活性维生素D3对糖尿病肾病大鼠足细胞的保护作用及其机制研究

发布时间：2018-03-25 15:46

本文选题：活性维生素D3　切入点：糖尿病肾病　出处：《东南大学》2015年博士论文

【摘要】：目的糖尿病肾病是糖尿病的严重并发症,是导致糖尿病肾病患者致残和死亡的主要原因之一。足细胞损伤被认为是引起糖尿病肾病蛋白尿和肾小球硬化的关键因素。因此,探讨足细胞损伤的机制并寻找有效可行的干预足细胞损伤的药物是解决问题的关键。活性维生素D3对糖尿病肾病具有显著的肾保护作用,但其机制尚未阐明。本研究通过构建STZ诱导的糖尿病肾病大鼠模型及体外足细胞培养模型两方面,观察活性维生素D3对糖尿病肾病足细胞的保护作用,同时探讨可能机制。方法将SD雄性大鼠随机分为四组：正常对照组(NC组)、活性维生素D3干预组(VD组,骨化三醇0.1μg·kg-1·d-1灌胃)、糖尿病肾病组(DN组,腹腔注射STZ 58mg·kg-1)和糖尿病肾病+活性维生素D3组(DN+VD组),定期检测血糖、体质量,收集尿标本,干预18w末处死动物,检测Scr、BUN、Ca、P和24小时尿蛋白变化,PAS和MASSON染色观察肾脏病理改变,电镜观察足细胞超微结构变化。体外研究采用体外培养小鼠永生系足细胞,对分化的足细胞予不同的刺激,观察1,25(OH)2D3、TRPC6抑制剂SKF-96365以及PI3K抑制剂LY294002对高糖诱导足细胞损伤的影响。免疫组化法检测肾组织WT-1、Nephrin、 Podocin、Desmin、VDR和p-Akt;免疫荧光法检测肾组织TRPC6表达以及足细胞Nephrin、Podocin、Desmin 及 TRPC6; Real-time PCR、Western Blot检测肾组织VDR、足细胞裂孔隔膜蛋白Nephrin、Podocin、TRPC6、足细胞损伤标志Desmin和PI3K/p-Akt信号通路相关分子表达。结果(1)体内研究表明,与DN组相比,DN+VD组蛋白尿减低达36%,肾脏病理损伤减轻,足细胞损伤减轻,血糖、体质量以及血尿素氮之间无差异(均P0.05)；血肌酐、钙、磷在四组之间未见差异(均P0.05)。与NC组相比,DN组Podocin、 Nephrin表达量均明显减低(均P0.05),足细胞损伤标志Desmin蛋白表达量显著增加(P0.05)。与DN组相比,DN+VD组Podocin、Nephrin表达量均明显增加(均P0.05),足细胞损伤标志Desmin表达量显著减低(P0.05)。WT-1在四组之间未见统计学差异。体外研究也证实,与对照组相比,30mmol/L高糖呈时间依赖的下调Nephrin以及Podocin的表达,上调Desmin的表达,且在24h达高峰。与高糖组相比,1,25(OH)2D3呈浓度依赖的上调Nephrin以及Podocin的表达,下调Desmin的表达,且100 nmol/L1,25(OH)2D3的浓度效果最佳。上述实验结果证实,足细胞损伤是糖尿病肾病的早期典型特征,活性维生素D3对高浓度葡萄糖状态下足细胞损伤有保护作用。 (2)进一步探讨Nephrin-PI3K/p-Akt信号通路在活性维生素D3保护足细胞中的作用发现,与NC组相比,DN组PI3K-p85、 p-Akt表达量均明显减低(均P0.05)。与DN组相比,DN+VD组PI3K-p85、p-Akt表达量均明显增加(均P0.05),相关性分析显示,Nephrin与PI3K-p85及p-Akt呈明显的正相关(r=0.736,P0.05;r=0.855,P0.05)。高糖干预足细胞24h后,PI3K、p-Akt蛋白表达明显减少,给予1,25(OH)2D3可显著上调PI3K, p-Akt蛋白的表达,而对总的Akt蛋白的表达无影响,在1,25(OH)2D3加用PI3K抑制剂LY294002后,足细胞的损伤尚未得到改善,与高糖组相似。(3)观察在活性维生素D3治疗糖尿病肾病足细胞损伤的一个可能的关键靶点TRPC6的表达变化发现,与NC组相比,DN组VDR表达量均明显减低(P0.05),而TRPC6表达显著升高。与DN组相比,DN+VD组VDR表达量明显增加(P0.05),而TRPC6下调(P0.05)。相关性分析显示,TRPC6与Nephrin(r=-0.752、P0.05)、VDR(r=-0.685、P0.05)呈负相关,而与24小时尿蛋白(r=0.739、P0.05)、Desmin (r=0.947、P0.05)呈正相关。高糖干预足细胞24h后,VDR mRNA以及蛋白表达明显减少,TRPC6 mRNA以及蛋白表达明显增加,给予1,25(OH)2D3可显著上调VDR,减少TRPC6表达。给予TRPC6抑制剂SKF96365干预后,足细胞的损伤得到改善,同时伴随PI3K、p-Akt蛋白的表达上调。结论1)足细胞损伤是糖尿病肾病的早期典型病理特征,活性维生素D3对高浓度葡萄糖状态下足细胞损伤有保护作用。2)高浓度葡萄糖环境下,Nehrin-PI3K/p-Akt信号通路的下调介导足细胞损伤,活性维生素D3能够通过稳定该信号通路减轻足细胞损伤,从而缓解DN的进展。3)高浓度葡萄糖状态下TRPO6表达增高在足细胞损伤过程中起着重要作用,阻断THPO6的高表达可能是活性维生素D3调节Nephrin-PI3K-p-Akt信号通路发挥足细胞保护作用的关键环节。
[Abstract]:Objective: diabetic nephropathy is a serious complication of diabetes mellitus, which is the main cause of death and disability in patients with diabetic nephropathy. Podocyte injury is considered to be the key factor to cause diabetic nephropathy proteinuria and glomerular sclerosis. Therefore, to explore the mechanism of podocyte injury and drug intervention for injury of podocyte effective is the key to solve the problem. The activity of vitamin D3 has significant protective effect on kidney of diabetic nephropathy, but its mechanism has not been elucidated. This study constructed two culture model induced by STZ model and in vitro in podocytes of diabetic nephropathy rats, to observe the protective effect of vitamin D3 on diabetic nephropathy podocyte, and explore the possible mechanism. Methods: SD male rats rats were randomly divided into four groups: normal control group (group NC), the activity of vitamin D3 intervention group (VD group, 0.1 g ossification in three alcohol kg-1 D-1 gavage), sugar Diabetic nephropathy group (DN group, intraperitoneal injection of STZ 58mg - kg-1) and diabetic nephropathy + vitamin D3 group (group DN+VD), regular blood glucose, body weight, urine samples were collected at the end of the intervention of 18W killed animal, detection of Scr, BUN, Ca, P and 24 hours urinary protein changes, renal pathological changes PAS and MASSON staining, electron microscope observation of podocyte ultrastructure changes. In vitro studies using cultured immortalized mouse podocytes in vitro differentiation of podocytes to different stimuli, observation of 1,25 (OH) 2D3, TRPC6 inhibitor SKF-96365 and PI3K inhibitor effect of LY294002 on high glucose induced podocyte injury. Immunohistochemistry in renal tissue WT-1, Nephrin, Podocin, Desmin, VDR and p-Akt; immunofluorescence detection of the expression of TRPC6 and Nephrin in podocyte, Podocin, Desmin and TRPC6; Real-time PCR Western Blot VDR, renal tissue detection, podocyte protein Nephrin, Podocin, The expression of TRPC6, Desmin and PI3K/p-Akt signal pathway related molecular markers podocytes. Results (1) in vivo, compared with the DN group, DN+VD group, urinary protein decreased up to 36%, reduce the renal pathological damage, reduce blood glucose, podocyte injury, no difference between body mass and blood urea nitrogen (P0.05); serum creatinine calcium, phosphorus, there was no difference between the four groups (all P0.05). Compared with NC group, DN group, Podocin, Nephrin expression was significantly decreased (P0.05), the amount of protein expression of Desmin in podocyte injury markers increased significantly (P0.05). Compared with DN group, DN group, +VD Podocin, Nephrin expression was significantly increase (P0.05), the amount of expression of Desmin in podocyte injury markers were significantly lower (P0.05).WT-1 there was no significant difference between the four groups. In vitro studies confirmed that, compared with the control group, high glucose 30mmol/L in a time dependent downregulation of Nephrin and Podocin expression, upregulating the expression of Desmin, And reached the peak at 24h. Compared with the high glucose group, 1,25 (OH) 2D3 showed a concentration dependent upregulation of Nephrin and Podocin expression, down regulating the expression of Desmin, and 100 nmol/L1,25 (OH) concentration of 2D3 have the best effect. The experimental results showed that podocyte injury is characteristic of early diabetic nephropathy, active vitamin D3 the protective effect of high glucose induced podocyte injury. (2) to further explore the role of Nephrin-PI3K/p-Akt signaling pathway in the activity of vitamin D3 protects podocytes found that compared with the NC group, DN group, PI3K-p85, p-Akt expression was significantly decreased (P0.05). Compared with DN group, DN+VD group, PI3K-p85, p-Akt expression increased significantly (P0.05), correlation analysis showed that Nephrin was significantly correlated with PI3K-p85 and p-Akt (r=0.736, P0.05; r=0.855, P0.05). High glucose foot cells after 24h, PI3K, p-Akt protein expression was significantly reduced to 1,25 (OH 2D3) significantly increased PI3K, p-Akt protein expression, but had no effect on the expression of total Akt protein, 1,25 (OH) PI3K inhibitor LY294002 2D3, podocyte injury has yet to be improved, similar to the high glucose group. (3) found to observe the expression change of a key target of TRPC6 the damage of active vitamin D3 treatment of diabetic kidney disease podocyte, compared with NC group, DN group, VDR expression was significantly decreased (P0.05), and the expression of TRPC6 was significantly increased. Compared with the DN group, DN+VD group, VDR expression was significantly increased (P0.05), and down-regulation of TRPC6 (P0.05). Correlation analysis showed that TRPC6 and Nephrin (r=-0.752, P0.05), VDR (r=-0.685, P0.05) was negatively correlated with 24 hours urine protein (r=0.739, P0.05), Desmin (r=0.947, P0.05) were positively correlated to high glucose. Podocyte 24h, expression of VDR mRNA and protein significantly reduced expression of TRPC6 protein and mRNA increased significantly, given the 1,25 (OH 2D3) significantly increased VDR, reduced the expression of TRPC6. With the TRPC6 inhibitor SKF96365 intervention, podocyte injury improved, accompanied by PI3K, increase the expression of p-Akt. Conclusion: 1) the podocyte injury is the typical pathological features of early diabetic nephropathy, active vitamin D3 has a protective effect of.2 on high glucose under foot cell injury) high glucose environment, Nehrin-PI3K/p-Akt signaling pathway mediated down-regulation of podocyte injury, the activity of vitamin D3 can reduce podocyte injury by stabilizing the signaling pathway, so as to relieve the progress of.3 DN) high glucose increased expression of TRPO6 plays an important role in podocyte injury in the process of blocking high expression of THPO6 the activity of vitamin D3 may be a key step in regulating Nephrin-PI3K-p-Akt signaling pathway play a protective effect of podocytes.

【学位授予单位】：东南大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R587.2;R692.9

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