Introduction

Periodontal disease is recognized as the sixth most prevalent condition globally, affecting approximately 10% of the adult population. This condition, if left untreated, can result in progressive destruction of the tooth-supporting apparatus, including the gingiva, cementum, periodontal ligament, and alveolar bone, eventually leading to tooth loss. Such outcomes not only compromise mastication and aesthetics, but also significantly impact patients’ quality of life [1-4]. Recent periodontal disease classification recognizes various disease entities, combining
diagnosis with prevention and treatment, thus ultimately enabling precision and personalized dentistry [5-12]. The relationship between periodontitis and systemic diseases has been extensively studied over the past two decades. Periodontitis has been linked to systemic diseases, including diabetes and atherosclerosis, through inflammatory and microbial pathways [13-18].

Chronic kidney disease (CKD), affecting over 10% of the worldwide population, is another major public health concern and a leading cause of mortality; it is more prevalent in elderly individuals, women, racial minorities, and those with diabetes or hypertension. CKD is described by deterioration of kidney function or the presence of kidney damage, for more than
3 months or prolonged. The global effect of CKD is seriously high, constituting up to 1.2 million deaths per year, and asserting it as the fifth leading cause of death by 2040 [19].

According to the National Kidney Foundation, CKD is classified into five stages based on estimated glomerular filtration rate (eGFR), also referred to as G1-G5:

• G1 (stage 1): ≥ 90 ml/min/1.73 m² (normal or high),

• G2 (stage 2): 60-89 ml/min/1.73 m² (mildly decreased),

• G3a (stage 3a): 45-59 ml/min/1.73 m² (mild to mo­derate decrease),

• G3b (stage 3b): 30-44 ml/min/1.73 m² (moderate to severe decrease),

• G4 (stage 4): 15-29 ml/min/1.73 m² (severely decreased),

• G5 (stage 5): < 15 ml/min/1.73 m² (kidney failure).

Evidence suggests that periodontitis and CKD may interact through systemic inflammation and shared risk factors, yet their relationship remains poorly understood [18].

Kidney injury molecule-1 (KIM-1), also called T-cell immunoglobulin mucin receptor-1 (TIM-1), is a type 1 membrane protein and a sensitive biomarker of renal tubular injury, with emerging roles in inflammation and immune regulation. KIM-1 is found widely in the kidney, liver, and spleen, showing its significance for these organs [19, 20]; it is mostly used in autoimmunity, immune tolerance, and allergic diseases. The wide-ranging perspectives of KIM-1 in these processes highlight its importance in both the immune system’s responses to pathogens and its balanced contributions to the state of immune homeostasis [20, 21].

Objectives

To our knowledge, there are no published reports about the association between KIM-1 levels in serum and saliva with periodontal health status in patients with CKD. Therefore, this study aimed to investigate the relationship between serum and salivary KIM-1 levels with periodontal health status in Iraqi CKD patients on and not on hemodialysis (HD).

Material and methods

Study design

This cross-sectional study was conducted among eligible CKD patients undergoing HD at Al-Hussein Teaching Hospital within the Al-Muthanna Health Directorate, disregarding the etiology of their disease. Acceptable subjects were aged more than 18 years, both males and females, with a diagnosis of CKD proven by clinical, biochemical, or ultrasonic findings, who consented for and were compliant with an oral examination. Patients were divided into two groups; first group: CKD patients on HD, and second group: CKD patients not on HD, and both groups were matched in age, sex, and medical history. This was a cross-sectional study with consecutively enrolled participants who met eligibility criteria. Randomization was not performed due to the observational nature of the study design.

Inclusion criteria: patients having at least 10 teeth, being an adult aged over 18 years, diagnosed with CKD under conservative treatment, and patients with end-stage renal disease (ESRD), who have been on HD for 6 months to 1 year, and receiving it twice per week, for 3-4 hours.

Patients with severe conditions, i.e., cancer, HIV, or hepatitis B and C, kidney transplant recipients, those who had received periodontal therapy within prior three months, pregnant or nursing women, and patients who refused to participate, were excluded.

Ethical considerations

This study was conducted between January 2023 and July 2023. Ethical approval was obtained from the Research Ethical Committees of the College of Dentistry, University of Baghdad, and the study followed the guidelines of the Declaration of Helsinki and the Declaration of Tokyo for humans (1964), with protocol number: 825623 issued on June 1, 2023.

Sample size calculation

KIM-1 in serum was employed as the primary outcome of the study to calculate sample size. A pilot study used 10 samples obtained from each group. The sample size was calculated using the following formula:

n = (Z₁ – α/₂ + Z₁ – β)² × (σ₁² + σ₂²) / (μ₁ – μ₂)²,

where Z₁ – α/₂ = 1.96 (for α = 0.05), Z₁ – β = 0.84 (for β = 0.20, power = 80%), σ₁, σ₂ = standard deviations from pilot study, and μ₁, μ₂ = means from pilot study.

In laboratory testing, ELISA was applied to examine the samples, while the concentration of KIM-1 from the pilot study was used to determine sample size, according to Sharma et al. [22]. The study required 60 subjects per group, and after considering a 10% dropout, the minimum sample required in each group was 70, with an allocation ratio of 1 : 1.

Serum collection

Between 9:00 and 11:00 AM, 5 ml of blood were drawn from patient’s double lumen or cubital fossa.  Every sample was assigned a number based on patient’s name and group. Before receiving heparin, samples were collected and connected to a kidney dialysis machine. To reduce protein denaturation, the samples were put in a cooling box after collection; they were centrifuged at 3,000 rpm for 15 minutes at 2-8°C, and kept at < –20°C in an aliquot.

Saliva collection

Between 9:00 and 11:00 AM, saliva was passively drooled into tubes as per the University of Southern California School of Dentistry guidelines. Before being collected, participants washed their mouths with ste­rile water. Samples tainted with blood were discarded. The samples were centrifuged for 10 minutes at 3,000 rpm and stored at –20^oC until further analysis. Human ELISA quantitative (Biotech, USA) kit was used to detect the presence of KIM-1 in serum and saliva samples.

Periodontal parameters

All teeth, except for wisdom teeth, were examined for periodontal parameters by a calibrated examiner using a UNC-15 periodontal probe, with bleeding on probing (BOP), probing pocket depth (PPD), and clinical attachment loss (CAL) at six locations per tooth measured. Using the O’Leary index, plaque index (PLI) recorded plaque presence on all teeth’s on mesial, distal, buccal, and lingual surfaces. A disclosing agent was applied to all tooth surfaces, then rinsed with water to remove excess discoloration. Stained dentogingival junction surfaces scored 1, whereas unstained surfaces scored 0 [23].

For BOP, periodontal probe was carefully placed into the periodontal pocket or gingival sulcus at six different sites per tooth. Surfaces bleeding for 10-30 seconds received a score of 1, while those that did not, received a score of 0. PPD was calculated by measuring the depth of probe penetration in the pocket or sulcus from the gingival margin. Similar to PPD, CAL was determined as the distance between the pocket base and cementoenamel junction (CEJ). CAL value was greater if apical and lesser if coronal from CEJ, and it matched PPD when the gingival border and CEJ overlap [24, 25].

Statistical analysis

Data description, analysis, and presentation were performed with Statistical Graph Pad Prism 9.5.1 software. Mean with standard deviation (SD) for quantitative variables, and frequency and percentage for qualitative variables were calculated using descriptive statistical analysis. Shapiro-Wilk test for normality of distribution, Levene’s test for group variance homogeneity, and c² test for correlations between two qualitative variables were included in inferential study. Multiple comparisons adjustment was not applied due to the exploratory nature of this study. Finally, multiple linear regression was employed for biomarker-periodontal health status-CKD relationship.

Results

Out of 151 patients evaluated for eligibility requirements, 140 were included in the current study and divided into two groups. Eleven patients were excluded from the trial due to their failure to meet the requirements for inclusion, as illustrated in Figure 1.

Periodontal health status and demographic data

In Table 1, the distribution of periodontal health status is presented comprehensively, showing a significant increase in generalized gingivitis in CKD patients on HD, while localized gingivitis overruled patients not on HD. Also, periodontitis was observed in CKD patients, but this was not statistically significant neither in patients on nor not on HD. Regarding demographic data, i.e., age and sex, the distribution of age between groups was statistically non-significant, while sex distribution was significant, with CKD males not on HD being affected with generalized periodontitis (GP). Still, the distribution of patients according to sex among groups was non-significant.

Periodontal parameters

The results demonstrated that in both groups, there were high mean values of periodontal parameters (PLI, PPD, and CAL) in periodontitis-affected CKD patients compared to gingivitis-affected CKD patients. However, statistical analysis revealed a significant increase in the mean PPD among CKD periodontitis patients on HD compared to CKD cases in the non-HD group (Table 2).

Biomarker concentration

The mean concentration of KIM-1 in ng/ml for serum and saliva is shown in Table 3 and Figure 2. A higher significant concentration of the biomarker (KIM-1) was found in serum compared to saliva. Moreover, the level of KIM-1 in serum was not significantly higher in perio­dontitis-affected CKD patients on HD compared to those not on HD. However, the concentration of KIM-1 in saliva was significantly higher in periodontitis-affected CKD patients on HD.

Regression analysis for biomarker

Multiple linear regression analysis was performed in different models to evaluate the association of the biomarker with disease progression. Since CKD was consider a categorical variable, patients on HD were deemed as 1, while non-HD cases were measured as 0. Moreover, periodontal health status was calculated in the same way, giving score 1 to periodontitis-affected CKD patients, while score 0 to gingivitis-affected cases. The first model is illustrated in Table 4, where KIM-1 in serum was considered a dependent variable. The result showed a non-significant impact of CKD cases on HD and other variables on KIM-1 serum level.

Furthermore, considering the concentration of KIM-1 in saliva as a dependent variable, the result demonstrated a significant association of the biomarker with CKD patients on HD and with CAL, as illustrated in Table 5.

Finally, the third model of multiple linear regressions was performed considering CAL as a dependent variable. The result showed a significant association of CKD and the level of the biomarker in saliva with PPD and periodontal health status, as presented in Table 6.

Discussion

Summary of key findings

The current study investigated the association between serum and salivary KIM-1 levels with periodontal health status in CKD patients, both on and not on HD. The main findings are:

• salivary KIM-1 levels were significantly higher in CKD patients on HD,

• salivary KIM-1 showed significant associations with both CKD and clinical attachment loss (CAL), suggesting a role of kidney disease in periodontal disease progression,

• CKD patients, regardless of HD status, showed a higher prevalence of periodontitis,

• periodontal indicator (PLI, BOP, PPD, and CAL) levels were generally worse in the HD group, though not all differences were statistically significant.

The observed increase in periodontitis among CKD patients aligns with previous reports showing higher periodontal disease prevalence in this population, including patients on HD [26, 27]. Our findings are consistent with those of Duran et al. [26], who also noted increased periodontitis in end-stage renal disease patients on HD. Similarly, earlier studies reported greater pocket depth and attachment loss in HD patients [27].

Furthermore, poor periodontal health may lead to an increased systemic dissemination of periodontal pathogens and inflammatory mediators, which can exacerbate kidney dysfunction and affect the efficacy of HD by changing the body’s response to treatment, and potentially leading to complications, such as vascular access problems and inadequate dialysis dose due to inflammation-related changes in the body [28].

The rise in salivary KIM-1 biomarker among CKD patients not on HD, may reflect renal tubular injury, as KIM-1 is an early marker of proximal tubule damage. Its overexpression contributes to inflammation, fibrosis, and glomerulosclerosis, main processes that lead to CKD progression [29-33].

The link between periodontal disease and systemic inflammation might explain the observed associations. Pathogens and endotoxins from periodontal lesions can enter the bloodstream, activating inflammasomes and triggering cytokine release [28, 34-36]. Additionally, inflammatory mediators from periodontal tissues, such as TNF and caspase, contribute to systemic inflammatory burden [37], which is particularly significant in CKD.

Shared risk factors, such as diabetes, smoking, age, and genetics, further complicate this relationship. Circulating periodontal pathogens can damage kidney endothelium, similar to mechanisms described for cardiovascular diseases [38-40]. This could contribute to the elevated KIM-1 levels observed in CKD periodontitis-affected patients compared to those with gingivitis, though the difference was not statistically significant.

Also, poor oral hygiene behaviors and reduced motivation for selfcare among HD patients are likely to contribute to higher PLI and BOP percentages [41, 42].

Clinical implications

The findings emphasize the importance of perio­dontal health assessment in CKD patients. This study suggests that salivary KIM-1 could potentially serve as a non-invasive biomarker for monitoring periodontal health in CKD patients, particularly those on HD. This may have important implications on periodontal care diagnostic procedures, as salivary biomarkers are easier to collect and less invasive than traditional periodontal assessments. However, further research is needed to establish the diagnostic accuracy and clinical utility of salivary KIM-1 in this context. Periodontal disease may exacerbate systemic inflammation, compromise HD efficacy, and increase complications. Incorporating periodontal care into CKD patients’ management could help reduce systemic inflammation, improve treatment outcomes, and enhance patients’ quality of life [28].

Limitations of the study

The present study was cross-sectional in nature, limiting causal inference in terms of the bidirectional relationship between CKD and periodontal disease. Potential confounders, such as diabetes, smoking, and genetic predispositions, may have influenced the observed associations. Although elevated KIM-1 levels were linked to CKD and periodontal parameters, the exact mechanistic pathways remain unclear. Furthermore, the sample size may not have been sufficient to detect subtle differences in some periodontal measures between groups.

Conclusions

This study demonstrates a significant association between salivary KIM-1 levels and periodontal health status of CKD patients on HD. While salivary KIM-1 shows promise as a potential biomarker, the cross-sectional nature of this study does not allow for conclusions regarding its diagnostic utility. Within the limitations of this study, the fundamental mechanism, through which periodontal diseases increase the salivary level of KIM-1 and its potential in the diagnosis or screening of periodontal diseases, demands further longitudinal studies to determine this protein as a reliable biological marker for periodontal diseases in CKD patients.

Disclosures

Author contributions: Conceptualization: A.M., M.S.M.,
and H.M.H.; Methodology: A.M., M.S.M., and H.M.H.; Investigation and data collection: A.M. and R.F.A.; Formal analysis: A.M., H.M.H., and M.S.M.; Data curation: A.M. and R.F.A.; Writing – original draft: A.M.; Writing – review and editing: M.S.M., H.M.H., R.F.A., and A.A.M.; Supervision: M.S.M.; Project administration: M.S.M. All authors have read and agreed to the published version of the manuscript.

Funding: This research received no external funding.

Institutional Review Board statement: The researchers obtained ethical approval for the current study from the Research Ethical Committees of the College of Dentistry, University of Baghdad (approval number: 825623, issued on 1 June 2023).

Informed consent statement: Informed consent was obtained from all subjects involved in the study.

Data availability statement: The data presented in this study are available within the article. Additional information is available from the corresponding author and can be provided upon reasonable request.

Acknowledgments: None.

Conflicts of interest: The authors declare no conflicts of interest.

AI use statement: No artificial intelligence tools were used in the preparation of this manuscript.

References