Effects Of Endoplasmic Reticulum Stress On The Expression Of CDK5 In High Glucose Cultured Podocytes

Objective: Diabetic nephropathy is one of the most common chronic microvascular complications in patients with diabetes mellitus,which is one of the important causes of end-stage renal disease.Podocytes are highly specific terminally differentiated cells,which attached on the outside of the glomerular basement membrane and formed glomerular filtration barrier.Podocyte plays an important role in the filtration of protein and the renewal of filtration barrier.Podocyte loss is closely related to the occurrence and development of DN,and apoptosis may be one of the important reasons.Endoplasmic reticulum stress(ERS)is one of the major cellular responses to stress,and plays an important role in determining the outcome of cellular stress such as resistance,adaptation,injury,or apoptosis.Endoplasmic reticulum stress plays an important role in podocyte apoptosis of diabetic nephropathy.The proline-directed serine threonine kinase,CDK5,is an unusual molecule that belongs to the well-known large family of proteins,cyclin-dependent kinases(Cdks).Ischemia,oxidative stress and other factors can activate CDK5,and result in a variety of pathological changes by phosphorylation of its substrates.When ERS occurs,it can over activate CDK5 and cause tissue lesion.ERS can induce podocyte apoptosis through activation of CDK5 in high glucose state.This study intends to observe the effect of ERS on the expression of CDK5 in podocyte,and to explore the role of CDK5 on podocyte apoptosis induced by ERS.Methods: The conditionally immortalized mouse podocytes were cultured at 33℃ with γ-IFN to induce proliferation,and then cells were cultured at 37℃ without γ-IFN for 10-14 days to induce quiescence and the differentiated phenotype.The podocytes cultured at 37℃ for 10 days were randomly divided as follows:(1)Control group;mannitol group(M);Highglucose group(HG).The podocytes cultured with high glucose for 6,12,24 and 48 hours respectively.(2)Control group;High glucose group(HG);High glucose + Salubrinal group(HG + Sal).The podocytes were treated with salubrinal(20 and 50 μM)for 24 and 48 hours.(3)Control group;Tunicamycin group(TM);Tunicamycin + CDK5 mi RNA group(TM + miCDK5);Tunicamycin +Roscovitine group(TM + ROS).The concentration of tunicamycin is 1.0 μM.All groups were cultured for 12 hours.The expression of GRP78,Caspase-12,CHOP and CDK5 were detected by Western blot,and the expression of CDK5 was also detected by immunofluorescence.The apoptosis was detected by TUNEL and flow cytometry.Results: Western blot results showed that,compared with Conntrol group and M group,the protein expression of GRP78,Caspase-12 and CHOP,the ERS markers,was significantly increased in a time-dependent manner in HG group;CDK5 expression was also increased in time-dependen manner.The expression of GRP78,Caspase-12,CHOP and CDK5 was decreased in a concentration-and time-dependent manner after the intervention of salubrinal,an inhibitor of ERS.Immunofluorescence assay showed that salubrinal could also significantly decrease the fluorescence intensity of CDK5 expression in podocytes after high glucose stimulation.Flow cytometry and TUNEL assay showed that salubrinal can significantly reduce podocyte apoptosis caused by high glucose;the inhibition of CDK5 by miRNA and roscovitine could reduce the apoptosis of podocyte induced by tunicamycin.Conclusion:1 High glucose can induce the occerrnce of endoplasmic reticulum stress: the ERS markers,GRP78,Caspase-12 and CHOP proteins,were significantly increased.High glucose can enhance the expression of CDK5 in podocytes by inducing endoplasmic reticulum stress,which leads to the podocyte apoptosis.2 Inhibition of endoplasmic reticulum stress can significantly reduce podocyte apoptosis induced by high glucose and can significantly reduce the expression of CDK5 in podocytes stimulated by high glucose.3 Inhibition of CDK5 could significantly decrease the apoptosis of podocytes induced by endoplasmic reticulum stress.