Fig. 4. Adriamycin treatment induced podocyte apoptosis in the progressive stage of FSGS.

A TUNEL staining was used to label apoptotic cell and DAPI to label nuclei, showing markedly increased apoptotic cells in peripheral area of glomeruli in mice sacrificed at 10th week after Adriamycin treatment compared to other groups. B Apoptotic cells in peripheral area of glomeruli were calculated from 25 randomly captured images at each time point (5 images per mouse, 5 mice per group). Only TUNEL-positive nuclei in peripheral area of glomeruli was counted for the numerator, which was divided by the sum of DAPI-positive nuclei and TUNEL-positive nuclei within glomeruli. C Double immunofluorescence staining with activated Caspase 3 and podocyte cytoplasm marker podocin. Representative apoptotic podocytes were marked with arrow (yellow). D Immunoblotting of glomeruli protein showed upregulated protein expression of activated Caspase 3 with the progression of Adriamycin-induced FSGS. Shown are representative blots from at least three separate experiments with similar results. E Podocyte number was measured and calculated using the dissector/fractionator principle that nuclei present in the first section of each dissector pair but not in the second section were counted. F Glomerular volume was not change significantly during FSGS progression, measured by the Cavalieri principle. G Podocyte density was calculated by dividing podocyte number by glomerular volume (n = 5 mice, 50 total glomeruli per group). Data are expressed as means ± SEM, *P < 0.05, **P < 0.01 (Scale bar: 20 μm in A, C).