肾脏缺血再灌注损伤通过上调TNF-α激活IkB/NF-kB通路增强BK病毒复制机制的研究

Polyomavirus BK infection and BK virus (BKV) associated nephropathy (BKVAN) have become a major complication in kidney transplant recipients, which affect the graft survival greatly. Previously, we found renal ischemia reperfusion injury(IRI) was an independent risk factor for BKV infection and BKVAN. However, the mechanism of the enhancement of BKV replication due to renal IRI is still not clear. Our pre-experiment results showed that BKV replication was enhanced greatly after renal tubular epithelial cells (RPTECs) with hypoxia-reoxygenation, which was associated with the increased expression of TNF-α and activation of IkB/NF-kB signal pathway. Thus, we hypothesize firstly that the new mechanism of the enhancement of BKV replication and antigen expression due to renal IRI involves activation of IkB/NF-kB signal pathway as a result of the increased expression of TNF-α. So our purposes are as the followings: ① we will observe the influence of renal IRI on BKV replication and antigen expression and determine whether this is through the increased expression of TNF-α and activation of IkB/NF-kB signal pathway; ② To detect the regulatory pathways of renal IRI －TNF-α－NF-kB－BKV replication and antigen expression in vivo and in vitro, we will use the lentiviral vector silencing and overexpressing TNF-α to treat RPTECs with hypoxia-reoxygenation and BKV infection, and use TNF-α shRNA (or anti-TNF-αmonoclonal antibody) and TNF-α to treat the mice with renal IRI and BKV infection, and then verify the activation of NF-kB and the BKV replication and antigen expression via Western Blot and real time - PCR methods.The aim of this project is to reveal the mechanism of the enhancement of BKV replication and antigen expression due to renal IRI. So that we might provide a scientific basis for the clinical application of prevention and treatment of BKV infection and BKVAN.

BK 病毒(BKV)相关性肾病(BKVAN)严重影响肾移植受者预后。前期研究表明,肾缺血再灌注损伤(IRI)是BKV感染及BKVAN独立危险因素。但肾IRI增强BKV复制机制尚不清楚。预实验显示肾小管上皮细胞(RPTECs)缺氧复氧后BKV复制明显增加，且与TNF-α和NF-kB增多有关。由此，提出肾IRI通过上调TNF-α激活IkB/NF-kB通路增强BKV复制的新机制。本课题首先观察肾IRI对BKV复制的影响及是否通过上调TNF-α和激活IkB/NF-kB完成的。然后通过慢病毒载体阻断和过表达TNF-α预处理缺氧复氧的RPTECs及TNF-α shRNA和TNF-α预处理肾IRI的小鼠并感染BKV，检测NF-kB水平及BKV复制和抗原表达水平，体内外验证肾IRI－TNF-α－NF-kB－BKV调控通路的存在。本项目将揭示肾脏IRI增强BKV复制的机制，为临床防治BKVAN提供科学依据。

多瘤病毒(PyV)肾病(PyVN)严重影响肾移植受者预后，但肾移植后PyV复制机制尚不清楚更没有相关的动物模型。我们通过小鼠的动物实验建立PyV感染的动物模型探讨相关机制，整个实验过程层层深入递进。首先，比较小鼠多瘤病毒（MPyV）胃肠道和呼吸道途径感染小鼠，MPyV可以通过口服途径和呼吸道途径感染小鼠。与呼吸道途径相比，口服途径，病毒明显扩散延迟并且峰值水平低，病毒载量在103-106 copies/mg（组织）之间。这两种途径造成的感染均为亚临床性。其次，MPyV胃肠道途径感染免疫抑制[每天均口服MMF (200 mg/Kg)和Tacro (0.2 mg/Kg)]的小鼠，可以造成感染加重，但尚未造成病毒相关性肾病的情况。进一步的，MPyV腹腔注射途径感染肾脏缺血再灌注损伤的免疫抑制的小鼠，MPyV在所有时间点均可以在很多器官被检测到，包括不常感染的器官如肺，主动脉弓，心脏，肝脏，等。感染峰值通常出现在3,7,10天。感染水平为105-107copies/mg（组织）之间。脾脏和主动脉弓水平最高。左肾病毒清除时间是第21天。其他器官均有10-1000倍的下降。尽管缺血再灌注损伤的左肾病毒水平高，但变异性很大，有些并未发生感染，组织学检测未发现与人类类似的多瘤病毒肾病。推测异体免疫反应产生的细胞因子连同免疫抑制和缺血再灌注损伤可能将是移植肾多瘤病毒肾病发生的根本机制。同时，细胞水平，我们发现缺氧复氧组肾小管上皮细胞感染BK病毒（BKV）后，病毒复制明显增加，而且培养液中的TNF-α浓度明显大于对照组(p<0.05)。相应的缺氧复氧组NF-kB核内表达量高于对照组。说明缺氧复氧后肾小管上皮细胞产生的TNF-α促进了BKV的大量复制，而且与IkB/NF-kB信号通路激活有关。最后，我们结合临床数据发现DCD供肾组织内C5aR水平较活体供肾高；发生BKPyVN（BK型多瘤病毒肾病）的供肾组织内C5aR分布范围较非BKPyVN广，且与PyV感染范围成正比；小鼠肾缺血再灌注损伤（IRI）感染PyV后肾组织内PyV含量与C5aR成正比，因此推测C5a-C5aR有可能是DCD肾移植TNF-α→IkB/NF-kB→BKPyV复制增强的上游信号，并有能构建出多瘤病毒肾病的动物模型用以今后相关机制的研究和抗病毒药物的开发。