Effects Of Calcitriol On Epithelial-Mesenchymal Transition Of NRK52E Cells Against Advanced Glycation End Products

Objective: It is known that oxidative stress and Epithelial-Mesenchymal Transition(EMT) induced by advanced glycation end products(AGEs) causes irreparable harm to kidney. Till now, Calcitriol have an important role to play in protecting kidney, but the precise mechanism remains unclear. In this study, EMT model by AGEs with or without Calcitriol pretreatment was used to observe the changes of receptor for advanced glycation end products(RAGE) and Phosphorylated NF-κB, To investigate the role of Calcitriol on Epithelial-Mesenchymal Transition in tubular epithelial cells against Advanced Glycation End products.Methods: 1. NRK52 E cells was incubated with Different concentrations(0, 100, 200, 300 g / ml) of AGE-BSA and BSA for 48 h, the level of E-cadherin 、α-SMA and RAGE were detected by Wstern blot.2. Groups: control group, 10nmol/LCalcitriol group and 100nmol/LCalcitriol group. E-cadherin、α-SMA was determined by Western blot.3. Groups: control group, AGEs group, 10nmol/LCalcitriol+AGEs group and 100nmol/LCalcitriol+AGEs group. The expression of E-cadherin、α-SMA were measured by Western blot, Fluorescence quantitative PCR( RT-PCR) and Immunofluorescence. And The level of RAGE and Phosphorylated NF-κB were detected by Western blot. The morphological changes in cells was observed under the Ordinary microscope.4. Groups: control group, AGEs group, 50g/ml Anti-RAGE neutralizing antibody+ AGEs group. E-cadherin、α-SMA were measured by Western blot.5. Groups: control group, AGEs group, 100nmol/L Calcitriol+AGEs and 100nmol/LCalcitriol+AGEs+IMD0354 group. The level of protein of RAGE was assessed by Western blot and m RNA was detected by RT-PCR.6. SPSS 20.0 software was used for a one-way analysis of variance(ANOVA).Results: 1. The expression of epithelial markers in AGEs group,E-cadherin, was lower than that in control group, while RAGE and the mesenchymal marker, α-SMA, were higher when compared with control group(P<0.05). No apparent change was observed in BSA group.2. No significantly differences were found in the level of E-cadherin and α-SMA among control group, 10nmol/L Calcotriol group and 100nmol/L Calcitriol group.3. E-cadherin remarkably decreased and α-SMA significantly increased in AGEs group when compared AGEs group with control group(P<0.05). Calcitriol up-regulated the expression of E-cadherin in renal tubular epithelial cells while reduced α-SMA against AGEs( P<0.05).RAGE and Phosphorylated NF-κB significantly increased when treated with AGEs compared the treated group with control group(P<0.05). Calcitriol reduced the expression of RAGE and Phosphorylated NF-κB against AGEs(P<0.05). Under ordinary microscope, the cells in the blank control group were polygonal, While in AGEs group was spindle shaped, and the Calcitriol+AGEs group was restored to polygon shape.4. E-cadherin remarkably decreased and α-SMA significantly increased in AGEs group when compared it with control group(P<0.05). Anti RAGE neutralizing antibody could reverse the result caused by AGEs(P<0.05)5. The level of RAGE in NRK52 E cells treated by AGEs was higher than that in control group. But the rising RAGE was inhibited by 100nmol/L Calcitriol, and the inhibition was reversible when pretreated with IMD0354.Conclusions: 1. The model of epithelial mesenchymal transition in NRK52 E cells was successfully constructed by AGEs.2. Calcitriol alone to stimulate the cells did not affect the level of cell marker expression.3. Calcitriol could reverse AGEs-induced EMT in NRK52 E cells, possibly by reverse AGEs-induced RAGE and phosphorylation NF kappa B.4. When RAGE was blocked, the degree of EMT induced by AGEs significantly decreased, Which shows AGEs-induced EMT is caused by RAGE.5. Calcitriol could reduce the expression of RAGE and the level of phosphoryled NF-κB, phosphorylation NF-κB inhibitor pretreatment could lessen the level of RAGE decreased by Calcitriol, meaning the change in RAGE induced by Calcitriol is caused by Phosphorylated NF-κB...