Bone turnover markers in oral and gingival crevicular fluid in children with end-stage chronic kidney disease

Chronic kidney disease (CKD) is a persistent organ damage for three months or more due to various etiologic factors. The pathologic basis of the disease is the process of replacement of normal anatomical structures by fibrosis, which leads to organ dysfunction. CKD is evidenced by a decrease in estimated glomerular filtration rate (eGFR) and/or albuminuria and other markers of kidney damage [1]. The prevalence of CKD in the world population is more than 800 million people [2]. The global mortality from CKD reached 1.2 million in 2017 and is projected to increase [3].

In the world pediatric population, the prevalence of CKD reaches 18.5–58.3 cases per 1 million children [4]. In Russia since 2012, the overall incidence of CKD in children continues to grow [3]. Approaches to CKD diagnosis are unified for both children and adults. However, due to the predominance of non-glomerular etiology of CKD in children, albuminuria is detected less frequently than in adults [5]. According to the European Pediatric Registry, congenital anomalies of the kidney and urinary tract and genetic diseases are the leading etiologic factors of CKD in children, accounting for 40–60% and 20–30% of detected cases, respectively; glomerulonephritis makes an etiologic contribution in less than 10% of cases [5].

End-stage kidney disease (ESCKD) requires renal replacement therapy (hemodialysis, peritoneal dialysis or renal transplant). ESCKD is associated with life quality decline and unfavorable outcomes [6]. Moreover, ESCKD in children is accompanied by significant mineral and bone disorders (CKD-MBD) [7–9], arising as a result of hyperparathyroidism and impaired calcium-phosphorus (Ca-P) metabolism [10][11]. In CKD-MBD children are observed with a decrease in growth [12], a high tendency to fractures [13][14], as well as multiple structural changes in bone tissue, including cortical loss, demineralization, bone trabeculae rarefaction, which are associated with increased osteoclast activity [13].

Bone turnover markers in CKD-MBD are deoxypyridinoline (DPD) and osteocalcin (OC) [15]. DPD is a compound formed during collagen breakdown, it is released into the bloodstream, and then excreted in the urine. DPD reflects osteoclasts activity; DPD level increasing directly correlates to the severity of renal dysfunction in experimental study on rats [16]. OC is a vitamin K-dependent protein synthesized by osteoblasts, reflects impaired bone mineralization in CKD-associated hyperparathyroidism [17][18].

CKD patients are prone to various maxillofacial bone changes such as decreased density of cortical bone and increased jawbone porosity [19], shortened mandible branches, increased gonial angle, decreased posterior facial height [8][20][21], structural and functional temporomandibular joint changes [22][23], along with a significant slowdown in teething [22]. These changes require a personalized approach to orthodontic treatment in CKD children and objective markers for making a medical decision.

Today there are still some open questions about optimal timing for initiating orthodontic treatment and its types in CKD children, and monitoring bone remodeling during this treatment. Thus, searching for biomarkers reflecting specific bone changes including maxillofacial bones in CKD patients remains in demand.

Study objective: to investigate bone turnover markers in different biological fluids (urine, blood serum, oral fluid (OF) and gingival crevicular fluid (GCF) at the stage of planning an orthodontic strategy in children with end-stage chronic kidney disease (ESCKD).

MATERIALS AND METHODS

This pilot cross-sectional multicenter study based on Russian Federal Law No. 323-FZ dated 21.11.2021 “On the Fundamentals of Public Health Protection in the Russian Federation”, (Legislation Bulletin of the Russian Federation, 2011, No. 48, Art. 6724). The required number of patients in groups was determined before the study. The sample size was sufficient given the power of 80%.

Patient enrollment

The study was conducted from March 1 to June 30, 2024, at the following clinical centers: E.V. Borovsky Institute of Dentistry, Sechenov University; Surgical Department No. 1, Academician V.I. Shumakov National Medical Research Center of Transplantology and Artificial Organs, Ministry of Health of the Russian Federation. A continuous recruitment of patients was carried out from those who applied to the above-mentioned medical institutions.

Inclusion criteria:

• age from 7 to 17 years;
• established diagnosis of CKD (ICD-10 codes¹: N18 Chronic kidney disease; T86.1 Renal transplant dysfunction);
• dental anomalies, including bite anomalies;
• availability of written informed voluntary consent of parents/legal representatives for the child’s participation in the study.

Non-inclusion criteria:

• active/current orthodontic treatment (n= 5);
• concomitant acute/chronic diseases affecting bone metabolism:

– endocrine and metabolic diseases (n = 10),

– autoimmune diseases (n = 2),

– genetic diseases (n = 4),

– oncological diseases (n = 1),

– diseases of the gastrointestinal tract (n = 3);

– chronic liver diseases (n = 7),

– drug-induced disorders of bone metabolism (n = 5).

A total of 65 children and adolescents were assessed for participation in the study. Exclusion criteria were identified in 37 patients (Fig.). The study included 28 CKD children, who were divided into two groups: Group 1 – 14 ESCKD patients with eGFR according to CKiD U25 with a constant creatinine coefficient ≤ 25 ml/min/1.73m²; Group 2 – 14 patients with kidney graft dysfunction (RTD) with eGFR according to the CKiD U25 with a constant creatinine coefficient > 25 ml/min/1.73 m².

The control group consisted of 20 practically healthy children and adolescents with no general medical pathology, matched by sex and age to the group of children with CKD, who underwent dental examination at the Department of Pediatric, Preventive Dentistry and Orthodontics in E.V. Borovsky Institute of Dentistry during the study period.

Determination of bone metabolism biomarkers

Biological fluids from patients were taken once in the morning before the breakfast and diagnostic and therapeutic procedures. Blood samples of 5 mL each were collected from cubital vein or from the hand back veins, stabilized with heparin (25 IU/ml), urine sample of 50 mL each, oral fluid sample at least 5 mL.

Biochemical blood analysis was performed by photocolorimetric methods to determine the level of total (Ca) and ionized (Ca²⁺) calcium, phosphorus (P). Calculation of total blood plasma calcium with correction for albumin was performed according to the formula: measured plasma calcium level (mmol/L) + 0.02*(40 – measured plasma albumin level (g/L).

Urinary DPD was measured by solid-phase chemiluminescent immunoassay.

OC in serum, GCF and OF was measured by using commercial Osteocalcin ELISA kits for solid-phase enzyme-linked immunosorbent assay (BioVendor, USA).

The Milwaukee PH56 (Milwaukee Instruments, USA) device was used to determine oral pH.

Bone density was assessed using cone-beam computed tomography (CBCT), expressed in Hounsfield units (HU) according to C. Mish classification [24], based on the mathematical reconstruction of X-ray attenuation coefficients assigned to each pixel. The X-ray assessment was performed in four sections: the anterior and posterior sections of the upper jaw; the anterior and posterior sections of the lower jaw.

Statistical analysis

Quantitative features are presented as median and interquartile range, qualitative ones – as proportion. The studied features of patient groups were tested for normal distribution using Shapiro-Wilk test and for homogeneity of variances using Levene test. Variables corresponding to normal distribution and having homogeneous variances are presented as mean values and standard deviation, mean values were compared using one-way analysis of variance (ANOVA). Other variables are presented as median and interquartile range (25th; 75th percentiles), for their comparison the Kruskal–Wallis method was used. For post-hoc analysis the Tukey test was used. The results of statistical analysis were considered significant at p < 0.05. The experimental results were processed using Prism 8.0.1 (GraphPad Software, USA) and R language. 4.4.2 in the R-Studio software environment.

RESULTS

The study included patients without pronounced clinical manifestations of CKD-MBD and osteoporosis. The main characteristics of the study groups are presented in Table 1.

Mean age in study groups were 12.7 ± 2.9 years and girls’ number in RTD group was lower than in ESCKD group and control, but the differences were not statistically significant (Table 1).

Serum creatinine in ESCKD group was significantly higher and eGFR was lower compared to RTD group and control. ESCKD patients received hemodialysis or peritoneal dialysis treatment for an average from 6 months to 3 years.

Total and ionized serum calcium did not differ between the study groups (Table 1). Serum P was significantly higher in ESCKD group compared to RTD group and control. While there were not found any statistically significant differences in phosphorus levels in RTD group and control (Table 1).

Summarized markers results in studied groups are presented in Table 2.

Urinary DPD concentration was higher in CKD groups compared to control (Table 2). However, no statistically significant differences were found between urinary DPD concentration in ESCKD and RTD patients.

Serum OC concentration was increased in ESCKD patients compared to control (p < 0.05) and did not differ from RTD group. OC in GCF was higher in control compared to ESCKD (p < 0.001) and RTD (p < 0.001) groups. Meanwhile salivary OC was comparable in all groups (Table 2).

OF pH was statistically significantly higher in both ESCKD and RTD children compared to control (Table 2). Moreover, there were no differences between oral pH in RTD and ESCKD groups, thus oral acidity was similar in these two groups.

Bone density radiographical assessment shows that Hounsfield Index of posterior maxilla was higher in RTD group compared to ESCKD group (p < 0.01), and there was no difference with control. Hounsfield Index of anterior maxilla in control was higher than in ESCKD and in RTD groups. A similar pattern was found in Hounsfield index of both anterior and posterior mandible where the control group levels were statistically significantly higher than in ESCKD and RTD patients (Table 2).

DISCUSSION

Study results demonstrated CBCT changes in bone turnover markers and bone density were the most pronounced in ESCKD children. Urinary DPD increase, a decrease in serum and GCF OC and a decrease in the Hounsfield index in both anterior and posterior regions of the maxilla and mandible were revealed. Similar changes were noted in RTD patients, but OC level was reduced only in GCF. Bone density assessment had no significant differences between ESCKD and RTD groups, except Hounsfield index in posterior maxilla.

Kidneys play a crucial role in Ca-P metabolism by almost complete tubular reabsorption of these ions. Ca-P homeostasis in CKD patients is disrupted, so serum P in ESCKD patients had been increasing with eGFR decreasing in our study. It’s important to check serum P in CKD-MBD patients for maintaining bone homeostasis, and our results are consistent with Rastogi A. et al. [25]. Serum P in KDG group was close to control group, and inversely correlates with a higher eGFR level, because of kidney filtration improvement after transplantation, but due graft dysfunction P level remained high. These trends are consistent with other studies data on mineral metabolism effect on bone remodeling in patients after kidney transplantation [26][27].

Our study results showed that serum Ca does not statistically differ between the groups, which indicates the stability of this marker regardless of kidney and graft function. However, ESCKD markers are less homogeneous and it still has a greater spread. No expressed calcium metabolism disorders were detected as well as in Liu J. et al. and Hasanzamani B. et al. studies [28][29]. It should be noted that the levels of parathyroid hormone and bone fraction of alkaline phosphatase were not taken into account.

Urinary DPD was found as highly sensitive marker of bone metabolism disorders in ESCKD and RTD patients. Thus, we found clear and significant differences in this marker between the study groups. DPD increase in CKD children compared to control may indicate high osteoclast activity and bone resorption activation. DPD changes were recorded simultaneously with a Hounsfield index decrease in jawbones. It indicates bone collagen breakdown, type I mainly, the end products of bone metabolism excretion with urine and it confirms persistent bone metabolism changes in CKD children. However, literary data did not confirm that urinary DPD can reflect bone metabolism in CKD-MBD patients [30]. On the other hand, DPD level is known as one of the leading biochemical markers of bone remodeling and is used in osteoporosis early diagnosis [31]. Thus, urinary DPD determination in CKD patients may become promising for assessing osteoclasts activity and bone resorption and requires further study on a larger CKD patient sample.

Presented study results convincingly demonstrate that OC is an informative marker of osteoblast activity in ESCKD and RTD children, and GCF is the best biological fluid for its determination, since the most significant OC changes are registered in GCF, despite the limited sample. OC decrease in GCF was found in ESCKD and RTD children compared to control, which indicates a violation of bone metabolism. Serum OC increase was detected only in ESCKD group probably due to the limited sample. OC in OF did not statistically significantly differ between three groups possibly due to high protease activity in OF [32]. We didn’t find any information about OC measurement in GCF in CKD patients in the reviewed literature. However, Fadli N. et al. used GCF for the assessment of OC and proinflammatory markers [33]. Interest in GCF exertion as a fluid for various markers detection in patients with systemic diseases, including CKD, is growing due to its sufficient informativeness and minimally invasive nature.

OF pH increase with its alkaline tendency may be associated with a disturbance in general metabolism, including a change in the acid-base balance in ESCKD patients [34].

In addition, the obtained data indicate significant disturbances in bone structure in ESCKD and RTD children, which is manifested in a significant decrease in Hounsfield index compared to control, especially in the anterior and posterior mandible. These changes are consistent with previous studies [23], confirming disturbances in bone metabolism and decreased bone mineralization in patients with renal dysfunction.

Limitations and directions for future research

Result interpretations have several limitations due to pilot study design: small sample size, one observation point. The levels of parathyroid hormone and bone fraction of alkaline phosphatase were not taken into account when assessing CKD-MBD. Perhaps due to insufficient study power, there were no statistically significant differences in total serum calcium adjusted for albumin, as well as OC in oral fluid. To draw conclusions on these markers, it is necessary to conduct longitudinal studies on large samples using probability selection of observation units.