Calcitriol Relieves Kidney Fibrosis In 5/6 Nephrectomized Rats By Upregulating Klotho

Chronic Kidney Disease(CKD)patients generally have lower vitamin D levels.It is believed that the decrease of active vitamin D is associated with the secondary hyperparathyroidism and decreased bone mineral density.As a kind of active vitamin D,calcitriol is widely used in chronic kidney disease,which can delay the process of sHPT and renal fibrosis.However,the mechanism of calcitriol is still unclear.In this study,we discussed whether the calcitriol can inhibit renal injury by up regulating the anti-aging protein Klotho.In order to prove this hypothesis,we established rat renal failure model,to observed the relationship between renal fibrosis and the level of Klotho protein in serum and tissues of rats after administration of vitamin D.In this study,we explained the potential mechanism of calcitriol in inhibiting renal fibrosis,and provided a reference for the further utilization of calcitriol.(1)Animal experiment:To establish animal model of renal failure,all 5/6 nephrectomy rats were randomly divided into 5/6 nephrectomy combined with high phosphorus diet group(Nx-Hp,n = 11)and 5/6 nephrectomy combined with high phosphorus diet and drug intervention group(Nx-Hp + Cal,n = 10).Respectively at the 4th,12th week detection line of eyeground vein blood and postoperative at the end of the experiment to be put to death all the experimental rats,kidney tissue,observed the pathological morphology of kidney tissue and collagen deposition.Detect Klotho mRNA expression in the rat kidney by qRT-PCR,Elisa to detect serum-Klotho,FGF-23,TGF-beta 1,using immunohistochemistry and western blot technique respectively to observe the Klotho protein expression in renal tissue in rat,and comparative analysis of three groups of rats Klotho expression intensity difference.1)By HE and Masson staining method observed the pathological changes of kidney tissue,basic normal operation control group in the rat kidney tissue,in Nx-Hp group kidney focal glomerular sclerosis,renal tubular epithelial cells degeneration and atrophy and necrosis,renal interstitial fibrosis significantly with a large number of inflammatory cells infiltration,calcitriol intervention group glomerular and renal tubular lesion was significantly lighter than experimental group.2)the Nx group rats serum creatinine and iPTH level increased significantly(P<0.05),serum creatinine and iPTH die along with the extension of time showed a trend of increase,at the same time of Calcitriol intervention group can significantly reduce serum iPTH(P<0.05),and can significantly decrease the rat serum creatinine level(P<0.05).3)respectively to compare the groups of rats serum Klotho,FGF-23,TGF-beta 1 level,Nx-Hp group Klotho decline in serum,serum FGF-23,TGF-beta 1 factor is on the rise,and found that serum 1,25(OH)2 d3 and serum TGF-beta 1 factor was significantly negative correlation,we speculated that by lowering the TGF-beta 1 Calcitriol levels play a role.4)were compared between groups compared to the rat kidney tissue of Klotho,E cadherin,TGF beta and alpha 1-SMA,such as index,the results show that the Nx-Hp group rats kidney Klotho most obvious reduction,Calcitriol has partial raised anti-aging Klotho protein expression,partial inhibition of renal tubular epithelial-mesenchymal transdifferentiation(EMT)process.(2)cell type experiment:1)To explore the TGF-beta 1 intervention of renal tubular epithelial cells(NRK-52 e),the influence of the expression of cell Klotho found in the process of Klotho expression reduced at the same time accompanied by EMT.Calcitriol part blocking EMT process by raising the Klotho.2)Inhibiting the expression of Klotho in renal tubular epithelial cells,alpha SMA and fibrosis factor significantly increased at the same time,the ability of Calcitriol anti fibrosis function significantly weakened at the sametime.Experimental conclusion:these data show that Calcitriol has partial delay renal fibrosis and the effects may be mediated by some raised klotho expression in renal tissue and inhibiting renal tubular epithelial-(EMT)implementation of conversion of interstitial accordingly for the clinical application of Calcitriol and its renal protective mechanism provides strong evidence.