Fig. 3 Intrarenal and RDLN lymphangiogenesis accompanied by lymphocyte expansion in the kidney, RDLN, and spleen. Intrarenal and RDLN lymphangiogenesis.

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Fig. 3 Intrarenal and RDLN lymphangiogenesis accompanied by lymphocyte expansion in the kidney, RDLN, and spleen. Intrarenal and RDLN lymphangiogenesis accompanied by lymphocyte expansion in the kidney, RDLN, and spleen. (A) Morphological changes in mouse kidney [periodic acid–Schiff (PAS) staining], RDLN, and spleen following UUO or IRI. (B) Change in weight of RDLN (left) and spleen (right) following UUO or IRI (primary axis, RDLN; secondary axis, spleen) relative to mouse body weight (BW). (C) Representative immunofluorescence images (40×) showing I-AD+ cells in the kidney, RDLN, and spleen of UUO or IRI model mice at day 3 after surgery. (D) Representative immunofluorescence images (10×) showing B220+ (green) B and CD3+ (red) T lymphocytes in kidneys, RDLN, and spleen of UUO or IRI model mice at day 3 after surgery. (E) CD45+CD11c+ cell numbers in the RDLN and spleen of UUO model mice analyzed by flow cytometry. (F to H) CD45+CD3+CD4+ cells, CD45+CD3+CD8+ cells, and CD45+CD19+CD3− cells analyzed by flow cytometry: (F) whole kidney, (G) RDLN, and (H) spleen. (I) LYVE-1+ LV (green) and CCL21 (red) colocalization in the kidney and RDLN of normal control, UUO (day 3 after surgery), and IRI (day 3 after surgery) mice. (J) Representative immunofluorescence images showing LYVE-1 and CCR7 staining in the kidney and RDLN of normal control, UUO (day 3 after surgery), and IRI (day 3 after surgery) mice. (K) Percentage of CCR7+ cells in the kidney, RDLN, and spleen at different time points after UUO analyzed by flow cytometry. (L and M) I-AD+ splenocytes (1 × 104) of GFP mice were sorted and injected under the renal capsule of UUO (day 3 after surgery) mice. Tissue was collected 6 hours later. (L) Co-identification of extrinsic GFP-positive cells with LVs (red) showed GFP+ cells in the lumen of LVs (white arrows) in kidneys and RDLN. (M) Colocalization of extrinsic GFP and CCR7 (red). n = 5 mice per group. Statistical analysis was performed using the Mann-Whitney U test. n.s. > 0.05, *P < 0.05, **P < 0.01. Values are mean ± SEM. Guangchang Pei et al. Sci Adv 2019;5:eaaw5075 Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).