Smad3/mTOR/PGC-1α介导线粒体片段化在CRP抑制急性肾损伤小管上皮细胞增殖的机制研究

Mitochondrial fragmentation(MF) is closely associated with the acute kidney injury(AKI) repair demonstrated by inhibiting MF can promote AKI, but the mechanisms remain abscure. mTOR/PGC-1α is the important signaling pathway which can inhibit MF. We demonstrated that CRP exacerbates AKI via TGF-β1/Smad3 signaling pathway inhibiting tubular epithelial cell proliferation. Moreover, our pilot study found that CRP induced MF accompanied with the inhibition of phosphorylation of mTOR whereas knocking out Smad3 could upregulate the expression of phosphorylation of mTOR. Futhermore, we found that the gene of mTOR has the Smad3 binding site through the database of promoter and transcription factor binding site which means that CRP maybe inhibit AKI repair via the MF mediated by Smad3 pathway. Based on aboved results, we hypothesize that CRP induces MF via the inactivation of mTOR/PGC-1α pathway which is Smad3 signaling pathway dependent therefore inhibits the proliferation and repair of tubular epithelial cell in AKI. This project plans to utilize the techniques including transgenic mice, interfering RNA and ultrasound microbubbles to explore the role and mechanisms of the MF on the CRP inhibiting tubular epithelial cell proliferation in AKI. We hope to provide new idea for the tubular epithelial cell proliferation in AKI and more abundant evidence to demonstrate the MF can be targeted as the new target of AKI repair treatment.

线粒体片段化（MF）与急性肾损伤（AKI）修复密切相关，抑制MF能促进AKI修复，但机制不明。mTOR/PGC-1α是调控MF的重要通路，激活mTOR/PGC-1α能抑制MF。我们证实CRP通过激活TGF-β1/Smad3抑制肾小管上皮细胞增殖，加剧AKI。我们还发现CRP可引起小管上皮细胞MF并抑制mTOR磷酸化，敲除Smad3则促mTOR磷酸化；通过启动子及转录因子结合位点数据库分析发现mTOR基因上有Smad3结合位点。这提示CRP可能通过Smad3途径促MF而参与AKI。为此我们推测CRP通过激活Smad3抑制mTOR/PGC-1α活化而致MF，进而抑制AKI小管上皮细胞增殖修复。本项目拟以转基因鼠、RNA干扰及超声微泡等技术明确MF在CRP抑制AKI小管上皮细胞增殖修复中的作用及其调控机制，为深入认识AKI小管上皮细胞修复提供新思路，并为将MF作为AKI修复的新靶点提供理论依据。

急性肾损伤是慢性肾脏病的重要危险因素，如何早期识别急性肾损伤的发生及相关的发病机制具有十分重要的意义。临床资料显示C反应蛋白（CRP）与急性肾损伤的发生进展及恢复关系密切，是急性肾损伤的重要生物学标志物之一。我们前期已发表的研究证实CRP不仅仅是急性肾损伤生物学标志，且能加剧急性肾损伤，延缓肾脏修复，其中Smad3信号通路在此过程中发挥了重要作用，敲除Smad3后可显著减轻CRP对于急性肾损伤肾小管上皮细胞增殖的抑制，促进修复。大量的研究已经证实线粒体片段化在急性肾损伤早期已经出现，与肾脏损伤密切相关，抑制片段化可促进肾脏修复。我们此次项目的研究主要是进一步探讨Smad3/mTOR/PGC-1α介导线粒体片段化在CRP抑制急性肾损伤小管上皮细胞增殖的机制研究。我们研究结果发现：1、CRP的确能够显著促进线粒体片段化，抑制片段化后可减轻CRP 对肾小管上皮细胞增殖的抑制作用，促进肾小管上皮细胞增殖；2、SIS3抑制Smad3活性后可上调线粒体融合蛋白表达，减轻CRP所引起的小管上皮细胞线粒体片段化，促进细胞增殖；3、缺氧肾小管上皮细胞mTOR/PGC-1a表达水平显著下降，Smad3敲除后蛋白水平PGC-1a表达升高，证实Smad3通过调控PGC-1a促进线粒体片段化，抑制细胞增殖。