The Mechanism Research Of Bmps Signal Pathway During Chronic Kidney Disease Vascular Calcification

Objective: Vascular calcification(VC)is common complication of chronic kidney disease(CKD),and is the main cause of the increased morbidity and mortality of cardiovascular disease(CVD).Vascular calcification is associated with many factors,recently,the attention is focus on BMPs signal pathway,but the specific mechanism has not been elucidated.The study took adenine gavage integrating high phosphorus diets to establish the rat model of CKD vascular calcification,exploring the expression and activation of BMPs signal pathway(BMP-2、BMP-4、BMPR-IA and MGP)during vascular calcification,for early prevention and treatment,providing strategies to reduce cardiovascular mortality.Methods:(1)Establishing CKD vascular calcification rats model: 55 male(specific pathogen free)Sprague-Dawley rats(190-270 g weight),randomly divided into the control group(CON,n=20)and CKD group(CKD,n=35)according to weight after adaptively feed for 10 days,from the first to the 4th week,the CKD group were given 2.5% adenine(220-250mg/kg/d)daily by gavage,and high phosphorus diets(P 1.8%);the 5th to 8th week,the adenine was given once every other day.Meanwhile,the control group rats were given normal saline(10 ml/kg)and normal forage.All rats were free to eat and drink.The experimental time were 8 weeks.(2)Detection of indicator: At the end of the second,4th,6th and 8th week,6 rats and 5 rats were randomly selected from each group to collect specimens.24-hour urine was reserved by metabolic cages to test 24 hours urinary albumin(24-h Upro).Therats were anaesthetized by 2% sodium pentobarbital(30-60mg/kg)through intraperitoneal injection,collecting blood from abdominal artery,detecting the concentration of serum urea nitrogen(BUN),creatinine(Scr),cystatin C(Cysc),calcium(Ca2+),phosphorus(P3-)by automatic biochemical analyzer;Detecting the level of serum BMP-2,BMP-4 by enzyme-linked immunosorbent(ELISA);Measuring the kidney weight/body weight index;Part of the kidney were fixed in 10% formalin liquid for Hematoxylin and eosin(HE)staining after isolated;the aorta was quickly isolated and divided into three parts on-demand,one part were fixed with 10% formaldehyde solution for Von Kossa staining、alizarin red staining,and Immunohistochemical detection of BMP-2,BMP-4,BMPR-IA(ALK3),MGP protein expression;the other part were collected in EP tube with1 ml RNA protective liquid,saved in-20℃,for Quantitative Real-time PCR detection of BMP-2,BMP-4mRNA expression;the rest were stored in-80℃for the determination of calcium content.Results: 1、General index:(1)deaths:The control group rats all survived;the mortality of the CKD group rats was relatively high,the rats were found to die from the second week,and a total of 9rats were sacrificed.(2)Body weight、Kidney weight、kidney weight/body weight index: Compared to the control group,the weight of the CKD group rats significantly decreased(P <0.05);while the kidney weight、kidney weight/body weight index both obviously increased(P < 0.05,P < 0.05).2、24h urinary protein(24h-Upro): the 24h-Upro of the CKD group increased significantly while compared to the control group(P < 0.01);Within the CKD group,the24h-Upro increased gradually with time(P < 0.01).3、Serum indicators:(1)BUN、Scr、Cycs: The level of serum BUN、Scr and Cycs in the CKD group increased significantly compared to the control group(P < 0.01,P < 0.01,P <0.01).(2)serum Ca2+、P3-、Ca2+×P3-: Compared to the control group,the level of serum P3-、Ca2+×P3-in the CKD group increased significantly(P < 0.01,P <0.01);while the level of serum Ca2+ decreased obviously since the 4th week(P< 0.05).4、Serum BMP-2 and BMP-4 by ELISA:(1)BMP-2: Compared to the control group,the serum BMP-2 concentration of CKD group rats increased significantly(P < 0.05);within the CKD group,the serum BMP-2concentration increased gradually(P < 0.05).(2)BMP-4: The serum BMP-4concentration of the CKD group increased significantly since the 4th week compared to the control group(P < 0.01);Within the CKD group,the level of serum BMP-4 in the 6th and 8th week was relatively decreased compared to the4 th week(P < 0.01).5、Renal pathological changes:(1)The appearance: Kidney size of the control group was moderate,red color,smooth surface,soft,clear boundary between cortex and medulla;while the CKD group was called "large white kidney",which was grey white color,unsmooth surface,poor elasticity.(2)Kidney HE staining: There was no abnormal changes in the control group.while,in the CKD group,renal tubular expansion began to emerge since the second week,and tan material deposition in the renal tubules,bowman’s capsule expansion,interstitial fibrosis,glomerular partial atrophy,inflammatory cells infiltration,the blood vessels reduction since the 4th,6th,8th week as time went by.6、Aortic morphology: The aorta of the control group was normal appearance,smooth,good elasticity.While,since the 4th week,the aorta of theCKD group gradually became bended,expanded,poor elasticity,aneurysmal change,and vascular stiffness become serious,calcified nodule formation in the6 th and 8th week.7、Aortic calcium staining:(1)Von Kossa staining: There were no black particles deposition in the control group,while,in the CKD group,the deposition of black particles were gradually increasing since the 4th week,accompanying with smooth muscle fiber fracture,especially the area where the calcified nodule formation.(2)Alizarin red staining: There were no orange particles deposition in the control group,while,in the CKD group,the deposition of orange particles were gradually increasing since the 4th week,accompanying with smooth muscle fiber fracture,especially the area where the calcified nodule formation.8 、 Aortic calcium content: The aortic calcium content in the CKD group was obviously increased compared to the control group(P < 0.01);within the CKD group,the aortic calcium content gradually increasing as time went by(P < 0.01).9、The expression of BMP-2,BMP-4,BMPR-IA(ALK3),MGP protein in the aorta: There was almost no expression of BMP-2,BMPR-IA,MGP protein in the control group,while,the expression of BMP-4 was a little bit more.Compared to the control group,the expression of BMP-2,BMPR-IA,MGP protein in the CKD group increased significantly since the 4th week(P < 0.01),positive staining was gradually deepened,cytoplasm was tan,distributed in the tunica media of smooth muscle cell layer;while the expression of BMP-4 protein in the CKD group in the 6th and 8th week was relatively decreased compared to the 4th week,but still higher than that in the control group(P < 0.01).10、The expression of BMP-2mRNA、BMP-4mRNA in the aorta by Quantitative RT-PCR:(1)BMP-2mRNA: The expression of BMP-2mRNA in the control group rarely increased.While,the expression of BMP-2mRNA in the CKD group increased significantly compared to the control group(P<0.01),within the CKD group,the expression of BMP-2mRNA increased gradually(P < 0.01).(2)BMP-4mRNA: The expression of BMP-4mRNA in CKD group obviously increased since the 4th week compared to the control group(P<0.01),but the 6th and 8th week was relatively decreased compared to the 4th week(P<0.01).Conclusion:(1)According to the pathogenesis of vascular calcification,adenine gavage joint high phosphorus feed can quickly establish the rat model of CKD vascular calcification.(2)The expression of gene and protein about BMP-2,BMP-4,BMPR-IA,MGP increased obviously in the early stage of CKD vascular calcification,suggesting BMPs signal pathway may play an important role during vascular calcification.(3)The level of serum BMP-2 and BMP-4significantly increased,and was positively correlated with the degree of calcification,which may be used as a marker of CKD vascular calcification.(4)The osteogenic activity and specificity of BMP-4 maybe worse than BMP-2 in the process of CKD vascular calcification.