多器官功能障碍综合征急性肾损伤水通道蛋白2的表达

发布时间：2018-06-20 05:06

本文选题：多器官功能障碍综合征 + 肾损伤　；参考：《吉林大学》2016年博士论文

【摘要】：目的:危重病人特别是合并严重感染、缺血-再灌注损伤及中毒者,极易出现多器官功能障碍综合征(Multiple Organ Dysfunction Syndromes,MODS),其中肾脏是最易受影响的器官之一。MODS发生肾损伤时,其继发的严重水电解质酸碱平衡代谢紊乱,可使病情进展迅速,导致病死率明显增加。水通道蛋白(aquaporin,AQP)是水和尿素跨膜主动转运机制的结构基础,为监测肾功能的动态变化及早期干预治疗肾损伤提供了新的靶点。水通道蛋白2(aquaporin 2,AQP2)在肾脏表达,通过加压素(Vasopressin,AVP)对集合管水通透性进行调节,在肾脏水代谢中发挥了重要的作用。AQP2异常表达可导致多种先天性或后天性肾脏疾病。肾脏钠-钾-三磷酸腺苷酶(Na~+-K~+-ATPase),又称钠钾泵,镶嵌在肾小管细胞膜的脂质双分子层中,除对钠、钾离子有转运功能外,还可以分解ATP使之释放能量,并利用此能量进行钠、钾的主动转运,其功能异常与疾病的严重程度呈正相关,其功能的恢复先于疾病的好转,这对病情及预后的判断有重要意义。本实验通过研究脂多糖及百草枯致多器官功能障碍大鼠模型炎症介质、肾脏AQP2及Na~+-K~+-ATPase的表达特点,从炎症反应、肾小管功能及能量代谢方面研究肾损伤的可能发病机制。材料与方法:1.采用大鼠腹腔注射脂多糖(Lipopolysaccharide,LPS)5mg/kg或百草枯20mg/kg灌胃复制多器官功能障碍急性肾损伤动物模型,同时应用20mg/kg赖氨酸阿司匹林分别干预LPS和百草枯MODS模型。2.免疫组织化学染色测定肾脏AQP2蛋白表达。3.RT-PCR测定肾脏AQP2m RNA表达。4.常规病理切片HE染色进行肾脏形态学检查。5.Na~+-K~+-ATPase试剂盒测定肾小管上皮细胞Na~+-K~+-ATPase含量及活性。6.ELSIA法测定百草枯大鼠外周血IL-1,IL-10,TNF-ɑ细胞因子表达及流式细胞仪测定外周血淋巴细胞凋亡比率。结果:1.LPS组大鼠肌酐、尿素氮水平逐渐增高,在48小时达高峰,此后逐渐降低;LPS组大鼠尿量早期(12h,24h)逐渐减少,此后明显增多。百草枯组大鼠肌酐、尿素氮水平逐渐增高,尿量无明显减少。2.LPS组大鼠肾脏病理切片可见明显的肾小管上皮细胞肿胀,肾小管管腔变小;肾小球肿胀,炎症细胞浸润;部分肾小管细胞坏死。百草枯组大鼠肾小管上皮细胞肿胀明显,但细胞坏死很少。3.LPS组大鼠肾小管上皮细胞AQP2m RNA表达在6h时增高,随后迅速降低,在2d降至最低,尔后逐渐增高。百草枯组大鼠肾小管上皮细胞AQP2m RNA表达表达明显降低,在2~3d时达最低水平,赖氨酸阿司匹林则可以减轻这种改变。4.LPS组大鼠肾脏组织在实验12h出现肾小管上皮细胞AQP2蛋白表达减少,在2d肾脏AQP2蛋白表达减少最为明显,此后逐渐增加;百草枯组大鼠肾小管上皮细胞AQP2蛋白表达在24h后明显降低,在2~3d时达最低水平,赖氨酸阿司匹林则可以减轻这种改变。5.各组大鼠Na~+-K~+-ATP酶含量无明显差异;但LPS及百草枯组大鼠Na~+-K~+-ATP酶活性在造模成功后明显降低,此后逐渐增高,但仍低于对照组,赖氨酸阿司匹林则可减轻这种改变。6.百草枯中毒组大鼠淋巴细胞凋亡比率明显增高,而赖氨酸阿司匹林可以减轻百草枯中毒导致的淋巴细胞凋亡。百草枯组TNF-ɑ、IL-1、IL-10表达明显增加,赖氨酸阿司匹林可以抑制TNF-ɑ、IL-1的表达增加,但促进IL-10的表达增加。结论:1.LPS致多器官功能障碍综合征大鼠尿素氮、肌酐增高,APQ2表达明显减少,肾小管上皮细胞肿胀、功能障碍甚至凋亡与炎症反应有关。2.LPS致多器官功能障碍综合征肾功能恢复阶段,尿量的多少与AQP2的表达呈负相关。AQP2是肾脏重吸收功能恢复的结构基础,Na~+-K~+-ATPase是肾脏功能恢复的能量代谢基础,Na~+-K~+-ATPase通过直接参与及间接调控的方式提示肾脏的能量代谢状态,只有在AQP2蛋白表达及能量代谢状态接近正常水平后受损的肾脏功能才逐渐好转。3.早期应用赖氨酸阿司匹林可通过抑制炎症反应的机制改善LPS致MODS肾损伤的预后。4.百草枯可引起全身炎症反应及多器官功能障碍,但这些表现较LPS引起者是延迟出现的,提示百草枯导致的MODS并非毒物直接引起的,而是由过度的炎症反应导致的。5.无论是炎症反应直接导致的MODS还是百草枯通过炎症反应间接引起的MODS,均可通过应用赖氨酸阿司匹林阻断或减轻炎症反应的途径,对受损的靶器官起到保护作用。
[Abstract]:Objective: critically ill patients, especially with severe infection, ischemic reperfusion injury and poisoning, are extremely susceptible to Multiple Organ Dysfunction Syndromes (MODS), in which the kidney is one of the most susceptible organs, one of the most vulnerable organs,.MODS with renal injury, and the secondary severe acid-base metabolic disorder of electrolyte acid-base metabolism, which can make it possible Aquaporin (AQP) is the structural basis for the active transmembrane mechanism of water and urea, which provides a new target for monitoring the dynamic changes of renal function and early intervention in the treatment of renal injury. The expression of aquaporin 2 (aquaporin 2, AQP2) in the kidney, through the vasopressin (Vasopressin, AVP) Regulating the water permeability of the collection tube and playing an important role in the water metabolism of the kidney, the abnormal expression of.AQP2 can lead to a variety of congenital or acquired renal diseases. The renal sodium potassium triphosphate ATPase (Na~+-K~+-ATPase), also known as the sodium potassium pump, is embedded in the lipid bilayer of the renal tubular cell membrane, with the exception of the sodium and potassium ions. In addition, it can also decompose ATP to release energy, and use this energy to carry on the active transport of sodium and potassium. The dysfunction of the disease is positively related to the severity of the disease. The recovery of its function is preceded by the improvement of the disease, which is of great significance to the judgement of the condition and prognosis. Rat model inflammatory mediators, renal AQP2 and Na~+-K~+-ATPase expression characteristics, from inflammatory response, renal tubule function and energy metabolism to study the possible pathogenesis of renal injury. Materials and methods: 1. rat intraperitoneal injection of lipopolysaccharide (Lipopolysaccharide, LPS) 5mg/kg or paraquat 20mg/kg replicating multiple organ dysfunction acute kidney Damage animal models and 20mg/kg lysine aspirin interfered with LPS and paraquat MODS model respectively,.2. immunohistochemical staining was used to determine the expression of renal AQP2 protein,.3.RT-PCR, AQP2m RNA, AQP2m RNA,.4. routine pathological section, HE staining, HE staining, renal morphological examination,.5.Na~+-K~ +-ATPase kit for renal tubular epithelial cells ~+-K~+-ATPase content and active.6.ELSIA method were used to determine IL-1, IL-10, TNF- cytokine expression and flow cytometry for peripheral blood lymphocyte apoptosis ratio in peripheral blood of paraquat rats. Results: the level of creatinine and urea nitrogen increased gradually in the 1.LPS group, reached the peak at 48 hours, and gradually decreased after this, and the early urine volume in the LPS group (12h, 24h) gradually increased. The level of creatinine and urea nitrogen in the paraquat group increased gradually, and the urine volume did not decrease obviously in the kidney pathological sections of the.2.LPS group. The renal tubular epithelial cells were swelling, the renal tubule cavity became smaller, glomerular swelling, inflammatory cells infiltrated, and some renal tubular cells were necrotic. The renal tubule epithelium of paraquat group was fine. The cell swelling was obvious, but the expression of AQP2m RNA in the renal tubular epithelial cells in the.3.LPS group was higher, then decreased rapidly, and then decreased to the lowest in 2D, and then gradually increased. The expression of AQP2m RNA in the renal tubular epithelial cells of the paraquat group decreased significantly, and the lowest level was at 2~3d, and lysine aspirin could reduce this. The expression of AQP2 protein in renal tubular epithelial cells decreased in experimental 12h and the expression of AQP2 protein in 2D kidney decreased most obviously, and then increased gradually. The expression of AQP2 protein in renal tubular epithelial cells in paraquat group decreased significantly after 24h and the lowest level in 2~3d, and lysine aspirin could decrease. There was no significant difference in the Na~+-K~+-ATP enzyme content of.5. rats in each group, but the activity of Na~+-K~+-ATP enzyme in LPS and paraquat rats decreased obviously after the mold making, and then gradually increased, but still lower than that of the control group. Lysine aspirin could reduce the rate of lymphocyte apoptosis in the.6. paraquat group. Lysine aspirin can reduce the apoptosis of lymphocyte caused by paraquat poisoning. The expression of TNF-, IL-1 and IL-10 in paraquat group is significantly increased. Lysine aspirin can inhibit the expression of TNF-, increase the expression of IL-1, but increase the expression of IL-10. Conclusion: the expression of urea nitrogen, creatinine, APQ2 expression in multiple organ dysfunction syndrome rats induced by 1.LPS Significantly decreased, renal tubular epithelial cells swelling, dysfunction or even apoptosis related to the inflammatory response to.2.LPS induced renal function recovery phase of multiple organ dysfunction syndrome, the amount of urine is negatively related to the expression of AQP2.AQP2 is the structural basis for the recovery of renal reabsorption function, Na~+-K ~+-ATPase is the basis of energy metabolism of renal function recovery. Na~+-K~+-ATPase indicates the energy metabolic state of the kidney through direct participation and indirect regulation. Only after the expression of AQP2 protein and the state of energy metabolism are near the normal level, the impaired renal function is gradually improved. The early application of lysine aspirin can improve the preconditioning of LPS induced MODS renal injury by inhibiting the mechanism of inflammation. The post.4. paraquat can cause systemic inflammatory response and multiple organ dysfunction, but these manifestations are delayed than those caused by LPS, suggesting that the MODS caused by paraquat is not a direct cause of the poison, but is caused by excessive inflammatory response, whether the MODS or paraquat directly caused by the inflammatory reaction, or by the inflammatory reaction. MODS can protect the damaged target organs by using lysine aspirin to block or reduce the inflammatory response.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R459.7

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