*2.2. Patients*

The study included 30 children with CKD treated in the Department of Pediatrics and Nephrology of the Medical University of Bialystok, Poland. Patients were divided into two subgroups based on the rate of non-stimulated salivary flow (NWS): normal salivary secretion (normal salivation, CKD NS) and reduced salivary secretion (hyposalivation; CKD HS). Hyposalivation was defined as NWS flow below 0.2 mL/min [16,26,28].

CKD was defined according to the Kidney Disease Improving Global Outcomes (KDIGO) criteria based on different eGFR distribution: Stage 1: >90 mL/min/1.73 m2; Stage 2: 60–89 mL/min/1.73 m2; Stage 3: 30–59 mL/min/1.73 m2; Stage 4: 15–29 mL/min/1.73 m2; and Stage 5: <15 mL/min/1.73 m2 [1]. The estimated glomerular filtration rate (eGFR) was calculated using the updated Schwartz formulaeGFR (mL/min/1.73 m2) <sup>=</sup> 0.413 <sup>×</sup> (height in cm/serum creatinine (Cr)) [29]. Upon the diagnosis of CKD, all patients were on a renal diet that was low in sodium and/or phosphorous and/or protein depending on patients' condition and CKD stage [30]. Office blood pressure (BP) was measured by means of either the manual auscultatory technique or an automated oscillometric device after the subject had rested for 5 min in a sitting position. The average values of the second and third measurements of systolic and diastolic BP were used. Hypertension was defined when the average value of the systolic and/or diastolic BP were ≥95th percentile for age, gender, and height [31].

The causes of CKD were urological defects (33.3%), glomerulopathies (33.3%), congenital nephropathies (13.3%), kidney dysplasia (13.3%), and undetermined etiology (6.8%).

The control group consisted of 30 healthy children attending the Specialist Dental Clinic of the Medical University of Bialystok, Poland for regular check-ups. The control was matched by age and gender to the study group. All patients in the control group had an NWS flow > 0.2 mL/min.

The exclusion criterion in the study and control groups was the occurrence of general diseases: metabolic (insulin resistance, type 1 and 2 diabetes), autoimmune (thyroiditis, systemic sclerosis, arthritis, lupus erythematosus, Crohn's disease, ulcerative colitis), infectious, gastrointestinal and pulmonary diseases. Patients taking antibiotics, non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticosteroids, vitamins and dietary supplements for at least 1 months before saliva collection were excluded from the study, similarly to children with acute inflammatory states. Subjects with poor oral hygiene (Approximal Plaque Index, API > 20) and gingivitis (Sulcus Bleeding Index, SBI > 0.5; Gingival Index, GI > 0.5) were also excluded from the experiment (see: dental examination).

Since pharmacotherapy significantly affects saliva secretion [16,27], patients with CKD taking 5 and more drugs were eliminated from the study.

Detailed characteristics of the patients and the control group are presented in Table 1.
