足细胞Cx43介导的细胞间miR-150传递在高胆红素血症加重肾缺血再灌注损伤中的作用研究

Acute kidney injury following cardiac surgery is a severe complication, and the hyperbilirubinemia in the perioperative period of cardiac surgery is a risk factor to acute kidney injury. Our previous data demonstrated that hyperbilirubinemia induced the apoptosis of renal tubular epithelial cell and aggravated renal ischemia reperfusion injury (RIRI). However, the mechanism remains elusive. The latest study found that injury podocytes expressed miR-150. Applicant’s preliminary experiment showed that hyperbilirubinemia leaded to early impairment of podocytes such as foot processes effaced and effacement and induced the expression of Cx43 on podocytes and that gap junction composed by Cx43 can delivery miR-150 mimics. It is hypothesized that hyperbilirubinemia up-regulates the expression of miR-150 and Cx43 in early injury podocytes and that the miR-150 is delivered to podocytes via Cx43 and to tubular epithelial cells, which inhibits autophagy, and thus aggravates RIRI. The aim of this study is to elucidate the permeability and activity of Cx43 to miR-150 by plasmid transfection and microinjection and the role of Cx43 on podocyte delivering miR-150 to inhibit autophagy in hyperbilirubinemia aggravated RIRI in vitro and in vivo, and finally the Cx43 blocker mimic peptides were investigated to achieve the mechanism-based treatment.

急性肾损伤是心脏手术围术期的严重并发症，而围术期高胆红素血症是急性肾损伤的危险因素。我们前期报道高胆红素血症会增加肾小管上皮细胞调亡并且加重肾缺血再灌注损伤（RIRI），但机制不明。近期有研究发现足细胞损伤会高表达miR-150。申请者预实验发现高胆红素血症引起足细胞足突融合、足突消失等早期损伤改变，同时会上调足细胞Cx43的表达，Cx43组成的缝隙连接可以传递miR-150模拟物。据此假设，高胆红素血症诱导足细胞早期损伤，上调Cx43，足细胞损伤产生的miR-150通过Cx43传递至足细胞及肾小管上皮细胞，抑制自噬，加重RIRI。本研究拟通过离体实验利用质粒转染和显微注射等方法验证Cx43对miR-150的通透性及活性，并在细胞和动物水平探讨Cx43传递miR-150抑制自噬在高胆红素血症加重RIRI中的机制，再通过Cx43阻滞模拟肽进行干预，为实现基于机制的治疗提供理论基础。

高胆红素血症是心脏手术围术期常见并发症，会加重急性肾损伤，但是机制不明。本研究通过腹腔注射胆红素建立SD大鼠高胆红素血症模型，发现胆红素增加肾脏氧化应激和自噬水平，引起足细胞损伤，肾小球损伤，增加Cx43的表达。细胞实验发现，胆红素能够诱导肾小管上皮细胞自噬，增加PINK1和Parkin表达，增加肾小管上皮细胞缺氧复氧的自噬水平，降低细胞缺氧复氧线粒体膜电位，增加细胞缺氧复氧凋亡，降低细胞活性。通过C57BL/6小鼠模型研究发现高胆红素血症诱导肾小管上皮细胞自噬，增加肾缺血再灌注PINK1和Parkin表达，加重肾缺血再灌注自噬水平和凋亡水平，加重肾缺血再灌注肾脏损伤和纤维化。抑制自噬或沉默PINK1会减轻高胆红素血症加重的肾缺血再灌注损伤程度、氧化应激、凋亡和纤维化水平。胆红素增加肾脏自噬，损伤肾小球和肾小管上皮细胞。胆红素通过PINK-Parkin介导的线粒体自噬加重肾缺血再灌注损伤。自噬或PINK1可能是治疗高胆红素血症加重的肾缺血再灌注损伤的靶点，为临床诊疗提供新的思路。