Reactive Oxygen Species-Scavenging Mesoporous Poly(tannic acid) Nanospheres Alleviate Acute Kidney Injury by Inhibiting Ferroptosis

Abstract

Acute kidney injury (AKI), predominantly associated with the excess production of endogenous ROS, is a serious renal dysfunction syndrome. Ferroptosis characterized by iron-dependent regulated cell death has significant involvement in AKI pathogenesis. As symptomatic treatment of AKI remains clinically limited, a new class of effective therapies has emerged, which is referred to as nanozyme. In our research, a natural mesoporous poly(tannic acid) nanosphere (referred to as PTA) was developed that can successfully mimic the activity of superoxide dismutase (SOD) by Mussel-inspired interface deposition strategy, for effective ROS scavenging and thus inhibition of ferroptosis to attenuate AKI. As anticipated, PTA mitigated oxidative stress and inhibited ferroptosis, as opposed to other modes of cell death such as pyroptosis or necrosis. Furthermore, PTA exhibited favorable biocompatibility and safeguarded the kidney against ferroptosis by enhancing the expression of SLC7a11/glutathione peroxidase 4(GPX4) and Nrf2/HO-1, while reducing the levels of ACSL4 protein in the ischemia and reperfusion injury (IRI)-induced AKI model. Moreover, PTA effectively suppressed aberrant expression of inflammatory factors. Overall, this study introduced antioxidative nanozymes in the form of mesoporous polyphenol nanospheres, showcasing exceptional therapeutic efficacy in addressing ROS-related diseases. This novel approach holds promise for clinical AKI treatment and broadens the scope of biomedical applications for nanozymes.

Keywords: acute kidney injury; ferroptosis; ischemia/reperfusion; mesoporous polyphenol nanospheres; oxidative stress.

Figure 1

Synthesis and characterization of the mesoporous poly(tannic acid) nanospheres (PTA) (A) SEM image and (B) cyro-TEM image of PTA. (C) Particle size distribution of PTA. (D) XPS spectrum and (E) corresponding C 1s spectrum of PTA. (F) Zeta potential of PTA. (G) ABTS·⁺ and (H) ·OH scavenging capacity of PTA (n = 3). (I) SOD inhibitor rate of PTA (n = 3).

Figure 2

Biosafety validation and renal targeting properties of PTA nanoparticles. (A) The cell viability of HK-2 cells incubated with PTA nanozymes at various concentrations for 24 h. (B–H) Blood biochemical analysis of mice at day 1, day 7, and day 14 following i.v. administration of PTA (5 mg/kg), and the results showed no significant difference. (I) H&E staining images of the major organs (heart, lung, liver, kidney, and spleen) obtained from mice following i.v. administration of PTA at a dosage of 5 mg/kg for 1 day, 7 days, and 14 days revealed no significant differences. Scale bar = 100 μm. (J) The distribution of PTA in major organs of mice at different times after intravenous injection. Data are presented as mean ± SD. NS means not significant, *P < 0.05, **P < 0.01.

Figure 3

Antioxidative performance and ROS scavenging ability of PTA. (A) PTA protects HK-2 cells from the oxidative stress caused by H/R (n = 3). (B) PTA protects HK-2 cells from the oxidative stress caused by H₂O₂ (250 μM H₂O₂) (n = 3). (C) Flow cytometry results of ROS of PTA in the H/R model. (D) In vitro TUNEL assay. Scale bars = 50 μm. (E) Flow cytometric analysis of apoptosis in HK-2 cells. (F) SOD, CAT (G), and GPX (H) activity in renal (n = 4). (I) ROS scavenging capability of PTA in vivo. Scale bars = 100 μm. Data has been presented as mean ± SD. NS implies nonsignificance, *P < 0.05, **P < 0.01.

Figure 4

PTA attenuated ferroptosis and mitochondrial injury induced by AKI. (A) Influence of PTA on cell proliferation of HK-2 cells after RSL-3 induced ferroptosis (n = 3). (B) Influence of PTA on cell proliferation of HK-2 cells after H/R (n = 3). (C) MDA levels in HK-2 cells under various conditions (n = 3). (D) Fe²⁺ levels in HK-2 cells under various conditions (n = 3). (E) Western blots of kidney Nrf2, HO-1, SLC7a11, GPX4, and ACSL4 proteins. (F) MDA level in renal tissue (n = 3). (G) Representative TEM micrographs of mitochondrial injury under indicated treatment (autophagic vesicles (AP), autophagic lysosomes (ASS), basement membrane (BM), lipid droplets (LD), mitochondria (M), nucleus (N), nucleolus (Nu), rough endoplasmic reticulum (RER), plasma membrane inner folds (PMI)). The scale bar in the top panel of (G) is set to 10 μm, while the scale bar in the bottom panel is set to 5 μm. (H) 4-Hydroxynonenal (4-HNE) staining in mice with I/R after PTA treatment. Scale bars = 100 μm. Data has been presented as mean ± SD. NS implies nonsignificance, *P < 0.05, **P < 0.01.

Figure 5

Attenuation of IRI induced-AKI in vivo. (A, C) HE and PAS staining after PTA treatment on IRI (Enlarged red frame shows characteristic renal tubular injury). Scale bars = 100 μm. (B, D) tubule injury score based on HE and PAS staining (n = 3). (E) TUNEL staining (green) showing TECs apoptosis. Scale bars = 100 μm. (F) Quantitative analysis of TUNEL staining (n = 3). (G) Quantitative analysis of Ki-67 immunostaining (n = 3). (H) Representative images show Ki-67 immunofluorescence staining (red) detected in renal tubular epithelial cells. Scale bars = 100 μm. (I, J) Graph showing the levels of urea nitrogen and serum creatine in mice treated with PTA following IRI (n = 3). Data are presented as mean ± SD. NS means not significant, *P < 0.05, **P < 0.01.

Figure 6

PTA attenuates inflammatory cell infiltration and suppresses the expression of inflammatory factor induced by AKI. (A) Representative images show Ly6G+ neutrophil (green) and F4/80+ macrophage (red) in kidney sections, Scale bar = 100 μm. (B–E) Renal mRNA levels of IL-1β, TNFα, IL-6, and IL-10 were assayed by qRT-PCR and quantified as fold changes. (F–H) IL-1β, TNFα, and IL-6 protein levels were assayed by ELISA. (I) Levels of M1 and M2 macrophages are measured by flow cytometry; CD86 for M1 type and CD206 (J) for M2 type. Data has been presented as mean ± SD. NS implies nonsignificance, *P < 0.05, **P < 0.01 compared with IRI.