#1766 Mincle receptor in macrophage and neutrophil contributes to the unresolved inflammation during the transition from AKI to CKD

Abstract

Abstract Background and Aims Recent studies have demonstrated a strong association between acute kidney injury (AKI) and chronic kidney disease (CKD). Unresolved inflammation may contribute to the AKI-to-CKD transition. As a transmembrane pattern recognition receptor, Mincle (macrophage-inducible C-type lectin, Clec4e) was previously reported to be critical in the early immune response after AKI. However, the impact of Mincle on the chronic transition from AKI remains poorly understood. Method We performed single-cell RNA sequencing (scRNA-seq) with the clinically relevant unilateral ischemia-reperfusion (UIR) murine model of AKI at days 1, 3, 14 and 28 after injury. Potential effects and mechanism of Mincle on renal inflammation and fibrosis were further validated in vivo utilizing Mincle knockout mice. Results Single-cell transcriptome analysis showed predominant Mincle expression in macrophages and neutrophils during the AKI-to-CKD transition. For both cell types, Mincle was significantly upregulated on day 1 after AKI which decreased gradually in later stages, and exhibited a secondary peak at day 14. Flow cytometry confirmed the increased infiltration of Mincle-positive macrophages and neutrophils post-UIR. Next, we identified distinct subclusters of Minclehigh neutrophils and Minclehigh macrophages, both displaying time-dependent influx with dual peaks on day 1 and day 14 and significant pro-inflammatory and pro-fibrotic phenotypes as indicated by functional gene scoring. Spatial transcriptomics data showed a pronounced expression of Mincle at the outer medulla in the kidney similar to myeloid cells, possibly correlating with fibrotic areas represented by α-SMA patterns. To further elucidate the mechanisms underlying AKI-to-CKD transition mediated by Mincle-expressing macrophages and neutrophils, we identified 54 common genes from the up-regulated genes of these two cell subsets, which were involved in immune-inflammatory responses and extracellular matrix synthesis. Importantly, TNF-α was revealed as a pivotal hub gene and strongly associated with Mincle in these two Mincle-expressing subclusters. Correspondingly, a prominent increase in TNF-α expression primarily in macrophages and neutrophils on days 1 and 14 was confirmed, displaying consistent patterns with Mincle expression. Meanwhile, significant TNF-α reduction specifically in macrophages and neutrophils on day 14 was observed in Mincle knockout mice. Remarkably, Mincle-deficient mice showed improved renal pathology and reduced extracellular matrix deposition, accompanied by notable mRNA down-regulation of pro-inflammatory factors (TNF-α, IL-1β, Ccl2) and fibrosis associated factors (TGF-β, α-SMA, COL1A1) as well as decreased immune cells infiltration. Conclusion Together, the present findings have unveiled combined persistence of Minclehigh neutrophils and macrophages during AKI-to-CKD transition, contributing to unresolved inflammation followed by fibrosis via TNF-α as a central pro-inflammatory cytokine. Targeting Mincle may offer a novel therapeutic strategy for preventing the transition from AKI to CKD.