#4549 INTEGRATIVE SPATIAL AND SINGLE-CELL ANALYSIS IDENTIFIES MACROPHAGE HETEROGENEITY AND A UNIQUE MULTI-IDENTITY SUBSET IN AKI TO CKD TRANSITION
Authors: Zhang Yilin , Tang Tao-Tao , Wen Yi , Yin Qing , Jiang Wei , Zhang Yue , Li Zuolin , Wang Bin , LV Linli , Liu Bicheng 
Published: June 2023

Abstract

Background and Aims
The pathogenesis of acute kidney injury (AKI) transformed to chronic kidney disease (CKD) is still largely unclear. Previous studies have suggested the heterogeneity of macrophages might play a critical role in this pathological process. Here we demonstrated the spatiotemporal dynamics of macrophages and its potential function in AKI to CKD in unilateral ischemia-reperfusion injury (UIR) mice model.

Method
We generated an integrative high-resolution map of kidney injury and repair in a mouse model of failed repaired UIR using single-cell RNA-sequencing (scRNA-seq) and spatial transcriptomic profiling of multiple zones at five time points. We identified cell types through unsupervised clustering analysis. Differential gene expression, enrichment analysis and main function scoring were applied to define functional heterogeneity of macrophages. Next, pseudotime analysis of differentiation transitions was performed to create the diverse macrophage lineages. Finally, using ligand-receptor analysis, we identified the interaction between macrophage and other cells.

Results
We identified 13 main cell clusters and the dynamic changes in the process of AKI to CKD transition were analysed. A remarkable increasing proportion of macrophages after injury at day 1 followed with second peak at day 14 post AKI. Spatiotemporal profiles of main cells showed injured tubules and macrophages co-localized in outer stripe of outer medullary region early after AKI, whereas in late chronic stages macrophages had spatial proximity to fibroblasts. The subtypes of macrophage after AKI were identified as pro-inflammatory, pro-repair, proliferative, and immature populations. Notably, a novel multi-identity macrophage cluster which is recruited to the kidney early after AKI and remained in the kidney until chronic stages. In the multi-identity cluster, elevated expression of genes such as Trem2, Ctsd, Fn1, and Ccl7 was observed. Importantly, the upregulation of Trem2 is a marker of transition from M1 to M2 type. Multi-identity cluster was characterized with the highest fibrosis and phagocytosis scores as well as second high inflammation scores among the diverse clusters. In the early phase of UIR (days 1 and 3), GO terms for multi-identity cluster were more enriched in acute inflammatory response and phagocytosis; however, its functions were more turned to fibrosis and extracellular matrix in the late phase of UIR (days 14 and 28). These data indicated that multi-identity cluster was rapidly adopting a pro-inflammatory phenotype early after AKI, which present a profibrotic phenotype in late chronic stages. We retrieved two distinct trajectories of macrophage populations, tissue-resident and monocyte-recruited. Starting at monocyte, multi-identity cluster progressed along lineage 1 towards pro-inflammatory cluster, suggesting that the monocyte-derived infiltrating multi-identity macrophages significantly contributed to the formation of the pro-inflammatory M1 cells. On the other hand, proliferative and immature cluster branched off along lineage 2 towards an endpoint of pro-repair cells, indicating that tissue-resident macrophages contribute to repair of kidney by local proliferation. Furthermore, ligand-receptor analysis identified enhanced interaction between diverse macrophage clusters and neighboring cells in the failed repaired stages after AKI, supporting that intercellular communication drives kidney fibrosis. Importantly, we identified that the Igf1-Igf1r interaction was highly specific in multi-identity macrophage and fibroblast intercellular communication.

Conclusion
Our study demonstrated the spatiotemporal dynamics of macrophage heterogeneity and the transitionary functions of multi-identity macrophages in the process of AKI to CKD transition.