#4976 DIFFERENTIAL ROLES OF REGNASE3 IN RESIDENT MACROPHAGE VERSUS TUBULAR EPITHELIAL CELLS IN THE HEALTHY OR POSTISCHEMIC KIDNEY

Abstract

Abstract Background and Aims Acute kidney injury (AKI) is a prevalent yet severe condition that occurs in clinical settings. The prognosis for AKI is poor, with many patients progressing to AKD, CKD, and even ESKD especially in those who were admitted to the ICU. RNA-binding proteins (RBPs) are a class of proteins that play a vital role in regulating gene expression and have been implicated in a wide range of diseases. Regnase3 is a member of the Regnase RBPs family and has been shown to promote inflammation by increasing TNF in macrophages. Despite this knowledge, the full extent of the role played by Regnase3 in AKI remains unknown. Therefore, we hypothesized that Regnase3 plays a role in both macrophages and tubular epithelial cells, influencing inflammation and tubular repair in AKI. Method In order to investigate the role of Regnase3 in kidney injury, a series of genetically-modified mice were developed on the C57BL/6J genetic background. These mice included the paired box gene 8 (Pax8)-reverse tetracycline transactivator (rTtA, Pax8rTtA), tetracycline resistance protein (TetO)-Cre (TetOCre), transgenic receptor activator of nuclear factor kappa-Β (Rank)-Cre (RankCre), and Regnase3 floxed (Regnase-3fl/fl) mice. Using these genetically modified mice, we applied a range of experimental animal models to study the effects of Regnase3 on kidney injury, including unilateral kidney ischemia-reperfusion surgery with or without nephrectomy and nephrocalcinosis-related kidney injury. Additionally, we employed in vitro models utilizing primary cells to study Regnase3 on the cellular level. Furthermore, we utilized scRNA-seq and RNA-seq to investigate the underlying mechanisms of Regnase3’s function. Results We conducted a thorough investigation and discovered that the Regnase3 is highly expressed in macrophages located within the kidney after AKI. The scRNA-seq showed the expression Regnase3 positively correlated with the phagocytosis, chemokines, and monocyte maturation. Furthermore, Rank-Regnase3 mice suffered from more inflammation and kidney injury after AKI, characterized by an increase in CCR2 positive leukocyte infiltration. Additionally, in vitro experiments revealed that Regnase3 is involved in modulating macrophage behavior, acting as a regulator of macrophage polarization towards an M1 and M2 phenotype and influencing the capacity of cell migration. These findings indicate that Regnase3 is essential in the migration of macrophages after AKI, which contribute to both early phase inflammation and progression towards AKI- CKD. Next, we aimed to examine the role of Regnase3 in the context of renal tubule injury. We observed that Regnase3 is highly expressed in healthy renal tubules, but its expression is reduced following injury. Through in silico, in vivo and in vitro experiments, we found that Regnase3 controls early apoptosis, cell death, proliferation, and wound healing capability of tubular epithelial cells. These effects contribute to kidney injury in postischemic kidneys. Additionally, we discovered that the deletion of Regnase3 provides protection against ischemic AKI (Figure 1). Mechanistically, Regnase3 targets pre-RNA and modulates alternative splicing by increasing the proportion of targeted skipped exon events and decreasing the probability of targeted retained intron events. Overall, our findings indicate that Regnase3 plays a vital role in tubular epithelial cells in the response to kidney injury by regulating alternative splicing. Conclusion In summary, our findings indicate that Regnase3 contributes to kidney injury in postischemic kidneys. However, the impact of Regnase3 is contingent upon the specific cell lineage in question. The Rank-Regnase3 leads to an exacerbation of kidney injury by increasing macrophage recruitment, whereas the Pax8-Regnase3 leads to an improvement in kidney injury through its effects on cell death and wound healing capability of tubular epithelial cells. Therefore, the role of Regnase3 in kidney injury is dependent on the cell lineage, which ultimately determines the detrimental or beneficial impact on kidney injury.