FIG. 6.

Glial cell line-derived neurotrophic growth factor (GDNF) secreted by hASCs ameliorated podocyte injury. (A) GDNF concentration was measured in conditioned hASCs culture media (CM) by enzyme-linked immunosorbent assays (ELISA). The results revealed that 24-h ASC-CM contained a 44.6-fold increased GDNF level as compared with CM from WI38 cells. High glucose did not affect GDNF secretion. Three independent samples were measured in triplicate. *P<0.05 as compared with WI38-CM; ^▵P<0.05 as compared with ASC-CM. The expression and location of the podocytic cytoskeletal protein synaptopodin (red) were evaluated by confocal microscopy (B). Nuclei were stained with DAPI (blue). The expression of podocytic synaptopodin in the high-glucose group was reduced and rearranged as compared with normal glucose group. ASC-CM and recombinant human GDNF (rhGDNF) rescued the synaptopodin downregulation, but WI38-CM, neutralizing antibody of GDNF (GDNF-NtAb) did not. To further assess the role of GDNF secreted by ASCs in the reduction of podocyte injury, GDNF expression in ASCs was knocked down by transient transfection with GDNF-siRNA. The GDNF siRNA knockdown efficiency was approximately 67.6% (A). ASCs cocultured with podocytes injured by high glucose treatment significantly restored the down-regulation of synaptopodin expression. There was a significant reduction in podocytic synaptopodin expression in the GDNF-siRNA group as compared with negative control group. Original magnification, 600×. These results were confirmed by western blotting (C). Data are means±SD (n=3–5). *P<0.05 versus NG; ^▵P<0.05 versus HG.