Epithelial-to-mesenchymal Transition In Renal Tubular Cells Research Articles

#2222 Suppression of miR-199a-3p ameliorates TGF-β-induced EMT in renal tubular cells

Abstract Background and Aims Various kidney conditions, such as acute kidney injury and fibrosis, cause dysregulation of miRNAs. TGF-β is one of the most potent inducer of epithelial-to-mesenchymal transition (EMT) and fibrosis. We recently reported increased miR-199 expression in TGF-β-induced kidney fibrosis mouse model (doi: 10.3390/ijms242115520). However, the exact role of miR-199 in fibrosis and EMT development is still unknown. Method Primary tubular cells were isolated from a 4-week-old C57BL6 mouse and kept in DMEM/F12 medium containing 2% serum, supplemented with insulin/transferrin/selenium. Cells were treated for 24 hours with 30 nM miR-199-3p miRNA inhibitor (anti-miR), with or without TGF-β (10 ng/ml). The expression of profibrotic genes, as well as miR-199a-3p, was assessed by qPCR. Western blot was used to evaluate the protein expression. Results are presented as mean fold expression relative to calibrator. Statistical significance was assessed using the Kruskal-Wallis test, and the level of significance was set to p < 0.05. Results The miR-199a-3p expression in the anti-miR transfected control and TGF-β groups decreased by 93.5% and 85.5%, respectively, as compared to non-transfected groups (p < 0.05). Morphologically, cells transfected with miR-199a-3p and TGF-β1 showed no difference from cells exposed to only TGF-β1 but appeared more elongated compared to the control groups. However, the suppression of miR-199a-3p alleviated TGF-β1-induced fibrotic cell responses as shown by downregulated fibronectin mRNA (Fn1) and protein expression by 30.2% (p < 0.05) and by 4.3%, respectively. This was accompanied by reduced Acta2 (α-SMA) and Tgfb1 mRNA expressions by 30.2% and 17.5%, respectively (p < 0.05). Conclusion Our findings suggest that in murine renal tubular epithelial cells, miR-199a-3p suppresses TGF-β1-induced EMT by inhibiting FN1, Acta2, and Tgfb1 both at mRNA and protein levels. Further studies are needed to elucidate the underlying molecular interactions.

P0028THE ACTIVATION OF P75NTR RECEPTOR SIGNALING PROMOTES EMT IN ADPKD CELL LINE WT9-12

Abstract Background and Aims ADPKD cystic epithelial cells have been shown to display an undifferentiated phenotype and to undergo epithelial to mesenchymal transition (EMT) changes as part of their phenotype. Thus, in ADPKD kidneys there is upregulation of EMT-related genes and increased fibrosis that support the proliferation of the cyst-lining cells and the progression of ADPKD. In our previous study, we provided that TGFβ1-induced p75NTR receptor activation promotes EMT in renal tubular cells, HK2. p75NTR is the common receptor for all the neurotrophins and their precursors. Since its identification and cloning in the 1980s, p75NTR has been reported to be involved in different and often contradictory functions such as survival, apoptosis, differentiation, proliferation and migration, depending on the cell type and cellular context. Furthermore, p75NTR is known to be overexpressed in some human cancer, where it participates in the stimulation of tumor growth and dissemination. p75NTR, through a neurotrophin-dependent mechanism, dramatically enhances migration and invasion of melanoma and glioma cells. Therefore, starting from these pieces of evidence and since WT9-12 cells exhibit higher constitutive expression of p75NTR and TGFβ1 than that of HK-2, the aim of this study is to carry out in vitro studies to investigate whether p75NTR is involved in EMT of cyst-lining cells and in renal fibrosis in ADPKD. Method Immortalized tubular renal cells homozygous for a truncating PKD1 mutation (WT9-12); Western Blot (WB); Real-Time PCR; Scratch and Trans-well migration assays; Immunofluorescence (IF); RNA interference (RNAi); Transfection assay. Results WB and IF assays showed that in WT9-12 cells TGFβ1 (5ng/ml) increased protein expression of p75NTR and of mesenchymal markers Fibronectin, N-Cadherin, α-SMA, Vimentin while reduced the expression of the epithelial marker E-Cadherin. This biological effect is significantly reversed in cells transfected with RNAi-p75NTR (Figure 1). Functional studies revealed that increased p75NTR protein expression was given to greater p75NTR mRNA stability. Time course assays, performed in cells transfected with RNAi-p75NTR and TGFβ1-treated, demonstrated downregulation of pERK1/2 and pJNK, p75NTR downstream pathways and involved in the non-canonical TGFβ1 pathway promoting pro-fibrotic genes transcription Figure 2A). Results of the analysis of SMAD pathway showed that SMAD3 was not significantly activated in cells untreated with TGFβ1. Moreover, selective ERK1/2 inhibition with PD98059 (10mM) reduced significantly the levels of mesenchymal markers N-Cadherin and α-SMA respect to the controls of untreated cells. Interestingly, in TGFβ1-treated cells in the presence of PD98059 the reduction of mesenchymal markers expression was minor respect to TGFβ1-untreated cells. These findings emphasize the importance of the ERK1/2 pathway in ADPKD. Our results revealed that the ERK1/2 pathway also plays a pivotal role in non-SMAD pathways downstream of TGFβ1 in fibrotic processes in ADPKD. Finally, Scratch and Trans-well migration assays showed a reduced cell motility in p75NTR-knocked-down cells (Figure 2B). Conclusion Although more basic studies are clearly needed to understand the mechanism of action of p75NTR, our results emphasized the crucial role of p75NTR in EMT changes in ADPKD cells which may contribute to the cystic phenotype and to fibrotic process.

ETS2 promotes epithelial-to-mesenchymal transition in renal fibrosis by targeting JUNB transcription

Epithelial-to-mesenchymal transition (EMT) plays an important role in the progression of renal tubulointerstitial fibrosis, a common mechanism leading to end-stage renal failure. V-ets erythroblastosis virus E26 oncogene homolog 2 (ETS2), a transcription factor, exhibits diverse roles in pathogenesis; however, its role in renal fibrosis is not yet fully understood. In this study, we detected the expression of ETS2 in an animal model of renal fibrosis and evaluated the potential role of ETS2 in tubular EMT induced by TGF-β1. We found that ETS2 and profibrogenic factors, alpha-smooth muscle actin (α-SMA) and fibronectin (FN), were significantly increased in the unilateral ureteral obstruction (UUO)-induced renal fibrosis model in mice. In vitro, TGF-β1 induced a high expression of ETS2 dependent on Smad3 and ERK signaling pathway in human proximal tubular epithelial cells (HK2). Knockdown of ETS2 abrogated TGF-β1-mediated expression of profibrogenic factors vimentin, α-SMA, collagen I, and FN in HK2 cells. Mechanistically, ETS2 promoted JUNB expression in HK2 cells after TGF-β1 stimulation. Furthermore, luciferase and Chromatin Immunoprecipitation (ChIP) assays revealed that the binding of ETS2 to three EBS motifs on the promoter of JUNB triggered its transcription. Notably, silencing JUNB reversed the ETS2-induced upregulation of the profibrogenic factors in HK2 cells after TGF-β1 stimulation. These findings suggest that ETS2 mediates TGF-β1-induced EMT in renal tubular cells through JUNB, a novel pathway for preventing renal fibrosis.

De-Glycyrrhizinated Licorice Extract Attenuates High Glucose-Stimulated Renal Tubular Epithelial-Mesenchymal Transition via Suppressing the Notch2 Signaling Pathway.

Tubulointerstitial fibrosis is a major pathological hallmark of diabetic nephropathy. Increasing evidence has shown that epithelial-to-mesenchymal transition (EMT) of renal proximal tubular cells plays a crucial role in tubulointerstitial fibrosis. Herein, we aimed to elucidate the detailed mechanism of EMT in renal tubular cells under high glucose (HG) conditions, and to investigate the potential of licorice, a medicinal herb, to inhibit HG-induced EMT. Our results showed that renal tubular epithelial cells (normal rat kidney cell clone 52E; NRK-52E) exposed to HG resulted in EMT induction characterized by increased fibronectin and α-SMA (alpha-smooth muscle actin) but decreased E-cadherin. Elevated levels of cleaved Notch2, MAML-1 (mastermind-like transcriptional coactivator 1), nicastrin, Jagged-1 and Delta-like 1 were also concomitantly detected in HG-cultured cells. Importantly, pharmacological inhibition, small interfering RNA (siRNA)-mediated depletion or overexpression of the key components of Notch2 signaling in NRK-52E cells supported that the activated Notch2 pathway is essential for tubular EMT. Moreover, we found that licorice extract (LE) with or without glycyrrhizin, one of bioactive components in licorice, effectively blocked HG-triggered EMT in NRK-52E cells, mainly through suppressing the Notch2 pathway. Our findings therefore suggest that Notch2-mediated renal tubular EMT could be a therapeutic target in diabetic nephropathy, and both LE and de-glycyrrhizinated LE could have therapeutic potential to attenuate renal tubular EMT and fibrosis.

Zinc Attenuates Tubulointerstitial Fibrosis in Diabetic Nephropathy Via Inhibition of HIF Through PI-3K Signaling.

Evidence has demonstrated that hypoxia may have a central pathogenic mechanism in the development of diabetic nephropathy (DN). Epithelial-to-mesenchymal transition (EMT) of mature tubular epithelial cells in kidney is a contributor to the renal accumulation of matrix protein in DN and is highly associated with the progression of tubulointerstitial fibrosis. Zinc (Zn) has anti-fibrosis effects in liver and lungs. In the present study, we aimed to investigate the effect of Zn on renal tubulointerstitial fibrosis especially under hypoxic conditions and its association with DN. We found that Zn treatment blockaded tubular EMT and attenuated renal tubulointerstitial fibrosis by downregulation of hypoxia-inducible factor alpha (HIF-1α) in the kidneys of diabetic streptozotocin-treated mice. High glucose (HG)/hypoxic conditions stimulated EMT in renal tubular cells as indicated by the significant decrease in epithelial marker E-cadherin and ZO-1 while the increase in mesenchymal markers α-smooth muscle actin (α-SMA). Zn supplement mainly prevented HG/hypoxic-induced HIF-1α accumulation and EMT marker changes. In co-treatment Zn with PI3K/Akt/GSK-3β signaling pathway, inhibitor LY294002 prevented HG/hypoxic-induced HIF-1α increase and EMT changes, suggesting that Zn may mediate HG/hypoxic-induced EMT through PI3K/Akt/GSK-3β pathway. Therefore, we concluded that Zn had an important anti-fibrosis role under HG/hypoxic conditions, and a novel mechanism contributing to Zn protection on renal tubular epithelial cells from HG/hypoxia-induced EMT through activation of PI3K/Akt/GSK-3β signaling pathway, which subsequently leads to the downregulation of the expression of HIF-1α.

Resveratrol prevents high glucose-induced epithelial–mesenchymal transition in renal tubular epithelial cells by inhibiting NADPH oxidase/ROS/ERK pathway

Resveratrol (RSV) is reported to have renoprotective activity against diabetic nephropathy, while the mechanisms underlying its function have not been fully elucidated. In this study, we investigate the effect and related mechanism of RSV against high glucose-induced epithelial to mesenchymal transition (EMT) in human tubular epithelial cells (HK-2). A typical EMT is induced by high glucose in HK-2 cells, accompanied by increased levels of reactive oxygen species (ROS). RSV exhibits a strong ability to inhibit high glucose-induced EMT by decreasing intracellular ROS levels via down-regulation of NADPH oxidase subunits NOX1 and NOX4. The activation of extracellular signal-regulated kinase (ERK1/2) is found to be involved in high glucose-induced EMT in HK-2 cells. RSV, like NADPH oxidase inhibitor diphenyleneiodonium, can block ERK1/2 activation induced by high glucose. Our results demonstrate that RSV is a potent agent against high glucose-induced EMT in renal tubular cells via inhibition of NADPH oxidase/ROS/ERK1/2 pathway.

Augmenter of liver regeneration inhibits TGF-β1-induced renal tubular epithelial-to-mesenchymal transition via suppressing TβR II expression in vitro

Tubular epithelial-to-mesenchymal transition (EMT) plays a crucial role in the progression of renal tubular interstitial fibrosis (TIF), which subsequently leads to chronic kidney disease (CKD) and eventually, end-stage renal disease (ESRD). We propose that augmenter of liver regeneration (ALR), a member of the newly discovered ALR/Erv1 protein family shown to ameliorate hepatic fibrosis, plays a similar protective role in renal tubular cells and has potential as a new treatment option for CKD. Here, we showed that recombinant human ALR (rhALR) inhibits EMT in renal tubular cells by antagonizing activation of the transforming growth factor-β1 (TGF-β1) signaling pathway. Further investigation revealed that rhALR suppresses the expression of TGF-β receptor type II (TβR II) and significantly alleviates TGF-β1-induced phosphorylation of Smad2 and nuclear factor-κB (NF-κB). No apparent adverse effects were observed upon the addition of rhALR alone to cells. These findings collectively suggest that ALR plays a role in inhibiting progression of renal tubular EMT, supporting its potential utility as an effective antifibrotic strategy to reverse TIF in CKD.

Everolimus-induced epithelial to mesenchymal transition in immortalized human renal proximal tubular epithelial cells: key role of heparanase.

BackgroundEverolimus (EVE) is a drug widely used in several renal transplant protocols. Although characterized by a relatively low nephrotoxicity, it may induce several adverse effects including severe fibro-interstitial pneumonitis. The exact molecular/biological mechanism associated to these pro-fibrotic effects is unknown, but epithelial to mesenchymal transition (EMT) may have a central role. Additionally, heparanase, an enzyme recently associated with the progression of chronic allograft nephropathy, could contribute to activate this machinery in renal cells.MethodsSeveral biomolecular strategies (RT-PCR, immunofluorescence, zymography and migration assay) have been used to assess the capability of EVE (10, 100, 200 and 500 nM) to induce an in vitro heparanase-mediated EMT in wild-type (WT) and Heparanase (HPSE)-silenced immortalized human renal epithelial proximal tubular cells (HK-2). Additionally, microarray technology was used to find additional biological elements involved in EVE-induced EMT.ResultsBiomolecular experiments demonstrated a significant up-regulation (more than 1.5 fold increase) of several genes encoding for well known EMT markers [(alpha-smooth muscle actin (α-SMA), Vimentin (VIM), Fibronectin (FN) and matrix metalloproteinase-9 (MMP9)], enhancement of MMP9 protein level and increment of cells motility in WT HK2 cells treated with high concentrations of EVE (higher than 100 nM). Similarly, immunofluorescence analysis showed that 100 nM of EVE increased α-SMA, VIM and FN protein expression in WT HK2 cells. All these effects were absent in both HPSE- and AKT-silenced cell lines. AKT is a protein having a central role in EMT. Additionally, microarray analysis identified other 2 genes significantly up-regulated in 100 nM EVE-treated cells (p < 0.005 and FDR < 5%): transforming growth factor beta-2 (TGFβ2) and epidermal growth factor receptor (EGFR). Real-time PCR analysis validated microarray.ConclusionsOur in vitro study reveals new biological/cellular aspects of the pro-fibrotic activity of EVE and it demonstrates, for the first time, that an heparanase-mediated EMT of renal tubular cells may be activated by high doses of this drug. Additionally, our results suggest that clinicians should administer the adequate dosage of EVE in order to increase efficacy and reduce adverse effects. Finally heparanase could be a new potential therapeutic target useful to prevent/minimize drug-related systemic fibrotic adverse effects.

Uric acid-induced phenotypic transition of renal tubular cells as a novel mechanism of chronic kidney disease

Recent experimental and clinical studies suggest a causal role of uric acid in the development of chronic kidney disease. Most studies have focused on uric acid-induced endothelial dysfunction, oxidative stress, and inflammation in the kidney. The direct effects of uric acid on tubular cells have not been studied in detail, and whether uric acid can mediate phenotypic transition of renal tubular cells such as epithelial-to-mesenchymal transition (EMT) is not known. We therefore investigated whether uric acid could alter E-cadherin expression and EMT in the kidney of hyperuricemic rats and in cultured renal tubular cells (NRK cells). Experimental hyperuricemia was associated with evidence of EMT before the development of significant tubulointerstitial fibrosis at 4 wk, as shown by decreased E-cadherin expression and an increased α-smooth muscle actin (α-SMA). Allopurinol significantly inhibited uric acid-induced changes in E-cadherin and α-SMA with an amelioration of renal fibrosis at 6 wk. In cultured NRK cells, uric acid induced EMT, which was blocked by the organic anion transport inhibitor probenecid. Uric acid increased expression of transcriptional factors associated with decreased synthesis of E-cadherin (Snail and Slug). Uric acid also increased the degradation of E-cadherin via ubiquitination, which is of importance since downregulation of E-cadherin is considered to be a triggering mechanism for EMT. In conclusion, uric acid induces EMT of renal tubular cells decreasing E-cadherin synthesis via an activation of Snail and Slug as well as increasing the degradation of E-cadherin.

Proteinuria and Epithelial-to-Mesenchymal Transition

The extent of injury to the tubulointerstitial compartment has been recognized for several decades as closely linked to declining renal function in glomerular diseases. In addition to secondary inflammatory injury and ischemia as mechanisms of injury in the tubulointerstitium downstream from diseased glomeruli, proteinuria itself is now recognized as a pathogenic factor and an independent risk factor for the progression of kidney disease.1–3 Reduction of proteinuria is now a major therapeutic goal in reducing risk for renal progression.4 Current strategies to reduce proteinuria are largely focused on treatment of the underlying glomerular disease and by alteration of glomerular hemodynamics and filtration. With improved understanding of basic mechanisms of proteinuria-induced injury, however, more refined and proximate strategies based on molecular pathogenesis of proteinuria-induced tubulointerstitial injury may be possible. The damaging effects of proteinuria on the renal tubulointerstitial compartment involve a variety of mechanisms.5 These include tubular cell toxicity, when the lysosomal capacity to degrade proteins reabsorbed in excess by the tubules is overwhelmed, the downstream exposure to chemokines in tubular fluid, or the expression of surface neo-antigens and adhesion molecules with proinflammatory action. In addition, tubular epithelial cells respond to exposure to filtered serum proteins, as to other forms of injury, by undergoing epithelial-to-mesenchymal transition (EMT), with reduction of epithelial phenotype and functions and induction of increased cell motility and matrix production. Transition toward a mesenchymal phenotype has been demonstrated in epithelial and endothelial cells by in vitro as well as in vivo studies and is recognized as an important contributor to the development of fibrosis,6 not only in the kidney but also in lung and liver. The mechanisms involved in proteinuria-induced EMT in renal tubular cells have not been clearly identified.5 As an important component of innate immunity, the complement system is activated in proteinuric states. Tang et al.7 in this issue of JASN examine the clinical and experimental evidence that complement activation plays a role in tubulointerstitial injury and dysfunction during proteinuria. Existing data suggest roles for both the membrane attack complex, C5b-9, and the anaphylotoxin C5a in injury and dysfunction in proteinuric states. There is also recent evidence that C3- or C6-deficient mice have reduced EMT in models of proteinuria. The elegant studies by Tang et al.7 provide a new focus on another complement activation product, the anaphylotoxin C3a, and its role in altering proximal tubular epithelial cell (PTEC) phenotype in vitro and presumptively in vivo. The HKC-8 in vitro cell system is capable of activating complement and undergoing EMT on exposure to serum C3a but not C5b-9. An antagonist of the C3a receptor expressed on the cells inhibits the effect and blocks an increase in the mRNA encoding collagen I. The antagonist also blocks EMT and collagen I–induced by exposure to serum. Extending these observations in vivo in an adriamycin model of proteinuria, C3a receptor null mice develop less albuminuria, lower mortality, and less severe renal failure compared with wild-type mice. C3aR null mice also have less glomerular injury and less severe tubulointerstitial disease, as measured by tubular diameter/cell height ratio and interstitial volume, with less interstitial collagen and fewer myofibroblasts and macrophages. Although in vitro evidence of C3aR-mediated injury induced by proteinuria in human renal proximal tubular cells reported by Tang et al.7 is compelling, it is difficult to define the role of complement and C3a directly at the level of the tubular epithelium in the in vivo studies, because glomerular injury was less and urinary albumin lower in the C3aR null mice, and interstitial macrophage infiltration was reduced as well. The diminished tubulointerstitial injury in C3aR null mice could be related to less glomerular injury and consequent diminution of ischemic injury downstream from sclerosing glomeruli, compared with wild-type mice (which have induction of C3aR in the mesangium as well as the tubular epithelium) and/or could be due to less macrophage infiltration, because macrophages can induce fibrogenesis.8 The role of the complement system in proteinuria, as in other forms of renal injury, is complex and difficult to define precisely in vivo. In vitro studies allow more precision in defining mechanisms but remove the important influences of the in vivo milieu. Regardless, the studies by Tang et al.7 provide convincing evidence of a role for C3a in tubulointerstitial changes with proteinuria, elegantly expanding previous observations of a role for other complement components. These studies provide yet another potential target for therapies that might be capable of abrogating both tubulointerstitial and glomerular injury mediated by complement activation in proteinuric states, with the goal of improving long-term outcome. Disclosures None.