背景:细胞内pH(pHi)稳态的破坏,导致生理范围的偏差,会损伤肾上皮细胞。然而,是否存在使pHi恢复正常的适应性机制尚不清楚。早期研究确定H+是缺血预处理(IPC)的关键介质,导致了酸性预处理(AP)的概念。这个概念提出了短期,重复的酸性刺激可以增强细胞承受随后不利压力的能力。虽然AP在各种缺血再灌注(I/R)损伤模型中显示出保护作用,其在肾损伤中的应用仍未被探索。
方法:通过用酸性培养基处理人肾(HK2)细胞12小时,建立了AP模型,然后用正常培养基恢复期6小时。为了诱导缺氧/复氧(H/R)损伤,HK2细胞缺氧24h,复氧1h。通过夹闭双侧肾蒂15min建立IPC小鼠模型,然后再灌注4天。相反,I/R模型包括夹闭双侧肾蒂35分钟,再灌注24小时。采用蛋白质印迹法评估cleavedcaspase3,cleavedcaspase9,NHE1,KIM1,FAK的表达水平,NOX4pH敏感的荧光探针用于测量pHi,而Hemin/CNF微电极监测肾组织pH。进行免疫荧光染色以显示NHE1,NOX4和FAK的定位,与HK2细胞中的肌动蛋白细胞骨架结构一起。进行细胞粘附和划痕测定以评估细胞运动性。
结果:我们的研究结果表明,AP可以有效减轻HK2细胞的H/R损伤。AP的这种保护作用和维持pHi稳态涉及Na/H交换剂1(NHE1)表达和活性的上调。NHE1的活性受Y397处的粘着斑激酶(FAK)的pHi依赖性磷酸化的动态变化调节。此过程与NOX4介导的活性氧(ROS)的产生有关。此外,AP诱导FAK的共同定位,NOX4和NHE1在粘着斑中,促进细胞骨架重塑,增强细胞粘附和迁移能力。
结论:本研究提供了令人信服的证据,表明AP通过FAK/NOX4/NHE1信号传导维持pHi稳态并促进细胞骨架重塑。该信号通路最终有助于减轻HK2细胞中的H/R损伤。
BACKGROUND: Disruptions in intracellular pH (pHi) homeostasis, causing deviations from the physiological range, can damage renal epithelial cells. However, the existence of an adaptive mechanism to restore pHi to normalcy remains unclear. Early research identified H+ as a critical mediator of ischemic preconditioning (IPC), leading to the concept of acidic preconditioning (AP). This concept proposes that short-term, repetitive acidic stimulation can enhance a cell\'s capacity to withstand subsequent adverse stress. While AP has demonstrated protective effects in various ischemia-reperfusion (I/R) injury models, its application in kidney injury remains largely unexplored.
METHODS: An AP model was established in human kidney (HK2) cells by treating them with an acidic medium for 12 h, followed by a recovery period with a normal medium for 6 h. To induce hypoxia/reoxygenation (H/R) injury, HK2 cells were subjected to hypoxia for 24 h and reoxygenation for 1 h. In vivo, a mouse model of IPC was established by clamping the bilateral renal pedicles for 15 min, followed by reperfusion for 4 days. Conversely, the I/R model involved clamping the bilateral renal pedicles for 35 min and reperfusion for 24 h. Western blotting was employed to evaluate the expression levels of cleaved caspase 3, cleaved caspase 9, NHE1, KIM1, FAK, and NOX4. A pH-sensitive fluorescent probe was used to measure pHi, while a Hemin/CNF microelectrode monitored kidney tissue pH. Immunofluorescence staining was performed to visualize the localization of NHE1, NOX4, and FAK, along with the actin cytoskeleton structure in HK2 cells. Cell adhesion and scratch assays were conducted to assess cell motility.
RESULTS: Our findings demonstrated that AP could effectively mitigate H/R injury in HK2 cells. This protective effect and the maintenance of pHi homeostasis by AP involved the upregulation of Na+/H+ exchanger 1 (NHE1) expression and activity. The activity of NHE1 was regulated by dynamic changes in pHi-dependent phosphorylation of Focal Adhesion Kinase (FAK) at Y397. This process was associated with NOX4-mediated reactive oxygen species (ROS) production. Furthermore, AP induced the co-localization of FAK, NOX4, and NHE1 in focal adhesions, promoting cytoskeletal remodeling and enhancing cell adhesion and migration capabilities.
CONCLUSIONS: This study provides compelling evidence that AP maintains pHi homeostasis and promotes cytoskeletal remodeling through FAK/NOX4/NHE1 signaling. This signaling pathway ultimately contributes to alleviated H/R injury in HK2 cells.