The Analysis Of The Expression Of ABCG2in Renal Proximal Tubular Epithelial Cells By IL-1β

Gout is an inflammatory arthritis characterized by abrupt self-limiting attacks of inflammation caused by precipitation of monosodium urate crystals (MSU) in the joint. It’s associated with hyperuricemia owing to purine metabolism disorders and/or reduced excretion of urate. Patients with gout are significantly more than before. Serum uric acid presents a higher frequency of cardiovascular disease, hypertension, diabetes, dyslipidemia and chronic nephrosis, which should be paid more attention to.In mammals, the proximal tubule is the major site of renal urate handling. The reabsorption and secretion of urate are completed by different protein transporters. Several genes with a potential role in reabsorbing and secreting urate have been discovered. Among them, ATP-binding cassette superfamily G member2(ABCG2), which promotes the urate excretion of the renal tubular epithelial cell, is one of the most important transporters so far. Some genetic loci of ABCG2is related to urate and gout. The recent research on ABCG2might provide data that could potentially lead to the development of improved therapies for hyperuricemia and gout. On the contrary, some gout patients’urate level is lower during acute attack according to the clinical finding. We assume that the function of ABCG2enhances for acute gout flares so that the urine uric acid excretion increases, which leads to the decrease of serum urate level. When the feedback mechanism attenuates, serum urate level rises.Interleukin(IL)-1β is a pivotal pro-inflammatory cytokines, and there are numerous researchs of its impact on ABCG2in different kinds of cells. However, the report of its effect of ABCG2in human renal tubular epithelial cell is rarely seen.Objective:To investigate the influence of ABCG2on the human renal proximal tubular epithelia (HK-2cells) with the stimulation of IL-β1and assess its related underlying mechanisms.Methods:1.The protein expression of ABCG2and subcellular localization in cells were evaluated using immunocytochemistry under confocal laser scanning microscopy.2.The HK-2cells were cultured with different concentrations (0,0.1,1,5, lOng/ml) of IL-1β and for different time (0,1,2,4,8,16,24,48h). The mRNA levels of ABCG2were observed by Quantitative real-time PCR. The protein expressions were measured by Western blot accordingly.3.After incubating with IL-1β for1hour,HK-2cells were stimulated with10ng/ml IL-1β,10ng/ml IL-10,10ng/ml TGF-β,25ng/ml TNF-α,10ng/ml IL-1β jointed with10ng/ml IL-10,10ng/ml IL-1β jointed with10ng/ml TGF-β and10ng/ml IL-1β jointed with25ng/ml TNF-α for24hours. And ABCG2proteins were detected by western blot Then HK-2cells were stimulated with inhibitor of JNK (10uM Sp600125), inhibitor of ERK (5uM U-0126), inhibitor of NF-κB (100uM PDTC), inhibitor of PI3K (1uMWortmannin), inhibitor of JAK (10uM AG-490) and5uM ubiquitin-proteasome inhibitor MG132;24hours later the expressions of ABCG2protein were measured by western blot Then the nucleoprotein and cytoplasmic protein expressions of P50, P52, P65, AKT, JNK, ERK, JAK were detected by western blot after HK-2cells were stimulated with0,1,10ng/ml IL-1β for24h.Results:Compared with MCF-7、HEK293and T47D cells, ABCG2expression of HK-2cells was moderate. As the concentration of IL-1β or stimulation time increased, the mRNA level of ABCG2decreased, but the protein expression rised. The protein location didn’t change. IL-1P jointed with TNF-a stimulation significantly enhanced the ABCG2expression. TNF-a, IL-10, TGF-β, IL-1β jointed with IL-10or TGF-β didn’t work. Inhibitor of NF-κB could inhibit the expression of ABCG2in HK-2cells after treating with IL-1β. Inhibitor of JNK, ERK, PI3K, JAK, AKT and ubiquitin-proteasome didn’t impact. What’s more, compared with control group, P50and P65expressions in HK-2cells were higher after treating with IL-1β, which were the subunits of NF-κB. It mainly happened in nucleus and the most effective dose was1ng/ml. Expressions of P52, JNK, ERK, AKT and JAK didn’t change.Conclusion:ABCG2expression of HK-2cells is moderate. IL-1β could reduce the ABCG2mRNA expression of HK-2cells, but rise the protein expression. The protein location does’t change. And the proteins do not undergo ubiquitin-mediated protein degradation or there are other ways of doing this. IL-1β jointed with TNF-a can induce the expression of ABCG2significantly. The upregulation of IL-1β in HK-2cells is mediated by NF-κB pathway. Among the subunits of NF-κB, P50and P65play the dominant role. It mainly happens in nucleus.