AngⅡ联合TNF-α促使肾小管上皮细胞发生程序性坏死在CKD进展中的作用机制

Chronic kidney disease (CKD) is a main cause and an ultimate common pathway of end-stage renal disease. It is widely recognized that tubulointerstitial fibrosis (TIF), an important pathological change caused by the ongoing loss of renal tubular cells and their progressive replacement by fibroblasts and amorphous fibrous material forming extracellular matrix, correlates better with the impairment of renal function and serves as a more reliable indicator of renal prognosis than glomerular damage. Therefore, it has great significance for early prevention of CKD that we explore the mechanism of renal tubular epithelial cell excessive death. In our earlier studies, we found that necroptosis mediated by RIPK1 and RIPK3 might be an important mode of renal tubular cell death, thus favoring the progression of tubular atrophy and CKD in rats submitted to subtotal nephrectomy (SNx), and it might correlate with TNF-α and AngⅡ. However, the role of necroptosis mediated by RIPK3 in the clinical progression of CKD and its mechanism fail to be fully elucidated. In the present study, renal tubular epithelial cells from the patients with CKD will be investigated to explore whether necroptosis mediated by RIP3 leads to the progressive depletion of tubular epithelial cells using transmission electron microscopy and Immunofluorescence for Caspase-3 and in situ fluorescence TUNEL staining. We would build up the cell models to assess whether AngⅡ combined TNF-α might cause necroptotic cell death of human kidney proximal tubular epithelial cells and its potential mechanism using molecular cytological methods. This will also be test in mouse model in vivo. We hope what we get from this study will illuminate how AngⅡ combined TNF-α causes necroptotic cell death in renal tubular and contributes to the progression of CKD. It will be new insights for early preventing the progression of CKD.

慢性肾脏病（CKD）是引起终末期肾病最主要的原因。其发病与肾小管上皮细胞的进行性死亡，导致肾小管间质纤维化不断进展密切相关。为此，探讨肾小管上皮细胞过度死亡的机制对CKD的早期防治具有重大意义。前期我们发现RIPK1/3依赖的程序性坏死可能是促使肾大部切除大鼠慢性肾损害进展的重要原因，且该程序性坏死可能与炎症介质TNF-α、AngⅡ相关。但该程序性坏死在CKD患者肾小管上皮细胞过度死亡中的作用及机制尚未清楚。本项目以不同时期CKD患者为研究对象，探讨RIPK3依赖的程序性坏死是否是引起CKD进展中肾小管上皮细胞过度死亡的重要方式；采用分子生物细胞学方法探讨该程序性坏死是否与CKD患者肾组织中AngⅡ、TNF-α过度活化相关及可能机制，并在动物模型上加以验证。以期回答AngⅡ是否可以联合TNF-α促使肾小管上皮细胞发生程序性坏死进而加速CKD进展这一科学问题，为尽早防治CKD找出新的靶点。

肾素-血管紧张素系统（RAS）及炎症介质TNF-α的异常激活在慢性肾脏病（CKD）的发生、发展中起着重要作用。课题组前期研究表明:慢性肾损伤大鼠肾小管上皮细胞的丢失过程中，肾小管上皮细胞程序性坏死可能比细胞凋亡起着更重要的作用，但其发生机制尚不清楚。本研究采用不同时期 CKD 患者肾组织标本，阐明在CKD进展中，CKD患者肾小管上皮细胞发生了程序性坏死，且该程序性坏死可能是导致CKD患者肾小管上皮细胞过度死亡，慢性肾损伤不断进展的重要因素；并且该肾小管上皮细胞程序性坏死可能与肾组织中高表达的AT2R及TNF-α 相关；并结合体外细胞实验与慢性肾损伤动物模型证实AngⅡ和/或TNF-α 可促使肾小管上皮细胞发生程序性坏死；且AngII的细胞毒性可能是促发慢性肾损伤进展中肾小管上皮细胞发生程序性坏死重要因素；通过使用 AngII 受体抑制剂、FasL 阻断剂及 RIP1/3 和 MLKL 相应的抑制剂均可阻断 AngII 可诱导肾小管上皮细胞程序性坏死，提示AngII 可能通过 AT2R 及下游信号分子 Fas 和 FasL 诱导肾小管上皮细胞发生程序性坏死。阻断程序性坏死、TNF-α、AT2R及其下游信号分子是尽早挽救肾小管上皮细胞过度死亡、防治 CKD 不断进展的重要措施。