A-Lipoic Acid Alleviates Folic Acid-Induced Renal Damage Through Inhibition of Ferroptosis

Abstract

Folic acid (FA)-induced acute kidney injury (AKI) is characterized by the disturbance of redox homeostasis, resulting in massive tubular necrosis and inflammation. Α-lipoic acid (LA), as an antioxidant, has been reported to play an important role in renal protection, but the underlying mechanism remains poorly explored. The aim of this study is to investigate the protective effect of LA on FA-induced renal damage. Our findings showed that LA could ameliorate renal dysfunction and histopathologic damage induced by FA overdose injection. Moreover, FA injection induced severe inflammation, indicated by increased release of pro-inflammatory cytokines tumor necrosis factor (TNF)-α and IL-1β, as well as infiltration of macrophage, which can be alleviated by LA supplementation. In addition, LA not only reduced the cellular iron overload by upregulating the expressions of Ferritin and ferroportin (FPN), but also mitigated reactive oxygen species (ROS) accumulation and lipid peroxidation by increasing the levels of antioxidant glutathione (GSH) and glutathione peroxidase-4 (GPX4). More importantly, we found that LA supplementation could reduce the number of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive tubular cells caused by FA, indicating that the tubular cell death mediated by ferroptosis may be inhibited. Further study demonstrated that LA supplementation could reverse the decreased expression of cystine/glutamate antiporter xCT (SLC7A11), which mediated GSH synthesis. What is more, mechanistic study indicated that p53 activation was involved in the inhibitory effect of SLC7A11 induced by FA administration, which could be suppressed by LA supplementation. Taken together, our findings indicated that LA played the protective effect on FA-induced renal damage mainly by inhibiting ferroptosis.

Keywords: A-lipoic acid; ferroptosis; folic acid; p53; renal damage.

Figure 1

The protective effects of lipoic acid (LA) supplementation on folic acid (FA)-induced acute kidney injury (AKI). FA group mice are given an intraperitoneal injection of FA at 250mg/kg body weight once, without or with LA-L (50mg/kg body weight) or LA-H (100mg/kg body weight) supplementation. (A) Representative images of hematoxylin and eosin (H&E) staining, showing the histological changes in FA-induced AKI and the LA-treated groups. Bar=50μm. Asterisks for dilated tubule; arrows for necrotic tubular epithelial cells. (B) Renal damage is assessed by Periodic acid-Schiff (PAS) staining. Bar=50μm. Asterisks for tubular ectasia and arrows for tubules with cells in necrosis and cellular debris. (C) Renal tubular injury scores on the basis of H&E staining. The renal function is evaluated by (D) serum creatinine and (E) serum blood urea nitrogen (BUN) levels. For the FA group vs. the control group, *indicates p<0.05, and ^**indicates p<0.01. For the LA-treated groups vs. the FA group, ^#indicates p<0.05, and ^##indicates p<0.01.

Figure 2

The effect of LA supplementation on acute tubular damage caused by FA injection. Mice are treated as described in Figure 1. (A) Representative images of immunohistochemical (IHC) staining for KIM-1. Bar=50μm. (B) Renal tubular damage marker, KIM-1 expression, as determined by western blotting. (C) Semi-quantitative assessments of KIM-1. (D) The expression of KIM-1 gene, as determined by relative mRNA expression. For the FA group vs. the control group, *indicates p<0.05, and **indicates p<0.01. For the LA-treated groups vs. the FA group, ^#indicates p<0.05, and ^##indicates p<0.01.

Figure 3

Lipoic acid supplementation reduces release of TNFα and IL-1β, as well as macrophage infiltration caused by FA injection. Mice are treated as described in Figure 1. (A) Representative images of IHC staining for TNF-α (brown), IL-1β (brown), and macrophages (brown). Bar=50μm. Black arrow heads for F4/80-positive interstitial macrophages. (B) The expression of inflammatory marker TNF-α, as determined by western blotting. (C) The expression of inflammatory marker IL-1β, as determined by western blotting. (D) Semi-quantitative assessments of TNF-α. (E) Semi-quantitative assessments of IL-1β. (F) The number of macrophages per high-power field (Hpf). For the FA group vs. the control group, *indicates p<0.05, and **indicates p<0.01. For the LA-treated groups vs. the FA group, ^#indicates p<0.05, and ^##indicates p<0.01.

Figure 4

Lipoic acid supplementation alleviates iron accumulation caused by FA injection. Mice are treated as described in Figure 1. (A) Representative images of Perl’s staining. Arrows for iron accumulation. Bar=50μm. (B) Quantitative assessment of iron staining. (C) Iron content of kidney tissue. (D) Representative images of IHC staining for Ferritin (brown). (E) The expression of iron storage marker Ferritin, as determined by western blotting. (F) The expression of iron exporter marker ferroportin (FPN), as determined by western blotting. (G) Semi-quantitative assessments of Ferritin. (H) Semi-quantitative assessments of FPN. For the FA group vs. the control group, ^*indicates p<0.05, and ^**indicates p<0.01. For the LA-treated groups vs. the FA group, ^#indicates p<0.05, and ^##indicates p<0.01.

Figure 5

Lipoic acid supplementation decreases oxidative stress caused by FA injection. Mice are treated as described in Figure 1. (A) Reactive oxygen species (ROS) level in the kidney. (B) Representative images of IHC staining for 4-HNE (brown). Bar=50μm. (C) The expression of lipid peroxidation marker 4-HNE, as determined by western blotting. (D) Semi-quantitative assessments of 4-HNE. For the FA group vs. the control group, ^*indicates p<0.05, and ^**indicates p<0.01. For the LA-treated groups vs. the FA group, ^#indicates p<0.05, and ^##indicates p<0.01.

Figure 6

Lipoic acid supplementation increases FA-induced reduction in antioxidants. Mice are treated as described in Figure 1. (A) Representative images of IHC staining for GPX4 (brown) and xCT (brown). Bar=50μm. (B) The expression of anti-oxidative enzyme marker GPX4, as determined by western blotting. (C) The expression of cystine/glutamate transporter xCT, as determined by western blotting. (D) Semi-quantitative assessments of GPX4. (E) Semi-quantitative assessments of xCT. (F) The levels of antioxidant glutathione (GSH) content. For the FA group vs. the control group, ^*indicates p<0.05, and ^**indicates p<0.01. For the LA-treated groups vs. the FA group, ^#indicates p<0.05, and ^##indicates p<0.01.

Figure 7

Lipoic acid supplementation decreases ferroptosis-mediated tubular cells death caused by FA injection. Mice are treated as described in Figure 1. (A) Representative images by TUNEL staining, showing the ferroptosis-mediated tubular cell death. White arrowheads for TUNEL-positive tubular cells (green) on kidney tissue sections; nuclei are labeled with DAPI (blue). Bar=50μm. (B) The number of TUNEL-positive nuclei per high-power field (Hpf). For the FA group vs. the control group, ^*indicates p<0.05, and ^**indicates p<0.01. For the LA-treated groups vs. the FA group, ^#indicates p<0.05, and ^##indicates p<0.01.

Figure 8

Lipoic acid supplementation inhibits the activation of p53 caused by FA injection. Mice are treated as described in Figure 1. (A) The expression for p53, as determined by western blotting. (B) Semi-quantitative assessments of p53. (C) The expression of p53 gene, as determined by relative mRNA expression. (D) Representative images of IHC staining for p53 activation. Bar=50μm. Black arrow heads for p53 nuclear staining; black arrows for folic acid crystals in tubular lumen. For the FA group vs. the control group, ^*indicates p<0.05, and **indicates p<0.01. For the LA-treated groups vs. the FA group, ^#indicates p<0.05, ^##indicates p<0.01.