GPR120调节肾巨噬细胞表型在肾间质纤维化损伤修复中的作用

Renal interstitial fibrosis (RIF) is a polyetiological pathological process with complex pathogenesis and in shortage in effective therapy. One of the pathological features of RIF is macrophage infiltration. Macrophages can be divided into M1 and M2 phenotypes, which involve in tissue damage or repair respectively. A study showed that fatty acids receptor GPR120 is expressed on macrophages in adipose tissue and could induce macrophages to M2 types, which type has anti-inflammatory effects. We first observed the specific distribution of GPR120-positive cells in the kidney of RIF patients and of RIF mouse models, and the preliminary results showed that GPR120-positive cells are macrophages. We proposed that GPR120 activation can induce the transition of renal macrophages into M2 phenotype and en route to the anti-inflammation and tissue repair of RIF. This project plans to clarify the phenotypes of renal macrophages that are GPR120-positive using double-staining immunohistochemical method. Then the effects of GPR120 activation on the changes in renal macrophage types are designed to be demonstrated in isolated macrophages by observing the expression of markers for different types of macrophages and secretion of cytokines. Finally, RIF mice in wild type and in GPR120 deficient type will be treated by GPR120 agonists in vivo, and then the states of renal fibrosis and the changes in macrophage types will be observed to further demonstrate the role of GPR120 in regulation of macrophage types and in alleviation of renal fibrosis. This project is hoped to develop insight into the pathogenesis of RIF and to provide new drug target for RIF therapy.

肾间质纤维化（RIF）发病机制复杂，现尚无有效的治疗方法。肾间质巨噬细胞浸润是RIF的一个重要特征。巨噬细胞可分为M1和M2两种表型，分别参与炎症的损伤和损伤修复。在脂肪组织的研究表明，脂肪酸受体GPR120表达于巨噬细胞，并可促其转变为M2表型，起到抗炎作用。本研究组率先在人和小鼠的肾脏组织观察到GPR120特异性分布于肾间质的巨噬细胞。我们推测，GPR120激活可促进肾巨噬细胞向M2表型转变，进而抗炎和修复受损组织，减轻RIF。为此，本项目将首先明确GPR120阳性肾巨噬细胞的表型，随后离体培养肾巨噬细胞，观察GPR120激活对其表型的调节作用，最后通过给予不饱和脂肪酸等激活GPR120，观察野生型和GPR120基因敲除的RIF小鼠的RIF改善情况和肾巨噬细胞表型变化，进而确证GPR120调节肾巨噬细胞表型转变和减轻RIF。本项目有望为治疗RIF提供药物靶点，并加深对RIF的认识。

本课题以临床慢性肾脏病患者的资料和大鼠为研究对象，对肾巨噬细胞参与肾间质纤维化的作用及GPR120调节巨噬细胞在抗纤维化中作用展开研究，主要完成的内容包括：1）分析了巨噬细胞在各种慢性肾脏病患者肾脏中的浸润程度及其与临床参数、肾纤维化程度之间的关系。2）研究了GPR120激动剂TUG891的抗炎及抗肾间质纤维化的作用。.本研究收取慢性肾脏病患者的肾活检后标本及相关临床资料，将标本进行免疫组织化学染色，观察不同肾脏病理类型中巨噬细胞浸润的位置及程度，对各种慢性肾脏病的纤维化程度、临床参数与巨噬细胞浸润程度进行相关性分析。结果如下：各种慢性肾脏病均可见肾巨噬细胞浸润，各种慢性肾脏病患者的肾纤维化程度和肾巨噬细胞浸润程度之间存在显著正相关。随着巨噬细胞浸润程度的增加，不同慢性肾脏病患者血肌酐、尿素氮、胱抑素C相应增加，而eGFR相应减小。.本研究随后进行大鼠实验，首先分离培养大鼠腹腔原代巨噬细胞，经TUG891等处理，24小时后收取RNA，应用qRT-PCR检测细胞的M1和M2表型标记物表达水平。结果显示TUG891处理后巨噬细胞的M1表型标记物下降， M2表型标记物升高。这说明GPR120激动剂具有改变巨噬细胞表型的作用。随后制备大鼠UUO模型并分组处理。在UUO手术之前，每只大鼠腹腔巨噬细胞被分离培养并经TUG891等处理24小时。大鼠在UUO术中进行TUG891等处理后巨噬细胞的自身肾脏注射，于术后7天处死大鼠，计算结扎侧和对侧肾脏体积，收集术侧标本，RT-PCR和免疫染色观察纤维化相关蛋白水平。结果显示：所有大鼠输尿管结扎侧肾脏明显胀大，TUG891处理巨噬细胞回输组术侧肾脏较M1巨噬细胞回输组的肾脏体积胀大程度明显减弱；TUG891处理巨噬细胞回输组的纤维化蛋白的基因表达明显减少，染色显示纤维化程度减轻。.总之，慢性肾脏病纤维化程度和巨噬细胞浸润程度正相关。GPR120激动剂处理可显著减少巨噬细胞的炎症因子水平。GPR120激动剂处理的巨噬细胞自体肾脏注射具有显著抗纤维化作用，这为临床肾脏纤维化治疗提供了新的思路。