Background The type 1a glycogen storage disease, also known as von Gierke's disease, is a rare inherited metabolic disorder caused by a mutation in the G6PC gene which leads to a deficiency in the enzyme glucose-6-phosphatase, vital for converting glycogen to glucose in the liver. The resulting accumulation of glycogen in tissues results in various consequences, such as hepatomegaly, hypoglycemia, growth delay, lactic acidosis, and nephropathy. As for the latter, patients display a condition similar to diabetic nephropathy that can progress to renal failure. Most patients over the age of 25 present microalbuminuria and more than 50% have proteinuria. In animal models with a kidney-specific deletion of G6Pase, early-onset nephropathy has been observed with an increase in oxidative stress levels due to the activation of angiotensin II and TGF-b1 systems. Regarding methodologies based on the use of primary human cell cultures, literature has described the use of human URECs renal epithelial cell cultures for studying renal involvement in some conditions, such as inherited cystic kidney diseases, propionic aciduria, methylmalonic aciduria, and Fabry's disease. Conversely, to date, it seems that this methodology has not yet been used for the study of renal involvement in GSD1a, which is the subject of this thesis. Aim of the study The aim of the study is to develop a methodology based on the in vitro culture of epithelial cells derived from the urine (URECs) of patients with GSD1a, to assess whether they retain their biological characteristics and can be used as a model for studying the renal and metabolic phenotypes of this genetic condition. Materials and Methods The studies were conducted on cells extracted from urine samples of three individuals with GSD1a and three healthy subjects, and cultured in vitro. The URECs were characterized after the fourth passage using transmission electron microscopy and immunofluorescence. Additionally, the production of reactive oxygen species (ROS) was measured using immunofluorescence at various glucose concentrations Results By the fourth passage, the URECs from patients with GSD1a continued to exhibit substantial glycogen accumulation. Both the GSD1a and control cells displayed the characteristic ciliary structures of the renal tubular epithelium, as well as the expression of specific markers for epithelial cells, such as E-cadherin, and renal tubular cells, like aquaporin 1 and 2. URECs from both groups adjusted their ROS levels in response to changes in glucose concentrations: notably, the GSD1a cells exhibited a heightened response compared to control cells across all glucose concentrations, particularly in the presence of low levels. This in vitro situation accurately replicates the metabolic disorder typically seen in patients with GSD1a. Conclusions This study represents the first demonstration that URECs, obtained from patients with GSD1a and cultured in vitro, retain their morphological and functional characteristics and could serve as a promising experimental model to study the renal complications of GSD1a, and specifically the molecular mechanisms leading to kidney damage
Background La glicogenosi tipo 1a, conosciuta anche come malattia di von Gierke, è una malattia metabolica ereditaria rara causata da una mutazione nel gene G6PC che provoca una deficienza dell'enzima glucosio-6-fosfatasi, essenziale per convertire il glicogeno in glucosio nel fegato. Il risultante accumulo di glicogeno nei tessuti porta a una serie di conseguenze, come epatomegalia, ipoglicemia, ritardo della crescita, acidosi lattica e nefropatia. Per quanto riguarda quest’ultima, i pazienti manifestano una malattia simile alla nefropatia diabetica che può evolvere in insufficienza renale. La maggior parte dei pazienti di età superiore ai 25 anni presentano microalbuminuria e oltre il 50% proteinuria. Nei modelli animali con delezione specifica per il rene di G6Pase, è stata osservata una nefropatia ad insorgenza precoce con un incremento dei livelli di stress ossidativo dovuto all’attivazione dei sistemi dell’angiotensina II e del TGF-b1. Per quanto riguarda le metodiche basate sull’uso di colture cellulari primarie umane, in letteratura è stato descritto l’utilizzo di colture di cellule epiteliali renali URECs umane per lo studio dell’interessamento renale in alcune patologie, come ad esempio le malattie renali cistiche ereditarie, l’aciduria propionica, l’aciduria metilmalonica e la malattia di Fabry. Al contrario ad oggi, non risulta che tale metodica sia stata ancora utilizzata per lo studio dell’interessamento renale nella GSD1a, che rappresenta l’oggetto della presente tesi. Scopo dello Studio Scopo dello studio è mettere a punto una metodica basata sulla coltura in vitro di cellule epiteliali derivate dall'urina (URECs) di pazienti con GSD1a, per valutare se conservano le loro caratteristiche biologiche e possono essere utilizzate come modello per lo studio dei fenotipi renali e metabolici di questa condizione genetica. Materiali e Metodi Gli studi sono stati condotti su cellule estratte da campioni di urina di tre soggetti con GSD1a e tre soggetti sani e coltivate in vitro. Le URECs sono state caratterizzate dopo il quarto passaggio tramite microscopia elettronica a trasmissione e immunofluorescenza. E’ stata inoltre misurata con immunofluorescenza la produzione delle specie reattive dell'ossigeno (ROS), a diverse concentrazioni di glucosio. Risultati Al quarto passaggio, le URECs provenienti dai pazienti affetti da GSD1a continuavano a presentare un massivo accumulo di glicogeno. Sia le cellule GSD1a sia quelle di controllo presentavano le tipiche strutture ciliate dell'epitelio tubulare renale, nonché l'espressione di marcatori specifici delle cellule epiteliali, come l'E-caderina, e delle cellule tubulari renali, come l'acquaporina 1 e 2. Le URECs di entrambi i gruppi adattavano i loro livelli di ROS in risposta alle variazioni delle concentrazioni di glucosio: in particolare, le cellule GSD1a erano caratterizzate da una risposta maggiore rispetto alle cellule ottenute dai controlli a tutte le concentrazioni di glucosio, in particolare in presenza di basse concentrazioni. Questa situazione riproduce fedelmente in vitro il disordine metabolico tipico dei pazienti con GSD1a. Conclusioni Questo studio rappresenta la prima dimostrazione che le URECs, ottenute da pazienti con GSD1a e coltivate in vitro, mantengono le proprie caratteristiche morfologiche e funzionali e potrebbero rappresentare un modello sperimentale promettente per studiare le complicanze renali della GSD1a e in particolare i meccanismi molecolari che portano al danno renale.
Malattie metaboliche rare: nuovi modelli cellulari per lo studio delle complicanze d'organo
SCANNAPIECO, FEDERICO
2022/2023
Abstract
Background The type 1a glycogen storage disease, also known as von Gierke's disease, is a rare inherited metabolic disorder caused by a mutation in the G6PC gene which leads to a deficiency in the enzyme glucose-6-phosphatase, vital for converting glycogen to glucose in the liver. The resulting accumulation of glycogen in tissues results in various consequences, such as hepatomegaly, hypoglycemia, growth delay, lactic acidosis, and nephropathy. As for the latter, patients display a condition similar to diabetic nephropathy that can progress to renal failure. Most patients over the age of 25 present microalbuminuria and more than 50% have proteinuria. In animal models with a kidney-specific deletion of G6Pase, early-onset nephropathy has been observed with an increase in oxidative stress levels due to the activation of angiotensin II and TGF-b1 systems. Regarding methodologies based on the use of primary human cell cultures, literature has described the use of human URECs renal epithelial cell cultures for studying renal involvement in some conditions, such as inherited cystic kidney diseases, propionic aciduria, methylmalonic aciduria, and Fabry's disease. Conversely, to date, it seems that this methodology has not yet been used for the study of renal involvement in GSD1a, which is the subject of this thesis. Aim of the study The aim of the study is to develop a methodology based on the in vitro culture of epithelial cells derived from the urine (URECs) of patients with GSD1a, to assess whether they retain their biological characteristics and can be used as a model for studying the renal and metabolic phenotypes of this genetic condition. Materials and Methods The studies were conducted on cells extracted from urine samples of three individuals with GSD1a and three healthy subjects, and cultured in vitro. The URECs were characterized after the fourth passage using transmission electron microscopy and immunofluorescence. Additionally, the production of reactive oxygen species (ROS) was measured using immunofluorescence at various glucose concentrations Results By the fourth passage, the URECs from patients with GSD1a continued to exhibit substantial glycogen accumulation. Both the GSD1a and control cells displayed the characteristic ciliary structures of the renal tubular epithelium, as well as the expression of specific markers for epithelial cells, such as E-cadherin, and renal tubular cells, like aquaporin 1 and 2. URECs from both groups adjusted their ROS levels in response to changes in glucose concentrations: notably, the GSD1a cells exhibited a heightened response compared to control cells across all glucose concentrations, particularly in the presence of low levels. This in vitro situation accurately replicates the metabolic disorder typically seen in patients with GSD1a. Conclusions This study represents the first demonstration that URECs, obtained from patients with GSD1a and cultured in vitro, retain their morphological and functional characteristics and could serve as a promising experimental model to study the renal complications of GSD1a, and specifically the molecular mechanisms leading to kidney damageFile | Dimensione | Formato | |
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https://hdl.handle.net/20.500.12608/51306