Stable Expression of Hypoxia-Inducible Factor-1α in Human Renal Proximal Tubular Epithelial Cells Promotes Epithelial to Mesenchymal Transition

doi:10.1016/j.transproceed.2013.06.024

Transplantation Proceedings

Volume 46, Issue 1, January–February 2014, Pages 130-134

https://doi.org/10.1016/j.transproceed.2013.06.024 Get rights and content

Abstract

Background

Late kidney allograft dysfunction is becoming a significant problem and tubular atrophy and interstitial fibrosis are main causes. It was reported that hypoxia could induce epithelial—mesenchymal transition (EMT) in renal tubular epithelial cells (TECs), and hypoxia-inducible factor-1 (HIF-1) is one of the important regulators of cellular adaptive to hypoxia.

Methods

In this study, we used an HIF-1αΔODD–expressing adenovirus, which could stably and functionally express HIF-1α under normoxia conditions, and used a hypoxia/reoxygenation cell model to simulate ischemia/reperfusion (I/R) injury in vitro, to investigate the effect of HIF-1α on EMT-related gene expressions.

Results

Our results demonstrated that HIF-1α could significantly upregulate α-smooth muscle actin expression, and reduced the E-cadherin expression in HK-2 cells during I/R injury. Moreover, miR-21 expression had a positive correlation with HIF-1α in this process.

Conclusion

These results suggest that HIF-1α may promote the EMT development through regulating fibrotic gene expression during I/R injury in human renal TECs, and miR-21 could be among the important regulatory pathways in the process.

Section snippets

Gene Cloning, Modification, and Adenoviral Vectors Construction

Ad.CMV.HIF-1α, Ad.CMV.HIF-1αΔODD, and Ad.CMV.LacZ (recombinant adenovirus carrying β-galactosidase DNA, generated as a control adenoviral construct), which encode the HIF-1α, HIF-1αΔODD, and β-galactosidase, respectively, were constructed as described previously [7]. The transduction protocol was performed as previously described [8].

Cell Culture and Hypoxia Protocol

Human renal proximal TECs (HK-2) were purchased from American Type Culture Collection (ATCC, Manassas, VA). Hypoxia protocol was performed as previously described

HIF-1α Mediates the Upregulation of α-SMA Expression in Response to Hypoxia on HK-2 Cells

Our previous study demonstrated that Ad.CMV.HIF-1αΔODD could mediate stable and functional HIF-1 transcriptional activity, regardless of oxygen tension on HK-2 cells [8]. In this study, our real-time polymerase chain reaction results showed that α-SMA mRNA expression level in Ad.CMV.HIF-1αΔODD group was significantly increased when compared with Ad.CMV.LacZ and control groups 72 hours after transduction (P < .05; Figure 1A), and kept increasing at 96, 120, and 144 hours. And α-SMA mRNA

Discussion

I/R injury is an inevitable process during transplantation. EMT plays an important role in the initiation of fibrogenesis during I/R injury [1]. E-cadherin, as a key component of cell–cell adhesion junctions, is essential for the formation of epithelia during maintenance of adult epithelial homeostasis. Downregulated expression of E-cadherin is consistently detected during EMT and thus represents an important molecular event during this process [10]. In this study, our results demonstrated that

Acknowledgments

Supported by the Guizhou Province Science and Technology Agency Fund ([2012]2232).

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However, prolonged HIF-1α activation exerts deleterious effects on the kidney, even if the causal mechanism is hypoxia or ischemia (Figure 2) (72). Sustained upregulation of HIF-1α promotes epithelial to mesenchymal transition in renal tubular epithelial cells (73). In chronic kidney disease, sustained signaling through HIF-1α promotes proinflammatory and profibrotic pathways in the glomerulus and renal tubules (72,74,75), which is characterized by activation of inflammation-related cytokines, profibrotic gene transcription, macrophage infiltration and collagen deposition, mesangial cell proliferation, and tubulo-interstitial inflammation (74−77).
Hypoxia-inducible factor (HIF)-1α and HIF-2α promote cellular adaptation to acute hypoxia, but during prolonged activation, these isoforms exert mutually antagonistic effects on the redox state and on proinflammatory pathways. Sustained HIF-1α signaling can increase oxidative stress, inflammation, and fibrosis, actions that are opposed by HIF-2α. Imbalances in the interplay between HIF-1α and HIF-2α may contribute to the progression of chronic heart failure, atherosclerotic and hypertensive vascular disorders, and chronic kidney disease. These disorders are characterized by activation of HIF-1α and suppression of HIF-2α, which are potentially related to mitochondrial and peroxisomal dysfunction and suppression of the redox sensor, sirtuin-1. Hypoxia mimetics can potentiate HIF-1α and/or HIF-2α; ideally, such agents should act preferentially to promote HIF-2α while exerting little effect on or acting to suppress HIF-1α. Selective activation of HIF-2α can be achieved with drugs that: 1) inhibit isoform-selective prolyl hydroxylases (e.g., cobalt chloride and roxadustat); or 2) promote the actions of the redox sensor, sirtuin-1 (e.g., sodium-glucose cotransporter 2 inhibitors). Selective HIF-2α signaling through sirtuin-1 activation may explain the effect of sodium-glucose cotransporter 2 inhibitors to simultaneously promote erythrocytosis and ameliorate the development of cardiomyopathy and nephropathy.
Ischemia-reperfusion injury in renal transplantation: 3 key signaling pathways in tubular epithelial cells
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Luo et al.49 demonstrated that HIF-1α upregulated α-smooth muscle actin expression and reduced E-cadherin expression in an in vitro model of IRI. The microRNA miR-21 was positively correlated with HIF-1α, suggesting that miR-21 may be a regulatory factor in the process by which HIF-1α promotes epithelial to mesenchymal transition in IRI.49 PHD inhibitors have been studied therapeutically to protect against ischemia through their mechanism of HIF stabilization.50
Renal ischemia-reperfusion injury (IRI) is a significant clinical challenge faced by clinicians perioperatively in kidney transplantation. Recent work has demonstrated the key importance of transmembrane receptors in the injured tubular epithelial cell, most notably Toll-like receptors, activated by exogenous and endogenous ligands in response to external and internal stresses. Through sequential protein-protein interactions, the signal is relayed deep into the core physiological machinery of the cell, having numerous effects from upregulation of pro-inflammatory gene products through to modulating mitochondrial respiration. Inter-pathway cross talk facilitates a co-ordinated response at an individual cellular level, as well as modulating the surrounding tissue’s microenvironment through close interactions with the endothelium and circulating leukocytes. Defining the underlying cellular cascades involved in IRI will assist the identification of novel interventional targets to attenuate IRI with the potential to improve transplantation outcomes. We present a focused review of 3 key cellular signalling pathways in the injured tubular epithelial cell that have been the focus of much research over the past 2 decades: toll-like receptors, sphingosine-1-phosphate receptors and hypoxia inducible factors. We provide a unique perspective on the potential clinical translations of this recent work in the transplant setting. This is particularly timely with the recent completion of phase I and ongoing phase 2 clinical trials of inhibitors targeting specific components of these signaling cascades.
Slit2 ameliorates renal inflammation and fibrosis after hypoxia-and lipopolysaccharide-induced epithelial cells injury in vitro
2017, Experimental Cell Research
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Hypoxia is an important micro-environmental factor that induces the expression of Epithelial-Mesenchymal Transition (EMT) regulators [1,2].
Hypoxic acute kidney injury (AKI) is often incompletely repaired and leads to chronic kidney disease (CKD), which is characterized by tubulointerstitial inflammation and fibrosis. The Slit2 family of secreted glycoproteins is expressed in the kidney, it has been shown to exert an anti-inflammatory activity and prevent ischemic renal injury in vivo. However, whether Slit2 reduces renal fibrosis and inflammation after hypoxic and inflammatory epithelial cells injury in vitro remains unknown. In this study, we aimed to evaluate whether Slit2 ameliorated fibrosis and inflammation in two renal epithelial cells line challenged with hypoxia and lipopolysaccharide (LPS). Renal epithelial cells were treated with hypoxia and LPS to induce cell injury. Hoechst staining and Western blot analysis was conducted to examine epithelial cells injury. Immunofluorescence staining and Western blot analysis was performed to evaluate tubulointerstitial fibrosis. Real-time polymerase chain reaction (PCR) tested the inflammatory factor interleukin (IL)−1β and tumor necrosis factor (TNF)-α, and Western blot analysis determined the hypoxia-inducible factor (HIF)−1α, Toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB. Results revealed that hypoxia induced epithelial cells apoptosis, inflammatory factor IL-1β and TNF-α release and tubulointerstitial fibrosis. LPS could exacerbate hypoxia -induced epithelial cells apoptosis, IL-1β and TNF-α release and fibrosis. Slit2 reduced the expression of fibronectin, the rate of epithelial cell apoptosis, and the expression of inflammatory factor. Slit2 could also inhibit the expression of TLR4 and NF-κB, but not the expression of HIF-1α. Therefore, Slit2 attenuated inflammation and fibrosis after LPS- and hypoxia-induced epithelial cells injury via the TLR4/NF-κB signaling pathway, but not depending on the HIF-1α signaling pathway.
Pharmacological models and approaches for pathophysiological conditions associated with hypoxia and oxidative stress
2016, Pharmacology and Therapeutics
Citation Excerpt :
Second, as a consequence of capillary loss and capillary hypoperfusion, tissue oxygen tensions usually decline in a diseased kidney (Schrier, 2010). Third, low oxygen tensions may not only impair energy generation, but also act as a regulator of cellular functions and as a specific stimulus for the induction of certain genes (Luo et al., 2014). Altogether, these findings suggest that hypoxia is an important factor in the progression of kidney disease.
Hypoxia is the failure of oxygenation at the tissue level, where the reduced oxygen delivered is not enough to satisfy tissue demands. Metabolic depression is the physiological adaptation associated with reduced oxygen consumption, which evidently does not cause any harm to organs that are exposed to acute and short hypoxic insults. Oxidative stress (OS) refers to the imbalance between the generation of reactive oxygen species (ROS) and the ability of endogenous antioxidant systems to scavenge ROS, where ROS overwhelms the antioxidant capacity. Oxidative stress plays a crucial role in the pathogenesis of diseases related to hypoxia during intrauterine development and postnatal life. Thus, excessive ROS are implicated in the irreversible damage to cell membranes, DNA, and other cellular structures by oxidizing lipids, proteins, and nucleic acids. Here, we describe several pathophysiological conditions and in vivo and ex vivo models developed for the study of hypoxic and oxidative stress injury. We reviewed existing literature on the responses to hypoxia and oxidative stress of the cardiovascular, renal, reproductive, and central nervous systems, and discussed paradigms of chronic and intermittent hypobaric hypoxia. This systematic review is a critical analysis of the advantages in the application of some experimental strategies and their contributions leading to novel pharmacological therapies.
HIF-1α participates in the regulation of S100A16-HRD1-GSK3β/CK1α pathway in renal hypoxia injury
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: L.L. and G.L. are considered co-first authors of this paper.

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New vistas in transplantationRenal transplantationStable Expression of Hypoxia-Inducible Factor-1α in Human Renal Proximal Tubular Epithelial Cells Promotes Epithelial to Mesenchymal Transition

Abstract

Background

Methods

Results

Conclusion

Section snippets

Gene Cloning, Modification, and Adenoviral Vectors Construction

Cell Culture and Hypoxia Protocol

HIF-1α Mediates the Upregulation of α-SMA Expression in Response to Hypoxia on HK-2 Cells

Discussion

Acknowledgments

Plasmid

BMC Cell Biol

Am J Pathol

J Biol Chem

Kidney Int

Evidence that fibroblasts derive from epithelium during tissue fibrosis

J Clin Invest

New vistas in transplantation
Renal transplantation
Stable Expression of Hypoxia-Inducible Factor-1α in Human Renal Proximal Tubular Epithelial Cells Promotes Epithelial to Mesenchymal Transition