METHODS: We conducted in vitro and in vivo experiments using CoCl2-induced hypoxic human kidney-2 (HK-2) cells and mice with renal IRI, respectively. Lrg1 was silenced using siRNA and lentiviral vectors in HK-2 cells and mouse kidneys. Rapamycin (Rapa) and methyladenine were applied to regulate autophagy in renal IRI models.
RESULTS: Increased Lrg1 expression was observed in hypoxic HK-2 cells and in the kidneys of mice with renal IRI. Silencing of Lrg1 through siRNA and lentiviral approaches restored autophagy and suppressed apoptosis in CoCl2-induced hypoxic HK-2 cells and renal IRI models. Additionally, reduced Lrg1 expression alleviated kidney damage caused by renal IRI. The downregulation of Lrg1 expression restrained the TGFβ-Smad1/5 signaling pathway in hypoxic-induced HK-2 cells and renal IRI by reducing ALK1 expression. Lastly, the enhancement of autophagy, achieved through Rapa treatment, provided protection against renal IRI in mice.
CONCLUSIONS: Our findings suggest that Lrg1 silencing can be applied as a potential therapeutic target to inhibit the TGFβ1-Smad1/5 pathway, thereby enhancing autophagy and decreasing apoptosis in patients with acute kidney injury.
方法:我们使用CoCl2诱导的低氧人肾-2(HK-2)细胞和具有肾IRI的小鼠进行了体外和体内实验,分别。在HK-2细胞和小鼠肾脏中使用siRNA和慢病毒载体沉默Lrg1。雷帕霉素(Rapa)和甲基腺嘌呤用于调节肾IRI模型中的自噬。
结果:在低氧HK-2细胞和肾IRI小鼠的肾脏中观察到Lrg1表达增加。通过siRNA和慢病毒方法沉默Lrg1可恢复CoCl2诱导的低氧HK-2细胞和肾IRI模型中的自噬并抑制凋亡。此外,Lrg1表达降低减轻肾IRI引起的肾损害。Lrg1表达下调通过降低ALK1表达抑制低氧诱导的HK-2细胞TGFβ-Smad1/5信号通路和肾IRI。最后,自噬的增强,通过Rapa治疗,对小鼠肾脏IRI提供保护。
结论:我们的研究结果表明,Lrg1沉默可以作为抑制TGFβ1-Smad1/5通路的潜在治疗靶点,从而增强急性肾损伤患者的自噬和减少细胞凋亡。