靶向上调脊髓LncTCONS_00058568在肾脏缺血再灌注损伤中的作用及机制研究

Renal ischemic reperfusion injury (RIRI) is the main risk factor of acute kidney injury (AKI), until now wihch is lack of effective approaches for preventing or treating. It hasbeen reported that lower thoracic spinal cord electrical stimulation affected renal function. Data from preliminary study revealed that lncRNAs were differently expressed in the lower thoracic spinal cord after RIRI. The LncTCONS_00058568 expression level was closely related to the renal damage. Furthermore, we found that overexpression of LncTCONS_00058568 down-regulated P2X7 receptor (P2X7R) expression in vitro and in vivo. Additionally, studies have reported that the P2X7R expression was related to sympathetic nerve activity. Therefore, we raise a hypothesis that LncTCONS_00058568–regulated P2X7R expression in the spinal cord might improve RIRI via decreasing the renal sympathetic nerve activity. In the present proposal, by establishing a rat model of renal ischemic reperfusion, LncTCONS_00058568 lentivirus will be given to the lower spinal cord, observing the protective effects of targeting up-regulated LncTCONS_00058568 on the RIRI and its mechanism. The mechanism of LncTCONS_00058568 regulating P2X7R expression will be elucidated by the RIP and CLIP experiments. This study is expected to provide a new target and theoretical basis for treatment of ischemic-related kidney injury.

肾脏缺血再灌注是急性肾损伤的主要危险因素，目前缺乏有效的防治手段。已有报道下胸节脊髓电刺激影响肾功能。申请者前期研究发现肾脏缺血再灌注损伤大鼠下胸节脊髓lncRNAs表达明显变化，其中TCONS_00058568表达与肾损伤程度密切相关，过表达TCONS_00058568在体内外实验中均下调P2X7R表达。此外，有研究报道P2X7R表达可影响交感神经活性。因此提出假说：脊髓特异性lncRNA调控P2X7R表达，降低肾交感神经活性，改善缺血性肾损伤。本项目拟建立大鼠肾缺血再灌注模型，在下胸节脊髓给予TCONS_00058568慢病毒，观察其在缺血性肾损伤中作用及机制，并通过RIP、CLIP实验阐明TCONS_00058568调控P2X7R表达的机制。本研究有望为临床治疗缺血性肾损伤提供新的靶点及理论依据。

在急性肾损伤(AKI)发病机制的研究中，中枢神经系统（CNS）对肾脏损伤的影响越来越受到关注且机制复杂。本人前期研究发现肾脏缺血再灌注（I/R）损伤大鼠下胸节脊髓lncRNAs 表达明显变化，其中LncTCONS_00058568表达与肾损伤程度密切相关。有研究报道微炎症状态下脊髓组织P2X7R高表达，并可影响交感神经活性。本课题构建LncTCONS_00058568过表达慢病毒，在下胸节脊髓立体定向注射LncTCONS_00058568过表达慢病毒后，建立大鼠肾脏I/R诱导的AKI模型，发现脊髓过表达LncTCONS_00058568可改善大鼠肾功能和减轻肾组织病理损伤。此外，过表达LncTCONS_00058568可抑制促炎性细胞因子释放以及降低交感神经活性标志物酪氨酸羟化酶（TH）和去甲肾上腺素（NE）的表达水平。利用RT-PCR和Western blot技术从mRNA和蛋白两个水平明确了过表达LncTCONS_00058568可抑制脊髓P2X7R表达以及PI3K/Akt通路磷酸化。此外，肾I/R模型中脊髓小胶质细胞活化明显增加，免疫荧光双标法发现脊髓P2X7R与小胶质细胞共表达。由此，我们推测脊髓过表达LncTCONS_00058568可抑制小胶质细胞P2X7R表达，通过PI3K/Akt信号通路介导交感神经活性传导途径，从而缓解I/R诱导的肾损伤。我们的研究从分子水平证实了CNS对AKI的调控作用，这将为AKI的临床治疗提供新的治疗靶点与新的途径。