Research Paper Volume 15, Issue 14 pp 6658—6689

Figure 3. Podocyte lifespan and glomerular ultrastructure were improved by inhibiting and deleting NLRP3. (A–F) Immunostaining for p57 (black, nuclear) and Collagen IV (brown) comparing glomeruli of young, middle-aged saline-treated, MCC950-treated mice as well as NLRP3 null mice. Quantification of the number of p57-positive podocytes was used to determine podocyte density (E), Collagen IV staining to assess glomerular scaring (F) and their nuclear size was used to determine hypertrophy (G). Samples were compared using Student’s t-test and significance is indicated. Representative images are shown; the scale bars in the images correspond to 25 μm. (H–O) Analysis of glomerular ultrastructure using FLARE coupled to confocal microscopy; hydrogel-expanded mouse kidney tissue has been stained for primary amines (red, labels proteins), oxidized carbohydrates (green, labels basement membrane and mesangial matrix), and DNA (blue, labels nuclei). Representative images comparing glomeruli of young, middle-aged saline-treated, MCC950-treated mice as well as NLRP3 null mice are shown. All scale bars correspond to 5 μm pre-expansion. Abbreviations: P: podocyte; E: endothelial cells; M: mesangial cells. (P, Q) Quantification of glomerular basement membrane (GBM) thickness by measuring the average thickness of the oxidized carbohydrate stain in capillary loops (P) and of foot process width by determining the average thickness of the amine-stained foot processes at half-maximum (Q).