肾损伤分子-1表达在移植肾损伤中的变化和意义

发布时间：2018-04-21 15:35

本文选题：肾损伤分子-1 + 肾移植　；参考：《吉林大学》2014年博士论文

【摘要】：肾损伤分子（KIM-1）是新颖、特异性高的判断肾小管损伤的生化指标，尿液和组织标本中均可以检测出其表达；当肾近曲小管发生急慢性损伤时，KIM-1在肾小管细胞中表达，成为凋亡细胞被吞噬与被清除的受体；KIM-1可能在肾小管损伤和修复过程中发挥重要病理生理作用。在移植肾脏中，肾小管损伤的相关因素有缺血、免疫抑制剂毒性、急性排斥反应等，，各种因素联合作用或单独作用均可以导致慢性移植肾肾病，最终导致移植肾脏丢失。肾组织穿刺活检，是判断和识别移植肾脏损伤的重要检测手段。肾穿标本病理形态学观察可见到：肾小管上皮刷状缘的改变、上皮细胞的凋亡/坏死、小管纤维化/萎缩、上皮脱落等，但因形态诊断是存在主观因素的影响，需要寻求一个早期敏感度高的客观判断肾小管轻微损伤的指标。本选题以KIM-1为研究的核心，探索其在移植肾损伤中的变化和意义。我们选取2009年-2011年69例移植肾穿刺活检的组织标本，应用免疫组织化学方法标记移植肾组织中KIM-1的表达情况，分析移植肾组织中KIM-1表达水平和其他血液生化指标的关系、移植肾组织中KIM-1表达与移植肾功能的相关性。结果显示：（1）在不同病理诊断分组中可见KIM-1在近端小管上皮出现不同程度的表达；（2）在急性T细胞性排斥反应中，KIM-1表达量随Banff07分级升高而呈现增高趋势，同时KIM-1表达水平与小管炎严重程度相一致。在小管炎出现可见的形态改变之前，KIM-1蛋白已出现可见的阳性表达。（3）慢性活动性抗体介导排异反应中，KIM-1呈强阳性表达，强烈提示活动性排异反应所致肾损伤的存在；（4）在交界性病变时，KIM-1出现不同程度阳性表达，提示肾小管上皮处于损伤状态；（5）未见排斥反应发生和免疫抑制剂中毒组，27.27%（3/11）病例血肌酐在正常范围内，但肾组织中KIM-1呈弱阳性表达。本研究表明：（1）在移植肾组织中KIM-1表达水平与移植肾损伤程度呈正相关，提示肾小管细胞损伤存在。（2）在未见血生化指标升高提示肾损伤时、未见形态学可见肾损伤改变时、未见肾小管炎症反应时，均可见KIM-1蛋白在肾小管上皮细胞表达，提示KIM-1蛋白在移植肾活检组织中表达可以成为早期、敏感、特异判定肾小管上皮细胞损伤的生化指标。本研究通过采用ELISA方法检测肾移植患者血清中KIM-1的表达，研究和探索早期血清中与组织内KIM-1的表达能否成为早期判断移植肾功能异常的应用指标。结果表明：（1）在移植肾患者血清中KIM-1表达的增强可以提示移植肾损伤程度的强弱。（2）慢性活动性排斥造成的损伤可以出现血清KIM-1的高表达，提示移植肾脏功能状态恶化。（3）早期血清中肾损伤分子-1水平升高可能成为提示移植肾功能状态的生化指标。在本次实验研究中，采用体外实验方法研究环孢素损伤肾小管细胞后导致其产生KIM-1的表达，并初步探索其中机制，寻找保护和延缓环孢素对移植肾损伤的药物。在实验中首先建立环孢素损伤肾近曲小管上皮细胞的体外研究模型，采用MTT实验方法筛选环孢素的损伤作用的适宜浓度；采用MTT实验法筛选可能的药物干预改善环孢素对肾小管上皮细胞的损伤；筛选出促红细胞生成素（EPO）可以保护肾小管细胞减少环孢素对细胞的损伤。采用丝裂原活化蛋白激酶系统阻断剂(p38通路阻断剂SB202190、ERK1/2通路阻断剂PD98059)，阻断相应酶的表达，以期推测生化过程发生的机制。采用ELISA方法鉴定KIM-1在细胞上清液中的表达，统计KIM-1表达量的变化。采用流式细胞仪测定在环孢素损伤肾小管中细胞凋亡发生特点。环孢素使肾近曲小管细胞损伤时KIM-1表达升高并导致细胞凋亡，在这一过程中丝裂原活化蛋白激酶中的p38通路、ERK1/2通路在其中发挥作用。阻断以上2条通路可以减少CsA所致HKC细胞的凋亡比率（P0.05），增加活细胞比率（P0.05），减少细胞的坏死比率（P0.05），同时减少KIM-1蛋白的表达量（P0.05）。加入EPO可以减少HKC细胞在CsA作用下产生KIM-1蛋白（P0.05）；选用EPO15U/ml和30U/ml进行实验，随EPO作用量的增加，可以减少HKC细胞在CsA损伤中产生KIM-1的量值，提示EPO可以拮抗CsA对肾小管上皮的损伤，减少CsA所致肾小管上皮细胞凋亡和坏死的发生。肾损伤分子-1在移植肾中的表达和变化具有重要的研究价值，可以成为判定移植肾损伤早期、敏感、特异性判定肾小管上皮细胞损伤的生化指标；早期血清中肾损伤分子-1水平升高可能成为提示移植肾功能状态的生化指标。环孢素使肾近曲小管细胞损伤时KIM-1表达升高，在这一过程中丝裂原活化蛋白激酶中的p38通路、ERK1/2通路在其中起到作用。阻断p38通路、阻断ERK1/2通路、加入EPO三种因素均可以减少肾小管细胞在CsA作用下产生KIM-1蛋白，有效保护和延缓环孢素对移植肾小管上皮损伤的过程。本次选题的实验结果与结论，为肾损伤分子-1在移植肾领域中的应用提供有利的基础实验依据，同时为减少环孢素对移植肾损伤方面继续探索提供可参考的实验模型与有效的实验药物。
[Abstract]:Renal injury molecule (KIM-1) is a novel and highly specific biochemical indicator of renal tubular injury, which can be detected in urine and tissue specimens. When acute and chronic renal tubule injury occurs, KIM-1 is expressed in renal tubular cells and becomes the receptor for phagocytosis and removal of apoptotic cells; KIM-1 may be damaged in renal tubules and in renal tubules. During the repair process, it plays an important role in pathophysiology. In the transplanted kidney, the related factors of renal tubular injury are ischemia, immunosuppressant toxicity, acute rejection and so on. Various factors combined or alone can lead to chronic renal transplant kidney disease and eventually lead to the loss of kidney transplantation. Renal tissue biopsy is a judgment and recognition shift. The pathological changes of the brush shaped edge of the renal tubular epithelium, the apoptosis / necrosis of the epithelial cells, the tubule fibrosis / atrophy, the epithelia and so on are observed, but the morphological diagnosis is influenced by the subjective factors, and it is necessary to seek an objective judgement of the renal tubules with high early sensitivity. The index of damage.
This topic takes KIM-1 as the core of the study to explore its changes and significance in renal allograft injury.
69 cases of renal transplant biopsy in 2009 -2011 were selected. The expression of KIM-1 in transplanted renal tissue was marked by immunohistochemistry. The relationship between the expression of KIM-1 and other blood biochemical indexes in the transplanted kidney tissue was analyzed. The correlation between the KIM-1 table and the function of the transplanted kidney in the transplanted kidney tissue was analyzed. The results showed: (1 Different levels of expression of KIM-1 in proximal tubule epithelium were found in different pathological diagnosis groups; (2) in acute T cell rejection, the expression of KIM-1 increased with the increase of Banff07 grading, and the expression level of KIM-1 was consistent with the severity of tubulitis. Before the appearance of visible morphological changes in canaliculitis, KIM-1 The positive expression of the protein has been seen. (3) in the reaction of chronic active antibody mediated rejection, KIM-1 is strongly positive and strongly suggests the existence of renal injury caused by active rejection; (4) in borderline lesions, the positive expression of KIM-1 appears in different degrees, suggesting that the renal tubular epithelium is in a state of injury; (5) no rejection occurs. The serum creatinine in 27.27% (3/11) cases was in the normal range, but the expression of KIM-1 in the renal tissue was weakly positive. The study showed that (1) the expression of KIM-1 in the transplanted kidney was positively correlated with the degree of renal transplant injury, suggesting that the renal tubular cells were damaged. (2) when no blood biochemical index was raised, the renal injury was not seen. The expression of KIM-1 protein in renal tubular epithelial cells can be seen in the renal tubular epithelial cells when the renal tubule inflammation is not seen in the morphological changes. It is suggested that the expression of KIM-1 protein in the transplanted renal biopsy tissue can be an early, sensitive and specific biochemical index to determine the injury of renal tubular epithelial cells.
In this study, the expression of KIM-1 in the serum of renal transplant patients was detected by ELISA method. The study and exploration of the early serum and intra tissue KIM-1 expression could be used as an indicator of early renal allograft dysfunction. The results showed that: (1) the enhancement of KIM-1 in the serum of patients with renal allograft could indicate the strong degree of renal transplant injury. (2) the high expression of serum KIM-1 can be found in the injury caused by chronic active rejection, which suggests the deterioration of the function of the transplanted kidney. (3) the increase in the level of renal injury molecules in the early sera may be a biochemical indicator of the function of the transplanted kidney.
In this experimental study, the expression of cyclosporin damaged renal tubule cells in vitro was studied in vitro, and the mechanism of KIM-1 was preliminarily explored to find the protection and delay of cyclosporin in the treatment of renal transplant injury.
In the experiment, we first set up an in vitro model of cyclosporin injury in renal proximal convoluted tubule epithelial cells. The suitable concentration of cyclosporin was screened by MTT method. The possible drug intervention was used to screen the damage of cyclosporin on renal tubular epithelial cells by MTT test, and the screening of erythropoietin (EPO) could protect the epithelial cells of renal tubules. Renal tubule cells reduce cyclosporine damage to cells. The mitogen activated protein kinase blocker (p38 pathway blocker SB202190, ERK1/2 pathway blocker PD98059) was used to block the expression of the corresponding enzyme, in order to speculate on the mechanism of biochemical process. ELISA method was used to determine the expression of KIM-1 in the cell supernatant, and the expression of KIM-1 was statistically expressed. The characteristics of apoptosis in renal tubules damaged by cyclosporine were determined by flow cytometry.
Cyclosporin increases the expression of KIM-1 in renal proximal tubular cells and causes apoptosis. In this process, the p38 pathway in mitogen activated protein kinase and the ERK1/2 pathway play a role in this process. Blocking the above 2 pathways can reduce the apoptosis ratio (P0.05) of HKC cells caused by CsA, increase the living cell ratio (P0.05), and reduce cell damage. The death rate (P0.05) and the decrease of the expression of KIM-1 protein (P0.05). The addition of EPO can reduce the production of KIM-1 protein (P0.05) in HKC cells under the action of CsA, and use EPO15U/ml and 30U/ml to carry out the experiment. Injury can reduce the occurrence of apoptosis and necrosis of renal tubular epithelial cells induced by CsA.
The expression and changes of renal injury molecule -1 in the transplanted kidney have important research value, which can be used to determine the early, sensitive and specific biochemical markers of renal tubular epithelial cell damage in the early stage of renal transplantation. The increase of the level of -1 in the early sera may be a biochemical indicator of the function of the transplanted kidney. Cyclosporin makes the kidney. The expression of KIM-1 increased during the injury of proximal tubule cells. In this process, the p38 pathway in mitogen activated protein kinase and the ERK1/2 pathway play a role in this process. Blocking the p38 pathway, blocking the ERK1/2 pathway, and adding EPO three factors can reduce the formation of KIM-1 protein in the renal tubule cells under the action of CsA and effectively protect and delay the cyclosporin transplantation. The experimental results and conclusions of this topic provide a favorable basis for the application of renal injury molecular -1 in the field of renal transplantation, and provide a reference experimental model and effective experimental drugs for the reduction of the continued exploration of cyclosporin.

【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R699.2

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