G. Ding, Division of Nephrology, Department of Medicine, Renmin Hospital of Wuhan University, No. 238, Jiefang Road, Wuhan, China. E-mail: firstname.lastname@example.org
Surfactant protein D (SP-D), a member of the C-type lectin (collectin) protein family, plays a critical role in innate host defence against various microbial pathogens and in the modulation of inflammatory responses in the lung. However, little is known about its expression and biological function in the kidney. In this work, we studied SP-D expression in human kidney and cultured human renal proximal tubular epithelial cells (HK-2), and examined the effect of SP-D on proinflammatory cytokine production after lipopolysaccharide (LPS) stimulus. We observed the expression of both SP-D mRNA and protein in human kidney and in-vitro HK-2 cells by immunohistochemistry, Western blot analysis, reverse transcription–polymerase chain reaction (RT–PCR) and real-time PCR. To explore the potential role of SP-D in the pathogenesis of tubulointerstitial fibrosis in kidney infection, we examined the production of monocyte chemoattractant protein-1 (MCP-1) in HK-2 cells after LPS treatment. Results showed that the level of MCP-1 in the conditioned medium increased significantly when HK-2 cells were cultured with LPS (>0·1 µg/ml) for 8 h. Of interest, LPS treatment inhibited SP-D expression in HK-2 cells. Furthermore, over-expression of SP-D reduced significantly the LPS-induced expression of MCP-1 in transfected cells. These findings suggest that SP-D in the kidney functions as an anti-inflammatory factor in renal tubular epithelial cells and may modulate tubulointerstitial fibrosis in kidney.
Tubulointerstitial fibrosis is a common pathway to end-stage renal failure [1,2]. Monocyte chemoattractant protein-1 (MCP-1), a cytokine, is expressed at the site of kidney injury and inflammation and can direct macrophage recruitment. Recent studies have revealed that MCP-1 plays a pivotal role in the pathogenesis of progressive tubulointerstitial lesions in various animal models of renal damage and human renal diseases [3,4]. Up-regulation of MCP-1 may be a common pathway involved in the progression of tubulointerstitial lesions . The renal tubular epithelial cell is one of the important sources of MCP-1 production in the kidney [6,7]. Human renal proximal tubular epithelial cells (HK-2) cells, an immortalized cell line derived from human proximal tubules, express a low basal level of MCP-1 but, remarkably, increase MCP-1 expression after stimulation with lipopolysaccharide (LPS) .
LPS is a cell wall component of Gram-negative bacteria such as Escherichia coli and can induce a variety of inflammatory mediators. There are two distinct serotypes of LPS, smooth and rough LPS. The presence or absence of O-antigen determines whether the LPS are considered smooth or rough, respectively .
Surfactant protein D (SP-D), a member of the C-type lectin (collectin) protein family, plays an important role in innate host defence and regulation of inflammatory processes in the lung , where SP-D protein is expressed and secreted by alveolar type II pneumocytes and bronchiolar Clara cells. The structure of SP-D consists of four domains: (a) an N terminus, (b) a triple-helical collagen-like domain, (c) a neck region and (d) a carbohydrate recognition domain [10,11]. Although SP-D expression was observed originally in the lung, it has been found recently in several extrapulmonary tissues , including kidney [13,14]. Recently, our study showed that urinary SP-D level was decreased in female patients with recurrent urinary tract infection compared to healthy controls , suggesting that SP-D may be implicated in the physiology and/or pathophysiology in kidney disease as in lung. Because SP-D is a potent modulator of inflammation according to in-vitro and in-vivo studies , it is logical to speculate that SP-D may contribute to the innate immune mechanism in the pathogenesis of renal inflammatory diseases. To our knowledge, this is the first study of SP-D expression and its potential role in the pathogenesis of kidney tubulointerstitial fibrosis. In the present study, we measured the expression of SP-D in human kidney tissue and renal proximal tubular epithelial cells and evaluated the effects of SP-D to modulate inflammatory MCP-1 level after LPS stimulation in HK-2 cells.
Materials and methods
Cell culture and human kidney tissues
HK-2 was purchased from the American Type Culture Collection (Manassas, VA, USA), and the cells were grown in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). HK-2 is a renal proximal tubular epithelial cell line derived from healthy human kidney after immortalization by transduction with human papilloma virus 16 E6/E7 genes. HK-2 cells respond in a similar fashion to primary human proximal tubular cells and are therefore a good model for studying proximal tubular cell biology . In this study, we chose to use the HK-2 cell line rather than isolated primary human proximal tubular epithelial cells, which are more expensive and more difficult to maintain in culture. All in-vitro experiments were performed using cells from passage 15 or less. Normal human renal and lung tissues were obtained from patients who had undergone operations for either kidney or lung cancer; tubulointerstitial fibrosic human kidney tissue was from one patient who underwent kidney biopsy. This study was carried out according to the protocol approved by the human ethics committee of the Renmin Hospital of Wuhan University in China.
Immunohistochemistry was performed on paraffin-embedded normal human kidney sections and tubulointerstitial fibrosic human kidney sections, as our previously published protocol . Slides were deparaffinized and treated with 3% H2O2 for 20 min. Antigen retrieval was performed in microwaved citrate buffer (0·01 mol/l, pH 6·0) for 20 min. Endogenous peroxidase was blocked with 5% bovine serum albumin in 0·01 mol/l phosphate-buffered saline (PBS, pH 7·4) for 20 min. Sections were incubated with SP-D antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4°C followed by biotinylated secondary antibody and avidin–biotin peroxidase complex (Dako, Glostrup, Denmark) for 30 min. After rinsing, the peroxidase activity was visualized by diaminobenzidine (DAB) (Dako), and the sections were then counterstained with haematoxylin. Negative controls were performed by non-immune serum instead of SP-D antibody.
Cell treatment with LPS
HK-2 cells were maintained in RPMI-1640 medium with 10% FBS at 37°C and 5% CO2. Cells were then incubated in serum-free medium with or without smooth LPS from E. coli O111:B4 (0–10 µg/ml; Sigma Chemical Co., St Louis, MO, USA) for 0 to 24 h. The LPS from E. coli O111:B4 cannot band to SP-D .
In brief, cells were fixed in 4% paraformaldehyde for 5 min. The cells were then immunostained using the primary polyclonal SP-D antibody (Santa Cruz Biotechnology) followed by incubation in fluorescein isothiocyanate (FITC)-labelled secondary antibody (Pierce Biochemicals, Tampa, FL, USA). Negative controls were performed by non-immune serum instead of SP-D antibody.
RT–PCR for SP-D
Total RNA of HK-2 cells, human renal and human lung tissue samples were extracted using Trizol (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. Total RNA (2 µg) was used to synthesize the first-strand cDNA with oligo-dT primers, and served as a template for amplification of SP-D and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). PCR for SP-D was then performed with sense primer 5′-AAAACCATTTACGGAGGC-3′ and anti-sense primer 5′-AACTCGCAGACCACAAGA-3′. GAPDH (sense 5′-GGATTTGGTCGTATTGGG-3′, anti-sense 5′-GGAAGATGGTGATGGGATT-3′) was used as control. PCR conditions were as follows: 30 cycles of denaturation at 94°C for 45 s, annealing at 54°C for 45 s and extension at 72°C for 45 s. The PCR product was analysed on a 2% agarose gel, and the 312 base pairs (bp) SP-D band and 205 bp GAPDH band were visualized with ultraviolet (UV) illumination. Human lung tissue sample was used as a positive control.
Total RNA of HK-2 cells were isolated as described above. Two µg of total RNA from each sample were used for reverse transcription using oligo-dT primers, and 10% of the cDNAs were used as a template for analysis. Real-time PCR was performed using SYBR Green real-time PCR Master Mix (Toyobo, Osa, Japan) and ABI 7300 real-time PCR system following the manufacturer's directions . β-actin (sense 5′- GTCCACCGCAAATGCTTCTA-3′, anti-sense 5′-TGCTGTCACCTTCACCGTTC-3′) was used as a housekeeping gene with which to normalize SP-D (sense 5′-CAATGGCAAGTGGAATGACAG-3′, anti-sense 5′-GGGTCTAAGCCTTGACTTCTGG-3′) and MCP-1 (sense 5′-CTCATAGCAGCCACCTTCATTC-3′, antisense 5′-CAAGTCTTCGGAGTTTGGGTTT-3′) expression. The relative levels were assessed using the 2-ΔΔCT method . Each experiment was performed in duplicate and repeated independently at least three times. Negative control reactions without RT reaction and template were also included.
Western blot analysis
HK-2 cells and human renal tissue samples were lysed with buffer [50 mm Tris, pH 8·0, 150 mm sodium chloride, 0·5% sodium deoxycholate, 0·1% sodium dodecyl sulphide (SDS), 1 mmol/l phenylmethylsulphonyl fluoride (PMSF), 1 mmol/l Na3VO4], centrifuged at 12 000 g for 15 min at 4°C, and the supernatant collected. The total protein content of the samples was determined using the bicinchoninic acid (BCA) protein assay kit (Pierce Biochemicals). Samples containing 10 µg of protein were separated by one-dimensional gel electrophoresis on a 10% polyacrylamide-SDS minigel and transferred to nitrocellulose membranes. Non-specific proteins binding was blocked by incubating the membranes with 5% milk in PBS with 0·1% Tween 20 for 1 h at room temperature, and then incubated overnight at 4°C with anti-SP-D antibody (Santa Cruz Biotechnology). Membranes were washed with Tris buffer and incubated with horseradish peroxidase (HRP)-conjugated immunoglobulin (Ig)G (Pierce Biochemicals) for 1 h at room temperature. Membranes were then incubated in ECL solution (Santa Cruz Biotechnology). The intensity of each band was quantitated by Gel-pro image software. Human lung tissue sample was used as a positive control.
Human SP-D (hSP-D) cDNA fragments were generated by PCR amplification with two hSP-D-specific primers (sense 5′-CCTAAGCTTGCCATGCTGCTCTTCCTCCTCTCTG-3′ and anti-sense 5′-GGTCTA AGCCTTGAATTCTTGCCAAACTCCT-3′). The sense primer is located in the hSP-D 5′untranslated region (UTR) and start site region and the anti-sense prime is within the 3′UTR region. The template of the PCR amplification was used with a human multiple tissue cDNA kit (cat. no. K14201) purchased from BD Biosciences (Palo Alto, CA, USA). About 1·4 kB PCR products were purified and then digested with restriction enzymes HindIII and EcoRI. The digested hSP-D cDNA fragment was cloned into the expression vector pEE14 K1. DNA sequences of recombinant plasmid were verified by DNA sequencing. The correct plasmid and vector were used in the following transfection experimentation.
Generation of stable transfection expressing human SP-D
HK-2 cells capable of constitutively over-expressing the SP-D gene were created by transfection with lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA), according to the manufacturer's instructions. After 2 days, the cells were transferred to glutamine-free RPMI-1640 culture medium containing 10% FBS supplemented with 25 µm glutamine inhibitor methionine sulphoximine (Sigma Chemical Co.). Individual clones were then tested for expression of SP-D by Western blot. HK-2 cells transfected with the control vector pEE14 were used as controls.
Enzyme-linked immunosorbent assay (ELISA)
MCP-1 in the conditioned culture medium was measured by ELISA (Bender MedSystems, Vienna, Austria), according to the manufacturer's protocol. In brief, 96-well plates containing anti-MCP-1 monoclonal antibody were incubated with conditioned media for 1 h at room temperature with shaking. After three washes with buffer and 1 h incubation at room temperature with streptavidin–HRP, the plates were incubated with 3,3′,5,5′-tetramethylbenzidine (TMB) substrate solution for 10 min at room temperature. When the samples became blue, the reaction was stopped by adding 100 µl stop solution to each well and the absorbance at 450 nm read with a spectrophotometer.
Data are expressed as mean ± standard deviation (s.d.). One-way analysis of variance (anova) with appropriate post-hoc analysis was used to assess the statistical significance of differences. P < 0·05 was considered statistically significant.
SP-D expression in human kidney tissue and HK-2 cells
To study SP-D expression in human kidney, we examined SP-D protein and mRNA with human kidney tissue and a human tubular renal epithelial cell line (HK-2). Results showed that SP-D mRNA expression occurred in human kidney tissue and HK-2 cells by RT–PCR with PCR products about 312 bp, the same size as the positive control in human lung (Fig. 1a). SP-D protein was also detected in human kidney tissue and HK-2 cells by Western blot analysis, migrating as a single band at 43 kD, the same molecular weight as SP-D of human lung tissue (Fig. 1b). These data indicate that human kidney and HK-2 cells expressed SP-D protein with the same molecular weight as human lung tissue in the reduced condition. However, it is difficult to quantify SP-D level in human lung and kidney due to the limitation of the quality of human tissues. These human tissues were obtained from clinically pathological department with different forms, i.e. fresh or fixed, the quality of which limited us to perform quantitative analysis for SP-D expression in these human tissues. Furthermore, we examined the localization of SP-D protein in HK-2 cells by immunostaining with SP-D antibody and FITC-labelled secondary antibody and examination by fluorescence microscopy. We found SP-D protein in HK-2 cells localized to the cytoplasmic and perinuclear area (Fig. 1c). Immunohistochemical analysis showed both normal human kidney and tubulointerstitial fibrosic kidney tissues showed expression of SP-D (Fig. 1e,f). SP-D protein expression was detected in the tubular epithelial cells of kidney. Negative controls showed no SP-D immunostaining in HK-2 cells and human kidney tissues (Fig. 1d,g).
Effect of LPS on SP-D expression in HK-2 cells
Although SP-D binds to bacterial pathogens and suppresses inflammatory responses in the lung, little is known about its biological effect in the kidney. In this study, we examined the effect of LPS treatment on SP-D expression in HK-2 cells. The concentration of LPS used was 0·1–10 µg/ml, as in previous studies 10 µg/ml LPS was used to treat HK-2 cells to study kidney interstitial inflammation [6,21]. When HK-2 cells were treated with different concentrations of LPS for 8 h, we observed that LPS (more than 0·1 µg/ml) significantly inhibited SP-D protein and mRNA expression in HK-2 cells (Figs 2a and 3a). In a time–course experiment, SP-D expression was decreased at 2 h, and maintained a low level for at least 24 h following stimulation with 5 µg/ml LPS (Figs 2b, 3b).
Effect of LPS treatment on MCP-1 expression in HK-2 cells
As MCP-1 is a critical mediator in the pathogenesis of renal tubulointerstitial fibrosis in various kidney diseases, we analysed MCP-1 content in the HK-2 culture medium after LPS treatment with concentrations from 0·1 to 10 µg/ml for 8 h. The results indicated that MCP-1 content in the conditioned culture medium increased significantly when LPS concentration was greater than 1 µg/ml (Fig. 4a). These results indicate that MCP-1 expression in HK-2 cells occurs in response to LPS in a dose-dependent manner. Similarly, a time–course analysis of MCP-1 expression after LPS treatment was carried out; results showed that MCP-1 expression increased significantly after 2 h of LPS stimulation (Fig. 4b). A similar pattern of MCP-1 mRNA levels in HK-2 cells was observed.
Generation of stably transfected HK-2 cell lines over expressing SP-D
To explore the biological effect of SP-D on suppression of the inflammatory response in HK-2 cells, we constructed a pEE14-hSP-D plasmid and transfected the plasmid into HK-2 cells. The control vector pEE14 K1 without hSP-D cDNA was transfected into HK-2 cells as a negative control. After transfection and selection, we obtained a stable pEE14-hSP-D transfection clone that over-expressed SP-D protein as detected by Western blot (Fig. 5). Over-expression of hSP-D was stable, suggesting that hSP-D cDNA was integrated stably into the genome of the cell line. The stable transfected cell line was used in subsequent experiments.
Effects of over-expression of SP-D by HK-2 cells on LPS-induced MCP-1 production
We examined whether over-expression of SP-D in transfected cells altered proinflammatory MCP-1 production following LPS treatment. Based on a previous publication, the LPS from E. coli O111:B4 could not bind to SP-D directly . SP-D transfected cells (pEE14-hSP-D), normal HK-2 cells (control) and vector pEE14 transfected cells (pEE14) were treated with 5 µg/ml LPS for 8 h. MCP-1 content in medium was then measured by ELISA and MCP-1 mRNA in the cells detected by real-time PCR. The results showed that basal MCP-1 expression was the same among normal HK-2 cells and hSP-D transfected cells, suggesting that transfection of recombinant construct or the vector did not affect MCP-1 expression. However, LPS treatment (5 µg /ml) of all three types of cells exhibited a higher level of MCP-1 compared with untreated cells (P < 0·05) (Fig. 6). Cells over-expressing SP-D showed less MCP-1 expression of both protein and mRNA compared with normal HK-2 cells or vector transfected cells (P < 0·05) (Fig. 6). These results indicate that over-expressed SP-D significantly inhibited MCP-1 expression and that SP-D plays a role in the regulation of proinflammatory process in renal tubular epithelial cells.
In the present study, we have demonstrated the expression of SP-D in human kidney tissue and cultured human renal tubular epithelial cells (HK-2). LPS treatment increased MCP-1 production significantly, but down-regulated SP-D expression in HK-2 cells. Over-expression of SP-D inhibited MCP-1 release from the HK-2 cells stimulated with LPS. These results indicate that SP-D plays a role in modulation of inflammatory processes in human renal tubular epithelial cells.
LPS, one cell wall component of Gram-negative bacteria, is a ligand known to interact with Toll-like receptor 4 (TLR-4) [22,23] and to cause proinflammatory cytokine production in renal tubular epithelial cells [23,24]. Previous work revealed that LPS stimulation increases MCP-1 expression through the activation of the NF-κB signalling pathway via binding to TLR-4 on the surface of the tubular epithelial cells [25–27]. MCP-1 is a major proinflammatory cytokine that is secreted by mononuclear cells and various non-leucocytic cells, including renal tubular epithelial cells [25–27]. Compared with wild-type mice, MCP-1-deficient mice exhibit less interstitial macrophage infiltration and tubulointerstitial lesions , suggesting that MCP-1 dependent processes commonly contribute to progressive kidney fibrosis.
SP-D is a surfactant-associated protein that provides a surface tension reduction at the air–liquid interface of the lung and preserves alveolar patency during expiration . As a member of the collectin family, SP-D also plays a key role in innate immunity in the lung [9,29,30]. SP-D can opsonize pathogens and enhance pathogen uptake by macrophages , binds directly to rough LPS present on the surface of Gram-negative bacteria and inhibits the growth of Gram-negative bacteria by increasing membrane permeability [18,32]. More importantly, SP-D modulates inflammatory processes through influencing nuclear factor (NF)-κB activity by blocking LPS binding to its TLR-4 receptor and/or CD14 [33,34]. SP-D(−/−) knock-out mice exhibit chronic pulmonary inflammation leading to emphysema and other lung maladies , and have shown more susceptibility to intratracheal LPS than wild-type mice . Children with absent SP-D in bronchoalveolar lavage have a higher incidence of pneumonia . As a significant correlation has been observed between the level of SP-D in serum of patients with idiopathic pulmonary fibrosis or systemic sclerosis , SP-D level in serum has been used as a biomarker for pulmonary fibrosis . This in-vitro, in-vivo and clinical relevance in pulmonary disease indicate SP-D is an important modulator of inflammatory processes in the lung.
Although SP-D was identified initially in the lung, research has revealed that SP-D is present in extrapulmonary tissue [40,41], including the kidneys [13,14]. Thus, besides its long-standing role in the lung, SP-D appears to play an important role in extrapulmonary tissues [40,41]. To our knowledge, SP-D expression, localization and potential function in the kidney have not yet been evaluated. In the present study, SP-D mRNA and protein expression were observed in kidney tissue and cultured HK-2 cells; LPS inhibited SP-D expression and stimulated MCP-1 production in HK-2 cells. This observation is different from some findings in the lung, where an increase is found. It is due probably to the lack of CD14 receptor on the surface of human renal tubular epithelial cells . CD14, one important receptor in innate immunity, is expressed in lung epithelial cells and is involved in the interaction of LPS and SP-D . Other factors, such as different types of LPS used in different studies, are not excluded. Of interest, a decrease of SP-D level in the lung lavage fluid was observed at the 6-h time-point in one in-vivo model of LPS instillation . However, over-expression of SP-D with transfected recombinant hSP-D decreased the MCP-1 production significantly in response to LPS. Because SP-D can bind directly to some kinds of LPS but not to smooth LPS from E. coli O111:B4 [18,29], LPS from E. coli O111:B4 was employed to avoid SP-D binding to LPS directly in the present study. Because CD14 is absent in human renal tubular epithelial cells , the protective effect of SP-D against LPS-mediated inflammatory response is mediated primarily by SP-D binding to extracellular TLR-4 via the carbohydrate recognition domain and subsequent blockage of NF-κB signalling pathways [26,27]. These indicate that SP-D may bind to TLR-4 and/or inhibit LPS binding to its receptor TLR-4, as in the TLR-4-expressing human embryonic kidney 293 cells , and then down-regulated LPS induced the MCP-1 expression. It is possible that the SP-D regulatory effect may contribute to initiating inhibition and/or progression of tubulointerstitial fibrosis by modulating the release of cytokines such as MCP-1 in kidney disease.
MCP-1 plays a crucial role in the pathogenesis of progressive tubulointerstitial lesions via recruitment and activation of monocytes and macrophages in animal models of renal damage and human renal diseases . Given these biological effects of MCP-1, the level of MCP-1 in urine has been used as a biomarker for tubulointerstitial lesions, and blockade of MCP-1 has served as a beneficial therapeutic application for renal injury and fibrosis [45–47]. Therefore, SP-D expression in human kidney and the inhibitory effect of SP-D on MCP-1 production after LPS treatment in renal tubular epithelial cells has a potential to increase our understanding of the pathogenesis of progressive tubulointerstitial lesions in kidney disease. However, the detailed mechanisms whereby SP-D exerts its anti-inflammatory effects in renal tubular epithelial cells remain to be determined. Further studies are needed to ascertain the role of SP-D in regulating inflammatory signalling pathways associated with the progression of tubulointerstitial fibrosis in kidney disease.
In summary, this study demonstrated that SP-D is expressed in human kidney tissue and renal epithelial HK-2 cells. SP-D may function to modulate inflammation and host defence in the kidney, which may affect the development or progression of tubulointerstitial fibrosis of kidney disease.
These studies were supported by grants from the National Science Foundation of China (30670985, 81070556) and NIH grant HL096007.
The authors declare that they have no conflict of interest related to the publication of this manuscript.