The Uremic Toxin Hippurate Promotes Endothelial Dysfunction Via The Activation Of Drpl-mediated Mitochondrial Fission

Objective:Cardiovascular disease(CVD)is a major factor affecting the prognosis of patients with chronic kidney disease(CKD),among whom the morbidity and mortality are respectively 10-to 20-fold higher than those in age-and sex-matched healthy people.Endothelial dysfunction,including inappropriate regulation of vascular tone,activated inflammatory response and impaired antithrombogenic property,is a key step in the development of atherosclerotic CVD.Although CKD patients have increased levels of traditional cardiovascular risk factors,i.e.,hypertension,diabetes and hyperlipidemia,these factors cannot fully explain the high risk of CVD in patients with CKD.Accumulating evidence shows that the accumulation of hippurate also play a vital role in CKD-associated endothelial dysfunction.In this study,we investigated the entire coagulation process especially endothelial function in CKD patients,examined the relationship between hippurate and biomarkers of endothelial function,and assessed the endothelial toxicity of hippurate and the contribution of altered mitochondrial dynamics to hippurate-induced endothelial dysfunction.Methods:1.A total of 95 CKD patients and 20 healthy controls who met the inclusion criteria were consecutively recruited.The markers of platelet,endothelial function,the major blood coagulation pathways,natural coagulation inhibitors,standard coagulation tests,and thromboelastography were measured in the CKD patients and controls.Associations between the estimated glomerular filtration rate(eGFR)and hemostatic biomarkers were tested using multivariable linear regression.The incidence of thromboembolic events in the CKD patients and healthy controls were recorded during the two-year of follow-up.2.The cross-sectional associations of hippurate with biomarkers of endothelial function were evaluated for 151 consecutive CKD patients.We collected the basic information and measured serum hippurate levels with commercially available ELISA kits according to the manufacturer's specifications.software.The correlations between hippurate and sICAM-1,sVCAM-1,vWF were assessed.3.We treated HAECs with increasing concentrations(0,1,2,4mM)of hippurate at different stimulation time to test whether hippurate promotes endothelial dysfunction in a time-and dose-dependent manner.We detected the cellular and mitochondrial ROS levels in hippurate-treated HAECs and measured the expression of endothelial function markers after Mito-TEMPO pretreatment.For animal experiment,a CKD rat model(5/6 nephrostomy)and a hippurate-treated rat model(50 mg/kg hippurate ip for 6 weeks)were established.The serum hippurate levels in two models were determined by high performance liquid chromatography.Endothelium-dependent vasodilation were examined on isolated aortic rings at the end of the experiment.4.We observed mitochondrial morphology in hippurate-stimulated endothelial cells by electron transmission microscopy and Mitotracker fluorescence staining and examined the expression of mitochondrial division-associated proteins(Drpl,Fis1,Mff)in both cell and animal models.After inhibiting Drp1 of the vascular endothelium(Mdivi-1 pretreatment or Drp1 siRNA transfection),changes in mitochondrial morphology,oxidative stress levels,mitochondrial membrane potential,and endothelial function were examined.Results:1.Levels of FVII,FVIII,vWF,fibrinogen,and D-dimer were significantly higher in the CKD patients than the levels in the controls after adjustment for traditional cardiovascular risk factors.Pearson correlation analysis revealed that higher vWF and FVIII were significantly associated with a decreased eGFR.Cardiovascular risk in CKD patients appeared to increase compared with healthy subjects,but the differences were not statistically significant.2.Compared with healthy subjects,the hippurate levels,IL-2 and IL-6 were significantly elevated in CKD patients,and SOD levels were decreased.Spearman correlation analysis showed that hippurate levels in patients with CKD were positively correlated with sICAM-1,sVCAM-1 and vWF concentrations.3.Hippurate reduced the protein and mRNA levels of eNOS and increased ICAM-1 and vWF expression in a time-and dose-dependent manner.After stimulation with 4 mM hippurate for 48 h,mitoROS levels were significantly elevated.When HAECs were pretreated with MitoTEMPO,mitoROS production was reduced to a relatively low level,and cell injury was mitigated.In animal experiment,CKD rats and hippurate-treated rats displayed higher hippurate levels than control rats.Compared with aortic rings from control rats,aortic rings from two model rats exhibited impaired Ach-mediated vasodilation.4.We observed an increase in Drpl levels and mitochondrial fission in hippurate-treated HAECs and in aortas of CKD rats and hippurate-treated rats.Inhibition of Drpl reduced mitochondrial fragmentation and ROS production,and improved mitochondrial membrane potential.Finally,Drp1 inhibition markedly improved endothelial-dependent vasodilationand eNOS expression and reduced vascular ICAM-1 and vWF expression.Conclusions:Serum hippurate levels are significantly raised in CKD patients than healthy subjects and the hippurate concentration was correlated with sICAM-1,sVCAM-1 and vWF.Both cellular and animal experiments have confirmed that hippurate cause endothelial oxidative damage.Increased mitochondrial fission plays an active role in hippurate-induced endothelial dysfunction via an increase in mitoROS.