Engineered naringenin-loaded nanoscale coordination polymers for targeted treatment of acute kidney injury

Abstract

Acute kidney injury (AKI) is a severe clinical condition lacking targeted and effective pharmacotherapies. Dysregulated inflammation, largely driven by the nuclear factor-κB (NF-κB) signaling pathway, is a primary mediator of tubular damage in AKI. While bioinformatic and preliminary analyses identified the natural flavonoid naringenin (Nag) as a potent NF-κB inhibitor, its therapeutic utility is severely restricted by poor aqueous solubility, rapid systemic metabolism, and insufficient renal accumulation. To address these pharmacokinetic limitations, we synthesized ultra-small (<10 nm) iron-naringenin nanoscale coordination polymers (Fe-Nag NPs). This sub-10 nm design exploits glomerular filtration for passive targeting, specifically leveraging the enhanced vascular permeability of injured kidneys. In both ischemia-reperfusion (IR) and cisplatin-induced murine AKI models, Fe-Nag NPs achieved significantly higher renal drug concentrations compared to free naringenin administration. This targeted delivery substantially improved therapeutic outcomes, as evidenced by restored renal function, attenuated acute histopathological lesions, and the profound suppression of pro-inflammatory cascades. Furthermore, Fe-Nag NPs provided sustained renoprotection, effectively mitigating the functional transition from severe AKI to chronic kidney disease (CKD). By seamlessly integrating transcriptomic insights with functional nanomaterial design, this metal-organic platform not only validates targeted NF-κB inhibition for AKI treatment but also offers a robust strategy for the precise delivery of poorly soluble bioactive natural products.

Graphical abstract

Highlights

• •Ultra-small Fe-Nag NPs overcome pharmacokinetic limits of free naringenin to achieve targeted renal accumulation.
• •Fe-Nag NPs outperform the free drug in ischemic and cisplatin AKI models, restoring function and limiting injury.
• •Transcriptomics reveal Fe-Nag NPs confer renoprotection via targeted inhibition of the NF-κB signaling pathway.

1. Introduction

Acute kidney injury (AKI) represents a critical syndrome marked by an abrupt deterioration of renal function, which is independently associated with substantial morbidity, mortality, and heightened risks of progression to chronic kidney disease (CKD) and end-stage renal disease (ESRD) [1,2]. Common triggers include major surgery, sepsis, nephrotoxic agents (e.g., cisplatin), and ischemia-reperfusion (I/R) injury, events that collectively impose a heavy burden on global healthcare systems [3,4]. Decades of research have elucidated the multifaceted pathophysiology of AKI, which encompasses interrelated processes of vascular dysfunction, tubular epithelial cell damage, sustained inflammation, oxidative stress, and programmed cell death [5,6]. Despite these advances, the clinical management of AKI remains predominantly supportive. Renal replacement therapy (RRT), while life-sustaining, functions as an extemporaneous measure to correct fluid overload, electrolyte imbalances, and uremia; it does not address the underlying molecular mechanisms of injury or facilitate renal repair [7,8]. This stark discrepancy between our understanding of AKI pathogenesis and the lack of targeted therapeutics underscores an urgent, unmet clinical need.

Among the key pathological drivers, dysregulated inflammation has emerged as a central perpetuating factor in AKI. Initial tubular insult activates a deleterious cascade, promoting vasoconstriction, microvascular thrombosis, and aggravated tissue ischemia [9,10]. The ensuing hypoxic milieu potently activates the innate immune system, leading to the robust production of pro-inflammatory cytokines and chemokines. At the molecular epicenter of this response lies the transcription factor nuclear factor-κB (NF-κB), a master regulator that, upon activation, orchestrates the expression of a vast network of genes governing inflammation, immune cell recruitment, and apoptosis, thereby cementing a vicious cycle of renal damage [11]. Consequently, targeted inhibition of the NF-κB pathway presents a rational and promising therapeutic strategy for mitigating AKI.

Naringenin (Nag), a naturally occurring flavonoid abundant in citrus fruits, has garnered considerable interest for its pleiotropic pharmacological activities, including potent anti-inflammatory, antioxidant, and anti-apoptotic effects [[12], [13], [14]]. Critically, naringenin has been demonstrated to directly suppress NF-κB signaling, attenuating inflammatory injury in preclinical models of cerebral I/R and drug-induced AKI [15,16]. However, the translational potential of naringenin is severely hampered by its unfavorable pharmacokinetics, notably its poor aqueous solubility, rapid systemic metabolism, and low bioavailability, which collectively preclude the attainment of therapeutic concentrations at the target site.

To overcome these delivery challenges, we turned to nanotechnology. Metal-organic nanoscale coordination polymers (NCPs) represent a versatile class of materials characterized by tunable structures, high loading capacity, and biocompatibility, making them excellent candidates for drug delivery [17]. Of particular relevance for renal targeting are ultra-small nanoparticles (<10 nm), which exhibit a unique propensity for passive accumulation in the kidneys via glomerular filtration—a phenomenon that is often enhanced in AKI due to increased vascular permeability and impaired renal clearance [18].

Based on these considerations, we hypothesized that constructing ultra-small, iron-based NCPs using naringenin itself as the organic ligand would simultaneously enhance the drug's solubility and stability while enabling targeted delivery to the injured kidney. To test this hypothesis, we engineered iron-naringenin nanocomposites (Fe-Nag NPs) through a simple coordination reaction between Fe³⁺ ions and naringenin, stabilized with the biocompatible polymer polyvinylpyrrolidone (PVP). The primary objectives of this study were to: (1) synthesize and physicochemically characterize the Fe-Nag NPs; (2) evaluate their renal targeting efficiency; and (3) rigorously assess their therapeutic efficacy in well-established murine models of I/R-induced and cisplatin-induced AKI. We anticipate that our Fe-Nag NP platform will not only overcome the pharmacokinetic barriers of naringenin but also synergize its intrinsic bioactivity with targeted delivery, thereby effectively disrupting the inflammatory cascade in AKI and offering a novel nanotherapeutic paradigm for this devastating condition.

2. Materials and method

2.1. Materials

FeCl₃ was purchased from Adamas. Naringenin (Nag) was purchased from MACKLIN. Polyvinylpyrrolidone (PVP) and polyvinylpyrrolidone-COOH (PVP-COOH) were purchased from Xi'an Ruixi Biotechnology Co., Ltd. Anti-p-IKKα/β (AF3800), anti-p-RELA (AF2006), and anti-RELA (AF5006) were purchased from Affinity. Anti-IKKβ (15649-1-AP), anti-p-IκBα (82349-1-RR), anti-IκBα (10268-1-AP), anti-NGAL (30700-1-AP), anti-KIM-1 (30948-1-AP), anti-TGF-β1 (21898-1-AP), anti-E-cadherin (20874-1-AP), and anti-α-SMA (14395-1-AP) antibodies were purchased from Proteintech. TNF-α and IL-6 ELISA assay kits were purchased from Boster Biological Technology.

2.2. Transcriptome sequencing data analysis

Transcriptome sequencing data of the mouse bilateral kidney IR-AKI model (GSE98622) were downloaded from the Gene Expression Omnibus (GEO) database [PMID: 28931758], yielding pre-processed gene expression profiles (FPKM). This dataset includes transcriptome sequencing data for the mouse bilateral kidney IRI model at 10 time points, along with data for 3 time points from a sham surgery model and 3 time points from a control model, with each group comprising 3 technical replicates. Gene Set Variation Analysis (GSVA) was employed, utilizing the mouse Hallmark signaling and metabolic pathway gene set (MsigDB database) to assess variations in the signal and metabolic pathway activities across the samples. Visualization was performed using the pheatmap package in R. Differential gene expression between the 4-h IRI group and the 4-h SHAM group was further analyzed using the limma and edgeR packages in R, with a selection threshold of adj.P.Val < 0.05 and an absolute log2FC > 1. The ggVolcano package in R was used for visualization. Functional enrichment analysis of the upregulated differentially expressed genes in the 4-h IRI group was conducted using Metascape [PMID: 30944313].

2.3. Transcription factor prediction and molecular docking

From the Research Collaboratory for Structural Bioinformatics Protein Data Bank (R CSB PDB) to download the three-level structure of the hub gene and its transcription factor corresponding protein, and to construct the hub gene transcription factor protein library of IR-AKI by using the SWISS-MODEL database for homologous modeling if the three-level structure is unknown. The three-dimensional structure of Naringin was downloaded from PubChem compound database. Autodock software was used to calculate the one-to-one reverse docking of key protein library and Naringin, and the binding energy between the selected protein and Naringin was analyzed and ranked. The docking molecules were visualized and their amino acid residues were displayed by Pymol software.

2.4. Synthesis and characterization of Fe-Nag NPs

Fe-Nag NPs were synthesized as follows. First, iron trichloride (FeCl₃) was dissolved in 1 mL of methanol. This solution was then added dropwise to 5 mL of a methanol solution containing polyvinylpyrrolidone (PVP) under constant stirring. After stirring for 5 min, 1 mL of a methanol solution of Nag was introduced dropwise into the mixture, and the reaction was allowed to proceed for 1 h with continuous stirring. The resulting product was dialyzed against water overnight to yield water-soluble Fe-Nag NPs, which were stored for subsequent use.

The synthesized Fe-Nag NPs were characterized using multiple techniques. Their hydrodynamic diameter and zeta potential were determined by dynamic light scattering (DLS). Morphology was examined using transmission electron microscopy (TEM). The chemical state of iron was analyzed by X-ray photoelectron spectroscopy (XPS), while the coordination between Fe and Nag was investigated by Fourier-transform infrared (FTIR) spectroscopy. Finally, the molar ratio of Fe to Nag in the complex was determined by thermogravimetric analysis (TGA).

2.5. CCK-8

To evaluate the cytocompatibility of Fe-Nag nanoparticles (NPs), HK-2 cells (renal proximal tubular cells) were seeded in 96-well plates at a density of 5 × 10³ cells per well and allowed to adhere overnight. The cells were then exposed to serial dilutions of Fe-Nag NPs (ranging from 0 to 200 μg/mL) in complete medium for 24 h under standard culture conditions (37°C, 5% CO₂). Following this incubation period, 10 μL of CCK-8 reagent was added directly to each well. The plates were then incubated for an additional 2 h at 37°C, protected from light. Absorbance at 450 nm was measured using a microplate reader, and cell viability was subsequently calculated [19,20]. In addition, the morphological changes of cells were recorded.

2.6. Cellular uptake

FITC was conjugated to PVP-COOH to prepare Fe-Nag(F) NPs, enabling the investigation of their cellular uptake by HK-2 cells treated with H₂O₂ or cisplatin. HK-2 cells were seeded onto sterile coverslips in 24-well plates and allowed to adhere overnight. Following treatment with H₂O₂ (200 μM for 2 h) or cisplatin (10 μM for 12 h), the cells were incubated with Fe-Nag(F) NPs in complete medium for 2, 4, or 6 h. After incubation, the cells were washed with cold PBS, fixed in 4% paraformaldehyde, and counterstained with DAPI. Coverslips were then mounted onto slides using antifade medium, and images were captured with an inverted fluorescence microscope. Additionally, the cellular uptake of Fe-Nag NPs by HK-2 cells was analyzed using flow cytometry.

2.7. Animal experiment

All experimental procedures involving animals were approved by the Institutional Animal Care and Use Committee of Ningbo University (approval number: NBU20230162). These procedures were performed in compliance with the ARRIVE guidelines and the ethical standards for laboratory animal use established by the U.S. National Institutes of Health. Balb/c mice, sourced from Beijing Vital River Laboratory Animal Technology Co., Ltd., were used in this study.

IR-AKI: Mice weighing approximately 20-22 g were selected and fasted for 12 h prior to surgery, with free access to water. Anesthesia was induced via intraperitoneal injection of 3% sodium pentobarbital (80 mg/kg). The mice's backs were shaved and disinfected. A skin and muscle incision were made 0.5 cm lateral to the spine and 0.5 cm below the rib cage to expose the kidneys. The renal arteries on both sides were carefully isolated and clamped to induce ischemia for 45 min. After this period, the clamps were removed to restore blood flow, and the recovery of the kidneys was observed. The incision was closed in two layers. Once the mice regained consciousness, they were returned to clean cages and housed in the animal facility [21]. Cis-AKI: An animal model of chemotherapy-induced acute kidney injury was established by a single intraperitoneal injection of cisplatin solution at a dose of 15 mg/kg.

2.8. In vivo imaging

To investigate the biodistribution of Fe-Nag NPs, we loaded the near-infrared dye indocyanine green (ICG) into the nanoparticles (denoted as Fe-Nag(I) NPs) via conjugation with PVP-COOH. These NPs were administered to mice via tail vein injection. The mice comprised three models: sham-operated controls, IR-AKI, and Cis-AKI. At 2 h post-injection, the mice were euthanized, and their major organs (kidneys, liver, lungs, and heart) were harvested, rinsed with PBS, and immediately imaged using an In Vivo Imaging System to evaluate the initial biodistribution. Furthermore, to analyze the intra-renal distribution of the NPs, the harvested kidneys were embedded in OCT compound and sectioned into frozen slices. The tissue sections were then stained with DAPI and visualized under a fluorescence microscope to determine the precise localization of the ICG signal within the kidney tissue.

2.9. Nag distribution in renal tissues

The renal distribution of naringenin (Nag) was quantified using high-performance liquid chromatography (HPLC). Mice were randomly allocated into experimental groups: (1) sham + free Nag, (2) sham + Fe-Nag NPs, (3) IR-AKI + free Nag, (4) IR-AKI + Fe-Nag NPs, (5) Cis-AKI + free Nag, and (6) Cis-AKI + Fe-Nag NPs. All formulations were administered via tail vein injection at a Nag-equivalent dose of 10 mg/kg. Two hours post-injection, the mice were euthanized, and the kidneys were promptly harvested. The renal tissues were rinsed with cold PBS, weighed, and homogenized in a pre-chilled buffer. The homogenates were centrifuged at 12,000 × g for 15 min at 4°C to obtain the supernatant. The supernatant was then diluted with the HPLC mobile phase (a mixture of 90% acetonitrile and 10% water containing 0.1% formic acid), filtered through a 0.22 μm membrane, and injected into the HPLC system. The analysis was performed using a reversed-phase C18 column with UV detection at 270 nm. The Nag concentration in each sample was determined from a standard calibration curve and expressed as nanograms per gram of tissue (ng/g).

2.10. Experimental group

The mice were randomly divided into four groups: (1) Sham, (2) AKI + PBS, (3) AKI + Nag (10 mg/kg), (4) AKI + Fe-Nag NPs (Nag, 10 mg/kg). All treatments were administered via tail vein injection at 2 h post-AKI induction. At 24 h after treatment, blood samples were collected for the assessment of renal function (serum creatinine and blood urea nitrogen), and kidney tissues were harvested for subsequent histological and molecular analyses.

2.11. Kidney function tests

Blood samples were allowed to clot at room temperature for at least 30 min. Subsequently, they were centrifuged at 3000 × g for 10 min to separate the serum. The levels of serum creatinine (Scr) and blood urea nitrogen (BUN) were measured using a fully automated biochemical analyzer.

2.12. Pro-inflammatory cytokines

Renal tissue samples were homogenized on ice in RIPA lysis buffer using an electric homogenizer. The homogenates were then centrifuged at 15,000 rpm for 15 min at 4°C to collect the supernatant, which constituted the total protein extract. The protein concentration of each sample was determined using a bicinchoninic acid (BCA) assay kit. Subsequently, the levels of pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), were quantified in the protein extracts using specific enzyme-linked immunosorbent assay (ELISA) kits, strictly following the manufacturers' protocols [22,23].

2.13. H&E and Masson staining

Renal tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at a thickness of 4-5 μm. Following deparaffinization in xylene and hydration through a graded ethanol series, the sections were subjected to standard hematoxylin and eosin (H&E) or Masson-trichrome staining [24]. Finally, the stained sections were observed under a light microscope to assess the degree of histopathological injury, and representative images were captured for each group.

2.14. Western blot

Total protein was extracted from renal tissues by homogenization in ice-cold RIPA lysis buffer, followed by centrifugation to collect the supernatant. The protein concentration was determined using a BCA protein assay kit. Equal amounts of protein from each sample were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto PVDF membranes. After blocking with 5% non-fat milk, the membranes were incubated overnight at 4°C with specific primary antibodies against NGAL, p-IKKα, IKKβ, p-IκBα, IκBα, p-RELA, RELA, α-SMA, and TGF-β1. Following extensive washing, the membranes were probed with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies at room temperature for 1 h. The protein bands were visualized using an enhanced chemiluminescence (ECL) substrate and captured with a gel imaging system. GAPDH was used as an internal loading control for normalization, and the relative expression levels of the target proteins were quantified using ImageJ software [25,26].

2.15. Immunofluorescence

Immunofluorescence (IF) staining was performed on paraffin-embedded kidney sections. Briefly, sections were deparaffinized in xylene and rehydrated through a graded ethanol series. Antigen retrieval was then carried out by heating the sections in sodium citrate buffer (pH 6.0). After cooling, non-specific binding sites were blocked with 10% normal serum from the secondary antibody host species. The sections were subsequently incubated overnight at 4°C in a humidified chamber with primary antibodies against KIM-1, NGAL, p-RELA (p-p65), p-IκBα, TGF-β1, α-SMA, and E-Cadherin. Following PBS washes, the sections were incubated with appropriate fluorophore-conjugated secondary antibodies for 1 h at room temperature, protected from light. Cell nuclei were counterstained with DAPI. Finally, the sections were mounted with an anti-fade mounting medium and imaged using a fluorescence microscope [27,28].

2.16. Hydroxyproline levels

The hydroxyproline content in renal tissues, as a marker of collagen deposition, was quantified using a hydroxyproline assay kit. Briefly, tissue samples were hydrolyzed in concentrated hydrochloric acid at high temperature to liberate hydroxyproline from collagen. The hydrolysates were neutralized, centrifuged, and the resulting supernatant was collected. The supernatant was then subjected to oxidation with chloramine-T, followed by reaction with p-dimethylaminobenzaldehyde at 60°C to develop a chromogenic product. The absorbance was measured at 560 nm using a microplate reader. The hydroxyproline concentration in each sample was determined by interpolation from a standard curve and expressed as micrograms per milligram of tissue wet weight (μg/mg) [29,30].

2.17. Hemolysis assay

A hemolysis assay was conducted to evaluate the blood compatibility of the nanoparticles. Red blood cells (RBCs) were isolated from whole blood via centrifugation and washed with PBS. The packed RBCs were then resuspended in 0.9% NaCl to prepare a 2% (v/v) suspension. Aliquots of this suspension were incubated for 3 h at 37°C with the following: 0.9% NaCl (negative control), deionized water (positive control), and serial concentrations of Fe-Nag NPs diluted in 0.9% NaCl. After incubation, the samples were centrifuged, and the absorbance of the supernatants was measured at 541 nm [31,32]. The hemolysis percentage was calculated using the following formula: Hemolysis (%) = [(Abs_sample - Abs_negative)/(Abs_positive - Abs_negative)] × 100.

2.18. Biosafety

The in vivo biosafety of Fe-Nag NPs was systematically evaluated in healthy mice. The mice were randomly divided into groups and intravenously administered either PBS (as a control) or Fe-Nag NPs at the therapeutic dose. To assess systemic toxicity, blood samples were collected from the retro-orbital plexus on day 7 and day 30 post-injection. These samples were subjected to comprehensive hematological analysis and clinical biochemistry tests to evaluate liver and kidney function. Subsequently, the mice were euthanized, and major organs (heart, liver, spleen, lungs, and kidneys) were harvested. The organs were fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with H&E for histopathological examination. Tissue sections were meticulously observed under a light microscope for any signs of damage, inflammation, or morphological abnormalities.

2.19. Statistics

Data are presented as the mean ± standard error of the mean (SEM). Statistical analyses were performed using GraphPad Prism software (version 8.0). Differences between two groups were analyzed using an unpaired, two-tailed Student's t-test. A P-value of less than 0.05 was considered statistically significant.

3. Results

3.1. NF-κB is a key early target of naringenin in kidney injury

Reanalysis was conducted on the transcriptome sequencing data of the IR-AKI model, sham operation model, and normal control model (NORM) at different time points. GSVA analysis revealed that in the early stage of the IRI model (0-4h), signaling pathways such as TNF-α signaling via NF-κB, Hypoxia, and TGF-β signaling were significantly activated (Fig. 1A). From 24h to 7d, the cells showed strong activity in DNA damage repair pathways, suggesting that some cells entered a proliferative state. After 72h, inflammatory and immune-related pathways in the IR-AKI model were significantly activated and maintained for a long period, including Il6 jak stat3 signaling, Inflammatory response, and Interferon alpha/gamma response. Previous studies have shown that after 6-48 h of IR-AKI, neutrophil-mediated inflammatory responses induce the release of various pro-inflammatory chemokines and cytokines [PMID: 21233280], which is consistent with the results of this study. In contrast, in the sham group, TNF-α and inflammation-related pathways were in a low-activity state (Fig. 1A).

Fig. 1.

NF-κB is a key early target of naringenin in kidney injury. (A) GSVA pathway analysis based on gene expression profiles of IR-AKI, sham, and NORM at different time points. (B) The differential genes between the IR-AKI 4 h and sham 4 h models by volcano plot. (C) Functional enrichment analysis of genes upregulated at IR-AKI 4 h. (D) Prediction of transcription factors regulating upregulated genes. (E) Molecular docking analysis of the RELA protein with naringenin.

To explore the gene expression patterns in the early stage of IR-AKI, the differentially expressed genes between the IR-AKI 4h and sham 4h samples were analyzed, and it was found that genes such as Fosb, Il6, and Nfkbiz were significantly upregulated (Fig. 1B). Functional enrichment analysis revealed that the upregulated genes were mainly involved in signaling pathways such as TNF signaling pathway and regulation of interleukin-1 production (Fig. 1C). Previous studies have shown that in the IR-AKI model, the activation of Kupffer cells leads to the production and release of ROS, which in turn promotes the release of cytokines such as TNF-α and IL-6 and activates ROS-sensitive transcription factors such as the Ap-1 family (Fosb, etc.) and NF-κB [PMID: 20637940].

The study further used a transcriptional regulatory database to analyze the transcription factors that regulate the upregulated genes and obtained 10 potential transcriptional regulatory factors, with Nfkb1 showing the most significant regulatory effect (Fig. 1D). Therefore, finding appropriate drugs to regulate the activity of transcription factors such as Nfkb1 has the potential for preventing or alleviating renal injury. Naringenin has anti-inflammatory properties and is a potential drug. To comprehensively evaluate the binding potential and specificity of naringenin, quantitative molecular docking simulations were systematically conducted between naringenin and the 10 key predicted transcription factors using the CDOCKER module. As summarized in Table S1 (Supplementary Information), naringenin exhibited highly favorable binding scores with specific targets. Most notably, it showed an excellent binding affinity for RELA (CDOCKER Energy: −22.081; CDOCKER Interaction Energy: −29.6671), ranking it among the highest compared to other evaluated transcription factors. The highly specific and advantageous binding model of naringenin within the active pocket of RELA is illustrated in Fig. 1E. These robust quantitative comparisons strongly suggest that naringenin may effectively participate in the prevention and treatment of renal injury through targeted interference with the NF-κB signaling pathway.

Guided by these bioinformatic findings that pinpointed NF-κB as a pivotal early target and identified naringenin as a potential inhibitor, we hypothesized that enhancing the delivery of Nag to the injured kidney could be a promising therapeutic strategy for AKI. To test this hypothesis and overcome the inherent pharmacokinetic limitations of free Nag, we developed the iron-based nanoscale coordination polymer Fe-Nag NPs. The design and application of Fe-Nag NPs for AKI therapy were, therefore, rationally derived from the integrative analysis of public database mining and preliminary experimental validation, aiming to translate these computational insights into effective targeted treatment.

3.2. Preparation and characterization of Fe-Nag NPs

Fe-Nag NPs were synthesized via a facile one-pot coordination reaction at room temperature. Briefly, a methanolic solution of Nag - a molecule rich in phenolic hydroxyl groups—was added dropwise to a methanol solution containing Fe³⁺ ions and the stabilizing agent PVP (Fig. 2A). An immediate color shift from light yellow to deep brown-black provided macroscopic evidence of Fe³⁺-Nag coordination. Subsequent overnight dialysis against water yielded a stable aqueous dispersion of Fe-Nag NPs, notably overcoming the poor water solubility of native Nag. DLS (Fig. 2B) and TEM (Fig. 2C) revealed that the NPs were near-spherical and uniformly distributed, with an average diameter of less than 10 nm - a critical size threshold for efficient renal filtration and targeted delivery. Furthermore, DLS measurements revealed that the Fe-Nag NPs possess a slightly negative surface charge with a zeta potential of −3.97 ± 2.12 mV. This near-neutral zeta potential, primarily attributed to the non-ionic steric shielding provided by the PVP stabilizer, is highly advantageous for in vivo applications. It minimizes non-specific protein adsorption and mitigates rapid clearance by the reticuloendothelial system (RES), thereby ensuring prolonged systemic circulation and favorable biocompatibility.

Fig. 2.

Preparation and characterization of Fe-Nag NPs. (A) The schematic diagram of Fe-Nag for AKI. (B) The dynamic light scattering of Fe-Nag and its dispersion (embedded image). (C) The transmission electron microscopy image of Fe-Nag (Scale bar, 50 nm). (D) XPS analysis results of Fe-Nag. (E) FTIR analysis results of Nag and Fe-Nag. (F) TGA analysis results of Fe-Nag. (G) Proposed schematic structural model of the Fe-Nag coordination complex, illustrating a central Fe³⁺ ion coordinating with the phenolic hydroxyl and carbonyl oxygen atoms of two Nag molecules (1:2 M ratio). (H) In vitro cumulative release profiles of Nag from Fe-Nag NPs in neutral (pH 7.4) and acidic (pH 5.0) PBS over 24 h. Data are presented as mean ± SD (n = 3).

Spectroscopic analyses further elucidated the chemical composition and coordination structure. XPS analysis (Fig. 2D) of the Fe-Nag NPs showed two strong binding energy peaks at 711 eV and 724 eV, which are characteristic of Fe 2p_3/2 and Fe 2p_1/2 core levels of Fe³⁺, respectively, confirming the presence of iron in the +3 oxidation state. FTIR spectroscopy provided direct evidence of the coordination bond formation. As shown in Fig. 2E, the intensity of the HO-C stretching vibration band of Nag in the 1150-1200 cm⁻¹ region was significantly diminished in the Fe-Nag NP spectrum, indicating the involvement of the phenolic hydroxyl groups in coordinating with Fe³⁺ ions. Additionally, TGA indicated a final Fe-to-Nag molar ratio of approximately 1:2 (Fig. 2F). Integrating these compositional and stoichiometric findings, we proposed a structural model for the Fe-Nag NPs (Fig. 2G). In this schematic, each Fe³⁺ ion serves as a coordination center, linking with the oxygen atoms from the phenolic hydroxyl and carbonyl groups of two adjacent Nag molecules. This 1:2 coordination network is further sterically stabilized by PVP chains, ensuring the structural integrity of the nanoscale assemblies.

To evaluate whether this coordination structure confers controlled drug release, we investigated the in vitro release behavior of Fe-Nag NPs under physiological (pH 7.4) and pathological acidic (pH 5.0) conditions. As shown in Fig. 2H, the cumulative release of Nag was highly pH-dependent. At pH 7.4, the nanoparticles exhibited excellent stability, with only ∼25.3% of the drug leaking over 24 h. Conversely, the release was significantly accelerated at pH 5.0, reaching ∼55.0% within the first 4 h and peaking at an almost complete release of ∼92.0% by 24 h. This responsive behavior is driven by the protonation of the phenolic hydroxyl groups under acidic conditions, which competitively weakens the metal-polyphenol coordination bonds. Such a pH-responsive dissociation profile is highly advantageous, as it prevents premature drug leakage during systemic circulation while triggering rapid payload release upon accumulation in the acidic microenvironment typically associated with AKI metabolic acidosis or intracellular endo/lysosomes. Finally, we tracked the hydrodynamic diameter of Fe-Nag at 4°C over 7 days to assess storage stability (Fig. S1). The particle size exhibited only minor fluctuations throughout the observation period, indicating that the intra-particle coordination interactions remain stable over time, assuring the reliability of Fe-Nag for practical storage and handling.

3.3. Efficient cellular internalization of Fe-Nag NPs by renal tubular epithelial cells

Prior to investigating cellular uptake, we first assessed the cytocompatibility of Fe-Nag NPs with HK-2 cells (a human renal proximal tubular epithelial cell line) to ensure that any subsequent observations of internalization were not confounded by overt toxicity. As determined by the CCK-8 assay, treatment with Fe-Nag NPs at concentrations ranging from 5 to 100 μg/mL for 24 h did not induce significant cytotoxicity. Cell viability remained consistently high, exceeding 90% across all tested concentrations compared to the untreated control (Fig. S2). Furthermore, phase-contrast microscopy observations confirmed that the cellular morphology of HK-2 cells remained normal and unchanged following exposure to these concentrations of Fe-Nag NPs, showing no signs of shrinkage, detachment, or membrane blebbing (Fig. S2).

Having established the excellent cytocompatibility of Fe-Nag NPs, we next investigated their cellular uptake behavior under both normal and injury-mimicking conditions to verify whether the compromised cellular environment of AKI affects the internalization efficiency of the nanoparticles. HK-2 cells were subjected to oxidative stress (200 μM H₂O₂ for 2 h) or nephrotoxic injury (10 μM cisplatin for 12 h) to simulate the pathological microenvironment of AKI, prior to incubation with Fe-Nag(F) NPs. Both fluorescence microscopy (Fig. 3A) and quantitative flow cytometry (Fig. 3B) consistently demonstrated that the cellular uptake of Fe-Nag(F) NPs was time-dependent across all three cellular states: untreated control, H₂O₂-treated, and cisplatin-treated cells. The intracellular fluorescence intensity significantly increased from 2 to 6 h of incubation. Notably, HK-2 cells subjected to H₂O₂ or cisplatin injury remained capable of effectively internalizing Fe-Nag(F) NPs. While the absolute uptake efficiency in injured cells showed a slight variation compared to healthy controls at certain time points, the overall capacity for nanoparticle uptake was robustly preserved under these pathological conditions. This key finding confirms that Fe-Nag NPs can be efficiently internalized by renal tubular cells even within a compromised cellular environment, which is a fundamental prerequisite for their subsequent intracellular therapeutic action.

Fig. 3.

The internalization of Fe-Nag NPs by renal tubular epithelial cells. (A) Fluorescence imaging results of Fe-Nag after co-incubation with HK-2 cells for 2, 4, or 6 h under normal, H₂O₂-induced, or cisplatin-induced conditions (Scale bar, 50 μm). (B) Flow cytometry results of Fe-Nag after co-incubation with HK-2 cells for 2, 4, or 6 h.

3.4. The biodistribution of Fe-Nag NPs in kidney tissues

To evaluate the renal targeting capability of our nanoplatform, we systematically investigated the in vivo biodistribution of Fe-Nag NPs. The experimental design for this study is schematically illustrated in Fig. 4A. Fe-Nag(I) NPs were intravenously injected into three mouse models: sham-operated controls, IR-AKI, and Cis-AKI. The ex vivo fluorescence imaging of major organs harvested at 2 h post-injection revealed that Fe-Nag(I) NPs were effectively delivered to the kidneys. As shown in Fig. 4B, conspicuous fluorescence signals were observed in the kidneys across all three experimental groups. Most importantly, quantitative analysis of the fluorescence intensity (Fig. 4C) demonstrated that the renal accumulation of Fe-Nag(I) NPs in both IR-AKI and Cis-AKI models was comparable to that in the sham group. This indicates that the pathological state of acute kidney injury did not diminish the renal distribution of our ultra-small nanoparticles, ensuring a sustained and efficient delivery—a fundamental prerequisite for achieving therapeutic efficacy. Concurrently, while observable signals were also detected in the liver, which is consistent with the typical clearance pathways of systemically administered nanomaterials, the therapeutic efficacy was predominantly localized to the kidneys. This renal-specific action can be attributed to the synergy between efficient organ accumulation and site-specific drug release. Specifically, our ultra-small Fe-Nag NPs can effectively penetrate the renal filtration barrier to achieve high accumulation in the kidneys (as evidenced in Fig. 4B–C). More importantly, the coordination structure is designed to be highly pH-sensitive; the NPs remain stable in the neutral environment (pH 7.4) of the systemic circulation and normal tissues - thereby minimizing off-target release and systemic toxicity - but undergo rapid dissociation in the acidic microenvironment typically associated with AKI-induced metabolic acidosis and intracellular lysosomal compartments. This dual-responsive mechanism ensures that the active naringenin is released specifically within the injured renal tissue, maximizing the therapeutic window.

Fig. 4.

The biodistribution of Fe-Nag NPs in kidney tissues. (A) Schematic diagram of the in vivo distribution experiment of Fe-Nag. (B) In vivo imaging results of indocyanine green-labeled Fe-Nag in major organs, and (C) quantitative analysis results of fluorescence signals (n = 3). (D) Representative immunofluorescence co-staining images demonstrating the specific intra-renal localization of Fe-Nag NPs. FITC-labeled Fe-Nag NPs are shown in green, and proximal tubular epithelial cells are immunostained with LRP2 (Megalin) in red. (Scale bar, 100 μm). (E) Quantitative detection results of Nag in kidney tissue (n = 3). Data are presented as mean ± SD. **P < 0.01.

Building upon the efficient cellular internalization observed in vitro, we further sought to confirm the specific intra-renal targeting potential of the platform in vivo. To precisely determine the intra-renal distribution of the nanoparticles, we performed immunofluorescence co-staining on frozen kidney sections using LRP2 (a specific marker for proximal tubular epithelial cells). As shown in Fig. 4D, the green fluorescence signal from FITC-labeled Fe-Nag NPs exhibited a high degree of spatial overlap with the red fluorescence of LRP2-positive renal tubules in both healthy and AKI models. This highly specific tubular localization, combined with the robust cellular uptake observed in injured HK-2 cells, collectively validates the robust targeting potential of the Fe-Nag NP platform toward its primary therapeutic site in AKI. The precise delivery of Fe-Nag NPs to the injured site ensures that the therapeutic agent (naringenin) is concentrated where it is most needed, thereby providing a solid foundation for its potent renoprotective effects.

To unequivocally quantify the delivery efficiency of the therapeutic agent, we directly measured the renal concentration of naringenin using HPLC. Mice from all three models (Sham, IR-AKI, Cis-AKI) were administered either free Nag or Fe-Nag NPs at an equivalent Nag dose (10 mg/kg). As quantitatively demonstrated in Fig. 4E, the renal concentration of Nag was significantly higher in mice treated with Fe-Nag NPs compared to those receiving free Nag in every experimental group. This result provides direct and conclusive evidence that the nano-formulation dramatically enhances the renal accumulation of the active drug.

3.5. Fe-Nag NPs treatment ameliorates kidney injury in IR-AKI mice

To evaluate the therapeutic efficacy of Fe-Nag NPs, we employed a murine model of ischemia-reperfusion acute kidney injury (IR-AKI). The experimental timeline is outlined in Fig. 5A. Mice were randomly assigned to four groups and received their respective treatments via tail vein injection at 2 h post-IRI induction. Renal function and tissue integrity were assessed 24 h later. The renoprotective effect of Fe-Nag NPs was first evident in the marked improvement of renal function. As shown in Fig. 5B, Scr levels were significantly elevated in the IR-AKI + PBS group compared to the Sham group. Treatment with free Nag provided a moderate but significant reduction in Scr levels compared to the PBS control (P < 0.05). Importantly, administration of Fe-Nag NPs resulted in a dramatically more pronounced decrease in Scr, achieving a significantly lower level than that in the free Nag group (P < 0.001). A parallel, and even more striking, therapeutic advantage was observed in the levels of BUN. As quantified in Fig. 5C, free Nag treatment effectively lowered BUN levels compared to the PBS control (P < 0.01). However, Fe-Nag NP treatment drove a far more robust reduction in BUN, outperforming free Nag with an extremely high level of statistical significance (P < 0.0001).

Fig. 5.

Fe-Nag NPs treatment ameliorates kidney injury and inflammation in IR-AKI mice. (A) Schematic diagram of the pharmacodynamic experiment of Fe-Nag. The changes of (B) serum creatinine (Scr) and (C) blood urea nitrogen (BUN) (n = 6). (D) H&E staining images of kidney tissues (Scale bar, 100 μm). (E) The expression levels of NGAL protein in kidney tissues measured by Western blotting, and (F) gray scale analysis results (n = 3). Immunofluorescence staining images of (G) KIM-1 and (H) NGAL in kidney tissue (Scale bar, 100 μm). The changes of (I) TNF-α and (J) IL-6 measured by ELISA. Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

To further explore the therapeutic window of Fe-Nag NPs and address their potential for clinical translation, we evaluated their efficacy under both prophylactic and delayed administration scenarios. First, we administered the treatments 30 min prior to IRI induction to assess the preventive potential. As shown in Fig. S4, pre-treatment with Fe-Nag NPs almost entirely abrogated the elevation of Scr and BUN, maintaining renal function at levels nearly comparable to the Sham group, which suggests that early intervention effectively intercepts the initial burst of oxidative stress and inflammatory cascades. Furthermore, we investigated the remedial capacity of Fe-Nag NPs by delaying administration to 12 h post-IRI, a time point characterized by established tubular necrosis. Remarkably, even with this delayed intervention, Fe-Nag NPs still conferred significant protection, as evidenced by substantially lower Scr and BUN levels compared to the PBS and free Nag groups (Fig. S5). Although the absolute levels of renal dysfunction markers were higher than those in the 2-h treatment group, the superior performance of Fe-Nag NPs over the free drug at this late stage underscores the robustness and broad therapeutic window of our nano-delivery system. A comparative analysis of these different administration schedules reveals a time-dependent efficacy profile for Fe-Nag NPs. While the prophylactic group (−0.5h) achieved near-complete preservation of renal function, this protocol serves primarily as a 'proof-of-principle' for early intervention rather than a common clinical scenario, as AKI onset is rarely predictable in practice. Conversely, the delayed group (+12h) showed a significant but reduced therapeutic benefit, likely due to the progression of irreversible tubular necrosis which limits the scope of rescue therapy. Consequently, the 2-h post-IRI administration was selected as our primary therapeutic model because it represents the critical 'golden hour' for intervention. At this stage, the initial molecular injury has been triggered, but widespread histological damage has not yet become irreversible. The robust protection observed at 2 h demonstrates that Fe-Nag NPs can effectively intercept the transition from acute stress to established organ failure, striking a pragmatic balance between experimental rigor and clinical relevance.

We next sought to confirm these functional improvements with histopathological evidence. H&E staining of kidney tissues (Fig. 5D) revealed severe tubular damage in the IR-AKI + PBS group, characterized by tubular dilation, epithelial cell necrosis, and extensive cast formation. While treatment with free Nag attenuated these pathological changes to a certain extent, the kidneys from the Fe-Nag NP-treated group exhibited a notably superior preservation of renal histoarchitecture. The tubules appeared more intact, with significantly reduced necrosis and cast formation, demonstrating that Fe-Nag NPs provide significantly stronger protection against I/R-induced structural injury than free Nag.

To further substantiate the renoprotective effects at the molecular level, we assessed the expression of established biomarkers of tubular injury, Kidney Injury Molecule-1 (KIM-1) and Neutrophil Gelatinase-Associated Lipocalin (NGAL). Immunofluorescence staining revealed a pronounced upregulation of both KIM-1 (Fig. 5G) and NGAL (Fig. 5H) in the renal tubules of the IR-AKI + PBS group. This injury signature was moderately attenuated in the free Nag-treated group. Critically, treatment with Fe-Nag NPs resulted in a far more substantial reduction in the expression of both damage markers. This striking decrease was further confirmed and quantified at the protein level for NGAL via Western blot (Fig. 5E and F). The concerted downregulation of KIM-1 and NGAL by Fe-Nag NPs provides compelling molecular evidence that our nanoformulation effectively mitigates the underlying tubular epithelial cell damage, an effect that is significantly more potent than that achieved by free naringenin.

Given the central role of inflammation in the pathogenesis of AKI, we further investigated the anti-inflammatory efficacy of the treatments by quantifying the renal levels of key pro-inflammatory cytokines. ELISA revealed that the renal concentrations of both tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) were sharply elevated in the IR-AKI + PBS group (Fig. 5I and J). While free Nag treatment significantly reduced the levels of these cytokines compared to the PBS control, Fe-Nag NP treatment induced a markedly more potent suppression. The reduction in TNF-α (Fig. 5I) by Fe-Nag NPs was significantly greater than that achieved by free Nag (P < 0.001). Similarly, Fe-Nag NPs were substantially more effective than free Nag in attenuating IL-6 (Fig. 5J, P < 0.01).

Given the profound anti-inflammatory effect observed, we sought to elucidate the underlying molecular mechanism by focusing on the NF-κB pathway, a master regulator of inflammation in AKI. We specifically examined the activation of the canonical NF-κB cascade through both immunofluorescence and Western blot analyses. Immunofluorescence staining revealed a robust elevation in the expression of phosphorylated RELA (p-RELA) and its upstream functional marker p-IκBα in the renal tissues of the IR-AKI + PBS group (Fig. 6A), indicating widespread activation of this pathogenic pathway. This activation was moderately suppressed by traditional free Nag treatment. Strikingly, targeted administration of Fe-Nag NPs resulted in a far more effective inhibition, markedly reducing the robust fluorescent signals of both phosphorylated proteins, indicative of a potent in vivo blockade of NF-κB pathway activation.

Fig. 6.

The anti-inflammatory mechanism of Fe-Nag NPs. Immunofluorescence staining images of (A) p-RELA and p-IκBα in kidney tissue (Scale bar, 100 μm). (B) The expression levels of p-IKKα, IKKβ, p-IκBα, IκBα, p-RELA, and RELA in kidney tissues measured by Western blotting. (C-E) Quantitative densitometric analysis of the relative phosphorylation ratios of p-IKKα/IKKβ (C), p-IκBα/IκBα (D), and p-RELA/RELA (E) (n = 3). Data are presented as mean ± SD. **P < 0.01, ***P < 0.001, and ****P < 0.0001.

To address the precise mechanisms driving this robust NF-κB blockade and to move beyond downstream phenotypic observations, we further investigated the upstream regulatory events of the canonical NF-κB cascade. Typically, NF-κB activation is triggered by the phosphorylation of the IKK complex, which subsequently phosphorylates the inhibitor IκBα, leading to its degradation and the release of active, phosphorylated RELA (p-RELA). Our in vivo Western blot analysis confirmed this pathogenic cascade in the IR-AKI model. The ratios of p-IKKα/IKKβ, p-IκBα/IκBα, and p- RELA/RELA were all sharply elevated in the renal tissues of the IR-AKI + PBS group (Fig. 6B–E), elucidating the upstream molecular reasons for the observed NF-κB nuclear translocation. Although traditional free Nag treatment resulted in a moderate decrease in these functional upstream markers, targeted intervention with Fe-Nag NPs achieved a remarkably deep and comprehensive suppression of the entire phosphorylation cascade. The phosphorylation levels of IKKα, IκBα, and the RELA subunit in the Fe-Nag NP-treated group were curtailed to levels significantly lower than those in the free Nag group. Together, these deeper mechanistic insights demonstrate that Fe-Nag NPs exert their potent anti-inflammatory effects by intercepting the NF-κB pathway at its upstream IKK/IκBα signaling nexus, thereby preventing downstream transactivational cascades and cytokine bursts in AKI.

3.6. Fe-Nag NPs treatment ameliorates kidney injury in Cis-AKI mice

To validate the broad-spectrum therapeutic potential of our nanoplatform, we further evaluated its efficacy in a Cis-AKI model, which represents a common form of clinical nephrotoxic injury. The renoprotective effect of Fe-Nag NPs was first confirmed by the assessment of renal function. As expected, cisplatin administration led to a sharp increase in Scr and BUN levels. Treatment with Nag modestly mitigated this functional impairment. Strikingly, mice treated with Fe-Nag NPs exhibited a far more significant recovery. Quantitative analysis demonstrated that the reduction in Scr (Fig. 7A) achieved by Fe-Nag NPs was significantly greater than that by free Nag (P < 0.01). An even more pronounced superior effect was observed in BUN levels (Fig. 7B), where Fe-Nag NPs outperformed free Nag with a high level of statistical significance (P < 0.001).

Fig. 7.

Fe-Nag NPs treatment ameliorates kidney injury and inflammation in Cis-AKI mice. The changes of (A) serum creatinine (Scr) and (B) blood urea nitrogen (BUN) (n = 6). (C) H&E staining images of kidney tissues (Scale bar, 100 μm). Immunofluorescence staining images of (D) KIM-1 in kidney tissue (Scale bar, 100 μm). The changes of (E) TNF-α and (F) IL-6 measured by ELISA (n = 6). (G) The expression levels of p-IKKα, IKKβ, p-IκBα, IκBα, p-RELA, and RELA in kidney tissues measured by Western blotting. (H-J) Quantitative densitometric analysis of the relative phosphorylation ratios of p-IKKα/IKKβ (H), p-IκBα/IκBα (I), and p-RELA/RELA (J) (n = 3). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

The functional improvement conferred by Fe-Nag NPs was corroborated by histopathological examination. H&E staining of kidney tissues from the Cis-AKI model (Fig. 7C) revealed severe tubular injury in the PBS-treated group, characterized by extensive tubular necrosis, epithelial cell vacuolization, and cast formation. While free Nag treatment alleviated these pathological changes to a moderate degree, the renal architecture in the Fe-Nag NP-treated group was notably better preserved. The tubules displayed significantly less necrosis and maintained greater structural integrity, demonstrating that Fe-Nag NPs provide superior histoprotection against cisplatin-induced injury compared to the free drug.

To molecularly substantiate the attenuation of tubular damage, we assessed the expression of the specific injury biomarker KIM-1 via immunofluorescence staining. As depicted in Fig. 7D, a pronounced induction of KIM-1 was observed in the renal tubules of cisplatin-injured mice treated with PBS. This signal was moderately reduced in the free Nag-treated group. In stark contrast, treatment with Fe-Nag NPs resulted in a far more effective suppression of KIM-1 expression, underscoring its superior capacity to mitigate the specific molecular markers of cisplatin-induced tubular epithelial injury.

Furthermore, to investigate the anti-inflammatory effects of the treatments in the Cis-AKI model, we quantified the renal levels of key pro-inflammatory cytokines. ELISA results confirmed a significant upregulation of TNF-α and IL-6 following cisplatin injury (Fig. 7E and F). Consistent with the trends observed in the IR-AKI model, Fe-Nag NP treatment induced a markedly more potent suppression of these cytokines compared to free Nag. The reduction in TNF-α (Fig. 7E) achieved by Fe-Nag NPs was significantly greater than that of the free drug (P < 0.001). A parallel, significant enhancement in IL-6 suppression was also observed in the Fe-Nag NP group (Fig. 7F, P < 0.05).

We subsequently explored whether the superior anti-inflammatory effect of Fe-Nag NPs in the Cis-AKI model was likewise mechanistically linked to the blockade of the canonical NF-κB signaling cascade. Western blot analysis of renal tissues demonstrated a robust activation of this pathogenic pathway following cisplatin-induced injury, as evidenced by the sharp elevation in the phosphorylation levels of IKKα/β, IκBα, and the RELA (p65) subunit (Fig. 7G). While treatment with free Nag moderately mitigated this phosphorylation cascade, intervention with Fe-Nag NPs achieved a remarkably deeper and more comprehensive suppression. Quantitative densitometric analysis revealed that Fe-Nag NPs significantly curtailed the relative phosphorylation ratios of p-IKKα/IKKβ (Fig. 7H), p-IκBα/IκBα (Fig. 7I), and p-RELA/RELA (Fig. 7J) to levels dramatically lower than those in the free Nag group. The concerted inhibition of the upstream IKK/IκBα regulatory complex and the downstream transactivator RELA in the Cis-AKI model underscores a consistent and key mechanism of action for Fe-Nag NPs. This result, perfectly corroborating our findings in the IR-AKI model, firmly establishes the targeted nanoformulation as a potent and broad-spectrum interceptor of NF-κB-driven inflammation in acute kidney injury.

3.7. Fe-Nag NPs treatment prevents the progression to chronic kidney disease

Given the critical link between severe AKI and subsequent progression to chronic kidney disease (CKD), we investigated the long-term renoprotective potential of Fe-Nag NPs. As schematically outlined in Fig. 8A, the therapeutic efficacy was evaluated over an extended period following IR-AKI induction. Strikingly, the initial functional advantage conferred by Fe-Nag NPs translated into a sustained protection against the progression of renal dysfunction. Assessment of renal function at the endpoint revealed that mice treated with Fe-Nag NPs maintained significantly lower levels of Scr and BUN compared to those receiving free Nag. Quantitative analysis demonstrated that the reduction in Scr (Fig. 8B) achieved by Fe-Nag NPs was significantly greater than that by free Nag (P < 0.001). A parallel, significant enhancement was observed in BUN levels (Fig. 8C), where Fe-Nag NPs outperformed free Nag (P < 0.01).

Fig. 8.

Fe-Nag NPs treatment prevents the progression to chronic kidney disease. (A) Schematic diagram of the pharmacodynamic experiment of Fe-Nag. The changes of (B) serum creatinine (Scr) and (C) blood urea nitrogen (BUN) (n = 6). (D) The changes of hydroxyproline content in renal tissues. (E) Masson staining images of kidney tissues (Scale bar, 100 μm). (F) The expression levels of α-SMA and TGF-β1proteins in kidney tissues measured by Western blotting, and (G-H) gray scale analysis results (n = 3). Immunofluorescence staining images of (I) TGF-β1, (J) E-Cadherin and (K) α-SMA in kidney tissue (Scale bar, 100 μm). *P < 0.05, **P < 0.01, and ***P < 0.001.

We next assessed renal fibrosis, a hallmark of progressive CKD, by quantifying the hydroxyproline content, a direct measure of collagen deposition. As shown in Fig. 8D, a significant increase in hydroxyproline was observed in the kidneys of the IR-AKI model vehicle group, indicating the initiation of maladaptive fibrotic processes. While free Nag treatment showed a moderate anti-fibrotic effect, Fe-Nag NP administration resulted in a dramatically greater reduction of hydroxyproline content (P < 0.001 vs. free Nag). The anti-fibrotic efficacy was further corroborated by histopathological examination using Masson's trichrome staining. As depicted in Fig. 8E, extensive blue-stained collagen fiber deposition was observed in the interstitial areas of the vehicle-treated AKI group. While free Nag treatment modestly reduced this fibrotic area, the kidneys from the Fe-Nag NP-treated group exhibited a markedly superior reduction in collagen deposition, demonstrating significantly better preservation of renal architecture.

To elucidate the molecular mechanism underlying this anti-fibrotic effect, we analyzed key proteins driving renal fibrosis. Western blot analysis (Fig. 8F) and subsequent grayscale quantification (Fig. 8G and H) demonstrated a marked upregulation of α-SMA (a marker of activated myofibroblasts) and TGF-β1 (a master profibrotic cytokine) in the AKI vehicle group. While free Nag moderately downregulated their expression, Fe-Nag NP treatment induced a substantially stronger suppression of both proteins. This potent inhibition of the profibrotic pathway was further confirmed and spatially resolved by immunofluorescence staining. As shown in Fig. 8I–K, Fe-Nag NPs more effectively attenuated the expression of TGF-β1 and the pathological induction of α-SMA in the interstitial area, while better preserving the expression of E-Cadherin (an epithelial marker), compared to the free drug. This pattern indicates that Fe-Nag NPs more effectively counteract the progression of epithelial-to-mesenchymal transition (EMT) and myofibroblast activation. In summary, by integrating biochemical, histological, and molecular evidence, we conclusively demonstrate that Fe-Nag NPs confer superior long-term renoprotection by robustly targeting the core TGF-β1/α-SMA fibrotic axis and mitigating the maladaptive processes that drive the transition from AKI to CKD.

3.8. Safety evaluation of Fe-Nag NPs treatment

Prior to considering any in vivo application, the hemocompatibility of Fe-Nag NPs was assessed using a hemolysis assay. Red blood cells (RBCs) were incubated with various concentrations of Fe-Nag NPs, with 0.9% NaCl and deionized water serving as the negative and positive controls, respectively. The hemolysis ratio was calculated based on the absorbance of the supernatants at 541 nm. As shown in Fig. S6, the positive control (deionized water) induced complete hemolysis (∼99.5%), while the negative control (0.9% NaCl) showed a negligible background hemolysis ratio. Critically, even at the highest tested concentration of 2 mg/mL, the hemolysis ratio induced by Fe-Nag NPs remained exceptionally low at approximately 2.45%. Across the entire concentration range (0.25 - 2 mg/mL), all hemolysis ratios were well below the 5% threshold, which is the internationally recognized standard for excellent hemocompatibility in biomaterials [33].

We further evaluated the systemic biosafety of Fe-Nag NPs following a single intravenous administration in healthy mice. Key indicators of hepatic and renal function, including AST, ALT, Scr, and BUN, were measured 24 h post-injection. As shown in Fig. S7, all these biochemical parameters in the Fe-Nag NP-treated group were comparable to those in the PBS control group, showing no significant differences. Moreover, histopathological examination of major organs (heart, liver, spleen, lungs, and kidneys) by H&E staining (Fig. S8) revealed no apparent signs of toxicity, inflammation, or morphological damage in the Fe-Nag NP-treated group. The tissue structures and cellular integrity were well-preserved and indistinguishable from those in the PBS control group.

To further assess the safety of Fe-Nag NPs under a clinically relevant multi-dose regimen, we evaluated systemic toxicity after repeated administration, following the same treatment protocol as the long-term efficacy study in Section 3.7 (normal mice). Biochemical analysis of serum markers for hepatic (AST, ALT) and renal (Scr, BUN) function, collected at the end of the study, confirmed that all parameters in the Fe-Nag NP-treated group remained at levels comparable to those in the PBS control group (Fig. S9). Consistently, histopathological assessment of major organs (heart, liver, spleen, lungs, and kidneys) via H&E staining (Fig. S10) revealed normal tissue architecture and cellular morphology, with no evidence of chronic inflammation, fibrosis, or other pathological changes attributable to the repeated administration of Fe-Nag NPs. Furthermore, to specifically address potential concerns regarding long-term iron accumulation, Prussian blue staining was performed on the kidney tissues. The results (Fig. S11) demonstrated no detectable Fe³⁺ deposition or retention in the kidneys of the Fe-Nag NP-treated group compared to the PBS control, effectively ruling out the risk of renal iron overload and associated oxidative damage following repeated administration.

Together with the single-dose safety data, these results from a multi-dose regimen provide a comprehensive safety profile, demonstrating that Fe-Nag NPs exhibit no appreciable systemic or organ-specific toxicity even upon repeated administration. This robust biosafety evidence strongly supports the feasibility of further clinical translation for chronic kidney disease management.

4. Discussion

The primary finding of this study is that the metal-organic nanocomposite Fe-Nag NPs, rationally designed based on transcriptomic insights, serve as a highly effective and targeted nanotherapeutic platform for AKI by specifically delivering naringenin to injured kidneys to potentently suppress the NF-κB signaling pathway. This work distinguishes itself by seamlessly integrating bioinformatics prediction with advanced nanotechnology, offering a novel solution to the long-standing challenge of inefficient renal delivery for bioactive natural products.

Our findings robustly confirm that naringenin is a direct inhibitor of the NF-κB pathway, a master regulator of inflammation in AKI [34]. This interaction is supported by our molecular docking analysis, which yielded a significant binding energy, demonstrating a high affinity between naringenin and the Rela subunit compared to other candidate transcription factors (Table S1). While the anti-inflammatory properties of naringenin have been previously documented in various disease models [35,36], its therapeutic efficacy in AKI has been severely limited by poor pharmacokinetics. Our approach leverages the unique advantages of ultra-small NPs. The sub-10 nm size of Fe-Nag NPs is a critical design feature, enabling passive targeting via glomerular filtration - a mechanism that is particularly enhanced in the leaky vasculature of injured kidneys [37].

It is important to distinguish between cellular internalization and systemic targeting potential. Our in vitro data (Fig. 3) demonstrated that even under pathologically compromised conditions, HK-2 cells maintain a robust capacity for internalizing Fe-Nag NPs, which serves as a necessary prerequisite for intracellular drug action. However, the true "targeting potential" of our platform is more conclusively evidenced by the in vivo biodistribution and histological analysis (Fig. 4). The significantly higher renal accumulation of Nag from Fe-Nag NPs compared to the free drug, as quantitatively demonstrated by HPLC and the precise localization within renal tubules observed in frozen sections, directly translates to its superior efficacy in two distinct AKI models. Our results are in agreement with the growing recognition of nanotechnology in drug delivery, but we specifically advance the field by demonstrating the successful application of a targeted NP platform for therapy in acute renal diseases, an area less explored compared to cancer or chronic fibrosis.

Despite the promising results, several limitations of this study should be acknowledged. First, while we have established the central role of NF-κB inhibition, the comprehensive downstream mechanisms, such as its interplay with other regulated pathways like TGF-β, warrant further investigation. Second, the long-term fate and potential biodegradation pathways of the Fe-Nag NPs in the body remain to be fully elucidated, although our repeated-dose safety data provides strong initial support for their biocompatibility. Third, the current models, while well-established, primarily reflect the initiation and acute phases of AKI; evaluating the therapeutic window and efficacy of Fe-Nag NPs in a model of established AKI or in a more complex comorbid setting would be valuable for future clinical translation.

Looking forward, the Fe-Nag NP platform opens several exciting avenues for future research. The inherent coordinative nature of NCPs allows for the integration of other metal ions or therapeutic agents, enabling combination therapy and diagnostic imaging (e.g., MRI with Fe³⁺), paving the way for theranostic applications. Furthermore, the "data-driven" design principle demonstrated here - from public database mining to targeted nanoplatform construction - establishes a translatable paradigm that can be adapted to discover and deliver novel therapeutics for other inflammation-driven diseases.

In conclusion, this study not only presents Fe-Nag NPs as a potent and safe nanotherapeutic for AKI but also provides a broadly applicable framework for overcoming the delivery challenges of natural products, thereby unlocking their full therapeutic potential for precise medical interventions.

CRediT authorship contribution statement

Li-Bin Zhou: Writing – original draft, Methodology, Formal analysis. Min-Zhi Zhou: Formal analysis, Data curation. Yan-Guo Li: Writing – original draft, Formal analysis. Zong-Jian Wu: Software, Methodology, Investigation. Xuan-Ze Peng: Methodology, Formal analysis. Yang Sun: Methodology, Formal analysis. Hai-Bo Mao: Methodology, Investigation, Formal analysis, Data curation. Hui-Min Long: Supervision. Min Yin: Supervision. Xue-Ying Tan: Supervision, Funding acquisition. Jian-Ting Xu: Writing – review & editing, Funding acquisition. Jing-Bo Hu: Writing – review & editing, Conceptualization.

Funding

The financial support received for this article was provided by the Nature Science Foundation of Zhejiang province (LGF22H090023), Ningbo Public Welfare Science and Technology Project (2023S188, 2024S163), and Ningbo Key Clinical Speciality Construction Project (2023-BZZ).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the Supplementary data to this article:

Data availability

Data will be made available on request.