抑制miR-150对狼疮肾炎肾纤维化的防治作用和机制研究

MicroRNAs (miRs) are promising biomarkers and involved in the pathogenesis of many kidney diseases. However, the mechanism and therapeutic role of miRs in renal fibrosis in lupus nephritis (LN) still remains unknown. Recently we have identified by microRNA microarray and validated by RT-PCR that miR-150 significantly increased in kidney biopsies from LN patients with high renal chronicity index (CI) compared to those with low CI. In addition, renal miR-150 is positively correlated with CI, profibrotic proteins TGF-β1, fibronectin (FN), collagen1 (COL1), and collagen3 (COL3) but negatively correlated with antifibrotic protein, suppressor of cytokine signal 1 (SOCS1). In the present study, we aim to answer the following three questions: 1) Can miR-150 inhibitor (LNA-anti-miR-150) prevent the development of LN and further block the progress of renal fibrosis of LN? 2) Can renal miR-150 be downregulated by a known effective treatment agent in renal fibrosis? 3) Can renal miR-150 be a useful biomarker to predict the treatment effect in a clinical setting? To answer these questions, LNA-anti-miR-150 is administrated (2mg/kg, sc, twice per week) to Fcgr2b-/- mice (a spontaneous LN mouse model) before and after LN development and Adriamycin-injected mice (a well defined progressive renal fibrosis model used as LN disease control) that are known to respond to vitamin D3 treatment. Renal miR-150 level, the expression of TGF-β1, FN, COL1, COL3, and SOCS1 at both mRNA and protein level are measured, and renal fibrosis severity is scored in all animal kidneys 6-16 weeks after the treatments in both animal models. The effect of LNA-anti-miR-150 on the above parameters and the traditional clinical indices such as serum ANA, BUN, and urinary albumin/creatinine were compared in different animal groups and the correlation between miR-150 and pro-/anti-fibrotic genes are analyzed. In addition, to evaluate the usefulness of miR-150 in clinical practice, a retrospective study is conducted in a small numbers of archived renal biopsies from LN patients grouped into treatment responders and non-responders based on the records of a 5-year follow up. Renal miR-150 level is examined in LN renal biopsies and logistic regression and receiver characteristic (ROC) is analyzed to verify whether renal miR-150 can predict responders and non-responders to LN treatment. Any possible positive findings from animal models will likely provide a novel insight to understand the pathogenesis of LN or discover a new therapeutic approach for LN. Any possible positive result from LN patients will likely help clinical decision making so that toxic therapeutic agents can be used sparingly or stopped if they are ineffective.

microRNAs(miR)被证实参与多种肾脏病发生机制，但在狼疮肾炎（LN）肾纤维化发生发展中的机制及防治应用尚无报道。本组新近应用microRNA microarray发现并用RT-PCR证实在LN病人肾活检中miR-150与肾纤维化程度显著正相关。本实验拟用自发性LN Fcgr2b-/-小鼠，在LN发生前、后分别给与miR-150抑制剂(LNA-anti-miR-150)，用阿霉素诱导的肾纤维化作为LN疾病对照模型，给与有效的VitD3 或miR-150抑制剂。检测肾miR-150、抗纤维化因子SOCS1、促纤维化因子TGF-β1及肾纤维化程度变化；并回顾分析LN病人肾miR-150与五年后疗效的相关性。旨在探明：1）miR-150在LN肾纤维化发生发展中作用机制、2）miR-150抑制剂能否防治LN、3）miR-150在LN临床治疗和预后评估中的应用价值。为LN临床防治提供新方法。

肾纤维化是慢性肾脏病的最终病理形式，抑制肾脏纤维化进而抑制慢性肾脏病的进展为终末期肾病对国家、社会、家庭、患者都具有重要意义。我们前期工作发现，在狼疮性肾炎(LN)患者重复肾活检组织中微小RNA-150（miR-150）的表达与肾脏纤维化程度正相关。本项目旨在该前期工作的基础上探明抑制miR-150在肾脏纤维化中的肾保护作用及其机制。通过给予Fcgr2b-/-自发LN小鼠及与阿霉素诱导的局灶节段肾小球硬化（FSGS）小鼠miR-150抑制剂（LNA-anti-miR-150）观察LNA-anti-miR-150治疗肾脏纤维化的治疗效果，对促炎性与促纤维化基因及抗炎与抗纤维化基因的调节效果。研究结果发现：1）LNA-anti-miR-150皮下注射可被肾组织充分吸收并有效抑制了Fcgr2b-/-自发LN与阿霉素诱导的局灶节段肾小球硬化（FSGS）肾组织miR-150表达增高。2）在两种肾脏纤维化动物模型中，LNA-anti-miR-150与miR-150 scrambled LNA（与LNA-anti-miR-150具有相同核酸数但是碱基无序排列无功能作为LNA-anti-miR-150 阴性对照）比较结果为：a)LN小鼠血清抗核抗核(ANA)水平及尿蛋白排泄水平显著降低，肾小球及间质纤维化程度明显改善；FSGS小鼠蛋白尿、低蛋白血症及高脂血症显著改善，肾小球硬化减轻。b）LN与FSGS肾组织中的促炎因子IL-6A,IFN,TNF表达降低，促纤维化因子TGF-β，α-SMA，FN表达降低；抗炎与抗纤维化因子SOCS1表达上调。3）初发未治LN和FSGS患者的肾活检组织中miR-150的表达水平显著增高。上述结果提示：1）抑制miR-150可以缓解继发LN与原发FSGS导致的肾脏纤维化。2）LNA-anti-miR-150在肾脏纤维化中的保护作用可能通过下调促炎性及促纤维化基因，上调抗炎及抗纤维化基因的表达。3）LNA-anti-miR-150可能作为治疗肾纤维化的新型治疗方法，为肾纤维化精准治疗提供理论基础。