N6 Methyladenosine-Modified CircNfix Attenuates Sepsis-Induced AKI by Facilitating the Ubiquitination of YTHDF2 and Increasing DAPK2 mRNA Stability

Background: Increasing evidence shows that circRNAs play an important role in sepsis-induced acute kidney injury (SAKI). However, the mechanisms of circRNAs in SAKI have not been reported except the way of miRNA sponges. Notably, the function of N6-methyladenosine (m6A)-modified circRNA in SAKI remains undiscovered. Therefore, global profiles of circRNAs in kidneys with SAKI are needed, which will contribute to uncovering novel mechanisms underlying circRNAs in pathophysiology of SAKI. Methods: SAKI-associated circRNAs were screened via circRNA microarray and examined using in situ hybridization (ISH) in mouse renal tissue. The role of circNfix in YTHDF2-induced pro-inflammatory or pro-apoptotic response of tubular epithelial cells (TECs) was assessed by luciferase reporter assay and Annexin V/ PI staining assay in vitro. Adeno-associated virus was used to deliver circNfix to the kidneys of SAKI mice to study the function of circNfix in vivo. The mechanism underlying circNfix-mediated activation of NF-κB signaling was examined by Mass spectrometry, RNA pull-down, MeRIP-qPCR and ISH. To evaluate the biomarker potential of circNfix to predict SAKI, expression level of circNfix in the urine and plasma of 52 sepsis patient was examined using qPCR. Results: circNfix expression was downregulated in TECs following SAKI, leading to apoptosis and inflammatory injury in TECs in vitro and in vivo. Mechanistically, circNfix was found to provide a scaffold for the interaction between YTHDF2 and HECTD1, which was augmented by circNfix m6A modification, facilitating YTHDF2 ubiquitination and degradation. By decreasing the expression of YTHDF2, circNfix promoted the stabilization of DAPK2 mRNA, a negative regulator of NF-κB signaling, to protect LPS-induced TECs injury. Restoring circNfix in an experimentally-induced mouse SAKI model suppressed the activation of NF-κB signaling, thereby improving renal function and ameliorating tubular injury. Spearman's test demonstrated a remarkable negative correlation between urinary circNfix and serum creatinine in sepsis patients. Conclusion: Collectively, m6A modified-circNfix suppresses NF-κB-driven renal inflammation via a YTHDF2-dependent mechanism during SAKI, and restoring circNfix might represent a therapeutic strategy for SAKI. Funding: Government Support – Non-U.S.