Progranulin Protects Against Podocyte Injury In Diabetic Nephropathy

Objective:Diabetic nephropathy(DN)is one of the main inducement of end stage renal disease(ESRD),seriously threats human health.The pathogenesis of diabetic nephropathy is complicated,including hyperglycemia,hyperlipidemia,haemodynamics change,oxidative stress,inflammation and heredity,ect.Podocyte is a major player in maintaining the homeostasis of the glomeruli.Glomerular podocytes,endothelial cells and glomerular basement membrane(GBM)constitute the glomerular filtration barrier.Podocyte injury is an early event of diabetic nephropathy.Podocyte injury leads to filtration barrier damage,results in proteinuria and exacerbates renal injury.However,the mechanism of podocyte injury in diabetic nephropathy is not yet clear.Progranulin(PGRN)is an autocrine growth factor.widely expressed in various tissues and organs,mainly distributed in epithelial cells,neurones,immune cells and chondrocytes.PGRN has multiple biological functions.is widely involved in a variety of pathophysiology processes,including tumorigenesis,autoimmune disease,tissue injury repair,and host defense.Our previous studies have demonstrated that PGRN protects against acute kidney injury by negatively regulating NOD2-mediated signaling pathways.But,the function of PGRN in diabetic nephropathy is still unknown.Studies have shown that autophagy plays a crucial role in the pathogenesis of diabetic nephropathy.Under normal physiological conditions,the podocytes maintain a high level of autophagy,and reducing the level of podocytes autophagy leads to podocyte injury further cause renal damage in the pathogenesis of diabetic nephropathy.Studies have elucidated that AMPK is a stimulator of autophagy.AMPK protects against kidney injury by direct phosphorylating ULK1(an autophagy initiate protein)and enhances autophagy under diabetic conditions.Therefore,our research aims to explore the role of PGRN in diabetic nephropathy and the involvement of autophagy and AMPK signaling pathway.Methods and Results:1.The expression of PGRN in diabetic nephropathyMale C57BL/6J mice were randomly divided into two group,and one week after uninephrectomized,mice were administrated with streptozotocin(STZ,100mg/kg)by intraperitoneal injection for three days to construct diabetic nephropathy model.The expression level and distribution of PGRN in renal cortex were detected by western blotting(WB),Real-time RT-PCR,immunofluorescence(IF),immunohistochemistry(IHC),the results showed that PGRN were significantly decreased in glomerular podocyte from diabetic nephropathy mice compared with control group.Human podocytes(HPC),glomerular endothelial cells(GEnC),rat mesangial cells(RMC)were stimulated by high glucose to mimic the pathogenesis of diabetic nephropathy in vitro,the results indicated that PGRN were decreased in podocytes and GEnCs.2.The role of PGRN in diabetic nephropathyWild type male C57BL/6J mice and Grn knockout mice(B6(Cg)-Grntml.lAidi/J)were randomly divided into four groups:two diabetic nephropathy groups and two normal groups.Kidney paraffin slides were stained with periodic acis-schiff(PAS).Transmission Electron Microscope(TEM)was employed to observe podocyte injury.The results indicated that the deficiency of PGRN exacerbated agglutination of glomerular glycogen and mesangial matrix proliferation.The results of TEM showed that PGRN knowout aggravate foot process fusion and podocyte injury.Podocyte specific protein Nephrin and Podocin were marked by immunofluorescence,the results showed that the deficiency of PGRN exacerbated the podocyte functional protein damage.Real time RT-PCR to detect the change of inflammatory factor and chemotactic factor in renal cortex,the results showed that the deficiency of PGRN in diabetic mice led to more severe inflammatory response compared with wild type group.Using flow cytometry to detect the apoptotic index of podocyte,podocyte treated with recombinant PGRN(rhPGRN)decreased high glucose-induced cell apoptosis.Western blot was carried out to detect the levels of functional protein Nephrin,the results indicated that rhPGRN can recover high glucose-decreased Nephrin expression.3.The mechanism of PGRN in diabetic nephropathyWestern blot was used to detect the level of autophagy-related protein in renal cortex of diabetic mice,shown that the autophagy level of Grn-/-group was significantly decreased compared with wild type diabetic mice.In vitro,the results from WB showed that autophagy level was significantly decreased in high glucose-treated podocytes and rhPGRN can restored it.Autophagic flux was detected by Tandem Sensor,results showed that rhPGRN restored autophagic flux in podocytes with high glucose treatment.WB was used to detect autophagy initial protein,the results showed that the phosphorylation of ULK1 at Ser555 was decreased in high glucose treated podocytes,which was rescued by rhPGRN.Further study revealed that activation of AMPK signaling was further weakened in Grn-/-diabetic mice than wild type diabetic mice.Conclusions:1.PGRN expression is significantly decreased in kidney from diabetic mice,and the deficiency of PGRN exacerbates diabetic renal injury.2.PGRN activates the AMPK signaling pathway to promote the phosphorylation of ULK1 at Ser555 and then initiate autophagy in podocytes.And this find may provide an important basis for the design of PGRN as a target for diabetic nephropathy.