| 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 2019 Dec 10];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.|
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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.|
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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.|
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|Figure 2: Intra-class correlation and multiple dot plots of estimated glomerular filtration rate.|
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|Figure 3: Receiver operating characteristic analysis of salivary parameters.|
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| 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).|
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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.
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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]