Aucubin ameliorates diabetic kidney disease by restoring hGENCs autophagy through promoting phosphorylation of ATG4B protein

Abstract

Aucubin is a major component of Eucommia ulmoides, a traditional Chinese medicine used to treat diabetic kidney disease (DKD). However, the protective effect and mechanism of action of aucubin in DKD remains unclear. In this study, we found that aucubin decreased proteinuria in a DKD mouse model and alleviated human glomerular endothelial cells (hGENCs) damage caused by high glucose (HG). We labeled and quantified the total proteome and phosphorylated proteome of hGENCs using mass spectrometry, and the subsequent direct-data-independent acquisition analysis results showed that ATG4B protein phosphorylation is a prospective target of aucubin. We found that aucubin increased the phosphorylation level of ATG4B, restored autophagy, and weakened endothelial-mesenchymal transformation to protect against DKD in vivo and in vitro. Importantly, specific deletion of p-ATG4B aggravated HG-induced damage and eliminated the effects of aucubin-mediated protection in hGENCs. In conclusion, our study demonstrated that aucubin has protective effects against HG-induced hGENCs injury and in a DKD mouse model by upregulating p-ATG4B levels and restoring autophagy. This establishes p-ATG4B as a potential target for delaying DKD progression.

Keywords: ATG4B; Aucubin; autophagy; diabetic kidney disease; glomerular endothelial cells; phosphorylation.

Figure 1.

Effects of aucubin on physical and biochemical parameters in DKD mice. (A) The workflow of animal experiments. The changes of (B) body weight and (C) fasting glucose levels for each group at weeks −6, −4, −2, 0, 2, 4, 6, and 8. The changes of (D) fasting glucose, (E) 24hUTP, (F) BUN, and (G) SCr for each group at the end of this experiment. *p < .05; **p < .01. STZ: streptozotocin; DKD: diabetic kidney disease; 24UTP: 24-h urinary total protein; AU: aucubin.

Figure 2.

Effects of aucubin on structure of renal tissue in DKD mice. (A) HE: HE staining images, original magnifications of 200×. (B) PAS: PAS staining images, original magnifications of 400×. (C) TEM: transmission electron microscope images, the bar in the lower left represents 1 μm. The red arrows indicate glomerular basement membrane. DKD: diabetic kidney disease; AU: aucubin; HE: hematoxylin-eosin; PAS: periodic acid–Schiff; TEM: transmission electron microscope.

Figure 3.

Effects of different concentrations of aucubin on proliferation and apoptosis of hGENCs. (A) Cell proliferation was detected by CCK-8 assay. (B,D) Cell proliferation was detected by EdU staining. (B) Comparison of positive rate of EdU staining. (C,E) Apoptosis was detected by flow cytometry. *p < .05; **p < .01. FITC-H: fluoresceine isothiocyanate-Height; PE-H: phycoerythrin-Height.

Figure 4.

Protective effect of aucubin on high glucose-induced hGENCs injury. (A,C) Cell proliferation detected by EdU staining. (B, E) Apoptosis images were detected by flow cytometry. (D) Cell proliferation was detected by CCK-8 assay. (F) The protein expression levels detected by WB. *p < .05; **p < .01. DKD: diabetic kidney disease; AU: aucubin; HG: high glucose.

Figure 5.

Protective effect of aucubin on high glucose-induced hGENCs injury by promoting ATG4B protein phosphorylation and increasing autophagy. (A) Electrophoretic map of cells in each group. 1, 2, 3, 4, and 5 represent Con group, HG group, HG-AU-L group, HG-AU-M group, and HG-AU-H group, respectively. (B) Mass spectrogram of protein identification. Venn diagram of (C) phosphorylated proteins, (D) phosphorylated protein sites and (E) protein identification, AU1, AU2 and AU3 represent HG-AU-L group, HG-AU-M group, and HG-AU-H group, respectively. (F) Representative images of expression levels of p-ATG4B and ATG4B detected by WB. (G) Representative images of the expression levels of autophagy protein LC3, p62, ATG5, and ATG7 in each group detected by WB, and the statistical analysis result of the proteins expression levels. *p < .05; **p < .01. AU: aucubin; HG: high glucose.

Figure 6.

Protective effect of aucubin on DKD mice by promoting ATG4B protein phosphorylation and increasing autophagy. The expression levels of p-ATG4B proteins, ATG4B proteins, autophagy protein (LC3, p62, ATG5, and ATG7), apoptosis-related proteins (Bax, cleaved caspase3, and Bcl-2), EndMT marker proteins (CD31 and α-SMA), and nephrin protein in each group were detected by WB and the statistical analysis result of the proteins expression levels. *p < .05; **p < .01. DKD: diabetic kidney disease; AU: aucubin.

Figure 7.

The effect of specific reduction of ATG4B protein phosphorylation levels on related proteins expression in hGENCs under high glucose culture condition and high glucose + aucubin culture condition. (A) Three sh-ATG4B sequences. (B) ATG4B protein expression level was detected by WB after transfection of three sh-ATG4B plasmids into hGENCs. (C) The multiple proteins expression levels detected by WB. a, b, c, and d represent NC group, sh-ATG4B + OVC group, sh-ATG4B + ATG4B WT group, and sh-ATG4B + ATG4B S383A group, respectively. *p < .05; **p < .01. AU: aucubin; HG: high glucose.

Figure 8.

The effect of specific reduction of ATG4B protein phosphorylation levels on proliferation and apoptosis of hGENCs. (A) Cell proliferation was detected by CCK-8 assay. (B, D) The apoptosis were detected by flow cytometry. (C, E) Cell proliferation were detected by EdU staining. **p < .01. AU: aucubin; HG: high glucose; NC: negative control.