CXCL8 Mediated The Crosstalk Between Valvular Interstitial Cell And Endothelial Cell In Chronic Kidney Disease With Valvular Calcification

Objective:Valvular calcification is one of the common complications in patients with chronic kidney disease(CKD),characterised with the accumulation of extracellular matrix,multicellular activation and synthesis of osteoid-like substances.The incidence of aortic calcification and mitral valvular calcification is about 55%and 59%respectively;the incidence of valvular stenosis is 6-13%.Valvular calcification is closely related to the high incidence of cardiovascular events and high all-cause mortality in patients with CKD,which seriously affects the survival rate and quality of life of patients.Current studies found that,compared with patients with chronic kidney disease with normal renal function,patients with dialysis have a faster progression of valvular stenosis and a higher risk of cardiovascular adverse events.Therefore,studying the pathogenesis of valvular calcification in CKD is of great significance for understanding the occurrence and development of the disease.Therefore,the project was aimed to study the pathogenesis of valvular calcification in chronic kidney disease in vivo and in vitro.Part one:A Rat Model with Multivalve Calcification Induced by Subtotal Nephrectomy and High-Phosphorus DietMethod:In the first part,we established a CKD model in Sprague-Dawley rats by performing5/6 nephrectomy(5/6Nx)followed by feeding with chow containing different phosphate concentrations for 8,12,or 16 weeks.The rats were divided into 4 groups:Sham+normal phosphate(NP,0.9%P),Sham+high phosphate(HP,2.0%P),5/6Nx+NP,and 5/6Nx+HP.Serum creatinine(Scr),blood urea nitrogen(BUN),parathyroid hormone(PTH),calcium,phosphorus,and 24h urine protein levels were investigated.Pathological examinations included histological characterization,safranin staining,Alcian blue staining,and von Kossa staining at different time points.By nanoanalytical electron microscopy,we examined valves from rats in the 5/6Nx+HP and Sham+HP groups and detected spherical particles using energy-dispersive spectroscopy(EDS)to observe microscopic changes in the valvulars.In addition,the calcified tissues were analyzed for phase and crystallization properties using an X-ray powder diffractometer.Result:In the first part,the rats in the 5/6Nx+HP and 5/6Nx+NP groups presented with increased levels of Scr,BUN,and 24h urine protein compared with those of the rats in the sham+HP and sham+NP groups.High levels of PTH were observed,and hematoxylin and eosin staining and immunohistochemistry for proliferating cell nuclear antigen showed parathyroid hyperplasia in rats in the 5/6Nx+HP group but not in the 5/6Nx+NP group.In rats in the 5/6Nx+HP group,extracellular matrix glycosylation was observed in the aortic valvular in the 12th week and the mitral valvular in the 16th week.In the 16th week,chondrocytes appeared in the aortic valvular,as confirmed by immunofluorescence and Western blotting.Calcified particles mainly composed of phosphorus and calcium were observed in both the aortic and mitral valvulars by transmission electron microscopy(TEM)and scanning electron microscopy(SEM).The main mineral component of the calcified aortic valvular particles was hydroxyapatite[Ca₅(PO4)₃(OH)],as shown by X-ray diffraction.However,there were no obvious difference in heart function between rats in the 5/6Nx+HP and sham+HP groups.Conlusion:Our findings demonstrate that multi-valvular calcification is involved in CKD following 16-week HP and that hydroxyapatite[Ca₅(PO₄)₃(OH)]is the main component of the calcified aortic valvular particles of rats in the 5/6Nx+HP group.Part two:CXCL8 mediated the crosstalk between valvular interstitial cell and endothelial cell upon high phosphorus stimulation in chronic kidney disease with valvular calcificationMethod:In the second part,we studied the contribution of HP in valvular calcification.Canine valvular interstitial cells(VICs),canine valvular endothelial cells(VECs)and cultured human umbilical vein endothelial cells(HUVECs)were used to study the crosstalk between VICs and VECs.Real-time PCR,Western Blotting,immunofluorescence and other technologies were used to study the changes of makers of VICs activation and endothelial mesenchymal transition(End MT);The mouse with endothelial fluorescence-specific label were designed,which was fed with 0.2%adenine diet and HP diet to construct the model of CKD.And adeno-associated virus with mesenchymal/endothelial specific expression was interfere with mouse to study the role of CXCL8.Result:In the second part,VICs was activated after the intervention of HP,which was manifested by increased m RNA levels of?-SMA,Vimentin,and Smmothelin.In the co-culture transwell,activated VICs(a VICs)could induce VECs End MT.Blocking TGF?-1receptor,CXCL8 receptor,CXCL6 receptor and TNF-?receptor respectively,and detecting the concentration of the cytokines secreted by VICs,CXCL8 was thought as the main cause of VECs End MT.micro RNA sequencing of HUVECs in the co-culture transwell showed that CXCL8 mediates VECs End MT mainly through the regulation of mi R-214-3p/PTEN/Akt pathway.In vivo,inhibiting CXCL8 synthesis,blocking CXCL8 receptors,and specifically inhibiting endothelial expression of mi R-214-3p respectively could block End MT.End MT is an important way to participate in valvular calcification.We also found that blocking End MT can relieve valvular calcification in CKD.Conculsion:HP could induce activation of VICs.a VICs mediates VECs End MT by releasing CXCL8 to regulate the VECs mi R-214-3p/PTEN/Akt pathway.Inhibition of End MT could alleviate valvular calcification in CKD.