Fig. 2.

Dgke KO mice develop glomerular thrombotic microangiopathy when injected with nephrotoxic serum (NS). A: representative images of Dgke KO mice glomeruli (left) after injection of subtoxic doses of rabbit NS compared with WT littermates (right). Glomeruli of KO mice show increased mesangial matrix (arrowheads), diffuse congestion of the capillary loops (black arrow), and numerous entrapped erythrocytes (white arrow). Capillary obliteration is not detectable in WT littermates (right) after injection of the same dose of NS (arrows indicate patent capillary loops) and in Dgke KOs treated with nonimmune isotypic serum (NIS; center). H&E staining, scale bars = 20 μm. Bottom: immunofluorescence microscopy images of glomeruli of the same mice probed with an antibody against rabbit IgG (rIgG), showing that the animals received comparable amount of NS or no NS in the NIS control. Mice were 6 wk of age at the date of the experiment. Scale bars = 20 μm. B: quantification of the average patent capillary area in glomeruli of WT and Dgke KO mice after injection of NS or of isotypic NIS as a control, obtained by digital image analysis. Values are means ± SE; n = 2 mice per group, with 10 optical fields per mouse. P values were calculated by Student's t-test. C: TEM images of Dgke KO after NS or isotypic NIS injections and WT littermate glomeruli after NS injection. In kidneys of Dgke KOs after NS injection (left) mesangial cells (m) are focally increased in number and infiltrate segments of the GBM (white asterisk). Endothelial cells (black asterisks) are swollen, and capillary rims are irregular and lack the typical fenestrations (black arrows). Lumens are partially obliterated by swollen endothelium and trapped irregular-shaped erythrocytes (stars). Notice that podocytes' foot processes (black arrowhead and inset) are relatively well conserved. Capillary loops of Dgke KOs treated with NIS and of WT littermates after NS injections (stars; middle and right) are patent, endothelial fenestrations are clearly detectable (arrows and insets), and podocytes' foot processes appear normal (arrowhead). Scale bars = 10 μm. D and E: TEM images at higher magnification of Dgke KO glomeruli after injection of NS that show bulky endothelial cells (black asterisks) with almost completely obstructed capillary lumens and the interposition of a mesangial cell (E; white asterisk), which results in the splitting of the basal membrane (E; black arrowheads). Scale bars = 2 μm. F: quantification of the number of stressed endothelial cells per number of capillary loops in glomeruli of Dgke KO and WT mice 14 days after injection with NS. Values are means ± SE; n = 20 consecutive capillary loops form multiple glomeruli, with 2 mice per experimental group.