Effect Of Klotho On Autophagy Induced In Diabetic Kidney Disease And Its Mechanism

BackgroundDiabetic kidney disease(DKD)is one of the serious complications of diabetes mellitus,is also the main cause of end-stage renal disease(ESRD).At the same time,it is also considered as an independent risk of many cardiovascular diseases.However,the pathophysiology of its occurrence and development is not cleared fully.Autophagy is one of the intracellular lysosomal degradation pathways,with the goal of removing excess protein aggregates within the cell while removing damaged or redundant organelles and thereby maintaining the cell's homeostasis.Autophagy can be activated by a variety of stress conditions such as hypoxia,endoplasmic reticulum stress or oxidative stress,whereas obesity or diabetes-induced metabolic dysfunction can impair autophagy.Studies have shown that DKD state significantly inhibited the level of renal autophagy,and activation of autophagy can play a role in protecting the kidneys.Klotho is an anti-aging factor,and it is expressed high in the kidneys selectively,especially the renal tubular epithelial cells.The current general view sees it as a hormone secreted mainly by the kidneys,playing a protective effect in a variety of acute and chronic kidney diseases.In the early stages of DKD,the Klotho protein in the patient's serum has been declining and its decline diminishes as DKD progresses,so the level of serum Klotho protein decline is an important biomarker for the progression of DKD.Currently,there is no study on the relationship between klotho and autophagy in diabetic nephropathy.This study aims to investigate the relationship between klotho and autophagy in DKD state and to explore whether klotho can improve renal autophagy via AMPK and ERK pathways and thus play a role in renal protection,providing a new theoretical basis for further understanding the relationship between klotho and autophagy in diabetes mellitus nephrotic pathogenesis.Methods1.Animal experimentsThe diabetic mice(DM group),which has been constructed in the laboratory by the STZ/HFD method,are controlled with the normal mice of the same type(control group).The mice were all purchased from Guangdong Provincial Department of Medicine Laboratory Animal Center.All mice were housed in the Experimental Animal Center of Nanfang Hospital of Southern Medical University.Kidney tissues from DM group and control group of 1w,6w and 18w groups were detected by RT-qPCR.and western blot.The expression of LC3B protein in renal cortex was detected by immunohistochemistry.At the same time,a model of kidney klotho overexpression was constructed by injecting plasmid through the tail vein at 12 weeks in DM group.Then,RT-qPCR and western blot were used to detect the molecular biological indexes of their Kinsey tissues.LC3B protein expression in renal cortex was detected by immunohistochemistry.2.Cell experimentThe laboratory-preserved human renal tubular epithelial cell line(HK2)(purchased from Shanghai Chinese Academy of Sciences)was cultured in a cell culture medium made of 1640 basic medium(2g/L glucose)and 10%FBS,and placed in a 5%CO2 incubator at 37 ? until the medium turns yellow or replace the medium every 2-3 days.When it grew up to about 80%on a 10 cm cell culture dish,it was digested and inoculated into a 6-well plate.The transfected plasmids and time gradient were grouped as follows:24h/empty plasmid control group(pcDNA-Vector)+ klotho overexpression group(pcDNA-klotho);48h/pcDNA-Vector + pcDNA-klotho;72h/pcDNA-Vector +pcDNA-klotho.After 12-24 hours of cell starvation,the corresponding plasmids were transfected and cultured in high glucose(Glu 30mM)medium.RT-qPCR and western blot were used to detect the molecular biological indexes after receiving the plate,and the autophagosome morphology was observed by electron microscope.3.Real-time fluorescence quantitative PCR(RT-qPCR)Total mRNA of tissues and cells was extracted by Trizol method.M-MLV reverse transcriptase reagent was added to enable reverse transcription of mRNA into cDNA.Real-time PCR was carried out on the Roche 480 PCR instrument using the SYBR method.The target gene expression was calculated by the method of 2-??Ct.4.Western BlotThe total protein of tissues and cells was extracted by RIPA method.Prepare SDS-PAGE separation gel;add protein samples;separate by electrophoresis;transmit to PVDF membrane;block PVDF membrane.After primary antibody incubation at 4?overnight,the secondary antibody is incubated for 1-2h and then rinsed with TBST.Tanon 5500 fully automated chemiluminescence imaging analysis system was used to acquire images and the results were analysed by Image J software.5.ImmunohistochemistryFresh mouse kidney specimens were taken,fixed with 4%paraformaldehyde,embedded in paraffin and sectioned.After dewaxing,hydration,antigen retrieval and blocking,they were incubated with rabbit anti-LC3 polyclonal antibody,incubated with secondary antibody,stained with DAB,stained with hematoxylin,stained with neutralized gelatin after bluing and dehydrated.Set the Upright Metallurgical Microscope to observe it,and take a picture.6.Electron microscopeAfter fixation of cell samples with electron microscope fixation solution,osmium tetroxide and the like,the gradient of alcohol and acetone were dehydrated,and acetone and 812 embedding agent were infiltrated,followed by embedding and sectioning.After ultrathin section were stained with uranyl acetate-lead citrate staining methods,photographs were taken by using a transmission electron microscope and images were collected for analysis.7.Statistical analysisAll experimental data was analyzed by SPSS 19.0 and expressed as mean ±standard deviation(x±SD).One-way analysis of variance(ANOVA)was used to compare multiple sets of data,and two independent samples t-test.All statistical results of the P value<0.05,were considered statistically significant.Results1.The expression differences of Klotho and LC3B in diabetic mice(1)Compared with the control group,the expression of klotho in DM group decreased slightly after modeling(1w),but there was no significant difference.The expression of klotho decreased significantly at 6w and further decreased at 18w.(2)Compared with the control group,the autophagy level of DM group increased slightly at 1w,and the autophagy was significantly decreased in DM group at 6w and 18w,whose autophagy was lessened than that at 6w.(3)Immunohistochemistry showed that compared with the control group,the expression of LC3B protein in the renal cortex of 18w mice was significantly reduced,especially in the tubular region,the main expression area of klotho.2.Changes of renal cortex autophagy in diabetic mice after overexpressing klotho(1)The klotho mRNA in renal cortex of pCMV-Klotho mice constructed by tail vein injection of plasmid was significantly higher than that of pCMV-Vector mice.(2)Compared with the pCMV-Vector group,the LC3B protein content in the kidney cortex of the pCMV-Klotho group was significantly increased.(3)Immunohistochemistry showed that the expression of LC3B protein in the renal cortex of pCMV-Klotho group was significantly increased in the tubular region,which is consistent with the expression region of klotho.3.Changes of autophagy in high glucose cultured HK2 cells after overexpressing klotho(1)Compared with pcDNA-Vector,klotho mRNA in HK2 cells of pcDNA-Klotho were significantly increased expression after transfecting plasmids and high glucose culturing for 24 hours.(2)In the pcDNA-Vector group,the content of LC3B in 72h group was significantly lower than groups in high glucose for 24h and 48h.(3)Compared with pcDNA-Vector,the content of LC3B in pcDNA-Klotho group increased,and the most obvious difference between the two was at 72h.(4)Observing the HK2 cells cultured in high glucose and transfected with plasmid for 72h under electron microscope indicates that the cytoplasm of pcDNA-Klotho group showed significant autophagosomes which contained damaged organelles and other cytoplasmic components,while pcDNA-Vector did not show integral autophagosome structure.4.The mechanism of klotho on DKD renal autophagy(1)Compared with pCMV-Vector group,diabetic mice in pCMV-Klotho group had higher AMPK phosphorylation level and lower ERK phosphorylation level(2)Increased AMPK phosphorylation and decreased ERK phosphorylation were still observed in the pcDNA-Klotho cells.Conclusions1.Under the condition of DKD,the expression of klotho mRNA in mouse renal cortex gradually decreased,and the decreasing degree of autophagy was more and more large,which was consistent with the trend of klotho expression.2.With the extension of high glucose stimulation,human renal tubular epithelial cells(HK2 cells)' inhibition of autophagy was more and more obvious.3.Overexpression of klotho can induce the up-regulation of autophagy in renal cortex of diabetic mice,and still up-regulate the level of autophagy in the high-glucose-cultured HK2 cells.4.Klotho can improve the inhibition of renal autophagy under DKD by up-regulating AMPK phosphorylation and down-regulating ERK phosphorylation.