Proteomic analysis of inhibitory protein profiles in the urine of children with nephrolithiasis: implication for disease prevention.

PURPOSE: In this study we aimed to screen for the presence of biomarkers that are downregulated in children with nephrolithiasis (RS) compared to healthy controls (HC) using a proteomic approach. We hypothesized that RS and HC would display unique inhibitory protein profiles that could be used for comparative pathway analysis. METHODS: This is a prospective, controlled, pilot study of pooled urine from RS (N = 30, 24 females, mean age 12.95 ± 4.03 years) versus age- and gender-matched HC, using liquid chromatography-mass spectrometry. The criteria for protein selection were: (1) patient/control abundance ratio of < 0.5; and (2) ≤ 0.05 p-value for the Fisher's Exact Test. Results were confirmed by ELISA testing in individual samples. RESULTS: 67 proteins were downregulated in RS group, and 17 of those were significantly different compared to controls. Of those seventeen proteins, five (two actins, annexin A5, keratin 6B, and serpin B4) were completely absent in the urine of stone patients but were found in controls. The remaining twelve proteins were significantly less abundant in the patient's urine compared to healthy controls. Protein-protein interaction modeling of significant proteins identified syndecan-1 as the key node, a protein associated with adhesion pathways. ELISA analysis by subgroups showed statistically significant difference in the urinary excretion of osteopontin (5.1 ± 3.22 ng/mg creatinine vs 14.1 ± 9.5 ng/mg creatinine, p = 0.046) between stone patients with hypocitraturia and controls. Urinary osteopontin concentration was positively correlated with urinary citrate excretion (r = 0.417, p = 0.03). CONCLUSIONS: Children with RS have a different urinary inhibitory polypeptide profile compared to HC. Decreased urinary excretion of these proteins indicates their potential inhibitory role in renal stone formation, especially of the adhesion phase. Lower concentration of urinary osteopontin in children with nephrolithiasis and hypocitraturia suggests its potential involvement in the pathogenesis of this disease. Further characterization of these proteins in a larger sample is imperative.

Diagnosis and Management of Nephrolithiasis in Children.

The incidence of kidney stones in children is increasing. Approximately two-thirds of pediatric cases have a predisposing cause. Children with recurrent kidney stones have an increased higher risk of developing chronic kidney. A complete metabolic workup should be performed. Ultrasound examination is the initial imaging modality recommended for all children with suspected nephrolithiasis. A general dietary recommendation includes high fluid consumption, dietary salt restriction, and increased intake of vegetables and fruits. Depending on size and location of the stone, surgical intervention may be necessary. Multidisciplinary management is key to successful treatment and prevention.

Cystatin C, Neutrophil Gelatinase-associated Lipocalin, and Lysozyme C: Urinary Biomarkers for Detection of Early Kidney Dysfunction in Children With Urolithiasis.

OBJECTIVE: To screen for the presence of biomarkers involved in tubular injury and kidney damage in children with urolithiasis (RS), and to validate these proteins by ELISA. METHODS: Prospective-controlled pilot study of children with urolithiasis and their age- and gender-matched controls (HC). Initial screening test was done by quantitative proteomic comparison of pooled urine from RS versus HC, using liquid chromatography-mass spectrometry. Proteins of interest were selected using the following criteria: (1) ≥5 spectral counts; (2) ≥2-fold difference in spectral counts; and (3) ≤.05 P value for the Fisher's Exact Test. Validation was performed by ELISA testing. Statistical analysis was performed by Student t-test and Mann-Whitney U test. RESULTS: Proteomic analysis identified 3 proteins of interest, Cystatin C (CYTC), neutrophil gelatinase-associated lipocalin (NGAL) and lysozyme C that were significantly over-represented in RS group versus HC. ELISA analysis revealed significantly increased urinary levels of CYTC and NGAL, and nearly significantly increased urinary levels of lysozyme C in RS group (N = 24) compared to controls (N = 13). Subgroup analysis showed significantly higher urinary levels of CYTC in both hypercalciuria (N = 14) and hypocitraturia (N = 10) versus HC (P <.05). CONCLUSION: Children with urolithiasis showed significant increase in urinary CYTC and NGAL irrespective of their normal serum creatinine. These biomarkers indicate tubular injury and early kidney damage and represent valid tools for early screening when traditional tests are normal.

Urine proteomic profiling in patients with nephrolithiasis and cystinuria.

PURPOSE: The purpose of the study was to assess the differences in the concentration and function of urinary proteins between patients with cystine stones (CYS) and healthy controls (HC). We postulated that CYS and HC groups would demonstrate different proteomic profiles. METHODS: A pilot study was performed comparing urinary proteomes of 10 patients with CYS and 10 age- and gender-matched HC, using liquid chromatography-mass spectrometry. Proteins which met the selection criteria (i) ≥ 2 unique peptide identifications; (ii) ≥ twofold difference in protein abundance; and (iii) ≤ 0.05 p value for the Fisher's Exact Test were analyzed using Gene Ontology classifications. RESULTS: Of the 2097 proteins identified by proteomic analysis, 398 proteins were significantly different between CYS and HC. Of those, 191 were involved in transport processes and 61 in inflammatory responses. The majority were vesicle-mediated transport proteins (78.5%), and 1/3 of them were down-regulated; of those, 12 proteins were involved in endosomal transport (including 6 charged multivesicular body proteins (CHMP) and 3 vacuolar sorting-associated proteins) and 9 in transmembrane transport. Myosin-2 and two actin-related proteins were significantly up-regulated in the vesicle-mediated transport group. CONCLUSION: We provide proteomic evidence of impaired endocytosis, dysregulation of actin and myosin cytoskeleton, and inflammation in CYS. Endosomal transport proteins were down-regulated mainly through defective CHMP. These findings may contribute to further understanding of the pathogenesis of CYS, potentially affecting its management.

24 Hour urine metabolic differences between solitary and multiple stone formers: Results of the Collaboration on Urolithiasis in Pediatrics (CUP) working group.

INTRODUCTION: Specific factors associated with the risk of developing pediatric urinary stone disease remain unclear, especially those that may be associated with recurrent stone disease. OBJECTIVE: We compared the results of 24-h urine collections in children with a solitary stone episode to those with multiple stone episodes to determine if there is a difference that may be associated with multiple stone formation in children. STUDY DESIGN: A multi-institutional retrospective analysis was completed to assess 24-h urinary metabolic profiles in children with urolithiasis aged 2-18 years old. Differences in mean urine collections between the two groups were assessed using chi-square tests to test the associations among gender, stone type, and multiple stone status, as well as multivariate analyses using general linear models. RESULTS: We analyzed 142 solitary stone patients and 136 multiple stone patients from four centers were included. Multiple stone patients were older than solitary stone patients (mean 13.4 ± 3.6 years vs. 12 ± 3.9 years, p = 0.002). Females were more likely to have multiple stones (58% vs. 39%, p = 0.002). BMI was not associated with multiple stones (p = 0.8467). Multiple stone formers had lower urine volumes, although this did not reach statistical significance when compared with solitary stone formation (20.4 mL/kg/day ± 11.5 vs. 22.9 ± 13.0, p = 0.0880). Higher values for super-saturation of calcium oxalate were associated with multiple stone disease in univariate (p = 0.0485) and multivariate analysis (p = 0.0469) (Figure). Centers located in the Southeast of the United States saw a higher proportion of children with multiple stones (Tennessee 62.7%, Virginia 44.4%, Oregon 31.6%, Michigan 27.3%, p < 0.0001). DISCUSSION: In a large multi-institutional retrospective analysis we found that multiple stone disease was associated with higher super-saturations of calcium oxalate. Many urinary parameters changed with patient age, highlighting that the values should be interpreted with respect to patient age. The inability to comment on follow-up because of the nature of our dataset is a limitation of this study. CONCLUSION: Multiple stone disease in children is associated with higher super-saturation calcium oxalate, while lower urinary volume may also be associated with multiple stones; however, further study is required. Early metabolic evaluation may help risk stratify children likely to form multiple stones.

Adenotonsillectomy Normalizes Hormones and Urinary Electrolytes in Children With Nocturnal Enuresis and Sleep-Disordered Breathing.

OBJECTIVE: To assess (1) plasma levels of antidiuretic hormone (ADH) and brain natriuretic peptide (BNP) and urinary levels of electrolytes in children with sleep disordered breathing (SDB), with or without nocturnal enuresis (NE), and (2) the effect of adenotonsillectomy (T&A) on urinary electrolytes and the secretion of ADH and BNP in children with NE and SDB. We previously reported post-T&A improvements in plasma levels of BNP and ADH in children with SDB and NE. However, the differences in plasma concentration of these hormones in SDB children with and without NE, and their relationships with urinary electrolytes, have not yet been addressed. METHODS: This prospective study compared concentrations of urinary electrolytes and plasma ADH and BNP in (1) children with SDB and NE (study group) and an age- and sex-matched control group of children with SDB without NE, and (2) the study group before and 1-month after T&A. RESULTS: Compared with the control group (n = 31), the study group (n = 37) exhibited significantly lower ADH (P = .04) and higher BNP (P = .009) plasma levels. The differences in urinary electrolytes were not significant. Post-T&A, the study group showed significantly decreased BNP (P = .018), urinary sodium-to-creatinine ratio (P = .02), and urinary calcium-to-creatinine ratio (P = .007) compared with the pre-T&A values. Post-T&A changes in urinary calcium were significantly correlated with changes in sodium excretion (P = .002) and in plasma levels of BNP (P <.001). CONCLUSION: The presence of NE is associated with altered ADH and BNP levels in children with SDB. T&A led to normalization of ADH and BNP, probably through a calcium- and sodium-dependent mechanism.

Adenotonsillectomy improves quality of life in children with sleep-disordered breathing regardless of nocturnal enuresis outcome.

BACKGROUND: Nocturnal enuresis (NE) and sleep-disordered breathing (SDB) have both been associated with impaired health-related quality of life (HRQoL). The following were investigated: (1) whether tonsillectomy and/or adenoidectomy (T&A) significantly affect the HRQoL in children with NE and SDB, and 2) differences in HRQoL between children with NE persistence versus resolution post-T&A. METHODS: This was a prospective study comparing the HRQoL of children with SDB and NE (study group) pre- and 4 weeks post-T&A, and the HRQoL of children with SDB without NE (control group) (independent t-tests). HRQol was assessed using the Obstructive Sleep Apnea Quality of Life 18 questionnaire (OSAS-18), a validated measure containing five subscales that combine to create a total score. Individual items were scored on a Likert-type scale ranging from 1 (none of the time) to 7 (all of the time). Symptoms of SDB were evaluated using the validated Pediatric Sleep Questionnaire (PSQ). Mixed ANOVA was conducted to evaluate changes in the measures between the wet and dry children post-T&A. Pre- and post-T&A change scores were calculated for both the PSQ and the OSAS-18. RESULTS: There were 30 children in the study group (18 male, mean age 9.07 years, SD 2.19), and 30 age-matched controls (16 male). There were no statistically significant differences between the two groups in regards to OSAS-18 total, PSQ total, BMI, diagnosis of snoring or OSAS on sleep study, or race. Overall, OSAS-18 and PSQ scores significantly improved in all children post-surgery (p < 0.001; p < 0.001, respectively), with no significant differences between dry and wet children post-T&A. The correlation between the pre- and post-T&A change scores on the OSAS-18 and PSQ was significant (r(29) = 0.58, p = 0.001), suggesting that a reduction in SDB symptoms post T&A is related to improved HRQoL. CONCLUSIONS: T&A significantly improved HRQoL in all children with SDB and NE, regardless of NE outcomes. These findings support recommendations for T&A in children with SDB with or without NE.

Urine proteomic analysis in cystinuric children with renal stones.

INTRODUCTION: The gene mutations responsible for cystinuria do not fully explain kidney stone activity, suggesting that specific proteins may serve as promoters of cystine precipitation, aggregation or epithelial adherence. In this study we assessed (1) the differences in the urinary proteins between children with cystinuria and kidney stones (CYS) and healthy controls (HC), with particular attention to the fibrosis-related proteins, and (2) the presence of diagnostic biomarkers for CYS. MATERIAL AND METHODS: We conducted a pilot study comparing individual urinary proteomes of 2 newly diagnosed children with CYS and 2 age- and gender-matched HC, using liquid chromatography-mass spectrometry. Relative protein abundance was estimated using spectral counting. Proteins of interest in both CYS and HC were selected using the following criteria: i) ≥5 spectral counts; ii) ≥2-fold difference in spectral counts; and iii) ≤0.05 p-value for the Fisher's Exact Test. DISCUSSION: This study demonstrates a different urinary polypeptide profile in two children with CYS compared to two HC. Of the 623 proteins identified by proteomic analysis, 180 exhibited at least a 2-fold increased relative abundance in CYS compared to HC. Of these, 39 were involved in response to stress, 26 in response to wounding, 21 in inflammatory response, 18 in immune response, and 4 in cellular response to oxidative stress. 133 proteins were found only in children with CYS, 33 of which met the selection criteria. Of these 33 unique proteins, six are known to be associated with fibrosis pathways (Table). The major limitation of this study is the small number of samples that were analyzed. Validation using highly specific methods such as ELISA is needed. CONCLUSION: We provide proteomic evidence of oxidative injury, inflammation, wound healing and fibrosis in two children with CYS. We speculate that oxidative stress and inflammation may cause remodeling via actin and vimentin pathways, leading to fibrosis. Additionally, we identified ITIH and MMP-9 as potential diagnostic biomarkers and novel therapeutic targets in CYS. These proteins merit further investigation.

Why does adenotonsillectomy not correct enuresis in all children with sleep disordered breathing?

PURPOSE: We analyzed the outcome of nocturnal enuresis after adenotonsillectomy in children with sleep disordered breathing. We also evaluated differences in demographic, clinical, laboratory and polysomnography parameters between responders and nonresponders after adenotonsillectomy. MATERIALS AND METHODS: We prospectively evaluated children 5 to 18 years old diagnosed with sleep disordered breathing (snoring or obstructive sleep apnea syndrome) on polysomnography and monosymptomatic primary nocturnal enuresis requiring adenotonsillectomy to release upper airway obstruction. Plasma antidiuretic hormone and brain natriuretic peptide were measured preoperatively and 1 month postoperatively. RESULTS: Sleep studies were done in 46 children and 32 also underwent blood testing preoperatively and postoperatively. Mean ± SD patient age was 8.79 ± 2.41 years and the mean number of wet nights weekly was 6.39 ± 1.26. Polysomnography revealed obstructive sleep apnea syndrome in 71.7% of patients and snoring in 28.3%. After adenotonsillectomy 43.5% of patients became dry. Preoperative polysomnography findings indicated that responders, who were dry, had significantly more arousals and obstructive apnea episodes but fewer awakenings than nonresponders, who were wet. Significant increases in plasma antidiuretic hormone and significant decreases in plasma brain natriuretic peptide were seen in all children with no difference between responders and nonresponders. No difference between the groups was noted in age, gender, race, body mass index, constipation, preoperative number of wet nights weekly or type of sleep disordered breathing. CONCLUSIONS: Nocturnal enuresis resolved after adenotonsillectomy in almost half of the children with sleep disordered breathing. Those who became dry had more frequent arousal episodes caused by apnea events than those who remained wet.

Sleep architecture parameters that predict postoperative resolution of nocturnal enuresis in children with obstructive sleep apnea.

OBJECTIVES: We performed a prospective cohort study in a pediatric tertiary care center to determine whether preoperative sleep architecture is associated with complete resolution of nocturnal enuresis (NE) after adenotonsillectomy. METHODS: Thirty-seven pediatric patients with primary NE who underwent adenotonsillectomy for obstructive sleep apnea (OSA) were evaluated. Preoperative polysomnograms, as well as preoperative and postoperative reports of NE, were recorded. We performed chi2 analysis, Fisher's exact test (for p values), and t-tests to evaluate the impact of multiple demographic characteristics on sleep architecture, comparing children with resolved NE to those with unresolved NE after adenotonsillectomy. RESULTS: The patients' mean age was 8.0 years (SD, 2.32 years). All children had presurgical primary NE. No age or gender differences were identified between children with resolved NE and those with unresolved NE. After surgery, more than half of the participants had resolution of NE. A higher percentage of boys had unresolved NE (chi2 = 3.63; p = 0.06). Improvement of NE was identified in children with a higher obstructive apnea-hypopnea index and more desaturation events. Eleven of the 12 children with prolonged stage 2 sleep reported resolution of NE (p = 0.001). Children with an obstructive apnea-hypopnea index of greater than 10 had a significantly greater rate of resolution of NE (p = 0.01). Logistic regression demonstrated that an elevated body mass index and the interaction of severe OSA and prolonged stage 2 sleep predicted resolution of NE. All 10 children with severe OSA and an abnormal total time spent in stage 2 sleep had resolution of NE. CONCLUSIONS: Adenotonsillectomy is a treatment option for children with OSA and NE. Postoperative resolution of NE was seen in 51.4% of patients who underwent adenotonsillectomy. The children with both severe OSA and prolonged stage 2 sleep were 3.4 times as likely to have postoperative resolution of NE. These results suggest that there are significant differences in preoperative sleep architecture between children whose NE resolves after adenotonsillectomy and those whose NE does not resolve.

Enuretic children with obstructive sleep apnea syndrome: should they see otolaryngology first?

OBJECTIVES: To study: (1) the prevalence of diurnal urinary incontinence (DI) and nocturnal enuresis (NE) in children with obstructive sleep apnea syndrome (OSAS) who underwent surgery for their upper airway symptoms, (2) the postoperative rate of enuresis resolution, and (3) factors that may predict lack of improvement post surgery. PATIENTS AND METHODS: An observational, pilot study of children 5-18 years of age with OSAS and NE who underwent tonsillectomy and/or adenoidectomy (T&A) between 2008 and 2010 was performed. Study consisted of a phone interview and chart review. Severity of NE and DI, frequency, arousal and sleeping disturbances were assessed pre and post T&A. Factors associated with failure to respond were analyzed using a logistic regression model. RESULTS: Among the 417 children who underwent T&A, 101 (24%) had NE (61 males, mean age 7.8 ± 2.5 years), and of these 24 had associated DI (6%). Mean postoperative follow-up was 11.7 months. Of the 49 whose NE responded to T&A (49%), 30 resolved within 1 month postoperatively. DI resolved in 4 children (17%). There was a statistically significant difference between responders and non-responders regarding the presence of prematurity, obesity, family history of NE, type of enuresis, enuresis severity, and ability to be easily aroused. CONCLUSION: NE was present in about one fourth of children with OSAS undergoing surgery, and resolved in about half. Lower response rate was associated with prematurity, obesity, family history of NE, presence of non-monosymptomatic NE, severe NE preoperatively, and arousal difficulties.

Effect of grape seed extract on puromycin-aminonucleoside-induced nephrosis in rats.

Recent studies indicate that an excessive production of oxidants plays an important role in the pathogenesis of glomerular disease. Grape seed extract (GSE) is a potent antioxidant, and the aim of this pilot study was to evaluate its effect on puromycin-aminonucleoside (PAN)-induced nephrosis in rats. Fifty Sprague-Dawley rats were divided into five groups. Groups 1 and 2 rats received water from day 0 to day 30. Rats in groups 3, 4, and 5 received GSE at 10 mg/100g of body weight (BW), which was started on day 0, 6, and 3 of the experiment, respectively. In group 5 animals the GSE dose was increased (40 mg/100g BW) on day 9. Intraperitoneal dextrose (group 1) or PAN 15 mg/100g BW (groups 2-5) was administered on day 3. Urine and blood specimens were collected at regular intervals, and the comparison between the various groups was made by analysis of variance (ANOVA). Rats in all study groups (groups 3-5) showed a decrease in urine protein and serum cholesterol and triglyceride levels, which was statistically significant in group 3 animals. No significant changes were noted in serum albumin and creatinine levels. In conclusion, GSE administration decreases urine protein excretion and serum cholesterol and triglyceride levels in rats with PAN-induced nephrosis.

Renal papillary necrosis induced by naproxen.

A 17-year-old healthy girl was admitted to our hospital with diffuse abdominal pain and decreased oral intake of about 11 days duration. About a week prior to admission, she had taken naproxen, 250 mg four times a day for 4 days. Physical examination was normal except for diffuse abdominal tenderness on deep palpation. Investigations revealed high serum BUN (42 mg/dl) and creatinine (4.0 mg/dl). Serum electrolytes and complement (C3, C4) levels and urinalysis were normal. Antinuclear-antibody and anti-dsDNA were negative. Kidney biopsy revealed renal papillary necrosis, acute tubular necrosis, and focal interstitial nephritis. A diagnosis of nonoliguric acute renal failure due to naproxen nephrotoxicity was made. She received intravenous hydration, and oral steroids, which was gradually discontinued in 3 months. A follow-up at 4 months revealed normal renal function with a serum creatinine of 1.1 mg/dl, BUN 7 mg/dl, and normal urinalysis. The report highlights a need for caution while using naproxen or any other nonsteroidal anti-inflammatory drugs, even for a short duration.