Department of Diagnostics, School of Medicine, Hunan University of Medicine, Huaihua, Hunan, China.
Guangdong Provincial Key Laboratory of Speed Capability, The Guangzhou Key Laboratory of Precision Orthopedics and Regenerative Medicine, Department of Sports Medicine, The First Affiliated Hospital, Jinan University, Guangzhou, Guangdong, China.
J Cell Biochem. 2024 Oct;125(10):e30634. doi: 10.1002/jcb.30634. Epub 2024 Aug 2.
In this study, we investigate the effect of neuregulin 4 (NRG4) on podocyte damage in a mouse model of diabetic nephropathy (DN) and we elucidate the underlying molecular mechanisms. In vivo experiments were conducted using a C57BL/6 mouse model of DN to determine the effect of NRG4 on proteinuria and podocyte injury, and in vitro experiments were performed with conditionally immortalized mouse podocytes treated with high glucose and NRG4 to assess the protective effects of NRG4 on podocyte injury. Autophagy-related protein levels and related signaling pathways were evaluated both in vivo and in vitro. The involvement of the adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway was detected using chloroquine or AMPK inhibitors. The results showed that the AMPK/mTOR pathway was involved in the protective roles of NRG4 against high glucose-mediated podocyte injury. Also, NRG4 significantly decreased albuminuria in DN mice. PAS staining indicated that NRG4 mitigated glomerular volume and mesangium expansion in DN mice. Consistently, western blot and RT-PCR analyses confirmed that NRG4 decreased the expression of pro-fibrotic molecules in the glomeruli of DN mice. The immunofluorescence results showed that NRG4 retained expression of podocin and nephrin, whereas transmission electron microscopy revealed that NRG4 alleviated podocyte injury. In DN mice, NRG4 decreased podocyte apoptosis and increased expression of nephrin and podocin, while decreasing the expression of desmin and HIF1α. Overall, NRG4 improved albuminuria, glomerulosclerosis, glomerulomegaly, and hypoxia in DN mice. The in vitro experiments showed that NRG4 inhibited HG-induced podocyte injury and apoptosis. Furthermore, autophagy of the glomeruli decreased in DN mice, but reactivated following NRG4 intervention. NRG4 intervention was found to partially activate autophagy via the AMPK/mTOR signaling pathway. Consequently, when the AMPK/mTOR pathway was suppressed or autophagy was inhibited, the beneficial effects of NRG4 intervention on podocyte injury were diminished. These results indicate that NRG4 intervention attenuates podocyte injury and apoptosis by promoting autophagy in the kidneys of DN mice, in part, by activating the AMPK/mTOR signaling pathway.
在这项研究中，我们研究了神经调节蛋白 4（NRG4）对糖尿病肾病（DN）小鼠模型中足细胞损伤的影响，并阐明了其潜在的分子机制。体内实验使用 C57BL/6 糖尿病肾病小鼠模型来确定 NRG4 对蛋白尿和足细胞损伤的影响，体外实验则使用条件永生化的小鼠足细胞进行，这些细胞经高糖和 NRG4 处理以评估 NRG4 对足细胞损伤的保护作用。体内和体外实验均评估了自噬相关蛋白水平和相关信号通路。使用氯喹或 AMPK 抑制剂检测腺苷单磷酸激活蛋白激酶（AMPK）/雷帕霉素靶蛋白（mTOR）通路的参与情况。结果表明，AMPK/mTOR 通路参与了 NRG4 对高糖介导的足细胞损伤的保护作用。此外，NRG4 显著降低了糖尿病肾病小鼠的白蛋白尿。PAS 染色表明 NRG4 减轻了糖尿病肾病小鼠的肾小球体积和系膜扩张。同样，western blot 和 RT-PCR 分析证实 NRG4 降低了糖尿病肾病小鼠肾小球中促纤维化分子的表达。免疫荧光结果表明 NRG4 保留了足细胞中 podocin 和 nephrin 的表达，而透射电镜显示 NRG4 减轻了足细胞损伤。在糖尿病肾病小鼠中，NRG4 减少了足细胞凋亡，增加了 nephrin 和 podocin 的表达，同时降低了 desmin 和 HIF1α 的表达。总的来说，NRG4 改善了糖尿病肾病小鼠的白蛋白尿、肾小球硬化、肾小球肥大和缺氧。体外实验表明，NRG4 抑制了高糖诱导的足细胞损伤和凋亡。此外，糖尿病肾病小鼠的肾小球自噬减少，但 NRG4 干预后重新激活。NRG4 干预被发现部分通过 AMPK/mTOR 信号通路激活自噬。因此，当 AMPK/mTOR 通路被抑制或自噬被抑制时，NRG4 干预对足细胞损伤的有益作用就会减弱。这些结果表明，NRG4 通过促进糖尿病肾病小鼠肾脏中的自噬来减轻足细胞损伤和凋亡，部分是通过激活 AMPK/mTOR 信号通路。
