Angiopoietin2 Increases Early Albuminuria In Diabetic Kidney Disease By Regulating P-Caveolin1/Caveolin1 Mediated Albumin Transcytosis Across Renal Tubular Epithelial Cells

Objective: Diabetic kidney disease(DKD)is one of the important microvascular complications of diabetes mellitus(DM),it is also the main cause of end-stage renal disease(ESRD)in China.Albuminuria is one of the criteria for evaluating the severity and clinical staging of DKD,but its pathogenesis in DKD has not been fully elucidated.It has been reported that glomerular filtration barrier(GFB)injury is the main mechanism of albuminuria generation in DKD,and the related roles of renal tubular epithelial cells are poorly understood.Recently studies have shown that albumin can be transported across cells by transcytosis dependent on the structure of caveolae on the cell membrane.Caveolin 1(CAV1)is the scaffold protein of caveolae,which can regulate the structure and function of caveolae.Angiopoietin2(ANGPT2)can regulate vascular reconstruction and endothelial permeability.The levels of ANGPT2 in serum and renal tissues of diabetic patients are significantly higher than the physiological levels,and it is related to the level of albuminuria,but its role in albuminuria is still unclear.The purpose of this study was to explore the role of ANGPT2 in DKD albuminuria and its possible mechanism.Methods:(1)C57BL/6 mice were intraperitoneally injected with streptozotocin(STZ)to establish a DKD model,and the blood glucose,renal weight-to-weight ratio,24-hour urinary albumin creatinine ratio and renal pathological changes were detected.(2)Immunohistochemistry was used to detect the expression levels of ANGPT2 and phosphorylated CAV1(P-CAV1)in kidney of DKD mice.(3)Human renal tubullar epithelial cells(HK-2)were cultured and inoculated in the upper compartment of transwell to establish in vitro transcytosis model.Bovine serum albumin(BSA)and fluorescein isothiocyanate(FITC)were co-incubated to form FITC-BSA complex,which was used to label the albumin.FITC-BSA was added into the upper ventricle,and the fluorescence intensity of FITC-BSA in the lower ventricle was observed to reflect the albumin transcytosis volume,and the albumin penetrating cell of HK2 cells under normal glucose(NG,5.6 m M)and high glucose(HG,30 m M)conditions was detected by in vitro transcytosis model.(4)Western blot was used to detect the expression level of ANGPT2 in HK2 cells under normal glucose(NG,5.6 m M)and high glucose(HG,30 m M)conditions.(5)The expression levels of P-CAV1 and CAV1 in HK2 cells under normal glucose(NG,5.6 m M)and high glucose(HG,30 m M)conditions were detected by Western blot.(6)ANGPT2-RNAi lentivirus was constructed to knock down the expression of ANGPT2 in HK2 cells,and the albumin penetrating cell of HK2 cells under normal glucose(NG,5.6 m M)and high glucose(HG,30 m M)conditions was detected by in vitro transcytosis model.The expression levels of P-CAV1 and CAV1 in HK2 cells under normal glucose(NG,5.6 m M)and high glucose(HG,30 m M)conditions were detected by Western blot.(7)CAV1-si RNA was constructed to knock down the expression of CAV1 in HK2 cells,and in vitro transcytosis model was used to detect the albumin penetrating cell under normal glucose(NG,5.6 m M)and high glucose(HG,30 m M)conditions.(8)The expression of CAV1 in HK2 cells was knocked down,and the expression level of ANGPT2 in HK2 cells under normal glucose(NG,5.6 m M)and high glucose(HG,30 m M)conditions was detected by Western blot.Results:(1)STZ induced DKD in mice;(2)The expression levels of ANGPT2 and P-CAV1 in renal tissues of DKD mice were increased;(3)Endocytosis and transcytosis of albumin in renal tubular epithelial cells stimulated by high glucose in vitro;(4)The protein expression of ANGPT2 in renal tubular epithelial cells was up-regulated by high glucose in vitro;(5)The phosphorylation level of CAV1 in renal tubular epithelial cells was up-regulated by high glucose in vitro;(6)Knocking ANGPT2 can promote the transcytosis of albumin in renal tubular epithelial cells and inhibit the phosphorylation of CAV1;(7)After knockdown of CAV1,CAV1 phosphorylation was down-regulated and albumin transcytosis of renal tubular epithelial cells was promoted;(8)CAV1 knockdown had no effect on the expression of ANGPT2 in renal tubular epithelial cellsConclusion: In diabetic nephropathy,the increased expression of ANGPT2 in renal tubular epithelial cells increases the progression of albuminuria by inhibiting the transcytosis of albumin in renal tubular epithelial cells by promoting CAV1 phosphorylation.