E3泛素连接酶HUWE1参与调节肾小管间质纤维化的分子机制研究

Renal fibrosis, particularly tubulointerstitial fibrosis, is the common final outcome of almost all progressive chronic kidney diseases. Renal tubulointerstitial fibrosis is also a reliable predictor of prognosis and a major determinant of renal insufficiency. Ubiquitin E3 ligases are involved in various diseases by mediating the specific protein degradation. However, the study of ubiquitin E3 ligases in renal tubulointerstitial fibrosis is very rare. The preliminary data from our proposed project has shown that the expression of ubiquitin E3 ligase HUWE1 was significantly decreased in the mice model of unilateral ureteral obstruction (UUO)-induced renal tubulointerstitial fibrosis, and the mRNA expression of HUWE1 was upregulated by Angiotensin II (AngII) in vitro. Of the interest, the data from our preliminary study indicated the interaction between HUWE1 and epidermal growth factor receptor (EGFR)，which is key molecular related to renal fibrosis. Based on these preliminary data, we’ll further study the three aspects in the future: 1) the mechanism mediating the interaction of HUWE1 and EGFR, and mechanism of HUWE1-mediated EGFR ubiquitylation and degradation; 2) the effect of the interaction of HUWE1 and EGFR on renal tubulointerstitial fibrosis; 3) the mechanism of AngII on HUWE1 transcriptional expression. The findings of our study will demonstrate the effects of E3 ligases mediated protein degradation on renal tubulointerstitial fibrosis.

肾小管间质纤维化是慢性肾脏病发展的重要病理过程，也是反映肾功能下降程度的主要决定因素。由E3泛素连接酶介导的特定蛋白质降解对不同疾病发生发展的影响是近年来医学研究的热点，但是有关E3连接酶对肾小管间质纤维化的研究较少。本课题的前期研究表明，E3泛素连接酶HUWE1在小鼠肾小管间质纤维化模型中表达明显降低；HUWE1与表皮生长因子受体EGFR之间存在直接相互作用，上调HUWE1的表达可促进EGFR降解；血管紧张素II (AngII) 显著上调HUWE1的mRNA表达。本课题将利用动物和细胞实验，围绕以下层面进行深入研究：1) HUWE1-EGFR直接相互作用以及介导后者泛素化降解的分子机制；2) HUWE1-EGFR相互作用对肾小管间质纤维化的影响；3) AngII对HUWE1表达的转录调控机制。深入探讨E3连接酶介导的泛素化降解对肾小管间质纤维化的影响，可为今后开发治疗策略奠定理论基础。

中国慢性肾脏疾病发病率已经高达10.8%，因此研究慢性肾脏疾病的发病机制意义重大。肾小管间质纤维化是各种不同病因的慢性肾脏疾病进展到终末期肾病的主要病理过程。研究表明，表皮生长因子受体 (epidermal growth factor receptor, EGFR) 的过度激活与肾小管间质纤维化密切相关。而泛素化修饰是蛋白降解的重要途径，E3连接酶起到了特异性控制泛素修饰途径的作用。但介导EGFR泛素化降解的E3连接酶至今研究很少。课题组前期研究结果提示HUWE1 (HECT, UBA, WWE domain containing 1) 参与肾脏集合管钠离子通道(ENaC)的表达和功能调控，与盐敏感性高血压密切相关。所以我们提出实验假设：1. HUWE1是否参与肾小管间质纤维化的进程？2. EGFR在肾小管间质纤维化中是否受到HUWE1的调控? 选用野生型C57BL/J6雄性小鼠行左侧输尿管结扎手术 (Unilateral Ureteral Obstruction, UUO) 建立肾小管间质纤维化模型。苏木素-伊红染色 (hematoxylin-eosin staining，HE)、马松染色证明了UUO肾脏间质发生纤维化，通过Western blot检测纤维化指标进一步证明。我们发现UUO组肾组织HUWE1表达降低，EGFR表达增高。在细胞实验中，利用TGF-β刺激HK-2细胞，可见纤维化指标随着药物浓度增加表达增加，随着HK-2细胞损伤的进展，HUWE1逐渐表达减少，EGFR表达增加。时间梯度上结果同浓度梯度一致。通过免疫共沉淀证实EGFR和HUWE1存在相互结合；免疫荧光染色证实EGFR和HUWE1在胞浆中存在共定位。进一步的细胞实验中过表达HUWE1后发现EGFR表达明显下调，通过MG132实验，放线菌酮示踪实验 (cycloheximide, CHX)，泛素化实验都证明了HUWE1是EGFR的E3泛素连接酶，介导EGFR的泛素化降解，并且HUWE1可以缓解因TGF-β所致的肾小管上皮细胞损伤。因此，本课题通过体内、体外多层次实验阐明HUWE1是EGFR的E3泛素连接酶，并且通过介导EGFR的泛素化降解来缓解肾脏间质纤维化进程，从而为肾脏间质纤维化提供了新的上游药物治疗靶点。

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