CHEMICAL REPROGRAMMING OF MACROPHAGES TO FACILITATE RENAL REPAIR AFTER AKI

Acute kidney injury causes kidney function decrease and renal cell loss, leading to chronic kidney disease. In AKI, tubular epithelial cells and vascular endothelial cells are damaged, some cells are repaired through proliferation, and others enter an abnormal state, secreting pro-inflammatory factors to attract immune cells, exacerbating damage and fibrosis. Promoting adaptive repair cell proliferation may improve the prognosis of kidney injury. The heterogeneity of macrophages enables their pivotal roles in renal injury and repair, which involves reaching the vicinity of tubular epithelial and vascular endothelial cells to provide regenerative growth factors, making them prime therapeutic targets for kidney disease. However, their diversity and function in the transition from acute kidney injury (AKI) to chronic kidney disease (CKD) remain poorly understood. Here, we employed single-cell RNA-sequencing (scRNA-seq) to identify key macrophage subpopulations involved in renal repair after AKI. We also explored a chemical approach to reprogram macrophages for AKI recovery.

A mouse model of renal ischemia-reperfusion AKI (IR-AKI) was established. Renal tissue was collected at different time points post-AKI for scRNA-seq analysis. Macrophages were identified and clustered based on gene expression profiles. Gene enrichment analysis and Cellchat were performed for the identification of pro-repair macrophage. AddModuleScore was used to analyze the scores of damage and repair functions in different subpopulations of macrophages, and to depict the dynamic changes of kidney repair and damage over time. To further validate the findings of single-cell sequencing, flow cytometry was used to observe and verify the proportion changes of DREAM cells during AKI progression. Additionally, flow sorting was employed to obtain DREAM cell populations, which were then co-cultured with tubular epithelial cells or vascular endothelial cells to verify their repair functions. Key transcription factors (TFs) were predicted to understand the gene regulatory network of pro-repair macrophages. To induce a pro-repair macrophage phenotype, small molecule compounds targeting key TFs and repair-associated factors were applied to RAW cells in various combinations. To screen the optimal reprogramming drug combinations, we utilized gene expression data after drug intervention in the CMAP database to predict preferred compounds, and use DRUG-SEQ sequencing for detailed drug screening and observation of gene expression patterns. The efficacy of these chemical cocktails was evaluated by repair factors levels and transcriptome sequencing analysis. The repair function of chemically reprogrammed macrophages was validated in vitro using co-culture systems with tubular epithelial cells or vascular endothelial cells. Finally, chemical cocktails were administered to AKI mice to reprogram macrophages into a pro-repair functional phenotype to facilitate renal repair. 

We identified 2241 macrophages through renal scRNA-seq, which clustered into seven distinct subgroups. A pro-repair macrophage subpopulation with unique renal repair functions was defined, characterized by high expression of epithelial repair factors (Hbegf, Gdf15, Spink1) and angiogenic factors (Vegfa, Ccn1, Timp3, Adamts1). Therefore, it was named the DREAM subgroup (Dual-repair Epithelial-Endothelial Macrophage). Cellchat analysis showed that pro-repair macrophages have strong interactions with damaged tubular epithelial cells and endothelial cells. Furthermore, DREAM acts on these damaged cells through growth factor-related signaling pathways, with the potential to achieve their regeneration and repair. We used Hbegf as a cell marker to sort DREAM macrophages and non-DREAM cells from the kidneys of AKI mice. After co-culturing with tubular epithelial cells or vascular endothelial cells, we also found that DREAM has the ability to promote the proliferation of epithelial and endothelial cells, confirming the existence of a DREAM cell subpopulation with repair functions in vivo. However, single-cell sequencing analysis and flow cytometry showed that the proportion of DREAM cells decreases as AKI progresses, which may lead to limited regeneration and repair. Therefore, we proposed the hypothesis that promoting kidney regeneration and repair could be achieved by reprogramming DREAM-like macrophages. Gene regulatory network analysis identified Cebpb, Ppara and Egr1 as core transcription factors governing pro-repair macrophage function. Furthermore, we designed a drug screening strategy targeting DREAM key transcription factors and repair factors, predicting a drug combination capable of in vitro reprogramming DREAM-like repair phenotype macrophages. We screened different combinations of small molecule drugs and found that the FDRC chemical cocktail (FDRC, a combination of four small-molecule compounds) effectively activates Cebpb, Ppara and EGR1 in RAW cells, leading to significant expression of Hbegf, Gdf15, and Vegfa. RNA-seq analysis showed that FDRC-reprogrammed macrophages display a unique gene expression profile distinct from M0 or M2 macrophages, which significantly increased gene expressions related to angiogenesis, anti-inflammatory, and wound healing. FDRC-reprogrammed RAW or BMDM cells can significantly promote their proliferation when co-cultured with endothelial cells and tubular epithelial cells. Notably, FDRC successfully reprogrammed macrophages toward a DREAM-like phenotype in IR-AKI mice, which significantly ameliorated tubular injury and peritubular capillary rarefaction, and potently prevented the AKI-to-CKD transition. 

Our study uncovers a novel pro-repair macrophage subset during AKI and introduces a promising chemical reprogramming cocktail to manipulate macrophage phenotype in vivo, facilitating the repair of renal tubules and peritubular capillaries.