Figure 1

High glucose (HG) induces apoptosis and injury of mouse podocyte clone (MPC5) cells. A) AnnexinV/PI double-staining-labeled cells in each group (n = 3 per group). The number of apoptotic or necrotic cells was quantified by FACS analysis after staining with annexin V and PI. The cytograms show viable cells that did not bilnd annexin V or PI in the D3 quadrant. Cells at early stages of apoptosis that bound annexin V but that still had intact cell membranes and excluded PI are shown in the D4 quadrant. Cells with advanced stages of apoptosis or necrotic were both annexin V and PI positive and are shown in the D2 quadrant. Cells lost its intact cell membranes that bound PI and excluded annexin V are shown in the D1 quadrant. The results showed that podocytic apoptosis rate was significantly higher at all time points in HG group than in normal glucose (NG) group, and was time-dependent. B) Western blot was used to detect the expression of cleaved caspase-3 at three time points (24, 48 and 72 hours). The expression of cleaved caspase-3 was increased with the prolonged stimulation of HG. All of the experiments were repeated three times (n = 3). *P <0.05, HG group versus NG group or NG+mannitol group; ^#P <0.05, 48-hour HG group or 72-hour HG group versus 24-hour HG group. C) The expression and the location of podocytic cytoskeletal protein synaptopodin (red) were measured by confocal microscopy. The expression of podocytic synaptopodin in the HG group was reduced and rearranged. Nuclei were stained with DAPI (blue). Magnification = 600×, 1800×. D) Using flow cytometry with TUNEL staining to measure the apoptosis rate of podocytes under treatment with NG, NG+Ma and HG at three time points (24, 48 and 72 hours) (n = 3 each group). Cells analyzed under marker ‘A’ are apoptotic (TUNEL positive).