Abstract
IntroductionThe apoptosis and subsequent injury of podocytes plays a pathogenic role in diabetic nephropathy (DN). Mesenchymal stem cells (MSCs) are promising therapeutic cells for preventing apoptosis and reducing cellular injury. Our previous study found that MSCs could protect kidneys from diabetes-induced injury without obvious engraftment. So we evaluated the effects of human adipose-derived MSCs (hAd-MSCs) on podocytic apoptosis and injury induced by high glucose (HG) and the underlying mechanisms.MethodsWe used flow cytometry, Western blot and confocal fluorescence microscopy to study podocytic apoptosis and injury induced by HG at 24 hours, 48 hours, and 72 hours in the presence or absence of MSC-conditioned medium (CM). An antibody-based cytokine array was used to identify the mediating factor, which was verified by adding the neutralizing antibody (NtAb) to block its function or adding the recombinant cytokine to the medium to induce its function.ResultshAd-MSC-CM reduced podocytic apoptosis in a dose-dependent manner, decreased the expression of podocytic cleaved caspase-3, and prevented the reduced expression and maintained the normal arrangement of podocytic synaptopodin and nephrin. However, human embryonic lung cell (Wi38)-CM failed to ameliorate podocytic apoptosis or injury. Twelve cytokines with concentration ratios (MSC-CM/Wi38-CM) >10-fold were identified. Epithelial growth factor (EGF) was singled out for its known ability to prevent apoptosis. Recombinant human EGF (rhEGF) prevented podocytic apoptosis and injury similarly to hAd-MSC-CM but, upon blockade of EGF, the beneficial effect of hAd-MSC-CM decreased dramatically.ConclusionshAd-MSCs prevent podocytic apoptosis and injury induced by HG, mainly through secreting soluble EG.
Highlights
The apoptosis and subsequent injury of podocytes plays a pathogenic role in diabetic nephropathy (DN)
Using human adiposederived (hAd)-Mesenchymal stem cells (MSCs)-conditioned medium (CM) with a model of podocytic apoptosis induced by high glucose (HG), we investigated whether MSCs-CM could itself inhibit HG-induced podocytic apoptosis, to identify the effect or molecule(s) and to provide novel insights into the treatment of DN
Podocytic apoptosis and injury was induced by HG mouse podocyte clone 5 (MPC5) cells were cultured in vitro and glucose (30 mM) was added to induce apoptosis to establish a model of podocytic apoptosis and injury
Summary
The apoptosis and subsequent injury of podocytes plays a pathogenic role in diabetic nephropathy (DN). Mesenchymal stem cells (MSCs) are promising therapeutic cells for preventing apoptosis and reducing cellular injury. After high passage of these cells, only a low level of senescence will occur [16] These cells have the ability to secrete a large number of protective cytokines [17,18] and show other MSCs characteristics, such as self-renewal and multiple lineage differentiation. We found that human adiposederived (hAd)-MSCs injected via the tail vein into rats with DN alleviated kidney injury, reduced proteinurea, and prevented the downregulation of synaptopodin (data not shown); the gross presence of stem cells was not found in the kidney. We speculated that hAd-MSCs protected the kidney via paracrine action
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