Maria Szczepańska

Genetic screening in adolescents with steroid-resistant nephrotic syndrome

Genetic screening paradigms for congenital and infantile nephrotic syndrome are well established; however, screening in adolescents has received only minor attention. To help rectify this, we analyzed an unselected adolescent cohort of the international PodoNet registry to develop a rational screening approach based on 227 patients with nonsyndromic steroid-resistant nephrotic syndrome aged 10-20 years. Of these, 21% had a positive family history. Autosomal dominant cases were screened for WT1, TRPC6, ACTN4, and INF2 mutations. All other patients had the NPHS2 gene screened, and WT1 was tested in sporadic cases. In addition, 40 sporadic cases had the entire coding region of INF2 tested. Of the autosomal recessive and the sporadic cases, 13 and 6%, respectively, were found to have podocin-associated nephrotic syndrome, and 56% of them were compound heterozygous for the nonneutral p.R229Q polymorphism. Four percent of the sporadic and 10% of the autosomal dominant cases had a mutation in WT1. Pathogenic INF2 mutations were found in 20% of the dominant but none of the sporadic cases. In a large cohort of adolescents including both familial and sporadic disease, NPHS2 mutations explained about 7% and WT1 4% of cases, whereas INF2 proved relevant only in autosomal dominant familial disease. Thus, screening of the entire coding sequence of NPHS2 and exons 8-9 of WT1 appears to be the most rational and cost-effective screening approach in sporadic juvenile steroid-resistant nephrotic syndrome.

Skeletal Status in Children, Adolescents and Young Adults with End-Stage Renal Failure Treated with Hemo- or Peritoneal Dialysis

Abstract: The skeletal status in 30 children, adolescents and young adults (18 females, 12 males) with end-stage renal failure (ESRF) aged 9-23 years (mean 15.8 ± 3.6 years) was evaluated using measurements of bone mineral density (BMD, g/cm2) at the spine and total body (TB) (Lunar DPX-L, USA), quantitative ultrasound (QUS) of the hand phalanges (DBM Sonic 1200, IGEA, Italy) and laboratory investigations (parathyroid hormone, serum total and ionized calcium, serum phosphate). Eleven subjects were treated with hemodialysis and 19 with peritoneal dialysis. The mean value of the amplitude-dependent speed of sound (Ad-SoS, m/s) measured by QUS was significantly decreased in comparison with the value obtained in a group of 686 age-matched controls (1942 ± 74 m/s vs 2050 ± 77 m/s, p<0.0001). BMD measurements were also decreased in comparison with mean values for the healthy population (Z-scores for spine −1.47, and for TB −1.53). Duration of dialysis correlated significantly with spine-BMD, TB-BMD and Ad-SoS (r=−0.37, r = −0.45, r=−0.55, respectively, p<0.05), while duration of ESRF did not have such an influence. Laboratory investigations did not correlate with skeletal parameters. Ad-SoS correlated significantly with spine-BMD (r= 0.45, p<0.05) and TB-BMD (r= 0.56, p<0.01). Both QUS and BMD values correlated significantly with Tanner stages (r ranged from 0.59 to 0.69, p<0.001) and did not increase with age except for correlation between age and TB-BMD. In conclusion, skeletal status in the population studied is strongly affected by ESRF. Both QUS and BMD measurements show an ability to express skeletal changes in a similar manner, though the QUS parameter seems to be more sensitive at revealing changes due to renal failure.

Genotype-phenotype associations in WT1 glomerulopathy.

WT1 mutations cause a wide spectrum of renal and extrarenal manifestations. Here we evaluated disease prevalence, phenotype spectrum, and genotype-phenotype correlations of 61 patients with WT1-related steroid-resistant nephrotic syndrome relative to 700 WT1-negative patients, all with steroid-resistant nephrotic syndrome. WT1 patients more frequently presented with chronic kidney disease and hypertension at diagnosis and exhibited more rapid disease progression. Focal segmental glomerulosclerosis was equally prevalent in both cohorts, but diffuse mesangial sclerosis was largely specific for WT1 disease and was present in 34% of cases. Sex reversal and/or urogenital abnormalities (52%), Wilms tumor (38%), and gonadoblastoma (5%) were almost exclusive to WT1 disease. Missense substitutions affecting DNA-binding residues were associated with diffuse mesangial sclerosis (74%), early steroid-resistant nephrotic syndrome onset, and rapid progression to ESRD. Truncating mutations conferred the highest Wilms tumor risk (78%) but typically late-onset steroid-resistant nephrotic syndrome. Intronic (KTS) mutations were most likely to present as isolated steroid-resistant nephrotic syndrome (37%) with a median onset at an age of 4.5 years, focal segmental glomerulosclerosis on biopsy, and slow progression (median ESRD age 13.6 years). Thus, there is a wide range of expressivity, solid genotype-phenotype associations, and a high risk and significance of extrarenal complications in WT1-associated nephropathy. We suggest that all children with steroid-resistant nephrotic syndrome undergo WT1 gene screening.

Clinical InvestigationGenotype–phenotype associations in WT1 glomerulopathy

WT1 mutations cause a wide spectrum of renal and extrarenal manifestations. Here we evaluated disease prevalence, phenotype spectrum, and genotype-phenotype correlations of 61 patients with WT1-related steroid-resistant nephrotic syndrome relative to 700 WT1-negative patients, all with steroid-resistant nephrotic syndrome. WT1 patients more frequently presented with chronic kidney disease and hypertension at diagnosis and exhibited more rapid disease progression. Focal segmental glomerulosclerosis was equally prevalent in both cohorts, but diffuse mesangial sclerosis was largely specific for WT1 disease and was present in 34% of cases. Sex reversal and/or urogenital abnormalities (52%), Wilms tumor (38%), and gonadoblastoma (5%) were almost exclusive to WT1 disease. Missense substitutions affecting DNA-binding residues were associated with diffuse mesangial sclerosis (74%), early steroid-resistant nephrotic syndrome onset, and rapid progression to ESRD. Truncating mutations conferred the highest Wilms tumor risk (78%) but typically late-onset steroid-resistant nephrotic syndrome. Intronic (KTS) mutations were most likely to present as isolated steroid-resistant nephrotic syndrome (37%) with a median onset at an age of 4.5 years, focal segmental glomerulosclerosis on biopsy, and slow progression (median ESRD age 13.6 years). Thus, there is a wide range of expressivity, solid genotype-phenotype associations, and a high risk and significance of extrarenal complications in WT1-associated nephropathy. We suggest that all children with steroid-resistant nephrotic syndrome undergo WT1 gene screening.

Serum VCAM-1, ICAM-1, and L-selectin levels in children and young adults with chronic renal failure.

Children and young adults with chronic renal failure (CRF) present with an impaired immune response. Our aim was to analyze whether leukocyte migration, determined by adhesion molecules, is disturbed in the course of CRF, hemodialysis (HD), and peritoneal dialysis (PD). Soluble (s) VCAM-1, ICAM-1, and L-selectin serum levels were evaluated by ELISA in 15 patients with CRF, 22 patients on cuprophane membrane HD, 24 patients on PD, and in 15 controls. The sVCAM-1 levels in all groups were significantly elevated compared with controls. The levels in HD patients were higher than in CRF patients (P <0.05), while levels in PD patients were higher than in CRF and HD (P <0.001 and P <0.01, respectively). The sICAM-1 concentrations in CRF and PD patients were significantly elevated compared with controls (P <0.001 and P <0.0001, respectively); in PD patients sICAM-1 levels were higher than in HD patients (P <0.001), but there were no differences between other groups. sL-selectin levels were decreased in all groups compared with controls. The levels in HD patients were the lowest and the differences, compared with CRF and PD patients, were significant (P <0.05 and P <0.01, respectively). Children and young adults with CRF and on maintenance dialysis have altered concentrations of soluble adhesion molecules, resulting from either inadequate clearance or disturbed synthesis and release. The differences in sVCAM-1 levels between CRF and both groups of patients on dialysis, as well as the differences in sL-selectin concentrations between HD and CRF patients, indicate that these disturbances are aggravated by maintenance dialysis, particularly HD.

Skeletal status in adolescents with end-stage renal failure: a longitudinal study

In a longitudinal study, bone status was assessed in adolescents and young adults aged 15.3±3.4 years at the onset of the study with end-stage renal failure (ESRF). The group consisted of 18 subjects (11 females and seven males), of whom nine patients were on hemodialysis and nine patients on peritoneal dialysis. Six patients were previous or current glucocorticoid (GCS) users. Renal failure was recognized before 6.1±4.1 years, and dialysis was performed for 3.0±2.0 years. Follow-up took placed 8.6±0.8 and 21.7±2.5 months later, and the following data were collected: bone mineral density (BMD) at the spine (s-BMD) and total body (TB-BMD) using DPX-L (Lunar, USA); quantitative ultrasound by DBM 1200 (IGEA, Italy) at the hand phalanges (Amplitude-dependent Speed of Sound, Ad-SoS), serum concentration of i-PTH, total calcium, ionized calcium and phosphate. Tanner stages were also evaluated. The mean values of BMD measurements and Ad-SoS were stable during a period of observation, and a mean Z-score for TB-BMD was significantly lower at the third versus baseline value (−1.87±1.75 versus −1.49±1.53, P<0.05). Z-scores for s-BMD and Ad-SoS decreased non-significantly. Changes in s-BMD and TB-BMD Z-scores were influenced by changes in body size and changes in biochemical parameters, and a change in Ad-SoS Z-score was not dependent on these factors. The values of second (P<0.05) and third (P<0.01) s-BMD Z-score were significantly lower in GCS treated subjects, and longitudinal change in spine Z-score was greater in GCS treated patients versus others (P<0.05). Duration of ESRF, duration and type of dialysis and gender did not influence skeletal variables. Skeletal measurements correlated significantly with Tanner stages (besides the correlation with Ad-SoS in the first measurement, r ranged from 0.5 to 0.72, P<0.05), and changes in Tanner stages observed over a period of observation did not correlate with changes in skeletal variables. Among laboratory variables, the following non-significant tendencies to change were observed: serum concentration of i-PTH and phosphate increased, and total and ionized calcium decreased. In conclusion, adolescent subjects with ESRF treated with dialysis showed stable mean values of skeletal measurements, and these were expressed as Z-scores, a tendency to drop was observed. The lack of an increase observed in normal healthy subjects of the same age, and low values in Z-scores, indicates that skeletal status is seriously affected in subjects with ESRF.

Serum Concentration of IL-2, IL-6, TNF-Alpha and Their Soluble Receptors in Children on Maintenance Hemodialysis

In chronic renal failure patients a state of immunodeficiency paradoxically coexists with the activation of immune effector cells, including monocytes and lymphocytes. The activation of these cells leads to the release of cytokines. The aim of this study was to estimate the serum concentrations of IL-2, IL-6, TNF-α and their soluble receptors: IL-2 sRα, IL-6 sR, sTNF RI in children with chronic renal failure and young adults on maintenance hemodialysis (HD). The study included 16 HD patients (11 females, 5 males) aged 11–22 (mean 16.1 ± 3.1) years and a control group of 15 age-matched healthy children. Only the mean concentration of IL-6 was similar in HD patients and the control group. The levels of the other cytokines were significantly higher in patients undergoing HD compared to the healthy subjects. No significant differences were observed between the pre- and post-dialysis values or between the values obtained using various dialyzer membranes. These data suggest that immune cells in HD children are in an activated state and that neither a single dialysis session nor the type of dialyzer membrane has an influence on the cytokines examined.

Evaluation of Adipokines: Apelin, Visfatin, and Resistin in Children with Atopic Dermatitis

Very little is known about the role of adipokines in atopic dermatitis (AD) in children. This study aimed at analyzing the serum levels of resistin, apelin, and visfatin in children with AD in relation to body weight, AD severity, and gender. Serum concentration of adipokines was measured in 27 children with AD and in 46 healthy subjects. Selected biochemical parameters were evaluated and skin prick test was performed. Serum levels of resistin and apelin were significantly higher, whereas serum visfatin concentration was significantly lower in children with AD versus healthy controls, although an increase in resistin levels was exclusively demonstrated in boys. In AD group, a significant increase in apelin levels in girls was documented. There was no relationship between adipokines levels and the degree of allergic sensitization. Receiver operating characteristic curve analysis demonstrated that the serum apelin cutoff value differentiating children with AD from those without was >137.8 pg/mL. Resistin and visfatin cutoff values were >3.8 ng/mL and ≤ 2.13 ng/mL, respectively. Apelin and visfatin can serve as excellent indicators to distinguish children with AD from those without disease.

THE HEAT SHOCK PROTEIN PROFILE IN CHILDREN WITH CHRONIC KIDNEY DISEASE

♦ Objectives: Chronic kidney disease (CKD) due to inflammation, lipid disorders, and endothelial dysfunction predisposes to accelerated atherosclerosis. Elevated levels of heat shock proteins (HSPs) and antibodies against them have been described in adults with atherosclerotic lesions and cardiovascular events. However, there are no investigations of these variables in children with CKD treated conservatively or on peritoneal dialysis. Therefore, we decided to evaluate the profile of HSPs and their potential role as markers of atherosclerosis in these groups of patients. ♦ Methods: The study group consisted of 37 children with CKD treated conservatively and 19 children and young adults on automated peritoneal dialysis (APD). The control group comprised 15 age-matched subjects with normal kidney function. HSP-60, HSP-70, HSP-90alpha, anti-HSP-60, anti-HSP-70, sE-selectin, and interleukin (IL)-4 serum concentrations were assessed by ELISA; high-sensitivity C-reactive protein (hs-CRP) serum levels were assessed by nephelometry. Serum lipid profiles (total cholesterol, high density lipoprotein cholesterol, low density lipoprotein cholesterol, triglycerides) were also estimated. ♦ Results: HSP-90α, anti-HSP-60, and sE-selectin concentrations in the CKD and APD patients were higher than in the controls and were lower in the predialysis subjects than in the children on dialysis. Median values of anti-HSP-70 were higher in the CKD patients than in the control group. Levels of IL-4 were increased in all patients versus controls. Median values of HSP-60 were decreased in the CKD and APD children versus controls. HSP-70 and hs-CRP concentrations were comparable in all groups. ♦ Conclusions: The altered HSP and anti-HSP concentrations may imply that the response to stress conditions in the course of CKD is disturbed in children; APD does not aggravate that dysfunction in a significant way. Relationships between HSPs, lipid profile, and markers of inflammation suggest a possible role of the selected HSPs as markers of atherosclerosis in children with CKD.

Soluble adhesion molecules in children and young adults on chronic hemodialysis

Children on chronic hemodialysis (HD) present with impaired immunity that may result from disturbances in leukocyte migration, caused by changes in expression of adhesion molecules on endothelium and immunocompetent cells. However, it is still not clear whether the type of dialyzer or a single dialysis session influences the concentrations of soluble adhesion molecules in these patients. We evaluated by ELISA serum levels of soluble (s) VCAM-1, ICAM-1, L-selectin, and P-selectin in 22 patients on cuprophane HD (CU), 8 on polysulfone HD (PS), 10 on vitamin E-modified cellulose HD (VE), and 15 controls. In all HD patients, sVCAM-1 levels were elevated compared with controls and were higher in CU than in VE. The sICAM-1 concentrations were increased in VE compared with controls, but remained unchanged in CU and PS. The sL-selectin levels were reduced in all HD patients. The mean values of sP-selectin were comparable in CU, PS, and controls. The lowest levels were observed in VE. In CU patients, sVCAM-1, sICAM-1, and sP-selectin concentrations rose after HD. A single PS session had no impact on adhesion molecules, whereas a VE session increased the level of sVCAM-1. The type of dialysis membrane may change the profile of adhesion molecule concentrations, thus influencing the immune system of a child on HD. The increase in levels of adhesion molecules in the course of a single HD session, which was pronounced in CU and VE patients, suggests poor biocompatibility of these dialyzers.