The Positive Feedback Regulation of NF-κB/miR-21c/IL-9 Axis in Septic Acute Kidney Injury

BACKGROUND: Sepsis-associated acute kidney injury (septic AKI) is a leading cause of mortality in critically ill patients. Despite its high prevalence, no specific therapies are currently available, primarily because the molecular mechanisms that sustain renal inflammation and tubular damage remain poorly understood. METHODS: A murine model of LPS-induced septic AKI and cultured murine proximal tubular (BUMPT) cells were used. In vivo, anti-miR-21c or miR-21c mimic was administered via tail vein injection prior to LPS challenge. Molecular interactions were assessed by chromatin immunoprecipitation (ChIP), luciferase reporter assay, qPCR, Western blotting, immunohistochemistry, and fluorescence in situ hybridization. Renal function and injury were evaluated by serum creatinine, blood urea nitrogen (BUN), histopathology, and TUNEL staining. RESULTS: miR-21c was significantly upregulated in renal proximal tubules during LPS-induced septic AKI, coinciding with peak renal dysfunction and tubular damage. This upregulation was driven by NF-κB, as LPS induced p65 nuclear translocation, and pharmacologic inhibition of NF-κB blocked miR-21c induction. ChIP assays confirmed direct binding of p65 to the miR-21c promoter. Subsequent dual-luciferase reporter assays specifically validated the binding site as the functional response element. Functional studies showed that inhibition of miR-21c attenuated kidney injury, reducing serum creatinine and BUN levels, alleviating histological damage, decreasing tubular apoptosis, and suppressing proinflammatory cytokines. Conversely, overexpression of miR-21c exacerbated all injury parameters. Mechanistically, miR-21c directly targeted the 3'-UTR of interleukin-9 (IL-9), as validated by luciferase reporter assay, resulting in reduced IL-9 protein expression. Restoration of IL-9 in tubular cells suppressed LPS-induced apoptosis and inflammation. Importantly, IL-9 overexpression specifically inhibited phosphorylation of NF-κB p65. CONCLUSIONS: Our study identified a pathogenic NF-κB/miR-21c/IL-9 feedback loop that amplified renal inflammation and injury in septic AKI. Targeting miR-21c or enhancing IL-9 signaling may offer novel therapeutic strategies to mitigate kidney damage and improve outcomes in septic patients.