The presented case–control study is a part of the project that included clinical examination of oral health parameters, sociodemographic questionnaires and assessment of selected salivary components in idiopathic steroid-sensitive nephrotic syndrome (iNS) patients and controls.
The iNS participants were treated in the Department and Clinic of Pediatric Nephrology, and controls were healthy outpatients attending the dental clinic at the Department of Conservative Dentistry and Pedodontics of Wroclaw Medical University, Poland. The examinations were performed from May 2018 to April 2019. The detailed data on oral health parameters and questionnaire items were published earlier . In this paper, we used only several basic oral health parameters, which could potentially influence the studied salivary components.
The STROBE guidelines for case–control studies were followed .
The recruited participants (n = 110), both genders, were between the ages of 4 and 17. Nevertheless, 9.1% (n = 10) of the parents refused to consent to the study, and 5.4% (n = 6) of the children refused to be examined. Finally, 94 participants were enrolled in the study. Half of them were patients suffering from iNS and were either in remission (n = 26) or in the relapse phase of the disease (n = 21), being treated in the Clinic of Pediatric Nephrology, and the remaining participants were healthy (n = 47). The inclusion criteria for the iNS patients were the disease diagnosed at least two years earlier than this study and no other acute systemic diseases present at that moment. The iNS phase was recognized by clinical symptoms and laboratory blood tests.
The control group included clinically healthy participants (n = 47) who were outpatients of the dental clinic without a history of impaired renal function or proteinuria or acute or chronic systemic diseases (based on medical interviews of a parent and a child’s health record book) and at the age range and gender corresponding to the iNS patients.
All participants involved in the study had to provide written informed consent from a parent (in addition to a separate written consent from participants age 16 and over), be willing to undergo the oral clinical examination and salivary sample collection, and had to respond to questionnaire items. Participants who did not meet the inclusion criteria were excluded from the study.
Approval to conduct the study was obtained from the Bioethics Committee of Wroclaw Medical University (permission no. KB-343/2016). All parents of the recruited participants provided their written consent to participate in the study, and the participants were willing to submit to the investigation. Participation in the study was voluntary and anonymous, and the collected data were treated confidentially.
Sample size estimation
Sample size determination was based on a t-test for independent groups using a special computer program . The expected difference between means for the two groups for salivary urea content was set at 10.0 (variance equal to 220). The power of the test was set at 90%, and the confidence level was set at 95%. With such assumptions, the required sample size for each group was equal to n = 47.
Data on gender, age, frequency of toothbrushing, use of fluoridated toothpaste and topical professional application of fluoride specimens, and diet were reported by the parents of the participants. The detailed questionnaire items were published earlier .
Oral health status
The clinical oral examination was carried out with the use of artificial light, a plain mirror and a ball-ended dental probe (WHO CPI probe). This process included the assessment of the number of primary and permanent caries-affected teeth (dmft and DMFT values) according to World Health Organization criteria (WHO) , oral hygiene using the Approximal Plaque Index—API by Lange et al., 1974  and gingival condition according to the Gingival Index—GI, by Löe and Silness, 1963) . Additional oral health parameters had been analyzed previously .
Salivary sample collection
The mixed saliva was collected after rinsing the mouth with distilled water without external stimulation at least 2 h after breakfast. The child sat with the head bent down and the mouth open and deposited saliva by spitting into a graded test tube that was stored on crushed ice. The salivary flow rate was calculated as ml/min (V) based on the measurement of the volume of the collected saliva sample and the time needed for its collection. The saliva samples were centrifuged for 15 min at a speed of 3500 rpm before the biochemical assays.
The following salivary parameters were assessed: pH (by potentiometric method), buffer capacity (BC, by titration method), total protein (P, by Lowry’s et al. method) , α-amylase (Amy, by the assay kit based on Caraway’s method), salivary peroxidase (SPO, by Nbs-SCN− method) , calcium (Ca, by the assay kit based on the formation of the chromogenic complex between calcium ions and o-cresolphthalein), magnesium (Mg, by the assay kit based on the reaction of magnesium with xylidyl blue-I), inorganic phosphate (iPh, by the assay kit based on the formation of the chromogenic complex of ammonium molybdate with phosphate), fluoride (F, by the ionic selective electrode Orion 9609), urea (U, by the assay kit based on urease activity), and uric acid (UA, by the assay kit based on uricase activity).
Assessment of selected components in the blood
Venous blood samples were collected from ill subjects after an overnight fast. After centrifugation, the protein, albumin, urea, uric acid, calcium, magnesium, inorganic phosphate, total cholesterol, triglycerides and creatinine were measured in plasma using an automated standardized multichannel analyzer (Konelab 30i; Thermo Fisher Scientific Inc., bioMerieux, Marcy l’Etoile, France). The estimated glomerular filtration rate (eGFR) was also determined . The blood tests were performed on the same day as salivary sample collection.
The consistency of the obtained results with a normal distribution was verified with the use of the Shapiro–Wilk test and the homogeneity of variance using the Brown–Forsythe test. The quantitative continuous variables with distributions that were not significantly different from normal are presented as the mean and standard deviation, and the qualitative variables are presented as numbers and percentages. Significant between-group differences in the mean values of the variables with a normal distribution were verified using Student’s t-test, ANOVA and Tukey’s test. The association between variables was tested with the chi square Pearson’s test and Spearman’s or Pearson’s correlation coefficients. Logistic bivariate and multivariate regression analyses for dependent variables (presence or absence of the disease) and independent variables (salivary parameters) were carried out. Receiver operating characteristic (ROC) curve analysis was used to evaluate the diagnostic potential of selected salivary constituents to classify the iNS participants compared to controls and the patients with relapse compared to remission. The overall results were assessed by the area under the ROC curve (AUC) and the cutoff values, which were determined based on the best trade-off between sensitivity and specificity. The level of significance was set at P < 0.05. All analyses were computed with Statistica 13 software (PL StatSoft).