Damage Effect Of Protein-bound Uremic Toxins On Vascular Endothelial Cells

Background:The latest statistics shows that the morbidity of chronic kidney disease(CKD) in the adult is more than 10%, and patients with end-stage kidney disease caused by CKD are also increased year by year[1]. with the continuous loss of renal function at the same time, this kind of patients are easy to have various syndromes. The epidemiological results shows that cardiovascular disease(CVD) is the most common complication and death causes of patients with uremia, compared to those person who has no kidney disease in the same gender and age[2-4]. The researchers found that uremia toxins may be the independent risk factors which cause the high prevalence of CVD in patients[5,6]. The uremia toxins contains protein-bound uremic toxins(PBUTs) and water-soluble uremic toxins, the latter is cleaned easily, however PBUTs is harder to be removed through regular hemodialysis and accumulated in the body for a long time, so they have much opportunity to contact the vascular endothelial cell that very likely to cause endothelial cell damage.Endothelial cell is the structure and functional unit of vascular endothelial, so the integrity of the structure and the normal function is the foundation of avoiding all kinds of vascular disease; the structure damage and insufficiency of endothelial cell is the most critical factor[7]. Numerous studies at home and abroad have confirmed that the PBUTs such as p-cresol sulfate(PCS) and indoxyl sulfate(IS) can cause endothelial cell damage through response mechanisms such as oxidative stress, so that it makes the happening of vascular disease[8-11]. However, there is no literature reported that PBUTs combined with serum protein can cause the damage of endothelial cell or not.Objectives:To investigate the injury effects of PBUTs on human umbilical vein endothelial cell(HUVEC), Cultured human umbilical vein endothelial cell is made as experiment object in this study, and protein solution of serum protein extracted from uremic patients with different concentration is used to intervene and stimulus HUVEC so that to detect the proliferation of HUVEC, nitric oxide production, nitric oxide synthase activity, loss rate and apoptosis index of the endothelial microparticles(EMPs).Methods:Every 10 ml venous blood extracted from 20 healthy volunteers and 20 maintenance hemodialysis uremic patients, hospitalized in Xinqiao hospital of TMMU was separated, dialyzed and freeze-dried with low temperature in a vacuum to get the serum protein[12]. HUVEC were cultured in growth medium with the addition of serum protein collected from the uremic patients or the normal persons in different concentrations(0.4%,1.0%,2.0% and 4.0%) for different time(24h,48 h and 72h),and the blank control group was established. Then the following indicators were observed and detected.1. The morphology of HUVEC affected by uremic serum protein was observed by inverted phase contrast microscope.2. The proliferation rate of HUVEC affected by PBUTs was measured by CCK-8.3. The secretion of nitric oxide(NO) affected by uremic serum protein was detected by assay kit of NO(nitric acid deoxidize enzyme method).4. The activity of nitric oxide synthase(NOS) affected by uremic serum protein was detected by assay kit of NOS(fluorescence method).5. The EMPs loss rate of HUVEC affected by uremic serum protein was detected by flow cytometry(FCM) of CD144.6. The apoptosis index of HUVEC affected by uremic serum protein was detected by(FCM) of Annexin V/PI double staining Situ cell death detection kit and DAPI dyeing method of nuclear.Results:1. The proliferation rate of HUVECs affected by uremic serum protein In the same time(24h/48h/72h), the OD value of HUVECs in uremic group is obviously lower than the OD value of HUVECs of the blank control group and the normal group at the same concentration(P<0.05); with the increase of concentration,compared to the same concentration of the normal one, the OD value of HUVECs in uremic group is lower apparently, it is concentration dependence(P<0.05); in the same concentration, as to the normal group, the OD value of HUVECs in uremic group declines apparently, it is time dependence(P<0.05).2. The morphology of HUVECs affected by uremic serum protein The HUVECs in the blank control group show uniform sizes, clear boundary, arrangement of single layer paving stone, short spindle shape; the normal group show little poor growth state, slightly varying sizes, thinly irregular surface, antennary cells, increased protuberances; the phenomenon that the plasma granules secreted by HUVECs increased could be observed; the HUVECs in uremic group show poor growth state, obviously varying sizes, different shape, obviously widened intercellular space, rough surface, antennary cells, distinctly increased protuberances, part of antenna, intertwined net-like protuberances, and the phenomenon that the plasma granules secreted by HUVECs increased could be observed too.3. The NO secretion of HUVECs affected by uremic serum protein After 72 h, The NO secretion of HUVECs in the blank control group is(13.24±0.32)umol/L, the NO secretion of HUVECs in the normal group(0.4%,1.0%,2.0% and 4.0%) is(12.68±0.55)umol/L、(10.10±0.30)umol/L、(7.86±0.49)umol/L、(7.40±0.14)umol/L respectively, the NO secretion of HUVECs in the uremic group is(9.46±0.29)umol/L、(6.64±0.28)umol/L、(5.42±0.50)umol/L、(3.92±0.34)umol/L respectively, the uremic group is obviously less than the blank one and the normal one(P<0.05); and presents concentration dependence(P<0.05).4.The activity of NOS affected by uremic serum protein After 72 h, the OD value of NOS activity of HUVECs in the blank control group is(192.39±3.69), the OD value of NOS activity of HUVECs in the normal group(0.4%,1.0%,2.0% and 4.0%) is(184.39±4.58)、(169.33±7.33)、(151.83±3.95)、(148.68±3.20)respectively, the OD value of NOS activity of HUVECs in the uremic group(0.4%,1.0%,2.0% and 4.0%) is(142.91±4.50)、(128.67±5.10)、(113.98±3.17)、(59.42±3.74)respectively, compared to the blank group and the normal one, the OD value of NOS activity of HUVECs in the uremic one is least(P<0.05). it is concentration dependence(P<0.05).5. The EMPs loss rate of HUVECs affected by uremic serum protein After 72 h,the EMPs loss rate of HUVEC is(7.30±0.57)%,(13.0±0.94)%,(62.57±1.59)% in the blank control group, the normal group and the uremic group respectively. The EMPs loss rate of uremic group is obviously more than the blank control group and the normal group(P<0.05).6. The apoptosis index of HUVEC affected by uremic serum protein The apoptosis index of HUVEC is(4.44±1.74)%,(5.49±1.57)%,(6.2±1.17)% in the blank control group, the normal group and the uremic group respectively, and in the statistical, there was no significant difference between groups.Conclusions:The uremic serum protein inhibited the proliferation rate and the NO secretion of HUVEC, affected the activity of NOS, accelerated the EMPs loss rate of HUVEC, so caused function of HUVEC, it supposed that PBUTs is the important inducement of CVD.