Correlation Between Salivary and Serum Biomarkers of Kidney Function in Chronic Kidney Disease Patients

: The present study was designed to determine the correlation between salivary and serum levels of creatinine and urea in patients with chronic kidney disease (CKD) and to propose the use of saliva as an alternative medium for the analysis of biochemical parameters of kidney function. Accordingly, an analytical cross-sectional study was carried out using 100 CKD patients attending the Nephrology Clinics at Teaching Hospital Karapitiya, Sri Lanka. Saliva and serum samples were collected from the patients for the estimation of creatinine and urea using spectrophotometric assay kits. Of the total, the majority of the patients were found within CKD stage 3 (82%). The salivary and serum concentrations of creatinine varied within the range of 0.20 - 2.10 mg/dL and 1.54 – 11.6 mg/dL, whereas the salivary and serum concentrations of urea varied within the range of 68.04 – 127.59 mg/dL and 60.14 – 168.25 mg/dL respectively within the study population. Significant positive correlations were observed between the salivary and serum concentrations of creatinine (r= 0.985, p=0.01), and urea (r= 0.942, p=0.01), and between salivary concentrations of creatinine and urea (r=0.887, p=0.01). A significant difference was observed in the salivary biomarkers between mild to moderate stages of CKD patients and severe to end-stage CKD patients. No significant association was observed in the salivary markers with the age and sex of the patients. The area under curve values in the receiver operating characteristic analysis was>0.9 (95% CI). These findings revealed high levels of diagnostic accuracy in salivary biomarkers suggesting the potential use of saliva for the assessment of kidney function in CKD patients.


INTRODUCTION
Chronic kidney disease (CKD) is a serious health problem worldwide.Diagnosis and management of CKD patients are done often based on an estimated value of glomerular filtration rate (eGFR) in the current healthcare system.The eGFR is derived from different equations, based on the clearance of endogenous filtration markers in which serum creatinine and urea are the most common (Levin et al., 2008;Bjornstad et al., 2018).
Serum creatinine and urea are nitrogenous end products of muscle and protein metabolism respectively.Accurate measurement of these parameters is often affected by the variations in body muscle mass, protein intake, endogenous protein catabolism, state of hydration, hepatic urea synthesis, renal urea excretion, methods of laboratory diagnosis, etc. (Hosten, 1990).
Proper management of patients with CKD requires frequent collection of blood samples for the assessment of kidney function parameters, such as serum creatinine, and urea.However, blood sample collection is an invasive procedure, which brings anxiety and discomfort to the patients.Further, repeated collection of blood samples is associated with a certain amount of blood loss in patients with CKD in addition to their blood loss during hemodialysis treatment (Arun et al., 2012).These potential drawbacks associated with repeated collection of blood samples for the assessment of serum creatinine and urea in the management of CKD patients permit the search of alternative diagnostic media for frequent analysis of kidney function parameters.
Saliva is an extracellular fluid produced and secreted by the salivary glands in the mouth and is composed of various biomarkers that are beneficial in the detection of systemic and local diseases (Cui et al., 2022).There are elevated levels of creatinine and urea in the saliva of patients with CKD due to passive diffusion of those waste products from blood to saliva, and alterations in the permeability of salivary glands.Therefore, at a stage of compromised kidney function, saliva offers an alternative method for the excretion of nitrogenous waste products (Suresh et al., 2014).
Collecting saliva is not an invasive procedure and does not cause discomfort for the patients.Suitability for all age groups, potentially less occupational risk for healthcare workers, and low cost for sample collection are a few additional advantages of the use of saliva as a potential diagnostic medium in patients with CKD (Kaufman and Lamster, 2000).
Numerous studies have been carried out to assess the suitability of salivary biomarkers for the diagnosis and treatment of diseases, and the findings are quite questionable concerning creatinine and urea.While the studies by Venkatapathy et al. (2014), Pandya et al. (2016), Lasisi et al. (2016) and Temilola et al. (2019) reported favorable findings on the potential correlation between salivary and serum biomarkers of kidney function, the findings by Nogalcheva et al. (2018) and Bhuvaneswari et al. (2023) revealed contrasting findings.Moreover, the majority of the studies have been reported based on late-stage CKD patients, and the applicability of salivary biomarkers at the early and moderate stages of CKD is quite uncertain.Therefore, the present study aims to evaluate the correlation between salivary and serum concentrations of creatinine and urea in patients with CKD, particularly at the early and moderate stages of the disease, and to propose to use of saliva as an alternative tool for the diagnosis and management of patients with CKD.

Study Design and Ethical Aspects
Hundred patients who attended the Nephrology Clinics at the Teaching Hospital, Karapitiya, Galle, Sri Lanka were recruited for this study using the convenience sampling technique.The sample size was determined based on the equation, N = [(Zα+Zβ)/C]2+3, considering an expected correlation coefficient of 0.30, when β and α values were 0.20 and 0.05 respectively, and by adding 10% of dropout rate (Kohn and Senyak, 2021;Hulley et al., 2013).Both male and female CKD patients, between the age group 20 -80 years and already categorized under one of the CKD stages based on the guidelines of the National Kidney Foundation were included in the study (Levey et al., 2005).Patients who were critically ill or unconscious and patients who appeared with a history of kidney transplantation, recent surgeries, or hospitalizations were excluded from the study.Ethical clearance was granted from the Ethics Review Committee of the Faculty of Allied Health Sciences, University of Ruhuna, Galle, Sri Lanka (Reference No. 2023.08.11).

Sample Collection Procedure
After obtaining the informed written consent, fresh saliva samples of the participants at the Nephrology Clinics were collected by spitting method.The samples were collected into clean leak-proofed wide-mouthed screw-capped containers provided by the investigators, followed by providing them proper instructions on sample collection (Venkatapathy et al., 2014).The patients were instructed to wash their mouths thoroughly with water before the sample collection.They were further instructed to avoid swallowing and other oral movements, and pool saliva on the floor of their mouth.until they experienced an urge to swallow the accumulated saliva.The samples were then collected by spitting into the containers and the participants were asked to continue the procedure until reached the required sample volume of 2 mL.The collected samples were stored at 4 -8 °C until processed for biochemical analysis.
The retained blood samples of the respective patients were collected from the Chemical Pathology Laboratory at the Teaching Hospital Karapitiya, Galle, Sri Lanka.The serum was separated by centrifugation of the samples at 3,500 rpm for 15 min within two hours of sample collection and stored at 4 -8 °C until the samples were used in biochemical assays.The laboratory investigations were carried out within eight hours of sample collection.

Biochemical Measurements
Saliva and serum samples collected were tested for the concentrations of creatinine (Bartels, et al., 1972; Biorex diagnostics, UK) and urea (Bablock, et al., 1988; SPECTRUM Diagnostics, Egypt) by spectrophotometric assay methods using commercially available test kits.

Statistical Analysis
Salivary and serum concentrations of urea and creatinine were expressed as mean ±SD.Data were analyzed using a statistical package for social science software (SPSS 29).The normality of the data was checked using the Kolmogorov-Smirnov test initially.Pearson's correlation, paired t-test, and chi-square test of independence were carried out to evaluate the relationship between variables.Receiver operating characteristic (ROC) curve analysis was conducted and the area under the curve (AUC) values were calculated for the assessment of diagnostic accuracy of salivary concentrations of creatinine and urea.

Baseline Data for the Study Population
A total number of 100 patients (20 -80 years in both sexes) attending the Nephrology Clinics at Teaching Hospital Karapitiya, Sri Lanka were enrolled for the study during the period from August 2023 to October 2023.Baseline characteristics of the participants and kidney function parameters are shown in Tables 1 and 2, respectively.

Correlation
Between Salivary and Serum

Concentrations of Creatinine and Urea
Pearson's correlation analysis revealed a positive correlation between serum and salivary creatinine (r= 0.985, p=0.01) and a significantly high positive correlation between serum and salivary urea (r= 0.942, p=0.01) concentrations.A significant high positive correlation was observed between salivary creatinine and urea concentrations as well (r= 0.887, p=0.01).The scatter plots for the assessment of serum and salivary biomarkers are shown in Figure 1 (A  -C).

Comparison of Serum and Salivary Markers Based on the Stage of CKD, Age, and Sex
Considering the absence/ few number of participants in CKD stages 1, 2, 4, and 5 the comparisons on serum and salivary markers based on CKD stages were done considering the CKD stages 2, 3, and stages 4, 5 communally.This categorization further supported the separation of mild to moderate CKD patients from severe to end-stage CKD patients.The results revealed significant differences in the values of both serum and salivary markers between the patients of two selected groups based on the stage of the disease (p<0.05).The findings are shown in Table 3.
Further, a comparison of the serum and salivary concentrations of creatinine based on the age of the CKD patients in the age groups of 20 -39 years (n=20), 40 -59 years (n=20), and 60 -80 years (n=21) revealed controversial findings (p>0.5).Significant differences could be observed between the 40 -59-years age group concerning serum creatinine whereas salivary creatinine showed a significant difference between the 20 -39 years age group (p=0.01).However, no significant differences in values were observed between either age group relevant to both serum and salivary urea (p>0.05).Similarly, no gender-wise differences were observed in the serum and salivary markers among the study population (p>0.05).The results are presented in Table 3.
The chi-square test of independence was performed to assess the relationship between serum and salivary biomarkers and the age and sex of the patients.A significant association was observed only between the age of the patients with serum creatinine concentration (χ2=2322.01,p=0.015).However, no significant association was observed in the age of the patients concerning concentrations of serum urea (χ2=3110.42,p=0.585), salivary creatinine (χ2=1872.14,p=0.273), and salivary urea (χ2=3112.79,p=0.061).

Diagnostic Accuracy of Salivary Creatinine and Urea in the Assessment of Kidney Function
The diagnostic accuracy of salivary creatinine and urea concentrations was assessed using ROC curves based on the CKD stage.The findings are shown in Figure 2. The AUC values provide a measure of discrimination between group 1 (CKD stage 2+3) and group 2 (CKD stage 4+5) CKD patients.In the present study AUC values of serum creatinine, salivary creatinine, serum urea, and salivary urea were 1.00 (1.00-1.00),1.00 (1.00-1.00),0.99 (0.97-1.00) and 0.99 (0.97-1.00) respectively at 95% confidence interval, indicating a high level (AUC > 0.9) of diagnostic accuracy in both salivary creatinine and urea estimations.

DISCUSSION
The present study was an attempt to determine the possibility of using saliva as an alternative medium to blood for the estimation of kidney function parameters in patients with CKD.The present findings revealed that there were high positive correlations between serum and salivary creatinine, serum and salivary urea, and salivary creatinine and urea, among CKD patients, corroborating the findings of Venkatapathy et al. (2014) and Lasisi et al. (2016).A study conducted by Goll and Mookerjee (1978)    revealed that there was no significant correlation between salivary and serum urea nitrogen in CKD patients after hemodialysis treatment, however in the present study, the findings were contradictory, showing a significant and high positive correlation between serum and salivary urea concentrations in study subjects on hemodialysis treatments.The investigations were carried out in samples collected before hemodialysis and the samples after hemodialysis were not subjected to biochemical estimations which could be a limitation of the study.
A comparison of salivary creatinine and urea concentrations based on the CKD stage of the participants revealed that there were significant differences between the values of the two groups selected.These findings substantiate the fact that salivary creatinine and urea could be used to differentiate mild to moderate CKD from severe to endstage CKD.However, there was no similar distribution of the participants to different CKD stages in the present study, and the majority was of the CKD stage 3, which is a limitation to the study in assessing the variability of salivary markers based on the CKD stage.
However, a comparison of serum and salivary markers based on age revealed controversial findings in the present study.Even though a significant difference in serum creatinine was observed between the age groups 20-39 and 60-80 years, contradictory findings were observed for salivary creatinine between the said age groups.Instead, a significant difference was observed between the 20-39 and 40-59 years age groups concerning salivary creatinine.An evenly distributed sample was observed in the three age groups selected in the present study and therefore, the observed variation in the findings needs further analysis using a large sample size.However, no significant differences between serum and salivary urea concentrations of patients in different age groups were observed in the present study, indicating that serum and salivary urea concentrations are independent of the age of the patients.Further assessment of the association of serum and salivary markers with age and sex revealed significantly high positive associations between age and serum creatinine concentration.Even though, generally, we report two different reference ranges for males and females with reference to serum creatinine considering the androgens present in the males, no statistically significant association was observed between the sex and the serum creatinine concentrations in the present study indicating the levels are independent of the sex (O' Leary et al., 2017).Further studies using healthy individuals would be beneficial to support this fact.However, the absence of a statistically significant association in salivary markers with age and sex is a potential plus point in defining reference ranges for this particular age group (20-80 years) in salivary estimations.Yet, both serum and salivary markers showed approximately similar chi-square values in the present study even though they were not statistically significant, indicating similar impacts on CKD diagnosis.
The findings of ROC analysis revealed excellent performance in both salivary creatinine and urea values.
Both salivary creatinine and urea demonstrated a high level of diagnostic accuracy as depicted by the AUC values.However, both serum and salivary creatinine values showed higher AUC values compared to urea indicating better diagnostic accuracy in estimation of creatinine level in CKD patients.

CONCLUSIONS
The findings of the present study revealed that there were positive correlations between serum and salivary biomarker levels of creatinine and urea.The salivary creatinine and urea values were independent of the age and sex of the patients.Further, the salivary markers revealed high levels of diagnostic accuracy in patients with CKD, suggesting the potential use of saliva for the assessment of kidney function in CKD patients.However, further studies are warranted to validate salivary estimations using healthy adults and to establish reference ranges for salivary measurements based on the respective stages of CKD patients.

Figure 1 :
Figure 1: Scatter plots for assessing the relationship between serum and salivary concentrations of creatinine (A), urea (B), and salivary concentrations of creatinine and urea (C).Scatter plots demonstrate a positive correlation between serum and salivary creatinine concentrations (A), significant high positive correlation between serum and salivary urea concentrations (B) and significant high positive correlation between salivary creatinine and salivary urea concentrations (C).

Figure 2 :
Figure 2: The receiver operating characteristic curves for the comparison of salivary creatinine and urea with serum creatinine and urea.The AUC values of serum creatinine, salivary creatinine, serum urea, and salivary urea were ≥0.99.AUC: area under curve.

Table 1 :
Baseline characteristics of study subjects with chronic kidney disease (n=100).

Table 2 :
Variation of kidney function parameters among the study population based on the stage of chronic kidney disease (n=100).

Table 3 :
This table presents the mean and standard deviation (SD) values for Serum and Salivary Biomarkers, serum creatinine, serum urea, salivary creatinine, and salivary urea, along with their associated p-values, categorized by CKD stage, age group, and sex.Significant differences are indicated by p-values less than 0.05.