ATF4 alleviates palmitic acid-induced apoptosis of glomerular podocytes by regulating Parkin

Abstract:

Objective：To investigate the role of activating transcription factor-4（ATF4） in palmitic acid（PA）-induced apoptosis of glomerular podocytes by regulating Parkin. Methods：Twenty Sprague Dawley rats were randomly divided into two groups：normal diet group with 10 rats and high-fat diet group with 10 rats. After 12 weeks of feeding，serum was collected for measurement of triglyceride（TG） and total cholesterol（TC） levels. Cell apoptosis in renal tissues was evaluated by Tunel staining. Immunohistochemical method and Western blot were used to measure the protein expression of cleaved cysteinyl aspartate specific proteinase-3（Cleaved caspase-3），ATF4，and Parkin in renal tissues. PA was used to stimulate glomerular podocytes in vitro，plasmids were trans－fected with overexpressed Parkin，or adenovirus was used to knock down the expression of ATF4. Western blot was used to measure the expression of Cleaved caspase-3，and flow cytom－etry was used to determine the apoptosis rate of glomerular podocytes. Results：The high-fat diet group had significantly higher expression of TG and TC compared with the normal diet group（2.700±0.565 vs. 0.780±0.165，t=-7.987，P=0.000；2.620±0.565 vs. 1.540±0.377，t=-3.895，P=0.003）. The protein expression of Cleaved caspase-3，Parkin，and ATF4 were significantly higher in the high-fat diet group than in the normal diet group（2.257±0.326 vs. 1.000±0.190，P=0.000；1.587±0.162 vs. 1.000±0.323，P=0.003；1.851±0.347 vs. 1.000±0.186，P=0.000）. After being stimu－lated with different concentrations of PA，compared with the control group，the expression of ATF4，Parkin，and Cleaved caspase-3 in podocytes was significantly increased in the 150 μmol/L PA group（1.689±0.235 vs. 1.000±0.030，P=0.002；1.529±0.057 vs. 1.000±0.066，P=0.000；1.773±0.186 vs. 1.000±0.039，P=0.000） and in the 300 μmol/L PA group（1.702±0.228 vs. 1.000±0.030，P=0.002；2.047±0.086 vs. 1.000±0.066，P=0.000；2.282±0.155 vs. 1.000±0.039，P=0.000）. After the Parkin overexpression plas－mid was transfected into podocytes，the expression of Parkin in the Parkin overexpression group was significantly increased compared with the control group（105.242±24.108 vs. 1.000±0.253，P=0.000）. After stimulation with PA，the expression of Cleaved caspase-3 in podocytes and PA-induced apoptosis rate in PA+Parkin overexpression group were significantly reduced compared with the PA+empty plasmid group[1.943±0.200 vs. 3.398±0.164，P=0.000；（9.590±0.418）% vs. （15.140±1.227）%，P=0.000]. After adenovirus transfection and knockdown of ATF4，the protein expression of ATF4 and Parkin in the shATF4 group was significantly reduced and the expression of Cleaved caspase-3 was significantly increased compared with the control group（0.175±0.064 vs. 1.000±0.112，P=0.000；0.518±0.130 vs. 1.000±0.104，P=0.004；2.305±0.150 vs. 1.000±0.279，P=0.000）. The cell apoptosis rate of podocytes was significantly higher in the shATF4 group than in the control group[（14.230±0.278）% vs.（7.020±0.773）%，P=0.000]. After podocytes were stimulated with ATF knockdown，the expression of Cleaved caspase-3 and apoptosis rate were significantly higher in the PA+shATF4 group than in the PA+shRNA group[2.483±0.114 vs. 1.926±0.215，P=0.007；（46.643±10.675）% vs.（20.943±4.194）%，P=0.018]. Conclusion：ATF4 can alleviate PA-induced apoptosis of glomerular podocytes by regulating Parkin.