Academic literature on the topic 'Primary Hyperoxaluria Type I (PHI)'

Primary hyperoxaluria type 1 (PH1) and type 2 (PH2) are rare genetic diseases that result from deficiencies in glyoxylate metabolism. The increased oxalate synthesis that occurs can lead to kidney stone formation, deposition of calcium oxalate in the kidney and other tissues, and renal failure. Hydroxyproline (Hyp) catabolism, which occurs mainly in the liver and kidney, is a prominent source of glyoxylate and could account for a significant portion of the oxalate produced in PH. To determine the sensitivity of mouse models of PH1 and PH2 to Hyp-derived oxalate, animals were fed diets containing 1% Hyp. Urinary excretions of glycolate and oxalate were used to monitor Hyp catabolism and the kidneys were examined to assess pathological changes. Both strains of knockout (KO) mice excreted more oxalate than wild-type (WT) animals with Hyp feeding. After 4 wk of Hyp feeding, all mice deficient in glyoxylate reductase/hydroxypyruvate reductase (GRHPR KO) developed severe nephrocalcinosis in contrast to animals deficient in alanine-glyoxylate aminotransferase (AGXT KO) where nephrocalcinosis was milder and with a lower frequency. Plasma cystatin C measurements over 4-wk Hyp feeding indicated no significant loss of renal function in WT and AGXT KO animals, and significant and severe loss of renal function in GRHPR KO animals after 2 and 4 wk, respectively. These data suggest that GRHPR activity may be vital in the kidney for limiting the conversion of Hyp-derived glyoxylate to oxalate. As Hyp catabolism may make a major contribution to the oxalate produced in PH patients, Hyp feeding in these mouse models should be useful in understanding the mechanisms associated with calcium oxalate deposition in the kidney.

The primary hyperoxalurias type 1 (PH1) and type 2 (PH2) are autosomal recessive calcium oxalate kidney stone diseases caused by deficiencies of the metabolic enzymes alanine:glyoxylate aminotransferase (AGT) and glyoxylate/hydroxypyruvate reductase (GR/HPR), respectively. Over 50 mutations have been identified in the AGXT gene (encoding AGT) in PH1, associated with a wide variety of effects on AGT, including loss of catalytic activity, aggregation, accelerated degradation, and peroxisome-to-mitochondrion mistargeting. Some of these mutations segregate and interact synergistically with a common polymorphism. Over a dozen mutations have been found in the GRHPR gene (encoding GR/HPR) in PH2, all associated with complete loss of glyoxylate reductase enzyme activity and immunoreactive protein. The crystal structure of human AGT, but not human GR/HPR, has been solved, allowing the effects of many of the mutations in PH1 to be rationalised in structural terms. Detailed analysis of the molecular aetiology of PH1 and PH2 has led to significant improvements in all aspects of their clinical management. Enzyme replacement therapy by liver transplantation can provide a metabolic cure for PH1, but it has yet to be tried for PH2. New treatments that aim to counter the effects of specific mutations on the properties of the enzymes could be feasible in the not-too-distant future.

Background: Primary hyperoxaluria type 1 (PH1) and idiopathic hypercalciuria (IHC) are stone-forming diseases that may result in the formation of calcium (Ca) oxalate (Ox) stones, nephrocalcinosis, and progressive chronic kidney disease (CKD). Poorer clinical outcome in PH1 is segregated by the highest urine (Ur)-Ox (UrOx), while IHC outcomes are not predictable by UrCa. We hypothesized that differences would be found in selected Ur-protein (PRO) patterns in PH1 and IHC, compared to healthy intra-familial sibling controls (C) of PH1 patients. We also hypothesized that the PRO patterns associated with higher UrOx levels would reflect injury, inflammation, biomineralization, and abnormal tissue repair processes in PH1. Methods: Twenty four-hour Ur samples were obtained from 3 cohorts: PH1 (n = 47); IHC (n = 35) and C (n = 13) and were analyzed using targeted platform-based multi-analyte profile immunoassays and for UrOx and UrCa by biochemical measurements. Results: Known stone matrix constituents, osteopontin, calbindin, and vitronectin were lowest in PH1 (C > IHC > PH1; p < 0.05). Ur-interleukin-10; chromogranin A; epidermal growth factor (EGF); insulin-like growth factor-1 (IGF-1), and macrophage inflammatory PRO-1α (MIP-1α) were higher in PH1 > C (p = 0.03 to p < 0.05). Fetuin A; IGF-1, MIP-1α, and vascular cell adhesion molecule-1 were highest in PH1 > IHC (p < 0.001 to p = 0.005). Conclusion: PH1 Ur-PROs reflected overt inflammation, chemotaxis, oxidative stress, growth factors (including EGF), and pro-angiogenic and calcification regulation/inhibition compared to the C and IHC cohorts. Many of the up- and downregulated PH1-PROs found in this study are also found in CKD, acute kidney injury, stone formers, and/or stone matrices. Further data analyses may provide evidence for PH1 unique PROs or demonstrate a poorer clinical outcome.

Oxalobacter colonization of rat intestine was previously shown to promote enteric oxalate secretion and elimination, leading to significant reductions in urinary oxalate excretion (Hatch et al. Kidney Int 69: 691–698, 2006). The main goal of the present study, using a mouse model of primary hyperoxaluria type 1 (PH1), was to test the hypothesis that colonization of the mouse gut by Oxalobacter formigenes could enhance enteric oxalate secretion and effectively reduce the hyperoxaluria associated with this genetic disease. Wild-type (WT) mice and mice deficient in liver alanine-glyoxylate aminotransferase (Agxt) exhibiting hyperoxalemia and hyperoxaluria were used in these studies. We compared the unidirectional and net fluxes of oxalate across isolated, short-circuited large intestine of artificially colonized and noncolonized mice. In addition, plasma and urinary oxalate was determined. Our results demonstrate that the cecum and distal colon contribute significantly to enteric oxalate excretion in Oxalobacter-colonized Agxt and WT mice. In colonized Agxt mice, urinary oxalate excretion was reduced 50% (to within the normal range observed for WT mice). Moreover, plasma oxalate concentrations in Agxt mice were also normalized (reduced 50%). Colonization of WT mice was also associated with marked (up to 95%) reductions in urinary oxalate excretion. We conclude that segment-specific effects of Oxalobacter on intestinal oxalate transport in the PH1 mouse model are associated with a normalization of plasma oxalate and urinary oxalate excretion in otherwise hyperoxalemic and hyperoxaluric animals.

Primary hyperoxaluria (PH) Types I, II, and III is an autosomal recessive inherited disorder of defect in glyoxylate metabolism due to specific hepatic enzyme deficiencies causing renal damage due to deposition of oxalate crystals that induce renal epithelial cell injury, and inflammation resulting in reduced renal oxalate elimination leading to extra renal deposition of calcium oxalate crystals. PH is under diagnosed because of phenotypic heterogeneity masquerading as infantile nephrocalcinosis (NC) with or without renal failure or renal calculus disease in adults. We present three children with genetically proven PH1 seen over last 2 years along with a brief review of the literature. In this series all cases were female. Two girls had infantile onset of symptoms and one presented in childhood. Renal failure in all with varying sonography features including small size kidneys, multiple renal calculi, bulky kidneys with loss of corticomedullary differentiation were seen. Extrarenal affection was seen in one child. Renal replacement therapy was provided in all. Awareness of PH and early diagnosis by measurement of plasma and urinary oxalate and molecular characterization helps in prompt aggressive therapy, preventing extrarenal manifestations and plan long term management.

BackgroundPrimary hyperoxaluria type 1 (PH1) is an inborn error of glyoxylate metabolism, characterized by increased endogenous oxalate production. The metabolic pathways underlying oxalate synthesis have not been fully elucidated, and upcoming therapies require more reliable outcome parameters than the currently used plasma oxalate levels and urinary oxalate excretion rates. We therefore developed a stable isotope infusion protocol to assess endogenous oxalate synthesis rate and the contribution of glycolate to both oxalate and glycine synthesis in vivo.MethodsEight healthy volunteers and eight patients with PH1 (stratified by pyridoxine responsiveness) underwent a combined primed continuous infusion of intravenous [1-13C]glycolate, [U-13C2]oxalate, and, in a subgroup, [D5]glycine. Isotopic enrichment of 13C-labeled oxalate and glycolate were measured using a new gas chromatography–tandem mass spectrometry (GC-MS/MS) method. Stable isotope dilution and incorporation calculations quantified rates of appearance and synthetic rates, respectively.ResultsTotal daily oxalate rates of appearance (mean [SD]) were 2.71 (0.54), 1.46 (0.23), and 0.79 (0.15) mmol/d in patients who were pyridoxine unresponsive, patients who were pyridoxine responsive, and controls, respectively (P=0.002). Mean (SD) contribution of glycolate to oxalate production was 47.3% (12.8) in patients and 1.3% (0.7) in controls. Using the incorporation of [1-13C]glycolate tracer in glycine revealed significant conversion of glycolate into glycine in pyridoxine responsive, but not in patients with PH1 who were pyridoxine unresponsive.ConclusionsThis stable isotope infusion protocol could evaluate efficacy of new therapies, investigate pyridoxine responsiveness, and serve as a tool to further explore glyoxylate metabolism in humans.

1. The activity of alanine:glyoxylate aminotransferase (AGT; EC 2.6.1.44) has been measured in the unfractionated livers of 20 patients with primary hyperoxaluria type 1 (PH1), three patients with other forms of primary hyperoxaluria and one PH1 heterozygote. The subcellular distribution of AGT activity was examined in four of the PH1 livers and in the liver of the PH1 heterozygote. 2. The mean AGT activity in the unfractionated PH1 livers was 12.6% of the mean control value. The activities of other aminotransferases and the peroxisomal marker enzymes were normal. When corrected for cross-over from glutamate:glyoxylate aminotransferase (GGT; EC 2.6.1.4), the mean AGT activity in the PH1 livers was reduced to 3.3% of the control values. 3. The livers from a patient with primary hyperoxaluria type 2 (d-glycerate dehydrogenase deficiency) and one with an undefined form of primary hyperoxaluria (possibly oxalate hyperabsorption) had normal AGT levels. The livers of a very mild PH1-type variant and a PH1 heterozygote had intermediate levels of AGT activity. 4. Subcellular fractionation of four PH1 livers by sucrose gradient isopycnic centrifugation demonstrated a complete absence of peroxisomal AGT activity. The subcellular distribution of the residual AGT activity was very similar to that of GGT activity (i.e. mainly cytosolic with a small amount mitochondrial). There were no alterations in the subcellular distributions of any of the peroxisomal marker enzymes. The subcellular distribution of AGT activity in the PH1 heterozygote liver was similar to that of the control (i.e. mainly peroxisomal). 5. The residual AGT activity in two of the PH1 livers, which could be accounted for largely by cross-over from GGT, was only slightly dependent on substrate (glyoxylate and alanine) concentration and virtually independent of cofactor (pyridoxal phosphate) concentration. 6. These data confirm our previous findings (C. J. Danpure & P. R. Jennings, FEBS Letters, 1986, 201, 20–24), but on a much larger number of patients, that AGT deficiency is pathognomic for PH1, and is not found in other forms of hyperoxaluria.

AbstractA retrospective statistical analysis of primary hyperoxaluria type 1 (PH1) in children from June 2016 to May 2019 was carried out to discover its clinical and molecular biological characteristics. Patients were divided into two groups (infant and noninfant) according to clinic type. There were 13 pediatric patients (male:female = 6:7) with PH1 in the cohort from 11 families (four of which were biological siblings from two families), whose median age of symptom onset was 12 months and median confirmed diagnosis age was 14 months. Infant type (6 patients) was the most common type. The infant type mortality rate (100%) was higher than the noninfant (14.3%) (p = 0.029). The incidence of renal failure in infant patients was 67%, while the noninfant was 14.3%. 8 of 10 patients with nephrocalcinosis (NC) (76.92%, 10/13) were diagnosed by radiological imaging examinations, including X-ray (3 patients), CT (4 patients) and MRI (1 patient). NC was an independent risk factor for renal insufficiency [OR 3.33, 95% CI (0.7–1.2)], p < 0.05). Nine types of AGXT gene mutations were found; 1 type, c.190A > T, were first reported here. The most common AGXT gene mutation was c.679_680del, which occurred in exon 6 (5 patients). The infant type is the most common type of pediatric PH, with a relatively higher ratio of renal failure at symptom onset and poor prognosis. NC is an independent risk factor leading to renal failure, and radiological imaging examination is recommended for patients with abnormal ultrasound examination to identify NC. AGXT gene detection is important for the diagnosis and treatment of PH1 in children.

Background: Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disease caused by a mutation in the AGXT gene, resulting in deficiency of the alanineglyoxylate:aminotransferase enzyme. It is characterized by accumulation of oxalate in the kidneys and other organs. Case Presentation: A Syrian woman with a history of nephrolithiasis and heterozygosity for factor V Leiden and prothrombin gene mutations presented with postpartum renal failure. She required initiation of renal replacement therapy at 14 weeks postpartum. Kidney biopsy showed severe acute and chronic crystalline deposition consistent with oxalate nephropathy. Genetic testing revealed a Gly170Arg mutation in the AGXT gene, confirming the diagnosis of PH1. Conclusions: The diagnosis of PH should be considered in patients with severe, recurrent calcium oxalate nephrolithiasis. Early treatment with pyridoxine reduces urinary oxalate excretion and can delay progression to end-stage renal disease (ESRD). After ESRD, intensive dialysis is needed to prevent systemic oxalate accumulation and deposition. Combined liver and kidney transplantation is curative. In our patient, we anticipate that liver transplantation will cure both the hyperoxaluria and the hypercoagulable state.

Background. Primary hyperoxaluria belongs to a group of rare metabolic disorders with autosomal recessive inheritance. It results from genetic mutations of theAGXTgene, which is more common due to higher consanguinity rates in the developing countries. Clinical features at presentation are heterogeneous even in children from the same family; this study was conducted to determine the clinical characteristics, type ofAGXTmutation, and outcome in children diagnosed with PH1 at a tertiary referral center in Oman.Method. Retrospective review of children diagnosed with PH1 at a tertiary hospital in Oman from 2000 to 2013.Result. Total of 18 children were identified. Females composed 61% of the children with median presentation age of 7 months. Severe renal failure was initial presentation in 39% and 22% presented with nephrocalcinosis and/or renal calculi. Family screening diagnosed 39% of patients. Fifty percent of the children underwent hemodialysis. 28% of children underwent organ transplantation. The most common mutation found in Omani children was c.33-34insC mutation in theAGXTgene.Conclusion. Due to consanguinity, PH1 is a common cause of ESRD in Omani children. Genetic testing is recommended to help in family counseling and helps in decreasing the incidence and disease burden; it also could be utilized for premarital screening.

Primary Hyperoxaluria Type 1 (PH1) is a rare autosomal recessive disorder characterized by the deposition of insoluble calcium oxalate crystals at first in the kidneys and urinary tract and then in the whole body. PH1 is caused by the deficiency of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT). AGT is a pyridoxal 5'-phosphate (PLP)-dependent enzyme, which converts glyoxylate to glycine, thus preventing glyoxylate oxidation to oxalate and calcium oxalate formation. Only two curative therapeutic approaches are currently available for PH1: the administration of pyridoxine (PN), a precursor of PLP, which is only effective in a minority of patients (25- 35%), and liver transplantation, a very invasive procedure. AGT is encoded by the AGXT gene, which is present in humans as two polymorphic forms: the major allele (encoding AGT-Ma) and the minor allele (encoding AGT-Mi). PH1 is a very heterogeneous disease with respect to the clinical manifestations, the response to treatment and the pathogenic mechanisms. In fact, more than 200 pathogenic mutations have been identified so far and the molecular mechanisms by which missense mutations cause AGT deficiency span from functional, to structural and to subcellular localization defects or to a combination of them. Several lines of evidence at both molecular and cellular level, indicate that many disease-causing missense mutations interfere with AGT dimer stability and/or aggregation propensity. However, neither the dimerization nor the aggregation process of AGT have been analyzed in detail. Therefore, we engineered a mutant form of AGT stable in solution in the monomeric form and studied its biochemical properties and dimerization kinetics. We found that monomeric AGT is able to bind PLP and that the coenzyme stabilizes the dimeric structure. Moreover, the identification of key dimerization hot-spots at the monomer-monomer interface allowed us to unravel the mechanisms at the basis of the aberrant mitochondrial mistargeting of two of the most common PH1-causing variants. We also elucidated the molecular and cellular consequences of the pathogenic mutations R36H, G42E, I56N, G63R and G216R, involving residues located at the dimer interface, and tested their in-vitro responsiveness to the treatment with PN. The latter results allowed us to suggest a possible correlation between the structural defect of a variant and its degree of responsiveness to PN. Finally, by combining bioinformatic and biochemical approaches, we analyzed in detail the tendency of AGT to undergo an electrostatically-driven aggregation. We found that the polymorphic changes typical of the minor allele have opposite effect on the aggregation propensity of the protein, and we predicted the possible effect/s of pathogenic mutations of residues located on the AGT surface. Overall, the results obtained allow not only to better understand PH1 pathogenesis, but also to predict the response of the patients to the available therapies as well as to pave the way for the development of new therapeutic strategies.

L’iperossaluria primaria di tipo 1 (PH1) è una malattia autosomica recessiva rara caratterizzata dal deposito di cristalli insolubili di ossalato di calcio prima nei reni e nel tratto urinario ed in seguito, in assenza di un appropriato trattamento, in tutto il resto del corpo. PH1 è causata da un deficit funzionale di alanina:gliossilato aminotransferasi umana (AGT), un enzima piridossal 5’-fosfato (PLP) dipendente che converte il gliossilato in glicina, prevenendo in tal modo la ossidazione del gliossilato ad ossalato e quindi la formazione di cristalli insolubili di ossalato di calcio. L’AGT normale è codificato dal gene AGXT che esiste nella popolazione umana in 2 varianti polimorfiche: l’allele maggiore (AGT-Ma) e l’allele minore (AGT-Mi), quest’ultimo caratterizzato da due mutazioni puntiformi, che portano alla sostituzione della prolina 11 con una leucina e della isoleucina 340 con una metionina, e da una duplicazione di 74 paia di basi nell’introne 1. Sebbene la presenza dell’allele minore non sia in sè patogenica, questa rende però l’enzima più suscettibile all’effetto di alcune mutazioni che non sarebbero patogeniche se associate all’allele minore. Perciò, c’è un grande interesse nel definire le proprietà dell’AGT-Mi, in quanto punto di partenza per capire il meccanismo molecolare alla base del sinergismo tra l’AGT-Mi e le mutazioni patogeniche che cosegregano con esso. In questo lavoro, tramite un approccio “in vitro” su proteine purificate, sono stati studiati gli effetti delle 2 mutazioni combinate polimorfiche proprie dell’allele minore, sulle caratteristiche biochimiche dell’AGT, così come di 2 mutazioni che provacono PH1 se associate all’allele minore: F152I e G170R. I dati ottenuti hanno evidenziato che: 1) AGT-Mi mostra caratteristiche spettroscopiche, parametri cinetici, e affinità per il PLP simili a quelle di AGT-Ma. Tuttavia, la sua struttura dimerica è caratterizzata da una bassa resistenza allo stress sia chimico che termico. Questi effetti sembrano essere dovuti alla mutazione P11L dal momento che tale variante mostra un profilo di denaturazione comparabile con quello di AGT-Mi; 2) La mutazione patgenica F152I porta ad una diminuzione di ca. 200 volte nell’affinità dell’AGT per la piridossamina 5-fosfato (PMP) e quando associata all’allele minore, anche ad una inattivazione tempo dipendente ed ad una aggregazione a temperatura fisiologica.; 3) La mutazione patogenica G170R non incide né sulle proprietà spettroscopiche né su quelle cinetiche dell’AGT-Mi in condizioni native. D’altro canto, rende la struttura dimerica dell’apoG170R-Mi più sucettibile alla dissociazione rispetto al corrispondente apoAGT-Mi. Riassumendo i dati ottenuti: (i) rivelano le differenze tra AGT-Ma e AGT-Mi; (ii) gettano luce sul difetto molecolare associato alle varianti F152I-Mi e G170R-Mi; (iii) permettono di fare ipotesi sulla risposta alla terapia con piridossina osservata nei pazienti recanti queste 2 mutazioni.
Primary hyperoxaluria type 1 (PH1) is a rare autosomal recessive disorder characterized by the deposition of insoluble calcium oxalate crystals at first in the kidneys and urinary tract and then, in the absence of appropriate treatments, in the whole body. PH1 is caused by the deficiency of human liver peroxisomal alanine:glyoxylate aminotransferase (AGT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme that converts glyoxylate to glycine, thus preventing glyoxylate oxidation to oxalate and therefore the formation of calcium oxalate. Normal human AGT is encoded by the AGXT gene that exists in human populations in two polymorphic forms: the major allele (AGT-Ma) and the minor allele (AGT-Mi), which is characterized by two point mutations, leading to the Pro11Leu and Ile340Met substitutions, and a 74 bp-duplication in intron 1. Although the presence of the minor allele polymorphism is not pathogenic “per se”, it makes AGT more susceptible to the effect of some PH1-causing mutation that are expected to be not pathogenic when associated with the major allele. Thus, there is a great interest in defining the properties of AGT-Mi, as the base to unravel the molecular mechanism underlying the synergism between AGT-Mi and the pathogenic mutations that cosegregate with it. In this work, by an “in vitro” approach on purified proteins, we studied the effects on the biochemical features of AGT of the two combined polymorphic mutations typical of the minor allele as well as of two PH1-causing mutations associated with the minor allele, Phe152Ile and Gly170Arg. The data obtained have shown that: 1) AGT-Mi displays spectral features, kinetic parameters, and PLP binding affinity similar to those of AGT-Ma. However, its dimeric structure is characterized by a low resistance to both chemical and thermal stress. This appears to be due to the P11L mutation since the P11L variant exhibits a denaturation pattern comparable to that of AGT-Mi; 2) The PH1-causing F152I mutation leads to a ~200 fold decrease in the affinity of AGT for pyridoxamine 5’-phosphate and, when associated with the minor allele polymorphism, to a time-dependent inactivation and aggregation at physiological temperature; 3) The pathogenic mutation G170R does not affect neither the spectroscopic nor the kinetic properties of AGT-Mi under native conditions. However, it makes the dimeric structure of apoG170R-Mi more susceptible to dissociation than the corresponding apoAGT-Mi. Overall, the obtained data: (i) reveal the biochemical differences between AGT-Ma and AGT-Mi; (ii) allow to shed light on the molecular defect associated with the F152-Mi and the G170R-Mi variants; (iii) permit to speculate on the responsiveness to pyridoxine therapy of the patients bearing these mutations.

Iperossaluria Primaria tipo I ( PH1 ) è una rara malattia autosomica recessiva caratterizzata da un elevato livello di ossalato nelle urine , che provoca la formazione di cristalli insolubili di ossalato di calcio dapprima nei reni e delle vie urinarie e , in assenza di un adeguato trattamento , in tutto il corpo . PH1 è causata da un deficit dell'enzima epatico Alanina: Gliossilato aminotransferasi ( AGT ). AGT è un enzima piridossal 5' - fosfato perossisomi ( PLP )-dipendente che converte il gliossilato in glicina, impedendo così l'ossidazione gliossilato di ossalato e la successiva formazione di ossalato di calcio. AGT è codificata dal gene AGXT, che presenta nell'uomo, due forme polimorfe : l'allele maggiore (codifica AGT - Ma) e l'allele minore (codificante AGT - Mi). Finora sono state identificate più di 150 mutazioni associate a PH1. Diversi studi hanno consentito interessanti progressi nella comprensione dei meccanismi molecolari per cui ciascuna mutazione conduce alla carenza di AGT . Tuttavia, molto spesso i pazienti affetti da PH1 sono eterozigoti composti e il loro fenotipo enzimatico potrebbe dipendere da fenomeni di complementazione interallelic ( IC ). Fino ad ora , la patogenesi di PH1 è stata studiata solo tramite approcci che mimano la situazione cellulare di pazienti omozigoti , mentre il fenotipo clinico relazione genotipo- fenotipo enzimatico di pazienti eterozigoti composti è completamente sconosciuto . Durante il mio dottorato di ricerca , ho condotto studi volti a chiarire il fenotipo enzimatico legata alla mutazione S81L su AGT -Ma , concernente un residuo di interazione con il PLP , sia in omozigosi sia in eterozigosi composta con la mutazione G170R , la variante più comune in AGT – Mi. G170R è nota per determinare il mistargeting mitocondriale di AGT senza alterare le proprietà funzionali dell'enzima stesso. Utilizzando un vettore di espressione eucariotico bicistronico abbiamo dimostrato che ( i) S81L -Ma ha una significativa ocalizzazione perossisomale, e ( ii ) l'interazione dei monomeri S81L e G170R si verifica nella cellula formando un eterodimero G170R-Mi/S81L-Ma, che viene importato in perossisomi e presenta una funzionalità migliorata rispetto agli enzimi parentali . Questi dati , integrati con i risultati della caratterizzazione biochimica dell' eterodimero purificato ottenuti da un vettore di espressione procariotico, sostengono l'ipotesi di un IC positiva tra i monomeri S81L e G170R. Questo studio rappresenta la prima indagine della patogenesi della PH1 in pazienti eterozigoti composti a livello molecolare. PH1 è una malattia molto difficile da curare. Solo due approcci terapeutici sono attualmente a disposizione: la somministrazione di piridossina , un precursore di PLP, che è efficace solo in una minoranza di pazienti, e il trapianto di fegato, una procedura molto invasiva . Ne consegue che lo sviluppo di nuove strategie di trattamento , meno invasive ed efficaci per tutti i pazienti , sarebbe altamente desiderabile. Poiché PH1 è originata dal deficit di un singolo enzima , la possibilità di ripristinare la capacità catalitica degli epatociti somministrando enzima esogeno è una prospettiva intrigante. Uno dei problemi principali per lo sviluppo di una terapia somministrazione enzima è l'ingresso intracellulare della proteina esogena. Durante il mio dottorato di ricerca ho utilizzato un duplice approccio per ottenere una forma AGT in grado di attraversare il plasma membrana: ( i) la costruzione di una proteina di fusione tra AGT e la Tat peptide sfruttando le capacità di attraversamento di membrana del domino Tat , e ( ii ) la coniugazione di AGT con un nanocarrier polimerico in grado di trasportare l'enzima funzionale attraverso la membrana plasmatica. Entrambe le strategiehanno dimostrato di essere efficaci nella trasduzione di AGT in un modello cellulare permettendo di ripristinare la capacità di disintossicazione gliossilato senza alterare significativamente le proprietà strutturali e funzionali di AGT. Questi risultati possono essere considerati un incoraggiante punto di partenza per lo sviluppo di una terapia somministrazione enzima per PH1.
Primary Hyperoxaluria Type I (PH1) is a rare autosomal recessive disorder characterized by a high level of oxalate in the urine, which in turn results in the formation of insoluble calcium oxalate crystals at first in the kidneys and urinary tract and then, in absence of an appropriate treatment, in the whole body. PH1 is caused by the deficiency of human liver alanine:glyoxylate aminotransferase (AGT), a peroxisomal pyridoxal 5'-phosphate (PLP)-dependent enzyme. AGT detoxifies glyoxylate to glycine, thus preventing glyoxylate oxidation to oxalate and the subsequent calcium oxalate formation. AGT is encoded by the AGXT gene, which presents, in humans, two polymorphic forms: the major allele (encoding AGT-Ma) and the minor allele (encoding AGT-Mi). At the time of writing, more than 150 mutations associated with PH1 have been reported and several studies allowed for interesting progresses in the understanding of the molecular mechanisms by which each mutation leads to AGT deficiency. However, quite often patients affected by PH1 are compound heterozygous and their enzymatic phenotype could depend on interallelic complementation (IC) effects. Until now, the pathogenesis of PH1 has been only studied by approaches mimicking homozygous patients, while the genotype-enzymatic phenotype-clinical phenotype relationship of compound heterozygous patients is completely unknown. During my PhD, we elucidated the enzymatic phenotype linked to the S81L mutation on AGT-Ma, concerning a PLP binding residue, and how it changes when the most common mutation G170R on AGT-Mi, known to cause AGT mistargeting without affecting the enzyme functional properties, is present in the second allele. By using a bicistronic eukaryotic expression vector we demonstrated that (i) S81L-Ma has a significant peroxisomal localization, and (ii) the interaction of the S81L and G170R monomers occurs in the cell yielding the G170R-Mi/S81L-Ma heterodimer, which is imported into peroxisomes and exhibits an enhanced functionality with respect to the parental enzymes. These data, integrated with the biochemical features of the recombinant purified heterodimer compared with those of the homodimeric counterparts obtained by a dual vector prokaryotic expression strategy, provided evidence for a positive IC between the S81L and G170R monomers. This study represents the first investigation of the pathogenesis of PH1 in compound heterozygous patients at molecular level. PH1 is a very difficult-to-treat disease. Only two curative therapeutic approaches are currently available: the administration of pyridoxine, a precursor of PLP that is only effective in a minority of patients, and liver transplantation, a very invasive procedure. It follows that the development of new treatment strategies, less invasive and effective for all the patients, would be highly desirable. In this regard, since PH1 originates from the deficit of a single enzyme, the opportunity to restore the catalytic pool of the hepatocytes by administering exogenous enzyme is an intriguing perspective. One of the major issues for the development of an enzyme administration therapy is the intracellular delivery of the exogenous protein. During my PhD, to obtain an AGT form able to cross the plasma membrane, a dual approach was used: (i) the construction of a fusion protein between AGT and the Tat peptide exploiting the membrane crossing capabilities of the Tat moiety, and (ii) the conjugation of AGT with a polymeric nanocarrier able to deliver the functional enzyme across the plasma membrane. Both strategies did not significantly alter the structural and functional properties of AGT and proved to be effective in transducing active AGT into a cellular disease model and in restoring their glyoxylate detoxification ability. These results can be considered an encouraging starting point for the development of an enzyme administration therapy for PH1.