赖氨酸特异性去甲基化酶调控乙肝相关性肾炎进展的作用研究

Mechanisms of Hepatitis B virus-associated glomerulonephritis (HBV-GN) are still elusive and no effective therapy can delay the development and progression of HBV-GN till now. Mechanisms involved are focused on upstream of immune linking reaction and downstream of signaling pathway transduction, as well as inflammatory eruption effection. However, rare report about mechanisms mediated in regulating gene transcription was demonstrated. Our studies have shown that Lysine specific demethylase 1(LSD1) expression were upregualted in renal biopsies samples from HBV-GN patients and Histone methylation modification was descreased. Moreover, we have shown that LSD1 can modulate proliferation of renal immanence cell and regulate activation of Toll-like receptor 4 (TLR4). In our another pivotal study, TLR4 expression are upregualted in renal biopsies samples from HBV-GN patients and TLR4 can be co-localized with HBsAg by immunofluorescence. Further, we found that exposure of cultured tubular cell to HBV serum resulted in cell proliferation and cell phenotype transition, concomitance with TLR4 high expression. Thus, it is extremely possible that TLR4 signaling pathway can mediate progression of HBV-GN. We suppose that LSD1 promotes development of HBV-GN through activating TLR4 signaling pathway. To test this hypothesis, we will first utilize gene transfection, epigenetics and other molecular techniques to clarify the role of LSD1 in regulating podocyte injury, tubular cell proliferation and phenotype transition exposed to HBV serum. Next, we will explore the mechanism by which LSD1 regulates HBV serum–induced production of profibrotic factors/proinflammatory cytokines as well as activation of the TLR4 signaling pathway after damage of podocyte and tubular cell. Finally, we will elucidate the therapeutic effect of pharmacologic LSD1 blockade on the development of HBV-GN and define the mechanism involved in a mouse model of HBV-GN. Successful completion of this project may provide a novel therapeutic approach for prevention and treatment of renal injury in HBV-GN patients.

乙肝相关性肾炎（HBV-GN）机制研究主要集中在其上游的免疫级联反应和下游的信号转导及炎症促发阶段，对基因转录调控研究鲜有报道。我们发现，HBV-GN肾组织赖氨酸特异性去甲基化酶1（LSD1）表达明显升高，组蛋白甲基化降低。进一步研究发现LSD1可介导肾脏固有细胞增殖，调控Toll样受体4（TLR4）活化。而前期研究已经证实HBV-GN患者肾脏TLR4表达显著上调；HBV感染血清刺激的肾小管细胞大量增殖，且TLR4高表达，TLR4在HBV-GN中发挥重要作用。据此推测，LSD1可通过调控TLR4通路介导HBV-GN进展。本研究拟应用基因转染、蛋白组学等技术，检测LSD1对肾小球足细胞损伤，肾小管细胞增殖及表型转化的影响，探讨LSD1调控TLR4通路活化、介导促纤维化和炎症因子分泌的机制，评估LSD1在HBV-GN小鼠肾脏损伤中的作用。为新型抗HBV-GN药物研发提供重要依据和靶标。

乙型肝炎病毒相关性肾炎（HBV-GN）发生机制不清，尚缺乏有效手段延缓其进展。虽然目前针对HBV-GN有一定的研究，但其机制及干预研究主要集中在其上游的免疫级联反应和下游的信号转导及炎症促发阶段，对基因转录调控研究却鲜有报道。结合我们前期研究发现，HBV-GN肾组织赖氨酸特异性去甲基化酶1（LSD1）表达明显升高，组蛋白甲基化降低，故本项目从表观遗传学调控角度系统的研究：1）LSD1对HBV感染的肾小球足细胞损伤及肾小管上皮细胞增殖、表型转化的影响；2）LSD1对HBV感染的肾小球足细胞及肾小管上皮细胞TLR4信号通路及炎症因子分泌的影响；3）利用LSD1新型抑制剂进一步评估LSD1在HBV-GN小鼠肾脏损伤中的作用及机制。. 在本项目执行期间，我们发现：1）HBV感染肾小球足细胞及肾小管上皮细胞后可引起LSD1表达增加，促进肾小球足细胞凋亡，诱导肾小管上皮细胞发生上皮-间充质细胞转分化（EMT）；过表达/沉默LSD1基因后可促进/抑制HBV诱导的肾小球足细胞凋亡及肾小管上皮细胞转分化。2）HBV可诱导肾小球足细胞及肾小管上皮细胞炎症因子分泌；同样过表达/沉默LSD1基因后可促进/抑制HBV诱导的上述细胞炎症因子分泌。3）RNA-seq分析发现TLR4是LSD1的下游靶基因；LSD1可直接结合到TLR4基因启动子上介导H3K9me1/2去甲基化，进而调控其表达；LSD1遂通过直接调控TLR4的表达介导NF-κB和JNK通路活化，从而促进HBV诱导的肾小球足细胞及肾小管上皮细胞炎症因子分泌。4）HBV-GN小鼠动物模型肾组织中LSD1表达明显上调，主要在肾小球和肾小管细胞核中表达；采用LSD1抑制剂反苯环丙胺（TCP）抑制LSD1表达，增强组蛋白甲基化，能抑制肾脏炎症因子及TLR4的表达，同时能明显改善HBV-GN小鼠肾脏损伤的程度。本研究深入探讨了LSD1调控HBV-GN进展的可能机制，为临床治疗HBV-GN提供了新的潜在靶点及理论依据。