Effect Of Febuxostat On Epithelial-to-mysenchymal Transition In Hyperuricemia Rats

BackgroundThe persistence of hyperuricemia will cause the urate accumulation,so that cause the kidney disease,which is known as uric acid nephropathy.Many kinds of chronic kidney disease have a common pathological process-tubulointerstitial fibrosis,and renal tubular epithelial mesenchymal transition(EMT)is considered to be the early manifestations of tubulointerstitial fibrosis.The EMT of renal tubular maens the process of renal tubular epithelial cell lose its phenotype characteristics and obtain the characteristics of interstitial cell phenotypic characteristics,which includes the epithelial cell polarity disappeared,E-cadherin,ZO-1 protein levels decreased,increased expression of a-smooth muscle actin(a-SMA),vimentin,collagen type ?/?,and so on.EMT was first proposed by Greenberg and Hay,the epithelial cells they cultured in the gellens lose their polarity and changed into mesenchymal cells with pseudo-foot-like.EMT is thought to play an important role in the formation and development of embryos,tumor formation and migration,tissue healing and organ fibrosis.Various growth factors,cytokines and hormone can affect the forming of EMT,such as epidermal growth factor(EGF),transforming growth factor(TGF-?)factor,fibroblast growth factor(FGF),insulin-like growth factor(IGF)angiotensin ?(Ang-?)and so on,and the TGF-? plays an important role in EMT in embryonic development,tumor metastasis and organ fibrosis,it is the most important factor.Recently a new statement proposed that uric acid is the cause of the occurrence and development of CKD,uric acid can directly stimulate renal tubular epithelial cells,increases the expression of TGF-?1,promotes and reduce the expression of related transcription factors to reduce the synthesis of E-cadherin,to increase the expression of a-SMA,TGF-?1 also stimulates the formation of fibroblasts by inhibiting matrix metalloproteinase which induce production of natural matrix metalloproteinase inhibitors,thereby inhibiting the degradation of extracellular matrix.In addition,uric acid can increase the degradation of E-cadherin through ubiquitination,then lead to the disappearance of the polarity of renal tubular epithelial cells.The xanthine oxidase inhibitors(XOI)can reduce uric acid levels,and reduce the condition of CKD,however,it still lack large-scale prospective clinical trials to provide verification.ObjectiveTo observe the changes of renal function and EMT of renal tubular in normal and hyperuricemic rats,to explore the possible mechanism of uric acid-induced EMT in renal tubular.Use the Xanthine oxidase inhibitor-Febuxostat and the urinary excretion drug-Benzbromarone to intervent the hyperuricemia rats,then observe and compare the effects of Febuxostat and Benzbromarone on renal tubular epithelial-mesenchymal transition in hyperuricemia rats,and further explore the role of Febuxostat in the prevention of renal fibrosis.Methods(1)Forty-eight male Sprague-Dawley(SD)rats were randomly divided into normal control group,model group,Febuxostat group and Benzbromarone group,there were 12 rats in each group.The potassium oxonate was dissolved with 0.5%sodium carboxymethylcellulose solution,all rats used potassium oxonate by intragastric administration once a day to establish hyperuricemia model in addition to the normal control group,and the Febuxostat group and Benzbromarone group gave Febuxostat(7.2mg/(kg.d))and Benzbromarone(9mg/(kg.d))respectively at the same time,the normal control group gave the same value of 0.5%sodium carboxymethylcellulose solution.Rats were weighted twice a week at the weekend,then put into the metabolic cage and fasting without water for 24 hours and collected the urine.The orbital venous plexus blood was collected about 1.5 ml/min at each rat.Used the Beckman automatic biochemical analyzer to detected serum uric acid,creatinine and urea nitrogen,the urinary protein was quantified by biuret method,IL-6,Cys-C,TGF-?1 and BMP-7 were measured by enzyme-linked immunosorbent assay.Four rats in each group were sacrificed at the end of the 0th,4th and 8th week,rats were anesthetized by intraperitoneal injection of 10%chloral hydrate,and separated the kidneys after the implementation of cardiac blood collection,then fixed in 10%formalin,embedded in paraffin,cut into 4?m for HE and Masson stain.Used immunohistochemical staining to tested the expression of E-cadherin,a-SMA,collagen ? and TGF-?1.When completed the above operations,observed the pathological changes of the kidneys under the light microscope,and selected ten non-overlapped fields randomly at high magnification(x400).After all,the ratio of tubulointerstitial fibrosis area in the total area of interstitial tubulointerstitium and the expression of each protein in renal tissue were determined by Image Pro-Plus version 6.0 image analysis software.ResultsAt the 4th week,the UA,Scr,BUN,IL-6,Cys-C and TGF-?1 of the Hyperuricemia group were significantly higher than those in normal control group(P<0.05 or P<0.01),combined with the decrease of BMP-7(P<0.01);After using Febuxostat,the levels of IL-6 and TGF-?1 were decreased significantly then the Hyperuricemia group(both P<0.01);UA and IL-6,Cys-c and TGF-?1 was positively correlated(r=0.908,P=0.000;r=0.759,P=0.000;r=0.850,P=0.000),and a negative correlation with BMP-7(r=-0.826,P=0.000).HE staining showed that Febuxostat treatment can significantly improved the renal injury;Masson staining reminder the degree of renal fibrosis of Hyperuricemia group were greater than the other three groups,and the fibrosis remission of Febuxostat group is superior then Benzbromarone group;Immunohistochemical found that,normal control group did not express a-SMA but high express E-cadherin;Compared with Hyperuricemia group,the expression of a-SMA and Collage ? were obviously decrease(P<0.01),and the E-cadherin was improved(P<0.01)?ConclusionsHyperuricemia will induce tubulointerstitial EMT in rats,and the phenotypic transformation of renal tubular epithelial cells occurs before renal interstitial fibrosis;Cys-c was more sensitive than the traditional renal functions,which can be used as a predictor of early renal injury;Febuxostat can effectively reduce the level of uric acid,then reduced the expression of IL-6/TGF-?1,increased the role of BMP-7 antagonize TGF-?1,so that to reverse the occurrence of early tubular fibrosis.But the specific regulation mechanism of IL-6/TGF-?1 and BMP-7/TGF-?1 is still need a further study.