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Original Report: Laboratory Investigation

Free Access

High Uric Acid-Induced Epithelial-Mesenchymal Transition of Renal Tubular Epithelial Cells via the TLR4/NF-kB Signaling Pathway

Liu H. · Xiong J. · He T. · Xiao T. · Li Y. · Yu Y. · Huang Y. · Xu X. · Huang Y. · Zhang J. · Zhang B. · Zhao J.

Author affiliations

Department of Nephrology, Institute of Nephrology of Chongqing and Kidney Center of PLA, Xinqiao Hospital, Third Military Medical University, Chongqing, China

Corresponding Author

Jinghong Zhao, MD, PhD

Department of Nephrology

Xinqiao Hospital, Third Military Medical University

Chongqing 400037 (China)

E-Mail zhaojh@tmmu.edu.cn

Related Articles for ""

Am J Nephrol 2017;46:333–342

Abstract

Background: Hyperuricemia is an independent risk factor for causing chronic kidney disease and contributes to kidney fibrosis. After urate crystals get deposited in the kidney, they can cause hyperuricemia nephropathy, leading to glomerular hypertrophy and renal tubular interstitial fibrosis. Recent data showed that uric acid (UA) could induce epithelial mesenchymal transition (EMT) of renal tubular cells, in which NRLP3 inflammatory pathway was involved. However, whether TLR4/NF-κB signaling pathway is also involved in EMT of renal tubular cells induced by UA is not clear. Methods: Human renal tubular epithelial cells (HK-2) were directly treated with UA and the phenotypic transition was detected by morphological changes and the molecular markers of EMT. The activation of the TLR4/NF-κB signaling pathway induced by UA was measured by Western blot and its involvement was further confirmed by the inhibition of NF-κB activation or knockdown of toll like receptor 4 (TLR4) expression. Results: UA induced obvious morphological changes of HK-2 cell, accompanied with altered molecular markers of EMT including fibronectin, α-SMA and E-cadherin. In addition, UA significantly upregulated the gene expression of interleukin-1β and tumor necrosis factor-α in a time- and dose-dependent manner. Furthermore, UA significantly activated the TLR4/NF-κB signaling pathway in HK-2 cells, while the inhibition of the TLR4 expression by siRNA and NF-κB activation by PDTC significantly attenuated EMT induced by UA in HK-2 cells. Conclusions: UA can induce EMT in renal tubular epithelial cells by the activation of the TLR4/NF-κB signaling pathway, and the targeted intervention of the TLR4/NF-κB signaling pathway might effectively inhibit UA-induced renal interstitial fibrosis mediated by EMT.

© 2017 S. Karger AG, Basel


Introduction

Uric acid (UA) is a biochemical product of purine metabolism, which is associated with gout and kidney calculi [1]. Hyperuricemia is a very common occurrence in chronic kidney disease (CKD) and associated with the development and progression of CKD [2]. At the same time, hyperuricemia is an independent risk factor for causing cardiovascular disease. CKD patients with hyperuricemia have higher risk for cardiovascular and all-cause mortality [3], while lowering UA can delay the progression of CKD. When UA gets deposited in the kidney for a long time, it can cause hyperuricemia nephropathy, leading to glomerular hypertrophy and renal tubular interstitial fibrosis [4]. Thus, UA is likely an important mediator and risk marker in CKD. Recent studies suggested a role of UA in the causation and progression of kidney fibrosis, a final common pathway in CKD [5].

Nowadays, multiple mechanisms leading to hyperuricemia nephropathy have been reported, such as endothelial dysfunction, renal angiotensin system activation, oxidative stress, and tubular epithelial cell transition [6]. Among them, epithelial mesenchymal transition (EMT) plays an important role in the renal interstitial fibrosis. EMT refers to a phenotypic transition of renal tubular cells, in which renal tubular cells lose their epithelial phenotypes and acquire new characteristic features of mesenchymal phenotypes. Studies have shown that long time deposition of UA in the kidney can cause renal tubular EMT and renal interstitial fibrosis [7]. Interestingly, EMT induced by UA was found to be associated with NLRP3 inflammatory pathway in a rat model of renal fibrosis [8]. More recently, a traditional Chinese recipe was confirmed to reduce serum UA levels and ameliorate renal fibrosis by the inhibition of EMT in a rat model [9]. Thus, EMT is one of the earliest phenomena of renal fibrosis and has been considered a therapeutic target due to its reversible characteristic features [10].

Both chronic inflammation and EMT is closely associated with renal interstitial fibrosis, while inflammation can be triggered by hyperuricemia [11]. Recent studies have found that toll like receptor 4 (TLR4) plays an essential role in the regulation of inflammation in diabetic nephropathy [12, 13], but whether it is involved in renal tubular EMT induced by hyperuricemia is not clear. The NF-κB is an important target gene of TLR4, which is responsible to produce inflammatory factors at the transcriptional level [14]. NF-κB signaling is the central cause of pathogenesis in many disease conditions, and it is one of the major targets for therapeutic intervention.

To further understand the mechanisms of UA-associated kidney disease, we investigated the effect of UA on phenotypic transition of renal tubule epithelial cells and elucidated the molecular mechanism in this work. We found that UA could induce EMT by activating the TLR4/NF-κB signaling pathway in HK-2 cells. This helps to understand the detrimental effects of UA and might provide a therapeutic target for CKD patients.

Materials and Methods

Human renal tubular epithelial cells (HK-2) were purchased from American Type Culture Collection. Antibody against α-SMA was obtained from Millipore (MA, USA) and other antibodies were purchased from BOSTER Biological Technology (Wuhan, China). Trizol RNA extraction reagent, reverse transcription kits, and SYBR Green polymerase chain reaction (PCR) Master Mix were obtained from Takara (Kyoto, Japan). PDTC was obtained from Beyotime Biotechnology (Nantong, China). All the primers were obtained from Shanghai Sangon Biotechnology (Shanghai, China).

HK-2 Cell Culture and Treatment

HK-2 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and grown in Dulbecco’s modified Eagle’s Medium/F-12 supplemented with 10% heat-inactivated fetal bovine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin (GIBCO, CA, USA) in a humidified atmosphere of 5% CO2 at 37°C.

For UA treatment, exponentially growing HK-2 cells were seeded at 4 × 105 cells/well in 6-well culture plates. UA (Ultrapure, Sigma) was dissolved in warm media and filtered. Before UA treatment, HK-2 cells were kept in serum-free media for 12 h prior to stimulation, and then changed with complete media with increasing concentration of UA for another 2 days. As for PDTC, HK-2 cells were pretreated with PDTC (20 μM) for 12 h and then further treated with UA.

Cell Morphological Observation

HK-2 cells were treated with UA for 48 h, and morphological changes were examined under an inverted phase contrast microscope (Axiovert 200; CarlZeiss, Oberkochen, Germany), and the images were obtained by a digital camera (AxioCam HRC; Carl Zeiss).

Total RNAs Extraction

Total RNAs were isolated from HK-2 cells using Trizol reagent according to the manufacturer’s instructions. The quality of the extracted RNA was monitored by formaldehyde agarose gel electrophoresis and the quantity was measured by Nanodrop spectrophotometer.

Real-Time RT-PCR

An amount of 0.5 µg RNA was reverse transcribed to cDNA according to the manufacturer’s directions (Roche). Real-time PCR (RT-PCR) was performed as previously described [15]. Sequence of primers could be available upon demand. Gene expression was normalized to the corresponding β-actin level and was presented as fold change relative to that of the control.

Western Blot Analysis

UA-treated HK-2 cells and controls were homogenized in ice-cold RIPA buffer containing complete protease inhibitors cocktail (Roche). The mixture was then centrifuged at 12,000 g at 4°C for 15 min and the protein concentration in the supernatant was determined using Bradford assay. Western blotting was performed as previously described [16]. The expression of each protein was normalized by that of β-actin.

Immunofluorescence Staining

Immunofluorescence staining was performed as previously described [16]. HK-2 cells were washed and fixed in 4% phosphate-buffered paraformaldehyde (25 min at room temperature) and permeabilized with 0.2% Triton X-100 in PBS (15 min). After being washed with PBS and blocking with 5% BSA for 1 h, cells were incubated with primary antibodies against E-cadherin or α-SMA in 5% BSA overnight at 4°C, followed by an incubation with goat anti-mouse IgG-FITC-conjugated secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature in the dark. The nucleus was counterstained with DAPI and the cells were visualized under the Axiovert 200 fluorescence microscope (Carl Ziss).

Transfection of siRNA

HK-2 cells were transfected with specific siRNA against TLR4 or control siRNA using Lipofectamine 2000 (Roche) according to the manufacturer’s instructions. Then cells were recovered and further cultured in media with or without UA for an indicated period.

Statistical Analysis

SPSS 20.0 was used for statistical analysis. All data are presented as means ± SD. The differences between 2 groups were determined by t test. Intergroup comparisons were made using one-wayanalysis of variance. A p value less than 0.05 was considered significant.

Results

High UA Induced Morphological Changes and EMT in HK-2 Cells

HK-2 cells were treated with increasing concentration of UA (0, 9, 12, and 15 mg/dL) for 48 h, and the morphology of HK-2 cells were observed by inverted light microscope. Compared with normal cultured HK-2 cells, UA-treated HK-2 cells were remodeled and the normal morphology was found to be with increased branches and intercellular space instead of a spindle shape (Fig. 1a). Furthermore, these changes were much apparent at higher concentration, showing a dose-dependent manner. In order to detect EMT induced by UA, the protein expression of fibronectin, α-SMA (mesenchymal cell marker) and E-cadherin (epithelial marker) were detected by Western blot. The results showed that the protein levels of fibronectin and α-SMA expression were significantly increased, while the epithelial marker E-cadherin expression was reduced, which was in a dose-dependent manner. This suggested that UA could induce EMT in HK-2 cells (Fig. 1b).

Fig. 1.

High UA induced morphological changes and EMT in HK-2 cells. Human HK-2 cells were treated with an increasing concentration of UA for 48 h, and the morphology of HK-2 observed by inverted light microscope (a). Total proteins were isolated for Western blot to detect the protein expression of fibronectin, α-SMA, and E-cadherin (b), and the density of bands were analyzed by quantity one (c). The scale bar: 100 μm. β-actin protein expression was determined as a control for equal loading. Western blotting was repeated at least 3 times. * p < 0.05, ** p < 0.01, *** p < 0.001.

/WebMaterial/ShowPic/884305

The Expression of Inflammatory Cytokines was Induced by High UA in HK-2 Cells

HK-2 cells were treated with increasing concentration of UA (0, 9, 12, and 15 mg/dL), and total RNA was harvested for quantitative RT-PCR analysis. As shown in Figure 2, mRNA expression of interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α) were upregulated by UA in a dose-dependent manner, compared with that of the control group, both at 24 and 48 h after treatment. Time course response showed that the upregulation of IL-1β and TNF-α could be detected at 12 h after treatment, with expression rising at later time points, which was clearly in a time-dependent manner. These results showed that high UA could induce the expression of inflammatory cytokines in HK-2 cells.

Fig. 2.

Gene expression of inflammatory cytokines was induced by high UA in HK-2 cells. Human HK-2 cells were treated with increasing concentration of UA for 24 h, and the total RNAs were isolated for real-time RT-PCR to detect the gene expression of IL-1β and TNF-α (a). To analyze the time-course response, HK-2 cells were treated with UA (15 mg/dL) for indicated periods, and then RNAs were isolated for real-time RT-PCR (b). * p < 0.05, ** p < 0.01, *** p < 0.001.

/WebMaterial/ShowPic/884303

NF-κB Was Involved in High UA-Induced EMT in HK-2 Cells

It was reported that the phosphorylated NF-κB expression was increased when NF-κB signal pathway was activated [17]. To detect whether NF-κB could be activated by UA, HK-2 cells were treated with UA for 48 h and the protein level of pNF-κB was measured by Western blot. Compared with that of the control group, the pNF-κB expression was significantly increased after incubated with 12 and 15 mg/dL UA (Fig. 3a). To further confirm the role of NF-κB in UA-induced EMT, PDTC, an inhibitor of NF-κB, was applied to pretreated HK-2 cells, followed by the incubation of 15 mg/dL UA. As shown in Figure 3b, the activation of NF-κB could be obviously inhibited by PDTC. Moreover, the altered protein levels of fibronectin, α-SMA, and E-cadherin induced by UA were also reversed by PDTC pretreatment. Furthermore, the expression of cytokeratin (a marker of epithelial cell) and Vimentin (an indicator of mesenchymal phenotype) was also detected by immunofluorescence. As shown in Figure 3d, e, the increased expression of Vimentin and the decreased expression of cytokeratin induced by UA were significantly reversed by PDTC pretreatment. In addition, we also measured the effect of PDTC on the expression of inflammatory cytokines. Compared with the levels of the control group, the elevated mRNA levels of IL-1β and TNF-α induced by UA were significantly attenuated by PDTC (Fig. 3f). All these data clearly showed that NF-κB was involved in UA-induced EMT in HK-2 cells.

Fig. 3.

NF-κB was involved in high UA-induced EMT in HK-2 cells. Human HK-2 cells were treated with an increasing concentration of UA for 24 h, and the total proteins were isolated for Western blot to detect the protein level of pNF-κB (a). To confirm the involvement of NF-κB, HK-2 cells were pretreated with PDTC (20 μM) and then further treated with UA (15 mg/dL) for 24 h, and the protein levels of pNF-κB, fibronectin, α-SMA, and E-cadherin were determined by Western blot (b), and the density of bands was analyzed by quantity one (c). Immunofluorescence staining of Vimentin and cytokeratin was carried out on similar treated HK-2 cells as described in materials and methods (d, e). For real-time RT-PCR, similar treated HK-2 cells were harvested and the gene expression of IL-1β and TNF-α was performed as described in materials and methods (f). The scale bar: 10 μm. Total NF-κB or β-actin protein expression was determined as a control for equal loading. Western blotting was repeated at least 3 times. * p < 0.05, ** p < 0.01, *** p < 0.001.

/WebMaterial/ShowPic/884301

Knockdown of TLR-4 Suppressed High UA-Induced EMT in HK-2 Cells

NF-κB can be activated by many upstream signals. Under the condition of chronic inflammation, TLR4 signaling cascade contributes to renal dysfunction [17]. Thus, we further investigated whether TLR4 was involved in high UA-induced EMT in HK-2 cells. The protein expression of TLR4 was detected by Western blot and result showed that UA induced TLR4 expression in a dose- and time-dependent manner (Fig. 4a, b). To further confirm the role of TLR4 in UA-induced EMT, we used siRNA to knockdown the expression of TLR4 in HK-2 cells prior to UA treatments. As shown in Figure 4c, the protein level of TLR4 could be efficiently downregulated by specific siRNA, and the phosphorylated NF-κB expression induced by UA was suppressed subsequently. Similarly, the protein markers of EMT induced by UA were also attenuated by TLR4 siRNA, as measured by Western blot (Fig. 4c, d). In addition, the elevated mRNA levels of IL-1β and TNF-α induced by UA were also significantly attenuated by TLR4 siRNA, while TLR4 siRNA alone had no such effects (Fig. 4e). These data suggest that the TLR4/NF-κB signal pathway plays an important role in UA-induced EMT in HK-2 cells.

Fig. 4.

Knockdown of TLR-4 suppressed high UA-induced EMT in HK-2 cells. Human HK-2 cells were treated with an increasing concentration of UA for 24 h (a) or with UA (15 mg/dL) for indicated periods (b). Total proteins were isolated for Western blot to detect the protein level of TLR4. To knockdown the expression of TLR4, HK-2 cells were transfected with TLR4 siRNA or control siRNA, and then further treated with UA for 24 h. Then the protein levels of TLR4, pNF-κB, fibronectin, α-SMA, and E-cadherin were detected by Western blot (c) and the density of bands were analyzed by quantity one (d). Gene expression of IL-1β and TNF-α was detected by real-time RT-PCR (e). Total NF-κB or β-actin protein expression was determined as a control for equal loading. Western blotting was repeated at least 3 times. * p < 0.05, ** p < 0.01, *** p < 0.001.

/WebMaterial/ShowPic/884299

Discussion

In CKD, hyperuricemia was previously thought to be a consequence of the declined glomerular filtration rate, rather than a risk factor for development or progression of kidney disease. However, recent epidemiological studies have shown that in healthy subjects UA itself can also promote the development of CKD [18]. When UA excretion is impaired, urate crystal deposition in kidneys, which was similar to gouty arthritis, can cause renal tubular epithelial cells injury, endothelial dysfunction, activation of renin angiotensin system and other pathological changes such as oxidative stress. All these alterations eventually lead to renal tubular interstitial fibrosis and glomerular sclerosis [19]. Chronic renal fibrosis is the common feature of CKD progression, and delaying or preventing renal fibrosis is the key intervention of CKD patients [20]. In recent years, studies of renal interstitial fibrosis are becoming more and more interesting. The EMT of renal tubule cells during the process of renal interstitial fibrosis plays a vital role [21]. However, the detailed molecular mechanisms have not been fully understood.

This study confirmed that UA could significantly induce EMT of renal tubular epithelial cells, including morphological changes as well as upregulation of mesenchymal markers. In a recent study, soluble UA was found to cause renal interstitial fibrosis closely associated with the NLRP3 inflammatory pathway [22]. Renal inherent cells and immune-regulating cells are the targets of inflammation. After injury, the renal inherent cells can produce a variety of cytokines and adhesion molecules as well as other inflammatory mediators, which mediated the infiltration and activation of CD4+ T cells, dendritic cells, and neutrophils. These immunological cells migrate to the kidney, and subsequently secreted IL-6, IL-1β, TNF-α, IL-18, IL-17, MCP-1, and chemokines, leading to amplify the inflammatory response [23]. A variety of inflammatory cytokines and chemokines are associated with all kinds of kidney diseases, including acute kidney injury and chronic renal fibrosis. A pervious study showed that soluble UA could stimulate monocytes produce IL-1β, IL-6, and TNF-α, and promote renal fibrosis [24]. TNF-α is a very important proinflammatory factor, which stimulates renal tubular epithelial cells to produce chemokines and recruitment of inflammatory cells [25, 26]. IL-1β and IL-6 are the vital cytokines secreted by inflammatory cells in the process of renal interstitial fibrosis, and is closely related to the development of renal fibrosis [27]. We detected the expression of IL-1β, IL-6, and TNF-α mRNA by R-T PCR, and the results showed that the expression of IL-1β and TNF-α mRNA was increased in a dose- and time-dependent manner except IL-6. The discrepancy of IL-6 gene expression might be related to UA concentration or the genetic background of the cell line. Except IL-6, the gene expression of other cytokines induced by UA in HK-2 cells is consistent with the finding of previous studies [28, 29].

TLR4, which is mainly expressed in glomerular mesangial cells and renal tubular epithelial cells, is a pattern recognition receptor, which is responsible for the detection and recognition of exogenous pathogen-associated molecular patterns and endogenous damage-associated molecular patterns. It is an important upstream regulatory factor of immunological and inflammatory response. As UA is thought to be a kind of damage-associated molecular pattern released from ischemic tissues and dying cells [22, 30], we speculate that UA can activate TLR4 directly. Studies have confirmed that TLR4 can promote the expression of a variety of inflammatory factors, and then activate inflammatory response by regulating the NF-κB signals. The NF-κB is an important transcription factor, involved in the immune regulation and differentiation [31]. During normal physiological conditions, the NF-κB is mainly expressed in the cell cytoplasm and inhibited by IκB. However, when cells were stimulated by cytokines, virus and oxidative stress, NF-κB was activated and then transferred to the nucleus to regulate gene transcription. Previous studies have showed that the NF-κB is closely related to a variety of signal transduction in cells, and also plays an important role in the process of development of renal interstitial fibrosis [32]. The NF-κB is extensively expressed in renal glomerular cells and renal tubular epithelial cells. The activation of NF-κB can mediate inflammatory response, promote renal tubular epithelial cells turn into fibroblasts cells, and upregulate the expression of a variety of inflammatory factors and chemokines including IL-6, IL-1β, and TNF-α, which results in the promotion of immune inflammatory reaction and renal tubule interstitial fibrosis [33, 34]. Our results found that UA could activate TLR4/NF-кB signaling pathways and induce EMT of renal tubular epithelial cells. At the same time, immunofluorescence also confirmed that UA can induce the upregulation of Vimentin and cytokeratin in HK-2 cells, while PDTC or TLR4 siRNA pretreatment could reverse the EMT induced by UA. The results showed that inhibition of TLR4/NF-κB signaling could reverse EMT and renal interstitial fibrosis induced by UA.

In summary, we demonstrated that UA could activate the TLR4/NF-κB signaling pathway, which in turn promoted the production of inflammatory cytokines. Subsequently, these cytokines contributed to the EMT of renal tubular epithelial cells. The inhibition of TLR4/NF-κB activation attenuated UA-induced EMT and inflammatory cytokines expression in HK-2 cells, which might be a potential therapeutic target for hyperuricemia nephropathy.

Acknowledgments

This study was supported by research grants from the National Key Technology Research and Development (R&D) Program (No. 2015BAI12B05), the National Natural Science Foundation of China (No.81600569, 81500561, 81500544), and the project for Chongqing basic science and advanced technology research (cstc2015jcyjBX0028).

Disclosure Statement

The authors declare that there are no conflicts of interest to disclose.


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Author Contacts

Jinghong Zhao, MD, PhD

Department of Nephrology

Xinqiao Hospital, Third Military Medical University

Chongqing 400037 (China)

E-Mail zhaojh@tmmu.edu.cn


Article / Publication Details

First-Page Preview
Abstract of Original Report: Laboratory Investigation

Received: July 17, 2017
Accepted: September 20, 2017
Published online: October 10, 2017
Issue release date: October 2017

Number of Print Pages: 10
Number of Figures: 4
Number of Tables: 0

ISSN: 0250-8095 (Print)
eISSN: 1421-9670 (Online)

For additional information: https://www.karger.com/AJN


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