Effects and mechanism of high ARNO expression on the permeability of human renal glomerular endothelial cells under high glucose condition
10.11855/j.issn.0577-7402.2020.01.05
- Author:
Xiao-Long WANG
1
Author Information
1. Department of Nephrology, Xinqiao Hospital, Army Medical University
- Publication Type:Journal Article
- Keywords:
Arf6;
ARNO;
High glucose;
Human renal glomerular endothelial cells;
Permeability
- From:
Medical Journal of Chinese People's Liberation Army
2020;45(1):55-61
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To explore the effects and mechanism of high expression of ARNO (ARF nucleotide-binding-site opener) on the permeability of human renal glomerular endothelial cells (HRGECs) under high glucose conditions. Methods: HRGECs were cultured in vitro, and ARNO expression and endothelial permeability were detected in high glucose time gradient (15, 30, 45 and 60 min) and concentration gradient (10, 20, 30 and 40 mmol/L) experiments, respectively. Cell lines of HRGECs with ARNO and Arf6 gene silencing were obtained by infecting HRGECs with ARNO siRNA and Arf6 siRNA recombinant lentivirus vectors, respectively. The effects of ARNO and Arf6 gene silencing on endothelial permeability were observed. Results: In the time gradient experiments, compared with control group, ARNO expressions increased significantly in the groups with high glucose for 15, 30, 45 and 60 min (0.670±0.051, 0.960±0.106, 0.716±0.026, 0.531±0.030 vs. 0.242±0.029. P<0.05). Compared with high glucose 15 min group, ARNO expression increased in high glucose 30 min group (P<0.05); Compared with high glucose 30 min group, ARNO expression decreased in high glucose 45 min group (P<0.05); Compared with high glucose 45 min group, ARNO expression decreased in high glucose 60 min group (P<0.05). Compared with control group, endothelial permeability increased significantly in the groups with high glucose for 15, 30, 45 and 60 min (1.196±0.004, 1.399±0.012, 1.301±0.052, 1.184±0.030 vs. 1.000, P<0.05). Compared with high glucose 15 min group, endothelial permeability increased in high glucose 30 min group (P<0.05); Compared with high glucose 30 min group, endothelial permeability decreased in high glucose 45 min group (P<0.05); Compared with high glucose 45 min group, endothelial permeability decreased in high glucose 60 min group (P<0.05). In the concentration gradient experiments, compared with normal concentration glucose group, ARNO expressions increased significantly in the groups with high glucose of 20, 30 and 40 mmol/L (0.632±0.031, 0.927±0.041, 1.183±0.098 vs. 0.169±0.033, P<0.05), and the ARNO expression level was increased along with the increase of glucose concentration (P<0.05). Compared with normal concentration glucose group, endothelial permeability increased significantly in the groups with high glucose of 10, 20, 30 and 40 mmol/L (1.147±0.015, 1.237±0.023, 1.351±0.015, 1.444±0.019 vs. 1.000, P<0.05), and the endothelial permeability increased along with the increase of glucose concentration (P<0.05). After transfection with lentivirus, compared with normal concentration glucose untransfected group and normal concentration glucose empty vector group, ARNO mRNA transcription reduced significantly (0.255±0.056 vs. 1.000, 1.183±0.297), and ARNO protein expression also reduced significantly (0.088±0.005 vs. 0.246±0.011, 0.237±0.009) in normal concentration glucose ARNO siRNA group (P<0.05). In addition, compared with those groups, Arf6 mRNA transcription was reduced significantly (0.314±0.090 vs. 1.000, 1.140±0.236), and Arf6 protein expression was also reduced significantly (0.690±0.012 vs. 0.917±0.009, 0.919±0.009) in the normal concentration glucose Arf6 siRNA group (P<0.05). After silencing ARNO, compared with high-glucose untransfected group and high-glucose empty vector group, the ARNO protein expression was reduced significantly (0.572±0.021 vs. 0.915±0.005, 0.916±0.012), Arf6 activity was reduced significantly (0.263±0.007 vs. 0.484±0.014, 0.490±0.008), and endothelial permeability was also decreased (0.718±0.017 vs. 1.000, 0.978±0.040) in high-glucose ARNO siRNA group (P<0.05). Besides, after silencing Arf6, compared with high-glucose untransfected group and high-glucose empty vector group, Arf6 protein expression was reduced significantly (0.673±0.015 vs. 0.932±0.020, 0.899±0.022), and endothelial permeability was also decreased (0.768±0.050 vs. 1.000, 0.978±0.040) in high-glucose Arf6 siRNA group (P<0.05). Conclusion: High-glucose induced high permeability of HRGECs is related to the activation of ARNO/Arf6 signal, and inhibition of ARNO expression and Arf6 activity may inhibit the glomerular endothelial hyperpermeability in diabetic nephropathy, and is expected to be a new direction for the treatment of glomerular endothelial dysfunction in diabetic nephropathy.
