Fig. 6. Mobilization of neutrophils to the site of kidney damage via CXCR4 is mediated by NGAL–metformin–Fe.

a The Cxcr family mRNAs were measured in neutrophils obtained from scRNA-seq data. b Kaplan–Meier survival curves of the I/R+Met100 group and I/R+Met100+SCH group. c Representative image of kidneys removed from a mouse following I/R, a mouse in I/R+Met100, a mouse in I/R+Met100+AMD (5 mg/kg), and a mouse in I/R+Met100+WZ (4 mg/kg) group. d Summary of the kidney to body weight ratio measured in the indicated mice. e–h Summary of serum BUN (e), creatinine (f), LDH (g), and AST (h) measured in the indicated mice. i Summary of the Lcn2 mRNA levels measured in the indicated mice, expressed relative to I/R. j Summary of the renal MDA levels measured in the indicated mice. k Summary of the renal MPO levels measured in the indicated mice. l Representative H&E-stained, Sirius Red-stained, Masson’s Trichrome-stained, MPO IHC-stained, Neutrophil Elastase IF-stained, and electron micrographs showing kidney sections from the indicated mice. m Cryo-EM density of CXCR4 (green) and NGAL (orange). NGAL binds to an extracellular facing cavity in CXCR4. n CXCR4 and NGAL were either co-expressed (Top panel) or only expressing CXCR4 and then co-cultured with recombinant human NGAL (rhNGAL) (Bottom panel) in HEK293T cells; 48 h after transfection, the cell lysates were immunoprecipitated using protein A/G beads to pull-down CXCR4 or NGAL (IP), respectively, followed by immunoblotting (IB) with the other antibody. Immunoglobulin G was used as a negative control, and the input was used a positive control. o Green asterisk labels the NGAL, red asterisk labels the CXCR4, then analyzed using immunofluorescence. *P < 0.05, **P < 0.01, ***P < 0.001 and n.s., not significant (One-way ANOVA). Survival curve comparison was analyzed by a log-rank Mantel-Cox test. In this and subsequent figures, summary data are represented as the mean ± SEM. See also Supplementary Figs. S18 and S19.