【作者】 韦正峥；

【导师】 徐顺清；

【作者基本信息】 华中科技大学 ， 劳动卫生与环境卫生， 2013， 博士

【摘要】 胚胎期和哺乳期是机体发育的重要时期,从生命的起源到发育成型要经历细胞增殖、分化、迁移和程序性调亡等一系列复杂的过程。上世纪90年代末,英国学者提出“成人疾病的宫内起源”假说,该观点发布后引起国内外广泛关注,学者们认为围产期经历的环境对于成年后健康影响巨大,可能影响机体成年后患心血管疾病、代谢性疾病和肾脏疾病等各种慢性非传染性疾病的风险。有研究发现宫内发育进程中胎盘功能不全、母体低蛋白质饮食、糖皮质激素暴露和高脂饮食等因素都会影响先天肾脏发育,可能会导致胎儿和新生儿肾单位数量降低,增加成年后疾病负担。邻苯二甲酸二(2-乙基己基)酯(Di-(2-ethylhexyl)-phthalate, DEHP),是生产聚氯乙烯产品中广泛使用的一种增塑剂,可以通过消化道、呼吸道及皮肤接触等途径进入人体。DEHP及其在体内的主要代谢产物邻苯二甲酸单乙基己基酯(Mono(2-ethylhexyl) phthalate, MEHP),均可以通过胎盘和乳汁进入子代体内,影响胎儿和新生儿的健康。既往关于DEHP的研究主要集中在短期高剂量暴露上,发现DEHP可以诱发肝毒性、睾丸毒性、肾脏毒性、发育障碍和生殖毒性等。考虑到MEHP是PPARs受体的激活剂,MEHP通过与PPARa结合可以影响能量代谢,与PPARy结合可能影响细胞分化。另外,PPARs可以影响糖皮质激素和肾素-血管紧张素系统(Renin-angiotensin system, RAS)的功能,从而直接或间接影响输尿管芽(ureteric bud, UB)在肾发生过程中的分支,干扰肾脏发育进程。因此,我们推测围产期暴露于DEHP可能会对子代大鼠肾脏发育产生影响。本研究首先在胚胎期和哺乳期通过母体将子代间接暴露于DEHP,观察DEHP对肾脏发育的影响,并观察子代大鼠成年后肾脏和血压的改变；然后,我们在断乳后给予部分大鼠高脂饮食,观察围产期DEHP暴露和成年高脂饮食对大鼠肾脏损害的协同作用；此外,选择肾脏发育窗口期的胚胎肾脏做基因表达谱分析,进一步探索DEHP损害肾脏发育的机制。第一部分围产期暴露于DEHP影响子代大鼠肾脏发育目的：探讨围产期通过母体间接暴露于DEHP对子代大鼠肾脏发育的影响及可能的机制。方法：将孕鼠随机分为三组：对照组、低剂量组和高剂量组。从受孕0天开始直至仔鼠出生后第21天断乳,分别进行玉米油和不同剂量的DEHP(0.25和6.25mg/kg body weight/day)灌胃染毒。测量子代大鼠体重、肾重、血压、肾功能等变化；通过H&E染色法、PAS染色和masson染色观察子代大鼠肾脏病理学变化,并且通过电镜观察肾脏超微结构变化；采用ELISA、Griss法和免疫组化切片法分别检测成年子代大鼠血清肾素、血管紧张素Ⅱ、内皮素-1和一氧化氮等血压调节相关指标；同时,采用Q-PCR、western blotting和免疫组化切片法分别检测幼年子代大鼠RAS、PPARs、肾脏早期发育相关基因和结构相关基因的变化。结果：与对照组相比,围产期通过母体间接暴露于DEHP会使断乳期子代大鼠肾单位数量降低、肾小球体积增大、肾小囊缩小等病理学改变,成年大鼠逐渐出现肾小球硬化、肾间质纤维化、足细胞足突肿胀甚至消失等病理学变化。并且,母体暴露于DEHP的子代大鼠成年后血压显著升高,RAS功能激活,肾功能显著降低,表现为内生肌酐清除率降低、血尿素氮升高、尿总蛋白升高。并且,肾脏肾素和血管紧张素Ⅱ的蛋白表达水平在出生时显著降低,但在断乳期显著升高。此外,母体暴露于DEHP同样也会导致肾脏分化关键基因Foxdl, Gdnf, Pax2和Wnt11的mRNA表达水平在出生时显著降低；肾脏发育结构相关基因Bmp4, Cdh11, Calm1和Ywhab的mRNA表达水平在出生时显著升高。结论：母体DEHP暴露将会影响子代大鼠肾脏发育,导致肾单位数量降低,以致成年后大鼠肾功能损害和血压升高。研究表明母体暴露于DEHP通过抑制子代RAS功能,影响肾单位发育和成年肾脏疾病。第二部分围产期暴露于DEHP加剧子代子代高脂饮食诱导的肾损害目的：第一部分研究显示围产期暴露于DEHP可以影响子代大鼠肾脏发育,在本部分,我们引入高脂高糖的暴露因素,进一步探讨围产期暴露于DEHP与子代断乳后高脂饮食暴露的协同效应及可能的影响机制。方法：将孕鼠随机分为对照组和试验组,从受孕0天开始直至仔鼠出生后第21天断乳,分别进行玉米油和DEHP (1.25mg/kg body weight/day)灌胃染毒。每组断乳后,子代被随机分为两个亚组,给予正常饮食(Normal diet, ND)或高脂饮食(High fat diet, HFD)。测量子代大鼠体重、肾重、血压、肾功能等的变化；通过H&E染色法、PAS染色和masson染色观察子代大鼠肾脏病理学变化；同时,采用Q-PCR、western blotting和免疫组化切片分别检测成年及幼年子代大鼠RAS系统、PPARs、肾脏早期发育相关基因和结构相关基因的变化。结果：研究发现,围产期暴露于DEHP会降低出生时子代大鼠肾单位的数量,增加肾脏PPARs蛋白质的表达,降低肾素-血管紧张素系统(RAS)蛋白质的表达。在成年期,如预期一样,高脂饮食显著增加大鼠的体重和血压。另外,DEHP或高脂饮食均可引起子代大鼠肾功能不全和肾小球硬化,并且同时暴露于DEHP和高脂饮食的子代大鼠肾脏损害现象最为严重。此外,围产期DEHP暴露会使高脂饮食暴露的子代大鼠肾脏PPARs蛋白的表达降低,RAS和TGFβ1蛋白的表达增加。结论：通过围产期间接暴露于DEHP与断乳后高脂饮食对于子代大鼠的肾损害具有协同效应。其影响原因可能是暴露于DEHP后,一方面降低子代肾单位数量,使肾脏功能过度代偿；另一方面使子代PPARs功能失调,肾脏自我保护能力降低。第三部分胚胎期DEHP暴露对大鼠肾脏发育窗口期肾脏基因表达谱的影响目的：分析暴露于DEHP对子代大鼠肾脏发育窗口期肾脏基因表达谱的影响,进一步探索胚胎期DEHP暴露对肾脏发育可能的影响机制。方法：将孕鼠随机分为对照组和试验组,从受孕0天开始分别进行玉米油和DEHP (0.25mg/kg body weight/day)灌胃染毒。在胚胎期14.5天和16.5天分别处死一批孕鼠取胚胎,分别标记为E14.5-C(胚胎期14.5天对照组)、E14.5-D(胚胎期14.5天DEHP组)、E16.5-C(胚胎期16.5天对照组)与E16.5-D(胚胎期16.5天DEHP组)。部分胚胎做HE染色观察肾脏的病理学改变,部分胚胎分离肾脏,提取肾脏RNA,制成混合样本应用表达谱分析技术,筛选差异表达基因,并对其进行聚类分析、Gene Ontology功能显著性富集分析和Pathway显著性富集分析。结果：胚胎期暴露于DEHP会导致子代大鼠肾脏发育不良。DEHP组与对照组相比,在E14.5天子代大鼠肾脏基因表达差异比E16.5天显著。并且,E14.5-D与E14.5-C的差异基因,和E16.5-C与E14.5-C与之间的差异基因变化规律相似；E16.5-D与E16.5-C的差异基因,和E16.5-D与E14.5-D之间的差异基因变化规律相似。另外,E14.5-D与E14.5-C的差异基因主要集中在细胞内,E16.5-D与E16.5-C的差异基因主要集中在细胞外区域,E16.5-C与E14.5-C的差异基因主要集中在细胞内,E16.5-D和E14.5-D的差异基因主要集中在收缩纤维。另外,E14.5-D与E14.5-C、E16.5-C与E14.5-C在PPAR信号转导、wnt信号转导、代谢等通路发生了变化,E16.5-D与E16.5-C在凋亡、黏附等通路发生了变化,E16.5-D与E14.5-D在蛋白质的消化吸收等通路发生了变化。结论：我们认为E14.5比E16.5肾脏发育更敏感,胚胎期DEHP暴露通过参与肾脏发育关键期PPAR通路、Wnt通路、糖脂代谢、凋亡等及相关信号通路,降低了基因的敏感性,影响了细胞、器官及系统的发育进程,导致胚胎大鼠肾脏发育速度加快,肾单位发育不足。更多还原

【Abstract】 Embryonic and lactation are critical for organism development. From the origin to formation, the life undergoes cell proliferation, migration, differentiation, procedural apoptosis and other complex processes. During the late1990s, the British scholar put forward "the intrauterine origins of adult disease" hypothesis, and then this view had aroused worldwide attention from scholars. The researchers believed that conditions experienced during perinatal played powerful roles in later life, and leading to certain chronic diseases in adulthood, including cardiovascular disease, metabolic disease and chronic kidney disease. Some studies indicate that placental insufficiency, maternal low protein diet, glucocorticoid and high-fat diet are all known to affect kidney development, which would cause reduced fetal and neonatal nephron number, leading to enhanced adult disease burden.Di-(2-ethylhexyl) phthalate (DEHP), a widely used plasticizer in many types of polyvinyl chloride consumer products, continually enters into human body via food, water, the atmosphere and medical devices. Both DEHP and Mono (2-ethylhexyl) phthalate (MEHP), a major metabolite of DEHP in vivo, were translocated across the placenta of pregnant rodents and distributed into the milk of lactating rat dams, leading to the fetus and neonate to be at risk. Previous animal studies have demonstrated short-term exposure to high-dosage DEHP induced hepatotoxicity, testicular toxicity, renal toxicity, developmental disturbance, reproductive toxicity, and teratogenicity. MEHP is believed as a peroxisome proliferator-activated receptor a (PPARa) activator, and PPARa is verified to be required for the metabolic toxicity of DEHP in the fetuses and pups. Interestingly, it can also transactivate PPARy which correlates with the ability to induce fibroblasts and adipocyte differentiation. Moreover, PPAR could influence the renin-angiotensin system (RAS) and glucocorticoid, which could directly and indirectly induce ureteric bud (UB) branching during nephrogenesis, which determines the nephron numbers. Therefore, we hypothesize that perinatal exposure to DEHP might affect kidney development in rat offspring.In this study, we have exposed maternal to DEHP during the embryos and lactating, and examine the effect of perinatal DEHP exposure on nephron formation, adult renal disease and blood pressure in offspring. To further confirm this damage, we gave parts of them high-fat diet (HFD) after weaning and examined the effects of maternal DEHP exposure combined with HFD on the development of kidney damage in the offspring. Otherwise, in order to explore the possible mechanisms of perinatal DEHP exposure on nephron formation, we detected the gene expression profiles of embryonic kidneys during critical periods in development, and analyzed the gene otology and pathway of the differentially expressed genes.Part1:Perinatal exposure to DEHP affects kidney development in offspringObjective:This study investigated the consequences of perinatal exposure to DEHP on nephron formation, examined the programming of renal function and blood pressure and explored the mechanism in offspringMethods:Two mature females and one male were placed together in one cage for breeding. Mating was confirmed by the appearance of a vaginal plug in the following morning. And then, maternal rats were treated with vehicle,0.25and6.25mg/kg body weight/day DEHP respectively from gestation day0to postnatal day21. Body weight and kidney weight were carefully recorded at birth day and week3,15and21. The H&E staining, PAS staining and Masson staining were used to detect the histopathological changes. The ELISA and Griss method were used to detect the concentration of serum rennin, angiotensin Ⅱ, ET-1and NO. Moreover, Q-PCR, western blotting and immunohistochemistry were used to detect the expression of RAS, PPARs and other kidney development-related genes.Results:Maternal DEHP exposure resulted in lower number of nephrons, higher glomerular volume and smaller Bowman’s capsule in the DEHP-treated offspring at weaning, as well as glomerulosclerosis, interstitial fibrosis and effacement of podocyte foot processes in adulthood. In the DEHP-treated offspring, the renal function was lower and the blood pressure was higher. The renal protein expression of renin and angiotensin Ⅱ was reduced at birth day and increased at weaning. Maternal DEHP exposure also led to reduced mRNA expression of some renal development involved genes at birth day, including Foxdl, Gdnf, Pax2and Wntll. While, the mRNA expression of some genes was raised, including Bmp4, Cdhll, Calml and Ywhab.Conclution:These data show that maternal DEHP exposure impairs the offspring renal development, resulting in a nephron deficit, and subsequently elevated blood pressure later in life. Our findings suggest that DEHP exposure in developmental periods may affect the development of nephrons and adult renal disease through inhibition of the renin-angiotensin system. Part2:Perinatal exposure to DEHP exacerbates high fat diet-induced kidney damage in offspringObjective:The first part suggested that perinatal exposure to DEHP induced renal toxicity in offspring rats. And, high-fat diet induced obesity has emerged as a strong independent risk factor for kidney disease. In this study, we investigated the effects of perinatal DEHP exposure on the kidney development of the offspring and confirm whether the effects could exacerbate high fat diet-induced renal damage in offspring.Methods:Pregnant Wistar rats were exposed to vehicle or1.25mg/kg body weight/day DEHP throughout gestation and lactation by oral gavage, and then the offspring were randomLy assigned to a normal diet (ND) or a high-fat diet (HFD) after weaning. Body weight, blood pressure, renal morphology and renal function were evaluated in the offspring. The H&E staining, PAS staining and Masson staining were used to detect the histopathological changes. Moreover, Q-PCR, western blotting and immunohistochemistry were used to detect the expression of RAS, PPARs and other kidney development-related genes.Results:Maternal DEHP exposure decreased the number of nephrons, increased renal protein expression of peroxisome roliferator-activated receptors (PPARs) and reduced renin-angiotensin system (RAS) expression in the offspring at birth day. In adulthood, as expected, HFD significantly increased weight and blood pressure. In addition, DEHP or HFD induced renal dysfunction and glomerulosclerosis, and the DEHP/HFD offspring had the most serious phenomenon. Moreover, maternal DEHP exposure reduced PPARs expression, increased RAS expression and TGFβ1expression in kidney of HFD-fed offspring.Conclution:Our data suggest that the maternal DEHP exposure induces congenital dysplasia of the kidney in the offspring, which exacerbates HFD-induced kidney damage in offspring. The mechanism probably involves reducing nephron numbers and impairing PPARs expression. Part3:The influence on offspring gene expression profile in kidney developmental window by prenatal DEHP exposureObjective:Analyze the influence of offspring expression profile in kidney developmental window induced by prenatal DEHP exposure, to further explore the mechanism of the early life DEHP exposure on kidney development.Methods:Pregnant Wistar rats were exposed to vehicle or0.25mg/kg body weight/day DEHP respectively from gestation day0. Pregnant rats were scarified in embryonic14.5days (E14.5) and16.5days (E16.5) respectively. The H&E staining was used to detect the histopathological changes of embryonic kidney. Moreover, we separated the embryonic kidney and extraction of kidney RNA, and used gene chips to detect gene expression profiling. The differentially expressed genes (DEGs), cluster analysis, the Gene Ontology features significant enrichment and Pathway significant enrichment were analyzed.Results:Exposure to DEHP in embryonic period caused kidney dysphasia. Comparing with the control and the exposure group, the DEGs of E14.5were significant higher than E16.5. And, the DEGs of E14.5-D vs E14.5-C were similar with E16.5-C vs E14.5-C, while the DEGs of E16.5-D vs E16.5-C were similar with E16.5-D vs E16.5-C. Moreover, the DEGs of E14.5-D vs E14.5-C mainly concentrated in the cell, the DEGs of E16.5-D vs E14.5-D mainly concentrated in the extracellular region, the DEGs of E16.5-C vs E14.5-C also mainly concentrated in the cell, and the DEGs of E16.5-D vs E14.5-D mainly concentrated in the contractile fibers. Additionally, the DEGs of E14.5-D vs E14.5-C and E16.5-C vs E16.5-D were related with PPAR signaling pathways, Wnt signaling pathways, metabolic pathways and others. While, the DEGs of E16.5-D vs E16.5-C were related with apoptosis, adhesion, etc. and the DEGs of E16.5-D vs E14.5-D were related with protein digestion and absorption.Conclution:We believe that kidney development in E14.5is more sensitive than in E16.5. Prenatal DEHP exposure could affect kidney development by influencing the PPARs pathway, Wnt signaling pathway, glucose and lipid metabolism, apoptosis and other related signaling pathways. And thus, these changes could reduce the sensitivity of the genes and affect the development process of cells, organs and systems, resulting in accelerated programing of kidney development, leading to nephron hyperplasia.更多还原