Alberto Magnasco

Autoimmunity in Membranous Nephropathy Targets Aldose Reductase and SOD2

Glomerular targets of autoimmunity in human membranous nephropathy are poorly understood. Here, we used a combined proteomic approach to identify specific antibodies against podocyte proteins in both serum and glomeruli of patients with membranous nephropathy (MN). We detected specific anti-aldose reductase (AR) and anti-manganese superoxide dismutase (SOD2) IgG(4) in sera of patients with MN. We also eluted high titers of anti-AR and anti-SOD2 IgG(4) from microdissected glomeruli of three biopsies of MN kidneys but not from biopsies of other glomerulonephritides characterized by IgG deposition (five lupus nephritis and two membranoproliferative glomerulonephritis). We identified both antigens in MN biopsies but not in other renal pathologies or normal kidney. Confocal and immunoelectron microscopy (IEM) showed co-localization of anti-AR and anti-SOD2 with IgG(4) and C5b-9 in electron-dense podocyte immune deposits. Preliminary in vitro experiments showed an increase of SOD2 expression on podocyte plasma membrane after treatment with hydrogen peroxide. In conclusion, our data support AR and SOD2 as renal antigens of human MN and suggest that oxidative stress may drive glomerular SOD2 expression.

Short-Term Effects of Rituximab in Children with Steroid- and Calcineurin-Dependent Nephrotic Syndrome: A Randomized Controlled Trial

BACKGROUND AND OBJECTIVES Prednisone and calcineurin inhibitors are the mainstay therapy of idiopathic nephrotic syndrome (INS) in children. However, drug dependence and toxicity associated with protracted use are common. Case series suggest that the anti-CD20 monoclonal antibody rituximab (RTX) may maintain disease remission. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This open-label randomized controlled trial was powered to show that a strategy based on RTX and lower doses of prednisone and calcineurin inhibitors was noninferior to standard doses of these agents in maintaining 3-month proteinuria as low as baseline or up to 1 g/d greater (noninferiority margin). Participants were stratified by the presence of toxicity to prednisone/calcineurin inhibitors and centrally assigned to add RTX (Mabthera, 375 mg/m(2) intravenously) to lower doses of standard agents or to continue with current therapy alone. The risk of relapse was a secondary outcome. RESULTS Fifty-four children (mean age 11 ± 4 years) with INS dependent on prednisone and calcineurin inhibitors for >12 months were randomized. Three-month proteinuria was 70% lower in the RTX arm (95% confidence interval 35% to 86%) as compared with standard therapy arm (intention-to-treat); relapse rates were 18.5% (intervention) and 48.1% (standard arm) (P = 0.029). Probabilities of being drug-free at 3 months were 62.9% and 3.7%, respectively (P < 0.001); 50% of RTX cases were in stable remission without drugs after 9 months. CONCLUSIONS Rituximab and lower doses of prednisone and calcineurin inhibitors are noninferior to standard therapy in maintaining short-term remission in children with INS dependent on both drugs and allow their temporary withdrawal.

Rituximab in Children with Resistant Idiopathic Nephrotic Syndrome

Idiopathic nephrotic syndrome resistant to standard treatments remains a therapeutic dilemma in pediatric nephrology. To test whether the anti-CD20 monoclonal antibody rituximab may benefit these patients, we conducted an open-label, randomized, controlled trial in 31 children with idiopathic nephrotic syndrome unresponsive to the combination of calcineurin inhibitors and prednisone. All children continued prednisone and calcineurin inhibitors at the doses prescribed before enrollment, and one treatment group received two doses of rituximab (375 mg/m(2) intravenously) as add-on therapy. The mean age was 8 years (range, 2-16 years). Rituximab did not reduce proteinuria at 3 months (change, -12% [95% confidence interval, -73% to 110%]; P=0.77 in analysis of covariance model adjusted for baseline proteinuria). Additional adjustment for previous remission and interaction terms (treatment by baseline proteinuria and treatment by previous remission) did not change the results. In conclusion, these data do not support the addition of rituximab to prednisone and calcineurin inhibitors in children with resistant idiopathic nephrotic syndrome.

Posttransplant De Novo Donor-Specific HLA Antibodies Identify Pediatric Kidney Recipients at Risk for Late Antibody-Mediated Rejection

The emerging role of humoral immunity in the pathogenesis of chronic allograft damage has prompted research aimed at assessing the role of anti‐HLA antibody (Ab) monitoring as a tool to predict allograft outcome. Data on the natural history of allografts in children developing de novo Ab after transplantation are limited. Utilizing sera collected pretransplant, and serially posttransplant, we retrospectively evaluated 82 consecutive primary pediatric kidney recipients, without pretransplant donor‐specific antibodies (DSA), for de novo Ab occurrence, and compared results with clinical–pathologic data. At 4.3‐year follow up, 19 patients (23%) developed de novo DSA whereas 24 had de novo non‐DSA (NDSA, 29%). DSA appeared at a median time of 24 months after transplantation and were mostly directed to HLA‐DQ antigens. Among the 82 patients, eight developed late/chronic active C4d+ antibody‐mediated rejection (AMR), and four C4d‐negative AMR. Late AMR correlated with DSA (p < 0.01), whose development preceded AMR by 1‐year median time. Patients with DSA had a median serum creatinine of 1.44 mg/dL at follow up, significantly higher than NDSA and Ab‐negative patients (p < 0.005). In our pediatric cohort, DSA identify patients at risk of renal dysfunction, AMR and graft loss; treatment started at Ab emergence might prevent AMR occurrence and/or progression to graft failure.

Rituximab is a safe and effective long-term treatment for children with steroid and calcineurin inhibitor–dependent idiopathic nephrotic syndrome

In children with idiopathic nephrotic syndrome, rituximab can maintain short-term remission with withdrawal of prednisone and calcineurin inhibitors. Long-term effects including the number of repeated infusions to maintain remission are unknown. To test this, we treated 46 consecutive children with idiopathic nephrotic syndrome lasting for at least 1 year (mean 6.3 years), maintained in remission with oral prednisone and calcineurin inhibitors. They received 1-5 rituximab courses during a median follow-up of 3 years. Oral agents were tapered after each infusion, and completely withdrawn within 45 days. Rituximab was well tolerated. Six-month probabilities of remission were 48% after the first infusion and 37% after subsequent infusions. One- and 2-year-remission probabilities were, respectively, 20 and 10%. Median time intervals between complete oral-agent withdrawal and relapse were 5.6 and 8.5 months, respectively, following the first and subsequent courses. The time to reconstitution of CD20 cells correlated with the duration of remission, but was not associated with variation in FcyR, CD20, or SMPDL-3B polymorphisms. Podocyte Src phosphorylation was normal. Thus, rituximab can be safely and repeatedly used as a prednisone and calcineurin inhibitor-sparing therapy in a considerable proportion of children with dependent forms of idiopathic nephrotic syndrome. Further study is needed to identify patients who will benefit most from rituximab therapy.In children with idiopathic nephrotic syndrome rituximab can maintain short-term remission with withdrawal of prednisone and calcineurin-inhibitors. Long-term effects including number of repeated infusions to maintain remission are unknown. We treated with rituximab 46 consecutive children with idiopathic nephrotic syndrome lasting for at least one year (6.3±4.1 years), who were maintained in remission with oral prednisone and calcineurin inhibitors. They received 1–5 rituximab courses during a median follow-up of three years (range 1–5). Oral agents were tapered after each infusion, and completely withdrawn within 45 days. Rituximab was well tolerated. Six-month probabilities of remission were 48% after the first infusion and 37% after subsequent infusions. One- and two-year-remission probabilities were respectively 20% and 10%. Median time intervals between complete oral-agent withdrawal and relapse were 5.6 and 8.5 months respectively following the first and subsequent courses. Time to reconstitution of CD20 cells correlated with the duration of remission, but was not associated with variation in FcyR, CD20 or SMPDL-3B polymorphisms. Podocyte Src phosphorylation was normal. Rituximab can be safely and repeatedly used as prednisone and calcineurin-inhibitor-sparing therapy in a considerable proportion of children with dependent forms of idiopathic nephrotic syndrome. Further research is needed to identify patients who will benefit most from rituximab therapy.

Mesenchymal stem cells protective effect in adriamycin model of nephropathy.

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.

Cyclosporin and Organ Specific Toxicity: Clinical Aspects, Pharmacogenetics and Perspectives

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.

Acquisition of C3d‐Binding Activity by De Novo Donor‐Specific HLA Antibodies Correlates With Graft Loss in Nonsensitized Pediatric Kidney Recipients

Alloantibody‐mediated graft injury is a major cause of kidney dysfunction and loss. The complement‐binding ability of de novo donor‐specific antibodies (dnDSAs) has been suggested as a prognostic tool to stratify patients for clinical risk. In this study, we analyzed posttransplant kinetics of complement‐fixing dnDSAs and their role in antibody‐mediated rejection development and graft loss. A total of 114 pediatric nonsensitized recipients of first kidney allograft were periodically monitored for dnDSAs using flow bead assays, followed by C3d and C1q assay in case of positivity. Overall, 39 patients developed dnDSAs, which were C1q+ and C3d+ in 25 and nine patients, respectively. At follow‐up, progressive acquisition over time of dnDSA C1q and C3d binding ability, within the same antigenic specificity, was observed, paralleled by an increase in mean fluorescence intensity that correlated with clinical outcome. C3d‐fixing dnDSAs were better fit to stratify graft loss risk when the different dnDSA categories were evaluated in combined models because the 10‐year graft survival probability was lower in patients with C3d‐binding dnDSA than in those without dnDSAs or with C1q+/C3d− or non‐complement‐binding dnDSAs (40% vs. 94%, 100%, and 100%, respectively). Based on the kinetics profile, we favor dnDSA removal or modulation at first confirmed positivity, with treatment intensification guided by dnDSA biological characteristics.

DQ molecules are the principal stimulators of de novo donor‐specific antibodies in nonsensitized pediatric recipients receiving a first kidney transplant

Data on the different HLA‐antibody (Ab) categories in pediatric kidney recipients developing de novo donor‐specific Abs (DSA) after transplantation are scarce. We retrospectively evaluated 82 consecutive nonsensitized pediatric recipients of a first kidney graft for de novo HLA Ab occurrence and antigen specificity. At a median follow‐up of 6 years, 29% of patients developed de novo DSA, while 45% had de novo non‐DSA. DSA appeared at 25‐month median time post‐transplant and were mostly directed toward HLA‐DQ antigens. Considering each HLA antigen, the estimated rate of DQ DSA (7.55 per 100 person‐years) was much higher than the rates observed for non‐DQ DSA. The HLA‐DQ Ab recognized determinants of the DQβ chain in 70% of cases, α chain in 25% of cases, and both chains in one patient. Non‐DSA peaked earlier than DSA, and were largely directed against HLA class I specificities that belonged to HLA‐A‐ and HLA‐B‐related cross‐reacting epitope groups (CREG) in 56% of cases. Our results indicate a need for evaluating HLA‐DQ compatibilities in kidney allocation, in order to minimize post‐transplant development of de novo DSA, known to be responsible for antibody‐mediated rejection and graft loss.

Thresholds for significant decrease in hemodialysis access blood flow.

During hemodialysis access surveillance, referral for evaluation and correction of stenosis is based upon determination that a significant decrease in blood flow (Q) has occurred. However, criteria for determining when a decrease is statistically significant have not yet been established. In this study we established such criteria by analyzing Q variation with the glucose pump test (GPT). We took nine Q measurements in each of 25 patients (18 grafts, 7 fistulas) during three dialysis sessions within a 2‐week period (predialysis and during hours 1 and 3). We determined thresholds that define a significant percentage decrease in Q (ΔQ) for various p values. In order to confirm the general applicability of these thresholds, we computed the average within‐patient Q variation during the three sessions (computed as a coefficient of variation and referred to as short‐term variation). We then determined the relative influences of biological (true) variation and analytical error on short‐term variation. We found that ΔQ must be > 33% to be significant at p < 0.05, whereas the threshold is > 17% for p < 0.20. Measuring Q at uniform versus different times during the sessions did not significantly reduce these thresholds. We also found that biological variation was nearly as large as short‐term Q variation, whereas analytical error contributed minimally to short‐term variation. In conclusion, this study defines thresholds for a significant ΔQ that have wide application in determining access referral for evaluation and correction of stenosis. Selection of a particular threshold should consider the relative importance of avoiding thrombosis versus avoiding unnecessary procedures. If avoiding unnecessary procedures is a priority, then we recommend a threshold of > 33%. These thresholds apply to other methods of measuring Q, provided analytical error is significantly less than biological variation.