Hyperphosphatemia Causes Vascular Smooth Muscle Rhythm Disturbance In Vitro Study And Establishment Of A Mouse Model Of Hyperphosphatemia

Objective:Cardiovascular disease(CVD)is a principal cause of mortality and is most prevalent in chronic kidney disease patients.The onset of CVD and death often variy by time-of-day,which occur most frequently during early morning.It is now accepted that hyperphosphatemia,a distinct syndrome associated with CKD,plays vital role in abnormal cardiovascular circadian rhythms.It has been confirmed that peroxisome proliferator-activated receptor ?(PPARy)and transforming growth factor?1(TGF-?1)play an important role in vascular smooth muscle cells(VSMC)by acting as a peripheral factor in regulation of circadian rhythms.The purpose of this study was to investigate the changes of PPAR? and clock gene in human aortic vascular smooth muscle cell(HASMC)under high phosphate environment in vitro Besides,the relationship between TGF-?1 pathway and PPAR? will be analyzed.This study will determine the treatment value of controlling serum phosphate levels and supplemental PPARy agonist for circadian rhythm disorders in cardiovascular systems The findings will be of great help in the establishment of a novel chronotherapeutic approach of cardiovascular disorders in CKD patientsMethods:The experiments were divided into(1)normal control group(1.0 mmol/L phosphorus),high phosphorus group(2.5 mmol/L phosphorus),rosiglitazone group(10 ?mol/L),and high phosphorus+rosiglitazone group(10 ?mol/L)(n=3);(2)normal control group(1.0 mmol/L phosphorus),high-phosphorus group(2.5 mmol/L phosphorus),TGF-?1 neutralizing antibody group(10 ?g/ml),and high phosphorus+TGF-?1 neutralizing antibody group(10 ?g/ml)(n=3).The timing of the beginning stimulated was counted as time 0.Thereafter,cells were collected every 4 hours for a total of 28 hours.The mRNA expressions of PPARy,Bmall,Per2,Rev-erba and TGF-?1 in different groups of cells at different time points were detected by quantitative polymerase chain reaction(qRT-PCR).Results:At normal conditions,the mRNA expressions of PPAR? and clock genes Bmal1,Per2,Rev-erba showed clear circadian rhythm in HASMC.A high-phosphorus environment could inhibit the expression of the above genes,thereby destroying the normal circadian rhythm of HASMC.After the activation of PPAR? stimulated by rosiglitazone,the expression of clock genes were up-regulated,and the inhibition of the above genes by high-phosphorus environment could be reversed.In addition,the expression of TGF-?1 in HASMC was also presented rhythmic under normal conditions,and the high-phosphorus environment promoted the secretion of TGF-?1.When the TGF-?1 neutralizing antibody blocked its signal pathway,the expression of PPAR? and clock genes were all up regulated,and the inhibitory effects of high-phosphorus environment on PPARy and Per2 could be blocked.Conclusion:Under normal circumstances,the mRNA expression of PPARy and clock gene Bmall,Per2,Rev-erba in the HASMC has a circadian rhythm.The high phosphorus environment destroys HASMC normal biological rhythms by inhibiting PPARy.Besides,high phosphorus environment promotes the secretion of HASMC TGF-?1,TGF-?1 neutralizing antibody can aderse the inhibition affect of high phosphorus on PPAR? and Per2,but it has no obvious effect on Bmall and Rev-erb?,the related mechanism needs further research.Objective:By adjusting the phosphorus content in adenine diet,we aimed to establish an effective chronic kidney disease mice model accompanied by hyperphosphatemia for further studiesMethods:Male C57BL/6 mice were divided into four groups:simple adenine dietary:normal group(1.0%calcium,0.6%phosphorus),simple adenine group(0.2%adenine,1.0%calcium,0.6%phosphorus).Adenine integrating high phosphorus dietary:normal group(0.8%calcium,0.6%phosphorus),adenine integrating high phosphorus dietary group(0.2%adenine,0.6%calcium,1.0%phosphorus)(n=7).The weight,the level of blood urea nitrogen(BUN)?calcium(Ca)and phosphorus(P)in serum were measured at 0 and 4 weeks after the start of adenine diet.The gene expression of fibrosis markers Collagen I,FN and PAI-1,as well as inflammatory markers TNF?,IL-1? and ICAM-1 in kidney were detected by RT-PCRResults:Compared to the control group,their bodyweights were decreased and the serum BUN level(41.15±4.59 mmol/L)was significantly increased at 4 weeks in simple adenine group,while the levels of serum Ca(2.62±0.16 mmol/L)and P(2.22±0.26 mmol/L)were slightly increased(P<0.05).In adenine integrating high phosphorus dietary group,their weights were reduced and the level of serum P(2.97±0.29 mmol/L)showed significant increase,whereas the level of serum BUN(14.68±3.57 mmol/L)were slightly increased at 4 weeks(P<0.05).There was no suggestive difference in serum Ca levels.Pathological results showed that two model groups all appeared remarkable histological injury in the kidney.RT-PCR results showed that the expression of Collagen I,FN,PAI-1,TNF?,IL-1? and ICAM-1 were increased in two model groups.Conclusions:Adenine combined with high phosphorus diet feding C57BL/6 mice for 4 weeks can establish a hyperphosphatemia model,accompanied by mild renal dysfunction and renal fibrosis,which is an effective method to set up the mice with hyperphosphatemia.