| Background:Acute kidney injury(AKI)is a serious clinical condition characterized by a rapid decline in renal function within hours or days.AKI can be caused by various factors,among which cisplatin is commonly observed.Ferroptosis has been recently identified as an important pathological process in cisplatin-induced AKI(CP-AKI).Ferritinophagy,as the initiating event of ferroptosis,refers to a process where nuclear receptor coactivator 4(NCOA4)mediates the degradation of ferritin in lysosomes to generate free iron.Protein arginine methyltransferase 4(PRMT4)catalyzes the methylation of arginine residues of its targeted proteins to participate in various biological processes.Previous studies have demonstrated the regulatory role of PRMT4 in the injuries of various organs,such as hearts and lungs.However,its role in AKI remains unclear.The interaction between PRMT4 and NCOA 1-3 has been well recognized,but the relationship between PRMT4 and NCOA4 has not been investigated.Therefore,it’s crucial to explore the interaction between PRMT4 and NCOA4 in CP-AKI,as well as elucidate their regulatory roles in ferroptosis.Objective:This study aimed to investigate the regulatory role of PRMT4 in the pathogenesis of CP-AKI and its underlying molecular mechanisms.Methods:This study analyzed the transcriptional levels of the PRMT family members in the mouse kidney using quantitative reverse transcription PCR(qRT-PCR),and the expression pattern of PRMT4 in the kidney was explored by immunofluorescence staining.To investigate the role of PRMT4 in vitro,lentivirus was used to intervene the expression profile of PRMT4 in BUMPT cells.Cell viability and ferroptosis-related markers were detected using various processes,including CCK8,Western blot studies,reactive oxygen species(ROS)detection,and free iron assay.Furthermore,intraparenchymal injection of adenovirus was applied to mice kidneys in in vivo studies,and intraperitoneal injection of cisplatin was used for the induction of AKI.The expression of PRMT4,kidney injuries and ferroptosis-related markers were detected by western blot studies,hematoxylin and eosin(H&E)staining,periodic acid-Schiff(PAS)staining,immunohistochemistry staining,and immunofluorescence staining.To further explore the mechanism involved in PRMT4 and ferroptosis,co-immunoprecipitation(CO-IP)was performed to analyze the interaction pattern between PRMT4 and NCOA4.In addition,their exact binding site was further investigated by molecular modeling,machine learning and site-directed mutagenesis.Results:PRMT4 was extensively expressed in renal proximal tubules,and its expression was obviously downregulated in cisplatin-treated BUMPT cells and mouse kidneys.Both in vitro and in vivo experiments demonstrated that PRMT4 overexpression mitigated cisplatin-induced damage to BUMPT cells and renal tissue.Further investigation revealed that this protective effect was associated with the regulation of ferroptosis,while PRMT4 knockdown exacerbated above process.These results were validated by cell viability assays,ROS detection,morphological analyses,tubular injury markers,and ferroptosis-related markers.Additionally,CO-IP analysis indicated that the PH and SAM domains of PRMT4 interact with the ARA70-1 domain of NCOA4,thus inhibiting ferritinophagy to decelerate ferroptosis.Conclusion:Our study demonstrated that PRMT4 played a critical role in the pathogenesis of CP-AKI.PRMT4 overexpression alleviated the renal injuries,while its gene knockdown exacerbating it.Mechanistically,our studies showed that the key effect was mediated by the interaction between PH&SAM domains of PRMT4 and ARA70-1 domain of NCOA4,inhibiting ferritinophagy and thus mitigating ferroptosis.These findings provided new insights into the mechanism of ferroptosis in CP-AKI and suggested PRMT4 as a potential therapeutic target for AKI.29 figures,16 tables,129 references... |
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