本文關鍵詞：HIF-1α在早期糖尿病腎臟纖維化易感性中的作用機制研究　出處：《第二軍醫(yī)大學》2017年博士論文　論文類型：學位論文

  更多相關文章： 高糖 糖尿病腎病 纖維化 HIF1α

【摘要】：研究背景及目的糖尿病腎病(diabetic nephropathy,DN)作為糖尿病的嚴重并發(fā)癥,已經(jīng)成為導致終末期腎病(end-stage renal disease,ESRD)的主要病因之一。據(jù)報道,在發(fā)達國家,糖尿病腎病是導致慢性腎臟疾病(chronic kidney disease,CKD)的首要原因。而在我國,糖尿病腎病是僅次于慢性腎小球腎炎導致CKD的第二大因素。這主要由兩方面造成:一是糖尿病發(fā)病率日趨升高以及發(fā)病年齡年輕化;二是人口老齡化。腎臟纖維化是DN的主要病理表現(xiàn)形式,可能的病理機制包括:(1)高血糖導致血流動力學紊亂和代謝紊亂;(2)轉(zhuǎn)化生長因子、血管內(nèi)皮生長因子等細胞因子的激活;(3)血管活性物質(zhì)代謝異常,比如一氧化氮合酶活性降低導致一氧化氮合成減少;(4)腎臟高濾過導致的氧耗增加以及細胞外基質(zhì)增多導致的氧氣彌散障礙。雖然目前臨床上有大量控制血壓、血糖、血脂及腎素-血管緊張素系統(tǒng)的相關藥物,但是仍舊不能延緩疾病進展。其高致殘率和致死率,給患者家庭和社會帶來了沉重的精神和經(jīng)濟負擔。有趣的是,有部分患者盡管血糖控制較好,仍無法避免進展為ESRD。而有些患者即使血糖控制不佳,腎功能卻能維持在穩(wěn)定的水平。一方面與個體的異質(zhì)性有關;另一方面可能與一些促纖維化始動因素的存在有關。本研究利用鏈脲佐菌素(streptozocin,STZ)誘導的1型糖尿病和Akita自發(fā)性1型糖尿病鼠(6-8周齡)合并單側輸尿管梗阻(unilateral ureteral obstruction,UUO)。研究結果證實,同樣是UUO手術,糖尿病組小鼠腎臟損傷程度明顯高于非糖尿病組,包括纖維化、細胞凋亡和炎細胞浸潤。體外實驗同樣證實,高糖刺激下的小鼠腎小管上皮細胞在缺氧條件下fibronectin蛋白表達量高于其他各組。從這些現(xiàn)象可以看出,無論是體內(nèi)還是體外條件下,高糖組更易于發(fā)生纖維化。那么究竟是何種因素促使這種現(xiàn)象發(fā)生?其潛在的作用機制是什么?為了闡述這個問題,我們將目光聚焦于明星分子--缺氧誘導因子(hypoxia inducible factors,HIFs)。腎臟血流大約占心輸出總量的20%,但是由于腎臟特殊的生理解剖結構以及豐富的代謝活動,導致腎臟極易出現(xiàn)缺氧。缺氧誘導因子是機體組織為適應缺氧而產(chǎn)生的一類因子,主要包括HIF1、HIF2和HIF3,其中研究較多的為前兩種。HIF1和HIF2(統(tǒng)稱為HIF)屬于PAS(per/arylhydrocarbon-receptor nuclear translocator/Sim,PAS)家族,是含有堿性螺旋-環(huán)-螺旋結構(basic helix-loop-helix,bHLH)的一類轉(zhuǎn)錄因子。HIF是一種異原二聚體蛋白,包括α亞基β亞基。由于β亞基結構與芳香羥受體核轉(zhuǎn)位蛋白(hydrocarbon-receptor nuclear translocator,ARNT)結構相同,故又稱為ARNT。正常情況下,hifβ亞基在細胞漿中穩(wěn)定表達,hifα在翻譯后即被泛素-蛋白酶體復合物降解。當機體處于缺氧環(huán)境時,hifα無法經(jīng)泛素化途徑降解,轉(zhuǎn)移到細胞核中,與hifβ以及cbp/p300結合形成穩(wěn)定復合物,調(diào)控下游相關靶基因表達,比如調(diào)節(jié)血管生成、紅細胞生成、糖酵解、細胞增殖、細胞凋亡,調(diào)整氧氣的分布和提高機體對缺氧的耐受力。腎臟包含有多種細胞類型,并且在表達hif方面存在差異。研究證實hif1主要在腎小管和腎小球上皮細胞內(nèi)表達,而hif2主要表達于血管內(nèi)皮細胞和間質(zhì)纖維化細胞中。有許多研究表明hif在調(diào)控多種腎臟疾病具有重要作用,比如缺血/再灌注相關急性腎損傷,5/6腎切除導致的慢性腎臟病等。對于hif在調(diào)控糖尿病腎病纖維化方面所起的作用,目前仍存在諸多爭議。nayak等學者證實糖尿病動物模型引起的腎小球及間質(zhì)纖維化中有hif表達,rosenberger等學者同樣發(fā)現(xiàn)hif1α不僅表達于糖尿病動物模型,在糖尿病腎病患者的病理標本中同樣有hif1α的表達。本研究證實hif1α在早期糖尿病纖維化中高表達,在一定程度上反映了hif1α可能在調(diào)控早期糖尿病纖維化方面起一定作用。研究方法首先,利用stz誘導的1型糖尿病小鼠模型和c57bl/6j-ins2akita/+小鼠模型(由于6-8周齡年齡小鼠血糖升高僅有2-3周,可視為早期糖尿病模型),進行uuo手術,通過馬松三色染色及四型骨膠原熒光染色觀察腎臟組織的骨膠原沉積,利用westernblot觀察纖維化指標和hif1α表達情況;利用巨噬細胞markerf4/80染色來評估腎臟組織內(nèi)的炎癥浸潤情況;腎臟組織細胞的凋亡主要依賴原位末端轉(zhuǎn)移酶標記法(tdt-mediateddutpmick-endlabeling,tunel)。同時,運用體外細胞模型--小鼠腎小管上皮細胞(themouseptcell,bumpt-306cell),進一步證實hif1α在缺氧條件下高糖組的表達情況。明確hif1α是否在糖尿病早期參與腎臟纖維化的形成。其次,利用hif1α的特異性抑制劑3-(5'-羥甲基-2'-呋喃基)-1-苯甲基吲唑{3-(5-hydroxymethl-2-furyl)-1-benzylindazole,yc-1}干預細胞,通過westernblot觀察其對hif1α和纖維化指標表達的影響。另外利用hif1αshrna轉(zhuǎn)染大鼠腎小管上皮細胞(renalproximaltubularcell,rptc)和人胚腎細胞(humanembryonickidney,hek)293,沉默hif1α在上述細胞系內(nèi)的表達,進一步觀察hif1α和纖維化指標的表達。最后,利用hif抑制劑yc-1干預行uuo后的akita糖尿病小鼠,觀察纖維化相關指標及hif1α的表達。為了深入研究hif1α的作用,我們利用近端小管hif1α特異性敲除(pt-hif1αko)小鼠和對照組(pt-hif1αwt)小鼠,連續(xù)5天低劑量注射stz誘導1型糖尿病模型,觀察發(fā)現(xiàn)pt-hif1αko小鼠較PT-HIF1αWT小鼠而言,纖維化程度及腎損傷程度均較輕,進一步證實HIF1α與早期糖尿病腎病纖維化敏感性的關系。研究結果(1)Akita小鼠和STZ誘導的1型糖尿病小鼠分別行UUO手術后,腎臟組織內(nèi)骨膠原沉積呈時間依賴性上升,纖維化指標fibronectin,α-SMA表達上升,同時伴隨HIF1α表達升高,且糖尿病組明顯高于同期對照組。此外,糖尿病組腎臟組織細胞凋亡程度明顯高于同期對照組,炎癥指標F4/80結果顯示手術后炎癥細胞浸潤明顯增加,且糖尿病組高于對照組。(2)HIF1α的特異性抑制劑YC-1干預BUMPT細胞,HIF1α表達下調(diào)的RPTC和HEK293細胞系都顯示一致結果,fibronectin的表達降低,且降低程度與HIF1α表達抑制程度一致。(3)YC-1干預UUO后的Akita糖尿病小鼠模型及PT-HIF1αKO和PTHIF1αWT糖尿病小鼠模型顯示,抑制HIF1α不僅可以降低骨膠原沉積、細胞外基質(zhì)纖維化等指標的表達,還可以減少腎臟組織細胞凋亡和間質(zhì)炎癥細胞浸潤。結論本課題通過體內(nèi)和體外實驗證實了早期高糖可提高腎臟對纖維化的敏感性,并且這種易感性受HIF1α的調(diào)節(jié)。
[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.

【學位授予單位】：第二軍醫(yī)大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：R587.2;R692.9

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