Department of Nephrology, Zhongshan Hospital, Fudan University, Shanghai Medical Center of Kidney, Shanghai Institute of Kidney and Dialysis, Shanghai Key Laboratory of Kidney and Blood Purification, Hemodialysis Quality Control Center of Shanghai, Shanghai, China.
Department of Nephrology, Xiamen Branch, Zhongshan Hospital, Fudan University, Xiamen, China.
Cell Commun Signal. 2024 Aug 8;22(1):393. doi: 10.1186/s12964-024-01773-w.
Disruptions in intracellular pH (pH) homeostasis, causing deviations from the physiological range, can damage renal epithelial cells. However, the existence of an adaptive mechanism to restore pH 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.
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 pH, 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.
Our findings demonstrated that AP could effectively mitigate H/R injury in HK2 cells. This protective effect and the maintenance of pH 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 pH-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.
This study provides compelling evidence that AP maintains pH homeostasis and promotes cytoskeletal remodeling through FAK/NOX4/NHE1 signaling. This signaling pathway ultimately contributes to alleviated H/R injury in HK2 cells.
细胞内 pH 值（pH）稳态的破坏会导致偏离生理范围，从而损害肾上皮细胞。然而，目前尚不清楚是否存在恢复 pH 值正常的适应机制。早期研究发现 H+是缺血预处理（IPC）的关键介质，由此产生了酸性预处理（AP）的概念。该概念提出，短期、重复的酸性刺激可以增强细胞耐受后续不利应激的能力。虽然 AP 在各种缺血再灌注（I/R）损伤模型中表现出保护作用，但在肾损伤中的应用仍在很大程度上尚未得到探索。
通过用酸性培养基处理人肾（HK2）细胞 12 小时，然后用正常培养基恢复 6 小时，建立 AP 模型。为了诱导缺氧/复氧（H/R）损伤，将 HK2 细胞缺氧 24 小时，复氧 1 小时。在体内，通过夹闭双侧肾蒂 15 分钟建立 IPC 小鼠模型，然后再灌注 4 天。相反，I/R 模型涉及夹闭双侧肾蒂 35 分钟，再灌注 24 小时。采用 Western blot 法检测裂解的半胱天冬酶 3、裂解的半胱天冬酶 9、NHE1、KIM1、FAK 和 NOX4 的表达水平。使用 pH 敏感荧光探针测量 pH 值，使用血红素/CNF 微电极监测肾组织 pH 值。免疫荧光染色用于观察 HK2 细胞中 NHE1、NOX4 和 FAK 的定位以及肌动蛋白细胞骨架结构。进行细胞黏附和划痕实验以评估细胞迁移能力。
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 pH 稳态涉及 Na+/H+交换器 1（NHE1）表达和活性的上调。NHE1 的活性受 FAK 上 Y397 位 pH 依赖性磷酸化的动态变化调节。该过程与 NOX4 介导的活性氧（ROS）产生有关。此外，AP 诱导 FAK、NOX4 和 NHE1 在粘着斑中的共定位，促进细胞骨架重塑并增强细胞黏附和迁移能力。
这项研究提供了有力的证据，证明 AP 通过 FAK/NOX4/NHE1 信号通路维持 pH 稳态并促进细胞骨架重塑，从而减轻 HK2 细胞的 H/R 损伤。
