Figure 4

Epithelial growth factor (EGF) is the main factor in human adipose-derived mesenchymal stem cells (hAd-MSCs)-conditioned medium (CM) to reduce apoptosis and injury of mouse podocyte clone (MPC5) cells induced by high glucose (HG). A) AnnexinV/PI double staining labeled cells in each group (n = 3 per group) and flow cytometry to determine podocytic apoptosis rate. Recombinant human (rh) EGF group significantly reduced podocytic apoptosis but EGF-neutralizing antibody (NtAb) did not. B) Western blot was used to detect the expression of cleaved caspase-3 at three time points (24, 48 and 72 hours). rhEGF reduced the expression of cleaved caspase-3, but EGF-NtAb did not. All of the experiments were repeated three times (n = 3). ^*P <0.05 HG group versus normal glucose (NG) group, MSC-CM group or rhEGF group; ^#P <0.05, EGF-NtAb versus NG group, MSC-CM group or rhEGF group. C) The expression and the location of the podocytic cytoskeletal protein synaptopodin (red) were measured by confocal microscopy. rhEGF prevented the downregulation and rearrangement of synaptopodin but EGF-NtAb did not. Nuclei were stained with DAPI (blue). Magnification = 600×, 1800×. D) The expression of the podocytic membrane protein nephrin (red) was measured by confocal microscopy. rhEGF prevented the downregulation and disorder of nephrin but EGF-NtAb did not. Nuclei were stained with DAPI (blue). Magnification = 600×, 1800 ×. DAPI, 4',6-diamidino-2-phenylindole; PI, propidium iodide.