GDF15在肾移植缺血-再灌注损伤中的作用及机制研究

Role and mechanism of GDF15 in ischemia-reperfusion injury during kidney transplantation

目的探讨生长与分化因子(GDF)15在肾移植缺血-再灌注损伤(IRI)中的作用及机制。方法选取野生型供体小鼠9只,野生型受体小鼠9只,分别于术后4、24、72 h取3只受体小鼠的移植肾,进行GDF家族转录组学分析,检测各组肾组织GDF15的表达情况。选取野生型供体小鼠5只,GDF15敲除型供体小鼠5只,野生型受体小鼠10只,根据实验方案将小鼠分为野生型假手术组、野生型移植组、GDF15敲除假手术组、GDF15敲除移植组,于术后72 h提取血清及肾组织样本,对比各组肾功能、肾小管损伤情况、炎症细胞浸润、炎症因子及Toll样受体4(TLR4)、核因子(NF)-κB表达水平。选取野生型供体小鼠9只,GDF15敲除型供体小鼠9只,野生型受体小鼠18只,根据实验方案将小鼠分为野生型移植组、GDF15敲除移植组,观察肾移植术后两组生存率。结果GDF15是移植肾转录组学中上调最多的GDF家族基因,主要在肾小管中表达。与假手术组比较,移植组小鼠肾功能下降;与野生型移植组比较,GDF15敲除移植组小鼠血清肌酐、血尿素氮水平升高(均为P<0.05)。野生型移植组小鼠术后1周生存率为87.6%,GDF15敲除移植组小鼠术后1周生存率为41.8%。GDF15敲除移植组肾损伤分子(KIM)-1表达增多,肾小管损伤评分更高。野生型移植组肾小管可见溶解或坏死,髓外和皮质中可见管型形成,而GDF15敲除移植组肾小管坏死及管型更加明显。移植组髓过氧化物酶(MPO)和F4/80表达增多,且GDF15敲除移植组炎症细胞浸润加重。与假手术组比较,移植组肿瘤坏死因子(TNF)-α、白细胞介素(IL)-1β和IL-6表达水平升高;与野生型移植组比较,GDF15敲除移植组TNF-α、IL-1β和IL-6表达水平升高(均为P<0.05)。移植组肾组织中TLR4、NF-κB表达较假手术组增多;GDF15敲除移植组肾组织中TLR4和NF-κB表达较野生型移植组增多。结论GDF15可减轻移植肾IRI,其作用机制可能与抑制TLR4-NF-κB信号通路有关。

Objective To investigate the role and mechanism of growth differentiation factor(GDF)15 in ischemia-reperfusion injury(IRI)during kidney transplantation.Methods Nine wild type donor mice and 9 wild type recipient mice were selected.The renal graft of 3 recipient mice were harvested at 4,24 and 72 h after transplantation.GDF family transcriptome analysis was carried out,and the expression of GDF15 in renal tissues of each group were detected.Five wild type donor mice,5 GDF15 knockout donor mice and 10 wild type recipient mice were selected.According to the experimental scheme,the mice were divided into wild type sham operation group,wild type transplantation group,GDF15 knockout sham operation group and GDF15 knockout transplantation group.Serum and renal tissue samples were extracted 72 h after transplantation.The renal function,renal tubular injury,inflammatory cell infiltration,inflammatory factors,Toll-like receptor 4(TLR4)and nuclear factor(NF)-κB expression level were compared in each group.Nine wild type donor mice,9 GDF15 knockout donor mice and 18 wild type recipient mice were selected.According to the experimental scheme,the mice were divided into wild type transplantation group and GDF15 knockout transplantation group,and the survival rate of two group after kidney transplantation was observed.Results Transcriptome sequencing of renal graft tissues indicated that GDF15 was the most up-regulated GDF family gene,which was mainly expressed in renal tubules.Compared with the sham operation group,the renal function of mice was declined in the transplantation group.Compared with the wild type transplantation group,the serum creatinine and blood urea nitrogen levels of mice were significantly up-regulated in the GDF15-knockout transplantation group(both P<0.05).The 1-week survival rate of mice was 87.6%in the wild type transplantation group and 41.8%in the GDF15 knockout transplantation group.In the GDF15 knockout transplantation group,the expression level of kidney injury molecule(KIM)-1 was up-regulated,and the renal tubule injury score was increased.In the wild type transplantation group,the renal tubules were dissolved or necrotized,and tubular formation was seen in the extramedullary and cortex area,whereas tubular necrosis and tubular formation were more evident in the GDF15 knockout transplantation group.The expression levels of myeloperoxidase(MPO)and F4/80 were up-regulated in the transplantation group,and the inflammatory cell infiltration was aggravated in the GDF15 knockout transplantation group.Compared with the sham operation group,the expression levels of tumor necrosis factor(TNF)-α,interleukin(IL)-1βand IL-6 in the transplantation group were up-regulated.Compared with the wild type transplantation group,the expression levels of TNF-α,IL-1βand IL-6 were also up-regulated in the GDF15 knockout transplantation group(all P<0.05).In the transplantation group,the expression levels of TLR4 and NF-κB in the renal graft tissues were higher than those in the sham operation group.In the GDF15 knockout transplantation group,the expression levels of TLR4 and NF-κB in the renal graft tissues were higher compared with those in the wild type transplantation group.Conclusions GDF15 may alleviate the IRI of renal graft probably via inhibiting the TLR4-NF-κB signaling pathway.