Degradation Of Perilipin2 By Chaperone-mediated Autophagy Ameliorates Palmitic Acid-induced Lipid Accumulation And Apoptosis In Podocytes In Podocytes In Vitro

Background: Chronic kidney disease(CKD)and its cause of end-stage renal disease(ERSD),has become a global health problem.CKD,including diabetic nephropathy(DN),is often associated with lipid metabolism disorders,mainly manifested as hypertriglyceridemia,hypercholesterolemia.Previous studies have found that hyperlipidemia itself can lead to kidney damage,such as mesangial cell proliferation,glomerulosclerosis,renal tubular interstitial fibrosis and so on.Podocytes,an important part of the glomerular filtration barrier,are highly specialized cells with limited ability to regenerate.Loss of podocytes can lead to glomerular injury,which eventually leads to proteinuria and glomerular filtration dysfunction.Excessive lipids in podocytes can cause cell dysfunction and cell death.In the early stage of hyperlipidemia,foot process fusion,abnormal cytoskeleton,cell apoptosis and shedding can occur.Chaperone-mediated autophagy(CMA)is the mechanism ofselective degradation of intracellular protein with KFERQ sequence by the lysosomal pathway.It recognizes proteins with KFERQ sequence through hsc70 and transports them to the surface of the lysosomal membrane to bind to the specific CMA protein receptor,LAMP2 A,to make the proteins unfold and then enter the lysosomal cavity to be degraded by lysosomal enzymes.Studies have found that the PAT family,a lipid-coated protein family,is present on the surface of Lipid drop(LD),including perilipin1,perilipin2,perilipin3,perilipin4 and perilipin5,which have a protective effect on Lipid drop and participate in the formation and degradation of lipids.Perilipin 2(PLIN2)is highly expressed in lipid droplets,and its expression level is correlated with triglyceride(TG)content and lipid droplet density,which has been proved to be a protein marker of lipid droplets.PLIN2 is widely expressed in liver,intestine,kidney and other tissues.Recent studies have found that PLIN2 also expresses KEFRQ sequence and is the substrate protein of CMA.CMA regulates lipolysis in the liver by degrading lipid droplet associated protein PLIN2.However,the exact role of CMA in the podocytes of DN patients with dyslipidemia is still uncertain.Herein,we aimed to find the role of CMA in palmitic acid(PA)-induced lipid accumulation and apoptosis in podocytes using an in vitro model.Methods: We treated podocytes with PA,in the absence or presence of hsc70 inhibitor VER155008,LAMP2 siRNA,or Torin1.Then,intracellular lipid content was detected using Oil Red O staining test and boron-dipyrromethene lipid probes,the expression of PLIN2 was detected by western blotting and immunofluorescence,podocyte apoptosis was measured by flow cytometry.The expression of apoptosis-related protein cleaved-caspase3 and CMA-related proteins,including hsc70 and LAMP2 A was detected by western blotting.Results:(1)Compared with the control group,PA can induce the increase of PLIN2 protein expression in podocytes in a concentration-dependent manner,and the accumulation of lipids also increases.(2)Compared with the control group,PA induced the expression of LAMP2 A and hsc70 proteins in podocytes significantly increased within a certain concentration range,and the co-localization of PLIN2 and hsc70,PLIN2 and lysosomal co-localization increased.(3)Using VER155008 to inhibit hsc70 or siRNA transfection podocytes to knock down LAMP2,PA-induced PLIN2 expression and lipid accumulation in podocytes increased.(4)PA-induced PLIN2 expression and lipid accumulation in podocytes were decreased when CMA inducer Torin1 was used to enhance CMA activity.(5)PA can increase the podocytes apoptosis and the expression of Cleaved-caspase3 protein in a concentration-dependent manner.UsingVER155008 to inhibit hsc70 or siRNA transfection to knock down LAMP2 in podiocytes,the expression of Cleaved-caspase3 stimulated by PA was increased and its apoptosis was more obvious.When CMA activity was enhanced by CMA inducer Torin1,we found PA-induced apoptosis and expression of Cleaved caspase-3 protein reduced.Conclusion:(1)PA induced lipid accumulation and increased PLIN2 expression in podocytes(2)PA can induce the increase of CMA activity of podocytes within a certain concentration range and participate in the degradation of PLIN2(3)CMA can improve PA-induced podocyte lipid accumulation and apoptosis by degrading PLIN2.