Possible involvement of tubular interleukin-34 in macrophage recruitment during colistin-induced nephrotoxicity in rats

Abstract

Colistin is a cationic cyclic lipopeptide antibiotic used against multidrug-resistant Gram-negative bacteria; however, its clinical application is limited by nephrotoxicity. Although oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum stress are implicated in the primary cytotoxic mechanisms, the secondary inflammatory pathways remain poorly understood. In this study, we aimed to elucidate the mechanisms underlying colistin-induced nephrotoxicity by analyzing the molecular responses of injured renal tubules isolated by laser microdissection (LMD) in a rat model. Six-week-old male Sprague–Dawley rats were subcutaneously administered colistin (0, 15, or 30 mg/kg/day) for 28 days. Increases in serum creatinine and blood urea nitrogen were associated with tubular vacuolation, single-cell necrosis, and regenerative changes in proximal tubules. Immunohistochemistry revealed increased expression of kidney injury molecule-1 (KIM-1) and presence of cleaved caspase-3 in the injured tubules, indicating epithelial regeneration and apoptosis, respectively. LMD-based microarray analysis identified 486 upregulated and 472 downregulated genes in vacuolated/regenerative tubules compared with normal tubules. Pathway analysis indicated activation of immune-related processes, particularly associated with macrophage activation and trafficking, including interleukin-34 (IL-34). In situ hybridization confirmed Il34 mRNA expression in the cytoplasm of the injured tubules, and accumulation of CD68-positive macrophages around KIM-1-positive tubules. Conversely, no significant change was observed in CD163-positive macrophages following colistin treatment, suggesting proinflammatory M1 macrophage predominance. These findings indicate that tubular IL-34 induction and subsequent macrophage recruitment amplify colistin-induced nephrotoxicity at the secondary level, suggesting that proximal tubules function as both primary targets and effectors of inflammation. Targeting the IL-34-related signaling could thus serve as a potential approach to alleviate colistin-induced renal injury.

INTRODUCTION

Colistin (polymyxin E) is a cationic cyclic lipopeptide antibiotic that was introduced in the 1950s, but later discontinued for clinical use due to its nephrotoxic and neurotoxic effects (Heybeli et al., 2019). However, in recent decades, the rapid global emergence and spread of multidrug-resistant Gram-negative pathogens have necessitated its reintroduction as a last-resort therapeutic agent (Li et al., 2006). Colistin exhibits potent bactericidal activity against carbapenem-resistant Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae, for which few or no alternative treatments exist (Li et al., 2006). Consequently, its clinical importance has grown in parallel with the escalating antimicrobial resistance crisis. Despite its critical role, nephrotoxicity remains the major dose-limiting adverse effect, with an incidence reported in approximately 20–40% of treated patients (Falagas and Kasiakou, 2006; Gai et al., 2019). Clinical manifestations range from mild elevations in serum creatinine (sCre) to acute tubular necrosis and acute kidney injury, which may necessitate dose adjustment or discontinuation of therapy (Falagas and Kasiakou, 2006). These adverse effects significantly compromise therapeutic efficacy and patient outcomes. Therefore, elucidating the molecular mechanisms underlying colistin-induced nephrotoxicity is essential to advance the understanding of drug-induced kidney injury and to facilitate the development of strategies that mitigate toxicity while preserving antibacterial efficacy.

Accumulating evidence indicates that the proximal tubular epithelium is the principal site of colistin-induced renal injury, where the drug preferentially accumulates and initiates cytotoxic responses (Gai et al., 2019). Several primary mechanisms of injury have been proposed, including oxidative stress, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress (Azad et al., 2019). However, despite these mechanistic insights, effective clinical interventions to prevent or mitigate colistin-induced nephrotoxicity remain unavailable, suggesting that additional unrecognized pathogenic mechanisms may contribute to the renal injury. Notably, injured proximal tubules can actively secrete inflammatory mediators that recruit immune cells and amplify tissue damage (Baker and Cantley, 2025). The involvement of such secondary inflammatory processes in colistin toxicity remains poorly defined but may represent a critical amplifying mechanism. To elucidate these complex pathways, site-specific transcriptomic approaches offer a powerful means of dissecting molecular events at the site of injury. In our previous studies, laser microdissection (LMD) was used to isolate proximal tubules from rat kidneys, followed by microarray analysis, which successfully revealed toxicological mechanisms at the precise site of injury (Matsushita et al., 2018, 2024a, 2024b). Building on this established approach, we applied the same strategy to colistin-induced nephrotoxicity to identify both primary and secondary pathogenic processes, thereby offering new insights into the propagation of inflammation and the exacerbation of renal injury associated with proximal tubular damage.

In the present study, we aimed to elucidate the mechanisms underlying colistin-induced kidney injury, by analyzing the global gene expression profiles of injured renal tubules isolated from colistin-treated rats using an LMD-based microarray approach. Our findings suggest a secondary injury mechanism mediated by interleukin-34 (IL-34), which is produced by injured tubular epithelial cells.

MATERIALS AND METHODS

Chemicals

Colistin sulfate (lot no. WTH2798; purity, 83.4%) and saline were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and Otsuka Pharmaceutical Factory, Inc. (Tokyo, Japan), respectively.

Experimental animals and housing conditions

Male, 5-week-old, specific pathogen-free Sprague–Dawley rats (Crl:CD(SD)) were obtained from The Jackson Laboratory Japan, Inc. (Kanagawa, Japan) and acclimated for one week before experimentation. The animals were housed in polycarbonate cages containing soft chip bedding (Sankyo Labo Service, Tokyo, Japan) under barrier system conditions with a controlled 12 hr light/dark cycle, ventilation rate of 20 air exchanges per hour, temperature maintained at 23 ± 1°C, and relative humidity of 50% ± 5%. The animals had ad libitum access to water and a basal diet (CRF-1; Oriental Yeast Co., Ltd., Tokyo, Japan). All experimental protocols were approved by the Animal Care and Utilization Committee of the National Institute of Health Sciences (approval no. 908), and all procedures complied with institutional guidelines for animal care and use.

Animal treatments

Rats were subcutaneously administered saline (vehicle) or colistin sulfate at doses of 15 or 30 mg/kg (5 mL/kg) once daily for 28 consecutive days (n = 5/group). One day after the final administration, the rats were euthanized by exsanguination via transection of the abdominal aorta under deep isoflurane anesthesia. At necropsy, blood samples were collected from the abdominal aorta for serum biochemical analysis of blood urea nitrogen (BUN) and sCre. The kidneys were excised, weighed, and processed for histological and molecular analyses. Kidney tissues were sliced into pieces and fixed in 10% neutral-buffered formalin (FUJIFILM Wako Pure Chemical Corporation) at room temperature overnight, followed by paraffin embedding, and sectioning at a thickness of 4 μm for hematoxylin and eosin (HE) staining, immunohistochemistry, in situ hybridization, and immunofluorescence. Histopathological grading was performed as previously described (Watanabe et al., 2017): (±) minimal; (+) mild; (++) moderate; (+++) marked; and (++++) severe. Additional kidney specimens were embedded in O.C.T. compound (Sakura Fintech Japan, Tokyo, Japan) and stored at −80°C for subsequent microarray analysis following LMD. The remaining kidney samples were snap-frozen in liquid nitrogen and stored at −80°C for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis.

Immunohistochemistry

For immunohistochemistry, the following primary antibodies were used: against kidney injury molecule-1 (KIM-1; 1:1000, polyclonal; #AF3689; R&D Systems, Minneapolis, MN, USA), anti-cleaved-caspase 3 (c-caspase 3; 1:400, polyclonal; #9661S; Cell Signaling Technology, Danvers, MA, USA), and CD68 (1:100, clone ED1; #MCA341R; Bio-Rad, Hercules, CA, USA). Antigen retrieval for c-caspase 3 was performed in pH 9.0 antigen retrieval solution (Dako, Glostrup, Denmark), whereas KIM-1 or CD68 antigen retrieval was performed in pH 6.0 citrate buffer, using an autoclave at 121°C for 15 min. The kidney sections were immersed in 3% H₂O₂ in methanol to inactivate endogenous peroxidase activity. After blocking nonspecific binding with 10% normal goat serum (c-caspase 3 and CD68: NICHIREI BIOSCIENCES, Tokyo, Japan) or 10% normal rabbit serum (KIM-1: NICHIREI BIOSCIENCES), sections were incubated with each primary antibody overnight at 4°C. Visualization was performed using the Histofine Simple Stain Rat MAX PO Kit (NICHIREI BIOSCIENCES) and 3,3′-diaminobenzidine (DOJINDO LABORATORIES, Tokyo, Japan) as the chromogen. Positive areas for KIM-1 and CD68 across entire kidney sections were quantified using an image analyzer (HALO, Albuquerque, NM, USA). All c-caspase 3-positive cells in kidney sections were counted, and the number of positive cells per unit area was calculated.

In situ hybridization

The localization of Il34 mRNA was detected using a specific probe (probe number: 1056011-C1) and the RNAscope 2.5 HD Reagent Kit-Brown (Advanced Cell Diagnostics, Newark, NJ, USA), according to the manufacturer's instructions.

Immunofluorescence

To analyze co-localization of CD68 and KIM-1, sections were autoclaved at 121°C for 15 min in 10 mM citrate buffer (pH 6.0) for antigen retrieval. After blocking with 10% normal donkey serum, sections were incubated with mouse anti-CD68 antibody (1:100) at 4°C overnight. Then, the sections were incubated with secondary anti-mouse immunoglobulin G (IgG) antibodies (1:200; Alexa Fluor 647; #ab150111; Abcam, Cambridge, UK) at room temperature for 2 hr. Subsequently, sections were incubated with goat antibodies against KIM-1 (1:500) at 37°C for 1 hr, followed by incubation with secondary anti-goat IgG antibodies (1:500; Alexa Fluor 488; #ab150133; Abcam). To eliminate autofluorescence, sections were treated with the Vector TrueVIEW Autofluorescence Quenching Kit (Vector Laboratories, Burlingame, CA, USA) and, subsequently, mounted using Vibrance Antifade Mounting Medium containing DAPI (Vector Laboratories). Finally, immunofluorescent images were captured using the All-in-One Fluorescence Microscope BZ-X710 (Keyence Corp., Osaka, Japan).

RT-qPCR for analysis of mRNA expression

Total RNA was extracted from freshly frozen kidney tissues using the RNeasy Mini Plus Kit (Qiagen GmbH, Hilden, Germany). To detect the mRNA expression of Havcr1 (KIM-1) and Il34 (IL-34), RT-qPCR assays were performed using specific primers for rat Havcr1 (Rn00597703_m1; Thermo Fisher Scientific, Waltham, MA, USA) and Il34 (Rn01432377_m1; Thermo Fisher Scientific). Primers for eukaryotic 18S rRNA (Hs99999901_s1; Thermo Fisher Scientific) were used as the endogenous reference. RT-qPCR was performed on the QuantStudio 3 Real-Time PCR System (Thermo Fisher Scientific) using TaqMan Fast Universal PCR Master Mix and TaqMan Gene Expression Assays (Thermo Fisher Scientific). Expression levels of target genes were calculated using the relative standard curve method and normalized to 18S rRNA expression. Data were expressed as fold-change values relative to the control group, comparing gene expression in the kidneys of colistin-treated rats with that in controls.

LMD-based sampling of renal tubules and total RNA extraction

Frozen kidney sections embedded in O.C.T. were sectioned at 15-μm thickness using a cryotome (Leica Microsystems, Wetzlar, Germany), fixed with 5% acetic acid in ethanol, and subjected to HE staining. Images of normal proximal tubules (control group) and vacuolated/regenerative tubules (30 mg/kg colistin-treated group) were captured using the LMD6000 system (Leica Microsystems). Total RNA was extracted using the RNeasy Micro Plus Kit (Qiagen), and its concentration and integrity were evaluated with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). The RNA integrity number (RIN) was used as an indicator of RNA quality. Samples with RIN values >7.0 were amplified and converted to Single Primer Isothermal Amplification (SPIA) cDNA using the Ovation PicoSL WTA System V2 (NuGEN, San Carlos, CO, USA) for microarray analysis.

Microarray and data analysis

SPIA cDNA was labeled with cyanine-3 dye using the SureTag DNA Labeling Kit (Agilent Technologies). cDNA concentration, dye incorporation efficiency, and quality were verified using an ultraviolet-visible spectrophotometer. Fluorescently-labeled cDNA was hybridized to Agilent 4 × 44K Whole Rat Genome Microarray gene expression chips (version 3.0; Agilent Technologies), according to the manufacturer’s instructions. Hybridized arrays were scanned using the Agilent Microarray Scanner (Model G2565BA; Agilent Technologies). Image processing and raw data extraction were performed using the Feature Extraction software (Agilent Technologies). Normalization of gene expression data and filtering of probe sets by expression levels, flags, and errors were performed using the GeneSpring software (Agilent Technologies). Differentially expressed genes (DEGs) between normal proximal tubules and vacuolated/regenerative tubules were identified using analysis of variance (ANOVA; cutoff: P < 0.05) and t-test with Benjamini–Hochberg false discovery rate correction, applying fold-change thresholds of >2.0 or <−2.0. Functional significance and pathway associations of DEGs were analyzed using Ingenuity Pathway Analysis^® (IPA; Qiagen).

Statistical analysis

All statistical analyses were performed using GraphPad Prism version 9 (GraphPad Software Inc., San Diego, CA, USA). Data are presented as means ± standard deviations (S.D.s). Significant differences between groups were assessed by one-way ANOVA followed by Dunnett’s multiple comparison test. Statistical significance was set at P < 0.05.

RESULTS

Body and kidney weights and serum biochemical parameters

The body weights of rats in both colistin-treated groups (15 and 30 mg/kg) significantly decreased compared with those in the control group throughout the experiment (Fig. 1A). The absolute kidney weights were significantly reduced for both treatment groups, whereas relative kidney weights were significantly increased for the 30 mg/kg group (Fig. 1B). BUN and sCre levels were significantly elevated for both colistin-treated groups (Fig. 1C).

Fig. 1

Systemic and biochemical effects of colistin. A. Body weight changes during the experimental period. B. Absolute and relative kidney weights. C. Serum biochemical parameters, including blood urea nitrogen (BUN) and serum creatinine (sCre). Values are presented as means ± standard deviations (S.D.s; n = 5) for panels (A, B, and C). * and ** indicate significant differences from the control group at P < 0.05 and P < 0.01, respectively.

Histopathological and immunohistochemical analysis

As summarized in Table 1, histopathological changes, including vacuolation, single-cell necrosis, tubular necrosis, and regeneration, were observed in the proximal tubules of rats in both colistin-treated groups. Representative images are shown in Fig. 2A. The vacuolated tubules exhibited high nuclear density and prominent nucleoli, indicating a regenerative response. Tubular dilatation is considered a secondary change following proximal tubular injury. Immunohistochemistry and RT-qPCR analyses revealed an increase in KIM-1, a marker for renal tubular regeneration (Bailly et al., 2002), in rats in the colistin-treated groups (Fig. 2B and C). The number of c-caspase 3-positive cells, a marker for apoptosis, was also increased significantly (Fig. 2B and C).

Table 1. Histopathology for kidneys of rats treated with 15 and 30 mg/kg of colistin.

	Colistin (mg/kg)
Findings	0	15	30
No. of animals	5	5	5
Vacuolation, proximal tubules (±/+/++)	-	5 (5/0/0)	5 (0/1/4)
Necrosis, single cell, proximal tubules (±/+/++)	-	5 (3/2/0)	5 (0/1/4)
Necrosis, proximal tubules (±)	-	-	2
Regeneration, tubules (±/+)	-	2 (2/0)	5 (1/4)
Dilatation, tubules (±/+)	-	-	3 (1/2)

±: Minimal, +: Mild, ++: Moderate, +++: Marked, ++++: Severe

Fig. 2

Histopathological analysis of kidneys in a rat model of colistin-induced nephrotoxicity. A. Representative photographs of histopathological findings observed in the kidney. Hematoxylin and eosin (HE) staining. B. Immunohistochemical detection of kidney injury molecule-1 (KIM-1) and cleaved-caspase 3 (c-caspase 3). C. Quantitative image analysis of KIM-1 and c-caspase 3, and quantitative PCR analysis for Havcr1 (encoding KIM-1). Arrowhead indicates single cell necrosis; arrows indicate c-caspase 3-positive cells. Values represent means ± standard deviations (S.D.s; n = 5) for panel C. ** indicates significant differences from the control group at P < 0.01. Bars indicate 20 µm (A) or 100 µm (B).

LMD-based microarray and pathway analysis

Fig. 3A shows representative images of LMD sampling of normal proximal tubules in rats from the control group and vacuolated/regenerative tubules in those from the 30 mg/kg colistin-treated group. As depicted in Fig. 3B, 486 genes were upregulated and 472 genes were downregulated in the vacuolated/regenerative tubules compared with those in the normal proximal tubules. Fig. 3C presents the results of IPA based on the “Diseases and Functions” category. A notably high z-score was observed for the “Immune cell trafficking” category, revealing the upregulation of multiple genes related to macrophage activation. Among these, IL-34 was selected for further analysis because it is closely associated with macrophage migration and proliferation (Baghdadi et al., 2018).

Fig. 3

Laser microdissection (LMD)-based microarray and pathway analyses in a rat model of colistin-induced nephrotoxicity. A. Representative LMD sampling images showing normal proximal renal tubules in rats from the control group and injured tubules in those from the 30 mg/kg colistin-treated group. B. Volcano plot depicting differential gene expression in the microarray dataset. C. “Diseases and Functions” annotation in Ingenuity Pathway Analysis^® highlighting the “Immune cell trafficking.” The illustrated pathway was constructed using genes classified under “Immune cell trafficking.” Arrowhead indicates normal proximal tubule; arrows indicate vacuolated/regenerative tubule. The rectangle indicates Il34 mRNA. Bars indicate 50 µm.

Expression of Il34 mRNA in injured tubules and macrophage infiltration

In situ hybridization revealed increased Il34 mRNA expression in the cytoplasm of injured tubules in rats from the colistin-treated groups, which exhibited high nuclear density, a feature of regenerative tubules (Fig. 4A). RT-qPCR analysis of whole kidney tissues showed an increase in Il34 mRNA expression (Fig. 4A). CD68-positive macrophages (M1-type, proinflammatory macrophages; Yamate et al., 2023) were observed in the interstitium, surrounding injured tubules (Fig. 4B). Immunofluorescence confirmed that CD68-positive macrophages accumulated around KIM-1-positive tubules (Fig. 4C). In contrast, CD163-positive macrophages (M2-type, reparative macrophages; Yamate et al., 2023) were rarely detected in rats from both the control and colistin-treated groups (Fig. 4D).

Fig. 4

Induction of interleukin-34 (IL-34) in injured tubules and peritubular macrophage accumulation in a rat model of colistin-induced nephrotoxicity. A. In situ hybridization showing Il34 mRNA expression in injured tubules and quantitative PCR quantification of Il34 mRNA in whole kidney tissues. B. Immunohistochemical detection of CD68-positive macrophages. C. Immunofluorescence for kidney injury molecule-1 (KIM-1)-positive injured tubules and CD68-positive macrophages. D. Immunohistochemistry for CD163-positive macrophages. Insets show CD163-positive macrophages in the renal pelvis. Values represent means ± standard deviations (S.D.s; n = 5) for panels A, B, and D. ** indicates significant difference from the control group at P < 0.01. Bars indicate 20 µm (A) or 50 µm (B, C, and D).

DISCUSSION

In this study, we demonstrated that colistin-induced nephrotoxicity in rats was characterized by proximal tubular injury accompanied by increased KIM-1 expression, enhanced apoptosis, and IL-34 upregulation in injured tubular epithelial cells. LMD-based transcriptomic analysis revealed activation of immune-related pathways, particularly those associated with macrophage activation and trafficking. Immunohistochemical and in situ hybridization analyses confirmed that IL-34 was produced by injured tubular cells and that CD68-positive macrophages accumulated around these lesions. These findings suggest that proximal tubular epithelial cells are not only the primary targets of colistin toxicity but also active mediators of interstitial inflammation through IL-34-associated macrophage recruitment.

The body weight of rats in the 30 mg/kg colistin-treated group decreased by week 4, suggesting that the maximal tolerated dose was reached. Moreover, increases in relative kidney weight, BUN, and sCre levels indicated substantial renal injury. Typical histopathological features of colistin-induced nephrotoxicity were also detected (Ghlissi et al., 2013; Kabel and Salama, 2021; Ozkan et al., 2013). Collectively, these systemic, biochemical, and histopathological findings confirm that our experimental protocol effectively reproduced colistin-induced renal toxicity.

Histopathological examination revealed vacuolar degeneration in the proximal tubular epithelial cells of rats in colistin-treated groups. Previous ultrastructural studies have reported ER dilatation in tubular cells exposed to colistin (Dai et al., 2014), suggesting that the vacuolation observed in the present study may partly correspond to ER stress. ER stress, together with oxidative stress and mitochondrial dysfunction, has been implicated as one of the principal mechanisms of colistin-induced cytotoxicity (Lee et al., 2019; Worakajit et al., 2022; Xie et al., 2024). These interrelated stress responses can trigger apoptosis, consistent with the increased levels of c-caspase 3 observed in our study. Accordingly, the vacuolar and apoptotic changes observed in our study likely represent downstream outcomes of multiple overlapping cellular stress pathways rather than a single causative mechanism. Further studies would be valuable in demonstrating that these mechanisms are occurring under the present experimental conditions.

In addition to direct epithelial cytotoxicity, our data suggest the presence of a secondary inflammatory component that may further amplify renal damage. Transcriptomic and histological findings demonstrated the induction of IL-34 in injured tubules and peritubular accumulation of CD68-positive M1 macrophages, which promote inflammation and tissue injury primarily through the production of IL-1β or reactive oxygen species (Yamate et al., 2023). IL-34 is known to interact with multiple receptors or binding partners, including the colony-stimulating factor 1 receptor, protein tyrosine phosphatase receptor type Z1, and syndecan-1, through which it may exert diverse biological effects in immune and tissue microenvironments (Baghdadi et al., 2018). The relative contribution of each receptor or binding partner to tubular injury and inflammatory responses in colistin-induced nephrotoxicity remains unclear and warrants further investigation. Previous studies using ischemia–reperfusion models have shown that tubular IL-34 promotes macrophage infiltration and aggravates both acute kidney injury and its progression to chronic disease (Baek et al., 2015; Sanchez-Niño et al., 2016). Although evidence for IL-34 involvement in drug-induced kidney injury remains limited, a recent study demonstrated that IL-34 blockade attenuated cisplatin-induced nephrotoxicity in mice (Wada et al., 2021), suggesting that IL-34-mediated mechanisms may also contribute to drug-induced kidney injury. However, the present findings should be interpreted as demonstrating an association between tubular IL-34 induction and macrophage accumulation, rather than definitive evidence of a causal relationship.

In our rat model, IL-34 expression was localized to KIM-1-positive regenerative tubules and was accompanied by peritubular accumulation of CD68-positive M1 macrophages, whereas CD163-positive M2 macrophages were scarce. This pattern indicates a predominance of proinflammatory responses that may hinder repair and exacerbate tissue injury. The concurrent expression of KIM-1, induction of IL-34, and macrophage infiltration suggests a complex epithelial–immune crosstalk, in which injured tubules both attempt regeneration and signal for immune cell recruitment. Thus, the outcome of colistin-induced nephrotoxicity may depend on the balance between repair and inflammation.

Although our data implicate IL-34 as a potential mediator of inflammatory amplification in colistin nephrotoxicity, a definitive causal relationship has yet to be established. Functional studies using IL-34 knockout animals and neutralizing antibodies, or pharmacological inhibition of the IL-34-related signaling pathways are required to clarify whether IL-34 directly contributes to the progression of renal injury. Furthermore, time-course analyses examining the temporal relationship between tubular injury, IL-34 induction, macrophage infiltration, and inflammatory mediator production would be essential to distinguish causal effects from secondary responses.

In conclusion, the overall results of this study indicate that secondary inflammatory responses are associated with tubular IL-34 induction and macrophage recruitment, with a predominance of proinflammatory macrophage phenotypes, in a rat model of colistin-induced nephrotoxicity. These findings suggest that the IL-34-related signals may be involved in the inflammatory processes accompanying tubular injury and therefore warrant further investigation as a potential therapeutic target for antibiotic-induced renal injury.

ACKNOWLEDGMENTS

We thank Ayako Saikawa and Yoshimi Komatsu for expert technical assistance in processing histological materials.

Funding

This work was supported by a Grant-in-Aid from the Ministry of Health, Labour, and Welfare, Japan (Grant No. 24KD2002).

Conflict of interest

All authors declare that they have no conflicts of interest.

Data availability

The data in this study are included in the article. Contact the corresponding author directly to request the underlying data. Complete microarray data have been submitted and are readily retrievable from the public database National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) with accession number GSE313541.

Author contributions

Conceptualization: K.M.

Funding acquisition: K.M.

Investigation: K.M., H.A., J.A., T.M., and Y.M.

Supervision: T.T.

Visualization: K.M.

Writing – original draft: K.M.

Writing – review & editing: H.A., J.A., T.M., Y.M., K.O., and T.T.

Ethical approval and consent to participate

All experimental protocols were approved by the Animal Care and Utilization Committee of the National Institute of Health Sciences (approval no. 908), and all procedures complied with institutional guidelines for animal care and use.

Patient consent for publication

Not applicable.

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