Overexpression of FOXO1 ameliorates the podocyte epithelial-mesenchymal transition induced by high glucose in vitro and in vivo.

Accumulating evidence has suggested that the epithelial-mesenchymal transition (EMT) is a pathway that potentially leads to podocyte depletion and proteinuria in diabetic nephropathy (DN). Therefore, this study was designed to investigate the protective effects of forkhead transcription factor O1 (FOXO1) on podocyte EMT, under high-glucose (HG) conditions in vitro and under diabetic conditions in vivo. The results showed that HG-induced podocyte EMT was associated with FOXO1 inactivation, which was accompanied by activation of the transforming growth factor (TGF)-β1/SMAD3/integrin-linked kinase (ILK) pathway. Accordingly, constitutive FOXO1 activation suppressed the TGF-β1/Smad3/ILK pathway and partially reversed EMT, similar to the effects observed after treatment with SIS3 or QLT0267, which are selective inhibitors of TGF-β1-dependent SMAD3 phosphorylation and ILK, respectively. In addition, lentiviral-mediated FOXO1 overexpression in the kidneys of diabetic mice considerably increased FOXO1 expression and activation, while decreasing proteinuria and renal pathological injury. These data suggested that forced FOXO1 activation inhibited HG-induced podocyte EMT and ameliorated proteinuria and renal injury in diabetic mice. Our findings further highlighted that FOXO1 played a protective role against diabetes in mice and may potentially be used as a novel therapeutic target for treating diabetic nephropathy.

Du M ，Wang Q ，Li W ，Ma X ，Wu L ，Guo F ，Zhao S ，Huang F ，Wang H ，Qin G ... -  《-》

Emodin ameliorates high glucose induced-podocyte epithelial-mesenchymal transition in-vitro and in-vivo.

Epithelial-to-mesenchymal transition (EMT) is a potential pathway leading to podocyte depletion and proteinuria in diabetic kidney disease (DKD). Here, we investigated the protective effects of Emodin (EMO) on high glucose (HG) induced-podocyte EMT in-vitro and in-vivo. Conditionally immortalized mouse podocytes were exposed to HG with 30 μg /ml of EMO and 1 μmol/ml of integrin-linked kinase (ILK) inhibitor QLT0267 for 24 h. Streptozotocin (STZ)-induced diabetic rats were treated with EMO at 20 mg· kg(-1)· d(-1) and QLT0267 at 10 mg· kg(-1)· w(-1) p.o., for 12 weeks. Albuminuria and blood glucose level were measured. Immunohistochemistry, immunofluorescence, western blotting and real-time PCR were used to detect expression of ILK, the epithelial marker of nephrin and the mesenchymal marker of desmin in-vitro and in-vivo. HG increased podocyte ILK and desmin expression while decreased nephrin expression. However, EMO significantly inhibited ILK and desmin expression and partially restored nephrin expression in HG-stimulated podocytes. These in-vitro observations were further confirmed in-vivo. Treatment with EMO for 12 weeks attenuated albuminuria, renal histopathology and podocyte foot process effacement in diabetic rats. EMO also repressed renal ILK and desmin expression, preserved nephrin expression, as well as ameliorated albuminuria in STZ-induced diabetic rats. EMO ameliorated glucose-induced EMT and subsequent podocyte dysfunction partly through ILK and desmin inhibition as well as nephrin upregulatiotion, which might provide a potential novel therapeutic option for DKD.

Chen T ，Zheng LY ，Xiao W ，Gui D ，Wang X ，Wang N ... -  《-》

Astragaloside IV protects against podocyte apoptosis by inhibiting oxidative stress via activating PPARγ-Klotho-FoxO1 axis in diabetic nephropathy.

Podocyte apoptosis plays an important role in the pathogenesis of diabetic nephropathy (DN). Astragaloside IV (AS-IV) has been shown to protect against podocyte apoptosis. Here we aim to investigate the mechanism responsible for the protective effects of AS-IV. Diabetic db/db mice and high glucose (HG)-cultured podocytes were treated with AS-IV. Renal function and histopathological changes were measured to evaluate the therapeutic effects of AS-IV against DN. Adenovirus-mediated Klotho overexpression, Klotho siRNA, and PPARγ inhibitor were applied in vitro to investigate the potential mechanism. The expression levels of mRNA and proteins were analyzed by qRT-PCR, western blot or immunofluorescence. Intracellular ROS and mitochondrial superoxide were detected by DHE and MitoSOx Red, respectively. Cell apoptosis was evaluated by TUNEL staining and flow cytometry. AS-IV improved renal function and ameliorated podocyte injury in db/db mice accompanied with enhanced Klotho expression in glomerular podocytes. In vitro, AS-IV inhibited HG-induced podocyte apoptosis and restored HG-inhibited Klotho expression, whereas Klotho knockdown abrogated the anti-apoptosis action of AS-IV. Further study showed that adenovirus-mediated Klotho overexpression enhanced Forkhead transcription factor O1 (FoxO1)-dependent antioxidant activity and attenuated HG-evoked oxidative stress and apoptosis. AS-IV prevented HG-induced FoxO1 inhibition and oxidative stress, whereas Klotho knockdown reversed these effects. Cotreatment with PPARγ inhibitor T0070907 abolished AS-IV-induced Klotho expression and anti-apoptosis action. These data suggested that AS-IV attenuated podocyte apoptosis presumably by inhibiting oxidative stress via activating PPARγ-Klotho-FoxO1 signaling pathway, thereby ameliorating DN. This study provided new insights into the molecular mechanisms of AS-IV against DN.

Xing L ，Fang J ，Zhu B ，Wang L ，Chen J ，Wang Y ，Huang J ，Wang H ，Yao X ... -  《-》

Astragaloside IV ameliorates diabetic nephropathy involving protection of podocytes in streptozotocin induced diabetic rats.

Podocyte loss and dysfunction play key role during the development of diabetic nephropathy (DN). The aim of this study was to observe the protective effects of astragaloside IV on podocyte in diabetic rats and explore its mechanisms preliminary. Healthy male Sprague-Dawley (SD) rats were randomized into normal control group, diabetic nephropathy group and diabetic nephropathy with AS-IV treatment group. DN was induced by intraperitoneal injection of streptozotocin (STZ). AS-IV treatment started 2 weeks before STZ injection and lasted 14 weeks. 24h Urinary proteins were measured 4, 8 and 12 weeks after STZ injection. Body weight, blood glucose, blood urea nitrogen (BUN), creatinine (Cr), alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured 12 weeks after STZ injection. Renal pathology, podocyte morphological changes, podocyte density, protein and mRNA expression of integrin α3, integrin β1 and integrin-linked kinase (ILK) were detected by histopathology, electron microscopy, immunohistochemistry, western blot and real-time PCR, respectively. Hyperglycemia, proteinuria, mesangial expansion and podocyte loss, increased protein expression of ILK and decreased protein expression of integrin α3 and integrin β1 were detected in diabetic rats. AS-IV treatment ameliorated podocyte loss, renal histopathology and podocyte foot process effacement, decreased proteinuria, partially restored protein expression of integrin α3, integrin β1 and ILK. These findings suggested that AS-IV may protect podocyte and ameliorate diabetic nephropathy by inhibiting the expression of ILK and restoring the expression of integrin α3β1 in diabetic rats.

Chen J ，Chen Y ，Luo Y ，Gui D ，Huang J ，He D ... -  《-》

The roles of connective tissue growth factor and integrin-linked kinase in high glucose-induced phenotypic alterations of podocytes.

Emerging evidence has suggested that podocytes undergo epithelial-mesenchymal transition (EMT) in diabetic nephropathy (DN). Connective tissue growth factor (CTGF) and integrin-linked kinase (ILK) are involved in the progression of DN. However, the underlying mechanisms of EMT are not well understood. The study aimed to investigate the roles of CTGF and ILK in high glucose-induced phenotypic alterations of podocytes and determine whether ILK signaling is downstream of CTGF. The epithelial marker of nephrin and the mesenchymal marker of desmin were investigated by real-time RT-PCR and Western blotting. The results demonstrated that podocytes displayed a spreading, arborized morphology in normal glucose, whereas they had a cobblestone morphology in high glucose conditions, accompanied by decreased nephrin expression and increased desmin expression, suggesting podocytes underwent EMT. In response to high glucose, CTGF and ILK expression in podocytes were increased in a dose- and time-dependent manner, whereas the increase did not occur in the osmotic control. Furthermore, the inhibition of CTGF with anti-CTGF antibody prevented the phenotypic transition, as demonstrated by the preservation of epithelial morphology, the suppression of high glucose-induced desmin overexpression and the restoration of nephrin. Of note, the upregulation of ILK induced by high glucose was partially blocked by the inhibition of CTGF. In summary, these findings suggested that CTGF and ILK were involved in high glucose-induced phenotypic alterations of podocytes. ILK acted as a downstream kinase of CTGF and high glucose-induced ILK expression might occur through CTGF-dependent and -independent pathways.

Dai HY ，Zheng M ，Lv LL ，Tang RN ，Ma KL ，Liu D ，Wu M ，Liu BC ... -  《-》