小肽 MRF 通过桥联ATF2 和CDK9复合物整合炎症和纤维化信号通路促进糖尿病肾病的发生和发展

Diabetic kidney disease is increasingly becoming one of the leading causes of end-stage renal disease in China. Currently we lack the specific treatments to delay the progression of diabetic kidney disease, making this unmet clinical need. It is believed that inflammation and fibrosis are the two major determinants in the development and progression of diabetic kidney disease. Recently studies demonstrated that long non-coding RNA (lncRNA) is a novel active regulator in the inflammatory and fibrotic networks. The role of lncRNA in the pathogenesis of diabetic kidney disease is worth exploring. To address this important question, we performed a microarray analysis and bioinformatics screen of lncRNA expressed in biopsy tissue from patients with progressive diabetic kidney disease compared to those with stable disease and other control tissues. Follow-up analysis using knockdown of lncRNAs in human mesangial cells identified a lead candidate lncRNA transcript that may be involved in kidney fibrosis. This lncRNA contains a short 168 nucleotide open reading frame which encodes a highly conserved 56 amino acid micropeptide which, based on our considerable preliminary data, we have named Micropeptide Regulator of Fibrosis (MRF). ..We used immunohistochemistry to confirm up-regulation of MRF protein expression in human progressive diabetic kidney disease, and up-regulation of MRF protein was also evident in mouse models of interstitial fibrosis (unilateral ureteral obstruction - UUO) and diabetic kidney disease (streptozotocin-induced diabetes in susceptible eNOS deficient mice). Next, we created mice lacking the MRF gene. These MRF-/- mice are viable and healthy and demonstrate profound protection against renal fibrosis in the UUO model. As a pharmacologic approach to knock-down MRF expression, we developed Locked Nucleic Acids antisense oligonucleotides targeting the MRF gene (LNA-MRF). Administration of LNA-MRF, but not control LNA, inhibited MRF expression and attenuated fibrosis in the UUO model. Finally, our biochemical analysis indicates a physical association of MRF with both ATF2 (a stress-induced transcription factor that promotes inflammation) and CDK9 (a positive regulator of TGF-β induced fibrosis). Therefore, MRF appears to play a unique role in regulating pro-inflammatory and pro-fibrotic signalling in progressive kidney disease. Based upon our compelling preliminary data, we hypothesize that MRF plays a central role in coordinating the inflammatory and fibrotic response in progressive kidney disease...The functional role of MRF will be determined by genetic (MRF knockout mice) and pharmacologic (LNA-MRF) approaches in moue model of progressive type I diabetic kidney disease. We will also perform mechanistic studies to explore how MRF integrates inflammatory and fibrotic signalling pathways to promote fibrosis through bridging ATF2 and CDK9 and what phosphorylation sites of MRF are related with its ability to bridge ATF2 and CDK9. This study presents a novel mechanism for the pathogenesis of DKD and provides a potential new therapeutic target for diabetic kidney disease.

我国糖尿病肾病（DKD）已快速成终末期肾病的主要病因之一，目前缺乏特异性的治疗手段。炎症和纤维化是其发病和进展的两个重要环节，我们从进展期的DKD肾活检中筛选到1个高度保守的长链非编码RNA （lncRNA），体外转录和翻译实验显示这个lncRNA含有一段168个碱基序列可编码56个氨基酸 （ 小肽）的开放阅读框，这个小肽我们称为MRF。利用我们制备的抗MRF抗体检测到人和小鼠进展期的DKD，MRF高度上调。在小鼠单侧输尿管结扎致纤维化模型中，敲除MRF基因（MRF-/-）或者用锁核酸LNA-MRF抑制MRF的表达明显减轻肾脏纤维化，从而证实MRF 的促纤维化作用。本研究利用我们制备的MRF-/-,eNOS-/-鼠和LNA-MRF在I 型 DKD模型中检测MRF的功能，同时探讨MRF磷酸化对CDK9和炎性转录因子ATF2侨联作用的影响，有可能为治疗DKD提供潜在新靶点。

2013年中国糖尿病(DM)患病人数为9840万，居全球首位。WHO预测，到2025年，中国将有1.3 亿的DM患者， 将消耗医疗预算的40%，严重阻碍经济的发展。糖尿病肾脏病(DKD）是糖尿病常见的微血管病发症之一，是目前引起终末期肾病（ESRD）的重要病因。终末期肾病患者需要接受透析或移植等肾脏替代治疗才能维持生命。DKD起病隐匿，一旦进入大量蛋白尿期后，进展至ESRD的速度大约为其他肾脏病变的14倍，而控制DKD的发生发展还缺乏特异性的治疗手段，是全球性难题。.我们从进展期的DKD肾活检中筛选到1个高度保守的长链非编码RNA （lncRNA），体外转录和翻译实验显示这个lncRNA含有一段168个碱基序列可编码56个氨基酸 （ 小肽）的开放阅读框，这个小肽我们称为MRF。利用我们制备的抗MRF抗体检测到人和小鼠进展期的DKD，MRF高度上调。在小鼠单侧输尿管结扎致纤维化模型中，敲除MRF基因（MRF-/-）或者用锁核酸LNA-MRF抑制MRF的表达明显阻断肾脏纤维化，从而证实MRF 的促纤维化作用。本研究我们利用制备的MRF-/-,eNOS-/-鼠和LNA-MRF在I 型 DKD模型中检测到MRF基因敲除和给小鼠腹腔注射LNA-MRF能保护肾功能 （PAS，Masson’s Trichrome, serum Creatinine，cystatin C，和尿蛋白） 减少肾脏纤维化（ɑ-SMA，Collagen IV和 Fibronectin）以及 F4/80(+)炎症细胞的侵润，延缓DKD的发生和发展，同时发现在DKD的发生发展过程中， MRF发生T49磷酸化， T49的磷酸化促进与 CDK9和炎性转录因子ATF2的相互作用， MRF从而在CDK9和ATF2中起侨联作用。 MRF和MRF T49磷酸化有可能成为治疗DKD潜在的新靶点。