ABSTRACT
Objective To investigate the role of BMP-2/Smad signaling pathway in the osteogenic differentiation of human aortic smooth muscle cells (HASMCs) caused by hyperphosphatemia -induced calcium phosphate (CaP) crystals.Methods High-phosphate medium was incubated at 37℃ for 3 days.CaP crystals and supernatant were isolated by ultracentrifugation.Scanning electron microscope and energy dispersive X-ray spectroscopy were performed for analysis of physicochemical characteristics of CaP crystals.HASMCs were cultured in vitro,and divided into high-phosphate,control,crystals and supernatant groups.Calcification was visualized by Alizarin red staining.Calcium loads in cells were quantified by o-cresolphthalein complexone method.Protein expression of bone morphogenetic protein-2 (BMP-2),Runt-related transcription factor 2 (RUNX2),osteopontin (OPN),phospho-Smad1/5/9 (p-Smad1/5/9) were quantified by Western blotting.After knockdowns of BMP-2 and Smad1 with small hairpin RNA (shRNA) interfering respectively in HASMCs,protein expressions were measured by Western blotting.Results High-phosphate medium induced the formation of CaP crystals.Compared with the cells in control group,CaP crystals significantly induced HASMCs calcification,increased calcium loads and up-regulated the levels of BMP-2,RUNX2 and OPN proteins (all P ＜ 0.05).After the addition of CaP crystals into HASMCs,the level of p-Smad 1/5/9 protein peaked at 30 min (P ＜ 0.05).After BMP-2 was knocked down in HASMCs,the expression of p-Smad1 caused by CaP crystals was blocked completely,and the expressions of RUNX2 and OPN caused by CaP crystals were reduced significantly (all P ＜ 0.05).After Smad1 was knocked down in HASMCs,the expressions of RUNX2 and OPN caused by CaP crystals were decreased significantly (all P ＜ 0.05).Conclusions Hyperphosphatemia-induced CaP crystals promoted osteogenic differentiation of HASMCs through the BMP-2/Smad signaling pathway.
ABSTRACT
Objective To compare the outcomes of patients starting peritoneal dialysis (PD)within two weeks and more than two weeks after catheter implantation.Methods All the patients undergoing Tenckhoff catheter implantation and initiating PD in Renji Hospital from January 2001 to December 2010 were enrolled in the study.Patients started PD within 2 weeks after catheter insertion were defined as urgent group,and those started PD 2 weeks later were defined as planned group.Kaplan-Meier curves and Log-rank tests were used to compare outcomes between two groups.Results Among 657 patients in this study,median break-in period was 6 days of 469 (71.4％)patients in urgent group and 26 days of 188 (28.6％) patients in planned group.Compared to planned group,patients of urgent group were younger [(52.6± 17.3) vs (56.1± 15.3) year,P =0.017],had less eGFR [(5.36±2.03) vs (6.50±2.50) ml· min-1 · (1.73 m2)-1,P ＜ 0.01],lower serum albumin [(34.0±5.7) vs (36.2±5.9) g/L,P ＜ 0.01] and hemoglobin [(76.9± 18.8) vs (80.8 ± 17.9) g/L,P =0.018],and higher phosphate [(2.19±0.67) vs (1.98±0.52) mmol/L,P＜ 0.01].Urgent group presented more catheter dysfunctions needed to transfer to hemodialysis (2.1％ vs 0％,P =0.044).The 1-,2-,3-and 5-year technique survival rates of urgent and planned group were 94％ vs 98％,92％ vs 94％,90％ vs 92％and 86％ vs 85％ respectively.There was no significant difference in technique survival (Log-rank =1.536,P =0.22) and peritonitis-free survival (Log-rank =0.035,P =0.85) between two groups.The 1-,2-,3-and 5-year patient survival rates of urgent and planned group were 90％ vs 95％,81％ vs 90％,74％ vs 79％ and 67％ vs 74％ respectively,and no significant difference was found (Log-rank =2.364,P =0.12).Conclusions Although patients needing urgent initial PD have poorer residual renal function and nutritional condition compared to those of planned initial PD,their outcomes are similar.Peritoneal dialysis may be a feasible and safe dialysis modality for patients who need urgent start.
ABSTRACT
Objective To investigate the impact of calcium phosphate crystals induced by uremic serum on calcification of human aortic smooth muscle cells (HASMCs).Methods Uremic serum was incubated at 37℃ for 3 days.Calcium phosphate crystals and uremic supernatant were isolated from uremic serum by uhracentrifugation.Scanning electron microscope (SEM) and energy dispersive X-ray spectroscopy (EDX) were performed for analysis of morphological and chemical characteristics of the crystals.HASMCs were treated in vitro with control medium,uremic serummedium,calcium phosphate crystals-medium and uremic supernatant-medium.Calcification was visualizcd by Alizarin red staining.Calcium loads in cells were quantified by o-cresolphthalein complexone method.Gene expression of bone morphogenetic protein-2 (BMP-2),osteopontin (OPN) and core-binding factor α1 (Cbfα1),alkaline phosphate (ALP) and matrix gamma carboxyglutamic acid protein (MGP) were quantified by real-time PCR.Cbfα1,OPN and BMP-2 protein levels were determined by Western blotting or ELISA.Results Calcium phosphate crystals which induced by uremic serum displayed laminated shapes containing crystallized needle-like projections and ranged from 30-500 nm,with Ca/P ratios of 1.41 ±0.05.Compared with the cells in control group,uremic serum induced HASMCs calcification,increased calcium loads (P ＜ 0.05),up-regulated BMP-2,OPN,Cbfα1 mRNA and protein expression (all P＜ 0.01).Similar to uremic serum,calcium phosphate crystals also induced HASMCs calcification,increased calcium loads (P＜0.05),and up-regulated BMP-2,OPN,Cbfα1 mRNA and protein expression (all P ＜ 0.01).However,there was no significant difference between HASMCs growing in uremic supernatant and control medium in calcium loads or the expression levels of these osteogenic proteins (P ＞ 0.05).Conclusions Calcium phosphate crystals induced by uremic serum promote HASMCs calcification,which might be one of the mechanisms of uremic vascular calcification.