Usnic Acid alleviates Acute Kidney Injury by mitigating oxidative stress and inflammation via the Keap1/Nrf2 axis

Acute Kidney Injury (AKI) is a critical clinical syndrome characterized by a rapid decline in renal function, leading to the accumulation of waste products and electrolyte imbalances. The pathophysiology of AKI is complex, with key hallmarks being sustained inflammatory responses, profound oxidative stress, and direct injury to renal tubular epithelial cells. This creates a vicious cycle: inflammatory cytokines and reactive oxygen species (ROS) promote tubular cell apoptosis and necrosis, while the release of cellular debris from damaged tubules further exacerbates inflammation and oxidative stress, ultimately contributing to functional impairment and potential progression to chronic kidney disease.

Despite its significant morbidity and mortality, the therapeutic arsenal for AKI remains severely limited. Current management is predominantly supportive, focusing on renal replacement therapy and addressing underlying causes. Crucially, there are no widely effective, targeted small-molecule pharmacological therapies approved to directly interrupt the core pathogenic pathways of inflammation and oxidative stress or to promote tubular repair. This glaring unmet medical need underscores the urgency to explore novel therapeutic agents.

In this context, Traditional Chinese Medicine(TCM) offer a rich source of potential drug candidates. UA, a unique dibenzofuran derivative found in lichens, has emerged as a compound of considerable interest. While historically noted for its antimicrobial properties, growing preclinical evidence highlights its potent anti-inflammatory and antioxidant activities. It has been shown to modulate key signaling pathways, such as NF-κB and Nrf2, which are central to the regulation of inflammation and the cellular antioxidant response. We hypothesize that the small-molecule properties of Usnic Acid position it as a promising candidate to disrupt the pathogenic cycle in AKI by simultaneously suppressing inflammatory cascades and mitigating oxidative damage, thereby protecting renal tubular epithelial cells from injury.

To comprehensively investigate the protective effects and underlying mechanisms of UA against Acute Kidney Injury (AKI), we established well-recognized in vivo and in vitro injury models. In vivo AKI mouse models were induced by cisplatin administration and renal ischemia-reperfusion (I/R). Correspondingly, in vitro injury models of mouse renal tubular epithelial cells were established using cisplatin treatment and hypoxia-reoxygenation (H/R), respectively.

Building upon these models, we employed a multi-faceted experimental strategy to decipher the mechanism of UA. RNA sequencing of renal tissues provided an unbiased overview of the global transcriptomic changes induced by UA, identifying critical pathways and potential targets. Molecular docking simulations offered structural insights into the plausible interactions between UA and key proteins implicated in these pathways. The direct engagement of these targets was then experimentally validated using the Cellular Thermal Shift Assay (CETSA), confirming drug-target binding within a cellular context.

The downstream molecular events were meticulously examined: alterations in key protein expression and phosphorylation were quantified by Western blot (WB), while corresponding changes in mRNA levels were assessed using quantitative PCR (qPCR). Furthermore, immunofluorescence (IF) staining visually demonstrated the compound's efficacy in attenuating oxidative stress and tubular damage, a finding corroborated by quantitative measurement of reactive oxygen species using fluorescent ROS probes.

This synergistic application of physiological models, bioinformatics, biophysical binding assays, and functional molecular biology techniques robustly delineates how UA ameliorates renal injury.

Our data indicated that UA mitigated renal damage, inflammation, and oxidative stress levels in mouse models of Acute Kidney Injury (AKI) induced by cisplatin and ischemia/reperfusion. Furthermore, we observed that UA also demonstrated a renal-protective effect in human kidney tubular epithelial cells treated with cisplatin and hypoxia/reoxygenation. Therefore, these results suggest that the administration of UA improves kidney function, reduces inflammatory responses, and inhibits the level of cellular oxidative stress. Mechanistic studies suggest that UA improves cisplatin-induced kidney inflammation and oxidative stress levels by negatively regulating the activation of the NRF2 pathway through suppression of KEAP1 expression. In summary, these results suggest that UA could have therapeutic agent for ameliorating cisplatin-induced renal dysfunction.

 The results demonstrate that UA could serve as a promising therapeutic agent for the management of AKI, potentially exerting its effects via the KEAP1/NRF2 signaling pathway.