Charge-dependent mitochondrial targeting of cerium-doped polypyrrole nanoparticles to injured renal tubules for acute kidney injury therapy

Abstract

Acute kidney injury (AKI) is a critical medical condition with limited treatment options and frequently progresses to chronic kidney disease (CKD). This study introduces a novel nanotherapeutic approach based on positively charged cerium-doped polypyrrole nanoparticles [(+)CePPy] for AKI treatment. The positive surface charge of these nanoparticles enhances their cellular uptake and mitochondrial targeting in proximal tubular epithelial cells (PTECs), which play a central role in the pathophysiology of AKI. Comprehensive in vitro and in vivo evaluations demonstrate that (+)CePPy effectively scavenges reactive oxygen species (ROS), ameliorates mitochondrial dysfunction, attenuates apoptosis, and prevents AKI-to-CKD progression. The nanoparticles exhibit excellent biocompatibility and undergo efficient renal clearance within 14 days. Mechanistically, the renoprotective effects involve both direct elimination of ROS and the reinforcement of endogenous antioxidant defenses through upregulation of the Mpv17 mitochondrial membrane protein-like (Mpv17l) pathway. Furthermore, we report that early endosome antigen 1 (EEA1) upregulation mediates the enhanced renal targeting of (+)CePPy under AKI conditions. This research highlights the potential of charge-modulated nanotherapeutics as a translatable strategy for renal protection and presents a promising approach for AKI treatment.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-025-03967-x.

Introduction

Acute kidney injury (AKI) is a severe medical condition characterized by a sudden decline in kidney function, resulting in significant morbidity and mortality [1]. Although current treatments, such as renal replacement therapy and nutritional supplementation, provide supportive care, effective pharmacological therapies remain limited [2]. This underscores the urgent need for novel therapeutic approaches to treat AKI.

Oxidative stress plays a central role in the pathogenesis of AKI [3–5]. Proximal tubular epithelial cells (PTECs), which are rich in mitochondria and have high oxygen demand, are particularly susceptible to oxidative damage [3, 6]. During ischemia‒reperfusion injury (IRI), cellular redox homeostasis is severely disrupted, leading to the excessive production of reactive oxygen species (ROS) within renal tissues. These ROS, including superoxide anion radicals (O₂⁻), hydrogen peroxide (H₂O₂), highly reactive hydroxyl radicals (•OH), and other free radicals, collectively contribute to kidney injury. Mitochondria, which generate nearly 90% of total cellular ROS [6], are the primary source of these reactive species. Excessive ROS production triggers a cascade of pathological events in IRI-induced AKI (IRI-AKI), including mitochondrial dysfunction, energy depletion, and tubular cell death. As a result, antioxidant-based therapies have emerged as promising strategies for treating AKI.

Advancements in nanomedicine have enabled the development of innovative treatments for diseases associated with ROS through the use of functional nanoparticles [7]. For instance, cerium-based (Ce-based) nanomedicines, which have tunable ROS-scavenging capabilities, have been widely explored in biomedicine, including in the treatment of neurological disorders [8] and other ROS-related diseases [9]. This is due to their ability to interconvert between Ce³⁺/Ce⁴⁺ ions during redox reactions, demonstrating excellent ROS-scavenging activity.

Beyond their intrinsic properties, the surface characteristics of engineered nanoparticles, particularly charge heterogeneity, can influence their therapeutic efficacy. Recent studies have shown that compared with their anionic or neutral counterparts, cationic nanoparticles exhibit superior cell penetration and therapeutic outcomes in AKI therapy [10]. This enhanced uptake is primarily attributed to the electrostatic interaction between positively charged nanoparticles and the negatively charged plasma membranes of cells [11, 12]. Furthermore, the negatively charged glomerular basement membrane supports the hypothesis that glomerular filtration is charge-dependent [13]. Notably, compared with their negatively charged counterparts, positively charged dextran nanoparticles exhibit more than tenfold greater glomerular retention [14]. These findings highlight the potential of positively charged antioxidant nanoparticles as a promising platform for AKI treatment.

Polypyrrole (PPy), a positively charged conductive polymer, has garnered significant attention in biomedical research because of its excellent biocompatibility, tunable particle size, and versatile doping capacity, which enhances its catalytic activity [15–17]. In this study, we developed a green one-step synthesis method for PPy preparation by using Ce(SO₄)₂ as an oxidizing catalyst. The resulting Ce-doped PPy nanoparticles with a positive surface charge, referred to as (+)CePPy, exhibited remarkable antioxidant properties. Additionally, we prepared negatively charged particles, termed (-)CePPy, through PEGylation as a control group to investigate the influence of surface charge on nanoparticle uptake and therapeutic efficacy in AKI treatment.

The primary objectives of this study are to: (1) elucidate the role of nanoparticle surface charge in determining cellular uptake efficiency and biodistribution; (2) characterize the renal-specific uptake mechanisms that underlie the selective accumulation of (+)CePPy in injured PTECs; (3) explore the renoprotective effects of CePPy and the mechanisms involved in AKI progression; and (4) comprehensively assess the systemic biosafety and biocompatibility of (+)CePPy using integrated in vivo and in vitro models.

Our findings demonstrate that compared with their negatively charged counterparts, the positive charge significantly enhances the cellular uptake efficiency and enables the mitochondrial-targeted delivery of CePPy nanoparticles. (+)CePPy preferentially accumulates in injured PTECs, a process mediated by the upregulation of early endosome antigen 1 (EEA1), which enhances endocytic efficiency. This renal accumulation occurs via transcytosis, in which the nanoparticles are transported from the peritubular capillaries to the basal compartment of the tubular epithelium. In AKI models, (+)CePPy exhibits remarkable antioxidative protection by preserving mitochondrial homeostasis while simultaneously demonstrating excellent biocompatibility. Mitochondrial homeostasis is maintained through two key synergistic mechanisms: (1) efficient scavenging of ROS by mimicking superoxide dismutase (SOD) and catalase (CAT) activities and (2) enhancement of endogenous mitochondrial antioxidant defenses via activation of the Mpv17 mitochondrial membrane protein-like (Mpv17l) pathway. These dual actions effectively mitigate AKI and prevent its progression to chronic kidney disease (CKD), positioning (+)CePPy as a promising charge-modulated therapeutic strategy.

Materials and methods

Synthesis of (+)CePPy and (-)CePPy

To prepare a 10% PVA solution, 1 g of PVA was dissolved in 10 mL of deionized water and stirred at 92 °C for 1 h. A mixed solution was prepared by combining 100 µL pyrrole with the PVA solution and stirring at room temperature for 1 h. Subsequently, a freshly prepared Ce(SO₄)₂ aqueous solution (200 mg in 10 mL) was slowly added dropwise to the mixed solution and stirred for 20 h. The resulting suspension was dialyzed for 24 h to remove any unreacted materials. After dialysis, the (+)CePPy was collected by ultrafiltration and stored at 4 °C.

To obtain (-)CePPy, (+)CePPy was PEGylated to reverse its surface charge. Specifically, (+)CePPy was redispersed in DMSO and mixed with DMSO containing DSPE-PEG2000 (100 mg/mL). This mixture was then added to ten times its volume of DI water under vigorous stirring for 5 min. The resulting (-)CePPy was collected via ultrafiltration and stored at 4 °C.

Characterization of differently charged CePPy

The morphology and dimensions of the nanoparticles were characterized via transmission electron microscopy (TEM, HT7800-120 kV, Hitachi, Japan). Dynamic light scattering (DLS, 90Plus PALS, Brookhaven, UK) was employed to assess the particle size distribution and zeta potential. The content of Ce in the nanoparticles was measured via inductively coupled plasma‒optical emission spectrometry (ICP‒OES).

The ROS scavenging ability of differently charged CePPy

SOD-mimetic activity was assessed by dispersing (+)CePPy or (-)CePPy in deionized water at concentrations of 0, 10, 20, 40, 80, and 160 µg/mL. The superoxide scavenging ability was determined via a total SOD assay kit with WST-8.

For the CAT-like activity assay, nanoparticles at the aforementioned concentrations were analyzed via a catalase assay kit to evaluate their H₂O₂ scavenging ability.

To assess the •OH scavenging ability, methylene blue (MB) was used as a probe. An •OH-generating system was prepared by mixing H₂O₂ solution (1 mM) with FeCl₂ aqueous solution (0.1 mM). Various concentrations of (+)CePPy or (-)CePPy were added to this mixture and stirred for 10 min. MB (7 µg/mL) was then introduced to evaluate the changes in the •OH content via a UV‒vis spectrometer (LAMBDA365, PerkinElmer, USA).

The 2,2’-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS) method was used to assess the free radical scavenging ability of the CePPy nanoparticles. After dispersing the nanoparticles in DI water at various concentrations, the total antioxidant capacity was evaluated using an antioxidant assay kit based on the ABTS method.

Preparation and treatment of a renal IRI mouse model

Male wild-type C57BL/6 mice, aged 7–8 weeks, were maintained under specific pathogen-free conditions at the Animal Center of the Chinese PLA General Hospital. To assess the protective effects of CePPy against AKI, we employed two IRI models: a 23-minute bilateral renal IRI model [18] for early protective efficacy and a 28-minute unilateral IRI (uIRI) model [19] for chronic progression studies. Both models incorporated core temperature maintenance at 37 °C via a thermoregulated heating system during ischemia. The uIRI model was specifically chosen for long-term assessment because of its optimal balance between renal injury severity and survival rates, enabling observation of AKI-to-CKD progression.

The treatment groups received a single intravenous (IV) injection of either (+)CePPy or (−)CePPy (5 mg/kg) immediately after IRI was induced. The control group received an equivalent volume of saline vehicle. Sham-operated animals underwent identical procedures without vascular clamping. For functional assessment in the chronic model, contralateral nephrectomy was performed 24 h prior to terminal sampling. All procedures were randomized and powered using effect size estimates from our prior AKI characterization study [19].

In vivo imaging and biodistribution

To investigate the in vivo tissue distribution, CePPy-Cy5 (5 mg/kg) was administered intravenously to both sham-operated and renal IRI mice immediately following reperfusion. The animals were sacrificed at 1, 6, 12, or 24 h post-injection for fluorescence imaging. Major organs (heart, lungs, liver, spleen, and kidneys) were collected and analyzed using an IVIS^® Lumina system (Caliper Life Sciences), with tissue-specific fluorescence quantified as photon flux (p/sec/cm²/sr) within defined regions of interest (ROIs) [20].

For renal clearance analysis, the Ce content in the kidneys was measured via inductively coupled plasma‒mass spectrometry (ICP-MS) at 1 h, 24 h, and 14 days after administration. The kidneys were weighed and subsequently dissolved in nitric acid to prepare for ICP‒MS analysis. The percentage of the injected dose in the kidneys (%ID) was determined via the following equation: %ID = (Ce content in the kidney/total administered Ce dose) × 100.

In vivo biocompatibility and toxicity evaluation

A hemolysis assay was conducted to evaluate the compatibility of (+)CePPy with red blood cells (RBCs). Briefly, 0.5 mL of whole blood was centrifuged at 3000 rpm for 10 min to collect RBCs, which were rinsed with PBS. Then, 20 µL of the diluted RBC suspension was mixed with 1 mL of the (+)CePPy dispersion at various concentrations, incubated at 37 °C for 3 h, and centrifuged at 3500 rpm for 5 min. The hemolysis ratio was quantified by measuring the absorbance of the supernatant at 540 nm with a microplate reader. Deionized water and PBS were used as positive and negative controls, respectively.

To evaluate the acute systemic toxicity of (+)CePPy in vivo, the mice were administered a single intravenous dose of 5, 10, or 20 mg/kg and euthanized 3 days post-injection for analysis. To assess long-term toxicity, the mice received three intravenous administrations of (+)CePPy at the same dose and were sacrificed 14 days after the final injection for evaluation. Major organs were collected for histopathological assessment via hematoxylin‒eosin (H&E) and periodic acid‒Schiff (PAS) staining. Concurrently, blood samples were collected for the serum biochemical analysis of markers related to renal (creatinine, BUN) and hepatic (AST, ALT) function.

In vivo two-photon imaging settings

Renal accumulation and excretion of CePPy-Cy5 were monitored in real time via a two-photon microscope system (TSP-SP5, Leica, Germany). The mice underwent surgical implantation of an abdominal imaging window under anesthesia [21] for intravital observation. Cy5 fluorescence (red channel) was excited at 640 nm, with emission collected at 660–670 nm, whereas tissue autofluorescence (green channel) was detected at 520–530 nm. Dynamic tracking involved sequential 5-minute scans with 5 μm Z-step intervals.

Histological analysis

After collection, renal tissues were subjected to fixation, dehydration, embedding, and sectioning. The prepared sections were then stained with PAS, Masson’s trichrome, and Sirius red to assess tubular injury and interstitial fibrosis. Tubular injury was quantified via blinded evaluation of PAS-stained sections at the corticomedullary junction by blinded analysis of ten randomly selected fields per sample (200×). Acute tubular necrosis (ATN) was scored from 0 to 4 on the basis of the extent of tubular necrosis, cast formation, dilation, and brush border loss and was graded as follows: 0 (normal), 1 (1–25%), 2 (26–49%), 3 (50–75%), and 4 (> 75%).

Immunofluorescence staining

Renal tissues were fixed in 4% paraformaldehyde and dehydrated through a sucrose gradient before being embedded in OCT. Prepared 5 μm sections were permeabilized with 0.25% Triton X-100, blocked with goat serum, and then incubated with primary antibodies overnight (details in Table S1). Afterward, the sections were treated with FITC- or Cy3-conjugated secondary antibodies for 2 h at room temperature and counterstained with DAPI. For apoptosis assessment, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was performed on HK-2 cells and kidney samples according to the manufacturer’s instructions (Beyotime Biotechnology). Confocal microscopy was used for image acquisition.

Immunohistochemical staining

Immunohistochemical staining was performed on paraffin-embedded kidney sections after deparaffinization, rehydration, heat-induced antigen retrieval, and blocking. The sections were then incubated with primary antibodies at 4 °C (details in Table S1) overnight. The signals were detected via an HRP-based kit.

Cell culture and treatment

HK-2 cells were purchased from the American Type Culture Collection (Manassas, VA, USA). PTECs were isolated from mice as previously described [18]. To simulate IRI, the cells were subjected to hypoxia/reoxygenation (H/R) injury. Specifically, the cells were cultured under hypoxic conditions (1% O₂, 5% CO₂, and 94% N₂) for 24 h in glucose-free and serum-free media. After this hypoxic exposure, the cells were returned to normoxic conditions (21% O₂, 5% CO₂, 74% N₂) in complete medium for an additional 3 h. CePPy treatment was initiated under H/R conditions.

For small interfering RNA (siRNA) or plasmid transfection experiments, PTECs or HK-2 cells were transfected via Lipofectamine RNAiMAX or Lipofectamine 3000, respectively. At 12 h post-transfection, the cells were subjected to H/R injury followed by (+)CePPy treatment. The Mpv17l overexpression plasmid was constructed by cloning the Mpv17l sequence into a pReceiver-M35 vector (GeneCopoeia) under the control of a cytomegalovirus (CMV) promoter. The sequences of the siRNAs used in this study (GenePharma, China) are listed in Table S2.

Endocytic pathway of CePPy

To investigate the endocytic pathways mediating (+)CePPy internalization in PTECs, pharmacological inhibition assays were systematically conducted via the following pathway-specific inhibitors: (1) chlorpromazine (30 µM), which blocks clathrin-mediated endocytosis; (2) amiloride (100 µM), which suppresses micropinocytosis; (3) filipin III (5 µg/mL), which inhibits caveolin-dependent endocytosis; and (4) dynasore (100 µM), which impedes dynamin-dependent endocytosis [22, 23]. PTECs were preincubated with each inhibitor in serum-free medium for 1 h, followed by incubation with (+)CePPy-Cy5 (20 µg/mL). After 2 h, intracellular fluorescence was captured via a confocal laser scanning microscope.

Intracellular ROS measurements

Intracellular ROS levels were detected via DCFH-DA (S0033S, Beyotime Biotechnology). Following treatment, the cells were exposed to 10 µM DCFH-DA diluted in serum-free DMEM/F-12 and incubated for 30 min at 37 °C in a 5% CO₂ environment protected from light. Following two washes with prewarmed PBS, live-cell fluorescence imaging was performed via a confocal microscope.

Mitochondrial ROS detection

Mitochondrial ROS levels in cells were detected via the fluorescent probe MitoSOX Red (M36008; Invitrogen, USA). Following treatment, the cells were exposed to 5 µM MitoSOX Red diluted in serum-free medium for 20 min under light-protected conditions and then washed twice with PBS. Fluorescence was observed by microscopy and quantified with ImageJ.

Mitochondrial membrane potential

The mitochondrial membrane potential in the cells was measured via a JC-1 Kit (C2003SB, Beyotime Biotechnology). After the specified treatments, the cells were incubated in medium containing 5 µM JC-1 dye at 37 °C for 40 min under light-protected conditions. Following two washes with prewarmed PBS, live-cell fluorescence imaging was performed via a confocal microscope.

Electron microscopy

The tissues were initially fixed with 2.5% glutaraldehyde solution at 4 °C, subsequently treated with 1% osmium tetroxide, dehydrated through methanol gradients, and embedded in epoxy resin. The mitochondrial ultrastructure was observed via TEM with a JEO-1400.

Protein extraction and Western blot

Kidney tissues or HK-2 cells were mechanically lysed via radioimmunoprecipitation assay (RIPA) buffer supplemented with phenylmethylsulfonyl fluoride (PMSF) as a protease inhibitor. Following high-speed centrifugation (12,000 rpm), the supernatants were collected, and protein concentrations were subsequently quantified. Western blotting was performed with 20–30 µg of protein per lane. Antibody specifics are documented in Table S1.

Quantitative real-time polymerase chain reaction (qRT‒PCR) assay

Total RNA was extracted from PTECs lysed in TRIzol reagent (Invitrogen, Carlsbad, CA) following the manufacturer’s protocol. Transcript levels were quantified via TransStart^® Top Green qPCR SupeWBrMix (AQ131, TransGen, Beijing, China) on an Applied Biosystems 7500 system PCR cycler (Applied Biosystems, Foster City, CA, USA). The data were normalized to 18 S rRNA levels and expressed relative to those of the control group. The primers used were synthesized by BGI Genomics (China); the sequences are listed in Table S3.

RNA sequencing

To elucidate the underlying mechanism of (+)CePPy, kidney tissues were obtained from sacrificed experimental mice. RNA isolation was accomplished via TRIzol Reagent (Life Technologies, USA) according to the provided extraction guidelines. RNA sequencing was performed by Biomarker Technologies Co., Ltd. (Beijing, China). The raw sequencing data were submitted to the GEO database (GSE313051).

To elucidate the underlying mechanisms targeting injured PTECs, a reanalysis was performed on a publicly available scRNA-seq dataset (GSE190887) derived from a uIRI model [24] and two bulk transcriptomic datasets profiling acute liver injury (GSE160880) [25] and acute lung injury (GSE216943).

Statistical analysis

Every experiment presented was carried out on at least three separate occasions. The data are presented as the means ± standard deviations (SDs). Analyses were performed via GraphPad Prism version 9.0. The normality of the data was assessed via the Shapiro‒Wilk test, whereas variance homogeneity was evaluated via the Brown‒Forsythe test. Two-group comparisons were performed via unpaired two-tailed Student’s t tests (with Welch’s correction if variances were unequal). For more than two groups, one-way ANOVA with Dunnett’s post hoc test. Statistical significance was set at p < 0.05.

Results

Synthesis, characterization and antioxidative ability of differently charged CePPy

(+)CePPy was synthesized using an in situ chemical oxidative polymerization method. Briefly, Ce(SO₄)₂ acted as an oxidizing catalyst, while PVA served as a stabilizer to initiate the polymerization of the pyrrole monomer at room temperature (Fig. 1A). Moreover, to preserve the surface characteristics of (+)CePPy, DSPE-PEG2000 was applied to modify (+)CePPy, resulting in the preparation of (-)CePPy. As shown in the TEM images, both (+)CePPy and (-)CePPy exhibited monodispersed, sphere-like structures (Fig. 1B, C). Dynamic light scattering revealed hydrodynamic diameters of 86.87 ± 2.45 nm for (+)CePPy and 96.77 ± 1.04 nm for (-)CePPy (Fig. 1D). The corresponding zeta potential of (+)CePPy was determined to be 7.29 ± 0.35 mV. Furthermore, following PEGylation, the zeta potential of (-)CePPy transitioned from a positive to a negative value, recorded at − 14.06 ± 1.39 mV (Fig. 1E). Moreover, the energy dispersive X-ray spectra demonstrated that C, N, O, and Ce were present in CePPy (Fig. S1). The content ratio of Ce in CePPy was 1 µg Ce/mg CePPy, as determined by ICP‒MS analysis. During I/R injury, the generation of O₂⁻, H₂O₂, •OH, and other free radicals leads to oxidative stress in the kidney, resulting in significant damage to renal tissue. Consequently, we investigated the antioxidant activity of (+)CePPy and (-)CePPy. As shown in Fig. 1F, G, both species exhibited significant O₂⁻- and H₂O₂-scavenging activity in a concentration-dependent manner according to the results of the relevant assay kits, suggesting that CePPy possesses SOD- and CAT-like activities. Next, the •OH-scavenging activities of the differently charged CePPy species were evaluated using MB as an indicator, which undergoes oxidation by •OH, resulting in color loss. Conversely, when exposed to antioxidants that effectively neutralize •OH, MB retains its blue color with a strong absorption peak at approximately 664 nm. As illustrated by the UV‒vis spectra of (+)CePPy and (-)CePPy at various concentrations, the absorbance increased with increasing concentration after mixing with MB plus •OH solution, indicating that CePPy has a significant ability to clear •OH (Fig. 1H, I). Furthermore, we evaluated free radical scavenging using an ABTS assay kit, which confirmed the concentration-dependent clearance capacity of free radicals by CePPy (Fig. 1J). These results validated the favorable antioxidative activity of both (+)CePPy and (-)CePPy against various ROS in vitro, which is crucial for treating IRI-induced AKI.

Fig. 1.

Characterization and antioxidative ability of CePPy with varied charge states. A Schematic illustration of CePPy preparation. TEM images of B (+)CePPy and C (-)CePPy. D Hydrodynamic sizes and E zeta potentials of different charges of CePPy. The ability of (-)CePPy and (+)CePPy at different concentrations to scavenge F O₂⁻ and G H₂O₂. The UV‒vis spectra of H (+)CePPy and I (-)CePPy inhibiting MB degradation by scavenging •OH. J Capacity of (-)CePPy and (+)CePPy at diverse concentrations to scavenge free radicals. For statistical analysis, the data represent the means ± SDs, n = 3 for E, F, G, and J. ns were assessed using a two-tailed unpaired Student’s t test (ns: not significant)

Positive surface charge determines the proximal tubular uptake and mitochondrial targeting of CePPy

To investigate the impact of the surface charge of CePPy on tubular cellular uptake, we intravenously administered fluorescently labeled (+)CePPy-Cy5 and (-)CePPy-Cy5 to healthy C57BL/6 mice and performed a systematic in vivo biodistribution analysis. In vivo imaging revealed prominent Cy5 fluorescence accumulation in the liver, with significant secondary renal localization at 1 h after injection (Fig. 2A–D). Quantitative ex vivo organ fluorescence analysis demonstrated that, compared with (-)CePPy, (+)CePPy exhibited a 3.05-fold greater renal uptake efficiency (p = 0.007), confirming charge-dependent renal targeting. To validate this charge-dependent uptake in vitro, we performed time-course kinetic studies in HK-2 cells incubated with (+)/(-)CePPy-Cy5, which were monitored via high-content imaging. (+)CePPy exhibited rapid internalization, reaching its peak mean fluorescence intensity (MFI) at an early time point (within 40 min) before plateauing. In contrast, (-)CePPy uptake was significantly delayed and attenuated, resulting in a substantially lower peak MFI at a much later time point (120 min) (Fig. 2E, F; Supplementary Videos 1, 2). These results confirm that a positive surface charge dramatically enhances both the speed and extent of proximal tubular cell uptake. A similar charge-dependent uptake pattern was also observed in hepatocytes (Fig. S2, S3), which explains why compared with (-)CePPy, (+)CePPy showed stronger liver accumulation in vivo imaging. To elucidate the subcellular localization of internalized (+)CePPy, we assessed its colocalization with organelle markers in PTECs via confocal laser scanning microscopy. After a 1-hour incubation, (+)CePPy-Cy5 fluorescence strongly colocalized with MitoTracker but minimally overlapped with LysoTracker, indicating preferential mitochondrial accumulation (Fig. 2G–I). Conversely, pure Cy5 dye (control) displayed a diffuse cytoplasmic distribution without specific organelle targeting (Fig. S4).

Fig. 2.

The positive surface charge on CePPy determines proximal tubular uptake and mitochondrial targeting. A Fluorescence imaging of major organs in healthy mice following (+)CePPy-Cy5 or (-)CePPy-Cy5 administration (5 mg/kg). B Kidney fluorescence imaging at 1, 6, 12, and 24 h post-administration. C Quantitative fluorescence intensity analysis of the data in (A). D Fluorescence intensity quantification of the data in (B). E Dynamic uptake of CePPy-Cy5 (20 µg/mL) with varying charges by HK-2 cells. F Fluorescence intensity quantification from (E) using ImageJ. G Colocalization imaging of (+)CePPy-Cy5 with MitoTracker (green) and LysoTracker (green) in PTECs. H, I Line-scanning plots for fluorescence intensity quantitative analysis of (G) via Image J. Data are presented as means ± SDs, n = 3 for C and D. Statistical significance between groups was evaluated using a two-tailed unpaired Student’s t test

Mechanistic insights into renal CePPy uptake and excretion

To identify the renal cell types responsible for (+)CePPy uptake, kidney sections harvested 2 h after injection were costained with Lotus tetragonolobus lectin (LTL; a proximal tubule marker) and endomucin (an endothelial marker). As shown in Fig. 3A, (+)CePPy-Cy5 predominantly accumulated in the proximal tubules (LTL⁺), with negligible glomerular uptake. To elucidate the in vivo uptake mechanism within PTECs, intravital microscopy was used to track dynamic (+)CePPy-Cy5 trafficking (Fig. 3B). Within 5 min of intravenous injection, (+)CePPy-Cy5 rapidly localized to the basal compartment of the tubular epithelium, with the cytoplasmic fluorescence intensity progressively increasing. After 1 h, granular fluorescence extended from the basolateral to the luminal side of the PTECs. In stark contrast, free Cy5 dye diffused uniformly within 10 s (Fig. S5, Video 3). Given the potential compromise of the glomerular filtration barrier in AKI, we further investigated whether (+)CePPy could access renal tubules via direct glomerular filtration. Since the deep anatomical location of the glomeruli limits the resolution by two-photon intravital microscopy, we performed histological analysis of kidney sections from sham and AKI mice collected at multiple time points after injecting (+)CePPy-Cy5 or free Cy5. No red fluorescence was detected in the glomeruli of either the sham or AKI mice injected with (+)CePPy-Cy5, whereas the free Cy5-injected mice presented diffuse red fluorescent speckles in the glomeruli (Fig. S6). These results demonstrate that (+)CePPy does not reach renal tubules through direct glomerular filtration, even under AKI conditions. Collectively, our findings confirm that (+)CePPy is primarily delivered to renal tubules via peritubular capillary uptake.

Fig. 3.

Mechanistic insights of (+)CePPy uptake and elimination by kidneys. A Colocalization images of (+)CePPy-Cy5 with LTL and endomucin in kidney tissues at 2 h post-injection. PT: proximal tubule. G: glomerulus. B Representative intravital microscopy images of mouse kidneys at 5 min and 1 h after intravenous injection of (+)CePPy-Cy5. Red indicates Cy5 fluorescence, and green indicates proximal tubule autofluorescence. The Cy5 signal rapidly localized to the basal compartment of the tubular epithelium within 5 min, and by 1 h, it appeared granulated and covered nearly the entire tubule from the basolateral to the luminal side. C Representative TEM images at 24 h post-injection, demonstrating elimination of (+)CePPy by the kidneys and its secretion into the tubular lumen for urinary excretion. D Schematic illustration depicting the uptake and elimination process of (+)CePPy

Furthermore, we tracked the postuptake excretion of (+)CePPy by PTECs. Ultrastructural imaging at 24 h after endocytosis revealed (+)CePPy nanoparticles within intracellular vesicles and extracellular vesicles in the tubular lumen (Fig. 3C), suggesting excretion via transcellular vesicular transport. This result was further corroborated by the detection of CePPy nanoparticles in the urine, confirming renal excretion. Collectively, these findings establish a complete pathway from renal uptake to transcellular vesicular excretion (Fig. 3D), highlighting the critical role of PTECs in regulating CePPy clearance.

EEA1 enhances (+)CePPy targeting to injured renal tubules

To investigate the renal targeting efficacy of (+)/(-)CePPy in AKI, we performed in vivo fluorescence imaging in IRI-AKI mice. The intravenous injection of (+)/(-)CePPy-Cy5 resulted in rapid accumulation in the kidneys and liver, with significantly higher renal uptake in AKI mice than in healthy controls (Fig. 4A, B), demonstrating preferential targeting to injured kidneys. The accumulation of (+)CePPy in the kidneys peaked at 6 h after injection, and substantial fluorescence was still visible in the injured kidney after 24 h (Fig. 4C, D). Immunofluorescence confirmed that (+)CePPy preferentially localized to injured tubules, as confirmed by kidney injury molecule-1 (KIM-1) positivity (Fig. 4E). Consistent with the in vivo findings, compared with normal cells, H/R-injured PTECs exhibited enhanced (+)CePPy uptake in vitro (Fig. 4F–H). To explore the mechanism of uptake in PTECs, we inhibited endocytic pathways and found that dynasore potently suppressed (+)CePPy uptake (by 70% in normal PTECs and 67% in H/R PTECs), whereas amiloride and filipin had minimal effects (Fig. 3I, J). These findings indicate that clathrin-dependent endocytosis is the primary uptake route, regardless of injury status. To elucidate the mechanism underlying the specific targeting of injured PTECs by (+)CePPy, we further investigated injury-induced alterations in the expression of endocytosis-related genes upon injury. qPCR revealed that Eea1 was the most upregulated endocytosis-related gene in injured PTECs (4.46-fold increase, Fig. 3K), which was confirmed at the protein level (Fig. 3L). Further analysis of a publicly available single-cell RNA-sequencing dataset from uIRI mouse kidneys (GSE190887) [24] confirmed that Eea1 expression was markedly upregulated during the acute phase of AKI, primarily in injured PTECs (Fig. S7A, B). Importantly, this increase was negligible in models of acute liver injury (GSE160880) [25] or acute lung injury (GSE216943), suggesting that EEA1 upregulation may represent a kidney-specific response to AKI (Fig. S7C, D).

Fig. 4.

EEA1 mediates CePPy targeting to injured renal tubules. A Fluorescence imaging of major organs in sham and AKI mice following (+)CePPy-Cy5 or (-)CePPy-Cy5 administration (5 mg/kg). B Quantitative fluorescence intensity analysis of the data in (A). C Kidney fluorescence imaging at 1, 6, 12, and 24 h post-administration. D Fluorescence intensity quantification of the data in (C). E Representative immunofluorescence images of the colocalization of KIM-1 (green) with (+)CePPy-Cy5 (red) in the kidney. F Representative fluorescence images of H/R-injured PTECs. KIM-1 was labeled in green, and (+)CePPy-Cy5 was visualized in red. G Flow cytometry analysis was conducted to determine (+)CePPy-Cy5 uptake with or without H/R injury. H Relative fluorescence intensity quantification of the data in (G). I Representative fluorescence microscopy images of (+)CePPy-Cy5 uptake in normal and injured PTECs following treatment with amiloride, filipin III, or dynasore. J Fluorescence intensity quantification from (I) via Harmony^® analysis software. K Changes in the transcription of endocytic genes in injured PTECs, as determined by qPCR analysis. L Western blot detection of EEA1 in H/R-injured PTECs after Eea1 knockdown. M Representative fluorescence microscopy images of (+)CePPy-Cy5 uptake in H/R-injured PTECs after Eea1 knockdown. Data are presented as means ± SDs, n = 3 for H, J and K. Statistical significance between groups was evaluated using a two-tailed unpaired Student’s t test

Given EEA1’s critical role in early endosome formation and vesicle trafficking [26, 27], we propose that its upregulation in injured PTECs enhances (+)CePPy endocytic efficiency and accelerates transcytosis by promoting early endosomal maturation, thereby facilitating targeted delivery and renoprotection. Critically, siRNA-mediated knockdown of Eea1 significantly impaired (+)CePPy-Cy5 uptake in injured PTECs (Fig. 3L, M), directly indicating that Eea1 upregulation enhances (+)CePPy targeting to injured renal tubules.

CePPy attenuates H/R–mediated mitochondrial oxidative damage

Considering the robust antioxidant capabilities and critical role of CePPy in AKI, we systematically evaluated its protective effects against H/R injury in vitro. Cytotoxicity screening revealed excellent biocompatibility for both CePPy species, with (+)CePPy (160 µg/mL) maintaining 100.1% ± 2.49% cell viability and 94.68% ± 2.35% for (-)CePPy (160 µg/mL) compared with the control (Fig. 5A, B). We subsequently assessed the protective effects of CePPy on HK-2 cells subjected to H/R injury. H/R insult significantly increased cell death; however, (+)CePPy enhanced cell viability in a dose-dependent manner (Fig. 5C). In contrast, (-)CePPy exhibited minimal therapeutic effects (Fig. 5D).

Fig. 5.

In vitro antioxidant and antiapoptotic properties of (+)CePPy and (-)CePPy. A, B The cell viability analysis of HK-2 cells following 24 h of incubation with (+)CePPy and (-)CePPy. C, D Cell viability analysis of HK-2 cells with H/R injury after coculture with (+)CePPy and (-)CePPy. E Representative fluorescence microscopy images showing the ROS levels (DCFH-DA probe) after CePPy treatment and H/R injury. F Quantification of the mean DCFH-DA fluorescence intensity via ImageJ. G Representative images of mitochondrial ROS (mitoSOX probe) in HK-2 cells subjected to H/R injury after CePPy treatment. H Quantification of the mean MitoSOX fluorescence intensity via ImageJ. I Representative fluorescence microscopy images of the mitochondrial membrane potential in the indicated treatment groups were assessed via JC-1 dye. J Quantification of the mitochondrial membrane potential. K Representative fluorescence microscopy images showing the release of cytochrome c from mitochondria into the cytoplasm of HK-2 cells with H/R injury after being treated with (+)CePPy and (-)CePPy. L Apoptosis in each group was assessed via TUNEL staining. M Quantitative analysis of TUNEL-positive cells. N The expression levels of BAX and cleaved caspase3 in the indicated treatment groups were analyzed by Western blotting. O, P The relative protein levels in (N) were quantified. The data are presented as the means ± SDs. n = 3 for A, B, C, D, F, H, J, M, O, and P. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test

Mechanistic investigations revealed the superior ROS-scavenging capacity of (+)CePPy. DCFH-DA assays revealed significant attenuation of H/R-induced oxidative stress by (+)CePPy (Fig. 5E, F), with (-)CePPy producing only marginal reductions. As a further control, blank PPy (without Ce doping) exhibited no significant ROS-scavenging activity compared with the H/R group (Fig. S8). Since CePPy predominantly localizes to mitochondria, we further evaluated its mitochondrial protective effects. MitoSOX assays demonstrated that (+)CePPy effectively scavenges mitochondrial ROS, leading to a significant reduction in H/R-induced mitochondrial oxidative stress. In contrast, (-)CePPy only slightly decreased mitochondrial ROS levels (Fig. 5G, H). Given that oxidative stress impairs mitochondrial function, we also evaluated the mitochondrial membrane potential (ΔΨm). In the control group, ΔΨm remained high, reflected by JC‑1 aggregation and strong red fluorescence. H/R injury severely decreased ΔΨm, whereas treatment with (+)CePPy restored nearly 50% of the potential. Neither (-)CePPy nor blank PPy provided significant protection against ΔΨm loss (Fig. 5I, J and Fig. S9). Mitochondria-mediated apoptosis is a major pathway of tubular cell death following ischemic injury [6, 28]. In the H/R group, we observed the translocation of cytochrome c from the mitochondria to the cytosol, indicating the activation of apoptotic processes. Treatment with (+)CePPy effectively reduced this cytochrome c release (Fig. 5K). Further evidence of the antiapoptotic effect of CePPy was provided by TUNEL staining (Fig. 5L, M) and Western blot analysis of key apoptotic markers, including BAX and cleaved caspase-3 (Fig. 5N–P).

In summary, our findings show that H/R-induced oxidative stress causes substantial mitochondrial damage, as reflected by the loss of mitochondrial membrane potential, increased cytochrome c release, and enhanced cell death. Importantly, (+)CePPy markedly mitigated mitochondrial oxidative injury and suppressed apoptosis.

Protective effects of (+)CePPy on IRI-AKI in vivo

To investigate the therapeutic potential of CePPy in AKI, we intravenously administered both (+)CePPy and (-)CePPy to IRI mice at the onset of reperfusion, with treatment continuing for 24 h (Fig. 6A). Prior to formal experiments, we conducted dose-ranging studies using 5, 10, and 20 mg/kg to identify the optimal therapeutic dose. These preliminary experiments revealed that (+)CePPy at 5 mg/kg already achieved significant therapeutic efficacy in AKI treatment, with no clear dose-dependent effects observed across the tested range (Fig. S10). We therefore selected 5 mg/kg as the optimal dose for subsequent in vivo studies to maximize therapeutic benefit while ensuring safety. Following the administration of 5 mg/kg CePPy, TEM revealed significant improvements in the mitochondrial ultrastructure, characterized by reduced swelling, structural disorganization, and vacuolation, in the CePPy-treated groups (Fig. S11). Renal function assessments demonstrated that serum creatinine (Scr) levels were markedly elevated in IRI mice compared with those in sham-operated controls (Fig. 6B). Treatment with CePPy led to significant reductions in the Scr concentration in both treatment groups, with the most pronounced improvement observed in the (+)CePPy group. PAS staining and ATN scoring revealed that (+)CePPy substantially reduced renal tubular injury in AKI mice (Fig. 6C, D). In contrast, the degree of tubular damage in the (-)CePPy group did not significantly differ from that in the untreated AKI group. Consistent with these observations, both forms of CePPy significantly decreased the expression of KIM-1, a key marker of tubular injury (Fig. 6E–H), and reduced the levels of apoptosis-related proteins such as cleaved caspase-3 and BAX. The greatest reduction in these markers was observed in the (+)CePPy-treated group (Fig. 6G–I). Additionally, TUNEL assays confirmed a notable decrease in apoptosis in AKI mice following CePPy treatment (Fig. S12). Taken together, these results indicate that CePPy, especially its positively charged form, effectively protects against AKI in vivo.

Fig. 6.

(+)CePPy alleviates IRI-induced AKI in mice. A Schematic illustration of the AKI model and its treatment with (+)CePPy and (-)CePPy (5 mg/kg). B Scr levels in each group at 1 day after IRI. C Representative micrographs of PAS staining in IRI mice treated with control or CePPy. D Quantification of tubular injury via ATN scoring. E Immunofluorescence images of KIM-1 (red) across groups. F Quantitative detection of the KIM-1-positive area in (E). G-J Western blot analysis of KIM-1, BAX, and cleaved caspase-3 protein levels in kidney tissue (G) with corresponding quantification (H‒J). The data of each group are expressed as the mean ± SDs. n = 5–6 samples in B, D. n = 3 samples in F, H, I, J. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test

(+)CePPy enhances endogenous mitochondrial antioxidant defense via Mpv17l-dependent mechanisms

To elucidate the molecular mechanisms by which (+)CePPy protects against AKI, a transcriptomics sequencing analysis was performed using kidneys from AKI mice treated with either vehicle or (+)CePPy. RNA sequencing revealed significant changes in the mRNA expression profiles following (+)CePPy treatment compared with those of the vehicle control, with 316 genes upregulated and 149 genes downregulated. Genes were considered differentially expressed if they exhibited a fold change of ≥ 1.5 for upregulation or ≤ − 1.5 for downregulation (Fig. 7A).

Fig. 7.

(+)CePPy enhances endogenous mitochondrial antioxidant defense via Mpv17l-dependent mechanisms. A-D RNA-sequencing analysis of kidneys from AKI mice treated with vehicle or (+)CePPy. A Volcano plot revealing the DEGs between the (+)CePPy treatment group and the vehicle treatment group. B Gene Ontology (GO) enrichment of the significantly altered cellular components. C Enriched GO biological process (GOBP) terms related to the regulation of the apoptotic process. D Heatmap of DEGs involved in the apoptotic process. Among these genes, Mpv17l presented the most significant changes in expression. E, F Mpv17l protein expression in kidney tissues by Western blot analysis (E) and quantitative analysis (F) across sham, vehicle-treated, and (+)CePPy-treated AKI mice. G‒M Mechanistic studies in H/R-injured HK-2 cells after Mpv17l knockdown and (+)CePPy treatment. G ROS levels were measured with a DCFH-DA fluorescent probe. H‒K Western blot detection of Mpv17l, BAX, and cleaved caspase-3 protein expression and quantitative analysis of protein levels. L TUNEL staining for apoptosis assessment. M Quantitative analysis of TUNEL-positive cells. Data are presented as the means ± SDs. n = 3 samples in F, I, J, K, M. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test

Given prior findings indicating that CePPy protects kidneys by mitigating mitochondrial oxidative damage and reducing apoptosis, we focused our bioinformatics analysis on genes associated with apoptosis and mitochondrial function. Gene Ontology analysis revealed that the differentially expressed genes (DEGs) were primarily linked to the endoplasmic reticulum, intracellular membrane-bound organelles, and mitochondria (Fig. 7B) and were involved in regulating the apoptotic process (Fig. 7C). Among these genes, Mpv17l presented the most significant changes in expression (Fig. 7D). Mpv17l is known to prevent the formation of ROS [29] and is localized in the mitochondria of PTECs [30]. The downregulation of Mpv17l is correlated with increased mitochondrial superoxide production, a hallmark of renal injury [31–33]. Based on these previous findings, we hypothesized that CePPy may enhance mitochondrial antioxidant defense through Mpv17l-dependent mechanisms. To investigate this hypothesis, we first assessed the expression of Mpv17l in AKI mice treated with either vehicle or (+)CePPy. The results revealed significant downregulation of Mpv17l expression in AKI mice, whereas treatment with (+)CePPy significantly upregulated its expression (Fig. 7E, F). We then transfected siRNAs targeting Mpv17l (si Mpv17l) into cells and subsequently treated them with (+)CePPy in the H/R model. Following Mpv17l silencing, CePPy partially failed to attenuate the H/R-induced increase in ROS levels (Fig. 7G) and mitigate apoptosis, as detected by apoptosis-associated proteins (BAX and cleaved caspase-3) (Fig. 7H–K) and TUNEL staining (Fig. 7L, M). Conversely, overexpression of Mpv17l in PTECs reduced mitochondrial ROS production and decreased the number of apoptotic cells (Fig. S13). These results suggest that CePPy enhances endogenous mitochondrial antioxidant defense through Mpv17l-dependent mechanisms.

(+)CePPy attenuates the transition of AKI to CKD

To evaluate the impact of CePPy on the progression from AKI to CKD, the mice were treated with CePPy starting on day 1 following uIRI and were sacrificed for analysis on day 28 (Fig. 8A). Compared with the vehicle-treated group, the (+)CePPy-treated group presented markedly lower Scr levels (Fig. 8B). By Day 28, vehicle-treated uIRI mice demonstrated focal tubular atrophy, interstitial fibrosis, and chronic inflammation, which are indicators of the AKI-to-CKD transition. In contrast, (+)CePPy treatment markedly alleviated tubular atrophy and chronic inflammatory responses (Fig. S14). Masson’s trichrome and Sirius red staining revealed less extracellular matrix deposition and a reduced fibrotic area in the (+)CePPy group than in the vehicle group (Fig. 8C, D; Fig. S15). Furthermore, immunofluorescence staining for vimentin (Fig. 8E, F) and Western blot analysis of α-smooth muscle actin (α-SMA) and vimentin expression (Fig. 8G–I) corroborated the antifibrotic effects of (+)CePPy. Immunohistochemical staining for F4/80, a macrophage marker, indicated that (+)CePPy significantly suppressed immune-inflammatory responses during renal fibrosis (Fig. 8J, K). Collectively, these results demonstrate that (+)CePPy effectively inhibits the transition from AKI to CKD.

Fig. 8.

(+)CePPy attenuates the transition of AKI to CKD. A Schematic overview of the AKI-to-CKD model establishment and treatment regimens with (+)CePPy and (−)CePPy. B Serum creatinine levels in each group at 28 days post-uIRI. C, D Representative Masson’s staining images (C) 28 days after uIRI, with corresponding quantitative analysis of the fibrotic area (D). E, F Representative images of immunofluorescence staining for vimentin (E) across groups, with quantitative analysis of vimentin-positive areas (F). G-I Western blot analysis of α-SMA and vimentin expression in kidney tissues (G) and the corresponding quantitative results (H, I). J, K Representative immunohistochemical staining for F4/80 with quantitative analysis. The data are presented as the means ± SDs. n = 5 for panels B and D; n = 3 for panels F, H, I, and K. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s post hoc test

In vitro and in vivo toxicity assessment of (+)CePPy

We first performed a RBC compatibility assay to verify the safety of intravenous administration of (+)CePPy. As illustrated in Fig. S16, the hemolysis rate of (+)CePPy remained below 2%, even at a high concentration of 320 µg/mL, confirming its good RBC compatibility. To evaluate the toxicity of (+)CePPy in vivo, we conducted a study in which healthy mice were intravenously injected with high doses of (+)CePPy. Tissue samples from major organs, including the kidney, liver, lung, heart, and spleen, were collected at 3 days for acute toxicity evaluation and at 14 days for long-term toxicity assessment. Histological examination revealed no significant tissue damage compared with that in the control group (Fig. 9A, Fig. S17A), indicating that CePPy did not exhibit adverse effects. Additionally, liver and kidney function tests, along with hematological indices, remained within normal ranges, similar to those of the control group (Fig. 9B, Fig. S17B). These findings collectively confirm the excellent biocompatibility and negligible toxicity of CePPy in vivo, suggesting its promising potential for clinical applications. The clearance of CePPy from the kidneys was investigated using ICP‒MS to measure Ce levels at different time points (1 h, 24 h, and 14 days) following the injection of (+)CePPy in both AKI and sham mice. Notably, almost 90% of the accumulated (+)CePPy was eliminated from the kidneys within 14 days (Fig. S18), indicating low toxicity and efficient clearance from the body. Furthermore, because CePPy is charged and tends to accumulate around mitochondria due to charge attraction, we investigated its impact on the mitochondrial membrane potential in normal cells at various concentrations. Compared with the control treatment, (+)CePPy did not significantly affect the mitochondrial membrane potential at different concentrations (Fig. S19). This result suggests that although (+)CePPy is localized to mitochondria, it does not disrupt mitochondrial function or integrity in terms of membrane potential, further supporting its safety profile and potential for therapeutic application.

Fig. 9.

In vivo acute toxicity evaluation of (+)CePPy. A Normal C57BL/6J mice were intravenously injected with a single dose of (+)CePPy at 5, 10, or 20 mg/kg and euthanized 3 days post-injection for subsequent analysis. Following treatment, major organs were harvested and subjected to histological assessment via H&E and PAS staining. B Serum samples from each group were analyzed for routine blood parameters, as well as liver and renal function markers. The data are presented as the means ± SDs, with n = 4 per group. For B, statistical comparisons were performed via one-way ANOVA

Discussion

AKI remains a critical global health challenge, with limited therapeutic interventions available. Excessive ROS contribute to renal damage by inducing progressive cellular dysfunction and apoptosis, highlighting the urgent need for novel renoprotective strategies. In this study, we designed a novel (+)CePPy nanoparticle that exhibits broad-spectrum antioxidant activity and provides significant protection against AKI. The cationic surface of (+)CePPy promotes renal uptake and facilitates mitochondria-specific targeting. Specifically, the selective accumulation of (+)CePPy in injured PTECs is likely mediated by an EEA1-dependent mechanism. The renoprotective effects are driven by two main mechanisms: (1) efficient ROS scavenging by mimicking SOD and CAT activities and (2) enhancement of endogenous mitochondrial antioxidant defenses through activation of the Mpv17l pathway. Together, these dual mechanisms effectively attenuate AKI and prevent its progression to CKD, positioning (+)CePPy as a promising charge-directed nanotherapeutic strategy (Fig. 10).

Fig. 10.

Schematic illustration of (+)CePPy in renal uptake and alleviating AKI via endogenous-exogenous regulation of oxidative stress

A key innovation of our study lies in the synthesis of CePPy. We established a green, one-step synthesis method using Ce(SO₄)₂ as an oxidizing catalyst, avoiding the use of organic solvents and simplifying the preparation process compared with conventional methods for ceria-based nanomaterials [8, 34, 35]. This approach not only enhances the practicality and scalability of CePPy production but also improves its biocompatibility. Notably, the Ce content in CePPy is only 0.1% of the total nanoparticle weight, which is significantly lower than those of most existing cerium-based nanozymes [8, 23, 35]. This low cerium loading minimizes the risk of metal aggregation and long-term toxicity, addressing major safety concerns associated with heavy-metal-doped nanomaterials and favorably positioning CePPy for potential clinical translation. Beyond its synthetic simplicity and safety, CePPy has several unique advantages over existing antioxidant nanoformulations. It possesses potent multienzymatic ROS-scavenging capabilities, mimicking both SOD and CAT activities, which allows for efficient elimination of a broad spectrum of ROS, including O₂⁻, H₂O₂, and •OH. Furthermore, the positive surface charge of (+)CePPy significantly enhances renal cellular uptake and promotes mitochondrial targeting. This charge-dependent targeting mechanism represents a comprehensive advantage. Unlike conventional cationic nanoparticles (e.g., chitosan-based systems), which predominantly function as drug carriers [36, 37], (+)CePPy integrates intrinsic targeting and therapeutic functions into a single entity. This integrated therapy offers a novel solution for AKI, combining efficient targeting, potent ROS scavenging, and endogenous regulation.

The enhanced renal uptake and mitochondrial targeting conferred by the positive surface charge of the (+)CePPy nanoparticles can be attributed to several sequential mechanisms. First, electrostatic interactions between cationic nanoparticles and the anionic membranes of PTECs promote strong adhesion to the cell surface. This close contact facilitates engagement with membrane components and enhances endocytic internalization, which is a well-established advantage of cationic nanoparticles, as supported by previous studies [10, 12, 38]. After cellular uptake, the positive charge further facilitates endosomal escape, thereby reducing lysosomal sequestration and degradation [39, 40], which helps maintain the structural and functional integrity of (+)CePPy. Finally, driven by the highly negative mitochondrial membrane potential [41], the nanoparticles accumulate efficiently within the mitochondria via electrophoretic attraction. This “charge-driven targeting” mechanism is essential for achieving highly efficient mitochondrial antioxidant therapy.

Our results demonstrate that (+)CePPy accumulates in the kidneys through transcellular transport from the peritubular capillaries to the basolateral membrane of tubular epithelial cells under both healthy and AKI conditions. However, the specific molecular mechanisms underlying its transendothelial transport remain to be elucidated. Recent studies have emphasized that nanoparticles primarily enter tissues through active transcellular pathways rather than through passive paracellular gaps [42]. In tumor tissues, the transendothelial transport of nanoparticles is predominantly mediated by nonreceptor-dependent macropinocytosis [43]. In contrast, in normal lung tissue, nanoparticles rely on specific ligand‒receptor interactions with lung microvascular endothelial cells to initiate active vesicular transport, enabling rapid entry from the bloodstream into lung tissue [44]. Therefore, we hypothesize that the transport of (+)CePPy across peritubular capillary endothelial cells in the kidney may involve a similar receptor- or transporter-mediated mechanism. Future studies should focus on identifying specific receptors on renal endothelial cells that may participate in CePPy recognition.

The observed renal targeting specificity of (+)CePPy toward injured PTECs is likely mediated through injury-induced upregulation of EEA1, a well-characterized regulator of early endosome biogenesis [26, 27]. Importantly, this EEA1 upregulation manifests as a kidney-specific injury response, as confirmed by transcriptomic and immunohistochemical analyses showing marked Eea1induction during the acute phase of AKI, predominantly localized to damaged PTECs [24]. In contrast, only minimal Eea1 upregulation was detected in models of acute liver injury [25] or acute lung injury (GSE216943), supporting an EEA1-driven mechanism for the targeted renal accumulation of (+)CePPy. However, it should be noted that the kidney has multiple complex pathways for nanoparticle uptake. While our data indicate that (+)CePPy predominantly accumulates in renal tissue through transcellular transport from peritubular capillaries, a process that may involve vesicular transcytosis or transporter-mediated mechanisms, the current experimental design cannot completely rule out potential minor contributions from glomerular filtration or paracellular transport under pathological conditions. Therefore, although the EEA1-mediated pathway appears to be the predominant mechanism, the proposed uptake model requires additional validation and should be interpreted with caution.

Regarding the mechanisms by which (+)CePPy mitigates AKI, in addition to mimicking the direct exogenous antioxidant effects of CAT and SOD, we found that (+)CePPy also confers renal protection by upregulating Mpv17l expression. As an inner mitochondrial membrane protein, Mpv17l plays a critical role in maintaining mitochondrial physiology [32, 33, 45, 46]. Previous studies have reported a marked reduction in Mpv17l in AKI, and its deficiency or dysfunction leads to impaired oxidative phosphorylation (OXPHOS), mitochondrial oxidative damage, and apoptosis [29–31]. Our results confirmed that (+)CePPy alleviates mitochondrial oxidative stress and apoptosis by upregulating Mpv17l expression. Mechanistically, Mpv17l contributes to mitochondrial antioxidant defense, probably by selectively activating HtrA2 protease, a key component in mitigating oxidative stress [30]. Furthermore, transcriptomic analysis revealed that (+)CePPy treatment induced substantial reprogramming of energy metabolism (Fig. S20). Several DEGs were enriched in processes critical for ATP generation, including acyl-CoA metabolism, fatty acid β-oxidation, and ADP metabolic processes. The consistent changes in genes such as Aadat, Acot11, and Aldoc indicated the restoration of energy homeostasis. This regulation of energy metabolism-related genes and the consequent increase in ATP synthesis are consistent with the established role of Mpv17l in restoring mitochondrial energy production, suggesting a coordinated mechanism through which (+)CePPy promotes mitochondrial functional recovery.

Several limitations of the present study should be acknowledged. First, while our data demonstrate renal accumulation of CePPy via transcytosis from peritubular capillaries to the basolateral domain of tubular epithelium, the exact mechanism underlying its transendothelial transport remains undefined. Second, although vesicle-mediated exocytosis is implicated in the clearance of internalized CePPy, the potential involvement of lysosomes or other organelles in this process remains unclear. Furthermore, although we demonstrate that (+)CePPy upregulates Mpv17l expression and thereby enhances endogenous antioxidant defenses, the specific upstream regulatory mechanism responsible for this effect has not been elucidated. Future studies should employ integrated experimental approaches to elucidate the precise molecular mechanism through which (+)CePPy upregulates Mpv17l. This could include protein pull-down assays combined with mass spectrometry to identify direct or indirect protein interactors of (+)CePPy, or Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) to map genome-wide chromatin accessibility changes and pinpoint the enhancer or promoter regions regulating Mpv17l upon (+)CePPy treatment. Resolving this signaling cascade will establish a complete molecular pathway linking (+)CePPy internalization to mitochondrial antioxidant reprogramming.

Conclusions

In summary, we developed a charge-modulated nanotherapeutic strategy using (+)CePPy nanoparticles for targeted AKI intervention. We demonstrated that the positive surface charge of (+)CePPy determines proximal tubular uptake and mitochondrial targeting. (+)CePPy preferentially accumulates in injured PTECs, and the underlying targeting mechanism revealed that the upregulation of EEA1 enhances nanoparticle endocytosis. These findings not only establish (+)CePPy as a promising AKI therapeutic but also elucidate a charge-dependent targeting paradigm, providing a mechanistic foundation for mitochondrial nanomedicine in kidney diseases.

Abbreviations

AKI

Acute kidney injury

PTECs

Proximal tubular epithelial cells

IRI

Ischemia‒reperfusion injury

uIRI

Unilateral IRI

ROS

Reactive oxygen species

Mpv17l

Mpv17 mitochondrial membrane protein-like

O₂⁻

Anion radicals

H₂O₂

Hydrogen peroxide

•OH

Highly reactive hydroxyl radical

PPy

Polypyrrole

CKD

Chronic kidney disease

EEA1

Early endosome antigen 1

SOD

Superoxide dismutase

CAT

Catalase

ABTS

2,2’-Azinobis-(3-ethylbenzthiazoline-6-sulfonate)

IV

Intravenous

ICP‒MS

Inductively coupled plasma‒mass spectrometry

MB

Methylene blue

ROIs

Regions of interest

ID

Injected dose

H&E

Hematoxylin‒eosin

PAS

Periodic acid-Schiff

ATN

Acute tubular necrosis

TUNEL

Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling

H/R

Hypoxia/reoxygenation

RIPA

Radioimmunoprecipitation assay

PMSF

Buffer with phenylmethylsulfonyl fluoride

qRT‒PCR

Quantitative real-time polymerase chain reaction

SD

Standard deviation

LTL

Lotus tetragonolobus lectin

siRNA

Small interfering RNA

ΔΨm

Mitochondrial membrane potential

KIM-1

Kidney injury molecule-1

Scr

Serum creatinine

DEGs

Differentially expressed genes

α-SMA

α-smooth muscle actin

OXPHOS

Oxidative phosphorylation

Author contributions

H.M., F.Z., W.M., and H.Q. conceived the study and wrote and revised the manuscript. L.Y., and H.Y.carried out the nanomaterial synthesis and characterization. Q.P., conducted bioinformatics analyses related to this study and prepared figures. H.M., X.T., Q.P., S.W., F.B., J.H. performed the in vitro and in vivo experiments, analyzed the data, and prepared the manuscript. A.Q,. H.Y., C.G., and C.X. provided input into the design of the experiments. All authors reviewed and approved the final manuscript.

Funding

This work was supported by Noncommunicable Chronic Diseases-National Science and Technology Major Project (2025ZD0547600, 2025ZD0547603, 2025ZD0547601); Open Project Program of the State Key Laboratory of Medical Proteomics (SKLP-O202403); Young Elite Scientist Sponsorship Program by CAST (YESS20200400); Young Talent Project of Chinese PLA General Hospital (20230404); National Key Research and Development Program of China (2023YFC3605500, 2023YFC3605501).

Data availability

The sequencing raw data generated in this study have been deposited in the NCBI database (GSE313051). All relevant data that support the findings of this work are available in the Supplementary Information or Source Data file. Source data are provided with this paper.

Declarations

Ethics approval

All animal experiments were performed in accordance with the protocols sanctioned by the Institutional Animal Care and Use Committee at the Chinese PLA General Hospital (Approved Number: SQ2022490).

Consent for publication

All authors have approved the manuscript and agree with the submission.

Competing interests

The authors declare no competing interests.