TRPM4介导钠离子失衡驱动急性肾小管损伤

doi:10.3969/j.issn.1674-8115.2026.09.005

摘要/Abstract

摘要：

目的·探讨瞬时受体电位M亚族成员4（transient receptor potential melastatin 4，TRPM4）在急性肾损伤（acute kidney injury，AKI）中的表达变化及其与肾小管上皮细胞死亡和炎症反应的关系，并评估体内抑制或体外敲低Trpm4对肾小管的保护作用。方法·建立小鼠缺血再灌注（ischemia-reperfusion，IR）及顺铂分别诱导的AKI模型，使用苏木精-伊红（hematoxylin-eosin，H-E）染色评估小鼠肾小管损伤并检测TRPM4在组织中的表达；在IR模型中给予TRPM4抑制剂格列本脲（glibenclamide，GLB）干预。采用Western blotting检测肾组织损伤标志物T细胞免疫球蛋白和黏蛋白结构域蛋白-1（T cell immunoglobulin and mucin domain-containing protein 1，TIM-1）、中性粒细胞明胶酶相关脂质运载蛋白（neutrophil gelatinase-associated lipocalin，NGAL），以及炎症小体通路分子核苷酸结合域富含亮氨酸重复序列和含热蛋白结构域受体3（nucleotide-binding domain leucine‑rich repeat and pyrin domain‑containing receptor 3，NLRP3）、裂解型半胱氨酸蛋白酶-1（cleaved caspase-1，c-CASP-1）、白细胞介素-18（interleukin-18，IL-18）。体外实验以人近端肾小管上皮细胞系（proximal tubular epithelial cell line，PTEC）与小鼠肾小管上皮细胞系（mouse renal tubular epithelial cell line，TCMK-1）建立顺铂、过氧化氢（hydrogen peroxide，H₂O₂）及缺氧复氧（hypoxia/reoxygenation，H/R）损伤模型，检测TRPM4及相关分子表达，并通过实时荧光定量聚合酶链式反应（quantitative polymerase chain reaction，qPCR）评估顺铂剂量梯度下Trpm4与神经损伤诱导蛋白1（ninjurin 1，Ninj1）的转录变化；在TCMK-1中以小干扰RNA（small interfering RNA，siRNA）敲低Trpm4，采用碘化丙啶（propidium iodide，PI）染色与CoroNa-AM探针，并通过流式细胞术检测细胞膜损伤与胞内Na⁺水平；对siRNA-Trpm4处理的细胞进行转录组测序（RNA sequencing，RNA-seq）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）通路富集分析。结果·① 2种AKI模型均出现显著肾小管损伤，同时TRPM4在肾小管上皮细胞中显著上调。② GLB干预显著降低了IR诱导的血清肌酐（serum creatinine，sCr）与尿素氮（blood urea nitrogen，BUN）水平，减轻IR肾小管损伤且降低TIM-1、NGAL水平，并抑制NLRP3-CASP-1-IL-18轴活化。③ 体外顺铂及H/R刺激均伴随TRPM4上调及损伤相关信号的增强，顺铂随剂量增加可进一步诱导Trpm4与Ninj1 mRNA升高。④ 敲低Trpm4可降低顺铂诱导的PI阳性细胞比例，并在PI⁻细胞群中显著下调CoroNa-AM信号［包括CoroNa-AM^high细胞比例及几何平均荧光强度（geometric mean fluorescence intensity，GeoMFI）］，提示Na⁺过载减轻。结论·TRPM4介导的钠离子稳态失衡是驱动肾小管上皮细胞损伤的强效促发因素，且胞内钠过载参与了下游NLRP3炎症小体的放大激活。靶向抑制TRPM4可能构成阻断AKI中“损伤—炎症”恶性循环的关键制动机制。

关键词: 急性肾损伤, 瞬时受体电位M亚族成员4, 肾小管上皮细胞, 格列本脲

Abstract:

Objective ·To investigate dynamic changes in the expression of transient receptor potential melastatin 4 (TRPM4) in acute kidney injury (AKI), its associations with renal tubular epithelial cell death and inflammatory responses, and the protective effects of TRPM4 inhibition in vivo or small interfering RNA (siRNA)-mediated Trpm4 knockdown in vitro on renal tubular injury. Methods ·Murine ischemia-reperfusion (IR) and cisplatin-induced AKI models were established, respectively. Tubular injury was evaluated by hematoxylin-eosin (H-E) staining, and renal TRPM4 expression was assessed. In the IR model, mice received the TRPM4 inhibitor glibenclamide. Western blotting was performed to quantify renal injury markers [T cell immunoglobulin and mucin domain-containing protein 1 (TIM-1) and neutrophil gelatinase-associated lipocalin (NGAL)] and inflammasome pathway components [nucleotide-binding domain leucine‑rich repeat and pyrin domain‑containing receptor 3 (NLRP3), cleaved caspase-1 (c-CASP-1), and interleukin-18 (IL-18)]. In vitro, a human proximal tubular epithelial cell line (PTEC) and a mouse renal tubular epithelial cell line (TCMK-1) were subjected to injury stimuli including cisplatin, hydrogen peroxide (H₂O₂), and hypoxia/reoxygenation (H/R), followed by assessment of TRPM4 and injury-associated signaling. Quantitative PCR (qPCR) was used to examine Trpm4 and ninjurin 1 (Ninj1) transcriptional responses across a cisplatin dose gradient. In TCMK-1 cells, Trpm4 was silenced using siRNA; plasma membrane injury and intracellular Na⁺ load were quantified by propidium iodide (PI) staining and the CoroNa-AM probe using flow cytometry. RNA sequencing was conducted in siRNA-Trpm4-treated cells, with Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis performed to characterize transcriptional programs impacted by Trpm4 knockdown. Results ·① Both AKI models exhibited marked tubular injury accompanied by significant upregulation of TRPM4 in renal tubular epithelial cells. ② Glibenclamide treatment significantly reduced serum creatinine (sCr) and blood urea nitrogen (BUN) levels, alleviated IR-induced tubular damage, decreased TIM-1 and NGAL levels, and suppressed activation of the NLRP3-CASP-1-IL-18 axis. ③ In vitro exposure to cisplatin or H/R induced TRPM4 upregulation and enhanced injury-associated signaling, while increasing cisplatin doses further elevated Trpm4 and Ninj1 mRNA levels. ④ Trpm4 knockdown reduced the proportion of PI-positive cells after cisplatin challenge and significantly decreased CoroNa-AM signals [including the proportion of CoroNa-AM^high cells and geometric mean fluorescence intensity (GeoMFI)] within the PI⁻ gate, indicating attenuation of intracellular Na⁺ overload. Conclusion ·TRPM4-mediated sodium dysregulation is a critical driver of renal tubular injury, and intracellular Na⁺ overload is involved in the amplification of NLRP3 inflammasome activation. Targeting TRPM4 may constitute a key brake on the injury-inflammation cycle in AKI.

Key words: acute kidney injury (AKI), transient receptor potential melastatin 4 (TRPM4), renal tubular epithelial cell, glibenclamide

中图分类号:

R692.5

王佳玲, 王彦哲, 缪乃俊, 王筱霞. TRPM4介导钠离子失衡驱动急性肾小管损伤[J]. 上海交通大学学报（医学版）, 2026, 46(9): 1199-1211.

Wang Jialing, Wang Yanzhe, Miao Naijun, Wang Xiaoxia. TRPM4-mediated sodium dysregulation drives acute renal tubular injury[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026,(9): 1199-1211.

图/表 4

图1 顺铂与缺血再灌注诱导建立AKI模型，模型伴随肾小管损伤加重、TRPM4上调及细胞凋亡增加Note： A. Schematic illustration of the experimental design and sampling timeline for cisplatin-induced and IR-induced AKI models[19]. B. Representative H-E staining images of renal cortex sections from control (Con) and cisplatin-treated mice. C. Quantitative analysis of tubular injury scores in B. D. Representative H-E staining images of renal cortex sections from sham and IR mice. E. Quantitative analysis of tubular injury scores in D. F. Immunohistochemistry (IHC) staining of TRPM4 in kidneys from control and cisplatin-treated mice. G. Quantification of the TRPM4-positive area in F. H. IHC staining of TRPM4 in kidneys from sham and IR mice. I. Quantification of TRPM4-positive area in H. J. Representative immunofluorescence images of TUNEL staining (green) and DAPI (blue) in renal tissues from control and cisplatin-treated mice. (Scale bar: 50 μm). K. Representative immunofluorescence images of TUNEL staining in sham and IR groups. L. Quantification of TUNEL fluorescence ratio in the Con and cisplatin groups. M. Quantification of the TUNEL fluorescence ratio in sham and IR groups. ①P<0.001, compared with the corresponding control group.

Fig 1 Cisplatin- and IR-induced AKI is accompanied by aggravated tubular injury, upregulated TRPM4 expression, and increased cell apoptosis

图2 体外损伤诱导TRPM4上调，敲低 Trpm4 缓解膜损伤并减轻Na⁺过载Note： A. Representative Western blotting bands showing TRPM4, TIM-1, and cleaved caspase-1 (c-CASP-1) expression in PTEC under control and cisplatin-treated conditions. B. Densitometric quantification of TRPM4, TIM-1, and c-CASP-1 protein levels in A, normalized to β-actin. C. Representative Western blotting bands showing TRPM4 and TIM-1 expression in PTEC under control and H/R conditions. D. Densitometric quantification of TRPM4 and TIM-1 protein levels in C, normalized to β-actin. E. qPCR analysis of Trpm4 and Ninj1 mRNA levels in TCMK-1 cells treated with increasing doses of cisplatin (cis15, cis30, and cis45 represent 15, 30, and 45 μmol·L-1 cisplatin, respectively), presented relative to the control group. F. Flow cytometry analysis in TCMK-1 cells transfected with si-Trpm4 under cisplatin treatment: plasma membrane injury was assessed by PI uptake (PI vs SSC-A), and intracellular Na⁺ levels were assessed by CoroNa-AM (CoroNa-AM vs SSC-A) within the PI⁻ gate, with representative CoroNa-AM geometric mean fluorescence intensity (GeoMFI) shown. G. Quantification of PI-positive cell rate. H. Quantification of CoroNa-AMhigh cells in PI⁻ cells. I. Quantification of GeoMFI within the PI⁻ gate. ①P<0.001, compared with the corresponding control group; ②P=0.006, compared with the control group in the cisplatin stimulation experiment; ③P=0.004, compared with the Con group in the cisplatin dose-gradient qPCR; ④P=0.002, compared with the cisplatin group; ⑤P=0.011, compared with the cisplatin group.

Fig 2 In vitro injury induces TRPM4 upregulation, and Trpm4 knockdown alleviates membrane injury and reduces Na⁺ overload

图3 抑制TRPM4减轻体内IR诱导的肾功能衰竭、肾小管损伤与炎症相关信号Note： A/B. Serum levels of creatinine (sCr; A) and blood urea nitrogen (BUN; B) in mice from the control (Con), glibenclamide (GLB), ischemia-reperfusion (IR), and IR+GLB groups. C. Representative Western blotting bands showing TRPM4, NINJ1, TIM-1, and NGAL expression in kidneys from the Con, GLB, IR, and IR+GLB groups. D. Densitometric quantification of TRPM4, NINJ1, TIM-1, and NGAL protein levels in (C), normalized to β-actin. E. Representative Western blotting bands showing NLRP3, cleaved caspase-1 (c-CASP-1), and IL-18 expression in kidneys from the Con, GLB, IR, and IR+GLB groups. F. Densitometric quantification of NLRP3, c-CASP-1, and IL-18 protein levels in E, normalized to β-actin. ①P<0.001, ②P=0.003.

Fig 3 TRPM4 inhibition attenuates IR-induced renal dysfunction, tubular injury, and inflammation-related signaling in vivo

图4 Trpm4 敲低在转录组水平重塑氧化应激-炎症/焦亡与离子转运相关转录程序Note： A. Principal component analysis (PCA) of transcriptomic profiles from the control (Con), si-Trpm4, and si-Trpm4+H2O2 groups. B. TRPM4 mRNA expression levels [log2(FPKM+1)] in the Con and si-Trpm4 groups. C. Heatmap showing the expression of representative stress/inflammation- and ion-transport-related genes across groups [row Z-score of log2(FPKM+1)]. D. Volcano plot of DEGs in si-Trpm4 vs Con. E. Volcano plot of DEGs in si-Trpm4+H2O2vs Con. F. KEGG pathway enrichment dot plot for upregulated DEGs in si-Trpm4vs Con. G. KEGG pathway enrichment dot plot for upregulated DEGs in si-Trpm4+H2O2vs Con. H. qPCR validation of inflammation- and injury-related genes (Nlrp3, Il1b, and Havcr1) in TCMK-1 cells treated with H2O2 and si-Trpm4. I. qPCR analysis of the same genes in kidney tissues from sham, IR, and IR+GLB mice to verify the biological relevance of the transcriptomic findings. ①P=0.040, ②P<0.001.

Fig 4 Trpm4 knockdown reshapes transcriptional programs associated with oxidative stress, inflammation/pyroptosis, and ion transport at the transcriptomic level

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