Saliva as a non-invasive biomarker for chronic kidney disease: Challenges and potential in disease monitoring

Abstract

Chronic kidney disease (CKD) is a degenerative disorder that affects millions of people throughout the world, causing considerable morbidity and healthcare burden. Frequent blood sampling is the current gold standard for monitoring CKD to evaluate biochemical and mineral indicators. However, there are drawbacks to frequent blood draws, such as pain for patients, the possibility of infection, and higher medical expenses. Saliva-based diagnostics offer advantages such as ease of collection, reduced invasiveness, and improved patient compliance. A comprehensive literature review was conducted to analyze studies evaluating the diagnostic utility of salivary creatinine, urea, calcium, and parathyroid hormone (PTH) in patients with CKD. Various saliva collection methods, including stimulated and unstimulated approaches, were investigated for efficiency and reliability, and a correlation was shown between serum and salivary creatinine, urea, PTH, and calcium levels, indicating their potential as CKD biomarkers. Despite these promising findings, challenges such as standardization of collection methods, variability in salivary flow rates, and predictive value in association with blood parameters are addressed to ensure clinical applicability. This review explores the potential and challenges of saliva as a non-invasive alternative for CKD diagnostics.

Key Words: Chronic kidney disease; Salivary biomarkers; Non-invasive diagnostics; Saliva-based monitoring

Core Tip: Saliva-based diagnostics offer a promising, non-invasive alternative for monitoring chronic kidney disease (CKD), particularly in resource-limited or outpatient settings. This review highlights that salivary urea and creatinine, especially via the spitting method, show consistent correlations with serum levels, supporting their potential use in CKD screening and monitoring. However, salivary parathyroid hormone and calcium exhibit weaker associations due to physiological and analytical limitations. Standardization of collection methods and further mechanistic and clinical research are essential to optimize the clinical utility of salivary biomarkers in CKD.

INTRODUCTION

Chronic kidney disease (CKD) is a long-term condition in which the kidneys gradually lose their ability to function effectively[1]. The disease is commonly associated with underlying conditions like hypertension, diabetes, and glomerulonephritis, which accelerate its progression[1,2]. Instances of diabetes-related end-stage renal disease (ESRD) increased dramatically (40%-700%) between 2002 and 2015 in nations including Scotland, Philippines, Malaysia, Singapore, Australia, Taiwan, Jalisco, Bosnia, Herzegovina, and Republic of Korea[3]. As CKD progresses, kidney function deteriorates, often marked by elevated levels of serum creatinine, urea, and disturbances in mineral metabolism, particularly calcium and parathyroid hormone (PTH)[4]. Early identification and monitoring are essential for preventing the progression of disease, and such markers are often measured using blood samples. However, invasive blood collection procedures have issues in terms of patient compliance and pain, especially for individuals who require regular monitoring[5].

Researchers have recently investigated saliva as a non-invasive way to diagnose and monitor disease[6,7]. Rodrigues et al[8] noted that saliva contains biomarkers such as proteins, fatty acids, and carbohydrates, which, like blood, can reflect changes in physiological activities, making saliva a viable alternative for the early identification and monitoring of disease. Studies have shown that biomarkers such as creatinine, urea, calcium, and PTH, which are typically assessed in blood, may also be detected in saliva and correlate with kidney function[9,10]. While previous reviews have summarized the role of saliva in CKD diagnostics, this review differentiates itself by not only summarizing biochemical correlations for creatinine, urea, calcium, and PTH but also evaluating methodological challenges, comparing salivary collection techniques, and proposing translational use cases for saliva diagnostics in nephrology. Additionally, we critically appraise the variability in biomarker consistency and reflect on the physiological and analytical reasons for these discrepancies, particularly in salivary calcium and PTH studies. This comprehensive synthesis aims to bridge laboratory findings with potential clinical applications.

LITERATURE SEARCH STRATEGY

A comprehensive literature search was conducted across PubMed, Scopus, and Web of Science databases. Search terms included combinations of "saliva", "salivary biomarkers", "chronic kidney disease", "CKD", "creatinine", "urea", "parathyroid hormone", and "calcium". Studies were included if they were original research articles published in English between January 2014 and December 2024, involved human subjects, and specifically evaluated salivary biomarkers in relation to CKD. Review articles, conference abstracts, animal studies, and case reports were excluded. The selection process involved initial title and abstract screening, followed by full-text review to ensure relevance and methodological adequacy. Approximately 120 studies were initially screened, of which 12 original research articles directly assessing salivary biomarkers in patients with CKD were included in this review.

CKD: EPIDEMIOLOGY AND CLINICAL BURDEN

CKD is a progressive condition marked by a gradual decrease in kidney function over time[10]. It is defined by the presence of a low estimated glomerular filtration rate [estimated glomerular filtration rate (eGFR) < 60 mL/min/1.73 m²] and/or persistently (at least 3 months) elevated urinary albumin excretion [albumin-to-creatinine ratio (ACR) ≥ 30 mg/g][11]. CKD is classified into five stages based on the eGFR, which measures kidney function. Stage 1 indicates normal kidney function (eGFR > 90 mL/min/1.73 m²) but with other signs of kidney damage. Stage 2 involves mild kidney damage with a slight decrease in eGFR (60-89 mL/min/1.73 m²). Stage 3 is divided into two subcategories: Stage 3a (eGFR 45-59 mL/min/1.73 m²) and Stage 3b (eGFR 30-44 mL/min/1.73 m²). Stage 4 indicates severe kidney damage with a significant reduction in eGFR (15-29 mL/min/1.73 m²). Finally, Stage 5 represents kidney failure (eGFR < 15 mL/min/1.73 m²) and patients need regular dialysis[12].

The global prevalence of CKD, including all five stages, is estimated to be about 13.4%, accounting for approximately 700 million patients and causing 1.2 million deaths and losing 28.0 million years of life annually[13-15]. With approximately 10.6% having CKD in stages 3-5, it is expected to become the fifth highest cause of death worldwide by 2040, representing one of the most significant anticipated rises of any major cause of mortality[16]. The incidence varies greatly across Asia, ranging from 7.0% to 34.3%, with an estimated 434.3 million individuals afflicted, including up to 65.6 million with advanced CKD[17]. In Malaysia, the prevalence of CKD has been rising. Countrywide population-based research in 2018 indicated that 15.48% of Malaysian adults had CKD, compared to 9.07% in 2011. The study also found that 3.85% had stage 1 CKD, 4.82% had stage 2 CKD, 6.48% had stage 3 CKD, and stages 4 and 5 (advanced CKD) were estimated to be around 0.33%[18].

Complications of CKD

CKD is a diminished capacity to filter waste and maintain fluid, electrolyte, and acid-base balance, and these consequences can lead to several complications[1]. CKD significantly raises the risk of cardiovascular disorders such as coronary artery disease, heart failure, arrhythmias, and sudden cardiac death. The relationship between poor kidney function and cardiovascular health is complicated, with each problem possibly increasing the other[19]. Hypertension is generally caused by impaired renal function, which leads to salt retention and increased fluid content. On the other hand, hypertension can exacerbate renal damage, starting a vicious cycle that hastens the development of CKD. Anemia is another common issue due to decreased erythropoietin production, leading to fatigue and reduced functional capacity[2].

Another serious consequence of chronic kidney disease mineral and bone disorder (CKD-MBD) is that when kidney function deteriorates, the kidney's capacity to eliminate phosphate is compromised, resulting in buildup in the blood (hyperphosphatemia)[20]. Fibroblast growth factor 23 (FGF23) rises as a result, which lowers the synthesis of calcitriol (vitamin D), the active form of the vitamin. Hypocalcemia is the outcome of reduced intestinal calcium absorption due to a drop in calcitriol levels. Secondary hyperparathyroidism results from the parathyroid glands producing more PTH in reaction to low calcium levels. Elevated PTH stimulates the release of calcium and phosphate from bones, increasing bone turnover and alkaline phosphatase levels, an enzyme linked to bone metabolism. This bone resorption can lead to renal osteodystrophy, encompassing various bone disorders associated with CKD[21,22]. Additionally, hyperphosphatemia and elevated calcium levels can cause vascular and soft tissue calcification, increasing the risk of cardiovascular disease[23]. For maintaining fluid balance, muscle function, nerve transmission, and metabolic health in patients with diabetes, serum electrolytes, including sodium, potassium, chloride, calcium, and magnesium, PTH play crucial roles[24]. Electrolyte abnormalities, particularly hyperkalemia, can result in potentially fatal cardiac arrhythmias, whereas fluid retention can cause lung congestion. Furthermore, metabolic acidosis, induced by the kidneys' decreased capacity to eliminate hydrogen ions, worsens bone demineralization and muscle atrophy[19,25].

Monitoring CKD progression: Current practices and limitations

A decreasing glomerular filtration rate suggests that the kidneys become less able to filter waste materials from the blood, such as urea and creatinine. Significant markers of this deterioration include elevated blood urea nitrogen and serum creatinine levels, that indicate the accumulation of metabolic waste as a result of compromised renal excretory function[26]. Increased creatinine and urea levels signify compromised renal function, highlighting the significance of routine evaluations to efficiently monitor and control the progression of the disease[26-29]. CKD related abnormalities in bone mineral metabolism, including elevated levels of PTH and calcium monitoring, is also essential[21].

In the early stage of CKD (stage 1-3), these markers are typically monitored every 3 to 6 months, while in advanced stages (stage 4), the frequency increases to every 1 to 3 months[30]. For patients with stage 5 CKD who require dialysis, the levels are monitored even more frequently, often before each dialysis session, which can be up to three times per week[30]. Frequent blood sampling, the current gold standard for monitoring, poses significant challenges. These repetitive blood draws are not only inconvenient and disruptive to daily routines, causing discomfort, but they also have the potential to impact quality of life negatively. It was reported that approximately 46% of patients experienced physical discomfort after venous phlebotomy, while 16% felt dependent on others, and 12% reported anxiety in anticipation of phlebotomy appointments[31]. In patients undergoing hemodialysis, frequent venous access increases the risk of complications such as access malfunction and infections[32].

Studies also have demonstrated that frequent phlebotomy is a major contributor to anemia in patients in intensive care[33-35]. To assure the accuracy of the process and reduce problems like infection or excessive bleeding, blood sampling requires the intervention of competent experts, such as trained phlebotomists or physicians[36]. This requirement adds to the healthcare burden, particularly in regions where access to such professionals is limited. Moreover, the process requires consumables that add to the total cost, including needles, vacutainer tubes, tourniquets, alcohol swabs, gloves, cotton balls or gauze, and adhesive bandages[31]. Moreover, patients with CKD often have multiple comorbidities, which complicates their treatment and further elevates healthcare costs[37]. These expenses, together with the requirement for skilled staff, provide major obstacles to access in healthcare settings with low resources and may have a negative impact on the treatment of patients with CKD. Therefore, improving patient care in these vulnerable populations requires tackling these issues using inexpensive sampling techniques. However, frequent venipuncture is associated with patient discomfort, increased infection risks, and elevated healthcare costs, necessitating exploring alternative diagnostic methods.

Saliva as an alternative tool

Saliva is a promising medium for non-invasive biomarker assessment in systemic diseases. Kaufman and Lamster emphasized the utility of saliva in diagnostic applications, particularly in identifying metabolic and systemic disorders[38]. Through passive diffusion, ultrafiltration, and active transport, biomarkers such as creatinine, urea, PTH, and calcium are transferred from blood to saliva, depending on their molecular characteristics, including size, charge, lipophilicity, and protein-binding affinity. The three major salivary glands: Parotid, submandibular, and sublingual, as well as numerous minor glands, contribute to salivary production, and the composition of saliva varies based on factors such as flow rate, stimulation method, and collection technique[39,40] (Figure 1).

Figure 1 Transport of molecules from blood to saliva. PTH: Parathyroid hormone.

The efficiency of biomarker transport is influenced by the interaction of multiple factors. Small, hydrophilic molecules like urea and creatinine can cross epithelial barriers by passive diffusion or paracellular ultrafiltration, whereas larger or charged molecules such as PTH and calcium often rely on active transport mechanisms involving specialized carrier proteins or ion channels. The expression and functionality of these transporters differ across salivary glands and may be modulated by systemic disease states[41,42]. For instance, tight junction permeability and transporter expression may be altered in CKD, potentially affecting biomarker excretion into saliva. Furthermore, glandular physiology, including acinar cell type, vascular supply, and innervation, plays a crucial role in modulating the transfer of serum constituents into saliva. The parotid gland, which primarily secretes serous fluid, favors the ultrafiltration of smaller solutes like urea, while the submandibular gland, with mixed serous and mucous secretion, supports a broader range of transport mechanisms, including active uptake of calcium and PTH. The sublingual gland plays a lesser role in biomarker secretion due to its predominantly mucous composition and lower flow rate[41,43,44].

Generally, saliva collection methods can be broadly divided into two main groups that are unstimulated and stimulated techniques. Unstimulated methods include the draining method, where saliva is allowed to drip passively from the lower lip into sterile tubes. This technique is reliable but slow and requires skilled personnel[45]. Spitting is a commonly utilized saliva collection method due to its simplicity and non-invasiveness. However, this approach may often result in insufficient sample volumes, particularly in individuals with reduced salivary flow rates, such as those experiencing xerostomia or elderly patients with CKD. Additionally, variations in participant technique, such as forceful expectoration vs gentle expulsion, as well as differences in body posture during collection, can contribute to inconsistencies in sample characteristics[5,46,47]. Another one is that the suction method continuously aspirates saliva from the floor of the mouth using micropipettes or syringes, enabling continuous collection. The passive drool method involves allowing participants to accumulate saliva and drool it into a collection container, making it non-invasive, easy to perform, and suitable for diverse populations, including children and the elderly[48,49].

On the other hand, stimulated methods include parotid saliva collection, submandibular and sublingual saliva collection, and minor salivary gland collection. Parotid saliva collection, which often involves citric acid stimulation, is valuable for studying parotid-specific secretion but is more invasive. Submandibular and sublingual saliva collection can be performed through cannulation or suction methods, with suction being the simplest approach. Minor salivary gland collection uses absorbent materials or filter paper, often utilized to assess oral mucosal health[45,49,50]. Paraffin chewing, a mechanical stimulation technique where participants chew inert paraffin wax to increase salivary flow. This method is simple and cost-effective, producing higher saliva volumes, but risks contamination from oral debris and requires participant compliance[51].

A commercially available device, the Salivette^®, has been designed specifically for standardized cortisol measurement. This device minimizes common challenges associated with saliva collection, such as variability and contamination, ensuring more reliable and consistent sample quality[52,53]. Another one is that the Lashley cup is specific for the parotid gland and is ideal for studying parotid function, but it is invasive and uncomfortable. SpitKit^® and OraGene^® devices offer excellent DNA preservation, but they are unsuitable for immediate biochemical analyses due to stabilizers. SalivaBio^® Swabs and OralFluids^® are useful for hormone assays, though they may collect limited volumes. Finally, minor salivary gland collection strips are non-invasive and localized but yield insufficient saliva for extensive biomarker studies[54,55]. Table 1 below summarizes key saliva collection methods, their biomarker reliability, feasibility, and limitations.

Table 1 Comparison of saliva collection methods based on biomarker reliability, feasibility, and limitations.

Collection method	Biomarker reliability	Feasibility	Major limitations
Spitting	High	Easy and patient-friendly	Inconsistent flow, variable technique
Passive drool	Moderate to high	Non-invasive, simple	Slow collection, risk of evaporation
Stimulated (paraffin chewing)	Moderate (flow-dependent dilution)	Simple, cost-effective	Contamination risk, requires compliance, time-sensitive processing
Stimulated (parotid)	Variable, biomarker-specific	Targeted gland sampling	Invasive, discomfort, specialized equipment needed
Oral rinse	Low (due to dilution)	Very easy and rapid	Dilution effect, unreliable concentrations
Salivette®	Moderate	Standardized device available	Absorption variability, potential contamination

One of the main obstacles to using saliva for diagnostics is the unpredictability brought about by the saliva collection process, which can significantly affect the precision, consistency, and repeatability of biomarker analysis. The absence of a standardized procedure for saliva collection has also been identified as a key contributor to inconsistent findings in research, underscoring the need for uniformity in methodology.

CORRELATION BETWEEN SALIVARY AND SERUM BIOMARKERS IN CKD

The findings from the reviewed studies collectively highlight the potential utility of salivary biomarkers, particularly creatinine and urea, as noninvasive alternatives to serum-based diagnostics for CKD. Most studies reported a strong positive correlation between serum and salivary creatinine and urea levels. Several studies using the spitting method, such as those by Renda[56], Lasisi et al[57], and Shrestha et al[58], have demonstrated that salivary creatinine and urea levels strongly correlate with their serum counterparts, with correlation coefficients frequently exceeding 0.8. Additionally, receiver operating characteristic (ROC) analysis in these studies reported area under the curve (AUC) values greater than 0.9, indicating high diagnostic accuracy of salivary biomarkers in detecting chronic conditions such as CKD. Lasisi et al[57] also showed significantly elevated salivary creatinine (median: 2.60 mg/dL) and urea (median: 92.00 mg/dL) levels in patients with CKD compared to controls, with strong correlations to serum levels. Nagarathinam et al[59] also used the spitting method, found an AUC of 0.917 for salivary urea, with high sensitivity (88%) and specificity (84%), reinforcing its potential as a CKD biomarker. Another study also using the same collection methods reveals that the elevated levels of salivary creatinine correspond with increased serum creatinine levels, indicating that as CKD progresses, both biomarkers rise concurrently[60]. Table 2 provides a comparative overview of various studies analyzing salivary biomarkers and their correlation with CKD conditions, with a focus on assessing creatinine and urea biomarkers in different populations[61-64].

Table 2 Summary of studies on salivary creatinine and urea for chronic kidney disease diagnosis.

Ref.	Biomarker(s)	Sample size (CKD/control)	CKD stage(s)	Collection method	Statistical analysis	Correlation (r)	AUC	Assay type	Key findings	Limitations
Venkatapathy et al[61], 2014	Creatinine	105/37	Stage 4-5	Spitting	T-test, pearson, linear regression, ROC	0.73	0.96 (97.1% sensitivity and 86.5% specificity)	Enzymatic	High diagnostic accuracy in advanced CKD	No early-stage CKD data
Lasisi et al[57], 2016	Creatinine, urea	49/50	Mostly stage 4–5	Spitting	Mann-Whitney U, Spearman, ROC	Creatinine = 0.69, urea = 0.51	Creatinine: 0.97 (94% sensitivity and 85% specificity, urea: 0.89 (86% sensitivity and 93% specificity)	Enzymatic	Reflects serum levels, high diagnostic accuracy	Lacked early-stage representation
Pandya et al[62], 2016	Creatinine, urea	90/30	Not specified	Spitting	Kruskal-Wallis, Mann-Whitney U, Spearman	Creatinine = 0.97; urea = 0.97	NR	Enzymatic	Significant serum-saliva correlation	Variability in flow rate, method inconsistency
Pham[60], 2017	Creatinine, urea	112/108	All stages	Spitting	Spearman, linear regression, ROC	Creatinine = 0.90; urea = 0.73	Creatinine: 0.92 (86.5% sensitivity 87.2% and specificity), urea: 0.76 (82.9% sensitivity and 57.8% specificity	Spectrophotometric	Validated diagnostic utility in large sample	Cross-sectional, urban-based sampling bias
Bagalad et al[63], (2017)	Creatinine, urea	41/41	Not specified	Spitting	T-test, pearson, ROC	Creatinine = 0.65; urea = 0.81	Creatinine: 0.9 (93.0% sensitivity and 93.0% specificity), urea: 0.9 (93.0% sensitivity and 90.0% specificity)	Enzymatic	High diagnostic accuracy demonstrated	Inconsistencies in collection and assay protocols
Renda[56], 2017	Creatinine	35/28	Not specified (Pediatric)	Spitting	Kolmogorov-Smirnov, pearson, linear regression, ROC	r = 0.79	0.80 (82.9% sensitivity and 78.6% specificity)	Enzymatic	Strong serum-saliva correlation	Pediatric-only, small sample, no adult comparison
Nagarathinam et al[59], 2023	Urea	150/30	Stage 1–5	Spitting	T-test, ANOVA, pearson, ROC	0.51 ≈ 0.75	0.91 (88% sensitivity and 84% specificity)	Enzymatic	Strong stage-wise diagnostic potential	Periodontal status, diet, hydration not controlled
Liyanage et al[64], 2024	Creatinine, urea	100/0	Stage 2–5	Spitting	Pearson, paired t-test, χ2, ROC	Creatinine = 0.98; urea = 0.94	≥ 0.99 (sensitivity and specificity: NR)	Enzymatic	Correlations unaffected by age/sex	No healthy controls included for comparison

AUC: Area under the curve; CKD: Chronic kidney disease; NR: Not reported; PTH: Parathyroid hormone; ROC: Receiver operating characteristic.

While the studies summarized in Table 2 consistently report strong correlations between salivary and serum levels of creatinine and urea, several methodological limitations are notable. Most studies employed small to moderate sample sizes, often focused predominantly on patients with advanced CKD (stages 4 and 5), which may limit generalizability to earlier stages. Variability in saliva collection methods, unaccounted influences of oral health conditions, hydration status, and comorbidities such as diabetes may have introduced bias. Differences in analytical platforms and assay sensitivities across studies further complicate cross-comparison of results. Furthermore, although many studies report strong correlation coefficients (e.g., r > 0.8 for creatinine), details regarding statistical power, confidence intervals, or sample size justification are frequently lacking, weakening the robustness of these findings. Selection bias may also be present due to recruitment from specific clinical settings. ROC analyses, while promising, are often presented without a critical discussion of sensitivity and specificity thresholds or their relevance in real-world diagnostic settings.

Rodrigues et al[65] using the stimulated saliva observed weak correlations between serum PTH and salivary PTH in patients with CKD. Rajolani et al[66] also explored no significant relationship between serum and salivary levels of calcium and PTH (P > 0.05). The mean calcium levels in saliva (2.03 ± 1.08 mEq/L) were substantially lower than in serum (8.37 ± 0.41 mEq/L), and PTH levels in saliva (1.16 ± 0.32 pg/dL) were much lower than in serum (579.75 ± 562.23 pg/dL). For calcium, only 46.2% of the normal range salivary samples matched serum levels, and none in the higher-than-normal range were consistent. Regarding PTH, all salivary samples in the higher-than-normal range matched serum levels, but only 10% of samples in the normal range were consistent. The study recommends further research, including healthy individuals to compare metabolites in both saliva and serum and to investigate the broader diagnostic potential of saliva.

Additionally, Al Habobe et al[67] evaluated the effects of three distinct saliva collecting techniques on biomarker measures in healthy individuals that are unstimulated, chew-stimulated, and oral rinse. The levels of all biomarkers measured using the oral rinse method significantly differed from those obtained through unstimulated and chew-stimulated saliva. In contrast, unstimulated and chew-stimulated saliva provided comparable biomarker levels. The discrepancy in results from the oral rinse method is likely due to the diluted nature of the saliva extract. It is also suggested to increase the dependability of saliva-based biomarker analysis, more research is needed. These studies also highlight the significance of examining the relationship between calcium and PTH in blood and saliva further, as inconsistent findings imply that more studies are necessary to evaluate the reliability of saliva for these indicators in a single method. Here, Table 3 provides a comparative overview of various studies analyzing salivary biomarkers and their correlation with CKD conditions, with a focus on assessing calcium and PTH biomarkers in different populations.

Table 3 Summary of studies on salivary calcium and parathyroid hormone for chronic kidney disease diagnosis.

Ref.	Biomarker(s)	Sample size (CKD/control)	CKD stage(s)	Collection method	Statistical analysis	Correlation (r)	AUC	Assay type	Key findings	Limitations
Rodrigues et al[65], 2016	Calcium, PTH	60/37	Stage 5	Stimulated (paraffin chewing)	T-test, pearson, simple linear regression	Calcium: -0.13, PTH: 0.03	NR	Colorimetric	No significant correlation between serum and saliva	Small sample size
Rajolani et al[66], 2024	Calcium, PTH	29/0	NR	Spitting method	Paired t-test, pearson's correlation	NR	NR	Enzymatic	No significant serum-saliva correlation for calcium and PTH	Healthy controls are needed for better comparison

AUC: Area under the curve; CKD: Chronic kidney disease; NR: Not reported; PTH: Parathyroid hormone; ROC: Receiver operating characteristic.

Table 3 highlights significant inconsistencies across studies regarding the correlation between salivary and serum concentrations of calcium and PTH. These discrepancies can be attributed to several factors, including small sample sizes, heterogeneity in CKD staging, and variability in saliva collection methods. Notably, the use of stimulated saliva or spitting techniques may dilute analyte concentrations, thereby complicating direct comparisons with serum levels.

Physiological and biochemical barriers further limit the accurate detection of these biomarkers in saliva. PTH, a relatively large peptide (approximately 9.5 kDa), exhibits restricted diffusion into saliva compared to smaller molecules like creatinine or urea. Once in the oral environment, PTH is susceptible to enzymatic degradation by salivary proteases, and both calcium and PTH may bind to salivary proteins, reducing the levels of freely detectable analytes[68]. Moreover, secretion patterns vary across different salivary glands, and this gland-specific variation may influence the concentrations of biomarkers detected. Another technical limitation lies in the analytical sensitivity of current assays, many of which are designed for serum analysis and may not reliably detect the lower concentrations typically found in saliva[69]. Figure 2 provides a schematic representation of the key physiological and methodological barriers that hinder the transfer and accurate detection of calcium and PTH from the bloodstream into saliva.

Figure 2 Physiological and methodological barriers that impede the transfer and accurate detection of calcium and parathyroid hormone from the bloodstream into saliva. PTH: Parathyroid hormone.

Additionally, the potential for publication bias must be considered. Studies reporting positive correlations are more likely to be published, while negative or inconclusive findings may be underrepresented in the literature. This bias can lead to an overestimation of the diagnostic reliability of salivary calcium and PTH.

Advantages of saliva over urine in CKD diagnostics

Given the promising potential of salivary biomarkers, it is essential to contextualize their utility by comparing them with established non-invasive fluids such as urine. Urine more directly reflects renal physiology, particularly glomerular filtration and tubular function, and is routinely used to assess biomarkers such as albumin, proteinuria, creatinine clearance, and the ACR. These metrics are well-established and validated for staging and monitoring CKD progression[4]. In contrast, salivary diagnostics remain an emerging field. While analytes like creatinine and urea show promising correlations with serum levels, salivary biomarker concentrations can be influenced by factors unrelated to renal filtration, including salivary flow rate, glandular function, oral health status, and circadian variation[57]. Additionally, the lack of standardization in collection methods and assay sensitivity further limits direct clinical translation at this stage. However, urine testing is not without limitations. It can be confounded by hydration status, diurnal variation, and may be impractical in patients who are anuric or oliguric, such as those undergoing dialysis[70,71]. Furthermore, proper urine sample collection requires privacy and compliance, which may not be feasible in elderly, pediatric, or bedridden populations[72]. In such cases, saliva offers a practical alternative for frequent or community-based screening, especially in low-resource or rural settings.

STANDARDIZATION AND RESEARCH GAPS IN SALIVA-BASED CKD DIAGNOSTICS

Since much of the research has focused on certain stages of CKD, larger, controlled studies that incorporate several stages of CKD and take age- and sex-specific reference values into consideration are required. Standardizing saliva analysis by resolving differences in salivary flow and collecting methods is also essential. More study is needed to validate the diagnostic accuracy of salivary biomarkers, set appropriate reference ranges, and assess their clinical usefulness across a range of patient demographics. There is a recognized gap in systematically comparing saliva collection methods to evaluate biomarker reliability, particularly in understanding the impact of salivary biomarker levels in populations. Existing research often focuses on single biomarkers, overlooking comprehensive biochemical and mineral marker panels.

CONCLUSION

Saliva-based diagnostics present a viable, non-invasive method of tracking CKD, with the potential to lower medical expenses while simultaneously increasing patient comfort. Particularly when obtained by the spitting method, which guarantees the most accurate representation of systemic concentrations, salivary urea and creatinine have continuously shown positive correlations with serum levels among the biomarkers assessed. Salivary urea and creatinine may be useful markers for CKD screening and monitoring, according to these findings, especially in outpatient or resource-constrained settings. Beyond conventional healthcare environments, saliva testing holds promise for integration into home-based monitoring systems, enabling early detection and longitudinal disease tracking without the need for frequent clinic visits, particularly beneficial for elderly patients or those with limited mobility. In rural and underserved areas, saliva-based point-of-care devices could facilitate population-level screening and timely intervention. Additionally, pediatric patients and individuals undergoing dialysis, who often experience discomfort or complications from repeated blood sampling, could benefit significantly from this less invasive alternative. PTH and salivary calcium, on the other hand, have shown weak or inconsistent correlations with serum levels across a number of studies. This is probably because of things like low analyte concentrations in saliva, variations in transcellular transport, and limitations in assay sensitivity, particularly for larger molecules like PTH. Therefore, to improve the integration of saliva-based diagnostics into regular CKD monitoring, future research should concentrate on large-scale clinical trials, the development of standardized protocols, and the improvement of saliva collection techniques. Physiological and biochemical barriers that impact the precise detection of calcium and PTH in saliva must also be identified through mechanistic studies, which also investigate novel approaches to improve the diagnostic reliability of these substances. While the saliva is flowing.