The Expression And Function Of HIF-1? Signal Pathway During Chronic Kidney Disease Vascular Calcification

objective:Vascular calcification?VC?,which could occur in all stages of chronic kidney disease?CKD?,is a common complication of CKD and seriously affects the life and health of patients with CKD.However,its'specific mechanism has not been fully clarified,and the clinical prevention and treatment measures for it are limited.So this study took adenine lavage with high phosphorus diets to induce the rat model of CKD Vascular calcification,observing the expression of hypoxia inducible factors 1 alpha?HIF-1??,Vascular endothelial growth factor A?VEGFA?and Notch1 in the rat aorta during VC,exploring the role of HIF-1?-VEGFA-Notch1 signaling pathway in VC,further providing the new strategies for the study of the specific mecha-nism and clinical prevention and treatment of VC in CKD.Methods:60 Spra-gue-Dawley?SD?rats were randomly divided into Control group?CON group,n=15?,only adenine lavage group?CKD group,n=20?,adenine lavage with high phosphorus diet group?CKD+HP group,n=25?.The CON and CKD group were fed with normal rat forage,and the CKD+HP group was fed with high phosphorus diets?P 1.2%?.The CKD and CKD+HP group was daily giv-en 2.5%adenine suspension at a dose of 250mg/?kg·d?from first to 4^thh week,and the 5^th to 8^th week,the adenine was given once every other day;simultane-ously,the CON group rats were given normal saline?10 ml/kg?.At the end of4^th,6^th and 8^th week after establishing the model,5 rats were randomly selected from each group to collect the specimens:24-hour urine was reserved to test24-h urinary protein?24-h Upro?;Blood samples were collected from ab-dominal artery to detect the level of serum Urea,creatinine?Scr?,cystatin C?Cys C?,calcium(Ca²⁺)and serum phosphorus(P^3-);The level of serum HIF-1?was tested by enzyme-linked immunosorbent?ELISA?;The body weight,kid-ney weight and kidney/body weight index was measured;The pathological changes of kidney and blood vessel were observed by hematoxylin-eosin?HE?staining;The calcification characteristic was determined by Von kossa staining,alizarin red staining and aortic calcium Content detection;Immunohistochemis-try?IHC?was applied to detect the protein expression of?-SMA,Runx2,HIF-1?,VEGFA and Notch1 in the aorta;Real-time PCR?RT-PCR?was used to test the gene expression levels of?-SMA,Runx2,HIF-1?,VEGFA and Notch1mRNA.Results:1.Body weight,Kidney weight and kidney/body weight in-dex:Compared with CON group,CKD and CKD+HP group rats were signifi-cantly lower in body weight at all time points?P<0.001?,and kidney weight and kidney/body weight index were significantly increased?P<0.001?;Com-pared with CKD group,the body weight was lower at 4^th and 8^th week?P<0.01?,kidney weight was higher at 6^th week?P<0.001?and kidney/body weight index was higher at 4^th and 6^th week?P<0.05,P<0.001;respectively?.2.Serum indicators:?1?Urea,Scr and Cys C:Compared with CON group,the level of serum Urea,Scr and Cys C were significantly increased in CKD and CKD+HP group?P<0.01?;Compared with CKD group,the CKD+HP group rats had higher level of Scr?P<0.05?and there was no significant difference in Urea and Cys C at each time point.?2?serum Ca²⁺,P^3-and Ca²⁺×P^3-:Compared with CON group,the level of serum Ca²⁺in CKD+HP group decreased at 4^thh and 8^thh week?P<0.05?,and serum P^3-and Ca²⁺×P^3-was significantly increased in CKD and CKD+HP group at each time point?P<0.01,P<0.05;respectively?,and the level of serum P^3-and Ca²⁺×P^3-was more higher in CKD+HP group than CKD group at 6^th and 8^th week?P<0.01,P<0.05;respectively?.3.24-h Upro:the 24-h Upro in CKD and CKD+HP group was significantly augmented than CON group?P<0.01?and no significant difference existed between CKD and CKD+HP group,furthermore,the 24-h Upro of both CKD and CKD+HP group rats increased gradually over time?P=0.009,P=0.003;respectively?.4.Serum HIF-1?:Within the CKD and CKD+HP group,the serum HIF-1?level gradually increased as time went by?P=0.009,P=0.033;respectively?;Serum HIF-1?level was significantly increased at 8^th week in CKD and CKD+HP group than CON group?P<0.05?,while,there was no significant difference be-tween CKD and CKD+HP group at all time points.5.Renal pathological changes:?1?macrography:CON group:normal size,kermesinus color,smooth surface,soft,clear boundary between cortex and medulla in bilateral kidneys;CKD and CKD+HP group:markedly increased size,gray white color?so-called“great white kidney”?,unsmooth surface,poor elasticity in bilateral kidneys.?2?HE staining:The structure and morphology of glomeruli,renal tubules and re-nal interstitium were normal in CON group;CKD and CKD+HP group:partial glomerular atrophy,bowman's capsule expansion,renal tubules dilation,tan substance deposition in renal tubules and renal interstitial fibrosis began to ap-pear from the 4^th week and the lesion gradually worsened with time.Moreover,the lesions appeared more earlier and severe in CKD+HP group than CKD group.6.aorta pathological changes:?1?macrography:The aorta morphology of CON group rats was normal with good elasticity at each time point;CKD group:The rats aorta were smooth and poor elasticity at 4^th week,and gradual-ly became crooked,intumescent,very poorly elastic with calcified nodule for-mation and increased vascular fragility since 6^th week;CKD+HP group:The rats aorta became obviously crooked,intumescent,poorly elastic,breakable with unsmooth surface and calcified nodule formation since 4^th week which gradually increased over time.?2?HE staining:The structure and morphology of aortas were normal in CON group at every time point;CKD group:The ar-rangement of elastic fibers in the aorta media was disordered at 4^th week,and the aorta media ruptured segmentally with elastic fibers reduction at 6^thh week and broken Continuously with fewer elastic fibers at 8^thh week;CKD+HP group:The aorta of rats showed the thick wall,Continuously linear rupture of the me-dia and significantly reduced elastic fibers since 4^th week and these lesions gradually worsened as time went by.7.Detection of aortic calcification:?1?Von kossa staining of whole aorta:The aorta showed no black substance depo-sition in CON group,while segmentary deposition of black substance was ob-served in the aorta at 6^th week and significantly increased at 8^th week in CKD group;However,diffuse deposition of black substance was observed in the whole aorta at 6^th and 8^th week in CKD+HP group.?2?Alizarin red staining:No orange particles deposition was observed in CON group;while,segmentary deposition of orange particles was observed since 6^th week and significantly in-creased at 8^th week in CKD group,and in CKD+HP group,diffuse deposition of orange particles was observed since 4^th week and increased over time ac-companying with smooth muscle fiber rupture.?3?Aortic calcium Content:The aortic calcium Content in CKD+HP group was significantly higher than that in CON and CKD group at all time points?P<0.001,P<0.05;respectively?,and the aortic calcium Content in CKD group was more higher than that in CON group at 6^th and 8^th week?P<0.001?;Within the CKD and CKD+HP group,the aortic calcium Content gradually increased as time went by?P=0.002,P<0.001;respectively?.8.Aortic protein expression:?1??-SMA:Compared with CON group,aortic?-SMA protein expression was significantly decreased in CKD group at 6^th and 8^th week and CKD+HP group at each time point?P<0.01?;Compared with CKD group in which aortic?-SMA protein expression gradual-ly reduced over time?P=0.001?,CKD+HP group showed lower expression at4^th and 6^th week?P<0.05?.?2?Runx2:The aortic Runx2 protein expression in CKD+HP group was significantly higher than that in CON and CKD group at all time points?P<0.001,P<0.01?,and the aortic Runx2 protein expression was significantly higher in CKD group than CON group at 6^th and 8^th week?P<0.01?;Within CKD and CKD+HP group,aortic Runx2 protein expression gradually increased as time went by?P<0.001,P=0.017?.?3?HIF-1?:Aortic HIF-1?protein expression was significantly higher in CKD+HP group than CON and CKD group at each time points?P<0.001,P<0.05;respectively?,And HIF-1?protein expression was more higher in CKD group than CON group at6^th and 8^th week?P<0.01?.Also within the CKD group,aortic HIF-1?protein expression gradually increased over time?P<0.001?.?4?VEGFA:Compared with CON group,aortic VEGFA protein expression was significantly increased in CKD group at 6^th and 8^th week,and in CKD+HP group at each time point?P<0.05,P<0.001?;Compared with CKD group,its'expression was higher in CKD+HP group at 4^th and 8^th week?P<0.05?;Within the CKD and CKD+HP group,aortic VEGFA protein expression gradually increased over time?P=0.001,P=0.007?.?5?Notch1:Compared with CON group,aortic Notch1protein expression was significantly increased in CKD group at 8^th week and CKD+HP group at all time points?P<0.01,P<0.001?;Compared with CKD group in which aortic Notch1 protein expression gradually increased over time?P=0.001?,CKD+HP group showed higher expression at 4^th and 6^thh week?P<0.05?.9.Aortic mRNA expression:?1??-SMA and Runx2 mRNA:Compared with CON group,aortic?-SMA mRNA expression reduced signifi-cantly in CKD group at 8^th week and CKD+HP group at each time point?P<0.01,P<0.05;respectively?;Compared with CKD group in which aortic?-SMA mRNA expression gradually reduced over time?P=0.014?,CKD+HP group showed lower expression at 4^th week?P<0.05?.The aortic Runx2 mRNA expression was significantly higher in CKD+HP group than CON and CKD group at each time point?P<0.05?,also,it's expression was more higher in CKD group than CON group at 6^th and 8^th week?P<0.05?.?2?HIF-1?,VEGFA and Notch1 mRNA:Compared with CON group,the aortic HIF-1?and Notch1mRNA expression increased significantly at 8^th week and VEGFA at 6^th and 8^thh week in CKD group?all P<0.05?,and these mRNA expression increased mark-edly at each time point in CKD+HP group?P<0.01,P<0.05,P<0.05;respec-tively?;Compared with CKD group,aortic HIF-1?mRNA expression was more higher at 4^th and 6^th week,VEGFA mRNA at 4^th and 8^th week and Notch1mRNA at 4^th week in CKD+HP group?P<0.05;P<0.05,P<0.01;P<0.05;re-spectively?;Moreover,the mRNA expression of HIF-1?and Notch1 in CKD group and VEGFA in CKD+HP group increased gradually with time?P=0.007,P=0.001,P<0.001?.10.Correlation analysis:Aortic calcium Content was posi-tively correlated to serum P^3-?r=0.618,P<0.001?,Ca²⁺×P^3-?r=0.590,P<0.001?,HIF-1??r=0.706,P<0.001?and aortic protein expression of HIF-1??r=0.852,P<0.001?,VEGFA?r=0.747,P<0.001?and Notch1?r=0.813,P<0.001?,and not significantly correlated to serum Ca²⁺?r=-0.246,P=0.104?.Serum P^3-was correlated to serum Urea,Scr,Cys C and 24h-Upro?r=0.559,P<0.001;r=0.676,P<0.001;r=0.537,P<0.001;r=0.539,P<0.001;respectively?.Among aortic proteins expression,VEGFA was markedly corre-lated to HIF-1??r=0.738,P<0.001?,and Notch1 correlated to HIF-1??r=0.865,P<0.001?and VEGFA?r=0.699,P<0.001?.Conclusion:?1?Compared with adenine gavage alone,adenine gavage combined with high phosphorus di-et could establish more rapidly a stable animal model of CKD vascular calcifi-cation.?2?Hyperphosphatemia could promote VC and aggravate the progres-sion of the kidney disease in CKD.?3?The HIF-1?-VEGFA-Notch1 signaling pathway was activated during aortic calcification in CKD rats,suggesting that the HIF-1?-VEGFA-Notch1 signaling pathway might be involved in the devel-opment of VC in CKD.?4?Serum HIF-1?level was significantly increased in CKD rats with VC,and was positively related to the severity of aortic calcifica-tion,which was expected to be one of serological markers for the assessment of VC severity in CKD.