ROLE AND MECHANISM OF PGE2-EP2 SIGNALING-MEDIATED MACROPHAGE SENESCENCE IN RENAL AGING

This study aims to clarify the senescence of macrophages and the activation status of the prostaglandin E2 (PGE2)-prostaglandin E receptor 2 (EP2) signaling pathway in the aging kidney. Using both in vitro cell models and in vivo animal models, the study systematically investigates the regulatory role of PGE2-EP2 signaling in macrophage senescence and renal aging.

Young (8-week-old) and aged (24-month-old) male C57BL/6J mice were used. The natural aging model was validated through histological staining (HE, PAS, Masson, and Sirius Red) and molecular analyses (RT-qPCR and Western blot). Immunofluorescence co-staining of F4/80 and p21 was used to assess the senescence of renal macrophages, while PGE2 levels were measured by ELISA and EP2 expression evaluated via immunofluorescence and RT-qPCR. Bone marrow-derived macrophages (BMDMs) were isolated to assess cellular senescence and EP2 receptor expression. In vitro, PGE2/EP2 signaling was modulated in BMDMs and THP-1 macrophages to examine its effects on cellular senescence, inflammation, and phagocytosis. A co-culture system with renal tubular epithelial cells was established to evaluate the impact of macrophage senescence on tubular epithelial cell aging. In vivo, a macrophage-specific EP2 knockdown model was generated using AAV-shEP2, allowing the evaluation of its effects on renal function, tissue injury, senescence markers, and SASP factors. Furthermore, transcriptomic sequencing and pathway enrichment analysis were performed, and the downstream mechanisms of EP2 signaling were validated using PI3K/Akt/STAT3 pathway blockade and factor intervention.

Compared to the young group, kidneys from aged mice exhibited a marked accumulation of F4/80⁺/p21⁺ senescent macrophages, elevated PGE2 levels, and an increased proportion of EP2⁺ macrophages, with upregulated expression of p16, p21, and EP2 in BMDMs. Treatment with PGE2 or the EP2 agonist Butaprost significantly induced senescence, a pro-inflammatory phenotype, and cytokine secretion in both murine and human macrophages, while also impairing phagocytic capacity. These effects were reversed by the EP2 antagonist PF04418948. In the co-culture system, macrophages treated with PGE2 or the EP2 agonist significantly increased SA-β-gal positivity and upregulated the expression of p16, p21, and SASP factors in renal tubular epithelial cells. In vivo, EP2 knockdown notably reduced the accumulation of senescent macrophages in aged kidneys, improved renal function and tissue structure, decreased senescence and SASP marker expression, and delayed the senescence and functional decline of BMDMs, alleviating their pro-senescent effects on mPTCs. Transcriptomic analysis revealed that differentially expressed genes were enriched in the PI3K/Akt and JAK-STAT pathways. Further validation confirmed that EP2 activates the PI3K/Akt/STAT3 signaling axis, promoting STAT3 phosphorylation. Blockade of the PI3K/Akt pathway or knockdown of STAT3 inhibited EP2-mediated macrophage senescence and its pro-senescent effects on renal tubular epithelial cells.

Senescent macrophages accumulate in aging kidneys, accompanied by activation of the PGE2-EP2 signaling pathway. PGE2-EP2 signaling promotes macrophage senescence through the PI3K/Akt/STAT3 pathway, enhancing the senescence-associated inflammatory phenotype while impairing macrophage phagocytic function. These changes contribute to tubular epithelial cell senescence and the progression of renal aging.