| BackgroundDiabetic nephropathy(DN) is a microvascular complication of diabetes and is clinically characterized by proteinuria. Recent findings have shown that podocyte is a pivotal factor in the induction of proteinuria and contributes to the DN. Podocytes reside at the glomerular basement membrane outside the glomerular capillaries. The most plausible explanation for proteinuria podocytes detaches from the glomerular basement membrane after apoptosis. However, recent experimental evidence demonstrated that podocyte detachment and apoptosis significantly lag behind the proteinuria. This point aginst a causative role of podocyte loss in the genesis of proteinuria. Liu et al. suggested that podocyte dysfunction through the epithelial-to-mesenchymal transition(EMT) could serve as a pathway contributing to proteinuria. Sam et al. demonstrated blocking EMT in its early phase can reverse podocyte apoptosis. Thus, there is an urgent need to develop novel treatments to prevent DN.Forkhead transcription factor O1(FoxO1) regulates cell cycle progression, stress resistance, and glucose metabolism. Previous studies have provided evidence thatupregulated FoxO1 expression can improve podocyte and mesangial cell, and alleviate glomerular pathological changes in diabetic rats. Emerging evidence indicates that when podocytes are exposed to HG conditions, they undergo EMT process, with epithelial marker nephrin increase and mesenchymal indicator desmin decrease. It is suggested that TGF-?/Smad/ILK plays an important role in promoting tubular EMT. Our previous data showed that FoxO1 plays a crucial role in downregulating activation of the TGF-?1 pathway in mesangial cells in diabetic rats.However, the effect of FoxO1 on the TGF-? pathway may depend on the cell type and the nature of the stimulus. Collectively, these findings lead us to speculate that FoxO1 plays an important role in the EMT of podocytes through activating the TGF-?1/SMAD3/ILK signal pathway.ObjectiveTo study the roles and mechanisms of FoxO1 on TGF-?1/Smad3/ILK pathway and podocyte EMT under high-glucose(HG) conditions and under diabetic conditions.MethodsThe podocytes were transfected with lentiviral vectors containing the sequences of constitutively active FoxO1(LV-CA-FoxO1), the lentiviral vectors of the FoxO1 short hairpin RNAs(LV-FoxO1-sh RNA) and empty lentiviral vectors(LV-NC). After transduction with LV-Foxo1 short hairpin RNA(sh RNA), podocytes were exposed to SIS3 or QLT0267. The podocytes were divided into seven groups of normal glucose(5.6mmol/L)(NG), high glucose(25mmol/L)(HG), HG plus LV-CA-Foxo1(CA),NG plus LV-Foxo1-sh RNA(sh RNA), NG-LV-Foxo1-sh RNA plus SIS3(SIS3),NG-LV-Foxo1-sh RNA plus QLT0267(QLT), or HG plus the empty LV vector(NC).After treatment 72 h, the m RNA levels of podocytes were measured through real time PCR in each group, including FoxO1, TGF-?1, Smad3, ILK, nephrin and desmin. The protein expressions of FoxO1, p-FoxO1, TGF- ?1, Smad3, p-Smad3, ILK, nephrin,and desmin were assessed by western blot. The distribution of FoxO1 in podocyteswas detected by immunofluorescence. In addition, the TGF-?1 secretion in cell culture supernatants was analyzed by ELISA.To establish the diabetes mouse model,mice were injected once intraperitoneally with 130 mg/kg streptozotocin.Then, 50 ?L recombinant LV(LV-NC or LV-CA-FoxO1) or normal saline was injected at different sites of the dorsal region of the right renal cortex, forming the NC, CA, and DM groups, respectively. At 8 weeks post-injection, urine was collected for the determination of 24-h urinary protein and urinary albumin levels in enzyme-linked immunosorbent assays, and blood was collected from the orbital vein to test serum creatinine and blood urea nitrogen levels. The m RNA levels of glomerulus were measured through real time PCR in each group, including FoxO1, TGF-?1, Smad3,ILK, nephrin and desmin. The protein expressions of FoxO1, p-FoxO1, TGF- ?1,Smad3, p-Smad3, ILK, nephrin, and desmin were assessed by western blot. The expression of nephrin and desmin were determined by immunohistochemistry.Kidney pathology in HE sections was examined by light microscopy. Ultrastructure of renal cortex was observed with electron microscope.Results1. LV vectors mediated FoxO1 expression in podocytesFoxO1 m RNA and protein expression levels were not significantly different in podocytes from the NG, HG, and NC groups(P>0.05), but were increased in the CA group(P<0.05). Under the HG condition, the p-FoxO1/FoxO1 ratio was increased compared with NG groups(P<0.05). Furthermore, the p-FoxO1/FoxO1 ratio was decreased in the CA group compared to the HG group(P<0.05). In the sh RNA group,FOXO1 levels decreased, but the p-FoxO1/FoxO1 ratio increased compared to the NG group( all P<0.05). There were no significant differences in any indices among the sh RNA, SIS3, and QLT groups, or between podocytes from the NC and HG groups(all P>0.05).In podocytes, FoxO1 nuclear translocation was decreased in podocytes in the HG group(P<0.05). However, FoxO1 nuclear translocation increased following CA-FoxO1 overexpression. Infection with the LV-FoxO1-sh RNA caused the oppositeeffect(P<0.05).2. Effects of FoxO1 on the TGF-?1/SMAD3/ILK signaling pathway in podocytesThe m RNA and protein levels of TGF-?1, Smad3 and ILK were elevated in the HG group compared with the NG group, and p-Smad3/Smad3 radio were significantly increased(all P<0.05). The m RNA and protein levels of TGF-?1 and ILK were decreased in the CA group compared with the HG group, and p-Smad3/Smad3 radio were decreased(all P<0.05), but the m RNA and protein levels of Smad3 were not significantly different(P>0.05). Podocytes in sh RNA group showed increased of levels of TGF-?1, SMAD3, ILK and the ratio of p-Smad3/Smad3 compared with the NG group(all P<0.05). By contrast, in the SIS3 group, the expression levels of ILK and the ratio of p-Smad3/Smad3 decreased.Similar results were obtained when the cells were treated with QLT0267(all P<0.05),except that the ratio of p-SMAD3/Smad3 level was unchanged(P>0.05).3. Effects of FoxO1 on the expression of nephrin and desmin in podocytesCompared with NG group, exposure to HG condition reduced and elevated the nephrin and desmin m RNA and protein expression levels in podocytes, respectively(all P<0.05). CA group showed decrease in desmin m RNA and protein levels and increase in nephrin expression.Compared with NG group, sh RNA group showed increase in desmin m RNA and protein levels and decrease in nephrin expression(all P<0.05). Compared with sh RNA group, SIS3 group and QLT group showed increase in nephrin m RNA and protein levels and decrease in desmin expression(all P<0.05).4. Expression of FoxO1 in the glomeruli of diabetic miceIn the glomeruli of mouse, the m RNA and protein expression of FoxO1 did not differ between the NG, DM, and NC groups(P>0.05), but increased in the CA group(all P<0.05). The p-FOXO1/FOXO1 ratio increased in the DM group, but decreased in the CA group(all P<0.05).5. Effects of FoxO1 on the TGF-?1/Smad3/ILK signaling pathway in vivoThe m RNA and protein levels of TGF-?1, Smad3, and ILK and the ratio of p-Smad/Smad3 were elevated in the glomeruli from STZ-induced diabetic mice when compared with the NG group(all P<0.05). However, the m RNA and proteinexpression of TGF-?1 and ILK were partially restored by CA-Foxo1 treatment(all P<0.05). There were no differences in the m RNA and protein levels of Smad3 between the DM group and CA group(P>0.05), but the CA group was lower than DM group in the ratio of p-Smad3/Smad3(all P<0.05).6.FoxO1 overexpression ameliorates the podocyte Epithelial-Mesenchymal Transition in vivoCompared with NG group, the nephrin m RNA and protein levels reduced and the desmin levels elevated in the glomeruli from STZ-induced diabetic mice, as compared with the corresponding levels measured in control mice(all P<0.05).Compared with DM group, CA group showed decrease in desmin m RNA and protein levels and increase in nephrin expression(all P<0.05). Similarly,immunohistochemistry assay revealed desmin upregulation and nephrin downregulation in the glomeruli of diabetic mice(all P<0.05). However, these abnormalities were partially restored in diabetic rats injected with LV-CA-FoxO1(all P<0.05).7. FoxO1 overexpression protected the kidney function of STZ-induced diabetic miceThe levels of blood glucose, 24-h urinary protein, urinary albumin, serum creatinine, and blood urea nitrogen markedly increased in the diabetic mice,compared to mice in the NG group(all P<0.05). FoxO1 overexpression significantly decreased the levels of all of these biological parameters except for blood glucose at the end of the eighth week in comparison to diabetic rats. Histopathological analysis with hematoxylin and eosin staining of the kidneys showed that the structures of glomeruli were clear in NG group. The glomerular basement membrane was uniform thickness with evenly distributed foot process and mesangial matrix accumulations were observed by electron microscopy in control group. HE staining showed a larger volume of the renal glomeruli, expansed mesangial cells, and thickened glomerular basement membrane in the kidneys of diabetic mice. Conversely, renal pathology was ameliorated in the CA group of mice. Moreover, electron microscopy showed that the basement membrane became thickening or not smooth and foot process fusion gradually increased, while mesangial matrix became nodular hyperplasia in diabeticmice. In contrast, FoxO1 overexpression promoted the recovery of the injured podocytes to some degree.Conclusions1. High glucose induces the inactivation of FoxO1, leading to podocyte EMT and subsequent podocyte dysfunction.2. Increased FoxO1 activity suppressed HG-induced stimulation of EMT in podocytes, which may be associated with the HG-dependent TGF-?1/SMAD3/ILK upregulation.3. Under diabetic conditions in vivo, HG-induced podocyte EMT was associated with FoxO1 inactivation. Forced FoxO1 activation with LV vectors ameliorates HG-induced podocyte EMT, thereby preventing podocyte dysfunction and DN progression. |
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