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From June 1985 to December 1998, 173 pediatric renal transplants were carried out in 170 patients at our center. From this pool, 73 patients (34 males and 39 females) with a follow‐up of 48 months were examined. In all patients, ureteroneocystostomy was performed according to the Lich‐Grégoire procedure. All patients were treated with cyclosporin A (CsA)‐based immunosuppression, including prednisone and sometimes azathioprine (AZA). Six months after transplantation, voiding cystography (VCU) was performed in all patients and reflux was classified from Grade I to Grade IV. The patients were divided into two groups: those with reflux (Group A: 25 patients) and those without (Group B: 48 patients). Grade I reflux was found in four patients, Grade II in seven patients, Grade III in seven patients, and Grade IV in seven patients. All the patients with severe reflux (Grade IV) underwent a corrective surgical procedure. Both groups were examined for immunologic and non‐immunologic risk factors and no significant differences were found. Analysis of patient and graft survival rates revealed no statistical differences (NS) between Groups A and B. Mean serum creatinine (mg/dL) was 1.06 ± 0.28 and 1.12 ± 0.41 at 4 yr in Groups A and B, respectively (NS). Mean calculated creatinine clearance (cCrC; ml/min) was 76.74 ± 15.92 and 77.96 ± 15.66 in Groups A and B, respectively (NS). The analysis was further extended by considering the grade of reflux (I to IV). Again, no significant differences in the above parameters emerged between the reflux sub‐groups; only in the Grade IV sub‐group was a slight decrease in cCrC detected, although this difference was not statistically significant when compared with the other sub‐groups. In conclusion, vesico‐ureteral reflux (VUR) does not seem to negatively affect graft function. However, as all severe reflux patients (Grade IV) were surgically corrected, no... [ABSTRACT FROM AUTHOR]

Background.The nephronophthisis-medullary cystic disease (NPH/MCD) complex represents a heterogeneous group of hereditary tubulointerstitial nephritis. The most common variant is juvenile recessive NPH, for which a gene locus (NPH1) has been mapped on chromosome 2q13. MCD is a less common dominant condition usually recognized later in life, which resembles NPH in many aspects, still presenting remarkable clinical differences. Nothing is known about the chromosome locus of MCD. [ABSTRACT FROM PUBLISHER]

Peritoneal fibrosis in patients on peritoneal dialysis is the result of interstitial collagen accumulation within the peritoneal membrane and in mural spaces. Hypothetically, collagen expression by target cells may be regulated by specific endoperitoneal factors, though the existence of such factors has not yet been demonstrated.We evaluated the effects of cell-free peritoneal effluents obtained from six children undergoing peritoneal dialysis on several mesothelial cell functions in vitro. Human peritoneal mesothelial cells (MC) were obtained from the omental tissue of six uraemic children who were undergoing surgery for insertion of a peritoneal catheter. Cells at confluence were utilized to determine cytotoxicity (LDH release), viability (trypan blue), proliferation (3H-thymidine incorporation), collagen expression (3H-proline incorporation, SDS-Page) and mRNA (dot-blot). A preliminary series of experiments, was undertaken to define which of the successive fluid collections during a dialytic procedure induces the greatest changes; this revealed maximal effects of the effluent from the long stasis period. Exposure to peritoneal effluents obtained from four patients with acute peritonitis induced marked changes in cell morphology, stimulated by (3H)-thymi-dine incorporation into DNA by 300% and upregulated the expression and transcription of type III collagen (6-fold increment in COL3Al mRNA). Qualitatively but not quantitatively comparable changes in cell proliferation (+100%) and collagen expression were induced by peritoneal effluents from patients without peritonitis. In an effort to reproduce the effect of peritoneal effluents in vitro, we exposed mesothelial cells to various cytokines putatively present in infected peritoneal effluents, namely IL-2, TGFβ and TNFα; in no case did we find stimulation of cell proliferation. Finally TGFβ but not TNFα or IL2 upregulated collagen synthesis by these cells.These findings demonstrate a direct influence of cell-free peritoneal effluents on mesothelial cell functions, including stimulation of interstitial collagen expression.All these changes were more evident upon exposure to effluents collected during acute peritonitis, which suggests a link between recurrent peritoneal infection and collagen deposition, the most typical precursor of peritoneal fibrosis. [ABSTRACT FROM PUBLISHER]

Nutritional status, assessed by anthropometric and biochemical methods, protein and amino-acid (AA) composition and muscle water were evaluated in 11 kidney transplanted children and in a control group of 10 children with normal renal function who were undergoing elective surgery. Samples of the rectus abdominis muscle were taken when surgery was performed in the control children and when the peritoneal catheter was removed in the transplanted children. The mean time from the transplantation to the study time was 97 ± 14 days (range 72–114 days). Height was reduced in the transplanted children compared to the controls but skinfold thickness, arm muscle circumference and serum proteins (total protein, albumin, transferrin, pseudocholinesterase) were normal. The body mass index was over the 50° percent ile in nine of the eleven children. The muscle contents of total, extracellular, and intracellular water, alkali-soluble protein (ASP), DNA and the ASP/DNA ratio were not significantly different in transplanted children from those in the controls. Plasma leucine and taunine levels were significantly decreased, whereas plasma citrulline and alanine levels and the glycine/serine ratio were increased. Muscle threonine, alanine+taurine, glycine and aspartic acid levels as well as the glycine/senine ratio were increased in the transplanted children. Transplanted children show an almost normal muscle AA profile and a plasma AA pattern that seems to be related to the prednisone therapy. [ABSTRACT FROM PUBLISHER]

Mesothelial cells (MC) from human peritoneal omentum fragments obtained during surgical insertion of peritotical catheters for continuous peritoneal dialysis in end stage renal failure (ESRF) patients were cultured in vitro. MC exhibited a phenotype different from macrophages, but MHC class II molecules were well expressed. Therefore MC lines were tested for antigen presenting capacity by pulsing with soluble antigens (tetanus toxoid and purified protein derivative (PPD)) or with a corpusculate antigen (Candida albicans bodies). Autologous peripheral blood mononuclear cells (PBMC) depleted of adherent monocytes and cloned T cells generated from an individual matched for the MHC class II antigen DR2 were used to test antigen-presenting function. MC effectively presented the soluble and corpusculate antigens to autologous and MHC-compatible allogeneic lymphocytes, indicating that they are endowed with both endocytic/phagocytic activity and with processing/presenting capacity. Preincubation of MC with human recombinant interferon-gamma (IFN-γ) up-regulated MHC class II and intercellular adhesion molecuIe-1 (lCAM-1) expression, but the effect on antigen-presenting function was not consistent. Since MC are an important component of the peritoneal environment, they may participate, along with macrophages. in activation of specific T cells and in the generation of local cell-mediated immunity to various pathogens. [ABSTRACT FROM AUTHOR]

Alfa-1-antitrypsin (alpha 1AT) was purified by pseudoligand chromatography and preparative electrophoresis from the serum of a patient with alpha 1AT deficiency. The combination of the two techniques yielded a high grade batch of alpha 1AT monomer and this was successfully used to purify the protein from the serum of PiMIM1, PiMIM2, and PiZZ phenotype subjects. This procedure should facilitate structural studies of alpha 1AT variants susceptible to intracellular accumulation. [ABSTRACT FROM PUBLISHER]

Presents abstracts on two studies involving kidney diseases. Use of apolipoproteins to prevent glomelular albumin permeability alterations induced by serum from patients with chronic renal failure caused by focal segmental glumerulosclerosis; Description of the function of the most commen congenital nephrotic syndrome gene mutations.

Apolipoprotein A1 (apoA1) is a component of the high density lipoproteins (HDL) that regulates the transport of cholesterol between the liver and peripheral cells and modulates the removal of any excess of cholesterol from membranes. Any variation in apoA1 composition may modify the plasma lipid profile and be involved in atherogenesis. We investigated apoA1 composition in plasma of 6 children with nephrotic syndrome, a condition characterized by high levels of cholesterol in plasma and by a high risk to develop atherosclerosis. Non-denaturing two-dimensional electrophoresis (Nat/SDS-PAGE), mass spectrometry, western blot and pull down experiments were done to characterize proteins and define putative interactions. ApoA1 was resolved in 12 variants, 6 of which had a slightly lower molecular weight (18-19 KDa) and migrated on the same axes of the β chain of haptoglobin (Hp). Low molecular weight apoA1 were observed in carriers of different Hp haplotypes (including one homozygous for the rare ββα1α1) ruling out any contaminant effect of co-migration of apoA1 with Hp α2 chain. Overall, apoA1 isoforms were much more present in plasma of nephrotic patients compared to a normal profile. These findings show that apoA1 plasma in nephrotic syndrome is heterogeneous in terms of molecular weight. Low molecular weight fragments lack internal structural domains and likely form macro-aggregates with Hp. Fragmentation and transport of apoA1 may be involved in the general disorder of lipid metabolism that characterizes nephrotic syndrome.

Unlabelled: Acute stress responses of women are typically more reactive than that of men. Women, compared to men, may be more vulnerable to posttraumatic stress disorder (PTSD). Whether there are differences between women and men with PTSD in levels of the stress hormone, cortisol, was investigated in a pilot study., Methods: women (n=6) and men (n=3) motor vehicle accident (MVA) survivors, with PTSD, had saliva collected at 1400 h, 1800 h, and 2200 h. Cortisol levels in saliva were measured by radioimmunoassay. An interaction between gender and time of sample collection was observed due to women's cortisol levels being lower and decreasing over time, whereas men's levels were higher and increased across time of day of collection. Results of this pilot study suggest a difference in the pattern of disruption of glucocorticoid secretion among women and men with PTSD. Women had greater suppression of their basal cortisol levels than did men; however, the diurnal pattern for cortisol levels to decline throughout the day was observed among the women but not the men., (2009 Elsevier Inc. All rights reserved.)

In humans, an increased synthesis of reactive oxygen species (ROS) may be a relevant cause of amplification of physiologic processes resulting in inflammatory organ damage or neoplasia. Efficient anti-oxidative systems targeting oxidative stress are thus essential to prevent tissue damage. In plasma, proteins proved to be the first line of defence against ROS and albumin, the highest concentration plasma protein, has a key role in this antioxidant function. Recent studies have clearly documented that albumin oxido-redox potential changes upon oxidation by different oxidants thus becoming a deputy biomarker of this process. ROS react primarily with the free (34)Cysteine ((34)Cys) residue of albumin to form two reversible intermediate derivatives, sulfenic-(SOH-alb) and sulfinic acid (SO(2)H-alb), resulting in sulfonic acid (SO(3)H-alb), the final stable product of the reaction. Upon stable oxidation (SO(3)H-alb), albumin properties are altered: the protein becomes more susceptible to trypsin digestion and is degraded faster compared to the non-oxidized counterpart. The present review focuses on the characterization of albumin chemical changes induced by ROS, their relevance in human pathology and the most recent advances in the approach to oxidation adduct analysis.

Proteome treatments with peptide libraries in view of reducing high-abundance proteins and increasing the concentration of rare species involve the adsorption on solid-phase material. Subsequent elution of captured proteins may not be fully effective except when sequences of eluting agents are used. The standard way utilized up to the present has been a three- to four-step, sequential elution system consisting of various agents mixed together such as urea, thiourea, CHAPS, sodium chloride, citric or acetic acid and some polar solvents such as ACN and isopropanol. Elution sequences produce distinct fractions adding to the burden of having to analyze all of them. An alternative, highly effective, single elution to reduce the workload is here reported for the first time, namely elution in boiling 10% SDS added with 3% DTE. This single step elutes almost quantitatively the adsorbed proteins, thus ensuring, for all practical purposes, a full recovery. This high efficiency is believed to be due to the fact that the SDS micelles bury the polypeptide chains within their hydrophobic core, thus shielding them from the surroundings and impeding accidental adsorption to surfaces. Suggestions for selecting the best method to eliminate the excess of SDS for further protein analysis are also evaluated. The merits and limits of this novel system are assessed and discussed.

Two new iodoacetamide-substituted cyanines, C3NIASO3 and C5NIASO3, were synthesized starting from hemicyanine and were utilized for labeling plasma proteins. Specificity, sensitivity and feasibility for SH residues was tested utilizing an equimolar mixture of standard proteins and with normal plasma. Oxidized plasma proteins following H(2)O(2 )exposure and plasma from patients with focal glomerulosclerosis were analyzed as models of altered protein oxido-redox status. Following optimization of the assay (dye/protein ratio, pH), C3NIASO3 and C5NIASO3 gave a sensitivity slightly better than N-hydroxysuccinimidyl dyes for plasma proteins and were successfully employed for differential display electrophoresis (DIGE). Twenty-nine proteins were detected in normal plasma after 2-DE while less proteins were detected in plasma of patients with glomerulosclerosis. Following massive 'in vitro' oxidation with H(2)O(2), C3NIASO3 and C5NIASO3 failed to detect any residual SH, implicating massive oxidation. In conclusion, this study describes the synthesis of two new iodoacetamide cyanines that can be utilized for the analysis of plasma proteins with 2-DE and DIGE. They are also indicated for the definition of the oxido-redox status of proteins and were successfully utilized to extend the analysis of oxidation damage in patients with glomerulosclerosis.

Cyclosporine (CsA) has considerably modified the graft survival in solid organ and bone marrow transplantations. It is also the treatment of choice in chronic diseases such as steroid resistance and/or dependence nephrotic syndrome and autoimmune-diseases, especially in those cases that require long term treatments. Renal toxicity is the major adverse effect of chronic CsA administration. Deterioration of renal function and renal histopathology are the basic elements of the diagnosis. Overall, available studies suggest a good degree of safety related to appropriate drug dosages even if they require an adequate degree of surveillance in case of rapid changes of renal functions and long term evaluation of renal pathology. CsA neurotoxicity is the second major problem that seems underestimated especially in case of subtle manifestations in children. The full blown picture of the acute form is characterized by convulsion and sudden alteration of mental function that are reversible upon drug withdrawal. The diagnosis is based on typical CT and MRI aspects of extensive bilateral white-matter abnormalities in the occipital region of the brain that mimics the posterior encephalopathy syndrome. Prospective evaluations of drug tolerance include renal histology in case of chronic renal toxicity and neuro-imaging to identify and block acute neurotoxicity.

Little is known on both the composition and mechanism(s) of proteinuria associated with the use of mTOR inhibitors, in particular of Everolimus (E). We characterized urinary proteins utilizing an integrated proteomics approach (quantitative essays, 2-DE, MALDI-TOF, Western blot) in 48 renal transplant recipients who were alternatively treated with E (n = 31) or with enteric coated mycophenolic acid (EC-MPA) (n = 17). Twelve E patients (39%) developed high (>3 g/day) or intermediate proteinuria (1-3 g) compared to four (23%) of the EC-MPA group. Urinary proteins (p<0.001), β2 microglobulin (p<0.001) and α1microglobulin (p<0.025) were higher in E than in EC-MPA, appeared more rapidly and were inversely correlated with the day of treatment. Proteomics showed a marked increase of all urinary components in E and EC-MPA patients, major changes involving typical components of glomerular damage (albumin, α1-Zn glycoprotein, α2HS glycoprotein, leucin-richα2-glycoprotein) and specific bio-markers for E (clusters of α1-antitrypsin fragments and monoclonal λ chains). Finally, inter-α-trypsin-inhibitor heavy chain H4 precursor was decreased in E and EC-MPA urine compared to normal urine. In conclusion, E induced massive and generalized proteinuria of mixed glomerular and tubular origin that was correlated with the start of treatment and reached a nephrotic range in few cases. Specific urinary markers reflect renal alterations related to the transplant or specific alterations associated with the drug., (Copyright © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Proteinuria is the hallmark of renal diseases and the characterization of the urinary protein composition may become an important source of information for diagnosis and research. So far, protein analysis in urine has been utilized for a generic individuation of site-specific defects (glomerular vs. tubular) but there is a need for an extension of proteomics to specific urinary biomarkers in selected clinical conditions. The identification of fragments of proteins in plasma and urine may increase the spectrum of urinary biomarkers. The unique speculative application so far proposed for protein fragments is nephrotic syndrome, and specifically focal segmental glomerulosclerosis, in which case they reflect intrinsic proteolysis occurring in plasma and represent surrogate biomarkers of the disease activity. Albumin is probably the most studied protein. Several of the albumin fragments present a peculiar distribution of the fingerprint peptide pattern containing both the N-terminal region and the C-terminal domain with a complete lack of any MS signals for the internal sequence region. Their characterization utilizing new strategies based on 2-D nondenaturing electrophoresis is now in progress. Studies on a direct characterization of proteases in plasma and urine will also define the participation of proteases to the genesis of renal diseases., (Copyright © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The oxido-redox status of plasma albumin in patients treated with hemodialysis was characterized with LC-ESI-MS/MS and was compared with models of oxidative stress. Oxidised albumin was characterized by sulfonation (SO3-) of the SH at Cys 34, unfolding and acidification of the molecule. Albumin in hemodialysis patients presented, instead, only intermediate oxidation products such as sulfenic (SO2), sulfonic (SO)and methionine sulfoxide (C5H9NO2S) involving Cys 165-269 and Met 329-548 but did not present SO3- at Cys 34. Absence of charge and structural alterations compared to the oxidised templates was also confirmed with electrophoretic titration and calorimetry. In conclusion, the oxido-redox status of plasma albumin in hemodialysis patients lacks the hallmarks of the advanced oxidation products. LC-ESI-MS/MS was crucial to characterize albumin in conditions of oxidation stress; surrogate techniques can mirror conformational changes induced by oxidation.

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.

Mesenchymal stem cells (MSCs) may be of value in regeneration of renal tissue after damage; however, lack of biological knowledge and variability of results in animal models limit their utilization. We studied the effects of MSCs on podocytes in vitro and in vivo utilizing adriamycin (ADR) as a model of renal toxicity. The in vivo experimental approach was carried out in male Sprague-Dawley rats (overall 60 animals) treated with different ADR schemes to induce acute and chronic nephrosis. MSCs were given a) concomitantly to ADR in tail vein or b) in aorta and c) in tail vein 60 days after ADR. Homing was assessed with PKH26-MSCs. MSCs rescued podocytes from apoptosis induced by ADR in vitro. The maximal effect (80% rescue) was obtained with MSCs/podocytes coculture ratio of 1:1 for 72 h. All rats treated with ADR developed nephrosis. MSCs did not modify the clinical parameters (i.e., proteinuria, serum creatinine, lipids) but protected the kidney from severe glomerulosclerosis when given concomitantly to ADR. Rats given MSCs 60 days after ADR developed the same severe renal damage. Only a few MSCs were found in renal tubule-interstitial areas 1-24 h after injection and no MSCs were detected in glomeruli. MSCs reduced apoptosis of podocytes treated with ADR in vitro. Early and repeated MSCs infusion blunted glomerular damage in chronic ADR-induced nephropathy. MSCs did not modify proteinuria and progression to renal failure, which implies lack of regenerative potential in this model.

We describe a child presenting with oligoclonal plasma IgM (1.2 g%) and nephrotic syndrome with focal segmental glomerulosclerosis. Oligoclonality was demonstrated by the analysis of the complementary determining region 3 (CDR 3) on immunoglobulin heavy chains and by two dimensional electrophoresis and Western blot analysis that showed the bulk of isoforms having a cationic muU chain compared with the normal homologue (pI 7.5 vs 6.5). Urinary light chains were absent, and bone marrow aspirate was normal. Usual therapies for nephrotic syndrome with steroids and cyclosporin were useless. At the age of 9 years the patient was treated with plasmapheresis plus cyclophosphamide (2 mg/kg per day for 60 days), which temporarily reduced plasma IgM, and proteinuria was normal for 3 years. After this period, due to new recurrence of nephrotic syndrome, the patient received a cycle with anti-CD20 antibodies (500 mg/m(2) every week for a month) associated with a cycle of plasmapheresis that normalized proteinuria again, and, after 3 years, the proteinuria is still in remission. This is the first case of nephrotic syndrome associated with oligoclonal plasma IgM and mesangial IgM deposits. Both cyclophosphamide and anti-CD20 antibodies associated with plasmapheresis induced, at different stages, stable and protracted remission of proteinuria without evident side effects. Long term efficacy and safety of the association are still to be determined.

We randomized, at two sites, 210 patients with Rome II diagnosed irritable bowel syndrome (IBS), of at least moderate severity, to one of three conditions: group-based cognitive therapy (CT; n=120), psychoeducational support groups (n=46) as an active control, or intensive symptom and daily stress monitoring (n=44). One hundred eighty-eight participants completed the initial treatment. Those in symptom monitoring were then crossed over to CT. For an intent to treat analysis on a composite GI symptom measure derived from daily symptom diaries, both CT and the psychoeducational support groups were significantly more improved than those in the intensive symptom monitoring condition, but the CT and psychoeducational support group did not differ. Among treatment completers on the same composite measure of GI symptoms, again, both CT and psychoeducational support groups were statistically superior to symptom monitoring but did not differ on the symptom composite, or on any other measure. On individual IBS symptoms, both CT and psychoeducational support were statistically superior to symptom monitoring on reductions in abdominal pain and tenderness and for flatulence. Patient global ratings at the end of treatment showed the two active conditions statistically superior to symptom monitoring on change in Bowel Regularity, with CT superior to symptom monitoring on reduction in overall pain and in improvement in sense of well-being. Three-month follow-up data on 175 patients revealed maintenance of significant improvement or continued significant improvement on all IBS symptoms, including the McGill Pain Questionnaire. Group CT and psychoeducational support groups continued not to differ on any measure. We thus conclude that group CT is not superior to an attention placebo control condition.