【作者】 刘萍；

【导师】 邱明才；

【作者基本信息】 天津医科大学 ， 内科学， 2012， 博士

【摘要】 研究背景目的：糖尿病肾脏疾病是糖尿病重要的微血管并发症之一,是终末期肾病最主要的病因。高血糖状态下肾小管结构功能的损伤早于肾小球是DKD主要的病理基础。免疫损伤参与糖尿病肾脏疾病的发生及发展是目前尚存争议的理论。TLR-NFκB激活途径是机体固有免疫的第一道屏障,参与机体天然防御反应。但过度激活会造成机体炎性反应,促进组织纤维化进程。目前关于TLRs固有免疫激活途径是否参与糖尿病肾脏肾小管间质纤维化进程的研究很少,有待于探讨。1,25(OH)2D3是一种具有免疫调节作用的激素。在固有免疫反应中,研究表明1,25(OH)2D3可能对TLRs具有双向调节作用。同时1,25(OH)2D3具有肾脏保护作用已经得到公认。研究的交叉点集中在对NF-KB的下调作用。1,25(OH)2D3是否通过对TLRs激活途径的抑制作用起到下调NF-KB的表达,有关这方面的研究内容目前国内外未见报道。因此,本课题的研究目的包括：(1)明确TLR是否通过MyD88依赖途径激活NF-κB,参与糖尿病大鼠肾小管间质纤维化的发生发展,证实DKD是固有免疫参与的免疫性肾病；(2)1,25(OH)2D3能否通过对TLR过度激活的固有免疫下调作用达到肾脏保护作用；(3)1,25(OH)2D3肾脏免疫治疗的剂量及安全性评估。依据上述实验结果,我们可以为DKD免疫损伤机制做一理论铺垫,并为1,25(OH)2D3的免疫调节机制奠定一定的理论基础,以便可以在临床上得以推广。研究方法：(1)动物实验：STZ诱导的雄性SD大鼠作为糖尿病动物模型。将观察大鼠随机分为7组：正常对照组(C组)、糖尿病未干预组(D组)、小剂量1,25(OH)2D3干预组(L组,0.025ug/kg/d)、中剂量1,25(OH)2D3干预组(M组,0.15ug/kg/d)、大剂量1,25(OH)2D3干预组(H组,0.3ug/kg/d)、氯沙坦钾干预组(A组,10.4mg/kg/d)及精蛋白锌胰岛素干预组(Y组,16U/kg/d)。糖尿病成模后每日1,25(OH)2D3及氯沙坦钾灌胃给药,胰岛素治疗组于颈后皮下注射给药。16周后留取24小时尿及随机尿样进行尿mALB及尿NAG检测；股动脉取血测定生化指标；左侧肾脏固定包埋,进行HE,Masson染色观察肾小管间质纤维化程度并进行间质损伤评分；免疫组织化学方法检测MCP-1)、α-SMA、NF-κB的肾小管间质表达情况。右侧肾脏称重冻存进行RT-PCR)、Western blot检测MCP-1)、α-SMA)、NF-κB)、TLR4)、MyD88的mRNA)、蛋白表达,并行冰冻切片免疫荧光检测观察TLR4)、MvD88的肾脏表达。(2)细胞实验：体外培养正常大鼠肾小管上皮细胞(NRK-52E)。三个葡萄浓度进行干预培养(5.5mmol/L葡萄糖、25mmol/L葡萄糖、50mmol/L葡萄糖)分别于0小时、2小时、4小时、6小时、8小时、24小时点收集细胞观察TLR4及MyD88的mRNA及蛋白表达。确定表达量达峰的葡萄糖浓度及时间点进行1,25(OH)2D3的干预实验。分组5.5mmol/L葡萄糖培养(LG组)、25mmol/L葡萄糖培养(HG组)、25mmol/L葡萄糖培养+1×10-9mmol/L1,25(OH)2D3(HG+10-9)、25mmol/L葡萄糖培养+1×10mol/L1,25(PH)2D3(HG+108)、25mol/L葡萄糖培养+1×10-7mol/L1,25(OH)2D3;(HG+10-7)、25mmol/L葡萄糖培养+1×10-5mol/L氯沙坦钾(HG+ARB)。于6小时收集细胞进行TLR4及MyD88的mRNA检测,24小时收集细胞行Western blot及细胞免疫荧光检测观察TLR4及MyD88的蛋白表达。研究结果：1、动物实验结果：(1)STZ诱导的糖尿病大鼠成型16周后,未干预组糖尿病大鼠血糖、尿mALB、尿NAG、尿素氮(BUN)、肌苷(Cr)、肾体比、间质损伤评分、肾脏MCP-1及α-SMA的表达较正常对照组均明显升高；大剂量1,25(OH)2D3及胰岛素、ARB治疗组能显著降低尿mALB、尿NAG、尿素氮(BUN)、肌苷(Cr)水平,间质损伤评分,降低MCP-1及α-SMA的表达,从而起到肾脏保护作用。中小剂量1,25(OH)2D3作用不显著。(2)未干预组糖尿病大鼠肾脏TLR4、MyD88及NF-κB的mRNA及蛋白表达较正常对照组均明显升高,大剂量1,25(OH)2D3及胰岛素、ARB治疗组能显著降低TLR4.MvD88及NF-κB的表达。TLR4、MvD88及NF-κB与尿mALB、BUN、SCr、间质损伤评分、肾脏MCP-1及α-SMA的表达存在正相关。(3)各组大鼠血钙磷、肝功能比较没有显著性差异,1,25(OH)2D3各干预组的尿钙排出较正常对照组显著增高。2、细胞实验结果：(1)NRK-52E细胞TLR4、MyD88的mRNA表达在25mmol/L葡萄糖培养6小时达到高峰,蛋白表达在25mmol/L葡萄糖培养24小时达到高峰,与其他葡萄糖浓度及时间点比较均存在显著性差异。(2)1×10-7mol/L的1,25(OH)2D3能够显著降低TLR4、MyD88的mRNA及蛋白表达,而1×10-8mol/L,1×10-9mol/L作用不明显。结论：1、肾小管间质病变是糖尿病肾脏疾病发生发展过程中的重要组成部分,TLR4-NF-κB介导的固有免疫反应过度激活造成的肾小管间质炎性反应参与了糖尿病肾脏疾病的发生发展。2、1,25(OH)2D3能够抑制STZ诱导糖尿病大鼠肾脏TLR4、MyD88及NF-κB的表达,从而抑制NF-κB对MCP-1及a-SMA的上调作用减轻间质免疫炎症反应,阻抑EMT的发生,减少尿蛋白的漏出,延缓肾小管间质纤维化的进程。3、体外实验得到相同结果,25mmol/L高糖培养的肾小管上皮细胞TLR4及MyD88表达增高,1×10-7mol/L浓度的1,25(OH)2D3能够抑制高糖培养的肾小管上皮细胞TLR4及MyD88的表达,抑制固有免疫的过度激活。4、本实验中,1,25(OH)2D3对STZ诱导的糖尿病大鼠肾病的干预剂量相对安全,但1,25(OH)2D3所引发的高尿钙状态仍需要长期观察和探讨。更多还原

【Abstract】 Objective:Diabetic kidney disease (DKD) is one of the important microvascular complications of diabetes mellitus and is believed to be the major causes of end stage renal disease. The injury of kidney tubules before glomerulus in DM is also thought of the primary pathologic abnormality of the DKD. The theory of the immuno-injury participate in the development of DKD is still being debated for the time being. Meanwhile, that the pathway of TLR-NF-κB activation may be the first barricade of the inherent immunity and participate in the natural defensive reaction also under wide investigation. But over-activation will result in inflammatory reaction which would promote fibrosis of the target tissues. There are few study up till now, on the relationship between the inherent immunity and the renal tubulointerstitial fibrosis in DKD. On the other hand,1,25-dihydroxycholecalciferol is a hormone, however, which is widely recognized to be involeved in the immuno-regulation. Some studies showed that by two-ways,1,25(OH)2D3had regulating effects on the TLRs in the inherent immunity. At the same time, many studies indicated that the1,25(OH)2D3had the renoprotection through the down-regulating effects on NF-KB expression.. However, there has been no any report in the literature about whether or not1,25(OH)2D3had a down-regulating effect on NF-κB expression by inhibiting the activation of the TLRs. Under such a circumstances, our investigation was launched and focused on (1) To make sure that whether or not the TLRs-MyD88-NF-κB pathway might be involved in the development of RIF in DKD. To elucidate the DKD is a immunity kidney disease in which the inherent immunity participative.(2) Whether the1,25-dihydroxycholecalciferol can ameliorate the development of DKD through down-regulating the over-activated of the TLRs.(3) To evaluate the safety of1,25-dihydroxycholecalciferol and look for an optimal dose in immunosurpressive therapy of kidney. Through this experiment, the theory for the immuno-injury in DKD could be established, while a concored theoretical basis could also be established for1,25(OH)2D3which was involved in the immune-regulating mechanism besides those effects on calcium and phosphorus metabolism. Methods:(1) Animal experiment:The DM rats models were made by intravenous injection of STZ. Then, they were randomly devided into seven groups, including control group(group C), diabetic unintervention group (group D), diabetic group intervention orally with a low dose1,25(OH)2D3(group L,0.025ug/kg/d),a middle dose1,25(OH)2D3(group M,0.15ug/kg/d), a high dose1,25(OH)2D3(group H,0.3ug/kg/d), diabetic group intervention orally with losartan potassium (group A,10.4mg/kg/d), diabetic group intervention injection with insulin(group Y,16U/kg/d). Sixteen weeks later, the urinary microalbumin, urinary NAG and blood biochemical indicator were determined. Renal pathological changes were studied by both HE and Masson staining. The tubulointerstitial injury score were assessed by optical microscope. Besides these, the expression of MCP-1、α-SMA and NF-κB in tubulointerstitial were also investigated by immunohistochemisty. The expression of MCP-1、α-SMA. NF-κB、TLR4and MyD88were detected by Real-time quantitive polymerase chain reaction and Western blot. The expression of both TLR4and MyD88in tubulointerstitial was studied by immunofluorescence.(2) Cellular experiment:The renal tubular epithelial cells of normal rats were incubated in vitro. NRK-52E cells were exposed to three concentrations of glucose (including5.5mmol/L,25mmol/L,50mmol/L respectively) and harvested at the0.2,4,6,8.24hour. The expression of TLR4and MyD88were examined by Western blot and Real-time PCR. In order to determine the glucose concentration and the time point for the harvesting based upon the expression peak of the TLR4and MyD88, the experiment treated with1,25(OH)2D3was subsequently carried out. The cells were divided into six groups according to the experimental conditions, with DMEM as the medium:Group normal glucose Control (5.5mmol/L, LG group); Group high glucose (25mmol/L, HG group); Groups1,25(OH)2D3, cultured with high concentration of glucose (25mmol/L)DMEM and with various concentrations of1,25(OH)2D3(10-9mol/L,10-8mol/L,10-7mol/L)(HG+10-9, HG+10-8, HG+10-7) Group ARB, cultured with high glucose DMEM+Losartan potassium (HG+ARB). The expression of TLR4and MyD88mRNA were studied by Real-time PCR at hour6and the expression of TLR4and MyD88protein were examined by Western blot and cellular immunofluorescence at hour24. Results:1、The results of animal experiment:(1)In the rats of diabetic group without intervention, blood glucose, urinary microalbumin, urinary NAG, blood urea nitrogen, serum creatinine, kidney weight/body weight, tubulointerstitial injury score, the expression of MCP-1and α-SMA in renal tissue was significantly increased compared with the normal control group. The urinary microalbumin, urinary NAG, blood urea nitrogen, serum creatinine, tubulointerstitial injury score and the expression of MCP-1and α-SMA in renal tissue of the diabetic group intervented orally with a high dose1,25(OH)2D3, diabetic group intervented by the injection of insulin and diabetic group intervented orally with losartan potassium were significantly lower than the diabetic wihtout any intervention. These expression in the diabetic group intervented orally by both a low dose and a middle dose of1,25(OH)2D3showed no difference with the diabetic without intervention.(2) In the rats of diabetic without intervention, the expression of TLR4, MyD88and NF-κB in the renal tissue significantly increased compared with normal control group. The expression of TLR4, MyD88and NF-κB in the renal tissue of the diabetic group intervented orally by a high dose of1,25(OH)2D3, diabetic group intervented injection with insulin and diabetic group intervented orally with losartan potassium were significantly lower than the diabetic group without any intervention. The expression of TLR4, MyD88, NF-κB were positively correlated with urinary microalbumin blood urea nitrogen, serum creatinine, tubulointerstitial injury score while the expression of MCP-1and a-SMA in renal tissue was nearly same.(3) Among each groups, the serum calcium, phosphate and the liver functions showed no difference.2、The results of the cellular experiment:(1) The peak times of the TLR4、MyD88mRNA expression and the protein expression were hour6and hour24in the culture with high glucose of25mmol/L and showed significantly difference in comparison with other glucose at various glucose concentrations and time points.(2) The expression of TLR4and MyD88in the group treated with the1,25(OH)2D3of1×10-/mol/L significantly decreased compared with the high glucose group. There had no difference compared with the high glucose group in the groups that the1,25(OH)2D3concentration were1×10-9mol/L and1×10-8mol/L.Conclusions:1、The kidney tubulointerstitial disease is the important component in the development of the DKD. The inflammatory reaction induced by the over-activation of the TLR4-NF-κB pathway, one of the inherent immunity, was involved in the development of the DKD.2、The over-activation of the TLR4-MyD88-NF-KB pathway in the kidney of STZ induced diabetic rats could be suppressed by1,25(OH)2D3, down-regulating the expression of the MCP-1and a-SMA. So the process of the renal tubulointerstitial fibrosis could be delayed by1,25(OH)2D3through alleviating the inflammatory reaction, repressing the generation of EMT and reducing the leakage of the urine protein consequently.3、The same results were also derived as well in vitro experiment. The expression of TLR4and MyD88was increased in the culture with25mol/L concentration glucose and was also suppressed by the1,25(OH)2D3. The over-activation of the inherent immunity could be suppressed by1,25(OH)2D3.4、In our experiment, relatively speaking, the intervention dose of1,25(OH)2D3we designed is currently believed to be safe while the hypercalciuria induced by1,25(OH)2D3needs to be further investigated.更多还原