糖尿病肾间质病变中巨噬细胞极化对肾组织干细胞的影响及miRNA-125b调控机制初探

According to our recent research, the diminished kidney functional reserve capacity in diabetes patients may result from the due to the impaired function of the kidney stem cells (KSCs) which is resident in the renal papilla interstitial in the high glucose environment, then influence the recovery of renal tubules from injury. Thus, the aim of our study is to test the hypothesis that the high glucose elicits the unbalanced of the bone marrow derived macrophage (BMDM) polarization in the diabetic papilla interstitial injury, then influence the repair function of KSCs, miRNA125b is very important in the process of BMDM polarization, through regulating miRNA125b we could change BMDM polarization situation, thus protecting KSCs repair function. First, in vitro cell culture studies will be applied to determine the influence of high glucose on the mice BMDM to polarize into MI or M2 macrophages. Mean while, the regulated effects of miRNA125b and its pathophysiological mechanism on the BMDM polarization are observed. Second, the BMDM co-culturing with KSCs via Transwell will be used to test the impacts of macrophage phenotype from M1 to M2 differentiation on the repair function of KSCs, and the regulating effect of miRNA125b will also be evaluated. Finally, the regulated effects of miRNA125b on the macrophage polarization and the kidney functional reserve capacity will be investigated in the rat model of diabetes. The blood and urine biochemistry analysis, the renal pathologic lesions, and the renal immunohistologic changes will be tested in the rat model. The regulation of the macrophage polarization status may be the target in protection of KSCs function, this target may provide a pathway to investigated the early pathogenesis of diabetic kidney disease and to find the new therapies on diabetic renal function protection.

我们前期研究发现:糖尿病（DM）患者肾储备功能差的源头在于肾乳头间质部位的肾组织干细胞（KSCs）在高糖的环境下对肾小管上皮细胞的修复功能受损。我们在此基础上提出“高糖可能通过引起骨髓来源的巨噬细胞（BMDMs）在肾间质部位聚集并发生极化作用（M1/M2比例失调），从而影响KSCs修复功能；通过调控miRNA-125b改变BMDMs极化状态，可能保护KSCs的修复功能。” 本项目将在体外观察高糖对BMDMs极化的影响，从细胞因子和miRNA-125b表达等方面探讨其病理生理机制；利用细胞共培养观察BMDMs极化改变对KSCs修复功能的影响及miRNA-125b的调控作用。在体内通过DM动物模型观察肾间质BMDMs的极化，并通过对miRNA-125b的调控评估DM大鼠肾脏储备功能改善情况。为探讨DM肾病早期发病机制及肾功能保护奠定理论基础。

项目背景：我们前期研究发现，糖尿病患者肾储备功能差的源头在于肾乳头间质部位的肾组织干细胞（KSCs）在高糖的微环境下对肾小管上皮细胞对修复功能受损，而有研究表明单核巨噬细胞在肾组织中的浸润可能是早期糖尿病肾小管间质病变的重要病理生理改变之一，那么巨噬细胞在高糖的微环境是否会发生极化作用，而这种极化作用是否能影响KSCs的修复能力成为本研究的关注点。.研究内容：1、小鼠骨髓来源巨噬细胞（BMDMs）分离及诱导培养方法的建立并评估高糖对巨噬细胞极化作用的影响。2、评估BMDMs发生的极化改变后是否影响KSCs的修复功能；3、建立2型糖尿病大鼠动物模型，观察糖尿病性肾小管间质早期病变，并初步探讨其损伤机制。.重要结果：细胞实验部分，我们成功的分离培养出小鼠骨髓来源BMDMs并证实其向M1和M2细胞亚型分化能力。BMDMs经过高糖刺激48h后向M1亚型极化。随后我们评估了巨噬细胞极化改变对肾组织干细胞（KSCs）修复功能的影响，首先建立RTEC缺氧/复氧H/R模型，缺氧后，RTEC凋亡率升高，上清的LDH和MDA水平升高，SOD水平降低。进行KSCs上清干预24h后，上述损伤减轻。而当KSCs经过M1型BMDMs预处理和高糖BMDMs预处理后，与未干预组相比，RTEC的凋亡率、LDH和MDA水平均高于正常组，而SOD低于正常组，提示预处后的KSCs对缺氧损伤的RTEC的修复作用明显减弱。动物实验部分，建立T2DM大鼠模型，在STZ注射后6周即可出现尿NAG和KIM-1的升高，光镜及电镜病理也证实存在肾小管间质病变。蛋白质组学筛查发现，与NC组相比，DM组有388个差异表达蛋白，其中上调173个，下调215个；TLR2/4炎症通路相关蛋白髓样分化因子88 (MyD88)表达明显上调，之后我们通过免疫组化等方法，再次验证T2DM大鼠早期肾小管间质病变与TLR2/4炎症通路的激活密切相关。.科学意义：本研究的结果可以更新目前有关对糖尿病性肾脏疾病的病理生理机制的认识，为发现糖尿病性肾脏疾病早期病变并发掘早期防治的靶点提供一定的科学依据。