The Effect And Mechanism Of Phosphorus Concentration On Microinflammatory And Oxidative Stress Response In Maintenance Hemodialysis Patients

Background:Maintained haemodiaysis(MHD)is one of the replacement therapy for end-stage renal disease(ESRD). Cardiovascular complications are the leading cause of death in MHD patients. Alteration of mineral metabolism is a prevalent condition in MHD patients. Among abnormalities of mineral metabolism, the most prominent and relevant one is hyperphosphatemia. High phosphorus is considered to be the main factor leading to CVD. It is now known that inflammation and Oxidative stress is a prevalent condition in MHD patients. Inflammation and oxidative stress is also nontraditional risk factors for CVD in MHD patients. However, it has not been defined, so far, whether and how hyperphosphatemia、micro-inflammatory states、oxidative stress are related to each other. The activation of the oxidant-sensitive nuclear factor (NF)-kB transcription factor family is one of the major pathways resulting in inflammation. NADPH oxidase (NOX) is the main enzyme responsible for reactive oxygen species (ROS) production.We hypothesized that hyperphosphatemia may promote the expression of inflammatory mediators and oxidative stress product by activation of NF-κB and NOX2in PBMCs. For this reason we will explore the relationship among hyperphosphatemia, micro-inflammatory states and oxidative stress and its possible mechanism.Objective:1. To investigate the effect of high phosphate on mRNA and protein expression of intracellular NF-κB and NOX2/gp91in PBMC of MHD patients. 2. To investigate the effects of high phosphate on serum inflammation mediators and MDA production, to understand the possible relationship among hyperphosphatemia, micro-inflammatory states and oxidative stress.Method:1. Blood was collected from stable MHD patients prior to dialysis, and normal person as controls. PBMCs were isolated over Ficoll. NF-κB and NOX2/gp91mRNA was detected by Real Time PCR, NF-κB and NOX2/gp91protein was detected by Western Blot.Serum was assayed for IL-6and MDA.2. Blood was collected from stable MHD patients. PBMCs were isolated over Ficoll. Cells were cultured at37℃in a humidified atmosphere of5%CO2using RPMI-1640containing10%FCS, penicillin(100units/ml), and streptomycin(100g/ml). PBMCs were incubated with different concentration of inorganic phosphate(NaH2PO41.5、2.5、5.5mM) for24h to observe the effect of phosphate on the expression of NF-κB and NOX2/gp91by Real Time PCR and Western Blot. Then PBMCs were treated with high concentration of phosphate(5.5mmol/l NaH2PO4)for different time to detect the expression of NF-κB and NOX2/gp91.3. The concentration of IL-6in the culture supematants collected under various exposure conditions was quantified by the enzyme linked immunosorbent assay(ELISA)using the ELISA kit. The concentration of MDA in the culture supematants collected under various exposure conditions was quantified by the TBA assay kit.Result:1. Compared with healthy person (control),the NF-κB p65mRNA and Phospho-NF-KBp65protein were increased significantly.Both expression of NOX2/gp91mRNA and protein in PBMCs of MHD pateints increased significantly(P<0.01). Compared with normal control(0.005±0.002ng/ml), serum IL-6production of MHD. pateints is (0.014±0.005ng/ml). Compared with normal control(2.534±0.853),serum MDA production of MHD pateints is (4.986±1.001nmol/ml).2. Compared with control,incubation of PBMCs with high phosphate)stimulated Intracellular NF-κB p65mRNA and Phospho-NF-κBp65protein expression(P<0.01); Both expression of NOX2/gp91mRNA and protein increased significantly(P<0.01).High phosphate increased NF-κB and NOX2/gp91mRNA and protein expression dose-and time-dependent manners totally.3. Effects of high phosphate on IL-6production.Compared with control(1.5mM0.223±0.049ng/ml), treatment with high phosphate(2.5mM0.326±0.044ng/ml,5.5mM0.442±0.022ng/ml); Compared with control(6h0.118±0.009ng/ml), treatment with high phosphate at different time(12h0.168±0.019ng/ml,24h 0.393±0.013ng/ml).IL-6increases significantly4. Effects of high phosphate on MDA production. Compared with control(1.5mM0.548±0.324ng/ml), treatment with high phosphate(2.5mM1.370±0.300nmol/ml,5.5mM2.215±0.493nmol/ml); Compared with control(6h1.164±0.375nmol/ml), treatment with high phosphate at different time(12h1.849±0.284nmol/ml,24h2.650±0.374nmol/ml).MDA increased significantly.5. Linear correlation analysis showed that NF-κB p65mRNA and Phospho-NF-κBp65protein were positively correlated with IL-6production. NOX2/gp91mRNA and protein expreesion were positively correlated with MDA production.Conclusion:1. NF-κB and NOX2signal pathway in PBMCs show abnormal activation status in MHD patients2. Treatment with high phosphate can upregulate NF-κB p65mRNA and Phospho-NF-KBp65protein expression. Both expression of NOX2/gp91mRNA and protein levels increased dose-and time-dependent manners totally. And it can upregulate IL-6and MDA production in supernatant dose-and time-dependent manners totally.3. High phosphate induction of supernatant IL-6and MDA production may correlate with the activtion of NF-κB and NOX2signalling pathway in PBMCs. 4. High phosphorus may be a risk factor for microinflammatory and oxidative stress status of MHD patients.