Knockdown of thioredoxin-interacting protein ameliorates high glucose-induced epithelial to mesenchymal transition in renal tubular epithelial cells
Introduction
Diabetic nephropathy (DN) is a leading worldwide cause of chronic kidney disease (CKD) and end-stage renal disease. The crucial pathology underlying progressive CKD in diabetes is tubulointerstitial fibrosis [1], [2]. Epithelial to mesenchymal transition (EMT), the transdifferentiation of tubular epithelial cells into myofibroblasts, is important in tubulointerstitial fibrosis [3], [4]. EMT of renal tubular epithelial cells, characterized by loss of epithelial phenotype and gain of profibrotic features that are characteristic of mesenchymal cells, has been implicated in the accelerated fibrogenesis that is associated with diabetic nephropathy [5], [6].
High glucose itself has been reported to be associated with most high glucose-induced effects and functional and structural changes of renal proximal tubular cells [7], [8], [9]. One of the potential mechanisms of high glucose-induced renal dysfunction is the EMT, which contributes to the generation of renal fibrosis [6]. Reactive oxygen species (ROS) are well recognized as important signaling molecules mediating renal injury in diabetes [10]. High glucose, advanced glycation end products (AGEs), angiotensin II (Ang II), and TGF-β1 all increase intracellular ROS in renal cells and contribute to the development and progression of diabetic renal injury [11], [12], [13], [14]. Previous studies have shown that ROS mediated high glucose, Ang II, and TGF-β1-induced EMT in renal tubular epithelial cells [14], [15], [16]. Therefore, approaches to reduce ROS generation may have therapeutic potential in renal injuries associated with diabetes.
Thioredoxin interacting protein (TXNIP) also known as vitamin D3 up-regulated protein-1 (VDUP-1) or thioredoxin binding protein-2 (TBP-2), is the endogenous inhibitor of cellular thioredoxin (TRX), inactivating its anti-oxidative function by binding to the redox-active cysteine residues [17]. Shah A et al. [18] reported that TXNIP mediated HG-induced ROS generation by mitochondria and the NADPH oxidase, Nox4, in mesangial cells. Our previous study showed that the expression of TXNIP mRNA and protein was increased in mouse mesangial cells under high glucose conditions, and, meanwhile, knockdown of TXNIP reversed high glucose-induced reduction of TRX activity and inhibited high glucose-induced ROS production and increased synthesis of TGF-β1 and fibronectin [19]. Previous studies have demonstrated that TXNIP was upregulated in renal tubular epithelial cells under high glucose conditions and in the kidneys of diabetic animals [20], [21]. Our recent study showed that TXNIP was involved in high glucose-induced ROS production and apoptosis in mouse mesangial cells [22]. However, the role of TXNIP in high glucose-induced EMT in tubular epithelial cells is unknown.
In the present study, we examined the expression of TXNIP in high glucose-treated HK-2 cells and the effects of knockdown of TXNIP with shRNA on high glucose or TGF-β1-induced EMT in vitro.
Section snippets
Reagents
d-glucose, mannitol and N-acetylcysteine (NAC) were obtained from Sigma (St. Louis, MO). Recombinant human TGF-β1 was purchased from Abgent (San Diego, CA). TXNIP and α-SMA antibodies were obtained from Abcam (Cambridge, UK). Antibodies for p38 mitogen-activated protein kinase (p38 MAPK), extracellular signal-regulated kinase1/2 (ERK1/2), p-p38 MAPK, p-ERK1/2 and E-cadherin were purchased from Cell Signaling Technology (Beverly, MA). TGF-β1 antibody was obtained from Proteintech (Chicago, IL).
High glucose induces TXNIP expression and EMT in HK-2 cells
To determine the role of high glucose in TXNIP expression in vitro, HK-2 cells were treated with high concentrations of glucose for varying periods in vitro and the levels of TXNIP protein and mRNA expression were determined. As shown in Fig. 1, HK-2 cells incubated with high glucose showed a time-dependent expression of TXNIP. The expression of TXNIP protein and mRNA significantly increased within 6 h and continued to increase up to 72 h. To evaluate high glucose-induced EMT, we observed the
Discussion
Excessive deposition of extracellular matrix (ECM) in the glomerular mesangium and tubulointerstitium is closely associated with progressive decline in renal function in diabetes [26], [27]. Tubulointerstitial lesions were more important in prognosis of diabetic nephropathy rather than glomerular lesions and EMT was commonly believed as one of the most important factors that could lead to renal fibrosis in diabetic nephropathy [6], [27]. It has been reported that EMT induced by high glucose or
Disclosures
All authors declare that no conflicts of interest exist.
Acknowledgment
This study was supported by the National Natural Science Foundation of China (No. 81170673; 81270804), Doctoral Program of Higher Education (20091323120001), Hebei Provincial Natural Science Fund (C2011206147), and Key Project of Hebei Education Department (ZD200906; 2010134).
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2020, Experimental Cell ResearchCitation Excerpt :Gao, K et al. [28] indicated that 5′-AMPK (adenosine monophosphate activated protein kinase) could prevent oxidative injury in podocytes by suppressing p38 MAPK signaling pathway via Trx, while, another literature illustrated that hyperglycemia could induce TXNIP in human aortic smooth muscle cells mediated by p38 MAPK, thus repress the scavenging function of Trx-ROS [29]. In our previous study, we knocked down TXNIP in human renal tubular cells and showed prevention of HG-induced epithelial to mesenchymal transition (EMT) through repressing p38 MAPK activation and TXNIP silencing in mouse mesangial cells reversed p38 MAPK activation induced by HG, and that, deletion of TXNIP reduced apoptosis of renal cells and p38 MAPK activation in kidneys of mouse with unilaterally obstructed ureter [30–32]. As such, we will explore whether TXNIP silencing could alleviate HG-induced apoptosis through p38 MAPK signaling in podocytes.
- 1
Contributed equally to this work.