Mechanism of Nicotinamide N-methyltransferase (NNMT) Regulating Heterogeneous Nuclear Ribonucleoprotein (HnRNP) D Promoting Ferroptosis in Renal Tubular Epithelial Cells and Aggravating Acute Kidney Injury

Background: The study has shown that AKI is a significant risk factor for CKD and ESRD, seriously endangering human health. Exploring the mechanisms of AKI and finding effective treatment and intervention measures is of great clinical significance. Recently, Xu et al. used single-cell mRNA sequencing (scRNA-seq) to show that the ferroptosis pathway was significantly activated in proximal renal tubular epithelial cells (PTCs) of AKI mouse models. Therefore, ferroptosis could be a potential therapeutic target, which can provide new strategies for the treatment of AKI. Methods: First, we screened the human GEO AKI database GSE30718, and enrichment analysis based on differential genes revealed significant enrichment of the ferroptosis pathway. Further screening of candidate gene nicotinamide N-methyltransferase (NNMT) in the ferroptosis pathway through machine learning for further research. Through combined human and mouse AKI scRNA-seq analysis, we found that NNMT is highly expressed in renal tubular epithelial cells of AKI renal tissue. Then, we overexpressed or silenced NNMT protein in HK-2 cells, changes in cell ferroptosis markers and release of inflammatory factors were detected by Western blot, real-time PCR, ELISA, immunofluorescence, and flow cytometry. Next, we used LC-MS/MS to detect potential binding proteins of NNMT and then validated it by using CO-IP and immunofluorescence. Results: Silencing or overexpression NNMT can improve or exacerbates H/R and Cis-induced ferroptosis and inflammatory cytokine release in HK-2 cells. By using LC-MS/MS analysis, it was found that there is a potential interaction between HnRNPD and NNMT. Further validation of the binding between NNMT and HnRNPD was conducted through CO-IP and immunofluorescence, and NNMT increased the expression of HnRNPD protein by inhibiting the degradation of HnRNPD ubiquitinated proteasomes. RIP experiments confirmed that HnRNPD can stabilize the expression of ATF3 protein expression and lead to ferroptosis. Conclusion: Our findings indicate an upregulation of NNMT expression in renal tubular epithelial cells during AKI. NNMT serves to stabilize ATF3 protein expression by impeding the ubiquitination and proteasome-mediated degradation of HnRNPD. This mechanism leads to lipid peroxidation and ferroptosis in renal tubular epithelial cells, thereby fostering the onset and progression of AKI