NLRP3炎症体在对比剂所致急性肾损伤发病中的作用和机制研究

Following the widely applications of iodinated contrast-media in disease diagnosis and treatment, the incidence of contrast-induced acute kidney injury (CI-AKI) markedly increases in both ambulatory and hospitalized patients. The pathogenesis of CI-AKI is not fully understood yet, and our previous research indicated that the administration of iodinated contrast could trigger local innate immune response in the kidney and urinary IL-18 levels are significantly elevated in patients after receiving coronary angiography. The NOD-like receptor protein 3 (NLRP3) is a newly identified member of the innate immune system sensors. NLRP3 can be triggered by diverse stimuli and lead to a cascade of events including the formation of the NLRP3 inflammasome, which results in processing and secretion of mature IL-18 and IL-1β by activated caspase-1. The present study is aiming to investigate the role of NLRP3/inflammasome axis in the pathogenesis of CI-AKI. Human renal proximal tubular epithelial cells (PTEC) and murine CI-AKI model will be used in the in vitro and in vivo experiments, respectively. We will firstly determine whether the administration of iodinated contrast could induce the expression of NLRP3, formation of inflammasome, activation of caspase-1, and secretion of mature IL-18 and IL-1β in both human PTEC and murine kidney tissue. Then we will silence the NLRP3 gene expression or interfere the inflammasome formation by RNA interference in cells and gene knockout in animals, and further investigate whether blocking the NLRP3/inflammasome axis could inhibit the activation of caspase-1 and the secretion of mature IL-18 and IL-1β, thus ameliorate cell inflammatory responses, EMT and apoptosis, as well as murine renal tubular injury, necrosis, and fibrosis induced by the administration of iodinated contrast, finally preserve the kidney function and delay the progression of CI-AKI. These data will allow us to gain further insight into the mechanism of CI-AKI, which is prerequisite to designing novel therapeutic strategies to preserve the structural and functional properties of the kidney after the administration of iodinated contrast.

含碘对比剂在疾病诊断和治疗中广泛应用使得对比剂所致急性肾损伤（CI-AKI）发病率急剧上升。本课题组既往研究发现对比剂可激发肾脏局部先天免疫反应，且尿液IL-18浓度显著升高。NLRP3是近年发现的胞内先天免疫系统模式识别受体，激活后可形成炎症体并活化caspase-1，引起IL-18和IL-1β的成熟和释放。本研究拟通过体外和体内两部分实验，探讨对比剂可否激发人近端肾小管上皮细胞或CI-AKI小鼠模型肾组织中NLRP3的表达、炎症体的形成、caspase-1活化、IL-1β和IL-18释放。通过细胞RNA干扰及小鼠基因敲除沉默NLRP3表达或影响炎症体形成，进一步探讨阻断NLRP3/炎症体轴可否抑制caspase-1活化及IL-18、IL-1β的释放，减轻细胞EMT和凋亡、肾小管损伤坏死，从而延缓CI-AKI的发生发展。本研究结果可为CI-AKI发病机制和治疗靶点提供新的理论依据。

研究背景：.对比剂引起的急性肾损伤（contrast-induced acute kidney injury，CI-AKI）已成为院内获得性急性肾损伤的第三大原因，CI-AKI的发病机制尚未完全明确，目前研究发现炎症反应可能参与了CI-AKI的发生与发展，核苷酸结合寡聚化结构域样受体蛋白3（nucleotide-binding oligomerization domain [NOD]-like receptor protein 3， NLRP3）是新发现的一种模式识别受体，参与炎症反应，有研究发现NLRP3参与了急性肾损伤的发病过程。.研究内容：.本研究拟通过体外（人肾小管上皮细胞（HK-2细胞株））和体内（建立CI-AKI小鼠模型）两部分实验，探讨NLRP3炎症体在CI-AKI发生发展中的作用和机制。.研究数据与结果：.1.对比剂可以引起HK-2细胞凋亡，低渗对比剂欧乃派克可引起HK-2细胞内NLRP3炎症体相关蛋白NLRP3、ASC mRNA升高。渗透压不是引起NLRP3、ASC mRNA升高的因素。 .2.低渗对比剂欧乃派克可激活HK-2细胞内NLRP3炎症体通路，沉默通路的NLRP3基因或ASC基因可阻断炎症体活化；低渗对比剂欧乃派克诱导HK-2细胞凋亡、沉默NLRP3基因或ASC基因可减轻细胞凋亡。 .3.单侧肾切除+脱水24h+速尿10ul/g+欧乃派克是一种较好的低渗对比剂造CI-AKI小鼠模型的方法。 .4. NLRP3-/-小鼠可更好地抵御CI-AKI引起的肾脏损伤。.科学意义：.低渗对比剂欧乃派克通过活化NLRP3炎症体通路诱导肾小管上皮细胞的损伤，阻断NLRP3炎症体通路可以缓解肾脏损伤。因此，对NLRP3炎症体通路的调控可能成为治疗或预防CI-AKI发生的新靶点。该研究结果可为保护造影术后患者肾功能、改善CI-AKI患者预后提供新的理论依据。