Severe sepsis and septic shock are associated with significantly increased morbidity and mortality in critically ill patients.1 In addition, severe sepsis and septic shock remain the most important causes of acute kidney injury (AKI) and act as independent risk factors for mortality and morbidity in the Intensive Care Unit (ICU). The mortality rate in sepsis-induced AKI patients is significantly higher than that in patients with AKI alone (70% vs. 45%).2 During sepsis, release of bacterial endoor exotoxins can occur. Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, induces expression of various inflammatory mediators, including tumor necrosis factor-α (TNF-α), interleukin-1, and interferon.3 However, it remains to be determined whether AKI progression is induced by elevated levels of inflammatory mediators or of cytokines secreted from damaged organ cells and inflammatory cells.
Pulmonary surfactant, a complex mixture of lipids and proteins, is produced and secreted by alveolar type II cells.4 Approximately 10% of the surfactant is formed by proteins, which contain the hydrophilic surfactant proteins A and D (SP-A and SP-D respectively) as well as the hydrophobic surfactant proteins B and C (SP-B and SP-C respectively).5 SP-A has characteristic structures similar to those of the C-type lectin superfamily, which forms a flower bouquet structure composed of six trimeric subunits, each of which consists of 26 kDa-35 kDa monomers.6 Alveolar type II and Clara cells in the lungs are the primary cells that secrete SP-A, which plays a role in preventing pulmonary alveoli from collapsing after expiration.7 SP-A participates in the pathophysiological and physiological regulation of the inflammatory process in the lungs.8 Compared to wild-type (WT) mice, SP-A-/- mice are more susceptible to a variety of viral and bacterial infections.9 SP-A plays important roles in modulating inflammation, apoptosis, and epithelial integrity during response to acute challenges in the lungs. Moreover, SP-A gene knockout mice have been shown to have increased synthesis of pro-inflammatory cytokines and enhanced inflammation.
LPS is one of the major causes of septic shock and can lead to AKI. Our previous study showed that SP-A can be expressed by human proximal tubular epithelial cells (HK-2 cells). However, the role of SP-A in septic AKI and the associated mechanisms are still unknown. Therefore, in the present study, we investigated the effect of SP-A on LPS-induced TNF-α expression and the underlying mechanism in HK-2 cells.
Renal tubular epithelial cells (HK-2, a proximal tubular cell line derived from normal kidney; purchased from ATCC (USA), CRL-2190) were cultured at 37°C in a humidified atmosphere of 5% CO2 and then treated with Dulbecco's modified Eagle's medium (DMEM, Hyclone Pierce, USA) in a mixture of 10% fetal bovine serum (Invitrogen, USA) and 1% ampicillin. The culture medium was changed every 2 days until the cells reached 70%-80% confluency, at which point they were dissociated with 0.25% trypsin/EDTA. Subsequently, the cells were incubated for 24 and 72 hours to determine mRNA and protein expressions respectively.
Indirect immunofluorescence experiment
Indirect immunofluorescence assay was performed to determine the SP-A expression and localization in HK-2 cells. The cells were seeded and the section was fixed by 4% paraformaldehyde. Non-specific sites were blocked with goat serum at 37°C for 30 minutes, and then the sections were incubated with primary antibody (rabbit anti-human monoclonal SP-A at a dilution of 1:200) overnight at 4°C. Normal rabbit IgG (at a dilution of 1:150) was used as a negative control. The sections were washed and treated with PBS at 37°C for 45 minutes to block the autofluorescence. FITC-labeled secondary antibody was added in the dark at 37°C for 45 minutes. Sections were then washed, air-dried, and mounted with fluoromount-G, and radiography was conducted under a fluorescence microscope.
Cultured HK-2 cells were used for RT-PCR analysis of SP-A1, SP-A2, and GADPH mRNA. An oligonucleotide dT primer and primer pairs of the target and control genes respectively for RT-PCR were purchased from Invitrogen Corporation. Total RNA was extracted from each sample using Trizol (Invitrogen) following the manufacturer's instructions. Total RNA (1 μg) extracted from HK-2 cells was used for the RT reaction (Takara, China), and then 1 μl of cDNA was used for amplification at a final volume of 20 μl according to the supplier's protocol (Fermentas, Germany). Each PCR product (6 μl) was subjected to electrophoresis on 2% agarose gel. SP-A1 primer: F: 5′-TGGGCAAGGTAATCAGTG-3′, R: 5′-GTCCAGGAAGATGGTTT-3′; SP-A2 primer: F: 5′-TATTGACC GAGCACATACC-3′, R: 5′-GTCCAG GAAGATGGGTTT-3′.
Normal human renal tissues and cultured HK-2 cells were used for Real-time PCR analysis of TNF-α mRNA. Total RNA was extracted from each sample using Trizol (Invitrogen) following the manufacturer's instructions. Total RNA (1 μg) extracted from the tissue was used for the RT reaction (Takara), and then 2.5 μl of cDNA was used for amplification at a final volume of 25 μl according to the supplier's protocol (Fermentas). Then, the amplified PCR product was used for the melting curve analysis. TNF-α primer: F: 5′-GACAAGCCTGTAGCCCATGTTGTA-3′, R: 5′-CAGCCTTGGCCCTTGAAGA-3′.
Proteins from HK-2 cells were extracted using RIPA lysate (150 mmol/L sodium chloride, 1.0% Triton X-100, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulphate (SDS), and 50 mmol/L Tris at pH 8.0). The total protein concentrations were determined using the bicinchoninic acid protein assay (Hyclone Pierce). Total protein (50 μg) was resolved by reducing 12% SDS-polyacrylamide gel electrophoresis and then transferred electrophoretically at 60 mA onto nitrocellulose membranes at 4°C overnight (Bio-Rad, USA). After the samples were blocked in 5% non-fat milk in Tris-buffered saline, immunoblotting was conducted using a primary antibody against SP-A (rabbit anti-human monoclonal SP-A at a dilution of 1:150) or NF-κB P65 (goat anti-human monoclonal SP-A at a dilution of 1:180 (Abcam, Britain, ab32536) and an anti-rabbit or anti-goat secondary antibody conjugated with horseradish peroxidase. Cellular β-actin protein was immunodetected using a human monoclonal antibody against human β-actin (Sigma, USA) as the internal standard. Immunoproducts were detected using enhanced chemiluminescence peroxidase detection reagents (Amersham, Sweden).
Enzyme-linked immunosorbent assay
HK-2 cell culture supernatant samples were centrifugated (5000 ×g/min for 3 minutes), and the supernatants were collected for the measurement of TNF-α concentration by enzyme-linked immunosorbent assay (ELISA) using the human TNF-α ELISA kit following the manufacturer's instructions (Austria, Bender).
SP-A siRNA transfection
HK-2 cells were incubated in 12-well plates until the cells reached 90%-95% confluence; then, the cells were washed twice with serum-free medium. A mixture of serum-free medium (200 μl), lipofectamine 2000 (10 μl), and siRNA (4 μg) were added to empty plates, mixed, and incubated at 37°C in the presence of 5% CO2 for six hours. The mixture was then replaced by serum-containing culture medium for 24 hours, and the medium (at a dilution of 1:10) was added to the culture plates containing the cells. Finally, antibiotics were added to choose stably transfected cell lines. HK-2 cells were divided into six groups: negative control group, LPS group, blank SP-A siRNA transfection group, blank SP-A siRNA transfection + LPS group, SP-A siRNA transfection group, and SP-A siRNA transfection + LPS group, and the HK-2 cells were stimulated by treating them with 5 mg/L LPS for 8 hours.
LPS-induced expression of SP-A1, SP-A2, TNF-α mRNA, and protein in HK-2 cells
HK-2 cells were incubated with various concentrations (0, 0.1, 1, 2, 5, and 10 μg/ml) of LPS from Escherichia coli 0111:B4 (Sigma) for 8 hours and then with 5 μg/ml of LPS for different durations (0, 2, 4, 8, 16, and 24 hours). The cells were collected to detect the expressions of SP-A1, SPA2, TNF-α mRNA, and protein as described above.
Data are expressed as mean ± standard error (SE). Results were analyzed by one-way analysis of variable (ANOVA) using the SPSS 13.0 software (SPSS Inc., USA). P<0.05 was considered to indicate statistical significance.
Localization of SP-A in HK-2 cells
Indirect immunofluorescence experiment using a specific SP-A antibody revealed the localization of SP-A in HK-2 cells. SP-A (Figure 1) was found to be expressed in the membrane and cytoplasm of HK-2 cells as indicated by significant green positive staining in these regions.
LPS-induced expression of SP-A1 and SP-A2 mRNA and protein in HK-2 cells
The effects of LPS on SP-A1 and SP-A2 mRNA and protein syntheses were determined by RT-PCR and Western blotting analysis. In untreated cells, expression of SP-A1 and SP-A2 mRNA and protein could be easily detected. HK-2 cells exposed to 1, 2, 5, and 10 μg/ml of LPS for 8 hours had significantly increased SP-A1 and SP-A2 mRNA and SP-A protein synthesis levels compared to those exposed to 0 and 0.1 μg/ml of LPS (P<0.05). LPS-induced (5 μg/ml) increase in expression of SP-A1 and SP-A2 mRNA and protein occurred within 2 hours (P<0.05) and was maintained beyond 24 hours (Figure 2). Thus, LPS was found to induce SP-A1 and SP-A2 mRNA and protein syntheses in a time- and concentration-dependent manner.
LPS-induced expression of TNF-α mRNA and protein in HK-2 cells
The effects of LPS on TNF-α mRNA and protein syntheses were determined by real-time PCR and ELISA. In untreated cells, mRNA and protein of TNF-α and NF-κB P65 were detected. HK-2 cells exposed to 1, 2, 5, and 10 μg/ml of LPS for 8 hours had significantly increased TNF-α mRNA and protein levels compared to those exposed to 0 and 0.1 μg/ml of LPS (P<0.05). The LPS-induced (5 μg/ml) increase in expression of TNF-α occurred within 2 hours (P<0.05) and was maintained beyond 24 hours (Figure 3). Thus, LPS was found to induce TNF-α mRNA and protein syntheses in a time- and concentration-dependent manner.
Effect of SP-A siRNA transfection on SP-A expression
Expression of SP-A protein could be detected both in the normal control group and the blank SP-A siRNA transfection group, but was significantly decreased in the SP-A siRNA transfection group (Figure 4, P<0.01).
Effect of SP-A siRNA transfection on NF-κB P65 and TNF-α expression induced by LPS
Expressions of NF-κB P65 and TNF-α in the LPS group and blank SP-A siRNA transfection + LPS group were significantly increased compared to those in the negative control group and blank SP-A siRNA transfection groups (P<0.01). Expression levels of NF-κB P65 and TNF-α in the SP-A siRNA transfection + LPS group was significantly increased compared to those in the LPS group and blank SP-A siRNA transfection + LPS group (P<0.01). There was no significant difference in NF-κB P65 and TNF-α expression between the negative control group and the blank SP-A siRNA transfection group (P>0.05; Figure 5).
The present study demonstrated that SP-A plays an important role in protecting renal tubular epithelial cells in septic AKI by inhibiting NF-κB activity to modulate TNF-α expression induced by LPS.
SP-A is the most abundant surfactant protein in the lungs and contributes to lung host defense. SP-A has been shown to act as an indicator for several lung diseases and their clinical outcomes. In patients with bacterial pneumonia, SP-A expression is decreased in the alveoles.10 SP-A knock-out mice are susceptible to several lung diseases. In addition, SP-A knock-out mice have higher degree of neutrophil-dominant cell recruitment and expression of inflammatory cytokines in BAL fluid, increased lung edema, and apoptosis in lung sections challenged with bleomycin.11 Consistent with these data, levels of SP-A1 and SP-A2 mRNA and protein were found to be significantly increased after LPS treatment in HK-2 cells in the present study.
It is well known that LPS is the major component of Gram-negative bacteria such as E. coli. LPS can cause systemic inflammatory responses and organ dysfunction, such as AKI. Thus, the severity and time course of kidney injury may depend on the level of LPS. In the pathogenesis of sepsis-induced AKI, inflammatory processes are now known to play an important role. Many inflammatory mediators and cytokines, such as TNF-α, are released by inflammatory cells, which are critical mediators of inflammatory tissue damage resulting from LPS-induced cytotoxic effects.12 In endotoxin- and cisplatin-induced AKI models, inhibition of the release or action of TNF-α could protect the kidney from nephrotoxicity.13 An increase in plasma pro-inflammatory cytokine levels predicts mortality in patients with AKI.14 NF-κB is a well-known transcription factor that contributes to the mediation of several signaling pathways in response to stimulation by LPS.15 In the resting state, NF-κB and inhibitor of κB (IκB) remain bound as a P50-P65-IκB trimer, which exists in the cytoplasm in a potentially inactive state. When cells are stimulated, IκB is dissociated from the complex, and P50-P65 is translocated from the cytoplasm to the nucleus, rendering NF-κB active and able to participate in transcriptional regulation. NF-κB is sequestered in the cytoplasm by the binding of inhibitory molecules such as IκB or NF-κB P65.16 It has been shown that LPS increases NF-κB in a dose- and time-dependent manner, similar to its regulation of MCP-1 mRNA expression in rat tubular epithelial cells.17 In the present study, LPS was found to increase NF-κB mRNA and protein levels in a dose- and time-dependent manner in HK-2 cells, similar to its effects on TNF-α expression in rat tubular epithelial cells.
The anti-inflammatory activity of surfactants can be used in therapy for endotoxin-induced respiratory distress. It has been shown that LPS-induced oxidative burst and apoptosis at 72 hours are reduced by both porcine and synthetic surfactants. Thus, exogenous application of surfactants can inhibit LPS-induced inflammation in the lung.18 A previous study showed that in alveolar macrophages from Ikappa B-alpha knockout/Iκ B-beta knockin mice, SP-A failed to inhibit LPS-induced TNF-α production and nuclear translocation of p65.19 In this study, we show that LPS can induce NF-κB expression in a dose- and time-dependent manner, which parallels its effects on TNF-α expression. In addition, our results show that LPS can induce SP-A expression in a dose- and time-dependent manner. Upon knockdown of SP-A expression by SP-A siRNA transfection, increases in NF-κB and TNF-α expression induced by LPS were both significantly enhanced, indicating that SP-A can play a protective role against sepsis-induced AKI.
In summary, we found that exposure of HK-2 cells to LPS increased SP-A expression in a dose- and time-dependent manner. In addition, LPS increased the transactivation of NF-κB as well as biosynthesis of TNF-α in a dose- and time-dependent manner in HK-2 cells. Upon siRNA-mediated knockdown of SP-A, significant enhancement in LPS-induced NF-κB and TNF-expression were observed. Taken together, our results suggest that SP-A can inhibit inflammatory responses of HK-2 cells to LPS by suppressing NF-κB expression and TNF-α synthesis.
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