To investigate the mechanism of jagged canonical Notch ligand 2 (Jag2) mediating early inflammation in acute kidney injury via crosstalk between renal vascular endothelial cells and mononuclear macrophages.

Male C57BL/6J mice were used to establish a bilateral renal ischemia-reperfusion injury (IRI) model. The mice were randomly divided into three groups with 6 mice in each group: the sham operation group, the model group (administered with 10 mg/kg isotype control IgG via tail vein injection at 24 h before IRI modeling and immediately after modeling), and the Jag2 blockade group (administered with an equal dose of Jag2 neutralizing antibody via tail vein injection at 24 h before IRI modeling and immediately after modeling). Renal injury and protein expression were assessed using histopathological staining, measurement of serum creatinine and urea nitrogen, and western blotting. Human umbilical vein endothelial cells (HUVEC) were used to establish a hypoxia/reoxygenation (H/R) model. After the H/R treatment, HUVEC in control group and Jag2 gene-knockdown group were co-cultured with human monocytic leukemia cell line tohoku hospital pediatrics-1 (THP-1), respectively. Then flow cytometry was applied to compare the proportion of CD86⁺ cells among the groups. After stimulating the mouse monocyte-macrophage leukemia cell line Raw264.7 with exogenous recombinant Jag2 protein, transcriptome sequencing was used to screen for changes in related signaling pathways, and the results were verified by Western blotting and qPCR.

After the renal IRI modeling, renal tubular injury at the corticomedullary junction of the mice kidneys was significant, with elevated levels of serum creatinine and blood urea nitrogen. The expressions of kidney injury molecule-1 and interleukin-1β were markedly upregulated (all P<0.05). Immunofluorescence results showed that Jag2 colocalized with the endothelial cell marker endomucin, and the fluorescence positive area of Jag2 in renal tissues of the model group was obviously larger than that in the sham operation group. Intervention experiments showed that Jag2 neutralizing antibody significantly alleviated the renal tubular necrosis, attenuated the deterioration of renal function, and downregulated the expression of kidney injury molecule-1 and interleukin-1β in the IRI mice (all P< 0.05). Co-culture experiments after the H/R treatment showed that the proportion of CD86⁺ cells was significantly lower in the HUVEC of Jag2 knockout group co-cultured with THP-1 cells than that in the HUVEC of the control group co-cultured with THP-1 cells (P<0.05). Transcriptome sequencing revealed that Jag2 activated multiple inflammation-related signaling pathways including the phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway. Experiments on Raw264.7 cells also demonstrated that Jag2 could upregulate the protein level of phosphorylated AKT, as well as the mRNA levels of interleukin-1β and interleukin-6 in the mouse mononuclear macrophages (all P<0.05).

Jag2 can induce the transformation of mononuclear macrophages into a pro-inflammatory phenotype through the crosstalk between renal vascular endothelial cells and mononuclear macrophages, activate inflammation-related signaling pathways such as PI3K-AKT, and promote the production of inflammatory cytokines, thereby driving the progression of inflammation in the early stage of acute kidney injury.