二氢生物蝶呤还原酶下调TGFβ1/Smad3信号传导通路参与糖尿病肾病发生发展的实验研究

【作者】 古艳婷；

【导师】 李平；

【作者基本信息】 北京协和医学院 ， 药理学， 2013， 博士

【摘要】 背景：多项流行病学调查显示,糖尿病肾病(diabetic nephropathy, DN)已成为全球慢性肾脏病(chronic kidney disease, CKD)和终末期肾病(end-stage renal disease, ESRD)的主要构成原因。DN的发病机制复杂,仍未完全阐明,目前认为DN的发生与多元醇通路激活、氧化应激、血流动力学改变、多种细胞因子的异常表达及遗传等多种因素密切相关。其中转化生长因子(transforming growth factor betal, TGF-β1)的表达增多是DN发生发展的重要因素,它可以诱导系膜外基质增加以及氧化应激反应从而促进肾小球硬化和纤维化。而TGF-β1作用于效应细胞的信号转导主要是通过激活Smad3通路实现。课题组在前期差异蛋白质组学中发现,二氢生物喋呤还原酶(dihydropteridine reductase, QDPR)在自发性Ⅱ型糖尿病模型OLETF大鼠肾脏损害时,编码此蛋白的基因发生了点突变,QDPR的主要功能是促进四氢生物喋呤(tetrahydrobiopterin, BH4)再生,而BH4又是3种一氧化氮合酶(nitric oxide synthase, NOS)生成的辅助因子继而会影响一氧化氮的合成。一氧化氮(nitric oxide, NO)在体内有着广泛的生理作用,在糖尿病肾病发病过程中它参与了肾小球硬化和氧化应激状态的形成过程,机制与调节TGF β1的生成,舒张血管,降低氧化应激反应,抑制中性粒细胞NADPH氧化酶活性等有关。QDPR作为调节体内BH4水平的关键代谢酶,在DN发生发展中的作用尚未明确。目前还没有文献报道QDPR与DN发生发展之间的关系。本研究通过观察QDPR在糖尿病肾病大鼠中的表达水平,以及野生型和突变型QDPR对TGF β1/Smad3信号通路的影响,深入揭示了DN的发病机制,为探索药物的作用靶点提供线索,研究思路具有较强的新颖性。目的：探讨二氢生物喋呤还原酶(dihydropteridine reductase, QDPR)是否能通过TGF-β1/Smad3信号转导通路参与糖尿病肾病的发生发展过程,为进一步深入了解DN的发生发展过程并为其分子诊断和基因治疗提供新的科学依据。方法：1.QDPR在1型糖尿病肾病大鼠模型肾皮质中表达变化的研究本部分研究首先制备了链脲佐菌素腹腔注射合并单侧肾切除诱导的1型糖尿病肾病的动物模型,20周后将动物处死取肾皮质提取总RNA及蛋白,然后采用RT-PCR和Western blot方法检测正常Wistar大鼠以及模型组大鼠肾皮质QDPR的表达变化,此外我们还检测了正常组和模型组TGF-β1, Smad3和NOXs基因和蛋白水平的变化。2.野生型QDPR基因对TGF β1/Smad3信号通路的影响本部分研究通过构建野生型QDPR重组质粒并转染至细胞,观察了在QDPR高表达时TGF-β/Smad通路以及相关基因的变化,质粒构建的具体步骤如下：根据GeneBank中大鼠QDPR mRNA序列设计引物,上游引物引入EcoR V酶切位点,下游引物引入Xba I酶切位点,以正常LETO大鼠肾脏皮质cDNA为模板,采用高保真PCR试剂盒扩增野生型QDPR cDNA片段,对PCR产物用1.2%琼脂糖凝胶电泳进行分离,分离后克隆至pUM-T载体并测序,然后亚克隆至真核表达载体pcDNA3.1/V5-His,酶切鉴定后测序,如果测序结果正确则证明QDPR/pcDNA3.1/V5-His (rQDPR)重组质粒构建成功。随后用磷酸钙共沉淀法瞬时转染HEK293T细胞建立QDPR高表达的细胞模型,以空载体(control vector)作为对照,转染72h后收集细胞上清和细胞蛋白,先用Western blot方法来验证融合蛋白在细胞中是否成功表达,再采用Western blot和RT-PCR技术检测当QDPR过表达时三种NOS, TGF-β/Smad通路,NADPH氧化酶各亚基的表达量,此外,用ELISA技术检测BH4在空载体和rQDPR组中的含量。另一方面,我们通过敲降实验观察了QDPR在低表达时TGF-p等基因的变化。具体步骤如下：我们首先将QDPR mRNA的序列给公司,随后吉玛公司设计了3种大鼠QDPR基因siRNA Oligo干扰序列,分别为QDPR1: GCCAGCGUGAUUGUUAAGATT UCUUAACAAUCACGCUGGCTT; QDPR2: CCAAAUCCAAGUCACUCUUTT AAGAGUGACUUGGAUUUGGTT; QDPR3: AGCCUUGCAGGGAAGAACATT UGUUCUUCCCUGCAAGGCUTT。每段序列加入150u1水配置成20uM的溶液,分别取4ug和野生型rQDPR重组质粒DNA共转染至293T细胞,48h后收集细胞上清以及细胞,检测TGF-β1以及NADPH氧化酶各亚基的含量。3.突变型QDPR对TGF-β1/Smad通路及自噬作用的影响本部分研究通过构建突变型QDPR重组质粒并转染至细胞,观察了在QDPR基因突变时TGF-β/Smad通路以及相关基因的变化,质粒构建的具体步骤如下：根据GeneBank中大鼠QDPR mRNA序列设计引物,上游引物引入EcoR V酶切位点,下游引物引入Xba I酶切位点,以OLETF2型糖尿病自发性大鼠的肾脏皮质cDNA为模板,采用高保真PCR试剂盒扩增突变型QDPR cDNA片段,对PCR产物用1.2%琼脂糖凝胶电泳进行分离,-20℃保存。用T4DNA连接酶连接目的片段与载体,构建QDPR (mut)/pcDNA3.1/V5-His(rQDPR(mut))重组质粒。随后用磷酸钙共沉淀法瞬时转染HEK293T细胞,实验分为空载体组(control vector),野生型rQDPR组,和突变型rQDPR(mut)组,转染72h后收集细胞上清和细胞蛋白,先用Vestern blot方法来验证融合蛋白在细胞中是否能成功表达,再采用Western blot和RT-PCR技术研究野生型和突变型QDPR对三种NOS, TGF-β/Smad通路,NADPH氧化酶各亚基的影响,此外,用ELISA技术来检测三组中BH4的含量。此外,我们还将野生型和突变型重组质粒转染至细胞观察了QDPR对自噬相关基因的影响,具体实验步骤如下：将成功构建好的野生型和突变型QDPR重组质粒分别转染至正常培养的HEK293T细胞,研究分为空载体组(control vector),野生型rQDPR组,和突变型rQDPR(mut)三组,转染72h后,采用RT-PCR及Western blot方法检测空载体组,野生型QDPR组和突变型QDPR组自噬相关基因LC3和Beclin1的表达量变化。结果：1. QDPR在STZ+单侧肾切诱导糖尿病大鼠肾脏的表达研究：造模成功后,即实验第0周模型组大鼠血糖与空白组相比显著升高(P<0.01),在之后的第4,8,12,16,20周各时间点均与空白组有极显著差异,均为P<0.01；在尿蛋白方面我们发现与空白组比较,模型组尿蛋白在实验第20周时显著升高(P<0.01)。20周龄Wistar大鼠以及单侧肾切除合并链脲佐菌素腹腔注射诱导的1型DN大鼠肾皮质与正常Wistar大鼠相比QDPR的mRNA及蛋白的水平都有所降低,TGF-β1,Smad3和NADPH氧化酶亚基的基因和蛋白水平都有所升高,提示在糖尿病肾病状态下QDPR的功能受损并影响了下游的一些蛋白和基因的表达。2.野生型QDPR对TGF β1/Smad3信号通路的影响：酶切和测序结果都显示QDPR的cDNA序列正确,测序正确后将构建好的重组质粒转染至293T细胞后,发现融合蛋白在细胞中能成功表达,并旦与空载体对照组相比,QDPR高表达组TGF-β1和smad3的基因和蛋白水平以及NADPH氧化酶亚基NoX1, NOX4的表达都有所降低,而nNOS的基因水平与BH4的含量在QDPR高表达时有所升高,初步推测QDPR的高表达影响了这些蛋白和基因的变化。此外,将设计的三对siRNA序列与野生型QDPR重组质粒DNA共转染至293T细胞中,Western blot结果显示有一条siRNA序列的基因敲除率达80%,可用于下一步研究,在随后的结果中我们发现,QDPR基因被敲除后,TGF-β1的表达相应升高,以及NOX1, NOX4的表达都有所升高。RNAi结果更证实了QDPR基因有下调TGF-β1表达的功能。3.突变型QDPR对TGF-β1/Smad通路及自噬作用的影响：在质粒构建中,测序结果显示突变型QDPR的cDNA序列与基因组的序列对比在93位氨基酸的位置突变,测序后将构建好的重组质粒转染至293T细胞后,发现融合蛋白在细胞中能成功表达,表明我们成功构建了突变型QDPR重组质粒,在进一步的检测中我们发现,与野生型QDPR重组质粒组相比,QDPR基因点突变后TGF-β1和Smad3的基因和蛋白水平以及NADPH氧化酶亚基的表达都明显增加,而nNOS的基因水平与BH4的含量却没有明显变化。此外,突变型研究中我们还描述了QDPR基因突变后对HEK293T细胞自噬相关基因的影响。RT-PCR结果显示,与空载体组相比,野生型QDPR组LC3基因水平明显上调(P<0.05),突变型QDPR组LC3基因水平与空载体组相比无统计学差异；与空载体组相比,野生型和突变型Beclinl基因水平无统计学差异；Vestern blot结果显示,与空载体组相比,野生型QDPR组LC3-II和Beclinl的蛋白表达量明显上调(P<0.05),而LC3-I的蛋白表达量无统计学差异。结论：1. QDPR的表达量在I型糖尿病肾病大鼠的肾皮质明显降低,提示糖尿病肾病的发生影响了QDPR的功能,由此我们推测QDPR可能参与了糖尿病肾病的发病过程并在其中有重要作用。2.野生型QDPR重组质粒细胞实验结果显示QDPR蛋白可以通过影响BH4来调节TGF-β1/Smad通路的变化。对于QDPR基因功能的深入研究将有助于揭示DN肾小球硬化的分子生物学机制,为研究糖尿病肾病的发病机制研究提供新的思路。3.突变型QDPR重组质粒细胞实验结果显示QDPR基因的点突变没有影响QDPR在BH4再生循环中的作用,但基因点突变后QDPR的结构有可能发生了变化,并通过调节其他途径影响了TGF-β1/Smad3通路,由此可见QDPR基因点突变对于QDPR调节TGF-β1/Smad3通路的功能有重要作用,这值得我们围绕QDPR的结构和功能做进一步研究。我们还发现突变型QDPR重组质粒能激活HEK293T细胞的自噬作用,激活过程可能和自噬相关基因的表达增强有关。更多还原

【Abstract】 Backgroud:Diabetic nephropathy (DN) has surpassed other major diseases to become the most common cause of the end-stage renal disease. The major cause of DN is the disorder of microcirculation in the glomerular, which eventually develops into glomerulosclerosis. Although the mechanisms of glomerulosclerosis are still under investigation, cytokines such as transforming growth factor beta1(TGF-β1) appear to play animportant role in pathogenesis of DN. TGF-β1is considered as a key mediator in diabetic nephropathy by inducing glomerulosclerosis and fibrosis. In our previous study, we have reported that there was the modification of QDPR gene in renal cortex of spontaneous OLETF diabetic rats, which suggests a role of QDPR in diabetic nephropathy. Dihydropteridine reductase (QDPR) plays an important role in the metabolism of biopterin, especially in the recycling of tetrahydrobiopterin (BH4), which is an essential cofactor of nitric oxide synthase (NOS). NOS can catalyze the conversion of arginine to citrulline releasing nitric oxide (NO). Increased NO production in the kidney generated from eNOS/nNOS has been documented to be involved in development of diabetic hyperfiltration. Moreover, decreased NO production will lead to an increase in TGF-β1expression, in the current study, we investigated the role of QDPR on TGF-β1/Smad pathway in diabetic rats and293T kidney cells.Objective:To observe the expression of QDPR in diabetic nephropathy rats and the regulation of QDPR in TGF-β1/Smad pathway.Methods:1. The expression of QDPR in type1diabetic nephropathy ratsDiabetic nephropathy model was induced by a single intraperitoneal injection of STZ at a dose of40mg·kg-1diluted in citrate buffer. Seventy-two hours after STZ injection, rats for blood glucose over16.7mmol·L-1were confirmed to be in diabetic state. After20weeks, renal cortex was isolated immediately and stored for the following study. RT-PCR and Western blot were adopted to observe the expression of QDPR, TGF-β1, Smad3and NOXs.2. The effect of wide type QDPR on regulating TGF-β1/Smad pathwayFirstly, we constructed a recombinant plasmid to observe the effect of wide type QDPR on regulating TGF-β1/Smad pathway. We amplified PCR fragments with QDPR specific primers (forward primer with EcoRV enzyme site AGATATCATGGCGGCTTCGGGCGAGGC; reverse primer with XbaI enzyme site ATCTAGAGAAATAGGCTGGAGTAAGCT) using the cDNA of rat renal cortex as a template and rat QDPR cDNA fragment was confirmed by sequence analysis. Then, rat QDPR cDNA was subcloned into pcDNA3.1/V5-His-A vector to form recombinant plasmid DNA rQDPR/pcDNA3.1/V5-His-A (rQDPR). Vector pcDNA3.1/V5-His-A (no cDNA) was used as the control vector group and the recombinant cDNA rQDPR (rQDPR group) was transfected into the293T cells. All sets of transfections were performed at least in triplicate. Cells were collected at72h for RT-PCR and Western blots analysis. RT-PCR and Western blot were adopted to observe the expression of QDPR, TGF-β1, Smad3, nNOS and NOXs. BH4levels were determined using an ELISA assay.The knockout experiment was adopted to identify the regulation of QDPR on TGF-β1/Smad pathway. Co-Transfection of rQDPR/pcDNA3.1/V5-His-A DNA and Rat QDPR Small Interfering RNA. The target sequences of rat QDPR siRNA (GCCAGCGUGAUUGUUAAGATT, UCUUAACAAUCACGCUGGCTT) was designed. The cells were divided into three groups:rQDPR group, rQDPR+siRNA group, and rQDPR+control siRNA group. Two days following transfection, cells in the6-well plates were harvested for immunoblotting to determine the degree of rat QDPR protein knockdown. Then, TGF-β1was determined by ELISA kit from culture media.3. The study of QDPR with mutation on regulating TGF-β1/Smad pathwayFirstly, we constructed a a recombinant plasmid to observe the effect of QDPR with mutation on regulating TGF-β1/Smad pathway. QDPR cDNA was amplified with specific primers using the cDNA from renal cortex of OLETF diabetic rat as a template. Rat QDPR cDNA fragments was then subcloned into pcDNA3.1/V5-His-A vector to form recombinant plasmids DNA of rQDPRmut/pcDNA3.1/V5-His-A (rQDPRmut). Control vector, recombinant rQDPR plasmid and rQDPRmut plasmid were transfected into the293T cells. Cells were collected at72h for mRNA and protein analyses. TGF-β1was determined by ELISA kit from culture media. Smad3and BH4levels was analysed by Western blot and ELISA assay, respectively.In addition, the effect of QDPR on autophagy was observed as well. HEK293T cells were transiently transfected with recombinant plasmid DNA rQDPR and recombinant plasmid DNA rQDPR (mut) by calcium phosphate. After72h, the expression of rat QDPR in293T cells was detected by Western Blot. Then, the effects of QDPR on autophagy related gene (including LC3and Beclinl) were analyzed by RT-PCR, and Western blot was used to monitor the changes in autophagy associated protein level.Results:1. Significantly decreased expression of QDPR was observed in STZ-induced diabetic rat kidneys at20weeks after STZ injection (p<0.05). At the same time, there were significantly increased expression of TGF-β1, Smad3, NOX1and NOX4in the renal cortex of diabetic group (p<0.01), however, there was no difference in NOX2and NOX3mRNA abundance between control and diabetic groups.2. After transfection of rQDPR in293T cells, there was a significant elevation of QDPR protein in293T cells. Moreover, with increase in QDPR expression, there were decreased expression of TGF-β1and Smad3at both mRNA and protein levels. BH4level in the rQDPR group was significantly higher than that of the control vector group, indicating that BH4production was upregulated in response to the QDPR overexpression. Since BH4is an important cofactor of NOS and there was an elevation of BH4level after QDPR overexpression, we further examined the mRNA level of NOS in293T cells. After transfection, QDPR appeared to induce expression of nNOS (p<0.05) in293T cells. Both the mRNA levels and the protein expression of NOX1and NOX4were significantly decreased in the rQDPR transfected cells as compared to that of control vector transfected cells (p<0.05)Co-transfection with rQDPR/pcDNA3.1/V5-His-A DNA and rat QDPR siRNA reduced rat QDPR protein levels by more than80%. The result showed that the expression of TGF-β1and NOX4in rQDPR+siRNA group increased compared to rQDPR group.3. After transfection of rQDPR(wt) and rQDPR(mut), there was a significant elevation of QDPR protein in293T cells. Moreover, with increase in QDPR expression, there were decreased expression of TGF-β1and Smad3at both mRNA and protein levels. the expression of NOX4was significantly decreased in the rQDPR(wt) transfected cells as compared to that of control vector group (p<0.05) while NOX4was increased in rQDPR(mut) group compared to control vector group (p<0.05).Besides, we discovered that QDPR had effect on autophagy, and the results show that the recombinant plasmid DNA rQDPR and recombinant plasmid DNA rQDPR (mut) were successfully constructed. The fusion protein can also express in HEK293T cell. In addition, compared with control vector group, the mRNA expression of LC3was significantly up-regulated in rQDPR group (P<0.05), and the mRNA expression of Beclinl showed no significant difference among the3groups (P>0.05). The Western blot analysis revealed that LC3II and Beclinl increased in rQDPR group when compared with control group, and there were no difference of LC3I protein levels among the3groups.Conclusions:1. The expression of QDPR was injured in diabetic nephropathy rats. The decrease in QDPR and the increase in TGF-β1and Smad3expression in diabetic rats suggest a link between QDPR and TGF-β1signaling pathway.2. The current study reveals a correlation between QDPR and NOXs/TGF-β1signaling pathway in the kidney of diabetic nephropathy. Moreover, that transfection of QDPR directly reduced the gene expression of TGF-β1/Smad3and NOX1/NOX4in293T cells indicates a function of QDPR in the kidney, which may be useful for development of a new therapeutic strategy for patients with diabetic nephropathy.3. Together with the previous observation that transfection of QDPR directly reduced the expression of TGF-β1/Smad3, we observed the levels of TGF-β1and Smad3increased when there was a point mutation, indicating that this mutation played a crucial role in regulating TGF-β1/Smad3signaling pathway. Whether this mutation is linked with the structure of QDPR may be undertaken as a next step using protein structural modeling. Additionally, QDPR may activate the autophagy of HEK293T cells by increasing the expression of autophagy associated genes of HEK293T cells.更多还原