MCP-1排泄和肾组织MCP-1mRNA表达的影响,探讨其保护肾损害的机制。
方法将Wistar大鼠随机分为正常对照(C组,n=8)、糖尿病组(D组, n=8)、罗格列酮治疗组(R组,n=8)。大鼠禁食24h后,D、R组单次腹腔注射链脲霉素[0.1mol/L无菌枸橼酸缓冲液配制为6.5mg/ml(PH 4.3)]65 mg/kg。48h后采空腹尾静脉血测血糖,以德国罗氏血糖仪测血糖≥16.7mmol/L者,入选糖尿病大鼠模型。糖尿病大鼠模型建立一周后,R组以马来酸罗格列酮(5mg?kg-1?d-1)灌胃,D组和C组灌以相应量的生理盐水。第2,4,8周检测各组大鼠尾外周血糖,第8周时以代谢笼收集12小时的尿液,测定尿白蛋白(ALB)、视黄醇结合蛋白(RBP)和MCP-1的排泄水平,计算其排泄率。之后,处死大鼠,在宰杀大鼠的同时从股动脉取血用于测定糖化血红蛋白(HbA1c)。留取肾脏标本,左肾用于称重、计算肾脏肥大指数=左肾重/体重(mg·g-1),右肾部分用于病理学检查,部分立即放置于液氮中待提取RNA,用于逆转录聚合酶链反应(RT-PCR)和实时定量聚合酶链反应(Real-time PCR),对肾脏组织中的MCP-1进行半定量及相对定量测定,分别采用MCP-1/GAPDH的光密度比值和??Ct相对定量法计算目的基因的含量(目的基因量=2-??Ct)。
结果①血糖和糖化血红蛋白:第2,4,8周,D、R组的血糖与C组的血糖比较,明显升高( P<0.01),而D组和R组两组间无统计学差异( P>0.05)。与C组比较,D组和R组第8周的糖化血红蛋白水平均明显升高( P<0.01),但后两组之间的数值差异无统计学意义。②尿ALB、RBP和MCP-1排泄率和肾脏肥大指数:第8周,D、R组的尿ALB排泄率(UAER),尿RBP排泄率和MCP-1排泄率与C组各值间有统计学差异(P<0.01);第8周,D、R组肾脏肥大指数也明显高于C组(P<0.01);与D组比较,R组中上述四项指标均明显降低( P< 0.01)。此外,尿MCP-1排泄率与UAER(r=0.945 ,P<0. 01)、尿RBP排泄率(r=0.879,P<0.01)及肾脏肥大指数(r=0.934,P<0.01)呈正相关。③电镜结果:C组结构清晰,无异常病理表现;D组肾小球毛细血管基底膜增厚,系膜细胞肿胀,系膜基质增多,系膜区扩大;R组较D肾脏病理改变明显减轻,同C组相比仅有少量病变,基底膜基本均匀一致,足突融合率明显降低。④RT-PCR和Real-time PCR结果:在RT-PCR扩增产物的电泳条带中,D、R组条带亮度明显强于C组条带,其与管家基因GAPDH的光密度比值结果提示与C组相比,D组大鼠肾脏MCP-1mRNA表达明显上调(P<0.01),R组MCP-1mRNA表达也增加(P<0.05),但较D组有明显的下调(P<0.05)。Real-time PCR法同样得出相似的结论,与C组比较,D、R组MCP-1mRNA基因的表达量高于C组(P<0.01或P< 0.05)。经罗格列酮治疗后,MCP-1在肾脏中的表达明显下调(与C组比较,P<0.05或P<0.01)。
结论罗格列酮对糖尿病大鼠肾脏具有确切的保护作用,可以降低ALB和RBP在尿液中的排泄,降低肾脏肥大指数,减轻肾脏的病理改变,其机制可能部分与其抑制肾脏局部MCP-1过度表达,减少尿MCP-1排泄有关,而且其作用是独立于降血糖效应的。
Aim Diabetic nephropathy is increasingly considered as an inflammatory disease characterized by leukocyte infiltration in the renal tissues at every stage in the development and progression of the disease. Chemokines recruit the special subpopulations of inflammatory cells to the local renal tissues. Monocyte chemoattractant protein-1 (MCP-1) has been identified as having a key role in monocyte/macrophage (M/M) recruitment in animal models of diabetic nephropathy, as well as in renal biopsies from patients with type 1 and 2 diabetes. Infiltrated M/M released various substances such as lysosomal enzyme, nitric oxide, reactive oxygen mediators or transforming growth factor-β, which are essential mediators of renal damage, leading to the hypertrophy and proliferation of renal instinct cells, accumulation of extracecullar matrix, with or without fibrosis of tubuloinstertitium, and eventually resulted in glomerular sclerosis. Thiazolidinediones (TZDs) are a class of anti-diabetes drugs that improve the insulin sensitivity and act as selective and potent agonists of peroxisome proliferators-activated receptor-γ. TZDs also have been reported to protect against diabetic kidney injury through various mechanisms, such as reducing blood glucose, lowering the blood pressure, improvement of endothelial function, antiproliferative action and inference of the rennin-angiotensin system and so on. It was reported that TZDs attenuated the IL-1-mediated increase in the expression of IL-6 and tumor necrosis factor-α, and the stretch-induced increase in monocyte chemoattractant activity, by decreasing nuclear factor-κB activation and MCP-1 production in renal mesangial cells. Another possible mechanism of the renoprotective effect of TZDs is the attenuation of oxidant injury at the kidney level. This experiment was designed to investigate the renoprotective effects of rosiglitazone (one anti-diabetic drug of the TZDs) and its influence on the urinary MCP-1 excretion and the expression of MCP-1 in the renal tissues of streptozocin (STZ)-induced diabetic rats and to analyze whether its renoprotective effect is related to the inhibition of inflammatory reaction in local renal tissue.
Methods 24 Wistar Rats were assigned into normal control group (group C, n=8), STZ-induced diabetes mellitus group (group D, n=8) and rosiglitazone (5mg?kg-1?d-1) treatment group (group R, n=8). Each rat in groups D and groups R received an intraperitoneal injection of STZ (65 mg/kg; Sigma Chemical, St. Louis, MO) in 0.1mol/L citrate buffer (pH 4.3) after a 24-hour fasting. 48 hours later, blood was harvested from vena caudalis to assess the blood glucose. Animals with fasting blood glucose more than 16.7mmol/L were considered diabetic. Since the modeling was completed for 1 week, rosiglitazone (5mg?kg-1?d-1) in group R or 0.9% saline in group C and D were administered via oral gavage once daily for 8 weeks. The blood was obtained from vena caudalis for the measurement of blood glucose at the second, 4th and 8th week. At the 8th week, 24h urine was collected and the urinary excretion levels of albumin (ALB), retinal-binding protein (RBP) and MCP-1 were examined, then the urinary excretion rates of the three parameters were calculated. When the rats were killed at the 8th week, femoral arterial blood was drawn simultaneously for testing the glycosylated hemoglobin A1c (HbA1c). After animals were sacrificed, the left kidney was used to calculate kidney hypertrophy index and a little part of the fresh right kidney cortices were stored for electron microscopic measurement and the rest of the kidney were stored in the liquid nitrogen using for the reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR. Photodensity ratio was used to semiquantitative determine the levels of transcript MCP-1 relative to the control transcript GAPDH in RT-PCR and the comparative ??Ct method was applied to comparative quantitative determine the gene in Real-time PCR.
Results①At the 2nd, 4th and 8th week, the peripheral blood glucose levels of groups D and R were significantly higher compared with those of group C at the same period (P<0.01). The HbA1c of group D or R was much higher than it of group C, however, there was no significance between the values of group D and group R.②At the 8th week, not only the urinary excretion rates of ALB, RBP and MCP-1 (P<0.01), but also the relative kidney index were markedly increased in groups D and R compared with those in group C. However, the parameters above in group R were much lower than those in group D (P<0.01). In addition, the urinary excretion of MCP-1 was positively correlated to the urinary ALB excretion(r=0.945,P<0.01), the urinary RBP excretion(r=0.879,P<0.01) and kidney/body weigh(tr=0.934,P<0.01).③The observation of the electronic microscope: the thickness of GBM, the width of epithelial foot processes and mesangial region were normal in group C. Compared to normal control, the GBM was thickened partly with some epithelial cells foot processes fused, the ultrastructure of glomeruli and blood capillary were ambiguity, and the mesangial cells swelled and the extracellular matrix expanded. After treatment with rosiglitazone, the histobiological changes were much lighter in group R. The GMB was uniform in general and a little of the foot processes fused together.④Both the results of semi-quantitive and the comparative quantitive PCR showed that in comparison with group C, the expression of MCP-1mRNA was significantly up-regulated in group D and group R (P< 0.05or P<0.01), however, treatment with rosiglitazone could decrease the expression of MCP-1 in the renal tissues (vs. D, P< 0.05or P<0.01).
Conclusions Rosiglitazone has a renoprotective effect through down-regulating the over-expression of MCP-1 in local renal tissues and reducing urinary excretion of MCP-1 in STZ-induced diabetic rats, which is independent on its effect on glycemic control.
引文
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