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. 2018 Jul 12;3(13):e120546. doi: 10.1172/jci.insight.120546

Figure 2. Repetitive renal IRI induces senescence in KLN-resident FRCs.

Figure 2

KLNs were harvested 2 weeks after the induction of the third IRI (KLN: IRI(rep)). (A) Microscopy shows marked hypocellularity and loss of differentiation between cortical and subcortical zones. Both ER-TR7 and fibronectin staining insets illustrate areas of lobulated and thickened ECM. Arrowheads in ER-TR7 and fibronectin staining demonstrate areas of excessive ECM deposits with interstitial swelling. Collagen I matrix is increased, especially in subcortical areas, and ECM is expanded in the subcortical T cell zone, proximal to the B cell zone in costaining of KLN with Coll I and F4/80 (see dashed lines in inset). FRC staining demonstrates enlarged cytoplasms (inset in PDPN). Many KLN interstitial cells are stained with αSMA. Several of these cells are senescent (p16INK4A+), especially in the subcortex (next to the dashed line); inset: close-up of senescent cells. HEV structure is markedly thickened (close-up image in inset). Lyve1 stain reveals regression of the lymphatic endothelial network. Scale bars: 500 μm and 200 μm (inset) for H&E; 200 μm and 100 μm (inset) for ER-TR7, PDPN, and αSMA+p16INK4A; 500 μm and 100 μm (inset) for fibronectin and CollI+F4/80; 100 μm and 50 μm (inset) for MECA79; 200 μm for Lyve1. (B) Flow cytometric analysis of CD45– cells from KLNs following repetitive IRI (KLN: IRI(rep)) or without repetitive IRI (KLN: Ctrl) shows significantly decreased percentages of CD45–PDPN+CD31– FRCs and proliferating (Ki67+) FRCs, along with representative flow cytometry plots (n = 4–6/group, mean ± SEM). (C) Electron microscopy of naive KLN shows HEV endothelium (white asterisks) and supporting perivascular FRCs (white arrowheads). Arrows indicate lymphocytes in active diapedesis into the interstitium of the KLN (KLN: Ctrl). KLN following repetitive IRI (KLN: IRI(rep)) shows obliteration of the HEV lumen with detachment of HEV cells (white asterisks) from the vessel’s basement membrane and intrusion into the lumen. White arrowheads indicate perivascular FRCs. Some HEV cells show denser and smaller nuclei, along with shrunken cytoplasm, and HEV has intraluminal ECM deposits (black arrowheads). (D) FRCs from KLNs of mice with or without repetitive IRI were cultured in vitro and stained with β-galactosidase (senescence marker). Micrograph of FRCs from KLN following repetitive IRI showed senescent cells with lysosome-like vacuoles (white arrowhead) at a significantly higher percentage than FRCs from the KLNs of naive mice (n = 6/group, mean ± SEM). **P < 0.01 by Student’s t test.