SOCS1/Wnt/IL-1β axis negatively regulates macrophage extracellular traps and M1 polarization through the TNF pathway in crystalline kidney injury

Abstract

Background

Calcium oxalate (CaOx) crystals are known to induce kidney damage and trigger inflammatory responses. However, the role of macrophage extracellular traps (METs) in CaOx-induced kidney injury and the relationship between METs and the M1 polarization phenotype of macrophages remain unclear.

Methods

A CaOx crystal deposition model was established in C57BL/6J mice using a high-calcium diet supplemented with vitamin D. Renal tissues from patients with CaOx stones and healthy controls were also collected. In vitro, mouse RAW264.7 macrophages (RAW cells) were stimulated with calcium oxalate monohydrate (COM) crystals or IL-1β. Immunofluorescence, flow cytometry, qRT-PCR, and western blotting were used to assess METs formation and M1 polarization. Genes regulating IL-1β expression were screened using TCMK-1 renal tubular epithelial cells. mRNA sequencing was performed in both cell types to identify relevant signaling pathways. In TCMK-1 cells and mouse kidneys, SOCS1 was overexpressed or knocked down using lentivirus or siRNA. CHIR-99021 and TNFR2-Fc were used to activate the Wnt signaling pathway and inhibit the TNF signaling pathway, respectively. In the in vivo model with regulated SOCS1 expression, METs levels and M1 polarization were quantified, and the potential feedback loop between METs and the TNF signaling pathway was examined through co-culture and sequential stimulation experiments.

Results

We found that renal tissues from patients with calcium oxalate stones and from mice with crystal deposition exhibited marked accumulation of macrophage extracellular traps (METs), identified as citH3⁺/SYTOX-Green⁺ extracellular structures, accompanied by significantly increased M1-polarized (iNOS⁺) macrophage infiltration. Calcium oxalate monohydrate (COM) crystals directly triggered MET formation and M1 polarization in RAW264.7 macrophages, and IL-1β stimulation alone was sufficient to reproduce these effects. METs themselves further promoted M1 polarization. In TCMK-1 renal tubular epithelial cells, we identified SOCS1 as a negative regulator of IL-1β transcription acting through the Wnt signaling pathway; accordingly, SOCS1 overexpression in mice markedly reduced renal MET burden and M1 macrophage infiltration in oxalate nephropathy. Mechanistically, IL-1β activated the TNF signaling pathway, which was both necessary and sufficient for MET release and subsequent M1 polarization. In turn, METs sustained TNF pathway activity, establishing a positive feedback loop that amplified both MET formation and M1 polarization. Disruption of TNF signaling abrogated this loop and significantly attenuated M1 polarization in vitro and in vivo.

Conclusion

In the renal microenvironment of CaOx crystal deposition, IL-1β induces METs formation by activating the TNF signaling pathway and promotes M1 polarization. In turn, METs sustain the TNF signaling pathway, forming a positive feedback amplification loop. SOCS1 negatively regulates IL-1β expression through the Wnt signaling pathway and, in vivo, effectively suppresses both METs formation and M1 polarization. These findings not only elucidate a macrophage-driven inflammatory amplification mechanism in crystalline kidney disease but also identify SOCS1 and the TNF signaling pathway as potential therapeutic targets for interrupting this positive feedback loop and mitigating sterile kidney injury.