Figure 5.

Genetic deletion or pharmacological inhibition of GR decreases proliferation and migration of primary PECs. (A) In vitro model for cellular crescents.¹⁶ Capsulated glomeruli were isolated from GR KO or Cre–negative littermate controls. Cellular outgrowths were analyzed on day 6. (B1 and B2) Cellular outgrowth was impaired by GR KO (arrows). *Glomerulus (phase contrast image). (B3 and B4) Actin stress fibers were reduced by KO of GR (arrows show phalloidin staining). (B5 and B6) Cellular outgrowths consisted exclusively of glomerular epithelial cells (i.e., PECs and podocytes; WT1 immunofluorescence staining). Scale bars, 400 μm in B1 and B2; 200 μm in B3–B6. (C) Outgrowths from GR KO animals were significantly larger and contained more cells (n=4; 10–15 glomeruli per animal). **P<0.01. (D) Immunoblot analysis of lysates of cellular outgrowths. GR was undetectable in KOs. PEC activation marker (CD44) and the phosphorylated form of S6 ribosomal protein (p-S6) of the mammalian target of rapamycin pathway were reduced in GR KO outgrowths. (E) Pharmacologic treatment of outgrowing cells from WT capsulated glomeruli. A dose-dependent inhibition of cellular outgrowth was observed for partial GR antagonist mifepristone and the agonist dexamethasone (dexa.) on day 8 (n=4; ten capsulated glomeruli per experiment; vehicle, 0.1% DMSO). (F and G) Effects of pharmacologic manipulation of GR activity in primary PECs and podocytes. (F) Primary cultures of murine PECs or podocytes¹⁷ were treated with increasing doses of mifepristone (n=5) for 24 hours and subjected to a WST assay to determine total metabolic cellular activity. Increasing concentrations of mifepristone inhibited proliferation and cellular activity in PECs but not in podocytes. Note that a dose of 100 μm mifepristone reduced cellular viability in PECs (A) but not in podocytes. **P<0.01. (G) When treating primary PECs or podocytes with increasing doses of prednisolone and mifepristone simultaneously, prednisolone inhibited proliferation in both cell types, but this trend did not reach significance. Mifepristone inhibited proliferation specifically in PECs (red arrow; n=5). Data are expressed as mean±SD.