趋化因子CX3CL1/CX3CR1在糖尿病肾脏纤维化的作用机制研究

Diabetic nephropathy (DN) is one of the most common diseases requiring renal replacement therapy. DN is characterized by infiltration of inflammatory mediators and activation of fibroblasts resulting in the accumulation of extracellular matrix in both the glomerular mesangium and tubular interstitum. But the underlying mechanism of the inflammation in DN is still poorly understood. Chemokines are synthesized at sites of inflammation and are known to be major regulatory proteins for leukocyte recruitment and activation.CX3CL1/CX3CR1 is involved in many inflammatory condition, such as infection and atherosclerosis, even in the fibrosis of liver and lung. Recently, it was demonstrated the expression of CX3CL1/CX3CR1 is upregulated in DN. But the specific role of CX3CL1/CX3CR1 in DN is still unclear. Recent evidences indicate that fibroblast in the kidney may originate from epithelial/endothelial-to-mesenchymal transition (EMT) and bone marrow-derived progenitor cells. It was demonstrated that macrophage can induce epithelial-to-mesenchymal transition by the secretion of transforming growth factor-beta and matrix metalloproteinases. And the expression of CX3CR1 on the surface of bone marrow-derived fibroblast precursors indicate that CX3CL1 may be involved in the infiltration of bone marrow-derived fibroblast into the kidney. In the light of the available data reviewed above, we hypothesized that the upregulation of CX3CL1 induced by high glucose and advanced glycation end products plays an important role in the progression of diabetic nephropathy by recruiting bone marrow-derived fibroblast precursors and inflammatory cells which can release inflammatory factors to induce epithelial-mesenchymal transition in renal proximal tubular epithelial cells. We therefore propose to investigate the role of CX3CL1/CX3CR1 in the streptozotocin-induced diabetic kidneys. The expression of CX3CL1/CX3CR1 and the markers of the fibrosis and EMT will be analyzed by real-time PCR, Western blot and histology. And flow cytometry and immunohistochemistry will serve to characterize the different type of inflammatory leukocyte and bone marrow-derived fibroblast. To determine whether or not CX3CL1/CX3CR1 has functional role in diabetic nephropathy will be essential to evaluate its use in further therapeutic interventions.

随着对发病机制的深入研究，糖尿病肾病逐渐被认为是一种炎症性疾病，肾小球和肾间质炎症细胞浸润导致成纤维细胞激活、细胞外基质积聚。但是炎症反应导致肾脏纤维化的具体机制至今不完全清楚。研究表明趋化因子CX3CL1具有趋化、黏附以及激活炎症细胞的功能，在糖尿病、动脉粥样硬化以及肝脏、肺组织纤维化中起重要作用。其在糖尿病肾脏纤维化中的作用机制有待研究。本项目拟采用高糖和糖基化终产物刺激肾脏系膜细胞和肾小管上皮细胞的离体模型以及链脲佐菌素诱导糖尿病肾病的动物模型为研究对象，通过siRNA阻断CX3CL1合成或采用CX3CR1基因敲除小鼠，观察肾脏损伤、纤维化、炎症细胞浸润、炎症因子表达、上皮细胞转分化以及骨髓来源的成纤维细胞浸润情况，探讨CX3CL1/CX3CR1在糖尿病肾脏纤维化的具体作用机制，从而丰富糖尿病肾病发病理论，积极探索阻断糖尿病肾脏纤维化新的靶点。

按照国家自然科学基金研究计划，我们成功构建了糖尿病肾病小鼠动物模型，并且探讨了趋化因子CX3CL1和CX3CR1在糖尿病肾脏纤维化中的作用。我们发现糖尿病肾病模型小鼠肾脏中趋化因子CX3CL1和CX3CR1以及纤维化相关指标Fibronectin、Collagen I和TGF-β1 的mRNA和蛋白水平显著升高，给予辛伐他汀后明显降低。高糖刺激系膜细胞后趋化因子CX3CL1、CX3CR1、Fibronectin、Collagen I和TGF-β1明显升高，给予辛伐他汀后明显降低。有趣的是，CX3CL1不仅可以作为趋化因子募集炎症细胞参与肾脏病变，而且可以直接刺激系膜细胞产生Fibronectin、Collagen I和TGF-β1，辛伐他汀同样具有保护作用。.同时我们发现单侧输尿管梗阻UUO肾脏纤维化模型中的CX3CL1和CX3CR1的蛋白水平明显增加。与野生型小鼠相比，CX3CR1基因敲除小鼠UUO模型的肾脏纤维化程度和SMA的表达明显下降，F4/80+巨噬细胞数量明显减少。.我们利用ELISA方法检测了107例IgA肾病患者血清CX3CL1的水平。结果表明IgA肾病患者血清CX3CL1水平与患者血肌酐、血钾成正相关，与白蛋白水平负相关，与肾脏病理牛津分型中系膜增殖（M）、节段硬化（S）、肾小管萎缩和间质纤维化（T）相关，与毛细血管内增殖（E）无关。.在UUO模型构建过程中，我们发现梗阻肾脏中有大量巨噬细胞浸润，其表面有IL-17RA受体的表达。我们利用IL-17RA基因敲除小鼠构建骨髓移植模型和单侧输尿管梗阻模型，发现单核/巨噬细胞表面的白介素17受体可以调控单核细胞亚型介导肾脏炎症和纤维化。结果发表在2014年Plos One杂志上。.我们利用以ApoE和LDLr基因敲除小鼠建立动脉粥样硬化模型，部分小鼠给与单侧肾脏切除，模拟慢性肾脏病 3-4 期的患者临床情况。结果发现白介素-17A参与肾功能不全加速动脉粥样硬化形成和血管壁内白细胞积聚过程。结果发表在2013年Circulation Research杂志上。.综上所述，在本项青年基金的支持下，我们深入研究了趋化因子CX3CL1/CX3CR1和IL-17RA在肾脏纤维化中作用机制，丰富了肾脏纤维化理论，积极探索阻断糖尿病肾脏纤维化新的靶点。