巨噬细胞清除对补体C3缺失的单侧输尿管梗阻小鼠肾间质纤维化的影响

目的探讨清除肾组织巨噬细胞对补体C3缺失单侧输尿管梗阻(UUO)小鼠肾间质纤维化结局的影响及其机制。方法8~12周龄的C57BL/6野生型(WT)和补体C3基因敲除(C3KO)小鼠用随机数字表法分为:野生型小鼠假手术组(WT/sham);野生型小鼠UUO模型组(WT/UUO);补体C3基因敲除小鼠假手术组(C3KO/sham);补体C3基因敲除小鼠UUO模型组(C3KO/UUO),每组18只。左侧输尿管结扎法建立UUO模型。免疫组化法检测UUO术后梗阻侧肾组织补体C3的表达;免疫荧光法检测肾间质F4/80阳性巨噬细胞的分布和数量;Masson及HE染色观察肾组织病理改变,比较肾间质胶原纤维面积和肾小管-间质损伤指数评分。分别用巨噬细胞清除剂氯磷酸二钠(CLO)脂质体在UUO早期或晚期处理WT/UUO和C3KO/UUO组小鼠,观察肾间质巨噬细胞数量和肾间质纤维化的改变。F4/80和iNOS免疫双荧光、F4/80和CD206免疫双荧光观察WT/UUO和C3KO/UUO组术后巨噬细胞表型及M1/M2型巨噬细胞的比例。Western印迹法检测肾组织巨噬细胞标志蛋白诱导型一氧化氮合酶(iNOS)、精氨酸酶-1(Arg-1)、甘露糖受体(CD206)的表达。结果与WT/UUO组比较,C3KO/UUO组小鼠肾间质胶原纤维面积减少,肾小管-间质损伤指数评分降低(均P<0.01),肾间质纤维化程度减轻。巨噬细胞清除剂早期或晚期处理后,WT/UUO组肾脏纤维化程度均较空白脂质体组减轻;C3KO/UUO组肾脏纤维化程度与空白脂质体组相比无明显改善。C3KO/UUO组早期肾组织M1型巨噬细胞占比及其标志蛋白iNOS较WT/UUO组表达降低,M2型巨噬细胞占比及其标志蛋白Arg-1、CD206表达增加(均P<0.01)。结论补体C3基因敲除的UUO小鼠清除巨噬细胞后肾脏纤维化程度无明显改善,其机制与补体C3缺失影响肾组织巨噬细胞极化表型有关。

Effect of renal fibrosis after macrophage depletion in C3-deficient unilateral ureteral obstruction mice

Objective To investigate the effect and mechanism of renal fibrosis after macrophage depletion in C3-deficient unilateral ureteral obstruction mice. Methods Renal interstitial fibrosis model was established by unilateral ureteral obstruction (UUO) in male C3-deficient mice and age-matched C57BL/6 WT mice (8-12 weeks of age). Mice were randomly divided into 4 groups, including sham operation in wild type group（WT/sham）(n=18), UUO operation in wild type group（WT/UUO）(n=18), sham operation in C3-deficient group（C3KO/sham）(n=18), and UUO operation in C3-deficient group（C3KO/UUO）(n=18). The expression of complement C3 was detected by immunohistochemical staining and renal interstitial macrophages were assessed by immunofluorescence staining. Tubulointerstitial fibrosis was observed by both HE staining and Masson staining after 14 days of UUO. Collagen accumulation and score of tubulointerstitial injury were obtained. Wild type and C3-deficient UUO mice were treated by liposome clodronate in early or late stage respectively and then interstitially infiltrated macrophages and renal fibrosis were analysed. Mice were sacrificed randomly at 3,7,14 days after UUO and obstructed kidneys were collected. Macrophage phenotype was detected by double-labeling immunofluorescence with F4/80 and iNOS for the M1, F4/80 and CD206 for the M2 macrophage subpopulation. iNOS, Arg-1 and CD206 were also detected by western blot. Results C3 deficient mice exhibited attenuated renal fibrosis, reduced collagen accumulation and tubulointerstitial injury score compared with WT mice (P＜0.01). Meanwhile, macrophage depletion in early or late stage of UUO reduced renal fibrosis in WT mice, but had no effect on C3-deficient UUO mice. Decreased accumulation of M1 macrophages and expression of iNOS, increased accumulation of M2 macrophages and expression of Arg-1, CD206 were found in C3 deficient mice compared with WT mice in early stage of UUO (P＜0.01). Conclusion Renal fibrosis is not reduced after depletion of macrophages in C3 deficient UUO mice due to the altered macrophage polarization.