General Control of Amino Acid Synthesis 5-Like 1-Mediated Acetylation of Manganese Superoxide Dismutase Regulates Oxidative Stress in Diabetic Kidney Disease

Abstract

Diabetic kidney disease (DKD) is the major cause of end-stage renal disease (ESRD). In the past few decades, there has been a large amount of evidence to highlight the pivotal role of oxidative stress in the development and progression of DKD. However, the detailed molecular mechanisms are not fully elucidated. A new sight has been established that the mitochondrial acetyltransferase GCN5L1 participates in cellular redox homeostasis maintenance in DKD. Firstly, we found that the expression of GCN5L1 is significantly elevated both in human and mouse kidney tissues with DKD and in hyperglycemic renal tubular epithelial cells (TECs), while deletion of GCN5L1 could effectively ameliorate oxidative stress-induced renal injury in DKD. Furthermore, deletion of GCN5L1 could reduce MnSOD acetylation on lysine 68 and activate its activity, thereby scavenging excessive ROS and relieving oxidative stress-induced renal inflammation and fibrosis. In general, GCN5L1-mediated acetylation of MnSOD exacerbated oxidative stress-induced renal injury, suggesting that GCN5L1 might be a potential intervention target in DKD.

Figure 1

The expression of GCN5L1 is significantly elevated in kidney tissues from diabetic kidney disease patients and mouse models. (a) Immunohistochemistry staining for GCN5L1 expression in human renal biopsies. Scale bars = 50 μm. (b) Urine albumin-to-creatine ratio (UACR) in STZ-induced diabetic mice. Independent experiments were performed in triplicate. (c) Hematoxylin and eosin staining, Masson's trichrome staining, and periodic acid-Schiff staining in the kidneys from STZ-induced diabetic mice. Scale bars = 20 μm. (d) Immunohistochemistry staining for GCN5L1 expression in mouse kidney tissues. Scale bars = 50 μm. (e, f) Western blotting analysis of GCN5L1 protein expression in the kidneys from STZ-induced diabetic mice. Data are presented as the mean ± SD. n = 6; ^∗∗∗P < 0.001.

Figure 2

Reduction of GCN5L1 ameliorates renal tubulointerstitial injury and proteinuria in DKD mice. (a) Experimental design for STZ-induced diabetic GCN5L1 knockdown mice. (b, c) Western blotting analysis showing the expression of GCN5L1 in the kidneys from diabetic GCN5L1 knockdown mice. Data are presented as the mean ± SD. (d, e) Immunofluorescent staining and fluorescence intensity for GCN5L1 expression in kidney sections. Scale bars = 100 μm. (f) Urine albumin-to-creatinine ratio (UACR) in the kidneys from diabetic GCN5L1 knockdown mice. Independent experiments were performed in triplicate. (g, h) Immunohistochemistry staining and relative staining intensity for GCN5L1 expression in kidney sections. Scale bars = 100 μm. (i) Hematoxylin and eosin staining, Masson's trichrome staining, and periodic acid-Schiff staining in the kidneys from diabetic GCN5L1 knockdown mice. Scale bars = 20 μm. n = 6‐8; ^∗∗P < 0.01 and ^∗∗∗P < 0.001.

Figure 3

Downregulation of GCN5L1 activates MnSOD to scavenge ROS by reducing MnSOD acetylation on lysine 68 in TECs treated with high glucose. (a, b) Immunofluorescent staining and western blotting analysis of GCN5L1 protein expression in TECs exposed to high glucose. Scale bars = 50 μm. (c, d) Coimmunoprecipitation of MnSOD and GCN5L1 in TECs. (e) Interaction of MnSOD and GCN5L1 in TECs visualized by Duolink proximity ligation assay. (f, g) Western blotting analysis of the protein levels of MnSOD and Ac-MnSOD K68 in TECs exposed to high glucose following GCN5L1 knockdown. (h) MnSOD activity was assessed in TECs exposed to high glucose following GCN5L1 knockdown. Independent experiments were performed in triplicate. (i) Cellular ROS was detected by MitoSOX staining in GCN5L1 knockdown TECs treated with high glucose. Scale bars = 50 μm. Independent experiments were performed in triplicate. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 4

Suppression of GCN5L1 alleviates inflammation and EMT through the MnSOD/ROS pathway in TECs under high glucose. (a) Protein levels of NLRP3, caspase-1, IL18, and IL1beta were detected by western blotting in TECs exposed to high glucose following knockdown of GCN5L1. (b) Protein levels of NLRP3, caspase-1, IL18, and IL1beta were detected by western blotting in GCN5L1 overexpressed TECs treated with NAC. (c) Western blotting analysis of the protein levels of NLRP3, caspase-1, IL18, and IL1beta after cotransfected with MnSODK68-R mutant plasmid and GCN5L1 silencing siRNA. (d, e) Western blotting analysis and immunofluorescent staining for E-cadherin and α-SMA in TECs exposed to high glucose following knockdown of GCN5L1. Scale bars = 100 μm. (f) Protein levels of E-cadherin and α-SMA were detected by western blotting in GCN5L1 overexpressed TECs treated with NAC. (g) Western blotting analysis of the protein levels of E-cadherin and α-SMA after cotransfected with MnSODK68-R mutant plasmid and GCN5L1 silencing siRNA. Independent experiments were performed in triplicate.

Figure 5

Downregulation of GCN5L1 protects the kidney from hyperglycemia-induced EMT and inflammation via the MnSOD/ROS pathway in vivo. (a) Immunohistochemistry staining for acetylation level of MnSODK68 in the kidneys from diabetic GCN5L1 knockdown mice. Scale bars = 100 μm. (b) DHE fluorescence to detect ROS in the kidneys from diabetic GCN5L1 knockdown mice. Scale bars = 50 μm. (c) Immunohistochemistry staining for E-cadherin, α-SMA, and NLRP3 expression in the kidneys of mice. Scale bars = 100 μm. (d–h) Western blotting analysis showing the expressions of E-cad, α-SMA, and NLRP3 in the kidneys of mice. Data are presented as the mean ± SD. n = 6‐8; ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001.

Figure 6

STZ treatment increases GCN5L1 expression by reducing its ubiquitination. (a) RNA levels of GCN5L1 were detected by RT-qPCR in the mouse kidneys. (b) The ubiquitination level of GCN5L1 was detected by immunoprecipitation assay. n = 6; ns: not significant.