Angiopoietin2-mediated caveolin1 phosphorylation regulating transcytosis of renal tubular epithelial cell contributes to the occurrence of albuminuria under high glucose exposure

Figures

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  Fig. 1. High glucose reduced albumin transcytosis in HK-2 cell. A Representative IF (immunoflurescence) images of albumin endocytosis. B FITC intensity in upper chamber of Transwell determined by Microplate System (n ≥ 3). *P < 0.05 vs. NG (normal glucose). C FITC intensity in the lower chamber of the Transwell detected by Microplate System (n ≥ 3). *P < 0.05 vs. NG

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  Fig. 2. ANGPT2 upregulation during HG exposure. A Representative immunoblot of ANGPT2, STX4 and DNM2. B Densitometric analysis of ANGPT2 immunoblot signals (n ≥ 3). *P < 0.05 vs. NG (normal glucose). C Densitometric analysis of STX4 immunoblot signals (n ≥ 3). No statistical significance vs. NG. D Densitometric analysis of DNM2 immunoblot signals (n ≥ 3). No statistical significance vs. NG. E Representative immunohistochemical (IHC) staining of ANGPT2 in diabetic kidney. F Statistical data of IHC staining of ANGPT2 (n ≥ 5). **P < 0.01 vs. Control

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  Fig. 3. CAV1 phosphorylation was induced in HG-cultured HK-2 in vitro and diabetic kidney in vivo. A Representative immunohistochemical (IHC) staining images of P-CAV1. B Statistical data of IHC staining of P-CAV1 (n ≥ 5). ** P < 0.01 vs. Control. C Representative immunoblot result of CAV1 and P-CAV1. D Densitometric analysis of phosphorylated-CAV1/ CAV1 ratio (n ≥ 3). *P < 0.05 vs. NG. E Representative immunofluorescence (IF) images of CAV1 and absorbed FITC-BSA in HK-2 after cultured in high glucose (HG) medium

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  Fig. 4. ANGPT2 downregulation promoted albumin transcytosis. HK-2 was transfected with shANGPT2, then the effect on albumin transcytosis was detected. A Representative immunoblot of ANGPT2 with shANGPT2 for 72 h. B Densitometric analysis of ANGPT2 immunoblot signals (n ≥ 3). *P < 0.05 vs. scra (scramble shRNA). C Statistical IF signals in the upper chambers of Transwell (n ≥ 3). **P < 0.01 vs. NG + scra, ##P < 0.01 vs. NG + scra, &P < 0.05 vs. HG + scra. D Statistical immunofluorescence (IF) signals in in the lower chambers of Transwell (n ≥ 3). **P < 0.01 vs. NG + scra, ## P < 0.01 vs. NG + scra, &&P < 0.01 vs. HG + scra

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  Fig. 5. Inhibition of CAV1 facilitated albumin transcytosis in HK-2 cell. A Immunoblot of CAV1 and P-CAV1 in HK-2 after transfection with siCAV1 for 72 h. B Densitometric analysis of P-CAV1/CAV1 immunoblot signals (n ≥ 3). *P < 0.05 vs. NC. C Statistical FITC intensity in the upper chamber of Transwell (n ≥ 3). ##P < 0.01 vs. NG + NC, ns vs. NG + NC, &&P < 0.01 vs. HG + NC. D FITC intensity in the lower chamber of Transwell (n ≥ 3). ##P < 0.01 vs. NG + NC, **P < 0.01 vs. NG + NC, &&P < 0.01 vs. HG + NC. HG: high glucose; NC: normal control; NG: normal glucose

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  Fig. 6. ANGPT2 downregulation suppressed CAV1 phosphorylation. A Immunoblot of P-CAV1 and CAV after transfection with shANGPT2 is shown in the figure. B Densitometric analysis ratio of P-CAV1/GAPDH (n ≥ 3). **P < 0.01 vs. NG + scra, #P < 0.05 vs. NG + scra, &P < 0.05 vs. HG + scra. C HK-2 was transfected with shANGPT2, the immunoblot of TIE2 and Src. D Densitometric analysis of TIE2 and Src (n ≥ 3). No statistical significance was observed. E Densitometric analysis of Src (n ≥ 3). No statistical significance was observed. F Immunoblot of P-CAV1 and ANGPT2 after transfection with siCAV1. G. Densitometric analysis ratio of P-CAV1/GAPDH (n ≥ 3). **P < 0.01 vs. NG + NC, ##P < 0.01 vs. NG + NC, &&P < 0.01 vs. HG + NC. H Densitometric analysis of ANGPT2/GAPDH (n ≥ 3). **P < 0.01 vs. NG + NC, ns vs. NG + NC, HG + NC. HG: high glucose; NC: normal control; NG: normal glucose

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  Fig. 7. Schematic model for ANGPT2-mediated CAV1 phosphorylation in the inhibition of albumin transcytosis. Here we depict the model for albumin transcytosis under the regulation of ANGPT2 and CAV1. A ANGPT2/P- CAV1 signaling pathways involved in albumin transcytosis in HK-2 cells in HG. B Summary chart of this research findings. Following HG exposure, ANGPT2 is upregulated, which lead to increased CAV1 phosphorylation in tubular cells. CAV1 phosphorylation inhibited the albumin transcytosis. Angiopoietin2-mediated Caveolin1 phosphorylation blocked albumin transcytosis, which participates in the occurrence of diabetic kidney disease (DKD)-related albuminuria