微小RNA和Toll 7/8受体在糖尿病肾病炎症发病中的作用

Inflammatory lesions and fibrosis are the hallmark of progressive diabetic nephropathy (DN). The underlying molecular mechanism for the development and continual presence of inflammatory lesions in DN is not clear. Receptors for damage associated molecular pattern sense cell/tissue injury and have been implicated in the pathogenesis of DN inflammation. Toll like receptor 7 and 8 (TLR7/8) are endosomal receptors that bind single strand RNA to transduce anti-viral responses. Serial mapping has revealed a 20 nucleotides length U/GU rich single strand RNA as one of core components in viruses to ligate and activate TLR7/8. Since matured microRNA is a single strand RNA with the length of approximately 20 nucleotides, we have explored microRNA databases for U/GU rich sequence as a likely candidate of endogenous ligand for TLR7/8. We have identified 6 microRNAs that are U/GU rich. Among them, miR574-5p and miR-32-3p are evolutionarily conserved and thus, are the focus of our study. Our preliminary data show that miR-574-5p binded directly to extracellular domain of mouse TLR7 and human TLR8 and the binding was localized in the endosomes. Importantly, miR-574-5p caused a mTLR7/hTLR8 and myD88 dependent increase in STAT1 phosphorylation and IRF and NF-κB transcriptional activation, resulting in increased proinflammatory cytokines and chemokines. In this proposal, we will further determine the affinity and binding equilibrium constant of miR-574-5p to mTLR7/hTLR8 and examine the dynamic interaction between miR-32-3p and mTLR7/hTLR8 in vitro and in vivo. Additionally, the proinflammatory action of miR-574-5p and miR32-3p will be further investigated in both cells and mice. Since we have found that the levels of miR-574-5p were increased in diabetic kidney and that the expression of miR-574-5p in renal cells and monocytes was upregulated by high glucose, we will examine the contribution of proinflammatory actions of miR-574-5p and miR-32-3p to diabetic kidney inflammatory lesions. TLR7/8 are normally presence in human kidney and their expression was reported to be elevated in DN. Since we hypothesize that miR-574-5p and miR-32-3p act as endogenous ligands for mTLR7/hTLR8 to induce inflammation, we will examine a role of mTLR7 in DN in mice. In summary, the purpose of this proposal is to determine the novel function of miR-574-5p and miR-32-3p as endogenous ligands for TLR7/8 to cause inflammation and contributes to the inflammatory lesions in DN. Our hypothesis, if confirm, will add knowledge of the molecular basis of inflammatory diabetic kidney lesions.

炎症是糖尿病肾病进展的关键因素。高糖等所致细胞损伤启动炎症。 Toll受体是感受细胞损伤，转导炎症的重要分子。其中TLR7/8可被富含U/GU，长20个核苷酸的病毒单链RNA活化。 microRNA是约20个核苷酸的单链RNA。我们从数据库找到6个富含U/GU的microRNA，其中miR-574-5-p和 miR-32-3p进化上高度保守。前期研究发现，miR-574-5-p可以和鼠TLR7及人TLR8结合并经它们诱导炎症信号、IRF 和NF-κB转录、及炎症因子产生。因此我们推测miR-574-5-p和 miR-32-3p是mTLR7/hTLR8的内源性配体。由于糖尿病肾病miR-574-5-p表达增加，同时TLR7/8存在于正常肾脏并在糖尿病肾病时升高，我们推测由miR-574-5和miR32-3p连接活化mTLR7/hTLR8诱导的炎症可能在糖尿病肾病炎症发病中起到重要作用。

炎症是糖尿病肾病进展的关键因素。高糖等所致细胞损伤启动炎症。Toll受体是感受细胞损伤，转导炎症的重要分子。研究发现，miR-574-5-p可以和鼠TLR7及人TLR8结合并经它们诱导炎症信号、IRF 和NF-κB转录、及炎症因子产生，而肾脏纤维化是不同原因慢性肾脏病进行性恶化的病理表现。我们的研究发现，TLR7表达在肾脏纤维化模型中进行性升高。而且临床糖尿病肾病和IgA肾病伴纤维化的病人肾脏TLR7染色增加。敲除TLR7可以减轻小鼠由输尿管梗阻(UUO)引起的肾脏纤维化及炎症。而用TLR7激动剂治疗野生UUO小鼠则显著加重纤维化。miR-574-5p等 microRNA在纤维化肾脏表达显著增加，可以活化TLR7。它们可能不是通过经典途径，而是直接和TLR7结合起作用。研究结果将有助于增加我们对microRNA 作用和糖尿病肾病炎症及纤维化机制的认识。为今后制定有针对性的防治措施提供理论和实践的基础。