全反式维甲酸对抗肾小球基底膜肾炎小鼠模型肾脏损害的保护作用

Protective effect of all-trans retinoic acid against renal damage in the mouse model of anti-glomerular basement membrane nephritis

目的探讨全反式维甲酸对肾毒性血清肾炎(NTSN)小鼠模型的作用及机制。方法使用肾毒性血清(NTS)尾静脉注射构建NTSN模型,设置对照组、NTS组、NTS+维甲酸组。NTS注射前5 d给予羊免疫球蛋白和完全弗氏佐剂腹腔注射预处理,预处理后24 h给予16 mg/kg全反式维甲酸或溶媒腹腔注射,NTS注射后7 d处死小鼠。ELISA法检测小鼠白蛋白尿/肌酐比值、小鼠BUN;肾组织石蜡切片HE染色,定量分析新月体肾炎、肾小球节段硬化的百分比;肾组织冰冻切片行CD8^+、CD4^+、CD68免疫荧光染色,并定量分析;QPCR方法分析肾皮质炎症细胞趋化因子如人单核细胞趋化蛋白-1(MCP-1)、细胞间黏附分子-1(ICAM-1)、淋巴细胞趋化因子(LTN)的表达量。结果维甲酸处理组显著减轻肾毒性血清肾炎小鼠模型的蛋白尿[NTS组和NTS+维甲酸组尿微量白蛋白/肌酐比值(UACR)分别为:(4. 52±0. 36)×10^-3mg/g、(2. 63±0. 18)×10^-3mg/g;q=18. 45,P <0. 01]、肾功能[NTS组和NTS+维甲酸组BUN分别为:(16. 81±1. 23) mmol/L、(13. 33±0. 62) mmol/L;q=6. 155,P <0. 01]、减轻肾组织病变[NTS组和NTS+维甲酸组含新月体的肾小球百分比分别为:[(36±1. 58)%、(22. 2±1. 92)%;q=21. 46,P <0. 01]。维甲酸处理组每低倍镜视野下CD8^+淋巴细胞计数、CD4^+淋巴细胞计数、每高倍镜视野下CD68阳性细胞面积较NTS组均明显减少,差异有统计学意义(q值分别为:9. 545、8. 610、11. 08;P <0. 01);维甲酸可以显著抑制炎症细胞趋化因子(MCP-1、ICAM-1、LTN)的基因表达。结论维甲酸通过抑制炎症细胞趋化因子的表达、减少炎症细胞的浸润,减轻NTSN小鼠模型的蛋白尿、新月体形成等肾损害。

ObjectiveTo investigate the effect and mechanism of all- trans retinoic acid in the mouse model of nephrotoxic serum nephritis (NTSN). MethodsThe NTSN model was constructed by tail vein injection of nephrotoxic serum (NTS), and the mice were divided into the control group, NTS group, and NTS+retinoic acid group. Five days before the injection of NTS, goat immunoglobulin and complete Freund′s adjuvant were intraperitoneally injected as pretreatment. 24 h after the pretreatment, 16 mg/kg all- trans retinoic acid or dissolvant was intraperitoneally injected. 7 days after the NTS injection, the mice were sacrificed. The albuminuria/creatinine ratio and serum BUN of the mice were measured by ELISA. HE staining of paraffin sections of renal tissue was performed, and quantitative analysis was made of the percentage of crescentic and glomerular segmental sclerosis lesions. Frozen sections of renal tissue were stained with immunofluorescence for quantitative analysis of CD8^+, CD4^+ and CD68. The QPCR method was used to analyze the expression levels of inflammatory cytokines such as human monocyte chemoattractant protein- 1 (MCP- 1), intercellular adhesion molecule- 1 (ICAM- 1), and lymphotactin (LTN) in the renal cortex tissue. ResultsCompared with the NTS group, the NTS+retinoic acid group had significantly lower proteinuria. The urinary microalbumin/creatinine ratio (UACR) for NTS group and NTS+retinoic acid group was (4.52±0.36)×10^-3 mg/g,(2.63±0.18)×10^-3 mg/g, respectively (q=18.45, P< 0.01), while the BUN level for NTS group and NTS+retinoic acid group was (16.81±1.23)mmol/L,(13.33± 0.62) mmol/L;respectively (q=6.155, P<0.01). And the percentage of glomeruli with crescents in the NTS group and NTS+retinoic acid group was 36.58% and 22.2%, respectively (q= 21.46 , P< 0.01). Compared with the NTS group, the NTS+retinoic acid group had significantly smaller CD8^+ lymphocyte count, CD4^+ lymphocyte count, and area of CD68 positive cells (q value: 9.545, 8.610 , and 11.08, respectively, P<0.01). The expression of MCP- 1, ICAM- 1, and LTN was significantly inhibited by retinoic acid. ConclusionRetinoic acid inhibited the expression of inflammatory chemokines, reduced the infiltration of inflammatory cells, and alleviated the renal damage such as proteinuria and crescent formation in the mouse model of nephrotoxic serum nephritis.