| ObjectiveThe purpose of clinical study confirmed that high phosphorus diets can accelerate diabetic nephropathy(DN)in patients with chronic renal tubular interstitial fibrosis,but its mechanism is not completely clear.Aging inhibitor Klotho mainly expressed in renal tubular epithelial cells.Recently discovered,Klotho can inhibit renal fibrosis by increasing renal tubular epithelial cell autophagy activity.Due to the high phosphorus diet is a major factor inhibiting renal tubular epithelial cells klotho expression,we hypothesized Klotho-autophagy signaling activity change is accelerating high-phosphorus diet in renal interstitial fibrosis progression important molecular mechanisms of diabetic nephropathy.In this study,high-phosphorus diet intervention rats with diabetic nephropathy,renal tubular epithelial cells were observed signal Klotho-autophagy activity and renal tubular epithelial-mesenchymal transition in the relationship,and low doses of paricalcitol above process intervention effect.MethodsThirty male Sprague-Dawley rats,of which 24 to give 60 mg / kg STZ intraperitoneal injection model of diabetes mellitus,after molding into DN group P + normal diet group(phosphorus content of 0.75%),DN + high phosphate diet group(phosphorus content 1.2%),DN group + paricalcitol group(intraperitoneal injection,0.4 ?g · kg-1 · d-1),DN + high phosphate diet + paricalcitol group each six;the rest 6 as the control group.After 12 weeks,measured Scr and blood levels of calcium and phosphorus,24 h urinary protein,the rats were sacrificed and the renal tissue were performed by light microscopy,electron microscopy,and fluorescence immunohistochemistry,western blotting and other tests.Pathological changes in renal interstitial HE and PASM staining,immunohistochemistry between renal tubular epithelial-mesenchymal transition flag molecule E-cadherin,?-SMA expression,immunofluorescence Klotho LC3 expression and autophagy molecules,electron microscopy autophagy super micro-structural changes,western blotting renal cortex p62,LC3-II / LC3-and other expression changed.Results 1.For each group of diabetic rats Scr nephropathy and 24-hour urine protein were significantly increased in rats with high phosphorus highest minimum rats paricalcitol group,the difference was statistically significant(P <0.05,).Paricalcitol group rat serum calcium and phosphorus levels compared with other diabetic rats,no significant difference(P> 0.05).2.The results of immunohistochemistry showed that compared with the control rats,rats at each diabetic nephropathy quality E-cadherin expression was significantly decreased,significantly increased ?-SMA expression,consistent with renal tubular epithelial-mesenchymal transition characterized.Compared with other diabetic rats,paricalcitol group were the highest expression of E-cadherin,?-SMA expression minimum,the difference was statistically significant(P <0.05).3.Immunofluorescence results showed that compared with control rats,diabetic rats each tubular epithelial cells Klotho significantly decreased(P <0.05),significantly reduced LC3 protein lumpy distribution(all P <0.05).Compared with other diabetic rats,paricalcitol rats Klotho highest expression,LC3 protein up to agglomerate distribution,and high phosphorus diet diabetic rats on the contrary,the difference was statistically significant(P <0.05).Electrical inspection results with the results of the expression of LC3 immunofluorescence microscopy consistent.4.Western blot results showed that compared with the control rats,the expression of p62 in renal cortex of rats with diabetic nephropathy group were significantly increased,LC3-II / LC3-I ratio was significantly decreased(P <0.05).Compared with other diabetic rats,rat p62 paricalcitol group was minimal,LC3-II / LC3-I ratio of the highest and high phosphorus diet diabetic rats on the contrary,the difference was statistically significant(P <0.05).5.Correlation analysis showed that,klotho expression level of p62 expression was negatively correlated with LC3-II / LC3-I ratio was positively correlated(r =-0.722,P <0.01;r = 0.551,P <0.05).Conclusions: 1.High-phosphorus diet can significantly inhibit the renal tubular epithelial cell signaling activity Klotho-autophagy rats with diabetic nephropathy,which may be a high phosphorus diet accelerate renal tubular epithelial-mesenchymal transition process is an important molecular mechanism;2.Low-dose of paricalcitol may not increase blood levels of calcium and phosphorus,the significantly increased tubular epithelial klotho-autophagic cell signaling activity,thereby inhibiting renal tubular epithelial-mesenchymal transition,reduce proteinuria,improve renal function and delay Advances in renal failure. |
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