Effect Of Indoxyl Sulfate On The Chemotaxis Of Monocytes

BackgroundChronic kidney disease(CKD)incidence increased year by year,affecting 10% of the population worldwide,is now viewed as part of the rising global non-communicable disease burden.With the progression of CKD,some patients will go into end-stage renal disease(ESRD),need rely on blood purification therapy or a kidney transplant to maintain life.The retention of uremic toxins in ESRD patients,is the basic cause of uremic syndrome.The European uremic toxin work group(EUTox)divided those uremic toxins into three categories according to molecular weight and protein binding capacity:(1)Free water-soluble low-molecular-weight solutes(<500Da);(2)Protein-bound uremic toxins(PBUTs);(3)the larger “middle molecules”(>500Da).Dialysis presents as the primary method for solute removal in ESRD patients.Present techniques allow for the removal of compounds up to 20 k Da(the majority of low and middle weight molecules);however,albumin(66.5 k Da)bound solutes are unable to be dialytically cleared due to their augmented size.Uremic toxins is a major risk factor for cardiovascular disease(CVD),and CVD is a common comorbidity and a major cause of mortality in ESRD patients,even when they enters the dialysis therapy.Thus,PBUTs as the non-dialyzable uremic toxins,are likely one of the potential missing culprits in cardiovascular events.Previous studies have demonstrated that PBUTs,especially indoxyl sulfate(IS),can induce endothelial dysfunction and vascular smooth muscle cells proliferation.The damaged vascular endothelial can produce monocyte chemotactic protein-1(MCP-1),a protein belonging to type CC chemokine family,that mediates monocyte recruitment in proximity of endothelial lesions after being combined with its specific receptor C-C chemokine receptors 2(CCR2),by creating of a chemotactic gradient towards the inflammatory site.Monocytes/macrophages play an important role in the pathogenesis of atherosclerosis.Previous studies on the cause of atherosclerosis by PBUTs,are more focused on the release of inflammatory factors after vascular injury,and then activate and induce monocytes/macrophages infiltration and adhesion to the inflammatory site.But whether PBUTs have toxicity on monocytes,or can enhance the chemotaxis of monocytes/macrophages.There is a lack of relevant reports and need to be further studied.Objectives:In this study,we used albumin-bound IS(IS bound to human serum albumin)and free IS to stimulate the THP-1 cells,and investigated the effects of albumin-bound IS and free IS on the activity of chemotaxis in monocytes.Methods:1.Different concentrations of IS were bound to albumin with 4% concentration,evaluate the protein binding rate by HPLC.2.THP-1 cells were cultured in vitro,treated with different concentrations of free IS(IS 100,200,400,800?mol/L)and albumin-bound IS(4%HSA+IS 100?mol/L,4%HSA+IS 200?mol/L,4%HSA+IS 400?mol/L,4%HSA+IS 800?mol/L)for 12 h,24h and 48 h,CCK-8 assay was employed to evaluate the proliferation of THP-1 cells in each group.3.Experimental groups: blank control group(PBS),negative control group(4%HSA solution),albumin-bound IS group(4%HSA+IS 200?mol/L)and free IS group(IS 200 ?mol/L)group.4 groups respectively incubation THP-1 cells for 24 h,Transwell chemotaxis chamber was used to measure the chemotaxis.4.Albumin-bound IS(4%HSA+IS 200?mol/L)and free IS(IS 200?mol/L)respectively incubation THP-1 cells for 24 h,ELISA kits was employed to evaluate the concentrates of MCP-1.5.Albumin-bound IS(4%HSA+IS200?mol/L)and free IS(IS 200?mol/L)respectively incubation THP-1 cells for 24 h.Flow cytometry was employed to evaluate the protein expression of CCR2.Results:1.The protein binding rate of ISThe free rates of IS in different concentrations of albumin-bound IS(4%HSA+IS 100?mol/L,4%HSA+IS 200?mol/L,4%HSA+IS 400?mol/L,4%HSA+IS 800?mol/L)were 11.5%,12.8%,9.2% and 7.85%,and the protein binding rate of IS were 88.5%,87.2%,90.8% and 92.15%.2.Effect of IS on cell proliferationFree IS can inhibit the proliferation of THP-1 cells.With the increase of drug concentration,the proliferation of THP-1 cells inhibitory effects gradually increased in a concentration dependent manner(P < 0.05);with the extension of the duration of drug action,the proliferation of THP-1 cells inhibition by gradually enhanced with time dependent(P < 0.05).Albumin-bound IS also can inhibit the proliferation of THP-1 cells.With the increase of drug concentration,the proliferation of THP-1 cells inhibitory effects gradually increased,but there was no significant difference between the concentration groups of more than 200?mol/L.At the same time,with the prolongation of the time of drug action,the proliferation inhibition of THP-1 cells was gradually increased,but there was no significant difference between the time points after 24 h.3.Effect of IS on the chemotaxis of monocytesThe results of chemotaxis experiments show that: the average number of penetrating cells of the blank control group(PBS)was(13.2 ± 5.6)cells/field,negative control group(4%HSA solution)was(14.5 ± 6.4)cells/field,albumin-bound IS group(4%HSA+IS 200 ?mol/L)was(85.2 ± 26.8)cells/field,and free IS group(IS 200 ?mol/L)group was(123.5 ± 48.6)cells/field.Compared with the two control groups,the number of cells in albumin-bound IS group and free IS group increased significantly,with significant difference(P < 0.05).Comparison between the two experimental groups,the number of transmembrane cells in free IS group was significantly higher than that in albumin-bound IS group,and the difference was statistically significant(P < 0.05).4.Effect of IS on the expression of MCP-1Compared with the two control groups,the MCP-1 concentrations of the free IS group(IS 200?mol/L)and albumin-bound IS group(4%HSA+IS 200?mol/L)were significantly increased,with a significant difference(P < 0.05);Comparison between the two experimental groups,the concentration of MCP-1 in free IS group(IS 200?mol/L)was higher than that in albumin-bound IS group(4%HSA+IS 200?mol/L)were significantly increased,with a significant difference(P<0.05).5.Effect of IS on the expression of CCR2Compared with the two control groups,the expression of CCR2 in free IS group(IS 200 ?mol/L)was significantly higher,and the difference was statistically significant(P < 0.05).Compared with the negative control group,the expression of CCR2 in albumin-bound IS group(4%HSA+IS 200?mol/L)was higher,and the difference was statistically significant(P < 0.05).Comparison between the two experimental groups,the expression of CCR2 in free IS group(IS 200 ?mol/L)was higher than albumin-bound IS group(4%HSA+IS 200 ?mol/L),and the difference was statistically significant(P < 0.05).Conclusions:Both albumin-bound IS and free IS could depressed the proliferation ability of THP-1 in a dose-dependent manner.Both albumin-bound IS and free IS could enhance the chemotaxis of monocytes by increasing the protein expression of MCP-1 and CCR2.The concentration of albumin-bound IS in the blood circulation is 9 times as much as the free toxins,So trying to remove the albumin-bound indoxyl sulfate is helpful to improve the long-term prognosis in patients with CKD.