Introduction

Chronic kidney disease (CKD) is a global health concern characterized by progressive loss of renal function, affecting approximately 10% of the adult population worldwide [1,2]. Among its numerous complications, vascular calcification is particularly prevalent in CKD patients and significantly contributes to their increased cardiovascular morbidity and mortality [3]. In CKD, the disruption of mineral metabolism, particularly elevated serum phosphate levels, triggers the phenotypic transformation of vascular smooth muscle cells (VSMCs) into osteoblast-like cells, leading to accelerated calcium deposition in the vessel wall [4]. While traditional risk factors such as mineral metabolism disorders, uremia, and inflammation are well-established contributors to CKD-induced vascular calcification, emerging evidence suggests that periodontal diseases may contribute to both CKD progression and its associated vascular complications [5].

Porphyromonas gingivalis, a gram-negative anaerobic bacterium and key periodontal pathogen, is increasingly recognized for its systemic impact beyond well-established role in periodontal disease, including cardiovascular disease, diabetes mellitus, and rheumatoid arthritis [6]. Current research indicates that increased levels of serum antibodies against P. gingivalis correlate significantly with reduced kidney function, and experimental studies confirm that oral administration of P. gingivalis leads to CKD development in mice [7,8]. P. gingivalis infection or its virulent factors such as outer membrane vesicles and lipopolysaccharide promoted the calcification of VSMCs [9-11].

To better understand the link between P. gingivalis infection and CKD-associated vascular calcification, we aimed to identify differentially expressed proteins in the serum of P. gingivalis-infected CKD mice compared to non-infected CKD controls, focusing specifically on interleukin-7 (IL-7) among these altered proteins, and to investigate the potential role of IL-7 in vascular calcification.

Materials and Methods

1. Bacterial strain and growth conditions

P. gingivalis strain 381 was cultured in Gifu Anaerobic Medium (GAM) broth (Nissui Pharmaceutical), which contained vitamin K (5 µg/mL) and hemin (5 µg/mL) at 37℃ in an anaerobic chamber in an atmosphere containing 90% N₂, 5% H₂, and 5% CO₂. To prepare the bacteria for infection, an overnight culture was diluted to an optical density of 1.0 at 660 nm in GAM, then washed and resuspended in either phosphate-buffered saline (PBS) for cell infections or PBS with carboxymethylcellulose (CMC) (Sigma-Aldrich) for animal infections.

2. Induction of CKD in the mice model

Eight-week-old male C57BL/6 mice (20–25 g, Samtako) were randomly divided into two groups: CKD and CKD + P. gingivalis. CKD was induced by feeding mice a diet supplemented with 0.2% (w/w) adenine for 8 weeks [12]. The CKD + P. gingivalis group received oral inoculations of live P. gingivalis (10⁹ CFU/100 μL) suspended in PBS containing 2% CMC. The bacterial inoculations were administered directly into the oral cavity three times per week for 8 weeks. The control group (CKD only) received the same volume of 2% CMC without P. gingivalis. All oral administration was performed at the same time for both groups. All animals were sacrificed with CO₂, on the last day of their feed period. Institutional Animal Care and Use Committee (PNU-IACUC) and conformed to the guidelines issued by the Animal Care Committee of the Institute of Laboratory Animal Resources of Pusan National University (PNU-2023-0401).

3. Antibody array assay

Total protein concentration in serum samples collected from both CKD mice and P. gingivalis -inoculated CKD mice was quantified using a bovine serum albumin protein assay kit (Pierce) with Multi-Skan FC (Thermo Fisher Scientific), and sample purity was verified by ultraviolet spectroscopy. The Antibody Array slide (RayBiotech) was air-dried for 2 hours at room temperature and blocked with blocking solution for 30 minutes. Diluted samples were applied to each subarray and incubated for 2 hours at room temperature. Arrays were washed three times with 1X wash buffer I for 5 minutes with shaking, followed by two advanced washing steps with 1X wash buffer II for 10 minutes. The arrays were then incubated with 1X biotin-conjugated anti-cytokine antibodies for 2 hours, followed by 1X Cy3-conjugated streptavidin for 2 hours at room temperature with gentle shaking. After two final washes with 1X wash buffer I for 10 minutes, slides were rinsed with deionized water and centrifuged at 1,000 rpm for 3 minutes to remove excess water.

4. Data acquisition and analysis

The slide scanning was performed using GenePix 4100A Scanner (Axon Instrument). The slides were absolutely dried before the scanning and scanned within 24–48 hours. The slides were scanned at 10 μm resolution, optimal laser power and photomultiplier tube. After got the scan image, they were grided and quantified with GenePix Software (Axon Instrument). After analyzing, the data about protein information was annotated using UniProt DB. Data mining and graphic visualization were performed using ExDEGA (Ebiogen Inc.).

5. Enzyme-linked immunosorbent assay

Cytokine secretions in the conditioned medium or serum samples collected from mice were assayed using an enzymelinked immunosorbent assay (ELISA) kit according to the procedure recommended by the supplier (Arigo Biolaboratories). Absorbance readings of the samples at 450 nm were taken using a Microplate reader (BIOAND). The IL-7 amounts were calculated by interpolating the values onto a standard curve generated as outlined in the manufacturer’s instructions.

6. Cell culture and P. gingivalis infection

A7r5 cells (American Type Culture Collection, ATCC) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Thermo Fisher Scientific) containing 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific) and 1% penicillin streptomycin (Thermo Fisher Scientific) at 37℃ in a humidified atmosphere containing 95% air and 5% CO₂. A7r5 cells were cocultured with live P. gingivalis strain 381 at 37℃ with a multiplicity of infection of 1:100. After 3 hours, the cells were washed with PBS and then cultured in fresh medium. For the control, A7r5 cells underwent a medium change and PBS wash but were not exposed to bacteria.

7. Gene knockdown by small interfering RNA

The small interfering RNA (siRNA) duplexes for rat IL-7 and a negative control siRNA were bought from GenePharma. A7r5 cells were transfected using Amaxa Nucleofector (Lonza) according to the manufacturer’s instructions.

8. Induction and quantification of calcification

A solution of inorganic phosphate (Pi; Na₂HPO₄ and NaH₂PO₄, pH 7.4) was added to serum-supplemented DMEM at a final concentration of 1.4, 2.6, and 3.5 mM. After the indicated incubation period, cellular calcium deposition was observed an alizarin red staining.

9. Alizarin red S staining

Cells were fixed with 4% paraformaldehyde at room temperature for 20 minutes and stained with 2% alizarin red S (ARS) solution (pH 4.2, adjusted with 1.0% NH₄OH) for 10 minutes at room temperature. After staining, calcium deposits were photographed. After imaging, stained cells were destained with 10% acetic acid for 20 minutes, and the calcium concentration was measured based on the absorbance at 420 nm using an ELISA reader (BIOAND).

10. Quantitative real-time reverse transcription-polymerase chain reaction

Total RNA was isolated using a RiboEx kit (GeneAll), and reverse-transcribed with a reverse transcription kit (Promega), followed by real-time reverse transcription-polymerase chain reaction (RT-PCR) with SYBR Green premix (Enzynomics) using oligonucleotide primers as follows: β-actin : 5′-AGGG AAATCGTGCGTGAC-3′ and 5′-CGCTCATTGCCGATAGTG-3′; IL-7 : 5′-CCCTGATCCTTGTGCTGCTG-3′ and 5′-TCAGCACACTCCCAAAGGCT- 3′; Alkaline phosphatase (ALP): 5′-TGCT TTGTGTGTGCTGACTGTA-3′ and 5′-AGTGACGGTGTCGTAGC CTTCT-3′; Runt-related transcription factor 2 (Runx2): 5′-GCCGGGAATGATGAGAACTA- 3′ and 5′-TGGGGAGGATTTGTGA AGAC-3′. Cycling parameters included 1 cycle at 95℃ for 10 minutes, followed by amplification for 40 cycles at 95℃ for 15 seconds, 60℃ for 60 seconds, and 72℃ for 7 seconds. The entire cycling process was monitored using the Applied Biosystems.

11. Western blot analysis

Cell lysates were prepared in buffer containing 40 mM Tris- Cl, 10 mM EDTA, 120 mM NaCl, 0.1% Nonidet P-40, and protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was determined by bicinchoninic acid assay, and 30 μg of total protein was separated by 10% SDS-PAGE and transferred to nitrocellulose membranes. After blocking with 5% skim milk, membranes were incubated with primary antibodies against IL-7 (Santa Cruz Biotechnology) and β-actin (Bioworld Technology) overnight at 4℃, followed by HRP-conjugated secondary antibodies (Thermo Fisher Scientific). Proteins were visualized using enhanced chemiluminescence solution (Amersham Pharmacia Biotech) on an Azure 300 Imaging system (Azure Biosystems).

12. Statistical analysis

Data are represented as mean ± standard deviation of at least three independent experiments. Data were subjected to a one-way analysis of variance with Tukey’s honest significant difference post-hoc test and Student’s t-test using IBM SPSS v27 (IBM Corp.).

Results

1. Oral administration of P. gingivalis alters serum protein expression profiles in a CKD mouse model

To investigate the effects of oral P. gingivalis administration on CKD, we performed comprehensive serum protein profiling using antibody array analysis in CKD mice with or without P. gingivalis administration. The analysis revealed distinct protein expression patterns with both upregulated and downregulated proteins across multiple biological function categories (Fig. 1A). Among the differentially expressed proteins, IL-7 showed notable upregulation in categories associated with inflammatory response and apoptotic processes. Subsequent validation by ELISA confirmed significantly elevated serum IL-7 levels in P. gingivalis-administered CKD mice compared to CKD controls (Fig. 1B).

2. Expression of IL-7 in P. gingivalis -infected vascular calcification of VSMCs

Accelerated vascular calcification is a characteristic pathological feature in patients with CKD [13]. The disruption of mineral homeostasis in CKD, notably increased serum phosphate, drives the phenotypic switch of VSMCs toward an osteoblast-like state [4]. We evaluated calcium deposition in A7r5 cells exposed to different phosphate concentrations using ARS staining (Fig. 2A). While cells maintained at physiological phosphate levels (1.4 mM Pi) showed no calcium deposition, exposure to elevated phosphate concentrations (2.6 mM and 3.5 mM Pi) induced concentration-dependent calcification (Fig. 2B). To investigate the involvement of IL-7 in P. gingivalis - stimulated calcification of VSMCs, we analyzed expression level of IL-7. IL-7 mRNA expression and serum protein levels were significantly elevated in the VSMCs under calcifying medium (2.6 mM high phosphate). Markedly, P. gingivalis infection further enhanced the phosphate-induced IL-7 mRNA expression and protein secretion compared to control cells (Fig. 2C and 2D).

3. Validation of IL-7 knockdown efficiency in VSMCs

To further investigate the functional role of IL-7 in calcification of VSMCs, we performed siRNA-mediated knockdown of IL-7 in A7r5 cells. The efficiency of IL-7 knockdown was confirmed at both mRNA and protein levels. Real-time RT-PCR analysis revealed that IL-7 siRNA significantly reduced IL-7 mRNA expression compared to control siRNA-transfected cells (Fig. 3A). Western blot analysis further confirmed the successful knockdown of IL-7 , showing significantly decreased IL-7 protein levels in siRNA-transfected cells. Densitometric analysis of the western blot data demonstrated substantial reduction in IL-7 protein expression when normalized to β-actin levels (Fig. 3B).

4. Effect of IL-7 silencing on P. gingivalis -stimulated VSMC calcification and osteogenic marker expression

To determine whether IL-7 is required for P. gingivalis - stimulated VSMC calcification, we examined the effect of IL-7 knockdown on calcification in the presence of P. gingivalis . Following IL-7 siRNA transfection, VSMCs were cultured in calcifying medium with or without P. gingivalis infection. ARS staining revealed that IL-7 silencing significantly attenuated the P. gingivalis-enhanced calcium deposition in VSMCs under high-phosphate conditions (Fig. 4A). Quantitative analysis of calcium deposition confirmed the significant reduction in mineralization in IL-7 knockdown cells compared to control siRNA-transfected cells (Fig. 4B). To further investigate the effect of IL-7 inhibition in P. gingivalis-stimulated osteogenic differentiation of VSMCs, we examined the expression of key osteogenic markers, ALP and Runx2. Quantitative real-time RT-PCR analysis demonstrated that P. gingivalis -stimulated upregulation of these osteogenic markers was markedly attenuated in IL-7-silenced VSMCs under calcification condition (Fig. 4C).

Discussion

Mounting evidence suggests a significant association between periodontal disease and renal dysfunction [14]. The presence of periodontal pathogens and their virulent factors in systemic circulation may directly impact renal function [8,15]. Gingipain, extracellular proteases secreted from P. gingivalis, disrupted renal tubular epithelial integrity in P. gingivalis-associated acute kidney injury [15]. The increased oxidative stress associated with periodontal disease creates a destructive cycle that may accelerate tissue damage in kidney [8,16]. Moreover, the inflammatory burden of periodontal disease appears to contribute to CKD progression [17]. The systemic inflammatory cascade initiated by periodontal disease results in elevated circulating pro-inflammatory cytokines, potentially contributing to renal dysfunction [17]. Clinical research has shown that plasma levels of IL-6 and tumor necrosis factor-alpha (TNF-α) serve as sensitive markers for detecting periodontal inflammation in CKD patients [18]. We demonstrated in this study IL-7 identified from serum P. gingivalis-infected CKD mice model can play a critical role in P. gingivalis-stimulated calcification of VSMCs. Based on these observations, we identified IL-7 as a novel mediator potentially bridging periodontal disease and CKD-associated vascular calcification. Our findings suggest that IL-7 may serve as a crucial inflammatory intermediary in the periodontal-renal axis, providing new mechanistic insights into the pathogenic relationship between periodontal inflammation and vascular complications in CKD.

The bidirectional relationship between CKD and periodontal diseases represents a complex interplay of inflammatory mediators and systemic conditions [19]. Periodontal diseases may accelerate CKD progression through systemic inflammatory burden [20]. Moreover, periodontal treatment has been shown to improve markers of kidney function, including reduced levels of C-reactive protein and enhanced glomerular filtration rate in CKD patients [21,22]. Conversely, patients with CKD exhibit increased prevalence and severity of periodontal diseases, which can be attributed to several factors including disturbed calcium-phosphorus metabolism, compromised immune response, and uremia-associated inflammation [23,24]. In this context, our findings suggest a novel role for IL-7 in mediating this bidirectional relationship. IL-7, traditionally known for its crucial role in T-cell homeostasis and development [25,26], may serve as a key inflammatory mediator linking periodontal inflammation to vascular complications in CKD. Recent proteomic analyses have revealed elevated levels of IL-7 in the serum of patients with CKD [27]. Notably, increased IL-7 concentrations were detected in both the culture media of peripheral blood mononuclear cells (PBMCs) from periodontitis patients and their corresponding serum samples [28]. The presence of elevated IL-7 in both types of samples points to a possible link between periodontal disease and CKD. Further research is warranted to elucidate the potential role of IL-7 as a mediating factor in the bidirectional relationship between these two conditions.

In conclusion, this study demonstrated that P. gingivalis accelerates vascular calcification in CKD through IL-7 upregulation, as evidenced by enhanced calcium deposition and osteogenic marker expression in VSMCs. Our findings reveal IL-7 as a potential therapeutic target for preventing vascular calcification in CKD patients with periodontal infection, although further investigation is needed to fully understand the underlying molecular mechanisms and evaluate clinical applications.