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Aldighieri, Enrico (2008) Human primary aldosteronism; investigation of the mechanism leading to autonomous aldosterone excess. [Tesi di dottorato]

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Abstract (inglese)

Objective: Due to its unique expression of aldosterone synthase cytocromo P450 (CYP11B2), the enzyme required for the final steps of aldosterone biosynthesis aldosterone is exclusively expressed in the adrenalcortical zona glomerulosa cell. Some studies suggested that the CYP11B2 gene expression can be higher in APAs than in normal adrenocortical tissues, leading to postulate a transcriptional modulation of aldosterone overproduction in these tumors but antibodies capable to identify this protein do not exist. Thus we performed studies of in-situ hybridization (ISH) to analyze CYP11B2 localization. Another aim of our study is to understand whether the occurrence of micronodularity peripheral to the main adenomatous mass in some of our patient could be involved in the pathophysiology of the adenoma. Our hypothesis is that a similar humoral stimulating factor could play a role in the genesis of APA.
Method: We developed a novel non radioactive in-situ hybridization technique based on use of a CYP11B2 specific oligo probe to localize the specific transcripts of the CYP11B2. Adrenocortical tissue from patients was studied. As housekeeping gene, we used PBGD, (NM_000190) probe labeled with Digoxigenin. The oligonucleotide probe for the CYP11B2 was 5' conjugated with DIG. Negative controls was detected with probe not labeled. The second approach was measuring of serum auto-antibodies anti AT1 in patients with APA and their localization on adrenal gland tissue by IHC.
Results: At hystopathology intensely labeled areas called "nodules producing aldosterone" and negative areas were detected in the APA analyzed. ISH showed specific staining of all nodules. The staining was predominantly nuclear, a finding consistent with a good preservation of the transcript at this level. The AAbodies' anti AT1 presence has been demonstrated with ELISA test but the tissue localization still did not show appreciable results.
Conclusion: this novel non radioactive ISH technique unequivocally demostrated the presence of aldosterone-producing microsatellites. High levels of AutoAntibodies in the serum of patients with APA allow us to hypothesize that this adrenal gland disorder is autoimmune.

Abstract (italiano)

Obiettivo: Alcuni studi hanno suggerito che l'espressione genica CYP11B2 è più elevata in pazienti con APA rispetto ai soggetti normali, tuttavia non esistono anticorpi in grado di identificare questa proteina. Quindi abbiamo deciso di effettuare studi di ibridazione in situ (ISH) per localizzare CYP11B2 a livello tessutale. Un altro obiettivo del nostro studio è quello di capire se la presenza di micronoduli possa influire sulla fisiopatologia dell'adenoma. La nostra ipotesi è che un fattore umorale stimolante potrebbe svolgere un ruolo nella genesi di APA.
Metodo: Abbiamo sviluppato un metodo non radioattivo basato sull'uso di uno specifica sonda oligo per localizzare CYP11B2. Per la rilevazione dell'housekeeping gene, abbiamo utilizzato una sonda a doppio filamento per il gene PBGD, (NM_000190). Entrambe le sonde sono state marcate con Digoxigenin. Abbiamo inoltre misurato i livelli di anticorpi anti-AT1 nel siero di pazienti con APA e la loro localizzazione tessutale tramite IHC nella ghiandola surrenalica.
Risultati: all'istopatologia si sono evidenziati noduli satelliti che presentavano lèespressione del CYP11B2. L'ISH ha mostrato una colorazione specifica di tutti i noduli a livello prevalentemente nucleare. La presenza di Auto anticorpi anti AT1 è stata dimostrata con test ELISA nel siero, ma la localizzazione nel tessuto non ha ancora mostrato risultati apprezzabili.
Conclusione: questa tecnica non radioattiva di ISH dimostra in modo inequivocabile la presenza di micro satelliti producenti aldosterone. Elevati livelli di autoanticorpi nel siero dei pazienti con APA ci permettono di ipotizzare che questo disordine della ghiandola surrenale possa essere di origine autoimmune.

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Tipo di EPrint:Tesi di dottorato
Relatore:Rossi, Gian Paolo
Dottorato (corsi e scuole):Ciclo 21 > Corsi per il 21simo ciclo > IPERTENSIONE ARTERIOSA E BIOLOGIA VASCOLARE
Data di deposito della tesi:NON SPECIFICATO
Anno di Pubblicazione:13 Marzo 2008
Parole chiave (italiano / inglese):Aldosteronismo, APA, Ibridazione in situ, Autoimmune
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/13 Endocrinologia
Struttura di riferimento:Dipartimenti > Dipartimento di Medicina Clinica e Sperimentale
Codice ID:2030
Depositato il:03 Lug 2009 10:47
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