Layered double hydroxide nanocarriers loaded with butylphthalide attenuate the AKI-CKD transition by regulating mitophagy

Abstract

Acute kidney injury (AKI) caused by ischemia-reperfusion (IR) is an independent risk factor for the progression of chronic kidney disease (CKD), yet there is a lack of effective clinical interventions. Although butylphthalide (NBP) has been proven to have multi-organ protective potential, its rapid in vivo metabolism and low bioavailability limit its clinical application. To overcome these limitations, we rationally designed and synthesized a layered double hydroxide (LDHs)-based nanocarrier system for NBP delivery (LDHs@NBP) via hydrothermal co-precipitation. Comprehensive characterization confirmed successful nanocomplex formation. Critically, LDHs@NBP exhibited accelerated NBP release under mildly acidic conditions, matching the pathological acidosis of injured and fibrotic renal tubules—thereby achieving pH-responsive drug release. Using both an in vivo rat model of unilateral renal ischemia-reperfusion injury (uIRI) and an in vitro TGF-β1-stimulated HK-2 cell model, we demonstrated that LDHs@NBP significantly attenuated renal dysfunction, suppressed interstitial fibrosis, and improved mitochondrial function. Importantly, all protective effects were abolished upon co-treatment with Mdivi-1, confirming mitophagy as the central mechanistic axis. Collectively, this study successfully constructed LDHs@NBP nanocomplexes with pH-responsive drug release properties. This system enhances mitophagy by activating the PINK1-Parkin pathway, thereby effectively blocking AKI-CKD transition. It provides a new strategy with good translational prospects for clinical intervention in kidney diseases.

Graphical abstract

Highlights

• •This study developed a novel layered double hydroxides (LDHs)-based nanocarrier system with precise pH-responsive drug release properties.
• •LDHs@NBP significantly enhances the therapeutic efficacy of NBP and attenuates the transition from AKI to CKD.
• •LDHs@NBP exerts renal protective effects by activating the PINK1-Parkin pathway and restoring mitophagy activity.

1. Introduction

Acute kidney injury (AKI) is a prevalent and life-threatening clinical syndrome associated with substantial morbidity and mortality [1]. Clinical studies have shown that even if AKI patients recover smoothly during the acute phase, more than 20% of them will progress to chronic kidney disease (CKD) [2,3]. Accumulating evidence implicates renal ischemia, hypoxia, oxidative stress, and consequent maladaptive fibrotic remodeling as central pathogenic mechanisms underlying the AKI-CKD transition [4]. However, no effective therapy currently exists to prevent or delay this progression in clinical practice.

Mitochondrial dysfunction is a central pathogenic driver of the AKI-CKD transition [5]. During renal ischemia-reperfusion (IR) injury, renal tubular epithelial cells (RTECs) undergo rapid loss of mitochondrial membrane potential and excessive reactive oxygen species (ROS) generation, triggering apoptosis, inflammation, and tubular damage [6]. To maintain mitochondrial homeostasis, cells rely on antioxidant defense systems and mechanisms such as mitophagy to ensure normal mitochondrial function [7]. Mitophagy is a selective pathway for clearing damaged mitochondria and is an important mechanism for balancing normal cellular functions [8]. Recent studies have shown that mitophagy plays a protective role in kidney diseases; thus, restoration of mitophagy flux in RTECs represents a mechanistically grounded and therapeutically promising strategy to halt the AKI-CKD transition [9].

Butylphthalide (NBP) is a novel synthetic neuroprotective agent independently developed in China and exhibits significant anti-inflammatory and antioxidant effects [10]. Our previous studies have confirmed that NBP plays a protective role in sepsis-induced AKI; however, its role in the AKI-CKD transition and the underlying mechanisms remain unclear [11]. Critically, NBP has the characteristics of low bioavailability and rapid metabolism in the body, which limit its therapeutic potential in kidney diseases.

Layered double hydroxides (LDHs) are a novel class of inorganic nanomaterials, featuring high drug loading capacity and excellent biocompatibility, and are widely applied in disease treatment and drug delivery [[12], [13], [14]]. Our previous research has confirmed that LDHs loaded with sorafenib can exert anti-liver fibrosis effects by regulating the PI3K/AKT pathway. Moreover, nanomedicine currently shows promising prospects for intervening in the AKI-CKD transition [15,16]. Therefore, this study developed an LDH-based nanocomposite for NBP delivery (LDHs@NBP), aiming to achieve the efficient delivery of NBP to damaged RTECs, thereby alleviating renal fibrosis and inhibiting AKI-CKD transition. We first synthesized and comprehensively characterized LDHs@NBP, and then evaluated its efficacy in both in vitro and in vivo models. Finally, we delved into the specific molecular mechanism by which it regulates mitophagy. This study is expected to provide a new strategy for the treatment of renal diseases with NBP.

2. Materials and methods

2.1. Chemicals and reagents

MgCl₂·6H₂O, AlCl₃·6H₂O, and NaOH were purchased from Aladdin Reagent Co., Ltd. DMEM/F12 and FBS were obtained from Procell Life Science & Technology Co., Ltd. Rats were obtained from Jiangsu Qinglongshan Biotechnology Co., Ltd. HK-2 cells were obtained from Cellcook Biotech Co., Ltd. The SCr, BUN, ALT, and AST detection kits were purchased from Njjcbio. Mito-Tracker Red and MitoSOX Red were obtained from Apexbio. The BCA assay kit, glutathione (GSH), adenosine triphosphate (ATP), ROS assay reagents, and the JC-1 mitochondrial membrane potential detection kit were all purchased from Beyotime. TGF-β1 (HY-P70543) and Mitochondrial division inhibitor-1 (Mdivi-1) (HY-15886) were purchased from MedChemExpress, while NBP was obtained from CSPC-NBP Pharmaceutical Co., Ltd. Fibronectin (FN) (AF5335), α-smooth muscle actin (α-SMA) (AF1032), LC3-I/II (AF5402), P62 (AF5384), PINK1 (DF7742), and Parkin (AF0235) were bought from Affinity Biosciences.

2.2. Animal grouping and model establishment

Male Sprague-Dawley rats (6-8 weeks of age) were housed under standard conditions for 7 days before experimentation and then randomly divided into sham operation (Sham group), unilateral ischemia-reperfusion injury (uIRI group), uIRI + LDHs (LDHs group), uIRI + NBP (NBP group), uIRI + LDHs@NBP (LDHs@NBP group), and uIRI + LDHs@NBP + Mdivi-1 (LDHs@NBP + M group). Before modeling, each group of rats was intraperitoneally injected with an equal volume of normal saline, LDHs (15 mg/kg), NBP (5 mg/kg) [17], and LDHs@NBP (20 mg/kg) for 7 consecutive days (the doses of LDHs and LDHs@NBP were converted based on the drug loading rate). Rats were anesthetized with isoflurane. In the Sham group, only the left kidney was exposed without clamping, while in the other groups, the left renal pedicle was clamped for 45 min using a non-invasive arterial clamp, and reperfusion was achieved by removing the clamp [18]. At 2 days, 7 days, and 14 days after the operation, the rats were euthanized, and their serum and organs were collected (Fig. 2A). The study protocol received ethical approval from the Animal Ethics Committee of Bengbu Medical University (Approval Number: 2025753).

Fig. 2.

Evaluation of drug safety. (A) Flowchart of rat model establishment (Created in BioRender. Yan, Y). (B-E) Levels of ALT, AST, SCr, and BUN in rats (n = 5). (F) HE staining of rat heart, liver, spleen, lung, and kidney (scale: 200 μm) (n = 6). (G) SCr levels in rats (n = 6). (H) BUN levels in rats (n = 6). Data are presented as mean ± SD, *p < 0.05, **p < 0.01 and ***p < 0.001.

2.3. Cell culture and treatment

HK-2 cells were routinely cultured in complete DMEM/F12 medium at 37 °C and 5% CO₂. The experimental groups were set as Control (Control group), TGF-β1 (TGF-β group), TGF-β1 + LDHs (LDHs group), TGF-β1 + NBP (NBP group), TGF-β1 + LDHs@NBP (LDHs@NBP group), and TGF-β1 + LDHs@NBP + Mdivi-1 (LDHs@NBP + M group). A fibrotic HK-2 cell model was induced by stimulating the cells with 10 ng/mL TGF-β1 for 48 h [19]. The Control group only had the culture medium replaced, while the other groups were treated with different drugs. In the LDHs@NBP + M group, cells were pre-incubated with Mdivi-1 (5 μM) for 1 h before co-treatment with TGF-β1 and LDHs@NBP [20].

2.4. Synthesis of LDHs and LDHs@NBP

LDHs were synthesized via hydrothermal coprecipitation. Briefly, solutions containing 272 mg of NaOH and a mixed metal salt solution containing 241.4 mg of AlCl₃·6H₂O and 609.9 mg of MgCl₂·6H₂O were prepared, respectively. The solutions were stirred in a water bath at 60 °C for 1 h, then transferred to a reaction vessel (100 °C, 16 h). After centrifugation, resuspension, and freeze-drying, the product was stored for later use [21]. Then, 10 mg of LDHs was added to a solution containing 20 mg of NBP, and the mixture was reacted at 37 °C for 24 h. Subsequently, it was freeze-dried for storage.

2.5. Characterization

The LDHs and LDHs@NBP were imaged and observed by transmission electron microscopy (FEI Talos F200X G2). The particle size distribution and Zeta potential were determined by a Malvern particle size analyzer (Malvern). The structure and chemical composition were characterized by Fourier transform infrared spectroscopy (FTIR) (Thermo Fisher Scientific Nicolet iS20) and X-ray diffraction (XRD) (Rigaku SmartLab SE).

2.6. Drug loading detection

The ultraviolet maximum absorption peak of NBP was obtained by using a UV spectrophotometer (Shimadzu UV-3600i Plus) within the range of 200-800 nm. A stock solution of NBP was serially diluted to yield concentrations of 7.8, 15.6, 31.2, 62.5, 125, and 250 μg/mL, and the absorbance was measured at the ultraviolet maximum absorption peak to obtain the standard curve. The concentration and drug loading were calculated by measuring the ultraviolet absorption peak of the supernatant after centrifugation during the synthesis of LDHs@NBP using a UV spectrophotometer and substituting the values into the standard curve.

2.7. Cell viability assays

Cells were plated in 96-well microplates and treated with a concentration gradient of either LDHs or LDHs@NBP; cell viability was then quantified using the CCK-8 assay.

2.8. ROS assays

After the cells were successfully modeled, they were digested with trypsin and collected. A DCFH-DA solution was prepared, and the cells were analyzed using a flow cytometer (CytoFLEX, Beckman Coulter).

2.9. JC-1 and MitoSOX red

After the cells were inoculated in confocal culture dishes and the modeling was completed, they were incubated with the JC-1 probe or MitoSOX Red in the dark for 30 min. Then, they were observed and photographed.

2.10. Reagent kit testing

According to the operation instructions of the reagent kit, the contents of ATP and GSH in renal tissues and cells, as well as the contents of SCr, BUN, AST, and ALT in rat serum, were determined, respectively, and the absorbance values were read by an enzyme-labeled instrument.

2.11. Cellular immunofluorescence

Cells were seeded in confocal culture dishes. After model establishment, they were fixed with 4% paraformaldehyde (PFA) and blocked for 30 min. They were then incubated overnight with α-SMA (1:200) and FN (1:200), respectively. The next day, they were incubated with secondary antibodies. Fluorescence microscopy was used for observation and photography.

2.12. HE staining, masson staining, and immunofluorescence

Animal tissues were fixed in 4% PFA, paraffin-embedded, and sectioned at 4 μm. HE staining and Masson staining were performed, respectively. Renal injury severity was assessed by certified pathologists at our hospital using the Paller scoring system. For co-staining of LC3 and VDAC, the slides were incubated overnight at 4 °C with LC3 (1:150) and VDAC (1:150), followed by incubation with secondary antibodies.

2.13. Transmission electron microscopy (TEM)

Fresh renal tissue (1 mm³) was pre-fixed with glutaraldehyde and then fixed in 1% osmium tetroxide. It was dehydrated through an ethanol gradient and embedded in hard resin, followed by ultrathin sectioning. The sections were stained and observed under a TEM.

2.14. Western blot (WB)

Cell and renal tissue proteins were prepared according to the standard protocol. Proteins were resolved by SDS-PAGE, electrotransferred to PVDF membranes, and blocked with a commercial rapid-blocking buffer for 30 min. The membranes were incubated with α-SMA (1:2000), Fibronectin (FN) (1:1500), LC3-I/II (1:1000), P62 (1:1200), PINK1 (1:1000), and Parkin (1:1500). After incubation with secondary antibodies, the membranes were developed, and the gray values were analyzed.

2.15. Bioinformatic analysis

The AKI transcriptome data from GSE139061 and the CKD transcriptome data from GSE236379 on the GEO website were downloaded for differential analysis. The limma-voom process was adopted, with |logFC| > [mean(|logFC|) + 2sd(|logFC|)] as the threshold criterion and p < 0.05 as the standard to screen for significantly differentially expressed genes. Volcano plots were drawn using ggplot2, and heatmaps were drawn using pheatmap. The clusterProfiler was used to conduct gene ontology (GO) enrichment analysis on the gene set. With all genes as the background, hypergeometric tests were performed to screen for significant items with p < 0.05 and draw graphs.

2.16. Statistical analysis

Data are expressed as mean ± SD. Group comparisons between two conditions were conducted using a t-test, while differences across three or more groups were assessed by one-way analysis of variance with Tukey's multiple comparisons test. Statistical analyses and figure generation were carried out using SPSS and GraphPad Prism. p < 0.05 was considered statistically significant.

3. Results

3.1. Preparation and characterization of LDHs@NBP

LDHs@NBP were successfully prepared by the hydrothermal coprecipitation method (Fig. 1A). The results show that LDHs are a regular hexagon. With the successful loading of NBP, its layered structure can still be identified (Fig. 1B). Elemental mapping shows that magnesium and aluminum are well dispersed (Fig. 1F–G). Dynamic light scattering results indicated that the average particle sizes of LDHs and LDHs@NBP were 211.6 ± 2.3 nm and 243 ± 6.2 nm, respectively, and the Zeta potential decreased from 41.7 ± 0.7 mV to 33.3 ± 2.7 mV (Fig. 1C). XRD patterns showed that LDHs and LDHs@NBP had similar diffraction peaks, and FTIR spectra indicated that LDHs@NBP simultaneously possessed the characteristic peaks of NBP and LDHs, demonstrating that NBP was successfully loaded through coprecipitation (Fig. 1D and E). UV spectra revealed that NBP had a maximum absorption peak at 221 nm (Fig. 1H), and based on the standard curve, the drug loading capacity of LDHs@NBP was approximately 24.3% (Fig. 1I). In vitro drug release experiments showed that LDHs@NBP exhibited significantly accelerated NBP release in acidic environments, confirming the pH-responsive property of LDHs@NBP (Fig. 1J).

Fig. 1.

Preparation and characterization of LDHs@NBP. (A) Synthetic roadmap of LDHs@NBP (Created in BioRender. Yan, Y). (B) TEM images of LDHs and LDHs@NBP (scale bar: 100 nm). (C) Particle size distribution and Zeta potential of LDHs and LDHs@NBP. (D) XRD spectra of LDHs, NBP, and LDHs@NBP. (E) FTIR spectra of LDHs, NBP, and LDHs@NBP. (F) Element mapping images of LDHs (scale bar: 100 nm). (G) Element mapping images of LDHs@NBP (scale bar: 50 nm). (H) NBP ultraviolet full wavelength scanning curve. (I) NBP standard curve. (J) Drug release curves of LDHs@NBP in phosphate buffer solutions with different pH values.

3.2. Safety evaluation

After the administration of drugs to each group of rats was completed, the functions of their vital organs were evaluated before modeling. The levels of blood SCr, BUN, AST, and ALT in the Sham, LDHs, NBP, and LDHs@NBP groups of rats showed no significant fluctuations (Fig. 2B–E). HE staining results showed that no obvious pathological changes occurred in the heart, liver, spleen, lung, and kidney of rats in each group, indicating that LDHs and LDHs@NBP have good biocompatibility (Fig. 2F).

3.3. LDHs@NBP suppressed the AKI-CKD transition in vivo

After the completion of modeling in each group of rats, blood samples and kidneys were collected for detection at 2 days, 7 days, and 14 days post-operation. It was found that the renal function of the uIRI group was significantly impaired, while LDHs@NBP could improve renal function (Fig. 2G and H). Additionally, from 2 days to 14 days post-operation, progressive fibrosis occurred in the kidneys of the uIRI group, indicating the successful construction of our AKI-CKD model. LDHs@NBP could significantly reduce the degree of renal fibrosis (Fig. 3A–F). The above results suggest that compared with NBP, LDHs@NBP can more effectively alleviate renal fibrosis and thereby suppress the transition from AKI to CKD in vivo.

Fig. 3.

LDHs@NBP suppressed AKI-CKD transition in vivo. (A) HE staining and Masson staining of rat kidneys (scale bar: 200 μm) (n = 6). (B) Quantitative analysis of renal tubular injury score. (C) Quantitative analysis of renal fibrosis area. (D) Representative western blot images of renal fibrosis proteins and (E, F) Quantitative analysis of protein levels (n = 3). Data are presented as mean ± SD, *p < 0.05, **p < 0.01 and ***p < 0.001.

3.4. LDHs@NBP attenuated fibrosis in HK-2 cells

Cell viability of HK-2 cells treated with TGF-β1 and different concentrations of LDHs, NBP, and LDHs@NBP was detected by CCK8 assay. The results showed that 10 μg/mL LDHs@NBP significantly promoted cell proliferation compared with the same concentration of NBP, and this concentration of LDHs had no obvious toxicity to HK-2 cells. Therefore, this concentration was selected for subsequent experiments (Fig. 4A and B). Immunofluorescence and WB results indicated that TGF-β1 treatment led to cell fibrosis, suggesting that the in vitro model was successfully constructed. LDHs@NBP could significantly alleviate cell fibrosis, and its effect was better than that of NBP (Fig. 4C–F).

Fig. 4.

LDHs@NBP alleviated fibrosis in HK-2 cells. (A, B) Comparison of the survival rates of HK-2 cells treated with different concentrations of LDHs and LDHs@NBP (n = 3). (C) Representative western blot images of fibrosis markers and (D, E) Quantitative analysis of protein levels (n = 3). (F) Representative immunofluorescence images of fibrosis markers (scale bar: 50 μm) (n = 3). Data are presented as mean ± SD, *p < 0.05, **p < 0.01 and ***p < 0.001.

3.5. LDHs@NBP inhibited the AKI-CKD transition by activating mitophagy in vivo

Relevant data were downloaded from the GEO website for differential enrichment analysis, which indicated that mitophagy and the PINK1 pathway might play a role in AKI-CKD (Fig. 5A–E). WB was used to detect the levels of mitophagy markers in the Sham group and the uIRI group at different time points. After uIRI occurred in the kidneys, the level of mitophagy significantly decreased, and the PINK1 pathway was inactivated (Fig. 5F–J). Previous results showed that the degree of renal fibrosis was the most severe at 14 days after uIRI (Fig. 3A and C). To explore the mechanism more precisely, this time point was selected for subsequent verification. Immunofluorescence co-staining of the outer mitochondrial membrane protein VDAC and LC3, and TEM were used to detect the level of mitophagy in the kidneys of each group of rats (Fig. 6A and B); immunofluorescence was used to detect the expression of PINK1 (Fig. 6A); WB was used to detect the expression of mitophagy-related factor proteins in each group of rats (Fig. 6C–G). The above results indicated that the level of mitophagy in the uIRI group decreased and the PINK1 pathway was inactivated, while LDHs@NBP could restore the activity of the PINK1-Parkin pathway and increase the level of mitophagy, and the effect was better than that of NBP. In addition, by detecting the ATP and GSH levels in rat kidney tissues, it was found that LDHs@NBP significantly improved the mitochondrial function of rat kidneys (Fig. 7A and B).

Fig. 5.

Bioinformatics analysis and WB detection. (A) Heatmap display of the differential analysis of gene sets related to AKI and CKD. (B) Volcano plot illustrating differential expression between AKI- and CKD-associated gene sets, generated using a dynamically determined log₂FC threshold of 4.051292. (C) Heatmap showing the differential analysis of PINK1 groups. (D) Volcano plot showing the differential analysis of PINK1 groups, with a dynamic log2FC threshold of 2.173318 calculated. (E) GO enrichment analysis of PINK1-related genes. (F) Representative western blot images of mitophagy-related proteins and (G-J) Quantitative analysis (n = 3). Data are presented as mean ± SD, **p < 0.01 and ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6.

LDHs@NBP enhanced mitophagy levels. (A) Representative images of VDAC and LC3 co-staining in the kidney (scale bar: 50 μm) (n = 6), representative images of mitochondria in the kidney (scale bar: 500 nm) (n = 3), and immunofluorescence staining of PINK1 in the kidney (scale bar: 50 μm) (n = 6). (B) Quantitative analysis of VDAC and LC3 co-staining. (C) Representative western blot images of mitophagy-related proteins and (D-G) Quantitative analysis (n = 3). Data are presented as mean ± SD, *p < 0.05, **p < 0.01 and ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7.

LDHs@NBP enhanced mitophagy and improved mitochondrial function. (A) The content of ATP in renal tissue (n = 5). (B) The content of GSH in renal tissue (n = 5). (C) Masson staining of the kidney (scale bar: 200 μm) (n = 6), representative images of mitochondria in the kidney (scale bar: 500 nm) (n = 3), representative images of VDAC and LC3 co-staining (scale bar: 50 μm) (n = 6), and immunofluorescence staining of PINK1 in the kidney (scale bar: 50 μm) (n = 6). (D) Quantitative analysis of renal fibrosis area. (E) Quantitative analysis of VDAC and LC3 co-staining. (F) Representative western blot images of fibrosis proteins and mitophagy proteins, and (G-L) Quantitative analysis (n = 3). Data are presented as mean ± SD, *p < 0.05, **p < 0.01 and ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

To further verify the role of mitophagy, rats were treated with Mdivi-1. The Mdivi-1 group was administered the drug via intraperitoneal injection starting from the postoperative period (twice a week, 10 mg/kg) [22]. Rats were divided into the Sham-14d group, the uIRI + LDHs@NBP-14d (LDHs@NBP-14d group), and the uIRI + LDHs@NBP-14d + Mdivi-1 treatment (LDHs@NBP + M-14d group). The results showed that after Mdivi-1 treatment, mitophagy activity decreased, renal fibrosis in rats worsened, and the protective effect of LDHs@NBP was blocked (Fig. 7C–L). These results collectively indicate that LDHs@NBP can improve mitochondrial function by activating the PINK1-Parkin pathway, enhance mitophagy activity, and inhibit the AKI-CKD transition.

3.6. LDHs@NBP alleviated fibrosis in HK-2 cells by activating mitophagy

WB was used to detect the expression of LC3-I/II, PINK1, Parkin, and p62. The results showed that TGF-β1 treatment led to a decrease in mitophagy activity in HK-2 cells, while LDHs@NBP could restore the activity of the PINK1-Parkin pathway and increase the level of mitophagy (Fig. 8A–E). In addition, the mitochondrial function of cells treated with LDHs@NBP was significantly improved (Fig. 8F–J). HK-2 cells were treated with Mdivi-1 and divided into Control group, TGF-β1 + LDHs@NBP (LDHs@NBP group) and TGF-β1 + LDHs@NBP + Mdivi-1 (LDHs@NBP + M group). The levels of mitophagy and fibrosis indicators were detected. The results showed that after Mdivi-1 treatment, the level of mitophagy in the LDHs@NBP + M group decreased, and the degree of cell fibrosis increased, indicating that Mdivi-1 blocked the protective effect of LDHs@NBP (Fig. 9A–H). The above results collectively indicated that LDHs@NBP improved mitochondrial function and reduced cell fibrosis in RTECs in vitro by activating the PINK1-Parkin pathway and increasing the level of mitophagy.

Fig. 8.

LDHs@NBP enhanced the level of mitophagy in HK-2 cells. (A) Representative western blot images of mitophagy-related proteins in HK-2 cells and (B-E) Quantitative analysis (n = 3). (F) The content of ATP in HK-2 cells (n = 3). (G) The level of ROS in HK-2 cells and (H) quantitative analysis (n = 3). (I) Quantitative analysis of mitochondrial membrane potential in HK-2 cells. (J) Representative images of mitochondrial membrane potential (scale bar: 50 μm) and MitoSOX (scale bar: 20 μm) in HK-2 cells (n = 3). Data are presented as mean ± SD, *p < 0.05, **p < 0.01 and ***p < 0.001.

Fig. 9.

LDHs@NBP improved mitochondrial function and enhanced mitophagy in cells. (A) Representative western blot images of fibrotic proteins and mitophagy proteins in HK-2 cells and (B-G) Quantitative analysis (n = 3). (H) Representative immunofluorescence images of fibrotic markers in HK-2 cells (scale bar: 50 μm) (n = 3). Data are presented as mean ± SD, *p < 0.05, **p < 0.01 and ***p < 0.001.

4. Discussion

The transition from IR-induced AKI to CKD represents a critical unmet clinical challenge. Accumulating evidence implicates persistent mitochondrial dysfunction in RTECs as a central driver of this maladaptive progression. Nanomedicine-based delivery systems have emerged as promising tools for intervention in complex pathophysiological processes [23,24]. Our preliminary study found that LDHs@NBP can improve renal IR injury and hypoxia-reoxygenation-induced HK-2 cell damage by suppressing oxidative stress and apoptosis [21]. In this study, both in vitro and in vivo experiments confirmed that LDHs@NBP could effectively inhibit the AKI-CKD transition by activating the PINK1-Parkin pathway, enhancing mitophagy levels, and improving mitochondrial function (Fig. 10). This discovery not only reveals a new mechanism of NBP in renal protection but also provides a new nano-delivery strategy to overcome its metabolic limitations.

Fig. 10.

Schematic representation of LDHs@NBP attenuating the AKI-CKD transition by regulating mitophagy (Created in BioRender. Yan, Y). Left panel: In the rat uIRI model, impaired mitophagy leads to the accumulation of damaged mitochondria and promotes the transition from AKI to CKD. Right panel: LDHs@NBP treatment activates the mitophagy, restores mitochondrial function, and markedly attenuates renal interstitial fibrosis.

Mitophagy deficiency is a pivotal pathogenic driver underlying the maladaptive transition from AKI to CKD. As the core organelle for energy metabolism in RTECs, mitochondria are often damaged in IR injury [25]. Prior work has documented an initial, transient upregulation of mitophagy following IR insult, interpreted as a compensatory response; however, sustained reperfusion leads to progressive impairment of autophagic flux, resulting in defective clearance of damaged mitochondria [26]. Consistently, our study demonstrates that uIRI in rats triggers a marked decline in mitophagy, culminating in the accumulation of dysfunctional mitochondria, ROS burst, and subsequent fibrosis of the tubulointerstitial tissue. Critically, LDHs@NBP treatment could significantly reverse the above changes, while the Mdivi-1 could counteract its protective effect, suggesting that mitophagy deficiency may be an important driver for the AKI-CKD transition. Similarly, Cobaltosic oxide-polyethylene glycol-triphenylphosphine nanoparticles have been independently shown to suppress the AKI-CKD transition via BNIP3-dependent mitophagy induction, which is consistent with the results of this study [27].

The PINK1-Parkin pathway is one of the classic mechanisms regulating mitophagy. When mitochondrial membrane potential decreases, PINK1 accumulates stably on the outer mitochondrial membrane and recruits Parkin, which then ubiquitinates mitochondrial substrate proteins to tag damaged mitochondria, thereby initiating selective autophagy for their clearance [28]. Studies have shown that in PINK1 or Parkin knockout mouse models, mitochondrial damage and the severity of AKI or CKD are more severe, indicating that this pathway plays an important role in kidney diseases [[29], [30], [31]]. Consistent with this, our study demonstrates that uIRI induces progressive inactivation of the PINK1-Parkin axis, concomitant with declining mitophagic levels, accumulation of dysfunctional mitochondria, and eventual transition to tubulointerstitial fibrosis. Strikingly, LDHs@NBP treatment not only restored PINK1 and Parkin protein expression but also reactivated downstream mitophagy, rescued mitochondrial function, and markedly attenuated renal fibrosis. In contrast, Mdivi-1 treatment suppressed the activity of the PINK1-Parkin pathway and mitophagy levels, thereby blocking the kidney-protective effects of LDHs@NBP. Mdivi-1 is a small-molecule drug that specifically inhibits Drp1-dependent excessive mitochondrial fission. Previous studies have shown that Mdivi-1 can suppress Drp1-mediated mitophagy in the kidneys and inhibit the activity of the PINK1-Parkin pathway. This study also observed that Mdivi-1 inhibits the activity of the PINK1-Parkin pathway and mitochondrial autophagy levels, further confirming that the protective effect of LDHs@NBP depends on this pathway [20,22,32].

NBP exhibits diverse biological activities and has demonstrated organ-protective effects in the heart, brain, and kidneys [33]. However, its rapid in vivo metabolism and low bioavailability severely restrict its clinical application. Recently, some scholars have developed NBP Nano-therapy to enhance its targeting and release performance in the treatment of ischemic stroke [34]. The LDHs nanocarrier used in this study offers several advantages: as a two-dimensional layered nanomaterial, LDHs can efficiently load drugs via ion exchange and achieve pH-responsive drug release in acidic microenvironments. Previous studies have shown that LDHs nanoparticles loaded with resveratrol exhibit enhanced release efficiency under acidic conditions and demonstrate promising effects in breast cancer treatment [35,36]. Similarly, the LDHs@NBP constructed in this study also exhibited higher drug release efficiency in acidic environments. In the IR-induced AKI model, hypoxia-driven enhanced glycolysis leads to substantial lactate accumulation, resulting in significant acidification of the tubular microenvironment and a drop in cortical pH to 6.5-6.8, enabling LDHs@NBP to respond and release at sites of RTECs damage, thereby overcoming metabolic defects [37]. Additionally, their surface positive charge promotes cellular uptake, while excellent biocompatibility and biodegradability ensure safety for in vivo applications [[38], [39], [40]]. This study further confirms that after continuous administration of LDHs and LDHs@NBP in vivo, no abnormalities were observed in rat liver and kidney function indicators or histopathological findings, indicating both formulations exhibit good safety and biocompatibility. At the same time, we also measured the data of LDHs@NBP in serum-containing buffer solution. The results showed that the particle size increased by 4.1% and the PdI increased to 0.179, indicating its good stability (Fig. S1). In this study, LDHs@NBP demonstrated significant anti-fibrotic and kidney-protective effects in both in vitro cell models and in vivo AKI-CKD models, confirming the feasibility and therapeutic potential of this pH-responsive nano-delivery strategy. Previous studies have reported an LDH@miR-182 nanosystem based on LDHs, which not only exhibits excellent biocompatibility but also significantly alleviates myocardial IR injury and prevents myocardial fibrosis by efficiently scavenging ROS and restoring mitochondrial membrane potential. More recently, several independent studies have further revealed that LDH-based nanoplatforms can exert anti-inflammatory and antioxidant effects in cross-organ pathological models such as cerebral IR injury and inflammatory bowel disease. Together, these findings broaden the application prospects of LDH nanoplatforms in organ protection [[41], [42], [43]].

Early intervention in the pathological cascade following AKI holds substantial clinical significance for mitigating long-term morbidity and mortality. The AKI-CKD transition is a dynamic evolution process involving maladaptive repair after acute injury, persistent inflammation, and fibroblast activation [[44], [45], [46]]. This study confirmed in the uIRI model that LDHs@NBP can improve renal function and inhibit fibrosis in the long term, suggesting its effectiveness in chronic intervention. This finding echoes recent studies on drugs such as Silibinin in delaying CKD progression after IR injury, jointly emphasizing the importance of the early treatment window after AKI [47]. Additionally, NBP has been used in the clinical treatment of ischemic cerebrovascular diseases with sufficient safety data. LDHs nanocarriers can be simply synthesized by hydrothermal coprecipitation, showing good production feasibility; these advantages provide feasibility for future clinical translation.

This study still has the following limitations. While the key mediating role of the PINK1-Parkin pathway has been clarified, the inhibitor used, Mdivi-1, primarily targets Drp1 and may have off-target effects, potentially weakening the specificity of causal inference. To address this, future studies will employ conditional knockout mouse models for the PINK1 or Parkin genes to rigorously validate the necessity and sufficiency of this pathway. Furthermore, this study only examined the release data of LDHs@NBP in neutral phosphate buffer solution, but did not verify the stability data of LDHs@NBP in serum or protein-containing buffer solutions. And the biodistribution of LDHs@NBP in the kidney has not yet been precisely characterized using techniques such as in vivo fluorescence imaging and tissue elemental quantification. This study mainly adopted a preventive administration strategy before injury and has not systematically defined the differential therapeutic windows of LDHs@NBP during the recovery phase of AKI, the early fibrosis initiation stage of CKD, and the late irreversible scarring phase. Future research will further explore these issues to provide more robust theoretical support for clinical translation.

5. Conclusion

This study successfully constructed LDHs@NBP with pH-responsive properties and verified its efficacy (Fig. 10). It was found that LDHs@NBP could effectively alleviate renal fibrosis and block the AKI-CKD transition. Further research revealed that LDHs@NBP might exert its effects by restoring mitophagy activity and improving mitochondrial function. The LDHs@NBP constructed in this study not only enhanced the bioavailability of NBP but also provided a reference for clinical research and clinical translation in the treatment of kidney diseases.

CRediT authorship contribution statement

Ke Wang: Investigation, Methodology, Visualization, Writing – original draft. Xing-chun Zhu: Data curation, Methodology. Yi Xia: Data curation, Methodology. Yan Yan: Investigation, Writing – original draft. Jun-jie Ma: Formal analysis. Jing-yi Xia: Conceptualization. Yuan Xu: Data curation. Meng-han He: Validation. Feng-le Guo: Software. Wen-rui Wang: Conceptualization, Methodology, Validation. Li Ren: Resources, Writing – review & editing. Cong-li Zhang: Funding acquisition, Methodology, Project administration, Writing – review & editing.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Cong-li Zhang reports financial support was provided by Anhui Provincial Health Commission. If there are other authors, they 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 are the Supplementary data related to this article:

figs1.

Data availability

Data will be made available on request.