棕色脂肪通过EVs/miRNAs调控肾脏FIH信号防治糖尿病肾病的作用及分子机制

Diabetic kidney disease (DKD) is one of the most common causes of chronic kidney disease (CKD), leading to premature death and end-stage renal disease (ESRD). In our prior study we found that the protective roles of miR-455 in the progression of DKD via AMPK/PGC1α pathway activation. Then, we proved that CL 316,243, a β3-adrenergic receptor agonist, significantly activated brown fat and led to marked decreases in urinary levels of albumin and 8-hydroxy-deoxyguanosine (8-OH-dG) in diabetic mice induced by high fat diet and STZ. Brown adipose tissue (BAT) has been regarded as a potential target organ for the treatment of obesity and related metabolic disorders. However, the protective concrete mechanism of BAT on DKD remains unclear. First we will use Cre/loxP technique to make a transgenic mice model with Dicer gene specifically knock out in adipose tissue (adipo-Dicer-KO). The adipo-Cre mice will be provided by Prof. Hongbin Zhang from Copenhagen University, Denmark. And the Dicerflox/flox mice will be introduced from Jackson Lab. Then, both adipo-Dicer-KO transgenic mice and wild type (wt) mice will be induced into diabetes mellitus by high fat diet (HFD) combined with STZ. The BAT from transgenic and wt mice will be transplanted into these diabetic mice. The development of renal disease will be monitored. The circulating extracellular vesicles (EVs) and the enclosed miRs will be assayed. Also we will detect the expressions of miRs, FIH, AMPK in the kidney. In vitro, we will employ podocytes to verify the function of factor inhibiting HIF (FIH) in cell apoptosis and degeneration under the high glucose condition. Then, the role of miRs targeting FIH will be researched in podocytes. Finally, we will use Dil-C18 flurochrome to mark the EVs from the supernatant of brown adipocytes, which will be injected into diabetic mice by tail vein. Then, the fluorescence and the miRs expression of the kidney will be analysed. The current study will clarify the function and the mechanism of BAT in the development of diabetic nephropathy by excreting EVs/miRs targeting kidney FIH/AMPK signaling pathway.

我们在前期研究中发现，调控棕色脂肪（BAT）分化发育的miR-455通过激活AMPK拮抗高糖引起的系膜细胞损害，而BAT激活剂能使糖尿病小鼠肾脏AMPK激活，天冬酰胺β羟化酶FIH表达改变，减少尿白蛋白。为明确BAT干预糖尿病肾病（DKD）的机制，我们拟采用脂肪组织特异性Dicer基因敲除小鼠，糖尿病造模后予BAT移植/移除，观察肾病进展，分析循环细胞外囊泡（EVs）miRNAs（miRs）谱及其靶基因、肾miRs/FIH/AMPK表达；进一步体外观察分别转染了目标miRs mimic及inhibitor的BAT细胞来源的EVs对相应靶基因沉默/过表达足细胞的影响；最后以Dil-C18荧光标记的分别包裹miRs mimic及inhibitor的BAT来源EVs尾静脉注射糖尿病小鼠，观察肾病进展及荧光分布。本课题将揭示BAT分泌EVs/miRs作用于肾脏FIH干预DKD的作用及分子机制。

基于前期研究成果我们发现，促进棕色脂肪（BAT）分化可以激活AMPK信号来改善糖尿病肾脏病中出现的能量代谢紊乱，减少尿白蛋白。为了明确糖尿病肾脏病中BAT的重要作用及相关分子机制，我们采用BAT移植/移除的方法干预糖尿病肾脏病（DKD）小鼠，并对循环中的细胞外囊泡（EVs）包含的miRNAs进行表达谱的分析，明确了BAT分泌的包含miR-30b的EVs是BAT移植改善DKD的重要miRNA，BAT移植的DKD小鼠中的miR-30b明显上调；进一步通过对DKD小鼠进行过表达/敲除miR-30b及其靶向基因位点分析验证，我们发现miR-30b的过表达可以通过靶向肾小管上皮细胞中的RUNX1、SNAIL1基因来显著改善DKD小鼠的尿白蛋白水平、胰岛素抵抗，从而明确BAT移植改善DKD的作用机制。在初步明确了miR-30b对DKD中肾小管上皮细胞中的靶基因RUNX1对的作用后，我们在DKD小鼠模型中使用腺相关病毒携带的过表达/敲减RUNX1的质粒进行干预治疗，发现靶向RUNX1可以减轻DKD小鼠肾脏炎症和纤维化，提示RUNX1可能是靶向DKD的重要靶点。本课题通过体内外实验及分子机制探讨，阐明了通过BAT激活和移植的代谢手段干预对DKD防治的应用价值及作用机制，为DKD防治的代谢性治疗提供了依据和理论基础；并对BAT分泌的miR-30b作用于肾小管上皮细胞的靶基因RUNX1进行了研究，为DKD的防治提供了潜在的新靶点。