DNA-Damaged PTEC-Derived Exosomes Induce DNA Methylation Changes in Macrophages

In an aging society, the number of patients with chronic kidney disease (CKD) is increasing. CKD is associated with adverse clinical outcomes such as cardiovascular events, infections, and mortality, as well as nutritional issues including weight loss, sarcopenia, and malnutrition. Elucidating the molecular mechanisms underlying CKD is a crucial issue. Recently, epigenomic change, including DNA damage, has attracted attention as a common driver of cellular senescence and the disruption of inter-organ networks. Previous epigenome-wide association studies (EWAS) have revealed correlations between DNA methylation in peripheral blood cells and kidney function, and we also reported a link between DNA damage in urinary proximal tubular epithelial cells (PTECs) and renal prognosis (Sci rep. 2024). Recently, we created a mouse model with PTEC-specific expression of the I-PpoI endonuclease, which induces non-mutagenic DSBs, and reported that DNA damage in PTECs led not only to macrophage activation in the renal cortex but also to systemic metabolic alterations by promoting macrophage activation and impairing adipocyte differentiation, resulting in systemic metabolic dysfunction and weight loss. Reduced representation of bisulfite sequencing (RRBS) analysis of peripheral blood cells revealed hypomethylation of the binding sites of KLF9 and its downstream genes including GSDMD, both of which are reported to be associated with diabetic complications and the activation of macrophage, suggesting that DNA damage in PTECs may induce changes in DNA methylation and activation of macrophages in the peripheral blood, resulting in systemic metabolic alterations. (Nat Commun. 2025). However, the mechanism by which PTEC-induced DNA damage causes gene-specific DNA methylation changes in hematopoietic cells remains unclear. 

To investigate the mechanism by which PTEC-induced DNA damage causes gene-specific DNA methylation changes, an in vitro analysis was conducted using cultured murine macrophage RAW 264.7 cells that had been transferred into the medium of murine PTECs following transfection with I-PpoI. 

Murine macrophages in the culture medium from PTECs overexpressing I-PpoI for 48 hours, showed increased expression of inflammatory cytokines such as IL-1β, TNFα and Ccr2. As shown in I-PpoI mice, GSDMD and KLF9 expressions were increased in the activated macrophages, as supported by the decreased DNA methylation levels of the GSDMD promoter region and the binding site of KLF9. It is known that exosome release is enhanced in cells subjected to DNA damage stress, and we also observed decreased mRNA expressions in DNA methyltransferase (DNMT)1 and DNMT3A in exosomes released by cultured PTECs and GW4868, an exosome inhibitor, suppressed the change in the methylation levels. Then we focused on exosomes released by injured PTECs as the factors mediating changes in DNA methylation and performed comprehensive microRNA-seq analysis, which revealed changes in the microRNA-29 and 30 family, which are known to target DNMTs and KLF9, respectively. This suggests that altered exosome properties may be a crucial factor as the entity of intercellular communication between internal organs by mediating DNA methylation changes.

It is suggested that exosomes may be a key factor in the process whereby PTECs with DNA damage induce gene-specific DNA methylation changes in peripheral blood cells. Our findings could lead to elucidating the detailed epigenetic mechanisms of metabolic dysfunction originating from renal DNA damage, identifying new biomarkers for renal complications, and developing novel therapeutic approaches.