Exosomes derived from a mesenchymal-like endometrial regenerative cells ameliorate renal ischemia reperfusion injury through delivery of CD73

Background: Renal ischemia reperfusion (I/R) injury is a major contributor to graft dysfunction and inflammation leading to graft loss. The deregulation of purinergic signaling has been implicated in the pathogenesis of renal I/R injury. CD73 and the generation of adenosine during purine metabolism to protect against renal I/R injury. A mesenchymal-like endometrial regenerative cell (ERC) has demonstrated a significant therapeutic effect on renal I/R injury. CD73 is a phenotypic marker of human endometrial regenerative cell exosomes (ERC-Exo). However, its immunosuppressive function in regulating purinergic metabolism has been largely neglected. Here, we investigate the protective effects and mechanism of ERC-Exo against renal I/R injury.

Methods: Lentivirus-mediated CRISPR-Cas9 technology was employed to obtain CD73-specific knockout ERC-Exo (CD73^-/-ERC-Exo). C57BL/6 mice who underwent unilateral ureteral obstruction were divided into the Untreated, ERC-Exo-treated, and CD73^-/-ERC-Exo-treated groups. Renal function and pathological injury were assessed 3 days after renal reperfusion. The infiltration of CD4⁺ T cells and macrophages was analyzed by flow cytometry and immunofluorescence staining in kidneys. CD73-mediated immunosuppressive activity of ERC-Exo was investigated by bone marrow-derived macrophages (BMDM) co-culture assay in vitro. Flow cytometry determined macrophage polarization. ELISA and Treg proliferation assays detected the function of macrophages. Furthermore, the role of the MAPK pathway in CD73-positive Exo-induced macrophage polarization was also elucidated.

Results: Compared with Untreated and CD73^-/-ERC-Exo-treated groups, CD73-positive Exo effectively improved the serum creatinine (sCr), blood urea nitrogen (BUN), and necrosis and detachment of tubular epithelial cells, necrosis and proteinaceous casts induced by ischemia. CD73 improved the capacity of ERC-Exo on CD4⁺ T cell differentiation in the renal immune microenvironment. Surprisingly, ERC-Exosomal CD73 significantly decreased the populations of M1 cells but increased the proportions of M2 in kidneys. Furthermore, CD73-positive Exo markedly reduced the levels of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and increased anti-inflammatory factors (IL-10) level in kidneys. ERC-Exosomal CD73 improved macrophage immunoregulatory function associated with the MAPK pathway (including ERK1/2 and p38 pathways), which exerted a potent therapeutic effect against renal I/R.

Conclusions: These data collected insight into how ERC-Exo facilitated the hydrolysis of proinflammatory ATP to immunosuppressive ADO via CD73. CD73 is a critical modulator of the MAPK signaling pathway, inducing a polarization shift of macrophages towards an anti-inflammatory phenotype. This study highlights the significance of ERC-Exosomal CD73 in contributing to the therapeutic effects against renal I/R.

CD73; Exosome; Human endometrial regenerative cells; Macrophage; Renal ischemia–reperfusion injury.