The Effect of Allograft Inflammatory Factor-1 on Inflammation, Oxidative Stress, and Autophagy via miR-34a/ATG4B Pathway in Diabetic Kidney Disease

Abstract

Increasing evidence suggests that disorders of inflammation, oxidative stress, and autophagy contribute to the pathogenesis of diabetic kidney disease (DKD). This study attempted to clarify the effect of allograft inflammatory factor-1 (AIF-1), miR-34a, and ATG4B on inflammation, oxidative stress, and autophagy in DKD both in vitro and in vivo experiments. In vivo, it was found that the levels of AIF-1, miR-34a, oxidative stress, and inflammatory factors were significantly increased in blood and urine samples of DKD patients and mouse models and correlated with the level of urinary protein. In vitro, it was also found that the expressions of AIF-1, miR-34a, ROS, and inflammatory factors were increased, while ATG4B and other autophagy related proteins were decreased in human renal glomerular endothelial cells (HRGECs) cultured with high concentration glucose medium (30 mmol/L). When AIF-1 gene was overexpressed, the levels of miR-34a, ROS, and inflammatory factors were significantly upregulated, and autophagy-related proteins such as ATG4B were downregulated, while downregulation of AIF-1 gene had the opposite effect. In addition, miR-34a inhibited the expression of ATG4B and autophagy-related proteins and increased the levels of ROS and inflammation. Furthermore, the result of luciferase reporter assay suggested that ATG4B was the target gene of miR-34a. When ATG4B gene was overexpressed, the level of autophagy was upregulated, and inflammatory factors were downregulated. Conversely, when ATG4B gene was inhibited, the level of autophagy was downregulated, and inflammatory factors were upregulated. Then, autophagy inducers inhibited the levels of inflammation and ROS, whereas autophagy inhibitors had the opposite function in HRGECs induced by glucose (30 mmol/L). In conclusion, the above data suggested that AIF-1 regulated the levels of inflammation, oxidative stress, and autophagy in HRGECs via miR-34a/ATG4B pathway to contribute to the pathogenesis of diabetic kidney disease.

Figure 1

Schematic diagram of possible mechanism of AIF-1 in oxidative stress, inflammation, and autophagy of glomerular endothelial cell induced by glucose.

Figure 2

The levels of AIF-1, miR-34a, ATG4B, autophagy, and inflammation in kidney of db/db mice. (a) The expression of AIF-1 in db/db mice was evaluated by immunohistochemical staining. (b) The expression of AIF-1, ATG4B, LC3II, p62, and NLRP3 protein in db/db mice was detected by western blot. Versus db/m mice group, ^∗P < 0.01. (c) The expression of miR-34a in db/db mice was detected by qPCR. Versus db/db mice group, ^∗P < 0.001. AIF-1: allograft inflammatory factor-1; miR-34a: microRNA 34a; ATG4B: autophagy related 4 homolog B; LC3II and p62: autophagy-associated protein; NLRP3: NOD-like receptor thermal protein domain associated protein 3.

Figure 3

The effect of AIF-1 or miR-34a on autophagy and inflammation in DKD mice. (a) The expression of AIF-1 in db/db mice treatment with miR-34a agonist or antagonist was evaluated by immunohistochemical staining. (b)The effect of miR-34a on the expression of AIF-1, ATG4B, LC3II, p62, and NLRP3 protein in db/db mice was detected by western blot. Versus db/m mouse group, ^∗P < 0.01. (c) The expression of ATG4B, LC3II, p62, and NLRP3 protein in AIF-1 transgenic mice was detected by western blot. Versus wild mice group, ^∗P < 0.001. (d) The expression of miR-34a in AIF-1 transgenic mice was detected by qPCR. Versus wild mice group, ^∗P < 0.001. AIF-1: allograft inflammatory factor-1; miR-34a: microRNA 34a; ATG4B: autophagy related 4 homolog B; LC3II and p62: autophagy-associated protein; NLRP3: NOD-like receptor thermal protein domain associated protein 3.

Figure 4

The effect of glucose on autophagy, inflammation, and ROS of HRGECs with a time- and concentration-dependent manner. (a–c) The effect of high concentration glucose (30 mmol/L) on AIF-1, miR-34a, ATG4B, LC3II, p62, NLRP3, and ROS level in HRGECs with a time-dependent manner. Versus 0 h group, ^∗P < 0.001. (d–f) The effect of glucose on AIF-1, miR-34a, ATG4B, LC3II, p62, NLRP3, and ROS level in HRGECs with a concentration-dependent manner for 48 h. All the relative levels of results were corrected by total protein. Versus 5.6 mmol/L glucose group, ^∗P < 0.01. HRGECs: human renal glomerular endothelial cells; AIF-1: allograft inflammatory factor-1; miR-34a: microRNA 34a; ATG4B: autophagy related 4 homolog B; LC3II and p62: autophagy-associated protein; NLRP3: NOD-like receptor thermal protein domain associated protein 3; ROS: reactive oxygen species.

Figure 5

The effect of AIF-1 or miR-34a on autophagy, inflammation, and ROS in HRGECs. (a, b) The effect of AIF-1 on the expression of miR-34a, ATG4B, LC3II, p62, and NLRP3 protein in HRGECs. Versus control group, ^∗P < 0.001. (c, d) The effect of miR-34a on the expression of ATG4B, LC3II, p62, NLRP3, and ROS in HRGECs. All the relative levels of results were corrected by total protein. Versus control group, ^∗P < 0.001. HRGECs: human renal glomerular endothelial cells; AIF-1: allograft inflammatory factor-1; miR-34a: microRNA 34a; ATG4B: autophagy related 4 homolog B; LC3II and p62: autophagy-associated protein; NLRP3: NOD-like receptor thermal protein domain associated protein 3; ROS: reactive oxygen species.

Figure 6

The effect of ATG4B on autophagy and inflammation in HRGECs cultured with high concentration glucose. (a) The relationship between ATG4B and miR-34a was analyzed via luciferase reporter assay. Versus ATG4B-MUT group, ^∗P < 0.05. (b) The effect of ATG4B on the level of NLRP3 and IL18 in HRGECs cultured in 30 mmol/L glucose for 48 h. Versus high glucose control group, ^∗P < 0.01. (c, d) The effect of ATG4B on the expression of LC3II and p62 in HRGECs cultured in 30 mmol/L glucose for 48 h. All the relative levels of results were corrected by total protein. Versus 30 mmol/L glucose control group, ^∗P < 0.001. HRGECs: human renal glomerular endothelial cells; miR-34a: microRNA 34a; ATG4B: autophagy related 4 homolog B; LC3II and p62: autophagy-associated protein; NLRP3: NOD-like receptor thermal protein domain associated protein 3.

Figure 7

The effect of autophagy on inflammation and ROS in HRGECs cultured with high concentration glucose. (a) The expression of AIF-1 protein was detected by western blot. Versus control group, ^∗P > 0.05. But compared with control group, the expression of NLRP3 protein was upregulated, ^∗P < 0.001. (b) The expression of miR-34a was detected by qPCR. All the relative levels of results were corrected by total protein. Versus control group, ^∗P > 0.05. (c) The effect of autophagy on ROS level in HRGECs cultured in 30 mmol/L glucose for 48 h. Versus high glucose control group, ^∗P < 0.001. HRGECs: human renal glomerular endothelial cells; AIF-1: allograft inflammatory factor-1; miR-34a: microRNA 34a; NLRP3: NOD-like receptor thermal protein domain associated protein 3; ROS: reactive oxygen species.