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Akt signaling pathway
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Knockdown of NLRC5 attenuates renal I/R injury in vitro through the activation of PI3K/Akt signaling pathway
T
Feng Hana,1, Yi Gaoa,d,1, Chen-guang Dinga,b,1, Xin-xin Xiaa,c, Yu-xiang Wanga, Wu-Jun Xuea,b, ⁎ Xiao-Ming Dinga,b, Jin Zhenga,b, Pu-Xun Tiana,b, a
Department of Kidney Transplantation, Hospital of Nephropathy, First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an, Shaanxi Province, China Institute of Organ Transplantation, Xi’an Jiaotong University, Xi’an, Shaanxi Province, China c Department of traditional Chinese medicine, First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an, Shaanxi Province, China d Department of nephrology, Xi’an Third Hospital, Xi’an, Shaanxi Province, China b
A R T I C LE I N FO
A B S T R A C T
Keywords: NLRC5 Kidney injury Oxidative stress Apoptosis
NLRC5, as the largest member of nucleotide-binding domain and leucine-rich repeat (NLR) family, was involved in various physiological processes, such as inflammation, fibrosis, innate immunity and diabetic nephropathy. However, the role of NLRC5 in acute kidney injury remains unclear. The aim of this study was to investigate the role of NLRC5 in human renal proximal tubular epithelial cells (HK-2) exposed to hypoxia/reoxygenation (H/R). Our results demonstrated that the expression of NLRC5 was significantly up-regulated in HK-2 cells exposed to H/R. Knockdown of NLRC5 significantly improved the viability of HK-2 cells exposed to H/R. In addition, knockdown of NLRC5 efficiently inhibited H/R-induced oxidative stress and apoptosis in HK-2 cells. Mechanistically, knockdown of NLRC5 markedly enhanced the activation of PIK3/Akt signaling pathway in H/Rstimulated HK-2 cells. In summary, our findings indicate that knockdown of NLRC5 attenuates renal I/R injury in vitro through the activation of PI3K/Akt signaling pathway.
1. Introduction Acute kidney injury is a common complication in clinical practice [1]. Renal ischemia-reperfusion (I/R) injury is a major cause of acute renal dysfunction. Although numerous efforts had been made to alleviate renal I/R injury, patients with renal I/R injury have a poor prognosis [2,3]. Renal tubular epithelial cells represent the major cell type constituting the entire tubulointerstitium. The injury of renal tubular epithelial cells is a prominent and characteristic feature of acute kidney injury [4]. Currently, the exact molecular mechanisms underlying renal I/R injury have not yet been completely elucidated. However, the pathophysiology of renal I/R injury involves a complex events, including inflammation, oxidative stress, apoptosis and autophagy [5]. Previous studies have demonstrated that oxidative stress plays critical roles in the process of renal I/R injury [6–8]. Thus, modulating oxidative stress is an effective therapeutic strategy for renal I/R injury. Nucleotide-binding domain and leucine-rich repeat (NLR) proteins are pattern-recognition receptors that play important roles in
inflammasome assembly, innate immune response, transcription activation and autophagy [9,10]. NLRC5, as the largest member of NLR family, is composed of three structural domains including the N-terminal atypical caspase activation and recruitment domain (CARD), the centrally located NACHT (named after NAIP, CIITA, HET-E, and TP-1 proteins) and 27 leucine-rich repeats (LRRs) at the C-terminal. Previous studies demonstrated that NLRC5 was involved in various physiological processes, such as inflammation, fibrosis and innate immunity [11–13]. A recent study by Luan et al. reported that knockdown of NLRC5 significantly fibrosis and inflammatory response during diabetic nephropathy [14]. However, the role of NLRC5 in acute kidney injury remains unclear. The aim of this study was to investigate the role of NLRC5 in human renal tubular epithelial cells exposed to hypoxia/reoxygenation (H/R). Our findings indicated that knockdown of NLRC5 attenuates renal I/R injury in vitro via the activation of PI3K/Akt signaling pathway.
Abbreviations: H/R, hypoxia/reoxygenation; I/R, ischemia-reperfusion; CARD, caspase activation and recruitment domain; siRNA, small interfering RNA; qRT-PCR, Quantitative realtime PCR; CCK-8, Cell-Counting Kit 8 ⁎ Corresponding author at: Department of Kidney Transplantation, Hospital of Nephropathy, First Affiliated Hospital of Medical College of Xi’an Jiaotong University, 277 Yanta West Road, Xi’an, Shaanxi Province, 710061, China. E-mail address: [email protected] (P.-X. Tian). 1 First authors (Feng Han, Yi Gao, and Chenguang Ding). https://doi.org/10.1016/j.biopha.2018.04.040 Received 5 March 2018; Received in revised form 5 April 2018; Accepted 5 April 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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2. Materials and methods 2.1. Cell culture Human renal tubular epithelial cells (HK-2) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; GibCo BRL, Grand Island, NY) with 10% fetal bovine serum (FBS; GibCo BRL), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a humidified incubator containing 5% (v/v) CO2. 2.2. H/R model HK-2 cells were incubated in 6-well plates with glucose-free DMEM, and then exposed to hypoxia in a humidified N2 flushed hypoxic chamber for 4 h. Subsequently, cells were maintained in complete DMEM and 21% O2 for reoxygenation. Control cells were maintained in DMEM in the incubator under normoxic conditions (95% air/5% CO2). 2.3. RNA interference and transfection The small interfering RNA (siRNA) targeting NLRC5 (si-NLRC5) and its negative control (si-NC) were synthesized by Sangon Biotech (Shanghai, China). For in vitro transfection, HK-2 cells were transfected with si-NLRC5 (2 μg) or si-NC (2 μg) using 4 μl Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Fig. 1. NLRC5 was upregulated in HK-2 cells subjected to H/R. HK-2 cells at a density of 1 × 104 cells/well were suffered with hypoxia/reoxygenation injury for 4 h/2 h. A and B, The expression of NLRC5 was measured using qRT-PCR and western blot analysis. All data are expressed as mean ± SD, n = 3. * p < 0.05 vs. control group.
2.4. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from HK-2 cells using TRIzol reagent (Invitrogen). The First-strand cDNA was synthesized from 1 μg of total RNA using PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Japan). The following primers were used to perform PCR: NLRC5, forward 5′-CAGATGGTGGAAACTTTTAGCC-3′ and reverse 5′-AACTTC CTTAGCACC TGGATCA-3′; GAPDH forward 5′-CTGCACCACCAACTGC TTAG-3′ and reverse 5′-AGGTCCACCACTGACACGTT-3′. We analyzed the relative quantity of mRNA using the 2−ΔΔCt method and the internal control to GAPDH.
values were expressed as a percentage of control.
2.7. Measurement of ROS content Intracellular ROS production was detected using the fluorescence probe 2′,7′-dichlorodihydrofluorescein diacetate (DCHF-DA, Jiancheng Biotech, Nanjing, China). Briefly, after treatment, HK-2 cells were washed twice with PBS, and then stained with 10 μM DCFH-DA for 30 min at 37 °C in the dark. Fluorescence of DCFH-DA was measured with a fluorescence microscope at 485 nm excitation and 535 nm emission.
2.5. Western blot Total proteins were extracted from HK-2 cells using RIPA lysis buffer (Takara Biotechnology, Dalian, China). A total of 20 μg proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on polyvinylidene difluoride membranes (Millipore Corp., Billerica, MA, USA). After blocking in TBS buffer (50 mmol/L NaCl, 10 mmol/L Tris, pH 7.4) containing 5% nonfat milk, the membranes were reacted with primary antibodies at 4 °C overnight. The primary antibodies were anti-Bcl-2, anti-Bax, anti-PI3K, anti-p-PI3K, anti-Akt, anti-p-Akt and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) at room temperature for 1 h. Finally, the immunoreactive protein bands were visualized using enhanced chemiluminescence reagents (Amersham, Little Chalfont, UK).
2.8. Measurement of malondialdehyde (MDA) level and SOD activity After treatment, the cell culture medium was centrifuged, and the supernatant was collected. The levels of MDA and SOD in the supernatant were measured using commercial kits (Beyotime, Jiangsu, China) according to the manufacturer’s instruction. The values of different MDA and SOD activities were expressed as a percentage of the control, respectively.
2.9. Apoptosis analysis by flow cytometry 2.6. Cell viability assay HK-2 cells were seeded in 6-well plates at a density of 1 × 105 cells/ well. After treatment, the cells were trypsinized and incubated in 500 μL of binding buffer containing 5 μL of Annexin V-FITC and 5 μL of propidium iodide (BD Biosciences, San Jose, CA, USA) for 30 min in the dark. Afterwards, the cells were analyzed with a flow cytometer (Invitrogen).
Cell viability was measured using a Cell-Counting Kit 8 (CCK-8; Dojindo, Kumamoto, Japan). In brief, the transfected HK-2 cells at a density of 1 × 104 cells/well were suffered with H/R injury for 4 h/2 h. Then, 10 μl CCK-8 solution was added into each well, and the plate was incubated for 3 h at 37 °C. The absorbance was read at 490 nm using a microplate reader (Bio-Rad, San Diego, CA, USA). The absorbance 223
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Fig. 2. Knockdown of NLRC5 improves the viability of HK-2 cells exposed to H/ R. HK-2 cells at a density of 1 × 104 cells/well were transfected with si-NC or siNLRC5 for 48 h, respectively. A and B, The expression of NLRC5 was measured using qRT-PCR and western blot analysis. C, The transfected HK-2 cells were suffered with hypoxia/reoxygenation injury for 4 h/2 h. Cell viability was measured using the CCK-8 assay. All data are expressed as mean ± SD, n = 3. * p < 0.05 vs. control group; # p < 0.05 vs. H/R group.
Fig. 3. Knockdown of NLRC5 inhibits H/R-induced oxidative stress in HK-2 cells. The transfected HK-2 cells were suffered with hypoxia/reoxygenation injury for 4 h/2 h. A, Intracellular ROS production was detected using the fluorescence probe 2′,7′-dichlorodihydrofluorescein diacetate (DCHF-DA). B and C, The levels of MDA and SOD were measured using commercial kits. All data are expressed as mean ± SD, n = 3. *p < 0.05 vs. control group; #p < 0.05 vs. H/R group.
2.10. Caspase-3 activity assay
3. Results
After treatment, the caspase-3 activity was detected using a caspase3 cellular activity assay kit (Merck, Darmstadt, Germany). The fluorescence released by active caspase-3 was measured by reading the plate using a wavelength of 405 nm.
3.1. NLRC5 was upregulated in HK-2 cells subjected to H/R We first examined the mRNA expression of NLRC5 in HK-2 cells exposed to H/R stimulation by qRT-PCR. As shown in Fig. 1A, the mRNA expression level of NLRC5 was significantly increased in H/Rstimulated HK-2 cells compared with that in un-treated control group. Similarly, the results of western blot analysis indicated that the protein expression of NLRC5 was also remarkably higher in H/R-stimulated HK2 cells (Fig. 1B).
2.11. Statistical analysis Statistical analysis was performed using the SPSS software (version 13.0; SPSS Inc., Chicago, IL, USA). Data are presented as mean ± SD. Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Bonferroni test for multiple groups or Student t test between two groups. P < 0.05 was considered significant.
3.2. Knockdown of NLRC5 improves the viability of HK-2 cells exposed to H/R To examine the effect of NLRC5 on HK-2 cell viability, we employed 224
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Fig. 4. Knockdown of NLRC5 inhibits H/R-induced HK-2 cell apoptosis. The transfected HK-2 cells were suffered with hypoxia/reoxygenation injury for 4 h/2 h. A, Cell apoptosis was detected using a flow cytometry assay after Annexin V-FITC/PI staining. B, Quantification of cell apoptosis. C, The expression of Bax and Bcl-2 was determined using western blot analysis. D, The relative protein expression levels of Bax and Bcl-2 were quantified using Image-Pro Plus 6.0 software. E, The caspase-3 activity was detected using a caspase-3 cellular activity assay kit. All data are expressed as mean ± SD, n = 3. *p < 0.05 vs. control group; #p < 0.05 vs. H/R group.
viability of HK-2 cells exposed to H/R (Fig. 2C).
siRNA against NLRC5 to knock down NLRC5 in HK-2 cells. As shown in Fig. 2A, the mRNA expression of NLRC5 was markedly decreased in HK2 cells transfected with si-NLRC5, compared to the si-NC group. Similarly, the results of western blot analysis indicated that si-NLRC5 efficiently reduced the protein expression of NLRC5 in HK-2 cells (Fig. 2B). Furthermore, we performed the CCK-8 assay to investigate the effect of NLRC5 on cell viability in HK-2 cells exposed to H/R. HK-2 cells exposed to H/R exhibited significantly decreased cell viability compared with the control. However, knockdown of NLRC5 notably improved the
3.3. Knockdown of NLRC5 inhibits H/R-induced oxidative stress in HK-2 cells To evaluate the effect of NLRC5 on oxidative damage in HK-2 cells exposed to H/R, we examined the levels of ROS, MDA and SOD using ELISA assay kits. The data revealed that a significant elevation in the ROS level in H/R-stimulated HK-2 cells compared to the control group. 225
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Fig. 5. Knockdown of NLRC5 enhanced the activation of PIK3/Akt signaling pathway in H/ R-stimulated HK-2 cells. A, The transfected HK2 cells were suffered with hypoxia/reoxygenation injury for 4 h/2 h. The levels of p-PI3K, PI3K, p-Akt and Akt were determined using western blot analysis. The transfected HK-2 cells were suffered with hypoxia/reoxygenation injury in the presence or absence of wortmannin (0.1 μM) for 30 min. B, ROS production was detected using the fluorescence probe DCHF-DA. C, The caspase-3 activity was detected using a caspase-3 cellular activity assay kit. All data are expressed as mean ± SD, n = 3. *p < 0.05 vs. control group; #p < 0.05 vs. H/R group, & p < 0.05 vs. H/R+si-NLRC5 group.
cells exposed to H/R.
However, knockdown of NLRC5 efficiently suppressed H/R-induced ROS level in HK-2 cells (Fig. 3A). Similarly, H/R-induced MDA level was prevented by si-NLRC5 in HK-2 cells (Fig. 3B). In contrast, knockdown of NLRC5 markedly reversed H/R-inhibited SOD activity in HK-2 cells (Fig. 3C).
4. Discussion To our best knowledge, this study was the first to show that the expression of NLRC5 was significantly up-regulated in HK-2 cells exposed to H/R. Knockdown of NLRC5 significantly improved the viability of HK-2 cells exposed to H/R. In addition, knockdown of NLRC5 efficiently inhibited H/R-induced oxidative stress and apoptosis in HK-2 cells. Mechanistically, knockdown of NLRC5 remarkably enhanced the activation of PIK3/Akt signaling pathway in H/R-stimulated HK-2 cells. H/R injury HK-2 cell model has been widely used as an in vitro renal I/R injury model. In this study, an in vitro H/R cell model was employed to evaluate the effect of NLRC5 on renal I/R injury. Our results found that NLRC5 was highly expressed in HK-2 cell exposed to H/R. These results suggest that NLRC5 might be involved in the progression of renal I/R injury. The excessive generation of ROS is related with the onset and development of renal I/R injury [15]. MDA is the end product of lipid peroxidation [16]. SOD is an antioxidant enzyme that acts against superoxide, and reduced levels of antioxidant enzymes including SOD were found in I/R-induced renal tissues [17]. Moreover, H/R is through to be a major stimulus contributing to oxidative stress in renal tubular epithelial cells [18,19]. In this study, we observed that H/R treatment significantly induced the levels of ROS and MDA, suppressed SOD activity in HK-2 cells, and these effects were attenuated by NLRC5 knockdown. These data strongly suggest that si-NLRC5 attenuated renal I/R injury in vitro through regulating the oxidative stress in HK-2 cells. Many studies have demonstrated that apoptosis is involved in the pathological process in renal I/R injury [20–22]. Bcl-2, known as a potent inhibitor of apoptosis, inhibits the mitochondria disruption and
3.4. Knockdown of NLRC5 inhibits H/R-induced HK-2 cell apoptosis Apoptosis is involved in the pathogenic process of renal I/R injury. Thus, we investigated the effect of NLRC5 on cell apoptosis in HK-2 cells in response to H/R. As indicated in Fig. 4A, HK-2 cells subjected to H/R showed a higher percentage of apoptotic cells than the control group; whereas knockdown of NLRC5 could repress H/R-induced cell apoptosis in HK-2 cells. In addition, as compared with the control group, H/R treatment significantly reduced the expression of Bcl-2 and increased the expression of Bax in HK-2 cells. However, these changes were significantly reversed by NLRC5 knockdown (Fig. 4C). Furthermore, we observed that knockdown of NLRC5 markedly reduced H/Rinduced caspase-3 activity in HK-2 cells (Fig. 4E). 3.5. Knockdown of NLRC5 enhanced the activation of PIK3/Akt signaling pathway in H/R-stimulated HK-2 cells PI3K/Akt signaling pathway plays an important role in renal I/R injury, thus, we investigated the effect of NLRC5 on PI3K/Akt pathway in HK-2 cells exposed to H/R. As shown in Fig. 5A, the phosphorylation levels of PI3K and Akt were moderately increased in H/R-stimulated HK-2 cells. Furthermore, knockdown of NLRC5 remarkably increased the phosphorylation of PI3K and Akt in H/R-stimulated HK-2 cells. And the PI3K inhibitor wortmannin remarkably counteracted the si-NLRC5attenuated oxidative stress (Fig. 5B) and cell apoptosis (Fig. 5C) in HK-2 226
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the subsequent Cyt c release, which further activates caspases activity [23]. Caspase-3 is a crucial mediator of programmed cell apoptosis [24]. It was reported that Bcl-2 protects tubular epithelial cells against I/R injury through the inhibition of apoptosis [25]. Furthermore, renal I/R injury can initiate complex events within the kidney that culminate in renal cell apoptosis [26]. Consistent with these previous results, the present study observed that H/R treatment significantly reduced the expression of Bcl-2 and increased the expression of Bax and caspase-3 activity in HK-2 cells, which were potently prevented by NLRC5 knockdown. These data imply that knockdown of NLRC5 attenuates renal I/R injury in vitro through suppressing cell apoptosis in HK-2 cells. The PI3K/Akt signaling pathway was involved in the pathogenesis of renal I/R injury [27–29]. Akt is a downstream kinase of PI3K that is activated by phosphorylation and plays an important role in cell survival and apoptosis [30]. Several studies demonstrated that I/R can induce the activation of PI3K/Akt, which promotes the viability of renal tubular epithelial cells [31,32]. In accordance with the previous studies, in the present study, we observed that H/R treatment moderately induced the phosphorylation levels of PI3K and Akt in HK-2 cells; knockdown of NLRC5 remarkably enhanced the phosphorylation of PI3K and Akt in H/R-exposed HK-2 cells. These results suggest that knockdown of NLRC5 attenuates renal I/R injury in vitro via the PI3K/ Akt pathway.
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5. Conclusion In summary, our findings indicate that knockdown of NLRC5 attenuates renal I/R injury in vitro through the activation of PI3K/Akt signaling pathway. Conflicts of interest The authors declare that they have no conflicts of interest. Acknowledgments This work was supported by the major basic research project of Shaanxi Province (No. 2017ZDJC-09); and the National Nature Science Foundation of China (No. 81670681). References [1] R. Bellomo, J.A. Kellum, C. Ronco, Acute kidney injury, Lancet 380 (2012) 756–766. [2] P.K. Chatterjee, Novel pharmacological approaches to the treatment of renal ischemia-reperfusion injury: a comprehensive review, Naunyn Schmiedebergs Arch. Pharmacol. 376 (2007) 1–43. [3] S. Helgadottir, M.I. Sigurdsson, R. Palsson, D. Helgason, G.H. Sigurdsson, T. Gudbjartsson, Renal recovery and long-term survival following acute kidney injury after coronary artery surgery: a nationwide study, Acta Anaesthesiol. Scand. 60 (2016) 1230–1240. [4] X.J. Liu, Q. Hong, Z. Wang, Y.Y. Yu, X. Zou, L.H. Xu, MicroRNA-34a suppresses autophagy in tubular epithelial cells in acute kidney injury, Am. J. Nephrol. 42 (2015) 168–175. [5] A.A. Sharfuddin, B.A. Molitoris, Pathophysiology of ischemic acute kidney injury, Nat. Rev. Nephrol. 7 (2011) 189–200. [6] V.G. Li, V. Sorrenti, P. Murabito, F. Galvano, M. Veroux, A. Gullo, R. Acquaviva, A. Stacchiotti, F. Bonomini, L. Vanella, Pharmacological induction of heme oxygenase-1 inhibits iNOS and oxidative stress in renal ischemia-reperfusion injury,
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Knockdown of NLRC5 attenuates renal I/R injury in vitro through the activation of PI3K/Akt signaling pathway
T
Feng Hana,1, Yi Gaoa,d,1, Chen-guang Dinga,b,1, Xin-xin Xiaa,c, Yu-xiang Wanga, Wu-Jun Xuea,b, ⁎ Xiao-Ming Dinga,b, Jin Zhenga,b, Pu-Xun Tiana,b, a
Department of Kidney Transplantation, Hospital of Nephropathy, First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an, Shaanxi Province, China Institute of Organ Transplantation, Xi’an Jiaotong University, Xi’an, Shaanxi Province, China c Department of traditional Chinese medicine, First Affiliated Hospital of Medical College of Xi’an Jiaotong University, Xi’an, Shaanxi Province, China d Department of nephrology, Xi’an Third Hospital, Xi’an, Shaanxi Province, China b
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A B S T R A C T
Keywords: NLRC5 Kidney injury Oxidative stress Apoptosis
NLRC5, as the largest member of nucleotide-binding domain and leucine-rich repeat (NLR) family, was involved in various physiological processes, such as inflammation, fibrosis, innate immunity and diabetic nephropathy. However, the role of NLRC5 in acute kidney injury remains unclear. The aim of this study was to investigate the role of NLRC5 in human renal proximal tubular epithelial cells (HK-2) exposed to hypoxia/reoxygenation (H/R). Our results demonstrated that the expression of NLRC5 was significantly up-regulated in HK-2 cells exposed to H/R. Knockdown of NLRC5 significantly improved the viability of HK-2 cells exposed to H/R. In addition, knockdown of NLRC5 efficiently inhibited H/R-induced oxidative stress and apoptosis in HK-2 cells. Mechanistically, knockdown of NLRC5 markedly enhanced the activation of PIK3/Akt signaling pathway in H/Rstimulated HK-2 cells. In summary, our findings indicate that knockdown of NLRC5 attenuates renal I/R injury in vitro through the activation of PI3K/Akt signaling pathway.
1. Introduction Acute kidney injury is a common complication in clinical practice [1]. Renal ischemia-reperfusion (I/R) injury is a major cause of acute renal dysfunction. Although numerous efforts had been made to alleviate renal I/R injury, patients with renal I/R injury have a poor prognosis [2,3]. Renal tubular epithelial cells represent the major cell type constituting the entire tubulointerstitium. The injury of renal tubular epithelial cells is a prominent and characteristic feature of acute kidney injury [4]. Currently, the exact molecular mechanisms underlying renal I/R injury have not yet been completely elucidated. However, the pathophysiology of renal I/R injury involves a complex events, including inflammation, oxidative stress, apoptosis and autophagy [5]. Previous studies have demonstrated that oxidative stress plays critical roles in the process of renal I/R injury [6–8]. Thus, modulating oxidative stress is an effective therapeutic strategy for renal I/R injury. Nucleotide-binding domain and leucine-rich repeat (NLR) proteins are pattern-recognition receptors that play important roles in
inflammasome assembly, innate immune response, transcription activation and autophagy [9,10]. NLRC5, as the largest member of NLR family, is composed of three structural domains including the N-terminal atypical caspase activation and recruitment domain (CARD), the centrally located NACHT (named after NAIP, CIITA, HET-E, and TP-1 proteins) and 27 leucine-rich repeats (LRRs) at the C-terminal. Previous studies demonstrated that NLRC5 was involved in various physiological processes, such as inflammation, fibrosis and innate immunity [11–13]. A recent study by Luan et al. reported that knockdown of NLRC5 significantly fibrosis and inflammatory response during diabetic nephropathy [14]. However, the role of NLRC5 in acute kidney injury remains unclear. The aim of this study was to investigate the role of NLRC5 in human renal tubular epithelial cells exposed to hypoxia/reoxygenation (H/R). Our findings indicated that knockdown of NLRC5 attenuates renal I/R injury in vitro via the activation of PI3K/Akt signaling pathway.
Abbreviations: H/R, hypoxia/reoxygenation; I/R, ischemia-reperfusion; CARD, caspase activation and recruitment domain; siRNA, small interfering RNA; qRT-PCR, Quantitative realtime PCR; CCK-8, Cell-Counting Kit 8 ⁎ Corresponding author at: Department of Kidney Transplantation, Hospital of Nephropathy, First Affiliated Hospital of Medical College of Xi’an Jiaotong University, 277 Yanta West Road, Xi’an, Shaanxi Province, 710061, China. E-mail address: [email protected] (P.-X. Tian). 1 First authors (Feng Han, Yi Gao, and Chenguang Ding). https://doi.org/10.1016/j.biopha.2018.04.040 Received 5 March 2018; Received in revised form 5 April 2018; Accepted 5 April 2018 0753-3322/ © 2018 Elsevier Masson SAS. All rights reserved.
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2. Materials and methods 2.1. Cell culture Human renal tubular epithelial cells (HK-2) were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM; GibCo BRL, Grand Island, NY) with 10% fetal bovine serum (FBS; GibCo BRL), 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA) at 37 °C in a humidified incubator containing 5% (v/v) CO2. 2.2. H/R model HK-2 cells were incubated in 6-well plates with glucose-free DMEM, and then exposed to hypoxia in a humidified N2 flushed hypoxic chamber for 4 h. Subsequently, cells were maintained in complete DMEM and 21% O2 for reoxygenation. Control cells were maintained in DMEM in the incubator under normoxic conditions (95% air/5% CO2). 2.3. RNA interference and transfection The small interfering RNA (siRNA) targeting NLRC5 (si-NLRC5) and its negative control (si-NC) were synthesized by Sangon Biotech (Shanghai, China). For in vitro transfection, HK-2 cells were transfected with si-NLRC5 (2 μg) or si-NC (2 μg) using 4 μl Lipofectamine 2000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Fig. 1. NLRC5 was upregulated in HK-2 cells subjected to H/R. HK-2 cells at a density of 1 × 104 cells/well were suffered with hypoxia/reoxygenation injury for 4 h/2 h. A and B, The expression of NLRC5 was measured using qRT-PCR and western blot analysis. All data are expressed as mean ± SD, n = 3. * p < 0.05 vs. control group.
2.4. Quantitative real-time PCR (qRT-PCR) Total RNA was extracted from HK-2 cells using TRIzol reagent (Invitrogen). The First-strand cDNA was synthesized from 1 μg of total RNA using PrimeScript RT reagent Kit with gDNA Eraser (TaKaRa, Japan). The following primers were used to perform PCR: NLRC5, forward 5′-CAGATGGTGGAAACTTTTAGCC-3′ and reverse 5′-AACTTC CTTAGCACC TGGATCA-3′; GAPDH forward 5′-CTGCACCACCAACTGC TTAG-3′ and reverse 5′-AGGTCCACCACTGACACGTT-3′. We analyzed the relative quantity of mRNA using the 2−ΔΔCt method and the internal control to GAPDH.
values were expressed as a percentage of control.
2.7. Measurement of ROS content Intracellular ROS production was detected using the fluorescence probe 2′,7′-dichlorodihydrofluorescein diacetate (DCHF-DA, Jiancheng Biotech, Nanjing, China). Briefly, after treatment, HK-2 cells were washed twice with PBS, and then stained with 10 μM DCFH-DA for 30 min at 37 °C in the dark. Fluorescence of DCFH-DA was measured with a fluorescence microscope at 485 nm excitation and 535 nm emission.
2.5. Western blot Total proteins were extracted from HK-2 cells using RIPA lysis buffer (Takara Biotechnology, Dalian, China). A total of 20 μg proteins were separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and blotted on polyvinylidene difluoride membranes (Millipore Corp., Billerica, MA, USA). After blocking in TBS buffer (50 mmol/L NaCl, 10 mmol/L Tris, pH 7.4) containing 5% nonfat milk, the membranes were reacted with primary antibodies at 4 °C overnight. The primary antibodies were anti-Bcl-2, anti-Bax, anti-PI3K, anti-p-PI3K, anti-Akt, anti-p-Akt and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Subsequently, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology) at room temperature for 1 h. Finally, the immunoreactive protein bands were visualized using enhanced chemiluminescence reagents (Amersham, Little Chalfont, UK).
2.8. Measurement of malondialdehyde (MDA) level and SOD activity After treatment, the cell culture medium was centrifuged, and the supernatant was collected. The levels of MDA and SOD in the supernatant were measured using commercial kits (Beyotime, Jiangsu, China) according to the manufacturer’s instruction. The values of different MDA and SOD activities were expressed as a percentage of the control, respectively.
2.9. Apoptosis analysis by flow cytometry 2.6. Cell viability assay HK-2 cells were seeded in 6-well plates at a density of 1 × 105 cells/ well. After treatment, the cells were trypsinized and incubated in 500 μL of binding buffer containing 5 μL of Annexin V-FITC and 5 μL of propidium iodide (BD Biosciences, San Jose, CA, USA) for 30 min in the dark. Afterwards, the cells were analyzed with a flow cytometer (Invitrogen).
Cell viability was measured using a Cell-Counting Kit 8 (CCK-8; Dojindo, Kumamoto, Japan). In brief, the transfected HK-2 cells at a density of 1 × 104 cells/well were suffered with H/R injury for 4 h/2 h. Then, 10 μl CCK-8 solution was added into each well, and the plate was incubated for 3 h at 37 °C. The absorbance was read at 490 nm using a microplate reader (Bio-Rad, San Diego, CA, USA). The absorbance 223
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Fig. 2. Knockdown of NLRC5 improves the viability of HK-2 cells exposed to H/ R. HK-2 cells at a density of 1 × 104 cells/well were transfected with si-NC or siNLRC5 for 48 h, respectively. A and B, The expression of NLRC5 was measured using qRT-PCR and western blot analysis. C, The transfected HK-2 cells were suffered with hypoxia/reoxygenation injury for 4 h/2 h. Cell viability was measured using the CCK-8 assay. All data are expressed as mean ± SD, n = 3. * p < 0.05 vs. control group; # p < 0.05 vs. H/R group.
Fig. 3. Knockdown of NLRC5 inhibits H/R-induced oxidative stress in HK-2 cells. The transfected HK-2 cells were suffered with hypoxia/reoxygenation injury for 4 h/2 h. A, Intracellular ROS production was detected using the fluorescence probe 2′,7′-dichlorodihydrofluorescein diacetate (DCHF-DA). B and C, The levels of MDA and SOD were measured using commercial kits. All data are expressed as mean ± SD, n = 3. *p < 0.05 vs. control group; #p < 0.05 vs. H/R group.
2.10. Caspase-3 activity assay
3. Results
After treatment, the caspase-3 activity was detected using a caspase3 cellular activity assay kit (Merck, Darmstadt, Germany). The fluorescence released by active caspase-3 was measured by reading the plate using a wavelength of 405 nm.
3.1. NLRC5 was upregulated in HK-2 cells subjected to H/R We first examined the mRNA expression of NLRC5 in HK-2 cells exposed to H/R stimulation by qRT-PCR. As shown in Fig. 1A, the mRNA expression level of NLRC5 was significantly increased in H/Rstimulated HK-2 cells compared with that in un-treated control group. Similarly, the results of western blot analysis indicated that the protein expression of NLRC5 was also remarkably higher in H/R-stimulated HK2 cells (Fig. 1B).
2.11. Statistical analysis Statistical analysis was performed using the SPSS software (version 13.0; SPSS Inc., Chicago, IL, USA). Data are presented as mean ± SD. Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Bonferroni test for multiple groups or Student t test between two groups. P < 0.05 was considered significant.
3.2. Knockdown of NLRC5 improves the viability of HK-2 cells exposed to H/R To examine the effect of NLRC5 on HK-2 cell viability, we employed 224
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Fig. 4. Knockdown of NLRC5 inhibits H/R-induced HK-2 cell apoptosis. The transfected HK-2 cells were suffered with hypoxia/reoxygenation injury for 4 h/2 h. A, Cell apoptosis was detected using a flow cytometry assay after Annexin V-FITC/PI staining. B, Quantification of cell apoptosis. C, The expression of Bax and Bcl-2 was determined using western blot analysis. D, The relative protein expression levels of Bax and Bcl-2 were quantified using Image-Pro Plus 6.0 software. E, The caspase-3 activity was detected using a caspase-3 cellular activity assay kit. All data are expressed as mean ± SD, n = 3. *p < 0.05 vs. control group; #p < 0.05 vs. H/R group.
viability of HK-2 cells exposed to H/R (Fig. 2C).
siRNA against NLRC5 to knock down NLRC5 in HK-2 cells. As shown in Fig. 2A, the mRNA expression of NLRC5 was markedly decreased in HK2 cells transfected with si-NLRC5, compared to the si-NC group. Similarly, the results of western blot analysis indicated that si-NLRC5 efficiently reduced the protein expression of NLRC5 in HK-2 cells (Fig. 2B). Furthermore, we performed the CCK-8 assay to investigate the effect of NLRC5 on cell viability in HK-2 cells exposed to H/R. HK-2 cells exposed to H/R exhibited significantly decreased cell viability compared with the control. However, knockdown of NLRC5 notably improved the
3.3. Knockdown of NLRC5 inhibits H/R-induced oxidative stress in HK-2 cells To evaluate the effect of NLRC5 on oxidative damage in HK-2 cells exposed to H/R, we examined the levels of ROS, MDA and SOD using ELISA assay kits. The data revealed that a significant elevation in the ROS level in H/R-stimulated HK-2 cells compared to the control group. 225
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Fig. 5. Knockdown of NLRC5 enhanced the activation of PIK3/Akt signaling pathway in H/ R-stimulated HK-2 cells. A, The transfected HK2 cells were suffered with hypoxia/reoxygenation injury for 4 h/2 h. The levels of p-PI3K, PI3K, p-Akt and Akt were determined using western blot analysis. The transfected HK-2 cells were suffered with hypoxia/reoxygenation injury in the presence or absence of wortmannin (0.1 μM) for 30 min. B, ROS production was detected using the fluorescence probe DCHF-DA. C, The caspase-3 activity was detected using a caspase-3 cellular activity assay kit. All data are expressed as mean ± SD, n = 3. *p < 0.05 vs. control group; #p < 0.05 vs. H/R group, & p < 0.05 vs. H/R+si-NLRC5 group.
cells exposed to H/R.
However, knockdown of NLRC5 efficiently suppressed H/R-induced ROS level in HK-2 cells (Fig. 3A). Similarly, H/R-induced MDA level was prevented by si-NLRC5 in HK-2 cells (Fig. 3B). In contrast, knockdown of NLRC5 markedly reversed H/R-inhibited SOD activity in HK-2 cells (Fig. 3C).
4. Discussion To our best knowledge, this study was the first to show that the expression of NLRC5 was significantly up-regulated in HK-2 cells exposed to H/R. Knockdown of NLRC5 significantly improved the viability of HK-2 cells exposed to H/R. In addition, knockdown of NLRC5 efficiently inhibited H/R-induced oxidative stress and apoptosis in HK-2 cells. Mechanistically, knockdown of NLRC5 remarkably enhanced the activation of PIK3/Akt signaling pathway in H/R-stimulated HK-2 cells. H/R injury HK-2 cell model has been widely used as an in vitro renal I/R injury model. In this study, an in vitro H/R cell model was employed to evaluate the effect of NLRC5 on renal I/R injury. Our results found that NLRC5 was highly expressed in HK-2 cell exposed to H/R. These results suggest that NLRC5 might be involved in the progression of renal I/R injury. The excessive generation of ROS is related with the onset and development of renal I/R injury [15]. MDA is the end product of lipid peroxidation [16]. SOD is an antioxidant enzyme that acts against superoxide, and reduced levels of antioxidant enzymes including SOD were found in I/R-induced renal tissues [17]. Moreover, H/R is through to be a major stimulus contributing to oxidative stress in renal tubular epithelial cells [18,19]. In this study, we observed that H/R treatment significantly induced the levels of ROS and MDA, suppressed SOD activity in HK-2 cells, and these effects were attenuated by NLRC5 knockdown. These data strongly suggest that si-NLRC5 attenuated renal I/R injury in vitro through regulating the oxidative stress in HK-2 cells. Many studies have demonstrated that apoptosis is involved in the pathological process in renal I/R injury [20–22]. Bcl-2, known as a potent inhibitor of apoptosis, inhibits the mitochondria disruption and
3.4. Knockdown of NLRC5 inhibits H/R-induced HK-2 cell apoptosis Apoptosis is involved in the pathogenic process of renal I/R injury. Thus, we investigated the effect of NLRC5 on cell apoptosis in HK-2 cells in response to H/R. As indicated in Fig. 4A, HK-2 cells subjected to H/R showed a higher percentage of apoptotic cells than the control group; whereas knockdown of NLRC5 could repress H/R-induced cell apoptosis in HK-2 cells. In addition, as compared with the control group, H/R treatment significantly reduced the expression of Bcl-2 and increased the expression of Bax in HK-2 cells. However, these changes were significantly reversed by NLRC5 knockdown (Fig. 4C). Furthermore, we observed that knockdown of NLRC5 markedly reduced H/Rinduced caspase-3 activity in HK-2 cells (Fig. 4E). 3.5. Knockdown of NLRC5 enhanced the activation of PIK3/Akt signaling pathway in H/R-stimulated HK-2 cells PI3K/Akt signaling pathway plays an important role in renal I/R injury, thus, we investigated the effect of NLRC5 on PI3K/Akt pathway in HK-2 cells exposed to H/R. As shown in Fig. 5A, the phosphorylation levels of PI3K and Akt were moderately increased in H/R-stimulated HK-2 cells. Furthermore, knockdown of NLRC5 remarkably increased the phosphorylation of PI3K and Akt in H/R-stimulated HK-2 cells. And the PI3K inhibitor wortmannin remarkably counteracted the si-NLRC5attenuated oxidative stress (Fig. 5B) and cell apoptosis (Fig. 5C) in HK-2 226
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the subsequent Cyt c release, which further activates caspases activity [23]. Caspase-3 is a crucial mediator of programmed cell apoptosis [24]. It was reported that Bcl-2 protects tubular epithelial cells against I/R injury through the inhibition of apoptosis [25]. Furthermore, renal I/R injury can initiate complex events within the kidney that culminate in renal cell apoptosis [26]. Consistent with these previous results, the present study observed that H/R treatment significantly reduced the expression of Bcl-2 and increased the expression of Bax and caspase-3 activity in HK-2 cells, which were potently prevented by NLRC5 knockdown. These data imply that knockdown of NLRC5 attenuates renal I/R injury in vitro through suppressing cell apoptosis in HK-2 cells. The PI3K/Akt signaling pathway was involved in the pathogenesis of renal I/R injury [27–29]. Akt is a downstream kinase of PI3K that is activated by phosphorylation and plays an important role in cell survival and apoptosis [30]. Several studies demonstrated that I/R can induce the activation of PI3K/Akt, which promotes the viability of renal tubular epithelial cells [31,32]. In accordance with the previous studies, in the present study, we observed that H/R treatment moderately induced the phosphorylation levels of PI3K and Akt in HK-2 cells; knockdown of NLRC5 remarkably enhanced the phosphorylation of PI3K and Akt in H/R-exposed HK-2 cells. These results suggest that knockdown of NLRC5 attenuates renal I/R injury in vitro via the PI3K/ Akt pathway.
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5. Conclusion In summary, our findings indicate that knockdown of NLRC5 attenuates renal I/R injury in vitro through the activation of PI3K/Akt signaling pathway. Conflicts of interest The authors declare that they have no conflicts of interest. Acknowledgments This work was supported by the major basic research project of Shaanxi Province (No. 2017ZDJC-09); and the National Nature Science Foundation of China (No. 81670681). References [1] R. Bellomo, J.A. Kellum, C. Ronco, Acute kidney injury, Lancet 380 (2012) 756–766. [2] P.K. Chatterjee, Novel pharmacological approaches to the treatment of renal ischemia-reperfusion injury: a comprehensive review, Naunyn Schmiedebergs Arch. Pharmacol. 376 (2007) 1–43. [3] S. Helgadottir, M.I. Sigurdsson, R. Palsson, D. Helgason, G.H. Sigurdsson, T. Gudbjartsson, Renal recovery and long-term survival following acute kidney injury after coronary artery surgery: a nationwide study, Acta Anaesthesiol. Scand. 60 (2016) 1230–1240. [4] X.J. Liu, Q. Hong, Z. Wang, Y.Y. Yu, X. Zou, L.H. Xu, MicroRNA-34a suppresses autophagy in tubular epithelial cells in acute kidney injury, Am. J. Nephrol. 42 (2015) 168–175. [5] A.A. Sharfuddin, B.A. Molitoris, Pathophysiology of ischemic acute kidney injury, Nat. Rev. Nephrol. 7 (2011) 189–200. [6] V.G. Li, V. Sorrenti, P. Murabito, F. Galvano, M. Veroux, A. Gullo, R. Acquaviva, A. Stacchiotti, F. Bonomini, L. Vanella, Pharmacological induction of heme oxygenase-1 inhibits iNOS and oxidative stress in renal ischemia-reperfusion injury,
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