HG-induced Epithelial-mesenchymal Transition Research Articles

P4HB regulates the TGFβ/SMAD3 signaling pathway through PRMT1 to participate in high glucose-induced epithelial-mesenchymal transition and fibrosis of renal tubular epithelial cells

BackgroundDiabetic nephropathy (DN) is a common complication of diabetes mellitus, and Prolyl 4-Hydroxylase Subunit Beta (P4HB) expression is increased in high glucose (HG)-induced renal tubular epithelial cells (TECs). But it’s role in HG-induced TECs remains to be elucidated.MethodsThe HK-2 cells were induced using HG and transfected with SiRNA-P4HB. DCFH-DA staining was utilized for the detection of cellular levels of ROS. WB and immunofluorescence were utilized to detect the expression of P4HB, epithelial-mesenchymal transition (EMT), fibrosis, and TGFβ/SMAD3-related proteins in HK-2 cells. Online databases were utilized for predicting the interaction target of P4HB, and immunoprecipitation (IP) experiments were employed to validate the binding of P4HB with the target. SiRNA and overexpression vectors of target gene were used to verify the mechanism of action of P4HB.ResultsHG induced an increase in the expression of P4HB and TGFβ, p-SMAD3, and ROS in HK-2 cells. Furthermore, HG downregulated the expression of E-cadherin and upregulated the expression of N-cadherin, Vimentin, α-SMA, Fibronectin, Collagen IV, SNAIL, and SLUG in HK-2 cells. Interfering with P4HB significantly reversed the expression of these proteins. Database predictions and IP experiments showed that P4HB interacts with PRMT1, and the expression of PRMT1 was increased in HG-induced HK-2 cells. Interfering with PRMT1 inhibited the changes in expression of EMT and fibrosis related proteins induced by HG. However, overexpression of PRMT1 weakened the regulatory effect of P4HB interference on the EMT, fibrosis, and TGFβ/SMAD3-related proteins in HK-2 cells.ConclusionP4HB regulated the TGFβ/SMAD3 signaling pathway through PRMT1 and thus participates in HG-induced EMT and fibrosis in HK-2 cells.

Colquhounia root tablet improves diabetic kidney disease by regulating epithelial-mesenchymal transition via the PTEN/PI3K/AKT pathway.

Diabetic kidney disease (DKD) is a severe microvascular complication of diabetes mellitus that can lead to end-stage renal disease. Colquhounia root tablet (CRT) has shown therapeutic potential in treating DKD, but its efficacy and underlying mechanisms remain to be elucidated. A randomized controlled clinical trial was conducted on 61 DKD patients. The treatment group received CRT in addition to standard therapy, while the control group received standard therapy alone. Treatment efficacy and adverse events were evaluated after 3months. Additionally, in vitro experiments using human renal tubular epithelial cells (HK-2) were performed to investigate the effect of CRT on high glucose (HG)-induced epithelial-mesenchymal transition (EMT) and the involvement of the PTEN/PI3K/AKT signaling pathway. CRT treatment significantly improved proteinuria and increased the effective treatment rate in DKD patients compared to the control group, with no significant difference in adverse events. Moreover, CRT reversed HG-induced EMT in HK-2 cells, as evidenced by the downregulation of α-SMA and upregulation of E-cadherin at both mRNA and protein levels. Mechanistically, CRT increased PTEN expression and inhibited the PI3K/AKT pathway, similar to the effects of the PI3K inhibitor LY29400. The combination of CRT and LY29400 further enhanced PTEN mRNA expression under HG conditions. CRT effectively improves proteinuria in DKD patients and ameliorates HG-induced EMT in HK-2 cells. The underlying mechanism may involve the upregulation of PTEN and subsequent inhibition of the PI3K/AKT signaling pathway. These findings provide new insights into the therapeutic potential of CRT for DKD treatment.

Cordyceps cicadae polysaccharides attenuate diabetic nephropathy via the miR-30a-3p/TRIM16 axis.

The molecular mechanism of the protective effect of Cordyceps cicadae polysaccharides (CCPs) on renal tubulointerstitial fibrosis in diabetic nephropathy (DN) is still unclear. This study aims to further understand the molecular mechanisms behind the therapeutic benefits of CCP on diabetic nephropathy. Mice were randomly assigned into six groups (n = 8). Cordyceps cicadae polysaccharide dissolved in 5% dimethyl sulfoxide was administered by gavage for 12 consecutive weeks. The CCP doses were divided into low, medium, and high, 75, 150, and 300 mg/kg/day, respectively. The efficacy of CCP was determined by assessing the renal function and histological alterations in diabetic db/db mice. The degree of glomerular mesangial dilatation and sclerosis was evaluated using semiquantitative markers. Cell viability, apoptosis, epithelial-mesenchymal transition (EMT), inflammation, oxidative stress, and mitochondrial reactive oxygen species (ROS) in high glucose (HG)-cultured MPC5 podocytes were determined. The interaction of miR-30a-3p and tripartite motif-containing protein 16 (TRIM16) was examined by luciferase reporter assay. Western blotting, reverse transcription-polymerase chain reaction, and immunofluorescence were used to analyze gene and protein expressions. The in vivo findings illustrated that CCP may protect mice with type 2 diabetes from inflammation and oxidative damage (P < 0.05). Furthermore, CCP has a therapeutic value in protecting renal function and morphology in diabetic nephropathy by reversing podocyte EMT. The in vitro results indicated that CCP dose-dependently inhibited HG-induced apoptosis, EMT, inflammation, oxidative stress, and mitochondrial ROS levels in MPC5 podocytes (P < 0.05). Luciferase reporter assay confirmed the interaction between miR-30a-3p and TRIM16 in MPC5 podocytes cultured in high glucose (P < 0.05). The protective effect of CCP on HG-induced MPC5 can be achieved by miR-30a-3p/TRIM16 axis.

The antioxidative effects of empagliflozin on high glucose‑induced epithelial-mesenchymal transition in peritoneal mesothelial cells via the Nrf2/HO-1 signaling

High glucose (HG)-induced epithelial-mesenchymal transition (EMT) and oxidative stress play an important role in peritoneal fibrosis, which could be regulated by the nuclear factor erythroid-2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway. This study aimed to investigate whether empagliflozin could inhibit HG-induced EMT and oxidative stress via activating the Nrf2/HO-1 signaling pathway. We used HG-based peritoneal dialysis (PD) solution in rats and HG in human peritoneal mesothelial cells (HPMCs) to induce EMT in vivo and in vitro respectively. The peritoneal structure and function were evaluated by hematoxylin and eosin, Masson’s trichrome staining, and the peritoneal equilibrium test. Oxidative stress was measured by assay kits. EMT was analyzed using immunohistochemistry and western blot. The PD rats showed decreased ultrafiltration capacity and increased levels of oxidative stress. Histopathological analysis revealed markedly peritoneal thickening, excessive collagen deposition, increased expression of α-SMA, Collagen-I, and Fibronectin, and decreased expression of E‑cadherin. Empagliflozin significantly ameliorated the aforementioned changes. The protein expression levels of nuclear Nrf2 (N-Nrf2) and HO-1 increased in PD rats, which were further promoted by treatment with empagliflozin. In in vitro experiments, the EMT of HPMCs was induced with 60 mM glucose for 24 h and inhibited by empagliflozin. Empagliflozin suppressed oxidative stress and promoted the protein expression of N-Nrf2 and HO-1 in HG‑stimulated HPMCs, which was reversed by the Nrf2 inhibitor. In conclusion, empagliflozin exerted a protective effect against HG-induced EMT and suppressed oxidative stress in PMCs by activating the Nrf2/HO-1 signaling pathway.

MDM2-mediated ubiquitination of LKB1 contributes to the development of diabetic cataract

Diabetic cataract (DC) is a common complication of diabetes mellitus. The epithelial-mesenchymal transition (EMT) of lens epithelial cells (LECs) is a crucial event in the development of DC. Murine double minute 2 (MDM2) is an E3 ubiquitin ligase that promotes EMT by regulating diverse targets. However, little is known about how MDM2 is involved in the pathogenesis of DC. We found the mRNA and protein levels of MDM2 were up-regulated in the lens of DC patients and rats. Thus, high glucose (HG)-induced human lens epithelial cells (HLECs) were constructed for further investigation. The results showed that the level of MDM2 was increased in HG-cultured HLECs, and the MDM2 knockdown alleviated HG-induced abnormal migration, EMT, and oxidative stress damage. Moreover, co-immunoprecipitation and ubiquitination assays demonstrated that MDM2 down-regulated LKB1 expression by ubiquitination degradation. LKB1 was found to be lower expressed in human and rat DC lenses, and HG-stimulated HLECs. Also, LKB1 overexpression mitigated HG-induced dysfunction of HLECs. Finally, our data showed that the changes related to EMT and oxidative stress induced by MDM2 knockdown were restored by down-regulation of LKB1. Together, MDM2 may involve in the pathogenesis of DC through down-regulating LKB1. MDM2 might be an effective therapeutical target of DC.

MiR-195 inhibits the ubiquitination and degradation of YY1 by Smurf2, and induces EMT and cell permeability of retinal pigment epithelial cells

The dysregulated microRNAs (miRNAs) are involved in diabetic retinopathy progression. Epithelial mesenchymal transition (EMT) and cell permeability are important events in diabetic retinopathy. However, the function and mechanism of miR-195 in EMT and cell permeability in diabetic retinopathy remain largely unclear. Diabetic retinopathy models were established using streptozotocin (STZ)-induced diabetic mice and high glucose (HG)-stimulated ARPE-19 cells. Retina injury was investigated by hematoxylin–eosin (HE) staining. EMT and cell permeability were analyzed by western blotting, immunofluorescence, wound healing, and FITC-dextran assays. MiR-195 expression was detected via qRT-PCR. YY1, VEGFA, Snail1, and Smurf2 levels were detected via western blotting. The interaction relationship was analyzed via ChIP, Co-IP, or dual-luciferase reporter assay. The retina injury, EMT, and cell permeability were induced in STZ-induced diabetic mice. HG induced EMT and cell permeability in ARPE-19 cells. MiR-195, YY1, VEGFA, and Snail1 levels were enhanced, but Smurf2 abundance was reduced in STZ-induced diabetic mice and HG-stimulated ARPE-19 cells. VEGFA knockdown decreased Snail1 expression and attenuated HG-induced EMT and cell permeability. YY1 silence reduced VEGFA and Snail1 expression, and mitigated HG-induced EMT and cell permeability. YY1 could bind with VEGFA and Snail1, and it was degraded via Smurf2-mediated ubiquitination. MiR-195 knockdown upregulated Smurf2 to decrease YY1 expression and inhibited HG-induced EMT and cell permeability. MiR-195 targeted Smurf2, increased expression of YY1, VEGFA, and Snail1, and promoted HG-induced EMT and cell permeability. MiR-195 promotes EMT and cell permeability of HG-stimulated ARPE-19 cells by increasing VEGFA/Snail1 via inhibiting the Smurf2-mediated ubiquitination of YY1.

Knockdown of the Long Noncoding RNA LUCAT1 Inhibits High-Glucose-Induced Epithelial-Mesenchymal Transition through the miR-199a-5p-ZEB1 Axis in Human Renal Tubular Epithelial Cells.

Renal fibrosis, the leading cause of end-stage renal disease and in which epithelial-mesenchymal transition (EMT) plays a central role, has a complex pathogenesis that is not fully understood. Therefore, we investigated the role of the long noncoding RNA LUCAT1 in the EMT of renal tubular epithelial cells under high-glucose (HG) conditions and the underlying mechanism involved. In this study, we established HG and normal glucose groups of HK-2 cells by treating HK-2 cells 30.0 or 5.5 mmol/L glucose, respectively. To investigate the roles of LUCAT1 and miR-199a-5p in HG-induced EMT, we transfected the HG group with negative control small interfering RNA (siRNA), siRNA targeting LUCAT1, negative control microRNA, or an miR-199a-5p mimic. The results of the quantitative reverse transcription PCR indicated that the LUCAT1 level in the HG group was increased, whereas the miR-199a-5p level was decreased. The EMT in the cells was induced by treatment with HG but was weakened by LUCAT1 knockdown or miR-199a-5p overexpression, which both also inhibited the HG-induced phosphorylation of SMAD3. Moreover, LUCAT1 and ZEB1 mRNA comprised the same microRNA response elements of miR-199a-5p. LUCAT1 knockdown had no effect on the miR-199a-5p level but decreased the HG-induced upregulation of ZEB1. In conclusion, HG conditions induced the upregulation of LUCAT1, and LUCAT1 knockdown inhibited the EMT in HG-treated HK-2 cells. LUCAT1 likely promotes HG-induced EMT through ZEB1 by sponging miR-199a-5p.

MiR-30c-5p inhibits high glucose-induced EMT and renal fibrogenesis by down-regulation of JAK1 in diabetic nephropathy.

Diabetic nephropathy (DN) is one of the most serious complications of diabetes mellitus (DM) and has become the major cause of end-stage renal failure. MicroRNAs (miRs) play key roles in many pathologic processes for initiating and progressing, including DN. Epithelial-mesenchymal transition (EMT) and renal fibrogenesis are important features of DN. However, the role of miR-30c-5p in high glucose (HG)-induced EMT and renal fibrogenesis is not clear. This study was aimed at determining the regulatory network of miR-30c-5p and JAK1 in DN. Quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot assays were performed to detect expressions of miR-30c-5p, JAK1, vimentin, α-SMA, and E-cadherin. The possible binding sites between miR-30c-5p and JAK1 were predicted by TargetScan online database and verified by Luciferase report assay. The secretion of fibronectin (FN) andCollagen IV (Col IV) in the supernatant was detected by Enzyme-linked immunosorbent (ELISA) assay. MiR-30c-5p was downregulated and JAK1 was upregulated in renal fibrosis tissue and HG stimulated HK2 cells. Transfection of miR-30c-5p inhibited HG-induced EMT and renal fibrogenesis in HK2 cells, which was reversed by miR-30c-5p inhibitor. Moreover, JAK1 was confirmed as a direct target of miR-30c-5 and knockdown of JAK1 markedly inhibited HG-induced renal fibrogenesis and EMT in HK2 cells. Furthermore, overexpression of JAK1 attenuated the inhibitory effect of miR-30c-5p on HG-induced EMT and renal fibrogenesis in HK2 cells. MiR-30c-5p evidently inhibited HG-induced EMT and renal fibrogenesis by down-regulation JAK1 in DN, providing a promising therapeutic strategy for the treatment of DN.

Inactivation of TSC1 promotes epithelial-mesenchymal transition of renal tubular epithelial cells in mouse diabetic nephropathy.

Epithelial–mesenchymal transition (EMT) of renal tubular epithelial cells is one of the potential mechanisms of renal fibrosis, which promotes the development of diabetic nephropathy (DN). However, the molecular mechanisms of EMT remain largely unknown. Tuberous sclerosis proteins TSC1 and TSC2 are key integrators of growth factor signaling, and the loss of TSC1 or TSC2 function leads to a spectrum of diseases that underlie abnormalities in cell growth, proliferation, differentiation, and migration. In this study, we investigated the effects of TSC1 on high glucose (HG)-induced EMT of human proximal tubular epithelial HK-2 cells in vitro and renal fibrosis in TSC1−/− and db/db mice. We found that the exposure of HK-2 cells to HG (30 mM) time-dependently decreased TSC1 expression, increased the phosphorylation of mTORC1, P70S6K, and 4E-BP-1, and promoted cell migration, resulting in EMT. Transfection of the cells with TSC1 mimic significantly ameliorated HG-induced EMT of HK-2 cells. The tubules-specific TSC1 knockout mice (TSC1−/−) displayed a significant decline in renal function. TSC1−/− mice, similar to db/db mice, showed greatly activated mTORC1 signaling and EMT process in the renal cortex and exacerbated renal fibrosis. Overexpression of TSC1 through LV-TSC1 transfection significantly alleviated the progression of EMT and renal fibrosis in the renal cortex of db/db mice. Taken together, our results suggest that TSC1 plays a key role in mediating HG-induced EMT, and inhibition of TSC1-regulated mTORC1 signaling may be a potential approach to prevent renal fibrosis in DN.

LncRNA MALAT1 facilities high glucose induced endothelial to mesenchymal transition and fibrosis via targeting miR-145/ZEB2 axis.

Diabetic nephropathy (DN) is one of the most common complications of diabetes mellitus (DM), but the pathophysiology of DN is complex and not fully understood. Renal tubal epithelial-mesenchymal transition (EMT) has been shown to be the critical mechanism of glomerulosclerosis and tubulointerstitial fibrosis. However, the precise mechanisms underlying EMT are not clear. MALAT1 was found induced by hyperglycemia in kidney but whether MALAT1 is involved in renal tubal EMT remains unknown. The objective of our study is to explore the role of MALAT1 in hyperglycemia-induced EMT and fibrosis. We used db/db mouse and high glucose (HG)-stimulated HK-2 cells as in vivo and in vitro model of DN, respectively. qRT-PCR was used to measure levels of MALAT1 and miR-145. In addition, we validated interactions of MALAT1-miR-145 and miR-145-ZEB2 by dual luciferase reporter assays. Western blot was used to examine expressions of proteins involved in EMT and fibrosis. MALAT1 was upregulated while miR-145 was downregulated in renal tissues of db/db mice. Consistently, hyperglycemia significantly increased the level of MALAT1 but decreased miR-145 expression in a time-dependent manner in HK-2 cells. Furthermore, miR-145 binds to both MALAT1 and ZEB2. Knockdown MALAT1 or ZEB2 inhibited HG-induced EMT and fibrosis, similar to miR-145 overexpression. Our study is the first to show that MALAT1 and miR-145 regulate HG-induced EMT and fibrosis. Mechanistically, MALAT1 functions as a sponge RNA for miR-145 to derepress the expression of target gene ZEB2, thereby inducing EMT and fibrosis. These results provide a novel potential target for DN therapy in the future.

Triptolide Attenuates Renal Tubular Epithelial-mesenchymal Transition Via the MiR-188-5p-mediated PI3K/AKT Pathway in Diabetic Kidney Disease.

Triptolide possesses the trait of renal protection. Epithelial-mesenchymal transition (EMT) is closely linked to the pathogenesis of diabetic kidney disease (DKD). MicroRNAs have recently emerged as critical regulators of DKD. However, it is poorly understood whether triptolide alleviates renal EMT by regulating microRNAs in DKD. In this study, we found that triptolide decreased albuminuria, improved the renal structure and reduced renal EMT in rats with DKD. Furthermore, activation of the PI3K/AKT signaling pathway was increased in diabetic rats, which was partly reversed by triptolide. Triptolide also alleviated glucose-induced EMT in HK-2 cells in vitro. PI3K/AKT signaling pathway activation was reduced after triptolide treatment. Moreover, triptolide decreased the increase in miR-188-5p expression stimulated by high glucose levels in HK-2 cells. miR-188-5p inhibited PTEN expression by directly interacting with the PTEN 3'-untranslated region. Additionally, downregulation of miR-188-5p, which imitates the effects of triptolide, attenuated the activation of the PI3K/AKT pathway and HG-induced EMT, whereas miR-188-5p overexpression reversed the effects of triptolide on the PI3K/AKT pathway and EMT. In conclusion, we demonstrated that triptolide ameliorates renal EMT via the PI3K/AKT signaling pathway through the interaction between miR-188-5p and PTEN, indicating that miR-188-5p may be a therapeutic target of triptolide in DKD.

MiRNA-23 regulates high glucose induced epithelial to mesenchymal transition in human mesotheial peritoneal cells by targeting VDR

Epithelial–mesenchymal transition(EMT) is the main reason for peritoneal fibrosis and the mechanism underlying peritoneal EMT were extensively studied in recent years. Recent researches showed that miRNAs were so important in the development of organ EMT and fibrosis, the role of microRNAs on peritoneal dialysis have also been studied. In the current study, we investigated microRNA-23(miR-23) expression in high glucose(HG) induced EMT in human mesotheial peritoneal cells(HPMCs). We found that HG promoted EMT, which was characterized by the upregulation of mesenchymal markers α-SMA and FN and downregulation of epithelial marker E-cadherin. The expression miR-23 showed a significant upregulation when treated with HG. Enhanced expression of miR-23 could aggravate HG induced EMT by targeting VDR, inhibition of miR-23 in HPMCs could reverse HG induced EMT by targeting VDR. Furthermore, VDRshRNA exacerbated the EMT process and reversed miR-23 inhibitor-attenuated EMT process in HPMCs. These data manifested that miR-23 played a key role in HG-induced EMT of HPMCs by targeting VDR.

Rapamycin inhibits epithelial-to-mesenchymal transition of peritoneal mesothelium cells through regulation of Rho GTPases.

Epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) is a key process of peritoneal fibrosis. Rapamycin has been previously shown to inhibit EMT of PMCs and prevent peritoneal fibrosis. In this study, we investigated the undefined molecular mechanisms by which rapamycin inhibits EMT of PMCs. To define the protective effect of rapamycin, we initially used a rat PD model which was daily infused with 20 mL of 4.25% high glucose (HG) dialysis solution for 6 weeks to induce fibrosis. The HG rats showed decreased ultrafiltration volume and obvious fibroproliferative response, with markedly increased peritoneal thickness and higher expression of α-smooth muscle actin (α-SMA) and transforming growth factor-β1. Rapamycin significantly ameliorated those pathological changes. Next, we treated rat PMCs with HG to induce EMT and/or rapamycin for indicated time. Rapamycin significantly inhibited HG-induced EMT, which manifests as increased expression of α-SMA, fibronectin, and collagen I, decreased expression of E-cadherin, and increased mobility. HG increased the phosphorylation of PI3K, Akt, and mTOR. Importantly, rapamycin inhibits the RhoA, Rac1, and Cdc42 activated by HG. Moreover, rapamycin repaired the pattern of F-actin distribution induced by HG, reducing the formation of stress fiber, focal adhesion, lamellipodia, and filopodia. Thus, rapamycin shows an obvious protective effect on HG-induced EMT, by inhibiting the activation of Rho GTPases (RhoA, Rac1, and Cdc42).

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 invitro and under diabetic conditions invivo. 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.

Silencing of angiotensin II type-1 receptor inhibits high glucose-induced epithelial–mesenchymal transition in human renal proximal tubular epithelial cells via inactivation of mTOR/p70S6K signaling pathway

The epithelial–mesenchymal transition (EMT) plays a significant role in renal tubulointerstitial fibrosis (TIF), which is one of hallmark pathological feature of diabetic nephropathy (DN). Angiotensin II via its type-1 receptor AT1R is involved in the development of TIF. The purpose of our study was aimed to investigate the effect of silencing of AT1R on EMT and elucidate the possible mechanism underling these effects. EMT was induced by high glucose (HG) in human proximal tubular epithelial cell line HK-2 cells. The mRNA levels of AT1R were determined. The expression of AT1R was silenced by small interfering RNA (siRNA) technology and confirmed by quantitative real time PCR (qRT-PCR). After transfection with siAT1R, cell viability and expression levels of epithelial cell marker (epithelial (E)-cadherin), mesenchymal cell marker (alpha-smooth muscle actin (α-SMA)), four transcriptional factors (snail, slug, twist, and ZEB-1) were determined, as well as the roles of mechanistic target of rapamycin (mTOR)/p70S6K signaling pathway. The levels of AT1R were significantly higher after exposure to HG (P < 0.05). Transfection with siAT1R had no effect on cell viability, but reversed HG-induced EMT by up-regulation of E-cadherin expression and decrease of α-SMA, snail, and twist levels. MTOR/p70S6K signaling pathway was highly activated in HK-2 cells cultured under HG, but was inhibited by transfection with siAT1R. Our results suggest that silencing of AT1R inhibits EMT induced by HG in HK-2 cells via inactivation of mTOR/p70S6K signaling pathway. Silencing of AT1R might be a new strategy to treat DN.

Smad Anchor for Receptor Activation Regulates High Glucose-Induced EMT via Modulation of Smad2 and Smad3 Activities in Renal Tubular Epithelial Cells

Background: SARA is an essential adaptor protein in TGF-β1 signaling and it is also involved in the process of Epithelial-mesenchymal transition (EMT) and fibrosis. Our aim was to investigate the effect of SARA on high glucose (HG)-induced EMT and extracellular matrix (ECM) synthesis in renal tubular epithelial cells. Methods: The cells were transfected with the following plasmids: wild-type SARA (SARA-WT), SARA-dSBD (SARA with Smad binding domain deletion) and then subjected to HG ambience (30 m<smlcap>M</smlcap>). The expression levels were assessed by Western-blot, real-time PCR and immunofluorescence. Results: HG-induced EMT phenotype with increased expression of ECM protein in HK-2 cells. This was associated with the decreased expression of SARA and Smad2. In comparison with the HG group, overexpression of SARA in HK-2 cells, a relatively high upregulation of ZO-1 expression was seen, while that of Vimentin, fibronectin and collagen I was decreased. The Smad2 protein expression was increased in HK-2 cells after transfection with SARA (WT) plasmid. Interestingly, the overexpression of SARA prolonged the activity period of Smad2 and shortened that of Smad3, which seemed consistent with the change of EMT phenotype and ECM changes in HK-2 cell induced by HG. Conclusions: SARA regulates HG-induced EMT and ECM excretion via modulation of the activation of Smad2 and Smad3 in renal tubular epithelial cells. In view of these findings, it is conceivable that SARA may serve as a potential novel target in pre-EMT states for the amelioration renal fibrosis seen in chronic kidney diseases.

Down-regulated miR-15a mediates the epithelial-mesenchymal transition in renal tubular epithelial cells promoted by high glucose.

High glucose (HG) has been reported to be associated with renal dysfunction. And one potential mechanism underlining the dysfunction is the epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells. Present study showed that EMT was induced in the HG-treated renal tubular epithelial cells by promoting the expression of mesenchymal phenotype molecules, such as α-SMA and collagen I, and down-regulating the expression of epithelial phenotype molecule E-cadherin. Moreover, we have identified the down-regulation of miR-15a which was accompanied with the HG-induced EMT. And the miR-15a overexpression inhibited the α-SMA, collagen I expression, and the promotion of E-cadherin expression by targeting and down-regulating AP4 which was also significantly promoted by the HG in the renal tubular epithelial cells. Thus, this study revealed that the weakening regulation on the AP4 expression by miR-15a might contribute to the HG-induced EMT in the renal tubular epithelial cells.

GSK-3β and Vitamin D Receptor are Involved in β-Catenin and Snail Signaling in High Glucose-Induced Epithelial-Mesenchymal Transition of Mouse Podocytes

Background: Epithelial-mesenchymal transition (EMT) is recognized to play an important role in diabetic nephropathy (DN). Objective: To analyze the roles of glycogen synthase kinase 3β (GSK-3β), β-catenin and Snail signaling in high glucose (HG)-induced mouse podocytes EMT. Methods: Differentiated podocytes were divided into: the normal glucose group (NG: glucose 5.6mM), the HG groups (12.5HG: 12.5mM; 25HG: 25mM; and 50HG: 50mM of glucose), and the osmotic control group (NG+M: glucose 5.6mM and mannitol 44.4mM). GSK-3β, β-catenin and Snail were assessed using semi-quantitative RT-PCR, western blot and immunofluorescence. β-catenin and Snail pathways were assessed after down-regulating GSK-3β expression using an inhibitor (LiCl) or a small-interfering RNA (siRNA). Results: HG increased GSK-3β, β-catenin and Snail expressions, and promoted EMT, as shown by decreased nephrin expression (epithelial marker), and increased α-SMA expression (mesenchymal marker). GSK-3β inhibitor and GSK-3β siRNA decreased β-catenin and Snail expressions, and reversed HG-induced EMT. Immunofluorescence showed that GSK-3β and β-catenin did not completely overlap; β-catenin was transferred to the nucleus in the 25HG group. VDR seems to be involved in HG-induced β-catenin nuclear translocation. Conclusion: Down-regulating GSK-3β expression decreased β-catenin and Snail expression and reversed HG-induced podocytes EMT. Thus, modulating GSK-3β might be a target to slow or prevent DN.

Troglitazone ameliorates high glucose-induced EMT and dysfunction of SGLTs through PI3K/Akt, GSK-3β, Snail1, and β-catenin in renal proximal tubule cells

Peroxisome proliferator-activated receptor-γ (PPARγ) agonists ameliorate renal fibrotic lesions in diabetic nephropathy. However, the effects of the agonists on the epithelial-mesenchymal transition (EMT) linked to membrane transport dysfunction are unknown. The present study aimed to verify the effects of the PPARγ agonist troglitazone on high glucose (HG)-induced EMT in primary cultured renal proximal tubular epithelial cells (PTCs). HG (25 mM) as well as hydrogen peroxide (H(2)O(2)) and transforming growth factor-β1 (TGF-β1) decreased expression of epithelial cell marker E-cadherin and increased the expression of the mesenchymal markers vimentin and α-smooth muscle actin (α-SMA). HG, H(2)O(2), and TGF-β1 decreased Na(+)/H(+) exchangers (NHEs) or Na(+)-glucose cotransporters (SGLTs) and glucose uptake, showing membrane transport dysfunction. HG stimulated the production of cellular reactive oxygen species (ROS), and antioxidants blocked the HG-induced increase in phosphatidylinositol 3-kinase (PI3K)/Akt activation. Antioxidants and inhibitors of PI3K/Akt reversed HG-induced EMT protein expression. Inhibition of PI3K/Akt also blocked HG-induced glycogen synthase kinase-3β (GSK-3β) phosphorylation. HG and lithium chloride (GSK-3β inhibitor) blocked Snail1 and β-catenin activation. Moreover, transfection with Snail1 or β-catenin small interfering RNA (siRNA) reversed HG-induced EMT protein expression. Importantly, HG decreased PPARγ activation and troglitazone reversed HG-induced expression of PI3K/Akt, GSK-3β, Snail1, and β-catenin as well as EMT proteins. Finally, inhibitors of PI3K/Akt, Snail1/β-catenin siRNA, and troglitazone blocking the HG-induced EMT restored glucose uptake in PTCs. In conclusion, HG induces EMT through ROS, PI3K/Akt, GSK-3β, Snail, and β-catenin. Subsequently, HG-induced EMT may result in SGLT dysfunction that is restored by the PPARγ agonist troglitazone in primary cultured PTCs.