C/EBP同源蛋白及其相关基因导致急性老年性肾损伤的分子机制研究

Acute kidney injury associates with hospitalization, mortality risk, progression to chronic kidney disease, which affects human healthy severly. Other studys and our researches indicated that renal tissues of old mice were more easily be developed endoplasmic reticulum stress (ERS) and more severe injury than that of young mice under tunicamycin stimulated. And c/EBP homologous protein (CHOP) plays a very important role in this ERS-related damage. However, the molecular mechanism of CHOP induces acute kidney injury (AKI) in elder is unclear. We will observe the pathological change and detect apoptosis cells in renal tissues of CHOP knockout old mice and wild-type old mice with tunicamycin treated. The expression of CHOP and CHOP-related genes in this passway of unfolded protein reaction will be measured using RT-PCR and western blot in above mice with same stimulus. Apoptosis cells will be detected after CHOP gene will be up-regulated or down-regulated in renal proximal tubular cells (PTCs) of young mice with overexpression, or of old mice with knockdown. The expression of CHOP gene and its down-stream genes will be measured after PKR-like eukaryotic initiation factor 2α kinase (PERK) and eukaryotic translational initiation factor 2α (eIF2α) genes are knockdowned transferred with lentivirus vector. We will illustrate that PERK/eIF2α/CHOP passway plays a key role in acute kidney injury of endoplasmic reticulum stress in old mice from these experiments. The change of ERS-related gene, inflammatory factors and apoptosis factors will be detected after tumor necrosis factor (TNF) receptor knockout mice (TNFR1, TNFR2, TNFR1R2) with tunicamycin or cisplatin intra-peritoneal injection. And CHOP and other ERS markers are be measured after pre-treated with hydrogen peroxide (H2O2) for seven days and with tunicamycin stimulated. In vitro, the expression of PERK, CHOP, eIF2α, XBP-1 and apoptosis cells in renal PTCs of CHOP knockout and wild-type mice will be detected after pre-treated with low-dose lipopolysaccharide (LPS), TNFα, or H2O2 and tunicamycin or cisplatin stimulated. Taken together, we will proof that oxidative stress and/or inflammatory factors could increase the susceptibility of ERS-inducible AKI. For illustrated the mechanism of CHOP-induced AKI, we will correct renal paraffins of acute kidney injury in old patients, and perform immunohistochemistry stain of CHOP, eIF2α, XBP-1, and analyse the relationship of these changes and pathogenesis, prognosis of patients.

急性肾损伤（AKI）严重影响人类健康。前期研究发现，衣霉素作用后老年小鼠明显比年轻小鼠更易产生内质网应激反应和引起肾组织损伤，同时C/EBP同源蛋白（CHOP）在介导内质网应激所致损伤中具有更重要作用，但CHOP引起老年性AKI的分子机制尚不明确。本研究首先检测衣霉素刺激后CHOP基因敲除和野生型老年小鼠的肾组织病理变化、细胞凋亡和CHOP所在信号通路相关基因的表达。然后应用过表达和基因沉默技术，分别上调和下调老年小鼠肾小管细胞CHOP基因；应用慢病毒载体，下调老年小鼠CHOP的上游基因，观察CHOP及其下游基因的表达变化，阐明CHOP及该基因所在的信号通路，是引起老年小鼠对内质网应激性AKI易感的关键。利用肿瘤坏死因子受体缺陷小鼠、过氧化氢及低剂量的脂多糖或肿瘤坏死因子α，证明氧化应激和/或炎症，在AKI机制中起加重损伤的作用。最后，在老年AKI患者的肾组织切片进一步验证该机制。

急性肾损伤（AKI）是引起老年性慢性肾损伤、肾病综合症的主要因素之一。研究发现，老年小鼠明显比年轻小鼠更易产生内质网应激反应和引起肾组织损伤，同时C/EBP同源蛋白（CHOP）在介导内质网应激所致损伤中具有更重要作用，但CHOP引起老年性AKI的分子机制尚不明确。本研究应用衣霉素腹腔注射老年C57小鼠后，其肾功能改变明显，肾组织损伤更为严重，凋亡细胞明显增多；但CHOP-/-老年小鼠的肾组织损伤和细胞凋亡不明显。老年小鼠的XBP1、CHOP基因mRNA表达均有不同程度的升高，尤其是CHOP的表达上升了23倍，但CHOP-/-小鼠的XBP1表达升高不明显；同时老年小鼠的磷酸化eIF2α表达显著升高，凋亡因子caspase-12的表达也显著上升。应用针对eIF2α基因的特异性siRNA序列可导致该基因下游的CHOP基因表达下调，但针对IRE1和ATF6的特异性siRNA序列，未显示出对CHOP基因及其下游基因产生下调或上调作用，进一步阐明CHOP及该基因所在的信号通路，是引起老年小鼠对内质网应激性AKI易感的关键。老年小鼠的磷酸化eIF2α、CHOP和GRP94表达显著升高；而TNFR1-/-老年小鼠的磷酸化eIF2α和CHOP升高不明显。经衣霉素处理后，老年小鼠的磷酸化eIF2α和CHOP蛋白明显高于TNFR1基因敲除小鼠，进一步说明TNFR1对eIF2α/CHOP信号通路的重要性。另外在炎症因子表达上，小鼠肾组织的MCP-1 mRNA表达上调，而TNFR1基因敲除小鼠的肾组织经衣霉素腹腔注射后，未出现表达改变。利用BHA的抗氧化特性，人为阻断老年小鼠体内的氧化应激作用后，分析小鼠肾组织的氧化蛋白、MDA等改变，结果显示：老年小鼠经衣霉素干预48小时后其肾组织的总谷胱甘肽、GSH表达明显比年青小鼠的要高，但经BHA处理4月后老年小鼠肾组织总谷胱甘肽、GSH等氧化蛋白表达下降，且比老年小鼠和年青小鼠组的都要低。证明了过度炎症和高氧化状态导致 PERK/eIF2α信号通路的调节异常，是引起老年小鼠 CHOP 基因表达异常升高，证实氧化应激和/或炎症，在AKI机制中起加重损伤的作用。