Adaptive Antioxidant Nanomedicines Inhibit Ferroptosis in Renal Tubular Epithelial Cells to Alleviate Diabetic Kidney Disease

Abstract

Diabetic kidney disease (DKD) imposes a heavy medical burden worldwide due to the lack of effective treatment. High levels of mtROS and mitochondrial damage in the renal tubules are the initiating and core factors driving the progression of DKD. However, the effectiveness of current antioxidant drugs is greatly limited, mainly due to the difficulty of simultaneously breaching the glomerular barrier and targeting tubular mitochondria, as well as their limited ability to sustain treatment of chronic DKD. Here, this study reports a Se embedded adaptive antioxidant nanodrug (AAN) with negative surface charge and high mitochondrial targeting that can pass through the renal tubules and be highly enriched in the affected renal tubular mitochondria in DKD. AAN can eliminate mtROS to release soluble Se, which is then converted into the key bioactive enzymes -GPX4, effectively inhibiting ferroptosis and protecting mitochondria by exerting adaptive antioxidant effects. In the DKD mouse model, AAN treatment can effectively restore renal function, and the therapeutic effect at a dose of 10 mg kg^-1 every 4 days is significantly better than Metformin administered at a dose of 200 mg kg^-1 per day. In conclusion, this study provides a promising strategy to enhance the effects of antioxidant therapy to break the pathological barriers in DKD treatment.

Keywords: GPX4; adaptive antioxidant nanodrug; diabetic kidney disease; ferroptosis; mtROS.

Scheme 1

Synthesis of AAN, mitochondrial targeting, and DKD genesis. A) In vitro antioxidation and mitochondrial targeting of AAN. B) AAN is slowly released and becomes the raw material for GPX4 synthesis. C) AAN enters renal tubules with its very small particle size, targets mitochondria to eliminate ROS, and then releases Se to synthesize GPX4 in vivo, which further eliminates ROS, inhibits lipid peroxidation, and thus alleviates ferroptosis. DKD is accompanied by inflammation and fibrosis, which interacts with ferroptosis, AAN can also alleviate the occurrence of inflammation and fibrosis at the same time.

Figure 1

Synthesis and characterization of AAN. A) TEM image of AAN, Scale bar: 10 nm. B‐F) XPS spectrum of AAN (B), Se3d(C), C1s(D), O1s(E), N1s(F) in AAN. G) Schematic diagram of the inheritance of the selenocysteine‐derived aminoacetic acid group by AAN. H) AAN docks with the mitochondrial outer membrane protein TOM20 molecule. I) Mitotracker‐stained with AAN‐FITC colocalization and Pearson coefficient in HK‐2 cells, EX/EM = 579/599nm. J) Zeta potential distribution intensity of AAN. K‐L) Elimination of O₂ ^∙−(K), ∙OH (L) by different concentrations of AAN. M) The ability of AAN to eliminate different concentrations of H₂O₂. N) Release degree of Se in AAN with or without H₂O₂.

Figure 2

Distribution of AAN in vivo. A) C57BL/6J construction of diabetic kidney disease process and administration of AAN‐FITC process. B) Distribution of AAN‐FITC in the renal cortex and medulla of C57BL/6J mice, Scale bar: 5 mm. C‐D) Representative fluorescence images of different organs (C), and quantitative analysis (D) in DKD mice 9 h after intravenous injection of AAN, Scale bar: 5 mm. E‐G) Representative fluorescence images of kidneys in DKD mice (E), Control mice (F) and quantitative analysis (G) at various time points (0‐96 h) after intravenous injection of AAN‐FITC. H) TEM image of glomerulus after intravenous injection of AAN, Scale bar: 2 µm and 200 nm. *P < 0.05, **P < 0.01, ***P < 0.001, ns means P > 0.05.

Figure 3

The therapeutic effect of AAN in DKD mice. A) Schematic illustration of the establishment and treatment schedule of DKD mice. B‐G) 24h urinary albumin excretion rates (B), blood creatinine (C), blood urea nitrogen (D), blood cholesterol (E), urinary creatinine/albumin ratio (F), and renal index (G) in different groups of mice. H) Western Blotting of KIM‐1 and NGAL in kidney tissues of different groups of mice. (I‐L) HE‐staining I), PAS glycogen‐staining J), TEM images of renal glomerular K), TEM images of renal tubular (L) in different groups of mice. Data represent means ± S.D. from three or six independent replicates. ^### P < 0.01 vs Control group; **P < 0.01, ***P < 0.001 vs DKD group.

Figure 4

AAN inhibits tubular ferroptosis in DKD. A) Schematic diagram of ferroptosis mechanism. B‐C) representative images of DHE (B) and quantitative analysis (C), Scale bar: 50 µm. D) the iron content in the kidney tissue of different mice. E‐H) Western blot analysis of ferroptosis‐related protein expression levels in the kidney tissue of different mice (E) and quantitative analysis of FPN (F), TFR1 (G), GPX4 (H). I) MDA content in the kidney tissue of different mice. Data represent means ± S.D. from three independent replicates. ^## P < 0.01, ^### P < 0.001 vs Control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs DKD group. ns means P > 0.05.

Figure 5

AAN inhibits high glucose‐induced ferroptosis in HK‐2 cells. A) Cell viability of high glucose‐induced HK‐2 cells treated with different concentrations of AAN and positive Control Fer‐1. B‐C) Representative images of FeRhoNox‐1(FeR)‐staining in high glucose‐induced HK‐2 cells treated with AAN and positive Control Fer‐1 (B), and quantitative analysis (C), Scale bar: 20 µm, EX/EM = 545/585nm. D‐G) Western blot analysis of ferroptosis‐related protein expression levels in HK‐2 cells(D) and quantitative analysis of FPN (E), TFR1 (F) and GPX4 (G). H‐J) MDA contents (H), GSH (I), GSSG/GSH ratio (J) in HK‐2 cells. K‐L) Representative images of DCFH‐DA‐staining in high glucose‐induced HK‐2 cell treated with AAN and positive Control Fer‐1 (K), and quantitative analysis (L), Scale bar: 100 µm, EX/EM = 488/525 nm. M‐O) Western blot analysis of kidney injury‐related protein expression levels in high glucose‐induced HK‐2 cells treated with different concentrations of AAN and positive Control Fer‐1 (M) and quantitative analysis of NGAL (N) and KIM‐1(O). Data represent means ± S.D. from three independent replicates. ^##P < 0.01, ^###P < 0.001 vs Control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs HG group. ns means P > 0.05.

Figure 6

Mitochondrial protection of AAN. A‐B) Representative images of MitoSox‐stained in high glucose‐induced HK‐2 cell treat with AAN and positive Control Fer‐1 (A), and quantitative analysis (B), Scale bar: 15 µm, EX/EM = 396/610 nm. C‐D) Representative images of JC‐1‐stained in high glucose‐induced HK‐2 cell treated with AAN and positive Control Fer‐1 (C), and quantitative analysis (D), Scale bar: 15 µm, EX/EM = 490/530 nm. E) ATP level in high glucose‐induced HK‐2 cell treat with different concentrations of AAN. Data represent means ± S.D. from three independent replicates. ^###P < 0.001 vs Control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs HG group.

Figure 7

AAN inhibits inflammation and fibrosis in DKD. A‐B) F4/80 immunohistochemical staining images (A) and F4/80 positive cells rate (B) of mice in different groups, Scale bar: 50 µm. C‐E) Enzyme‐linked immunosorbent assays of TNF‐α (C), IL‐6 (D) and IL‐1β (E) in renal tissue homogenates of mice in different groups. F) Western blot analysis of INOS and COX‐2 expression levels in LPS‐induced HK‐2 cells treated with different concentrations of AAN. G‐H) Representative images of Masson‐stained in kidney of different mice (G), and Collagen fiber area statistics (H), Scale bar: 50 µm. I‐N) Western blot analysis of fibrosis‐related protein expression levels in kidney (I), and quantitative analysis of α‐SMA (J), Vimentin (K), E‐cadherin (L), Collagen I (M) and Fibronectin (N). O) Representative images of Collagen I‐stained in kidney of different mice, Scale bar: 50 µm. Data represent means ± S.D. from three independent replicates. ^###P < 0.001 vs Control group; *P < 0.05, **P < 0.01, ***P < 0.001 vs HG group. ns means P > 0.05.

Figure 8

The excellent biocompatibility of AAN. A) Schematic illustration of short‐term and long‐term treatment protocol for biosafety evaluation of AAN. B) Representative H&E‐stained images of major organs from C57BL/6J mice after short‐term (1 day) and long‐term (28 days) administration of AAN or 1× PBS. C‐D) Liver function index of short‐term (C) treatment and long‐term treatment (D) in different groups of mice. E‐F) Renal function index of short‐term (E) treatment and long‐term treatment (F) in different groups of mice. G‐H) blood routine examination of short‐term (G) treatment and long‐term treatment (H) in different groups of mice.