维生素D受体在细胞核水平拮抗糖尿病肾病炎症反应的机制

Inflammation plays an important role in the pathegenesis and progression of diabetic nephropathy (DN), and vitamin D receptor (VDR) is widely recognized as a key reno-protective factor through its complex anti-inflammatory effects. Our previous clinical research has found significant down-regulation of nuclear VDR expression in type 2 DN patients, which is negatively associated with the expressions of several pro-inflammatory factors, including NF-κB and MCP-1. Our preliminary in vitro studies have further demonstrated that both VDR and NF-κB bind to nuclear pore proteins 214 (Nup214) for nuclear transportation, and that either VDR or NF-κB has shown differential nuclear or cytoplasmic distributions under high glucose conditions. Here, we intend to establish three high glucose-stimulated cell line models, including podocyte，mesangial and renal tubular epithelial cells, transiently transfect these cell models with epitope-tagged full-length, N-terminal, C-terminal constructs or siRNAs of VDR, NF-κB, Smad2 or Nup214, and investigate with CoIP whether VDR could competitively inhibit Nup214 binding of NF-κB or Smad2 and their subsequent nuclear transportion; we will use ChIP and EMSA assays to evaluate whether VDR, as a nuclear transcription factor, specifically recognizes and binds to putative vitamin D response elements on the promoter of two anti-inflammatory factors, MIP-1β and PTPN2, and regulates their transcriptions; we will further explore novel molecular mechanisms involved in the anti-inflammatory actions of nuclear VDR with STZ-induced diabetic mice and VDR-/- mice based on our in vitro findings in order to provide new clues and evidence basis for the prevention and treatment of DN.

炎症在糖尿病肾病(DN)发生发展中起重要作用，VDR通过抗炎发挥肾保护作用，但其机制尚未完全阐明。我们前期研究发现2型DN患者存在核内VDR下调，且与NF-κB、MCP-1等促炎因子呈负相关，细胞研究发现VDR和NF-κB均能与核孔蛋白214(Nup214)结合，且在高糖刺激下呈现核内外差异性表达。本研究拟构建不同片段VDR、NF-κB、Smad2和Nup214高表达质粒以及siRNA质粒，分别转染高糖诱导的足细胞/系膜细胞/肾小管上皮细胞，采用CoIP等方法证实VDR可竞争性抑制NF-κB、Smad2与Nup214结合及入核转运，采用ChIP、EMSA等方法证实VDR可通过特异性识别抑炎因子MIP-1β、PTPN2启动子上的维生素D反应元件调控转录；拟在db/db和STZ诱导糖尿病小鼠及VDR-/-小鼠模型上证实VDR在胞核水平拮抗DN炎症的分子机制，为早期防治DN提供新线索和实验支持。

本课题基本按计划完成，1.首次报道了2型糖尿病肾病患者肾活检组织中肾小管上皮细胞VDR表达减少，血中单个核细胞VDR表达下调且与白蛋白尿和PBMCs核内NF-κB/p65表达呈显著负相关，TNF-α可通过上调miR-346抑制人PBMCs的VDR表达，该论文发表在国际内分泌临床顶尖杂志J Clin Endocrinol Metab；2.成功构建了VDR敲除小鼠和VDR肾小管上皮细胞特异过表达小鼠，筛选出1932个差异mRNA和24个差异miRNA，以此为基础取得了2018 年国家自然科学基金面上项目（81870498）；3.本课题发现VDR敲除STZ糖尿病小鼠炎症反应增加、线粒体自噬紊乱加重，VDR激动剂可减轻STZ糖尿病小鼠肾组织的炎症和间质纤维化，其机制与VDR上调靶基因抗炎因子PTPN2、修复肾小管上皮细胞线粒体自噬紊乱以及抑制RhoA / ROCK信号通路有关；4.本课题观察到VDR在胞浆中可通过与NF-κB/p65的结合以及竞争性的抑制NF-κB/p65与核膜转运蛋白Nup214的结合减少NF-κB/p65入核，这是VDR发挥抗炎效应的重要机制之一，该项成果目前在投稿中；5.我们也首次报道了在自身免疫性疾病系统性红斑狼疮患者中，肾组织及外周血单个核细胞VDR表达下调。总之，通过该课题的完成，共发表SCI论文4篇，CSCD论文1篇，美国肾脏病年会壁报3篇，获得国家自然科学基金面上项目1项。