MO067PHASE-SPECIFIC IRF4 AND IRF8 REGULATION/EXPRESSION OF MONONUCLEAR PHAGOCYTES DURING ISCHEMIC ACUTE KIDNEY INJURY/DISEASE

Abstract

Abstract Background Ischemic acute tubular necrosis is a common cause of acute kidney disease (AKD) and subsequent chronic kidney disease (CKD). The different phases of AKD involve a greater functional diversity of mononuclear phagocytes (MPCs) including resident and infiltrating macrophages and conventional dendritic cells (cDCs). In addition, hematopoietic transcription factors such as IRF4 and IRF8 play an important role in immune cell maturation and polarization that contribute to tissue inflammation and remodeling. However, their role on MPCs polarization and function during AKD are not well understood. Hence, we hypothesized that the dynamically altered MPC subsets contribute to the progression of AKD, accompanied with varied expression of IRF4 and IRF8. Methods AKD was induced by transient unilateral renal pedicle clamping in C57BL/6N mice. After 1, 3, and 7 days kidneys, renal lymph nodes and spleens were collected. Flow cytometry was performed to identify different MPC subsets. GFR was measured and mRNA expression of inflammatory, anti-inflammatory transcription factors determined via RT-PCR. For in vitro experiments, bone marrow-derived macrophages and cDCs, tubular epithelial cells (TECs), renal and splenic resident CD11b+ immune cells isolated from naïve mice were stimulated with LPS or cultured under hypoxic condition for 3 and 10 hours. Bone marrow-derived monocytes were differentiated into macrophages or cDCs, and stimulated with LPS for 3 and 10 hours. After stimulation, cells were harvested for mRNA analysis via RT-PCR. Results We identified four renal phenotypically distinct MPC subsets with diverse expression patterns of CD11b/CD11c during the different phases of post-ischemic AKD. During the early (day 1) and late injury phase (day 3), the number of infiltrating CD11bhiCD11clow R2 and CD11bhiCD11chi R3 macrophage-like subsets increased, along with a significant GFR decline compared with sham-operated mice (see Figure). During the repair phase (day 7), the number of CD11blowCD11clow R1 (resident macrophage-like MPCs) and CD11blowCD11chi R4 (infiltrating cDC-like MPCs) subsets significantly increased (see Figure). Both resident macrophage-like (R1) and cDC-like (R4) MPCs significantly upregulated the expression of IRF8, whereas cDCs-like (R4) MPCs were also positive for IRF4 during the repair phase. This pattern of MPCs was consistent in spleen and renal lymph node. In vitro stimulation of renal and splenic CD11b+ cells from naïve mice with LPS or under hypoxic condition induced a significant upregulation of IRF4 and IRF8 compared to untreated cells. This was also observed in bone marrow-derived macrophages and cDCs but not in TECs. Conclusions Our data indicate that infiltrating macrophage-like and cDC-like MPCs appear in high numbers during the early and late injury phase. Furthermore, both resident macrophage-like and cDC-like MPCs are predominately present during the late injury and recovery phase in AKD with altered IRF4 and IRF8 expression pattern. Further studies are needed to unravel a potential role of IRF4 and IRF8 during the progression of AKD and CKD by e.g. using fate mapping approaches.