Protein-bound P-cresol Inhibits Human Umbilical Vein Endothelial Cell Proliferation By Cell Cycle Arrest At G₀/G₁

BackgroundChronic kidney disease (CKD) is a global public health concern. The incidence ofadult CKD is as high as10%. Cardiovascular disease (CVD) is highly prevalent in CKDpatients and is associated with exposure to uremic toxins. Cardiovascular andcerebrovascular events account for over50%of deaths in patients with end-stage renalfailure, despite adequate dialysis. Why enough dialysis could not modify the incidence andmortality of cardiovascular and cerebrovascular events? Are there any toxins which cannoteliminate by dialysis play important role in the mechanism of cardiovascular andcerebrovascular damage? Dialysis cannot efficiently eliminate the protein-bound uremictoxins. Our previous studies showed that uremic serum damaged the vascular endothelialcells and resulted in accelerated atherosclerosis. Therefore, a key question relates to the roleof protein-bound toxins in the pathogenesis of cardio-and cerebro-vascular disease.Previous studies associated protein-bound uremic toxins (e.g., indoxyl sulfate,homocysteine, etc.) with endothelial dysfunction in patients with CKD, but not with theincreased incidence of cardiovascular disease. Recent experimental studies showedsignificant increases in serum levels of P-cresol in the mid-to-late stage of chronic kidneydisease (CKD) and were associated with cardiovascular mortality. Free P-cresol sulphate isa predictor of mortality in patients at different stages of chronic kidney disease. Studieshave mostly focused on the toxicity of unbound fraction of P-cresol. It is not knownwhether protein-bound P-cresol was correlated with cardiovascular diseases.ObjectiveIn this study, we used P-cresol bound to human serum albumin (HSA, with4%concentration) and unbound P-cresol to stimulate the cultured human umbilical veinendothelial cells (HUVEC), and investigated the effects of unbound P-cresol andprotein-bound P-cresol on HUVEC proliferation and cell cycle. MethodHUVEC were treated with20,40, and80μg/ml HSA-bound P-cresol and unboundP-cresol respectively. Equivalent volume of methanol solvent of P-cresol was added to theculture medium as control. After treating for24h,48h and72h, the cells were harvestedfor these experiments:1. The effects of of P-cresol on cell proliferation were determined by CCK-8assay.2. The effects of P-cresol on HUVEC apoptosis were determined by AnnexinV-FITC/PI staining kit.3. The effects of of P-cresol on Cell cycle were analysised on a FACS flow cytometer.4. The protein expressions of cell cycle-regulatory proteins of all cultured cells weredetermined by western blot.5. Effects of P-cresol on the expression of p21Cip1and cyclin D1proteins weredetected by i Double labeling immunofluorescence of all cultured cells.Results1. Effect of P-cresol on cell proliferation.Both protein-bound and unbound P-cresol inhibited HUVEC proliferation. The80μg/ml unbound P-cresol induced inhibition of HUVEC proliferation by23.56%±3.24%,48.55%±5.05%and80.68%±1.69%at24,48and72hr, respectively, compared withcontrol medium (P <0.05). The80μg/ml protein-bound P-cresol induced inhibition ofHUVEC proliferation by22.39%±4.79%,41.38%±2.10%and77.56%±2.06%at24,48and72hr, respectively, compared with control medium (P <0.05). P-cresol inhibitedHUVEC proliferation in a dose-and time-dependent manner.2. Effect of P-cresol on HUVEC apoptosis.To determine whether P-cresol induced HUVEC apoptosis, we measured thepercentage of annexin V-positive cells after solute stimulation. This percentage was notincreased after incubation with protein-bound and unbound P-cresol, showing that P-cresoldid not induce HUVEC apoptosis.3. P-cresol induces HUVEC cell cycle arrest at G0/G1.HUVEC were arrested at G0/G1by unbound and protein-bound P-cresolin a dose-dependent manner. With unbound P-cresol, when cultured for72hr, the G0/G1phasepercentage of HUVEC was significantly higher (68.68±3.44%at20μg/ml,75.26±1.37%at 40μg/ml, and81.27±1.15%at80μg/ml) compared with the control (62.37±0.39%, P <0.05).With protein bound P-cresol, when cultured for72hr, the G0/G1phase percentage ofHUVEC was significantly higher (69.87±2.57%at20μg/ml,74.88±1.56%at40μg/ml, and79.63±1.18%at80μg/ml) compared with the control (62.45±0.72%, P <0.05).4. Effect of P-cresol on the expression of cell cycle-regulatory proteins.HUVEC were treated with protein-bound and unbound P-cresol (2080μg/ml) for72hr, and then measured by immunoblotting analysis using specific antibodies. Both unboundand protein-bound P-cresol markedly diminished the expression of cyclin D1. Bothunbound and protein-bound P-cresol markedly increased the expression of p21Cip1.However, CDK4, p16INK4A, p15INK4B, and p27Kip1levels were not affected by P-cresoltreatment.5. Effects of P-cresol on the expression of p21Cip1and cyclin D1proteins.To further evaluate the influence of P-cresol on the expression of p21Cip1and cyclinD1proteins, double immunofluorescent labeling technique was performed in HUVEC.Cultured cells were treated with protein-bound and unbound P-cresol (80μg/ml) for72hr.The results indicated that p21Cip1staining was apparently increased, while cyclin D1staining was significantly decreased compared with the control.Conclusion1. Both unbound and protein-bound P-cresol inhibited HUVEC proliferation in a dose-and time-dependent manner.2. Both unbound and protein-bound P-cresol introduced HUVEC cell cycle arrest atG0/G1phase.3. P-cresol-introduced HUVEC cell cycle arrest was associated with the upregulationof p21Cip1expression and downregulation of cyclin D1expression.