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.     

TDAG51 mediates epithelial-to-mesenchymal transition in human proximal tubular epithelium

Epithelial-to-mesenchymal transition (EMT) contributes to renal fibrosis in chronic kidney disease. Endoplasmic reticulum (ER) stress, a feature of many forms of kidney disease, results from the accumulation of misfolded proteins in the ER and leads to the unfolded protein response (UPR). We hypothesized that ER stress mediates EMT in human renal proximal tubules. ER stress is induced by a variety of stressors differing in their mechanism of action, including tunicamycin, thapsigargin, and the calcineurin inhibitor cyclosporine A. These ER stressors increased the UPR markers GRP78, GRP94, and phospho-eIF2α in human proximal tubular cells. Thapsigargin and cyclosporine A also increased cytosolic Ca(2+) concentration and T cell death-associated gene 51 (TDAG51) expression, whereas tunicamycin did not. Thapsigargin was also shown to increase levels of active transforming growth factor (TGF)-β1 in the media of cultured human proximal tubular cells. Thapsigargin induced cytoskeletal rearrangement, β-catenin nuclear translocation, and α-smooth muscle actin and vinculin expression in proximal tubular cells, indicating an EMT response. Subconfluent primary human proximal tubular cells were induced to undergo EMT by TGF-β1 treatment. In contrast, tunicamycin treatment did not produce an EMT response. Plasmid-mediated overexpression of TDAG51 resulted in cell shape change and β-catenin nuclear translocation. These results allowed us to develop a two-hit model of ER stress-induced EMT, where Ca(2+) dysregulation-mediated TDAG51 upregulation primes the cell for mesenchymal transformation via Wnt signaling and then TGF-β1 activation leads to a complete EMT response. Thus the release of Ca(2+) from ER stores mediates EMT in human proximal tubular epithelium via the induction of TDAG51.     

Inhibitor of growth 2 regulates the high glucose-induced cell cycle arrest and epithelial-to-mesenchymal transition in renal proximal tubular cells

Journal of Physiology and Biochemistry - The epithelial-to-mesenchymal transition (EMT)-based tubulointerstitial fibrosis is the major pathological feature of diabetic kidney disease (DKD). While...     

Correction to: Inhibitor of growth 2 regulates the high glucose-induced cell cycle arrest and epithelial-to-mesenchymal transition in renal proximal tubular cells

Journal of Physiology and Biochemistry - The above article was published online first with the following errors which have now been corrected.     

Hepatocyte growth factor induces tubulogenesis of primary renal proximal tubular epithelial cells

Hepatocyte growth factor (HGF)-induced tubulogenesis has been demonstrated with renal epithelial cell lines grown in collagen gels but not with primary cultured renal proximal tubular epithelial cells (RPTEs). We show that HGF selectively induces proliferation and branching morphogenesis of primary cultured rat RPTEs. Additional growth factors including fibroblast growth factor (FGF)-1, epidermal growth factor (EGF), FGF-7, or insulin-like growth factor-1 (IGF-1) did not selectively induce tubulogenesis. However, when administered in combination, these factors initiated branching morphogenesis comparable to HGF alone and greatly augmented HGF-induced proliferation and branching. Microscopic analysis revealed that branching RPTEs were undergoing tubulogenesis and formed a polarized epithelium. TGF-β1 blocked HGF- or growth factor cocktail (GFC; HGF, FGF-1, EGF, IGF-1)-induced proliferation and branching morphogenesis. Adding TGF-β1 after GFC-induced tubulogenesis had occurred caused a progressive regression of the tubular structures, a response associated with an increase in apoptosis of the RPTEs. Primary cultured RPTEs are capable of undergoing HGF-induced tubulogenesis. Unlike cell lines, combinations of growth factors differentially augment the response. J. Cell. Physiol. 180:81–90, 1999. © 1999 Wiley-Liss, Inc.     

Distal tubular epithelial cells of the kidney: Potential support for proximal tubular cell survival after renal injury

The tubular epithelium of the kidney is susceptible to injury from many causes, such as ischemia-reperfusion and the associated oxidative stress, nephrotoxins, inflammatory and immune disorders and many others. The outcome is often acute kidney injury, which may progress to chronic kidney disease and fibrosis. Acute kidney injury involves not only direct injury to the distal tubular (DT) and proximal tubular (PT) epithelium during and immediately following the injurious event, but the closely-associated and sometimes dysfunctional renal vascular endothelium also plays an important part in modulating the tubular epithelial injury. In comparison with the PT, the DT epithelium is less sensitive to cell death, especially after ischemic injury. It is more prone to apoptosis than necrosis when it dies, and has key paracrine and autocrine functions in secreting an array of inflammatory, reparative, and survival cytokines that include chemotactic cytokines, polypeptide growth factors, and vasoactive peptides. In a neighborly way, the cytokines and growth factors secreted by the DT epithelium may then act positively on the ischemia-sensitive PT that has receptors to many of these proteins, but may not be able to synthesize them. A more complete understanding of these cellular events will allow protection against nephron destruction, regeneration leading to re-epithelialization of the injured tubules, or prevention of progression to chronic kidney disease. This review looks at these functions in the DT epithelial cells, specifically the cells in the medullary thick ascending limb of the loop of Henle, in contrast with those of the straight segment of the PT.     

MiR-4756 promotes albumin-induced renal tubular epithelial cell epithelial-to-mesenchymal transition and endoplasmic reticulum stress via targeting Sestrin2

Accumulating evidence indicates that proteinuria promotes the progression of diabetic kidney disease (DKD) and induces renal epithelial tubular cell epithelial-to-mesenchymal transition (EMT) and endoplasmic reticulum (ER) stress, but the mechanism remains unclear. In our previous research, we found that miR-4756 levels were increased in the urinary extracellular vesicles of type 2 diabetes mellitus patients with macroalbuminuria. In a preliminary study, we found that miR-4756 may be derived from renal tubular epithelial cells, but its role has not been elucidated. Albumin stimulation significantly increased miR-4756 levels in HK-2 cells. In addition, an miR-4756 mimic accelerated albumin-stimulated HK-2 cell EMT and ER stress, and an miR-4756 inhibitor suppressed these events. We then found that miR-4756 targeted the 3′-untranslated region (UTR) of Sestrin2 and directly suppressed Sestrin2 expression. Furthermore, the induction of EMT and ER stress by the overexpression of miR-4756 was abolished by Sestrin2 overexpression. Moreover, the overexpression of miR-4756 increased ERK1/2 activation and decreased 5′ monophosphate-activated protein kinase activation. Thus, our study provides evidence that miR-4756 accelerates the process of DKD through Sestrin2, suggesting that targeting miR-4756 may be a novel strategy for DKD treatment.     

Functional expression of intrinsic factor-cobalamin receptor by renal proximal tubular epithelial cells.

Previous studies from our laboratory (Seetharam, B., Levine, J. S., Ramasamy, M., and Alpers, D. H. (1988) J. Biol. Chem. 263, 4443-4449; Fyfe, J. C., Ramanujam, K. S., Ramaswamy, K., Patterson, D. F., and Seetharam, B. (1991) J. Biol. Chem. 266, 4489-4494) have identified and isolated a 230-kDa receptor from rat and canine kidney which binds with high affinity [57Co]cyanocobalamin (Cbl) complexed to gastric intrinsic factor (IF). Although these studies have identified a renal receptor which binds intrinsic factor-cobalamin (IFCR), it is not known whether the binding is specific for IF-Cbl and whether renal cells internalize [57Co]Cbl bound to IF and transport [57Co]Cbl across the cell. Using a variety of renal cells, our results show that IF-[57Co]Cbl binding activity is detected in proximal tubular-derived epithelial cells from opossum (OK) and porcine kidney (LLC-PK1) but not in distal tubular-derived cells from canine kidney cells (MDCK). Metabolic labeling studies with Tran 35S-label confirmed the presence of a 230-kDa IFCR in OK and LLC-PK1 cells. Cell surface labeling and binding studies demonstrated that IFCR is targeted to the apical membrane. This apical expression of IFCR in OK cells is inhibited by the microtubule-disruptive drugs, colchicine and nocodazole. Opossum kidney cells when grown on culture inserts are polarized and transport [57Co]Cbl only when bound to IF and not to other Cbl binders. Furthermore, the transport of [57Co]Cbl occurred unidirectionally from the apical to the basolateral surface. Treatment of cells with colchicine or nocodazole inhibited the surface binding of IF-[57Co]Cbl as well as the transcytosis of [57Co]Cbl by 70-75%. IFCR retained intracellualarly by incubation of cells with colchicine or nocodazole is degraded by leupeptin-sensitive proteases. Based on these results, we suggest that proximal tubular-derived epithelial cells transport [57Co]Cbl bound to IF in a saturable way via receptor-mediated endocytosis.     

Caveolar endocytosis is critical for BK virus infection of human renal proximal tubular epithelial cells

In recent years, BK virus (BKV) nephritis after renal transplantation has become a severe problem. The exact mechanisms of BKV cell entry and subsequent intracellular trafficking remain unknown. Since human renal proximal tubular epithelial cells (HRPTEC) represent a main natural target of BKV nephritis, analysis of BKV infection of HRPTEC is necessary to obtain additional insights into BKV biology and to develop novel strategies for the treatment of BKV nephritis. We coincubated HRPTEC with BKV and the cholesterol-depleting agents methyl beta cyclodextrin (MBCD) and nystatin (Nys), drugs inhibiting caveolar endocytosis. The percentage of infected cells (detected by immunofluorescence) and the cellular levels of BKV large T antigen expression (detected by Western blot analysis) were significantly decreased in both MBCD- and Nys-treated HPRTEC compared to the level in HRPTEC incubated with BKV alone. HRPTEC infection by BKV was also tested after small interfering RNA (siRNA)-dependent depletion of either the caveolar structural protein caveolin-1 (Cav-1) or clathrin, the major structural protein of clathrin-coated pits. BKV infection was inhibited in HRPTEC transfected with Cav-1 siRNA but not in HRPTEC transfected with clathrin siRNA. The colocalization of labeled BKV particles with either Cav-1 or clathrin was investigated by using fluorescent microscopy and image cross-correlation spectroscopy. The rate of colocalization of BKV with Cav-1 peaked at 4 h after incubation. Colocalization with clathrin was insignificant at all time points. These results suggest that BKV entered into HRPTEC via caveolae, not clathrin-coated pits, and that BKV is maximally associated with caveolae at 4 h after infection, prior to relocation to a different intracellular compartment.     

Inhibition of the Unfolded Protein Response by metformin in renal proximal tubular epithelial cells

All-trans retinoic acid (ATRA) induces cellular senescence via up-regulation of p16 and p21; however, the action mechanism of ATRA is unknown. Here, we show that ATRA induces promoter hypomethylation of p16 and p21 via down-regulation of DNA methyltransferases 1, 3a, and 3b to facilitate binding of Ets1/2 to the p16 promoter and p53 to the p21 promoter, resulting in up-regulation of their expression and subsequent induction of cellular senescence in HepG2 cells. These effects were mediated by retinoic acid receptor β₂ whose promoter was also hypomethylated in the presence of ATRA. Therefore, ATRA can be considered as an epi-drug in cancer therapy.     

Inhibition of the Unfolded Protein Response by metformin in renal proximal tubular epithelial cells

Metformin (Met), an AMP-activated protein kinase (AMPK) inducer, is primarily transported by organic cation transporters expressed at the surface of renal proximal tubular epithelial cells. However, the implication of Met in renal function remains poorly understood. Interestingly, AICAR, another AMPK inducer, has been shown to inhibit the Unfolded Protein Response (UPR) generated by tunicamycin in cardiomyocytes in an AMPK-kinase dependent fashion suggesting metformin may also block the UPR. In this work, we have examined the effect of metformin on the expression of UPR-related markers (GRP94 and CHOP) induced by glucosamine (GlcN), 2-deoxyglucose (2-DOG) and tunicamycin (TUNI) in renal proximal tubular epithelial cells and in murine mesangial cells. Met attenuated GRP94 and CHOP expression induced by GlcN and 2-DOG, but not TUNI only in renal epithelial cells, even though the AMPK activation was observed in both renal epithelial and mesangial cells. Met did not require the contribution of its AMPK kinase inducing activity to block UPR markers expression. This report has identified a novel inhibitory function of metformin on UPR, which may have a beneficial impact on kidney homeostatic function.     

Mitochondrial superoxide plays a crucial role in the development of mitochondrial dysfunction during high glucose exposure in rat renal proximal tubular cells

Diabetic nephropathy is the leading cause of end-stage renal disease in the United States. Despite several studies indicating a role for mitochondrial oxidative stress and mitochondrial dysfunction in the development of diabetic complications, the precise mechanisms underlying renal mitochondrial dysfunction and renal cell injury remain unclear. The hypothesis of the current study was that high-glucose-mediated generation of mitochondrial superoxide is a key early event that leads to mitochondrial injury in renal proximal tubular cells. To ascertain the role of mitochondrial superoxide we have tested whether overexpression of the primary mitochondrial antioxidant, manganese superoxide dismutase (MnSOD), protects against hyperglycemia-induced renal injury using normal rat renal proximal tubular cells (NRK). NRK cells were exposed to high glucose (25 mM) and the changes in the mitochondrial membrane potential, ATP levels, and superoxide generation and the loss of cell viability were measured at 24 and 48 h after high glucose exposure. Our results indicate that high glucose first induced superoxide generation and hyperpolarization in the mitochondria, followed by a secondary event, which involved a decline in ATP levels, partial Complex III inactivation, and loss of cell viability. These high-glucose-induced changes were completely prevented by overexpression of MnSOD in NRK cells. However, MnSOD activity was not changed after high glucose exposure in vitro or during the early stages of diabetes using the streptozotocin rat model. These findings show for the first time that hyperglycemic induction of superoxide production within the mitochondria initiates specific mitochondrial injury (i.e., Complex III) via a mechanism independent of MnSOD inactivation.     

Knockdown of thioredoxin-interacting protein ameliorates high glucose-induced epithelial to mesenchymal transition in renal tubular epithelial cells

Epithelial to mesenchymal transition (EMT) of tubular cells contributes to the renal accumulation of matrix protein that is associated with diabetic nephropathy. Both high glucose and transforming growth factor-β (TGF-β) are able to induce EMT in cell culture. In this study, we examined the role of the thioredoxin-interacting protein (TXNIP) on EMT induced by high glucose or TGF-β1 in HK-2 cells. EMT was assessed by the expression of α-smooth muscle actin (α-SMA) and E-cadherin and the induction of a myofibroblastic phenotype. High glucose (30 mM) was shown to induce EMT at 72 h. This was blocked by knockdown of TXNIP or antioxidant NAC. Meanwhile, we also found that knockdown of TXNIP or antioxidant NAC inhibited high glucose-induced generation of reactive oxygen species (ROS), phosphorylation of p38 MAPK and ERK1/2 and expression of TGF-β1. HK-2 cells that were exposed to TGF-β1 (4 ng/ml) also underwent EMT. The expression of TXNIP gene and protein was increased in HK-2 cells treated with TGF-β1. Transfection with TXNIP shRNA was able to attenuate TGF-β1 induced-EMT. These results suggested that knockdown of TXNIP antagonized high glucose-induced EMT by inhibiting ROS production, activation of p38 MAPK and ERK1/2, and expression of TGF-β1, highlighting TXNIP as a potential therapy target for diabetic nephropathy.     

Compartmentation of ATP within renal proximal tubular cells

Temperature-dependent spin changes of the heme iron atom on cytochrome P-450scc were studied by optical absorption and circular dichroism measurements. The optical absorption and circular dichroism spectra of cholesterol-free cytochrome P-450scc did not change between 10 and 26 degrees C. In contrast, the absorbance at 390 nm and the ellipticity at 330 nm of cholesterol-bound cytochrome P-450scc decreased upon temperature elevation, and the absorbance at 424 nm correspondingly increased. These spectral changes were reversible in respect of temperature. The far-ultraviolet circular dichroism spectra of both cholesterol-bound and -free cytochrome P-450scc were not affected by temperature. In addition, bound cholesterol molecule is not released from the cytochrome molecule by increasing temperature. From these results, we propose that temperature modulates specific interactions between the heme protein and bound cholesterol rather than the gross secondary structural changes of the protein.     

Reactive oxygen species-mediated endoplasmic reticulum stress contributes to aldosterone-induced apoptosis in tubular epithelial cells

Apoptosis contributes to tubular epithelial cell death and atrophy in aldosterone (Aldo)-induced renal injury. This study aimed to determine mechanisms underlying Aldo-induced reactive oxygen species (ROS) and endoplasmic reticulum (ER) stress in tubular epithelial cells. Intracellular ROS generation was evaluated by 2',7'-dichlorofluorescin diacetate fluorescence. Apoptosis was detected by annexin V/propidium iodide staining and flow cytometry. ER stress induced protein and mRNA were evaluated by Western blot and real-time PCR, respectively. Aldo promoted tubular epithelial cell apoptosis, increased intracellular ROS production and induced ER stress, as evidenced by increased expression of glucose-regulated protein 78 (GRP78) and CAAT/enhancer-binding protein homologous protein (CHOP) in a dose- and time-dependent manner. Additionally, siRNA knockdown of CHOP and antioxidant N-acetyl-l-cysteine (NAC) attenuated ER stress-mediated apoptosis. NAC also could inhibit Aldo-induced expression of GRP78 and CHOP. Altogether, these observations suggest that Aldo induces apoptosis via ROS-mediated, CHOP-dependent activation in renal tubular epithelial cells.     

Elevated oxidized glutathione in cystinotic proximal tubular epithelial cells

Cystinosis, the most frequent cause of inborn Fanconi syndrome, is characterized by the lysosomal cystine accumulation, caused by mutations in the CTNS gene. To elucidate the pathogenesis of cystinosis, we cultured proximal tubular cells from urine of cystinotic patients (n = 9) and healthy controls (n = 9), followed by immortalization with human papilloma virus (HPV E6/E7). Obtained cell lines displayed basolateral polarization, alkaline phosphatase activity, and presence of aminopeptidase N (CD-13) and megalin, confirming their proximal tubular origin. Cystinotic cell lines exhibited elevated cystine levels (0.86 +/- 0.95 nmol/mg versus 0.09 +/- 0.01 nmol/mg protein in controls, p = 0.03). Oxidized glutathione was elevated in cystinotic cells (1.16 +/- 0.83 nmol/mg versus 0.29 +/- 0.18 nmol/mg protein, p = 0.04), while total glutathione, free cysteine, and ATP contents were normal in these cells. In conclusion, elevated oxidized glutathione in cystinotic proximal tubular epithelial cell lines suggests increased oxidative stress, which may contribute to tubular dysfunction in cystinosis.