HE4在缺氧诱导的肾小管上皮细胞细胞外基质沉积及肾脏纤维化的作用机制

Hypoxia play important roles in the development of renal fibrosis of patients with chronic kidney disease. HE4 acts as a matrix metaloproteinases (MMP) inhibitor in ECM induced renal fibrosis, but the specific biological function of HE4 is not clear. We found for the first time that HE4 was strongly expressed in the cytoplasm of tubular epithelial cells from kidneys of patients with CKDs, and HE4 was high expression in human renal epithelia induced by hypoxia, HIF-1α associates with the HRE sites in HE4 promoter by bioinformatics analysis. Thus, we postulated that HIF-1α may directly regulate the expression of HE4 and regulate its downstream target genes. Thereby the HIF1α-HE4-MMP might be a new signaling in renal fibrosis. The aim of this study was to examine whether HIF-1α associates with the HRE sites in HE4 promoter by chromatin immunoprecipitation (ChIP) assay, and to confirmed the role of HE4 in regulation of downstream target of NF-κB and MMPs using PCR, Western, coprecipitation technology, also to confirm that HE4 can activate proliferation of myofibroblast by co-culture and the cell MTT technology. The findings of this study will provide a novel insight into the pathogenesis of hypoxia-induced kidney injury and lay a foundation for the future development of new treatment modalities.

缺氧是导致慢性肾脏病肾脏纤维化发生的重要原因。金属蛋白酶（MMP）抑制剂HE4能促进肾小管上皮细胞胞外基质沉积增加，导致肾脏纤维化，但其分子体机制尚不清楚。我们在缺氧性肾病组织中首次发现肾小管上皮细胞胞浆高表达促纤维化因子HE4, 体外缺氧诱导肾小管上皮细胞，也可发现HE4高表达；生物信息学分析，HE4启动子区存在HIF-1α结合位点。我们推测，缺氧后HIF-1α直接调节HE4的表达，进而调控其下游靶基因的表达，存在HIF1α-HE4-MMPs信号通路。因此本研究拟通过EMSA和ChIp实验明确HE4和HIF-1α的结合作用；利用qPCR、Western、共沉淀技术证实HE4对下游靶分子NF-κB和-MMPs的调控作用；利用细胞共培养、MTT等验证HE4表达刺激肌成纤维细胞的增殖作用。从而为阐明缺氧性肾损伤机制和肾脏纤维化提供全新的视角，为探讨新的治疗手段提供重要的线索。

肾间质纤维化是各种CKD引起的异常损伤修复反应，主要病理学表现是肌成纤维细胞增殖和大量ECM（extracellular matrix）沉积构成的网状瘢痕结构，是过度修复的表现。缺氧引起的细胞外基质（ECM）沉积是肾纤维化的重要原因，缺氧引发ECM沉积的机制尚不清楚。人附睾蛋白4（HE4）是小分子分泌性蛋白，据报道，在卵巢癌患者的血清中甚至在早期疾病中都发现了HE4的过度表达，是一种新发现的能引起ECM沉积的关键分子。本研究发现缺氧可诱导肾小管上皮细胞内源性HE4的合成和分泌增加。过表达的HE4可通过磷酸化NF-κB转录活性基团p65激活NF-κB信号通路。NF-κB通路的激活可上调靶基因TIMP1的表达，抑制MMP2的活性，从而抑制ECM的降解。沉默HE4可抑制单侧输尿管梗阻模型（unilateral ureteral obstruction，UUO，是一个经典的肾纤维化模型）小鼠体内缺氧诱导的ECM沉积，减轻纤维化，阻断NF-κB通路的活化，也可以抑制肌成纤维细胞的增殖。同时我们发现，肾小管上皮细胞和血清中HE4蛋白的表达与慢性肾脏病患者肾间质纤维化程度呈正相关。因此，揭示缺氧诱导肾小管上皮细胞中HE4过表达的新功能和分子机制为理解肾纤维化的病理和抗纤维化的诊疗策略提供了重要的思路。