HIF-1α在早期糖尿病肾脏纤维化易感性中的作用机制研究

发布时间：2018-01-01 14:16

本文关键词：HIF-1α在早期糖尿病肾脏纤维化易感性中的作用机制研究　出处：《第二军医大学》2017年博士论文　论文类型：学位论文

【摘要】：研究背景及目的糖尿病肾病(diabetic nephropathy,DN)作为糖尿病的严重并发症,已经成为导致终末期肾病(end-stage renal disease,ESRD)的主要病因之一。据报道,在发达国家,糖尿病肾病是导致慢性肾脏疾病(chronic kidney disease,CKD)的首要原因。而在我国,糖尿病肾病是仅次于慢性肾小球肾炎导致CKD的第二大因素。这主要由两方面造成:一是糖尿病发病率日趋升高以及发病年龄年轻化;二是人口老龄化。肾脏纤维化是DN的主要病理表现形式,可能的病理机制包括:(1)高血糖导致血流动力学紊乱和代谢紊乱;(2)转化生长因子、血管内皮生长因子等细胞因子的激活;(3)血管活性物质代谢异常,比如一氧化氮合酶活性降低导致一氧化氮合成减少;(4)肾脏高滤过导致的氧耗增加以及细胞外基质增多导致的氧气弥散障碍。虽然目前临床上有大量控制血压、血糖、血脂及肾素-血管紧张素系统的相关药物,但是仍旧不能延缓疾病进展。其高致残率和致死率,给患者家庭和社会带来了沉重的精神和经济负担。有趣的是,有部分患者尽管血糖控制较好,仍无法避免进展为ESRD。而有些患者即使血糖控制不佳,肾功能却能维持在稳定的水平。一方面与个体的异质性有关;另一方面可能与一些促纤维化始动因素的存在有关。本研究利用链脲佐菌素(streptozocin,STZ)诱导的1型糖尿病和Akita自发性1型糖尿病鼠(6-8周龄)合并单侧输尿管梗阻(unilateral ureteral obstruction,UUO)。研究结果证实,同样是UUO手术,糖尿病组小鼠肾脏损伤程度明显高于非糖尿病组,包括纤维化、细胞凋亡和炎细胞浸润。体外实验同样证实,高糖刺激下的小鼠肾小管上皮细胞在缺氧条件下fibronectin蛋白表达量高于其他各组。从这些现象可以看出,无论是体内还是体外条件下,高糖组更易于发生纤维化。那么究竟是何种因素促使这种现象发生?其潜在的作用机制是什么?为了阐述这个问题,我们将目光聚焦于明星分子--缺氧诱导因子(hypoxia inducible factors,HIFs)。肾脏血流大约占心输出总量的20%,但是由于肾脏特殊的生理解剖结构以及丰富的代谢活动,导致肾脏极易出现缺氧。缺氧诱导因子是机体组织为适应缺氧而产生的一类因子,主要包括HIF1、HIF2和HIF3,其中研究较多的为前两种。HIF1和HIF2(统称为HIF)属于PAS(per/arylhydrocarbon-receptor nuclear translocator/Sim,PAS)家族,是含有碱性螺旋-环-螺旋结构(basic helix-loop-helix,bHLH)的一类转录因子。HIF是一种异原二聚体蛋白,包括α亚基β亚基。由于β亚基结构与芳香羟受体核转位蛋白(hydrocarbon-receptor nuclear translocator,ARNT)结构相同,故又称为ARNT。正常情况下,hifβ亚基在细胞浆中稳定表达,hifα在翻译后即被泛素-蛋白酶体复合物降解。当机体处于缺氧环境时,hifα无法经泛素化途径降解,转移到细胞核中,与hifβ以及cbp/p300结合形成稳定复合物,调控下游相关靶基因表达,比如调节血管生成、红细胞生成、糖酵解、细胞增殖、细胞凋亡,调整氧气的分布和提高机体对缺氧的耐受力。肾脏包含有多种细胞类型,并且在表达hif方面存在差异。研究证实hif1主要在肾小管和肾小球上皮细胞内表达,而hif2主要表达于血管内皮细胞和间质纤维化细胞中。有许多研究表明hif在调控多种肾脏疾病具有重要作用,比如缺血/再灌注相关急性肾损伤,5/6肾切除导致的慢性肾脏病等。对于hif在调控糖尿病肾病纤维化方面所起的作用,目前仍存在诸多争议。nayak等学者证实糖尿病动物模型引起的肾小球及间质纤维化中有hif表达,rosenberger等学者同样发现hif1α不仅表达于糖尿病动物模型,在糖尿病肾病患者的病理标本中同样有hif1α的表达。本研究证实hif1α在早期糖尿病纤维化中高表达,在一定程度上反映了hif1α可能在调控早期糖尿病纤维化方面起一定作用。研究方法首先,利用stz诱导的1型糖尿病小鼠模型和c57bl/6j-ins2akita/+小鼠模型(由于6-8周龄年龄小鼠血糖升高仅有2-3周,可视为早期糖尿病模型),进行uuo手术,通过马松三色染色及四型骨胶原荧光染色观察肾脏组织的骨胶原沉积,利用westernblot观察纤维化指标和hif1α表达情况;利用巨噬细胞markerf4/80染色来评估肾脏组织内的炎症浸润情况;肾脏组织细胞的凋亡主要依赖原位末端转移酶标记法(tdt-mediateddutpmick-endlabeling,tunel)。同时,运用体外细胞模型--小鼠肾小管上皮细胞(themouseptcell,bumpt-306cell),进一步证实hif1α在缺氧条件下高糖组的表达情况。明确hif1α是否在糖尿病早期参与肾脏纤维化的形成。其次,利用hif1α的特异性抑制剂3-(5'-羟甲基-2'-呋喃基)-1-苯甲基吲唑{3-(5-hydroxymethl-2-furyl)-1-benzylindazole,yc-1}干预细胞,通过westernblot观察其对hif1α和纤维化指标表达的影响。另外利用hif1αshrna转染大鼠肾小管上皮细胞(renalproximaltubularcell,rptc)和人胚肾细胞(humanembryonickidney,hek)293,沉默hif1α在上述细胞系内的表达,进一步观察hif1α和纤维化指标的表达。最后,利用hif抑制剂yc-1干预行uuo后的akita糖尿病小鼠,观察纤维化相关指标及hif1α的表达。为了深入研究hif1α的作用,我们利用近端小管hif1α特异性敲除(pt-hif1αko)小鼠和对照组(pt-hif1αwt)小鼠,连续5天低剂量注射stz诱导1型糖尿病模型,观察发现pt-hif1αko小鼠较PT-HIF1αWT小鼠而言,纤维化程度及肾损伤程度均较轻,进一步证实HIF1α与早期糖尿病肾病纤维化敏感性的关系。研究结果(1)Akita小鼠和STZ诱导的1型糖尿病小鼠分别行UUO手术后,肾脏组织内骨胶原沉积呈时间依赖性上升,纤维化指标fibronectin,α-SMA表达上升,同时伴随HIF1α表达升高,且糖尿病组明显高于同期对照组。此外,糖尿病组肾脏组织细胞凋亡程度明显高于同期对照组,炎症指标F4/80结果显示手术后炎症细胞浸润明显增加,且糖尿病组高于对照组。(2)HIF1α的特异性抑制剂YC-1干预BUMPT细胞,HIF1α表达下调的RPTC和HEK293细胞系都显示一致结果,fibronectin的表达降低,且降低程度与HIF1α表达抑制程度一致。(3)YC-1干预UUO后的Akita糖尿病小鼠模型及PT-HIF1αKO和PTHIF1αWT糖尿病小鼠模型显示,抑制HIF1α不仅可以降低骨胶原沉积、细胞外基质纤维化等指标的表达,还可以减少肾脏组织细胞凋亡和间质炎症细胞浸润。结论本课题通过体内和体外实验证实了早期高糖可提高肾脏对纤维化的敏感性,并且这种易感性受HIF1α的调节。
[Abstract]:Background and objective: diabetic nephropathy (diabetic nephropathy DN) is a serious complication of diabetes mellitus, has become the leading cause of end-stage renal disease (end-stage renal, disease, ESRD) is one of the main causes. It is reported that in developed countries, diabetic nephropathy is the leading cause of chronic kidney disease (chronic kidney, disease, CKD). The primary reason in my in China, diabetic nephropathy is a chronic glomerulonephritis after the second major factors leading to CKD. This is mainly caused by two aspects: one is the incidence of diabetes is rising and younger age of onset; two is the aging of the population. Renal fibrosis is the main pathological manifestations of DN, including the possible pathological mechanisms: (1) high blood lead hemodynamic disorder and metabolic disorders; (2) transforming growth factor activation, vascular endothelial growth factor and other cytokines; (3) vasoactive substances such as nitric oxide metabolism. Reduce the synthase activity leads to reduced nitric oxide synthesis; (4) the renal filtration leads to increased oxygen consumption and extracellular matrix increased oxygen diffusion barriers caused. Although there are a lot of blood pressure control, blood glucose and blood lipid in clinic at present, renin angiotensin system hormone related drugs, but still can not delay the progression of the disease. The high disability and death rates, brought heavy mental and economic burden to patients, families and society. Interestingly, although some patients with good glycemic control, still can not avoid progress to ESRD. and some even patients with poor glycemic control, the renal function was maintained at a stable level. On the one hand, heterogeneity and individual; on the other hand may be associated with some initial profibrotic factors exist. This study used streptozotocin (streptozocin, STZ) - induced type 1 diabetes and Akita spontaneous type 1 diabetes rats (6-8 Week old) with unilateral ureteral obstruction (unilateral ureteral obstruction, UUO). The results confirmed that UUO is the same operation, the degree of renal injury in diabetic group were significantly higher than that in non-diabetic group, including fibrosis, apoptosis and inflammatory cell infiltration. In vitro experiments also confirmed that the mouse renal tubular epithelial cells stimulated by high glucose was higher than that of the other in each group of fibronectin protein under anoxic conditions. From these phenomena can be seen either in vivo or in vitro under the condition of high glucose group were more prone to fibrosis. So what exactly is what factors lead to this phenomenon? What is the underlying mechanism? To address this issue, we will star molecules -- focus (hypoxia inducible factor hypoxia inducible factors, HIFs). Renal blood flow accounted for about 20% of the total output of the heart, but because of the special students understand kidney structure and cesarean section The metabolic activity of the rich, causes the kidney prone to hypoxia. Hypoxia inducible factor is a factor of body tissue and to adapt to hypoxia, mainly including HIF1, HIF2 and HIF3, more research for the first two types of.HIF1 and HIF2 (referred to as HIF) belongs to PAS (per/ arylhydrocarbon-receptor nuclear translocator/Sim, PAS) family. There is a basic helix loop helix structure (basic helix-loop-helix bHLH) is a transcription factor.HIF is a different original two dimeric protein, including alpha subunit beta subunit. The beta subunit structure and aryl hydrocarbon receptor nuclear translocator (hydrocarbon-receptor nuclear, translocator, ARNT) the same structure, so it is also called ARNT. normally, HIF beta subunit expression in the cytoplasm, HIF alpha is the ubiquitin proteasome complex degradation in translation. When the body is in a hypoxic environment, the HIF alpha ubiquitination pathway to degradation. Move to the nucleus, combined with the HIF beta and cbp/p300 to form stable complexes, regulate expression of downstream target genes, such as regulating erythropoiesis, angiogenesis, glycolysis, cell proliferation, cell apoptosis, and improve the distribution of adjusting oxygen tolerance to hypoxia. The body kidney contains a variety of cell types, and there the expression of HIF in difference. The study confirmed that HIF1 mainly expressed in renal tubular and glomerular epithelial cells, whereas hif2 is mainly expressed in vascular endothelial cells and interstitial cells. Many studies have indicated that HIF plays an important role in the regulation of a variety of kidney diseases, such as ischemia / reperfusion for acute kidney injury, resulting in 5/6 nephrectomy the chronic kidney disease. For HIF in the regulation of the role of fibrosis in diabetic nephropathy, there are still many disputes.Nayak scholars confirmed that the animal model of diabetes induced renal small The ball and interstitial fibrosis in the expression of HIF, Rosenberger and other scholars also found HIF1 expressed not only in animal models of diabetes mellitus, the pathological specimens in patients with diabetic nephropathy in the same expression of HIF1. This study demonstrated that HIF1 a high expression in early diabetic fibrosis, to a certain extent reflects HIF1 alpha may play a role in the regulation of early diabetic fibrosis. The first research method, using the model and c57bl/6j-ins2akita/+ mice with type 1 diabetes mice model induced by STZ (6-8 weeks old age mice due to blood glucose increased only 2-3 weeks, can be regarded as the early diabetic model), UUO operation, through the Masson trichrome stain and type four collagen fluorescence staining in kidney tissue collagen deposition, observation of fibrosis and HIF1 alpha expression by Westernblot; using markerf4/80 staining to evaluate the macrophages in the kidney tissue inflammation In the infiltration; apoptosis of kidney cells depends mainly on the TDT mediated dUTP nick end labeling method (tdt-mediateddutpmick-endlabeling, TUNEL). At the same time, the use of renal tubular epithelial cells in vitro cell model mice (themouseptcell, bumpt-306cell), further confirmed the expression of HIF1 alpha glucose under hypoxia group. Whether in the form of clear HIF1 alpha early in diabetes kidney fibrosis. Secondly, using a specific inhibitor of 3- HIF1 alpha (5'- hydroxymethyl -2'- furyl) -1- phenyl methyl indazole {3- (5-hydroxymethl-2-furyl) -1-benzylindazole, yc-1} cells by Westernblot intervention, to observe its effect on expression of HIF1 alpha and fibrosis index. In addition to the use of HIF1 alpha shRNA transfection of rat renal tubular epithelial cells (renalproximaltubularcell, RPTC) and human embryonic kidney cells (humanembryonickidney, HEK) 293, HIF1 silencing in the alpha cells, into the The expression step observed HIF1 alpha and fibrosis index. Finally, using HIF inhibitor YC-1 intervention after UUO Akita diabetic mice, observe the expression indexes related to fibrosis and HIF1 alpha. In order to study the role of HIF1 alpha, we use proximal tubule specific knockout HIF1 alpha (pt-hif1 alpha KO) mice and control group (pt-hif1 a WT) mice, 5 consecutive days of low doses of STZ induced type 1 diabetic model, we found that pt-hif1 KO mice with alpha PT-HIF1 alpha WT mice, the degree of fibrosis and renal damage were mild, further confirmed the relationship between HIF1 alpha and early diabetic nephropathy fibrosis sensitivity. Results (1) and Akita mice STZ induced type 1 diabetic mice were treated with UUO after surgery, renal tissue collagen deposition showed time-dependent increase, fibrosis index fibronectin, -SMA expression increased, accompanied by increased expression of HIF1 alpha, and diabetic group Significantly higher than the control group. In addition, diabetic renal cell apoptosis was significantly higher than those in the control group, inflammation index F4/80 showed postoperative inflammatory cell infiltration increased significantly, and the diabetic group than control group. (2) cell specific inhibitor YC-1 intervention BUMPT HIF1 alpha, HIF1 expression down-regulation of RPTC and HEK293 cells the department shows consistent results, reduce the expression of fibronectin, and reduce the degree of HIF1 expression inhibition degree. (3) showed that YC-1 intervention after UUO Akita diabetic mice and PT-HIF1 alpha KO and PTHIF1 alpha WT diabetic mice, inhibition of HIF1 can not only reduce the collagen deposition and expression of extracellular matrix fibrosis, also can reduce renal cell apoptosis and interstitial infiltration of inflammatory cells. Conclusion: in vivo and in vitro experiments showed that high glucose can improve the early kidney fibrosis Sensitivity, and this susceptibility is regulated by HIF1 alpha.

【学位授予单位】：第二军医大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R587.2;R692.9

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