Neutrophil extracellular traps in urinary tract infection.

Background: Urinary tract infections (UTI) are common types of bacterial infection in children. UTI treatment is aimed to prevent complications including hypertension, proteinuria, and progression to chronic kidney disease. Activated neutrophils release chromatin-based structures associated with antimicrobial proteins called neutrophil extracellular traps (NETs). We aimed to describe the role of NET-associated markers in children with UTI as well as the role of NETs formation in a mouse model of UTI. Materials and methods: Markers of NETs including extracellular DNA (ecDNA), myeloperoxidase (MPO) and cathelicidin were analyzed in children with febrile UTI caused by E. coli (n = 98, aged 0.3-1.3 years) and in healthy controls (n = 50, 0.5-5.2 years). Moreover, an acute experimental model of UTI was performed on PAD4 knock-out mice with diminished NETs formation (n = 18), and on wild-type mice (n = 15). Results: Children with UTI had significantly higher urinary NETs markers including total ecDNA, nuclear DNA and mitochondrial DNA, altogether with MPO and cathelicidin. The concentrations of MPO and cathelicidin positively correlated with ecDNA (r = 0.53, p ≤ 0.001; r = 0.56, p ≤ 0.001, respectively) and the number of leukocytes in the urine (r = 0.29, p ≤ 0.05; r = 0.27, p ≤ 0.05, respectively). Moreover, urinary MPO was positively associated with cathelicidin (r = 0.61, p ≤ 0.001). In the experimental model, bacterial load in the bladder (20-fold) and kidneys (300-fold) was significantly higher in PAD4 knock-out mice than in wild-type mice. Conclusion: Higher urinary NETs makers-ecDNA, MPO and cathelicidin and their correlation with leukocyturia in children with UTI confirmed our hypothesis about the association between NETs and UTI in children. Higher bacterial load in mice with diminished NETs formation suggests that NETs are not only a simple consequence of UTI, but might play a direct role in the prevention of pyelonephritis and other UTI complications.

De novo weekly and biweekly darbepoetin alfa dosing in pediatric patients with chronic kidney disease.

BACKGROUND: Darbepoetin alfa is a commonly prescribed erythropoiesis-stimulating agent (ESA) for correcting anemia in pediatric chronic kidney disease (CKD) patients. However, little information exists on its use in ESA-naïve patients. This study evaluated the efficacy and safety of darbepoetin alfa in pediatric patients initiating ESA therapy. METHODS: One-hundred sixteen pediatric ESA-naïve subjects (aged 1-18 years) with CKD stages 3-5D and hemoglobin (Hb) <10 g/dl from 43 centers in the US, Europe, and Mexico were randomized by age (three groups) and dialysis status (yes vs. no) to receive darbepoetin alfa once weekly (QW) or every 2 weeks (Q2W) subcutaneously (not on dialysis and peritoneal dialysis subjects) and intravenously (hemodialysis subjects). The drug was titrated to achieve Hb levels of 10.0-12.0 g/dl over 25 weeks. Patient- and parent-reported health-related outcomes were measured by the Pediatric Quality of Life Inventory (PedsQL™) in children ≥2 years. RESULTS: In both groups, mean Hb concentrations increased to ≥11.0 g/dl over the first 3 months of treatment and remained stable within the 10.0-12.0 g/dl target range. The median time to achieve hemoglobin ≥10 g/dl was slightly longer for subjects <12 years (QW and Q2W, both 28 days) vs. those ≥12 years (23 and 22 days, respectively). Adverse event profiles were similar between groups, with QW, four (7%) and Q2W, five (9%). PedsQL™ scores showed modest increases. CONCLUSIONS: Darbepoetin alfa can be safely administered either QW or Q2W to ESA-naïve pediatric patients with CKD-related anemia to achieve Hb targets of 10.0-12.0 g/dl.

KANK deficiency leads to podocyte dysfunction and nephrotic syndrome.

Steroid-resistant nephrotic syndrome (SRNS) is a frequent cause of progressive renal function decline and affects millions of people. In a recent study, 30% of SRNS cases evaluated were the result of monogenic mutations in 1 of 27 different genes. Here, using homozygosity mapping and whole-exome sequencing, we identified recessive mutations in kidney ankyrin repeat-containing protein 1 (KANK1), KANK2, and KANK4 in individuals with nephrotic syndrome. In an independent functional genetic screen of Drosophila cardiac nephrocytes, which are equivalents of mammalian podocytes, we determined that the Drosophila KANK homolog (dKank) is essential for nephrocyte function. RNAi-mediated knockdown of dKank in nephrocytes disrupted slit diaphragm filtration structures and lacuna channel structures. In rats, KANK1, KANK2, and KANK4 all localized to podocytes in glomeruli, and KANK1 partially colocalized with synaptopodin. Knockdown of kank2 in zebrafish recapitulated a nephrotic syndrome phenotype, resulting in proteinuria and podocyte foot process effacement. In rat glomeruli and cultured human podocytes, KANK2 interacted with ARHGDIA, a known regulator of RHO GTPases in podocytes that is dysfunctional in some types of nephrotic syndrome. Knockdown of KANK2 in cultured podocytes increased active GTP-bound RHOA and decreased migration. Together, these data suggest that KANK family genes play evolutionarily conserved roles in podocyte function, likely through regulating RHO GTPase signaling.

Prevalence and predictors of the sub-target Hb level in children on dialysis.

BACKGROUND: Anaemia is a common and potentially treatable co-morbidity of end-stage renal disease. We aimed to determine the prevalence of the sub-target haemoglobin (Hb) level among European children on dialysis and to identify factors associated with a low Hb level. METHODS: From the European Society for Paediatric Nephrology (ESPN)/European Renal Association-European Dialysis Transplant Association (ERA-EDTA) registry, data were available on 2351 children between 1 month and 18 years of age, totalling 5546 measurements from 19 countries. RESULTS: The mean Hb level was 10.8 g/dL (5th-95th percentiles, 7.4-13.9). Among those above 2 years of age, the mean Hb level was 10.9 g/dL (11.4% below 8.5 g/dL), while it was 10.3 g/dL among those below 2 years (11.2% below 8.0 g/dL). A total of 91.2% of the patients were on an erythropoiesis-stimulating agent (ESA). Hb levels increased with age and were higher in peritoneal dialysis compared with haemodialysis patients. Patients with congenital anomalies of the kidney and urinary tract showed the highest Hb levels, and those with cystic kidney diseases or metabolic disorders the lowest ones. Ferritin levels between 25 and 50 ng/mL were associated with the highest Hb levels. We found a weak inverse association between parathyroid hormone (PTH) and Hb. Whereas standardized blood pressure (BP) was not elevated in patients with above-target Hb, elevated systolic BP z-score was noted in those with sub-target Hb levels. CONCLUSIONS: Sub-target Hb levels remain common in children on dialysis, in spite of virtually all children being treated with ESA; although we cannot exclude under-dosing. Optimal ferritin levels seemed to be slightly lower in children (25-50 ng/mL) than those in adults. Other risk factors for sub-target Hb are dialysis modality and a high PTH level.

Methylenetetrahydrofolate reductase gene polymorphisms and neural tube defects epidemiology in the Slovak population.

BACKGROUND: Folate deficiency is a known factor contributing to the formation of neural tube defects (NTDs). Many folate metabolism gene variants have been investigated, but only a few substantial associations have been established, the C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR) gene being one of the most significant. METHODS: We determine the MTHFR C677T and A1298C genotypes in 93 Slovak NTD patients and 290 control newborns with respect to sex and ethnicity. Furthermore, we summarize current data on the incidence and types of NTDs in Slovakia. RESULTS: The Slovak population frequencies of T allele and TT genotype of the C677T MTHFR gene polymorphism were 0.25 and 6.9%, respectively; similarly, those of the C allele and CC genotype of the A1298C polymorphism were 0.35 and 13.8%, respectively. No differences between the sexes and within ethnic groups were observed. In NTD patients, genotype analysis of the C677T polymorphism revealed 0.29 and 9.8% for T allele and TT genotype frequencies, respectively (p = 0.26; OR, 1.23; 95% CI, 0.84-1.81; resp. p = 0.36; OR, 1.46; 95% CI, 0.56-3.52) compared to the controls. The frequencies of C allele and CC genotype of A1298C polymorphism were 0.34 and 6.5%, respectively (p = 0.81; OR, 0.96; 95% CI, 0.66-1.38; resp. p = 0.06; OR, 0.44; 95% CI, 0.15-1.09). There were also no sex-related differences in genotypes distribution in NTD patients. CONCLUSIONS: No significant associations between the C677T and A1298C MTHFR gene polymorphisms and NTDs and no differences between the two main ethnic groups (white-Caucasians, Roma) were found in Slovakia. The total incidence of NTDs in Slovakia is, according to the official sources, 0.53/1000, and the incidence among liveborn newborns is 0.28/1000.

A new link between steroid resistance, glucocorticoid receptor and nuclear factor kappa B p65 in idiopathic nephrotic syndrome.

OBJECTIVES: The aim of our study was to investigate levels of glucocorticoid receptor (GRalpha), nuclear factor kappa B (NFkappaB) p65/p50 and inhibitor of NFkappaB alpha (IkappaBalpha) in the peripheral mononuclear blood cells (PMBC) of children with idiopathic nephrotic syndrome (INS). METHODS: The expression of GRalpha, NFkappaBp65/p50 and IkappaBalpha was determined in 59 patients (age 10.2+/-5.1) and 25 healthy controls (CO) (age 13.1+/-3.4) using Western blot analysis. Patients were labeled according to their clinical sensitivity to glucocorticoids (GCs) as responders (RE), partial responders (PR), and non-responders (NR). RESULTS: Significantly higher expressions of GRalpha were observed in RE than in PR, NR (p<0.01) and even CO (p<0.05). Similarly, expression of NFkappaBp65 was higher in RE in comparison to PR, NR and CO (p<0.05). These differences were more emphasized in the relapse: levels of GRalpha were significantly lower in PR than in RE and CO (p<0.01). Significant differences were also observed in expression of NFkappaB: RE showed significantly higher expression of NFkappaBp65 in comparison to PR, NR and even CO (p<0.01). CONCLUSIONS: Lower levels of both GRalpha and NFkappaBp65 are associated with poor or no response to GCs and the difference is more pronounced in patients experiencing relapse of INS.

The antimicrobial peptide cathelicidin protects the urinary tract against invasive bacterial infection.

The urinary tract functions in close proximity to the outside environment, yet must remain free of microbial colonization to avoid disease. The mechanisms for establishing an antimicrobial barrier in this area are not completely understood. Here, we describe the production and function of the cathelicidin antimicrobial peptides LL-37, its precursor hCAP-18 and its ortholog CRAMP in epithelial cells of human and mouse urinary tract, respectively. Bacterial contact with epithelial cells resulted in rapid production and secretion of the respective peptides, and in humans LL-37/hCAP-18 was released into urine. Epithelium-derived cathelicidin substantially contributed to the protection of the urinary tract against infection, as shown using CRAMP-deficient and neutrophil-depleted mice. In addition, clinical E. coli strains that were more resistant to LL-37 caused more severe urinary tract infections than did susceptible strains. Thus, cathelicidin seems to be a key factor in mucosal immunity of the urinary tract.

Intra-individual variation of cystatin C and creatinine in pediatric solid organ transplant recipients.

There is controversy about the feasibility of cystatin C (CysC) as a marker of glomerular filtration rate (GFR) post-transplant (Tx). We studied intra-patient variability of CysC in comparison with serum creatinine (SCr) in 20 children (11 males, mean age 11.5 +/- 6.4 yr) with solid organ transplants (14 kidney, four liver, and two combined liver + kidney transplants). The mean age at Tx was 7.0 +/- 5.6 yr. A total of 178 simultaneous SCr and CysC measurements (median 8 per patient) were analyzed. In addition, GFR was calculated using the Schwartz and a novel CysC-based formula. Intra-individual coefficient of variations (CV) was calculated as ratio of standard deviation over mean. The mean CV was significantly lower for SCr (7.71 +/- 4.16%) when compared with CysC (10.27 +/- 4.87, p = 0.04), but was no longer significantly different when excluding patients with a bladder augment. The CV of the GFR estimated by Schwartz formula (7.44 +/- 3.77) was significantly lower than GFR calculated from CysC (12.52 +/- 7.37), p = 0.001. The mean ratio between the Schwartz GFR and the GFR calculated from CysC was 102.6 +/- 12.8%, not significantly different from 100% (p = 0.3796). The only potential confounding factors to explain increased CV after Tx were gender and bladder augmentation, whereas calcineurin inhibitors or steroids did not influence CV. With the limitation of a small number of subjects, our data suggest that the CysC and the CysC-calculated GFR is equivalent but not better than SCr and Schwartz formula. We therefore conclude that measurement of CysC can be used for longitudinal intra-individual follow-up of renal function post-Tx.