【作者】 王沛；

【导师】 刘章锁；

【作者基本信息】 郑州大学 ， 内科学， 2010， 博士

【摘要】 持续低氧状态促进肾脏纤维化进展的观点已被学界广泛接受。低氧促进CKD进展的作用主要通过低氧诱导因子(Hypoxia inducible factor, HIF)/低氧反应元件(hypoxia responsive element,HRE)途径。HIF-1是在低氧状态下发挥活性的特异性转录因子,由低氧敏感的α亚单位和结构性的p亚单位组成的异二聚体。HIF-1α在低氧条件下进入细胞核内,与β亚单位组成稳定的HIF-1异二聚体,与HRE结合并启动靶基因转录而发挥生物学效应。持续稳定表达HIF-1α的基因敲除小鼠肾脏纤维化加重,提示HIF-1α是二个新的调节肾脏纤维化的因子。传统上认为蛋白尿是慢性肾脏病(Chronic Kideny Disease,CKD)进展的最重要危险因素之一,与CKD患者的生存率及肾存活率都有明确的相关性。持续蛋白尿可导致严重的小管间质损害及肾脏疾病的进展,做为蛋白尿的主要成分——白蛋白对小管间质损害起到决定性作用。肾小球疾病导致尿中增加的白蛋白在肾小管重吸收和分解的过程中消耗大量能量,而由于肾脏本身结构的因素,肾小管所在区域的氧分压又比较低,故临床上低氧张力常同白蛋白过负荷同时存在,因此很自然考虑到白蛋白过负荷与低氧张力在促进肾脏纤维化方面是否存在某些联系。事实上,HIF-1的很多下游基因在白蛋白过负荷诱导的肾小管上皮细胞损伤中发挥重要作用,如CTGF和TIMP-1等。白蛋白诱导肾小管上皮细胞表达CTGF和TIMP-1,进而促进胶原蛋白的合成,并抑制胶原蛋白的降解。基于以上背景,本研究假设白蛋白通过HIF/HRE途径启动靶基因转录,发挥致肾小管上皮细胞损伤的作用,设计如下：首先在构建HRE-Luc报告基因质粒的基础上,检测无脂肪酸的牛血清白蛋白(BSA)对大鼠肾小管上皮细胞系(NRK-52E)HIF/HRE转录活性的影响；然后观察BSA对NRK-52E细胞凋亡、前纤维化因子的合成和转分化三个方面的影响,并应用RNAi技术沉默HIF-1α,深入探讨HIF-1α在其中的作用。目的应用HRE报告基因质粒检测不同浓度白蛋白孵育不同时间对NRK-52E细胞HIF/HRE转录活性的影响。方法pGL3-Epo-HRE-Luc报告基因质粒转染的NRK-52E细胞在含BSA(0,5,10及20mg/ml)的培养基中孵育24h,48h及72h,双荧光素酶法检测各组的相对荧光强度,Western Blotting法检测相应条件下HIF-1 a的表达。结果小剂量(5mg/ml) BSA孵育24h就能够增加NRK-52E细胞的HIF/HRE转录活性,并且HIF/HRE转录活性随BSA剂量增加和刺激时间延长而增强。BSA(20mg/ml)呈时间依赖性增强NRK-52E细胞HIF-1 a蛋白表达。小结表明BSA增强肾小管上皮细胞HIF/HRE转录活性。实验一shRNAHIF-1。转染对BSA过负荷致肾小管上皮细胞凋亡的影响目的探讨BSA过负荷对NRK-52E细胞凋亡的影响及HIF-1 a在其中的作用。方法NRK-52E细胞在含不同浓度BSA的培养基中孵育不同时间的凋亡情况应用TUNEL和Annexin V-FITC/PI双染流式细胞学技术检测,同时应用Western Blotting方法检测Bax蛋白表达。然后观察应用shRNA沉默HIF-1 a对NRK-52E细胞凋亡的影响。结果抗BSA抗体与dUTP双染细胞免疫荧光技术检测到NRK-52E细胞在含BSA20mg/ml的培养基中发生凋亡细胞的位置与摄取BSA细胞的位置一致；流式细胞学技术检测到低浓度BSA(5mg/ml)对NRK-52E细胞凋亡影响较小,而高浓度(>10mg/ml)长时间(>48h)刺激则显著加重凋亡；Western Blotting方法检测NRK-52E细胞在BSA(20mg/ml,72h)刺激下总Bax蛋白表达无显著改变,而6A7单克隆抗体检测到的活化Bax蛋白表达显著增高。shRNA-Hiflα转染细胞在BSA(20mg/ml,72h)刺激下的凋亡率和活化Bax蛋白表达显著低于空质粒转染的细胞。小结BSA诱导NRK-52E细胞凋亡需要较大浓度和较长刺激时间,HIF/HRE转录活性增加参与了较大剂量BSA诱导的NRK-52E细胞凋亡。目的探讨巨噬细胞趋化因子-1(MCP-1)、结缔组织生长因子(CTGF)和组织型金属蛋白酶组织抑制剂-1(TIMP-1)等前纤维化因子在BSA诱导NRK-52E细胞合成细胞外基质中的作用,及HIF-1 a在其中的作用。方法NRK-52E细胞在含不同浓度BSA的培养基中孵育不同时间,层粘连蛋白(FN)、MCP-1、CTGF和TIMP-1的表达应用Western Blotting、RT-PCR和ELISA等方法检测。结果较低浓度BSA(5mg/ml)在较短的刺激时间(24h)即诱导NRK-52E细胞MCP-1蛋白高表达,BSA浓度愈高、刺激时间愈长,MCP-1表达愈高；在BSA10mg/ml刺激72h时,NRK-52E细胞FN,CTGF和TIMP-1的表达显著高于BSA(Omg/ml)组。shRNA-Hifl a转染细胞在BSA(10mg/ml,72h)刺激下FN,MCP-1,CTGF和TIMP-1的表达显著低于空质粒转染的细胞。小结BSA呈剂量和时间依赖性诱导NRK-52E细胞前纤维化因子和细胞外基质蛋白的表达,并且这种作用部分通过HIF/HRE途径实现。目的探讨BSA对NRK-52E细胞转分化的影响,及HIF-1α在其中的作用。方法NRK-52E细胞在含不同浓度BSA的培养基中孵育不同时间,细胞间紧密连接(Z0-1)、α平滑肌肌动蛋白(α-SMA)、转化生长因子β1(TGF-β1)的表达应用Western Blotting、RT-PCR和ELISA等方法检测。结果在BSA 10mg/ml刺激72h时,NRK-52E细胞ZO-1表达显著高于BSA(Omg/ml)组,而α-SMA则显著高于BSA(Omg/ml)组,shRNA-Hifl a转染部分逆转BSA的作用。在BSA10mg/ml刺激24h,48h和72h时,NRK-52E细胞TGF-β1蛋白含量都显著高于BSA(Omg/ml)组,shRNA-Hifl a转染细胞TGF-β1蛋白含量则显著低于空质粒转染对照组。小结较小剂量的BSA即可诱导NRK-52E细胞转分化为肌成纤维细胞及TGF-β1高表达,HIF-1α在其中发挥重要作用。结论BSA增强NRK-52E细胞HIF/HRE转录活性；BSA诱导NRK-52E细胞凋亡、转分化及前纤维化因子高表达等损伤至少部分是通过激活HIF-1α途径而发挥更多还原

【Abstract】 It’s well been accepted by the majority of nephrologists that renal fibrosis could be accelerated by sustained hypoxia. Hypoxia promoted chronic kidney disease (CKD) through Hypoxia inducible factor/hypoxia responsive element (HIF/HRE) pathway. As a specific transcription factor stimulated by low oxygen tension, HIF-1 was composed by two subunits, the oxygen sensitive (HIF-1 a) and the constitutive (HIF-1β) subunit. Under low oxygen condition, stable HIF-1αsubunit translocated into the nucleus, where it dimerized with HIF-1 P subunit, recognized and combined with HRE, and then triggered a number of target genes transcription. It’s proved that HIF-1αmight be a new regulator of renal fibrosis by the evidence that interstitial fibrosis was promoted by sustained and stable expression of HIF-1 a in tubular epithelial cells of gene knock-out mice.Early, it’s been believed that proteinuria was the key factor accelerating renal fibrosis, and that it had a direct-propotional relationship with CKD patients’kidney survival.Sustained proteinuria induced severe tubulointerstitial damage and disease progression. Albumin was the key protein leading to tubulointerstitial damage as albumin comprises the major portion of proteinuria. Large amount of energy was consumed in the process of increased albumin reabsorption and dissolution in tubular epithelial cells (TEC).Another wise, peritubule low oxygen tension existed because of kidney structure weakness. Thus, low oxygen tension along with albumin overload might have a positive relation with the pathogenesis of renal fibrosis. In fact, many target genes of HIF-1,such as Connective Tissue Growth Factor(CTGF) and Tissue Inhibitor of Metalloproteinases-1 (TIMP-1),played crucial roles in the pathogenesis of tubular damage resulted from albumin overload. CTGF and TIMP-1 overexpression induced by albumin in TEC increased the production of extra cellular matrix (ECM) and decreased their degeneration.Based on these evidences, we assumed that it’s through the HIF/HRE pathway that bovine serum albumin (BSA) induced TEC damage, then triggered target genes expression and promoted renal fibrosis. Experiment protocol:the effects of fatty acid free bovine serum albumin (BSA) overload on rat NRK-52E cells HIF/HRE transcription activity was detected by a HRE-Luc reporter plasmid. And then, the effects of BSA overload on NRK-52E cells apoptosis, profibrosis factors expression and epithelial-myofibroblast transdifferentiation (EMT) were observed, and the role of HIF-1αin these pathological phenomenons was explored by silence HIF-la with RNAi technique.Objective To explore the effects of BSA at varying concentration in varying stimulus duration on HIF/HRE transcription activity of NRK-52E cells with HRE-Luc reporter plasmid. Methods Luciferase activity NRK-52E cells incubated in a medium contained BSA in varying concentration (0,5,10 or 20mg/ml) and stimulus duration (24h,48h or 72h) was detected by dual luciferase detecting system based on HRE-Luc reporter plasmid and HIF-la expression was detected by Western Blotting. Results HIF/HRE transcription activity of NRK-52E cells was increased by BSA at a relatively low concentration, and increased depended on BSA concentration and incubating duration. HIF-la expression was also increased depended on BSA concentration and incubating duration. Conclusions Our study showed that albumin increased the HIF/HRE transcription activity of TEC.Objective To explore the role HIF-la in BSA induced NRK-52E cells apoptosis. Methods NRK-52E cells apoptosis induced by BSA at varying concentration (0,5, 10 or 20mg/ml) in varying stimulus duration (24h,48h or 72h) was detected by TUNEL and Annexin V-FITC/PI dual labeling flow cytometry, as well as, Bax expression was detected by Western Blotting. And then, we observed the effects of shRNA-HIF-la transfection on BSA induced NRK-52E cells apoptosis.Results A large proportion of the cells that were positive for albumin staining also displayed positive dUTP-FITC staining of their nuclei.A low concentration of BSA (5mg/ml) had little effects on NRK-52E cells apoptosis, however, BSA at a higher concentration(>10mg/ml) or in a longer duration (>48h) promoted apoptosis. BSA (20mg/ml,72h) had no effects on total Bax expression, but elevated active Bax expression detected by 6A7 monoclone antibody. After incubated in a medium contained BSA at a concentration of 20mg/ml incubated for 72h, apoptosis cell ratio and Bax expression in shRNA-HIF-1αtransfected NRK-52E cells were lower than that in naked plasmid transfected cells. Conclusions Our study showed that a relatively higher concentration and longer incubating duration were needed for BSA induced NRK-52E cells apoptosis, and HIF/HRE transcription pathway took part in BSA induced NRK-52E cells apoptosis.Objective Profibrosis factors, such as MCP-1,CTGF and TIMP-1,played crucial role in BSA induced ECM production in NRK-52E cells. This study was designed to explore the role of HIF-1αin BSA induced profibrosis factors expression in NRK-52E cells.Methods Expressions of Fibronectin(FN), MCP-1,CTGF and TIMP-1 in NRK-52E cells induced by BSA in varying concentration and stimulus duration was detected by Western Blotting, RT-PCR or ELISA. Results MCP-1 production in NRK-52E cells was incerased by BSA at a low concentration of 5mg/ml in a short duration of 24h. BSA increased MCP-1 production in NRK-52E cells in a dosage and duration dependent manner. BSA(10mg/ml,72h) induced higher expression of FN, CTGF and TIMP-1 than BSA(0mg/ml,72h).After incubated in a medium contained BSA at a concentration of 10mg/ml incubated for 72h,FN,MCP-1, CTGF and TIMP-1 expression in shRNA-HIF-la transfected NRK-52E cells were lower than that in naked plasmid transfected cells. Conclusions Our study showed that BSA increased these profibrosis factors expression in NRK-52E cells in a dosage and duration dependent manner, and this effect partially depended on HIF/HRE pathway.Objective To explore the role HIF-1αin BSA induced NRK-52E cells EMT. Methods Expressions of tight junction protein ZO-1,myofibroblast marker a-smooth muscle actin (a-SMA) and transforming growth factorβ1(TGF-(31) expression in NRK-52E cells induced by BSA in varying concentration and stimulus duration was detected by Western Blotting, RT-PCR or ELISA. Results BSA (10mg/ml,72h) induced a significantly higher expression of ZO-1 and a lower expression of a-SMA than BSA (0mg/ml,72h), and these effects were partially reversed by shRNA-HIF-1αtransfection. BSA (10mg/ml) induced a significantly higher production of TGF-β1 than BSA (Omg/ml) in varying duration (24h,48h or 72h), and these effects were partially reversed by shRNA-HIF-1αtransfection.Conclusions Our study showed that NRK-52E cells EMT and TGF-β1 overexpression were induced by BSA, and HIF-1αplayed a crucial role in this pathological phenomenon. Above all, all these study showed that albumin increased the HIF/HRE transcription activity of TEC, and indicated that HIF-1αat least partially involved in the effects of BSA on NRK-52E cells damage.更多还原