Fig 2: CCL21 binds to the surface of human DCs and forms discrete puncta; a feature not matched by tailless-CCL21. Fluorescence microscopy pictures of DCs incubated (A) with100 nM CCL21, (B) 100 nM tailless-CCL21, or (C) in the absence of ligand obtained on LSM 780 confocal microscope using 63× oil-objective (ligands were stained with Alexa 488 and the cell nucleus visualized with Hoecst DNA staining). (D) Zoom in on DC incubated with 100 nM CCL21 to visualize that anti-CCL21 staining followed the villi-like surface of non-adherent DCs. (E) Visualization of two different types of CCL21 puncta. (F) The anti-CCL21 antibody also recognized tailless-CCL21.

Fig 3: CCL21 expression correlated with CD3 + T cell infiltration in DN. A Representative micrographs of immunofluorescent staining of CCL21 (green) in renal tissue from controls (normal kidney tissue from patients with renal carcinoma) and patients with DN. Nuclei were stained with DAPI (blue). Scale bar: 50 µm. Representative micrographs (B) and the quantification (C) of CD3-positive cells, CD4-positive cells and CD8-positive cells in renal tissues from DN patients with low (< median) and high (> or = median) expression of urinary small EVs derived CCL21. Scale bar: 50 µm. D The correlation between CD3-positive cells and the expression of urinary small EVs derived CCL21 (r = 0.6427, p = 0.0054). E The correlation between the number of CD3-positive cells with ESR levels in DN patients (r = 0.8511, p = 0.004). F Representative micrographs of immunofluorescent staining for CD3 (green) and CCL21 (red) in renal tissue from patients with DN. Nuclei were stained with DAPI (blue). Scale bar: 20 µm

Fig 4: Urinary small EVs derived CCL21 mRNA increased in STZ-induced DN rats and correlated with the alteration in kidney. A, B Blood glucose and body weight of rats with or without STZ treatment at week 20 after injection. Significant increase of blood glucose (A) and body weight (B) were found in STZ-injected rats, compared with control rats. C–E Levels of albumin-to-creatinine ratio (ACR), serum creatinine (SCr) and blood urea nitrogen (BUN) in STZ-injected and control rats (n = 8 for each group). F Representative micrographs of histological analysis (PAS staining) of renal tissue from rats with or without STZ treatment. Scale bar: 50 µm. G Western blotting analysis of protein (Alix, CD81 and CD63) in urinary small EVs fraction from Ctrl and DN rats. H Representative size distribution and particle number of urinary small EVs analyzed by nanoparticle tracking analysis (NTA). The NTA experiment has been performed for three samples in each group. Relative CCL21 mRNA expression in renal cortex (I) and urinary small EVs (J) from rats with or without STZ injection. K The correlation between renal CCL21 mRNA expression and urinary small EVs derived CCL21 mRNA expression in STZ-injected DN rats (r = 0.6935, p = 0.0103). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, compared with Ctrl-rats. Ctrl control; DN diabetic nephropathy

Fig 5: Urinary small EVs derived CCL21 mRNA in different stages of DN divided by histological damage. A The expression of CCL21 mRNA in urinary small EVs of DN patients with normal (0, n = 7), mild (< 25%, n = 11) or severe (> 25%, n = 10) tubulointerstitial inflammation. Urinary small EVs derived CCL21 mRNA expression increased progressively with the deterioration of tubulointerstitial inflammation. (****p < 0.0001, compared with normal group; ####p < 0.0001, compared with mild group). B Urinary small EVs derived CCL21 expression in DN patients based on glomerular classification (*p < 0.05 compared with patients with class II)