Macrophage Subtypes Affect the Damage and Repair of Sepsis-Associated AKI by Regulating the Phagocytic Function of Kidney Epithelial Cells
- AKI: Mechanisms - III
November 04, 2023 | Location: Exhibit Hall, Pennsylvania Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Acute Kidney Injury
- 103 AKI: Mechanisms
- Huang, Lili, Affiliated Hospital of Nantong University, Nantong, China
- Wu, Yuanyuan, Nantong University, Nantong, China
- Yang, Bin, Affiliated Hospital of Nantong University, Nantong, China
Different subtype macrophages play different roles in acute kidney injury, but how they work is still not fully defined. This research highlighted the mechanism underpinning the clearance of dying cells and its impact on the surrounding environment including the phagocytotic function of kidney epithelial cells.
Mouse RAW264.7 cells (peritoneal macrophage cell line) were cultured, in which LPS or IL-4 was used to induce the M1 or M2 phenotype macrophages. The changes of phenotype were verified by Western Blot and flow cytometry. M1 and M2 macrophages were then co-cultured with TCMK-1 cells (kidney epithelial cell line) with or without LPS treatment for 24 h in a trans-well system. Inflammation cytokines were detected by quantitative real-time PCR (qPCR). Apoptotic cell death was accessed by Annexin V/Proidium iodid staining, while the phagocytotic capacity of TCMK-1 cells was evaluated by the uptake of FITC-labelled pHrodo E.coli Bioparticles® Conjugate, and then detected by a flow cytometer.
RAW264.7 cells were successfully induced into M1 and M2, which were verified by increased iNOS expression, F4/80+CD86+ cells and F4/80+iNOS+ cells in M1 macrophages, and increased Arg-1 expression, F4/80+CD206+ cells and F4/80+Arg-1+ cells in M2 macrophages. In trans-well culture, M1 macrophages increased pro-inflammatory mediators including IL-1β, IL-6 and TNF-α in TCMK-1 cells, especially with LPS stimulation, but M2 macrophages raised anti-inflammation mediators. A similar change trend was revealed in the percentage of apoptotic TCMK-1 cells, together with Bax and EPOR protein expression. Most importantly, regardless of LPS, the phagocytotic function of TCMK-1 cells was decreased by co-cultured M1 macrophages, but it was enhanced by co-cultured M2 macrophages.
M1 macrophages affect adjacent kidney epithelial cells by releasing inflammatory mediators, inducing apoptotic cell death, compromising phagocytotic function and further repair, whereas M2 macrophages have opposite impacts.