Epithelial-mesenchymal Transition Of Tubular Cells Research Articles

Kidney Fibrosis and Matrix Metalloproteinases (MMPs).

Chronic kidney disease (CKD) is a disorder that causes changes in both the structure and function of the kidneys, causing complications such as hypertension, edema, and oliguria. Renal fibrosis is also a common pathological feature of CKD. Matrix metalloproteinases (MMPs) are endopeptidases that degrade extracellular matrix (ECM) proteins. The proteinase domain consists of a zinc ion in the active site, which contributes to its stabilization with another zinc and three calcium structural ions. Many cellular processes are controlled by MMPs, such as cell-cell interactions and various signaling pathways, while they are also involved in degrading substrates on cell surfaces. Tissue inhibitors of metalloproteinases (TIMPs) are key regulators of metalloproteinases, and both are involved in regulating cell turnover, the regulation, and the progression of fibrosis and apoptosis in the tissue. MMPs play a role in renal fibrosis, such as the tubular cell epithelial-mesenchymal transition (TEM), activation of resident fibroblasts, endothelial-mesenchymal transition (EndoMT), and pericyte-myofibroblast transdifferentiation. This review aims to show the mechanisms through which MMPs contribute to renal fibrosis, paying particular attention to MMP-9 and the epithelial-mesenchymal transition.

FGF2 isoforms play distinct roles in tubular epithelial-to-mesenchymal transition in diabetic nephropathy

IntroductionThe role of different isoforms of Fibroblast growth factor-2 (FGF2) in tubular epithelial-to-mesenchymal transition (EMT) in diabetic nephropathy remains unknown. We aimed to evaluate the role of FGF2 isoforms in the pathogenesis of EMT. Materials and methodsWestern blot and immunofluorescence were used to assess the expression of FGF2 isoforms in db/db mice and high glucose-stimulated HK2 cells. The effects of specific FGF2 isoforms on EMT were explored via overexpression or knockdown of the corresponding isoform in HK2 cells cultivated in high glucose. ResultsExpression of low molecular weight (LMW) FGF2 was up-regulated while high molecular weight (HMW) FGF2 was down-regulated in the kidney of db/db mice and HK2 cells cultured in high glucose that underwent EMT. Overexpression of the LMW FGF2 enhanced EMT changes, while overexpression of the HMW FGF2 attenuated EMT. Knockdown of HMW FGF2 in HK2 cells promoted the EMT process. ConclusionsThe expression and function of LMW and HMW FGF2 differed in the process of EMT in tubular cells. LMW FGF2 contributed to EMT, while HMW FGF2 played a protective role in the EMT process.

Lysophosphatidic Acid Promotes Epithelial-Mesenchymal Transition in Kidney Epithelial Cells via the LPAR1/MAPK-AKT/KLF5 Signaling Pathway in Diabetic Nephropathy.

The epithelial–mesenchymal transition (EMT) is a differentiation process associated with fibrogenesis in diabetic nephropathy (DN). Lysophosphatidic acid (LPA) is a small, naturally occurring glycerophospholipid implicated in the pathogenesis of DN. In this study, we investigated the role of LPA/LPAR1 signaling in the EMT of tubular cells as well as the underlying mechanisms. We observed a decrease in E-cadherin and an increase in vimentin expression levels in the kidney tubules of diabetic db/db mice, and treatment with ki16425 (LPAR1/3 inhibitor) inhibited the expression of these EMT markers. Ki16425 treatment also decreased the expression levels of the fibrotic factors fibronectin and alpha-smooth muscle actin (α-SMA) in db/db mice. Similarly, we found that LPA decreased E-cadherin expression and increased vimentin expression in HK-2 cells, which was reversed by treatment with ki16425 or AM095 (LPAR1 inhibitor). In addition, the expression levels of fibronectin and α-SMA were increased by LPA, and this effect was reversed by treatment with ki16425 and AM095 or by LPAR1 knockdown. Moreover, LPA induced the expression of the transcription factor, Krüppel-like factor 5 (KLF5), which was decreased by AM095 treatment or LPAR1 knockdown. The expression levels of EMT markers and fibrotic factors induced by LPA were decreased upon KLF5 knockdown in HK-2 cells. Inhibition of the extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and serine-threonine kinase (AKT) pathways decreased LPA-induced expression of KLF5 and EMT markers. In conclusion, these data suggest that LPA contributes to the pathogenesis of diabetic nephropathy by inducing EMT and renal tubular fibrosis via regulation of KLF5 through the LPAR1.

Immunité et dysfonctions tubulaires au cours des connectivites

The immune renal tubular diseases are known since five decades, but their prevalence remains to be defined. They are caused by humoral and cellular effectors of innate and adaptative immunities on several targets of the renal tubule: protein channels, co or counter transporters, luminal or cytosolic enzymes, tight junctions. Genetic or epigenetic variations are also involved. Clinical manifestations are various and make the diagnosis difficult. They can precede the causal affection and they worsen the prognosis. The classical model consists in hypokalemic tubular distal acidosis observed in Sjögren's syndrome which illustrates the auto-immune epithelitis concept. Cellular immunity can act through other ways, like tertiary lymphoid neogenesis in systemic lupus. Humoral immunity through autoantibodies targets several membrane, cytosolic or nuclear proteins, causing specific tubular dysfonctions. It is also implied in the epithelial-mesenchymal transition of tubular cells. Innate immunity through cytokines may be involved. Treatment consists in electrolytic disorders correction and immunosupppressive medication: the choice should be guided at best by physiopathology.

Bixin Confers Prevention against Ureteral Obstruction-Caused Renal Interstitial Fibrosis through Activation of the Nuclear Factor Erythroid-2-Related Factor2 Pathway in Mice.

Bixin is a natural carotenoid isolated from the seeds of Bixa orellana, with numerous important pharmacological activities, including antioxidant and antifibrotic effects. The nuclear factor erythroid-2-related factor2 (Nrf2) signaling pathway induced by bixin is involved in the process. Excessive reactive oxygen species generation in tubular cells contributes to kidney interstitial fibrosis. The potential therapeutic strategy for bixin in alleviating kidney fibrosis remains largely unclear. In this study, we used unilateral ureteral obstruction (UUO) to establish a renal fibrotic model. Dramatic oxidative DNA damage occurs in kidneys, especially in tubular cells after UUO. In cultured tubular cells, bixin could induce Nrf2 signaling activation by suppressing Nrf2 ubiquitination and increasing its protein stability. Transforming growth factor beta 1-induced epithelial-to-mesenchymal transition (EMT) and extracellular matrix production were suppressed by bixin, and blockade of Nrf2 activation by small interfering RNA could largely reverse the protective effect of bixin. In vivo studies showed that administration of bixin induces Nrf2 signaling activation in tubular cells and markedly attenuates partial EMT of tubular cells and kidney interstitial fibrosis after subjecting to UUO. Together, this study implies that bixin may protect against kidney interstitial fibrosis through stimulating Nrf2 activation in renal tubular cells.

Novel AXL-specific inhibitor ameliorates kidney dysfunction through the inhibition of epithelial-to-mesenchymal transition of renal tubular cells.

Chronic kidney diseases affect more than 800 million people globally and remain a high unmet need. Various therapeutic targets are currently under evaluation in pre-clinical and clinical studies. Because the growth arrest specific gene 6 (Gas6)/AXL pathway has been implicated in the pathogenesis of kidney diseases, we generated a novel selective and potent AXL inhibitor, CH5451098, and we evaluated its efficacy and elucidated its mechanism in an NEP25 mouse model that follows the clinical course of glomerular nephritis. In this model, CH5451098 significantly ameliorated the excretion of urinary albumin and elevation of serum creatinine. Additionally, it also inhibited tubulointerstitial fibrosis and tubular damage. To elucidate the mechanism behind these changes, we analyzed the effect of CH5451098 against transforming growth factor β1 (TGFβ1) and Gas6, which is a ligand of AXL receptor, in NRK-52E renal tubular epithelial cells. CH5451098 inhibited epithelial-to-mesenchymal transition (EMT) caused by the synergistic effects of TGFβ1 and Gas6 in NRK-52E cells. This inhibition was also observed in NEP25 mice. Taken together, these results suggest that CH5451098 could ameliorate kidney dysfunction in glomerular nephritis by inhibiting EMT in tubular cells. These results reveal that AXL strongly contributes to the disease progression of glomerular nephritis.

Blockade of ERK1/2 by U0126 alleviates uric acid-induced EMT and tubular cell injury in rats with hyperuricemic nephropathy.

Extracellular signal-regulated kinases 1 and 2 (ERK1/2) are serine/threonine kinases and function as regulators of cellular proliferation and differentiation. Recently, we demonstrated that inhibition of ERK1/2 alleviates the development and progression of hyperuricemia nephropathy (HN). However, its potential roles in uric acid-induced tubular epithelial-mesenchymal transition (EMT) and tubular epithelial cell injury are unknown. In this study, we showed that hyperuricemic injury induced EMT as characterized by downregulation of E-cadherin and upregulation of vimentin and Snail1 in a rat model of HN. This was coincident with epithelial cells arrested at the G2/M phase of cell cycle, activation of Notch1/Jagged-1 and Wnt/β-catenin signaling pathways, and upregulation of matrix metalloproteinase-2 (MMP-2) and MMP-9. Administration of U0126, a selective inhibitor of ERK1/2, blocked all these responses. U0126 was also effective in inhibiting renal tubular cell injury, as shown by decreased expression of lipocalin-2 and kidney injury molecule-1 and active forms of caspase-3. U0126 or ERK1/2 siRNA can inhibit tubular cell EMT and cell apoptosis as characterized with decreased expression of cleaved caspase-3. Moreover, ERK1/2 inhibition suppressed hyperuricemic injury-induced oxidative stress as indicated by decreased malondialdehyde and increased superoxide dismutase. Collectively, ERK1/2 inhibition-elicited renal protection is associated with inhibition of EMT through inactivation of multiple signaling pathways and matrix metalloproteinases, as well as attenuation of renal tubule injury by enhancing cellular resistance to oxidative stress.

Blockade of enhancer of zeste homolog 2 alleviates renal injury associated with hyperuricemia.

Hyperuricemia has been identified as an independent risk factor for chronic kidney disease (CKD) and is associated with the progression of kidney diseases. It remains unknown whether enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27 methyltransferase, can regulate metabolism of serum uric acid and progression of renal injury induced by hyperuricemia. In this study, we demonstrated that blockade of EZH2 with 3-DZNeP, a selective EZH2 inhibitor, or silencing of EZH2 with siRNA inhibited uric acid-induced renal fibroblast activation and phosphorylation of Smad3, epidermal growth factor receptor (EGFR), and extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) in cultured renal fibroblasts. Inhibition of EZH2 also suppressed proliferation of renal fibroblasts and epithelial-mesenchymal transition of tubular cells. In a mouse model of renal injury induced by hyperuricemia, EZH2 and trimethylation of histone H3 at lysine27 expression levels were enhanced, which was coincident with renal damage and increased expression of lipocalin-2 and cleaved caspase-3. Inhibition of EZH2 with 3-DZNeP blocked all these responses. Furthermore, 3-DZNeP treatment decreased the level of serum uric acid and xanthine oxidase activity, alleviated renal interstitial fibrosis, inhibited activation of transforming growth factor-β/Smad3, EGFR/ERK1/2, and nuclear factor-κB signaling pathways, as well as reduced expression of multiple chemokines/cytokines. Collectively, EZH2 inhibition can reduce the level of serum uric acid and alleviate renal injury and fibrosis through a mechanism associated with inhibition of multiple signaling pathways. Targeting EZH2 may be a novel strategy for the treatment of hyperuricemia-induced CKD.

Membrane raft redox signalling contributes to renal tubular cell epithelial-mesenchymal transition and fibrosis and inflammation of the kidneys.

Membrane raft redox signalling contributes to renal tubular cell epithelial-mesenchymal transition and fibrosis and inflammation of the kidneys.

The influence of fasudil on renal proximal tubular cell epithelial–mesenchymal transition induced by parathormone

Background: Renal fibrosis is a common pathway through which a variety of chronic kidney diseases progress to end-stage renal disease. Epithelial–mesenchymal transition (EMT) of renal proximal tubular cells is one of the most important factors in renal fibrosis. This study investigates if fasudil could influence EMT of renal proximal tubular cells.Methods: HK-2 cells in passage 3–4 were used for all experiments. The cells were divided into five groups and treated with different concentrations of PTH and then observe cellular morphological changes at 0, 24 and 48 h using an inverted microscope and investigate the expression of the epithelial cell marker E-cadherin and the renal fibroblast marker α-smooth muscle actin (α-SMA).Results: PTH significantly induced EMT, fasudil-inhibited EMT induced by PTH to different degrees, and the inhibitory effect of fasudil was most pronounced at 20 μmol/L.Conclusion: Monitoring PTH levels, early prevention and control of hyperparathyroidism and reducing the concentration of PTH are important means to improve prognosis and delay the progression of chronic kidney disease. Fasudil can restrain EMT induced by PTH; this conclusion provides experimental data for the application of fasudil in the clinical prevention and treatment of renal fibrosis.

Is there any role of epithelial to mesenchymal transition in the pathogenesis of contrast nephropathy?

Aim: Contrast medium-induced nephropathy is one of the major complications of intravenous contrast medium use. But its pathogenesis is unclear. Epithelial mesenchymal transition (EMT) is defined as the transformation of the primer epithelial cells to mesenchymal cells. EMT in tubular cells might cause tubulointerstitial damage. In this study, we investigated whether or not EMT has a role in radiocontrast-induced nephropathy. Radiocontrast medium might be triggering reversible EMT via serum and glucocorticoid-regulated kinase 1 (SGK 1). We investigated the effect of different concentrations of the contrast agent iopromide on human proximal tubule cell (HK-2) culture by measuring the level of SGK1, snail family zinc finger 1 (SNAIL1), connective tissue growth factor (CTGF), and collagen type I alpha 1 (COL1A1).Methods: We conducted a scratch assay and qPCR. HK-2 cells were cultured in the petri dishes/flasks and starved with serum-free medium. The 40, 20, and 10 mg/mL doses of iopromide were administrated to cells. The scratches were photographed immediately and again at the 20th hour. The levels of gene expression of SGK1, SNAIL1, CTGF, and COL1A1 were measured using the real-time qPCR system at the end of the 24th hour.Results: Iopromide caused the breaking of intercellular connections, the disappearance of the cobblestone appearance of cells, and the migration of cells at the 20th hour in the scratch assay. It also increased the expression of SGK1, SNAIL1, CTGF, and COL1A1 genes.Conclusion: Our study concluded that certain important markers of EMT increase in different concentrations of the contrast agent. High osmolality might trigger EMT. The relationship between contrast agent and EMT has not been defined before. Further in vivo and in vitro studies are required.

Screening and identification of the differentially expressed proteins in neonatal rat kidney after partial unilateral ureteral obstruction.

Renal fibrosis, considered to be a common consequence of progressive renal disease, involves glomerulosclerosis and/or tubulointerstitial fibrosis. Currently, research is focused on investigating potential mechanisms to prevent or reverse the damage caused by fibrosis. Under the influence of cytokines, chemokines and other signaling molecules, the cellular interactions that regulate the development of interstitial fibrosis are complex. Epithelial-mesenchymal transition (EMT) has emerged as an important pathway leading to the generation of matrix-producing fibroblasts and myofibroblasts in diseased kidneys. The proteomics study compared the protein profiles between the time points of podocyte EMT and tubular cell EMT in a partial unilateral ureteral obstruction (PUUO) model in rats. Proteins isolated from the PUUO group and corresponding sham rat kidney tissues were subjected to 2-D gel electrophoresis and were then identified by mass spectrometry. In total, 43 proteins with differential expression were identified, which were reported to be involved in the regulation of the cytoskeleton and actin, glucose metabolism, cell apoptosis, mitochondrial energy metabolism, oxidative stress and endoplasmic reticulum stress. Electron transfer flavoprotein, β polypeptide was detected by immunoblot analysis and its mRNA levels were determined in renal tissues. The results demonstrate protein alterations that reflect the pathology of the obstructed kidneys, and thus may aid in understanding the pathogenesis of obstructive nephropathy.

Cyclic helix B peptide inhibits ischemia reperfusion-induced renal fibrosis via the PI3K/Akt/FoxO3a pathway.

Renal fibrosis is a main cause of end-stage renal disease. Clinically, there is no beneficial treatment that can effectively reverse the progressive loss of renal function. We recently synthesized a novel proteolysis-resistant cyclic helix B peptide (CHBP) that exhibits promising renoprotective effects. In this study, we evaluated the effect of CHBP on renal fibrosis in an in vivo ischemia reperfusion injury (IRI) model and in vitro TGF-β-stimulated tubular epithelial cells (TCMK-1 and HK-2) model. In the IRI in vivo model, mice were randomly divided into sham (sham operation), IR and IR + CHBP groups (n = 6). CHBP (8 nmol/kg) was administered intraperitoneally at the onset of reperfusion, and renal fibrosis was evaluated at 12 weeks post-reperfusion. Our results showed that CHBP markedly attenuated the IRI-induced deposition of collagen I and vimentin. In the in vitro model, CHBP reversed the TGF-β-induced down-regulation of E-cadherin and up-regulation of α-SMA and vimentin. Furthermore, CHBP inhibited the phosphorylation of Akt and Forkhead box O 3a (FoxO3a), whose anti-fibrotic effect could be reversed by the 3-phosphoinositide-dependent kinase-1 (PI3K) inhibitor wortmannin as well as FoxO3a siRNA. These findings demonstrate that CHBP attenuates renal fibrosis and the epithelial-mesenchymal transition of tubular cells, possibly through suppression of the PI3K/Akt pathway and thereby the inhibition FoxO3a activity.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0699-2) contains supplementary material, which is available to authorized users.

TNF-related weak inducer of apoptosis (TWEAK) regulates junctional proteins in tubular epithelial cells via canonical NF-κB pathway and ERK activation.

The tubular epithelium may be intrinsically involved in promoting kidney injury by junctional instability, epithelial-mesenchymal transition (EMT) and extracellular matrix remodelling. In this work, we investigated whether the pleiotropic and proinflammatory cytokine tumor necrosis factor-like weak inducer of apoptosis (TWEAK), could be able to disturb junctional protein expression and to induce EMT of tubular cells. In cultured murine proximal tubular cells TWEAK induced phenotypic changes that were accompanied by F-actin redistribution, loss of epithelial adherent (E-cadherin, Cadherin-16, β-catenin) and tight junction (ZO-1) proteins, and re-expression of the mesenchymal protein Vimentin. The transcriptional repressors Snail and HNF1β were also modulated by TWEAK. In a murine model of obstructive renal pathology, TWEAK expression correlated with the appearance of the mesenchymal marker αSMA in kidney tubular cells. Mechanistically, the epithelial changes induced by TWEAK, including loss of epithelial integrity and EMT, via Fn14 were TGF-β1 independent, but mediated by several intracellular signaling systems, including the canonical NF-κB, ERK activation and the vitamin D receptor modulation. These results highlight potential contributions of TWEAK-induced inflammatory mechanisms that could unveil new pathogenic effects of TWEAK starting tubulointerstitial damage and fibrosis.

Implication of Bcl-2-associated athanogene 3 in fibroblast growth factor-2-mediated epithelial-mesenchymal transition in renal epithelial cells.

The epithelial-mesenchymal transition (EMT) of tubular epithelial cells to myofibroblast-like cells plays a substantial role in renal tubulointerstitial fibrosis, which is a common pathological character of end-stage renal disease (ESRD). Fibroblast growth factor-2 (FGF-2) triggers EMT in tubular epithelial cells and increases Bcl-2-associated athanogene 3 (BAG3) expression in neural progenitor and neuroblastoma cells. In addition, a novel role of regulation of EMT has been ascribed to BAG3 recently. These previous reports urged us to study the potential involvement of BAG3 in EMT triggered by FGF-2 in renal tubular epithelial cells. The current study found that FGF-2 induced EMT, simultaneously increased BAG3 expression in human kidney 2 (HK2) cells. Although FGF-2 induced EMT in nontransfected or scramble short hairpin RNA (shRNA) transfected HK2 cells, it was ineffective in BAG3-silenced cells, indicating a favorable role of BAG3 in EMT of tubular cells induced by FGF-2. Knockdown of BAG3 also significantly suppressed motion and invasion of HK2 cells mediated by FGF-2. Furthermore, we confirmed that BAG3 was upregulated in kidney of unilateral ureteral obstruction (UUO) rats, a well-established renal fibrosis model, in which EMT is supposed to exert a substantial influence on renal fibrosis. Importantly, upregulation of BAG3 was limited to tubular epithelial cells. Results of the current study identify BAG3 as a potential player in EMT of tubular epithelial cells, as well as renal fibrosis.

Heparanase is a key player in renal fibrosis by regulating TGF-β expression and activity

Epithelial–mesenchymal transition (EMT) of tubular cells is one of the mechanisms which contribute to renal fibrosis and transforming growth factor-β (TGF-β) is one of the main triggers. Heparanase (HPSE) is an endo-β-D-glucuronidase that cleaves heparan-sulfate thus regulating the bioavailability of growth factors (FGF-2, TGF-β). HPSE controls FGF-2-induced EMT in tubular cells and is necessary for the development of diabetic nephropathy in mice.The aim of this study was to investigate whether HPSE can modulate the expression and the effects of TGF-β in tubular cells.First we proved that the lack of HPSE or its inhibition prevents the increased synthesis of TGF-β by tubular cells in response to pro-fibrotic stimuli such as FGF-2, advanced glycosylation end products (AGE) and albumin overload.Second, since TGF-β may derive from sources different from tubular cells, we investigated whether HPSE modulates tubular cell response to exogenous TGF-β. HPSE does not prevent EMT induced by TGF-β although it slows its onset; indeed in HPSE-silenced cells the acquisition of a mesenchymal phenotype does not develop as quickly as in wt cells. Additionally, TGF-β induces an autocrine loop to sustain its signal, whereas the lack of HPSE partially interferes with this autocrine loop.Overall these data confirm that HPSE is a key player in renal fibrosis since it interacts with the regulation and the effects of TGF-β. HPSE is needed for pathological TGF-β overexpression in response to pro-fibrotic factors. Furthermore, HPSE modulates TGF-β-induced EMT: the lack of HPSE delays tubular cell transdifferentiation, and impairs the TGF-β autocrine loop.

A microRNA-30e/mitochondrial uncoupling protein 2 axis mediates TGF-β1-induced tubular epithelial cell extracellular matrix production and kidney fibrosis

Mitochondria dysfunction has been reported in various kidney diseases but how it leads to kidney fibrosis and how this is regulated is unknown. Here we found that mitochondrial uncoupling protein 2 (UCP2) was induced in kidney tubular epithelial cells after unilateral ureteral obstruction in mice and that mice with ablated UCP2 resisted obstruction-induced kidney fibrosis. We tested this association further in cultured NRK-52E cells and found that TGF-β1 remarkably induced UCP2 expression. Knockdown of UCP2 largely abolished the effect of TGF-β1, whereas overexpression of UCP2 promoted tubular cell phenotype changes. Analysis using a UCP2 mRNA-3′-untranslated region luciferase construct showed that UCP2 mRNA is a direct target of miR-30e. MiR-30e was downregulated in tubular cells from fibrotic kidneys and TGF-β1-treated NRK-52E cells. A miR-30e mimic significantly inhibited TGF-β1-induced tubular-cell epithelial–mesenchymal transition, whereas a miR-30e inhibitor imitated TGF-β1 effects. Finally, genipin, an aglycone UCP2 inhibitor, significantly ameliorated kidney fibrosis in mice. Thus, the miR-30e/UCP2 axis has an important role in mediating TGF-β1-induced epithelial–mesenchymal transition and kidney fibrosis. Targeting this pathway may shed new light for the future of fibrotic kidney disease therapy.

Polyphenols of Hibiscus sabdariffa Improved Diabetic Nephropathy via Attenuating Renal Epithelial Mesenchymal Transition

We previously reported that Hibiscus sabdariffa polyphenol extracts (HPE) are beneficial for diabetic nephropathy. Since an epithelial to mesenchymal transition (EMT) is critical in renal fibrosis, the present study aimed to investigate whether HPE could prevent EMT of tubular cells. Treatment of HPE reduced angiotensin II receptors (AT)-1 and transforming growth factor β1 (TGF-β1) evoked by high glucose and recovered the increased vimentin and decreased E-cadherin. HPE decreased fibronectin, thus avoiding EMT and accompanying fibrosis. AT-1 was upstream to TGF-β1, while there were recruitment signals between AT-1 and TGF-β1. Scan electron microscopy (SEM) and immunohistochemistry (IHC) revealed that the interacting filaments of tubular cells disappeared when treated with high glucose, and type IV collagen of tubulointerstitial decreased in diabetic kidneys. Treatment of HPE recovered morphological changes of cell junction and basement membrane. We suggest that HPE has the potential to be an adjuvant for diabetic nephropathy by regulating AT-1/TGF-β1 and EMT.

Astragaloside IV attenuates glycated albumin-induced epithelial-to-mesenchymal transition by inhibiting oxidative stress in renal proximal tubular cells

In diabetic kidney disease (DKD), epithelial-to-mesenchymal transition (EMT) is a classic pathological process in tubular damage. Oxidative stress is considered to play an important role in DKD. Astragaloside IV (A-IV), one of the main active ingredients of Astragalus membranaceus, exhibits a wide range of biological activities. However, the effect of A-IV on regulating EMT in tubular cells is unclear. This study aims to determine whether A-IV could attenuate glycated albumin (GA)-induced EMT in the NRK-52E cell line by inhibiting oxidative stress. GA and A-IV-induced cytotoxicity were assayed by CCK-8. The intercellular reactive oxygen species (ROS) level was detected by H2DCFDA. The activity of NADPH oxidase was assayed by adding exogenous NADPH oxidase, and the superoxide dismutase (SOD) units were observed by NBT. We used a microscope to examine the morphology of the NRK-52E cell line. We conducted a wound healing assay to measure cell mobility. To determine mRNA and protein expressions of α-SMA and E-cadherin, we used real-time polymerase chain reaction (real-time PCR), immunofluorescence, and western blot analysis. A-IV significantly attenuated GA-induced amplification of ROS, lowered the increased level of NADPH oxidase activity, and elevated the decreased level of SOD units. The GA-induced NRK-52E cell line showed increased expression of α-SMA and decreased expression of E-cadherin in mRNA and protein levels, whereas A-IV alleviated the expression of α-SMA and increased the expression of E-cadherin. Our data demonstrate that GA could induce NRK-52E cell line EMT through oxidative stress. This effect could be attenuated by A-IV via regulation of the impaired redox balance.

Matrix metalloproteinase-9 of tubular and macrophage origin contributes to the pathogenesis of renal fibrosis via macrophage recruitment through osteopontin cleavage

A pro-fibrotic role of matrix metalloproteinase-9 (MMP-9) in tubular cell epithelial–mesenchymal transition (EMT) is well established in renal fibrosis; however studies from our group and others have demonstrated some previously unrecognized complexity of MMP-9 that has been overlooked in renal fibrosis. Therefore, the aim of this study was to determine the expression pattern, origin and the exact mechanism underlying the contribution of MMP-9 to unilateral ureteral obstruction (UUO), a well-established model of renal fibrosis via MMP-9 inhibition. Renal MMP-9 expression in BALB/c mice with UUO was examined on day 1, 3, 5, 7, 9, 11 and 14. To inhibit MMP-9 activity, MMP-2/9 inhibitor or MMP-9-neutralizing antibody was administered daily for 4 consecutive days from day 0–3, 6–9 or 10–13 and tissues harvested at day 14. In UUO, there was a bi-phasic early- and late-stage upregulation of MMP-9 activity. Interestingly, tubular epithelial cells (TECs) were the predominant source of MMP-9 during early stage, whereas TECs, macrophages and myofibroblasts produced MMP-9 during late-stage UUO. Early- and late-stage inhibition of MMP-9 in UUO mice significantly reduced tubular cell EMT and renal fibrosis. Moreover, MMP-9 inhibition caused a significant reduction in MMP-9-cleaved osteopontin and macrophage infiltration in UUO kidney. Our in vitro study showed MMP-9-cleaved osteopontin enhanced macrophage transwell migration and MMP-9 of both primary TEC and macrophage induced tubular cell EMT. In summary, our result suggests that MMP-9 of both TEC and macrophage origin may directly or indirectly contribute to the pathogenesis of renal fibrosis via osteopontin cleavage, which, in turn further recruit macrophage and induce tubular cell EMT. Our study also highlights the time dependency of its expression and the potential of stage-specific inhibition strategy against renal fibrosis.