Appropriate intracellular pH is essential for the proper functioning of cells. Acidic pretreatment can induce intracellular acidic adaptation and improve cells' ability to withstand harmful stress, a process known as acidic preconditioning (AP). Sodium-hydrogen exchanger 1 (NHE1) is commonly found in renal tubular epithelial cells and serves as a key channel for the excretion of H⁺. Nevertheless, the role of NHE1 in renal AP are still not fully understood.

This study developed an AP model in the human renal tubular epithelial cell line (HK2) that effectively mitigates hypoxia/reoxygenation (H/R) injury. The research also clarified the mechanism of AP in regulating pHi homeostasis via regulating NHE1, which was further validated in a mouse ischemic preconditioning(IPC) model.

The experiment was conducted from June to December 2023 at the Laboratory of Nephrology, Zhongshan Hospital, Fudan University. HK2 cells were subjected to hypoxia for 24 hours and then reoxygenation for 1 hour to establish an in vitro model of acute kidney injury(AKI); C57BL/6 mice were performed 35 minutes occlusion of bilateral renal artery following reperfusion for 24 hours to establish in vivo model of AKI; Mice were clamped on both sides of the renal artery for 15 minutes and then perfused for 4 days since I/R. C57BL/6 mice were randomly divided into sham group (Sham), IPC group, I/R group and IPC+I/R group, n=6 in each group, among which the mice in the IPC+I/R group referred to bilateral renal arteries of the mice were occluded for 15 minutes and then perfused for 4 days before I/R was performed. The in vitro experiment was divided into three parts. Part 1: to establish the AP model, HK-2 cells were treated with pH 6.6 acidic medium (A) followed by pH 7.4 normal medium (R) before subjecting them to H/R. Part 2: small interfering RNA against NHE1 (siNHE1) was used to knock down NHE1 expression; Part 3: HK2 cells were treated with low chloride medium at pH 6.6 in phase A and normal chloride medium at pH 7.4 in phase R. Cell viability was evaluated using the CellTiter-Lumi luminescence method, apoptosis levels were assessed through Western Blotting, Tunel staining, and Annexin V-FITC/PI double staining. The activity of NHE1 and pHi were detected by BCECF-AM probe and NH4Cl-prepulse technology. The ability of maintenancing steady-state of pHi was evaluated by extracellular pH(pHe)-pHi curve, and the expression of NHE1 was detected through immunofluorescence in kidney tissue and HK2 cells.

Treatment with acidic conditions for 12 hours and subsequent recovery in normal medium for 6 hours significantly decreased the expression levels of cleaved caspase 3 and cleaved caspase 9 following hypoxia/reoxygenation (H/R) treatment, as well as reduced the proportion of Annexin V and Tunel positive cells (P<0.05). Furthermore, AP mitigated intracellular acidification post H/R treatment (P<0.05), increased NHE1 expression and activity, and shifted the pHe-pHi curve upwards; silencing NHE1 expression resulted in a downward shift of the pHe-pHi curve after AP treatment. Knocking down NHE1 attenuated the protective effects of AP against H/R injury, leading to higher expression levels of cleaved caspase 3 and cleaved caspase 9 (P<0.05). Treatment with low-chloride medium abolishes the impact of acidic stimulation on pHi, while also reversing the upregulation of NHE1 expression and the upward shift of the pHe-pHi curve post AP (P<0.05). There was a decrease in pHi fluctuations after ischemia/reperfusion (I/R) (P<0.05) and an upward shift in the pHe-pHi curve after IPC. IPC also significantly decreased the expression levels of KIM1 and cleaved caspase 3, while upregulating NHE1 expression after I/R (P<0.05). Additionally, IPC facilitated the colocalization of NHE1 and LTL after I/R.

AP promotes the maintenance of pHi homeostasis by increasing NHE1 expression and activity via transient fluctuations in pHi, leading to intracellular acidic adaptation and mitigating H/R injury.

适宜的细胞内pH（pHi）是细胞发挥功能的基础。酸性预处理有利于诱导细胞内液酸适应，增强细胞应对损伤性应激的能力，被称为酸性预适应（AP）。钠-氢交换体1（NHE1）在肾小管上皮细胞广泛表达，也是细胞排H⁺的主要通道之一。然而，NHE1在肾脏AP中的作用及其机制尚不明确。

本研究首先在人肾小管上皮细胞系（HK2）中建立可有效减轻缺氧/复氧（H/R）损伤的AP模型，围绕NHE1阐明AP调控pHi稳态的作用机制，并在小鼠缺血预适应（IPC）模型中加以验证。

实验于2023年6—12月在复旦大学附属中山医院肾内科实验室完成。在体外实验，采用缺氧24 h后复氧1 h建立HK2细胞的H/R模型。应用pH 6.6酸性培养基处理细胞，构建AP模型。根据不同实施方案分为3部分，（１）AP模型的建立：将AP过程分为pH 6.6酸性培养基处理阶段（A）和pH 7.4正常培养基恢复阶段（R），HK2细胞先后经过不同时间的A和R处理后再进行H/R，探索最佳AP模型及其对NHE1和pHi的调控作用；（2）使用NHE1的小干扰RNA（siNHE1）敲低NHE1，观察AP保护效应的改变；（3）应用低氯离子培养基抑制pHi的波动，观察AP对NHE1调控作用和保护效应的改变。在体内实验，选取24只SPF级雄性野生型C57BL/6小鼠，分为假手术组（Sham组）、肾缺血再灌注组（I/R组）、肾缺血预适应组（IPC组）、肾缺血预适应+肾缺血再灌注组（IPC+I/R组），每组各6只。I/R模型采用夹闭双侧肾动静脉35 min后复灌流24 h。IPC模型采用夹闭双侧肾动静脉15 min后复灌流4 d。IPC+I/R组则是在IPC后进行I/R处理。使用CellTiter-Lumi Ⅱ发光法检测细胞活力。使用Western Blotting法检测NHE1、活化型半胱氨酸天冬氨酸蛋白酶（cleaved caspase）3、肾脏损伤分子1（KIM1）和cleaved caspase 9表达水平；使用流式细胞仪和Tunel染色评估细胞凋亡水平；采用BCECF-AM探针和NH₄Cl冲击法检测pHi和NHE1活力；采用细胞外液pH值（pHe）-pHi曲线评估pHi稳态维持能力；使用免疫荧光染色检测NHE1在HK2细胞和肾脏组织中的定位。

酸性处理12 h，正常培养基恢复6 h可显著降低H/R处理后cleaved caspase 3和cleaved caspase 9表达水平，减少Annexin V和Tunel阳性细胞比例（P<0.05）。AP缓解H/R处理后的细胞内酸化（P<0.05），上调NHE1表达和活性，并上移pHe-pHi曲线；敲减NHE1表达下移AP后的pHe-pHi曲线。敲减NHE1减弱AP对H/R损伤的保护作用，表现为cleaved caspase 3和cleaved caspase 9表达水平升高（P<0.05）。低氯培养消除了AP早期pHi的波动，同时抑制了AP后NHE1表达上调和pHe-pHi曲线上移。IPC缓解I/R后的pHi降低（P<0.05）并上移pHe-pHi曲线。IPC显著降低了I/R后KIM1和cleaved caspase 3表达水平，上调I/R后NHE1表达水平（P<0.05）。免疫荧光显示I/R后NHE1在近端肾小管上皮细胞表达增多。

本研究在肾脏细胞H/R模型中证实AP的保护作用。AP通过诱导pHi的短暂波动上调NHE1表达和活性，进而诱导细胞内酸适应，最终减轻H/R损伤。