Introduction
Chronic kidney disease (CKD) is a growing health problem worldwide (1), which manifests as a progressive loss of renal function. Numerous factors, including oxidative stress, systemic inflammation, hypertension, and dyslipidemia, contribute to the onset and progression of CKD (2). Lipid abnormalities in patients with CKD usually consist of reduced high-density lipoprotein (HDL) concentrations and elevated plasma triglyceride, low-density lipoprotein (LDL), and oxidized lipids and lipoproteins (3). HDL facilitates cholesterol efflux from peripheral tissues and delivers cholesterol to the liver for excretion, therefore it has a key role in reverse cholesterol transport (4). HDL confers protection from cardiovascular disease due to its high anti-oxidant and anti-inflammatory activities. LDL has converse roles to HDL in the regulation of cholesterol transport. Oxidized LDL is capable of inducing inflammatory responses and is therefore implicated in the progression of numerous inflammatory disorders, such as atherosclerosis (5) and CKD (3).
The endoplasmic reticulum (ER) is a central organelle of eukaryotic cells and has principle functions in lipid synthesis and protein folding, maturation, and transport. Disturbances to the normal functions of the ER (e.g. due to redox dysregulation, aberrant calcium regulation, and glucose deprivation) may result in the accumulation of unfolded or misfolded proteins in the ER, a state known as ER stress (6). Three major ER stress response transducers have previously been identified: Activating transcription factor-6, inositol-requiring enzyme-1α (IRE1α), and protein kinase RNA-like endoplasmic reticulum kinase (7). IRE1α is activated by homodimerization and autophosphorylation under ER stress. It functions as an endoribonuclease which splices X-box-binding protein-1 (XBP-1) mRNA, yielding a potent transcriptional activator. Splicing of XBP-1 has previously been shown to initiate the transcription of genes involved in protein folding, transport and ER-associated protein degradation (7).
ER stress initially serves as an adaptive response but may lead to cell death, if severe or prolonged ER dysfunction occurs. Previous research has demonstrated the importance of ER stress in the pathophysiology of both acute and chronic kidney diseases (8). Chiang et al (9) reported that overwhelming ER stress may induce renal cell apoptosis and subsequent fibrosis. However, numerous studies have recently supported a protective role of the induction of ER stress in cell damage (10–12). Li et al (10) reported that ER stress preconditioning mitigated retinal endothelial inflammation, which was mediated through activation of the XBP-1-mediated unfolded protein response (UPR) and inhibition of NF-κB signaling. In a rat model, preconditioning with ER stress ameliorated mesangioproliferative glomerulonephritis (12). These previous findings suggest that transiently targeting ER stress may have therapeutic benefits in the treatment of inflammatory diseases.
Inflammation is an important mechanism leading to glomerular damage in CKD (13). Native and modified LDL have previously been shown to increase the production of numerous inflammatory mediators, including interleukin-6 (IL-6), CD40, and macrophage migration inhibitory factor, in human mesangial cells (HMCs) (14). The present study aimed to investigate whether ER stress preconditioning could attenuate LDL-induced inflammatory responses in HMCs, and to explore the associated molecular mechanisms.
Materials and methods
Chemical reagents and antibodies
LDL was purchased from ProSpec (East Brunswick, NJ, USA) and tunicamycin from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-human monoclonal antibodies anti-XBP1 and anti-β-actin were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA), anti-phospho-NF-κB p65 (Ser536), and anti-phospho-IκB kinase (IKK)α/β (Ser176/180) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA), and anti-phospho-IRE1α and anti-IRE1α were purchased from Novus Biologicals (Littleton, CO., USA).
Cell culture and drug treatment
Primary HMCs were obtained from the Key Laboratory of Infectious Diseases of Chongqing Medical University (Chongqing, China). The cells were cultured in Dulbecco’s Modified Eagle’s Medium, supplemented with 10% fetal bovine serum, 100 μg/ml streptomycin, and 100 U/ml penicillin (Invitrogen Life Technologies, Carlsbad, CA, USA), at 37°C in an atmosphere containing 5% CO2. The experiments were performed with cells taken from passages 3–8.
The HMCs were treated with LDL (200 nm) for various durations (1–24 h), and then subjected to gene expression analysis. To assess the effects of ER stress preconditioning on the LDL-induced inflammatory responses, the cells were exposed for 8 h to a low dose (0.1 μg/ml) of tunicamycin, an ER stress inducer, followed by treatment with LDL.
Plasmids, small interfering RNA (siRNA) oligonucleotides, and transfection
The full-length human XBP1 cDNA was amplified and subcloned into an expression vector pEGFP-C1 (ClonTech, Palo Alto, CA, USA), which encoded a C-terminal green fluorescent protein tag. Specific siRNAs targeting human IRE1α and XBP1 were synthesized by Invitrogen Life Technologies, with the following sense sequences: IRE1α, 5′-GUCCCACUUUGUGUCCAAUTT-3′; and XBP1, 5′-CCAAGCUGGAAGCCAUUAATT-3′. A scrambled siRNA sequence, 5′-UUCUCCGAACGUGUCACGUTT-3′, was used as a negative control. The final concentration of each siRNA was 2 μM. The HMCs were seeded in 6-well plates, at a density of 5×104 cells/well, 24 h prior to transfection. The siRNA molecules were transfected into the cells using Lipofectamine® 2000 reagent, according to the manufacturer’s instructions (Invitrogen Life Technologies). The transfection efficiency was determined by western blot analysis of the corresponding protein levels, 24 h following siRNA transfection.
Quantitative polymerase chain reaction (qPCR)
Total cellular RNA was extracted using the RNeasy kit, according to the manufacturer’s instructions (Qiagen, Hilden, Germany). Reverse transcription was performed using the AMV First Strand cDNA Synthesis kit (Bio Basic Inc., Amhurst, NY, USA). The qPCR was conducted using an Applied Biosystems StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) and the SYBR® green PCR master mix (Applied Biosystems). GAPDH was amplified in a parallel reaction, and was used as an internal quantitative control. The cycle threshold (Ct) was calculated for each gene tested. The relative mRNA expression levels were normalized to those of GAPDH, using the 2−ΔΔCt method (15). Primers are listed in Table I.
Western blot analysis
Following LDL treatment, the cells were lysed in lysis buffer (50 mm Tris, pH 7.4, 150 mm NaCl, 1% NP-40, and 0.1% SDS), supplemented with protease inhibitors (2 μg/ml aprotinin, 2 μg/ml leupeptin, and 1 mm phenylmethylsulfonyl fluoride). The protein samples were separated by electrophoresis on polyacrylamide gels containing 0.1% SDS, and then transferred to nitrocellulose membranes (Pierce Biotechnology, Inc., Rockford, IL, USA). The membranes were incubated with the individual primary antibodies (goat anti-mouse NF-κB p65 antibody, sc-166748; Santa Cruz Biotechnology, Inc; dilution, 1:1,000) overnight at 4°C, followed by incubation for 1 h with the appropriate secondary antibodies (Alexa Fluor® 488-labeled goat anti-rabbit immunoglobulin G, #4412; Cell Signaling Technology, Inc.; dilution, 1:200), at room temperature. Immune complexes were visualized using enzyme-linked chemiluminescence (GE Heakthcare Life Sciences, Chalfont, UK). The band density was densitometrically analyzed using the Quantity One software (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and normalized against the density of β-actin.
Immunocytochemistry staining
The cells were washed with phosphate-buffered saline (PBS; pH 7.4) and fixed in 4% formaldehyde in PBS for 15 min. The cells were blocked by preincubation with 10% normal goat serum in PBS for 1 h at room temperature, and were then incubated with anti-NF-κB p65 antibodies, at 4°C overnight. Following further washes with PBS, the cells were incubated with Alexa Fluor® 488-labeled goat anti-rabbit immunoglobulin G (1:200 dilution), at room temperature for 1 h. The cells were counterstained with 4,6-diamidino-2-phenylindole, in order to visualize the nuclei. The cells were visualized under a fluorescent microscope (LeicaTCS-SP5, Leica, Mannheim, Germany).
Statistical analysis
The data are expressed as the means ± standard deviation. The statistical differences among the numerous groups were calculated using a one-way analysis of variance, followed by the Tukey post hoc test. A P<0.05 was considered to indicate a statistically significant difference. All statistical tests were performed using SPSS version 13.0 software package for Windows (SPSS, Inc., Chicago, IL, USA)
Results
LDL treatment stimulates expression of CD40 and proinflammatory cytokines in HMCs
HMCs were exposed to LDL for various durations, and any changes to gene expression levels were determined by qPCR. LDL treatment caused a significant increase in the relative mRNA expression levels of IL-6, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-α (TNF-α), and CD40, as compared with the untreated control cells (P<0.05; Fig. 1). Furthermore, this induction was time-dependent, peaking at 24 h of culture.
Activation of the IRE1α/IKK/NF-κB pathway is involved in LDL-induced proinflammatory cytokine expression
Treatment with LDL induced a time-dependent increase in the phosphorylation of IRE1α, IKK, and NF-κB p65, as determined by western blot analysis (Fig. 2A). This result is indicative of activation of the IRE1α/IKK/NF-κB pathway. However, the total protein expression levels of IRE1α, IKK, and NF-κB p65 remained unchanged (data not shown). To further assess the activation of NF-κB signaling, immunocytochemistry was used to examine the nuclear translocation of NF-κB p65. Treatment with LDL markedly promoted NF-κB p65 nuclear translocation, in a time-dependent manner (Fig. 2B).
To confirm the essential role of IRE1α in LDL-induced inflammatory responses, HMCs were transfected with IRE1α specific siRNA, or scrambled control siRNA, 24 h before treatment with LDL. The delivery of the IRE1α specific siRNA, but not control siRNA, markedly reduced the mRNA expression levels of endogenous IRE1α in HMCs, as compared with the non-transfected cells (data not shown). Depletion of IRE1α expression significantly blocked the induction of IL-6, MCP-1, TNF-α, and CD40 exerted by LDL, as determined by qPCR (P<0.05; Fig. 3A). Furthermore, knockdown of IRE1α expression markedly reduced the phosphorylation levels of IKK and NF-κB p65 (Fig. 3B).
ER stress preconditioning attenuates the LDL-induced inflammatory response
The effects of ER stress preconditioning were also determined on LDL-induced inflammatory responses in HMCs. Pretreatment with tunicamycin significantly attenuated the induction of IL-6, MCP-1, TNF-α, and CD40 by LDL (P<0.05; Fig. 4A). Furthermore, the phosphorylation levels of IRE1α, IKK, and NF-κB p65 were markedly reduced in the LDL-treated HMCs with tunicamycin pretreatment (Fig. 4B).
Tunicamycin pretreatment upregulates the expression levels of XBP1 in HMCs
Treatment with tunicamycin alone markedly increased the protein expression levels of XBP1 in the HMCs, as determined by western blot analysis (Fig. 5A). However, the mRNA expression levels of IL-6, MCP-1, TNF-α, and CD40 remained unchanged in the tunicamycin-treated cells (Fig. 5B).
Depletion of XBP1 reverses the anti-inflammatory effects of ER stress preconditioning on LDL-treated HMCs
The role of XBP1 on the anti-inflammatory effects of ER stress preconditioning in HMCs was then determined. Transfection with specific XBP1 siRNA resulted in a marked reduction in endogenous XBP1 protein expression levels in the HMCs, as compared with the cells transfected with control siRNA (data not shown). Notably, silencing XBP1 expression significantly restored the mRNA expression of IL-6, MCP-1, TNF-α, and CD40, in the LDL-treated HMCs with ER stress preconditioning (P<0.05; Fig. 6A). Furthermore, the phosphorylation levels of IRE1α, IKK, and NF-κB p65 were markedly increased in the XBP1-depleted HMCs with ER stress preconditioning (Fig. 6B).
Overexpression of XBP1 antagonizes LDL-induced inflammation
The present study also examined whether overexpression of XBP1 could block LDL-induced inflammatory responses in HMCs. The overexpression of XBP1 in the HMCs transfected with the XBP1-expressing plasmid was confirmed by western blot analysis (data not shown). Overexpression of XBP1 significantly reduced the mRNA expression levels of IL-6, MCP-1, TNF-α, and CD40 in LDL-treated HMCs, as determined by qPCR (Fig. 7A). Furthermore, the phosphorylation levels of IRE1α, IKK, and NF-κB p65 in LDL-treated cells were diminished in response to overexpression of XBP1 (Fig. 7B).
Discussion
Native and modified LDL has previously been shown to possess potent proinflammatory activities in various biological contexts. Meng et al (16) reported that oxidized LDL triggers inflammatory responses in cultured human mast cells by activating the Toll-like receptor 4-dependent signaling pathway. Oxidized LDL has also been shown to stimulate proinflammatory responses in alternatively activated M2 macrophages (17). The present study showed that LDL treatment promoted inflammatory responses in HMCs, as evidenced by the increased expression levels of proinflammatory cytokines IL-6, MCP-1, and TNF-α. These results are concordant with previous studies (14,18). Systemic inflammation is regarded as a key factor in the pathogenesis of CKD (13). The proinflammatory activity of LDL in HMCs may partially explain the association between dyslipidemia and the progression of CKD (2).
CD40 is expressed in a wide range of cells, including macrophages, lymphocytes, endothelial cells, vascular smooth muscle cells, and mesangial cells (19,20). CD40/CD40 ligand interactions between infiltrating mononuclear cells and resident renal cells, are associated with the pathogenesis of immune-mediated glomerulonephritis (21). Increasing evidence has indicated that activation of the CD40/CD40 ligand pathway is associated with the initiation of inflammatory responses (19). Tanaka et al (22) reported that human platelets stimulate mesangial cells to produce MCP-1, via the CD40/CD40 ligand pathway. The results of the present study demonstrated that upregulation of CD40 expression occurs in LDL-induced inflammatory responses in HMCs. In addition to initiation of inflammatory responses, activation of CD40-dependent signaling has been found to protect HMCs from oxidized LDL-induced apoptosis (23). These findings suggest that LDL-mediated toxic effects on HMCs are associated with the regulation of CD40 signaling.
There is known to be a close association between ER stress and the inflammatory response (24). Li et al (25) showed that induction of ER stress contributes to retinal inflammation in diabetic retinopathy (24). The results of the present study showed that, in parallel with induction of proinflammatory cytokines, LDL treatment resulted in a time-dependent increase in the phosphorylation of IRE1α, IKK, and NF-κB p65. IRE1α is a major sensor of unfolded proteins in the ER, and is activated by autophosphorylation (7). In response to ER stress, activated IRE1α may bind to the IKK complex and activate NF-κB (10,26). In the present study siRNA technology was used to induce a specific depletion of IRE1α expression. The loss of IRE1α expression antagonized the induction of IL-6, MCP-1, TNF-α, and CD40 exerted by LDL, coupled with the suppression of NF-κB activation. These findings indicate that ER stress has a mediating role in LDL-induced inflammatory responses in HMCs, which involves the activation of the IRE1α/IKK/NF-κB pathway. Consistently, phospholipolyzed LDL has been found to induce an inflammatory response in endothelial cells, by activating ER stress signaling pathways (27).
Accumulating evidence indicates that ER stress preconditioning confers protection against cellular injury in various biological settings (12,28). Hung et al (28) reported that ER stress preconditioning attenuated hydrogen peroxide-induced oxidative injury in LLC-PK1 renal epithelial cells. Li et al (10) previously demonstrated that preconditioning with ER stress mitigated retinal endothelial inflammation. The present study showed that LDL-induced inflammatory cytokine production was significantly diminished in ER stress-primed HMCs, suggesting a protective role for ER stress preconditioning. Notably, the results of the present study revealed that pretreatment with the ER stress inducer tunicamycin, caused an upregulation of XBP1, but not of the proinflammatory cytokines. In addition, siRNA-mediated inhibition of XBP1 attenuated the protective effects of tunicamycin pretreatment on LDL-induced inflammation, which was coupled with an enhanced activation of the IRE1α/IKK/NF-κB pathway. Numerous studies have shown that XBP1 ablation triggers feedback activation of its upstream enzyme IRE1α (29,30). Furthermore, the present study revealed that XBP1 overexpression inhibited LDL-induced activation of the IRE1α/IKK/NF-κB pathway. These results collectively indicate that ER stress preconditioning ameliorates LDL-induced inflammation in HMCs, which is largely mediated through activation of XBP1 signaling and blockade of the IRE1α/IKK/NF-κB pathway.
In conclusion, activation of the IRE1α/IKK/NF-κB pathway is required for LDL-induced inflammation in HMCs. ER stress preconditioning resulted in the upregulation of XBP1 expression and subsequent inhibition of the IRE1α/IKK/NF-κB pathway, which consequently interfered with the LDL-induced inflammatory responses. These findings warrant further investigation of the therapeutic potential of ER stress preconditioning in inflammatory disorders, such as CKD.