Objectives: The binding of CX3C chemokine receptor 1 (CX3CR1) and its unique ligand CX3C chemokine ligand 1 (CX3CL1) can promote the migration of inflammatory cells to the lesion and affect the progression of renal interstitial fibrosis, but the underlying mechanisms remain unclear. This study aims to investigate whether CX3CR1 affects renal interstitial fibrosis by macrophage polarization.
Methods: A mouse model of renal interstitial fibrosis was established by unilateral ureteral obstruction (UUO). C57/B6 mice were divided into a CX3CR1 inhibitor group (injected with CX3CR1 inhibitor AZD8797) and a model group (injected with physiological saline). After continuous intraperitoneal injection for 5 days, the ligated lateral kidneys of mice were obtained on the 7th day. Hematoxylin and eosin (HE) staining and Masson staining were used to observe the infiltration of inflammatory cells and the collagen fiber deposition in renal interstitium, respectively. The mRNA and protein expressions of CX3CR1, alpha-smooth muscle actin (α-SMA) and fibronectin (FN) in the kidneys were detected by reverse transcription PCR (RT-PCR) and Western blotting, respectively. Differentially expressed genes in kidney of the 2 groups were identified by whole genome sequencing and the differential expression of arginase-1 (Arg-1) was verified by RT-PCR. Flow cytometry was used to detect the proportion of M2 type macrophages in kidneys of the 2 groups.
Results: The infiltration of inflammatory cells and the collagen fiber deposition in renal interstitium were significantly reduced in the CX3CR1 inhibitor group. The mRNA and protein levels of CX3CR1 and the mRNA levels of α-SMA and FN in the CX3CR1 inhibitor group were significantly lower than those of the model group (all P<0.05). Whole genome sequencing showed that the top 5 differentially expressed genes in kidney of the 2 groups were Ugt1a6b, Serpina1c, Arg-1, Retnla, and Nup62. RT-PCR verified that the expression level of Arg-1 in kidney of the CX3CR1 inhibitor group was significantly higher than that of the model group (P<0.001). Flow cytometry showed that the proportion of Arg1+CD206+M2 macrophages in kidney of the CX3CR1 inhibitor group was significantly higher than that of the model group (P<0.01).
Conclusions: Inhibiting CX3CR1 can effectively prevent the progression of renal interstitial fibrosis. The mechanism may be related to macrophage polarization towards M2 type and upregulation of Arg-1 expression.
目的: CX3C趋化因子受体1(CX3C chemokine receptor 1，CX3CR1)与CX3C趋化因子配体1(CX3C chemokine ligand 1，CX3CL1)结合后促使巨噬细胞等炎症细胞向受损部位迁移，巨噬细胞极化与肾间质纤维化相关。本研究旨在探讨CX3CR1是否通过调控巨噬细胞极化来影响肾间质纤维化的进展。方法: 建立C57/B6小鼠单侧输尿管梗阻(unilateral ureteral obstruction，UUO)肾间质纤维化模型，并将其分为CX3CR1抑制剂组与模型组，分别连续 5 d腹腔注射CX3CR1抑制剂AZD8797或生理盐水，于建模后第7天取小鼠建模侧肾脏。分别采用苏木精-伊红(hematoxylin and eosin，HE)染色和Masson染色观察肾间质炎症细胞浸润和胶原纤维沉积情况。采用反转录聚合酶链反应(reverse transcription PCR，RT-PCR)和蛋白质印迹法检测小鼠肾组织中α-平滑肌肌动蛋白(alpha-smooth muscle actin，α-SMA)、纤维粘连蛋白(fibronectin，FN)、CX3CR1的mRNA表达和CX3CR1蛋白质表达情况。采用全基因组测序鉴别2组肾组织的差异表达基因并用RT-PCR验证精氨酸酶-1(arginase-1，Arg-1)的差异表达。采用流式细胞术检测2组肾组织M2型巨噬细胞占比。结果: CX3CR1抑制剂组小鼠肾间质炎症细胞浸润明显减少，胶原纤维沉积明显减轻。CX3CR1抑制剂组小鼠肾组织中CX3CR1 mRNA及蛋白质水平、α-SMA和FN mRNA水平均明显低于模型组(均P<0.05)。全基因组测序显示2组肾组织中差异表达前5位的基因依次为Ugt1a6b、Serpina1c、Arg-1、Retnla和Nup62，RT-PCR证实CX3CR1抑制剂组Arg-1 mRNA水平明显高于模型组(P<0.001)。流式细胞术结果显示CX3CR1抑制剂组小鼠肾脏中Arg1+CD206+M2型巨噬细胞占比明显高于模型组(P<0.01)。结论: 抑制CX3CR1的表达可有效缓解小鼠肾间质纤维化，其机制可能与巨噬细胞向M2型极化及Arg-1的表达上调有关。.
Keywords: CX3C chemokine receptor 1; arginase-1; macrophage polarization; renal interstitial fibrosis.