Introduction

Chronic kidney disease (CKD) and chronic periodontitis are inflammatory conditions with a bidirectional relationship, and inflammation plays a key role in influencing and exacerbating the progression of each condition [1]. While there is ample literature linking chronic periodontitis to various systemic inflammatory diseases, such as cardiovascular disease, diabetes, and rheumatoid arthritis, available research exploring the direct impact of chronic periodontitis on CKD is relatively limited [2]. Although common risk factors and systemic inflammation associated with chronic periodontitis may have implications for CKD, further investigation is necessary to establish a clearer understanding of the specific relationship between the two conditions. The identification of a clinical biomarker is crucial at the earliest stage for individuals who are at risk.

Although many biomarkers have been studied, including inflammatory cytokines, growth factors, and enzymes, highly specific biomarkers for the definitive diagnosis and prognosis of CKD and chronic periodontitis have not yet been identified.

Galectins are a family of beta-galactoside-binding proteins that have both pro- and anti-inflammatory effects. Several studies have shown that galectin-3 (Gal-3) is the most important member of this family and a possible biomarker for the early detection of inflammatory diseases [3]. Gal-3 is systemically released at sites of active inflammation in CKD and chronic periodontitis, promoting the activation and adhesion of neutrophils at the site of inflammation, the differentiation of monocytes into macrophages, the stimulation of T-cell apoptosis, and an increased immune response to many pathogens [4].

Gal-3 is involved in angiogenesis, fibroblast activation, fibrosis, and modulation of immune-inflammatory responses. As Gal-3 levels are high in CKD and chronic periodontitis, estimating Gal-3 levels could indicate the severity of these conditions. Although Gal-3 has been described as a mediator of inflammation and fibrosis in CKD and periodontitis, to the best of our knowledge, a link between these two conditions based on Gal-3 has not been assessed. Measuring Gal-3 levels in patients with chronic periodontitis and CKD could validate the biological plausibility of both diseases and the validity of Gal-3 as a marker of inflammatory burden in these patients.

Therefore, the present study aimed to assess salivary Gal-3 concentrations in patients with chronic periodontitis and CKD.

Methods

Ethics statement: The study was conducted in accordance with the Declaration of Helsinki on Medical Research, reviewed in 2016. Ethical approval was obtained from the Institutional Ethics Committee of Sri Ramachandra University (REF: CSP/22/JUL/114/428). Informed consent was obtained from all participating patients, who were notified of the purpose and risks of the study.

1. Patients and treatment

A total of 75 patients who reported to the Departments of Nephrology and Periodontology at the Sri Ramachandra Institute of Higher Education in Chennai, India, were screened and enrolled in this study. The enrolled patients were divided into three groups: Group I, patients with CKD and chronic periodontitis (n=25); Group II, patients with chronic periodontitis who are systemically healthy (n=25); and Group III, patients with CKD without chronic periodontitis (n=25).

Patients who have a history of other systemic malignancies, take anti-inflammatory or immunosuppressant drugs, are pregnant or lactating, consume alcohol and smoke, or underwent periodontal therapy in the last 6 months were excluded from the study.

The patients’ demographic details were recorded, and they were subjected to a comprehensive periodontal assessment that included evaluation of the Simplified Oral Hygiene Index [5], plaque index (PI) [6], bleeding on probing (BOP), periodontal probing depth (PPD), and clinical attachment loss (CAL) using a sterilized UNC-15 probe. Periodontal inflamed surface area (PISA), developed by Nesse et al. [7] in 2008, was also calculated. This estimation measures the area (in mm²) of inflamed periodontal tissue, using PPD, CAL, and BOP values recorded in a Microsoft Excel spreadsheet.

2. Definition of chronic periodontitis and chronic kidney disease

Patients in the chronic periodontitis group (Groups I and II) were classified under Page & Eke Criteria (2012) [8,9] using the following inclusion criteria: (1) the presence of ≥15 teeth; (2) ≥40% of sites with BOP; and (3) ≥2 interproximal sites with AL ≥6 mm (not on the same tooth) and ≥1 interproximal site with PD ≥5 mm.

CKD diagnosis was confirmed by evaluating renal parameters such as serum creatinine (SCr) and blood urea nitrogen (BUN) levels. The estimated glomerular filtration rate (eGFR) was calculated using an equation that incorporates creatinine according to the Chronic Kidney Disease Epidemiology Collaboration guidelines [10]. Patients with CKD were classified according to the National Kidney Foundation Kidney Disease Outcomes Quality Initiative 2002 criteria.

3. Power sample size analysis

Power sample analysis was performed using G*Power software (ver. 3.1.9.7; Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany) to determine the required sample size for the study. The sample size was determined based on the mean data from relevant previous studies [11], resulting in an estimated n=25 for each group with a power of 95% and α=0.05.

4. Diagnostic laboratory measurements

Saliva samples were collected from all patients between 8:00 am and 10:00 am before periodontal assessment. The patients were instructed to abstain from eating, drinking, smoking, tooth brushing, or any oral hygiene activities for 12 hours before saliva collection. The passive drooling method was used to collect 0.5 to 1.5 mL of pooled unstimulated saliva. During saliva collection, the participants were seated quietly with their heads tilted downwards and their mouths open, allowing saliva to flow passively from the lower lip into sterile Eppendorf tubes. The salivary samples were then immediately centrifuged at 4°C (1,000×g for 20 minutes) and were stored at −20°C.

5. Measurement of galectin-3 levels in salivary samples

Salivary Gal-3 levels were measured using an enzyme-linked immunosorbent assay kit (Elabscience Biotechnology Inc., Houston, Texas, USA) following the manufacturer’s guidelines. Wells for the diluted standard, blank, and samples were included. Standard, blank, and sample solutions were pipetted into the respective wells, with a volume of 100 μL each. The plate was sealed and incubated at 37°C for 90 minutes. Next, 100 μL of biotinylated detection antibody working solution was added to each well, followed by another round of sealing and 1-hour incubation at 37°C. After decanting the solution, each well was washed with 350 μL of wash buffer. This process was repeated three times. Horseradish peroxidase conjugate working solution was then added to each well; the plate was covered and incubated for 30 minutes at 37°C. The solution was decanted and the washing procedure was repeated five times. Subsequently, 90 μL of substrate reagent was added to each well, and the plate was covered and incubated for approximately 15 minutes at 37°C, followed by the addition of 50 μL of stop solution to each well. The absorbance of each well was measured at 450 nm using a microplate reader.

6. Statistical analysis

Data were entered into a Microsoft Excel spreadsheet and analyzed using SPSS for Windows ver. 17.0 (SPSS Inc., Chicago, IL, USA). Statistical analysis was conducted following an assessment of variable distribution using the Shapiro-Wilk normality test. Mean and standard deviation are used to describe quantitative data. One-way analysis of variance with Tukey post-hoc test was used to determine significant differences in the multivariate analysis. Spearman correlation was used to assess the relationships between variables. Throughout the study, a p-value of <0.05 was considered statistically significant.

Results

1. Clinical characteristics

There were no statistically significant differences in the age or sex of the participants between the groups, as they were age- and sex-matched (Table 1). In Groups I and III, the renal parameter values of patients with CKD were compared and analyzed. Neither group exhibited statistically significant differences in eGFR, SCr, or BUN values (Table 2). Thus, renal parameters were similar between the two groups. The correlation between Gal-3 and CKD stage based on eGFR values was significantly higher in stage 4 than in stages 3a and 3b, suggesting an increase in Gal-3 with CKD severity (Table 3). Considering periodontal characteristics, Groups I and II displayed significantly higher periodontal indices than Group III (Table 4).

2. Primary endpoint: galectin-3 analysis

Group I had the highest mean Gal-3 levels (Table 4, Fig. 1). Bivariate correlations showed a significant correlation between Gal-3 levels and periodontal parameters, such as PPD, CAL, gingival bleeding index, and PI in Groups I and II. The strongest correlation was observed between Gal-3 levels and CAL in Group II (r=0.902, p<0.001). This suggests that Gal-3 levels are strongly associated with CAL, a measure of periodontal disease severity, in patients with chronic periodontitis. There was a significant correlation between Gal-3 levels and renal parameters such as eGFR, SCr, and BUN in Groups I and III. The strongest correlation was observed between Gal-3 levels and eGFR in Group III (r=0.997, p<0.001) (Table 5). The correlation between Gal-3 and PISA was significant in all three groups, with the strongest correlation observed in Group I (r=0.876, p<0.001). Intergroup comparisons of Gal-3, PISA, and periodontal parameters revealed statistically significant intergroup differences among all three groups (Table 4).

Discussion

In this study, we analyzed the effects of chronic periodontitis and CKD on salivary Gal-3 levels. The results demonstrated that patients with chronic periodontitis and CKD and those with chronic periodontitis had higher salivary Gal-3 levels than those with CKD alone. To the best of our knowledge, this is the first research investigating the levels of Gal-3 in saliva samples from patients with renal disease and periodontitis.

The levels of Gal-3 are estimated to be higher in systemic inflammatory diseases, such as coronary heart disease, CKD, rheumatoid arthritis, osteoarthritis, atopic dermatitis, diabetes mellitus, Behçet disease, systemic lupus erythematosus, and asthma [12]. Because periodontitis is a chronic inflammatory condition, it is very likely that the levels of this protein will be high in such inflammatory conditions.

Several cell studies have demonstrated the role of Gal-3 in inflammation. Gal-3 induces the synthesis of proinflammatory cytokines (interleukin [IL]-6, and tumor necrosis factor-α) by synovial fibroblasts in infection-induced acute inflammation [13] and acts as a promoter of host IL-1β responses in epithelial inflammation [14]. It induces the release of superoxide by neutrophils and monocytes [15] and promotes leukocyte recruitment [16]. Considering the role of Gal-3 in inflammation, we investigated its potential involvement and association with periodontitis and renal disease.

In this research, salivary Gal-3 levels were higher in the periodontitis plus CKD group, followed by the periodontitis group, than in the CKD group. The increased Gal-3 levels in the periodontitis group may have been due to Gal-3 production by chondrocytes, osteoblasts, and osteoclasts. Gal-3 plays a crucial role in bone metabolism through osteoblast differentiation and osteoclast activity [17]. Furthermore, it is abundantly expressed in areas with severe cartilage and bone destruction [18].

These findings in the literature suggest that the presence of Porphyromonas gingivalis in patients with chronic periodontitis enhances local Gal-3 production. Gal-3 is highly expressed in various membrane proteins, such as toll-like receptor-2- and 4-expressing cells or oral epithelial cells during periodontitis or in periapical lesions, and has been observed to function as a key modulator of the inflammatory response [19].

Gal-3 levels increased as CKD progressed to advanced stages, with the highest mean in stage 4, based on eGFR values, indicating that as the rate of eGFR decreases, Gal-3 levels increase. This indicates that Gal-3 is a possible diagnostic marker for assessing CKD severity. The results of the present study concur with those of Tang et al. [20], who found a positive correlation between plasma Gal-3 elevation and poor kidney function, including lower eGFR. Similarly, O’Seaghdha et al. [21] found that higher plasma levels of Gal-3 were associated with a more rapid decline in eGFR. The increased levels of Gal-3 in patients with renal impairment further predispose them to the risk of rapidly declining renal function, incident CKD, and all-cause mortality [22].

Gal-3 was previously shown to be an independent risk factor for CKD and was also independently associated with endothelial dysfunction in patients with CKD [23]. Several studies have indicated that periodontal disease is a novel risk factor for CKD progression [24,25]. In patients with both periodontitis and CKD, the manifestation of high endothelial risk factors may be related to the activation of common inflammatory and immune pathways directed against known active periodontal pathogens, particularly involving Gal-3 [11].

Furthermore, the association between PISA scores and Gal-3 levels in patients with both periodontitis and CKD, as well as in those with only periodontitis, implies that elevated Gal-3 levels are associated with a higher inflammatory burden. Therefore, we hypothesize that due to the inflammatory and infectious burden on periodontal tissues caused by periodontitis alone or in combination with CKD, an increase in Gal-3 is an additional risk factor for the development or progression of CKD.

The present study suggests a strong link between chronic periodontitis and CKD, with Gal-3 playing a key role. In all three groups, both renal and periodontal parameters showed significantly elevated levels, indicating that Gal-3 is a potent biomarker for the progression of both diseases. Gal-3 modulates immune cell activation and migration and potentially amplifies inflammatory responses. It influences both the innate and adaptive immune responses, thereby affecting immune homeostasis. Its elevation in periodontitis may disrupt the immune balance, contributing to CKD deterioration. The elevated levels of Gal-3 at sites of periodontitis may enter the systemic circulation, reach renal tissues, and exacerbate inflammatory processes in CKD [26]. Gal-3 also facilitates fibroblast proliferation and collagen synthesis leading to tissue fibrosis. Increased Gal-3 levels due to periodontitis could promote renal tissue fibrosis and accelerate CKD progression [27].

In the future, further longitudinal studies should be conducted with increased sample sizes to assess the effect of Gal-3 over time. Additionally, the estimation of Gal-3 levels after periodontal intervention could indicate the role of Gal-3 in periodontal diseases.

Elevated levels of Gal-3 in the saliva of the patients with periodontitis and CKD suggest a potential link between renal and periodontal diseases within the scope of this study. Therefore, based on the results of this study, it is appropriate to conclude that salivary Gal-3 levels may be related to the severity of periodontitis or the extent of renal impairment. Additionally, Gal-3 is an effective marker of CKD severity. Despite the promising results of the current study, further research is required to better understand the role of Gal-3 in periodontitis and CKD.

Article information

Conflicts of interest

No potential conflict of interest relevant to this article was reported.

Funding

None.

Author contributions

Conceptualization, Formal analysis: SVM, DA, MS, RE, Sandhya S, Sathya S, RD, RB; Data curation, Project administration: SVM, DA, MS; Validation: MS, RE, Sandhya S, Sathya S, RD, RB; Methodology, Supervision: DA, MS, RE, Sandhya S, Sathya S, RD, RB; Investigation: SVM, DA, MS, Sathya S, RB; Resources: SVM, DA, MS, RE, Sandhya S, Sathya S; Software: SVM, MS; Visualization: DA, MS; Writing-original draft: SVM, DA; Writing-review & editing: DA, MS, RD, RB.

Fig. 1.

The box and whisker plot shows the comparison of galectin-3 (Gal-3) levels in all three groups. Group I shows statistically significant Gal-3 levels (F=286.064, ^*p<0.001).

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Figure & Data

References

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