Salivary and Serum Inflammatory Biomarkers in Diabetic Nephropathy by Periodontal Status

Salivary and Serum Inflammatory Biomarkers Based on Periodontal Status in Individuals With Diabetic Nephropathy

Chronic inflammation underlies the bidirectional relationship between diabetes and periodontitis, a process that may be further exacerbated in the presence of diabetic nephropathy. While the roles of inflammatory cytokines such as TNF-α and IL-10 in both periodontal tissue destruction and diabetes-related microvascular complications remain unclear, NGAL is recognized as a biomarker for diabetic nephropathy but its association with periodontal disease is not well established. This study aimed to comparatively evaluate salivary and serum levels of NGAL, TNF-α, and IL-10 according to different periodontal conditions in individuals with newly diagnosed diabetes and those with diabetic nephropathy.

Study Overview

• Status: Completed
• Conditions: Periodontitis; Gingivitis; Type 2 Diabetes; Diabetic Nephropathy Type 2; Periodontal Health
• Intervention / Treatment: Diagnostic test: clinical periodontal measurements; Diagnostic test: serum samples; Diagnostic test: saliva samples

Detailed Description

The newly diagnosed diabetes mellitus group consisted of patients attending the Department of Endocrinology who had been diagnosed with type 2 diabetes mellitus for less than 5 years and had no evidence of diabetic vascular complications. The diabetic nephropathy group comprised patients followed in the Department of Endocrinology for nephropathy, defined as a urinary albumin-to-creatinine ratio >30 mg/g.

At baseline, height, body weight, and waist circumference were measured, and body mass index (BMI) was calculated. The body mass index (BMI) was calculated by taking the weight, in kilograms divided by the height in meters squared. A complete physical examination was also performed. In patients evaluated for diabetes mellitus, routinely requested laboratory parameters, including fasting plasma glucose, insulin, HOMA-IR, glycated haemoglobin (HbA1c), complete blood count, liver and renal function tests, erythrocyte sedimentation rate, C-reactive protein (CRP), estimated glomerular filtration rate (eGFR), triglycerides, total cholesterol, HDL, LDL, TSH, and urinary albumin-to-creatinine ratio, were retrieved from the hospital information management system and recorded. Subsequently, all patients were referred to the Faculty of Dentistry, Pamukkale University for further periodontal evaluation.

Following periodontal examination, participants were diagnosed as periodontally healthy, gingivitis, or periodontitis, and were allocated to the corresponding study groups according to their periodontal status. The periodontal status of patients was determined based on the Classification of Periodontal and Peri-Implant Diseases and Conditions stated in 2017 World Workshop. Periodontally health was considered if the volunteers have clinically healthy gingiva on an intact periodontium who had BOP < 10% and PD ≤ 3 mm, no sites with attachment loss, no radiographic sign of alveolar bone destruction and no history of periodontitis. Gingivitis was characterized by the absence of clinical attachment loss and radiographic bone loss, with a BOP score ≥10% (gingivitis on an intact periodontium). Patients with periodontitis were defined as individuals presenting with periodontal disease characterized by PD ≥6 mm, interdental clinical attachment loss (CAL) ≥5 mm, and radiographic bone loss extending to the middle third of the root or beyond in ≥30% of teeth. In these patients, the number of teeth lost due to periodontitis was ≤4.

In Endocrinology clinic, peripheral venous blood samples were collected from all included participants via the antecubital vein. A total of 10 mL of venous blood was obtained using separator vacutainer tubes and centrifuged at room temperature. Unstimulated whole saliva was collected by passive drooling into a sterile test tube over a 10-minute period in Periodontology clinic. The collected serum and saliva samples were transferred into Eppendorf tubes and stored at -80°C until the day of analysis.

The concentrations of NGAL, TNF-α, and IL-10 in serum and saliva samples were measured using the enzyme-linked immunosorbent assay (ELISA) method at the Department of Histology and Embryology.

The power analyse of the study was performed for sample size calculation. Sample size was calculated using the G*Power software program (G*Power; Universitat, Dusseldorf, Germany) for α = 0.05 and f = 0.4. The analyses revealed that 19 subjects per group achieved a power of 80 % with 95% confidence.

The data were analysed with the SPSS 25 program (SPSS Inc., Chicago, IL). Continuous variables were presented as mean ± standard deviation and categorical variables as numbers and percentages. Shapiro-Wilk test was used to detect data's normality. For the comparison of the parameters of the study groups, One-way ANOVA test was used for normally distributed data while Kruskal-Wallis test was performed as non-parametric test. For pairwise group comparisons, Student's t-test or the Mann-Whitney U test was applied. Chi-square test was used for the comparison of the categorical variables. Statistical significance value was considered as p < 0.05.

• Study Type: Observational
• Enrollment (Actual): 126

Contacts and Locations

Study Locations

• Turkey (Türkiye)
  • Denizli, Turkey (Türkiye), 20160
    • Pamukkale University Faculty of Dentistry

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: No

• Sampling Method: Non-Probability Sample

Study Population

Newly diagnosed type 2 diabetic subjects were recruited from patients diagnosed with type 2 diabetes for less than 5 years attending the outpatient clinic of the Department of Endocrinology, Pamukkale University Hospital and subsequently referred to the Department of Periodontology.

Following periodontal examination, participants were diagnosed as periodontally healthy, gingivitis, or periodontitis, and were allocated to the corresponding study groups according to their periodontal status.

Description

Inclusion Criteria:

• Newly diagnosed diabetes: Diagnosed with type 2 diabetes mellitus for less than 5 years and the absence of any diabetic vascular complications
• Diabetic nephropathy: Urinary albumin-to-creatinine ratio >30 mg/g

Exclusion Criteria:

• Having fewer than 15 natural teeth
• Pregnancy or lactation
• History of malignancy
• Use of systemic antibiotics or anti-inflammatory drugs within the 6 months prior to study enrollment
• Periodontal treatment within the previous 6 months
• Type 1 diabetes mellitus, gestational diabetes mellitus, or secondary diabetes mellitus

Study Plan

How is the study designed?

Number of groups / cohorts

6

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
newly diagnosed type 2 diabetes and periodontal health; 21 participants with newly diagnosed type 2 diabetes and periodontal health were recruited in group 1. The clinical periodontal parameters taken are as follows: plaque index (PI), gingival index (GI), bleeding on brobing (BOP), probing depth (PD) and clinical attachment level (CAL). The present number of teeth in participants was recorded. Serum and saliva samples were collected for the analysis of biomarkers such as NGAL, IL-10, and TNF-α.	Diagnostic test: clinical periodontal measurements; Periodontal parameters such as PI, GI, BOP, PD, and CAL were measured using the Williams periodontal probe (Hu-Friedy, Chicago IL). PD and CAL were calculated at six surfaces per tooth, whereas PI and GI were measured at four surfaces per tooth. the periodontal status of patients was determined based on the Classification of Periodontal and peri-implant diseases and conditions stated in 2017 World Workshop; Diagnostic test: serum samples; Peripheral blood samples were obtained from the antecubital vein. A total of 10 mL of venous blood was collected using separator vacutainer tubes and centrifuged at room temperature. The obtained serum was transferred into separate Eppendorf tubes. The tubes were sealed with parafilm, labeled, and stored at -80°C until the day of analysis. NGAL, TNF-alpha and IL-10 levels were analyzed with ELISA method; Diagnostic test: saliva samples; Unstimulated whole saliva samples were collected using the passive drooling method into a sterile collection tube over a 10-minute period.The obtained saliva was transferred into separate Eppendorf tubes. The tubes were sealed with parafilm, labeled, and stored at -80°C until the day of analysis. NGAL, TNF-alpha and IL-10 levels were analyzed with ELISA method
newly diagnosed type 2 diabetes and gingivitis; 20 participants with newly diagnosed type 2 diabetes and gingivitis were recruited in group 2. The clinical periodontal parameters taken are as follows: plaque index (PI), gingival index (GI), bleeding on brobing (BOP), probing depth (PD) and clinical attachment level (CAL). The present number of teeth in participants was recorded. Serum and saliva samples were collected for the analysis of biomarkers such as NGAL, IL-10, and TNF-α.	Diagnostic test: clinical periodontal measurements; Periodontal parameters such as PI, GI, BOP, PD, and CAL were measured using the Williams periodontal probe (Hu-Friedy, Chicago IL). PD and CAL were calculated at six surfaces per tooth, whereas PI and GI were measured at four surfaces per tooth. the periodontal status of patients was determined based on the Classification of Periodontal and peri-implant diseases and conditions stated in 2017 World Workshop; Diagnostic test: serum samples; Peripheral blood samples were obtained from the antecubital vein. A total of 10 mL of venous blood was collected using separator vacutainer tubes and centrifuged at room temperature. The obtained serum was transferred into separate Eppendorf tubes. The tubes were sealed with parafilm, labeled, and stored at -80°C until the day of analysis. NGAL, TNF-alpha and IL-10 levels were analyzed with ELISA method; Diagnostic test: saliva samples; Unstimulated whole saliva samples were collected using the passive drooling method into a sterile collection tube over a 10-minute period.The obtained saliva was transferred into separate Eppendorf tubes. The tubes were sealed with parafilm, labeled, and stored at -80°C until the day of analysis. NGAL, TNF-alpha and IL-10 levels were analyzed with ELISA method
newly diagnosed type 2 diabetes and periodontitis; 22 participants with newly diagnosed type 2 diabetes and gingivitis were recruited in group 3. The clinical periodontal parameters taken are as follows: plaque index (PI), gingival index (GI), bleeding on brobing (BOP), probing depth (PD) and clinical attachment level (CAL). The present number of teeth in participants was recorded. Serum and saliva samples were collected for the analysis of biomarkers such as NGAL, IL-10, and TNF-α.	Diagnostic test: clinical periodontal measurements; Periodontal parameters such as PI, GI, BOP, PD, and CAL were measured using the Williams periodontal probe (Hu-Friedy, Chicago IL). PD and CAL were calculated at six surfaces per tooth, whereas PI and GI were measured at four surfaces per tooth. the periodontal status of patients was determined based on the Classification of Periodontal and peri-implant diseases and conditions stated in 2017 World Workshop; Diagnostic test: serum samples; Peripheral blood samples were obtained from the antecubital vein. A total of 10 mL of venous blood was collected using separator vacutainer tubes and centrifuged at room temperature. The obtained serum was transferred into separate Eppendorf tubes. The tubes were sealed with parafilm, labeled, and stored at -80°C until the day of analysis. NGAL, TNF-alpha and IL-10 levels were analyzed with ELISA method; Diagnostic test: saliva samples; Unstimulated whole saliva samples were collected using the passive drooling method into a sterile collection tube over a 10-minute period.The obtained saliva was transferred into separate Eppendorf tubes. The tubes were sealed with parafilm, labeled, and stored at -80°C until the day of analysis. NGAL, TNF-alpha and IL-10 levels were analyzed with ELISA method
diabetic nephropathy and periodontal health; 21 participants with diabetic nephropathy and periodontal health were recruited in group 4. The clinical periodontal parameters taken are as follows: plaque index (PI), gingival index (GI), bleeding on brobing (BOP), probing depth (PD) and clinical attachment level (CAL). The present number of teeth in participants was recorded. Serum and saliva samples were collected for the analysis of biomarkers such as NGAL, IL-10, and TNF-α.	Diagnostic test: clinical periodontal measurements; Periodontal parameters such as PI, GI, BOP, PD, and CAL were measured using the Williams periodontal probe (Hu-Friedy, Chicago IL). PD and CAL were calculated at six surfaces per tooth, whereas PI and GI were measured at four surfaces per tooth. the periodontal status of patients was determined based on the Classification of Periodontal and peri-implant diseases and conditions stated in 2017 World Workshop; Diagnostic test: serum samples; Peripheral blood samples were obtained from the antecubital vein. A total of 10 mL of venous blood was collected using separator vacutainer tubes and centrifuged at room temperature. The obtained serum was transferred into separate Eppendorf tubes. The tubes were sealed with parafilm, labeled, and stored at -80°C until the day of analysis. NGAL, TNF-alpha and IL-10 levels were analyzed with ELISA method; Diagnostic test: saliva samples; Unstimulated whole saliva samples were collected using the passive drooling method into a sterile collection tube over a 10-minute period.The obtained saliva was transferred into separate Eppendorf tubes. The tubes were sealed with parafilm, labeled, and stored at -80°C until the day of analysis. NGAL, TNF-alpha and IL-10 levels were analyzed with ELISA method
diabetic nephropathy and gingivitis; 20 participants with diabetic nephropathy and gingivitis were recruited in group 5. The clinical periodontal parameters taken are as follows: plaque index (PI), gingival index (GI), bleeding on brobing (BOP), probing depth (PD) and clinical attachment level (CAL). The present number of teeth in participants was recorded. Serum and saliva samples were collected for the analysis of biomarkers such as NGAL, IL-10, and TNF-α.	Diagnostic test: clinical periodontal measurements; Periodontal parameters such as PI, GI, BOP, PD, and CAL were measured using the Williams periodontal probe (Hu-Friedy, Chicago IL). PD and CAL were calculated at six surfaces per tooth, whereas PI and GI were measured at four surfaces per tooth. the periodontal status of patients was determined based on the Classification of Periodontal and peri-implant diseases and conditions stated in 2017 World Workshop; Diagnostic test: serum samples; Peripheral blood samples were obtained from the antecubital vein. A total of 10 mL of venous blood was collected using separator vacutainer tubes and centrifuged at room temperature. The obtained serum was transferred into separate Eppendorf tubes. The tubes were sealed with parafilm, labeled, and stored at -80°C until the day of analysis. NGAL, TNF-alpha and IL-10 levels were analyzed with ELISA method; Diagnostic test: saliva samples; Unstimulated whole saliva samples were collected using the passive drooling method into a sterile collection tube over a 10-minute period.The obtained saliva was transferred into separate Eppendorf tubes. The tubes were sealed with parafilm, labeled, and stored at -80°C until the day of analysis. NGAL, TNF-alpha and IL-10 levels were analyzed with ELISA method
diabetic nephropathy and periodontitis; 22 participants with diabetic nephropathy and gingivitis were recruited in group 6. The clinical periodontal parameters taken are as follows: plaque index (PI), gingival index (GI), bleeding on brobing (BOP), probing depth (PD) and clinical attachment level (CAL). The present number of teeth in participants was recorded. Serum and saliva samples were collected for the analysis of biomarkers such as NGAL, IL-10, and TNF-α.	Diagnostic test: clinical periodontal measurements; Periodontal parameters such as PI, GI, BOP, PD, and CAL were measured using the Williams periodontal probe (Hu-Friedy, Chicago IL). PD and CAL were calculated at six surfaces per tooth, whereas PI and GI were measured at four surfaces per tooth. the periodontal status of patients was determined based on the Classification of Periodontal and peri-implant diseases and conditions stated in 2017 World Workshop; Diagnostic test: serum samples; Peripheral blood samples were obtained from the antecubital vein. A total of 10 mL of venous blood was collected using separator vacutainer tubes and centrifuged at room temperature. The obtained serum was transferred into separate Eppendorf tubes. The tubes were sealed with parafilm, labeled, and stored at -80°C until the day of analysis. NGAL, TNF-alpha and IL-10 levels were analyzed with ELISA method; Diagnostic test: saliva samples; Unstimulated whole saliva samples were collected using the passive drooling method into a sterile collection tube over a 10-minute period.The obtained saliva was transferred into separate Eppendorf tubes. The tubes were sealed with parafilm, labeled, and stored at -80°C until the day of analysis. NGAL, TNF-alpha and IL-10 levels were analyzed with ELISA method

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
NGAL analysis	Mean NGAL levels (ng/ml) in serum and saliva	baseline

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
TNF-alpha analysis	Mean TNF-alpha levels (ng/L)in serum and saliva	baseline
IL-10 analysis	Mean IL-10 levels (ng/ml) in serum and saliva	baseline

Collaborators and Investigators

• Sponsor: Pamukkale University

Study record dates

Study Major Dates

• Study Start (Actual): June 10, 2024
• Primary Completion (Actual): December 12, 2025
• Study Completion (Actual): December 12, 2025

Study Registration Dates

• First Submitted: March 23, 2026
• First Submitted That Met QC Criteria: March 23, 2026
• First Posted (Actual): March 27, 2026

Study Record Updates

• Last Update Posted (Actual): March 27, 2026
• Last Update Submitted That Met QC Criteria: March 23, 2026
• Last Verified: March 1, 2026

More Information

Terms related to this study

• Keywords: periodontitis; cytokine; diabetic nephropathy; NGAL; saliva
• Additional Relevant MeSH Terms: Urogenital Diseases; Endocrine System Diseases; Periodontal Diseases; Mouth Diseases; Stomatognathic Diseases; Male Urogenital Diseases; Kidney Diseases; Urologic Diseases; Female Urogenital Diseases; Female Urogenital Diseases and Pregnancy Complications; Metabolic Diseases; Infections; Glucose Metabolism Disorders; Diabetes Mellitus; Diabetes Complications; Gingival Diseases; Nutritional and Metabolic Diseases; Periodontitis; Diabetes Mellitus, Type 2; Diabetic Nephropathies; Gingivitis
• Other Study ID Numbers: 2024HZDP005

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No