【作者】 宋文霞；

【导师】 王建伟；

【作者基本信息】 吉林大学 ， 病理学与病理生理学， 2012， 硕士

【摘要】 蛋白尿提示各种慢性肾脏病的发生发展，是病程进展中的一个独立的危险因素，可以反映肾损伤程度。具有高水平蛋白尿的患者比低水平蛋白尿患者更容易导致慢性肾小球、肾小管及间质疾病的发生，最终形成慢性肾功能衰竭。但是目前蛋白尿的治疗尚无有效治疗方法，因此，控制蛋白尿仍为目前研究的热点。生理状态下，滤过蛋白质在肾小管上皮细胞中的重吸收主要依靠受体介导的内吞作用来完成，小泡内的pH值可以直接影响受体-配体的解离、小泡运输、内体溶合、配体蛋白分选过程、降解酶的活性等。因而转运小泡的酸化对蛋白内吞及降解起至关重要的作用，发生蛋白尿时，肾小球滤液中含量最丰富的是白蛋白，白蛋白可以直接导致肾小管上皮细胞损伤。在大量蛋白尿模型中可以见到肾小管上皮细胞凋亡现象，但是其引起凋亡的机制尚无统一定论。本实验以小泡酸化为切入点，通过复制过蛋白负荷大鼠肾近端小管上皮细胞(NRK-52E)模型，研究不同实验条件对滤过蛋白内吞及降解的影响，同时检测凋亡通路中Caspase-3、Caspase-8和Caspase-9蛋白变化情况，探讨其发生凋亡的机制。目的：本研究通过过蛋白负荷NRK-52E细胞，模拟体内蛋白尿环境，研究诱导或抑制小泡酸化对蛋白质内吞和降解的影响，及其对细胞凋亡的影响，探讨过度蛋白负荷时蛋白代谢障碍的机制，及蛋白尿引起肾小管上皮细胞凋亡的机制。为进行性肾损伤治疗靶的发现奠定技术和理论基础，更有效控制临床蛋白尿患者肾脏疾病的进展。方法：1.CCK-8法检测NRK-52E细胞生存率；2.吖啶橙(AO)染色观察酸性小泡；3.间接免疫荧光组织化学染色检测FITC-BSA分别与EEA1、TfR、CathepsinB的共定位；4.Hoechst33342染色观察细胞凋亡；5.Westernblot方法检测CleavedCaspase-8、CleavedCaspase-9和CleavedCaspase-3的表达情况。结果：过量白蛋白引起NRK-52E细胞损伤呈时间和剂量依赖性，诱导小泡酸化可以提高过蛋白负荷NRK-52E细胞的生存率，而抑制小泡酸化会使细胞生存率进一步下降。AO染色可见诱导小泡酸化后过蛋白负荷NRK-52E细胞内出现较多的红色荧光，表明酸性小泡增多。间接免疫荧光染色可见诱导小泡酸化可使细胞内绿色荧光减少，而抑制小泡酸化将使细胞内绿色荧光增多，胞内白蛋白蓄积；FITC-BSA与EEA1、TfR、CathepsinB的共定位可见，小泡酸化受阻时EEA1与BSA双色荧光强度及TfR与BSA的双色荧光强度较高，而CathepsinB表达量较低，诱导小泡酸化时EEA1与BSA双色荧光强度及TfR与BSA的双色荧光强度较低，而CathepsinB表达量较高。小泡酸化受阻时CleavedCaspase-3的表达量较高，证明凋亡较为严重，而诱导小泡酸化时CleavedCaspase-3的表达量较低。CleavedCaspase-9的表达趋势与CleavedCaspase-3相同，而Caspase-8没有出现活性裂解片段。结论：诱导小泡酸化可以增强溶酶体活性，促进内吞蛋白降解，减轻过蛋白负荷所致NRK-52E细胞损伤，而小泡酸化受阻时内吞蛋白蓄积在胞内，主要聚集在早期内体和再循环内体中，会加重细胞损伤。过负荷蛋白引起的NRK-52E细胞凋亡可能与内源性凋亡途径有关。更多还原

【Abstract】 In chronic kidney disease, proteinuria is one of the significant signs of end-stage renal failure. Proteinuria is an independent risk factor in the progression of renal diseases, which can indicate the degree of glomerular and tubular injury. Patients with high levels of proteinuria were more likely to develop into chronic kidney diseases than the low level of proteinuria, and into chronic renal failure in the end. However, nowadays, there is no effective treatment method in proteinuria. Therefore, treatment of proteinuria has caught much attention of the whole world.In the physiological condition, the reabsobtion of filtered proteins in the renal tubular epithelial cells mainly rely on receptor-mediated endocytosis. Due to the pH of transport vesicles can directly affect receptor-ligand dissociation, vesicular transport, sorting of ligand proteins, activity of enzymes, so the acidification of transport vesicles plays a critical role in normal maintenance of function. When proteinuria occurs, albumin is most abundant in glomerular filtrate and can lead to renal tubular epithelial cells injury directly. Renal tubular epithelial cell apoptosis can be seen in massive proteinuria models, but there is no unified conclusion of its mechanism.The present experiments take vesicular acidification as the starting point, through model of albumin overload NRK-52E cell, albumin induced cell injury was studied through obseving the effect of vesicular acidification on filtered albumin endocytosis and degradation, and the mechanism of apoptosis was also discussed by determining the expression of Caspase-3, Caspase-8and Caspase-9. Objective:Through albumin overload NRK-52E cell, this study simulates proteinuria environment in vivo to investigate filtered proteins’endocytosis and degradation with induction or inhibition of vesicular acidification, and to approach to mechanism of protein overload-induced protein metabolism disorders, and to define mechanism of cell apoptosis induced by proteinuria. In order to lay the technical and theoretical basis to therapeutic target of progressive renal damage, this study maybe can contribute to more effective control and treatment of kidney diseases’progression in patients with clinical proteinuria.Methods:1. CCK-8detection of cell viability in NRK-52E;2.The AO staining of acidic vesicles;3. The colocalization between FITC-BSA and EEA1, TfR, Cathepsin B individually by immunofluorescence staining;4. The Hoechst33342staining of nuclei;5. Cleaved Caspase-8、Cleaved Caspase-9and Cleaved Caspase-3were detected by western blot.Results:Albumin’s inhibition on NRK-52E cell viability had time and dose dependence. The induction of vesicular acidification could improve the viability of NRK-52E which is of protein overload, while the inhibition would decline cell viability. In the AO staining, we could see that red fluorescence vesicles were more in BSA+NaH2PO4group, which indicated that there were more acdic vesicles. From immunofluorescence staining we knew that the green fluorescence particles were fewer when we induced vesicular acidification, while inhibit the acidification of vesicles would allow cells increase in green fluorescence, and it means proteins’ acumulation in the cells. The colocalization between FITC-BSA and EEA1, TfR, Cathepsin B individually indicated that the inhibition of vesicular acidification made increase of EEA1’s and TfR’s colocalization with BSA, reduction of Cathepsin B, the induction of vesicular acidification had the opposite consequence. Protein from western blots showed Cleaved Caspase-3and Cleaved Caspase-9’s expression was more in BSA+CEP group than in BSA+NaH2PO4group, while Caspase-8had no active cleaved fragments.Conclusion:Induced acidification of the vesicles can enhance lysosomal activity, promote endocytosis protein degradation and reduce protein overload-induced cell injury in NRK-52E cells. When the acidification is blocked, albumin will accumulate in the early endosomes and recycling endosomes, increasing the cell damage. Protein overload-induced apoptosis in NRK-52E cells maybe relate to the endogenous apoptosis pathway.更多还原