Inhibition of Lipopolysaccharide-Induced Expression of Fractalkine by Methylprednisolone via NF-Κb in Human Renal Tubular Epithelial Cells

Objective: to study the effect of the glucocorticoid, methylprednisolone (MP), in lipopolysaccharide (LPS)-induced fractalkine (FKN) expression in HK-2 cells and to determine the role of NF-κB in this signaling pathway. Methods: HK-2 cells were stimulated by LPS to set up an in vitro inflammation model. The concentration of FKN in cell culture supernatant was measured by ELISA. FKN and p65 mRNA expression were detected by RT-PCR. FKN, p65 protein expression and the activity of the NF-κB were detected by immunofluorescence staining and western blotting. The effect of MP and SC-514 (a selective and reversible inhibitor of IKK beta) in FKN expression and NF-κB activation induced by LPS were evaluated. Results: LPS induced FKN expression and secretion in HK-2 cells occurred in a timeand dose-dependent manner and correlated with the activation of NF-κB. MP was able to inhibit FKN expression and secretion as well as the NF-κB induced activation of LPS, whereas SC-514 abolished this effect. Conclusions: MP inhibited FKN expression induced by LPS through the NF-κB pathway in human renal tubular epithelial cells in vitro. Use of HK-2 cells to study the renal inflammatory process will allow the further elucidation of the pathways involved in kidney disease. *Corresponding author: Liao Pinhu, Department of Intensive Care Medicine, Affiliated Hospital of Youjiang Medical University for Nationalities, No.18 Zhongshan Road II, Baise 533000, Guangxi Zhuang Autonomous Region, China, Tel: 008615277695188; E-mail: liaopinhu@163.com Received January 28, 2015; Accepted February 28, 2015; Published March 03, 2015 Citation: You YW, Qin YQ, Lin X, Yang FF, Li J, et al. (2015) Inhibition of Lipopolysaccharide-Induced Expression of Fractalkine by Methylprednisolone via NF-Κb in Human Renal Tubular Epithelial Cells. J Pulm Respir Med 5: 249. doi:10.4172/2161-105X.1000249 Copyright: © 2015 You YW, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


Introduction
Extensive studies have confirmed that inflammatory mechanisms play significant roles in the pathogenesis of kidney disease [1,2]. Fractalkine (FKN) is a unique member of the chemokines CX3C superfamily [3]. TNF-α, IL-1 and IFN-γ is known to induce the expression of FKN in endothelial cells [4]. FKN is involved in the acute inflammatory response [5] where it acts as an adhesion molecule mediating inflammatory lesions and participates in the occurrence and progression of a variety of renal diseases [6][7][8] and damage processes of renal cells [9][10][11], in particular, renal tubular epithelial cells [12,13]. The expression of FKN has been shown to be increased in renal diseases [14][15][16]. It is produced as a precursor protein in cells and transfers to the cell surface as a mature protein after glycosylation [16,17].
Lipopolysaccharide (LPS) is a proinflammatory factor which plays an important role in the regulation of endogenous expression of FKN in endotoxemia. LPS induces the expression of inflammation factors such as prostaglandin E2 and TNF-α in RAW264.7 cells. The expression of inflammatory factors and the activity of NF-κB are blocked by rosemary, when used as a medicinal herb [18]. LPS also stimulates the activation of NF-κB in human renal tubular epithelial cells [19]. The expression of MCP-1 increases and the expression of surface active protein D (SP-D) are reduced after incubation of human renal tubular epithelial cells with LPS. In addition, the LPS-induced MCP-1 expression is reduced by over-expression of SP-D in transfected cells [20]. LPS induces the expression of endogenous FKN protein in human umbilical vein endothelial cells whereas alpha-lipoic acid inhibits LPSinduced expression of endogenous FKN [21]. These results confirm that LPS induces the inflammatory reaction and FKN expression.
The activity of NF-κB, which includes Rel A (p65), p50, p52, Rel B and c-Rel, can be induced by pathogenic microorganisms, tumor growth factors and inflammatory factors. Activated NF-κB transfers from the cytoplasm to the nucleus, and then binds to specific DNA promoter regions and encodes proteins that regulates cell adhesion, proliferation and apoptosis [22]. Microarray analysis of renal tubular epithelial cells (HK-2) show that NF-κB plays a major role in the network structure of normal cells and that aristolochic acid has an inhibitory effect on the NF-κB activity [23]. Several studies have confirmed that the NF-κB signaling pathway is involved in the first step of inflammatory reaction induced by LPS [24]. TNF-α, stimulates the expression of FKN in an NF-κB dependent and autocrine manner in arterial smooth muscle cells [25] and the NF-κB pathway has been shown to be associated with expression of FKN. The binding activity of NF-κB DNA has been correlated with the expression of FKN induced by LPS in rat endothelial cells and the NF-κB inhibitor, an adenovirusmediated mutant of IkappaB inhibits the expression of FKN, suggesting that LPS induces expression of FKN in NF-κB dependent manner [26].
The glucocorticoid, methylprednisolone (MP), is a clinically effective drug that is not only used for the treatment of inflammatory diseases, but also the preferred drug against inflammation in kidney disease. But the mechanism for the beneficial effect of MP in renal inflammatory disease has still to be elucidated. Glucocorticoids have an inhibitory effect on FKN by reducing NF-κB gene promoter recruitment in respiratory epithelial A549 cells [27].
There is a need to investigate the protective effect of MP on the expression of FKN and the NF-κB pathway in the renal inflammatory

Measurement of FKN level in cell culture supernatant
The level of FKN in the supernatant was determined using enzymelinked immunosorbent assay (ELISA) kits (RayBiotech, Inc., Norcross, GA 30092, USA) according to the manufacturer's instructions.

Immunofluorescence staining for the detection of FKN and NF-κB p65 in HK-2 Cells
Immunofluorescence staining of cultured cells was performed as described previously [9]. The cells were incubated with the primary antibodies anti-FKN antibody (ab25088, Abcam Ltd, Hong Kong, 1:200 dilution) or anti-NF-κB p65 antibody (ab31481, Abcam Ltd, Hong Kong, 1:100 dilution) overnight at 4°C and then incubated with secondary antibody Alexa Fluor 488-conjugated goat anti-rabbit IgG (Beyotime Institute of Biotechnology, China, 1:200 dilution) for 60 min at 37°C. Cells stained with Alexa Fluor 488 conjugates of goat anti-mouse IgG antibody in order to detect HK-2 cells probed with human FKN or NF-κB p65 antibodies. Alexa fluor 488 appeared as green fluorescence and cells nuclei stained with PI appeared as red fluorescence. The image of immunofluorescence staining was scanned by Quantity One software and the original intensity was quantified with freeware image analysis software, NIH Image (National Institute of Health, Bethesda, Md., USA).

Statistical analysis
Data are reported as means ± SEM for normally distributed data and median (range) for nonparametric data. The comparisons of gene expression levels and demographic characteristics of the participants between groups were performed by using the one-way ANOVA for parametric data, F-test for equality of variances and Newman-Keuls test for heterogeneity of variance. All analyses were conducted with SPSS software, version 18.0. P value <0.05 was considered statistically significantly.

Effect of MP or SC-514 on FKN and p65 expression in LPS-Induced HK-2 cells
Immunofluorescence staining of FKN and p65 expression in HK-2 cells at 24 h is shown in Figures 4 and 5

Effect of MP or SC-514 on FKN, p65 protein and phospho p65 expression in LPS-Induced HK-2 cells
Western blot analysis of FKN in HK-2 cells at 24 h is shown in Figure 6. Compared with the normal HK-2 cells, the expression of FKN protein in LPS-induced HK-2 cells was increased significantly at 24 h (P<0.05). MP or SC-514 inhibited the increasing expression of FKN protein induced by LPS at 24 h (P<0.05).
Western blot analysis of p65 protein and phospho p65 in HK-2 cells is shown in Figure 7. Compared with the normal HK-2 cells, the ratio of phospho p65/p65 in LPS-induced HK-2 cells was increased significantly (P<0.05). MP or SC-514 down-regulated the increased ratio of phospho p65/p65 induced by LPS (P<0.05).

Discussion
Progressive renal interstitial fibrosis is the histological hallmark of chronic renal disease ultimately necessitating renal replacement therapy. Important biological roles of FKN in renal inflammation have been well documented in several tissues including the pancreas [31], joints and arteries [32] and more details are known for tubular and interstitial inflammation [33]. Because FKN has critical roles in renal inflammation, factors affecting its epithelial expression may be important in regulating inflammatory processes in renal interstitial fibrosis.
NF-κB is the predominant transcription factor that regulates a wide array of genes that encode pro-inflammatory cytokines, adhesion molecules and chemokines in kidney injury [34,35]. It is now well established that there are large numbers of genes activated via the NF-κB signaling pathway. Activation of NF-κB could play a central role in inflammatory cytokines-induced FKN expression at the transcriptional level [36]. LPS is a component of gram-negative bacteria that is known to cause pro-inflammatory cytokines production in renal tubular epithelial cells [37]. Previous work revealed that LPS stimulation increases FKN expression through the activation of the NF-κB signaling pathway in vitro [21].    The activation of NF-κB by induction with LPS was detected in HK-2 cells. Both MP and SC-514 was able to inhibit the activation of NF-κB induced by LPS. This suggests that the NF-κB signaling pathway was involved in the LPS induced FKN expression in HK-2 cells and that MP was able to prevent FKN-mediated renal inflammatory injury. The effect of MP was similar to NF-κB inhibitor, SC-514, suggesting that glucocorticoid MP may inhibit LPS-induced FKN expression via NF-κB pathway.
In summary, LPS induces the expression of FKN and activation of NF-κB in HK-2 cells. MP inhibits the expression of FKN and NF-κB activation induced by LPS in vitro. The inhibition of MP is consistent with NF-κB inhibitor, SC-514. MP inhibits LPS-induced FKN expression via the NF-B pathway in vitro and this cell culture model may be used to further explore ways of inhibiting inflammation in kidney cells.