雷公藤甲素对Heymann肾炎模型足细胞病变的影响

Therapeutic effect of triptolide on podocyte injury in passive Heymann nephritis

目的:利用被动型Heymann肾炎模型(passive Heymann nephritis,PHN)研究雷公藤甲素(triptolide)对膜性肾病的疗效及其对足细胞病变的影响。方法:实验动物分为正常对照组、PHN组和雷公藤甲素治疗组。雷公藤甲素(200μg/kg·d)灌胃治疗,分别在观察7天、14天、21天和28天时宰杀大鼠,留取血清和尿标本,观察尿蛋白、血清白蛋白、肝酶、肌酐和外周血白细胞等指标的变化;留取肾组织标本,行光镜、免疫病理、电镜和免疫电镜检查,并对肾组织IgG、C5b-9沉积以及Nephrin和Podocin的分布进行观察。结果:(1)尿蛋白:雷公藤甲素治疗7天即可显著降低PHN大鼠的蛋白尿,至14天、21天和28天时,治疗组大鼠的尿蛋白进一步降低,与Heymann肾炎大鼠之间差异有统计学意义(P<0·01)。在尿蛋白显著降低的同时,血浆白蛋白显著增加。(2)肾小球上皮侧免疫沉积物:电镜观察结果显示,雷公藤甲素治疗组上皮侧免疫复合物、钉突以及基膜反应性增殖均较Heymann肾炎组似有所改善,治疗后上皮侧电子致密物减少,基膜反应减轻,但在尿蛋白明显减少的同时,上皮侧电子致密物始终存在。(3)肾小球IgG和C5b-9的沉积:经雷公藤甲素治疗后肾小球IgG和C5b-9的沉积与Heymann肾炎大鼠相比,荧光强度有所减少,尤其在治疗后的第7天,减少较为明显。(4)足细胞病变:经雷公藤甲素治疗7天、14天、21天和28天后,均可见足细胞的足突损伤比对照组明显减轻,足突融合显著改善,足突宽度显著降低,观察至第28天时,治疗组可见大部分足细胞的足突形态基本恢复正常。(5)足细胞裂孔膜蛋白的变化:经雷公藤甲素治疗7天后,足细胞表面Nephrin和Podocin的表达比Heymann肾炎大鼠有明显增加,分布上的异常开始得到纠正,至第21天基本恢复成连续的线样分布。免疫电镜的结果再次显示了治疗后Nephrin的上述修复过程。结论:雷公藤甲素对被动型Heymann肾炎具有显著的治疗作用,能有效地减少蛋白尿,减轻肾组织免疫损伤,促进足细胞病变和裂孔膜蛋白结构的修复。雷公藤甲素疗效机制除了其免疫抑制和抗炎作用外,还与它能显著地改善和修复足细胞病变有关。

Objective：To explore the therapeutic effect and mechanism of triptolide on membranous nephropathy using passive Heymann nephritis （PHN） in rats. Methodology：Passive Heymann nephritis was induced in SD rats using rabbit antiserum against FxIA antigen. SO rats were divided into three groups, including normal controls, PHN rats with or without treatment with triptolide. PHN rats were administrated orally with triptolide （200μg/kg per day）. After the treatment with triptolide for 7, 14, 21 and 28 days ,the 24hr urine and blood were collected to examine the levels of proteinuria, serum albumin, aminotransferases, serum creatinine and white blood cell counts. The renal tissue was processed for the examinations of light microscopy, electron microscopy, immunefluorescene and immune electron microscopy. The deposition of IgG and C5b-9 along capillary wall was observed. The distribution of nephrin and podocin as the expression of slit diaphragm proteins was also determined. Results： （ 1 ） Urinary protein： Triptolide could significantly reduce proteinuria in rats with passive Heymann nephritis. This effect could be seen at day 7 after the treatment and got more prominent at day 14 and persistent on day 21 and 28. At the same time, the plasma albumin was also significantly increased（P 〈 0. 01 ）. （2） Subepithelial immune deposits： The examination of electron microscopy showed that the subepithelial immune deposits, spikes formation and basement membrane proliferation were improved after treatment with triptolide. The subepithelial immune deposits decreased and the reaction of basement membrane was also ameliorated. However, the subepithelial immune deposits still existed although the urinary protein decreased significantly. （3） Glomerular deposition of IgG and C5b-9： The immunefluorescene observation indicated that the deposition of IgG and C5b-9 decreased after treatment with triptolide. Compared with Heymann nephritis, the intensity of fluorescene decreased, especially after treatment with triptolide for 7 days. （4） Injuries on podocytes： After treatment with triptolide for 7, 14, 21 and 24 days, the injuries on foot process of podocyte were recovered compared with that in Heymann nephritis. The effacement of the podocyte foot processes was improved. The mean width of foot processes was decreased. Most foot processes was restored to normal shape after treatment with triptolide for 28 days. （5） Change of slit diaphragm related protein： After treatment with triptolide for 7 days, the expression of nephrin and podocin was significantly increased compared with that in Heymann nephritis. The abnormal distribution of nephrin and podocin started to be corrected. After treatment with triptolide for 21 days, the expression of nephrin and podocin was mostly restored to normal continuous linear expression. The immune electron microscopy confirmed the therapeutic effect of triptolide on the restoration of nephrin expression. Conclusion： Triptolide could effectively reduce proteinuria and alleviate the glomerular immune injuries, and also remarkably improve the podocyte lesion and help to restore the normal structure of slit diaphragm. In addition to its immunosuppression and anti-inflammation activity, the therapeutic effect of triptolide might be due to its direct activity on the treatment with podocyte injuries.