Tubular Epithelial-mesenchymal Transition Research Articles

85 - Nitro-Oleic Acid Inhibits Soluble Epoxide Hydrolase Via Adduction at Cys521 to Provide Cardioprotection Against Ischemia and Reperfusion Injury

Soluble epoxide hydrolase (sEH) plays an essential role in chronic kidney disease by hydrolyzing renoprotective epoxyeicosatrienoic acids to the corresponding inactive dihydroxyeicosatrienoic acids. However, there have been few mechanistic studies elucidating the role of sEH in epithelial-mesenchymal transition (EMT). The present study investigated, in vitro and in vivo, the role of sEH in proteinuria-induced renal tubular EMT and the underlying signaling pathway. We report that urinary protein (UP) induced EMT in cultured NRK-52E cells, as evidenced by decreased E-cadherin expression, increased alpha-smooth muscle actin (α-SMA) expression, and the morphological conversion to a myofibroblast-like phenotype. UP incubation also resulted in upregulated sEH, activated phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/Akt) signaling and increased phosphorylated glycogen synthase kinase-3β (GSK-3β). The PI3K inhibitor LY-294002 inhibited phosphorylation of Akt and GSK-3β as well as blocking EMT. Importantly, pharmacological inhibition of sEH with 12-(3-adamantan-1-yl- ureido)-dodecanoic acid (AUDA) markedly suppressed PI3K-Akt activation and GSK-3β phosphorylation. EMT associated E-cadherin suppression, α-SMA elevation and phenotypic transition were also attenuated by AUDA. Furthermore, in rats with chronic proteinuric renal disease, AUDA treatment inhibited PI3K-Akt activation and GSK-3β phosphorylation, while attenuating levels of EMT markers. Overall, our findings suggest that sEH inhibition ameliorates proteinuria-induced renal tubular EMT by regulating the PI3K-Akt-GSK-3β signaling pathway. Targeting sEH might be a potential strategy for the treatment of EMT and renal fibrosis.

Chrysin inhibits diabetic renal tubulointerstitial fibrosis through blocking epithelial to mesenchymal transition.

Renal fibrosis is a crucial event in the pathogenesis of diabetic nephropathy (DN). The process known as epithelial to mesenchymal transition (EMT) contributes to the accumulation of matrix proteins in kidneys, in which renal tubular epithelial cells play an important role in progressive renal fibrosis. The current study investigated that chrysin (5,7-dihydroxyflavone) present in bee propolis and herbs, inhibited renal tubular EMT and tubulointerstitial fibrosis due to chronic hyperglycemia. Human renal proximal tubular epithelial cells (RPTEC) were incubated in media containing 5.5 mM glucose, 27.5 mM mannitol (as an osmotic control), or 33 mM glucose (HG) in the absence and presence of 1-20 μM chrysin for 72 h. Chrysin significantly inhibited high glucose-induced renal EMT through blocking expression of the mesenchymal markers vimentin, α-smooth muscle actin, and fibroblast-specific protein-1 in RPTEC and db/db mice. Chrysin reversed the HG-induced down-regulation of the epithelial marker E-cadherin and the HG-enhanced N-cadherin induction in RPTEC. In addition, chrysin inhibited the production of collagen IV in tubular cells and the deposition of collagen fibers in mouse kidneys. Furthermore, chrysin blocked tubular cell migration concurrent with decreasing matrix metalloproteinase-2 activity, indicating epithelial cell derangement and tubular basement membrane disruption. Chrysin restored the induction of the tight junction proteins Zona occludens protein-1 (ZO-1) and occludin downregulated in diabetic mice. Chrysin inhibited renal tubular EMT-mediated tubulointerstitial fibrosis caused by chronic hyperglycemia. Therefore, chrysin may be a potent renoprotective agent for the treatment of renal fibrosis-associated DN. • Glucose increases renal tubular epithelial induction of vimentin, α-SMA and FSP-1. • Glucose enhances renal EMT by blocking tubular epithelial E-cadherin expression. • Chrysin inhibits tubular EMT-mediated tubulointerstitial fibrosis in mouse kidneys. • Chrysin restores renal tubular induction of ZO-1 and occludin downregulated in diabetic mice. • Chrysin blocks glucose-induced renal tubular cell migration with reducing MMP-2 activity.

Soluble epoxide hydrolase inhibition ameliorates proteinuria-induced epithelial-mesenchymal transition by regulating the PI3K-Akt-GSK-3β signaling pathway

Soluble epoxide hydrolase (sEH) plays an essential role in chronic kidney disease by hydrolyzing renoprotective epoxyeicosatrienoic acids to the corresponding inactive dihydroxyeicosatrienoic acids. However, there have been few mechanistic studies elucidating the role of sEH in epithelial-mesenchymal transition (EMT). The present study investigated, in vitro and in vivo, the role of sEH in proteinuria-induced renal tubular EMT and the underlying signaling pathway. We report that urinary protein (UP) induced EMT in cultured NRK-52E cells, as evidenced by decreased E-cadherin expression, increased alpha-smooth muscle actin (α-SMA) expression, and the morphological conversion to a myofibroblast-like phenotype. UP incubation also resulted in upregulated sEH, activated phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB/Akt) signaling and increased phosphorylated glycogen synthase kinase-3β (GSK-3β). The PI3K inhibitor LY-294002 inhibited phosphorylation of Akt and GSK-3β as well as blocking EMT. Importantly, pharmacological inhibition of sEH with 12-(3-adamantan-1-yl- ureido)-dodecanoic acid (AUDA) markedly suppressed PI3K-Akt activation and GSK-3β phosphorylation. EMT associated E-cadherin suppression, α-SMA elevation and phenotypic transition were also attenuated by AUDA. Furthermore, in rats with chronic proteinuric renal disease, AUDA treatment inhibited PI3K-Akt activation and GSK-3β phosphorylation, while attenuating levels of EMT markers. Overall, our findings suggest that sEH inhibition ameliorates proteinuria-induced renal tubular EMT by regulating the PI3K-Akt-GSK-3β signaling pathway. Targeting sEH might be a potential strategy for the treatment of EMT and renal fibrosis.

Arctigenin suppresses transforming growth factor-β1-induced expression of monocyte chemoattractant protein-1 and the subsequent epithelial–mesenchymal transition through reactive oxygen species-dependent ERK/NF-κB signaling pathway in renal tubular epithelial cells

Transforming growth factor-β1 (TGF-β1) induces expression of the proinflammatory and profibrotic cytokine monocyte chemoattractant protein-1 (MCP-1) in tubular epithelial cells (TECs) and thereby contributes to the tubular epithelial–mesenchymal transition (EMT), which in turn leads to the progression of tubulointerstitial inflammation into tubulointerstitial fibrosis. Exactly how TGF-β1 causes MCP-1 overexpression and subsequent EMT is not well understood. Using human tubular epithelial cultures, we found that TGF-β1 upregulated the expression of reduced nicotinamide adenine dinucleotide phosphate oxidases 2 and 4 and their regulatory subunits, inducing the production of reactive oxygen species. These reactive species activated a signaling pathway mediated by extracellular signal-regulated kinase (ERK1/2) and nuclear factor-κB (NF-κB), which upregulated expression of MCP-1. Incubating cultures with TGF-β1 was sufficient to induce hallmarks of EMT, such as downregulation of epithelial marker proteins (E-cadherin and zonula occludens-1), induction of mesenchymal marker proteins (α-smooth muscle actin, fibronectin, and vimentin), and elevated cell migration and invasion in an EMT-like manner. Overexpressing MCP-1 in cells exposed to TGF-β1 exacerbated these EMT-like changes. Pretreating cells with the antioxidant and anti-inflammatory compound arctigenin (ATG) protected them against these TGF-β1-induced EMT-like changes; the compound worked by inhibiting the ROS/ERK1/2/NF-κB pathway to decrease MCP-1 upregulation. These findings suggest ATG as a new therapeutic candidate to inhibit or even reverse tubular EMT-like changes during progression to tubulointerstitial fibrosis, and they provide the first clues to how ATG may work.

Nerve growth factor exposure promotes tubular epithelial–mesenchymal transition via TGF-β1 signaling activation

Clinical studies showed that renal expression and serum levels of nerve growth factor (NGF) are increased in renal diseases characterized by progressive fibrosis, a pathologic process in which TGF-β1 mediates most of the key events leading to tubular epithelial–mesenchymal transition (EMT). However, the pathogenic role of high NGF levels has not yet been elucidated. In this study, we found that in tubular renal cells, HK-2, NGF transcriptionally up-regulated TGF-β1 expression and secretion and enhanced cell motility by activating EMT markers via its receptors, TrkA and p75NTR. Interestingly, we observed that TGF-β1–SMAD pathway activation and the up-regulation of EMT markers NGF-induced were both prevented when knockdown of TGF-β1 gene occurred and that the pretreatment with an antibody anti-NGF reversed the nuclear translocation of pSMAD3/SMAD4 complex. Collectively, our results demonstrated that NGF promotes renal fibrosis via TGF-β1-signaling activation, suggesting that in kidney diseases high NGF serum levels could contribute to worsen renal fibrosis.

Beta-casomorphin-7 prevents epithelial-mesenchymal transdifferentiation of NRK-52E cells at high glucose level: Involvement of AngII-TGF-β1 pathway

BackgroundHyperglycemia is the most important risk factor in the progression of renal fibrosis in diabetic kidney. Based on previous studies, β-casomorphin-7 may exert anti-fibrotic activities in diabetic rats. However, the role of β-casomorphin-7 in the pathogenesis of renal tubulointerstitial fibrosis remains unclear. Thus, this study was designed to investigate the protective effect of β-casomorphin-7 on epithelial-mesenchymal transition (EMT) of NRK-52E cells treated under hyperglycemic condition and to explore the possible mechanism. Research design and methodsNRK-52E cells were cultured in high glucose (30mM) for 3 days. Different concentrations of β-casomorphin-7, naloxone (antagonist of opioid receptor) and losartan (antagonist of angiotensin II type I receptor) were added in the culture. Expression of α-smooth muscle actin (α-SMA), E-cadherin, vimentin and cytokeratin19 mRNA were determined by real-time PCR. Protein levels of E-cadherin and α-SMA were analyzed by Western blotting. The concentrations of angiotensin (Ang) II and transforming growth factor β1 (TGF-β1) in the culture medium were determined. ResultsHigh glucose-induced up-regulation of vimentin mRNA and α-SMA mRNA and protein were significantly inhibited by β-casomorphin-7. On the contrary, high glucose-induced down-regulation of cytokeratin19 mRNA and E-cad mRNA and protein was significantly reversed by β-casomorphin-7. β-casomorphin-7 significantly alleviate high glucose induced increase of AngII and TGF-β1 in the culture. Moreover, losartan significantly attenuated the expression of TGF-β1 and EMT of NRK-52E cells treated under hyperglycemic condition. But naloxone did not affect the EMT of NRK-52E cells treated by high glucose and β-casomorphin-7. ConclusionWe demonstrate that β-casomorphin-7 has the potential to inhibit high glucose-induced renal proximal tubular EMT partly by modulating AngII-TGF-β1 pathway, but not by opioid receptor.

Connective tissue growth factor induces tubular epithelial to mesenchymal transition through the activation of canonical Wnt signaling in vitro

Background: Overwhelming evidences suggest epithelial to mesenchymal transition (EMT) of tubular epithelial cells contributes to renal fibrosis of chronic kidney disease (CKD). Connective tissue growth factor (CTGF) plays an important role in the pathogenesis of EMT. However, the molecular mechanisms that regulate cell behaviors are not clear. Objective: The purpose of this study was to investigate whether CTGF induces EMT via activation of canonical Wnt signaling in renal tubular epithelial cells. Methods: Human renal proximal tubular epithelial cells (HK-2) were divided into control group, CTGF group and dickkopf (Dkk)-1 plus CTGF group. We assessed the biological changes of canonical Wnt signaling, including phosphorylation of low-density lipoprotein receptor-related protein (LRP6) and glycogen synthase kinase-3β (GSK-3β) and accumulation and nuclear localization of β-catenin. Meanwhile, morphological changes of the three groups were observed and tubular EMT was further confirmed by detecting the expression of α-SMA and E-cadherin. Results: The phosphorylation levels of LRP6 and GSK-3β and the expression of β-catenin in CTGF group were higher than control group (p < 0.05). The accumulation and nuclear localization of β-catenin was induced in CTGF group. Meanwhile, CTGF group cells showed a mesenchymal morphological phenotype and exhibited increased expressions of E-cadherin and decreased expressions of α-SMA compared to control group (p < 0.05), suggesting tubular EMT. Furthermore, we also found that Dkk-1 blocked the above CTGF’s effects by binding with LRP6. Conclusion: CTGF induces EMT via activation of canonical Wnt signaling in HK-2 cells in vitro, which may play an important role in the renal fibrosis of CKD.

The regulatory peptide apelin: a novel inhibitor of renal interstitial fibrosis

Epithelial-mesenchymal transition (EMT) of tubular epithelial cells is a key event in renal interstitial fibrosis and the progression of chronic kidney disease (CKD). Apelin is a regulatory peptide involved in the regulation of normal renal hemodynamics and tubular functions, but its role in renal fibrosis remains unknown. In this study, we examined the inhibitory effects of apelin on transforming growth factor-β1 (TGF-β1)-induced EMT in HK-2 cells, and evaluated its therapeutic efficacy in mice with complete unilateral ureteral obstruction (UUO). In vitro, apelin inhibited TGF-β1-mediated upregulation of α-smooth muscle actin (α-SMA) and downregulation of E-cadherin. Increased levels of phosphorylated Smad-2/3 and decreased levels of Smad7 in TGF-β1-stimulated cells were reversed by apelin co-treatment. In the UUO model, administration of apelin significantly attenuated renal interstitial fibrosis, as evidenced by the maintenance of E-cadherin and laminin expression, and markedly suppressed expression of α-SMA, TGF-β1 and its type I receptor, as well as interstitial matrix components. Interestingly, in UUO mice, there was a reduction in the plasma level of apelin, which was compensated by upregulation of APJ expression in the injured kidney. Exogenous supplementation of apelin normalized the level of plasmatic apelin and renal APJ. In conclusion, our study provides the first evidence that apelin is able to ameliorate renal interstitial fibrosis by suppression of tubular EMT through a Smad-dependent mechanism. The apelinergic system itself may promote some compensatory response in the renal fibrotic process. These results suggest that apelin has potential renoprotective effects and may be an effective agent for retarding CKD progression.

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.

Egr-1 Mediates Chronic Hypoxia-Induced Renal Interstitial Fibrosis via the PKC/ERK Pathway

Background: Chronic hypoxia-induced epithelial-to-mesenchymal transition (EMT) is a crucial process in renal fibrogenesis. Egr-1, as a transcription factor, has been proven to be important in promoting EMT. However, whether it functions in hypoxia-induced renal tubular EMT has not been fully elucidated. Methods: Egr-1 were detected at mRNA and protein levels by qPCR and Western blot analysis respectively after renal epithelial cells were subjected to hypoxia treatment. Meanwhile, EMT phenotype was also observed through identification of relevant EMT-specific markers. siRNA was used to knock down Egr-1 expression and subsequent changes were observed. Specific PKC and MAPK/ERK inhibitors were employed to determine the molecular signaling pathway involved in Egr-1-mediated EMT phenotype. In vivo assays using rat remnant kidney model were used to validate the in vitro results. Furthermore, Egr-1 expression was examined in the samples of CKD patients with the clinical relevance revealed. Results: Hypoxia treatment enhanced the mRNA and protein levels of Egr-1 in HK-2 cells, which was accompanied by a reduced expression of the epithelial marker E-cadherin and an enhanced expression of the mesenchymal marker Fsp-1. Downregulation of Egr-1 with siRNA reversed hypoxia-induced EMT. Using the specific inhibitors to protein kinase C (calphostin C) or MAPK/ERK (PD98059), we identified that hypoxia induced Egr-1 expression through the PKC/ERK pathway. In addition, the upregulation of Egr-1 raised endogenous Snail levels, and the downregulation of Snail inhibited Egr-1-mediated EMT in HK-2 cells. Through in vivo assays using rat remnant kidney and CKD patients' kidney tissues, we found that Egr-1 and Snail were overexpressed in tubular epithelial cells with EMT. Conclusion: Egr-1 may be an important regulator of the development of renal tubular EMT induced by hypoxia through the PKC/ERK pathway and the activation of Snail. Targeting Egr-1 expression or activity might be a novel therapeutic strategy to control renal fibrosis.

Mesenchymal Stem Cells Modulate Albumin-Induced Renal Tubular Inflammation and Fibrosis

Bone marrow-derived mesenchymal stem cells (BM-MSCs) have recently shown promise as a therapeutic tool in various types of chronic kidney disease (CKD) models. However, the mechanism of action is incompletely understood. As renal prognosis in CKD is largely determined by the degree of renal tubular injury that correlates with residual proteinuria, we hypothesized that BM-MSCs may exert modulatory effects on renal tubular inflammation and epithelial-to-mesenchymal transition (EMT) under a protein-overloaded milieu. Using a co-culture model of human proximal tubular epithelial cells (PTECs) and BM-MSCs, we showed that concomitant stimulation of BM-MSCs by albumin excess was a prerequisite for them to attenuate albumin-induced IL-6, IL-8, TNF-α, CCL-2, CCL-5 overexpression in PTECs, which was partly mediated via deactivation of tubular NF-κB signaling. In addition, albumin induced tubular EMT, as shown by E-cadherin loss and α-SMA, FN and collagen IV overexpression, was also prevented by BM-MSC co-culture. Albumin-overloaded BM-MSCs per se retained their tri-lineage differentiation capacity and overexpressed hepatocyte growth factor (HGF) and TNFα-stimulating gene (TSG)-6 via P38 and NF-κB signaling. Albumin-induced tubular CCL-2, CCL-5 and TNF-α overexpression were suppressed by recombinant HGF treatment, while the upregulation of α-SMA, FN and collagen IV was attenuated by recombinant TSG-6. Neutralizing HGF and TSG-6 abolished the anti-inflammatory and anti-EMT effects of BM-MSC co-culture in albumin-induced PTECs, respectively. In vivo, albumin-overloaded mice treated with mouse BM-MSCs had markedly reduced BUN, tubular CCL-2 and CCL-5 expression, α-SMA and collagen IV accumulation independent of changes in proteinuria. These data suggest anti-inflammatory and anti-fibrotic roles of BM-MSCs on renal tubular cells under a protein overloaded condition, probably mediated via the paracrine action of HGF and TSG-6.

Salt-induced epithelial-to-mesenchymal transition in Dahl salt-sensitive rats is dependent on elevated blood pressure

Dietary salt intake has been linked to hypertension and cardiovascular disease. Accumulating evidence has indicated that salt-sensitive individuals on high salt intake are more likely to develop renal fibrosis. Epithelial-to-mesenchymal transition (EMT) participates in the development and progression of renal fibrosis in humans and animals. The objective of this study was to investigate the impact of a high-salt diet on EMT in Dahl salt-sensitive (SS) rats. Twenty-four male SS and consomic SS-13BN rats were randomized to a normal diet or a high-salt diet. After 4 weeks, systolic blood pressure (SBP) and albuminuria were analyzed, and renal fibrosis was histopathologically evaluated. Tubular EMT was evaluated using immunohistochemistry and real-time PCR with E-cadherin and alpha smooth muscle actin (α-SMA). After 4 weeks, SBP and albuminuria were significantly increased in the SS high-salt group compared with the normal diet group. Dietary salt intake induced renal fibrosis and tubular EMT as identified by reduced expression of E-cadherin and enhanced expression of α-SMA in SS rats. Both blood pressure and renal interstitial fibrosis were negatively correlated with E-cadherin but positively correlated with α-SMA. Salt intake induced tubular EMT and renal injury in SS rats, and this relationship might depend on the increase in blood pressure.

Tongxinluo ameliorates renal structure and function by regulating miR-21-induced epithelial-to-mesenchymal transition in diabetic nephropathy

Diabetic nephropathy (DN) is one of the most important diabetic microangiopathies. The epithelial-to-mesenchymal transition (EMT) plays an important role in DN. The physiological role of microRNA-21 (miR-21) was closely linked to EMT. However, it remained elusive whether tongxinluo (TXL) ameliorated renal structure and function by regulating miR-21-induced EMT in DN. This study aimed to determine the effect of TXL on miR-21-induced renal tubular EMT and to explore the relationship between miR-21 and TGF-β1/smads signals. Real-time RT-PCR, cell transfection, in situ hybridization (ISH), and laser confocal microscopy were used, respectively. Here, we revealed that TXL dose dependently lowered miR-21 expression in tissue, serum, and cells. Overexpression of miR-21 can enhance α-smooth muscle actin (SMA) expression and decrease E-cadherin expression by upregulating smad3/p-smad3 expression and downregulating smad7 expression. Interestingly, TXL also increased E-cadherin expression and decreased α-SMA expression by regulating miR-21 expression. More importantly, TXL decreased collagen IV, fibronectin, glomerular basement membrane, glomerular area, and the albumin/creatinine ratio, whereas it increased the creatinine clearance ratio. The results demonstrated that TXL ameliorated renal structure and function by regulating miR-21-induced EMT, which was one of the mechanisms to protect against DN, and that miR-21 may be one of the therapeutic targets for TXL in DN.

Inhibition of the tubular epithelial-to-mesenchymal transition in vivo and in vitro by the Uremic Clearance Granule (尿毒清颗粒)

To investigate the effect of the Uremic Clearance Granule (UCG, ), a Chinese patent medicine, on tubular epithelial-to-mesenchymal transition (EMT) in a unilateral ureteral obstruction (UUO) model in vivo and transforming growth factor (TGF)-β1 induced EMT of HK-2 cells in vitro. In vivo study, 50 Sprague Dawley rats were divided into three groups: a sham operation group (n=10), a UUO group (n=20), and a UUO with UCG treatment group (n=20). The UCG was given at a dose of 4.5 g/kg body weight per day by gavage after surgery. In vitro study, HK-2 cells were cultured in 10% fetal bovine serum (FBS), 10% healthy rat serum, 10% FBS and TGF-β1 (10 ng/mL), 10% healthy rat serum and TGF-β1, or 10% rat serum containing the uremic clearance granule and TGF-β1. The expression of the epithelial marker E-cadherin and the mesenchymal markers vimentin and α-smooth muscle actin (α-SMA) in kidney tissues and HK-2 cells were investigated by Western blot analysis and immunofluorescence staining. The rats of the UUO group showed obvious tubulointerstitial fibrosis, compared with the sham operation group rats. Tubulointerstitial fibrosis score was reduced by 17.5%±1.1% at day 7 and by 20.0%±1.2% at day 14 in the UCG-treated group, compared with the UUO group. The UCG could maintained expression of E-cadherin and suppressed expression of vimentin and α-SMA in kidney tissues of UUO rats at days 7 and 14, as determined by Western blot analysis and immunofluorescence staining. Rat serum containing the UCG partially inhibited TGF-β1-induced fibroblast phenotype of HK-2 cells and maintained the epithelial morphology of HK-2 cells in vitro. This occurred partially through a reduction of vimentin expression and an increase of E-cadherin expression. These results suggest that the UCG prevents tubular EMT and may be a promising agent for treating tubulointerstitial fibrosis.

Kindlin-2 regulates renal tubular cell plasticity by activation of Ras and its downstream signaling

Kindlin-2 is an adaptor protein that contributes to renal tubulointerstitial fibrosis (TIF). Epithelial-to-mesenchymal transition (EMT) in tubular epithelial cells was regarded as one of the key events in TIF. To determine whether kindlin-2 is involved in the EMT process, we investigated its regulation of EMT in human kidney tubular epithelial cells (TECs) and explored the underlying mechanism. In this study, we found that overexpression of kindlin-2 suppressed epithelial marker E-cadherin and increased the expression of fibronectin and the myofibroblast marker α-smooth muscle actin (SMA). Kindlin-2 significantly activated ERK1/2 and Akt, and inhibition of ERK1/2 or Akt reversed kindlin-2-induced EMT in human kidney TECs. Mechanistically, kindlin-2 interacted with Ras and son of sevenless (Sos)-1. Furthermore, overexpression of kindlin-2 increased Ras activation through recruiting Sos-1. Treatment with a Ras inhibitor markedly repressed kindlin-2-induced ERK1/2 and Akt activation, leading to restraint of EMT. We further demonstrated that knockdown of kindlin-2 inhibited EGF-induced Ras-Sos-1 interaction, resulting in reduction of Ras activation and suppression of EMT stimulated by EGF. Importantly, we found that depletion of kindlin-2 significantly inhibited activation of ERK1/2 and Akt signaling in mice with unilateral ureteral obstruction. We conclude that kindlin-2, through activating Ras and the downstream ERK1/2 and Akt signaling pathways, plays an important role in regulating renal tubular EMT and could be a potential therapeutic target for the treatment of fibrotic kidney diseases.

Association of URG11 and Twist with clinical pathological characteristics and prognosis in patients with IgA nephropathy

Our previous studies demonstrated that URG11 is involved in hypoxia-induced tubular epithelial-mesenchymal transition and the development of kidney fibrosis in cellular and animal models. The objective of this study was to determine the expression levels of URG11 in kidneys with IgA nephropathy (IgAN), and the association of URG11 with various clinical parameters. We analysed the degree of expression and localization of URG11 in biopsies from kidneys with IgAN, and correlated their immunostaining levels with various clinical and histological parameters. We also analysed the correlation between the expression of URG11 and Twist in the renal interstitium with renal survival. URG11 was strongly expressed in the cytoplasm of tubular epithelial cells obtained from kidneys of patients with IgAN. However, there was little positive staining for URG11 in the renal tubules of normal kidneys (P = 0.024). URG11 protein levels in the tubulointerstitium were inversely correlated with estimated glomerular filtration rates (eGFRs) (r = -0.305, P = 0.038) and the percentage of tubulointerstitial fibrosis (r = 0.350, P = 0.023). Moreover, a high level of URG11 correlated with the activation of Twist expression and E-cadherin repression in patients with IgAN (P = 0.000 and 0.041, respectively). Multivariate analyses indicated that a combination of high URG11 and Twist expression was an independent prognostic factor [relative ratio, RR 4.738 (95% CI: 1.040, 21.591), P = 0.044] of IgAN. Our findings suggest that URG11 staining in renal biopsy specimens might be a novel histological marker for progression in IgAN patients.

Norcantharidin Inhibits Renal Interstitial Fibrosis by Blocking the Tubular Epithelial-Mesenchymal Transition

Epithelial–mesenchymal transition (EMT) is thought to contribute to the progression of renal tubulointerstitial fibrosis. Norcantharidin (NCTD) is a promising agent for inhibiting renal interstitial fibrosis. However, the molecular mechanisms of NCTD are unclear. In this study, a unilateral ureteral obstruction (UUO) rat model was established and treated with intraperitoneal NCTD (0.1 mg/kg/day). The UUO rats treated with NCTD showed a reduction in obstruction-induced upregulation of α-SMA and downregulation of E-cadherin in the rat kidney (P<0.05). Human renal proximal tubule cell lines (HK-2) stimulated with TGF-β1 were treated with different concentrations of NCTD. HK-2 cells stimulated by TGF-β1 in vitro led to downregulation of E-cadherin and increased de novo expression of α-SMA; co-treatment with NCTD attenuated all of these changes (P<0.05). NCTD reduced TGF-β1-induced expression and phosphorylation of Smad2/3 and downregulated the expression of Snail1 (P<0.05). These results suggest that NCTD antagonizes tubular EMT by inhibiting the Smad pathway. NCTD may play a critical role in preserving the normal epithelial phenotype and modulating tubular EMT.

Human Wharton’s jelly-derived mesenchymal stromal cells reduce renal fibrosis through induction of native and foreign hepatocyte growth factor synthesis in injured tubular epithelial cells

IntroductionBased on some well-documented reports, we attempted to clarify the antifibrotic mechanisms of human Wharton’s-jelly-derived mesenchymal stromal cells (WJ-MSCs) from the perspective of induction of hepatocyte growth factor (HGF) expression in tubular epithelial cells (TECs).MethodsA rat model of acute kidney injury (AKI) was established through unilateral renal ischemia for 1 hour. Two days later, a single intravenous cell or vehicle injection, or contralateral nephrectomy, was performed. Rats were sacrificed at 1 day, 1 week, 4 weeks, or 6 weeks after the intervention. Renal fibrosis was evaluated by Masson trichrome staining and Sircol collagen assay. The upregulation of α-smooth muscle actin (α-SMA) versus E-cadherin expression was adopted as an indicator of tubular epithelial-mesenchymal transition (EMT). Gene and protein expression of HGF or transforming growth factor-beta1 (TGF-β1) was determined by real-time polymerase chain reaction (RT-PCR) and Western blot, respectively. HGF expression in TECs was detected with immunostaining. In vitro, rat TECs subjected to hypoxia injury were incubated with or without conditioned medium (CM) from WJ-MSCs for 1, 3, 24, or 48 hours. Rat or human HGF synthesis in TECs was assessed with immunostaining, RT-PCR, or ELISA.ResultsCell delivery or nephrectomy led to abrogation of renal scarring. At the incipient period of AKI, through induction of HGF expression, either of them remarkably promoted the upregulation of HGF versus TGF-β1 expression in damaged kidney. Rat TECs were not only the principal cells expressing HGF but also exhibited human HGF expression after cell infusion. During fibrogenesis, the downregulation of HGF versus TGF-β1 expression was greatly prevented by WJ-MSCs or kidney removal, thereby resulting in tubular EMT delay. In vitro, after 24 or 48 hours of incubation, CM not only robustly induced the upregulation of rat HGF gene expression in TECs but substantially amplified the release of rat HGF. Under the induction of CM, human HGF mRNA and protein were detected in rat TECs.ConclusionsWJ-MSCs contribute to tubular EMT delay and the alleviation of renal fibrosis. Induction of native and foreign HGF synthesis in damaged TECs at the initial stage of AKI leads to recovery of the disturbed balance of HGF/TGF-β1 during scar formation, being one of the vital mechanisms.

Paricalcitol Attenuates 4-Hydroxy-2-Hexenal-Induced Inflammation and Epithelial-Mesenchymal Transition in Human Renal Proximal Tubular Epithelial Cells

4-Hydroxy-2-hexenal (HHE), the aldehyde product of lipid peroxidation, may be responsible for the pathogenesis of progressive renal disease. Recently, paricalcitol (19-nor-1,25-dihydroxyvitamin D2) was shown to be renoprotective through its anti-inflammatory and antifibrotic effects in various experimental nephropathy models. In this study, we investigated the effects of paricalcitol on inflammation and epithelial-mesenchymal transition (EMT) after HHE-induced renal tubular epithelial cell injury. To investigate the molecular mechanisms underlying HHE-induced renal tubular cell injury, the human proximal tubular epithelial (HK-2) cells cultured with 10 µM HHE in the presence or absence of paricalcitol. In HK-2 cells, paricalcitol attenuated the HHE-induced expression of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase, and prevented nuclear factor-κB (NF-κB) activation. The expression of the inflammatory proteins inducible nitric oxide synthase and cyclooxygenase-2 was attenuated by paricalcitol pretreatment. In addition, HHE increased the expression of the transforming growth factor (TGF)-β/Smad signaling proteins and fibrotic proteins, such as α-smooth muscle actin and connective tissue growth factor; this inducible expression was suppressed by pretreatment with paricalcitol. Treatment with HHE resulted in the activation of the β-catenin signaling pathway, and paricalcitol pretreatment reduced the expression of β-catenin in HHE-treated HK-2 cells. Coimmunoprecipitation shows that paricalcitol induced vitamin D receptor (VDR)/β-catenin complex formation in HK-2 cells. Also immunofluorescence staining revealed that co-localization of VDR and β-catenin in the nuclei. ICG-001, an inhibitor of β-catenin, decreased the expression of TGF-β1 and attenuated HHE-induced tubular EMT. These results show that paricalcitol attenuated HHE-induced renal tubular cell injury by suppressing inflammation and EMT process through inhibition of the NF-κB, TGF-β/Smad, and β-catenin signaling pathways.

Epithelial–mesenchymal transition of renal tubules: Divergent processes of repairing in acute or chronic injury?

Epithelial–mesenchymal transition (EMT) of tubular epithelial cells (TECs) is commonly considered as the major mechanism leading to renal fibrosis in chronic kidney diseases (CKD) injury. We raise the hypothesis that EMT in adult kidney may be an event of “atavistic” phenotypic transition, which mimics but reverses the genetic and cellular processes of development of renal tubules. Transformed TECs may be regarded as induced mesenchymal stem-like cells, representing a cellular self-adaptation when in acute or chronic injury. The reasons are as follows: (1) Embryonic gene WT1 and Pax2, which govern tubule development, have been found to re-express during tubular EMT when facing injury. (2) The common factors that induce EMT in vitro, like IL-1, angiotension II and hypoxia could also promote WT1 and/or Pax2 re-expression. (3) Expression of WT1 and Pax2 are found to be associated with progenitor cells. (4) Beside embryonic gene WT1 and Pax2, we also found that some stem cell markers like CD133 were expressed during EMT process. (5) The process of EMT is not only take place in chronic kidney injury (CKD), but also in acute kidney injury (AKI) as well. (6) The phenotype transition of TECs and genetic event during AKI are entirely consistent with what happened in CKD, but the outcome is completely different. Thus, we thought tubular injury of CKD and AKI may share a common initiative repair mechanism: tubular EMT, that is TECs are transformed into induced mesenchymal stem-like cells, and then interpret the injurious signal differently in acute versus chronic conditions, so as to possess a divergent fates, tubular regeneration or fibrosis formation, depending on a different microenvironment or the duration of the injury. In this sense, tubular EMT could be purposefully orientated into a constructively pathway that repair kidney injury via tubular regeneration, matrix remodeling and tissue structure and function restoration.