Idiopathic Nephrotic Syndrome (INS) in children is characterized by protein loss in urine, related to the disruption of the integrity of glomerular filtration barrier (GBM). Children with steroid-resistant INS (SRNS) have a great risk of end-stage renal disease (ESRD). Although evidence suggests the presence of an immune-related circulating factor responsible for glomerular barrier dysfunction, any previous attempt to unravel these mechanisms has been inconclusive. The ability to identify the underlying mechanisms of the disease and/or steroid-resistance would lay the foundations for a potential, much-needed target therapy, since 50% of drug-resistant cases progress to ESRD, requiring renal replacement therapy and may suffer from relapse after renal transplantation. This project aim is to clarify the mechanisms responsible of INS in children. During the 3 years of this PhD project we have applied both a clinical and laboratory approach. We performed a retrospective, multicentre, national cohort study to address the long-term prognosis of renal graft, the risk factors for recurrence and the predictors for response to therapy following recurrence in paediatric patients undergoing renal transplantation because of a SRNS. We identified all children with SRNS who underwent renal transplantation in Italy, between 2005 and 2017. 101 patients were identified. According to our results, the absence of a causative mutation represents the major risk factor for post-transplant recurrence in children with SRNS, while transplant can be curative in genetic SRNS. A prolonged time spent on dialysis before transplantation has no protective effect on the risk of relapse and should not be encouraged. Retransplantation represents a second chance after graft loss for recurrence. At the bench side, we identify patients with different forms of SRNS, collected their sera and tested the samples by means of a novel method which assess the permeability to bovine serum albumin (BSA) through a three-layer device (3LD). The 3 layers comprise: 1. conditionally immortalized human podocytes (HCiPodo), 2. collagen type IV coated porous membrane and 3. human glomerular endothelial cells (HCiGEnC). Seven recurrent SRNS (rSRNS), 5 non-recurrent SRNS (nrSRNS), and 5 genetic SRNS (gSRNS) were collected. Using this model, we were able to demonstrate that sera from all rSRNS patients recurred after transplantation increased albumin permeability, while sera from nrSRNS and gSRNS did not. Furthermore, we also found that the plasmapheresis eliminated the increasing BSA permeability effect in sera from rSRNS patients. These preliminary results confirm the hypothesis that a circulating permeability factors is responsible for SRNS in patients recurring after renal transplantation, without underlying causative gene mutations. If these results will be confirmed on larger cohorts, this GBM model could be a useful tool to predict the probability of disease relapse on transplantation, monitor the therapy or exclude a genetic diagnosis at the INS onset. A preliminary proteomic analysis of sera tested on the GBM model showed different profiles for each category.

La sindrome nefrosica idiopatica (INS) in età pediatrica è caratterizzata da proteinuria massiva, secondaria alla alterazione della struttura della membrana basale glomerulare (GBM). I pazienti pediatrici affetti da sindrome nefrosica cortico-resistente (SRNS) sono ad elevato rischio di insufficienza renale terminale (ESRD). Sebbene evidenze suggeriscano la presenza di un fattore circolante di permeabilità correlato al sistema immunitario, tutti i precedenti tentativi di comprensione degli esatti meccanismi alla base della patologia sono stati inconclusivi. L'identificazione dei meccanismi alla base della INS e della cortico-resistenza potrebbe favorire lo sviluppo di terapie mirate, in grado di arrestare la progressione verso del danno renale in pazienti con SRNS, nei quali l'ESRD è un evento comune ed in cui è necessario ricorrere a dialisi o trapianto di rene, con elevato rischio di recidiva su rene trapiantato. Questo progetto mira a identificare i meccanismi responsabili della INS in età pediatrica. Durante i 3 anni di progetto, abbiamo a tal fine applicato un approccio clinico e di laboratorio. Abbiamo effettuato uno studio retrospettivo, multicentrico di corte nazionale per identificare la prognosi renale, i fattori di rischio per la recidiva e quelli predittivi di risposta alla terapia post-recidiva, in tutti i pazienti pediatrici sottoposti a trapianto di rene per SRNS in Italia dal 2005 al 2017. Sono stati identificati 101 pazienti. In base ai risultati ottenuti, l'assenza di mutazioni causative per SRNS rappresenta il più importante fattore di rischio per la recidiva nei bambini con SRNS. Il trapianto è invece curativo per le SRNS genetiche. Prolungare il tempo di dialisi prima del trapianto non ha un effetto protettivo sul rischio di recidiva ed è una strategia da non incoraggiare. Il ritrapianto è possibile dopo la perdita di un primo rene trapiantato. Contemporaneamente è stato eseguito uno studio di laboratorio identificando pazienti con differenti forme di SRNS, raccogliendone i sieri e testandoli su un nuovo modello di laboratorio in grado di determinare la permeabilità della albumina sierica bovina (BSA) attraverso un sistema a 3 strati (3LD). I 3 strati comprendono: 1. Podociti umani immortalizzati (HCiPodo), 2. una membrana rivestita di collagene tipo IV e 3. cellule endoteliali glomerulari umane (HCiGEnC). I sieri di 7 pazienti con SRNS recidivata su trapianto (rSRNS), 5 SRNS non recidivate (nrSRNS), e 5 SRNS genetiche (gSRNS) sono stati testati sul sistema. E' stato possibile dimostrare che i sieri di tutte i pazienti con rSRNS aumentano la permeabilità all'albumina nel modello, mentre i sieri dei pazienti con nrSRNS e gSRNS non ne determinano alterazioni significative. Inoltre, la plasmaferesi elimina la capacita dei sieri di incrementare la permeabilità all'albumina nei pazienti rSRNS. Questi risultati confermano l'ipotesi di un fattore circolante di permeabilità responsabile della SRNS nei pazienti recidivati dopo trapianto, senza mutazioni genetiche causative. Se questi risultati verranno confermati su casistiche più ampie, il modello di GBM potrà essere utilizzato per predire la probabilità di recidiva su trapianto, monitorare la terapia ed escludere la presenza di mutazioni genetiche alla diagnosi di INS. Una analisi preliminare di proteomica dei sieri ha evidenziato differenti profili proteici nelle diverse forme con differente attività sul modello di GBM.

(2020). Underlying mechanisms of Idiopathic Nephrotic Syndrome in children: evidence of a circulating permeability factor. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2020).

Underlying mechanisms of Idiopathic Nephrotic Syndrome in children: evidence of a circulating permeability factor

MORELLO, WILLIAM
2020

Abstract

Idiopathic Nephrotic Syndrome (INS) in children is characterized by protein loss in urine, related to the disruption of the integrity of glomerular filtration barrier (GBM). Children with steroid-resistant INS (SRNS) have a great risk of end-stage renal disease (ESRD). Although evidence suggests the presence of an immune-related circulating factor responsible for glomerular barrier dysfunction, any previous attempt to unravel these mechanisms has been inconclusive. The ability to identify the underlying mechanisms of the disease and/or steroid-resistance would lay the foundations for a potential, much-needed target therapy, since 50% of drug-resistant cases progress to ESRD, requiring renal replacement therapy and may suffer from relapse after renal transplantation. This project aim is to clarify the mechanisms responsible of INS in children. During the 3 years of this PhD project we have applied both a clinical and laboratory approach. We performed a retrospective, multicentre, national cohort study to address the long-term prognosis of renal graft, the risk factors for recurrence and the predictors for response to therapy following recurrence in paediatric patients undergoing renal transplantation because of a SRNS. We identified all children with SRNS who underwent renal transplantation in Italy, between 2005 and 2017. 101 patients were identified. According to our results, the absence of a causative mutation represents the major risk factor for post-transplant recurrence in children with SRNS, while transplant can be curative in genetic SRNS. A prolonged time spent on dialysis before transplantation has no protective effect on the risk of relapse and should not be encouraged. Retransplantation represents a second chance after graft loss for recurrence. At the bench side, we identify patients with different forms of SRNS, collected their sera and tested the samples by means of a novel method which assess the permeability to bovine serum albumin (BSA) through a three-layer device (3LD). The 3 layers comprise: 1. conditionally immortalized human podocytes (HCiPodo), 2. collagen type IV coated porous membrane and 3. human glomerular endothelial cells (HCiGEnC). Seven recurrent SRNS (rSRNS), 5 non-recurrent SRNS (nrSRNS), and 5 genetic SRNS (gSRNS) were collected. Using this model, we were able to demonstrate that sera from all rSRNS patients recurred after transplantation increased albumin permeability, while sera from nrSRNS and gSRNS did not. Furthermore, we also found that the plasmapheresis eliminated the increasing BSA permeability effect in sera from rSRNS patients. These preliminary results confirm the hypothesis that a circulating permeability factors is responsible for SRNS in patients recurring after renal transplantation, without underlying causative gene mutations. If these results will be confirmed on larger cohorts, this GBM model could be a useful tool to predict the probability of disease relapse on transplantation, monitor the therapy or exclude a genetic diagnosis at the INS onset. A preliminary proteomic analysis of sera tested on the GBM model showed different profiles for each category.
BIONDI, ANDREA
Sindrome nefrosica; steroido-resistenza; trapianto di rene; Modelli in vitro; fattor permeabilità
Nephrotic syndrome; steroid-resistance; kidney transplant; In vitro model; fattor permeabilità
MED/38 - PEDIATRIA GENERALE E SPECIALISTICA
English
4-feb-2020
MEDICINA TRASLAZIONALE E MOLECOLARE - DIMET
32
2018/2019
open
(2020). Underlying mechanisms of Idiopathic Nephrotic Syndrome in children: evidence of a circulating permeability factor. (Tesi di dottorato, Università degli Studi di Milano-Bicocca, 2020).
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10281/263395
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