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Background and objectives ANCA-associated vasculitis is extremely rare in children. We report the clinicopathologic features, long-term outcomes, and prognostic factors of a large pediatric cohort of patients with ANCA-associated kidney vasculitis. Design, setting, participants, & measurements This retrospective study included 85 consecutive patients with kidney biopsy specimen–proven ANCA-associated vasculitis from tertiary referral centers in Italy and Canada. Kidney biopsy specimens were categorized as focal, crescentic, sclerotic, or mixed, according to the Berden classification. The prognostic significance of baseline clinical, laboratory, and histologic findings was analyzed with respect to kidney failure or CKD stage 3–5/kidney failure. Results A total of 53 patients had microscopic polyangiitis (62%), and 32 had granulomatosis with polyangiitis (38%). Rapidly progressive GN was the most frequent presentation (39%); a third of the patients also had nephrotic-range proteinuria. Kidney biopsy specimens were classified as focal in 21% of the patients, crescentic in 51%, sclerotic in 15%, and mixed in 13%. Remission-induction therapies included cyclophosphamide in 78% of patients. A total of 25 patients (29%) reached kidney failure. The median (interquartile range) time to kidney failure or last follow-up was 35 (6–89) months in the whole cohort, and 73 (24–109) months among the patients who did not reach this outcome. Patients whose biopsy specimens showed sclerotic histology had significantly shorter kidney survival (hazard ratio, 11.80; 95% confidence interval, 2.49 to 55.99) and survival free of CKD stage 3–5 (hazard ratio, 8.88; 95% confidence interval, 2.43 to 32.48), as compared with those with focal/mixed histology. Baseline eGFR, low serum albumin, hypertension, central nervous system complications, and sclerotic histology, which reflected severe kidney involvement, were associated with both kidney failure and CKD stage 3–5/kidney failure at unadjusted analysis; no independent prognostic factors emerged at multivariable analysis. Conclusions Children with ANCA-associated kidney vasculitis often have aggressive presentation; a third of such children progress to kidney failure and this usually occurs early during follow-up. A severe clinical presentation is associated with the development of CKD or kidney failure.

To assess the efficacy of the vascular hitch procedure in extrinsic ureteropelvic junction obstruction (UPJO) by crossing vessels in children, both by open and mini-invasive approach. Retrospective analysis on patients treated by open or mini-invasive vascular hitch during the period 2006–2020. Preoperative imaging included renal ultrasound (US) and functional studies by magnetic resonance or renogram. Crossing vessels obstruction and UPJ anatomy were evaluated at surgery. Success was defined as the resolution of symptoms and improvement of hydronephrosis at US or functional studies. Forty-eight patients were included with mean age at surgery of 7.6 years and 25% had an antenatal diagnosis. Aberrant vessels were suspected preoperatively in 72.9%; mean hydronephrosis and renal function were 30.9 mm and 38% respectively. Seven cases were treated by open approach, 37 by laparoscopy and 4 were robotassisted. Mean operative time was 58.3 min and mean hospital stay was 2 days. Mean follow-up was 16.9 months; success rate was 91.6% and 5 patients required secondary surgery. Vascular hitch shows to be a safe, feasible and effective alternative to pyeloplasty. Careful pre and intra-operative patients’ selection is essential to exclude any intrinsic cause of UPJO. Mini-invasive surgery could be helpful for better evaluation of UPJ anatomy and its relationships with crossing vessels. Complications and secondary surgeries could be related to incorrect patients’ selection.

Childhood-onset idiopathic nephrotic syndrome affects 1.15-3.4 children/100,000 children/year in Western Countries. Immune-mediated mechanisms, particularly T cell-mediated, are thought to play a key pathogenic role. The genetic basis of the disease is still poorly understood. We tested the association between single nucleotide polymorphisms (SNPs) of four genes encoding Fc gamma receptors (FCGR2A, FCGR2B, FCGR3A, FCGR3B) and idiopathic nephrotic syndrome in a case-control study of paediatric patients. Children with idiopathic nephrotic syndrome (aged 1-16 years) were included. FCGR2A rs1801274 and FCGR3A rs396991 SNPs were genotyped using real-time PCR with the TaqMan method, while FCGR2B rs1050501 and FCGR3B NA1/NA2 were genotyped using Sanger sequencing. Fisher's exact test was used to explore genetic association. We enrolled 103 idiopathic nephrotic syndrome patients and 181 healthy controls. A significant association was found between idiopathic nephrotic syndrome and FCGR2A rs1801274 SNP (both with the T allele and the TT genotype, p value=0.0009, OR 1.81, 95% CI 1.27-2.59 and p value=0.0007, OR 2.39, 95% CI 1.44-3.99, respectively). No associations were found for the remaining SNPs. Fc gamma receptors might modulate response to rituximab; since 60 of the enrolled patients were treated with rituximab, we also tested the association between the studied SNPs and rituximab efficacy in this patient subgroup, but found only a weak association with FCGR2A CC genotype (p value=0.03). The FCGR2A rs1801274 SNP in the gene encoding the activating receptor CD32A confers susceptibility to idiopathic nephrotic syndrome.

A 2-DE system has been devised in which proteins are first separated in their native state followed by separation according to mass under denaturing conditions (Nat/SDS-PAGE). Hydrophilic properties of the gel and the presence of dihydroxybisacrylamide in the first dimension allowed a good resolution for high-molecular-weight proteins and maintained interactions. With this method 252 plasma spots have been resolved and 140 have been characterized by MS as isoforms of 60 proteins, a relevant part of which (12) were not detected by traditional 2-D gels or by other nondenaturing 2-D techniques. The list includes complement factors (C4d, C7), coagulation factors (coagulation factor II, fibrin beta), apolipoproteins (apolipoprotein B) and cell debris (vinculin, gelsolin, tropomyosin, dystrobrevin beta, fibrinectin I). Nat/SDS PAGE also allowed separation of nicked forms of albumin, Apo B100 and alpha2-macroglobulin and showed the presence of atypical albumin adducts corresponding to post-translational and oxidation products. Our system provides therefore new tools for resolving proteins, protein aggregates and complexes and amplifies the potentiality of traditional electrophoretic analysis.

PubMedID: 23138488 Overexpression of soluble urokinase receptor (suPAR) causes pathology in animal models similar to primary FSGS, and one recent study demonstrated elevated levels of serum suPAR in patients with the disease. Here, we analyzed circulating suPAR levels in two cohorts of children and adults with biopsy-proven primary FSGS: 70 patients from the North America-based FSGS clinical trial (CT) and 94 patients from PodoNet, the Europe-based consortium studying steroid-resistant nephrotic syndrome. Circulating suPAR levels were elevated in 84.3% and 55.3% of patients with FSGS patients in the CT and PodoNet cohorts, respectively, compared with 6% of controls (P

In this review, we report the evolution on experimental conditions for the analysis of normal urine based on combinatorial peptide ligand library (CPLL) treatment and successive 2-DE and 2-DE/MS analysis. The main topics are (i) definition of the urine sample requirements, (ii) optimization of the urine/ligand ratio, (iii) essay conditions, (iv) en bloc elution. Overall, normal urine protein composition as studied by 2-DE includes over 2600 spots. Relevant data on inter and intraessay reproducibility obtained by the analysis of different normal urines repeated several times are also here presented. We found a 73% reproducibility upon analysis of the same sample and 68% correspondence of protein composition among different normal urine samples. Based on the above results, we are completing the characterization with LC-MS of 249 spots. The composition of normal urine proteins after CPLLs is finally shown with the indication of those spots which are currently under identification. This map will be completed in a near future; in the meantime this would represent the basic reference sample for newly developed studies on human diseases.

Primary membranous glomerulonephritis (MGN) is a major glomerular disease causing proteinuria in humans (Jones, 1957). It is the prototype of an autoimmune disease (Couser, et al., 1978) characterized by sub-epithelial immune deposits within glomeruli. Its pathogenesis remains still unknown. Immune deposits are formed by IgG4, their respective antigen and complement. The definition of the immune deposit architecture has been a main focus of the pathology research for years but advances were restricted, until recently, to animal models of the disease, in particular to Heymann nephritis (HN) (Heymann, et al., 1959; Heymann, et al., 1952; Van Damme, et al., 1978). Unfortunately, results from experimental HN cannot be readily exported to human MGN since the major antigen of immune deposits in rat is not present in human glomeruli. Therefore different podocyte antigens are involved in human MGN and their identification is a fundamental step in understanding human pathology. Technology problems, mainly concerning dissection of glomeruli and purification/characterization of glomerular antibodies from human biopsies, have limited the experimental approach in humans for years. In the last 5 years, human MGN has become the topic of renewed nephrologic research. Debiec et al. (Debiec, et al., 2002, 2004) first showed that neutral endopeptidase (NEP) emerges as podocyte antigen in a rare form of congenital MGN due to maternal NEP deficiency and alloimmunization during pregnancy. More recently, the existence of three new glomerular auto-antigens, i.e. phospholipase A2 receptor (PLA2R), aldose reductase (AR) and superoxide dismutase 2 (SOD2) have been proposed by independent groups (Beck, et al., 2009; Prunotto, et al., 2010). Technology evolution in the field of laser capture and proteomics allowed a direct experimental approach in humans, a crucial step for a direct analysis ‘in vivo’. It is now clear that IgG4 eluted from glomeruli of MGN patients recognize a panel of podocyte proteins

To identify gene loci associated with steroid-resistant nephrotic syndrome (SRNS), we utilized homozygosity mapping and exome sequencing in a consanguineous pedigree with three affected siblings. High-density genotyping identified three segments of homozygosity spanning 33.6Mb on chromosomes 5, 10, and 15 containing 296 candidate genes. Exome sequencing identified two homozygous missense variants within the chromosome 15 segment; an A159P substitution in myosin 1E (MYO1E), encoding a podocyte cytoskeletal protein; and an E181K substitution in nei endonuclease VIII-like 1 (NEIL1), encoding a base-excision DNA repair enzyme. Both variants disrupt highly conserved protein sequences and were absent in public databases, 247 healthy controls, and 286 patients with nephrotic syndrome. The MYO1E A159P variant is noteworthy, as it is expected to impair ligand binding and actin interaction in the MYO1E motor domain. The predicted loss of function is consistent with the previous demonstration that Myo1e inactivation produces nephrotic syndrome in mice. Screening 71 additional patients with SRNS, however, did not identify independent NEIL1 or MYO1E mutations, suggesting larger sequencing efforts are needed to uncover which mutation is responsible for the phenotype. Our findings demonstrate the utility of exome sequencing for rapidly identifying candidate genes for human SRNS.

Ciliopathies are an expanding group of rare conditions characterized by multiorgan involvement, that are caused by mutations in genes encoding for proteins of the primary cilium or its apparatus. Among these genes, CEP290 bears an intriguing allelic spectrum, being commonly mutated in Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), Senior-Loken syndrome and isolated Leber congenital amaurosis (LCA). Although these conditions are recessively inherited, in a subset of patients only one CEP290 mutation could be detected. To assess whether genomic rearrangements involving the CEP290 gene could represent a possible mutational mechanism in these cases, exon dosage analysis on genomic DNA was performed in two groups of CEP290 heterozygous patients, including five JSRD/ MKS cases and four LCA, respectively. In one JSRD patient, we identified a large heterozygous deletion encompassing CEP290 C -terminus that resulted in marked reduction of mRNA expression. No copy number alterations were identified in the remaining probands. The present work expands the CEP290 genotypic spectrum to include multiexon deletions. Although this mechanism does not appear to be frequent, screening for genomic rearrangements should be considered in patients in whom a single CEP290 mutated allele was identified.

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Side effects such as cutaneous vasculitis, which occur during prolonged levamisole treatment, may discourage the utilization of the drug in relapsing nephrotic syndrome. We describe a child who developed disseminated vasculitis during prolonged treatment with levamisole. The acute phase was characterized by hepatosplenomegaly, hemolytic anemia, IgM anticardiolipin and p-antineutrophil cytoplasmic antibodies. One month after withdrawal of therapy all symptoms had disappeared and tests normalized. This case report, together with other reports on cutaneous vasculitis, suggest caution and close monitoring during prolonged levamisole therapy.

Background IgA nephropathy (IgAN) or Berger's disease, is the most common glomerulonephritis in the world diagnosed in renal biopsied patients. The involvement of genetic factors in the pathogenesis of the IgAN is evidenced by ethnic and geographic variations in prevalence, familial clustering in isolated populations, familial aggregation and by the identification of a genetic linkage to locus IGAN1 mapped on 6q22–23. This study seems to imply a single major locus, but the hypothesis of multiple interacting loci or genetic heterogeneity cannot be ruled out. The organization of a multi-centre Biobank for the collection of biological samples and clinical data from IgAN patients and relatives is an important starting point for the identification of the disease susceptibility genes. Description The IgAN Consortium organized a Biobank, recruiting IgAN patients and relatives following a common protocol. A website was constructed to allow scientific information to be shared between partners and to divulge obtained data (URL: http://www.igan.net). The electronic database, the core of the website includes data concerning the subjects enrolled. A search page gives open access to the database and allows groups of patients to be selected according to their clinical characteristics. DNA samples of IgAN patients and relatives belonging to 72 multiplex extended pedigrees were collected. Moreover, 159 trios (sons/daughters affected and healthy parents), 1068 patients with biopsy-proven IgAN and 1040 healthy subjects were included in the IgAN Consortium Biobank. Some valuable and statistically productive genetic studies have been launched within the 5th Framework Programme 1998–2002 of the European project No. QLG1-2000-00464 and preliminary data have been published in "Technology Marketplace" website: http://www.cordis.lu/marketplace. Conclusion The first world IgAN Biobank with a readily accessible database has been constituted. The knowledge gained from the study of Mendelian diseases has shown that the genetic dissection of a complex trait is more powerful when combined linkage-based, association-based, and sequence-based approaches are performed. This Biobank continuously expanded contains a sample size of adequately matched IgAN patients and healthy subjects, extended multiplex pedigrees, parent-child trios, thus permitting the combined genetic approaches with collaborative studies.

With the aid of ultrathin-layer isoelectric focusing, rat serum albumin was studied in a group of 6 normal rats and 4 rats affected by an experimentally induced nephrotic syndrome. In normal rats the albumin was microheterogeneous with pI values ranging from 4.7 to 6.1. In animals with the nephrotic syndrome the albumin was almost homogeneous, consisting only of the isoform with the lowest pI of 4.7. The patterns were altered by performing IEF in low denaturing media in presence of 0.5–2 Murea. Under these conditions two rat serum albumin bands with pI's close to 6.1 were observed. Urinary albumin was highly microheterogeneous in nephrotic rats (pI 4.7–6.1) and less heterogeneous in the normal group(pI4.7). Pooled urinary albumin from nephrotic rats was fractionated by preparative IEF in granulated gels in four isoforms (A1–A4) with decreasing pI. Both for serum and urinary albumin the fatty acid content in normal and nephrotic syndrome animals was higher in isoforms with low pI.