Effect And Mechanism Of Vitamin D On Vascular Endothelial Cell Activation In Patients With Chronic Kidney Disease

Objective To investigate vitamin D levels and the association between hypovitaminosis D and vascular endothelial cells activation in patients with non-dialysis chronic kidney disease(CKD).The possible mechanism involved in this process was also explored through in vitro experiments.Methods In vivo study:A total of 117 non-dialysis CKD patients in a stable clinical state were enrolled.Markers of endothelial cells activation including vascular cell adhesion molecular(VCAM)and E-selectin were measured by enzyme linked immunosorbent assay(ELISA).25-hydroxyvitamin D was measured by chemiluminescence.The correlation between serum 25-hydroxyvitamin D levels and various indicators was observed.In vitro study:Human umbilical vein endothelial cells were cultured and treated with tumor necrosis factor-α(TNF-α)(40ng/ml),1α,25-dihydroxyvitamin D3(10-8mol/L),and SN50 as indicated.The protein and RNA expressions of VCAM and E-selectin were detected by Western blot and Real-time PCR.In the study of mechanism,nuclear factor kappa B(NF-κB)signaling pathway was used as the entry point,and Western blot,Real-time PCR,immunofluorescence assay and chromatin immunoprecipitation(ChIP)methods were used to evaluate the effects of lα,25-dihydroxyvitamin D3 on its early activation(including I-κBα and NF-κB p65 phosphorylation),nuclear transport and binding to VCAM and E-selectin promoters.Results In vivo study:In 117 non-dialysis CKD patients,there were 50.4%cases of vitamin D deficiency,43.6%cases of vitamin D insufficiency and 6.0%cases of vitamin D sufficiency.The levels of VCAM and E-selectin were higher in vitamin D-deficient and insufficient groups versus vitamin D-sufficient group,and the highest value was observed in vitamin D-deficient group.Values of VCAM(P=0.002)and E-selectin(P=0.016)were statistically different between vitamin D-deficient and vitamin D-sufficient group.There was a negative correlation between both VCAM(r=-0.549,P<0.001)and E-selectin(r=-0.360,P<0.001)and 25-hydroxyvitamin D.Multiple regression analysis revealed that vitamin D deficiency was an independent risk factor for endothelial cells activation.In vitro study:1)Western blot and Real-time PCR showed that both 1α,25-dihydroxyvitamin D3 and SN50 could significantly inhibit TNF-α induced upregulation of VCAM and E-selectin.2)Western blot showed that TNF-α can induce I-κBα phosphorylation,thereby activating NF-κB p65 subunit.1α,25-dihydroxyvitamin D3 inhibited I-κBα and NF-κB p65 phosphorylation.Immunofluorescence showed that TNF-α can induce the nuclear translocation of NF-κB p65 subunit,1α,25-dihydroxyvitamin D3 significantly inhibited the nuclear translocation of NF-κB p65 subunit.ChIP analysis showed that TNF-α can induce the binding of NF-κB p65 to VCAM and E-selectin promoters,1α,25-dihydroxyvitamin D3 inhibited the binding of NF-κB p65 to VCAM and E-selectin promoters and thus affected gene expression.Conclusions Vitamin D deficiency or insufficiency is common in non-dialysis CKD patients.Hypovitaminosis D is an important factor affecting the activation of vascular endothelial cells in CKD patients.The mechanism may be related to the inhibition of TNF-α-induced NF-κB early activation,nuclear transport and the binding of NF-κB p65 to VCAM and E-selectin promoters by vitamin D.Vitamin D may help to inhibit vascular endothelial cell activation in CKD patients,improve vascular endothelial cell function,reduce cardiovascular complications,improve quality of life and long-term prognosis.