Multiscale molecular modeling–directed ROS-responsive nanotherapy for dual-axis regulation of fibrotic and inflammatory signaling in alport nephropathy

Abstract

Background

In Alport nephropathy, tightly interconnected fibrotic, inflammatory, and oxidative cascades are activated, elevating reactive oxygen species (ROS) that intensify renal injury. The broad activation of stress-responsive and profibrotic pathways further induces disease progression and limits the efficacy of monotherapies.

Results

We performed structure-based docking and long-timescale molecular dynamics simulations to identify mechanistically complementary agents, enabling the assessment of ligand stability, specificity, and suitability for selecting an effective drug combination. These analyses revealed stable histone deacetylase binding by ivaltinostat and sustained JNK1 engagement by genistein, supporting their selection as complementary antifibrotic and anti-inflammatory agents. To translate these insights, we engineered PEG-TK-C18/DSPE-PEG-maleimide nanomixed micelles that are functionalized with the proximal-tubule-targeting peptide (KKEEE)₃K-C (PPCK), co-loading both drugs, to yield PPCK + IG. Thioketal linkages conferred ROS-responsive cleavage and controlled release, while (KKEEE)₃K-C enhanced proximal tubule targeting. In Col4a3^–/– mice, PPCK + IG exhibited selective renal accumulation, oxidative activation, and robust suppression of fibrotic (α-SMA, fibronectin, and p-Smad2/3) and inflammatory markers (p-JNK, IL-6, and MCP-1), as well as downstream ERK attenuation, significantly improving renal function.

Conclusions

The findings of our study demonstrate precision nanotherapy that exploits pathological oxidative stress for targeted delivery and the coordinated modulation of epigenetic and MAPK pathways, offering a promising strategy for Alport nephropathy and other chronic kidney diseases.

Graphical abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12951-026-04451-w.

Background

The kidneys play central roles in maintaining systemic homeostasis by regulating blood filtration, electrolyte and fluid balance, acid-base equilibrium, and metabolic waste excretion [1]. The structural disruption of the glomerulus leads to chronic kidney disease (CKD), a major global health burden. The glomerular basement membrane (GBM) becomes destabilized in Alport syndrome (AS), an inherited nephropathy caused by COL4A3/A4/A5 mutations, resulting in progressive proteinuria, glomerulosclerosis, and interstitial fibrosis, which are often accompanied by hearing and ocular abnormalities [2]. Current therapies for AS, including ACE inhibitors, ARBs, and SGLT2 inhibitors, provide only partial renoprotection and do not address the complex molecular pathology of AS. Investigational agents such as bardoxolone methyl, lademirsen, and sparsentan also showed limited clinical success, underscoring the need for mechanism-driven strategies targeting multiple pathogenic pathways [3–5].

At the molecular level, fibrotic, inflammatory, and oxidative cascades, which are tightly interconnected, become activated in the kidneys in AS, collectively driving disease progression. Elevated reactive oxygen species (ROS) amplify glomerular and tubular injury, disrupt cellular redox balance, and potentiate inflammatory and profibrotic signaling. Several central pathways, including TGF-β/Smad-mediated fibroblast activation, TNF-α-driven inflammatory amplification, and downstream MAPK phosphorylation, are consistently upregulated in Alport nephropathy, converging on pathological markers such as α-SMA, fibronectin, phosphorylated Smad2/3, IL-6, MCP-1, and MAPK mediators [6]. Since these pathways operate synergistically, dual-axis modulation that simultaneously addresses epigenetic dysregulation underlying fibrosis and MAPK-mediated inflammatory amplification is necessary to effectively treat AS and more comprehensively interrupt the reinforcing loops of injury that characterize Alport nephropathy [7–9]. Moreover, histone deacetylase (HDAC) inhibition has emerged as a powerful strategy to counteract fibrotic remodeling in chronic kidney diseases. Transcriptome-guided drug repurposing has repeatedly identified HDAC inhibitors as top candidates for reversing disease-associated gene signatures in Col4a3^–/– mice, underscoring the central role of epigenetic dysregulation in progression of Alport nephropathy. Ivaltinostat (CG200745), a clinically advanced HDAC inhibitor, showed strong antifibrotic potential by suppressing TGF-β signaling, reducing fibroblast-to-myofibroblast transition, and modulating extracellular matrix deposition [8]. Despite these benefits, ivaltinostat has a narrow therapeutic window, dose-limiting toxicity, and limited renal selectivity [10]. In parallel, genistein, a naturally occurring isoflavone with MAPK/JNK-modulating, antioxidant, and anti-inflammatory properties, has emerged as a complementary agent capable of attenuating inflammatory and oxidative stress responses. Genistein can inhibit proinflammatory cytokine release, modulate MAPK and downstream ERK signaling, and mitigate oxidative injury. However, its therapeutic impact is limited by its rapid systemic clearance, poor renal retention, and a lack of responsiveness to disease-specific oxidative cues. These limitations undermine its efficacy in vivo and prevent the sustained modulation of inflammatory pathways in the kidneys [9–11].

Because empirically pairing of drugs does not ensure mechanistic complementarity, computational validation is essential for a rational combination therapy design. Structure-based molecular docking enables rapid identification of favorable binding orientations and key intermolecular interactions, while long-timescale molecular dynamics (MD) simulations rigorously assess conformational stability, binding pocket retention, and dynamic ligand-target engagement under physiologically relevant conditions. By applying this multiscale modeling framework, we confirmed that ivaltinostat stably occupies the catalytic pocket of class I HDAC, whereas genistein engages the ATP-binding cleft of JNK1 with sustained stability (Supplementary Figure S1). Therefore, HDAC inhibition and JNK1/MAPK modulation represent mechanistically distinct but converging therapeutic axes that can jointly suppress fibrosis and inflammation, the two central processes driving Alport nephropathy.

To translate this dual-target strategy into an effective therapeutic modality and overcome the pharmacokinetic limitations of the agents, we engineered a precision nanocarrier platform tailored to the oxidative pathology of AS kidneys. Specifically, we developed ROS-responsive PEG-thioketal-C18/DSPE-PEG-maleimide nanomixed micelles that are functionalized with the proximal-tubule-targeting peptide (KKEEE)₃K-C. The elevated ROS levels, characteristic of AS kidneys, promote thioketal bond cleavage, enabling the microenvironment-triggered release of the encapsulated agents, while the targeting ligand enhances selective renal accumulation. This dual-drug ROS-activated, kidney-targeted nanotherapy was designed to achieve the coordinated suppression of fibrotic and inflammatory signaling, thereby addressing core pathogenic mechanisms and offering a promising therapeutic strategy for AS.

Results

Molecular docking and molecular dynamics of genistein and ivaltinostat

Molecular docking and MD simulations demonstrated that genistein and ivaltinostat exhibit stable and robust binding engagement, comparable in interaction profile to their respective co-crystallized reference ligands, SCH772984 for JNK1 and vorinostat for HDAC (Supplementary Table S1; Fig. 1). Genistein was bound tightly within the ATP-binding cleft of JNK1 with a CDOCKER interaction energy of − 52.63 kcal/mol, supported by van der Waals contacts (ALA-36, GLY-35, ASN-33, SER-34), hydrogen bonds (SER-34, ASN-114, ASN-156), π-alkyl interactions (LEU-168, VAL-40, ILE-32, MET-108, ALA-53), and a π-sulfur interaction with MET-108 (Fig. 1A–B). Ivaltinostat demonstrated the strongest binding affinity toward HDAC, yielding a CDOCKER score of − 74.58 kcal/mol, which is mediated by the classical hydroxamate-Zn²⁺ coordination, hydrogen bonding (HIS-145, TYR-308, GLY-305), hydrophobic contacts (PHE-210, LEU-276, PRO-34), and a salt bridge with ASP-104 (Fig. 1C–D). In contrast, the co-crystallized ligands SCH772984 and vorinostat showed weaker binding energies in both docking and MM/GBSA analyses. Consistent with these findings, 500-ns MD simulations confirmed stable backbone RMSDs across all systems, while ligand RMSD values highlighted a markedly better pose retention for genistein (4.2 ± 0.6 Å) compared with the co-crystallized SCH772984 (28.7 ± 1.7 Å). In the HDAC complex, ivaltinostat displayed deeper and more persistent anchoring than co-crystallized vorinostat. RMSF analyses showed no major perturbations in protein flexibility, and SMD simulations further validated the strength of binding, with genistein requiring a higher rupture force (~ 410 pN) than SCH772984 (~ 327 pN) and ivaltinostat resisting dissociation more strongly (~ 342 pN) than vorinostat (~ 291 pN) (Supplementary Figure S2&S3). Collectively, these in silico results provide robust mechanistic support for the stable binding and mechanical resilience of the drug-target complexes, justifying the selection of genistein and ivaltinostat for the coordinated nanotherapeutic platform as optimized modulators of MAPK and HDAC signaling involved in kidney fibrosis.

Fig. 1.

CDOCKER docking poses and interaction maps of Genistein and Ivaltinostat with their respective protein targets. (A–B) Genistein is bound to JNK1 (PDB: 4QTD) with a CDOCKER interaction energy of − 52.63 kcal/mol. The ligand is stabilized by van der Waals interactions (ALA-36, GLY-35, ASN-33, SER-34), conventional hydrogen bonding (SER-34, ASN-114, ASN-156), π-alkyl interactions (LEU-168, VAL-40, ILE-32, MET-108, ALA-53), and a π-sulfur contact with MET-108, collectively supporting its stable binding at the ATP pocket. (C–D) Ivaltinostat is bound to HDAC (PDB: 4LXZ), showing a CDOCKER interaction energy of − 74.58 kcal/mol. Key interactions include hydrogen bonds (HIS-145, TYR-308, GLY-305), metal-acceptor interaction between the hydroxamate group and Zn²⁺, π-alkyl and hydrophobic contacts (PHE-210, LEU-276, PRO-34), and a salt bridge with ASP-104, indicating strong and selective binding within the catalytic pocket

Synthesis & physicochemical characterization of PPCK nanomicelle

¹H NMR spectra confirmed the successful synthesis of the amphiphilic copolymer through the conjugation of mPEG amine, thioketal (TK) linker, and C18 chains, with distinct peaks corresponding to each segment (Fig. 2A). DLS analysis revealed that the final PPCK formulation exhibited a hydrodynamic diameter of 238.9 ± 84.6 nm and a slightly negative zeta potential of − 9.4 ± 1.18 mV (Fig. 2B, C). The PPCK nanomicelles underwent marked structural disruption within 30 min upon treatment with hydrogen peroxide (H₂O₂), confirming their oxidative sensitivity and H₂O₂-triggered disassembly. These values varied moderately with changes in the formulation composition. FE-TEM images corroborated the DLS findings, displaying spherical micelles with uniform morphology (Fig. 2D). Furthermore, an in vitro release study was conducted using a dialysis-based system under oxidative (1 mM H₂O₂) and non-oxidative (PBS) conditions to assess drug release kinetics. PPCK + IG nanomicelles exhibited accelerated drug release under oxidative conditions, with approximately 35% of the payload liberated within 12 h, after which the release proceeded in a sustained manner (Fig. 2E). In contrast, the drug release in PBS was significantly lower. These findings validate the ROS-responsive nature of PPCK nanomicelles and highlight their potential for controlled, pathology-responsive drug delivery in oxidative stress–associated kidney diseases such as Alport nephropathy.

Fig. 2.

Characterization of ROS-responsive peptide-guided nanomicelles (PPCK). (A) 1 H NMR spectra confirming the successful synthesis of the amphiphilic polymer PEG-TK-C18 in CDCl₃. (B) The hydrodynamic size and surface charge of blank micelles (PP and PPCK) were measured by dynamic light scattering (DLS). (C) Time-dependent size variation of PPCK micelles with or without exposure to hydrogen peroxide (H₂O₂). (D) Transmission electron microscopy (TEM) images of PPCK micelles before and after H₂O₂ exposure. The insets show magnified views. (E) Drug release patterns of ivaltinostat and genistein from PPCK micelles in PBS (pH 7.4) were examined in the absence and presence of 1 mM H₂O₂

Assessment of cytotoxicity and cellular internalization

The cytocompatibility and internalization profile of the PPCK + IG nanomicelles were systematically evaluated to ensure their safety and target efficacy in renal cells. The HK-2 cells exposed to the escalating doses of ivaltinostat and genistein (0.004–10 µg/mL) encapsulated in PPCG and PPCK + IG nanomicelles exhibited no significant reduction in cell viability after 24 h, as assessed by the MTT assay (Fig. 3A). These results confirm the excellent biocompatibility of the nanomicelles in kidney epithelial cells and indicate minimal nephrotoxic effects at therapeutic concentrations.

Fig. 3.

Biocompatibility and cellular uptake of PPCK + IG nanomicelles. (A) Viability of HK-2 cells exposed to PPCG or PPCK + IG micelles containing ivaltinostat and genistein, as determined by the MTT assay after a 24-hour incubation (5–10 µg/mL). (B) Representative fluorescence microscopy images showing internalization of free IR780, PP-IR780, and PPCK-IR780 in HK-2 (top row) and L929 (bottom row) cells after a 4-hour incubation. IR780 fluorescence is visualized in red, and nuclei are counterstained with DAPI (blue). (C) Quantification of mean fluorescence intensity (MFI) in HK-2 and L929 cells relative to untreated control cells

Strong IR780 fluorescence was detected in the HK-2 cells incubated with PPCK-IR780 for 4 h, whereas significantly lower uptake was observed with free IR780 and PP-IR780 (Fig. 3B). In contrast, L929 fibroblast cells exhibited markedly reduced fluorescence signals across all formulations, including PPCK-IR780, demonstrating cell-type selectivity. The quantitative analysis of mean fluorescence intensity (MFI) further confirmed the enhanced internalization of PPCK-IR780 in the HK-2 cells compared to L929 cells and other nanoparticle formulations, underscoring the targeting efficiency conferred by the CK peptide (Fig. 3C). In addition to the selective uptake, the ROS-responsive behavior of PPCK nanomicelles was validated through a comparative intracellular release study using non-ROS-responsive formulations (PS-IR780 and PSCK-IR780) (Supplementary Figure S4). The PPCK-IR780 formulation exhibited significantly greater IR780 release under oxidative conditions, confirming that drug release is triggered in a ROS-rich microenvironment, such as that present in diseased renal tissues.

Assessment of in vivo biodistribution and renal-targeting efficiency

The biodistribution profile of PPCK was evaluated in healthy mice using PPCK-IR780 as a model compound and compared with the non-targeted formulation, PP-IR780. Both targeted and non-targeted formulations exhibited distribution in the kidneys similar to that of healthy mice (Fig. 4A and B). Then, in vivo studies were conducted in 7-week-old Col4a3^⁻/⁻ (KO) Alport mice and WT mice to further investigate biodistribution under pathological conditions. The fluorescence signal detected in KO mouse kidneys was markedly greater compared to WT animals, indicating enhanced renal accumulation (Fig. 4C and D). Additionally, distinct differences in PPCK distribution were observed across major internal organs between KO and WT mice, with KO mice demonstrating greater kidney-specific accumulation of PPCK-IR780 compared to WT mice. This enhanced renal deposition of PPCK observed in the diseased group underscores the potential of the nanoplatform for targeted renal delivery.

Fig. 4.

Organ distribution and renal-targeting capability of CK peptide-modified nanomicelles in mice. (A) Fluorescence imaging of major organs collected 24 h after the intravenous injection of PP-IR780 or PPCK-IR780 micelles in healthy mice. (B) Quantification of fluorescence intensity (MFI) in organs shown in panel A. (C) Comparative fluorescence imaging of organ distribution in Col4a3^⁻/⁻ (KO) and wild-type (WT) mice 24 h after the PPCK-IR780 administration. (D) Quantification of kidney fluorescence from panel C

Anti-inflammatory and anti-apoptotic effects of PPCK + IG in renal epithelial cells

We analyzed the impact of PPCK + IG on TNF-α-induced MAPK signaling and apoptosis in HK-2 cells to investigate its protective role under inflammatory stress. Western blotting combined with densitometric quantification was used to assess ERK, JNK, and p38 activation, along with apoptosis-associated markers (Fig. 5A). Upon TNF-α stimulation, ERK (P-ERK), JNK (P-JNK), and p38 (P-p38) phosphorylation was markedly elevated compared with untreated controls, confirming the robust activation of pro-inflammatory MAPK signaling. Notably, PPCK + IG co-treatment substantially suppressed phosphorylation of P-ERK, P-JNK, and P-p38, while total protein levels of ERK, JNK, and p38 remained unchanged (Fig. 5B). This inhibition was statistically significant for all three pathways (p < 0.01), confirming that PPCK + IG attenuates MAPK-driven inflammatory signaling. In addition to the regulation of the MAPK pathway, we assessed apoptosis-related changes. TNF-α increased the Bax/Bcl-2 ratio (Fig. 5B), indicating a shift toward pro-apoptotic signaling. This effect was mitigated by PPCK + IG, which significantly lowered the Bax/Bcl-2 ratio compared to TNF-α alone. Moreover, cleaved caspase-3 levels, a hallmark of apoptotic activation, were also elevated by TNF-α but significantly suppressed upon PPCK + IG co-treatment (Fig. 5B), further demonstrating the anti-apoptotic potential of the compound. Therefore, PPCK + IG effectively attenuates TNF-α-induced inflammatory and apoptotic signaling in HK-2 cells by inhibiting MAPK phosphorylation and restoring the balance between pro- and anti-apoptotic markers. Annexin V/PI dual-staining flow cytometry was performed to further confirm the anti-apoptotic effects of PPCK + IG, quantifying early and late apoptotic cell populations under TNF-α-induced inflammatory conditions (Fig. 5C). In the control group, most HK-2 cells remained viable, with only a small proportion undergoing early (1.65%) or late (1.26%) apoptosis. TNF-α stimulation markedly increased apoptotic cells, as evidenced by elevated levels of both early (9.33%) and late (3.33%) apoptotic cell populations. This result supports the strong pro-apoptotic influence of TNF-α in renal epithelial cells. PPCK + IG treatment alone showed no evidence of apoptosis, with cell survival levels comparable to untreated controls. Importantly, PPCK + IG co-treatment significantly reduced TNF-α-induced apoptosis, with early and late apoptotic cell populations decreasing to 4.45% and 1.18%, respectively (Fig. 5C). This protective effect further corroborates the anti-apoptotic capacity of PPCK + IG, which was previously observed via the suppression of cleaved caspase-3 and normalization of Bax/Bcl-2 ratios. Thus, these flow cytometry results reinforce the conclusion that PPCK + IG protects HK-2 cells from TNF-α-induced apoptosis by reducing both early and late apoptotic events.

Fig. 5.

PPCK + IG attenuates TNF-α-induced MAPK activation and apoptosis in HK-2 cells. (A) Representative western blot image of phosphorylated and total ERK, JNK, p38, Bax, Bcl-2, Cleaved caspase 3, and Caspase 3. (B) Quantification of western blots and ratio of phosphorylated to total MAPK, Bax/Bcl-2 and cleaved caspase-3/total caspase-3 ratios. Data are presented as mean ± standard deviation (SD; n = 3). Statistical significance was analyzed by one-way ANOVA with Tukey’s post hoc test: p < 0.05, p < 0.01, p < 0.001, p < 0.0001; ns, not significant. (C) Representative annexin V/PI flow cytometry plots and quantification of apoptotic cells with Annexin V+/PI- cells proportion (n = 2)

Effect of PPCK + IG nanomicelles on fibrosis in TGF-β-stimulated HK-2 cells

PPCK + IG was tested for its antifibrotic activity in HK-2 cells exposed to TGF-β (Fig. 6A). TGF-β stimulation increased α-SMA expression, a hallmark of epithelial-to-mesenchymal transition (EMT), Smad4 and elevated phosphorylation of Smad2/3, while total Smad2/3 remained unchanged. Quantitative densitometry confirmed these findings: α-SMA expression was strongly upregulated in response to TGF-β, while the co-treatment with PPCK + IG nanoparticles significantly reduced α-SMA levels (Fig. 6B). Similarly, TGF-β-induced Smad2/3 phosphorylation was suppressed by the nanoparticles, indicating that the therapeutic formulation interferes with the canonical TGF-β/Smad signaling cascade. Additionally, Smad4 expression was significantly elevated by TGF-β stimulation but was attenuated by the co-treatment, further supporting its inhibitory effect on TGF-β downstream signaling. Moreover, endogenous TGF-β expression was also elevated upon exogenous TGF-β stimulation, suggesting a feed-forward loop that may exacerbate fibrosis. Importantly, this feedback was significantly blunted by the nanoparticles (Fig. 6B). Thus, PPCK + IG nanoparticles strongly inhibit TGF-β-driven fibrotic signaling and EMT marker expression in HK-2 cells.

Fig. 6.

PPCK + IG nanomicelles inhibit TGF-β-induced profibrotic signaling in HK-2 cells. (A) Western blots depicting p-Smad2/3, total Smad2/3, α-SMA, Smad4, and TGF-β, with β-actin serving as a loading control. (B) Quantification of protein levels normalized to β-actin or total protein: α-SMA/β-actin, p-Smad2/3/Smad2/3, Smad4/β-actin, and TGF-β/β-actin. Data are presented as mean ± standard deviation (SD; n = 3). Statistical significance was determined using one-way ANOVA followed by Tukey’s post hoc test: p < 0.05, p < 0.01, p < 0.001; ns, not significant

Effect of PPCK + IG nanomicelles on inflammation, apoptosis, and fibrosis markers in the alport nephropathy animal model

Finally, we assessed the therapeutic efficacy of PPCK + IG nanoparticles in an Alport nephropathy mouse model. The nanoparticles were evaluated for their ability to reduce renal injury. Urinary neutrophil gelatinase-associated lipocalin (NGAL) was measured as a marker of kidney damage, which was strongly increased in Col4a3^−/− mice but was substantially lowered after PPCK + IG treatment (Table 1). We examined immunoblotting for inflammation and apoptosis markers. MAPKs involved in inflammation and apoptosis, such as P-ERK, P-JNK, and pP38, were up-regulated in the Col4a3^−/− group, which was attenuated by PPCK + IG nanoparticles (Fig. 7). Apoptosis markers such as Bax/Bcl2 and cleaved caspase-3/caspase-3 ratio were up-regulated in the Col4a3^−/− group, which was attenuated by PPCK + IG nanoparticles (Fig. 7). We also conducted immunoblotting for fibrosis markers such as α-SMA and TGF-β-Smad signaling, which were up-regulated in the Col4a ^−/− group, which was attenuated by PPCK + IG nanoparticles (Fig. 8).

Table 1.

Effects of PPCK + IG nanomicelles on the functional status and kidney damage markers of mice

	WT + placebo	WT + PPCK + IG	KO + placebo	KO + PPCK + IG
BW (g)	20.69 ± 0.99	18.17 ± 1.38	18.47 ± 1.41	16.88 ± 0.80*
LK/BW (g/kg)	6.66 ± 0.32	7.61 ± 0.48	9.11 ± 0.50	8.70 ± 0.39*
RK/BW (g/kg)	6.63 ± 0.28	7.60 ± 0.49	9.31 ± 0.62	8.68 ± 0.53*
Urine output (µL/day)	937 ± 279	730 ± 146	2750 ± 763	1480 ± 305
Urinary NGAL (ng/ml)	155 ± 12	172 ± 17	753 ± 79*	406 ± 89#

Mean ± standard error of the mean, * p < 0.05, compared to WT + placebo, # p < 0.05, compared to KO + placebo; BW, body weight; KO, knock out; LK, left kidney; RK, right kidney; WT, wild type

Fig. 7.

PPCK + IG nanoparticles attenuate inflammation, apoptosis, and fibrosis in the kidneys of Col4a3^⁻/⁻ mice. (A) Western blots showing phosphorylated MAPKs (ERK, JNK, and p38), apoptotic proteins (Bax, Bcl-2, cleaved caspase-3, and total caspase-3) in kidney tissues from WT, WT+PPCK + IG, Col4a3^⁻/⁻, and Col4a3^⁻/⁻ + PPCK + IG groups. (B) Densitometric quantification of MAPK proteins: p-ERK/ERK, p-JNK/JNK and p-p38/p38, Quantification of apoptotic proteins: Bax/Bcl-2 ratio and cleaved caspase-3/total caspase-3. Data are presented as mean ± SD (n = 3–4 mice per group). Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test: p < 0.05, p < 0.01, p < 0.001; ns, not significant

Fig. 8.

PPCK + IG nanoparticles attenuate inflammation, apoptosis, and fibrosis in the kidneys of Col4a3^⁻/⁻ mice. (A) Western blots fibrotic markers (α-SMA, TGF-β, Smad2/3, and Smad4) in kidney tissues from WT, WT+PPCK + IG, Col4a3^⁻/⁻, and Col4a3^⁻/⁻ + PPCK + IG groups. β-actin was used as a loading control. (B) Densitometric quantification of fibrosis marker quantification: α-SMA/β-actin, TGF-β/β-actin, p-Smad2/3/Smad2/3, and Smad4/β-actin. Data are presented as mean ± SD (n = 3–4 mice per group). Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test: p < 0.05, p < 0.01, p < 0.001; ns, not significant

Furthermore, the expression of pro-inflammatory and profibrotic genes, such as genes for IL-6, TNF-α, and MCP-1, as well as genes for α-SMA, fibronectin, and TGF-β, were analyzed by real-time PCR (Fig. 9). TGF-β-driven inflammatory and fibrotic responses were significantly elevated in KO animals, with PPCK + IG co-treatment substantially attenuating these pathological changes (Fig. 9A–F). Genes for inflammatory cytokines, including IL-6, TNF-α, and MCP-1, were markedly upregulated in KO mice, reflecting a robust inflammatory state. In all three cases, the co-treatment with PPCK + IG nanoparticles significantly reduced cytokine expression compared to the untreated KO group, indicating an effective anti-inflammatory response. Similarly, profibrotic genes, such as those encoding α-SMA, fibronectin, and TGF-β, were significantly elevated in the KO group. Administering PPCK + IG nanoparticles reduced the expression of all three markers, indicating the suppression of fibrogenic genes. In summary, PPCK + IG treatment effectively suppressed the upregulation of these inflammatory and fibrotic genes in Col4a3^−/− mice. Notably, the WT + PPCK + IG group did not show significant deviations from the WT group across any of the markers, suggesting that the formulation is non-toxic and does not trigger inflammatory or fibrotic gene expression under normal physiological conditions. Collectively, these transcriptional analyses provide robust evidence that PPCK + IG nanoparticles mitigate both inflammation and fibrosis at the molecular level in the kidneys of AS mice.

Fig. 9.

PPCK + IG nanoparticles suppress inflammatory and fibrotic gene expression in Col4a3^⁻/⁻ mouse kidneys. (A–C) Relative mRNA expression of pro-inflammatory cytokines: (A) IL-6, (B) TFN-α, and (C) MCP-1. (D–F) Fibrosis-associated genes: (D) α-SMA, (E) fibronectin, and (F) TGF-β. Gene expression values are shown as fold change compared with WT controls. Data are shown as mean ± standard deviation (SD; n = 3–4 per group). Statistical comparisons were performed using one-way ANOVA with Tukey’s post hoc test: p < 0.05, p < 0.01, p < 0.001, p < 0.0001; ns, not significant

Histopathological evaluation of renal injury and fibrosis

A histopathological evaluation was conducted to assess the extent of renal injury and fibrosis across treatment groups, further validating the molecular and biochemical findings. The WT group exhibited a well-preserved renal cortical architecture with normal glomeruli and intact tubular epithelium (Fig. 10). The WT + PPCK + IG group showed a comparable histological pattern, indicating that the formulation itself did not induce structural abnormalities. In contrast, Col4a3^−/− mice displayed extensive renal injury characterized by tubular dilation, epithelial desquamation, interstitial expansion with inflammatory cell infiltration, and glomerular sclerosis, consistent with severe nephropathy. These degenerative changes were markedly attenuated in the KO + PPCK + IG group, which showed a partial restoration of the tubular structure and reduced inflammatory infiltration. Masson’s trichrome staining further revealed extensive collagen deposition and interstitial fibrosis in KO kidneys, whereas PPCK + IG treatment diminished fibrotic regions and collagen accumulation, confirming its potent antifibrotic activity. Similarly, PAS staining demonstrated thickened glomerular basement membranes and disrupted brush borders in KO mice, while these abnormalities were ameliorated in the PPCK + IG group, indicating structural preservation of the tubular basement membrane and glomerular matrix. Collectively, these histological findings substantiate biochemical and molecular data, demonstrating that PPCK + IG alleviates glomerular and tubular damage, reduces collagen deposition, and restores renal architecture in Alport nephropathy.

Fig. 10.

Representative histological images of kidney tissues from WT, WT + PPCK + IG, Col4a3^-/- (KO), and Col4a3^-/- + PPCK + IG mice stained with H&E, Masson’s trichrome, and PAS. WT, wild type; KO, knock out

Discussion

This study introduces PPCK + IG nanoparticle, a kidney proximal tubule–targeting, ROS-responsive polymeric drug delivery system for the treatment of Alport nephropathy. We achieved enhanced renal accumulation, optimized therapeutic efficacy, and minimized systemic toxicity by integrating HDAC inhibitor ivaltinostat and natural isoflavone genistein into a polymeric nanocarrier functionalized with the (KKEEE)₃K peptide. Pharmacokinetic analyses confirmed that PPCK + IG significantly increased kidney-specific retention of both drugs compared to free drug administration, resulting in a pronounced therapeutic benefit. When co-delivered, ivaltinostat and genistein exerted complementary effects: they epigenetically reduced HDAC-driven profibrotic gene expression while concurrently modulating TGF-β/Smad and oxidative stress–linked MAPK pathways. This dual-action strategy allows the coordinated inhibition of both fibrotic and inflammatory responses, offering therapeutic benefits beyond those of either agent alone. Importantly, the elevated ROS levels characteristic of Alport nephropathy provide a disease-specific trigger for an intelligent drug release. By encapsulating both drugs within a ROS-sensitive polymeric nanocarrier, delivery can be directed specifically to the injured kidney tissue, where pathological signals trigger their release. Such targeted delivery increases local bioavailability, optimizes therapeutic benefit, and limits systemic side effects, helping address challenges in translating combination therapy for Alport nephropathy into clinical use.

Progression of Alport nephropathy is driven by interconnected fibrotic and inflammatory signaling cascades, with disease severity often aggravated by the excess production of ROS [6, 12]. The activation of the TGF-β/Smad and MAPK pathways, which synergistically promote fibrosis and inflammation, is central to Alport nephropathy. These processes reinforce one another in a pathogenic loop, limiting the efficacy of monotherapies directed at a single axis. Beyond amplifying TGF-β/Smad-driven fibrosis and MAPK-mediated inflammation, ROS directly contribute to glomerular basement membrane injury and proximal tubular epithelial stress, reinforcing disease progression. This dual role makes ROS both a pathological hallmark and a therapeutic trigger. Importantly, TK linkers offer a unique advantage in this setting by undergoing selective cleavage in oxidative environments, enabling on-demand drug release while simultaneously scavenging ROS. Such a mechanism provides not only spatially controlled delivery but also an intrinsic antioxidant benefit, thereby addressing two critical drivers of Alport nephropathy in a single design. To enable kidney-specific targeting, we exploited the affinity between lysine residues and megalin receptors located on the apical membrane of proximal tubular cells [13, 14]. The (KKEEE)₃K peptide, derived from ε-polylysine, demonstrated favorable renal accumulation and pharmacokinetic characteristics, which were validated through molecular imaging, biodistribution, and immunohistochemical analysis [15].

To mechanistically substantiate this dual-drug strategy, we performed structure-based docking and long-timescale all-atom MD simulations using HDAC (PDB 4LXZ) and JNK1 (PDB 4QTD). Ivaltinostat displayed a stable, low-energy pose within the HDAC catalytic pocket, with its hydroxamate group maintaining a tight bidentate coordination with the catalytic Zn²⁺ throughout the simulation. MD trajectories confirmed minimal RMSD drift and favorable MM/PBSA binding energies, while SMD revealed high rupture forces, indicating robust inhibition of HDAC activity. Genistein similarly formed persistent hydrogen bonds and hydrophobic interactions within the ATP-binding cleft of JNK1, maintaining the stable binding across 500-ns simulations. Although the co-crystallized synthetic inhibitor SCH772984 exhibited the most negative docking and MMGBSA energies as expected for an optimized, high-affinity ATP-competitive kinase inhibitor, genistein still demonstrated a markedly favorable and thermodynamically stable binding profile. This validates SCH772984 as a positive control and confirms that genistein engages JNK1 with sufficient affinity to modulate MAPK activation while offering superior biocompatibility and translational suitability for chronic kidney disease. According to these in silico results, ivaltinostat effectively blocks the HDAC-mediated epigenetic activation, whereas genistein directly suppresses MAPK signaling through the high-affinity engagement of JNK1. Together, the complementary molecular actions of both drugs provide strong justification for their co-delivery to the kidney to maximize therapeutic synergy while minimizing systemic toxicity. It should be noted that the computational superiority in metrics such as rupture force or RMSD does not inherently guarantee enhanced biological potency. However, our multiscale modeling framework confirms that both agents maintain stable and persistent engagement within their respective target pockets under dynamic conditions. This stable anchoring provides a rational mechanistic basis for their inclusion in a ROS-responsive delivery system, ensuring that once released in the oxidative microenvironment of the Alport kidney, the drugs can effectively engage their intended therapeutic axes.

In vitro studies demonstrated the biocompatibility and selective cellular uptake of PPCK + IG nanomicelles by renal epithelial cells, underscoring the efficacy of the (KKEEE)₃K targeting moiety. The ROS-responsive behavior of the micelles, triggered by oxidative conditions characteristic of kidney injury, facilitated controlled and timely drug release. In TNF-α- and TGF-β-stimulated HK-2 cells, PPCK + IG effectively suppressed inflammation, apoptosis, and fibrotic responses, as evidenced by the downregulation of the ERK, JNK, and p38 MAPK pathways, Bax/Bcl-2 ratio, cleaved caspase-3, and fibrotic markers including α-SMA, fibronectin, and TGF-β/Smad signaling.

In vivo efficacy was further validated in Col4a3^−/− Alport mice, in which treatment with PPCK + IG nanoparticles significantly reduced serum creatinine and urinary NGAL levels, accompanied by a marked attenuation of the histopathological markers of kidney damage. Histopathological analysis provided direct evidence of structural recovery in PPCK + IG-treated Alport mice. H&E and PAS staining confirmed notable improvements in tubular integrity and glomerular morphology, while Masson’s trichrome staining demonstrated pronounced attenuation of interstitial collagen deposition. These morphological improvements complement the molecular suppression of TGF-β/Smad and MAPK pathways, indicating that PPCK + IG not only modulates profibrotic and inflammatory signaling but also translates these effects into tangible preservation of renal architecture. Biodistribution studies further confirmed superior renal targeting, particularly in fibrotic kidneys, reinforcing the specificity and therapeutic potential of this delivery platform. The dual-drug strategy employed in PPCK + IG leverages their complementary mechanisms of action: ivaltinostat modulates the local renin-angiotensin system and inhibits the fibroblast-to-myofibroblast transition to reduce fibrosis, while genistein provides antioxidant and anti-inflammatory effects, including the suppression of profibrotic cytokines. The co-delivery within a single nanoparticle enhances therapeutic synergy while minimizing systemic exposure and off-target toxicity. Although the PPCK nanomicelles exhibited a relatively broad hydrodynamic size distribution (238.9 ± 84.6 nm), this variability likely reflects the dynamic assembly characteristics of amphiphilic polymeric micelles in aqueous environments rather than structural instability. Importantly, the observed size range remains well above the glomerular filtration threshold (< 8 nm), thereby preventing rapid renal clearance via passive filtration. Instead, kidney accumulation is mediated through (KKEEE)₃K peptide–facilitated proximal tubule targeting and enhanced retention within the oxidative microenvironment. Consistent with this mechanism, biodistribution studies demonstrated selective renal enrichment despite the moderate size heterogeneity. Furthermore, particles within the 150–300 nm range are known to exhibit favorable circulation properties while avoiding immediate hepatic clearance, supporting the translational feasibility of this formulation. Collectively, the significant improvements observed across in silico, in vitro, and in vivo evaluations underscore the potential of PPCK + IG nanomicelles as an advanced therapeutic strategy for chronic kidney disease, particularly for genetic conditions such as AS.

It is important to acknowledge certain limitations of the present study. First, while our results demonstrate the attenuation of oxidative stress-linked pathways, direct assessments of mitochondrial function were not performed. Given that mitochondrial dysfunction is a key driver of podocyte injury in Alport syndrome [16, 17], future studies should focus on the impact of PPCK + IG on mitochondrial bioenergetics. Second, our ROS-responsiveness was validated using 1 mM H₂O₂ as an accelerated proof-of-concept; although this exceeds typical endogenous levels, the localized and sustained generation of ROS in diseased kidneys likely facilitates the triggered release observed in vivo. Lastly, our study demonstrates mechanistic complementarity targeting dual pathogenic axes, rather than formal pharmacological synergy, as comparisons with single-drug-loaded nanomicelles were not conducted.

Conclusions

In conclusion, our findings establish PPCK + IG as a safe, effective, and precision-driven nanomedicine approach capable of co-delivering ivaltinostat and genistein to attenuate inflammation and fibrosis in Alport nephropathy. Notably, the pathophysiologic derangements identified in the AS model, such as ROS-induced inflammation, fibrosis, and the activated MAPK pathway, reflect common pathological mechanisms involved in the progression of chronic kidney disease. This strategy opens promising avenues for the development of next-generation, kidney-targeting nanotherapeutics for diseases associated with oxidative stress and fibrotic remodeling.

Methods

Molecular docking and complex preparation

The crystal structures of HDAC (PDB ID: 4LXZ) and MAPK kinase JNK1 (PDB ID: 4QTD) were retrieved from the Protein Data Bank. PDB 4LXZ provides the high-resolution, ligand-bound conformation of HDAC, a molecule central to profibrotic transcriptional regulation that is the primary enzymatic target of ivaltinostat. PDB 4QTD offers a well-resolved ATP-competitive inhibitor–bound structure of JNK1, a key MAPK mediator of inflammatory signaling and the predicted molecular target of genistein. The use of experimentally validated, active-state structures ensured the accurate representation of the catalytic pocket geometry for docking and MD simulations. Protein structures were prepared by removing crystallographic water molecules, correcting bond orders, adding missing hydrogens, and minimizing side chains using the Prepare Protein protocol in Discovery Studio 2022 (BIOVIA). Geometry-optimized ligand structures of ivaltinostat and genistein were docked using the CDOCKER algorithm, and top-scoring poses were advanced to MD simulations.

Molecular dynamics simulations

All-atom MD simulations were performed using GROMACS 2025.3 with the AMBER6 force field for proteins and ligands. Ligand topology and parameters were generated using the antechamber server and validated for penalty scores. Each protein-ligand complex was solvated in a cubic box with TIP3P water molecules and neutralized with Na⁺/Cl⁻ ions. Energy was minimized using the steepest descent algorithm up to the maximum force of < 1000 kJ/mol/nm. The system was equilibrated under NVT (100 ps) and NPT (100 ps) ensembles with positional restraints on heavy atoms. Production MD was performed for 500 ns at 310 K using the velocity-rescale thermostat and Parrinello-Rahman barostat (1 bar). Periodic boundary conditions were applied in all directions, and long-range electrostatics were treated with the particle mesh Ewald (PME) method (cutoff = 1.2 nm). A 2-fs integration step was used, and trajectories were recorded every 10 ps.

Steered molecular dynamics

Moreover, 0.5-ns steered molecular dynamics (SMD) simulations were performed in GROMACS using the pull code to probe the unbinding forces and dissociation pathways. A harmonic spring constant of 650 kJ/mol/nm² was applied to the center of mass of the ligand, while pulling was conducted along the vector connecting the ligand and protein binding pocket at a constant velocity of 0.009 nm/ps. Force-distance profiles were extracted to evaluate the binding strength and unbinding pathway.

Trajectory analysis

Trajectory analysis was performed using built-in GROMACS tools and PyMOL 2.5. Root mean square deviation (RMSD) and root mean square fluctuation (RMSF) were calculated to assess conformational stability and interaction persistence. Binding free energies were estimated using the MM/GBSA method implemented in gmx_MMPBSA 1.6.4. Representative snapshots and interaction maps were visualized in PyMOL.

Chemicals and reagents

Methoxy-polyethylene glycol amine (mPEG–NH₂, 2 kDa), stearylamine (C18), 3-mercaptopropionic acid, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), triethylamine (TEA), and genistein were obtained from Sigma-Aldrich. N, N-dimethylformamide (DMF) was purchased from Merck. Ivaltinostat (CG200745) was provided by Crystal Genomics. The CK peptide [Cys(KKEEE)₃K] was purchased commercially.

Preparation of PTC and PPCK + IG micelles

The thioketal (TK) linker, PEG-TK, and PEG-TK-stearylamine (PTC) were synthesized as previously reported [18] with minor modifications. Briefly, mPEG-amine and TK were coupled with TEA, EDC, and NHS in DMF under nitrogen at 80 °C for 3 h using microwave irradiation. The PEG-TK intermediate was isolated by diethyl-ether precipitation and dialysis. Then, PEG-TK was conjugated with stearylamine under identical conditions to yield PTC, which was purified, lyophilized, and confirmed by ¹H NMR (Bruker, 400 MHz).

Characterization of micelles

Micelles were prepared by the thin-film hydration method; their particle size and zeta potential were determined using a Zetasizer Nano Z (Malvern, UK). Morphology was examined by field-emission transmission electron microscopy (TEM; JEOL JEM-2100 F, Japan).

Cell culture

In vitro studies were conducted as previously described [6]. Human proximal tubular epithelial cells (HK-2) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) and Ham’s F-12 medium (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin at 37 °C in 5% CO₂. The HK-2 cells were sub-cultured until 70%–80% confluence. The HK-2 cells were plated onto 60-mm dishes in a medium containing 10% FBS and incubated for 24 h. Then, the cells were incubated in the DMEM-F12 medium with serum-free FBS and treated with rhTGF-β (2 ng/mL; R&D Systems, Minneapolis, MN, USA) or TNF-α (20 ng/mL; R&D Systems, Minneapolis, MN, USA) for 24 h in the presence or absence of PPCK + IG micelles (25 ng). TNF-α treatment lasted 15 min. PPCK + IG micelles were added 1 h before rhTGF-β or TNF-α treatment. Mouse fibroblast cells (L929) were cultured in a high-glucose DMEM (Gibco, USA) supplemented with 10% FBS and 1% penicillin-streptomycin under standard culture conditions (37 °C, 5% CO₂). The L929 cells were employed to evaluate the cytocompatibility and nanoparticle uptake behavior of PPCK + IG micelles.

In vitro drug release

Micelle suspensions were placed in dialysis bags and incubated in phosphate-buffered saline (PBS; pH 7.4) with or without 1 mM H₂O₂ at 37 °C under gentle shaking. At predetermined intervals, samples were withdrawn, and drug release was quantified by HPLC.

Cytotoxicity assay

Cell viability was evaluated using the WST-1 kit (Abfrontier, Korea) following the manufacturer’s instructions. The HK-2 cells were treated with different concentrations of PPCK + IG micelles, free ivaltinostat, or genistein for 24 h. Untreated cells served as negative controls, whereas 0.1% Triton X-100 was used as a positive control.

Cellular uptake

Uptake studies were performed using IR780-loaded micelles as previously described [18]. The HK-2 cells were incubated with PPCK-IR780 or control PP-IR780 micelles (lacking CK peptide), fixed, stained with Hoechst, and visualized by fluorescence microscopy.

Flow cytometry

Flow cytometry analysis was conducted as previously described [19]. An annexin V FLUOS staining kit (Sigma-Aldrich) was used to measure annexin V binding according to the manufacturer’s instructions. After treatment with 0 or 20 ng/mL TNF-α for 24 h with or without 25 ng of PPCK-IG pretreatment, the HK-2 cells were harvested and washed twice with pre-cooled PBS and resuspended in a binding buffer containing annexin V. To ensure data stability and minimize technical noise, cells from four individual culture plates per group were pooled for each independent biological replicate. After incubation in the dark for 15 min, the cells were analyzed by flow cytometry (Becton-Dickinson, San Jose, CA, USA). Several controls were used to optimize the instrument settings and determine the gating for the Windows-based platform. Apoptotic cells were defined as PI-negative, while annexin V-FITC was marked positive. Data from two independent biological experiments were analyzed, and the results are presented as the percentage of apoptotic cells. Quantitative results were integrated with other protein-level apoptosis markers (Bax, Bcl-2, cleaved caspase-3, and Caspase-3) to confirm the consistency of the anti-apoptotic effects.

Experimental animals and treatment protocol

Wild-type (WT) and Col4a3^⁻/⁻ mice (129XI/SvJ background, Jackson Laboratory) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA) and housed under standard conditions. The sample size for animal studies (n = 3 for wild-type groups; n = 4 for Col4a3^−/− groups) was justified using the resource equation method, an alternative approach for exploratory research where effect sizes are not predetermined [20]. For our design, the calculated error degrees of freedom (E = N - k = 10) fell within the statistically acceptable range of 10 to 20, ensuring sufficient power while minimizing the number of animals used in accordance with ethical guidelines. The mice were maintained in a 12-hour light/dark cycle and given free access to standard chow and tap water. Genotyping was performed by polymerase chain reaction (PCR) using reported primers [9]. For treatment, four-week-old mice received PPCK formulations via the tail vein every three days for three weeks and were sacrificed at seven weeks. Urine samples were collected from metabolic cages 2 days prior to the sacrifice, and the mice aged 4 or 7 weeks were euthanized for plasma collection and metabolomic analysis. Plasma was collected from the cardiac puncture and centrifuged at 2000 × g for 5 min. Urine samples were collected from metabolic cages to examine the metabolites two days before the mice were sacrificed. Urine samples were centrifuged immediately after the collection at 8000 × g for 5 min. Organs were collected for histology, PCR, or Western blotting. The CNUH IACUC approved all procedures (CNUHIACUC-22026).

Biodistribution in vivo

For biodistribution, the mice received an intravenous injection of IR780-loaded PPCK micelles. At 24 and 72 h after the injection, their organs were collected, while fluorescence signals were recorded using a FOBI imaging system (NeoScience, Korea).

Plasma creatinine and urinary NGAL

Plasma creatinine levels were measured using the Jaffe method (Olympus 5431; Olympus Optical, Tokyo, Japan). Urinary NGAL levels were determined using commercial ELISA kits (R&D Systems, USA) according to the manufacturer’s protocol, with a 1:4000 dilution for NGAL.

Semi-quantitative immunoblotting

Western blot analysis was performed as previously described [6]. Kidney tissues were homogenized in an isolation buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, 8.5 mM leupeptin, 1 mM PMSF, pH 7.2) and centrifuged at 4000 × g for 15 min at 4 °C. Protein concentrations were determined by the BCA assay (Pierce, Rockford, IL, USA). Equal protein amounts were separated on 9%–12% SDS-PAGE, transferred to nitrocellulose membranes (Amersham Pharmacia Biotech, UK), blocked with 5% milk in PBS-T, and probed with primary and HRP-conjugated secondary antibodies. Bands were visualized by enhanced chemiluminescence and quantified by densitometry (Scion Corporation, MD, USA). Supplementary Table S2 provides a list of primary and secondary antibodies used in immunoblotting.

Real-time qPCR

PCR analysis was performed as previously described [6]. Total RNA was extracted from the kidney cortex using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and reverse-transcribed into cDNA with oligo(dT) primers and Superscript II (Invitrogen, USA). Quantitative PCR (qPCR) was performed using SYBR Green Premix Ex Taq (Takara Bio Inc., Japan) on a Smart Cycler II (Cepheid, Sunnyvale, CA, USA) under standard cycling conditions. Gene expression was normalized to GAPDH and expressed as fold change relative to controls. Supplementary Table S3 provides the list of the primers used in real-time qPCR.

Histology

Preparation and staining of the kidney tissue proceeded as previously described [6]. The kidneys were fixed in 4% paraformaldehyde, paraffin-embedded, and sectioned at 3 μm. Sections were stained with hematoxylin and eosin (H&E) and Masson’s trichrome using standard protocols. Collagen, nuclei, and muscle fibers were visualized as blue, black, and red, respectively. Periodic acid–Schiff (PAS) staining followed the manufacturer’s instructions (Abcam, Cambridge, MA, USA).

Statistical analysis

Data are presented as mean ± standard error of the mean (SEM). Statistical comparisons were made by one-way ANOVA, followed by Tukey’s post hoc test. A p < 0.05 indicated statistical significance.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

#H.S.C. and #A.S. contributed equally to this work. H.S.C.: Writing - Original Draft, Visualization, Funding acquisition; A.S.: Formal analysis, Investigation, Resources, Writing - Original Draft, Visualization; A.V.: Investigation, Resources, Writing - Review & Editing; A.P.M.: Writing - Review & Editing; P.S.: Writing - Review & Editing; I.J.K.: Formal analysis, Investigation, Resources, Writing - Review & Editing; S.H.S.: Writing - Review & Editing; C.S.K.: Writing - Review & Editing; S.K.M.: Writing - Review & Editing; S.W.K.: Writing - Review & Editing; I.-K.P.: Conceptualization, Methodology, Resources, Writing - Review & Editing, Supervision, Project administration; E.H.B.: Conceptualization, Methodology, Investigation, Resources, Writing - Review & Editing, Supervision, Project administration, Funding acquisition. All authors have read and agreed with the published version of the manuscript.

Funding

This research was supported by the National Research Foundation of Korea (NRF) funded by the Korea government, MSIT (RS-2023-00217317, RS-2025-00513306, and RS-2025-02213506), the grant from Chonnam National University Hospital Biomedical Research Institute (No. BCRI26034), and the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI) funded by the Ministry of Health and Welfare, Republic of Korea (RS-2024-00439029).

Data availability

All data generated or analyzed during this study are included in this published article. Data Availability Statement: Data available on reasonable request from the authors.

Declarations

Ethics approval and consent to participate

All animal use procedures were carried out in accordance with the Animal Care Regulations Committee of Chonnam National University Hospital.

Consent for publication

All authors read and approve the final manuscript.

Competing interests

The authors declare no competing interests.