To Investigate The Regulatory Factors Of FGF23 And Their Molecular Mechanisms In Chronic Renal Failure

Fibroblast growth factor 23(fibroblast growth factor 23,FGF23)is the most important pathogenic factor in Chronic Kidney Disease-Mineral and Bone disorder(CKD-MBD).In the moderate and late stage of chronic renal failure,abnormally elevated serum FGF23 may lead to adverse events including myocardial hypertrophy and is positively associated with all-cause mortality in patients,so it is recognized as one of the markers of poor prognosis.Therefore,it is of great significance to find out the regulatory factors of abnormally elevated FGF23 and solutions for reducing its serum level in chronic renal failure.The aim of this study is to compare difference of serum FGF23 level between patients undergoing different dialysis modalities and to analyze the regulatory factors of FGF23.In addition,the effect of calcium on fibroblast growth factor 23(FGF23)in vitro and vivo was investigated,and the possible mechanism of calcium regulating the FGF23 expression of osteoblasts together with the intervention efficacy were explored.The results of this study might further enrich the mechanism of the elevated serum FGF23 and provide theoretical basis to lower the serum FGF23 level in chronic renal failure patients.To compare the difference of serum FGF23 level in patients undergoing different dialysis modalities,the serum phosphorus,serum calcium,ferritin,hemoglobin,albumin,low-density lipoprotein,high-density lipoprotein,cholesterol and triglyceride,blood urea nitrogen and creatinine before and after dialysis were detected by automatic biochemical analyzer in hemodialysis patients,the adequacy of dialysis were calculated by the urea clearance index(Kt/V).Intact PTH level and 25 hydroxyl vitamin D in dialysis patients were detected by chemiluminescence method.Serum intact FGF23 level in hemodialysis patients was detected by ELISA.The regulatory factors of FGF23 were analyzed by multivariate regression analysis.The phosphorus and calcium-phosphate product in patients undergoing INHD were significantly lower than patients undergoing CHD.Kt/V of INHD is higher than that of CHD.The standard-obtained rate of phosphorus and PTH in INHD patients was higher than that in CHD patients.The levels of calcium,ferritin,hemoglobin,albumin,low-density lipoprotein,high-density lipoprotein,cholesterol and triglyceride were compared.There was no significant difference between INHD group and HF-CHD group,and there was no difference between HFCHD and LF-CHD.The serum FGF23 level in patients undergoing INHD was significantly lower than that in patients undergoing CHD,but there was no significant difference between HF-CHD and LF-CHD.Multivariate linear regression analysis showed that serum FGF23 levels was positively correlated with serum calcium,phosphorus and calcium-phosphate product.To explore the effect of potential regulatory factors on FGF23 expression and find out their possible mechanism in vitro,FGF23 expression was observed by Real-time PCR and Immunofluorescence staining after osteoblast cells were stimulated by different regulatory factors.FGF23 expression was detected by Real-time PCR after calcium-sensing receptor(CaSR)was down-regulated by siRNA in osteoblasts.Mineralization of osteoblasts stimulated by calcium and phosphorus was detected by Alizarin red staining.Mineralization related proteins including alkaline phosphatase(ALP)and bone gammacarboxyglutamic-acidcontaining proteins(BGP)mRNA were detected by Real-time PCR.The expression of P-ERK and β-catenin protein in osteoblast cells stimulated by calcium and phosphorus was detected by Western blot.The effect of ERK pathway inhibitor(U0126)on FGF23 expression was observed by Real-time PCR and Western Blot.The result showed that 1,25(OH)2D3 could stimulate the experssion of FGF23 in osteoblasts.After osteoblast cells were stimulated by calcium 、phosphorus 、 rhPTH.The expression of FGF23 in osteoblasts remained unchanged when compared with the blank control group.When the osteoblasts were stimulated by calcium together with phosphorus,the expression of FGF23 was up-regulated compared with the blank control group.FGF23 mRNA expression was the highest in 5mM phosphorus and 6mM calcium chloride group.FGF23 protein expression was also increased by immunofluorescence compared to blank control group.When CaSR was down-regulated by CaSR-siRNA,the effect of high calcium together with phosphorus on the expression FGF23 could not be influenced.When UMR106 was stimulated by 5mM phosphorus and 6mM CaCl2,the P-ERK protein expression was up-regulated and β-catenin protein expression remained unchanged,the ALP,BGP mRNA expression and mineralization of osteoblasts were promoted compared with control group.ERK pathway inhibitor(U0126)inhibited the expression of P-ERK protein and decreased the mineralization and FGF23 mRNA expression in osteoblasts.To explore the effect of different calcium diet on FGF23 expression,bone morphology,myocardial hypertrophy and cardiovascular calcification in vivo,CKD and wild type rats were divided into different calcium load diet group(high calcium,normal calcium and low calcium).The tibial morphology of rats on different diets were detected by Micro-CT.The ALP、BGP、FGF23 mRNA expression of tibias were detected by Real-time PCR.The myocardial hypertrophy was observed by HE staining and myocardium cell membrane were observed by Wheat germ agglutinin staining.Calcification of aorta and myocardium were observed by Vonkossa staining.After 8 weeks of high calcium diet,rats were intraperitoneally injected with a calcimimetic compound NPS R568 for 2 weeks.Serum creatinine,blood urea nitrogen,phosphorus,calcium,PTH,iFGF23 were detected by automatic biochemical analyzer and ELISA before and after NPS R568 intervention.In CKD rats,calcium load was positive correlated with FGF23.Serum calcium in CKD group on high calcium diet was higher than that in CKD rats on normal calcium,and serum FGF23 was elevated.Serum calcium in CKD group on low calcium diet dropped and serum FGF23 was also reduced.There was no difference for serum calcium between the low calcium diet group and normal calcium group of WT rats,but serum FGF23 level was lower in low calcium diet group.CKD rats on low calcium diet and normal calcium diet did harm to bone morphology compared with CKD rats on high calcium diet.Bone quality of CKD rats on high calcium diet was improved than the other two CKD groups.ALP,BGP and FGF23 mRNA expression in the tibia of the high calcium CKD group was higher than that in normal calcium CKD group.These parameters of low calcium diet CKD group were lower than those of normal calcium group.CKD rats on high calcium diet had better bone quality than the other two CKD groups.Compared with WT rats,the myocardial hypertrophy in CKD rats was obvious.The myocardial hypertrophy of CKD rats on high calcium diet significantly aggravated compared with CKD rats on normal calcium diet.Cardiovascular calcification was only observed in CKD rats on high calcium diet,and there were no positive results in other groups.After NPS R568 was injected intraperitoneally,serum calcium、iPTH、iFGF23 level in the intervention group were significantly reduced compared with the control group.In conclusion,INHD can significantly modify the metabolism of calcium and phosphorus and reduce the serum iFGF23 level when compared with CHD.The serum iFGF23 level in patients undergoing hemodialysis is positively correlated with serum calcium,phosphorus and calcium-phosphate product.High calcium together with phosphorus can stimulate the expression of FGF23 in a CaSR and Wnt/β-catenin independent pathway in vitro.ERK pathway is involved in the process and participate in promoting the osteoblast mineralization and stimulating the expression of FGF23.High calcium diet leads to the up-regulation of FGF23 in CKD rats,mediates the myocardial hypertrophy and aggravates calcification,FGF23 production is affected by bone quality and mineralization.Intervention by diet and drugs can reduce calcium load and calcium-phosphate product,thus subsequent significantly reduce serum FGF23 level,which was of potential clinical application value.