BK-des9在狼疮及肾脏损伤中的作用机制研究

Lupus is a common autoimmune disease, often seen in middle-aged women, with the highest prevalence in Asia. So far there is no cure for lupus,and the prevention and treatment of this diseasehas been a great clinical challenge. Our previous studies have demonstrated that KLK1 and KLK3 in the kallikrein system are two lupus-susceptibility genes; Kallikreins, kinins,and the blockade of bradykinin (BK) receptor B1 could all ameliorate kidney damage in murine lupus,whereas the inhibition of B2 receptor aggravated kidney damage. These results suggested that the kallikrein-kinin system may play an important role in the pathogenesis of lupus nephritis. More recently, our metabolomics studies demonstrated that serum levels of BK and its degradation product, BK-des9 were significantly increased in lupus patients. Random Forest Analysis indicated that both BK and BK-des9 are critical metabolites in human lupus. Compared to normal controls, serum levelsof BK-des9 and BK are 21-fold and 2.4-fold higher in lupus patients, respectively. Compared to normal B6 mice, serum BK-des9 level is 80-fold, 91-fold and 167-fold higher in the murine lupus strains BWF1, B6.Sle1.Sle3 and MRL.lpr,respectively. We know that BK-des9 metabolite binds the B1 receptor, and B1 receptor is an inflammation-inducing molecule. Hence we speculate that “BK-des9 is a key player in the pathogenesis of lupus and kidney damage”. In this project, we propose to determine the molecular mechanism by which BK-des9 acts in the pathogenesis of lupus and kidney damage using B1 (and B2) receptor knockout mice. This will provide experimental basis for the design of novel therapeutic strategies for the treatment of lupus nephritis, especially in exploring highly selective antagonists of the B1 receptor.

狼疮是常见自身免疫疾病，其防治仍是临床难题。我们的研究证实：激肽释放酶KLK1和KLK3基因是狼疮易感基因；激肽释放酶、激肽和阻断缓激肽B1受体治疗均减轻狼疮小鼠肾损伤，阻断B2受体加重肾损害。表明激肽释放酶激肽系统在狼疮发病中有重要作用。近来，代谢组学检测意外发现狼疮患者血液缓激肽(BK)和其降解分子BK-des9均增高；随机森林分析示BK和BK-des9为关键分子；血液水平狼疮患者BK-des9较对照高21倍，BK增高2.4倍；BWF1, B6.Sle1.Sle.3和MRL.lp自发性狼疮小鼠较对照分别增高80, 91和167倍。由于BK-des9可与B1受体结合，B1受体为诱导型炎症受体。据此认为BK-des9是狼疮发病和肾损伤的关键分子。为此，采用B受体敲除小鼠系统研究BK-des9在狼疮及肾损伤的作用机理，为探索BK-des9及其关键通路分子阻断剂联合治疗狼疮新方案提供实验依据。

为了研究des-Arg9-BK在狼疮性肾炎的作用机制，我们使用LC / MS和GC / MS对SLE患者和健康对照者以及狼疮性肾炎的小鼠模型和正常小鼠的血液进行了代谢筛查，用多反应监测（Multiple Reaction Monitoring ，MRM）的方法对以上的筛选结果进行了验证。发现与健康对照组相比，SLE患者均显示较高的血清des-Arg9-BK水平。我们也对研究des-Arg9-BK其相应受体B1R和B2R在狼疮性肾炎小鼠的肾脏表达情况，发现在狼疮肾炎模型中，肾脏B1R 蛋白表达较正常对照增加；B2R表达较正常对照减少。接着，我们研究了缓激肽1型受体（B1R）拮抗剂SSR240612对狼疮肾炎的实验性治疗作用，发现B1R抑制剂治疗12周，狼疮肾炎模型中多种自身免疫抗体，如dsDNA，组蛋白抗体，IgG，明显高于对照组；经治疗后小鼠血压较对照组下降，自身免疫抗体浓度有所下降，蛋白尿和肾功能都得到了改善。我们也用流式细胞术检测了两组小鼠的脾脏和肾脏的主要免疫细胞的变化，发现经过B1R受体的抑制剂治疗后脾脏总B细胞数量，边缘区B细胞数量以及肾脏浸润的CD4+ T细胞，巨噬细胞及中性粒细胞均较对照组有所下降。B1R抑制剂治疗12周以后和对照组相比狼疮小鼠的尿中和肾脏的CCL2,CCL5都有了明显降低，提示B1R抑制剂对狼疮性肾炎的肾脏保护作用可能是通过CCL2，5介导完成的。我们认为，在狼疮性肾炎的发病过程中，des-Arg9-BK通过激活B1R，从而激活脾脏和肾脏的免疫细胞，肾脏CCL2,5表达增加，加重肾脏的炎症反应，从而引起狼疮性肾炎；而阻断B1R可以通过抑制脾脏和肾脏的免疫反应，减少肾脏CCL2,5表达，减轻肾脏的炎症反应，从而减轻狼疮性肾炎的蛋白尿和肾脏损伤。.本课题为研究des-Arg9-BK及B1R在狼疮性肾炎中的发病机制提供了实验基础，为狼疮性肾炎的治疗提供了新的治疗策略和靶点。