The Role And Mechanism Of PPAR? Inhibiting CKD Vascular Calcification

The incidence of chronic kidney disease(CKD)in China and the world is increasing year by year,and most of the patients will eventually develop into end-stage renal disease(ESRD)and receive renal replacement therapy.Compared with the general population,the incidence and mortality of cardiovascular disease in CKD patients were significantly increased.And rapid progression of vascular calcification is an important feature of cardiovascular disease in such patients.Therefore,it is of great significance to intervene the occurrence and development of vascular calcification in patients with CKD.Normal calcification occurs only in the bones and teeth,in addition to any place of calcification are abnormal calcification.The main feature of vascular calcification is the pathological deposition of calcium phosphate in vascular tissue.Vascular calcification can occur in two areas of the vessel wall,the intima and the media.While patients with CKD can develop both types of vascular calcification,calcification of the media is more specific to CKD.The media is made up of smooth muscle cells in a framework of loose connective tissue.The main reason for the calcification of the media is the differentiation of vascular smooth muscle cells(VSMCs)into osteoblasts.It was thought that CKD vascular calcification mainly occurred in ESRD and dialysis phase,but new studies have found that CKD vascular calcification can occur in the early stage 2 of CKD.In the late CKD,especially in uremic patients with severe mineral metabolic disorders,high phosphate and Ca × P product abnormalities are the characteristic inducement of VSMCs calcification.Many clinical studies have confirmed that high phosphate is an independent risk factor for CVD and all-cause mortality of CKD patients.In high phosphorus environment,VSMCs sodium-dependent phosphate transporter(Pit)expression was up-regulated and promote cell inflow phosphorus increased,followed by the activation of osteoblast specific transcription factor(Runx2)and core binding factor alpha1(Cbfa1)and its downstream osteogenesis program,and eventually lead to VSMCs calcification.Although the prevention and treatment of hyperphosphatemia is the key to prevention of CKD vascular calcification,but even through the restriction of dietary phosphorus and the use of non-calcium-based phosphate binders,CKD patients especially in dialysis patients is still difficult to avoid the influence of hyperphosphatemia on vascular calcification and CVD.Therefore,to explore the mechanism of VSMCs in high phosphorus environment and the prevention and treatment of CKD is an important breakthrough to inhibit vascular calcification.Peroxisome proliferator activated receptor gamma(PPAR?)is a subtype of peroxisome proliferator activated receptors,is expressed mainly in adipose tissue.PPAR? may be involved in the regulation of insulin sensitivity,glucose metabolism and promote adipocyte differentiation and lipogenesis,it is the target of thiazolidinediones.Recent studies have indicated that PPAR? is closely related to calcium and phosphate metabolism and vascular calcification,and it has important protective effect on cardiovascular system,and its protective effect is not achieved by regulating blood glucose.It was found that the expression of PPAR? was down regulated in VSMCs of spontaneously hypertensive rats,and the expression of VSMCs phenotype markers were decreased,including contractile protein,?-SMA and SM22?.But PPAR gamma agonist could restore the expression of VSMCs phenotype markers and inhibit the remodeling of arterial in spontaneously hypertensive rats.This indicates that PPAR? is an important transcription factor to maintain the phenotype of VSMCs.Whether PPAR gamma plays an important role in the vascular calcification of CKD is still unknown.At present,the mechanism of PPAR involved in vascular calcification has not been reported at home and abroad.To clarify the key role of PPAR? in CKD vascular calcification and whether the high phosphate induced the differentiation of VSMCs into osteoblast and calcification by inhibiting PPAR?,and whether the PPAR? activation could inhibit the high phosphate induced VSMCs calcification and CKD vascular calcification,we studied the calcified vessel of CKD patients undergoing arteriovenous fistula and renal transplant,the CKD model mice,the vascular smooth muscle cell line(VSMCs),and the Klotho gene knockout mice(kl/kl).The main research contents and results are as follows:1.The expression of PPAR? was down regulated in the calcified vessel of CKD patientsAfter the consent of CKD patients and through ethical review,we selected 12 pairs of renal arteries of donor and recipient in renal transplant recipients,and 37 cases of primary of CKD patients of radial artery in arteriovenous fistula operation and these vessels were used for calcium staining and immunohistochemical staining.Von kossa staining found that 0%(0/12)of renal arteries from healthy donate and 100%(12 of 12)of renal arteries from CKD patients undergoing a renal transplant showed medial calcification.However,only 37.83 %(14 of 37)medial calcification of radial arteries were identified in CKD patients undergoing arteriovenous fistula,indicating the medial calcification is more likely to occur in large artery vessels.The immunohistochemical staining result showed that the expression of PPAR? was decreased in the calcified radial arteries,accompanied by the increased levels of Runt-related transcription factor 2(Runx2)and bone morphogenic proteins-2(BMP-2)and the decreased levels of SM22?,suggesting osteogenic transformation of vascular cells in comparison with non-calcificated of radial arteries.Clinical examination results analysis showed that,radial artery vascular calcification in CKD patients with CKD stage 5 serum phosphorus level(2.16±0.52)was significantly higher than that of non-vascular calcification in patients with CKD stage 5(1.60 + 0.54)(P < 0.05),and parathyroid hormone(PTH)level(534.63 + 292.77)was also significantly higher than non-vascular calcification in patients with CKD stage 5(242.01 + 188.74)(P < 0.01).But there was no significant difference in serum calcium level and eGFR level.The results showed that hyperphosphatemia and the down-regulation of PPAR? were closely related to vascular calcification in patients with CKD.2.PPAR? expression was decreased in calcified vascular vessels of CKD mice fed a high phosphate dietIn order to observe the relationship between the expression of PPAR? and CKD vascular calcification,we used DBA/2J female mice to produce CKD mouse model(single kidney excised +2/3 kidney damage),and fed with high phosphate diet(1.2%Pi)for 12 weeks.At the time of experimental termination,mouse serum was used for the determination of urea nitrogen(BUN),calcium and phosphate content,the aorta was used for Von Kossa staining,immunohistochemistry staining,immunofluorescence staining and western blot.The results showed that BUN and serum phosphorus were significantly higher in CKD+HP group mice.Von Kossa staining identified the obvious medial calcification of aorta in CKD+HP group mice.Immunohistochemistry,immunofluorescence and blot Western results showed that,compared with other groups,the expression of PPAR?,Klotho,SM22? in CKD+HP group mice was significantly decreased,Runx2,BMP2 expression was significantly higher,there was no significant difference between the other groups.This further suggests that there is a close relationship between CKD vascular calcification and PPAR?3.High phosphate induces the reduction of PPAR? in VSMCs.In order to observe the effect of high phosphorus on the expression of PPAR,we incubated VSMCs 5 days with normal medium and high phosphate medium(2.6mmol/L).Alizarin red S staining showed that high phosphate induced VSMCs calcification clearly,and Western blot results show that high phosphate significantly reduced the expression of PPAR?,Klotho,and SM22?,and the expression of Runx2,BMP2 increased significantly.The above results confirmed that high phosphate can not only induce differentiation of VSMCs into osteoblasts and vascular calcification,but also down-regulation the expression of PPAR?.4.PPAR? agonist inhibits the high phosphate-induced VSMCs calcificationIn order to elucidate the key role of PPAR? in high phosphate-induced VSMCs calcification,we used PPAR? agonist rosiglitazone(RGL)(10?mol/L)to pre incubation VSMCs for 1h,and observe the effects of high phosphate on VSMCs calcification.Alizarin Red S staining was used to observe the cell calcification in each group.The expression of SM22?,Runx2,BMP2 and PPAR? were detected by Western blot.The results showed that RGL pre incubation could significantly inhibit the high phosphate-induced VSMCs calcification,and RGL pre incubation could significantly inhibit the high phosphate-induced the down-regulation of PPAR?,SM22?and the up-regulation of Runx2,BMP2.These results suggest that PPAR? could significantly inhibit the high phosphate-induced VSMCs calcification.5.Silence of Klotho gene attenuates the inhibitory effect of PPAR? agonist on high phosphate-induced VSMCs calcificationAlthough PPAR? activation could inhibit the calcification of VSMCs induced by high phosphate,but its mechanism is still not clear.Studies have demonstrated that Klotho is a factor that inhibits vascular calcification,which has been demonstrated to be expressed in VSMCs.Klotho could inhibit the calcification of VSMCs by down-regulating the phosphate transporter 1(Pit-1).To this end,we intend to investigate whether the transcriptional regulation factor PPAR? inhibits vascular calcification by regulating the transcription of Klotho gene.We observed the effects of high phosphorus on the expression of Klotho and Pit1,and it was clear that the high phosphorus could induce the decrease of Kloth and the increase of Pit-1 in VSMCs.Then we studied the effect of RGL pre incubation on the expression of Klotho.The results showed that RGL could significantly inhibit the decrease expression of Klotho induced by high phosphate,and inhibit the increase expression of Pit-1 induced by high phosphate.In the follow-up study,we used Klotho si RNA to knock down the expression of Klotho in VSMCs and then observed the inhibitory effect of PPAR? agonist on the calcification of VSMCs induced by high phosphate.The results showed that the inhibition effect of RGL on the high phosphate-induced VSMCs calcification was significantly weakened after Klotho siRNA interference.And the inhibition effect of RGL on the high phosphate-induced down-regulation of SM22? and up-regulation of Runx2,BMP2,Pit-1 was significantly attenuated.This indicates that the inhibitory effect of PPAR? on high phosphate-induced VSMCs calcification may be by regulating its downstream target gene Klotho.6.PPAR? agonist could not inhibit vascular calcification in kl/kl mice induced by high phosphorusTo further confirm PPAR gamma inhibits vascular calcification induced by high phosphorus is through the regulation of Klotho,we put the kl/kl mice as the research object,observe when Klotho Gene deletion,PPAR? agonist whether can also inhibit vascular calcification induced by high phosphate.We directly use high phosphate medium to cultivate the aorta of kl/klmice in vitro for 10 days.At the time of experimental termination,protein was extracted and Western blot was used to detect the expression of related proteins in each group.The results showed that PPAR? could reverse the decreased expression of SM22?and increased expression of Runx2 and BMP2 in artery of wild-type mice induced by high phosphate.But in kl/kl mice,the effect of PPAR? agonist was significantly impaired.7.PPAR? agonist can significantly improve vascular calcification in CKD miceWe used CKD model of DBA/2J female mice as the research object,and fed the high phosphate diet(1.2%Pi)while feeding the PPAR? agonist rosiglitazone(10mg/kg/d and 20mg/kg/d)for 12 weeks.At the time of experimental termination,mouse serum was used for the determination of urea nitrogen(BUN),calcium and phosphate content,the aorta was used for Von Kossa staining,immunohistochemistry staining,immunofluorescence staining and western blot.It was found that PPAR? agonist rosiglitazone significantly inhibited vascular calcification in CKD mice.Immunohistochemistry staining,immunofluorescence staining and Western blot detection found that rosiglitazone feeding can up regulate the expression of PPAR?,Klotho and SM22?in the aorta of CKD mice,and inhibit the expression of Runx2 and BMP2.This shows that PPAR? agonist can significantly improve CKD vascular calcification.In summary,our study found that high phosphorus may down regulating the expression of PPAR?,and inhibition the target gene transcription,resulted in Klotho expression decreased,leads to increased expression of Pit,intake of phosphate increased in VSMCs and subsequently started the program of osteogenic cell differentiation in CKD status.This may be a key mechanism for vascular calcification in CKD.