Background Vitamin Deb is considered to exert a protective effect on

Background Vitamin Deb is considered to exert a protective effect on various renal diseases but its underlying molecular mechanism remains poorly understood. of Akt. The production of proinflammatory cytokines GDC-0349 and the number of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells were attenuated by paricalcitol in LPS uncovered HK-2 cells. The cotreatment with an EP4 antagonist abolished these anti-inflammatory and antiapoptotic effects. Conclusion EP4 plays a pivotal role in anti-inflammatory and antiapoptotic effects through Akt and NF-B signaling after paricalcitol pretreatment in LPS-induced renal proximal tubule cell injury. for 15 minutes. The pellet was resuspended in solubilization buffer and the solubilized membrane fraction was harvested after centrifugation for 30 minutes at 4C. Enzyme immunoassay HK-2 cells were homogenized by PRO-PREPTM Protein Extraction Answer (iNtRON Biotechnology, Seongnam, Korea) and then centrifuged for 10 minutes at 12,000test. All analyses were performed using SPSS software (ver. 20.0; IBM Corp., Armonk, NY, USA). A < 0.05 was considered statistically significant. Results Paricalcitol upregulates COX-2 GDC-0349 and PGE2 manifestation in LPS-exposed HK-2 cells GDC-0349 Fig. 1 shows the effects of paricalcitol on VDR, COX-2, and PGE2 in LPS-exposed HK-2 cells. Paricalcitol pre-treatment significantly increased the manifestation of VDR and LPS exposure caused the suppression of VDR. Paricalcitol pretreatment largely restored the manifestation of VDR in LPS-exposed HK-2 cells (Fig. 1A). The manifestation of COX-2 was increased in paricalcitol-pretreated HK-2 cells and further enhanced in LPS-exposed cells treated with paricalcitol (Fig. 1B). HK-2 cells treated with paricalcitol produced a significantly higher level of PGE2 than control cells. LPS exposure alone increased PGE2 manifestation than control cells and paricalcitol further enhanced PGE2 manifestation in LPS-exposed cells (Fig. 1C). Physique 1 Paricalcitol upregulates the VDR, COX-2 and PGE2 manifestation in LPS-exposed HK-2 cells Paricalcitol activates the PGE2-EP4 signaling pathway in LPS-exposed HK-2 cells We examined the induction of PGE2 receptor subtypes in paricalcitol-treated HK-2 cells. Paricalcitol significantly increased the mRNA level of in HK-2 cells with or without LPS exposure. However, paricalcitol did not affect mRNA levels of (Fig. 2A). Paricalcitol increased the manifestation of EP4 protein dose-dependently, and the highest increase in EP4 manifestation was observed at 2 ng/mL paricalcitol (Fig. 2B). The cellular membrane/ cytoplasm ratio of EP4 was significantly increased in LPS-exposed cells compared with the control group, and it was similarly increased in response to TNF- treatment (2 ng/mL) instead of LPS (Supplementary fig. 1). Membrane/cytoplasm ratio of EP4 was further enhanced when paricalcitol-pretreated cells were uncovered LPS injury (Fig. 2C). The conversation between PGE2 and EP4 was investigated by immunoprecipitation. PGE2/EP4 complex formation GDC-0349 was increased after LPS GDC-0349 exposure, and paricalcitol pretreatment further increased their interactions (Fig. 2D). Physique 2 Paricalcitol activates PGE2-EP4 signaling pathway in LPS-exposed HK-2 cells Paricalcitol improves cell survival after LPS injury via EP4 The viability of cells treated with 0.5% NaHCO3 was similar to that of control cells without any treatment (> 0.05). LPS treatment decreased the cell viability to around 80% of the control group, and EP4 siRNA treatment further decreased the viability of LPS-exposed cells (86.9 1.7 vs. 81.7 1.1; = 0.031). Paricalcitol pretreatment significantly increased the viability of LPS-exposed cells, and this protective effect of paricalcitol was offset by AH-23848 cotreatment or an EP4 siRNA blockade (Fig. 3). Physique 3 Paricalcitol protects HK-2 cells against endotoxin-induced death via an EP4-dependent pathway EP4 regulates the inhibitory effects of paricalcitol on p65 NF-B activation LPS activation increased the phosphorylation of p65 NF-B (Fig. 4A). Paricalcitol suppressed phosphorylation of p65 NF-B, and the inhibitory effect of paricalcitol was reversed by AH-23848 cotreatment in LPS-exposed HK-2 cells. We further examined the subcellular localization of p65 NF-B (Fig. 4B). p65 NF-B was translocated to the nuclei in response to LPS activation. Paricalcitol decreased p65 IL1R NF-B translocation, and cotreatment with AH-23848 and paricalcitol promoted the nuclear translocation of p65 NF-B in LPS-exposed cells. To evaluate whether phosphorylation of p65 NF-B was associated with the specific activation of EP4, we performed further experiments for manifestation using EP4 siRNAs (Fig. 4C). As with AH-23848 treatment, EP4 siRNA enhanced the nuclear translocation of p65 NF-B in LPS-stimulated cells treated with paricalcitol. Physique 4 Paricalcitol inhibits p65 NF-B activation after LPS treatment via an EP4-dependent pathway The EP4-dependent pathway contributes to the inhibitory effects.