Klotho启动子超甲基化介导肾脏纤维化表观遗传机制及干预研究

Renal fibrosis is a common feature of chronic kidney disease (CKD), characterized by myofibroblast trans-differentiation and excess extracellular matrix deposition. Epidemiology studies indicate that CKD patients with identical etiology display various susceptibilities, severities and prognosis of renal fibrosis and epigenetic DNA methylation modification is a major cause for the differences, but the underlining mechanism is not fully revealed. Klotho is a renal enriched anti-aging protein with multiple renoprotective and anti-fibrogenic functions. Klotho promoter contains typical CpG islands and Klotho distinct renal expression and silence in other tissue are controlled by epigenetic mechanisms. Our preliminary study discovered that in addition to the elevated TGF-beta and the reduced Klotho, mouse fibrotic kidney exhibited aberrant DNA methyltransferase (DNMT) induction and Klotho promoter DNA hypermethylation. Since TFG-beta has strong DNA methylation-inducing capacity we therefore hypothesize that Klotho is an important DNA methylation target and TGF-beta-induced aberrant DNA methylation constitutes a critical regulatory pathway leading to Klotho repression and renal fibrosis. In this study we propose to investigate the aberrant epigenetic DNA methylation that causes Klotho repression in animal model of renal fibrosis and to investigate the potential of epigenetic intervention of Klotho repression in preventing renal fibrosis. The findings of this study should offer new insights into the Klotho-targeted therapeutic strategy in the potential treatment of renal fibrosis-associated kidney diseases.

肾脏纤维化是慢性肾脏病共有的核心病理改变。流行病学调查发现，病因相同的慢性肾脏病患者发生肾脏纤维化的易感性，严重性，及最终是否出现肾衰存在极大差异，表观遗传DNA甲基化可能是造成这种差异的主要原因，但甲基化靶点和机制不明。Klotho是肾脏特异表达的抗衰老基因，具有维护肾脏稳态及多位点抗肾脏纤维化功能，其启动子含有大片CpG岛区域。我们的前期研究发现小鼠纤维化肾脏TGFb升高，Klotho下调，还出现DNA甲基化酶水平异常及Klotho启动子超甲基化。TGFb具有强烈的DNA甲基化诱导功能，据此我们推测Klotho是重要的纤维化早期甲基化靶点，TGFb诱导的DNA甲基化异常是导致Klotho缺失促进肾脏纤维化的重要调控通路。本课题将探索Klotho下调的表观遗传机制及对肾脏纤维化的作用，并尝试表观遗传干预Klotho下调抗肾脏纤维化，为治疗肾脏纤维化相关疾病提供有效的干预靶点和设计思路。

肾脏纤维化是慢性肾脏病的特征性病理改变，临床缺乏有效治疗措施。肾脏特异抗衰老蛋白Klotho具有显著的抗肾脏纤维化作用，但在肾脏纤维化发生发展中受异常表观遗传修饰影响而下调，是防治肾脏纤维化的关键潜在靶点。我们课题组针对由于DNA甲基化异常导致的Klotho 表观遗传下调特征和靶向干预Klotho缺失抗肾脏纤维化进行了一系列研究。我们采用腺嘌呤辅食和单侧输尿管结扎两种肾脏纤维化小鼠模型，首先确定了TGF是导致肾脏纤维化Klotho下调的主要致病因子，异常升高的促纤维化细胞因子TGF-通过抑制miR-152和miR-30诱导DNA甲基化酶1/3a在纤维化肾脏异常高表达，导致Klotho启动子超甲基化和Klotho沉默。接着，我们采用DNA甲基化酶抑制剂地西他滨处理UUO诱导的肾脏纤维化小鼠，发现地西他滨能够有效减轻Klotho启动子甲基化水平和Klotho下调，Klotho依赖性的缓解小鼠的肾脏纤维化病理改变，表明由于DNA甲基化异常所致的Klotho下调是促进肾脏纤维化发生发展的主要诱因。另外，我们还发现中草药成分大黄酸和大豆成分异黄酮具有显著的去DNA甲基化作用，能够抑制异常高表达的DNA甲基化酶1/3a并缓解Klotho启动子超甲基化，逆转Klotho下调，并Klotho依赖性地有效缓解肾脏纤维化及相关的慢性肾脏病和骨质病理损害。本系列研究揭示了肾脏纤维化Klotho下调的表观遗传特征和有效干预策略，探讨了传统医药有效成分治疗肾脏纤维化的潜在应用前景，共发表xx篇相关论文和两篇综述，为揭示Klotho表观遗传下调特征和靶向干预Klotho下调治疗肾脏纤维化及相关疾病提供了理论依据和新的设计思路。