Twist通过调控脂肪酸代谢介导慢性缺氧性肾损伤的机制研究

Renal tubular injury induced by chronic hypoxia is an important cause of renal interstitial fibrosis. However, the mechanism of renal interstitial fibrosis induced by hypoxia has not been fully elucidated. In the early study, we found that Twist regulated renal fibrosis induced by hypoxia via Epithelial-MesenchymalTransition (EMT) . However, in the later study we found that partial tubular epithelial cells were not involved in the EMT process even though they had a high expression of Twist and profibrotic factors. Insteadly, we observed a number of lipid droplets deposition in tubular cells, which revealed that Twist might play other important role in the renal interstitial fibrosis through any other mechanisms. We further found that some metabolism related transcription factors including PGC-1α、PGC-1β、PPARγ and PPARα significantly decreased in the hypoxia induced tubular epithelial cells accompanied by the increasing expression of Twist and accumulation of lipid droplets. The reporter gene assay proved that Twist can directly regulate the expression of PGC-1α／PPARα. Therefore, we assume that Twsit may play an important role in the tubular fatty acids metabolism handicap by inhibiting the expression of PGC-1α／PPARα and consequently lead to renal interstitial fibrosis. We will prove this hypothesis through the in vivo and in vitro assays and provide the theoretical basis for clarifying the mechanism of the hypoxia induced renal damage and seeking the new prevention methods.

慢性缺氧性肾小管损伤是导致肾间质纤维化的重要原因，但其机制尚不清楚。我们前期发现Twist可通过上皮细胞转分化参与缺氧导致肾间质纤维化，但在后续实验中我们发现：有一部分高表达Twist的肾小管上皮细胞分泌大量的促纤维化因子,但并未观察到转分化表型，而是在胞内看到有大量脂滴的聚集，提示Twist可能通过其他途径介导慢性缺氧性肾损伤。进一步研究发现，代谢相关转录因子PGC-1α、PGC-1β、PPARα、PPARγ等的表达在缺氧性肾小管上皮细胞中显著降低，且与Twist的表达和脂滴聚集呈负相关。报告基因实验也证实Twist可以直接结合PGC-1α/PPARα，调节其活性。由此，我们推测：“在缺氧微环境下Twist可能通过抑制PGC-1α/PPARα的表达，导致肾小管脂肪酸代谢障碍，进而导致肾间质纤维化”。本课题将通过体内外实验证实这一推论，为阐明缺氧性肾损伤机制和寻找新的防治手段提供理论依据。

慢性缺氧性肾小管损伤是导致肾间质纤维化的重要原因，但其中的机制尚未完全阐明。通过研究我们发现在缺氧微环境下Twist可通过抑制脂肪酸氧化关键转录因子PGC-1α，进而抑制了PPARα，CPT1，ACOX1 等分子的表达和活性，导致肾小管上皮细胞脂肪酸氧化障碍，细胞内脂滴聚集，甘油三酯含量增加ATP减少，大量细胞凋亡，并释放炎症因子、促纤维化因子。常氧条件下体外过表达 Twist可以抑制肾小管上皮细胞脂肪酸代谢水平，促进纤维化因子的释放，体外沉默Twist可以缓解缺氧诱导的脂肪酸代谢障碍。进一步采用荧光素酶报告实验和ChIP 实验证实Twist可以直接转录抑制PGC-1α，Twist通过抑制PGC-1α干扰了PPARα的激活，并且通过 PGC-1α抑制 PGC1α的多个与脂肪酸氧化相关的靶基因如CPT1，ACOX1等。成功构建了肾小管Twist基因敲除鼠，采用此基因鼠构建了UUO和UIRI 经典的肾纤维化模型，发现在UUO模型中，与对照鼠构建的UUO模型肾组织相比，肾小管Twist基因敲除鼠的肾组织中脂滴的堆积明显减少，甘油三酯含量减少，纤维化程度减轻，进一步检测发现，肾组织中ATP含量增加，参与脂肪酸代谢的关键酶表达升高，促纤维化因子的表达降低。因此沉默肾小管上皮细胞的Twist可以从恢复肾小管上皮细胞脂肪酸代谢平衡的角度，减缓了肾间质纤维化的进展。