Mesenchymal Stem Cells Protect Podocytes From Apoptosis Induced By High Glucose Via Secreting EGF

Objective:Diabetic nephropathy (DN) is the main disease leading to end stage renal disease (ESRD). The apoptosis and the subsequent injury of podocytes play a pathogenic role in the process of DN. Mesenchymal stem cells (MSCs) are promising therapeutic cells efficient for preventing apoptosis and reducing celluar injury. Our previous data suggested that MSCs could protect kidney from diabetes-inducing injury. Unfortunately, the underlyling mechanism remains obscure. The purpose of our research is to evaluate the effect of human adipose-derived MSCs (hAd-MSCs) on podocytic apoptosis and injury induced by high glucose, and to clarify the specific mechanisms of this effect.Methods:hAd-MSCs or human embryonic lung cells (Wi38) were cultured in serum-free dulbecco minimum essential medium (DMEM) for24h, conditioned medium (CM) were concentrated to1×,2×or4×by ultracentrifugation. Matured mouse podocyte clone (MPC5) cultured in vitro were randomly divided into5groups, i.e. normal glucoses (NG) control group, mannitol (MAN) control group, RPMI-1640medium containing30mM dextroglucose (high glucose, HG) group, serum-free Wi38-CM treated group, and serum-free hAd-MSCs-CM treated group. MCP5were cultured in high glucose for12h, then the above-mentioned CM was added to the medium respectively for another12h,36h, or60h. Fluorescence activated cell sorting (FACS) was used to analysize AnnexinV/PI double staining for apoptosis of MPC5, Western blot was used to demonstrate the protein expression of cleaved caspase-3of MPC5, light microscopy was used to observe the morphological changes of MPC5, Confocal fluorescence microscopy was used to evaluate the expression and the arrangement of skeleton protein-synaptopodin in MPC5. Antibody based cytokine array was used to analyize the elements in CM, the candidates were selected, and Enzyme-linked immunosorbent assay (ELISA) was followed for further test. Specific neutralizing antibody (NtAb)was used to block the function of the selected cytokine in CM. or the recombinant cytokine was added to the blank medium, then the above-mentioned analyses were used again to evaluate the apoptosis and the injury induced by high glucose in MPC5. Results:In MPC5of HG group, compared with NG group, the respective rates of apoptosis at24h,48h,72h were increased dramatically (P<0.05), the protein expressions of cleaved caspase-3were also increased significantly (P<0.05), while the expressions of synaptopodin were diminished with disordered arrangements. On the contrary, in MPC5of HG group, compared with HG group hAd-MSC-CM reduced the rate of apoptosis with a dose-dependent manner (P<0.05), decreased the protein expressions of cleaved caspase-3, prevented the expression of synaptopodin from diminishment, and maintained the normal arrangements of synaptopodin. But Wi38-CM failed to ameliorate the apoptosis or the injury of MPC5.12cytokines with ratios of MSC-CM/Wi38-CM higher than5-fold were identified by Cytokine antibody array and ELSIA, such as, bFGF, DGF-BB, EGF, GDNF, PIGF, Leptin, TGF-β, et al. Epithelial growth factor (EGF) was singled out as the candidate for its capacity of preventing apoptosis. Recombinant human EGF could prevent the apoptosis and the injury induce by high glucose in MPC5similar to hAd-MSC-CM, but with EGF blocked, the beneficial effects of hAd-MSC-CM were decreased dramatically.Conclusion:Collectively, our data suggested that hAd-MSCs could prevent the apoptosis and the injury induced by high glucose in podocytes, through secreting soluble EGF.