| Abstract|| |
Diagnosis of renal diseases by assessing renal parameters in saliva. Biochemical investigations using serum form important component of monitoring patients with renal disease. Utility of saliva, in diagnosis and monitoring of patients with renal disease and for calculation of estimated glomerular filtration rate (eGFR), was studied. Sixty patients with renal disease and sixty ageand sex-matched healthy controls were studied. Urea, creatinine, sodium, potassium, uric acid, calcium, and phosphorus were measured in both serum and saliva. eGFR was calculated using salivary creatinine. Data were expressed as mean ± standard deviation. Comparison and correlation between groups were assessed by Student's t-test and Pearson correlation, respectively. Bland-Altman plot, mountain plot, and intra-class correlation coefficient were used to test agreement. A P <0.05 was considered statistically significant. Statistical analysis was done using Microsoft excel spreadsheets, Medcalc Version 10.0, and SPSS version 11.5. Salivary levels of urea, creatinine, uric acid, sodium, potassium, and phosphorus were higher in patients compared to controls. Potassium and phosphorus levels were higher (P = 0.001) and creatinine, sodium, calcium, and uric acid levels were lower (P = 0.001) in saliva compared to serum in both patients and controls. Positive correlation was observed between serum and salivary urea and creatinine (P < 0.0001). eGFR values calculated from salivary creatinine showed good agreement with those calculated form serum creatinine. Salivary urea (>6 mmol/L) and creatinine (>14.6 μmol/L) and eGFR calculated from salivary creatinine can be used to identify patients with renal disease.
|How to cite this article:|
Yajamanam N, Vinapamula KS, Sivakumar V, Bitla AR, Rao PS. Utility of saliva as a sample to assess renal function and estimated glomerular filtration rate. Saudi J Kidney Dis Transpl 2016;27:312-9
|How to cite this URL:|
Yajamanam N, Vinapamula KS, Sivakumar V, Bitla AR, Rao PS. Utility of saliva as a sample to assess renal function and estimated glomerular filtration rate. Saudi J Kidney Dis Transpl [serial online] 2016 [cited 2020 Jan 18];27:312-9. Available from: http://www.sjkdt.org/text.asp?2016/27/2/312/178549
| Introduction|| |
Salivary investigations are used for oral patho logy such as periodontal disease. Recently, saliva is being considered as an alternate biological sample to blood in the management of systemic diseases in view of noninvasive collection method and providing similar information. Sources of various analytics in saliva may be endogenous synthesis in acinar cells or plasma. Passive diffusion, ultrafiltration, transudation, and selective transport are the mechanisms that explain the movement of constituents from plasma to saliva. , Saliva was found to reflect the changes that occur in plasma in case of endocrine, cardiovascular, autoimmune, and infectious diseases.  Saliva also exhibits biochemical indices of renal function and studies have shown association between Salivary and serum creatinine levels. ,,,,, As per Kidney Diseases Initiatives and Global Outcome (KDIGO) guidelines, acute kidney injury (AKI), and chronic kidney disease (CKD) are diagnosed by calculating estimated glomerular filtration rate (eGFR) from serum creatinine levels. However, no studies were found validating salivary creatinine for calculation of eGFR (PubMed search using MESH terms "salivary creatinine" and "glomerular filtration rate"). Hence, this study was carried out to assess utility of saliva as a biological sample in monitoring renal disease and calculation of eGFR.
| Subjects and Methods|| |
Sixty patients with either AKI or CKD as per KDIGO guidelines aged 20-65 years [male/ female: 35/25; mean ± standard deviation (SD): 41.45 ± 13.3 years] attending nephrology department and sixty age-and sex-matched healthy controls (male/female: 35/25; mean ± SD: 49.10 ± 12.3 years) were included in the study. Subjects with oral pathology and H/O of smoking or betel nut/tobacco chewing were excluded from the study. Written informed consent was obtained from all the subjects. The study was approved by the Institutional Ethics Committee.
Five milliliters of venous blood was collected after overnight fast. Serum was separated and stored at −80°C. Five milliliters of unstimulated whole saliva was collected by spitting method on the same day between 9 and 11 am, centrifuged at 2000 rpm for 10 min and supernatant stored at −80°C.
All parameters were assayed using commercial reagents on Synchron CX5 autoanalyzer, Beckman, Minnesota, USA. Salivary creatinine was estimated by modified Jaffe's method.  The intraand inter-assay coefficient of variation for salivary creatinine were 7% and 14% at 12.01 μmol/L, 6%, and 6% at 50.28 μmol/L, respectively. Sodium and potassium were estimated using ion selective electrodes on ECS2000 analyzer from SM diagnostics, India. eGFR was calculated using modified Cockcroft-Gault (multiplied by 0.89 for females), modification of diet in renal disease (MDRD) and CKD Epidemiology collaboration (EPI) formule. ,,
The parameters studied were expressed as mean ± SD. Data not normally distributed as per Kolmogorov-Smirnov test were logarithmically transformed. Student's t-test was used to compare groups. Correlation was assessed using Pearson correlation analysis. Agreement between two estimations was tested using Bland-Altman plot, mountain plot, and intra-class correlation coefficient. Diagnostic utility of parameters was assessed using receiver operating characteristic (ROC) analysis. A P <0.05 was considered statistically significant. Statistical analysis was done using Microsoft excel spreadsheets (Microsoft, Redmond, WA USA), Medcalc Version 10.0 (MedCalc Software bvba, Ostend Belgium), and SPSS version 11.5 (SPSS Inc., Chicago IL, USA).
| Results|| |
[Table 1] shows a comparison of parameters between patients and controls. Concentrations of urea, creatinine, uric acid, and phosphorus were significantly higher in patient group in both serum and saliva. Serum calcium was lower in patient group, whereas salivary calcium did not show any change. Serum sodium was lower and potassium higher in patients compared to controls. However, both salivary sodium and potassium were higher in the patient group. eGFR based on serum creatinine as well as salivary creatinine were significantly lower in the patient group.
|Table 1: Comparison of serum and salivary parameters in control and patient groups.|
Click here to view
Comparison of serum and salivary parameters studied showed that concentrations of potassium and phosphorus were significantly higher in saliva than serum in both groups. Salivary urea was significantly higher than serum in controls but lower in patients. Concentrations of creatinine, sodium, calcium, and uric acid were significantly lower in saliva compared to serum in both groups [Table 2].
|Table 2: Comparison of serum and salivary parameters in control and patient groups.|
Click here to view
A positive correlation (P <0.0001) was found between serum and salivary urea (r = 0.843), creatinine (r = 0.958), and eGFR (r = 0.967). There was good agreement between eGFR values calculated using serum creatinine and salivary creatinine [Figure 1] and [Figure 2]. ROC analysis gave significant area under curve (AUC) for salivary urea, creatinine, sodium, and potassium [Figure 3]. A cut-off value of >6 mmol/L for salivary urea (96.7% sensitivity and 90% specificity) and >14.6 μmol/L for salivary creatinine (100% sensitivity and 98.3% specificity) was found to be useful to identify patients with renal failure.
|Figure 1: Bland–Altman and mountain plots of glomerular filtration rate calculated from serum and salivary creatinine.|
Click here to view
|Figure 2: Intra-class correlation and multiple dot plots of estimated glomerular filtration rate.|
Click here to view
|Figure 3: Receiver operating characteristic analysis of salivary parameters.|
Click here to view
| Discussion|| |
CKD is an important contributor to the poor health outcomes for major non-communicable diseases.  Elevated urea, creatinine, potassium, phosphorus, uric acid, and decreased calcium are the biochemical parameters estimated repeatedly in the management of these patients. The inconvenience caused due to repeated blood collections led to search for alternative biological fluid. Saliva is considered alternative diagnostic fluid of choice for many clinical situations , including renal disease.
In this study, biochemical parameters used in monitoring renal failure were evaluated in saliva with simultaneous estimations in serum. Both salivary and serum creatinine levels were higher in patients with renal failure when compared to controls (P = 0.001). Similar results were observed in earlier studies. , The increase in serum creatinine in patients reflects with higher salivary creatinine levels.  Accordingly, a positive correlation was observed between salivary and serum creatinine concentrations (r = 0.958; P <0.0001). However, Salivary creatinine levels were found to be significantly lower when compared to serum in both patient and control group (P = 0.001) which is in accordance with other reports. ,, Creatinine enters saliva from blood by ultrafiltration. However, due to relative nonpolar nature of creatinine resulting in incomplete filtration, salivary levels are only 10-15% of serum levels. , These findings suggest that salivary creatinine levels can be used to monitor renal function similar to serum creatinine and a cut-off value of >14.6 μmol/L is proposed for salivary creatinine (100% sensitivity and 98.3% specificity) to identify patients with renal failure. This is similar to 11.33 μmol/L and 16.8 μmol/L proposed in earlier studies. , Salivary and serum urea levels were found to be higher in patients than controls (P = 0.001). Similar findings were reported in other studies. , In this study, salivary urea levels were lower than serum in patient group (P = 0.001) but higher in the control group which is in contradiction to literature reports.  However, this finding may not have clinical relevance as both the values are close (3.65 ± 0.66 mmol/L in serum and 4.48 ± 1.33 mmol/L in saliva) and within expected normal range. Urea enters by the process of passive diffusion from blood into saliva through acini of salivary glands.  Accordingly, our study (r = 843; P <0.0001) as well as other reports , showed a positive correlation between salivary and serum urea concentrations. ROC analysis showed a cut-off value of >6 mmol/L (96.7% sensitivity and 90% specificity) to identify patients with renal failure. This is in agreement with earlier studies showing 6.57 mmol/L  and 7.5 mmol/L. 
Salivary and serum uric acid concentrations were higher in patients with renal failure when compared to controls (P = 0.001) which are in agreement with other reports. , Salivary uric acid was lower than serum in both patient and control groups (P = 0.001). Hadi and Al-Jubouri also reported lower salivary uric acid levels compared to serum.  However, correlation (r = 0.109; P = 0.236) and ROC analysis to identify cut-off value did not yield significant results.
Salivary sodium and potassium were higher in patients compared to controls (P = 0.001) in accordance with Tomás et al  and in contradiction to others.  Potassium was higher and sodium lower in saliva when compared to serum in both patient and control groups (P = 0.001). There was no significant correlation between serum and saliva with respect to sodium and potassium which might be due to factors such as active reabsorption of sodium and active secretion of potassium by Na-K-Cl symporters apart from passive diffusion. , Hence, although ROC analysis in this study showed significant AUC (P = 0.0002 and 0.010 for sodium and potassium, respectively), these parameters are not likely to be useful to monitor renal failure patients.
Salivary calcium did not show significant change while phosphorus levels were increased (P = 0.014) in patients compared to controls. Similar results were reported earlier. , Salivary calcium levels were significantly lower than serum, whereas phosphorus levels were significantly higher than serum levels in both patients and controls (P = 0.001). Higher Salivary phosphorus concentrations may be due to secretion of phosphorus from ductal cells.  No correlation was found between serum and salivary for calcium and phosphorus levels. ROC analysis was found to be insignificant. Hence, salivary calcium and phosphorus may be of limited use in monitoring patients with renal disease.
Utility of creatinine estimation lies in its relationship to GFR. If salivary creatinine is to be useful in renal disease, then eGFR calculated from salivary creatinine should be available. There are no studies on estimation of eGFR based on salivary creatinine estimation (PubMed search including MESH terms "salivary creatinine" and "glomerular filtration rate"). A modified Cockcroft-Gault formula for calculation of eGFR based on salivary creatinine levels has been proposed  but not confirmed. In this study, salivary creatinine was equated to serum creatinine by deducing a factor based on regression equation between the two parameters [Figure 4]a. Regression analysis showed a significant correlation between serum and salivary creatinine values and a factor of "8" was obtained from regression equation. The calculated serum creatinine (Cscr) using this factor of "8" and measured serum creatinine (M-scr) values showed good agreement [Figure 4]b. The eGFR values derived from M-scr and C-scr using modified Cockcroft-Gault, MDRD, and CKD-EPI formulae also showed good agreement [Figure 1] and [Figure 2]. Thus, the simplified factor of "8" was found to be adequate to equate salivary creatinine to the corresponding serum levels for the purpose of calculating eGFR. Since Cockcroft-Gault, MDRD, and CKD-EPI formulae are the commonly used formula,  it is proposed that salivary creatinine is useful to calculate eGFR.
|Figure 4: Linear regression between serum and salivary creatinine (a), Bland–Altman plot of calculated and measured serum creatinine (b).|
Click here to view
Due to increasing prevalence rates of renal failure, there is a need for early diagnosis. The main thrust lies on imaging techniques that visualize the kidney and biochemical investigations. Saliva has distinctive advantages as alternate biological sample to blood for monitoring patients of renal disease. Though Salivary composition is under the influence of the method of collection,  hydration status, body posture, lighting, circadian rhythm, seasonal variations, gland size, smoking, stimulants, and medications, , it is noninvasive and causes less inconvenience to participants of repeat sample analysis. Based on the results of the this study, it is proposed that estimation of urea and creatinine in saliva and eGFR calculated from salivary creatinine are likely to be useful as biochemical markers to differentiate patients with renal failure from healthy subjects. With respect to other salivary parameters, further studies are needed to establish their role.
| Acknowledgment|| |
The present work was supported by grants from Sri Balaji Arogya Vara Prasadini Scheme (AS/21/SBAVP-RG/SVIMS/2012), Sri Venkateswara Institute of Medical Sciences, Tirupati, India, and Indian Council of Medical Research (3/2/2012-13/PG-thesis-HRD-13), New Delhi, India.
Conflict of interest: None declared.
| References|| |
Kaufman E, Lamster IB. The diagnostic applications of saliva - A review. Crit Rev Oral Biol Med 2002;13:197-212.
Chiappin S, Antonelli G, Gatti R, De Palo EF. Saliva specimen: a new laboratory tool for diagnostic and basic investigation. Clin Chim Acta 2007;383:30-40.
Malamud D. Saliva as a diagnostic fluid. Dent Clin North Am 2011;55:159-78.
Xia Y, Peng C, Zhou Z, et al. Clinical significance of saliva urea, creatinine, and uric acid levels in patients with chronic kidney disease. Zhong Nan Da Xue Xue Bao Yi Xue Ban 2012;37:1171-6.
de Almeida Pdel V, Grégio AM, Machado MA, de Lima AA, Azevedo LR. Saliva composition and functions: a comprehensive review. J Contemp Dent Pract 2008;9:72-80.
Davidovich E, Davidovits M, Peretz B, Shapira J, Aframian DJ. Elevated salivary potassium in paediatric CKD patients, a novel excretion pathway. Nephrol Dial Transplant 2011;26:1541-6.
Lloyd JE, Broughton A, Selby C Salivary creatinine assays as a potential screen for renal disease. Ann Clin Biochem 1996;33(Pt 5):428-31.
Venkatapathy R, Govindarajan V, Oza N, Parameswaran S, Pennagaram Dhanasekaran B, Prashad KV. Salivary creatinine estimation as an alternative to serum creatinine in chronic kidney disease patients. Int J Nephrol 2014; 2014:742724.
Cockcroft DW, Gault MH. Prediction of creatinine clearance from serum creatinine. Nephron 1976;16:31-41.
Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med 1999;130:461-70.
Levey AS, Stevens LA, Schmid CH, et al. A new equation to estimate glomerular filtration rate. Ann Intern Med 2009;150:604-12.
Couser WG, Remuzzi G, Mendis S, Tonelli M. The contribution of chronic kidney disease to the global burden of major noncommunicable diseases. Kidney Int 2011;80:1258-70.
Hofman LF. Human saliva as a diagnostic specimen. J Nutr 2001;131:1621S-5S.
Ladell WS. Creatinine losses in the sweat during work in hot humid environments. J Physiol 1947;106:237-44.
Chiou WL, Pu FS. Creatinine VIII: saliva levels of endogenous "true" creatinine in normal subjects. Clin Pharmacol Ther 1979;25: 777-82.
Cardoso EM, Arregger AL, Tumilasci OR, Elbert A, Contreras LN. Assessment of Salivary urea as a less invasive alternative to serum determinations. Scand J Clin Lab Invest 2009;69:330-4.
Carco P, Canciullo D. Urea excretion through the human salivary glands. Ann Otol Rhinol Laryngol 1958;67:1050-65.
Hadi BA, Al-Jubouri RH. Salivary and plasma analysis of oxidative stress biomarkers in end stage renal failure patients. J Baghdad Coll Dent 2011;23:46-50.
Tomás I, Marinho JS, Limeres J, Santos MJ, Araújo L, Diz P. Changes in salivary composition in patients with renal failure. Arch Oral Biol 2008;53:528-32.
Manley KJ, Haryono RY, Keast RS. Taste changes and saliva composition in chronic kidney disease. Ren Soc Australas J 2013;8:56-60.
Whelton HP. Introduction: The anatomy and physiology of salivary glands. In: Edgar M, O'Mullane DM, eds. Saliva and Oral Health. 2nd ed. London: British Dental Association; 1996. p. 1-16.
Savica V, Calò LA, Caldarera R, et al. Phosphate salivary secretion in hemodialysis patients: implications for the treatment of hyperphosphatemia. Nephron Physiol 2007;105:p52-5.
Heflin L, Walsh S, Bagajewicz MJ. Design of medical diagnostics products: A case-study of a saliva diagnostics kit. Comput Chem Eng 2009;33:1067-76.
Sakhuja V, Dheerendra PC. eGFR: What the internist should know. Medicine 2012;22:615-7.
Pfaffe T, Cooper-White J, Beyerlein P, Kostner K, Punyadeera C. Diagnostic potential of saliva: current state and future applications. Clin Chem 2011;57:675-87.
P. V. L. N. Srinivasa Rao
Department of Biochemistry, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh - 517 507
[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]