Limonoid walrobsin A alleviates LPS-induced septic acute kidney injury through modulating inflammatory microenvironment by targeting G protein-coupled receptor 75

Background: Septic acute kidney injury (S-AKI) is associated with high morbidity and mortality, and dysregulation of oxidative stress and remodeling of the inflammatory microenvironment are emerging as central pathogenic mechanisms. Alleviating inflammation and oxidative stress are the primary effects of limonoids found in nature, and our previously discovered walrobsin A (WA) from Walsura robusta has exhibited significant anti-inflammatory properties. Moreover, bioactive natural products with novel skeletons are ideal molecular probes for identifying potential novel molecular targets for diseases using chemical biology methods.

Purpose: To explore the therapeutic effect and mechanism of WA for treating S-AKI and to identify previously unrecognized targets in S-AKI.

Methods: The effect of WA was detected by establishing LPS-induced mouse S-AKI in vivo. Functional and pathological changes in murine kidneys were evaluated by measuring serum creatinine (CRE), Cystatin C (Cys C), blood urea nitrogen (BUN), NGAL levels, and HE pathology. A RAW264.7/mTEC coculture system was established to elucidate the target and mechanism of WA by techniques such as RNA-seq, gene interference, DARTS, LC-MS/MS, molecular docking, BLI, and SPR.

Results: The screening results revealed that WA, a limonoid, significantly alleviated LPS-induced mouse S-AKI, and a RAW264.7/mTEC coculture experiment revealed that WA directly acted on macrophages. Transcriptomic analysis and further research revealed that WA promoted the expression of the antioxidant proteins HMOX1 and SOD2 and inhibited IL-1β, IL-6, and TNF-α secretion. G protein-coupled receptor 75 (GPR75), which was highly expressed in the renal macrophages of S-AKI mice, was subsequently identified as the direct target of WA. In the RAW264.7/mTEC coculture system, siGPR75 inhibited macrophage oxidative stress and restrained IL-1β, IL-6, and TNF-α secretion, thereby reducing mTEC injury, and overexpression of GPR75 resulted in the opposite effects. Moreover, siGPR75 reversed the WA-mediated remission of macrophage oxidative stress and the inflammatory microenvironment in vitro, and coadministration of WA failed to increase the therapeutic efficacy of 20-SOLA (a GPR75 inhibitor) in S-AKI in vivo. These results indicate that the WA-GPR75 axis mitigates S-AKI by regulating oxidative stress in macrophages and the inflammatory microenvironment.

Conclusion: In this study, we elucidated the novel role of GPR75 as a potential target for S-AKI using a bioactive limonoid-type natural product and identified a natural GPR75 inhibitor (WA) as a potential candidate for S-AKI treatment. This study firstly identifies GPR75 in pathological renal conditions and demonstrates its direct role in driving renal injury, thus establishing it as a novel therapeutic target for kidney diseases.

GPR75; Inflammation microenvironment; Limonoids; Oxidative stress; Septic acute kidney injury; Walrobsin A.