Revealing novel biomarkers for diagnosing chronic kidney disease in pediatric patients.

Pediatric chronic kidney disease (CKD) is a clinical condition characterized by progressive renal function deterioration. CKD diagnosis is based on glomerular filtration rate, but its reliability is limited, especially at the early stages. New potential biomarkers (citrulline (CIT), symmetric dimethylarginine (SDMA), S-adenosylmethionine (SAM), n-butyrylcarnitine (nC4), cis-4-decenoylcarnitine, sphingosine-1-phosphate and bilirubin) in addition to creatinine (CNN) have been proposed for early diagnosis. To verify the clinical value of these biomarkers we performed a comprehensive targeted metabolomics study on a representative cohort of CKD and healthy pediatric patients. Sixty-seven children with CKD and forty-five healthy children have been enrolled in the study. Targeted metabolomics based on liquid chromatography-triple quadrupole mass spectrometry has been used for serum and plasma samples analysis. Univariate data analysis showed statistically significant differences (p < 0.05) in the concentration of CNN, CIT, SDMA, and nC4 among healthy and CKD pediatric patients. The predictive ability of the proposed biomarkers was also confirmed through specificity and sensitivity expressed in Receiver Operating Characteristic curves (AUC = 0.909). In the group of early CKD pediatric patients, AUC of 0.831 was obtained, improving the diagnostic reliability of CNN alone. Moreover, the models built on combined CIT, nC4, SDMA, and CNN allowed to distinguish CKD patients from healthy control regardless of blood matrix type (serum or plasma). Our data demonstrate potential biomarkers in the diagnosis of early CKD stages.

Association of Ischemia-Modified Albumin (IMA) in Saliva, Serum, and Urine with Diagnosis of Chronic Kidney Disease (CKD) in Children: A Case-Control Study.

BACKGROUND Ischemia-modified albumin (IMA) is a secreted biomarker for ischemic oxidative stress. This case-control study aimed to evaluate the association of ischemia-modified albumin (IMA) in saliva, serum, and urine with diagnosis of chronic kidney disease (CKD) in 24 children. MATERIAL AND METHODS The study involved 24 children with CKD. CKD was defined according to the Kidney Disease Improving Global Outcomes (KDIGO) diagnostic criteria. The control group consisted of 24 healthy children who were matched for age and gender to the experimental group. The concentration of IMA was determined by the colorimetric method in non-stimulated whole saliva (NWS), stimulated whole saliva (SWS), serum, and urine of children with CKD. The Mann-Whitney U test was used for inter-group comparisons. RESULTS IMA levels were significantly higher in NWS (P=0.0082) and SWS (P=0.0014) of children with CKD than in the control group. The concentration of IMA in NWS was correlated with standard indicators of kidney function, including the estimated glomerular filtration rate (r=-0.798, P≤0.0001), stage of CKD (r=0.814, P≤0.0001), and serum creatinine (r=0.711, P≤0.0001) and urea levels (r=0.738, P≤0.0001). CONCLUSIONS Salivary IMA concentration depends on renal function in children. Salivary IMA discriminates children with end-stage kidney disease from children with mild and moderate CKD and healthy children with high sensitivity and specificity. Further research is required, including assessment of the diagnostic usefulness and validation of the biomarker in a clinical diagnostic study.

Mitochondrial Redox Balance of Fibroblasts Exposed to Ti-6Al-4V Microplates Subjected to Different Types of Anodizing.

Despite the high biocompatibility of titanium and its alloys, the need to remove titanium implants is increasingly being debated due to the potential for adverse effects associated with long-term retention. Therefore, new solutions are being sought to enhance the biocompatibility of titanium implants. One of them is to increase the thickness of the passive layer of the implant made of titanium dioxide. We were the first to evaluate the effect of hard-anodized (type II) Ti-6Al-4V alloy discs on the cytotoxicity, mitochondrial function, and redox balance of fibroblasts mitochondria compared to standard-anodized (type III) and non-anodized discs. The study used fibroblasts obtained from human gingival tissue. The test discs were applied to the bottom of 12-well plates. Cells were cultured for 24 h and 7, 14, and 21 days and mitochondria were isolated. We demonstrated the occurrence of oxidative stress in the mitochondria of fibroblasts of all tested groups, regardless of the presence and type of anodization. Type II anodization prevented changes in complex II activity (vs. control). The lowest degree of citrate synthase inhibition occurred in mitochondria exposed to titanium discs with type II anodization. In the last phase of culture, the presence of type II anodization reduced the degree of cytochrome c oxidase inhibition compared to the other tests groups and the control group, and prevented apoptosis. Throughout the experiment, the release of titanium, aluminium, and vanadium ions from titanium discs with a hard-anodized passive layer was higher than from the other titanium discs, but decreased with time. The obtained results proved the existence of dysfunction and redox imbalance in the mitochondria of fibroblasts exposed to hard-anodized titanium discs, suggesting the need to search for new materials perhaps biodegradable in tissues of the human body.

Association Between Salivary Cytokines, Chemokines and Growth Factors and Salivary Gland Function in Children with Chronic Kidney Disease.

Introduction: Chronic kidney disease (CKD) is a systemic inflammatory disease that leads to multiple organ complications not only in the kidneys and the cardiovascular system, but also in the oral cavity. CKD children experience reduced saliva secretion (hyposalivation), which leads to increased incidence of dental caries and significant impairment of patients' quality of life. However, the causes of salivary gland dysfunction in children with CKD are unknown. The present study is the first to evaluate the inflammatory and anti-inflammatory profile in the saliva of children with CKD at different stages of renal failure with normal and reduced salivary gland function. Methods: Thirty children with CKD (age 9-16) and thirty age- and gender-matched healthy children were classified for the study. Salivary inflammatory and anti-inflammatory profile were assayed using the multiplex ELISA assay. Results: We demonstrated statistically significant changes in salivary pro-inflammatory (↑TNF-α, ↓IL-7), anti-inflammatory (↑IL-10), Th1 (↑INF-γ, ↑IL-15), Th2 (↑IL-4, ↑IL-5, ↑IL-6, ↑IL-9) and Th17 (IL-17) cytokines as well as chemokines (↑MCP-1/CCL-2, ↑MIP-1α/CCL3, ↓MIP-1ß/CCL4, ↓EOTAXIN/CCL11) and growth factors (↑G-CSF, ↑FGF) in unstimulated saliva of children with CKD compared to the controls. Although the evaluation of the salivary inflammatory profile does not indicate a particular dominance of any of the branches of the immune system, we observed a statistically significant increase in the concentration of all Th2 cytokines assayed. The multivariate regression analysis showed that the content of salivary cytokines, chemokines and growth factors depends on the secretory function of the salivary glands, ie, salivary flow, total protein concentration and amylase activity in the saliva. Salivary MIP-1α/CCL3 was the most effective to differentiate children with CKD and hyposalivation from patients with normal saliva secretion. Discussion: Inflammation is involved in salivary gland dysfunction in children with CKD, although further studies on in vitro and in vivo models are necessary to confirm this hypothesis.

Oxidation, Glycation, and Carbamylation of Salivary Biomolecules in Healthy Children, Adults, and the Elderly: Can Saliva Be Used in the Assessment of Aging?

Background: Aging is inextricably linked to oxidative stress, inflammation, and posttranslational protein modifications. However, no studies evaluate oxidation, glycation, and carbamylation of salivary biomolecules as biomarkers of aging. Saliva collection is non-invasive, painless, and inexpensive, which are advantages over other biofluids. Methods: The study enrolled 180 healthy subjects divided into six groups according to age: 6-13, 14-19, 20-39, 40-59, 60-79, and 80-100 years. The number of individuals was determined a priori based on our previous experiment (power of the test = 0.8; α = 0.05). Non-stimulated saliva and plasma were collected from participants, in which biomarkers of aging were determined by colorimetric, fluorometric, and ELISA methods. Results: The study have demonstrated that modifications of salivary proteins increase with age, as manifested by decreased total thiol levels and increased carbonyl groups, glycation (Nε-(carboxymethyl) lysine, advanced glycation end products (AGE)) and carbamylation (carbamyl-lysine) protein products in the saliva of old individuals. Oxidative modifications of lipids (4-hydroxynonenal) and nucleic acids (8-hydroxy-2'-deoxyguanosine (8-OHdG)) also increase with age. Salivary redox biomarkers correlate poorly with their plasma levels; however, salivary AGE and 8-OHdG generally reflect their blood concentrations. In the multivariate regression model, they are a predictor of aging and, in the receiver operating characteristic (ROC) analysis, significantly differentiate children and adolescents (under 15 years old) from the working-age population (15-64 years) and the older people (65 years and older). Conclusion: Salivary AGE and 8-OHdG have the most excellent diagnostic utility in assessing the aging process. Saliva can be used to evaluate the aging of the body.

Salivary Gland Dysfunction, Protein Glycooxidation and Nitrosative Stress in Children with Chronic Kidney Disease.

This study is the first to evaluate protein glycooxidation products, lipid oxidative damage and nitrosative stress in non-stimulated (NWS) and stimulated whole saliva (SWS) of children with chronic kidney disease (CKD) divided into two subgroups: normal salivary secretion (n = 18) and hyposalivation (NWS flow < 0.2 mL min-1; n = 12). Hyposalivation was observed in all patients with severe renal failure (4-5 stage CKD), while saliva secretion > 0.2 mL/min in children with mild-moderate CKD (1-3 stage) and controls. Salivary amylase activity and total protein content were significantly lower in CKD children with hyposalivation compared to CKD patients with normal saliva secretion and control group. The fluorescence of protein glycooxidation products (kynurenine, N-formylkynurenine, advanced glycation end products), the content of oxidative damage to lipids (4-hydroxynonneal, 8-isoprostanes) and nitrosative stress (peroxynitrite, nitrotyrosine) were significantly higher in NWS, SWS, and plasma of CKD children with hyposalivation compared to patients with normal salivary secretion and healthy controls. In CKD group, salivary oxidation products correlated negatively with salivary flow rate, -amylase activity and total protein content; however, salivary oxidation products do not reflect their plasma level. In conclusion, children with CKD suffer from salivary gland dysfunction. Oxidation of salivary proteins and lipids increases with CKD progression and deterioration of salivary gland function.

High-Fat Diet Affects Ceramide Content, Disturbs Mitochondrial Redox Balance, and Induces Apoptosis in the Submandibular Glands of Mice.

This is the first study to investigate the relationship between ceramides, the mitochondrial respiratory system, oxidative stress, inflammation, and apoptosis in the submandibular gland mitochondria of mice with insulin resistance (IR). The experiment was conducted on 20 male C57BL/6 mice divided into two equal groups: animals fed a high-fat diet (HFD; 60 kcal% fat) and animals fed a standard diet (10 kcal% fat). We have shown that feeding mice HFD induces systemic IR. We noticed that HFD feeding was accompanied by a significant increase in ceramide production (C18 1Cer, C18 Cer, C22 Cer, C24 1Cer, C24 Cer), higher activity of pro-oxidant enzymes (NADPH oxidase and xanthine oxidase), and weakened functioning of mitochondrial complexes in the submandibular glands of IR mice. In this group, we also observed a decrease in catalase and peroxidase activities, glutathione concentration, redox status, increased concentration of protein (advanced glycation end products, advanced oxidation protein products) and lipid (malondialdehyde, lipid hydroperoxide) peroxidation products, and enhanced production of tumor necrosis factor alpha (TNFα) and interleukin 2 (IL-2) as well as pro-apoptotic Bax in the submandibular gland mitochondria. In summary, HFD impairs salivary redox homeostasis and is responsible for enhanced oxidative damage and apoptosis in the submandibular gland mitochondria. The accumulation of some ceramides could boost free radical formation by affecting pro-oxidant enzymes and the mitochondrial respiratory chain.

Salivary FRAP as A Marker of Chronic Kidney Disease Progression in Children.

Chronic kidney disease (CKD) is one of the most common modern-age diseases in children. Kidney failure does not reveal any symptoms for a long time; therefore, new biomarkers are sought, preferably those reflecting an early stage of CKD. The aim of our study was to evaluate total antioxidant potential as a biomarker differentiating the degree of CKD advancement. The study included 30 children with CKD and a control group matched by age and gender. Non-stimulated saliva (NWS), stimulated saliva (SWS), plasma and urine were used as study material. Total antioxidant potential was determined spectrophotometrically using the FRAP method (ferric ion reducing antioxidant parameter) by measuring total FRAP and uric acid (UA)-independent FRAP (FRAP-UA). We demonstrated that total FRAP, FRAP-UA and UA were significantly higher in stimulated saliva, as well as urine of CKD patients compared to the controls. These biomarkers increase with the progression of chronic kidney disease and their concentration in SWS reflects their content in urine. Interestingly, salivary FRAP and uric acid clearly differentiate between various stages of CKD as well as between healthy and ill children. Special attention should be paid to total FRAP which-measured in SWS-distinguishes patients with mildly to moderately decreased kidney function from those with severe renal impairment (AUC = 1, sensitivity = 100%, specificity = 100%). Although salivary FRAP may be a potential CKD biomarker in children, further studies are needed in a larger group of patients.

Salivary Biomarkers of Oxidative Stress in Children with Chronic Kidney Disease.

There are still missing non-invasive biomarkers of chronic kidney disease (CKD) in children. Therefore, the aim of the study was to evaluate oxidative stress indicators in the non-stimulated (NWS) and stimulated saliva (SWS) of CKD children (n = 25) and healthy controls (n = 25). Salivary antioxidants (catalase (CAT), peroxidase (Px), superoxide dismutase (SOD), uric acid (UA), reduced glutathione (GSH), albumin), redox status (total antioxidant capacity (TAC), total oxidant status (TOS), oxidative stress index (OSI)), and oxidative damage products (advanced glycation end products (AGE), advanced oxidation protein products (AOPP), malondialdehyde (MDA)) were evaluated. We have demonstrated the significantly higher activity of SWS GPx and SOD, as well as elevated concentrations of UA and albumin in NWS and SWS of CKD children vs. the control group. TAC, TOS and OSI were significantly higher only in SWS, while oxidative damage products (AGE, AOPP and MDA) were significantly higher in both NWS and SWS of CKD children. ROC analysis showed a considerably high diagnostic value of AOPP in both NWS and SWS of CKD children compared to controls (AUC = 0.92; 0.98). CKD is responsible for disturbances in salivary antioxidant systems and oxidative damage to proteins and lipids. Salivary AOPP can be a potential biomarker of CKD in children.

Exposure to Ti4Al4V Titanium Alloy Leads to Redox Abnormalities, Oxidative Stress, and Oxidative Damage in Patients Treated for Mandible Fractures.

Due to the high biotolerance, favourable mechanical properties, and osseointegration ability, titanium is the basic biomaterial used in maxillofacial surgery. The passive layer of titanium dioxide on the surface of the implant effectively provides anticorrosive properties, but it can be damaged, resulting in the release of titanium ions to the surrounding tissues. The aim of our work was to evaluate the influence of Ti6Al4V titanium alloy on redox balance and oxidative damage in the periosteum surrounding the titanium miniplates and screws as well as in plasma and erythrocytes of patients with mandibular fractures. The study included 31 previously implanted patients (aged 21-29) treated for mandibular fractures and 31 healthy controls. We have demonstrated increased activity/concentration of antioxidants both in the mandibular periosteum and plasma/erythrocytes of patients with titanium mandibular fixations. However, increased concentrations of the products of oxidative protein and lipid modifications were only observed in the periosteum of the study group patients. The correlation between the products of oxidative modification of the mandible and antioxidants in plasma/erythrocytes suggests a relationship between the increase of oxidative damage at the implantation site and central redox disorders in patients with titanium miniplates and screws.

Lysosomal Exoglycosidase Profile and Secretory Function in the Salivary Glands of Rats with Streptozotocin-Induced Diabetes.

Before this study, there had been no research evaluating the relationship between a lysosomal exoglycosidase profile and secretory function in the salivary glands of rats with streptozotocin- (STZ-) induced type 1 diabetes. In our work, rats were divided into 4 groups of 8 animals each: control groups (C2, C4) and diabetic groups (STZ2, STZ4). The secretory function of salivary glands-nonstimulated and stimulated salivary flow, α-amylase, total protein-and salivary exoglycosidase activities-N-acetyl-ß-hexosaminidase (HEX, HEX A, and HEX B), ß-glucuronidase, α-fucosidase, ß-galactosidase, and α-mannosidase-was estimated both in the parotid and submandibular glands of STZ-diabetic and control rats. The study has demonstrated that the activity of most salivary exoglycosidases is significantly higher in the parotid and submandibular glands of STZ-diabetic rats as compared to the healthy controls and that it increases as the disease progresses. Reduced secretory function of diabetic salivary glands was also observed. A significant inverse correlation between HEX B, α-amylase activity, and stimulated salivary flow in diabetic parotid gland has also been shown. Summarizing, STZ-induced diabetes leads to a change in the lysosomal exoglycosidase profile and reduced function of the salivary glands.

Salivary: flow and proteins of the innate and adaptive immunity in the limited and diffused systemic sclerosis.

BACKGROUND: However, there are some informations on the salivary glands involvement in systemic sclerosis, and there is a lack of any data about salivary glands function depending on systemic sclerosis subsets. METHODS: The unstimulated and stimulated salivary flow, the activity of peroxidase, the total amount of lactoferrin, lysozyme and sIgA were determined in two subgroups of systemic sclerosis and healthy controls. RESULTS: In the unstimulated saliva of both patients groups, the salivary flow, the output of total protein and peroxidase activity were significantly lower; the total: sIgA and lactoferrin were significantly higher as compared with the control. In the stimulated saliva of the patients with limited form, the total lysozyme and peroxidase activity were significantly higher than in the control. In the stimulated saliva of the patients with diffused form, the salivary flow was significantly lower and the total sIgA and peroxidase activity were significantly higher than in the control. CONCLUSION: Systemic sclerosis regardless of its subset affects salivary defense system of human unstimulated and stimulated saliva. Patients with the limited form experience the same impairment of the submandibular glands function as compared with patients with the diffused form. Only patients with the diffused form are deficient in respect of stimulated saliva secretion; the parotid glands of the patients with the limited form have a good secretory capacity in comparison with the healthy control.

Salivary exoglycosidases in the detection of early onset of salivary gland involvement in rheumatoid arthritis.

INTRODUCTION: The aim of the present study was to evaluate the possibility of making use of the specific activity of N-acetyl-ß-hexosaminidase, its isoenzymes and ß-glucuronidase--potential indicators of salivary gland damage--in the detection of early onset of salivary gland impairment in RA, which is also demonstrated by xerostomia. MATERIAL/METHODS: For this purpose RA xerostomic salivary patients (unstimulated salivary flow >0.1 mL/min) were compared with RA xerostomic hyposalivary patients (unstimulated salivary flow ≤0.1 mL/min), RA patients without xerostomia (unstimulated salivary flow >0.1 mL/min) and generally healthy controls (unstimulated salivary flow >0.1 mL/min, without xerostomia). Salivary N-acetyl-ß-hexosaminidase, its isoenzymes A and B, and ß-glucuronidase specific activity were determined according to the Marciniak et al. method. The protein content in the unstimulated saliva was determined by the bicinchoninic acid method. RESULTS: In xerostomic rheumatoid arthritis patients, the specific activity of salivary ß-glucuronidase and isoenzyme A was significantly higher than in the healthy controls but the specific activity of salivary N-acetyl-ß-hexosaminidase, its isoenzyme B and ß-glucuronidase was significantly lower than in xerostomic hyposalivary rheumatoid arthritis patients. CONCLUSIONS: We suggest a simple, safe and cheap method for the determination of exoglycosidases as a useful tool for the diagnosis of early stages of salivary gland involvement in rheumatoid arthritis.