TRPM4介导钠离子失衡驱动急性肾小管损伤OA

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-AMhigh 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.